Analysis of Medium-Chain-Length Polyhydroxyalkanoate-Producing Bacteria in Activated Sludge Samples Enriched by Aerobic Periodic Feeding.

Analysis of mixed microbial populations responsible for the production of medium-chain-length polyhydroxyalkanoates (MCL-PHAs) under periodic substrate feeding in a sequencing batch reactor (SBR) was conducted. Regardless of activated sludge samples and the different MCL alkanoic acids used as the sole external carbon substrate, denaturing gradient gel electrophoresis analysis indicated that Pseudomonas aeruginosa was the dominant bacterium enriched during the SBR process. Several P. aeruginosa strains were isolated from the enriched activated sludge samples. The isolates were subdivided into two groups, one that produced only MCL-PHAs and another that produced both MCL- and short-chain-length PHAs. The SBR periodic feeding experiments with five representative MCL-PHA-producing Pseudomonas species revealed that P. aeruginosa has an advantage over other species that enables it to become dominant in the bacterial community.

Enhanced Photosensing and Photodynamic Treatment of Colon Cancer Cells Using Methoxy Poly(ethylene glycol)-Conjugated Chlorin e6.

Nanophotosensitizer composed of methoxy poly(ethylene glycol) (MePEG) and chlorin e6 (Ce6) (abbreviated as Pe6) was synthesized for efficient delivery of Ce6 to the colon cancer cells. Pe6 nanophotosensitizer has small diameter less than 100 nm with spherical shape and core-shell structure. They were activated in aqueous solution while Ce6 was quenched due to its poor aqueous solubility. They showed no intrinsic cytotoxicity against normal cells and colon cancer cells. Pe6 nanophotosensitizers showed enhanced cellular uptake, phototoxicity, and reactive oxygen species (ROS) generation at in vitro cell culture experiment. Furthermore, they showed improved tumor tissue penetration and accumulation in vivo animal studies. We suggested Pe6 nanophotosensitizers as an ideal candidate for PDT of colon cancer.

Biocatalytic characterization of an endo-ß-1,4-mannanase produced by Paenibacillus sp. strain HY-8.

OBJECTIVES: To evaluate the biocatalytic characteristics of a new endo-ß-1,4-D-mannan-degrading enzyme (ManP) from Paenibacillus sp. strain HY-8, a gut bacterium of the longicorn beetle Moechotypa diphysis. RESULTS: Purified ManP (32 kDa) with an N-terminal amino acid sequence of APSFAVGADFSYVPG displayed the greatest degree of biocatalytic activity toward locust bean gum (LBG) at 55 °C and pH 7.0. The enzyme degraded LBG, guar gum, ivory nut mannan, and mannooligosaccharides (M2-M5), but did not exhibit any hydrolytic activity against structurally unrelated substrates. The biocatalytic activity of ManP against LBG and guar gum was 695 and 450 U mg-1, respectively. Especially, enzymatic hydrolysis of mannobiose yielded a mixture of mannose (16.6 %) and mannobiose (83.4 %), although the degree of mannobiose degradation by ManP with was relatively limited. CONCLUSION: The present results suggest that ManP is an endo-ß-1,4-mannanase and is distinct from various other characterized endo-ß-1,4-mannanases.

Dual Stimuli-Responsive Vesicular Nanospheres Fabricated by Lipopolymer Hybrids for Tumor-Targeted Photodynamic Therapy.

Smart delivery system of photosensitizer chlorin e6 (Ce6) has been developed for targeted photodynamic therapy (PDT). Simple self-assemblies of the mixtures comprising soybean lecithin derived phosphatidylcholine (PC), phosphatidylethanolamine-poly(L-histidine)40 (PE-p(His)40), and folic acid (FA) conjugated phosphatidylethanolamine-poly(N-isopropylacrylamide)40 (PE-p(NIPAM)40-FA) in different ratios yield smart nanospheres characterized by (i) stable and uniform particle size (∼100 nm), (ii) positive surface charge, (iii) high hydrophobic drug (Ce6) loading efficiency up to 45%, (iv) covalently linked targeting moiety, (v) low cytotoxicity, and (vi) smartness showing p(His) block oriented pH and p(NIPAM) oriented temperature responsiveness. The Ce6-encapsulated vesicular nanospheres (Ce6@VNS) were used to confirm the efficiency of cellular uptake, intracellular distribution, and phototoxicity against KB tumor cells compared to free Ce6 at different temperature and pH conditions. The Ce6@VNS system showed significant photodynamic therapeutic efficiency on KB cells than free Ce6. A receptor-mediated inhibition study proved the site-specific delivery of Ce6 in targeted tumor cells.

