以聚乙烯亞胺修飾之石墨烯攜帶DNA佐以外在刺激釋放之標靶載體應用於神經基因治療

Abstract

在過去，因為神經細胞 (neuron) 特殊的形態及性質增加了現今療法對於神經退化性疾病 (neurodegenerative disease) 的治療困難度，然而，最近數年來，基因治療 (gene therapy) 漸漸發展為具有潛力的解決辦法之一，為神經退化性疾病提供另一種分子層面的治療方式。此外，結合標靶性治療及物理性刺激釋放，將有機會大大地增加藥物的治療效率。因此，本篇研究主要開發一基因奈米載體系統，並探討其在細胞內的輸送行為以及治療效果。 第一部分是利用化學剝離法所合成的石墨烯 (reduced graphene oxide, rGO)，先藉由多正電荷高分子─聚乙烯亞胺 (polyethylenimine) 來進行石墨烯表面的改植，使表面覆蓋聚乙烯亞胺並帶有高正電荷，接著在聚乙烯亞胺的一級胺基 (primary amino group) 嫁接神經標靶胺基酸─神經調壓素 (neurotensin)，使奈米載體具有神經細胞專一性，將奈米載體與帶有綠螢光蛋白 (green fluorescence protein, GFP)基因之質體DNA (pDNA) 混合後，送入神經細胞內進行轉染。根據文獻，聚乙烯亞胺擁有優秀的轉染效率及逃脫溶酶體的能力 (endo/lysosome escaping ability)，而且具有高正電荷，可吸附甚至保護負電荷的質體DNA，藉由此材料的改植，賦予了石墨烯可進行吸附質體DNA與轉染的能力，然而聚乙烯亞胺的生物毒性卻不低，因此我們以神經調壓素加以修飾石墨烯上的聚乙烯亞胺，結果顯示不僅降低了奈米載體的細胞毒性，也增加了對於神經細胞的標靶能力，從螢光顯微鏡及共軛焦雷射顯微鏡之影像可以觀察到，奈米載體有效地大量累積在已分化的 (differentiation) 類神經細胞株─大屬腎上腺嗜鉻細胞瘤 (PC-12) ，從最終細胞轉染實驗結果顯示，轉染效率也有所提升。也進行了活體小鼠 (mice) 腦部的實驗，證明神經調壓素可確實提供幫助來針對神經細胞的轉染。 在第二部分，除了第一部分所製備之奈米載體外，也施加了外在物理性近紅外光雷射 (near infrared laser) 的協助，藉由雷射與石墨烯之間特別的協同效應所產生的熱震動，不僅可以使細胞膜的通透性暫時增加，同時可以促使奈米載體所攜帶的藥物釋放，更重要的是可以增加奈米載體逃脫溶酶體的機率，避免攜帶的質體DNA被DNA分解酶I降解掉。實驗中，我們設計了兩步驟的雷射施加，第一步驟雷射 (laser-step-1) 主要功用是暫時增加細胞的膜通透性，增加奈米載體進入細胞的數量，藉由細胞流氏儀可以觀察到，在有施加近紅外光雷射的情況下，細胞吞入奈米載體的數量增加了七倍。第二步驟雷射 (laser-step-2) 主要功用則是增加奈米載體逃離溶酶體的機率以及質體DNA的釋放，當奈米載體被胞吞入細胞體內後，大部分的奈米載體都會被侷限在溶酶體內而無法執行功用，然而，如果施加了近紅外光雷射之後，可以發現載體漸漸地從溶酶體內釋放到細胞質內，證實近紅外光雷射確實增加了奈米載體逃離溶酶體的機率。整體來說，不論在細胞外或細胞內，近紅外光雷射確實增加了藥物運輸的效率，解決了影響轉染效率的因素中最大的障礙之一。從細胞的轉染結果也可以發現，第二步驟雷射的施加大大地影響了轉染結果，也就是說，真正的關鍵步驟即是在於溶媒體的逃離。

In the past few years, the current therapies for neurodegenerative diseases are not efficient because the morphology and properties of neurons are very different from normal cells. However, recently gene therapy is gradually developed as a new solution that have the potential for to cure neurodegenerative diseases at molecular level. Addition, the combination of targeting therapy and external stimulation will have the opportunity to increase the drug efficacy. Therefore, our research is to develop a gene delivery system and to investigate its behavior and transfection efficiency. In the first part of this study, a neuron-specific gene delivery system is developed by conjugating a neurotensin and poly(ethylenimine)-modificated reduced graphene oxide (rGO) via electrostatic force. This rGO-PEI-NT nanoparticle which stably protects plasmid DNA (pDNA) from digesting performs great targeting ability toward neuron-like cells. According to the literature, polyethyleneimine (PEI) has excellent transfection ability, the endosomal escaping power, and the ability to protect plasmid DNA from digesting; however, cell toxicity of PEI is high. In order to fix this problem, we conjugated neurotensin on PEI. The results show that neurotensin not only reduces the cytotoxicity of nanoparticles, but also increased the targeting ability toward neurons. We can observed that a large number of rGO-PEI-NT effectively accumulate in differentiated PC-12 by photoluminescence (PL) microscopy and confocal laser scanning microscopy (CLSM). From the transfection experiments in vitro and in vivo, the transfection efficiency are exactly improved by neurotensin. In the second part, in addition to the nanoparticles we mention in part one, we also combine it with external NIR laser. The thermal-vibration effect generated by rGO under NIR irradiation not only temporarily increase the permeability of cell membrane but also increase the possibility to escape from digesting in endo/lysosome. In our experiment, there are two steps of NIR laser, the function of first step laser (laser-step-1) is to increase the amount of nanoparticles internalization by temporarily increasing the permeability of the cell membrane. On the other hand, the second step of the laser (laser-step-2) to increase the chances of nanoparticles escaping from endo/lysosome. After the NIR irradiation, it can be found that nanoparticles gradually released from the lysosome into the cytoplasm, confirming the near-infrared laser light did increase the chance to escape from endo/lysosomes. Overall, both in extracellular or intracellular part, near-infrared laser does increase the efficiency of drug delivery and solve one of the factors that affect the transfection efficiency most. Besides, from the results of transfection experiment, we can conclude that second step of laser play a crucial role more than first step of laser, namely, the real key point affect the transfection is not captured by endo/lysosome.