奈米金柱鍵結上單株抗體對腫瘤細胞進行光熱療法

Abstract

奈米金柱在癌症治療領域上展現出無窮的潛力。因為奈米金柱經由精準的控制長寬比，可吸收位於生物光學窗範疇之近紅外光，進而轉變成熱能，故能在光熱治療中使用。製作奈米金柱的過程中，大多使用溴化十六烷三甲基銨(CTAB)做為介面活性劑。由於CTAB的生物不相容性，所以使用CTAB來製備奈米金柱，必須對奈米金柱的表面進行改質，以利應用於細胞相關之研究。生物相容性極高的甲殼素接上硫基後與奈米金柱混合，硫基會在奈米金柱的表面產生Au-S鍵結。因此，藉由ligand exchange程序可使硫基化的甲殼素將CTAB自原始的奈米金柱表面置換出來，以提昇生物相容性。甲殼素在接枝硫基後剩餘之胺基，可用來進一步與抗體鍵結。本論文中，將兩種單株抗體，anti-GD2以及anti-CD133，分別與奈米金柱鍵結；接著將標記上單株抗體之奈米金柱分別置入含有神經母細胞瘤(neuroblastoma cells)及 神經膠母細胞瘤 (glioblastoma cells)的培養皿內，觀察腫瘤細胞以胞飲作用將標記上單株抗體之奈米金柱噬入之情形。接著使用808 奈米波長的二極體雷射系統，令奈米金柱吸收近紅外光波段的光，進而轉變成可摧毀腫瘤細胞之熱能。

Gold nanorod (GNR) has great potential in cancer therapy field, because of its unique photophysical property in converting near-infrared (NIR) laser light into heat. GNRs fabricated by seed-mediated growth method with the aid of surfactant hexadecyltrimethylammonium bromide (CTAB) have been widely used. However, due to strong cytotoxicity of CTAB, it is necessary to modify the surface of GNRs for cell-related studies. In this study, thiolated chitosan was synthesized and harnessed to replace CTAB originally used for stabilizing gold nanorods. The degree of thiol groups immobilized on chitosan was quantitatively determined by Ellman’s analysis. The average size of GNR and thiolated chitosan-modified GNR (CGNR) determined by dynamic light scattering (DLS) was 66 nm and 84.9 nm, respectively which are consistent with the images obtained by transmission electron microscopy (TEM). X-ray photoelectron spectroscopy (XPS) was used to confirm the existence of Au-S binding energy at 162.4 eV. Cytotoxicity study revealed that CGNR was much biocompatible than CTAB-stabilized GNR. CGNRs were further conjugated with two different kinds of monoclonal antibodies: anti-GD2 and anti-CD133. The former antibody can specifically target neuroblastoma cells and the latter one is able to recognize glioblastoma stem-like cells. Our results showed that CGNRs functionalized with specific monoclonal antibody could be internalized by its corresponding cancer cells via receptor-mediated endocytosis. NIR laser was then used to irradiate CGNR-laden cells for pre-determined power intensity and exposure time. The viability of NIR laser treated cells examined by calcein-AM dye demonstrated that cancer cells ingested with antibody-tagged CGNRs and irradiated with 808-nm NIR laser light were all found to undergo necrosis.