Laura Ravani

Nanoparticulate lipid dispersions for bromocriptine delivery: characterization and in vivo study.

The physico-chemical properties and in vivo efficacies of two nanoparticulate systems delivering the antiparkinsonian drug bromocriptine (BC) were compared in the present study. Monoolein Aqueous Dispersions (MADs) and Nanostructured Lipid Carriers (NLCs) were produced and characterized. Cryogenic transmission electron microscopy (cryo-TEM) and X-ray diffraction revealed the morphology of MAD and NLC. Dimensional distribution was determined by Photon Correlation Spectroscopy (PCS) and Sedimentation Field Flow Fractionation (SdFFF). In particular, BC was shown to be encapsulated with high entrapment efficiency both in MAD and in NLC, according to SdFFF combined with HPLC. Two behavioral tests specific for akinesia (bar test) or akinesia/bradykinesia (drag test) were used to compare the effects of the different BC formulations on motor disabilities in 6-hydroxydopamine hemilesioned rats in vivo, a model of Parkinsons disease. Both free BC and BC-NLC reduced the immobility time in the bar test and enhanced the number of steps in the drag test, although the effects of encapsulated BC were longer lasting (5h). Conversely, BC-MAD was ineffective in the bar test and improved stepping activity in the drag test to a much lower degree than those achieved with the other preparations. We conclude that MAD and NLC can encapsulate BC, although only NLC provide long-lasting therapeutic effects possibly extending BC half-life in vivo.

Clotrimazole nanoparticle gel for mucosal administration.

In this study a formulation suitable to be applied on oral and/or vaginal mucosa has been developed for the treatment of fungal infections. The aim of the research is a comparison between clotrimazole (CLO) containing semisolid formulations based on monoolein aqueous dispersion (MAD) or nanostructured lipid carrier (NLC). MAD and NLC have been characterized in terms of morphology and dimensional distribution by cryogenic Transmission Electron Microscopy (cryo-TEM) and Photon Correlation Spectroscopy (PCS). CLO was encapsulated with high entrapment efficiency both in MAD and in NLC, according to Sedimentation Field Flow Fractionation (SdFFF) combined with HPLC. CLO recovery in MAD and NLC has been investigated by time. In order to obtain formulations with suitable viscosity for mucosal application, MAD was diluted with a carbomer gel, while NLC was directly viscosized by the addition of poloxamer 407 in the dispersion. The rheological properties of MAD and NLC after viscosizing have been investigated. Franz cell has been employed to study CLO diffusion from the different vehicles, evidencing diffusion rates from MAD and NLC superimposable to that obtained using Canesten(®). An anticandidal activity study demonstrated that both CLO-MAD and CLO-NLC were more active against Candida albicans with respect to the pure drug.

Size characterization by Sedimentation Field Flow Fractionation of silica particles used as food additives.

Four types of SiO2, available on the market as additives in food and personal care products, were size characterized using Sedimentation Field Flow Fractionation (SdFFF), SEM, TEM and Photon Correlation Spectroscopy (PCS). The synergic use of the different analytical techniques made it possible, for some samples, to confirm the presence of primary nanoparticles (10 nm) organized in clusters or aggregates of different dimension and, for others, to discover that the available information is incomplete, particularly that regarding the presence of small particles. A protocol to extract the silica particles from a simple food matrix was set up, enriching (0.25%, w w(-1)) a nearly silica-free instant barley coffee powder with a known SiO2 sample. The SdFFF technique, in conjunction with SEM observations, made it possible to identify the added SiO2 particles and verify the new particle size distribution. The SiO2 content of different powdered foodstuffs was determined by graphite furnace atomic absorption spectroscopy (GFAAS); the concentrations ranged between 0.006 and 0.35% (w w(-1)). The protocol to isolate the silica particles was so applied to the most SiO2-rich commercial products and the derived suspensions were separated by SdFFF; SEM and TEM observations supported the size analyses while GFAAS determinations on collected fractions permitted element identification.

Clotrimazole-loaded nanostructured lipid carrier hydrogels: Thermal analysis and in vitro studies

