CN104525151A - Endotoxin adsorbent used in hemoperfusion, and preparation method thereof - Google Patents
Endotoxin adsorbent used in hemoperfusion, and preparation method thereof Download PDFInfo
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- CN104525151A CN104525151A CN201410717344.4A CN201410717344A CN104525151A CN 104525151 A CN104525151 A CN 104525151A CN 201410717344 A CN201410717344 A CN 201410717344A CN 104525151 A CN104525151 A CN 104525151A
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
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Abstract
The invention discloses an endotoxin adsorbent used in hemoperfusion, and a preparation method thereof. The preparation method comprises the following steps: a porous carrier is prepared; the carrier is polystyrene divinylbenzene resin or polymethyl methacrylate resin, wherein the pore size of the resin is 5-50nm and the specific surface area of the resin is no less than 500m<2>/g; residual double bonds of the carrier resin are subjected to epoxidation modification; and ligands are immobilized. According to the scheme of the invention, a synthesis process route is changed, such that adsorbent harmful substance residue is avoided, and adsorbent adsorption capacity is improved. Specifically, residual double bonds of the adsorption resin are subjected to epoxidation modification, such that the epoxy group amount is increased; with the epoxy groups, hydrophobic tethers are added, such that ligand movement capacity is improved, and the adsorbent is provided with a good endotoxin adsorption effect; and resin pore size and specific surface area are controlled, such that the adsorption amount and removal rate of the adsorbent upon cytokines are ensured.
Description
Technical field
The present invention relates to a kind of endotoxin absorbent for blood perfusion and preparation method thereof.
Background technology
Endotoxemia can appear in the various diseases of multisystem, and usually cause septicopyemia, lethal septic shock, MOF, disseminated intravascular coagulation etc., case fatality rate is high, more than more than 40%.And blood perfusion endotoxin absorbent can be used for removing the endotoxin in the patient bodies such as septicopyemia, septic shock and MODS, reach the object improving patient survival and improve its clinical condition.
Blood perfusion endotoxin absorbent normally with macroreticular resin or active carbon etc. for carrier, activated loading aglucon and obtaining.The aglucon that current absorption endotoxin is conventional mainly contains PB, polylysine, human serum albumins, polymine PEI, diethylaminethyl DEAE, anti-rHu-TNF-alpha monoclonal antibodies etc.
The preparation of conventional neutral macroporous absorbent resin, is after styrene-divinylbenzene polymerization, adopts chloromethyl ether activation, then increase specific area through the fourier cross-linking reaction of nitrobenzene.Adopt this synthetic technology route easily to cause the residual of intense stimulus chemical substance chloromethyl ether and nitrobenzene, these two kinds of materials are strong carcinogenic substance, there is great potential safety hazard to the health of patient.
And absorbent resin blood compatibility is poor usually, itself and blood directly contact and can destroy visible component as red blood cell, leucocyte and blood platelet.At the beginning of the seventies, professor Zhang Mingrui of McGill University of Canada takes the lead in using albumin collodion coating active charcoal for blood perfusion, makes adsorbent surface more smooth, improves blood compatibility, prevent particles from getting loose, absorption property there is no significant change simultaneously.After this, under scholars makes joint efforts, have developed a lot of new coated fertilizers and new coating method, to raising blood compatibility, promote blood perfusion and play important function.
But because the coatings such as pyroxylin are thinner, inevitably in use occur that disintegration comes off, produce particulate, add the Blood occlusion in Clinical practice process and blood coagulation risk.Domesticly report is had to point out collodion coating blood perfusion device HCT(hematocrit value) non-limiting increases and synchronous with decrease of platelet, illustrates that hemosorber blood compatibility is poor; Invest the WBC(leucocyte of vascular wall) can dissociate in blood, the quick transient rising of inflammatory factor, in body during application, because biocompatibility missionary society impels inflammatory factor Fast back-projection algorithm and the release of various cell, coated film aperture significantly reduces absorber to medium molecular substance adsorption capacity without unified specific aim, and this also may be another reason.In addition, film-coating technique generally can block the duct (and its duct is for adsorb cell factor design) of adsorbent, thus reduces adsorbent to the adsorption capacity of cell factor.And the ether not easy-clear in the solvent used when preparing coating, harmful.
