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標題: | 功能性氫氧基磷灰石奈米顆粒於癌症治療之研究 Functional Hydroxyapatite Nanoparticles for Cancer Treatment |
作者: | Min-Hua Chen 陳民樺 |
指導教授: | 林?輝(Feng-Huei Lin) |
關鍵字: | 氫氧基磷灰石,含有CpG雙核?酸序列模組的寡?酸,鉿離子,活性氧化物質, hydroxyapatite,CpG oligodeoxynucleotides,hafnium ions,Reactive oxygen species, |
出版年 : | 2015 |
學位: | 博士 |
摘要: | 由於磷酸鈣陶瓷(calcium phosphate)具有優越的生物相容性、生物活性,與人體骨骼或牙齒組成分類似等特性,因此目前它被視為較合適的骨骼植入物,並在組織工程上有許多應用。近年來已有許多不同結晶相的磷酸鈣陶瓷被應用在生物醫學領域,而其中又以氫氧基磷灰石的應用最為普遍,最廣為人知,這是因為氫氧基磷灰石具有高度穩定性,其材料特性不易受酸鹼值、溫度及生理環境影響而產生變化。
在先前的研究中,我們已觀察到,利用濕式化學沉澱法,結晶性奈米氫氧基磷灰石可在水溶液中合成,而不需要添加任何的介面活性劑。合成出的粒子是屬於B型含碳的氫氧基磷灰石,且能長時間穩定地懸浮在培養液中,這樣的特性非常合適作為生物應用。有這樣令人振奮的結果,且考量到癌症仍是現今社會主要的致死疾病。因此,本研究我們將致力於氫氧基磷灰石奈米顆粒對癌症治療的研究和探討,包括: (1) 以氫氧基磷灰石奈米顆粒攜帶含有胞嘧啶(cytosine)-鳥糞鹼(guanine)雙核苷酸序列模組的寡苷酸(oligodeoxynucleotides; CpG ODN),作為癌症免疫治療的載體。 (2) 藉由參雜四價鉿離子(Hf4+)於氫氧基磷灰石中(Hf:HAp),探討經放射線照射後,作為癌症治療的可行性評估。 在前半部的研究裡,我們利用濕式化學沉澱法,合成帶有CpG ODN的氫氧基磷灰石奈米顆粒。藉由分析其在溶液中的穩定性、顆粒大小、表面型態、CpG ODN載附量、及其刺激免疫細胞產生細胞激素的表現,來評估未來應用於癌症免疫治療的可行性。此部份的研究,我們觀察到結晶性氫氧基磷灰石奈米顆粒可增加CpG ODN在融酶體上的釋放,進而使CpG ODN與Toll-like receptor(TLR9)進行交互作用。相較於非晶型磷酸鈣奈米顆粒及奈米微脂體,結晶性氫氧基磷灰石可促使免疫細胞產生較多的細胞激素(IL-12及IL-6)。這些細胞激素的對未來癌症免疫治療扮演重要角色。 在後半部的研究裡,我們同樣經由式化學沉澱法,合成具有四價鉿離子摻雜的氫氧基磷灰石奈米顆粒。並以人類肺腺癌A549細胞株作為此奈米顆粒在動物體外(in-vitro)及體內(in-vivo)的療效評估。本研究證實摻雜四價鉿離子的氫氧基磷灰石經放射線照射後,可增加細胞內活性氧化物質(reactive oxygen species; ROS)的量並促進細胞凋亡。此研究結果可作為未來治療深層癌細胞的參考,以取代目前光動力治療中,可見光源穿透組織深度的限制。 Calcium phosphates, because of their superior biocompatibility, bioactivity and compositionally similar to the mineral phases of the bone, are preferred as bone grafts and tissue engineering. Several phases of calcium phosphate have currently been used in the biomedical field. Among those, HAp, due to the highest stability, is the most popular and well-known phase of calcium phosphate, which is the most stable with the variation of pH, temperature or composition of the physiological fluid. In our preliminary study, we observed crystalline nano-sized HAp could be easily synthesized by wet chemical precipitation in water without addition of any surfactant, showing the particles belonged to biodegradable B type carbonated HAp and could be well-suspended in culture medium for a relatively long period of time, indicating it could be an ideal candidate for biological application. In recent years, cancer treatment has been always a global and imperative health issue. With these encouraging results, in this study, we ask whether functional crystalline HAp can treat cancer, involving (1) Enhancing immune-activation of macrophage by delivering CpG ODN-loaded HAp nanoparticles; (2) Reactive oxygen species (ROS)-enhanced cancer treatment by hafnium-doped HAp nanoparticles with ionizing radiation. In the first part of our study, we synthesized HAp nanoparticles by wet chemical precipitation method. HAp nanoparticles loaded with CpG ODN were characterized for their stability, size, morphology, loading capacity, and cytokine induction behavior. The results revealed TLR9 dependent cytokine (IL-12 and IL-6) was enhanced by loading CpG ODN onto crystalline HAp nanoparticles, indicating HAp facilitated the retaintion of ODNs in endolysosome, giving rise to specific CpG ODN/ TLR9 interactions. The results suggest HAp may be an appropriate vehicle for ODN delivery and cancer immunotherapy. In the second part, 15% of hafnium ions were successfully doped into HAp crystal by wet chemical precipitation method. The human lung epithelia cell line A549 was selected as the in-vitro and in-vivo model. The results suggest Hf4+-doped HAp nanoparticles enhance the quantity of ROS in cells and induce cell apoptosis by bombarding with ionizing radiation, indicating a possible approach instead of photodynamic therapy to treat deeper tumor. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54703 |
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