CN102580683B - Endotoxin synergistic adsorbent and preparation method thereof - Google Patents
Endotoxin synergistic adsorbent and preparation method thereof Download PDFInfo
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- CN102580683B CN102580683B CN201210023935.2A CN201210023935A CN102580683B CN 102580683 B CN102580683 B CN 102580683B CN 201210023935 A CN201210023935 A CN 201210023935A CN 102580683 B CN102580683 B CN 102580683B
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Abstract
The invention discloses an endotoxin synergistic adsorbent and a preparation method thereof. The endotoxin synergistic adsorbent comprises a porous vector and a genin immobilized on the vector, wherein the genin at least comprises deoxycholic acid genin and amino compound genin. By the synergistic effect of two types of genin on the vector, the adsorption capacity of the adsorbent for endotoxin is significantly enhanced.
Description
Technical field
The present invention relates to a kind of medical endotoxin synergistic adsorbent and preparation method thereof, particularly endotoxin synergistic adsorbent and preparation method thereof for blood perfusion.
Background technology
A lipoid polysaccharose substance that discharges when endotoxin is Gram-negative bacteria growing or produced by cell membrane cracking when dead, denier (nanogram level) endotoxin enters human body will cause high heat, blood vessel dilatation is even fainted or dead.Endotoxemia is to cause because the endotoxin on the outer cell membrane of Gram-negative bacteria is discharged in blood, can come across in various diseases process, as: the diseases such as large-area burns, serious hepatitis, cirrhosis.Endotoxemia is one of clinical modal lethal disease, it is badly damaged body's immunity, and can cause a series of serious pathological changes such as shock, disseminated intravascular coagulation, MOF, finally cause organ necrosis, irreversible shock and death.The death rate can reach 40%-90%.
There is no clinically the method for effective treatment endotoxemia, one of existing method is to have the adsorbent of PB to carry out blood perfusion by bonding.The polypeptide antibiotics that PB is made up of 10 amino acid, can deactivation ET and have an effect of anti-Gram-negative bacteria.As, 200510046452.4 number Chinese invention patent application discloses a kind of preparation method of the endotoxin absorbent for blood perfusion, be matrix by the spherical Ago-Gel of good biocompatibility, adopt epoxychloropropane, hexamethylene diamine, 1, after the activation of 1 '-carbonyl dimidazoles, after bonding PB aglucon, through NaBH4 reduction, obtain endotoxin synergistic adsorbent.6.12 milligrams of/gram of adsorbents of adsorbent PB supported quantity of preparing by the method, endotoxin adsorbance reaches 22 ng/g adsorbents.
Use in addition other positively charged quaternary ammonium salt, amino acid or polymer to do the research of aglucon.Jia Lingyun etc. are grafting quaternary ammonium salt on agarose, can reach 50%(CN200710012501.1 to Endotoxin removal rate in blood).Taking the agarose carrier of fixing lysine as adsorbent, the endotoxin in adsorbed proteins solution, to albumen without obvious absorption.Adopt macropore cellulose balls carrier, fixing polymine, blood perfusion absorption endotoxin, also can make endotoxin significantly decline.
But, be the adsorbent of immobilized PB no matter above-mentioned, still use other positively charged quaternary ammonium salt, amino acid or polymer to make the adsorbent of aglucon, they only have good adsorption capacity to endotoxin free under low concentration conventionally.But due to the endotoxin complex shape in blood, most applications exists with coherent condition, above-mentioned adsorbent is unsatisfactory to the endotoxin Molecular Adsorption effect in state of aggregation under high concentration, therefore can not effectively remove the endotoxin in blood.
The people such as Johoson once studied with deoxycholic acid aglucon.As on polyethylene support, do arm with diamines, grafting deoxycholic acid, although can adsorb the endotoxin of removing in blood, adsorbance is extremely limited.
Summary of the invention
It is larger that the technical problem to be solved in the present invention is to provide a kind of induced by endotoxin adsorption capacity, removes more thoroughly endotoxin absorbent.
For solving the problems of the technologies described above, technical scheme provided by the invention is: endotoxin synergistic adsorbent, comprise carrier and the immobilized aglucon on described carrier of porous, and wherein, described aglucon at least comprises deoxycholic acid aglucon and amino-compound aglucon.
Preferred scheme is that immobilized deoxycholic acid and the mol ratio of amino-compound are 0.05:1-20:1.
Preferred scheme is that the supported quantity of deoxycholic acid is 0.1umol-50umol/g adsorbent.
Technique scheme is immobilized deoxycholic acid of while and two kinds of aglucons of amino-compound on porous carrier, utilize deoxycholic acid that endotoxin is dissociated, and utilize amino-compound that the endotoxin after dissociating is adsorbed and removed simultaneously.Above-mentioned adsorbent, by the synergy of two kinds of aglucons on carrier, makes the adsorption capacity of its induced by endotoxin far above the endotoxin absorbent with single aglucon.
