多功能奈米複合還原氧化石墨烯/二氧化矽載體之結構特性及光熱效應於腫瘤治療之研究

Abstract

近年來，以氧化態石墨烯(GO)為奈米載體用於光熱治療對癌細胞影響的探討與研究越來越熱門，主要因為GO具有許多的優點。(1)其結構上具有許多官能基，例如氫氧基(OH)和羧酸(COOH)，可以藉由化學鍵結的方式去攜帶藥物;(2) GO烯由苯環結構組成，因為許多抗癌藥物具有苯環結構，所以可以用物理方式藉由π-π作用力把藥物攜帶再氧化石墨烯的上面，進行癌細胞藥物治療研究;(3)還原氧化石墨烯(rGO)可以把吸收的近紅外光轉換成熱的型式散發，進而達到定點殺死癌細胞的效用。如此一來，藥物治療結合光熱治療不僅可以減少藥物劑量使用進而改善傳統化療上藥物對正常細胞產生的副作用問題;抑或可以藉由等待其藥物載體達到病灶位置再用光去產生熱與藥物協同作用提高投藥的成功率。然而，目前有關以rGO為藥物載體的研究，仍然有許多地方需要改善，例如:單純以rGO為載體並攜帶藥物的能力有限，或無法判定藥物載體是否已達到病灶位置。 為了改善這些問題，於本論文第一部分研究為解決疏水性藥(CPT)不易溶於水而被人體所吸收，用化療結合光熱治療來提高癌細胞治療效果。首先，我們發展出一個以rGO為基材覆蓋一層多功能孔洞的二氧化矽奈米載體(rGO/SiO2)，二氧化矽洞不僅可以改善rGO的生物相容性，其孔洞內碳化的高分子，可以提高疏水性藥物吸附量，同時在特定波長(808nm)雷射的照射下，由於石墨稀受到熱效應的影響而振動，把光能轉換成熱能，達到特定時間藥物釋放兼具熱療的效果。研究結果顯示，此藥物載體具有攜帶高濃度的疏水性藥物、階段性釋藥、並針對病灶位置作熱療的能力，癌細胞治療效果明顯優於傳統化學藥物治療效果。 由於治療結合診斷功用變得越來越重要，傳統上用螢光染劑來標示細胞位置，但螢光強度會隨著照光時間的增加而減弱，造成使用上的不方便。所以近年來以拉曼影像來追蹤藥物分子的技術逐漸受到重視，雖然拉曼影像具有良好的光穩定性，但傳統的拉曼影像強度太低，無法用於細胞顯影辨識，另一方面，以rGO為熱療的載體需用高能量的雷射，而且因為熱會擴散，如果在長時間的照射下會造成癌細胞周圍正常細胞的損害。所以於本論文的第二部分，調控在rGO/SiO2孔隙的大小，藉由溶劑在抽真空的環境下逐漸揮發，使得金奈米粒子可以依序地填入孔內。透過簡單的承載方式，不僅改善金奈米粒子在生物體內易團聚的問題，且奈米金粒子具有拉曼表面增強效應可克服拉曼強度太弱的問題，同時紅外光吸收能力的增加，提升了載體的熱轉換效率，並進一步結合標靶分子的作用，對於特定腫瘤細胞的標靶治療有明顯的差異，降低負作用的產生，達到新一代載體具標靶顯影與治療效果。 第三部分實驗，主要是針對目前臨床上對光動力的應用侷限在皮膚或表層癌細胞的治療，因為光對生物體的穿透能力有限，所以本實驗藉由在rGO/SiO2載體藉由化學鍵結方式修飾上氧化鐵奈米粒子與Rose bengal(sonodynamic agent)，結合磁引導的作用，在聚焦式超音波的照射下，對特定部位的腫瘤細胞達到以超音波產生自由基與熱療殺死癌細胞的效果。實驗證明，用聚焦式超音波對修飾具有rose Bengal的載體進行照射比單純用可見光對rose bengal照射所產生的自由基還要高，所以此載體不僅提高自由基產生的效率，且結合氧化鐵奈米粒子，不僅提供熱療的效果，加上磁引導的作用，大大增加自由基殺癌細胞的效果。未來期望臨床上藉由結合多功能的奈米粒子達到診斷與快速治療的效果。

