Maria Angela VANDELLI - personale UniMoRe

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Maria Angela VANDELLI

Professore Ordinario
Dipartimento Scienze della Vita sede ex Scienze Farmaceutiche Via Campi 103

Pubblicazioni

- Drug Delivery Peptides [Brevetto]
Costantino, Luca; Forni, Flavio; Vandelli, Maria Angela
abstract

Disclosed is a peptide comprising the sequence: H2N-Gly-Phe-D-Thr-Gly-Phe-Leu-Ser-CONH2, wherein the serine residue may be functionalized with sugar residues; other aminoacids can replace the first 2 amino acids of the N-terminal portion, the order of which can be reversed, and their no. may be different from 2. Thus, a data anal. obtained from the in vivo study with fluorescence microscopy clearly shows that the nanoparticles obtained with PLGA modified with the peptide H2N-Gly-Phe-D-Thr-Gly-Phe-Leu-Ser-CONH2, and to a greater extent with the peptide H2N-Gly-Phe-D-Thr-Gly-Phe-Leu-SerO-.beta.-D-glucose-CONH2 are able to cross the BBB and penetrate into the brain parenchyma. However, the nanoparticles only constituted by PLGA are unable to cross the blood-brain barrier, and remain in the blood vessels.

- Peptidi per la veicolazione di farmaci [Brevetto]
Costantino, Luca; Vandelli, Maria Angela; Forni, Flavio
abstract

Sintesi e coniugazione di sequenze peptidiche per la veicolazione di farmaci al sistema nervoso centrale

- Sistema di veicolazione e rilascio di farmaci antiparassitari e loro uso in campo medico [Brevetto]
Pozio, Edoardo; Ludovisi, Alessandra; Gómez Morales Maria, Angeles; Tosi, Giovanni; Ruozi, Barbara; Vandelli, Maria Angela
abstract

Molte delle infezioni di origine parassitaria sia umana che animale sono orfane di farmaci di scelta per la mancanza di molecole efficaci verso questi patogeni. A tale proposito abbiamo voluto verificare se l’utilizzo di nanoparticelle modificate caricate con un farmaco aumentasse l’efficacia del principio attivo nei confronti di questi patogeni. Come modello sperimentale sono stati utilizzati parassiti del genere Cryptosporidum agenti eziologici di zoonosi e che infettano un gran numero di ospiti vertebrati, costituendo una delle cause più importanti di enteriti nell’uomo e negli animali, per i quali non esiste al giorno d’oggi una terapia efficace. E’ stata dimostrata precedentemente la capacità degli inibitori delle proteasi, e in particolare dell’Indinavir (IND), di ridurre l’infezione da Cryptosporidium in modelli sperimentali in vitro e in vivo. Tuttavia esistono limitazioni all’uso di questi inibitori, dovute all’alto grado di degradazione metabolica e alla grave tossicità riscontrata quando vengono usati a dosi elevate. Abbiamo usato nanoparticelle biodegradabili (Poly(D,L-lactide-co-glycolide, Np), come sistema capace di proteggere l’IND da un metabolismo intenso che porta ad una sua rapida degradazione. Inoltre, la successiva modifica della superficie delle Np mediante legame con un anticorpo policlonale specifico verso questo patogeno (Cryptosporidium), ha originato un sistema di delivery del farmaco altamente selettivo nei confronti del parassita. Le Np caricate con IND e modificate non hanno mostrato alcuna modificazione nè dimensionale nè nella carica superficiale, ma hanno esibito una elevata affinità verso il bersaglio. L’utilizzo delle NP così modificate e caricate con l’IND ha indotto una riduzione dell’infezione da Cryptosporidium tempo dipendente, così che dopo solo 96 ore di trattamento già si osserva una riduzione del 70% dei parassiti rispetto al controllo non trattato.

2023 - "Combo" Multi-Target Pharmacological Therapy and New Formulations to Reduce Inflammation and Improve Endogenous Remyelination in Traumatic Spinal Cord Injury [Articolo su rivista]
Moretti, Marzia; Caraffi, Riccardo; Lorenzini, Luca; Ottonelli, Ilaria; Sannia, Michele; Alastra, Giuseppe; Baldassarro, Vito Antonio; Giuliani, Alessandro; Duskey, Jason Thomas; Cescatti, Maura; Ruozi, Barbara; Aloe, Luigi; Vandelli, Maria Angela; Giardino, Luciana; Tosi, Giovanni; Calzà, Laura
abstract

Spinal cord injury (SCI) is characterized by a cascade of events that lead to sensory and motor disabilities. To date, this condition is irreversible, and no cure exists. To improve myelin repair and limit secondary degeneration, we developed a multitherapy based on nanomedicines (NMeds) loaded with the promyelinating agent triiodothyronine (T3), used in combination with systemic ibuprofen and mouse nerve growth factor (mNGF). Poly-L-lactic-co-glycolic acid (PLGA) NMeds were optimized and loaded with T3 to promote sustained release. In vitro experiments confirmed the efficacy of T3-NMeds to differentiate oligodendrocyte precursor cells. In vivo rat experiments were performed in contusion SCI to explore the NMed biodistribution and efficacy of combo drugs at short- and long-term post-lesion. A strong anti-inflammatory effect was observed in the short term with a reduction of type M1 microglia and glutamate levels, but with a subsequent increase of TREM2. In the long term, an improvement of myelination in NG2-IR, an increase in MBP content, and a reduction of the demyelination area were observed. These data demonstrated that NMeds can successfully be used to obtain more controlled local drug delivery and that this multiple treatment could be effective in improving the outcome of SCIs.

2023 - Chronic cholesterol administration to the brain supports complete and long-lasting cognitive and motor amelioration in Huntington's disease [Articolo su rivista]
Birolini, Giulia; Valenza, Marta; Ottonelli, Ilaria; Talpo, Francesca; Minoli, Lucia; Cappelleri, Andrea; Bombaci, Mauro; Caccia, Claudio; Canevari, Caterina; Trucco, Arianna; Leoni, Valerio; Passoni, Alice; Favagrossa, Monica; Nucera, Maria Rosaria; Colombo, Laura; Paltrinieri, Saverio; Bagnati, Renzo; Duskey, Jason Thomas; Caraffi, Riccardo; Vandelli, Maria Angela; Taroni, Franco; Salmona, Mario; Scanziani, Eugenio; Biella, Gerardo; Ruozi, Barbara; Tosi, Giovanni; Cattaneo, Elena
abstract

: Evidence that Huntington's disease (HD) is characterized by impaired cholesterol biosynthesis in the brain has led to strategies to increase its level in the brain of the rapidly progressing R6/2 mouse model, with a positive therapeutic outcome. Here we tested the long-term efficacy of chronic administration of cholesterol to the brain of the slowly progressing zQ175DN knock-in HD mice in preventing ("early treatment") or reversing ("late treatment") HD symptoms. To do this we used the most advanced formulation of cholesterol loaded brain-permeable nanoparticles (NPs), termed hybrid-g7-NPs-chol, which were injected intraperitoneally. We show that one cycle of treatment with hybrid-g7-NPs-chol, administered in the presymptomatic ("early treatment") or symptomatic ("late treatment") stages is sufficient to normalize cognitive defects up to 5 months, as well as to improve other behavioral and neuropathological parameters. A multiple cycle treatment combining both early and late treatments ("2 cycle treatment") lasting 6 months generates therapeutic effects for more than 11 months, without severe adverse reactions. Sustained cholesterol delivery to the brain of zQ175DN mice also reduces mutant Huntingtin aggregates in both the striatum and cortex and completely normalizes synaptic communication in the striatal medium spiny neurons compared to saline-treated HD mice. Furthermore, through a meta-analysis of published and current data, we demonstrated the power of hybrid-g7-NPs-chol and other strategies able to increase brain cholesterol biosynthesis, to reverse cognitive decline and counteract the formation of mutant Huntingtin aggregates. These results demonstrate that cholesterol delivery via brain-permeable NPs is a therapeutic option to sustainably reverse HD-related behavioral decline and neuropathological signs over time, highlighting the therapeutic potential of cholesterol-based strategies in HD patients. DATA AVAILABILITY: This study does not include data deposited in public repositories. Data are available on request to the corresponding authors.

2023 - Corrigendum to “Quantitative comparison of the protein corona of nanoparticles with different matrices” [Int J Pharm X 2022 Oct 21;4: 100136] (International Journal of Pharmaceutics: X (2022) 4, (S2590156722000251), (10.1016/j.ijpx.2022.100136)) [Articolo su rivista]
Ottonelli, I.; Duskey, J. T.; Genovese, F.; Pederzoli, F.; Caraffi, R.; Valenza, M.; Tosi, G.; Vandelli, M. A.; Ruozi, B.
abstract

: [This corrects the article DOI: 10.1016/j.ijpx.2022.100136.].

2023 - Enantioseparation of chiral phytocannabinoids in medicinal cannabis [Articolo su rivista]
Russo, Fabiana; Tolomeo, Francesco; Vandelli, Maria Angela; Biagini, Giuseppe; Laganà, Aldo; Laura Capriotti, Anna; Cerrato, Andrea; Carbone, Luigi; Perrone, Elisabetta; Cavazzini, Alberto; Maiorano, Vincenzo; Gigli, Giuseppe; Cannazza, Giuseppe; Citti, Cinzia
abstract

The evaluation of the chiral composition of phytocannabinoids in the cannabis plant is particularly important as the pharmacological effects of the (+) and (-) enantiomers of these compounds are completely different. Chromatographic attempts to assess the presence of the minor (+) enantiomers of the main phytocannabinoids, cannabidiolic acid (CBDA) and trans-Δ9-tetrahydrocannabinolic acid (trans-Δ9-THCA), were carried out on heated plant extracts for the determination of the corresponding decarboxylated species, cannabidiol (CBD) and trans-Δ9-tetrahydrocannabinol (trans-Δ9-THC), respectively. This process produces an altered phytocannabinoid composition with several new and unknown decomposition products. The present work reports for the first time the stereoselective synthesis of the pure (+) enantiomers of the main phytocannabinoids, trans-CBDA, trans-Δ9-THCA, trans-CBD and trans-Δ9-THC, and the development and optimization of an achiral-chiral liquid chromatography method coupled to UV and high-resolution mass spectrometry detection in reversed phase conditions (RP-HPLC-UV-HRMS) for the isolation of the single compounds and evaluation of their actual enantiomeric composition in plant. The isolation of the peaks with the achiral stationary phase ensured the absence of interferences that could potentially co-elute with the analytes of interest in the chiral analysis. The method applied to the Italian medicinal cannabis variety FM2 revealed no trace of the (+) enantiomers for all phytocannabinoids under investigation before and after decarboxylation, thus suggesting that the extraction procedure does not lead to an inversion of configuration.

2023 - Nanowired Delivery of Curcumin Attenuates Methamphetamine Neurotoxicity and Elevates Levels of Dopamine and Brain-Derived Neurotrophic Factor [Capitolo/Saggio]
Ottonelli, Ilaria; Sharma, Aruna; Ruozi, Barbara; Tosi, Giovanni; Duskey, Jason Thomas; Vandelli, Maria Angela; Lafuente, José Vicente; Nozari, Ala; Muresanu, Dafin Fior; Buzoianu, Anca Dana; Tian, Z Ryan; Zhang, Zhiqiang; Li, Cong; Feng, Lianyuan; Wiklund, Lars; Sharma, Hari Shanker
abstract

: Curcumin is a well-known antioxidant used as traditional medicine in China and India since ages to treat variety of inflammatory ailments as a food supplement. Curcumin has antitumor properties with neuroprotective effects in Alzheimer's disease. Curcumin elevates brain-derived neurotrophic factor (BDNF) and dopamine (DA) levels in the brain indicating its role in substance abuse. Methamphetamine (METH) is one of the most abused substances in the world that induces profound neurotoxicity by inducing breakdown of the blood-brain barrier (BBB), vasogenic edema and cellular injuries. However, influence of curcumin on METH-induced neurotoxicity is still not well investigated. In this investigation, METH neurotoxicity and neuroprotective effects of curcumin nanodelivery were examined in a rat model. METH (20 mg/kg, i.p.) neurotoxicity is evident 4 h after its administration exhibiting breakdown of BBB to Evans blue albumin in the cerebral cortex, hippocampus, cerebellum, thalamus and hypothalamus associated with vasogenic brain edema as seen measured using water content in all these regions. Nissl attaining exhibited profound neuronal injuries in the regions of BBB damage. Normal curcumin (50 mg/kg, i.v.) 30 min after METH administration was able to reduce BBB breakdown and brain edema partially in some of the above brain regions. However, TiO2 nanowired delivery of curcumin (25 mg/kg, i.v.) significantly attenuated brain edema, neuronal injuries and the BBB leakage in all the brain areas. BDNF level showed a significant higher level in METH-treated rats as compared to saline-treated METH group. Significantly enhanced DA levels in METH-treated rats were also observed with nanowired delivery of curcumin. Normal curcumin was able to slightly elevate DA and BDNF levels in the selected brain regions. Taken together, our observations are the first to show that nanodelivery of curcumin induces superior neuroprotection in METH neurotoxicity probable by enhancing BDNF and DA levels in the brain, not reported earlier.

2023 - Optimization of an Injectable Hydrogel Depot System for the Controlled Release of Retinal-Targeted Hybrid Nanoparticles [Articolo su rivista]
Ottonelli, I.; Bighinati, A.; Adani, E.; Loll, F.; Caraffi, R.; Vandelli, M. A.; Boury, F.; Tosi, G.; Duskey, J. T.; Marigo, V.; Ruozi, B.
abstract

A drawback in the development of treatments that can reach the retina is the presence of barriers in the eye that restrain compounds from reaching the target. Intravitreal injections hold promise for retinal delivery, but the natural defenses in the vitreous can rapidly degrade or eliminate therapeutic molecules. Injectable hydrogel implants, which act as a reservoir, can allow for long-term drug delivery with a single injection into the eye, but still suffer due to the fast clearance of the released drugs when traversing the vitreous and random diffusion that leads to lower pharmaceutic efficacy. A combination with HA-covered nanoparticles, which can be released from the gel and more readily pass through the vitreous to increase the delivery of therapeutic agents to the retina, represents an advanced and elegant way to overcome some of the limitations in eye drug delivery. In this article, we developed hybrid PLGA-Dotap NPs that, due to their hyaluronic acid coating, can improve in vivo distribution throughout the vitreous and delivery to retinal cells. Moreover, a hydrogel implant was developed to act as a depot for the hybrid NPs to better control and slow their release. These results are a first step to improve the treatment of retinal diseases by protecting and transporting the therapeutic treatment across the vitreous and to improve treatment options by creating a depot system for long-term treatments.

2023 - Recent Advances on Surface-Modified GBM Targeted Nanoparticles: Targeting Strategies and Surface Characterization [Articolo su rivista]
Rodà, Francesca; Caraffi, Riccardo; Picciolini, Silvia; Tosi, Giovanni; Vandelli, Maria Angela; Ruozi, Barbara; Bedoni, Marzia; Ottonelli, Ilaria; Duskey, Jason Thomas
abstract

Glioblastoma multiforme (GBM) is the most common malignant brain tumor, associated with low long-term survival. Nanoparticles (NPs) developed against GBM are a promising strategy to improve current therapies, by enhancing the brain delivery of active molecules and reducing off-target effects. In particular, NPs hold high potential for the targeted delivery of chemotherapeutics both across the blood-brain barrier (BBB) and specifically to GBM cell receptors, pathways, or the tumor microenvironment (TME). In this review, the most recent strategies to deliver drugs to GBM are explored. The main focus is on how surface functionalizations are essential for BBB crossing and for tumor specific targeting. We give a critical analysis of the various ligand-based approaches that have been used to target specific cancer cell receptors and the TME, or to interfere with the signaling pathways of GBM. Despite the increasing application of NPs in the clinical setting, new methods for ligand and surface characterization are needed to optimize the synthesis, as well as to predict their in vivo behavior. An expert opinion is given on the future of this research and what is still missing to create and characterize a functional NP system for improved GBM targeting.

2023 - Synthesis and pharmacological activity of the epimers of hexahydrocannabinol (HHC) [Articolo su rivista]
Russo, Fabiana; Vandelli, Maria Angela; Biagini, Giuseppe; Schmid, Martin; Luongo, Livio; Perrone, Michela; Ricciardi, Federica; Maione, Sabatino; Laganà, Aldo; Capriotti, Anna Laura; Gallo, Alfonso; Carbone, Luigi; Perrone, Elisabetta; Gigli, Giuseppe; Cannazza, Giuseppe; Citti, Cinzia
abstract

Cannabis is a multifaceted plant with numerous therapeutic properties on one hand, and controversial psychotropic activities on the other hand, which are modulated by CB1 endocannabinoid receptors. Δ9-Tetrahydrocannabinol (Δ9-THC) has been identified as the main component responsible for the psychotropic effects, while its constitutional isomer cannabidiol (CBD) has shown completely different pharmacological properties. Due to its reported beneficial effects, Cannabis has gained global popularity and is openly sold in shops and online. To circumvent legal restrictions, semisynthetic derivatives of CBD are now frequently added to cannabis products, producing "high" effects similar to those induced by Δ9-THC. The first semi-synthetic cannabinoid to appear in the EU was obtained through cyclization and hydrogenation of CBD, and is known as hexahydrocannabinol (HHC). Currently, there is limited knowledge regarding HHC, its pharmacological properties, and its prevalence, as it is not commonly investigated in routine toxicological assays. In this study, synthetic strategies were explored to obtain an excess of the active epimer of HHC. Furthermore, the two epimers were purified and individually tested for their cannabinomimetic activity. Lastly, a simple and rapid chromatographic method employing a UV detector and a high-resolution mass spectrometer was applied to identify and quantify up to ten major phytocannabinoids, as well as the HHC epimers, in commercial cannabis samples.

2022 - Applications of the ROS-Responsive Thioketal Linker for the Production of Smart Nanomedicines [Articolo su rivista]
Rinaldi, A.; Caraffi, R.; Grazioli, M. V.; Oddone, N.; Giardino, L.; Tosi, G.; Vandelli, M. A.; Calza, L.; Ruozi, B.; Duskey, J. T.
abstract

Reactive oxygen species (ROS)-sensitive drug delivery systems (DDS) specifically responding to altered levels of ROS in the pathological microenvironment have emerged as an effective means to enhance the pharmaceutical efficacy of conventional nanomedicines, while simultaneously reducing side effects. In particular, the use of the biocompatible, biodegradable, and non-toxic ROS-responsive thioketal (TK) functional group in the design of smart DDS has grown exponentially in recent years. In the design of TK-based DDS, different technological uses of TK have been proposed to overcome the major limitations of conventional DDS counterparts including uncontrolled drug release and off-target effects. This review will focus on the different technological uses of TK-based biomaterials in smart nanomedicines by using it as a linker to connect a drug on the surface of nanoparticles, form prodrugs, as a core component of the DDS to directly control its structure, to control the opening of drug-releasing gates or to change the conformation of the nano-systems. A comprehensive view of the various uses of TK may allow researchers to exploit this reactive linker more consciously while designing nanomedicines to be more effective with improved disease-targeting ability, providing novel therapeutic opportunities in the treatment of many diseases.

2022 - Cis-Δ9-tetrahydrocannabinolic acid occurrence in Cannabis sativa L. [Articolo su rivista]
Tolomeo, Francesco; Russo, Fabiana; Kaczorova, Dominika; Vandelli, Maria Angela; Biagini, Giuseppe; Laganà, Aldo; Laura Capriotti, Anna; Paris, Roberta; Fulvio, Flavia; Carbone, Luigi; Perrone, Elisabetta; Gigli, Giuseppe; Cannazza, Giuseppe; Citti, Cinzia
abstract

Cannabidiolic acid (CBDA) and trans-Δ9-tetrahydrocannabinolic acid (trans-Δ9-THCA) are known to be the major phytocannabinoids in Cannabis sativa L., along with their decarboxylated derivatives cannabidiol (CBD) and trans-Δ9-tetrahydrocannabinol (trans-Δ9-THC). The cis isomer of Δ9-THC has been recently identified, characterized and quantified in several Cannabis sativa varieties, which had been heated (decarboxylated) before the analysis. Since decarboxylation alters the original phytocannabinoids composition of the plant, this work reports the identification and characterization of the carboxylated precursor cis-Δ9-THCA. The compound was also synthesized and used as analytical standard for the development and validation of a liquid chromatography coupled to high resolution mass spectrometry-based method for its quantification in ten Cannabis sativa L. samples from different chemotypes. The highest concentrations of cis-Δ9-THCA were found in CBD-rich varieties, lower levels were observed in cannabigerol (CBG)-rich varieties (chemotype IV) and in those varieties with a balanced level of both CBD and THC (chemotype III), while its levels were not detectable in cannabichromene (CBC)-rich varieties (chemotype VI). The presence of the cis isomer of THC and THCA raises the question on whether to include or not this species in the calculation of the total amount of THC to classify a cannabis variety as a drug-type or a fiber-type (hemp).

2022 - Editor's Note: Nanoparticles Engineered with Rituximab and Loaded with Nutlin-3 Show Promising Therapeutic Activity in B-Leukemic Xenografts [Articolo su rivista]
Voltan, R.; Secchiero, P.; Ruozi, B.; Forni, F.; Agostinis, C.; Caruso, L.; Vandelli, M. A.; Zauli, G.
abstract

2022 - Glioblastoma Multiforme Selective Nanomedicines for Improved Anti-Cancer Treatments [Articolo su rivista]
Duskey, J. T.; Rinaldi, A.; Ottonelli, I.; Caraffi, R.; De Benedictis, C. A.; Sauer, A. K.; Tosi, G.; Vandelli, M. A.; Ruozi, B.; Grabrucker, A. M.
abstract

Glioblastoma Multiforme (GBM) is a devastating disease with a low survival rate and few efficacious treatment options. The fast growth, late diagnostics, and off-target toxicity of currently used drugs represent major barriers that need to be overcome to provide a viable cure. Nanomedicines (NMeds) offer a way to overcome these pitfalls by protecting and loading drugs, increasing blood half-life, and being targetable with specific ligands on their surface. In this study, the FDA-approved polymer poly (lactic-co-glycolic) acid was used to optimise NMeds that were surface modified with a series of potential GBM-specific ligands. The NMeds were fully characterised for their physical and chemical properties, and then in vitro testing was performed to evaluate cell uptake and GBM cell specificity. While all targeted NMeds showed improved uptake, only those decorated with the-cell surface vimentin antibody M08 showed specificity for GBM over healthy cells. Finally, the most promising targeted NMed candidate was loaded with the well-known chemotherapeutic, paclitaxel, to confirm targeting and therapeutic effects in C6 GBM cells. These results demonstrate the importance of using well-optimised NMeds targeted with novel ligands to advance delivery and pharmaceutical effects against diseased cells while minimising the risk for nearby healthy cells.

2022 - Kynurenine and kynurenic acid: two human neuromodulators found in Cannabis sativa L [Articolo su rivista]
Russo, Fabiana; Tolomeo, Francesco; Vandelli, Maria Angela; Biagini, Giuseppe; Paris, Roberta; Fulvio, Flavia; Laganà, Aldo; Capriotti, Anna Laura; Carbone, Luigi; Gigli, Giuseppe; Cannazza, Giuseppe; Citti, Cinzia
abstract

L-Kynurenine (KYN) and kynurenic acid (KYNA) are products of the metabolism of L-tryptophan (TRP) in the central nervous system of animals, but they are not commonly found in plants. In particular, KYNA is known for its interesting pharmacological properties (anti-oxidative, anti-inflammatory, hypolipidemic, and neuroprotective), which suggest a potential functional food ingredient role. The three compounds were identified in samples of Cannabis sativa L. by means of high-performance liquid chromatography coupled to high-resolution mass spectrometry using an untargeted metabolomics approach. Their concentrations were evaluated using a targeted metabolomics method in three organs of the plant (roots, stem, and leaves) in soil at two different growth stages and in hydroponics conditions. The distribution of TRP, KYN and KYNA was found tendentially higher in leaves compared to stem and roots and changed over time. Moreover, the levels of KYNA found in this study are unprecedentedly high compared to those found so far in other plant species, suggesting that Cannabis sativa L. could be a promising alternative source of this metabolite.

2022 - Quantitative comparison of the protein corona of nanoparticles with different matrices [Articolo su rivista]
Ottonelli, Ilaria; Duskey, Jason Thomas; Genovese, Filippo; Pederzoli, Francesca; Caraffi, Riccardo; Valenza, Marta; Tosi, Giovanni; Vandelli, Maria Angela; Ruozi, Barbara
abstract

: Nanoparticles (NPs) are paving the way for improved treatments for difficult to treat diseases diseases; however, much is unknown about their fate in the body. One important factor is the interaction between NPs and blood proteins leading to the formation known as the "protein corona" (PC). The PC, consisting of the Hard (HC) and Soft Corona (SC), varies greatly based on the NP composition, size, and surface properties. This highlights the need for specific studies to differentiate the PC formation for each individual NP system. This work focused on comparing the HC and SC of three NPs with different matrix compositions: a) polymeric NPs based on poly(lactic-co-glycolic) acid (PLGA), b) hybrid NPs consisting of PLGA and Cholesterol, and c) lipidic NPs made only of Cholesterol. NPs were formulated and characterized for their physico-chemical characteristics and composition, and then were incubated in human plasma. In-depth purification, identification, and statistical analysis were then performed to identify the HC and SC components. Finally, similar investigations demonstrated whether the presence of a targeting ligand on the NP surface would affect the PC makeup. These results highlighted the different PC fingerprints of these NPs, which will be critical to better understand the biological influences of the PC and improve future NP designs.

2022 - Tunneling Nanotubes: A New Target for Nanomedicine? [Articolo su rivista]
Ottonelli, I.; Caraffi, R.; Tosi, G.; Vandelli, M. A.; Duskey, J. T.; Ruozi, B.
abstract

Tunneling nanotubes (TNTs), discovered in 2004, are thin, long protrusions between cells utilized for intercellular transfer and communication. These newly discovered structures have been demonstrated to play a crucial role in homeostasis, but also in the spreading of diseases, infections, and metastases. Gaining much interest in the medical research field, TNTs have been shown to transport nanomedicines (NMeds) between cells. NMeds have been studied thanks to their advantageous features in terms of reduced toxicity of drugs, enhanced solubility, protection of the payload, prolonged release, and more interestingly, cell-targeted delivery. Nevertheless, their transfer between cells via TNTs makes their true fate unknown. If better understood, TNTs could help control NMed delivery. In fact, TNTs can represent the possibility both to improve the biodistribution of NMeds throughout a diseased tissue by increasing their formation, or to minimize their formation to block the transfer of dangerous material. To date, few studies have investigated the interaction between NMeds and TNTs. In this work, we will explain what TNTs are and how they form and then review what has been published regarding their potential use in nanomedicine research. We will highlight possible future approaches to better exploit TNT intercellular communication in the field of nanomedicine.

2021 - Glioblastoma: State of the Art of Treatments and Applications of Polymeric and Lipidic Nanomedicines [Capitolo/Saggio]
Sgarbi, V.; Duskey, J. T.; Ottonelli, I.; Da Ros, F.; Oddone, N.; Vandelli, M. A.; Forni, F.; Tosi, G.; Ruozi, B.
abstract

Glioblastoma multiforme (GBM) is one of the most devastating tumors affecting more than 5 in 100,000 people. Unfortunately, its diagnosis is often discovered in late stages and is normally deadly, having a life expectancy of 12–15 months and a mere 3% of the affected patients living 3 years or more independent of race, sex, and age. Sadly, current treatments (i.e., chemotherapy, radiation, surgery) are extremely aggressive and extend the patient’s life by little more than a year on average. Even when treatment appears successful, relapse is often experienced. These extreme treatments, combined with their lack of long-term success, call for new innovations. Among them, nanomedicine becomes one of the most promising approaches regarding possible applications in advancing or ameliorating GBM management. In this chapter, we will therefore analyze the state of the art and the most novel and outstanding innovation in terms of diagnosis and treatment options.

2021 - HPLC-UV-HRMS analysis of cannabigerovarin and cannabigerobutol, the two impurities of cannabigerol extracted from hemp [Articolo su rivista]
Tolomeo, Francesco; Russo, Fabiana; Vandelli, Maria Angela; Biagini, Giuseppe; Capriotti, Anna Laura; Laganà, Aldo; Carbone, Luigi; Gigli, Giuseppe; Cannazza, Giuseppe; Citti, Cinzia
abstract

A sensitive and straightforward HPLC-UV method was developed for the simultaneous quantification of the two main impurities in "pure" commercial cannabigerol (CBG) samples. The identification of such impurities, namely cannabigerovarin (CBGV) and cannabigerobutol (CBGB), the propyl and butyl homologs of CBG, respectively, was accomplished employing the high-resolution mass spectrometry (HRMS) technique, and subsequently confirmed by comparison with the same compounds obtained by chemical synthesis. Complete spectroscopic characterization (NMR, FT-IR, UV, and HRMS) of both impurities is reported in the present work. The method was validated in terms of linearity, which was assessed in the range 0.01-1.00 μg/mL, sensitivity, selectivity, intra- and inter-day accuracy and precision, and short-term stability, which all satisfied the acceptance criteria of the ICH guidelines. Application of the method to the analysis of four commercial CBG samples highlighted a certain variability in the impurity profile that might be ascribed to the hemp variety of the starting plant material. With these new analytical standards in hand, it would be interesting to investigate their concentrations in different hemp varieties and expand the scope of a phytocannabinomics approach for a comprehensive profiling of this remarkable class of natural compounds.

2021 - Insights into kinetics, release, and behavioral effects of brain-targeted hybrid nanoparticles for cholesterol delivery in Huntington's disease [Articolo su rivista]
Birolini, Giulia; Valenza, Marta; Ottonelli, Ilaria; Passoni, Alice; Favagrossa, Monica; Duskey, Jason T; Bombaci, Mauro; Vandelli, Maria Angela; Colombo, Laura; Bagnati, Renzo; Caccia, Claudio; Leoni, Valerio; Taroni, Franco; Forni, Flavio; Ruozi, Barbara; Salmona, Mario; Tosi, Giovanni; Cattaneo, Elena
abstract

Supplementing brain cholesterol is emerging as a potential treatment for Huntington's disease (HD), a genetic neurodegenerative disorder characterized, among other abnormalities, by inefficient brain cholesterol biosynthesis. However, delivering cholesterol to the brain is challenging due to the blood-brain barrier (BBB), which prevents it from reaching the striatum, especially, with therapeutically relevant doses. Here we describe the distribution, kinetics, release, and safety of novel hybrid polymeric nanoparticles made of PLGA and cholesterol which were modified with an heptapeptide (g7) for BBB transit (hybrid-g7-NPs-chol). We show that these NPs rapidly reach the brain and target neural cells. Moreover, deuterium-labeled cholesterol from hybrid-g7-NPs-chol is released in a controlled manner within the brain and accumulates over time, while being rapidly removed from peripheral tissues and plasma. We confirm that systemic and repeated injections of the new hybrid-g7-NPs-chol enhanced endogenous cholesterol biosynthesis, prevented cognitive decline, and ameliorated motor defects in HD animals, without any inflammatory reaction. In summary, this study provides insights about the benefits and safety of cholesterol delivery through advanced brain-permeable nanoparticles for HD treatment.

2021 - Microfluidic technology for the production of hybrid nanomedicines [Articolo su rivista]
Ottonelli, I.; Duskey, J. T.; Rinaldi, A.; Grazioli, M. V.; Parmeggiani, I.; Vandelli, M. A.; Wang, L. Z.; Prud'Homme, R. K.; Tosi, G.; Ruozi, B.
abstract

Microfluidic technologies have recently been applied as innovative methods for the production of a variety of nanomedicines (NMeds), demonstrating their potential on a global scale. The capacity to precisely control variables, such as the flow rate ratio, temperature, total flow rate, etc., allows for greater tunability of the NMed systems that are more standardized and automated than the ones obtained by well-known benchtop protocols. However, it is a crucial aspect to be able to obtain NMeds with the same characteristics of the previously optimized ones. In this study, we focused on the transfer of a production protocol for hybrid NMeds (H-NMeds) consisting of PLGA, Cholesterol, and Pluronic® F68 from a benchtop nanoprecipitation method to a microfluidic device. For this aim, we modified parameters such as the flow rate ratio, the concentration of core materials in the organic phase, and the ratio between PLGA and Cholesterol in the feeding organic phase. Outputs analysed were the chemico–physical properties, such as size, PDI, and surface charge, the composition in terms of %Cholesterol and residual %Pluronic® F68, their stability to lyophilization, and the morphology via atomic force and electron microscopy. On the basis of the results, even if microfluidic technology is one of the unique procedures to obtain industrial production of NMeds, we demonstrated that the translation from a benchtop method to a microfluidic one is not a simple transfer of already established parameters, with several variables to be taken into account and to be optimized.

2021 - Nanomedicines for brain diseases: Where we are and where we are going [Articolo su rivista]
Tosi, G.; Thomas Duskey, J.; Angela Vandelli, M.; Ruozi, B.
abstract

Graphical abstract [Formula: see text].

2021 - Origin of Δ9-tetrahydrocannabinol impurity in synthetic cannabidiol [Articolo su rivista]
Citti, C.; Russo, F.; Linciano, P.; Strallhofer, S. S.; Tolomeo, F.; Forni, F.; Vandelli, M. A.; Gigli, G.; Cannazza, G.
abstract

Introduction: Cannabidiol (CBD), the nonintoxicating constituent of cannabis, is largely employed for pharmaceutical and cosmetic purposes. CBD can be extracted from the plant or chemically synthesized. Impurities of psychotropic cannabinoids Δ9-tetrahydrocannabinol (Δ9-THC) and Δ8-THC have been found in extracted CBD, thus hypothesizing a possible contamination from the plant. Materials and Methods: In this study, synthetic and extracted CBD samples were analyzed by ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry and the parameters that can be responsible of the conversion of CBD into THC were evaluated by an accelerated stability test. Results: In synthetic and extracted CBD no trace of THC species was detected. In contrast, CBD samples stored in the dark at room temperature on the benchtop for 3 months showed the presence of such impurities. Experiments carried out under inert atmosphere in the absence of humidity or carbon dioxide led to no trace of THC over time even at high temperature. Conclusions: The results suggested that the copresence of carbon dioxide and water from the air could be the key for creating the acidic environment responsible for the cyclization of CBD. These findings suggest that it might be appropriate to review the storage conditions indicated on the label of commercially available CBD.

2021 - The novel heptyl phorolic acid cannabinoids content in different Cannabis sativa L. accessions [Articolo su rivista]
Linciano, Pasquale; Russo, Fabiana; Citti, Cinzia; Tolomeo, Francesco; Paris, Roberta; Fulvio, Flavia; Pecchioni, Nicola; Vandelli, Maria Angela; Laganà, Aldo; Capriotti, Anna Laura; Biagini, Giuseppe; Carbone, Luigi; Gigli, Giuseppe; Cannazza, Giuseppe
abstract

The recent discovery of the novel heptyl phytocannabinoids cannabidiphorol (CBDP) and Δ9-tetrahydrocannabiphorol (Δ9-THCP) raised a series of questions relating to the presence and abundance of these new unorthodox compounds in cannabis inflorescence or derived products. As fresh inflorescence contains mainly their acid precursors, which are not commercially available, an ad hoc stereoselective synthesis was performed in order to obtain cannabidiphorolic acid (CBDPA) and Δ9-tetrahydrocannabiphorolic acid (THCPA) to be used as analytical standards for quantitative purposes. The present work reports an unprecedented targeted analysis of both pentyl (C5) and heptyl (C7) CBD- and THC-type compounds in forty-nine cannabis samples representing four different chemotypes. Moreover, the ultrahigh performance liquid chromatography coupled to highresolution mass spectrometry-based method was applied for the putative identification of other heptyl homologs of the most common phytocannabinoid acids, including cannabigerophorolic acid (CBGPA), cannabichromephorolic acid (CBCPA), cannabinophorolic acid (CBNPA), cannabielsophorolic acid (CBEPA), cannabicyclophorolic acid (CBLPA), cannabitriophorolic acid (CBTPA), and cannabiripsophorolic acid (CBRPA).

2021 - Tween® preserves enzyme activity and stability in PLGA nanoparticles [Articolo su rivista]
Duskey, J. T.; Ottonelli, I.; Rinaldi, A.; Parmeggiani, I.; Zambelli, B.; Wang, L. Z.; Prud'Homme, R. K.; Vandelli, M. A.; Tosi, G.; Ruozi, B.
abstract

Enzymes, as natural and potentially long-term treatment options, have become one of the most sought-after pharmaceutical molecules to be delivered with nanoparticles (NPs); however, their instability during formulation often leads to underwhelming results. Various molecules, including the Tween® polysorbate series, have demonstrated enzyme activity protection but are often used uncontrolled without optimization. Here, poly(lactic-co-glycolic) acid (PLGA) NPs loaded with β-glucosidase (β-Glu) solutions containing Tween® 20, 60, or 80 were compared. Mixing the enzyme with Tween® pre-formulation had no effect on particle size or physical characteristics, but increased the amount of enzyme loaded. More importantly, NPs made with Tween® 20:enzyme solutions maintained significantly higher enzyme activity. Therefore, Tween® 20:enzyme solutions ranging from 60:1 to 2419:1 mol:mol were further analyzed. Isothermal titration calorimetry analysis demonstrated low affinity and unquantifiable binding between Tween® 20 and β-Glu. Incorporating these solutions in NPs showed no effect on size, zeta potential, or morphology. The amount of enzyme and Tween® 20 in the NPs was constant for all samples, but a trend towards higher activity with higher molar rapports of Tween® 20:β-Glu was observed. Finally, a burst release from NPs in the first hour with Tween®:β-Glu solutions was the same as free enzyme, but the enzyme remained active longer in solution. These results highlight the importance of stabilizers during NP formulation and how optimizing their use to stabilize an enzyme can help researchers design more efficient and effective enzyme loaded NPs.

2020 - Enzyme Stability in Nanoparticle Preparations Part 1: Bovine Serum Albumin Improves Enzyme Function [Articolo su rivista]
Duskey, Jason Thomas; da Ros, Federica; Ottonelli, Ilaria; Zambelli, Barbara; Vandelli, Maria Angela; Tosi, Giovanni; Ruozi, Barbara
abstract

Enzymes have gained attention for their role in numerous disease states, calling for research for their efficient delivery. Loading enzymes into polymeric nanoparticles to improve biodistribution, stability, and targeting in vivo has led the field with promising results, but these enzymes still suffer from a degradation effect during the formulation process that leads to lower kinetics and specific activity leading to a loss of therapeutic potential. Stabilizers, such as bovine serum albumin (BSA), can be beneficial, but the knowledge and understanding of their interaction with enzymes are not fully elucidated. To this end, the interaction of BSA with a model enzyme B-Glu, part of the hydrolase class and linked to Gaucher disease, was analyzed. To quantify the natural interaction of beta-glucosidase (B-Glu,) and BSA in solution, isothermal titration calorimetry (ITC) analysis was performed. Afterwards, polymeric nanoparticles encapsulating these complexes were fully characterized, and the encapsulation efficiency, activity of the encapsulated enzyme, and release kinetics of the enzyme were compared. ITC results showed that a natural binding of 1:1 was seen between B-Glu and BSA. Complex concentrations did not affect nanoparticle characteristics which maintained a size between 250 and 350 nm, but increased loading capacity (from 6% to 30%), enzyme activity, and extended-release kinetics (from less than one day to six days) were observed for particles containing higher B-Glu:BSA ratios. These results highlight the importance of understanding enzyme:stabilizer interactions in various nanoparticle systems to improve not only enzyme activity but also biodistribution and release kinetics for improved therapeutic effects. These results will be critical to fully characterize and compare the effect of stabilizers, such as BSA with other, more relevant therapeutic enzymes for central nervous system (CNS) disease treatments.

2020 - Identification of a new cannabidiol n-hexyl homolog in a medicinal cannabis variety with an antinociceptive activity in mice: cannabidihexol [Articolo su rivista]
Linciano, P.; Citti, C.; Russo, F.; Tolomeo, F.; Lagana, A.; Capriotti, A. L.; Luongo, L.; Iannotta, M.; Belardo, C.; Maione, S.; Forni, F.; Vandelli, M. A.; Gigli, G.; Cannazza, G.
abstract

The two most important and studied phytocannabinoids present in Cannabis sativa L. are undoubtedly cannabidiol (CBD), a non-psychotropic compound, but with other pharmacological properties, and Δ9-tetrahydrocannabinol (Δ9-THC), which instead possesses psychotropic activity and is responsible for the recreative use of hemp. Recently, the homolog series of both CBDs and THCs has been expanded by the isolation in a medicinal cannabis variety of four new phytocannabinoids possessing on the resorcinyl moiety a butyl-(in CBDB and Δ9-THCB) and a heptyl-(in CBDP and Δ9-THCP) aliphatic chain. In this work we report a new series of phytocannabinoids that fills the gap between the pentyl and heptyl homologs of CBD and Δ9-THC, bearing a n-hexyl side chain on the resorcinyl moiety that we named cannabidihexol (CBDH) and Δ9-tetrahydrocannabihexol (Δ9-THCH), respectively. However, some cannabinoids with the same molecular formula and molecular weight of CBDH and Δ9-THCH have been already identified and reported as monomethyl ether derivatives of the canonical phytocannabinoids, namely cannabigerol monomethyl ether (CBGM), cannabidiol monomethyl ether (CBDM) and Δ9-tetrahydrocannabinol monomethyl ether (Δ9-THCM). The unambiguously identification in cannabis extract of the n-hexyl homologues of CBD and Δ9-THC different from the corresponding methylated isomers (CBDM, CBGM and Δ9-THCM) was achieved by comparison of the retention time, molecular ion, and fragmentation spectra with those of the authentic standards obtained via stereoselective synthesis, and a semi-quantification of these cannabinoids in the FM2 medical cannabis variety was provided. Conversely, no trace of Δ9-THCM was detected. Moreover, CBDH was isolated by semipreparative HPLC and its identity was confirmed by comparison with the spectroscopic data of the corresponding synthetic standard. Thus, the proper recognition of CBDH, CBDM and Δ9-THCH closes the loop and might serve in the future for researchers to distinguish between these phytocannabinoids isomers that show a very similar analytical behaviour. Lastly, CBDH was assessed for biological tests in vivo showing interesting analgesic activity at low doses in mice.

2020 - Investigating Novel Syntheses of a Series of Unique Hybrid PLGA-Chitosan Polymers for Potential Therapeutic Delivery Applications [Articolo su rivista]
Duskey, Jason Thomas; Baraldi, Cecilia; Gamberini, Maria Cristina; Ottonelli, Ilaria; Da Ros, Federica; Tosi, Giovanni; Forni, Flavio; Vandelli, Maria Angela; Ruozi, Barbara
abstract

Discovering new materials to aid in the therapeutic delivery of drugs is in high demand. PLGA, a FDA approved polymer, is well known in the literature to form films or nanoparticles that can load, protect, and deliver drug molecules; however, its incompatibility with certain drugs (due to hydrophilicity or charge repulsion interactions) limits its use. Combining PLGA or other polymers such as polycaprolactone with other safe and positively-charged molecules, such as chitosan, has been sought after to make hybrid systems that are more flexible in terms of loading ability, but often the reactions for polymer coupling use harsh conditions, films, unpurified products, or create a single unoptimized product. In this work, we aimed to investigate possible innovative improvements regarding two synthetic procedures. Two methods were attempted and analytically compared using nuclear magnetic resonance (NMR), fourier-transform infrared spectroscopy (FT-IR), and dynamic scanning calorimetry (DSC) to furnish pure, homogenous, and tunable PLGA-chitosan hybrid polymers. These were fully characterized by analytical methods. A series of hybrids was produced that could be used to increase the suitability of PLGA with previously non-compatible drug molecules

2020 - Isolation of a High-Affinity Cannabinoid for the Human CB1 Receptor from a Medicinal Cannabis sativa Variety: Δ9-Tetrahydrocannabutol, the Butyl Homologue of Δ9-Tetrahydrocannabinol [Articolo su rivista]
Linciano, P.; Citti, C.; Luongo, L.; Belardo, C.; Maione, S.; Vandelli, M. A.; Forni, F.; Gigli, G.; Lagana, A.; Montone, C. M.; Cannazza, G.
abstract

The butyl homologues of Δ9-tetrahydrocannabinol, Δ9-tetrahydrocannabutol (Δ9-THCB), and cannabidiol, cannabidibutol (CBDB), were isolated from a medicinal Cannabis sativa variety (FM2) inflorescence. Appropriate spectroscopic and spectrometric characterization, including NMR, UV, IR, ECD, and HRMS, was carried out on both cannabinoids. The chemical structures and absolute configurations of the isolated cannabinoids were confirmed by comparison with the spectroscopic data of the respective compounds obtained by stereoselective synthesis. The butyl homologue of Δ9-THC, Δ9-THCB, showed an affinity for the human CB1 (Ki = 15 nM) and CB2 receptors (Ki = 51 nM) comparable to that of (-)-trans-Δ9-THC. Docking studies suggested the key bonds responsible for THC-like binding affinity for the CB1 receptor. The formalin test in vivo was performed on Δ9-THCB in order to reveal possible analgesic and anti-inflammatory properties. The tetrad test in mice showed a partial agonistic activity of Δ9-THCB toward the CB1 receptor.

2020 - Novel peptide-conjugated nanomedicines for brain targeting: In vivo evidence [Articolo su rivista]
Duskey, J. T.; Ottonelli, I.; Da Ros, F.; Vilella, A.; Zoli, M.; Kovachka, S.; Spyrakis, F.; Vandelli, M. A.; Tosi, G.; Ruozi, B.
abstract

Central nervous system (CNS) compartments remain one of the most difficult districts for drug delivery. This is due to the presence of the blood–brain barrier (BBB) that hampers 90% of drug passage, dramatically requiring non-invasive treatment strategies. Here, for the first time, the use of opioid-derived deltorphin-derivative peptides to drive biodegradable and biocompatible polymeric (i.e. poly-lactide-co-glycolide, PLGA) nanomedicines delivery across the BBB was described. Opioid-derived peptides were covalently conjugated to furnish activated polymers which were further used for fluorescently tagged nanoformulations. Beyond reporting production, formulation methodology and full physico-chemical characterization, in vivo tests generated clear proof of BBB crossing and CNS targeting by engineered nanomedicines opening the research to further applications of drug delivery and targeting in CNS disease models.

