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In this work, for the first time, films were obtained based on biodegradable polyhydroxyalkanoates (PHAs) - a copolymer of 3-hydroxybutyrate-co-3-hydroxyvalerate P(3HB-co-3HV) and a copolymer of 3-hydroxybutyrate-co-4-hydroxybutyrate P(3HB-co-4HB), containing drugs of different chemical structure and action - antibiotics (ceftazidime, doripenem), antiseptic (chlorhexidine) and tissue regeneration stimulator (Actovegin) for drug delivery and cell culture. Using SEM, AFM and measuring the contact angles of water wetting, it is shown how PHAs composition and loading of films of P(3HB-co-3HV) and P(3HB-co-4HB) with drugs affect the porosity, roughness indicators and adhesive properties of the surface. The constructed films are a depot form of drug, the release of which in vitro is realized for a long time, without burst releases, corresponds to Korsmeyer-Peppas and Higuchi models with diffusion character. Films loaded with antibiotics have pronounced antibacterial activity and suppress the development of pathogens S. aureus and E. coli, without a pronounced negative effect on the adhesion and proliferation of epidermal cells. In the cultures of human keratinocytes HaCaT it was shown that the presence of actovegin, as well as ceftazidime in the films, exhibits a stimulating effect, increasing their number. The results demonstrate the suitability of the depot-films for cellular technologies and are promising for the reconstruction of tissues complicated by infection.

The features of the degradation of the ''green'' plastic poly(3-hydroxybutyrate) [P(3HB)] in the soil of various geographical regions were studied: in red ferralitic soil under tropical conditions (Kerala, India) and in chernozem soil under conditions of a sharply continental climate (Eastern Siberia, Russia). Significant differences in the chemical composition, temperature, and humidity of the studied soils were revealed. The number of bacteria and mycelial fungi in the Siberian chernozem was higher than in the red soil of India, from 2-3 to 10 or more times. The degradation of P(3HB) films in the chernozem occurred faster than in the red soil, which was drier, with a low content of humus and minerals, and fewer microorganisms than the chernozem. The half-life of polymer samples in Siberia and India was 64.8 and 126.4 days, respectively. During degradation, a decrease in the molecular weight and an increase in the degree of crystallinity of polymer samples were revealed, which indicates a more active biodegradation of the amorphous phase of the polymer by soil microorganisms. The primary degraders of the polymer have been isolated and identified, and it has been shown that the complexes of degrading bacteria and fungi in different types of soils did not have common species. Despite the presence of species with pronounced depolymerase activity, the rate of film degradation in red ferralitic soils was slowed down by unfavorable environmental conditions. The obtained results confirm the importance of studying the process of PHA degradation in natural conditions.

Biodegradable polyhydroxyalkanoates, biopolymers of microbiological origin, formed by 3- and 4-hydroxybutyrate monomers P(3HB-co-4HB), were used to obtain nanomembranes loaded with drugs as cell carriers by electrospinning. Resorbable non-woven membranes from P(3HB-co-4HB) loaded with ceftazidime, doripinem, and actovegin have been obtained. The loading of membranes with drugs differently affected the size of fibers and the structure of membranes, and in all cases increased the hydrophilicity of the surface. The release of drugs in vitro was gradual, which corresponded to the Higuchi and Korsmeyer-Peppas models. Antibiotic-loaded membranes showed antibacterial activity against S. aureus and E. coli, in which growth inhibition zones were 41.7 ± 1.1 and 38.6 ± 1.7 mm for ceftazidime and doripinem, respectively. The study of the biological activity of membranes in the NIH 3T3 mouse fibroblast culture based on the results of DAPI and FITC staining of cells, as well as the MTT test, did not reveal a negative effect despite the presence of antibiotics in them. Samples containing actovegin exhibit a stimulating effect on fibroblasts. Biodegradable polyhydroxyalkanoates formed by 3-hydroxybutyrate and 4-hydroxybutyrate monomers provide electrospinning non-woven membranes suitable for long-term delivery of drugs and cultivation of eukaryotic cells, and are promising for the treatment of wound defects complicated by infection.

