The Application Of Novel Dual-targeted Fluorescent Probes In Prostate Cancer Surgery And Novel Photodynamic Nanoparticles In Prostate Cancer Chemotherapy And Immunotherapy

Objective Prostate cancer has become a major urological tumor that threatens men’s health and lives.Surgery is the primary method for treating localized prostate cancer,but three major problems are faced in prostate cancer surgery:positive margins,positive lymph node dissection,and damage to nerve and blood vessel bundles.Fluorescenceguided intraoperative navigation provides good help to solve these problems,but traditional intraoperative navigation using fluorescence dyes faces problems such as low sensitivity,poor specificity,low resolution,and low signal-to-noise ratio,which reduces the effectiveness of surgical navigation.Therefore,there is an urgent need to develop a new type of probe for surgical guidance to solve these problems and ultimately achieve the goal of precise surgical navigation.Method Firstly,novel probes will be designed and synthesized,followed by analyzing their physical and optical properties.Secondly,we will utilize a comprehensive approach,incorporating multiple levels(in vitro and in vivo),multiple perspectives(cellular,animal,and clinical samples),and multiple techniques(flow cytometry,fluorescence confocal microscopy,near-infrared fluorescence imaging,etc.)to validate the sensitivity,specificity,stability,safety,imaging capability,and signal-to-noise ratio of the new probes.Subsequently,we will perform a real-time excision of animal prostate tumors using fluorescence probe guidance and finally evaluate the imaging performance of the probes using clinical prostate samples.real-time removal and efficacy evaluation of animal prostate tumors were performed using fluorescence probe guidance.Results In this study,a novel near-infrared fluorescent small molecule intelligent selfassembled nanoprobe,Cy-KUE-OA,was designed and synthesized with dual membrane targeting(targeting prostate-specific membrane antigen and oleic acid).Before reaching the targeting site,Cy-KUE-OA self-assembled into～160 nm nanospheres nanosphere,accompanied by fluorescence quenching.After reaching the targeting site,Cy-KUE-OA specifically bound to prostate-specific membrane antigen(PSMA)and anchored to the phospholipids of prostate cell membranes,achieving specific fluorescence activation.Cy-KUE-OA exhibited extremely high affinity and specificity for PSMA-overexpressing prostate cancer cells,with fluorescence intensity 13.2 times higher than that of the singletargeting PSMA probe,Cy-KUE.In a mouse prostate tumor model,the fluorescence signal of Cy-KUE-OA reached its peak 24 hours after injection and maintained a high signal-to-noise ratio for up to 48 hours in the tumor tissue.During simulated clinical fluorescence-guided laparoscopic prostate cancer resection surgery,Cy-KUE-OA provided real-time guidance for precise removal of prostate tumors in mice(including tiny lesions that cannot be discerned by the naked eye).Furthermore,Cy-KUE-OA can accurately locate,illuminate,and image prostate cancer lesions and metastatic lymph nodes in clinical samples with a fluorescence signal strength approximately 10 times higher than that of benign prostate tissue,indicating promising clinical translation potential.Conclusion This study has innovatively designed and synthesized a prostate cancer cell membrane dual-targeted smart self-assembling nanoprobe,Cy-KUE-OA.The probe exhibits remarkably high sensitivity and specificity towards PSMA-expressing prostate cancer cells,while demonstrating excellent blood stability and safety.Cy-KUE-OA exhibits excellent sensitivity and specificity in fluorescence-guided real-time,precise,and complete excision of prostate cancer under fluorescence guidance,as well as outstanding imaging performance in clinical prostate cancer specimens and metastatic lymph nodes.This study provides important theoretical and experimental foundations for fluorescence-guided prostate surgery and builds an important bridge for preclinical research and clinical trials in prostate cancer.Objective Chemotherapy is an important treatment method for advanced prostate cancer,but traditional cisplatin chemotherapy has poor efficacy and significant systemic side effects,mainly due to poor solubility,low bioavailability,and high hematotoxicity of platinum drugs.In addition,cisplatin chemotherapy is prone to drug resistance,and one of the main reasons is the overexpression of glutathione S-transferase(GST)in prostate cancer cells,which reacts with glutathione(GSH)to cause cisplatin inactivation and ultimately drug resistance.In addition,prostate cancer is an immunologically "cold tumor" with low PD-L1 expression,which limits the application of immune checkpoint inhibitors.Therefore,it is urgent to develop new nanomedicines to address these issues simultaneously,ultimately improving the therapeutic efficacy of advanced prostate cancer and achieving the goal of precision treatment for advanced prostate cancer.Method Firstly,novel nanomedicines will be designed and synthesized,followed by analysis of their nanoparticle characterization.Then,a combination of multilevel(in vitro and in vivo),multi-dimensional(cell and animal),and multi-modal approaches(flow cytometry,confocal fluorescence analysis,near-infrared fluorescence imaging,apoptosis detection,etc.)will be utilized to verify the internalization capability of the novel nanomedicines in prostate cancer cells,platinum drug release capacity,ability to produce reactive oxygen species(ROS)and anti-tumor activity.Subsequently,immunogenic cell death markers and immune response indicators will be assessed and evaluated.Finally,the effectiveness of combining the novel nanomedicines with PD-L1 antibodies will be evaluated.Results In this study,a novel ROS-responsive and four-valent cisplatin prodrugdelivering photodynamic nanoparticle(NP2)with Michael addition receptor was designed and synthesized.In vitro,NP2 can be rapidly internalized and taken up by prostate cancer cells,and under near-infrared light(808 nm)irradiation(NP2+L),a large amount of ROS can be rapidly produced,leading to the dissociation and release of the four-valent cisplatin prodrug from NP2,thereby precisely delivering and killing prostate cancer tumor cells.Meanwhile,the Michael addition receptor contained in NP2 reduced GST activity by 87.6%,greatly reducing the inactivation of platinum and enhancing the sustained ability of platinum to kill prostate cancer cells,ultimately significantly improving the effect of platinum chemotherapy.Additionally,NP2+L can effectively kill prostate cancer cells through photodynamic therapy and chemotherapy,with an IC50 value of only 0.94 μM.In vivo,NP2 can effectively target mouse prostate tumors,and after 14 days of NP2+L treatment,the tumor volume inhibition rate reached 77.1%.When NP2+L was combined with PD-L1 monoclonal antibody,the tumor volume inhibition rate further increased to 85.9%.Furthermore,NP2+L can induce immunogenic cell death of prostate cancer cells,further accelerate DC maturation,promote CD8+T cell infiltration,transform macrophages from M2 to M1 type,inhibit the expression of immunosuppressive Tregs cells,increase the expression of PD-L1 on the surface of prostate cancer cells,and transform "cold tumors" into "hot tumors",finally,improve the effect of immunotherapy for prostate cancer.Conclusion The novel photodynamic nanoparticles,under NIR light exposure,not only enhance the therapeutic efficacy of platinum for prostate cancer and reduce systemic toxicity,but also induce immunogenic cell death of prostate cancer,stimulate a stronger anti-tumor immune response,improve the response rate of PD-L1 monoclonal antibody,and ultimately achieve synergistic treatment of chemotherapy,photodynamic therapy,and immunotherapy,resulting in the optimal anti-prostate tumor effect.This provides new ideas and strategies for clinical multimodal anti-tumor therapy.