Physico-chemical analysis of cationic liposome–DNA complexes (lipoplexes) with respect to in vitro and in vivo gene delivery efficiency†

Abstract

Cationic liposomes are potential vectors for gene therapy applications. In previous work, our first generation cationic liposome system, formulated from cytofectin 3β-[N-(N′,N′-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol) and the neutral phospholipid dioleoyl-L-α-phosphatidylethanolamine (DOPE), was shown to transfect the lungs of mice in vivo. More recently, we described second generation cationic liposome systems including one formulated from DOPE and the novel pentaamine cytofectin N15-cholesteryloxycarbonyl-3,7,12-triazapentadecane-1,15-diamine (CTAP). As a result of formulation changes, CTAP–DOPE cationic liposomes are shown here to be approximately 400-fold more efficient at mediating gene delivery to mouse lung in vivo than DC-Chol–DOPE liposomes (2000-fold more effective than plasmid DNA alone). Physico-chemical analyses were performed on CTAP–DOPE, other second generation cationic liposome systems and DC-Chol–DOPE to determine how differences in the structural and physical properties of cytofectins, cationic liposomes and lipoplex mixtures might affect the efficiency of transfection both in vitro and in vivo. The data suggest that CTAP–DOPE cationic liposomes are effective in vivo for two reasons. (1) They are able to efficiently neutralise, condense and encapsulate nucleic acids into lipoplex particles; (2) they present unprotonated amine functional groups (pKa < 8) at neutral pH that could have the capacity for endosome buffering, thereby facilitating nucleic acid escape from endosome compartments into the cytosol following cell entry, like polyethylenimine. Weak, inefficient neutralisation, condensation and encapsulation of nucleic acids and the presence of unprotonated amine functional groups appear to be desirable liposome characteristics for in vitro transfection. The inclusion of “natural” propylene and butylene spacings between the amine functional groups of cytofectin head groups appears to promote efficient neutralisation, condensation and encapsulation. The inclusion of some “unnatural” ethylene spacings appears to be a useful way of lowering amine pKa values.