CN102671637B - Biomimetic specific immune adsorption material with PAMAM (Polyamidoamine) as spacer arm, and preparation method and application thereof - Google Patents
Biomimetic specific immune adsorption material with PAMAM (Polyamidoamine) as spacer arm, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a biomimetic specific immune adsorption material with PAMAM (Polyamidoamine) as a spacer arm, and a preparation method and the application thereof. The preparation process comprises the following steps of: firstly, obtaining sepharose gel azide by reaction of agarose epoxide and sodium azide; preparing a series of PAMAM dendrimer-like polymers in different generations by repeated addition reaction with methyl acrylate and amination reaction with ethidene diamine; after preparing amino acid methyl ester hydrochloride with the amino acid as the raw material, modifying the PAMAM dendrimer-like polymers with the amino acid methyl ester hydrochloride to prepare the amino acid modified PAMAM; coupling the amino acid modified PAMAM with sepharose gel azide through a click reaction so as to obtain the biomimetic specific immune adsorption material with PAMAM as the spacer arm. The material disclosed in the invention is prepared by simple synthetic steps, and the preparation is safe; products have the characteristics of strong specificity, high adsorbing efficiency to the immunoglobulin and excellent regeneration performance, and can be used for IgG (immunoglobulin G) separation and clinical immune adsorption treatment.
Description
Technical field
The invention belongs to biomedical devices, the preparation method of a kind of blood purification with bionical thing specific immunity sorbing material is provided especially, such sorbing material is take polyamide-amine type branch-shape polymer (PAMAM) as spacerarm.
Technical background
As everyone knows, with albumin A, G etc. have good biologic specificity avidity for the protein immune absorption material of aglucon to immunoglobulin (Ig) (IgG), and the reactive force between protein aglucon and IgG relates to hydrophobic interaction and electrostatic interaction.Can remove the autoantibody antibody purification and treat various autoimmune disorders thereby the immune absorption material that contains this class biologic specificity aglucon has proved, thereby at biologically pure and being widely applied clinically.Yet there are some defectives that can't ignore in the protein aglucon: protein is difficult to stand harsh sterilizing process and more easily come off from carrier, and these aglucons that come off can cause safety problem; Protein immune absorption material fancy price has caused heavy economical load to the patient; The protein aglucon is difficult to carry out chemical functional group's modification, thereby can not be coupled on the carrier with only angle.In order to address these problems, a collection ofly can be have optionally obtained widely research and development in conjunction with the imitative protein-based small molecules aglucon of IgG.For example, these classes such as amino acid, polypeptide, dyestuff, metal ion have the small molecules of degree of physical chemical stability and nominal price, can be used for as the substitute of protein aglucon absorption IgG.In these biomimetic ligands, amino acid is because it is nontoxic, and is inexpensive and replaced protein to be used for the affinity chromatography aspect to the highly selective of IgG.There is report proof amino acid from human plasma or serum, to isolate IgG efficiently.
In the preparation method of immune absorption material commonly used, aglucon is coupled on the carrier by reactive groups such as amino, hydroxyl, carboxyl, aldehyde radicals.But when having too much chemical functional group on the aglucon or have the group that other can participate in linked reaction, the selectivity that carrier and aglucon are used between the functional group of linked reaction can lower, thereby the avidity of aglucon is affected.Therefore, increase carrier and the selectivity of aglucon when linked reaction and just seem particularly important.If it is inertia to other group that a pair of functional group has very high reaction preference and they to each other, so such functional group is to just greatly improving the preparation efficiency of immune absorption material.And azido-and alkynyl such functional group pair just, the Huisgen1 of the two, the 3-Dipolar Cycloaddition is most widely used in the click chemistry reaction.Cu (the I)-azido-of catalysis and the cycloaddition reaction of alkynyl have highly selective, and are inertia to a lot of other chemical functional groups, and (solvent, temperature, pH etc.) can both keep stable under very wide reaction conditions.These features are so that click chemistry is demonstrating unique advantage, for example preparation of affinity chromatography reagent aspect the biological lotus root connection.Based on this, there is the commercial amination agarose of human to prepare the sepharose ball of azide and alkynyl, successfully be used for the affinity chromatography aspect after using again the carrier of this " can click " and aglucon coupling.Undoubtedly, in order to develop safe, effective and cheap immune absorption material, the carrier and the aglucon that prepare similar " can click " with common agarose and cheap aglucon are particularly important.
If aglucon is to be bonded directly to carrier surface fixedly the time, then can cause avidity to descend even forfeiture owing to sterically hindered.Therefore, aglucon and target molecule are formed the impact of mixture time space steric hindrance for reducing carrier, increase the mobility of aglucon and the probability that combines with target molecule, usually need to be between carrier and aglucon covalent bonding the preceding paragraph spacerarm, especially when aglucon be small molecules and purifying object when being macromole, the introducing of spacerarm is just more necessary.The spacerarm that immune absorption material is commonly used is linear spacerarm, and such spacerarm can twine with biomacromolecule when the separating bio macromole, thereby reduces the effect of immunosorption.
Summary of the invention
The first purpose of the present invention provides a kind of safe, with low cost, the bionical thing specific immunity sorbing material take PAMAM as spacerarm stable and reliable for performance.The present invention's the second purpose is to provide the preparation method of the bionical thing specific immunity sorbing material take PAMAM as spacerarm.The present invention's the 3rd purpose is to provide the application of such bionical thing specific immunity sorbing material take PAMAM as spacerarm in IgG separates.
