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Cancer Research

Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube (SWCNT)-delivered MALAT1 Antisense Oligos

Published: December 13th, 2018



1Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, 2Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School
* These authors contributed equally

This manuscript describes the synthesis of a single-wall carbon nanotube (SWCNT)-conjugated MALAT1 antisense gapmer DNA oligonucleotide (SWCNT-anti-MALAT1), which demonstrates the reliable delivery of the SWCNT and the potent therapeutic effect of anti-MALAT1 in vitro and in vivo. Methods used for synthesis, modification, conjugation, and injection of SWCNT-anti-MALAT1 are described.

The single-wall carbon nanotube (SWCNT) is a new type of nanoparticle, which has been used to deliver multiple kinds of drugs into cells, such as proteins, oligonucleotides, and synthetic small-molecule drugs. The SWCNT has customizable dimensions, a large superficial area, and can flexibly bind with drugs through different modifications on its surface; therefore, it is an ideal system to transport drugs into cells. Long noncoding RNAs (lncRNAs) are a cluster of noncoding RNA longer than 200 nt, which cannot be translated to protein but play an important role in biological and pathophysiological processes. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a highly conserved lncRNA. It was demonstrated that higher MALAT1 levels are related to the poor prognosis of various cancers, including multiple myeloma (MM). We have revealed that MALAT1 regulates DNA repair and cell death in MM; thus, MALAT1 can be considered as a therapeutic target for MM. However, the efficient delivery of the antisense oligo to inhibit/knockdown MALAT1 in vivo is still a problem. In this study, we modify the SWCNT with PEG-2000 and conjugate an anti-MALAT1 oligo to it, test the delivery of this compound in vitro, inject it intravenously into a disseminated MM mouse model, and observe a significant inhibition of MM progression, which indicates that SWCNT is an ideal delivery shuttle for anti-MALAT1 gapmer DNA.

The SWCNT is a novel nanomaterial that can deliver various types of drugs, such as proteins, small molecules, and nucleic acids, stably and efficiently with ideal tolerability and minimum toxicity in vitro1 and in vivo2. A functionalized SWCNT has great biocompatibility and water solubility, can be used as a shuttle for smaller molecules, and can carry them to penetrate the cell membrane3,4,5.

lncRNAs are a cluster of RNA (>200 nt) that are transcribed from the genome to mRNA but cannot be transla....

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All experiments involving animals were pre-approved by the Cleveland Clinic IACUC (Institutional Animal Care and Use Committee).

1. Synthesis of Functionalized SWCNTs

  1. Mix 1 mg of SWCNTs, 5 mg of DSPE-PEG2000-Amine, and 5 mL of sterilized nuclease-free water in a glass scintillation vial (20 mL). Shake it well to dissolve all reagents completely.
  2. Sonicate the vial in a water bath sonicator at a power level of 40 W for 1 h at room temperature (RT, 20 min x 3, change the wat.......

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To demonstrate the inhibition effect of anti-MALAT1 gapmer DNA in MM, we knocked down the expression of MALAT1 and used it in H929 and MM.1S cells. Forty-eight hours later, cells were collected for the analysis of knock-down efficiency and the apoptosis status in cells transfected with anti-MALAT1 gapmer or control DNA. qRT-PCR results showed that anti-MALAT1 gapmer DNA knocked down the MALAT1 expression in H929 and MM.1S cells efficiently (Figure 2A<.......

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Evidence has shown that lncRNAs take part in the regulation of numerous physiological and pathophysiological procedures in cancers, including MM7,8,9; they have the potential to be targeted for cancer treatment, which can be realized by antisense oligonucleotides20,21,22. The U.S. Food and Drug Administration (FDA) has approved several .......

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The authors thank the Lerner Research Institute proteomic, genomic, and imaging cores for their assistance and support. Funding: This work was financially supported by NIH/NCI grant R00 CA172292 (to J.J.Z.) and start-up funds (to J.J.Z.) and the Clinical and Translational Science Collaborative (CTSC) of Case Western Reserve University Core Utilization Pilot Grant (to J.J.Z.). This work utilized the Leica SP8 confocal microscope that was purchased with funding from National Institutes of Health SIG grant 1S10OD019972-01.


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Name Company Catalog Number Comments
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
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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