Dye adsorption characteristics of magnetite nanoparticles coated with a biopolymer poly(γ-glutamic acid)

Magnetite nanoparticles coated with an anionic biopolymer poly(γ-glutamic acid) (PGA-MNPs) were synthesized and characterized for their methylene blue dye adsorption capability. Both bare- and dye-loaded PGA-MNPs were characterized by FTIR, TEM and VSM measurements, revealing the PGA-MNPs to be superparamagnetic with average particle diameter being 12.4 nm and magnetization value 59.2 emu/g. The synthesized PGA-MNPs were stable in deionized, tap and river waters as well as in acidic and basic media. Redlich-Peterson and Langmuir models precisely described the isotherm and the maximum adsorption capacity was 78.67 mg/g. A pseudo-second-order equation best predicted the kinetics with a maximum adsorption attained within 5 min. Incorporation of sodium or calcium ions reduced the dye adsorption, while a raise in pH enhanced adsorption and a complete desorption occurred at pH 1.0. Dye removal mechanism by PGA-MNPs was probably due to electrostatic interaction through exchange of protons from side-chain α-carboxyl groups on PGA-MNPs surface.     

Induction of endoplasmic reticulum-endosome fusion for antigen cross-presentation induced by poly (γ-glutamic acid) nanoparticles

We previously reported that poly (γ-glutamic acid)-based nanoparticles (γ-PGA NPs) are excellent vaccine carriers for inducing efficient cross-presentation in dendritic cells, thereby producing strong antitumor immunity in vivo. Analyzing the mechanism of cross-presentation induced by γ-PGA NPs will be useful toward designing novel vaccine carriers. In this study, we show an intracellular mechanism of efficient cross-presentation induced by OVA-loaded γ-PGA NPs. Cross-presentation induced by γ-PGA NPs depended on cytoplasmic proteasomes and TAP, similar to the classical MHC class I presentation pathway for endogenous Ags. Intracellular behavior analyzed by confocal laser scanning microscopy revealed that encapsulated OVA and γ-PGA accumulated in both the endoplasmic reticulum (ER) and endosome compartments within 2 h. At the same time, electron microscopy analysis clearly showed that intracellular γ-PGA NPs and encapsulated Au NPs were enveloped in endosome-like vesicles, not in the ER. These findings strongly suggest that γ-PGA NPs enhance ER-endosome fusion for cross-presentation. Moreover, inhibition of ER translocon sec61 significantly decreased the γ-PGA NP/OVA-mediated cross-presentation efficiency, indicating that sec61 is important for transporting Ags from the fused ER-endosome to the cytoplasm. These findings imply that the ER-endosome complex is key for the efficient cross-presentation of Ags encapsulated in γ-PGA NPs.     

Antifreeze activities of poly(γ-glutamic acid) produced by Bacillus licheniformis

Various enantiomeric isomers, metals salts and molecular sizes of poly(-glutamic acid), -PGA, produced by Bacillus licheniformis CCRC 12826, were prepared and their antifreeze activities were studied by differential scanning calorimetry. The antifreeze activity of -PGA increased as its molecular weight decreased but was indifferent to its d/l-glutamate composition. The antifreeze activity was cation dependent decreasing in the order Mg²⁺>>Ca ²⁺Na ⁺>>K ⁺ which follows that of inorganic chlorides in that high ionic charge leads to high antifreeze activity. The mechanism by which the cryoprotective effects of -PGA can be explained is still yet to be determined.     

Biodistribution of vaccines comprised of hydrophobically-modified poly(γ-glutamic acid) nanoparticles and antigen proteins using fluorescence imaging

Fluorophores-modified nanoparticles comprised of poly(γ-glutamic acid)-phenylalanine (γ-PGA-Phe-633) and ovalbumin (OVA-750) termed NPs-633/OVA-750 were prepared to assess their biodistribution using an in vivo fluorescence imager. Dynamic light scattering measurements indicated that NPs-633/OVA-750 were about 200 nm in diameter. The release of encapsulated OVA from NPs-633 in PBS was negligible (～10%) for a week. When subcutaneously injected, the localization period of OVA-750-encapsulated into NPs-633 at the site of injection (SOI) was much longer than that of free OVA-750, but was shorter as compared to a mixture with aluminum hydroxide. The NPs-633 disappeared at the SOI and major organs within 1 month after administration. Moreover, intravenously and intraperitoneally administered NPs-633 were mainly observed at the liver, and there was more rapid clearance from all organs as compared with non-biodegradable NPs. These fast clearance and degradation characteristics of γ-PGA-Phe NPs will be important not only for avoiding undesired adverse effects, but also for inducing a strong vaccine effect.     

