Chemical modification of chitosan for developing of new hemostatic materials: A review.

Uncontrolled bleeding that occurs during surgery, trauma, and in combat conditions is critical and require immediate action. Chitosan is a polysaccharide, obtained from natural sources with unique biological properties. It is often used as basis for local hemostatic agents (LHA). We summarized the data on hemostatic properties of chitosan, commercially available chitosan-based products with focus in the field of chemical modification of chitosan. Various approaches are used to enhance hemostatic activity of chitosan-based materials. The approach with chemical modification of chitosan allows changing the properties of the polymer in order to obtain an active macromolecule that contributes to hemostasis. Ongoing research on the mechanism of interaction with blood components in the case of different chitosan derivatives will make it possible to identify promising directions for chemical modification to obtain an effective LHA.

Effect of Oligochitosan on Experimental Venous Thrombosis in Guinea Pigs.

Oligochitosan Сh10/85 with a molecular weight of 10 kDa and a deacetylation degree of 85% prevented the development of experimental venous thrombosis in guinea pigs after intravenous administration in a dose of 30 mg/kg. In a concentration of 0.005-0.5 mg/ml, oligochitosan Ch10/85 did not provoke hemolysis of human red blood cells in in vitro experiments. The antithrombotic effect of oligochitosan Ch10/85 that exhibits low anticoagulant activity (by two orders of magnitude lower than that of unfractionated heparin) can be associated with inhibition of platelet aggregation.

Physicochemical and biological characteristics of chitosan/κ-carrageenan thin layer-by-layer films for surface modification of nitinol.

Thin films based on the natural polysaccharides κ-carrageenan (Carr) and chitosan (Chit) were formed by layer-by-layer deposition technique. Surface topography and mechanical characteristics (Young's modulus, adhesion strength) of the polymer films with different number of layers were determined using various modes of atomic force microscopy (AFM). Polymer films were used to deposit on the surface of nitinol (NiTi) plates. The creation of polysaccharide coatings on nitinol led to a change in surface properties, such as hydrophilicity and root mean square roughness. in vitro cytotoxicity assay for nitinol plates with and without polymer coating by the MG-63 osteoblast-like cell line was conducted and was shown that all the studied samples are not toxic. A decrease in cytotoxicity for samples with a polymer film consisting of 4 layers of chitosan and carrageenan was shown as compared to an uncoated nitinol plates.

Hemocompatibility of Silver Nanoparticles Based on Conjugate of Quaternized Chitosan with Gallic Acid in In Vitro Experiments.

We studied hemocompatibility of silver nanoparticles synthesized on the basis of a conjugate of quaternized chitosan with gallic acid (QChit-Gal). For the three variants of silver particles (Nos. 1, 2, and 3), the QChit-Gal:AgNO3 ratio was 5:1, 5:3, and 1:1, respectively. Anticoagulant activity of all samples of silver nanoparticles was lower than that of the conjugate. Samples of nanoparticles Nos. 1 and 2 in a concentration of 0.0233 mg/ml did not affect plasma clotting time and can be used for intravenous administration. However, their concentration in the blood should not exceed 0.01 mg/ml, because in this concentration they do not affect erythrocyte membrane, do not induce platelet aggregation, and do not affect platelet aggregation induced by ADP.

Chitin/Chitosan and Its Derivatives: Fundamental Problems and Practical Approaches.

In this review, we present the data on the natural occurrence of chitin and its partially or fully deacetylated derivative chitosan, as well as their properties, methods of modification, and potential applications of derivatives with bactericidal, fungicidal, and antioxidant activities. The structure and physicochemical characteristics of the polymers, their functions, and features of chitin microbial synthesis and degradation, including the processes occurring in nature, are described. New data on the hydrolytic microorganisms capable of chitin degradation under extreme conditions are presented. Special attention is focused on the effect of physicochemical characteristics of chitosan, including molecular weight, degree of deacetylation, polydispersity index, and number of amino group derivatives (quaternized, succinyl, etc.) on the antimicrobial and antioxidant properties of modified polymers that can be of particular interest for biotechnology, medicine, and agriculture. Analysis of the available literature data confirms the importance of fundamental research to broaden our knowledge on the occurrence of chitin and chitosan in nature, their role in global biosphere cycles, and prospects of applied research aimed at using chitin, chitosan, and their derivatives in various aspects of human activity.

Analysis of the Action of Quaternized Chitosans with Different Molecular Weight on Anticoagulant Activity of Heparins In Vitro.

