奈米碳材做為靶向藥物載體之製備及其效果之研究

Preparation and Properties of Carbon Nanomaterials as targeted drug carriers

李仕明(Shin-Ming Li)

指導教授 ： 馬振基

國立清華大學/工學院/化學工程學系/碩士(2011年)

摘要

本研究的主要目的為兩種藥物釋放系統的製備、檢測以及它們在生物體外的癌細胞毒殺測試。由於傳統對癌細胞的藥物治療不但浪費藥物還容易引起副作用，故本研究利用兩種不同的碳材作為藥物載體，期望能改善傳統藥物治療的缺點。第一種係以多壁碳奈米管為主體的藥物載體，第二種則為以奈米氧化石墨烯為主體的藥物載體。
在多壁碳奈米管藥物載體的部分，結合多壁碳奈米管、磁性粒子Fe3O4 、抗癌藥物DOX、標靶分子FA和螢光染料FITC形成多功能的多壁碳奈米管藥物載體。此研究主要包含以下三個步驟 :
1. 將多壁碳奈米管酸化並純化之，以減低毒性、增加生物可相容性和親水性。
2. 在多壁碳奈米管表面接枝上聚丙烯酸PAA形成PAA-CNTs以增進生物相容性，同時增加爾後修飾奈米磁性粒子的方便性。
3. 透過在PAA-CNTs上修飾之FA、FITC及Fe3O4使載體獲得磁性和主動標靶的能力，再加上多壁碳奈米管的高度藥物承載量，將此多功能性載體和U87MG神經膠質瘤細胞作共同培養，測試細胞存活率並評估其治療能力。
將PAA-CNTs以高解析電子能譜儀(XPS)做定性分析，再以熱重損失分析儀(TGA)做定量分析，另外再以TEM觀測修飾了磁性粒子的PAA-CNTs之表面型態。
由TGA的分析，可得知接枝在多壁碳奈米管上的PAA含量大約為23.1wt%。經過XPS的分析，得知在PAA-CNTs上比多壁碳奈米管多出了兩個訊號峰，分別在285.2eV處和286.1eV處，這都是源自於PAA主鏈上的特徵峰，說明了PAA有成功接枝在多壁碳奈米管上。而在TEM的觀測下則可發現磁性粒子Fe3O4均勻的分佈在多壁碳奈米管表面上。
PAA-CNTs的DOX承載量比純多壁碳奈米管要來的高，分別是187.6 wt%和95 wt%，這是由於PAA-CNTs上的PAA有帶負電的官能基，可和在水溶液中帶正電的DOX有電荷相吸的現象，因此PAA-CNTs可大幅增加DOX的承載量。此外，從PAA-CNTs上釋放的DOX受制於釋放環境的pH值，在酸性的環境下DOX釋放快，而在中性的環境下釋放慢，由於細胞內部呈酸性，所以此現象有助於藥物載體進入細胞後的釋放。
而為了要確定標靶分子FA對於載體藉由胞吞作用進入細胞的助益，將沒接FA的載體PAA-CNTs-Fe-FITC與有接FA的載體PAA-CNTs-FA-Fe-FITC分別與U87MG細胞做培養，並在共軛焦顯微鏡下觀察兩者進入細胞的情形。結果顯示兩者皆可進入細胞內部，但PAA-CNTs-FA-Fe-FITC進入細胞的量比PAA-CNTs-Fe-FITC要來的多，說明了FA對於載體進入細胞有額外的幫助。
在後續用多壁碳奈米管藥物載體對細胞的毒殺實驗中，可發現PAA-CNTs-FA-Fe此組接上DOX對於U87MG細胞的毒殺效果最好，在這個實驗中使用了IC50 (half-maximum inhibitory concentration)這個概念來評估載體效能，IC50就是指使細胞存活率降到一半所需的DOX濃度。PAA-CNTs-FA-Fe-DOX的IC50是28μg/ml，而純DOX的IC50則為89μg/ml，顯示PAA-CNTs-FA-Fe-DOX的細胞毒殺效果遠比純DOX來的高。
最後將藥物載體PAA-CNTs-Fe-DOX、PAA-CNTs-FA-Fe-DOX和U87MG標共同培養並施與一外加磁場來測試磁導集中對細胞毒殺是否有影響。結果顯示在有磁場施加的培養皿區域，其細胞的生長情形遠低於其他沒有磁場施加的區域，這現象說明了在磁場的作用下將載體集中在一區使此區的藥物濃度變得比其他區域來的高，因此可以有效壓抑癌細胞的生長。綜合上述結果可以說明本研究製備的多功能碳奈米管載體在未來癌細胞的治療上有很大的應用潛力。
