Polyethyleneimine/poly-(γ-glutamic acid)/poly(lactide-co-glycolide) nanoparticles for loading and releasing antiretroviral drug

Nanoparticles (NPs) with ternary components of polyethyleneimine (PEI), poly-(γ-glutamic acid) (γ-PGA), and poly(lactide-co-glycolide) (PLGA) were applied to carry and release saquinavir (SQV). Hydrophobic SQV was encapsulated in the particle core composed of PLGA to form SQV-PLGA NPs, and the surface of SQV-PLGA NPs was grafted successively with hydrophilic γ-PGA and PEI (PEI/γ-PGA/SQV-PLGA NPs). The morphological images revealed that PEI/γ-PGA/SQV-PLGA NPs were spheroid-like, in general. An increase in the concentration of didecyl dimethylammonium bromide and a reduction in the dose of SQV enhanced the entrapment efficiency of SQV in PLGA NPs. In addition, an increment in the molecular weight of γ-PGA reduced the grafting efficiency of PEI on γ-PGA/SQV-PLGA NPs. An increase in the weight percentage of PEI enhanced the average particle diameter. However, the grafting efficiency of PEI on γ-PGA/SQV-PLGA NPs and the dissolution rate of SQV from PEI/γ-PGA/SQV-PLGA NPs reduced when the weight percentage of PEI increased. PEI/γ-PGA/SQV-PLGA NPs are an innovative drug delivery system and can be used for antiretroviral trials.     

Graft polymerization of methyl methacrylate initiated by pendant azo groups introduced onto γ-poly(glutamic acid)

The grafting of poly(methyl methacrylate) (PMMA) onto biosynthesized γ-poly(glutamic acid) (γ-PGA) initiated by pendant azo groups introduced onto γ-PGA was performed. The introduction of pendant azo groups onto γ-PGA was achieved by the reaction of carboxyl groups of γ-PGA with azo initiators having hydroxyl or amino groups, such as 2,2-azobis[2-(hydroxymethyl)propionitrile] (AHP), 2,2′-azobis[2-methyl-N-(2-hydroxyethyl)propionamide] (AMHP), and 2,2′-azobis[2-(2-imidazolin-2-yl)propane] (AIP), using N,N′-dicyclohexylcarbodiimide. The amount of pendant AHP groups introduced onto γ-PGA was estimated to be 0.15 mmol/g. Untreated γ-PGA failed to initiate the polymerization of MMA. On the contrary, the polymerization of MMA was found to be initiated in the presence of γ-PGA having azo groups: the polymerization rate was proportional to the square root of the concentration of γ-PGA having pendant azo groups. During the polymerization PMMA was grafted onto γ-PGA; the percentage of grafting of PMMA onto γ-PGA obtained from the graft polymerization initiated by pendant AHP, AMHP, and AIP groups was evaluated to be 65.0, 53.1, and 29.0%, respectively. Differential scanning calorimetric analysis shows that the endotherm transition point of γ-PGA at 220°C disappears by the grafting of PMMA onto the polymer. © 1993 John Wiley & Sons, Inc.     

Enhanced Low Molecular Weight Poly-γ-Glutamic Acid Production in Recombinant Bacillus subtilis 1A751 with Zinc Ion

Applied Biochemistry and Biotechnology - Poly-γ-glutamic acid (γ-PGA) is a novel biodegradable polyamide material. Microbial fermentation is the only way to produce γ-PGA, but the...     

Expression of ornithine decarboxylase during the transport of saquinavir across the blood-brain barrier using composite polymeric nanocarriers under an electromagnetic field

The expression of human ornithine decarboxylase (ODC) and permeability of saquinavir (SQV) across the blood-brain barrier were studied using nanoparticles (NPs) composed of poly(lactide-co-glycolide) (PLGA), poly-(γ-glutamic acid) (γ-PGA), and polyethyleneimine (PEI). SQV was encapsulated in the particle core to traverse a monolayer of human brain-microvascular endothelial cells (HBMECs) with the regulation of human astrocytes under an electromagnetic field (EMF). An increase in the weight percentage of PEI enhanced the particle size, zeta potential, and permeability of SQV. However, the viability of HBMECs reduced when the weight percentage of PEI increased. In addition, an increment in the molecular weight of γ-PGA enhanced the particle size and viability of HBMECs, and reduced the zeta potential. The permeability of SQV and expression of ODC were in the order: an EMF with amplitude modulation (AM)>an EMF with frequency modulation>no EMF. At 0.04% PEI, the AM EMF increased 2.38 times the uptake of NPs and 2.72 times the expression of ODC. The combination of PEI/γ-PGA/PLGA NPs and EMF can be an innovative strategy for delivering SQV into the brain.     

