Mucoadhesive hydroxyethyl-cellulose sponges as lipoplexes delivery system for vaginal application

It is well known that the vagina is an excellent route of mucosal drug delivery for local applications. However, an important parameter to consider and which could interfere with the performance of a delivery system is the vaginal mucus and its natural clearance process. This study has a double objective. The first consists on the elaboration of a solid mucoadhesive system which will be retained in the vagina. We focused on hydrogels formed with a mucoadhesive polymer, hydroxylethyl-cellulose (HEC). These hydrogels are lyophilized in order to obtain a solid form, with a spongy texture. Sponges must meet different criteria: adhere to the vaginal cavity and be rehydrated with a little amount of mucus to reform hydrogel with an appropriated viscosity. Different freezing temperatures during lyophilization have been tested and sponges have been characterized in terms of mucoadhesion and rehydration ability. The second objective is the incorporation of lipoplexes, formed by complexation of siRNA and cationic liposomes, into this system in order to treat vaginal diseases by the RNA interference mechanism. These lipoplexes have to diffuse through the rehydrated system and through the vaginal mucus to reach vaginal cells. For that, they have been pegylated. It has been demonstrated that a dense coating of low molecular PEG could facilitate the diffusion through the mucus. Sponges containing lipoplexes will be prepared by lyophilization of hydrogels containing the lipoplexes. We first studied the physicochemical characteristics of lyophilized lipoplexes. In order to protect lipidic membrane during freezing process and to avoid an excessive increase in size, a cryoprotectant (trehalose) was added. Keyword(s): Vaginal; Sponge; Lyophilization; Lipoplexes. 1. EXPERIMENTAL METHODS 1.1 Preparation of HEC hydrogels and sponges Hydrogels (6 g) were prepared by a homogenous aqueous dispersion of HEC 250M and PEG 400 (Table 1). Sponges were then prepared by lyophilization of hydrogels. Three freezing temperatures have been tested; -15°C, -25°C or -35°C. Table 1. Composition of hydrogels HEC 250M (mg) PEG 400 (mg) A 100 25 B 200 25 C 200 50 1.2 Rehydration study The ability of sponges to be rehydrated was evaluated. To simulate the dilution that may occur after a vaginal application, 2 mL of synthetic cervicovaginal mucus (CVM) were added on sponges. 1.3 Evaluation of mucoadhesive strength Mucoadhesive strength of hydrogels and sponges was determined using a texture analyzer (TA), as described in figure 1. For that, a mucin disc was attached to the probe of the TA and the mucoadhesion (N) was determined from the force of detachment between the disc and the hydrogel/sponge. Figure 1: Determination of mucoadhesive strength (N). 1.4 Preparation of liposomes, lipoplexes and pegylated lipoplexes Liposomes were prepared from a mixture of DOTAP, DOPE and cholesterol at 1/0.75/0.5 molar ratio (5.6 mM), by the hydration of lipid film method. Scramble siRNA was added to liposomes to form lipoplexes at optimal N/P 2.5. Then, 30% of DSPE-PEG2000, PEG750 or C8-PEG2000ceramide (% mol/DOTAP) were added to preformed lipoplexes. Finally, 1, 3, 5, 7 or 10% of trehalose were added to the suspension. 1.5 Measurement of particle size and zeta potential The sizes (nm) of lipoplexes and pegylated lipoplexes were determined before and after lyophilization (-35°C), using a Zetasizer Nano ZS (Malvern Instruments, UK). 2. RESULTS AND DISCUSSION 2.1 Sponges The microstructure of the sponges was analyzed by SEM microscopy. All sponges are formed by a dense network of interconnected pores. On figure 2a, it clearly appears that the sizes of the pores depend on the freezing temperature (1C at -15°C, 2C at -25°C and 3C at -35°C). Freezing at -15°C has created big ice crystals that have disrupted the hydrogel and made large pores. Conversely, freezing at -35°C has generated small ice crystals and thus small pores. These observations were confirmed comparing the surface areas of the pores (μm2), calculated using ImageJ® software (figure 2b). Figure 2. (a) SEM images of sponges C obtained after the 3 freezing conditions. (b) Median surface areas (μm2) of pores in sponges. -25 mm = step 3 +1 mm, 60 sec = step 2 0 = step 1