Osteogenic Potential and Long-Term Enzymatic Biodegradation of PHB-based Scaffolds with Composite Magnetic Nanofillers in a Magnetic Field.

Millions of people worldwide suffer from musculoskeletal damage, thus using the largest proportion of rehabilitation services. The limited self-regenerative capacity of bone and cartilage tissues necessitates the development of functional biomaterials. Magnetoactive materials are a promising solution due to clinical safety and deep tissue penetration of magnetic fields (MFs) without attenuation and tissue heating. Herein, electrospun microfibrous scaffolds were developed based on piezoelectric poly(3-hydroxybutyrate) (PHB) and composite magnetic nanofillers [magnetite with graphene oxide (GO) or reduced GO]. The scaffolds' morphology, structure, mechanical properties, surface potential, and piezoelectric response were systematically investigated. Furthermore, a complex mechanism of enzymatic biodegradation of these scaffolds is proposed that involves (i) a release of polymer crystallites, (ii) crystallization of the amorphous phase, and (iii) dissolution of the amorphous phase. Incorporation of Fe3O4, Fe3O4-GO, or Fe3O4-rGO accelerated the biodegradation of PHB scaffolds owing to pores on the surface of composite fibers and the enlarged content of polymer amorphous phase in the composite scaffolds. Six-month biodegradation caused a reduction in surface potential (1.5-fold) and in a vertical piezoresponse (3.5-fold) of the Fe3O4-GO scaffold because of a decrease in the PHB ß-phase content. In vitro assays in the absence of an MF showed a significantly more pronounced mesenchymal stem cell proliferation on composite magnetic scaffolds compared to the neat scaffold, whereas in an MF (68 mT, 0.67 Hz), cell proliferation was not statistically significantly different when all the studied scaffolds were compared. The PHB/Fe3O4-GO scaffold was implanted into femur bone defects in rats, resulting in successful bone repair after nonperiodic magnetic stimulation (200 mT, 0.04 Hz) owing to a synergetic influence of increased surface roughness, the presence of hydrophilic groups near the surface, and magnetoelectric and magnetomechanical effects of the material.

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.

Magnetoactive Composite Conduits Based on Poly(3-hydroxybutyrate) and Magnetite Nanoparticles for Repair of Peripheral Nerve Injury.

Peripheral nerve injury poses a threat to the mobility and sensitivity of a nerve, thereby leading to permanent function loss due to the low regenerative capacity of mature neurons. To date, the most widely clinically applied approach to bridging nerve injuries is autologous nerve grafting, which faces challenges such as donor site morbidity, donor shortages, and the necessity of a second surgery. An effective therapeutic strategy is urgently needed worldwide to overcome the current limitations. Herein, a magnetic nerve guidance conduit (NGC) based on biocompatible biodegradable poly(3-hydroxybutyrate) (PHB) and 8 wt % of magnetite nanoparticles modified by citric acid (Fe3O4-CA) was fabricated by electrospinning. The crystalline structure of NGCs was studied by X-ray diffraction, which indicated an enlarged ß-phase of PHB in the composite conduit compared to a pure PHB conduit. Tensile tests revealed greater ductility of PHB/Fe3O4-CA: the composite conduit has Young's modulus of 221 ± 52 MPa and an elongation at break of 28.6 ± 2.9%, comparable to clinical materials. Saturation magnetization (σs) of Fe3O4-CA and PHB/Fe3O4-CA is 61.88 ± 0.29 and 7.44 ± 0.07 emu/g, respectively. The water contact angle of the PHB/Fe3O4-CA conduit is lower as compared to pure PHB, while surface free energy (σ) is significantly higher, which was attributed to higher surface roughness and an amorphous phase as well as possible PHB/Fe3O4-CA interface interactions. In vitro, the conduits supported the proliferation of rat mesenchymal stem cells (rMSCs) and SH-SY5Y cells in a low-frequency magnetic field (0.67 Hz, 68 mT). In vivo, the conduits were used to bridge damaged sciatic nerves in rats; pure PHB and composite PHB/Fe3O4-CA conduits did not cause acute inflammation and performed a barrier function, which promotes nerve regeneration. Thus, these conduits are promising as implants for the regeneration of peripheral nerves.

