Inhibition Of MCF-7 On Breast Cancer Cells In Vitro Based On HSA-ABD-iTEP-PTX Nanoparticles

Objective:To construct an immunocompatible elastic polypeptide（ABD-iTEPA）nanoparticle targeted drug delivery system loaded with paclitaxel,to inhibit the proliferation of breast cancer cells MCF-7 in vitro,and to evaluate the effect of nano-drug HSA-ABD-iTEPA-PTX on breast cancer cells.Methods:The cDNA designed and synthesized by genetic engineering technology in our laboratory was transfected,transcribed and translated to prepare an immunocompatible elastic polypeptide（albumin-binding domain immune-toleran elastin-like polypeptide,ABD-iTEPA）,and synthesized with the modified The paclitaxel（PTX-LEV-EMCH）self-assembles through sulfhydryl linkage to form nano-drug particles（albumin-binding domain immune-toleran elastin-like polypeptide nano-particles,ABD-iTEPA-PTX NPs）;utilizes the albumin domain at the hydrophilic end（albumin-binding domain,ABD）combined with human serum albumin（HSA）to form nano-drugs（human serum albumin albumin-binding domain immune-toleran elastin-like polypeptide PTX-LEV-EMCH nano-particles,HSA-ABD-iTEPA-PTX NPs）.The nanodrugs were identified by Nano Brook Omni particle size and Zeta potential determination and transmission electron microscopy,and their structure was confirmed by Nano Measurer random counting of the particle size distribution of the nanodrugs in TEM images.The standard curve of paclitaxel was established by ultraviolet spectrophotometer and the encapsulation efficiency and uploading efficiency of paclitaxel in nanomedicines were detected.The drug-loaded nanoparticles were tested for in vitro acid release by simulating the difference between the normal human tissue internal environment and the acidic surrounding tissue environment.Taking MCF-7 breast cancer cells cultured in vitro as a model,the CCK-8 method was used to investigate the inhibition of breast cancer cell MCF-7proliferation by ABD-iTEPA-PTX NPs in vitro,and to evaluate the effect of nano-drug HSA-ABD-iTEPA-PTX on breast cancer cells.Results:The cDNA encoding the nanocarrier polypeptide was transfected with DH5αcompetent cells,and the plasmid was extracted to measure the OD260/OD280 of2.073±0.043,and the concentration was 104.05 ug/m L±9.95 ug/m L;the repeatability of the extracted plasmid sequence and the original plasmid sequence was more than98%,139.17 mg±7.42 mg of carrier polypeptide can be extracted per liter of TB culture medium after being expressed by BL21 competent cells.The particle size of the synthesized nanomedicine ABD-iTEPA-PTX NPs is 87.09 nm±7.63 nm（PDI:0.20）;its charge potential is not shown;the particle size of HSA-ABD-iTEPA-PTX NPs is 117.24 nm±5.42 nm（PDI:0.28）,which carries a charge potential of-21.26m V±1.37 m V.The HSA-ABD-iTEPA-PTX NPs showed a typical ellipsoidal shape under the electron microscope.The established standard curve of paclitaxel is:Y=0.2173 X+0.0064（R2=0.9999）,and the linear relationship is good in the concentration range of 1.875～7.5μg/m L.The encapsulation rate and upload rate of ABD-iTEPA-PTX NPs were 52.52%and 19.11%,respectively.HSA-ABD-iTEPA-PTX NPs had a higher release rate in the acidic microenvironment of tumors;no obvious cytotoxicity was observed in the prepared ABD-iTEPA and HSA-ABD-iTEPA nanocarriers at 31.25-500μg/m L.Compared with nab-paclitaxel,HSA-ABD-iTEPA-PTX NPs significantly inhibited the proliferation of breast tumor MCF-7 cells,and the drug concentration of 2.5-40μg/m L was significantly higher than that of nab-paclitaxel（P<0.05）,IC₅₀ is 9.205μg/m L.Conclusion:A new type of self-assembled nanoparticles（HSA-ABD-iTEPA-PTX NPs）with immunocompatible elastin peptides loaded with paclitaxel were successfully constructed,and the MCF-7 breast cancer cell model was established.The results of in vitro cytotoxicity test showed that HSA-ABD-iTEPA-PTX NPs had good ability to kill breast cancer tumor cells.It lays a foundation for further research on HSA-ABD-iTEPA-PTX NPs in vivo and clinical trials in mice.