Nano-carrier material suitable for exogenous gene transfection and preparation method thereof

A carrier and nano-technology, applied in the field of nano-carrier materials and their preparation, can solve the problems of restricting virus vectors and questioning application safety, and achieve the effects of expanding the scope of application, improving biological safety, and being easy to operate

Inactive Publication Date: 2019-03-01
SHANGHAI UNIV OF MEDICINE & HEALTH SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the competition in the field of biomedicine and health is fierce. Although the transfection efficiency of viral vectors is very high, its application safety has been questioned for a long time. There is no conclusion yet, and the requirements for medical approval are very strict, so it limits viral vectors. Applications

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  • Nano-carrier material suitable for exogenous gene transfection and preparation method thereof
  • Nano-carrier material suitable for exogenous gene transfection and preparation method thereof
  • Nano-carrier material suitable for exogenous gene transfection and preparation method thereof

Examples

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preparation example Construction

[0071] The method for preparing the cationic nanomaterial of efficient transfection of the present invention comprises the following steps:

[0072] A, prepare nano-carrier precursor by chemical cross-linking method;

[0073] B. Preparation of highly efficient transfection nanocarriers by nanometer self-assembly method.

[0074] The specific step A is: take 1mL of 50mg / mL cationic polymer in a clean glass bottle, throw a small stirring bar, and slowly stir in a constant temperature water bath at 28°C; blow nitrogen, slowly add 0.809mL of 1mg / mL mL of crosslinker and continue stirring for 2 hours.

[0075] The step B is specifically: take a dialysis bag with a length of 5 cm, the molecular weight is 25KD, transfer the reaction product obtained in step A to the dialysis bag, dialyze with deionized water, replace the deionized water once in 12 hours, and after 24 hours Recover, characterize its physical and chemical properties such as particle size and potential, and electron m...

Embodiment 1

[0091] Preparation of Nanotransfection Vectors

[0092] Take 1mL of 50mg / mL polymer polyisopropylacrylamide or polyethyleneimine or polylysine (molecular weight at 10 3 ~10 6 g / mol), or the composition of two polymers (according to the ratio of 0.05 to 0.85) in a clean glass bottle, throw a small stirring bar, and stir slowly in a constant temperature water bath at 28°C; Add 0.809mL of 1mg / mL DTSP (3,3'-dithiodipropionate bis(N-hydroxysuccinimide) ester), and continue to stir for 2 hours; take a dialysis bag with a length of 5cm (molecular weight: 30KD) , transfer the obtained reaction product into a dialysis bag, dialyze with deionized water, replace the deionized water once at 12h, recover after 24h, and characterize its particle size, potential, electron microscope and other physical and chemical properties.

Embodiment 2

[0094] The hydrodynamic radius of the nano transfection reagent JH-ZRR2000 synthesized in Example 1 was characterized by ALV-DLS dynamic and static light scattering instrument. The specific operation process is to take 10 μl of the original solution of the nano-transfection reagent JH-ZRR2000 in Example 1, dissolve it in 1 mL of 18.2Ω ultrapure water, and carry out the detection on the machine after mixing. The set temperature is 20°C, the wavelength is 632.8nm, the solution viscosity coefficient is 0.8900, and the detection signal time is 300s. The test results (seefigure 2 ) shows that the particle radius is about 186nm.

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Abstract

The invention provides a cationic nanometer material used for cell transfection and a preparation method thereof. According to the invention, a macromolecular crosslinking and nanometer self-assemblymethod is firstly utilized to prepare the cationic nanometer material with high transfection efficiency; and then, DNA electrophoresis is utilized to detect the capacity of transfection material to carry plasmid DNA; lastly, an inverted fluorescence microscope and a flow cytometry are utilized to detect the transfection efficiency of the transfection material. The transfection material not only can directly instant transfect cells but also can be used as a carrier for stable cell transfection; the transfection material especially has a transfection capacity under the existence of serum and canachieve a better transfection effect; the transfection efficiency thereof can be adjusted through temperature; the nanometer transfection material acquired according to the invention can realize instant transfection and stable transfection of different eukaryotes, can be used for replacing a virus vector for transfection, can promote the biosecurity of exogenous gene transfection and has a broadapplication prospect.

Description

technical field [0001] The invention belongs to the fields of cell molecular biology and nanobiology, and in particular relates to a nano-carrier material suitable for exogenous gene transfection eukaryotic cells and a preparation method thereof. Background technique [0002] Since the completion of the human gene map, human beings have entered the post-genome era. With the deepening of gene research in recent years, gene editing and cell transformation have become the main theme of the life era, and gene transfection technology is the main means to study the expression of the target gene in cells. The gene transfection technology is exactly to send the purified plasmid DNA containing the target gene into the cell, and express it in the cell (Transfection, at the US National Library of Medicine Medical Subject Headings (MeSH), 2011, Tree Number: E05.393.350.810 ). This technology has not only revolutionized the research on many fundamental issues in life sciences and medic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87C12N15/79C12N15/85
CPCC12N15/79C12N15/85C12N15/87
Inventor 李威梁蓓蓓黄勇黄钢孟继鸿董霞
Owner SHANGHAI UNIV OF MEDICINE & HEALTH SCI
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