Recovery of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from Ralstonia eutropha cultures with non-halogenated solvents

Reduced downstream costs, together with high purity recovery of polyhydroxyalkanoate (PHA), will accelerate the commercialization of high quality PHA-based products. In this work, a process was designed for effective recovery of the copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) (P(HB-co-HHx)) containing high levels of HHx (>15 mol%) from Ralstonia eutropha biomass using non-halogenated solvents. Several non-halogenated solvents (methyl isobutyl ketone, methyl ethyl ketone, and butyl acetate and ethyl acetate) were found to effectively dissolve the polymer. Isoamyl alcohol was found to be not suitable for extraction of polymer. All PHA extractions were performed from both dry and wet cells at volumes ranging from 2 mL to 3 L using a PHA to solvent ratio of 2% (w/v). Ethyl acetate showed both high recovery levels and high product purities (up to 99%) when using dry cells as starting material. Recovery from wet cells, however, eliminates a biomass drying step during the downstream process, potentially saving time and cost. When wet cells were used, methyl isobutyl ketone (MIBK) was shown to be the most favorable solvent for PHA recovery. Purities of up to 99% and total recovery yields of up to 84% from wet cells were reached. During polymer recovery with either MIBK or butyl acetate, fractionation of the extracted PHA occurred, based on the HHx content of the polymer. PHA with higher HHx content (17–30 mol%) remained completely in solution, while polymer with a lower HHx content (11–16 mol%) formed a gel-like phase. All PHA in solution could be precipitated by addition of threefold volumes of n-hexane or n-heptane to unfiltered PHA solutions. Effective recycling of the solvents in this system is predicted due to the large differences in the boiling points between solvent and precipitant. Our findings show that two non-halogenated solvents are good candidates to replace halogenated solvents like chloroform for recovery of high quality PHA. Biotechnol. Bioeng. 2013; 110: 461–470. © 2012 Wiley Periodicals, Inc.Malaysia-MIT Biotechnology Partnership Programm

Construction of Microparticles Based on Resorbable Polymers Bioplastotan Using Spray Drying Method

Исследована возможность применения метода распылительного высушивания для конструирования микрочастиц на основе поли-3-гидроксибутирата (П(3ГБ)). Установлена зависимость характеристик микрочастиц (выход, средний диаметр, дзета-потенциал) от параметров процесса получения (температура на входе в систему, скорость подачи полимерного раствора и концентрация раствора полимера). Показано, что на величину дзета- потенциала микрочастиц достоверное влияние оказывала концентрация раствора полимера, а на средний диаметр – температура на входе в систему. Также в работе оптимизированы условия получения П(3ГБ) микрочастиц с минимальной потерей продукта (до 10 %)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 %

Microparticles prepared from biodegradable polyhydroxyalkanoates as matrix for encapsulation of cytostatic drug

Microparticles made from degradable polyhydroxyalkanoates of different chemical compositions a homopolymer of 3-hydroxybutyric acid, copolymers of 3-hydroxybutyric and 4-hydroxybutyric acids (P3HB/4HB), 3-hydroxybutyric and 3-hydroxyvaleric acids (P3HB/3HV), 3-hydroxybutyric and 3-hydroxyhexanoic acids (P3HB/3HHx) were prepared using the solvent evaporation technique, from double emulsions. The study addresses the influence of the chemical compositions on the size and ξ-potential of microparticles. P3HB microparticles loaded with doxorubicin have been prepared and investigated. Their average diameter and ξ-potential have been found to be dependent upon the level of loading (1, 5, and 10 % of the polymer mass). Investigation of the in vitro drug release behavior showed that the total drug released from the microparticle into the medium increased with mass concentration of the drug. In this study mouse fibroblast NIH 3T3 cells were cultivated on PHA microparticles, and results of using fluorescent DAPI DNA stain, and MTT assay showed that microparticles prepared from PHAs of different chemical compositions did not exhibit cytotoxicity to cells cultured on them and proved to be highly biocompatible. Cell attachment and proliferation on PHA microparticles were similar to those on polystyrene. The cytostatic drug encapsulated in P3HB/3HV microparticles has been proven to be effective against HeLa tumor cells.Russia (Federation) (Agreement 11.G34.31.0013)Program of the President of Russia for Young Doctors of Sciences (Grant MD-3112.2012.4

