Carbonylative Polymerization of Epoxides Mediated by Tri-metallic Complexes: A Dual Catalysis Strategy for Synthesis of Biodegradable Polyhydroxyalkanoates.

Polyhydroxyalkanoates (PHAs) are a unique class of commercially manufactured biodegradable polyesters with properties suitable for partially substituting petroleum-based plastics. However, high costs and low volumes of production have restricted their application as commodity materials. In this study, tri-metallic complexes were developed for carbonylative polymerization via a dual catalysis strategy, and 17 products of novel PHAs with up to 38.2 kg mol-1 Mn values were discovered. The polymerization proceeds in a sequential fashion, which entails the carbonylative ring expansion of epoxide to ß-lactone and its subsequent ring-opening polymerization that occurs selectively at the O-alkyl bond via carboxylate species. The wide availability and structural diversity of epoxide monomers provide PHAs with various structures, excellent functionalities, and tunable properties. This study represents a rare example of the preparation of PHAs using epoxides and carbon monoxide as raw materials.

Fabrication of biopolymer polyhydroxyalkanoate/chitosan and 2D molybdenum disulfide-doped scaffolds for antibacterial and biomedical applications.

Antibiotic resistance in pathogenic bacteria is a major health challenge, as Infectious Diseases Society of America (IDSA) has recognized that the past simply drugs susceptible pathogens are now the most dangerous pathogens due to their nonstop growing resistance towards conventional antibiotics. Therefore, due to the emergence of multi-drug resistance, the bacterial infections have become a serious global problem. Acute infections feasibly develop into chronic infections because of many factors; one of them is the failure of effectiveness of antibiotics against superbugs. Modern research of two-dimensional nanoparticles and biopolymers are of great interest to attain the intricate bactericidal activity. In this study, we fabricated an antibacterial nanocomposite consisting of representative two-dimensional molybdenum disulfide (2D MoS2) nanoparticles. Polyhydroxyalkanoate (PHA) and chitosan (Ch) are used to encapsulate MoS2 nanoparticles into their matrix. This study reports the in vitro antibacterial activity and host cytotoxicity of novel PHA-Ch/MoS2 nanocomposites. PHA-Ch/MoS2 nanocomposites were subjected to time-dependent antibacterial assays at various doses to examine their antibacterial activity against multi-drug-resistant Escherichia coli K1 (Malaysian Type Culture Collection 710859) and methicillin-resistant Staphylococcus aureus (MRSA) (Malaysian Type Culture Collection 381123). Furthermore, the cytotoxicity of nanocomposites was examined against spontaneously immortalized human keratinocyte (HaCaT) cell lines. The results indicated significant antibacterial activity (p value < 0.05) against E. coli K1 and MRSA. In addition, PHA-Ch/MoS2 showed significant host cytocompatibility (p < 0.05) against HaCaT cells. The fabricated PHA-Ch/MoS2 nanocomposites have demonstrated effective antibacterial activity against both Gram-positive and -negative bacteria and exhibited better biocompatibility. Finally, PHA-Ch/MoS2 nanocomposites are shown to be suitable for antibacterial applications and also hold potential for further biomedical studies. Graphical Abstract.

[Advances in synthesis of polyhydroxyalkanoates by using residual activated sludge].

Pure microbial fermentation is the primary means of polyhydroxyalkanoates (PHA) production at present, but high cost limits its large-scale application. In recent years, the synthesis of PHA by mixed microbial culture of activated sludge has been extensively studied. Combining the residual sludge treatment with PHA synthesis not only eliminates the sterilization process necessary for pure culture, but also utilizes residual sludge as resource. The hydrolysis and acidification of the residual sludge, the enrichment and acclimation of the consortium and the PHA synthesis are affected by environmental factors. The intensive biosynthesis mechanism study is helpful for the application of mixed culture to synthesize PHA. This paper introduces the feasibility of using residual sludge to synthesize PHA, the factors affecting the hydrolysis and acidification of residual sludge, the accumulation and domestication of PHA by sludge microbial consortium and its mechanism.

Polyhydroxyalkanoates based copolymers.

Polyhydroxyalkanoates (PHAs) belong to a family of natural polyesters and are produced under unbalanced growth conditions as intracellular carbon and energy reserves by a wide variety of microorganisms. Being biodegradable, biocompatible and environmental friendly thermoplastics, the PHAs are considered as future polymers to replace petrochemicals based plastics. In this review, the introduction section deals with the brief discussion on PHA nature, availability, raw materials for production, processing etc. This is followed by the discussions on modifications. The copolymer syntheses by bacterial and chemical methods have been discussed. Under chemical methods, unsaturated side chains and their derivatives, oligomer, coupling, macro-initiating, trans-esterification, radiation grafting, click chemistry, ring opening and several miscellaneous polymerization methods have been elaborated. A brief discussion on applications has been incorporated. The last section includes conclusion and future perspectives.