In this work, delivery systems in the form of microparticles and films containing 1,2,4-trioxolane (ozonide, OZ) based on polyhydroxyalkanoates (PHAs) were developed. Main systems’ characteristics were investigated: the particle yield, average diameter, zeta potential, surface morphology, loading capacity, and drug release profile of microparticles, as well as surface morphology and release profiles of OZ-containing films. PHA-based OZ-loaded microparticles have been found to have satisfactory size, zeta potential, and ozonide loading-release behavior. It was noted that OZ content influenced the surface morphology of obtained systems.

The needs of modern regenerative medicine for biodegradable polymers are wide and varied. Restoration of the viability of the vascular tree is one of the most important components of the preservation of the usefulness of organs and tissues. The creation of vascular implants compatible with blood is an important task of vascular bioengineering. The function of the endothelial layer of the vessel, being largely responsible for the development of thrombotic complications, is of great importance for hemocompatibility. The development of surfaces with specific characteristics of biomaterials that are used in vascular technologies is one of the solutions for their correct endothelialization. Linear polyhydroxyalkanoates (PHAs) are biodegradable structural polymeric materials suitable for obtaining various types of implants and tissue engineering, having a wide range of structural and physicomechanical properties. The use of PHA of various monomeric compositions in endothelial cultivation makes it possible to evaluate the influence of material properties, especially surface characteristics, on the functional state of cells. It has been established that PHA samples with the inclusion of 3-hydroxyhexanoate have optimal characteristics for the formation of a human umbilical vein endothelial cell, HUVEC, monolayer in terms of cell morphology as well as the levels of expression of vinculin and VE-cadherin. The obtained results provide a rationale for the use of PHA copolymers as materials for direct contact with the endothelium in vascular implants.

From a series of biodegradable natural polymers of polyhydroxyalkanoates (PHAs)—poly-3-hydroxybutyrate (P(3HB) and copolymers containing, in addition to 3HB monomers, monomers of 3-hydroxyvalerate (3HV), 3-hydroxyhexanoate (3HHx), and 4-hydroxybutyrate (4HB), with different ratios of monomers poured—solvent casting films and nanomembranes with oriented and non-oriented ultrathin fibers were obtained by electrostatic molding. With the use of SEM, AFM, and measurement of contact angles and energy characteristics, the surface properties and mechanical and biological properties of the polymer products were studied depending on the method of production and the composition of PHAs. It has been shown in cultures of mouse fibroblasts of the NIH 3T3 line and diploid human embryonic cells of the M22 line that elastic films and nanomembranes composed of P(3HB-co-4HB) copolymers have high biocompatibility and provide adhesion, proliferation and preservation of the high physiological activity of cells for up to 7 days. Polymer films, namely oriented and non-oriented nanomembranes coated with type 1 collagen, are positively evaluated as experimental wound dressings in experiments on laboratory animals with model and surgical skin lesions. The results of planimetric measurements of the dynamics of wound healing and analysis of histological sections showed the regeneration of model skin defects in groups of animals using experimental wound dressings from P(3HB-co-4HB) of all types, but most actively when using non-oriented nanomembranes obtained by electrospinning. The study highlights the importance of nonwoven nanomembranes obtained by electrospinning from degradable low-crystalline copolymers P(3HB-co-4HB) in the effectiveness of the skin wound healing process.

The morphotypes of human macrophages (MPh) were studied in the culture on nano-structured biopolymer substrates, made from polyhydroxyalcanoates (PHAs) of five various monomer compositions, followed by the solvent evaporation. Its surface relief, which was further in direct contact with human cells in vitro, was analyzed by atomic force microscopy (AFM) and scanning electron microscopy (SEM). It was shown, that the features of the micro/nano relief depend on the monomeric composition of the polymer substrates. Monocytes (MN) of patients with atherosclerosis and cardiac ischemia, undergoing stenting and conventional anti-atherosclerotic therapy, were harvested prior and after stenting. MN were isolated and cultured, with the transformation into MPh in direct contact with biopolymer culture substrates with different monomer composition and nano-reliefs, and transformed into MPh, in comparison with the same process on standard culture plastic. Sub-populations of cells with characteristic morphology in each phenotypic class were described, and their quantitative ratios for each sample of polymers were counted as an intermediate result in the development of “smart” material for cardiovascular devices.The results obtained allow us to assume, that the processes of MPh differentiation and polarization in vitro depend not only on the features of the micro/nano relief of biopolymer substrates, but also on the initial state of MN in vivo and general response of patients. Keywords: Monocytes, Macrophages, Cell morphology, Polyhydroxyalkanoates, Atherosclerosis, Intravascular stenting