Herbicidal activity of slow-release herbicide formulations in wheat stands infested by weeds

This study addresses herbicidal activity of experimental formulations of metribuzin and tribenuron-methyl embedded in the degradable matrix of natural poly-3-hydroxybutyrate [P(3HB/MET) and P(3HB)/TBM] in stands of soft spring wheat (Triticum aestivum, cv. Altaiskaya 70) infested by weeds – white sweet clover Melilotus albus and lamb’s quarters Chenopodium album – under laboratory conditions. Indicators of herbicidal activity were the density and weight of the vegetative organs of weeds measured during 30-day and 50-day experiments. Wheat crop yield was estimated as dependent on the method of herbicide delivery and the decrease in weed density. The experimental MET and TBM formulations showed pronounced herbicidal activity against the weed species used in the study. The effectiveness of the experimental formulations in inhibiting weed growth was comparable to and, sometimes, higher than that of the commercial formulations (positive control). The amount of the biomass of the wheat treated with the experimental herbicide formulations was significantly greater than that of the wheat treated with commercial formulations

Effects of Fullerenes and Nanodiamonds in Hydroponic Culture of Triticum aestivum

В настоящее время перспективы повышения эффективности различных агротехнологий связывают с использованием наноматериалов в качестве нанопестицидов, наноудобрений, наноадаптогенов, агентов для праймирования семян. Успешное развитие наноагротехнологий сдерживается недостаточной изученностью механизмов реализации биологической активности наноматериалов на различных этапах развития растительного организма. В связи с этим в гидропонной культуре пшеницы изучали влияние углеродных наночастиц, фуллеренов (С60Ф) и наноалмазов (НА), на активность прорастания зерновок и функциональный статус корня 2-суточных проростков пшеницы. Эффект праймирования был выявлен только для С60Ф (5 мкг/мл). Углеродные наночастицы не стимулировали рост корня. Для НА (25 и 50 мкг/мл) отмечалось ингибирование роста корня. Увеличение численности популяции пограничных клеток в корневом апексе было выявлено только для С60Ф (50 мкг/мл). В этом случае увеличение численности пограничных клеток сопровождалось повышением содержания белка в гелевом чехле апекса. Уменьшение численности пограничных клеток без изменения содержания белка в гелевом чехле апекса было выявлено для НА (50 мкг/мл) и С60Ф (25 мкг/мл). Оба типа наночастиц вызывали дозозависимое увеличение секреторной активности пограничных клеток. Углеродные наночастицы обладали дозозависимой прооксидантной активностью. НА (5 мкг/мл) и С60Ф (5 и 25 мкг/мл) увеличивали содержание карбонилированных белков в корнях 2-дневных проростков. Увеличение содержания малонового диальдегида наблюдалось только для НА (50 мкг/мл). Выявленные дозозависимые эффекты углеродных наночастиц не связаны с их проникновением в клетки (размеры наночастиц значительно превышают размеры нанопор в клеточной стенке пшеницы). Предполагается, что биологическая активность С 60Ф и НА может реализоваться через индукцию окислительного стресса в клеточной стенке и апопласте. Полученные результаты позволяют рассматривать углеродные наночастицы (определенных размерных классов в определенных концентрациях) как перспективные регуляторы физиологии корневой системы, реализующие свою активность «экстраклеточно», без транслокации в клеткуCurrently, the prospects for increasing the efficiency of various agricultural technologies are associated with the use of nanomaterials as nanopesticides, nanofertilizers, nanoadaptogens, and seed priming agents. The successful development of nanoagricultural technologies is hampered by insufficient data on the mechanisms through which the biological activity of nanomaterials is realized at various stages of development of the plant organism. Therefore, in the present work, the effects of carbon nanoparticles – fullerenes (C 60F) and nanodiamonds (ND) – on the germination activity of grains and the functional status of the roots of 2-day-old wheat seedlings were studied in hydroponic wheat culture. The priming effect was detected only for C 60P (5 μg/mL). Carbon nanoparticles did not stimulate root growth. In the ND (25 and 50 μg/mL) treatments, root growth was inhibited. An increase in the population of border cells in the root apex was detected only in the C 60F (50 μg/mL) treatment, where the increase in the number of border cells was accompanied by an increase in the protein content in the mucilage cap of the root apex. A decrease in the number of border cells without changes in the protein content in the apical mucilage cap was detected in the ND (50 μg/mL) and C 60F (25 μg/mL) treatments. Both types of nanoparticles caused a dose-dependent increase in the secretory activity of border cells. Carbon nanoparticles had dose- dependent pro- oxidant activity. ND (5 μg/mL) and C 60F (5 and 25 μg/mL) increased the content of carbonylated proteins in the roots of 2-day-old seedlings. An increase in malondialdehyde content was observed only in the ND (50 μg/mL) treatment. The detected dose-dependent effects of carbon nanoparticles are not associated with their penetration into cells (the sizes of nanoparticles significantly exceed the sizes of nanopores in the cell wall of wheat). It is assumed that the biological activity of C 60F and ND can be realized through the induction of oxidative stress in the cell wall and apoplast. Results of the current study suggest that carbon nanoparticles (of certain size classes in certain concentrations) can be regarded as promising regulators of the physiology of the root system, realizing their activity “extracellularly”, without translocation into the cel

