Effect of Green Tea Extract/Poly-gamma-Glutamic Acid Complex in Obese Type 2 Diabetic Mice

BACKGROUND: The increasing prevalence of type 2 diabetes mellitus (T2DM) is associated with the rapid spread of obesity. Obesity induces insulin resistance, resulting in beta-cell dysfunction and thus T2DM. Green tea extract (GTE) has been known to prevent obesity and T2DM, but this effect is still being debated. Our previous results suggested that circulating green tea gallated catechins (GCs) hinders postprandial blood glucose lowering, regardless of reducing glucose and cholesterol absorption when GCs are present in the intestinal lumen. This study aimed to compare the effect of GTE with that of GTE coadministered with poly-gamma-glutamic acid (gamma-PGA), which is likely to inhibit the intestinal absorption of GCs. METHODS: The db/db mice and age-matched nondiabetic mice were provided with normal chow diet containing GTE (1%), gamma-PGA (0.1%), or GTE+gamma-PGA (1%:0.1%) for 4 weeks. RESULTS: In nondiabetic mice, none of the drugs showed any effects after 4 weeks. In db/db mice, however, weight gain and body fat gain were significantly reduced in the GTE+gamma-PGA group compared to nondrug-treated db/db control mice without the corresponding changes in food intake and appetite. Glucose intolerance was also ameliorated in the GTE+gamma-PGA group. Histopathological analyses showed that GTE+gamma-PGA-treated db/db mice had a significantly reduced incidence of fatty liver and decreased pancreatic islet size. Neither GTE nor gamma-PGA treatment showed any significant results. CONCLUSION: These results suggest that GTE+gamma-PGA treatment than GTE or gamma-PGA alone may be a useful tool for preventing both obesity and obesity-induced T2DM.

Accelerated healing of diabetic wound using artificial dermis constructed with adipose stem cells and poly (L-glutamic acid)/chitosan scaffold / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Diabetic wound is one of the most serious complications of diabetes mellitus. There are no significantly effective therapies for chronic non-healing diabetes ulcer so far. This study aimed to explore the feasibility of healing impaired wound using artificial dermis constructed with human adipose derived stem cells (ASCs) and poly (L-glutamic acid)/chitosan (PLGA/CS) scaffold in streptozotocin-induced diabetic mice.</p><p><b>METHODS</b>ASCs were isolated from fresh human lipoaspirates and expanded ex vivo for three passages, and then cells were seeded onto PLGA/CS scaffold to form artificial dermis. Expression of VEGF and TGFβ1 by ASCs presented in artificial dermis was determined. The artificial dermis was transplanted to treat the 20 mm × 20 mm full-thickness cutaneous wound created on the back of diabetic mice. Wound treated with scaffold alone and without treatment, and wound in normal non-diabetic mice served as control.</p><p><b>RESULTS</b>Cells growing within scaffold showed great proliferation potential, depositing abundant collagen matrix. Meanwhile, expression of VEGF and TGF-β1 by seeded ASCs maintained at a consistent high level. After treated with ASC based artificial dermis, diabetic wounds exhibited significantly higher healing rate compared with wounds treated with scaffold alone or without treatment. Histological examination also demonstrated an improvement in cutaneous restoration with matrix deposition and organization. Further quantitative analysis showed that there was a significant increase in dermis thickness and collagen content on artificial dermis treated wounds.</p><p><b>CONCLUSION</b>ASC/PLGA artificial dermis can effectively accelerate diabetic wound healing by promoting angiogenic growth factors and dermal collagen synthesis.</p>

Preparation, characterization and Calu-3 cellular uptake of three kinds of poly(b-benzyl-L-amino)block-poly(ethylene glycol) nanoparticles / 药学学报

