Stefano Barbieri

Lecithin/chitosan nanoparticles of clobetasol-17-propionate capable of accumulation in pig skin

In this study, clobetasol-17-propionate (CP) loaded lecithin/chitosan nanoparticles were studied with special attention to the transport of the active agent across the skin in vitro. Nanoparticles were characterized by measuring particle size, zeta potential, polydispersity index and encapsulation efficiency. The morphology of nanoparticles was evaluated by transmission electron microscopy. Encapsulation experiments with CP showed high encapsulation efficiency (92.2%). To assess the advantages of this carrier-based formulation in topical administration, the accumulation in and permeation across pig ear skin were compared with chitosan gel and commercially available cream of CP. The results obtained indicate that the incorporation of drug into nanoparticles induced an accumulation of CP especially in the epidermis without any significant permeation across the skin. Dilution of CP loaded nanoparticles with chitosan gel (1:9) produced the same amount of CP in the skin compared with commercial cream, although the former contained ten times less CP. This is a remarkable point for the reduction of the side effects of CP. These results demonstrated the suitability of lecithin/chitosan nanoparticles to induce epidermal targeting and to improve the risk-benefit ratio for topically applied CP.

Structure of self-organized multilayer nanoparticles for drug delivery.

The combined use of cryo-TEM, dynamic light scattering, and small-angle X-ray and neutron scattering techniques allows a detailed structural model of complex pharmaceutical preparations of soybean lecithin/chitosan nanoparticles used as drug vectors to be worked out. Charge-driven self-organization of the lipid(-)/polysaccharide(+) vesicles occurs during rapid injection, under mechanical stirring, of an ethanol solution of soybean lecithin into a chitosan aqueous solution. We conclude that beyond the charge inversion region of the phase diagram, i.e., entering the redissolution region, the initial stages of particle formation are likely to be affected by a re-entrant condensation effect at the nanoscale. This behavior resembles that at the mesoscale which is well-known for polyion/amphiphile systems. Close to the boundary of the charge inversion region, nanoparticle formation occurs under a maximum condensation condition at the nanoscale and the complexation-aggregation process is driven toward a maximum multilamellarity. Interestingly, the formulation that maximizes vesicle multilamellarity corresponds to that displaying the highest drug loading efficiency.

IN13 Backscattering Spectrometer at ILL: Looking for Motions in Biological Macromolecules and Organisms

In 1998, three partner groups (the French institutions Institut de Biologie Structurale and the Léon Brillouin Laboratory and the Italian Istituto Nazionale per la Fisica della Materia, now merged with the Consiglio Nazionale delle Ricerche, INFM-CNR) applied to operate the thermal backscattering spectrometer IN13, at the Institut Laue Langevin, as a French-Italian Collaborative Research Group (CRG). The plan was to have access to a dedicated spectrometer in order to explore how far neutron scattering could contribute to the understanding of dynamics in biological macromolecules: how “flexible” must be a biological object to perform its function?

Liposome sensing and monitoring by organic electrochemical transistors integrated in microfluidics

BACKGROUNDnOrganic electrochemical transistors (OECTs), which are becoming more and more promising devices for applications in bioelectronics and nanomedicine, are proposed here as ideally suitable for sensing and real time monitoring of liposome-based structures. This is quite relevant since, currently, the techniques used to investigate liposomal structures, their stability in different environments as well as drug loading and delivery mechanisms, operate basically off-line and/or with pre-prepared sampling.nnnMETHODSnOECTs, based on the PEDOT:PSS conductive polymer, have been employed as sensors of liposome-based nanoparticles in electrolyte solutions to assess sensitivity and monitoring capabilities based on ion-to-electron amplified transduction.nnnRESULTSnWe demonstrate that OECTs are very efficient, reliable and sensitive devices for detecting liposome-based nanoparticles on a wide dynamic range down to 10(-5)mg/ml (with a lowest detection limit, assessed in real-time monitoring, of 10(-7)mg/ml), thus matching the needs of typical drug loading/drug delivery conditions. They are hence particularly well suited for real-time monitoring of liposomes in solution. Furthermore, OECTs are shown to sense and discriminate successive injection of different liposomes, so that they could be good candidates in quality-control assays or in the pharmaceutical industry.nnnGENERAL SIGNIFICANCEnDrug loading and delivery by liposome-based structures is a fast growing and very promising field that will strongly benefit from real-time, highly sensitive and low cost monitoring of their dynamics in different pharma and biomedical environments, with a particular reference to the pharmaceutical and production processes, where a major issue is monitoring and measuring the formation and concentration of liposomes and the relative drug load. The demonstrated ability to sense and monitor complex bio-structures, such as liposomes, paves the way for very promising developments in biosensing and nanomedicine. This article is part of a Special Issue entitled Organic Bioelectronics-Novel Applications in Biomedicine.