The existence of change to sepsis patient of endotoxin and cell factor (as tumor necrosis factor TNF-alpha, interleukins IL-1, IL-6) level plays an important role.Therefore, the specific adsorption effect of adsorbent induced by endotoxin and cell factor seems particularly important.
But at present and for the research of endotoxin absorbent, great majority just study the absorption research of its induced by endotoxin molecule, and do not have adsorbent to the Study on adsorption properties of cell factor.Such as, in Japanese patent application publication No. 1-16389, polymyxins is fixed in suitable carrier and makes endotoxin absorbent.Japanese patent application publication No. 3-35974 discloses basic nitrogen function base in a kind of adsorbent and polymyxins is fixed on polyvinyl.This adsorbent by polymyxins aglucon task decomposition granularity, and allows basic nitrogen function base to adsorb endotoxin catabolite.But the part run preparing this adsorbent is very expensive, as polymyxins is expensive.Japanese Laid-Open Patent publication number 6-211900 discloses a kind of sorbing material, and polyanionic polymer is fixed to a carrier (as cellular glass, silica gel scribbles porous organic polymer, crosslinked carbohydrate) and has suitable aperture and the excluded ranges of molecule.But it is less that this sorbing material adsorbs endotoxic ability.And the existence of the change of cytokine levels to sepsis patient plays an important role, therefore, adsorbent induced by endotoxin molecule and cell factor have adsorption removal effect simultaneously and seem particularly important.
Summary of the invention
For solving the problem, the present invention selects absorbent resin, the synthetic route of adsorbent is improved simultaneously, prepare residual few, little to human body side effect, there is good blood compatibility also has good adsorbance and the clearance rate simultaneously endotoxin absorbent for blood perfusion to cell factor, endotoxin.
The solution that the present invention solves its technical problem is: for the preparation method of the endotoxin absorbent of blood perfusion, comprise the following steps,
Prepare porous carrier; Described carrier is polystyrene divinylbenzene resin or plexiglass, and the aperture of described resin is 5 ~ 50nm, specific area>=500m
2/ g;
Epoxidation modification is carried out to the residual double bonds of vector resin;
Immobilized aglucon.
In order to improve the blood compatibility of polystyrene divinylbenzene resin, as the further improvement of such scheme, can increase the step of graft modification, particularly, a kind of preparation method of the endotoxin absorbent for blood perfusion, comprises the following steps:
Prepare porous carrier; Described carrier is polystyrene divinylbenzene resin, and the aperture of described resin is 5 ~ 50nm, specific area>=500m
2/ g;
Graft modification is carried out to vector resin;
Epoxidation modification is carried out to the residual double bonds of the modified vector resin of grafting;
Immobilized aglucon.
In order to improve adsorption efficiency, as the further improvement of such scheme, can also increase the step of adding linking arm, particularly, a kind of preparation method of the endotoxin absorbent for blood perfusion, comprises the following steps:
Prepare porous carrier; Described carrier is polystyrene divinylbenzene resin, and the aperture of described resin is 5 ~ 50nm, specific area>=500m
2/ g;
Epoxidation modification is carried out to the residual double bonds of vector resin;
Carrier adds linking arm;
Immobilized aglucon.
Or a kind of preparation method of the endotoxin absorbent for blood perfusion, comprises the following steps:
Prepare porous carrier; Described carrier is polystyrene divinylbenzene resin, and the aperture of described resin is 5 ~ 50nm, specific area>=500m
2/ g;
Graft modification is carried out to vector resin;
Epoxidation modification is carried out to the residual double bonds of the modified vector resin of grafting;
Vector resin after epoxidation modification adds linking arm;
Immobilized aglucon.
By adding linking arm, effectively can improve the locomitivity of aglucon, and the hydrophobic effect of linking arm can be utilized to strengthen the adsorption selection ability of induced by endotoxin molecule.
