CN101596422B - Method for preparing polyvinylidene fluoride affinity membrane using amino acid as ligand - Google Patents

Abstract

The invention discloses a method for preparing a polyvinylidene fluoride affinity membrane using amino acid as a ligand. The affinity membrane is prepared by hydrophilically modifying a polyvinylidene fluoride hollow fiber membrane and grafting the amino acid ligand. The grafting amount of the amino acid is 150 to 250mg/g for each membrane. The invention also discloses application of the affinity membrane in removing endotoxin in blood plasma, which removes the endotoxin in the blood plasma by a dynamic absorption means. The polyvinylidene fluoride affinity membrane prepared by the method has stable performance, good bio-compatibility and high endotoxin removing efficiency, and can be used for whole blood perfusion as well as blood plasma perfusion.

Description

Preparation method of polyvinylidene fluoride affinity membrane with amino acid as ligand

technical field

The invention relates to a preparation method of a polyvinylidene fluoride affinity membrane with amino acid as a ligand, in particular to a method for preparing a polyvinylidene fluoride affinity membrane for removing blood plasma through hydrophilic modification and grafting of an amino acid ligand to a polyvinylidene fluoride hollow fiber membrane Polyvinylidene fluoride affinity membrane method for endotoxin.

Background technique

At present, endotoxemia has three high phenomena of high morbidity, high mortality, and high treatment costs, which have constituted a serious threat to human health and a huge burden on economic development. Endotoxin is one of the structural components of the outer membrane of the cell wall of Gram-negative bacteria, and its chemical essence is lipopolysaccharide (LPS). Since the intracellular signal transduction of cell activation induced by LPS is extremely complex, once the cascade effect initiated by the binding of LPS to the corresponding receptors of inflammatory response cells occurs, it will be difficult to effectively control.

In recent years, although the application of a large number of broad-spectrum and high-efficiency antibiotics has provided powerful measures for the clinical treatment of infectious diseases, it has not fundamentally changed the current situation of high mortality in patients with sepsis. The reason is that antibiotics cannot curb the systemic uncontrolled inflammatory response caused by the interaction between endotoxin and the body, and may cause endotoxin release while killing bacteria, thereby aggravating the inflammatory response. At the same time, there are also defects such as large dosage, high cost, and high toxicity, which affect its further clinical application.

Many experiments at home and abroad have shown that it is an effective method to use affinity technology with the characteristics of high selectivity, fast separation speed, low energy consumption, and easy amplification to remove endotoxin. Romi Lamb et al. added silica particles to chitosan to prepare chitosan microporous blend membranes to absorb and remove endotoxins in prepared human immunoglobulin G (IgG) solutions (Machado RL, et al., Process Biochemistry, 41( 11): 2252-2257 (2006)). Sundberg et al. used immobilized metal affinity chromatography (chromatography) technology to remove endotoxin from proteins (U.S.Pat.20050186218). Affinity membrane separation is a separation technology that combines affinity chromatography and membrane separation. It has the characteristics of high selectivity, fast separation speed, high product activity, and easy scale-up. The application research of this technology is very active at home and abroad, involving The range is wide. The endotoxin adsorbent for hemoperfusion with polyvinylidene fluoride membrane as the carrier has not been reported.

Contents of the invention

The invention provides a method for preparing a polyvinylidene fluoride affinity membrane with amino acid as a ligand. By hydrophilically modifying a polyvinylidene fluoride (PVDF) membrane and grafting an amino acid ligand, a stable performance is prepared. PVDF affinity membrane with good biocompatibility and high endotoxin removal rate. Can be used for whole blood perfusion, can also be used for plasma perfusion.

A preparation method of a polyvinylidene fluoride affinity membrane with amino acid as ligand, the steps are:

1) Pretreatment of basement membrane

After rinsing the PVDF hollow fiber membrane with 30% ethanol by mass percentage, soak it in deionized water to wash away the impurities on the surface of the membrane;

2) Hydrophilic modification

Send the mixed solution of KMnO ₄ and KOH into the PVDF hollow fiber membrane module with a constant flow pump at 60-80°C to react for 30-90 minutes, and then reduce it with the mixed solution of H ₂ SO ₄ and NaHSO ₃ ;

The concentration of KMnO ₄ in the mixed solution of KMnO ₄ and KOH is 40-80g/l, the concentration of KOH is 3-5mol/l; the concentration of H ₂ SO ₄ in the mixed solution of H ₂ SO ₄ and NaHSO ₃ is 50-100g /l, the concentration of _NaHSO3 is 50-100g/l;

