CN109010806A - A kind of degradable internal uric acid simultaneously realizes the collaboration complex enzyme and its preparation method and application that by-product is removed - Google Patents
A kind of degradable internal uric acid simultaneously realizes the collaboration complex enzyme and its preparation method and application that by-product is removed Download PDFInfo
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Abstract
A kind of degradable internal uric acid simultaneously realizes the collaboration complex enzyme and its preparation method and application that by-product is removed, first two kinds of enzymes have been carried out with chemical modification in situ, the operation had not only made UOx and Cat remain higher activity, but also provided reaction site for the further assembling of Nano capsule.The PEG molecule of adamantane and cyclodextrin sealing end further modifies the Nano capsule prepared, and by Ad, the Subjective and Objective between CD has been self-assembly of compound enzyme system with synergy.The nanocluster system can be stabilized long period, the more efficiently uric acid content in degradation blood, and toxicity caused by generated H2O2 in the significant reduction uric acid degradation process of energy in vivo.
Description
Technical field
The invention belongs to biomedicine field, being related to one kind can the internal uric acid of long-acting degradation and to be able to achieve efficient by-product clear
Collaboration complex enzyme removed and its preparation method and application.
Background technique
Gout is one of most common form of inflammatory arthritis, and the illness rate of global adult is between 1-2%, 65 years old
The above prevalence has been even more than 7%.Urarthritis is a kind of spy for joint monosodium urate (MSU) deposition
The inflammatory reaction of sign property.In essence, gout is primarily due to the uric acid content in blood and is chronically at saturation point or more, and from
It is gradually precipitated in blood and forms MSU crystal.Therefore for patient with gout, by inhibiting the generation of uric acid or reducing uric acid
Reabsorption with the uric acid content reduced in blood be extremely effective treatment means.However both treatment means to patient with
The potential side effect (cardiovascular disease, allergy either lithangiuria) come significantly limits their application.Recently, it is based on
The pegloticase of uricolytic enzyme UOx has been used for the treatment of chronic gout by FDA approval.Although polyethylene glycol weight
Group uricase can effectively degrade uric acid, but generated H during uric acid of degrading2O2For G-6-P dehydrogenation
Enzyme deficiency disease (G6PD) patient can generate hemolytic disease that may be lethal.Therefore, it is based on to be used safely when treating gout
The therapy of UOx, urgent need, which finds one kind, may be implemented efficiently to remove H2O2Strategy.
Internal at us, many metabolism and decomposable process can all generate toxic products.For example, oxidizing ferment as in human body most
For important class of enzymes, multiple important metabolism in vivo and the synthesis of necessary product are participated in.In this process, oxidizing ferment exists
By-product H can be generated while consuming oxygen2O2.Oxidizing ferment and detoxication enzyme (such as Cat) are limited in a limited sky by the Nature
In and dexterously solve this problem because narrow space limits H2O2Movement, Cat is completed pair
H2O2Efficient-decomposition.It is inspired by nature, a kind of UOx and Cat is limited in receiving in lesser spatial dimension if can construct
Rice corpuscles will be a kind of ideal gout treatment method, because such an approach achieves do not generating toxic byproduct H2O2The case where
Under remain uricolytic ability.So far, it has been reported that the method for a variety of building multienzyme structures, including a variety of enzymes
Chemical crosslinking, the self assembly etc. for the enzyme that the covalent fixation of a variety of enzymes and template-directed are realized on pedestal.These strategies
Transport of the reaction intermediate between a variety of enzymes is effectively optimized, more preferably controlling and mentioning to intermediate product transport is realized
High whole catalytic performance.However, the chemical modification being related to mostly to enzyme in these strategies, this may damage UOx's
Uric acid degrading activity.In addition, current strategy improves the stability of multienzyme structure almost without consideration, especially for albumen water
Solution is as a result, such multi-enzyme system vulnerable to protease attack, limits them for therapeutic purposes.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, and providing one kind the internal uric acid of long-acting degradation and can be able to achieve efficiently
The collaboration complex enzyme and its preparation method and application that by-product is removed.Cooperate with complex enzyme during preparation, first to two kinds
Enzyme has carried out chemical modification (in-situ polymerization, in situpolymerization) in situ, which protected UOx and Cat
Higher activity has been stayed, and has provided reaction site for the further assembling of Nano capsule.Adamantane (Ad) and cyclodextrin (CD)
The PEG molecule of sealing end further modifies the Nano capsule prepared, and by Ad, the Subjective and Objective between CD, which has been self-assembly of, to be had
The compound enzyme system of synergistic effect.The nanocluster system can be stabilized the long period in vivo, blood of more efficiently degrading
Uric acid content in liquid, and generated H in the significant reduction uric acid degradation process of energy2O2Caused toxicity.
