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.

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.

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.

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.

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.

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.

Robust, active tumor-targeting and fast bioresponsive anticancer nanotherapeutics based on natural endogenous materials.

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.

Methoxy poly (ethylene glycol)-block-poly (glutamic acid)-graft-6-(2-nitroimidazole) hexyl amine nanoparticles for potential hypoxia-responsive delivery of doxorubicin.

Tumor microenvironment-responsive nano drug delivery vehicles are gaining mounting attention in the field of biomedical sciences. The hypoxic response of the tumorous cells due to very low partial pressure of oxygen (some time less than 2.5 mm of Hg) in the tumor tissues makes hypoxia-responsive drug delivery system as the more appealing in cancer chemotherapy. Based on these considerations, we synthesized hypoxia-responsive polymeric materials methoxy poly (ethylene glycol)-block-poly (glutamic acid)-graft-6-(2-nitroimidazole) hexyl amine (mPEG-b-PLG-g-NID) by conjugation of the hydrophobic nitro imidazole derivative (NID)[6-(2-nitroimidazole) hexyl amine] with the pendant carboxylic group of poly (ethylene glycol)-block-poly (L-glutamic acid)(mPEG-b-PLG). The structure and degree of substitution were confirmed by proton NMR, FTIR, and UV-Vis spectroscopy. The degree of substitution was found to enhance with the increase in NID to polymer ratio. The hypoxia response of the material was evaluated by UV-Vis spectroscopy and zeta potential measurements. Doxorubicin was hydrophobically encapsulated in the micellar core of the hypoxia-responsive nanoparticles. The drug-loaded micelles showed faster release in hypoxic condition as compared to normoxic conditions. Moreover, the developed polymeric system was found non-toxic to MCF-7 cell line, thus suggesting its biocompatibility and suitability as drug delivery device.

Well-Defined Star-Shaped Polyglutamates with Improved Pharmacokinetic Profiles As Excellent Candidates for Biomedical Applications.

There is a need to develop new and innovative polymer carriers to be used as drug delivery systems and/or imaging agents owing to the fact that there is no universal polymeric system that can be used in the treatment of all diseases. Additionally, limitations with existing systems, such as a lack of biodegradability and biocompatibility, inevitably lead to side effects and poor patient compliance. New polymer therapeutics based on amino acids are excellent candidates for drug delivery, as they do not suffer from these limitations. This article reports on a simple yet powerful methodology for the synthesis of 3-arm star-shaped polyglutamic acid with well-defined structures, precise molecular weights (MW), and low polydispersity (D = <1.3). These were synthesized by ring-opening polymerization (ROP) of N-carboxyanhydrides (NCA) in a divergent method from novel multifunctional initiators. Herein, their exhaustive physicochemical characterization is presented. Furthermore, preliminary in vitro evaluation in selected cell models, and exhaustive in vivo biodistribution and pharmacokinetics, highlighted the advantages of these branched systems when compared with their linear counterparts in terms of cell uptake enhancement and prolonged plasma half-life.

Polypeptide-b-Poly(Phenyl Isocyanide) Hybrid Rod-Rod Copolymers: One-Pot Synthesis, Self-Assembly, and Cell Imaging.

Hybrid rod-rod diblock copolymers, poly(γ-benzyl L-glutamate)-poly(4-cyano-benzoic acid 2-isopropyl-5-methyl-cyclohexyl ester) (PBLG-PPI), with determined chirality are facilely synthesized through sequential copolymerization of γ-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA) and phenyl isocyanide monomers bearing chiral menthyl pendants using a Ni(cod)(bpy) complex as the catalyst in one-pot. Circular dichroism and absorption spectra reveal that each block of the block copolymers possesses a stable helical conformation with controlled helicity in solution due to the induction of chiral pendants. The two diastereomeric polymers self-assemble into helical nanofibrils with opposite handedness due to the different chiral induction of the L- and D-menthyl pendants, confirmed by transmission electron microscopy (TEM). Deprotection of the benzyl groups of the PBLG segment affords biocompatible amphiphilic diblock copolymers, poly(L-glutamic acid)-poly(4-cyano-benzoic acid 2-isopropyl-5-methyl-cyclohexyl ester) (PLGA-PPI), that can self-assemble into well-defined micelles by cosolvent induced aggregation. Very interestingly, a chiral rhodamine chromophores RhB(D) can be selectively encapsulated into the chiral polymeric micelles, which is efficiently internalized into living cells when directly monitored with a confocal microscope. This contribution will be useful for developing novel rod-rod biocompatible hybrid block copolymers with a controlled helicity, and may also provide unique chiral materials for potential bio-medical applications.

pH-Responsive chimaeric pepsomes based on asymmetric poly(ethylene glycol)-b-poly(l-leucine)-b-poly(l-glutamic acid) triblock copolymer for efficient loading and active intracellular delivery of doxorubicin hydrochloride.

