Preparation Of Herceptin-gold Nanoprobe And Its Inhibitory Effect On Breast Cancer Cells

Objective:Construct an innovative biocompatible nanoprobe of Herceptin-gold nanoparticle conjugates, to study its inhibitory on breast cancer cells in vitro, to explore new targeted therapies in HER2 overexpressed breast cancer.Methods:The gold nanoparticles (GNPs) (10 nm,20 nm and 40 nm) were synthesized by the classic citrate reduction solution, characterd them by TEM and characterd 40 nm GNPs by UV-Vis absorption spectra, hydrodynamic size, zeta potential. The Herceptin conjugated to GNPs by electrostatic adsorption, charactered them with the same methods of 40 nm GNPs, and the amount of conjugated antibody measured by BCA method. The HER2 receptor of the human breast cancer cell lines of BT474 and MCF-7 cells were detected by immunocytochemical technique. The nanoprobes located on the BT474 cells membrane surface were verified using SEM. Setting 4 concentration gradients of GNPs (10nm,20nm and 40nm) from 12.5 μg/mL to 100 μg/mL, Setting 5 concentration gradients of Herceptin-GNPs and Herceptin from 0.625 μg/mL to 10 μg/mL along with their control groups to detect the cytotoxicity by MTT assay. Setting 3 concentration gradients of Herceptin-GNPs and Herceptin from 1.25 μg/mL to 5 μg/mL along with their control groups to detect apoptosis, cell cycle changes by flow cytometry and the expression of p-AKT, p-MAPK, Bcl-2 and HER2 proteins were detected by western blot, respectively.Results:(1) The GNPs (10 nm,20 nm and 40 nm) aqueous solution obtained were fuchsia and clear, TEM characterization showed that GNPs approximately round in the average diameter of 10.2±1.2 nm,20.9±2.0 nm and 40.7±2.2 nm. The UV-Vis absorption spectra, hydrodynamic size, zeta potential and TEM characterizations showed successful preparation of Herceptin-GNPs nanoprobes, the average amount of conjugated antibody was 11.3 μg/mL, the coupling rate of Herceptin on gold nanoparticles was 37.67%, and each 40 nm GNP surface can be connected with about 633 Herceptin molecules.(2) Immunohistochemistry results showed that HER2 negative expression on MCF-7 cells and BT474 cells HER2 positive expression. Our findings demonstrated 10 nm GNPs can inhibit the proliferation of BT474 cells in 50 μg/mL, and inhibit the proliferation of MCF-7 cells in 100 μg/mL, while GNPs with diameters 20 μm and 40 nm were no significant proliferation inhibition of both BT474 and MCF-7 cells at low concentrations, suggesting that GNPs cells with low toxicity, which displayed good biocompatibility.(3) Both Herceptin-GNPs and Herceptin can inhibit BT474 cells proliferation in a dose dependent, increase cell apoptosis and arrest cell in the G0/G1 cycle, Herceptin-GNPs had better effect. Moreover, for western blot analysis, both Herceptin-GNPs and Herceptin could inhibit the expression of p-AKT, p-MAPK, Bcl-2 and HER2 protein, and Herceptin-GNPs had better inhibitory effect.Conclusions:Gold nanoparticles aqueous solution obtained by citric acid reduction method of low cell toxicity, good biocompatibility. Herceptin-GNPs can significantly inhibit the proliferation of HER2 positive breast cancer BT474 cells, inhibit downstream signal transduction, induce cell apoptosis, increase intracellular uptake of HER2, which can reduce dosage of Herceptin and improve the treatment efficacy, it would provide a nanoparticle-based targeted delivery system for the treatment of HER2 overexpressing breast cancer.