CN102247817A - Endotoxin adsorbent using molecular cluster as functional group and preparation method thereof - Google Patents

Abstract

The invention discloses an endotoxin adsorbent using a molecular cluster as a functional group and a preparation method thereof, and belongs to the fields of bioseparation and biomedical engineering. By taking agarose gel as a vector, the endotoxin adsorbent is subjected to epoxy activation to be directly coupled with a molecular sieve frame epsilon-polylysine or polyaspartic acid, then the coupled mixture is ingrafted with micromolecular lysine or lycine by virtue of a condensation reaction to prepare the adsorbent. The adsorbent prepared by the method has higher removal rate to endotoxin in serum of a human, but adsorbs less serum protein. Meanwhile, the preparation process of the adsorbent has the advantages of mild conditions, fewer synthesis steps and relatively low material cost.

Description

A kind of endotoxin adsorbent with molecular cluster as functional group and preparation method thereof

technical field

The invention belongs to the fields of biological separation and biomedical engineering, and in particular relates to an endotoxin adsorbent with molecular clusters as functional groups and a preparation method thereof for the treatment of endotoxemia.

Background technique

Endotoxin, also known as lipopolysaccharides (Lipopolysaccharides, LPS), is a component of the outer membrane of Gram-negative bacteria. In a normal human body, a certain amount of endotoxin can be removed by the liver so that the endotoxin content in the blood can be kept at a low level. If the liver function is severely damaged or the content of endotoxin exceeds the detoxification ability of the liver, the endotoxin absorbed through the intestine can enter the internal circulation through the liver, forming endotoxemia, and then leading to multiple organ failure and even death. According to reports, at least hundreds of thousands of people suffer from endotoxemia in the United States every year, and its fatality rate can reach 40%-90%. In Japan, 30,000 to 50,000 people die from endotoxemia every year. Therefore, developing a method that can efficiently remove excess endotoxin in the patient's blood will have great practical significance for saving the lives of patients with endotoxemia.

So far, there have been various research reports on the removal of endotoxin. At present, more studies mainly focus on the direct therapeutic removal of anti-endotoxin drugs and hemoperfusion removal in blood purification therapy.

Anti-endotoxin drugs mainly include: lipid A antagonists, polymyxin B, anti-endotoxin proteins and polypeptides, and endotoxin antibodies. Lipid A is the center of toxicity and biological activity of LPS. Loppbow et al. found that its biosynthetic precursors and non-toxic lipid A derivatives can antagonize the toxic response of LPS. As an antibiotic, polymyxin B has a strong binding ability to endotoxin, but it has toxic effects on the central nervous system and kidney, so it cannot be used directly. Jian-Dong Ren et al. (JD Renet al.Int Immunopharmacol.2008 Jun; 8(6):775-81.Epub 2008 Feb 25.) synthesized a cyclic peptide (CRKPTFRRLKWKIKFKFKC, CLP-19), with the help of positively charged amino acids and Ring structure to achieve the removal of negatively charged endotoxins. Mouse experiments showed that the survival rate of mice with endotoxemia was 60-80%. US6395717B1 discloses the therapeutic effect of sialic acid and its polymer on endotoxemia in mice, the survival rate of mice is over 80%, and compared with traditional antibiotic treatment, the body will not produce drug resistance and is safer High, but its mechanism of action with endotoxin is not yet clear.

In addition, studies have shown that phospholipid complexes, lipid A analogs, and algae lipopolysaccharides all have varying degrees of neutralization of endotoxins. All of the above are molecules designed for the negative charge of the phosphate group or the hydrophobicity of the aliphatic chain in the endotoxin molecule, but due to the high cost of synthesis of these molecules, there is still a long time before clinical application.

Endotoxin antibodies mainly include anti-endotoxin polyclonal antibodies, anti-lipid A monoclonal antibodies, anti-endotoxin-binding protein antibodies, and anti-endotoxin receptor CD14. However, these antibodies also face problems such as difficulty in preparation, high cost, specificity of bacterial species, easy to cause allergic reactions, and may lose their effect on the damaged immune system of patients.