Another technical problem that the present invention will solve is to provide a kind of method of preparing endotoxin synergistic adsorbent, and simultaneously immobilized on the carrier of this endotoxin synergistic adsorbent have deoxycholic acid and two kinds of aglucons of amino-compound.
For solving the problems of the technologies described above, technical scheme provided by the invention is: the method for preparing endotoxin synergistic adsorbent, comprise carrier and the immobilized aglucon on described carrier of porous, wherein said aglucon at least comprises deoxycholic acid aglucon and amino-compound aglucon, said method comprising the steps of: step 1: prepare porous carrier; Step 2: immobilized deoxycholic acid on porous carrier; Step 3: at immobilized immobilized amino-compound again on the porous carrier of deoxycholic acid.
Preferred scheme is: step 2 comprises the following steps:
Step 2.1: carrier is carried out to epoxy activation;
Step 2.2: the carrier to epoxy activation carries out ammonification;
Step 2.3: immobilized deoxycholic acid on the carrier of ammonification.
Another preferred scheme is that step 2 comprises the following steps:
Step 2.1 is carried out epoxy activation to carrier:
By volume part, in 5-15 part carrier, add sodium hydroxide solution and 10-20 part one end band active group that 5-10 part concentration is 1-3M, the epoxide of one end band epoxy radicals, at 30-60 DEG C of stirring reaction 1-5h, then ethanolic solution wash vehicle 5-10 time of water and 50%-95% successively, obtains the carrier of epoxy activation;
Step 2.2 is carried out ammonification to the carrier of epoxy activation:
What obtain to step 2.1 adds 10-20mL ammoniacal liquor in the carrier through epoxy activation, stirs anti-1-5h at 30-60 DEG C, and amino is connected on carrier;
Step 2.3 is immobilized deoxycholic acid on the carrier of ammonification:
NaTDC is dissolved in to 40% the methyl-sulfoxide aqueous solution, being mixed with concentration is the deoxycholic acid sodium solution of 1-5mmol/L, by volume part, get the described deoxycholic acid sodium solution of 200-400 part, add the carrier 5-15 part after ammonification, stir, by the HCl regulation system pH value of 0.2-0.4M to 4-6, to the methyl-sulfoxide aqueous solution that slowly adds 5-10mM1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate in system, the volume ratio of the methyl-sulfoxide aqueous solution of the volume of wherein said carrier and described 1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate is 1:2, stirring reaction 1-5h, in course of reaction, drip the HCl of 0.2-0.4M, make system pH remain on 4-6, react rear use 95% ethanolic solution washing, removed unreacted NaTDC.
Preferred scheme is: step 3 comprises the steps:
The immobilized carrier that has deoxycholic acid that step 3.1 obtains step 2 carries out re-activation;
Immobilized amino-compound on the immobilized carrier that has deoxycholic acid that step 3.2 obtains to step 3.1.
The endotoxin synergistic adsorbent of preparing by said method, simultaneously immobilized on carrier have deoxycholic acid and two kinds of aglucons of amino-compound, and the adsorption capacity of induced by endotoxin is far longer than the endotoxin synergistic adsorbent of single aglucon.
The average grain diameter of the carrier of endotoxin synergistic adsorbent of the present invention is 50um-1500um, is better 300um-800um, takes this, and endotoxin synergistic adsorbent of the present invention not only can have good adsorption rate, and can adsorb for whole blood.The pore diameter range of carrier of the present invention can be 2nm-300nm, and preferably 10nm-100nm, can ensure to have larger specific area on the one hand, is applicable to again on the other hand endotoxic absorption.The carrier of endotoxin synergistic adsorbent of the present invention can be cellulose, agarose, polyvinyl alcohol, styrene-divinylbenzene copolymer or polymethyl methacrylate, preferred cellulose, polymethylacrylic acid, and preferred carrier is cellulose carrier.Because cellulose carrier has the following advantages: the mechanical strength that (1) is relatively high and toughness, can meet the requirement of whole blood perfusion.(2) have good biocompatibility, security is good.(3) source is abundant, with low cost.(4) environmental friendliness, can natural degradation after use.
The endotoxic carrier of the present invention can be that commercial sources is bought, and can be also oneself preparation.For example, can be by the cellulose acetate solution that adds a certain proportion of pore-foaming agent be dispersed in polyvinyl alcohol (PVA) aqueous solution, after being distributed to appropriate particle size, the balling-up that is heating and curing, prepares the satisfactory cellulose diacetate carrier in particle diameter aperture.About the preparation of carrier, in prior art, existing a lot of methods for the purpose of saving, repeat no more herein.