The aim of the present work is to design a new formulation containing clotrimazole (CTZ) loaded into nanostructured lipid carriers (NLC) for the treatment of fungal vaginal infections. In order to obtain formulations with suitable viscosity for mucosal application, NLC containing CTZ produced by the ultrasonication method were viscosized by the addition of poloxamer P407 in the NLC dispersion (CTZ-NLC-gel). These systems exhibit well-known thermogelling properties. The rheological characterization of the CTZ-NLC hydrogel using a controlled stress rheometer evidenced that the presence of NLC or CTZ did not affect gelling temperature (Tgel). Dilution with simulated vaginal fluid (SVF) increased the Tgel from 17.4 to 29.6°C. For these thermogelling systems, micro-calorimetric assays conducted by a Micro-DSC III confirmed that the hydrogel-containing CTZ-NLC was able to change its structure with a rapid passage from non-crystalline (liquid) to crystalline (semi-solid) form. Furthermore, when a local application is considered, no drug should pass through the vaginal mucosa, limiting thus the systemic diffusion and toxicity. For this purpose, Franz cell has been employed to investigate the ex vivo permeation of CTZ through pig vaginal mucosa. The results showed no CTZ diffusion. The toxicological experiments performed on HeLa cells after a 24h incubation time confirmed that CTZ-NLC-gel at a concentration of 1mg/mL showed a low toxicity profile resulting in a cell vitality of 77.2%. Interestingly, anti-candida activity studies demonstrated that CTZ-NLC gel was 4-fold more active than Fungizone(®) against Candida albicans. These encouraging results suggest that the hydrogel containing CTZ-NLC could be proposed as an innovative system to administer CTZ to treat vaginal infections.

Evaluation of Percutaneous Absorption of Naproxen from Different Liposomal Formulations

The present study concerns the percutaneous absorption of naproxen (NPX), as model anti-inflammatory drug, included in liposome formulations constituted of different lipids: stratum corneum lipids (SCL) and phosphatidylcholine/cholesterol (PC/CHOL). Liposome dispersions were produced using two different methods: reverse-phase evaporation (REV) and thin layer evaporation (TLE). Morphology and dimensions of the disperse phase were characterized by cryo-transmission electron microscopy (cryo-TEM) and photon correlation spectroscopy, respectively. X-ray diffraction was employed to determine the structural organization of the vesicles. In vitro diffusion was studied by Franz cell on liposome dispersions viscosized by carbomer. Tape stripping was performed to investigate in vivo the performance of differently composed liposomes as NPX delivery system. Cryo-TEM showed spherical vesicles and bigger irregular elongated nanoparticles for TLE SCL liposomes. REV resulted in spherical and elongated multilamellar vesicles. Also X-ray diffraction evidenced L alpha or L beta multilamellar vesicles for PC/CHOL and SCL liposome respectively. The in vitro study showed a lower NPX flux for SCL with respect to PC/CHOL liposome. Tape stripping corroborate the in vitro findings regarding SCL, suggesting that liposomes create a drug reservoir mixing with SC lipids, whilst PC/CHOL liposome promoted NPX permeation through the skin. Liposome lipid composition seems to affect NPX permeation through the skin.

Curcumin containing monoolein aqueous dispersions: a preformulative study.

The present study describes the production and characterization of monoolein aqueous dispersions (MAD) as drug delivery systems for curcumin (CR). MAD based on monoolein and different emulsifiers have been produced and characterized. Morphology and dimensional distribution have been investigated by Cryogenic Transmission Electron Microscopy (cryo-TEM), X-ray and Photon Correlation Spectroscopy (PCS). Monoolein in different mixtures with sodium cholate, sodium caseinate, bentonite and poloxamer resulted in heterogeneous dispersions constituted of unilamellar vesicles, cubosomes and sponge type phases, depending on the employed components, as found by cryo-TEM and X-ray studies. CR was encapsulated with entrapment efficiencies depending on the MAD composition, particularly the highest was reached in the case of monoolein/poloxamer/sodium cholate mixture. The same mixture was able to maintain CR stability also after 6 months. CR release modalities were in vitro investigated in order to mimic a possible subcutaneous administration of MAD. It was found that MAD constituted of monoolein/poloxamer and monoolein/poloxamer/sodium cholate mixtures were able to sustain CR release. MAD viscous vehicles were produced by xanthan gum. CR percutaneous absorption has been studied in vitro using excised human skin membranes [stratum corneum epidermis (SCE)] mounted into Franz cells. It was found that fluxes (Fn) of CR incorporated in MAD are influenced by the presence of monoolein based nanosystems. In particular xanthan gum based MAD better control CR diffusion from MAD.

Cannabinoid antagonist in nanostructured lipid carriers (NLCs): design, characterization and in vivo study

This study describes the preparation, characterization, and in vivo evaluation in rats of nanostructured lipid carriers (NLCs) encapsulating rimonabant (RMN) as prototypical cannabinoid antagonist. A study was conducted in order to optimize NLC production by melt and ultrasonication method. NLCs were prepared by alternatively adding the lipid phase into the aqueous one (direct protocol) or the aqueous phase into the lipid one (reverse protocol). RMN-NLCs have been characterized by cryogenic transmission electron microscopy (cryo-TEM), X-ray, photon correlation spectroscopy (PCS) and sedimentation field flow fractionation (SdFFF). Reverse NLCs were treated with polysorbate 80. RMN release kinetics have been determined in vitro by dialysis method. In vivo RMN biodistribution in rats was evaluated after intranasal (i.n.) administration of reverse RMN-NLC. The reverse protocol enabled to prevent the lost of lipid phase and to achieve higher RMN encapsulation efficacy (EE) with respect to the direct protocol (98% w/w versus 67% w/w). The use of different protocols did not affect NLC morphology and dimensional distribution. An in vitro dissolutive release rate of RMN was calculated. The in vivo data indicate that i.n. administration of RMN by reverse NLC treated with polysorbate 80 increased RMN concentration in the brain with respect to the drug in solution. The nanoencapsulation protocol presented here appears as an optimal strategy to improve the low solubility of cannabinoid compounds in an aqueous system suitable for in vivo administration.

