以聚乙烯亞胺官能化奈米碳管作為小分子干擾RNA傳遞載體

Polyethyleneimine-Functionalized Carbon Nanotube as a Delivery Vector for Small Interfering RNA

林憶柔(I-Jou Lin)

指導教授 ： 李孟娟

長榮大學/健康科學學院/生物科技學系(所)/碩士(2013年)

https://doi.org/10.6833/CJCU.2013.00208

摘要

奈米碳管(carbon nanotubes, CNTs)具有穿透細胞膜的特性，可作為基因或藥物的載體。由於奈米碳管上的C–C與C=C鍵結不具極性，須經過官能化(functionalization)才可與基因或藥物結合，同時提高其生物相容性。本研究將單壁奈米碳管(SWNT)、直徑20-40 nm的多壁奈米碳管(MWNT20-40 nm)、直徑100 nm的多壁奈米碳管(MWNT100 nm)與工業級多壁奈米碳管(MWNTindustrial)，以兩種官能化方法，於奈米碳管表面接枝聚乙烯亞胺(polyethyleneimine, PEI)，使奈米碳管表面充滿帶正電的胺基(–NH2)，期能藉以吸附小干擾RNA (small interfering RNA, siRNA)結構中帶負電的磷酸根，做為基因傳遞載體。我們的研究結果顯示，以PEI直接胺化的多壁奈米碳管(PEI-NH-MWNT)，較間接胺化所得之PEI官能化多壁奈米碳管(PEI-COCl-MWNT)的懸浮性佳，且增加離心的程序以去除較大的奈米碳管團聚物(agglomerates)，可提高PEI官能化奈米碳管的均質性(homogeneity)。以穿透式電子顯微鏡(transmission electron microscopy, TEM)與掃描式電子顯微鏡(scanning electron microscopy, SEM)觀察PEI官能化奈米碳管的表面形態，顯示PEI成功接枝於單壁或多壁奈米碳管表面。利用熱重分析法(thermogravimetry analysis, TGA)定量官能化奈米碳管表面PEI的接枝比例，可得到PEI-NH-SWNT、PEI-NH-MWNTindustrial、PEI-NH-MWNT20-40 nm與PEI-NH-MWNT100 nm的PEI接枝比例分別為19.478% (w/w)、24.584% (w/w)、6.125% (w/w)和10.6% (w/w)。利用動態光散射(dynamic light scattering)的原理分析官能化奈米碳管的平均粒徑，可得到PEI-NH-SWNT、PEI-NH-MWNTindustrial、PEI-NH-MWNT20-40 nm與PEI-NH-MWNT100 nm的平均粒徑分別為229±8-290±34、210±8-343±37、287±8-433±102和219±4-269±22 nm，並且可觀察到在4℃、25℃以及37℃，PEI官能化奈米碳管的表面皆帶有正電荷。為了解PEI官能化奈米碳管是否具有細胞毒性，我們將0至100 μg/ml的PEI官能化奈米碳管與人類子宮頸癌細胞HeLa-S3培養48小時，以細胞存活率分析(MTT assay)檢測，發現HeLa-S3細胞的存活率隨著PEI官能化奈米碳管濃度提高而降低，而在相同濃度下，PEI較PEI官能化奈米碳管的毒性高，故藉由將PEI接枝於奈米碳管，可降低PEI的細胞毒性。將PEI官能化奈米碳管與抑制甘油醛-3-磷酸脱氫酶(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)的siRNA (siGAPDH)以不同質量比(w:w)結合，並以瓊脂凝膠電泳(agarose gel electrophoresis)分析，顯示siGAPDH與PEI-NH-SWNT作用比例達1:80 (w:w)以上可完全結合，而PEI-NH-MWNTindustrial、PEI-NH-MWNT20-40 nm及PEI-NH-MWNT100 nm與siGAPDH完全結合的質量比分別為1:80、1:160及1:20。此外，將siGAPDH與PEI官能化奈米碳管以三種質量比(1:1、1:10及1:20，w:w)結合，與HeLa-S3細胞培養48小時後分析GAPDH的基因表現，顯示除了PEI-NH-MWNTindustrial之外，以PEI-NH-SWNT、PEI-NH-MWNT20-40 nm與PEI-NH-MWNT100 nm作為傳遞siGAPDH的載體，皆可達到抑制HeLa-S3細胞GAPDH基因表現的效果，證實PEI官能化奈米碳管可成功結合siRNA，傳遞siRNA至細胞內並達到抑制基因表現的目的，其效果與泛用的siRNA轉殖試劑DharmaFECT相當。

