Research Of Doxorubicin Encapsulated By Microparticles Derived From Tumor Cells In The Treatment Of Liver Malignant Tumors

Rational:Under natural conditions or in response to stimuli,tumor cells can change their cytoskeleton,leading to encapsulation of cytosolic contents within cellular membrane to form vesicles that are subsequently released into extracellular spaces.Such subcellular vesicles with 100-1000nm sizes are called tumor cell derived microparticles（TMPs）.In tumor microenvironment,TMPs are common subcellular structures.TMPs not only participate in the transportation of biologically materials,but also play an important role in the exchange of information between cells.After special handled,TMPs can combine with drugs and participate in the transportation and delivery of drugs in body.Based on this,our study aims to explore the safety,therapeutic effect and partial mechanisms of TMPs as a new type of carrier to deliver the chemotherapy drug doxorubicin（DOX）in the treatment of liver malignant tumors.Methods:（1）Anti-tumor drug selection:we compared the killing effects of doxorubicin,cisplatin,5-fluorouracil and gemcitabine on different liver malignant tumor cells in vitro.（2）Preparation and characterization of doxorubicin encapsulated by tumor cell-derived microparticles（DOX-TMPs）:tumor cells are exposed to UVB for 1 hour,and then placed in a 37°C constant temperature incubator for 24 hours.The supernatant was collected and separated by differential centrifugation to obtain and purificat TMPs,and then DOX was added and incubated to obtain DOX-TMPs.The characterization of TMPs or DOX-TMPs including:the total number of TMPs produced by 1×10⁸ tumor cells after UVB irradiation and the particle size of TMPs and DOX-TMPs by NTA.（3）Safety study of TMPs as anti-tumor drug carrier:First,we study the effects of TMPs on cell viability of various liver malignant tumors in vitro;then,we injected TMPs into normal BALB/c mice intraperitoneally to observe whether the mice developed malignant ascites or other solid tumors.Furthermore,we injected TMPs into H22 BALB/c mice hepatocellular carcinoma ascites model to observe whether TMPs could promote ascites production,and we record the survival time of mice.（4）The killing effects of DOX-TMPs in vitro:First,PKH-26 labeled TMPs are co-cultured with tumor cells,laser confocal or flow cytometry are used to detect the uptake of TMPs by tumor cells;secondly,DOX-TMPs are incubated with various liver malignant tumor cells including H22,RBE,HepG2 and Huh-7,and then detects the killing effect of DOX-TMPs on tumor cells in vitro.（5）The efficacy and safety of DOX-TMPs in animal models:establish mice hepatocellular carcinoma ascites model or mice liver malignant subcutaneous tumor model by intraperitoneal injection or subcutaneous injection of H22 tumor cells,and then give corresponding interventions for consecutive five days.The main observation phenomenon is the volume of ascites or the volume of subcutaneous tumor.Construct mice hepatocellular carcinoma ascites model and give corresponding interventions for survival analysis.After treatment,venous blood of mice is tested for liver and kidney function.（6）Influence of DOX-TMPs on tumor immune microenvironment of mice hepatocellular carcinoma ascites model:Take mice ascites and label each immune cells,including tumor cells（CD45^-）,T cells（CD3⁺T cells,CD3⁺CD4⁺T cells,CD3⁺CD8⁺T cells）and macrophages（CD11b⁺F4/80⁺）,and then the absolute quantity or proportion of each group is detected by flow cytometry.Main results:（1）After expose to UVB irradiation,1×10⁸ tumor cells could produce approximately 8×10¹⁰-1×10¹¹ TMPs.The D10 of TMPs is about 134.2±1.5nm,the D90 of TMPs is about 350.8±6.6nm,and the average particle size of TMPs is about225.7±2.1 nm.The D10 of DOX-TMPs is about 130±3.6nm,the D90 of DOX-TMPs is about 350.8±10.2nm,and the average particle size of DOX-TMPs is about226.7±14.2nm.（2）Compared to other anti-tumor drugs,including CIS,GEM and5-FU,DOX has stronger inhibitory and killing effect on liver tumor cells.And TMPs have no significant effect on tumor cell activity in virto.Our experiment results show tumor cells could take up DOX-TMPs efficiently and then be killed.（3）Animal experiments show that TMPs do not have tumorigenic effects in normal BALB/c mice.And at the same time,TMPs neither promote the formation of ascites in mice hepatocellular carcinoma ascites model,nor will it affect the survival time of mice.In the animal model effectiveness experiment,our results show DOX-TMPs can effectively inhibit the growth of subcutaneous tumors and reduce the production of ascites.Survival analysis shows the survival period of mice in the DOX-TMPs intervention group is prolonged.（4）After DOX-TMPs treatment,the number of tumor cells in ascites was significantly reduced.And the results of the immune cell subsets in ascites showed that compared to the control group,the ratio of CD3⁺T cells,CD3⁺CD4⁺T cells,CD3⁺CD8⁺T cells and CD11b⁺F4/80⁺cells in the ascites of the DOX-TMPs group increased,and the difference was statistically significant.Conclusions:DOX-TMPs could be one of the comprehensive treatment options for liver tumors.The results of our experiments show that DOX-TMPs could kill various liver tumor cells in virto.In BALB/c mouse liver malignant tumor ascites and subcutaneous tumor models,DOX-TMPs can significantly inhibit tumor growth and have a good therapeutic effect.Analysis of the tumor immune microenvironment of mouse ascites found that after DOX-TMPs treatment,the ratio of lymphocytes and macrophages in mouse ascites increased.Our study uses TMPs as a biologically-derived carrier combined with traditional chemotherapeutics,and utilizes the killing effect of DOX-TMPs on tumor cells and has a certain regulatory effect on the tumor immune microenvironment,which is expected to provide a new strategy for the clinical treatment of liver malignant tumors.