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Preparation method and application of aminated graphene quantum dot

A technology of aminated graphene and graphene quantum dots, applied in the field of biomedicine, can solve the problems of limited gene loading, antigenic tumorigenic risk, low transfection efficiency, etc., and achieve high transfection rate, low cost, and high technology simple steps

Active Publication Date: 2019-03-29
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Viral vectors are widely used due to their high transduction efficiency and expression efficiency, but they have a series of defects such as antigenicity, potential tumorigenic risk, and limited gene loading, which restrict the use of such vectors in gene expression. Developments in delivery
Non-viral gene delivery vectors have safety and stability characteristics that viral vectors do not have, but their transfection efficiency is usually low
Therefore, it is still one of the challenges in this field to find non-viral gene delivery vectors with high efficiency, low toxicity and a wide range of targets.

Method used

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  • Preparation method and application of aminated graphene quantum dot
  • Preparation method and application of aminated graphene quantum dot
  • Preparation method and application of aminated graphene quantum dot

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] 1) The preparation of graphene is as follows: first use strong oxidant concentrated sulfuric acid, concentrated nitric acid, potassium permanganate, etc. to oxidize graphite into graphite oxide. During the oxidation process, some oxygen-containing functional groups are interspersed between graphite layers, thereby increasing the distance between graphite layers , and then after ultrasonic treatment for a period of time, a single layer or several layers of graphene oxide can be formed, and then the graphene oxide can be reduced to graphene with strong reducing agents such as hydrazine hydrate and sodium borohydride.

[0049] 2) Aminated graphene quantum dot synthesis method is as follows: add 5ml of graphene uniform dispersion liquid and 3.0ml ammoniacal liquor (28wt%) and 5ml deionized water in glass bottle, after stirring for 30min, the mixture is transferred to polytetrafluoroethylene (PTFE)-lined autoclave (50Ml), heated at 70-150°C for 5h, after cooling to room tempe...

Embodiment 2

[0055] Aminated graphene quantum dots are used as an in vivo gene delivery carrier, so the influence of aminated graphene quantum dots on the biological activity of targeted cells and its possibility of gene delivery must be considered. For this reason, the present invention aims at implementing The aminated graphene quantum dots prepared by Example 1 have carried out the following tests:

[0056] 1. Detection of cytotoxic activity CCK-8 by aminated graphene quantum dots

[0057] 1) The mouse macrophage RAW264.7 was resuscitated and passaged into a 96-well plate, and 100ul was inoculated in each well, and the number of cells was about 4000; 2) After the cells were cultured for 12 hours, the medium was replaced, 100ul in each well; 3 ) Add 10ul graphene quantum dots of different concentrations to each well, and set 9 concentration gradients, which are 0ug / ml, 10ug / ml, 50ug / ml, 100ug / ml, 200ug / ml, 400ug / ml, 600ug / ml ml, 800ug / ml, 1mg / ml, 5 complexes for each concentration; 4) A...

Embodiment 3

[0067] Example 3: GQDs-NH 2 cross-linked miRNA

[0068] The present invention uses disulfide bonds to combine miRNA with the GQDs-NH prepared in Example 1 2 connected to achieve GQDs-NH 2 The purpose of carrying miRNA, the specific steps are as follows: use sulfo-lc-spdp bifunctional cross-linking agent-the sulfhydryl-modified miRNA and aminated graphene quantum dots (GQDs-NH 2 ) cross-linking, so that the miRNA is bound to the graphene quantum dots through a disulfide bond. Take 1ml of aminated graphene quantum dots (GQDs-NH 2 ) stock solution was diluted 10 times with PBS, adjusted the pH to about 7.44 with dilute HCL and NaOH, dissolved 2.5mg sulfo-lc-spdp in it, stirred gently at room temperature for 2h, centrifuged with a 100KDa ultrafiltration centrifuge tube, 2000rpm, 15min, twice Second-rate. Collect the liquid after centrifugation, take 1ml, add 200pmol miRNA, and react overnight in a refrigerator at 4°C.

[0069] The reaction steps are as follows:

[0070] ...

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Abstract

The invention discloses a preparation method and application of an aminated graphene quantum dot. The preparation method includes: blending a uniform dispersion liquid of prepared graphene, ammonia water and deionized water, then transferring the mixture into an autoclave for heating, filtering out insoluble fragments by a porous inorganic membrane, then conducting heating to remove redundant amine, and then using a centrifugal filtration device of molecular weight interception membrane for ultrafiltration of the supernatant, and subjecting the obtained yellow suspension liquid to further dialysis by a Spectra / Por CE dialysis tube membrane, thus obtaining aminated graphene quantum dot. The aminated graphene quantum dot prepared by the method provided by the invention can be applied to gene delivery vectors, has no inhibiting effect on RNAnase, has protective effect on to-be-delivered miRNA, and has good selectivity and universality.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a preparation method and application of aminated graphene quantum dots. Background technique [0002] A gene delivery vector (gene delivery vector) is sometimes referred to as a gene carrier for short, and refers to the necessary medium for assembling genes, introducing them into target cells or corresponding host cells, and expressing genes. In a broad sense, it generally refers to all carriers for delivering nucleic acid molecule drugs (including plasmids, miRNA, siRNA, shRNA, etc.). Gene delivery vectors are mainly divided into two categories: viral vectors and non-viral vectors. [0003] Viral vectors package exogenous genes into the shell of natural viruses, and use the infectivity of viruses to host cells to introduce exogenous genes into cells. Viral vectors are widely used due to their high transduction efficiency and expression efficiency, but they have a series of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C01B32/184C01B32/194C01B32/196C12N15/87
CPCC12N15/87C01B32/184C01B32/194C01B32/196
Inventor 刘非拉魏可毓朱楚洪
Owner ARMY MEDICAL UNIV
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