To increase the availability and expand the raw material base, the production of polyhydroxyalkanoates (PHA) by the wild strain Cupriavidus necator B-10646 on hydrolysates of sugar beet molasses was studied. The hydrolysis of molasses was carried out using β-fructofuranosidase, which provides a high conversion of sucrose (88.9%) to hexoses. We showed the necessity to adjust the chemical composition of molasses hydrolysate to balance with the physiological needs of C. necator B-10646 and reduce excess sugars and nitrogen and eliminate phosphorus deficiency. The modes of cultivation of bacteria on diluted hydrolyzed molasses with the controlled feeding of phosphorus and glucose were implemented. Depending on the ratio of sugars introduced into the bacterial culture due to the molasses hydrolysate and glucose additions, the bacterial biomass concentration was obtained from 20–25 to 80–85 g/L with a polymer content up to 80%. The hydrolysates of molasses containing trace amounts of propionate and valerate were used to synthesize a P(3HB-co-3HV) copolymer with minor inclusions of 3-hydroxyvlaerate monomers. The introduction of precursors into the medium ensured the synthesis of copolymers with reduced values of the degree of crystallinity, containing, in addition to 3HB, monomers 3HB, 4HB, or 3HHx in an amount of 12–16 mol.%.

The synthesis of bacterial cellulose (BC) by Komagataeibacter xylinus strain B-12068 was investigated on various C-substrates, under submerged conditions with stirring and in static surface cultures. We implemented the synthesis of BC on glycerol, glucose, beet molasses, sprat oil, and a mixture of glucose with sunflower oil. The most productive process was obtained during the production of inoculum in submerged culture and subsequent growth of large BC films (up to 0.2 m2 and more) in a static surface culture. The highest productivity of the BC synthesis process was obtained with the growth of bacteria on molasses and glycerol, 1.20 and 1.45 g/L per day, respectively. We obtained BC composites with silver nanoparticles (BC/AgNPs) and antibacterial drugs (chlorhexidine, baneocin, cefotaxime, and doripenem), and investigated the structure, physicochemical, and mechanical properties of composites. The disc-diffusion method showed pronounced antibacterial activity of BC composites against E. coli ATCC 25922 and S. aureus ATCC 25923.

Bacterial cellulose (BC) is widely used in medicine as a dressing material due to its good biological properties – high biocompatibility, low adhesion and the ability to absorb wound exudate. The BC does not have antimicrobial activity itself, which limits the use of products in infected wounds, and also treatment of wounds in hospitals. A method for producing a two-component composite material based on bacterial cellulose (BC) synthesized in culture of Komagataeibacter xylinus B-12068 with silver nanoparticles [BC/AgNps], by hydrothermal synthesis of AgNO3 in the layer of BC at different temperatures and concentrations of AgNO3, is proposed. The antibacterial activity of BC/AgNps samples against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, and Staphylococcus aureus has been confirmed in vitro with the disc diffusion method. It is shown, that the antibacterial activity of samples is mostly expressed in cultures of P. aeruginosa and S. aureus

Non-woven experimental samples of wound dressings, made from natural degradable polymers, were produced and investigated. High-porous membranes were obtained by electrostatic formation of solutions of copolymers of 3-hydroxybutyrate-co-4-hydroxybutyrate [P(3HB-co-4HB)]. The set of pre-clinical assessments was performed, included testing of membranes in vitro and in vivo. Study was carried out by staining of blood cells with vital fluorochromic dyes, followed by analysis of activation of platelet adhesion, the ratio of the main morphological types of erythrocytes, measurements of changes of hemoglobin content in human blood, and hemolytic activity. The general toxic effect was investigated in experiment with white mice after the intraabdominal injection of water extracts of membranes. It was established, that direct contact with membranes had no negative influence towards cells of blood and showed no toxic effects in animal experiments

The paper presents the results of clinical trials of wound dressing based on cellulose in comparison with wound dressing “Gidrokoll” in patients with acute purulent soft tissue infections. It is proved that the wound covering based on cellulose more efficient, contribute to the rapid and intensive purification and epithelialization of infected wounds