The aim of this paper is to compare the cytotoxicity and cellular uptake efficiency of three kinds of poly(b-benzyl-L-amino) block-poly(ethylene glycol) nanoparticles (PXA-PEG-NPs) using Calu-3 cells, and select one as a nasal drug delivery vector for curcumin (Cur). Poly(gamma-benzyl-L-glutamate) block-poly(ethylene glycol) nanoparticles (PBLG-PEG-NPs), poly(gamma-benzyl-L-lysine) block-poly(ethyleneglycol) nanoparticles (PZLL-PEG-NPs) and poly(gamma-benzyl-L-aspartate) block-poly(ethylene glycol) nanoparticles (PBLA-PEG-NPs) were prepared by emulsion-solvent evaporation method. MTT assays were used to evaluate the cytotoxicity of PXA-PEG-NPs against Calu-3 cells. The cellular uptake of nanoparticles was visualized by an inverted fluorescence microscope and quantified by a flow cytometer. The results indicated that even at high concentration of 2 mg x mL(-1) the three nanoparticles had no cytotoxicity on Calu-3 cells. Compared to the curcumin solution, the three curcumin-loaded PXA-PEG-NPs showed significantly higher cellular uptake efficiency on Calu-3 cells (at equal concentration of curcumin with 5 microg x mL(-1) Cur solution), PBLG-PEG-NPs group was the highest. The cellular uptake increased with incubation time, and has positive correlation with nanoparticle concentration. In brief, PXA-PEG-NPs are conducive to delivery Cur into cells, and PBLG-PEG-NPs might be provided as a good nasal drug delivery carrier.

Synthesis and characterization of PEG-b-(PG-g-PEI) for gene delivery / 南方医科大学学报

<p><b>OBJECTIVE</b>To synthesize a biodegradable non-viral gene carrier with a high transfection efficiency and a low cytotoxicity.</p><p><b>METHODS</b>Poly(ethylene glycol)-block-(poly(L-glutamic acid)-graft-polyethylenimine) was prepared via ammonolysis of poly(ethylene glycol)-block-poly (γ-benzyl L-glutamate) with the low-molecular-mass polyethylenimine (600 Da). The synthesized copolymer was characterized by 1H nuclear magnetic resonance spectroscopy and gel permeation chromatography. The polyplex micelle from PEG-b-(PG-g-PEI) and plasmid DNA (pDNA) was studied using dynamic light scattering, zeta-potential measurements, and gel retardation assay. The in vitro cytotoxicity and transfection efficiency of PEG-b-(PG-g-PEI) were tested by MTT assay and luciferase assay in HEK 293T cells using PEI (25 kDa) as the control.</p><p><b>RESULTS</b>PEG-b-(PG-g-PEI) could efficiently condense DNA into nanosized particles with positive surface charges when the N/P ratio of polymer and DNA was above 5:1. The zeta potential of the polyplexes was about 25 mV, and the particle size was 120 nm at a N/P ratio of 10. The cell toxicity and gene transfection evaluations showed a lower cytotoxicity and a higher gene transfection efficiency of the copolymer than PEI 25000 in HEK 293T cells.</p><p><b>CONCLUSIONS</b>The polymer can be used as a potential non-viral gene carrier for gene therapy.</p>

Hidden Questions on Methotrexate

Since the 1950's, methotrexatehas been the most widely used for the treatment of rheumatoid arthritis among various disease-modifying anti-rheumatic drugs (DMARDs). In this review, several hidden questions on methotrexate were discussed. First, so far, methotrexate has been considered to improve rheumatoid arthritis by inhibiting cell proliferation through the reduction of synthesis regarding purine and pyrimidine. Recently, a new concept was proposed that methotrexate could increase the release of adenosine, which subsequently decreases the inflammatory function of immune cells, and can finally quench the inflammation in affected joints of rheumatoid arthritis. Second, there were only three clinical trials done to directly compare the efficacy between methotrexate and biologics. With these results, methotrexate showed comparable therapeutic efficacy to biologics, but did not prevent radiological progression. In the future, clinical trials to directly compare the efficacy of methotrexate to biologics will be needed. Third, measuring the serum concentration of methotrexate is not appropriate, since circulating methotrexate is rapidly cleared by cellular uptake or renal excretion. Methotrexate polyglutamate is a more stable compound than methotrexate and it is more likely to relate to efficacy or adverse effects of methotrexate. Recently, the efforts to measure methotrexate polyglutamate in red blood cells have been done to increase therapeutic efficacy and reduce its adverse effects. Fourth, NSAIDs can decrease the excretion of methotrexate though renal tubular cells and it may increase the serum concentration of methotrexate and the risk of its toxicity, suggesting that physicians should pay close attention to dose adjustments concerning methotrexate combined with NSAIDs.

