Eliana Grazia LEO - personale UniMoRe

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Eliana Grazia LEO

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

Pubblicazioni

2023 - Combination of Nanodelivery Systems and Constituents Derived from Novel Foods: A Comprehensive Review [Articolo su rivista]
Truzzi, E.; Bertelli, D.; Bilia, A. R.; Vanti, G.; Maretti, E.; Leo, E.
abstract

: Novel Food is a new category of food, regulated by the European Union Directive No. 2015/2283. This latter norm defines a food as "Novel" if it was not used "for human consumption to a significant degree within the Union before the date of entry into force of that regulation, namely 15 May 1997". Recently, Novel Foods have received increased interest from researchers worldwide. In this sense, the key areas of interest are the discovery of new benefits for human health and the exploitation of these novel sources of materials in new fields of application. An emerging area in the pharmaceutical and medicinal fields is nanotechnology, which deals with the development of new delivery systems at a nanometric scale. In this context, this review aims to summarize the recent advances on the design and characterization of nanodelivery systems based on materials belonging to the Novel Food list, as well as on nanoceutical products formulated for delivering compounds derived from Novel Foods. Additionally, the safety hazard of using nanoparticles in food products, i.e., food supplements, has been discussed in view of the current European regulation, which considers nanomaterials as Novel Foods.

2023 - Inhaled lipid nanoparticles: a feasible tool for a challenging route [Articolo su rivista]
Leo, Eliana; Maretti, Eleonora
abstract

2023 - Linking endocannabinoid system, palmitoylethanolamide, and sarcopenia in view of therapeutic implications [Capitolo/Saggio]
Molinari, S.; Maretti, E.; Battini, R.; Leo, E.
abstract

Sarcopenia, a debilitating skeletal muscle disease closely connected with elderly, is becoming a major public health problem with the increasing of life expectancy. In the aim to found effective, targeted and side-effect-free therapies, understanding the endocannabinoid system (ECS) role in muscle homeostasis is of strategic importance. The skeletal muscle expresses all the ECS elements; in particular, a central role is played by the nuclear receptor PPARα and its main endogenous ligand, palmitoylethanolamide (PEA), an endocannabinoid-like molecule with an important anti-inflammatory effect. It is worth highlighting that in muscle the expression level of both PPARα receptor and its coactivator PGC1a, decreases with age, suggesting a causative relation between the lower PPARα function and sarcopenia. Therefore, the administration of PEA to the muscle can be a promising approach to counteract sarcopenia. In this regard, to promote the muscle targeting, innovative drug delivery systems, such as solid lipid nanoparticles, can be considered.

2023 - Nanoparticulate systems for the delivery of Palmitoylethanolamide to muscle cells [Poster]
Maretti, Eleonora; Molinari, Susanna; Gioia, Federica; Rustichelli, Cecilia; Leo, Eliana Grazia
abstract

2022 - Design and physicochemical characterization of novel hybrid SLN-liposome nanocarriers for the smart co-delivery of two antitubercular drugs [Articolo su rivista]
Truzzi, Eleonora; Capocefalo, Angela; Meneghetti, Fiorella; Maretti, Eleonora; Mori, Matteo; Iannuccelli, Valentina; Domenici, Fabio; Castellano, Carlo; Leo, Eliana
abstract

2022 - Design, Characterization, and In Vitro Assays on Muscle Cells of Endocannabinoid-like Molecule Loaded Lipid Nanoparticles for a Therapeutic Anti-Inflammatory Approach to Sarcopenia [Articolo su rivista]
Maretti, Eleonora; Molinari, Susanna; Battini, Renata; Rustichelli, Cecilia; Truzzi, Eleonora; Iannuccelli, Valentina; Leo, Eliana
abstract

Inflammatory processes play a key role in the pathogenesis of sarcopenia owing to their effects on the balance between muscle protein breakdown and synthesis. Palmitoylethanolamide (PEA), an endocannabinoid-like molecule, has been well documented for its anti-inflammatory properties, suggesting its possible beneficial use to counteract sarcopenia. The promising therapeutic effects of PEA are, however, impaired by its poor bioavailability. In order to overcome this limitation, the present study focused on the encapsulation of PEA in solid lipid nanoparticles (PEA-SLNs) in a perspective of a systemic administration. PEA-SLNs were characterized for their physico-chemical properties as well as cytotoxicity and cell internalization capacity on C2C12 myoblast cells. Their size was approximately 250 nm and the encapsulation efficiency reached 90%. Differential scanning calorimetry analyses demonstrated the amorphous state of PEA in the inner SLN matrix, which improved PEA dissolution, as observed in the in vitro assays. Despite the high internalization capacity observed with the flow cytometer (values between 85 and 94% after 14 h of incubation), the Nile Red labeled PEA-SLNs showed practically no toxicity towards myoblasts. Confocal analysis showed the presence of SLNs in the cytoplasm and not in the nucleus. These results suggest the potentiality provided by PEA-SLNs to obtain an innovative and side-effect-free tool in the medical treatment of sarcopenia

2022 - Palmitoylethanolamide-loaded Solid Lipid Nanoparticles for a therapeutic anti-inflammatory approach of Sarcopenia involving ECS [Relazione in Atti di Convegno]
Leo, Eliana Grazia; Maretti, Eleonora; Molinari, Susanna; Battini, Renata; Rustichelli, Cecilia; Iannuccelli, Valentina
abstract

2021 - Development of solid lipid nanoparticles for the delivery of an anti- inflammatory drug to muscle skeletal cells [Poster]
Maretti, Eleonora; Truzzi, Eleonora; Molinari, Susanna; Battini, Renata; Leo, Eliana Grazia
abstract

2021 - Drug delivery strategies of Geraniol via nose-to-brain for the treatment of neuronal disorders [Abstract in Atti di Convegno]
Truzzi, Eleonora; Dalpiaz, Alessandro; Rustichelli, Cecilia; Ferraro, Luca; de Oliveira Junior Edilson, Ribeiro; Fogagnolo, Marco; Pavan, Barbara; Beggiato, Sarah; Lima, Eliana; Leo, Eliana
abstract

Parkinson’s disease (PD) is a neurodegenerative disorder which affects 1% of people over 65 years. Neuroinflammation and mitochondrial dysfunction play a central role in the disorder progression. Therefore, anti-oxidants, promoters of neurotrophic factor expression and mitochondrial rescuers could prevent the disease progression. Geraniol (GER) is a natural compound with demonstrated anti-oxidant and neuroprotective activities in PD models. However, its activity is impaired by a fast metabolism following oral administration, with a half-life of about 12 minutes. To overcome this limitation, nose-to-brain administration is a promising strategy because it allows the direct delivery of drugs beyond the blood-brain barrier avoiding the first-pass metabolism. However, GER is highly volatile and irritant. Thus, an appropriate delivery system for its nose-to-brain administration is required. Two different strategies were developed in order to achieve a successful nose to brain delivery. The first one aimed the encapsulation of pure GER and GER prodrug in solid lipid nanoparticles (SLNs), while the second one, displayed here, yearns to complex GER with cyclodextrins (CDs). The inclusion complexes were designed in order to reduce GER volatility and to obtain long-term stable formulations as powders.

2021 - Formulazione per veicolare principi attivi lipofili [Brevetto]
Maretti, Eleonora; Leo, Eliana Grazia; Brighenti, Virginia
abstract

2021 - In vivo β-carotene skin permeation modulated by Nanostructured Lipid Carriers [Articolo su rivista]
Maretti, E.; Leo, E.; Rustichelli, C.; Truzzi, E.; Siligardi, C.; Iannuccelli, V.
abstract

Nanostructured Lipid Carriers (NLC) were investigated with the purpose of promoting skin permeation of the highly lipophilic β-carotene (BC) across the stratum corneum (SC) barrier so that it may perform its antioxidant properties in photo-aging and epithelial skin cancer prevention. Two differently sized NLC samples were developed using stearic acid and squalene as lipid matrix and evaluated in comparison with Microstructured Lipid Carriers (MLC). The carriers were characterized for morphology, size, Z-potential, BC loading and release as well as physical state by means of DSC and XRPD analyses. In vivo penetration of the carriers was assessed on humans by determining BC concentrations within the SC stratum disjunctum and stratum compactum layers removed by means of the tape stripping test in comparison with pure BC. Unlike MLC and pure BC that were mostly retained within the outermost layers of the SC, the NLC sample having the smallest size (about 200 nm) has proved to penetrate more deeply into the SC barrier. Accordingly, the goal of providing β-carotene actions against oxidative damages within the looser skin viable tissues could be envisaged.

2021 - Nasal biocompatible powder of geraniol oil complexed with cyclodextrins for neurodegenerative diseases: Physicochemical characterization and in vivo evidences of nose to brain delivery [Articolo su rivista]
Truzzi, Eleonora; Rustichelli, Cecilia; de Oliveira Junior, Edilson Ribeiro; Ferraro, Luca; Maretti, Eleonora; Graziani, Daniel; Botti, Giada; Beggiato, Sarah; Iannuccelli, Valentina; Lima, Eliana Martins; Dalpiaz, Alessandro; Leo, Eliana
abstract

Recently, many studies have shown that plant metabolites, such as geraniol (GER), may exert anti-inflammatory effects in neurodegenerative diseases and, in particular, Parkinson's disease (PD) models. Unfortunately, delivering GER to the CNS via nose-to-brain is not feasible due to its irritant effects on the mucosae. Therefore, in the present study β-cyclodextrin (βCD) and its hydrophilic derivative hydroxypropyl-beta-cyclodextrin (HPβCD) were selected as potential carriers for GER nose-to-brain delivery. Inclusion complexes were formulated and the biocompatibility with nasal mucosae and drug bioavailability into cerebrospinal fluid (CSF) were studied in rats. It has been demonstrated by DTA, FT-IR and NMR analyses that both the CDs were able to form 1:1 GER-CD complexes, arising long-term stable powders after the freeze-drying process. GER-HPβCD-5 and GER-βCD-2 complexes exhibited comparable results, except for morphology and solubility, as demonstrated by SEM analysis and phase solubility study, respectively. Even though both complexes were able to directly and safely deliver GER to CNS, GER-βCD-2 displayed higher ability in releasing GER in the CSF. In conclusion, βCD complexes can be considered a very promising tool in delivering GER into the CNS via nose-to-brain route, preventing GER release into the bloodstream and ensuring the integrity of the nasal mucosa.

2020 - A Peptidic Thymidylate-Synthase Inhibitor Loaded on Pegylated Liposomes Enhances the Antitumour Effect of Chemotherapy Drugs in Human Ovarian Cancer Cells [Articolo su rivista]
Marverti, Gaetano; Gozzi, Gaia; Maretti, Eleonora; Lauriola, Angela; Severi, Leda; Sacchetti, Francesca; Losi, Lorena; Pacifico, Salvatore; Ferrari, Stefania; Ponterini, Glauco; Leo, Eliana Grazia; Costi, Maria Paola; D'Arca, Domenico
abstract

There is currently no effective long-term treatment for ovarian cancer (OC) resistant to poly-chemotherapy regimens based on platinum drugs. Preclinical and clinical studies have demonstrated a strong association between development of Pt-drug resistance and increased thymidylate synthase (hTS) expression, and the consequent cross-resistance to the hTS inhibitors 5-fluorouracil (5-FU) and raltitrexed (RTX). In the present work, we propose a new tool to combat drug resistance. We propose to treat OC cell lines, both Pt-sensitive and -resistant, with dual combinations of one of the four chemotherapeutic agents that are widely used in the clinic, and the new peptide, hTS inhibitor, [D-Gln4]LR. This binds hTS allosterically and, unlike classical inhibitors that bind at the catalytic pocket, causes cell growth inhibition without inducing hTS overexpression. The dual drug combinations showed schedule-dependent synergistic antiproliferative and apoptotic effects. We observed that the simultaneous treatment or 24h pre-treatment of OC cells with the peptide followed by either agent produced synergistic effects even in resistant cells. Similar synergistic or antagonistic effects were obtained by delivering the peptide into OC cells either by means of a commercial delivery system (SAINT-PhD) or by pH sensitive PEGylated liposomes. Relative to non-PEGylated liposomes, the latter had been previously characterized and found to allow macrophage escape, thus increasing their chance to reach the tumour tissue. The transition from the SAINT-PhD delivery system to the engineered liposomes represents an advancement towards a more drug-like delivery system and a further step towards the use of peptides for in vivo studies. Overall, the results suggest that the association of standard drugs, such as cDDP and/or 5-FU and/or RTX, with the novel peptidic TS inhibitor encapsulated into PEGylated pH-sensitive liposomes can represent a promising strategy for fighting resistance to cDDP and anti-hTS drugs.

2020 - Chitosan/heparin polyelectrolyte complexes as ion-paring approach to encapsulate heparin in orally administrable SLN: In vitro evaluation [Articolo su rivista]
Maretti, E.; Pavan, B.; Rustichelli, C.; Montanari, M.; Dalpiaz, A.; Iannuccelli, V.; Leo, E.
abstract

Enhancing oral bioavailability of hydrophilic drugs by encapsulation in lipid-based nanocarriers, including Solid Lipid Nanoparticles (SLN), has been well documented. In this work, high molecular weight heparin was “insolubilized” by an “ion-paring” approach, forming Chitosan/Heparin Polyelectrolyte Complexes (PEC) to promote its encapsulation in SLN. Hybrid PEC-SLN, heparin-loaded SLN (H-SLN) as well as naked PEC were prepared and characterized regarding size, Z potential, morphology, drug loading and drug release. Physicochemical characterization of the nanoparticles was also performed by differential scanning calorimetry (DSC), and Fourier Transform Infra-Red (FTIR) analysis. FITC-labeled naked PEC along with Nile Red labeled PEC-SLN were assessed on CaCo-2 cells to study cytotoxicity as well as cell internalization ability by cytometric and confocal analysis. Transepithelial electrical resistance (TEER) was measured on NCM460 cell monolayers to evaluate whether chitosan may induce a modification of tight junctions’ integrity at epithelial level. Results showed that the minimum size of PEC (around 170 nm) was at pH 5.5 with a positive surface charge and after encapsulation in SLN produced hybrid PEC-SLN with a size of about 370 nm and a negative zeta potential. In comparison to both H-SLN and naked PEC, PEC-SLN were able to achieve a pH-controlled drug release and showed on CaCo-2 cells low toxicity and rapid internalization. Finally, TEER measurements highlighted that the hybrid nanocarriers were internalized without interference in the membrane resistance. Therefore, PEC-SLN could be considered valuable candidate for further in vivo investigations about the systemic bioavailability of oral heparin.

2020 - Formulazione per veicolare principi attivi lipofili [Brevetto]
Maretti, Eleonora; Leo, Eliana Grazia; Brighenti, Virginia
abstract

2020 - In Vivo Biodistribution of Respirable Solid Lipid Nanoparticles Surface-Decorated with a Mannose-Based Surfactant: A Promising Tool for Pulmonary Tuberculosis Treatment? [Articolo su rivista]
Truzzi, Eleonora; Leite Nascimento, Thais; Iannuccelli, Valentina; Costantino, Luca; Martins Lima, Eliana; Leo, Eliana Grazia; Siligardi, Cristina; Gualtieri, Eva Magdalena; Maretti, Eleonora
abstract

The active targeting to alveolar macrophages (AM) is an attractive strategy to improve the therapeutic efficacy of ‘old’ drugs currently used in clinical practice for the treatment of pulmonary tuberculosis. Previous studies highlighted the ability of respirable solid lipid nanoparticle assemblies (SLNas), loaded with rifampicin (RIF) and functionalized with a novel synthesized mannose-based surfactant (MS), both alone and in a blend with sodium taurocholate, to efficiently target the AM via mannose receptor-mediated mechanism. Here, we present the in vivo biodistribution of these mannosylated SLNas, in comparison with the behavior of both non-functionalized SLNas and bare RIF. SLNas biodistribution was assessed, after intratracheal instillation in mice, by whole-body real-time fluorescence imaging in living animals and RIF quantification in excised organs and plasma. Additionally, SLNas cell uptake was determined by using fluorescence microscopy on AM from bronchoalveolar lavage fluid and alveolar epithelium from lung dissections. Finally, histopathological evaluation was performed on lungs 24 h after administration. SLNas functionalized with MS alone generated the highest retention in lungs associated with a poor spreading in extra-pulmonary regions. This effect could be probably due to a greater AM phagocytosis with respect to SLNas devoid of mannose on their surface. The results obtained pointed out the unique ability of the nanoparticle surface decoration to provide a potential more efficient treatment restricted to the lungs where the primary tuberculosis infection is located.

2020 - Nasal administration of nanoencapsulated geraniol/ursodeoxycholic acid conjugate: Towards a new approach for the management of Parkinson's disease [Articolo su rivista]
de Oliveira Junior, Edilson Ribeiro; Truzzi, Eleonora; Ferraro, Luca; Fogagnolo, Marco; Pavan, Barbara; Beggiato, Sarah; Rustichelli, Cecilia; Maretti, Eleonora; Lima, Eliana Martins; Leo, Eliana; Dalpiaz, Alessandro
abstract

The combined use of different therapeutic agents in the treatment of neurodegenerative disorders is a promising strategy to halt the disease progression. In this context, we aimed to combine the anti-inflammatory properties of geraniol (GER) with the mitochondrial rescue effects of ursodeoxycholic acid (UDCA) in a newly-synthesized prodrug, GER-UDCA, a potential candidate against Parkinson's disease (PD). GER-UDCA was successfully synthetized and characterized in vitro for its ability to release the active compounds in physiological environments. Because of its very poor solubility, GER-UDCA was entrapped into both lipid (SLNs) and polymeric (NPs) nanoparticles in order to explore nose-to-brain pathway towards brain targeting. Both GER-UDCA nanocarriers displayed size below 200 nm, negative zeta potential and the ability to increase the aqueous dissolution rate of the prodrug. As SLNs exhibited the higher GER-UDCA dissolution rate, this formulation was selected for the in vivo GER-UDCA brain targeting experiments. The nasal administration of GER-UDCA-SLNs (1 mg/kg of GER-UDCA) allowed to detect the prodrug in rat cerebrospinal fluid (concentration range = 1.1 to 4.65 μg/mL, 30–150 min after the administration), but not in the bloodstream, thus suggesting the direct nose to brain delivery of the prodrug. Finally, histopathological evaluation demonstrated that, in contrast to the pure GER, nasal administration of GER-UDCA-SLNs did not damage the structural integrity of the nasal mucosa. In conclusion, the present data suggest that GER-UDCA-SLNs could provide an effective and non-invasive approach to boost the access of GER and UDCA to the brain with low dosages.

