Multifunctional Electrospinning Polyhydroxyalkanoate Fibrous Scaffolds with Antibacterial and Angiogenesis Effects for Accelerating Wound Healing.

To treat large-scale wounds or chronic ulcers, it is highly desirable to develop multifunctional wound dressings that integrate antibacterial and angiogenic properties. While many biomaterials have been fabricated as wound dressings for skin regeneration, few reports have addressed the issue of complete skin regeneration due to the lack of vasculature and hair follicles. Herein, an instructive poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB) fibrous wound dressing that integrates an antibacterial ciprofloxacin (CIP) and pro-angiogenic dimethyloxalylglycine (DMOG) is successfully prepared via electrospinning. The resultant dressings exhibit suitable flexibility with tensile strength and elongation at break up to 4.08 ± 0.18 MPa and 354.8 ± 18.4%, respectively. The in vitro results revealed that the groups of P34HB/CIP/DMOG dressings presented excellent biocompatibility on cell proliferation and significantly promote the spread and migration of L929 cells in both transwell and scratch assays. Capillary-like tube formation is also significantly enhanced in the P34HB/CIP/DMOG group dressings. Additionally, dressings from the P34HB/CIP and P34HB/CIP/DMOG groups show a broad spectrum of antimicrobial action against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. In vivo studies further demonstrated that the prepared dressings in the P34HB/CIP/DMOG group not only improved wound closure, increased re-epithelialization and collagen formation, as well as reduced inflammatory response but also increased angiogenesis and remodeling, resulting in complete skin regeneration and hair follicles. Collectively, this work provides a simple but efficient approach for the design of a versatile wound dressing with the potential to have a synergistic effect on the rapid stimulation of angiogenesis as well as antibacterial activity in full-thickness skin repair.

In Search of Effective Anticancer Agents-Novel Sugar Esters Based on Polyhydroxyalkanoate Monomers.

Cancer is one of the deadliest illness globally. Searching for new solutions in cancer treatments is essential because commonly used mixed, targeted and personalized therapies are sometimes not sufficient or are too expensive for common patients. Sugar fatty acid esters (SFAEs) are already well-known as promising candidates for an alternative medical tool. The manuscript brings the reader closer to methods of obtaining various SFAEs using combined biological, chemical and enzymatic methods. It presents how modification of SFAE's hydrophobic chains can influence their cytotoxicity against human skin melanoma and prostate cancer cell lines. The compound's cytotoxicity was determined by an MTT assay, which followed an assessment of SFAEs' potential metastatic properties in concentrations below IC50 values. Despite relatively high IC50 values (63.3-1737.6 µM) of the newly synthesized SFAE, they can compete with other sugar esters already described in the literature. The chosen bioactives caused low polymerization of microtubules and the depolymerization of actin filaments in nontoxic levels, which suggest an apoptotic rather than metastatic process. Altogether, cancer cells showed no propensity for metastasis after treating them with SFAE. They confirmed that lactose-based compounds seem the most promising surfactants among tested sugar esters. This manuscript creates a benchmark for creation of novel anticancer agents based on 3-hydroxylated fatty acids of bacterial origin.

Eugenol-piperine loaded polyhydroxy butyrate/polyethylene glycol nanocomposite-induced apoptosis and cell death in nasopharyngeal cancer (C666-1) cells through the inhibition of the PI3K/AKT/mTOR signaling pathway.

Nasopharyngeal cancer is a malignancy developing from the nasopharynx epithelium due to smoking and nitrosamine-containing foods. Nasopharyngeal cancer is highly endemic to Southeast Asia. Eugenol and piperine have shown many anticancer activities on numerous cancer types, like colon, lung, liver, and breast cancer. In this study, we amalgamated eugenol and piperine loaded with a polyhydroxy butyrate/polyethylene glycol nanocomposite (Eu-Pi/PHB-PEG-NC) for better anticancer results against nasopharyngeal cancer (C666-1) cells. In the current study, nasopharyngeal cancer cell lines C666-1 were utilized to appraise the cytotoxic potential of Eug-Pip-PEG-NC on cell propagation, programmed cell death, and relocation. Eu-Pi/PHB-PEG-NC inhibits cellular proliferation on C666-1 cells in a dose-dependent manner, and when compared with 20 µg/ml, 15 µg/ml of loaded mixture evidently restrained the passage aptitude of C666-1 cells, this was attended with a downregulated expression of mitochondrial membrane potential. Treatment with 15 µg/ml Eu-Pi/PHB-PEG-NC suggestively amplified cell apoptosis in the C666-1 cells. Furthermore, its cleaved caspase-3, 8, and 9 and Bax gene expression was augmented and Bcl-2 gene expression was diminished after Eu-Pi/PHB-PEG-NC treatment. Additionally, our data established that the collective effect of Eu-Pi/PHB-PEG-NC loaded micelles inhibited the expansion of C666-1 cells augmented apoptosis connected with the intrusion of PI3K/Akt/mTOR signaling pathway.

