ELEONORA MARETTI - personale UniMoRe

Nuova ricerca

ELEONORA MARETTI

Assegnista di ricerca
Dipartimento di Scienze della Vita sede ex-Scienze Farmaceutiche

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 - Epitranscriptomics as a New Layer of Regulation of Gene Expression in Skeletal Muscle: Known Functions and Future Perspectives [Articolo su rivista]
Imbriano, Carol; Moresi, Viviana; Belluti, Silvia; Renzini, Alessandra; Cavioli, Giorgia; Maretti, Eleonora; Molinari, Susanna
abstract

Epitranscriptomics refers to post-transcriptional regulation of gene expression via RNA modifications and editing that affect RNA functions. Many kinds of modifications of mRNA have been described, among which are N6-methyladenosine (m6A), N1-methyladenosine (m1A), 7-methylguanosine (m7G), pseudouridine (Ψ), and 5-methylcytidine (m5C). They alter mRNA structure and consequently stability, localization and translation efficiency. Perturbation of the epitranscriptome is associated with human diseases, thus opening the opportunity for potential manipulations as a therapeutic approach. In this review, we aim to provide an overview of the functional roles of epitranscriptomic marks in the skeletal muscle system, in particular in embryonic myogenesis, muscle cell differentiation and muscle homeostasis processes. Further, we explored high-throughput epitranscriptome sequencing data to identify RNA chemical modifications in muscle-specific genes and we discuss the possible functional role and the potential therapeutic applications.

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 - 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 - Aerodynamic performance of lipid-based nanocarrier functionalized by novel mannose derivatives for Rifampicin intramacrophagic delivery [Abstract in Atti di Convegno]
Maretti, Eleonora; Costantino, Luca; Rustichelli, Cecilia; Buttini, Francesca; Iannuccelli, Valentina
abstract

Introduction. Alveolar macrophages (AM) are sites of infection by pathogens such as Mycobacterium tuberculosis. On the basis of the presence of mannose receptors on AM membranes, mannose moieties have been considered as ligands for macrophage active targeting. Mannosylated derivatives were synthesized to decorate the surface of Rifampicin (RIF) loaded Solid Lipid Nanoparticle assemblies (SLNas) for an inhaled anti-tuberculosis (TB) therapy by Dry Powder Inhaler (DPI) device with the goal to relate surface functionality with respirability performance. Methods. Biocompatible lipid components such as fatty acids and their derivatives were processed using the melt emulsification technique. SLNas surface decoration was obtained by means of four newly synthesized mannose derivatives having surfactant effect. The obtained mannosylated carriers were examined for their intrinsic properties (size, morphology and shape, surface charge, bulk and tap density, aerodynamic diameter, physical state of the components, drug loading and in vitro release) associated to respirability features assessed by Next Generation Impactor (NGI). Mannosylation occurrence was investigated using X-ray Photoelectron Spectroscopy for Chemical Analysis. Furthermore, SLNas cytotoxicity, macrophage internalization, and ability to provide RIF intramacrophagic transport were evaluated on J774 cell line by MTT test, flow cytometry, and confocal microscopy. Results and Discussion. SLNas exhibited adequate drug payload, in vitro release, and cell internalization (Fig. 1, Confocal microscopy images of J774 cells incubated for 6 h with Nile Red labeled SLNas after blue nuclei and green lysosome staining) with consequent efficient RIF translocation inside macrophages (about 70%). A relevant increase in drug content within macrophages provided by SLNas compared to free RIF solution (Fig. 2, Intracellular RIF percentage inside J774 cell line) confirmed both RIF difficulty to diffuse across macrophage membranes and SLNas efficacy to promote drug transport into cells. Furthermore, it is noteworthy that the presence of these new surface-active agents reduced powder cohesiveness without impairing respirability (fine particle fraction ranging from 30% to 50%) (Fig. 3, SLNas aerodynamic distribution by NGI upon powder aerosolisation). SLNas proposed in this research can be prepared by a green-technology avoiding organic solvents. Thus, these new SLNas may represent an encouraging opportunity in a perspective of an efficacious anti-TB inhaled therapy.

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 - 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.

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.

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 - 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 - PASSIVE AND ACTIVE TARGETING TO MACROPHAGES USING LIPID NANOPARTICLES ASSEMBLIES FOR ANTI-TB THERAPY BY PULMONARY ROUTE [Abstract in Atti di Convegno]
Maretti, Eleonora; Iannuccelli, Valentina
abstract

Tuberculosis is caused by M. tuberculosis, which may attack mainly the lungs surviving within alveolar macrophages (AM). Pulmonary route appears the most promising way to reach promptly the infected site. To achieve effective drug delivery inside AM, drugs are required to reach alveoli and to be taken up by AM. In order to fulfil this goal, respirable powders of rifampicin loaded Solid Lipid Nanoparticle assemblies (SLNas) able to be internalized by AM were developed using the melt emulsifying technique and modifying conveniently pre-freezing parameters by means of Design of Experiments approach. The most favourable impact on powder respirability (>50% respirable fraction) was exerted by quick-freezing combined with a certain grade of sample dilution without the use of cryoprotectants [1, 2]. SLNas surface functionalization by methyl α-D-mannopyranoside (MP) recognizing mannose receptors located on infected AM was realized to facilitate cell internalization (active targeting). Mannosylated SLNas exhibited physical properties suitable for AM passive targeting, adequate rifampicin payloads (10-15%), and feasible drug maintenance within SLNas along the respiratory tract before AM internalization. Concerning the active targeting, the presence of MP on SLNas surface caused a quicker internalization rate by AM. Nevertheless, respirability was impaired by the powders cohesiveness [3]. Therefore, novel more balanced amphiphiles bearing mannose residues differently arranged on SLNas surface were considered reaching improved respirability performance.

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 - Inhaled micro- or nanoparticles: which are the best for intramacrophagic anti-infectious therapies? [Articolo su rivista]
Iannuccelli, Valentina; Maretti, Eleonora
abstract

The editorial aims to consider the impact of the particle size of inhaled powders administered to treat intramacrophagic infectious diseases on breathability and phagocytosis by alveolar macrophages.

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 - 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.

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.

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 - 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.

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.

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 - 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 - 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 - 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.

Università degli studi di Modena e Reggio Emilia

Pubblicazioni