CN102580683A - Endotoxin synergistic adsorbent and preparation method thereof - Google Patents
Endotoxin synergistic adsorbent and preparation method thereof Download PDFInfo
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- CN102580683A CN102580683A CN2012100239352A CN201210023935A CN102580683A CN 102580683 A CN102580683 A CN 102580683A CN 2012100239352 A CN2012100239352 A CN 2012100239352A CN 201210023935 A CN201210023935 A CN 201210023935A CN 102580683 A CN102580683 A CN 102580683A
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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 collaborative adsorbent of a kind of medical endotoxin and preparation method thereof, particularly blood perfusion with collaborative adsorbent of endotoxin and preparation method thereof.
Background technology
A lipoid polysaccharose substance that discharges when endotoxin is the Gram-negative bacteria growing or produce by the cell membrane cracking when dead, denier (nanogram level) endotoxin gets into human body will cause high heat, blood vessel dilatation, even faint or dead.Endotoxemia is to cause owing to the endotoxin on the outer cell membrane of Gram-negative bacteria is discharged in the blood, can come across in the multiple lysis, as: diseases such as large-area burns, serious hepatitis, cirrhosis.Endotoxemia is one of clinical modal deadly 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%.
Still do not have the effectively method of treatment endotoxemia clinically, one of existent method is to have the adsorbent of PB to carry out blood perfusion through 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 who is used for the endotoxin absorbent of blood perfusion; Spherical Ago-Gel through good biocompatibility is a matrix, adopts epoxychloropropane, hexamethylene diamine, 1, after the activation of 1 '-carbonyl dimidazoles; Behind the bonding PB aglucon, through NaBH
4Reduction promptly obtains the collaborative adsorbent of endotoxin.Through 6.12 milligrams/gram of the adsorbent PB supported quantity adsorbent of this method preparation, the endotoxin adsorbance reaches 22 ng/g adsorbents.
Use other positively charged quaternary ammonium salt, amino acid or polymer to do the research of aglucon in addition.Jia Lingyun etc. are the grafting quaternary ammonium salt on agarose, can reach 50% (CN200710012501.1) to endotoxin clearance rate in the blood.With the fixing agarose carrier of lysine is adsorbent, and the endotoxin in the adsorbed proteins solution does not have obvious absorption to albumen.Adopt macropore cellulose balls carrier, fixing polymine, blood perfusion adsorbs endotoxin, and endotoxin is significantly descended.
But, though above-mentioned be the adsorbent of immobilized PB, also be to use other positively charged quaternary ammonium salt, amino acid or polymer to make the adsorbent of aglucon, their usually only have the good adsorption ability to the free down endotoxin of low concentration.But because the endotoxin complex shape in the blood, most applications exists with coherent condition, and above-mentioned adsorbent is unsatisfactory to the endotoxin Molecular Adsorption effect that is in state of aggregation under the high concentration, therefore can not effectively remove the endotoxin in the blood.
People such as Johoson once studied with the deoxycholic acid aglucon.As on polyethylene support, do arm with diamines, the grafting deoxycholic acid, though can adsorb the endotoxin of removing in the blood, adsorbance is extremely limited.
Summary of the invention
The technical problem that the present invention will solve provides a kind of bigger to the endotoxin adsorption capacity, removes endotoxin absorbent more completely.
For solving the problems of the technologies described above, technical scheme provided by the invention is: endotoxin is worked in coordination with adsorbent, comprises the carrier and the immobilized aglucon on said carrier of porous, and wherein, said aglucon comprises deoxycholic acid aglucon and amino-compound aglucon at least.
Preferred scheme be immobilized deoxycholic acid and the mol ratio of amino-compound be 0.05: 1-20: 1.
Preferred scheme is that the supported quantity of deoxycholic acid is the 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 simultaneously and remove.Above-mentioned adsorbent is through the synergy of two kinds of aglucons on the carrier, make its to endotoxic adsorption capacity far above the endotoxin absorbent that has single aglucon.
Another technical problem that the present invention will solve provides a kind of method for preparing the collaborative adsorbent of endotoxin, and simultaneously immobilized on the carrier of the collaborative adsorbent of this endotoxin 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 the collaborative adsorbent of endotoxin; The carrier and the immobilized aglucon on said carrier that comprise porous; Wherein said aglucon comprises deoxycholic acid aglucon and amino-compound aglucon at least, said method comprising the steps of: step 1: prepare porous carrier; Step 2: immobilized deoxycholic acid on porous carrier; Step 3: at immobilized immobilized again amino-compound on the porous carrier of deoxycholic acid.
