Pingsheng Huang

cRGD-Modified Benzimidazole-based pH-Responsive Nanoparticles for Enhanced Tumor Targeted Doxorubicin Delivery

Finding a smart cancer drug delivery carrier with long blood circulation, enhanced cancer targeting, and quick drug release in tumors is critical for efficient cancer chemotherapy. Herein, we design a cRGD-polycarboxybetaine methacrylate-b-polybenzimidazole methacrylate (cRGD-PCB-b-PBBMZ) copolymer to self-assemble into smart drug-loaded nanoparticles (cRGD-PCM NPs) which can target αvβ3 integrin overexpressed cancer tissue by cRGD peptide unit and release drug quickly in cancer cells by protonation of benzimidazole groups. The outer PCB layer can resist protein adhesion, and there are only about 10% of proteins in mouse serum adhered to the surface of PCM NPs. With the pKa value of 5.08 of the benzimidazole units, DOX can be released from NPs in pH 5.0 PBS. cRGD-PCM NPs can bring more DOX into HepG2 cells than nontargeting PCM NPs, and there has high DOX release rate in HepG2 cells because of the protonation of benzimidazole groups in endosome and lysosome. MTT assay verifies that higher cellular uptake of DOX causes higher cytotoxicity. Furthermore, the results of ex vivo imaging studies confirm that cRGD-PCM/DOX NPs can successfully deliver DOX into tumor tissue from the injection site. Therefore, the multifunctional cRGD-PCM NPs show great potential as novel nanocarriers for targeting cancer chemotherapy.

Self-assembled PEG-b-PDPA-b-PGEM copolymer nanoparticles as protein antigen delivery vehicles to dendritic cells: preparation, characterization and cellular uptake

Antigen uptake by dendritic cells (DCs) is a key step for initiating antigen-specific T cell immunity. In the present study, novel synthetic polymeric nanoparticles were prepared as antigen delivery vehicles to improve the antigen uptake by DCs. Well-defined cationic and acid-responsive copolymers, monomethoxy poly(ethylene glycol)-block-poly(2-(diisopropyl amino) ethyl methacrylate)-block-poly(2-(guanidyl) ethyl methacrylate) (mPEG-b-PDPA-b-PGEM, PEDG) were synthesized by reversible addition-fragmentation chain transfer polymerization of 2-(diisopropylamino)ethyl methacrylate) and N-(tert-butoxycarbonyl) amino ethyl methacrylate monomers, followed by deprotection of tert-butyl protective groups and guanidinylation of obtained primary amines. 1H NMR, 13C NMR and GPC results indicated the successful synthesis of well-defined PEDG copolymers. PEDG copolymers could self-assemble into nanoparticles in aqueous solution, which were of cationic surface charges and showed acid-triggered disassembly contributed by PGEM and PDPA moieties, respectively. Significantly, PEDG nanoparticles could effectively condense with negatively charged model antigen ovalbumin (OVA) to form OVA/PEDG nanoparticle formulations with no influence on its secondary and tertiary structures demonstrating by far-UV circular dichroism and UV–vis spectra. In vitro antigen cellular uptake by bone marrow DCs (BMDCs) indicated using PEDG nanoparticles as antigen delivery vehicles could significantly improve the antigen uptake efficiency of OVA compared with free OVA or the commercialized Alum adjuvant. Moreover, as the surface cationic charges of OVA/PEDG nanoparticle formulations reduced, the uptake efficiency decreased correspondingly. Collectively, our work suggests that guanidinylated, cationic and acid-responsive PEDG nanoparticles represent a new kind of promising antigen delivery vehicle to DCs and hold great potential to serve as immunoadjuvants in the development of vaccines.

Targeted antigen delivery to dendritic cell via functionalized alginate nanoparticles for cancer immunotherapy

