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Recently, novel approaches for the delivery of therapeutic antibodies have attracted much attention, especially sustained release formulations. However, sustained release formulations capable of carrying a high antibody load remain a challenge for practical use. In this study, a novel injectable hydrogel composed of maleimide-modified γ-polyglutamic acid (γ-PGA-MA) and thiol end-functionalized 4-arm poly(ethylene glycol) (4-arm PEG-SH) was developed for the subcutaneous delivery of trastuzumab. γ-PGA-MA and 4-arm PEG-SH formed a hydrogel through thiol-maleimide reactions, which had shear-thinning properties and reversible rheological behaviors. Moreover, a high content of trastuzumab (>100 mg/mL) could be loaded into this hydrogel, and trastuzumab demonstrated a sustained release over several weeks through electrostatic attraction. In addition, trastuzumab released from the hydrogel had adequate stability in terms of its structural integrity, binding bioactivity, and antiproliferative effect on BT-474 cells. Pharmacokinetic studies demonstrated that trastuzumab-loaded hydrogel (Her-hydrogel-10, composed of 1.5% γ-PGA-MA, 1.5% 4-arm PEG-SH, and 10 mg/mL trastuzumab) and trastuzumab/Zn-loaded hydrogel (Her/Zn-hydrogel-10, composed of 1.5% γ-PGA-MA, 1.5% 4-arm PEG-SH, 5 mM ZnCl 2 , and 10 mg/mL trastuzumab) could lower the maximum plasma concentration (C max ) than the trastuzumab solution. Furthermore, Her/Zn-hydrogel-10 was better able to release trastuzumab in a controlled manner, which was ascribed to electrostatic attraction and formation of trastuzumab/Zn nanocomplexes. In a BT-474 xenograft tumor model, Her-hydrogel-10 had a similar tumor growth-inhibitory effect as that of the trastuzumab solution. By contrast, Her/Zn-hydrogel-10 exhibited a superior tumor growth-inhibitory capability due to the functionality of Zn. This study demonstrated that this hydrogel has potential as a carrier for the local and systemic delivery of proteins and antibodies. STATEMENT OF SIGNIFICANCE: Recently, novel sustained-release formulations of therapeutic antibodies have attracted much attention. However, these formulations should be able to carry a high antibody load owing to the required high dose, and these formulations remain a challenge for practical use. In this study, a novel injectable chemically cross-linked hydrogel was developed for the subcutaneous delivery of trastuzumab. This novel hydrogel possessed ideal characteristics of loading high content of trastuzumab (>100 mg/mL), sustained release of trastuzumab over several weeks, and maintaining adequate stability of trastuzumab. In vivo studies demonstrated that a trastuzumab-loaded hydrogel possessed the ability of controlled release of trastuzumab and maintained antitumor efficacy same as that of trastuzumab. These results implied that a γ-PGA-MA and 4-arm PEG-SH-based hydrogel has great potential in serving as a carrier for the local or systemic delivery of therapeutic proteins or antibodies., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Peptide-based vaccines have the potential to overcome the limitations of classical vaccines; however, their use is hampered by a lack of carriers and adjuvants suitable for human use. In this study, an efficient self-adjuvanting peptide vaccine delivery system was developed based on the ionic interactions between cationic trimethyl chitosan (TMC) and a peptide antigen coupled with synthetically defined anionic α-poly-(l-glutamic acid) (PGA). The antigen, possessing a conserved B-cell epitope derived from the group A streptococcus (GAS) pathogen and a universal T-helper epitope, was conjugated to PGA using cycloaddition reaction. The produced anionic conjugate formed nanoparticles (NP-1) through interaction with cationic TMC. These NP-1 induced higher systemic and mucosal antibody titers compared to antigen adjuvanted with standard mucosal adjuvant cholera toxin B subunit or antigen mixed with TMC. The produced serum antibodies were also opsonic against clinically isolated GAS strains. Further, a reduction in bacterial burden was observed in nasal secretions, pharyngeal surface and nasopharyngeal-associated lymphoid tissue of mice immunized with NP-1 in GAS challenge studies. Thus, conjugation of defined-length anionic polymer to peptide antigen as a means of formulating ionic interaction-based nanoparticles with cationic polymer is a promising strategy for peptide antigen delivery. STATEMENT OF SIGNIFICANCE: A self-adjuvanting delivery system is required for peptide vaccines to enhance antigen delivery to immune cells and generate systemic and mucosal immunity. Herein, we developed a novel self-adjuvanting nanoparticulate delivery system for peptide antigens by combining polymer-conjugation and complexation strategies. We conjugated peptide antigen with anionic α-poly-(l-glutamic acid) that in turn, formed nanoparticles with cationic trimethyl chitosan by ionic interactions, without using external crosslinker. On intranasal administration to mice, these nanoparticles induced systemic and mucosal immunity, at low dose. Additionally, nanoparticles provided protection to vaccinated mice against group A streptococcus infection. Thus, this concept should be particularly useful in developing nanoparticles for the delivery of peptide antigens., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Polysaccharides represent a versatile class of building blocks that are used in macromolecular design. By choosing the appropriate saccharide block, various physico-chemical and biological properties can be introduced both at the level of the polymer chains and the resulting self-assembled nanostructures. Here, we synthetized amphiphilic diblock copolymers combining a hydrophobic and helical poly(γ-benzyl-L-glutamate) PBLG and two polysaccharides, namely hyaluronic acid (HA) and laminarin (LAM). The copolymers could self-assemble to form particles in water by nanoprecipitation. In addition, hybrid particles containing both HA and LAM in different ratios were obtained by co-nanoprecipitation of the two copolymers. By controlling the self-assembly process, five particle samples with different morphologies and compositions were developed. The interaction between the particles and biologically relevant proteins for HA and LAM, namely CD44 and Dectin-1 respectively, was evaluated by surface plasmon resonance (SPR). We demonstrated that the particle-protein interaction could be modulated by the particle structure and composition. It is therefore suggested that this method based on nanoprecipitation is a practical and versatile way to obtain particles with controllable interactions with proteins, hence with the appropriate biological properties for biomedical applications such as drug delivery.