Water-Soluble Poly(3-hydroxyalkanoate) Sulfonate: Versatile Biomaterials Used as Coatings for Highly Porous Nano-Metal Organic Framework.

Water-soluble poly(3-hydroxyalkanoate) containing ionic groups were designed by two successive photoactivated thiol-ene reactions. Sodium-3-mercapto-1-ethanesulfonate (SO3-) and poly(ethylene glycol) (PEG) methyl ether thiol were grafted onto poly(3-hydroxyoctanoate-co-3-hydroxyundecenoate) PHO(67)U(33) to introduce both ionic groups and hydrophilic moieties. The grafted copolymers PHO(67)SO3-(20)PEG(13) were then used as biocompatible coatings of nano-metal organic frameworks (nanoMOFs) surfaces. Scanning electron microscopy and scanning transmission electron microscopy coupled with energy dispersive X-ray characterizations have clearly demonstrated the presence of the copolymer on the MOF surface. These coated nanoMOFs are stable in aqueous and physiological fluids. Cell proliferation and cytotoxicity tests performed on murine macrophages J774.A1 revealed no cytotoxic side effect. Thus, biocompatibility and stability of these novel hybrid porous MOF structures encourage their use in the development of effective therapeutic nanoparticles.

Methods of synthesis, properties and biomedical applications of polyhydroxyalkanoates: a review.

This study aims to provide an overview of the main polyhydroxyalkanoates (PHAs) used in medical applications. In this review, it has been demonstrated that the properties of PHAs can be controlled both by varying the concentration of units in the copolymer and the substrate for PHA production. Another way of controlling the mechanical properties of PHAs is varying the 3HV content, such as the case of P(3HB-co-3HV). A higher 3HV content in the structure of this polyester will lead to a lower cristallinity and, therefore, to greater flexibility, strength and elongation at break. PHAs are biocompatible, completely biodegradable, and non-toxic. Considering the state of the art technologies and their ideal properties, PHAs (especially the short-chain-length PHA polymers) can be tailored for specific medical applications, such as surgical suture, scaffolds, grafts and heart valves or drug delivery systems. Nowaday, random copolyesters of 3HB and 4HB (P(3HB-co-4HB)) are used for the development of biodegradable implants loaded with antibiotics for therapeutic treatment of chronic osteomyelitis. It is also believed that these biodegradable materials can be efficient alternatives for reducing the pollution produced by the medical waste consisting of replacing plastic handles, packs, syringes or tubes that are trashed into the homes, clinics and hospitals around the world.

Highly Efficient Fluorescent Material Based on Rare-Earth-Modified Polyhydroxyalkanoates.

Fluorescent materials play an important role in biomedical fields. However, the main types of fluorescent materials suffer from several disadvantages especially the biotoxicity, which largely restrict its wider applications in biological fields. In this study, a highly efficient rare-earth-modified fluorescent material was successfully designed and fabricated based on polyhydroxyalkanoates, which are known as biodegradable and biocompatible materials. A new Functional-PHA polymer was microbially synthesized by engineered Halomonas bluephagenesis and was used as a basal matrix to generate the rare-earth-modified PHA. N-Acetyl-l-cysteine-grafted PHA (NAL-grafted-PHA) was first produced via a UV-initiated thiol-ene click reaction and the rare earth metal ions (Eu3+ and Tb3+) were subsequently chelated onto the NAL-grafted-PHA through the coordination effect. The composite material exhibited intense photoluminescence properties under UV laser excitation, indicating the excellent features as fluorescent material. The enhanced hydrophilicity and superior biocompatibility of rare-earth-chelated PHA were confirmed, suggesting its great potential application value in biomedical fields.

Polyhydroxyalkanoates as biomaterial for electrospun scaffolds.

