Synergistic Treatment Of TACE And TARE For Rabbit Liver VX2 Tumor Based On ¹³¹I-labeled BaGdF₅@PDA Nanoparticles

Background:The incidence rate of hepatocellular carcinoma(HCC)is high in China.The prognosis of HCC is poor.Trans-arterial chemoembolization(TACE)is the first choice for unresectable HCC without metastasis.However,conventional TACE(c-TACE)and drug-eluting bead TACE(DEB-TACE)have high recurrence rates and unsatisfactory long-term efficacy.Radioactive drugs are injected into the hepatic artery by an interventional method in trans-arterial radioembolization(TARE).Radioactive drugs are trapped into the tumor and emitted?-ray for the treatment of tumors.As a novel trans-arterial therapy,TARE is promising to cooperate with TACE to improve tumor prognosis.Objective:To design and characterize the novel nanocomposites ¹³¹I-BaGdF₅@PDA-CDDP.Furtherly,the efficacy of synergistic treatment of TACE combined with TARE based on ¹³¹I-BaGdF₅@PDA-CDDP was explored in vivo and in vitro.Methods:This study includes three parts:(1)synthesis and characterization of ¹³¹I-BaGdF₅@PDA-CDDP nanocomposites,(2)evaluation of the synergistic effect of chemo-radiotherapy at the cellular level,(3)synergistic treatment of TACE combined with TARE in rabbit liver VX2 tumor,including the preparation of animal model,exploration of imaging conditions,implementation of synergistic treatment,and evaluation of therapeutic effect.The procedure is as follows:BaGdF₅ nanoparticles possessing CT/MR imaging function were synthesized by a hydrothermal method.BaGdF₅ nanoparticles were coated by polydopamine(PDA)and then loaded with cisplatin(CDDP)and radio-labeled with ¹³¹I to synthesize ¹³¹I-BaGdF₅@PDA-CDDP eventually.Cell counting kit-8(CCK-8)tests were used for evaluating the cytotoxicity of ¹³¹I-BaGdF₅@PDA-CDDP in hepatoma cell line Huh7.Cell immunofluorescence was used to observe the expression level of DNA double strands break related protein?-H2AX in normal oxygen conditions and in chemical simulated hypoxia conditions.The nanocomposites were emulsified with lipiodol.The mixture was injected into rabbit liver VX2 tumor via hepatic artery to realize the selective drug delivery and efficient embolization treatment.¹⁸F-FDG PET/CT was used to observe the changes of the mean value of standard uptake value(SUVmean)of the tumor lesions at3 and 5 days after treatment.The extent of cell apoptosis in tumor samples was analyzed by TUNEL immunofluorescence.SPECT/CT observed the deposition of ¹³¹I and the regional hypoxia in the tumor.MRI reflected the post-TAE deposition of BaGdF₅@PDA nanoparticles by the effect of T1 signal enhancement at the tumor site.Bio-TEM observed The locations of lipidol and nanoparticles in the tumor cell were observed by Bio-TEM.The biosafety evaluations of nanoparticles included the biochemical blood test and H&E pathological staining.Results:(1)The diameter of BaGdF₅ nanoparticles was about 10 nm,and the diameter of self-assembled BaGdF₅@PDA was about 160 nm.The X-ray attenuation coefficient of BaGdF₅@PDA was 4.73.The longitudinal relaxation rate in MRI imaging was 0.45.The loading efficiency of CDDP in nanoparticles was 10.3%.The radiochemical purity of ¹³¹I-labeled BaGdF₅@PDA was more than 90%.The radiolabeled stability in PBS was better than in fetal bovine serum,saline.(2)The results of CCK-8 experiment proved that the survival rate of Huh7 cells was 7.58%±1.06%after 48h incubation with ¹³¹I-BaGdF₅@PDA-CDDP.The results of cellular immunofluorescence showed that the fluorescence intensity of?-H2AX was 199.41±44.65 in BaGdF₅@PDA-CDDP group,171.16±44.40 in ¹³¹I-BaGdF₅@PDA group,higher than that in CDDP group(115.31±34.77)and ¹³¹I group(96.44±26.88).These results indicated the therapeutic effects of nanoparticles loaded with CDDP or labeled with ¹³¹I were better than that of single drug CDDP or ¹³¹I.In hypoxic conditions,the fluorescence intensity of?-H2AX expression in¹³¹I-BaGdF₅@PDA-CDDP group was 201.51±49.67,which was higher than that in other groups.(3)PET/CT were performed 5 days after TAE.The postoperative SUVmean of the tumor was 16.03%±5.67%of preoperative SUVmean in ¹³¹I-BaGdF₅@PDA-CDDP group,then followed by the BaGdF₅@PDA-CDDP group(32.96%±3.54%).Apoptosis of VX2tumor cells was evaluated by TUNEL,for ¹³¹I-BaGdF₅@PDA-CDDP group,the positive rate was 101.47%±4.77%,which was higher than that of BaGdF₅@PDA-CDDP(81.70%±3.40%)and ¹³¹I-BaGdF₅@PDA(46.64%±7.83%).SPECT/CT results showed the radioactive count of ¹³¹I in ¹³¹I-BaGdF₅@PDA-CDDP group was significantly higher than that in ¹³¹I-BaGdF₅@PDA group and ¹³¹I group at 72 hours post-operation.In BaGdF₅@PDA group and ¹³¹I-BaGdF₅@PDA-CDDP group,the postoperative MRI T1signal ratio of tumor to liver increased by 8.00%±1.29%,6.71%±1.42%,respectively.The biosafety of nanoparticles was acceptable.^99mTc-HL-91 hypoxic imaging preliminarily explored the tumoral hypoxia after TAE,which was significant than that of untreated VX2tumor.Conclusion:The nanoparticles ¹³¹I-BaGdF₅@PDA-CDDP were synthesized successfully.The synergistic therapeutic effects of TARE with TACE based on ¹³¹I-BaGdF₅@PDA-CDDP were confirmed preliminarily in Huh7 cell line and rabbit liver VX2tumor model.The results proved that ¹³¹I-BaGdF₅@PDA-CDDP had integrated functions of radiotherapy and chemotherapy combined with imaging successfully.