Tunable pDNA/DODAB:MO lipoplexes: the effect of incubation temperature on pDNA/DODAB:MO lipoplexes structure and transfection efficiency

Abstract

tDioctadecyldimethylammonium bromide (DODAB):1-monooleoyl-rac-glycerol (MO) cationic liposomeswere reported as a promising alternative to common transfection agents, showing superior effective-ness on the transfection of the 293T mammalian cell line with pSV- -gal plasmid DNA. The study ofDODAB:MO aggregates in the absence of DNA has indicated that their morphology depends on the balancebetween DODAB’s tendency to form bilayer structures and MO’s propensity to form inverted non-lamellarstructures. Other parameters, such as the temperature have proved to be crucial in the definition of themorphology of the developed nanocarrier. Therefore, in this work, a step forward to the current genecarrier system will be given by studying the effect of the tunable parameters (incubation temperatureand MO content) on the structure of pDNA:DODAB:MO lipoplexes. More importantly, the implicationsthat these tunable parameters could have in terms of lipoplex transfection efficiency will be investigated.Dynamic light scattering (DLS), zeta ( ) potential, cryo-transmission electron microscopy (cryo-TEM) andethidium bromide (EtBr) exclusion were used to assess the formation, structure and destabilization ofpDNA:DODAB:MO lipoplexes at DODAB molar fractions of (1:1) and above equimolarity (2:1, 4:1) pre-pared at incubation temperatures from 25 to 50◦C. Experimental results indicate that pDNA:DODAB:MO’sstructure is sensitive to the lipoplex incubation temperature, resulting in particles of distinct size, super-ficial charge and structure. These variations are also visible on the complexation dynamics of pDNA, andsubsequent release upon incubation with the model proteoglycan heparin (HEP), at 25 and 50◦C. Increasein temperature leads to re-organization of DODAB and MO molecules within the liposomal formulation,causing a positive charge re-localization in the lipoplex surface, which not only alters its structure but alsoits transfection efficiency. Altogether, these results confirm that in the DODAB:MO carriers, an increasein the incubation temperature has a similar effect on aggregate morphology as the observed with anincrease in MO content. This conclusion is extended to the pDNA:DODAB:MO lipoplexes morphologyand subsequent transfection efficiency defining new strategies in lipoplexes preparation that could beused to modulate the properties of other lipid formulations for nonviral gene delivery applications