2020 - PLGA-PEG-ANG-2 Nanoparticles for Blood-Brain Barrier Crossing: Proof-of-Concept Study [Articolo su rivista]
Hoyos-Ceballos, Gina P; Ruozi, Barbara; Ottonelli, Ilaria; Da Ros, Federica; Vandelli, Maria Angela; Forni, Flavio; Daini, Eleonora; Vilella, Antonietta; Zoli, Michele; Tosi, Giovanni; Duskey, Jason T; López-Osorio, Betty L
abstract

The treatment of diseases that affect the central nervous system (CNS) represents a great research challenge due to the restriction imposed by the blood-brain barrier (BBB) to allow the passage of drugs into the brain. However, the use of modified nanomedicines engineered with different ligands that can be recognized by receptors expressed in the BBB offers a favorable alternative for this purpose. In this work, a BBB-penetrating peptide, angiopep-2 (Ang-2), was conjugated to poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles through pre- and post-formulation strategies. Then, their ability to cross the BBB was qualitatively assessed on an animal model. Proof-of-concept studies with fluorescent and confocal microscopy studies highlighted that the brain-targeted PLGA nanoparticles were able to cross the BBB and accumulated in neuronal cells, thus showing a promising brain drug delivery system.

2020 - Pitfalls in the analysis of phytocannabinoids in cannabis inflorescence [Articolo su rivista]
Citti, Cinzia; Russo, Fabiana; Sgrò, Salvatore; Gallo, Alfonso; Zanotto, Antonio; Forni, Flavio; Vandelli, Maria Angela; Laganà, Aldo; Montone, Carmela Maria; Gigli, Giuseppe; Cannazza, Giuseppe
abstract

The chemical analysis of cannabis potency involves the qualitative and quantitative determination of the main phytocannabinoids: Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), etc. Although it might appear as a trivial analysis, it is rather a tricky task. Phytocannabinoids are present mostly as carboxylated species at the aromatic ring of the resorcinyl moiety. Their decarboxylation caused by heat leads to a greater analytical variability due to both reaction kinetics and possible decomposition. Moreover, the instability of cannabinoids and the variability in the sample preparation, extraction, and analysis, as well as the presence of isomeric forms of cannabinoids, complicates the scenario. A critical evaluation of the different analytical methods proposed in the literature points out that each of them has inherent limitations. The present review outlines all the possible pitfalls that can be encountered during the analysis of these compounds and aims to be a valuable help for the analytical chemist. Graphical abstract.

2020 - Synthesis, Characterization, and In Vitro Studies of an Reactive Oxygen Species (ROS)-Responsive Methoxy Polyethylene Glycol-Thioketal-Melphalan Prodrug for Glioblastoma Treatment [Articolo su rivista]
Oddone, N.; Boury, F.; Garcion, E.; Grabrucker, A. M.; Martinez, M. C.; Da Ros, F.; Janaszewska, A.; Forni, F.; Vandelli, M. A.; Tosi, G.; Ruozi, B.; Duskey, J. T.
abstract

Glioblastoma (GBM) is the most frequent and aggressive primary tumor of the brain and averages a life expectancy in diagnosed patients of only 15 months. Hence, more effective therapies against this malignancy are urgently needed. Several diseases, including cancer, are featured by high levels of reactive oxygen species (ROS), which are possible GBM hallmarks to target or benefit from. Therefore, the covalent linkage of drugs to ROS-responsive molecules can be exploited aiming for a selective drug release within relevant pathological environments. In this work, we designed a new ROS-responsive prodrug by using Melphalan (MPH) covalently coupled with methoxy polyethylene glycol (mPEG) through a ROS-cleavable group thioketal (TK), demonstrating the capacity to self-assembly into nanosized micelles. Full chemical-physical characterization was conducted on the polymeric-prodrug and proper controls, along with in vitro cytotoxicity assayed on different GBM cell lines and “healthy” astrocyte cells confirming the absence of any cytotoxicity of the prodrug on healthy cells (i.e. astrocytes). These results were compared with the non-ROS responsive counterpart, underlining the anti-tumoral activity of ROS-responsive compared to the non-ROS-responsive prodrug on GBM cells expressing high levels of ROS. On the other hand, the combination treatment with this ROS-responsive prodrug and X-ray irradiation on human GBM cells resulted in an increase of the antitumoral effect, and this might be connected to radiotherapy. Hence, these results represent a starting point for a rationale design of innovative and tailored ROS-responsive prodrugs to be used in GBM therapy and in combination with radiotherapy.

2019 - A novel phytocannabinoid isolated from Cannabis sativa L. with an in vivo cannabimimetic activity higher than Δ9-tetrahydrocannabinol: Δ9-Tetrahydrocannabiphorol [Articolo su rivista]
Citti, C.; Linciano, P.; Russo, F.; Luongo, L.; Iannotta, M.; Maione, S.; Lagana, A.; Capriotti, A. L.; Forni, F.; Vandelli, M. A.; Gigli, G.; Cannazza, G.
abstract

(-)-Trans-Delta(9)-tetrahydrocannabinol (Delta(9)-THC) is the main compound responsible for the intoxicant activity of Cannabis sativa L. The length of the side alkyl chain influences the biological activity of this cannabinoid. In particular, synthetic analogues of Delta(9)-THC with a longer side chain have shown cannabimimetic properties far higher than Delta(9)-THC itself. In the attempt to define the phytocannabinoids profile that characterizes a medicinal cannabis variety, a new phytocannabinoid with the same structure of Delta(9)-THC but with a seven-term alkyl side chain was identified. The natural compound was isolated and fully characterized and its stereochemical configuration was assigned by match with the same compound obtained by a stereoselective synthesis. This new phytocannabinoid has been called (-)-trans-Delta(9)-tetrahydrocannabiphorol (Delta(9)-THCP). Along with Delta(9)-THCP, the corresponding cannabidiol (CBD) homolog with seven-term side alkyl chain (CBDP) was also isolated and unambiguously identified by match with its synthetic counterpart. The binding activity of Delta(9)-THCP against human CB1 receptor in vitro (K-i = 1.2 nM) resulted similar to that of CP55940 (K-i = 0.9 nM), a potent full CB1 agonist. In the cannabinoid tetrad pharmacological test, Delta(9)-THCP induced hypomotility, analgesia, catalepsy and decreased rectal temperature indicating a THC-like cannabimimetic activity. The presence of this new phytocannabinoid could account for the pharmacological properties of some cannabis varieties difficult to explain by the presence of the sole Delta(9)-THC.

2019 - Analysis of impurities of cannabidiol from hemp. Isolation, characterization and synthesis of cannabidibutol, the novel cannabidiol butyl analog [Articolo su rivista]
Citti, C.; Linciano, P.; Forni, F.; Vandelli, M. A.; Gigli, G.; Lagana, A.; Cannazza, G.
abstract

Cannabidiol (CBD), one of the two major active principles present in Cannabis sativa, is gaining great interest among the scientific community for its pharmaceutical, nutraceutical and cosmetic applications. CBD can be prepared either by chemical synthesis or extraction from Cannabis sativa (hemp). The latter is more convenient from several points of view, including environmental and economic, but mainly for the absence of harmful organic solvents generally employed in the chemical synthesis. Although CBD produced by hemp extraction is the most widely employed, it carries two major impurities. The first one is the already known cannabidivarin (CBDV), whereas the second one is supposed to be the butyl analog of CBD with a four-term alkyl side chain. In this work, we report the isolation by semi-preparative liquid chromatography and the unambiguous identification of this second impurity. A comprehensive spectroscopic characterization, including NMR, UV, IR, circular dichroism and high-resolution mass spectrometry (HRMS), was carried out on this natural cannabinoid. In order to confirm its absolute configuration and chemical structure, the stereoisomer (1R,6R) of the supposed cannabinoid was synthesized and the physicochemical and spectroscopic properties, along with the stereochemistry, matched those of the natural isolated molecule. According to the International Nonproprietary Name, we suggested the name of cannabidibutol (CBDB) for this cannabinoid. Lastly, an HPLC-UV method was developed and validated for the qualitative and quantitative determination of CBDV and CBDB in samples of CBD extracted from hemp and produced according to Good Manufacturing Practices regulations for pharmaceutical and cosmetic use.

2019 - Cannabinoid profiling of hemp seed oil by liquid chromatography coupled to high-resolution mass spectrometry [Articolo su rivista]
Citti, Cinzia; Linciano, Pasquale; Panseri, Sara; Vezzalini, Francesca; Forni, Flavio; Vandelli, Maria Angela; Cannazza, Giuseppe
abstract

Hemp seed oil is well known for its nutraceutical, cosmetic and pharmaceutical properties due to a perfectly balanced content of omega 3 and omega 6 polyunsaturated fatty acids. Its importance for human health is reflected by the success on the market of organic goods in recent years. However, it is of utmost importance to consider that its healthy properties are strictly related to its chemical composition, which varies depending not only on the manufacturing method, but also on the hemp variety employed. In the present work, we analyzed the chemical profile of ten commercially available organic hemp seed oils. Their cannabinoid profile was evaluated by a liquid chromatography method coupled to high-resolution mass spectrometry. Besides tetrahydrocannabinol and cannabidiol, other 30 cannabinoids were identified for the first time in hemp seed oil. The results obtained were processed according to an untargeted metabolomics approach. The multivariate statistical analysis showed highly significant differences in the chemical composition and, in particular, in the cannabinoid content of the hemp oils under investigation.

2019 - Chemical and spectroscopic characterization data of ‘cannabidibutol’, a novel cannabidiol butyl analog [Articolo su rivista]
Citti, C.; Linciano, P.; Forni, F.; Vandelli, M. A.; Gigli, G.; Lagana, A.; Cannazza, G.
abstract

Cannabidibutol (CBDB), a novel butyl analog of cannabidiol, was identified as impurity of commercial cannabidiol (CBD) extracted from hemp (for full data and results interpretation see “Analysis of impurities of cannabidiol from hemp. Isolation, characterization and synthesis of cannabidibutol, the novel cannabidiol butyl analog” Citti et al, 2019). The compound was isolated from a CBD sample and subject to a full characterization. First, a complete spectroscopic characterization was performed by Nuclear Magnetic Resonance (NMR): in particular, 1H-NMR, 13C-NMR, COSY, HSQC and HMBC, which were followed by UV absorption and circular dichroism (CD) spectra. In order to confirm the structural identity and stereochemistry of the compound, a stereoselective synthesis of the trans isomer (1R,6R) was carried out and all the chemical and spectroscopic properties were analyzed. The synthesized compound was characterized by NMR (1H-NMR, 13C-NMR, COSY, HSQC and HMBC), Infra-Red spectroscopy (IR), UV and CD absorption, matching the results obtained for the natural isolated compound. With the analytical standard in hand, a simple high-performance liquid chromatography method coupled to UV detection (HPLC-UV) was developed and validated in house in terms of linearity, accuracy, precision, dilution integrity and stability. The present data might be useful to any researcher or industry that may run into a very common impurity of CBD extracted from hemp, so it can be easily compared with their own experimental data.

2019 - Nanomedicine against Aβ aggregation by β–sheet breaker peptide delivery: In vitro evidence [Articolo su rivista]
Pederzoli, F.; Ruozi, B.; Duskey, J.; Hagmeyer, S.; Sauer, A. K.; Grabrucker, S.; Coelho, R.; Oddone, N.; Ottonelli, I.; Daini, E.; Zoli, M.; Vandelli, M. A.; Tosi, G.; Grabrucker, A. M.
abstract

The accumulation of amyloid β (Aβ) triggers a cascade of toxic events in Alzheimer’s disease (AD). The KLVFF peptide can interfere with Aβ aggregation. However, the peptide suffers from poor bioavailability and the inability to cross the blood–brain barrier. In this work, we study the possibility of adopting nanomedicine to overcome KLVFF limits in biodistribution. We produced new engineered polymeric nanoparticles (NPs), and we evaluated the cellular toxicity of these NPs and validated that KVLFF peptides released by NPs show the same promising effects on AD pathology. Our results revealed the successful generation of KVLFF loaded NPs that, without significant effects on cell heath, are even more potent in reversing Aβ-induced pathologies compared to the free peptide. Therefore, NPs will significantly advance KVLFF treatment as a therapeutic option for AD.

2019 - Nanomedicine in Alzheimer's disease: Amyloid beta targeting strategy [Capitolo/Saggio]
Tosi, Giovanni; Pederzoli, Francesca; Belletti, Daniela; Vandelli, Maria Angela; Forni, Flavio; Duskey, Jason Thomas; Ruozi, Barbara
abstract

The treatment of Alzheimer's disease (AD) is up to today one of the most unsuccessful examples of biomedical science. Despite the high number of literature evidences detailing the multifactorial and complex etiopathology of AD, no cure is yet present on the market and the available treatments are only symptomatic. The reasons could be ascribed on two main factors: (i) lack of ability of the majority of drugs to cross the blood-brain barrier (BBB), thus excluding the brain for any successful therapy; (ii) lack of selectivity and specificity of drugs, decreasing the efficacy of even potent anti-AD drugs. The exploitation of specifically engineered nanomedicines planned to cross the BBB and to target the most “hot” site of action (i.e., β-amyloid) is one of the most interesting innovations in drug delivery and could reasonably represent an promising choice for possible treatments and even early-diagnosis of AD. In this chapter, we therefore outline the most talented approaches in AD treatment with a specific focus on the main advantages/drawbacks and future possible translation to clinic application.

2019 - ROS-responsive “smart” polymeric conjugate: Synthesis, characterization and proof-of-concept study [Articolo su rivista]
Oddone, N.; Pederzoli, F.; Duskey, J. T.; De Benedictis, C. A.; Grabrucker, A. M.; Forni, F.; Vandelli, M. A.; Ruozi, B.; Tosi, G.
abstract

New approaches integrating stimuli-responsive linkers into prodrugs are currently emerging. These “smart” prodrugs can enhance the effectivity of conventional prodrugs with promising clinical applicability. Oxidative stress is central to several diseases, including cancer. Therefore, the design of prodrugs that respond to ROS stimulus, allowing a selective drug release in this condition, is fairly encouraging. Aiming to investigate the ROS-responsiveness of prodrugs containing the ROS-cleavable moiety, Thioketal (TK), we performed proof-of-concept studies by synthesizing ROS-responsive conjugate, namely mPEG-TK-Cy5, through exploiting Cy5 fluorescent dye. We demonstrated that, differently to non-ROS-responsive control conjugate (mPEG-Cy5), mPEG-TK-Cy5 shows a selective release of Cy5 in response to ROS in both, ROS-simulated conditions and in vitro on glioblastoma cells. Our results confirm the applicability of TK-technology in the design of ROS-responsive prodrugs, which constitutes a promising approach in cancer treatment. The translatability of this technology for other diseases treatment makes this a highly relevant and promising approach.

2019 - Targeting Brain Disease in MPSII: Preclinical Evaluation of IDS-Loaded PLGA Nanoparticles [Articolo su rivista]
Rigon, Laura; Salvalaio, Marika; Pederzoli, Francesca; Legnini, Elisa; Duskey, Jason Thomas; D'Avanzo, Francesca; De Filippis, Concetta; Ruozi, Barbara; Marin, Oriano; Vandelli, Maria Angela; Ottonelli, Ilaria; Scarpa, Maurizio; Tosi, Giovanni; Tomanin, Rosella
abstract

Mucopolysaccharidosis type II (MPSII) is a lysosomal storage disorder due to the deficit of the enzyme iduronate 2-sulfatase (IDS), which leads to the accumulation of glycosaminoglycans in most organ-systems, including the brain, and resulting in neurological involvement in about two-thirds of the patients. The main treatment is represented by a weekly infusion of the functional enzyme, which cannot cross the blood-brain barrier and reach the central nervous system. In this study, a tailored nanomedicine approach based on brain-targeted polymeric nanoparticles (g7-NPs), loaded with the therapeutic enzyme, was exploited. Fibroblasts from MPSII patients were treated for 7 days with NPs loaded with the IDS enzyme; an induced IDS activity like the one detected in healthy cells was measured, together with a reduction of GAG content to non-pathological levels. An in vivo short-term study in MPSII mice was performed by weekly administration of g7-NPs-IDS. Biochemical, histological, and immunohistochemical evaluations of liver and brain were performed. The 6-weeks treatment produced a significant reduction of GAG deposits in liver and brain tissues, as well as a reduction of some neurological and inflammatory markers (i.e., LAMP2, CD68, GFAP), highlighting a general improvement of the brain pathology. The g7-NPs-IDS approach allowed a brain-targeted enzyme replacement therapy. Based on these positive results, the future aim will be to optimize NP formulation further to gain a higher efficacy of the proposed approach.

2018 - A Metabolomic Approach Applied to a LiquidChromatography Coupled to High-ResolutionTandem Mass Spectrometry Method (HPLC-ESI-HRMS/MS): Towards the ComprehensiveEvaluation of the Chemical Composition ofCannabis Medicinal Extracts [Articolo su rivista]
Citti, Cinzia; Battisti, Umberto Maria; Braghiroli, Daniela; Ciccarella, Giuseppe; Schmid, Martin; Vandelli, Maria Angela; Cannazza, Giuseppe
abstract

Introduction – Cannabis sativa L. is a powerful medicinal plant and its use has recently increased for the treatment of several pa-thologies. Nonetheless, side effects, like dizziness and hallucinations, and long-term effects concerning memory and cognition,can occur. Most alarming is the lack of a standardised procedure to extract medicinal cannabis. Indeed, each galenical prepara-tion has an unknown chemical composition in terms of cannabinoids and other active principles that depends on the extractionprocedure.Objective – This study aims to highlight the main differences in the chemical composition of Bediol® extracts when the extractionis carried out with either ethyl alcohol or olive oil for various times (0, 60, 120 and 180 min for ethyl alcohol, and 0, 60, 90 and120 min for olive oil).Methodology.Cannabis medicinal extracts (CMEs) were analysed by liquid chromatography coupled to high-resolution tandem mass spec-trometry (LC–MS/MS) using an untargeted metabolomics approach. The data sets were processed by unsupervised multivariateanalysis.Results – Our results suggested that the main difference lies in the ratio of acid to decarboxylated cannabinoids, which dramat-ically influences the pharmacological activity of CMEs. Minor cannabinoids, alkaloids, and amino acids contributing to this differ-ence are also discussed. The main cannabinoids were quantified in each extract applying a recently validated LC–MS and LC-UVmethod.Conclusions – Notwithstanding the use of a standardised starting plant material, great changes are caused by different extractionprocedures. The metabolomics approach is a useful tool for the evaluation of the chemical composition of cannabis extracts.

2018 - Analysis of cannabinoids in commercial hemp seed oil and decarboxylation kinetics studies of cannabidiolic acid (CBDA) [Articolo su rivista]
Citti, Cinzia; Pacchetti, Barbara; Vandelli, Maria Angela; Forni, Flavio; Cannazza, Giuseppe
abstract

Hemp seed oil from Cannabis sativa L. is a very rich natural source of important nutrients, not only polyunsaturated fatty acids and proteins, but also terpenes and cannabinoids, which contribute to the overall beneficial effects of the oil. Hence, it is important to have an analytical method for the determination of these components in commercial samples. At the same time, it is also important to assess the safety of the product in terms of amount of any psychoactive cannabinoid present therein. This work presents the development and validation of a highly sensitive, selective and rapid HPLC-UV method for the qualitative and quantitative determination of the main cannabinoids, namely cannabidiolic acid (CBDA), tetrahydrocannabinolic acid (THCA), cannabidiol (CBD), tetrahydrocannabinol (THC), cannabinol (CBN), cannabigerol (CBG) and cannabidivarin (CBDV), present in 13 commercial hemp seed oils. Moreover, since decomposition of cannabinoid acids generally occurs with light, air and heat, decarboxylation studies of the most abundant acid (CBDA) were carried out in both open and closed reactor and the kinetics parameters were evaluated at different temperatures in order to evaluate the stability of hemp seed oil in different storage conditions.

2018 - Development of a simple and sensitive liquid chromatography triple quadrupole mass spectrometry (LC–MS/MS) method for the determination of cannabidiol (CBD), Δ9-tetrahydrocannabinol (THC) and its metabolites in rat whole blood after oral administration of a single high dose of CBD [Articolo su rivista]
Palazzoli, Federica; Citti, Cinzia; Licata, Manuela; Vilella, Antonietta; Manca, Letizia; Zoli, Michele; Vandelli, Maria Angela; Forni, Flavio; Cannazza, Giuseppe
abstract

The investigation of the possible conversion of cannabidiol (CBD) into Δ 9 -tetrahydrocannabinol (THC) in vivo after oral administration of CBD is reported herein since recent publications suggested a rapid conversion in simulated gastric fluid. To this end, single high dose of CBD (50 mg/kg) was administered orally to rats and their blood was collected after 3 and 6 h. A highly sensitive and selective LC–MS/MS method was developed and fully validated in compliance with the Scientific Working Group of Forensic Toxicology (SWGTOX) standard practices for method validation in forensic toxicology. This method also involved the optimization of cannabinoids and their metabolites extraction in order to remove co-eluting phospholipids and increase the sensitivity of the MS detection. Neither THC nor its metabolites were detected in rat whole blood after 3 or 6 h from CBD administration. After oral administration, the amount of CBD dissolved in olive oil was higher than that absorbed from an ethanolic solution. This could be explained by the protection of lipid excipients towards CBD from acidic gastric juice.

2018 - Hybrid nanoparticles as a new technological approach to enhance the delivery of cholesterol into the brain [Articolo su rivista]
Belletti, Daniela; Grabrucker, Andreas Martin; Pederzoli, Francesca; Menerath, Isabel; Vandelli, Maria Angela; Tosi, Giovanni; Duskey, Thomas Jason; Forni, Flavio; Ruozi, Barbara
abstract

Restoration of the Chol homeostasis in the Central Nervous System (CNS) could be beneficial for the treatment of Huntington's Disease (HD), a progressive, fatal, adult-onset, neurodegenerative disorder. Unfortunately, Chol is unable to cross the blood–brain barrier (BBB), thus a novel strategy for a targeted delivery of Chol into the brain is highly desired. This article aims to investigate the production of hybrid nanoparticles composed by Chol and PLGA (MIX-NPs) modified with g7 ligand for BBB crossing. We described the impact of ratio between components (Chol and PLGA) and formulation process (nanoprecipitation or single emulsion process) on physico-chemical and structural characteristics, we tested MIX-NPs in vitro using primary hippocampal cell cultures evaluating possible toxicity, uptake, and the ability to influence excitatory synaptic receptors. Our results elucidated that both formulation processes produce MIX-NPs with a Chol content higher that 40%, meaning that Chol is a structural particle component and active compound at the same time. The formulation strategy impacted the architecture and reorganization of components leading to some differences in Chol availability between the two types of g7 MIX-NPs. Our results identified that both kinds of MIX-NPs are efficiently taken up by neurons, able to escape lysosomes and release Chol into the cells resulting in an efficient modification in expression of synaptic receptors that could be beneficial in HD.

2018 - Pharmaceutical and biomedical analysis of cannabinoids: A critical review [Articolo su rivista]
Cittia, Cinzia; Braghiroli, Daniela; Vandelli, Maria Angela; Cannazza, Giuseppe
abstract

Cannabis products have recently regained much attention due to the high pharmacological potential of their cannabinoid content. In this review, the most widely used sample preparation strategies for the extraction of cannabinoids are described for the specific application to either plant materials or biological matrices. Several analytical techniques are described pointing out their respective advantages and drawbacks. In particular, chromatographic methods, such as TLC, GC and HPLC, are discussed and compared in terms of selectivity and sensitivity. Various detection methods are also presented based on the specific aim of the cannabinoids analysis. Lastly, critical considerations are mentioned with the aim to deliver useful suggestions for the selection of the optimal and most suitable method of analysis of cannabinoids in either biomedical or cannabis derived samples.

2018 - Qualitative and semiquantitative analysis of the protein coronas associated to different functionalized nanoparticles [Articolo su rivista]
Pederzoli, Francesca; Tosi, Giovanni; Genovese, Filippo; Belletti, Daniela; Vandelli, Maria Angela; Ballestrazzi, Antonio; Forni, Flavio; Ruozi, Barbara
abstract

The investigation on protein coronas (PCs) adsorbed onto nanoparticle (NP) surface is representing an open issue due to difficulties in detection and clear isolation of the adsorbed proteins. In this study, we investigated protocols able to isolate the compositions of PCs of three polymeric NPs.

2018 - Reduced plaque size and inflammation in the APP23 mouse model for Alzheimer's disease after chronic application of polymeric nanoparticles for CNS targeted zinc delivery [Articolo su rivista]
Vilella, Antonietta; Belletti, Daniela; Sauer, Ann Katrin; Hagmeyer, Simone; Sarowar, Tasnuva; Masoni, Martina; Stasiak, Natalia; Mulvihill, John J. E; Ruozi, Barbara; Forni, Flavio; Vandelli, Maria Angela; Tosi, Giovanni; Zoli, Michele; Grabrucker, Andreas M.
abstract

A local dyshomeostasis of zinc ions in the vicinity of amyloid aggregates has been proposed in Alzheimer's disease (AD) due to the sequestration of zinc in senile plaques. While an increase in zinc levels may promote the aggregation of amyloid beta (Aβ), increased brain zinc might also be beneficial rescuing some pathological alterations caused by local zinc deficiency. For example, increased Aβ degradation by metalloproteinases, and a reduction in inflammation can be hypothesized. In addition, zinc may allow a stabilization of the number of synapses in AD brains. Thus, to evaluate whether altering zinc-levels within the brain is a promising new target for the prevention and treatment of AD, we employed novel zinc loaded nanoparticles able to deliver zinc into the brain across the blood-brain barrier. We performed in vivo studies using wild type (WT) and APP23 mice to assess plaque load, inflammatory status and synapse loss. Furthermore, we performed behavioral analyses. After chronically injecting these nanoparticles for 14 days, our results show a significant reduction in plaque size and effects on the pro-inflammatory cytokines IL-6 and IL-18. On behavioral level we could not detect negative effects of increased brain zinc levels in APP23 mice and treatment with g7-NP-Zn normalized the observed hyperlocomotion of APP23 mice. Therefore, we conclude that a targeted increase in brain zinc levels may have beneficial effects in AD.

2018 - Untargeted rat brain metabolomics after oral administration of a single high dose of cannabidiol [Articolo su rivista]
Citti, Cinzia; Palazzoli, Federica; Licata, Manuela; Vilella, Antonietta; Leo, Giuseppina; Zoli, Michele; Vandelli, Maria Angela; Forni, Flavio; Pacchetti, Barbara; Cannazza, Giuseppe
abstract

Cannabidiol (CBD), for long time considered as a minor cannabinoid of Cannabis sativa, has recently gained much attention due to its antioxidant, anti-inflammatory, analgesic and anticonvulsant properties. A liquid chromatography coupled to mass spectrometry based method was developed for the quantitative determination of CBD and other cannabinoids (Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC and 11-nor-9-carboxy-THC) in rat brain samples after oral administration of a single high dose (50 mg/kg) of CBD. The main challenge of the present work was to study CBD pharmacokinetics in rat cortex: the identification of its metabolites and pharmacodynamics through the study of variations in endogenous compounds’ concentrations following CBD administration. An untargeted metabolomics approach revealed the formation of some CBD metabolites that are not commonly found in other body tissues or fluids. Lastly, the changes in some endogenous compounds’ concentrations were correlated with some of the pharmacological properties of this cannabinoid.

2017 - Anticancer drug-loaded quantum dots engineered polymeric nanoparticles: Diagnosis/therapy combined approach [Articolo su rivista]
Belletti, Daniela; Riva, Giovanni; Luppi, Mario; Tosi, Giovanni; Forni, Flavio; Vandelli, Maria Angela; Ruozi, Barbara; Pederzoli, Francesca
abstract

Primary Effusion Lymphoma (PEL) is an HHV-8-related non Hodgkin lymphoma localized in body cavities (as pleural, peritoneal and pericardial) presenting lymphomatous effusion that, until now, lack of an effective therapy. Curcumin was reported to display pro-apoptotic effect via the inhibition of the JAK/STAT pathway, that is overexpressed in PEL cells, as consequence of virus infection. The administration of curcumin is severely restricted by its physicochemical properties, mainly its low solubility in biological fluid and consequently low bioavailability. Encapsulation into biocompatible and biodegradable PLGA nanoparticles (NPs) could be a strategy to overcome biological limits of curcumin, offering a valuable step forward for its clinical application. In this study we described single-emulsion process for curcumin loading into NPs (encapsulation efficiency about 35%). We applied a post-formulation strategy (NHS/EDC reaction) to decorate the surface of the curcumin-loaded NPs with quantum dots (QDs) as imaging agents (QDs-NPs-Cur, 24pmol of QDs per 100mg of NPs) obtaining tools useful for possible application in theranostic approach. Bifunctionalized NPs were tested in vitro on two PEL's cell line (BCBL-1 and HBL-6). The efficacy of the treatment was evaluated by cytofluorimetric assay by measuring both cell viability and cell density. We found that the NPs significantly improve the cellular effect of curcumin (respect to free drug). Moreover, by means of confocal microscopy, both the localization of bifunctional NPs and of the released drug were easily detectable. Thus, we conclude that the delivery of curcumin using bifunctional traceable NPs is a promising future approach for the diagnosis and the treatment of PEL.

2017 - Apoferritin nanocage as streptomycin drug reservoir: Technological optimization of a new drug delivery system [Articolo su rivista]
Ruozi, Barbara; Veratti, Patrizia; Vandelli, Maria Angela; Tombesi, Andrea; Tonelli, Massimo; Forni, Flavio; Pederzoli, Francesca; Belletti, Daniela; Tosi, Giovanni
abstract

The aim of this study is to formulate and characterize streptomycin-loaded apoferritin nanoparticles (ApoStrep NPs) for their potential therapeutic use in bacterial resistant infections (i.e. tuberculosis). ApoStrep NPs were prepared by disassembly/reassembly process via pH method and changing apoferritin/drug molar ratio, purified by dialyses process also associated with gel filtration chromatography and characterized in their chemico-physical and technological parameters as yield, size distribution, polidispersivity, morphology, internal structure, zeta potential and loading efficacy. The results showed that spherical reproducible NPs could be obtained by using apoferritin/drug molar ratio lower than 1:25 and purification based on the combination of dialysis and gel filtration chromatography. Photon correlation spectroscopy, Uv–visible detection and electron microscopy showed the maintenance of the native apoferritin chemico-physical properties and structure. When formulated with apoferritin/drug 1:10 and 1:25 molar ratio, ApoStrep NPs showed remarkable encapsulation efficacy (35% and 28%, respectively) along with kinetic profile of drug delivery, approximately 15% at 37 °C in 72 h, as evidenced by “in vitro” release experiments.

2017 - Corrigendum to: EXPLOITING THE VERSATILITY OF CHOLESTEROL IN NANOPARTICLES FORMULATION (International Journal of Pharmaceutics (2016) 511 (1) (331-340) (S0378517316306512) (10.1016/j.ijpharm.2016.07.022)) [Articolo su rivista]
Belletti, D.; Grabrucker, A. M.; Pederzoli, F.; Menrath, I.; Cappello, V.; Vandelli, M. A.; Forni, F.; Tosi, G.; Ruozi, B.
abstract

The authors regret that Table 1 was incorrect. The correct table is given below. The authors would like to apologise for any inconvenience caused. DOI of original article: 10.1016/j.ijpharm.2016.07.022

2017 - Current Strategies for the Delivery of Therapeutic Proteins and Enzymes to Treat Brain Disorders [Capitolo/Saggio]
Duskey, Jason T.; Belletti, Daniela; Pederzoli, Francesca; Vandelli, Maria Angela; Forni, Flavio; Ruozi, Barbara; Tosi, Giovanni
abstract

Brain diseases and injuries are growing to be one of the most deadly and costly medical conditions in the world. Unfortunately, current treatments are incapable of ameliorating the symptoms let alone curing the diseases. Many brain diseases have been linked to a loss of function in a protein or enzyme, increasing research for improving their delivery. This is no easy task due to the delicate nature of proteins and enzymes in biological conditions, as well as the many barriers that exist in the body ranging from those in circulation to the more specific barriers to enter the brain. Several main techniques are being used (physical delivery, protein/enzyme conjugates, and nanoparticle delivery) to overcome these barriers and create new therapeutics. This review will cover recently published data and highlights the benefits and deficits of possible new protein or enzyme therapeutics for brain diseases.

2017 - How does “Protein Corona” Affect the In vivo Efficiency of Polymeric Nanoparticles? State of Art [Capitolo/Saggio]
Pederzoli, F.; Galliani, M.; Forni, F.; Vandelli, M. A.; Belletti, D.; Tosi, G.; Ruozi, B.
abstract

Nanomedicine is increasingly considered as one of the most promising ways to overcome the limits of traditional medicine and conventional pharmaceutical formulations. In particular, polymeric nanoparticles (NPs) represent one of the most important tools in the nanomedicine field due to their potential in a wide range of biomedical applications such as imaging, drug targeting and drug delivery. However, their application is strongly hampered by limited knowledge and control of their interactions with complex biological systems. In biological environments, NPs are enshrouded by a layer of biomolecules, predominantly proteins, which tend to associate with NPs, forming a new surface named 'protein corona' (PC). Thus, the resulting nano-structure is a new entity, defined as PC-NP complex, featured by new characteristics, different from the original features of the bare NPs. In this chapter, starting from the definition of PC, we critically discuss the physico-chemical properties of polymeric NPs (e.g., size, shape, composition, surface functional groups, surface charge, hydrophilicity/hydrophobicity) and the environmental biological parameters (blood concentration, plasma gradient, temperature) affecting PC formation and composition. We further discuss how the new “entity” generated by the interactions between NPs and proteins in vivo mediates the ability of all the nanosystems to circulate, biodistribute and selectively release the drugs to the target site. We conclude by highlighting the gaps in the knowledge of the PC in relation to polymeric NPs and by discussing the main issues to be addressed and investigated in order to speed up the translatability of NPs into clinical protocols.

2017 - Novel Curcumin loaded nanoparticles engineered for Blood-Brain Barrier crossing and able to disrupt Abeta aggregates [Articolo su rivista]
Ruozi, Barbara; Belletti, Daniela; Pederzoli, Francesca; Masoni, Martina; Keller, JOHANNES ROBIN; Ballestrazzi, Antonio; Vandelli, Maria Angela; Tosi, Giovanni; Grabrucker, Andreas M.
abstract

The formation of extracellular aggregates built up by deposits of β-amyloid (Aβ) is a hallmark of Alzheimer's disease (AD). Curcumin has been reported to display anti-amyloidogenic activity, not only by inhibiting the formation of new Aβ aggregates, but also by disaggregating existing ones. However, the uptake of Curcumin into the brain is severely restricted by its low ability to cross the blood-brain barrier (BBB). Therefore, novel strategies for a targeted delivery of Curcumin into the brain are highly desired. Here, we encapsulated Curcumin as active ingredient in PLGA (polylactide-co-glycolic-acid) nanoparticles (NPs), modified with g7 ligand for BBB crossing. We performed in depth analyses of possible toxicity of these NPs, uptake, and, foremost, their ability to influence Aβ pathology in vitro using primary hippocampal cell cultures. Our results show no apparent toxicity of the formulated NPs, but a significant decrease of Aβ aggregates in response to Curcumin loaded NPs. We thus conclude that brain delivery of Curcumin using BBB crossing NPs is a promising future approach in the treatment of AD.

2017 - Protein cage nanostructure as drug delivery system: magnifying glass on apoferritin [Articolo su rivista]
Belletti, Daniela; Pederzoli, Francesca; Forni, Flavio; Vandelli, Maria Angela; Tosi, Giovanni; Ruozi, Barbara
abstract

New frontiers in nanomedicine are moving towards the research of new biomaterials. Apoferritin (APO), is a uniform regular self-assemblies nano-sized protein with excellent biocompatibility and a unique structure that affords it the ability to stabilize small active molecules in its inner core. Areas covered: APO can be loaded by applying a passive process (mainly used for ions and metals) or by a unique formulative approach based on disassemby/reassembly process. In this article, we aim to organize the experimental evidence provided by a number of studies on the loading, release and targeting. Attention is initially focused on the most investigated antineoplastic drug and contrast agents up to the most recent application in gene therapy. Expert opinion: Various preclinical studies have demonstrated that APO improved the potency and selectivity of some chemotherapeutics. However, in order to translate the use of APO into therapy, some issues must be solved, especially regarding the reproducibility of the loading protocol used, the optimization of nanocarrier characterization, detailed understanding of the final structure of loaded APO, and the real mechanism and timing of drug release.

2017 - Protein corona and nanoparticles: How can we investigate on? [Articolo su rivista]
Pederzoli, Francesca; Tosi, Giovanni; Vandelli, Maria Angela; Belletti, Daniela; Forni, Flavio; Ruozi, Barbara
abstract

Nanoparticles (NPs) represent one of the most promising tools for drug-targeting and drug-delivery. However, a deeper understanding of the complex dynamics that happen after their in vivo administration is required. Particularly, plasma proteins tend to associate to NPs, forming a new surface named the 'protein corona' (PC). This surface is the most exposed as the 'visible side' of NPs and therefore, can have a strong impact on NP biodistribution, targeting efficacy and also toxicity. The PC consists of two poorly delimited layers, known as 'hard corona' (HC) and 'soft corona' (SC), that are affected by the complexity of the environment and the formed protein-surface equilibrium during in vivo blood circulation. The HC corona is formed by proteins strongly associated to the NPs, while the SC is an outer layer consisting of loosely bound proteins. Several studies attempted to investigate the HC, which is easier to be isolated, but yielded poor reproducibility, due to varying experimental conditions. As a consequence, full mapping of the HC for different NPs is still lacking. Moreover, the current knowledge on the SC, which may play a major role in the 'first' interaction of NPs once in vivo, is very limited, mainly due to the difficulties in preserving it after purification. Therefore, multi-disciplinary approaches leading to the obtainment of a major number of information about the PC and its properties is strongly needed to fully understand its impact and to better support a more safety and conscious application of nanotechnology in medicine.

2016 - EXPLOITING THE VERSATILITY OF CHOLESTEROL IN NANOPARTICLES FORMULATION [Articolo su rivista]
Belletti, Daniela; Grabrucker, A. M; Pederzoli, Francesca; Menrath, I; Cappello, V; Vandelli, M. A; Forni, Flavio; Tosi, Giovanni; Ruozi, Barbara
abstract

The biocompatibility of polymers, lipids and surfactants used to formulate is crucial for the safe and sustainable development of nanocarriers (nanoparticles, liposomes, micelles, and other nanocarriers). In this study, Cholesterol (Chol), a typical biocompatible component of liposomal systems, was formulated in Chol-based solid nanoparticles (NPs) stabilized by the action of surfactant and without the help of any other formulative component. Parameters as type (Solutol HS 15, cholic acid sodium salt, poly vinyl alcohol and Pluronic-F68), concentration (0.2; 0.5 and 1% w/v) of surfactant and working temperature (r.t. and 45°C) were optimized and all samples characterized in terms of size, zeta potential, composition, thermal behavior and structure. Results demonstrated that only Pluronic-F68 (0.5% w/v) favors the organization of Chol chains in structured NPs with mean diameter less than 400nm. Moreover, we demonstrated the pivotal role of working temperature on surfactant aggregation state/architecture/stability of Chol-based nanoparticles. At room temperature, Pluronic-F68 exists in solution as individual coils. In this condition, nanoprecipitation of Chol formed the less stable NPs with a 14±3% (w/w) of Pluronic-F68 prevalently on surface (NP-Chol/0.5). On the contrary, working near the critical micelle temperature (CMT) of surfactant (45°C), Chol precipitates with Pluronic-F68 (9±5% w/w) in a compact stable matricial structure (NP-Chol/0.5-45). In vitro studies highlight the low toxicity and the affinity of NP-Chol/0.5-45 for neuronal cells suggesting their potential applicability in pathologies with a demonstrated alteration of neuronal plasticity and synaptic communication (i.e. Huntington's disease).

2016 - Medicinal cannabis: Principal cannabinoids concentration and their stability evaluated by a high performance liquid chromatography coupled to diode array and quadrupole time of flight mass spectrometry method [Articolo su rivista]
Citti, Cinzia; Ciccarella, Giuseppe; Braghiroli, Daniela; Parenti, Carlo; Vandelli, Maria Angela; Cannazza, Giuseppe
abstract

In the last few years, there has been a boost in the use of cannabis-based extracts for medicinal purposes, although their preparation procedure has not been standardized but rather decided by the individual pharmacists. The present work describes the development of a simple and rapid high performance liquid chromatography method with UV detection (HPLC-UV) for the qualitative and quantitative determination of the principal cannabinoids (CBD-A, CBD, CBN, THC and THC-A) that could be applied to all cannabis-based medicinal extracts (CMEs) and easily performed by a pharmacist. In order to evaluate the identity and purity of the analytes, a high-resolution mass spectrometry (HPLC-ESI-QTOF) analysis was also carried out. Full method validation has been performed in terms of specificity, selectivity, linearity, recovery, dilution integrity and thermal stability. Moreover, the influence of the solvent (ethyl alcohol and olive oil) was evaluated on cannabinoids degradation rate. An alternative extraction method has then been proposed in order to preserve cannabis monoterpene component in final CMEs.

2016 - Nanoparticle transport across the blood brain barrier [Articolo su rivista]
Grabrucker, Andreas M.; Ruozi, Barbara; Belletti, Daniela; Pederzoli, Francesca; Forni, Flavio; Vandelli, Maria Angela; Tosi, Giovanni
abstract

ABSTRACT: While the role of the blood-brain barrier (BBB) is increasingly recognized in the (development of treatments targeting neurodegenerative disorders, to date, few strategies exist that enable drug delivery of non-BBB crossing molecules directly to their site of action, the brain. However, the recent advent of Nanomedicines may provide a potent tool to implement CNS targeted delivery of active compounds. Approaches for BBB crossing are deeply investigated in relation to the pathology: among the main important diseases of the CNS, this review focuses on the application of nanomedicines to neurodegenerative disorders (Alzheimer, Parkinson and Huntington's Disease) and to other brain pathologies as epilepsy, infectious diseases, multiple sclerosis, lysosomal storage disorders, strokes.

2016 - PEGylated siRNA lipoplexes for silencing of BLIMP-1 in Primary Effusion Lymphoma: In vitro evidences of antitumoral activity [Articolo su rivista]
Belletti, Daniela; Tosi, Giovanni; Forni, Flavio; Lagreca, Ivana; Barozzi, Patrizia; Pederzoli, Francesca; Vandelli, Maria Angela; Riva, Giovanni; Luppi, Mario; Ruozi, Barbara
abstract

Silencing of the B lymphocyte-induced maturation protein 1 (Blimp-1), a pivotal transcriptional regulator during terminal differentiation of B cells into plasma cells with siRNAs is under investigation as novel therapeutic approach in Primary Effusion Lymphoma (PEL), a HHV-8 related and aggressive B cell Lymphoma currently lacking of an efficacious therapeutic approach. The clinical application of small interfering RNA (siRNA) in cancer therapy is limited by the lack of an efficient systemic siRNA delivery system. In this study we aim to develop pegylated siRNA lipoplexes formed using the cationic lipid DOTAP and DSPE-PEG2000, capable to effectively stabilize anti-Blimp-1 siRNA and suitable for systemic administration. Two types of pegylated lipoplexes using a classic (C-PEG Lipoplexes) or a post-pegylation method (P-PEG-Lipoplexes) were formulated and compared in their physicochemical properties (size, zeta potential, morphology and structure) and efficiency on PEL cell lines. A stable siRNAs protection was obtained with post pegylation approach (2% molar of DSPE-PEG2000 with respect to lipid) resulting in structures with diameters of 300 nm and a complexation efficiency higher that 80% (0.08 nmol/10 nmol of lipid). In vitro studies on PEL cell lines suggested that empty liposomes were characterized by a low cell toxicity also after PEG modification (cell viability and cell density over 85% after treatment with 10 μM of lipid). We demonstrated that P-PEG-Lipoplexes were able to significantly reduce the levels of BLIMP-1 protein leading to reduction of viability (less that 15% after transfection with 100 nM of complexed siRNAs) and activation of apoptosis. In vitro efficiency encourages us to further test the in vivo potential of P-PEG-Lipoplexes in PEL therapy.

2016 - Potential Use of Nanomedicine for Drug Delivery Across the Blood-Brain Barrier in Healthy and Diseased Brain [Articolo su rivista]
Ruozi, Barbara; Belletti, Daniela; Pederzoli, Francesca; Forni, Flavio; Vandelli, Maria Angela; Tosi, Giovanni
abstract

The research of efficacious non-invasive therapies for the treatment of brain diseases represents a huge challenge, as people affected by disorders of the central nervous system (CNS) will significantly increase. Moreover, the blood-brain barrier is a key factor in hampering a number of effective drugs to reach the CNS. This review is therefore focusing on possible interventions of nanomedicine-based approaches in selected diseases affecting the CNS. A wide overview of the most outstanding results on preclinical evaluations of the potential of nanomedicine in brain diseases (i.e. brain tumor, Alzheimer, Parkinson, epilepsy and others) is given, with highlights on the data with relevant interest and real possibility in translation from bench-to-bedside. Moreover, a critical evaluation on the rationale in planning nanosystems to target specific brain pathologies is described, opening the path to a more structured and pathology-tailored design of nanocarriers.

2016 - Targeted polymeric nanoparticles for brain delivery of high molecular weight molecules in lysosomal storage disorders [Articolo su rivista]
Salvalaio, Marika; Rigon, Laura; Belletti, Daniela; D'Avanzo, Francesca; Pederzoli, Francesca; Ruozi, Barbara; Marin, Oriano; Vandelli, Maria Angela; Forni, Flavio; Scarpa, Maurizio; Tomanin, Rosella; Tosi, Giovanni
abstract

Lysosomal Storage Disorders (LSDs) are a group of metabolic syndromes, each one due to the deficit of one lysosomal enzyme. Many LSDs affect most of the organ systems and overall about 75% of the patients present neurological impairment. Enzyme Replacement Therapy, although determining some systemic clinical improvements, is ineffective on the CNS disease, due to enzymes' inability to cross the blood-brain barrier (BBB). With the aim to deliver the therapeutic enzymes across the BBB, we here assayed biodegradable and biocompatible PLGA-nanoparticles (NPs) in two murine models for LSDs, Mucopolysaccharidosis type I and II (MPS I and MPS II). PLGA-NPs were modified with a 7-aminoacid glycopeptide (g7), yet demonstrated to be able to deliver low molecular weight (MW) molecules across the BBB in rodents. We specifically investigated, for the first time, the g7-NPs ability to transfer a model drug (FITC-albumin) with a high MW, comparable to the enzymes to be delivered for LSDs brain therapy. In vivo experiments, conducted on wild-type mice and knockout mouse models for MPS I and II, also included a whole series of control injections to obtain a broad preliminary view of the procedure efficiency. Results clearly showed efficient BBB crossing of albumin in all injected mice, underlying the ability of NPs to deliver high MW molecules to the brain. These results encourage successful experiments with enzyme-loaded g7-NPs to deliver sufficient amounts of the drug to the brain district on LSDs, where exerting a corrective effect on the pathological phenotype.