Preferred scheme is: step 2 may further comprise the steps:
Step 2.1: carrier carried out the epoxy activation;
Step 2.2: the carrier to own epoxy activation carries out ammonification;
Step 2.3: immobilized deoxycholic acid on the carrier of ammonification.
Another preferred scheme is that step 2 may further comprise the steps:
Step 2.1 pair carrier carry out the epoxy activation:
By volume part; In 5-15 part carrier, adding 5-10 part concentration is sodium hydroxide solution and the 10-20 part one end band active group of 1-3M; The epoxide of one end band epoxy radicals; At 30-60 ℃ of stirring reaction 1-5h, ethanolic solution wash vehicle 5-10 time of water and 50%-95% successively then obtains the carrier of epoxy activation;
The carrier of step 2.2 pair own epoxy activation carries out ammonification:
In the carrier of the epoxy of the process activation that step 2.1 obtained, add 10-20mL ammoniacal liquor, stir anti-1-5h, amino is connected on the carrier at 30-60 ℃;
Step 2.3 is immobilized deoxycholic acid on the carrier of ammonification:
NaTDC is dissolved in 40% the methyl-sulfoxide aqueous solution, and being mixed with concentration is the deoxycholic acid sodium solution of 1-5mmol/L; By volume part; Get the said deoxycholic acid sodium solution of 200-400 part, the carrier 5-15 part after the adding ammonification is stirred; With the HCl regulation system pH value of 0.2-0.4M to 4-6; In system, slowly add the methyl-sulfoxide aqueous solution of 5-10mM 1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate, the volume ratio of the methyl-sulfoxide aqueous solution of the volume of wherein said carrier and said 1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate is 1: 2, stirring reaction 1-5h; Drip the HCl of 0.2-0.4M in the course of reaction, make system pH remain on 4-6; Reaction is accomplished anti-back with the washing of 95% ethanolic solution, removes unreacted NaTDC.
Preferred scheme is: step 3 comprises the steps:
Pair step 2 obtained step 3.1 immobilizedly has the carrier of deoxycholic acid to carry out re-activation;
Step 3.2 is immobilized amino-compound on step 3.1 obtained the immobilized carrier that deoxycholic acid arranged.
The collaborative adsorbent of endotoxin through method for preparing, simultaneously immobilized on the carrier have deoxycholic acid and two kinds of aglucons of amino-compound, and the endotoxin that endotoxic adsorption capacity is far longer than single aglucon is worked in coordination with adsorbent.
The average grain diameter of the carrier of the collaborative adsorbent of endotoxin of the present invention is 50um-1500um, is 300um-800um better, takes this, and the collaborative adsorbent of endotoxin of the present invention not only can have the good adsorption rate, and can be used for whole blood absorption.The pore diameter range of carrier of the present invention can be 2nm-300nm, and preferred 10nm-100nm can guarantee to have bigger specific area on the one hand, is fit to endotoxic absorption on the other hand again.The carrier of the collaborative adsorbent of endotoxin of the present invention can be cellulose, agarose, polyvinyl alcohol, styrene-divinylbenzene copolymer or polymethyl methacrylate, and preferred cellulose, polymethylacrylic acid, preferred carrier are cellulose carrier.Because cellulose carrier has the following advantages: mechanical strength that (1) is high relatively and toughness, can satisfy the requirement of whole blood perfusion.(2) have excellent biological compatibility, security is good.(3) source is abundant, and is with low cost.(4) environmental friendliness can natural degradation after the use.
The endotoxic carrier of the present invention can be that commercial sources is bought, and also can be oneself preparation.For example, can be through the cellulose acetate solution that adds a certain proportion of pore-foaming agent be dispersed in polyvinyl alcohol (PVA) aqueous solution, after waiting to be distributed to appropriate particle size, the satisfactory cellulose diacetate carrier in particle diameter aperture is prepared in the balling-up that is heating and curing.About the preparation of carrier, existing a lot of methods for the purpose of saving, repeat no more here in the prior art.
In the present invention, consider that deoxycholic acid is more stable on chemical property, should not receive the influence of subsequent chemistry process, therefore adopt the at first immobilized deoxycholic acid and then the method for immobilized cationic compound.
