In this study, we first identified three effective MDR1 siRNAs from four candidate siRNA sites by qRT-PCR. The three siRNA plasmids were pooled at an equal molar concentrations

and transfected into L2-RYC cells. All three siRNAs were specific for MDR1 target gene but at different mRNA degradation sites, so increased the target gene knock-down efficiency of random-designed siRNAs. The decreased concentration of individual siRNAs could reduce potential off-target effects. Our result confirmed that the pooled siRNAs have higher inhibition efficacy than that of potent individual siRNAs. Effective siRNA DNA delivery into cells and in vivo has been a great challenge for the broad use of RNAi therapeutics. The most commonly used JQ-EZ-05 in vivo carriers for delivering nucleic acids into mammalian cells are non-viral and viral vectors. Liposome-mediated Luminespib transfection is simple and powerful, but has cytotoxic side effects [26]. Calcium phosphate co-precipitation has rigorous conditions of transfection and a small range of target cells [42, 43]. Virus-mediated transfection is high efficient and available to achieve sustainable transgene expression. However the

biosafety for in vivo use remains a concern [44]. Recently, ultrasound contrast agents (in a form of microbubble) have been used to deliver gene and drug in vitro and in vivo, providing a new and efficient therapeutic technique [22–25]. Ultrasound microbubble-mediated destruction has been shown to enhance cell membrane permeability and improve gene and drug delivery. It has been shown that ultrasound microbubble-mediated destruction can transfect DNA into a variety of mammalian cells [22, 24, 26, 45]. The change of cell membrane permeability is recoverable when ultrasound energy and exposure time are within a suitable range. Thus ultrasound exposure will not cause permanent damage to cells [45, 46]. We first determined the optimal ultrasound parameters of acoustic intensity and exposure time for L2-RYC cell transfection. When cultured L2-RYC cells

were exposed to ultrasound with intensity Unoprostone of 0.75 W/cm2 and 1 W/cm2, the survival rates was too low to be used in the study. Although ultrasound with intensity of 0.25 W/cm2 did not affect cell viability, plasmids DNA delivery into cells was poor. Fortunately, we found out ultrasound with intensity of 0.5 W/cm2 for 30 s could effectively transfect plasmids into cells without causing significant amount of cell death. Our previous study on bone marrow mononuclear cells also reported gene delivery by ultrasound with intensity of 0.5 W/cm2 did not reduce cell viability and not destroy membrane of treated cells [45]. Under the chosen condition, we found that 30% GFP-positive cells can be achieved by gene transfection using ultrasound microbubble-mediated delivery.