Changes in the Gut Microbiota Composition during Implantation of Composite Scaffolds Based on Poly(3-hydroxybutyrate) and Alginate on the Large-Intestine Wall.

The development of biopolymer scaffolds for intestine regeneration is one of the most actively developing areas in tissue engineering. However, intestinal regenerative processes after scaffold implantation depend on the activity of the intestinal microbial community that is in close symbiosis with intestinal epithelial cells. In this work, we study the impact of different scaffolds based on biocompatible poly(3-hydroxybutyrate) (PHB) and alginate (ALG) as well as PHB/ALG scaffolds seeded with probiotic bacteria on the composition of gut microbiota of Wistar rats. Implantation of PHB/ALG scaffolds on the large-intestine wall to close its injury showed that alpha diversity of the gut microbiota was not reduced in rats implanted with different PHB/ALG scaffolds except for the PHB/ALG scaffolds with the inclusion of Lactobacillus spheres (PHB/ALG-L). The composition of the gut microbiota of rats implanted with PHB/ALG scaffolds with probiotic bacteria or in simultaneous use of an antimicrobial agent (PHB/ALG-AB) differed significantly from other experimental groups. All rats with implanted scaffolds demonstrated shifts in the composition of the gut microbiota by individual operational taxonomic units. The PHB/ALG-AB construct led to increased abundance of butyrate-producing bacteria: Ileibacterium sp. dominated in rats with implanted PHB/ALG-L and Lactobacillus sp. and Bifidobacterium sp. dominated in the control group. In addition, the PHB/ALG scaffolds had a favourable effect on the growth of commensal bacteria. Thus, the effect of implantation of the PHB/ALG scaffold compared to other scaffolds on the composition of the gut microbiota was closest to the control variant, which may demonstrate the biocompatibility of this device with the microbiota.

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.

Poly(3-hydroxybutyrate) 3D-Scaffold-Conduit for Guided Tissue Sprouting.

Scaffold biocompatibility remains an urgent problem in tissue engineering. An especially interesting problem is guided cell intergrowth and tissue sprouting using a porous scaffold with a special design. Two types of structures were obtained from poly(3-hydroxybutyrate) (PHB) using a salt leaching technique. In flat scaffolds (scaffold-1), one side was more porous (pore size 100-300 µm), while the other side was smoother (pore size 10-50 µm). Such scaffolds are suitable for the in vitro cultivation of rat mesenchymal stem cells and 3T3 fibroblasts, and, upon subcutaneous implantation to older rats, they cause moderate inflammation and the formation of a fibrous capsule. Scaffold-2s are homogeneous volumetric hard sponges (pore size 30-300 µm) with more structured pores. They were suitable for the in vitro culturing of 3T3 fibroblasts. Scaffold-2s were used to manufacture a conduit from the PHB/PHBV tube with scaffold-2 as a filler. The subcutaneous implantation of such conduits to older rats resulted in gradual soft connective tissue sprouting through the filler material of the scaffold-2 without any visible inflammatory processes. Thus, scaffold-2 can be used as a guide for connective tissue sprouting. The obtained data are advanced studies for reconstructive surgery and tissue engineering application for the elderly patients.

Adhesion of Escherichia coli and Lactobacillus fermentum to Films and Electrospun Fibrous Scaffolds from Composites of Poly(3-hydroxybutyrate) with Magnetic Nanoparticles in a Low-Frequency Magnetic Field.

The ability of materials to adhere bacteria on their surface is one of the most important aspects of their development and application in bioengineering. In this work, the effect of the properties of films and electrospun scaffolds made of composite materials based on biosynthetic poly(3-hydroxybutyrate) (PHB) with the addition of magnetite nanoparticles (MNP) and their complex with graphene oxide (MNP/GO) on the adhesion of E. coli and L. fermentum under the influence of a low-frequency magnetic field and without it was investigated. The physicochemical properties (crystallinity; surface hydrophilicity) of the materials were investigated by X-ray structural analysis, differential scanning calorimetry and "drop deposition" methods, and their surface topography was studied by scanning electron and atomic force microscopy. Crystal violet staining made it possible to reveal differences in the surface charge value and to study the adhesion of bacteria to it. It was shown that the differences in physicochemical properties of materials and the manifestation of magnetoactive properties of materials have a multidirectional effect on the adhesion of model microorganisms. Compared to pure PHB, the adhesion of E. coli to PHB-MNP/GO, and for L. fermentum to both composite materials, was higher. In the magnetic field, the adhesion of E. coli increased markedly compared to PHB-MNP/GO, whereas the effect on the adhesion of L. fermentum was reversed and was only evident in samples with PHB-MNP. Thus, the resultant factors enhancing and impairing the substrate binding of Gram-negative E. coli and Gram-positive L. fermentum turned out to be multidirectional, as they probably have different sensitivity to them. The results obtained will allow for the development of materials with externally controlled adhesion of bacteria to them for biotechnology and medicine.