The paper describes research of biomedical applications of experimental polymer devices prepared from polyhydroxyalkanoates (PHAs) in the Institute of Biophysics SB RAS and Siberian Federal University (Krasnoyarsk, Russia) between 2000 and 2012. The high-purity PHA specimens were investigated and then used to prepare surgical sutures, 2D and 3D dense and porous matrices, fully resorbable tubular stents, and polymer coatings. The polymer devices that differed in their shape and mass were introduced into muscles, bones, blood vessels, and internal organs in order to investigate the response of tissues with different structure to the implants. The studies showed that PHAs were highly biocompatible with different tissues and their implantation for extended periods of time did not induce adverse responses of the blood system, cells, tissues, or the entire organism. The authors proved that PHAs can be effectively used as prosthetic implants and bone-replacing implants

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The paper presents the analysis of the results of application of monofilament suture based on polyhydroxyalkanoates (PHAs) in comparison with suture materials “Vicryl” and “Kaprofil” in experiment with Chinchilla rabbits. It was proved that the use of the PHA-based suture is accompanied by minimally pronounced local tissue response

Increasing number of industrial and household injuries and burns lead to the necessity of research and development of new materials and resources for damaged skin regeneration. In this study results of experimental investigation of wound dressing made of natural degradable polyesters polyhydroxyalkanoates (PHAs) using electrospinning are presented. Studies on laboratory animals with model skin burns showed that the developed PHA membranes provide regeneration of the skin after 7 days and it is comparable with the control collagen dressings used in clinical practice. Developed nonwoven membranes may be used as the equivalent of collagen dressings in the treatment of II degree burns

The present review summarizes the literature data on the degradation dynamics of polyhydroxyalkanoates (PHAs) in vivo and the authors’ results obtained over a more than ten years of research, which included implantation of experimental polymer items shaped as microparticles, films, filaments, and 3D constructs subcutaneously, intramuscularly, and into internal organs of animals for long periods of time. The study shows that PHA degradation is determined by the chemical structure of the polymer, the shape of the implant, and implantation site; PHA degradation is slow, occurring via humoral and cellular pathways, usually beginning on the surface of the implants, with no local defects developing or strength decreasing dramatically. PHA biodegradation involves macrophages and foreign body giant cells. PHA products are resistant to the effects of biological media and are capable of functioning in vivo for between several months and one year or longer

The study investigated the resorbable polymeric 3D plates based on polyesters of hydroxybutyric acid – poly-3-hydroxybutyrate (P(3HB)) in comparison with the commercial preparation Ltd. “Plastic” (allocartilage) for regeneration of bottom wall orbit defect of rabbits. The studied 3D plates don’t induce inflammatory reactions after implantation. They can perform function of insulating elements and have osteoprotective properties; they contribute to shortening of the period of injured bone tissue restoration, and complete closure of defect for 60 days

These studies suggest that manipulation of the structural and surface characteristics of synthetic scaffold materials can affect macrophage phenotype. Specifically, some of these manipulations appear to alter the macrophage response and are also associated with improved outcomes. Another manner in which biomaterials can be tailored to promote shifts in macrophage phenotype is through the use of biologically active molecules such as growth factors and cytokines

The possibility of using the spray drying method for the construction of poly-3-hydroxybutyrate (P3HB) based microparticles was studied. The dependence of characteristics of microparticles (yield, average diameter, zeta-potential) from processing parameters (temperature at the inlet of the system, the feed rate of polymer solution and concentration of the polymer solution) was found. It is shown that zeta potential of microparticles was influenced by concentration of the polymer solution, and the average diameter was determined by temperature of the inlet. Also, conditions of obtaining P3HB microparticles were optimized to minimize polymer loss (approximately 10 %)

Reconstruction and optimization of the process of healing of bone tissue defects using new materials and technologies is an urgent problem in medicine. This is due to a high level of orthopedic traumas and prevalence of dental and other socially significant diseases of bone tissue, leading to the dramatic incapacitation of population and accompanied by huge financial costs. This review analyzes the published data on osteoplastic materials for bone reconstructive surgery from traditional allo- and xenografts, titanium alloys, calcium phosphates and bioceramics to modern materials based on biocompatible and biodegradable polymers; the results obtained by the SFU Laboratory of New Biomaterials Biotechnology are presented

