Abstract:

Based on the biocompatibility and excel affinity for single-strand nucleic acids of graphene， we investigated the transfection ability of graphene-shelled ferro-nitride magnetic beads （GFeNMB） carrying the antisense oligonucleotides （ASO） to target Survivin， and the inhibitory effect on tumor cells. Firstly， the ASO sequences were designed based on the secondary structure of Survivin mRNA predicted by RNA Draw software， and FAM-labeled and unlabeled ASOs were synthesized. The affinity of GFeNMB for single-strand nucleic acids was detected based on fluorescence with FAM-ASO. Based on the adsorbability of graphene to single-stranded nucleic acid and the quenchability of fluorophore， the fluorescence intensity detection method was used to detect the adsorption capacity of GFeNMB to ASO， and GFeNMB and GFeNMB-ASO were characterized with transmission electron microscope and laser granulometer. Survivin ASO was magnetically transfected into human non-small cell lung cancer cell A549 with a magnetic pole. The changes of Survivin expression were tested by Western blot， apoptosis by reactive oxygen species （ROS） kit and flow cytometry， cell proliferation and migration ability by CCK-8 assay and scratch assay. The results confirmed the excel affinity of GFeNMB for ASO， and the maximum adsorption amount （0.44 μg·mg^-1） was reached after 20 min of mixing GFeNMB with ASO. FAM-ASO was magnetically transfected into cells， which was confirmed by intracellular fluorescence focused on the nucleus observed under confocal microscope. Moreover， GFeNMB mediated transfection into nucleus was significantly superior to the liposome transfection. The down-regulated level of Survivin protein expression after magnetic loading ASO was better than those of the untreated and liposome transfected groups， followed by an increase in the proportion of apoptotic cells， an increase of intracellular ROS level， and inhibition of cell proliferation and migration. The results suggested that GFeNMB could transfect Survivin ASO into tumor cells， thus inhibit tumor cell proliferation and induce apoptosis. The down-regulation of gene expression after GFeNMB-ASO magnetofection of cells also indicated that GFeNMB may be a useful transfection medium for single-strand oligonucleotides.

Key words: graphene-shelled ferro-nitride magnetic beads, survivin, antisense oligonucleotide, magnetofection

摘要：

基于石墨烯的生物相容性和对单链核酸的吸附性，研究烯壳铁氮磁珠(graphene-shelled ferro-nitride magnetic beads, GFeNMB)运载靶向Survivin的反义寡核苷酸(antisense oligonucleotide, ASO)及其对肿瘤细胞的抑制作用。首先用RNA Draw针对Survivin mRNA的二级结构设计ASO，合成FAM标记和未标记的ASO；基于石墨烯对单链核酸的吸附性和对荧光团的淬灭性，采用酶标仪检测荧光强度方法检测GFeNMB对ASO的吸附能力，并对GFeNMB和GFeNMB-ASO表征；磁极作用下将Survivin ASO磁转染至人非小细胞肺癌细胞A549中，荧光显微镜观察GFeNMB将ASO载入细胞的能力；ASO转染后，采用Western blot检测Survivinr的表达，活性氧（reactive oxygen species, ROS）试剂盒、流式细胞仪检测细胞凋亡，CCK-8和划痕实验检测细胞增殖和迁移能力。结果表明，GFeNMB对ASO具有良好的吸附性，GFeNMB与ASO混合20 min达最大吸附量(0.44 μg·mg^-1)；FAM-ASO经磁性载入细胞内呈绿色荧光且集中于细胞核，GFeNMB介导的核转染能力显著优于脂质体；ASO经磁转染至细胞后，Survivin蛋白的降低水平优于未处理组和脂质体转染组；磁转染Survivin ASO后，肿瘤细胞凋亡比例增大，细胞内ROS升高，细胞增殖和迁移能力受到抑制。综上，GFeNMB可将Survivin ASO转染至肿瘤细胞中，能够抑制细胞增殖，诱导细胞凋亡。GFeNMB-ASO磁转染细胞后下调靶基因表达的情况表明，GFeNMB有望成为单链寡核苷酸的转染介质。

关键词: 烯壳铁氮磁珠, Survivin, 反义寡核苷酸, 磁转染