Tumor Microenvironment-responsive Polypeptide Nanomedicine For Prostate Cancer Chemoimmunotherapy

Background:In recent years,the morbidity of prostate cancer in China is increasing rapidly.Since 2008,the incidence of prostate cancer has been the first rank male urinary system tumors,which should arouse more attention from the domestic medical community.At present,the best treatment for prostate cancer is radical surgery or radical radiotherapy and postoperative adjuvant radiotherapy and chemotherapy.If standardized treatment is performed,the prognosis is favorable.However,due to the insidious onset of prostate cancer,and China has not carried out large-scale screening yet,70%of the new cases have developed into locally advanced or metastatic prostate cancer,which is no longer suitable for radical treatment.Androgen deprivation therapy is the main systemic treatment for advanced prostate cancer,but its adverse reactions are serious and have a high probability of occurrence.The adverse reactions will continue to increase with the prolonged treatment time.As the disease progresses,prostate cancer will eventually develop into castration resistant prostate cancer（CRPC）.At this time,the available treatment methods have lost the benefits of survival,and there is an urgent need to develop new treatment methods to improve the quality of life of the group and to extends the survival period.In recent studies,immunotherapy,especially immune checkpoint blockade represented by anti-PD-L1,has shown excellent performance against a variety of hematological tumors and solid tumors.The development of immunotherapy for prostate cancer has also become a research focus.With the advancement of research,the problem that prostate cancer immunotherapy has to face has gradually appeared.Half of prostate cancers are"cold tumors".The lack of sufficient immune cell infiltration in the tumor microenvironment greatly limits the efficacy.How to realize the transformation from"cold tumor"to"hot tumor"has become a breakthrough in prostate cancer immunotherapy.Objective:Based on the unique microenvironment of the tumor,we designed and prepared[methoxy poly（ethylene glycol）-poly（L-glutamic acid-co-L-cystine）],(m PEG-P（LG-co-LC）,nanogel with pH and reductive dual responsiveness,in order to improve the immunotherapy effect of prostate cancer and reduce the side effects caused by treatment.This nanogel was used for in vivo delivery of the anti-tumor drug doxorubicin（DOX）to achieve synergistic therapy of prostate cancer with anti-PD-L1 antibodies（αPD-L1）.During the circulation in the body,m PEG-p（LG-co-LC）nanogel can effectively protect DOX,and achieve the aggregation in tumor sites by the enhanced permeability and retention effect（EPR）.DOX was trigged to release under low pH and high glutathione（GSH）conditions in the tumor cells,due to dissociation of electrostatic interaction between DOX and nanogel,and the broken of S-S,respectively.While DOX induces tumor cell apoptosis,it can also cause immunogenic cell death（ICD）.ICD induce tumor cells to release tumor associated antigen（TAA）and damage associated molecular patterns（DAMPs）,thereby promote the infiltration of immune cells in tumor tissues and realize the conversion of non-immunogenic tumor microenvironment into immunogenicity,improvingαPD-L1 sensitivity to tumor.Finally,the tumor suppressor effect was significantly enhanced by immune checkpoint.Methods:In the first part,we realized the efficient and stable loading of DOX by nanoparticle（DOX NP）by using the principle of positive and negative electric interaction.First,systemically characterize the assembly performance,drug loading and release behavior of DOX NP.Subsequently,the cytotoxicity of DOX NP to tumors was evaluated by the MTT.The uptake of DOX NP by tumor cells was evaluated by flow cytometry.The in vivo imaging system was used to observe the distribution of different pharmaceutical preparations in mice.The C57BL/6 mouse subcutaneous allograft prostate cancer tumor model was established,and the tumor suppressive ability of DOX NP was evaluated by in vivo tumor suppression experiments and immunohistochemical staining.Finally,the safety of nanomedicine was systematically evaluated through weight monitoring,biochemical indicators and histopathological testing of major organs.In the second part,we evaluated the efficacy of DOX NP combined withαPD-L1 chemoimmunotherapy on prostate cancer.First,the ability of DOX NP to induce ICD was evaluated by detecting the exposure of CRT on the surface of tumor cells and the release of ATP and HMGB1.In vivo tumor suppression experiments and immunohistochemical staining were used to systemically evaluate the tumor suppression ability of DOX NP+αPD-L1 combined therapy.The immune status and serum cytokine levels of the treated mice were evaluated by flow cytometry and ELISA.Results:In the first part,the results showed that the hydrodynamic diameter of NP was 94.6±0.6 nm in PBS（pH 7.4）,the particle size increased to 127.8±2.3nm after loading DOX,and the zeta potential increased from-20.1±1.0 m V to-6.8±0.4 m V after loading DOX,which indicates that the positively charged-NH₂of DOX and the negatively charged-COOH in NP are combined through positive and negative interactions.The drug loading capacity is 17.3%and the drug loading efficiency is 86.5%.DOX NP can exist relatively stable in a physiological environment（pH 7.4）,while in acidic environment,the cumulative release of DOX gradually increases as the pH value decrease.When pH 5.3 with GSH,the cumulative release of DOX was the highest,which proved that DOX NP has good pH and reduction responsiveness.The in vitro results showed that cytotoxicity of DOX NP in GSH-pretreating cells was similar to that of free DOX,and both groups were higher than that of the DOX NP group,indicating that high GSH concentration promoted the release of DOX NP.In the cell uptake experiment,the DOX NP group shows the weakest fluorescence intensity,and the DOX group has the strongest.This is because free DOX enters the cell through free diffusion,while DOX NP enters the cell through endocytosis.After 6 hours and 12 hours of intravenous injection,free DOX accumulated more in the liver but less at the tumor site than DOX NP group.The fluorescence intensity of DOX decreased with time.On the contrary,the fluorescence intensity of DOX NP group increased over time at the tumor site.This result indicated that the NP effectively reduced the accumulation of the drug in the liver,and at the same time increased its concentration at the tumor site through the EPR effect.DOX NP showed the strongest tumor suppression ability in vivo.TUNEL and immunohistochemical staining results showed that the most of tumor cell apoptosis in the DOX NP treatment group.The pathological examination of major organs in DOX NP group showed no obvious toxicity,and the body weight did not fluctuate significantly during the treatment,which proved that DOX NP had more reliable safety.In the second part,the results showed that both DOX and DOX NP can effectively induce ICD in tumor cells,and DOX NP is slightly stronger than DOX.DOX NP+αPD-L1 had the strongest tumor suppressor effect in vivo.The proportion of CD8⁺T cells in tumor infiltrating lymphocytes and tumor draining lymph nodes is higher than that in the DOX+αPD-L1 and PBS group.The recruitment of Treg was effectively reduced at the same time.After the treatment,the level of immunosuppressive cytokine TGF-βin the DOX NP+αPD-L1 treatment group was significantly reduced compared with other treatment groups.TUNEL and immunohistochemical staining of tumor tissues also confirmed the excellent tumor suppression ability of DOX NP+αPD-L1treatment group.Conclusion:（1）We successfully prepared dual-responsive m PEG-p（LG-co-LC）drug-loading nanoplatform,DOX NP.（2）DOX NP can selectively accumulate at the tumor site and effectively reduce systemic side effects.（3）DOX NP can not only inhibit tumor growth,but also induce ICD in tumor cells.（4）The combination of DOX NP andαPD-L1 has stronger tumor suppression ability,which provides a theoretical basis for the clinical application of chemoimmunotherapy.