Abstract
The consequences for the transfection efficiencies of different lipoplexes preparation methods, largely remain to be explored, but the knowledge of how different experimental approaches can affect the physicochemical properties and transfection efficiency is essential for a proper tailoring of transfection complexes to particular applications. Therefore, the influence of the number of mixing steps (one-step addition versus multi-step addition of liposomes to plasmid DNA (pDNA)) and lipoplex incubation temperature on the final physicochemical properties and transfection efficiency of pDNA/ Dioctadecyldimethylammonium Bromide (DODAB):1-monooleoyl-rac-glycerol (MO) complexes was studied in three distinct DODAB:MO molar ratios: 4:1, 2:1 and 1:1. Dynamic Light Scattering (DLS), Zeta (ζ) Potential, Ethidium Bromide (EtBr) exclusion assays were used to assess the formation, structure and destabilization of the lipoplexes, whereas in vitro transfection assays with pSV-β-gal plasmid DNA were performed to evaluate their transfection efficiency on the 293T mammalian cell line. Results indicate that the morphology of pDNA/DODAB:MO complexes is dependent on the lipoplex preparation method, resulting in particles of distinct size, surface charge and membrane fluidity. These variations are visible during the complexation dynamics of pDNA and continue throughout the profile of pDNA release from pDNA/DODAB:MO lipoplexes upon incubation with Heparin (HEP), as well as in the in vitro transfection assays. The stepwise addition of DODAB:MO vesicles to pDNA decreases the transfection efficiency of the lipoplexes, while the effect of the lipoplex preparation methods is dependent on the MO content.
Highlights
Cationic lipid complexes have several advantages as nucleic acid delivery systems over viral vectors, which are difficult to prepare, can be mutagenic and have low loading capacity [1,2,3,4,5,6,7]
The stepwise addition of Dioctadecyldimethylammonium Bromide (DODAB):MO vesicles to pDNA decreases the transfection efficiency of the lipoplexes, while the effect of the lipoplex preparation methods is dependent on the MO content
It was found that the addition of cationic liposomes to pDNA leads to a dual-lipoplex phase diagram, with prevalence of lamellar structures at DODAB molar fractions above 0.5 and inverted bicontinuous cubic mesophases at DODAB molar fractions below 0.5 [40]
Summary
Cationic lipid complexes (lipoplexes) have several advantages as nucleic acid delivery systems over viral vectors, which are difficult to prepare, can be mutagenic and have low loading capacity [1,2,3,4,5,6,7]. The final transfection efficiency of the lipoplexes has been reported in the literature as dependent of the preparation strategies used in numerous aspects that include: (i) the type and proportions of main lipid and helper lipids chosen (e.g. changing lipoplex composition affects physicochemical properties, such as, size, surface charge and fluidity [8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25]); (ii) the type of aggregates formed (e.g. in comparison to multilamellar vesicles, lowest molecular weighted small unilamellar vesicles require more aggregates to complex the same amount of DNA) [26]; (iii) the chosen addition procedure Given the reported impact that the optimization of experimental procedures (such as the vesicle preparation method or the stepwise addition of cationic liposomes to DNA) may have on the lipoplex. The physicochemical properties of the lipoplexes (size, charge and structure) prepared by the different methods were studied by Dynamic and Electrophoretic Light Scattering (DLS and ELS) and Cryo-Transmission Electron Microscopy (Cryo-TEM). In vitro transfection assays were performed in 293T human cell line using the pSV--gal plasmid, in order to investigate the correlation between these different lipoplex preparation methods and the final lipofection efficiency
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