Experimental Study Of Antisense Oligodeoxynucleotides Transduction Mediated By Combined Ultrasound And Liposome Microbubbles

Objectives: To study the efficiency and safety of transduction of antisense oligodeoxynuleotides by liposome microbubbles mediated by ultrasound and to explore the mechanism of enhanced transduction by ultrasound combined with microbubles.Materials and Methods:1. in vitro experiment: Cultured human breast cancer cells in 12 holes plate were grouped to receive the following eight treatment separately, (1)ultrasound irradiation alone;(2)contrast agent alone;(3)HA2741 alone;(4)ultrasound irradiation and HA2741;(5)contrast agent and HA2741;(6)contrast agent and HA2741 and ultrasound irradiation;(7)liposome and HA2741;(8)liposome and HA2741and ultrasound irradiation. The probe emission frequency of ultrasound machine was 1.3 MHz with sound wave intensity of-18.0-0dB and exposure time of 5-240s The concentration of microbubbles was 0.5-30%.The concentration of oligodeoxynuleotides HA2741 was 12-200 nmol/L. After experimental treatment, the fluorescence microscope was used to dynamically observe the transferring rate of 5' -FAM -modified HA2741 and cell activity after PI staining. And immuno-histochemical staining was used to detect the expression of HER-2 protein and RT-PCR was used to detect the level of HER-2 mRNA. AFM was used to observe the shape change of the cellular membrane.2. in vivo experiment: healthy rats were divided into the following 5 groups: control group, simple cy5ODN group, contrast agent plus cy5ODN group, ultrasound plus cy5ODN group, ultrasound plus contrast agent plus cy5ODN group.With change of concentration of contrast agent and ultrasound exposure time, laser focal concentration microscope was used to detect expression of florescence, and RT-PCR was used to detect mRNA level of CTGF, and Westen Blot was used to detect protein expression products of CTGF, and serum GPT^ GOT> BU1SK Cr level was measured to evaluate the side effects of this method.Results:1. in vitro experiment: Transfection rate of HA2741 is 94.6% in group 6, 22.5 times of group 4, 30.5times of group 5, 8.3 times of group7. Transfection rate is highest at 2 %and 5% concentration of contrast agent. The increased contrast agent would lead to decreased cellular activity. The transfection efficiency is highest with emission intensity of - 6.0 — 3.0dB and exposure time of 30 ~ 60s. The HER-2 expression of breast cancer cells decreased with decreased protein expression of HER-2 protein expression in negative correlation with HA2741 transfection. No difference in cell shape among simple ultrasound irradiation group and contrast agent group and control group. Micro-holes of different size appeared on cell membrane in contrast agent plus ultrasound irradiation group which increased with elevated contrast agent and cell survival rate decreased significantly.2. in vivo experiment: cy5ODN transefection was significantly higher than other groups with significant decrease of mRNA level and protein expression of CTGF, and GPT and GOT were transiently elevated and no change of BUN and Cr.Conclusions: As a new type of gene introduction measure, ultrasound-activated microbubbles could significantly improve the transefection efficiency of gene and it is safe and easy to use with good targeting ability and should be used widely.