Synthesis and characterization of poly-α,β-[N-(2-hydroxyethyl)-L-aspartamide]-g-poly(glycolide) amphiphilic graft copolymers as potential drug carriers

A series of biodegradable amphiphilic graft polymers were successfully synthesized by grafting poly(glycolide) (PGA) sequences onto a water-soluble poly-α,β-[N-(2-hydroxyethyl)-_L-aspartamide] (PHEA) backbone. These novel graft polymers were synthesized by the ring-opening polymerization initiated by the macroinitiator PHEA bearing hydroxyl groups without adding any catalyst. The graft polymers were characterized by Fourier transform infrared spectroscopy (FTIR), ¹H nuclear magnetic resonance spectroscopy (¹H NMR), combined size-exclusion chromatography (SEC) and multiangle laser light scattering (MALLS) analysis, and differential scanning calorimetry (DSC). By controlling the feed ratio of the macroinitiator to the monomer, graft polymers with different branch lengths can be obtained. The degradation behaviors of the copolymers were studied. Based on the amphiphilicity of the graft copolymers, nanoparticle drug delivery systems were prepared by the direct dissolution method and the dialysis method, and the in vitro drug release behavior was investigated. Transmission electron microscopy (TEM) images demonstrated that these nanoparticles were regularly spherical in shape. The particle size and distribution of the nanoparticles were measured.     

γ-poly(glutamic acid) as an initiator of cationic polymerization of N-vinylcarbazole and N-vinyl-2-pyrrolidone

The cationic polymerization of vinyl monomers initiated by biosynthesized γ-poly(glutamic acid) (γ-PGA) powder surface was carried out in a heterogeneous system. It was found that the polymerization of N-vinylcarbazole (NVC) and N-vinyl-2-pyrrolidone (NVPD) is initiated by γ-PGA powder. The grafting of polymers onto γ-PGA surface was scarcely observed. The apparent activation energy of the polymerization of NVC and NVPD was estimated to be 20.2 and 24.8 kJ/mol, respectively. The polymerization was totally inhibited by the addition of triethylamine and pyridine, but not hydroquinone. The polymerization rate in nitrobenzene was larger than that in toluene. These results indicated the cationic nature of the polymerization. γ-PGA treated with KOH did not show the initiating activity of the polymerization. Therefore, carboxyl groups on γ-PGA powder surface play an important role in the initiation of the polymerization. It may be suggested that the polymerization is initiated by proton addition to monomer from carboxyl groups on γ-PGA powder surface and the propagation proceeds with carboxylate anion on the surface as counter ion. © 1993 John Wiley & Sons, Inc.     

Convenient esterification of poly(γ-glutamic acid) produced by microorganism with alkyl halides and their thermal properties

The esterification of poly(γ-glutamic acid) (γ-PGA) produced by Bacillus subtilis F2-01 with alkyl halides was carried out at 60°C in N-methyl-2-pyrrolidinone (NMP) in the presence of sodium bicarbonate to obtain the corresponding esterified γ-PGA. The thermal properties of these γ-PGA esters were examined by differential scanning calorimetry and thermogravimetry. γ-PGA esters were more stable than free acid type γ-PGA, which decomposed at 210°C. Melting temperature (T_m) of γ-PGA esters could be observed at 230-250°C. T_m of γ-PGA n-alkyl esters reached a maximum at an alkyl chain length of n = 3. © 1995 John Wiley & Sons, Inc.     

Production of Ultra-high Molecular Weight Poly-γ-Glutamic Acid with Bacillus licheniformis P-104 and Characterization of its Flocculation Properties

A novel strain of Bacillus licheniformis P-104 was isolated from Chinese soybean paste to produce a bioflocculant. The bioflocculant was confirmed as ultra-high molecular weight poly-γ-glutamic acid (γ-PGA) using Fourier transform infrared spectrum, high-performance liquid chromatography, and gel permeation chromatography with multi-angle laser light scattering. The production technology and flocculation properties of γ-PGA were investigated. By fed-batch fermentation in a 7-L bioreactor, the maximum γ-PGA yield reached 41.6 g L^?1 with a productivity rate of 1.07 g L^?1 h^?1. The flocculating activity of γ-PGA for kaolin suspension was 33.5?±?1.6 1/OD under the optimized flocculation conditions (6 mM Ca²⁺, 1.5 mg L^?1 γ-PGA, and pH 6.0). The optimized dosage of γ-PGA for flocculation was just about 30 % of that of reported γ-PGA produced by other strains. Moreover, the flocculation activity of γ-PGA produced by strain P-104 was much higher than commercial γ-PGA with the molecular weight ranging 200–500 kDa and 1,500–2,500 kDa. This study provided a promising strain and an efficient method for production of ultra-high molecular weight γ-PGA which could be used as a potential green bioflocculant.     