Take all factors into consideration factors such as cost and adsorption effect, the amount of immobilized deoxycholic acid is preferably the 0.1umol-50umol/g adsorbent on the collaborative adsorbent of endotoxin of the present invention, more preferably the 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, the amount of deoxycholic acid aglucon reaches after a certain plateau value, increases the deoxycholic acid amount again, and endotoxic adsorption capacity does not have too big raising yet, and cost will be higher.Among the present invention; The method that can adopt peptide coupling covalent bond deoxycholic acid is deoxycholic acid fixedly; As: the carrier and an end that will have hydroxyl have active group, and the compound that the other end has epoxide group carries out the epoxy priming reaction, uses the ammoniacal liquor ammonification again; Introduce amino, pass through the method grafting deoxycholic acid of peptide coupling then.Wherein, an end has active group, and the compound that the other end has epoxide group can be epoxychloropropane or the compound of being with diepoxy group etc.
Amino-compound on the collaborative adsorbent of endotoxin 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., preferred PMX-B.The positive charge of amino-compound can relative specificity the ground absorption surface have the endotoxin molecule of phosphate groups.Amino-compound among the present invention on the collaborative adsorbent of endotoxin can adopt the method for epoxy radicals or aldehyde radical and amino reaction immobilized, preferred epoxy reaction.Take all factors into consideration factors such as cost and adsorption effect, the amount of immobilized amino-compound is preferably the 0.1umol-100umol/g adsorbent on the collaborative adsorbent of endotoxin of the present invention, is more preferably the 1umol-10umol/g adsorbent.Considering after through the method grafting deoxycholic acid of peptide coupling on the carrier maybe also residual a large amount of amino, can select to use dialdehyde or diepoxides, through the reaction between aldehyde radical and the amino, removes aminoly, accomplishes the grafting of cationic compound.Wherein dialdehyde can be glutaraldehyde or hexandial, preferred glutaraldehyde.Bis-epoxy can be 1, ammediol glycidol ether, 1, and 4-butanediol glycidol ether or 1,5-pentanediol glycidol ether, preferred 1,4-butanediol glycidol ether.
On the collaborative adsorbent of endotoxin of the present invention the ratio of immobilized two kinds of aglucons the effect and the cost of absorption had certain influence; Preferably; Deoxycholic acid and amino-compound mol ratio are 0.05: 1-20: between 1, and preferred 0.1: 1-10: between 1, more preferably 0.5: 1-1: between 3.Wherein the amount of deoxycholic acid can not be very little, otherwise adsorbent endotoxicly dissociates limited in one's abilityly to assembling, and influences 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, increases the deoxycholic acid amount again, can not increase endotoxic adsorption capacity.The excess of ammonia based compound has neurotoxicity and renal toxicity like PMX-B.Therefore, the supported quantity of amino-compound can not be too high, preferably is controlled at below the 20mg/mL adsorbent.
The specific embodiment
Embodiment 1
The collaborative preparation of adsorbent method of present embodiment endotoxin is following:
Step 1: preparation macropore cellulose carrier
The 12g cellulose diacetate is dissolved in the mixed solvent of being made up of 80mL carrene and 20mL ethanol, and the mass fraction of cellulose solution is 9%.
In above-mentioned solution, add ethyl acetate 100mL,, mix as pore-foaming agent.The PVA aqueous solution of preparation 400mL5% is as decentralized photo.The slow impouring of cellulose solution is equipped with in the decentralized photo, and the control mixing speed is 100-160r.p.m, makes cellulose solution fully be dispersed into uniform droplet; Keep mixing speed then; Be heated to 35 ℃, carrene is volatilized fully, cellulose grain thoroughly solidifies.Water and 75% washing with alcohol are thoroughly removed PVA and pore-foaming agent, 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
In the 10mL carrier, add sodium hydroxide solution and the 15mL epoxychloropropane of 6mL 3M, stir anti-2h at 50 ℃.Water and washing with alcohol carrier successively obtain the carrier of epoxy activation.
The ammonification of step 2.2 carrier
In the carrier of the own epoxy activation that step 2.1 obtained, add 20mL ammoniacal liquor, stir anti-2h, amino is connected on the carrier at 50 ℃.
Step 2.3 is immobilized deoxycholic acid (DOC) on carrier
1mmol deoxycholic acid sodium solution is dissolved in the methyl-sulfoxide aqueous solution of 300mL 40%.Carrier 10mL after the adding ammonification stirs, with the HCl regulation system pH value to 4.8 of 0.3M.The methyl-sulfoxide aqueous solution (volume ratio of the carrier and the methyl-sulfoxide aqueous solution is 1: 2) that in system, slowly adds 6mM 1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate (WCCM).Stirring reaction 3h, the HCl of dropping 0.3M makes system pH remain on 4.8 in the process.Reaction is used washing with alcohol after accomplishing, and removes unreacted NaTDC.
