Synthesis of ultrasound contrast agent containing siRNA and chemotherapeutic drug for theragnosis and in vitro evaluation in prostate cancer model

Abstract

Introduction: Prostate cancer is the leading cause of mortality in male population. For androgen-refractory prostate cancer, targeted gene therapy using RNA interference as well as chemotherapy such as doxorubicin is being highlighted. Using small interfering RNA, specific proteins for tumor cell growth can be inhibited and cell apoptosis can be induced. For effective intracellular delivery of small interfering RNA and chemotherapeutic drug for prostate cancer cell, we synthesized nanocomplex in combination of ultrasound contrast agent, microbubble, and liposome to work as a carrier. We aimed to enhance intracellular delivery of small interfering RNA and chemotherapeutic drug via ultrasound-guided delivery of microbubble liposome complex. Methods: The human prostate cancer cell lines PC-3 and LNCaP were obtained from the manufacturer and incubated. Predesigned survivin-targeted small interfering RNA and doxorubicin were purchased. By mixing lipids and sulfur hexafluoride gas core, microbubbles and liposomes were made, and were shaken together to form the complex. The complex was conjugated with anti-Her2 antibody to target prostate cancer cells. The complex was fluorescent-labeled, incubated with prostate cancer cells, and then evaluated under confocal laser scanning microscopy for intracellular delivery. Next, small interfering RNA, doxorubicin and both were conjugated with the complexes, respectively, and incubated with prostate cancer cells to assess cell apoptosis induced by therapeutic agents. Concurrently, ultrasound exposure was additionally performed in selected cell lines to investigate the effect of ultrasound-guided delivery of the complex. Results: Under microscopy, significant intracellular uptake of the complex into LNCaP cells which expressed high level of Her2 was confirmed, suggesting effective target-specific delivery of the complex. In LNCaP cells with effective uptake of complex, small interfering RNA and doxorubicin conjugated complex showed significant increase in cell death after the exposure of ultrasound. Subgroup analysis of complexes with different conjugated therapeutics revealed that small interfering RNA plus doxorubicin conjugated complex showed the highest level of cell death after ultrasound guiding, followed by doxorubicin, and siRNA conjugated complexes in order. No significant cell death was confirmed without ultrasound exposure. Conclusions: Microbubble liposome complex can effectively target prostate cancer cell lines by the conjugation of anti-Her2 antibody. It can also effectively transfer small interfering RNA and doxorubicin into target cell via ultrasound guidance, resulting in significant cancer cell death. Therefore, it can provide promising method of targeted gene and drug delivery using microbubbles.