Multidrug resistance (MDR) in cancer remains a significant challenge for curing cancer by chemotherapy. In this work, a kind of glutathione (GSH)-responsive polymer prodrug of SO 2 was designed and synthesized, which presented synergistic effect with doxorubicin (DOX) for combating MCF-7 ADR human breast cancer cell. Firstly, a small molecular prodrug of SO 2 , N-(3-azidopropyl)-2,4-dinitrobenzenesulfonamide (AP-DNs), was chemically conjugated onto the side chain of methoxy poly (ethylene glycol)-block-poly (γ-propargyl-l-glutamate) (mPEG-PPLG) block copolymer to generate an amphiphilic polymer prodrug of SO 2 , mPEG-PLG (DNs). The obtained mPEG-PLG (DNs) prodrug could self-assemble into micelles in aqueous media and release SO 2 rapidly in response to thiol compounds. Then, DOX was loaded into mPEG-PLG (DNs) nanoparticles with ultrahigh drug-loading efficiency (97.3%). In vitro drug release tests indicated that the DOX-loaded nanoparticles could simultaneously release SO 2 and DOX by GSH triggering. Moreover, the effective cellular uptake of the DOX-loaded nanoparticles and subsequent intracellular release of SO 2 and DOX were verified by confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) analyses. The released SO 2 could promote the reactive oxygen species (ROS) level in tumor cells, which thereby resulted in oxidative damages of cancer cells, together with restoration of MCF-7 ADR cells sensitivity to DOX. As a result, the released DOX and SO 2 showed synergistic therapeutic effect against MCF-7 ADR cells. In vivo antitumor evaluation further indicated that, compared with free DOX, the DOX-loaded nanoparticles exhibited better antitumor effect in a MCF-7 ADR-xenografted nude mice model while had lower system toxicity. Overall, we demonstrated, for the first time, that a SO 2 polymer prodrug, acting as a stimuli-responsive nanocarrier to codeliver DOX, can efficiently inhibit the proliferation of MDR tumor cells, which may offer a new weapon for combating MDR in cancer therapy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In this study, novel poly(γ-benzyl l-glutamate)/poly(lactic-co-glycolic acid)/bioglass (PBLG/PLGA/BG) composite scaffolds with different weight ratios were fabricated using a negative NaCl-templating method. The morphology, compression modulus and degradation kinetics of the scaffolds were characterized. The results showed that the PBLG/PLGA/BG composite scaffolds with a weight ratio of 5:5:1, namely PBLG5PLGA5BG composite scaffolds, displayed a pore size range of 50-500μm, high compressive modulus (566.6±8.8kPa), suitable glass transition temperature (46.8±0.2°C) and low degradation rate (>8weeks). The in vitro biocompatibility of the scaffolds was evaluated with MC3T3-E1 cells by live-dead staining, MTT and ALP activity assays. The obtained results indicated that the PBLG5PLGA5BG composite scaffolds were more conducive to the adhesion, proliferation and osteoblastic differentiation of MC3T3-E1 cells than PBLG and PBLG/PLGA composite scaffolds. The in vivo biocompatibility of the scaffolds was evaluated in both SD rat subcutaneous model and rabbit tibia defect model. The results of H&E, Masson's trichrome and CD34 staining assays demonstrated that the PBLG5PLGA5BG composite scaffolds allowed the ingrowth of tissue and microvessels more effectively than PBLG/PLGA composite scaffolds. The results of digital radiography confirmed that the PBLG5PLGA5BG composite scaffolds significantly improved in vivo osteogenesis. Collectively, the PBLG5PLGA5BG composite scaffolds could be a promising candidate for tissue engineering applications., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The clinical success of cancer nanomedicines critically depends on availability of simple, safe and highly efficient nanocarriers. Here, we report that robust and multifunctional nanoparticles self-assembled from hyaluronic acid-g-poly(γ-benzyl-l-glutamate)-lipoic acid conjugates achieve a remarkably high loading (up to 25.8wt.%) and active targeted delivery of doxorubicin (DOX) to human breast tumor xenograft in vivo. DOX-loaded nanoparticles following auto-crosslinking (DOX-CLNPs) are highly stable with little drug leakage under physiological conditions while quickly release ca. 92% DOX in 30h under a cytoplasmic-mimicking reductive environment. The in vitro assays reveal that DOX-CLNPs possess a superior selectivity and antitumor activity to clinically used pegylated liposomal doxorubicin hydrochloride (DOX-LPs) in CD44 receptor overexpressing MCF-7 human breast cancer cells. Strikingly, DOX-CLNPs exhibit a superb tolerated dose of over 100mg DOX equiv./kg, which is more than 5 times higher than DOX-LPs, and an extraordinary breast tumor accumulation of 8.6%ID/g in mice. The in vivo therapeutic studies in MCF-7 human breast tumor-bearing nude mice show that DOX-CLNPs effectively inhibit tumor growth, improve survival rate, and significantly decrease adverse effects as compared to DOX-LPs. DOX-CLNPs based on natural endogenous materials with high drug loading, great stability and CD44-targetability are highly promising for precision cancer chemotherapy., Statement of Significance: We demonstrate that with rational design, simple and multifunctional anticancer nanotherapeutics can be developed to achieve highly efficient and targeted cancer chemotherapy. Doxorubicin-loaded multifunctional nanoparticles based on hyaluronic acid-g-poly(γ-benzyl-l-glutamate)-lipoic acid conjugates exhibit a high drug loading, superior stability, fast bioresponsivity, high tolerability, and obvious selectivity toward CD44-overexpressing tumors in vivo. These nanotherapeutics achieve effective tumor suppression, drastically improved survival rate and reduced side effects as compared to clinically used pegylated liposomal doxorubicin in MCF-7 human breast tumor-bearing nude mice. Unlike previously reported multifunctional nanomedicines, the present nanotherapeutics primarily based on natural endogenous materials are simple and straightforward to fabricate, which makes them potentially interesting for clinical translation., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Glycopolypeptides are an emerging class of bioinspired polymers that mimic naturally occurring glycopeptides or glycoproteins, and therefore are expected to exhibit great potential for biomedical applications. In this study, a glycopolypeptide was synthesized by conjugation of poly(γ-propargyl-l-glutamate) (PPLG) with azido-modified mannose and 3-(4-hydroxyphenyl) propanamide (HPPA), via click chemistry. Injectable hydrogels based on the glycopolypeptide were developed through enzymatic crosslinking reaction in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The physicochemical properties of the hydrogels, such as gelation time, storage modulus, swelling and degradation time, could be controlled by varying the concentrations of HRP and H2O2. The glycopolypetide copolymer as well as the extracts of the glycopolypetide hydrogels displayed good cytocompatibility in vitro. After subcutaneous injection into rats, the glycopolypeptide hydrogels were rapidly formed in situ, and exhibited acceptable biocompatibility accompanying the degradation of the hydrogels in vivo. The rabbit chondrocytes inside the glycopolypeptide hydrogels showed spherical morphology with high viability during the incubation period of 3 weeks in vitro, and exhibited a higher proliferation rate than within the hydrogel counterparts of PPLG grafted with 2-(2-(2-methoxyethoxy)ethoxy)ethane (MEO3) and HPPA. Biochemical analysis demonstrated that the production of glycosaminoglycans (GAG) and type II collagen were significantly enhanced after incubation for 2 and 3 weeks in vitro. Moreover, the chondrocyte-containing glycopolypeptide hydrogels in subcutaneous model of nude mice maintained chondrocyte phenotype and produced the cartilaginous specific matrix. These results indicated that the biomimetic glycopolypeptide-based hydrogels hold potential as three-dimensional scaffolds for cartilage tissue engineering., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In this study, monomethoxy (polyethylene glycol)-b-P (d,l-lactic-co-glycolic acid)-b-P (l-glutamic acid) (mPEG-PLGA-PGlu) nanoparticles with the ability to rapidly respond to the endolysosomal pH and hydrolase were prepared and the pH-sensitivity was tuned by adjusting the length of the PGlu segment. The mPEG5k-PLGA20k-PGlu (60) nanoparticles were specifically responsive to an endosomal pH of 5.0-6.0 due to the configuration transition of the PGlu segment and rapidly initiated chemical degradation after incubation with proteinase k for 10 min. Doxorubicin hydrochloride (DOX), used as a model drug, was easily encapsulated into nanoparticles and the DOX-loaded nanoparticles (DOX-NPs) exhibited a pH-dependent and enzyme-sensitive release profile in vitro. The dual sensitivity enabled the rapid escape of DOX-loaded nanoparticles from the endolysosomal system to target cellular nuclei, which resulted in increased cell toxicity against MCF/ADR resistant breast cancer cells and a higher cellular uptake than free DOX. In Vivo Imaging studies indicated that the nanoparticles could continuously accumulate in the tumor tissues through EPR effects and Ex vivo Imaging biodistribution studies indicated that DOX-NPs increased drug penetration into tumors compared with normal tissues. The in vivo antitumor activity demonstrated that DOX-loaded NPs had less body loss and a significant regression of tumor growth, indicating the increased anti-tumor efficacy and lower systemic toxicity. Therefore, this dual sensitive nanoparticle system may be a potential nanocarrier to overcome the multidrug resistance exhibited by breast cancer., (Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Difficulties in the extraction of membrane proteins from cell membrane and their solubilization in native conformations have hindered their structural and biochemical analysis. To overcome these difficulties, an amphipathic polypeptide was synthesized by the conjugation of octyl and glucosyl groups to the carboxyl groups of poly-γ-glutamic acid (PGA). This polymer, called amphipathic PGA (APG), self-assembles as mono-disperse oligomers consisted of 4-5 monomers. APG shows significantly low value of critical micelle concentration and stabilization activity toward membrane proteins. Most of the sodium dodecyl sulfate (SDS)-solubilized membrane proteins from Escherichia coli remain soluble state in the presence of APG even after the removal of SDS. In addition, APG stabilizes purified 7 transmembrane proteins such as bacteriorhodopsin and human endothelin receptor Type A (ETA ) in their active conformations. Furthermore, ETA in complex with APG is readily inserted into liposomes without disrupting the integrity of liposomes. These properties of APG can be applied to overcome the difficulties in the stabilization and reconstitution of membrane proteins., (© 2014 The Protein Society.)

