Tumor Microenvironment Responsive Nanogel Coated With Erythrocyte Membrane For Combinatorial Antitumor Effect

In recent years,the strategy of combining chemotherapy and immunotherapy has shown synergistically enhanced antitumor effect,which has drawn great attention in the process of study on combination therapy.However,considering the quite different physicochemical properties and mechanisms of chemotherapy and immunotherapy agents,a reasonable delivery system is very important to control the dosage and release of both drugs.In this study,we constructed a tumor microenvironment responsive nanogel with membrane coated.The core was consisted of two opposite charged chitosan derivatives and hydroxypropyl beta cyclodextrin(HP-?-CD)derivative under crosslinking for PTX encapsulation.The coating red blood cell membrane(RBCm)was used for the adsorption of cytokine interleukin-2(IL-2).Methacryloyl CEC(CECm)and methacryloyl HTCC(HTCCm)were synthesized and then characterized by 'H NMR and FT-IR.HP-?-CD acrylate(HP-?-CD-A)was further synthesized and evidenced by 1H NMR.Stoichiometric ratio of CECm,HTCCm and HP-?-CD-A was explored by turbidity titration method.PTX loaded nanogel(NGp)was prepared and extruded with RBCm to prepare PTX loaded nanogel with RBCm envelope(NRp).NRP+I was prepared by adsorbing IL-2 in NRp.The RBCm coating structure of NRP+I was confirmed by transmission electron microscopy(TEM)and DLS.EE of PTX and IL-2 in NRP+I was measured by HPLC and ELISA,respectively.The pH responsive capability was then analyzed by DLS and TEM.Moreover,the stability and toxicity in vitro were evaluated.Subsequently,pH responsive drug release behaviors of NR in vitro and tumor microenvironment were evaluated.Meanwhile,the bioactivity of released IL-2 was validated.Pharmacokinetics and biodistribution studies were then exhibited.The optimal dosage of PTX in chemo-immunotherapy was explored on murine melanoma model.The therapeutic experiment was then conducted to compare the antitumor efficiency of mono-therapy and combination therapy of free drugs.,Expressions of CRT and CD8 were measured by immunofluorescence assay.Moreover,toxicity of NR mediated chemo-immunotherapy was evaluated in H&E section of main organs and blood biochemical examination.By immunohistochemically staining,immune cells(CD45),CD8+ cells and FOXP3' cells were further observed in treated tumors.The filtration of immune cells in tumor was further analyzed by flow cytometry and cytokines in tumor were also measured by ELISA.In order to explore the effect of combination of PTT with chemo-immunotherapy,DiR was adsorbed on membrane of NRP+I(NRP+I+D).The adsorption of DiR was characterized by TEM and UV spectrum.Then,the heating efficiency in vitro and in vivo were evaluated.The antitumor efficiency of NR mediated multi-therapies was studied by tumor inhibition experiment.Immune cells in proximal and distant lymph nodes were analyzed by flow cytometer.Besides,chemo-immunotherapy could also be combined with NO donor.Its antitumor effects and activation of immune cells were further studied.As results indicated,we have successfully synthesized CECm,HTCCm and HP-?-CD-A and the mass ratio of 5:1:1 was selected to prepare nanogel.The RBCm coating structure of NRP+I was confirmed by TEM and Triton X-100 treatment.EE of PTX and IL-2 in NRP+I was measured as 73.5±2.2%,and as 89.3 ± 5.4%,respectively.Through DLS and TEM analysis,NR showed a good pH responsive capability to weak acid tumor microenvironment.Moreover,NR exhibited good stability and low toxicity in vitro.NRP+I exhibited slow PTX and IL-2 release rate in physiological condition(pH 7.4)and faster release behavior in pH 6.5 buffer.Furthermore,NR presented an improved diug release and penetration in tumor microenvironment.Meanwhile,the bioactivity of released IL-2 was proved with unaffected bioactivity.In pharmacokinetics study,circulation time and half-life of PTX and IL-2 in NR were significantly extended.Moreover,NR exhibited an effective drug delivery to tumor in biodistribution experiment.In addition,the optimal dosage of PTX in chemo-immunotherapy was explored on murine melanoma model and selected as 4mg/kg with 2.5?g IL-2/kg for following investigation.In comparison with NGp+i and different treatments,NRP+I exhibited significantly enhanced antitumor effects.In immunofluorescence assay,expressions of CRT and CD8 were both enhanced by NRP+I in the boundary and inner regions of tumor.Moreover,in H&E section of main organs and blood biochemical examination,no significant toxicity was observed in NR mediated chemo-immunotherapy.By immunohistochemically staining,improved filtration of immune cells(CD45)and CD8+ cells and the reduction of FOXP3+ cells were further observed in NRP+I treated tumor.In flow cytometry analysis,maturation of dendritic cells(DCs)in draining and inguinal lymph node were promoted in NRP+I treated tumor.Moreover,the infiltration and activation of DCs,T helper cells,cytotoxic T lymphocytes(CTLs)and natural killer(NK)cells were significantly enhanced in NRP+I treated tumor meanwhile the ratio of activated CTLs/Tregs was increased.Immunostimulatory cytokines(IFN-y and IL-12)in tumor were increased in NR mediated chemo-immunotherapy.In contrast,the secretion of immunosuppressive cytokines(TGF-(3 and IL-10)in NRP+I was reduced compared with other groups.These results demonstrated that NR mediated chemo-immunotherapy could activate antitumor immunity and modulate the immunosuppressive tumor microenvironment.In the combination therapy of PTT with chemo-immunotherapy,as characterized by TEM and UV spectrum,DiR could adsorb on membrane without influencing construction of NR.By irradiation of 808 nm laser at 2 W/cm2,NRP+I+D+NIR exhibited obviously enhanced heating efficiency in vitro and in vivo.Results of tumor inhibition experiment indicated that NRp-I-D+NIR could significantly reduce tumor volume,extend mice survival time and delay tumor recurrence.Moreover,the maturation and number of DCs in proximal and distant lymph nodes were improved,and numbers of T helper cells and CTLs were also increased by NRP+I+D+NIR.The effect of PTT on chemo-immunotherapy was further evaluated in dual tumor model,which indicated an enhanced antitumor effect for distant tumor.Taken together,when combining PTT with chemo-immunotherapy,the antitumor effects could be remarkably improved with increased immune effector cells in proximal and distant lymph nodes.Besides,in the combination therapy of NO donor,antitumor efficiency of chemo-immunotherapy could also be improved with increasing mature DC,CD3+CD4 + CD3+CD8+ and NK cells in tumor.In summary,taking use of HP-?-CD and two opposite charged chitosan derivatives,a novel nanogel was formulated with pH responsive property in tumor microenvironment and RBCm envelope.This nanoplatform can achieve high loading capability of chemotherapeutic drug PTX and immune agent IL-2 with unaffected bioactivity,extend blood circulation time and enhance drug penetration in tumor.The nanogel mediated chemo-immunotherapy exhibited significantly synergistic antitumor effects with the promoted antitumor immunity and regulated immunosuppressive microenvironment.Further combination with PTT could obviously enhance antitumor effects and delay tumor recurrence.By uniting NO donor with chemo-immunotherapy,the toxicity of PTX to immune cells may be reduced and the percent of immune effector cells in tumor may be increased.Thus,antitumor effects could be further enhanced.This study demonstrates a combinatorial strategy through nano-delivery system to realize the synergistic antitumor effect and may provide inspiration for new cancer treatment in the clinic.