Endotoxin adsorption medium based on magnetic chitosan microballoon and its application method

A technology of chitosan microspheres and adsorption media, applied in the field of biomedical materials, can solve the problems of small processing capacity and large loss of active ingredients, and achieve high selectivity and easy operation

Inactive Publication Date: 2012-02-15
ZHEJIANG UNIV OF TECH
3 Cites 16 Cited by

AI-Extracted Technical Summary

Problems solved by technology

[0003] At present, the commonly used methods for removing endotoxin include activated carbon adsorption, ultrafiltration, extraction, affinity chromatography, etc., but the first three methods are only suitable for removing endotoxin from relatively stabl...
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Abstract

The invention discloses an endotoxin adsorption medium based on magnetic chitosan microballoon and an application method, wherein the preparation method of endotoxin adsorption medium comprises the following steps: (1) dispersing a magnetic material in an acetic acid solution, adding chitosan to prepare water phase I, transferring water phase I to liquid paraffin containing span 80, taking glutaraldehyde as a cross-linking agent, preparing chitosan microballoon by using an anti-phase suspension method; (2) dispersing magnetic chitosan microballoon in a mixed solution of dimethyl sulfoxide and sodium hydroxide, adding chloropropylene oxide for activating, washing by deionized water till neutralizing to obtain an activated product; (3) placing the activated product in an endotoxin affinity ligand solution for immobilizing to obtain the endotoxin adsorption medium based on the magnetic chitosan microballoon. The endotoxin adsorption medium of the present invention has high selectivity of endotoxin and simple operation, and can be used for removing the endotoxin in a plurality of biological products.

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Technology Topic

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  • Endotoxin adsorption medium based on magnetic chitosan microballoon and its application method
  • Endotoxin adsorption medium based on magnetic chitosan microballoon and its application method
  • Endotoxin adsorption medium based on magnetic chitosan microballoon and its application method

Examples

  • Experimental program(3)

Example Embodiment

[0024] Example 1: Preparation of Chitosan-Histidine Endotoxin Adsorption Medium (MCM-His)
[0025] (1) Disperse 1.5g of ferroferric oxide ultrasonically in 100mL of 2% acetic acid solution, and add 3g of chitosan (produced by Aoxing Company, with a molecular weight of 300,000, and a degree of deacetylation of 95%, that is, the amino group density is 5833μmol /g) Dissolve it completely and disperse it into 100mL liquid paraffin containing 1mL Span 80, stir for 30min, add 3mL glutaraldehyde solution with a volume fraction of 25%, react at 40°C for 2h, use a 10% mass fraction Adjust the pH of the NaOH solution to about 12, while heating to 70°C, continue to stir and react for 2h, the product is washed with petroleum ether and hot ethanol and dried in vacuum to obtain magnetic chitosan microspheres (MCM), which is determined by acid-base titration Amino density: Take 50mg of microspheres and disperse into 20ml of 10mmol/L hydrochloric acid solution, oscillate for 1h, apply a magnetic field to absorb the microspheres, take 15ml of clear liquid and titrate with 10mmol/L sodium hydroxide solution. The calculated results are shown in Table 1.
[0026] (2) Disperse 1.0 g of magnetic chitosan microspheres obtained in step (1) into 20 mL of a mixed solution of dimethyl sulfoxide and sodium hydroxide (volume ratio 1:1, sodium hydroxide concentration of 1mol/L), and add 3mL epichlorohydrin, shake and react at 40°C for 4h, then wash with deionized water until it is neutral and dry.
[0027] (3) Transfer 0.5 g of the product obtained in step (2) to 10 mL 0.1 mol/L histidine solution, adjust the pH to about 11 with 0.5 M sodium hydroxide solution, shake in a water bath at 40°C overnight, and wash with deionized water After drying and storing to neutrality, the magnetic chitosan-histidine endotoxin adsorption medium (MCM-His) is obtained. The amino group density is shown in Table 1. The scanning electron microscopy, particle size distribution, and hysteresis regression line are shown in Table 1. figure 1 , figure 2 , image 3 Shown.