Electrospun Magnetic Composite Poly-3-hydroxybutyrate/Magnetite Scaffolds for Biomedical Applications: Composition, Structure, Magnetic Properties, and Biological Performance.

Magnetically responsive composite polymer scaffolds have good potential for a variety of biomedical applications. In this work, electrospun composite scaffolds made of polyhydroxybutyrate (PHB) and magnetite (Fe3O4) particles (MPs) were studied before and after degradation in either PBS or a lipase solution. MPs of different sizes with high saturation magnetization were synthesized by the coprecipitation method followed by coating with citric acid (CA). Nanosized MPs were prone to magnetite-maghemite phase transformation during scaffold fabrication, as revealed by Raman spectroscopy; however, for CA-functionalized nanoparticles, the main phase was found to be magnetite, with some traces of maghemite. Submicron MPs were resistant to the magnetite-maghemite phase transformation. MPs did not significantly affect the morphology and diameter of PHB fibers. The scaffolds containing CA-coated MPs lost 0.3 or 0.2% of mass in the lipase solution and PBS, respectively, whereas scaffolds doped with unmodified MPs showed no mass changes after 1 month of incubation in either medium. In all electrospun scaffolds, no alterations of the fiber morphology were observed. Possible mechanisms of the crystalline-lamellar-structure changes in hybrid PHB/Fe3O4 scaffolds during hydrolytic and enzymatic degradation are proposed. It was revealed that particle size and particle surface functionalization affect the mechanical properties of the hybrid scaffolds. The addition of unmodified MPs increased scaffolds' ultimate strength but reduced elongation at break after the biodegradation, whereas simultaneous increases in both parameters were observed for composite scaffolds doped with CA-coated MPs. The highest saturation magnetization─higher than that published in the literature─was registered for composite PHB scaffolds doped with submicron MPs. All PHB scaffolds proved to be biocompatible, and the ones doped with nanosized MPs yielded faster proliferation of rat mesenchymal stem cells. In addition, all electrospun scaffolds were able to support angiogenesis in vivo at 30 days after implantation in Wistar rats.

Honeycomb-Structured Porous Films from Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyvalerate): Physicochemical Characterization and Mesenchymal Stem Cells Behavior.

Surface morphology affects cell attachment and proliferation. In this research, different films made of biodegradable polymers, poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-co-HV), containing different molecular weights, with microstructured surfaces were investigated. Two methods were used to obtain patterned films-water-assisted self-assembly ("breath figure") and spin-coating techniques. The water-assisted technique made it possible to obtain porous films with a self-assembled pore structure, which is dependent on the monomer composition of a polymer along with its molecular weight and the technique parameters (distance from the nozzle, volume, and polymer concentration in working solution). Their pore morphologies were evaluated and their hydrophobicity was examined. Mesenchymal stem cells (MSCs) isolated from bone marrow were cultivated on a porous film surface. MSCs' attachment differed markedly depending on surface morphology. On strip-formed stamp films, MSCs elongated along the structure, however, they interacted with a larger area of film surface. The honeycomb films and column type films did not set the direction of extrusion, but cell flattening depended on structure topography. Thus, stem cells can "feel" the various surface morphologies of self-assembled honeycomb films and change their behavior depending on it.

Competitive Biosynthesis of Bacterial Alginate Using Azotobacter vinelandii 12 for Tissue Engineering Applications.