The effect of surface relief profiles of alkanoate-based bionanofilms to the monocyte-macrophages (MN-MPhs) from peripheral blood of patients with atherosclerosis was studied in vitro. Patients were subjected to coronary stenting. Cell morphology and phenotype (expression of CD antigens, levels of production of marker cytokines) in vitro were analyzed before and after the installation of stents. It was shown, that the mean square roughness (Rq) of the bionanofilms determined the variability of cell morphology, CD antigens spectraand activity of production interleukins-6 and -10. Also, it was revealed, that the “activity” of the surface topography of biopolymer substrates depends on the functional state of MNs, isolated in different time points: Before and after stenting the ratios of cell morphotypes and production of cytokines in MN-MPhs differed significantly.

Results of construction and investigation of ultrafine fibers (UFF) made of resorbable poly(3 hydroxybutyrate) by electrostatic formation are presented in this study. Influence of concentration of polymer solutions on fiber diameters and physical-mechanical properties of nonwoven fabric (NF) was investigated. While concentration of polymer solutions increased from 2 to 8 % elongation at break of NF increased from 4,5 to 10,6 % and Young’s modulus decreased from 1,16 to 0,3 GPa. Murine fibroblasts NIH 3T3 were seeded on NF. Using DAPI staining and MTT assay UFF samples were estimated as good cell supporting carriers.

Transdermal drug delivery systems have become increasingly sophisticated over time. However, the greatest limitation for developing an effective drug delivery system is the highly impermeable outermost layer of the skin called the stratum corneum. Therefore, materials penetrating the skin must be of low molecular weight, and lipophilic. There are many techniques to safely pass the stratum corneum and one of the promising method of transdermal drug delivery is the use of micro and nano sized particles. The aim of this study was to develop three different micro and nano sized carriers to study their skin penetration and to judge their effectiveness within the skin. Polymeric micro/ nanocapsules carrying a fluorescent dye Nile Red, were prepared using poly(3-hydroxybutyrate-co 3-hydroxyvalerate) (PHBV) (5 %mol. hydroxyvalerate). The in vivo transdermal permeation of PHBV micro/nanoparticles were studied using a mouse model. According to the particle size analysis with Master Sizer and Zeta Potential Measurement System, the PHBV micro/nanocapsules were 1.9 µm, 426 nm and 166 nm in diameter. The particles were applied to healthy skin of the dorsal region of BALB/c mice. Penetration of the particles was determined by GC-MS analysis of the skin for PHBV. Scanning electron microscope (SEM) imaging was used to study their morphology. GC-MS results showed that the capsules penetrated into the skin in relation with their particle size, despite the highly impermeable outer skin layer. However, histology cross-section revealed that uncompromised skin could not penetrate; the transport of the polymeric particle was clearly impeded by the stratum corneum. It was thus shown in this study that control of penetration depth, and therefore, the target size within into the skin is possible by varying the size of the drug carrying nanocapsules.

The paper reports analysis of results of the first limited clinical trials of high technology medical devices prepared using polyhydroxyalkanoates (PHAs) – degradable microbial biopolymers: PHA coated mesh stents for surgical repair of inguinal hernia, endobiliary stents for treatment of the patients with obstructive jaundice caused by extrahepatic biliary obstruction due to different reasons, and biodegradable monofilament sutures for closing operative wounds on the abdominal wall. The devices were compared with their commercial Russian and foreign analogs widely used clinically. The choice of the device influenced the incidence of early and delayed postsurgical complications and laboratory and clinical parameters. The study proves the suitability of PHA-based devices for reconstructive surgery.

