Interaction of pDNA with reverse phase chitosome

https://doi.org/10.1016/j.colsurfa.2018.02.005Get rights and content

Abstract

The use of liposomes and polyelectrolytes is on scientific and technologic scrutiny for development of gene nano carriers. We produced a reverse phase lipid:chitosan “chitosome” to which plasmid DNA (pDNA) effectively interacts and compacts. Dynamic light scattering and zeta potential evidence the complexation between pDNA and lipid:chitosan particles with size increase and surface charge decrease with concentration of plasmid. Zeta potential shows a variation of −4 mV/μg plasmid. Cell culture media have weak or even absent effect on size and surface charge of the composite complexes denoting physical stability in application media. SAXS results show increased structural organization of the lipopolyplex, with a membrane repeat distance of 6.50 nm, and gel electrophoresis confirms effective interaction between chitosomes and pDNA. The thermodynamic energy involved in complexation provides 0.29 chitosan protonated monomers interacting to phosphate group of pDNA at saturation as determined by isothermal titration calorimetry, which is in accordance with the linear charge density between chitosan and DNA. The strong exothermic binding determines Gibbs energy around −13.45 kcal/mol of protonated chitosan denoting that the complex formation leads to a state of lower Gibbs energy, thus energetically favored with decrease in entropy, corroborating compaction of plasmid chains over surface of chitosomes. A model of the lipopolyplex is unveiled thanks to the complete physicochemical description. The results demonstrate perspectives for development of a promising gene deliver.

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).

References (30)

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