Abstract The purpose of the present study was to identify an “easy‐to‐adopt” strategy to enhance immune responses using functionalized alginate (ALG) nanoparticles (MAN‐ALG/ALG=OVA NPs), which were prepared by CaCl2 cross‐linking of two different types of ALG. The mannose (MAN) modified ALG (MAN‐ALG) was used for dendritic cell targeting. The other component, composed of ovalbumin (OVA), a model antigen, is conjugated to ALG (ALG=OVA) via pH sensitive Schiff base bond. Grafting of alginate was demonstrated by FT‐IR and 1H NMR, while the morphological structure, particle size, Zeta potential of MAN‐ALG/ALG=OVA NPs were measured using TEM and DLS. The OVA releasing behavior of MAN‐ALG/ALG=OVA NPs was determined as a function of pH. Antigen uptake was examined by flow cytometry and confocal laser scanning microscopy in vitro using mouse bone marrow dendritic cells (BMDCs). The results showed that MAN‐ALG/ALG=OVA NPs facilitated antigen uptake of BMDCs and cytosolic release of the antigen. Significant up‐regulation of cytokine secretion and expression levels of the surface co‐stimulatory molecules were also observed in MAN‐ALG/ALG=OVA NPs‐treated BMDCs, compared to free OVA. In vivo bio‐distribution study using Cy7 (a near‐infrared fluorescence dye) labeled MAN‐ALG/ALG=OVA NPs showed efficient in vivo trafficking of the nanoparticles from the injection site to the draining lymph nodes. Moreover, MAN‐ALG/ALG=OVA NPs were found to enhance cross‐presentation of OVA to B3Z T cell hybridoma in vitro. Subcutaneous administration of MAN‐ALG/ALG=OVA NPs also induced major cytotoxic T lymphocytes (CTL) response and inhibition of E.G7 tumor growth in C57BL/6 mice. In summary, we report here that the MAN‐ALG/ALG=OVA NPs have the potential as a potent nanovaccine for cancer immunotherapy. Graphical abstract Figure. No Caption available.

Self-assembled PEG–poly(L-valine) hydrogels as promising 3D cell culture scaffolds

Self-assembled polypeptide aggregates have shown great promise in biomedical fields including drug delivery, tissue regeneration and regenerative medicine. In this study, we report self-assembled hydrogels based on mPEG-block-poly(l-valine) (PEV) copolymers. PEV copolymers with varying poly(l-valine) chain lengths were prepared by the ring-opening polymerization of N-carboxy anhydrides of l-valine using mPEG-NH2 as the initiator. 1H NMR and GPC confirmed their well-defined chemical structures. FT-IR analysis and DSC curves indicated the combined α-helix and β-sheet secondary polypeptide conformation and the PEG crystallization microphase in bulk solid state, respectively. Moreover, the poly(l-valine) block restricted the crystallization of PEG segment. DLS, TEM and circular dichroism spectra were employed to study the self-assembly profiles of PEV copolymers in aqueous solution. The results manifested that in diluted solution, PEV copolymers showed a combination of typical β-sheet and α-helical polypeptide structures and self-assembled into nanostructures with diverse morphologies and sizes. For concentrated PEV solutions, clear hydrogel phases were observed and dynamic rheological analyses demonstrated that the hydrogel modulus was sensitive to the polypeptide length, angular frequency, shear strain and temperature. The hydrogel formation was possibly dominated by the physical aggregation of PEV nanoassemblies as well as driven by the formation of particular polypeptide secondary structures. Human fibroblast NIH/3T3 cells were encapsulated and cultured within the hydrogel scaffolds. The encapsulated cells exhibited high viability, suggesting that PEV hydrogels have excellent cytocompatibility and could be used as three-dimensional (3D) cell culture matrices. Collectively, self-assembled PEGylated poly(l-valine) conjugate hydrogels represented a new kind of biomaterial scaffold in biomedical fields including but not limited to 3D cell culture.

Thermosensitive micellar hydrogel for enhanced anticancer therapy through redox modulation mediated combinational effects