The technical scheme is that
A kind of degradable internal uric acid and the preparation method for realizing the collaboration complex enzyme that by-product is removed, including walk as follows
It is rapid:
1) synthesis step of nUOx and nCat,
Wherein catalase capsule nCat is acrylated catalase: acrylamide: N-3- aminopropyltriethoxy third
Acrylamide: N, N'- methylene-bisacrylamide: ammonium persulfate: tetramethylethylenediamine=1:9000:1000:1000:450:900
Molar ratio；
Wherein urate oxidase Nano capsule nUOx is acrylated urate oxidase: acrylamide: N-3- aminopropyl first
Base acrylamide: N, N'- methylene-bisacrylamide: ammonium persulfate: tetramethylethylenediamine=1:8000:2000:1000:450:
900 ratio preparation, obtains urate oxidase Nano capsule nUOx.
2) preparation step of nUOx-PEG-Ad and nCat-PEG-CD,
It will be added drop-wise to the solution of nUOx or nCat after PEG-CD NHS and EDC activation, removes unreacted through dialysis after reaction
Molecule obtain nUOx-PEG-Ad or nCat-PEG-CD；
3) preparation of complex enzyme nanocluster n (UOx-Cat) is cooperateed with
The nUOx-PEG-Ad of above-mentioned preparation and nCat-PEG-CD is mixed to get n (UOx-Cat) with the molar ratio of 1:4.
Further, the synthetic method of step 1) nUOx and nCat is:
Firstly, 40mg catalase is added into plastic centrifuge tube, it is dissolved in 4mL borate buffer, by N- acryloyl-oxy
Base succinimide is dissolved in super dry dimethyl sulfoxide, and it is sub- that above-mentioned N- acryloxy succinyl is slowly added dropwise in 4 DEG C of ice-water bath stirrings
Amine aqueous solution is added dropwise, and keeps this thermotonus 2 hours, immediately collects this reaction solution and in sodium bicarbonate buffer liquid
In 4 DEG C of dialysed overnights, using the concentration of the acrylated catalase of BCA protein concentration detection method calibration gained, and be diluted to
5mg/mL is saved in 4 DEG C of refrigerators；
Then, by above-mentioned acrylated catalase: acrylamide: N-3- aminopropyl methacrylamide: N, N'- are sub-
Above-mentioned monomer and crosslinking agent is added in bisacrylamide=1:9000:1000:1000 molar ratio, after mixing, is added four
Methyl ethylenediamine and ammonium persulfate cause home position polymerization reaction, and molar ratio is catalase: ammonium persulfate: tetramethyl second two
Amine=1:450:900, the reaction are reacted 1 hour at 4 DEG C, and after reaction, 4 DEG C of dialysed overnights, are adopted in phosphate buffer
1mg/mL is concentrated into BCA protein concentration detection method super filter tube to save in 4 DEG C of refrigerators, obtains catalase capsule nCat；
40mg uricolytic enzyme is added into plastic centrifuge tube, 4mL borate buffer is dissolved in, by N- acryloxy amber
Acid imide is dissolved in super dry dimethyl sulfoxide, and above-mentioned N- acryloxy succinimide solution is slowly added dropwise in 4 DEG C of ice-water bath stirrings,
It is added dropwise, keeps this thermotonus 2 hours, immediately this reaction solution collected and 4 DEG C thoroughly in sodium bicarbonate buffer liquid
Analysis overnight, using the concentration of the acrylated urate oxidase of BCA protein concentration detection method calibration gained, and be diluted to 5mg/mL in
4 DEG C of refrigerators save；
According to urate oxidase: acrylamide: N-3- aminopropyl methacrylamide: N, N'- methylene-bisacrylamide:
Ammonium persulfate: the preparation of tetramethylethylenediamine=1:8000:2000:1000:450:900 ratio obtains urate oxidase nanometer
Capsule nUOx.
