The Experimental Research Of The Ultrasonic Cavitation Sensitize Chemotherapy Of Hepatic Carcinoma With RGD-Targeted And Loading DOX Nanobubbles

Objective: Hepatic carcinoma is a common malignancy of the digestive system.The treatment is based on surgery.However,only a few patients achieve surgery because of occult nature of the disease.Chemotherapy is the main palliative treatment for advanced hepatic carcinoma,The clinical application still remains limited because of its systemic side effects.In recent years,studies have found that ultrasound contrast agents can be used as carriers to achieve targeted drug delivery,thereby reducing the toxic side effects of drugs.Therefore,this article studies the preparation of RGD-targeted and doxorubicin-loaded nanobubbles to reduce the side effects of chemotherapy and to sensitize the advanced hepatocellular carcinoma.Methods:(1)Preparation of blank phospholipid nanobubbles(DP),RGD phospholipid nanobubbles(RDP)and RGD with DOX phospholipid nanobubbles(RDPD)were done by thin film hydration.Their morphological differences were observed under a microscope and the particle size distribution was detected by particle size detector.And the distribution of potentialwas noted,measurement of RDPD drug loading with the use of microplate reader and the rate of entrapment,evaluation of its drug carrying capacity was seen.(2)Evaluate the differences between DP nanobubbles,RDP nanobubbles and RDPD nanobubbles of ultrasound image in vitro.The stability of DP?RDP and RDPD nanobubbles in vitro was measured using the cell counting principle and the in vitro stability curve was drawn.(3)in vivo stability was measured and TIC curve determined,the peak intensity,time to peak and area under curve(AUC)of three contrast agents assessed,the differences between the three groups were compared..(4)Using cell culture techniques,the acute toxicity and cytotoxicity of DP,RDP and RDPD nanobubbles were examined and the biosafety of the above three nanobubbles was evaluated.(5)A nude mouse model ofhepatocarcinoma subcutaneous tumor was established,ultrasound diagnostic apparatus was used to evaluate the tumor targeting of DP,RDP and RDPD nanobubbles.The positive expression of ?v?3 integrin in the liver and subcutaneous tumors of normal nude mice was detected by immunohistochemical double staining.(6)Successfully modeled tumor-bearing mice were divided into 5 groups and were given the same dose of physiological saline,blank nanobubbles,RGD nanobubbles,RGD with DOX nanobubbles,and DOX solution.Tumor size and ultrasound elastography were compared before and after administration and histopathological differences.Results:(1)The blank phospholipid nanobubbles(DP),RGD phospholipid nanobubbles(RDP)and RGD-loaded DOX phospholipid nanobubbles(RDPD)were successfully prepared by thin-film hydration.The particle sizes were 292 nm,303 nm,and 322 nm.Their potentials were-3.69 mv,-2.69 mv,-2.68 mv.The RDPD drug loading was 3.02%,the entrapment efficiency was 71%,and the drug loading capacity was good.(2)Both DP,RDP and RDPD nanobubbles can achieve ultrasound Imaging in vitro,and the results are good.There was no significant difference in initial nanobubble concentration between the three groups(t=2.457P>0.05).(3)There was no significant difference in peak intensity,time to peak,and area under curve(AUC)for the three contrast agents of DP,RDP and RDPD(P=0.176,0.100,0.733>0.05).The difference between in vivo stability DP and RDP,RDPD was statistically significant(P=0.046,0.038<0.0.5).(4)DP and RDP had no obvious cytotoxicity,but DOX solution and RDPD were cytotoxic.There was a statistically significant difference in cytotoxicity between the DOX solution group and the RDPD group(P<0.001).DP and RDP had no obvious toxicity in vivo,and both DOX solution and RDPD had in vivo toxicity.The difference was statistically significant between the RDPD and DOX group in vivo toxicity(P=0.0493<0.05).Pathological findings showed that the DOX group had cardiotoxicity and the remaining pathological findings were normal.(5)The numerical value of db before and after cavitation in the RDP and RDPD groups were statistically significant(P=0.007,0.004<0.05).The numerical value of db before and after cavitation in the RDP and RDPD groups were statistically different from those in the DP group(P=0.032,0.008<0.05).The results of immunohistochemistry showed that the expression of ?v?3was positive in tumor tissues,and the expression of ?v?3 was negative in tumor tissues(6)There was a significant difference in tumor volume between the RDPD group and the DOX group compared with the control group(P<0.001),and there was a statistically significant difference in the tumor volume change between the RDPD group and the DOX group(P=0.008<0.05).The hardness of the tumor in RDPD group and DOX group was significantly different from that in control group(P=0.001,0.002 <0.05).The difference in hardness of RDPD group compared with DOX group was statistically significant(P=0.011 <0.05).The pathological results of tumors in each group showed that the tumor necrosis was most obvious in the RDPD group.Partial necrosis was seen in the DOX group,and necrosis was rare in the RDP group.No necrotic area was found in the DP group and the control group.Conclusions:1.The blank phospholipid nanobubbles,RGD-targeted nanobubbles and RGD with DOX phospholipid nanobubbles can be prepared by thin film hydration.The particle size distribution is uniform,and the stability and imaging effects are good in vivo and in vitro.The encapsulation rate is still good.Blank nanobubbles and RGD nanobubbles have no in vivo toxicity and cytotoxicity,but RGD-loaded DOX nanobubbles have strong cytotoxicity.Compared with DOX solution,its toxicity in vivo is lower,and attenuating chemotherapy drugs can be achieved in vivo.2.The expression level of av?3 receptor in the neovascularization of hepatocellular carcinoma is high,but the expression of av?3 receptor in normal liver tissue is extremely negative.The RGD-loaded peptide nanobubble carrier has liver cancer targeting and can target hepatic neovascularization.Combined with UTMD and ultrasound cavitation effects,targeted delivery of chemotherapeutic drugs can be achieved,thereby increasing local plasma concentration of the tumor.Plays a role in sensitization of advanced liver cancer chemotherapy.However,its mechanism of action still needs further study.