2016 - The “fate” of polymeric and lipid nanoparticles for brain delivery and targeting: Strategies and mechanism of blood–brain barrier crossing and trafficking into the central nervous system [Articolo su rivista]
Tosi, Giovanni; Musumeci, Teresa; Ruozi, Barbara; Carbone, Claudia; Belletti, Daniela; Pignatello, Rosario; Vandelli, Maria Angela; Puglisi, Giovanni
abstract

Drug delivery to the brain represents one of the most important challenges in the field of nanomedicine, as the study and planning of nanocarriers able to cross the blood brain barrier are topics at the cutting edge of technology and innovation. In this review, we analyze the interventions and progresses in the field, by analyzing the choice of the polymer, of the ligands and the main relevant in vivo and in vitro experiments. A critical overview of these aspects will help in better understanding the status of nanomedicine in the application to central nervous system pathologies along with the future directions

2015 - Antineoplastic effects of liposomal siRNA treatment targeting BLIMP1/PRDM1 in primary effusion lymphoma [Articolo su rivista]
Riva, Giovanni; Lagreca, Ivana; Mattiolo, Adriana; Belletti, Daniela; Lignitto, Laura; Barozzi, Patrizia; Ruozi, Barbara; Vallerini, Daniela; Quadrelli, Chiara; Corradini, Giorgia; Forghieri, Fabio; Marasca, Roberto; Narni, Franco; Tosi, Giovanni; Forni, Flavio; Vandelli, Maria Angela; Amadori, Alberto; Chieco Bianchi, Luigi; Potenza, Leonardo; Calabro', Maria Luisa; Luppi, Mario
abstract

RNA interference (RNAi) has been suggested to represent a promising therapeutic approach in different disease settings. Primary effusion lymphoma (PEL) is a plasmablastic lymphoma consistently expressing B lymphocyte-induced maturation protein 1 (Blimp-1), a pivotal transcriptional regulator during terminal differentiation of B cells into plasma cells. Here we report, for the first time, that transient knockdown of the BLIMP1 gene (also known as PR Domain Containing 1 with ZNF Domain, or PRDM1) using small interfering RNA (siRNA) delivered by liposomes, induced remarkable killing in PEL cell lines. Furthermore, in a murine model of PEL, significantly prolonged survival was achieved by intraperitoneal treatment with such anti-BLIMP1 lipoplexes, while no vector-induced toxicity was observed. This effective and safe RNAi strategy, based on liposomal siRNA targeting a master transcription factor of post-germinal center B cells, may indeed be a potential treatment against plasmablastic lymphoma

2015 - Application of Polymeric Nanoparticles for CNS Targeted Zinc Delivery In Vivo [Articolo su rivista]
Chhabra, Resham; Ruozi, Barbara; Vilella, Antonietta; Belletti, Daniela; Mangus, Katharina; Pfaender, Stefanie; Sarowar, Tasnuva; Boeckers, Tobias Maria; Zoli, Michele; Forni, Flavio; Vandelli, Maria Angela; Tosi, Giovanni; Grabrucker, Andreas Martin
abstract

A dyshomeostasis of zinc ions has been reported for many psychiatric and neurodegenerative disorders including schizophrenia, attention deficit hyperactivity disorder, depression, autism, Parkinson's and Alzheimer's disease. Furthermore, alterations in zinc-levels have been associated with seizures and traumatic brain injury. Thus, altering zinclevels within the brain is emerging as a new target for the prevention and treatment of psychiatric and neurological diseases. However, given the restriction of zinc uptake into the brain by the blood-brain barrier, methods for controlled regulation and manipulation of zinc concentrations within the brain are rare. Here, we performed in vivo studies investigating the possibility of brain targeted zinc delivery using zinc-loaded nanoparticles which are able to cross the blood-brain barrier. After injecting these nanoparticles, we analyzed the regional and time-dependent distribution of zinc and nanoparticles within the brain. Moreover, we evaluated whether the presence of zinc-loaded nanoparticles alters the expression of zinc sensitive genes and proteins such as metallothioneins and zinc transporters and quantified possible toxic effects. Our results show that zinc loaded g7 nanoparticles offer a promising approach as a novel non - invasive method to selectively enrich zinc in the brain within a small amount of time.

2015 - Cholesterol-loaded nanoparticles ameliorate synaptic and cognitive function in Huntington's disease mice [Articolo su rivista]
Valenza, Marta; Chen, Jane Y.; Di Paolo, Eleonora; Ruozi, Barbara; Belletti, Daniela; Ferrari Bardile, Costanza; Leoni, Valerio; Caccia, Claudio; Brilli, Elisa; Di Donato, Stefano; Boido, Marina M.; Vercelli, Alessandro; Vandelli, Maria Angela; Forni, Flavio; Cepeda, Carlos; Levine, Michael S.; Tosi, Giovanni; Cattaneo, Elena
abstract

Brain cholesterol biosynthesis and cholesterol levels are reduced in mouse models of Huntington's disease (HD), suggesting that locally synthesized, newly formed cholesterol is less available to neurons. This may be detrimental for neuronal function, especially given that locally synthesized cholesterol is implicated in synapse integrity and remodeling. Here, we used biodegradable and biocompatible polymeric nanoparticles (NPs) modified with glycopeptides (g7) and loaded with cholesterol (g7-NPs-Chol), which per se is not blood-brain barrier (BBB) permeable, to obtain high-rate cholesterol delivery into the brain after intraperitoneal injection in HD mice. We report that g7-NPs, in contrast to unmodified NPs, efficiently crossed the BBB and localized in glial and neuronal cells in different brain regions. We also found that repeated systemic delivery of g7-NPs-Chol rescued synaptic and cognitive dysfunction and partially improved global activity in HD mice. These results demonstrate that cholesterol supplementation to the HD brain reverses functional alterations associated with HD and highlight the potential of this new drug-administration route to the diseased brain.

2015 - Endocytosis of Nanomedicines: The Case of Glycopeptide Engineered PLGA Nanoparticles [Articolo su rivista]
Vilella, Antonietta; Ruozi, Barbara; Belletti, Daniela; Pederzoli, Francesca; Galliani, Marianna; Semeghini, Valentina; Forni, Flavio; Zoli, Michele; Vandelli, Maria Angela; Tosi, Giovanni
abstract

The success of nanomedicine as a new strategy for drug delivery and targeting prompted the interest in developing approaches toward basic and clinical neuroscience. Despite enormous advances on brain research, central nervous system (CNS) disorders remain the world's leading cause of disability, in part due to the inability of the majority of drugs to reach the brain parenchyma. Many attempts to use nanomedicines as CNS drug delivery systems (DDS) were made; among the various non-invasive approaches, nanoparticulate carriers and, particularly, polymeric nanoparticles (NPs) seem to be the most interesting strategies. In particular, the ability of poly-lactide-co-glycolide NPs (PLGA-NPs) specifically engineered with a glycopeptide (g7), conferring to NPs' ability to cross the blood brain barrier (BBB) in rodents at a concentration of up to 10% of the injected dose, was demonstrated in previous studies using different routes of administrations. Most of the evidence on NP uptake mechanisms reported in the literature about intracellular pathways and processes of cell entry is based on in vitro studies. Therefore, beside the particular attention devoted to increasing the knowledge of the rate of in vivo BBB crossing of nanocarriers, the subsequent exocytosis in the brain compartments, their fate and trafficking in the brain surely represent major topics in this field.

2015 - Exploiting Bacterial Pathways for BBB Crossing with PLGA Nanoparticles Modified with a Mutated Form of Diphtheria Toxin (CRM197): In Vivo Experiments [Articolo su rivista]
Tosi, Giovanni; Vilella, Antonietta; Veratti, P; Belletti, Daniela; Pederzoli, F; Ruozi, Barbara; Vandelli, Maria Angela; Zoli, Michele; Forni, Flavio
abstract

Drugs can be targeted to the brain using polymeric nanoparticles (NPs) engineered on their surface with ligands able to allow crossing of the blood-brain barrier (BBB). This article aims to investigate the BBB crossing efficiency of polymeric poly lactide-co-glycolide (PLGA) NPs modified with a mutated form of diphtheria toxin (CRM197) in comparison with the results previously obtained using PLGA NPs modified with a glycopeptide (g7-NPs). Different kinds of NPs, covalently coupled PLGA with different fluorescent probes (DY405, rhodamine-B base and DY675) and different ligands (g7 and CRM197) were tested in vivo to assess their behavior and trafficking. The results highlighted the possibility to distinguish the different kinds of simultaneously administered NPs and to emphasize that CRM-197 modified NPs and g7-NPs can cross the BBB at a similar extent. The analysis of BBB crossing and of the neuronal tropism of CRM197 modified NPs, along with their BBB crossing pathways were also developed. In vivo pharmacological studies performed on CRM197 engineered NPs, loaded with loperamide, underlined their ability as drug carriers to the CNS.

2015 - Functionalization of liposomes: microscopical methods for preformulative screening [Articolo su rivista]
Belletti, Daniela; Vandelli, Maria Angela; Tonelli, Massimo; Zapparoli, Mauro; Forni, Flavio; Tosi, Giovanni; Ruozi, Barbara
abstract

The development of smart delivery systems able to deliver and target a drug to the site of action is one of the major challenges in the field of pharmaceutical technology. The surface modification of nanocarriers, such as liposomes, is widely investigated either for increasing the blood circulation time (by pegylation) or for interacting with specific tissues or cells (by conjugation of a selective ligand as a monoclonal antibody, mAb). Microscopical analysis thereby is a useful approach to evaluate the morphology and the size owing to resolution and versatility in defining either surface modification or the architecture and the internal structure of liposomes. This contribution aims to connect the outputs obtained by transmission electron (TEM) and atomic force (AFM) microscopical techniques for identifying the modifications on the liposomal surface. To reach this objective, we prepared liposomes applying two different pegylation technologies and further modifying the surface by mAb conjugation. This work demonstrates the feasibility to apply the combined approach (TEM and AFM analysis) in the evaluation of the efficacy of a surface engineering process.

2015 - Nanoimaging: photophysical and pharmaceutical characterization of poly-lactide-co-glycolide nanoparticles engineered with quantum dots [Articolo su rivista]
Pederzoli, F.; Ruozi, Barbara; Pracucci, E.; Signore, G.; Zapparoli, Mauro; Forni, Flavio; Vandelli, Maria Angela; Ratto, G.; Tosi, Giovanni
abstract

Quantum dots (QDs) and polymeric nanoparticles (NPs) are considered good binomials for the development of multifunctional nanomedicines for multimodal imaging. Fluorescent imaging of QDs can monitor the behavior of QD-labeled NPs in both cells and animals with high temporal and spatial resolutions. The comprehension of polymer interaction with the metallic QD surface must be considered to achieve a complete chemicophysical characterization of these systems and to describe the QD optical properties to be used for their unequivocal identification in the tissue. In this study, by comparing two different synthetic procedures to obtain polymeric nanoparticles labeled with QDs, we investigated whether their optical properties may change according to the formulation methods, as a consequence of the different polymeric environments. Atomic force microscopy, transmission electron microscopy, confocal and fluorescence lifetime imaging microscopy characterization demonstrated that NPs modified with QDs after the formulation process (post-NPs-QDs) conserved the photophysical features of the QD probe. In contrast, by using a polymer modified with QDs to formulate NPs (pre-NPs-QDs), a significant quenching of QD fluorescence and a blueshift in its emission spectra were observed. Our results suggest that the packaging of QDs into the polymeric matrix causes a modification of the QD optical properties: these effects must be characterized in depth and carefully considered when developing nanosystems for imaging and biological applications.

2015 - Nanomedicine and neurodegenerative disorders: So close yet so far [Articolo su rivista]
Tosi, Giovanni; Vandelli, Maria Angela; Forni, Flavio; Ruozi, Barbara
abstract

This editorial provides an overview of the main advantages of the use of nanomedicine-based approach for innovation in the treatment of neurodegenerative diseases. Besides these aspects, a critical analysis on the main causes that slow the application of nanomedicine to brain disorders is given along with the identification of possible solutions and possible interventions. Better communication between the main players of research in this field and a detailed understanding of the most critical issues to be addressed should help in defining future directions towards the improvement and, finally, the clinical application of nanomedicine to neurodegenerative diseases.

2015 - Nanomedicine in neurodegenerative disorders: Understanding the journey [Relazione in Atti di Convegno]
Tosi, G.; Ruozi, B.; Vilella, A.; Grabrucker, A. M.; Belletti, D.; Vandelli, M. A.; Boeckers, T. M.; Forni, F.; Zoli, M.; Sharma, A.; Muresanu, D. F.; Sharma, H. S.
abstract

Nanocarriers can be useful tools for delivering drugs to the central nervous system (CNS). Their distribution within the brain and their interaction with CNS cells must be assessed accurately before they can be proposed for therapeutic use. We investigated these issues by employing poly-lactide-co- glycolide nanoparticles (NPs) specifically engineered with a glycopeptide (g7) conferring to NPs the ability to cross the blood brain barrier (BBB) at a concentration of up to 10% of the injected dose. g7- NPs display increased in vitro uptake in neurons and glial cells, in vivo administration of g7-NPs leads to a region- And cell type-specific enrichment of NPs within the brain. Moreover, g7-NPs are endocytosed in a clathrin-dependent manner and transported into a specific subset of early endosomes positive for Rab5 in vitro and in vivo. Moreover, in order to understand the journey of NPs, we demonstrated that g7-NPs can be transported intra- And intercellularly inside vesicles. Cell-to-cell transport is mediated by tunneling-nanotube (TNT)-like structures in cell lines and most interestingly in glial as well as neuronal cells in vitro. These in vitro findings were in part confirmed by in vivo evidence after i.p. administration in mice. We also tested Ab-modified g7-NPs both in vitro and in vivo to investigate the possibility of a specific targeting.

2015 - Nutlin-3 loaded nanocarriers: Preparation, characterization and in vitro antineoplastic effect against primary effusion lymphoma [Articolo su rivista]
Belletti, Daniela; Tosi, Giovanni; Riva, Giovanni; Lagreca, Ivana; Galliania, M; Luppi, Mario; Vandelli, Maria Angela; Forni, Flavio; Ruozi, Barbara
abstract

In this investigation, Nutlin-3 (Nut3), a novel antitumor drug with low water solubility (<0.1mg/L at 25°C), was loaded into liposomes (Lipo-Nut3), polymeric nanoparticles (NPs-Nut3) and nanoparticles engineered with an antibody direct against Syndecan-1/CD 138 (Syn-NPs-Nut3) to obtain carriers targeted to PEL (primary effusion lymphoma). The physicochemical properties of these carriers were determined. Atomic force microscopy showed that all the particles were well formed and spherical in shape. The presence of the antibody on surface led to a significant increase of mean diameter (280 ± 63 nm), PDI (0.3) and the shift of zeta potential towards neutrality (-1 mV). The entrapment efficiency of Lipo-Nut3, NPs-Nut3 and Syn-NPs-Nut3 was 30, 52 and 29%, and drug loading was 1.4, 4.5 and 2.6%, respectively. By performing cytofluorimetric analyses and bromodeoxyuridine (BrdU) assay, the efficacy of nanocarriers to deliver the antineoplastic drug into a PEL cell line namely BCBL-1 (immortalized body cavity B-cell lymphoma) was investigated. Two days after the treatment with 20 μM of Syn-NPs-Nut3, the cell density decreased at about 60% while the cell viability decreased at 56% only 5 days after transfection, when compared with untreated cells. A cell cycle arrest was observed with a significant decrease of cells in S-phase and increasing of apoptotic cell, if compared with untreated control. These results confirms the potential of nanocarriers approaches to deliver antitumor drug with unfavorable chemico-physical properties. Moreover, this study strongly suggests that Syn-NPs-Nut3 can be a valuable drug carrier system for the treatment of PEL lymphoma.

2015 - PEG-g-chitosan nanoparticles functionalized with the monoclonal antibody OX26 for brain drug targeting [Articolo su rivista]
Monsalve, Yuliana; Tosi, Giovanni; Ruozi, Barbara; Belletti, Daniela; Vilella, Antonietta; Zoli, Michele; Vandelli, Maria Angela; Forni, Flavio; López, Betty L.; Sierra, Ligia
abstract

Aim: Drug targeting to the CNS is challenging due to the presence of blood-brain barrier. We investigated chitosan (Cs) nanoparticles (NPs) as drug transporter system across the blood-brain barrier, based on mAb OX26 modified Cs. Materials & methods: Cs NPs functionalized with PEG, modified and unmodified with OX26 (Cs-PEG-OX26) were prepared and chemico-physically characterized. These NPs were administered (intraperitoneal) in mice to define their ability to reach the brain. Results: Brain uptake of OX26-conjugated NPs is much higher than of unmodified NPs, because: long-circulating abilities (conferred by PEG), interaction between cationic Cs and brain endothelium negative charges and OX26 TfR receptor affinity. Conclusion: Cs-PEG-OX26 NPs are promising drug delivery system to the CNS.

2015 - PLGA Nanoparticles Loaded Cerebrolysin: Studies on Their Preparation and Investigation of the Effect of Storage and Serum Stability with Reference to Traumatic Brain Injury [Articolo su rivista]
Ruozi, Barbara; Belletti, Daniela; Sharma, Hari S.; Sharma, Aruna; Muresanu, Dafin F.; Mössler, H.; Forni, Flavio; Vandelli, Maria Angela; Tosi, Giovanni
abstract

Cerebrolysin is a peptide mixture able to ameliorate symptomatology and delay progression of neurological disorders such as Alzheimer’s disease and dementia. The administration of this drug in humans presents several criticisms due to its short half-life, poor stability, and high doses needed to achieve the effect. This paper investigates the potential of polylactic-co-glycolide (PLGA) nanoparticles (NPs) as sustained release systems for iv administration of cerebrolysin in normal and brain injured rats. NPs were prepared by water-in-oil-in-water (w/o/w) double emulsion technique and characterized by light scattering for mean size and zeta potential and by scanning electron microscopy (SEM) for surface morphology. The NPs produced by double sonication under cooling at 60 W for 45 s, 12 mL of 1 % w:v of PVA, and 1:0.6 w:w drug/PLGA ratio (C-NPs4) displayed an adequate loading of drug (24 ± 1 mg/100 mg of NPs), zeta potential value (−13 mV), and average diameters (ranged from 250 to 330 nm) suitable to iv administration. SEM images suggested that cerebrolysin was molecularly dispersed into matricial systems and partially adhered to the NP surface. A biphasic release with an initial burst effect followed by sustained release over 24 h was observed. Long-term stability both at room and at low temperature of freeze-dried NPs was investigated. To gain deeper insight into NP stability after in vivo administration, the stability of the best NP formulation was also tested in serum. These PLGA NPs loaded with cerebrolysin were able to reduce brain pathology following traumatic brain injury. However, the size, the polydispersivity, and the surface properties of sample were significantly affected by the incubation time and the serum concentration

2015 - Use of Polylactide-Co-Glycolide-Nanoparticles for Lysosomal Delivery of a Therapeutic Enzyme in Glycogenosis Type II Fibroblasts [Articolo su rivista]
Tancini, Brunella; Tosi, Giovanni; Bortot, Barbara; Dolcetta, Diego; Magini, Alessandro; De Martino, Eleonora; Urbanelli, Lorena; Ruozi, Barbara; Forni, Flavio; Emiliani, Carla; Vandelli, Maria Angela; Severini, Giovanni Maria
abstract

Glycogenosis type II, or Pompe Disease, is a lysosomal storage disease caused by the deficiency of acid alpha-glucosidase (GAA), leading to glycogen accumulation in muscles. A recombinant human GAA (rhGAA, Myozyme®) is currently used for enzyme replacement therapy. Despite its efficacy in most of patients, some of them show a diminished response to the treatment with rapidly progressive clinical deterioration, due to immuno-mediated enzyme inactivation. To demonstrate that Nanoparticles (NPs) could be profitably exploited to carry macromolecules, PLGA NPs loaded with rhGAA (GAA-NPs) were prepared by double emulsion solvent evaporation. Their surface morphology, particle size, zeta-potential and biochemical activity were assessed. “Pulse and chase” experiments were made by administrating GAA-NPs on patients’ fibroblasts. Biochemical activity tests showed a more efficient cellular uptake of rhGAA loaded to NPs and a more significant stability of the enzyme (up to 7 days) in vitro, if compared to the same amount of rhGAA free enzyme. This data allows to envision in vivo experiments, in significant animal models, to further characterize lysosomal enzyme loaded-NPs’ efficacy and toxicity.

2014 - AFM/TEM complementary structural analysis of surface-functionalized nanoparticles [Articolo su rivista]
Ruozi, Barbara; Belletti, Daniela; Vandelli, Maria Angela; Tonelli, Massimo; Zapparoli, Mauro; Pederzoli, Francesca; Veratti, Patrizia; Forni, Flavio; Tosi, Giovanni
abstract

In the field of nanomedicine, the characterization of functionalized drug delivery systems, introduced on market as efficacious and selective therapeutics, represents a pivotal aspect of great importance. In particular, the morphology of polymeric nanoparticles, the most studied nanocarriers, is frequently assessed by transmission electron microscopy (TEM). Despite of TEM high resolution and versatility, this technology is frequently hampered by both the complicated procedure for sample preparation and the operative condition of analysis. Considering the scanning probe microscopies, atomic force microscopy (AFM) represents an extraordinary tool for the detailed characterization of submicron-size structure as the surface functionalization at the atomic scale. In this paper we discussed the advantage and limits of these microscopies applied to the characterization of PLGA nanoparticles functionalized with three different kinds of ligands (carbohydrate ligand, an antibody and quantum dots crystals) intentionally designed, created and tailored with specific physico-chemical properties to meet the needs of specific applications (targeting or imaging).

2014 - Characterization of lysosome-destabilizing DOPE/PLGA nanoparticles designed for cytoplasmic drug release [Articolo su rivista]
Resham, Chhabra; Andreas M., Grabrucker; Veratti, Patrizia; Belletti, Daniela; Tobias M., Boeckers; Vandelli, Maria Angela; Forni, Flavio; Tosi, Giovanni; Ruozi, Barbara
abstract

Polymeric nanoparticles (NPs) offer a promising approach for therapeutic intracellular delivery of proteins, conventionally hampered by short half-lives, instability and immunogenicity. Remarkably, NPs uptake occurs via endocytic internalization leading to NPs content’s release within lysosomes. To overcome lysosomal degradation and achieve NPs and/or loaded proteins release into cytosol, we propose the formulation of hybrid NPs by adding 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as pH sensitive component in the formulation of poly-lactide-co-glycolide (PLGA) NPs. Hybrid NPs, featured by different DOPE/PLGA ratios, were characterized in terms of structure, stability and lipid organization within the polymeric matrix. Experiments on neuronal cells and rat primary cultures highlighted the safety profile of hybrid NPs. Moreover, after internalization, NPs are able to transiently destabilize the integrity of lysosomes in which they are taken up, speeding their escape and favoring cytoplasmatic localization. Thus, these DOPE/PLGA-NPs configure themselves as promising carriers for intracellular protein delivery

2014 - Detection of PLGA-based nanoparticles at single cell level by Synchrotron Radiation FTIR Spectromicroscopy and correlation with X-Ray Fluorescence Microscopy [Articolo su rivista]
Lorella, Pascolo; Barbara, Bortot; Nuria Benseny, Cases; Alessandra, Gianoncelli; Tosi, Giovanni; Ruozi, Barbara; Clara, Rizzardi; Eleonora De, Martino; Vandelli, Maria Angela; Giovanni Maria, Severini
abstract

Poly-lactide-co-glycolide (PLGA) is one of the few polymers approved by the Food and Drug Administration (FDA) as carrier for drug administration in humans and, therefore, it is one of the most used materials in the formulation of polymeric nanoparticles (NPs) for therapeutic purposes. Since the cellular uptake of polymeric NPs is a highlight topic in the nanomedicine field, the development of techniques able to assure an incontrovertible evidence of NPs presence in the cells plays a key role for gaining understanding of their therapeutic potential. On the strength of this premise, this paper aims to evaluate the application of the Synchrotron Radiation-based FTIR (SR-FTIR) spectromicroscopy and the Synchrotron Radiation X-Ray Fluorescence (SR-XRF) microscopy in the study of the in vitro interaction of PLGA NPs with cells. To reach this aim, we used PLGA NPs, sizing around 200 nm and loaded with superparamagnetic iron oxide nanoparticles (Fe3O4; size 10-15 nm) (PLGA-IO-NPs). After having exposed human mesothelial (MeT5A) cells to PLGA-IO-NPs (0.1 mg/ml), the cells were analyzed after fixation both by SR-FTIR spectromicroscopy and SR-XRF microscopy setups. SR-FTIR-SM enabled the detection of PLGA NPs at single cell level allowing the polymer detection inside the biological matrix by the characteristic band in the 1700–2000 cm− 1 region. The precise PLGA IR-signature (1750 cm− 1 centered pick) was clearly evident also within an area of high amide density. SR-XRF microscopy performed on the same cells investigated under SR-FTIR microscopy allowed to put in evidence the Fe presence in the cells and to emphasize the intracellular localization of the PLGA-IO-NPs. These findings suggest that SR-FTIR and SR-XRF techniques could be two valuable tools to follow the PLGA NPs fate in the in vitro studies on cell cultures .

2014 - Insight on the fate of CNS-targeted nanoparticles. Part I: Rab5-dependent cell-specific uptake and distribution [Articolo su rivista]
Vilella, Antonietta; Tosi, Giovanni; Andreas M., Grabrucker; Ruozi, Barbara; Belletti, Daniela; Vandelli, Maria Angela; Tobias M., Boeckers; Forni, Flavio; Zoli, Michele
abstract

Nanocarriers can be useful tools for delivering drugs to the central nervous system (CNS). Their distribution within the brain and their interaction with CNS cells must be assessed accurately before they can be proposed for therapeutic use. In this paper, we investigated these issues by employing poly-lactide-co-glycolide nanoparticles (NPs) specifically engineered with a glycopeptide (g7) conferring to NPs the ability to cross the blood brain barrier (BBB) at a concentration of up to 10% of the injected dose. g7-NPs display increased in vitro uptake in neurons and glial cells. Our results show that in vivo administration of g7-NPs leads to a region- and cell type-specific enrichment of NPs within the brain. We provide evidence that g7-NPs are endocytosed in a clathrin-dependent manner and transported into a specific subset of early endosomes positive for Rab5 in vitro and in vivo. The differential Rab5 expression level is strictly correlated with the amount of g7-NP accumulation. These findings show that g7-NPs can cross the BBB and target specific brain cell populations, suggesting that these NPs can be promising carriers for the treatment of neuropsychiatric and neurodegenerative diseases.

2014 - Insight on the fate of CNS-targeted nanoparticles. Part II: Intercellular neuronal cell-to-cell transport [Articolo su rivista]
Tosi, Giovanni; Vilella, Antonietta; Resham, Chhabra; Michael J., Schmeisser; Tobias M., Boeckers; Ruozi, Barbara; Vandelli, Maria Angela; Forni, Flavio; Zoli, Michele; Andreas M., Grabrucker
abstract

The application of polymeric nanoparticles (NPs) has a promising future for targeting and delivering drugs into the central nervous system (CNS). However, the fate of NPs once entered in the brain after crossing the blood-brain barrier (BBB) and taken up into neuronal cells is a neglected area of study. Thus, here, we investigate the possible mechanisms of a cell-to-cell transport of poly-lactide-co-glycolide (PLGA) NPs modified with a glycopeptide (g7-NPs), already demonstrated to be able to cross the BBB after in vivo administration in rodents. We also tested antibody (Ab) -modified g7-NPs both in vitro and in vivo to investigate the possibility of a specific targeting. Our results show that g7-NPs can be transported intra- and intercellularly inside vesicles. Moreover, cell-to-cell transport is mediated by tunneling-nanotube (TNT)-like structures in cell lines and most interestingly in glial as well as neuronal cells in vitro. The transport is dependent on F-actin and can be increased by induction of TNT-like structures overexpressing M-Sec, a central factor and inducer of TNT formation. Moreover, cell-to-cell transport occurs independently from NP surface modification with antibodies. These in vitro findings were in part confirmed by in vivo evidence after i.p. administration in mice.

2014 - Nanoparticles as Blood–Brain Barrier Permeable CNS Targeted Drug Delivery Systems [Capitolo/Saggio]
Andreas M., Grabrucker; Resham, Chhabra; Belletti, Daniela; Forni, Flavio; Vandelli, Maria Angela; Ruozi, Barbara; Tosi, Giovanni
abstract

Research in the field of nano-neuroscience is becoming a promising future direction given the advantages presented by nanosystems for central nervous system (CNS) drug delivery. Since the blood–brain barrier (BBB) represents an invincible obstacle for the majority of drugs such as antineoplastic agents and a variety of psychoactive drugs such as neuropeptides, “smart” CNS drug delivery systems with high ability to deliver substances across the BBB are highly desired and will not only enable drugs to reach the CNS but also target specific areas of the CNS. Thus, injectable biodegradable nanoparticles have an important potential application in the treatment of a variety of neurological and psychiatric disorders. Therefore, in the following, we will highlight the requirement and importance of CNS drug delivery systems with particular emphasis on nano-scale systems. It is the objective of this article to offer a perspective on the complexity and challenges in fabrication of nanostructures, in vivo nano–bio interactions and also to highlight some of the most used nanosystems for drug delivery into the CNS.

2014 - Nanotechnology and Alzheimer’s disease: What has been done and what to do’ [Articolo su rivista]
Ruozi, Barbara; Belletti, Daniela; Pederzoli, Francesca; Veratti, Patrizia; Forni, Flavio; Vandelli, Maria Angela; Tosi, Giovanni
abstract

Up to date, Alzheimer’s Disease (AD) is considered as an “urgency” for public health, since it represents one of the most dramatic causes of death in adults. The drugs currently used for AD are only symptomatic, thus not curing the pathology, but only trying to slow or delay the progression of the pathology. Moreover, there is a total lack of early identification, with only “probable’’ or ‘‘possible’’ diagnosis of AD patients. With this review, we aimed to individuate and to highlight the most promising approaches for AD therapy and diagnosis. In this view, at the cutting-edge of innovation, nanocarriers as polymeric nanoparticles, liposomes, nanoassembly and dendrimers, have been studied and investigated in order to ameliorate the detection (in vitro and in vivo) and/or the therapeutic options in AD. In this review, the most outstanding nanomedicine-driven approaches in AD imaging/detection and treatments are summarized in order to help in individuating values and criticisms. Moreover, an overview of one of the most innovative strategies in AD management, namely theranostic nanomedicine, is reported and commented.

2014 - Poly (D,L-lactide-co-glycolide) nanoparticles loaded with Cerebrolysin display neuroprotective activity in a rat model of closed head injury [Articolo su rivista]
Ruozi, Barbara; Belletti, Daniela; Forni, Flavio; Sharma, A.; Muresanu, D.; Mössler, H.; Vandelli, Maria Angela; Tosi, Giovanni; Sharma, H. S.
abstract

Cerebrolysin (CBL) is a neuroprotective agent in central nervous system (CNS) injury and stimulates neurorepair processes. Several studies in our laboratory suggest that CBL administered through nanowired technology may have superior neuroprotective efficacy in CNS trauma. In this investigation, we compared the neuroprotective efficacy of poly-lactide-co-glycolide nanoparticles (NPs) loaded with CBL vs. unloaded CBL in a rat model of closed head injury (CHI). Free CBL or CBL loaded NPs was administered 1 h after CHI and animals sacrificed 4 h later. Changes in blood-brain barrier and brain edema formation were measured as parameters of neuroprotection in CHI after giving CBL alone or as the nanodelivered compound. Our results clearly show that delivery of CBL by NPs has superior neuroprotective effects following CHI as compared to normal CBL. This suggests that CBL delivered by NPs could have strong neuroprotective ability in CNS trauma. These findings have potential clinical relevance with regard to nanodelivery of CBL, a feature that requires further investigation.

2013 - A Nanoparticle-based approach for drug delivery to the brain in Lysosomal Storage Disorders [Abstract in Atti di Convegno]
M., Salvalaio; Tosi, Giovanni; L., Rigon; Belletti, Daniela; F., D’Avanzo; Ruozi, Barbara; Vandelli, Maria Angela; Forni, Flavio; M., Scarpa; R., Tomanin
abstract

Lysosomal Storage Disorders (LSDs) are a group of more than 50 different hereditary diseases, mostly due the deficit of activity of one or more acid hydrolases in lysosomes. About 70% of LSD patients present a neurological impairment which is still untreatable, since recombinant corrective lysosomal enzymes, where available, cannot cross the blood-brain barrier (BBB). Among LSDs, Mucopolysaccharidosis type I (MPS I, Hurler Disease) and type II (MPS II, Hunter Disease) are both characterized by a totally or partially defective activity of lysosomal enzymes involved in the catabolism of the mucopolysaccharides (or glycosaminoglycans, GAGs) heparan- and dermatan-sulfate which, therefore, heavily accumulate within cellular compartment and in the extracellular matrix. Enzyme Replacement Therapy (ERT), applied to both diseases in the last few years, has shown to determine some clinical improvements, but it has also shown some limitations. In addition to the elevated costs and to the weekly administration in a day-hospital regimen, the low level of the BBB transport system for acid hydrolases and the high molecular weight of these enzymes make any paracellular or transcellular diffusion of these proteins across the BBB almost non-existent. Therefore, alternative methods to achieve transcytosis into the CNS need to be explored. Thus, we combined the experience of clinical-based skills with pharmaceutical nanotechnology-based skills in order to create nanocarriers, biodegradable and biocompatible, able to deliver the recombinant enzymes across the BBB and to both assure a prolonged drug circulation and release, and a protection from metabolic drug inactivation. With this aim, we produced polymeric nanoparticles (PLGA-NPs) modified with 7-aminoacid glycopeptides (g7), able to drive the NPs across the BBB after administration in rodents. Before going into functional and efficacy study, we studied the ability of PLGS-NPs in carrying across the BBB the FITC-albumin, as a model drug with a high molecular weight, comparable to that of the enzymes using in ERT. In vivo experiments on both WT and knock-out (KO) mouse models for MPS I and MPS II were performed by i.v.-injecting g7-NPs loaded with FICT-albumin together with a plethora of control samples (i.e. un-modified NPs, FITC-albumin solution) in order to have a broad preliminary view. The results clearly showed that g7-NPs are able to deliver FITC-albumin to the brain, crossing the BBB, in all treated mice (WT and KO models); interestingly, we found qualitative and semi-quantitative evidences of a higher grade of brain accumulation of g7-NPs loaded with Albumin in the KO-brains with respect to WT ones. These results lay the basis for a possible successful set of pilot experiments on the ability of enzyme-loaded g7-NPs to deliver sufficient amount of the drug to the brain district, hopefully exerting a corrective effect on the cellular pathological GAG deposits.

2013 - AFM and TEM characterization of siRNAs lipoplexes: a combinatory tools to predict the efficacy of complexation [Articolo su rivista]
Belletti, Daniela; Tonelli, Massimo; Forni, Flavio; Tosi, Giovanni; Vandelli, Maria Angela; Ruozi, Barbara
abstract

This work aims to evaluate the effects of two different surface modification strategies: PEG conventional coupling (PEG-Lpx) and postpegylation technique (postPEG-Lpx), on lipoplexes obtained between liposomes and siRNAs. Photon correlation spectroscopy (PCS) and gel electrophoreses (as conventional techniques), and atomic force microscopy (AFM) and transmission electron microscopy (TEM) (proposed to complete the assessments of lipoplexes) were employed to investigate reorganization, structure, qualitative-quantitative stabilization of siRNAs, and PEG covering of lipoplexes. The results suggested that postPEGLpx exhibited high level of homogeneity with a mean diameter (Z-Average) of about 320 nm, low tendency to aggregation (a polydispersion index, PDI, close to 0.06) and high loading efficiency (E.E. 82%). Otherwise, PEG-Lpx showed a Z-Average greater than 1m, high aggregation rate (PDI > 0.3) and a low E.E. (10%). The definition of the architecture by using optimized microscopical procedure allows to suggest postpegylation technique as a promising technology for the preparation of applicable complexes. This formulation strategy lead to a stable siRNA condensation and full compaction of gene material, moreover the PEG coverage generated a homogeneous hydrated surface, well described by the ―phase‖ AFM approach. The microscopical techniques can provide a predictive and useful tool to use in the preformulative technological studies of complicated gene complexes.

2013 - Biodegradable device applied in flatfoot surgery: Comparative studies between clinical and technological aspects of removed screws [Articolo su rivista]
Ruozi, Barbara; Belletti, Daniela; Giuseppe, Manfredini; Tonelli, Massimo; Paola, Sena; Vandelli, Maria Angela; Forni, Flavio; Tosi, Giovanni
abstract

Poly-L-lactide (PLLA) is one of the most used polymers for biomedical application; its use in sutures and other implants has been widely investigated. Although the knowledge of PLLA biodegradation and biocompatibility features is deep, PLLA screws used to correct the flat foot deformity have deserved attention since they are not degraded inmost of cases after a long period of years (3–7) fromthe implantation. In this article, a clinical and radiological evaluation (NMR, histological and clinical outcomes) on patients was correlatedwith physico-chemical characterization (by SEM, DSC, GPC and XRD analysis at different temperatures) on both native and patientrecovered screws together with the theoretical degradation processes of PLLA-based implants. The data demonstrated the need for crossing the biodegradation and bioabsorption of the polymer with the characteristics of both the device (geometry, structure and fabrication process) and the implantation site.

2013 - Blood-Brain Barrier crossing of high molecular weight molecules mediated by nanoparticles: a potential approach to treat neurological Lysosomal Storage Disorders [Abstract in Atti di Convegno]
Tosi, Giovanni; L., Rigon; M., Salvalaio; Belletti, Daniela; F., D’Avanzo; Ruozi, Barbara; Vandelli, Maria Angela; Forni, Flavio; M., Scarpa; R., Tomanin
abstract

Enzyme Replacement Therapy (ERT) is the most common therapeutic strategy applied to several Lysosomal Storage Disorders (LSDs) including Mucopolysaccharidoses (MPSs), as MPS type I (MPS I, Hurler Disease) and type II (MPS II, Hunter Disease). Both diseases are characterized by a totally or partially defective activity of lysosomal enzymes involved in the catabolism of the mucopolysaccharides (or glycosaminoglycans, GAGs) heparan- and dermatan-sulfate which therefore heavily accumulate within cellular compartment and in the extracellular matrix. Although presenting several forms of clinical severity and disease progression, both pathologies affect most of the organ systems, are mostly life-threatening and about two-thirds of the patients also present neurological and cognitive impairment. Enzyme Replacement Therapy, applied to both diseases in the last few years, has shown to determine some clinical improvements, but it has also shown some limitations. In addition to the elevated costs of intervention, ERT presents the need of weekly administrations in a day-hospital regimen, this reducing patients’compliance, and the inefficacy of the recombinant enzymes in treating the CNS impairment due to their inability in blood-brain barrier (BBB) crossing. Thus, we combined the experience of clinical-based skills with pharmaceutical nanotechnology-based skills in order to create nanocarriers, biodegradable and biocompatible, able to deliver the recombinant enzymes across the BBB and to both assure a prolonged drug circulation and release, and a protection from metabolic drug inactivation. With this aim, we produced polymeric nanoparticles (PLGA-NPs) modified with 7-aminoacid glycopeptides (g7), yet demonstrated to be able to drive the NPs across the BBB after administration in rodents. Before going into functional and efficacy study, we developed several preliminary experiments in order to explore the ability of PLGA-NPs in transferring across the BBB a model drug (FITC-albumin), with a high molecular weight, comparable to that of the enzymes to be delivered across the BBB. In vivo experiments on both WT and knock-out (KO) mouse models for MPS I and MPS II were performed by i.v.-injecting g7-NPs loaded with FICT-albumin together with a plethora of control samples (i.e-. un-modified NPs, FITC-albumin solution) in order to have a broad preliminary view. The results clearly showed that g7-NPs are able to cross the BBB in all treated mice (WT and KO models) and to deliver FITC-albumin to the brain; interestingly, we found qualitative and semi-quantitative evidences of a higher grade of brain accumulation of g7-NPs loaded with Albumin in the KO -brains with respect to WT ones. Taken together, these results pave the way to a possible successful set of pilot experiments on the ability of enzyme-loaded g7-NPs to deliver sufficient amount of the drug to the brain district, hopefully exerting a corrective effect on the pathological cellular GAG deposits.

2013 - Brain targeting with polymeric nanoparticles: which administration route should we take? [Articolo su rivista]
Tosi, Giovanni; Ruozi, Barbara; Vandelli, Maria Angela
abstract

Polymeric nanoparticles (NPs) represent one of the most studied carriers for drug delivery, due to their ability in overcoming problems related to the instability of drugs and their widespread biodistribution as well as for the possibility of targeting cells or organs [1]. In particular, drug delivery to the brain is a challenge for scientists, since the Blood Brain Barrier (BBB) allows the entrance of specific and selected molecules, but it hampers the passage of a remarkable number of pharmacologically active molecules. A plethora of strategies aims to create, characterize and test in vitro and in vivo NPs able to cross the BBB, in health or diseased state, i.e. with physiological or enhanced permeability [2]. Nearly all the strategies involved are based on moieties, as surfactants, antibodies, ligands for receptors and peptides [3-7] linked on the NPs surface (engineered NPs). Beside the “BBB crossing aim”, NPs targeted to the Central Nervous System (CNS) have to be planned considering their “journey into the body” [8], as the biodistribution of the nanocarriers and the route of administration is another aspect to be taken into account.

2013 - Brain-targeted polymeric nanoparticles: in vivo evidences after different routes of administration in rodents. [Articolo su rivista]
Tosi, Giovanni; Ruozi, Barbara; Belletti, Daniela; Vilella, Antonietta; Zoli, Michele; Vandelli, Maria Angela; Forni, Flavio
abstract

The capacity of polymeric nanoparticles (NPs) to reach the target regardless to the administration route is a neglected field of investigation in the pharmaceutical nanotechnology. Therefore, after having demonstrated in previous studies that glycopeptide-engineered NPs (g7-NPs) were able to reach the brain after intravenous administrations in rodents, this paper aimed to evaluate if they can reach the Central Nervous System (CNS) also when administered by different routes. The confocal microphotographs on murine brain sections showed the capability of g7-NPs to reach the target also after intraperitoneal, intranasal and oral administrations. These highlights could open new vistas to a future application of the g7-NPs in the therapeutic treatments of CNS diseases.

2013 - Ligand- based strategies to modified NPs surface for blood-brain barrier crossing [Abstract in Atti di Convegno]
Tosi, Giovanni; Ruozi, Barbara; Belletti, Daniela; Vandelli, Maria Angela; Forni, Flavio
abstract

The main limitation in the treatment of neurological diseases consists of the presence of the blood–brain barrier (BBB), which precludes the entry of therapeutic molecules from blood to brain. Specifically engineered nanoparticles (NPs) have gained interest as drug carriers able to ensure an effective brain targeting, overcoming the BBB and carrying drugs to the central nervous system (CNS). Our research group focused on biocompatible and biodegradable poly(d,l-lactide-co-glycolide) (PLGA) nanocarriers, engineered with different specific ligands able to promote the brain targeting taking advantage of the BBB crossing pathways, such as endocytosis or transcytosis (1-2). In particular, we explored different ligand to favor the BBB crossing and the cellular interaction; i) a g7 peptide ( H 2 N – Gly- L -Phe- D -Thr-Gly- L -Phe- L -Leu – L -Ser – O- - D -glucose-CONH 2) ii) a sequence 12–32 (g21) of leptin iii) both glycopeptide (g7) for blood brain barrier (BBB) crossing and SA residue for interaction with brain receptors. The brain localization of engineered nanoparticles NPs was evaluated in rats after intravenous administration, by confocal microscopy, fluorescence microscopy and electron microscopy. Studies to evaluate the biodistribution of modified NPs in comparison to the unmodified NPs were also carried out. Results: i) g7-NPs were able to cross the BBB (2): in particular, the biodistribution of these NPs showed a localization into the CNS in a quantity about two orders of magnitude greater than that found with the other known NP drug carriers. Not only, the results obtained by quantitative brain biodistribution of Rhodamine-123 loaded g7-NPs (15% of the injected dose) are comparable with the results obtained by antinociceptive assays with loperamide loaded into g7-NPs (at least 13% of the injected dose inferred by ICV studies). ii) After intravenous administration in rats, the g21-NPs were able to cross the BBB and to enter the brain parenchyma. The biodistribution studies of both unmodified and modified NPs pointed out an uptake at liver and spleen level, whereas only the g21-NPs showed brain localization. The food-intake experiments pointed out that the intravenous administration of g21 conjugated to the NP surface did not produce any anorectic effect in the rats. iii) the double-covered NPs (with SA and glycopeptide) crossed the BBB owing to the presence of glycopeptide on the NPs’ surface, followed by endocytosis as the BBB crossing mechanism. Then, as a consequence of the presence of SA moiety on the NPs’ surface, the double-covered NPs could interact with brain SA-specific receptors, thus explaining both the prolonged activity of loperamide delivered by NPs and the prolonged NP brain residence time. Biodistribution studies showed high NP localization (6% of the injected dose into the CNS) over a prolonged time (24 h) along with the qualitative evaluation of the NPs’ visualization within the brain, kidney, liver, spleen and lung tissue parenchyma

2013 - Nanomedicine in Neuroscience: the potential of targeted nanoparticles in crossing the Blood-Brain Barrier [Relazione in Atti di Convegno]
Tosi, Giovanni; Ruozi, Barbara; Vilella, Antonietta; Belletti, Daniela; Veratti, Patrizia; Baraldi, Elisa; Zoli, Michele; A., Grabrucker; Forni, Flavio; Vandelli, Maria Angela
abstract

Non-invasive strategies for treatment of Central Nervous System (CNS) diseases based on colloidal carriers represent a huge potential to efficiently transport drug across the BBB, since nanocarriers can protect drugs (or gene material) and deliver them to target specific populations of brain cells. The efficacy of the nanotechnological approach for brain targeting has been proved by several papers and widely reviewed. Literature contributions mainly deal with several kind of nanometric carriers such as polymeric nanoparticles (NPs), liposomes, solid-lipid NPs, micelles, nanogels and dendrimers. However, these nanocarriers, target and reach the brain poorly, if not engineered in their surface to take advantage of BBB transport mechanisms. Recent studies demonstrated the efficacy of the medicinal chemistry approach, based on the modification of the physico-chemical properties of drugs and the biological approach, based on the conjugation of molecules with antibodies or ligands targeting the BBB. In this contest, polymeric nanoparticles (NPs) and liposomes (LPs) were formulated and specifically engineered to cross the BBB and arrive to CNS and proposed to encapsulate an deliver cholesterol an BDNF to the CNS. Our attention point on the use of polymeric nanoparticles engineered on surface by a selective ligand able to promote the NPs crossing of BBB. In fact, preliminary studies demonstrated the ability of new targeted polymeric poly-lactide-co-glycolide (PLGA) NPs modified with a short peptide (H2N-Gly-L-Phe-D-Thr-Gly-L-Phe-L-Leu-L-Ser(O-β-D-Glucose)-CONH2 (g7-NPs) to create BBB interaction and trigger an efficacious BBB crossing delivering of active. In particular, several in vivo biodistribution studies and pharmacological proof-of-evidence of brain delivery of model drugs (not able by themselves to reach the brain, as Rhodamine-123 and Loperamide) demonstrated the ability of g7-NPs to create BBB interaction and trigger an efficacious BBB crossing. A total biodistibution of g7-NPs, obtained after i.v. administration in rats, evidenced a strong and significant localization of the g7-NPs into CNS in a quantity about two orders of magnitude greater (10-15%) than that found with the other known NP drug carriers. More recently, the g7-NP BBB crossing mechanism was investigated, pointing out an interaction between g7-NPs and BBB and endocytosis/macropinocytosis pathways for BBB crossing. Same results were pointed out also in vitro on neurons/glia cell coltures, evidencing the endocytotic pathways as g7-NPs cell entrance as well as the assessing of the safety of g7-NPs not creating any damage to cells even at high doses.