Blood purification therapy has a unique curative effect on some serious and difficult diseases, among which hemoperfusion has attracted extensive attention of researchers due to its low cost. The therapy is mainly based on the physical and chemical interaction between the special functional group molecules of the adsorbent and the toxic molecules, and removes the toxic or pathogenic substances in the blood through adsorption, so as to achieve the purpose of purifying the blood and treating diseases. Traditional activated carbon and macroporous resin adsorbents have no selectivity for adsorption of blood endotoxin, and their blood compatibility is poor, so the removal effect is not obvious. Therefore, the development and clinical application of new and efficient endotoxin adsorbents are imminent.

Due to its excellent antibacterial properties, polymyxin B has always been the focus of researchers. In order to solve the problem of neurotoxicity and nephrotoxicity in direct medication, many scholars have considered immobilizing it on various carriers to make different kinds of adsorption materials. In 1994, Aoki and others immobilized polymyxin B on a fiber carrier for the first time to treat endotoxemia. Japan's Toray Industries Inc. has immobilized polymyxin B on fibers to make an adsorption carrier-polymyxin B fiber column, and has achieved commercial production (PMX-F, trade name Toraymyxin).

Nankai University (Chinese patent CN1493368A) discloses a kind of agarose as a carrier, after epoxy activation, amination, glutaraldehyde cross-linking coupling polymyxin B, α-polylysine (Mr=10,000- 40,000Da) and other ligands to prepare a series of adsorbents for plasma endotoxin removal, which have good blood compatibility and good removal effect. However, the adsorbent has many synthesis steps, and the polymyxin B ligand is expensive, and once it falls off, it will cause potential danger to the patient's nerves and kidneys. In addition, some studies (D. Petsch, F. B. Anspach: Journal of Biotechnology 76 (2000) 97-119) have shown that polymyxin B also has about 20% adsorption of BSA. Relatively speaking, although α-polylysine has less adsorption to BSA, it is expensive (7,000RMB/g). These deficiencies above limit the wide clinical application of polymyxin B and α-polylysine as ligand adsorbents.

In addition, in order to improve the selectivity of the adsorbent to endotoxin, U.S. Patent US2005/0238641A1 discloses a kind of agarose gel etc. as the carrier, epichlorohydrin is activated, and branched branch (tris(2-aminoethyl) Amine) obtained adsorbent, in the endotoxin-containing HSA solution, the removal rate of endotoxin is high, and the adsorption amount of HSA is very small. However, the removal effect of endotoxin in actual plasma has not been reported, and only adsorbents with good removal rate in actual plasma (or serum) have potential clinical application value.

Therefore, in order to solve the current research on endotoxin removal (1) the difficulty in preparing anti-endotoxin drugs and α-polylysine, etc., the price is expensive; (2) the potential toxicity of polymyxin B; (3) the traditional The hemoperfusion adsorbent has defects such as poor blood compatibility, low selectivity and unsatisfactory removal effect. The present invention provides a cheap and efficient endotoxin adsorbent and its preparation method, which is expected to be used in the clinical treatment of endotoxemia treat.

Contents of the invention

The invention provides an endotoxin adsorbent with molecular clusters as functional groups and a preparation method thereof. The idea of the present invention is: use agarose gel with good blood compatibility as the matrix, directly couple with non-toxic and cheap ε-polylysine or polyaspartic acid after epoxy activation, and use Molecular clustering of macromolecular polymers, and then grafting of small molecule lysine or betaine that can be combined with endotoxin molecules mainly through electrostatic interaction, and different functions can be synthesized by controlling the density of epoxy activation and the addition of molecular cluster functional groups Adsorbent with base density to achieve selective removal of endotoxin in serum.

Technical scheme of the present invention is as follows:

An endotoxin adsorbent with molecular clusters as functional groups, composed of solid-phase matrix agarose gel and functional group molecules, the adsorbent uses ε-polylysine or polyaspartic acid as functional group skeleton molecules, Molecular cluster functional group of endotoxin adsorbent formed by coupling with lysine or betaine. The molecular weight of the ε-polylysine is 3600-4300Da, and the molecular weight of the polyaspartic acid is 1000-5000Da.

