Anti-EGFR Conjugated Hollow Gold Nanospheres Enhance Radio-cytotoxic Targeting Of Cervical Cancer At Megavoltage Radiation Energies

Objective:To confirm that the anti-epidermal growth factor receptor monoclonal antibodies conjugated hollow gold nanospheres (anti-EGFR/HGNs) can be selectively uptaked by cervical cancer cells and induce its apoptosis when combined with radiotherapy as a result enhancing radiosensitivity of cervical cancer cells.Methods:Immunofluorescence staining was adopted to further demonstrate the high expression of EGFR on cytomembrane of CaSki cells. The diameter and morphology of the particles were viewed by transmission electron microscopy (TEM). Bifunctional SH-PEG-COOH was used as a linker to conjugate antibodies to HGNs. Spectrophotometry was performed to confirm conjugation of anti-EGFR to HGNs. Cell uptake of naked HGNs and anti-EGFR/HGNs and the concentration of HGNs was assayed by inductively coupled plasma atomic emission spectroscopy(ICP-AES).The cytotoxicity of naked HGNs and anti-EGFR/HGNs on CaSki cells, which were used in our experiment, was assessed by CCK-8 assay. Cell cycle and apoptosis were examined by a propidium iodide(PI)kit with flow cytometry(FCM).The expression of several critical apoptosis-related proteins including Bcl-2,Bax,Bad,active caspase 3, was tested by western-blot analysis.Results:Immunofluorescence staining confirmed the high expression of EGFR on cytomembrane of CaSki cells. The HGNs were with a mean diameter of(54.6±7.11 nm) and wall thickness of(5.01±2.23 nm). A characteristic red-shift (≈20 nm) of the peak of surface plasma resonance (SPR) demonstrated successful conjugation of anti-EGFR on HGNs. Cells treated by anti-EGFR/HGNs showed an obvious increase in nanoparticles uptake compared to naked HGNs. Neither naked HGNs (1,3 and 5 nM) nor anti-EGFR/HGNs (1,3 and 5 nM) induce remarkable cytotoxicity on CaSki cells. Anti-EGFR/HGNs combined with radiation resulted in a significant growth inhibition, compared with radiation combined with naked HGNs. Anti-EGFR/HGNs remarkably increased the ratio of CaSki cells in the G2/M phase and induced more apoptosis by an obvious deregulation of Bcl-2 and upregulation of Bax,Bad,caspase-3 when combined with radiation.Conclusions:The study demonstrated that anti-EGFR/HGNs significantly increase the cytotoxicity of 6 MV X-ray on CaSki cells, and anti-EGFR/HGNs combined with radiotherapy may possess large therapeutic potential in cervical cancer. We can also conclude that anti-EGFR/HGNs may be involved in the increased apoptosis of CaSki cells by regulating expression of Bcl-2 family proteins and activating caspase 3. In addition, anti-EGFR/HGNs manipulate the cancer cell cycle to enhance radiation susceptibility.