Biomimetic Selenium-black Phosphorus Nanomaterials For Photothermal Combined Immunotherapy Of Prostate Cancer

In recent years,the incidence and mortality probability of malignant tumors have been high and rising year by year,which has caused a very large economic burden to the society and a heavy impact on patients and families.Therefore,malignant tumors have become a well-deserved "killer" at present.Among them,the incidence of prostate cancer is more and more significant,accounting for the second place of male cancer deaths,and is the most common malignant tumor of the male reproductive system.According to statistics,its incidence accounts for 7% of all newly diagnosed male cancers in the world(15% in developed regions).There is no large-scale screening for prostate cancer in China,which also leads to the fact that 70% of the new incidence of prostate cancer is found when the patients are already in the advanced stage and present with metastatic disease.In general,most of the current treatments for prostate cancer include single or multiple treatments such as surgical resection,radiotherapy,immunotherapy,and endocrine therapy,but only metastatic Castration-resistant prostate cancer(m CRPC)has a certain effect.After treatment,the 5-year survival rate of patients is 50-70%.However,there is no unified and effective treatment for m CRPC at present,so there is an urgent need to find more effective treatment methods in order to treat m CRPC and prolong the survival rate of patients.Recent studies have shown that photothermal therapy(PTT)is a good means of treating prostate cancer.In simple terms,PTT uses materials with high photothermal conversion efficiency,inject them into the body,and collect them near the tumor tissue using targeted recognition technology.And under the irradiation of an external light source(usually near-infrared light)to convert light energy into heat energy to kill cancer cells.This requires materials with good photothermal conversion efficiency combined with laser to convert light energy into heat to kill tumor cells.In addition,tumor immunotherapy can effectively activate and protect the immune system to resist the invasion of tumor cells and effectively prevent its development.This therapeutic approach can use a variety of methods,such as monoclonal antibodies,therapeutic antibodies,tumor vaccines,biological macromolecular drugs and small molecule inhibitors,through the use of a series of immune checkpoint inhibitors,can effectively turn prostate cancer "cold tumor" into "hot tumor".Therefore,in this study,two-dimensional black phosphorus nanosheets with high photothermal conversion efficiency were selected to be constructed into Se@BPNSs material through a series of synthetic methods and liquid phase stripping,and combined with Anti-PD1 at the cellular and animal levels in order to achieve the effect of photothermal combined immunotherapy to activate macrophage reprogramming.It brings certain clinical theoretical significance for the treatment of prostate cancer.Objective:In order to achieve the purpose of photothermal combined with immune checkpoint inhibitors in the treatment of castration-resistant prostate cancer,and realize the conversion process from "cold tumor" to "hot tumor",this study designed and synthesized a two-dimensional material,Se-doped black phosphorus,from photothermal materials,and formed Se@BPNSs with higher photothermal conversion efficiency by liquid phase exfoliation.In addition,to achieve Enhanced permeability andretention effect(EPR)in solid tumors,we encapsulated the murine prostate cancer cell membrane RM-1 in the outer layer of Se@BPNSs,which can effectively achieve homologous targeting.By injecting a certain dose of Se@BPNSs into the tumor body and under the irradiation of 808 nm laser,Se@BPNSs converts the light energy into heat energy to achieve a high temperature to kill tumor cells,and at the same time releases antigen to be captured by DC cells.At the same time,we combined Anti-PD1,an immune checkpoint inhibitor,to amplify immunotherapy and promote immune cell infiltration.In summary,this study confirmed the successful preparation of Se@BPNSs in the synthesis and characterization of the material,and verified it in both cell and animal levels to achieve the purpose of photothermal immunotherapy,which will bring certain clinical guidance for the treatment of m CRPC.Methods:Part I: The Se-doped black phosphorus nanosheets(Se@BPNSs)were prepared by liquid phase stripping method.A series of characterization methods,including transmission electron microscopy(TEM),XRD,XPS and AFM,were used to confirm the successful preparation of the materials.The addition of selenium can improve the air stability of BPNSs and delay its degradation.At the same time,by taking advantage of the specific recognition ability of the cell membrane surface,the