Further, the preparation method of step 2) nUOx-PEG-Ad and nCat-PEG-CD is:
PEG-CD is stayed overnight with NHS and EDC in 4 degree of activation first, the solution of nCat is then added drop-wise to, was reacted in 4 degree
Unreacted molecule is removed with the bag filter dialysis of 10kDa molecular weight after night, concentration is finally set to 1mg/ with the method for BCA
ML, it is spare to be placed in 4 degree of refrigerators, obtains nCat-PEG-CD；
PEG-CD is stayed overnight with NHS and EDC in 4 degree of activation, is then added drop-wise to the solution of nUOx, after reaction overnight in 4 degree
Unreacted molecule is removed with the bag filter dialysis of 10kDa molecular weight, concentration is finally set to 1mg/mL with the method for BCA, is put
It is spare to be placed in 4 degree of refrigerators, obtains nUOx-PEG-Ad.
Further, the preparation method of step 3) collaboration complex enzyme nanocluster n (UOx-Cat) is:
NUOx-PEG-Ad and nCat-PEG-CD is blended under 4 degree with the molar ratio of 1:4 and stirs 2h, obtains n (UOx-
Cat), with sodium carbonate buffer by final UOx concentration dilution to 1mg/mL, be placed in 4 degree it is spare.
A kind of degradable internal uric acid and the application for realizing the collaboration complex enzyme that by-product is removed, it is characterized in that including following
Aspect: the 1) application in terms of antitrypsin degradation and protein adsorption；2) in H2O2Generation and degradation kinetics in terms of answer
With；3) application in terms of avoiding erythrocyte haemolysis；4) application in terms of reducing cytotoxicity；5) degradation in vivo uric acid
And H2O2Remove the application of aspect.
The invention has the advantages that
This nanoparticle possesses dirt resistant surfaces, and the monokaryon system (MPS) in blood that can minimize is removed and trypsase drop
Solution, can possess longer circulation time in blood can realize long-acting degradation to uric acid.Meanwhile this multienzyme cluster system
Since two kinds of enzymes with synergistic effect being limited in a lesser spatial dimension, enable catalase to uric acid
By-product caused by during oxidizing ferment degradation uric acid is efficiently removed, therefore the collaboration complex enzyme is improving uric acid degradation capability
While reduce H2O2Caused toxic reaction.And this collaboration system preparation is simply, easily operated, cost is relatively low, is extremely easy to
It promotes and applies.
Detailed description of the invention
Fig. 1 is that collaboration complex enzyme prepares schematic diagram.
Fig. 2 is collaboration complex enzyme to by-product H2O2Efficient removing figure.
Fig. 3 is that collaboration complex enzyme high-efficiency continuous degradation uric acid and reduces H in vivo2O2Caused toxic reaction datagram.
Specific embodiment
Below by way of non-limiting example, present invention be described in more detail.
Embodiment 1:
Attached drawing 1 shows present invention collaboration complex enzyme nanocluster and prepares schematic diagram, and the collaboration of the method for the present invention preparation is multiple
Synthase nanocluster system is 28 ± 2nm and two kinds of enzyme nanogel institute with synergy groups being evenly distributed by partial size
At the Subjective and Objective molecular self-assembling that collaboration complex enzyme of the invention is formed by nanogel surface by being respectively present in two kinds of enzymes
Driving force formed.Referring to attached drawing 1, the degradable internal uric acid of the present invention is simultaneously able to achieve the collaboration complex enzyme that by-product is removed
Preparation method includes the following steps:
1) synthesis of nUOx and nCat
Firstly, 40mg catalase is added into plastic centrifuge tube, it is dissolved in 4mL borate buffer, by N- acryloyl-oxy
Base succinimide is dissolved in super dry dimethyl sulfoxide, and it is sub- that above-mentioned N- acryloxy succinyl is slowly added dropwise in 4 DEG C of ice-water bath stirrings