The present invention is take sepharose as carrier, amino acid is biomimetic ligands, adopt the click chemistry design and prepare a kind of immune absorption material take polyamide-amide dendrimer material (PAMAM) as spacerarm, IgG had highly selective, and with low cost, stable and reliable for performance, effect is suitable with the albumin A sorbing material.Can overcome the shortcoming of linear spacerarm take branch-shape polymer as spacerarm and increase carrier to the supported quantity of aglucon.Polyamidoamine dendrimers had both had the general character of dendrimer, it is take quadrol as core that self-character: PAMAM is arranged again, was undertaken by Michael addition and amidation condensation reaction, and the productive rate of whole reaction is very high; Connect PAMAM type dendritic macromole at gel carrier, the advantage that takes full advantage of its a large amount of avtive spots is carried out the immobilization of aglucon, the outside surface of the carrier of the carrier antithesis on line shape spacerarm behind this dendroid spacerarm of coupling has more avtive spot, be easier to the bonding of aglucon, greatly improve the immobilized efficient of aglucon, increase the adsorptive power of immune absorption material.Sepharose has good chemical stability and physical strength, and protein is chemical inert, and its wetting ability and blood compatibility are also relatively good, activates easily and can carry out under relatively gentle, simpler condition the immobilized reaction of aglucon.
Purpose of the present invention is achieved through the following technical solutions:
A kind of bionical thing specific immunity sorbing material take PAMAM as spacerarm has the chemical structure of following Sep-PAMAM-AA:
Wherein, Sep represents sepharose; The AA represented amino acid; N=0,1,2 or 3; PAMAM is polyamide-amide type dendrimer, and described amino acid is L-Histidine, phenylalanine or tryptophane.
The preparation method of described bionical thing specific immunity sorbing material comprises the steps and processing condition:
(a) preparation of azide sepharose
The aqueous solution that adds sodiumazide in the epoxidation agarose adds sodiumazide 3～15mmol in every gram epoxidation agarose, behind 20～60 ℃ of reaction 6～60h, product is drained to get in washing;
(b) the PAMAM branch-shape polymer is synthetic
Be under-10～10 ℃ of conditions in temperature, drip propargylamine in the methanol solution of methyl acrylate, the mol ratio of methyl acrylate and propargylamine is 5～15:1, behind insulation reaction 0.5～3h, be warming up to 20～50 ℃ of reaction 12～50h, reaction product is carried out underpressure distillation, get G0.5PAMAM; Under-10～10 ℃, drip G0.5PAMAM in the methanol solution of quadrol, the mol ratio of quadrol and G0.5PAMAM is 10～30:1, behind insulation reaction 0.5～3h, be warming up to 20～50 ℃ of reaction 12～50h, reaction product carried out underpressure distillation get first-generation product G1.0PAMAM;
Be under-10～10 ℃ of conditions in temperature, in the methanol solution of methyl acrylate, drip G1.0PAMAM, the mol ratio of methyl acrylate and G1.0PAMAM is 10～30:1, behind insulation reaction 0.5～3h, be warming up to 20～60 ℃ of reaction 15～60h, reaction product is carried out underpressure distillation, get G1.5PAMAM; Under-10～10 ℃, drip G1.5PAMAM in the methanol solution of quadrol, the mol ratio of quadrol and G1.5PAMAM is 30～50:1, behind insulation reaction 0.5～3h, be warming up to 20～60 ℃ of reaction 15～60h, reaction product carried out underpressure distillation get s-generation product G2.0PAMAM;
Be under-10～10 ℃ of conditions in temperature, in the methanol solution of methyl acrylate, drip G2.0PAMAM, the mol ratio of methyl acrylate and G2.0PAMAM is 30～50:1, behind insulation reaction 0.5～3h, be warming up to 20～70 ℃ of reaction 18～66h, reaction product is carried out underpressure distillation, get G2.5PAMAM; Under-10～10 ℃, drip G2.5PAMAM in the methanol solution of quadrol, the mol ratio of quadrol and G2.5PAMAM is 70～90:1, behind insulation reaction 0.5～3h, be warming up to 20～70 ℃ of reaction 18～66h, reaction product carried out underpressure distillation get third generation product G3.0PAMAM;
Be under-10～10 ℃ of conditions in temperature, in the methanol solution of methyl acrylate, drip G3.0PAMAM, the mol ratio of methyl acrylate and G3.0PAMAM is 70～90:1, behind insulation reaction 0.5～3h, be warming up to 20～80 ℃ of reaction 24～72h, reaction product is carried out underpressure distillation, get G3.5PAMAM; Under-10～10 ℃, drip G3.5PAMAM in the methanol solution of quadrol, the mol ratio of quadrol and G3.5PAMAM is 150～170:1, behind insulation reaction 0.5～3h, be warming up to 20～80 ℃ of reaction 24～72h, reaction product carried out underpressure distillation get the 4th generation product G4.0PAMAM;
(c) preparation of amino acid methyl ester hydrochloride
Amino acid is placed methanol solution, add trimethylchlorosilane, described trimethylchlorosilane and amino acid whose mol ratio are 1～3:1, are back to that to revolve evaporate to dryness after solution is clarified dry; Described amino acid is L-Histidine, phenylalanine or tryptophane; Described amino acid places methanol solution, and the methyl alcohol add-on is 1～3mL in every 1mmol amino acid;
(d) preparation of amino acid modified PAMAM
Amino acid methyl ester hydrochloride is soluble in water, and add in triethylamine and first～the 4th generation PAMAM product any one the aqueous solution, the mol ratio of amino acid methyl ester hydrochloride, triethylamine and first～the 4th generation PAMAM product is 1～10:1～10:1; 20～90 ℃ of reaction 24～96h under the magnetic agitation, dialysis and lyophilize get amino acid modified PAMAM product, are expressed as PAMAM-AA; Described amino acid methyl ester hydrochloride is Histidine methyl ester hydrochloride, phenylalanine methyl ester hydrochloride or tryptophan methyl ester hydrochloride; Described the first～the 4th generation PAMAM product is G1.0PAMAM, G2.0PAMAM, G3.0PAMAM and G4.0PAMAM; Described amino acid methyl ester hydrochloride is soluble in water, and every 1mmol amino acid methyl ester hydrochloride adds entry 0.25～4mL.
