CN109999008A - Compound agarose microbeads and its preparation method and application - Google Patents
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Abstract
The invention discloses a kind of compound agarose microbeads and its preparation method and application, compound agarose microbeads include enzyme A, enzyme B, nitric oxide precursors derivative and agarose, enzyme A, enzyme B and nitric oxide precursors derivative, which are wrapped in agarose, forms compound agarose microbeads, and enzyme A is glucose oxidase or class glucose oxidase, the enzyme B are peroxidase or peroxidase.Preparation method is that enzyme A and enzyme B is added in agarose solution to form water phase；Water phase is added dropwise in organic phase, centrifugation product is collected in stirring centrifugation；Nitric oxide precursors derivative is added in centrifugation product to be incubated for obtain compound agarose microbeads.Compound agarose microbeads of the invention can stimulate gel complex microsphere controlled catalytic to generate nitric oxide by reaction substrate of endogenous glucose, and drug treating time is long, and biological safety is good.Preparation method is easy to operate, is easy to be extended and applied.The compound agarose microbeads can be applied to the drug that preparation prevents and treats cardiovascular disease.
Description
Technical field
The present invention relates to field of material technology, in particular to compound agarose microbeads and its preparation method and application.
Background technique
Nitric oxide (nitric oxide, NO) is a kind of new bio informational molecule, has expansion blood vessel, adjusts blood
Pressure inhibits the effects of platelet adhesion reaction, participates in the mechanism such as body protective, adjusting and reverse, especially in adjusting cardiovascular system
Numerous physiology and pathophysiological process.The gel micro-ball of two kinds of enzymes and gel rubber system heterozygosis building coating cascade enzyme can be filled
The advantage that the two is waved in distribution generates nitric oxide, is expected to be applied to the fields such as biological medicine, analysis catalysis.
Currently, supplement supply nitric oxide, raising blood nitric oxide concentration are that the cardiovascular diseases such as thrombus, hypertension are pre-
A kind of anti-and treatment important measures.It is nitric oxide production to supply the supplement or blood for depending on Nitric oxidedonating compounds
The nitricoxide synthase transgene expression of endothelial cell.That there are drug treating times is too short, biological for nitric oxide donors drug
Stability is poor, human body toxic side effect is big, lacks the deficiencies of targeting and tissue specificity.Nitricoxide synthase transgenic technology is deposited
It is difficult to control in expression quantity, there is the disadvantages of drug slow effect of expression lag.The research of nitric oxide supplement supply mainly collects
In Nitric oxidedonating compounds exploitation and vascular endothelial cell nitricoxide synthase transgene expression.However,
Nitric oxide production generation research is applied to also using glucose oxidase and peroxidase cascade enzyme building bionic catalysis system
It is very rare.
Agarose (Agarose) is dehydrated α-L- galactopyranosyl glycosyl unit by β-D- galactopyranose (1-4) connection 3,6-
It constitutes, is the gel rubber system with porous structure, good biocompatibility, is widely used in affinity chromatography and immunoassay.Enzyme is
A kind of particularly important biocatalyst (biocatalyst).Due to the effect of enzyme, the intracorporal chemical reaction of biology is extremely warm
It also can efficiently and specifically be carried out under conditions of.
Summary of the invention
The technical problem to be solved by the present invention is that there are drug treating times is too short, biological steady for nitric oxide donors drug
Qualitative poor, human body toxic side effect is big, lacks the deficiencies of targeting and tissue specificity.For overcome the deficiencies in the prior art, originally
Invention provides a kind of compound agarose microbeads, can stimulate gel complex microsphere controlled catalytic by reaction substrate of endogenous glucose
Nitric oxide is generated, drug treating time is long, and biological safety is good.Preparation method is easy to operate, is easy to be extended and applied.It should
Compound agarose microbeads can be applied to the drug that preparation prevents and treats cardiovascular disease.
The present invention is to solve above-mentioned technical problem by the following means:
A kind of compound agarose microbeads, the compound agarose microbeads include enzyme A, enzyme B, nitric oxide precursors derivative and agar
Sugar, the enzyme A, enzyme B and nitric oxide precursors derivative, which are wrapped in the agarose, forms compound agarose microbeads, the enzyme
A is glucose oxidase or class glucose oxidase, the enzyme B are peroxidase or peroxidase.
Above-mentioned compound agarose microbeads, it is preferred that the glucose oxidase is β-D-Glucose oxidoreducing enzyme；Institute
Stating class glucose oxidase is palladium nano-particles or gold nano grain.
