Study On The Anti-Tumor Effects Of A Triple Drug Co-Delivery System Based On Chemoimmunotherapy

Metal complexes,represented by platinum drugs,are an important class of chemical antitumor drugs,and metal complexes have become one of the hot spots in anticancer drug research.Among many transition metals,Cu²⁺has good coordination properties and the copper complexes demonstrate excellent anticancer activity and are considered to be the best choice to replace platinum-based drugs;however,like other classes of chemotherapeutic drugs,these drugs have low uptake rates and resistance due to the metal ion homeostasis mechanism of their free drugs in cells.To address this issue,in this study,we constructed a co-delivery system for tumor combination therapy based on chemoimmunotherapy and chemosensitization effects.The targeted delivery of Cu²⁺complexes to tumor cells can induce immunogenic cell death by triggering ROS through the Fenton effect;Olaparib（Ola）selectively acts on nuclear PARPase after delivery to the nucleus,synchronizing with the cytotoxic effect of copper complexes and inhibiting the repair of DNA damage of Cu²⁺that occurs in tumor cells,achieving chemosensitization.In addition,Polyethylenimine（PEI）,a cationic gene vector for co-loading olaparib and Cu²⁺,can condense PD-L1 trap plasmids for targeted delivery and intracellular in situ and transient expression to achieve PD-1/PD-L1 pathway blockage.With the synthesis of the above vector system,it is hoped that the shortcomings of metal complexes can be overcome to some extent,providing a new idea to improve the combined anti-tumor therapy based on metal chemotherapy treatment.Chapter 1 firstly reviewd the main effects of Ola and the existing research prospects;secondly,it showed the recent progress in the combination of Ola with other drugs,and an innovative and implementable synthetic pathway was designed after extensive reading of the relevant literature in order to achieve the chemical linkage between Ola and PEI.Chapter 2 showed the synthesis and characterization of the three-drug co-delivery system.First of all,the main pharmacodynamic moiety of chemotherapy sensitizer Ola was condensed with low molecular weight PEI by aldehyde amine to obtain Ola-PEI（OP）,which was a derivative hydrolyzable at 37°C.Then,a bifunctional peptide containing TAT and NLS fusion sequence with targeting cell nucleus and promoting nuclear transport was designed and synthesized,which was named R15,and R15 was modified on the surface of OP to obtain Ola-PEI-R15（OPR）;then,Cu²⁺ligated OPR was used to The novel metal complex Cu@Ola-PEI-R15（Cu@OPR）with gene carrier function was obtained,and content of Cu²⁺was measured by inductively coupled plasma mass spectrometry（ICP-MS）as 3.73%.The results of gel electrophoresis experiments showed that Cu@OPR could perfectly condense PD-L1 trap plasmid when the mass ratio（Cu@OPR:p PD-L1 trap）was 1.0;finally,for long circulation and tumor targeting in vivo,Cu@Ola-PEI-R15/p PD-L1 trap（Cu@OPR/P）used tumor cell membrane Encapsulation of 4T1/Cu@Ola-PEI-R15/p PD-L1 trap（4T1/Cu@OPR/P）was obtained,and transmission electron microscopy（TEM）and dynamic light scattering（DLS）images showed that the final complex was a spherical shape with a particle size of 115.8 nm and a zeta potential of-2.4 m V.Western blotting experiments as well as co-localization results from cellular uptake demonstrated that the nanoparticles were successfully encapsulated and that the cell membrane was able to deliver the drug efficiently into the tumor cells.The hemolysis experiments showed that the hemolysis rate of 4T1/Cu@OPR/P complexes was less than 3%,which had a certain biosafety.Chapter 3 showed the in vitro antitumor effects of the three-drug co-delivery system.4T1/Cu@OPR/P complex was co-incubated with 4T1 cells,MSC cells,A549cells and RAW264.7 cells,and the results showed that 4T1/Cu@OPR/P complex had significant homologous targeting ability and good macrophage escape ability;the results of cytotoxicity assay and apoptosis assay showed that the Cu@OPR complex was able to trigger apoptosis and Ola had a chemosensitizing effect.The results of dissolved oxygen and intracellular hydrogen peroxide assay showed that Cu@OPR complex could catalyze the decomposition of H₂O₂,reduce the intracellular H₂O₂content to produce O₂,and alleviate the hypoxia of tumor microenvironment.The results of ROS and ICD experiments showed that the Cu@OPR complex was able to trigger a significant rise in intracellular ROS,while inducing ICD and promoting the upregulation of ICD markers such as calreticulin（CRT）,high mobility group protein（HMGB-1）and ATP.In addition,the 4T1/Cu@OPR/P complex was able to enhance the efficiency of transfection of GFP reporter genes as well as PD-L1 trap plasmids to some extent under the dual conditions of bifunctional peptide R15 and cell membrane encapsulation.Chapter 4 showed the in vivo antitumor effects of the three-drug co-delivery system.In vivo experiments in tumor-bearing mice showed that the 4T1/Cu@OPR/P complex showed good tumor suppression,Ola exhibited significant chemo-sensitization,and the encapsulation of 4T1 cell membrane prolonged the drug circulation and successfully targeted intracellularly;lung metastasis assay and T cell infiltration assay showed the potential of the complex to suppress lung metastases and enhance the infiltration of CD8⁺T cells in tumor tissues.In summary,based on the strategy of combining chemotherapy and PD-1/PD-L1blockade,this study constructed a cancer cell membrane-encapsulated Cu@OPR/P co-delivery system based on bionic camouflage,homologous adhesion,nucleus targeting,nucleoplasmic transport,in situ blockade,metal ligand,environmental response,and synthetic lethality to address the co-delivery problem,with the aim of delivering to the same tumor cells in vivo in a certain ratio and simultaneously.This study aimed to better exploit the synergistic effect of chemoimmunotherapy,to lay the foundation for solving the current problems of chemoimmunotherapy and its practical application,and to provide a new idea for multi-drug combination anti-tumor therapy.