MRI/PAI-guided PTT/CDT Synergistic Therapy For Prostate Cancer Based On Carbon-MMT-PEG

Prostate cancer(PCa)is one of the most common malignant tumors in men and the second leading cause of cancer-related mortality in men.At present,surgical resection is the main treatment for early-stage prostate cancer,while chemotherapy and castration are still the main clinical treatments for advanced-stage prostate cancer.However,these traditional methods face many problems such as limited therapeutic effect,severe side effects and drug resistance,and most of them eventually transform into castration-resistant prostate cancer.Therefore,for the treatment of advanced prostate cancer,there is a great need to explore an alternative therapeutic strategy with high anticancer efficiency and low side effects.Photothermal therapy(PTT),as an emerging treatment method,uses an external light source(usually near-infrared light)to irradiate a material with high photothermal conversion efficiency(PCE)to convert light energy into heat energy to kill cancer tumors,and it has attracted widespread attention in tumor therapy due to its high efficiency,local targeted therapy,and low systemic toxicity.Chemodynamic therapy(CDT)utilizes Fenton or Fenton-like reaction to promote endogenous hydrogen peroxide(H2O2)into highly cytotoxic hydroxyl radicals(·OH)to kill cancer cells,which showed potential therapeutic value in tumors.In addition,Photothermal therapy agents(PTA)with an imaging modality can be enriched into tumors through EPR effect after systemic administration,monitor drug accumulation in tumors in real time through imaging,and provide guidance for PTT,called imaging guide the treatment of tumors.Therefore,in this study,a new nanosystem Carbon-MMT-PEG was constructed,which was verified by in vitro and in vivo experiments to have magnetic resonance imaging(MRI)and photoacoustic imaging(PAI)and anti-tumor effects.MRI /PAI guides PTT to cooperate with CDT in the treatment of prostate cancer,realizes the integration of multimodal diagnosis and treatment,and provides a new potential treatment plan for prostate cancer precision medicine.This research is mainly divided into three parts:Part Ⅰ Construction,Characterization and Properties of Carbon-MMT-PEG NanosystemsObjective: To construct a novel nanosystem Carbon-MMT-PEG and explore its properties.Methods: 1.Synthesize PIDA co-crystal from1,4-bis(trimethylsilyl)-1,3-butadiyne,obtain black carbonized product after vacuum carbonization at 600℃,pulverize it to fine powder to obtain two Two-dimensional nanocarbon sheets(2D-Carbon)and C18-PMH-PEG were sonicated in an ice-water bath for 4 hours(the black suspension was lyophilized and resuspended to the corresponding concentration before use),and finally mixed with two-dimensional MMT and PEG.After sonication in an ice bath for 4 h,Carbon-MMT-PEG was finally obtained.2.Through Energy Dispersive Spectroscopy(EDS),Raman Spectroscopy,Fourier Transform Infrared Spectrometer(FTIR),X-ray Diffraction(XRD)Spectroscopy,Atomic Force Microscope(AFM),Dynamic Light Scattering(DLS),Transmission Electron Microscope(TEM),Scanning Electron Microscope(SEM),Zeta Potential Analysis,UV-visible spectrophotometer(UVs)to characterize it.3.The photothermal conversion efficiency of Carbon-MMT-PEG nanosystem solution was tested by 1064 nm laser,and bacon of different thickness was used to simulate tumor layers of different depths in vitro to test the penetration ability of 1064 nm laser.4.Application of 3,3’,5,5’-tetramethylbenzidine(TMB)to evaluate the generation of reactive oxygen species(ROS)in the catalytic process of Carbon-MMT-PEG cascade,reflecting chemodynamic therapy(Chemodynamic therapy,CDT).Results: 1.EDS and Raman analysis showed that the PIDA carbonization products mainly contained carbon elements at a high temperature of 600 ℃.2.AFM,TEM,SEM and DLS showed that a circular two-dimensional carbon nanomaterial Carbon with a particle size of about 40 nm and a thickness of about 4mm was obtained by ultrasonic exfoliation and UVs showed strong absorption in the near-infrared Ⅱ region.3.TEM,DLS,and Zeta potential analysis showed that 2D-Carbon and2D-MMT together were constructed into 2D-2D(two-dimensional,2D)Carbon-MMT-PEG nanoclusters under PEG modification,with a particle size of about 300 nm.Zeta potential analysis is negatively charged and has strong absorption in the near-infrared Ⅱ region.3.The photothermal conversion efficiency of Carbon-MMT-PEG in the solution is 34.6%,and the relative decay rate in the near-infrared Ⅱ region(1064nm laser)is better than that in the near-infrared region I(808 nm laser).2.In weakly acidic conditions,Carbon-MMT-PEG can generate reactive oxygen and hydroxyl radicals(·OH)through the Fenton