Polyhydroxyalkanoates (PHA) are natural polyesters produced by microorganisms under carbon source excess and limiting nutrient conditions. However, these biopolymers possess low mechanical and thermal properties, decreasing their potential applications in the medical field. Electrospinning is a technique that forms fibers from different polymers. PHA electrospun fibers improve the mechanical properties and decrease the crystallinity of PHA, including poly-3-hydroxybutyrate and its copolymers, which is attributed to the metastable structure (ß-form) formation. Therefore, the mechanical properties of fibers are intrinsically related to their plane orientation. Aligned fibers present better mechanical properties than randomly oriented fibers. However, randomly oriented fibers promote cell-fiber interaction and cell infiltration. Fibers produced with PHA blended with other polymers have shown improved mechanical and biological properties. Gelatin, zein and cellulose acetate are the main natural polymers that have been blended with PHA for electrospun scaffolds. For scaffold production by coaxial electrospinning, gelatin has been used as a shell and PHA as the core. PHA have been combined with different synthetic polymers and plasticizers resulting in an increase in the PHA miscibility. Therefore, the use of electrospinning in the development of PHA-based scaffolds seems to be an attractive method to change the intrinsic polymer features, increasing and enhancing PHA applications in tissue engineering.

Synthesis of Short-Chain-Length and Medium-Chain-Length Polyhydroxyalkanoate Blends from Activated Sludge by Manipulating Octanoic Acid and Nonanoic Acid as Carbon Sources.

The effects of octanoic acid/nonanoic acid and acclimation time on the synthesis of short-chain-length and medium-chain-length PHA blends from activated sludge were investigated. An increased concentration (847-1366 mg/L) of PHAs resulted from 4-month acclimation compared with the concentration derived from 2-month acclimation (450-1126 mg/L). The content of octanoic acid had a positive linear relationship with the content of even-numbered carbon monomers among the PHAs. The blending products were identified mainly with scl-PHAs during the 2-month acclimation period and were thereafter dominated by mcl-PHAs until 4 months of acclimation. Thermal properties analysis demonstrated that the products derived from 4-month acclimation were a mixture of scl-PHAs and mcl-PHAs rather than a copolymer of scl-PHAs and mcl-PHAs. High-throughput sequencing results indicated that Pseudofulvimonas, Paracoccus, and Blastocatella were the dominant genera that might be responsible for scl-PHAs production during the 2-month acclimation period, whereas Comamonas and Pseudomonas that were responsible for mcl-PHAs production then became the dominant genera after 4-months acclimation.

Design of functionalized biodegradable PHA-based electrospun scaffolds meant for tissue engineering applications.

Modification of electrospun nanofibrous poly(3-hydroxyalkanoate) (PHA)-based mats was implemented through two routes to obtain biomimetic scaffolds meant for tissue engineering applications. The first strategy relied on a physical functionalization of scaffolds thanks to an original route which combined both electrospinning and electrospraying, while the second approach implied the chemical modification of fiber surface via the introduction of reactive functional groups to further conjugate bioactive molecules. The degree of glycidyl methacrylate grafting on PHA reached 20% after 300s under photoactivation. Epoxy groups were modified via the attachment of a peptide sequence, such as Arg-Gly-Asp (RGD), to obtain biofunctionalized scaffolds. SEM and TEM analysis of mats showed uniform and well-oriented beadless fibers. The electrospinning/electrospraying tandem process afforded highly porous scaffolds characterized by a porosity ratio up to 83% and fibers with a surface largely covered by the electrosprayed bioceramic, i.e. hydroxyapatite. Gelatin was added to the latter PHA-based scaffolds to improve the hydrophilicity of the scaffolds (water contact angle about 0°) as well as their biological properties, in particular cell adhesion, proliferation, and osteogenic differentiation after 5days of human mesenchymal stromal culture. Human mesenchymal stromal cells exhibited a better adhesion and proliferation on the biofunctionalized scaffolds than that on non-functionalized PHA mats.

Chemical modification of functionalized polyhydroxyalkanoates via "Click" chemistry: A proof of concept.

A novel approach to the post-biosynthetic chemical modification of bromo and alkyne functionalized poly(3-hydroxyalkanoates), (PHAs), via copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain promoted azide alkyne cycloaddition (SPAAC) is reported. Optimum conditions for the biosynthesis of the PHA copolymers, poly(3-hydroxynonanoate-co-3-hydroxy-11-bromoundecanoate) (PHNUBr) and poly(3-hydroxynonanoate-co-3-hydroxy-10-undecynoate) (PHNUD), using Pseudomonas oleovorans as cell factories were 20h of fermentation time and a total carbon substrate concentration of 40mM. Percent incorporation of brominated repeat units and alkyne repeat units were 38.5% and 50% respectively, as determined by 1H NMR. PHNUBr was converted into an azido-terminated precursor for the polymer analogous "click" reactions via SN2 nucleophilic substitution reaction using sodium azide with a yield of 96.7% and analyzed by FTIR and 1H NMR. CuAAC reactions were used to attach propargyl benzoate and methyl-2-azidoacetate to the PHAs with terminal azido and alkyne functional side groups respectively, with yields of 78.2% and 65.4% respectively. FTIR analysis of the products showed the disappearance of the azide peak at 2093.5cm-1 and the alkynyl CCH stretch at 3292cm-1. 1H NMR analysis confirmed the formation of the expected triazole linkage; showing the expected proton chemical shift corresponding to the triazole proton at 7.73 and 7.47ppm respectively. A strain promoted azide-alkyne reaction was used to attach (1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN-OH) to the azido-terminated PHA with an average yield of 94.5%. Products were analyzed by FTIR and 1H NMR.

