New N-Methylimidazole-Functionalized Chitosan Derivatives: Hemocompatibility and Antibacterial Properties.

Novel imidazole derivatives of the low molecular weight chitosan N-(2-hydroxypropyl)-1H-1,2,3-triazol-4-yl)methyl)-1-methyl-1H-imidazol-3-ium chitosan chloride (NMIC) were synthesized using copper-catalyzed azide-alkyne cycloaddition (CuAAC). The degrees of substitution (DSs) for the new derivatives were 18-76%. All chitosan derivatives (2000 µg/mL) were completely soluble in water. The antimicrobial activity of the new compounds against E. coli and S. epidermidis was studied. The effect of chitosan derivatives on blood and its components was studied. NMIC samples (DS 34-76%) at a concentration <10 µg/mL had no effect on blood and plasma coagulation. Chitosan derivatives (DS 18-76%) at concentrations of ≥83 µg/mL in blood and ≥116.3 µg/mL in plasma resulted in a prolongation of the clotting time of blood and plasma, positively related to the DS. At concentrations up to 9.1 µg/mL, NMIC did not independently provoke platelet aggregation. The degree of erythrocyte hemolysis upon contact with NMIC samples (2.5-2500 µg/mL) was below 4%. The inhibition of blood/plasma coagulation indicates the promising use of the studied samples to modify the surface of medical materials in order to achieve thromboresistance.

Synthesis of Selenium Nanoparticles Modified by Quaternary Chitosan Covalently Bonded with Gallic Acid.

Quaternary chitosan derivative with covalently bonded antioxidant (QCG) was used as media for synthesis of selenium nanoparticles (SeNPs). SeNPs were characterized using AFM, TEM, and DLS methods. The data confirmed the formation of stable nanoparticles with a positive charge (34.86-46.73 mV) and a size in the range 119.5-238.6 nm. The antibacterial and fungicidal activity of SeNPs occurred within the range of values for chitosan derivatives. In all cases, the highest activity was against C. albicans (MIC 125 µg/mL). The toxicity of the modified selenium nanoparticles to eukaryotic cells was significantly higher. Among nanoparticle samples, SeNPs that were synthesized at 55 °C demonstrated the highest toxicity against Colo357 and HaCaT cell lines. Based on these results, SeNPs loaded with doxorubicin were obtained. DOX loading efficiency was about 18%. QCG-SeNPs loaded with DOX at a concentration of 1.25 µg/mL inhibited more than 50% of hepatocarcinoma (Colo 357) cells and about 70% of keratinocytes (HaCaT).

Protective Properties of Copper-Loaded Chitosan Nanoparticles against Soybean Pathogens Pseudomonas savastanoi pv. glycinea and Curtobacterium flaccumfaciens pv. flaccumfaciens.

Soybeans are a valuable food product, containing 40% protein and a large percentage of unsaturated fatty acids ranging from 17 to 23%. Pseudomonas savastanoi pv. glycinea (Psg) and Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) are harmful bacterial pathogens of soybean. The bacterial resistance of soybean pathogens to existing pesticides and environmental concerns requires new approaches to control bacterial diseases. Chitosan is a biodegradable, biocompatible and low-toxicity biopolymer with antimicrobial activity that is promising for use in agriculture. In this work, a chitosan hydrolysate and its nanoparticles with copper were obtained and characterized. The antimicrobial activity of the samples against Psg and Cff was studied using the agar diffusion method, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. The samples of chitosan and copper-loaded chitosan nanoparticles (Cu2+ChiNPs) significantly inhibited bacterial growth and were not phytotoxic at the concentrations of the MIC and MBC values. The protective properties of chitosan hydrolysate and copper-loaded chitosan nanoparticles against soybean bacterial diseases were tested on plants in an artificial infection. It was demonstrated that the Cu2+ChiNPs were the most effective against Psg and Cff. Treatment of pre-infected leaves and seeds demonstrated that the biological efficiencies of (Cu2+ChiNPs) were 71% and 51% for Psg and Cff, respectively. Copper-loaded chitosan nanoparticles are promising as an alternative treatment for bacterial blight and bacterial tan spot and wilt in soybean.

Effect of Seed Priming with Chitosan Hydrolysate on Lettuce (Lactuca sativa) Growth Parameters.

