Dark-field Detection And Inhibitory Effect Of Breast Cancer Cells By Gold Nanoparticles Probe That Absorbing Trastuzumab In Vitro

Breast cancer is the major disease for women health because of the highincidence and mortality around the world. It is easily misdiagnosed according to theconventional diagnostic techniques and may lead variety of adverse problems aftertreatment with the patients who has an overexpression of HER-2protein. With theadvancement of nanotecnology theory and improvement of the nanoparticlessynthesis with the uique characterics of gold nanoparticles, it is necessary tointroduce the nanotechnology to medical science for an innovative diagnosis andeffective treatment with breast cancer.ObjectiveSynthensizing the gonanoparticles and constructing a new nano-bio-probe inthe fields of nano-medicine were used to explore the cytotoxicity of goldnanoparticles, the dark field application and their effects with breast cancer cells.The purpose of this study is to provide the experimental basis for breast cancerdetection kand treatment using the nanotecnology.MethodsThe gold nanoparticles (GNPs) was synthesized by citrate reduction methodand the probe of GNP@Herceptin was obtained using the method of electrostaticadsorption. TEM and UV spectral detector were used to describe the characteristicsof GNPs and GNP@Herceptin and confirm the stability of them. Before observingthe GNP@Herceptin in the dark-field, the probe that located on the cell membranesurface was verified using SEM. The human breast cancer cell lines of MCF-7and SK-BR-3were bought from cell bank of Shanghai of chinese academy of science,and the HER-2protein of which was detected by immunohistochemical. Sevenconcentration gradients of GNPs from zero to400mg/mL were designed to detectthe cytotoxicity of GNPs by the kit of cell proliferation inhibition. Sevenconcentration gradients of GNPs, Herceptin and GNP@Herceptin from zero to200ng/mL were designed to investigate the proliferation inhibition of MCF-7andSK-BR-3. Four concentration gradients of Herceptin and GNP@Herceptin from zeroto1.0μg/mL were used to study the apoptosis with the cell apoptosis detection kitand the cycle arrest by the cell cycle detection kit. The data of cytotoxicity,proliferation inhibition, apoptosis and cycle arrest with the different concentrationsfor the same cell were analyzed by the method of analysis of variance and the date ofcytotoxicity and the optimal dose between different cells for the same concentrationwere analyzed by the method of dependent two-sample t-test by using SPSS21.0.The α of test level is0.05.ResultsThe gold nanoparticles were approximately round in the diameter of(14.11±1.13) nm and clarifying after absorbing Herceptin. The GNPs and Herceptinhad a perfect coupling with the combing rate of2.25:1and they had an excellentstability. With the help of dark-field we could find the orange biological probearound the SK-BR-3cells. The survival of MCF-7was43.9%(t=39.709,P=0.001)when the concentration of GNPs was400μg/mL, and when it was100μg/mL thesurvival of SK-BR-3was65.1%(t=6.796,P=0.002), which suggested it had thetoxicity to breast cancer cells. But when the concentration of GNPs was50μg/mLthe survival of SK-BR-3and MCF-7was82.8%(t=1.979,P=0.119) and99.3%(t=0.177,P=0.868) respectively, they didn’t show any toxicity to breast cancer. Thelow concentration of GNPs had a good biocompatibility. The optimal dose ofHerceptin was7.143ng/1×104cell if it treated with SK-BR-3cells andGNP@Herceptin could decrease the optimal dose from50ng/mL to25ng/mL, andthe difference had a statistical significance (t=14.774, P<0.001). When the SK-BR-3was dealt with highest concentration of GNP@Herceptin, the probe could led the effect of apoptosis from9.37%to16.87%(t=6.537, P=0.001), and block the G1phase of cell cycle from74.70%to83.12%(t=5.286, P=0.006), and reduce the cellnumber of the G2/M phase of cell cycle from14.14%to6.22%(t=6.732, P=0.003)while comparing with Herceptin.Conclusions1. GNPs has a cytotoxicity threshold limits and GNP@Herceptin can anchor tothe surface of cell membrace steady for the breast cancer rapidly detection;2. The GNP@Herceptin can improve the sensitivity of trastuzumab when dealwith the SK-BR-3and can expand the impact of apoptosis and cycle arrest.