In the present invention, consider that deoxycholic acid is more stable in chemical property, should not be subject to the impact of subsequent chemistry process, therefore adopt first immobilized deoxycholic acid and then the method for immobilized cationic compound.
Consider the factor such as cost and adsorption effect, on endotoxin synergistic adsorbent of the present invention, the amount of immobilized deoxycholic acid is preferably 0.1umol-50umol/g adsorbent, more preferably 0.5umol-20umol/g adsorbent.The amount of deoxycholic acid aglucon very little, adsorbent to assemble endotoxic dissociate limited in one's ability.Under the amount of a certain amount of amino-compound aglucon, after the amount of deoxycholic acid aglucon reaches a certain plateau value, then increase deoxycholic acid amount, endotoxic adsorption capacity does not have too large raising yet, and cost will be higher.In the present invention, can adopt the fixing deoxycholic acid of method of peptide coupling covalent bond deoxycholic acid, as: by the carrier with hydroxyl and one end with active group, the other end carries out epoxy priming reaction with the compound of epoxide group, use again ammoniacal liquor ammonification, introduce amino, then by the method grafting deoxycholic acid of peptide coupling.Wherein, one end is with active group, and the other end can be epoxychloropropane or the compound with diepoxy group etc. with the compound of epoxide group.
Amino-compound on endotoxin synergistic adsorbent of the present invention is to contain amino compound, can be amino acid or polyaminoacid, as: PB (PMX-B), lysine, arginine, histidine or polylysine etc., preferably PMX-B.The positive charge of amino-compound can relative specificity ground absorption surface with the endotoxin molecule of phosphate groups.Amino-compound in the present invention on endotoxin synergistic adsorbent can adopt the method for epoxy radicals or aldehyde radical and amino reaction immobilized, preferably epoxy reaction.The factor such as considering cost and adsorption effect, on endotoxin synergistic adsorbent of the present invention, the amount of immobilized amino-compound is preferably 0.1umol-100umol/g adsorbent, is more preferably 1umol-10umol/g adsorbent.Consider after the method grafting deoxycholic acid by peptide coupling on carrier also residual a large amount of amino, can choice for use dialdehyde or diepoxides, by the reaction between aldehyde radical and amino, remove aminoly, complete the grafting of cationic compound.Wherein dialdehyde can be glutaraldehyde or hexandial, preferably glutaraldehyde.Bis-epoxy can be 1,3-PD glycidol ether, BDO glycidol ether or 1,5-PD glycidol ether, preferably BDO glycidol ether.
On endotoxin synergistic adsorbent of the present invention, the ratio of two kinds of immobilized aglucons has a certain impact to effect and the cost of absorption, preferably, deoxycholic acid and amino-compound mol ratio are between 0.05:1～20:1, preferably between 0.1:1～10:1, more preferably between 0.5:1-1:3.Wherein the amount of deoxycholic acid can not be very little, otherwise adsorbent endotoxicly dissociates limited in one's abilityly to assembling, and affects adsorption capacity.Deoxycholic acid aglucon amount does not too much have positive effect yet, under a certain amount of PMX-B aglucon amount, after a certain plateau value, then increases deoxycholic acid amount, can not increase the adsorption capacity of induced by endotoxin.Excess of ammonia based compound, as PMX-B has neurotoxicity and renal toxicity.Therefore, the supported quantity of amino-compound can not be too high, is preferably controlled at below 20mg/mL adsorbent.
Detailed description of the invention
Embodiment 1
The preparation method of the present embodiment endotoxin synergistic adsorbent is as follows:
Step 1: prepare macropore cellulose carrier
12g cellulose diacetate is dissolved in the mixed solvent being made up of 80mL carrene and 20mL ethanol, the mass fraction of cellulose solution is 9%.
In above-mentioned solution, add ethyl acetate 100mL, as pore-foaming agent, mix.The PVA aqueous solution of preparation 400mL5% is as decentralized photo.Slow cellulose solution impouring is equipped with in decentralized photo, and control mixing speed is 100-160r.p.m, makes cellulose solution fully be dispersed into uniform droplet, then keep mixing speed, be heated to 35 DEG C, carrene is volatilized completely, cellulose grain thoroughly solidifies.PVA and pore-foaming agent are thoroughly removed in water and the washing of 75% ethanol, obtain the cellulose carrier that pore diameter range is 50-200nm.
Step 2: immobilized deoxycholic acid on the prepared porous carrier of step 1
The epoxy activation of step 2.1 carrier
To the sodium hydroxide solution and the 15mL epoxychloropropane that add 6mL3M in 10mL carrier, stir anti-2h at 50 DEG C.Water and ethanol wash vehicle successively, obtains the carrier that epoxy activates.