Genetic and functional characterization of an extracellular modular GH6 endo-ß-1,4-glucanase from an earthworm symbiont, Cellulosimicrobium funkei HY-13.

The gene (1608-bp) encoding a GH6 endo-ß-1,4-glucanase (CelL) from the earthworm-symbiotic bacterium Cellulosimicrobium funkei HY-13 was cloned from its whole genome sequence, expressed recombinantly, and biochemically characterized. CelL (56.0 kDa) is a modular enzyme consisting of an N-terminal catalytic GH6 domain (from Val57 to Pro396), which is 71 % identical to a GH6 protein (accession no.: WP_034662937) from Cellulomonas sp. KRMCY2, together with a C-terminal CBM 2 domain (from Cys429 to Cys532). The highest catalytic activity of CelL toward carboxymethylcellulose (CMC) was observed at 50 °C and pH 5.0, and was relatively stable at a broad pH range of 4.0-10.0. The enzyme was capable of efficiently hydrolyzing the cellulosic polymers in the order of barley ß-1,3-1,4-D-glucan > CMC > lichenan > Avicel > konjac glucomannan. However, cellobiose, cellotriose, p-nitrophenyl derivatives of mono- and disaccharides, or structurally unrelated carbohydrate polymers including ß-1,3-D-glucan, ß-1,4-D-galactomannan, and ß-1,4-D-xylan were not susceptible to CelL. The enzymatic hydrolysis of cellopentaose resulted in the production of a mixture of 68.6 % cellobiose and 31.4 % cellotriose but barley ß-1,3-1,4-D-glucan was 100 % degraded to cellotriose by CelL. The enzyme strongly bound to Avicel, ivory nut mannan, and chitin but showed relatively weak binding affinity to lichenan, lignin, or poly(3-hydroxybutyrate) granules.

Nano-Self Assembled Photosensitizer Composed of Methoxy Poly(ethylene glycol)-Conjugated Chlorin e6 for Enhanced Photosensing of HCT116 Cells.

We synthesized methoxy poly(ethylene glycol) (MPEG)-chlórin e6 (Ce6) conjugates to increase aqueous solubility of Ce6, to fabricate nanoparticles, and to improve tumor targetability of Ce6. MPEG-Ce6 conjugates (abbreviated as Pe6) associated in the aqueous solution as a nanoparticle. Pe6 nanoparticles have small diameter less than 100 nm, spherical shape, and core-shell structure in the aqueous environment. They have improved photophysical properties compared to Ce6 itself. Photosensitivity of Pe6 nanoparticles were studied using HCT116 human colon carcinoma cells. Pe6 nanoparticles practically have no dark-toxicity against HCT116 human colon carcinoma cells while they showed enhanced cellular uptake, phototoxicity, and ROS generation at in vitro cell culture experiment. Furthermore, they showed improved tumor tissue penetration and accumulation in vivo animal studies. We suggested Pe6 nanoparticles as an ideal candidate for PDT of colon cancer.

Lipo-poly(L-histidine) hybrid materials with pH-sensitivity, intracellular delivery efficiency, and intrinsic targetability to cancer cells.

Biocompatible lipo-histidine hybrid materials conjugated with IR820 dye show pH-sensitivity, efficient intracellular delivery of doxorubicin (Dox), and intrinsic targetability to cancer cells. These new materials form highly uniform Dox-loaded nanosized vesicles via a self-assembly process showing good stability under physiological conditions. The Dox-loaded micelles are effective for suppressing MCF-7 tumors, as demonstrated in vitro and in vivo. The combined mechanisms of the EPR effect, active internalization, endosomal-triggered release, and drug escape from endosomes, and a long blood circulation time, clearly prove that the IR820 lipopeptide DDS is a safe theranostic agent for imaging-guided cancer therapy.

