Chunsheng Xiao

One-step preparation of reduction-responsive poly(ethylene glycol)-poly (amino acid)s nanogels as efficient intracellular drug delivery platforms†

A series of disulfide-core-cross-linked poly(ethylene glycol)-poly(amino acid)s star copolymers were synthesized through one-step ring-opening polymerization of L-phenylalanine N-carboxyanhydride (L-Phe NCA) and L-cystine N-carboxyanhydride (L-Cys NCA) with amino group terminated poly(ethylene glycol) monomethyl ether (mPEG-NH2) as macroinitiator. The reduction-responsive PEG-poly(amino acid)s nanogels (NGs) were prepared by directly dispersing the resultant PEG-poly(amino acid)s in phosphate buffer solution at pH 7.4. Dynamic light scattering (DLS) measurements showed that the reducible NG swelled in response to 10 mM glutathione (GSH). Doxorubicin (DOX), an anthracycline anticancer drug, was loaded into the NGs. The in vitro release results revealed that the release behaviors could be adjusted by GSH concentration, and poly(amino acid)s content and composition. The intracellular DOX release results showed that enhanced intracellular DOX release occurred in GSH pretreated Henrietta Lacks (HeLa) cells. In vitro methyl thiazolyl tetrazolium (MTT) assays indicated that the NGs were biocompatible, and DOX-loaded NG showed higher cellular proliferation inhibition towards GSH pretreated HeLa cells than that of non-pretreated cells. Therefore, the NGs can efficiently deliver anticancer drugs into tumor cells and inhibit cell proliferation, rendering highly promising for targeted intracellular delivery of operative chemotherapeutic drugs in cancer therapy.

Biocompatible reduction-responsive polypeptide micelles as nanocarriers for enhanced chemotherapy efficacy in vitro

To develop biocompatible reduction-responsive micellar systems for efficient intracellular drug delivery, disulfide-linked block copolymers of methoxyl poly(ethylene glycol) (mPEG) and poly(ε-benzyloxycarbonyl-l-lysine) (PZLL) were synthesized through ring-opening polymerization of ε-benzyloxycarbonyl-l-lysine N-carboxyanhydride with amino group terminated disulfide functionalized mPEG as macroinitiator. The copolymers self-assembled into micelles in phosphate buffered saline (PBS) at pH 7.4 through direct dissolution and dialysis methods. The micelles were revealed to have excellent hemocompatibilities, and cell and tissue compatibilities, which rendered them with potential for drug delivery applications. Doxorubicin (DOX), an anthracycline antitumor drug, was loaded into the micelles through nanoprecipitation with a drug loading efficiency of about 30 wt%. The in vitro DOX release from all DOX-loaded micelles was accelerated in PBS with 10.0 mM GSH, mimicking intracellular reductive conditions. DOX-loaded micelles showed higher cellular proliferation inhibition towards glutathione monoester pretreated HeLa (a human cervical cell line) and HepG2 cells (a human hepatoma cell line) as compared to unpretreated or buthionine sulfoximine pretreated cells. The above results indicated that the biocompatible reduction-responsive micelles have vast potential in targeted intracellular delivery of antitumor drugs to achieve enhanced efficacy in malignancy therapy.

Intracellular microenvironment responsive PEGylated polypeptide nanogels with ionizable cores for efficient doxorubicin loading and triggered release

Two kinds of reduction and pH responsive disulfide-cross-linked poly(ethylene glycol)-polypeptide copolymers were prepared through one-step ring-opening polymerization of γ-benzyl-L-glutamate N-carboxyanhydride (BLG NCA) or e-benzyloxycarbonyl-L-lysine N-carboxyanhydride (ZLL NCA) and L-cystine N-carboxyanhydride (LC NCA) with amino group terminated monomethoxy poly(ethylene glycol) (mPEG-NH2) as macroinitiator. Then, the copolymers were deprotected and dispersed in phosphate buffered saline, yielding PEG-polypeptide nanogels. Doxorubicin (DOX), a model anticancer drug, was effectively loaded into nanogels via electrostatic and hydrophobic interactions. The DOX release from all DOX-loaded nanogels was accelerated in intracellular reductive and acidic conditions, which controlled by Fickian diffusion and nanogels swelling. The enhanced intracellular DOX release was observed in glutathione monoester (GSH-OEt) pretreated HeLa cells. DOX-loaded nanogels showed higher cellular proliferation inhibition towards GSH-OEt pretreated HeLa and HepG2 cells than to unpretreated or buthionine sulfoximine (BSO) pretreated cells. Hemolysis tests indicated that nanogels were hemocompatible, and the presence of nanogels could reduce the hemolysis ratio (HR) of DOX significantly. These features suggest that the nanogels can efficiently load and deliver DOX into tumor cells and enhance the inhibition of cellular proliferation in vitro, providing a favorable platform to construct an efficient drug delivery system for cancer therapy.