Step 3: at immobilized immobilized again amino-compound on the porous carrier of deoxycholic acid
Pair step 2 obtained step 3.1 immobilizedly has the carrier of deoxycholic acid to carry out re-activation
Still have a lot of residual amino on the carrier behind the immobilized DOC.In this carrier of 10mL, add 1 of 10mL, the sodium hydroxide solution 15mL of 4-butanediol-diglycidyl ether and 0.1M is stirring reaction 18h at room temperature.Reaction back water is rinsed well.
Step 3.2 is immobilized PB on step 3.1 obtained the immobilized carrier that deoxycholic acid arranged
The 0.1g PB is dissolved in the 10mL water; With sodium hydroxide solution the pH value is transferred to 11; Adding is by the carrier 5mL that step 3.1 obtained, and room temperature reaction 2h is then with the sodium chloride flushing of 1M; Acquisition is a carrier with the macropore cellulose, contains the collaborative adsorbent of endotoxin of deoxycholic acid and PB aglucon.
Embodiment 2
The collaborative preparation of adsorbent process of present embodiment endotoxin is following:
Step 1: preparation 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
In the 10mL carrier, add sodium hydroxide solution and the 15mL epoxychloropropane of 6mL 3M, stir anti-2h at 50 ℃.Water and washing with alcohol carrier successively obtain the carrier of epoxy activation.
The ammonification of step 2.2 carrier
In the carrier of the own epoxy activation that step 2.1 obtained, add 20mL ammoniacal liquor, stir anti-2h, amino is connected on the carrier at 50 ℃.
Step 2.3 is immobilized deoxycholic acid (DOC) on carrier
The 1mmol NaTDC is dissolved in the methyl-sulfoxide aqueous solution of 300mL 40%.Carrier 10mL after the adding ammonification stirs, with the HCl regulation system pH value to 4.8 of 0.3M.The methyl-sulfoxide aqueous solution (volume ratio is 1: 2) that in system, slowly adds 6mM 1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate (WCCM).Stirring reaction 3h, the HCl of dropping 0.3M makes system pH remain on 4.8 in the process.Reaction is used washing with alcohol after accomplishing, and removes unreacted NaTDC.
Step 3: at immobilized immobilized again amino-compound on the porous carrier of deoxycholic acid
Pair step 2 obtained step 3.1 immobilizedly has the carrier of deoxycholic acid to carry out re-activation
Still have a lot of residual amino on the carrier behind the immobilized DOC.Adding 25mL 0.05M pH is 7.4 phosphate buffer solution in this carrier of 10mL, under stirring condition, drips the glutaraldehyde of 8mL 25%, at room temperature stirring reaction 2h.(aldehyde group content is about 74umol/g).The reaction back is with phosphate buffer and the water flushing of 0.1M.
Step 3.2 is immobilized PB on step 3.1 obtained the immobilized carrier that deoxycholic acid arranged
The 0.1g PB is dissolved in the 10mL water, the pH value is transferred to 11 with sodium hydroxide solution, the carrier 5mL behind the adding re-activation, room temperature reaction 2h, the sodium chloride with 1M washes then.With the two keys of sodium borohydride reduction, washing is to neutral.Acquisition is a carrier with the macropore cellulose, contains the collaborative adsorbent of endotoxin of deoxycholic acid and PB aglucon.
Embodiment 3
The collaborative preparation of adsorbent process of present embodiment endotoxin is following:
Step 1: preparation 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
In the 10mL carrier, add sodium hydroxide solution and the 15mL epoxychloropropane of 6mL 3M, stir anti-2h at 50 ℃.Water and washing with alcohol carrier successively obtain the carrier of epoxy activation.
The ammonification of step 2.2 carrier
In the carrier of the own epoxy activation that step 2.1 obtained, add 20mL ammoniacal liquor, stir anti-2h, amino is connected on the carrier at 50 ℃.
Step 2.3 is immobilized deoxycholic acid (DOC) on carrier
The 1mmol NaTDC is dissolved in the methyl-sulfoxide aqueous solution of 300mL 40%.Carrier 10mL after the adding ammonification stirs, with the HCl regulation system pH value to 4.8 of 0.3M.The methyl-sulfoxide aqueous solution (volume ratio is 1: 2) that in system, slowly adds 6mM 1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate (WCCM).Stirring reaction 3h, the HCl of dropping 0.3M makes system pH remain on 4.8 in the process.Reaction is used washing with alcohol after accomplishing, and removes unreacted NaTDC.
