Colloids and Surfaces A: Physicochemical and Engineering Aspects
Interaction of pDNA with reverse phase chitosome
Graphical Abstract
Introduction
Gene transference technology is widely used for overexpression, knock-down or knock-out of a specific gene to gene function study, gene therapy or recombinant protein production [[1], [2], [3], [4], [5]]. Plasmid DNA (pDNA) alone does not possess the accurate physicochemical properties to enter cells. Consequently cationic liposomes (lipoplexes) or cationic polymers (polyplexes) are frequently used as pDNA compacting agent and used for transfection [[6], [7], [8], [9]]. These complexes have shown to be less immunogenic and oncogenic than viral vectors and are easier to formulation, modification, production and purification than viral vectors because these are chemically well-defined and much more stable in working and storing conditions [1,10,11].
Lipopolyplex formed with chitosan and cationic liposomes has been studied by several authors because both components have already been extensively explored as gene carriers [[10], [11], [12], [13], [14]]. Chitosan, which is a biodegradable, biocompatible and low immunogenic polysaccharide, acquires positive charges below physiological pH, which interact with the pDNA negative charges, leading to polyplex formation. However, the role of chitosan valence on lipopolyplex production and performance remains ambiguous due to ionization of chitosan amine groups which is strongly pH dependent and which modulates electrostatic interactions and hydrolysis [[15], [16], [17]]. Transfection is a complex process, which involves internalization of vectors by endocytosis, the escape from endosome into the cytosol and, finally, the particles have to pass through the nucleus membrane to start expression; therefore, detailed physicochemical studies can be useful in the comprehension of transfection and for elaboration of more efficient gene carriers. For instance, Liu et al. [13] showed that the lipopolyplex made with pDNA-chitosan-cationic liposome increased transfection efficiency; however, it was not conclusive about the existence of a correlation between the positive charge density provided by chitosan and the transfection efficiency. Wang et al. [14] showed that alteration of synthesis order to make lipopolyplex with pDNA-chitosan-cationic liposome can form structurally different lipoplexes. Two lipopolyplexes produced by these authors were more efficient in transfection than polyplex and lipoplex tested separately. The ternary complex internalization monitored by confocal microscopy showed that liposomes are detached from the complexes and remained in the cytoplasm, and pDNA moved toward the nucleus. Therefore, development of lipopolyplexes as gene carriers is an emerging area, so, new ternary complexes deserve to be analyzed deeply about physicochemical properties before testing in biological systems.
In the present study, we used zwitterionic lipid to make ternary complexes pDNA-chitosan-zwitterionic lipid and detailed physicochemical studies of these lipopolyplexes were reported here.
Section snippets
Materials
Phospholipid was 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC, 99%; Avanti Polar Lipids, Alabaster, USA). Chitosan was purchased from Primex (Iceland), presenting 95% degree of deacetylation (DDA), average molecular weight of 130 kDa and low viscosity (<20 cP). The polysaccharide was dissolved in aqueous solution of sodium acetate/acetic acid (40 mM, pH 4.95) under vigorous overnight stirring at 1 mg/mL as stock solution. All solutions were prepared with purified water from MilliQ
Size and surface charge of plasmid composite complexes
Fig. 1a shows sizes as hydrodynamic diameters of lipid particle along with composite lipid:chitosan particles (chitosomes) containing different concentrations of plasmid. The sole lipid particle presents average diameter of 127 nm. With inclusion of chitosan in a constant w/w proportion of 10:0.3 (lipid:chitosan), a very slight increase in average diameter to 135 nm was observed. Of notice, for this specific chitosan concentration, size increase was not significant, since polymer concentration
Conclusion
In the present contribution, we developed a new plasmid carrier with specific structure where the association of chitosan to single zwitterionic lipid liposomes is prone by reverse phase evaporation method, leading to efficient coverage of the lipids bilayers and producing a chitosome to which pDNA effectively interacts and compacts over the surface. We employed a range of plasmid and chitosan concentrations whose interactions were effectively established, leading to complexation of pDNA and
Acknowledgments
The authors thank the Brazilian Synchrotron Light Laboratory (LNLS) for allowing SAXS experiments. O.M. and S.W.H. thank São Paulo Research Foundation, FAPESP, for research grants (2015/23948-5; 2016/13368-4; and 2015/20206-8).
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