Proliferative Inhibition Of AIPC Cells By AR SiRNA Loaded Nanoscale Microbubbles Under Ultrasonic Irradiation In Experimental Study

Background:Prostate cancer is one of the most prevalent malignancies around the world. Mostcases treated by androgen ablation ultimately relapse to more aggressive AIPC which hasno effective method to cure as yet. Previously, it has been reported that the androgenreceptor plays a critical role in the progression of the disease. It has been shown thattransfection of AIPC cells with the siRNA targeting androgen receptor (AR) gene caninhibit the growth of human AIPC cells expressing AR in vitro. Nevertheless, thetherapeutic effect of gene therapy for AIPC has been unsatisfactory like other cancers,primarily due to lack of a safe, efficient, controllable in vivo delivery systems available todeliver this siRNA to tumor tissue intravenously, which limits the therapeutic effects ofsiRNA. Ultrasound microbubbles is a novel approach for an in vivo gene delivery systemdeveloped quickly in recent years. Ultrasonic microbubble-mediated gene therapy canfacilitate gene transfection in cancer cells, while also achieve the goal of targeted genedelivery. Therefore, it is ultrasound microbubble that is superior to other gene deliverymodalities, such as viral vectors, liposomes and electroporation, owing to the merits of itssafety, effectiveness and strong targeting function. With the development of nanomedicineand ultrasonic contrast-enhanced technology, the particle size of an ultrasound microbubblewill necessarily be measured in nanometers. Compared with micro-scale ultrasoundmicrobubbles, nano-scale microbubbles have excellent properties, such as goodpenetrability, long half-life time in blood circulation and high efficiency of genetransfection. Nano-scale microbubbles are promising for targeted tumor imaging andtherapy. Relevant studies of nano-scale microbubbles have become an intriguing issue inultrasonic molecular imaging over the recent years. Objectives:Based on these findings, we hypothesized the AR siRNA-loaded nanoscalemicrobubble as a novel modality for gene therapy, investigated its property in vitro and invivo, transfected prostate cancer cells under the condition of ultrasonic irradiation andestimated the effect of AR siRNA inhabiting the expression AR gene and the cell growth.Thus we provided a basis for using nanoscale microbubbles as an in vivo delivery systemfor tumor treatment, and AIPC gene therapy.Methods:1. Three pairs of DNA template coding siRNA against AR were designed andsynthesized, which were transfected using liposome mediated gene transfer into C4-2cellsto determine the optimal concertration. RT-PCR and western blot methods were appliedrespectively to detect the AR mRNA and protein expression level. The cytoactive wasevaluated with CCK-8analysis and cell growth curve, and screened the most effectivesiRNA sequence.2. Blanked nanoscale microbubbles were prepared using mechanical vibration andlow-speed centrifugation method. AR siRNA-loaded nanoscale microbubbles wereprepared using the Poly-L-Lysine approach, and measured particle diameter, surface zetapotential and the binding capacity. The coupling of AR Cy3-siRNA and microbubbles wereobserved under fluorescence microscopy. The contrast-enhanced performance of ARsiRNA-loaded nanoscale microbubbles was investigated in prostate cancer xenografttumors compared with the conventional microbubbles (SonoVue).3. We made use of AR siRNA-loaded nanoscale microbubbles to transfectLNCap,C4-2and PC-3prostate cancer cells combined with ultrasonic irradiation. In orderto acquire the optimal condition of ultrasound irradiation and the optimal concentration ofnanoscale microbubbles, we used fluorescence microscopy and flow cytometry to evaluatethe effect and efficiency of the cell transfection, and the cell proliferation assay wasanalysed by CCK-8. The levels of AR mRNA and AR expression were measured byRT-PCR and Western blot respectively after the AR-siRNA silencing AR gene.Results:1. The AR mRNA and protein expression level decreased significantly in C4-2cellstransfected by AR siRNA compared with the control groups and the C4-2cytoactivity wassuppressed significantly in the groups transfected by AR siRNA with the optimum concentration100nM.2. The mean particle diameter was (608.2±13.3) nm, and the Zeta potential was(-29.9±6.5) mV. Fluorescence microscopy showed the tight binding of AR siRNA to themicrobubbles. The siRNA loading increased with increasing siRNA-added concentrations.Compared to SonoVue, nanoscale microbubbles showed superior in vivo contrast-enhancedperformance with significantly prolonged visualization time (P<0.05) and better penetrationinto tumor tissue especially regions of lack of the blood vessels.3. The optimum conditions of ultrasound irradiation in vivo: MI=1.2, Durationtime=2mim，microbubbles counts/cell numbers=100:1(siRNA loading=18.94×10-9nmol/microbubble). About24hours post-transfection，we observed Morphology changes both inC4-2and LNCap cells, and after48h，this changes became obviously， such as outline blur,form atrophy, slow growth of the cells and so on，On the other hand, PC-3cells have notchanged significantly at the same time in the six groups. The growth inhibition effects ofthe three groups including nude AR siRNA+ultrasound irradiation, nude AR siRNA+naoscale microbubbles+ultrasound irradiation and AR siRNA loaded nanoscalemicrobubbles+ultrasonic irradiation of C4-2and LNCap cells were obvious. The highestproliferation inhibition rate obtaining from the group of AR siRNA loaded nanoscalemircobubble+ultrasound irradiation in C4-2cells was64.7%by CCK-8method and has aremarkable statistic difference with other groups(P<0.05). The AR mRNA and ARexpression were downregulated in the above-mentioned three groups of C4-2and LNCapcells by RT-PCR and Western blot. The most obvious downregulation was obtained in thegroup of AR siRNA loaded naoscale mircobubble+ultrasound irradiation in C4-2andLNCap cells.Conclusion:1. The novel AR siRNA can inhibit C4-2cellular proliferation by down-regulatingandrogen receptor expression.2. The novel AR siRNA-loaded nanoscale microbubble which was prepared with thePoly-L-Lysine approach has small particle size, high siRNA loading capacity and superiorcontrast-enhanced performance in xenograft tumors in vivo,which provides anexperimental basis for the nanoscale microbubbles mediated release of siRNA.3. We transfected AR siRNA to prostate cancer cells under ultrasound irradiation using nanoscale microbubbles as gene delivery vectors. It is observed that the expression ofAR gene was downregulated and the proliferation of prostate cancer cell LNCap and C4-2cells was inhabited efficiently.