Step 3: at immobilized immobilized again amino-compound on the porous carrier of deoxycholic acid
Pair step 2 obtained step 3.1 immobilizedly has the carrier of deoxycholic acid to carry out re-activation
Still have a lot of residual amino on the carrier behind the immobilized DOC.Adding 25mL 0.05M pH is 7.4 phosphate buffer solution in this carrier of 10mL, under stirring condition, drips the glutaraldehyde of 8mL 25%, at room temperature stirring reaction 2h.(aldehyde group content is about 74umol/g).The reaction back is with phosphate buffer and the water flushing of 0.1M.
Step 3.2 is immobilized lysine on step 3.1 obtained the immobilized carrier that deoxycholic acid arranged
0.2g lysine is dissolved in the 10mL water, solution solution is transferred to alkalescence with NaOH, the carrier 5mL behind the adding re-activation, room temperature reaction 2h, the sodium chloride with 1M washes then.With the two keys of sodium borohydride reduction, washing is to neutral.Acquisition is a carrier with the macropore cellulose, contains the collaborative adsorbent of endotoxin of deoxycholic acid and lysine.
Embodiment 4
The collaborative preparation of adsorbent process of present embodiment endotoxin is following:
Step 1: preparation 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
In the 10mL carrier, add sodium hydroxide solution and the 15mL epoxychloropropane of 6mL 3M, stir anti-2h at 50 ℃.Water and washing with alcohol carrier successively obtain the carrier of epoxy activation.
The ammonification of step 2.2 carrier
In the carrier of the own epoxy activation that step 2.1 obtained, add 20mL ammoniacal liquor, stir anti-2h, amino is connected on the carrier at 50 ℃.
Step 2.3 is immobilized deoxycholic acid (DOC) on carrier
The 1mmol NaTDC is dissolved in the methyl-sulfoxide aqueous solution of 300mL 40%.Carrier 10mL after the adding ammonification stirs, with the HCl regulation system pH value to 4.8 of 0.3M.The methyl-sulfoxide aqueous solution (volume ratio is 1: 2) that in system, slowly adds 6mM 1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate (WCCM).Stirring reaction 3h, the HCl of dropping 0.3M makes system pH remain on 4.8 in the process.Reaction is used washing with alcohol after accomplishing, and removes unreacted NaTDC.
Step 3: at immobilized immobilized again amino-compound on the porous carrier of deoxycholic acid
Pair step 2 obtained step 3.1 immobilizedly has the carrier of deoxycholic acid to carry out re-activation
Still have a lot of residual amino on the carrier behind the immobilized DOC.Adding 25mL 0.05M pH is 7.4 phosphate buffer solution in this carrier of 10mL, under stirring condition, drips the glutaraldehyde of 8mL 25%, at room temperature stirring reaction 2h.(aldehyde group content is about 74umol/g).The reaction back is with phosphate buffer and the water flushing of 0.1M.
Step 3.2 is immobilized arginine on step 3.1 obtained the immobilized carrier that deoxycholic acid arranged
The 0.2g arginine is dissolved in the 10mL water, solution solution is transferred to alkalescence with NaOH, the carrier 5mL behind the adding re-activation, room temperature reaction 2h, the sodium chloride with 1M washes then.With the two keys of sodium borohydride reduction, washing is to neutral.Acquisition is a carrier with the macropore cellulose, contains the collaborative adsorbent of deoxycholic acid and arginic endotoxin.
Embodiment 5:
The collaborative preparation of adsorbent process of present embodiment endotoxin is following:
Step 1: preparation 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
In the 10mL carrier, add sodium hydroxide solution and the 15mL epoxychloropropane of 6mL 3M, stir anti-2h at 50 ℃.Water and washing with alcohol carrier successively obtain the carrier of epoxy activation.
The ammonification of step 2.2 carrier
In the carrier of epoxy activation, add 20mL ammoniacal liquor, stir anti-2h, amino is connected on the carrier at 50 ℃.
Step 2.3 is immobilized deoxycholic acid (DOC) on carrier
The 0.5mmol NaTDC is dissolved in the methyl-sulfoxide aqueous solution of 300mL 40%.Carrier 10mL after the adding ammonification stirs, with the HCl regulation system pH value to 4.8 of 0.3M.The methyl-sulfoxide aqueous solution (volume ratio is 1: 2) that in system, slowly adds 6mM 1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate (WCCM).Stirring reaction 3h, the HCl of dropping 0.3M makes system pH remain on 4.8 in the process.Reaction is used washing with alcohol after accomplishing, and removes unreacted NaTDC.