reaction.Conclusions: 1.In this study,a new 2D-2D nanocluster Carbon-MMT-PEG system was successfully constructed.2.Carbon-MMT-PEG system has the potential to become a photothermal material in the near-infrared Ⅱ region to realize photothermal therapy(PTT),and also has the value of chemodynamic therapy(CDT).3.Through the characterization and performance testing of the Carbon-MMT-PEG system,the Carbon-MMT-PEG nanosystem has the potential for biomedical applications.Part Ⅱ Imaging properties of Carbon-MMT-PEG systemObjective: To evaluate the imaging performance of Carbon-MMT-PEG nanosystems in vitro and in vivo.Methods: 1.Magnetic resonance imaging(MRI)and Photoacoustic Imaging(PAI)were used to detect MRI and PAI signals in Carbon-MMT-PEG solutions with different concentrations.2.Construct a mouse prostate cancer subcutaneous tumor model,monitor the MRI and PAI signals in the mouse tumor at different time points(0h,1h,2h,6h,12 h,24h)by tail vein injection,and evaluate the the best irradiation time of 1064 nm laser.3.Carbon-MMT-PEG was labeled with DIR,injected through the tail vein,and the distribution in major organs and tumors at different time points was detected by in vivo imaging of small animals.Results: 1.Carbon-MMT-PEG solution exhibited concentration-dependent MRI and PAI signal responses.2.Carbon-MMT-PEG has obvious contrast enhancement signal of MRI and PAI in mouse tumor.When injected through tail vein for 12 h,the enhanced signal of MRI/PAI is the strongest,indicating that Carbon-MMT-PEG is most enriched at this time.3.In vivo imaging of small animals showed that the fluorescence was mainly distributed in the liver and spleen,and there was obvious fluorescence in the tumor at 12 hours.Conclusions: 1.Carbon-MMT-PEG has the performance of MRI and PAI contrast-enhanced imaging in vitro and in vivo,and has the potential to become a dual-modal imaging nanomaterial.2.MRI and PAI showed that Carbon-MMT-PEG was most enriched in the tumor at 12 h after injection through the tail vein.This time point can be used as the best time point for 1064 nm laser irradiation during treatment.Part Ⅲ The antitumor effect of Carbon-MMT-PEG nanosystemObjective: To evaluate the antitumor effect and biosafety of Carbon-MMT-PEG nanosystem in vitro and in vivo.Methods: 1.The effect of Carbon-MMT-PEG nanosystem on tumor cell viability was detected by MMT method,and the PTT/CDT efficacy of Carbon-MMT-PEG nanosystem was evaluated.2.The generation of intracellular ROS was detected by fluorescence confocal microscopy with ROS probe DCFH-DA.3.Construct mouse prostate cancer subcutaneous tumor with mouse-derived prostate cancer cell RM-1,and draw the tumor growth curve and survival curve by monitoring tumor size and survival period by intratumoral injection and tail vein injection.The anti-tumor effect of Carbon-MMT-PEG was evaluated in vivo,and the tumor tissue was stained with H&E and Tunel to detect tissue necrosis and apoptosis.4.After 24 hours of tail vein injection of Carbon-MMT-PEG,the ocular venous blood of mice was collected to monitor liver and kidney functions(ALT,AST,ALP,CREA).Biosafety of Carbon-MMT-PEG was evaluated by hemolysis reaction,mouse body weight change and HE staining of major organs(heart,liver,spleen,lung,kidney).Results: 1.MTT cell proliferation experiments showed that,compared with PTT or CDT alone,Carbon-MMT-PEG could obviously synergize CDT with PTT to achieve the best inhibitory effect on tumor cell growth.2.Carbon-MMT-PEG can generate ROS at the cellular level by confocal fluorescence microscopy.3.In the mouse prostate cancer subcutaneous tumor model,compared with the PBS group,the synergistic effect of Carbon-MMT-PEG in inhibiting tumor growth is better than that of PTT or CDT alone,and can significantly prolong the survival time of mice.HE and Tunel Staining resulted in tumor cell necrosis as well as induction of apoptosis.3.In the mouse prostate cancer subcutaneous tumor model,compared with the PBS group,there was no statistically significant difference in the liver and kidney function indexes(ALT,AST,ALP,CREA)of the Carbon-MMT-PEG group.The hemolysis reaction induced by-MMT-PEG is basically negligible.5.After the treatment,there was no significant difference in the body weight of the mice in each group,and no obvious necrosis of the main organs was found by HE staining.Conclusions: 1.In vitro and in vivo experiments show that Carbon-MMT-PEG can inhibit the growth of mouse prostate tumor and prolong the survival period of mice through PTT and CDT.2.Carbon-MMT-PEG has good biological safety.3.Carbon-MMT-PEG can be used as an integrated platform for multimodal diagnosis and treatment,providing a new treatment option for prostate cancer precision medicine.