Determination of physical and chemical parameters that can be used to predict the hemostatic activity of chitosan without conducting in vivo experiments

Bleeding is the main cause of death as a result of injuries and wounds, so the development of effective hemostatics remains an urgent task of emergency medicine. Currently, local hemostatic agents (LGA) based on the chitosan biopolymer are widely used as means of providing emergency care for bleeding. At the same time, there is no single standard for evaluating the physicochemical and biological properties of chitosan. None of its traditionally controlled parameters, such as molecular weight and the degree of deacetylation, is crucial in determining the ability to initiate hemostasis. In this regard, the most reliable test for evaluating the effectiveness is still conducting in vivo experiments on large animals. In our work, using NMR spectroscopy, spectrophotometry, viscometry, inductively coupled plasma mass spectrometry, as well as methods for assessing hemostasis by in vitro and in vivo experiments, we determined the physicochemical parameters of chitosan, which are related with its hemostatic properties. These indicators were tested when evaluating the quality of chitosan raw materials, as well as LGA in the form of bandages and granules obtained on its basis. It was found that the ability to initiate hemostasis was shown by those samples of chitosan in which, at pH values of the solution close to physiological, a significant part of the molecules passed from the conformational state «rigid rod» to the state «globule», which was accompanied by an abrupt change in the light transmission of the solution. For effective samples, the value of light transmission determined by spectrophotometry at pH 7.36 was 42.5–66.1 %, and for inefficient samples >85.0 %. In this case, the viscosity average molecular weight and the degree of deacetylation affect the dynamics of changes in the conformation of the chitosan molecule, but the relation is not linear and depends on the viscosity of solutions and their temperature. The parameters of the molecular mass distribution determined by the DOSY NMR method did not demonstrate a connection with the manifestation of hemostatic properties of chitosan. The results obtained are consistent with the results of in vitro and in vivo tests. (author)