Above-mentioned compound agarose microbeads, it is preferred that the peroxidase is horseradish peroxidase；The class peroxide
Compound enzyme is ferroferric oxide nano granules, di-iron trioxide nano particle, prussian blue nano particle, fullerene nanometer
One of the G- tetramer that grain, manganese dioxide nano particle and hemin combine.
Above-mentioned compound agarose microbeads, it is preferred that the nitric oxide precursors derivative is hydroxycarbamide, L-arginine
Or N- hydroxyl L-arginine.
In a total technical concept, the present invention also provides a kind of preparation sides of above-mentioned compound agarose microbeads
Method, the preparation method comprises the following steps:
S1, water phase will be formed in enzyme A and enzyme B addition agarose solution；
S2, water phase is added dropwise in organic phase, centrifugation product is collected in stirring centrifugation；
S3, addition nitric oxide precursors derivative is incubated for obtain compound agarose microbeads in the centrifugation product.
The preparation method of above-mentioned compound agarose microbeads, it is preferred that organic phase described in the S2 be include toluene, three
The mixed solution of chloromethanes and sorbitan fatty ester.
The preparation method of above-mentioned compound agarose microbeads, it is preferred that the toluene, chloroform and Sorbitan
The volume ratio of alcohol fatty acid ester is 15 ~ 20 ︰, 5 ~ 10 ︰ 0.1 ~ 0.5.
The preparation method of above-mentioned compound agarose microbeads, it is preferred that the concentration of the enzyme A is 1 mg/mL of μ g/mL ~ 5；
The concentration of the enzyme B is 1 mg/mL of μ g/mL ~ 5.
The preparation method of above-mentioned compound agarose microbeads, it is preferred that the temperature of the water phase is 30 DEG C ~ 55 DEG C, described
The temperature of organic phase is 50 DEG C ~ 80 DEG C, and in the S2, it is 50 DEG C ~ 55 DEG C that water phase, which is added dropwise to reaction temperature in organic phase,.
In a total technical concept, the present invention also provides a kind of above-mentioned compound agarose microbeads to prevent in preparation
With the application in the drug for the treatment of cardiovascular disease.
Compared with the prior art, the advantages of the present invention are as follows:
(1) the present invention provides a kind of compound agarose microbeads, glucose oxidase and peroxidase are wrapped in agarose
In gel, the hydrogen bond action based on enzyme and Ago-Gel, enzyme, which is greatly concentrated and is coated in agarose, forms compound fine jade
Lipolysaccharide microballoon.Compound agarose microbeads are under glucose stimulation, the cascade catalysis based on glucose oxidase and peroxidase
Effect generates nitric oxide.The nitric oxide cascade that this agarose microbeads generates is controllable, and mode is novel, and precursor substance easily obtains,
There is huge application prospect in chemical industry catalysis, biomedicine.
(2) the present invention provides a kind of compound agarose microbeads, glucose oxidase and peroxidase are wrapped up simultaneously
In gel, the ordered fabrication of multienzyme is cascaded, can not only to form substrate channels between cascade enzyme, improve catalytic efficiency,
And the stability of enzyme can be enhanced.
(3) by microemulsion method that grape is glycoxidative the present invention provides a kind of preparation method of compound agarose microbeads
Enzyme and peroxidase are wrapped in agarose microbeads.Based on the reaction and physically trapping suction-operated between grease phase liquid level,
Two kinds of enzyme coatings enter in agarose microlayer model；The agarose microbeads of two kinds of enzymes of coating, this method packet are obtained by control temperature
It is big, easy to operate by measuring.Meanwhile agarose is inexpensively cheap, biological safety is good, is easy to be extended and applied.
(4) the present invention provides a kind of applications of compound agarose microbeads, and compound agarose microbeads can be with endogenous grape
Sugar is that reaction substrate stimulates gel complex microsphere controlled catalytic to generate nitric oxide, and supplement supply nitric oxide is conducive to improve
Blood nitric oxide concentration prevents and treats thrombus, the cardiovascular diseases such as hypertension to reach, can be applied to preparation prevention and
The drug for treating cardiovascular disease.
Detailed description of the invention
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, the technical scheme in the embodiment of the invention is clearly and completely described.
Fig. 1 is the schematic diagram of the agarose microbeads of coating cascade enzyme.
Fig. 2 is the nitric oxide of the compound agarose microbeads generation of the embodiment of the present invention 1 after Griess reagent is added
Ultraviolet characteristic light spectrogram.
Fig. 3 be the embodiment of the present invention 1 compound agarose microbeads catalysis generate nitric oxide, nitric oxide output and when
Between curve graph.