Antimicrobial PHAs coatings for solid and porous tantalum implants.

Biomaterial-associated infections (BAI) are the major cause of failure of indwelling medical devices. The risk of BAI can end dramatically in the surgical removal of the affected device. Therefore, a major effort must be undertaken to guarantee the permanence of the implant. In this regard, we have developed antimicrobial coatings for tantalum (Ta) implants, using polyhydroxyalkanoates (PHAs) as matrices for carrying an active principle. The dip-coating technique was successfully used for covering solid Ta discs. An original PHA emulsion flow process was developed for the coating of porous Ta structures, specially for the inner surfaces. The complete characterization of the biopolymer coatings, their antibacterial properties, toxicity and biointegration were analyzed. Thus, non-toxic, well-biointegrated homogeneous biopolymer coatings were attained, which showed antibacterial properties. By using biodegradable PHAs, the resulting drug delivery system assured the protection of Ta against bacterial infections for a period of time.

Integrating Molecular Network and Culture Media Variation to Explore the Production of Bioactive Metabolites by Vibrio diabolicus A1SM3.

Vibrio diabolicus A1SM3 strain was isolated from a sediment sample from Manaure Solar Saltern in La Guajira and the produced crude extracts have shown antibacterial activity against methicillin-resistant Staphylococcus aureus and cytotoxic activity against human lung cell line. Thus, the aim of this research was to identify the main compound responsible for the biological activity observed and to systematically study how each carbon and nitrogen source in the growth media, and variation of the salinity, affect its production. For the characterization of the bioactive metabolites, 15 fractions obtained from Vibrio diabolicus A1SM3 crude extract were analyzed by HPLC-MS/MS and their activity was established. The bioactive fractions were dereplicated with Antibase and Marinlit databases, which combined with nuclear magnetic resonance (NMR) spectra and fragmentation by MS/MS, led to the identification of 2,2-di(3-indolyl)-3-indolone (isotrisindoline), an indole-derivative antibiotic, previously isolated from marine organisms. The influence of the variations of the culture media in isotrisindoline production was established by molecular network and MZmine showing that the media containing starch and peptone at 7% NaCl was the best culture media to produce it. Also, polyhydroxybutyrates (PHB) identification was established by MS/MS mainly in casamino acids media, contributing to the first report on PHB production by this strain.

Polyhydroxyalkanoates (PHA) for therapeutic applications.

As intracellular carbon and energy storage materials, polyhydroxyalkanoates (PHA) are a diverse biopolyesters synthesized by many bacteria. PHA have been produced in large quantity for various application research including medical implants for approximately 30years. Many studies demonstrated that PHA are promising implant materials due to their diverse and ascendant mechanical, biodegradable and tissue compatible properties. Importantly, common PHA biodegradation products including oligomers and monomers are also not toxic to the cells and tissues. Pharmaceutical applications of some PHA degradation products also have been reported. So far, no study has been reported to have any carcinogenesis result induced by any PHA or their biodegradation products. All results suggest that PHA could be developed into various bio-implant products.

Biodegradable and Biocompatible Polyhydroxy-alkanoates (PHA): Auspicious Microbial Macromolecules for Pharmaceutical and Therapeutic Applications.

Polyhydroxyalkanoates (PHA) are bio-based microbial biopolyesters; their stiffness, elasticity, crystallinity and degradability are tunable by the monomeric composition, selection of microbial production strain, substrates, process parameters during production, and post-synthetic processing; they display biological alternatives for diverse technomers of petrochemical origin. This, together with the fact that their monomeric and oligomeric in vivo degradation products do not exert any toxic or elsewhere negative effect to living cells or tissue of humans or animals, makes them highly stimulating for various applications in the medical field. This article provides an overview of PHA application in the therapeutic, surgical and tissue engineering area, and reviews strategies to produce PHA at purity levels high enough to be used in vivo. Tested applications of differently composed PHA and advanced follow-up products as carrier materials for controlled in vivo release of anti-cancer drugs or antibiotics, as scaffolds for tissue engineering, as guidance conduits for nerve repair or as enhanced sutures, implants or meshes are discussed from both a biotechnological and a material-scientific perspective. The article also describes the use of traditional processing techniques for production of PHA-based medical devices, such as melt-spinning, melt extrusion, or solvent evaporation, and emerging processing techniques like 3D-printing, computer-aided wet-spinning, laser perforation, and electrospinning.