Cisplatin-incorporated nanoparticles of methoxy poly(ethylene glycol)-b-poly(L-glutamic acid) copolymer.

We fabricated cisplatin-incorporated nanoparticles using block copolymer composed of methoxy poly(ethylene glycol) (MPEG) and poly(L-glutamic acid) (PGA) (abbreviated as GE). For synthesis of block copolymer, MPEG was directly conjugated to the terminal amine of PGA. Cisplatin-incorporated nanoparticles was prepared by ion complex formation of cisplatin and PGA domain of block copolymer. Size of cisplatin-incorporated nanoparticles was less than 200 nm at particle size measurement and their shapes were spherical at TEM observation. To study drug release properties, cispaltin release from nanoparticles were performed at phosphate-buffered saline (PBS, 0.01 M, pH 7.4) and distilled water, indicating that cisplatin release rate at PBS was approximately three times higher than that of deionized water. The cell growth inhibition of cisplatin incorporated nanoparticles in vitro was tested with HuCC-T1 cells. Cisplatin-incorporated nanoparticles were effectively inhibited tumor cell growth as similar as cisplatin itself. In other words, nanoparticles showed decreased inherent cytotoxicity compared to cisplatin against normal cells. We suggest that cisplatin incorporated GE nanoparticles is a good candidate for antitumor drug targeting.

Effect of 5-aminolevulinic acid-encapsulate liposomes on photodynamic therapy in human cholangiocarcinoma cells.

Intracellular protoporphyrin IX (PpIX) generation following administration of 5-aminolevulinic acid (ALA) has been used in photodynamic therapy (PDT). Subsequent irradiation can lead to selective damage to photosensitizer-treated cells or tissues. In the present work, we describe the enhancement of ALA-induced PpIX accumulation using a liposome carrier. ALA-containing liposomes (Lipo-ALA) were prepared using dipalmitoyl-phosphatidyl choline and in vitro PDT effect was investigated against human cholangiocarcinoma HuCC-T1 cells. Lipo-ALA increased the uptake efficiency into tumor cells compared to ALA itself, which increased the phototoxic effect. A positive relationship was evident between small particle size, PpIX accumulation and cell death after Lipo-ALA based PDT.

Dual stimuli-responsive poly(N-isopropylacrylamide)-b-poly(L-histidine) chimeric materials for the controlled delivery of doxorubicin into liver carcinoma.

A series of dual stimuli responsive synthetic polymer bioconjugate chimeric materials, poly(N-isopropylacrylamide)55-block-poly(L-histidine)n [p(NIPAM)55-b-p(His)n] (n=50, 75, 100, 125), have been synthesized by employing reversible addition-fragmentation chain transfer polymerization of NIPAM, followed by ring-opening polymerization of α-amino acid N-carboxyanhydrides. The dual stimuli responsive properties of the resulting biocompatiable and membrenolytic p(NIPAM)55-b-p(His)n polymers are investigated for their use as a stimuli responsive drug carrier for tumor targeting. Highly uniform self-assembled micelles (∼55 nm) fabricated by p(NIPAM)55-b-p(His)n polymers display sharp thermal and pH responses in aqueous media. An anticancer drug, doxorubicin (Dox), is effectively encapsulated in the micelles and the controlled Dox release is investigated in different temperature and pH conditions. Antitumor effect of the released Dox is also assessed using the HepG2 human hepatocellular carcinoma cell lines. Dox molecules released from the [p(NIPAM)55-b-p(His)n] micelles remain biologically active and have stimuli responsive capability to kill cancer cells. The self-assembling ability of these hybrid materials into uniform micelles and their efficiency to encapsulate Dox makes them a promising drug carrier to cancer cells. The new chimeric materials thus display tunable properties that can make them useful for a molecular switching device and controlled drug delivery applications needing responses to temperature and pH for the improvement of cancer chemotherapy.

Biotransformation of d-Xylose-Rich Rice Husk Hydrolysate by a Rice Paddy Soil Bacterium, Priestia sp. Strain JY310, to Low Molecular Weight Poly(3-hydroxybutyrate).