Quaternized derivatives of chitosan with a substitution degree of 85-98% (highly substituted) synthesized from chitosans with a molecular weight of 5, 10, 20 kDa, with a degree of deacetylation of 89-98%, and the code numbers of QChit 5, QChit 10, QChit 20, respectively, completely neutralize antithrombin activity of unfractionated heparin and partially neutralize aXa activity of low-molecular-weight heparin (clexane), similar to protamine sulfate. The advantages of QChit 5 and QChit 10 over QChit 20 and protamine sulphate are the follows: the effect is achieved at lower concentrations and in greater concentration range; they do not promote platelet aggregation; in a concentration of 0.0072 mg/ml they do not destroy the erythrocyte membranes.

Effects of Chitosan Derivative N-[(2-Hydroxy-3-Trimethylammonium)Propyl]Chloride on Anticoagulant Activity of Guinea Pig Plasma.

Intravenous injection of protamine sulfate or quarternized chitosan derivative to guinea pigs after injection of 70 aIIa U/kg non-fractionated heparin shortened plasma clotting time (shown by partial activated thromboplastin time, thrombin time, and prothrombin time). Intravenous injection of protamine sulfate or quarternized chitosan derivative to guinea pigs after injection of 1 mg/kg (100 aXa U/kg) low-molecular-weight heparin (clexane) led to shortening of plasma clotting time in the ReaClot Heparin test and to prolongation of plasma amidolytic activity in the factor Xa chromogenic substrate test.

[The study of the process of enzymatic hydrolysis of yeast biomass to generate food ingredients with the specified fractional composition of protein substances].

With the use of enzyme systems (ES) the directed biocatalytic destruction of subcellular structures of the yeast biomass Saccharomyces cerevisiaе has been conducted for obtaining products of the specified structural-fractional composition. The composition of ES-1 included the enzymes catalyzing the hydrolysis of cell wall polysaccharides of yeast. Enzymes were dosed out at the rate of ß-glucanase - 300 units of ß-GcS/g of yeast, mannanase - 28.9 units of MS/g of yeast. ES-2, along with the enzymatic composition of ES-1, also contained a proteolytic complex, which included enzymes of bacterial origin, which were neutral, serine and metal-depended proteases (in a dosage of 2 units of PS/g of yeast). ES-3 consisted of the enzymes with ß-glucanase, mannanase, proteolytic activities and was further reinforced by high dose of proteases of fungal origin (10 units PS/g of yeast) for the implementation of deep hydrolysis of protein substances of yeast cell protoplasm to low molecular weight peptides and free amino acids. The action of enzymatic systems with different substrate specificity on the degree of destruction of subcellular structures of yeast was illustrated by electron microscopy. The resulting degradation products had different fractional composition and structural features. The results showed that ЕS-1 treatment of yeast led to deformation of the cell walls, but did not affect the composition of the protein fractions, represented by peptides with different molecular weight (20-60 kDa) that were characteristic for the starting material. The use of ES-2 has provided a deeper degradation of the protein-polysaccharide matrix of the cell walls and partial hydrolysis of proteins with the formation of soluble protein components with molecular weight less than 14 kDa. ES-3 treatment of yeast cells allowed to obtain composition with predominant content (89%) of free amino acids and short peptides with molecular weight up to 300 Da. The efficacy of targeted destruction of subcellular structures of Saccharomyces cerevisiae with getting of fermentation biomass with the specified fractional composition of protein substances for the production of food ingredients with special functional effects has been shown.

[Antibacterial Activity of Alkylated and Acylated Derivatives of Low-Molecular Weight Chitosan].

A number of alkylated (quaternized) and acylated derivatives of low-molecular weight chitosan were obtained. The structure and composition of the compounds were confirmed by the results of IR and PMR spectroscopy, as well as conductometric titration. The effect of the acyl substituent and the degree of substitution of N-(2-hydroxy-3-trimethylammonium) with the propyl fragment appended to amino groups of the C2 atom of polymer chains on antibacterial activity against typical representatives of gram-positive and gram-negative microorganisms (Staphylococcus epidermidis and Escherichia coli) was studied. The highest activity was in the case of N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride with the maximal substitution (98%). The minimal inhibitory concentration of the derivative was 0.48 µg/mL and 3.90 µg/mL for S. epidermis and E. coli, respectively.

[Neutralization of anticoagulant activity of heparin by N-[(2-hydroxy-3-trimethylammonium) propyl] chloride derivatives of chitosan].