Amine aqueous solution is added dropwise, and keeps this thermotonus 2 hours, immediately collects this reaction solution and in sodium bicarbonate buffer liquid
In 4 DEG C of dialysed overnights, using the concentration of the acrylated catalase of BCA protein concentration detection method calibration gained, and be diluted to
5mg/mL is saved in 4 DEG C of refrigerators；
Then, by above-mentioned acrylated catalase: acrylamide: N-3- aminopropyl methacrylamide: N, N'- are sub-
Above-mentioned monomer and crosslinking agent is added in bisacrylamide=1:9000:1000:1000 molar ratio, after mixing, is added four
Methyl ethylenediamine and ammonium persulfate cause home position polymerization reaction, and molar ratio is catalase: ammonium persulfate: tetramethyl second two
Amine=1:450:900, the reaction are reacted 1 hour at 4 DEG C, and after reaction, 4 DEG C of dialysed overnights, are adopted in phosphate buffer
1mg/mL (BCA protein concentration detection method) is concentrated into super filter tube to save in 4 DEG C of refrigerators, obtains catalase capsule nCat；
The preparation of urate oxidase Nano capsule follows the above method: 40mg uric acid being added into plastic centrifuge tube and decomposes
Enzyme is dissolved in 4mL borate buffer, and N- acryloxy succinimide is dissolved in super dry dimethyl sulfoxide, 4 DEG C of ice-water bath stirrings
Above-mentioned N- acryloxy succinimide solution is slowly added dropwise, is added dropwise, keeps this thermotonus 2 hours, immediately
This reaction solution is collected and 4 DEG C of dialysed overnights in sodium bicarbonate buffer liquid, using BCA protein concentration detection method calibration gained third
The concentration of the acylated uricolytic enzyme of alkene, and be diluted to 5mg/mL and saved in 4 DEG C of refrigerators；
According to urate oxidase: acrylamide: N-3- aminopropyl methacrylamide: N, N'- methylene-bisacrylamide:
Ammonium persulfate: the preparation of tetramethylethylenediamine=1:8000:2000:1000:450:900 ratio obtains urate oxidase nanometer
Capsule nUOx.
2) preparation of nUOx-PEG-Ad and nCat-PEG-CD
First by PEG-CD with NHS and EDC 4 degree activation overnight, be then added drop-wise to nCat solution (pH 8.5,
NaHCO3, 10mM), unreacted molecule is removed with the bag filter dialysis of 10kDa molecular weight after reaction overnight in 4 degree.It is final to use
Concentration is set to 1mg/mL by the method for BCA, and it is spare to be placed in 4 degree of refrigerators, obtains nCat-PEG-CD.
The preparation of nUOx-PEG-Ad follows method identical with nCat-PEG-CD: PEG-CD being existed with NHS and EDC first
4 degree of activation overnight, are then added drop-wise to the solution (pH 8.5, NaHCO of nUOx3, 10mM), in 4 degree after reaction overnight with 10kDa points
The bag filter dialysis of son amount removes unreacted molecule.Concentration is finally set to 1mg/mL with the method for BCA, is placed in 4 degree of ice
Case is spare, obtains nUOx-PEG-Ad.
3) preparation of complex enzyme nanocluster n (UOx-Cat) is cooperateed with
The nUOx-PEG-Ad of above-mentioned preparation and nCat-PEG-CD is blended under 4 degree with the molar ratio of 1:4 and stirs 2h, with
N (UOx-Cat) sodium carbonate buffer is prepared by final UOx concentration dilution to 1mg/mL, be placed in 4 degree it is spare.
Embodiment 2: application of the observation collaboration complex enzyme in terms of antitrypsin degradation and protein adsorption.
Firstly, we will collaboration complex enzyme and trypsase co-cultured under 60 degree, specific time (5-60min) from mix
It closes and takes out 10 microlitres of solution in liquid, return back to room temperature after the activity of trypsase is quenched in ice water, pass through dynamic (dynamical) method
Monitor the fall off rate absorbed at ultraviolet 290nm.Ultraviolet fall off rate and 0 moment at 290nm by calculating different time points
The ratio of fall off rate come determine with trypsase co-culture mixed liquor in, cooperate with the activity of complex enzyme.