This study investigated the effect of various cultivation conditions (sucrose/phosphate concentrations, aeration level) on alginate biosynthesis using the bacterial producing strain Azotobacter vinelandii 12 by the full factorial design (FFD) method and physicochemical properties (e.g., rheological properties) of the produced bacterial alginate. We demonstrated experimentally the applicability of bacterial alginate for tissue engineering (the cytotoxicity testing using mesenchymal stem cells (MSCs)). The isolated synthesis of high molecular weight (Mw) capsular alginate with a high level of acetylation (25%) was achieved by FFD method under a low sucrose concentration, an increased phosphate concentration, and a high aeration level. Testing the viscoelastic properties and cytotoxicity showed that bacterial alginate with a maximal Mw (574 kDa) formed the densest hydrogels (which demonstrated relatively low cytotoxicity for MSCs in contrast to bacterial alginate with low Mw). The obtained data have shown promising prospects in controlled biosynthesis of bacterial alginate with different physicochemical characteristics for various biomedical applications including tissue engineering.

Poly(3-hydroxybutyrate)/hydroxyapatite/alginate scaffolds seeded with mesenchymal stem cells enhance the regeneration of critical-sized bone defect.

A critical-sized calvarial defect in rats is employed to reveal the osteoinductive properties of biomaterials. In this study, we investigate the osteogenic efficiency of hybrid scaffolds based on composites of a biodegradable and biocompatible polymer, poly(3-hydroxybutyrate) (PHB) with hydroxyapatite (HA) filled with alginate (ALG) hydrogel containing mesenchymal stem cells (MSCs) on the regeneration of the critical-sized radial defect of the parietal bone in rats. The scaffolds based on PHB and PHB/HA with desired shapes were prepared by two-stage salt leaching technique using a mold obtained by three-dimensional printing. To obtain PHB/HA/ALG/MSC scaffolds seeded with MSCs, the scaffolds were filled with ALG hydrogel containing MSCs; acellular PHB/ALG and PHB/ALG filled with empty ALG hydrogel were prepared for comparison. The produced scaffolds have high porosity and irregular interconnected pore structure. PHB/HA scaffolds supported MSC growth and induced cell osteogenic differentiation in a regular medium in vitro that was manifested by an increase in ALP activity and expression of the CD45 phenotype marker. The data of computed tomography and histological studies showed 94% and 92%, respectively, regeneration of critical-sized calvarial bone defect in vivo at 28th day after implantation of MSC-seeded PHB/HA/ALG/MSC scaffolds with 3.6 times higher formation of the main amount of bone tissue at 22-28 days in comparison with acellular PHB/HA/ALG scaffolds that was shown at the first time by fluorescent microscopy using the original technique of intraperitoneal administration of fluorescent dyes to living postoperative rats. The obtained in vivo results can be associated with the MSC-friendly microstructure and in vitro osteogenic properties of PHB/HA base-scaffolds. Thus, the obtained data demonstrate the potential of MSCs encapsulated in the bioactive biopolymer/mineral/hydrogel scaffold to improve the bone regeneration process in critical-sized bone defects.

Comparative Structure-Property Characterization of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate)s Films under Hydrolytic and Enzymatic Degradation: Finding a Transition Point in 3-Hydroxyvalerate Content.

The hydrolytic and enzymatic degradation of polymer films of poly(3-hydroxybutyrate) (PHB) of different molecular mass and its copolymers with 3-hydroxyvalerate (PHBV) of different 3-hydroxyvalerate (3-HV) content and molecular mass, 3-hydroxy-4-methylvalerate (PHB4MV), and polyethylene glycol (PHBV-PEG) produced by the Azotobacter chroococcum 7B by controlled biosynthesis technique were studied under in vitro model conditions. The changes in the physicochemical properties of the polymers during their in vitro degradation in the pancreatic lipase solution and in phosphate-buffered saline for a long time (183 days) were investigated using different analytical techniques. A mathematical model was used to analyze the kinetics of hydrolytic degradation of poly(3-hydroxyaklannoate)s by not autocatalytic and autocatalytic hydrolysis mechanisms. It was also shown that the degree of crystallinity of some polymers changes differently during degradation in vitro. The total mass of the films decreased slightly up to 8-9% (for the high-molecular weight PHBV with the 3-HV content 17.6% and 9%), in contrast to the copolymer molecular mass, the decrease of which reached 80%. The contact angle for all copolymers after the enzymatic degradation decreased by an average value of 23% compared to 17% after the hydrolytic degradation. Young's modulus increased up to 2-fold. It was shown that the effect of autocatalysis was observed during enzymatic degradation, while autocatalysis was not available during hydrolytic degradation. During hydrolytic and enzymatic degradation in vitro, it was found that PHBV, containing 5.7-5.9 mol.% 3-HV and having about 50% crystallinity degree, presents critical content, beyond which the structural and mechanical properties of the copolymer have essentially changed. The obtained results could be applicable to biomedical polymer systems and food packaging materials.

