Ionization behavior of chitosan and chitosan-DNA polyplexes indicate that chitosan has a similar capability to induce a proton-sponge effect as PEI.

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.

[1]  M. Buschmann,et al.  One-step analysis of DNA/chitosan complexes by field-flow fractionation reveals particle size and free chitosan content. , 2010, Biomacromolecules.

[2]  S. W. Kim,et al.  Lactose-poly(ethylene glycol)-grafted poly-L-lysine as hepatoma cell-tapgeted gene carrier. , 1998, Bioconjugate chemistry.

[3]  M. Buschmann,et al.  High efficiency gene transfer using chitosan/DNA nanoparticles with specific combinations of molecular weight and degree of deacetylation. , 2006, Biomaterials.

[4]  A. Katchalsky,et al.  Dissociation of weak polymeric acids and bases , 1954 .

[5]  M. Buschmann,et al.  Intracellular trafficking and decondensation kinetics of chitosan-pDNA polyplexes. , 2010, Molecular therapy : the journal of the American Society of Gene Therapy.

[6]  M. Buschmann,et al.  New insights into chitosan-DNA interactions using isothermal titration microcalorimetry. , 2009, Biomacromolecules.

[7]  Xian‐Zheng Zhang,et al.  Chitosan-graft-polyethylenimine with improved properties as a potential gene vector , 2010 .

[8]  D. Putnam,et al.  Competitive reactions in solutions of poly-L-histidine, calf thymus DNA, and synthetic polyanions: determining the binding constants of polyelectrolytes. , 2003, Journal of the American Chemical Society.

[9]  M. Buschmann,et al.  Complete physicochemical characterization of DNA/chitosan complexes by multiple detection using asymmetrical flow field-flow fractionation. , 2010, Analytical chemistry.

[10]  S. Armes,et al.  Effect of polymer ionization on the interaction with DNA in nonviral gene delivery systems. , 2003, Biomacromolecules.

[11]  K. Leong,et al.  Interactions of phospholipid bilayer with chitosan: effect of molecular weight and pH. , 2001, Biomacromolecules.

[12]  M. Borkovec,et al.  Ising models and acid‐base properties of weak polyelectrolytes , 1996 .

[13]  K. Vårum,et al.  siRNA delivery with chitosan nanoparticles: Molecular properties favoring efficient gene silencing. , 2012, Journal of controlled release : official journal of the Controlled Release Society.

[14]  E. Urbansky,et al.  Understanding, Deriving, and Computing Buffer Capacity , 2000 .

[15]  C. Culmsee,et al.  Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer , 2004, The journal of gene medicine.

[16]  M. Buschmann,et al.  Excess polycation mediates efficient chitosan-based gene transfer by promoting lysosomal release of the polyplexes. , 2011, Biomaterials.

[17]  T. Andresen,et al.  Polycation cytotoxicity: a delicate matter for nucleic acid therapy-focus on polyethylenimine , 2010 .

[18]  Y. Li,et al.  Characterization of commercially available and synthesized polyethylenimines for gene delivery. , 2000, Journal of controlled release : official journal of the Controlled Release Society.

[19]  K. Gupta,et al.  Linear polyethylenimine-graft-chitosan copolymers as efficient DNA/siRNA delivery vectors in vitro and in vivo. , 2012, Nanomedicine : nanotechnology, biology, and medicine.

[20]  F. Szoka,et al.  Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. , 1993, Bioconjugate chemistry.

[21]  Marie C. M. Lin,et al.  Revisit complexation between DNA and polyethylenimine - Effect of uncomplexed chains free in the solution mixture on gene transfection. , 2011, Journal of controlled release : official journal of the Controlled Release Society.

[22]  J. Isaacs,et al.  Activated polyamidoamine dendrimers, a non-viral vector for gene transfer to the corneal endothelium , 1999, Gene Therapy.

[23]  K. Vårum,et al.  Influence of chitosan structure on the formation and stability of DNA-chitosan polyelectrolyte complexes. , 2005, Biomacromolecules.

[24]  Y Wang,et al.  Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency. , 2001, Journal of controlled release : official journal of the Controlled Release Society.