2020 - Physicochemical characterization of SLN/Liposomes hybrid nanoparticles for the co-delivery of two antitubercular drugs: focus on SANS analysis [Poster]
Truzzi, Eleonora; Meneghetti, Fiorella; Mori, Matteo; Maretti, Eleonora; Domenici, Fabio; Castellano, Carlo; Capocefalo, Angela; Leo, Eliana Grazia
abstract

2019 - Bile salt-coating modulates the macrophage uptake of nanocores constituted by a zidovudine prodrug and enhances its nose-to-brain delivery [Articolo su rivista]
Dalpiaz, A.; Fogagnolo, M.; Ferraro, L.; Beggiato, S.; Hanuskova, M.; Maretti, E.; Sacchetti, F.; Leo, E.; Pavan, B.
abstract

We have previously demonstrated that the ester conjugation of zidovudine (AZT) with ursodeoxycholic acid (UDCA) allows to obtain a prodrug (U-AZT) which eludes the active efflux transporters (AET). This allows the prodrug to more efficiently permeates and remains in murine macrophages than the parent compound. Here we demonstrate that U-AZT can be formulated, by a nanoprecipitation method, as nanoparticle cores coated by bile acid salt (taurocholate or ursodeoxycholate) corona, without any other excipients. The U-AZT nanoparticles appeared spherical with a mean diameter of ∼200 nm and a zeta potential of ∼−55 mV. During the incubation (5 h) in fetal bovine serum, the ursodeoxycholate-coated nanoparticle size did not change. Differently, taurocholate-coated particle size was firstly reduced and then increased up to 800 µm, thus suggesting the high aptitude of these nanoparticles to interact with serum proteins. The in vitro uptake of taurocholate coated particles by murine macrophages was strongly higher than that of ursodeoxycholate-coated particles or free U-AZT (∼500% and ∼7000%, respectively). AZT was also detected in macrophages following the prodrug uptake, with the greatest amounts observed after the taurocholate-coated nanoparticle incubation. As macrophages in the subarachnoid spaces of cerebrospinal fluid (CSF) constitute one of the most unreachable HIV sanctuaries in the body, we also tested the ability of taurocholate-coated nanoparticles (i.e., nanoparticles highly internalized by macrophages) to reach them after their nasal administration in the presence or absence of chitosan. The results indicate that chitosan allowed to obtain a relatively high uptake (up to 4 µg/ml) of U-AZT in CSF. Taking into account that chitosan may promote the direct brain nanoparticle uptake, these findings can be considered an initial step toward the in vivo targeting of the subarachnoid macrophages by U-AZT prodrug.

2019 - Drugs/lamellae interface influences the inner structure of double-loaded liposomes for inhaled anti-TB therapy: An in-depth small-angle neutron scattering investigation [Articolo su rivista]
Truzzi, Eleonora; Meneghetti, Fiorella; Mori, Matteo; Costantino, Luca; Iannuccelli, Valentina; Maretti, Eleonora; Domenici, Fabio; Castellano, Carlo; Rogers, Sarah; Capocefalo, Angela; Leo, Eliana
abstract

With the aim of developing new drug carriers for inhalation therapy, we report here an in depth investigation of the structure of multilamellar liposomes loaded with two well-established anti-tubercular (anti-TB) drugs, isoniazid (INH) and rifampicin (RIF), by means of small-angle neutron-scattering (SANS) analysis. Unloaded, single drug-loaded and co-loaded liposomes were prepared using different amounts of drugs and characterized regarding size, encapsulation efficiency and drug release. Detailed information on relevant properties of the investigated host-guest structures, namely the steric bilayer thickness, particle dispersion, number of lamellae and drug localization was studied by SANS. Results showed that RIF-liposomes were less ordered than unloaded liposomes. INH induced a change in the inter-bilayer periodical spacing, while RIF-INH co-loading stabilized the multilamellar liposome architecture, as confirmed by the increment of the drug loading capacity. These findings could be useful for the understanding of in vitro and in vivo behavior of these systems and for the design of new drug carriers, intended for inhaled therapy.

2019 - Newly synthesized surfactants for surface mannosylation of respirable SLN assemblies to target macrophages in tuberculosis therapy [Articolo su rivista]
Maretti, Eleonora; Costantino, Luca; Buttini, Francesca; Rustichelli, Cecilia; Leo, Eliana Grazia; Truzzi, Eleonora; Iannuccelli, Valentina
abstract

The present study reports about new Solid Lipid Nanoparticle assemblies (SLNas) loaded with rifampicin (RIF) surface-decorated with novel mannose derivatives, designed for anti-tuberculosis (TB) inhaled therapy by dry powder inhaler (DPI). Mannose is considered a relevant ligand to achieve active drug targeting being mannose receptors (MR) overexpressed on membranes of infected alveolar macrophages (AM), which are the preferred site of Mycobacterium tuberculosis. Surface decoration of SLNas was obtained by means of newly synthesized functionalizing compounds used as surfactants in the preparation of carriers. SLNas were fully characterized in vitro determining size, morphology, drug loading, drug release, surface mannosylation, cytotoxicity, macrophage internalization extent and ability to bind MR, and intracellular RIF concentration. Moreover, the influence of these new surface functionalizing agents on SLNas aerodynamic performance was assessed by measuring particle respirability features using Next Generation Impactor. SLNas exhibited suitable drug payload, in vitro release, and more efficient ability to enter macrophages (about 80%) compared to bare RIF (about 20%) and to non-functionalized SLNas (about 40%). The involvement of MR specific binding has been demonstrated by saturating MR of J774 cells causing a decrease of RIF intracellular concentration of about 40%. Furthermore, it is noteworthy that the surface-decoration of particles produced a poor cohesive powder with an adequate respirability (fine particle fraction ranging from about 30% to 50%). Therefore, the proposed SLNas may represent an encouraging opportunity in a perspective of an efficacious anti-TB inhaled therapy.

2019 - Novel engineered lipid-based nanoparticles for pulmonary tuberculosis inhalation therapy [Poster]
Maretti, Eleonora; Truzzi, Eleonora; Costantino, Luca; Rustichelli, Cecilia; Martins Lima, Eliana; Leite Nascimento, Thais; Siligardi, Cristina; Gualtieri, Eva Magdalena; Miselli, Paola; Buttini, Francesca; Leo, Eliana Grazia; Iannuccelli, Valentina
abstract

Priorities to achieve the WHO goal of ending tuberculosis (TB) epidemic by 2030 include new drug treatments to simplify and shorter conventional drug regimens. TB is caused by Mycobacterium tuberculosis residing and surviving inside alveolar macrophages (AM). Considering that 75-80% of cases of infection remain localized in the lungs, the easiest and most successful therapy could involve the inhalation route offering benefits in terms of patient’s autonomy and compliance, by-passing hepatic metabolism, reducing dose amount, dose frequency, and treatment duration, thus minimising the risk of drug-resistant mutants, toxicity, and side effects. Inhalable powder formulations of repurposed drugs entail engineering techniques such as micro- or nanoparticulate carriers enabling drug emission by Dry Powder Inhaler devices, deposition onto alveolar epithelia, and transport into AM. Within this context, Solid Lipid Nanoparticle assemblies (SLNas) loaded with rifampicin, a clinically useful anti-TB drug, were produced by processing accepted excipients for DPI formulations through an optimized methodology that avoids organic solvents and is suitable for a large-scale production. The prototypes were functionalized by means of newly synthesized AM receptor-specific targeting agents as the ligands anchored on SLNas surface without chemical reactions. In vitro and in vivo preclinical studies highlighted functionalized SLNas with adequate respirability performance, safety, AM internalization ability, and mice lung deposition in an encouraging perspective of a potential efficacious pulmonary TB therapy. This research was supported by a grant on the project “FAR interdisciplinare 2017” from the University of Modena and Reggio Emilia, Modena, Italy (PI Prof. Luca Costantino)

2019 - Self-assembled organogelators as artificial stratum corneum models: key-role parameters for skin permeation prediction [Articolo su rivista]
Maretti, Eleonora; Rustichelli, Cecilia; Miselli, Paola; Leo, Eliana Grazia; Truzzi, Eleonora; Iannuccelli, Valentina
abstract

Self-assembled organogelators were explored as artificial stratum corneum (SC) models for the in vitro skin permeation assessment. Four SC models consisting of binary (organogels) or ternary (microemulsion-based organogels) mixtures were developed using stearic acid, tristearin, or sorbitan tristearate, at two different concentrations, gelled in squalene. The permeation of lipophilic butyl-methoxydibenzoylmethane and hydrophilic methylene blue as the permeant compounds across the SC models was compared with ex vivo experiments using excised porcine ear skin. A multi-analytical approach was adopted to provide detailed understanding about organogelator organization within the SC models and find possible parameters playing key-roles in SC permeation prediction. The SC models were investigated for gelling properties and microstructure. Parameters such as gel occurrence, organogelator concentration, and rheological properties appeared as negligible conditions for skin permeation prediction. Conversely, arrangement packing, interactions, and crystallinity extent of the self-assembled organogelator were found to play a fundamental role in the simulation of SC barrier function according to the permeant feature.

2019 - The Impact of Lipid Corona on Rifampicin Intramacrophagic Transport Using Inhaled Solid Lipid Nanoparticles Surface-Decorated with a Mannosylated Surfactant [Articolo su rivista]
Maretti, Eleonora; Rustichelli, Cecilia; Gualtieri, Eva Magdalena; Costantino, Luca; Siligardi, Cristina; Miselli, Paola; Buttini, Francesca; Montecchi, Monica; Leo, Eliana Grazia; Truzzi, Eleonora; Iannuccelli, Valentina
abstract

The mimicking of physiological conditions is crucial for the success of accurate in vitro studies. For inhaled nanoparticles, which are designed for being deposited on alveolar epithelium and taken up by macrophages, it is relevant to investigate the interactions with pulmonary surfactant lining alveoli. As a matter of fact, the formation of a lipid corona layer around the nanoparticles could modulate the cell internalization and the fate of the transported drugs. Based on this concept, the present research focused on the interactions between pulmonary surfactant and Solid Lipid Nanoparticle assemblies (SLNas), loaded with rifampicin, an anti-tuberculosis drug. SLNas were functionalized with a synthesized mannosylated surfactant, both alone and in a blend with sodium taurocholate, to achieve an active targeting to mannose receptors present on alveolar macrophages (AM). Physico-chemical properties of the mannosylated SLNas satisfied the requirements relative to suitable respirability, drug payload, and AM active targeting. Our studies have shown that a lipid corona is formed around SLNas in the presence of Curosurf, a commercial substitute of the natural pulmonary surfactant. The lipid corona promoted an additional resistance to the drug diffusion for SLNas functionalized with the mannosylated surfactant and this improved drug retention within SLNas before AM phagocytosis takes place. Moreover, lipid corona formation did not modify the role of nanoparticle mannosylation towards the specific receptors on MH-S cell membrane.

2018 - Organo-modified bentonite for gentamicin topical application: interlayer structure and in vivo skin permeation [Articolo su rivista]
Iannuccelli, Valentina; Maretti, Eleonora; Bellini, Alessia; Malferrari, Daniele; Ori, Guido; Montorsi, Monia; Bondi, Moreno; Truzzi, Eleonora; Leo, Eliana Grazia
abstract

Recent biomedical applications of clay materials have included organically modified clays or clay minerals with the purpose of modifying and improving drug biological activity. The present research aims to explore the potential benefits provided by a raw bentonite (Bt) modified by gentamicin (GM) adsorbed within montmorillonite interlayers in the management of cutaneous infectious diseases. Information arisen from controlled X-ray powder diffraction, thermogravimetry coupled with evolved gas mass spectrometry, and molecular dynamics simulations pointed out GM monolayer arrangement within montmorillonite framework without producing substantial effects on the layer periodicity. Concerning skin biomedical application, unlike the pure antibiotic permeating along the trans-follicular pathway across stratum corneum, the organo-modified Bt/GM would favor the trans-epidermal route along inter-cluster corneocyte region, as in vivo skin penetration studies by means of tape stripping test indicated. Based on the results obtained, GM intercalation could represent a potential advantageous approach allowing a long-term Bt/GM reservoir for sustained antibacterial activity.

2018 - Polymeric and Solid Lipid Nanoparticles for nose-to-brain delivery of geraniolursodeoxycholic acid conjugate: development and characterization studies [Abstract in Atti di Convegno]
Edilson Oliveira Junior, ; Truzzi, Eleonora; Maretti, Eleonora; Leo, Eliana Grazia; Dalpiaz, Alessandro; Rustichelli, Cecilia; Marco, Fogagnolo; Eliana, Lima
abstract

Neurodegenerative disorder treatment is a challenge mainly due to the difficulty of drug transport across the blood-brain barrier [1]. Intranasal administration of nanoparticles as carrier system may increase drug concentration into the brain [2]. Geraniol (GER) has demonstrated antioxidant and neuroprotective activities in Parkinson’s disease animal models [3]. However, due to its volatility, GER is hardly incorporated into freezedrying nanoparticles. On the other hand, GER-ursodeoxycholic acid conjugate (GER-UDCA) is a non-volatile derivative with high potentiality to be incorporated into nanocarriers. Therefore, in this work GER-UDCA-loaded Solid Lipid Nanoparticles (SLNs) and PLGA nanoparticles (NPs) intended for nose-to-brain delivery were developed and characterized. SLNs were prepared by emulsion/solvent evaporation method and NPs by nanoprecipitation method. Briefly, formulations were optimized considering various processing variables and nanoparticle characterization was performed in terms of morphology, size, surface charge, drug loading (DL%), encapsulation efficiency (EE%) and in vitro drug release. Finally, the stability of free and encapsulated GERUDCA was evaluated in enzymatic medium from rat liver homogenates. GER-UDCA-SLN and GER-UDCA-NPs showed spherical shape, mean size of 120/180 nm with polydispersity index < 0.2, and zeta potential around −22/-26 mV, respectively. After freeze-drying, the DL% was 6% for SLN and 12% for NPs with EE% values of 89.3% and 60.1%, respectively. Preliminary data regarding in vitro release of GER-UDCA from the nanoparticles evidenced a higher dissolution rate than the free drug, probably due to the increase of surface contact. Results in liver rat homogenate suggested a contribution of the nanoparticles in the stability of the prodrug in physiologic environments. In conclusion, these GER-UDCA-loaded nanocarriers demonstrated a possible application in further in vivo studies of nose-to-brain drug delivery.

2018 - Self-assembling organogelators for artificial stratum corneum models: key-role parameters in skin permeation prediction [Poster]
Iannuccelli, V.; Maretti, E.; Rustichelli, C.; Miselli, P.; Truzzi, E.; Leo, E.
abstract

The development of in vitro methods to predict in vivo percutaneous absorption of bioactive molecules is a challenge to which the researchers are called in order to eliminate or reduce the pharmacological and toxicological tests on animal models. Artificial stratum corneum (SC) models obtained by self-assembled oganogelators were designed for skin permeation assessment of butyl methoxydibenzoylmethane (BMDBM, log Po/w = 4.68) and methylene blue (MB, log Po/w = 0.91). A multi-analytical approach was adopted to provide detailed understanding about the gelator organization within the models and find possible parameters playing a key-role in in vivo and ex vivo SC permeation prediction. The evaluation of in vitro skin permeation data compared with those obtained ex vivo and previously in vivo on humans for BMDBM showed good correlations vitro/ex vivo and vitro/vivo for both butyl BMDBM, as the lipophilic permeant, and MB, as the hydrophilic permeant, by using TS20 as well as both STS and ME models. With the aim of providing detailed understanding about the organogelator behaviour and organization within the models and find possible parameters playing a key-role in SC permeation prediction a multi-analytical approach was adopted. All the models did not flow upon tube tilting and could be described as gels, with the exception of STS10 model that appeared as thick liquid being gelator concentration lower than mgc value. Unlike SA and TS models that exhibited networks capable of immobilizing completely the solvent, STS and ME10 models revealed the syneresis phenomenon according to gelator concentration. The actual presence of water within STS aggregates (reverse micelles) of ME models was demonstrated by means of TG/DTA analysis showing two thermal events in the range of about 50-130°C related to removal of water molecules. Unlike the pure gelators, XRPD profiles from all the SC models exhibited a broad peak at about 20° 2θ indicating the presence of a networked structure of the gelators where the width of the peak at half maximum is dependent on the crystallinity of the sample, which in turn is dependent on non-covalent interactions amongst the gelator molecules responsible for the formation of an ordered structure. Intermolecular interactions also arisen from FT-IR spectra showing subsided ester group stretching in TS, STS, and ME models. Architectural arrangements of the organogelators within TS, STS, and ME models, as outlined by microscopy analyses, involved round or worm-like architectures of spherulitic clusters. Under polarized light, the occurrence of birefringence revealed the so-called “maltese crosses” in STS models that are characteristic of liquid crystals with lamellar structures. The results demonstrated the relevant role of both the arrangement of gelator packing and crystallinity extent in mimicking SC in vivo/ex vivo skin permeation of both lipophilic and hydrophilic compounds. These findings could account for the behaviour and development of other artificial skin models involving different materials for the skin permeation prediction.

2018 - Small-angle neutron scattering characterization of liposomes for anti-tuberculosis inhaled therapy. [Poster]
Truzzi, Eleonora; Angela, Capocefalo; Fabio, Domenici; Carlo, Castellano; Fiorella, Meneghetti; Iannuccelli, Valentina; Maretti, Eleonora; Leo, Eliana Grazia; Costantino, Luca
abstract

The present investigation studied the effects of two first-line anti-tuberculosis (TB) drugs, rifampicin (RIF) and isoniazid (INH), on the structure of multilamellar liposomes. Liposomes have been shown to be a promising system for inhaled therapy. (1) The study of liposome-drug interactions is essential, and small-angle neutron scattering (SANS) technique provides valuable and unique data about steric bilayer thickness, particle dispersion, number of lamellae and drug localization under physiological conditions. (2) Unloaded, single drug-loaded and co-loaded liposomes were prepared using different amounts of drugs by reverse phase evaporation method. Liposomal suspensions were prepared using D2O, in order to emphasize the contrast between the aqueous and the lipid/drug phases. The samples were characterized by dynamic light scattering, atomic force microscopy and finally by SANS technique (Rutherford Appleton Laboratory, U.K.). Neutron scattering curves were analyzed using a multi-shell spherical model of the fitting routine SASView 2.2.0. Liposomes have been shown to be physico-chemically stable during the experiments, efficiently drug-loaded, and able to control drug release. Dimensional analysis demonstrated that particle sizes are in the range of SANS dimensional detection. SANS curves exhibited Bragg peaks for all samples, confirming the multilamellar liposome structure. By fitting the data, significant differences among the samples have been highlighted. RIF-liposomes were less ordered than unloaded liposomes: a reduction of the lamellae number was observed and the periodicity of the lipid bilayers slightly increased with the increment of the drug loading, may be due to RIF interaction with phospholipid tails, which can destabilize liposome lamellarity, since RIF is a hydrophobic drug. In INH-liposomes, the drug payloads did not change vesicle structure, because INH is a hydrophilic drug. However, INH induced a change in the inter-bilayer periodical spacing, which could be compatible with the formation of drug-liposome complexes at the water-lipid interface. Finally, the RIF-INH co-loaded liposomes exhibited the same characteristics of unloaded liposomes, suggesting that INH and RIF together have a stabilizing effect on the structure. In fact, no destabilization and no changing in inter-bilayer periodical spacing were observed. In conclusion, SANS analysis provides fundamental information about drug-liposome interactions to comprehend the relation between system structure behaviour and its biological activity. Moreover, data suggest that the co-encapsulation of the two anti-TB drugs may have a synergic effect on liposome stability. 1. A. Elhissi, Curr. Pharm. Des., 2017, 23, 362-372. 2. PC. Lin, S. Lin, PC. Wang, R. Sridhar, Biotechnol. Adv., 2014, 32, 711– 726.

