具有磁性及標靶功能藥物載體的合成與分析用於腦瘤治療

Abstract

由於腦血屏障（the blood-brain barrier, the BBB）的存在，它嚴格控管各種物質進入腦部，導致運送抗癌藥物到腦部並維持其高藥物濃度及劑量成為相當困難的工作[1]。本篇論文研究重點在於開發新型又獨特的藥物載體，稱之為「可調控式磁性藥物載體」（Lf-PCDA-Cur-Carriers），該載體藥物能協助將藥物跨越前述之屏障並運送到腦部以治療腦瘤，加以其標靶特性，能使載體具備專一性，到達預期之特定部位，達成治療功效。 本次研究中，我們將聚乙烯醇（polyvinyl alcohol, PVA）、超順磁性氧化鐵（superparamagnetic iron oxide, SPIO）奈米粒子以及10,12-二十五烷基二炔酸（10,12-pentacosadiynoic acid, PCDA）設計合成奈米載體。利用超順磁性氧化鐵的特性，賦予載體十分獨特的能力，像是磁引導與磁共振造影（magnetic resonance image, MRI）。最後，磁性藥物載體在表面修飾乳鐵蛋白（lactoferrin, Lf），讓載體可以穿越腦血屏障，有效提升進入腦部的效率。載體大小在一百到一百五十奈米之間；而載體的表面電荷大約在負十三到負五之間，蛋白質和奈米載體的嫁接情形則用紅外光譜儀和蛋白質定量分析獲得鑑定。此外，經由不同的聚合度（degree of polymerization）可調控薑黃素（curcumin, Cur）的釋放速率。並以大鼠惡性腦膠質瘤細胞（rat malignant glioma, RG2）作為細胞實驗和動物實驗的模型，並給予治療。最後，在動物實驗方面，以大鼠原位腫瘤實驗測試奈米載體的效果，並藉由磁共振顯影，可精確地追蹤載體進入體內的動向，偵測腫瘤的位置。 「可調控式藥物載體」的動物實驗結果顯示，植入大鼠惡性腦膠質瘤細胞的大鼠大腦，雖然可以有效降低腫瘤的成長，但卻因為單一藥物的能力不佳，不能延長大鼠的壽命。為獲取更好的效果，在本文第二部分，更進一步改良了載體，將聚丙烯酸（polyacrylic acid, PAA）、聚乙烯醇和超順磁性氧化鐵合成了雙層結構的奈米載體，可包覆兩種不同的藥物。相較之前的治療藥物，使用了薑黃素以及小紅莓（doxorubicin, Dox），並將載體取名為「雙層奈米載體」。同樣以掃描式電子顯微鏡、穿透式電子顯微鏡和動態光散射儀偵測載體大小，也大約在一百到一百五奈米之間。乳鐵蛋白被修飾在奈米載體的表面，用來標靶腦血屏障及癌細胞，以順利將藥物運送到腦部。最後，經由生物實驗測試雙層奈米載體，一方面共同培養載體和大鼠惡性腦膠質瘤細胞，另一方面治療患有腦癌細胞的小鼠。最後結果顯示，嫁接乳鐵蛋白的載體將有效地標靶進入癌細胞，配合外部磁鐵吸引也能提高載體在體內局部的累積，由數據得知，包覆雙重藥物的治療效果也較單純使用薑黃素來的優異。

It is difficult that maintain a high concentration of therapeutic agents at the tumor site due to the blood-brain barrier (BBB). Especially in chemotherapy for brain tumor, we have to provide against the spread of cancer cell into healthy tissue [1]. The primary aim of this study is to develop a special delivery system which could transport drugs across the BBB and target the brain glioma. Here, we designed a novel drug nanocarrier system which is composed of polyvinyl alcohol (PVA), superparamagnetic iron oxide (SPIO) nanoparticles and 10,12-pentacosadiynoic acid (PCDA). SPIO nanoparticles were encapsulated in the carriers that could be used as magnetic guide and magnetic resonance image (MRI) contrast agent for tracking the position of carriers and tumor. The magnetic PCDA-carriers were further surface-modified by the lactoferrin (Lf) to enhance the transportation of drug-carrier across the BBB by targeting brain glioma. Several characterizations will be performed on the nanocarriers. The particle size and nanostructure of Lf-PCDA-Carriers were about 100 to 150 nanometers. The Zeta-potential was -13.5 ± 3 to -5.33 ± 0.68 mV and Bradford analysis as well as infrared were used for proving the conjugation between Lf and PCDA-Carriers. Besides, we could control the rate of cumulative release by various degree of polymerization. We used rat malignant glioma (RG2) cells as a cancer model in cell culture as well as animal experiment and curcumin (Cur) as a therapeutic agent for treatment in this investigation. In vivo targeting effect and MRI tracing were evaluated on RG2 tumor-bearing rats of orthotopic sites. The results show that Lf-PCDA-Cur-Carriers could reduce the growth of tumor on the rats implanted RG2 into the brain of striatal region. But we couldn’t prolong significantly the survival time of rats by only using Cur as the therapeutic drug. Therefore, in the second part of the thesis, to improve the therapeutic efficiency. PAA-Carriers which composed of PVA, SPIO nanoparticles and polyacrylic acid (PAA) were further synthesized for encapsulating two type of drugs which are Cur and doxorubicin (Dox) into nanoparticles simultaneously. The size of PAA-Carriers were still 100 to 150 nanometers measured by SEM, TEM and DLS. Lf was also modified in the surface of our carriers to transport drugs into the BBB. Finally, Lf-PAA-Dox-Cur-Carriers were confirmed on biological experiments by incubating with RG2 cells and injecting into brain tumor-bearing mice. All these results show that drug carriers coated with Lf had high efficiency of entering brain tumor cells and the external magnet guided high accumulation of drug carriers in specific location. Two types of drugs encapsulated in the nanocarriers made synergistic effects and better performance compared to single drug.