The polystyrene divinylbenzene resin used in such scheme can be prepared by the divinylbenzene suspension polymerisation of purity >=78wt%; Plexiglass also can be prepared by suspension polymerisation.
Carry out for the ease of epoxidation reaction, the more resin of preferred residual double bonds content is as carrier, and the resin as content >=1.0 mmol/g that can adopt residual double bonds carries out epoxidation modification.
Aglucon can be the aglucon being usually used in endotoxin absorbent, and preferably, described aglucon is one or both the combination in polylysine, glycine, arginine, aerosporin, polymine, hexamethylene diamine, human serum albumins; Further preferably, be polylysine, polylysine and glycine, arginine, aerosporin, polymine, hexamethylene diamine or human serum albumins; Further, under the condition of pH=10 ~ 14, introduce polylysine, polylysine and glycine.
Epoxidation utilizes the residual double bonds on resin to carry out, and to improve epoxy group content, increases ligand content for carrying out follow-up series reaction.Preferably, epoxy group content >=0.7mmol/g after described epoxidation modification, preferably, adopts metachloroperbenzoic acid or hydrogen peroxide to carry out epoxidation modification.
Preferably, described graft modification be adopt in vinylacetate, vinyl pyrrolidone, hydroxyethyl methacrylate and methylacryoyloxyethyl Phosphorylcholine one or more graft modification is carried out to vector resin.
Preferably, described interpolation linking arm is the vector resin after first introducing diamines activation epoxidation modification, and then adds linking arm, and described linking arm has the group that can react with amino, to be combined with aglucon.Further preferably, described linking arm is butanedioic acid or glutaraldehyde.
In addition, in above-mentioned preparation method, in order to prevent the residual of the active epoxy radicals of unreacted, in perfusing course, may risk be there is to human body, also comprise the step of closed residual epoxy base.
Present invention also offers a kind of endotoxin absorbent for blood perfusion prepared by said method.
The invention has the beneficial effects as follows: the present invention program is by changing synthesis route, adsorbent harmful substance is avoided to remain, particularly, the residual double bonds of polymeric adsorbent is first utilized to carry out epoxidation modification to it, improve epoxy base unit weight, then utilize epoxy radicals to add hydrophobic linking arm, improve aglucon locomitivity, thus make adsorbent have good Endotoxin adsorption effect; And control the aperture of resin and specific area to ensure that adsorbent is to the adsorbance of cell factor and clearance rate.
The present invention utilizes the residual double bonds of resin to carry out epoxidation activation to it, effectively can improve the reactivity of resin.The such as conventional hydroxyl on resin that utilizes carries out epoxidation with epichlorohydrin reaction, epoxide modified epoxy group content only has hundreds of μm ol/g, the epoxy group content that this law obtains significantly improves, and even up to more than 1.0mmol/g, can increase ligand content to carry out follow-up series reaction.
Further selection induced by endotoxin has the aglucon of specific effect to strengthen the absorption of adsorbent induced by endotoxin, thus endotoxic object in removing blood of human body can be reached, and polylysine, glycine, arginine and human serum albumins etc. are harmless, and the absorption of polylysine and glycine induced by endotoxin has synergy.
Further, p-poly-phenyl ethene divinyl benzene adsorbent carries out graft modification, plays the object improving blood compatibility.Improved the hydrophily of polystyrene divinylbenzene by grafting hydrophilic monomer, and then improve the blood compatibility of polymeric adsorbent, make its hemolysis rate be reduced to less than 1%, far below national standard require lower than 5%.
Further, to the vector resin carried out after epoxidation modification, add linking arm, improve adsorbent performance further.
Detailed description of the invention
Clear, complete description is carried out, to understand object of the present invention, characteristic sum effect fully below with reference to the technique effect of embodiment to design of the present invention, concrete structure and generation.Obviously; described embodiment is a part of embodiment of the present invention, instead of whole embodiment, based on embodiments of the invention; other embodiments that those skilled in the art obtains under the prerequisite not paying creative work, all belong to the scope of protection of the invention.