在奈米氧化石墨烯藥物載體的部分，本研究則結合奈米氧化石墨烯和抗癌藥物BCNU形成藥物載體，研究主要包含以下四個步驟 :
1. 用酸氧化石墨烯形成氧化石墨烯，以超音波粉碎的方法減小其尺寸並加以純化，以減低毒性、增加生物可相容性和親水性。
2. 在奈米氧化石墨烯表面接枝上聚丙烯酸PAA形成PAA-GOs以增進生物相容性，增加爾後於生物培養液中的穩定性。
3. 在PAA-GOs表面接上一種螢光染料Cyanine-5(Cy5) ，並和另一種神經膠質瘤細胞GL261做培養，觀測PAA-GOs進入GL261細胞的情形。
4. 將BCNU接於PAA-GOs上形成PAA-GOs-BCNU，並和GL261細胞作共同培養，測試細胞存活率並評估其治療能力。
以高解析電子能譜儀(XPS)對PAA-GOs做定性分析，再以熱重損失分析儀(TGA)做定量分析，另外再以TEM和AFM觀測其表面型態和尺寸大小。
由TGA的分析，可得知接枝在奈米氧化石墨烯上的PAA含量大約為15.7%。由XPS的分析，得知在PAA-GOs上比奈米氧化石墨烯多出了兩個訊號峰，分別在285.1eV處和285.9eV處，這都是源自於PAA主鏈上的特徵峰，說明了PAA已成功接枝在奈米氧化石墨烯上。而在TEM下觀測GOs和PAA-GOs可明顯的發現GOs為一層薄且具摺皺的材料，在PAA-GOs上則可觀察到GOs的部分以及高分子覆蓋的部分。最後由AFM的數據可發現PAA-GOs的厚度大約是GOs的兩倍左右，再度說明了PAA已成功接於GOs上。此外，PAA-GOs和GOs的尺寸大約在60nm以內，顯示以超音波粉碎的方式可以有效減小兩者的大小。
為了證明PAA-GOs能否被GL261細胞吞噬，將PAA-GOs接上Cy5形成PAA-GOs-Cy5並和GL261細胞共同培養。藉由共軛焦顯微鏡的影像可觀察到PAA-GOs確實能被GL261細胞吞噬並均勻分布在其細胞質中。
在進行後續奈米氧化石墨烯藥物載體對GL261的毒殺實驗前，必須先確定純PAA-GOs本身對於癌細胞是否有毒性，因此將PAA-GOs和GL261做共同培養。結果顯示即使PAA-GOs的濃度達到200μg/ml，GL261細胞的存活率仍有七成以上，說明PAA-GOs並不會對GL261的生長造成太大影響。再將BCNU接於PAA-GOs其上形成PAA-GOs-BCNU，並和GL261細胞共同培養，再以純BCNU和GL261細胞共同培養作為對照組，比較兩組的細胞存活率。結果顯示當BCNU的濃度達到50μg/ml，PAA-GOs-BCNU此組的細胞數只剩約百分之二十；而純BCNU則有約百分之七十五存活，此現象說明以PAA-GOs作為藥物載體能在較低的藥物濃度下達到較高的細胞毒殺效果，如此能有效避免藥物的浪費。
本研究發展出以多壁碳奈米管為主體的藥物載體與以奈米氧化石墨烯為主體的藥物載體，它們皆可在攜帶較低濃度藥物的情況下來達到較好的癌細胞毒殺效果。在多壁碳奈米管藥物載體方面，本研究設計出了一多功能的藥物載體PAA-CNTs-FA-Fe-DOX，它可在磁性和標靶分子的雙重效果有效集中毒殺癌細胞並可避免藥物浪費。在奈米氧化石墨烯藥物載體方面則是純粹將PAA-GOs及BCNU結合形成PAA-GOs-BCNU來進行癌細胞毒殺實驗，結果說明了在攜帶較低濃度BCNU的情況下就能達到較好的癌細胞毒殺效果，也就是當PAA-GOs-BCNU中的BCNU濃度達到50μg/ml便能使GL261細胞的死亡率上升至八成左右，相對而言，相同濃度的free BCNU只能造成約二成五的細胞死亡率。這兩種載體在未來的癌症治療上可望有很大的應用潛力。

