Construction Of Multifunctional Nanosystems Loaded With Biguanides For Amplifying The Photodynamic Immunotherapy Efficacy Of Bladder Cancer

Photodynamic therapy（PDT）is an emerging tumor treatment strategy in recent decades.It has the advantages of minimal invasion,fewer complications,elimination of tiny lesions,preservation of organ function and no drug resistance.Moreover,the bladder itself is a hollow organ,and the urethra is directly connected with the outside world,which is very convenient for the introduction of endoscopes and laser fibers.Therefore,PDT has been regarded as one of the most promising therapies for bladder cancer.In recent years,with the development of various new photosensitizers and the continuous progress of optical technology,the clinical application of PDT for the treatment of bladder cancer has become more and more promising.However,the curative effect of PDT is still not very satisfactory at present,and the most important reasons are that it is limited by the hypoxic microenvironment of bladder cancer,insufficient tumor targeting of photosensitizers and the adaptive immune escape mediated by PDT.Therefore,the development of a new treatment strategy that can solve the above drawbacks of PDT will be of great significance to the clinical application of PDT in the treatment of bladder cancer.In this study,to solve the key scientific problems faced by PDT,two different multifunctional nanosystems containing biguanides with both immune and oxygen regulation capacity were constructed.A series of experiments in vitro and in vivo were then designed to study their mechanism for amplifying the photodynamic immunotherapy efficacy of bladder cancer.Part Ⅰ Construction of metformin-loaded liposome nanosystems for amplifying the photodynamic immunotherapy efficacy of bladder cancerObjective: To prepare a liposome nanosystem（IR775@Met@Lip）co-loaded with metformin（Met）and photosensitizer IR775,and to explore its mechanism for amplifying the photodynamic immunotherapy efficacy of bladder cancer,thereby bringing new ideas for the clinical application of PDT in the treatment of bladder cancer.Methods:（1）Liposome nanoparticles co-loaded with Met and IR775,namely IR775@Met@Lip,were synthesized by a thin-film hydration method.Then,the particle size,potential,morphology,ultraviolet and fluorescence spectra of the nanoparticles were characterized,and their in vitro stability and drug sustained-release properties were also investigated.（2）The ability of IR775@Met@Lip to reverse hypoxia in bladder tumors was evaluated using the oxygen consumption rate experiment of MB49 cells and immunofluorescent staining of HIF-1α and Pimonidazole（Pimo）.（3）The DCFH-DA probe and CCK-8 assay were used to evaluate the ability of IR775@Met@Lip to promote the PDT effect of bladder cancer in vitro.（4）The bladder tumor-targeting ability of IR775@Met@Lip was evaluated using in vivo small-animal imaging.（5）The MB49 mouse bladder cancer model was established to evaluate the effect of PDT on the expression of IFN-γ and PD-L1 protein in bladder tumors in vivo.（6）The MB49 mouse bladder cancer model was used to detect the effect of IR775@Met@Lip-mediated photodynamic immunotherapy on PD-L1 protein expression and T cell infiltration in bladder tumor tissues.（7）The MB49 mouse bladder cancer model was used to assess the ability of IR775@Met@Lip-mediated photodynamic immunotherapy to inhibit the growth of bladder cancer in vivo.（8）The bilateral MB49 tumor-bearing mouse model was used to evaluate the ability of IR775@Met@Lip-mediated photodynamic immunotherapy to inhibit the growth of distal bladder tumors.（9）The biosafety of IR775@Met@Lip was evaluated using healthy C57BL/6 mice in a simulated pharmacodynamic study.Results:（1）A liposome nanosystem co-loaded with Met and IR775,namely IR775@Met@Lip,was successfully prepared.The nanoparticles had a uniform spherical structure with a particle size of 152 ± 2.2 nm and potential of-26.4 ± 1.4 m V.In addition,the cumulative release rates of Met and IR775 in the nanoparticles were 40.5 ± 2.9% and 14.8% ± 3.2% in 24 h,respectively.Moreover,the nanoparticles also had good stability in vitro.（2）IR775@Met@Lip could effectively reduce the oxygen consumption rate of MB49 cells.Moreover,it also significantly reduced HIF-1α expression and Pimo accumulation in MB49 bladder tumor sites.（3）Compared with other groups,IR775@Met@Lip with laser irradiation could generate the most ROS in MB49 cells and had the strongest MB49 cell killing ability.（4）Compared with free IR775,IR775@Met@Lip had a better tumor-targeting ability.（5）In vivo,PDT could induce increased secretion of IFN-γ in bladder tumor tissues and upregulate PD-L1 expression on the tumor cells.（6）IR775@Met@Lip could significantly reduce the expression of PD-L1 in MB49 cells in vitro and in vivo.Moreover,it could also reverse the upregulation of PD-L1 protein in MB49 cells caused by PDT in vivo and then effectively increase the infiltration and distribution of T cells in MB49 bladder tumor tissues.（7）Compared with other groups,the IR775@Met@Lip + laser group could most effectively inhibit the growth of bladder tumors in vivo.（8）IR775@Met@Lip-mediated photodynamic immunotherapy could effectively inhibit the growth of distal bladder tumors.