Gemini Amphiphile-Based Lipoplexes for Efficient Gene Delivery:Synthesis, Formulation Development, Characterization, GeneTransfection, and Biodistribution Studies

摘要

Some quaternary gemini amphiphiles (GAs) were synthesized asnonviral gene delivery carriers. The critical miceller concentration values of theseamphiphiles are indicative of their superior surface-active properties. All of thesynthesized GAs, alone or along with lipids like cholesterol and/or dioleoylphosphatidyl ethanolamine (DOPE), were formulated as liposomes. Formulations of GAswith DOPE showed average particle diameters of 326?400 nm with positive ζ-potential (30.1?46.4 mV). The lipoplexes of theses formulations showed completepDNA retention at the base at a N/P ratio higher than 1.0 in gel retardation study.The GAs were effective in condensing pDNA into a ψ-phase, as indicated by circulardichroism study, and provided complete protection of the pDNA against the enzymeDNase at a N/P ratio more than 1. In vitro cell line studies showed that GA liposomalformulations caused β-gal expression and offered a higher transfection efficiency thanthat of liposomes prepared with the help of N-[1-(2,3-dioleoyloxy)propyl]-N,N,Ntrimethylammonium methyl-sulfate (DOTAP)/DOPE and dicyclocarbodiimide(DCC)/DOPE but comparable to those of Lipofectamine 2000 in A549 and HeLa cell lines. Modulation of head grouppolarity significantly affected the transfection efficacy of GAs. The cell viabilities of almost all of the formulations werecomparable to those of the standards (DCC/DOPE and DOTAP/DOPE liposomes). Incorporation of cholesterol [GA/DOPE/cholesterol in the ratio of 1:1:1] further improved the serum compatibility of the formulations and improved thetransfection efficacy when evaluated in A549 and HeLa cell lines. Fluorescence-assisted cell sorting studies showed comparablenumber of transfected cells to Lipofectamine 2000 in the HeLa cell line. Intracellular trafficking studies using confocalmicroscopy indicated transfection of the HeLa cells with the reporter gene within 30 min of lipoplex treatment. γ-Scintigraphyusing 99mTc-labeled lipoplexes showed higher concentrations of the lipoplexes in vital tissues like liver, spleen, lungs, andkidneys.