Sugar-based tertiary amino gemini surfactants with a vesicle-to-micelle transition in the endosomal pH range mediate efficient transfection in vitro

Novel reduced sugar gemini amphiphiles linked through their tertiary amino head groups via alkyl spacers of 4 or 6 carbons, and with varying (unsatura ted) alkyl tail lengths of 12-18, have been synthesized and tested for tran sfection in vitro in an adherent Chinese hamster ovary cell line (CHO-K1). Transfection efficiencies peaked at 2.7 times that of the commercial standa rd Lipofectamine Plus/2000 for pure solutions of the compound bearing unsat urated (oleyl) alkyl tails. For those compounds bearing saturated alkyl tai ls, transfection efficiency peaked at a tail length of 16, at a level simil ar to Lipofectamine Plus/2000. All of the amphiphiles formed bilayer vesicl es at physiological pH. Some of the amino groups at the surface were proton ated, and vesicles therefore bore a positive charge. Increased protonation with reduced pH resulted in greatly increased monomer solubility and a morp hology change from vesicle to micelle at characteristic pH values, dependen t on the tail length. For the compounds promoting high transfection efficie ncy, this characteristic pH was within the range found in the endosomal com partment (7.4-4.0). Formation of mixed micelles between gemini surfactant a nd membrane phospholipids at reduced pH may therefore provide a method of e ndosome rupture and subsequent escape of entrapped DNA, thus discarding the need for extra fusogenic or endosomolytic agents. The positive charge on t he vesicles at physiological pH drives the colloidal association with DNA. Small angle X-ray scattering measurements indicate that lamellar aggregates are formed, which have a d spacing of 48-54 Angstrom. Preliminary differen tial scanning calorimetric measurements suggest that reduction of pH causes a disordering of the hydrocarbon region of the DNA-surfactant complex.