Eleonora Maretti

Inhaled Solid Lipid Microparticles to target alveolar macrophages for tuberculosis.

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.

Solid Lipid Nanoparticle assemblies (SLNas) for an anti-TB inhalation treatmentA Design of Experiments approach to investigate the influence of pre-freezing conditions on the powder respirability

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.

Gastroretentive montmorillonite-tetracycline nanoclay for the treatment of Helicobacter pylori infection

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 Ca(2+)) 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.

Enhanced anti-hyperproliferative activity of human thymidylate synthase inhibitor peptide by solid lipid nanoparticle delivery

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.

In vivo penetration of bare and lipid-coated silica nanoparticles across the human stratum corneum.

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±51nm in size) and hydrophobic lipid-coated silica (LC-silica, 363±74nm 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.

Self-Assembled Lipid Nanoparticles for Oral Delivery of Heparin-Coated Iron Oxide Nanoparticles for Theranostic Purposes

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.

Conveying a newly designed hydrophilic anti-human thymidylate synthase peptide to cisplatin resistant cancer cells: are pH-sensitive liposomes more effective than conventional ones?

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 (hTS) 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.

Surface engineering of Solid Lipid Nanoparticle assemblies by methyl α-d-mannopyranoside for the active targeting to macrophages in anti-tuberculosis inhalation therapy

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.

CHARACTERIZATION OF NATURAL CLAYS FROM ITALIAN DEPOSITS WITH FOCUS ON ELEMENTAL COMPOSITION AND EXCHANGE ESTIMATED BY EDX ANALYSIS: POTENTIAL PHARMACEUTICAL AND COSMETIC USES

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.

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

PurposeTo 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.MethodsPeptide-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.ResultsAlthough 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.ConclusionPpHL can be considered as efficient delivery systems for the new promising anti-cancer peptide.