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

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 applicationsPortuguese Foundation for Science and Technology (FCT) -project PEst-OE/BIA/UI4050/2014 (CBMA) and PEst-C/FIS/UI0607/2013 (CFUM