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Magnetic composite nanoparticles and preparation method and application thereof

A magnetic composite nanometer and nanoparticle technology is applied in the field of avidin-modified nucleic acid functionalized magnetic composite nanoparticles and their preparation, which can solve the problems of lack of cancer cell release regulation, difficult nuclease activity analysis and in situ imaging, etc. , to achieve the effect of uniform particle size and good dispersion

Active Publication Date: 2017-03-15
PEKING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Such methods are mainly used for hybridization with intracellular miRNA or mRNA (Zhang P, ChengF, Zhou R, et al. DNA‐hybrid‐gated multifunctional mesoporous siliconanocarriers for dual‐targeted and microRNA‐responsive controlled drug delivery. 2014,53(9):2371-2375.), due to the protective effect of silicon nanoparticles on DNA (He X, Wang K, Tan W, et al. Bioconjugated nanoparticles for DNA protection from cleavage. Journal of the American Chemical Society, 2003 ,125(24):7168-7169.), these nanoparticles usually inhibit the reaction of DNA with intracellular nucleases, making it difficult to use for nuclease activity analysis and in situ imaging
In addition, the current use of nanoparticles to introduce miRNA drugs lacks specific release regulation for cancer cells (Mura S, Nicolas J, Couvreur P. Stimuli-responsive anocarriers for drug delivery. Nature materials, 2013, 12(11): 991-1003. )

Method used

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  • Magnetic composite nanoparticles and preparation method and application thereof
  • Magnetic composite nanoparticles and preparation method and application thereof
  • Magnetic composite nanoparticles and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1, preparation magnetic composite nanoparticle Fe 3 o 4 @SiO 2 @AVD-AP-DNA (Nanoprobe A)

[0049] The process of preparing magnetic composite nanoparticles in the present invention is as follows: figure 1 As shown, it specifically includes the following steps:

[0050] 1.0mg Fe 3 o 4 @SiO 2 At room temperature, under 40KHz (this ultrasonic frequency is used in the present invention, it can be adjusted to a different ultrasonic time as required) ultrasonic for 20 minutes, dispersed in 2.0mL pure water to form a 0.5mg / mL solution, add 4.0mg EDC and 10.0 mg NHS to activate the carboxyl group, shake the reaction at room temperature for 30 minutes. Add 0.1 mL of avidin with a concentration of 2.0 mg / mL, and shake and react at room temperature for 8 hours. Magnetic separation, removal of unreacted substances, washing with deionized water three times, re-sonication dispersion in 2.0 mL of pure water. Add biotinylated double-stranded DNA containing U base at...

Embodiment 2

[0052] Embodiment 2, the APE1 activity in the solution is directly detected

[0053]In this embodiment, the DNA probe sequence of design is as follows:

[0054] 5'-GGGGACGACTCCATCGACCTCAAGCAGTTGATCCTTTGGAAAAAAA-Biotin-3'

[0055] 5'-GTCGATGGAGXCGTCCCC-FAM-3'

[0056] Among them, X represents the abasic site, Biotin represents biotin labeling, and FAM represents carboxycarboxyfluorescein labeling.

[0057] The experimental steps are as follows:

[0058] 1. In a 200 μL PCR tube, add 10 μL of the 0.5 mg / mL magnetic composite nanoparticle solution prepared in Example 1, 5 μL of 10× Buffer1.1 buffer, add deionized water to make the total volume 48 μL, and then add 2 μL Different concentrations of APE1 solutions, so that the final concentrations of APE1 were 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1U / mL, and the response of the composite nanoparticles was detected.

[0059] Stratagene Mx3000P fluorescent PCR instrument was used to collect fluorescence every 5s at 37°C, the excitation wave...

Embodiment 3

[0064] Example 3. Composite nanoparticles are quickly introduced into cells under the action of a magnetic field and release fluorescent signals

[0065] The prepared magnetic composite nanoparticles have good stability and biocompatibility, and can be quickly imported into Hela cells through endocytosis under the action of a magnetic field to image APE1.

[0066] The experimental steps are as follows:

[0067] 1. Cultivate cells in DMEM medium containing 1% penicillin-streptomycin double antibody and 10% inactivated fetal bovine serum, the incubator conditions are 37°C and 5% CO 2 / 95% air. When the cells grow to cover 70%-80% of the culture flask, add 0.25% trypsin and treat at 37°C for 4 minutes, then transfer the cells to a 96-well flat-bottom glass plate, each well contains 0.1mL cell culture medium, about 10 4 cells. The cells were cultured for 24 h to allow them to adhere to the wall before the experiment.

[0068] 2. The cells were washed with DPBS buffer, and the...

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Abstract

The invention discloses magnetic composite nanoparticles and a preparation method and an application thereof. The magnetic composite nanoparticles have uniform particle size and have the diameter of 40-60 nm; an abasic site-containing DNA double-chain connected on the surface through avidin and biotin can be specifically identified and cut by a target protein abasic endonuclease I (APE1), and fluorophores or drug molecules are released; at the same time, the obtained magnetic composite nanoparticles have good dispersity, stability and biocompatibility, thereby being used for in-situ fluorescent imaging of the APE1 in living cells. In addition, because cancer cells usually overexpress the APE1, the magnetic composite nanoparticles can more effectively release the drug in the cancer cells and are expected to achieve accurate treatment of the cancer cells.

Description

technical field [0001] The invention relates to a multifunctional composite nanoparticle and its preparation method and application. More specifically, it relates to an avidin-modified nucleic acid functionalized magnetic composite nanoparticle and its preparation method and application. The nanoparticle can be used as a gene carrier, in-situ imaging of nucleic acid repair enzymes in living cells and a drug carrier, and belongs to the technical field of nanoparticle preparation. Background technique [0002] Nanomaterials have excellent electrical, optical, and mechanical properties, with controllable morphology and easy surface modification. Biomolecules have the characteristics of strong specificity and abundant binding sites (Eugenii Katz, Itamar Willner. Integrated Nanoparticle-Biomolecule Hybrid Systems: Synthesis, Properties, and Applications. Angew. Chem. Int. Ed (2004), 43, 6042–6108). With the vigorous development of nanotechnology and biotechnology, the combinati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87C12Q1/00A61K49/00A61K9/51A61K47/42A61K47/30A61K47/02A61P35/00
CPCA61K9/5115A61K9/513A61K9/5169A61K49/005A61K49/0054A61K49/0056A61K49/0093C12N15/87C12Q1/00
Inventor 赵美萍翟筠秋刘艺斌黄山方思敏
Owner PEKING UNIV
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