LIPOSOME PRODUCTION BY INNOVATIVE METHOD USING SUPERCRITICAL FLUIDS

INTRODUCTION
Liposomes are nanoparticles made of phospholipids, able to encapsulate many active molecules, protecting them and transporting them in a targeted way. Liposomes are thus widely studied as vectors of numerous active molecules, improving their therapeutic window. However, the usual production methods often involve the use of organic solvents. Moreover, traditional methods used at the laboratory scale are generally difficult to transfer to the industrial scale under GMP condition [1], [2]. Supercritical fluids are increasingly used in the pharmaceutical industry. Depending on the pressure and the temperature, this fluid develops some properties between those of liquids and gases suitable for particle engineering [3].

OBJECTIVES
The aim of the research is to develop an innovative method using supercritical fluids technology to allow the large-scale production of liposomes in GMP conditions, in one step and without the use of organic solvents.

MATERIALS AND METHODS
Formulation: A dispersion of soybean phosphatidylcholine and cholesterol 70/30 % (m/m) was prepared in HEPES (acide 4-(2-hydroxyéthyl)-1-pipérazine éthane sulfonique) buffer 0,01 M pH 7,38 at 65°C and stirred at 1200 rpm for 15 minutes.
Supercritical process: CO2 was chosen as a supercritical fluid. The involved parameters were: pressure, temperature, agitation rate, contact time, concentration in lipids of the dispersion and volume of the lipid dispersion.
Experimental design: 20 experiments varying the 6 influential parameters over definite intervals of values were performed: the lipids concentration (from 5 mM to 50 mM), the dispersion volume introduced into the reactor (from 10 mL to 30 mL), the contact time with the supercritical fluid (from 0.5 h to 2 h), the agitation rate (from 400 rpm to 700 rpm), the temperature (from 35°C to 80°C) and the pressure (from 120 bars to 250 bars).
Physico-chemical properties: Size, PdI and zeta potential were characterized before and after the supercritical process using Dynamic Light Scattering (DLS) (n=3).
RESULTS
The lipid concentration, the temperature, the pressure and, to a lesser extent, the dispersion volume and the stirring speed have a real impact on the physicochemical characteristics of the liposomes produced, whereas the contact time seemed to have only a weak influence.

CONCLUSION AND PERSPECTIVES
This initial analysis helped to refine the experimental plan. Indeed, the ranges of values of the influential parameters were refined in order to be closer to the values giving interesting results. Regarding the contact time, a negligible parameter, it was set at 30 minutes. A new design of experiment is in progress and will allow a complete analysis of the influential parameters.