The Growth of 3T3 Fibroblasts on PHB, PLA and PHB/PLA Blend Films at Different Stages of Their Biodegradation In Vitro.

Over the past century there was a significant development and extensive application of biodegradable and biocompatible polymers for their biomedical applications. This research investigates the dynamic change in properties of biodegradable polymers: poly(3-hydroxybutyrate (PHB), poly-l-lactide (PLA), and their 50:50 blend (PHB/PLA)) during their hydrolytic non-enzymatic (in phosphate buffered saline (PBS), at pH = 7.4, 37 °C) and enzymatic degradation (in PBS supplemented with 0.25 mg/mL pancreatic lipase). 3T3 fibroblast proliferation on the polymer films experiencing different degradation durations was also studied. Enzymatic degradation significantly accelerated the degradation rate of polymers compared to non-enzymatic hydrolytic degradation, whereas the seeding of 3T3 cells on the polymer films accelerated only the PLA molecular weight loss. Surprisingly, the immiscible nature of PHB/PLA blend (showed by differential scanning calorimetry) led to a slower and more uniform enzymatic degradation in comparison with pure polymers, PHB and PLA, which displayed a two-stage degradation process. PHB/PLA blend also displayed relatively stable cell viability on films upon exposure to degradation of different durations, which was associated with the uneven distribution of cells on polymer films. Thus, the obtained data are of great benefit for designing biodegradable scaffolds based on polymer blends for tissue engineering.

Evaluation of eliciting activity of peptidil prolyl cys/trans isomerase from Pseudonomas fluorescens encapsulated in sodium alginate regarding plant resistance to viral and fungal pahogens.

Use of chemical pesticides poses a threat for environment and human health, so green technologies of crop protection are of high demand. Some microbial proteins able to activate plant defense mechanisms and prevent the development of resistance in plant pathogens, may be good alternative to chemicals, but practical use of such elicitors is limited due to need to protect them against adverse environment prior their delivery to target receptors of plant cells. In this study we examined a possibility to encapsulate heat resistant FKBP-type peptidyl prolyl cis-trans isomerase (PPIase) from Pseudomonas fluorescens, which possesses a significant eliciting activity in relation to a range of plant pathogens, in sodium alginate microparticles and evaluated the stability of resulted complex under long-term UV irradiation and in the presence of proteinase K, as well as its eliciting activity in three different "plant-pathogen" models comparing to that of free PPIase. The obtained PPIase-containing microparticles consisted of 70% of sodium alginate, 20% of bovine serum albumin, and 10% of PPIase. In contrast to free PPIase, which lost its eliciting properties after 8-h UV treatment, encapsulated PPIase kept its eliciting ability unchanged; after being exposed to proteinase K, its eliciting ability twice exceeded that of free PPIase. Using "tobacco-TMV", "tobacco-Alternaria longipes", and "wheat-Stagonospora nodorum" model systems, we showed that encapsulation process did not influence on the eliciting activity of PPIase. In the case of the "wheat-S. nodorum" model system, we also observed a significant eliciting activity of alginate-albumin complex and almost doubled activity of encapsulated PPIase as compared to the free PPIase. As far as we know, this is the first observation of a synergistic interaction between alginate and other compound possessing any bioactive properties. The results of the study show some prospects for a PPIase use in agriculture.

Preclinical Toxicity of Paclitaxel Biopolymer Formulation.