Alkylated derivatives of low molecular weight chitosan with different substitution degrees of 98, 40, and 9% (I, II, and III respectively) have been synthesized. The structure of the obtained derivatives was defined by spectral assays (IR-spectroscopy and proton magnetic resonance). Chitosan derivatives were characterized with positive zeta-potential (33­51 mV) and solubility from 2 to 100 mg/mL in pH 7.4 and 25°C. It was shown that, at a concentration of 0.0014­0.0029 mg/mL, derivative I, as well as protamine sulfate, could be used to neutralize the anticoagulant activity of unfractionated or low molecular weight heparin. At a concentration of 0.0029­0.58 mg/mL, derivative I enhanced platelet aggregation, which would be necessary when hemostatic compounds or materials were used. Derivatives II and III enhanced platelet aggregation to a lesser extent.

[Determination of residual protein and endotoxins in chitosan ].

Methods for the determination of residual protein and endotoxins in chitosan preparations, which can be used as vectors for biologically active compounds delivery, are discussed. The limits of their use for the determination of residual impurities in chitosan preparations associated with the structure of the biopolymer are estimated.

[Analysis of toxicity and biocompatibility of chitosan derivatives with different physico-chemical properties].

A comparative study of the toxicity and hemocompatibility of chitosan and its derivatives with different acetylation degrees, molecular masses, charges, and hydrophobicity has been performed. It has been shown that only positively charged chitosan derivatives activate platelets and suppress cell proliferation, regardless of the acetylation degree, molecular mass, and hydrophobicity. Chitosan quaternization decreases toxicity at a low degree of substitution and abruptly increases it at a high one. Negatively charged chitosan derivatives were nontoxic and compatible with blood components. It was concluded that the toxicity of chitosan and its derivatives is defined by their charge and solubility at a neutral pH.

[Antibacterial effect of peptide conjugates with a quaternized chitosan derivative and its estimation by the method of atomic force microscopy].

We obtained a number of conjugates based on a quaternized chitosan derivative and antimicrobial peptides (melittin and warnerin) crosslinked by microbial transglutaminase. We determined the optimal conditions for the synthesis (30 minutes, with a mole ratio of peptides and chitosan derivative of 1.4: 100) and studied the antibacterial properties of obtained conjugates. The antibacterial effect of the conjugates was found to be greater than that of their components. The antibacterial activity of the conjugates was determined by the double-dilution method and by atomic force microscopy.

[Comparative evaluation of antimicrobial activity of oligochitosans against Klebsiella pneumoniae].

Antibacterial activity of chitosan with different molecular weight at different pH values against gram-negative Klebsiella pneumoniae was studied. It was found that the dependence of inhibition activity of chitosan on its molecular weight was inversed when the medium pH was increased above 7.0. In acidic media higher molecular weight chitosan had the higher antibacterial activity was occured while and in weak alkaline media oligomeric forms of chitosan displayed the inhibition effect only. Our results showed that the antibacterial activity of chitosan against Klebsiella pneumoniae was closely connected with its polycationic nature, and was dependent on the degree of protonation of chitosan amino groups, which at the same time depended on the degree of polymerization and solution pH values.

Intracellular sorting of differently charged chitosan derivatives and chitosan-based nanoparticles.

Chitosan (Chi) is a biodegradable nontoxic polycation with multiple reactive groups that is easily used to obtain derivatives with a desired charge and hydrophobic properties. The aim of this work was to study the intracellular traffic of positively charged hexanoyl-chitosan (HC) or HC-based nanoparticles (HCNPs) and negatively charged succinoyl-chitosan (SC) and SCNPs in epithelial and macrophage cell lines. By using flow cytometry we demonstrated that positively charged HC adhered to cell membranes quicker and more efficiently than negatively charged SC or NPs. However confocal studies showed that SC and SCNPs penetrated cells much more efficiently than HC while HCNPs did not enter the epithelial cells. Macrophages also phagocyted better negatively charged material but were able to engulf both HC and HCNPs. Upon entering the cells, SC and SCNPs were co-localized with endosomes and lysosomes while HC was found in mitochondria and, to a lesser extent, in lysosomes of epithelial cells. Macrophages, RAW264.7, more efficiently transported all Chi samples to the lysosomal compartment while some positively charged material was still found in mitochondria. Incubation of Chi derivatives and ChiNPs at pH specific to mitochondria (8.0) and lysosomes (4.5) demonstrated the neutralization of Chi charge. We concluded that epithelial cells and, to a lesser extent, macrophages sort charged material to the organelles neutralizing Chi charge.