Seed priming increases germination, yield, and resistance to abiotic factors and phytopathogens. Chitosan is considered an ecofriendly growth stimulant and crop protection agent. Chitosan hydrolysate (CH) is an unfractionated product of hydrolysis of high-molecular-weight crab shell chitosan with a molecular weight of 1040 kDa and a degree of deacetylation of 85% with nitric acid. The average molecular weight of the main fraction in CH was 39 kDa. Lettuce seeds were soaked in 0.01-1 mg/mL CH for 6 h before sowing. The effects of CH on seed germination, plant morphology, and biochemical indicators at different growth stages were evaluated. Under the 0.1 mg/mL CH treatment, earlier seed germination was detected compared to the control. Increased root branching was observed, along with 100% and 67% increases in fresh weight (FW) at the 24th and 38th days after sowing (DAS), respectively. An increase in the shoot FW was found in CH-treated plants (33% and 4% at the 24th and 38th DAS, respectively). Significant increases in chlorophyll and carotenoid content compared to the control were observed at the 10th DAS. There were no significant differences in the activity of phenylalanine ammonia-lyase, polyphenol oxidase, ß-1,3-glucanase, and chitinase at the 24th and 38th DAS. Seed priming with CH could increase the yield and uniformity of plants within the group. This effect is important for commercial vegetable production.

The Application of Chitosan for Protection of Cultural Heritage Objects of the 15-16th Centuries in the State Tretyakov Gallery.

Microorganisms are one of the main factors in the deterioration of cultural heritage, in particular art paintings. The antiseptics currently used in painting have significant limitations due to insufficient effectiveness or increased toxicity and interaction with art materials. In this regard, the actual challenge is the search for novel materials that effectively work against microorganisms in the composition with painting materials and do not change their properties. Chitosan has pronounced antimicrobial properties but was not used previously as an antiseptic for paintings. In our study we developed a number of mock layers based on sturgeon glue, supplemented which chitosan (molecular weight 25 kDa or 45 kDa), standard antiseptics for paintings (positive controls) or without additives (negative control). According to Fourier transform infrared spectroscopy and atomic force microscopy, the addition of chitosan did not significantly affect the optical and surface properties of this material. The ability of chitosan to effectively protect paintings was shown after inoculation on the created mock-up layers of 10 fungi-destructors of tempera painting, previously isolated from cultural heritage of the of the 15-16th centuries in the State Tretyakov Gallery, on the created mock layers. Our study demonstrated the principled opportunity of using chitosan in the composition of painting materials to prevent biodeterioration for the first time.

Investigation of the Antioxidant Properties of the Quaternized Chitosan Modified with a Gallic Acid Residue Using Peroxidase that Produces Reactive Oxygen Species.

Chitosan modified with a (2-hydroxy-3-trimethylammonium) propyl group and gallic acid residue, or quaternized chitosan with gallic acid (QCG), was synthesized. Antioxidant properties of the produced QCG have been investigated. Peroxidase in combination with NADH and salicyl hydroxamate (SHAM) caused consumption of oxygen and production of H2O2 in aqueous solution as a result of O2 reduction in the peroxidase-oxidase reactions. The rates of O2 consumption and H2O2 generation were reduced in the presence of QCG. The antioxidant propyl gallate (PG) and superoxide dismutase (SOD) had the same effect, but not the quaternized chitosan (QC) without gallic acid. The effect of chitosan derivatives on the production of reactive oxygen species (ROS) in the cells of pea leaf epidermis and on the cell death detected by the destruction of cell nuclei, was investigated. QCG, QC, and SOD had no effect, while PG decreased the rate of ROS generation in the cells of the epidermis, which was induced by NADH with SHAM or by menadione. QCG and QC prevented destruction of the guard cell nuclei in the pea leaf epidermis that was caused by NADH with SHAM or by KCN. SOD had no effect on the destruction of nuclei, while the effect of PG depended on the inducer of the cell death. Suppression of the destruction of guard cell nuclei by chitosan derivatives was associated not with their antioxidant effect, but with the disruption of the plasma membrane of the cells. The results obtained have shown that QCG exhibits antioxidant properties in solutions, but does not prevent generation of ROS in the plant cells. The mechanism of antioxidant effect of QCG is similar to that of PG and SOD.

Simple method for ultrasound assisted «click¼ modification of azido-chitosan derivatives by CuAAC.

New quaternized chitosan derivatives HT-TMC were synthesized as a result of copper catalyzed azide-alkyne [3 + 2] cycloaddition (CuAAC). The structure of the HT-TMC was verified by 2D NMR. The synthesis was carried out as a result of the formation of Cu(I) in situ, under the action of ultrasound in aerobic conditions in the presence of acetic acid and metallic copper (copper turnings). The new derivatives were characterized by increased pH range of solubility (DS range 18-76%) and the presence of antibacterial and fungicidal activities. The proposed catalytic system makes it possible to easily and efficiently obtain new derivatives of chitosan as a result of ultrasound-promoted CuAAC.

Antifungal action of chitosan in combination with fungicides in vitro and chitosan conjugate with gallic acid on tomatoes against Botrytis cinerea.