Layer-by-layer self-assembly of chitosan and poly(γ-glutamic acid) into polyelectrolyte complexes

Chitosan (Ch) is a nontoxic and biocompatible polysaccharide extensively used in biomedical applications. Ch, as a polycation, can be combined with anionic polymers by layer-by-layer (LbL) self-assembly, giving rise to multilayered complexed architectures. These structures can be used in tissue engineering strategies, as drug delivery systems, or artificial matrices mimicking the extracellular microenvironment. In this work, Ch was combined with poly(γ-glutamic acid) (γ-PGA). γ-PGA is a polyanion, which was microbially produced, and is known for its low immunogenic reaction and low cytotoxicity. Multilayered ultrathin films were assembled by LbL, with a maximum of six layers. The interaction between both polymers was analyzed by: ellipsometry, quartz crystal microbalance with dissipation, Fourier transform infrared spectroscopy, atomic force microscopy, and zeta potential measurements. Ch/γ-PGA polyelectrolyte multilayers (PEMs) revealed no cytotoxicity according to ISO 10993-5. Overall, this study demonstrates that Ch can interact electrostatically with γ-PGA forming multilayered films. Furthermore, this study provides a comprehensive characterization of Ch/γ-PGA PEM structures, elucidating the contribution of each layer for the nanostructured films. These model surfaces can be useful substrates to study cell-biomaterial interactions in tissue regeneration.     

Stimuli-responsive materials prepared from carboxymethyl chitosan and poly(γ-glutamic acid) for protein delivery

The development of stimuli-responsive materials in response to the molecules involved in biological processes has gained increased attentions. In this work, carboxymethyl chitosan (CM-chitosan) and poly(γ-glutamic acid) (pGlu) were reacted with a naturally occurring compound, genipin, leading to the formation of genipin-crosslinked CM-chitosan/pGlu conjugates with fluorescence emissions. The genipin-conjugated polymers were sensitive to the oxidation product of glucose, gluconic acid and hydrogen peroxide (H₂O₂). Fluorescence emissions of the polymers were quenched by gluconic acid and H₂O₂. An increase in the hydrodynamic diameter together with the quenching of fluorescence indicated that the genipin-conjugated polymers were self-aggregated into nanoparticles, in response to the stimulus of gluconic acid (but not for H₂O₂). Bovine serum albumin (BSA) could be loaded in the self-aggregated nanoparticles, and the incorporated BSA slowly released from the nanoparticles under hyper-gluconic acid conditions. This material is hence proposed as a stimuli-responsive material for optical sensing and protein delivery purposes.     

An integrated high-throughput strategy for rapid screening of poly(γ-glutamic acid)-producing bacteria

Poly(γ-glutamic acid) (γ-PGA) is a promising biomaterial with a wide range of unique applications. To extensively screen γ-PGA-producing bacteria with high yield and different molecular weight, we developed an integrated high-throughput strategy. Firstly, γ-PGA-producing bacteria were selected in a primary screen plate containing a basic dye (neutral red) based on the concentric zone formed through the electrostatic interaction between the dye and the secreted acidic polymer γ-PGA. Then, the isolates were cultured in 50 ml tubes instead of 250 ml flasks. A good correlation of fermentation results in 50 ml tubes and 250 ml flasks was observed. Thirdly, the γ-PGA yield and weight-average molecular weight (M _w) were simultaneously determined by spectrophotomic assay (UV assay) and neutral red plate assay. The results showed that the diameter of the concentric zone varied among isolates and was negatively correlated with the weight-average molecular weight of γ-PGA. The accuracy of the methods was comparable to that of high-performance liquid chromatography and gel permeation chromatography assay. Lastly, γ-PGA obtained from the target isolates was rapidly identified using thin layer chromatography assay. With this strategy, 13 bacteria with high yield and various molecular weights of γ-PGA from 500 obvious single colonies on the primary screen plate were obtained.     