Se@BPNSs prepared by the prostate cancer cell membrane is used to form the cell membrane biomimetic modified nanosheets.This nanodrug delivery system retains the good photothermal effect of BPNSs.The system is endowed with many characteristics of prostate cancer cells,which makes the nanodrug delivery system have the ability of immune escape and tumor targeting to enhance the therapeutic effect of prostate cancer.The second part was to explore the anti-proliferation activity of Se-doped black phosphorus compounds on prostate cancer cells and its potential mechanism at the cellular level.CCK-8 assay,cell invasion and migration assay,and detection of ROS generation were used to verify the anti-proliferation activity of Se-doped black phosphorus compounds.In the third part,the anti-cancer immunotherapy mechanism of Se-doped black phosphorus compound combined with anti-PD-1 checkpoint blocker was explored by delivering drugs to tumor sites at the animal level.Blood biochemical indexes,EPR effect of drugs and flow cytometry of tumor immune cells(DC,Treg)were used to further reveal the mechanism of anti-tumor activity.Results:1.In this study,we successfully prepared Se@BPNSs two-dimensional nanosheets by vapor deposition method combined with liquid phase ultrasonic peeling method,and constructed a novel cell membrane biomimetic modified nanodrug delivery system,namely Se@BPNSs@RM-1.The material size is about 100 nm.Elemental analysis profiles showed that Se was successfully doped into BP.AFM results show that the size of BP nanosheets after Se incorporation is more stable at about 100 nm than that without Se incorporation,and the thickness of two-dimensional nanosheets does not change,indicating that the introduction of Se can stabilize the active BP nanosheets.As seen from the XRD crystal structure,the fabricated Se@BP 2D nanosheets have a similar crystal structure to BP,indicating that Se doping does not change the stable interlayer structure of the black phosphorus nanosheets.Further XPS experiments showed that the enhancement of BP stability by Se doping may be due to the stabilization of the P network in BP due to the oxidation of the electron vacancies provided by Se with the solitary pair electrons on the black P surface.2.The antitumor activity of Se@BPNSs@RM-1 was evaluated at the cellular level.The results showed that Se@BPNSs@RM-1 could inhibit the proliferation activity of tumor cells,and the metastatic ability of prostate cancer cells under various administration methods was investigated by scratch test and cell invasion test.The results showed that Se@BPNSs@RM-1 could inhibit the invasive ability of black phosphorus to prostate cancer cells.On the other hand,the biologically active Se@BPNSs@RM-1 nanosheets also showed good anti-invasive activity,and with the help of flow cytometry,the death of prostate cancer cells under various administration modes was investigated.In general,Se@BPNSs@RM-1can effectively inhibit the proliferation of tumor cells,and greatly increase the content of CRT and HMGB1,which provides certain guiding significance for subsequent animal experiments.3.Animal experiments The in vivo anti-tumor activity of Se@BPNSs@RM-1 was evaluated using a unilateral prostate cancer mouse model.With the help of in vivo imaging of small animals,it was found that the selenium doped black phosphorus complex encapsulated in biological cell membrane could significantly increase the accumulation of drugs in the tumor of tumor-bearing mice,which was helpful to improve the targeted therapy of drugs.In addition,we examined Treg cells in the tumor body,Treg cells in the lymph nodes and D magnetic storm.We found that the combination of Se@BPNSs@RM-1 with Anti-PD1 and laser group had the lowest expression of Treg cells and the highest content of DC cells,indicating that the combination of PD-1 checkpoint blocker can further enhance its anti-tumor activity.Stimulate the body’s immune response ability.Conclusions:1.This project successfully designed,synthesized and prepared Se@BPNSs@RM-1,and carried out a series of characterization experiments to confirm the successful preparation of photothermal materials.2.Se@BPNSs@RM-1 can increase the level of reactive oxygen species in prostate cancer cells,thus inducing the death of tumor cells,and releasing a certain amount of CRT and HMGB1,and its ATP activity is also enhanced.With the help of flow cytometry,the DNA content of prostate cancer cells under various drug administration methods was investigated.Se@BPNSs@RM-1 can inhibit prostate cancer cells in the G0/G1 phase.3.The results of animal experiments show that Se@BPNSs@RM-1 can significantly inhibit the proliferation of tumor cells.Combined with PD-1 checkpoint blocker,it can further sensitizing its anti-tumor activity and realize the effect of photothermal combined immunotherapy.