Study On The Effect And Mechanism Of Targeted Inhibition Of Breast Cancer Cell Growth By Multimodal Imaging Nanobubbles Loaded With AMD070

BackgroundBreast cancer is one of the most common malignant tumors in females.Early diagnosis and timely treatment are important prerequisites for increasing the survival rate of breast cancer patients.It has been confirmed that ultrasound microbubbles are not only widely used in the diagnosis of breast cancer,but also as a good carrier of drugs.Ultrasound microbubbles can release drugs,promote drug absorption and promote the apoptosis of tumor cells under ultrasound irradiation.Because the particle size of traditional ultrasound microbubbles is in the order of micrometer,which is a kind of blood pool contrast agent,and lack of targeted ability at the same time,they can't go through tumor neovascularization and can't identify specific targets on tumor cells through tumor blood vessels.They can not carry therapeutic drugs through tumor neovascularization into tumor tissue,either.These shortcomings limit its in-depth study and clinical application in the diagnosis and treatment of breast cancer.There are gaps between tumor neovascularized endothelial cells.In principle,the nanoscale ultrasound microbubbles with particle size less than 700 nm can pass through the endothelial cell gaps of tumor neovascularization.At present,CXC chemokine receptor type4(CXCR4)has abnormally high expression on the cell membrane of most solid tumors(including breast cancer cells),but is poorly expressed or not expressed in normal tissues,which has acted as a highly specific target in tumor-specific imaging and targeted therapy.Its ligand is stromal cell-derived factor-1(SDF-1).The SDF-1/CXCR4 signal pathway plays an important role in tumor growth,proliferation,metastasis,and invasion.Blocking this signal pathway can effectively inhibit tumor growth and metastasis.AMD070 is a small molecular antagonist for CXCR4,which is simple in structure and easy to connect with lipid nanobubbles,and it makes nanobubbles have targeting properties.Indocyanine green(ICG),as a widely used fluorescent dye in clinic,is a good light absorbing material.Nanobubbles carrying ICG will have the ability of ultrasound,photoacoustic and fluorescence multimodal imaging at the same time.ObjectiveMultimodal imaging nanobubbles loading with AMD070(targeted nanobubbles carrying ICG and AMD070,ICG-TNBs)was constructed in this study;To detect the in vitro multimodal imaging ability of ICG-TNBs,and to study the effect of ICG-TNBs on targeting inhibiting the growth of CXCR4 positive breast cancer cells and promoting apoptosis;meanwhile,we further research and explore the mechanism of ICG-TNBs inhibiting the growth of breast cancer cells,especially the effect of ICG-TNBs on the activity of SDF-1/CXCR4 signal pathway.Material and methodsThis project is divided into three parts for experiment:1.Construction and identification of ICG-TNBs:(1)ICG-TNBs were prepared by carbodiimide reaction and mechanical oscillation.The particle size,Zeta potential,ICG carrying capacity and encapsulation rate were measured.The morphology of ICG-TNBs was observed and the fluorescence absorption characteristics of ICG-TNBs were detected.Study on the stability of ICG-TNBs was carried out.(2)The multimodal imaging ability of ICG-TNBs was detected on agarose model in vitro.(3)The specific binding ability of ICG-TNBs to breast cancer cells and the specific uptake of ICG-TNBs by breast cancer cells were observed by light microscopy and laser confocal microscopy.2.Study on the effects of ICG-TNBs targeted inhibition of breast cancer cells:(1)The ideal parameters of ultrasonic irradiation were selected,including ultrasonic power,irradiation time and concentration of nanobubbles.(2)To study the inhibition concentration range of ICG-TNBs on breast cancer cells.(3)CCK-8 and flow cytometry were used to detect the growth inhibition and pro-apoptotic effects of ICG-TNBs on breast cancer cells.3.Study on mechanism of ICG-TNBs targeted inhibition of breast cancer cells:(1)The cavitation effect of ICG-TNBs on breast cancer cells was observed by scanning electron microscopy(SEM).