Decoding the Kinetic Pathways toward a Lipid/DNA Complex of Alkyl Alcohol Cationic Lipids Formed in a Microfluidic...

Complexes of cationic liposomes with DNA have emerged as promising nonviral vectors for delivering genetic information into cells for gene therapy. Kinetics of the liposome/DNA complex (lipoplex) formation on a millisecond time scale are studied by monitoring time evolution of fluorescence of 8-anilino-1-naphthalene sulfonic acid (ANS) and ethidium bromide (EtBr) in a continuous flow microfluidic channel coupled to a fluorescence microscope. The formation of lipoplexes between calf thymus DNA and liposomes based on two novel cationic lipids (Lip1810 and Lip1814) are found to follow a two-step process with kinetic constants for the Lip1814/DNA complex (k₁ = 1120-1383 s^-1, k₂ = 0.227-1.45 s^-1) being significantly different from those (k₁ = 68.53-98.5 s^-1, k₂ = 32.3-60.19 s^-1) corresponding to formation of the Lip1810/DNA complex. The kinetic pathway leading to the formation of Lip1814/DNA complex is diffusion-controlled whereas the formation of Lip1810/DNA complex occurs by a conformational rearrangement-controlled pathway. The observed difference in the kinetics of lipoplex formation likely originates from different structures of the lipid/DNA complexes.