Amphiphilic Graft Copolymers of Hydroxypropyl Cellulose Backbone with Nonpolar Polyisobutylene Branches

The novel amphiphilic graft copolymers with hydrophilic hard polar hydroxypropyl cellulose(HPC) backbone and hydrophobic soft nonpolar polyisobutylene(PIB) branches have been successfully synthesized through nucleophilic substitution reaction of living PIB chains carrying oxonium ions with the-OH groups along HPC backbone. The PIB branch length in the graft copolymers could be designed by living cationic polymerization and the grafting density could be adjusted by PIB~+/-OH molar ratio. The living PIB chains carrying oxonium ion were prepared by transformation of allyl bromide end groups in the presence of AgClO_4 and silver nanoparticles(3.2±0.3 nm, 0.7 wt%-1.8 wt%)generated in situ from AgBr. The phase-separation morphology was formed in the graft copolymers due to their incompatibility between backbone and branches. The hydrophilicity on the surface of graft copolymer films could be turned to hydrophobicity by increasing grafting density or/and length of PIB branches. The soft PIB segments in graft copolymers provided an unique surface via self-assembly for anti-protein adsorption against bovine serum albumin. A small amount of Ag nanoparticles in the copolymers contributed to good antibacterial activities against Staphylococcus aureus or Escherichia coli.     

聚(L-谷氨酸γ-苄酯)-聚四氢呋喃-聚(L-谷氨酸γ-苄酯)三嵌段共聚物的合成与表征

聚氨基酸是一类低毒性、生物相容性良好、易被机体吸收和代谢的可降解合成高分子材料，在药物控释载体、组织工程支架、生物材料表面改性方面得到了广泛应用．但其降解周期及降解速度通常难以控制，应用受到一定限制．通过共聚方法将生物相容和亲水性良好的聚乙二醇（PEG）引入聚氨基酸链段中形成两亲性嵌段共聚物旧，研究其自组装行为，及作为基因转染和药物控释载体等已成为高分子科学领域新的研究热点．     

Chain-length dependence of diblock copolymer micellization kinetics studied by stopped-flow pH-jump

A series of well-defined poly(ethylene oxide)- b-poly(2-(diethylamino)ethyl methacrylate) (PEO- b-PDEA) diblock copolymers containing PEO block of identical chain length and PDEA block with varying degrees of polymerization (DP, in the range of 32-154) were prepared via atom transfer radical polymerization (ATRP) employing a PEO-based macroinitiator (DP = 113). Upon a pH-jump from 3 to 12 under highly efficient stopped-flow mixing conditions, PEO- b-PDEA copolymers spontaneously form spherical micelles of increasing sizes and aggregation numbers ( N agg) with increasing PDEA chain lengths. Stopped-flow light scattering technique was used to probe the pH-induced micellization kinetics of PEO- b-PDEA copolymers, aiming to elucidate the PDEA chain-length effects on the unimer-to-micelle transition process. Upon a stopped-flow pH-jump from 3 to 12, the obtained dynamic traces can be well-fitted with double exponential functions. The calculated fast and slow characteristic relaxation times (tau 1 and tau 2) can be ascribed to the formation of quasi-equilibrium micelles (fast process) and subsequent relaxation into final equilibrium micelles (slow process), respectively. For PEO 113- b-PDEA 32 and PEO 113- b-PDEA 61, tau 2 is almost independent of polymer concentrations, suggesting that the relaxation from quasi-equilibrium micelles into final equilibrium micelles mainly proceeds via insertion/expulsion of unimer chains. Upon increasing the DP of pH-responsive PDEA block to 89, 117, and 154, the obtained slow relaxation time, tau 2, tends to decrease with increasing polymer concentrations, suggesting that the slow process is dominated by the micelle fusion/fission mechanism. The apparent activation energy ( E a) associated with tau 2 has also been determined from temperature-dependent micellization kinetics for five PEO- b-PDEA copolymers. It was found that during micellization, copolymers with longer PDEA blocks exhibit much lower E a compared to those with shorter blocks. Thus, we observed experimentally for the first time that increasing the hydrophobic block length in double hydrophilic block copolymers (DHBCs) can transform the mechanism of the slow process from unimer insertion/expulsion to micelle fusion/fission.     

