Anna Lechanteur

Mucoadhesive cellulosic derivative sponges as drug delivery system for vaginal application

Vaginal delivery of active drugs has been largely studied for local and systemic applications. It is well known that vagina is a complex route, due to physiological and non-physiological changes. Therefore, in order to achieve a prolonged local effect, these variations have to be considered. The aim of this study was to formulate and to characterize a solid system, called sponges, obtained by lyophilization of cellulosic derivative (HEC 250M) hydrogels. These sponges have to meet particular criteria to be adapted for vaginal application: they have to adhere to the vaginal cavity and to be rehydrated by the small amount of vaginal fluids. Moreover, they have to be easily manipulated and to be stable. Three freezing temperatures have been tested to prepare sponges (-15°C, -25°C, -35°C). By SEM analyzes, it was observed that the pores into the sponges were smaller and numerous as the freezing temperature decreases. However, this temperature did not have any influence on the rehydration speed that was rather influenced by the HEC concentration. Viscosity and mucoadhesive strength of hydrogels and corresponding sponges were also measured. It appeared that these parameters are mainly dependent on the HEC concentration. These mucoadhesive sponges can be considered as potential drug delivery systems intended for vaginal application.

Development of anti-E6 pegylated lipoplexes for mucosal application in the context of cervical preneoplastic lesions

Cervical cancer induced by human papillomavirus (HPV) is the fourth highest mortality causing cancer in women despite the use of prophylactic vaccines. E6 targeting represents an attractive strategy to treat this cancer. Indeed, oncoprotein E6 is produced by keratinocytes infected by HPV and is partially responsible for carcinogenesis. E6 interferes with the apoptosis process in stressed cells by degradation of p53 tumor suppressor gene. Our strategy consists in using E6 siRNA complexed with pegylated lipoplexes. The addition of hydrophilic polymer around the nanoparticles is crucial to use them by vaginal application on account of cervicovaginal mucus. Physicochemical characteristics were evaluated and in vitro assays were performed to evaluate transfection potential, E6 mRNA extinction and p53 re-expression. Cationic liposomes DOTAP/Cholesterol/DOPE 1/0.75/0.5 (N/P 2.5) with or without 50% DSPE-PEG2000 and associated with siE6 have demonstrated good physicochemical characteristics in terms of complexation, size, surface charge and stability. Both lipoplexes have been tested on CaSki cell line (HPV 16+) with 50 nM and 100 nM of siE6. Lipoplexes formulations induce 30-40% of E6 mRNA extinction and induce the re-expression of p53. In conclusion, pegylated anti-E6 lipoplexes have demonstrated their efficiency to cross the cellular membrane and to release siRNA into the cytoplasm confirmed by final p53 protein production.

The role of mucus in cell-based models used to screen mucosal drug delivery

Abstract The increasing interest in developing tools to predict drug absorption through mucosal surfaces is fostering the establishment of epithelial cell‐based models. Cell‐based in vitro techniques for drug permeability assessment are less laborious, cheaper and address the concerns of using laboratory animals. Simultaneously, in vitro barrier models that thoroughly simulate human epithelia or mucosae may provide useful data to speed up the entrance of new drugs and new drug products into the clinics. Nevertheless, standard cell‐based in vitro models that intend to reproduce epithelial surfaces often discard the role of mucus in influencing drug permeation/absorption. Biomimetic models of mucosae in which mucus production has been considered may not be able to fully reproduce the amount and architecture of mucus, resulting in biased characterization of permeability/absorption. In these cases, artificial mucus may be used to supplement cell‐based models but still proper identification and quantification are required. In this review, considerations regarding the relevance of mucus in the development of cell‐based epithelial and mucosal models mimicking the gastro‐intestinal tract, the cervico‐vaginal tract and the respiratory tract, and the impact of mucus on the permeability mechanisms are addressed. From simple epithelial monolayers to more complex 3D structures, the impact of the presence of mucus for the extrapolation to the in vivo scenario is critically analyzed. Finally, an overview is provided on several techniques and methods to characterize the mucus layer over cell‐based barriers, in order to intimately reproduce human mucosal layer and thereby, improve in vitro/in vivo correlation. Graphical abstract Figure. No Caption available.