The bioactive polymer poly(L-glutamic acid)n-b-poly(D, L-lactic acid)m was synthesized and used to form doxorubicin-loaded hybrid polymeric micelles to treat melanoma. These polymers exhibited pH-responsive changes in conformation, which controlled the diverse functionalities of the micelles. During circulation, poly(L-glutamic acid)n-b-poly(D, L-lactic acid)m protected Tat peptides on the micelles from proteolysis. Under tumor-acidic conditions, polymers with shorter poly(l-glutamic acid) blocks underwent a conformational change to form channels that accelerated the release of doxorubicin. The conformational change also exposed the Tat peptides to tumor cells, thereby promoting cellular internalization of the micelles. Enhanced cellular uptake of the micelles induced significant apoptosis of A375 melanoma cells in tumor-acidic conditions. In vivo studies demonstrated that the micelles with shorter poly(L-glutamic acid) blocks could effectively accumulate in tumor tissues, suppress tumor growth and help maintain the body weight of tumor-bearing mice. However, micelles with longer poly(l-glutamic acid) blocks did not undergo a conformational change under acidic conditions and performed poorly in both in vitro and in vivo evaluations. Our work provides a strategy for applying bioactive polymers to the rational construction of pH-responsive delivery systems for solid tumors and lends insight into possible conformational effects on the bioactivity of drug carriers., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