關鍵字

單壁奈米碳管 ； 多壁奈米碳管 ； 聚乙烯亞胺 ； 小干擾RNA ； RNA靜默 ； 基因傳遞

並列摘要

Carbon nanotubes (CNTs) are capable of penetrating the cell membrane and are widely considered as potential carriers for gene or drug delivery. Because the C–C and C=C bonds in carbon nanotubes are nonpolar, functionalization is required for carbon nanotubes to interact with genes or drugs, as well as to improve their biocompatibility. In this study, single-walled carbon nanotube (SWNT), multi-walled carbon nanotube with a diameter of 20-40 nm (MWNT20-40 nm), multi-walled carbon nanotube with a diameter of 100 nm (MWNT100 nm) and industrial of multi-walled carbon nanotube (MWNTindustrial) were functionalized with polyethyleneimine (PEI) through two different strategies. PEI functionalization may increase the positive charge on the surface of CNTs, so that they can interact electrostatically with the negatively charged small interfering RNAs (siRNAs). Our results suggest that CNTs aminated directly with PEI (PEI-NH-CNTs) had a better solubility in water than those covalently attached to PEI through the –COCl– linkage (PEI-COCl-CNTs). The homogeneity of PEI-NH-CNTs was further improved by an additional centrifugation procedure that removed larger CNT agglomerates. Result from transmission electron microscopy (TEM) and scanning electron microscopy (SEM) suggested that, PEI was successfully grafted on the surface of single-walled or multi-walled carbon nanotubes. As determined by thermal gravimetric analysis (TGA), PEI graft ratio of PEI-NH-SWNT, PEI-NH-MWNTindustrial, PEI-NH-MWNT20-40 nm and PEI-NH-MWNT100 nm was 19.478% (w/w), 24.584% (w/w), 6.125% (w/w) and 10.6% (w/w), respectively. As determined by dynamic light scattering, the average particle size of PEI-NH-SWNT, PEI-NH-MWNTindustrial, PEI-NH-MWNT20-40 nm and PEI-NH-MWNT100 nm was 229±8-290±34、210±8-343±37、287±8-433±102 and 219±4-269±22 nm. We also found that the zeta potential of PEI-NH-CNTs was positive at 4, 25 and 37℃. To understand the cytotoxicity of PEI-NH-CNTs various concentrations (0-100 μg/ml) of PEI-NH-CNTs were incubated with human cervical cancer cells HeLa-S3 for 48 h. Using the MTT assay, we found that the viability of HeLa-S3 cells decreased with increasing PEI-NH-CNT or PEI concentration. Nevertheless, the cytotoxicity of PEI was reduced when the cationic polymer was attached to CNTs. As determined by electrophoretic mobility shift assay (EMSA), siRNA against glyceraldehydes 3-phosphate dehydrogenase (siGAPDH) was completely associated with PEI-NH-SWNT at a siGAPDH:PEI-NH-CNT mass ratio (w:w) of 1:80, while the ratio for complete binding of PEI-NH-MWNTindustrial, PEI-NH-MWNT20-40 nm and PEI-NH-MWNT100 nm with siGAPDH was 1:80, 1:160 and 1:20, respectively. HeLa-S3 cells were then treated with PEI-NH-CNTs complexed with siGAPDH at three mass ratio (1:1, 1:10 and 1:20, w:w) for 48 h, followed by real-time PCR to analyze the mRNA level of GAPDH. Except for PEI-NH-MWNT, the other three types of PEI-NH-CNTs, including PEI-NH-SWNT, PEI-NH-MWNT20-40 nm, and PEI-NH-MWNT100 nm, suppressed the mRNA level of GAPDH to various degrees when complexed with siGAPDH, and the transfection efficiency was comparable to that of the commercial transfection reagent, DharmaFECT. Our results indicate that the PEI-NH-CNTs produced in this study are capable of delivering siRNAs into HeLa-S3 cells to suppress gene expression, and may therefore considered as novel gene delivery reagent.

並列關鍵字

single-walled carbon nanotubes ； multiwalled carbon nanotubes ； polyethyleneimine ； small interfering RNA ； RNA interference ； gene deliver

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