Nano gold mir-885-5p conjugate and its preparation method and application

A technology of conjugates and gold nanoparticles, applied in the field of liver cancer, to achieve significant growth, good stability, and good pharmacological properties

Active Publication Date: 2016-01-20
TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on miR-885-5p in liver cancer is rarely reported. Our study found that miR-885-5p was significantly down-regulated in liver cancer and played a tumor suppressor role

Method used

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  • Nano gold mir-885-5p conjugate and its preparation method and application
  • Nano gold mir-885-5p conjugate and its preparation method and application
  • Nano gold mir-885-5p conjugate and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 The preparation and identification of nano-gold particles with a particle size of about 13nm

[0046] 1) Preparation of aqua regia: prepare according to the volume ratio of concentrated hydrochloric acid and concentrated nitric acid as 1:3, that is, measure 90ml of concentrated hydrochloric acid with a measuring cylinder and put it in a 500ml clean beaker, then measure 30ml of concentrated nitric acid, and slowly move along the glass rod under stirring Add it into concentrated hydrochloric acid, stir it evenly, and it turns yellowish brown (nitrosyl chloride is produced, and has a chlorine smell, all preparation and use must be carried out in a fume hood), seal the mouth of the beaker with a parafilm, and mark it as king with a marker pen Water back up.

[0047] 2) 10ml 25mM HAuCl 4 Solution preparation: weigh 0.0995g of HAuCl 4 .3H 2 Dissolve O in 10ml of ultrapure water, mix well, wrap in foil paper and store in dark at 4°C for later use.

[0048] 3) 100...

Embodiment 2

[0063] Example 2 The coupling method of nano-gold and miR-885-5p, the steps are as follows: 1) Preparation of 0.1% DEPC: Take 0.446ml of 1.12g / ml DEPC into 500ml of ultrapure water, place it in an ultrasonic cleaner for ultrasonic treatment for 2 hours until there are no oil droplets;

[0064] 2) Preparation of 10mMPBS: Accurately weigh NaCl8g, KCl0.2g, KH2PO40.2g, Na2HPO4.12H2O2.9g with an analytical balance, dissolve in 100ml enzyme-free sterile water, dissolve fully after ultrasonication, and then use enzyme-free sterile water to determine Capacity up to 1L;

[0065] 3) 0.18MPBS preparation: according to the PBS preparation plan, first prepare 0.2M, pH8.0 PBS with enzyme-free sterile water, and then dilute to 0.18M according to the proportion;

[0066] 4) Design and chemically synthesize miR-885-5pmimics, and connect Cy5 fluorescent dye to the 5' end of its sense strand, and connect six-carbon alkylthiol to the 5' end of its complementary strand. The sequence is as follows...

Embodiment 3

[0078] Example 3: Detection of miR-885-5p-AuNPs directly entering liver cancer cells and significantly upregulating miR-885-5p:

[0079] 1) Cell culture: Liver cancer cell lines Huh7, Hep3B, and HepG2 cells were placed in Dulbecco’s modified Eagle medium (DMEM) medium with 10% fetal bovine serum, 37°C, 5% CO 2and cultured in a cell incubator with saturated humidity; when the cells grow to about 90% confluent, discard the old culture medium in the culture bottle and wash once with sterilized PBS. Add 0.25% trypsin digestion solution for digestion, and lay the culture bottle flat so that the digestion solution covers the bottom of the bottle. Observe under an inverted microscope and find that the cytoplasm of the cells has retracted and the gap has increased, immediately discard the digestion solution, add 6-8ml of fresh DMEM culture solution containing 10% fetal bovine serum to stop the digestion, and repeat along the edge of the culture bottle Pipette adherent cells to form a...

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Abstract

The invention discloses a nano-gold miR-885-5p conjugate as well as a preparation method and an application thereof. The conjugate comprises nano-gold particles and double-stranded nucleotide, wherein the double-stranded nucleotide is double-stranded miR-885-5p of which the double strands both have 28 bases, or is double-stranded nucleotide which has a sequence homology of 95%-100% with the double-stranded miR-885-5p and has the same coding function as the double-stranded miR-885-5p, wherein the double-stranded nucleotide comprises two stem-loop structures, and a 5'-terminal of each antisense strand contains hexacarbon alkyl mercaptan and is coupled with the nano-gold particles. The nano-gold miR-885-5p conjugate has a remarkable growth inhibition effect on both liver cancer cells in vitro and liver cancer nude mouse transplanted tumors in vivo, and has a beneficial effect in the application for treating the liver cancer.

Description

technical field [0001] The invention relates to the field of liver cancer, in particular to a nano-gold miR-885-5p conjugate and its preparation method and application. Background technique [0002] Primary liver cancer (referred to as liver cancer) is a major disease that seriously endangers the health of our people, and has the characteristics of high incidence, high recurrence rate and high mortality rate. Exploring new and more effective therapeutic targets for liver cancer has always been the focus of research, and it is also a useful supplement and improvement to the current comprehensive treatment system for liver cancer that is dominated by surgical resection and liver transplantation. [0003] Drug nanocarriers have the advantages of high targeting, controlled drug release, and improved dissolution rate and absorption rate of insoluble drugs, thereby improving drug efficacy and reducing side effects. Nanoparticles have some significant advantages as gene carriers: ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K47/48A61K48/00A61K31/713A61P35/00
Inventor 何星星林菊生廖家智闫静君徐传瑞刘勇冉伟黎培员常莹田德安赵秋但自力
Owner TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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