Effects of poly-gamma-glutamic acid on serum and brain concentrations of glutamate and GABA in diet-induced obese rats

Poly-gamma-glutamic acid (gamma-PGA) is a mucilaginous and biodegradable compound produced by Bacillus subtilis from fermented soybeans, and is found in the traditional Korean soy product, cheongkukjang. This study was carried out to evaluate the effects of gamma-PGA from a food source on the concentration of the neurotransmitter GABA and its metabolic precursor glutamate in diet-induced obese rats. Eight-week old male Sprague-Dawley rats (n=60) were used. The rats were divided into two groups and obesity was induced by providing either a 10% control fat or 45% high fat diet for 5 weeks. The rats were then blocked into 6 groups and supplemented with a 0.1% gamma-PGA diet for 4 weeks. After sacrifice, brain and serum GABA and glutamate concentrations were analyzed by high performance liquid chromatography with fluorometric detection. The rats fed the high fat diet had significantly increased body weights. gamma-PGA supplementation significantly increased serum concentrations of glutamate and GABA in the control fat diet groups while this effect was not found in the high fat groups. In the brain, glutamate concentrations were significantly higher in the gamma-PGA supplemented groups both in rats fed the normal and high fat diets than in the no gamma-PGA controls. GABA concentrations showed the same tendency. The results indicated that gamma-PGA intake increased GABA concentrations in the serum and brain. However, the effects were not shown in obese rats.

The Effects of Poly-gamma Glutamic Acid on Broad Band Ultraviolet B-induced Skin Tumors in a Mice Model / 대한피부과학회지

BACKGROUND: Poly-gamma glutamic acid (gamma-PGA) is a natural polymer that is synthesized by Bacillus subtilis. Because of the reported anti-tumor effect, gamma-PGA and its derivatives have attracted enormous interest in the past few years. OBJECTIVE: The anti-tumor effects of gamma-PGA against ultraviolet B (UVB) were evaluated by irradiating hairless mice with UVB. METHODS: The experimental animals were 12-week-old male Albino hairless Skh:HR-1 mice. Thirty-two mice were divided into 4 groups: a chronologic aging (control); irradiation with UVB without gamma-PGA administration (T1); irradiation with UVB and 2,000 kDa gamma-PGA orally (T2); and irradiation with UVB and 5,000 kDa gamma-PGA orally (T3). The irradiation period was 12 weeks. The anti-tumor effect of gamma-PGA was evaluated with photographs and skin biopsy. A 51Cr release assay was performed to determine nutural killer cell (NK cell) activity. RESULTS: After 20 weeks of UVB irradiation, seborrheic keratosis, actinic keratosis, Bowen's disease, squamous cell carcinoma, and basal cell carcinoma were observed on the backs of mice. The most common skin lesion was seborrheic keratosis. gamma-PGA showed inhibitory effects on the total number of skin tumors and the premalignant and malignant skin lesions induced by UVB. The NK cell activities showed a tendency to increase based on the molecular weight of gamma-PGA. CONCLUSION: We suggest that gamma-PGA shows anti-tumor effects that inhibit the tumor generation induced by UVB, and the antitumor effects are related to the molecular weight of gamma-PGA.