Biosynthesis of poly(γ-glutamic acid) from l-glutamic acid,citric acid,and ammonium sulfate in Bacillus subtilis IFO3335

Poly(-glutamic acid) (PGA) production in Bacillus subtilis IFO3335 was studied. When l-glutamic acid, citric acid, and ammonium sulfate were used as carbon and nitrogen sources, a large amount of PGA without a by-product such as a polysaccharide was produced. The time courses of cell growth, PGA, glutamic acid, and citric acid concentrations during cultivation were investigated. It was found that glutamic acid added to the medium was apparently not assimilated. It can be presumed that the glutamic acid unit in PGA is mainly produced from citric acid and ammonium sulfate. The PGA productivity was investigated at various concentrations of ammonium sulfate in the media, which caused the depression of cell growth, high productivity of PGA, and the production of PGA with a high relative molecular mass. The yield of PGA determined by gel permeation chromatography (GPC) reached approximately 20 g/l. This yield was the highest value for PGA production by B. subtilis IFO3335, suggesting that B. subtilis IFO3335 was a bacterium that could produce PGA effectively. Time courses relative to the molecular mass of PGA at various concentrations of ammonium sulfate were investigated. It was suggested that B. subtilis IFO3335 excreted a PGA degradation enzyme with the progress of cultivation and that PGA was degraded by this enzyme. Correspondence to: M. Kunioka     

Biosynthesis of poly (γ-glutamic acid) from l-glutamine,citric acid and ammonium sulfate in Bacillus subtilis IFO3335

Poly(-glutamic acid) (PGA) production in Bacillus subtilis IFO3335 was studied. PGA was only slightly produced from medium (100 ml) containing 2 g citric acid and 0.5 g ammonium sulfate in B. subtilis IFO3335. When 0.01 g/100 ml l-glutamine was added to this medium, a large amount of PGA (0.45 g/100 ml), without any by-products such as polysaccharides, was produced. The changes in cell growth, and PGA, glutamic acid, citric acid and ammonium sulfate concentrations in this medium during cultivation were investigated. It was found that PGA was effectively produced for the short time of 20 h after an induction period and that glutamic acid was scarcely excreted during PGA production. PGA could be effectively produced using this medium containing l-glutamine, citric acid and ammonium sulfate. It is suggested that a small amount of l-glutamine added to the medium activated enzymes in the pathway of PGA synthesis in B. subtilis IFO3335. It can be presumed that the enzyme catalyzing the reaction from 2-oxoglutaric acid to l-glutamic acid was glutamate synthase in this bacterium.     

Studies on the UV spectrum of poly(γ-glutamic acid) based on development of a simple quantitative method

A simple and valid ultraviolet (UV) spectrophotometric method for the determination of poly(γ-glutamic acid) is developed. The method is based on the UV absorption spectrum of γ-PGA in aqueous solution, which exhibits a maximum absorption wavelength at 216 nm. The results obtained were comparable to those obtained with the reported high-performance liquid chromatography (HPLC) method according to ICH guidelines. Under the proposed procedure, the calibration graph is linear over the range of 20-200 μg/ml with regression correlation coefficient of 0.9997. Precision (%R.S.D.<1.50) and recovery (%R.>99.29%) are good. The limit of detection (LOD) and limit of quantitation (LOQ) are 0.39 and 1.19 μg/ml, respectively. These results agree well with those of HPLC method. Its spectrum properties studies showed that the spectrum of γ-PGA remarkably changed with an increase in temperature due to γ-PGA was digested into glutamate monomer. In spite of this, the determining procedure could carried out in a wide temperature range (25-50°C). In addition, the method is not influenced by the molecular weight, but the measurement system need to control in pH 3.0-10.0 and ionic strength not more than 0.5M. The proposed method is applied successfully for high-throughput quantification of poly(γ-glutamic acid) in biological samples. The advantages of the UV method are simplicity of operation, rapidity, sensitive, low-cost and high-throughput.     

The reaction of chicken liver fatty acid synthetase with 5,5′-dithiobis(2-nitrobenzoic acid)

Chicken liver fatty acid synthetase is rapidly inhibited by 5,5′-dithiobis(2-nitrobenzoi acid). The inhibition results from the reaction of 5,5′-dithiobis(2-nitrobenzoic acid) with the cysteine-SH residue of the β-ketoacyl synthetase site. The adjacent pantetheine-SH of the other subunit displaces 2-nitro-5-thiobenzoic acid from the mixed disulfide resulting in the formation of a disulfide bond between the two residues and thereby cross-linking the two subunits. Scatchard analysis of the 5,5′-dithiobis(2-nitrobenzoic acid) inhibition indicated that there are two β-ketoacyl synthetase sites in the homodimer. The mixed disulfide formed between the pantetheine-SH and the cysteine-SH was reduced by 2-mercaptoethanol resulting in restoration of enzyme activity.     

