Impact of various ß-ketothiolase genes on PHBHHx production in Cupriavidus necator H16 derivatives.

Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] (PHBHHx) is a type of biopolyester of the polyhydroxyalkanoate group (PHA). Due to a wide range of properties resulting from the alteration of the (R)-3-hydroxyhexanoate (3HHx) composition, PHBHHx is getting a lot of attention as a substitute to conventional plastic materials for various applications. Cupriavidus necator H16 is the most promising PHA producer and has been genetically engineered to produce PHBHHx efficiently for many years. Nevertheless, the role of individual genes involved in PHBHHx biosynthesis is not well elaborated. C. necator H16 possesses six potential physiologically active ß-ketothiolase genes identified by transcriptome analysis, i.e., phaA, bktB, bktC (h16_A0170), h16_A0462, h16_A1528, and h16_B0759. In this study, we focused on the functionality of these genes in vivo in relation to 3HHx monomer supply. Gene deletion experiments identified BktB and H16_A1528 as important ß-ketothiolases for C6 metabolism in ß-oxidation. Furthermore, in the bktB/h16_A1528 double-deletion strain, the proportion of 3HHx composition of PHBHHx produced from sugar was very low, whereas that from plant oil was significantly higher. In fact, the proportion reached 36.2 mol% with overexpression of (R)-specifc enoyl-CoA hydratase (PhaJ) and PHA synthase. Furthermore, we demonstrated high-density production (196 g/L) of PHBHHx with high 3HHx (32.5 mol%) by fed-batch fermentation with palm kernel oil. The PHBHHx was amorphous according to the differential scanning calorimetry analysis. KEY POINTS: • Role of six ß-ketothiolases in PHBHHx biosynthesis was investigated in vivo. • Double-deletion of bktB/h16_A1528 results in high 3HHx composition with plant oil. • Amorphous PHBHHx with 32.5 mol% 3HHx was produced in high density by jar fermenter.

Microbial Degradation Behavior in Seawater of Polyester Blends Containing Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx).

The microbial degradation behavior of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and its compound with several polyesters such as poly(butylene adipate-co-telephtharate) (PBAT), poly(butylene succinate) (PBS), and polylactic acid (PLA) in seawater was tested by a biological oxygen demand (BOD) method. PHBHHx showed excellent biodegradation in seawater in this study. In addition, the biodegradation rate of several blends was much influenced by the weight ratio of PHBHHx in their blends and decreased in accordance with the decrement of PHBHHX ratio. The surface morphology of the sheet was important factor for controlling the biodegradation rate of PHBHHx-containing blends in seawater.

Polyhydroxyalkanoate production from sucrose by Cupriavidus necator strains harboring csc genes from Escherichia coli W.

Cupriavidus necator H16 is the most promising bacterium for industrial production of polyhydroxyalkanoates (PHAs) because of their remarkable ability to accumulate them in the cells. With genetic modifications, this bacterium can produce poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), which has better physical properties, as well as poly(3-hydroxybutyrate) (PHB) using plant oils and sugars as a carbon source. Considering production cost, sucrose is a very attractive raw material because it is inexpensive; however, this bacterium cannot assimilate sucrose. Here, we used the sucrose utilization (csc) genes of Escherichia coli W to generate C. necator strains that can assimilate sucrose. Especially, glucose-utilizing recombinant C. necator strains harboring the sucrose hydrolase gene (cscA) and sucrose permease gene (cscB) of E. coli W grew well on sucrose as a sole carbon source and accumulated PHB. In addition, strains introduced with a crotonyl-CoA reductase gene (ccr), ethylmalonyl-CoA decarboxylase gene (emd), and some other genetic modifications besides the csc genes and the glucose-utilizing mutations produced PHBHHx with a 3-hydroxyhexanoate (3HHx) content of maximum approximately 27 mol% from sucrose. Furthermore, when one of the PHBHHx-producing strains was cultured with sucrose solution in a fed-batch fermentation, PHBHHx with a 3HHx content of approximately 4 mol% was produced and reached 113 g/L for 65 h, which is approximately 1.5-fold higher than that produced using glucose solution.