Preparation of photo-cross-linked pH-responsive polypeptide nanogels as potential carriers for controlled drug delivery

Diblock and triblock copolymers, including poly(ethylene glycol monomethyl ether)-b-poly(L-glutamic acid-co-γ-cinnamyl-L-glutamate) (mPEG-b-P(LGA/CLG)) and poly(L-glutamic acid-co-γ-cinnamyl-L-glutamate)-b-poly(ethylene glycol)-b-poly(L-glutamic acid-co-γ-cinnamyl-L-glutamate) (P(LGA/CLG)-b-PEG-b-P(LGA/CLG)), were synthesized by ring-opening polymerization (ROP) of γ-benzyl-L-glutamate-N-carboxyanhydride (BLG-NCA) monomer with PEG-based macroinitiator, deprotection of the benzyl groups and subsequent chemical modification with cinnamyl alcohol. The structures of copolymers were confirmed by 1H NMR and GPC analyses. Pyrene-probe-based fluorescence technique revealed that these diblock and triblock copolymers could self-assemble into micelles in aqueous solution at pH 7.4 spontaneously, with PEG shells and P(LGA/CLG) cores. Under UV-irradiation at λ = 254 nm, the P(LGA/CLG) blocks in the cores of the micelles were cross-linked through the photodimerization of the cinnamyloxy groups, yielding nanogels. The nanogels were characterized by 1H NMR, FT-IR, SEM, AFM and DLS. The nanogels were pH-responsive and their properties could be tuned by varying the compositions of block copolymers. In vitro MTT assay demonstrated that the nanogels were biocompatible to HeLa cells, rendering their potential for drug delivery applications. Rifampin as a model drug was loaded into the nanogels. The in vitro rifampin release behaviors of nanogels could be affected by both the compositions of block copolymers and solution pH. These properties indicated that the pH-responsive nanogels fabricated by photo-cross-linking polypeptide micelles can be used as drug carriers for intelligent drug delivery.

Injectable glycopolypeptide hydrogels as biomimetic scaffolds for cartilage tissue engineering

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.

Versatile synthesis of temperature-sensitive polypeptides by click grafting of oligo(ethylene glycol)

A series of novel temperature-sensitive polypeptides were synthesized by ring opening polymerization (ROP) of γ-propargyl-L-glutamateN-carboxyanhydride (PLG-NCA) and subsequent click reaction between the pendant alkyne groups and 1-(2-methoxyethoxy)-2-azidoethane (MEO2-N3) or 1-(2-(2-methoxyethoxy)ethoxy)-2-azidoethane (MEO3-N3). The efficient click grafting and structure of the resultant copolymers were verified by 1H NMR, 13C NMR and GPC. All the copolymers hold α-helix conformation, and could self-assemble into amphiphilic nanoparticles in aqueous solution with hydrodynamic radii (Rh) of 32.3–62.8 nm. The graft copolymers exhibited sharp temperature-dependent phase transitions, and the LCST could be adjusted from 22.3 to 74.1 °C by varying the molecular weight, the length of the OEG side chain, the polymer concentration and salt concentration. MTT assays revealed that the graft copolymers exhibited no detectable cytotoxicity at all test concentrations up to 1 mg mL−1. In vitrodegradation tests demonstrated that the graft copolymers could be degraded by proteinase K. The drug release behaviors from the PPLG112-g-MEO2 nanoparticles were evaluated at 37 °C and 15 °C using doxorubicin (DOX) as a model drug. The drug release behavior displayed thermosensitivity, and a sustained release profile was observed at physiological temperature. These results suggested that the novel biodegradable and biocompatible polypeptide derivatives with adjustable temperature sensitivity could be a promising material for biomedical applications.

Versatile preparation of intracellular-acidity-sensitive oxime-linked polysaccharide-doxorubicin conjugate for malignancy therapeutic.

Recently, chemotherapy has been one of the most important therapeutic approaches for malignant tumors. The tumor tissular or intracellular microenvironment-sensitive polymer-doxorubicin (DOX) conjugates demonstrate great potential for improved antitumor efficacy and reduced side effects. In this work, the acid-sensitive dextran-DOX conjugate (noted as Dex-O-DOX) was synthesized through the versatile efficient oximation reaction between the terminal aldehyde group of polysaccharide and the amino group in DOX in the buffer solution of sodium acetate/acetic acid. The insensitive one, i.e., Dex-b-DOX, was prepared similarly as Dex-O-DOX with a supplemented reduction reaction. The DOX release from Dex-O-DOX was pH-dependent and accelerated by the decreased pH. The efficient intracellular DOX release from Dex-O-DOX toward the human hepatoma HepG2 cells was further confirmed. Furthermore, Dex-O-DOX exhibited a closer antiproliferative activity to free DOX·HCl as the extension of time. More importantly, compared with Dex-b-DOX, Dex-O-DOX exhibited higher antitumor activity and lower toxicity, which were further confirmed by the systemic histological and immunohistochemical analyses. Hence, the facilely prepared smart polysaccharide-DOX conjugates, i.e., Dex-O-DOX, exhibited great potential in the clinical chemotherapy of malignancy.

