量子點與氫氧基磷灰石複合材料合成及與骨母細胞株交互作用之研究

Synthesis of Quantum Dots-Hydroxyapatite Composites and Its Interaction with MC3T3-E1 Cells

黃馨慧(Shin-Huie Huang)

指導教授 ： 張恆雄 ； 謝明發

中原大學/工學院/醫學工程研究所/碩士(2006年)

https://doi.org/10.6840/CYCU.2006.00509

摘要

本研究以量子點為支架，利用化學沉澱法合成氫氧基磷灰石，製備出量子點-氫氧基磷灰石之複合材料。藉由量子點的光學特性賦予氫氧基磷灰石標定之功能，並進一步了解此複合材料與骨母細胞間之交互作用。
實驗設計分為四階段：一、利用兩性高分子對有機相量子點進行表面修飾，使量子點由疏水性轉為親水性；二、改變三種參數，找出氫氧基磷灰石之最佳合成條件：溫度(25、40、55和70℃)、時間(4、3、2、1和0.5小時)、pH值(7.1、8.5、9.5、10.6和11.6)，分別以80℃乾燥和-80℃冷凍乾燥後，利用掃描式電子顯微鏡觀察材料表面形貌和X光粉末繞射分析材料之結晶性；三、利用第二階段的最佳參數，加入不同體積(0、1、5、25ml)量子點，以製備出量子點-氫氧基磷灰石之複合材料；四、將此複合材料(1mg~1ng/ml)與骨母細胞共培養1~24小時， 進行細胞存活率分析，並使用共軛焦顯微鏡觀察其與骨母細胞間的交互作用。
研究結果顯示：(1)利用兩性高分子可成功地將量子點改質為親水性奈米粒子，其粒徑大小約為17.9nm，表面電荷約為-38.8mV；(2)在溫度25℃下，反應溶液pH值為10.6，利用80℃乾燥法得到之氫氧基磷灰石結晶性最佳，呈圓球形，粒徑大小約25~30nm，反應時間並不會影響氫氧基磷灰石的生成；(3)可成功地製備量子點-氫氧基磷灰石複合材料，加入不同體積之量子點，並不會影響此複合材料的表面形貌與結晶性，且隨量子點的體積增加，複合材料的產量與螢光強度也會增加；(4)此複合材料的濃度為1mg/ml、100μg/ml和10μg/ml時會造成細胞毒性，培養24小時後，其細胞存活率分別約為39.1 %、57.6 %和81 %；此外，進一步地觀察骨母細胞可攝入此複合材料，且均勻的分散在細胞質中。
本研究合成之量子點-氫氧基磷灰石複合材料，量子點仍具其優異的光學特性，具有標定追蹤氫氧基磷灰石的功能。利用此複合材料可進一步暸解氫氧基磷灰石與骨母細胞間之交互作用，期望此複合材料可被應用在相關的研究領域，進一步地暸解材料與細胞組織間的交互作用。

關鍵字

量子點 ； 細胞存活率 ； 骨母細胞 ； 氫氧基磷灰石

並列摘要

Over the past decade, a large number of biomaterials have been proposed as artificial bone fillers for repairing bone defects. The material most widely used in clinical medicine was hydroxyapatite (HA). Luminescent colloidal semiconductor nanocrystals (QDs) were robust inorganic fluorophores that have the potential to circumvent some of the functional limitations encountered by organic dyes in sensing and biotechnological applications. Recently, QDs have been utilized for labeling different cellular organelles and tracking their functions as well. In this research, the surface-modified QDs were used as templates for the growth of HA nanoparticles and to track/image the movement of QDs-HA composites on osteoblast (OB, MC3T3-E1).
The experiments were divided into four parts. The first part was to modifly the surface of QDs with amphiphilic polymer, and QDs were changed to hydrophilic particles from hydrophobic particles. The second part was to find the best condition of nano-HA synthesis. There were three synthetic parameters : temperature (25, 40, 55 and 70℃), time (4, 3, 2, 1 and 0.5 hours), and pH value (7.1, 8.5, 9.5, 10.6 and 11.6). After the 80℃ or -80℃ freeze drying process, the materials were analyzed by scanning electron microscopy (SEM) and X-ray diffraction examination (XRD) to understand its morophology and crystallography. The third part was to synthesize QDs-HA composites by different volume (0, 1, 5, and 25ml) of QDs with the best parameters in the second part of the experiments. The forth part was to evaluate the cell viability of osteoblast after the QDs-HA composites (106, 105, 104, 103, 102, 10 and 1ng/ml) adding by MTT assay at the time point 1, 2, 4, 8, 12 and 24 hours, and observed the interaction between composites and osteoblast by confocal microscopy.
The results demonstrated that : (1)the hydrophilic QDs were successfully changed to hydrophilic particles by using amphiphilic polymer. The diameter of QDs size was 17.9nm, and the surface charge was -38.8mV ; (2) when the pH value of reaction solution was 10.6 at 25℃, the nano-HA crystal was round from 25 to 30nm. However, the reaction time was not the important factor on the growth of HA ; (3) the QDs-HA composites were synthesized successfully. The shape and crystallization of composites were not affected by the adding volume of QDs, but its yield and fluorescent intensity would be enhanced; (4) the cell viability rate would be decreased 39.1%, 57.6%, 81% respectively at the concentration 1mg/ml, 100μg/ml, and 10ug/ml of the composites after 24 hours culturing with osteoblast.
In this study, QDs-HA composites had excellent optical property and the interactions between composites and osteoblast could be investigated by confocal microscopy successfully. These composites can be applied in the field of relevant research to understand its interaction among cell and tissue.

並列關鍵字

Cell viability ； Ostoblast ； Hydroxyapatite ； Quantum dots

參考文獻

[1] Groot KD, “Bioceramics of Calcium Phosphate.” CRC Press. Inc., Bocaa Ration, Florida, 1983.

[2] Hench LL, “Bioceramics : from Concept to Clinic.” J. Am. Ceram. Soc., 74, 1487, 1991.

[3] 鍾仁傑, “以溶膠-凝膠法製備含鋅及銀之氫氧基磷灰石與其抗菌性質研究.” 國立清華大學材料科學工程所,碩士論文,2001.

[4] Niu JL, Zhang ZX, and Jiang DZ, “Synthesis and Characteristics of Porous Hydroxyapatite Bioceramic.” Shenwu Tixue Gongchengxue Zazhi, 9, 302-305, 2002.

[6] Panda RN, Hsieh MF, Chung RJ, and Chin TS, “X-Ray Diffractometry and X-Ray Photoelectron Spectroscopy Investigations of Nanocrytalline Synthesized by a Hydroxide Gek Technique.” Jpn. J. Appl. Phys., 40, 5030-5035, 2001.