Evaluation of gene expression cassettes and production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with a fine modulated monomer composition by using it in Cupriavidus necator.

BACKGROUND: Cupriavidus necator has attracted much attention as a platform for the production of polyhydroxyalkanoate (PHA) and other useful materials. Therefore, an appropriate modulation of gene expression is needed for producing the desired materials effectively. However, there is insufficient information on the genetic engineering techniques required for this in C. necator. RESULTS: We found that the disruption of a potential ribosome binding site (RBS) in the phaC1 gene in C. necator caused a small decrease in the PhaC1 expression level. We applied this result to finely regulate the expression of other genes. Several gene expression cassettes were constructed by combining three Escherichia coli derived promoters (PlacUV5, Ptrc and Ptrp) to the potential RBS of phaC1 or its disruptant, respectively. Their expression levels were then determined via a lacZ reporter assay in C. necator strains. The promoter strengths were both ranked similarly for the cells that were cultured with fructose or palm kernel oil as a sole carbon source (Ptrc ≥ PlacUV5 > Ptrp), both of which were much stronger than the phaC1 promoter. The disruption of RBS had minute attenuation effect on the expression level of these expression cassettes with E. coli promoters. Furthermore, they were used to finely regulate the (R)-3-hydroxyhexanoate (3HHx) monomer ratio in the production of poly[(R)-3-hydroxybutyrate-co-3-hydroxyhexanoate] (PHBHHx) via R-specific enoyl-CoA hydratases (PhaJs). The 3HHx composition in PHBHHx is crucial because it defines the thermal and mechanical properties of the resulting plastic material. The C. necator mutant strains, whose PhaJ expression was controlled under the gene expression cassettes, could be used to produce PHBHHx with various 3HHx compositions in the same culture conditions. CONCLUSIONS: We constructed and evaluated several gene expression cassettes consisting of promoters and RBSs that finely regulate transcription and translation. These were then applied to finely modulate the monomer composition in the production of PHBHHx by recombinant C. necator.

Concanavaline A conjugated bacterial polyester-based PHBHHx nanoparticles loaded with curcumin for breast cancer therapy.

The aim of this study was to evaluate therapeutic potential of curcumin-loaded poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) PHBHHx nanoparticles (CUR-NPs) and concanavaline A conjugated curcumin-loaded NPs (ConA-CUR-NPs) for breast cancer treatment. The size and zeta potential of prepared NPs were about 228 ± 5 nm and -23.3 mV, respectively. The entrapment efficiencies of polymer/drug weight ratios, 1.25CUR-NPs, 2.5CUR-NPs, 5CUR-NPs, ConA-1.25CUR-NPs, ConA-2.5CUR-NPs and ConA-5CUR-NPs were found to be ≈68, 55, 45, 70, 60 and 51%, respectively. Optimized NPs formulations in the freeze-dried form were assessed with their short-term stability for 30 days of storage at 4 °C and 25 °C. Anticancer activity of ConA-CUR-NPs was proved by MTT assay and reconfirmed by double staining and flow cytometry results. The anticancer activity of ConA-CUR-NPs was measured in human breast cancer cells (MDA-MB 231) in vitro, and the results revealed that the ConA-CUR-NPs had better tumor cells decline activity.

Thymopentin nanoparticles engineered with high loading efficiency, improved pharmacokinetic properties, and enhanced immunostimulating effect using soybean phospholipid and PHBHHx polymer.

