DEVELOPMENT OF PEG-COATED LIPOPLEXES TO BE INCORPORATED INTO MUCOADHESIVE HEC-SPONGES Tania Furst 1, Anna Lechanteur 1,2, Pascale Hubert 2, Brigitte Evrard.

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DEVELOPMENT OF PEG-COATED LIPOPLEXES TO BE INCORPORATED INTO MUCOADHESIVE HEC-SPONGES Tania Furst 1, Anna Lechanteur 1,2, Pascale Hubert 2, Brigitte Evrard 1, Géraldine Piel 1 1 Laboratory of Pharmaceutical Technology and Biopharmacy - CIRM, University of Liege (4000), Belgium 2 Laboratory of Experimental Pathology – GIGA Cancer, University of Liege (4000), Belgium 1. INTRODUCTION 2. RESULTS AND DISCUSSION 3. CONCLUSION AND PERSPECTIVES This research has a double objective: Firstly, the preparation of cationic nanovectors, liposomes, which are elaborated for a topical administration into vagina. Liposomes are complexed with siRNA to form lipoplexes. DSPE-PEG 2000, an hydrophilic polymer, is added to lipoplexes to facilitate their diffusion through cervico-vaginal mucus. These lipoplexes must have good physicochemical characteristics to be effective. They also have to be stable in acidic environment (vaginal pH ) and do not release the siRNA. Secondly, we would like to incorporate these lipoplexes into hydroxyethylcellulose ( HEC ) gels, which will be freeze-dried to form sponges. For that, we must first characterize these sponges. They have to be malleable to facilitate their handling. They have to adhere to the vaginal mucosa and to rehydrate in a short time to allow the diffusion of lipoplexes. A.Preparation and characterization of liposomes, lipoplexes and lipoplexes-PEG Liposomes are prepared from a mixture of 3 lipids: DOTAP/Chol/DOPE 1/0.75/0.5 + siRNA  lipoplexes with >95% of encapsulation at N/P=2.5 DSPE-PEG 2000 is added by the post-insertion technique (from 10 to 100 mol% of DOTAP) Table 1. physicochemical characteristics of liposomes and lipoplexes Fig.2. Size, PDI and zeta potential of PEG-coated lipoplexes (A) When the % of PEG increases, the diameter is ranged between 150 and 220nm. Up 50%, PDI 0.5. (B) The zeta potential decreases from +50mV to -20mV when the % of PEG increases. (n=3) B.Preparation and characterization of HEC hydrogels and sponges Lipoplexes DOTAP/Chol/DOPE 1/0.75/0.5 at N/P=2.5 with and without PEG have good physicochemical characteristics. Moreover, they seem to be stable in acidic pH, there is no leakage of siRNA and their sizes remain constant. Other studies will be realized to verify their efficacy and stability in mucus, in the gel and after lyophilization. Regarding sponges, HEC 250M seems to be an ideal polymer to form a mucoadhesive system. A balance between the viscosity and the mucoadhesion need to be selected to obtain an optimal system to further incoporate lipoplexes. The deformability and the hardness of the sponges will also be characterized with the Texture Analyzer DOTAP/Chol/DOPE 1/0,75/0,5N/PZ-average diameter (nm)PDIzeta potential (mV) liposomes0 163,6 ± 5,6 0,12 ± 0,03 53,15 ± 6,0 lipoplexes2,5219,9 ± 14,8 0,15 ± 0,05 48,0 ± 1,7 The gels (6g) are prepared by homogeneization of HEC 250M, PEG 400 and milliQ water The sponges are obtained after freeze-drying (24hours) The mucoadhesion is characterized with a Texture Analyzer  mucin discs (250mg) and synthetic cervical mucus are used Lipoplexes and PEG-coated lipoplexes in acidic environment Fig.3. Agarose gel electrophoresis There is no leakage of siRNA when lipoplexes 1/0.75/0.5 are in contact with acidic pH. Fig.4. Size and zeta potential of lipoplexes When the pH becomes increasingly acidic, there is no variation in size (around 200nm) and zeta potential (around 45mV) and lipoplexes are stable. (n=3) (A)(B) Fig.5. Size of PEG-coated lipoplexes The sizes of the different pegylated lipoplexes seem to be constant (around 200nm) when the pH is acidic. (n=1) F min = adhesion force Fig.5. Example of graph obtained with the TA to quantify the mucoadhesion. F min represents the adhesion force of the gel/sponge with the mucin disc. Fig.6. Mucadhesion of the gels and the sponges (A) The mucoadhesion of the gels is measured before and after lyophilization and rehydration with synthetic cervical mucus (2.5ml). When the viscosity of the gel increases the mucoadhesion increases too. (n=12) (B) The mucoadhesion of the sponges is consistent with the one of the gels. The more the viscosity of the gels increases, the more the mucoadhesion increases. (n=15) (A)(B) HEC 250M (mg)PEG400 (mg) A10025 B20025 C20050 Fig.7. SEM images of transversal section of sponges A, B and C respectively. The strucutre of the pores is also related to the viscosity of the gels before freeze-drying. When the viscosity increases, the pores are more numerous and organized.