| The ultimate goal of cancer immunotherapy is to boost antigen-specific immune responses and maintain a long-term immunologic effect to protect against rapid tumor progression. The most widely used Dendritic cells (DC)-based vaccine is still at high cost, extremely labor-intensive, and less reproducible. Moreover, transplanted DC which could home to the LN was only0.5-2.0%and thus not enough to induce efficient T cell responses. To avoid the tedious ex vivo DC-based procedure and present sufficient amount of antigen to DC for efficient induction of antitumor cytotoxicity T lymphocyte (CTL) responses, nanotechnology is applied for sustained and targeted antigen delivery to DC. In this study, we proposed a novel antigenic peptide delivery system with erythrocyte membrane-enveloped Poly (D,L-lactide-co-glycolide)(PLGA) NPs. The conjugation of cysteine-modified peptide with polymer via disulfide bond may be an alternative solution with enhanced entrapment efficiency and rapid intracellular release. We took advantage of red blood cells to increase uptake of nano-vaccine and promote the immune response, such as DC target, enhanced antigen presenting ability and activated T cells. In order to enhance the immune response, we also incorporated mannose into RBC membrane and combined with adjuvant.PLGA-SS-hgp was synthesized via a three-step reaction. The conjugation of hgp to PLGA was confirmed by1H-NMR. The response of PLGA-SS-hgp in reductive environment was evaluated under the condition of DL-Dithiothreitol (DTT) and the hgp content was calculated by HPLC measurement. RBC-membrane enveloped PLGA NP (RBC-NP) was fabricated by serial extrusion. DSPE-PEG-Mannose (DSPE-PEG-Man) was thus incorporated into RBC membrane to generate DSPE-PEG-Man-inserted-RBC (Man-RBC). In order to visualize the structure of RBC-NP, the particles were observed through transmission electron microscopy (TEM). We evaluated the stability of PLGA-NP and RBC-NP by suspending them in two commonly used biological media, pH7.4phosphate buffer solution (PBS) and pure fetal bovine serum (FBS). We also evaluated the retained membrane proteins of RBC in RBC-NP after extrusion through SDS-PAGE and examined the presence of specific antigens on the RBC-NP, CD58and CD59by Western blotting analysis. The cell uptake capability of RBC-NP and Man-RBC-NP was investigated by confocal laser scanning microscope and flow cytometry against DC2.4cells. To further uncover the possibility of Man-RBC-NP taking up by antigen presenting cells (APCs), we injected lipid dye DiD-loaded NP in the inguinal region of C57/BL6mice to analyze the cellular populations in draining LN after48h of intradermal injection. To explore the antigen depot effect and precise localization of Man-RBC-NP, mice were imaged through ex vivo fluorescence imaging with the IVIS Lumina XR system. We focused on the expression of co-stimulatory molecule, CD86and secretion of cytokines by ELISA kits for secretion of tumor necrosis factor-a (TNF-a), interleukin-12(p70)(IL-12p70) and interferon-γ (IFN-γ) to monitor DC maturation. Then, we used three different animal models (prophylactic, therapeutic and metastatic melanoma models) to study the antitumor effect of this nano-vaccine for murine melanoma cell line B16F10and investigate the mechanism. Finally, we combined this nano-vaccine with the chemotherapy drug to evaluate the antitumor effect for murine melanoma cell line B16F10. These results demonstrated the successful synthesis of PLGA-SS-hgp and reductive-cleavage behavior of integrity peptide under in vitro reductive environment. A spherical core-shell structure was exhibited as expected in RBC-NP. RBC-NP was stable in PBS and serum. The protein profiles of Man-RBC-NP and RBC-NP were as similar as natural RBC membranes. CD58and CD59molecules were both existed no matter pre-/post-membrane extrusion. The cell uptake assay suggested that Man-RBC-NP and RBC-NP have been effectively taken up by DC2.4cells. Moreover, the negligible cytotoxicity of Man-RBC-NP toward DC2.4cells implied the high biocompatibility of our antigen delivery system. The in vivo experiment showed that Man-RBC-NP and RBC-NP can be efficiently taken up by APCs, including DCs and macrophages. There was no fluorescent signal at the injection sites after24h injection with PLGA NP. The fluorescent signal of Man-RBC-NP was much higher than that of RBC-NP, especially at24h after injection. The RBC-NP and Man-RBC-NP accumulated mostly in the draining LN. The nano-vaccine caused an up-regulation of CD86expression and increased cytokines secretion. We found that the nano-vaccine prolonged tumor-occurring time, inhibited tumor growth and suppressed tumor metastasis in prophylactic, therapeutic and metastatic melanoma models, respectively. Additionally, we revealed that nano-vaccine effectively decreased the expression of CD31and MMP2, increased the contents of IgG in the serum and the percentage of CTL, enhanced IFN-y secretion and lymphocytotoxicity effect. The chemo-immunotherapy significantly inhibited tumor growth, increased the percentage of CTL and DC, decreased the percentage of CD4+CD25+Foxp3+T regulatory cells and enhanced IFN-y secretion.To sum up all the results, showed that we successfully synthesized reductive-cleavage PLGA-SS-hgp under reductive environment, constructed a novel nano-vaccine formulation composed of three key elements, antigenic peptide (hgp10025-33), adjuvant MPLA and delivery system of Man-RBC coated PLGA NP. The nano-vaccine had low cytotoxicity and was uptaken easily by APCs. It had a good depot effect, accumulated mostly in the draining LN, induced DC maturation and increased the secretion of cytokines. The prophylactic, therapeutic and metastatic melanoma assays proved that the nano-vaccine have the potential to be a safe and effective vaccine formulation in future.This study has successfully developed a novel nano-vaccine based on erythrocyte membrane-enveloped polymeric nanoparticles, and it provides theoretical basis for the exploring of new therapy for melanoma. |
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