Formulation of protein and peptide drugs with sustained release properties is crucial to enhance their therapeutic effect and minimize administration frequency. In this study, immunomodulating polymeric systems were designed by manufacturing PHBHHx nanoparticles (NPs) containing thymopentin (TP5). The release profile of the drug was studied over a period of 7 days. The PHBHHx NPs containing TP5-phospholipid (PLC) complex (TP5-PLC) displayed a spherical shape with a mean size, zeta potential, and encapsulation efficiency of 238.9 nm, -32.0 mV, and 72.81%, respectively. The cytotoxicity results showed the PHBHHx NPs had a relatively low toxicity in vitro. TP5 entrapped in the NPs could hardly release in vitro, while the NPs had longer than 7 days release duration after a single subcutaneous injection in Wistar rats. The immunodepression rat model was built to evaluate the immunomodulating effects of TP5-PLC-NPs in vivo. The results of T-lymphocyte subsets (CD3(+), CD4(+), CD8(+), and CD4(+)/CD8(+) ratio) analysis and superoxide dismutase (SOD) values suggested that TP5-PLC-NPs had stronger immunoregulation effects than TP5 solution. In conclusion, an applicable approach to markedly enhancing the loading of a water-soluble peptide into a hydrophobic polymer matrix has been introduced. Thus, TP5-PLC-NPs are promising nanomedicine systems for sustained release effects of TP5.

The effect of poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) (PHBHHx) and human mesenchymal stem cell (hMSC) on axonal regeneration in experimental sciatic nerve damage.

This study is designed to evaluate the treatment effect of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and human mesenchymal stem cells (hMSC) on axonal regeneration in experimental rat sciatic nerve damage, and compare the results of this modality with autologous nerve grafting. In Spraque-Dawley albino rats, 10-mm-long experimental nerve gaps were created. Three groups were constituted, the gap was repaired with autologous nerve graft (autograft group), PHBHHx nerve graft alone (PHBHHx alone group), and PHBHHx nerve graft with hMSCs inside (PHBHHx with hMSC group), respectively. The results were evaluated with functional recovery, electrophysiological evaluation, and histological evaluation either with light microscopy and transmission electron microscopy for axonal regeneration and myelin formation. In functional evaluation, autograft and PHBHHx with hMSC groups showed functional improvement with time, whereas PHBHHx alone group did not. Electrophysiological evaluation showed better results in autograft and PHBHHx with hMSC groups when compared to PHBHHx alone group. There was no statistical difference between autograft and PHBHHx with hMSC groups. Histological evaluation showed regenerated axons in each group. Autograft group was better than the others, and PHBHHx with hMSC group was better than PHBHHx alone group both for axonal regeneration and myelin formation. This study showed that the nerve grafts which were prepared from PHBHHx with oriented nanofiber three-dimensional surfaces aided to nerve regeneration, either used alone or with hMSC. PHBHHx provided better nerve regeneration when used with hMSCs inside than alone, and reached the same statistical treatment effect in functional evaluation and electrophysiological evaluation when compared to autografting.

A bionanocomposite of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)/ZnO-nanoparticles intended for food packaging.

Films-based bionanocomposites have gained a great importance in food plastic packaging because they are eco-friendly materials and have the potential to improve food protection, while limiting the accumulation of synthetic plastics on the planet. In this paper, biofilms were prepared using poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) reinforced with Zinc oxide nanoparticles (ZnO-NPs) to develop new bionanocomposite materials intended for food packaging. The samples were fabricated using first solvent casting method followed by melt compounding at various loading rates, i.e., 1.5, 3 and 6 wt%. The obtained results showed that the incorporation of ZnO-NPs to PHBHHx at 3 wt% leads to higher crystallinity, improved mechanical properties and antimicrobial activity, compared with neat polymer and other bionanocomposites. This was attributed to the finer and homogeneous nanofiller dispersion in the polymer matrix evidenced by scanning electron microscopy analysis. Whereas at 6 wt%, the bionanocomposite sample exhibited low mechanical properties due to the formation of ZnO-NPs aggregates. In view of the obtained results, the study highlights the potential of using the PHBHHx/ZnO-NPs bionanocomposite at 3 wt% in food packaging without any prior filler treatment.

Combined Effect of Poly(lactic acid)-Grafted Maleic Anhydride Compatibilizer and Halloysite Nanotubes on Morphology and Properties of Polylactide/Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Blends.

