GSH/pH Dual Response Drug Delivery System For Photothermal Enhanced Gene Immunotherapy

Photothermal therapy(PTT)is a local tumor-killing method developed in recent years.It has the advantages of small invasion,high accuracy,and integration of diagnosis and treatment.However,the excitation wavelength of traditional photothermal agents is mostly in the ultraviolet or visible light region,and the penetration of tumor tissue is insufficient,which is only suitable for epidermal therapy,thus limit the application of PTT in tumor therapy.Therefore,researchers have designed a series of near-infrared photothermal agents to enhance the penetration depth of tumor tissues.Indocyanine green(ICG)can be used as an excellent photothermal agent for thermal imaging and fluorescence imaging-guided photothermal therapy due to its near-infrared absorption and fluorescence emission characteristics.In addition,PTT can cause tumor cell immunogenic death(ICD),so the combination of photothermal and immunotherapy strategies can be considered to ablate tumors and inhibit tumor recurrence by initiating anti-tumor immune response.Blocking the PD-1/PD-L1 signaling pathway by directly knocking out the overexpressed PD-L1 on the surface of tumor cells through small interfering RNA(si RNA)can effectively avoid tumor immune escape.Therefore,according to the characteristics of slightly acidic and high glutathione concentration in the tumor microenvironment,a dual-responsive nano-drug delivery system with reduction-responsive disulfide bonds and p H-sensitive imine bonds was constructed for ICG-mediated photothermal therapy and si PD-L1 mediated gene therapy.The specific research contents of this thesis are as follows:Firstly,P123-SS-PEI(PSP)was synthesized by amidation method and dialysis method.The structure,particle size and morphology were characterized by nuclear magnetic resonance,dynamic light scattering(DLS)and transmission electron microscopy(TEM).The release rate of ICG stimulated by p H of PSP and the release rate of si RNA under GSH condition were detected by UV.The photothermal performance in vitro was investigated by infrared thermal imager and the optimal photothermal treatment conditions were determined.MTT,dead/live cell staining and cell apoptosis experiments were conducted to explore the cytotoxicity of PSP/ICG/si PD-L1.The uptake efficiency of 4T1 cells was investigated by laser confocal and flow cytometry in cytology.Flow cytometry was used to explore the release of molecular models related to its damage and the maturation of dendritic cells.The 4T1 mouse model was constructed,and thermal imaging and fluorescence imaging experiments were performed to prove that the drug targeted cancer cells and accumulated in the tumor site.To evaluate the synergistic anti-tumor effect and biological safety of PTT/GT in vivo,the expression of calreticulin,high mobility group protein B1 and PD-L1 protein are investigated by immunofluorescence.The ability of PSP/ICG/si PD-L1 to activate tumor CD4~+and CD8~+T cells and to reduce the expression of Treg cells to initiate immune response was also studied by flow cytometry.In summary,this immunogenic nanomedicine treatment strategy is expected to achieve complete ablation of 4T1 in situ tumors.