(2)To detect the effect of ICG-TNBs on the level of reactive oxygen species(ROS)in breast cancer cells.(3)Western blot was used to detect the expression of CXCR4,p-Akt/Akt,Caspase3and Cleaved-caspase3.Results1.Construction and identification of ICG-TNBs:(1)The particle size and zeta potential of ICG-TNBs were(497.0±29.2)nm and(-8.05±0.73)mV,respectively,and the carrying capacity and encapsulation rate of ICG were(5.0±0.29)ug/mg and(80.2±0.64)%,respectively.ICG-TNBs were round under light microscopy and transmission electron microscopy(TEM).Under the loading of ICG-TNBs,the fluorescence absorption characteristics of ICG did not change significantly.ICG-TNBs has good stability.(2)With the decrease of ICG-TNBs concentration,the intensities of ultrasonic,photoacoustic and fluorescence signals decrease.ICG-TNBs has good multimodal imaging ability in vitro.(3)ICG-TNBs can specifically bind to CXCR4 positive MCF-7 cells and be absorbed into the cell,while CXCR4 negative MDA-MB-468 cells cannot bind to or absorb ICG-TNBs.2.Study on the effects of ICG-TNBs targeted inhibition of breast cancer cells:(1)The optimal ultrasonic irradiation parameters were:ultrasonic power is 1.0 W/cm~2,irradiation time is 10 s and the range of ultrasonic nanobubbles concentration is 5×10~6-2×10~7/ml.(2)When the concentration of ICG-TNBs reached 5×10~6/ml,the growth of MCF-7cells was significantly inhibited and weakened with the further increase of the concentration,while they had no significant suppressive effect on the growth of MDA-MB-468 cells.(3)Compared with PBS group and other control groups,ICG-TNBs+US treatment group had the strongest growth inhibition and pro-apoptotic effects on breast cancer cells.3.Study on mechanism of ICG-TNBs targeted inhibition of breast cancer cells:(1)Compared with the control group,the number of pores and folds on MCF-7 cells in ICG-TNBs+US treatment group was the most,which indicated that the cavitation effect of ICG-TNBs+US was the most obvious.(2)Compared with other control groups,ICG-TNBs+US group had the highest level of ROS,indicating that ICG-TNBs+US treatment group promoted the production of ROS.(3)WB results showed that compared with the MDA-MB-468 cells in the control groups,ICG-TNBs+US could significantly reduce the expression of CXCR4,Akt phosphorylation(P<0.05),and could significantly increase the expression of Caspase 3 and Cleaved-caspase3 in MCF-7 cells(P<0.05),indicating that ICG-TNBs+US can effectively carry AMD070 and inhibit the growth of MCF-7 cells by inhibiting the activity of SDF-1/CXCR4/Akt pathway.Cavitation effect and AMD070 can effectively improve the ROS level in MCF-7 cells(P<0.05),and then increase the expression of Caspase3 and Cleaved-caspase3 and induce apoptosis.ConclusionIn this study,ultrasound,photoacoustic and fluorescence multimodal imaging nanobubbles loaded with AMD070(targeted nanobubbles carrying ICG and AMD070,ICG-TNBs)were successfully constructed,and confirmed that ICG-TNBs can bind specifically to breast cancer cell membrane with positive expression of CXCR4,as well as exert a significant effects of ICG-TNBs targeted inhibition of breast cancer cells.On this basis,mechanism of ICG-TNBs targeted inhibition of breast cancer cells was investigated in depth.Under ultrasound irradiation.ICG-TNBs can effectively improve the membrane permeability of tumor cells,promote AMD070 to effectively inhibit and block the activity of SDF-1/CXCR4/Akt pathway,improve the level of ROS in cells,promote the expression of Caspase3 and Cleaved-caspase3,which lead to the growth inhibition and apoptosis of tumor cells.This study not only provides a new idea for the construction of targeted multimodal diagnosis and treatment integration nanobubbles,but also has laid an elaborate foundation for further research on the in vivo process and mechanism of targeted imaging and tumor growth inhibition with multimodal imaging nanobubbles loaded with AMD070.