Evaluation And Mechanism Exploration Of Anti-tumor Therapy Based On Multifunctional Nano-photothermal Materials

Tumor is a chronic disease that seriously endangers human health.Metastasis and recurrence are the main factors affecting the survival of patients.Therefore,it is difficult to treat tumors while killing tumors in situ and destroying metastases.Surgery,chemoradiotherapy,and biological therapy are currently the main methods for treating tumors,but each method has limitations in clinical indications,curative effects,and adverse reactions.Exploring new methods for treating tumors is very important for tumor treatment.With the increasing requirements of people for quality of life and the continuous development of medical technology,minimally invasive and non-invasive treatment with minimal trauma,precise curative effect,targeted and fast recovery are the most promising technologies in the field of comprehensive tumor treatment.Photothermal Therapy（PTT）is the use of optical absorption material to effectively convert the energy of light irradiation in the near infrared region to generate heat to"burn"tumor cells.Compared with traditional cancer treatments,photothermal therapy has unique advantages,including high selectivity,low systemic toxicity,minimally invasive and precise space-time selectivity,and limited treatment tolerance.Recent studies have found that after killing tumors in situ with nano-PTT,tumor-specific biomolecular proteins,tumor-associated antigens（Desoxyribonucleic Acid,DNA）are exposed to the body,which can be recognized by the immune system.Supplement with immune adjuvants can increase the sensitivity of the body’s immunity to TAA stimulation,which can also effectively inhibit the growth of residual tumor cells in the body,thereby eliminating metastases while killing tumors in situ.Nano-PTT combined with immunotherapy has become a hot spot in the treatment of metastatic tumors.Achieving effective tumor PTT includes the following key points:First,finding nano-photothermal agents（PTA）that have both biological safety and high photothermal conversion efficiency（PCE）is the premise of achieving safe and efficient PTT.Secondly,clarifying the molecular mechanism of PTT action and the mechanism of activating the body’s tumor-specific immunity is the key to maximizing the efficacy of PTT.In order to solve the above two key issues,this subject conducts the following four parts of research:The first part is to develop PTA of double polyethylene glycol modified black porous silicon（DPEG-BPSi）with high light-to-heat conversion efficiency and verify its PTT efficacy and safety.The main research is as follows:（1）Using dynamic laser scattering（DLS）and other methods to characterize its physical and chemical characteristics;（2）Through CCK8 experiment,flow cytometry experiment and clone proliferation experiment verified its photothermal treatment effect and cytotoxicity;（3）In vivo,using mouse tumor-bearing experiments to validate DPEG-BPSi PTT effect,biosafety and ability to activate tumor specific immunity.In the second part,based on the research of DPEG-BPSi,the photothermal material polyethylene glycol-bismuth（PEG-Bi）with higher light-to-heat conversion efficiency and imaging enhancement function is developed and its PTT performance and safety are verified.The research methods include:（1）The morphology and physicochemical properties of nanoparticles are studied by transmission electron microscope（TEM）and other experiments;（2）The photothermal treatment effect and cells toxicity of PEG-Bi are verified by CCK8 experiment,flow cytometry experiment and clone proliferation experiments;（3）The tumor-bearing experiments in mice have verified the ability of PEG-Bi to passively targeting tumors in vivo,and the ability to warm up tumor,the PTT effect,biosafety,and the ability to activate mouse tumor-specific immunity.The third part takes DPEG-BPSi as the research object,explores its PTT mechanism,and finds ways to enhance its PTT efficacy.The main research methods are:（1）Gene sequencing is used as the basic method to study the changes of expressed genes in Hep G-2cells after DPEG-BPSi photothermal treatment;（2）Using RT-q PCR,Western blot and other experiments to verify the results of cell sequencing and cell autophagy activation;（3）Observing the effect of combined application of autophagy inhibitors or autophagy activators on the photothermotherapy effect of DPEG-BPSi and the effect of DPEG-BPSi on the pH value of lysosomal cells.In the fourth part,to find the evidence of the the third part starting point that increasing the lysosomal pH value can effectively inhibit the degradation of autophagy and enhance the PTT effect of DPEG-BPSi,the main research contents include:（1）It is planned to accurately measure the changes in the pH value of Hep G-2 cells before and after adding DPEG-BPSi through commercially available pH indicator probes such as PDMPO;（2）Due to the current lysosomes pH value indicator cannot accurately measure the lysosomal pH values and changes,we builded a self-dissociating MB@Si O₂ nano system,in which the release of MB in the nanoparticles is linearly related to the pH value;（3）Verifying that the nanoparticles mainly stay in the lysosome after entering the cell,this is the premise of measuring the lysosomal pH value with MB@Si