3) Hydroxyethyl cellulose (HEC) treatment

The PVDF hollow fiber membrane after reduction treatment is treated with a mixed solution of NaOH and HEC at 80-90°C for 10-30min to increase the hydrophilicity of the membrane surface; then dried at 80-90°C, after drying, use 0.5-2mol/l Wash with Na ₂ CO ₃ solution at 80-90°C to wash off the unreacted HEC on the membrane surface;

The concentration of NaOH in the mixed solution of NaOH and HEC is 1-3mol/l, and the concentration of HEC is 10-30g/l;

4) The first activation

The PVDF hollow fiber membrane treated in step 3) is activated with a mixed solution of epichlorohydrin (ECH) and NaOH at 50-70° C., and rinsed with deionized water after activation;

The mixed solution of epichlorohydrin and NaOH is a mixed solution prepared according to the volume ratio of epichlorohydrin: NaOH solution whose concentration is 1 mol/l;

5) Bonded hexamethylenediamine spacer

After the first activation, the PVDF hollow fiber membrane is reacted with 40-80g/l hexamethylenediamine (HDA) at 50-70°C for 2-3h, and rinsed with deionized water after the reaction;

6) Second activation

After step 5) the PVDF hollow fiber membrane connected with the spacer arm is reactivated with a mixed solution of epichlorohydrin (ECH) and NaOH at 50-70° C.;

The mixed solution of epichlorohydrin and NaOH is a mixed solution prepared according to the volume ratio of epichlorohydrin: NaOH solution whose concentration is 1 mol/l;

7) Bonded amino acid ligands

The PVDF hollow fiber membrane after the second activation reacts with 2-8g/l amino acid solution (0.2mol/l phosphate buffer with pH 7.2 as solvent) at 40-50°C for 20-24h to obtain grafted amino acid Ligand-based polyvinylidene fluoride affinity membrane.

In the preparation method, the amino acid is one of serine, asparagine, arginine, histidine, polylysine or glutamine. Endotoxin molecules are negatively charged due to their phosphate groups and can therefore be removed with positively charged sorbents. The amino and carboxyl groups of amino acids can bind to the anion and hydrophobic groups on the endotoxin molecule, respectively.

The amino acid grafting amount of the polyvinylidene fluoride affinity membrane prepared by the method of the invention is 150-250 mg/g membrane.

The polyvinylidene fluoride affinity membrane prepared by the above method is applied to remove endotoxin in plasma, and the membrane assembly of the affinity membrane is used for hemoperfusion, including the following steps: at room temperature, the blood of patients with endotoxemia is pumped into In the membrane module, the flow rate is 0.5-2.0mL/min, and the effluent is not circulated.

The polyvinylidene fluoride affinity membrane of the invention dynamically adsorbs the blood plasma of patients with endotoxemia, and the removal rate of endotoxin can reach 61.9%, and the adsorption efficiency is relatively high. It can be used in clinical whole blood perfusion to remove endotoxin in blood and treat diseases such as endotoxemia.

The advantages of the present invention are:

1) Using polyvinylidene fluoride as a carrier, the material has stable physical and chemical properties and has good blood compatibility.

2) Fill the membrane module with polyvinylidene fluoride hollow fiber membrane, which makes the process of membrane modification, grafting ligand and hemoperfusion simple and convenient, and because of the large specific surface area of the membrane, the grafting of ligand can be greatly improved. branch rate. And due to the advantages of the membrane module itself, the process is easy to scale up, and at the same time, it can be connected in series to meet the needs of clinical applications.

3) Amino acid is used as ligand, and it contains two functional groups of amine group and carboxyl group at the same time, which improves the specific adsorption of endotoxin by the adsorption material.

Description of drawings

Fig. 1 The experimental flow chart of removing endotoxin in blood plasma by amino acid affinity membrane;

1-blood; 2-pump; 3-flow meter; 4-affinity membrane module; 5-constant temperature water bath; 6-collection solution;

Fig. 2 is the breakthrough curve of endotoxin adsorbed by serine affinity membrane in Example 1.

Detailed ways

The membrane module contains 16 PVDF hollow fiber membranes, the mass of the membrane is 0.42g, the effective length is 16.70cm, and the effective area is 19.93cm ² .