Poly(3-hydroxybutyrate) (PHB) is a versatile thermoplastic with superior biodegradability and biocompatibility that is intracellularly accumulated by numerous bacterial and archaeal species. Priestia sp. strain JY310 that was able to efficiently biotransform reducing sugars in d-xylose-rich rice husk hydrolysate (reducing sugarRHH) to PHB was isolated from the soil of a rice paddy. Reducing sugarRHH including 12.5% d-glucose, 75.3% d-xylose, and 12.2% d-arabinose was simply prepared using thermochemical hydrolysis of 3% H2SO4-treated rice husk for 15 min at 121 °C. When cultured with 20 g/L reducing sugarRHH under optimized culture conditions in a batch bioreactor, Priestia sp. strain JY310 could produce PHB homopolymer up to 50.4% of cell dry weight (6.2 g/L). The melting temperature, heat of fusion, and thermal decomposition temperature of PHB were determined to be 167.9 °C, 92.1 J/g, and 268.1 °C, respectively. The number average and weight average molecular weights of PHB with a broad polydispersity index value (4.73) were estimated to be approximately 16.2 and 76.8 kg/mol, respectively. The findings of the present study suggest that Priestia sp. strain JY310 can be exploited as a good candidate for the low-cost production of low molecular weight PHB with improved biodegradability and reduced brittleness from inexpensive agricultural waste hydrolysates.

Novel Bi-Modular GH19 Chitinase with Broad pH Stability from a Fibrolytic Intestinal Symbiont of Eisenia fetida, Cellulosimicrobium funkei HY-13.

Endo-type chitinase is the principal enzyme involved in the breakdown of N-acetyl-d-glucosamine-based oligomeric and polymeric materials through hydrolysis. The gene (966-bp) encoding a novel endo-type chitinase (ChiJ), which is comprised of an N-terminal chitin-binding domain type 3 and a C-terminal catalytic glycoside hydrolase family 19 domain, was identified from a fibrolytic intestinal symbiont of the earthworm Eisenia fetida, Cellulosimicrobium funkei HY-13. The highest endochitinase activity of the recombinant enzyme (rChiJ: 30.0 kDa) toward colloidal shrimp shell chitin was found at pH 5.5 and 55 °C and was considerably stable in a wide pH range (3.5-11.0). The enzyme exhibited the highest biocatalytic activity (338.8 U/mg) toward ethylene glycol chitin, preferentially degrading chitin polymers in the following order: ethylene glycol chitin > colloidal shrimp shell chitin > colloidal crab shell chitin. The enzymatic hydrolysis of N-acetyl-ß-d-chitooligosaccharides with a degree of polymerization from two to six and colloidal shrimp shell chitin yielded primarily N,N'-diacetyl-ß-d-chitobiose together with a small amount of N-acetyl-d-glucosamine. The high chitin-degrading ability of inverting rChiJ with broad pH stability suggests that it can be exploited as a suitable biocatalyst for the preparation of N,N'-diacetyl-ß-d-chitobiose, which has been shown to alleviate metabolic dysfunction associated with type 2 diabetes.

Prevention of covered enteral stent migration in patients with malignant gastric outlet obstruction: a pilot study of anchoring with endoscopic clips.

OBJECTIVE: Placement of a self-expandable metal stent is a palliative treatment of choice for patients with malignant gastric outlet obstruction (GOO). Although covering an enteral stent with a membrane almost solves the problem of tumor ingrowth, stent migration continues to be a major unresolved problem. Our aim was to evaluate the clinical efficacy of endoscopic clipping for prevention of covered stent migration in the treatment of malignant GOO. MATERIAL AND METHODS: A total of 25 consecutive patients with malignant GOO were evaluated prospectively. After deployment of a double-layered combination stent (comprising an outer uncovered stent and an inner covered stent), three endoscopic clips were applied for fixation of the proximal end of the enteral stent to the gastric or duodenal mucosa. RESULTS: Technical and clinical success rates were 100% (25/25) and 88% (22/25), respectively. No stent migration was observed in any of the patients. Five patients (20%) experienced complications such as tumor overgrowth and stent compression. CONCLUSION: Endoscopic clipping for enteral stent placement seems to be effective for prevention of covered stent migration in patients with malignant GOO.