(e) click the standby immune absorption material take PAMAM as spacerarm of legal system
After amino acid modified PAMAM product is water-soluble, add the azide sepharose, every gram azide sepharose adds the amino acid modified PAMAM product of 1～10mmol, then add cupric sulfate pentahydrate successively and sodium ascorbate is made catalyzer, the mol ratio of cupric sulfate pentahydrate, sodium ascorbate and amino acid modified PAMAM product is 0.01～0.05:0.02～0.1:1; And in 20～90 ℃ of reaction 24～96h, target product Sep-PAMAM-AA is drained to get in washing.
Described epoxidation agarose prepares by the following method: the ratio that adds 2mL epoxy chloropropane and 1～2mL sodium hydroxide solution in every gram sepharose, sepharose, epoxy chloropropane and sodium hydroxide solution are mixed stirring reaction 3～4h under the rear room temperature, with water washing, drain until after in washings, adding 1.3M Sulfothiorine phenolphthalein do not redden, namely get the epoxidation agarose; The concentration of described sodium hydroxide solution is 1.5～3M.
The G1.0PAMAM end contains 2 amino, the structure in its chemical structure such as the general structure during n=0.
The G2.0PAMAM end contains 4 amino, the structure in its chemical structure such as the general structure during n=1.
The G3.0PAMAM end contains 8 amino, the structure in its chemical structure such as the general structure during n=2.
The G4.0PAMAM end contains 16 amino, the structure in its chemical structure such as the general structure during n=3.
Intermediate product when G0.5PAMAM, G1.5PAMAM, G2.5PAMAM and G3.5PAMAM are respectively preparation the first～the 4th generation PAMAM product G1.0PAMAM, G2.0PAMAM, G3.0PAMAM and G4.0PAMAM.
Among the present invention, aglucon is L-Histidine, phenylalanine or tryptophane, and the coupling method of carrier and aglucon is Huisgen1, the 3-Dipolar Cycloaddition.Take propargylamine as core, the PAMAM(of synthetic 1.0～4.0 generations end group ethynylation is G1.0PAMAM, G2.0PAMAM, G3.0PAMAM and G4.0PAMAM first); Again PAMAM and amino acid aglucon are combined into active compound; To be coupled to the aglucon of PAMAM spacerarm and end group ethynylation at last the agarose carrier surface preparation immune absorption material of azide.The reaction equation of each step is as follows:
(a) preparation of azide sepharose
(b) the PAMAM branch-shape polymer is synthetic
(c) preparation of amino acid methyl ester hydrochloride
(d) preparation of amino acid modified PAMAM
(e) click the standby immune absorption material take PAMAM as spacerarm of legal system
Wherein, Sep represents sepharose; The AA represented amino acid; N=0,1,2,3.
For further realizing the object of the invention, described amino acid places methanol solution, and the methyl alcohol add-on is preferably 1～3mL in every 1mmol amino acid; In the methanol solution of described methyl acrylate, the methyl alcohol add-on is preferably 0.2～1.0mL in every 1mmol methyl acrylate.
Described amino acid methyl ester hydrochloride is soluble in water, and every 1mmol amino acid methyl ester hydrochloride adds entry and is preferably 0.25～4mL.
The invention provides the application of bionical thing specific immunity sorbing material in IgG separates take PAMAM as spacerarm.
With respect to prior art, the present invention has following advantage and beneficial effect:
(1) with cheapness, safety, stablizing and having modifiable amino acid small molecules is that biomimetic ligands replaces albumin A commonly used to prepare immune absorption material, can avoid albumin A para-immunity sorbing material expensive, easily comes off the not high defective of security.And design and the modification that can suit to the structure of aglucon, make modified aglucon keep on its molecule being used for the complete of the avtive spot of being combined with the material generation specificity such as IgG and keep certain space structure.
(2) click chemistry reaction---Huisgen1 reaction conditions is gentle, efficient, highly selective is proposed; the 3-Dipolar Cycloaddition is used for the coupling of carrier-aglucon; design and prepare the Research Thinking of the immune absorption material of non-albumin A class; be expected greatly to improve the coupling effect between carrier and the aglucon; avoid the generation of side reaction; protect to greatest extent on the aglucon avtive spot to material generation specificity combinations such as IgG, for the new way with industrial prospect is explored in the preparation of the immune absorption material of non-albumin A class.
(3) take dendritic macromole PAMAM as spacerarm, make the carrier introduced behind such spacerarm have more avtive spot than the outside surface of the carrier of introducing linear spacerarm, be convenient to the more aglucon of coupling, thereby increased ligand density, improved the absorption property of prepared immune absorption material.
Above advantage shows as at clinical treatment and has improved clinical therapeutic efficacy, safety and reliability.External blood plasma adsorption test data show that bionical thing specific immunity sorbing material of the present invention has specific adsorption to immunoglobulin IgG in the human plasma.
Description of drawings
Fig. 1 is the infrared spectrogram of azide sepharose among the embodiment one.
Fig. 2 is the nucleus magnetic hydrogen spectrum figure of G3-His among the embodiment one.
Fig. 3 is the nucleus magnetic hydrogen spectrum figure of G2-Phe among the embodiment two.
Fig. 4 is the nucleus magnetic hydrogen spectrum figure of G4-Trp among the embodiment three.
Fig. 5 is the infrared spectrogram of immune absorption material among the embodiment one, two, three.
Fig. 6 is the gel electrophoresis figure of Sep-G3.0-His elutriant among the embodiment one.
Embodiment
In order to understand better the present invention, the present invention is further illustrated below in conjunction with embodiment, need to prove, embodiment is used for explanation the present invention rather than limitation of the present invention.The improvement that essence according to the present invention is carried out all belongs to the scope of protection of present invention.
Embodiment one:
The preparation of azide sepharose
In the 100mL Erlenmeyer flask, add sepharose, 8mL epoxy chloropropane and the 4mL3M sodium hydroxide solution that 4.0g drains.Mixture is at room temperature behind the stirring reaction 3h, with water washing drain until after in washings, adding 1.3M Sulfothiorine phenolphthalein do not redden, namely get the epoxidation agarose.The 1.82g sodiumazide is dissolved in 15mL water, adds again 4.0g epoxidation agarose, 20 ℃ of reaction 60h.React complete, the azide sepharose is drained to get in washing.Infrared spectrogram shown in the accompanying drawing 1 is at 2100cm
-1The charateristic avsorption band of azido-appears in the place, has proved the existence of azido-in the azide agarose.