Battle of GLP-1 delivery technologies

Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) belong to an important therapeutic class for treatment of type 2 diabetes. Six GLP-1 RAs, each utilizing a unique drug delivery strategy, are now approved by the Food and Drug Administration (FDA) and additional, novel GLP-1 RAs are still under development, making for a crowded marketplace and fierce competition among the manufacturers of these products. As rapid elimination is a major challenge for clinical application of GLP-1 RAs, various half-life extension strategies have been successfully employed including sequential modification, attachment of fatty-acid to peptide, fusion with human serum albu- min, fusion with the fragment crystallizable (Fc) region of a monoclonal antibody, sustained drug delivery sys- tems, and PEGylation. In this review, we discuss the scientific rationale of the various half-life extension strategies used for GLP-1 RA development. By analyzing and comparing different approved GLP-1 RAs and those in development, we focus on assessing how half-life extending strategies impact the pharmacokinetics, pharmacodynamics, safety, patient usability and ultimately, the commercial success of GLP-1 RA products. We also anticipate future GLP-1 RA development trends. Since similar drug delivery strategies are also applied for de- veloping other therapeutic peptides, we expect this case study of GLP-1 RAs will provide generalizable concepts for the rational design of therapeutic peptides products with extended duration of action

Dose-Dependent Effects of Nanoscale Forms of Humic Acids in a Hydroponic Culture of Triticum aestivum: Induction of Oxidative Stress and an Increase in the Number of Border Cells

