Elena D. Nikolaeva

A Glucose-Utilizing Strain, Cupriavidus euthrophus B-10646: Growth Kinetics, Characterization and Synthesis of Multicomponent PHAs

This study investigates kinetic and production parameters of a glucose-utilizing bacterial strain, C. eutrophus B-10646, and its ability to synthesize PHA terpolymers. Optimization of a number of parameters of bacterial culture (cell concentration in the inoculum, physiological activity of the inoculum, determined by the initial intracellular polymer content, and glucose concentration in the culture medium during cultivation) provided cell concentrations and PHA yields reaching 110 g/L and 80%, respectively, under two-stage batch culture conditions. Addition of precursor substrates (valerate, hexanoate, propionate, γ-butyrolactone) to the culture medium enabled synthesis of PHA terpolymers, P(3HB/3HV/4HB) and P(3HB/3HV/3HHx), with different composition and different molar fractions of 3HB, 3HV, 4HB, and 3HHx. Different types of PHA terpolymers synthesized by C. eutrophus B-10646 were used to prepare films, whose physicochemical and physical-mechanical properties were investigated. The properties of PHA terpolymers were significantly different from those of the P3HB homopolymer: they had much lower degrees of crystallinity and lower melting points and thermal decomposition temperatures, with the difference between these temperatures remaining practically unchanged. Films prepared from all PHA terpolymers had higher mechanical strength and elasticity than P3HB films. In spite of dissimilar surface structures, all films prepared from PHA terpolymers facilitated attachment and proliferation of mouse fibroblast NIH 3T3 cells more effectively than polystyrene and the highly crystalline P3HB.

Electrospinning of polyhydroxyalkanoate fibrous scaffolds: effects on electrospinning parameters on structure and properties

In this study, electrospinning was used to prepare ultrafine fibers from PHAs with different chemical compositions: P(3HB) and copolymers: P(3HB-co-4HB), P(3HB-co-3HV), and P(3HB-co-3HHx). The main process parameters that influence ultrafine fiber diameter and properties (polymer concentration, solution feeding rate, working distance, and applied voltage) have been investigated and their effects evaluated. The study revealed electrospinning parameters for the production of high-quality ultrafine fibers and determined which parameters should be varied to tailor the properties of the products. This study is the first to compare biological and physical-mechanical parameters of PHAs with different chemical compositions as dependent upon the fractions of monomers constituting the polymers and ultrafine fiber orientation. Mechanical strength of aligned ultrafine fibers prepared from different PHAs is higher than that of randomly oriented ones; no significant effect of ultrafine fiber orientation on surface properties has been found. None of the fibrous scaffolds produced by electrospinning from PHAs had any adverse effects on attachment, growth, and viability of NIH 3T3 mouse fibroblast cells, and all of them were found to be suitable for tissue engineering applications.

Surface wettability and energy effects on the biological performance of poly-3-hydroxybutyrate films treated with RF plasma.

The surface properties of poly-3-hydroxybutyrate (P3HB) membranes were modified using oxygen and an ammonia radio-frequency (RF, 13.56 MHz) plasma. The plasma treatment procedures used in the study only affected the surface properties, including surface topography, without inducing any significant changes in the crystalline structure of the polymer, with the exception being a power level of 250 W. The wettability of the modified P3HB surfaces was significantly increased after the plasma treatment, irrespective of the treatment procedure used. It was revealed that both surface chemistry and surface roughness changes caused by the plasma treatment affected surface wettability. A treatment-induced surface aging effect was observed and resulted in an increase in the water contact angle and a decrease in the surface free energy. However, the difference in the water contact angle between the polymers that had been treated for 4 weeks and the untreated polymer surfaces was still significant. A dependence between cell adhesion and proliferation and the polar component of the surface energy was revealed. The increase in the polar component after the ammonia plasma modification significantly increased cell adhesion and proliferation on biodegradable polymer surfaces compared to the untreated P3HB and the P3HB modified using an oxygen plasma.