Synthesizing of G3.0PAMAM branch-shape polymer
In the round-bottomed flask of 250ml, add first methyl alcohol 200ml, add again the 272mmol methyl acrylate, in ice-water bath, place for some time, make the temperature of system be approximately 0 ℃, then in 1h, drip continuously the methanol solution 30ml that contains the 27.2mmol propargylamine, dropwise rear continuation at 0 ℃ of lower reaction 1.5h, be warming up to afterwards 35 ℃ of reaction 36h.React complete, product is carried out underpressure distillation get G0.5PAMAM; In the round-bottomed flask of 250ml, add first methyl alcohol 100ml, add again the 510mmol quadrol, in ice-water bath, place for some time, make system temperature be approximately 0 ℃, then in 1h, drip continuously the methanol solution 30ml that contains 25.5mmol G0.5PAMAM, dropwise rear continuation at 0 ℃ of lower reaction 1.5h, be warming up to afterwards 35 ℃ of reaction 36h.React complete, product is carried out underpressure distillation get G1.0PAMAM.
In the round-bottomed flask of 250ml, add first methyl alcohol 140ml, add again the 284mmol methyl acrylate, in ice-water bath, place for some time, make the temperature of system be approximately 0 ℃, then in 1h, drip continuously the methanol solution 30ml that contains 14.2mmol G1.0PAMAM, dropwise rear continuation at 0 ℃ of lower reaction 1.5h, be warming up to afterwards 40 ℃ of reaction 42h.React complete, product is carried out underpressure distillation get G1.5PAMAM; In the round-bottomed flask of 250ml, add first methyl alcohol 100ml, add again the 500mmol quadrol, in ice-water bath, place for some time, make system temperature be approximately 0 ℃, then in 1h, drip continuously the methanol solution 30ml that contains 12.5mmol G1.5PAMAM, dropwise rear continuation at 0 ℃ of lower reaction 1.5h, be warming up to afterwards 40 ℃ of reaction 42h.React complete, product is carried out underpressure distillation get G2.0PAMAM.
In the round-bottomed flask of 250ml, add first methyl alcohol 120ml, add again the 288mmol methyl acrylate, in ice-water bath, place for some time, make the temperature of system be approximately 0 ℃, then in 1h, drip continuously the methanol solution 30ml that contains 7.2mmol G2.0PAMAM, dropwise 0 ℃ of lower reaction 1.5h of rear continuation, be warming up to afterwards 55 ℃ of reaction 54h.React complete, product is carried out underpressure distillation get G2.5PAMAM; In the round-bottomed flask of 250ml, add first methyl alcohol 70ml, add again the 440mmol quadrol, in ice-water bath, place for some time, make system temperature be approximately 0 ℃, then in 1h, drip continuously the methanol solution 30ml that contains 5.5mmol G2.5PAMAM, dropwise rear continuation at 0 ℃ of lower reaction 1.5h, be warming up to afterwards 55 ℃ of reaction 54h.React complete, product is carried out underpressure distillation.The viscous body of gained yellow is G3.0PAMAM.
The preparation of Histidine methyl ester hydrochloride
Get the 50mmol L-Histidine in the 500ml single necked round bottom flask, and add 150ml methyl alcohol and 150mmol trimethylchlorosilane.Be back to the solution becomes clarification, revolve steaming and be drying to obtain the Histidine methyl ester hydrochloride.
The preparation of the G3.0PAMAM that Histidine is modified
24mmol Histidine methyl ester hydrochloride is dissolved in 24ml water and places the 150ml single necked round bottom flask, adding 24mmol triethylamine and 22ml are dissolved with the aqueous solution of 1mmol G3.0PAMAM, 70 ℃ of successive reaction 70h under magnetic agitation, completely small molecules of unreacted is removed in the dialysis tubing dialysis, lyophilize obtains G3.0-His, and wherein His represents L-Histidine.Shown in the accompanying drawing 2
1H NMR figure shows alkynyl and the methylene radical characteristic peak of the characteristic peaks such as the methyne, methylene radical and the imidazole ring that contain simultaneously Histidine in the prepared sample and PAMAM, but and by the ratio of the integral area at each peak 6 Histidine molecules of 1 G3.0PAMAM molecule grafting as can be known.
Click the standby bionical thing specific immunity sorbing material take PAMAM as spacerarm of legal system
The aqueous solution 15ml of 4.0mmol G3.0-His is placed the 100ml single necked round bottom flask, add 1.0g azide agarose.After stirring, add the aqueous solution 2.5ml that is dissolved with the 48mg cupric sulfate pentahydrate and the aqueous solution 2.5ml that is dissolved with the 76mg sodium ascorbate.Behind 60 ℃ of successive reaction reaction 48h, the sepharose of gained is drained in water, EDTA and water washing successively, namely gets immune absorption material Sep-G3.0-His.Infrared spectrogram shown in the accompanying drawing 5 shows that immune absorption material take Histidine as aglucon is at 2100cm
-1The nitrine peak completely dissolve at place illustrates that the azido-on the azide agarose reacts completely, and successfully synthesizes target product in conjunction with Fig. 1 and Fig. 2 proof by click-reaction.