Microbial polyhydroxyalkanoates as medical implant biomaterials.

Polyhydroxyalkanoates (PHAs), a diverse biopolyester synthesized by many bacteria as intracellular carbon and energy storage materials, have been produced in large quantity for various application researches including medical implants for approximately 30 years. It has been demonstrated by many studies that PHAs possess the required mechanical, biodegradable and tissue-compatible properties for implant applications. Very importantly, common PHA biodegradation products including oligomers and monomers are also not toxic to the cells and tissues. Some PHA degradation products have been studied for pharmaceutical applications. Mechanisms of PHA that stimulate cell growth were revealed. So far, no study has been reported to have any carcinogenesis result induced by any PHA or their biodegradation products. All results point to the feasibility of PHA to be developed into various bio-implant products.

[Bio-modification of polyhydroxyalkanoates and its biocompatibility with chondrocytes].

OBJECTIVE: To study the hydrophilicity and the cell biocompatibility of the poly(3-hydroxybutyrate-co- 3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) coated with a fusion protein polyhydroxyalkanoates granule binding protein (PhaP) fused with Arg-Gly-Asp (RGD) peptide (PhaP-RGD). METHODS: PHBV and PHBHHx films were fabricated by solvent evaporation. Scanning electronic microscope (SEM) was used to study the morphology of the films. PhaP-RGD fusion proteins were expressed and purified by the technology of protein engineering; PHBV and PHBHHx films were immersed in the PhaP-RGD with an amount of 3.5 mg/mL protein/per sample respectively. The hydrophilicity of the surface were detected by the contact angle measurements. Septal cartilage cells obtained from human septal cartilage were cultured in vitro. The 2nd passage chondrocytes were incubated on PHBV unmodified with PhaP-RGD in group A1, PHBV modified with PhaP-RGD in group A2, PHBHHx unmodified with PhaP-RGD in group Bl, PHBHHx modified with PhaP-RGD in group B2, and on the cell culture plates in group C. After cultured for 3 days, the proliferation of cells was detected by the DAPI staining; the proliferation viability of cells was detected by the MTT assay after cultured for 3 and 7 days; after cultured for 7 days, the adhesion and morphology of the cells on the surface of the biomaterial films were observed by SEM and the matrix of the cells was detected through the toluidine blue staining. RESULTS: SEM observation showed that PHBV and PHBHHx films had porous structures. The contact angle of the surface of the PHBV and PHBHHx films modified with PhaP-RGD fusion proteins were significantly reduced when compared with the films unmodified with PhaP-RGD fusion proteins (P < 0.05). Chondrocytes of human nasal septal cartilage incubated on the films could grow in all groups. After 3 days of cultivation in vitro, the cell proliferation and viability of group B2 were the strongest among all groups (P < 0.05); the cell proliferation after cultured for 7 days was significantly stronger than that after cultured for 3 days in groups A1, A2, B1, and B2 (P < 0.05); and the cell proliferation was significantly stronger in groups B1 and B2 than groups A1, A2 and C, in group B2 than group B1, and in group A1 than group A2 (P < 0.05). The results of toluidine blue staining showed that blue metachromasia matrixes were observed in groups A1, A2, B1, and B2; group A1 and group A2 had similar staining degree, and the staining of group B2 was deeper than that of group B1. The adhesion of cells in all groups was good through SEM observation; and the connection of cells formed and stretched into the pores of the materials. CONCLUSION: The biomaterial films of PHBHHx modified with PhaP-RGD fusion protein can promote its biocompatibility with chondrocytes.

Reaction of the rat tissues to implantation of polyhydroxyalkanoate films and ultrafine fibers.

The reaction of various tissues of rats to implantation of polyhydroxyalkanoate films and ultrafine fibers was studied by optic microscopy. Implantation of polyhydroxyalkanoate films into the abdominal cavity caused a peritoneal reaction, leading after 1 month to the formation of fibrous adhesions between polyhydroxyalkanoate and intestinal loops. Under the skin and in the muscle tissue polyhydroxyalkanoate films were encapsulated in a thick fibrous capsule. Implantation of polyhydroxyalkanoate ultrathin fibers led to formation of foreign body granulomas in all tissues with perifocal inflammation and sclerosis of the adjacent tissues. The polymer was fragmented in these granulomas and phagocytosed by macrophages with the formation of giant foreign body cells. Hence, polyhydroxyalkanoate materials implanted in vivo caused chronic granulomatous inflammatory reaction and were very slowly destroyed by macrophages.

