利用螢光奈米鑽石在上皮生長因子受體 的新穎檢測及追蹤方法

Abstract

螢光奈米鑽石是一種有潛力與生物相容性的奈米材料，可被開發作為生物影像及生物檢測的應用。在此研究中我們發現專一性上皮生長因子受體的抗體cetuximab可被結合到螢光奈米鑽石表面，並可進行人類肺癌細胞的上皮生長因子受體之體外及體內追蹤。螢光奈米鑽石的表面經由羧酸基修飾使表面帶負電。螢光奈米鑽石與cetuximab的最佳結合比率約為1: 0.25，螢光奈米鑽石之平均粒徑大小為113.9±4.9 nm，螢光奈米鑽石連結cetuximab之複合體的平均粒徑大小增至 192.0±8.1 nm。利用免疫共沉澱法及共軛焦顯微技術可觀察螢光奈米鑽石-cetuximab會專一性結合到人類肺癌細胞上皮生長因子受體蛋白，在免疫共沉澱後之共軛焦顯微鏡下，可直接觀察到蛋白G珠上的單一螢光奈米鑽石-cetuximab結合上皮生長因子受體蛋白的複合體。此外螢光奈米鑽石-cetuximab複合體可辨識肺癌細胞的上皮生長因子受體與促進進入細胞的能力。螢光奈米鑽石-cetuximab結合上皮生長因子受體會被運送進入溶酶體中，進行上皮生長因子受體蛋白的分解。綜合以上結果，螢光奈米鑽石連接cetuximab可作為人類肺癌細胞之上皮生長因子受體的新穎標靶檢測及追蹤的方法。

Fluorescent nanodiamond (FND) is a promising biocompatible nanomaterial that is developed for bio-imaging and -detection. Here we show cetuximab, a specific epidermal growth factor receptor (EGFR) monoclonal antibody, which can be conjugated on the surface of FNDs for tracking the EGFR of human lung cancer cells in vitro and in vivo. The FND’s surface was modified with carboxylation, which contained negative charge. The optimal binding ratio of FND: cetuximab was approximate 1:0.25. The average size of FND was 113.9 ± 4.9 nm. The average size of FND-cetuximab complexes was around 192.0 ± 8.1 nm. FND-cetuximab can specifically bind on the EGFR proteins of human lung cancer cells using immunoprecipitation method and confocal microscopy. The binding image of single FND-cetuximab and EGFR on a protein G bead can be directly observed under confocal microscope after immunoprecipitation. Moreover, FND-cetuximab can recognize the EGFR to increase the uptake ability of lung cancer cells. FND-cetuximab-EGFR complexes were transported to lysosomes for EGFR degradation. Taken together, these findings demonstrate the FND-cetuximab complex that can be used for the novel targeting detection and tracking method on the EGFR in the human lung cancer cells.