2018 - pH-Promoted Release of a Novel Anti-Tumour Peptide by “Stealth” Liposomes: Effect of Nanocarriers on the Drug Activity in Cis-Platinum Resistant Cancer Cells [Articolo su rivista]
Sacchetti, Francesca; Marverti, Gaetano; D’Arca, Domenico; Severi, Leda; Maretti, Eleonora; Iannuccelli, Valentina; Pacifico, Salvatore; Ponterini, Glauco; Costi, Maria Paola; Leo, Eliana
abstract

Purpose: To evaluate the potential effects of PEGylated pH-sensitive liposomes on the intracellular activity of a new peptide recently characterized as a novel inhibitor of the human thymidylate synthase (hTS) over-expressed in many drug-resistant human cancer cell lines. Methods: Peptide-loaded pH-sensitive PEGylated (PpHL) and non-PEGylated liposomes (nPpHL) were carefully characterized and delivered to cis-platinum resistant ovarian cancer C13* cells; the influence of the PpHL on the drug intracellular activity was investigated by the Western Blot analysis of proteins involved in the pathway affected by hTS inhibition. Results: Although PpHL and nPpHL showed different sizes, surface hydrophilicities and serum stabilities, both carriers entrapped the drug efficiently and stably demonstrating a pH dependent release; moreover, the different behavior against J774 macrophage cells confirmed the ability of PEGylation in protecting liposomes from the reticuloendothelial system. Comparable effects were instead observed against C13* cells and biochemical data by immunoblot analysis indicated that PEGylated pH-sensitive liposomes do not modify the proteomic profile of the cells, fully preserving the activity of the biomolecule. Conclusion: PpHL can be considered as efficient delivery systems for the new promising anti-cancer peptide.

2017 - Conveying a newly designed hydrophilic anti-human thymidylate synthase peptide to cisplatin resistant cancer cells: are pH-sensitive liposomes more effective than conventional ones? [Articolo su rivista]
Sacchetti, Francesca; D'Arca, Domenico; Genovese, Filippo; Pacifico, Salvatore; Maretti, Eleonora; Hanuskova, Miriam; Iannuccelli, Valentina; Costi, Maria Paola; Leo, Eliana Grazia
abstract

Context: LR-peptide, a novel hydrophilic peptide synthetized and characterized in previous work, is able to reduce the multi-drug resistance response in cisplatin (cDPP) resistant cancer cells by inhibiting human thymidylate synthase overexpressed in several tumors, including ovarian and colon-rectal cancers, but it is unable to enter the cells spontaneously. Objective: The aim of this work was to design and characterize liposomal vesicles as drug delivery systems for the LR peptide, evaluating the possible benefits of the pH-responsive feature in improving intracellular delivery. Materials and methods For this purpose, conventional and pH-sensitive liposomes were formulated, compared regarding their physical-chemical properties (size, PDI, morphology, in vitro stability and drug release) and studied for in vitro cytotoxicity against a cDDP-resistant cancer cells. Results and discussion Results indicated that LR peptide was successfully encapsulated in both liposomal formulations but at short incubation time only LR loaded pH-sensitive liposomes showed cell inhibition activity while for long incubation time the two kinds of liposomes demonstrated the same efficacy. Conclusions Data provide evidence that acidic pH-triggered liposomal delivery is able to significantly reduce the time required by the systems to deliver the drug to the cells without inducing an enhancement of the efficacy of the drug.

2017 - DEVELOPMENT AND COMPARISON OF TWO LIPID-BASED NANOSYSTEMS FOR THE CO-DELIVERY OF RIFAMPICIN AND ISONIAZID: SMALL-ANGLE NEUTRON SCATTERING CHARACTERISATION [Abstract in Atti di Convegno]
Truzzi, Eleonora; Maretti, Eleonora; Meneghetti, Fiorella; Castellano, Carlo; Iannuccelli, Valentina; Leo, Eliana
abstract

Recently lipid-based drug delivery system (DDS), such as Solid Lipid Nanoparticles (SLNs) and liposomes, have been proposed in the inhaled therapy to improve the drug targeting and delivery. In this work SLNs and liposomes were studied for the co-delivery of two first-line anti-tuberculosis drugs, Rifampicin (RIF) and Isoniazid (INH). RIF and INH were chosen as model drugs, since they display different physicochemical features: RIF is a poor water soluble compound, while INH is extremely hydrophilic. Both lipid-based DDS (SLN and liposomes) have been characterized regarding size and zeta potential by DSL (dynamic light scattering) and regarding shape and external morphology by AFM (atomic force microscopy) and by SEM (scanning electron microscopy). Moreover, drug content, in vitro drug release and stability in suspension, were evaluated in order to highlight the effects of the co-encapsulation. In fact, drugs can interact with the delivery systems modifying its properties and in vitro behaviour. In order to comprehend and evaluate the possible interactions between drugs and DDS, loaded SLNs and liposomes has been further studied by small-angle neutron scattering (SANS). Small-Angle Neutron Scattering (SANS) is a useful tool in gaining a detailed understanding of the structure of macromolecular systems that make up SLN and liposomes. SANS is widely applicable, with the inherent advantage over visual techniques, of probing accurately structural features on nanometer length scales as well as yielding ensemble-averaged information on freely water-suspended membranes. Of note, the SANS D2O/H2O contrast variation methodology allows a well distinguished scattering from the shell with respect to the corresponding inner content. Besides, SANS do not entail the addition or inclusion of bulky fluorescent dyes or labels needed by other techniques (i.e. fluorescence and electron microscopy), which can perturb or affect the phase behavior observed.

2017 - Food and/or nutraceutical composition [Brevetto]
Leo, Eliana Grazia
abstract

No abstract

2017 - Metodo per modulare l'assorbimento fagocitario di un principio attivo o un suo precursore da parte di macrofagi [Brevetto]
Dalpiaz, Alessandro; Pavan, Barbara; Fogagnolo, Marco; Paganetto, Guglielmo; Leo, Eliana Grazia; Iannuccelli, Valentina; Maretti, Eleonora
abstract

La nostra invenzione propone nanoparticelle costituite da agenti terapeutici o da loro profarmaci insolubili in ambiente acquoso capaci di modulare la loro fagocitosi da parte di cellule macrofagiche con un semplice rivestimento, in assenza di componenti polimerici o lipidici

2017 - Nuove molecole funzionalizzanti per il targeting ai macrofagi di vettori lipidici [Brevetto]
Iannuccelli, Valentina; Maretti, Eleonora; Costantino, Luca; Leo, Eliana; Rustichelli, Cecilia; Truzzi, Eleonora
abstract

La presente invenzione ha per oggetto molecole derivate dal mannosio ed il loro uso per direzionare carrier farmacologici in maniera specifica verso i macrofagi. L’invenzione consiste nella sintesi di nuove molecole derivate dal mannosio utilizzate come funzionalizzanti di superficie per vettori micro- e nano- particellari progettati per attività intra-5 macrofagica di farmaci somministrabili per via inalatoria.

2017 - Self-Assembled Lipid Nanoparticles for Oral Delivery of Heparin-Coated Iron Oxide Nanoparticles for Theranostic Purposes [Articolo su rivista]
Truzzi, Eleonora; Bongio, Chiara; Sacchetti, Francesca; Maretti, Eleonora; Montanari, Monica; Iannuccelli, Valentina; Vismara, Elena; Leo, Eliana Grazia
abstract

Recently, solid lipid nanoparticles (SLNs) have attracted increasing attention owing to their potential as an oral delivery system, promoting intestinal absorption in the lymphatic circulation which plays a role in disseminating metastatic cancer cells and infectious agents throughout the body. SLN features can be exploited for the oral delivery of theranostics. Therefore, the aim of this work was to design and characterise self-assembled lipid nanoparticles (SALNs) to encapsulate and stabilise iron oxide nanoparticles non-covalently coated with heparin (Fe@hepa) as a model of a theranostic tool. SALNs were characterised for physico-chemical properties (particle size, surface charge, encapsulation efficiency, in vitro stability, and heparin leakage), as well as in vitro cytotoxicity by methyl thiazole tetrazolium (MTT) assay and cell internalisation in CaCo-2, a cell line model used as an indirect indication of intestinal lymphatic absorption. SALNs of about 180 nm, which are stable in suspension and have a high encapsulation efficiency (>90%) were obtained. SALNs were able to stabilise the heparin coating of Fe@hepa, which are typically unstable in physiological environments. Moreover, SALNs-Fe@hepa showed no cytotoxicity, although their ability to be internalised into CaCo-2 cells was highlighted by confocal microscopy analysis. Therefore, the results indicated that SALNs can be considered as a promising tool to orally deliver theranostic Fe@hepa into the lymphatic circulation, although further in vivo studies are needed to comprehend further potential applications.

2017 - Surface engineering of Solid Lipid Nanoparticle assemblies by methyl α-d-mannopyranoside for the active targeting to macrophages in anti-tuberculosis inhalation therapy [Articolo su rivista]
Maretti, Eleonora; Costantino, Luca; Rustichelli, Cecilia; Leo, Eliana Grazia; Croce, Maria Antonietta; Francesca, Buttini; Truzzi, Eleonora; Iannuccelli, Valentina
abstract

This study describes the development of new mannosylated Solid Lipid Nanoparticle assemblies (SLNas) delivering rifampicin for an inhaled treatment of tuberculosis. SLNas were surface engineered with mannose residues to recognize mannose receptors located on infected alveolar macrophages and facilitate cell internalization. Two sets of SLNas were produced by the melt emulsifying technique using biocompatible lipid components, i.e. cholesteryl myristate combined with palmitic acid (PA set) or tripalmitin (TP set), in the presence of the targeting moiety, methyl α-d-mannopyranoside. Mannosylated SLNas were examined for their physical properties, drug payloads and release, as well as respirability in terms of emitted dose and respirable fraction determined by Next Generation Impactor. The most appropriate formulations were assessed for mannosylation using FTIR, XPS, SEM coupled with EDX analysis, and wettability assay, in comparison with the respective non-functionalized SLNas. Besides, cytotoxicity and cell internalization ability were established on J774 murine macrophage cell line. Mannosylated SLNas exhibited physical properties suitable for alveolar macrophage passive targeting, adequate rifampicin payloads (10-15%), and feasible drug maintenance within SLNas along the respiratory tract before macrophage internalization. Despite respirability impaired by powder cohesiveness, surface mannosylation provided quicker macrophage phagocytosis, giving evidence of an active targeting promotion.

2016 - Anti-TB inhalation therapy: Design of mannose-based functionalised Solid Lipid Microparticles for an active targeting to alveolar macrophages [Abstract in Atti di Convegno]
Maretti, Eleonora; Rustichelli, Cecilia; Costantino, Luca; Truzzi, Eleonora; Sacchetti, Francesca; Leo, Eliana Grazia; Iannuccelli, Valentina
abstract

Human tuberculosis (TB) is mainly a disease of the lung characterised by a long chronic stage of infection and progressive pathology that compromise the respiratory system. This is a curable infectious bacterial disease caused by the Mycobacterium tuberculosis (Mtb). TB therapies have exploited conventional routes of administration, such as oral and intramuscular1. The pulmonary route appears the most reasonable and effective way to target the alveolar macrophages (AM) and eradicate surviving Mtb at the primary infected site of TB, especially considering that 75-80% of cases remain localised in the lungs. The anti-TB therapy by inhalation offers benefits compared with the current treatment in terms of patient’s compliance improvement, reduction in dose amount and frequency, treatment duration and TB diffusion in other organs, thus minimising the risk of drug-resistant mutants, toxicity and side effects. For a direct intramacrophagic antitubercular therapy using Dry Powder Inhaler (DPI) devices, Solid Lipid Microparticles (SLM), produced using the melt emulsifying technique followed by freeze-drying, were developed to load rifampicin, a first-line antitubercular drug. In the present project, SLM were modified to improve drug loading level and release as well as AM targeting. Several biocompatible lipid components such as fatty acids and their derivatives, diglycerides and triglycerides, were processed using mixtures of biocompatible stabilisers (sodium taurocholate and methyl mannopyranoside) in order to obtain SLM with maximum efficiency in terms of drug loading and release in simulated lung fluid. Lipids in the liquid physical state embedded into SLM provided Microstructured Lipid Carriers (MLC) that are known to exhibit superior advantages over SLM such as enhanced drug loading capacity and prevention of drug expulsion intended to maximise the drug concentration at the primary site of TB infection. The obtained microcarriers were examined for their intrinsic properties such as size and size distribution, morphology and shape, surface charge, bulk and tap density, aerodynamic diameter, physical state of the components, wettability, drug loading and release. Macrophages, as is common knowledge, possess mannose-specific membrane receptors (MR) that can be recognised by carriers bearing mannose residues, facilitating their internalisation 2, 3. Therefore, the functionalisation of SLM surface by mannose derivatives used as the co- stabiliser in the SLM formulation was used to achieve an active targeting. The actual presence of mannose on SLM surface was investigated by means of X-ray Photoelectron Spectroscopy for Chemical Analysis (XPS) and Energy Dispersive X-ray Analysis (EDX).

2016 - Application of the "in-oil nanoprecipitation" method in the encapsulation of hydrophilic drugs in PLGA nanoparticles [Articolo su rivista]
Dalpiaz, Alessandro; Sacchetti, Francesca; Baldisserotto, Anna; Pavan, Barbara; Maretti, Eleonora; Iannuccelli, Valentina; Leo, Eliana Grazia
abstract

Three hydrophilic model drugs with different characteristics and molecular weights, namely protamine sulphate, diclofenac sodium and N6-Cyclopentyladenosine (CPA), were nano-encapsulated in poly(d,l-lactide-co-glycolide) (PLGA) using a novel “in-oil nanoprecipitation” method recently developed for the purpose. Although the same settings were used for all three model drugs, the drug loading efficiency was greatly dependent on their chemical–physical characteristics, being considerably higher for protamine (roughly 93%), intermediate for diclofenac (roughly 50%), and very low for CPA (roughly 7%). The resulting particle size and drug release rates were also strictly model-drug dependent. In the attempt to improve the characteristics of the CPA-loaded nanoparticles, the respective effects of adding an excipient (lauric acid) and substituting PLGA with poly(d,l-lactide) polymer (PLA) were investigated by measuring in vitro drug release and drug degradation kinetics in human whole blood. The results indicate that the proposed method seems promising for the nanoencapsulation of hydrophilic drugs in hydrophobic polymers, and easily modifiable to suit molecules that are difficult to incorporate into a polymeric matrix.

2016 - Characterization of natural clays from italian deposits with focus on elemental composition and exchange estimated by edx analysis: potential pharmaceutical and cosmetic uses [Articolo su rivista]
Iannuccelli, Valentina; Maretti, Eleonora; Sacchetti, Francesca; Romagnoli, Marcello; Bellini, Alessia; Truzzi, Eleonora; Miselli, Paola; Leo, Eliana Grazia
abstract

Purification processes performed on natural clays to select specific clay minerals are complex and expensive and can lead to over-exploitation of some deposits. The present study aimed to examine physicochemical (mineralogy, morphology, size, surface charge, chemical composition, cation exchange capacity [CEC], and pH) and hydration (swelling, wettability, water sorption, and rheological behavior) properties of three native clays from Italian deposits for potential pharmaceutical and cosmetic uses due to the presence of phyllosilicate minerals. Particular emphasis was placed on energy dispersive X-ray (EDX) microanalysis coupled with the ‘cesium method’ to assay clay elemental composition and CEC. One bentonite of volcanic origin (BNT) and two kaolins, one of hydrothermal origin (K-H) and another of lacustrine-fluvial origin (K-L), were evaluated in comparison with a commercial, purified bentonite. The CEC assay revealed the complete substitution of exchangeable cations (Na+ and Ca2+) by Cs+ in BNT samples and CEC values consistent with those of typical smectites (100.64 7.33 meq/100 g). For kaolins, partial substitution of Na+ cations occurred only in the K-L samples because of the interstratified mineral component which has small CEC values (11.13 5.46 meq/100 g for the K-H sample and 14.75 6.58 meq/100 g for the K-L sample). The degree of isomorphous substitution of Al3+ by Mg2+ affected the hydration properties of BNT in terms of swelling, water sorption, and rheology, whereas both of the poorly expandable kaolins exhibited significant water-adsorption properties. The EDX microanalysis has proved to be of considerable interest in terms of providing more information about clay properties in comparison with other commonly used methods and to identify the role played by both chemical and mineralogical composition of natural clays for their appropriate use in pharmaceutical and cosmetic fields.

2016 - Development and in vitro characterization of SLN encapsulating magnetic Heparin coated Iron Oxide for theranostic application [Abstract in Atti di Convegno]
Truzzi, Eleonora; Mazza, Federica; Sacchetti, Francesca; Maretti, Eleonora; Iannuccelli, Valentina; Vismara, Elena; Leo, Eliana Grazia
abstract

Solid Lipid Nanoparticles (SLN) have been proposed for the oral delivery of drugs with poor oral bioavailability for their ability to be internalized directly by the lymphatic circulation, like chylomicrons, through intestinal absorption. Lymphatic system is considered an interesting target for anti-cancer drugs and contrast agents because exerts an active role in the cancer metastasis being the major route of the solid tumor spread. Recently, according to the potentiality of the iron oxide in diagnostic and of heparin in the cancer therapy, iron oxide nanoparticles non-covalently coated with heparin (Fe@hepa) have been proposed as delivery systems for theranostic application (1). The aim of this work was to encapsulateFe@hepa in a biocompatible solid lipid shell in order to obtain a nanotheranostic tool for promoting the oral absorption through the lymphatic route. SLN have been formulated by a modified self nanoemulsifying technique by using Gelucire50/13 and Geleol™. The resulted Fe@hepa-SLN were characterized regarding size, morphology, storage stability as well in vitro release of heparin and iron oxide. In addition, preliminary studies on Caco-2 cell line were carried out evaluating cytotoxicity by MTT tests and internalization by the direct quantification of Fe@hepa inside the cells. Fe@hepa-SLN displayed a mean diameter below 300 nm, suitable for the oral administration, and an incorporation efficiency of 75% ± 3.9. Morphology analyisis showed the lipid shell surrounding the Fe@hepa nanoparticles and the release studies demonstrated that this lipid envelop stabilized the heparin coating in physiological conditions. Finally, studies on Caco2 cells showed the low cytotoxicity of the Fe@hepa-SLN and their ability to be internalized in the cells used as intestinal permeability model. These results indicate that this novel nanotechnology strategy could be a promising tool for oral nanotheranostic approaches.

2016 - Medicinal Chemistry drives drug targeted delivery: a successful interplay [Abstract in Atti di Convegno]
Costantino, Luca; Maretti, Eleonora; Rustichelli, Cecilia; Truzzi, Eleonora; Sacchetti, Francesca; Leo, Eliana Grazia; Iannuccelli, Valentina
abstract

Targeted drug delivery is object of an intense research. A medicinal chemistry approach allowed us to modify an opioid peptide in order to remove the opioid activity and retain the ability to cross the blood-brain barrier. Polyester-based nanoparticles (Np) surface-decorated with this peptide were shown to be able to deliver loperamide (a model drug) into CNS, as well as cholesterol (for the treatment of Huntington’s disease) and albumin (a model of a cargo protein). With a view of clinical translation, however, polyester Np seems to be not well suited for CNS diseases. Thus, we decided to use our peptide in alternative ways other than as a conjugate with the carboxyl group of the polyester PLGA, starting material for the production of the Np. We develop a new synthetic procedure that allow the conjugation of the peptide with substrates containing hydroxyl groups, less reactive than the carboxy group of the polyester (we considered as a substrate the poly(vinyl alcohol)), as well as a linker that could be inserted between a cargo and the peptide targeting moiety. At the same time, we moved towards lipidic carriers, a kind of delivery agents already clinically available. To gain experience on these carriers, we designed rifampicin-loaded Solid Lipid Nanoparticle assemblies (SLNas), for a direct intramacrophagic antitubercular therapy using Dry Powder Inhaler (DPI) devices and, in the first instance, methyl mannopyranoside as targeting ligand, able to interact with mannose receptors present on macrophages. Results obtained will be presented and discussed.

