A novel low-molecular-weight chitosan/gamma-polyglutamic acid polyplexes for nucleic acid delivery into zebrafish larvae.

Many challenges, such as virus infection, extreme weather and long cultivation periods, during the development of fish larvae have been observed, especially in aquaculture. Gene delivery is a useful method to express functional genes to defend against these challengers. However, the methods for fish larvae are insufficient. In our earlier report, low-molecular-weight chitosan (LMWCS) showed a strong positive charge and may be useful for polyplex formulation. Herein, we present a simple self-assembly of LMWCS polyplexes (LMWCSrNPs) for gene delivery into zebrafish larvae. Different weight ratios of LMWCS/gamma-polyglutamic acid (γ-PGA)/plasmid DNA were analyzed by gel mobility assay. Delivery efficiency determined by green fluorescent protein (GFP) expression in zebrafish liver (ZFL) cells showed that delivery efficiency at a weight ratio of 20:8:1 was higher than others. Zeta potential and transmission electron microscopy (TEM) analysis showed that the round shape of the particle size varied. In our earlier reports, IRF9S2C could induce interferon-stimulated gene (ISG) expression to induce innate immunity in zebrafish and pufferfish. Further delivery of pcDNA3-IRF9S2C-HA plasmid DNA into ZFL cells and zebrafish larvae by LMWCSrNP successfully induced ISG expression. Collectively, LMWCSrNP could be a novel gene delivery system for zebrafish larvae and might be used to improve applications in aquaculture.

Mechanoadaptive injectable hydrogel based on poly(γ-glutamic acid) and hyaluronic acid regulates fibroblast migration for wound healing.

Injectable hydrogels have shown therapeutic effects on wound repair, but most of them exhibit poor mechanical strength. The impacts of stiff injectable hydrogels on cell behavior and wound healing remain unclear. Herein, an injectable hydrogel was developed based on thiolated poly(γ-glutamic acid) (γ-PGA-SH) and glycidyl methacrylate-conjuated oxidized hyaluronic acid (OHA-GMA). Thiol-methacrylate Michael chemistry-mediated post-stabilization and increase of polymer concentration were found to improve the mechanical strength of γ-PGA-SH/OHA-GMA hydrogel. Moreover, in vitro studies confirmed its biodegradability, biocompatibility, and self-healing property. Using the mechanically-tunable hydrogel, it further showed that fibroblasts migrated faster on the surface of stiffer hydrogel, but infiltrated slowly inside it compared with softer hydrogel. In animal experiments, the injectable hydrogel could promote wound healing by increasing collagen deposition and vascularization. In summary, γ-PGA-SH/OHA-GMA hydrogel is able to regulate migration and infiltration of fibroblasts by altering stiffness and offers effective in situ forming scaffolds towards skin tissue regeneration.

Mussel-Inspired Bisphosphonated Injectable Nanocomposite Hydrogels with Adhesive, Self-Healing, and Osteogenic Properties for Bone Regeneration.

Injectable hydrogels have received much attention because of the advantages of simulation of the natural extracellular matrix, microinvasive implantation, and filling and repairing of complex shape defects. Yet, for bone repair, the current injectable hydrogels have shown significant limitations such as the lack of tissue adhesion, deficiency of self-healing ability, and absence of osteogenic activity. Herein, a strategy to construct mussel-inspired bisphosphonated injectable nanocomposite hydrogels with adhesive, self-healing, and osteogenic properties is developed. The nano-hydroxyapatite/poly(l-glutamic acid)-dextran (nHA/PLGA-Dex) dually cross-linked (DC) injectable hydrogels are fabricated via Schiff base cross-linking and noncovalent nHA-BP chelation. The chelation between bisphosphonate ligands (alendronate sodium, BP) and nHA favors the uniform dispersion of the latter. Moreover, multiple adhesion ligands based on catechol motifs, BP, and aldehyde groups endow the hydrogels with good tissue adhesion. The hydrogels possess excellent biocompatibility and the introduction of BP and nHA both can effectively promote viability, proliferation, migration, and osteogenesis differentiation of MC3T3-E1 cells. The incorporation of BP groups and HA nanoparticles could also facilitate the angiogenic property of endothelial cells. The nHA/PLGA-Dex DC hydrogels exhibited considerable biocompatibility despite the presence of a certain degree of inflammatory response in the early stage. The successful healing of a rat cranial defect further proves the bone regeneration ability of nHA/PLGA-Dex DC injectable hydrogels. The developed tissue adhesive osteogenic injectable nHA/PLGA-Dex hydrogels show significant potential for bone regeneration application.