Experimental wound dressings of degradable PHA for skin defect repair

The present study reports construction of wound dressing materials from degradable natural polymers such as hydroxy derivatives of carboxylic acids (PHAs) and 3-hydroxybutyrate/4-hydroxybutyrate [P(3HB/4HB)] as copolymer. The developed polymer films and electrospun membranes were evaluated for its wound healing properties with Grafts—elastic nonwoven membranes carrying fibroblast cells derived from adipose tissue multipotent mesenchymal stem cells. The efficacy of nonwoven membranes of P(3HB/4HB) carrying the culture of allogenic fibroblasts was assessed against model skin defects in Wistar rats. The morphological, histological and molecular studies revealed the presence of fibroblasts on dressing materials which facilitated wound healing, vascularization and regeneration. Further it was also observed that cells secreted extracellular matrix proteins which formed a layer on the surface of membranes and promoted the migration of epidermal cells from the neighboring tissues surrounding the wound. The wounds under the P(3HB/4HB) membrane carrying cells healed 1.4 times faster than the wounds under the cell-free membrane and 3.5 times faster than the wounds healing under the eschar (control).The complete wound healing process was achieved at Day 14. Thus the study highlights the importance of nonwoven membranes developed from degradable P(3HB/4HB) polymers in reducing inflammation, enhancing angiogenic properties of skin and facilitating better wound healing process.

Constructing herbicide metribuzin sustained-release formulations based on the natural polymer poly-3-hydroxybutyrate as a degradable matrix.

ABSTRACT Polymer poly(3-hydroxybutyrate) [P(3HB)] has been used as a matrix in slow-release formulations of the herbicide metribuzin (MET). Physical P(3HB)/MET mixtures in the form of solutions, powders, and emulsions were used to construct different metribuzin formulations (films, granules, pellets, and microparticles). SEM, X-Ray, and DSC proved the stability of these formulations incubated in sterile water in vitro for long periods of time (up to 49 days). Metribuzin release from the polymer matrix has been also studied. By varying the shape of formulations (microparticles, granules, films, and pellets), we were able to control the release time of metribuzin, increasing or decreasing it.

Efficacy of tebuconazole embedded in biodegradable poly-3-hydroxybutyrate to inhibit the development of Fusarium moniliforme in soil microecosystems.

BACKGROUND An important line of research is the development of a new generation of formulations with targeted and controlled release of the pesticide, using matrices made from biodegradable materials. In this study, slow-release formulations of the fungicide tebuconazole (TEB) have been prepared by embedding it into the matrix of poly-3-hydroxybutyrate (P3HB) in the form of films, microgranules and pellets. RESULTS The average rates of P3HB degradation were determined by the geometry of the formulation, reaching, for 63 days, 0.095-0.116, 0.081-0.083 and 0.030-0.055 mg day-1 for films, microgranules and pellets respectively. The fungicidal activity of P3HB/TEB against the plant pathogen Fusarium moniliforme was compared with that of the commercial formulation Raxil Ultra. A pronounced fungicidal effect of the experimental P3HB/TEB formulations was observed in 2-4 weeks after application, and it was retained for 8 weeks, without affecting significantly the development of soil aboriginal microflora. CONCLUSION TEB release can be regulated by the process employed to fabricate the formulation and the fungicide loading, and the TEB accumulates in the soil gradually, as the polymer is degraded. The experimental forms of TEB embedded in the slowly degraded P3HB can be used as a basis for developing slow-release fungicide formulations.

Laser processing of polymer constructs from poly(3-hydroxybutyrate).

CO2 laser radiation was used to process poly(3-hydroxybutyrate) constructs – films and 3D pressed plates. Laser processing increased the biocompatibility of unperforated films treated with moderate uniform radiation, as estimated by the number and degree of adhesion of NIH 3T3 mouse fibroblast cells. The biocompatibility of perforated films modified in the pulsed mode did not change significantly. At the same time, pulsed laser processing of the 3D plates produced perforated scaffolds with improved mechanical properties and high biocompatibility with bone marrow-derived multipotent, mesenchymal stem cells, which show great promise for bone regeneration.

