Technological Aspects of Scalable Processes for the Production of Functional Liposomes for Gene Therapy

Abstract

The success of gene delivery systems in in vivo or in vitro applications depends on efficient transfection. Cationic liposomes remain a promising alternative for nonviral DNA carriers, mainly because they protect DNA from interstitial fluids and easily interact with cells (Gregoriadis, 1993; Lasic, 1997). However, in order to be effective in the immunological response, cationic liposomes must be functional and reach their specific target. Stability, reduced toxicity, efficiency in delivering genes to cells, and specific targeting to the nucleus are essential requirements for prophylactic and/or therapeutic performance. In order to achieve these standards, important physico-chemical parameters in liposomes must be controlled, such as the functionality of the lipids, the concentration of the cationic lipid, DNA loading (reflected by the R+/molar charge ratio), the zeta potential, size, and polydispersity. Several laboratory experiments have already explored DNA vaccines using cationic lipids. A classical investigation of lipid functionality and composition, as well as the efficiency of cationic liposomes as DNA carriers, was performed by Perrie and colleagues (Perrie & Gregoriadis, 2000; Perrie et al., 2001). The plasmid pRc/CMV HBS encoding the S (small) region of hepatitis B surface antigen was encapsulated in dehydrated-hydrated liposomes composed of egg phosphatidylcholine (EPC, bilayer-forming lipid), 1,2-dioleoyl-3trimethylammonium-propane (DOTAP, cationic lipid), 1,2-dioleoyl-sn-glycero-3phosphoethanolamine (DOPE, helper lipid) in a 50:25:25 percent molar ratio. The authors demonstrated that the encapsulation process protects the DNA vaccine against incubation with sodium dodecyl sulfate (SDS) due to DNA incorporation inside the liposome lamella (Perrie & Gregoriadis, 2000), and a better immunological response was obtained with cationic liposomes compared to naked DNA. Concerning size and polydispersity, different authors have reported that nanoparticle size is an important parameter for transfection success (Ma et al., 2007; Ogris et al., 1998; Rejman et al., 2004; Ross & Hui, 1999; Wiewrodt et al., 2002). Indeed, most of the variability in transfection procedures is a consequence of non-viral gene delivery systems with high polydispersity index values. The polydispersity index is related to the width of the particle