Ionization Behavior of Chitosan and Chitosan–DNA Polyplexes Indicate That Chitosan Has a Similar Capability to Induce a Proton-Sponge Effect as PEI

Abstract

Polycations having a high buffering capacity in the endosomal pH range, such as polyethylenimine (PEI), are known to be efficient at delivering nucleic acids by overcoming lysosomal sequestration possibly through the proton sponge effect, although other mechanisms such as membrane disruption arising from an interaction between the polycation and the endosome/lysosome membrane, have been proposed. Chitosan is an efficient delivery vehicle for nucleic acids, yet its buffering capacity has been thought to be significantly lower than that of PEI, suggesting that the molecular mechanism responsible for endolysosomal escape was not proton sponge based. However, previous comparisons of PEI and chitosan buffering capacity were performed on a mass concentration basis instead of a charge concentration basis, the latter being the most relevant comparison basis because polycation-DNA complexes form at ratios of charge groups (amine to phosphate), rather than according to mass. We hypothesized that chitosan has a high buffering capacity when compared to PEI on a molar basis and could therefore possibly mediate endolysosomal release through the proton sponge effect. In this study, we examined the ionization behavior of chitosan and chitosan-DNA complexes and compared to that of PEI and polylysine on a charge concentration basis. A mean field theory based on the use of the Poisson-Boltzmann equation and an Ising model were also applied to model ionization behavior of chitosan and PEI, respectively. We found that chitosan has a higher buffering capacity than PEI in the endolysosomal pH range, while the formation of chitosan-DNA complexes reduces chitosan buffering capacity because of the negative electrostatic environment of nucleic acids that facilitates chitosan ionization. These data suggest that chitosans have a similar capacity as PEI to mediate endosomal escape through the proton sponge effect, possibly in a manner which depends on the presence of excess chitosan.

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

“…This hypothesis proposes that endosomes are acidified and chitosan promotes the active transport of protons due to the presence of NH2 groups at D-glucosamine residues. The accumulation of protons in the vesicle must be balanced by an influx of counter ions (Cl -), which produces osmotic swelling, burst of the late endosome and release of the cargo (Pack et al, 2005;Richard et al, 2013). Chitosan may undergo biodegradation by enzymes present in the endosomal/lysosomal vesicles.…”

Polymeric micro/nanoparticles: Particle design and potential vaccine delivery applications

“…Considering the advantage of a facile preparation condition, particles that bear this merit but need no modification were intriguing candidates. Under the "proton-sponge" effect, the positive charge of NPs (as referred in P-NP) may trigger the influx of chloride ions to maintain charge neutrality, leading to osmotic swelling, physical rupture of lysosomes, and the final antigen escape to cytoplasm [63,64].…”

Polysaccharide-Based Nanocarriers for Drug Delivery

“…189,194 A recent study suggests that CS's buffering capacity may be closer to that of polyethlyeneimine (a well established proton sponge) than originally thought. 195 As with protein delivery, a wide variety of CS based vectors have been studied. The majority are based on direct complexation between CS and the nucleic acid, 187 although other sophisticated systems incorporating lipids or polymers, e.g.…”

“…The success of CS is based in the reported ability of CS to enhance its penetration through cellular and epithelial barriers, its cationic nature is capable of compacting gene medicines, and its buffering properties are important for endosomal escape. 189,195 A potential weakness of CS for gene therapy is its general cationic character that would drive opsonization upon parenteral administration. Due to this limitation, some of the most successful applications of CS in gene delivery are related to mucosal administration.…”