To achieve effective intracellular anticancer drug delivery, the polymeric vesicles supplemented with the pH-responsive outlayered gels as a delivery system of doxorubicin (DOX) were developed from self-assembly of the lipid/polypeptide adduct, distearin grafted poly(γ-glutamic acid) (poly(γ-GA)), followed by sequential deposition of chitosan and poly(γ-GA-co-γ-glutamyl oxysuccinimide)-g-monomethoxy poly(ethylene glycol) in combination with in situ covalent cross-linking on assembly surfaces. The resultant gel-caged polymeric vesicles (GCPVs) showed superior performance in regulating drug release in response to the external pH change. Under typical physiological conditions (pH 7.4 and 37 °C) at which the γ-GA/DOX ionic pairings remained mostly undisturbed, the dense outlayered gels of GCPVs significantly reduced the premature leakage of the uncomplexed payload. With the environmental pH being reduced from pH 7.4 to 4.7, the drug liberation was appreciably promoted by the massive disruption of the ionic γ-GA/DOX complexes along with the significant swelling of nanogel layers upon the increased protonation of chitosan chain segments. After being internalized by HeLa cells via endocytosis, GCPVs exhibited cytotoxic effect comparable to free DOX achieved by rapidly releasing the payload in intracellular acidic endosomes and lysosomes. This strongly implies the great promise of such unique GCPVs as an intracellular drug delivery carrier for potential anticancer treatment.