2013 - Nanoparticles Loaded with Nutlin-3 Display Cytotoxicity Towards p53wildtype JVM-2 But Not Towards p53mutated BJAB Leukemic Cells [Articolo su rivista]
R., Voltan; P., Secchiero; Ruozi, Barbara; L., Caruso; Forni, Flavio; M., Palomba; G., Zauli; Vandelli, Maria Angela
abstract

Abstract The small molecule Nutlin-3 is a potent antagonist of the murine double minute 2 (MDM2)/p53 interaction exhibiting promising therapeutic anti-cancer activity. Nutlin-3 has been proposed as an anti-neoplastic agent for the treatment of onco-hematological diseases characterized by a lower incidence of p53 mutation with respect to solid tumors. Indeed, based on its selective non-genotoxic p53 activation, Nutlin-3 might represent an alternative to the current cytotoxic chemotherapy. To overcome the poor bioavailability of Nutlin-3, we have assessed the potential efficacy of Nutlin-3 loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NP) against hematological malignancies. To test the specificity of the anti-leukemic activity, we have used leukemic cell lines characterized by different p53 status (JVM-2 and BJAB). NP loaded with Nutlin-3 (NP-Nutlin) were rapidly taken up by the leukemic cells and were as effective as native Nutlin-3 in promoting both induction of apoptosis and cell cycle arrest in p53(wild-type) JVM-2 cells, but not in p53(mutated) BJAB cells. Moreover, injection of NP-Nutlin, but not of free Nutlin-3, in a JVM-2-derived xenograft mouse model, reduced the subcutaneous tumor volume and promoted induction of apoptosis in the tumor mass. Overall, the chemical and structural characteristics of the NP-Nutlin-3, as well as their biological activity in vitro and in vivo, made them promising for further preclinical evaluations as potentially useful anti-leukemic carriers.

2013 - Nanoparticles engineered with rituximab and loaded with Nutlin-3 show promising therapeutic activity in B-leukemic xenografts. [Articolo su rivista]
Voltan, R; Secchiero, P; Ruozi, Barbara; Forni, Flavio; Agostinis, C; Caruso, L; Vandelli, Maria Angela; Zauli, G.
abstract

Abstract PURPOSE: Because the nongenotoxic inhibitor of the p53/MDM2 interactions Nutlin-3 has shown promising in vitro therapeutic activity against a variety of p53(wild-type) cancer cells, in this study we evaluated an innovative strategy able to specifically target Nutlin-3 toward CD20(+) malignant cells. EXPERIMENTAL DESIGN: The cytotoxic effects of Nutlin-3 encapsulated into poly(lactide-co-glycolide) nanoparticles (NP-Nut) and into rituximab (anti-CD20 antibody)-engineered NP (NP-Rt-Nut) as well as of NPs engineered with rituximab alone (NP-Rt) were initially analyzed in vitro in JVM-2 B-leukemic cells, by assessing both the functional activation of the p53 pathway (by Nutlin-3) and/or the activation of the complement cascade (by rituximab). Moreover, the potential therapeutic efficacy of NP-Nut, NP-Rt, and NP-Rt-Nut were comparatively assessed in vivo in CD20(+) JVM-2 leukemic xenograft SCID mice. RESULTS: Functional in vitro assays showed that NP-Nut and NP-Rt-Nut exhibited a comparable ability to activate the p53 pathway in the p53(wild-type) JVM-2 leukemic cells. On the other hand, NP-Rt and NP-Rt-Nut, but not NP nor NP-Nut, were able to promote activation of the complement cascade. Of note, the in vivo intratumoral injection in JVM-2 B-leukemic/xenograft mice showed that NP-Rt-Nut displayed the maximal therapeutic activity promoting a survival rate significantly higher not only with respect to control animals, treated either with vehicle or with empty NP, but also with respect to animals treated with NP-Nut or NP-Rt. CONCLUSIONS: Our data show for the first time the potential antileukemic activity of rituximab-engineered Nutlin-3-loaded NPs in xenograft SCID mice.

2013 - Nanotechnology and Central Nervous System Drug Delivery [Abstract in Atti di Convegno]
Tosi, Giovanni; Ruozi, Barbara; Vilella, Antonietta; Belletti, Daniela; Veratti, Patrizia; Baraldi, Elisa; Zoli, Michele; A., Grabrucker; A., Sharma; H. S., Sharma; Forni, Flavio; Vandelli, Maria Angela
abstract

In line with the overall increase in knowledge and nanotechnologies, surface engineering of nano-sized carriers is now representing the cutting edge of nanomedicine, leading to the production of selectively targeted therapies based on targeted nanocarriers. In fact, achieving nanocarriers able to be stable in the blood-stream, to protect the drug from metabolism and to promote a long-lasting release of the drug is still a pivotal pre-requisite for nanomedicine, but it is now to be considered as “not enough”. Active targeting to specific pathological cells is now the challenge of pharmaceutical nanotechnologists, who are facing with difficulties in colloidal chemistry and most of all in the characterization of the engineered nanocarriers from a technological and physiological points of view. As an example, the application of nanotechnology to brain-related disorders, called nanoneuromedicine, is surely representing one of the most stimulating challenge as well as one the most difficult due to the presence of biological barriers (BBB) and the great variability in BBB permeability depending on the chosen disease. Encouraging results have been obtained demonstrating the possibility of targeting the CNS up to an important percentage of brain localization. In this contest, polymeric nanoparticles (NPs) and liposomes (LPs) were formulated and specifically engineered to cross the BBB and arrive to CNS and proposed to encapsulate some drugs able to rescue from neurodegeneration, to the CNS. Our attention point on the use of polymeric nanoparticles engineered on surface by a selective ligand able to promote the NPs crossing of BBB. In fact, preliminary studies demonstrated the ability of new targeted polymeric poly-lactide-co-glycolide (PLGA) NPs modified with a short peptide (H2N-Gly-L-Phe-D-Thr-Gly-L-Phe-L-Leu-L-Ser(O-β-D-Glucose)-CONH2 (g7-NPs) to create BBB interaction and trigger an efficacious BBB crossing delivering of active. In particular, several in vivo biodistribution studies and pharmacological proof-of-evidence of brain delivery of model drugs (not able by themselves to reach the brain) demonstrated the ability of g7-NPs to create BBB interaction and trigger an efficacious BBB crossing. A total biodistibution of g7-NPs, obtained after i.v. administration in rats, evidenced a strong and significant localization of the g7-NPs into CNS in a quantity about two orders of magnitude greater (10-15%) than that found with the other known NP drug carriers. More recently, the g7-NP BBB crossing mechanism was investigated, pointing out an interaction between g7-NPs and BBB and endocytosis/macropinocytosis pathways for BBB crossing. Same results were pointed out also in vitro on neurons/glia cell coltures, evidencing the endocytotic pathways as g7-NPs cell entrance as well as the assessing of the safety of g7-NPs not creating any damage to cells even at high doses. Notwithstanding these outputs, it is our opinion that in order to obtain a real update of neurological disorders’ therapy based on innovative and non invasive protocols (i.e. nanomedicine), a team work is strongly needed. The interdisciplinar competences and skills of all the experts in Neuro-diseases and Nano-Technology (from neurobiologists to neurophysiologist, from nanotechnologists to physicians) must be shared, discussed, considered and applied, thus opening the pave to new vistas in treatments and most of all for the correct development of the research.

2013 - Nanovettori lipidici e polimerici per la somministrazione sito-specifica di farmaci antitumorali [Relazione in Atti di Convegno]
Ruozi, Barbara; Tosi, Giovanni; Belletti, Daniela; Forni, Flavio; Vandelli, Maria Angela
abstract

La ricerca di nuovi approcci per la realizzazione di terapie mirate verso diverse forme tumorali, trattate ancora in modo aspecifico e non risolutivo, rappresenta un importante obiettivo della ricerca medica e farmaceutica. Per la loro versatilità formulativa ed applicativa, i nanosistemi polimerici (nanoparticelle) e lipidici (liposomi), appaiono oggi come i migliori candidati per la veicolazione e il direzionamento sito specifico di antitumorali nell’uomo. La ricerca proposta descrive le fasi di progettazione, ottimizzazione e sviluppo di nanoparticelle polimeriche e liposomi per la somministrazione sito specifica di Nutlin 3, una molecola abile nell’indurre una selettiva non-genotossica attivazione di p53 con attività antitumorale vs le leucemie linfoidi. Per migliorare il direzionamento al target, anticorpi e molecole ligando specifiche sono state coniugate sulla superficie dei sistemi. Sarà riportato e discusso l’approccio tecnologico adottato per la formulazione, la funzionalizzazione e purificazione dei sistemi, le tecniche di caratterizzazione e i risultati ottenuti dopo somministrazione in vitro ed in vivo per dimostrare il direzionamento e l’efficacia del delivery. Accanto all’impiego di nanoparticelle polimeriche sarà considerata la versatilità formulativa del carrier liposomiale per l’approccio genico alla terapia antitumorale; saranno descritte le strategie adottate per la formulazione di lipoplessi stabili tra liposomi e siRNA con applicazione alla terapia di linfomi. Studi in vitro e in vivo saranno documentati per dimostrare la validità dell’approccio.

2013 - Potential use of polymeric nanoparticles for drug delivery across the blood-brain barrier. [Articolo su rivista]
Tosi, Giovanni; Bortot, B; Ruozi, Barbara; Dolcetta, D; Vandelli, Maria Angela; Forni, Flavio; Severini, G. M.
abstract

Nanomedicine is certainly one of the scientific and technological challenges of the coming years. In particular, biodegradable nanoparticles formulated from poly (D,L-lactide-co-glycolide) (PLGA) have been extensively investigated for sustained and targeted delivery of different agents, including recombinant proteins, plasmid DNA, and low molecular weight compounds. PLGA NPs present some very attractive properties such as biodegradability and biocompatibility, protection of drug from degradation, possibility of sustained release, and the possibility to modify surface properties to target nanoparticles to specific organs or cells. Moreover, PLGA NPs have received the FDA and European Medicine Agency approval in drug delivery systems for parenteral administration, thus reducing the time for human clinical applications. This review in particular deals on surface modification of PLGA NPs and their possibility of clinical applications, including treatment for brain pathologies such as brain tumors and Lysosomal Storage Disorders with neurological involvement. Since a great number of pharmacologically active molecules are not able to cross the Blood-Brain Barrier (BBB) and reach the Central Nervous System (CNS), new brain targeted polymeric PLGA NPs modified with glycopeptides (g7-NPs) have been recently produced. In this review several in vivo biodistribution studies and pharmacological proof-of-evidence of brain delivery of model drugs are reported, demonstrating the ability of g7-NPs to create BBB interaction and trigger an efficacious BBB crossing. Moreover, another relevant development of NPs surface engineering was achieved by conjugating to the surface of g7-NPs, some specific and selective antibodies to drive NPs directly to a specific cell type once inside the CNS parenchyma.

2013 - Rab5-dependent cell-specific uptake and distribution of engineered nanoparticles for CNS targeted drug delivery in vivo [Abstract in Atti di Convegno]
Vilella, Antonietta; Tosi, Giovanni; Grabrucker, A. M.; Ruozi, Barbara; Belletti, Daniela; Vandelli, Maria Angela; Boeckers, T. M.; Forni, Flavio; Zoli, Michele
abstract

Employment of brain-targeted nanocarriers as tools for drug delivery to the central nervous system (CNS) represents a pivotal step forward in the development of innovative therapeutic strategies. If nanocarriers are to be translated into the clinic, their distribution within the brain and interaction with CNS cells must be assessed accurately. Here, we investigated these issues by employing polylactide-co-glycolide nanoparticles (NPs) specifically engineered with g7, a glycopeptide conferring the ability to cross the blood brain barrier (BBB) at a concentration of up to 10% of the injected dose. g7-NPs display increased in vitro uptake in neurons and glia. Our results show that in vivo administration of g7-NPs leads to a region- and cell type-specific enrichment of NPs within the brain that is not dependent on the presence of the BBB. We provide evidence that g7-NPs are endocytosed in a clathrin-dependent manner and transported into a specific subset of early endosomes positive for Rab5 in vitro and in vivo. The differential Rab5 expression level is strictly correlated with the amount of g7-NP accumulation. These findings show that g7-NPs can cross the BBB and target specific brain cell populations, suggesting that these NPs are promising drug carriers for the treatment of neuropsychiatric diseases.

2013 - siRNA-BASED THERAPEUTICS: DELIVERY AND TARGETING TO PEL TUMOR BY USING CATIONIC LIPOSOMES [Abstract in Atti di Convegno]
Belletti, Daniela; Riva, Giovanni; Tosi, Giovanni; Lagreca, Ivana; Barozzi, Patrizia; Adriana, Mattiolo; Elena, Negri; Laura, Lignitto; Luigi Chieco, Bianchi; Forni, Flavio; Luppi, Mario; Vandelli, Maria Angela; Maria Luisa, Calabrò; Ruozi, Barbara
abstract

Aims of this research was to develop a “nanomedicine” approach based on siRNA delivery for the treatment of primary effusion lymphoma (PEL). The therapeutic use of antitumoral siRNA requires the development of specifically designed functional vectors, allowing improve¬ment of siRNA stability after systemic admin¬istration, and enabling targeted delivery directly into the neoplastic cells. In this context, liposomes, and particularly cationic liposomes, appears particu¬larly suitable to generate complexes with highly degradable siRNAs, as well as to specifically deliver siRNAs directly into the cytoplasm of the target tumor cells, where RNA interference processes take place. Generally, the electrostatic interaction between the positively charged lipids and the negatively charged nucleic acids leads to the formation of stable lipoplexes, protecting the cargo against nuclease attack and improving the cellular uptake and activity [1. In this context, we are investigating innovative target strategies to improve the treatment of human herpesvirus 8 (HHV8)-associated primary effusion lymphoma (PEL). Primary effusion lymphoma (PEL) is an aggressive B cell non-Hodgkin’s lymphoma, affecting the serous cavities (such as the pleu¬ral, pericardial and abdominal cavities) and preferentially arising in immunocompromised or elderly patients, typically affected by several comorbidities and organ function impairments. PEL therapy has been revealed to be unsuccessful in the vast majority of patients, who are invariably characterized by a poor prognoses. Recently, small interfering RNAs (siRNAs), able to knock-down viral oncogenic proteins, were shown to induce efficient PEL cell apoptosis in vitro and PEL regressions in mice treated with intracavitary injection of lentiviral vectors expressing siRNA precursors[2. Moving from our promising preliminary results in the field of nanotechnologies[3-4, we are developing different lipid-based nanocarriers (cationic and stealth-cationic liposomes), to deliver specific siRNAs to knock-down novel molecular targets (HHV8-encoded microRNAs, viral oncogenic proteins, or host transcription factors) with relevant functions in PEL pathogenesis [5. We are presently testing the delivery efficiency of these nanocarriers and the antineoplastic activity in vitro and in vivo using different PEL-derived cell lines and a previously established PEL mouse model[6. We performed several preliminary technological experiments aimed at optimizing the operative condition to obtain the efficient liposomes/siRNAs complexes. Chemic-physical properties of both liposomes and lipoplexes were evaluated by exploiting microscopic, spectroscopic and gel electrophoresis techniques. In vitro experiments demonstrated a high transfection efficiency of some of our carriers, which stably protected and efficaciously delivered siRNAs into PEL cells. Preliminary experiments using a mixture of siRNAs targeting a specific cellular gene showed a remarkable dose-dependent apoptosis, measured by annexin-V staining, in lipoplexes-transfected PEL cells. Moreover, the in vivo delivery of these therapeutic siRNAs significantly increased the survival time of treated mice compared with control treatment (log-rank test, lipoplexes vs empty liposomes, p=0.002), indicating that our lipoplexes exerted a significant antineoplastic activity. The empty carriers were not toxic in control mice and did not delay PEL development respect untraeted mice. Our data indicate that our lipoplexes may therefore be considered as the basis for the development of useful short interfering RNA delivery vectors to treat PEL tumor. Moreover, we identified a target gene whose suppression exerts a relevant tumoricidal activity on PEL cells in vitro and in vivo, opening new perspectives for PEL treatment.

2012 - Can leptin-derived sequence-modified nanoparticles be suitable tools for brain delivery? [Articolo su rivista]
Tosi, Giovanni; Badiali, Luca; Ruozi, Barbara; Vergoni, Anna Valeria; Bondioli, Lucia; Ferrari, Anna; Rivasi, Francesco; Forni, Flavio; Vandelli, Maria Angela
abstract

Aim: In order to increase the knowledge on the use of the nanoparticles (Np) in the brain targeting, this paper describes the conjugation of the sequence 12-32 (g21) of Leptin (Lp) to poly-lactide-co-glycolide (PLGA) Np. The capability of these modified Np to reach the brain was evaluated in rats after i.v. administration.Materials and Methods: The g21 was linked on the surface of Np labeled with tetramethylrhodamine (TRM) by means of the Avidin-Biotin technology. The g21 labeled Np were injected into the tail vein of rats and, after animal sacrifice, the brain localization was evaluated by confocal microscopy, fluorescence microscopy and electron microscopy. Studies to evaluate the biodistribution of the g21 modified Np in comparison to the un-modified Np were also carried out. Moreover, to confirm the absence of any anorectic effect of g21 linked on the surface of Np, appropriate studies were assess in the rats.Results: After i.v. administration, the g21 modified Np were able to cross the BBB and to enter the brain parenchyma. The biodistribution studies of both un-modified and modified Np pointed out an uptake at liver and spleen level, whereas only the g21 modified Np showed brain localization. The food-intake experiments pointed out that the i.v. administration of g21 conjugated to the Np surface did not produce any anorectic effect in the rats.Conclusion: g21-modified Np were able to cross the BBB. This new modified Np could be effectively considered as useful carrier systems for brain drug delivery.

2012 - Chemico-physical investigation of tenofovir loaded polymeric nanoparticles [Articolo su rivista]
Belletti, Daniela; Tosi, Giovanni; Forni, Flavio; Gamberini, Maria Cristina; Baraldi, Cecilia; Vandelli, Maria Angela; Ruozi, Barbara
abstract

Tenofovir (PMPA), an acyclic nucleoside phosphonate analog, is one of the most important drugs used for the HIV treatment. Unfortunately, several adverse reactions are related to its i.v. administration owing to the saturation of an anionic renal transporter. In order to improve the drug administration, the PMPA was embedded into a new type of nanocarriers based on poly-(d,l-lactide-co-glycolide) (PLGA) and/or chitosan (CH). The strategies for the preparation of nanoparticles (Nps) with a more efficient drug loading respect to the one reported in the literature for PMPA nanoencapsulation were investigated. CH was added in the first inner emulsion or in the external phase during the second emulsion of water/oil/water (W/O/W) Nps. The addition of CH in the first inner emulsion was the most promising technique. The Nps have a Z-average of 230 nm, a Z-potential of −3 mV and an EE% of 15 that was 2.5–3 times higher than that obtained with PLGA Nps or CH Nps. In vitro release studies showed a limited control on drug release in phosphate buffer (pH 7.4) while an initial burst effect followed by a slow drug release was observed in acidic receiving phase (pH 4.6). These results suggest the PLGA/CH Nps should be an effective and attractive anti-HIV drug carrier to study the cellular uptake and drug delivery on target cells such as macrophages.

2012 - Cholesterol nanoparticles partially rescue the alterations in synaptic phenotype of the R6/2 mouse model of Huntington's Disease [Abstract in Atti di Convegno]
J. Y., Chen; C., Cepeda; M., Valenza; Tosi, Giovanni; Ruozi, Barbara; Belletti, Daniela; Forni, Flavio; Vandelli, Maria Angela; E., Cattaneo; M. S., Levine
abstract

Previous studies in the R6/2 mouse model of Huntington's disease (HD) have demonstrated key alterations in membrane properties and synaptic transmission of striatal medium-sized spiny neurons (MSNs) in behaviorally phenotypic mice. These changes include: lower frequency of spontaneous excitatory postsynaptic currents (sEPSCs), higher frequency of spontaneous inhibitory postsynaptic currents (sIPSCs), increased membrane input resistance, and decreased cell capacitance. Enzymatic analyses show that brains of symptomatic R6/2 mice also have reduced total sterol mass and reduced translocation of sterol regulatory element-binding proteins, leading to a decrease in transcription of genes involved in the cholesterol (Chol) biosynthesis pathway. The present study examines how Chol supplementation using glycopeptide-modified nanoparticles (g7-Nps), specifically engineered to cross the blood-brain barrier after systemic administration in rodents, affects synaptic transmission and basic membrane properties of striatal MSNs in symptomatic R6/2 mice compared to their wildtype (WT) littermates. We show that g7-Nps injected systemically enter the brain and localize in neurons and glia in the cortex and striatum within 4 h after an i.p. injection and persist for several weeks in R6/2 and WT mice. In addition, MSNs of R6/2 mice injected with Chol-g7-Nps (8 injections over a four-week period) show a less pronounced increase in sIPSC frequency and a trend towards an increase in sEPSC frequency compared to R6/2 mice that received either empty-g7-Nps or saline injections. Furthermore, basic membrane properties of MSNs from R6/2 mice treated with Chol-g7-Nps show a trend towards decreased membrane input resistance and increased cell capacitance compared to R6/2 mice that did not receive Chol supplementation. There were no significant differences between WTs that received Chol-g7-Nps, empty-g7-Nps, or saline injections. These results suggest that repeated administrations of Chol-g7-Nps produce a partial rescue of the alterations in membrane properties and the synaptic phenotype of R6/2 mice. In conclusion, the results demonstrate the therapeutic potential of a nanomedicine-based approach using g7-Nps to deliver specific drugs that could modify HD symptoms, as well as the use of Chol to reverse the cascade of synaptic alterations associated with this disease

2012 - Derivatized nanoparticles for CNS-targeted drug delivery [Relazione in Atti di Convegno]
B., Tancini; B., Bortot; D., Dolcetta; Tosi, Giovanni; A., Magini; Vandelli, Maria Angela; Forni, Flavio; G. M., Severini; C., Emiliani
abstract

Neurodegenerative and cerebrovascular diseases exert a growing impact from the societal and economic point of view. Therefore the development of strategies for early detection as well as for effective and safe treatments of such diseases is becoming more important than ever. For these reasons, research the targeting of active molecules to CNS represents one of the most challenging drawbacks. The selectivity of blood-brain barrier (BBB) highly limits therapies for the cerebral diseases and in the recent years a great deal of efforts to develop strategies that aid drug passage across the BBB have been made. Nanotechnology-based approaches have gained increasing attention as the most promising strategies for CNS targeted drug delivery. Such approach involves the use of at least two components, one of which is a nanoparticle (NP), which serves as a carrier (nanocarrier), and the other is the therapeutic agent (cargo). Among NPs, those made of poly(lactic-co-glycolic acid) (PLGA) hold an uncommon biocompatibility and, when conjugated with an heptapeptide (g7) able to cross the BBB, they reach at high rate the cerebral tissue. Using in vitro cell models of lysosomal storage diseases, an heterogeneous group of rare inherited disorders characterized by the lysosomal accumulation of undigested or partially digested macromolecules, which ultimately results in cellular dysfunction and clinical abnormalities, with a strong neurological involvement, we demonstrate that PLGA-NPs loaded with the missing enzyme were able to reach lysosomes and to rescue 50% of the enzymatic deficiency after a single administration. Moreover the conjugation of the enzyme with NPs also might contribute to improve the stability/integrity of the enzyme thus prolonging its life span. Thus, g7-NPs seem to represent a promising tool for the treatment of diseases with neurological involvement. Work supported by ELA Foundation (Agreement n. 2011-037C1B: “Leading nanomedicine into the therapy for Leukodystrophies: nanoparticles overcoming the blood−brain barrier to treat the mouse model of Krabbe Disease”) and Fondazione Cassa di Risparmio di Perugia (Project n. 2010.011.0434: “Effetto sulla salute umana dell’esposizione a materiale nano strutturato: impiego di modelli cellulari per lo studio della nanotossicità”)

2012 - Immunonanosystems to CNS pathologies: State of the art [Capitolo/Saggio]
Tosi, Giovanni; Ruozi, Barbara; Badiali, Luca; Bondioli, Lucia; Belletti, Daniela; Forni, Flavio; Vandelli, Maria Angela
abstract

This chapter deals with an overall view of nanoparticulate systems opportunately engineered with antibodies for the delivery of active substance to the Central Nervous System. Concordantly to this aim, a brief introduction of the rationale undergoing the nanoparticulate approach to the brain targeting has been developed. Nanoparticles, liposomes and solid lipid nanoparticles, representing the most widely studied carriers for drug delivery and targeting, have been reviewed in their application to CNS targeting. In particular, antibody-mediated targeting represent one of the best choices in order to engineer and superficially modify the carriers, to finally target the therapeutic goal. Amongst the receptors to be usefully used for translocation into the CNS, the transferrin and insulin receptors seem to be the most selective systems for an active transcytosis of antibody-engineered nanosystems across the the Blood Brain Barrier.

2012 - LIPOPLEXES PER LA VEICOLAZIONE ED IL DIREZIONAMENTO DI siRNA AL PEL: VALUTAZIONE DELL’EFFICIENZA DI TRATTAMENTO CON SISTEMI LIPIDICI STABILIZZATI [Relazione in Atti di Convegno]
Belletti, Daniela; Riva, Giovanni; Tosi, Giovanni; Barozzi, Patrizia; Luppi, Mario; Forni, Flavio; Vandelli, Maria Angela; Ruozi, Barbara
abstract

Per la stabilizzazione e la somministrazione di siRNAs, sono sempre più studiati vettori non virali, ed in particolare liposomi cationici, capaci di stabilizzare, per interazione elettrostatica, materiale genico generando strutture note come lipoplexes (Lpx). In questo contesto, sono stati formulati e testati Lpx attivi nel trattamento del PEL (Primary Effusion Lymphoma), un linfoma altamente aggressivo e con scarse possibilità terapeutiche. Inizialmente, liposomi allestiti con DOTAP sono stati impiegati per complessare 3 differenti siRNAs anti-BLIMP-1 (fattore trascrizionale fondamentale per mantenere lo stato neoplastico del PEL). I Lpx ottenuti sono stati caratterizzati e ne è stata dimostrata l’elevata capacità trasfettiva sulla linea cellulare BCBL-1, modello di PEL. Per una somministrazione efficace, ed in previsione di studi in vivo, i complessi sono stati stabilizzati all’uptake macrofagico, attraverso modificazioni superficiali con PEG. Gli studi tecnologico-formulativi sono stati condotti sia partendo da liposomi pegilati che introducendo la pegilazione su complessi preformati. La complessazione tra liposomi cationici pegilati (preparati con DOTAP e DSPE-PEG) ed i siRNAs non è risultata efficace nello stabilizzare e trasferire il cargo alle cellule in coltura, presumibilmente a causa dell’ingombro e del tamponamento operato dalla copertura idrofila del liposoma, con conseguente limitata interazione tra il vettore ed il materiale genico. Incoraggianti risultatati sono invece derivati dall’applicazione della strategia “post complexation”, ovvero la pegilazione di Lpx cationici preformati (ottenuti tra liposomi DOTAP e siRNAs) mediante incubazione con micelle di DSPE-PEG. Tali “Post PEG-Lpx” hanno mostrato elevata capacità di stabilizzazione dell’attivo con conservata abilità transfettiva

2012 - NANOMEDICINE IN NEUROSCIENCE: THE POTENTIAL OF TARGETED NANOPARTICLES IN NEURODEGENERATIVE DISORDERS [Relazione in Atti di Convegno]
Tosi, Giovanni; Ruozi, Barbara; Vilella, Antonietta; Belletti, Daniela; Veratti, Patrizia; Baraldi, Elisa; Zoli, Michele; M., Schmeisser; A., Grabrucker; H. S., Sharma; A., Sharma; Forni, Flavio; Vandelli, Maria Angela
abstract

Pathologic conditions affecting the brain such as neurodegenerative diseases and neurological disorders (i.e. Parnkison’s disease, Alzheimer’s disease, Huntington disease, multiple sclerosis, brain tumors, etc.) are amongst the most un-treatable syndromes. A major obstacle for the application of therapeutics is that a great number of pharmacologically active molecules (estimated 98%) are not able to reach the Central Nervous System (CNS) and to exert their activity as they cannot cross the Blood-Brain Barrier (BBB). Thus, one of the challenges of pharmaceutical research nowadays is to discover tools enabling an effective and efficacious delivery of drugs into the CNS. Non-invasive techniques based on colloidal carriers (nanomedicine) could represent a huge potential and, in line with the overall increase in knowledge and nanotechnologies, surface engineering of nano-sized carriers is now representing the cutting edge of nanomedicine, leading to the production of selectively targeted therapies based on targeted nanocarriers. In fact, achieving nanocarriers able to be stable in the blood-stream, to protect the drug from metabolism and to promote a long-lasting release of the drug, is still a pivotal pre-requisite for nanomedicine, but it is now to be considered as “not enough”. Active targeting to specific pathological cells is now the challenge of pharmaceutical nanotechnologists, who are facing with difficulties in colloidal chemistry and most of all in the characterization of the engineered nanocarriers from a technological and physiological points of view. As an example, the application of nanotechnology to brain-related disorders, called nanoneuromedicine, is certainly representing one of the most stimulating as well as one of the most difficult challenges, due to the presence of biological barriers (BBB) and the great variability in BBB permeability depending on the chosen disease. Nevertheless, encouraging results have been obtained demonstrating the possibility of targeting the CNS up to reaching a significant percentage in brain localization of nanocarriers. As an example of targeted NPs, new targeted polymeric poly-lactide-co-glycolide (PLGA) NPs modified with glycopeptides (g7-NPs) have been recently demonstrated, by in vivo and in vitro experiments, to be able to trigger brain delivery of active substances (brain accumulation up to 10-15% of the injected dose). Moreover, BBB crossing of g7-NPs was recently assessed by our team, evidencing endocytosis/macropinocytosis pathways as preferential mechanisms for g7-NPs movements and interactions. With this work, we will also show new developments and insights of our research with highlights mainly on g7-NPs in vitro behavior on neurons/glia as well as in vivo (rodents) brain localization and trafficking after different routes of administration. Notwithstanding these results, it is our opinion that in order to obtain a real progress in neurological disorders’ therapy based on innovative and non invasive protocols (i.e. nanomedicine), a team effort is highly desired. The interdisciplinary competences and skills of all the experts in Neuro-diseases and Nano-Technology (from neurobiologists to neurophysiologists, from nanotechnologists to physicians) must be shared, discussed, considered and applied, thus paving the way to new vistas in treatments and most of all for the correct development of this field of research.

2012 - NANOPARTICELLE DIREZIONANTI AL CERVELLO: RIPRISTINO DEI LIVELLI DI COLESTEROLO CELLULARE NELLA TERAPIA DELLA COREA DI HUNTINGTON. [Abstract in Atti di Convegno]
Belletti, Daniela; Tosi, Giovanni; M., Valenza; E., Cattaneo; Forni, Flavio; Vandelli, Maria Angela; Ruozi, Barbara
abstract

Il colesterolo svolge un ruolo fondamentale sia strutturale che funzionale nel mantenimento delle attività cellulari cerebrali, con notevoli implicazioni nella regolazione del trafficking cellulare, della trasduzione del segnale e della sintesi della mielina. Alterazioni nella omeostasi del colesterolo sono spesso causa di patologie neurodegenerative, come la Corea di Huntington, una grave patologia del sistema nervoso centrale (SNC) che colpisce specificamente i neuroni del corpo striato e si manifesta con disfunzioni motorie, alterazioni cognitive e comportamentali. L’implementazione del colesterolo, con l’apporto di colesterolo esogeno a livello del SNC appare pertanto come un approccio proponibile nel trattamento combinato di tali patologie. Purtroppo, il colesterolo tal quale non supera la barriera ematoemcefalica (BEE) in quantità efficaci. Per veicolare colesterolo al SNC, è stata proposta la formulazione di nanoparticelle polimeriche (Nps) caricate con colesterolo (CHOL) e modificate in superficie con un epatpeptide (g7) capace di promuovere il superamento della BEE. Inizialmente sono state allestite Nps variando diversi parametri formulativi quali il rapporto CHOL/PLGA, la tecnica di purificazione, l’impiego di tensioattivo nella formulazione e di crioprotettori nella fase di stabilizzazione (tipo, quantità etc..); tale studio tecnologico ha permesso di ottenere un sistema riproducibile, con un adeguato caricamento, una buona resa e stabile alla conservazione. Successivamente le Nps, rese tracciabili attraverso l’impiego di piccole percentuali di polimero coniugato con rodamina e caricate con colesterolo a sua volta marcato con un diverso fluorocromo (CHOLNBD), sono state somministrate IP in topi sani e modelli di Huntington (R6/2). Studi di microscopia confocale hanno dimostrato la presenza di significative concentrazioni di Nps e di colesterolo a livello celebrale, nonchè l’abilità di tali sistemi nel promuovere un rilascio sostenuto del farmaco per almeno due settimane.

2012 - NANOPARTICELLE IBRIDE PLGA/DOPE: STUDI DI TRANSFEZIONE, ESCAPE ENDOSOMIALE E TRAFFICKING CELLULARE [Abstract in Atti di Convegno]
Veratti, Patrizia; Ruozi, Barbara; Belletti, Daniela; Forni, Flavio; Vandelli, Maria Angela; Andreas M., Grabrucker; Tosi, Giovanni
abstract

Studi in vivo e in vitro hanno dimostrato che nanoparticelle (Nps) di PLGA (co-polimero tra acido lattico e acido glicolico), coniugate in superficie con il glicopeptide g7, sfruttano meccanismi di endocitosi clatrina dipendenti per attraversare la barriera emato encefalica (BEE) e per entrare in cellule neuronali e gliari (Tosi et al., Nanomedicine, 2011; Grabrucker et al., PloS One, 2011). Tali studi hanno anche evidenziato la difficoltà di questi vettori di evadere dal compartimento endolisosomiale, pregiudicando il rilascio di farmaci attivi a livello citoplasmatico e nucleare. In questo contesto, sono state formulate Nps ibride, miscelando il PLGA con il DOPE (dioleoilfosfatidiletanolamina), un lipide con caratteristiche fusogeniche, già impiegato per la formulazione di liposomi, applicati particolarmente in terapia genica. Utilizzando la tecnica della doppia emulsione, sono state formulate Nps impiegando diverse quantità di DOPE (2.5, 5, 10 e 20% p/p rispetto al PLGA). Messa a punto la tecnica di purificazione dei campioni, per ogni tipologia è stato dosato il lipide effettivamente incorporato nella matrice ed ogni preparazione è stata caratterizzata dal punto di vista dimensionale e di carica superficiale (potenziale Z). Studi di microscopia elettronica a scansione (SEM) e a forza atomica (AFM) hanno mostrato come varia la morfologia dei campioni al variare della loro composizione. I primi studi in vitro, in rapporto alla tossicità cellulare,hanno dimostrato la migliorata capacità di escape endosomiale di tali sistemi rispetto alle Nps polimeriche. In particolare, incoraggianti risultati sono stati osservati dopo transfezione con sistemi ibridi allestiti con basse concentrazioni di DOPE (5%), confermando le potenzialità di questa strategia nel targeting subcellulare di farmaci.

2012 - NANOPARTICELLE POLIMERICHE PER LA VEICOLAZIONE DI MACROMOLECOLE PROTEICHE: STUDI PRELIMINARI DI CARATTERIZZAZIONE TECNOLOGICA [Abstract in Atti di Convegno]
Baraldi, Elisa; Belletti, Daniela; Tosi, Giovanni; Ruozi, Barbara; Forni, Flavio; Vandelli, Maria Angela
abstract

Tra le patologie cerebrali caratterizzate da grave deficit enzimatico sono sempre più attuali le malattie da accumulo lisosomiale (Lysosomial Storage Disease). La somministrazione di enzimi a livello sistemico presenta però numerose problematiche; accanto all’instabilità, vi è l’incapacità della molecola di superare barriere fisiologiche come la Barriera Emato Encefalica (BEE) e giungere al Sistema Nervoso Centrale (SNC) in concentrazioni adeguate e sufficienti. L’impiego di vettori nanoparticellari per la veicolazione (intesa come stabilizzazione e prolungamento) di enzimi e più in generale di macromolecole di natura proteica rappresenta, in tale ottica, una valida strategia per migliorare la biodisponibilità dell’attivo, oltre che permettere un targeting selettivo. Questo studio preliminare è volto a standardizzare i parametri formulativi per la preparazione di sistemi nanoparticellari (Nps) di polilattico- co-glicolico (PLGA) in grado di incapsulare efficacemente macromolecole di natura proteica. A tal proposito, Albumina bovina (Alb) e Albumina bovina coniugata con Fitc (Alb-Fitc) sono state scelte come farmaci modello e formulate in nanoparticelle (Nps-Alb e Nps- Alb-Fitc). I sistemi sono stati caratterizzati dal punto di vista chimicofisico e tecnologico. Lo studio ha permesso di standardizzare le condizioni ottimali per la formulazione (tecnica della doppia emulsione; sonication output, prima e seconda emulsione costante- 80Watt per 45”), ottenendo sistemi di dimensioni adeguate alla somministrazione (~200nm) e con buona resa. In virtù della sua maggiore idrofilia, Alb viene incapsulata con efficienza minore (~8%) rispetto all’Alb-Fitc (~30%), che però appare prevalentemente assorbita sulla superficie quindi rapidamente ceduta dalle Nps (90% di farmaco ceduto nelle 24h). Le Nps-Alb invece modulano il rilascio del farmaco per tempi più lunghi (60% di farmaco ceduto in 72 h).

2012 - Nano-Neuroscience: Targeted nanoparticles For CNS drug delivery [Relazione in Atti di Convegno]
Tosi, Giovanni; Ruozi, Barbara; Vilella, Antonietta; Belletti, Daniela; Veratti, Patrizia; Baraldi, Elisa; Zoli, Michele; M., Schmeisser; A., Grabrucker; Forni, Flavio; Vandelli, Maria Angela; A., Sharma; H. S., Sharma
abstract

2012 - Nanomedicine in Neuroscience: The potential of targeted nanoparticles in neurodegenerative disorders [Relazione in Atti di Convegno]
Tosi, Giovanni; Ruozi, Barbara; Vilella, Antonietta; Belletti, Daniela; Veratti, Patrizia; Baraldi, Elisa; Zoli, Michele; M., Schmeisser; A., Grabrucker; H. S., Sharma; A., Sharma; Forni, Flavio; Vandelli, Maria Angela
abstract

2012 - Nanotechnology in medicine: therapeutic strategies in neurological disorders [Relazione in Atti di Convegno]
Tosi, Giovanni; Ruozi, Barbara; Belletti, Daniela; Vandelli, Maria Angela; Forni, Flavio
abstract

In the last years, the application of "nanotechnology “to the field of “medicine” surely represented the most innovative strategy to cope with diseases and it could be named as nanomedicine applied to difficult-to-treat diseases. As known, in this field of research, the most important goal to be reached is an increase in selectivity and specificity of drug action. Several results with stimulating findings in preclinical or clinical phases have been reached by using nanocarriers, delivering agents to targeted pathologies, and among them, it is known that neuro-pathologies represent a stimulating issue. In fact, the pharmaceutical treatment of Central Nervous System (CNS) disorders is the second largest area of therapy, following cardiovascular diseases. Nowadays, non-invasive drug delivery systems for CNS are actively studied. In fact, the development of new delivery systems (nanoparticles and liposomes) started with the discovery that properly surface-engineered colloidal vectors, with a diameter around 200 nm, were shown to be able to cross the Blood-Brain Barrier without apparent damage, and to deliver drugs or genetic materials into the brain. During this talk, an overview will be presented considering the most recent literature results of nanomedicine applied to brain diseases, carried out with all the most popular kinds of nanoparticulate systems, focusing in particular on immune-nanoparticles and peptide-decorated nanosystems able to target the CNS, with in vivo and in vitro evidences investigating the pathway for BBB crossing and CNS localization of engineered nanoparticles. The brain localization and the multi-modal pathways for BBB crossing highlighted the endocytosis as preferential pathway; moveover, in vitro test on hippocampal neurons showed the presence of cell-to-cell transport of nanoparticles.References:•A.M. Grabrucker, C. C. Garner, T.M. Boeckers, L. Bondioli, B. Ruozi, F.Forni, M.A. Vandelli, G.Tosi , Development of novel Zn2+ loaded nanoparticles designed for cell-type targeted drug release in CNS neurons: in vitro evidences. PLOS ONE, 2011, Vol 6, e17851. •G. Tosi, R.A. Fano, L. Badiali, R. Benassi, F. Rivasi, B. Ruozi, F. Forni, M.A., Vandelli. Investigation on the mechanisms for Blood-Brain Barrier crossing of brain-targeted glycopeptides nanoparticles, Nanomedicine UK, 2011, 6(3), 423-436.•G. Tosi, AV Vergoni, B. Ruozi, L. Bondioli, L. Badiali, F. Rivasi, L. Costantino, F. Forni, M.A. Vandelli, Sialic-acid and glycopeptides conjugated PLGA nanoparticles for Central Nervous System targeting: in vivo pharmacological evidence and biodistribution, Journal of Controlled Release, 2010,145, 49–57.

2012 - STUDIO TECNOLOGICO DI OTTIMIZZAZIONE DI SISTEMI NANOPARTICELLARI ALLESTITI IN EMULSIONE SEMPLICE [Relazione in Atti di Convegno]
A., De Vita; Ruozi, Barbara; Tosi, Giovanni; Forni, Flavio; Vandelli, Maria Angela
abstract

Da diversi anni le nanoparticelle polimeriche vengono ampiamente studiate come vettori di farmaci e diagnostici; tale scelta è da ricercarsi in primis nella versatilità formulativa di tali sistemi, che possono essere allestiti con diversi polimeri sintetizzati ad hoc ed applicando varie metodiche in virtù delle esigenze applicative, nonché nella stabilità e nella capacità di modificare proprietà dimensionali e superficiali. Accanto alla preparazione di nanoparticelle (Nps) con polimeri modificati con sonde fluorescenti da utilizzare quali vettori per diagnostica (optical imaging) si è allestito anche uno studio tecnologico teso all’ottimizzazione di parametri formulativi per l’allestimento di Nps attraverso una tecnica meno indagata, ma utile per il caricamento di farmaci con caratteristiche anfifiliche, come l’emulsione semplice. Diversi parametri quali potenza e tempi di sonicazione, metodo e caratteristiche di purificazione, condizioni di conservazione con l’impiego di crioprotettore a differenti concentrazioni sono stati indagati. Impiegando potenza e tempi di sonicazione brevi (55 watt, 1 minuto) si ottengono nanoemulsioni omogenee e stabili che si traducono in preparazioni nanoparticellari monodisperse e monomodali, con diametro prossimo a 200 nm. Centrifugazioni protratte per non meno di 10 minuti a 15000 rpm portano alla pellettizzazione e risospensione dei sistemi, purificati dagli additivi di reazione; l’impiego di trealosio in quantità minime (rapporto inferiore a 1:1 tra polimero e crioprotettore) consente la risospensione dopo liofilizzazione. Sulla base di questi risultati, tali Nps sono state modificate in superficie con anticorpo monoclonale (Rituximab) ed impiegate per caricare un farmaco antitumorale, il Nutlin. Gli studi preliminari hanno dimostrato buone efficienze di caricamento, mantenendo le caratteristiche di somministrabilità del sistema. Studi in vitro ed in vivo sono in esecuzione per dimostrate la targettabilità del sistema e l’efficacia della veicolazione.

2012 - TERAPIA NUTRIZIONALE PARENTERALE: APPROCCIO MULTIDISCIPLINARE, ORGANIZZATIVO E TECNOLOGICO [Abstract in Atti di Convegno]
Belletti, Daniela; S., Paioli; S., Mantovani; I., Bosi; M., Cavana; M., Pantaleoni; G., Gavioli; Vandelli, Maria Angela
abstract

In diverse strutture ospedaliere, la nutrizione parenterale totale (TPN), è affidata a miscele commerciali standard. Tuttavia in alcune condizioni, quali patologie d’organo, nutrizione di neonati o pazienti compromessi che richiedono il monitoraggio dei parametri clinici e la frequente variazione dell’apporto nutizionale, non è possibile affidarsi a miscele industriali e diventa essenziale l’allestimento di miscele personalizzate da effettuarsi in farmacia ospedaliera. In questo contesto nasce il progetto ICL (Integrated Compounding Lab) nell’ambito della rete Alta tecnologia della regione Emilia Romagna che vede la collaborazione della ditta B-Braun Avitum e della ditta Lean per sviluppare un nuovo sistema automatizzato per il riempimento delle sacche. L’obiettivo del progetto consiste nel realizzare una piattaforma tecnologica, integrata e multidisciplinare, finalizzata alla produzione automatizzata in ambiente ospedaliero di composti diversamente complessi e personalizzati per TPN. In questa prima fase, sacche per TPN prescritte da alcuni reparti interni, sono state formulate presso la Camera Bianca dell’AOU Policlinico utilizzando inizialmente un sistema di riempimento automatico (Caretronic® Compounding System). Considerando la complessità di miscele personalizzate, sono stati messi a punto metodi di controllo delle formulazioni (determinazione del contenuto in nutrienti ed elettroliti, valutazione delle dimensioni delle micelle lipidiche e analisi di sterilità) con lo scopo finale di validare il sistema. La stabilità delle soluzioni (tempi, modalità di conservazione, caratteristiche di sicurezza microbiologica e particellare) è stata indagata e relazionata in conformità agli standard stabiliti dalla normativa vigente.

2012 - The bridge between Nanotechnology and Neuroscience: Neuro-Nanomedicine [Articolo su rivista]
Tosi, Giovanni; Ruozi, Barbara; Belletti, Daniela; Badiali, Luca; Vandelli, Maria Angela
abstract

The application of nanotechnology to the field of medicine is now at the cutting edge of scientific research. Named as Nanomedicine, this smart strategy is aimed to find new approaches for therapeutic application in the difficult-to-treat pathologies, as neurological diseases. Applied to Central Nervous System (CNS) pathologies, nanocarriers, engineered for the specific passage across the Blood-Brain Barrier (BBB), have been widely studied with stimulating and interesting results. The in vivo and in vitro experiments clearly demonstrated the potential of this kind of approach, that, now, clearly needs a higher grade of translation of the research to preclinical model of pathologies. This paper provides for an incisive description of the rationale and the development of nanomedicine applied to neuroscience, the neuro-nanomedicine.