Effects of oxygen vectors on the synthesis and molecular weight of poly(γ-glutamic acid) and the metabolic characterization of Bacillus subtilis NX-2

The effects of different oxygen vectors on the synthesis and molecular weight of poly(γ-glutamic acid) (PGA) were investigated in the batch fermentation of Bacillus subtilis NX-2. n-Hexane, n-heptane, and n-hexadecane enhanced the PGA concentration and molecular weight. The PGA concentration reached a maximum of 39.4 ± 0.19 g L^?1, and the highest molecular weight obtained was (19.0 ± 0.02) × 10⁵ Da with the addition of 0.3% n-heptane. However, n-dodecane decreased the PGA concentration and molecular weight to final values of 20.1 ± 0.10 g L^?1 and (8.4 ± 0.02) × 10⁵ Da, respectively. Analysis of the intracellular nucleotide levels of B. subtilis NX-2 with n-heptane and n-dodecane additives showed that the lowest NADH/NAD⁺ ratio and ATP levels were obtained with the n-dodecane additives, which can explain the decreased PGA yield and molecular weight. The metabolic flux distribution of B. subtilis NX-2 with n-heptane and n-dodecane additives was also investigated. Flux distribution was primarily directed to the EMP and TCA cycles with n-heptane additives. The flux of 2-oxoglutarate to intracellular glutamate and the flux distribution from extracellular to intracellular glutamate both increased to improve PGA production.     

Enhanced anti-tumor effects of HPV16E749–57-based vaccine by combined immunization with poly(I:C) and oxygen-regulated protein 150

Background: It is well known that both heat shock protein (HSP) and Toll-like receptor (TLR)3 agonist polyinosinic:polycytidylic acid (poly(I:C)) are capable of promoting the antigen-specific immune responses. In the current study, we assessed whether the anti-tumor effects of the HPV16E7_49–57-based vaccine can be elevated by combined applications of poly(I:C) and oxygen-regulated protein 150 (ORP150) in a mouse cervical cancer model. Methods: Recombinant mouse ORP150 and HPV E7_49–57 peptide were combined to passively form the ORP150–E7_49–57 complex under heat shock conditions. The effects of ORP150–E7_49–57 complex plus poly(I:C) adjuvant on lymphocyte proliferation and functional cytotoxic T cells were investigated by methyl thiazolyl tetrazolium (MTT), ELISPOT, and non-radioactive cytotoxicity assays. Finally, the complex's therapeutic anti-tumor effects with and without adjuvant therapy were observed in a tumor challenge experiment. Results: This combination vaccine approach significantly enhanced the proliferation of splenocytes and induced strong E7_49–57-speci?c CTL responses. More importantly, the ORP150–E7_49–57 complex plus poly(I:C) vaccine format demonstrated more potent anti-tumor effects than ORP150–E7_49–57 complex alone or E7_49–57 plus poly(I:C) in TC-1 tumor-bearing mice. Conclusion: Both poly(I:C) and ORP150 chaperone can synergistically enhance the anti-tumor effects of the HPV16E7_49–57-based vaccine in vitro and in vivo. This strategy provides a platform for the design of a tumor therapeutic vaccine capable of inducing an effective anti-tumor immune response.     

A new dendritic cell vaccine generated with interleukin-3 and interferon-β induces CD8+ T cell responses against NA17-A2 tumor peptide in melanoma patients

Dendritic cells derived from monocytes cultured in the presence of type I interferon were found to induce efficient T cell responses against tumor antigens in vitro. We vaccinated eight stage III or IV melanoma patients with dendritic cells generated with interferon-β and interleukin-3, activated by poly I: C, and pulsed with the tumor-specific antigen NA17.A2. This dendritic cell vaccine was well-tolerated with only minor and transient flu-like symptoms and inflammatory reactions at the injection sites. In most patients, isotopic imaging documented dendritic cells (DC) migration from the intradermal injection site to the draining lymph nodes. Finally, mixed lymphocyte-peptide culture under limiting dilution conditions followed by tetramer labeling indicated that three out of eight patients mounted a CD8 T cell response against the NA17.A2 antigenic peptide. We conclude that DC generated in type I-IFN represent an interesting alternative to DC generated in IL-4 and GM-CSF for cancer immunotherapy.     

