Influence of Chitosan Structure on the Formation and Stability of DNA−Chitosan Polyelectrolyte Complexes

Abstract

The interactions between DNA and chitosans varying in fractional content of acetylated units (FA), degree of polymerization (DP), and degree of ionization were investigated by several techniques, including an ethidium bromide (EtBr) fluorescence assay, gel retardation, atomic force microscopy, and dynamic and electrophoretic light scattering. The charge density of the chitosan and the number of charges per chain were found to be the dominating factors for the structure and stability of DNA-chitosan complexes. All high molecular weight chitosans condensed DNA into physically stable polyplexes; however, the properties of the complexes were strongly dependent on FA, and thereby the charge density of chitosan. By employing fully charged oligomers of constant charge density, it was shown that the complexation of DNA and stability of the polyplexes is governed by the number of cationic residues per chain. A minimum of 6-9 positive charges appeared necessary to provide interaction strength comparable to that of polycations. In contrast, further increase in the number of charges above 9 did not increase the apparent binding affinity as judged from the EtBr displacement assay. The chitosan oligomers exhibited a pH-dependent interaction with DNA, reflecting the number of ionized amino groups. The complexation of DNA and the stability of oligomer-based polyplexes became reduced above pH 7.4. Such pH-dependent dissociation of polyplexes around the physiological pH is highly relevant in gene delivery applications and might be one of the reasons for the high transfection activity of oligomer-based polyplexes observed.

Effect of ionic strength solution on the stability of chitosan–DNA nanoparticles

“…As expected, in general, when pH and ionic strength were kept low (pH 4.0, ionic strength 10 mM) nanoparticle sizes remained smaller than 250 nm as seen from Table 2, independent of the chitosan molecular weight and N/P ratio. However, the lower molecular weight sample (5 kDa) formed the smaller nanoparticles with diameters ranging from 120 to 140 nm and the higher molecular chitosan (CH 150 kDa) exhibited nanoparticle sizes ranging from 200 to 240 nm, which is in agreement with results previously published on calf thymus DNA using the same range of chitosan molecular weight [18].…”

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

“…OCS had greater complexation efficiency for pDNA than PCS (>90% compared to an average of 80% for PCS-pDNA), however, pDNA appears to be released from OCS faster than from PCS as evidenced by gel electrophoresis studies where there is evidence of faint bands seen at all N/P ratios, but more so at lower N/P ratios, suggesting that the pDNA is beginning to be released from the OCS. This result is in agreement with other reports which state that low MW chitosan forms less stable nanoparticles with pDNA than with high MW [23,25,49,50]. While stability is important extracellularly, release of the pDNA is critical in ensuring transfection so OCS-pDNA nanoparticles formulated at all the N/P ratios investigated were deemed suitable for transfection experiments.…”

Chitosan as a non-viral co-transfection system in a cystic fibrosis cell line

“…Driven by electrostatic interactions, chitosanpDNA complexes have been used for transfection of mammalian cells both in vitro and in vivo (Koping-Hoggard et al, 2001;Romøren et al, 2003;Vauthier et al, 2014). Nevertheless, results of transfection efficiency using chitosan-based systems are strongly dependent on chitosan properties (e.g., molecular weight and the relative amount of N-acetylglucosamine units, namely degree of acetylation (DA)) (Lavertu et al, 2006;Santos-Carballal et al, 2015;Strand et al, 2005). Chitosan has been reported as a suitable candidate for transmucosal administration of drugs (Grenha et al, 2010).…”