LIPIDS IN NON VIRAL VACCINE ENGINEERING: BIOCHEMICAL AND BIOPHYSICAL APPROACH

NADIA S. CHIARAMONI; LUCÍA SPERONI; CARLOS FACUNDO TEMPRANA; JULIETA GASPARRI; MARÍA C. TAIRA; SILVIA DEL V. ALONSO-ROMANOWSKI

La Havanna, Cuba

Congreso; 5th International congress Chemistry and Chemical Engineering; 2004

Abstract Liposomal vaccines can be made by associating microbes, soluble antigens, cytokines or deoxyribonucleic acid (DNA) with liposomes, the latter stimulating an immune response on expression of the antigenic protein. Cationic liposomes (CLs) are used as gene vectors (carriers) in worldwide human clinical trials of non-viral gene therapy. These lipid-gene complexes have the potential of transferring large pieces of DNA of up to 1 million base-pairs into cells. As our understanding of the mechanisms of action of CL-DNA complexes remains poor and transfection efficiencies are still low when compared to gene delivery with viral vectors. We describe recent studies with a combination of biophysical and biochemical techniques (CD, UV-Visible and Fluorescence for structure determination, along with hydrophobicity index, osmotic and hydrodynamic volume by light scattering, DSC, protein , DNA and phospholipids determinations, electronic and optical microscopy to probe the interactions of plasmid DNA molecules with membranes, stability and toxicity evaluated through peroxidation index, haemolysis, biodistribution, serum protein binding and immune response), which collectively are beginning to unravel the relationship between the distinctly structured CL-DNA complexes and their immune modulation. The work described here is applicable to gene delivery optimization in vitro and in vivo. Cationic lipid performance is compared with neutral and negative lipids. Polymeric lipids are also tested. Complexes containing the CL lipids show the highest DNA encapsulation efficiency with changes in the DNA conformation. In addition to the lipid structure, many other factors are known to influence transfection activity in vivo. Such variables include the stoichiometry of the various components of the complex, the rate and order of mixing of these components, their respective concentrations, the rate and order of mixing of these components, their respective concentrations, the selection of excipients used in the formation of the complexes. Depending on the parameters used, different-sized lipid/pDNA complexes with various net charges realized at the surfaces of these complexes can be attained. These variables can result in the generation of complexes with significantly different structure-activity relationships and transfection activities. Future directions are outlined, which make use of a strategy for liposomal formulation, characterization and evaluation in vitro and in vivo. This special drug delivery system is mented to carry viral epitopes (Vp7) from Rotavirus, responsible of immune activation, both cellular and humoral to avoid the effects of Rotavirus infection (childhood diarrhea). At present protein production and liposomal formulations containing plasmid DNA expressing VP7 viral proteins in stable liposomes, made of EPC, DMPE, CHOL,:DOPE,:DOTAP, and DC8,9PC, in different ratios are analyzed. In all cases lipid-DNA mass ratio was 300:1. The above studies demonstrate that DNA is efficiently encapsulated into liposomes carrying very different conformations and transfection and structure-activity relationships were evaluated.