Thermosensitive hydrogels based on polypeptides for localized and sustained delivery of anticancer drugs

Thermosensitive hydrogels based on poly(γ-ethyl-L-glutamate)-poly(ethylene glycol)-poly(γ-ethyl-L-glutamate) triblock copolymers (PELG-PEG-PELG) were prepared for localized and sustained delivery of anticancer drugs. The polypeptide-based hydrogels showed much lower critical gelation concentration than the traditional polyester-based hydrogels. In vivo biocompatibility studies revealed that the in situ formed gels in the subcutaneous layer last for ≈ 21 days, and H&E staining study suggested acceptable biocompatibility of our materials in vivo. Then the hydrogels were tried as injectable implants to encapsulate antitumor drug, paclitaxel (PTX), to assess the in situ anti-tumoral activity using liver cancer xenograft model. The results demonstrated that the PTX-incorporated hydrogels could efficiently suppress the tumor growth, and did not result in obvious damage to normal organs. Therefore, the polypeptide-based thermosensitive hydrogels designed in the present study have great potential to serve as an effective platform for localized anti-cancer drug delivery.

Highly efficient "grafting from" an α-helical polypeptide backbone by atom transfer radical polymerization.

Because of their favorable biodegradability, biocompatibility, regular secondary structures, and stimuli-responsiveness, synthetic polypeptides have attracted more and more attention in the biomedical material field. In this work, a novel thermo-responsive graft polypeptide, poly(L-glutamate)-graft-poly(2-(2-methoxyethoxy)ethyl methacrylate) (PLG-g-PMEO₂ MA), is prepared through a combination of ring-opening polymerization and atom transfer radical polymerization. The structure of PLG-g-PMEO₂ MA is confirmed by FT-IR, ¹H NMR, and GPC analyses. The phase transition temperature of PLG-g-PMEO₂ MA is adjustable by varying the NaCl concentration in aqueous solution. PLG-g-PMEO₂ MA adopts α-helical conformations both in aqueous solution at 25 and 60 °C and even in the solid state. In addition, PLG-g-PMEO₂ MA forms stimuli-responsive micelles with an α-helical core and a thermo-responsive shell in water.

Disulfide crosslinked PEGylated starch micelles as efficient intracellular drug delivery platforms

Novel reduction-responsive disulfide core-crosslinked micelles based on amphiphilic starch-graft-poly(ethylene glycol) (starch-g-PEG) were prepared and used for efficient intracellular drug delivery. The starch-g-PEG copolymers can be conveniently prepared by grafting starch with carboxyl group terminated PEG, and subsequently conjugated with lipoic acid for disulfide crosslinking. The self-assembled starch-g-PEG micelles and the corresponding disulfide core-crosslinked micelles were then characterized by transmission electron microscopy, dynamic laser scattering and fluorescence techniques. It is interesting to observe that the hydrodynamic radii of disulfide core-crosslinked micelles would increase gradually in phosphate buffered saline (PBS) due to the cleavage of the disulfide bond in the micellar core, caused by the presence of reductive glutathione (GSH). The glutathione-responsive behaviors of the disulfide core-crosslinked micelles should be attractive for intracellular drug delivery. Thus, a model anticancer drug doxorubicin (DOX) was loaded into micelles and the in vitro drug release in response to GSH was also studied. The results showed that only a small amount of loaded DOX was released from the core-crosslinked starch-g-PEG micelles in PBS solution without GSH, while quick release occurred in the presence of 10.0 mM GSH. Confocal laser scanning microscopy and flow cytometry analyses further demonstrate that the disulfide crosslinked micelles exhibited a faster drug release behavior in glutathione monoester (GSH-OEt) pretreated HeLa cells than that in the nonpretreated and buthionine sulfoximine (BSO) pretreated cells. In addition, the DOX-loaded crosslinked micelles show higher cellular proliferation inhibition against GSH-OEt pretreated HeLa and HepG2 than against the nonpretreated and BSO pretreated ones. These results suggest that such disulfide crosslinked starch-g-PEG micelles, which can efficiently release the loading drug in response to intracellular GSH concentration, may provide favorable platforms for cancer therapy.