Yilong Cheng

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.

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.

pH and reduction dual responsive polyurethane triblock copolymers for efficient intracellular drug delivery

A series of pH/reduction dual responsive poly(ethylene glycol)/polyurethane triblock copolymers containing tertiary amines and disulfide bonds are reported. The polyurethane block copolymers self-assembled into stable micelles in aqueous medium at pH 7.4, which responded rapidly to both a narrow pH change within the physiologically relevant pH range and a reduction environment mimicking the intracellular space. The in vitro drug release from doxorubicin (DOX)-loaded polyurethane micelles was significantly accelerated by reducing the pH or by addition of an intracellular reducing agent, glutathione (GSH). Confocal laser scanning microscopy (CLSM) and flow cytometry measurements revealed that the intracellular drug release from the DOX-loaded nanoparticles was increased in the HeLa cells with enhanced intracellular GSH level. In addition, even though the polyurethane block copolymers exhibited good cytocompatibility, the DOX-loaded polyurethane micelles displayed efficient growth inhibition of HeLa and HepG2 cells, which showed a dependence on the intracellular GSH concentration. Owing to their unique responsiveness to dual biological stimuli, the biocompatible and bioreducible polyurethane block copolymers have the potential to serve as a versatile platform for intracellular drug delivery.

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.

Decisive Role of Hydrophobic Side Groups of Polypeptides in Thermosensitive Gelation

Thermosensitive hydrogels based on PEG and poly(l-glutamate)s bearing different hydrophobic side groups were separately synthesized by the ring-opening polymerization (ROP) of l-glutamate N-carboxyanhydrides containing different alkyl protected groups, that is, methyl, ethyl, n-propyl, and n-butyl, using mPEG(45)-NH(2) as macroinitiator. The resulting copolymers underwent sol-gel transitions in response to temperature change. Interestingly, the polypeptides containing methyl and ethyl showed significantly lower critical gelation temperatures (CGTs) than those bearing n-propyl and butyl side groups. Based on the analysis of (13)C NMR spectra, DLS, circular dichroism spectra, and ATR-FTIR spectra, the sol-gel transition mechanism was attributed to the dehydration of poly(ethylene glycol) and the increase of β-sheet conformation content in the polypeptides. The in vivo gelation test indicated that the copolymer solution (6.0 wt %) immediately changed to a gel after subcutaneous injection into rats. The mass loss of the hydrogel in vitro was accelerated in the presence of proteinase K, and the MTT assay revealed that the block copolymers exhibited no detectable cytotoxicity. The present work revealed that subtle variation in the length of a hydrophobic side group displayed the decisive effect on the gelation behavior of the polypeptides. In addition, the thermosensitive hydrogels could be promising materials for biomedical applications due to their good biocompatibility, biodegradability, and the fast in situ gelation behavior.

In Vitro Study of Electroactive Tetraaniline-Containing Thermosensitive Hydrogels for Cardiac Tissue Engineering

Injectable hydrogels made of degradable biomaterials can function as both physical support and cell scaffold in preventing infarct expansion and promoting cardiac repair in myocardial infarction therapy. Here, we report in situ hydrogels consisting of thermosensitive PolyNIPAM-based copolymers and electroactive tetraaniline (TA). Studies showed that the addition of 2-methylene-1,3-dioxepane (MDO) provided the PolyNIPAM-based gel with biodegradability, and the introduction of tetraaniline endowed these copolymers with desirable electrical properties and antioxidant activities. The encapsulated H9c2 cells (rat cardiac myoblast) remained highly viable in the gel matrices. In vivo gel formation and histological analyses were performed in rats by subcutaneous injection and excellent biocompatibility was observed. Furthermore, the proliferation and intracellular calcium transients of H9c2 cells were also studied with (and without) electrical stimuli. Both in vitro and in vivo results demonstrated that electroactive hydrogel may be used as a promising injectable biomaterial for cardiac tissue engineering.

PLK1shRNA and doxorubicin co-loaded thermosensitive PLGA-PEG-PLGA hydrogels for osteosarcoma treatment.