Embodiment 1
The preparation process of the polymethacrylates adsorbent of the present embodiment is as follows:
1-1 polymethacrylates adsorbent
1-1-1 prepares porous carrier;
Prepare plexiglass, obtained by suspension polymerisation, aperture 5 ~ 50nm, specific area 600m
2/ g, residual double bonds content 1.2mmol/g.
1-1-2 epoxidation modification
Get plexiglass 10g, add the dichloroethanes soaked overnight of 40mL, then dropwise add the dichloroethane solution of 20mL 1% metachloroperbenzoic acid, (temperature controls at 0 ~ 4 DEG C) stirring reaction 24h, rotating speed 200rpm in ice-water bath.Rear alcohol, purified water cleaning are reacted; Obtain the plexiglass of epoxidation modification, test obtains the epoxy group content 0.7mmol/g of resin.
The immobilized aglucon of 1-1-3 (polylysine and glycine)
Get the aqueous solution 60mL that 1-1-2 resin 10g adds 1.0g polylysine, 0.1g glycine, in 40 DEG C of reactions 24 hours.Rear alcohol, injection water cleaning are reacted.
1-1-4 closes residual epoxy base
Get the resin of 10g 1-3 respectively, add the ethanolamine solutions of 20mL 1.0 mol/L, 30 DEG C of concussion reactions 2 hours.React rear alcohol, injection water cleaning, obtain polymethacrylates adsorbent S1-1.
1-2 is with linking arm polymethacrylates adsorbent
1-2-1 prepares porous carrier;
Prepare plexiglass, obtained by suspension polymerisation, aperture 5 ~ 50nm, specific area 600m
2/ g.
1-2-2 epoxidation modification
Get plexiglass 10g, add the dichloroethanes soaked overnight of 40mL, then dropwise add the dichloroethane solution of 20mL 1% metachloroperbenzoic acid, (temperature controls at 0 ~ 4 DEG C) stirring reaction 24h, rotating speed 200rpm in ice-water bath.Rear alcohol, purified water cleaning are reacted.
1-2-3 introduces diamines
Get 1-2-2 adds 10mL dichloroethanes soaked overnight through the resin 10g of epoxidation modification, then add the dichloroethane solution of 10mL 50% hexamethylene diamine, stirring reaction 24 hours in 60 DEG C, rotating speed 200rpm.Rear alcohol, purified water cleaning are reacted.
1-2-4 adds butanedioic acid (succinic acid) linking arm
Get 1-2-3 resin 10g, add the succinic acid solution of 20mL 10%, pH is regulated to be 4.8, add 0.6g NHS (N-hydroxy-succinamide), slowly add 0.5g 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) again, in 25 DEG C, shake reaction 4 hours.Rear alcohol, purified water cleaning are reacted.
The immobilized aglucon of 1-2-5 (polylysine and glycine)
Get the aqueous solution 60mL that 1-2-4 resin 10g adds 1.0g polylysine, 0.1g glycine, in 40 DEG C of reactions 24 hours.Rear alcohol, injection water cleaning are reacted.
1-2-6 closes residual epoxy base
Get the resin of 10g 1-2-5 respectively, add the ethanolamine solutions of 20mL 1.0 mol/L, 30 DEG C of concussion reactions 2 hours.React rear alcohol, injection water cleaning, obtain polymethacrylates adsorbent S1-2.
Embodiment 2
The preparation process of the polystyrene divinylbenzene resin sorbent of the present embodiment is as follows:
2-1 prepares porous carrier;
Prepare polystyrene divinylbenzene resin, use the divinylbenzene that purity is 80wt%, prepared by suspension polymerisation, the resin aperture 5 ~ 50nm obtained, specific area 800m
2/ g, residual double bonds content 2.1mmol/g.
2-2 epoxidation modification
Get the dichloroethanes soaked overnight that 10g polystyrene divinylbenzene resin adds 40mL, dropwise add the dichloroethane solution of 20mL 1% metachloroperbenzoic acid, (temperature controls at 0 ~ 4 DEG C) stirring reaction 24 hours in ice-water bath, rotating speed 200rpm.Rear alcohol, purified water cleaning are reacted.Obtain the plexiglass of epoxidation modification, test obtains the epoxy group content 1.3mmol/g of resin.