There are two important obstacles for the currently applied anti-cancer drug delivery systems. One is the conflict between long-circulation and cellular uptake while the other one is the achievement of ideal anti-cancer efficacy. To solve these problems, we designed a polypeptide-based micelle system that combined the advantages of receptor mediated endocytosis and multi-drug delivery. Firstly, an amphiphilic PLG-g-Ve/PEG graft copolymer was prepared by grafting α-tocopherol (Ve) and polyethylene glycol (PEG) to poly(l-glutamic acid) (PLG). Then docetaxel (DTX) and cisplatin (CDDP) were co-loaded into the PLG-g-Ve/PEG micelles via hydrophobic and chelation effect. After that, the surface of the dual-drug-loaded micelles was decorated with an αvβ3 integrin targeting peptide c(RGDfK). The targeted dual-drug-loaded micelles showed synergistic cytotoxicity and enhanced internalization rate in mouse melanoma (B16F1) cells. In vivo tests demonstrated that remarkable long circulation, anti-tumor and anti-metastasis efficacy could be achieved using this drug delivery system. This work revealed a strategy for the design and preparation of anti-cancer drug delivery systems with reduced side effect, enhanced anti-tumor and anti-metastasis efficacy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We prepared size-regulated nanoparticles (NPs) composed of amphiphilic poly(γ-glutamic acid) (γ-PGA). In this study, 40, 100 and 200 nm γ-PGA-graft-l-phenylalanine ethylester (γ-PGA-Phe) NPs were employed. The size of NPs significantly influenced the uptake and activation behaviors of antigen-presenting cells (APCs). When 40 nm γ-PGA-Phe NPs were applied to these cells in vitro, they were highly activated compared with 100 and 200 nm NPs, while cellular uptake was size dependent. The size of the γ-PGA-Phe NPs also significantly affected their migration to the lymph nodes and uptake behavior of NPs by dendritic cells (DCs) in vivo. The 40 nm γ-PGA-Phe NPs migrated more rapidly to the lymph nodes and were taken up by a greater number of DCs compared with 100 and 200 nm NPs. On the other hand, when the amount of γ-PGA-Phe NPs taken up per DC was evaluated, it was higher for 100 and 200 nm NPs than for 40 nm NPs, which suggests that the larger γ-PGA-Phe NPs can deliver a large amount of antigen to a single DC compared with smaller NPs. Furthermore, when examined the maturation of DCs in lymph nodes, 40 nm γ-PGA-Phe NPs efficiently stimulated DCs. These results suggest that the activation, uptake behavior by APCs, migration to lymph nodes, and DC maturation can be controlled by the size of γ-PGA-Phe NPs., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Objective: To synthesize a biodegradable non-viral gene carrier with a high transfection efficiency and a low cytotoxicity., Methods: 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., Results: 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., Conclusions: The polymer can be used as a potential non-viral gene carrier for gene therapy.

Biodegradable micelles with sheddable poly(ethylene glycol) shells were fabricated based on poly(ethylene glycol)-block-poly(γ-benzyl L-glutamate) (mPEG-SS-PBLG) diblock copolymer and applied as the carrier of 7-ethyl-10-hydroxy-camptothecin (SN-38) in order to enhance its solubility and stability in aqueous media. The diblock polymer was designed to have the hydrophilic PEG moiety and hydrophobic PBLG moiety linked by biodegradable disulfide bond, so in reducing environment the PEG shells can be detached. The polymer was able to form the micelles of nano-scale in aqueous media, suggesting their passive targeting potential to tumor tissue. Water-insoluble antitumor drug, SN-38, was easily encapsulated into mPEG-SS-PBLG nanomicelles by lyophilization method. When setting theoretical drug loading content at 10 wt%, the drug encapsulation efficiency (EE) was assayed as 73.5%. Owing to the disulfide bond in mPEG-SS-PBLG, intense release of SN-38 occurred in the presence of dithiothreitol (DTT) at the concentration of simulating the intracellular condition, however, micelles showed gradual release of SN-38 in the absence of DTT. Also, the mPEG-SS-PBLG micelles effectively protected the active lactone ring of SN-38 from hydrolysis under physiological condition. Compared with free SN-38, SN-38-loaded nanomicelles showed essentially decreased cytotoxicity against L929 cell line in 24h, bare mPEG-SS-PBLG nanomicelles showed almost non-toxicity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Self-assembly behavior of mixture systems containing poly(γ-benzyl-L-glutamate)-poly(ethylene glycol) graft (PBLG-g-PEG) and block (PBLG-b-PEG) copolymers in aqueous solution was investigated by both experiments and computer simulations. Pure graft copolymers self-assembled into vesicles, and pure block copolymers aggregated into spherical micelles or vesicles, while, for the mixture systems, hybrid cylindrical micelles were observed. In addition to the experimental observations, self-consistent field theory (SCFT) simulations were performed on the self-assembly behavior of graft/block copolymer mixtures. Simulation results reproduced the morphological transitions observed in the experiments. Moreover, from the SCFT simulations, the chain distributions of copolymers in the aggregates were obtained. For the hybrid cylindrical micelles, block copolymers were found to mainly locate at the ends of aggregates, which prevents the fusion of cylinders to vesicles. By combining experimental findings with simulation results, the mechanism regarding the morphological transition of the aggregates formed by graft/block copolymer mixtures is proposed.

