Investigation On Tumor Microenvironment-responsive Nano Drug Delivery System For Enhancing Immunotherapy

The complex immunosuppressive microenvironment of tumor is an important reason for tumor treatment failure and recurrence and metastasis.Bone marrow derived inhibitory cells(MDSCs),Tumor-associated macrophages(M2-TAMs)and regulatory T cells(Tregs)are the main forces of the tumor immunosuppressi've microenvironment,which can form an immunosuppressive "iron triangle" in the tumor microenvironment(TME)to inhibit the immune response of the body and promote the growth and metastasis of the tumor.In order to enhance the effect of tumor immunotherapy,it is urgent to reconstruct TME while inhibiting the growth of tumor cells,reduce the number of immunosuppressive cells and effectively activate the immune response to kill tumor cells.Based on this,a tumor microenvironmental response nano drug delivery system GW965@PAE-PEG-PEP(GW@PPP)for enhanced immunotherapy was constructed.We used the sensitive material PAE and polyethylene glycol(PEG)were connected to self-assembling nanoparticles to delivery of LXR agonist GW3965 promotes the transcriptional of lipid metabolizing proteins(ApoE)and consumption of MDSCs and Tregs.At the same time,the nanoparticles were loaded with an enzyme-sensitive M2-TAMs targeted apoptotic peptide(PEP)by mercaptans reaction.It is used to kill M2-TAMs to effectively activate the immune response and inhibit tumor growth.The system has the following characteristics:(1)the nanoparticles can target to tumor tissue because of the targeting of apoptotic peptide(PEP)of M2-TAMs;(2)nanoparticles releases GW3965 and PEP in response to matrix metalloproteinases(MMP)and pH of tumor microenvironment;(3)GW3965 can act on a variety cells of tumor microenvironments enhanced the transcriptional consumption of ApoE to kill tumor cells,MDSCs and Tregs and the fractured PEP was targeted at the M2-TAMs macrophage promotes its apoptosis through the mitochondrial pathway.Therefore,GW@PPP can be based on elimination tumor cells and immunosuppressive cells in TME,effectively activate the body's immune response and enhance anti-tumor activity.The specific contents and results of the research are as follows:1.Firstly,nanoparticles with TME dual response were synthesized.Then,the pH-sensitive polymer block PAE-PEG-Mal was synthesized with esterified Mal-PEG 2000,1,6-diol diacrylate(HDD)and 4,4-trimethyldipiperidine(TDP)by 1,4-michael addition reaction.The results of nuclear magnetic resonance imaging(NMR)showed that all the products could be prepared successfully.The basic nanoparticles GW3965@PAE-PEG(GW3965@PP)was prepared by thin film dispersion method and GW3965 was located in the lipid soluble core of the nanoparticles.TAMs-M2 macrophages targeted apoptotic peptide(PEP)was load by mercaptans reaction to gain GW3965@PPP.The results of the nano-particle size analyzer showed that the GW3965@PPP particle size distribution was 88.7±1.26 nm and the nanoparticles changed at pH 6.4,indicating that the nanoparticles had a good pH response in tumor microenvironment.In addition,in vitro drug release experiments also showed that the nanoparticles have good weak acid and enzyme response properties,which can prevent drug leakage before reaching the tumor site.2.In vitro experiments,melanoma B16F10 cells,BMDMs and HUVEC were used as model cells to evaluate the anti-tumor effect of GW3965@PPP in vitro.Cytotoxicity experiments showed that GW3965 and polypeptide PEP effectively inhibited the growth of B16F10 cells and M2 macrophages,respectively.The results of targeted experiments showed that the amount of PEP uptake by M2 macrophages at 1h was 1.6 times that of Ml macrophages,indicating that PEP has a good targeting ability of M2 macrophages.The results of cell apoptosis experiment and mitochondrial membrane potential detection showed that GW3965@PPP could induce the apoptosis of 62.3 ± 2.3%through the mitochondrial pathway.The experimental results of scratch and Transwell verified the anti-tumor metastasis and invasion ability of GW3965@PP in vitro.The results of angiogenesis experiment showed that GW3965@PP could inhibit 50.3±1.8%angiogenesis in vitro.In summary,the prepared GW3965@PPP drug delivery system can effectively inhibit the proliferation,invasion and migration of melanoma cells,and has significant anti-tumor activity in vitro.3.In vivo experiments,C57BL/6 black mice were used as animal models to comprehensively evaluate the pharmacodynamics and pathological characteristics of GW3965@PPP by investigating the distribution of the GW3965@PPP drug delivery system in mice,tumor volume changes,immunofluorescence experiments and flow experiments of tumor cells.In vivo imaging results of mice showed that after 2 hours of administration,the fluorescence signal intensity in the tumor site of the IR780@PPP group was significantly higher than that of theIR780@PP group,indicating that the M2 macrophage targeting peptide had a good tumor targeting effect.In vivo pharmacodynamics,the tumor volume in the GW3965@PPP group decreased by 68.8±1.7%compared with the normal saline group,which significantly inhibited tumor growth.The results of pathological features showed that there was no significant trend of weight loss in all groups,which indicated that the nanometer drug delivery system had good biological safety.Flow experiments on animals showed GW@PPP group compared with saline group,MDSCs decreased 71.3 ± 1.3%,M2-TAMs decreased 42.2 ± 0.8%,Tregs decreased 48.2 ± 2.2%,CD8+T cells increased 72.8 ± 1.4%,which indicating the nano drug delivery system can remove of the tumor microenvironment immunosuppression by killing the tumor microenvironment of immune cells to trigger a strong immune immunosuppression status,inhibit tumor growth.The results of immunofluorescence experiment were consistent with that of flow experiment.In summary,the GW@PPP drug delivery system can deliver drugs to the tumor site and trigger a strong anti-tumor immune response by removing TME immunosuppression,thus inhibiting the growth and metastasis of melanoma.In summary,the pH and enzyme-sensitive nanoparticles GW@PPP prepared in this study can be highly enriched in tumor sites,and enhance the anti-tumor effect by inhibiting tumor cells and the immunosuppressive cells of MDSCs,M2-TAMs and Tregs to promote the infiltration of cytotoxic T cells and the secretion of pro-inflammatory cytokines.This study provide a new way of thinking for tumor immunotherapy and has an important application prospect in tumor immunotherapy.