Study On The Combination Of MoSe₂ Nanomaterials With Abraxane For The Synergistic Photothermal Therapy And Chemotherapy In Pancreatic Ductal Adenocarcinoma

Part I:Establishment and identification of pancreatic cancer PDX modelObjective:To establish Patient-derived xenograft(PDX model),and to identify the successful establishment of pancreatic cancer PDX model through a variety of detection techniques,and to provide a reliable in vivo experiment for Synergistic photothermal and chemotherapy.Methods:67 cases of fresh tumor tissue from pancreatic cancer patients were planted on immunodeficient BALB/C nude mice to construct the PDX model.HE staining and immunohistochemistry were used to evaluate the consistency of tumor tissues from PDX nude mice and patient tumors.Proteomics and metabolomics detection technologies were used to compare the protein expression and metabolic changes of serum and tissue between different generations of mice to verify the stability of tumor biology during the passage of the PDX model.Results:29 cases of pancreatic cancer PDX models were successfully constructed(rate:43.28%).HE staining,?SMA,and vimentin immunohistochemical comparison showed that the primary tumor had consistent pathological characteristics with the tumor tissue samples of each generation of PDX models.(LC-MS/MS)detected the expression of proteins in nude mice of P1 and P3 generation PDX models.The correlation was 98%.NMR detection of serum metabolic changes were similar between generations.Conclusion:We have successfully constructed pancreatic cancer PDX model in nude mice.And the pathology,proteomics,and metabolomics analysis had verified its stability and consistency with the primary tumor,and It would be suitable for photothermal/Chemotherapy and anti-tumor research.Part II Successful preparation of photothermal/chemotherapy nanocomposite Abraxane@MoSe₂Objective:We plan to prepare the photothermal/chemotherapy dual-mode nanocomposite Abraxane@MoSe₂ and study its physical and chemical properties,photothermal conversion ability and synergistic photothermal/chemotherapy in vitro,so as to provide a basis for further verification of its photothermal/chemotherapy synergistic effect in PDX model of pancreatic cancer.Method:It is planned to use an ultrasonic-assisted liquid stripping method to directly prepare MoSe₂ nanosheets in aqueous solution,and to combine with Abraxane into a dual-mode nanotherapeutic agent Abraxane@MoSe₂ through electrostatic interaction.Using transmission electron microscope(TEM),X-ray photoelectron spectroscopy(EDS),DLS dynamic light scattering instrument,Fourier infrared spectrometer(FT-IR),X-ray photoelectron spectroscopy(XPS),ultraviolet-visible spectrophotometer,Fluorescence spectrometer to study the morphology,structure,size distribution,potential,material composition,solution dispersibility,solution stability and spectroscopic characteristics of Abraxane@MoSe₂,we verified its synergistic photothermal/chemotherapy antitumor effect in vitro.Results:(1)The prepared Abraxane@MoSe₂ NS is a two-dimensional sheet structure with a particle size of about 100-400 nm,the layer thickness of about 3.8±2.6 nm,and a potential of-10.04 m V.TEM,FT-IR,and ultraviolet-visible near-infrared absorption spectroscopy proved that the Abraxane@MoSe₂ composite was successfully prepared.The nano reagent has strong near infrared absorption and a high light-to-heat conversion efficiency of about 43.47%.(2)In vitro cell experiments showed that Abraxane@MoSe₂ has negligible cytotoxicity,and it could effectively kill BXPC-3 and PANC-1 cells under the irradiation of near infrared laser(808 nm,1W/cm2,5min).CCK-8 experiment showed that the survival rate of pancreatic cancer cells BXPC-3,PANC-1 and HUVEC cultured in high concentration MoSe₂NS(1000?g/ml)composite nanomaterials for24 hours was higher than 90%.After 808nm laser irradiation,the temperature of BXPC-3 cells treated by Abraxane@MoSe₂ reached 46.1?;The results of CCK-8and calcein AM/PI cells living/dead staining showed that Abraxane@MoSe₂composite nano reagent effectively killed pancreatic cancer cells,and the cell death rate was over 