[In vitro antitumor activity of heterochitooligosaccharides (a review)].

This review presents an analysis of the literature data over the last decade in order to reveal the relationship between the structure and composition of chitin/chitosan oligomers and their antitumor activity. Oligosaccharides consisting of N-acetylglucosamine and/or glucosamine units connected to each other by ß-1,4-O-glycoside bond are generally called chitooligosaccharides. Homochitooligosaccharides are the oligomers of N-acetylglucosamine (AGA) or glucosamine (GA). Heterooligosaccharides are a mixture of various oligomers differing in the degree of polymerization (DP), acetylation (DA), or deacetylation (DD), as well as in the location of N-acetyl residues in the oligomer chain. Heterochitooligomers with a polymerization degree of no more than 10 are usually water soluble. The solubility of chitooligosaccharides with a DP of more than 10 depends on the DA and pH of a solution. The pharmaceutical and food industries, as well as scientists engaged in basic research, are interested in the use of heterochitooligosaccharides. This is explained by their unique properties, such as good water solubility; minimal toxicity; biocompatibility; the ability to penetrate cell membranes, resulting in a high degree of absorption (unlike chitin and chitosan); and their biological activity. Therefore, in the last decade, researchers have focused their attention on studying the relationship of the structure of oligosaccharides and their specific activity, such as antitumor, antimicrobial, antioxidant, immunemodulatory, and other activities. This is shown by the number of publications, which has doubled compared to the number in 2001.

[Influence of Polycations on Antibacterial Activity of Lysostaphin].

The synergistic antibacterial activity of lysostaphin and polycations of different chemical structures against Staphylococcus aureus has been shown. Polycations improved the lytic activity of lysostaphin against the peptidoglycan of staphylococci. It is proposed that this resulted in decreased binding of positively charged lysostaphin with S. aureus cell-wall teichoic acids. These data provide an opportunity to search for polycations that would amplify the synergistic effect of lysostaphin or other antibacterial proteins against staphylococci.

[Hydrolysis of chitozan with an enzyme complex from Myceliophthora sp].

Samples of low-molecular weight chitosan with molecular masses of 8-24 kDa, identical deacetylation degrees (85%), and polydispersity indexes soluble at pH 5-7 were obtained by enzymatic hydrolysis using an enzyme complex from the micelial fungi Myceliophthora fergusii with yields of 50-80%. The optimal conditions for hydrolysis were found (pH 5.6, 37 degrees C, an enzyme/substrate ratio 1/800, 15-60 min). The obtainment of chitosan sample sets with different characteristics will enable the selection of the most efficient ones for comparison in in vitro/in vivo experiments.

[Replication-competent γ-retrovirus Mo-MuLV expressing green fluorescent protein gene as an efficient tool for screening of inhibitors of retroviruses that use heparan sulfate as the primary cell receptor].

The effect of sulfated polysaccharides on the efficiency of infection of mouse embryonic fibroblast cell lines SC-1 and NIH-3T3 by replication-competent recombinant Moloney murine leukemia virus (Mo-MuLV) carrying the eGFP gene was investigated. It was shown that used polysaccharides have no cytostatic and cytotoxic effects on SC-1 and NIH 3T3 cells inthe concentrations from 0.01 to 100 µg/ml and have virucidal activity against Mo-MuLV. Polysaccharides in the indicated concentrations inhibit cell infection by Mo-MuLV, that prevents further expansion of viral infection. It was detected that sulfated polysaccharides are effective inhibitors of other retroviruses, including lentiviruses, that use heparan sulfate as cell receptors for non-specific binding.

[Effect of polysaccharides on biological activity of human lactoferrin].

The influence of neutral and ionic polysaccharides on the antioxidant (AOA) and detoxifying activities of lactoferrin (LF) and the duration of its circulation in the body was studied. In addition to natural polymers, we studied artificial chitosan derivatives with different functional groups. On the basis ofAOA test, five polysaccharides were selected. The study of the detoxifying effect of LF in two models of induced toxicity revealed polysaccharides that maintained or increased the detoxifying activity of LF. We established that the formation of a complex of lactoferrin with two galactomannans and succinyl chitosan caused positive changes in LF properties: the detoxifying activity of the protein remained unchanged or increased, whereas its elimination from the body was decelerated.