Microbial polyhydroxyalkanoates (PHAs) have long been studied due to their potential for replacement of petroleum-based plastic. The study addresses the effect of different conditions of carbon nutrition on synthesis of polyhydroxyalkanoates by the bacterium Ralstonia eutropha. First and foremost, PHAs are carbon storage compounds for many organisms. There are still many aspects of the physiology of PHA accumulation and degradation that are still not understood. Measurements of key P3HBrelated enzyme activities throughout cell growth reveal correlations of acetoacetyl-CoA reductase and synthase enzyme activity maxima with P3HB biosynthesis. The investigation addressed kinetic parameters of growth and accumulation of polyhydroxyalkanoates and gas exchange parameters of the culture of the CO-resistant strain of the hydrogen bacteria Ralstonia eutropha B 5786 cultivated on synthesis gas - a product of gasification of brown coals. The results were compared with those obtained by growing the bacteria on electrolytic hydrogen and it was concluded that synthesis gas can be successfully used to produce PHAs. In experiments with wild-type strain it has been first found that under mixotrophic growth conditions - CO2 + co-substrate (alcanoic acids) - bacteria can synthesize multi-component PHAs, consisting of short- and medium-chain-length monomers with carbon chains containing 4 to 8 atoms. It has been shown that PHA composition is determined by the type of the co-substrate. Fatty acids with odd number of carbons induce bacteria to synthesize multi-component PHAs with 3-hydroxybutyrate, 3-hydroxyvalerate, and 3-hydroxyhexanoate as major monomers and 3-hydroxyheptanoate and 3-hydroxyoctanoate as minor, occasionally occurring, ones. Fatty acids with even number of carbons induce synthesis of not only their respective monomers (3-hydroxyhexanoate and 3-hydroxyoctanoate) but also 3-hydroxyvalerate, making possible synthesis of four-component PHAs, containing 3-hydroxybutyrate and 3-hydroxyhexanoate as major components. A family of short- and medium-chain-length four- and five-component PHAs has been synthesized and their physicochemical and biomedical-properties examined.

Resorbable polymer of hydroxybutyric acid labeled with 14C was used to prepare microparticles (diameter smaller than 3.8 μm ) that were then injected to laboratory animals (Wistar rats) via the tail vein, without causing any adverse effects on growth and development of the animals or altering the macroscopic and microscopic structure of the tissues of internal organs. Examination of the distribution of microparticles among the internal organs and the dynamics of accumulation of carbon-containing polymer degradation products in internal organs showed that the main targets for microparticles were tissues of the liver, kidneys, and spleen. The most rapid degradation of the polymer of microparticles occurred in the spleen and liver. The presence of high molecular weight polymer registered in internal organs suggested that the microparticles remained undecomposed and that the PHB microparticles could function in vivo for extended periods of time (up to 12 weeks).

We investigated the process of production of oriented fibers by melt spinning of biodegradable hydroxybutyrate and hydroxyvalerate (PHBV) copolymers. It was revealed that temperature has a large effect on the stability of extrusion and quality of fibers, and the optimal paramter values were found. We showed that the diameter of fibers and orientation degree depend on the spinning rate which can vary in a wide range (from 2-3 to 40 m/min), not affecting the quality of fibers. We studied the extensibility and deformability of fibers at different temperatures and found the conditions allowing to produce fibers with the diameter of 0.17-0.20 mm with high mechanical characteristics (strength 306 MPa, elasticity modulus 3 GPa, elongation at break 24%), that were stable during the 3 month period of observation. We obtained and analyzed extension curves on which three areas were fixed: linear elasticity, visible deformation, totally nonreversible deformation, which is typical for the elongation curves of polymer elastomers. It was stated that the produced fibers have high mechanical stability at static and cyclic loading conditions (up to 100 MPa); in the area of linear elasticity the deformation of fibers is practically totally reversible after 1000 cycles of loading.

Microspheres have been prepared from the resorbable linear polyester of β-hydroxybutyric acid (polyhydroxybutyrate, PHB) by the solvent evaporation technique and investigated in vitro and in vivo. Biocompatibility of the microspheres has been proved in tests in the culture of mouse fibroblast cell line NIH 3Т3 and in experiments on intramuscular implantation of the microspheres to Wistar rats for 3 months. Tissue response to the implantation of polymeric microspheres has been found to consist in a mild inflammatory reaction, pronounced macrophage infiltration that increases over time, involving mono- and poly-nuclear foreign body giant cells that resorb the polymeric matrix. No fibrous capsules were formed around polymeric microparticles; neither necrosis nor any other adverse morphological changes and tissue transformation in response to the implantation of the PHB microparticles were recorded. The results of the study suggest that polyhydroxybutyrate is a good candidate for fabricating prolonged-action drugs in the form of microparticles intended for intramuscular injection.