Cancer Research
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Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube (SWCNT)-delivered MALAT1 Antisense Oligos
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Summary December 13th, 2018
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.
Transcript
The method we are using here can help to package oligonucleotide with single-wall carbon nanotube, which is a new type of nanoparticle for drug delivery. The main advantage of this technique is more efficient delivery of oligonucleotide drugs into cells in vivo. The implications of this technique extend to research multiple myeloma, because it's introduced nanoparticle to improve the delivery of oligonucleotide drugs.
Through this method can provide insight into treatment for multiple myeloma. It can also be applied to other diseases and conjugated with other cargo, such as proteins and small molecules. First, mix one milligram of single-wall carbon nanotubes, five milligrams of DSPE-PEG 2000 amine, and five milliliters of sterilized nuclease-free water in a 20-milliliter glass scintillation vial.
Sonicate the vial in a waterbath sonicator at a power level of 90 watts for one hour at room temperature. Following sonication, centrifuge the vial at 24, 000 times g for six hours. Then collect the supernatant solution.
Add one milliliter of the single-wall carbon nanotube solution to a 100-kilodalton molecular weight cutoff centrifugal filter device followed by four milliliters of sterilized nuclease-free water. Centrifuge for 10 minutes at 4, 000 times g at room temperature to remove the excess amine. Add 0.5 milligrams of Sulfo-LC-SPDP to 450 microliters of the amine-functionalized single-wall carbon nanotube.
Then add 50 microliters of 10x PBS, and incubate for two hours at room temperature. Next, add the reaction mixture to a 100-kilodalton molecular weight cutoff centrifugal filter device, followed by four milliliters of nuclease-free water. Centrifuge the sample for 10 minutes at 4, 000 times g at room temperature to remove the excess Sulfo-LC-SPDP.
Collect the Sulfo-LC-SPDP-treated amine single-wall carbon nanotube solution left in the filter, and dilute it with 500 microliters of previously-prepared anti-MALAT1-Cy3 solution. Then incubate the sample at four degrees Celsius overnight. To achieve the best results of comparison, randomly arrange 14 mice into trial or control groups with seven in each group, and with the same male-to-female ratio in each group.
Next, clean the operation bench and sterilize it with 70%ethyl alcohol. Use a heating lamp to warm one of the mice to help the tail vein to appear. Restrain the mouse properly with a tail injection restrainer.
Inject MM.1S-luc/mCherry cells in 50 microliters of PBS through the tail vein. Press the injection site with an alcohol swab for 30 seconds. Then mark the injected mouse and return it to a clean cage.
On day seven, after the MM1S cell injection, inject 50 microliters of PBS containing single-wall carbon nanotube anti-MALAT1 into the tail vein of the mouse. Then press the injection site with an alcohol swab for 30 seconds. Observe the local bleeding on the tail and the general behavior of the mouse for one minute, and then return it to a clean cage.
Observe all injected mice again before returning the cage to the rack to make sure that the injections are tolerated well. Weigh each mouse before dissection. Collect peripheral blood from the heart for a complete blood count assay performed by a blood cell counter machine.
Next, collect the tissues of the bone marrow, spleen, lymph node, kidney, and the tissue with visible metastasis. Extract RNA and protein from the bone marrow samples. Fix all remaining tissues in formalin.
After 24 hours of fixation, immerse the bone samples in a 0.5-molar EDTA solution for five days for decalcification. Then immerse all samples in 75%ethyl alcohol for long-term storage. QRT PCR results showed that anti-MALAT1 gapmeR DNA knocked down the MALAT1 expression and induced apoptosis significantly in H929 and MM.1S cells.
Single-wall carbon nanotube anti-MALAT1-Cy3 was delivered into the nucleus of MM cells, and significantly suppressed the endogenous MALAT1 level in both H929 and MM.1S cells. The viability of BMEC-1 cell has no significant difference after treatment of single-wall carbon nanotube anti-MALAT1-Cy3 or control at different dosages and timepoints. This indicates that the toxicity of single-wall carbon nanotube anti-MALAT1-Cy3 has a limited and acceptable toxicity to normal cells at high dosage.
A human MM-disseminated mouse model was established to evaluate the treatment effects of single-wall carbon nanotube anti-MALAT1 in vivo. The luciferon signals of mice in the single-wall carbon nanotube anti-MALAT1 treatment group were remarkably lower compared to the control after 21 days of treatment. From these results, it was concluded that the single-wall carbon nanotube anti-MALAT1 treatment via intravenous injection could deliver the anti-MALAT1 oligos to tumor cells effectively.
No significant side effects of the treatment were observed, indicating that single-wall carbon nanotube anti-MALAT1 injection is a safe treatment for mice. While attempting this procedure, it is important to remember that the cell molecule composition will influence the delivery, efficiency, and therapeutic effect of the oligonucleotide drugs. Following this procedure, other methods like conjugation of proteins or small molecules can be performed in order to answer additional questions.
Like using single-wall carbon nanotubes to deliver other drugs.
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