The research of immune absorption material loading capacity
Used blood plasma thaws under 37 ℃, adds then that to use 0.02M, pH be the immune absorption material sample that 7.0 phosphoric acid buffer balance is crossed, 25 ℃ of whip attachment 2h.Then the sepharose washing is drained, use 0.02M again, pH is 2.5 citrate buffer solution wash-out.Get supernatant liquid behind the 2h and survey its absorption in 200～500nm wavelength region, the wherein absorbance A at 280nm place with ultraviolet spectrophotometer
280The concentration of IgG is washed * A by formula Q=(M in the corresponding elutriant
280)/(1.35 * M fine jade) calculate the amount of the IgG that every gram immune absorption material adsorbs, wherein Q is the loading capacity of agarose, and M washes the quality into elutriant, and the M fine jade is the quality of used agarose, the molar absorptivity of 1.35 expression IgG.Test result shows, A
280, M washes, the M fine jade is respectively 4.115,6.015g, 0.652g, thereby calculate to such an extent that the made bionical thing specific immunity sorbing material Sep-G3.0-His of present embodiment can reach 28.12mg/g to the loading capacity of IgG in the human plasma, be better than protein A immunoadsorption material (22.97mg/g).This is because it is can greatly improve the supported quantity of aglucon behind the spacerarm that Sep-G3.0-His adopts dendritic macromole, thereby its loading capacity is correspondingly improved.
The research of immune absorption material adsorption selectivity
Sodium lauryl sulphate-polyacrylamide gel electrophoresis (SDS – PAGE) is used for analyzing immune absorption material to the adsorption selectivity of human plasma IgG.Sample and sodium lauryl sulphate sample-loading buffer boil 5min after mixing with the volume ratio of 1:1 in 100 ℃ water, the applied sample amount of sample and protein molecular weight standard all is 10 μ L, and used staining agent is coomassie brilliant blue R_250.The the 1st, 2 swimming band in the accompanying drawing 6 represents respectively standard protein and Sep-G3.0-His elutriant, SDS – PAGE test shows that the bionical thing specific immunity sorbing material Sep-triazole-His that invents has higher selectivity to the IgG in the human plasma, and non-specific adsorption does not almost have.
The research of immune absorption material absorption stability
In order to estimate the repeat performance of immune absorption material, sample has been carried out absorption-desorption cycle 10 times.0.02M is still used in desorb, and pH is 2.5 citrate buffer solution, and then uses 0.02M, and pH is 7.0 phosphoric acid buffer balance, and the concentration to IgG is tested behind the absorption-desorption process each time.Test result show the bionical thing specific immunity sorbing material Sep-G3.0-His that invents use through 10 times after its IgG loading capacity slightly drop to 25.63mg/g from 28.12mg/g, illustrate that this immune absorption material can be reused to a certain extent and its adsorptive power to IgG can be kept stable substantially.
Embodiment two:
The preparation of azide sepharose
In the 100mL Erlenmeyer flask, add sepharose, 8mL epoxy chloropropane and the 8mL1.5M sodium hydroxide solution that 4.0g drains.Mixture is at room temperature behind the stirring reaction 4h, with water washing drain until after in washings, adding 1.3M Sulfothiorine phenolphthalein do not redden, namely get the epoxidation agarose.The 0.78g sodiumazide is dissolved in 10mL water, adds again 4.0g epoxidation agarose, 40 ℃ of reaction 30h.React complete, the azide sepharose is drained to get in washing.
Synthesizing of G2.0PAMAM branch-shape polymer
In the round-bottomed flask of 250ml, add first methyl alcohol 270ml, add again the 272mmol methyl acrylate, in ice-water bath, place for some time, make the temperature of system be approximately 10 ℃, then in 1h, drip continuously the methanol solution 30ml that contains the 54.4mmol propargylamine, dropwise rear continuation at 10 ℃ of lower reaction 0.5h, be warming up to afterwards 20 ℃ of reaction 12h.React complete, product is carried out underpressure distillation get G0.5PAMAM; In the round-bottomed flask of 250ml, add first methyl alcohol 100ml, add again the 255mmol quadrol, in ice-water bath, place for some time, make system temperature be approximately 10 ℃, then in 1h, drip continuously the methanol solution 30ml that contains 25.5mmol G0.5PAMAM, dropwise rear continuation at 10 ℃ of lower reaction 0.5h, be warming up to afterwards 20 ℃ of reaction 12h.React complete, product is carried out underpressure distillation get G1.0PAMAM.
In the round-bottomed flask of 250ml, add first methyl alcohol 70ml, add again the 142mmol methyl acrylate, in ice-water bath, place for some time, make the temperature of system be approximately 10 ℃, then in 1h, drip continuously the methanol solution 30ml that contains 14.2mmol G1.0PAMAM, dropwise rear continuation at 10 ℃ of lower reaction 0.5h, be warming up to afterwards 20 ℃ of reaction 15h.React complete, product is carried out underpressure distillation get G1.5PAMAM; In the round-bottomed flask of 250ml, add first methyl alcohol 100ml, add again the 375mmol quadrol, in ice-water bath, place for some time, make system temperature be approximately 10 ℃, then in 1h, drip continuously the methanol solution 30ml that contains 12.5mmol G1.5PAMAM, dropwise rear continuation and in ice-water bath, react 0.5h, be warming up to afterwards 20 ℃ of reaction 15h, aminolysis reaction is fully carried out.React complete, product is carried out underpressure distillation.The oily matter of gained yellowish brown is G2.0PAMAM.
The preparation of phenylalanine methyl ester hydrochloride
Get the 50mmol L-Phe in the 500ml single necked round bottom flask, and add 100ml methyl alcohol and 100mmol trimethylchlorosilane.Be back to the solution becomes clarification, revolve steaming and be drying to obtain phenylalanine methyl ester hydrochloride.
The preparation of the G2.0PAMAM that phenylalanine is modified
The 4mmol phenylalanine methyl ester hydrochloride is dissolved in 16ml water and places the 100ml single necked round bottom flask, add the 4mmol triethylamine, add the aqueous solution that 15ml is dissolved with 1mmol G2.0PAMAM behind the stir about 15min, 20 ℃ of successive reaction 24h under magnetic agitation, completely small molecules of unreacted is removed in the dialysis tubing dialysis, lyophilize gets G2.0-Phe, and wherein Phe represents L-Phe.Shown in the accompanying drawing 3
1H NMR figure shows alkynyl and the methylene radical characteristic peak of the characteristic peaks such as the methyne, methylene radical and the phenyl ring that contain simultaneously phenylalanine in the prepared sample and PAMAM, but and by the ratio of the integral area at each peak 4 phenylalanine molecules of 1 G2.0PAMAM molecule grafting as can be known.
