title: Tunable pDNA/DODAB:MO lipoplexes: the effect of incubation temperature on pDNA/DODAB:MO lipoplexes structure and transfection efficiency.
abstract: Dioctadecyldimethylammonium bromide (DODAB):1-monooleoyl-rac-glycerol (MO) cationic liposomes were reported as a promising alternative to common transfection agents, showing superior effectiveness on the transfection of the 293T mammalian cell line with pSV-β-gal plasmid DNA. The study of DODAB:MO aggregates in the absence of DNA has indicated that their morphology depends on the balance between DODAB's tendency to form bilayer structures and MO's propensity to form inverted non-lamellar structures. Other parameters, such as the temperature have proved to be crucial in the definition of the morphology of the developed nanocarrier. Therefore, in this work, a step forward to the current gene carrier system will be given by studying the effect of the tunable parameters (incubation temperature and MO content) on the structure of pDNA:DODAB:MO lipoplexes. More importantly, the implications that these tunable parameters could have in terms of lipoplex transfection efficiency will be investigated. Dynamic light scattering (DLS), zeta (ζ) potential, cryo-transmission electron microscopy (cryo-TEM) and ethidium bromide (EtBr) exclusion were used to assess the formation, structure and destabilization of pDNA:DODAB:MO lipoplexes at DODAB molar fractions of (1:1) and above equimolarity (2:1, 4:1) prepared at incubation temperatures from 25 to 50°C. Experimental results indicate that pDNA:DODAB:MO's structure is sensitive to the lipoplex incubation temperature, resulting in particles of distinct size, superficial charge and structure. These variations are also visible on the complexation dynamics of pDNA, and subsequent release upon incubation with the model proteoglycan heparin (HEP), at 25 and 50°C. Increase in 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 also its transfection efficiency. Altogether, these results confirm that in the DODAB:MO carriers, an increase in the incubation temperature has a similar effect on aggregate morphology as the observed with an increase in MO content. This conclusion is extended to the pDNA:DODAB:MO lipoplexes morphology and subsequent transfection efficiency defining new strategies in lipoplexes preparation that could be used to modulate the properties of other lipid formulations for nonviral gene delivery applications.
authors.affiliation: CFUM (Centre of Physics of the University of Minho), Department of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal; CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal.
authors.forename: João P Neves
authors.initials: JP
authors.lastname: Silva
authors.affiliation: CFUM (Centre of Physics of the University of Minho), Department of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal; CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal.
authors.forename: Ana C N
authors.initials: AC
authors.lastname: Oliveira
authors.affiliation: CFUM (Centre of Physics of the University of Minho), Department of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal.
authors.forename: Marlene
authors.initials: M
authors.lastname: Lúcio
authors.affiliation: CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal.
authors.forename: Andreia C
authors.initials: AC
authors.lastname: Gomes
authors.affiliation: CFUM (Centre of Physics of the University of Minho), Department of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal.
authors.forename: Paulo J G
authors.initials: PJ
authors.lastname: Coutinho
authors.affiliation: CFUM (Centre of Physics of the University of Minho), Department of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal. Electronic address: beta@fisica.uminho.pt.
authors.forename: M Elisabete C D Real
authors.initials: ME
authors.lastname: Oliveira
mesh_terms: D000818:Animals; D020285:Cryoelectron Microscopy; D004247:DNA / Q000737:chemistry*; D017930:Genes, Reporter; D005989:Glycerides / Q000737:chemistry*; D057809:HEK293 Cells; D006493:Heparin / Q000737:chemistry; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry / Q000648:ultrastructure; D010316:Particle Size; D010957:Plasmids / Q000737:chemistry*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D055672:Static Electricity; D013499:Surface Properties; D034421:Sus scrofa; D013696:Temperature; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D005989:Glycerides; D008055:Lipids; D008081:Liposomes; D000644:Quaternary Ammonium Compounds; C015831:dimethyldioctadecylammonium; D006493:Heparin; D004247:DNA; C471272:1-monooleoyl-rac-glycerol; D001616:beta-Galactosidase
keywords: EtBr exclusion; Monoolein-based lipoplexes; Transfection