The immobilized aglucon of 2-3
The immobilized polylysine of 2-3-1
Get the aqueous solution 20mL that 2-2 resin 10g adds 1.0g polylysine, regulate pH=12, in 60 DEG C of reactions 24 hours.React rear alcohol, injection water cleaning, obtained adsorbent, be designated as S2-1.
The immobilized glycine of 2-3-2
Get the aqueous solution 20mL that 2-2 resin 10g adds 1.0g glycine, regulate pH=12, in 60 DEG C of reactions 24 hours.React rear alcohol, injection water cleaning, obtained adsorbent, be designated as S2-2.
2-3-3 immobilized (polylysine and glycine)
Get the aqueous solution 20mL that 2-2 resin 10g adds 1.0g polylysine and 0.2g glycine, regulate pH=12, in 60 DEG C of reactions 24 hours.React rear alcohol, injection water cleaning, obtained adsorbent, be designated as S2-3.
Embodiment 3
The preparation process of the polystyrene divinylbenzene resin sorbent of the present embodiment is as follows:
3-1 prepares porous carrier
Prepare polystyrene divinylbenzene resin, use the divinylbenzene that purity is 80wt%, prepared by suspension polymerisation, the resin aperture 5 ~ 50nm obtained, specific area 800m
2/ g, residual double bonds content 2.2mmol/g.
3-2 graft modification
3-2-1 grafting vinylacetate
Get 10g polystyrene divinylbenzene resin, add 95% alcoholic solution 50mL, the dibenzoyl peroxide 0.12g of 1.5% vinylacetate.Start speed of agitator 200rpm.React 4 hours in 70 DEG C.Rear alcohol, purified water cleaning are reacted.
3-2-2 grafting vinyl pyrrolidones and hydroxyethyl methacrylate
Get 10g polystyrene divinylbenzene resin, add 1% vinyl pyrrolidone, 95% alcoholic solution 50mL of 1% hydroxyethyl methacrylate, dibenzoyl peroxide 0.09g.Start speed of agitator 200rpm.React 6 hours in 60 DEG C.Rear alcohol, purified water cleaning are reacted.
3-2-3 grafted methacrylic acid hydroxyl ethyl ester
Get 10g polystyrene divinylbenzene resin, add 95% alcoholic solution 50mL, the dibenzoyl peroxide 0.07g of 1.5% hydroxyethyl methacrylate.Start speed of agitator 200rpm.React 4 hours in 60 DEG C.Rear alcohol, purified water cleaning are reacted.
3-2-4 grafting methylacryoyloxyethyl Phosphorylcholine
Get 10g polystyrene divinylbenzene resin, add 95% alcoholic solution 50mL, the azodiisobutyronitrile 0.08g of 1.2% methylacryoyloxyethyl Phosphorylcholine.Start speed of agitator 200rpm.React 4 hours in 60 DEG C.Rear alcohol, purified water cleaning are reacted.
3-3 epoxidation modification
3-3-1 metachloroperbenzoic acid epoxidation
Get the dichloroethanes soaked overnight that 3-2-1 resin adds 40mL, dropwise add the dichloroethane solution of 20mL 1% metachloroperbenzoic acid, (temperature controls at 0 ~ 4 DEG C) stirring reaction 24 hours in ice-water bath, rotating speed 200rpm.Rear alcohol, purified water cleaning are reacted.
3-3-2 metachloroperbenzoic acid epoxidation
Get the dichloroethanes soaked overnight that 3-2-2 resin adds 40mL, dropwise add the dichloroethane solution of 20mL 1% metachloroperbenzoic acid, (temperature controls at 0 ~ 4 DEG C) stirring reaction 24 hours in ice-water bath, rotating speed 200rpm.Rear alcohol, purified water cleaning are reacted.
3-3-3 is hydrogen peroxide epoxidizing
Get the dichloroethanes soaked overnight that 3-2-3 resin adds 40mL, then add 5mL 30% hydrogen peroxide and 5g 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) stirring reaction 4 hours in 60 DEG C, rotating speed 200rpm.Rear alcohol, purified water cleaning are reacted.
