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Objective  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 α V β 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-α V β 3  integrin monoclonal antibodies into cationic microbubbles (CMBs αvβ3 ), and evaluated its killing effect in HCC cells.    Methods  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, α V β 3  integrin antibody was conjugated to CMBs, and CMBs αvβ3  was generated to specifically target to HepG2 cells. The morphology and physicochemical properties of the CMBs αvβ3  was detected by optical microscope and zeta detector. The conjugation of plasmid and the antibody in CMBs αvβ3  were examined by immunofluorescent microscopy and flow cytometry. The binding capacities of CMBs αvβ3  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 αvβ3  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 αvβ3 , HCC cells with CMBs αvβ3  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.    Results  CMBs αvβ3  had a regular shape and good dispersion. Compared to CMBs, CMBs αvβ3  had more stable concentrations of α V β 3  ligand and pEGFP-KDRP-CD/TK, and CMBs αvβ3  was much sticker to HepG2 HCC cells than normal liver L-02cells. Moreover, after exposed to anti-α V β 3  monoclonal antibody, the adhesion of CMBs αvβ3  to HepG2 cells and L-02 cells were significantly reduced. Also, CMBs αvβ3  demonstrated a substantially higher efficiency in pEGFP-KDRP-CD/TK plasmid transfection in HepG2 cells than CMBs. In addition, CMBs αvβ3  could significantly facilitate 5-FC/GCV-induced cell cycle arrest in S phase. Moreover, treatment of 5-FC/GCV combined with CMBs αvβ3  resulted in a marked apoptotic cell death in HepG2 and SK-Herp-1 HCC cells. In vitro, treatment of 5-FC/GCV combined with CMBs αvβ3  suppresed cell proliferation. In nude mice model, 5-FU + GCV combined with plasmid + CMBs αvβ3 were able to significantly suppress tumor volumes.    Conclusion  Through biotin-avidin mediation system, CMBs αvβ3  were successfully generated to specifically target HCC HepG2 cells. More importantly, CMBs αvβ3  could significantly facilitate 5-FC/GCV-induced cell cycle arrest and apoptotic cell death in HepG2 cells. Our study demonstrated a potential strategy that could be translated clinically to improve liver tumor gene delivery.

Effects of Cationic Microbubble Carrying CD/TK Double Suicide Gene and αVβ3 Integrin Antibody in Human Hepatocellular Carcinoma HepG2 Cells