氧化鐵/氧化石墨烯奈米複合載體結合磁和光熱刺激以增強基因轉染於人類懸浮型細胞之重新編程

Abstract

近年來，基因轉染在生物醫學研究中扮演一個重要的角色，其目的是將外在的遺傳訊息送入細胞內，並在細胞內表達基因的產物或改變細胞中內源性基因的表現，例如:細胞重新編程、基因編輯等。基於安全性的考量，目前多數研究採用非病毒載體攜帶基因以避免突變的可能性。然而，非病毒載體的低轉染效率主要是受限於難以有效的將基因導入到靶細胞。甚至是攜帶多個基因或是想要承載較大片段的外來 DNA時，往往很容易形成大型質體 (例如:游離型質體)，這種大型質體即使是透過病毒或是一般非病毒載體都受限於攜帶質體無法太大。然而文獻指出許多非病毒載體，包括脂質體、磷酸鈣已成功應用於貼附型細胞轉染為具有分化能力的幹細胞狀態，但是對於某些類型的細胞，像是初代細胞或懸浮型細胞，仍然是難以轉染，因此有效的利用非病毒載體解決這些問題是必要的。懸浮細胞像是體內的造血幹細胞、或是周邊血單核細胞，這些細胞的提取相當容易，相對於其他體細胞，能節省更多時間而不需要侵入性的取得。因此，在研究中主要開發多功能性非病毒載體，結合了磁和光熱刺激，目的以增強轉染效率於懸浮型細胞，且更進一步的應用在細胞重新編程中。 研究中非病毒載體主要以氧化石墨烯及氧化鐵為主，石墨烯因良好的裝載能力，在近幾年已廣泛應用於基因傳遞的材料。載體製備利用化學共沉澱方式將氧化鐵修飾於石墨烯上，並接上聚乙烯亞胺高分子 (Polyethylenimine，PEI)，根據文獻，聚乙烯亞胺高分子具有高正電荷，可吸附甚至保護負電荷質體DNA。結果顯示，nGO-Fe3O4-PEI接上質體形成之奈米複合材料，整體大小為200奈米，是細胞適合主動胞吞的一個合適範圍。此外也將功能性載體結合外在磁攪拌及近紅外光照射刺激，進而增加轉染效率。因懸浮式細胞受液體擾動的現象，不容易將細胞與奈米複合材料接觸，因此利用攪拌器旋轉方式增加正電荷奈米複合材料與帶負電荷細胞膜接觸機會，促使更多載體黏附於細胞表面上；接著藉由石墨烯及氧化鐵載體在波長700-1100 nm的近紅外光具有敏感的特性，當施加近紅外光雷射時，進而轉換熱使細胞膜產生震動而增加通透性，使靠近細胞膜外圍的載體進到細胞內的機會增加。 結合上述之特性，本研究先以人類單核球細胞株 (THP-1)，證明奈米複合載體應用於懸浮型細胞的可行性及安全性，再將奈米複合載體攜帶綠色螢光蛋白做為驗證，實驗結果顯示在轉染效率及細胞活性，奈米複合材料 (nGO-Fe3O4-PEI) 皆優越於市售轉染試劑 (lipofectamine 2000)，亦證實外在物理性刺激，磁和光熱效應將有效增加轉染效率的提升。實驗結果選擇n/p ratios 14k為載體與各個質體 (pCXLE-hSK, pCXLE-hUL, pCXLE-hOCT3/4- shp53)結合之最佳參數，應用於人類周邊血單核細胞 (PBMCs)之重新編程。聚合酶鏈反應在最佳條件下有觀察到內源性的Oct4，Sox2和Nanog表達。藉由免疫螢光實驗觀察，在體外分化後，也有外胚層 (Tuj-1) 及中胚層 (SMA) 標記物。總結，nGO-Fe3O4-PEI奈米複合載體成功結合磁攪拌和光熱效應，確實增加了轉染效率並且得以應用於初代的周邊血細胞，更進一步的將此非病毒系統應用在細胞重新編程中，並且達到個人化醫療的目標。

Recently, gene transfection plays an important role in biomedical researches, especially in the investigation of cell reprogramming. Utilizing non-viral gene carriers to promote the efficiency of cell reprogramming is a tendency based on the safety concerns. However, low transfection efficiency of non-viral vectors is the major restriction that is difficult to carry genes into the same host cells, not to mention the delivery of multiple genes or large plasmids (i.e., Episomal plasmids). In order to develop more safety manners for gene transfection, many non-viral vectors, including liposome, calcium phosphate have been successfully demonstrated to transfect various adherent cell for cell reprogramming. However, some important types of cells, such as primary cells and suspension cells, still hard to transfect. Therefore, the development of efficient non-viral carriers to solve those difficulties is necessary. Suspension cells, i.e. peripheral blood mononuclear cells, as target cells are accessible from human blood via non-invasive method, which is a time-saving manner for acquisition of target cells. Thus, we aimed to develop a multifunctional non-viral vector combined magnetic and photothermal stimulations to carry genes into suspension cells from human blood for the application of cell reprogramming. GO is a widely explored material applying in the field of gene delivery due to its excellent gene carrying capability. In this study, we modified nGO using Fe3O4 was prepared by chemical depositing onto nGO, and coated branch 25k polyethyleneimine (bPEI) through electrostatic interaction to create a multifunctional gene carrier using for gene transfection on suspension cells. According to previous researches, PEI is a positively charged polymer, which can be used to absorb and protect negatively charged DNA. The nGO-Fe3O4-PEI-pDNA nanocomposites were measured to be 200 nm in diameter that is easy-to-enter cell through endocytosis. Additionally, GO and Fe3O4 can introduce external stimulations, including magnetic stirring and NIR laser induced photothermal stimulation to enhance gene transfection efficiency. Fe3O4 drives the whole nanocomposites to stick on the surface of cells under magnetic stirring and cell sorting, whereas both GO and Fe3O4 can absorb NIR irradiation with wavelength 700 nm-1100 nm and transform the light into local heat on the cell membrane, which can facilitate nanocomposites to get into cells. In this study, THP-1 cell line (a surrogate for monocytes) was first used to demonstrate the possibility and safety of nanocomposites as a gene carrier by transfecting plasmid encoded with the green fluorescent protein (GFP) gene. Our results show the nGO-Fe3O4-PEI/pDNA nanocomposites exhibited lower cytotoxicity and better transfection efficiency than commercial vector lipofectamine 2000. Then, we further isolated PBMC cell from the healthy human donor and transfected three transcription factors encoded in episomal plasmids (pCXLE-hSK, pCXLE-hUL, pCXLE-hOCT3/4- shp53) into PBMCs. Polymerase chain reaction (PCR) analysis showed that the endogenous genes, including Oct4, Sox2 and Nanog, were expression under the optimal condition. In-vitro differentiation study revealed that specific ectoderm marker (Tuj-1) and mesoderm marker (SMA) could be detected by immunofluorescence analysis. In summary, these experimental results showed that the nGO-Fe3O4-PEI nanocomposites could be served as an appropriate non-viral gene vector to treat suspension cells. That means this multifunctional non-viral vector might be provided as a transfection tool for cell reprogramming possibility.