2016 - SURFACE ENGINEERING OF SOLID LIPID NANOASSEMBLIES FOR INHALED INTRAMACROPHAGIC ANTI-TB THERAPY [Abstract in Atti di Convegno]
Costantino, Luca; Maretti, Eleonora; Truzzi, Eleonora; Rustichelli, Cecilia; Leo, Eliana Grazia; Zapparoli, Mauro; Iannuccelli, Valentina
abstract

For an inhaled tuberculosis (TB) treatment, antibiotic aerosolization has to be produced by using drugs in their solid state administered by means of Dry Powder Inhaler (DPI) devices. In this regard, untreated drugs generally fail to reach alveolar epithelium and penetrate alveolar macrophages (AM) as the primary site of the infection.1 Therefore, the urgency to treat TB disease effectively may be addressed with approaches consisting of micro- or nanoparticulate carriers redeveloping existing drugs to reach the intended goal.2, 3 Specific modifications of the particulate carrier surface by conjugation with molecules that can specifically bind the receptors (active targeting) are expected to boost the particle avidity to cells increasing accumulation and intracellular uptake. Macrophages possess mannose-specific membrane receptors (MR) that can recognize and facilitate the internalization of carriers bearing mannose residues. In particular, the infected AM have an overexpression of MR.4 In the present study, surface engineered Solid Lipid Nanoparticle assemblies (SLNas) were developed as potential carriers of rifampicin, a first choice antitubercular drug, intended to maximize drug concentration at the primary site of TB infection. To increase specificity for macrophages and internalization potential, SLNas surface was functionalized by a mannosylated derivative to induce AM active targeting. Biocompatible lipid components such as fatty acids and their derivatives, diglycerides and triglycerides were processed by means of the melt emulsifying technique using biocompatible surfactants (sodium taurocholate and methyl mannopyranoside). Mannosylated SLNas were examined for their intrinsic properties (size and size distribution, shape, surface charge, bulk and tap density, aerodynamic diameter, porosity, flowability, physical state of the components). Powder breathability in terms of Emitted Dose and Fine Particle Fraction was assayed by Next Generation Impactor (NGI). This information on powder interparticle adhesion and deaggregation ability influencing powder dispersion and deposition onto alveolar epithelia. SLNas mannosylation was investigated by means of X-ray Photoelectron Spectroscopy for Chemical Analysis and Energy Dispersive X-ray Analysis. Prototypes of SLNas in terms of successful functionalization, optimal breathability and chemico-physical stability, were examined for cytotoxicity by MTT test on murine macrophages J774 cell lines.

2016 - Solid Lipid Nanoparticle assemblies (SLNas) for an anti-TB inhalation treatment-A Design of Experiments approach to investigate the influence of pre-freezing conditions on the powder respirability [Articolo su rivista]
Maretti, Eleonora; Rustichelli, Cecilia; Romagnoli, Marcello; Balducci, Anna Giulia; Buttini, Francesca; Sacchetti, Francesca; Leo, Eliana Grazia; Iannuccelli, Valentina
abstract

For direct intramacrophagic antitubercular therapy, pulmonary administration through Dry Powder Inhaler (DPI) devices is a reasonable option. For the achievement of efficacious aerosolisation, rifampicin-loaded Solid Lipid Nanoparticle assemblies (SLNas) were developed using the melt emulsifying technique followed by freeze-drying. Indeed, this drying method can cause freezing or drying stresses compromising powder respirability. It is the aim of this research to offer novel information regarding pre-freezing variables. These included type and concentration of cryoprotectants, pre-freezing temperature, and nanoparticle concentration in the suspension. In particular, the effects of such variables were observed at two main levels. First of all, on SLNas characteristics – i.e., size, polydispersity index, zeta-potential, circularity, density, and drug loading. Secondly, on powder respirability, taking into account aerodynamic diameter, emitted dose, and respirable fraction. Considering the complexity of the factors involved in a successful respirable powder, a Design of Experiments (DoE) approach was adopted as a statistical tool for evaluating the effect of pre-freezing conditions. Interestingly, the most favourable impact on powder respirability was exerted by quick-freezing combined with a certain grade of sample dilution before the pre-freezing step without the use of cryoprotectants. In such conditions, a very high SLNas respirable fraction (>50%) was achieved, along with acceptable yields in the final dry powder as well as a reduction of powder mass to be introduced into DPI capsules with benefits in terms of administered drug dose feasibility.

2015 - Enhanced anti-hyperproliferative activity of human thymidylate synthase inhibitor peptide by solid lipid nanoparticle delivery [Articolo su rivista]
Sacchetti, Francesca; Marraccini, Chiara; D'Arca, Domenico; Pela', Michela; Pinetti, Diego; Maretti, Eleonora; Hanuskova, Miriam; Iannuccelli, Valentina; Costi, Maria Paola; Leo, Eliana Grazia
abstract

Recently, octapeptide LSCQLYQR (LRp), reducing growth of cis-platinum (cDDP) resistant ovarian carcinoma cells by inhibiting the monomer–monomer interface of the human enzyme thymidylate synthase, has been identified. As the peptide is not able to cross the cell membrane it requires an appropriate delivery system. In this work the application of SLNs, biocompatible and efficient tools for the intracellular drug transport, applied especially for lipophilic drugs, was exploited for the delivery of the hydrophilic peptide LRp. SLNs formulated in the absence/presence of small amount of squalene showed dimensions below 150 nm, negative zeta potential and good stability to the freeze-drying process. Even though the particles formulated with squalene exhibited a less ordered crystal lattice and a lower surface hydrophobicity, a rapid drug release from these nanocarriers occurred as a result of the relevant expulsion of the drug from the lipid core during lipid crystallization. On the contrary, SLNs formulated in the absence of squalene were able to incorporate more stably the peptide showing considerable cytotoxic effect on cDDP resistant C13* ovarian carcinoma cell line at concentration 50 times lower than that used previously with a marketed delivery system. From the cell cycle analysis by the propidium iodide test in SLNs-peptide treated cancer cells an increase of apoptosis percentage was observed, indicating that SLNs were able to carry efficiently the peptide until its enzymatic target.

2015 - Gastroretentive montmorillonite-tetracycline nanoclay for the treatment of Helicobacter pylori infection [Articolo su rivista]
Iannuccelli, Valentina; Maretti, Eleonora; Montorsi, Monia; Rustichelli, Cecilia; Sacchetti, Francesca; Leo, Eliana Grazia
abstract

The paper aims to explore the potential benefits provided by an organically modified montmorillonite (nanoclay) in the problematic management of the Helicobacter pylori gastric infection that is one of the most prevalent infectious diseases worldwide. Two nanoclay samples were produced by the intercalation of tetracycline (TC) into the interlayer of montmorillonite (MM) under two different pH reaction conditions (pH 3.0 and 8.7). MM/TC nanoclays were characterized by EDX, XRD, FTIR, DSC, drug adsorption extent, in vitro mucoadhesiveness and desorption in simulated gastric media. The reaction between MM and TC led to a complete MM cation (Na+ and Ca2+) exchange process, an increase of MM characteristic interlayer spacing as well as an involvement of NHR3+ group of TC, regardless of the reaction pH value. However, MM/TC nanoclay obtained under alkaline conditions provided a lower TC adsorption as well as a drug fraction weakly linked to MM in comparison with the nanoclay obtained in acidic conditions. Both the nanoclays exhibited good mucoadhesion properties to porcine mucin and TC desorption occurring mainly via a cation exchange process by H+ ions. Based on the results obtained, TC intercalation into MM nanoplatelets could represent a potential advantageous approach allowing the antibiotic to distribute homogeneously on the gastric mucosa, diffuse through the gastric mucus layer and achieve the microorganism localization.

2015 - In vitro behaviour of hybrid lipid/chitosan nanoparticles for the oral delivery of heparin [Abstract in Atti di Convegno]
Sacchetti, Francesca; Raffaella, Aracri; Maretti, Eleonora; Iannuccelli, Valentina; Montanari, Monica; Barbara, Pavan; Alessandro, Dalpiaz; Leo, Eliana Grazia
abstract

2015 - Inhaled SLM for anti-TB therapy by DPI device: process parameters affecting freeze-drying and breathability [Abstract in Atti di Convegno]
Maretti, Eleonora; Porcheddu, Eleonora; Imbuluzqueta, Edurne; Balducci, Anna Giulia; Buttini, Francesca; Sacchetti, Francesca; Romagnoli, Marcello; Leo, Eliana Grazia; Iannuccelli, Valentina
abstract

The advantages of an inhaled anti-TB therapy over parenteral or oral administration are inherent to drug delivery directly to the alveolar macrophages, in which M. tuberculosis survives, bypassing gastrointestinal barriers and hepatic metabolism, so obtaining rapid clinical response, decreased dose, dose frequency, treatment period, side-effects and drug-resistance. Moreover, since 75-80% of TB cases remain localized in the lungs, inhalation therapy could also arrest TB dissemination to other organs by maximizing drug concentration at the infected sites in the lungs, also achieving therapeutic but non toxic systemic levels of drugs. Concerning inhaled anti-TB therapy, very limited marketed, pre-clinical and clinical trials are available, although successful results of few research studies on volunteers. Recently, the scientific research has revived an interest in the administration of anti-TB drugs by inhalation especially due to the advent of multi-drug-resistance (MDR-TB) and extensively drug resistant (XDR-TB) strains. However, studies with dried powder formulations are relatively scarce although the benefits of a DPI device compared with MDI or nebulizers: no propellants, no coordination between the patient and the device, drug stability owing to its dry state which makes DPIs suitable for developing countries in warm climates, higher drug payload delivery, portability, and patient compliance. The inhalation of antibiotics alone fails in its attempt to reach alveoli owing to negative powder physical properties. Moreover, inhaled antibiotics alone showed poor uptake by alveolar macrophages (AM) in cell line studies (Hirota et al., 2009). Technological approach to obtain powder fluidization, deaggregation and flowability with proper breathability to target the most distal lung airways, and capacity to be taken up by alveolar macrophages are needed. Among the strategies aiming to make antibiotics breathable, particle engineering on drug alone (controlled crystallization, different morphology by spray-drying technique), or on drug embedded into microcarriers (liposomes, microparticles) were proposed. Microparticles could modify drug flowability acting on their density, surface features and interparticle cohesive forces, drug release and AM phagocytosis. Microparticles were found also able to activate AM innate bactericidal mechanism. Among the breathable microparticulate systems, most of the studies have focused on polymeric microparticles or liposomes, and less attention has been paid to Solid Lipid Microparticles (SLM) although their advantages in terms of stability. Based on these assumptions, biocompatible, biodegradable and eco-friendly processable SLM loaded with rifampicin, a first-line anti-TB drug, able to be taken up by AM and induce intracellular bactericidal effect were designed in a perspective of an inhaled therapy by means a DPI device for the treatment of TB infection. SLM were previously in vitro characterized showing proper aerodynamic size, drug bactericidal activity maintenance, low cytotoxicity and good capacity to be taken up by murine macrophage cell lines J774 (Maretti et al., 2014). In the present work parameters affecting interparticle forces such as sample water dilution before the freeze-drying process, quick freezing at lower temperature, and cryoprotectant use were evaluated in order to improve the powder breathability.

2015 - Nanoencapsulation of an hTS inhibitor octapeptide against ovarian cancer in solid lipid matrix [Abstract in Atti di Convegno]
Sacchetti, Francesca; Marraccini, Chiara; Cannazza, Giuseppe; Iannuccelli, Valentina; Hanuskova, Miriam; Maretti, Eleonora; Costi, Maria Paola; Leo, Eliana Grazia
abstract

New octapeptides able to reduce the growth of platinum-resistant cells by inhibiting the enzyme human thymidylate synthase (hTS), cannot cross the cell membrane alone and require an appropriate delivery system. In the aim to transport hTS inhibiting LR octapeptide (LR-op) into the cells, Solid Lipid Nanoparticles (SLNs) were developed and evaluated in vitro. The optimized SLNs were formulated in the absence and presence of squalene (7S and 7Sq) both in the LR-op loaded and unloaded form. All the SLNs produced had dimensions below 150 nm, negative Zpotential and a good stability both in suspension and after freeze-drying. Only the sample obtained in the absence of squalene showed to stably incorporate the LR-op promoting its cell internalization, as demonstrated by in vitro studies on C13* ovarian carcinoma cell line.

2015 - PROGETTAZIONE E OTTIMIZZAZIONE DI SISTEMI MICROPARTICELLARI PER LA VEICOLAZIONE AI MACROFAGI ALVEOLARI DI FARMACI ANTITUBERCOLARI PER VIA INALATORIA [Poster]
Maretti, Eleonora; Rustichelli, Cecilia; Romagnoli, Marcello; Leo, Eliana Grazia; Iannuccelli, Valentina
abstract

La tubercolosi (TBC), causata dal Mycobacterium tuberculosis, è una patologia infettiva trasmissibile per via aerea che interessa un terzo della popolazione mondiale e rappresenta il principale fattore di mortalità per le persone colpite da HIV. E’ stata dichiarata dall'OMS “a major global health problem” con una elevata incidenza e un aumento dei casi di farmacoresistenza (MDR-TB). La strategia del piano proposto dall’OMS per ridurre l’impatto della TBC comprende lo sviluppo di strumenti nuovi ed efficaci al fine di prevenire, individuare e trattare la patologia (WHO, 2014). I limiti dall’attuale terapia di tipo convenzionale, per via orale o parenterale, risiedono nell’elevato dosaggio, nel lungo periodo di trattamento e nei numerosi effetti collaterali che possono essere evitati mediante lo sviluppo di Drug Delivey Systems innovativi in grado di modulare l’azione di farmaci già in uso. Poiché la tubercolosi polmonare è caratterizzata dal coinvolgimento dei macrofagi alveolari nei quali i bacilli rimangono vitali, la somministrazione di farmaci anti-TBC direttamente ai polmoni mediante carrier microparticellari consentirebbe il vantaggioso targeting ai macrofagi alveolari di antibiotici impossibilitati a diffondere attraverso le membrane cellulari. Ciò nonostante, non esistono, ad oggi, farmaci anti-TBC somministrabili per via inalatoria approvati per l’uso umano. Ancora meno studiati, nella progettazione di microcarrier per questa terapia, nonostante i benefici in termini di biocompatibilità, risultano i materiali naturali, quali i lipidi. Tra questi, quelli in grado di generare superfici microparticellari cariche o contenenti molecole coinvolte nel processo endocitico potrebbero promuovere l’uptake macrofagico del farmaco. Al fine di ottenere una polvere biocompatibile che, una volta inalata mediante Dry Powder Inhaler (DPI), favorisca la captazione del farmaco da parte dei macrofagi alveolari, il presente studio è stato finalizzato allo sviluppo e ottimizzazione di microparticelle caratterizzate da dimensioni nell'ambito della respirabilità (0.5-5 µm) per la veicolazione di un farmaco di prima linea quale la rifampicina. A tale scopo, sono stati impiegati materiali naturali, biocompatibili e biodegradabili, quali lipidi solidi, trattati con metodologie eco-friendly, in assenza di solventi organici. Sono state, pertanto, sviluppate Solid Lipid Microparticles (SLM) di acido stearico stabilizzate con sodio taurocolato al fine di direzionare il chemioterapico ai macrofagi alveolari. Il microcarrier formulato è stato caratterizzato dal punto di vista chimico-fisico per determinarne morfologia, dimensioni, carica superficiale, densità, diametro aerodinamico, livello di caricamento e rilascio in vitro del farmaco, attività antimicrobica, frazione respirabile, citotossicità e capacità di internalizzazione su linee cellulari macrofagiche J774. Il microcarrier è risultato idoneo per proprietà aerodinamiche, citotossicità e internalizzazione da parte di macrofagi murini (Maretti, 2014). Inoltre, l’analisi di alcuni parametri coinvolti nel processo di liofilizzazione, quali temperatura di congelamento, presenza di crioprotettori e diluizione del campione, eseguita mediante uno studio statistico di Design of Experiment (DOE), ha evidenziato la loro rilevante influenza sulla frazione respirabile del prodotto finale permettendone l’ottimizzazione.

2015 - SOLID LIPID MICROPARTICLES FOR INHALED ANTI-TB THERAPY BY DPI: INFLUENCE OF THE PRODUCTION PROCESS ON DRUG STABILITY AND POWDER BREATHABILITY [Poster]
Maretti, Eleonora; Bellani, Martina; Rustichelli, Cecilia; Sacchetti, Francesca; Romagnoli, Marcello; Balducci, Anna Giulia; Buttini, Francesca; Leo, Eliana Grazia; Iannuccelli, Valentina
abstract

According to the WHO global report, tuberculosis (TB) remains one of the world’s deadliest communicable diseases. The current therapy involves three/four drug oral regimen, long-term therapy, high and frequent doses so producing several side-effects [1]. To overcome these drawbacks and improve treatment efficacy, the new strategies could involve new formulation design for old drugs. Among these, the shortest-term goal is represented by Drug Delivery Systems (DDS). In this latter context, considering that 75-80% of TB cases remain localized in the lungs, pulmonary route appears the most strategical route [2]. For an efficient drug delivery by Dry Powder Inhaler (DPI) device, several powder properties (particle microsize, irregular shape, low tap density, surface charge, weak adhesion between particles, good flowability) contribute to determining powder aerodynamic performance and, consequently, deposition onto alveolar epithelium, and phagocytosis by alveolar macrophages [3]. Based on these assumptions, Solid Lipid Microparticles (SLM), known to be biocompatible, biodegradable and physically stable were designed as the carrier for rifampicin (RIF). The present research focused on the evaluation of both RIF stability during the production phases and the role of variables relating to freeze-drying process (freezing conditions, sample dilution, cryoprotectants) affecting the powder aerosolization. Considering the complexity of the factors involved in a successful breathable powder, a statistical Design of Experiments (DOE) was adopted to study the critical variables that influence the final product. SLM were obtained by the melt emulsification technique under sonication by using stearic acid and sodium taurocholate as lipid and surfactant, respectively [4]. Loaded SLM were prepared by adding RIF in the melted lipid. All the emulsions were rapidly cooled to room temperature providing SLM that were purified by dialysis and freeze-dried. Freeze-drying was carried out following dilution with water, mixture with cryoprotectant (trehalose or mannitol), and lowering temperature of freezing. SLM exhibited an irregular shape and the following value ranges: size (470 - 1700 nm), PDI (0.32 – 0.94), circularity (0.43 – 0.66), bulk density (0.02 – 0.24 g/cm3), tapped density (0.04 – 0.31 g/cm3) and drug loading level (11.85 – 15.88%). The Emitted Dose and the Fine Particle Mass were between 92.1-103.4% and 0.9-6.83 mg, respectively. DOE approach highlighted the combination of the water dilution before freezing with the cryoprotectant use as the most important parameter to obtain a highly breathable powder as well as the influence of water dilution and freezing temperature on SLM breathability. For a powder to be inhaled by a DPI device, many parameters can guarantee quality and efficacy of the product. The analysis performed demonstrated RIF stability and proved to be efficient in distinguishing the major contribution factors on the final product and identifying the key factors that are helpful for the improvement of SLM production process.