pH-Responsive chimaeric polypeptide-based polymersomes (refer to as pepsomes) were designed and developed from asymmetric poly(ethylene glycol)-b-poly(l-leucine)-b-poly(l-glutamic acid) (PEG-PLeu-PGA, PEG is longer than PGA) triblock copolymers for efficient encapsulation and triggered intracellular delivery of doxorubicin hydrochloride (DOX·HCl). PEG-PLeu-PGA was conveniently prepared by sequential ring-opening polymerization of l-leucine N-carboxyanhydride and γ-benzyl-l-glutamate N-carboxyanhydride using PEG-NH2 as an initiator followed by deprotection. Pepsomes formed from PEG-PLeu-PGA had unimodal distribution and small sizes of 64-71 nm depending on PLeu block lengths. Interestingly, these chimaeric pepsomes while stable at pH 7.4 were quickly disrupted at pH 5.0, likely due to alternation of ionization state of the carboxylic groups in PGA that shifts PGA blocks from hydrophilic and random coil structure into hydrophobic and α-helical structure. DOX·HCl could be actively loaded into the watery core of pepsomes with a high loading efficiency. Remarkably, the in vitro release studies revealed that release of DOX·HCl was highly dependent on pH, in which about 24.0% and 75.7% of drug was released at pH 7.4 and 5.0, respectively, at 37 °C in 24 h. MTT assays demonstrated that DOX·HCl-loaded pepsomes exhibited high antitumor activity, similar to free DOX·HCl in RAW 264.7 cells. Moreover, they were also potent toward drug-resistant MCF-7 cancer cells (MCF-7/ADR). Confocal microscopy studies showed that DOX·HCl-loaded pepsomes delivered and released drug into the cell nuclei of MCF-7/ADR cells in 4 h, while little DOX·HCl fluorescence was observed in MCF-7/ADR cells treated with free drug under otherwise the same conditions. These chimaeric pepsomes with facile synthesis, efficient drug loading, and pH-triggered drug release behavior are an attractive alternative to liposomes for targeted cancer chemotherapy.

Dual-responsive mPEG-PLGA-PGlu hybrid-core nanoparticles with a high drug loading to reverse the multidrug resistance of breast cancer: an in vitro and in vivo evaluation.

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.

Block copolypeptide nanoparticles for the delivery of ocular therapeutics.

Self-assembling block copolypeptides were prepared by sequential ring-opening polymerization of N-carboxyanhydride (NCA) derivatives of γ-benzyl-L-glutamic acid and ε-carbobenzyloxy-L-lysine, followed by selective deprotection of the benzyl glutamate block. The synthesized polymers had number average molecular weights close to theoretical values, and had low dispersities (DM = 1.15-1.28). Self-assembly of the amphiphilic block copolymers into nanoparticles was achieved using the "solvent-switch" method, whereby the polymer was dissolved in THF and water and the organic solvent removed by rotary evaporation. The type of nanostructures formed varied from spherical micelles to a mixture of spherical and worm-like micelles, depending on copolymer composition. The spherical micelles had an average diameter of 43 nm by dynamic light scattering, while the apparent diameter of the mixed phase system was around 200 nm. Reproducibility of nanoparticle preparation was demonstrated to be excellent; almost identical DLS traces were obtained over three repeats. Following qualitative dye-solubilization experiments, the nanoparticles were loaded with the ocular anti-inflammatory drug dexamethasone. Loading efficiency of the nanoparticles was 90% and the cumulative drug release was 94% over 16 d, with a 20% burst release in the first 24 h.

An amphipathic polypeptide derived from poly-γ-glutamic acid for the stabilization of membrane proteins.

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.

Extended self-assembled long periodicity and Zig-Zag domains from helix-helix diblock copolymer Poly(γ-benzyl-l-glutamate)-block-poly(O-benzyl-l-hydroxyproline).

We describe the synthesis and self-assembly of particularly high periodicity of diblock copolymers composed of poly(benzyl-l-hydroxyproline) (PBLHyP) and poly(γ-benzyl-l-glutamate) (PBLG), that is, two polypeptide blocks with dissimilar helical structures. The robust helicity of the PBLHyP block is driven by steric constraints of the repeat units, while PBLG forms α-helices driven by hydrogen bonding, allowing defects and deformations. Herein, high-molecular-weight diblock copolypeptides of PBLG-b-PBLHyP with three different volume fractions of the PBLHyP-blocks are discussed. For shorter PBLHyP blocks, hexagonal packing of PBLHyP helices is observed, while by increasing the length of the PBLHyP block, keeping at a similar PBLG block length, the packing is distorted. Zig-zag lamellar structures were obtained due to the mismatch in the packing periodicities of the PBLG and PBLHyP helices. The frustration that takes place at the interface leads the PBLHyP to tilt to match the PBLG periodicity. The zig-zag morphology is reported for the first time for high-molecular-weight helix-helix (rod-rod) copolypeptides, and the self-assembled periodicity is uncommonly large.