The ammonification of step 2.2 carrier
In the carrier of the activation of epoxy obtaining to step 2.1, add 20mL ammoniacal liquor, stir anti-2h at 50 DEG C, amino is connected on carrier.
Step 2.3 is immobilized deoxycholic acid (DOC) on carrier
1mmol deoxycholic acid sodium solution is dissolved in to the methyl-sulfoxide aqueous solution of 300mL40%.Add the carrier 10mL after ammonification, stir, by the HCl regulation system pH value to 4.8 of 0.3M.To the methyl-sulfoxide aqueous solution (volume ratio of carrier and the methyl-sulfoxide aqueous solution is 1:2) that slowly adds 6mM1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate (WCCM) in system.Stirring reaction 3h, drips the HCl of 0.3M in process, make system pH remain on 4.8.React rear with ethanol washing, removed unreacted NaTDC.
Step 3: at immobilized immobilized amino-compound again on the porous carrier of deoxycholic acid
The immobilized carrier that has deoxycholic acid that step 3.1 obtains step 2 carries out re-activation
On carrier after immobilized DOC, still have a lot of residual amino.In this carrier of 10mL, add BDO-diglycidyl ether of 10mL and at room temperature stirring reaction 18h of the sodium hydroxide solution 15mL of 0.1M.After reaction, water is rinsed well.
Immobilized PB on the immobilized carrier that has deoxycholic acid that step 3.2 obtains to step 3.1
0.1g PB is dissolved in 10mL water, pH value is adjusted to 11 with sodium hydroxide solution, add the carrier 5mL being obtained by step 3.1, room temperature reaction 2h, then rinse with the sodium chloride of 1M, obtain taking macropore cellulose as carrier the endotoxin synergistic adsorbent that contains deoxycholic acid and PB aglucon.
Embodiment 2
The preparation process of the present embodiment endotoxin synergistic adsorbent is as follows:
Step 1: prepare macropore cellulose carrier
This step is identical with embodiment 1, repeats no more.
Step 2: immobilized deoxycholic acid on the prepared porous carrier of step 1
The epoxy activation of step 2.1 carrier
To the sodium hydroxide solution and the 15mL epoxychloropropane that add 6mL3M in 10mL carrier, stir anti-2h at 50 DEG C.Water and ethanol wash vehicle successively, obtains the carrier that epoxy activates.
The ammonification of step 2.2 carrier
In the carrier of the activation of epoxy obtaining to step 2.1, add 20mL ammoniacal liquor, stir anti-2h at 50 DEG C, amino is connected on carrier.
Step 2.3 is immobilized deoxycholic acid (DOC) on carrier
1mmol NaTDC is dissolved in to the methyl-sulfoxide aqueous solution of 300mL40%.Add the carrier 10mL after ammonification, stir, by the HCl regulation system pH value to 4.8 of 0.3M.To the methyl-sulfoxide aqueous solution (volume ratio is 1:2) that slowly adds 6mM1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate (WCCM) in system.Stirring reaction 3h, drips the HCl of 0.3M in process, make system pH remain on 4.8.React rear with ethanol washing, removed unreacted NaTDC.
Step 3: at immobilized immobilized amino-compound again on the porous carrier of deoxycholic acid
The immobilized carrier that has deoxycholic acid that step 3.1 obtains step 2 carries out re-activation
On carrier after immobilized DOC, still have a lot of residual amino.Be 7.4 phosphate buffer solution to adding 25mL0.05M pH in this carrier of 10mL, under stirring condition, drip the glutaraldehyde of 8mL25%, at room temperature stirring reaction 2h.(aldehyde group content is about 74umol/g).Phosphate buffer and water with 0.1M after reaction rinse.
Immobilized PB on the immobilized carrier that has deoxycholic acid that step 3.2 obtains to step 3.1
0.1g PB is dissolved in 10mL water, pH value is adjusted to 11 with sodium hydroxide solution, add the carrier 5mL after re-activation, room temperature reaction 2h, then rinses with the sodium chloride of 1M.With the two keys of sodium borohydride reduction, be washed to neutrality.Obtain taking macropore cellulose as carrier the endotoxin synergistic adsorbent that contains deoxycholic acid and PB aglucon.
Embodiment 3
The preparation process of the present embodiment endotoxin synergistic adsorbent is as follows:
Step 1: prepare macropore cellulose carrier
This step is identical with embodiment 1, repeats no more.
Step 2: immobilized deoxycholic acid on the prepared porous carrier of step 1
The epoxy activation of step 2.1 carrier
To the sodium hydroxide solution and the 15mL epoxychloropropane that add 6mL3M in 10mL carrier, stir anti-2h at 50 DEG C.Water and ethanol wash vehicle successively, obtains the carrier that epoxy activates.
