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单壁碳纳米管 (sscnt) (mallat1 反义寡 3号) 抑制多骨髓瘤细胞在体内的生长

Published: December 13, 2018
doi:
10.3791/58598
, ,
1Department of Cancer Biology, Lerner Research Institute,Cleveland Clinic, 2Department of Medical Oncology, Dana-Farber Cancer Institute,Harvard Medical School

Summary

这篇手稿描述了单壁碳纳米管 (swcnt)-共轭 malat1 反义 gapgmer dna 寡核苷酸 (swcnt 反材料-malat1) 的合成, 这表明了 swcnt 的可靠交付和强大的治疗效果。抗马拉特1在体外和体内。介绍了用于 swcnt 抗 malat1 的合成、改性、共轭和注射的方法。

Abstract

单壁碳纳米管 (swcnt) 是一种新型的纳米粒子, 已被用于向细胞输送多种药物, 如蛋白质、寡核苷酸和合成小分子药物。swcnt 具有可定制的尺寸, 一个大的表面积, 并可以灵活地结合药物通过不同的修改, 在其表面;因此, 它是一个理想的系统, 运输药物到细胞。长非编码 rna (lnrna) 是一组时间超过200nt 的非编码 rna 群, 不能转化为蛋白质, 但在生物和病理生理过程中起着重要作用。转移性与肺腺癌相关的转录 1 (malat1) 是一种高度保守的 lncRNA。结果表明, 较高的 malat1 水平与各种癌症的预后不良有关, 包括多发性骨髓瘤 (mm)。我们发现, matr1 调节 dna 修复和细胞死亡在 mm;因此, matr1 可被视为 mm 的治疗靶点。然而, 反义寡核苷酸在体内有效传递给抑制-击倒 malat1 仍然是一个问题。在这项研究中, 我们修改了 swcnt 与 peg-2000, 并结合抗 mat1 寡核苷酸到它, 测试这种化合物的体外交付, 注入静脉注射 mm 小鼠模型, 并观察到一个显著的抑制 mm 进展, 这表明swcnt 是抗马拉特1口角 dna 的理想穿梭机。

Introduction

swcnt 是一种新型的纳米材料, 可提供各种类型的药物, 如蛋白质、小分子和核酸, 在体外 1和体内2具有理想的耐受性和最小毒性。功能化的 swcnt 具有很强的生物相容性和水溶性, 可作为小分子的穿梭点, 并可携带小分子穿透细胞膜 3,4,5.

lnrna 是一组 rna (gt;200 nt), 从基因组转录到 mrna, 但不能转化为蛋白质。越来越多的证据表明, ncrna 参与了基因表达的调节 6, 并参与了大多数类型癌症的发生和发展, 包括 mm7,8,9。mallat1 是一种富核非编码记录 2 (neat2) 和高度保守的 lncRNA 10。mat1 最初被认为是转移性非小细胞肺癌 (nsclc)11, 但已在许多肿瘤5,12,13过度表达;它是 mm8,14中表达最严重的信息之一, 与预后差有关。与仅确诊为 mm 15 的患者相比, 致命病程外髓质 mm 患者的 malat1 表达水平明显高于.

在先前的一项研究中, 我们已经证实, 抗 mat1 寡核苷酸在 mm 16 中使用 gapmer dna 反义寡核苷酸靶向 mm1 (抗 amalat1) 强致 mm 16 中的 dna 损伤和凋亡。gapmer dna 由反义 dna 组成, 并由 2 ‘-ome-rna 连接, 这可能会促使 mirat1 通过 rnase h 活性一旦结合 17.反义寡核苷酸的体内传递效率仍然限制了其临床应用。

为了测试 swcnt 对抗 amalat1 变指寡核苷酸的传递效果, 将抗 malat1 报的 dna 与 dspe-peg2000-胺功能化的 cnt 结合。然后将 swcnt 抗 matrat1 静脉注射到 mm 扩散小鼠模型中;经过四种治疗后观察到明显的抑制。

Protocol

所有涉及动物的实验都是由克利夫兰诊所 iacuc (机构动物护理和使用委员会) 预先批准的。 1. 功能化 swcnt 的合成 将1毫克的 swcnt、5毫克的 dspe-peg2000 胺和5毫升的灭菌无核水混合在玻璃闪烁小瓶中 (20 毫升)。摇匀, 完全溶解所有试剂。 将小瓶放入水浴声纳中, 在室温下功率为 40 w, 1小时 (rt, 20分钟 x 3, 每20分钟更换一次水, 以避免过热)。然后, 以 24, 000 x g离心…