80%;The results of annexin V-FITC/prodidium iodide flow cytometry showed that Abraxane@MoSe₂ nanocomposite could effectively induce apoptosis of BXPC-3 cells,the rate of apoptosis or necrosis was as high as 87.6%;annexin V-FITC/prodidium iodide flow cytometry showed that Abraxane@MoSe₂nanocomposite could effectively induce apoptosis of BXPC-3 cells,and the rate of apoptosis or necrosis was 87.6%;Conclusion:(1)The prepared Abraxane@MoSe₂ NS is a two-dimensional sheet-like nanomaterial.Transmission electron microscopy,infrared spectroscopy,and ultraviolet-visible near infrared absorption spectroscopy prove that the Abraxane@MoSe₂ composite is successfully prepared.(2)The nano reagent has strong nearinfrared absorption and high light-to-heat conversion efficiency.(3)In vitro experiments show that MoSe₂ has almost no cytotoxicity,and the synthesized Abraxane@MoSe₂ has good biocompatibility and can play an anti-tumor effect through photothermal/chemotherapy Synergistic treatment.Part III Abraxane@MoSe₂ for Synergistic Photothermal therapy and Chemotherapy in PDX Model of PDACObjective:The human-derived pancreatic cancer tissue was used to establish a PDX model(subcutaneous xenograft model)to study Abraxane@MoSe₂ organ enrichment,tumor enrichment,and its combined tumor photothermal/chemotherapy antitumor efficacy,to provide a new idea for the comprehensive treatment of pancreatic cancer and to evaluate the in vivo safety of Abraxane@MoSe₂.Method:(1)We used normal BALB/C mice as a model.After injecting Abraxane@MoSe₂through the tail vein.We performed blood routine and liver and kidney function tests,then killed the mice and collected the main organs of the mice(heart,liver,spleen,lung,kidney),and conducted the pathological examination(H&E staining)to investigate the in vivo bio-tissue compatibility of composite nanomaterials.(2)We used human-derived pancreatic cancer tissue to establish a PDX model(subcutaneous transplantation tumor model),and small animal fluorescence imaging technology to study the distribution of Abraxane@MoSe₂ in the main organs of nude mice.We studied the fluorescence signal of Abraxane@MoSe₂ to tumors Changes in time to provide the best exposure time for laser photothermal therapy.We randomly divided pancreatic cancer PDX model mice into the PBS group,PBS+Laser group,Abraxane group,Abraxane@MoSe₂ group,Abraxane@MoSe₂+Laser group,and studied the anti-tumor effect of Abraxane@MoSe₂ photothermal/chemotherapy through tumor volume,animal weight,survival time,histopathology.Results:In vivo experiments(PDX model of pancreatic cancer patients),the thermal imaging camera showed that the temperature of the tumor site of the mice in the Abraxane@MoSe₂ composite nanomaterial treatment group increased to 53.7?after808 nm laser irradiation.The tumor volume of mice in 21 days was significantly reduced,the pathological sections were stained by HE and Ki67,and the cell proliferation of the tumor site of the Abraxane@MoSe₂+Laser group was significantly inhibited;TUNEL staining apoptosis detection showed increasing tumor apoptosis and smooth muscle Immunofluorescence analysis of actin(?-SMA)and vimentin(Vimentin)showed a significant decrease in tumor-associated fibroblasts.In addition,the mice in the treatment group had no significant change in body weight after 21 days of treatment.The pathological results of the main organs(heart,liver,spleen,lung,kidney)of the mice showed no obvious damage to the organs.In vivo and in vitro experiments have shown that Abraxane@MoSe₂ has an efficient tumor targeting effect,good biocompatibility and synergistic photothermal therapy.Conclusion:(1)Abraxane@MoSe₂ will not cause obvious liver and kidney dysfunction,no obvious damage to the main organs of nude mice,and has good biological safety.(2)Abraxane@MoSe₂ can achieve the enrichment of nanomaterials in tumors through the EPR effect,and at the same time,it can enhance the stroma destruction in pancreatic cancer PDX model mice under laser irradiation,thereby enhancing the Abraxane chemotherapy effect of pancreatic cancer and realizing the synergistic anti-tumor effect of photothermal/chemotherapy.