Lipid-Polyglutamate Nanoparticle Vaccine Platform.

Peptide-based subunit vaccines are attractive in view of personalized cancer vaccination with neo-antigens, as well as for the design of the newest generation of vaccines against infectious diseases. Key to mounting robust antigen-specific immunity is delivery of antigen to antigen-presenting (innate immune) cells in lymphoid tissue with concomitant innate immune activation to promote antigen presentation to T cells and to shape the amplitude and nature of the immune response. Nanoparticles that co-deliver both peptide antigen and molecular adjuvants are well suited for this task. However, in the context of peptide-based antigen, an unmet need exists for a generic strategy that allows for co-encapsulation of peptide and molecular adjuvants due to the stark variation in physicochemical properties based on the amino acid sequence of the peptide. These properties also strongly differ from those of many molecular adjuvants. Here, we devise a lipid nanoparticle (LNP) platform that addresses these issues. Key in our concept is poly(l-glutamic acid) (PGA), which serves as a hydrophilic backbone for conjugation of, respectively, peptide antigen (Ag) and an imidazoquinoline (IMDQ) TLR7/8 agonist as a molecular adjuvant. Making use of the PGA's polyanionic nature, we condensate PGA-Ag and PGA-IMDQ into LNP by electrostatic interaction with an ionizable lipid. We show in vitro and in vivo in mouse models that LNP encapsulation favors uptake by innate immune cells in lymphoid tissue and promotes the induction of Ag-specific T cells responses both after subcutaneous and intravenous administration.

Poly(l-glutamic acid)-Based Zwitterionic Polymer in a Charge Conversional Shielding System for Gene Therapy of Malignant Tumors.

Recently, pH-sensitive polymers have received extensive attention in tumor therapy. However, the rapid response to pH changes is the key to achieving efficient treatment. Here, a novel shielding system with a rapidly pH-responsive polymer (PAMT) is synthesized by click reaction between poly(γ-allyl-l-glutamate) and thioglycolic acid or 2-(Boc-amino)ethanethiol. The zwitterionic biodegradable polymer PAMT, which is negatively charged at physiological pH, can be used to shield positively charged nanoparticles. PAMT is electrostatically attached to the surface of the positively charged PEI/pDNA complex to form a ternary complex. The zwitterionic PAMT-shielded complex exhibits rapid charge conversion when the pH decreases from 7.4 to 6.8. For the in vivo tumor inhibition experiment, PAMT/PEI/shVEGF injected intravenously shows a more significant inhibitory effect on tumor growth. The excellent results are mainly attributed to introduction of the zwitterionic copolymer PAMT, which can shield the positively charged PEI/shVEGF complex in physiological conditions, while the surface potential of the shielded complexes changes to a positive charge in the acidic tumor environment.

In situ chemically crosslinked injectable hydrogels for the subcutaneous delivery of trastuzumab to treat breast cancer.

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 ZnCl2, and 10 mg/mL trastuzumab) could lower the maximum plasma concentration (Cmax) 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.

Multivalent and multifunctional polysaccharide-based particles for controlled receptor recognition.

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.

Zn2+-binding in the glutamate-rich region of the intrinsically disordered protein prothymosin-alpha.