The anti-cancer activity of a cationic anti-microbial peptide derived from monomers of polyhydroxyalkanoate.

The biodegradable polymer medium chain length polyhydroxyalkanoate (mclPHA), produced by Pseudomonas putida CA-3, was depolymerised and the predominant monomer (R)-3-hydroxydecanoic acid (R10) purified. R10 was conjugated to a d-peptide DP18 and its derivatives. All peptides conjugated with R10 exhibited greater anti-cancer activity compared to the unconjugated peptides. Unconjugated and conjugated peptides were cytocidal for cancer cells. Conjugation of R10 to peptides was essential for enhanced anti-proliferation activity, as unconjugated mixes did not result in enhancement of anti-cancer activity. The conjugation of R10 resulted in more rapid uptake of peptides into HeLa and MiaPaCa cells compared to unconjugated peptide. Both unconjugated and R10 conjugated peptides localized to the mitochondria of HeLa and MiaPaCa cells and induced apoptosis. Peptide conjugated with a terminally hydroxylated decanoic acid (ω-hydroxydecanoic acid) exhibited 3.3 and 6.3 fold higher IC(50) values compared to R10 conjugated peptide indicating a role for the position of the hydroxyl moiety in enhancement of anti-cancer activity. Conjugation of decanoic acid (C10) to peptides resulted in similar or higher IC(50) values compared to R10 conjugates but C10 conjugates did not exhibit any cancer selectivity. Combination studies showed that R10DP18L exhibited synergy with cisplatin, gemcitabine, and taxotere with IC(50) values in the nanomolar range.

Comparison between aerobic and anoxic metabolism of denitrifying-EBPR sludge: effect of biomass poly-hydroxyalkanoates content.

Biomass with denitrifying phosphate uptake ability was tested under sequencing anaerobic-aerobic and anaerobic-anoxic conditions. The initial dose of acetate, under anaerobic conditions varied to achieve different PHA (poly-hydroxyalkanoates) saturation of PAO (polyphosphate accumulating organisms) cells. Increased acetate dosage under anaerobic conditions led to higher phosphate release and increased PHA storage by PAOs and, also, to greater phosphate uptake rates under the following aerobic and/or anoxic conditions. The experimental results also indicated that when organic carbon is limited under anaerobic conditions, more internal glycogen supplementary to polyphosphate cleavage is utilized by the biomass, resulting in less phosphate release and more PHA stored per acetate taken up. In the subsequent aerobic and/or anoxic phase PAOs demonstrate an improved EBPR (enhanced biological phosphorus removal) performance, with regard to PHA consumption per phosphate taken up, for reduced initial biomass PHA content under both aerobic and anoxic conditions. The examination of EBPR biomass under controlled operational conditions, where experimental analysis of the relevant compounds in the bulk phase (PO(4)(3-), NO(3)(-) and/or O(2)) in conjunction with the biomass intracellular products (PHA, glycogen), contributes to an improved understanding of the PAOs metabolic behavior, with regard to organic substrate availability.

The cytocompatability of polyhydroxyalkanoates coated with a fusion protein of PHA repressor protein (PhaR) and Lys-Gln-Ala-Gly-Asp-Val (KQAGDV) polypeptide.

Microbial polyhydroxyalkanoates (PHAs) are a family of polyesters with biodegradability, biocompatibility and adjustable mechanical properties that are under intensive development for bioimplant applications. In this research, a fusion protein of PHA repressor protein (PhaR) and Lys-Gln-Ala-Gly-Asp-Val (KQAGDV) oligopeptide (PhaR-KQAGDV) was utilized to enhance the PHA cytocompatability via a mechanism of PhaR hydrophobically binding to PHA coupled with KQAGDV oligopeptide, a specific ligand to the integrins on the cell surface, for promotion of cell adhesion. The PhaR-KQAGDV fusion protein successfully produced and purified from recombinant E. coli was used to coat the surfaces of several PHA including poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), respectively. The PhaR was observed to bind efficiently on all PHA surfaces measured by the fluorescence intensity of PhaR-EGFP as compared to the uncoated (PhaR negative) PHA films. The PHA surface hydrophilicity measured by water contact angles was significantly improved after PhaR-KQAGDV coating. Observations under confocal microscope and scanning electron microscopy, together with CCK-8 assays clearly demonstrated that adhesion and proliferation of human vascular smooth muscle cells (HvSMCs) inoculated on PHA films were much better on PhaR-KQAGDV coated surfaces than the non-coated control ones. The convenient physical coating approach for enhanced PHA cytocompatibility provides an advantage for PHA based tissue engineering.