The glass instruments touched by the test were dry-baked at 400°C for 4 hours, and the plastic utensils were soaked in 30% H ₂ O ₂ for 4 hours to remove the heat source. The affinity membrane module was washed with 0.1mol/L NaOH containing 20% ethanol, 1.5mol/L NaCl, and pyrogen-free water for 2 hours to remove the pyrogen.

Example 1

1) Rinse the PVDF hollow fiber membrane with 30% ethanol for 30 minutes, and soak it in deionized water for 1 hour to wash away impurities on the surface of the membrane.

2) Send 40g/l KMnO ₄ and 3mol/l KOH mixed solution into PVDF hollow fiber membrane module at 80°C with a constant flow pump to react for 30min, then use 60g/l H ₂ SO ₄ and 60g/l NaHSO ₃ mixed solution reduction.

3) The membrane after the reduction treatment was treated with a mixture of 1 mol/l NaOH and 10 g/l HEC at 90° C. for 15 min to increase the hydrophilicity of the membrane surface. Put it in a constant temperature hot air drying oven at 90°C and dry it. After drying, wash it with 0.5mol/l Na ₂ CO ₃ solution at 90°C for 10 minutes to wash off the unreacted HEC on the surface of the PVDF membrane.

4) The hydrophilized modified membrane was activated at 60° C. with a mixture of epichlorohydrin (ECH): 1 mol/l NaOH with a volume ratio of 1:4, and washed with deionized water for 1 h.

5) Then react with 50 g/l hexamethylenediamine (HDA) at 50° C. for 2 hours, and rinse with deionized water for 1 hour.

6) The PVDF membrane connected with the spacer arm was reactivated at 60° C. with a mixture of epichlorohydrin (ECH): 1 mol/l NaOH with a volume ratio of 1:4.

7) React with 3g/l serine solution (0.2mol/l phosphate buffer with pH 7.2 as solvent) at 45°C for 24h to obtain a polyvinylidene fluoride affinity membrane grafted with serine ligands.

According to the method shown in Figure 1, human plasma 1 is pumped into the affinity membrane module 4 by the pump 2 through the flow meter 3, the flow rate is 1.0mL/min, and the affinity membrane module 4 is provided with a constant temperature water bath 5. Take the collected solution 6 every 3 minutes to measure the content of endotoxin, draw the breakthrough curve, see Figure 2, and the effluent is not circulated. By measuring the initial concentration of endotoxin and the breakthrough curve, the removal efficiency of endotoxin can be obtained, as shown in Table 1.

The performance of the prepared affinity membrane of table 1 embodiment 1

Example 2

1) Rinse the PVDF hollow fiber membrane with 30% ethanol for 40 minutes, and soak it in deionized water for 1 hour to wash away impurities on the surface of the membrane.

2) Send 50g/l KMnO ₄ and 3mol/l KOH mixed liquid into PVDF hollow fiber membrane module with constant flow pump at 60°C for 30min reaction, then use 60g/l H ₂ SO ₄ and 60g/l NaHSO ₃ The mixed solution is restored.

3) The membrane after the reduction treatment was treated with a mixture of 1.5 mol/l NaOH and 15 g/l HEC at 80° C. for 10 min to increase the hydrophilicity of the membrane surface. Put it in a constant temperature hot air drying oven at 80°C and dry it. After drying, wash it with 0.5mol/l Na ₂ CO ₃ solution at 80°C for 10 minutes to wash off the unreacted HEC on the surface of the PVDF membrane.

4) The hydrophilized modified membrane was activated with a mixture of epichlorohydrin (ECH): 1mol/l NaOH with a volume ratio of 1:3 at 50°C, and rinsed with deionized water for 1 h.

5) Then react with 40 g/l hexamethylenediamine (HDA) at 60° C. for 2 hours, and rinse with deionized water for 1 hour.

6) The PVDF membrane connected with the spacer arm was reactivated at 50° C. with a mixture of epichlorohydrin (ECH): 1 mol/l NaOH with a volume ratio of 1:3.

7) React with 2 g/l serine solution (0.2 mol/l phosphate buffer with pH 7.2 as solvent) at 40° C. for 20 h to obtain a polyvinylidene fluoride affinity membrane grafted with serine ligands.

The experimental procedure for the removal of endotoxin in plasma is the same as in Example 1, and the graft density and endotoxin removal efficiency of the ligand are shown in Table 2.