Click the standby bionical thing specific immunity sorbing material take PAMAM as spacerarm of legal system
The aqueous solution 12ml of 1.0mmol G2.0-Phe is placed the 100ml single necked round bottom flask, add 1.0g azide agarose.After stirring, add the aqueous solution 2.5ml that is dissolved with the 2.5mg cupric sulfate pentahydrate and the aqueous solution 2.5ml that is dissolved with the 4.0mg sodium ascorbate.Behind 20 ℃ of successive reaction reaction 24h, the sepharose of gained is drained in water, EDTA and water washing successively, namely gets immune absorption material Sep-G2.0-Phe.Infrared spectrogram shown in the accompanying drawing 5 shows that immune absorption material take phenylalanine as aglucon is at 2100cm
-1The nitrine peak at place disappears, and illustrates that the azido-on the azide agarose reacts completely, and successfully synthesizes target product in conjunction with Fig. 1 and Fig. 3 proof by click-reaction.
The research of immune absorption material loading capacity
Testing method is with embodiment one.Test result shows, A
280, M washes, the M fine jade is respectively 3.655,6.005g, 0.692g, thereby calculate to such an extent that the made bionical thing specific immunity sorbing material Sep-G2.0-Phe of present embodiment can reach 22.98mg/g to the loading capacity of IgG in the human plasma, this conforms to the size of phenylalanine ligand content in this material.
The research of immune absorption material adsorption selectivity
Testing method is with embodiment one.SDS – PAGE test shows that the bionical thing specific immunity sorbing material Sep-G2.0-Phe that invents has higher selectivity to the IgG in the human plasma.
The research of immune absorption material absorption stability
Testing method is with embodiment one.Test result show the bionical thing specific immunity sorbing material Sep-G2.0-Phe that invents use through 10 times after its IgG loading capacity slightly drop to 19.86mg/g from 22.98mg/g, illustrate that this immune absorption material can be reused to a certain extent and its adsorptive power to IgG can be kept stable substantially.
Embodiment three:
The preparation of azide sepharose
In the 100mL Erlenmeyer flask, add sepharose, 8mL epoxy chloropropane and the 6mL2.5M sodium hydroxide solution that 4.0g drains.Mixture is at room temperature behind the stirring reaction 3.5h, with water washing drain until after in washings, adding 1.3M Sulfothiorine phenolphthalein do not redden, namely get the epoxidation agarose.The 3.9g sodiumazide is dissolved in 25mL water, adds again 4.0g epoxidation agarose, 60 ℃ of reaction 6h.React complete, the azide sepharose is drained to get in washing.
Synthesizing of G4.0PAMAM branch-shape polymer
In the round-bottomed flask of 250ml, add first methyl alcohol 180ml, add again the 300mmol methyl acrylate, in ice-water bath, place for some time, the temperature of system is approximately-10 ℃, then in 1h, drips continuously the methanol solution 30ml that contains the 20.0mmol propargylamine, dropwise rear continuation at-10 ℃ of lower reaction 1.5h, be warming up to afterwards 3h, be warming up to afterwards 50 ℃ of reaction 50h.React complete, product is carried out underpressure distillation get G0.5PAMAM; In the round-bottomed flask of 250ml, add first methyl alcohol 100ml, add again the 465mmol quadrol, in ice-water bath, place for some time, make system temperature be approximately-10 ℃, then in 1h, drip continuously the methanol solution 30ml that contains 15.5mmol G0.5PAMAM, dropwise rear continuation at-10 ℃ of lower reaction 3h, be warming up to afterwards 50 ℃ of reaction 50h.React complete, product is carried out underpressure distillation get G1.0PAMAM.
In the round-bottomed flask of 250ml, add first methyl alcohol 200ml, add again the 426mmol methyl acrylate, in ice-water bath, place for some time, the temperature of system is approximately-10 ℃, then in 1h, drip continuously the methanol solution 30ml that contains 14.2mmol G1.0PAMAM, dropwise rear continuation at-10 ℃ of lower reaction 3h, be warming up to afterwards 60 ℃ of reaction 60h.React complete, product is carried out underpressure distillation get G1.5PAMAM; In the round-bottomed flask of 250ml, add first methyl alcohol 100ml, add again the 500mmol quadrol, in ice-water bath, place for some time, make system temperature be approximately-10 ℃, then in 1h, drip continuously the methanol solution 30ml that contains 10.0mmol G1.5PAMAM, dropwise rear continuation at-10 ℃ of lower reaction 3h, be warming up to afterwards 60 ℃ of reaction 60h.React complete, product is carried out underpressure distillation get G2.0PAMAM.
In the round-bottomed flask of 250ml, add first methyl alcohol 150ml, add again the 360mmol methyl acrylate, in ice-water bath, place for some time, the temperature of system is approximately-10 ℃, then in 1h, drip continuously the methanol solution 30ml that contains 7.2mmol G2.0PAMAM, dropwise rear continuation-10 ℃ lower reaction 3h, be warming up to afterwards 70 ℃ of reaction 66h.React complete, product is carried out underpressure distillation get G2.5PAMAM; In the round-bottomed flask of 250ml, add first methyl alcohol 70ml, add again the 495mmol quadrol, in ice-water bath, place for some time, make system temperature be approximately-10 ℃, then in 1h, drip continuously the methanol solution 30ml that contains 5.5mmol G2.5PAMAM, dropwise rear continuation at-10 ℃ of lower reaction 3h, be warming up to afterwards 70 ℃ of reaction 66h.React complete, product is carried out underpressure distillation get G3.0PAMAM.