2011 - AFM, ESEM, TEM and CLSM in liposomal characterization: a comparative study [Articolo su rivista]
Ruozi, Barbara; Belletti, Daniela; Tombesi, Andrea; Tosi, Giovanni; Bondioli, Lucia; Forni, Flavio; Vandelli, Maria Angela
abstract

An outstanding aspect of pharmaceutical nanotechnology lies in the characterization of nanocarriers for targeting of drugs and other bioactive agents. The developments of the microscopical techniques allow to deepen the study of the surface and the architecture of the systems. In the field of the pharmaceutical nanosystems, the researchers pay special attention to the microscopy characterization of liposomes owing to the vital information on size, stability and bilayer organization. Therefore, this paper aims to compare the results obtained by atomic force microscopy (AFM), environmental scanning electron microscopy (ESEM), transmission electron microscopy (TEM), and confocal laser scanning microscopy (CLSM) in order to pointed out limits and advantages of these applications in the evaluation of the vesicular systems. Besides the comparative aim, the work aspires to propose a simple CLSM procedure to rapidly and easily detect the liposomal membrane.

2011 - Brain targeting by engineered nanoparticles [Relazione in Atti di Convegno]
Tosi, Giovanni; A., Grabrucker; L., Bondioli; Ruozi, Barbara; Zoli, Michele; Vilella, Antonietta; Forni, Flavio; Rivasi, Francesco; Vandelli, Maria Angela
abstract

In the last years, the application of "nanotechnology“ to the field of “medicine” surely represented the most innovative strategy to cope with difficult-to-treat diseases. Thus, nanotech-based drug delivery and targeting are nowadays some of the hottest topics in science and in particular in Neuroscience. The results of our research, based on in vitro and in vivo preclinical tests strongly indicate that specifically engineered nanoparticles, made of poly-lactide-co-glycolide (PLGA) polymer, are able to cross the Blood-brain barrier (BBB) and to deliver a variety of drugs or active molecules inside the Central Nervous System (CNS). A recent report from the World Health Organization (WHO) emphasizes that neurological disorders (brain injuries, neuroinfections, multiple sclerosis, epilepsy, stroke, Alzheimer and Parkinson disease) affect up to one billion people worldwide [World Health Organization, Neurological disorders : public health challenger, Geneva, 2006]. Until now, only 2% of the overall drugs are able to enter the brain as the BBB restricts the diffusion of substances from blood to the brain. Thus, one of the challenges of pharmaceutical research nowadays is to discover tools enabling an effective and efficacious delivery of drugs into the CNS. To improve the efficacy of drugs, a possible answer could be the nanomedicine approach, and its application on neuroscience (neuro-nanomedicine). Thereby, the perspective of introducing a tool, capable of directed delivery of every drug into the brain, is undoubtedly an attractive goal for researchers and practitioners. To that end, neuro -nanomedicine exploits pharmaceutical technology, using well-known nanocarriers such as liposomes and polymeric nanoparticles (NPs). These nanosystems, ranging from 100 nm to 250 nm, are able to protect loaded drugs from being metabolised and eliminated, to assure the controlled release of the embedded drugs and to target specific cell population if specifically engineered.To achieve this goal we planned, create and test specifically engineering the NPs surface able to take advantage of the BBB crossing pathways, such as endocytosis or transcytosis. We applied this approach modifying FDA-approved biodegradable NPs with two different peptides to produce highly selective nanosystems able to enter the brain after i.v. administration in the rodents model. The administration of engineered-NPs allowed a variety of drugs to cross the BBB at a rate of 15-20% of the injected dose. The mechanism of BBB crossing of those NPs were elucidated by means of several in vitro and in vivo experiments as the safety of NPs on neuron cell colture was proven. The potential impact of such nanotech-based innovations relies on the possible changes in treatments and cures of the most difficult-to-treat neurological diseases, opening the pave to a new vista on the future trend in medicine, which should strengthen the relationship between different field of research (from clinician-based to chemistry, nanotechnology, biology and pre-clinical study) becoming more and more translational and interdisciplinar.

2011 - Brain targeting by engineered nanoparticles: in vivo and in vitro evidences [Abstract in Atti di Convegno]
Tosi, Giovanni; A., Grabrucker; L., Bondioli; Ruozi, Barbara; Zoli, Michele; Vilella, Antonietta; Forni, Flavio; Rivasi, Francesco; Vandelli, Maria Angela
abstract

In the last years, the application of "nanotechnology“ to the field of “medicine” surely represented the most innovative strategy to cope with diseases and could be named as nanomedicine, which is mostly applied to difficult-to-treat diseases. In this field of research, the most important goal to be reached is an increase in selectivity and specificity of drug-action. Several results with stimulating findings in preclinical or clinical phases have been obtained using nanocarriers delivering agents to targeted pathologies, and among them, it is known that neuro-pathologies represent a stimulating issue. In fact, the pharmaceutical treatment of Central Nervous System (CNS) disorders is the second largest area of therapy, following cardiovascular diseases. Nowadays, non-invasive drug delivery systems for CNS are actively studied. The nano-technological approach consists of the use of nanosystems (colloidal carriers), which could be polymer-based (nanoparticles, Np) or solid lipid material made (solid lipid nanoparticles, SLNp) and lipid-based (liposomes, LP). In fact, the development of these new delivery systems started with the discovery that properly surface-engineered colloidal vectors, with a diameter around 200 nm, are able to cross the BBB without causing apparent damage, and to deliver drugs or genetic materials into the brain. During this talk, an overview will be presented considering the most recent literature results of nanomedicine applied to brain diseases, focusing in particular on peptide-decorated nanosystems able to target the CNS.In vitro and in vivo experiments allowed to establish a pathway through which engineered NPs can cross the BBB and showed the possible NPs’ transport from cell to cell inside the CNS and the possible tropism of NPs for specific neuronal cell populations.References• A.M. Grabrucker, C. C. Garner, T.M. Boeckers, L. Bondioli, B. Ruozi, F.Forni, M.A. Vandelli, G.Tosi , Development of novel Zn2+ loaded nanoparticles designed for cell-type targeted drug release in CNS neurons: in vitro evidences. PLOS ONE, 2011, Vol 6, e17851.• G. Tosi, R.A. Fano, L. Badiali, R. Benassi, F. Rivasi, B. Ruozi, F. Forni, M.A., Vandelli. Investigation on the mechanisms for Blood-Brain Barrier crossing of brain-targeted glycopeptides nanoparticles, Nanomedicine UK, 2011, 6(3), 423-436• G. Tosi, AV Vergoni, B. Ruozi, L. Bondioli, L. Badiali, F. Rivasi, L. Costantino, F. Forni, M.A. Vandelli, Sialic-acid and glycopeptides conjugated PLGA nanoparticles for Central Nervous System targeting: in vivo pharmacological evidence and biodistribution, Journal of Controlled Release, 2010,145, 49-57.

2011 - CHOLESTEROL LOADED NANOPARTICLES INJECTED SYSTEMICALLY REACH THE BRAIN AND LOCALIZE INTO SPECIFIC CELL TYPES [Abstract in Atti di Convegno]
Valenza, M; Tosi, Giovanni; Cepeda, C; Bondioli, Lucia; Brilli, E; Ruozi, Barbara; Joshi, Pr; Chen, Jy; Singh, S; Vandelli, Maria Angela; Levine, Ms; Cattaneo, E.
abstract

Changes in brain cholesterol biosynthesis have been reported in Huntington’s disease (HD) (Valenza et al., J. Neurosci. 2005; Valenza et al., J. Neurosci. 2010). In particular, biochemical and mass spectrometry analyses showed reduced levels of several cholesterol precursors in brain of multiple HD rodent models, leading to reduced content of sterols/cholesterol (Valenza et al., Hum Mol Genet. 2007; Valenza et al., J. Neurosci. 2010). Other authors have reported accumulation of sterols in HD brains and cell models, as measured by methods other than Mass Spectrometry (Trushina et al., Hum Mol genet. 2006; LuthiCarter et al., PNAS 2010; DelToro et al., J Neurochem. 2010), questioning whether decreased cholesterol biosynthesis in HD is a primary event leading to reduced content of sterols or a consequence of sterol accumulation. To test the impact of changes in cholesterol levels in HD brain, we injected systemically cholesterol loaded poly-lactide-co-glycolide (PLGA) Nanoparticles (Nps) modified with glycopeptides (M-Nps) to obtain high rate brain delivery (Tosi et al., Expert Opin Drug Deliv. 2008 ). Differently from unmodified Nps (C-Nps), we show that M-Nps loaded with cholesterol (M-Nps-chol) efficiently enter the brain, and localize in different brain cells within 4 hrs after i.p. injection while persisting for several weeks both in wt and HD (R6/2) mice. Additionally, a pilot study suggests that multiple injections of M-Nps-chol in R6/2 mice positively influence some electrophysiological parameters compared to animals that received empty M-Nps. These preliminary findings suggest that M-Nps may represent a novel route to deliver molecules (besides cholesterol) in the brain and that cholesterol released by M-Nps may modulate functional parameters in vivo. Further studies toward this direction will be critical for defining the impact of a cholesterol defect in HD pathogenesis and its possible efficacy from a therapeutical standpoint.

2011 - CHOLESTEROL LOADED NANOPARTICLES INJECTED SYSTEMICALLY REACH THE BRAIN AND LOCALIZE INTO SPECIFIC CELL TYPES [Abstract in Atti di Convegno]
Valenza, M; Tosi, Giovanni; Cepeda, C; Bondioli, Lucia; Brilli, E; Ruozi, Barbara; Joshi, Pr; Chen, Jy; Singh, S; Forni, Flavio; Vandelli, Maria Angela; Levine, Ms; Cattaneo, E.
abstract

Changes in brain cholesterol biosynthesis have been reported in Huntington’s disease (HD) (Valenza et al., J. Neurosci. 2005; Valenza et al., J. Neurosci. 2010). In particular, biochemical and mass spectrometry analyses have shown reduced levels of several cholesterol precursors in the brains of multiple HD rodent models, leading to reduced content of sterols/cholesterol (Valenza et al., Hum Mol Genet. 2007; Valenza et al., J.Neurosci. 2010). However, others have reported accumulation of sterols in HD brains and cell models, as measured by methods other than mass spectrometry (Trushina et al., Hum Mol Genet. 2006; LuthiCarter et al., PNAS 2010; DelToro et al., J Neurochem. 2010). To test the impact of changes in cholesterol levels in the HD brain, wild-type and HD (R6/2) mice have been injected systemically with poly-lactide-co-glycolide (PLGA) nanoparticles (Nps) loaded with cholesterol and modified with glycopeptides (M-Nps) to obtain high-rate brain delivery (Tosi et al., Nanomedicine 2011). In contrast to unmodified Nps (C-Nps), we show that M-Nps loaded with cholesterol (chol-M-Nps) enter the brain efficiently, and localize in neurons and glial cells both in striatum and cortex within 4 h after intraperitoneal injection while persisting for several weeks both in wild type and HD mice. Additionally, a pilot study suggested that multiple injections ofchol-M-Nps in R6/2 mice positively influence some electrophysiological parameters of striatal medium-sized spiny neurons compared to animals that received empty M-Nps. These preliminary findings suggest that M-Nps may represent a novel and efficient route to deliver molecules (besides cholesterol) into the brain and that cholesterol released by M-Nps may modulate functional parameters in vivo. They also suggest that increasing cholesterol in HD may be beneficial. Further studies will be necessary for defining the impact of abnormalities in cholesterol level in HD pathogenesis and its possible efficacy from a therapeutic standpoint.

2011 - Complessazione di SiRNA anti Blimp-1/PRDM con liposomi cationici: caratterizzazione chimico-fisica e validazione in vitro su cellule di PEL [Relazione in Atti di Convegno]
Belletti, Daniela; Riva, Giovanni; Forni, Flavio; Barozzi, Patrizia; Luppi, Mario; Tosi, Giovanni; Vandelli, Maria Angela; Ruozi, Barbara
abstract

La tecnologia siRNAs (Small Interfering RNAs) è una innovativa strategia di regolazione genica post-trascrizionale potenzialmente applicabile in diversi campi della medicina ed in particolare in campo oncologico. [1] I siRNAs sono piccole sequenze di RNA a doppio filamento che a livello citoplasmatico subiscono un processo di attivazione mediato da un complesso sistema enzimatico denominato RISC (RNA induced silencing complexes) e divengono capaci di riconoscere e “silenziare” l’RNA target. In particolare le nostre ricerche sono indirizzate al linfoma effusivo delle cavità sierose (PEL). Si tratta di un raro tipo di linfoma di tipo non Hodgkin il cui agente eziologico è l’oncovirus HHV-8; nonostante i differenti approcci chemioterapici tentati, ad oggi non esiste un trattamento farmacologico efficace [2]. La disregolazione specifica mediata da siRNA del network di trascrizione della cellula malata potrebbe rappresentare una possibile alternativa nel trattamento della patologia. Considerando l’instabilità in vivo dei siRNAs associata alla loro scarsa capacità di penetrazione cellulare, il successo della strategia di silenziamento è strettamente dipendente dall’utilizzo di sistemi di delivery capaci di proteggere e trasportare selettivamente l’attivo nella cellula malata [3]. I liposomi, ed in particolare quelli cationici, sono sistemi di veicolazione e direzionamento innovativi, studiati e utilizzati da diversi anni, per la loro capacità di stabilizzare e trasferire materiale genico al bersaglio; la modificazione superficiale con molecole stabilizzanti (es PEG) e ligando selettive quali anticorpi monoclonali, peptidi ed aptameri garantisce la veicolazione e selettività del sistema [4,5].In studi preliminari, abbiamo dimostrato come sistemi liposomiali “stealth” modificati con anticorpo anti CD-138 specificamente riconosciuto da proteoglicani largamente espressi sulle cellule linfomatose, risultino altamente efficienti nella complessazione e stabilizzazione di materiale genico. Tali immunoliposomi sono stati testati quali carriers di un oligonucleotide modello (ODN-FITC) su cellule BCBL-1 (linea cellulare di PEL) evidenziando una buona capacità di trasferimento e di targeting valutata mediante citofluorimetria e microscopia confocale [6]. Tali evidenze sono risultate basilari per intraprendere nuovi studi di ottimizzazione di sistemi veicolanti siRNAs specifici nel silenziamento di fattori trascrizionali dominanti dello stadio plasmacellulare (knock-down di BLIMP-1/PRDM) in cellule PEL.In questa ricerca sono stati dapprima formulati e caratterizzati vettori liposomiali (Lp) utilizzando lipidi cationici (Dotap e DC-Chol) e neutri (Dope e Pc). Sono stati così selezionati i vettori che presentavano migliori caratteristiche in termini di dimensioni, carica superficiale e stabilità ed è stata valutata la loro capacità di complessare e stabilizzare siRNAs anti BLIMP-1. I complessi ottimizzati sono stati caratterizzati dal punto di vista chimico fisico mediante PCS e AFM ed è stata valutata l’efficienza di complessazione mediante elettroforesi su gel di agarosio. I dati hanno evidenziato come i liposomi allestiti con il lipide cationico Dotap risultino quelli maggiormente idonei alla formazione di lipoplexes idonei alla somministrazione: caratterizzati da strutture definite e riproducibili, di dimensioni prossime a 350nm che efficientemente proteggono i siRNAs, mostrando inoltre una buona stabilità dopo incubazione in siero. I lipoplexes allestiti con Dotap sono inoltre stati testati in vitro sulla linea cellulare BCBL-1, tali vettori sono risultati atossici in un ampio range di concentrazioni ed abili nel trasferire il materiale genico a livello citoplasmatico. In particolare è stato valutato l’effetto mediato dall’inibizione della proteina bersaglio mediante determinazione della proliferazione cellulare (analisi citomorfologica e conta cellulare) e test annessina/propidio per valutare

2011 - Development of novel Zn2+ loaded nanoparticles designed for cell-type targeted drug release in CNS neurons: in vitro evidences. [Articolo su rivista]
Andreas M., Grabrucker; Craig C., Garner; Tobias M., Boeckers; Bondioli, Lucia; Ruozi, Barbara; Forni, Flavio; Vandelli, Maria Angela; Tosi, Giovanni
abstract

Intact synaptic function and plasticity are fundamental prerequisites to a healthy brain. Therefore, synaptic proteins are one of the major targets for drugs used as neuro-chemical therapeutics. Unfortunately, the majority of drugs is not able to cross the blood–brain barrier (BBB) and is therefore distributed within the CNS parenchyma. Here, we report the development of novel biodegradable Nanoparticles (NPs), made of poly-lactide-co-glycolide (PLGA) conjugated with glycopeptides that are able to cross the BBB and deliver for example Zn2+ ions. We also provide a thorough characterization of loaded and unloaded NPs for their stability, cellular uptake, release properties, toxicity, and impact on cell trafficking. Our data reveal that these NPs are biocompatible, and can be used to elevate intracellular levels of Zn2+. Importantly, by engineering the surface of NPs with antibodies against NCAM1 and CD44, we were able to selectively target neurons or glial cells, respectively. Our results indicate that these biodegradable NPs provide a potential new venue for the delivery Zn2+ to the CNS and thus a means to explore the influence of altered zinc levels linked to neuropsychological disorders such as depression.

2011 - Investigation on Mechanisms of Glycopeptide Nanoparticles for Drug Delivery across the Blood-Brain Barrier [Articolo su rivista]
Tosi, Giovanni; Fano, Rita Adriana; Bondioli, Lucia; Badiali, Luca; Benassi, Rois; Rivasi, Francesco; Ruozi, Barbara; Forni, Flavio; Vandelli, Maria Angela
abstract

AimsNano-neuroscience, based on the use polymeric nanoparticles (NPs), is representing an emerging field of research for achieving an effective therapy for neurodegenerative diseases. In particular, poly-lactide-co-glycolide (PLGA) glyco-heptapetide-conjugated NPs (g7-NPs) were shown to be able to cross the Blood-Brain Barrier (BBB). However, the in vivo mechanisms of the BBB crossing of this kind of NPs has not been investigated until now. This paper aimed to develop a deep investigation on the mechanism of BBB crossing of the modified NPs.Materials and MethodsLoperamide (LOP) and Rhodamine-123 (Rh-123) (model drugs unable to cross the BBB) were loaded into NPs, composed of a mixture of poly-lactide-co-glycolide (PLGA), differently modified with g7 or with a random sequence of the same aminoamids (random-g7). To study brain targeting of these model drugs, loaded NPs were administered via tail vein in rats in order to perform both pharmacological studies and biodistribution analysis along with fluorescent, confocal and electron microscopy analysis, in order to achieve NPs BBB crossing mechanism. Computational analysis on the conformation of the g7- and random-g7-NPs of the NPs surface was also developed. Results Only LOP delivered to the brain with g7-NPs created a high central analgesia, corresponding to the 14% of the injected dose, data confirmed by biodistribution studies. Electron photomicrographs showed the ability of g7-NPs in crossing the BBB as evidenced by several endocytotic vesicles and macropinocytotic processes. The computational analysis on g7 and random-g7 showed a different conformation (linear versus globular), thus suggesting a different interaction with the BBB. ConclusionTaken together, these evidences suggested that g7-NPs BBB crossing is owing to a multiple-pathway, mainly membrane-membrane interaction and macropinocytosis-like mechanisms. The results of the computational analysis showed the Biousian structure of the g7 peptide, on the contrary of random-g7 peptide (globular conformation), suggesting that this difference is pivotal in explaining the BBB crossing and to allow to hypothesize the mechanism of BBB crossing by g7-NPs.

2011 - KNOCK-DOWN DI BLIMP1/PRDM1 IN CELLULE PEL MEDIANTE siRNA; OTTIMIZZAZIONE DEL SISTEMA DI TRANSFEZIONE E STUDI IN VITRO [Abstract in Atti di Convegno]
Belletti, Daniela; Riva, Giovanni; Tosi, Giovanni; Barozzi, Patrizia; Luppi, Mario; Forni, Flavio; Vandelli, Maria Angela; Ruozi, Barbara
abstract

Il trattamento del cancro mediante la chemioterapia tradizionale è spesso ostacolato dall’alta tossicità sistemica e dalla scarsa selettività dei principi attivi. La tecnologia siRNA (Small Interfering RNAs) basata sulla transfezione di ODN RNA antisenso, disegnati ad hoc per riconoscere e bloccare l’RNA messaggero target, rappresenta un’innovazione nelle strategie attuate in gene-silencing mostrando evidenti vantaggi anche in campo oncologico rispetto ai tradizionali chemioterapici. [1]Il linfoma effusivo delle cavità sierose (PEL), oggetto della ricerca, è un particolare tipo di linfoma associato invariabilmente all’infezione di HHV-8, con opzioni terapeutiche convenzionali limitate e non efficaci.[2] La disregolazione specifica mediata da siRNA del network di trascrizione della cellula malata rappresenta, ad oggi, una possibile alternativa nel trattamento della patologia. Considerando l’instabilità in vivo dei siRNAs associata alla loro scarsa capacità di penetrazione cellulare, il successo della strategia di silenziamento è strettamente dipendente dall’utilizzo di sistemi di delivery capaci di proteggere e trasportare selettivamente l’attivo nella cellula malata[3]. I liposomi, ed in particolare quelli cationici, sono sistemi di veicolazione e direzionamento innovativi, studiati e utilizzati da diversi anni, per la loro capacità di stabilizzare e trasferire materiale genico al bersaglio; la modificazione superficiale con molecole stabilizzanti (es PEG) e ligando selettive quali anticorpi monoclonali, peptidi ed aptameri garantisce la veicolazione e selettività del sistema [4,5].In studi preliminari, abbiamo dimostrato come sistemi liposomiali “stealth” modificati con anticorpo anti CD-138 specificamente riconosciuto da proteoglicani largamente espressi sulle cellule linfomatose, risultino altamente efficienti nella complessazione e stabilizzazione di materiale genico. Tali immunoliposomi sono stati testati quali carriers di un oligonucleotide modello (ODN-FITC) su cellule BCBL-1 (linea cellulare di PEL) evidenziando una buona capacità di trasferimento e di targeting valutata mediante citofluorimetria e microscopia confocale [6]. Tali evidenze sono risultate basilari per intraprendere nuovi studi di ottimizzazione di sistemi veicolanti siRNA specifici nel silenziamento di fattori trascrizionali dominanti dello stadio plasmacellulare (knock-down di BLIMP1/PRDM1) in cellule PEL. In questa ricerca sono stati formulati e caratterizzati vettori liposomiali (Lp) utilizzando lipidi neutri e cationici (Dotap e DC-Chol). Tali liposomi sono stati complessati con siRNAs e caratterizzati (elettroforesi su gel di agarosio, PCS ed AFM); i complessi ottenuti utilizzando il lipide cationico Dotap risultano caratterizzati da strutture definite e riproducibili, di dimensioni prossime a 350nm che efficientemente proteggono i siRNAs, mostrando inoltre una buona stabilità dopo incubazione in siero. I vettori sono stati testati in vitro su cellule BCBL-1; i dati preliminari evidenziano come i lipoplessi siano abili nel trasferire il materiale genico a livello citoplasmatico. L’inibizione della proteina bersaglio produce un effetto dose dipendente a concentrazioni superiori a 50nM evidente dopo 48/72 ore dal trattamento. In particolare è stata osservata un elevata induzione di necrosi /apoptosi mediante test annessina/propidio associata ad una significativa inibizione della proliferazione cellulare. E’ stata inoltre valutata la peghilazione come strategia per aumentare il tempo di permanenza in circolo oltre ad offrire il supporto per il legame con anticorpi specifici verso il PEL (anti.CD-138).[1] Oh YK, Park TG (2009). Adv. Drug Deliv Rev. 61: 850–862.[2] Carbone A, Gloghini A (2007). BJH review. 140: 13–24.[3] Whitehea KA , Langer R, Anderson DG (2009). Nature Review. 8:129-138.[4] Ruozi B, Belletti D, Tombesi A, Tosi G, Bondioli L, Forni F, Vandelli M A (2011). Int J Nanomed. 6:557–563.[5] Ruo

2011 - Leptin-conjugated nanoparticles as drug delivery system to the brain [Relazione in Atti di Convegno]
Badiali, Luca; Belletti, Daniela; Veratti, Patrizia; Baraldi, Elisa; Ruozi, Barbara; Tosi, Giovanni; Vergoni, Anna Valeria; Vandelli, Maria Angela
abstract

Leptin-conjugated nanoparticles as drug delivery system to the brain

2011 - NANOPARTICELLE POLIMERICHE PER IL DIREZIONAMENTO CEREBRALE: DELIVERY DI ZINCO E STUDI PRELIMINARI IN VITRO SU CELLULE NEURONALI [Relazione in Atti di Convegno]
Bondioli, Lucia; Tosi, Giovanni; Ruozi, Barbara; Forni, Flavio; Vandelli, Maria Angela; Andreas M., Grabrucker; Tobias M., Boeckers; Craig C., Garner
abstract

Alterazioni morfologiche e funzionali a livello delle sinapsi cerebrali sono caratteristiche di numerose patologie del sistema nervoso centrale (SNC), come ad esempio la sindrome di Phelan-McDermid (o sindrome da delezione 22q13). Tale patologia è correlata a ritardi dello sviluppo fisico e mentale a causa della delezione di un segmento distale del cromosoma 22, in cui è incluso il gene SHANK3 (un membro della famiglia Shank “multidomain scaffold proteins” della densità post-sinaptica) che svolge un ruolo significativo nella connessione dei neuroni [1]. Studi recenti hanno mostrato una stretta relazione tra la presenza di Zn2+ e l’organizzazione morfologica e strutturale delle sinapsi. Sebbene lo ione metallico sia in grado di modulare, nell’arco di pochi secondi, l’impalcatura strutturale creata dalle proteine, la sua somministrazione non può risolvere i sintomi della malattia non essendo in grado di oltrepassare la barriera ematoencefalica (BEE) per distribuirsi nel SNC.In questo studio viene riportato lo sviluppo di un nuovo sistema nanoparticellare (NPs) che si è rivelato promettente nel veicolare Zn2+ verso cellule neuronali di ratto; tale sistema è costituito da polilattico-co-glicolico (PLGA) coniugato con un ligando glicopeptidico che promuove l’attraversamento della barriera emato-encefalica [2-4] e la localizzazione nel SNC. Differenti preparazioni nanoparticellari caricate e non caricate con Zn2+ (sia modificate con il glicopeptide che non modificate) sono state testate sia su colture di fibroblasti (HEK293 Cells) che su cellule neuronali ippocampali di ratto. Gli esperimenti hanno confermato l’assenza di tossicità del vettore anche a concentrazioni superori a quelle utilizzate per veicolare lo Zn2+ e la capacità delle NPs di promuovere un efficace rilascio dello ione a livello intracellulare. I dati raccolti hanno permesso altresì di formulare ipotesi riguardo al meccanismo di internalizzazione. A tale scopo è stato valutato il destino di NPs marcate (rodamina) in seguito all’incubazione con cellule neuronali (anch’esse trattate con FM1-43, marker utilizzato per il monitoraggio delle vescicole endocitotiche). La colocalizzazione del marker FM1-43 con le NPs, supporta l’ipotesi di avventa internalizzazione delle NPs mediante un meccanismo endocitotico, potenziato altresì dalla presenza del glicopeptide sulla superficie del vettore.Gli studi di rilascio condotti in vitro hanno dimostrato che lo ione incapsulato viene completamente e gradualmente rilasciato in circa 3 settimane. L’aumento di Zn2+ osservato all’interno delle cellule può pertanto essere dovuto ad un aumento dello Zn2+ extracellulare o al rilascio dello stesso da NPs endocitate.La modifica della superficie nanoparticellare con anticorpi diretti verso epitopi extracellulari presenti su cellule neuronali (NCAM1) e cellule gliali (CD44), ha migliorato la selettività e la direzionabilità del sistema (drug targeting).[1] Knight SJ, Flint J (2004). Methods Cell Biol. 75: 799–831.[2] Costantino L, Gandolfi F, Tosi G, Rivasi F, Vandelli MA, (2005). J Control Release. 108: 84–96.[3] Vergoni AV, Tosi G, Tacchi R, Vandelli MA, Bertolini A, Costantino L (2009). Nanomedicine: Nanotechnology, Biology and Medicine. 5:369-377. [4] Tosi G, Fano RA, Badiali L, Benassi R, Rivasi F, Ruozi B, Forni MA, Vandelli MA (2011), Nanomedicine UK. 6: 423-436.

2011 - NEUROTROPHIC FACTORS AND NEURODEGENERATIVE DISEASES: A DELIVERY ISSUE [Capitolo/Saggio]
Ruozi, Barbara; Belletti, Daniela; Bondioli, Lucia; A., De Vita; Forni, Flavio; Vandelli, Maria Angela; Tosi, Giovanni
abstract

Neurotrophic factors (NTFs) represent one of the most stimulating challenge in neurodegenerative diseases, due to their potential in neurorestoring and neuroprotection. Despite the large number of proofs-of-concept and evidences of their activity, most of the clinical trials, mainly regarding Parkinson’s Disease and Alzheimer Disease, demonstrated several failures of the therapeutic intervention.A large number of researches were conducted on this hot topic of neuroscience, clearly evidencing the advantages of NTFs approach, but evidencing the major limitations in its application. The inability in crossing the Blood-Brain Barrier and the lack of selectivity actually represent some of the most highlighted limits of NTFs-based therapy. In this review, beside an overview of NTFs activity versus the main neuropathological disorders, a summary of the most relevant approaches, from invasive to non-invasive strategies, applied for improving NTFs delivery to the Central Nervous Systems is critically considered and evaluated.

2011 - NIR-labeled nanoparticles engineered for brain targeting: in vivo optical imaging application and fluorescent microscopy evidences. [Articolo su rivista]
Tosi, Giovanni; Bondioli, Lucia; Ruozi, Barbara; Badiali, Luca; Gm, Severini; S., Biffi; A., De Vita; B., Bortot; D., Dolcetta; Forni, Flavio; Vandelli, Maria Angela
abstract

The presence of the blood–brain barrier (BBB)makes extremely difficult to develop efficacious strategiesfor targeting contrast agents and delivering drugs inside theCentral Nervous System (CNS). To overcome this drawback,several kinds of CNS-targeted nanoparticles (NPs)have been developed. In particular, we proposed polylactide-co-glycolide (PLGA) NPs engineered with a similopioidglycopeptide (g7), which have already proved to bea promising tool for achieving a successful brain targetingafter i.v. administration in rats. In order to obtain CNStargetedNPs to use for in vivo imaging, we synthesizedand administrated in mice PLGA NPs with double coverage:near-infrared (NIR) probe (DY-675) and g7. Theoptical imaging clearly showed a brain localization of thesenovel NPs. Thus, a novel kind of NIR-labeled NPs wereobtained, providing a new, in vivo detectable nanotechnologytool. Besides, the confocal and fluorescencemicroscopy evidences allowed to further confirm the abilityof g7 to promote not only the rat, but also the mouseBBB crossing.

2011 - Nanoparticles and the BBB crossing: in vivo and in vitro upcomings [Relazione in Atti di Convegno]
Tosi, Giovanni; Bondioli, Lucia; Ruozi, Barbara; Andreas, Grabrucker; Vilella, Antonietta; Zoli, Michele; Rivasi, Francesco; Vandelli, Maria Angela; Forni, Flavio
abstract

In the last years, the application of "nanotechnology “to the field of “medicine” surely represented the most innovative strategy to cop_20e with diseases and it could be named as nanomedicine applied to difficult-to-treat diseases. As known, in this field of research, the most important goal to be reached is an increase in selectivity and specificity of drug action. Several results with stimulating findings in preclinical or clinical phases have been reached by using nanocarriers, delivering agents to targeted pathologies, and among them, it is known that neuro-pathologies represent a stimulating issue. In fact, the pharmaceutical treatment of Central Nervous System (CNS) disorders is the second largest area of therapy, following cardiovascular diseases. Nowadays, non-invasive drug delivery systems for CNS are actively studied. In fact, the development of new delivery systems (nanoparticles and liposomes) started with the discovery that properly surface-engineered colloidal vectors, with a diameter around 200 nm, were shown to be able to cross the Blood-Brain Barrier without apparent damage, and to deliver drugs or genetic materials into the brain. During this talk, an overview will be presented considering the most recent literature results of nanomedicine applied to brain diseases, carried out with all the most popular kinds of nanoparticulate systems, focusing in particular on immune-nanoparticles and peptide-decorated nanosystems able to target the CNS, with in vivo and in vitro evidences investigating the pathway for BBB crossing and CNS localization of engineered nanoparticles. The brain localization and the multi-modal pathways for BBB crossing highlighted the endocytosis as preferential pathway; moveover, in vitro test on hippocampal neurons showed the presence of cell-to-cell transport of nanoparticles.

2011 - Nanoparticles for brain delivery of drugs: in vivo experiments and mechanism of BBB crossing [Articolo su rivista]
Tosi, Giovanni; Fano, Rita Adriana; Badiali, Luca; Bondioli, Lucia; Ruozi, Barbara; Vergoni, Anna Valeria; Rivasi, Francesco; Benassi, Rois; Vandelli, Maria Angela; Forni, Flavio
abstract

Specific ligands on the surface allowed the Np to cross the Blood-Brain Barrier (BBB) carrying model drugs within the brain district after their i.v. administration in experimental animals. It is known that sialic acid receptors are present in several organs, including in the brain parenchyma. Thus, we prepared PLGA NPs surface modified with a BBB-penetrating peptide (simil-opioid peptide) for BBB crossing and with a sialic acid residue (SA) for the interaction with brain receptors. This double coverage could allow to obtain novel targeted NPs with a prolonged residence within the brain parenchyma, thus letting to reach a long-lasting brain delivery of drugs. The central analgesic activity of Loperamide (opioid drug, unable to cross the BBB) loaded in these novel NPs was evaluated in order to point out the capability of the NPs to reach and to remain in the brain. The results showed that the pharmacological effect induced by loaded NPs administration remained significant over 24 hrs. Using confocal and fluorescent microscopy, the novel NPs were localized within the tissue parenchyma (brain, kidney, liver, spleen and lung). Finally, the biodistribution studies showed a localization of the 6% of the injected dose into the CNS over a prolonged time (24 hrs). Notwithstanding an increased accumulation of SA-covered NPs in those organs showing SA-receptors (liver, kidney, lung), the pharmacological and biodistribution results are proofs of the ability of double targeted NPs to enter the brain allowing the drug to be released over a prolonged time. Moreover, electron microscopy of brain sections after iv administration of modified NPs, allowed us to hypothesize a multiple-pathway mechanism of BBB crossing of modified NPs. NPs surface interaction with BBB membrane without a clear involvement of specific receptors, but possibly based on “biousian conformation” of the surface of NPs, along with ruffles of the membrane produced near to modified-NPs, seemed to mediate a BBB crossing process based on endocytosis

2011 - Novel polymeric/lipidic hybrid systems (PLHs) for effective Cidofovir delivery: preparation, characterization and comparative in vitro study with polymeric particles and liposomes [Articolo su rivista]
Belletti, Daniela; Riva, Giovanni; Tosi, Giovanni; Forni, Flavio; Barozzi, Patrizia; Luppi, Mario; Vandelli, Maria Angela; Ruozi, Barbara
abstract

Cidofovir is an antiviral drug active as antitumoral agent a high doses against the Primary Effusion Lymphoma, a herpesvirus HHV8-associated B-cell lymphoma. A novel polymeric/lipidic hybrid system, consisting in a specific combination of biocompatible materials, capable to build a crossbred betweenpolymeric particles and liposomes were prepared and used to stabilize and deliver the drug, unsuccessfully formulated into several types of carriers. This innovative cidofovir-delivering system has structurally been characterized in comparison to multilamellar liposomes and polymeric particles, and then testedfor antitumoral efficacy against tumor cells (BCBL-1 cell line). The results demonstrated the improving of drug stability and encapsulation efficiency and suggested that polymeric/lipidic hybrid system could be promising to improve the antitumoral effect of cidofovir even at lower doses.

2011 - Oral delivery of insulin loaded into polymeric nanoparticles in rats. [Articolo su rivista]
Tosi, Giovanni; Vergoni, Anna Valeria; Ruozi, Barbara; Bondioli, Lucia; Forni, Flavio; Vandelli, Maria Angela; Tacchi, Raffaella; Ferrari, Anna; Spaccapelo, Luca; Bertolini, Alfio
abstract

Diabetes prevalence is steadily increasing and both type 1 and type 2 contribute to this “diabetes epidemic”. Insulin is the sole therapeutic, life-saving option for type 1 diabetes with patients self-injecting the hormone every day for life. In this study, insulin has been loaded into polymeric nanoparticles (Np) of poly (D,L-lactide-co-glycolide) and different amounts of insulin-loaded Np (1, 3 or 10 I.U./kg) were administered by oral gavage to normal and diabetic rats, s.c. pre-treated with omeprazole (5 mg/Kg). In normal rats, the in vivo results highlighted a dose-related decrease of blood glucose levels in normal rats at the end of the observation period (20-50% depending on the doses of insulin delivered by Np). In diabetic rats, the dose of 3 I.U./kg produced a 50% decrease of the glycaemia 90 min after the treatment, with an effect stable up to the end of the observation period; a higher dose of insulin delivered by Np (10 I.U./kg) developed a significant decrease of glycaemia (65%). Correspondently, in diabetic rats, plasma insulin levels increased in a dose-related manner (3, 5, and 7-folds with respect to the basal level, depending on insulin doses delivered by NPs) with still significant values 2 h after administration. Thus, these Np are able, after pre-treatment with omeprazole, to transport insulin across the intestinal barrier, preserving the biological activity of the hormone in rats and if replicated in humans, it could suggest the concrete possibility of oral administration of insulin.

2011 - STUDI PRELIMINARI SUL BRAIN TARGETING MEDIANTE L’OPTICAL IMAGING E TERAPIA ENZIMATICA PER LA POMPE-DISEASE [Abstract in Atti di Convegno]
A., De Vita; Tosi, Giovanni; Bondioli, Lucia; Ruozi, Barbara; Badiali, Luca; G. M., Severini; S., Biffi; B., Bortolotti; D., Dolcetta; C., Emiliani; Forni, Flavio; Vandelli, Maria Angela
abstract

Nel campo delle neuroscienze, uno degli obiettivi più ambiti è rappresentato dal direzionamento di principi attivi al Sistema Nervoso Centrale (SNC). Le terapie proposte per la cura delle patologie cerebrali risultano spesso inefficaci; farmaci potenzialmente applicabili, mostrano limitata capacità di superamento della barriera emato-encefalica (BEE) e pertanto concentrazioni non terapeutiche (o permanenza troppo breve) al sito target. In tale ottica, l’utilizzo di sistemi nanoparticellari di rilascio opportunamente modificati, può migliorare l’applicabilità e la targettabilità dei farmaci al SNC [1-3]. Tale progetto di ricerca rappresenta uno studio preliminare sul delivery al SNC. In un primo momento sono state allestite nanoparticelle (Np) derivatizzate sulla solo superficie con un eptapeptide g7 in grado di attraversare la BEE e modificate con un marker fluorescente, il DY-675, allo scopo di visualizzarle in vivo a seguito di somministrazione i.v. in topi. Mediante l’ausilio del microscopio confocale e a fluorescenza è stato possibile visualizzare il DY-675 all’interno del cervello dimostrando l’avvenuto raggiungimento nell’area cerebrale dei nanosistemi.La seconda parte del progetto si è concentrata sugli studi di caricamento e di rilascio di un enzima (Myozime) in Np, successivamente si è proceduto allo studio dell’efficacia farmacologica del sistema su colture di fibroblasti affette dalla Pompe-Disease constatandone una correzione della deficienza enzimatica del 50% a seguito di una singola somministrazione, confermando così il possibile impiego di tali Np nella terapia enzimatica di patologie a carico del SNC.[1] Tosi G, Rivasi F, Gandolfi F, Costantino L, Vandelli M A, Forni F. Biomaterials 26, 4189-4195, 2005.[2] Tosi G, Costantino L, Ruozi B, Forni F, Vandelli M A. Expert Opinion on Drug Delivery 5, 155-174, 2008.[3] Tosi G, Vergoni A V, Ruozi B, Bondioli L, Badiali L, Rivasi F, Costantino L, Forni F, Vandelli M A. Journal of Controlled Release 145, 49–57, 2010.

2011 - Sialic Acid as a potential approach for the protection and targeting of nanocarriers [Articolo su rivista]
Bondioli, Lucia; Ruozi, Barbara; Belletti, Daniela; Forni, Flavio; Vandelli, Maria Angela; Tosi, Giovanni
abstract

IntroductionNanocarriers are considered as the most innovative drug delivery systems, due to their high potential in drug protection, delivery and mainly targeting to the diseased site; unfortunately, their applicability is mainly hampered by their uptake due to macrophagic recognition and lack of specificity if not properly engineered. Areas coveredSialic acid (SA) and its derivatives was deeply studied in order to govern the stealthness of carriers or, more recently, to act as targeting moiety. In this review, we summarized the most outstanding researches (in vitro and in vivo) dealing with the use of SAs or derivatives to modify the carriers surface in order to achieve targeted or stealth nanosystems. Moreover, we also consider the application of SA or derivatives as modifiers used for cancer targeting and therapy and for recognition purposes.Expert OpinionThe application of SA-based strategy for nanocarriers engineering represents one of the most stimulating challenge in drug delivery and drug targeting. The in vivo and in vitro ouptuts on stealth or targeted nanocarriers, modified with different kinds of SAs or SA-derivative, highlighted the great potential of this approach, both evidencing the advantages (stealth properties, targeting ability, cancer inhibition, viral and inflammation recognition, brain targeting) and the possible disadvantages (i.e. presence of possible multi-target side effects outputs), suggesting further investigations on this strategy.

2011 - Studi preliminari sul brain targeting mediante l’optical imaging e terapia enzimatica per la Pompe-Disease, Sci Ottobre 2011 [Abstract in Atti di Convegno]
De Vita, A.; Tosi, Giovanni; Baraldi, Elisa; Veratti, Patrizia; Ruozi, Barbara; Badiali, Luca; Severini, G. M.; Biffi, S.; Bortolotti, B.; Dolcetta, D.; Emiliani, C.; Vandelli, Maria Angela
abstract

Studi preliminari sul brain targeting mediante l’optical imaging e terapia enzimatica per la Pompe-Disease, Sci Ottobre 2011

2011 - The loading of labeled antibody engineered nanoparticles with Indinavir increases its in vitro efficacy against Cryptosporidium parvum [Articolo su rivista]
Bondioli, Lucia; A., Ludovisi; Tosi, Giovanni; Ruozi, Barbara; Forni, Flavio; E., Pozio; Vandelli, Maria Angela; M. A., Gómez Morales
abstract

Introduction: There are evidences indicating the Indinavir (IND) ability to reduce C. parvum infection in both in vitro and in vivo models. However, there are limitations to administrate IND as it, such as its renal toxicity and the high grade of metabolism and degradation. We aimed to encapsulate IND in biodegradable Poly (D,L-lactide-co-glycolide) nanoparticles (Np) and to engineer their surface by the conjugation with an anti-Cryptosporidium IgG polyclonal antibody (Ab).Methods: Tetramethylrhodamine labelled Np were loaded with IND and modified by conjugation with an Ab. The IND loaded modified Np (Ab-TMR-IND-Np) did not show any change, as shown by chemical analysis studies. Results: The treatment with 50µM of the Ab-TMR-IND-Np added to the culture at the same time with excysted oocysts, resulted in a complete inhibition of the infection. In C. parvum infected cells, the extent to which the infection decreased was found to have depended on the duration of treatment with the Ab-TMR-IND-Np. Discussion: The antibody engineered Np loaded with IND are able to target C. parvum oocysts in infected cells, could represent a novel therapeutic strategy against Cryptosporidium sp. infection. Moreover, the Np as IND delivery devices, allow the development of a more appropriate dose formulation reducing the IND side effects.

2011 - Tumor-targeted immunoliposomal nanosystems to deliver either Cidofovir or Antineoplastic SiRNA against Primary Effusion Lymphoma (PEL) [Relazione in Atti di Convegno]
Riva, Giovanni; Belletti, Daniela; Ruozi, Barbara; Barozzi, Patrizia; Vallerini, Daniela; Quadrelli, Chiara; Zanetti, Eleonora; Morselli, M; Forghieri, Fabio; Marasca, Roberto; Narni, Franco; Tosi, Giovanni; Forni, Flavio; Vandelli, Maria Angela; Potenza, Leonardo; Luppi, Mario
abstract

Therapeutic applications of siRNA-mediated gene silencing appear to be highly dependent on the use of pharmaco-technologic carrier systems, able to protect siRNAs from rapid degradation upon administration, as well as to specifically deliver them to target cells. Actually, while siRNA-expressing viral vectors are burdened with safety concerns for their clinical use, the development of modified liposomal nanocarriers may represent a feasible option to harness the therapeutic potential of targetedantineoplastic siRNAs. Recently, we have successfully developed and characterized effective immunoliposomal nanosystems (ILNs) for targeted delivery of Cidofovir (an anti-herpesviral nucleotideanalogue, also showing antitumor activity) against PEL cell lines, demonstrating a significant improvement of the antineoplastic activity of the drug, especially at lower doses (less than 1nM). Thus, we tried to adapt such PEL-specific ILNs (PEGilated nanovescicles made of cationic/neutral lipids, engineered with anti-CD138 moAb on their surface) to efficiently encapsulate siRNAs and deliver them into PEL cell lines (highly expressing CD138 membrane protein). Our preliminary data showed thatsingle treatments with anti-PEL ILNs, delivering specific siRNAs against Blimp1 (Prdm1), which is a master transcription factor in PEL (a plasmablast/pre-plasmacell lymphoma, consistently Bcl-6 neg, Blimp1 pos), were able to induce a dose-dependent (50-200nM) inhibition of Blimp1 production (as assessed by Blimp1 mRNA and protein levels using RT-PCR and Western Blot, respectively), and this was strongly associated with enhanced cell death (more than 80%, using Annexin V/PI test). In particular, we observed a massive reduction of PEL viability (mean viable cells 8%, range 3-15%) as soon as 48-72 hours after treatment with 100nM anti-Blimp1 siRNAs. Interestingly, these data may resemble those described in multiple myeloma cell lines, after transduction with lentiviral vector constitutively expressing anti-Blimp1 shRNAs. Further studies on PEL murine models are now warranted to assess the efficacy and toxicity profile of in-vivo treatment with PEL-specific ILNs, loadedwith either Cidofovir or anti-Blimp1 siRNAs.