In the present work, a positive effect was obtained by using low molecular weight chitosan compounds in combination with synthetic fungicides. Antifungal activity against Botrytis cinerea, determined by the radial growth method, was more than 75%, with a 25 × 10- 10 g/L concentration of fludioxonil or difenoconazole in compounds. Metabolic activity of B. cinerea fungus was about 15% when using a chitosan compound containing fludioxonil at a concentration of 25 × 10- 7 g/L. The combined action of chitosan with difenoconazole at a fungicide concentration of 25 × 10- 4 g/L is 2-3 times more effective than the action of each component separately. Results of studies for artificially inoculated B. cinerea tomato fruit when treated with low molecular chitosan and chitosan conjugate with gallic acid reduced the frequency of rotting fruit by 50 and 83%, respectively. Chitosan-gallic acid conjugate were obtained from chitosans with Mw of 28 kDa (Ch28GA) was proved to be effective as a preventive treatment for 3 days and can potentially be used as a biofungicide against B. cinerea on tomatoes in the post-harvest period.

Collagen/Chitosan Gels Cross-Linked with Genipin for Wound Healing in Mice with Induced Diabetes.

Diabetes mellitus continues to be one of the most common diseases often associated with diabetic ulcers. Chitosan is an attractive biopolymer for wound healing due to its biodegradability, biocompatibility, mucoadhesiveness, low toxicity, and hemostatic effect. A panel of hydrogels based on chitosan, collagen, and silver nanoparticels were produced to treat diabetic wounds. The antibacterial activity, cytotoxicity, swelling, rheological properties, and longitudinal sections of hydrogels were studied. The ability of the gels for wound healing was studied in CD1 mice with alloxan-induced diabetes. Application of the gels resulted in an increase in VEGF, TGF-b1, IL-1b, and TIMP1 gene expression and earlier wound closure in a comparison with control untreated wounds. All gels increased collagen deposition, hair follicle repair, and sebaceous glands formation. The results of these tests show that the obtained hydrogels have good mechanical properties and biological activity and have potential applications in the field of wound healing. However, clinical studies are required to compare the efficacy of the gels as animal models do not reproduce full diabetes pathology.

Synthesis of silver nanoparticles using gallic acid-conjugated chitosan derivatives.

In presented study, various chitosan derivatives containing covalently bounded gallic acid were obtained: chitosan with gallic acid (CG), quaternized chitosan with gallic acid (QCG), and succinylated chitosan with gallic acid (SCG). Chitosan derivatives were used as stabilizing and reducing agents in the synthesis of silver nanoparticles (AgNPs). The dimensional characteristics of nanomaterials were determined by transmission electron (TEM), dynamic light scattering (DLS) and atomic force (AFM) microscopy, antibacterial activity (against E. coli, S. epidermidis), cytotoxicity (HaCaT, Colo 357 cell lines) and hemocompatibility. Among all samples, QCG-AgNPs showed low toxicity in the range of studied concentrations (3.125-100 µg/ml) high stability of nanoparticle for 4 months (according to UV.spectroscopy data) the highest antibacterial activity against S. epidermidis (3.91 µg/ml). The high antibacterial activity, stability, and simplicity of the process of producing AgNPs based on the QCG derivative reveals that a new method for producing modified AgNPs deserves future consideration.

Investigation of the properties of N-[(2-hydroxy-3-trimethylammonium) propyl] chloride chitosan derivatives.

N-[(2-Hydroxy-3-Trimethylammonium) Propyl] Chitosan Chloride Derivatives (HTCC), based on low molecular weight crab chitosan, were synthesized by the alkylation reaction with a degree of substitution of 10-98%. The chemical structure was confirmed by 1H NMR and IR-spectra. Physical and chemical characteristics and a number of properties were defined. All HTCC derivatives were soluble at pH 7.4. HTCCs have an inhibitory capacity on the growth of the studied microorganisms. The introduction of quaternary ammonium groups into chitosan molecule contributed to the increase of antibacterial activity of derivatives. HTCC53 showed antifungal activity and at a concentration of 500 mg/ml completely inhibited the growth of mycelial fungi F. oxysporum, A. alternata and C. herbarum. When studying the ability of HTCCs to absorb DPPH radicals, it was found that samples of HTCC10 and HTCC40 showed high inhibitory capacity at a concentration of >15 mg/ml. It was shown that the chelating ability of HTCCs decreased by reducing the number of free amino groups. HTCC10-HTCC53 demonstrated the maximum values of chelating ability at a concentration of 4-10 mg/ml. Due to the solubility at neutral pH values and the properties shown, obtained chitosan derivatives can be used in clinical practice, pharmaceutical and food industries in the future.