Magnetic nanoparticle of layer-by-layer wrapping structure and preparation method and application thereof

A technology of magnetic nanospheres and coating structures, applied in other methods of inserting foreign genetic materials, recombinant DNA technology, etc., can solve problems such as difficult and effective gene transfection, achieve low cytotoxicity, improve cell entry efficiency, and avoid degradation Effect

Active Publication Date: 2016-07-27
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The present invention provides a magnetic nanosphere with a layer-by-layer structure, which solves the problem that the magnetic transfection reagent in the prior art is difficult to be used for effective gene transfection of stem cells such as bone marrow mesenchymal stem cells. dyed problem

Method used

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  • Magnetic nanoparticle of layer-by-layer wrapping structure and preparation method and application thereof
  • Magnetic nanoparticle of layer-by-layer wrapping structure and preparation method and application thereof
  • Magnetic nanoparticle of layer-by-layer wrapping structure and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0078] The preparation of the magnetic nanosphere of embodiment 1 layer-by-layer coating structure

[0079] 1. Preparation of Magnetic Nanospheres

[0080] (1) Take the prepared ED-pul and use 2 Double-distilled water to make 20mg / mL to obtain ED-pul aqueous solution; take 120μL ferric chloride solution (100mg / mL) and add it to ED-pul aqueous solution (1mL), vortex to dissolve, then continue to add 120μL ferrous chloride solution (41mg / mL), vortex vigorously to dissolve; then add 250 μL of concentrated ammonia water (28vol.%), and place in a 60°C water bath for 20min; the obtained suspension is used on a PD-10 (GE Healthcare Bio-Sciences Corp.) Double-distilled water elution removes excess ammonia water, and ferric oxide nanoparticles modified by cationic polymer ED-pul can be obtained, that is, the inner core (hereinafter referred to as IONPsED-pul);

[0081] (2) Dilute the purified IONPsED-pul to 250 μg / mL with 5% sucrose aqueous solution, take 100 μL, add double volume of p...

Embodiment 2

[0102] Example 2 Antiserum Gene Transfection Efficiency and Toxicity Evaluation of Magnetic Nanospheres

[0103] 1. Magnetic transfection of BMSCs

[0104] Taking human BMSC and rat BMSC as examples, the antiserum gene magnetic transfection efficiency of the present invention on BMSC is evaluated. In this embodiment, a blank control group (blank), a negative control group (NakepDNA, pDNA direct transfection), a non-viral gene carrier control group (Lipo2000 group or PEI group, and SP group), and a non-magnetic field control group were set at the same time.

[0105] Specific steps are as follows:

[0106] 1) Human BMSCs or rat BMSCs were mixed with 6×10 4 cells / well were seeded on a 24-well plate in 5% CO 2 In a cell culture incubator, culture at 37°C for 24 hours;

[0107] 2) Discard the culture medium, rinse twice with phosphate buffer (pH7.4), add 0.5mL DMEM low-sugar culture medium containing 10% fetal bovine serum to each well, prepare IONPsED-pul / pDNA / SP in Example 1 ...

Embodiment 3

[0142] Example 3 Magnetic transfection mechanism of magnetic nanospheres of the present invention

[0143] Taking IONPsED-pul / pDNA / SP as an example, using isothiocyanate-labeled pDNA (FITC-PGL-3), the distribution of pDNA and IONPs cores in magnetic nanospheres in rat BMSCs was investigated. Specific steps are as follows:

[0144] 1) Divide BMSC into 2×10 4 Each well was inoculated on a 20 mm diameter glass-bottom culture dish in 5% CO 2 , cultured at 37°C for 24 hours;

[0145] 2) Discard the culture medium, rinse twice with phosphate buffer (pH7.4), add DMEM low-sugar culture medium containing 10% fetal bovine serum and IONPsED-pul / pDNA / SP prepared and diluted according to Example 2; Place the cell culture dish on a cobalt-nickel magnetic field (the magnetic field strength is 2700Gs and 5000Gs respectively), 5% CO 2 , and incubated at 37°C for different time (0.5h, 2h, 4h and 6h);

[0146] 3) At the corresponding time point, remove the magnetic field, discard the cultur...

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Abstract

The invention discloses a magnetic nanoparticle of a layer-by-layer wrapping structure and a preparation method and application thereof.The magnetic nanoparticle is composed of an inner core, a middle layer and an outer layer, wherein the inner core and the middle layer are combined and the middle layer and the outer layer are combined by means of the electrostatic attraction effect, the inner core is a ferroferric oxide nanoparticle modified by a cationic polymer, the middle layer is a to-be-transfected gene wrapping the outside of the inner core, and the outer layer is a spermine and Pulullan copolymer wrapping the outside of the middle layer.According to the magnetic nanoparticle, the to-be-transfected gene is combined with the inner core first and then combined with the spermine and Pulullan copolymer, and therefore the to-be-transfected gene is tightly wrapped by the magnetic nanoparticle and prevented from being degraded in the transfection process; spermine and Pulullan can be recognized and absorbed by mesenchymal stem cells, and the cell entering efficiency of the magnetic nanoparticle is further improved; the spermine and Pulullan copolymer has little toxicity to mesenchymal stem cells, and the safety risks caused by transfection to stem cells are effectively reduced.

Description

technical field [0001] The invention relates to a magnetic transfection preparation, in particular to a magnetic nanosphere with a layer-by-layer coating structure and a preparation method and application thereof. Background technique [0002] Some stem cells, such as Bone-marrow mesenchymal stem cells (BMSC), are increasingly used in stem cell therapy, gene therapy and tumor therapy due to their potential for multi-directional differentiation and the characteristics of easy acquisition, isolation and purification. etc. research. [0003] However, in actual research and clinical applications, it is often necessary to carry out appropriate genetic modification on stem cells to induce more specific differentiation of stem cells into desired cells or to express specific therapeutic genes so as to better exert corresponding therapeutic effects in vivo. effect. [0004] Currently, gene transfection vectors used for genetic recombination in stem cells mainly include viral gene v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87
CPCC12N15/87
Inventor 高建青张添源吴佳禾
Owner ZHEJIANG UNIV
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