Reactive blends based on polyhydroxyalkanoates: Preparation and biomedical application.

Polyhydroxyalkanoates (PHAs) are a class of natural polyesters as carbon and energy reserves by >300 species of microorganisms. They are fully biodegradable, biocompatible and piezoelectric biopolymers that have attracted much attention recently as the biomaterial of choice for medical applications. However, the toughness, processability and hydrophilicity of PHAs need to tune to expand their applications as tissue engineering scaffolds or drug delivery systems. Reactive polymer blending is one of the most economic and versatile way to produce materials combining the desired properties via forming the compatibilizing agents in situ or inducing the chemico-physical interactions between polymer blends. This review focuses on the PHAs-based reactive blends aiming to present a brief introduction to the mechanism of reactive polymer blending technique, including the formation of H-bonding, branching/crosslinking copolymers, graft copolymers or complex copolymers during polymer blending process.

Study of Class I and Class III Polyhydroxyalkanoate (PHA) Synthases with Substrates Containing a Modified Side Chain.

Polyhydroxyalkanoates (PHAs) are carbon and energy storage polymers produced by a variety of microbial organisms under nutrient-limited conditions. They have been considered as an environmentally friendly alternative to oil-based plastics due to their renewability, versatility, and biodegradability. PHA synthase (PhaC) plays a central role in PHA biosynthesis, in which its activity and substrate specificity are major factors in determining the productivity and properties of the produced polymers. However, the effects of modifying the substrate side chain are not well understood because of the difficulty to accessing the desired analogues. In this report, a series of 3-(R)-hydroxyacyl coenzyme A (HACoA) analogues were synthesized and tested with class I synthases from Chromobacterium sp. USM2 (PhaCCs and A479S-PhaCCs) and Caulobacter crescentus (PhaCCc) as well as class III synthase from Allochromatium vinosum (PhaECAv). It was found that, while different PHA synthases displayed distinct preference with regard to the length of the alkyl side chains, they could withstand moderate side chain modifications such as terminal unsaturated bonds and the azide group. Specifically, the specific activity of PhaCCs toward propynyl analogue (HHxyCoA) was only 5-fold less than that toward the classical substrate HBCoA. The catalytic efficiency (kcat/Km) of PhaECAv toward azide analogue (HABCoA) was determined to be 2.86 × 10(5) M(-1) s(-1), which was 6.2% of the value of HBCoA (4.62 × 10(6) M(-1) s(-1)) measured in the presence of bovine serum albumin (BSA). These side chain modifications may be employed to introduce new material functions to PHAs as well as to study PHA biogenesis via click-chemistry, in which the latter remains unknown and is important for metabolic engineering to produce PHAs economically.

Development and characterization of bio-derived polyhydroxyalkanoate nanoparticles as a delivery system for hydrophobic photodynamic therapy agents.

In this study, we developed and investigated nanoparticles of biologically-derived, biodegradable polyhydroxyalkanoates (PHAs) as carriers of a hydrophobic photosensitizer, 5,10,15,20-Tetrakis(4-hydroxy-phenyl)-21H, 23H-porphine (pTHPP) for photodynamic therapy (PDT). Three PHA variants; polyhydroxybutyrate, poly(hydroxybutyrate-co-hydroxyvalerate) or P(HB-HV) with 12 and 50% HV were used to formulate pTHPP-loaded PHA nanoparticles by an emulsification-diffusion method, where we compared two different poly(vinyl alcohol) (PVA) stabilizers. The nanoparticles exhibited nano-scale spherical morphology under TEM and hydrodynamic diameters ranging from 169.0 to 211.2 nm with narrow size distribution. The amount of drug loaded and the drug entrapment efficiency were also investigated. The in vitro photocytotoxicity was evaluated using human colon adenocarcinoma cell line HT-29 and revealed time and concentration dependent cell death, consistent with a gradual release pattern of pTHPP over 24 h. This study is the first demonstration using bacterially derived P(HB-HV) copolymers for nanoparticle delivery of a hydrophobic photosensitizer drug and their potential application in PDT.