2010 - Cidofovir-loaded liposomes: an intro-study using BCBL-1 cell line as a model for primary effusion lymphoma [Articolo su rivista]
Ruozi, Barbara; Riva, Giovanni; Belletti, Daniela; Tosi, Giovanni; Forni, Flavio; Mucci, Adele; Barozzi, Patrizia; Luppi, Mario; Vandelli, Maria Angela
abstract

Cidofovir (HPMPC) was recently reported to exert a valuable antineoplastic activity against primary effusion lymphoma (PEL), a B-cell neoplasm associated with Human Herpesvirus-8 (HHV-8) infection. In this study, we developed and characterized liposomes encapsulating HPMPC to increase drug efficacy reducing the administered dose and the related toxicity, which actually hamper its clinical therapeutic use in patients affected with PEL. The liposomes, obtained using different formulations of neutral and cationic lipids, were analyzed by microscopical (AFM) and spectroscopical (PCS and NMR) techniques. Using an in vitro model of PEL (BCBL-1 cell line), the carrier toxicity and the antineoplastic efficacy of liposomes were evaluated by flow cytometry applying apoptosis and cell death analysis. The in vitro study showed the applicability of the liposomes within a restricted range of lipidic concentrations according to the lipids used during the preparation. The moderate increases in the percentage of apoptotic/necrotic cells suggests that liposomal delivery allows the release of HPMPC into BCBL-1 cells enabling an unexpectedantineoplastic activity of this drug even at lower doses.

2010 - Development and characterization of immunoliposomes for Cidofovir and SiRNA delivery: a new strategy for the treatment of Primary Effusion Lymphoma [Abstract in Atti di Convegno]
Belletti, Daniela; Ruozi, Barbara; Riva, Giovanni; Tosi, Giovanni; Barozzi, Patrizia; Luppi, Mario; Forni, Flavio; Vandelli, Maria Angela
abstract

Primary Effusion Lymphoma (PEL) is an aggressive and consistently lethal non-Hodgkin's B-cell lymphoma growing as lymphomatous effusions in serous body cavities and invariably associated with HHV-8 [1,2].The majority of the patients affected with PEL, either elderly HIV-negative or immunocompromised AIDS patients, are typically characterized by several age-related co-morbidities or opportunistic infectious diseases. Clinical efficacy of conventional anti-neoplastic chemotherapy is commonly hampered by the excessive grade of systemic toxicity and low drug levels in the tumor area [3].New therapeutic challenges may arise from the use of SiRNA technology, which is based on cellular transfection of antisense small RNAs, specifically designed to recognize the target mRNA and able to turn off the changed cellular mechanism of tumor cells, or by using antineoplastic drug (Cidofovir). Considering the high instability and the poor cellular uptake of both these actives, tumor-specific delivery by means of targeted nanocarriers is strongly required. These strategies represent an attractive approach to enhance intra-tumoral cytotoxic effects together with the reduction of “off-target” side effects, possibly offering a radical improvement in the treatment of such fragile oncologic patients. Among the colloidal carrier systems for drug delivery, liposomes have received considerable attention. They allow to protect the drug from rapid degradation, being particularly suitable to form complexes with highly-degradable ribonucleic acids.In this study, we formulated and characterized immunoliposomal formulation direct to PEL cell line (BCBL-1 cell line) using the cationic lipid DOTAP and a pegylated cholesterol funzionalized with a maleidoimide moiety capable to interact with anti CD-138 antibody. The formulation was characterized (size, zeta potential and morphology) in comparison with untargeted DOTAP liposomes and pegylated systems.These liposomal systems were used to transfect a model FITC-ODN into a model PEL cell line (BCBL-1). The studies on cellular binding and on the internalization of oligo by flow cytometry and confocal analysis confirmed the higher transfection efficiency of the immunoliposomes when compared with DOTAP and pegylated liposomes. This targeted formulation could be reasonably considered as optimal candidates for therapeutic siRNA delivery and more generally for gene encapsulation and delivery against the poor curable PEL tumor. Concerning cidofovir, it has been demonstrated that this antiviral drug is able to induce cell apoptosis in different tumor included PEL. Indeed, the high pro-apoptotic concentrations of cidofovir are never achievable in situ after full-dose systemic administration (5mg/Kg i.v.), and however, this systemic treatment can frequently cause severe nephrotoxicity [4]. We proposed liposomes encapsulating cidofovir by a modified reversed phase evaporation method (mREV) followed by extrusion. The characterization of samples suggested that cationic liposomes are more suitable for cidofovir stabilization, taking advantage of the charge interaction between the anionic drug and the cationic lipid moieties. Using the in vitro model of PEL (BCBL-1 cell line), the carrier toxicity and the antineoplastic efficacy of liposomes were evaluated by flow cytometry, applying apoptosis and cell death analysis. This in vitro study showed the applicability of the liposomes within a restricted range of lipidic concentrations, mainly depending on the lipids used during the preparation. The cidofovir transfection mediated by liposome composed of PC:DOTAP and PC:DC-CHOL caused a moderate increase in the percentage of apoptotic/necrotic PEL cells with respect to the controls (free drug and empty liposomes) suggesting that liposomal delivery allows the release of cidofovir into BCBL-1 cells enabling an unexpected antineoplastic activity of this drug even at lower doses.

2010 - Formulation and characterization of new Polymeric/Lipidic Hybrid systems for cidofovir delivery [Abstract in Atti di Convegno]
Belletti, Daniela; Riva, Giovanni; Tosi, Giovanni; Barozzi, Patrizia; Luppi, Mario; Forni, Flavio; Vandelli, Maria Angela; Ruozi, Barbara
abstract

Cidofovir is an antiviral drug with a remarkable antitumour activity in several animal models, associated or not with viral infections [1]. More recently, cidofovir has also been shown to be an effective treatment against Primary Effusion Lymphoma (PEL), a B cell non–Hodgkin lymphoma involving the serous cavities, invariably associated with Human HerpesVirus-8 (HHV8) and often with Epstein–Barr Virus (EBV) infection. A hindrance to the clinical applications of cidofovir is the high systemic toxicity, mainly the nephrotoxicity. Cidofovir encapsulation into specific micro- or nanocarriers, able to extend the release of the drug, may represent an effective strategy both to minimize the off-target organ exposure as well as to simultaneously increase the concentration of drug within the site of action. Unfortunately, the physical-chemical characteristics of the drug (low molecular weight, high solubility at different pH, unfavourable partition coefficient) limit the encapsulation into delivery systems [2,3]. Recently, we proposed the use of cationic liposomes to stably encapsulate cidofovir; unfortunately, the in vivo applicability of such cidofovir carriers is limited by the presence of cationic lipids, inducing a dose-related toxicity [4]. To overcome this problem, in this work we aimed to investigate a novel hybrid system, consisting in a specific combination of biocompatible materials, capable to build a crossbred between polymeric particles and liposomes. This innovative cidofovir-delivering systems (called PLHs, polymeric/lipidic hybrid systems) made of phosphatidilcholine (PC), cholesterol (CHOL) and polylactic acid (PLA) have been characterized (size, zeta potential, morphology, structure and thermal behaviour) in comparison to multilamellar liposomes and polymeric particles. Microscopical studies (atomic force and confocal microscopy), in agreement with the other characterizations, suggested that a rearrangement of the components has taken place to form a new matricial porous structure different both from liposomes and polymeric particles, with a wide dispersion of polymer in the lipidic bulk. This new crossbeald delivery systems was able to increased the encapsulation of cidofovir (encapsulation efficiency twice higher than liposomes and about 10 times higher than polymeric particles) and resulted atossic against PEL tumor cells (BCBL-1 cell line) revealing also a capability to better traslocate the drug into the cells causing and increased apoptosis respect to the free drug.

2010 - Immunoliposomal systems targeting the primary effusion lymphoma (PEL): in vitro study [Articolo su rivista]
Ruozi, Barbara; Riva, Giovanni; Belletti, Daniela; Tosi, Giovanni; Barozzi, Patrizia; Luppi, Mario; Forni, Flavio; Vandelli, Maria Angela
abstract

Aims: To develop an appropriate liposomal formulation for gene delivery against Primary Effusion Lymphoma (PEL), a herpesvirus HHV8-associated B-cell lymphoma. Materials and methods: Cationic, cationic pegylated and cationic pegylated anti-CD138 liposomes (ILp) linking a monoclonal antibody expressed on PEL cells were prepared by thin layer evaporation method followed by extrusion technique. The formulations were mixed with a model oligonucleotide to form the lipoplexes tested on BCBL-1 cell (a PEL cell line). The transfection efficiency was evaluated by flow cytometry and confocal laser scanning microscopy analysis. Results: Based on antigen–antibody interaction, ILp mediated a specific gene delivery as shown by a significant increase in the transfection rate and a localized internalization of the oligo, in comparison with cationic liposomes and cationic pegylated liposomes.Conclusion: ILp could be proposed as effective carriers for oligo transfer in BCBL-1 cells. In vitro experimental results encourage to further test the in vivo therapeutic potentials of ILp for specific delivery of antitumoral agents.

2010 - Immunoliposomes for the delivery of SiRNA and chemioterapeutic agent to primary effusion lymphoma [Abstract in Atti di Convegno]
Belletti, Daniela; Ruozi, Barbara; Riva, Giovanni; Barozzi, Patrizia; Luppi, Mario; Forni, Flavio; Vandelli, Maria Angela
abstract

In this study we have formulated and characterized immunoliposomes direct to PEL cells using the cationic lipid DOTAP and a pegylated cholesterol funzionalized with a maleidoimide moiety capable to stable interact with anti CD 138 antbody. The formulation was characterized in comparison with only pegylated systems and DOTAP liposomes. These liposomes were used to transfect a model of FITC-ODN into the target cells. The efficiency of these carriers was evaluated using flow cytometry and confocal analysis. The results confirm the higher transfection efficiency of the immunoliposomes if compared with conventional and pegylated liposomes.

2010 - Nanoparticelle per il delivery di farmaci al Sistema Nervoso Centrale [Articolo su rivista]
Tosi, Giovanni; Ruozi, Barbara; Vandelli, Maria Angela; Forni, Flavio
abstract

La ricerca e la messa punto di ulteriori studi su modelli patologici è ancora lunga, ma in continuo divenire, confidando che la nanotecnologia applicata alla medicina, secondo la terminologia inglese “Nanomedicine”, rappresenti dunque una delle più promettenti strategie, aprendo la strada verso nuove prospettive, con la possibilità di trasportare e rilasciare all’interno del sistema nervoso centrale una grande varietà di sostanze attive, di creare nuove terapie per contrastare patologie ad oggi di difficile approccio, come le malattie neurodegenerative, che rappresentano uno dei maggiori problemi di salute pubblica.

2010 - Nanoparticelle polimeriche per il direzionamento cerebrale di Zinco: studi preliminari in vitro su neuroni ippocampali e cellule gliali [Abstract in Atti di Convegno]
Bondioli, Lucia; Tosi, Giovanni; Ruozi, Barbara; Forni, Flavio; Vandelli, Maria Angela; Andreas M., Grabrucker; Tobias M., Boeckers; Craig C., Garner
abstract

Alterazioni morfologiche e funzionali a livello delle sinapsi cerebrali sono caratteristiche di numerose patologie cerebrali, come ad esempio la sindrome di Phelan-McDermid (o sindrome da delezione 22q13). Tale patologia, correlata a ritardi dello sviluppo fisico e mentale, è causata della delezione di un segmento distale del cromosoma 22, in cui è incluso il gene SHANK3, un membro della famiglia Shank “multidomain scaffold proteins” della densità post-sinaptica (PSD): tali proteine svolgono un ruolo significativo nella connessione dei neuroni. Studi recenti hanno mostrato una stretta relazione tra la presenza di Zn2+ e l’organizzazione morfologica e strutturale delle sinapsi. Sebbene lo ione metallico sia in grado di modulare, nell’arco di pochi secondi, l’impalcatura strutturale creata dalle proteine a livello della PSD, la sua somministrazione non può risolvere i sintomi della malattia in quanto lo Zn2+ non è in grado di oltrepassare la barriera ematoencefalica (BEE) per distribuirsi nel SNC.In questo studio viene riportato lo sviluppo di un nuovo sistema nanoparticellare (Nps) costituito da PLGA coniugato con un oppotuno glicopeptide, che in studi precedenti ha mostrato la capacità di attraversare la BEE. Sono stati effettuati studi “in vitro” su culture di neuroni ippocampali e cellule gliali, volti alla valutazione della tossicità, del meccanismo di uptake e della stabilità del sistema stesso. I risultati hanno dimostrato l’assenza di tossicità cellulare del sistema nanoparticellare sulle linee testate e la sua capacità di promuovere un efficace rilascio dello ione Zn2+ a livello intracellulare. Le nostre prove hanno permesso altresì di evidenziare come l’endocitosi sembri rappresentare il principale meccanismo di internalizzazione del sistema veicolante

2010 - Nanoparticles for brain delivery of drugs: in vivo experiments andmechanism of BBB crossing [Relazione in Atti di Convegno]
Tosi, Giovanni; Fano, Rita Adriana; Badiali, Luca; Bondioli, Lucia; Ruozi, Barbara; Vergoni, Anna Valeria; Rivasi, Francesco; Benassi, Rois; Vandelli, Maria Angela; Forni, Flavio
abstract

Specific ligands on the surface allowed the Np to cross the Blood-Brain Barrier (BBB) carrying model drugs within the brain district after their i.v. administration in experimental animals. It is known that sialic acid receptors are present in several organs, including in the brain parenchyma. Thus, we prepared PLGA Np surface modified with a BBB-penetrating peptide (simil-opioid peptide) for BBB crossing and with a sialic acid residue (SA) for the interaction with brain receptors. This double coverage could allow to obtain novel targeted Np with a prolonged residence within the brain parenchyma, thus letting to reach a long-lasting brain delivery of drugs. The central analgesic activity of Loperamide (opioid drug, unable to cross the BBB) loaded in these novel Np was evaluated in order to point out the capability of the Np to reach and to remain in the brain. The results showed that the pharmacological effect induced by loaded Np administration remained significant over 24 hrs. Using confocal and fluorescent microscopy, the novel Np were localized within the tissue parenchyma (brain, kidney, liver, spleen and lung). Finally, the biodistribution studies showed a localization of the 6% of the injected dose into the CNS over a prolonged time (24 hrs). Notwithstanding an increased accumulation of SA-covered Np in those organs showing SA-receptors (liver, kidney, lung), the pharmacological and biodistribution results are proofs of the ability of double targeted Np to enter the brain allowing the drug to be released over a prolonged time. Moreover, electron microscopy of brain sections after iv administration of modified Np, allowed us to hypothesize a multiple-pathway mechanism of BBB crossing of modified Np. Np surface interaction with BBB membrane without a clear involvement of specific receptors, but possibly based on “biousian conformation” of the surface of Np, along with ruffles of the membrane produced near to modified-Np, seemed to mediate a BBB crossing process based on endocytosis.

2010 - Nanosistemi lipidici targettizzati per il direzionamento di siRNA con attività antineoplastica al linfoma primitivo delle cavità sierose (PEL) [Abstract in Atti di Convegno]
Belletti, Daniela; Ruozi, Barbara; Riva, Giovanni; Tosi, Giovanni; Barozzi, Patrizia; Luppi, Mario; Forni, Flavio; Vandelli, Maria Angela
abstract

La tecnologia dei siRNA (Small Interfering RNAs) basata sulla transfezione di ODN RNA antisenso, disegnata ad hoc per riconoscere e bloccare l’RNA messaggero target, rappresenta l’innovazione nelle strategie attuate in gene-silencing con importanti prospettive per il trattamento di patologie complesse, come il linfoma primitivo delle cavità sierose (PEL), oggetto della ricerca.Il PEL è un particolare tipo di linfoma associato invariabilmente all’infezione di HHV-8, altamente aggressivo che presenta opzioni terapeutiche convenzionali limitate e non efficaci. La disregolazione specifica mediata da siRNA del network di trascrizione della cellula malata rappresenta, ad oggi, una possibile alternativa nel trattamento della patologia. I liposomi, ed in particolare quelli cationici, sono da diversi anni studiati per la loro capacità di stabilizzare materiale genico proteggendolo dalla degradazione in vivo ed offrendo inoltre la possibilità di coniugare molecole specifiche nel riconoscimento del target.In questa ricerca sono stati formulati e caratterizzati sistemi immunoliposomiali “stealth” e targettizzati con anticorpo CD-138 specificamente riconosciuto da proteoglicani largamente espressi sulle cellule linfomatose. Tali immunoliposomi sono stati testati in studi preliminari quali carriers di un oligonucleotide modello in una linea cellulare di PEL (BCBL-1); la capacità di trasferimento e di targeting è stata valutata mediante citofluorimetria e microscopia confocale. Successivamente sono stati allestiti immunoliposomi incorporanti siRNA per il silenziamento di fattori trascrizionali dominanti dello stadio plasmacellulare (knock-down di BLIMP1/PRDM1) e testati sulla stessa linea cellulare. I risultati evidenziano come i nostri sistemi siano altamente efficienti nella stabilizzazione del materiale genico promuovendo inoltre il trasferimento a livello citoplasmatico dove avviene il silenziamento della proteina bersaglio con conseguente apoptosi cellulare.

2010 - Nanotech approaches to CNS delivery: nanoparticle surface modification to obtain long-circulating polymeric drug carriers [Abstract in Atti di Convegno]
Bondioli, Lucia; Costantino, Luca; A., Balestrazzi; Tosi, Giovanni; Badiali, Luca; Vandelli, Maria Angela
abstract

There is a broad interest in the development of nanoparticles (NPs) carrying on their surface carbohydrates such as sialic acids. It is known that these carbohydrates influence the biological and physical properties of biopharmaceutical proteins and living cells. Macromolecular compounds containing these carbohydrates showed an anti-recognition effect, exert an antiviral effect and also are able to be recognized by the cell surface of some kind of cancer cells. Thus, in the present research we performed two different approaches in order to obtain polymeric (poly(d,l-lactide-co-glycolide), PLGA) NPs surface decorated with the sialic acid N-acetylneuraminic acid (Neu5Ac). The first strategy that has been followed is based on the derivatization of the polyester PLGA with the thioderivative of Neu5Ac, starting material for the preparation of the NPs; the second is based on the synthesis of compounds potentially able to insert their lipophilic moiety into the underivatized PLGA NPs during their preparation, and to display their hydrophilic moiety (Neu5Ac) on their surface. The first approach allowed the obtainment of NPs surface decorated with Neu5Ac, as evidenced by ESCA spectroscopy and interaction with the lectin Wheat Germ Agglutinin. Moreover, a formulation of these NPs suitable for in vitro assays showed that they are phagocytosed by human monocytes with an apparently different mechanism with respect of those made of underivatized PLGA. The second strategy led to NPs in which their surface appears to be very different with respect to the NPs obtained following the first strategy, with the carboxylic groups of Neu5Ac markedly shielded. Thus, the new Neu5Ac-modified PLGA polyester represent a useful starting material for the preparation of NPs surface decorated with this sialic acid.

2010 - Nanotechnology for the CNS drug delivery and targeting [Relazione in Atti di Convegno]
Tosi, Giovanni; Ruozi, Barbara; Bondioli, Lucia; Badiali, Luca; Vandelli, Maria Angela; Forni, Flavio
abstract

In the last years, the application of "nanotechnology “to the field of “medicine” surely represented the most innovative strategy to cope with diseases and it coule be named as nanomedicine applied to difficult-to-treat diseases. As known, in this field of research, the most important goal to be reached is an increase in selectivity and specificity of drug action. Several results with stimulating findings in preclinical or clinical phases have been reached by using nanocarriers, delivering agents to targeted pathologies, and among them, it is known that neuro-pathologies represent a stimulating issue. In fact, the pharmaceutical treatment of Central Nervous System (CNS) disorders is the second largest area of therapy, following cardiovascular diseases. Nowadays, non-invasive drug delivery systems for CNS are actively studied. The nano-technological approach consist of the use of nanosystems (colloidal carriers) which could be polymer-based (nanoparticles, Np) or solid lipid material made (solid lipid nanoparticles, SLNp) and lipid-based (liposomes, LP). In fact, the development of these new delivery systems started with the discovery that properly surface-engineered colloidal vectors, with a diameter around 200 nm, were shown to be able to cross the BBB without apparent damage, and to deliver drugs or genetic materials into the brain. During this talk, an overview will be presented considering the most recent literature results of nanomedicine applied to brain diseases, carried out with all the most popular kinds of nanoparticulate systems, focusing in particular on peptide-decorated nanosystems able to target the CNS.

2010 - Nanotechonology for drug targeting [Capitolo/Saggio]
Ruozi, Barbara; Tosi, Giovanni; Forni, Flavio; Vandelli, Maria Angela
abstract

Nanoparticles (Np) and liposomes (L) were engineered obtaining selective drug delivery systemsable to cross BBB and to treat cancer diseases, respectively. The first goal was achievedconjugating a specific epta-glucopeptide (g7) to polymeric nanoparticles (Np). The data related thenociceptive activity showed the ability of g7-Np to cross the BBB and to release loperamide in thebrain.To reach the second goal we have recently proposed the immunoliposomes (ILp) for tumor-targeteddelivery of gene material (particularly SiRNAs), which are selected in vitro for the specificantineoplastic activity against herpesvirus-associated B-cell lymphomas, particularly HHV8+Primary Effusion Lymphoma (PEL). In the preliminary study we have prepared and characterizedthe ILp direct to PEL cells (BCBL-1 cell line). The cellular trafficking of the encapsulated modelFITC-ODN obtained by flow cytometry and confocal microscopy was evaluated by the ability ofthe new carriers to selectively interact with cells. The data were compared with the differentbehaviour of these liposomes respect to the un-targeted cationic and pegylated liposomes.

2010 - PLGA nanoparticles surface decorated with the sialic acid, N-acetylneuraminic acid [Articolo su rivista]
Bondioli, Lucia; Costantino, Luca; Ballestrazzi, Antonio; Lucchesi, Davide; Boraschi, Diana; Pellati, Federica; Benvenuti, Stefania; Tosi, Giovanni; Vandelli, Maria Angela
abstract

2010 - Peptide-engineered polylactide-co-glycolide (PLGA) nanoparticles for brain delivery of drugs: in vivo experiments and proof of concept [Relazione in Atti di Convegno]
Tosi, Giovanni; Fano, Rita Adriana; Badiali, Luca; Bondioli, Lucia; Ruozi, Barbara; Vergoni, Anna Valeria; Rivasi, Francesco; Benassi, Rois; Vandelli, Maria Angela; Forni, Flavio
abstract

Drug delivery to the Central Nervous System (CNS) represents a huge challenge for all neuroscientists owing to the presence of the Blood-Brain Barrier (BBB) hampering the influx to the brain of most of the drugs, enzymes, gene materials. Nanotechnology, based on polymeric nanoparticles (Np) and liposomes, could be an useful tool for the delivery of the drugs in the brain if they are planned for crossing the BBB. This goal can be achieved specifically engineering the Np surface in order to take advantage of the BBB crossing pathways, such as endocytosis or transcytosis. We applied this approach modifying polylactide-co-glycolide (PLGA) Np with two different peptides to produce highly selective nanosystems able to enter the brain after i.v. administration in the rats [Costantino L. et al. (2005). J Control Rel 108, 84-96; Tosi G. et al. (2007) J. Control Rel 122, 1-9]. The administration of decorated Np with a simil-opioid peptide (planned and synthesized in our laboratories) allows a variety of P-glycoprotein substrate to cross the BBB at a rate of 15-20% of the injected dose, as microscopy technique (confocal, fluorescent), biodistribution and pharmacological studies proved [Vergoni A.V. et al. (2009) Nanomedicine (NBM) 5, 369-377] These systems cross the BBB via an endocytic mechanism pointed out by an electron microscopy procedure (fig. 1). On the contrary, the Np decorated with a Leptin fragment should be able to take advantage of specific BBB-leptin receptors (Ob-R). In vivo experiments pointed out the efficacy of these leptin modified Np in the brain delivery and the trancytosis mechanism of the BBB crossing (fig. 2). Any anoretic effect of the Leptin-fragment covering the Np was exclude by food-intake experiments.Figure 1. Left: Electron microscopy image of multiple mechanisms of simil-opioid-Np interaction with BBB endothelial cells; Right: Fig. 2. Brain images after iv administration of Leptin-derived peptide- Np. Red spots are due to Np labeled with TRICT and blue-spots are brain nuclei with DAPI.

2010 - Polymeric nanoparticles for CNS drug delivery: strategies and perspectives [Relazione in Atti di Convegno]
Tosi, Giovanni; Bondioli, Lucia; Badiali, Luca; Ruozi, Barbara; Vergoni, Anna Valeria; Rivasi, Francesco; Vandelli, Maria Angela; Forni, Flavio
abstract

The application of nanotechnology to health raises high expectations for a more efficient and affordable healthcare. Even if several areas of medical care could benefit from the advantages that nanotechnology can offer, a selective CNS drug delivery and targeting could improve the therapy of brain diseases which have a tremendous negative impact not only on the patient himself but also on the whole society and linked social and insurance systems. Polymeric nanoparticles (Np) have been considered as strategic carriers for the brain delivery and targeting. Specific ligands on the surface allowed the Np to cross the Blood-Brain Barrier (BBB) carrying model drugs within the brain district after their i.v. administration in experimental animals. A new strategy for Np brain targeting by using a simil-opioid peptide−derived PLGA, obtaining Np showing both the ligands for CNS targeting and the marker of fluorescence on their surface was found (M-Np). After administration, the M-Np were found to be able to cross the BBB and the ability of these M-Np to act as drug carriers has been shown (Tosi et al., 2007). Moreover, the biodistribution of M-Np showed a localization into the CNS in a quantity (15% of the injected dose) about two orders of magnitude greater than that found with the other known Np drug carriers (Vergoni et al., 2009). Moreover, it is known that sialic acid receptors are present in several organs, including in the brain parenchyma. Thus, PLGA Np modified on their surface with a BBB-crossing ligand (simil-opioid peptide) and with a sialic acid residue (SA) were prepared (SA-M-Np). This double targeting (for BBB crossing and for the interaction with brain receptors) allowed to obtain novel targeted Np with a prolonged residence within the brain parenchyma, thus letting to reach a long-lasting brain delivery of drugs. Notwithstanding an increased accumulation of SA-covered Np in those organs showing SA-receptors (liver, kidney, lung), the pharmacological and biodistribution results are proofs of the ability of double-targeted Np to enter the brain allowing the drug to be released over a prolonged time. References•Tosi G. et al., Targeting the Central Nervous System. In vivo experiments with peptide derivatized nanoparticles loaded with Loperamide and Rhodamine 123, J. Control. Release 122 (2007) 1-9.•Vergoni AV et al., Nanoparticles as drug delivery agents specific for CNS: in vivo biodistribution. Nanomedicine: Nanotechnology, Biology and Medicine 5 (2009) 369-377.

2010 - Sialic acid and glycopeptides conjugated PLGA nanoparticles for central nervous system targeting: In vivo pharmacological evidence and biodistribution [Articolo su rivista]
Tosi, Giovanni; Vergoni, Anna Valeria; Ruozi, Barbara; Bondioli, Lucia; Badiali, Luca; Rivasi, Francesco; Costantino, Luca; Forni, Flavio; Vandelli, Maria Angela
abstract

Polymeric nanoparticles (Np) have been considered as strategic carriers for brain targeting. Specific ligands on the surface allowed the Np to cross the Blood-Brain Barrier (BBB) carrying model drugs within the brain district after their i.v. administration in experimental animals.It is known that sialic acid receptors are present in several organs, including in the brain parenchyma. Thus, in this paper, we prepared PLGA Np surface modified with a BBB-penetrating peptide (similopioid peptide) for BBB crossing and with a sialic acid residue (SA) for the interaction with brain receptors. This double coverage could allow to obtain novel targeted Np with a prolonged residence within the brain parenchyma, thus letting to reach a long-lasting brain delivery of drugs. The central analgesic activity of Loperamide (opioid drug, unable to cross the BBB) loaded in these novel Np was evaluated in order to point out the capability of the Np to reach and to remain in the brain. The results showed that the pharmacological effect induced by loaded Np administration remained significant over 24 h. Using confocal and fluorescent microscopies, the novel Np were localized within the tissue parenchyma (brain, kidney, liver, spleen and lung). Finally, the biodistribution studies showed a localization of the 6% of the injected dose into the CNS over a prolonged time (24 h). Notwithstanding an increased accumulation of SA-covered Np in those organs showing SA-receptors (liver, kidney, and lung), the pharmacological and biodistribution results are proofs of the ability of double targeted Np to enter the brain allowing the drug to be released over a prolonged time.

2010 - Studies of optical imaging and fluorescence microscopy with nanoparticles functionalized for brain targeting with NIR [Abstract in Atti di Convegno]
A., De Vita; Tosi, Giovanni; Bondioli, Lucia; Ruozi, Barbara; Badiali, Luca; G. M., Severini; S., Biffi; B., Bortolotti; D., Dolcetta; Forni, Flavio; Vandelli, Maria Angela
abstract

Nowadays the Central Nervous System (CNS) is made object of studying from the international scientific community. The pathologies that affects this system are extremely disabling and many times chronicles therefore represent a big burden for the patient and his family. The study of cerebral diseases is very difficult because this district is characterized by a complex morphology; a very important structure in the CNS is represented by the BBB (blood-brain-barrier), one of the most drawback on the transit of the active principles and contrast agents. One of the strategies for crossing this barrier is the preparation of CNS-targeted nanoparticles (NPs), a nanometrical carrier modified on the surface with specific peptides, that allow the BBB crossing. The aim of this project is the development of poly-lactide-co-glycolide (PLGA) NPs engineered with a simil-opioid glycopeptide (g7) which is able to cross the BBB in order to use for in vivo imaging modifying the NPs with a marker, the near-infrared probe (DY-675). After i.v. administration in mice, a confocal and fluorescence microscopy study allowed to detect the presence of the DY-675 in the brain.

2009 - AFM phase imaging of soft-hydrated samples: A versatile tool to complete the chemical-physical study of liposomes [Articolo su rivista]
Ruozi, Barbara; Tosi, Giovanni; Tonelli, Massimo; Bondioli, Lucia; Mucci, Adele; Forni, Flavio; Vandelli, Maria Angela
abstract

Despite of the several approaches applied to the physicochemical characterization of liposomes, few techniques are really useful to obtain information about the surface properties of these colloidal drug-delivery systems. In this paper, we demonstrate a possible new application of tapping mode atomic force microscopy (AFM) to discriminate between conventional and pegylated liposomes. We showed that the differences on liposomal surface properties revealed by the phase images AFM approach well correlate with the data obtained using classical methods, such as light scattering, hydrodynamic, and nuclear magnetic resonance analysis.

2009 - CNS drug delivery by using polymeric nanoparticles [Relazione in Atti di Convegno]
Costantino, Luca; Bondioli, Lucia; Tosi, Giovanni; Ruozi, Barbara; Forni, Flavio; Vandelli, Maria Angela
abstract

Sono state discusse le possibili modificazioni alla superficie di nanoparticelle per ottenere stabilità e possibilità di direzionamento al cervello.

2009 - Collagen modified based membranes for tissue engineering: influence of type and molecular weight of GAGs on cell proliferation [Articolo su rivista]
Ruozi, Barbara; B., Parma; Croce, Maria Antonietta; Tosi, Giovanni; Bondioli, Lucia; S., Vismara; Forni, Flavio; Vandelli, Maria Angela
abstract

This study aims to evaluate the effects of the two most widely used glycosaminoglycans (dermatan sulphate and heparin) on both the structural and biological properties of collagen based modified membranes (COL/GAGs membranes) designed for tissue engineering. The molecular weight of dermatan sulphate and heparins was correlated with the membrane feasibility and the cell (fibroblasts and keratinocytes) ability to adhere and proliferate on the COL/GAG membranes.Microstructure and physico-chemical properties of COL/GAGs membranes were examined using scanning electron microscopy and differential scanning calorimetry; the free amino group content and the swelling properties were also detected. The morphology, proliferation and growth behaviour of keratinocytes and fibroblasts were investigated using microscopical approach and in vitro colorimetric assay. Both fibroblasts and keratinocytes are able to growth and proliferate on COL/dermatan sulphate membranes. Fibroblasts revealed significantly higher proliferation on the membranes prepared with heparin if compared to the proliferation on the membrane without heparin (COL membrane). Particularly, a combination of the membranes formulated adding high molecular weight dermatan sulphate and high molecular weight heparin could be suitable to be used as biomaterials for epidermal substitute.

2009 - Colloidal systems for CNS drug delivery [Capitolo/Saggio]
Costantino, Luca; Tosi, Giovanni; Ruozi, Barbara; Bondioli, Lucia; Vandelli, Maria Angela; Forni, Flavio
abstract

The pharmaceutical treatment of central nervous system (CNS) disorders is the second largest area of therapy, following cardiovascular diseases. Nowadays, non-invasive drug delivery systems for CNS are actively studied. The development of these new delivery systems started with the discovery that properly surface-engineered colloidal vectors, and in particular liposomes and polymeric nanoparticles, with a diameter around 200 nm, were shown to be able to cross the BBB without apparent damage, and to deliver drugs or genetic materials into the brain. However, even if this ability was confirmed by confocal microscopy and measured by biodistribution experiments or by means of the pharmacological effect exerted by the embedded drugs, a clear understanding of the main characteristics of the colloidal systems that are important for BBB crossing is still lacking. It is also shown that the presence of the drug is able to modify the surface of these systems, with unpredictable results on the colloidal systems biodistribution; thus, the results obtained in the absence of the loaded drug has to be taken cautiously. Moreover, since the loaded drug is only a fraction of the colloidal system that is administered, the presence of the carrier in the body and into CNS, especially in the case of long-term therapies, might cause adverse effects not yet fully understood. Thus, even if promising results have been obtained, and some colloidal systems loaded with a drug are FDA approved for human use (but not for brain targeting), a long way of research has to be done in order to use these drug delivery systems for the treatment of CNS pathologies.

2009 - Drug Delivery to the brain: in vivo evaluation on surface engeneered nanoparticles [Abstract in Atti di Convegno]
Badiali, Luca; Tosi, Giovanni; Bondioli, Lucia; Vandelli, Maria Angela; Forni, Flavio
abstract

Drug Delivery to the brain: in vivo evaluation on surface engeneered nanoparticles

2009 - Engineered nanoparticles for drug delivery to the CNS: in vivo experiments [Abstract in Atti di Convegno]
Badiali, Luca; Vergoni, Anna Valeria; Bertolini, Alfio; Tosi, Giovanni; Costantino, Luca; Bondioli, Lucia; Ruozi, Barbara; Belletti, Daniela; Forni, Flavio; Vandelli, Maria Angela
abstract

Engineered nanoparticles for drug delivery to the CNS: in vivo experiments. Biodistribution of drug and pharmacological activity

2009 - Engineered polylactide-co-glycolide(PLGA) Np as drug delivery systems for the Central Nervous System [Relazione in Atti di Convegno]
Tosi, Giovanni; Costantino, Luca; Rivasi, Francesco; Bondioli, Lucia; Ruozi, Barbara; Vergoni, Anna Valeria; Tacchi, Raffaella; Bertolini, Alfio; Vandelli, Maria Angela; Forni, Flavio
abstract

Nanoparticulate polymeric systems (nanoparticles, Np) have been widely studied for the delivery of drugs to a specific target site. Np have been recently considered for the therapy of various brain diseases. The major problem in accessing the central nervous system (CNS) is due to the presence of the Blood-Brain Barrier (BBB). Recently, it has been shown the possibility to reach the CNS district crossing the BBB using nanoparticles (Np) made of polylactide-co-glycolide (PLGA), modified with a simil-opioid sequence and different glycosidic moieties. Firstly, PLGA was modified with different glycol-heptapetides (Glucose, Lactose, Xylose, and Mannose as sugar moieties and with a single [P] or a triple sequence of heptapeptides [3P]). Then, after i.v. administration, Np, labeled with covalent linkage with a fluorescent dye, were demonstrated to be able to cross the BBB by using confocal microscopy. A strong analgesic effects due to the encapsulated Loperamide, a P-glycoprotein (P-gp) substrate model drug, demonstrated the ability of modified PLGA Np to cross the BBB, after i.v. administration. The effect was different in the intensity and in the time period according to the different surface modification, being the Glucose preferable when compared with the other ones. When 3P-PLGA Np were used, a different profile in the pharmacological activity was assessed, i.e. a sudden maximum analgesic effect followed by a fast decrease over the time. Finally, the biodistribution of Np loaded with Rhodamine-123 (P-gp substrate) was analyzed quantifying the fluorescent intensity in the different organs including brain, in order to better understand the fate of these modified Np.

2009 - Flow cytometry and live confocal analysis for the evaluation of the uptake and intracellular distribution of FITC-ODN into HaCaT cells [Articolo su rivista]
Ruozi, Barbara; Montanari, Monica; Vighi, Eleonora; Tosi, Giovanni; Tombesi, Andrea; Battini, Renata; Restani, Cinzia; Leo, Eliana Grazia; Forni, Flavio; Vandelli, Maria Angela
abstract

In this study the mechanism of the internalisation and the cellular distribution of 5’ fluorescein conjugated PS-ODN (FITC-ODN) after transfection with different mixed lipidic vesicles/oligo complexes (lipoplexes) have been investigated. Mixed lipidic vesicles were prepared with one of the most used cationic lipid (DOTAP) and different amount of a cholic acid (UDCA) to release the oligo into HaCaT cells. Using flow cytometry, the cellular uptake of the oligo was studied with and without different inhibitors able to block selectively the different pathways involved in the internalisation mechanism. The intracellular distribution of the oligo was analysed by confocal laser scanning microscopy (CLSM) treating the cells with the lipoplexes and directly observing without any fixing procedure. To better carry out the co-localization studies, fluorescent labelled markers, specific for the different cellular compartments, were co-incubated with FITC-ODN.The different lipidic vesicles affect the internalisation mechanism of FITC-ODN. After using the inhibitors, the uptake of complexes involved a different internalization mechanism. The live CLSM analysis demonstrated that, after 1h from the complex incubation, the oligo was transferred into cells and localized into the endosomes; after 24 h, oligo was intracellularly localized close to the nuclear structure in a punctuate pattern. However, the results from fusion experiments showed also a binding of a quite amount of oligo with the cell membranes.

2009 - Glycopeptide-decorated nanoparticles as drug carriers for CNS: effects of surface coverage and carbohydrate type [Articolo su rivista]
Tosi, Giovanni; Costantino, Luca; Rivasi, Francesco; Ruozi, Barbara; Bondioli, Lucia; Vergoni, Anna Valeria; Tacchi, Raffaella; Bertolini, Alfio; Vandelli, Maria Angela; Forni, Flavio
abstract

n order to study the ability of peptide-decorated PLGA Np to act as CNS drug delivery agents, the effect of various degrees of Np surface coverage by the peptide H2N-Gly-L-Phe-D-Thr-Gly-L-Phe-L-Leu-L-Ser(O-R)-CONH2 (R = -D-glucose) and of the type of carbohydrate present on Ser (R= H, -D-glucose, -D-xylose, -D-lactose, -D-Mannose) were evaluated. Loperamide was used as a model drug and its presence on rat CNS was evaluated by means of its pharmacological effect (antinociceptive assay, hot-plate test). The pharmacological effect exerted by loperamide loaded into Np demonstrated to be strongly dependent on the degree of peptidic surface coverage of Np. Thus, in the presence of a high surface coverage, a very short effect is observed, which appears early on (0.5 min) after Np iv administration. Moreover, the sugar moiety influences markedly the CNS effect of loperamide loaded into the peptide-decorated Np: among the carbohydrates here examined, the presence of Beta-D-glucose on the Ser of the peptide showed the best results, both in terms of the maximum effect and length.

2009 - Immunoliposomes for the delivery of SiRNA and chemioterapeutic agent to primary effusion lymphoma [Abstract in Atti di Convegno]
Belletti, Daniela; Ruozi, Barbara; Riva, Giovanni; Barozzi, Patrizia; Luppi, Mario; Forni, Flavio; Vandelli, Maria Angela
abstract

Breve descrizione delle metodologie attuate per promuovere il rilascio di farmaci e materiale genico al tumore. In particolare è stato discusso l'approccio con liposomi e sistemi lipidici

2009 - Immunoliposomes to target the Primary Effusion Lymphoma (PEL): in vitro study [Abstract in Atti di Convegno]
Belletti, Daniela; Ruozi, Barbara; Riva, Giovanni; Barozzi, Patrizia; Luppi, Mario; Forni, Flavio; Vandelli, Maria Angela
abstract

Preparazione e caratterizzazione di immunoliposomi per la veicolazione di materiale genico ai tumori (limfoma effusivo primario)

2009 - Nanoparticles as drug delivery agents specific for CNS: in vivo biodistribution [Articolo su rivista]
Vergoni, Anna Valeria; Tosi, Giovanni; Raffaella, Tacchi; Vandelli, Maria Angela; Alfio, Bertolini; Costantino, Luca
abstract

The pharmacological treatment of neurological disorders is often complicated by the inability of drugs to pass the Blood Brain Barrier. Recently we discovered that polymeric nanoparticles (Np) made of poly(D,L-lactide-co-glycolide) surface-decorated with the peptide Gly-L-Phe-D-Thr-Gly-L-Phe-L-Leu-L-Ser(O--D-Glucose)-CONH2 are able to deliver, after i.v. administration, the model drug loperamide into Central Nervous System (CNS). This new drug delivery agent is able to ensure a strong and long-lasting pharmacological effect, far greater than that previously observed with other nanoparticulate carriers. Here, we confirmed the effectiveness of this carrier for brain targeting comparing the effect obtained by the administration of loperamide-loaded Np with the effect of an intracerebroventricular administration of the drug; moreover, the biodistribution of these Np showed a localization into CNS in a quantity about two orders of magnitude greater than that found with the other known Np drug carriers. Thus, a new kind of Np that target CNS with a very high specificity was discovered

2009 - Nanotech approaches to CNS delivery: nanoparticles surface modification to obtain long circulating polymeric drug carriers [Abstract in Atti di Convegno]
Bondioli, Lucia; Costantino, Luca; A., Balestrazzi; Tosi, Giovanni; Badiali, Luca; Vandelli, Maria Angela
abstract

Nanotech approaches to CNS delivery: nanoparticles surface modification to obtain long circulating polymeric drug carriers

2009 - New perspectives in peptides- and antibodies- conjugated nanocarriers for brain delivery therapeutic purposes [Abstract in Atti di Convegno]
Tosi, Giovanni; Ruozi, Barbara; Vergoni, Anna Valeria; Bondioli, Lucia; Badiali, Luca; Belletti, Daniela; Forni, Flavio; Vandelli, Maria Angela
abstract

New perspectives in peptides- and antibodies- conjugated nanocarriers for brain delivery therapeutic purposes; description of therapeutic strategies for the treatment of brain phatologies ( especially non-invasive techniques)

2009 - Sialic acid as surface modifier of polymeric PLGA nanoparticles [Relazione in Atti di Convegno]
Bondioli, Lucia; Costantino, Luca; A., Balestrazzi; Tosi, Giovanni; Badiali, Luca; Forni, Flavio; Vandelli, Maria Angela
abstract

Nanoparticles for drug delivery to CNS

2009 - Surface decorated nanoparticles as drug delivery agents to the brain [Abstract in Atti di Convegno]
Badiali, Luca; Vergoni, Anna Valeria; Tosi, Giovanni; Ruozi, Barbara; Costantino, Luca; Bertolini, Alfio; Vandelli, Maria Angela; Forni, Flavio
abstract

Ingegnerizzazione della superficie di sistemi nanoparticellari per la veicolazione ed il direzionamento di farmaci al cervello

2008 - Immunoliposome for the delivery of drugs and SiRNA for the treatment of cancer [Abstract in Atti di Convegno]
Ruozi, Barbara; Riva, Giovanni; Barozzi, Patrizia; Tosi, Giovanni; Belletti, Daniela; Forni, Flavio; Luppi, Mario; Vandelli, Maria Angela
abstract

Immunoliposome for the delivery of drugs and SiRNA for the treatment of cancer

2008 - Nanoparticles for drug delivery to the CNS [Abstract in Atti di Convegno]
Tosi, Giovanni; Costantino, Luca; Bondioli, Lucia; Ruozi, Barbara; Badiali, Luca; Forni, Flavio; Vandelli, Maria Angela
abstract

Nanoparticles for drug delivery to the CNS

2008 - Peptide-decorated nanoparticles as carriers for drug delivery to CNS [Abstract in Atti di Convegno]
Costantino, Luca; Bondioli, Lucia; Tosi, Giovanni; Vergoni, Anna Valeria; Tacchi, Raffaella; Bertolini, Alfio; Vandelli, Maria Angela; Forni, Flavio
abstract

Peptide-decorated nanoparticles as carriers for drug delivery to CNS

2008 - Polymeric nanoparticles for the drug delivery to the central nervous system [Articolo su rivista]
Tosi, Giovanni; Costantino, Luca; Ruozi, Barbara; Forni, Flavio; Vandelli, Maria Angela
abstract

Background: Nanoparticulate polymeric systems (nanoparticles, Np) have been widely studied for the delivery of drugs to a specific target site. This approach has been recently considered for the therapy of brain diseases. The major problem in accessing the central nervous system (CNS) is linked to the presence of the Blood-Brain Barrier (BBB). Objective: The present review deals with the different strategies that have been developed in order to allow the Np drug carriers to entry into the CNS parenchyma. Among these, the use of magnetic Np, Np conjugation with ligands for BBB receptors, with antibodies, and the use of surfactants have been considered.Methods: All the literature available is reviewed in order to highlight the potentiality of this drug delivery system to be used as drug carrier for the treatment of CNS pathologies.Conclusions: Polymeric Np showed to be promising carriers for the CNS drug delivery, due to their potential both in encapsulating drugs, hence protecting them from the body excretion and metabolism, and in delivering active agents across the BBB without inflicting any damage to the BBB. Different polymers have been used and different strategies have been applied; among these, the use of specific ligands, in order to render the delivery of drugs to CNS more specific, has been recently considered. At present, some clinical trials appeared for the use of these drug carriers, but none is related to the treatment of CNS diseases.

2008 - Veicolazione liposomiale del cidofovir nel trattamento del PEL (Primary Effusion Lynphoma) [Abstract in Rivista]
Barozzi, Patrizia; Riva, Giovanni; Ruozi, Barbara; Tosi, Giovanni; Belletti, Daniela; Potenza, Leonardo; Quadrelli, Chiara; Vallerini, Daniela; Zanetti, Eleonora; M., Morselli; Forghieri, Fabio; Maccaferri, Monica; A., Paolini; F., Volzone; Vandelli, Maria Angela; Forni, Flavio; Torelli, Giuseppe; Luppi, Mario
abstract

Veicolazione liposomiale del cidofovir nel trattamento del PEL (Primary Effusion Lynphoma)

2007 - Application of atomic force microscopy to characterize liposomes as drug and gene carriers [Articolo su rivista]
Ruozi, Barbara; Tosi, Giovanni; Leo, Eliana Grazia; Vandelli, Maria Angela
abstract

At present, liposomes play a significant role as drug delivery vehicles being considered very promising for gene therapeutics. The in vivo application of these systems widely dependent on their physico-chemical and technological characteristics such as the structure, shape, size distribution, surface modification and drug interaction. To describe the liposomes, different analytical techniques were used. In this paper, we reviewed the application of the atomic force microscopy (AFM), one of the most commonly applied scanning probe microscopy (SPM) techniques, in the description of liposome. The advantages and limitations of these techniques are discussed comparing the reported data with those referred to other well-know microscopical and spectroscopical techniques such as trasmission electron microscopy (TEM) and photon correlation spectroscopy (PCS). A detailed description of the application of AFM to evaluate the formation and the geometry of liposomes/DNA complexes is presented.