The ammonification of step 2.2 carrier
In the carrier of the activation of epoxy obtaining to step 2.1, add 20mL ammoniacal liquor, stir anti-2h at 50 DEG C, amino is connected on carrier.
Step 2.3 is immobilized deoxycholic acid (DOC) on carrier
1mmol NaTDC is dissolved in to the methyl-sulfoxide aqueous solution of 300mL40%.Add the carrier 10mL after ammonification, stir, by the HCl regulation system pH value to 4.8 of 0.3M.To the methyl-sulfoxide aqueous solution (volume ratio is 1:2) that slowly adds 6mM1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate (WCCM) in system.Stirring reaction 3h, drips the HCl of 0.3M in process, make system pH remain on 4.8.React rear with ethanol washing, removed unreacted NaTDC.
Step 3: at immobilized immobilized amino-compound again on the porous carrier of deoxycholic acid
The immobilized carrier that has deoxycholic acid that step 3.1 obtains step 2 carries out re-activation
On carrier after immobilized DOC, still have a lot of residual amino.Be 7.4 phosphate buffer solution to adding 25mL0.05M pH in this carrier of 10mL, under stirring condition, drip the glutaraldehyde of 8mL25%, at room temperature stirring reaction 2h.(aldehyde group content is about 74umol/g).Phosphate buffer and water with 0.1M after reaction rinse.
Immobilized lysine on the immobilized carrier that has deoxycholic acid that step 3.2 obtains to step 3.1
0.2g lysine is dissolved in 10mL water, solution solution is adjusted to alkalescence with NaOH, add the carrier 5mL after re-activation, room temperature reaction 2h, then rinses with the sodium chloride of 1M.With the two keys of sodium borohydride reduction, be washed to neutrality.Obtain taking macropore cellulose as carrier the endotoxin synergistic adsorbent that contains deoxycholic acid and lysine.
Embodiment 4
The preparation process of the present embodiment endotoxin synergistic adsorbent is as follows:
Step 1: prepare macropore cellulose carrier
This step is identical with embodiment 1, repeats no more.
Step 2: immobilized deoxycholic acid on the prepared porous carrier of step 1
The epoxy activation of step 2.1 carrier
To the sodium hydroxide solution and the 15mL epoxychloropropane that add 6mL3M in 10mL carrier, stir anti-2h at 50 DEG C.Water and ethanol wash vehicle successively, obtains the carrier that epoxy activates.
The ammonification of step 2.2 carrier
In the carrier of the activation of epoxy obtaining to step 2.1, add 20mL ammoniacal liquor, stir anti-2h at 50 DEG C, amino is connected on carrier.
Step 2.3 is immobilized deoxycholic acid (DOC) on carrier
1mmol NaTDC is dissolved in to the methyl-sulfoxide aqueous solution of 300mL40%.Add the carrier 10mL after ammonification, stir, by the HCl regulation system pH value to 4.8 of 0.3M.To the methyl-sulfoxide aqueous solution (volume ratio is 1:2) that slowly adds 6mM1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate (WCCM) in system.Stirring reaction 3h, drips the HCl of 0.3M in process, make system pH remain on 4.8.React rear with ethanol washing, removed unreacted NaTDC.
Step 3: at immobilized immobilized amino-compound again on the porous carrier of deoxycholic acid
The immobilized carrier that has deoxycholic acid that step 3.1 obtains step 2 carries out re-activation
On carrier after immobilized DOC, still have a lot of residual amino.Be 7.4 phosphate buffer solution to adding 25mL0.05M pH in this carrier of 10mL, under stirring condition, drip the glutaraldehyde of 8mL25%, at room temperature stirring reaction 2h.(aldehyde group content is about 74umol/g).Phosphate buffer and water with 0.1M after reaction rinse.
Immobilized arginine on the immobilized carrier that has deoxycholic acid that step 3.2 obtains to step 3.1
0.2g arginine is dissolved in 10mL water, solution solution is adjusted to alkalescence with NaOH, add the carrier 5mL after re-activation, room temperature reaction 2h, then rinses with the sodium chloride of 1M.With the two keys of sodium borohydride reduction, be washed to neutrality.Obtain taking macropore cellulose as carrier, contain deoxycholic acid and arginic endotoxin synergistic adsorbent.
Embodiment 5:
The preparation process of the present embodiment endotoxin synergistic adsorbent is as follows:
Step 1: prepare macropore cellulose carrier
This step is identical with embodiment 1, repeats no more.