Given the global challenge of plastic pollution, the development of new bioplastics to replace conventional polymers has become a priority. It is therefore essential to achieve a balance in the performances of biopolymers in order to improve their commercial availability. In this topic, this study aims to investigate the morphology and properties of poly(lactic acid) (PLA)/ poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) (at a ratio of 75/25 (w/w)) blends reinforced with halloysite nanotubes (HNTs) and compatibilized with poly(lactic acid)-grafted maleic anhydride (PLA-g-MA). HNTs and PLA-g-MA were added to the polymer blend at 5 and 10 wt.%, respectively, and everything was processed via melt compounding. A scanning electron microscopy (SEM) analysis shows that HNTs are preferentially localized in PHBHHx nodules rather than in the PLA matrix due to its higher wettability. When HNTs are combined with PLA-g-MA, a finer and a more homogeneous morphology is observed, resulting in a reduction in the size of PHBHHx nodules. The presence of HNTs in the polymer blend improves the impact strength from 12.7 to 20.9 kJ/mm2. Further, with the addition of PLA-g-MA to PLA/PHBHHX/HNT nanocomposites, the tensile strength, elongation at break, and impact strength all improve significantly, rising from roughly 42 MPa, 14.5%, and 20.9 kJ/mm2 to nearly 46 MPa, 18.2%, and 31.2 kJ/mm2, respectively. This is consistent with the data obtained via dynamic mechanical analysis (DMA). The thermal stability of the compatibilized blend reinforced with HNTs is also improved compared to the non-compatibilized one. Overall, this study highlights the effectiveness of combining HNTs and PLA-g-AM for the properties enhancement of PLA/PHBHHx blends.

Production of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from CO2 via pH-Stat Jar Cultivation of an Engineered Hydrogen-Oxidizing Bacterium Cupriavidus necator.

The copolyester of 3-hydroxybutyrate (3HB) and 3-hydoxyhexanoate (3HHx), PHBHHx, is a biodegradable plastic characterized by high flexibility, softness, a wide process window, and marine biodegradability. PHBHHx is usually produced from structurally related carbon sources, such as vegetable oils or fatty acids, but not from inexpensive carbon sources such as sugars. In previous studies, we demonstrated that engineered strains of a hydrogen-oxidizing bacterium, Cupriavidus necator, synthesized PHBHHx with a high cellular content not only from sugars but also from CO2 as the sole carbon source in the flask culture. In this study, the highly efficient production of PHBHHx from CO2 was investigated via pH-stat jar cultivation of recombinant C. necator strains while feeding the substrate gas mixture (H2/O2/CO2 = 80:10:10 v/v%) to a complete mineral medium in a recycled-gas, closed-circuit culture system. As a result, the dry cell mass and PHBHHx concentration with the strain MF01/pBPP-ccrMeJAc-emd reached up to 59.62 ± 3.18 g·L-1 and 49.31 ± 3.14 g·L-1, respectively, after 216 h of jar cultivation with limited addition of ammonia and phosphate solutions. The 3HHx composition was close to 10 mol%, which is suitable for practical applications. It is expected that the autotrophic cultivation of the recombinant C. necator can be feasible for the mass production of PHBHHx from CO2.

Biosynthesis of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from CO2 by a Recombinant Cupriavidusnecator.

The copolyester of 3-hydroxybutyrate (3HB) and 3-hydoxyhexanoate (3HHx), PHBHHx, is one of the most practical kind of bacterial polyhydroxyalkanoates due to its high flexibility and marine biodegradability. PHBHHx is usually produced from vegetable oils or fatty acids through ß-oxidation, whereas biosynthesis from sugars has been achieved by recombinant strains of hydrogen-oxidizing bacterium Cupriavidus necator. This study investigated the biosynthesis of PHBHHx from CO2 as the sole carbon source by engineered C. necator strains. The recombinant strains capable of synthesizing PHBHHx from fructose were cultivated in a flask using complete mineral medium and a substrate gas mixture (H2/O2/CO2 = 8:1:1). The results of GC and 1H NMR analyses indicated that the recombinants of C. necator synthesized PHBHHx from CO2 with high cellular content. When 1.0 g/L (NH4)2SO4 was used as a nitrogen source, the 3HHx composition of PHBHHx in the strain MF01∆B1/pBBP-ccrMeJ4a-emd was 47.7 ± 6.2 mol%. Further investigation demonstrated that the PHA composition can be regulated by using (R)-enoyl-CoA hydratase (PhaJ) with different substrate specificity. The composition of 3HHx in PHBHHx was controlled to about 11 mol%, suitable for practical applications, and high cellular content was kept in the strains transformed with pBPP-ccrMeJAc-emd harboring short-chain-length-specific PhaJ.

