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ILARIA OTTONELLI

Ricercatore t.d. art. 24 c. 3 lett. A
Dipartimento Scienze della Vita sede ex Scienze Farmaceutiche Via Campi 103

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Pubblicazioni

2024 - C-end rule peptide-guided niosomes for prostate cancer cell targeting [Articolo su rivista]
D’Avanzo, Nicola; Sidorenko, Valeria; Simón-Gracia, Lorena; Rocchi, Antonella; Ottonelli, Ilaria; Ruozi, Barbara; Longo, Francesca; Celia, Christian; Teesalu, Tambet
abstract

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 - 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.

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 - Hyaluronic Acid Scaffolds for Loco-Regional Therapy in Nervous System Related Disorders [Articolo su rivista]
Djoudi, A.; Molina-Pena, R.; Ferreira, N.; Ottonelli, I.; Tosi, G.; Garcion, E.; Boury, F.
abstract

Hyaluronic acid (HA) is a Glycosaminoglycan made of disaccharide units containing N-acetyl-D-glucosamine and glucuronic acid. Its molecular mass can reach 10 MDa and its physiological properties depend on its polymeric property, polyelectrolyte feature and viscous nature. HA is a ubiquitous compound found in almost all biological tissues and fluids. So far, HA grades are produced by biotechnology processes, while in the human organism it is a major component of the extracellular matrix (ECM) in brain tissue, synovial fluid, vitreous humor, cartilage and skin. Indeed, HA is capable of forming hydrogels, polymer crosslinked networks that are very hygroscopic. Based on these considerations, we propose an overview of HA-based scaffolds developed for brain cancer treatment, central and peripheral nervous systems, discuss their relevance and identify the most successful developed systems.

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 - 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 - 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 - Autism-associated SHANK3 mutations impair maturation of neuromuscular junctions and striated muscles [Articolo su rivista]
Lutz, A. -K.; Pfaender, S.; Incearap, B.; Ioannidis, V.; Ottonelli, I.; Fohr, K. J.; Cammerer, J.; Zoller, M.; Higelin, J.; Giona, F.; Stetter, M.; Stoecker, N.; Alami, N. O.; Schon, M.; Orth, M.; Liebau, S.; Barbi, G.; Grabrucker, A. M.; Delorme, R.; Fauler, M.; Mayer, B.; Jesse, S.; Roselli, F.; Ludolph, A. C.; Bourgeron, T.; Verpelli, C.; Demestre, M.; Boeckers, T. M.
abstract

Heterozygous mutations of the gene encoding the postsynaptic protein SHANK3 are associated with syndromic forms of autism spectrum disorders (ASDs). One of the earliest clinical symptoms in SHANK3-associated ASD is neonatal skeletal muscle hypotonia. This symptom can be critical for the early diagnosis of affected children; however, the mechanism mediating hypotonia in ASD is not completely understood. Here, we used a combination of patient-derived human induced pluripotent stem cells (hiPSCs), Shank3∆11(−/−) mice, and Phelan-McDermid syndrome (PMDS) muscle biopsies from patients of different ages to analyze the role of SHANK3 on motor unit development. Our results suggest that the hypotonia in SHANK3 deficiency might be caused by dysfunctions in all elements of the voluntary motor system: motoneurons, neuromuscular junctions (NMJs), and striated muscles. We found that SHANK3 localizes in Z-discs in the skeletal muscle sarcomere and co-immunoprecipitates with α-ACTININ. SHANK3 deficiency lead to shortened Z-discs and severe impairment of acetylcholine receptor clustering in hiPSC-derived myotubes and in muscle from Shank3∆11(−/−) mice and patients with PMDS, indicating a crucial role for SHANK3 in the maturation of NMJs and striated muscle. Functional motor defects in Shank3∆11(−/−) mice could be rescued with the troponin activator Tirasemtiv that sensitizes muscle fibers to calcium. Our observations give insight into the function of SHANK3 besides the central nervous system and imply potential treatment strategies for SHANK3-associated ASD.

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 - 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 - 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.

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 - 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.