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Freeze-drying preparation of ternary gene transfer system and preparation method thereof

A technology of gene delivery system and freeze-dried preparation, which is applied in the field of pharmaceutical preparations, can solve the problems of limiting the prospect of in vivo application, the decrease of transfection efficiency, and the decrease of surface potential, and achieves significant antiserum transfection properties and improved gene transfection efficiency , the effect of improving transfection efficiency

Active Publication Date: 2017-05-31
GUANGZHOU ZHONGDA NANSHA TECH INNOVATION IND PARK +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, liposome is a relatively mature in vitro transfection carrier, but its stability is poor; cationic polymer carrier usually has a good in vitro gene transfection effect, but this type of material has obvious toxicity, and its transfection ability under serum conditions Poor, which limits the prospect of its in vivo application; Inorganic nanomaterials have good stability and are easy to modify, etc., and can be used as gene delivery carriers. The disadvantage is that the transfection efficiency is usually low
[0005] Among inorganic nanocarriers, mesoporous silica nanoparticles (MSNs) have unique advantages such as low cytotoxicity, good stability, easy modification and large specific surface area, pore volume and ordered pore structure. It has received extensive attention in the field of drug and gene delivery. The disadvantage is that the surface potential of nanoparticles loaded with genes will decrease, which is not conducive to the uptake of cells, and after the system enters cells, lysosomes will interfere with its transfection, causing transfection Efficiency drops
This method has been widely used in the preservation of proteins and probiotics, and it has also been used in the solidification of liposomes / genes and cationic polymers / genes. However, the method of inorganic nanoparticles / genes / organic polymers The freeze-drying preservation of the three-component gene delivery system is rarely studied

Method used

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  • Freeze-drying preparation of ternary gene transfer system and preparation method thereof
  • Freeze-drying preparation of ternary gene transfer system and preparation method thereof
  • Freeze-drying preparation of ternary gene transfer system and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1～3

[0034] Embodiments 1-3 Freeze-dried preparation of the three-way gene delivery system and its preparation method

[0035] The preparation method of the freeze-dried preparation of the ternary gene delivery system of Examples 1-3 comprises the following steps:

[0036] (1) Synthesis of mesoporous silica nanoparticles MSNs

[0037] a. Dissolve 1.0 g of cationic surfactant cetyltrimethylammonium bromide (CTAB, used as a template) and 0.28 g of NaOH in 480 mL of deionized water, stir and heat at 600 rpm to 80° C. to dissolve CTAB. After CTAB is completely dissolved to form a homogeneous system, add 5 mL silicon source tetraethyl orthosilicate (TEOS) drop by drop, hydrolyze for 2 hours, centrifuge to obtain a white solid, wash with a large amount of water until neutral, and then wash with ethanol for 2-3 times; to obtain mesoporous silica nanoparticles MSNs;

[0038] b. Use the acid reflux method to remove the template agent, specifically: weigh 1g of MSNs, disperse in 100mL of a...

Embodiment 4～6

[0046] Embodiments 4-6 Freeze-dried preparation of ternary gene delivery system and its preparation method

[0047] The preparation method of the freeze-dried preparation of the ternary gene delivery system of Examples 4-6 comprises the following steps:

[0048] (1), the synthesis of mesoporous silica nanoparticles MSNs, the steps are the same as in Example 1;

[0049] (2), the synthesis of aminated MSNs, the steps are the same as in Example 1;

[0050] (3), extract the plasmid DNA according to the kit instructions, prepare the MSNs suspension and mix it with the DNA solution in equal volumes, so that the mass ratio of MSNs to DNA is 10:1 (Example 4), 20:1 (Example 5), 30:1 (Example 6), mixed with a pipette, incubated at room temperature for 30min, then added a certain amount of cationic polymer carrier PEI solution and continued to incubate for 30min, wherein the mass ratio of PEI to DNA was 0.65: 1 (the corresponding N / P is about 5), constructing a three-way gene delivery ...

Embodiment 7～9

[0052] Examples 7-9 Freeze-dried preparation of ternary gene delivery system and its preparation method

[0053] The preparation method of the freeze-dried preparation of the ternary gene delivery system of Examples 7-9 comprises the following steps:

[0054] (1), the synthesis of mesoporous silica nanoparticles MSNs, the steps are the same as in Example 1;

[0055] (2), the synthesis of aminated MSNs, the steps are the same as in Example 1;

[0056] (3), extract plasmid DNA according to the kit instructions, prepare MSNs suspension and mix it with DNA solution in equal volume, so that the mass ratio of MSNs to DNA is 10:1 (Example 7), 20:1 (Example 8), 30:1 (Example 9), mixed with a pipette, incubated at room temperature for 30min, then added a certain amount of cationic polymer carrier PEI solution and continued to incubate for 30min, wherein the mass ratio of PEI to DNA was 1.3: 1 (the corresponding N / P is about 10), constructing a three-way gene delivery system of mesopo...

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Abstract

The invention discloses a freeze-drying preparation of a ternary gene transfer system and a preparation method thereof. The preparation method comprises the following steps of using 3-aminopropyltriethoxysilane to perform aminated modification on mesoporous silicon dioxide nanoparticles, so as to obtain MSHs-NH2; hatching the MSHs-NH2, DNA (deoxyribonucleic acid) and a cationic polymer carrier solution, and building the ternary gene transfer system; freezing and drying by a freeze-drying protection agent, so as to obtain the freeze-drying preparation of the ternary gene transfer system. The freeze-drying preparation of the ternary gene transfer system has the advantages that the transfection efficiency is higher, and the toxicity risk in body is decreased; the obvious antiserum transfection characteristic is realized, the transfection activity is better, the liquid preparation can be cured, the long-time storage under the non-cold chain condition is realized, and the stability and transfection activity are maintained.

Description

technical field [0001] The invention belongs to the technical field of pharmaceutical preparations. More specifically, the invention relates to a freeze-dried preparation of a three-component gene delivery system based on mesoporous silica and organic polymers and a preparation method thereof. Background technique [0002] Gene therapy refers to the transfer of exogenous genes with therapeutic effects into specific cells in a certain way to achieve the purpose of treatment. Among them, how to effectively deliver the target gene to cells and exert curative effect is the key to this therapy. [0003] Gene delivery usually requires the help of vectors, and the current gene vectors are often divided into two types: viral vectors and non-viral vectors. Although viral vectors can achieve high transfection efficiency, high immunogenicity, possible carcinogenicity and other safety issues limit the wide application of such vectors. Non-viral vectors have attracted the attention of ...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K9/19A61K47/26
CPCA61K47/26A61K48/0025A61K9/19
Inventor 吴传斌王倍张晓旭潘昕权桂兰陈航平
Owner GUANGZHOU ZHONGDA NANSHA TECH INNOVATION IND PARK
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