Complexing agents such as diethylene triamine pentaacetic acid (DTPA) are known to disrupt intestinal tight junctions and inhibit intestinal proteases by chelating divalent metal ions. This study attempts to incorporate these benefits of DTPA in functional nanoparticles (NPs) for oral insulin delivery. To maintain the complexing agent concentrated on the intestinal mucosal surface, where the paracellular permeation enhancement and enzyme inhibition are required, DTPA was covalently conjugated on poly(γ-glutamic acid) (γPGA). The functional NPs were prepared by mixing cationic chitosan (CS) with anionic γPGA-DTPA conjugate. The γPGA-DTPA conjugate inhibited the intestinal proteases substantially, and produced a transient and reversible enhancement of paracellular permeability. The prepared NPs were pH-responsive; with an increasing pH, CS/γPGA-DTPA NPs swelled gradually and disintegrated at a pH value above 7.0. Additionally, the biodistribution of insulin orally delivered by CS/γPGA-DTPA NPs in rats was examined by confocal microscopy and scintigraphy. Experimental results indicate that CS/γPGA-DTPA NPs can promote the insulin absorption throughout the entire small intestine; the absorbed insulin was clearly identified in the kidney and bladder. In addition to producing a prolonged reduction in blood glucose levels, the oral intake of the enteric-coated capsule containing CS/γPGA-DTPA NPs showed a maximum insulin concentration at 4 h after treatment. The relative oral bioavailability of insulin was approximately 20%. Results of this study demonstrate the potential role for the proposed formulation in delivering therapeutic proteins by oral route., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A polypeptide-based double hydrophilic graft copolymer was synthesized by the sequential grafting of poly(N-isopropylacrylamide) (PNIPAM) and 2-hydroxyethyl methacrylate (HEMA) onto poly(l-glutamic acid) (PGA) backbone. The copolymers were sensitive to both temperature and pH. The phase transition and aggregation behaviors of the graft copolymers in aqueous solutions were investigated by the turbidity measurements and dynamic laser scattering (DLS). The light transmittance decrease of the copolymers at temperature above lower critical solution temperature (LCST) was remarkably weakened at pH around 6.5 due to the coil to α helix change of PGA chain induced by pH. The copolymers can self-assembly into micelles with PNIPAM cores in the aqueous solution at pH 8.0 and 60°C. Subsequently, polymerization of HEMA led to the facile preparation of crosslinked micelles, which were observed directly by transmission electron microscopy (TEM). The temperature controlled shrinkage behaviors of crosslinked micelles highly depended on the pH values of the solution. The crosslinked micelles aggregated at pH 5.0 due to the increased hydrophobic interactions among them induced by the protonation of PGA component. These crosslinked micelles have promising applications as intelligent drug delivery vehicles., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A poly(gamma-glutamic acid) (gammaPGA)-cholesterol conjugate was synthesized and the properties of an aqueous solution were evaluated. The conjugate showed amphiphilic nature derived from the hydrophilic gammaPGA backbone and the hydrophobic cholesterol side chain. The conjugate spontaneously formed nanoparticles in the aqueous solution of the low concentration, and the high concentration resulted in the formation of the physical gel. By utilizing the self-aggregating properties of the conjugate in water, an artificial chaperone was developed. The complex of protein with the nanoparticles of the conjugate was formed and the protein was released upon the dissociation of the nanoparticles by the addition of beta-cyclodextrin. For denatured carbonic anhydrase, the activity was recovered in the artificial chaperone of the nanoparticle conjugate.

The three-dimensional (3D)-engineered tissues composed of only cells and extracellular matrices (ECM) were constructed by the hydrogel template approach. The disulfide-crosslinked poly(gamma-glutamic acid) hydrogels were prepared as a template hydrogel. These template hydrogels were easily decomposed under physiological conditions using reductants such as cysteine, glutathione and dithiothreitol by cleavage of disulfide crosslinkage to thiol groups. The decomposed polymers are soluble in cell culture medium. The cleaving of disulfide bond was determined by UV-vis and FT-IR spectroscopies. We successfully prepared the 3D-engineered tissues (thickness/diameter, 2mm/1cm) composed of mouse L929 fibroblast cells and ECM by the decomposition of only the template hydrogel with cysteine after 10 days 3D-cell culture on/in the template hydrogel. The size and thickness of the 3D-engineered tissues was completely transferred from the template hydrogel. The cultured L929 cells viability in the obtained engineered tissues was confirmed by a culture test, WST-1 method and LIVE/DEAD staining assay. The engineered tissue was self-standing and highly dense composite of the cultured cells and collagen produced by the cells. This hydrogel template approach may be useful as a new class of soft-tissue engineering technology to substitute a synthetic polymer scaffold to the ECM scaffold produced from the cultured cells.

