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.

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A procedure for the determination of total N-nitroso compounds (NOC) in human gastric juice was developed by modifying earlier methods. The gastric juice sample, treated with sulphamic acid to remove nitrite, is injected directly into refluxing ethyl acetate containing either acetic acid for determining thermo-and acetic acid labile thermal energy analyser (TEA) responsive compounds (TAC), or into hydrogen bromide for the determination of TAC and NOC. The nitric oxide (NO) levels released are measured using thermal energy analysis with chemiluminescence detection, and the difference between the two determinations represents the concentrations of NOC in gastric juice. This method is not affected by nitrate concentrations of up to 1000 pmol I-'.The method was found to be rapid, reproducible and sensitive (detection limit 0.02 ymol I-' NOC), requiring only small volumes of gastric juice and no prior extraction. Because the difficulties arising from the system response to the denitrosating agent and variability of NO release by acetic acid from nitrite were eliminated, this improved method can more accurately distinguish NOC from most other TEA-responsive species. Suitable techniques for stabilising gastric juice samples from duodenal ulcer and atrophic gastritis patients and the influence of the time and storage conditions on NOC concentrations have been studied.

Chitosan is a promising biocompatible polymer for regenerative engineering applications, but its processing remains challenging due to limited solubility and rigid crystalline structure. This work represents the development of electrospun chitosan/poly(ethylene oxide) blend nanofibrous membranes by means of a numerical analysis in order to identify and tailor the main influencing parameters with respect to accessible surface nitrogen functionalities which are of importance for the biological activity as well as for further functionalization. Depending on the solution composition, both gradient fibers and homogenous blended fiber structures could be obtained with surface nitrogen concentrations varying between 0 and 6.4%. Response surface methodology (RSM) revealed chitosan/poly(ethylene oxide) ratio and chitosan molecular weight as the main influencing factors with respect to accessible nitrogen surface atoms and respective concentrations. The model showed good adequacy hence providing a tool to tailor the surface properties of chitosan/poly(ethylene oxide) blends by addressing the amount of accessible chitosan.