The preparation of the above-mentioned endotoxin adsorbent comprises the following steps:

(1) Epichlorohydrin activated agarose gel: the amount of epichlorohydrin added is 0.01-0.04ml/ml gel, the concentration of NaOH in the system is 0.4M, and the reaction is shaken at 40°C and 170rpm for 1-2h;

(2) Coupling of the molecular cluster skeleton: ε-polylysine or polyaspartic acid added in an amount of 0.06-0.4g/ml gel, pH 9-11, 50-60°C and 300rpm stirring reaction for 3-4h;

(3) ε-polylysine coupled with lysine or betaine: the amount of lysine or betaine added is 0.01-0.02g/ml gel, pH4.5-7.5, 37°C 170rpm shaking reaction for 3-4h;

(4) Polyaspartic acid coupled with lysine or betaine: polyaspartic acid is first grafted with ethylenediamine spacer arm through grafting reaction, and the grafting condition is that the amount of ethylenediamine added is 0.1-0.2ml /ml gel, pH4.5-7.5, 37℃170rpm shaking reaction for 3-4h; re-coupling lysine or betaine, the coupling condition is lysine or betaine 0.01-0.02g/ml gel, pH4 .5-7.5, 37 ° C 170rpm shaking reaction 3-4h.

The density of the agarose gel activated by epichlorohydrin is 1-10 μmol/ml gel, and the coupling density of lysine or betaine is 10-100 μmol/ml gel.

After the endotoxin adsorbent is depyrogenated, it is mixed with human serum containing 1-5EU/ml endotoxin in a certain ratio (1:5-1:10), statically adsorbed for 2-3h, and the removal rate of endotoxin It is 35%-65%, and the serum protein adsorption rate is within 20%.

The beneficial effects of the present invention are:

1. Using agarose gel Sepharose CL-6B with good blood compatibility and stable physical and chemical properties as the matrix, the endotoxin adsorbent was prepared by directly coupling the molecular cluster skeleton and then connecting small molecules after epoxy activation. The invention overcomes the toxicity and instability of the cyanogen bromide method; the ester bond formed by the carbonyldiimidazole method may be broken during acid and alkali treatment, thereby causing the risk of ligand falling off; the glutaraldehyde bonding method has relatively many steps shortcoming. The use of less organic solvents in the preparation process increases the safety of the preparation, the reaction conditions are mild, and the steps are simple.

2. There are a large number of activatable groups on Sepharose CL-6B, and the density of epoxy activation can be controlled by controlling the amount of epichlorohydrin added; by controlling ε-polylysine and polyaspartic acid The addition amount and reaction temperature can obtain endotoxin adsorbents coupled with terminal functional groups of different densities, thereby selectively removing endotoxin in serum.

3. ε-polylysine or polyaspartic acid with a molecular clustering effect can solve the problem of epoxy-activated materials by virtue of the amino groups of multiple lysine residues or the carboxyl groups of aspartic acid in each molecule. The coupling density is low, and the molecular clusters formed by multiple amino or carboxyl groups can interact with small molecules of lysine or betaine through multi-point interactions and relatively firm interactions with endotoxin macromolecules.

4. The ε-polylysine, polyaspartic acid, lysine and betaine molecules used in the endotoxin adsorbent are cheaper and easier to obtain than polymyxin B and α-polylysine. Among them, ε-polylysine and polyaspartic acid are non-toxic, degradable and environmentally friendly polymer compounds with high thermal stability.

Detailed ways

Specific embodiments of the present invention will be described in detail below in conjunction with technical solutions.

Example 1: Preparation of ε-polylysine coupled with lysine as an adsorbent for molecular cluster functional groups

(1) Epichlorohydrin activated agarose gel

Take 10ml of the washed Sepharose CL-6B gel, add it to 10ml of an aqueous solution with a concentration of epichlorohydrin of 1% (v/v) and a concentration of NaOH of 0.4M, and shake it at 170rpm at 40°C for 2 hours. Wash the gel with deionized water until neutral;

(2) Coupling of ε-polylysine

Prepare 10ml of ε-polylysine aqueous solution with a concentration of 6% (w/v) and a pH of 11, add the gel after the reaction in the previous step and wash it, and stir it with a paddle at 50°C and 300rpm for 3-4h. The gel was thoroughly washed with deionized water;

(3) Fixation of terminal functional group lysine

Add a certain amount of lysine to 10ml of MES buffer with a pH of 4.8 to make the concentration 1% (w/v), then add the gel after the reaction in the previous step, adjust the pH to 4.8, and then add 0.2g of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) condensing agent, 37 ° C, 170rpm shaking reaction for 4h, after the reaction is fully washed with deionized water, you can get The final endotoxin adsorbent1.