Physicochemical properties of PBLG (poly(gamma-benzyl-l-glutamate))-PEO (poly(ethylene oxide)) diblock copolymers composed of PBLG as the hydrophobic rod component and PEO as the hydrophilic component were investigated at the air-water interface. Surface pressure-area isotherms obtained by the Wilhelmy plate method provide several variables such as molecular size, compressibility of PEO, and the free energy change of the PBLG-PEO block copolymer. GE-1 (M(w) of PBLG:PEO=103,700:12,000), with a relatively longer rod, has negative temperature effects and GE-3 (M(w) of PBLG:PEO=8400:12,000), with a relatively shorter rod, shows a positive temperature effect because of the large entropy loss. These competitions were based on the block size of PBLG and PEO and were affected by various microstructures of the PBLG-PEO diblock copolymer. Monolayer aggregations transferred onto mica from the air-water interface were analyzed with AFM. AFM images of GE-1 monolayers show cylindrical micelles, but the self-assembled structure has many large domains. The monolayer of GE-2 (M(w) of PBLG:PEO=39,800:12,000), which has a medium size rod, forms a spherical structure at the air-water interface. Monolayers of GE-3, with a short rod length, form bilayer structures. These results demonstrate that the microstructures of PBLG-PEO diblock copolymers are related to free energy changes between rod and coil blocks.

A novel structural triblock copolymer of poly(gamma-benzyl-l-glutamic acid)-b-poly(ethylene oxide)-b-poly(epsilon-caprolactone) (PBLG-PEO-PCL) was synthesized by a new approach in the following three steps: (1) sequential anionic ring opening polymerization (ROP) of ethylene oxide and epsilon-caprolactone with an acetonitrile/potassium naphthalene initiator system to obtain a diblock copolymer CN-PEO-PCL with a cyano end-group; (2) conversion of the CN end-group into NH2 end-group by hydrogenation to obtain NH2-PEO-PCL; (3) ROP of gamma-benzyl-l-glutamate-N-carboxyanhydrides (Bz-l-GluNCA) with NH2-PEO-PCL as macroinitiator to obtain the target triblock copolymer. The structures from CN-PEO precursor to the triblock copolymers were confirmed by FT-IR and 1H NMR spectroscopy, and their molecular weights were measured by gel permeation chromatography. The monomer of Bz-l-GluNCA can react almost quantitatively with the amino end-groups of NH2-PEO-PCL macroinitiator by ROP.

Cell modulators such as colchicine (CO), cytochalasin B (CY) and taxol (TX) loaded nanoparticles coated with carbohydrate-carrying polymers, as hepatocyte-specific targeting material using galactose ligands as recognition signals to asialoglycoprotein receptors were prepared by the diafiltration method. Effects of cell modulators from their loaded nanoparticles on morphology of hepatocytes were studied. Receptor-mediated endocytosis of the nanoparticles were examined by fluorescence and confocal laser microscopy. It was found that the shapes of most hepatocytes were changed for the CY-loaded, TX-loaded, or CO-loaded nanoparticles whereas their shapes were not changed in comparison with control when CY, TX, or CO were mixed with the nanoparticles. From the fluorescence and confocal laser microscopic studies, it is suggested that the nanoparticles coated with sugar-carrying polymers were internalized by the hepatocytes through the receptor-mediated mechanism.

Although fibrin glue has been widely used as a surgical adhesive, its components, fibrinogen and thrombin, obtained from human blood are not completely free from the risk of virus infection due to acquired immune deficiency and hepatitis. Recently, we have reported that a polymer pair composed of gelatin and poly(L-glutamic acid) (PLGA) promptly forms a gel and can firmly bond to soft tissues when crosslinked with the aid of water-soluble carbodiimide (WSC). The present study was undertaken to design a new PLGA-gelatin glue without using WSC. Two kinds of PLGA with molecular weights of 71 and 22 kDa were employed to prepare N-hydroxysuccinimide (NHS) activated derivatives. The NHS-activated PLGA could be synthesized at high yields and was found to be stable for an extended time without losing the ability to crosslink with gelatin when stored under a dry-cold condition. This NHS-activated PLGA could spontaneously form a gel with gelatin in an aqueous solution within a short time, comparable to a commercial fibrin glue, when gelation was allowed to proceed at pH 8.3. The NHS-activated PLGA prepared from PLGA with the molecular weight of 22 kDa could be readily dissolved at high concentrations and its ability to form a gel was maintained for more than 10 min when an acidic 8% NHS-activated PLGA solution was used. The bonding strength of PLGA gelatin glues with natural tissue was higher than that of fibrin glue. These findings strongly suggest that this combination of gelatin and NHS-PLGA is very promising as a surgical adhesive and may possibly replace fibrin glues prepared from human blood components.

Many natural polymeric materials (particularly structural proteins) display a hierarchy of structure over several length scales. Block copolymers are able to self-assemble into ordered nanostructures, but the random-coiled nature of their polymer chains usually suppresses any further levels of organization. The use of components with regular structures, such as rigid-rod polymers, can increase the extent of spatial organization in self-assembling materials. But the synthesis of such polymeric components typically involves complicated reaction steps that are not suitable for large-scale production. Proteins form hierarchically organized structures in which the fundamental motifs are generally alpha-helical coils and beta-sheets. Attempts to synthesize polypeptides with well-defined amino-acid sequences, which might adopt similar organized structures, have been plagued by unwanted side reactions that give rise to products with a wide range of molecular weights, hampering the formation of well-defined peptide block copolymers. Here I describe a polymerization strategy that overcomes these difficulties by using organonickel initiators which suppress chain-transfer and termination side reactions. This approach allows the facile synthesis of block copolypeptides with well-defined sequences, which might provide new peptide-based biomaterials with potential applications in tissue engineering, drug delivery and biomimetic composite formation.