Prothymosin-α is a small, multifunctional intrinsically disordered protein associated with cell survival and proliferation which binds multiple Zn2+ ions and undergoes partial folding. The interaction between prothymosin-α and at least two of its protein targets is significantly enhanced in the presence of Zn2+ ions, suggesting that Zn2+ binding plays a role in the protein's function. The primary sequence of prothymosin-α is highly acidic, with almost 50% comprised of Asp and Glu, and is unusual for a Zn2+-binding protein as it lacks Cys and His residues. To gain a better understanding of the nature of the Zn2+-prothymosin-α interactions and the protein's ability to discriminate Zn2+ over other divalent cations (e.g., Ca2+, Co2+, Mg2+) we synthesized a set of three model peptides and characterized the effect of metal binding using electrospray ionization mass spectrometry (ESI MS) and circular dichroism (CD) spectroscopy. ESI MS data reveal that the native peptide model of the glutamic acid rich region binds 4 Zn2+ ions with apparent, stepwise Kd values that are, at highest, in the tens of micromolar range. A peptide model with the same amino acid composition as the native sequence, but with the residues arranged randomly, showed no evidence of structural change by CD upon introduction of Zn2+. These results suggest that the high net negative charge of the glutamic acid-rich region of prothymosin-α is not a sufficient criterion for Zn2+ to induce a structural change; rather, Zn2+ binding to prothymosin-α is sequence specific, providing important insight into the behavior of intrinsically disordered proteins.

Polyglutamic acid-trimethyl chitosan-based intranasal peptide nano-vaccine induces potent immune responses against group A streptococcus.

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.

One-step synthesis of europium complexes containing polyamino acids through ring-opening polymerization and their potential for biological imaging applications.

Lanthanide-doped nanoprobes have received significant attentions for utilization in biological sensing and imaging due to their unique optical properties. However, only few works were focused on fabrication of europium complexes based fluorescence polymeric nanoparticles (FPNs) with excellent biocompatibility and biodegradability. In this study, we fabricated the FPNs (named as Eu(TTA)3Phen-GluEG FPNs) with encapsulated europium complexes which show low cytotoxicity, high sensitivity and deep penetration. Free amine group present on europium complexes initiated the ring-opening polymerization (ROP) of side-chain modified glutamic acid NCAs, offering a simple and effective method to prepare europium core FPNs with a uniform size distribution. Europium (III) chelates were furnished with a functional polyamino acid shell to fabricate biocompatible and biodegradable FPNs. Biological evaluation results demonstrate that such fabricated FPNs process excellent biocompatibility and dyeing performance. Therefore, we can expect that the fabrication approach will attract much research interest and effort on the preparation of biodegradable probes for various biological applications.

A glutathione-responsive sulfur dioxide polymer prodrug as a nanocarrier for combating drug-resistance in cancer chemotherapy.

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 SO2 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 SO2, 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 SO2, mPEG-PLG (DNs). The obtained mPEG-PLG (DNs) prodrug could self-assemble into micelles in aqueous media and release SO2 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 SO2 and DOX by GSH triggering. Moreover, the effective cellular uptake of the DOX-loaded nanoparticles and subsequent intracellular release of SO2 and DOX were verified by confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) analyses. The released SO2 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 SO2 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 SO2 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.

Multiarm-polyethylene glycol-polyglutamic acid peptide dendrimer: Design, synthesis, and dissolving thrombus.

Thrombotic events affect many individuals in a number of ways, all of which can cause significant morbidity and mortality. Nattokinase (NK), as a novel thrombolytic drug, has been used for thrombolytic therapy. It not only possesses plasminogen activator activity, but also directly digests fibrin through limited proteolysis. However, it may undergo inactivation and denaturation in the harsh external environment. In this study, a multiarm-polyethylene glycol-polyglutamic acid peptide dendrimer was fabricated and used as a carrier for NK protection and delivery. Different arm numbers of polyethylene glycol-polyglutamic acid peptide dendrimers (x-PEG(G3 )x , x = 2, 4, 6, 8) were designed, prepared, and characterized by 1 H NMR and FTIR. Then, x-PEG(G3 )x were loaded with NK to form nanocomposites. Their size and morphology were determined by dynamic light scattering and transmission electron microscopy. Enzyme activity was evaluated via UV-Vis absorbance spectra, fluorescence spectra, circular dichroism spectra, and zeta potential measurements. The study reveals that the obtained x-PEG(G3 )x /NK nanocomposites possess high enzyme activity. In addition, the nanocomposites show increased viability of rat macrophage cells, and excellent thrombolysis ability in vitro and in vivo. This work establishes a multiarm-polyethylene glycol-polyglutamic acid peptide dendrimer with potential application in NK carrier and thrombolytic therapy. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1687-1696, 2018.

Polypeptide Polymer Brushes by Light-Induced Surface Polymerization of Amino Acid N-Carboxyanhydrides.

