Method for preparing nano particles comprising protamine and genetic material

A protamine and nanoparticle technology, used in gene therapy, genetic material components, pharmaceutical formulations, etc., can solve problems such as reducing targeting effect, in vivo toxicity, in vivo immunogenicity, etc., and achieve the effect of improving targeting effect.

Inactive Publication Date: 2014-04-30
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a method for preparing nanoparticles comprising protamine and genetic material, so as to solve the problem of using protamine and cationic liposomes to compress and encapsulate DNA or siRNA. Unnatural cationic lipids have problems of in vivo toxicity and in vivo immunogenicity, and the positive charges on the surface of the cationic that fail to neutralize cause adhesion to non-target tissues in vivo and aggregate with plasma components, thereby reducing target tropism, and directly affects the technical issues of endocytosis of diseased cells in vivo

Method used

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  • Method for preparing nano particles comprising protamine and genetic material
  • Method for preparing nano particles comprising protamine and genetic material
  • Method for preparing nano particles comprising protamine and genetic material

Examples

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Embodiment 1

[0038] Example 1 Block polymer PEG 45 -PCL 30 -Synthetic method of DEX

[0039] The synthetic route of block polymer PEG-PCL-DEX is as follows figure 1shown. The whole reaction is carried out in an anhydrous and oxygen-free environment. Add PEG, polycaprolactone, and stannous octoate to a three-necked flask, add anhydrous toluene, and stir and react at 120°C for 24 hours. After the reaction is completed, add ether to precipitate , the filter cake was dissolved by adding a small amount of dichloromethane, and then precipitated with ether, and repeated three times to wash away small molecular impurities to obtain PEG 45 -PCL 30 The block polymer product was vacuum-dried and set aside.

[0040] Dextran and ethylenediamine were dissolved in DMSO, and reacted in an oil bath at 60° C. for 8 days. At the same time, 10 mg of sodium cyanoborohydride was added every day. After the reaction, add a large amount of methanol to precipitate, filter, add water to dissolve, methanol prec...

Embodiment 2

[0043] The preparation of the protamine / DNA binary complex of embodiment 2 PEG-PCL-DEX encapsulation

[0044] Prepare PEG (MW8000) external aqueous phase solution, the concentration of PEG is 10wt%, add genetic material, protamine and block polymer to the external aqueous phase solution, wherein, the sum of the mass of genetic material and protamine and embedded The mass ratio of high molecular weight segment is 7:30, and the mass ratio of genetic material to protamine is 1:1.5. Stir magnetically in an oil bath at 50°C for 24 hours to obtain a carrier structure in which the inner water phase is a protamine-DNA or siRNA complex. Finally, dialysis is performed using a dialysis bag with a molecular weight cut-off of 10,000 to remove PEG remaining in the water phase. For specific preparation methods, see Figure 4 .

Embodiment 3

[0045] The preparation of the protamine / DNA binary complex of embodiment 3 PEG-PCL-DEX encapsulation

[0046] Prepare PEG (MW8000) external aqueous phase solution, the concentration of PEG is 0.1wt%, add genetic material, protamine and block polymer to the external aqueous phase solution, wherein the sum of the mass of genetic material and protamine is equal to The mass ratio of block polymer is 1:0.1, and the mass ratio of genetic material and protamine is 0.1:1. Stir magnetically in an oil bath at 50°C for 24 hours to obtain a carrier structure in which the inner water phase is a protamine-DNA or siRNA complex. Finally, dialysis is performed using a dialysis bag with a molecular weight cut-off of 10,000 to remove PEG remaining in the water phase. For specific preparation methods, see Figure 4 .

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Abstract

The invention relates to the technical field of biology, in particular to a method for preparing nano particles comprising protamine and a genetic material. The preparation method comprises the following steps of: 1) preparing a hydrophilic phase system by using polyethylene glycol (PEG) as a continuous phase; 2) adding block macromolecules, the protamine and the genetic material into the hydrophilic phase system prepared in the step 1), and stirring or shaking to form nano particles; and 3) removing residual PEG in the aqueous phase of the nano particles by dialysis through a dialysis bag, and thus obtaining the nano particles. Compared with the prior art, the method has the advantages that the nano particles prepared by adopting the preparation method can realize delivery of the genetic material and does not have the problems of toxicity in vivo and immunogenicity in vivo; because the nano particles do not carry charges, adhesion of non-target tissues in vivo can be avoided; and the nano particles can be grafted with targeting groups, so that targeting of pathological cells in vivo is realized, the targeting effect is effectively improved, and endocytosis of the pathological cells in vivo is not affected.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing nano particles including protamine and genetic material. technical background [0002] siRNA is a 21-25 base pair RNA molecule found in the mechanism of single-celled organisms to resist viral invasion. Single-cell organisms synthesize a complementary siRNA to the mRNA sequence of the invading virus, which actively binds to the mRNA, thereby blocking the replication of the virus. Due to its unique target specificity, structural designability and metabolic safety, siRNA has become a promising new therapeutic drug in the scientific research community. However, in the past 20 years, researchers have designed a series of carriers for in vivo delivery of siRNA, but only a few have entered the clinical stage. , leading to the in vivo delivery of nucleic acid substances is still in the clinical bottleneck period. [0003] To deliver nucleic acid substances into the...

Claims

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Application Information

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Patent Type & AuthorityPatents(China)
IPC IPC(8): A61K48/00A61K47/42A61K47/36A61K47/34A61K9/14C08G81/00C08B37/02C08G63/08C08G63/91
Inventor袁伟恩金拓吴飞葛雪梅张奇昕蔡云鹏段诗悦
OwnerSHANGHAI JIAOTONG UNIV