The surface characteristics of A-B-A type triblock copolymer (GIG) membranes consisting of alpha-helical poly (gamma-benzyl-L-glutamate) (PBLG) as the A component and polyisoprene (PI) as the B component were investigated by X-ray photoelectron spectroscopy (XPS) and contact angle measurements. The XPS measurements showed the copolymer composition of the outermost surface to be quite different from the bulk composition. Results of contact angle measurements indicated the existence of an interfacial region between the alpha-helical A component and the B component at the surfaces of the block copolymer membranes. Finally, the results of in vivo tests on tissue compatibility indicate that the GIG block copolymer membranes have good biocompatibility.

Photodynamic therapy is a relatively new and potentially selective experimental approach to the treatment of malignant neoplasms. Its inherent dual selectivity is reinforced by the use of photosensitizer-monoclonal antibody conjugates. The goal of this study was to evaluate the phototoxicity and selectivity of an immunoconjugate (IC) synthesized from a chlorin derivative chlorin e6-monoethylenediamine monoamide (CMA) as the photosensitizer and an anti-ovarian carcinoma monoclonal antibody OC125. Binding efficiency and specificity of the IC were determined by enzyme-linked immunosorbent assay, and specific covalent linkage of the monoclonal antibody to the photosensitizer was demonstrated by fluorescence and electrophoresis. Phototoxicity was tested against ascites or pleural fluid cells from 15 patients with ovarian and nonovarian cancers. Tumor cells from the fluid were treated with the IC at 3 microM equivalent CMA concentration and irradiated at 654 nm (lambda max CMA in IC) at 25 J/cm2 from an argon ion-pumped dye laser. Phototoxic efficacy was assayed by [3H]thymidine incorporation. Ovarian cancer cells exhibited high cytotoxicity with [3H]thymidine incorporation of 2.4 +/- 2.2%, while nonovarian cancer cells under identical conditions exhibited none to reduced cytotoxicity with [3H]thymidine incorporation of 70 +/- 54%. Using a Wilcoxon test, there was a statistically significant difference between these two groups (P less than 0.001). Dose-response curves revealed reciprocity in photosensitizer concentration and fluence. These results demonstrate that photoimmunoconjugates retain significant antigen binding specificity and affinity, are effective in the selective photochemical eradication of target cells, and merit further evaluation as photochemotherapeutic agents.

We have synthesized and tested a new series of compounds that inhibit the polymerization of deoxyhemoglobin S by noncovalent interaction. They consist of three structural elements: A p-aminobenzoyl residue to anchor the compound in the central cavity between the beta chains, a number of glutamates in gamma linkage to provide tight binding, and one or two hydrophobic amino acid residues which block the intermolecular hydrophobic interaction of valine beta 6. The most active compound was p-aminobenzoyl-(gamma-Glu)5-Phe-Phe. It increases the solubility of deoxy-HbS by a factor of 1.3 at a concentration of only 5-6 mM and is effective even in the presence of physiological concentrations of 2,3-diphosphoglycerate.

A-B-A type block copolymers of poly(gamma-benzyl L-glutamate) (PBLG, A segment) and polydimethylsiloxane (PDMS, B segment) and PDMS (trunk)-PBLG (branch) graft copolymers were synthesized, and the permeation of oxygen in water and the permeation of oxygen and carbon dioxide in the dry state were investigated. The gas permeation coefficient (P) increased with increasing content of PDMS. However, PCO2/PO2 values of copolymer films were in the range 6-9, i.e. larger than 5.4 for PDMS film. The oxygen permeation in water suffered from the interfacial resistance, which was reduced by the hydrolysis of film surface. The Arrhenius plot of the gas permeation coefficient in the dry state of the block copolymer B showed a turning point at about 40 degrees C. This temperature is close to beta-peak temperature (39 degrees C) and may be ascribed to the molecular motion of the PBLG segment. Transmission electron microscopy showed that one of the block copolymer films (PDMS 46 mol%) appears to have PDMS segments dispersed in the PBLG matrix (island-in-sea structure) and one of the graft copolymer films (PDMS 58 mol%) appears to take a lamellar structure. The gas permeation across the graft copolymer film appears to occur through the continuous PDMS phase, leading to a near-negligible activation energy in this process.

A cis-diammineplatinum(II)-polyglutamic acid complex proved to have marked trypanocidal activity in vivo. When 5 mg of platinum per kg was administered daily for 5 days or when 20 mg of platinum per kg was administered on days 1, 3, and 6, five of five treated Trypanosoma congolense-infected mice were cured. In contrast, cis-platin was ineffective at all doses tested. We believe that the complex has a much higher therapeutic ratio than does uncomplexed drug because it is less toxic.