Preparation and properties of self-assemble paclitaxel-loaded core-shell type nano-micelles / 药学学报

Polyethylene glycol-polybenzyl-L-glutamate copolymer (PEG-PBLG) was synthesized and paclitaxel-loaded core-shell type nano-micelles with amphiphilic copolymer PEG-PBLG was prepared by the dialysis method. The drug loading content and entrapment efficiency were determined by HPLC. The average size and its distribution were determined by dynamic light scattering method. The paclitaxel release rate in vitro from micelles was measured by HPLC. The cell cytotoxicity in vitro was observed with MTT assay. The anti-tumor activity of paclitaxel-loaded micelles were evaluated in tumor-inhibiting test of nude mice using human liver cancer HepG-2. The results indicated that paclitaxel could be entrapped in PEG-PBLG copolymer micelles and its size was in the range of 80-265 nm which increased with an increase in molecular weight of PBLG in copolymer; in vitro the paclitaxel could be released sustainably from the micelles. In high concentration of paclitaxel (>20 microg x mL(-1)) the paclitaxel-loaded PEG-PBLG micelles displayed much less cell cytotoxicity than paclitaxel injections with Cremophor EL (P<0.05); the tumor inhibiting activity of paclitaxel-loaded PEG-PBLG micelles was similar to that of paclitaxel injections with Cremophor EL in the same paclitaxel concentration. It was concluded that the paclitaxel-loaded PEG-PBLG micelles had more uniform size and size distribution, excellent drug sustainable-release behavior, less cytotoxicity, good anti-tumor activity similar to paclitaxel injections with Cremophor EL. So paclitaxel-loaded PEG-PBLG micelles would be a novel paclitaxel preparation in clinic for the treatment of tumor.

Preparation and biological activity of poly (gamma-glutamic acid) -cisplatin conjugate / 药学学报

Preparation of a poly (gamma-glutamic acid)-cisplatin conjugate was introduced and its in vitro antitumor effect was investigated. Poly (gamma-glutamic acids) was obtained by using fermentation methods. The hydrolyzed small molecular weight of poly (gamma-glutamic acids) was prepared by acid hydrolysis. The interaction between poly (gamma-glutamic acids) -cisplatin conjugate (PGA-CDDP) and DNA was investigated by PCR model. MTT assay was used to investigate the in vitro anticancer activity of the conjugate. Apoptosis assay of the conjugate was investigated by FCM assay and the in vivo toxicity was also proceeded. The results showed that the poly (gamma-glutamic acids) -cisplatin conjugate was obtained successfully and its yield is 10% - 12%. It has obvious antitumor effects on human liver tumor BEL7404 cells, human lung tumor H446 cells and human colon tumor RKO cells. At the same time, it also has apoptosis effects on the three kinds of tumor cell lines. The in vivo toxicity of PGA-CDDP was examined in normal mice and the results showed that the in vivo toxicity of this conjugate was significantly lower than that of free CDDP. In conclusion, the poly (gamma-glutamic acids) -cisplatin conjuate could be used as a potential clinic antitumor drug. The poly (gamma-glutamic acids) obtained by fermentation can be used as a valuable drug carrier system.

