Intracellular Trafficking and Decondensation Kinetics of Chitosan–pDNA Polyplexes

Abstract

The transfection efficiency (TE) of chitosan-plasmid DNA (pDNA) polyplexes can be critically modulated by the polymer degree of deacetylation (DDA) and molecular weight (MW). This study was performed to test the hypothesis that the TE dependence on chitosan MW and DDA is related to the polyplex stability, hence their intracellular decondensation/unpacking kinetics. Major barriers to nonviral gene transfer were studied by image-based quantification. Although uptake increased with increased DDA, it did not appear to be a structure-dependent process affecting TE, nor was nuclear entry. Colocalization analysis showed that all chitosans trafficked through lysosomes with similar kinetics. Fluorescent resonant energy transfer (FRET) analysis revealed a distinct relationship between TE and polyplex dissociation rate. The most efficient chitosans showed an intermediate stability and a kinetics of dissociation, which occurred in synchrony with lysosomal escape. In contrast, a rapid dissociation before lysosomal escape was found for the inefficient low DDA chitosan whereas the highly stable and inefficient complex formed by a high MW and high DDA chitosan did not dissociate even after 24 hours. This study identified that the kinetics of decondensation in relation to lysosomal escape was a most critical structure-dependent process affecting the TE of chitosan polyplexes.

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.…”

“…25,26 We found that an intermediate stability of chitosan-DNA binding, attained through appropriate choice of DDA and MW, 22 produced the most efficient delivery vectors that are stable enough to condense and protect DNA extracellularly, but with low enough affinity to permit intracellular release of DNA cargo. 27 Thus, as the only natural polysaccharide with positive charge, chitosan has several unique properties as a carrier for gene therapy since chitosan: (1) is safe, non-toxic and biodegradable; (2) provides tunable electrostatic binding to negatively charged DNA; (3) is mucoadhesive to permit interaction between the delivered macromolecule and membrane epithelia; and (4) can open intercellular tight junctions to facilitate transport into cells. Compared with other cationic polymers such as polylysine and polyethleneimine, chitosan is less toxic and has better tolerability.…”

Real-time analysis of composite magnetic nanoparticle disassembly in vascular cells and biomimetic media

“…In the present studies, a FRET-based strategy is introduced for real-time in situ measurements of biodegradable NP disassembly, and is applied to examining the degradation patterns of composite PLA-based MNPs in liquid and semisolid biomimetic media and in cultured vascular cells. Because of their unique advantages, including nanometer-scale spatial resolution and subsecond time response (11), FRET-based techniques are particularly well-suited for studies of molecular disassembly processes, such as intracellular decondensation of gene vectors or drug release from colloidal systems (27)(28)(29). However, the use of FRET for investigating disassembly of biodegradable polymer-based nanocarriers poses several significant challenges, including (i) identifying chemically and spectrally compatible donor and acceptor fluorophores providing an adequate range of FRET response and exhibiting strong, environment-insensitive fluorescence; (ii) developing chemical strategies for covalently attaching the fluorophores to a particle-forming polymer without adversely affecting the colloidal stability and other properties of the carrier; (iii) identifying conditions providing rapid, effective, and synchronized cell uptake in studies focusing on intracellular disassembly of NPs; and (iv) enabling continuous, quantitative measurements in cells or biomimetic media on a protracted time scale ranging from several days to several weeks.…”

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

“…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].…”