BACKGROUND: Poly(hydroxyalkanoates) (PHA) have recently attracted increasing attention due to their biodegradability and high biocompatibility, which makes them suitable for the development of new prolong drug formulations. OBJECTIVE: A preclinical toxicology study of paclitaxel biopolymer formulation (PBF) (paclitaxel-loaded poly(3- hydroxybutyrate) (PHB) microparticles) was done in order to assess its safety and to forecast side and toxic effects in a clinical study on patients. METHOD: PHB microparticles loaded with antitumor cytostatic drug PTX were obtained by spray-drying method using Nano Spray Dryer B-90. The comprehensive study of cytotoxicity (on bone marrow stem cells), acute and chronic toxicity, allergenic and pyrogenic properties, histological investigation (in mice, rats and rabbits) of obtained PBF was carried out. RESULTS: The acute toxicity study showed that PBF is much less toxic in equivalent PTX-content doses than PTX in conventional formulation when administered intraperitoneally to mice and rats. However, the chronic toxicity study showed that at intraperitoneal administration PBF has distinct cumulative properties and toxic effects that prevent PBF from clinical testing in current composition. CONCLUSION: Thus, the PBF as a prolong drug needs to correct its parameters for further drug formulation development.

New Poly(3-hydroxybutyrate) Microparticles with Paclitaxel Sustained Release for Intraperitoneal Administration.

BACKGROUND: Poly(hydroxyalkanoates) (PHA) have recently attracted increasing attention due to their biodegradability and high biocompatibility, which makes them suitable for the development of new prolong drug formulations. OBJECTIVE: This study was conducted to develop new prolong paclitaxel (PTX) formulation based on poly(3- hydroxybutyrate) (PHB) microparticles. METHOD: PHB microparticles loaded with antitumor cytostatic drug PTX were obtained by spray-drying method using Nano Spray Dryer B-90. The PTX release kinetics in vitro from PHB microparticles and their cytotoxity on murine hepatoma cell line MH-22a were studied. Microparticles antitumor activity in vivo was studied using intraperitoneally (i.p.) transplanted tumor models: murine Lewis lung carcinoma and xenografts of human breast cancer RMG1. RESULTS: Uniform PTX release from PHB-microparticles during 2 months was observed. PTX-loaded PHB microparticles have demonstrated a significant antitumor activity versus pure drug both in vitro in murine hepatoma cells and in vivo when administered i.p. to mice with murine Lewis lung carcinoma and xenografts of human breast cancer RMG1. CONCLUSION: The developed technique of PTX sustained delivery from PHB-microparticles has therapeutic potential as prolong anticancer drug formulation.

Cell attachment on poly(3-hydroxybutyrate)-poly(ethylene glycol) copolymer produced by Azotobacter chroococcum 7B.

BACKGROUND: The improvement of biomedical properties, e.g. biocompatibility, of poly(3-hydroxyalkanoates) (PHAs) by copolymerization is a promising trend in bioengineering. We used strain Azotobacter chroococcum 7B, an effective producer of PHAs, for biosynthesis of not only poly(3-hydroxybutyrate) (PHB) and its main copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV), but also alternative copolymer, poly(3-hydroxybutyrate)-poly(ethylene glycol) (PHB-PEG). RESULTS: In biosynthesis we used sucrose as the primary carbon source and valeric acid or poly(ethylene glycol) 300 (PEG 300) as additional carbon sources. The chemical structure of PHB-PEG and PHB-HV was confirmed by 1H nuclear-magnetic resonance (1H NMR) analysis. The physico-chemical properties (molecular weight, crystallinity, hydrophilicity, surface energy) and surface morphology of films from PHB copolymers were studied. To study copolymers biocompatibility in vitro the protein adsorption and COS-1 fibroblasts growth on biopolymer films by XTT assay were analyzed. Both copolymers had changed physico-chemical properties compared to PHB homopolymer: PHB-HV and PHB-PEG had less crystallinity than PHB; PHB-HV was more hydrophobic than PHB in contrast to PHB-PEG appeared to have greater hydrophilicity than PHB; whereas the morphology of polymer films did not differ significantly. The protein adsorption to PHB-PEG was greater and more uniform than to PHB and PHB-PEG copolymer promoted better growth of COS-1 fibroblasts compared with PHB homopolymer. CONCLUSIONS: Thus, despite low EG-monomers content in bacterial origin PHB-PEG copolymer, this polymer demonstrated significant improvement in biocompatibility in contrast to PHB and PHB-HV copolymers, which may be coupled with increased protein adsorption and hydrophilicity of PEG-containing copolymer.