Conjugation of Polymer-Coated Gold Nanoparticles with Anti-EGFR Antibodies for Enhanced Radiation Therapy

Using optical microscopy techniques to assess nanoparticle-cell interactions in cervical and liver cancer cells incubated with gold nanoparticles (GNPs) of various surface coatings for applications in enhanced radiation therapy. The reduction of tetrachloroauric acid (HAuCl4) by trisodium citrate (Na3C6H5O7) to form GNPs was carried out by adding sodium citrate to a boiling gold chloride solution in a glass bottle. The mixture was heated and stirred for 10 minutes until the color of the solution changed to a wine red, which indicates the formation of citrate-capped GNPs. After the synthesis process, thiolated polyethylene glycol (mPEG-SH) of MW 7500 was attached to the surface of the GNPs via the gold-thiol bond by stirring for 2 hours. The excess PEG was removed by centrifugation at 8000 rpm for 10 minutes followed by decanting the supernatant and resuspending the pellet in DI water. Absorption spectra were obtained on a UV-vis spectrometer. C33A (cervical) and HEPG2 (liver) cancer cells were plated in 6-well plates with increasing concentration of PEGylated GNPs and allowed to incubate for 24 hours. After incubation, cells were fixed, and confocal fluorescence imaging was carried out with an excitation wavelength of 514 nm using a tunable argon-ion laser. Images were assessed to determine the conditions yielding the most favorable cell/nanoparticle interaction (i.e., maximum cellular uptake) for use in GNP-enhanced radiation therapy. The UV-vis spectra of the citrate-capped GNPs resulted in an absorption maximum of 534.17 nm, whereas the PEGylated GNPs exhibited an absorption maximum of 536.26 nm, indicating an increase in the local refractive index. SEM imaging of the GNPs revealed that they are spherical particles approximately 30 nm in diameter. Internalization of functionalized GNPs by C33A and HEPG2 cells has been observed using confocal fluorescence microscopy. Our findings show that we have successfully functionalized spherical gold nanoparticles 30 nm in diameter with the biocompatible polymer PEG, replacing the citrate layer on as-synthesized GNPs. A protocol for bioconjugation of functionalized GNPs with anti-EGFR antibodies has been presented. The investigation of GNP-cell interactions by confocal laser scanning microscopy shows 30 nm GNPs distributed inside the cell cytoplasm. Our future work is to apply these PEGylated GNPs with anti-EGFR antibodies to active targeting delivery in in vitro studies of nanoparticle-enhanced radiation therapy for cervical and liver cancer.