Step 2: immobilized deoxycholic acid on the prepared porous carrier of step 1
The epoxy activation of step 2.1 carrier
To the sodium hydroxide solution and the 15mL epoxychloropropane that add 6mL3M in 10mL carrier, stir anti-2h at 50 DEG C.Water and ethanol wash vehicle successively, obtains the carrier that epoxy activates.
The ammonification of step 2.2 carrier
In the carrier of epoxy activation, add 20mL ammoniacal liquor, stir anti-2h at 50 DEG C, amino is connected on carrier.
Step 2.3 is immobilized deoxycholic acid (DOC) on carrier
0.5mmol NaTDC is dissolved in to the methyl-sulfoxide aqueous solution of 300mL40%.Add the carrier 10mL after ammonification, stir, by the HCl regulation system pH value to 4.8 of 0.3M.To the methyl-sulfoxide aqueous solution (volume ratio is 1:2) that slowly adds 6mM1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate (WCCM) in system.Stirring reaction 3h, drips the HCl of 0.3M in process, make system pH remain on 4.8.React rear with ethanol washing, removed unreacted NaTDC.
Step 3: at immobilized immobilized amino-compound again on the porous carrier of deoxycholic acid
The immobilized carrier that has deoxycholic acid that step 3.1 obtains step 2 deaminizes
On carrier after immobilized DOC, still have a lot of residual amino.Be 7.4 phosphate buffer solution to adding 25mL0.05M pH in this carrier of 10mL, under stirring condition, drip the glutaraldehyde of 8mL25%, at room temperature stirring reaction 2h.(aldehyde group content is about 74umol/g).Phosphate buffer and water with 0.1M after reaction rinse.
Immobilized PB on the immobilized carrier that has deoxycholic acid that step 3.2 obtains to step 3.1
0.1g PB is dissolved in 10mL water, pH value is adjusted to 11 with sodium hydroxide solution, add the carrier 5mL after re-activation, room temperature reaction 2h, then rinses with the sodium chloride of 1M.
Comparative example 1
Prepare according to the following steps adsorbent:
Step 1: prepare carrier, method is with embodiment 1.
Step 2: immobilized PB
The epoxy activation of step 2.1 carrier
To the sodium hydroxide solution and the 15mL epoxychloropropane that add 6mL3M in 10mL carrier, at 50 DEG C of stirring reaction 2h.Water and ethanol wash vehicle more than three times, obtain the carrier of epoxy activation successively.
Step 2.2PMX-B's is immobilized
0.1g PB is dissolved in 10mL water, pH value is adjusted to 11 with sodium hydroxide solution, add the carrier 5mL after epoxy activation, room temperature reaction 2h, then rinses with the sodium chloride of 1M, obtains the immobilized adsorbent that has PB.
Comparative example 2:
Prepare according to the following steps adsorbent:
Step 1: prepare carrier, method is with embodiment 1.
Step 2: immobilized PB
The epoxy activation of step 2.1 carrier
To the sodium hydroxide solution and the 15mL epoxychloropropane that add 6mL3M in 10mL carrier, at 50 DEG C of stirring reaction 2h.Water and ethanol wash vehicle more than three times, obtain the carrier of epoxy activation successively.
Step 2.2PMX-B's is immobilized
0.3g PB is dissolved in 10mL water, pH value is adjusted to 11 with sodium hydroxide solution, add the carrier 5mL after once activation, room temperature reaction 2h, then rinses with the sodium chloride of 1M.Obtain the immobilized adsorbent that has PB.
Comparative example 3:
Prepare according to the following steps adsorbent:
Step 1: prepare carrier, method is with embodiment 1.
Step 2: immobilized PB
The epoxy activation of step 2.1 carrier
To the sodium hydroxide solution and the 15mL epoxychloropropane that add 6mL3M in 10mL carrier, at 50 DEG C of stirring reaction 2h.Water and ethanol wash vehicle more than three times, obtain the carrier of epoxy activation successively.
Step 2.2PMX-B's is immobilized
0.2g PB is dissolved in 10mL water, pH value is adjusted to 11 with sodium hydroxide solution, add the carrier 5mL after once activation, room temperature reaction 2h, then rinses with the sodium chloride of 1M.Obtain the immobilized adsorbent that has PB.
Comparative example 4:
Prepare according to the following steps adsorbent:
Step 1: prepare carrier, method is with embodiment 1.
Step 2: immobilized PB
The epoxy activation of step 2.1 carrier
To the sodium hydroxide solution and the 15mL epoxychloropropane that add 6mL3M in 10mL carrier, stir anti-2h at 50 DEG C.Water and ethanol wash vehicle successively, obtains the carrier that epoxy activates.