2007 - Dotap/Udca vesicles: novel approach in oligonucleotide delivery [Articolo su rivista]
Ruozi, Barbara; Battini, Renata; Montanari, Monica; Mucci, Adele; Tosi, Giovanni; Forni, Flavio; Vandelli, Maria Angela
abstract

The relatively hydrophilic bile acid, ursodeoxycholic acid (UDCA), was used as additive to DOTAP cationic liposomes to evaluate the effect on the cellular uptake of an oligonucleotide. Nuclear magnetic resonance studies were applied to estimate the relative amount of incorporated UDCA into the lipidic bilayers. The interaction between the new formulations and 5’fluorescein conjugated 29-mer phosphorothioate oligonucleotide (PS-ODN) was studied using gel electrophoresis experiments. Besides, DOTAP or DOTAP/UDCA vesicles (MixVes; DOTAP/UDCA 1:0.25, 1:0.5, 1:1 and 1:2 molar ratio) were complexed with PS-ODN and tested after transfections the cellular uptake and the localization of the oligo in HaCaT cell line by the use of cytofluorimetric and confocal microscopic analysis. DOTAP lipid formulated in presence of defined amount of UDCA forms more stable, flexible and active MixVes. In particular, the MixVes at 1:0.25 and 1:0.5 molar ratio increase and modify the cellular uptake of PS-ODN if compared with DOTAP liposomes 3 h after the transfection studies. Besides, the in vitro data suggest that these new formulations are not toxic.

2007 - Flow Cytometry and live confocal analysis in the evaluation of uptake and intracellular distribution of FITC-ODN into HaCaT cells [Abstract in Atti di Convegno]
Ruozi, Barbara; Tosi, Giovanni; Tombesi, Andrea; Montanari, Monica; Leo, Eliana Grazia; Restani, Cinzia; Forni, Flavio; Vandelli, Maria Angela
abstract

Flow Cytometry and live confocal analysis in the evaluation of uptake and intracellular distribution of FITC-ODN into HaCaT cells

2007 - Intact collagen and atelocollagen sponges: Characterization and ESEM observation [Articolo su rivista]
Ruozi, Barbara; Tosi, Giovanni; Leo, Eliana Grazia; B., Parma; S., Vismara; Forni, Flavio; Vandelli, Maria Angela
abstract

In this study we have investigated the chemical-physical and morphological properties of intact and atelocollagen sponges used for tissue engineering. The porous sponges were prepared by lyophilization and their physico-chemical characteristics (water binding capacity, denaturing temperature, amino group content) were investigated. Considering the importance of the “in vivo” interactions between these sponges and the tissue, our attention was addressed a) to clarify the relationships between the morphology and the amount of water absorbed and b) to evaluate the influence of pepsin-alkaline treatment on the reorganization of the atelocollagen fibres. Conventional scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) were employed to study the morphology and wetting behaviour of the intact and atelocollagen sponges. The observations by SEM indicated remarkable differences both in the structure and dimension of the pores between intact and atelocollagen sponges. At the data are related to a different water binding capacity. However, the ESEM observations, achieved by changing the relative humidity in the operative chamber, demonstrated that the water adsorbed can be removed with major difficulty from atelocollagen sponges than from intact ones

2007 - Modified PLGA Nanoparticles as drug carriers for CNS delivery [Relazione in Atti di Convegno]
Tosi, Giovanni; Costantino, Luca; Bondioli, Lucia; Ruozi, Barbara; Leo, Eliana Grazia; Rivasi, Francesco; Vergoni, Anna Valeria; Bertolini, Alfio; Tacchi, Raffaella; Forni, Flavio; Vandelli, Maria Angela
abstract

Modified PLGA Nanoparticles as drug carriers for CNS delivery

2007 - Nanoparticelle polimeriche per il direzionamento di farmaci al Sistema Nervoso Centrale [Relazione in Atti di Convegno]
Tosi, Giovanni; Costantino, Luca; Ruozi, Barbara; Bondioli, Lucia; Rivasi, Francesco; Vergoni, Anna Valeria; Bertolini, Alfio; Forni, Flavio; Vandelli, Maria Angela
abstract

Nanoparticelle polimeriche per il direzionamento di farmaci al Sistema Nervoso Centrale

2007 - Nanoparticles and Brain Targeting [Relazione in Atti di Convegno]
Tosi, Giovanni; Ruozi, Barbara; Costantino, Luca; Rivasi, Francesco; Leo, Eliana Grazia; Bertolini, Alfio; Vergoni, Anna Valeria; Forni, Flavio; Vandelli, Maria Angela
abstract

Nanoparticles and Brain Targeting

2007 - Nuove formulazioni liposomiali per la veicolazione di materiale genico in cellule cheratinocito simili [Abstract in Atti di Convegno]
Ruozi, Barbara; Montanari, Monica; Tosi, Giovanni; Mucci, Adele; Vighi, Eleonora; Battini, Renata; Leo, Eliana Grazia; Forni, Flavio; Vandelli, Maria Angela
abstract

Nuove formulazioni liposomiali per la veicolazione di materiale genico in cellule cheratinocito simili

2007 - Peptide-derivatized nanoparticles as carriers of drugs into the central nervous system [Relazione in Atti di Convegno]
Tacchi, Raffaella; Vergoni, Anna Valeria; Tosi, Giovanni; Costantino, Luca; Rivasi, Francesco; Bertolini, Alfio; Ruozi, Barbara; Forni, Flavio; Vandelli, Maria Angela
abstract

Peptide-derivatized nanoparticles as carriers of drugs into the central nervous system

2007 - Targeting the Central Nervous System: in vivo experiments with peptide-derivatized nanoparticles loaded with Loperamide and Rhodamine-123 [Articolo su rivista]
Tosi, Giovanni; Costantino, Luca; Rivasi, Francesco; Ruozi, Barbara; Leo, Eliana Grazia; Vergoni, Anna Valeria; Tacchi, Raffaella; Bertolini, Alfio; Vandelli, Maria Angela; Forni, Flavio
abstract

Polymeric nanoparticles (Np) represent one of the most innovative non-invasive approaches for the drug delivery to the central nervous system (CNS). It is known that the ability of the Np to cross the Blood Brain Barrier (BBB), thus allowing the drugs to exert their pharmacological activity in the central nervous district, is linked to their surface characteristics. Recently it was shown that the biocompatible polyester poly(D,L-lactide-co-glycolide) (PLGA) derivatized with the peptide H2N-Gly-L-Phe-D-Thr-Gly-L-Phe-L-Leu-L-Ser(O--D-Glucose)-CONH2 [g7] was an useful starting material for the preparation of Np (g7-Np); moreover, fluorescent studies showed that these Np were able to cross the BBB. In this research, g-7 Np were loaded with Loperamide in order to assess their ability as drug carriers for CNS, and with Rhodamine-123, in order to qualitatively determine their biodistribution in different brain macro-areas. A pharmacological evidence is given that g7-Np are able to cross the BBB, ensuring, for the first time, a sustained release of the embedded drug, and that these Np are able to reach all the brain areas here examined. The ability to enter the CNS appears to be linked to the sequence of the peptidic moiety present on their surface.

2006 - Applicazione delle tecniche microscopiche, spettroscopiche e calorimetriche nella caratterizzazione di nanoparticelle funzionalizzate [Abstract in Atti di Convegno]
Tosi, Giovanni; Ruozi, Barbara; Costantino, Luca; Leo, Eliana Grazia; Forni, Flavio; Vandelli, Maria Angela
abstract

Applicazione delle tecniche microscopiche, spettroscopiche e calorimetriche nella caratterizzazione di nanoparticelle funzionalizzate

2006 - Caratterizzazione chimico-fisica e studi di stabilità di sistemi lipidici per il transfer genico [Abstract in Atti di Convegno]
Ruozi, Barbara; Tosi, Giovanni; Vighi, Eleonora; Leo, Eliana Grazia; Mucci, Adele; Schenetti, Luisa; Forni, Flavio; Vandelli, Maria Angela
abstract

Caratterizzazione chimico-fisica e studi di stabilità di sistemi lipidici per il transfer genico

2006 - High density surface modified nanoparticles [Abstract in Atti di Convegno]
Costantino, Luca; Gandolfi, Francesca; Tosi, Giovanni; Vandelli, Maria Angela; Forni, Flavio
abstract

High density surface modified nanoparticles

2006 - Nanoparticelle con superficie modificata ad alta densità [Abstract in Atti di Convegno]
Tosi, Giovanni; Gandolfi, Francesca; Ruozi, Barbara; L., Bossy Nobs; R., Gurny; Rivasi, Francesco; Forni, Flavio; Vandelli, Maria Angela; Costantino, Luca
abstract

Nanoparticelle con superficie modificata ad alta densità

2006 - Nanoparticelle modificate per il drug targeting [Relazione in Atti di Convegno]
Tosi, Giovanni; Ruozi, Barbara; Costantino, Luca; Leo, Eliana Grazia; Rivasi, Francesco; M. A., Gomez; E., Pozio; Forni, Flavio; Vandelli, Maria Angela
abstract

Nanoparticelle modificate per il drug targeting

2006 - Nanoparticelle per il direzionamento cerebrale di farmaci [Articolo su rivista]
Tosi, Giovanni; Costantino, Luca; Gandolfi, Francesca; Ruozi, Barbara; Rivasi, Francesco; Vandelli, Maria Angela; Forni, Flavio
abstract

2006 - Nanoparticulate drug carriers based on hybrid poly(D,L-lactide-co-glycolide)-dendron structures [Articolo su rivista]
Costantino, Luca; Gandolfi, Francesca; L., BOSSY NOBS; Tosi, Giovanni; R., Gurny; Rivasi, Francesco; Vandelli, Maria Angela; Forni, Flavio
abstract

We describe a general method for incorporating target moieties in a well-defined arrangement into the surface of biocompatible polyester poly(D,L-lactic-co-glycolic acid) (PLGA) materials using dendrons. In this way it is possible to obtain nanoparticles (NPs) with a high degree of surface coverage. This new strategy was successfully applied to the preparation of peptide- and beta-D-glucose-covered NPs. The first application is based on the discovery of NPs made of conjugates between PLGA and short peptidic sequences able to cross the blood-brain barrier (BBB) after systemic administration. In this paper, we used a branched structure (dendron) in order to prepare a derivative of PLGA able to form, by simple nanoprecipitation, NPs with a higher degree of surface coverage than previously reported by us, characteristic that could influence the uptake by the liver and spleen. The NPs thus obtained retain the ability to cross the BBB and possess a core-shell structure, as evidenced from zeta-potential, X-ray photoelectron (ESCA) spectroscopy and elemental analyses. These results are comparable with the NPs obtained by the derivatization of preformed NPs. The same strategy, namely the use of a branched spacer (a dendron or a G1 dendrimer) inserted between one end of the PLGA chain and a derivatizing molecule, was also successfully applied to obtain beta-D-glucose-covered NPs; in this case, the surface analysis of the NPs was performed by using high resolution magic angle spinning (HRMAS) NMR spectroscopy and zeta-potential measurements.

2006 - PLA-microparticles formulated by means a thermoreversible gel able to modify protein encapsulation and release without being co-encapsulated [Articolo su rivista]
Leo, Eliana Grazia; Ruozi, Barbara; Tosi, Giovanni; Vandelli, Maria Angela
abstract

The aim of this work was to develop a novel strategy for the formulation of biodegradable PLA microspheres as delivery systems for proteins or peptides. The strategy is based on the exploitation of the gel-sol transition of the thermoreversible Pluronic F127 gel. The gel allows the formation of the particles without be co-entrapped in the matrix. The microspheres prepared using the novel technique (TG-Ms, or thermoreversible gel-method microspheres) were characterized in vitro (as concerns the size, the morphology, the protein encapsulation, the release and the protein distribution in the polymer matrix), in comparison with microspheres prepared using the classical double emulsion/solvent evaporation method (w/o/w-Ms). Two types of bovine serum albumin (BSA), with different water solubility, were used as model proteins. TG-Ms exhibited small size (7-50 m) and high protein content (8.6%, w/w) regardless of the BSA water solubility, in contrast with w/o/w-Ms, which revealed a size range of 100-130 mu m and a protein content related to the BSA water solubility. TG-Ms, in spite of their smaller size respect of the w/o/w-Ms, displayed a reduced initial burst effect and a higher rate in the second release phase that resulted in a quasi-constant profile. The release behavior of the TG-Ms may be attributable to both the localization of the protein in the particle core, as shown by the confocal laser scanning microscopy analysis on labeled-BSA loaded microspheres, and the few pores in the matrix, as shown by the scanning electron microscopy. A working hypothesis about the mechanism of the particle formation was also discussed. (c) 2006 Elsevier B.V. All. rights reserved.

2006 - Sistemi lipidici per il transfer genico: sintesi di nuove molecole per la progettazione di vettori sintetici efficaci [Relazione in Atti di Convegno]
Ruozi, Barbara; Costantino, Luca; Gandolfi, Francesca; Vighi, Eleonora; Leo, Eliana Grazia; Mucci, Adele; Tosi, Giovanni; Vandelli, Maria Angela
abstract

Sistemi lipidici per il transfer genico: sintesi di nuove molecole per la progettazione di vettori sintetici efficaci

2005 - Atomic force microscopy and photon correlation spectroscopy: Two techniques for rapid characterization of liposomes [Articolo su rivista]
Ruozi, Barbara; Tosi, Giovanni; Forni, Flavio; M., Fresta; Vandelli, Maria Angela
abstract

The direct evaluation of the heterogeneity of the particle population of nanometric drug delivery systems as liposomes is difficult to achieve owing to the dimension and the carrier characteristics. The influence of the lipidic ratio and composition on the physical stability of liposomes during their storage was investigated using atomic force microscopy (AFM) and photon correlation spectroscopy (PCs). Liposomes were made by a mixture of different lipids and obtained using distinct methods of preparation. AFM images, acquired immediately after the deposition of the sample on mica surface, clearly showed the spherical shape of the lipidie vesicles. In all the 7 months of the experiment, the average sizes of the different liposomes evaluated using the two techniques were comparable. According to PCs analysis, AFM images confirmed that almost all the diversified vesicular systems tended to form aggregates during their storage; this loss of stability was strengthened by the increase of polydispersity index value. The different behaviours observed were to ascribe to the lipidic composition more than the methods of liposome preparation. In conclusion, AFM technique owing to the relative simplicity cold be useful for the technological control of size distribution profile according to the preparative factors and moreover to the batch-to-batch reproducibility.

2005 - Caratterizzazione chimico-fisica di membrane e feltri di collagene [Abstract in Atti di Convegno]
Ruozi, Barbara; Tosi, Giovanni; B., Parma; S., Vismara; Leo, Eliana Grazia; Vandelli, Maria Angela
abstract

Caratterizzazione chimico-fisica di membrane e feltri di collagene

2005 - Conjugated poly(D,L-lactide-co-glycolide) for the preparation of in vivo detectable nanoparticles [Articolo su rivista]
Tosi, Giovanni; Rivasi, Francesco; F., Gandolfi; Costantino, Luca; Vandelli, Maria Angela; Forni, Flavio
abstract

Cellular localization of nanoparticles (Np) represents an important target in the understanding of their distribution after endovenous injection. The need of suitable devices and methodologies capable to detect Np in tissues or in cellular districts can be satisfied by Np which have to be easily recognizable by simple methods. Conjugations of poly(D,L-lactide-co-glycolide) with fluorescein and biotin allow fluorescent and immuno-histochemically active Np to be obtained. The fluorescein Np are detectable using fluorescent microscopy whereas biotin Np can be detected by optical microscopy after streptavidin-biotin-peroxidase complexation. In vivo experiments confirm the ability of these particles to be easily detected in the brain parenchyma or in the liver cell population according to the infusion pathway.

2005 - Development and characterization of biodegradable nanospheres as delivery systems of anti-ischemic adenosine derivatives [Articolo su rivista]
A., DAL PIAZ; Leo, Eliana Grazia; F., Vitali; B., Pavan; A., Scatturin; F., Bortolotti; S., Manfredini; E., Durini; Forni, Flavio; B., Brina; Vandelli, Maria Angela
abstract

We report a preliminary study concerning the encapsulation modalities in nanoparticles of the anti-ischemic drug N6-cyclopentyladenosine (CPA) and its pro-drug 5′-octanoyl-CPA (Oct-CPA). The release of these compounds and the related pro-drug stability effects in human whole blood have been tested. Moreover, the influence of the delivery systems on CPA interaction toward human adenosine A1 receptor has been analysed. The nanospheres were prepared by nanoprecipitation or double emulsion solvent evaporation method using poly(lactic acid) and recovered by gel filtration or ultracentrifugation or dialysis. Free and encapsulated Oct-CPA was incubated in fresh blood and its stability was analysed with HPLC. Quite spherical nanoparticles with mean diameters ranging between 210±50 and 390±90 nm were obtained. No encapsulation occurred when CPA was used. Satisfactory results concerning drug content (0.1–1.1% w/w) and encapsulation efficiency (6–56%) were achieved when Oct-CPA was employed. The controlled release of the pro-drug was achieved, being released within a range of 1–4 h, or very slowly, depending on nanoparticle preparations. The hydrolysis rate of Oct-CPA in human whole blood appeared stabilized in human whole blood with modalities related to the release patterns. The presence of all nanoparticle preparations did not interfere with CPA interaction at its action site.

2005 - Formulation of nanoparticles by means a technique based on pluronic gel for the entrapment of an antiischemic drug [Abstract in Atti di Convegno]
Leo, Eliana Grazia; Ruozi, Barbara; Tosi, Giovanni; Vandelli, Maria Angela; Forni, Flavio
abstract

Formulation of nanoparticles by means a technique based on pluronic gel for the entrapment of an antiischemic drug

2005 - Ketorolac tromethamine liposomes: Encapsulation and release studies [Articolo su rivista]
Ruozi, Barbara; Tosi, Giovanni; Forni, Flavio; Vandelli, Maria Angela
abstract

Liposomes loaded with ketorolac tromethamine salt were prepared by using a thin layer evaporation method. The physical properties of liposomes were studied by using atomic force microscopy (AFM) and transmission electron microscopy (TEM). The relationship between lipid composition, encapsulation efficiency, vesicle size, and the release of ketorolac tromethamine-loaded liposomes was studied. The drug content was found to be dependent on the lipidic composition used in the preparations and, in particular, vesicles containing both cationic lipids (dimethyldioctadecylammonium bromide and N-[1-(2,3-dioleoyloxy)prop],N,N,N-trimethylammonium chloride), and phosphatidylcholine had a higher entrapped efficiency than liposomes with phosphatidylcholine alone or in the presence of cholesterol. Finally, the cationic liposomes appear to be useful as carriers for ketorolac tromethamine to control its in vitro release.

2005 - Liposome evolution: novel approach in gene transfer [Abstract in Atti di Convegno]
Ruozi, Barbara; Battini, Renata; Mucci, Adele; Schenetti, Luisa; Tosi, Giovanni; Forni, Flavio; Vandelli, Maria Angela
abstract

Liposome evolution: novel approach in gene transfer

2005 - Liposome-oligonucleotides interaction for in vitro uptake by COSI and HaCaT cells [Articolo su rivista]
Ruozi, Barbara; Battini, Renata; Tosi, Giovanni; Forni, Flavio; Vandelli, Maria Angela
abstract

Liposomes are considered very promising delivery systems for antisense therapeutic approach, offering drug protection and facilitating oligonucleotide cell internalization. The present study was aimed to investigate the influence of phospholipid composition of the liposomal systems both on the encapsulation and on the oligonucleotide carrier capacity in vitro. Liposomes composed of neutral ( phosphatidylcholine, cholesterol and dioleoylphosphatidylethanolamine) and/or cationic lipids (N-(1-(2,3-dioleoyloxy) propyl)-N,N,N-trimethylammonium chloride salt, DOTAP) with different molar ratios were complexed with 50 fluorescein conjugated 29-mer phosphorothioate oligonucleotide (PS-ODN). The interaction was evaluated using atomic force microscopy (AFM), gel electrophoresis and HPLC analysis. Cytofluorimetric analysis and fluorescence microscopy were applied to evaluate the uptake and intracellular distribution of fluorescently labelled PS-ODN after transfection in two cell lines, COS I ( fibroblast cell) and HaCaT ( immortalized keratinocyte cell). The AFM studies reveal that the liposome/PS-ODN interaction leads the formation of a new irregular structure that completely hides the PS-ODN. Gel electrophoresis experiments and HPLC analysis have clearly demonstrated that also neutral liposomes are able to keep a little amount of PS-ODN but without strain to the complexation; the interaction was weak and rapidly destabilized when the complex was added to the cells. Transfection experiments performed with different incubation times show that DOTAP liposomes increase the rate of cellular uptake of PS-ODN and seem to influence its intracellular distribution in COS I cells where the oligonucleotide looks localized in nucleoli. Similar behaviour, at a lesser extent, is exhibited in HaCaT cells.

2005 - Nanoparticelle per il direzionamento cerebrale di farmaci [Relazione in Atti di Convegno]
Tosi, Giovanni; Costantino, Luca; Gandolfi, Francesca; Ruozi, Barbara; Rivasi, Francesco; Forni, Flavio; Vandelli, Maria Angela
abstract

Nanoparticelle per il direzionamento cerebrale di farmaci

2005 - Ovuli vaginali a rilascio prolungato come inquinanti in pap-test [Abstract in Atti di Convegno]
Tosi, Giovanni; Rivasi, Francesco; Ruozi, Barbara; Leo, Eliana Grazia; Vandelli, Maria Angela
abstract

Ovuli vaginali a rilascio prolungato come inquinanti in pap-test

2005 - Peptide-derivatized biodegradable nanoparticles able to cross the blood brain barrier [Abstract in Atti di Convegno]
Tosi, Giovanni; Costantino, Luca; Gandolfi, Francesca; Ruozi, Barbara; Leo, Eliana Grazia; Vandelli, Maria Angela; Forni, Flavio
abstract

Peptide-derivatized biodegradable nanoparticles able to cross the blood brain barrier

2005 - Peptide-derivatized biodegradable nanoparticles able to cross the blood-brain barrier [Articolo su rivista]
Costantino, Luca; Gandolfi, Francesca; Tosi, Giovanni; Rivasi, Francesco; Vandelli, Maria Angela; Forni, Flavio
abstract

Injectable nanoparticulate drug carriers (Np) able to cross the blood-brain barrier (131313) have important potential applications for the treatment of diseases that affect the central nervous system (CNS). With the aim to create a system able to address Np to the CNS, we synthesized conjugates between a biodegradable copolymer, poly(D,L-lactide-co-glycolide) (PLGA), and five short peptides, by means of an amidic linkage. These peptides, that are similar to synthetic opioid peptides, were synthesized in turn by means of Fmoc solid-phase peptide synthesis. The new five modified copolymers thus obtained turned out to be valuable starting material for the preparation of Np; these were made fluorescent, in order to allow their localization after their administration, by inclusion of a fluorescent probe. The Np thus prepared were characterized (morphology, size and z-potential) and were shown to possess the peptidic moieties on their surface, as evidenced by ESCA spectroscopy. Then, their ability to cross the BBB was assessed by the in vivo Rat Brain Perfusion Technique and, in one case, by means of a systemic administration (rat femoral vein injection). Fluorescent and confocal microscopy studies showed that while PLGA Np are unable to cross the BBB, for the first time these solid Np surface-modified with peptides were shown to be able to cross the BBB.

2004 - Characterization of the mechanism of interaction in ibuprofen-Eudragit RL100 (R) coevaporates [Articolo su rivista]
R., Pignatello; D., Spadaro; Vandelli, Maria Angela; Forni, Flavio; G., Puglisi
abstract

The present study is a preliminary exploration of the affinity between a carboxylic model drug, the nonsteroidal anti inflammatory agent ibuprofen (IBU) and Eudragit RL100 (RL) polymer. Due to the presence of a variable amount of quaternary ammonium groups in this matrix, physical and chemical interaction with the carboxylic drug can occur, which reinforces its scant mechanical dispersion in the polymer network and can ultimately affect its release profile in vitro and in vivo. To study these aspects, IBU was mixed at increasing weight ratios and in different chemical forms (free acid, sodium salt, and n-butyl ester), to investigate further the role of the carboxylic group in the interaction with the RL polymer. Therefore, IBU-RL solid dispersions (coevaporates) were obtained and fully characterized in the solid state through spectroscopic, calorimetric, and x-ray diffractometric analyses. The in vitro release pattern of the drug, in the different chemical states, was studied for the coevaporates, compared with drug-RL physical mixtures, along with drug adsorption profiles from aqueous solutions on the surface of the polymer granules.

2004 - In vitro evaluation of PLA nanoparticles containing a lipophilic drug in water-soluble or insoluble form [Articolo su rivista]
Leo, Eliana Grazia; Brina, Barbara; Forni, Flavio; Vandelli, Maria Angela
abstract

Cloricromene (AD6), an anti-ischemic drug, is rapidly metabolised into a stable and active metabolite (cloricromene acid, AD6-acid) poorly soluble in water and less lipophilic than cloricromene. The aim of this study was to evaluate which of the two forms has more possibility to be efficiently encapsulated in nanoparticles based on poly(D,L-lactide) and prepared using the nanoprecipitation method. Increasing the theoretical loading of AD6, an increase in drug actual loading and in the mean particle size occurred, while no formation of nanoparticles was observed when the highest theoretical loading (50 mg) was employed. Changing the pH of the aqueous phase the drug content dramatically increased. However, at a pH value of I I a more rapid hydrolysis of AD6 occurred. When AD6-acid was embedded in the nanoparticles, suitable results concerning both drug content and encapsulation efficiency were achieved. A good control in the release of AD6 from the AD6-loaded nanoparticles was observed while the liberation of AD6-acid from the AD6-acid-loaded nanoparticles was faster than the dissolution of the AD6-acid free. These results confirm that the most easy encapsulable form in nanoparticles is AD6-acid probably owing to its poor water solubility. Further studies will be carried out in order to evaluate if the increase in the liberation of AD6-acid by nanoencapsulation may have outcomes in its bioavaibility in vivo.

2004 - Liposome-oligonucleotides interaction; in vitro studies of cellular uptake [Abstract in Atti di Convegno]
Ruozi, Barbara; Battini, Renata; Forni, Flavio; Vandelli, Maria Angela
abstract

Liposome-oligonucleotides interaction; in vitro studies of cellular uptake

2004 - Microparticelle di PLGA per la somministrazione intrapleurica di Cidofovir [Abstract in Atti di Convegno]
Tosi, Giovanni; Vandelli, Maria Angela; Forni, Flavio
abstract

Microparticelle di PLGA per la somministrazione intrapleurica di Cidofovir

2004 - Microwave-treated gelatin microspheres as drug delivery system [Articolo su rivista]
Vandelli, Maria Angela; Romagnoli, Marcello; A., Monti; M., Gozzi; P., Guerra; F., Rivasi; Forni, Flavio
abstract

The crosslinking process of natural macromolecules with microwave energy should have the potentiality to overcome the problems due to the toxicity of the residuals of chemical crosslinking agents and moreover of the in vivo biodegradation products of the chemical crosslinked macromolecule. To evaluate the effective crosslinking of the gelatin forming the microspheres, the water-soluble fraction at 37 degreesC, the water absorption capability, the free amino and free carboxylic acid groups of the gelatin were determined. The structural change in the gelatin microspheres has been detected by the porosity studies. Moreover, both the in vitro biodegradability and the biocompatibility of the gelatin microspheres microwave-treated after a subcutaneous injection into female albino guinea pigs were tested. As the results suggest only the gelatin microspheres microwave-treated for 10 min at an inlet temperature of 250 degreesC could have been modified by the crosslink formation among the macromolecular chains. The gelatin microspheres treated with the microwave energy were very well biodegraded as indicated both by the in vitro enzymatic degradation studies and mainly by the histopathological examination. This latter study has also demonstrated the biocompatibility of the gelatin microspheres crosslinked with the microwave energy. In order to evaluate the feasibility of the microwave crosslinking process for pharmaceutical applications, both the drug loading and the drug release processes were evaluated using diclofenac as drug model, either as acidic form or as sodium salt. The microspheres were swollen in aqueous solution of diclofenac sodium salt, followed by a washing procedure with cool water to maintain the sodium salt into the microspheres or with pH 1.5 HCl to induce the diclofenac precipitation. To increase the amount of diclofenac acid form in the microspheres, the procedure was repeated three times washing with pH 1.5 HCl after each swelling process. Both the X-ray diffractometry and thermal analysis investigations showed a different physical state of the two drug forms in the microspheres, i.e. the amorphous state of the sodium salt and the crystalline state of the acidic form. According to the experimental results, the drug is released from gelatin microspheres according to the drug loading and the drug solubility.

2004 - Nuovi polimeri per la preparazione di nanoparticelle per il direzionamento di farmaci al cervello [Abstract in Atti di Convegno]
Costantino, Luca; Tosi, Giovanni; Gandolfi, Francesca; Vandelli, Maria Angela; Forni, Flavio
abstract

Nuovi polimeri per la preparazione di nanoparticelle per il direzionamento di farmaci al cervello

2004 - Sviluppo di nuovi sistemi liposomiali per la veicolazione di oligonucleotidi in cellule cheratinocito simili [Abstract in Atti di Convegno]
Ruozi, Barbara; Battini, Renata; Forni, Flavio; Mucci, Adele; Vandelli, Maria Angela
abstract

Sviluppo di nuovi sistemi liposomiali per la veicolazione di oligonucleotidi in cellule cheratinocito simili

2003 - Cationic liposomes for gene transfection [Articolo su rivista]
Ruozi, Barbara; Forni, Flavio; Battini, Renata; Vandelli, Maria Angela
abstract

Cationic liposomes spontaneously interact with the negatively charged DNA to form a stable complex that promotes the gene transfer to cells. The mode of formation and the size of cationic liposomes/DNA complexes were investigated using the atomic force microscopy (AFM). Also the most important physical-chemical factors involved in cationic liposome-mediated gene transfection, e.g. size and lipidic composition, were evaluated through the transfection of complexes with different liposomes/DNA molar ratio into three types of cultured cells. Cationic liposomes, composed of a neutral lipid (phosphatidilcoline), a cationic lipid dimethyldioctadecylammonium bromide (DDAB), a co-lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and a phospholipid derivative of polyethylene glycol (DSPE-mPEG) at different molar ratio, were mixed with a plasmid pCMVbeta to form liposomes/DNA complexes. We have demonstrated that the complexes were made by complicated structures in which the liposomes tend to aggregate and the DNA is surrounded by lipidic material. In vitro transfection efficiency by liposomes/plasmid pCMVbeta complexes was found to depend on the kind of lipid associated in the liposomes and the liposomes/DNA mixing ratio. The importance of associating DOPE in cationic liposomes was confirmed; this co-lipid is able to improve the ability of cationic liposomes to transfect cells but in addition, the AFM images and the EtBr fluorescence experiments have suggested that this lipid can also play an important role to facilitate the formation of stable liposomes, which efficaciously protect the DNA by nuclease digestion.

2003 - Encapsulation modalities of antiischemic adenosine derivatives in poly(lactic acid) nanoparticles [Abstract in Rivista]
DAL PIAZ, A.; F., Vitali; F., Bortolotti; A., Scatturin; S., Manfredini; F., Nalin; Leo, Eliana Grazia; B., Brina; M. A., Vandelli
abstract

Novel delivery of an antiischemic drug via nasal administration

2003 - Influence of preparation conditions on acyclovir-loaded poly-d,l-lactic acid nanospheres and effect of PEG coating on ocular drug bioavailability [Articolo su rivista]
C., Giannavola; C., Bucolo; A., Maltese; D., Paolino; Vandelli, Maria Angela; G., Puglisi; V. H. L., Lee; M., Fresta
abstract

Purpose. The evaluation of nanosphere colloidal suspensions containing acyclovir as potential ophthalmic drug delivery systems was carried out. The influence of polymer molecular weight and type and concentration of various surfactants on nanosphere properties was studied. The ocular pharmacokinetics of acyclovir- loaded nanoparticles was evaluated in vivo and compared with an aqueous suspension of the free drug. Methods. Nanospheres were made up of poly- d, l- lactic acid ( PLA). The colloidal suspension was obtained by a nanoprecipitation process. The surface properties of PLA nanospheres were changed by the incorporation of pegylated 1,2- distearoyl- 3- phosphatidylethanolamine. The mean size and zeta potential of the nanospheres were determined by light scattering analysis. The acyclovir loading capacity and release were also determined. In vivo experiments were carried out on male New Zealand rabbits. The ocular tolerability of PLA nanospheres was evaluated by a modified Draize test. The aqueous humor acyclovir levels were monitored for 6 h to determine the drug's ocular bioavailability for the various formulations. Results. A reduction of the mean size and a decrease of the absolute zeta potential of PLA nanospheres resulted from increasing the surfactant concentration. The higher the polymer molecular weight, the smaller the nanosphere mean size. PEG- coated and uncoated PLA nanospheres showed a sustained acyclovir release and were highly tolerated by the eye. Both types of PLA nanospheres were able to increase the aqueous levels of acyclovir and to improve the pharmacokinetics profile, but the efficacy of the PEG- coated nanospheres was significantly higher than that of the simple PLA ones. Conclusions. PEG- coated PLA nanospheres can be proposed as a potential ophthalmic delivery system for the treatment of ocular viral infections.

2003 - Preparation and characterization of biodegradable nanoparticles containing a lipophilic drug in water-soluble or insoluble form [Abstract in Rivista]
Leo, Eliana Grazia; Brina, Barbara; Ruozi, Barbara; Vandelli, Maria Angela; Forni, Flavio
abstract

Numerous studies have shown that cloricromene, a semi-synthetic non-anticoagulant coumarin derivative, exerts a clear protective action in several experimental models of ischaemia and shock (1). The antithrombotic and anti-ischemic effects of cloricromene are evident in the peripheral ischemia for the difficult of the drug to pass the brain-blood barrier (BBB). Recently, polymeric nanoparticles have been proposed as interesting alternative to the traditional approaches to overcome brain drug delivery obstacles (2). Among the techniques proposed in the preparation of nanoparticles from polyester polymers, such as poly (D,L lactide) (PLA) or poly (D,L lactide-co-glicolide) (PLGA), the nanoprecipitation method (3) represents an easy and reproducible technique to obtain nanoparticles below 200 nm. However, this method is basically applicable to lipophilic drugs and several attempts are developed in order to improve hydrophilic drug encapsulation. Cloricromene is rapidly metabolised in vitro or in vivo in the blood into a stable and active catabolite (Cloricromene acid, AD6-acid) through the hydrolysis of an ester bound within the molecule (Fig 1). Cloricromene (as chloride salt, AD6) is freely soluble in water even if it is very lipophilic (log P= 3.96); on the contrary the catabolite is poorly soluble in water and is less lipophilic than Cloricromene (log P = 3.12). The aim of this paper was to evaluate which of the two drug forms has more possibility to be encapsulated efficiently in the nanoparticles, according to the parameters used in the preparation.

2002 - Poly(lactic acid) microspheres for the sustained release of antiischemic agents [Articolo su rivista]
A., DAL PIAZ; A., Scatturin; B., Pavan; C., Biondi; Vandelli, Maria Angela; Forni, Flavio
abstract

We report a preliminary study evaluating the encapsulation modalities in microparticles of the antiischemic drug N(6)-cyclopentyladenosine (CPA). The effects of release systems have been evaluated on the stability in human whole blood of CPA and its affinity toward human adenosine A(1) receptors. The microspheres were prepared by an emulsion-solvent evaporation method (different CPA amounts and two stirring rates were employed) using poly(lactic acid). Free and encapsulated CPA was incubated in human blood and the drug stability was analyzed. The affinity of CPA to human A(1) receptor was also obtained in the presence and in the absence of unloaded microspheres. The microspheres obtained using 1200 rpm showed a broad size distribution and a mean diameter value of 21+/-9 microm. Using 1700 rpm the mean diameter decreased to 5+/-2 microm and a more homogeneous size distribution was obtained. The CPA release changed with the particle size and the different amounts of drug employed during the preparation of the microspheres. The degradation in human whole blood of CPA encapsulated in the microspheres was negligible, with respect to that of free CPA. Affinity values of CPA obtained in the absence and in the presence of unloaded microspheres were the same.

2002 - The application of a thermosensible gel in the formulation of BSA-loaded PLA microspheres [Abstract in Atti di Convegno]
Leo, Eliana Grazia; Ruozi, Barbara; B., Brina; Vandelli, Maria Angela
abstract

The application of a thermosensible gel in the formulation of BSA-loaded PLA microspheres

2001 - Comparison between Roesy and C-13 NMR complexation shifts in deriving the geometry of inclusion compounds: A study on the interaction between hyodeoxycholic acid and 2-hydroxypropyl-beta-cyclodextrin [Articolo su rivista]
Mucci, Adele; Schenetti, Luisa; Vandelli, Maria Angela; Ruozi, Barbara; Salvioli, Gianfranco; Forni, Flavio
abstract

The formation and geometry of the hyodeoxycholic acid (HDCA)/ 2-hydroxypropyl-beta -cyclodextrin (HP beta CD) complex in methanol-d(4) solution was determined through a rotating frame nuclear Overhauser (ROESY) experiment. The reported results confirmed those independently and previously obtained though the use of C-13 complexation shifts in the same solvent. The C-13 approach, which needs shorter experimental times and is currently used in the study of HP beta CD/bile acid systems, was then substantiated.

2001 - Evaluation of bioadhesive properties of microspheres using an "ex vivo" test [Articolo su rivista]
Leo, Eliana Grazia; Coppi, Gilberto; Guerra, Paolo; Vandelli, Maria Angela; Cameroni, Riccardo
abstract

The purpose of this work was to evaluate the bioadhesive properties of microspheres based on sodium alginate and BSA prepd. by a spray-drying technique. The microspheres were cross-linked using a two steps process with calcium chloride and various amts. of chitosan. The bioadhesiveness of these systems was evaluated using a "ex vivo" method that allowed quant. results to be drawn. It was concluded that the crosslinking step using different amts. of chitosan, even if involved the sites implicated in the bioadhesive process, did not modified the good bioadhesiveness of the calcium alginate microspheres.

2001 - Gelatin microspheres crosslinked with D,L-glyceraldehyde as a potential drug delivery system: preparation, characterisation, in vitro and in vivo studies [Articolo su rivista]
Vandelli, Maria Angela; Rivasi, Francesco; Guerra, P.; Forni, Flavio; Arletti, R.
abstract

To overcome the restriction in using crosslinked gelatin in the pharmaceutical field, D,L-glyceraldehyde (GAL), a non-toxic crosslinking agent, was proposed. Gelatin microspheres crosslinked with different concentrations of GAL (0.5, 1 or 2%, w/v) and for different time periods (1 or 24 h) were prepared. The effect of the preparation variables was evaluated analysing the extent of crosslinking, the morphological aspect, the particle size and the swelling behaviour. To evaluate the pharmaceutical properties, an antihypertensive drug, clonidine hydrochloride, was chosen as drug model and loaded into the microspheres. Either the increase of the crosslinker concentration or of the crosslinking time period decreased both the swelling and the in vitro drug release processes of the microspheres. After the subcutaneous injection, the loaded microspheres crosslinked with the lowest GAL concentration (0.5%. w/v) or for the shortest time period (1 h) showed a reduction of systolic blood pressure (SBP) similar to that recorded with a clonidine hydrochloride solution having the same drug concentration. Instead, the microspheres crosslinked for 24 h with concentrations of GAL higher than 0.5% (w/v) produced a more gradual and sustained SEP reduction and the antihypertensive effect was maintained until 52-72 h. The biocompatibility studies showed that the microspheres crosslinked with GAL are well tolerated in vivo. These results suggest the potential application of gelatin microspheres crosslinked with GAL as a suitable drug delivery system for the subcutaneous administration. (C) 2001 Elsevier Science B.V. All rights reserved.

2001 - Interazione tra liposomi cationici e DNA plasmidico: studi di transfezione genica in diverse linee cellulari [Relazione in Atti di Convegno]
Ruozi, Barbara; Battini, Renata; Forni, Flavio; Vandelli, Maria Angela
abstract

Interazione tra liposomi cationici e DNA plasmidico: studi di transfezione genica in diverse linee cellulari

2001 - Poly(lactic acid) microspheres for the sustained release of a selective A1 receptor agonist [Articolo su rivista]
A., DAL PIAZ; A., Scatturin; B., Pavan; C., Biondi; Vandelli, Maria Angela; Forni, Flavio
abstract

A study concerning the feasibility of microsphere use as sustained delivery systems for N(6)-cyclopentyladenosine (CPA) administration has been performed. The release of this drug and the related stability effects in human whole blood have been tested. Moreover, the impact of the delivery system on CPA interaction toward human adenosine A1 receptor and the related cellular responses has been analyzed. The microspheres were prepared by an emulsion-solvent evaporation method using poly(lactic acid). Free and encapsulated CPA was incubated in fresh blood and the drug stability was analyzed with HPLC. The affinity of CPA to human A1 receptor expressed by CHO cells was obtained by binding experiments. Activity was evaluated by measurements of the inhibition of forskolin-stimulated 3',5'-cyclic adenosine monophosphate (c-AMP) performing competitive binding assays. Encapsulated CPA was released within 72 h and its degradation in blood was negligible. Affinity and activity values of CPA obtained in the absence and in the presence of unloaded microspheres were the same. CPA encapsulation in microspheres allows its sustained release and its stabilization in human whole blood to be obtained. The presence of this release system does not interfere with the CPA activity at its action site.

2001 - Preformulative and formulative studies on inclusion complexes between cyclodextrins and ursodeoxycholic acid [Articolo su rivista]
Vandelli, Maria Angela; Ruozi, Barbara; Forni, Flavio; Sergi, Santo
abstract

Three different methods (evaporation, spray-drying and lyophilisation) were used to prepare the complex between ursodeoxycholic acid and cyclodextrin (beta CD or HP beta CD). A small fraction of free drug not included into the cyclodextrin cavity along with the complex was noticed only for the complexes prepared using the evaporation method. Preformulative parameters of at least three batches of each complex were evaluated in view of the industrial preparation method selected. All the methods guarantee good reproducibility, but the surface area and the density of the complex powders were chiefly affected. Given the low-density value, the spray-dried and lyophilised complexes need to be transformed into granules before tablet preparation. The formulative parameters of the tablets were also evaluated. The disintegration test of both uncoated and coated tablets prepared with the granules obtained form the different beta CD complexes was not satisfactory. In spite of the long disintegration time, the ursodeoxycholic acid dissolution rate from the tablets was similar to that observed in the case of the inclusion complex powders.

2001 - Preparation, characterisation and photosensitivity studies of solid dispersions of diflunisal and Eudragit RS100® and RL100® [Articolo su rivista]
R., Pignatello; M., Ferro; G., De Guidi; G., Salemi; Vandelli, Maria Angela; S., Guccione; M., Geppi; C., Forte; G., Puglisi
abstract

Solid dispersions of diflunisal (DIF) with Eudragit RS100 (RS) and RL100 (RL) with different drug-to-polymer ratios were prepared by a solvent method (coevaporates) and were characterised in the solid state in comparison with the corresponding physical mixtures. The work was aimed at characterising the interactions occurring between DIF and RS or RL polymers, along with their influence on the in-vitro drug-dissolution pattern. The findings suggest that the drug did not change its crystalline form within the polymer network. Drug dispersion in the polymer matrix strongly influences its dissolution rate, which appears slower and more gradual while increasing the polymer ratios. Moreover, DIF is known to be a photosensitive compound, and its photoproduct has been found to be a toxic agent. This can be evidenced by testing red blood cell membranes for their resistance to the osmotic shock induced by UVA irradiation in the presence of DIF. The presence of some DIF/RS coevaporates was shown to reduce significantly the drug photosensitization process towards cell membranes. This suggests the possibility of combining the design of a drug delivery system with a photoprotective strategy. (C) 2001 Elsevier Science B.V. All rights reserved.

2001 - Studio di stabilità nelle dimensioni delle vescicole liposomiali [Abstract in Atti di Convegno]
M., Fresta; Ruozi, Barbara; Leo, Eliana Grazia; Vandelli, Maria Angela
abstract

Studio di stabilità nelle dimensioni delle vescicole liposomiali

2001 - Sviluppo della formulazione di nanosfere polimeriche aventi dimensioni inferiori a 200 nm per la veicolazione di un farmaco anti-ischemico [Abstract in Atti di Convegno]
Leo, Eliana Grazia; Vandelli, Maria Angela; Ruozi, Barbara; Forni, Flavio
abstract

Sviluppo della formulazione di nanosfere polimeriche aventi dimensioni inferiori a 200 nm per la veicolazione di un farmaco anti-ischemico

2001 - Ursodeoxycholic acid complexation with 2-hydroxypropyl-beta-cyclodextrin increases ursodeoxycholic acid biliary excretion after single oral administration in rats [Articolo su rivista]
Ventura, Paolo; R., Panini; G., Montosi; C., Garuti; Vandelli, Maria Angela; G., Brunetti; Hd, Tauschel; Pietrangelo, Antonello; Salvioli, Gianfranco
abstract

Complexation of ursodeoxycholic acid (UDCA) with 2-hydroxypropyl-beta -cyclodextrin (HP beta CD) improves the water solubility and the dissolution rate of UDCA and may therefore increase its bioavailability. We compared the amount and the rate of biliary excretion of UDCA and biliary lipid secretion after a single oral administration of UDCA in 3 different pharmaceutical formulations [UDCA-HP beta CD (´urso-beta -cyclodextrin´), UDCA suspension and UDCA capsule] at 3 different dosages each, in 11 groups (2 control groups) of bile fistula rats. UDCA excretion increased with an increase in dose, biliary UDCA recovery and peak secretion were significantly higher after administration of UDCA-HP beta CD than after UDCA in suspension or capsule. This enhancement of biliary excretion may achieve greater UDCA enrichment in the bile acid pool than conventional pharmaceutical UDCA formulations, this giving to UDCA-HP beta CD a considerable therapeutical potential, Copyright

2000 - A New Physical Method To Crosslink The Gelatin Microspheres. [Relazione in Atti di Convegno]
Vandelli, Maria Angela; Forni, Flavio; M. T., Bernabei; R., Cameroni; Romagnoli, Marcello
abstract

New Physical Method To Crosslink The Gelatin Microspheres.

