Development of serum stable lipoplex for in vivo gene transfer and characterization of coformulation liposomes for increased gene expression

A method for formulation of cationic lipid/plasmid DNA complex has been devised that yields stable complexes with final DNA concentration of 0.2 mg/mL. The particles can be formed with a monocationic diacylglycerol lipid, like DOTAP, or cholesterol based cationic lipid, like DC-Chol. The resultant suspension is composed of particles with an average diameter of 70 nm. Electron microscopy revealed a multilamellar "finger print" configuration for the ethanolic complexes. In vitro transfection demonstrated 2-fold higher transfection efficiency in serum-containing media compared to serum-free media. The DC-Chol complex is able to transfect cells in serum-containing medium for 3 weeks on storage as suspension in 5% dextrose at 4°C. In vivo gene transfer studies in a rat brain tumor model showed that the DC-Chol formulation yielded gene transfer to rat brain tumor vasculature, whereas no expression was observed for DOTAP complexes.; To understand the difference on gene expression from different lipid formulations, decomplexation of the ethanolic complexes based on endosomal release model was used. The DC-Chol complexes released more plasmid DNA in a 10-fold excess of negatively charged liposomes (DOPS/DOPE 1:2 mol: mol) compared to DOTAP complexes based on 1% agarose gel electrophoresis and TO-PRO-1 assay.; The effect of decomplexation on gene transfer was tested. The A-172 cells were either incubated with transfection complex followed by negatively charged liposomes or incubated in the reverse order of addition. Higher expression was observed regardless of the order of the addition. Our study also illustrated the following. (1) Different anionic lipids (PS, PA, PG) could increase gene transfer of cationic complexes. (2) Different cationic complexes (DMRIE-C, DOTAP/DOPE, and DC-Chol/DOPE) gene transfection could be changed by anionic liposome. (3) Higher gene transfection was observed in anionic liposome containing DOPE and cholesterol. (4) Crystalline multilamellar anionic liposome with small particle size yielded higher gene expression. (5) The pH-sensitive anionic liposome formulation could also increase gene expression.; To further explore the mechanism for enhancement of gene transfer, a calcein release study was conducted and indicated possible endosome membrane disruption induced by anionic liposome. De-convolution microscopy indicated that anionic liposome could increase nuclear DNA uptake.