The ammonification of step 2.2 carrier
In the carrier of epoxy activation, add 20mL ammoniacal liquor, stir anti-2h at 50 DEG C, amino is connected on carrier, complete once activation
Step 2.3 is immobilized deoxycholic acid (DOC) on carrier
1mmol NaTDC is dissolved in to the methyl-sulfoxide aqueous solution of 300mL40%.Add the carrier 10mL after ammonification, stir, by the HCl regulation system pH value to 4.8 of 0.3M.To the methyl-sulfoxide aqueous solution (volume ratio is 1:2) that slowly adds 6mM1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate (WCCM) in system.Stirring reaction 3h, drips the HCl of 0.3M in process, make system pH remain on 4.8.React rear and removed unreacted NaTDC with ethanol washing, obtained the immobilized adsorbent that has deoxycholic acid.
Comparative example 5:
Prepare according to the following steps adsorbent:
Step 1: prepare carrier, method is with embodiment 1.
Step 2: immobilized PB
The epoxy activation of step 2.1 carrier
To the sodium hydroxide solution and the 15mL epoxychloropropane that add 6mL3M in 10mL carrier, stir anti-2h at 50 DEG C.Water and ethanol wash vehicle successively, obtains the carrier that epoxy activates.
The ammonification of step 2.2 carrier
In the carrier of epoxy activation, add 20mL ammoniacal liquor, stir anti-2h at 50 DEG C, amino is connected on carrier, complete once activation
Step 2.3 is immobilized deoxycholic acid (DOC) on carrier
2mmol NaTDC is dissolved in to the methyl-sulfoxide aqueous solution of 300mL40%.Add the carrier 10mL after ammonification, stir, by the HCl regulation system pH value to 4.8 of 0.3M.To the methyl-sulfoxide aqueous solution (volume ratio is 1:2) that slowly adds 6mM1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate (WCCM) in system.Stirring reaction 6h, drips the HCl of 0.3M in process, make system pH remain on 4.8.React rear with ethanol washing, removed unreacted NaTDC.Obtain the immobilized adsorbent that has deoxycholic acid.
Performance evaluation
1, the detection of aglucon supported quantity:
On adsorbent, the content of DOC can detect by deoxycholic acid kit (sigma).To embodiment 1 to 3, the testing result of the amount of the immobilized deoxycholic acid of adsorbent of comparative example 1-5 is specifically in table one.
Amino-compound is the polypeptide of amino acid or amino acid composition, can detect by ninhydrin method.The amino reaction of ninhydrin and α generates hepatic compound, and under variable concentrations, this compound, in the absorbance difference at 570nm place, can be depicted as calibration curve accordingly.Detect the ultraviolet absorptivity value of particular adsorbent at 570nm place, just can calculate amino acid whose supported quantity according to calibration curve.
Weigh 0.02g adsorbent, be placed in 25mL colorimetric cylinder, the 4.0mL that adds water, then add 2% ninhydrin solution and Na in each pipe
2hPO
4-KH
2pO
4(pH=8) individual 1.0 milliliters of cushioning liquid, mixes, and in 80 DEG C of reactions 30 minutes, is cooled to room temperature, adds water to 25mL, mixes, and leaves standstill 10min, detects the OD. value at 570nm place in sample cell.By above method, respectively to embodiment 1 to 3, the amount of the immobilized PB of adsorbent of comparative example 1-5 detects, and testing result is in table one.
2, the detection of adsorption capacity:
Detection method is as follows:
Standard liquid is that calf serum (BSA) solution adds endotoxin standard outward.The BSA aqueous solution of preparation 10mg/mL concentration, adds endotoxin standard to about 500Eu/mL.
Accurately take 1mL adsorbent in 50mL conical flask, with sterilized water for injection flushing three times, blot surface moisture, add the endotoxin BSA solution of 5mL500Eu/mL.This sample and blank sample are put into water bath with thermostatic control oscillator, 37 DEG C of absorption 2 hours.Detect endotoxic level by dynamic turbidimetric, calculate the adsorption rate of adsorbent.
By above method, respectively to embodiment 1 to 3, the adsorption capacity of the adsorbent induced by endotoxin of comparative example 1-5 detects, and testing result is in table one.