A Targeted Nano Drug Delivery System of AS1411 Functionalized Graphene Oxide Based Composites

A novel method for the preparation of antitumor drug vehicles has been optimized. Biological materials of chitosan oligosaccharide (CO) and γ-polyglutamic acid (γ-PGA) have previously been employed as modifiers to covalently modify graphene oxide (GO), which in turn loaded doxorubicin (DOX) to obtain a nano drug delivery systems of graphene oxide based composites (GO-CO-γ-PGA-DOX). The system was not equipped with the ability of initiative targeting, thus resulting into toxicity and side effects on normal tissues or organs. In order to further improve the targeting property of the system, the nucleic acid aptamer NH₂-AS1411 (APT) of targeted nucleolin (C23) was used to conjugate on GO-CO-γ-PGA to yield the targeted nano drug delivery system APT-GO-CO-γ-PGA. The structure, composition, dispersion, particle size and morphology properties of the synthesized complex have been studied using multiple characterization methods. Drug loading and release profile data showed that APT-GO-CO-γ-PGA is provided with high drug loading capacity and is capable of controlled and sustained release of DOX. Cell experimental results indicated that since C23 was overexpressed on the surface of Hela cells but not on the surface of Beas-2B cells, APT-GO-CO-γ-PGA-DOX can target Hela cells and make increase toxicity to Hela cells than Beas-2B cells, and the IC₅₀ value of APT-GO-CO-γ-PGA-DOX was 3.23±0.04 μg/mL. All results proved that APT-GO-CO-γ-PGA can deliver antitumor drugs in a targeted manner, and achieve the effect of reducing poison, which indicated that the targeted carrier exhibits a broad application prospect in the field of biomedicine.     

Syntheses of biodegradable graft copolymers from sodium caseinate and poly ɛ-caprolactone or poly lactic acid. Applications to the compatibilization of sodium caseinate/polyester blends

Casein (and its sodium salt, sodium caseinate, SC) is an inexpensive natural milk protein that is used as a biodegradable biomaterial, especially to produce packaging films. However, to enhance some of its properties, it needs to be blended with other polymers, which should preferably be biodegradable such as poly lactic acid (PLA) and poly ε-caprolactone (PCL). New SC-g-PLA and SC-g-PCL graft copolymers have been prepared and unambiguously characterized, in particular by ¹H and DOSY NMR. The grafting degrees are high (between 24 and 35% by weight) and result in variations of properties, such as hydrophobicity and thermal properties. The microstructures of SC/PLA and SC/PCL blends were studied and compared, with and without the addition of the SC-g-PLA and SC-g-PCL copolymers to test the compatibilization capacity of these new biodegradable copolymers.     

Improved Production of Poly-γ-Glutamic Acid by Bacillus subtilis D7 Isolated from Doenjang,a Korean Traditional Fermented Food,and Its Antioxidant Activity

The objectives of this study was to improve poly-γ-glutamic acid (γ-PGA) production by Bacillus subtilis D7 isolated from a Korean traditional fermented food and to assess its antioxidant activity for applications in the cosmetics and pharmaceutical industries. Strain D7 produced γ-PGA in the absence of l-glutamic acid, indicating l-glutamic acid-independent production. However, the addition of l-glutamic acid increased γ-PGA production. Several tricarboxylic acid cycle intermediates and amino acids could serve as the metabolic precursors for γ-PGA production, and the addition of pyruvic acid and d-glutamic acid to culture medium improved the yield of γ-PGA markedly. The maximum yield of γ-PGA obtained was 24.93?±?0.64 g/l in improved medium, which was about 5.4-fold higher than the yield obtained in basal medium. γ-PGA was found to have 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (46.8?±?1.5 %), hydroxyl radical scavenging activity (52.0?±?1.8 %), 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS) radical scavenging activity (42.1?±?1.8 %), nitric oxide scavenging activity (35.1?±?1.3 %), reducing power (0.304?±?0.008), and metal chelating activity (91.3?±?3.5 %). These results indicate that γ-PGA has a potential use in the food, cosmetics, and biomedical industries for the development of novel products with radical scavenging activity. As far as we are aware, this is the first report to describe the antioxidant activityof γ-PGA produced by bacteria.     

