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Electrostatic interactions between synthetic polyelectrolytes and proteins can lead to the formation of dense, macroion-rich liquid phases, with equilibrium microheterogeneities on length scales up to hundreds of nanometers. The effects of pH and ionic strength on the rheological and optical properties of these coacervates indicate microstructures sensitive to protein-polyelectrolyte interactions. We report here on the properties of coacervates obtained for bovine serum albumin (BSA) with the biopolyelectrolyte chitosan and find remarkable differences relative to coacervates obtained for BSA with poly(diallyldimethylammonium chloride) (PDADMAC). Coacervation with chitosan occurs more readily than with PDADMAC. Viscosities of coacervates obtained with chitosan are more than an order of magnitude larger and, unlike those with PDADMAC, show temperature and shear rate dependence. For the coacervates with chitosan, a fast relaxation time in dynamic light scattering, attributable to relatively unrestricted protein diffusion in both systems, is diminished in intensity by a factor of 3-4, and the consequent dominance by slow modes is accompanied by a more heterogeneous array of slow apparent diffusivities. In place of a small-angle neutron scattering Guinier region in the vicinity of 0.004 Å -1 , a 10-fold increase in scattering intensity is observed at lower q. Taken together, these results confirm the presence of dense domains on length scales of hundreds of nanometers to micrometers, which in coacervates prepared with chitosan are less solidlike, more interconnected, and occupy a larger volume fraction. The differences in properties are thus correlated with differences in mesophase structure.

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.

Abstract:Chitosan is a unique biopolymer in the respect that it is abundant, cationic, low-toxic, non-immunogenic and biodegradable. The relative occurrence of the two monomeric building units (N-acetyl-glucosamine and D-glucosamine) is crucial to whether chitosan is predominantly an ampholyte or predominantly a polyelectrolyte at acidic pH-values. The chemical composition is not only crucial to its surface activity properties, but also to whether and why chitosan can undergo a sol-gel transition. This review gives an overview of chitosan hydrogels and their biomedical applications, e.g., in tissue engineering and drug delivery, as well as the chitosan's surface activity and its role in emulsion formation, stabilization and destabilization. Previously unpublished original data where chitosan acts as an emulsifier and flocculant are presented and discussed, showing that highly-acetylated chitosans can act both as an emulsifier and as a flocculant.

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Chitosan is a promising biomaterial with an attractive safety profile; however, its application potential for gene delivery is hampered by poor compatibility at physiological pH values. Here we have tailored the molecular architecture of chitosan to improve the functional properties and gene transfer efficacy of chitosan oligomers and have developed self-branched glycosylated chitosan oligomer (SB-TCO) substituted with a trisaccharide containing N-acetylglucosamine, AAM. SB-TCO was prepared by controlled depolymerization of chitosan, followed by simultaneous branching and AAM substitution. The product was fully soluble at physiological pH and complexed plasmid DNA into polyplexes of high colloidal and physical stability. SB-TCO displayed high transfection efficacy in HEK293 cells, reaching transfection efficiencies of up to 70%, and large amounts of transgene were produced. Gene transfer efficacy was confirmed in HepG2 cells, where gene expression levels mediated by SB-TCO were up to 10 and 4 times higher than those obtained with unsubstituted and substituted linear oligomers, respectively. The rapid onset of transgene expression in both cell lines indicates efficient DNA release and transcription from SB-TCO polyplexes. In comparison with 22 kDa linear PEI-based transfection reagent used as the control, SB-TCO possessed higher gene transfer efficacy, significantly lower cytotoxicity, and improved serum compatibility.

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Electrostatic properties of three chitosans with fractions of N-acetylated units (F(A)) of 0.01, 0.13 and 0.49 were examined by electrophoretic light-scattering technique (ELS) and (1)H NMR spectroscopy. From the dependency of mean electrophoretic mobilities on pH, the pK(a) values were calculated. Despite their large differences in chemical composition, all chitosans had similar pK(a) values of 6.5-6.6. All chitosans also showed the same polyelectrolyte behavior when apparent pK(a) values were calculated according to Katchalsky and plotted as a function of the degree of ionization alpha. The intrinsic pK(a) values (pK(0)) extrapolated to zero charge were about 9. The results derived from an independent (1)H NMR study of the same chitosan samples showed no effect of F(A) on titration behavior of chitosan, confirming the results obtained by ELS.