Transfection with a novel cationic gene carrier: PEI-PBLG / 生物工程学报

This study mainly deals with cell transfection and cytotoxicity for PEI(10kD)-PBLG, a novel cationic copolymer, to observe its potential as a gene carrier. Size measurement and SEM were used to show the modality of the PEI-PBLG/pDNA complexes. Cytotoxicity of PEI (10kD)-PBLG was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and compared with PEI(25kD)-PBLG, PEI(10kD), and PEI(25kD). Furthermore, pEGFP that can express the enhanced green fluorescent protein was chosen as a reporter to observe the transfection efficiency directly. Then, PEI (10kD)-PBLG/pEGFP complexes were transfected into several cell lines, such as Hela, COS-7, Vero-E6, and ECV-304, and effects of the transfection conditions were evaluated. The efficiencies were measured by FACS. Size measurement of complex particles indicated that PEI-PBLG/pDNA tended to form smaller nanoparticles compared with PEI/pDNA. The representative size of the PEI(10kD)-PBLG/pDNA complex was approximately 100 - 200 nm. SEM images showed that the particles were condense and compact. This can be suitable for their entry into cells. Cytotoxicity studies suggested that PEI (10kD)-PBLG had considerably lower toxicity than the other three materials. In the transfection tests, PEI (10kD)-PBLG/pDNA complexes could be transfected into all the cell lines that were tested. These provided the highest level of EGFP expression (45.02%) in Hela cells, which was considerably higher than that of PEI(10kD)/pEGFP (29.16%). Being less affected by the serum during transfection, PEI-PBLG/pDNA complexes offered greater biocompatibility than PEI. PEI-PBLG copolymer reduces the cytotoxicity of PEI, improves the transfection efficiency, and offers greater biocompatibility than PEI. It shows considerable potential as an efficient nonviral carrier for gene delivery.

Synthesis and characterizations of poly (ethylene glycol)-block-poly (glutamate) / 生物医学工程学杂志

Amine-terminated poly (ethylene glycol) (PEG) was prepared by two steps. Firstly, potassium naphthalene was added to a solution of methoxypolyethylene glycol 5,000 in benzene until the solution maintains green in half of an hour, then excess tosylchloride was introduced; secondly, the conversion of the tosylate into an amine was carried out by Gabriel synthesis. The block copolymer poly (ethylene glycol)-co-poly (gamma-benzyl L-glutamate ) could be obtained by ring-opening polymerization of gamma-benzyl-L-glutamate N-carboxy anhydride with amine-terminated PEG as macroinitiator. And the benzyl group could be removed by sodium hydroxide. The product structure was characterized by IR, 1HNMR, GPC. The cisplatin-loaded micelle was observed by transmission electron microscope (TEM). And the block copolymer is expected to be useful as functional materials including carrier systems in drug controlled delivery applications.

Blood compatibility of block copolymer membranes of poly(benzyl L-glutamate)/poly(ethylene glycol) / 生物医学工程学杂志

The blood compatibility of block copolymer membranes of poly(benzyl L-glutamate)/poly(ethylene glycol) and the effect of on the blood compatibility of copolymer were evaluated by the clotting time test, the platelet adhesion and deformation test, and the protein adsorption test. The results showed that in terms of blood compatibility, homopolymer was better than glass and silicone, copolymer was better than homopolymer, and the more the PEG in the copolymer, the better the blood compatibility.

Study on adhesion of platelets to surface of copoly(benzyl glutamate-hydroxy ethyl glutamine) / 生物医学工程学杂志

Copoly (benzyl glutamate-hydroxy ethyl glutamine) were prepared by partially aminolysis of poly benzyl glutamate with hydroxyl ethylamine at 60 degrees C for predeternined period. As aminolysis was proceeding, the content of hydroxy ethyl glutamine in copolymer increased and the yield of aminolysis decreased. When aminolysis time reached 16.5 hours, a copolymer with 0.357 mole fraction of hydroxy ethyl glutamine was obtained. After 16.5 hours a soluble yellow viscous product of reaction was obtained. The aminolysized specimens displayed more swelling degree in water, that is, more hydrophilits. The meassurement of solid-liquid contact angles showed that an increase in critical surface tention with content of hydroxy ethyl glutamine in copolymer was observed. Likewise a obvious increase in polar component and a slight decrease in dispersive of surface free energy, thus an increase in total surface free energy with content of hydroxy ethyl glutamine was found. The platelet adhesion and deformation test indicated that less platelets were adhered to surfaces of all aminolysized specimens than that of both poly benzyl glutamate and polydimethylsilicone. The least adhered platelets on surface of copolymer with 0.133 mole fraction of content of hydroxy ethyl glutamine were observed. The partiall aminolysis of poly benzyl glutamate films is an effective method to improve its hydrophility and antithrombogenicity.