2015 - Solid Lipid Microparticles for inhaled anti-TB therapy by DPI: influence of the production process on drug stability and powder breathability [Abstract in Atti di Convegno]
Maretti, Eleonora; Sacchetti, Francesca; Romagnoli, Marcello; Balducci, Anna Giulia; Buttini, Francesca; Leo, Eliana Grazia; Iannuccelli, Valentina
abstract

According to the WHO global report 2014, tuberculosis (TB) remains one of the world’s deadliest communicable diseases, despite implementation of highly standard treatment regimen that has caused the number of cases began to decline from 1992. The current therapy following WHO guidelines involves three/four drug oral regimen (first-line drugs), long-term therapy (6/7 months), high and frequent doses so producing several side-effects, in particular hepatotoxicity (WHO, 2014). The long therapy, responsible for patient’s non-compliance that is the most common reason for treatment failure, is connected to Mycobacterium tuberculosis (Mtb) nature. Unlike most microbes, Mtb survives in AM phagosomes, has a slow growing and metabolism besides death, so that the drugs have to be taken for a long period. To overcome these drawbacks and improve treatment efficacy, the new strategies could involve vaccination (limited success), new drug development (no new drugs in the last 30 year) and new formulation design for old drugs. Among these, the shortest-term goal is represented by new technological approaches or Drug Delivery Systems (DDS). In this latter context, considering that 75-80% of TB cases remain localized in the lungs, pulmonary route appears the most logical route to reach promptly the primary infected site providing to reduced dose and dose frequency, treatment duration, TB dissemination in other organs risk of drug-resistant mutants and toxicity as well as improving patient’s compliance (Pham D-D., 2015). Among the portable inhalation devices, Dry Powder Inhaler (DPI) appears more advantageous than Metered Dose Inhaler (MDI), considering low water solubility of most anti-TB drugs, propellant absence and drug stability in its dry state (Hoppentocht, 2014). For an efficient drug delivery by DPI, several powder properties (particle microsize, irregular shape reducing particle contact area so favoring powder deaggragation, low tapped density, surface charge, weak adhesion between particles, good flowability) contribute to determining powder aerodynamic performance and, consequently, dose emission and dispersion, deposition onto alveolar epithelium, and phagocytosis by alveolar macrophages (Claus, 2014). Based on these assumptions, Solid Lipid Microparticles (SLM), constituted by a solid lipid core stabilized by a surfactant on their surface, are known to be biocompatible, biodegradable, physically stable, and obtainable by using low cost materials and eco-friendly processes without organic solvents. More, SLM are suitable to incorporate firmly high lipophilic drug loading levels, and they are not hygroscopic avoiding so powder flowability compromising. In a previous study, SLM loaded with rifampicin (RIF), a first-line anti-TB drug, were designed in a perspective of an inhaled therapy by means a DPI device and found capable of preserving drug antimicrobial activity and being taken up by murine macrophages cell lines (Maretti, 2014). The present research focused on the evaluation of both RIF stability during the production phases and the role of variables relating to freeze-drying process (freezing conditions, sample dilution, cryoprotectants) affecting the powder aerosolization. The freezing method can gave a significant effect on the ice structure affecting both water-vapor flow during the primary drying and the ice crystal size, so influencing the dry final product. Considering the complexity of the factors involved in a successful breathable powder, a statistical Design of Experiments (DOE) was adopted to study the critical variables that influence the final product.

2015 - The improvement of Solid Lipid Microparticle breathability for an anti-TB inhalation therapy [Abstract in Atti di Convegno]
Maretti, Eleonora; Eleonora, Porcheddu; Leo, Eliana Grazia; Edurne, Imbuluzqueta; Francesca, Buttini; Giulia, Balducci Anna; Sacchetti, Francesca; Iannuccelli, Valentina
abstract

For a strategy based on inhalation therapy for tuberculosis by a Dry Powder Inhaler (DPI) device, powder physical properties play a crucial role on powder deposition onto alveolar epithelium and macrophage phagocytosis. The study aimed to assess the role of parameters involved in freeze-drying process of Solid Lipid Microparticles (SLM) loaded with rifampicin, a first-line anti-TB drug. Excellent breathability in terms of both emitted dose (∼95%) and breathable fraction (∼90%), better than that obtained by cryoprotectants, was achieved by sample water dilution and freezing at a very low temperature before freeze-drying. These results indicate the relevant role of the ice crystal structure on SLM aggregation for a superior DPI performance.

2015 - pH sensitive PEGylated Liposomes delivering active hydrophilic peptide with anticancer activity: in vitro study on cDDP-resistant ovarian cell line [Abstract in Atti di Convegno]
Sacchetti, Francesca; Marraccini, Chiara; D'Arca, Domenico; Pinetti, Diego; Genovese, Filippo; Maretti, Eleonora; Iannuccelli, Valentina; Costi, Maria Paola; Leo, Eliana Grazia
abstract

Thymidylate synthase (TS) can be considered a very interesting molecular target for the therapy of the ovarian cancer.. Recently, specific octapeptides able to reduce the growth of platinum-resistant cells by inhibiting the enzyme human thymidylate synthase (hTS), have been identified. Similarly to the majority of peptides, they cannot cross the cell membrane and require a delivery system for transport into the cells and pH sensitive liposomes, destabilizing at mildly acidic pH, are considered efficient tools for delivering water-soluble drugs into the cell cytoplasm. In the present study in order to attain the peptide triggering in the cells promoting endosomal escape, stealth pH-sensitive liposomes were developed and characterized. Results suggested that pH sensitive liposomes seemed suitable carriers for the encapsulation of small hydrophilic molecules like peptides. The appreciable difference in cytotoxicity between loaded and unloaded liposomes demonstrated that the peptide, whose activity is held in the cytoplasm, was triggered in the proper biological site.

2014 - Brain uptake of a Zidovudine prodrug after nasal administration of solid lipid microparticles. [Articolo su rivista]
A., Dalpiaz; L., Ferraro; D., Perrone; Leo, Eliana Grazia; Iannuccelli, Valentina; B., Pavan; G., Paganetto; S., Beggiato; S., Scalia
abstract

Our previous results demonstrated that a prodrug obtained by the conjugation of the antiretroviral drug zidovudine (AZT) with ursodeoxycholic acid (UDCA) represents a potential carrier for AZT in the central nervous system, thus possibly increasing AZT efficiency as anti-HIV drug. Based on these results and in order to enhance AZT brain targeting, the present study focuses on solid lipid microparticles (SLMs) as a carrier system for the nasal administration of UDCA-AZT prodrug. SLMs were produced by the hot emulsion technique, using tristearin and stearic acid as lipidic carriers, whose mean diameters were 16 and 7 μm, respectively. SLMs were of spherical shape and their prodrug loading was 0.57 ±0.03% (w/w, tristearin based) and 1.84 ±0.02% (w/w, stearic acid based). The tristearin SLMs were able to control the prodrug release, whereas the stearic acid SLMs induced a significant increase of the dissolution rate of the free prodrug. The free prodrug was rapidly hydrolyzed in rat liver homogenates with a half-life of 2.7 ± 0.14 min (process completed within 30 min). The tristearin SLMs markedly enhanced the stability of the prodrug (75 % of the prodrug still present after 30 min), whereas the stabilization effect of the stearic acid SLMs was lower (14 % of the prodrug still present after 30 min). No AZT and UDCA-AZT were detected in the rat cerebrospinal fluid (CSF) after an intravenous prodrug administration (200 μg). Conversely, the nasal administration of stearic acid based SLMs induced the uptake of the prodrug in the CSF, demonstrating the existence of a direct nose – CNS pathway. In the presence of chitosan, the CSF prodrug uptake increased six times, up to 1.5 μg/mL within 150 minutes after nasal administration. The loaded SLMs appear therefore as a promising nasal formulation for selective zidovudine brain uptake.

2014 - Development and characterization of PLGA nanoparticles as delivery systems of a prodrug of zidovudine obtained by its conjugation with ursodeoxycholic acid [Articolo su rivista]
Dalpiaz, Alessandro; Contado, Catia; Mari, Lara; Perrone, Daniela; Pavan, Barbara; Paganetto, Guglielmo; Hanuskova, Miriam; Vighi, Eleonora; Leo, Eliana Grazia
abstract

The main purpose of this study was to investigate the effect of the Pluronic F68 coating on the loading, release and stability of PLGA nanoparticles embedded with a prodrug of zidovudine, an anti HIV agent, obtained by its conjugation with ursodeoxycholic acid. The mean diameter of the nanoparticles prepared by nanoprecipitation or emulsion/solvent evaporation methods was determined using both photon correlation spectroscopy and sedimentation field–flow fractionation (SdFFF) and resulted about 600 nm with a relatively high polidispersity. The nanoparticles obtained by emulsion/solvent evaporation method were not able to control the prodrug release while the nanoparticles obtained by nanoprecipitation were able to control the release of the prodrug, showing a burst release of about 50%. The presence of the Pluronic coating did not substantially modify the kinetics of the drug release nor the extent of the burst effect which was instead only influenced by the preparation parameters. The prodrug incorporated in the nanoparticles was more stable in the rat liver homogenates than the free prodrug and no influence of the Pluronic coating was observed. Considering the different potential applications of nanoparticles coated and uncoated with Pluronic, both of these nanoparticle systems could be useful in the therapies against HIV

2014 - Development of lipid nanocarriers as delivery systems for a small peptide with anti-ovarian activity [Abstract in Atti di Convegno]
Sacchetti, Francesca; Cazzato, ADDOLORATA STEFANIA; Marraccini, Chiara; Cannazza, Giuseppe; Iannuccelli, Valentina; Maretti, Eleonora; Costi, Maria Paola; Leo, Eliana Grazia
abstract

The encapsulation of a small peptide in SLN was achieved modifying the hot high shear homogenization method. The data obtained by the comparison of SLN to standard Liposomes suggested that even if Liposomes are more efficient carrier for hydrophilic peptides, it is possible to embed this kind of molecules in a solid lipid matrix achieving carriers with higher in vitro stability and lower cytotoxicity. Moreover, the ability of the loaded carriers to reduce the cell viability more efficiently than the unloaded vectors, indicates that the peptide was released inside the cell environment being able to exert its action.

2014 - In vitro evaluation on CaCo-2 cells of hybrid lipid/chitosan nanoparticles for the oral delivery of heparin. [Abstract in Atti di Convegno]
Sacchetti, Francesca; Aracri, Raffaella; Maretti, Eleonora; Iannuccelli, Valentina; Montanari, Monica; Leo, Eliana Grazia
abstract

Enhanced oral bioavailability of poorly aqueous soluble drugs encapsulated in a number of lipid-based formulations, including emulsions, micellar systems, self-emulsifying drug delivery systems, liposomes and solid lipid nanoparticles (SLN) via lymphatic delivery has been documented (1). In the present work, SLN were designed for the oral delivery of heparin in order to take advantage from the lymphatic intestinal transport pathway. In order to improve the incorporation of a high hydrophilic compound in a lipid matrix, heparin was “insolubilized” by the coupling with chitosan. In this aim we have developed chitosan/heparin Polyelectrolyte complexes (PEC). Such as systems are able to complex stably heparin (up to pH < 6.8) (2) and after pelletization by centrifugation were embedded in SLN obtaining a hybrid system lipid/chitosan nanoparticles (PEC-SLN). Since no in-vitro lymphoid tissue is currently available, CaCo-2 cell monolayer could be considered an alternative in vitro model to be used as a screening tool before animal studies are undertaken (1). Aim of the work In this work naked PEC, hybrid PEC-SLN as well as heparin-loaded SLN (Hep-SLN) were characterized as regard as the size, zeta potential, morphological characteristics, drug loading and in vitro drug release. Moreover FITC labeled PEC along with Red Nile labeled PEC-SLN and empty SLN were evaluated on CaCo-2 cell line in order to study their cytotoxicity by MTT test and their cell internalization ability by cytometric and confocal analysis. Experimentals Size and zeta potential were measured by Zetasizer Nano ZS (Malvern), morphological characteristics by SEM-FEI (SEM, Nova NanoSEM 450, Fei) and by AFM (Park Autoprobe Atomic Force Microscope (Park Instruments) Intenalization extent was visualized by confocal laser scanning microscopy (CLSM) (Leica DM IRE2) Results and discussion Results demonstrated that PEC size is highly influenced by pH, being 322 ±30 nm at the optimal pH 6.5. PEC-SLN and Hep-SLN displayed a size between 150 and 400 nm and a zeta potential from -20 to -36 mV. The drug loading was higher for PEC-SLN respect to Hep-SLN (50.4 ± 6 and 32.4 ± 4 UI/100 mg, respectively) while the heparin release rate was faster for Hep-SLN than for PEC-SLN, achieving a percentage of heparin released of 100% and 20%, respectively, in 6 h in simulated intestinal fluid. These data indicate that heparin was not well encapsulated in the Hep-SLN while after conjugation with chitosan stable heparin encapsulation was achieved in SLN. Finally naked PEC labeled with FITC along with PEC-SLN labeled with Red Nile was evaluated in vitro on CaCo-2 cells. MTT test showed that all the samples were poor cytotoxic even for long incubation time (overnight). Internalization data showed that PEC complexes (A) achieved a poor internalization level in the cells while PEC-SLN (B) have proved to be able to entry CaCo-2 cells in a time-dependent manner. Conclusions PEC-SLN being able to enter in the CaCo-2 cells unlike the naked PEC, probably due to their lipidic nature, can be considered promising carrier to be further studied as promoter for the lymphatic intestinal transport of heparin

2014 - In vivo penetration of bare and lipid-coated silica nanoparticles across the human stratum corneum [Articolo su rivista]
Iannuccelli, Valentina; Bertelli, Davide; Romagnoli, Marcello; S., Scalia; Maretti, Eleonora; Sacchetti, Francesca; Leo, Eliana Grazia
abstract

Skin penetration of silica nanoparticles (NP) currently used in pharmaceutical and cosmetic products is a topic of interest not only to evaluate their possible toxicity, but also to understand their behaviour upon contact with the skin and to exploit their potential positive effects in drug or cosmetic delivery field. Therefore, the present work aimed to elucidate the in vivo mechanism by which amorphous hydrophilic silica NP enter human stratum corneum (SC) through the evaluation of the role played by the nanoparticle surface polarity and the human hair follicle density. Two silica samples, bare hydrophilic silica (B-silica, 162 ± 51 nm in size) and hydrophobic lipid-coated silica (LC-silica, 363 ± 74 nm in size) were applied on both the volar and dorsal side of volunteer forearms. Twelve repetitive stripped tapes were removed from the human skin and evaluated for elemental composition by Energy Dispersive X-ray (EDX) analysis and for silicon content by Inductively Coupled Plasma quadrupole Mass Spectrometry (ICP-MS). All the stripped tapes revealed nanosized structures generally located in the broad spaces between corneocytes and characterized by the same elemental composition (relative weight percentage of silicon and silicon to oxygen weight ratio) than that of the applied samples. However, only about 10% B-silica permeated until the deepest SC layers considered in the study indicating a silica retention in the upper layers of SC, regardless of the hair follicle density. Otherwise, the exposure to LC-silica led to a greater silica skin penetration extent into the deeper SC layers (about 42% and 18% silica following volar and dorsal forearm application, respectively) indicating that the NP surface polarity played a predominant role on that of their size in determining the route and the extent of penetration.

2014 - Inhaled Solid Lipid Microparticles to target alveolar macrophages for tuberculosis [Articolo su rivista]
Maretti, Eleonora; Rossi, Tiziana; Bondi, Moreno; Croce, Maria Antonietta; Hanuskova, Miriam; Leo, Eliana Grazia; Sacchetti, Francesca; Iannuccelli, Valentina
abstract

The goal of the work was to evaluate an anti-tubercular strategy based on breathable Solid Lipid Microparticles (SLM) to target alveolar macrophages and to increase the effectiveness of the conventional tuberculosis (TB) therapy. Rifampicin loaded SLM composed of stearic acid and sodium taurocholate were characterized for aerodynamic diameter, surface charge, physical state of the components, drug loading and release as well as drug biological activity on Bacillus subtilis strain. Moreover, SLM cytotoxicity and cell internalization ability were evaluated on murine macrophages J774 cell lines by MTT test, cytofluorimetry and confocal laser microscopy. SLM exhibited aerodynamic diameter proper to be transported up to the alveolar epithelium, negative charged surface able to promote uptake by the macrophages and preserved drug antimicrobial activity. The negligible in vitro release of rifampicin indicated the capacity of the microparticle matrix to entrap the drug preventing its spreading over the lung fluid. In vitro studies on J774 cell lines demonstrated SLM non-cytotoxicity and ability to be taken up by cell cytoplasm. The microparticulate carrier, showing features suitable for the inhaled therapy and for inducing endocytosis by alveolar macrophages, could be considered promising in a perspective of an efficacious TB inhaled therapy by means of a Dry Powder Inhaler device.

2014 - Lipid-based microparticles for TB inhaled therapy: physical properties and cell internalization [Abstract in Atti di Convegno]
Maretti, Eleonora; Rossi, Tiziana; Leo, Eliana Grazia; Montanari, Monica; Romagnoli, Marcello; Sacchetti, Francesca; Iannuccelli, Valentina
abstract

Tuberculosis (TB) disease is caused by Mycobacterium tuberculosis that survives and replicates within human alveolar macrophages and is characterized by a long chronic stage of infection and progressive pathology mainly compromising (90% of cases) the respiratory system. Current TB therapies have exploited conventional routes of administration, such as oral or intramuscular, based on high and frequent dosages to maintain the drug therapeutic concentration in infection site because of poor drug permeability, poor drug bioavailability and pre-systemic clearance. An alternative acceptable therapy to systemic treatments involves inhalation route delivering the drug directly to the desired site, enabling a rapid onset of the action and avoiding the long period of the current treatment and the first-pass metabolism, as well as the use of high doses of drug resulting in drug resistance onset and in severe side effects on other organs. Inhaled TB therapy can presuppose the development of micro- or nanoparticles acting as drug carriers toward the alveolar region in the deepest lung so inducing the endocytosis process of alveolar macrophages being many antimicrobials difficult to cross cell membranes (1-3). Lipid-based particulate systems have been poorly investigated for TB inhaled therapy (4) though they were generally recognized as safe, poor liable to swell upon contact with the moisture located into the lungs and, consequently, to release the drug before the target site. Among the lipid-based particulate systems, Solid Lipid Microparticles (SLM), constituted by a solid lipid core stabilized by a surfactant at the surface, exhibit several favourable properties as production without organic solvents and long-term stability. In the present study, SLM loaded with rifampicin, a first-line anti-TB drug, were developed by the melt emulsification technique and evaluated in a perspective of an inhaled therapy for the treatment of TB infection. The lipid-based microparticles designed as rifampicin carrier showed features proper to be delivered from a DPI device, to deposit onto alveolar epithelium and to be internalized by macrophages in which Mycobacterium tuberculosis resides.

2013 - Cell internalization study of hybrid chitosan-heparin-PLGA nanocarriers on Caco-2 cells [Abstract in Atti di Convegno]
Leo, Eliana Grazia; R., Aracri; Sammak, Susan; Costantino, Luca; Sacchetti, Francesca; Vighi, Eleonora; Iannuccelli, Valentina
abstract

Purpose: Oral delivery of Heparin, the most potent anticoagulant drug, could have a great clinical impact, offering undoubted advantages over injectable formulations. The aim of this work was to evaluate the ability of hybrid Chitosan-Heparin-PLGA nanocarriers to be taken up by CaCo-2 cells, a model of mature enterocytes of the absorbent epithelium of intestine. Methods: Firstly, Chitosan/Heparin nanocoacervates were prepared by polyelectrolyte complexation method; subsequently, the coacervates were coated with PLGA to be stabilized at intestinal pH, resulting in hybrid Chitosan-Heparin-PLGA nanocarriers. Free Chitosan/Heparin nanocoacervates and hybrid nanoparticles were characterized regarding size, z-potential, morphology and Heparin release in gastric and intestinal simulated media. The influence of the size and of the time contact with cells on the ability of the carriers to be internalized was studied on Caco-2 cell monolayers by using fluorescence microscopy. In order to visualize the carriers, FITC-Chitosan and Rodamine- PLGA were employed. Results: It was found that in the case of free coacervates (from 300 to 500 nm) the smaller the size, the higher was the extent of cell internalization, regardless of the time contact with cells (4 or 18 h). On the contrary, in the case of hybrid Chitosan-Heparin-PLGA nanocarriers, showing an increased size (until 700 nm) due to the polymeric coating, the cell internalization increased according to the contact-time with cells. Conclusion: The size of nanocarriers greatly influences the cell internalization rate in CaCo-2 cells. Although hybrid systems showed a low internalization rate, being stable in the intestinal environment unlike the free coacervates, they seem a promising approach to be further investigated for the oral absorption of Heparin.

