Enhanced Gene Delivery Mediated by Low Molecular Weight Chitosan/DNA Complexes: Effect of pH and Serum

Abstract

This study was designed to systematically evaluate the influence of pH and serum on the transfection process of chitosan–DNA complexes, with the objective of maximizing their efficiency. The hydrodynamic diameter of the complexes, measured by dynamic light scattering (DLS), was found to increase with salt and pH from 243 nm in water to 1244 nm in PBS at pH 7.4 and aggregation in presence of 10% serum. The cellular uptake of complexes into HEK 293 cells assessed by flow cytometry and confocal fluorescent imaging was found to increase at lower pH and serum. Based on these data, new methodology were tested and high levels of transfection (>40%) were achieved when transfection was initiated at pH 6.5 with 10% serum for 8–24 h to maximize uptake and then the media was changed to pH 7.4 with 10% serum for an additional 24–40 h period. Cytotoxicity of chitosan/DNA complexes was also considerably lower than Lipofectamine™. Our study demonstrates that the evaluation of the influence of important parameters in the methodology of transfection enables the understanding of crucial physicochemical and biological mechanisms which allows for the design of methodologies maximising transgene expression.

Effective and safe gene-based delivery of GLP-1 using chitosan/plasmid-DNA therapeutic nanocomplexes in an animal model of type 2 diabetes

“…We have previously investigated the coupling effect of chitosan DDA and MW for plasmid delivery and found that maximum transgene expression occurs for DDA:MW values that run along a diagonal from high DDA/low MW to low DDA/high MW. 26 Our results here for GLP-1 are in general agreement with our previous in vitro 26,27,52 and in vivo 25 studies where chitosan 92-10-5 and 80-10-10 showed highest level of transfection efficiency and transgene expression, whereas the chitosan 80-80-5 showed the poorest expression level and was associated with the generation of neutralizing antibodies in vivo to fibroblast growth factor-2 and platelet-derived growth factor-BB. 25 We have previously determined that the latter formulation is suitable for DNA vaccination where it induces the highest antibody production against human fibroblast growth factor-2 and platelet-derived growth factor-BB in Balb/c mice.…”

Low Molecular Weight Chitosan (LMWC)-based Polyplexes for pDNA Delivery: From Bench to Bedside

“…According to some authors, the presence of serum enhances the transfection efficiency of Ch based polyplexes [41,67,68]. The reason for the increment of transfection in the presence of serum is not clear.…”

“…This parameter determines the colloidal stability of the polyplexes aquous suspension and it plays a crucial role in the viability, cellular uptake and transfection efficiency of LMWC-pDNA polyplexes, as it is widely reported in the literature [30,41].…”

“…As reported in the bibliography, an increment of the surface charge is observed when the pH of the medium is decreased from 7.4 to 6.5. Recently, our research group has described an increase of around 6 mV in the zeta potential of ultrapure LMWC [30,41]. Considering the zeta potential has an important influence on the cellular uptake of the vectors, as it is explained below, much attention must be paid to the elaboration of the polyplexes in order to get the appropriate surface charge.…”

“…Unless the polyplexes are modified with a specific targeting ligand, the binding of polyplexes to the cell membrane occurs by unspecific electrostatic interactions between the positive charge of the polyplexes and the negative charge of the cell surface [53]. The microenvironment around the target cells has to be considered, since it has been described that acidic environments favor the cellular binding and uptake of LMWC-pDNA polyplexes due to an increase in the zeta potential value of the polyplexes at low pH values as a consequence of the protonated amines [30,41,54].…”