Example Embodiment

[0028] Example 2: Preparation of Chitosan-Tetraethylenepentamine Endotoxin Adsorption Medium (MCM-TEPA)
[0029] (1) Disperse 2.0 g of ferroferric oxide ultrasonically in 100 mL of 5% acetic acid solution, and add 4 g of chitosan (produced by Aoxing Company, with a molecular weight of 300,000, and a degree of deacetylation of 95%, that is, the amino group density is 5833μmol /g) Dissolve it completely and then disperse it into 200mL liquid paraffin containing 3mL Span 80. After stirring for 30 minutes, add 4mL glutaraldehyde solution with a volume fraction of 25%, react at 40°C for 1 hour, and use a 10% mass fraction The pH of the NaOH solution was adjusted to about 12, while the temperature was raised to 70°C, and the stirring reaction was continued for 3 hours. The product was washed with petroleum ether and hot ethanol and dried in vacuum to obtain magnetic chitosan microspheres.
[0030] (2) Disperse 0.5 g of the magnetic chitosan microspheres obtained in step (1) into 10 mL of a mixed solution of dimethyl sulfoxide and sodium hydroxide (volume ratio 1:1, sodium hydroxide concentration 1mol/L), and add 3mL epichlorohydrin, shake and react at 40°C for 4h, then wash with deionized water until it is neutral and dry.
[0031] (3) Transfer 0.5g of the product obtained in step (2) to 10mL 0.2mol/L tetraethylenepentamine solution, adjust the pH to about 11 with 0.5M sodium hydroxide solution, shake in a 40℃ water bath overnight, and use deionized water After washing to neutrality and dry storage, the magnetic chitosan-tetraethylenepentamine endotoxin adsorption medium (MCM-TEPA) is obtained. The amino group density is shown in Table 1.
[0032] Table 1 Amino density of different adsorption media
[0033]

Example Embodiment

[0034] Example 3: Use of adsorption medium
[0035] Take magnetic chitosan microspheres (MCM prepared in Example 1), magnetic chitosan microspheres-histidine (MCM-His prepared in Example 1), and magnetic chitosan microspheres-tetraethylene five Amine (MCM-TEPA prepared in Example 2) 50mg was washed with a mass fraction of 1% sodium deoxycholate aqueous solution, suction filtration or magnetic field separation three times, then washed with pyrogen-free water three times, dried and dispersed into 2mL solution containing endotoxin (Tap water), shake and adsorb for 30 minutes, absorb the adsorption medium with a magnet, and take out the supernatant to determine the endotoxin content.
[0036] The endotoxin content is determined by the limulus reagent color matrix method, and the adsorption kinetic curve of the magnetic chitosan microsphere-histidine (MCM-His) on endotoxin is as follows Figure 4 As shown, the adsorption effect of different adsorption media is as Figure 5 Shown from Figure 4 It can be seen that the adsorption equilibrium time of the adsorption medium is relatively short, within 10 minutes, the sample can be treated with endotoxin efficiently and quickly. From Figure 5 It can be seen that the magnetic chitosan microsphere (MCM) itself also has a certain adsorption effect on endotoxin, because although the cross-linking reaction of glutaraldehyde and chitosan occurs between the aldehyde group and the amino group, it is impossible to complete it. The gel pores of the microspheres will have a certain adsorption effect on endotoxins. After the solid loading of histidine and tetraethylenepentamine, the adsorption capacity of the adsorption medium has been improved, and the adsorption capacity of the adsorption medium (MCM-TEPA) with tetraethylenepentamine as the ligand has increased to 554.1EU/g.
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PUM

PropertyMeasurementUnit
Concentration30.0 ~ 40.0g/l
tensileMPa
Particle sizePa
strength10

Description & Claims & Application Information

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