In the round-bottomed flask of 250ml, add first methyl alcohol 80ml, add again the 405mmol methyl acrylate, in ice-water bath, place for some time, the temperature of system is approximately-10 ℃, then in 1h, drip continuously the methanol solution 30ml that contains 4.5mmol G3.0PAMAM, dropwise rear continuation-10 ℃ lower reaction 3h, be warming up to afterwards 80 ℃ of reaction 72h.React complete, product is carried out underpressure distillation get G2.5PAMAM; In the round-bottomed flask of 250ml, add first methyl alcohol 100ml, add again the 510mmol quadrol, in ice-water bath, place for some time, make system temperature be approximately-10 ℃, then in 1h, drip continuously the methanol solution 30ml that contains 3.0mmol G3.5PAMAM, dropwise rear continuation and in ice-water bath, react 3h, be warming up to afterwards 80 ℃ of reaction 72h, aminolysis reaction is fully carried out.React complete, product is carried out underpressure distillation.The lurid viscous body of gained is G4.0PAMAM.
The preparation of tryptophan methyl ester hydrochloride
Get the 50mmol L-Trp in the 500ml single necked round bottom flask, and add 50ml methyl alcohol and 50mmol trimethylchlorosilane.Be back to the solution becomes clarification, revolve the dry tryptophan methyl ester hydrochloride that namely gets of evaporate to dryness.
The preparation of the G4.0PAMAM that tryptophane is modified
The 160mmol tryptophan methyl ester hydrochloride is dissolved in 40ml water and places the 250ml single necked round bottom flask, add the 160mmol triethylamine, add the aqueous solution that 30ml is dissolved with 1mmol G4.0PAMAM behind the stir about 15min, 90 ℃ of successive reaction 96h under magnetic agitation, completely small molecules of unreacted is removed in the dialysis tubing dialysis, lyophilize gets G4.0-Trp, and wherein Trp represents L-Trp.Shown in the accompanying drawing 4
1H NMR figure shows alkynyl and the methylene radical characteristic peak of the characteristic peaks such as the methyne, methylene radical and the indyl that contain simultaneously tryptophane in the prepared sample and PAMAM, but and by the ratio of the integral area at each peak 6 tryptophane molecules of 1 G4.0PAMAM molecule grafting as can be known.
Click the standby bionical thing specific immunity sorbing material take PAMAM as spacerarm of legal system
The aqueous solution 30ml of 10mmol G4.0-Trp is placed the 150ml single necked round bottom flask, add 1.0g azide agarose.After stirring, add the aqueous solution 6ml that is dissolved with the 250mg cupric sulfate pentahydrate and the aqueous solution 6ml that is dissolved with the 400mg sodium ascorbate.Behind 90 ℃ of successive reaction reaction 96h, the sepharose of gained is drained in water, EDTA and water washing successively, namely gets immune absorption material Sep-G4.0-Trp.Infrared spectrogram shown in the accompanying drawing 5 shows that immune absorption material take tryptophane as aglucon is at 2100cm
-1The place also has faint nitrine peak, may be because the molecular weight of G4.0-Trp is larger, sterically hindered also larger, thereby relatively be not easy to react with the azide agarose.
The research of immune absorption material loading capacity
Testing method is with embodiment one.Test result shows, A
280, M washes, the M fine jade is respectively 3.298,6.001g, 0.656g, thereby calculate to such an extent that the made bionical thing specific immunity sorbing material Sep-G4.0-Trp of present embodiment can reach 22.35mg/g to the loading capacity of IgG in the human plasma, this conforms to the ligand content size of tryptophane in this material.In addition, the excessive dendroid spacerarm of molecular weight is because it sterically hinderedly also has certain impact to the loading capacity of immune absorption material.
The research of immune absorption material adsorption selectivity
Testing method is with embodiment one.SDS – PAGE test shows that the bionical thing specific immunity sorbing material Sep-G4.0-Trp that invents has higher selectivity to the IgG in the human plasma.
The research of immune absorption material absorption stability
Testing method is with embodiment one.Test result show the bionical thing specific immunity sorbing material Sep-G4.0-Trp that invents use through 10 times after its IgG loading capacity slightly drop to 19.18mg/g from 22.35mg/g, illustrate that this immune absorption material can be reused to a certain extent and its adsorptive power to IgG can be kept stable substantially.
By above-mentioned example as can be known, the invention provides the bionical thing specific immunity sorbing material of a series of PAMAM take different algebraically as spacerarm, different aminoacids as aglucon, they can safety, selectivity, repeatability ground are adsorbed IgG from human plasma, the whole absorption property of part material is enough to match in excellence or beauty with protein A immunoadsorption material, the price that it is cheap and good performance make it have the potential that replaces protein A immunoadsorption material, and are expected to be applied to clinical.
Claims (6)
1. bionical thing specific immunity sorbing material take PAMAM as spacerarm is characterized in that having the chemical structure of following Sep-PAMAM-AA:
Wherein, Sep represents sepharose; The AA represented amino acid; N=0,1,2 or 3; PAMAM is polyamide-amide type dendrimer, and described amino acid is L-Histidine, phenylalanine or tryptophane.