Freeze-dried mucoadhesive polymeric system containing pegylated lipoplexes: towards a vaginal sustained released system for siRNA

Topical vaginal sustained delivery of siRNA presents a significant challenge due to the short residence time of formulations. Therefore, a drug delivery system capable to adhere to the vaginal mucosa is desirable, as it could allow a prolonged delivery and increase the effectiveness of the therapy. The aim of this project is to develop a polymeric solid mucoadhesive system, loaded with lipoplexes, able to be progressively rehydrated by the vaginal fluids to form a hydrogel and to deliver siRNA to vaginal tissues. To minimize adhesive interactions with vaginal mucus components, lipoplexes were coated with different derivatives of polyethylene glycol: DPSE-PEG2000, DPSE-PEG750 and ceramide-PEG2000. Based on stability and diffusion properties in simulated vaginal fluids, lipoplexes containing DSPE-PEG2000 were selected and incorporated in hydroxyethyl cellulose (HEC) hydrogels. Solid systems, called sponges, were then obtained by freeze-drying. Sponges meet acceptable mechanical characteristics and their hardness, deformability and mucoadhesive properties are not influenced by the presence of lipoplexes. Finally, mobility and stability of lipoplexes inside sponges rehydrated with vaginal mucus, mimicking in situ conditions, were evaluated by advanced fluorescence microscopy. The release rate was found to be influenced by the HEC concentration and consequently by the viscosity after rehydration. This study demonstrates the feasibility of entrapping pegylated lipoplexes into a solid matrix system for a prolonged delivery of siRNA into the vagina.

PEGylation of lipoplexes: The right balance between cytotoxicity and siRNA effectiveness.

The delivery of small interfering RNA (siRNA) is an attractive therapeutic approach to treat several pathologies, such as viral infections or cancers. However, the stability and the efficacy of these biotherapies are still a major obstacle to their use. Cationic liposomes (DOTAP/Chol/DOPE 1/0.75/0.5M ratio) have been complexed to siRNA (lipoplexes) in order to be administrated by the vaginal route, in the context of HPV16 induced cervical preneoplastic lesions. To overcome the constraint of the cervico-vaginal mucus, PEGylation is required to allow the diffusion of lipoplexes through it. Thereby, PEGylated lipoplexes coated with three types of polyethylene glycol (PEG) as DSPE-PEG2000, DSPE-PEG750 or C8-PEG2000-Ceramide (Ceramide-PEG2000) at different densities have been developed and characterized. PEGylated lipoplexes were successfully prepared and showed a hydrodynamic diameter around 200nm, appropriate for vaginal application. In vitro assays on HPV16 positive cell lines revealed that a positive charge of PEGylated lipoplexes allows a higher mRNA knockdown by siRNA. However, the cationic property is also associated to cytotoxicity. The addition of a high percentage of PEG prevented this toxicity but seemed also to reduce siRNA endosomal escape, probably by steric hindrance. The decreasing of PEG density of Ceramide-PEG2000 to 20% allows the release of siRNA and in consequence, biological activities, contrarily to DSPE-PEG. These results suggest that Ceramide-PEG is more appropriate for siRNA delivery compared to DSPE-PEG. In conclusion, the right balance between cytotoxicity and siRNA effectiveness has been found with the transfection of lipoplexes coated with 20% of Ceramide-PEG2000. This new nanovector could have a high potential against multiple mucosal diseases, such as human papillomavirus-induced genital lesions.

Promoting Vaginal Distribution of E7 and MCL-1 siRNA-Silencing Nanoparticles for Cervical Cancer Treatment

There is an urgent need to develop a less aggressive and more effective treatment against cervical lesions induced by different high-risk human papillomavirus (HR-HPV). We investigated the potential of a cocktail of small interfering RNA (siRNA) directed against the oncoprotein E6 (E6), the oncoprotein E7 (E7), or the antiapoptotic protein MCL-1 (MCL-1). The combination of siRNA anti-E7 and anti-MCL-1 demonstrated high efficacy on multiple HPV16 and HPV18 cell lines and no effects on healthy keratinocytes. This gene therapy has been considered for a vaginal administration since this route of application holds high potential for the treatment of diseases in the female reproductive tracts. Therefore, PEGylated lipoplexes have been designed and characterized to protect siRNA and to diffuse in the mucosal environment before they reach the cervico/vaginal epithelium. This new nanovector complexed to the combination of active siRNA induced an efficient mRNA knockdown since biological effects were obtained in vitro. This work also provided evidence that the PEGylated lipoplexes had appropriate physicochemical properties to diffuse into a mucin network according to size measurement experiments in artificial mucus. After demonstrating the distribution and the efficacy of siRNA into a 3D-cervical model lesion and through porcine vaginal mucosa, in vivo experiments in mouse have been performed under physiological conditions. This study revealed a complete and sustained coverage of the mucosal epithelium following an unique vaginal administration of fluorescent PEGylated lipoplexes. Overall, our results showed the potential of the PEGylated lipoplexes for the prolonged delivery of active siRNA to treat HPV-induced lesions.