Fig. 4 is the enzyme phenogram of the compound agarose microbeads coating modification fluorescein of the embodiment of the present invention 2.
Fig. 5 is that the compound agarose microbeads catalysis of the embodiment of the present invention 2 generates nitric oxide production fluorescence imaging figure.
Fig. 6 is the ultraviolet spectrogram of the compound agarose microbeads of sample 1 to 4 in experimental example 4 of the present invention.
Specific embodiment
Below in conjunction with Figure of description and specific preferred embodiment, the invention will be further described, but not therefore and
It limits the scope of the invention.
Material employed in following embodiment and instrument are commercially available.
Embodiment 1:
A kind of compound agarose microbeads of the invention, including hydroxycarbamide, β-D-Glucose oxidoreducing enzyme and horseradish peroxidase
Enzyme, hydroxycarbamide, β-D-Glucose oxidoreducing enzyme and horseradish peroxidase are wrapped in agarose that form compound agarose micro-
Ball.Fig. 1 is the schematic diagram of the agarose microbeads of coating cascade enzyme, and preparation method includes the following steps:
(1) organic phase is prepared: by 18 mL toluene, 7 mL chloroforms and 0.3 mL sorbitan fatty ester mixing structure
At organic phase.By it is above-mentioned it is organic be added in 100 mL flasks, 55 DEG C are preheated at 1000 rpm.
(2) it prepares water phase: taking 5 ml water that 84 mg agaroses are added, dissolve by heating, obtain agarose solution.Work as agarose
When the temperature of solution is down to 55 DEG C, it is 7.0 that 0.1 mL pH, which is added, and concentration is that β-D-Glucose oxidoreducing enzyme of 1 mg/mL is molten
Liquid and 0.5 mL pH are 7.0, and concentration is the horseradish peroxidase solution of 1 mg/mL, is uniformly mixed, and constitute water phase.
(3) it mixes: before the temperature of water phase is down to 50 DEG C, being added dropwise in organic phase, under the mixing speed of 1800 rpm
15 min are cultivated, after being cooled to room temperature, 5 min is centrifuged using 5000 rpm speed, removes supernatant, collect centrifugation product.Upper
State in centrifugation product and continuously add a certain amount of hydroxycarbamide, until final concentration of 200 mmol/l of solution hydroxycarbamide, be incubated for after 1h from
The heart obtains that nitric oxide production compound agarose microbeads can be generated.
Embodiment 2:
A kind of compound agarose microbeads of the invention, including hydroxycarbamide, β-D-Glucose oxidoreducing enzyme and ferroso-ferric oxide are received
Rice grain, hydroxycarbamide, β-D-Glucose oxidoreducing enzyme and ferroferric oxide nano granules be wrapped in agarose formed it is compound
Agarose microbeads.Preparation method includes the following steps:
(1) organic phase is prepared: by 18 mL toluene, 7 mL chloroforms and 0.3 mL sorbitan fatty ester mixing structure
At organic phase.By it is above-mentioned it is organic be added in 100 mL flasks, 55 DEG C are preheated at 1000 rpm.
(2) it prepares water phase: taking 5 ml water that 84 mg agaroses are added, dissolve by heating, obtain agarose solution.Work as agarose
When the temperature of solution is down to 55 DEG C, it is 7.0 that 0.1 mL pH, which is added, and concentration is that β-D-Glucose oxidoreducing enzyme of 1 mg/mL is molten
Liquid and 0.5 mL pH are 7.0, and concentration is the ferroferric oxide nano granules solution of 1 mg/mL, and ultrasonic disperse constitutes water phase.
(3) it mixes: before the temperature of water phase is down to 50 DEG C, being added dropwise in organic phase, under the mixing speed of 1800 rpm
15 min are cultivated, after being cooled to room temperature, 5 min is centrifuged using 5000 rpm speed, removes supernatant, collect centrifugation product.Upper
It states in centrifugation product and continuously adds a certain amount of hydroxycarbamide, until final concentration of 200 mmol/l of hydroxycarbamide in solution, after being incubated for 1h
Centrifugation, obtains that nitric oxide production compound agarose microbeads can be generated.
Experimental example 1:
Take 200 μ L embodiments 1 agarose composite microsphere (total gel quality be 0.3 mg, number containing microballoon 2.0 × 105) plus
Enter in the glucose solution that concentration is 10 mmol/L, react 30 min, is added Griess reagent (nitric oxide detection reagent),
It is put into ultraviolet specrophotometer and detects characteristic absorption peak, and be not coated with the agarose microbeads of enzyme with unsupported nitric oxide precursors
As blank control.