Manipulating cell behavior on a bacterial macro-polymer poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) via tuning the S-doped graphene ratio.

Graphene is a material with various application potentials Graphene is a unique material with superiorities and has been applied in various fields for different purposes. Although studies on the utility of graphene oxide in the biomedical field are available, no evaluation has yet been done regarding the utility of sulfur doped (S-doped) graphene. The study focuses on the effect of blending the poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) membrane with sulfur heteroatom doped graphene and the evaluation of biological responses to S-doped graphene/PHBHHx. PHBHHx membranes were blended with 1%, 0.5%, 0.1% (w/v) S-doped graphene. The morphological (SEM and Microscopy), chemical (FTIR and Raman spectroscopy), and surface area (BET) characterizations of S-doped graphene/PHBHHx membranes were performed. The presence of S groups on the surface was determined with the EDS results. Besides, the swelling profile and biodegradation tendency of the membranes were evaluated. The differentiation of protein adhesion, cell viability, cell adhesion, and cell proliferation by the increasing content of S-doped graphene was examined. The contact angle analysis revealed that modification of PHBHHx with S-doped Graphene reduced the free surface energy of PHBHHx membranes. Blending with S-doped Graphene has decreased the polarity of the PHBHHx membrane. The protein adsorption on the PHBHHx membrane was determined as 10.12 ± 0.247 mg/ml. Protein absorption on 1%, 0.5% and 0.1% S-doped graphene/PHBHHx membranes were determined as 11.34 ± 0.551 mg/ml, 9.91 ± 0.294 mg/ml and 9.48 ± 0.093 mg/ml, respectively. The cell attachment to the surface decreased with the increasing amount of S-doped graphene, however, PHBHHx membranes with graphene did not affect cytotoxicity. S-doped graphene blended PHBHHx membrane seems like a suitable patch for biomedical treatments as a hydrophobic membrane where less cell adhesion and proliferation are required like the prevention of peritoneal adhesion.

Implantable sandwich PHBHHx film for burst-free controlled delivery of thymopentin peptide.

Sustained release and non-parental formulations of peptides and protein drugs are highly desirable because of enhanced therapeutic effects as well as improved patient compliance. This is especially true for small peptides such as thymopentin (TP5). To this end, implantable sandwich poly (hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) films were designed to prolong release time and to inhibit burst release phenomenon of TP5 by a simple volatilization method. In vitro release studies revealed that sandwich films had nearly no burst release. In vivo release time of sandwich films was prolonged to 42 days. Pharmacodynamic evaluation demonstrated that TP5 sandwich films significantly increased survival rates in a rat immunosuppressive model and normalized CD4+/CD8+ values. These results suggest that TP5 released from sandwich films can attenuate cyclophosphamide's immunosuppressive activity, and possibly achieve results comparable to daily TP5 injection therapy. Thus, sandwich PHBHHx films show excellent potential as a sustained, burst-free release system for small molecular weight, hydrophilic peptide drugs.

A Specific Drug Delivery System for Targeted Accumulation and Tissue Penetration in Prostate Tumors Based on Microbially Synthesized PHBHHx Biopolyester and iRGD Peptide Fused PhaP.