The performance of the prepared affinity membrane of table 2 embodiment 2

Example 3

Step 1-6 is the same as embodiment 1

7) React with 3 g/l asparagine solution (0.2 mol/l phosphate buffer with a pH of 7.2 as solvent) at 45°C for 24 hours to obtain a polyvinylidene fluoride affinity membrane grafted with asparagine ligand .

The experimental procedure for the removal of endotoxin in plasma by the polyvinylidene fluoride affinity membrane with asparagine ligand is the same as that in Example 1, and the graft density and endotoxin removal efficiency of the ligand are shown in Table 3.

The performance of the prepared affinity membrane of table 3 embodiment 3

Example 4

Step 1-6 is the same as embodiment 1

7) React with 3 g/l arginine solution (0.2 mol/l phosphate buffer with a pH of 7.2 as solvent) at 45°C for 24 hours to obtain a polyvinylidene fluoride affinity membrane grafted with arginine ligands .

The experimental procedure for the removal of endotoxin in plasma by the polyvinylidene fluoride affinity membrane with arginine ligand is the same as in Example 1, and the graft density and endotoxin removal efficiency of the ligand are shown in Table 4.

The performance of the prepared affinity membrane of table 4 embodiment 4

Claims (7)

1. a kind of preparation method of the polyvinylidene fluoride affinity film taking amino acid as ligand, is characterized in that comprising the steps: 1) Pretreatment of basement membrane Rinse the polyvinylidene fluoride hollow fiber membrane with ethanol with a mass percent concentration of 30%, and soak it in deionized water; 2) Hydrophilic modification Send the mixed solution of KMnO ₄ and KOH into the membrane module composed of polyvinylidene fluoride hollow fiber membrane at 60-80°C, react for 30-90min, and then reduce it with the mixed solution of H ₂ SO ₄ and NaHSO ₃ ; 3) Hydroxyethyl cellulose treatment The polyvinylidene fluoride hollow fiber membrane after reduction treatment is treated with a mixed solution of NaOH and hydroxyethyl cellulose at 80-90°C for 10-30min; then dried at 80-90°C, and dried with 0.5-2mol/l Na ₂ CO ₃ solution is washed at 80-90°C; 4) The first activation The polyvinylidene fluoride hollow fiber membrane treated in step 3) is activated with a mixed solution of epichlorohydrin and NaOH at 50-70°C, and rinsed with deionized water after activation; 5) Bonded hexamethylenediamine spacer The polyvinylidene fluoride hollow fiber membrane after the first activation is reacted with 40-80g/l hexamethylenediamine at 50-70°C for 2-3h, and rinsed with deionized water after the reaction; 6) Second activation After step 5) the polyvinylidene fluoride hollow fiber membrane bonded with hexamethylenediamine spacer is reactivated with a mixed solution of epichlorohydrin and NaOH at 50-70°C, and rinsed with deionized water after activation; 7) Bonded amino acid ligands The polyvinylidene fluoride hollow fiber membrane after the second activation reacts with 2-8g/l amino acid solution at 40-50°C for 20-24h to obtain a polyvinylidene fluoride affinity membrane grafted with amino acid ligands. Described amino acid solution is solvent with phosphate buffer saline; The amino acid is serine, asparagine, arginine, histidine, polylysine or glutamine. 2. The preparation method according to claim 1, characterized in that: the amount of grafted amino acid is 150-250 mg/g film. 3. The preparation method according to claim 1, characterized in that: the concentration of KMnO ₄ in the mixed solution of KMnO ₄ and KOH is 40-80 g/l, and the concentration of KOH is 3-5 mol/l. 4. The preparation method according to claim 1, characterized in that: the concentration of H 2 SO ₄ in the mixed solution of said H ₂ SO ₄ and NaHSO ₃ is 50-100 g/l, and the concentration of NaHSO ₃ is 50-100 g/ _l . 100g/l. 5. preparation method as claimed in claim 1 is characterized in that: the concentration of NaOH in the described NaOH and hydroxyethyl cellulose mixed solution is 1-3mol/l, and the concentration of hydroxyethyl cellulose is 10-30g /l. 6. preparation method as claimed in claim 1 is characterized in that: the mixed solution of described epichlorohydrin and NaOH is according to epichlorohydrin: the volume ratio of the NaOH solution that concentration is 1mol/l is 1: 3 ~6 prepared mixed solution. 7. The application of the polyvinylidene fluoride affinity membrane prepared by the preparation method according to any one of claims 1-6 in removing endotoxin in blood plasma.

Images