2. the preparation method of the bionical thing specific immunity sorbing material take PAMAM as spacerarm claimed in claim 1 is characterized in that comprising the steps and processing condition:
(a) preparation of azide sepharose
The aqueous solution that adds sodiumazide in the epoxidation agarose adds sodiumazide 3～15mmol in every gram epoxidation agarose, behind 20～60 ℃ of reaction 6～60h, product is drained to get in washing;
(b) the PAMAM branch-shape polymer is synthetic
Be under-10～10 ℃ of conditions in temperature, drip propargylamine in the methanol solution of methyl acrylate, the mol ratio of methyl acrylate and propargylamine is 5～15:1, behind insulation reaction 0.5～3h, be warming up to 20～50 ℃ of reaction 12～50h, reaction product is carried out underpressure distillation, get G0.5PAMAM; Under-10～10 ℃, drip G0.5PAMAM in the methanol solution of quadrol, the mol ratio of quadrol and G0.5PAMAM is 10～30:1, behind insulation reaction 0.5～3h, be warming up to 20～50 ℃ of reaction 12～50h, reaction product carried out underpressure distillation get first-generation product G1.0PAMAM;
Be under-10～10 ℃ of conditions in temperature, in the methanol solution of methyl acrylate, drip G1.0PAMAM, the mol ratio of methyl acrylate and G1.0PAMAM is 10～30:1, behind insulation reaction 0.5～3h, be warming up to 20～60 ℃ of reaction 15～60h, reaction product is carried out underpressure distillation, get G1.5PAMAM; Under-10～10 ℃, drip G1.5PAMAM in the methanol solution of quadrol, the mol ratio of quadrol and G1.5PAMAM is 30～50:1, behind insulation reaction 0.5～3h, be warming up to 20～60 ℃ of reaction 15～60h, reaction product carried out underpressure distillation get s-generation product G2.0PAMAM;
Be under-10～10 ℃ of conditions in temperature, in the methanol solution of methyl acrylate, drip G2.0PAMAM, the mol ratio of methyl acrylate and G2.0PAMAM is 30～50:1, behind insulation reaction 0.5～3h, be warming up to 20～70 ℃ of reaction 18～66h, reaction product is carried out underpressure distillation, get G2.5PAMAM; Under-10～10 ℃, drip G2.5PAMAM in the methanol solution of quadrol, the mol ratio of quadrol and G2.5PAMAM is 70～90:1, behind insulation reaction 0.5～3h, be warming up to 20～70 ℃ of reaction 18～66h, reaction product carried out underpressure distillation get third generation product G3.0PAMAM;
Be under-10～10 ℃ of conditions in temperature, in the methanol solution of methyl acrylate, drip G3.0PAMAM, the mol ratio of methyl acrylate and G3.0PAMAM is 70～90:1, behind insulation reaction 0.5～3h, be warming up to 20～80 ℃ of reaction 24～72h, reaction product is carried out underpressure distillation, get G3.5PAMAM; Under-10～10 ℃, drip G3.5PAMAM in the methanol solution of quadrol, the mol ratio of quadrol and G3.5PAMAM is 150～170:1, behind insulation reaction 0.5～3h, be warming up to 20～80 ℃ of reaction 24～72h, reaction product carried out underpressure distillation get the 4th generation product G4.0PAMAM;
(c) preparation of amino acid methyl ester hydrochloride
Amino acid is placed methanol solution, add trimethylchlorosilane, described trimethylchlorosilane and amino acid whose mol ratio are 1～3:1, are back to that to revolve evaporate to dryness after solution is clarified dry; Described amino acid is L-Histidine, phenylalanine or tryptophane;
(d) preparation of amino acid modified PAMAM
Amino acid methyl ester hydrochloride is soluble in water, and add in triethylamine and first～the 4th generation PAMAM product any one the aqueous solution, the mol ratio of amino acid methyl ester hydrochloride, triethylamine and first～the 4th generation PAMAM product is 1～10:1～10:1; 20～90 ℃ of reaction 24～96h under the magnetic agitation, dialysis and lyophilize get amino acid modified PAMAM product, are expressed as PAMAM-AA; Described amino acid methyl ester hydrochloride is Histidine methyl ester hydrochloride, phenylalanine methyl ester hydrochloride or tryptophan methyl ester hydrochloride; Described the first～the 4th generation PAMAM product is G1.0PAMAM, G2.0PAMAM, G3.0PAMAM and G4.0PAMAM;
(e) click the standby immune absorption material take PAMAM as spacerarm of legal system
After amino acid modified PAMAM product is water-soluble, add the azide sepharose, every gram azide sepharose adds the amino acid modified PAMAM product of 1～10mmol, then add cupric sulfate pentahydrate successively and sodium ascorbate is made catalyzer, the mol ratio of cupric sulfate pentahydrate, sodium ascorbate and amino acid modified PAMAM product is 0.01～0.05:0.02～0.1:1; And in 20～90 ℃ of reaction 24～96h, target product Sep-PAMAM-AA is drained to get in washing.
3. the preparation method of the bionical thing specific immunity sorbing material take PAMAM as spacerarm claimed in claim 2, it is characterized in that: described epoxidation agarose prepares by the following method: the ratio that adds 2mL epoxy chloropropane and 1～2mL sodium hydroxide solution in every gram sepharose, sepharose, epoxy chloropropane and sodium hydroxide solution are mixed stirring reaction 3～4h under the rear room temperature, with water washing, drain until after in washings, adding 1.3M Sulfothiorine phenolphthalein do not redden, namely get the epoxidation agarose; The concentration of described sodium hydroxide solution is 1.5～3M.
4. the preparation method of the bionical thing specific immunity sorbing material take PAMAM as spacerarm claimed in claim 2, it is characterized in that: described amino acid places methanol solution, and the methyl alcohol add-on is 1～3mL in every 1mmol amino acid; In the methanol solution of described methyl acrylate, the methyl alcohol add-on is 0.2～1.0mL in every 1mmol methyl acrylate.
5. the preparation method of the bionical thing specific immunity sorbing material take PAMAM as spacerarm claimed in claim 2, it is characterized in that: described amino acid methyl ester hydrochloride is soluble in water, and every 1mmol amino acid methyl ester hydrochloride adds entry 0.25～4mL.
6. the application of the bionical thing specific immunity sorbing material take PAMAM as spacerarm claimed in claim 1 in IgG separates.
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| CN104492395B (en) * | 2014-11-28 | 2016-10-05 | 珠海健帆生物科技股份有限公司 | Bionical immunoadsorbent with PAMAM as spacerarm and preparation method and application |
| RU2017134828A (en) | 2015-04-13 | 2019-04-04 | Фуджифилм Корпорэйшн | METHOD FOR PRODUCING A MEDIA IMMOBILIZED ON A SHORT-CHAIN PEPTIDE, AND A MEDIA IMMOBILIZED ON A SHORT-CHAIN PEPTIDE |
| CN109337929A (en) * | 2018-09-11 | 2019-02-15 | 广东医科大学 | A kind of preparation method of the non-viral gene vector with core target function |
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