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Preparation method of gene targeting delivery system for tumors with high CD44 expression

A high expression, tumor technology, applied in the field of medicine, can solve the problems of long stirring reaction time, small mass concentration range and high reaction temperature, and achieve the effect of huge market application prospects, positive social effects and rich economic benefits.

Active Publication Date: 2020-03-31
青岛百迈德生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Yet, there is following problem in the method described in this application: (1) reaction temperature is high, is 40-70 ℃, easily causes the reduction of chitosan molecular weight; (2) the mass concentration range of hyaluronic acid in aqueous solution is little, only 1-5%, thereby limiting the amount of reaction; (3) after the completion of the reaction, repeated dialysis and washing treatments are required to obtain modified hyaluronic acid, and the operation is complicated; (4) the stirring reaction time is as long as 3-24h, resulting in The whole operation process takes a long time, which will easily lead to a decrease in the activity of the aldehyde group after the reaction
[0006] In addition, there is no report on the combination of oxidized hyaluronic acid and chitosan nanoparticles as a gene-targeted delivery system for tumors with high CD44 expression

Method used

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  • Preparation method of gene targeting delivery system for tumors with high CD44 expression
  • Preparation method of gene targeting delivery system for tumors with high CD44 expression
  • Preparation method of gene targeting delivery system for tumors with high CD44 expression

Examples

Experimental program
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Effect test

Embodiment 1

[0028] A method for preparing a gene-targeted delivery system for tumors with high CD44 expression, comprising the following steps:

[0029] (1) The oxidized hyaluronic acid HA is reacted with alcohol to obtain hyaluronic acid HAD with two aldehyde groups:

[0030] (a) Weigh 2g of hyaluronic acid (HA), and add it into 95% (v / v) ethanol under stirring to obtain an ethanol solution of 9% (w / v) hyaluronic acid; (b) avoid light Weigh 0.5310g NaIO 4 (M HA :M NaIO4 =4:1), it was completely dissolved in water to obtain 0.5M NaIO 4 Aqueous solution; at 25°C the dissolved NaIO 4 The solution was slowly added to the HA solution drop by drop, and the reaction was stirred at a constant speed in the dark for 6 hours until the oxidation reaction was completed; (c) the solution after the reaction was added to three times the volume of 95% (v / v) ethanol for precipitation, and after washing, 25-30 ℃ drying to obtain oxidized hyaluronic acid HAD.

[0031] (2) Preparation of chitosan nanopar...

Embodiment 2

[0055] Different from Example 1,

[0056] A method for preparing a gene-targeted delivery system for tumors with high CD44 expression, comprising the following steps:

[0057] (1) The oxidized hyaluronic acid HA is reacted with alcohol to obtain hyaluronic acid HAD with two aldehyde groups:

[0058] (a) take by weighing 1g hyaluronic acid (HA), add in 60% ethanol under stirring, obtain the ethanolic solution of the hyaluronic acid that concentration is 5% (w / v); (b) avoid light and take by weighing 0.5310g NaIO 4 (M HA :M NaIO4 =2:1), it was completely dissolved in water to obtain 0.1M NaIO 4 Aqueous solution; at 25°C the dissolved NaIO 4 The solution was slowly added to the HA solution drop by drop, and the reaction was stirred at a constant speed in the dark until the oxidation reaction was completed. (c) Add the reacted solution to three times the volume of absolute ethanol for precipitation, wash and dry to obtain oxidized hyaluronic acid HAD.

[0059] (2) Preparatio...

Embodiment 3

[0064] Different from Example 1,

[0065] A method for preparing a gene-targeted delivery system for tumors with high CD44 expression, comprising the following steps:

[0066] (1) The oxidized hyaluronic acid HA is reacted with alcohol to obtain hyaluronic acid HAD with two aldehyde groups:

[0067] (a) take by weighing 3g hyaluronic acid (HA), add in 85% ethanol under stirring, obtain the ethanol solution of the hyaluronic acid that concentration is 6% (w / v); (b) avoid light and take by weighing 0.5310g NaIO 4 , which was completely dissolved in water to obtain 1M NaIO 4 Aqueous solution; at 22°C the dissolved NaIO 4 The solution was slowly added to the HA solution drop by drop, and the reaction was stirred at a constant speed in the dark until the oxidation reaction was completed.

[0068] (2) Preparation of chitosan nanoparticles CSNps-siRNA wrapped with siRNA:

[0069] The mass ratio of CS and TPP is 6:1; the stirring time is 40min.

[0070] (3) Preparation of CSNps-s...

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Abstract

Based on the present situation that chitosan (CS) nanoparticle gene targeting delivery technology is recently adopted, the invention provides a preparation method of a gene targeting delivery system for CD44 high-expression tumors. The preparation method of the invention comprises the following steps: (1) modifying oxidized hyaluronic acid HA to obtain hyaluronic acid HAD with aldehyde groups; themodification is chemical modification and there are two aldehyde groups; (2) preparing siRNA-coated chitosan nanoparticle CSNps-siRNA; (3) carrying out covalent cross-linking of HAD prepared in the step (1) onto the CSNps-siRNA prepared in the step (2) through a reaction between the aldehyde groups and amino groups; the mass ratio of HAD to CS is 1:50-1:500. Problems caused by loss of HA in the prior art are solved, and the basis for opening up a new way to gene therapy of CD44 high-expression tumors in clinic is provided in the invention.

Description

technical field [0001] The invention belongs to the field of medicine and relates to a gene carrier, in particular to a targeting gene carrier obtained by modifying chitosan nanoparticles with modified hyaluronic acid. Background technique [0002] Modern molecular biology studies have shown that the occurrence and development of tumors is a complex evolution process with multiple genes, multiple stages, and multiple steps. The root cause may be abnormal changes in gene structure and expression inside cells, involving the abnormality and accumulation of various proto-oncogenes, tumor suppressor genes, growth factors and their receptors, cell adhesion factors, and DNA repair genes. For example, Bcl2 is a proto-oncogene and one of the most important genes that inhibit tumor cell apoptosis. Studies have shown that the translocation and high expression of Bcl2 gene are closely related to the occurrence, development and poor prognosis of tumors. Reducing or eliminating the overe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K47/61A61K47/69A61K31/713A61P35/00
CPCA61K31/713
Inventor 刘玉敏梁晔孙建辉
Owner 青岛百迈德生物科技有限公司
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