2013 - Design flexibility influencing the in vitro behavior of cationic SLN as a nonviral gene vector [Articolo su rivista]
Vighi, Eleonora; Montanari, Monica; Hanuskova, Miriam; Iannuccelli, Valentina; Coppi, Gilberto; Leo, Eliana Grazia
abstract

In this paper SLN were prepared using stearic acid as main lipid component, stearylamine as cationic agent and protamine as transfection promoter and adding phosphatidylcholine (PC), cholesterol (Chol) or both to obtain three different multicomponent SLN (SLN-PC, SLN-Chol and SLN-PC-Chol, respectively). Cytotoxicity and transfection efficiency of the obtained SLN:pDNA complexes were evaluated on three different immortalized cell lines: COS-I (African green monkey kidney cell line), HepG2 (human hepatocellular liver carcinoma cell line) and Na1300 (murine neuroblastoma cell line). Samples were characterized for the exact quantitative composition, particle size, morphology, zeta potential and pDNA binding ability. All the three SLN samples were about 250-300nm in size with a positive zeta potential, whereas SLN:pDNA complexes were about 300-400nm in size with a less positive zeta potential, depending on the SLN composition. Concerning the cell tolerance, the three samples showed a level of cytotoxicity lower than that of the positive control polyethylenimine (PEI), regardless of the cell lines. The best transfection performance was observed for SLN-PC-Chol on COS-I cells while a transfection level lower than PEI was observed on HepG2 cells, regardless the SLN composition. On Na1300 cells, SLN-Chol showed a double efficiency with respect to PEI. Comparing these results to those obtained with the same kind of SLN without PC and/or Chol, it is possible to conclude that the addition of Chol and/or PC to the composition of cationic SLN modify the cell tolerance and the transfection efficiency of the gene vector in a manner strictly dependent on the cell type and the internalization pathways.

2013 - In vivo detection of lipid-based nano- and microparticles in the outermost human stratum corneum by EDX analysis [Articolo su rivista]
Iannuccelli, Valentina; Coppi, Gilberto; Romagnoli, Marcello; Sergi, Santo; Leo, Eliana Grazia
abstract

Lipid-based particulate delivery systems have been extensively investigated in the last decade for both pharmaceutical and cosmetic skin application although their translocation across the skin is not yet clarified. The aim of this paper was to investigate on humans the ability of Solid Lipid Nanoparticles (SLN) and Solid Lipid Microparticles (SLM) to penetrate the outermost stratum corneum (SC) and to be modified upon contact with the cutaneous components by using the Tape Stripping Test coupled with the Energy Dispersive X-ray (EDX) analysis. SLN and SLM were prepared by the melt emulsification technique and loaded with nanosized titanium dioxide (TiO2) to become identifiable by means of X-ray emission. Following human skin application, the translocation of the particulate systems was monitored by the analysis of twelve repetitive stripped tapes using non-encapsulated metal dioxide as the control. Intact SLN as well as non-encapsulated TiO2 were recorded along the largest SC openings until the 12th stripped tape suggesting the intercluster region as their main pathway. Evidences of a concurrent biodegradation process of the lipid matrix, as the result of SLN interaction with the lipid packing between the corneocyte clusters, were found in the deepest SC layers considered. On the contrary, SLM were retained on the skin surface without undergoing biodegradation so preventing the leaching and the subsequent SC translocation of the loaded TiO2.

2013 - In vivo silica nanoparticle translocation across human stratum corneum: the effect of surface hydrophobization in relation to hair follicle density. [Abstract in Atti di Convegno]
Iannuccelli, Valentina; Bertelli, Davide; Romagnoli, Marcello; Maretti, Eleonora; Sacchetti, Francesca; Leo, Eliana Grazia
abstract

Insoluble nano- and submicron-scale metal dioxides are widely used in pharmaceutical and cosmetic formulations designed for being applied on the skin surface and acting mainly as thickeners, sunscreens and pigments. Since it’s primarily for the insoluble nanoparticles that health concerns related to possible skin uptake arise, the EU requires producers to submit a detailed safety report on the nanomaterials used as well as declaring their presence on the label. As noted in the 2007 FDA Nanotechnology Task Force report, there may be a higher degree of uncertainty associated with nanoscale materials compared to conventional chemicals, both with respect to knowledge about them and the way that testing is performed. Indeed, the issue of insoluble nanoparticle skin penetration, mainly focusing on nanosized TiO2 and ZnO, is still controversial, divided between a penetration limited to the hair follicles and the translocation across the lipid pathway within SC cells sometimes reaching living skin cells. Among the metal dioxide materials, colloidal silicon dioxide, a submicroscopic amorphous hydrophilic silica is one of the most used in terms of commercial production amounts together with TiO2, ZnO and silver nanoparticles. With regard to topical formulations, it is used to stabilize emulsions and as a thixotropic thickening or suspending agent in gels, microemulsions, and semisolid preparations. Although amorphous silica is generally regarded as an essentially nontoxic and non-irritant excipient [1] also proposed for implants, the nano-scale feature might alter the bioavailability of the cosmetic formulation [2]. Actually, silica nanoparticles have been shown in vitro to modulate the diffusion through animal skin of hydrophilic and hydrophobic model drugs (caffeine, retinol, quercetin) from topical emulsions to affect skin properties and also to penetrate up to the viable epidermis and upper dermis of excised porcine skin [3, 4]. Previously, we demonstrated on human beings that colloidal silica is able to penetrate in the upper region of the human stratum corneum (SC) and different mechanisms of translocation were hypothesized [4]. A similar finding arose from a research on ultra-fine TiO2 [5]. Therefore, the goal of the present work was to investigate in vivo the mechanism of silica entry into SC by means of qualitative and quantitative assays performed on stripped tapes removed from volunteers treated with silica incorporated in a standard cream. With this objective, the study took into account the influence of human hair follicle density by means of sample application on both volar and dorsal forearm. Moreover, by considering that skin surface is hydrophobic and skin penetration requires particle wetting by the SC lipids, the effect of silica surface hydrophobization was also investigated. Twelve repetitive tape strips removed from each forearm of the volunteers following sample application were evaluated by EDX analysis by points. All the tapes removed from the skin that has received silica and H-silica application showed nanosized and nearly spherical structures generally located in rather broad intercellular spaces. Their EDX spectra exhibited the characteristic peaks owing to X-ray emission from Si atoms (Fig. n. 1). The relative weight percentages of the metal and the metal to oxygen weight ratio were found to be equivalent to those detected in the samples before their application. These findings could give evidence of a movement of both the nanoparticles across the outermost part of SC reaching the corneum compactum along broad channels between the corneocytes. Silicon quantitative analysis carried out on the removed tapes indicated silica accumulation in the outer part of SC corresponding to the corneum disjunctum and its significant decrease inside the deepest tapes, regardless of the sample and the hair follicle density. Nevertheless, the higher silica level detect

2013 - Influence of secondary preparative parameters and aging effects on PLGA particle size distribution: a sedimentation field flow fractionation investigation [Articolo su rivista]
C., Contado; Vighi, Eleonora; A., Dalpiaz; Leo, Eliana Grazia
abstract

Poly(lactic-co-glycolic acid) particles in the 200-400-nm size range were formulated through nanoprecipitation and solvent evaporation methods. Different concentrations of the polymer and stabilizer (Pluronic® F 68) were tested in order to identify the best conditions for making poly(lactic-co-glycolic acid) particles of suitable size, stable in time, and to be used as carriers for brain-targeting drugs. The particles with the best characteristics for delivery system design were those formulated by nanoprecipitation with an organic/water phase ratio of 2:30, a polymer concentration of 25 mg/mL, and a surfactant concentration of 0.83 mg/mL; their surface charge was reasonably negative (approximately -27 mV) and the average size of the almost monodisperse population was roughly 250 nm. Particle characterization was obtained through ζ-potential measurements, scanning electron microscope observations, and particle size distribution determinations; the latter achieved by both photon-correlation spectroscopy and sedimentation field flow fractionation. Sedimentation field flow fractionation, which is considered more reliable than photon-correlation spectroscopy in describing the possible particle size distribution modifications, was used to investigate the effects of 3 months of storage at 4 °C had on the lyophilized particles

2013 - Inhalated drug delivery systems to target alveolar macrophages for tuberculosis therapy: design of safe SLM loaded with rifampicin [Abstract in Atti di Convegno]
Maretti, Eleonora; Iannuccelli, Valentina; Leo, Eliana Grazia; Bondi, Moreno; Croce, Maria Antonietta; Sacchetti, Francesca; Rossi, Tiziana
abstract

The present research aimed to improve the effectiveness of TB treatment by a non conventional therapy and using teh respiratory tract as a novel adminictration route for rifampicin. The study dealt with the design of Solid Lipid Microparticles (SLM) to be delivered by a Dry Powder Inhaler (DPI) device and to target the alveolar macrophages. The negligible in vitro drug release indicated the capacity of the matrix to firmly entrap the drug. Rifampicin maintained its biological activity during the preparation steps. Moreover, SLM were suitable to be taken up by murine J774 cells.

2013 - Solid Lipid Nanoparticles for the sustained release of a prodrug of Zidovudine by its conjugation with ursodeoxycholic acid [Abstract in Atti di Convegno]
A., Dalpiaz; Leo, Eliana Grazia; Iannuccelli, Valentina; S., Scalia
abstract

Purpose: We report a study of encapsulation and release from solid lipid microparticles (SLMs) of a prodrug of zidovudine (AZT), an antiviral agent, obtained by its ester conjiugation with a bile acid, the ursodeoxycholic acid (UDCA). We have demonstrated that this prodrug is able to elude the active efflux systems expressed on physiological barriers. Results: The mean diameters of tristerain and stearic acid loaded microparticles were 7 and 14 micron, respectively. The shape of the SLMs was spherical and their prodrug loading was 0.57% (tristearin based) and 1.84% (stearic acid based). The tristearin SLMs were able to control the release of the prodrug, whereas the stearic acid SLMs induced a significant increase of the dissolution rate of the free prodrug obtained in the presence of Twwen 60. Conclusions. The developed SLMs were represent a good candidate for the local delivery of UDCA-AZT in the nose and its potential uptake in the brain.

2013 - Tetracycline-Montmorillonite nanoclay for the treatment of H. pylori infection [Abstract in Atti di Convegno]
Iannuccelli, Valentina; Montorsi, Monia; Sacchetti, Francesca; Maretti, Eleonora; Coppi, Gilberto; Leo, Eliana Grazia
abstract

Purpose: To explore the potential benefit provided by an organically modified phyllosilicate in the problematic management of the Helicobacter pylori gastric infection that is one of the most prevalent infectious disease worldwide. Methods: An organically modified layered-silicate or nanoclay was produced by the intercalation of tetracycline (TC) into the interlayer of montmorillonite (MM) under two different pH reaction conditions (pH 3.0 and 8.7). MM-TC organoclays were characterized by EDX, XRD, FT-IR, DSC, drug content and in vitro release. Moreover, the organoclays were evaluated in vitro for mucoadhesiveness and buoyancy ability upon suspension in a sodium alginate hydrogel. Results: The reaction between clay and TC led to a complete MM cation (Na+ and Ca2+) exchange process, to an increase of MM characteristic interlayer spacing as well as to an involvement of NHR3+ group of TC, regardless of the medium pH value, as shown by EDX, XRD and FT-IR analyses. However, MM-TC obtained under alkaline condition determined a lower sorption of TC into clay in comparison with MM-TC obtained in acidic condition as well as a drug fraction weakly linked to MM and quickly released. Both the organoclays exhibited good mucoadhesion properties to mucin as well as to porcine gastric mucosa and good floatability in a sodium alginate water solution. Conclusion: TC intercalated into MM nanoplatelets and dispersed in an alginate hydrogel could represent an advantageous formulation allowing the antibiotic to distribute homogeneously on the gastric mucosa and to diffuse gradually into the gastric mucus layer where the microorganism is localized.

2013 - Vettori particellari per il rilascio sito-specifico di farmaci nel tratto gastro-intestinale [Abstract in Atti di Convegno]
Iannuccelli, Valentina; Leo, Eliana Grazia; Coppi, Gilberto
abstract

Nell’ambito della progettazione di Drug Delivery Systems rivolti alla somministrazione di farmaci per via orale, sono stati sviluppati dispositivi particellari biocompatibili e biodegradabili per il direzionamento di farmaci nel tratto gastro-intestinale superiore (stomaco e duodeno) utilizzando materiali polimerici polisaccaridici e semplici tecnologie produttive. Un dispositivo a unità multiple con prolungato Gastric Residence Time (GRT), in grado di veicolare un’ampia gamma di farmaci, è stato ottimizzato in vitro e valutato in vivo su volontari in condizioni pre- e postprandiali. Le unità progettate, costituite da un nucleo di calcio alginato separato, mediante un compartimento d’aria, da un rivestimento di calcio alginato/PVA, sono state monitorate mediante tecnica radiografica risultando in grado di galleggiare sul contenuto gastrico, senza lag time, in condizioni di stomaco pieno, anche dopo un pasto leggero, determinando un incremento del GRT (da 2 a 9 ore in funzione del regime nutrizionale). La somministrazione a volontari delle unità quali carrier di riboflavina, caratterizzata da una finestra biologica di assorbimento nel primo tratto intestinale, ha prodotto un aumento della sua biodisponibilità del 50-80%, in funzione del regime nutrizionale, e una diminuzione della variabilità individuale del tempo di transito gastrico. Un secondo dispositivo, costituito da microparticelle mucoadesive di calcio alginato/chitosano quali carrier di farmaci instabili in ambiente gastrico e a biodisponibilità orale critica (polimixina B, gentamicina, tamoxifene), ottenute mediante tecnica di spray-drying, è stato ottimizzato in vitro e valutato su linee cellulari e su modelli animali ex-vivo e in vivo. Le microparticelle, valutate mediante microscopi a fluorescenza e confocale, hanno permesso di direzionare i farmaci al Gut Associated Lymphoid Tissue (GALT) del primo tratto intestinale, proteggendoli dall’ambiente gastrico, per poi aderire alla mucosa intestinale ed essere captate dalle cellule M delle placche del Peyer con il coinvolgimento di meccanismi anche non linfoidi. La permanenza delle microparticelle a livello del GALT ha determinato nel ratto livelli serici di farmaco prolungati rispetto a quelli ottenibili somministrando la soluzione di farmaco come tale e diminuiti effetti tossici monitorati su diversi organi. Inoltre, studi effettuati su linee cellulari MCF-7 hanno evidenziato una tossicità selettiva e una attività dell’antitumorale tamoxifene in funzione del tipo di alginato impiegato.

2012 - Detection of solid lipid nanoparticles in human stratum corneum by EDX analys [Abstract in Atti di Convegno]
Iannuccelli, Valentina; Leo, Eliana Grazia; Sergi, Santo; Coppi, Gilberto
abstract

The purpose of the work was to examine in vivo the structural modifications and the translocation ability of Solid Lipid Nanoparticles (SLN) loaded with titanium or silicon dioxides across stratum corneum (SC) by EDX analysis. Intact SLN can translocate across SC until the corneum compactum where a biodegradation process can occur suggesting an interaction with the lipid packing between the corneocyte clusters supposed as the main pathway. Conversely, lipid microparticles were retained on the skin surface preventing the loaded metal dioxides from permeating the uppermost SC layers.

2012 - Influence of penetration enhancers and lipid nanoparticles on in vivo human skin penetration of quercetin [Abstract in Atti di Convegno]
S., Scalia; Leo, Eliana Grazia; Iannuccelli, Valentina
abstract

Quercetin has been shown in animal and human skin to inhibit, in vitro an in vivo, the adverse effects caused by the sun UV rays. However, the skin protective activity of quercetin is hampered by its inefficient percutaneous penetration. Object of this study was the comparative evaluation of emulsions containing penetration enhancer or lipid noanoparticles on the skin penetration of the flavonoid.The study indicated that the nanoparticles achieved a targeting effect, favouring the localisation of the flavonoid in the superficial skin layers.

2012 - Polymeric nanoparticles for the substained release of a prodrug of zidovudine obtained by its conjugation with the ursodeoxycholic acid [Abstract in Atti di Convegno]
A., Dalpiaz; M., Fogagnolo; D., Perrone; C., Contado; B., Pavan; G., Paganetto; Vighi, Eleonora; Iannuccelli, Valentina; Leo, Eliana Grazia
abstract

We report a study of encapsulation and release from PLGA nanoparticles of a prodrug of zidovudine obtained by its ester conjugation with a bile acid, the ursodeoxycholic acid. The free prodrug was hydrolizated in rat liver homogenates with an half life of 2.7 min, whereas the loaded NP nanoparticles appeared able to significantly stabilize the prodrug in this physiological fluid.

2012 - Release modulation of hydrophilic drugs from polymeric nanoparticles produced by in-oil nanoprecipitation process [Abstract in Atti di Convegno]
Leo, Eliana Grazia; Vighi, Eleonora; B., Pavan; A., Baldisserotto; Iannuccelli, Valentina; A., Dalpiaz
abstract

A novel in oil-nanoprecipitation method using a mixture of cottonseed oil and Tween-80 as non-solvent phase has been developed for the nanoincapsulation of water-soluble drugs in poly (D,L-lactide-coglicolide) (PLGA). In this work the method was applied to three hydrophilic drugs with different structure and molecular weight. The results indicate that the CPA stability in whole blood was increased after nanoincapsulation according to the in vitro profile release pattern. The proposed method appears promising for the nanoincapsulation of hydrophilic durgs in hydrophobic polymers.

2012 - Studying the in vitro behaviour of cationic solid lipid nanoparticles as a nonviral vector [Articolo su rivista]
Vighi, Eleonora; Leo, Eliana Grazia
abstract

SLNs are basically composed of a solid lipid core in nanometer ranges stabilized by a layer of emulsifier; they can be prepared by using lipids with a relatively high melting point (i.e., triglycerides, hard fat types, partial glycerides, steroids and waxes). Among these lipids, glycerides, which are composed of fatty acids, can be employed in injection form since they are already used in parenteral nutrition

2012 - The role of protamine amount in the transfection performance of cationic SLN designed as a gene nanocarrier [Articolo su rivista]
Vighi, Eleonora; Montanari, Monica; Ruozi, Barbara; Iannuccelli, Valentina; Leo, Eliana Grazia
abstract

Cationic solid lipid nanoparticles (SLN) have been recently proposed as non-viral vectors in systemic gene therapy. The aim of this study was to evaluate the effect of the protamine amount used as the transfection promoter in SLN-mediated gene delivery. Three protamine-SLN samples (Pro25, Pro100, and Pro200) prepared by adding increasing amounts of protamine were characterized for their size, zeta potential, and protamine loading level. The samples were evaluated for pDNA complexation ability by gel-electrophoresis analysis and for cytotoxicity and transfection efficiency by using different cell lines (COS-I, HepG2, and Na1300). The size of SLN was ~230 nm and only Pro200 showed few particle aggregates. Unlike the Pro25 sample with the lowest protamine loading level, the others SLN samples (Pro100 and Pro200) exhibited a good ability in complexing pDNA. A cell-line dependent cytotoxicity lower than that of the positive control PEI (polyethilenimmine) was observed for all the SLN. Among these, only Pro100, having an intermediate amount of protamine, appeared able to promote pDNA cell transfer, especially in a neuronal cell line (Na1300). In conclusion, the amount of protamine as the transfection promoter in SLN affects not only the gene delivery ability of SLN but also their capacity to transfer genes efficiently to specific cell types.