Example 2: Preparation of ε-polylysine-coupled betaine as an adsorbent for molecular cluster functional groups

The preparation method of the endotoxin adsorbent 2 with betaine at the end of ε-polylysine is the same as that in Example 1, except that the lysine in step (3) is replaced with betaine, and the other steps and conditions remain unchanged.

Example 3: Preparation of Adsorbent Using Polyaspartic Acid Coupled to Lysine as Molecular Cluster Functional Group

The preparation method of the adsorbent 3 of polyaspartic acid coupling lysine as molecular cluster functional group comprises four steps, and the first two steps are the same as (1) and (2) in Example 1, only need (2) The epsilon-polylysine in is changed into polyaspartic acid, and all the other conditions are unchanged, and steps (3) and (4) are as follows:

(3) Connection of the spacer arm

Add a certain volume of ethylenediamine to 10ml of MES buffer with a pH of 4.8 to make the concentration 10% (v/v), then add the gel after the reaction in the previous step, adjust the pH to 4.8, and then add 0.2g EDC condensing agent, 37°C, 170rpm shaking reaction for 4h, after the reaction is completed, fully wash with deionized water;

(4) Fixation of terminal functional group lysine

Add a certain amount of lysine to 10ml of MES buffer with a pH of 4.8 to make the concentration 1% (w/v), then add the gel after the reaction in the previous step, adjust the pH to 4.8, and then add 0.2g EDC condensing agent, 37°C, 170rpm shaking reaction for 4h. After the reaction is completed, the endotoxin adsorbent 3 is obtained by fully washing with deionized water.

Example 4: Preparation of polyaspartic acid-coupled betaine as an adsorbent for molecular cluster functional groups

The preparation method of the endotoxin adsorbent 4 with polyaspartic acid connected to betaine at the end is the same as in Example 3, except that the lysine in step (4) is replaced with betaine, and the other steps and conditions remain unchanged.

Embodiment 5: depyrogenation treatment of adsorption material and experimental equipment

All materials and utensils used in the experiment need to be depyrogenated: glass test tubes are cleaned repeatedly and placed in an oven at 250°C for more than 2 hours; plastic centrifuge tubes are soaked in 30% hydrogen peroxide overnight, and then rinsed with pyrogen-free water for several times Finally, dry; the adsorbent is first rinsed with normal saline for several times, then sequentially rinsed with 0.2M NaOH (containing 20% ethanol), 1.5M NaOH, and finally rinsed with normal saline and pyrogen-free water until neutral.

Example 6: Static adsorption removal of endotoxin in human serum

Take the adsorbent 1-4 with terminal functional group density of 10 μmol/ml after depyrogenation treatment, mix it with human serum containing 1.95 EU/ml endotoxin at a volume ratio of 1:5, and place it on a shaker at 37°C and 200 rpm Medium static adsorption for 3h. Centrifuge the supernatant to measure the remaining endotoxin concentration and protein concentration, and calculate the endotoxin removal rate and protein adsorption rate of the four adsorbents respectively.

Table 1 shows the removal rate of endotoxin in human serum and the adsorption rate of major proteins by endotoxin adsorbents with a terminal functional group density of 10 μmol/ml. The results showed that under the condition that the initial endotoxin concentration was 1.95 EU/ml, and the serum total protein and albumin concentrations were 60.23g/L and 41.67g/L respectively, the removal rate of endotoxin by the four endotoxin adsorbents was 48%. -53%, and the adsorption rate of serum protein is within 15%.