Fig. 2 is the ultraviolet spectrogram of agarose composite microsphere, it can be seen that agarose composite microsphere generates an oxidation
Nitrogen makes Griess reagent have characteristic absorption peak at 540nm.
Fig. 3 is the curve of nitric oxide output and time.As we know from the figure: over time, nitric oxide production
Content increases in S curve.
It was found from Fig. 2 and Fig. 3: agarose composite microsphere can produce nitric oxide.
Experimental example 2:
It is coated with enzyme modification fluorescein in the compound agarose microbeads of embodiment 2, is placed on fluorescence microscope and observes, Fig. 4 is fine jade
Picture of the lipolysaccharide complex microsphere in fluorescence field and light field.
As can be known from Fig. 4: having green fluorescence in the agarose composite microsphere of embodiment 2, illustrate that enzyme is wrapped up agar of keeping forging ahead
Sugared microballoon.
Experimental example 3:
Take 200 μ L embodiments 2 compound agarose microbeads (total gel quality be 0.3 mg, number containing microballoon 2.0 × 105) set
In the glucose solution that concentration is 10 mmol/l, 30 min are reacted, DAF-2 fluorescence probe is added and is placed on fluorescence microscope
Observation, observation result are as shown in Figure 5: (excitation wavelength 495nm) discovery is observed under laser confocal microscope at two kinds of coating
There is yellow fluorescence appearance around the agarose microbeads of enzyme, shows that agarose generates nitric oxide.
Experimental example 4:
200 μ L samples 1,2,3,4 are taken respectively, in which:
Sample 1 is compound agarose microbeads (the total gelling for being coated with β-D-Glucose oxidoreducing enzyme and horseradish peroxidase
Amount is 0.3 mg, number containing microballoon 2.0 × 105), preparation method is same as Example 1.
Sample 2 is only to be coated with the compound agarose microbeads of horseradish peroxidase (total gel quality be 0.3 mg, containing microballoon
Number 2.0 × 105)；Remaining preparation method is same as Example 1.
Sample 3 be only be coated with the compound agarose microbeads of β-D-Glucose oxidoreducing enzyme (total gel quality is 0.3 mg,
Number containing microballoon 2.0 × 105)；Remaining preparation method is same as Example 1.
Sample 4 is not to be coated with the agarose microbeads of glucose oxidase and horseradish peroxidase (total gel quality is 0.3
Mg, number containing microballoon 2.0 × 105), unsupported nitric oxide precursors derivative, remaining preparation method is same as Example 1.
It is added in sample 1,2,3,4 in the glucose solution that concentration is 10 mmol/l respectively, reacts 30 min, be added
Griess reagent (nitric oxide detection reagent), is put into ultraviolet specrophotometer and detects characteristic absorption peak, and with unsupported one
Oxidation nitrogen precursor is not coated with the agarose microbeads (sample 4) of two fermentoids as blank control, is derived with loading nitric oxide precursors
Object is only coated with the agarose microbeads (sample 2, sample 3) of class of enzymes as experiment contrast.
Fig. 6 is the ultraviolet spectrogram of the compound agarose microbeads of sample 1 to 4, it can be seen that agarose composite microsphere produces
Raw a large amount of nitric oxides, can react with Griess reagent and generate purplish red color substance, there is characteristic absorption peak at 540nm.And sample 2
The agarose microbeads for being only coated with class of enzymes with the compound agarose microbeads of sample 3 cannot generate a large amount of nitric oxides, and sample 1 is answered
It closes agarose microbeads and generates nitric oxide production efficiency is the agarose microbeads of a coating horseradish peroxidase in sample 2 6.5
Times, it is 10.2 times of the agarose microbeads of a coating glucose oxidase in sample 3.
The above described is only a preferred embodiment of the present invention, being not intended to limit the present invention in any form.Though
So the present invention is disclosed as above with preferred embodiment, and however, it is not intended to limit the invention.It is any to be familiar with those skilled in the art
Member, in the case where not departing from Spirit Essence of the invention and technical solution, all using in the methods and techniques of the disclosure above
Appearance makes many possible changes and modifications or equivalent example modified to equivalent change to technical solution of the present invention.Therefore,
Anything that does not depart from the technical scheme of the invention are made to the above embodiment any simple according to the technical essence of the invention
Modification, equivalent replacement, equivalence changes and modification, all of which are still within the scope of protection of the technical scheme of the invention.
