The role of protamine amount in the transfection performance of cationic SLN designed as a gene nanocarrier

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

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

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

A Peptidic Thymidylate-Synthase Inhibitor Loaded on Pegylated Liposomes Enhances the Antitumour Effect of Chemotherapy Drugs in Human Ovarian Cancer Cells

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

Sistemi nanoparticellari per la veicolazione e il direzionamento del farmaco

Dottorato di ricerca in chimica del farmaco. 7. ciclo. Coordinatore G. Cignarella. Tutore E. GalliConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Studying the in vitro behaviour of cationic solid lipid nanoparticles as a nonviral vector

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

Dynamic dialysis for the drug release evaluation from doxorubicin-gelatin nanoparticle conjugates

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

Vettori particellari per il rilascio sito-specifico di farmaci nel tratto gastro-intestinale

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

Influence of penetration enhancers and lipid nanoparticles on in vivo human skin penetration of quercetin

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

A hydroxypropylcellulose (HPC) system for the immediate and controlled release of diclofenac sodium

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