Interaction of tritium-labeled H2DIDS (4,4′-diisothiocyano-1,2,diphenyl ethane-2,2′disulfonic acid) with the ehrlich mouse ascites tumor cell

Summary The experiments reported in this paper were undertaken to explore the interaction of tritiated H₂DIDS (4,4-diisothiocyano-1,2,diphenyl ethane-2,2-disulfonic acid) with Ehrlich ascites tumor cells. Addition of (³H)H₂DIDS to tumor cell suspension at 21°C, pH 7.3, resulted in: (i) rapid reversible binding which increased with time and (ii) inhibition of sulfate transport. Tightly bound H₂DIDS, i.e., reagent not removed by cell washing, also increased with time. Binding of 0.02 nmol H₂DIDS/mg dry mass or less did not affect sulfate transport, but, at greater than 0.02 nmol and up to 0.15 nmol the relationship between tight binding and inhibition of transport is linear. The fact that H₂DIDS could bind to the cell and yet not affect anion transport suggests that binding sites exist unrelated to those concerned with the regulation of anion permeability. Support for this is the observation that H₂DIDS is spontaneously released from cells even after extensive washings by a temperature-sensitive process. The most important source of released H₂DIDS is the cell surface coat which labels rapidly (within 1 min) and is then spontaneously released into the medium. A second source is derived from H₂DIDS that slowly entered the cells. Consequently, at least four modes of interaction exist between H₂DIDS and ascites tumor cells. These include both reversible and irreversible binding to membrane components which regulate anion permeability, irreversible binding to cell surface proteins or glycocalyx, and finally incorporation of H₂DIDS into the intracellular phase.     

Simultaneous and selective production of levan and poly(γ-glutamic acid) by <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis(natto) Takahashi, used to prepare the fermented soybean product natto, was grown in a basal medium containing 5% (w/w) sucrose and 1.5% (w/w) l-glutamate and produced 58% (w/w) poly(-glutamic acid) and 42% (w/w) levan simultaneously. After 21 h, 40–50 mg levan ml^-1had been produced in medium containing 20% (w/w) sucrose but without l-glutamate. In medium containing l-glutamic acid but without sucrose, mainly poly(-glutamic acid) was produced. Revisions requested 28 August 2004/14 October 2004; Revisions received 11 October 2004/22 November 2004     

Enhanced production of poly (γ-glutamic acid) from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NCIM 2324 in solid state fermentation

This work reports on the optimization of PGA production by Bacillus licheniformis NCIM 2324 in solid state fermentation (SSF). In the first step, the one factor-at-a-time method was used to investigate the effect of solid substrates, initial moisture content, pH, and additional carbon and nitrogen source on PGA production; subsequently, response surface methodology (RSM) was used to establish the optimum concentrations of the key nutrients for higher PGA production. In the second step, the effects of amino acids and TCA cycle intermediates on the production of PGA were studied. The final optimized medium gave a maximum yield of 98.64 ± 1.61 mg gds⁻¹ of PGA, which is significantly higher than that reported in the literature.     

Improvement of poly(γ-glutamic acid) biosynthesis and redistribution of metabolic flux with the presence of different additives in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> CGMCC 0833

Tween-80, dimethyl sulfoxide (DMSO), and glycerol could be used as novel materials to regulate the central carbon metabolic pathway and improve gamma-PGA biosynthesis by Bacillus subtilis CGMCC 0833. With glycerol in the medium, the activity of 2-oxoglutarate dehydrogenase complex at the key node of 2-oxoglutarate was depressed, more carbon flux distribution was directed to synthesize glutamate, the substrate of gamma-PGA, which led to overproducing of gamma-PGA, reached 31.7 g/l, compared to the original value of 26.7 g/l. When Tween-80 or DMSO was in the medium, the activity of isocitrate dehydrogenase was stimulated, the branch flux from 2-oxoglutarate to glutamate was also enhanced due to the increasing of total flux from iso-citrate to 2-oxoglutarate, then a large amount of glutamate was produced, and formation of gamma-PGA was also improved, which was a different process compared with that of glycerol. Moreover, with the addition of Tween-80 or DMSO, cell membrane permeability was increased, which facilitated the uptake of extracellular substrates and the secretion of gamma-PGA by this strain; therefore, gamma-PGA production was further stimulated, and 34.4 and 32.7 g/l gamma-PGA were obtained, respectively. This work firstly employed additives to improve the biosynthesis of gamma-PGA and would be helpful in understanding the biosynthesis mechanism of gamma-PGA by Bacillus species deeply.