3-3-4 is hydrogen peroxide epoxidizing
Get the dichloroethanes soaked overnight that 3-2-4 resin adds 40mL, then add 5mL 30% hydrogen peroxide and 5g 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) stirring reaction 4 hours in 60 DEG C, rotating speed 200rpm.Rear alcohol, purified water cleaning are reacted.
The immobilized aglucon of 3-4
The immobilized arginine of 3-4-1
Get 3-3-1 resin 10g and add the arginic aqueous solution 60mL of 0.8g, in 40 DEG C of reactions 24 hours.Rear alcohol, injection water cleaning are reacted.
The immobilized aerosporin of 3-4-2
Get the aqueous solution 60mL that 3-3-1 resin 10g adds 2.1g aerosporin, in 40 DEG C of reactions 24 hours.Rear alcohol, injection water cleaning are reacted.
The immobilized polymine of 3-4-3
Get the aqueous solution 60mL that 3-3-2 resin 10g adds 2.4g polymine, in 40 DEG C of reactions 24 hours.Rear alcohol, injection water cleaning are reacted.
The immobilized hexamethylene diamine of 3-4-4
Get the aqueous solution 60mL that 3-3-3 resin 10g adds 1.7g hexamethylene diamine, in 40 DEG C of reactions 24 hours.Rear alcohol, injection water cleaning are reacted.
The immobilized human serum albumins of 3-4-5
Get the aqueous solution 60mL that 3-3-4 resin 10g adds 0.8g human serum albumins, in 40 DEG C of reactions 24 hours.Rear alcohol, injection water cleaning are reacted.
3-5 closes residual epoxy base
Get the resin of 10g 3-4-1,3-4-2,3-4-3,3-4-4,3-4-5 respectively, add the ethanolamine solutions of 10mL 1.0 mol/L, 30 DEG C of concussion reactions 2 hours.React rear alcohol, injection water cleaning, obtain adsorbent S3-1, S3-2, S3-3, S3-4, S3-5 respectively.
Embodiment 4
The preparation process of the polystyrene divinylbenzene resin sorbent of the present embodiment is as follows:
The selection of vector resin, grafting and epoxidised step are with embodiment 3.
4-4 introduces diamines
Get 3-3-1 adds 10mL dichloroethanes soaked overnight through the resin 10g of epoxidation modification, then add the dichloroethane solution of 10mL 50% hexamethylene diamine, stirring reaction 24 hours in 60 DEG C, rotating speed 200rpm.Rear alcohol, purified water cleaning are reacted.
4-5 adds linking arm
4-5-1 butanedioic acid (succinic acid) linking arm
Get 4-4 resin 10g, add the succinic acid solution of 20mL 10%, pH is regulated to be 4 ~ 6, add 0.6g NHS (N-hydroxy-succinamide), slowly add 0.5g 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) again, in 25 DEG C, shake reaction 4 hours.Rear alcohol, purified water cleaning are reacted.
4-5-2 glutaraldehyde linking arm
Get 4-4 resin, add the sodium bicarbonate solution of 20mL pH=8.0 ± 1.0, dropwise add the glutaraldehyde solution 5mL of 50%, in 30 DEG C, shake reaction 2 hours.Rear alcohol, purified water cleaning are reacted.
The immobilized aglucon of 4-6
The immobilized arginine of 4-6-1
Get 4-5-1 resin 10g and add the arginic solution 30mL of 0.7g, regulate pH to be 5.0, then add 0.6g NHS (N-hydroxy-succinamide), 0.5g EDC, in 30 DEG C of reactions 24 hours.Rear alcohol, injection water cleaning are reacted.
The immobilized aerosporin of 4-6-2
Get the solution 30mL that 4-5-1 resin 10g adds 1.8g aerosporin, regulate pH to be 5.0, then add 0.6g NHS (N-hydroxy-succinamide), 0.5g EDC, in 30 DEG C of reactions 24 hours.Rear alcohol, injection water cleaning are reacted.