MicroRNA regulation associated chondrogenesis of mouse MSCs grown on polyhydroxyalkanoates.

Microbial polyhydroxyalkanoates (PHA) including poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) were found to induce chondrogenesis of mesenchymal stem cells (MSCs) and preserve chondrocytic phenotype as well as support chondrocytes-specific extracellular matrix (ECM) secretion. In this study, mouse MSCs cultured on the PHBHHx films for 24 h showed up-regulated expression of chondrogenic marker genes including aggrecan, col2, sox9, col10 and pthrp. To further illustrate this phenomonon, chondrogenesis-related microRNA expression profiling was examined by quantitative real-time PCR (RT-PCR) based on results of microRNA array obtained from comparison between mouse MSCs and mature mouse chondrocytes. Among 44 microRNAs related to chondrogenesis on microrray studies, considering only broadly-conserved microRNAs, seven differentially-expressed microRNAs were selected to study their target genes related to chondrogenesis. Two microRNAs out of the seven, namely, miR-29a and miR-29b, were revealed to directly target 3' UTR of col2a1 encoding type II collagen by dual-luciferase assay, and their activity was under the regulation of Sox9, the SRY-related high mobility group-box gene 9. For the first time microRNAs were shown to regulate the stem cell differentiation processes mediated by cell-material interactions.

Chondrogenic differentiation of human bone marrow mesenchymal stem cells on polyhydroxyalkanoate (PHA) scaffolds coated with PHA granule binding protein PhaP fused with RGD peptide.

Hydrophobic polyhydroxyalkanoate (PHA) scaffolds made of a copolyester of 3-hydroxybutyrate-co-hydroxyhexanoate (PHBHHx) were coated with a fusion protein PHA granule binding protein PhaP fused with RGD peptide (PhaP-RGD). Human bone marrow mesenchymal stem cells (hBMSCs) were inoculated on/in the scaffolds for formation of articular cartilages derived from chondrogenic differentiation of hBMSCs for cartilage tissue engineering. PhaP-RGD coating led to more homogeneous spread of cells, better cell adhesion, proliferation and chondrogenic differentiation in the scaffolds compared with those of PhaP coated or uncoated scaffolds immerging in serum minus chondrogenic induction medium. In addition, more extracellular matrices were produced by the differentiated cells over a period of 14 days on/in the PhaP-RGD coated scaffolds evidenced by scanning electron microscopy imaging, enhanced expression of chondrocyte specific genes including SOX-9, aggrecan and type II collagen, suggesting the positive effect of RGD on extracellular matrix production. Furthermore, cartilage-specific extracellular substances sulphated glycosaminoglycans (sGAG) and total collagen content found on/in the PhaP-RGD coated scaffolds were significantly more compared with that produced by the control and PhaP only coated scaffolds. Homogeneously distributed chondrocytes-like cells forming cartilage-like matrices were observed on/in the PhaP-RGD coated scaffolds after 3 weeks. The results suggested that PhaP-RGD coated PHBHHx scaffold promoted chondrogenic differentiation of hBMSCs and could support cartilage tissue engineering.

Anticancer properties of highly purified L-asparaginase from Withania somnifera L. against acute lymphoblastic leukemia.

Withania somnifera L. has been traditionally used as a sedative and hypnotic. The present study was carried out for the purification, characterization, and in vitro cytotoxicity of L-asparaginase from W. somnifera L. L-Asparaginase was purified from the fruits of W. somnifera L. up to 95% through chromatography. The purified L-asparaginase was characterized by size exclusion chromatography, polyacrylamide gel electrophoresis (PAGE), and 2D PAGE. The antitumor and growth inhibition effect of the L-asparaginase was assessed using [3-(4, 5-dimethyl-thiazol-2yl)-2, 5-diphenyl-tetrazolium bromide] (MTT) colorimetric dye reduction method. The purified enzyme is a homodimer, with a molecular mass of 72 +/- 0.5 kDa, and the pI value of the enzyme was around 5.1. This is the first report of the plant containing L-asparaginase with antitumor activity. Data obtained from the MTT assay showed a LD(50) value of 1.45 +/- 0.05 IU/ml. W. somnifera L. proved to be an effective and a novel source of L: -asparaginase. Furthermore, it shows a lot of similarity with bacterial L-asparaginases EC-2.