Step 3: at immobilized immobilized again amino-compound on the porous carrier of deoxycholic acid
Pair step 2 obtained step 3.1 immobilizedly has the carrier of deoxycholic acid to deaminize
Still have a lot of residual amino on the carrier behind the immobilized DOC.Adding 25mL 0.05M pH is 7.4 phosphate buffer solution in this carrier of 10mL, under stirring condition, drips the glutaraldehyde of 8mL 25%, at room temperature stirring reaction 2h.(aldehyde group content is about 74umol/g).The reaction back is with phosphate buffer and the water flushing of 0.1M.
Step 3.2 is immobilized PB on step 3.1 obtained the immobilized carrier that deoxycholic acid arranged
The 0.1g PB is dissolved in the 10mL water, the pH value is transferred to 11 with sodium hydroxide solution, the carrier 5mL behind the adding re-activation, room temperature reaction 2h, the sodium chloride with 1M washes then.
Comparative example 1
Prepare adsorbent according to the following steps:
Step 1: the preparation carrier, method is with embodiment 1.
Step 2: immobilized PB
The epoxy activation of step 2.1 carrier
The sodium hydroxide solution and the 15mL epoxychloropropane that in the 10mL carrier, add 6mL 3M are at 50 ℃ of stirring reaction 2h.Water and washing with alcohol carrier obtain the carrier of epoxy activation more than three times successively.
Step 2.2PMX-B's is immobilized
The 0.1g PB is dissolved in the 10mL water, the pH value is transferred to 11 with sodium hydroxide solution, the carrier 5mL after the activation of adding epoxy, room temperature reaction 2h with the sodium chloride flushing of 1M, obtains the immobilized adsorbent that PB is arranged then.
Comparative example 2:
Prepare adsorbent according to the following steps:
Step 1: the preparation carrier, method is with embodiment 1.
Step 2: immobilized PB
The epoxy activation of step 2.1 carrier
The sodium hydroxide solution and the 15mL epoxychloropropane that in the 10mL carrier, add 6mL 3M are at 50 ℃ of stirring reaction 2h.Water and washing with alcohol carrier obtain the carrier of epoxy activation more than three times successively.
Step 2.2PMX-B's is immobilized
The 0.3g PB is dissolved in the 10mL water, with sodium hydroxide solution the pH value is transferred to 11, add the carrier 5mL after the activation, room temperature reaction 2h is then with the sodium chloride flushing of 1M.Obtain the immobilized adsorbent that PB is arranged.
Comparative example 3:
Prepare adsorbent according to the following steps:
Step 1: the preparation carrier, method is with embodiment 1.
Step 2: immobilized PB
The epoxy activation of step 2.1 carrier
The sodium hydroxide solution and the 15mL epoxychloropropane that in the 10mL carrier, add 6mL 3M are at 50 ℃ of stirring reaction 2h.Water and washing with alcohol carrier obtain the carrier of epoxy activation more than three times successively.
Step 2.2PMX-B's is immobilized
The 0.2g PB is dissolved in the 10mL water, with sodium hydroxide solution the pH value is transferred to 11, add the carrier 5mL after the activation, room temperature reaction 2h is then with the sodium chloride flushing of 1M.Obtain the immobilized adsorbent that PB is arranged.
Comparative example 4:
Prepare adsorbent according to the following steps:
Step 1: the preparation carrier, method is with embodiment 1.
Step 2: immobilized PB
The epoxy activation of step 2.1 carrier
In the 10mL carrier, add sodium hydroxide solution and the 15mL epoxychloropropane of 6mL 3M, stir anti-2h at 50 ℃.Water and washing with alcohol carrier successively obtain the carrier of epoxy activation.
The ammonification of step 2.2 carrier
In the carrier of epoxy activation, add 20mL ammoniacal liquor, stir anti-2h, amino is connected on the carrier, accomplish an activation at 50 ℃
Step 2.3 is immobilized deoxycholic acid (DOC) on carrier
1mmo l NaTDC is dissolved in the methyl-sulfoxide aqueous solution of 300mL 40%.Carrier 10mL after the adding ammonification stirs, with the HCl regulation system pH value to 4.8 of 0.3M.The methyl-sulfoxide aqueous solution (volume ratio is 1: 2) that in system, slowly adds 6mM 1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate (WCCM).Stirring reaction 3h, the HCl of dropping 0.3M makes system pH remain on 4.8 in the process.Reaction is used washing with alcohol after accomplishing, and removes unreacted NaTDC, obtains the immobilized adsorbent that deoxycholic acid is arranged.
Comparative example 5:
Prepare adsorbent according to the following steps:
Step 1: the preparation carrier, method is with embodiment 1.
