Volume 52, Nº 5 (2016)

Mechanisms of interaction between chitosan, various macromolecules or drug delivery systems and mammalian cells are reviewed. Modernly the role of different physicochemical properties of chitosan and chitosan nanoparticles on the mechanisms of cell bunding, endocytosis and redistribution are poorly understood.

A low-molecular derivative of the polysaccharide (5 kDa) was obtained and its cytokine-inducing and anti-inflammatory activity was studied by free radical depolymerization of chitosan (110 kDa). It was shown that high-molecular chitosan in vitro inhibited the synthesis of anti-inflammatory cytokine, the tumor necrosis factor alpha induced by endotoxin. In the case of peroral introduction to experimental animals, high- and low-molecular chitosans stimulated synthesis of the anti-inflammatory cytokine IL-10 in the blood serum of mice; in this case, the activity of the high-molecular derivative was two times higher as compared with the initial polysaccharide. With peroral introduction, the initial polysaccharide (50 mg/kg) and its derivative inhibited the development of chemically induced inflammation of experimental animals’ large intestines, which was manifested as a decrease in the affected area and the degree of damage to the large intestine wall, as well as a two-fold reduction of myeloperoxidase activity. According to morphological and biochemical characteristics, the effect of chitosans was similar to that of a hormone anti-inflammatory drug, prednisolone.

It was shown using various methods that the radical scavenging and radical scavenging activities of chitosan conjugates with phenolic antioxidants of plant origin significantly exceed the corresponding values of their low molecular weight analogs (gallic and syringic acids, quercetin and dihydroquercetin). Cytogenetic analysis of human peripheral blood lymphocytes demonstrated that the conjugates showed pronounced antimutagenic efficiency when the cells were γ-irradiated in vitro at a dose of 2 Gy.

A method for the determination of the antimicrobial activity of chitosan with the use of organic salts for the production of pH in the range of 5.5–8.2 was studied. The double-dilution method demonstrated the effectiveness of the determination of the antimicrobial activity of chitosan samples with different molecular weights and solubilities. It was found that the antibacterial activity increased at low pH values with increasing molecular weight, but chitosans with a molecular weight of 5–6 kDa showed higher activity at neutral and slightly alkaline pH levels. Determination of the antimicrobial activity of various chitosan samples at different pH values allowed a more reliable assessment of the potential biological activity of chitosan.

The influence of the conditions of the formation of chitosan hydrogels crosslinked with glutaraldehyde (GA) or genipin (the polysaccharide molecular weight, pH level, and concentration of the chitosan solution) on the gel time and the properties of biopolymer scaffolds for tissue engineering obtained by the freeze-drying of hydrogels was studied. The resulting scaffolds had different structures (morphology, degree of anisotropy, average pore size) and moisture-retaining capacities. The cytotoxicity of biodegradable scaffolds based on chitosan with a low content of genipin and GA was studied for the first time. Using the L929 mouse fibroblasts model line, we demonstrated that scaffolds based on chitosan with a molecular weight of 320 and 190 kDa crosslinked with genipin and GA (0.005 and 0.01 mol/mol of chitosan amino groups) are biocompatible. Using confocal laser microscopy, we demonstrated that the cells are uniformly distributed in all scaffold samples and they successfully grew and proliferated when cultured in vitro for 4 days.

A new strain Penicillium sp. IB-37-2, which actively hydrolyzes chitosan (SD ∼80–85%) but possesses low activity against colloidal chitin, was isolated. The fungus was observed to have a high level chitosanase biosynthesis (1.5–3.0 U/mL) during submerged cultivation at 28°C, with a pH of 3.5–7.0 and 220 rpm in nutrient media containing chitosan or chitin from shells of crabs. Purification of the chitosanase enzyme complex from Penicillium sp. IB-37-2 by ultrafiltration and hydrophobic chromatography, followed by denaturing electrophoresis, revealed two predominant proteins with molecular weights of 89 and 41 kDa. The purified enzyme complex demonstrated maximal activity (maximal rate of hydrolysis of dissolved chitosan) and stability at 50–55°C and a pH of 3.5–4.0. The enzyme preparation also hydrolyzed laminarin, β-(1,3)-(1,4)-glycan, and colloidal chitin. Exohydrolysis of chitosan by the preparation isolated from Penicillium sp. IB-37-2 resulted in the formation of single product, D-glucosamine.

The response of Triticum aestivum L. to infection by Septoria nodorum Berk, a pathogen causing speckled leaf blotch, was studied. The effect of salicylic acid (SA) and jasmonic acid (JA) signal molecules, as well as chitooligosaccharides (COSs) with different acetylation degrees (ADs), on the accumulation of hydrogen peroxide (Н₂О₂) in wheat leaves and the pathogenesis-related (PR) proteins of oxalate oxidase (AJ556991.1), peroxidase (TC 151917), and proteinase inhibitor (EU293132.1) was investigated. Treatment with the signal molecules inhibited S. nodorum growth and stimulated Н₂О₂ accumulation, as well as PR gene expression. SA and COS with 65% AD are found to be more efficient in Н₂О₂ induction and elevation of the transcriptional level of the oxalate oxidase and peroxidase genes. At the same time, JA and COS with 30% AD stimulated transcription of the proteinase inhibitor gene. The results suggest the existence of differential means of defense response induction by signal molecules with more prospects for the regulation of plant immunity.

High-molecular weight chitosan (200 kDa, 75% deacetylated) and N-succinoyl chitosan (300 kDa, 75% deacetylated) were shown to have a preadaptive effect and increase the lifespan of honeybees due to the induction of protective antioxidant and immune mechanisms. Chitosan with a molecular weight of 200 kDa had a fungistatic effect on a pathogenic fungus that causes ascospherosis, a disease of bee larvae and pupae.

The cDNA gene (BgL1), encoding GH3 family β-glucosidase (EC 3.2.1.21) from Aspergillus niger BE-2 (abbreviated to BgL1), was amplified and inserted into the yeast expression pPIC9K vector at the site of Bln I (Avr II) and NotI. The recombinant expression vector, designated as pPIC9K-BgL1, was transformed into Pichia pastoris GS115. The transformants were screened on minimal dextrose plates, which inoculated on geneticin G418-containing yeast extract-peptone-dextrose plates. The transformants expressed the high β-glucosidase activity of 22.6 U/mL. SDS-PAGE demonstrated that the BgL1 was extracellularly expressed with an apparent molecular weight of 90.0 kDa. The purified BgL1 displayed the maximum activity at pH 6.0 and 60°C. It was highly stable at a broad pH range of 4.0–7.5 and temperature of 60°C. The BgL1 displayed high similarity to the β-glucosidases of A. niger FN430671 and A. niger DQ655704, the members of the GH3 family. Its three-dimensional structure was predicted using http://swiss-model.expasy.org/ on-line programs based on the crystal structure of Aspergillus aculeatus β-glucosidase.