| Background:At present, hepatocellular carcinoma (HCC) is one of the most common malignant tumors with a high incidence and mortality in the world. This cancer severely threatens our health and needs to be diagnosed as early as possible, but differential diagnosis of HCC at its early stage is still difficult. With the progress of ultrasound contrast agents(UCA) and corresponding techniques, the importance of ultrasound contrast in diagnosis of liver tumors has been recognized increasingly. However, at present, the contrast agents used in diagnosis of liver tumors are non-targeted. Studies have demonstrated that human hepatocarcinoma specific monoclonal antibody(McAb) HAb18 could specifically bind to hepatocellular carcinoma cells and could be used for site targeting. If the antibody is used as a target carrier to prepare targeted ultrasound contrast agent, the contrast agent could assemble at a higher concentration of hepatoma tissues and the aim of target imaging could be achieved.Objective:First, to prepare human hepatocellular carcinoma-targeted liposome contrast agent and investigate its immunological properties in vitro. Second, to evaluate the enhancement effect of this targeted contrast agent on HCC in nude mice model by video intensity analysis.Methods:1. Human hepatocarcinoma specific monoclonal antibody HAb18 was attached to the surface of self-made liposome microbubbles by electrostatic attraction to prepare the targeted liposome microbubbles. The combination of HAb18 with liposome microbubbles was confirmed by the slide agglutination test and immunofluorescent assay. Their immunological activity was measured by ELISA. Rosette formation test, rosette formation blocking test and immunofluorescent assay were used to identify the specific binding of the targeted liposome microbubbles to HHCC hepatoma cells, and cytotoxicity assay was usedto detect their effect on human hepatocytes.2. Tumor cells HHCC of human HCC were inoculated subcutaneously into nude mice to develop implantation tumor. The targeted liposome ultrasound contrast agent or the non-targeted liposome contrast agent was injected into 8 human HCC-bearing nude mices via caudal veins. The tumors were imaged using second harmonic mode. The images were assessed qualitatively by three independent observers and quantitatively using video intensity analysis including time-intensity curves to determine the contrast enhancement effect of HCC.Results:1. The targeted liposome microbubbles were positive in the slide agglutination test and immunofluorescent assay. ELISA indicated that the immunological activity of HAbl8 on the liposome microbubbles was similar to that of free HAbl8. HHCC cells were surrounded by the targeting liposome microbubbles to form rosettes, while the control cells were not. Proliferation of HHCC cells and normal human hepatocytes was not influenced by the targeted liposome microbubbles.2. The subcutaneous implanted nude mice model of human HCC was established successfully. The tumors imaging enhanced increasingly after injection of the HCC-targeted contrast agent. There were differences in enhancement imaging effect of tumors of different size, and the smaller tumors enhanced more obviously. The peak of the tumor enhancement imaging appeared at about 8 min after injection of the HCC-targeted contrast agent, while the peak appeared at about 10 s after injection of the non-targeted contrast agent. The peak-gray scales of the tumors were 39.545 ± 10.099 dB and 22.438 ± 5.108 dB respectively. The difference was statistically significant (P<0.01).Conclusions: The targeted ultrasound contrast agent with high specific biological activity was prepared successfully. The contrast agent can bind to human hepatocarcinoma cells specifically and effectively in vitro. The human HCC-targeted liposome contrast agent was able to enhance ultrasound imaging of the tumors in nude mice.
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