關鍵字

藥物 ； 載體 ； 碳材 ； 靶向

並列摘要

The objectives of this research are the preparation, characterization and in vitro cell tests of two drug delivery systems. Since the conventional drugs belong to the dump system, people have to take more than the predicted amounts of drugs to reach the therapeutic effect. Hence, this study has developed two types of carbon materials as drug carriers to overcome the drawbacks of conventional drugs. The first one is multi-walled carbon nanotubes (MWCNTs) based drug delivery system and the second one is graphene oxides (GOs) based drug delivery system.
In the aspect of carbon nanotubes, multi-walled carbon nanotubes (MWCNTs) was combined with the iron oxide magnetic nanoparticles (Fe3O4), anti-cancer drug doxorubicin (DOX), targeting molecule folic acid (FA), and fluorescent dye (FITC) to form a multifunctional MWCNT drug carrier system. This study includes the following subjects:
1. Acid oxidation and purification of the MWCNTs to reduce toxicity and to increase the biocompatibility and aqueous solibility.
2. Supramolecular modification of the MWCNT surface with poly acrylic acid (PAA) was conducted to obtain the buffer dispersible PAA-CNT. The possibility of the following magnetic particle decoration was evaluated.
3. Conjugation with folic acid, FITC and Fe3O4 (magnetic particle) were grafted on the PAA-CNT surface to acquire the ability of magnetic guidance and primary targeting. Accompanying the high drug loading ability of the MWCNT, the chemotherapeutic ability during co-culture with the U87MG glioma cells was evaluated and the cell survival was investigated.
PAA-CNTs were analyzed by X-ray photoelectron (XPS) and thermogravimetric analysis (TGA). The morphology of PAA-CNTs-Fe was observed by Transmission electron microscope (TEM).
Through TGA analysis, the content of PAA on MWCNT was estimated about 23.1wt%. From XPS analysis, it was found that the additional XPS peaks of PAA-MWCNTs appeared at 285.2, 286.1eV, originated from the PAA main chain; these peaks confirmed that MWCNTs were successfully functionalized. Then from the TEM image of PAA-CNTs-Fe, one can observe the uniform dispersion of Fe3O4 nanoparticles on the PAA-MWCNT surface.
PAA-MWCNTs exhibit much higher DOX loading efficiency than that of pristine-MWCNTs. The DOX loading efficiencies of pristine-MWCNTs and PAA-MWCNTs were 95% and 187.6%, respectively. This suggests that PAA polymer chain on MWCNTs can provide excellent electrostatic interaction with positively charged DOX due to the negatively charged functional groups such as carboxyl group. Thus, PAA-MWCNTs can combine electrostatic and stacking interactions to increase the efficiency of DOX loading. Furthermore, the release of DOX from PAA-MWCNTs was pH-dependent. The release of DOX was fast in acid environment but slow in neutral environment. This phenomenon suggested that PAA-MWCNTs were promising delivery materials for anticancer drug DOX.
It is intend to investigate the function of the targeting molecules FA which plays the key role of receptor-mediated endocytosis. U87MG cells were incubated with PAA-CNTs-Fe-FITC and PAA-CNTs-FA-Fe-FITC, respectively. By confocal fluorescence microscopy imaging, one can observe that both PAA-CNTs-Fe and PAA-CNTs-FA-Fe can be transported into cells via endocytosis. However, PAA-CNTs-FA-Fe-FITC revealed intense green fluorescence than PAA-CNTs-Fe-FITC in U87MG cells. It means that the effect of FA can assist the PAA-CNTs to enter the cell efficiently. This important result will help to develop MWCNT functionalization schemes with specific materials for recognizing and targeting different types of tumor cell.
In the tumor cell toxic tests, PAA-CNTs-FA-Fe was designed to assist the antitumor drugs DOX to eliminate the brain tumor cells (U87MG) with less side reaction, and this drug delivery system exhibited the highest cytotoxicity against U87MG cells. In comparison with free drug, the IC50 (half-maximum inhibitory drug concentration) value for PAA-CNTs-FA-Fe-DOX (~28μg/ml) is much lower than that of free DOX (~89μg/ml), suggesting the PAA-CNTs-FA-Fe loaded with DOX can enter into cell effectively and release DOX into cell nuclei rapidly.
Finally, the magnetic targeting test was conducted to confirm the magnetism of PAA-CNTs based drug delivery system and its effect on cell toxic tests. For U87MG cells which was treated by PAA-CNTs-Fe-DOX or PAA-CNTs-FA-Fe-DOX with an external magnetic field, it can be observed that almost no U87MG cells were located on the outer of culture mediums and the number of tumor cell decreasing significantly in magnetic zones, which implies that these zones attracted more drug carriers and thus possessed higher drug concentration to inhibit the survival of tumor cells. These phenomena indicate that magnetic MWCNTs drug delivery system is a very promising nano-platform for future cancer therapeutics.
