整合石墨烯/兩性多醣類導電高分子之植入式生物電極應用於神經基因治療

Abstract

隨著醫學的進步和環境的改善，高齡化社會的現象伴隨許多老年化疾病的發生，其中，神經退化性疾病(neurodegenerative disease)例如阿茲海默症(Alzheimer's disease)、帕金森氏症(Parkinson's disease)在治療效果上仍有所限制。近年來，細胞移植(cell transplantation)、基因療法(gene therapy)或是深層腦部刺激(Deep brain stimulation, DBS)的發展逐漸成為具有潛力的治療方法。本研究致力於結合基因療法與深層腦部刺激的概念，開發出結合標靶性基因載體的水膠型生物電極，並進一步整合於微電極陣列晶片(Micro electrode arrays, MEA)以及植入式神經微電極，在給予電刺激下，可提升攜帶質體DNA載體的釋放量，並能同時增加細胞膜的通透性以提高細胞轉染之效果。研究中分為三個部分，第一部分利用化學剝離法所合成的石墨烯(reduced graphene oxide, rGO)與自我改質合成的兩性導電多醣體發展出一個新型的水膠生物性電極，名為PG，其具有奈米結構並展現良好的仿生特性、優越的導電能力、生物相容性及機械性質。第二部分是開發具神經標靶特性的基因載體，將帶有高正電荷的聚乙烯亞胺(primary amino group)嫁接上神經標靶胺基酸─神經調壓素 (neurotensin)，使載體對於神經細胞具有專一性，在與綠螢光蛋白 (green fluorescence protein, GFP)基因之質體DNA (plasmid DNA)結合後，可直接進入神經細胞內進行基因轉染。第三部分，我們將水膠生物性電極與攜帶質體DNA之標靶特性載體結合，名為PGEND，整合於微電極陣列晶片上進行體外轉染，發現在施加電流刺激下會形成一梯度電場而造成不同區域性的轉染效果。最後，將此搭載於具有十六個電極通道的軟性神經電極探針上，進行體內的研究，研究中發現施加電刺激可有效的提升轉染效率，証實此系統確實可增加載體的釋放與進入細胞內的效率。相較於傳統的轉染方式，本研究系統能更直接與細胞接觸以達到增強基因轉染的成效。本研究將基因載體與植入式元件結合發展出具有神經標靶特性、電刺激回應之可調控基因釋放與電穿孔之生物性電極，此一新概念期望為神經疾病治療帶來新的突破以提高治療功效。

With the advancement and improvement of biomedical systems which brings about the aging society, many neurodegeneration diseases like Alzheimer's and Parkinson's disease were generated. However, the therapeutic effect is limited in the current medical systems. Recently, new approaches including cell transplantation, gene therapy and deep brain stimulation (DBS), had become promising for treating neurodegenerative diseases. Therefore, in this thesis, we develop a targeting gene delivery system that is combined with the hydrogel-based nanostructural bioelectrode and gene vectors. According to the concept of DBS and gene therapy, the system demonstrated the synergistic functions including electrically responsive release neuron-targeting DNA vectors and electroporation to enable more precise gene transfection. In the first part, we developed a new type of hydrogel-based nanostructural bioelectrode, called PG, based on the reduced graphene oxide (rGO) and self-synthesized conductive amphiphilic chitosan polymer (PMSDT). The PG demonstrated bio-mimic characteristic, excellent ionic/electric conductivity, biocompatible and mechanism properties. In the second part, a DNA vector which owns specificity to neuronal cells was developed using polyethylenimine (PEI) conjugated neurotensin to load plasmid DNA with green fluorescent protein (GFP) for gene transfection in neuron cells. For the final part, by the combination of the hydrogel-based nanostructural bioelectrode and DNA vectors, a DNA-delivered bioelectrode, called PGEND, was produced to investigate gene transfection under external electrical stimulation. We further fabricated the PGEND onto microelectrode arrays and an implanted neural microprobe, followed by the in vitro and in vivo investigation on gene transfection under electrical stimulation. The electrical stimuli resulted in the enhancement on DNA release, even exhibiting the successful gene transfection both in vitro and in vivo. With the incorporation of both neurotensin and electrical stimulation, a localized gene transfection was achieved based on the effects in combination of targeting, stimuli-responsive controllable delivery and electroporation. Such bioelectrode is expected to provide more effective treatments in neurodegenerative diseases.