UV-induced graft copolymerization of monoacrylate-poly(ethylene glycol) onto poly(3-hydroxyoctanoate) to reduce protein adsorption and platelet adhesion.

Homogeneous solutions of poly(3-hydroxyoctanoate) (PHO) and the monoacrylate-poly(ethylene glycol) (PEGMA) monomer in chloroform were irradiated with UV light to obtain PEGMA-grafted PHO (PEGMA-g-PHO) copolymers. Variables affecting the degree of grafting (DG), such as the time of UV irradiation and the concentrations of the PEGMA monomer and initiator, were investigated. The PEGMA-g-PHO copolymers were characterized by measuring the water contact angle, molecular weight, thermal transition temperatures and mechanical properties, as well as by nuclear magnetic resonance spectroscopy. The results from all of these measurements indicate that PEGMA groups were present on the PHO polymer. The protein adsorption and platelet adhesion on the PEGMA-g-PHO surfaces were examined using poly(L-lactide) (PLLA) surfaces as the control. The proteins and platelets had a significantly lower tendency to adhere to the PEGMA-g-PHO copolymers than to PLLA. The graft copolymer with a high DG of PEGMA was very effective in reducing the protein adsorption and platelet adhesion and did not activate the platelets. The results obtained in this study suggest that PEGMA-g-PHO copolymers have the potential to be used as blood-contacting devices in a broad range of biomedical applications.

Preparation and hydrolytic degradation of semi-interpenetrating networks of poly(3-hydroxyundecenoate) and poly(lactide-co-glycolide).

Semi-interpenetrating networks (semi-IPNs), where poly(lactide-co-glycolide) (PLGA) molecules were entrapped in the crosslinked matrices of poly(3-hydroxyundecenoate) (PHU), were prepared by irradiating homogeneous solutions of PHU and PLGA in chloroform with UV light. Attenuated total reflectance infrared spectroscopy showed that the PLGA chains were entrapped in PHU networks. The semi-IPNs showed enhanced mechanical strength as the PLGA content increased. The semi-IPNs were incubated at 37 degrees C in a 0.01N NaOH solution, and the extent of hydrolytic degradation was investigated by monitoring changes in various parameters such as water uptake, pH, mass, and morphology. Hydrolysis of semi-IPNs were significantly affected by the presence of PLGA. A semi-IPN prepared from a 9:1 (by weight) mixture of PHU and PLGA lost 25% of its original weight in 12 weeks while a PHU sample containing no PLGA lost only 5% of its weight during the same period under identical conditions. The hydrolysis was most likely accelerated when the pH of the medium was lowered by the hydrolyzed products of PLGA, 2-hydroxyalkanoic acids. These results showed that hydrolysis of PHA could be enhanced by incorporating a second component that lowered the pH of the hydrolysis system.

Drug release from and hydrolytic degradation of a poly(ethylene glycol) grafted poly(3-hydroxyoctanoate).

Monoacrylate-poly(ethylene glycol)-grafted poly(3-hydroxyoctanoate) (PEGMA-g-PHO) copolymers were synthesized to develop a swelling-controlled release delivery system for ibuprofen as a model drug. The in vitro hydrolytic degradation of and the drug release from a film made of the PEGMA-g-PHO copolymer were carried out in a phosphate buffer saline (pH 7.4) medium. The hydrolytic degradation of the copolymer was strongly dependent on the degree of grafting (DG) of the PEGMA group. The degradation rate of the copolymer films in vitro increased with increasing DG of the PEGMA group on the PHO chain. The copolymer films showed a controlled delivery of ibuprofen to the medium in periods of time that depend on the composition, hydrophilic/hydrophobic characteristics, initial drug loading amount and film thickness of the graft copolymer support. The drug release rate from the grafted copolymer films was faster than the rate of weight loss of the films themselves. In particular, a combination of the low DG of the PEGMA group in the PHO chains with the low ibuprofen solubility in water led to long-term constant release from these matrices in vitro.