Modification of natural rubber by different grafting techniques

An attempt has been made to graft a hydrophylic monomer of N,N.dimethyl acrylamide (DMAA) onto natural rubber (NR) tube by simultaneous, per-oxidation and preirradiation grafting techniques. It was found that the grafting by simultaneous grafting technique results a maximum 29 wt% degree of grafting and by peroxidation and preirradiation techniques, results the maximum 42 wt% and 13 wt% degree of grafting, respectively. It was concluded that the peroxidation technique is the most appropriate to obtain a high degree of grafting in radiation copolymerization of DMAA onto NR.     

Silica nanoparticle dispersions in homopolymer versus block copolymer

Silica nanoparticles (17 ± 4 nm in diameter) were modified by grafting polystyrene chains to the surfaces using atom transfer radical polymerization (ATRP). The molecular weight of the grafted chains ranged from 8 to 48 kDa. These modified nanoparticles were mixed in solution with poly(styrene) homopolymer (18–120 kDa) and symmetric poly(styrene‐b‐butadiene) (PS‐PB) diblock copolymer (34–465 kDa) and the states of dispersion in the dried composites were characterized by transmission electron microscopy (TEM). In the so‐called wet brush limit, when the graft molecular weight equals or exceeds the matrix value, the silica particles form a uniform random dispersion in poly(styrene). Increasing the homopolymer matrix, molecular weight above the graft value results in particle clustering and macroscopic‐phase separation. Mixtures of the lamellar forming block copolymer and nanoparticles exhibit a very different trend, with particle clustering at the lower PS‐PB molecular weights and dispersion at the highest value. This latter finding is rationalized on the basis of packing constraints associated with lamellar order and the effective particle dimensions, and the degree of solvation at ordering, both of which favor higher molecular weight block copolymers. © 2007 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 45: 2284–2299, 2007     

Layer-by-layer films of enzymatically synthesized poly(aniline)/bacterial poly(γ-glutamic acid) for the construction of nanocapacitors

Poly(aniline) (PANi) and poly(γ-glutamic acid) (γ-PGA) have been synthesized by enzymatic catalysis and natural bacterial reactions, respectively. Layer-by-layer films have been prepared on glass or quartz slides by alternative immersions of the substrate in dilute solutions of γ-PGA and PANi, with several rinsing in between each deposition. UV-vis spectroscopy has been used to follow the evolution of the self-assembly process as well as to characterize the oxidative states of PANi. The linear dependence of the absorbance vs. the number of layers indicates a constant increase of thickness layer-by-layer. The morphology of the multilayer films, analyzed by atomic force microscopy, is granular type. Enzymatically synthesized PANi nanofilms present good electrical conductivity while γ-PGA acts as an insulating material. These differences in electrical properties and the possibility to obtain alternated multilayered films permit the construction of entirely “biological” nanocapacitors.     

Specific thermosensitive volume change of biopolymer gels derived from propylated poly(γ-glutamate)s

Thermosensitive biopolymers with an amphipathic structure were synthesized through the propyl esterification of the carboxyl groups of poly(γ-glutamic acid) (γ-PGA). The clouding temperature on heating was controlled by the addition of different amounts of NaCl and by the degree of esterification. The clearing temperature on cooling was independent of the aqueous milieu, presumably because of the strong multiple hydrogen bonds between the polymer chains formed in the collapsed state. The hydrogel of γ-PGA propylate crosslinked by a chemical reaction with hexamethylene diisocyanate also showed pH-responsive and thermoresponsive shrinking, but the volume recovery was incomplete during the cooling process. A Fourier transform infrared/attenuated total reflection study showed that the incomplete volume recovery might be associated with the amide hydrogen bonding being strengthened by the chemical crosslinkage. The addition of urea made the volume change complete. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4492–4501, 2004     

Nanoparticles prepared by self-assembly of Chitosan and poly-γ-glutamic acid

The present paper describes the preparation and characterization of novel biodegradable nanoparticles based on self-assembly of poly-gamma-glutamic acid (γ-PGA) and chitosan (CH). The nanosystems were stable in aqueous media at low pH conditions. Solubility of the systems was determined by turbidity measurements. Surface charge and mobility were measured electrophoretically. The particle size and the size distribution of the polyelectrolyte complexes were identified by dynamic light scattering and transmission electron microscopy (TEM). It was found that the size and size distribution of the nanosystems depends on the concentrations of γ-PGA and CH solutions and their ratio as well as on the pH of the mixture and the order of addition. The diameter of individual particles was in the range of 20–285 nm measured by TEM, and the average hydrodynamic diameters were between 150 and 330 nm. These biodegradable, self-assembling stable nanocomplexes might be useful for several biomedical applications.