Experimental Study Of HER2 Targeted Lipid Nanobubbles Carrying HSV-TK Suicide Gene Combined With Ultrasound Targeted Microbubble Destruction Technology On Transplanted Tumor Of Gastric Cancer In Nude Mice

Objective: The purpose of this study is to verify the antitumor effect of human epidermal growth factor receptor 2(HER2)targeted nanovesicles containing herpes simplex virus thymidine kinase(HSV-TK)gene on HER2 positive gastric cancer xenografts in nude mice,At the same time,the transfection efficiency of nanobubbles and the effect of low-intensity focused ultrasound(LIFU)on nanobubbles were investigated.Methods: The NCI-N87 cell line with high expression of HER2 was selected for culture,and the cells were collected at logarithmic growth stage for tumorigenesis in nude mice.Two kinds of nude mice models(unilateral and bilateral tumor models)were constructed respectively.Nude mice with unilateral tumorigenesis were divided into four groups:group A,blank control group;Group B,HER2 targeted empty nanobubbles group(without LIFU);Group C,HER2 targeted nanobubbles containing HSV-TK gene group(without LIFU);Group D,HER2 targeted nanovesicles containing HSV-TK gene(with LIFU).Nude mice with bilateral tumorigenesis were divided into two groups: targeted group and non-targeted group.The pre-drug ganciclovir(50mg/kg * d)was injected intraperitoneally on the 3rd and 4th day after tumor seeding(tumor formation).On the10 th day after tumor seeding,nanobubbles were injected into the tail vein of each group,and within 30 minutes after injection,LIFU was used to irradiate the transplanted tumor,and ultrasound targeted microbubble destruction(UTMD)technology was used to induce the substances carried by nanobubbles to further transfer into tumor cells.Three days after the first injection of nanobubbles,another tail vein injection of nanobubbles and LIFU irradiation was performed.Finally,on the 18 th day after the tumor was planted,the animal was dissected and collected,and the transplanted tumor and organs were taken out for subsequent verification.Western-blot method was used to detect the protein expression to verify the transfection of nanobubbles and the expression of target protein.TUNEL method was used to observe the apoptosis of tumor tissue after treatment.The tumor was observed by HE staining.In addition,the biological safety of HER2 targeted nanobubbles loaded with HSV-TK gene was studied by injection alone.The contrast enhancement ability of nanobubbles was observed with the aid of ultrasonic diagnostic instrument.Results: In the treatment experiment of unilateral tumor animal model,the transplanted tumor of group A and group B in nude mice showed natural growth,and the growth rate of tumor volume was not significantly inhibited after tail vein injection,and the rate of weight loss of nude mice was not slowed down.After treatment,the tumor volume of group C and group D decreased significantly or the transplanted tumor stopped growing.The results of Western-blot experiment showed that there was no or only a small amount of TK1 protein expression in the tumors of group A and B,while the content of TK1 protein expressed by HSV-TK gene in group C and D was much higher than that of the first two groups,and the expression of group D was higher than that of group C without LIFU,with a statistically significant difference(P<0.05).The expression results of caspase-3 protein related to apoptosis also showed that the targeted gene-loaded nanovesicles combined with LIFU group had the strongest effect on apoptosis of transplanted tumor,and the difference between the groups was statistically significant(P<0.05).The difference between group C and group D indicates that LIFU can promote the apoptosis induced by nanobubbles.In the animal bilateral tumor model experiment,both the non-target group and the targeted group can inhibit tumor growth to some extent,but the inhibition effect of the targeted group is better than the non-target group,and the difference is statistically significant(P<0.05).The results of TUNEL method showed that the HER2 targeted HSV-TK gene nanovesicles constructed in the experimental group in the early stage could lead to certain apoptosis of gastric cancer xenografts with high HER2 expression in vivo,and the apoptosis rate of HCPAN group reached(19.58 ±0.69)%;In the HCPAN+LIFU group,the apoptosis rate was further increased to(25.39± 0.83)% under the effect of LIFU,and the difference between the groups was statistically significant(P<0.05).HE staining showed that nanobubbles had a good effect on tumor apoptosis,and had little impact on other organs,and their biological safety was verified.At the same time,the prepared nanobubbles have been proved to have the ability to enhance the contrast.Conclusion: This experiment has successfully confirmed that the HER2 targeted lipid loaded HSV-TK gene nanobubbles constructed by the research group in the early stage have a certain anti-tumor effect on the nude mice gastric cancer xenografts with high HER2 expression in vivo,can effectively transfect,inhibit its growth and promote its apoptosis,and the tumor inhibition effect of the nanobubbles under the effect of LIFU is better,the experimental results and the differences between groups have statistical significance(P<0.05).It lays a certain theoretical basis for the research of nanobubbles used in the integration of clinical diagnosis and treatment in the future.