Chuangnian Zhang

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.

Effect of Resveratrol on Modulation of Endothelial Cells and Macrophages for Rapid Vascular Regeneration from Electrospun Poly(ε-caprolactone) Scaffolds

Rapid endothelialization is a key factor that determines the success of small-diameter vascular grafts as an artery substitute in the treatment of cardiovascular diseases. Aimed to facilitate vascular regeneration, we developed a vascular scaffold loaded with resveratrol, which is a natural compound extracted from plants and showed multifaceted effects in cardiovascular protection. The tubular poly(ε-caprolactone) (PCL) scaffold was prepared by electrospinning with resveratrol in the PCL solution. In vitro assay demonstrated that resveratrol could be released from the scaffolds in a sustained and controlled manner. Cell culture results indicated that the migration of endothelial cells (ECs), nitric oxide production, and the ability of tube formation increased in the resveratrol-containing PCL scaffold groups compared with the PCL control. Meanwhile, the level of tumor necrosis factor (TNF)-α, the main proinflammatory factor secreted from macrophages, was reduced, and the messenger RNA expressions of the M2 macrophage-related genes were increased in the resveratrol-containing group. Further, in vivo implantation was performed by replacing rat abdominal aorta. We observed fast endothelialization and enhanced vascular regeneration in rats with resveratrol-containing scaffolds. The presence of resveratrol also induced a large number of M2 macrophages to infiltrate into the graft wall. Taken together, the incorporation of resveratrol into the PCL grafts enhanced the vascular regeneration by modulation of ECs and macrophages.

Dendritic Cells Pulsed with Exosomes in Combination with PD-1 Antibody Increase the Efficacy of Sorafenib in Hepatocellular Carcinoma Model

Advanced hepatocellular carcinoma (HCC) has limited therapeutic options. Immunotherapy is a promising treatment, while sorafenib is a first-line drug-based treatment for advanced HCC. However, the efficacy of sorafenib and immunotherapy in combination, have not been clearly evaluated. Sorafenib treatment has been shown to promote immunosuppression by increasing hypoxia in orthotopic HCC models. Here, we found that sorafenib treatment in mice with orthotopic HCC increased the expression of inhibitor programmed death-ligand 1 (PD-L1) and T-regulatory cells in tumor tissues. We pulsed dendritic cells with exosomes derived from tumor cells (DC-TEX) and found that the number of T-regulatory cells decreased and the number of CD8+T cells increased. However, combining DC-TEX and sorafenib did not prolong survival in these mice. Moreover, we found that the number of PD-1+CD8+T cells significantly increased after DC-TEX treatment. Therefore, we next added PD-1 antibody (PD-1 Ab) to the treatment regimen to block the PD-1/PD-L1 pathway, and found that the exhausted CD8+T cells were restored, without affecting the number of T-regulatory cells. Thus, our data suggest that the combination of DC-TEX and PD-1 Ab enhanced the efficacy of sorafenib, but treatment with either DC-TEX or PD-1 Ab alone, did not.

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.

The regeneration of macro‐porous electrospun poly(ɛ‐caprolactone) vascular graft during long‐term in situ implantation

Long-term evaluation of vascular grafts is an essential step to facilitate clinical translation. In this study, we investigate the long-term performance of a macro-porous poly(ɛ-caprolactone) (PCL) electrospun vascular graft using the rat abdominal artery replacement model. Long-term patency, endothelialization, and smooth muscle cell regeneration were evaluated, as well as calcification and degradation. The data showed that all the grafts remained open and unobstructed. There was no evidence of aneurysm, stenosis, or calcification one year after implantation. Importantly, neo-vessel was regenerated on the luminal surface of the graft, and was composed of a complete endothelial layer and several layers of smooth muscle cells. The neo-vessel showed vascular physiological function, although not as good as that in native blood vessels, likely due to the remaining scaffold fibers. These data indicated that the PCL macro-porous electrospun vascular graft has potential to be an artery substitute for long-term implantation. Also, this work indicates that continued efforts are needed to develop advanced vascular grafts that exhibit the appropriate balance between the regeneration of the neo-vessel and the complete degradation of the graft materials.