Elucidation of the impact of cell culture conditions of Caco-2 cell monolayer on barrier integrity and intestinal permeability

Graphical abstract Figure. No Caption available.

Cationic Liposomes Carrying siRNA: Impact of Lipid Composition on Physicochemical Properties, Cytotoxicity and Endosomal Escape

In recent year, cationic liposomes have gained a lot of attention for siRNA delivery. Despite this, intracellular barriers as endosomal escape and cytosolic delivery of siRNA still represent a challeng, as well as the cytotoxicity due to cationic lipids. To address these issues, we developed four liposomal formulations, composed of two different cationic lipids (DOTAP and DC-Cholesterol) and different ratio of co-lipids (cholesterol and DOPE). The objective is to dissect these impacts on siRNA efficacy and cytotoxicity. Liposomes were complexed to siRNA at six different N/P molar ratios, physico-chemical properties were characterized, and consequently, N/P 2.5, 5 and 10 were selected for in vitro experiments. We have shown that cytotoxicity is influenced by the N/P ratio, the concentration of cationic lipid, as well as the nature of the cationic lipid. For instance, cell viability decreased by 70% with liposomes composed of DOTAP/Cholesterol/DOPE 1/0.75/0.5 at a N/P ratio 10, whereas the same formulation at a N/P ratio of 2.5 was safe. Interestingly, we have observed differences in terms of mRNA knock-down efficiency, whereas the transfection rate was quite similar for each formulation. Liposomes containing 50% of DOPE induced a mRNA silencing of around 80%. This study allowed us to highlight crucial parameters in order to develop lipoplexes which are safe, and which induce an efficient intracytoplasmic release of siRNA.

Capillary electrophoresis method to determine siRNA complexation with cationic liposomes

Small interfering RNA (siRNA) inducing gene silencing has great potential to treat many human diseases. To ensure effective siRNA delivery, it must be complexed with an appropriate vector, generally nanoparticles. The nanoparticulate complex requires an optimal physiochemical characterization and the complexation efficiency has to be precisely determined. The methods usually used to measure complexation in gel electrophoresis and RiboGreen® fluorescence‐based assay. However, those approaches are not automated and present some drawbacks such as the low throughput and the use of carcinogenic reagents. The aim of this study is to develop a new simple and fast method to accurately quantify the complexation efficiency. In this study, capillary electrophoresis (CE) was used to determine the siRNA complexation with cationic liposomes. The short‐end injection mode applied enabled siRNA detection in less than 5 min. Moreover, the CE technique offers many advantages compared with the other classical methods. It is automated, does not require sample preparation and expensive reagents. Moreover, no mutagenic risk is associated with the CE approach since no carcinogenic product is used. Finally, this methodology can also be extended for the characterization of other types of nanoparticles encapsulating siRNA, such as cationic polymeric nanoparticles.

Development and characterization of lipid-polymeric nanoparticles for oral insulin delivery

ABSTRACT Introduction: The oral route is widely accepted as the most physiological path for exogenous administration of insulin, as it closely mimic the endogenous insulin pathway. Thus, in this work it is proposed an innovative lipid-polymeric nanocarrier to delivery insulin orally. Areas covered: Nanoparticles were produced through a modified solvent emulsification-evaporation method, using ethyl palmitate and hydroxypropylmethylcellulose acetate succinate as matrix. Lipid-polymeric nanoparticles were around 300 nm in size, negatively charged (−20 mV) and associated insulin with efficiency higher than 80%. Differential scanning calorimetry suggested thermal stability of nanoparticles. In vitro release assays under simulated gastrointestinal conditions resulted in 9% and 14% of insulin released at pH 1.2 during 2 h and at pH 6.8 for 6 h, respectively, demonstrating the ability of those nanoparticles to protect insulin against premature degradation. Importantly, nanoparticles were observed to be safe at potential therapeutic concentrations as did not originate cytotoxicity to intestinal epithelial cells. Lastly, the permeability of nanoencapsulated insulin through Caco-2 monolayers and a triple Caco-2/HT29-MTX/Raji B cell model correlated well with slow release kinetics, and fosters the effectiveness of nanoparticles to promote intestinal absorption of peptidic drugs. Expert opinion: Lipid-polymeric nanoparticles were developed to encapsulate and carry insulin through intestine. Overall, nanoparticles provide insulin stability and intestinal permeability.