Cancer is increasingly viewed as an eco-system, a community in which tumor cells cooperate with other tumor cells and host cells in their microenvironment. The improved understanding of the intricate relationships in this eco-system has led to revolutionary treatments, which have evolved from relatively nonspecific cytotoxic agents to selective, mechanism-based therapeutics. Herein, from the view of dynamic equilibrium, a synergistic intracellular redox-regulation therapeutic strategy was proposed, in which combinational treatment of chemotherapeutic agents and ROS-elimination inhibitors was expected to effectively kill cancer cells and overcome redox adaptation mechanism associated drug resistance. To this end, a thermosensitive micellar hydrogel was prepared for co-delivery of nanomedicines in situ, which was capable of encapsulating and delivering multiple drugs with diverse therapeutic properties while maintaining the controlled synergistic ratio. Firstly, fluorescence resonance energy transfer (FRET) technology was adopted to track the real-time spatial pattern of drug presentation at a molecular level in this micellar hydrogel. Results suggested that the drug encapsulation in this micellar hydrogel platform proved to be a dynamic equilibrium process, during which free drug movement, drug exchange or penetration between micelles could occur. Furthermore, doxorubicin (DOX) and Zn(II) protoporphyrin IX (ZnPP) were used as the model chemotherapeutant and HO-1 inhibitor, respectively. In vitro and in vivo evaluation demonstrated that the intracellular redox-regulation mediated synergistic advantages of both two types of drugs translated into improved therapeutic outcomes. Consequently, such a thermosensitive micellar hydrogel formulation, which enabled precise control over the dosage and ratio of combination therapeutic agents to obtain the desired therapeutic effect with a single drug administration, holds great potential for spatiotemporal delivery of multiple bioactive agents for sustained combination therapy.

Engineering Dendritic-Cell-Based Vaccines and PD-1 Blockade in Self-Assembled Peptide Nanofibrous Hydrogel to Amplify Antitumor T-Cell Immunity

Dendritic cells (DCs) are increasingly used in cancer vaccines due to their ability to regulate T-cell immunity. Major limitations associated with the present DC adoptive transfer immunotherapy are low cell viability and transient duration of transplanted DCs at the vaccination site and the lack of recruitment of host DCs, leading to unsatisfactory T-cell immune response. Here, we developed a novel vaccine nodule comprising a simple physical mixture of the peptide nanofibrous hydrogel, anti-PD-1 antibodies, DCs, and tumor antigens. Upon subcutaneous injection, the vaccine nodule maintained the viability and biological function including the antigen uptake and maturation of encapsulated DCs and simultaneously recruited a number of host DCs and promoted the drainage of activated DCs to lymph nodes, resulting in enhanced proliferation of antigen-specific splenocytes and provoking potent cellular immune responses. Compared with adoptive transfer of DCs and subcutaneous administration of antigen vaccine, such a vaccine nodule shows superior antitumor immunotherapy efficiency in both prophylactic and therapeutic tumor models including delayed tumor growth and prolonged mice survival due to effective stimulation of antitumor T-cell immunity and increased infiltration of activated CD8+ effector T-cells in the tumor. Our findings provide a simple and robust vaccination strategy for DC-based vaccines and also a unique vaccine product for stimulating and enhancing T-cell immunity, holding great promise for immunotherapy against cancer and infectious diseases.

Co-delivery of doxorubicin and pheophorbide A by pluronic F127 micelles for chemo-photodynamic combination therapy of melanoma

Combined chemotherapy and photodynamic therapy (PDT) is a promising strategy to enhance the anticancer efficacy of both drugs via combination effects. In this work, doxorubicin (DOX)-loaded pheophorbide A (PheoA)-modified Pluronic F127 (F127) micelles (DOX/F127-PheoA micelles) were developed for combined chemo-photodynamic therapy of melanoma. DOX/F127-PheoA micelles were characterized in terms of size and size distribution, zeta potential, surface morphology, drug loading efficiency, and drug-releasing properties. It was observed that the DOX/F127-PheoA micelles were spherical, with a mean particle size of 146.5 nm and a zeta potential of -3.2 mV. Confocal laser scanning microscopy showed that DOX/F127-PheoA micelles were internalized by B16 melanoma cells and capable of dual-delivery of both DOX and PheoA into tumor cells. Upon light irradiation, DOX/F127-PheoA micelles could generate reactive oxygen species (ROS) both in vitro and in vivo. The in vitro cytotoxic activity of DOX/F127-PheoA micelles in B16 melanoma cells were evaluated by CCK-8 assay. In vivo antitumor efficacy was also assessed using C57 mice bearing B16 tumors, and the DOX/F127-PheoA micelles were administrated intravenously. Under light irradiation, DOX/F127-PheoA micelles significantly inhibited tumor growth compared with free DOX and DOX/F127-PheoA micelles without light irradiation. The mean tumor growth inhibition rate of DOX/F127-PheoA micelles with light irradiation was 73.5%, compared with 42.3% for DOX/F127-PheoA micelles without light irradiation and 26.5% for free DOX. These results suggest that DOX/F127-PheoA micelles are a versatile and effective drug delivery system for combinational chemo-photodynamic therapy against melanoma.