Lecithin/chitosan controlled release nanopreparations of tamoxifen citrate: loading, enzyme-trigger release and cell uptake.

Tamoxifen citrate (TAM), an anticancer drug with amphiphilic properties, was loaded in lecithin/chitosan nanoparticles (LCN) with a view to oral administration. The influence of tamoxifen loading on the physico-chemical properties of nanoparticles was studied. Size, surface charge and morphological properties of tamoxifen-loaded nanoparticles (LCN-TAM) were assessed. The increase in the tamoxifen amount in the LCN-TAM preparation up to 60 mg/100 ml maintained the positive zeta potential value of about +45 mV. A statistically significant decrease in particle size was observed for TAM amounts between 5 and 20mg. A strong influence of loaded tamoxifen on the structure of lecithin/chitosan nanoparticles was observed, supported by the quantification of free chitosan and morphological analysis. A loading of tamoxifen in nanoparticles of around 19% was obtained. The release of the drug from the LCN-TAM colloidal dispersion was measured, showing that tamoxifen citrate was released very slowly in simulated gastro-intestinal fluids without enzymes. When enzymes able to dismantle the nanoparticle structure were added to the dissolution medium, drug release was triggered and continued in a prolonged manner. Tamoxifen-loaded nanoparticles showed cytotoxicity towards MCF-7 cells comparable to that obtained with tamoxifen citrate solution, but the rate of this toxic effect was dependent on drug release. Caco-2 cells, used as a model of the intestinal epithelium, were shown to take up the TAM loaded nanoparticles extensively.

Flexibility and drug release features of lipid/saccharide nanoparticles

The effect of lipophilic additives (excipients and drugs) on the behavior of lipid/saccharide nanoparticles has been investigated by incoherent elastic neutron scattering. Temperature scans from 20 K to 350 K have been performed on lecithin/chitosan particles loaded with isopropyl myristate and cetyl-stearyl alcohol, two lipophilic molecules with different melting temperatures which are commonly added to improve drug loading efficiency. In a similar way the effect of tamoxifen citrate, a lipophilic drug frequently used in breast cancer therapy, has also been studied. The different melting points of the two excipients affect mostly the low-temperature behavior of the nanoparticles. At physiological temperatures they both improve the particle flexibility. On the other hand addition of tamoxifen leads to stiffer structures and to lower amounts of released drug. The macroscopic features of the drug release appear to be correlated to the microscopic flexibility determined by neutron scattering. The data confirm also the role of chitosan as a stiffening and stabilizing agent of the lipid particles.

Ex vivo permeation of tamoxifen and its 4-OH metabolite through rat intestine from lecithin/chitosan nanoparticles

Tamoxifen citrate is an anticancer drug slightly soluble in water. Administered orally, it shows great intra- and inter-patient variations in bioavailability. We developed a nanoformulation based on phospholipid and chitosan able to efficiently load tamoxifen and showing an enzyme triggered release. In this work the permeation of tamoxifen released from lecithin/chitosan nanoparticles across excised rat intestinal wall mounted in an Ussing chamber was investigated. Compared to tamoxifen citrate suspension, the amount of the drug permeated using the nanoformulation was increased from 1.5 to 90 times, in absence or in presence of pancreatin or lipase, respectively. It was also evidenced the formation of an active metabolite of tamoxifen, 4-hydroxy tamoxifen, however, the amount of metabolite permeated remained roughly constant in all experiments. The effect of enzymes on intestinal permeation of tamoxifen was shown only when tamoxifen-loaded nanoparticles were in intimate contact with the mucosal surface. The encapsulation of tamoxifen in lecithin/chitosan nanoparticles improved the non-metabolized drug passing through the rat intestinal tissue via paracellular transport.

Single Layer Transdermal Film Containing Lidocaine: Water and Lidocaine Mobility Determined using Neutron Scattering

The diffusivity of lidocaine through a polymer film developed for transdermal drug administration has been characterized by macroscopic permeation experiments and by neutron quasielastic spectroscopy that provides information on microscopic diffusivity parameters. It turns out that film hydration plays a key role on the performance and efficiency of the investigated system. Diffusion of lidocaine, at the microscopic level, is triggered by the presence of mobile water. At hydration levels below 15% (weight water/weight hydrated film) neither lidocaine nor water show any appreciable long-range diffusion. At higher hydration levels, the onset of water long-range diffusion triggers diffusion of lidocaine through the film. The use of neutron quasielastic scattering makes it possible to measure lidocaine mobility within the film without the need of any additional physical barrier.