O₂ nano system;（4）Measuring the changes of lysosomal pH value of Hep G-2 cells after phagocytosis of DPEG-BPSi nanoparticles with MB@Si O₂ nano system.The main experimental results of this subject are as follows:1.The DPEG-BPSi photothermal treatment system was successfully constructed.The DPEG-BPSi photothermal conversion efficiency was 33.6%.In in vitro experiments,50μg/m L nanoparticles irradiated with 808 nm laser for 10 min could increase the suspension temperature by 12.5℃.The cell survival rate of the DPEG-BPSi photothermal treatment group was significantly lower than that of the control group（P<0.05）.);In mice,0.75mg/m L DPEG-BPSi after irradiating 808 nm laser（power of 1 W/cm²）for 10 min can increase the tumor temperature by about 8℃.The tumor volume increase percentage of irradiation treatment group,0.5 mg/m L and above DPEG-BPSi combined 808 nm,was significantly lower than the control group（P DPEG-BPSi（0.5 mg/m L）+808 nm VS control group=0.004773）,and the DPEG-BPSi photothermal treatment group could increase mouse cytokines IL-2,IFN-γand TNF-αlevels,spleen cell killing ability,inhibit distant tumor growth,and provide experimental references for the combined application of DPEG-BPSi photothermal therapy and immunotherapy.In vivo and in vitro experiments did not find the toxicity of DPEG-BPSi to cells and mice within the effective photothermal concentration,and the biological safety was good.2.After DPEG-BPSi,we have found a material with higher photothermal conversion efficiency,PEG-Bi（54.8%）.50μg/m L nanoparticles with 808 nm laser irradiation10 min can increase the suspension temperature for 20.4℃.When the concentration of PEG-Bi reached 75μg/m L,the cell survival rate was only about 30%.When the concentration was100μg/m L,the high temperature effect mediated by PEG-Bi completely killed CT 26tumor cells.In vivo experiment,0.75 mg/m L PEG-Bi when irradiated with 808 nm laser（1W/cm² power）for 10 min can increase the tumor temperature by about 15°C,and PEG-Bi+808 nm treatment can decrease volume increase percentage,which is significantly lower than the control group and nanomaterial treatment group（P0.5mg/m L-PEG-Bi+laser VS control=0.016599,<0.05,P 1.0mg/m L-PEG-Bi+laser VS control=0.008222,<0.05）.In vivo and in vitro experiments,PEG-Bi did not have significant toxicity to cells or mice within the effective photothermal treatment concentration.But in the PEG-Bi photothermal treatment system,we did not find that the mouse immune system was activated.3.DPEG-BPSi can increase the expression level of TFEB,CTSK,ATP5J2-PTCD1,ATP2A1,CLCN6 and V-ATPase complexes in the TFEB-CLEAR gene network after photothermal treatment,and TFEB can promote the lysosomal synthesis and autophagy occur,so cells can be induced to undergo autophagy after PTT treatment.RT-q PCR,Western,and other experiments have verified that DPEG-BPSi can activate the gene network and induce cell autophagy after photothermal treatment,but confocal and flow cytometry experiments have found that the amine-PEG layer of DPEG-BPSi itself can improve the lysosome pH,inhibiting degradation of autophagy flow.At the same time,it was found that the autophagy inhibitor chloroquine increased the cell inhibition of DPEG-BPSi photothermal treatment,such as after undergoing 0.1 mg/m L DPEG-BPSi photothermal treatment,the cell survival rate was 27.2%±4.7%,and the survival rate was12.3%±4.8%when combined with chloroquine,P=0.019046,there was a statistical difference.Taking the relationship between autophagy and lysosomal pH as the starting point,a new approach to enhance the effect of DPEG-BPSi PTT was proposed.4.Currently,commercially available fluorescent probes cannot accurately detect the lysosomal pH value and its small changes.Based on the self-dissociating silica system,this study has developed the MB@Si O₂ nanoprobe system can accurate quantitatively detecte the lysosomal pH.In this system,the release rate of the dye Methylene Blue（MB）has a linear relationship with the pH value in the lysosomal pH range.For example,the relationship between the release rate of MB in NPs _6/100and the lysosomal pH value is y=0.07759x+0.44559,which can sensitively indicate the lysosomal pH value,and the sensitivity reaches 0.01 pH values,providing a characterization means for predicting the autophagy state of cells.In summary,in this study we constructe two PTT systems of DPEG-BPSi and PEG-Bi with high light-to-heat conversion efficiency and good photothermal treatment effects both in vivo and in vitro,and verify that DPEG-BPSi can activate the mouse immune system and inhibit distant tumor growth.The molecular mechanism of DPEG-BPSi photothermal therapy was initially explored at the molecular level.It was found that DPEG-BPSi can promote the genes of the TFEB-CLEAR lysosomal gene network transcripte.But the surface of DPEG-BPSi nanoparticles was coated with amine-PEG,which can increase the lysosomal pH value and block the degradation of autophagy flow,and can increase the effect of DPEG-BPSi photothermal treatment when combined with chloroquine.Finally,in order to find evidence of lysosomal alkalization,the MB@Si O₂ nano system was developed to accurately measure the lysosomal pH value to the nearest 2 decimal places,providing a new characterization method for predicting the state of cell autophagy.