Evaluation of Chemical and Biological Properties of Biodegradable Composites Based on Poly(3-hydroxybutyrate) and Chitosan.

In this study, composite films and scaffolds of polyester poly(3-hydroxybutyrate) and polysaccharide chitosan obtained via a simple and reproducible blending method using acetic acid as a solvent were considered. The degradation process of the films was studied gravimetrically in a model biological medium in the presence of enzymes in vitro for 180 days. The kinetics of weight reduction depended on the amount of chitosan in the composition. The biocompatibility of the films was evaluated using the Alamar blue test and fluorescence microscopy. The materials were non-cytotoxic, and the addition of poly(3-hydroxybutyrate) to chitosan improved its matrix properties on mesenchymal stem cells. Then, the 3D composites were prepared by freeze-drying. Their structure (using SEM), rheological behavior, moisture absorption, and porosity were investigated. The addition of different amounts of chitosan allowed us to vary the chemical and biological properties of poly(3-hydroxybutyrate) materials and their degradation rate, which is extremely important in the development of biomedical poly(3-hydroxybutyrate) materials, especially implantable ones.

Anti-Obesity Effects of Chitosan and Its Derivatives.

The number of obese people in the world is rising, leading to an increase in the prevalence of type 2 diabetes and other metabolic disorders. The search for medications including natural compounds for the prevention of obesity is an urgent task. Chitosan polysaccharide obtained through the deacetylation of chitin, and its derivatives, including short-chain oligosaccharides (COS), have hypolipidemic, anti-inflammatory, anti-diabetic, and antioxidant properties. Chemical modifications of chitosan can produce derivatives with increased solubility under neutral conditions, making them potential therapeutic substances for use in the treatment of metabolic disorders. Multiple studies both in animals and clinical trials have demonstrated that chitosan improves the gut microbiota, restores intestinal barrier dysfunction, and regulates thermogenesis and lipid metabolism. However, the effect of chitosan is rather mild, especially if used for a short periods, and is mostly independent of chitosan's physical characteristics. We hypothesized that the major mechanism of chitosan's anti-obesity effect is its flocculant properties, enabling it to collect the chyme in the gastrointestinal tract and facilitating the removal of extra food. This review summarizes the results of the use of COS, chitosan, and its derivatives in obesity control in terms of pathways of action and structural activity.

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.

Preparation and characterization of poly(3-hydroxybutyrate)/chitosan composite films using acetic acid as a solvent.

Poly(3-hydroxybutyrate) and chitosan are among the most widely used polymers for biomedical applications due to their biocompatibility, renewability and low toxicity. The creation of composite materials based on biopolymers belonging to different classes makes it possible to overcome the disadvantages of each of the components and to obtain a material with specific properties. Solving this problem is associated with difficulties in the selection of conditions and solvents for obtaining the composite material. In our study, acetic acid was used as a common solvent for hydrophobic poly(3-hydroxybutyrate) and chitosan. Mechanical, thermal, physicochemical and surface properties of the composites and homopolymers were investigated. The composite films had less crystallinity and hydrophobicity than poly(3-hydroxybutyrate), and the addition of chitosan caused an increase in moisture absorption, a decrease in contact angle and changes in mechanical properties of the poly(3-hydroxybutyrate). The inclusion of varying amounts of chitosan controlled the properties of the composite, which will be important in the future for its specific biomedical applications.

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.

Biocomposite Materials Based on Poly(3-hydroxybutyrate) and Chitosan: A Review.

One of the important directions in the development of modern medical devices is the search and creation of new materials, both synthetic and natural, which can be more effective in their properties than previously used materials. Traditional materials such as metals, ceramics, and synthetic polymers used in medicine have certain drawbacks, such as insufficient biocompatibility and the emergence of an immune response from the body. Natural biopolymers have found applications in various fields of biology and medicine because they demonstrate a wide range of biological activity, biodegradability, and accessibility. This review first described the properties of the two most promising biopolymers belonging to the classes of polyhydroxyalkanoates and polysaccharides-polyhydroxybutyrate and chitosan. However, homopolymers also have some disadvantages, overcome which becomes possible by creating polymer composites. The article presents the existing methods of creating a composite of two polymers: copolymerization, electrospinning, and different ways of mixing, with a description of the properties of the resulting compositions. The development of polymer composites is a promising field of material sciences, which allows, based on the combination of existing substances, to develop of materials with significantly improved properties or to modify of the properties of each of their constituent components.

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.

Evaluation of Antibacterial and Antifungal Properties of Low Molecular Weight Chitosan Extracted from Hermetia illucens Relative to Crab Chitosan.

