Efficient encapsulation of proteins in submicro liposomes using a supercritical fluid assisted continuous process

Recently a new supercritical assisted process was proposed in the literature for the production of liposomes, called Supercritical Assisted Liposome formation (SuperLip) process. It has been demonstrated that, using this new process, it is possible to produce liposomes based on phosphatidylcholine (PC) with a controlled particle size distribution. Also a preliminary encapsulation test revealed a high encapsulation efficiency of bovine serum albumin (BSA), used as model hydrophilic compound. Being convinced of the great potential of the SuperLip process, in this work it has been studied the possibility of producing liposomes with a different composition of the bilayer membrane using the SuperLip process. Phosphatidylcholine (PC) and phosphatidylglycerol (PG) were used in a mixture and the production of liposomes was tested under different operative conditions: pressure, temperature and lipid composition. Results obtained using only PC and PC/PG mixture have been systematically compared. Furthermore a deepened study about the possibility to encapsulate BSA as model compound was performed. SuperLip experiments using different amount of BSA dissolved in the water internal phase were performed and the relative encapsulation efficiencies of produced liposomes were estimated. A comparison of encapsulation efficiency of liposomes produced, at the same BSA loadings, using a conventional preparation process (Bangham method) was also performed. The stability of entrapped protein was investigated. Sub-micrometric liposomes of soybean phosphatidylcholine (PC) of different size and distribution ranging between 250 ± 58 nm and 330 ± 82 nm were successfully produced. When using PG coupled with PC, larger liposomes were produced, ranging between 280 ± 70 nm and 350 ± 101 nm. Both PC and PC/PG liposomes were stable over one month, thanks to the large and negative surface charge (zeta potential ranging between -20 mV and -30 mV). For the drug encapsulation tests different BSA theoretical loadings with respect to the lipid amount (10-30-60%, w/w) were tested. In the case of SuperLip process, very high encapsulation efficiencies (92-98%) were obtained at all the drug loadings; lower encapsulation efficiencies were instead obtained using the Bangham method (2-57%). Results reported in this work demonstrated that using SuperLip process, the protein contained in the water phase can be efficiently entrapped without damaging the protein structure as confirmed by FTIR analysis of processed BSA.