Combination cancer therapy has emerged as crucial approach for achieving superior anti-cancer efficacy. In this study, we developed a strategy by localized co-delivery of PLK1shRNA/polylysine-modified polyethylenimine (PEI-Lys) complexes and doxorubicin (DOX) using biodegradable, thermosensitive PLGA-PEG-PLGA hydrogels for treatment of osteosarcoma. When incubated with osteosarcoma Saos-2 and MG-63 cells, the hydrogel containing PLK1shRNA/PEI-Lys and DOX displayed significant synergistic effects in promoting the apoptosis of osteosarcoma cells in vitro. After subcutaneous injection of the hydrogel containing PLK1shRNA/PEI-Lys and DOX beside the tumors of nude mice bearing osteosarcoma Saos-2 xenografts, the hydrogels exhibited superior antitumor efficacy in vivo compared to the hydrogels loaded with PLK1shRNA/PEI-Lys or DOX alone. It is noteworthy that the combination treatment in vivo led to almost complete suppression of tumor growth up to 16 days, significantly enhanced PLK1 silencing, higher apoptosis of tumor masses, as well as increased cell cycle regulation. Additionally, ex vivo histological analysis of major organs of the mice indicated that the localized treatments showed no obvious damage to the organs, suggesting lower systemic toxicity of the treatments. Therefore, the strategy of localized, sustained co-delivery of PLK1shRNA and DOX by using the biodegradable, injectable hydrogel may have potential for efficient clinical treatment of osteosarcoma.

Versatile Biofunctionalization of Polypeptide-Based Thermosensitive Hydrogels via Click Chemistry

In this study, we report thermosensitive hydrogels based on poly(ethylene glycol)-block-poly(γ-propargyl-l-glutamate) (PEG-PPLG). (13)C NMR spectra, DLS, and circular dichroism spectra were employed to study the mechanism of the sol-gel phase transition. Mouse fibroblast L929 cells were encapsulated and cultured within the hydrogel matrices, and the encapsulated cells were shown to be highly viable in the gel matrices, suggesting that the hydrogels have excellent cytocompatibilities. The mass loss of the hydrogels in vitro was accelerated by the presence of proteinase K compared to the control group. 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. The presence of alkynyl side groups in the PEG-PPLG copolymers allowed convenient further functionalization with azide-modified bioactive molecules, such as biotin and galactose. The biofunctionalized PEG-polypeptide block copolymers showed sol-gel phase transitions similar to the parent copolymers. Interestingly, the incorporation of galactose groups into the hydrogels was found to improve cell adhesion, likely due to the adsorption of fibronectin (FN) in cell-extracellular matrix (ECM). Because bioactive materials have shown unique advantages in biomedical applications, especially tissue engineering and regenerative medicine applications, we believe our novel functionalizable thermosensitive hydrogels have potential to serve as a versatile platform for the development of new biofunctional materials, for example, bioadhesive and bioresponsive hydrogels.

Reduction-responsive cross-linked micelles based on PEGylated polypeptides prepared via click chemistry

This study aims to develop novel reduction-responsive cross-linked micelles (CMs) based on poly(ethylene glycol)-block-poly(γ-propargyl-L-glutamate) (PEG-PPLG) by click chemistry. 1H NMR spectroscopy, IR spectroscopy, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were performed to confirm the successful construction of the CMs. Doxorubicin (DOX) was loaded into the CMs as a model anticancer drug. The DOX-loaded CMs could hold the drug under physiological conditions, and release the payload quickly in the presence of glutathione (GSH). Confocal laser scanning microscopy (CLSM) and flow cytometry measurements revealed that the intracellular drug release from the DOX-loaded CMs was increased in the HeLa cells with an enhanced intracellular GSH level. In vitro methyl thiazolyl tetrazolium (MTT) assays indicated that the CMs were biocompatible, and DOX-loaded CMs showed higher cellular proliferation inhibition towards GSH-pretreated HeLa cells than non-pretreated cells. Due to their unique responsiveness, the biocompatible CMs show promise for the intracellular delivery of chemotherapeutic drugs in cancer therapy.

Localized Co-delivery of Doxorubicin, Cisplatin, and Methotrexate by Thermosensitive Hydrogels for Enhanced Osteosarcoma Treatment

Localized cancer treatments with combination drugs have recently emerged as crucial approaches for effective inhibition of tumor growth and reoccurrence. In this study, we present a new strategy for the osteosarcoma treatment by localized co-delivery of multiple drugs, including doxorubicin (DOX), cisplatin (CDDP) and methotraxate (MTX), using thermosensitive PLGA-PEG-PLGA hydrogels. The release profiles of the drugs from the hydrogels were investigated in vitro. It was found that the multidrug coloaded hydrogels exhibited synergistic effects on cytotoxicity against osteosarcoma Saos-2 and MG-63 cells in vitro. After a single peritumoral injection of the drug-loaded hydrogels into nude mice bearing human osteosarcoma Saos-2 xenografts, the hydrogels coloaded with DOX, CDDP, and MTX displayed the highest tumor suppression efficacy in vivo for up to 16 days, as well as led to enhanced tumor apoptosis and increased regulation of the expressions of apoptosis-related genes. Moreover, the monitoring on the mice body change and the ex vivo histological analysis of the key organs indicated that the localized treatments caused less systemic toxicity and no obvious damage to the normal organs. Therefore, the approach of localized co-delivery of DOX, CDDP, and MTX by the thermosensitive hydrogels may be a promising approach for enhanced osteosarcoma treatment.