Study On A Novel Ultrasound Contrast Agent Microbubble Based On CNTs Loading Drug And Targeting Property

Objective A novel multifunctional ultrasound contrast agent was designed and developed for targeting and chemotherapy based on carbon nanotubes drug delivery and targeting.By studying the preparation process,the loading rate of carbon nanotubes and paclitaxel in microbubbles,its anti-tumor activity in vitro,imaging performance in vitro and vitro,acute toxicity were respectively investigated.The preclinical study of composite microbubbles was systematically completed,which provided a safe and effective basic data for the clinical application of the novel multifunctional ultrasound contrast agent.Methods(1)Using sound vibration cavitation method,Span-PEG as membrane material,adding the complex folate-carbon nanotube-paclitaxel(FA-CNTs-PTX),the Span-PEG microbubble ultrasound contrast agent with FA-CNTs-PTX compound was prepared.The microbubble structure and morphology were characterized by infrared spectroscopy,ultraviolet spectrophotometer,thermogravimetry,scanning electron microscopy,transmission electron microscopy and laser particle size analyzer.(2)Using doppler ultrasound diagnostic apparatus,the ultrasound contrast effect of composite microbubble was investigated.(3)Selecting the Breast cancer MCF-7 cells and Hepatic carcinoma HepG2 cells as the experimental models,CCK-8 kit and AO/EB kit double staining method were used to detect the antitumor activity in vitro of composite microbubble contrast agent.Quantitative analysis of the apoptosis rate of breast cancer MCF-7 cells and normal human umbilical vein endothelial cells(HUVECs)by flow cytometry.(4)With Kunming mice as model,the maximum tolerable dose of the composite microbubble contrast agent was determined by acute toxicity test.Results(1)The prepared Span-PEG microbubbles compositing FA-CNTs-PTX were smooth and the average particle size was 442 nm.The FA-CNTs-PTX targeting drug-loaded complex was successfully coated in Span-PEG microbubbles,the composite microbubbles structure was a hollow sphere,its particle size distribution was uniform and nano-level.(2)Compared with the saline and Span-PEG microbubbles,the microbubble contrast agent compositing FA-CNTs-PTX significantly enhanced the ultrasound signal in vitro and vivo,it could improve the clarity of ultrasound imaging.(3)The obviously inhibitory rate of composite microbubbles with FA-CNTs-PTX on breast cancer MCF-7 cells appeared,which was correlated with the higher drug loading rate and microbubbles nano-size.The composite microbubbles with FA-CNTs-PTX affected the proliferation inhibition rate and cell apoptosis cycle of breast cancer MCF-7 cells,its inhibitory effect on the proliferation of MCF-7 cells was higher than that of HepG2 cells.The composite microbubbles with FA-CNTs-PTX resulting in the early apoptosis rate of breast cancer MCF-7 cells was 34.85%,which was much higher than that of normal human umbilical vein endothelial cells(HUVECs)was 14.99%.The composite microbubbles with FA-CNTs-PTX effect on breast cancer MCF-7 cells was significantly inhibited.(4)Kunming mices did not appear the death and adverse reactions under the maximal tolerant dose,and the maximum tolerating dose was 350 times of adults with 50 kg.Conclusions The microbubble ultrasound contrast agent compositing FA-CNTs-PTX was designed and prepared in this study,it was expected to be a good contrast agent for angiography and targeted therapy.Firstly,the composite microbubble structure satisfied the theoretical conditions as the ultrasound contrast agent,it had the ideal enhanced ultrasound imaging function in vivo and vitro.Secondly,the composite microbubbles successfully loaded drugs to achieve targeting inhibition of breast cancer MCF-7 cells proliferation.Finally,the maximum tolerated dose of the compound microbubbles in Kunming mice was much higher than that of adult doses.Therefore,the microbubble contrast agent compositimg FA-CNTs-PTX was safe and stable in vivo,it enhanced ultrasound imaging effect and should be a highly effective neoadjuvant chemotherapy reagent,providing the drug delivery pharmacological model for the next step.