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is an intracellular biopolyester synthesized by various bacteria. Polyhydroxyalkanoate granule-binding protein (PhaP), a natural biomacromolecule symbiotic with PHBHHx, can be steadily adsorbed into the PHBHHx matrix through hydrophobic interactions. In this study, PHBHHx nanoparticles (NPs) and iRGD peptide fused PhaP (iRGD-PhaP) were used in conjunction to build a specific drug delivery system for targeted accumulation and tissue penetration in prostate tumors. A proper presentation and high surface density of iRGD could be ensured within 1 h through a convenient coincubation method using a PhaP-mediated modification strategy. iRGD-PhaP-NPs showed a satisfactory particle size (182.9 ± 4.9 nm) and slightly negative surface charge (-17.2 ± 0.3 mV), with a uniformly spherical shape. In human prostate cancer cell line PC3, iRGD-PhaP-NPs displayed remarkably improved cellular uptake compared to naked NPs, which was attributed to iRGD receptor-mediated active endocytosis. Enhanced targeted accumulation and retention of iRGD-PhaP-NPs in prostate tumors were found in both the ex vivo tumor spheroid assay and in vivo real-time imaging. Moreover, slices of the tumor deep region demonstrated the favorable tumor penetration ability of iRGD-PhaP-NPs after intravenous administration. These results highlight the specificity and efficiency of iRGD-PhaP-NPs in future clinical use.

Enhanced cell affinity of PHBHHx composite scaffold with polylactide-graft-hydroxyapatite as compatibilizer.

Poor interfacial bonding and liability to aggregation were the major obstacles for designing of composite scaffolds. Herein, hydroxyapatite (HA) surface-grafted with poly (l-lactide) via ring-opening polymerization was prepared and introduced into PHBHHx-based complex scaffold serving as oligomer compatibilizer. The physicochemical properties, including superficial roughness and wettability, mechanical strength, as well as in vitro bioactivity were systematically investigated. Improved cellular response was acquired owing to the favourable uniformity and interfacial compatibility, as well as the decreased contact angle and elevated roughness, through surface-modification strategy. Moreover, some of the osteogenic marker genes (COL I, Runx2, OCN and OPN) were up-regulated, thus made for the osteogenic differentiation. Our study extrapolated that the PHBHHx-based composite scaffold combined with surface-graft HA compatibilizer might be a potential candidate for bone repairing.

[Development of tumor targeting PHBHHx nanoparticles by PhaP mediated immobilization of EGFR-targeting peptide].

PHA granule binding protein phasin (PhaP) has a high affinity for hydrophobic materials and can bind to hydrophobic polymers via strong hydrophobic interaction. In this study, an EGFR-targeting peptide (ETP) was fused with PhaP and the fusion protein ETP-PhaP was produced in recombinant Escherichia coli BL21 (DE3) (pPI-ETP-P) and then purified by Ni affinity purification. The tumor targeting PHBHHx nanoparticles were developed based on PhaP mediated ETP immobilization and the cellular uptake of the ETP-PhaP modified PHBHHx NPs and none modified PHBHHx NPs by cervical cancer cell lines SiHa (EGFR over expressed) and CaSKi (EGFR low expressed) were analyzed. The purified ETP-PhaP could be adsorbed onto the hydrophobic surface of PHBHHx NPs. The ETP-PhaP modified PHBHHx NPs could target to EGFR over expressed cervical cancer cells SiHa more efficiently than to the EGFR low expressed CaSKi cells. These results demonstrated the advantage in effectiveness and convenience of PhaP mediated ETP adsorption on PHBHHx nanoparticles, providing a novel strategy for hydrophobic nanocarrier surface modification.

Concentration-dependent protein adsorption at the nano-bio interfaces of polymeric nanoparticles and serum proteins.

AIM: A comprehensive understanding of nanoparticle (NP)-protein interaction (protein corona formation) is required. So far, many factors influencing this interaction have been investigated, like size and ζ potential. However, NPs exposure concentration has always been ignored. Herein, we aim to disclose the correlation of NPs exposure concentration with protein adsorption. MATERIALS & METHODS: Four polymeric NPs systems possessing similar sizes (230 ± 20 nm) but varied ζ potentials (-30 ∼ +40 mv) were prepared. Physicochemical properties and protein adsorption upon NP-protein interaction were characterized. RESULTS: Protein adsorption capacity and adsorbed protein types were NPs concentration-dependent. CONCLUSION: Considering the critical impacts of protein adsorption on NPs delivery, our work could be an urgent warning about the possible risks of dosage adjustment of nanoformulations.

Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from excess activated sludge as a promising substitute of pure culture.

This study aimed to investigate the feasibility and technology to harvest poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) by mixed culture. Copolymer PHBHHx, usually fermented by pure strains, was reported to be synthesized from activated sludge for the first time. Sodium laurate was used as the sole carbon substrate for sludge acclimation and PHBHHx accumulation. Batch experiments were designed to look into the impact of the carbon, nitrogen, phosphorus and oxygen supply on PHBHHx production. The results showed that the acclimated excess sludge was able to produce PHBHHx, and the maximum output (505.6 mg/L PHBHHx containing 6.34 mol% HHx) was achieved with conditions of the continuous aeration, nitrogen and phosphorus limitation, and adequate carbon source implemented by pulse feeding 0.5 g/L sodium laurate every 4h. Moreover, composition and structure of the PHBHHx from sludge were found similar to that from pure culture, according to literature, FTIR and NMR spectra. Finally, high-throughput sequencing technique characterized that phylum Chlorobi and genus Leadbetterella should be critical groups for PHBHHx synthesis in the sludge community.

Blood compatibility of a ferulic acid (FA)-eluting PHBHHx system for biodegradable magnesium stent application.

Magnesium stent has shown potential application as a new biodegradable stent. However, the fast degradation of magnesium stent limited its clinic application. Recently, a biodegradable and drug-eluting coating system was designed to prevent magnesium from fast degradation by adding ferulic acid (FA) in poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) by a physical method. In vitro study has demonstrated that the FA-eluting system exhibited strong promotion to the endothelialization, which might be a choice for the stent application. In this paper, the hemolysis rate, the plasma recalcification time (PRT), the plasma prothrombin time (PT) and the kinetic clotting time of the FA-eluting films were investigated and the platelet adhesion was observed in order to assess the blood compatibility of the FA-eluting PHBHHx films in comparison with PHBHHx film. The results have shown that the addition of FA had no influence on the hemolysis, but prolonged PRT, PT and the clotting time and reduced the platelet adhesion and activation, displaying that the FA-eluting PHBHHx exhibited better blood compatibility than PHBHHx. In addition, the effect of alkali treatment on the blood compatibility of FA-eluting PHBHHx was also studied. It was indicated that alkali treatment had no effect on the hemolysis and the coagulation time, but enhanced slightly the platelet adhesion. All these demonstrated that FA-eluting PHBHHx film had good blood compatibility and might be a candidate surface coating for the biodegradable magnesium stent.

A ferulic acid (FA)-eluting system for biodegradable magnesium stent: Cells response of HUVECs.

A new drug-eluting system was designed for biodegradable magnesium stents, in which ferulic acid (FA) was used as drug due to its promotion function to endothelial cells and PHBHHx as drug delivery due to its biodegradability and biocompatibility. A 5 and 10% FA were added in PHBHHx to prepare FA containing PHBHHX films. The cell adhesion, spreading and proliferation on these films was investigated in order to assess cell response of HUVECs. It was also found that FA enhanced the adhesion, spreading and proliferation of HUVECs in a dose-dependent manner. Cell viability after H2 O2 injury and NO production of HUVECs were also studied. The results indicated that FA effectively inhibited H2 O2 -induced injury and promotes NO production. It was also shown that alkali treatment improved the cell adhesion, spreading and proliferation while the treatment reduces the FA release and in turn reduces the inhibition on H2 O2 -induced injury and NO production. However, alkali treatment itself had no influence on the H2 O2 induced injure and NO production. The tensile shear strength between the FA containing coating and Mg substrate was also tested. All results demonstrated that FA containing PHBHHx films exhibited strong promotion to the endothelialization and could be a choice for surface modification of magnesium stent.