2000 - A solution and solid state study on 2-hydroxypropyl-beta-cyclodextrin complexation with hyodeoxycholic acid [Articolo su rivista]
Vandelli, Maria Angela; Ruozi, Barbara; Forni, Flavio; Mucci, Adele; Salvioli, Gianfranco; Galli, Ermanno
abstract

Hyodeoxycholic acid (HDCA) is a 6-alpha dihydroxylated natural bile acid capable of preventing gallstone formation by reducing the bile cholesterol saturation. However, any attempt to enrich the bile acid pool with HDCA have failed owing to the poor solubility of the molecule. To resolve the bioavailability problems, the complexation of HDCA into the HP beta CD cavity was studied in solution (solubility methods, C-13- and H-1-NMR spectroscopy and circular dichroism) and in the solid state (IR spectroscopy, X-ray diffractometry and thermal analysis). According to the results, the HDCA inclusion took place with 1 : 1 stoichiometry. The influence of different preparation methods of the solid complex was evaluated for its potential use in appropriate pharmaceutical formulations to improve the bioavailability of HDCA.

1999 - Biocompatibility of chemically and physically crosslinked gelatin microspheres [Abstract in Rivista]
Vandelli, Maria Angela; Ruozi, Barbara; Guerra, Paolo; Forni, Flavio
abstract

Biocompatibility of chemically and physically crosslinked gelatin microspheres

1999 - Evidence of the existence of 2:1 guest-host complexes between diclofenac and cyclodextrins in D2O solutions. A 1H and 13C NMR study on diclofenac/beta-cyclodextrin and diclofenac/2-hydroxypropyl-beta-cyclodextrin systems [Articolo su rivista]
Mucci, Adele; Schenetti, Luisa; Vandelli, Maria Angela; Ruozi, Barbara; Forni, Flavio
abstract

The interaction of diclofenac sodium salt (DCFNa) and two cyclodextrins, beta-cyclodextrin (CD) and 2-hydroxypropyl-beta-cyclodextrin (HPCD), studied in D2O solution with different NMR techniques (1H, 13C NMR, ROESY experiments, NMR titrations), shows the existence of multiple equilibria involving 1:1 and 2:1 guest–host complexes.

1999 - Microparticelle per il rilascio prolungato di melatonina per uso orale [Abstract in Atti di Convegno]
Vandelli, Maria Angela; Ruozi, Barbara; Forni, Flavio; Bernabei, Maria Teresa
abstract

Microparticelle per il rilascio prolungato di melatonina per uso orale

1999 - Microsfere di gelatina reticolata con le microonde: studio del caricamento e del rilascio del farmaco [Abstract in Atti di Convegno]
Vandelli, Maria Angela; Ruozi, Barbara; Forni, Flavio
abstract

Microsfere di gelatina reticolata con le microonde: studio del caricamento e del rilascio del farmaco

1999 - PLA microparticles for nimesulide prolonged release: effect of the preparative variables. [Articolo su rivista]
Vandelli, Maria Angela; Ruozi, Barbara; Forni, Flavio
abstract

PLA microparticles for nimesulide prolonged release: effect of the preparative variables.

1999 - Synthesis and characterisation of poly(D,L-lactic acid) idoxuridine conjugate [Articolo su rivista]
M. G., Rimoli; L., Avallone; P., de Caprariis; A., Galeone; Forni, Flavio; Vandelli, Maria Angela
abstract

A new polymeric prodrug was prepared coupling 5-iodo-2'-deoxyuridine (IDU) to poly(D,L-lactic acid) (PLA) via a succinic acid spacer. The PLA-IDU conjugate was characterised by thermal analysis, IR and H-1 and C-13 NMR spectroscopy. The IDU content (0.024 mequiv. g(-1) of PLA) was consistent with the carboxylic acid endgroup present in the polymer sample (0.025 mequiv. g(-1) of polymer). The PLA-IDU conjugate was susceptible to degradation in biological environments containing esterase, whereas IDU was not detected by chemical hydrolysis in pH 7.4 phosphate buffer. The conjugate should be used to prepare injectable microspheres and nanospheres containing IDU chemically coupled to the polymer carrier. (C) 1999 Elsevier Science B.V. All rights reserved.

1999 - The application of the Atomic Force Microscopy in the liposome evaluation [Abstract in Rivista]
Vandelli, Maria Angela; Ruozi, Barbara; Guerra, Paolo; Forni, Flavio
abstract

The application of the Atomic Force Microscopy in the liposome evaluation

1998 - 1H and 13C NMR Study on the Interaction between Diclofenac and 2-Hydroxypropyl-beta-Cyclodextrin [Abstract in Atti di Convegno]
Forni, Flavio; Mucci, Adele; Schenetti, Luisa; Vandelli, Maria Angela
abstract

The interaction bertween Diclofenac and 2-Hydroxypropyl-beta-Cyclodextrin were studied through 1H and 13C NMR spectroscopy.

1998 - Complexation of the sunscreen agent, butyl-methoxydibenzoylmethane, with hydroxypropyl-beta-cyclodextrin. [Articolo su rivista]
Scalia, S; Villani, S; Scatturin, A; Vandelli, Maria Angela; Forni, Flavio
abstract

The interaction between the sunscreen, butyl-methoxydibenzoylmethane (BM-DBM), and parent and modified alpha-, beta- or gamma-cyclodextrins was investigated in water by phase-solubility analysis. Among the available cyclodextrins, only hydroxypropyl-beta-cyclodextrin (HP-beta-CD) produced a significant increase in the aqueous solubility of BM-DBM. The complexation of the sunscreen agent with HP-beta-CD was studied by circular dichroism, differential scanning calorimetry and X-ray diffractometry. The data from the solubility and the circular dichroism studies suggested the formation of a 1:2 (sunscreen:cyclodextrin) complex. The photodegradation of BM-DBM was reduced by inclusion complexation with HP-beta-CD. Therefore the complex can be used to improve the photostability of the sunscreen agent. (C) 1998 Elsevier Science B.V. All rights reserved.

1998 - Drug release from perforated matrices containing hydroxypropylcellulose [Articolo su rivista]
Vandelli, Maria Angela; Leo, Eliana Grazia; F., Foni; Mt, Bernabei
abstract

Perforated matrices were obtained using as excipient hydroxypropylcelluloses having different viscosity degrees. Furosemide release was affected by the polymer viscosity, while it was not related to the releasing surface area. In fact, the matrix having the highest hole diameter, and consequently the lowest releasing surface area, showed the highest release rate. The application of an impermeable coating on all the surfaces, except the hole surface, restricted the releasing surface area reducing the release rate. Furosemide release from the perforated coated matrices was the same regardless of the hole diameter (surface) as the data were related to the unitary releasing surface. The comparative analysis of the drug release mechanism showed the decrease of the erosion (polymer dissolution) component and the increase of the drug diffusion component of the process as the perforated matrices were coated. These results can be justified by the restriction of the matrix swelling produced by the impermeable coating. (C) 1998 Elsevier Science B.V. All rights reserved.

1998 - Preparazione e caratterizzazione del complesso tra acido iodesossicolico e 2-idrossipropil-beta-ciclodestrina [Abstract in Atti di Convegno]
Vandelli, Maria Angela; Panini, Rossana; Mucci, Adele; Schenetti, Luisa; Ruozi, Barbara; Forni, Flavio
abstract

Preparazione e caratterizzazione del complesso tra acido iodesossicolico e 2-idrossipropil-beta-ciclodestrina

1998 - Studi preformulativi sul complesso di inclusione tra acido ursodesossicolico e beta-ciclodestrine [Abstract in Atti di Convegno]
Vandelli, Maria Angela; Ventura, Paolo; Ruozi, Barbara; Forni, Flavio; Bernabei, Maria Teresa
abstract

Studi preformulativi sul complesso di inclusione tra acido ursodesossicolico e beta-ciclodestrine

1998 - Studio di interazione Melatonina-Adenosina [Abstract in Atti di Convegno]
Vandelli, Maria Angela; Mucci, Adele; Ruozi, Barbara; Forni, Flavio; Galli, Ermanno
abstract

Studio di interazione Melatonina-Adenosina, XVI Simposio A.D.R.I.T.E.L.F.

1998 - Ursodeoxycholic acid complexation with 2-hydroxypropyl-beta-cyclodextrin increases ursodeoxycholic acid biliary excretion after single oral administration in rats [Abstract in Rivista]
Ventura, Paolo; Panini, Rossana; Montosi, Giuliana; Garuti, Cinzia; Vandelli, Maria Angela; G., Brunetti; Pietrangelo, Antonello; Salvioli, Gianfranco
abstract

Complexation of ursodeoxycholic acid (UDCA) with 2-hydroxypropyl-beta-cyclodextrin (HPbCD) (1:1 molar ratio) improves the water solubility and dissolution rate of UDCA and hence its bioavailability. We compared UDCA bile recovery (percentage of administered UDCA dose excreted in bile) after single oral (gavage) administration of UDCA in three different pharmaceutical forms in a bile fistula rat model. Male Sprague-Dawley rats were randomly assigned to 11 groups (6 rats per groups); the bile duct was cannulated after gavage to collect 4-h bile samples. Groups 1, 2 and 3 (fed 5, 10 and 50 mg/kg body weight UDCA/HPbCD complex, respectively) showed (mean values ± SD) 39.2 ± 5.9%, 25.7 ± 4.1% and 9.4 ± 3.1% UDCA bile recovery, respectively; in groups 4, 5 and 6 (fed UDCA suspension formulation containing 5, 10 and 50 mg/kg body weight, respectively) UDCA bile recovery was 33.2 ± 3.6%, 16.9 ± 4.7% and 6.3 ± 0.8%, respectively; groups 7, 8 and 9 (fed UDCA capsule formulation containing 5, 10 and 50 mg/kg body weight, respectively) showed 30.6 ± 9.0%, 21.7 ± 6.4% and 4.7 ± 1.8% UDCA recovery. Groups 10 and 11 (controls) were fed with HPbCD and saline, respectively. Results indicate a significantly (p<0.05) higher bile UDCA recovery in rats fed UDCA-HPbCD complex than in those fed UDCA suspension or UDCA capsule formulations at the same dose. In this model, oral UDCA/HPbCD complex increased UDCA biliary excretion making for greater UDCA enrichment of the bile acid pool than identical doses of common pharmaceutical formulations.

1997 - A new class of polymers for the preparation of ion-exchange delivery systems [Relazione in Atti di Convegno]
Vandelli, M. A.; Toselli, M.; Forni, F.; Bernabei, M. T.; Cameroni, R.
abstract

1997 - A slow release dosage form for the subcutaneous drug administration [Relazione in Atti di Convegno]
Vandelli, M. A.; Rivasi, F.; Guerra, P.; Scarpetta, G.; Arletti, R.; Forni, F.
abstract

1997 - Applicazione delle microonde all'essiccamento di granulati farmaceutici [Abstract in Atti di Convegno]
Vandelli, Maria Angela; Ruozi, Barbara; Sergi, Santo; Forni, Flavio
abstract

In numerosi campi industriali esiste attualmente un crescente interesse per le microonde a causa delle possibilità di risparmio energetico, nonché dell'opportunità di diversificazione delle fonti energetiche e del rispetto dellìambiente.

1997 - Doxorubicin-loaded gelatin nanoparticles stabilized by glutaraldehyde: Involvement of the drug in the cross-linking process [Articolo su rivista]
Leo, Eliana Grazia; Vandelli, Maria Angela; Cameroni, Riccardo; Forni, Flavio
abstract

The possible involvement of the primary amino group of doxorubicin (DXR) in the cross-linking process of gelatin nanoparticles stabilized by glutaraldehyde was investigated. Nanoparticles were characterized with regard to particle size, drug content, enzymatic degradation and cross-linking degree. The size of nanoparticles was around 100-200 nm and DXR was loaded with an entrapment efficiency of 42%. Upon the study of crosslinking rate, DXR-loaded nanoparticles showed a greater number of free amino groups than the unloaded ones. This should be due to a competition between the amino group of DXR and the amino groups of the gelatin chains during the cross-linking process. Hence, a binding of a drug fraction to the protein matrix via glutaraldehyde was hypothesized and confirmed by the results of a thin-layer chromatography (TLC) analysis. According to the in vitro study only a little fraction of DXR was released as free drug (8%) when the nanoparticles were put in saline solution. The addition of proteolytic enzymes disrupts the matrix structure producing the release of a further 10-15% of the drug loading which was entrapped in the nanoparticle matrix. The remaining part of the drug corresponds to DXR covalently linked to peptide residues produced by nanoparticle digestion.

1997 - Properties of tolmetin-loaded Eudragit RL100 and Eudragit RS100 microparticles prepared by different techniques [Articolo su rivista]
R., Pignatello; Vandelli, Maria Angela; P., Giunchedi; G., Puglisi
abstract

The feasibility of different techniques for the preparation of tolmetin-loaded microparticles of Eudragit RL100 and Eudragit RS100 was evaluated. The traditional emulsion-solvent evaporation method was compared to spray-drying and the quasi-emulsion-solvent diffusion technique. The results were analysed in terms of drug encapsulation and in vitro release (pH 1.2 and 6.8), as well as in terms of particle yield and size distribution. Preliminary physicochemical studies also indicated the possibility of a polymorphic change of the drug during the microsphere preparation using the first procedure.

1996 - A theoretical model for the calculation of the drug distribution profile in matrices of different shape to achieve the desired drug release kinetics [Articolo su rivista]
Forni, Flavio; Vandelli, Maria Angela; Borghi, Massimo
abstract

The drug release kinetics from a matrix is often affected by the matrix geometry. To compensate for the influence of the matrix geometry on the drug release, a nonhomogeneous drug distribution has been suggested. Keeping in mind this approach, a theoretical mathematical model to calculate the drug distribution profile according to The matrix geometry is proposed. This approach could be useful in achieving the desired drug release kinetics without varying the matrix geometry.

1996 - Application of perforated geometry for the development of a drug release system with differential speed [Relazione in Atti di Convegno]
Vandelli, M. A.; Forni, F.; Bernabei, M. T.; Cameroni, R.
abstract

1996 - Complexation of bile acids with 2-hydroxypropyl-beta-cyclodextrin: A C-13-NMR study [Articolo su rivista]
Mucci, Adele; Vandelli, Maria Angela; Salvioli, Gianfranco; L., Malmusi; Forni, Flavio; Schenetti, Luisa
abstract

The interaction of chenodeoxycholic acid (3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oic acid) 1; cholic acid (3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholan-24-oic acid) 2, deoxycholic acid (3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid) 3 and ursodeoxycholic acid (3 alpha,7 beta-dihydroxy-5 beta-cholan-24-oic acid) 4 with 2-hydroxypropyl-beta-cyclodextrin HP beta CD (partially functionalized) is studied by measuring the changes in C-13-NMR chemical shifts induced by complexation in methanol-d(4). The alkyl chain of the bile acids enters the cyclodextrin cavity. The interaction of 1 and 4 with HP beta CD is stronger, in this solvent, with respect that of 2 and 3.

1996 - In vitro evaluation of a potential colonic delivery system that releases drug after a controllable lag-time [Articolo su rivista]
Vandelli, Maria Angela; Leo, Eliana Grazia; Forni, Flavio; Mt, Bernabei
abstract

In vitro evaluation of a potential colonic delivery system that releases drug after a controllable lag-time

1996 - Multinuclear NMR spectroscopy structural properties of ofloxacin [Articolo su rivista]
Mucci, Adele; L., Malmusi; Vandelli, Maria Angela; M., Fresta; Schenetti, Luisa
abstract

An accurate 1-H, 13-C and 15-N NMR study on ofloxacin 1 is reported. The conformational properties of the two mobile regions (the benzoxazine and the piperazinyl ring) are studied and discussed. According to the spectroscopic data, the condensed benzoxazine ring shows a conformational consistent with the C-2 atom below the mean molecular plane and the 3-CH3 group in a pseudo-axial position and the piperazinyl group maintains a conformational favourable to N-1' conjugation with the aromatic ring. How multinuclear NMR spectroscopic studies may impact on biological activity and SAR investigations is discussed.

1996 - One- and two-dimensional NMR study of complexation of ursodeoxycholic acid with beta-cyclodextrin [Articolo su rivista]
Mucci, Adele; Schenetti, Luisa; Vandelli, Maria Angela; Forni, Flavio; Ventura, Paolo; Salvioli, Gianfranco
abstract

The interaction of ursodeoxycholic acid (UDCA) with beta-cyclodextrin (beta-CD) is investigated through one and two-dimensional (ROESY) NMR spectroscopy. The relative orientation of host and guest is unequivocally established: the aliphatic side chain of UDCA enters the torus of B-CD on the side of the secondary hydroxy groups.

1996 - Pharmaceutical evaluation of gelatin microspheres cross-linked with d,l-glyceraldehyde. Determinations of residuals of the preparation procedure. [Articolo su rivista]
A., Griffini; Vandelli, Maria Angela; R., Seghizzi; Mucci, Adele; G., Pifferi
abstract

Pharmaceutical evaluation of gelatin microspheres cross-linked with d,l-glyceraldehyde. Determinations of residuals of the preparation procedure.

1996 - Preparation and physico-chemical study of inclusion complexes between idebenone and modified beta-cyclodextrins [Articolo su rivista]
Puglisi, G; Ventura, Ca; Fresta, M; Vandelli, Maria Angela; Cavallaro, G; Zappala, M.
abstract

The inclusion properties of modified beta-cyclodextrins (trimethyl-beta-cyclodextrin, dimethyl-beta-cyclodextrin and hydroxypropyl-beta-cyclodextrin) towards idebenone were compared with natural beta-cyclodextrin. The inclusion complexes were prepared by different methods (coprecipitation, kneading, and freeze-drying) and characterized by differential scanning calorimetry, X-ray diffractometry, UV, CD and NMR spectroscopy. The results obtained by CD and NMR spectroscopy indicate a different orientation of idebenone in dimethyl-beta-cyclodextrin with respect to other cyclodextrins. Stability constants of the complexes were determined in water at various temperatures and consequently thermodynamic parameters were obtained. All cyclodextrins are able to significantly increase the water solubility of idebenone, particularly dimethyl-beta-cyclodextrin and hydroxypropyl-beta-cyclodextrin, as a result of complexation. Consequently, they enhance the dissolution rate of the complexed drug compared to the free drug.

1996 - THE INTERACTION OF BILIAR ACIDS WITH 2-HYDROXYPROPYL-CYCLODEXTRIN IN SOLUTION AND IN THE SOLID STATE [Articolo su rivista]
Mucci, Adele; Schenetti, Luisa; Salvioli, Gianfranco; Ventura, Paolo; Vandelli, Maria Angela; Forni, Flavio
abstract

The interaction of two biliar acids (chenodeoxycholic acid and cholic acid) with 2-hydroxypropyl-beta-cyclodextrin (HP beta CD) in solution and in the solid state was studied using different techniques. The formation of an inclusion complex with a 1 : 1 stoichiometry was suggested by the phase solubility studies. Both differential scanning calorimetry and X-ray diffractometry exhibited the amorphous state of the complex. The inclusion of both biliar acids into the HP beta CD cavity was confirmed by the C-13-NMR studies. Cholic acid showed a weaker affinity with respect to chenodeoxycholic acid probably owing to the presence of a hydroxyl group on C(12) (12 alpha) close to the complexation site.

1996 - The influence of 2-hydroxypropyl-beta-cyclodextrin on the haemolysis induced by bile acids [Articolo su rivista]
R., Panini; Vandelli, Maria Angela; Leo, Eliana Grazia; Salvioli, Gianfranco; R., Cameroni
abstract

Cyclodextrins improve the water-solubility of drugs and can mask their haemolytic effect in parenteral use. Because the mechanism by which bile acids induce haemolysis is poorly understood, it has been investigated in the presence of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD). The haemolytic effect of 1.8 mM solutions of cholic acid, chenodeoxycholic acid (CDCA), deoxycholic acid and ursodeoxycholic acid (UDCA) in isotonic buffer at pH 7.4 was investigated at 37 degrees C in the presence of HP-beta-CyD at concentrations from 0.18 to 32 mM. No haemolytic effect was evident for cholic acid and UDCA. The haemolytic effect of the other bile acids was reduced by addition of HP-beta-CyD and was prevented at a molar ratio of 1:1 owing to complex formation. An HP-beta-CyD:bile acid molar ratio greater than 5:1 had a different effect on the erythrocyte membrane, irrespective of the identity of the bile acid; the effect was in accordance with the complexion affinities. In the absence of HP-beta-CyD, the haemolytic effect of CDCA and deoxycholic acid appeared related to their capacity to form a surface monolayer and to solubilize the components of the erythrocyte membrane. The haemolytic effect observed after complexation of the bile acids appeared to be solely the effect of HP-beta-CyD, which was able to form a reversible inclusion complex with lipophilic components of the erythrocyte membranes at concentrations higher than 12 mM.

1995 - 2-HYDROXYPROPYL-BETA-CYCLODEXTRIN COMPLEXATION WITH URSODEOXYCHOLIC ACID [Articolo su rivista]
Vandelli, Maria Angela; Salvioli, Gianfranco; Mucci, Adele; Panini, R; Malmusi, L; Forni, Flavio
abstract

The complexation in aqueous medium and in the solid phase of ursodeoxycholic acid (UDCA) with a highly soluble cyclodextrin, 2-hydroxypropyl-beta-cyclodextrin, was studied by means of solubility methods, IR and C-13-NMR spectroscopy, X-ray diffractometry and thermal analysis. UDCA inclusion took place with 1:1 stoichiometry. C-13-NMR analysis suggested that the side chain was introduced into the cyclodextrin cavity. The UDCA/cyclodextrin complex showed better dissolution properties than plain drug crystals. Therefore, the complex may be used to improve the delivery and bioavailability of ursodeoxycholic acid.

1995 - 2-Hydroxypropyl-β-cyclodextrin in the protection of haemolysis induced by bile acid [Relazione in Atti di Convegno]
Vandelli, M. A.; Panini, R.; Salvioli, G.; Bernabei, M. T.; Cameroni, R.
abstract

1995 - A DELIVERY SYSTEM FOR THE CONCURRENT ADMINISTRATION OF HYDROCHLOROTHIAZIDE AND CAPTOPRIL [Articolo su rivista]
Vandelli, Maria Angela; Forni, Flavio; Bernabei, Mt
abstract

A delivery system for the concurrent administration of drugs was prepared inserting a conventional hydrochlorothiazide tablet in the hole of a perforated matrix of captopril coated on all the surfaces except the hole surface. The influence of both the hole diameter and the drug/polymer ratio on the release process was evaluated. According to the system design, hydrochlorothiazide was promptly liberated from the tablet inserted in the hole, whereas captopril was gradually released following a linear release profile. No significant differences were pointed out between the release behavior of both drugs in de-ionized water and in simulated gastrointestinal fluids.

1995 - A contribution on the complexation of ursodeoxycholic acid with β-cyclodextrin [Relazione in Atti di Convegno]
Mucci, A.; Vandelli, M. A.; Salvioli, G.; Schenetti, L.; Cameroni, R.
abstract

1995 - A contribution to the complexation of ursodeoxycholic acid with beta-cyclodextrin [Abstract in Atti di Convegno]
Mucci, Adele; Vandelli, Maria Angela; Salvioli, Gianfranco; Schenetti, Luisa; Cameroni, Riccardo
abstract

A contribution to the complexation of ursodeoxycholic acid with beta-cyclodextrin is reported

1995 - A hydroxypropylcellulose (HPC) system for the immediate and controlled release of diclofenac sodium [Articolo su rivista]
Vandelli, Maria Angela; Leo, Eliana Grazia; Forni, Flavio
abstract

Immediate and controlled release systems were prepared fitting a fast dissolution tablet in the hole of perforated matrices coated on all the surface excepting the hole. The release from the systems of a non steroidalanti-inflammatory drug, diclofenac sodium, was investigated both in water and in gastro-intestinal simulated flluids. A constant drug release was achieved after the complete dissolution of the tablet fitted in the hole. The dissolution of the initial dose was affected by the pH of the simulated gastric fluid. However, as the comparison with a sustained commercial product showed, a higher amount of drug was available from the system in thefirst 150 min.

1995 - A new crosslinked gelatin in the microsphere preparation [Relazione in Atti di Convegno]
Vandelli, M. A.; Forni, F.; Cameroni, R.; Bernabei, M. T.
abstract

1995 - Bead coating process via an excess of crosslinking agent [Articolo su rivista]
Iannuccelli, Valentina; Coppi, Gilberto; Vandelli, Maria Angela; Leo, Eliana Grazia; Bernabei, Maria Teresa
abstract

Coated beads were prepared by soaking in sodium alginate solutions spherical matrices (beads) of carboxymethylcellulose crosslinked with aluminum chloride (AlCl3) and loaded with ambroxol hydrochloride as a model drug. The residual amount of the crosslinker induced an interfacial crosslinking reaction of the sodium alginate. Therefore, an insoluble, smooth and uniform in thickness coat was formed around the beads. As the coating time increased, the coat thickness increased untill AlCl3 was present inside the beads. The rate of drug release from the coated beads was slower than that from the uncoated beads and decreased with the increase in coating time. Moreover, a constant rate phase, subsequent a burst period for the samples obtained with the highest coating times, was achieved. The dynamic swelling analysis allowed to exclude the influence of the polymer relaxation on the release process which appeared to be controlled by the alginate coat.

1995 - Complexation of ursodeoxycholic acid with beta-cyclodextrin: a ROESY study [Abstract in Atti di Convegno]
Mucci, Adele; Vandelli, Maria Angela; Schenetti, Luisa
abstract

A ROESY study on the inclusion compound formed by beta-cyclodextrin and ursodeoxycholic acid is reported.

1995 - Evaluation of in vitro absorption capacity of glyceraldehyde cross-linked gelatin microspheres [Articolo su rivista]
Vandelli, M. A.; Bulgarelli, E.; Forni, F.; Seghizzi, R.; Pifferi, G.
abstract

The absorption properties of glyceraldehyde cross-linked gelatin microspheres was compared in vitro with those of dextranomer or cadexomeriodine microspheres, which are used to draw up exudates and other particles away from wounds. The volume of the liquid absorbed was evaluated using both the microscopic observation of the particle swelling and the Enslin apparatus. Both the methods showed the highest absorption capacity of the glyceraldehyde cross-linked gelatin microspheres, which can be therefore proposed as a mechanical agent for cleansing the exudating wounds and ulcers.

1995 - IMPROVEMENT OF URSODEOXYCHOLIC ACID BIOAVAILABILITY BY 2-HYDROXYPROPYL-BETA-CYCLODEXTRIN COMPLEXATION IN HEALTHY-VOLUNTEERS [Articolo su rivista]
Panini, R; Vandelli, Maria Angela; Forni, Flavio; Pradelli, Jm; Salvioli, Gianfranco
abstract

Tablets containing the inclusion complex of ursodeoxycholic acid (UDCA) with 2-hydroxypropyl-beta-cyclodextrin were prepared by direct compression. Plasma concentrations of UDCA were determined in six healthy volunteers after oral administration of tablets containing the inclusion complex or UDCA alone (Ursacol). Following the administration of the complex tablets, the mean area under the plasma concentration curve (AUC) and the maximum UDCA plasma concentration (C-max) were significantly higher than those obtained after the administration of the commercial ones. Moreover, the time of maximum plasma concentration (t(max)) appeared at a shorter time. These results may be explained by the increase of the UDCA dissolution rate via complex formation.

1995 - METHOTREXATE INTERACTION WITH A LIPID-MEMBRANE MODEL OF DPPC [Articolo su rivista]
Puglisi, G; Fresta, M; Ventura, C; Mazzone, G; Vandelli, Maria Angela
abstract

Dipalmitoylphosphatidylcholine (DPPC) liposomes were employed as membrane models for the investigation of the interaction occurring between methotrexate (MTX) and bilayer lipid matrix. Liposomes were obtained by hydrating a lipid film with 50 mM Tris buffer (pH 7.4). The differential scanning calorimetry (DSC) evaluation of the thermotropic parameters associated with the phase transitions of DPPC liposomes gave useful information about the kind of drug-membrane interaction. The results showed an electrostatic interaction taking place with the negatively charged molecules of MTX and the phosphorylcholine head groups, constituting the outer part of DPPC bilayers. No interaction with the hydrophobic phospholipid bilayer domains was detected, revealing a poor capability of MTX to cross through lipid membranes to reach the interior compartment of a lipid bounded structure. These findings correlate well with in vitro biological experiments on MTX cell susceptibility.

1995 - The prescription of modified release oral dosage forms [Articolo su rivista]
Vandelli, M. A.; Bernabei, M. T.; De Rosa, M.; Covezzoli, A.; Bianchi, G.
abstract

The development of modified release oral dosage forms (retard dosage forms) is one of the main goals of pharmaceutical industry for drugs having a short half-life. The rationale of these dosage forms would be the reduction of interdose fluctuations throughout 24 hours owing to the decreased number of daily administrations. Consequently, the incidence of side effects is reduced and the patient compliance is improved. The drug prescription both in conventional and in retard dosage forms has been analysed in order to evaluate the impact of modified release dosage forms over the period from January 1990 to July 1993. As the analysis of the prescriptions shows, the consumption of the retard dosage forms is regularly increased. Usually, the preference toward retard dosage forms is different according to the drug and does not appear affected by the cost of the therapy.

1994 - AN INTERPRETATIVE ANALYSIS OF THE EFFECT OF THE SURFACTANTS USED FOR THE PREPARATION OF POLYALKYLCYANOACRYLATE NANOPARTICLES ON THE RELEASE PROCESS [Articolo su rivista]
Vandelli, Maria Angela; Fresta, M; Puglisi, G; Forni, Flavio
abstract

The release of fluorescein from polyethylcyanoacrylate (PECA) or polyisobutyl-cyanoacrylate (PICA) nanoparticles was affected by the surfactants used for the preparation. The different surfactants also modified the molecular weight, size and loading of the nanoparticles. However, these factors were not be responsible for the different release. According to the release profiles and the Baker-Lonsdale model, a portion of fluorescein was concentrated near the nanoparticle surface. Thus, a non-homogeneous distribution of the fluorescent probe inside the nanoparticles was hypothesized. This distribution could reflect the fluorescein position inside the micella during the polymerization stage, or could be reached during the washing stage as the consequence of a different effect of the surfactants on the porosity of the nanoparticle structure.

1993 - ANALYSIS OF RELEASE DATA IN THE EVALUATION OF THE PHYSICAL STATE OF PROGESTERONE IN MATRIX SYSTEMS [Articolo su rivista]
Vandelli, Maria Angela; Forni, Flavio; Cameroni, R.
abstract

Ethylene-vinyl acetate microspheres were prepared by an emulsion solvent-evaporation method as sustained delivery carriers of progesterone. Physical state of the drug in the microspheres was affected by drug loading level. As thermal analysis and mathematical release models showed, low payloads of the matrix supported the molecularly dispersed drug. Crystalline drug appeared only as the drug loading level increased. The release process of the crystalline drug produced a porosity increase in the polymeric system; thus, the drug could diffuse through pores and channels. Hence, the porosity of the matrix structure was affected by the payload. This hypothesis could justify the increasing goodness-of-fit of the release data to the square-root model as the drug loading level of the microspheres increased

1993 - Cilindric matrix with a central hole [Articolo su rivista]
Vandelli, M. A.; Forni, F.; Coppi, G.; Cameroni, R.
abstract

1993 - Effect of the loading method on the drug release from cross-linked carboxymethylcellulose beads [Articolo su rivista]
Iannuccelli, Valentina; Forni, Flavio; Vandelli, Maria Angela; Bernabei, Mt
abstract

Spherical matrices (beads) of cross-linked carboxymethylcellulose were prepared by the in-liquid curing coating process using AlCl3 as curing agent (cross-linker). The loading process was carried out either by adding the drug to the polymer solutions before the extrusion process or by swelling the cross-linked beads in a drug solution. The energy dispersive X-ray (EDS) analysis showed a homogeneous distribution of both the drug concentration and cross-linking density in the network regardless to the loading procedure. When the loading process was carried out before the extrusion process, a residual amount of AICl3 remained in the beads. The effects of the residual amount of AlCl3 on the matrix swelling and drug release processes were analyzed.

1993 - Release behaviour of polyethyl- and polyisobutylcyanoacrylate nanoparticles as a function of surfactant used in the preparation procedure [Relazione in Atti di Convegno]
Vandelli, M. A.; Fresta, M.; Puglisi, G.; Forni, F.
abstract

1993 - SELECTIVE COATING OF CYLINDRICAL MATRICES WITH A CENTRAL HOLE .1. AN INTERPRETATION OF THE SWELLING PROCESS [Articolo su rivista]
Vandelli, Maria Angela; Cameroni, R.
abstract

Cylindrical matrices were prepared by compression either of polyvinyl alcohol 100000 or mixtures of the excipient and a drug (sodium salicylate or theophylline). To modify the cylindrical shape, a hole was bored in the centre of the flat surface through both sides of the matrices. Different swellable systems were obtained applying an impermeable coating to one, two or three surfaces of the perforated matrices. The swelling of the perforated matrices was modified according to the number and the position of the coated surfaces (selective coating) and the loaded drug. Pseudo-zero order kinetics were obtained when the interior hole was the only uncoated surface.

1993 - Selective coating of cylindrical matrices with a central hole. II. An interpretation of the release process [Articolo su rivista]
Vandelli, Maria Angela; Coppi, Gilberto; R., Cameroni
abstract

A swellable perforated matrix was prepared and coated on one or more of its surfaces. Two drugs having different water solubility were chosen to load the matrix. To clarify the effect of the matrix swelling on the release process, the release rate was analysed according to the swelling area, i.e., the surface area from which the drugs could be released. According to the experimental release data, the drug solubility usually predominated at the beginning and the matrix swelling at the end of the release process. When the interior hole was the only uncoated surface, pseudo-zero order kinetics were obtained when the drug solubility allows the control of the drug release by the matrix swelling.

1992 - A coating process of spherical partticles via an excess of cross-linking agent [Abstract in Atti di Convegno]
Iannuccelli, Valentina; Vandelli, Maria Angela; Cameroni, Riccardo
abstract

A membrane-controlled drug delivery system has been developed by coating spherical crosslinked carboxymethylcellulose particles. The excess of crosslinking agent insed the core activated an interfacial crosslinking process with sodium alginate providing an insoluble wall around the particles.

1992 - Influence of drug loading level on drug release and dynamic swelling of crosslinked gelatin microspheres [Articolo su rivista]
Forni, Flavio; Vandelli, Maria Angela; R., Cameroni
abstract

The effect of drug loading level both on dynamic swelling and drug releae was evaluated using crosslinked gelatin microspheres. Owing to water penetration the microsphere diameter went first to a maximum value, wich was not affected by the payload; the diameter gradually approached to an equilibrium swollen value, which was affected by drug loading level. Water absorption increases and drug diffusion decreases the microsphere diameter. Obviolusly, the diameter variation depends on the factor (water absorption and drug diffusion) predominating in the process. As the payload affected only the equilibrium swollen level it is reasonable to hypothesize that drug loading level has a greater effect on drug diffusion than on polymer relaxation. This rationale could explain the increase of the diffusion component of the drug release process as the payload increased.

1992 - Matrici cilindriche con foro centrale: effetto della modificazione della superficie sul rigonfiamento e sul rilascio [Abstract in Atti di Convegno]
Vandelli, Maria Angela; Forni, Flavio; Coppi, Gilberto; Cameroni, Riccardo
abstract

Matrici cilindriche di polivinilalcool 100.000 contenenti sodio salicilato sono state preparate per compressione diretta e modificate mediante la formazione di un foro centrale. Si è così ottenuto un sistema matriciale le cui superfici di rilascio sono rappresentate dalle due basi di ugual area e da due superfici laterale, di cui una interna di area inferiore rispetto a quella esterna. Il rilascio del farmaco sembra influenzato dalla differente distribuzione dell'area effettiva tra le superfici di base e quelle laterali.

1992 - The control of the microcapsule wall by use of the Energy Dispersive X-ray Analysis [Articolo su rivista]
Coppi, Gilberto; Vandelli, Maria Angela; Forni, Flavio; Bernabei, Maria Teresa
abstract

The energy dispersive X-ray analytical procedure (EDS) was applied to the control of the microcapsule coating. Ethylcellulose (EC) was the coating material. The microcapsule core was either sulfadiazine or salbutamol sulfate. As the sulfur atom is not contained in the ethylcellulose molecule but only in the drug molecules, clearly the EDS emission of sulfur can be merely considered the evidence of the microcapsule core. The results of the analysis show a decrease in the intensity of the EDS emission from the microcapsule core in respect to that from both the uncoated drug crystals and the physical mixtures of drug and EC. Furthermore, the emission intensity decreases as the wall thickness increases. The EDS analysis could be therefore useful for the technological control of the actual drug microencapsulation and the batch-to-batch reproductibility of the wall thickness.

1991 - Drug release from spray-dried and spray-embedded microparticles of diltiazem hydrochloride [Articolo su rivista]
Forni, Flavio; Coppi, Gilberto; Vandelli, Maria Angela; R., Cameroni
abstract

Spray-dried and spray-embedded ethylcellulose microparticles were prepared using a spray-drying technique. The structure of the microparticles (microcapsules or microspheres) changed according to whether the drug (diltiazem hydrochloride) was dispersed or dissolved in the ethylcellulose solution to be spray-dried. The drug release rate from the microparticles was not affected by the physical state of the drug (amorphous or crystalline state), but by the microparticle structure.

1991 - Possibili applicazioni dell'analisi EDS in tecnologia farmaceutica [Abstract in Atti di Convegno]
Coppi, Gilberto; Iannuccelli, Valentina; Vandelli, Maria Angela
abstract

L'analisi EDS (Energy Dispersive X-Ray Analysis) è una tecnica poco utilizzata in campo farmaceutico, nonostante la sua versatilità. Vengono illustrate alcune applicazioni in campo tecnologico-farmaceutico, particolarmente alla analisi della distribuzione del principio attivo in una matrice, al controllo dei materiali di confezionamento in vetro, alla valutazione del rivestimento in sistemi ibridi quasi-reservoir e in microparticelle.

1991 - The EDS analysis as a tool for the coating evaluation of microcapsules [Abstract in Atti di Convegno]
Coppi, Gilberto; Vandelli, Maria Angela; Forni, Flavio; Cameroni, Riccardo
abstract

This work aimed to evaluate the use of EDS analysis in the control of the coating of microcapsules prepared by coacervation. Ethylcellulose was the coating material and sulfadiazine was the capsule core. As the sulfur atom is not contained in the ethylcellulose molecule but only in the drug molecule, clearly the EDS emission of sulfur can be merely considered the evidence of the microcapsule core. The results of the analysis show a decrease in the intensity of the EDS emission from the microcapsule core respect to the emission intensity from the uncoated drug crystals. Furthermore, the emission intensity decrease as the wall thickness increases.The EDS analysis could be therefore useful for the technological control of the actual drug microencapsulation and the batch to batch reproductibility of the wall thickness.

1991 - The concentration of the cross-linking agent as a tool for the control of release and swelling properties of gelatin microspheres [Articolo su rivista]
Vandelli, Maria Angela; Forni, Flavio; Iannuccelli, Valentina; Cameroni, Riccardo
abstract

The effect of the concentration of a cross-linking agent (gelatin hardener) on gelatin microspheres was evaluated. A concentration increase of the gelatin hardener (formaldehyde) produced a decrease of drug loading, swelling degree and drug release rate. The cross-linking agent concentration affected also the kinetics of water absorption and drug release. This paper suggests a decreasing significance of the diffusion-type mechanism of drug release as the concentration of the cross-linking agent increased.

1991 - The effect of the cross-linking time period upon the drug release and the dynamic swelling of gelatin microspheres [Articolo su rivista]
Vandelli, Maria Angela; Forni, Flavio; Coppi, Gilberto; R., Cameroni
abstract

The cross-linking time period affected both the swelling and release processes of cross-linked gelatin microspheres. In the dynamic swelling procedure, the combination of both phenomena of microparticle swelling and drug diffusion produced at first the increase and then the decrease of the diameter of a loaded microsphere. The increase of the cross-linking time period produced the shift of the penetrant transport from anomalous to super-case II kinetics. This behaviour could justify the decrease of the diffusion component of the drug release as the cross-linking time period increased.

1990 - An interpretation of the diffusion-type mechanism of drug release from microcapsules [Articolo su rivista]
Forni, Flavio; Coppi, Gilberto; Vandelli, Maria Angela; Mt, Bernabei
abstract

Salbutamol sulfate microcapsules were prepared by a spray-drying technique. The release data were calculated approximately via standard equations (square root and first-order relationships), but unequivocal evidence in support of the cumulative release mechanism was not obtained. Even more stringent mathematical or statistical (Durbin-Watson analysis) methods did not allow any distinction to be made between the two release mechanisms. In contrast, on application of the simple power law expression Mt/M∞ = ktn proposed by Peppas the value of the kinetic exponent of release (about 0.4) indicated a diffusion-type mechanism of the release process relative to the microcapsule shape. Therefore, it suggested here that this power law expression can be applied in order to confirm the actual diffusion-type mechanism of drug release from microcapsules.

1990 - Riscaldamento di un farmaco allo stato fuso: effetto sulla cristallizzazione delle sue forme polimorfe [Abstract in Atti di Convegno]
Forni, Flavio; Iannuccelli, Valentina; Coppi, Gilberto; Vandelli, Maria Angela
abstract

Nelle curve DSC, il punto di fusione della Forma A del fenilbutazone era influenzata velocità di riscaldamento (primo ciclo di riscaldamento). Le curve DSC del fenilbutazone cristallizzato fuso (secondo ciclo di riscaldamento) hanno mostrato i picchi endotermici della Forma C e della Forma A. Le condizioni del primo ciclo di riscaldamento hanno influenzato il valore del rapporto delle aree dei picchi delle due forme polimorfe. Il livello di energia che il fuso raggiunge per cristallizzare come forma metastabile C sarebbe maggiore di quello per cristallizzare nella forma stabile A.

1989 - Distribution of drugs in polymers loaded by swelling [Articolo su rivista]
Forni, Flavio; Coppi, Gilberto; Iannuccelli, Valentina; Vandelli, Maria Angela; Bernabei, Maria Teresa
abstract

Ethylene vinyl acetate pellets were loaded by swelling with chloroformic solutions of tolbutamide. The energy dispersive X-ray analysis showed different concentrations of the tolbutamide sulfur in the pellet sections according to the loading time. The concentration peaks might depend on a drug sieving process as the solution flow reaches a less swollen inner area. Another possible explanation of these concentration profiles is that they could be a result of thesolvent evaporation process. The concentration distribution of the drug becomes homogenous only after the complete swelling of all the polymer.

1989 - Microspheres de gelatine, etudes des variables de preparation et de la liberation de principe actif [Abstract in Atti di Convegno]
Forni, Flavio; Iannuccelli, Valentina; Vandelli, Maria Angela; Bernabei, Maria Teresa; Cameroni, Riccardo
abstract

On a aussi étudié les conditions de la methode de preparation influencantes le charhement du principe actif et les procedes de gonflement et de liberation.

1988 - Solid state transitions and CAP availability in surface solid dispersions of chloramphenicol stearate polymorphs [Articolo su rivista]
Forni, Flavio; Coppi, Gilberto; Iannuccelli, Valentina; Vandelli, Maria Angela; Bernabei, Maria Teresa
abstract

By grinding, chloramphenicol stearate Form III turns in Form I: then this form tends to become amorfous. So the grinding of both forms produces a "physically activated" form (FI*) with low crystallinity degree and high CAP availability.In the presence of microcrystalline cellulose, the transition from FIII to FI is accelerated, the amorphisation time does not change and the CAP availability increase.Therefore the results shown that CAP availability may be a function of the cristallinity degree and not of the polymorphic form of the ester.

1988 - The grinding of the polymorphic forms of chloramphenicol stearic ester in the presence of colloidal silica [Articolo su rivista]
Forni, Flavio; Coppi, Gilberto; Iannuccelli, Valentina; Vandelli, Maria Angela; Cameroni, Riccardo
abstract

The chloramphenicol stearate in the polymorphic Forms A and B was ground in the presence of colloidal silica. The grinding process turned Form A into Form B, and decreased the crystallinity degree of Form B. These conversion and amorphisation processes were related both to the ester/colloidal silica ratio and to the grinding time. After grinding in the presence of colloidal silica, the chloramphenicol stearate showed an in vitro enzymatic hydrolysis rate value higher than those of a commercial sample (Form B) and of Form B in the physical mixtures.

1987 - Montmorillonite as a drug carrier: surface deposition of papaverine on the papaverine-veegum complex [Articolo su rivista]
Forni, Flavio; Iannuccelli, Valentina; Coppi, Gilberto; Vandelli, Maria Angela; Cameroni, Riccardo
abstract

The surface deposition of papaverine on the previously emphasized papaverine-Veegum complex has been prepared. In this way, a simple therapeutic system as been obtained. In this system, the drug lays both in the interlayer, as in the complex, and on the carrier surface. Papaverine was released according to a zero order kinetics after the release of an initial rapid dose (priming dose).The in vitro results obtained with the Sartorius Simulator according to Stricker suggested both an absorption according to a zero order kinetics and the acievement in a short time of constant papaverine concentrations in the gastro-intestinal tract.

1987 - Study of solid phenylbutazone dispersions on colloidal silica [Articolo su rivista]
Forni, Flavio; Iannuccelli, Valentina; Vandelli, Maria Angela; Cameroni, Riccardo
abstract

Phenylbutazone was loaded on colloidal silicon dioxide in various proportions either by organic solvent evaporation and by grinding. Besides reducing the drug crystallinity, these preparation methods turned completely or partly the form IV of the drug into form III.

1985 - Montmorillonite come "carrier" di farmaci: studio dell'interazione con la papaverina [Articolo su rivista]
Forni, Flavio; Iannuccelli, Valentina; Coppi, Gilberto; Vandelli, Maria Angela; Cameroni, Riccardo
abstract

Allo scopo di verificare la possibilità d'impiego di "carrier" naturali nel rilascio controllato di farmaci, è stato studiato il "caricamento" della montmorillonite (Veegum) con papaverina cloridrato in funzione del pH e della concentrazione del principio attivo.L'interazione farmaco-argilla si realizza attraverso un processo di scambio ionico fino alla capacità totale di scambio della montmorillonite, mentre solo a valori basici di pH possono essere trattenute ulteriori quantità di papaverina.La diffrattometria dei raggi X ha permesso di localizzare il sito di interazione del farmaco con il Veegum nell'interstrato tra i pacchetti TOT della montmorillonite dove, come dimostrato dalla spettrofotometria I.R. e dall'analisi termica differenziale, la papaverina è presente come base.Sono stati infine discussi i meccanismi che ai vari pH considerati portano alla formazione del complesso papaverina-Veegum del quale è stato anche ipotizzato un modello strutturale.

Università degli studi di Modena e Reggio Emilia

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