В гидропонной культуре Triticum aestivum L. изучали биологическую активность гуминовых кислот в форме наночастиц размером 6,5 нм и 68 нм. Гуминовые наночастицы (мелкие и крупные) в изученных концентрациях не ингибировали рост корня и не вызывали деструктивных изменений корневого апекса 2-дневных проростков. На средах с гуминовыми наночастицами в корнях проростков наблюдалось дозозависимое увеличение содержания карбонилированных белков, малонового альдегида и пролина – маркеров индуцированного окислительного стресса. Для мелких наночастиц основным продуктом свободнорадикального окисления был малоновый диальдегид, для крупных – карбонилированные белки. Индуцированный гуминовыми наночастицами окислительный стресс сопровождался дозозависимым увеличением численности свободных пограничных клеток и размеров гелевого чехла в корневом апексе 2-дневных проростков. Для мелких наночастиц (6,5 нм) эти изменения были выражены в значительно большей степени, чем для крупных наночастиц (68 нм). Полученные результаты позволяют рассматривать гуминовые наночастицы в качестве адаптогенов: способствуя увеличению численности популяции пограничных клеток и системы гелевого чехла (как продукта экскреторной активности пограничных клеток), наночастицы увеличивают устойчивость корневого апекса к воздействию неблагоприятных биотических и абиотических факторовThe biological activity of humic acids in the form of nanoparticles with a size of 6.5 nm and 68 nm was studied in a hydroponic culture of Triticum aestivum L. Both smaller and larger humic nanoparticles at the studied concentrations neither inhibited root growth nor caused destructive changes in the root apexes of 2-day-old seedlings. A dose-dependent increase in the contents of markers of induced oxidative stress such as carbonylated proteins, malonaldehyde, and proline was observed in the roots of seedlings on media with humic nanoparticles. Malondialdehyde was the main product of free radical oxidation for smaller nanoparticles and carbonylated proteins – for larger ones. Oxidative stress induced by humic nanoparticles was accompanied by a dose-dependent increase in the number of free border cells and the size of the mucilaginous sheaths on the root apexes of 2-day-old seedlings. For smaller (6.5 nm) nanoparticles, these changes were much more pronounced than for larger (68 nm) nanoparticles. The results obtained allow us to consider humic nanoparticles as adaptogens: contributing to an increase in the population of border cells and the mucilaginous sheath system (as a product of the excretory activity of border cells), nanoparticles enhance the resistance of the root apex to the effects of unfavorable biotic and abiotic factor

Fatty Acids Composition in Cyanobacteria and Their Ability to Synthesize Degradable Polyhydroxyalkanoates (PHA)

Исследованы состав жирных кислот липидов и способность к синтезу разрушаемых полигидроксиалканоатов (ПГА) у ранее не изученных цианобактерий, выделенных из водоемов бассейна р. Енисей. Изучено 10 штаммов различных видов, относящихся к порядкам Chroococcales, Oscillatoriales, Nostocales и Leptolyngbyales. В составе липидов исследованных штаммов цианобактерий идентифицированы 33 жирные кислоты, соотношение которых в зависимости от видовой специфики цианобактерий значительно варьировало, оказывая влияние на насыщенность липидов, которая составляла от 0,4 до 9,5. Для всех штаммов в качестве основной жирной кислоты (ЖК) идентифицирована пальмитиновая кислота, содержание которой составляло от 24 до 47 % от суммы ЖК. Доминирующими моноеновыми ЖК были C16:1ɷ7 и C18:1ɷ9, содержание которых варьировало от 2 до 16 и от 2 до 24 % от суммы ЖК соответственно. Все штаммы синтезировали полиеновые кислоты C16- и C18 рядов, но в различной степени. Выявлены пять штаммов (Chroococcus limneticus ACCS019, Trichormus variabilis ACCS039, Trichormus variabilis ACCS060, Anabaena aequalis ACCS089 и Cylindrospermum stagnale ACCS051), обладающих способностью синтезировать полимер, который у всех штаммов представлен гомополимером 3-гидроксимасляной кислоты – поли(3- гидроксибутиратом). Модификация стандартной питательной среды (исключение из ее состава азота и фосфора) обеспечила многократное повышение внутриклеточного содержания полимера. Цианобактерии Chroococcus limneticus ACCS019, Anabaena aequalis ACCS089 и Cylindrospermum stagnale ACCS51, в которых содержание полимера составило, соответственно, 8,9, 4,7 и 3,2 %, можно рассматривать в качестве потенциальных продуцентов ПГА в процессах фотосинтезаThe composition of fatty acids of lipids and the ability of cyanobacteria to synthesize degradable polyhydroxyalkanoates (PHA) were examined in previously unstudied cyanobacteria isolated from a number of water bodies of the Yenisei River Basin. Ten strains of various species belonging to the orders Chroococcales, Oscillatoriales, Nostocales and Leptolyngbyales were investigated. Thirty-three fatty acids (FAs) were identified in the lipid composition of the studied cyanobacterial strains. The ratio of FAs was to a high extent species-specific with the lipid saturation ranging from 0,4 to 9,5. In all strains, palmitic acid was identified as the main FA with the content ranging from 24 to 47 % of the total FA. The dominant monoenoic FAs were C16:1ɷ7 and C18:1ɷ9; their content varied from 2 to 16 and from 2 to 24 % of the total FA, respectively. All strains synthesized polyenoic acids of the C16- and C18 series but in different amounts. Five strains (Chroococcus limneticus ACCS019, Trichormus variabilis ACCS039, Trichormus variabilis ACCS060, Anabaena aequalis ACCS089 and Cylindrospermum stagnale ACCS051) were determined to be able to synthesize a homopolymer of 3-hydroxybutyric acid – poly(3-hydroxybutyrate). A modification of the standard nutrient medium (the exclusion of nitrogen and phosphorus from its composition) ensured a multiple increase in the intracellular content of the polymer. The cyanobacteria Chroococcus limneticus ACCS019, Anabaena aequalis ACCS089 and Cylindrospermum stagnale ACCS051 with the polymer content of 8.9, 4.7 and 3.2 %, respectively, can be considered as potential producers of PHA in photosynthesi

