生醫載體用之氧化石墨烯的設計與製備

Design and Preparation of Graphene Oxides for Bio-Carrier

佘書漢(Shu-Han She)

指導教授 ： 馬振基

國立清華大學/工學院/化學工程學系/碩士(2012年)

摘要

本研究旨在製備全新多功能之生醫載體，用於克服傳統生醫載體承載藥物效率過低的問題，本研究選擇一新穎的奈米碳材-奈米石墨烯作為基本載體，因奈米石墨烯具有高表面積、高親水性以及輕量化等優點，也具備高修飾性、高吸藥率以及智慧型放藥等特性，使奈米石墨烯能具有極大的潛力成為下世代生醫載體材料。
i. 將奈米石墨烯應用於自由基之捕捉：
1. 將石墨氧化脫層為氧化石墨烯以增加其水相分散性，接著以長時間超音波震盪的方法使其碎裂成為奈米等級尺寸，最後接枝上水性高分子Poly acrylic acid(PAA)，以利材料進入血液中後具有更佳的穩定性及生物相容性。
2. 為了探討PAA-g-GOs的自由基捕捉能力，將PAA-g-GOs打入會釋放自由基的腫瘤細胞(GL261)，透過觀察PAA-g-GOs可以捕捉DCFH-DA所發出的螢光，可以證明PAA-g-GOs確實具有自由基捕捉的能力。
3. 最後將PAA-g-GOs與腫瘤細胞(GL261)做體外培養，以及將PAA-g-GOs直接打入腫瘤鼠的腫瘤上，因PAA-g-GOs能夠捕捉腫瘤細胞所釋放出的自由基，結果顯示可以有效的殺死腫瘤細胞及抑制腫瘤的成長。
ii. 將奈米石墨烯應用於生物分佈：
1. 將石墨氧化脫層為氧化石墨烯以增加其水相分散性，利用超音波細胞粉碎機去震碎氧化石墨烯，並透過離心及過濾的方式將氧化石墨烯分成三種平面的尺寸，分別為1000nm以上、400~1000nm以及200nm以下，最後接枝上水性高分子Polyethylene glycol (PEG)以增加其懸浮性與生物相容性。
2. 將GO、PEG-g-GOs與嗜中性白血球進行毒性與發炎反應測試，發現GO、PEG-g-GOs皆不會明顯對嗜中性白血球造成傷害，但GO會使嗜中性白血球相互聚集與黏附，並具有濃度相關性的刺激細胞內自由基的產生，但PEG-g-GOs卻沒有這樣的問題，代表PEG-g-GOs可以有效的避過嗜中性白血球的吞噬，更證明了PEG-g-GOs的高生物相容性與穩定性。
3. 將PEG-g-GOs吸附上放射性元素Tc-99m，並利用小動物單光子發射電腦斷層掃描系統(Nanospect/CT)與放射顯像法(Autoradiography)去追蹤PEG-g-GOs於生物體內的分佈，可得知不同尺寸的PEG-g-GOs於生物體器官內的分佈有很明顯的差異，並且在接枝上水性高分子後，材料於生物體內的分散性與穩定性皆有明顯的提升。PEG-g-GOs (<200 nm)分佈的器官以脾臟為主；PEG-g-GOs (400-1000 nm)分佈的器官以肝臟為主；PEG-g-GOs (>1000 nm)分佈的器官則以肺臟為主。了解各尺寸材料於器官的分佈情形後，除了可以利用尺寸控制去進行被動標靶(Passive targeting)的治療外，也可藉由分佈情形去了解何種尺寸的材料循環能力最佳，提供了製備成為主動標靶(Active targeting)生醫載體材料之未來性，更重要的一點是透過長時間的觀察，PEG-g-GOs會隨著時間漸漸代謝出體外，並不會有殘留於體內的疑慮，所以透過本研究可以更加確立了石墨烯在生醫載體領域上的應用性。

關鍵字

石墨烯 ； 生物分佈 ； 自由基 ； 生醫載體

並列摘要

The first object of this work is the preparation of novel multi-functional bio-carrier. To improve the low drug loading efficiency of traditional bio-carrier, this study used a rising star in carbon-based nanomaterials, graphene. Among these Graphene-based nanomaterials, graphene oxide (GO) has been proposed as the most promising bio-carriers due to its ultrahigh specific surface area and extraordinary chemical capability.
i. The application of superoxide scavenging by using graphene-based material
1. To increase dispersibility of bio-carrier in water, this study used modified Hummers method to prepare graphene oxide (GO) graphite powder. The graphene oxide was further modified by grafting poly acrylic acid(PAA) to increase its stability and biocompatibility in physiological solutions.
2. PAA-g-GOs was incubated with GL261 tumor cells. PAA-g-GOs would scavenge the fluorescent by using DCFH-DA method that confirmed the superoxide scavenge ability of PAA-g-GOs.
3. By in vitro and in vivo experiment, the growth of tumor cells would be inhibited due to the superoxide scavenging ability of PAA-g-GOs. These results indicated that PAA-g-GOs is a promising candidate for novel multi-functional bio-carrier of clinical tumor treatment.
ii. The application of biodistribution by using graphene-based material
1. The effects of various dimensions of GO on the organ distribution have been investigated in this work. The GO with three different dimensions can be fabricated by ultrasonication, centrifugation, and filtration method. The stability and biocompatibility of GO in physiological solutions were increased by grafting PEG on GO surface.
2. PEG-g-GOs was incubated with neutrophil cells to investigate its toxicity and inflammation. It was found that both GO and PEG-g-GOs will not apparently cause damage to neutrophil. However, GO will cause the mutually aggregation of neutrophil and dose-dependent stimulation inducing the induction of the free radicles while PEG-g-GOs will not, this represents that PEG-g-GOs can effectively avoid phagocytosis by neutrophil, moreover, it verifies the high biocompatibility and stability of PEG-g-GOs.
3. The radioactivity element Tc-99m was absorbed on PEG-g-GOs to form 99mTc-PEG-g-GOs that was tracked by Nanospect/CT and Autoradiography method to observe the biodistribution in vivo. The results of Nanospect/CT and Autoradiography showed that the dimension of GO is a major factor to control the biodistribution. PEG-g-GOs (<200nm) were mainly accumulated in spleen; PEG-g-GOs (400-1000nm) were mainly accumulated in liver; PEG-g-GOs (>1000nm) were mainly accumulated in lung. We can do the passive targeting as the treatment by the distribution of bio-carrier. Moreover, we are capable of discovering which material is the best in cycle ability and provide a probability of active targeting bio-carrier in the future. The most important thing is that in the long-term biodistribution result suggests the gradual clearance of PEG-g-GOs from mice. It means PEG-g-GOs would not residual in vivo for long time. Nevertheless, our results are highly encouraging and pave the way for future graphene based in vivo biomedical research.

參考文獻

62. 徐善慧, 奈米生醫材料. 中興大學化工系 組織工程與幹細胞研究中心.

2. 李仕明, 國立清華大學化學工程學系碩士論文. 馬振基教授指導, 2010.

20. 吳至彧, 國立清華大學工程與系統科學系碩士論文. 2009.

1. 楊士億, 國立清華大學化學工程學系碩士論文. 馬振基教授指導, 2009.

3. Novoselov, K.S., et al., Electric field effect in atomically thin carbon films. Science, 2004. 306(5696): p. 666-669.