Objective:To prepare the resiquimod-loaded lipid microbubbles R848-MBs, evaluate their enhanced ultrasound imaging and high intensity focused ultrasound (HIFU) ablation effects, and explore their ability to improve tumor immune microenvironment synergize with HIFU.Methods:R848-MBs were prepared by the thin film hydration-mechanical shock method; The basic characteristics and safety of R848-MBs were detected, the HIFU controlled-release characteristics were verified in vitro and the drug metabolism and biological distribution were investigated in vivo. The ability of enhancing ultrasound imaging was observed in vitro and in vivo. To investigate the enhanced HIFU ablation effect of R848-MBs, six EMT6 tumor-bearing mice were randomly divided into HIFU group and R848-MBs+ HIFU group, three mice in each group, the changes in contrast average sound intensity before and after ablation in mouse tumor areas and the change of ultrasound image gray value in tumor area were evaluated, the tumor were resected to observe the coagulative necresis by TTC staining and HE staining. Forty-five tumor-bearing mice were randomly divided into control group, Free R848 group, HIFU group, Blank-MBs+ HIFU group and R848-MBs+ HIFU group, nine mice in each group. On the third day after treatment, 3 mice in each group were randomly selected and killed, to evaluate the ability of R848-MBs to improve tumor immune microenvironment synergize with HIFU. The expression level of CRT on the surface of tumor cells were detected by immunofluorescence staining, the proportion of mature DC in lymph nodes, spleen, and CD8 + T cells in spleen were detected by flow cytometry. The treatment effectiveness of each group( n=6) were evaluated by measuring tumor volume, observing and drawing survival curves. Results:The R848-MBs lipid microbubbles with good safety were successfully prepared, with a concentration of 2.58×10 9/ml, as spherical bubbles under optical microscope and laser confocal microscopy, in a particle size of (1.72±0.11)μm, at a surface potential of (-10.16±0.73)mV. The cumulative drug release was up to 83.44% after HIFU (90 W, 3 s) in vitro. The concentration of R848 in plasma decreased rapidly, and the drug concentration in tumor tissue of the R848-MBs+ HIFU group was higher than that of the R848 group 24 hours after treatment ( P<0.01). The ultrasound imaging of R848-MBs was significantly enhanced in contrast mode in vitro and in vivo; R848-MBs can significantly enhance the HIFU ablation effect, the contrast average sound intensity change in the tumor area before and after ablation in the R848MBs+ HIFU group was greater than that in the R848 group ( P<0.05), and the immediate ultrasound grayscale value change in the HIFU+ R848-MBs group was 46.34±3.21, which was significantly greater than that in the HIFU group (10.67±1.53), with statistical significance ( P<0.000 1). Coagulation necrosis was observed in tumor HE staining and TTC staining. The results of treatment efficacy in vivo showed that R848-MBs+ HIFU group had the strongest therapeutic effect, and R848-MBs combined with HIFU treatment could significantly prolong the survival period of mice compared with intravenous injection of free R848 ( P<0.01). Immunofluorescence staining and flow cytometry results showed an increase in the expression level of CRT on the surface of tumor tissue in the R848-MBs combined with HIFU group, and the percentage of mature DC in tumor draining lymph nodes (58.53±1.04)% were significantly higher than those in the HIFU group (37.56±2.13)% ( P<0.001), and the percentage of mature DC in the spleen (70.65±1.91)% were significantly higher than those in the HIFU group (36.46±3.89)% ( P<0.001), the percentage of CD8 + T cells in the spleen (27.46±3.04)% was significantly higher than that in the HIFU group (18.69±0.29)% ( P<0.01). Conclusions:The HIFU controlled-release lipid microbubbles R848-MBs can not only enhance the efficiency of HIFU ablation, but also improve the tumor immune microenvironment.