Collaboration complex enzyme is marked into upper FITC fluorescent molecule, is determined by BCA and UV absorption on each albumen
The number (F/P) of fluorescent molecule.Then collaboration complex enzyme is co-cultured 30 minutes with bovine serum albumin at 37 degree, with 100kDa's
Ultra-filtration centrifuge tube removes the unadsorbed bovine serum albumin that dissociates on collaboration complex enzyme, and then measurement remains in ultra-filtration centrifuge tube
Tot Prot, the content of the bovine serum albumin of non-specific adsorption is the Tot Prot and F/P that BCA is determined on collaboration complex enzyme
Difference between the amount of determining collaboration complex enzyme.
Experimental result is as shown in Figure 2: collaboration complex enzyme is minimum to albumen non-specific adsorption amount compared with control group
's.This is consistent to the surface PEGization modification of nanoparticle with us.
Embodiment 3: observation collaboration complex enzyme is in H2O2Generation and degradation kinetics in terms of application.
Tetramethyl benzidine (TMB) is dissolved in DMSO first, is then added to the molten of horseradish peroxidase (HRP)
In liquid (acetate buffer solution, pH=5.0,100mM, TMB=1.6mM, HRP=1.33 μ g/mL) and by 30 microlitres of above-mentioned preparations
Mixed solution is added in 96 orifice plates.At the same time, uric acid and lithium carbonate are dissolved in borate buffer and prepare uric acid work
Liquid (30 μM).Collaboration complex enzyme is added into uric acid working solution to be added to above-mentioned mixed solution in advance at specific time point
It is added in 96 orifice plates of TMB and HRP.Sulfuric acid is added after five minutes and terminates the enzymatic reaction.Measure the UV absorption at 450nm
In order to determine ultraviolet absorptivity and H at 450nm2O2Relationship between concentration, we are by the H of series of concentrations2O2
(10mM, 5mM, 2.5mM, 1.25mM, 0.625mM and 0.3125mM) is added in the mixed solution of HRP and TMB, and room temperature is put
30 minutes are set to ensure H2O2Conversion completely, terminates the reaction then to isometric sulfuric acid is added in solution.
Experimental result is as shown in Figure 2: collaboration complex enzyme can efficiently remove by-product H compared with control group2O2。
Embodiment 4: application of the observation collaboration complex enzyme in terms of reducing cytotoxicity
Firstly, we are by HeLa cell with 104The density in every hole is seeded in 96 orifice plates, is trained in carbon dioxide incubator
12h is supported to ensure that cell is completely adherent, collaboration complex enzyme is added in 96 orifice plates after cultivating 1h in incubator, uric acid work is added
Make liquid, further cultivates 11h.Then the H generated after above-mentioned material is added using the observation of CCK-8 Apoptosis method of testing2O2To thin
The apoptosis situation that born of the same parents generate.
Experimental result is as shown in Figure 2: collaboration complex enzyme is compared with control group, and cell survival rate highest, this is primarily due to
Collaboration complex enzyme can efficiently remove toxic byproduct H2O2, so as to avoid H2O2Induce the cytotoxicity generated.
Embodiment 5: application of the observation collaboration complex enzyme in terms of avoiding erythrocyte haemolysis.
Firstly, we will be stored in anticoagulant tube from fresh blood, be placed in 4 degree it is spare.Then it is added into blood
Sodium azide is with peroxidase in Noncompetition inhibition erythrocyte to H2O2Degradation.Then it is centrifuged off free nitrine
Change sodium small molecule.Then mixed solution (1.6mL, 0.02mg/mL, the pH=of uric acid and lithium carbonate are added into erythrocyte
7.4, PBS, 10mM) and collaboration complex enzyme, it is stood overnight in 4 degree.It is then centrifuged for handling.By ultraviolet at monitoring 540nm
It absorbs and quantitative analysis is carried out with the degree of hemolysis to erythrocyte.
Attached drawing 2 gives collaboration complex enzyme to by-product H2O2Efficient removing figure, the figure explanation: collaboration complex enzyme protecting
While the high-efficiency catalytic activity for staying degradation urate oxidase to degrade uric acid, realize to H2O2Efficient removing.
Embodiment 6: observation collaboration complex enzyme degradation in vivo uric acid and H2O2Remove the application of aspect.