Silicon wafers are decorated with photoamine generator 4,5-dimethoxy-2-nitrobenzyl 3-(triethoxysilyl)propyl carbamate. UV-irradiation in the presence of benzyl-l-glutamate N-carboxyanhydride is carried out, resulting in the release of the surface-bound primary amines, making them viable N-carboxyanhydride (NCA) polymerization initiators. Successful polypeptide grafting is confirmed by water contact angle measurements as well as by ellipsometry, revealing a poly(benzyl-l-glutamate) (PBLG) layer of ≈3 nm. X-ray photoelectron spectroscopy confirms the presence of amide groups in the grafted PBLG while time-of-flight secondary ion mass spectroscopy provides additional evidence for the presence of PBLG on the surface. Evaluation of negative control samples confirms successful UV surface grafting. The approach is thus established as a viable general method for light exposure directable polypeptide functionalization of silicon surfaces.

Synthesis and cellular characterization of various nano-assemblies of cell penetrating peptide-epirubicin-polyglutamate conjugates for the enhancement of antitumor activity.

A new class of cell penetrating peptides (CPPs) named peptide amphiphile was designed to improve the intracellular uptake and the antitumor activity of epirubicin (EPR). Various amphiphilic CPPs were synthesized by solid phase peptide synthesis method and were chemically conjugated to EPR. Their corresponding nanoparticles (CPPs-E4 and CPPs-E8) were prepared via non-covalent binding of the peptides and polyanions. Cytotoxicity and anti-proliferative activity were evaluated by MTT assay. Cellular uptake was examined by flow cytometry and fluorescence microscopy. The CPPs exhibited slight cytotoxicity. Binding of polyglutamate to CPPs (CPPs-E4 and CPPs-E8 nanoparticles) decreased their cytotoxicity. CPPs-E8 nanoparticles showed lower cytotoxicity than CPPs-E4 nanoparticles. Cellular uptake of K3W4K3-E8, K2W4K2-E8 and W3K4W3-E8 reached 100% with no difference between each of the mentioned CPPs and its nanoparticles at 50 µM. The anti-proliferative activity of EPR was enhanced following conjugation to peptides and nanoparticles at 25 µM. CPPs-EPR-E4 and CPPs-E8-EPR nanoparticles displayed higher anti-proliferative activity than CPPs-EPR at 25 µM. CPPs-E8-EPR nanoparticles showed higher anti-proliferative activity than CPPs-E4-EPR. K3W4K3-E8-EPR nanoparticles exhibited the highest anti-proliferative activity at 25 µM. The synthesized peptide nanoparticles are proposed as suitable carriers for improving the intracellular delivery of EPR into tumor cells with low cytotoxicity and high antitumor activity.

Synthesis of controlled, high-molecular weight poly(l-glutamic acid) brush polymers.

We report the synthesis and characterization of high-molecular weight poly(l-glutamic acid) based brush polymers. Utilizing a combination of ring-opening metathesis polymerization of norbornene based monomers and ring-opening polymerization of γ-benzyl-l-glutamate N-carboxyanhydride, high-molecular weight γ-benzyl protected poly(l-glutamic acid) brush polymers are synthesized. Controlled and complete deprotection of the benzyl groups using trimethylsilyl iodide resulted in poly(l-glutamic acid) based brush polymers with molecular weights up to 3.6 MDa, which may potentially be used to prepare size-controlled unimolecular polymeric nanomedicine for drug delivery applications. Camptothecin brush poly(l-glutamic acid) conjugates were prepared and their stability, drug release kinetics, and in vitro toxicity were studied.

Synthesis of Poly-γ-S-2-methylbutyl-l-glutamate and Poly-γ-S-2-methylbutyl-d-glutamate and Their Use as Enantiodiscriminating Alignment Media in NMR Spectroscopy.

