Effects of Cationic Microbubble Carrying CD/TK Double Suicide Gene and [alpha].sub.V[beta].sub.3 Integrin Antibody in Human Hepatocellular Carcinoma HepG2 Cells

Hepatocellular carcinoma (HCC), mostly derived from hepatitis or cirrhosisis, is one of the most common types of liver cancer. T-cell mediated immune response elicited by CD/TK double suicide gene has shown a substantial antitumor effect in HCC. Integrin [alpha].sub.V [beta].sub.3 over expresssion has been suggested to regulate the biology behavior of HCC. In this study, we investigated the strategy of incorporating CD/TK double suicide gene and anti-[alpha].sub.V [beta].sub.3 integrin monoclonal antibodies into cationic microbubbles (CMBs.sub.[alpha]v[beta]3 ), and evaluated its killing effect in HCC cells. To improve the transfection efficiency of targeted CD/TK double suicide gene, we adopted cationic microbubbles (CMBs), a cationic delivery agent with enhanced DNA-carrying capacity. The ultrasound and high speed shearing method was used to prepare the non-targeting cationic microbubbles (CMBs). Using the biotin-avidin bridge method, [alpha].sub.V [beta].sub.3 integrin antibody was conjugated to CMBs, and CMBs.sub.[alpha]v[beta]3 was generated to specifically target to HepG2 cells. The morphology and physicochemical properties of the CMBs.sub.[alpha]v[beta]3 was detected by optical microscope and zeta detector. The conjugation of plasmid and the antibody in CMBs.sub.[alpha]v[beta]3 were examined by immunofluorescent microscopy and flow cytometry. The binding capacities of CMBs.sub.[alpha]v[beta]3 and CMBs to HCC HepG2 and normal L-02 cells were compared using rosette formation assay. To detect EGFP fluorescence and examine the transfection efficiencies of CMBs.sub.[alpha]v[beta]3 and CMBs in HCC cells, fluorescence microscope and contrast-enhanced sonography were adopted. mRNA and protein level of CD/TK gene were detected by RT-PCR and Western blot, respectively. To evaluate the anti-tumor effect of CMBs.sub.[alpha]v[beta]3, HCC cells with CMBs.sub.[alpha]v[beta]3 were exposed to 5-flurocytosine / ganciclovir (5-FC/GCV). Then, cell cycle distribution after treatment were detected by PI staining and flow cytometry. Apoptotic cells death were detected by optical microscope and assessed by MTT assay and TUNEL-staining assay. CMBs.sub.[alpha]v[beta]3 had a regular shape and good dispersion. Compared to CMBs, CMBs.sub.[alpha]v[beta]3 had more stable concentrations of [alpha].sub.V [beta].sub.3 ligand and pEGFP-KDRP-CD/TK, and CMBs.sub.[alpha]v[beta]3 was much sticker to HepG2 HCC cells than normal liver L-02cells. Moreover, after exposed to anti-[alpha].sub.