Novel spray-dried PHA microparticles for antitumor drug release

ABSTRACT The production of poly-3-hydroxybutyrate (P3HB) and poly-3-hydroxybutyrate/polyethylene glycol (PEG)-based microparticles, loaded with antitumor drugs paclitaxel (PTX) and 5-Fluorouracil (5-FU) by spray-drying technique, was investigated. The average diameter of microparticles was found to be 3.4 ± 0.5 µm and zeta potential was about −44 mV. The addition of surfactant PEG did not show any effect on the morphological characteristics of the particles. But the chemical structure of drug influenced on the properties. Microparticles had heterogeneous pores on the surface when the hydrophobic PTX was encapsulated. It was established that the addition of surfactant positively influenced on the properties of particles and led to the loading of 5-FU directly into the matrix. This is confirmed by the results of electron microscopy and dynamics of drug release in vitro. As a whole, the release profiles of PTX and 5-FU from composite P3HB/PEG microparticles were less than from P3HB microparticles. The results of the morphological evaluation of Hela cells demonstrated that the use of cytostatic drugs loaded in P3HB microparticles induces morphological changes associated with apoptosis (chromatin condensation, core fragmentation, margination of nucleus). Thus, the obtained results can serve as the basis for the development of new antitumor drugs of prolonged action, intended for various modes of administration.

Biocomposites Based on Degradable Materials as Biotechnological Dermal Equivalents

Hybrid tissue-engineered biosystems based on biodegradable polymers of microbiological origin – bacterial cellulose (BC) and a copolymer of 3-hydroxybutyric and 4-hydroxybutyric acid P(3HB/4HB) – were constructed using various methods: 1) the dried BC pellicles were soaked with a 2% P(3HB/4HB) solution in chloroform, kept for 24 h, and dried in a dust-free cabinet until the solvent had completely evaporated; 2) nonwoven membranes were placed into the K. xylinus B-12068 culture, and bacterial cellulose synthesized in it grew under static conditions; 3) powdered cellulose (particle size of 120 μm) was added to a 3% P(3HB/4HB) solution in chloroform and mixed ultrasonically to homogeneity; then, the films were produced by the solvent evaporation technique, at polymer to cellulose ratios of 2:1 and 1:1. Investigation of surface properties of the samples showed an increase in surface hydrophilicity of the composites produced by mixing a solution of P(3HB/4HB) with BC at ratios of 2:1 and 1:1 (the contact angle was 43.9±17.55° and 36.1±0.66°) relative to the starting materials. The physical/mechanical properties of the composites based on P(3HB/4HB) and BC were superior to the properties of the neat materials, and they were largely determined by the methods of production of the composite and the properties of the materials used. Based on the study of surface and physical/mechanical properties of the hybrids, films prepared by casting the polymer solution with BC powder (2:1 and 1:1) followed by solvent evaporation were chosen for studying cell adhesion. These scaffolds were loaded with drugs promoting wound healing (actovegin, solcoseryl) and tested in the culture of fibroblasts derived from the adipose tissue MSCs. The MTT assay showed that the most effective hybrid systems were polymer:bacterial cellulose powder (1:1) samples loaded with actovegin or solcoseryl at a concentration of 5%. Those systems produced a stimulating effect on fibroblasts, and, thus, they can be regarded as promising wound dressings to repair skin defects.

Differentiation of Mscs into Osteoblasts on a Porous 3D -carrier of Poly-3-Hydroxybutyrate

The study describes preparation and testing of porous 3D implants of natural degradable polymer of 3-hydroxybutyric acid (P(3HB)) for regeneration of bone tissue defects. The ability of the 3D carriers to favor attachment and facilitate proliferation and directed differentiation into osteoblasts of mesenchymal stem cells (MSCs) was studied in the culture of MSCs isolated from bone marrow and adipose tissue. MSC differentiation into osteoblasts was confirmed by measurements of the activity of alkaline phosphatase, gene expression of the marker of bone protein BGP (glutamic protein participating in mineralization) and measurements of intracellular precipitates of calcium and phosphorus salts.