Table one: embodiment 1 to 3, aglucon supported quantity and the adsorption capacity of comparative example 1-5 adsorbent
Can find out by table one, the adsorbent adsorption capacity of grafting PMX-B is general separately, the supported quantity that increases PMX-B can be in the adsorption capacity that to a certain degree increases adsorbent induced by endotoxin, but can reach after plateau value, increase again aglucon supported quantity, as shown in comparative example 1-3, the adsorption capacity of endotoxin synergistic adsorbent induced by endotoxin no longer improves, and also has a lot of endotoxins in association state to be adsorbed in explanation system.With reference to comparative example 4-5, the adsorbent of grafting DOC separately, the adsorption capacity of induced by endotoxin is very weak.And the adsorbent of the present invention while grafting DOC and two kinds of aglucons of PMX-B has shown obvious advantage on absorption property.Particularly, the supported quantity of DOC is at 3umol-4umol/g adsorbent, and the mol ratio of DOC and PMX-B is 0.6 when above, and adsorption capacity is very good, as embodiment 1-2.When DOC supported quantity is below 1umol/g, though the adsorption capacity of adsorbent induced by endotoxin is than the excellence of single aglucon, slightly aobvious undesirable, as embodiment 5.In the time that the PMX-B in aglucon uses lysine or arginine instead, though the adsorption capacity of induced by endotoxin is better than single aglucon, absorption property is slightly poorer than immobilized PMX-B aglucon, as embodiment 3-4.
Claims (4)
1. prepare the method for endotoxin synergistic adsorbent, comprise carrier and the immobilized aglucon on described carrier of porous, described aglucon at least comprises deoxycholic acid aglucon and amino-compound aglucon, it is characterized in that: said method comprising the steps of:
Step 1: prepare porous carrier;
Step 2: immobilized deoxycholic acid on porous carrier:
Step 2.1: carrier is carried out to epoxy activation;
Step 2.2: the carrier to epoxy activation carries out ammonification;
Step 2.3: immobilized deoxycholic acid on the carrier of ammonification;
Step 3: at immobilized immobilized amino-compound again on the porous carrier of deoxycholic acid, described amino-compound is selected from amino acid or polyaminoacid.
2. the method for preparing according to claim 1 endotoxin synergistic adsorbent, is characterized in that:
Described step 2 comprises the following steps:
Step 2.1 is carried out epoxy activation to carrier:
By volume part, in 5-15 part carrier, add sodium hydroxide solution and 10-20 part one end band active group that 5-10 part concentration is 1-3M, the epoxide of one end band epoxy radicals, at 30-60 DEG C of stirring reaction 1-5h, then ethanolic solution wash vehicle 5-10 time of water and 50%-95% successively, obtains the carrier of epoxy activation;
Step 2.2 is carried out ammonification to the carrier of epoxy activation:
What obtain to step 2.1 adds 10-20mL ammoniacal liquor in the carrier through epoxy activation, stirs anti-1-5h at 30-60 DEG C, and amino is connected on carrier;
Step 2.3 is immobilized deoxycholic acid on the carrier of ammonification:
NaTDC is dissolved in to 40% the methyl-sulfoxide aqueous solution, being mixed with concentration is the deoxycholic acid sodium solution of 1-5mmol/L, by volume part, get the described deoxycholic acid sodium solution of 200-400 part, add the carrier 5-15 part after ammonification, stir, by the HCl regulation system pH value of 0.2-0.4M to 4-6, to the methyl-sulfoxide aqueous solution that slowly adds 5-10mM1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate in system, the volume ratio of the methyl-sulfoxide aqueous solution of the volume of wherein said carrier and described 1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate is 1:2, stirring reaction 1-5h, in course of reaction, drip the HCl of 0.2-0.4M, make system pH remain on 4-6, react rear use 95% ethanolic solution washing, removed unreacted NaTDC.
3. the method for preparing according to claim 2 endotoxin synergistic adsorbent, is characterized in that:
Described step 3 comprises the steps:
The immobilized carrier that has deoxycholic acid that step 3.1 obtains step 2 carries out re-activation;
Immobilized amino-compound on the immobilized carrier that has deoxycholic acid that step 3.2 obtains to step 3.1.
4. according to the method for preparing endotoxin synergistic adsorbent described in claims 1 to 3 any one, it is characterized in that:
Described amino-compound is selected from PB, lysine, arginine, histidine or polylysine.
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| CN101157018A (en) * | 2007-08-17 | 2008-04-09 | 大连理工大学 | Endotoxin adsorption material for curing endotoxemia |
| CN101322933A (en) * | 2008-06-17 | 2008-12-17 | 珠海丽珠医用生物材料有限公司 | Endotoxin absorbent and preparation thereof |
| CN102247817A (en) * | 2011-05-03 | 2011-11-23 | 大连理工大学 | Endotoxin adsorbent using molecular cluster as functional group and preparation method thereof |
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| CN101157018A (en) * | 2007-08-17 | 2008-04-09 | 大连理工大学 | Endotoxin adsorption material for curing endotoxemia |
| CN101322933A (en) * | 2008-06-17 | 2008-12-17 | 珠海丽珠医用生物材料有限公司 | Endotoxin absorbent and preparation thereof |
| CN102247817A (en) * | 2011-05-03 | 2011-11-23 | 大连理工大学 | Endotoxin adsorbent using molecular cluster as functional group and preparation method thereof |
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