Cationic lipid nanosystems as carriers for nucleic acids

Solid lipid nanoparticles (SLNs) consisting of tristearin or tribehenin, and monoolein aqueous dispersions (MADs) consisting of glyceryl-monoolein have been studied as potential nanocarriers for nucleic acids. The cationic character of nanocarriers was obtained by adding cationic surfactants, such as diisobutylphenoxyethyl-dimethylbenzyl ammonium chloride (DEBDA) or PEG-15 Cocopolyamine (PCPA), to the lipid composition. The products were characterised in terms of size and morphology by Cryo-TEM and PCS. The charge properties were determined by measuring the zeta potential. Our experimental protocol enabled us to obtain homogeneous and stable cationic nanosystems within 3-6 months of production. Assessment of cytotoxicity on HepG2 cells by MTT assays indicated that MAD preparations were less toxic than SLN, and in general PCPA-containing formulations are less cytotoxic than DEBDA-containing ones. The formation of electrostatic complexes with salmon sperm or plasmid DNA, used as model nucleic acids, was evaluated by electrophoresis on agarose gel. The results confirmed that all the formulations studied are able to form the complex. Finally, we investigated the ability of SLN and MAD to deliver DNA into HepG2 cells, and to this purpose we exploited expression plasmids for green fluorescent protein or firefly luciferase. Although with reduced efficiency, the results showed that the produced nanocarriers are able to convey plasmids into cells. The data obtained encourage further study aimed at improving these new formulations and proposing them as novel in vitro transfection reagents with potential application to in vivo delivery of nucleic acids.

Biodistribution of nanostructured lipid carriers: A tomographic study

This study describes the preparation, characterization, and biodistribution of radiolabelled nanostructured lipid carriers (NLCs) especially designed for in vivo tomographic study. A preliminary formulative study was conducted in order to incorporate (99m)Tc based tracer in NLCs. At this aim a (99m)Tc complex containing a terminal (99m)Tc ≡ N multiple bond ([(99m)Tc]N-DBODC2) has been synthesized and included in NLCs produced by a stirring and ultrasonication method. The morphological and dimensional characteristics of the produced NLCs have been accurately investigated by a number of specific techniques, including: cryogenic transmission electron microscopy, X-ray, photon correlation spectroscopy and sedimentation field flow fractionation. The obtained NLCs were employed for achieving in vivo tomographic images of the rat body by small-animal SPECT scanner that enabled the investigation of NLC biodistribution after intraperitoneal, intravenous, intranasal and oral administration. NLC production protocol allowed to firmly encapsulate the radiotracer within the nanoparticles. In vivo studies evidenced that NLC remained stable in vivo, suggesting their suitability as controlled release system for drugs and radiochemical for therapeutic and diagnostic purposes. Moreover the high resolution images obtained by the SPECT technique allowed to detect NLC presence in brown fat tissue, suggesting NLC therapeutic application for treating human obesity and related metabolic disorders.

Intranasal immunization in mice with non-ionic surfactants vesicles containing HSV immunogens: A preliminary study as possible vaccine against genital herpes

The purpose of this study was to investigate the potential of intranasal immunization with non-ionic surfactant vesicles (NISV) containing either the secretory recombinant form of glycoprotein B (gBs) of herpes simplex virus type 1 or a related polylysine reach peptides (DTK) for induction of protective immunity against genital herpes infection in mice. NISV were prepared by lipid film hydration method. The mean diameter of vesicles was around 390 nm for DTK-containing NISV (DTK-NISV) and 320 nm for gB1s-containing NISV (gB1s-NISV). The encapsulation efficiency of the molecules was comprised between 57% and 70%. After 7-14 day NISV maintained stable dimensions and a drug encapsulation higher than 48%. We showed that intranasal immunization with gB1s-NISV induces gB-specific IgG antibody and lymphoproliferative responses, whereas vaccination with DTK-NISV was not able to generate a gB-specific immune response. Our results indicate that vaccination of BALB/c mice with gB1s-NISV induced Th1 responses, as characterized by increased titre of IG2a in plasma and IFN-production in CD4+ splenic cells. Intranasal immunization with gB1s-NISV could elicit 90% (almost complete) protection against a heterologous lethal vaginal challenge with herpes simplex virus type 2. These data may have implications for the development of a mucosal vaccine against genital herpes.