Lyotropic liquid crystalline (LLC) phases of polyglutamic acid derivatives, such as poly-γ-benzyl-l-glutamate, are suitable alignment media for organic structure elucidation by NMR spectroscopy. Their helical structure is responsible for enantiodiscrimination. As part of our ongoing investigations concerning the alignment mechanism(s) of these systems, we considered whether an additional chiral center in the side chain could improve enantiodiscrimination relative to the helical polymer with an achiral side chain. Therefore, the diastereoisomers poly-γ-S-2-methylbutyl-l-glutamate (PSMBLG) and poly-γ-S-2-methylbutyl-d-glutamate (PSMBDG) were synthesized. These two polymers were tested for their liquid crystallinity and suitability as alignment media. The spatial structure of the solutes (-)-curcumol and isopinocampheol (IPC) were validated by the residual dipolar coupling data obtained. Additionally, enantiodiscrimination of IPC was observed in the new alignment media and compared with the enantiodiscrimination of IPC in other homopolypeptides.

Preparation and mineralization of a biocompatible double network hydrogel.

Double network (DN) hydrogels have shown favorable toughness and strength and been harnessed as scaffolding materials in tissue engineering, particularly, mineralized DN hydrogels may be potential scaffolds in bone tissue engineering. In this paper, DN hydrogels were fabricated from poly(ethylene glycol) diacrylate (PEGDA) and methacrylated poly(γ-glutamic acid) (mPGA). This DN hydrogel not only showed excellent mechanical properties but also good cytocompatibility as evidenced by the characterizations in terms of swelling ratio, mechanical strength/modulus, and cytotoxicity. Further, the DN hydrogels were subjected to mineralization in simulated body fluid, during which hydroxyapatite or analogues formed within the DN hydrogels; the DN hydrogels may be potentially harnessed as bone tissue engineering scaffold.

Chemoenzyamtic synthesis and self-assembling gelation behavior of amylose-grafted poly(γ-glutamic acid).

In this study, we investigated chmemoenzymatic synthesis of amylose-grafted poly(γ-glutamic acid) (PGA) as a new artificial saccharide-peptide conjugate composed of two biological macromolecules. Maltooligosaccharide as a primer of enzymatic polymerization by phosphorylase catalysis was first introduced on the PGA main chain by the condensation reaction using the condensing agent in NaOH aq. Thermostable phosphorylase-catalyzed enzymatic polymerization of α-d-glucose 1-phosphate (G-1-P) as a monomer was then performed from the primer chain ends of the product to obtain amylose-grafted PGAs, which formed hydrogels in reaction media depending on the G-1-P/primer feed ratios. The powder X-ray diffraction patterns of lyophilized samples (cryogels) from the hydrogels suggested that the amylose graft chains formed double helixes, which acted as cross-inking points for self-assembling hydrogelation. The scanning electron microscopic images of the cryogels showed regularly controlled porous morphologies. Moreover, pore sizes of the cryogels increased with increasing the G-1-P/primer feed ratios, whereas the degrees of substitution of primer on the PGA main chain did not obviously affect pore sizes.

Preparation, physicochemical properties and biocompatibility of PBLG/PLGA/bioglass composite scaffolds.

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.

Co-delivery of polymeric metformin and cisplatin by self-assembled core-membrane nanoparticles to treat non-small cell lung cancer.

Clinically, combined therapy of cisplatin (CDDP) and metformin is an effective treatment for non-small cell lung cancer (NSCLC). The success is attributed to synergistic effects between the two drugs. Therefore, we hypothesize that co-encapsulation of CDDP and metformin will avoid the prominent toxicity of CDDP while maintaining the synergy between the regimens. CDDP was first conjugated to polyglutamic acid (PGA) to form anionic PGA-CDDP which was electrostatically complexed with the cationic polymeric metformin (polymet). The nano-sized complex was then stabilized with cationic liposomes composed of DOTAP (2, 3-Dioleoyloxy-propyl)-trimethylammonium/Cholesterol/DSPE-PEG-anisamide aminoethyl. Both in vitro and in vivo experiments confirmed the synergy between polymet and CDDP. CDDP delivered with nanoparticles (NPs) exhibited significantly increased tumor accumulation over free CDDP and suppressed tumor growth through apoptosis in NSCLC H460 tumor-bearing mice without nephrotoxicity. The synergistic effect of polymet alongside CDDP demonstrates that polymet-CDDP NPs can activate the AMP-activated protein kinase α (AMPKα) pathway and inhibit mammalian target rapamycin (mTOR) activity to enhance growth suppression. In all, this platform is the first to successfully co-load polymet, a polymeric metformin, and CDDP into the same nanoparticle for successful treatment of NSCLC.