Collaboration complex enzyme is injected into mouse tail vein according to the dosage of 2.5mg/kg by us, and subsequent intraperitoneal injection time is yellow
Purine (500mg/kg hypoxanthine is dissolved in 1% sodium carboxymethylcellulose), 1h, 2h, 3h rear molding venous blood sampling are surveyed in blood
Uric acid content.Same time repeats the hypoxanthine of intraperitoneal injection same dose within second to the 5th day.Then it repeats to take blood,
The step of surveying uric acid.5th day, after having surveyed uric acid in blood content, mouse is put to death, take whole blood, it is normal then to carry out blood to blood
Rule, blood biochemistry and ELISA detection.
Attached drawing 3, which gives collaboration complex enzyme, high-efficiency continuous degradation uric acid and reduces H in vivo2O2Caused toxicity data figure,
Figure explanation: collaboration complex enzyme can recycle for a long time in vivo and reduce H2O2Induce the system toxicity generated and to metabolic organ
It causes to damage.
Claims (7)
1. a kind of degradable internal uric acid and the preparation method for realizing the collaboration complex enzyme that by-product is removed, it is characterized in that including such as
Lower step:
1) synthesis step of nUOx and nCat,
Wherein catalase capsule nCat is acrylated catalase: acrylamide: N-3- aminopropyltriethoxy acryloyl
Amine: N, N'- methylene-bisacrylamide: ammonium persulfate: tetramethylethylenediamine=1:9000:1000:1000:450:900 moles
Than；
Wherein urate oxidase Nano capsule nUOx is acrylated urate oxidase: acrylamide: N-3- aminopropyltriethoxy third
Acrylamide: N, N'- methylene-bisacrylamide: ammonium persulfate: tetramethylethylenediamine=1:8000:2000:1000:450:900
Ratio preparation, obtain urate oxidase Nano capsule nUOx.
2) preparation step of nUOx-PEG-Ad and nCat-PEG-CD,
It will be added drop-wise to the solution of nUOx or nCat after PEG-CD NHS and EDC activation, removes unreacted point through dialysis after reaction
Son obtains nUOx-PEG-Ad or nCat-PEG-CD；
3) preparation of complex enzyme nanocluster n (UOx-Cat) is cooperateed with
The nUOx-PEG-Ad of above-mentioned preparation and nCat-PEG-CD is mixed to get n (UOx-Cat) with the molar ratio of 1:4.
2. degradable internal uric acid according to claim 1 and the preparation side for realizing the collaboration complex enzyme that by-product is removed
Method, it is characterized in that: the synthetic method of step 1) nUOx and nCat are,
Firstly, 40mg catalase is added into plastic centrifuge tube, it is dissolved in 4mL borate buffer, by N- acryloxy amber
Amber acid imide is dissolved in super dry dimethyl sulfoxide, and it is molten that above-mentioned N- acryloxy succinimide is slowly added dropwise in 4 DEG C of ice-water bath stirrings
Liquid is added dropwise, and keeps this thermotonus 2 hours, immediately collects this reaction solution and 4 DEG C in sodium bicarbonate buffer liquid
Dialysed overnight using the concentration of the acrylated catalase of BCA protein concentration detection method calibration gained, and is diluted to 5mg/mL
It is saved in 4 DEG C of refrigerators；
Then, by above-mentioned acrylated catalase: acrylamide: N-3- aminopropyl methacrylamide: N, N'- methylene
Above-mentioned monomer and crosslinking agent is added in bisacrylamide=1:9000:1000:1000 molar ratio, after mixing, tetramethyl is added
Ethylenediamine and ammonium persulfate cause home position polymerization reaction, and molar ratio is catalase: ammonium persulfate: tetramethylethylenediamine=
1 hour, after reaction, 4 DEG C of dialysed overnights in phosphate buffer, using BCA are reacted in 1:450:900, the reaction at 4 DEG C
Protein concentration detection method super filter tube is concentrated into 1mg/mL and saves in 4 DEG C of refrigerators, obtains catalase capsule nCat；
40mg uricolytic enzyme is added into plastic centrifuge tube, is dissolved in 4mL borate buffer, N- acryloxy succinyl is sub-
Amine is dissolved in super dry dimethyl sulfoxide, and 4 DEG C of ice-water bath stirrings are slowly added dropwise above-mentioned N- acryloxy succinimide solution, are added dropwise
Finish, keep this thermotonus 2 hours, immediately this reaction solution collected and in sodium bicarbonate buffer liquid 4 DEG C dialysed