Step 2: immobilized PB
The epoxy activation of step 2.1 carrier
In the 10mL carrier, add sodium hydroxide solution and the 15mL epoxychloropropane of 6mL 3M, stir anti-2h at 50 ℃.Water and washing with alcohol carrier successively obtain the carrier of epoxy activation.
The ammonification of step 2.2 carrier
In the carrier of epoxy activation, add 20mL ammoniacal liquor, stir anti-2h, amino is connected on the carrier, accomplish an activation at 50 ℃
Step 2.3 is immobilized deoxycholic acid (DOC) on carrier
The 2mmol NaTDC is dissolved in the methyl-sulfoxide aqueous solution of 300mL 40%.Carrier 10mL after the adding ammonification stirs, with the HCl regulation system pH value to 4.8 of 0.3M.The methyl-sulfoxide aqueous solution (volume ratio is 1: 2) that in system, slowly adds 6mM 1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate (WCCM).Stirring reaction 6h, the HCl of dropping 0.3M makes system pH remain on 4.8 in the process.Reaction is used washing with alcohol after accomplishing, and removes unreacted NaTDC.Obtain the immobilized adsorbent that deoxycholic acid is arranged.
Performance evaluation
1, the detection of aglucon supported quantity:
The content of DOC can detect through deoxycholic acid kit (sigma) on the adsorbent.To embodiment 1 to 3, the testing result of the amount of the immobilized deoxycholic acid of adsorbent of Comparative Examples 1-5 is specifically seen table one.
Amino-compound is the polypeptide that amino acid or amino acid are formed, and can detect through ninhydrin method.The amino reaction of ninhydrin and α generates hepatic compound, and this compound is different at the absorbance at 570nm place under the variable concentrations, can be depicted as calibration curve in view of the above.Detect the ultraviolet absorptivity value of particular adsorbent, just can calculate amino acid whose supported quantity according to calibration curve at the 570nm place.
Weighing 0.02g adsorbent places the 25mL colorimetric cylinder, adds water 4.0mL, in each pipe, adds 2% ninhydrin solution and Na2 again
HPO
4-KH
2PO
4(pH=8) cushioning liquid is individual 1.0 milliliters, and mixing in 80 ℃ of reactions 30 minutes, is cooled to room temperature, adds water to 25mL, and mixing leaves standstill 10min, the OD. value at 570nm place in the test sample pipe.By above method, respectively to embodiment 1 to 3, the amount of the immobilized PB of adsorbent of Comparative Examples 1-5 detects, and testing result is seen table one.
2, the detection of adsorption capacity:
Detection method is following:
Standard liquid is that calf serum (BSA) solution adds the endotoxin standard items outward.The BSA aqueous solution of preparation 10mg/mL concentration adds the endotoxin standard items to about 500Eu/mL.
Accurately take by weighing the 1mL adsorbent in the 50mL conical flask,, blot surface moisture, add the endotoxin BSA solution of 5mL 500Eu/mL with sterilized water for injection flushing three times.This sample and blank are put into the water bath with thermostatic control oscillator, 37 ℃ of absorption 2 hours.Detect endotoxic level with dynamic turbidimetric, calculate the adsorption rate of adsorbent.
Through above method, to embodiment 1 to 3, the adsorbent of Comparative Examples 1-5 detects endotoxic adsorption capacity respectively, and testing result is seen table one.
Table one: embodiment 1 to 3, the aglucon supported quantity and the adsorption capacity of Comparative Examples 1-5 adsorbent
Can find out through table one; The adsorbents adsorb ability of grafting PMX-B is general separately, and the supported quantity that increases PMX-B can to a certain degree increase adsorbent to endotoxic adsorption capacity, but after can reaching plateau value; Increase the aglucon supported quantity again; Shown in comparative example 1-3, the collaborative adsorbent of endotoxin no longer improves endotoxic adsorption capacity, and the endotoxin that much is in the association state in the explanation system in addition can't be adsorbed.With reference to comparative example 4-5, the independent adsorbent of grafting DOC, to endotoxic adsorption capacity very a little less than.And the adsorbent of the present invention while grafting DOC and two kinds of aglucons of PMX-B has shown remarkable advantages on absorption property.Particularly, the supported quantity of DOC is at the 3umol-4umol/g adsorbent, and the mol ratio of DOC and PMX-B is 0.6 when above, and adsorption capacity is very good, like embodiment 1-2.When the DOC supported quantity below 1umol/g, though that adsorbent to the excellence of endotoxic adsorption capacity than single aglucon, shows slightly is undesirable, like embodiment 5.When the PMX-B in the aglucon used lysine or arginine instead, though endotoxic adsorption capacity is better than single aglucon, absorption property was slightly poorer than immobilized PMX-B aglucon, like embodiment 3-4.