The 'PHAome'.

Polyhydroxyalkanoates (PHAs) are a family of polyesters synthesized by bacteria. Similarly to the genome, transcriptome, and proteome (the entire array of nucleic acids and proteins present in a cell or population of cells at a given time), the PHA spectrum exhibits diverse and dynamic modifications - the 'PHAome' - reflecting not only by the diversity of monomers, homopolymers, random and block copolymers, functional and graft polymers, molecular weights, and combinations of the above, but also the ranges of PHAs with various molecular weights and monomer ratios that are present at a particular timepoint in a bacterial cell. Echoing the Materials Genome Initiative (MGI) launched in 2011 to develop an infrastructure to accelerate advanced materials discovery and deployment, understanding the PHAome and ensuring an ample supply of PHAs based on it will promote the discovery of new properties and applications of this family of advanced materials.

Disassembly and reassembly of polyhydroxyalkanoates: recycling through abiotic depolymerization and biotic repolymerization.

An abiotic-biotic strategy for recycling of polyhydroxyalkanoates (PHAs) is evaluated. Base-catalyzed PHA depolymerization yields hydroxyacids, such as 3-hydroxybutyrate (3HB), and alkenoates, such as crotonate; catalytic thermal depolymerization yields alkenoates. Cyclic pulse addition of 3HB to triplicate bioreactors selected for an enrichment of Comamonas, Brachymonas and Acinetobacter. After each pulse, poly(3-hydroxybutyrate) (P3HB) transiently appeared: accumulation of P3HB correlated with hydrolysis of polyphosphate; consumption of P3HB correlated with polyphosphate synthesis. Cells removed from the cyclic regime and incubated with 3HB under nitrogen-limited conditions produced P3HB (molecular weight>1,000,000Da) at 50% of the cell dry weight (<8h). P3HB also resulted from incubation with acetate, crotonate, or a mixture of hydrolytic depolymerization products. Poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) resulted from incubation with valerate or 2-pentenoate. A recycling strategy where abiotic depolymerization of waste PHAs yields feedstock for customized PHA re-synthesis appears feasible, without the need for energy-intensive feedstock purification.

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.

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.

Development of Bio-PORec® system for polyhydroxyalkanoates (PHA) production and its storage in mixed cultures of palm oil mill effluent (POME).

High PHA production and storage using palm oil mill effluent (POME) was investigated using a laboratory batch Bio-PORec® system under aerobic-feeding conditions. Results showed that maximum PHA was obtained at a specific rate (q(p)) of 0.343 C-mol/C-molh when air was supplied at 20 ml/min. The PHA yield was found to be 0.80 C-mol/C-mol acetic acid (HAc) at microaerophilic condition and the mass balance calculation showed that PHA production increased up to 15.68±2.15 C-mmol/cycle. The experiments showed that short feeding rate, limited requirements for electron acceptors (e.g. O(2), NO(3)) and nutrients (N and P) showed lower tendency of glycogen accumulation and contributed more to PHA productivity.

Transient embolization with microspheres of polyhydroxyalkanoate renders efficient adenoviral transduction of pancreatic capillary in vivo.

BACKGROUND: Our previous study showed an efficient targeting of islets of Langerhans by adenoviral injection via the celiac trunk. Unexpectedly, none of the endothelial cells was infected given the direct contact between adenoviruses and the capillary wall. The present study intended to provide an efficient approach for adenoviral targeting of the microcapillary endothelial cells in the pancreas. METHODS: We prepared microspheres of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) with a size comparable to the diameter of capillary (5-10 µm). Scanning electron microscopy was applied to verify that adenoviruses carrying a green fluorescence protein gene were complexed with PHBHHx-microspheres after 30 min of co-incubation. The complexes were then injected into the pancreas of mice via the celiac trunk. RESULTS: Approximately 40% of endothelial cells in the pancreas were labeled 5 days after surgery. Islet cells were labeled occasionally, whereas labeling of the acinar and ductal tissues was barely detectable. Endothelium targeting was inefficient in other internal organs. Consistent with the reported superior tissue compatibility of PHBHHx, no discernable microspheres were found in all of the organs examined. Furthermore, splenocyte activation was dampened when adenoviruses were complexed with the microspheres. CONCLUSIONS: The present study has established an approach for efficient pancreatic capillary targeting by using microsphere-adenoviral complexes. This procedure could be invaluable for the treatment of capillary-related diseases.