2011 - In vitro behaviour of multicomposite cationic SLN as new platform for pDNA delivery [Relazione in Atti di Convegno]
Vighi, Eleonora; Iannuccelli, Valentina; Coppi, Gilberto; Leo, Eliana Grazia
abstract

The enhancement of non viral vectors is a priority in the field of gene therapy, in order to manage effective and safe vectorsi. Among non viral vectors, solid lipid nanoparticles (SLN) have emerged in the last years as an alternative to liposomes and PEI (poly-ethylene-imine) and many efforts have been spent in order to improve their performanceii. The present study aims to design and characterize in vitro multicomposite SLN as a novel platform for pDNA delivery: SLN matrix composition was modified by assembling several components able to optimize the carrier in terms of transfection efficieny and safety. In details, stearic acid was selected as the main lipid component along with stearylamine as its analogous cationic compound. Protamine and Pluronic F68 were included in the formulation as transfection promoter and surfactant, respectively. Cholesterol (Chol) and phosphatidylcholine (PC) were added to improve biocompatibility and plasticity of the carriers.

2011 - Structural investigation and intracellular trafficking of a novel multicomposite cationic solid lipid nanoparticle platform as a pDNA carrier [Articolo su rivista]
Vighi, Eleonora; Leo, Eliana Grazia; Montanari, Monica; Mucci, Adele; Hanuskova, Miriam; Iannuccelli, Valentina
abstract

Background: The ability to efficiently cross cellular barriers and accomplish high-level transgene expression is a critical challenge to broad application of nonviral vectors, such as cationic solid lipid nanoparticles (SLN).Aims: This study aims to design and characterize in vitro multicomposite SLN as a novel platform for pDNA delivery.Results/Discussion: The distribution of each component (stearic acid, stearylamine, phosphatidylcholine, cholesterol, protamine and Pluronic F68) in the SLN matrix was studied by electron spectroscopy for chemical analysis and NMR in order to establish its influence on SLN cytotoxicity and transfection efficiency. Multicomposite SLN mediated the expression of enhanced green fluorescent protein in a way comparable with the positive control,but inducing a lower cytotoxicity. Moreover, the carrier exhibited the ability to enter the nucleoli, probably as a result of the synergic action of the nuclear localization signal of protamine and the flexibility of the lipid matrix owing to the phosphatidylcholine. Conclusion: The multicomposite SLN showed good transfection efficiency and negligible cytotoxicity, both crucial factors for an efficient gene-delivery system. Considering the fact that nucleolihave emerged in recent years as important targets in many fields, this novel carrier could have significant future therapy involvements whenever there is a requirement to overcome subcellular barriers. However, further work needs to be carried out in order to fully characterize the formulation, to elucidate where alternative colloidal structures might exist and play a role in obtaining the results presented.

2010 - Nuclear localization of cationic solid lipid nanoparticles containing Protamine as transfection promoter [Articolo su rivista]
Vighi, Eleonora; Montanari, Monica; Ruozi, Barbara; Tosi, Giovanni; Magli, Alessandro; Leo, Eliana Grazia
abstract

Protamine has attracted much attention as DNA condenser and nuclear transfer enhancer although theexcess of hydrophilicity and the strong DNA pack restrain its potentialities. In order to overcome this lim-itation, we added Protamine in the composition of solid lipid nanoparticles (SLN-Protamine) and we com-pared this carrier with the same kind of SLN containing Esterquat 1 instead of Protamine (SLN-EQ1).Carriers cytotoxicity was assessed on COS-I cells evaluating the cell cycle by propidium iodide test, whilethe transfection efficiency was studied using pEGFP as plasmid model. The cell penetrating activity ofProtamine inside the lipid vectors was evaluated studying cell internalization by confocal microscopyusing Red Nile-labeled carriers. SLN-Protamine:pDNA showed a mean diameter five-times smaller thanthe size of SLN-EQ1:pDNA and a remarkably lesser cytotoxicity. Transfection by SLN-Protamine:pDNAwas seven-times more effective compared with the Protamine:pDNA polyplexes while no transfectioncapacity was observed for SLN-EQ1:pDNA complexes due to their inability to be internalized owing totheir larger dimension. Red Nile-SLN-Protamine were localized in endocytic-like vesicles into the nuclearmembrane suggesting the inclusion of Protamine in nano-lipophilic systems may enhance the reductionin the complex dimensions, the nuclear pDNA translocation and the pDNA release in the cell

2010 - Particulate adducts based on sodium risedronate and titanium dioxide for the bioavailability enhancement of oral administered bisphosphonates [Articolo su rivista]
V., Dissette; P., Bozzi; C. A., Bignozzi; A., Dalpiaz; L., Ferraro; S., Beggiato; Leo, Eliana Grazia; Vighi, Eleonora; L., Pasti
abstract

Adducts based on a bisphosphonate drug (sodium risedronate) and titanium dioxide (TiO2) particles have been developed and characterized in order to improve the bioavailability of orally administrated bisphosphonates. Nanocrystalline and colloidal TiO2, both characterized by powder X-ray diffraction, were used to obtain the adducts 1 and 2, respectively. Adducts 1 and 2 appeared constituted by nanoparticles of about 50 and 90 nm grouped in clusters of about 0,2 and 2,5 m, respectively. Higher amounts of drugs were adsorbed on adduct 2 (7,2 ± 0.3%) with respect to adduct 1 (4,0 ± 0.3%). In vitro studies demonstrate that the adducts were able to release the drug in the pH range 6-9, whereas they remained essentially stable in the pH range 0-5. In vivo studies indicate that after oral administration to male Wistar rats, the microparticles of adduct 2 were able to prolong the presence of risedronate in the bloodstream during an eight hours period, resulting in a relative bioavailability almost doubled with respect to the free drug. This behaviour allows envisioning an improvement of the risedronate therapeutic effects and/or a reduction of its frequency of administration with consequent reduction of gastrooesophageal injuries typically induced by oral administration of bisphosphonates.

2010 - The role of protamine in the gene delivery by cationic SLN on different cell lines [Abstract in Atti di Convegno]
Vighi, Eleonora; Montanari, Monica; Ruozi, Barbara; Battini, Renata; Leo, Eliana Grazia
abstract

In this study we produced cationic SLN using stearicacid, stearylamine and protamine sulfate astransfection promoter, in order to improve SLN transfection capacity without the use of adjuvantsubstances. In fact, protamine is a cationic smallprotein with high arginine content that is FDAapproved for the parenteral administration. Since protamine is a nuclear proteinthat helps DNA packaging in sperm cells, it is also used as transfection acceleratorin the gene delivery.Therefore, in this work we optimized the protamineamount in SLN formulation in order to evaluate therole of this protein in the transfection activity ofcationic SLN.

2010 - pDNA condensation capacity and in vitro gene delivery properties of cationic solid lipid nanoparticles [Articolo su rivista]
Vighi, Eleonora; Ruozi, Barbara; Montanari, Monica; Battini, Renata; Leo, Eliana Grazia
abstract

Cationic solid lipid nanoparticles (SLN) are promising nonviral gene delivery carriers suitable for systemic administration. The objective of this study was to investigate the relationship between the composition of cationic SLN and their ability to condense plasmid DNA (pDNA) and to transfer it in neuroblastoma cells. The SLN were prepared by using stearic acid and stearylamine as lipid core along with Esterquart 1 (EQ1) or Protamine obtaining two samples (SLN-EQ1 and SLN-Protamine, respectively). The cationic SLN were freeze-dried after preparation and their physical–chemical properties, including the surface composition and the transfection efficiency were investigated. The results showed that the two samples had similar size, zeta potential and pDNA binding properties but SLN-Protamine were able to condense pDNA more efficaciously than SLN-EQ1 forming smaller and less positive complexes. SLN-Protamine:pDNA complexes demonstrated to be less cytotoxic and more efficient in the transfection of Na1300 cell line than SLN-EQ1:pDNA. These findings were attributed to the different surface composition of the two samples and in particular to the localization of the Protamine on the surface of the particle while EQ1 in the lipid core. In conclusion the results here suggest that not only the z-potential but also the surface composition may affect the pDNA condensation proprieties and thus the transfection efficiency of nonviral gene nanocarriers.

2009 - Cationic solid lipid nanoparticles containing Protamine as transfection vector on neuroblastoma cell line [Relazione in Atti di Convegno]
Vighi, Eleonora; Ruozi, Barbara; Tosi, Giovanni; Battini, Renata; Montanari, Monica; Leo, Eliana Grazia
abstract

Sono stati presentati i risultati di studi effettuati in vitro sull'efficienza di transfezione di SLN allestite con diversi componenti tra i quali protamina.

2009 - Development of new formulations of cationic solid lipid nanoparticles (SLNs) for the drug delivery to the brain [Relazione in Atti di Convegno]
Vighi, Eleonora; Ruozi, Barbara; Montanari, Monica; Leo, Eliana Grazia
abstract

Development of new formulations of cationic solid lipid nanoparticles (SLNs) for the drug delivery to the brain

2009 - Fabrication Via a Nonaqueous Nanoprecipitation Method,Characterization and In Vitro Biological Behavior ofN6-Cyclopentyladenosine-Loaded Nanoparticles [Articolo su rivista]
A., Dalpiaz; E., Vighi; B., Pavan; Leo, Eliana Grazia
abstract

A novel nonaqueous nanoprecipitation method was proposed to achieve theencapsulation of a small weight hydrophilic drug (N6-cyclopentyladenosine, CPA) inPLGA nanoparticles using a mixture of cottonseed oil and Tween-80 as nonsolventphase. The nanoparticles were characterized in vitro as concerns size, morphology, drugloading, drug release, and drug stability in human blood. Human retinal pigmentepithelium (HRPE) cells were employed to study intracellular accumulation of encapsulatedor free CPA with and without unloaded particles, in the presence or absence of anequilibrative nucleoside transporter inhibitor. The particles displayed a mean size lowerthan 300 nm and a drug loading considerably higher than that found by conventionalencapsulation methods. The suitable in vitro release properties permitted to obtain gooddrug stabilization in the blood. Studies on HRPE cells suggested that CPA can permeatetheir membrane by both diffusive- and transport-mediated mechanisms. The loaded andunloaded nanoparticles appeared able to increase the permeation rate of the diffusivemechanism, without interfering with the transporter.

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 - Formulazione e caratterizzazione di nanoparticelle polimeriche per la somministrazione orale di eparina [Abstract in Atti di Convegno]
Vighi, Eleonora; Ruozi, Barbara; Montanari, Monica; Leo, Eliana Grazia
abstract

Nanoparticelle polimeriche per la somministrazione di eparina. studi di caratterizzazione tecnologica ed uptake in CACO-2

2008 - In vitro behaviour of N6-cyclopentyladenosine-loaded nanoparticles prepared via a non-aqueous nanoprecipitation method [Abstract in Atti di Convegno]
Leo, Eliana Grazia; S., Scalia; B., Pavan; Vighi, Eleonora; Ruozi, Barbara; Dalpiaz, Alessandro
abstract

In vitro behaviour of N6-cyclopentyladenosine-loaded nanoparticles prepared via a non-aqueous nanoprecipitation method

2008 - Preparation and characterization of particulate drug delivery systems for the brain targeting [Capitolo/Saggio]
A., Dalpiaz; C., Contado; Vighi, Eleonora; Tosi, Giovanni; Leo, Eliana Grazia
abstract

American Scientific Publishers

2008 - Veicolazione intracellualre di DNA plasmidico mediante solid lipid nanoparticles (SLN) cationiche [Relazione in Atti di Convegno]
Vighi, Eleonora; Ruozi, Barbara; Tosi, Giovanni; Montanari, Manuela; Battini, Renata; Leo, Eliana Grazia
abstract

Veicolazione intracellualre di DNA plasmidico mediante solid lipid nanoparticles (SLN) cationiche

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 - Characterization of cationic SLN/protamine complex as a non-viral transfer vector [Abstract in Atti di Convegno]
Vighi, Eleonora; Ruozi, Barbara; Tosi, Giovanni; Montanari, Monica; Leo, Eliana Grazia
abstract

Characterization of cationic SLN/protamine complex as a non-viral transfer vector

2007 - Complementary use of flow and sedimentation field-flow fractionation techniques for size characterizing biodegradable poly(lactic acid) nanospheres [Articolo su rivista]
Contado, C; Dalpiaz, A; Leo, Eliana Grazia; Zborowski, M; Williams, P. S.
abstract

Poly(lactic acid) (PLA) nanoparticles were synthesized using a modified evaporation method, testing two different surfactants (sodium cholateand Pluronic F68) for the process. During their formulation the prodrug 5-octanoyl-CPA (Oct-CPA) of the anti-ischemic N6-cyclopentyladenosine(CPA) was encapsulated. Three different purification methods were compared with respect to the influence of surfactant on the size characteristicsof the final nanoparticle product. Flow and sedimentation field-flow fractionation techniques (FlFFF and SdFFF, respectively) were used to sizecharacterize the five poly(lactic acid) particle samples. Two different combinations of carrier solution (mobile phase) were employed in the FlFFFanalyses, while a solution of poly(vinyl alcohol) was used as mobile phase for the SdFFF runs. The separation performances of the two techniqueswere compared and the particle size distributions (PSDs), derived from the fractograms, were interpreted with the support of observations byscanning electron microscopy. Some critical aspects, such as the carrier choice and the channel thickness determination for the FlFFF, have beeninvestigated. This is the first comprehensive comparison of the two FFF techniques for characterizing non-standard particulate materials. The twoFFF techniques proved to be complementary and gave good, congruent and very useful information on the size distributions of the five poly(lacticacid) particle samples.

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 - 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 - Re-dispersible cationic solid lipid nanoparticles (SLNs) freeze-dried without cryoprotectors: Characterization and ability to bind the pEGFP-plasmid [Articolo su rivista]
Vighi, Eleonora; Ruozi, Barbara; Montanari, Monica; Battini, Renata; Leo, Eliana Grazia
abstract

Cationic solid lipid nanoparticles (SLNs) have recently been suggested for non-viral gene delivery as a promising alternative to the liposomes. The aim of this study was to investigate the possibility to obtain re-dispersible cationic SLNs after a freeze-drying process in the absence of lyo- and/or cryoprotectors. The physical-chemical characteristics of cationic SLNs and their ability to bind gene material were investigated before and after the freeze-drying. To perform this study three samples of cationic SLNs, based on stearic acid, Compritol or cetylpalmitate, were prepared and characterized by PCS (photon correlation spectroscopy) and AFM (atomic force microscopy). The results indicated that solely the re-dispersed sample of stearic acid (SLN-SA) became very similar in terms of size and morphology to the fresh prepared sample, although it displayed a sensible reduction of the zeta potential (from 39.2 to 23.3 mV). By both the DSC (differential scanning calorimetry) and the ESCA (electron spectroscopy for chemical analysis) determinations, the reduction of the zeta potential was ascribed to the loss of the cationic lipids from the particle surface due to the rearrangement of the stearic acid lattice after the freeze-drying. Finally, the gel electrophoresis analysis demonstrated that SLN-SA re-suspended in PBS are unable to complex the DNA, while the SLN-SA re-dispersed in water displayed the same ability to bind DNA as the fresh prepared sample. We can conclude that cationic SLNs, based on stearic acid, retain the ability to complex DNA even after the freeze-drying in the absence of lyo- or cryoprotectors; thus, the powder form of this sample represents an attractive candidate to be investigated as in vivo DNA vector formulation.

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 - 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 - Nanoparticle formulation may affect the stabilization of an antiischemic prodrug [Articolo su rivista]
Leo, Eliana Grazia; C., Contado; F., Bortolotti; B., Pavan; A., Scatturin; Tosi, Giovanni; S., Manfredini; A., Angusti; A., Dalpiaz
abstract

The prodrug 5'-octanoyl-CPA (Oct-CPA) of the antiischemic N-6-cyclopentyladenosine (CPA) has been encapsulated by nanoprecipitation in poly(lactic acid) nanoparticles, which have been recovered by gel-filtration, ultra-centrifugation or dialysis. We have analysed how different surfactants and purification methods can influence the nanoparticle characteristics. The particle sizes have been obtained by scanning electron microscope, whereas a SdFFF system was employed to detect their distributions. The Oct-CPA release from nanoparticles and stabilities in human blood of free and encapsulated prodrug have been analysed by HPLC techniques. The effects of nanoparticles on CPA interaction toward adenosine A, receptor (its action site) have been analysed using radiolabelled drugs. The smallest nanoparticles and the best degree of homogeneity have been obtained using sodium cholate; the best recovery has been achieved by dialysis, whereas gel-filtration and ultra-centrifugation have induced the greatest removal of surfactants. The release of Oct-CPA was better controlled from the nanoparticles obtained using Pluronic F68 and purified by gel-filtration or ultra-centrifugation. Similarly, these nanoparticles better increased the stability of the prodrug in human blood. In particular, the nanoparticles purified by ultra-centrifugation induced a strong stability to a fraction of the encapsulated Oct-CPA. Any interference by unloaded nanoparticles has been registered for CPA-adenosine A(1) receptor interaction. (c) 2005 Elsevier B.V. All rights reserved.

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 - Polymeric nanoparticles as drug controlled release systems: A new formulation strategy for drugs with small or large molecular weight [Articolo su rivista]
Leo, Eliana Grazia; Scatturin, A; Vighi, E; Dalpiaz, A.
abstract

We report a study evaluating the encapsulation and release modalities from poly(D,L lactic acid) (PLA) or poly(D,L-lactide-co-glicolide) (PLGA) micro- and nano-particles of the antiischemic drug N-6-cyclopentyladenosine (CPA) and bovine serum albumin (BSA), chosen as protein model. The results obtained by classical preparation methods (nanoprecipitation, single or double emulsion/solvent evaporation) of the particles were compared with those obtained by their formulation with a novel method, employing a thermosensible gel of Pluronic F-127, whose aqueous solutions can be liquid when refrigerated, but gel upon warming. Our results indicate that CPA-loaded nanoparticles, obtained by classical methods, drastically reduce their drug content showing, moreover, any control of the drug release with respect to CPA-loaded microparticles. The novel preparation method allowed us to obtain, instead, CPA encapsulation values in nanoparticles similar to those obtained for microparticles, achieving also a weak control of the drug release. Any drastic reduction of BSA particle content was obtained by decreasing their size from micro- to nano-scales, independently on the employment of classical or novel preparation methods. Moreover, the size reduction induced only a weak increase of the BSA release rate. The patterns of protein released from micro- and nano-particles obtained by the same formulation method were similar. In particular, the micro- and nano-spheres prepared by double emulsion technique showed an incomplete BSA release, characterized by an elevated burst effect followed by a very slow phase. On the other hand, the release from micro- and nano-particles obtained by the novel method was complete and quite regular, being characterized by a little-burst release followed by a fast phase. These results have been related to the strong BSA distribution (observed by confocal laser scanning microscope) in the surface or in the core of microparticles obtained by the classical or novel methods, respectively.