Night using the concentration of the acrylated urate oxidase of BCA protein concentration detection method calibration gained, and is diluted to 5mg/mL in 4 DEG C
Refrigerator saves；
According to urate oxidase: acrylamide: N-3- aminopropyl methacrylamide: N, N'- methylene-bisacrylamide: over cure
Sour ammonium: the preparation of tetramethylethylenediamine=1:8000:2000:1000:450:900 ratio obtains urate oxidase Nano capsule
nUOx。
3. degradable internal uric acid according to claim 1 and the preparation side for realizing the collaboration complex enzyme that by-product is removed
Method, it is characterized in that: the preparation method of step 2) nUOx-PEG-Ad and nCat-PEG-CD are,
PEG-CD is stayed overnight with NHS and EDC in 4 degree of activation first, is then added drop-wise to the solution of nCat, after reaction overnight in 4 degree
Unreacted molecule is removed with the bag filter dialysis of 10kDa molecular weight, concentration is finally set to 1mg/mL with the method for BCA, is put
It is spare to be placed in 4 degree of refrigerators, obtains nCat-PEG-CD；
PEG-CD is stayed overnight with NHS and EDC in 4 degree of activation, the solution of nUOx is then added drop-wise to, is used after reaction overnight in 4 degree
The bag filter dialysis of 10kDa molecular weight removes unreacted molecule, and concentration is finally set to 1mg/mL with the method for BCA, is placed
It is spare in 4 degree of refrigerators, obtain nUOx-PEG-Ad.
4. degradable internal uric acid according to claim 1 and the preparation side for realizing the collaboration complex enzyme that by-product is removed
Method, it is characterized in that: the preparation method of step 3) collaboration complex enzyme nanocluster n (UOx-Cat) is,
NUOx-PEG-Ad and nCat-PEG-CD is blended under 4 degree with the molar ratio of 1:4 and stirs 2h, obtains n (UOx-Cat),
With sodium carbonate buffer by final UOx concentration dilution to 1mg/mL, be placed in 4 degree it is spare.
5. degradable internal uric acid according to claim 3 and the preparation side for realizing the collaboration complex enzyme that by-product is removed
Method, it is characterized in that: wherein the solution of the solution of nUOx or nCat are pH 8.5, NaHCO3,10mM。
6. a kind of degradable internal uric acid simultaneously realizes the collaboration complex enzyme that by-product is removed, it is characterized in that: the collaboration is compound
Enzyme is the collaboration complex enzyme nanocluster n (UOx-Cat) that any one of claim 1-5 the method obtains.
7. a kind of degradable internal uric acid and the application for realizing the collaboration complex enzyme that by-product is removed, it is characterized in that including with lower section
Face:
1) application in terms of antitrypsin degradation and protein adsorption；
2) in H2O2Generation and degradation kinetics in terms of application；
3) application in terms of reducing cytotoxicity；
4) application in terms of avoiding erythrocyte haemolysis；
5) degradation in vivo uric acid and H2O2Remove the application of aspect.
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| WO2021083278A1 (en) * | 2019-10-29 | 2021-05-06 | Westlake Therapeutics (Hangzhou) Co. Limited | Engineering red blood cells for treating gout and hyperuricemia diseases |
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| CN116920116A (en) * | 2022-04-14 | 2023-10-24 | 上海巴久巴生物技术有限公司 | Catalase-encapsulated nanocapsule, preparation method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021083278A1 (en) * | 2019-10-29 | 2021-05-06 | Westlake Therapeutics (Hangzhou) Co. Limited | Engineering red blood cells for treating gout and hyperuricemia diseases |
| CN114746546A (en) * | 2019-10-29 | 2022-07-12 | 西湖生物医药科技(杭州)有限公司 | Engineered erythrocytes for the treatment of gout and hyperuricemia |
| CN116920116A (en) * | 2022-04-14 | 2023-10-24 | 上海巴久巴生物技术有限公司 | Catalase-encapsulated nanocapsule, preparation method and application thereof |
| CN116272705A (en) * | 2023-02-07 | 2023-06-23 | 广东工业大学 | Preparation method and application of core-shelled nanocluster hydrogel microsphere |
| CN116272705B (en) * | 2023-02-07 | 2023-09-19 | 广东工业大学 | Preparation method and application of core-shelled nanocluster hydrogel microsphere |
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