6-O-N-trimethyl chitosan chloride-SOD (superoxide dismutase) modifier and preparation thereof

A technology of quaternized chitosan and superoxide, applied in the field of biomedicine, can solve the problem of poor activity retention rate of conjugates, low SOD efficiency, and restrictions on the research of quaternized chitosan-superoxide dismutase modifiers and application issues, to achieve the effect of improving the modification rate, high activity retention rate of the product, and high activity retention rate of the modified product

Inactive Publication Date: 2015-02-18
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] During the preparation of TMC, the 2-NH of chitosan 2 After being methylated, 2-NH(CH 3 ) or even 2-N(CH 3 ) 2 , and then the further formation of 2-N(CH 3 ) 3 + Quaternary ammonium groups, therefore, the number of free amino groups in TMC with good water solubility is very small (generally <10%), resulting in lower efficiency to SOD and poor activity retention of the conjugate, which restricts the quaternization of chitosan-superior Further Research and Application of Oxide Dismutase Modifiers

Method used

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  • 6-O-N-trimethyl chitosan chloride-SOD (superoxide dismutase) modifier and preparation thereof
  • 6-O-N-trimethyl chitosan chloride-SOD (superoxide dismutase) modifier and preparation thereof
  • 6-O-N-trimethyl chitosan chloride-SOD (superoxide dismutase) modifier and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The preparation of 6-O-quaternized chitosan (O-HTCC), the steps are as follows:

[0044] ① Dissolve 3g of chitosan in 100ml of 10% acetic acid aqueous solution, stir at 40°C for 30min, fully swell, add 15.8g of benzaldehyde, continue to stir for 1h to obtain a milky white reaction solution, and dry it in vacuum at 60°C for 12h , using a NaOH solution with a mass concentration of 10% to adjust the pH to neutral, filter, collect the precipitate, wash with methanol 3 times, and dry to obtain reactant A;

[0045] ② Grind the reactant A prepared in step ①, put it in a round bottom flask, add 50ml of isopropanol, stir for 30min, then add 9g of 2,3-epoxypropyltrimethylammonium chloride (GTMAC) dropwise ), reflux and stir in a water bath at 70°C for 16h, filter the reaction solution, wash the obtained solid with methanol, and dry it in vacuum to obtain reactant B;

[0046] ③Grind the reactant B prepared in step ②, put it in a round bottom flask, add 50ml of 0.25mol / L hydrochlo...

Embodiment 2

[0049] A kind of preparation method of 6-O-quaternized chitosan-superoxide dismutase modification, preparation steps are as follows:

[0050] (1) Superoxide dismutase (SOD) is dissolved in the PBS damping fluid of 50ml concentration 10mM, pH 6.0, makes superoxide dismutase solution, and the concentration of superoxide dismutase is 1.0mg / ml (0.03125 μ mol / ml); then add 50mg EDC and 10mg NHS, shake and react for 30min under the greenhouse to prepare the reaction solution A;

[0051] (2) taking by weighing the quaternized chitosan (O-HTCC) 100mg that embodiment 1 makes is dissolved in the PBS damping fluid that 50ml concentration is 10mM, pH6.0, makes chitosan solution, O-HTCC The concentration is 2.0mg / ml, then add the reaction solution A prepared in step (1), shake and react for 6h, separate and purify through 2.6×40cm DEAE Sepharose Fast Flow resin, use pH 8.0, concentration 10mmol / L~100mmol / L Phosphate buffer (pH 8.0) for gradient elution, flow rate 1.5ml / min, detection wav...

Embodiment 3

[0056] A kind of preparation method of 6-O-quaternized chitosan-superoxide dismutase modification, preparation steps are as follows:

[0057] (1) Superoxide dismutase (SOD) is dissolved in the PBS damping fluid of 50ml concentration 10mM, pH 6.0, makes superoxide dismutase solution, and the concentration of superoxide dismutase is 1.0mg / ml (0.03125 μ mol / ml); then add 30mg EDC and 10mg NHS, shake and react for 30min under the greenhouse to prepare reaction solution A;

[0058] (2) taking by weighing the quaternized chitosan (O-HTCC) 50mg that embodiment 1 makes is dissolved in the PBS buffer solution that 25ml concentration is 10mM, pH 6.0, makes chitosan solution, the concentration of O-HTCC 2.0mg / ml, then add the reaction solution A prepared in step (1), shake and react for 6h, separate and purify through 2.6×40cm DEAE Sepharose Fast Flow resin, use pH 8.0, concentration 10mmol / L~100mmol / L Phosphate buffer (pH 8.0) for gradient elution, flow rate 1.5ml / min, detection wavel...

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Abstract

The invention relates to a 6-O-N-trimethyl chitosan chloride-SOD modifier and a preparation thereof. The modifier is formed by combining a carboxyl on an SOD molecule with an amino group on an O-HTCC molecule through an amido bond; one O-HTCC molecule and one to ten SOD molecules are combined. The invention further relates to a preparation method of the 6-O-N-trimethyl chitosan chloride-SOD modifier. Compared with natural SOD, the O-HTCC-SOD modifier remains the activity of decomposing superoxide anions by SOD, integrates the favorable characteristics of O-HTCC, and has the characteristics of high stability, long half-life period and low immunogenicity; compared with TMC, the efficiency of modifying SOD by O-HTCC is high, and therefore, the 6-O-N-trimethyl chitosan chloride-SOD modifier has a better using effect and a wider application range.

Description

technical field [0001] The invention relates to a 6-O quaternized chitosan-superoxide dismutase modified product and its preparation, belonging to the technical field of biomedicine. Background technique [0002] Superoxide dismutase (SOD) is an important class of superoxide anion free radical scavenger, which can be used to prevent and treat diseases and body damage caused by excessive superoxide anion free radicals, such as radiation injury, ischemic refocus injury, autoimmune disease etc. At present, the main source of superoxide dismutase is extracted from animals and plants, which has the disadvantages of antigenicity, short half-life in vivo and poor stability, which limits its application. Chemical modification of its molecule is an effective method to overcome the above-mentioned shortcomings of superoxide dismutase. Modifiers commonly used include polyethylene glycol (PEG), dextran, low molecular weight heparin, chitosan, etc. Modified SOD Reduced immunogenicity, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/96C12N9/02
CPCC12N9/96C12N9/0089C12Y115/01001
Inventor 刘纯慧许玲华王凤山宋春霞曹吉超
Owner SHANDONG UNIV
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