Objective: Ultrasound targeted microbubble destruction(UTMD) has become a novel gene/drug delivery method in cancer therapeutic application. However, the gene transfection efficiency mediated by UTMD is still unsatisfactory. Here, we introduced a dual-targeted cationic microbubble(MBi RGD/CCR2) which was modified with PEI-600 and coated with i RGD peptides and anti-CCR-2 antibodies to improve the gene delivery efficiency and to be used for ultrasound molecular imaging-guided gene therapy of breast tumor.Method: The cationic MBi RGD/CCR2 were prepared through linking the anti-CCR-2 antibodies to the surface of the biotinylated cationic MBi RGD which was first fabricated by using of DSPC/DSPE-PEG2000/DSPE-PEG2000-i RGD/Stearic-PEI600(8.5 : 0.5 : 0.5 : 0.5 in molar ratios). The size, zeta-potential and i RGD/CCR2 amounts were characterized and the p GPU6/GFP/Neo-sh AKT2 plasmid was chosen for testing its DNA loading capacity and anti-tumor effects. The cell targeting ability of MBi RGD/CCR2 was examined by incubating them with HUVECs and MCF-7 breast cancer cells in vitro. Taking advantage of ultrasound targeted microbubble destruction technology, we compared the gene transfection efficiency of MBi RGD/CCR2 with untargeted cationic microbubbles(MBcontrol) and single-targeted cationic microbubbles(MBi RGDand MBCCR2) with flow cytometry. The knock-down of AKT2 gene was confirmed by western blotting. The cell viability and cell cycle arrest were examined by CCK-8 assays and flow cytometry. The tumor model of subcutaneous breast cancer in nude mice was developed to determine the in vivo ultrasound molecular imaging and gene therapy effects.Results: Our results showed that MBi RGD/CCR2 had no significant difference from the non-targeted MBcontrol and single-targeted MBi RGD or MBCCR2 in the bubble size and zeta-potential. The DNA loading capacity of MBcontrol and MBi RGD/CCR2 were significantly higher than that of MBnegative, with 27.85 ±1.56 ?g or 28.33 ±1.45 ?g per 5×108 microbubbles, indicating that the presence of the i RGD peptide and CCR2 antibody did not affect the DNA loading capability of the cationic bubbles. The cell targeting experiments showed the dual-targeted MBi RGD/CCR2 had a significant higher affinity to HUVECs in vitro than that of MBi RGD and MBCCR2. The gene transfection efficiency of MBi RGD/CCR2 combined with ultrasound was higher than that of MBi RGD, MBCCR2 and MBcontrol(P<0.05), resulting in a significant lower cell viability, stronger G1 arrest in the cell cycle in vitro(P < 0.01). These results were confirmed by western blotting experiments, revealing a significant decreased expression of AKT2 proteins. In the in-vivo experiments, a significant ultrasound molecular imaging effect was observed in the tumor of the mice which received with MBi RGD/CCR2 that of MBi RGD, MBCCR2 and MBcontrol(P<0.01). After loading the theranostic gene and combining with ultrasound irradiation, MBi RGD/CCR2 showed significantly stronger anti-tumor effects in these tumor-bearing mice than that of MBi RGD, MBCCR2, and MBcontrol. This was confirmed by histological analysis.Conclusion: In summary, our study provided a novel dual-targeting cationic contrast agent and plasmid carrier(MBi RGD/CCR2), which can be used not only for ultrasound molecular imaging, but also for targeted gene therapy of breast tumor. It would have a potential value to act as an ultrasound imaging probe for ultrasound image-guided tumor gene therapy. |