Claims (10)
1. a kind of compound agarose microbeads, which is characterized in that before the compound agarose microbeads include enzyme A, enzyme B, nitric oxide
Syntaxy object and agarose, the enzyme A, enzyme B and nitric oxide precursors derivative, which are wrapped in the agarose, forms compound fine jade
Lipolysaccharide microballoon, the enzyme A is glucose oxidase or class glucose oxidase, the enzyme B are peroxidase or class peroxidating
Object enzyme.
2. compound agarose microbeads according to claim 1, which is characterized in that the glucose oxidase is β-D- grape
Glycoxidative reductase；The class glucose oxidase is palladium nano-particles or gold nano grain.
3. compound agarose microbeads according to claim 1, which is characterized in that the peroxidase is horseradish peroxidating
Object enzyme；The peroxidase is ferroferric oxide nano granules, di-iron trioxide nano particle, prussian blue nano
One of the G- tetramer that grain, fullerene nano particle, manganese dioxide nano particle and hemin combine.
4. compound agarose microbeads according to any one of claim 1 to 3, which is characterized in that before the nitric oxide
Syntaxy object is hydroxycarbamide, L-arginine or N- hydroxyl L-arginine.
5. a kind of preparation method of compound agarose microbeads described in any one of Claims 1-4, which is characterized in that described
Preparation method the following steps are included:
S1, water phase will be formed in enzyme A and enzyme B addition agarose solution；
S2, the water phase is added dropwise in organic phase, centrifugation product is collected in stirring centrifugation；
S3, addition nitric oxide precursors derivative is incubated for obtain compound agarose microbeads in the centrifugation product.
6. the preparation method of compound agarose microbeads according to claim 5, which is characterized in that organic described in the S2
It is mutually the mixed solution for including toluene, chloroform and sorbitan fatty ester.
7. the preparation method of compound agarose microbeads according to claim 6, which is characterized in that the toluene, three chloromethanes
The volume ratio of alkane and sorbitan fatty ester is 15 ~ 20 ︰, 5 ~ 10 ︰ 0.1 ~ 0.5.
8. the preparation method of compound agarose microbeads according to any one of claims 5 to 7, which is characterized in that described
The concentration of enzyme A is 1 mg/mL of μ g/mL ~ 5；The concentration of the enzyme B is 1 mg/mL of μ g/mL ~ 5.
9. the preparation method of compound agarose microbeads according to any one of claims 5 to 7, which is characterized in that described
The temperature of water phase is 30 DEG C ~ 55 DEG C, and the temperature of the organic phase is 50 DEG C ~ 80 DEG C, and in the S2, water phase is added dropwise to organic phase
Middle reaction temperature is 50 DEG C ~ 55 DEG C.
10. a kind of compound agarose microbeads described in any one of Claims 1-4 prevent and treat cardiovascular disease in preparation
Drug in application.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112169717A (en) * | 2020-09-30 | 2021-01-05 | 深圳大学 | Microencapsulated hemin and preparation method and application thereof |
| CN112741930A (en) * | 2020-12-22 | 2021-05-04 | 四川大学 | Enzyme modified anticoagulation valve and preparation method thereof |
| CN113575851A (en) * | 2021-08-05 | 2021-11-02 | 汪秋兰 | Reproduced rice with reduced phosphorus and protein content and processing method thereof |
-
2019
- 2019-03-15 CN CN201910196474.0A patent/CN109999008B/en active Active
Non-Patent Citations (2)
| Title |
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| JINMING HUANG等: "Horseradish Peroxidase Catalyzed Nitric Oxide Formation from Hydroxyurea", 《J. AM. CHEM. SOC. 》 * |
| WENPEI FAN等: "Glucose-Responsive Sequential Generation of Hydrogen Peroxide and Nitric Oxide for Synergistic Cancer Starving-Like/Gas Therapy", 《ANGEW. CHEM. INT. ED.》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112169717A (en) * | 2020-09-30 | 2021-01-05 | 深圳大学 | Microencapsulated hemin and preparation method and application thereof |
| CN112741930A (en) * | 2020-12-22 | 2021-05-04 | 四川大学 | Enzyme modified anticoagulation valve and preparation method thereof |
| WO2022135453A1 (en) * | 2020-12-22 | 2022-06-30 | 吉林启明皓月生物科技有限公司 | Enzyme-modified anticoagulant valve and manufacturing method therefor |
| CN113575851A (en) * | 2021-08-05 | 2021-11-02 | 汪秋兰 | Reproduced rice with reduced phosphorus and protein content and processing method thereof |
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