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Aptamer-based targeted delivery microRNA nanometer carrier as well as preparation method and application thereof

A nanocarrier and carrier technology, which is applied in the field of nanomaterial biology, can solve problems such as harm caused to the human body, and achieve the effects of good effect, low cytotoxicity, and quick and convenient preparation.

Inactive Publication Date: 2015-07-22
BEIJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the most commonly used and effective method to treat cancer is still combined chemotherapy and radiotherapy, but this method will inevitably cause harm to the human body

Method used

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  • Aptamer-based targeted delivery microRNA nanometer carrier as well as preparation method and application thereof
  • Aptamer-based targeted delivery microRNA nanometer carrier as well as preparation method and application thereof
  • Aptamer-based targeted delivery microRNA nanometer carrier as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037]Example 1 Preparation of nano-diamond working fluid

[0038] The nanodiamond used in the present invention is a gel solution (15% w / v), purchased from NanoCarbon Research Institute Ltd. (catalogue number: 386-8567), with a particle size of 3.6±0.7nm.

[0039] Before use, a 150 mg / mL nanodiamond (Nanodiamond, ND) gel solution was dissolved in deionized water and diluted to a final concentration of 1 mg / mL. The diluted ND solution was sonicated at 100W overnight.

Embodiment 2

[0040] Example 2 Protamine Sulfate Modified Nanodiamonds (NPs)

[0041] The protamine sulfate used in the present invention was purchased from Sigma Adrich (Cat. No.: PS4020).

[0042] 50 mg of protamine sulfate (PS) was dissolved in 5 mL of deionized water, so that the final concentration of the protamine sulfate solution was 10 mg / mL. The nanodiamond gel solution and protamine sulfate solution were mixed at a mass ratio of 1:15 to form a NPs solution, and incubated at 37°C for 48 hours for self-assembly reaction.

[0043] After 48 hours, the mixture was centrifuged at 12000 rpm for 15 minutes. The supernatant was discarded, and the obtained NPs pellets were redispersed with deionized water, washed by centrifugation again, and this step was repeated three times. Finally, the precipitate was lyophilized and dissolved in deionized water to adjust the concentration to 0.6 mg / mL.

Embodiment 3

[0044] Example 3 Modification of miR-34a by SPDP cross-linking agent

[0045] The miR-34a mimics used in the present invention were synthesized by Guangzhou Ruibo Biotechnology Co., Ltd., and its sequence is: sense strand: 5'-UUCUCCGAACGUGUCACGUdTdT-3'; antisense strand: 5'-ACGUGACACGUUCGGAGAAdTdT-3', used in the present invention Nsuccinimidyl-3-(2-pyridyldithio)propionate (SPDP) was purchased from Thermo-Fisher Scientific (catalog number: 21857).

[0046] The SPDP cross-linker was dissolved in DMSO at a molar concentration of 1:20 and incubated for 2 hours, while 1.25 nmol of miR-34a mimics with amino groups at the 5' end were dissolved in 200 μL of PBS. After the reaction, the excess SPDP was centrifuged out of the membrane ultrafiltration centrifuge tube several times.

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Abstract

The invention relates to an aptamer-based targeted delivery microRNA nanometer carrier as well as a preparation method and application thereof. A targeting molecule is a sulfydryl-modified MUC-1molecule, and can be combined with cancer-inhibiting microRNA-34a, so that a MicroRNA-nanodiamond-protamine complex (MNPs) becomes AMNPs with an active targeting function; through electrostatic interaction, a negatively-charged aptamer-microRNA (Apt-miR) chimera is adsorbed to a cationic protamine-nanodiamond carrier to form a trinary compound carrier. The therapeutic effect of a nanotransport system on a non-small cell lung cancer is researched; the Apt-miR chimera is low in immunogenicity; the modified MUC-1 aptamer has high chemical stability in vivo, and can be chemically synthesized easily; in order to improve the transfection efficiency, a protamine-modified nanodiamond material (NPs) is used for helping miRNA to increase accumulation in a tumor part through permeability and retention enhancing effects.

Description

Technical field: [0001] The invention belongs to the field of nanometer material biology. The invention relates to a targeted microRNA-loaded nano-carrier and a preparation method thereof, in particular to a preparation method of a MUC-1-targeted protamine-modified nano-diamond carrier and its application in the treatment of lung cancer. Background technique: [0002] MicroRNA (miRNA) is a type of endogenous non-coding RNA with a length of 20-25 bases. MicroRNA binds to the 3'-untranslated region (3'-UTR) of mRNA to form local base pairing to inhibit the translation of mRNA or degrade it. A microRNA can bind to multiple sites of mRNA, and the expression result of a gene may also be regulated by different microRNAs. Many microRNAs are abnormally expressed in different cancers, suggesting that microRNAs are closely related to the occurrence and development of cancer. Different MicroRNAs play different roles in cancer. Some microRNAs have functions similar to oncogenes and a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K31/708A61K47/26A61K47/42A61K47/04A61P35/00A61P11/00
Inventor 张芳辛婧王怡慧邓雄威盛望
Owner BEIJING UNIV OF TECH
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