In the aspect of graphene oxides, GOs were combined with anti-cancer drug Carmustine (BCNU) to form a drug carrier system. This study includes the following subjects:
1. Preparation of the acid-oxided graphene (GOs), the length of GOs was shortened by ultrasonication, then the toxicity was also reduced and the biocompatibility will be increased via purification.
2. Supramolecular modification of the GO surface with poly acrylic acid (PAA) to obtain the biocompatible GO, rendering stability in physiological solutions including serum.
3. In order to reach the target of fluorescent detection during cell tests, Cyanine-5(Cy5) was decorated on the surface of PAA-GOs and carried on the incubation with GL261 glioma cells to observe cell endocytosis of PAA-GOs.
4. To form a drug delivery system, BCNU was attached on the PAA-GOs surface. The chemotherapeutic ability of PAA-GOs-BCNU during co-culture with the GL261 glioma cells was evaluated in comparison with free BCNU.
PAA-GOs were analyzed by X-ray photoelectron (XPS) and thermogravimetric analysis (TGA). The morphology and dimension of PAA-GOs were observed by Transmission electron microscope (TEM) and Atomic force microscope (AFM).
Through TGA analysis, the content of PAA on GO was estimated about 15.7 wt%. From XPS analysis, it was found that the additional XPS peaks of PAA-GOs appeared at 285.1, 285.9 eV, which were originated from the PAA main chain, these peaks confirmed GOs were successfully functionalized. Then from the TEM image of GO and PAA-GO, one can observe that GO was a thin material with wrinkles while PAA-GO showed obvious PAA-covered area. Finally, from the AFM image GO and PAA-GO one can find the dimensions of GO and PAA-GO were less than 60nm, suggesting that continued ultrasonication helped reduce the dimensions of both materials. The thickness of PAA-GO was about twice of GO, this also confirmed that PAA was grafted on GO successfully.
In order to confirm that PAA-GOs can be transported into glioma cells, GL261 cells were incubated with PAA-GOs-Cy5 to proceed the fluorescent detection. Cy5 (Cyanine-5) is a kind of synthetic red fluorescent dye which is often used in biomedical imaging. By confocal fluorescence microscopy imaging, one can observe that PAA-GOs were transported into GL261 cells via endocytosis and spread uniformly inside cells.
In order to confirm whether pure PAA-GOs were toxic to tumor cells or not, PAA-GOs were incubated with GL261 cells at first. Results showed that even when the concentration of PAA-GO reached up to 200μg/ml, cell survival was still very high (>70%). This suggested pure PAA-GOs did not induce cell death. Hence, PAA-GOs was combined with the antitumor drugs BCNU to proceed the following tumor cell toxic tests. In comparison with free BCNU, when the concentration of BCNU reached up to 50μg/ml, the number of cells treated with PAA-GOs-BCNU only remained 20%. At the same concentration of BCNU, the number of cells treated with free BCNU still remained 75%. These results suggest that PAA-GO drug delivery system with less BCNU can reach the better cancer therapeutics. This can reduce the usage of drugs and prevent the waste of drugs.
In this study, two types of carbon material (MWCNT and GO) based drug delivery systems have been developed to increase their effect in killing tumor cells and to decrease the usage of drugs at the same time. Regarding MWCNT-based drug delivery system, the hydrophilic, pH sensitive, and dual targeting drug vehicle, PAA-CNTs-FA-Fe-DOX was designed to eliminate U87MG cells in vitro. From the results it was found that doxorubincin could focus on the tumor region and decreased unnecessary peripheral toxicity by the magnetic guidance (primary targeting) and the affinity of FA (secondary targeting). Regarding GO-based drug delivery system, PAA-GO was combined with BCNU to form PAA-GO and it was used to eliminate GL261 cells in vitro. From the results it was found that PAA-GO with less BCNU could kill GL261 cells effectively in comparison with free BCNU. It is anticipated that both of these materials can become very promising drug delivery platforms for cancer therapeutics effectively.

參考文獻

[1] 楊士億. 國立清華大學化學工程學系論文 馬振基教授指導. 2009.

[2] Iijima S., Helical microtubules of graphitic carbon. Nature. 1991;354(6348):56-8.

[3] Sandler J., Shaffer M.S.P, Prasse T., Bauhofer W., Schulte K., Windle A.H., Development of a dispersion process for carbon nanotubes in an epoxy matrix and the resulting electrical properties. Polymer. 1999;40(21):5967-71.

[5] Subramoney S., Novel nanocarbons¡Xstructure, properties, and potential applications. Advanced Materials. 1998;10(15):1157-71.

[7] Treacy M.M.J, Ebbesen T.W, Gibson J.M., Exceptionally high Young's modulus observed for individual carbon nanotubes. 1996.