In recent year, the study of photothermal therapy with graphene oxide (GO) as nanocarrier is more and more popular. There are many merits on reduced graphene oxide. Firstly, one of the advantages is many functional group on its structure, such as hydroxyl group and carboxylic group. It can carry anti-cancer drug by chemical bonding with these group, or absorb the drug on its plane, which constructing from benzene unit, with physical interaction (π-π interaction) for cancer therapy. Secondly, some studies reported reduced graphene oxide (rGO) can absorb infrared radiation (IR) and transfer to thermal effect to kill cancer cell. Thus, combining photothermal effect with chemotherapy can reduce the dose of drug and reduction of side effect to normal cell comparing to conventional chemotherapy. Besides, with near IR irradiation, we can control drug release and simultaneously generate thermal effect when the rGO as drug nanocarrier reached the tumor site. This can not only improve drug performance but also cancer therapy. However, there are many obstacles need to solve, for example, the limit drug capability of pure reduced graphene oxide, or without imaging system we can not track the drug whether it get to target site. In order to improve the current obstacles, in the first part of this thesis is focused on the capacity of hydrophobic drug (CPT) on nanocarrier and combine chemotherapy with photothermal therapy to increase the therapeutic effect on tumor cell. First, we developed a reduced graphene oxide/silica oxide (rGO/SiO2) nanocarrier, which used rGO as template and cover with functional porous silica. Porous silica not only can improve the biocompatibility of rGO, but increase the hydrophobic drug capability due to the carbon layer into the porous silica. And with the specific wavelength of light irradiation (808nm), rGO can transfer the photo energy by vibration to thermal energy, and can further control the drug release in certain time. The result showed with high drug capability, control drug release, and photothermal effect in a system, the tumor killing effect is more excellent than conventional chemotherapy. Combining therapy with diagnosis system in biological field is becoming more and important. Conventionally, the intensity of fluorescent dye applied to track location of drug molecule in biological system will decay with the time of irradiation. Recently, the Raman imaging technique for tracking biological molecule has received a lot of attention. Although Raman imaging has a good photostability under long-term irradiation, the intensity of Raman spectrum is too weak to identify the cell image. On the other hand, it needs high power density or extending irradiation time under low power of near IR energy for using rGO as photothermal agent to kill cancer cell. Because heat will diffuse and affect the normal cell around tumor, there will be hazardous if irradiation time extends. In the second part of this thesis, we further controlled the pore size of rGO/SiO2 nanocomposite and loaded gold nanoparticles into the pore by solvent evaporation under vacuum. With this simple loading method, we successfully separated the gold nanoparticles and avoided aggregation in biological system, and enhance the Raman imaging intensity, and improve photothermal efficiency of rGO. And with conjugation of target protein on nanocomposite, we can promote the specific tumor therapeutic effect and reduce the side effect, achieving a new generation of nanocarrier combining targeting, imaging and therapy in a system. In the third part of this thesis, we focused on improving the photodynamic therapy system forward clinical application. So far, it is limited applying for skin cancer or superficial tumor therapy due to the limited penetration ability of light. We fabricated a Fe3O4 caped nano-reduced graphene oxide/ mesoporous silica (nrGO/MSN) nanocomposite with conjugation of rose bengal. This nanocomposite under focus ultrasound irradiation and magnetic guiding can increased specific cytotoxicity on tumor cell. Modification of rose bengal on nanocomposite under focused ultrasound irradiation can produce more singlet oxygen than under photo-irradiation. Besides, with the Fe3O4 capping, the synergistic effect of thermal and magnetic guiding can improve the efficiency of killing tumor cell. Finally, in the future, we hope drug modified functional nanoparticle can be applied in clinical usage to perform therapy and diagnosis effect.