Mechanism of cell transfection with plasmid/chitosan complexes

Abstract

Chitosan is useful as a non-viral vector for gene delivery. Although there are several reports supporting the use of chitosan for gene delivery, studies regarding effects on transfection and the chitosan-specific transfection mechanism remain insufficient. In this report, the level of expression with plasmid/chitosan was observed to be no less than that with plasmid/lipofectin complexes in SOJ cells. The transfection mechanism of plasmid/chitosan complexes as well as the relationship between transfection activity and cell uptake was analyzed by using fluorescein isothiocyanate-labeled plasmid and Texas Red-labeled chitosan. In regard to effects on transfection, there were several factors to affect transfection activity and cell uptake, for example: the molecular mass of chitosan, stoichiometry of complex, as well as serum concentration and pH of transfection medium. The level of transfection with plasmid/chitosan complexes was found to be highest when the molecular mass of chitosan was 40 or 84 kDa, ratio of chitosan nitrogen to DNA phosphate (N/P ratio) was 5, and transfection medium contained 10% serum at pH 7.0. We also investigated the transfection mechanism, and found that plasmid/chitosan complexes most likely condense to form large aggregates (5-8 microm), which absorb to the cell surface. After this, plasmid/chitosan complexes are endocytosed, and possibly released from endosomes due to swelling of lysosomal in addition to swelling of plasmid/chitosan complex, causing the endosome to rupture. Finally, complexes were also observed to accumulate in the nucleus using a confocal laser scanning microscope.

Development of a gene-activated scaffold platform for tissue engineering applications using chitosan-pDNA nanoparticles on collagen-based scaffolds

“…Chitosan is a versatile biomaterial derived from the exoskeleton of many crustaceans which can be used in the formulation of microparticles and nanoparticles suitable for drug delivery [15][16][17][18][19]. The amine groups on the chitosan chain are positively charged and can bind to negatively charged DNA and condense it into positively charged nanoparticles.…”

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.…”

Evaluation of cystamine-modified hyaluronic acid/chitosan polyplex as retinal gene vector

“…Other studies have shown an association between surface charge and increased transfection efficiency as well as a decrease in toxicity [24,25]. Also, cellular uptake is one of the major obstacles for a successful gene delivery strategy and polyplexes are thought to enter the cell via endocytosis in a process that may be dependent on polyplex charge [26]. A higher surface charge can facilitate electrostatic interactions with the negatively charged proteoglycans in cellular membranes and hence promote polyplex internalization [27].…”