[25]  Yuichi Yamasaki,et al.  In situ single cell observation by fluorescence resonance energy transfer reveals fast intra‐cytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine , 2004, The journal of gene medicine.

[26]  O. Smidsrod,et al.  Degradation of partially N-acetylated chitosans with hen egg white and human lysozyme , 1996 .

[27]  M. Borkovec,et al.  Proton Binding Characteristics of Branched Polyelectrolytes , 1997 .

[28]  D. V. Slyke ON THE MEASUREMENT OF BUFFER VALUES AND ON THE RELATIONSHIP OF BUFFER VALUE TO THE DISSOCIATION CONSTANT OF THE BUFFER AND THE CONCENTRATION AND REACTION OF THE BUFFER SOLUTION , 1922 .

[29]  M. Buschmann,et al.  Effective and safe gene-based delivery of GLP-1 using chitosan/plasmid-DNA therapeutic nanocomplexes in an animal model of type 2 diabetes , 2011, Gene Therapy.

[30]  P. Artursson,et al.  Chitosan as a nonviral gene delivery system. Structure–property relationships and characteristics compared with polyethylenimine in vitro and after lung administration in vivo , 2001, Gene Therapy.

[31]  F. Szoka,et al.  Chloride Accumulation and Swelling in Endosomes Enhances DNA Transfer by Polyamine-DNA Polyplexes* , 2003, Journal of Biological Chemistry.

[32]  M. Mandel,et al.  The influence of nearest- and next-nearest-neighbor interactions on the potentiometric titration of linear poly(ethylenimine) , 1993 .

[33]  Xian‐Zheng Zhang,et al.  Chitosan based oligoamine polymers: synthesis, characterization, and gene delivery. , 2009, Journal of controlled release : official journal of the Controlled Release Society.

[34]  Kristen N. Duthie,et al.  Wide varieties of cationic nanoparticles induce defects in supported lipid bilayers. , 2008, Nano letters.

[35]  S Moein Moghimi,et al.  The possible "proton sponge " effect of polyethylenimine (PEI) does not include change in lysosomal pH. , 2013, Molecular therapy : the journal of the American Society of Gene Therapy.

[36]  T. Bieber,et al.  Intracellular route and transcriptional competence of polyethylenimine-DNA complexes. , 2002, Journal of controlled release : official journal of the Controlled Release Society.

[37]  M. Buschmann,et al.  Ionization and solubility of chitosan solutions related to thermosensitive chitosan/glycerol-phosphate systems. , 2007, Biomacromolecules.

[38]  S. Ohkuma,et al.  Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages , 1981, The Journal of cell biology.

[39]  D. Fischer,et al.  A Novel Non-Viral Vector for DNA Delivery Based on Low Molecular Weight, Branched Polyethylenimine: Effect of Molecular Weight on Transfection Efficiency and Cytotoxicity , 1999, Pharmaceutical Research.

[40]  R. Langer,et al.  Exploring polyethylenimine‐mediated DNA transfection and the proton sponge hypothesis , 2005, The journal of gene medicine.

[41]  S. W. Kim,et al.  pH-sensitive cationic polymer gene delivery vehicle: N-Ac-poly(L-histidine)-graft-poly(L-lysine) comb shaped polymer. , 2000, Bioconjugate chemistry.

[42]  C. R. Middaugh,et al.  Biophysical characterization of PEI/DNA complexes. , 2003, Journal of pharmaceutical sciences.

[43]  D. Scherman,et al.  A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[44]  T. Delair,et al.  Polyelectrolyte complexes from polysaccharides: formation and stoichiometry monitoring. , 2007, Langmuir : the ACS journal of surfaces and colloids.

[45]  M. Buschmann,et al.  Enhanced Gene Delivery Mediated by Low Molecular Weight Chitosan/DNA Complexes: Effect of pH and Serum , 2010, Molecular biotechnology.

[46]  Kenneth A Howard,et al.  RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system. , 2006, Molecular therapy : the journal of the American Society of Gene Therapy.

[47]  Y. Osada,et al.  Formation of polyion complexes between polycarboxylic acids and polycations carrying charges in the chain backbone , 1974 .

[48]  O. Danos,et al.  Polyethylenimine‐mediated gene delivery: a mechanistic study , 2001, The journal of gene medicine.