Representative Results

为了证明抗 amat1 gapmer dna 在 mm 中的抑制作用, 我们击倒了菌与高级101的表达, 并将其应用于 h929 和 mm. 1s 细胞中。48小时后, 收集细胞, 分析细胞的击倒效率和细胞凋亡状态转染抗 mallat1 口吃或控制 dna。qrt-pcr 结果表明, 抗 makat1 间隙酶 dna 有效地降低了 h929 和 mm. 1s 细胞中的 halat1 表达 (图 2a)。流式细胞仪测定细胞凋亡的状态, 显示下调节的 …

Discussion

有证据表明, ncrna 参与调节癌症的多种生理和病理生理程序, 包括 mm7,8,9;它们有可能成为癌症治疗的目标, 而反义寡核苷酸20、21、22 可以实现。美国食品药品监督管理局 (fda) 已批准了几种反义寡核苷酸药物, 包括用于巨细胞病毒视网膜炎23的 fomivirsen、用于纯…

Declarações

The authors have nothing to disclose.

Acknowledgements

作者感谢勒纳研究所的蛋白质组学、基因组和成像核心的帮助和支持。资金: 这项工作得到了 NIH/NCI 赠款 r00 ca17292 (给 j. z.) 和启动资金 (jj. z.) 和凯斯西储备大学核心利用试点赠款 (j. z.) 临床和转化科学合作 (ctsc) 的财政支持。这项工作使用了 leica sp8 共聚焦显微镜, 该显微镜是在国家卫生研究院 sig 赠款1s10od01992-01 下购买的。

Materials

SWCNTs Millipore-Sigma 704113
DSPE-PEG2000-Amine Avanti Polar Lipids 880128
bath sonicator VWR 97043-992
4 mL centrifugal filter Millipore-Sigma Z740208-8EA
UV/VIS spectrometer Thermo Fisher Scientific accuSkan GO UV/Vis Microplate Spectrophotometer extinction coefficient of 0.0465 L/mg/cm at 808 nm
Sulfo-LC-SPDP ProteoChem c1118
DTT solution Millipore-Sigma 43815
NAP-5 column GE Healthcare 17-0853-01
in vivo imaging system PerkinElmer
NOD.CB17-Prkdcscid/J mice Charles River lab 250
Flow cytometer Becton Dickinso
Lipofectamine Invitrogen 11668019 Lipofectamine2000
Fetal bovine serum (FBS) Invitrogen 10437-028
RMPI-1640 medium Invitrogen 11875-093
MALAT1-QF: synthesized by IDT Company 5’- GTTCTGATCCCGCTGCTATT – 3’
MALAT1-QR: synthesized by IDT Company 5’- TCCTCAACACTCAGCCTTTATC – 3’
GAPDH-QF: synthesized by IDT Company 5’- CAAGAGCACAAGAGGAAGAGAG – 3’
GAPDH-QR: synthesized by IDT Company 5’- CTACATGGCAACTGTGAGGAG – 3’
Quantitative PCR using SYBR Green PCR master mix Thermo Fisher Scientific A25780
RevertAid first-stand cDNA synthesis kit Thermo Fisher Scientific K1621
anti-MALAT1 synthesized by IDT Company 5’-mC*mG*mA*mA*mA*C*A*T*T
*G*G*C*A*C*A*mC*mA*mG*mC*mA-3’
Cell Viability Assay Kit Promega Corporation G7570 CellTiter-GloLuminescent Cell Viability Assay Kit
accuSkan GO UV/Vis Microplate Spectrophotometer Thermo Fisher Scientific
centrifugal filter Millipore-Sigma UFC910008
SPSS software IBM version 24.0
D-Luciferin Millipore-Sigma L9504
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Tags

Multiple MyelomaSingle-wall Carbon Nanotube (SWCNT)MALAT1 Antisense OligoOligonucleotide DeliveryIn VivoNanoparticleDrug DeliveryTreatmentCentrifugal FiltrationSulfo-LC-SPDPAnti-MALAT1-Cy3Mouse Model

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Lin, J., Hu, Y., Zhao, J. Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube (SWCNT)-delivered MALAT1 Antisense Oligos. J. Vis. Exp. (142), e58598, doi:10.3791/58598 (2018).
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