Folic acid-tethered poly(N-isopropylacrylamide)-phospholipid hybrid nanocarriers for targeted drug delivery.

A series of temperature-responsive lipopolymers have been synthesized by bioconjugating poly(N-isopropylacrylamide)n (n = 25, 40, 60) onto three different phospholipids by the combination of reversible addition fragmentation chain transfer polymerization and azide-alkyne click reactions. To achieve the active targeting of cancer cells, folic acid (FA) has also been tethered to the resulting hybrid materials. The doxorubicin (Dox) encapsulated uniform nanocarriers (150 nm in diameter) fabricated by the self-assembly of the lipopolymers display temperature responsive controlled release. The FA receptor-mediated delivery of Dox was then assessed using KB cell lines, and the anti-cancer activity was assessed by the blocking of folic acid receptors. The FA-tethered lipopolymers showing temperature-responsiveness are advantageous for the cell-specific release of Dox, potentiating their anti-cancer activity.

Preparation and cell compatibility of acrylamide-grafted poly(3-hydroxyoctanoate).

Poly(3-hydroxyoctanoate) (PHO) films were treated with plasma of different discharge powers (10-50 W) and then treated with acryl amide solutions in order to prepare films with surfaces that contained different amounts of amide groups. The surfaces were characterized by contact angle measurement, attenuated total reflectance infrared spectroscopy, electron spectroscopy for chemical analysis, and scanning electron microscopy. Results from all these measurements indicated that amide groups were present on the surfaces. The amount of amide groups increased in proportion to the discharge power of the plasma. The interaction of Chinese hamster ovary cells with these grafted surfaces was investigated. The number of cells that adhered to and grew on the surfaces was highest for films grafted at 30 W of plasma discharge power, indicating that the moderate hydrophilicity was optimal for cells to adhere and grow. The present results support the suggestion that acryl amide-grafted PHO could be used as cell-compatible biomedical applications.

Poly(ethylene glycol)-grafted poly(3-hydroxyundecenoate) networks for enhanced blood compatibility.

Poly(ethylene glycol)-grafted poly(3-hydroxyundecenoate) (PEG-g-PHU) networks were prepared by irradiating homogeneous solutions of poly(3-hydroxyundecenoate) (PHU) and the monoacrylate of poly(ethylene glycol) (PEG) with UV light. The resulting polymer networks were characterized by measuring the water contact angle, water uptake, and mechanical properties and by performing attenuated total reflectance infrared spectroscopy and scanning electron microscopy. These measurements showed that the PEG chains were present in polymer networks. Adsorption of blood proteins and platelets on cross-linked PHU (CLPHU) and PEG-g-PHU were examined using poly(L-lactide) (PLLA) surfaces as control. Blood proteins and platelets had significantly lower tendency of adhesion to surfaces composed of CLPHU and PEG-g-PHU networks than to PLLA. Blood compatibility of polymer networks increased as the fraction of grafted PEG increased. The results of this study suggest that PEG-g-PHU networks might be useful for blood-compatible biomedical applications.

Defensive mechanism in cholangiocarcinoma cells against oxidative stress induced by chlorin e6-based photodynamic therapy.

In this study, the effect of chlorin e6-based photodynamic therapy (Ce6-PDT) was investigated in human intrahepatic (HuCC-T1) and extrahepatic (SNU1196) cholangiocarcinoma (CCA) cells. The amount of intracellular Ce6 increased with increasing Ce6 concentration administered, or with incubation time, in both cell lines. The ability to take up Ce6 and generate reactive oxygen species after irradiation at 1.0 J/cm(2) did not significantly differ between the two CCA cell types. However, after irradiation, marked differences were observed for photodamage and apoptotic/necrotic signals. HuCC-T1 cells are more sensitive to Ce6-PDT than SNU1196 cells. Total glutathione (GSH) levels, glutathione peroxidase and glutathione reductase activities in SNU1196 cells were significantly higher than in HuCC-T1 cells. With inhibition of enzyme activity or addition of GSH, the phototoxic effect could be controlled in CCA cells. The intracellular level of GSH is the most important determining factor in the curative action of Ce6-PDT against tumor cells.