The immobilized polymine of 4-6-3
Get the solution 30mL that 4-5-1 resin 10g adds 2.6g polymine, regulate pH to be 5.0, then add 0.6g NHS (N-hydroxy-succinamide), 0.5g EDC, in 30 DEG C of reactions 24 hours.Rear alcohol, injection water cleaning are reacted.
The immobilized hexamethylene diamine of 4-6-4
Get the solution 30mL that 4-5-1 resin 10g adds 1.2g hexamethylene diamine, regulate pH to be 5.0, then add 0.6g NHS (N-hydroxy-succinamide), 0.5g EDC, in 30 DEG C of reactions 24 hours.Rear alcohol, injection water cleaning are reacted.
The immobilized human serum albumins of 4-6-5 and arginine
Get 4-5-2 resin 10g and add 0.5g human serum albumins and the arginic aqueous solution 30mL of 0.5g, in 40 DEG C of reactions 24 hours.Rear alcohol, injection water cleaning are reacted.
4-7 closes residual epoxy base
Get the resin of 10g 4-6-1,4-6-2,4-6-3,4-6-4,4-6-5 respectively, add the ethanolamine solutions of 20mL 1.0 mol/L, 30 DEG C of concussion reactions 2 hours.React rear alcohol, injection water cleaning, obtain adsorbent S4-1, S4-2, S4-3, S4-4, S4-5 respectively.
Embodiment 5
5-1 adsorbs endotoxin experiment
The endotoxin concns of endotoxemia patient is generally less than 1EU/mL, and experiment setting absorption initial concentration is 1EU/mL.Get without thermal source test tube, add obtained adsorbent 0.05g respectively, add the blood plasma 1.5mL containing endotoxin 1EU/mL again, concussion absorption 2 hours (temperature 37 DEG C, concussion speed 100 ± 10rpm), then measure the rear endotoxin concns of absorption with chromogenic substrate tachypleus amebocyte lysate box, and calculate adsorbent to its adsorbance and clearance rate, shown in result table 1.
5-2 adsorb cell factor is tested
Get apyrogeneity test tube, add adsorbent 0.05g obtained in embodiment 1-4 respectively, add factor-containing interleukin-6 (IL-6) 13.40pg/mL again, the blood plasma 1.5mL of TNF (TNF-α) 31.80 pg/mL, concussion absorption 2 hours (temperature 37 DEG C, concussion speed 100 ± 10rpm), then IL-6 is detected, TNF-α concentration, and calculate adsorbent to its adsorbance and clearance rate, result is the clearance rate >70%(300 pg/g of S1 ~ S12 adsorbent to IL-6), clearance rate >50%(500pg/g to TNF-α).
Can be found out by test result, adsorbent in the embodiment of the present invention all has good Endotoxin removal rate, also has good clearance rate, further to cell factor simultaneously, according to the result of table 1, obtain adsorbent in comparative example 3 and 4, especially contrast S3-1 vs.S3-2, the adsorption effect of S3-2 vs. S4-2, can find out, by introducing diamines, after increasing linking arm, the clearance rate of the adsorbent induced by endotoxin of immobilized different aglucon is all significantly increased.And contrast S2-1, S2-2, S2-3, can find out that the absorption of combination and polylysine and glycine combination induced by endotoxin between aglucon has synergy, higher adsorption effect can be reached.
Embodiment 6
Hemolytic experiment
Hemolytic experiment is carried out according to " GB/T16886.4-2003 BiologicalEvaluationofMedicalDevice the 4th part and blood interact to test and selects ", " GB/T16175-2008 medical organic silicon material biological assessment test method ".
Sample thief group often pipe adds adsorbent 5g obtained in embodiment 1-4, then adds sodium chloride injection 10ml; Negative control group often pipe adds sodium chloride injection 10ml; Positive controls often pipe adds distilled water 10ml.Often organize operation repetitive 3 to manage.After water bath with thermostatic control (37 ± 1) DEG C insulation 30min put into by whole test tube, often prop up test tube and add 0.2ml dilution rabbit blood, mix gently, put (37 ± 1) DEG C warm 60 min of water-bath relaying continuation of insurance.Pour out liquid in pipe with centrifugal 5 min of 800g.Aspirate supernatant moves in cuvette, measures absorbance with spectrophotometer at 545nm wavelength place.Sample combination control group absorbance all gets the mean value of 3 pipes.The absorbance of negative control pipe should be not more than 0.03, and the absorbance of positive control pipe should be 0.8 ± 0.3, otherwise should again test.