Table 1: Adsorption effect of endotoxin and protein in human serum

Example 7: Static removal effect of endotoxin in human serum by adsorbents (5-14) with different functional group densities

By controlling the addition of epichlorohydrin (1%-4% (v/v)) and the addition of ε-polylysine (6%-40% (w/v)), the synthetic terminal coupling density is respectively 10 μmol/ml, 37 μmol/ml, 52 μmol/ml, 81 μmol/ml and lysine (adsorbent 5-9) and betaine adsorbent (adsorbent 10-14) of 100 μmol/ml, method is with embodiment 1 and 2 . After the obtained adsorbent was depyrogenated, it was mixed with serum with an initial endotoxin concentration of 3.81 EU/ml at a volume ratio of 1:5. Toxin removal rate.

Example 8 Adsorbents (15-24) with different functional group densities on the static removal effect of endotoxin in human serum

By controlling the addition of epichlorohydrin (1%-4% (v/v)) and the addition of polyaspartic acid (6%-40% (w/v)), the synthetic terminal coupling density is 10 μmol respectively /ml, 37 μmol/ml, 52 μmol/ml, 81 μmol/ml and 100 μmol/ml lysine (adsorbent 15-19) and betaine adsorbent (adsorbent 20-24), method is the same as embodiment 3 and 4. After the obtained adsorbent was depyrogenated, it was mixed with serum with an initial endotoxin concentration of 3.81 EU/ml at a volume ratio of 1:5. Toxin removal rate.

Table 2 shows the removal effects of various adsorbents on endotoxin at different functional base densities. Lysine with terminal coupling densities of 10 μmol/ml, 37 μmol/ml, 52 μmol/ml, 81 μmol/ml and 100 μmol/ml were synthesized by controlling the epoxy activation density and the amount of ε-polylysine or polyaspartic acid added. Amino acid or betaine adsorbents were used to investigate the effect of functional group density on endotoxin removal in serum with an initial endotoxin concentration of 3.81 EU/ml. The results showed that at a certain concentration of endotoxin, the removal rate of endotoxin by different adsorbents increased with the increase of functional group density.

Table 2: The removal effect of endotoxin in human serum by adsorbents with different functional group densities

Claims (4)

1. A kind of endotoxin adsorbent with molecular cluster as functional group, is made up of solid-phase matrix agarose gel and functional group molecule, it is characterized in that this adsorbent is made of ε-polylysine or polyaspartic acid as The functional group skeleton molecule forms the molecular cluster functional group of the endotoxin adsorbent through coupling with lysine or betaine. 2. The endotoxin adsorbent according to claim 1, characterized in that the polyaspartic acid has a molecular weight of 1000-5000Da. 3. the preparation method of endotoxin adsorbent described in claim 1 or 2, is characterized in that following steps: (1) Epichlorohydrin-activated agarose gel: Epichlorohydrin is 0.01-0.04ml/ml gel, the NaOH concentration of the system is 0.4M, 40°C and 170rpm shaking reaction for 1-2h; (2) ε-polylysine or polyaspartic acid and epoxy activated agarose gel: the amount of ε-polylysine or polyaspartic acid is 0.06-0.4g/ml gel, pH 9- 11. Stir at 50-60°C for 3-4 hours at 300rpm; (3) Immobilize lysine or betaine on the ε-polylysine skeleton: lysine or betaine 0.01-0.02g/ml gel, pH4.5-7.5, 37℃170rpm shaking reaction for 3-4h; Immobilize lysine or betaine on the polyaspartic acid skeleton: polyaspartic acid is first grafted with ethylenediamine spacer arm through grafting reaction, the grafting condition is 0.1-0.2ml/ml gel of ethylenediamine, pH4.5-7.5, 37℃170rpm shaking reaction for 3-4h; re-coupling lysine or betaine, the coupling conditions are lysine or betaine 0.01-0.02g/ml gel, pH4.5-7.5, 37°C 170rpm shaking reaction for 3-4h; The density of agarose gel activated by epichlorohydrin is 1-10 μmol/ml gel, and the coupling density of lysine or betaine is 10-100 μmol/ml gel. 4. Application of the endotoxin adsorbent according to claim 1 or 2, characterized in that: after the adsorbent is treated with depyrogenation, it is mixed with human serum containing 1-5EU/ml endotoxin in a ratio of 1:5-1:10 Mixing and static adsorption for 2-3 hours, the removal rate of endotoxin is 35%-65%, and the adsorption rate of serum protein is within 20%.