This study shows the research on the depolymerisation of insect and crab chitosans using novel enzymes. Enzyme preparations containing recombinant chitinase Chi 418 from Trichoderma harzianum, chitinase Chi 403, and chitosanase Chi 402 from Myceliophthora thermophila, all belonging to the family GH18 of glycosyl hydrolases, were used to depolymerise a biopolymer, resulting in a range of chitosans with average molecular weights (Mw) of 6-21 kDa. The depolymerised chitosans obtained from crustaceans and insects were studied, and their antibacterial and antifungal properties were evaluated. The results proved the significance of the chitosan's origin, showing the potential of Hermetia illucens as a new source of low molecular weight chitosan with an improved biological activity.

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.

Obtaining chitin, chitosan and their melanin complexes from insects.

Interest in insects as a source of valuable biologically active substances has significantly increased over the past few years. Insects serve as an alternative source of chitin, which forms up to 40% of their exoskeleton. Chitosan, a deacetylated derivative of chitin, attracts the attention of scientists due to its unique properties (sorption, antimicrobial, film-forming, wound healing). Furthermore, some insect species are unique and can be used to obtain chitin- and chitosan-melanin complexes in the later stages of ontogenesis. Due to the synergistic effect, chitosan and melanin can enhance each other's biological activity, providing a wide range of potential applications.

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.

Chitin and Cellulose Processing in Low-Temperature Electron Beam Plasma.

Polysaccharide processing by means of low-temperature Electron Beam Plasma (EBP) is a promising alternative to the time-consuming and environmentally hazardous chemical hydrolysis in oligosaccharide production. The present paper considers mechanisms of the EBP-stimulated destruction of crab shell chitin, cellulose sulfate, and microcrystalline cellulose, as well as characterization of the produced oligosaccharides. The polysaccharide powders were treated in oxygen EBP for 1-20 min at 40 °C in a mixing reactor placed in the zone of the EBP generation. The chemical structure and molecular mass of the oligosaccharides were analyzed by size exclusion and the reversed phase chromatography, FTIR-spectroscopy, XRD-, and NMR-techniques. The EBP action on original polysaccharides reduces their crystallinity index and polymerization degree. Water-soluble products with lower molecular weight chitooligosaccharides (weight-average molecular mass, Mw = 1000-2000 Da and polydispersity index 2.2) and cellulose oligosaccharides with polymerization degrees 3-10 were obtained. The ¹H-NMR analysis revealed 25-40% deacetylation of the EBP-treated chitin and FTIR-spectroscopy detected an increase of carbonyl- and carboxyl-groups in the oligosaccharides produced. Possible reactions of ß-1,4-glycosidic bonds' destruction due to active oxygen species and high-energy electrons are given.

Preparation and biocompatibility evaluation of pectin and chitosan cryogels for biomedical application.

Today, there is a need for the development of biomaterials with novel properties for biomedical purposes. The biocompatibility of materials is a key factor in determining its possible use in biomedicine. In this study, composite cryogels were obtained based on pectin and chitosan using ionic cryotropic gelation. For cryogel preparation, apple pectin (AP), Heracleum L. pectin (HP), and chitosan samples with different physical and chemical characteristics were used. The properties of pectin-chitosan cryogels were found to depend on the structural features and physicochemical characteristics of the pectin and chitosan within them. The addition of chitosan to cryogels can increase their mechanical strength, cause change in surface morphology, increase the degradation time, and enhance adhesion to biological tissues. Cryogels based on AP were less immunogenic when compared with cryogels from HP. Cryogels based on AP and HP were hemocompatible and the percentage of red blood cells hemolysis was less than 5%. Unlike cryogels based on HP, which exhibited moderate cytotoxicity, cryogels based on AP exhibited light cytotoxicity. Based on the results of low immunogenicity, light cytotoxicity data as well as a low level of hemolysis of composite cryogels based on AP and chitosan are biocompatible and can potentially be used in biomedicine. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 547-556, 2017.

Cross-presentation of lactoferrin encapsulated into chitosan-based nanoparticles.

Induction of CD8+ cytotoxic T-cell response is essential for the protection from intracellular pathogens. It requires major histocompatibility complex class I processing of newly synthesized proteins transported from the cytosolic pathway. Presentation of mature soluble proteins occurs via a cross-presentation (CP) pathway that is much less efficient in the activation of cytotoxic response. Encapsulation of proteins into polymeric nanoparticles (NPs) can modulate the efficacy of antigen CP. In this article, a model antigen lactoferrin (L) was encapsulated into polysaccharide NPs with different physicochemical properties (size, charge, and hydrophobicity) and used as an immunogen. CD8+ or CD4+ associated IgG2a or IgG1 subclasses of L-specific antibodies, respectively, served as a measure of CD8+ versus CD4+ T-cell activation. Among five types of NPs produced, only succinylchitosan-galactomannan (LSG) and succinylchitosan-PEG-chitosan (LSPC) NPs induced a significant IgG2a response. IgG1 production was comparable in all but hydrophobic succinyl-dodecyl-chitosan (LSD) NPs, where it was only marginal. Confocal studies demonstrated that galactomannan-equipped LSG-NPs induced vacuolar type of CP, while positively charged LSPC-NPs were transported mostly via the cytosolic CP pathway.