Hemolysis rate=(A-B)/(C-B) × 100%
Wherein A---sample sets absorbance;
B---negative control group absorbance;
C---positive controls absorbance.
Result obtains the hemolysis rate of adsorbent in embodiment 1-4 and is less than 1%, far below national standard require lower than 5%.For the plexiglass adsorbent S1-1 in embodiment 1, hemolysis rate 0.75 ± 0.2%.For polystyrene divinylbenzene resin sorbent, the S2-3 adsorbent hemolysis rate 3.71 ± 0.3% in embodiment 2, after grafting, in embodiment, the hemolysis rate of the resin of S3-1 is 0.45 ± 0.2%.Visible, before and after graft modification, adsorbent hemolysis rate has clear improvement, and illustrates that graft modification can well improve the blood compatibility of absorbent resin.
Embodiment 7
Blood compatibility is tested
Get each 1mL wet resin of adsorbent, load after physiological saline soaks 10 hours in perfusion device, inject 10mL through the rabbit whole blood of liquaemin anti-freezing with syringe, with the flow velocity perfusion 2 hours of 50mL/min, add an empty perfusion device simultaneously and carry out control experiment.The change of each component of blood before and after perfusion is measured through Beckman LH750 cellanalyzer.
Result shows, adsorbent in embodiment of the present invention 1-4, before and after perfusion, in blood, the change of each key component is little, and the percentage of decline is all within 5%, show that the adsorbent of the embodiment of the present invention has good blood compatibility thus, there is the prospect that can be applicable to whole blood perfusion.
Above better embodiment of the present invention is illustrated, but the invention is not limited to described embodiment, those of ordinary skill in the art also can make all equivalent modifications or replacement under the prerequisite without prejudice to spirit of the present invention, and these equivalent modification or replacement are all included in the application's claim limited range.
Claims (10)
1. for the preparation method of the endotoxin absorbent of blood perfusion, it is characterized in that, comprise the following steps:
Prepare porous carrier; Described carrier is polystyrene divinylbenzene resin or plexiglass, and the aperture of described resin is 5 ~ 50nm, specific area>=500m
2/ g;
Epoxidation modification is carried out to the residual double bonds of vector resin;
Immobilized aglucon.
2. preparation method according to claim 1, is characterized in that, described carrier is polystyrene divinylbenzene resin, and carrying out step, " to carry out epoxidation modification to the residual double bonds of vector resin " front, carries out graft modification to vector resin.
3. preparation method according to claim 2, is characterized in that, after carrying out epoxidation modification to the residual double bonds of vector resin, adds linking arm, then immobilized aglucon at vector resin.
4. preparation method according to claim 1, is characterized in that, carrying out, step " immobilized aglucon " is front, adds linking arm at vector resin.
5. the preparation method according to any one of claim 1-4, is characterized in that: described aglucon is one or both the combination in polylysine, glycine, arginine, aerosporin, polymine, hexamethylene diamine, human serum albumins.
6. the preparation method according to any one of claim 1-4, is characterized in that: adopt metachloroperbenzoic acid or hydrogen peroxide to carry out epoxidation modification.
7. the preparation method according to Claims 2 or 3, is characterized in that: described graft modification be adopt in vinylacetate, vinyl pyrrolidone, hydroxyethyl methacrylate or methylacryoyloxyethyl Phosphorylcholine one or more graft modification is carried out to carrier.
8. the preparation method according to claim 3 or 4, is characterized in that: described interpolation linking arm is the vector resin after first introducing diamines activation epoxidation modification, and then adds linking arm.
9. preparation method according to claim 8, is characterized in that: described linking arm is butanedioic acid or glutaraldehyde.
10. one kind adopts the endotoxin absorbent for blood perfusion that described in aforementioned any one of claim prepared by method.
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