Aligned nanofibres made of poly(3-hydroxybutyrate) grafted to hyaluronan for potential healthcare applications

In this work, a hybrid copolymer consisting of poly(3-hydroxybutyrate) grafted to hyaluronic acid (HA) was synthesised and characterised. Once formed, the P(3HB)-g-HA copolymer was soluble in water allowing a green electrospinning process. The diameters of nanofibres can be tailored by simply varying the Mw of polymer. The optimization of the process allowed to produce fibres of average diameter in the range of 100-150 nm and low polydispersity. The hydrophobic modification has not only increased the fibre diameter, but also the obtained layers were homogenous. At the nanoscale, the hybrid copolymer exhibited an unusual hairy topography. Moreover, the hardness and tensile properties of the hybrid were found to be superior compared to fibres made of unmodified HA. Particularly, this reinforcement was achieved at the longitudinal direction. Additionally, this work reports the use in the composition of a water-soluble copolymer containing photo cross-linkable moieties to produce insoluble materials post-electrospinning. The derivatives as well as their nanofibrous mats retain the biocompatibility of the natural polymers used for the fabrication

Получение и свойства микрочастиц поли(3-гидроксибутирата-со‑3- гидроксивалерата), нагруженных рифампицином

In this work, polyhydroxyalkanoates-based microparticles containing antibacterial drug rifampicin were obtained. The main characteristics of the microparticles were investigated: the particles yield, encapsulation efficiency and drug loading, average radius, surface morphology, polydispersity index, zeta potential and cytotoxicity towards HeLa cells. Loading of RIF into microparticles leads to the substantial decrease of its cytotoxicityВ настоящей р аботе о писан п роцесс п олучения м икрочастиц п оли(3-гидроксибутирата-со‑3- гидроксивалерата), содержащих антибактериальный препарат рифампицин. Определены основные параметры процесса получения и свойства микрочастиц: выход, эффективность инкапсулирования и включение препарата, средние размеры, морфология поверхности, индексы полидисперсности, дзета-потенциал, цитотоксичность в модельной культуре клеток HeLa. Установлено, что цитотоксичность полученной системы существенно ниже таковой для чистого РИ