- 學位論文
胞飲雙磷酸鹽磁性奈米複合粒子 蝕骨/造骨細胞之射頻輻射探討
The studies of radiofrequency on the osteoclasts/osteoblasts endocytosed magnetite-bisphosphonates nanoparticles
摘要
本研究是以射頻輻射蝕骨細胞所胞飲之磁性奈米粒子，藉熱效應裂解蝕骨細胞以達到治療骨質疏鬆症的目的。研究以共沈澱法製備磁性奈米粒子：飽和磁化強度為62.55 emu/g、粒徑約為8 nm。並以萄聚醣及氯乙酸進行表面修飾，再將改質後磁性奈米粒子與雙磷酸鹽類藥物進行接枝，完成粒徑約10 nm之雙磷酸鹽磁性奈米複合粒子。藉傅利葉轉換紅外線光譜儀分析，於圖譜上顯現屬於雙磷酸鹽類藥物所帶之胺基與磷-氧之特徵峰值，確認雙磷酸鹽類藥物能夠接枝於磁性奈米粒子上。體外試驗所使用之蝕骨細胞則是以RANKL分化劑將RAW 264.7老鼠單核球細胞分化所得之多核蝕骨前驅細胞，造骨細胞則是使用MC3T3-E1老鼠骨骼細胞。體外細胞試驗分為胞飲量試驗與熱裂解試驗，二項試驗都在盛有多核蝕骨前驅細胞與造骨細胞之培養盤中分別加入磁性奈米子與雙磷酸鹽磁性奈米複合粒子，共培養一天後，以42 kHz射頻輻射胞飲有磁性奈米粒子或雙磷酸鹽磁性奈米複合粒子之類蝕骨細胞30分鐘，探討不同輻射次數與週期對細胞存活率之差異，結果顯示間隔兩天之輻射時序能夠明顯降低類蝕骨細胞之存活率至55 %；對造骨細胞則仍保有93%之存活率。而體外動物實驗則於MRI影像中證實注射雙磷酸鹽磁性奈米複合粒子之老鼠腿骨影像訊號較高，並於骨骼切片染色中證實的確有磁性奈米粒子貼附於骨骼中。根據以上結果，證實本研究合成出之雙磷酸鹽磁性奈米複合粒子確實能貼附於骨骼表面並在蝕骨細胞胞飲後能運用射頻輻射磁性奈米粒子之熱效應熱裂解蝕骨細胞，將有助於控制骨質疏鬆症惡化的可行性。
並列摘要
The hypothesis of this study is to apply thermolysis technique to control osteoporosis. The technique is performed in a way that iron oxide (or magnetite) (Fe3O4) nanoparticles are engulfed by osteoclasts; which were lysed by the temperature increase of the nanoparticles induced by radio frequency (RF) exposures. Fe3O4 nanoparticles were first prepared by a co-precipitation method and then conjugated with bisphosphonates bridging by dextran. These synthesized Fe3O4 and Fe3O4-bisphosphonates nanoparticles were confirmed by FTIR spectroscopy. In performing the RF thermolysis studies, these nanoparticles were individually co-cultured with multinucleated giant osteoclast-like cells (OCLs) and osteoblast cells (OBs). OCLs and OBs were induced from RAW 264.7 mouse monocytes and MC3T3-E1 cell line, respectively. Endocytosis of the nanoparticles by the OCLs and OBs was confirmed by ICP-AES. The RF system was set to produce electromagnetic wave of 42-KHz and 12.5 W. Two RF exposure study protocols were applied to OCLs and OBs for a period of 7 days with each RF exposure for 30 minutes. These protocols included every single-day exposure and every three-day exposure. The results indicate that the protocol of every three-day RF exposure is more favorable because the ratio of survival rates of OBs and OCLs resulted from this protocol is greater. Intravenous injection of Fe3O4-bisphosphonates PBS was applied to four rats. The MRI images and bone slicing microscopic images evidence Fe3O4-bisphosphonates nanoparticles exist on the femur bones of these rats. In conclusion, there is a potential of using the thermolysis technique based on Fe3O4-bisphosphonates and RF to alleviate osteoporosis.