Polymeric films loaded with cisplatin for malignant pleural mesothelioma: a pharmacokinetic study in an ovine model

BackgroundnMalignant pleural mesothelioma (MPM) continues to be a distressing tumor due to its aggressive biologic behavior and scanty prognosis. Several therapeutic approaches have been tested both in clinical and preclinical settings, being intrapleural chemotherapy one of the most promising. Some years ago, our interest focused on polymeric films loaded with cisplatin for the adjuvant intrapleural treatment of surgical patients. After in vitro and in vivo studies in a rat recurrence model of MPM, the aim of this study was to evaluate the pharmacokinetics of the polymeric films in a sheep model in view of further studies in a clinical setting.nnnMethodsnAn ovine model was used. Animals were divided into four groups according to pharmacologic treatment: control group (three animals undergoing left pneumonectomy and saline-NaCl solution); intrapleural hyaluronate cisplatin films (HYALCIS) group (six animals undergoing left pneumonectomy and intrapleural application of polymeric films loaded with cisplatin); intrapleural cisplatin solution (six animals undergoing left pneumonectomy and intrapleural application of cisplatin solution); intravenous cisplatin (five animals undergoing left pneumonectomy and intravenous administration of cisplatin solution). The primary objective was the plasmatic and pleural concentration of cisplatin in the treatment groups. The secondary objective was the treatment-related toxicity evaluated by plasmatic analysis performed at prearranged time intervals and histological examinations of tissue samples collected during animal autopsy. Analysis of variance (ANOVA) was used for statistical analysis. Bonferroni correction was applied for comparison between all groups.nnnResultsnTwenty female Sardinian sheep with a mean weight of 45.1 kg were studied. All animals survived the surgical procedures. The whole surgical procedure had a mean duration of 113 minutes. Cisplatin blood levels obtained from polymeric films application were low during the first 24 hours after the application; then, the cisplatin blood level increased gradually and progressively until it reached significantly higher plasmatic concentrations after 120 hours compared to intrapleural cisplatin solution (P=0.004) and intravenous administration (P=0.001), respectively. Considering cisplatin concentration at 168 hours after the application, animals treated with polymeric films had higher plasmatic values than animals treated with intrapleural cisplatin solution and intravenous cisplatin (P=0.001). Despite the high cisplatin plasmatic concentrations, treatment related-toxicity towards kidneys and liver was comparatively lower compared to the intravenous and intrapleural cisplatin administration and closer to the control levels.nnnConclusionsnPolymeric films loaded with cisplatin allowed to reach significantly higher intrapleural and plasmatic cisplatin concentrations compared to intrapleural and intravenous cisplatin solution, providing at the same time, a significant reduction of treatment related toxicity.

Physicochemical and pharmacokinetic properties of polymeric films loaded with cisplatin for the treatment of malignant pleural mesothelioma

BackgroundnMalignant mesothelioma is an invasive neoplasm arising from mesothelial surfaces of the pleural and peritoneal cavities. Mesothelioma treatment is unsatisfactory and recurrence is common. Here an innovative locoregional treatment for malignant pleural mesothelioma is presented.nnnMethodsnChitosan- and hyaluronate-based films were loaded with 0.5% and 4% w/w cisplatin and were studied for their physicochemical, mechanical and drug release characteristics. The performance of the drug delivery systems was assessed in vitro on A549 cells and on an orthotopic model of MPM recurrence in rats.nnnResultsnPolysaccharide films produced were thin, flexible and resistant. Cisplatin was completely released from hyaluronic acid films within 96 hours, while drug release was found to be much more prolonged with chitosan films. The drug released from hyaluronate films was effective against A549 cell line, while for chitosan films the release was too slow to produce cytotoxicity. Similarly, cisplatin-loaded chitosan films in vivo released minimal quantities of cisplatin and induced inflammation and foreign body reaction. Cisplatin-loaded hyaluronate acid films on the contrary were able to prevent tumor recurrence. The cisplatin-loaded hyaluronate films provided higher Cmax and AUC compared to a solution of cisplatin administered intrapleurally, but did not show any sign of treatment related toxicity.nnnConclusionsnHyaluronate-based films appear as an optimal platform for the development of drug delivery systems suitable for the loco-regional post-surgical treatment of lung malignancies.