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

2006 - Solid lipid nanoparticles: a new cationic lipid matrix composition for gene transfer, [Abstract in Atti di Convegno]
Vighi, Eleonora; Ruozi, Barbara; Montanari, Monica; Battini, Renata; Forni, Flavio; Leo, Eliana Grazia
abstract

Solid lipid nanoparticles: a new cationic lipid matrix composition for gene transfer

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 - 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 - 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 - Preparation and physical stability evaluation of cationic solid lipid nanoparticles [Abstract in Atti di Convegno]
Leo, Eliana Grazia; Vighi, Eleonora; Ruozi, Barbara; Forni, Flavio
abstract

Preparation and physical stability evaluation of cationic solid lipid nanoparticles

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.

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 - 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 - Protein immobilization in crosslinked alginate microparticles [Articolo su rivista]
Coppi, Gilberto; Iannuccelli, Valentina; Leo, Eliana Grazia; Bernabei, Maria Teresa; Cameroni, Riccardo
abstract

Oral administration of peptide and protein drugs requires their protection from the acidic and enzymatic degradation in the gastro-intestinal environment and their targeting to the absorption zone. For this purpose, an alginate microsystem, as a carrier of bovine serum albumin (BSA), as a model protein, was developed using a spray-drying technique. A hardening process with Ca2+ and chitosan (CS) provided a system with resistance to the gastro-intestinal barriers and of appropriate size for targeting to the Peyer's patches. The present work aims to evaluate the effects of the ratio of sodium alginate (Na-A) and BSA as well as the pH of the crosslinking medium on the microsystem properties. Microparticle morphological and dimensional characteristics did not change significantly with the formulation variables. BSA loading at a pH value less than the protein isoelectric point (pI) was higher than that at a pH similar to the pI owing to an electrostatic interaction between the charged protein and the polyanionic alginate. The maximum encapsulation efficiency was obtained at the highest Na-A/BSA ratio. Protein release in a simulated gastro-intestinal fluid was not affected by the preparative variables, but was controlled by the pH-dependent nature of the polymer material. Polyacrylamide gel electrophoresis (PAGE) demonstrated the stability of the protein to both the preparative conditions and the gastro-intestinal pH values.

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 - Chitosan-alginate microparticles as a protein carrier [Articolo su rivista]
Coppi, Gilberto; Iannuccelli, Valentina; Leo, Eliana Grazia; Bernabei, Maria Teresa; Cameroni, Riccardo
abstract

The oral administration of peptidic drugs requires their protection from degradation in the gastric environment and the improvement of their absorption in the intestinal tract. For these requirements, a microsystem based on cross-lined alginate as the carrier of bovine serum albumin (BSA), used as a model protein, was proposed. A spray-drying technique was applied to BSA/sodium alginate solutions to obtain spherical particles having a mean diameter less than 10 mum. the microparticles were hardened using first a solution of calcium chloride and then a solution of chitosan (CS) to obtain stable microsystems. The cross-linking process was carried out at different CS concentrations and pH values of the cross-linking medium. The CS concentration affected the BSA loading in the micro particles prepared at a pH value less than the protein isoelectric point (pI). Moreover, the BSA loading at a pH value less than the pI was higher than that at a pH similar to the pI regardless of the CS concentration. This finding could be attributable to the formation of a BSA/alginate complex. The evaluation of the interaction between BSA and alginate at different pH values by means rheological measurements confirmed this hypothesis. This approach may represent a promising way to devise a microcarrier system with appropriate size for targeting the Peyer's patches, with appropriate immobilization capacity, and suitable for the oral administration of peptidic drugs.

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 - 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

2000 - Oral tolerance elicited in mice by b-lactoglobulin entrapped in biodegradable microspheres. [Articolo su rivista]
S., Pecquet; Leo, Eliana Grazia; R., Fritsche; A., Pfeifer; P., Couvreur; E., Fattal
abstract

Oral administration of antigen is knon to be appropriate for some vaccine purporses as well as oral tolerance induction. In the present study oral administration of beta lactoglobulin loaded PLGA microspheres induced tolerance was evaluated. A single feeding of five micrograms of encapsulated protein tolerised Balb/c mice to subsequent BLG parenteral challenge, suppressing the specific humoral, intestinal and cellular responses.

2000 - PVP solid dispersions for the controlled release of furosemide from a floating multiple-unit system. [Articolo su rivista]
Iannuccelli, Valentina; Coppi, Gilberto; Leo, Eliana Grazia; F., Fontana; Bernabei, Maria Teresa
abstract

The poor bioavailability of orally dosed furosemide (FUR) is due to the presenceof a biological window in the upper gastrointestinal tract. The purpose of the presentstudy was to develop and optimize in vitro a multiple-unit floating system with increasedgastric residence time for FUR. The incomplete release of FUR from theunits, related to its low water solubility, led to the preparation and evaluation ofdifferent FUR samples to be incorporated into the units. The complete dose releaseover the actual intragastric residence time of the system (about 8 hr) was achievedby loading both the core and the membrane forming the units with a 1:5 FUR/polyvinylpyrrolidone (FUR/PVP) solid dispersion. Physicochemical analyses suggestedthe predominant role of the amorphous state of FUR in producing enhanceddrug solubility and dissolution rate, which led to the desired release profile fromthe floating units.

2000 - Surface drug removal from ibuprofen-loaded PLA microspheres [Articolo su rivista]
Leo, Eliana Grazia; Forni, Flavio; M. T., Bernabei
abstract

The preparation. characterisation and drug release behaviour of ibuprofen loaded poly(D,L-lactic acid) (PLA) microspheres are described. Depending on the gelatin concentration in the aqueous external solution (1, 0.5, 0.1% w/v), microspheres with three different sizes (2.2. 4.1, 7.5 mu m) were obtained. The properties or. microspheres washed with water (Untreated microspheres) (Un-Ms) were compared to those of the microspheres washed with a sodium carbonate solution in order to remove the surface drug (treated microspheres) (T-Ms). The results indicate that the removal of the surface drug did not induce any change in the size of the microspheres whereas the morphology Of the smallest T-Ms appeared to be modified. The release profiles of both Un-Ms and T-Ms resulted in biphasic patterns. The initial burst effect (first release phase) of the T-Ms was lower than that of the Un-Ms. The rate of the second release phase did not change for the microspheres with the biggest size but increased for the smallest microspheres probably owing to the modification of the matrix porosity. (C) 2000 Published by Elsevier Science B.V. All rights reserved.

2000 - The effect of triglycerid/phospholipid ratio in liposphere preparation [Abstract in Atti di Convegno]
Iannuccelli, Valentina; Coppi, Gilberto; Leo, Eliana Grazia; Cameroni, Riccardo
abstract

Lipospheres for the cosmetic delivery of glycolic acid were prepared by the melt method using tristearin as the lipid phase and hydrogenated soybean phosphatidylcholine as the emulsifier. The most favourable conditions leading to the highest liposphere yield involved triglyceride/phospholipid ratios of 4:1 or 5:1 and a phospholipid concentration of at least 2%. The lipospheres, sized from 5 to 40 micron, contained a rather high glycolic acid loading level probably due to a partial polymorphic modification of the lipid and determined glycolic acid sustained release pattern.

1999 - A polysorbate-based non-ionic surfactant can modulate loading and release of beta-lactoglobulin entrapped in multiphase poly(DL-lactide-co-glycolide) microspheres [Articolo su rivista]
Rojas, J; Pinto Alphandary, H; Leo, Eliana Grazia; Pecquet, S; Couvreur, P; Gulik, A; Fattal, E.
abstract

Purpose. The goal of the present paper was to investigate the role of a surfactant, Tween 20, in the modulation of the entrapment and release of beta-lactoglobulin (BLG) from poly (DL-lactide-co-glycolide) microspheres. Methods. Poly(DL-lactide-co-glycolide) microspheres containing BLG were prepared by a water-in-oil-in-water emulsion solvent procedure. Tween 20 was used as a surfactant in the internal aqueous phase of the primary emulsion. BLG entrapment efficiency and burst release were determined. Displacement of BLG from microsphere surface was followed by confocal microscopy observations and zeta potential measurements, whereas morphological changes were observed by freeze-fracture electron microscopy. Results. Tween 20 was shown to increase 2.8 fold the encapsulation efficiency of BLG without any modification of the stability of the first emulsion and the viscosity of the internal aqueous phase. In fact, Tween 20 was shown to be responsible for removing the BLG molecules that were adsorbed on the particle surface or very close to the surface as shown by confocal microscopy and zeta potential measurements. Tween 20 reduced the number of aqueous channels between the internal aqueous droplets as well as those communications with the external medium. Thus, the more dense structure of BLG microspheres could explain the decrease of the burst release. Conclusions, These results constitute a step forward in the improvement of existing technology in controlling protein encapsulation and delivery from microspheres prepared by the multiple emulsion solvent evaporation method.

1999 - Changing the pH of the external aqueous phase may modulate protein entrapment and delivery from poly(lactide-co-glycolide) microspheres prepared by a w/o/w solvent evaporation method [Articolo su rivista]
Leo, Eliana Grazia; S., Pecquet; J., Rojas; P., Couvreur; E., Fattal
abstract

The milk model protein, beta lactoglobulin (BLG), was encapsulated into microspheres prepared by a multiple emulsion/solvent evaporation method. The effect of the pH of the outer aqueous phase on protein encapsulation and release as well as on microsphere morphology has been investigated. At all tested pH values, the encapsulation efficiency was shown to decrease with increasing the initial amout of BLG. This was correlated with the reduced stability of the primary emulsion as the initial BLG increased. In addition, reducing the solubility of BLG in the external aqueous phase by decreasing the pH to the isoelectric point of BLG (pI 5.2) resulted in an improved protein encapsulation. Moreover, it was shown that combining pH modification and optimal stability of the first emulsion yielded microspheres with a high encapsulation efficiency. However, release kinetic studies revealed that a significant burst release was observed with microspheres loaded with large amounts of BLG, especially when prepared in a medium at pH 5.2. This burst effect was attributed to morphology changes in the microsphere surface which was characterized by the presence of pores or channels able to accelerate the release of BLG. These pores were assumed to result from the presence of large amounts of protein molecules on the microsphere surface, that aggregate during microsphere formation at pH 5.2. Indeed, single adsorption experiments have shown that BLG had a higher affinity for the particle surface when the pH was close to the pi. Thus, reducing the solubility of a protein in the external aqueous phase allows the product of microspheres with a better encapsulation efficiency, although this benefit is provided by a strong adsorption of the protein on microsphere surface.

1999 - Dynamic dialysis for the drug release evaluation from doxorubicin-gelatin nanoparticle conjugates [Articolo su rivista]
Leo, Eliana Grazia; R., Cameroni; Forni, Flavio
abstract

The drug release from doxorubicin (DXR)-gelatin nanoparticle conjugates was evaluated by means of a dynamic dialysis technique. The study was carried out in absence and in presence of a proteolytic enzyme (trypsin) able to degrade the carrier. In a preliminary study the apparent permeability constant (K-cv) of the drug through the dialysis bag was evaluated in several media. On the basis of this screening, a saline solution (NaCl 0.9%, w/v) resulted appropriate to carry out the dialysis study since, in this medium, the K-cv did not depend on the drug concentration in the donor solution. In absence of the enzyme only a little fraction (from 9 to 13%, w/w of the drug content) was released from nanoparticles. This fraction was considered as the evidence of the free drug fraction. After the addition of trypsin, the diffusion of a further drug fraction was observed. This fraction is probably due to a fraction of the DXR-peptide conjugates characterised by a molecular weight lower than membrane cut-off (3500 Da). (C) 1999 Elsevier Science B.V. All rights reserved.

1999 - In vivo fate and immune pulmonary response after nasal administration of microspheres loaded with phosphorylcholine-thyroglobulin [Articolo su rivista]
S., Tolle; E., Caudron; Leo, Eliana Grazia; P., Couvreur; A., Andramont; E., Fattal
abstract

The effect of the protein concentration on antigen encapsulation and relase as well as on microsphere epithelial cells of the nasopharyngeal associated lymphoid tessue and induced a specific IgA and IgG responce in pulmonary secretions as well as a strong systemic immune response in Balb/c mice

1999 - Optimization of the encapsulation and release of beta-lactoglobulin entrapped poly(DL-lactide-co-glycolide) microspheres [Articolo su rivista]
J., Rojas; H., Pintoalphandary; Leo, Eliana Grazia; S., Pecquet; P., Couvreur; E., Fattal
abstract

The goal of the present paper was to optimize the encapsulation of beta-lactoglobulin (BLG) within poly(lactide-co-glycolide) (PLGA) microparticles prepared by the multiple emulsion solvent evaporation method. The role of the pH of the external phase and the introduction of the surfactant Tween 20, in the modulation of the entrapment and release of BLG from microparticles, was studied. Reducing the solubility of BLG by decreasing the pH of the external phase to a value close to the pi of BLG resulted in a better encapsulation with, however, a larger burst release effect. By contrast, Tween 20 was shown to increase the encapsulation efficiency of BLG and reduce considerably the burst release effect. In fact, Tween 20 was shown to be responsible for removing the BLG molecules that were adsorbed on the particle surface. In addition, Tween 20 reduced the number of aqueous channels between the internal aqueous droplets as well as those communicating with the external medium. Thus, the more dense structure of BLG microspheres could explain the decrease in the burst release. These results constitute a step ahead in the improvement of an existing technology in controlling protein encapsulation and delivery from microspheres prepared by the multiple emulsion solvent evaporation method. (C) 1999 Elsevier Science B.V. All rights reserved.

1999 - Studio della fotodegradazione di un complesso PABA/alginato per la formulazione di filtri solari [Abstract in Atti di Convegno]
Iannuccelli, Valentina; Coppi, Gilberto; Leo, Eliana Grazia; Bernabei, Maria Teresa
abstract

Grande attenzione è stata rivolta di recente agli effetti biologici a carico della cute da parte delle radiazioni solari. La tendenza da parte della popolazione ad un’eccessiva esposizione alla luce solare unitamente ad una maggiore conoscenza degli effetti nocivi derivanti ha portato ad un aumento dell’uso di prodotti contenenti differenti filtri solari, ad un crescente interesse nello studio della loro attività fotoprotettiva e alla necessità di ridurne gli effetti collaterali. E’ noto infatti che l’instabilità fotochimica di alcuni filtri solari può comportare una diminuita efficacia d’azione e la formazione di prodotti di degradazione fototossici. L’acido p-amino benzoico (PABA) rappresenta uno dei filtri solari maggiormente impiegati. Nonostante la mancanza di un parere univoco, è stato riportato che il PABA, per esposizione prolungata alla luce solare, si decompone con la formazione di fotoprodotti potenzialmente dannosi (1-3).Poiché le variazioni fotochimiche del PABA coinvolgerebbero il gruppo aminico, il presente studio è stato rivolto al tentativo di diminuire la fotoinstabilità del PABA mediante la formazione di un complesso con un polisaccaride di natura polianionica quale il sodio alginato (4). Mediante determinazioni del comportamento reologico basato sulla valutazione dell’effetto elettroviscoso tipico dei polielettroliti, è stata dimostrata la formazione di un complesso di natura elettrostatica tra il PABA e il sodio alginato in soluzione acquosa. La fotostabilità del complesso PABA/alginato è stata studiata esponendo i campioni a luce solare simulata e analizzandoli a tempi prefissati mediante spettrofotometria UV e TLC. La valutazione dello “shift” del massimo di assorbimento e dei coefficienti di estinzione molare del PABA in complesso con l’alginato rispetto a quelli del PABA come tale potrebbe indicare un effetto positivo della reazione di complessazione sulla fotostabilità e sulla capacità filtrante. Conformemente, lo studio cromatografico sembra dimostrare che il complesso ottenuto presenti una maggior stabilità all’azione della luce solare rispetto al PABA.

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 - Studio reologico del complesso alginato/chitosano/albumina per la preparazione di microsfere [Abstract in Atti di Convegno]
Iannuccelli, Valentina; Coppi, Gilberto; Leo, Eliana Grazia; Cameroni, Riccardo
abstract

La formazione di complessi macromolecolari tra polielettroliti aventi cariche opposte permette di ottenere strutture chimiche impiegabili in campo farmaceutico per la preparazione di microparticelle quali carrier di farmaci proteici.La struttura reticolata del Ca-alginato, quale polianione, può essere ulteriormente stabilizzata per interazione poliionica con chitosano che agisce quale policatione.Tale substrato polimerico è stato impiegato per la preparazione di microparticelle mediante tecnica di spray-drying abbinata al processo di reticolazione in presenza di albumina (BSA), quale farmaco modello.Nella preparazione di tali sistemi la presenza della proteina può influenzare il grado di complessazione tra i polielettroliti. Infatti, la BSA a valori di pH inferiori al proprio punto isoelettrico potrebbe indurre la formazione di complessi proteina-polianione con effetti diretti sulle caratteristiche del sistema (reticolazione, caricamento, rilascio, ...).Pertanto si sono valutate le modalità e il grado di interazione tra le macromolecole sotto diverse condizioni sperimentali di preparazione mediante analisi reologica.

1997 - A polysorbate-based non ionic surfactant improves β lactoglobulin delivery from PLGA microspheres [Abstract in Rivista]
Fattal, E.; Leo, E.; Rojas, J.; Pecquet, S.; Couvreur, P.
abstract

1997 - Chapter 9: Design of nanoparticles for vaccine delivery [Capitolo/Saggio]
Fattal, E.; Blanco-Príeto, M. J.; Leo, E.; Puisieux, F.; Couvreur, P.
abstract

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 - General and cardiac toxicity of doxorubicin-loaded gelatin nanoparticles [Articolo su rivista]
Leo, Eliana Grazia; R., Arletti; Forni, Flavio; R., Cameroni
abstract

General and cardiac toxicity of doxorubicin loaded gelatin nanoparticles cross-linked by glutaraldheyde were investigated in healthy rats. The rats were treated with free doxorubicin (DXR), unloaded nanoparticles (UNp), physical mixture of doxorubicin, and unloaded nanoparticles (DRX/UNp), and DXR-loaded nanoparticles (DXR-Np). Each group of animals received the same dose of DXR (3 mg/kg) via i.p. once a week. Both electrocardiogram (EGG) parameters and body weight were measured 24h before each administration. Rats treated with UNp behaved as controls. DXR/UNp provoked the same toxic effects as free DXR. On the contrary, DXR-Np resulted more toxic since significant variations of both the body weight and the ECG parameters were observed during the first week of treatment. In addition, the rats treated with DXR-Np died between the 3(rd) and the 5(th) day after the 2(nd) administration. These results demonstrate that, in these experimental conditions, the couplage of DXR to nanoparticles enhanced the cardiotoxicity of the drug. Since DXR was linked to the protein matrix of nanoparticles via glutaraldehyde, the high toxicity of DXR-loaded nanoparticles could be due to the covalent binding of the drug to the carrier.

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 - Study of the influence of several stabilizing agents on the entrapment and in vitro release of pBC 264 from poly(lactide-co-glycolide) microspheres prepared by a W/O/W solvent evaporation method. [Articolo su rivista]
M. J., BLANCO PRIETO; Leo, Eliana Grazia; F., Delie; A., Gulik; P., Couvreur; E., Fattal
abstract

In this report has been investigated the influence of different stabilitzing agents added to the internal aqueous phase as well as the concentration of the polymer in the organic pahse on the encapsulation efficiency and the in vitro release of pBC264

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 - 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 - 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.

Università degli studi di Modena e Reggio Emilia

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