Claims (10)
1. endotoxin is worked in coordination with adsorbent, comprises the carrier and the immobilized aglucon on said carrier of porous, it is characterized in that:
Said aglucon comprises deoxycholic acid aglucon and amino-compound aglucon at least.
2. endotoxin according to claim 1 is worked in coordination with adsorbent, it is characterized in that:
The mol ratio of said deoxycholic acid and said amino-compound is 0.05: 1-20: 1.
3. according to claim 1 or the collaborative adsorbent of 2 each described endotoxins, it is characterized in that:
The supported quantity of said deoxycholic acid is the 0.1umol-50umol/g adsorbent.
4. endotoxin according to claim 3 is worked in coordination with adsorbent, it is characterized in that:
Said amino-compound is selected from amino acid or polyaminoacid.
5. endotoxin according to claim 3 is worked in coordination with adsorbent, it is characterized in that:
Said amino-compound is selected from PB, lysine, arginine, histidine or polylysine.
6. prepare endotoxin and work in coordination with the method for adsorbent, comprise the carrier and the immobilized aglucon on said carrier of porous, said aglucon comprises deoxycholic acid aglucon and amino-compound aglucon at least, 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 3: at immobilized immobilized again amino-compound on the porous carrier of deoxycholic acid.
7. according to the said method for preparing the collaborative adsorbent of endotoxin of claim 6, it is characterized in that:
Said step 2 may further comprise the steps:
Step 2.1: carrier carried out the epoxy activation;
Step 2.2: the carrier to own epoxy activation carries out ammonification;
Step 2.3: immobilized deoxycholic acid on the carrier of ammonification.
8. according to the said method for preparing the collaborative adsorbent of endotoxin of claim 6, it is characterized in that:
Said step 2 may further comprise the steps:
Step 2.1 pair carrier carry out the epoxy activation:
By volume part; In 5-15 part carrier, adding 5-10 part concentration is sodium hydroxide solution and the 10-20 part one end band active group of 1-3M; The epoxide of one end band epoxy radicals; At 30-60 ℃ of stirring reaction 1-5h, ethanolic solution wash vehicle 5-10 time of water and 50%-95% successively then obtains the carrier of epoxy activation;
The carrier of step 2.2 pair own epoxy activation carries out ammonification:
In the carrier of the epoxy of the process activation that step 2.1 obtained, add 10-20mL ammoniacal liquor, stir anti-1-5h, amino is connected on the carrier at 30-60 ℃;
Step 2.3 is immobilized deoxycholic acid on the carrier of ammonification:
NaTDC is dissolved in 40% the methyl-sulfoxide aqueous solution, and being mixed with concentration is the deoxycholic acid sodium solution of 1-5mmol/L; By volume part; Get the said deoxycholic acid sodium solution of 200-400 part, the carrier 5-15 part after the adding ammonification is stirred; With the HCl regulation system pH value of 0.2-0.4M to 4-6; In system, slowly add the methyl-sulfoxide aqueous solution of 5-10mM 1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate, the volume ratio of the methyl-sulfoxide aqueous solution of the volume of wherein said carrier and said 1-cyclohexyl-2-(morpholine ethyl) carbodiimide methyl tosilate is 1: 2, stirring reaction 1-5h; Drip the HCl of 0.2-0.4M in the course of reaction, make system pH remain on 4-6; Reaction is accomplished anti-back with the washing of 95% ethanolic solution, removes unreacted NaTDC.
9. according to the said method for preparing the collaborative adsorbent of endotoxin of claim 6, it is characterized in that:
Said step 3 comprises the steps:
Pair step 2 obtained step 3.1 immobilizedly has the carrier of deoxycholic acid to carry out re-activation;
Step 3.2 is immobilized amino-compound on step 3.1 obtained the immobilized carrier that deoxycholic acid arranged.
10. according to each described method for preparing the collaborative adsorbent of endotoxin of claim 6 to 9, it is characterized in that:
Said amino-compound is selected from PB, lysine, arginine, histidine or polylysine.
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| CN112759563A (en) * | 2020-12-31 | 2021-05-07 | 苏州昊帆生物股份有限公司 | Preparation method of 1-cyclohexyl-2- (morpholinoethyl) carbodiimide methyl p-toluenesulfonate |
| CN112759563B (en) * | 2020-12-31 | 2022-03-04 | 苏州昊帆生物股份有限公司 | Preparation method of 1-cyclohexyl-2- (morpholinoethyl) carbodiimide methyl p-toluenesulfonate |
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