（9）The therapeutic dose of IR775@Met@Lip did not cause hemolysis and had no obvious toxic side effects on the main organs of the mice.Conclusions:（1）The IR775@Met@Lip was a very suitable nanodrug delivery system with ideal particle size,in vitro stability,drug release behavior,and biocompatibility.（2）IR775@Met@Lip could achieve good accumulation in MB49 bladder tumors in vivo and effectively reverse the hypoxia of MB49 cells both in vitro and in vivo.（3）IR775@Met@Lip could effectively down-regulate PD-L1 protein expression in MB49 cells both in vitro and in vivo.Moreover,it could also reverse the upregulation of PD-L1 protein in MB49 cells caused by PDT in vivo,thereby increasing T cell infiltration and activity in bladder tumor tissues.（4）IR775@Met@Lip-mediated photodynamic immunotherapy could effectively inhibit the growth of MB49 bladder tumor cells in vitro and in vivo.（5）IR775@Met@Lip-mediated photodynamic immunotherapy could effectively inhibit the growth of distal bladder tumors in vivo.Part Ⅱ Construction of buformin-loaded manganese dioxide nanosystems for amplifying the photodynamic immunotherapy efficacy of bladder cancerObjective: To prepare a manganese dioxide（MnO₂）multifunctional nanosystem（MB@Bu@MnO₂）co-loaded with buformin（Bu）and methylene blue（MB）,and to explore its mechanism for amplifying the photodynamic immunotherapy efficacy of bladder cancer,thereby providing a new way to improve the PDT effect of bladder cancer.Methods:（1）The multifunctional MnO₂ nanoparticles co-loaded with Bu and photosensitizer MB,namely MB@Bu@MnO₂,were synthesized by the bio-mineralization method.Then,the morphology,particle size,potential,ultraviolet and fluorescence spectra of the nanoparticles were characterized,and their in vitro stability and drug release behavior in response to the tumor microenvironment were detected.（2）The dual oxygen regulation ability of MB@Bu@MnO₂ was evaluated by detecting the oxygen consumption rate,ADP/ATP ratio and hypoxia markers（HIF-1α and Pimo）of MB49 cells.（3）The ROS probes（DPBF and DCFH-DA）and CCK-8 assay were used to evaluate the ability of MB@Bu@MnO₂ to promote the PDT effect of bladder cancer in vitro.（4）The bladder tumor-targeting ability of MB@Bu@MnO₂ was evaluated using in vivo small-animal imaging.（5）The effect of Bu on the expressions of AMPK,p AMPK and PD-L1 was evaluated in bladder cancer cell lines 5637 and MB49.（6）The MB49 mouse bladder cancer model was used to detect the effect of MB@Bu@MnO₂-mediated photodynamic immunotherapy on PD-L1 protein expression and T cell infiltration in bladder tumor tissues.（7）The MB49 mouse bladder cancer model was used to assess the ability of MB@Bu@MnO₂-mediated photodynamic immunotherapy to inhibit the growth of bladder cancer in vivo.（8）The biosafety of MB@Bu@MnO₂ was evaluated using healthy C57BL/6 mice in a simulated pharmacodynamic study.Results:（1）A MnO₂ nanosystem co-loaded with MB and Bu,namely MB@Bu@MnO₂,was successfully prepared.The nanoparticles showed a uniform spherical structure with a particle size of 22.4 ± 4.3 nm and a potential of-21.9 ± 2.6 m V,and they also had good stability in vitro and ideal tumor microenvironment-responsive release capabilities（acidic p H and high H2O2 concentration）.（2）MB@Bu@MnO₂ could significantly reduce the oxygen consumption rate and ATP production in MB49 cells.Moreover,it was also able to significantly reduce HIF-1α expression and Pimo accumulation in MB49 bladder tumor sites.（3）Compared with other groups,MB@Bu@MnO₂ with laser irradiation could generate the most ROS in MB49 cells and possessed the strongest MB49 cell killing ability.（4）Compared with free MB,MB@Bu@MnO₂ had better MB49 bladder tumor-targeting ability.（5）Bu could effectively induce AMPK phosphorylation and down-regulate PD-L1 expression in both 5637 and MB49 bladder cancer cells（6）MB@Bu@MnO₂ could significantly reduce the expression of PD-L1 in MB49 cells in vitro and in vivo.Moreover,it could also reverse the upregulation of PD-L1 protein in MB49 cells caused by PDT in vivo and then effectively increase the infiltration and distribution of T cells in MB49 bladder tumor tissues.（7）The MB49 bladder tumor growth in the MB@Bu@MnO₂ + Laser group was maximally inhibited compared with all other groups.（8）The therapeutic dose of MB@Bu@MnO₂ did not significantly increase the level of serum lactic acid in mice and had no obvious toxic side effects on the main organs of the mice.Conclusions:（1）MB@Bu@MnO₂ possessed ideal particle size,in vitro stability and drug release behavior in response to the tumor microenvironment,as well as excellent bladder tumor targeting ability and biocompatibility in vivo,so it was an excellent drug delivery nanosystem.（2）MB@Bu@MnO₂ could not only increase the oxygen production through catalyzing H2O2 in tumors by MnO₂,but also reduce the oxygen consumption of bladder tumor cells by releasing Bu,so it could improve the hypoxic microenvironment of bladder tumors to the greatest extent in vitro and in vivo.（3）MB@Bu@MnO₂ could effectively down-regulate PD-L1 expression in MB49 bladder tumors both in vitro and in vivo.Moreover,it could also reverse the upregulation of PD-L1 protein in MB49 bladder tumor cells caused by PDT in vivo and further increase T cell infiltration and activity in bladder tumors.（4）MB@Bu@MnO₂-mediated photodynamic immunotherapy could effectively inhibit the growth of MB49 bladder tumor cells both in vitro and in vivo.