CN105854026A - Basic protein entrapment vector, preparation method of entrapment vector and entrapment method - Google Patents
Basic protein entrapment vector, preparation method of entrapment vector and entrapment method Download PDFInfo
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- CN105854026A CN105854026A CN201610210632.XA CN201610210632A CN105854026A CN 105854026 A CN105854026 A CN 105854026A CN 201610210632 A CN201610210632 A CN 201610210632A CN 105854026 A CN105854026 A CN 105854026A
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- basic protein
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- carbonate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
- A61K38/4826—Trypsin (3.4.21.4) Chymotrypsin (3.4.21.1)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21001—Chymotrypsin (3.4.21.1)
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical & Material Sciences (AREA)
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- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a basic protein entrapment vector, a preparation method of the entrapment vector and an entrapment method. The preparation method comprises the following steps: at room temperature, stirring and mixing polyanion electrolyte and calcium salt; in a rapid stirring or ultrasonic process, quickly adding carbonate for co-precipitation to form microspheres; performing washing, centrifugation and drying to obtain polyanion electrolyte doped calcium carbonate microspheres; mixing a basic protein solution of certain concentration with the calcium carbonate microspheres; performing low-temperature stirring or vibration incubation for 15-90 minutes; and performing centrifugation, supernate removal and freeze drying to obtain basic protein-entrapped calcium carbonate microspheres, wherein the size distribution of the particles is narrow, the dispersity is good, and the biocompatibility is high. The advantage of the invention is that high-concentration and high-activity entrapped basic protein can be obtained under mild conditions.
Description
Technical field
The invention belongs to technical field of biological material, specifically refer to basic protein bag and carry carrier, bag load support preparation method and bag
Support method.
Background technology
Calcium carbonate is that a kind of consumption is very big, the new inorganic material having many uses, be widely used in coating, rubber, plastics,
Multiple fields such as papermaking, ink, food, cosmetics and medicine.The actual application of calcium carbonate and its character (as pattern, particle diameter,
Zeta potential, specific surface area etc.) closely related.
Biodegradable medical macromolecular materials, for diagnosing, treating and the material of neomorph, extensively should in recent years
Put the fields such as carrier for medicine controlled releasing, its research occupies highly important in fields such as biotechnology, life sciences and medical science
Position.
Along with the layer-by-layer (LBL) development in terms of masking, field of medicaments utilizes LBL technology to prepare microcapsule
Reach bag medicine carrying thing, while protection curative effect of medication, effectively realize the slow release of medicine.Calcium carbonate is utilized to prepare micro-glue for template
Capsule, adds diluted acid and decomposes or ethylenediaminetetraacetic acid (EDTA) complexing calcium ions, remove CaCO3Colloidal particle forms the micro-of high concentration
Capsule, reaches low bio-toxicity and efficient bag carries purpose.Li Junbai etc. (Chinese patent CN 102921014 A) disclose one
Planting and prepare the bio-compatible Nano medication complex carrier with synergistic antitumor effect, template is exactly porous, inorganic nanoparticle carbon
Acid calcium.Zhu Limins etc. (Chinese patent CN103585638 A), by sodium alginate, sodium carbonate and calcium chloride co-percipitation, are prepared
The calcium carbonate micron ball of doping alginate；Tang Hua etc. (Chinese patent CN 102992374 A) utilize add polystyrene with
The method of calcium carbonate co-precipitation prepares the calcium carbonate micron ball of good dispersion, and this absolutely proves that calcium carbonate granule can as template
Modify utilizing polyanion electrolyte to reach well.
Basic protein generally refers to isoelectric point, IP and is more than 7 than the protein of meta-alkali under common physiological condition, i.e. isoelectric point, IP
All albumen.In vivo, the effect of many basic proteins is particularly significant, such as aprotinin etc..
But high concentration and highly active bag can carry that to transmit these basic proteins particularly important.Document [De
Temmerman M L, Demeester J, De Vos F, et al. Encapsulation performance of
layer-by-layer microcapsules for proteins[J]. Biomacromolecules, 2011, 12(4):
1283-1289.] show, calcium carbonate can realize wrapping in a large number load for acidic protein, document [Balabushevich N G,
Lopez de Guerenu A V, Feoktistova N A, et al. Protein-Containing Multilayer
Capsules by Templating on Mesoporous CaCO3 Particles: POST- and PRE- Loading
Approaches [J]. Macromolecular bioscience, 2016,16 (1): 95-105.] and document [Hassani
L N, Hindré F, Beuvier T, et al. Lysozyme encapsulation into nanostructured
CaCO3 microparticles using a supercritical CO2 process and comparison with the
Normal route [J]. Journal of Materials Chemistry B, 2013,1 (32): 4011-4019.] also
All represent that calcium carbonate is difficult to a large amount of bag and carries basic protein.This is because in neutral aqueous solution, calcium carbonate and alkaline protein are equal
With electropositive, when co-precipitation or absorption bag carry, they are the most identical and mutually exclusive, it is difficult to reach to wrap in a large number load.
Therefore, by retrieval, utilize efficient calcium carbonate bag to carry the research of basic protein the most in a mild condition
Not report.
Summary of the invention
The invention aims to the shortcoming and defect overcoming prior art to exist, and provide one to can be suitably used in temperature
Under the conditions of with, high concentration and highly active bag carry the bag load carrier of basic protein.
Second object of the present invention is to provide a kind of basic protein bag and carries the preparation method of carrier.
Third object of the present invention is to provide the efficient packet support method of a kind of basic protein.
For realizing first purpose of the present invention, the technical scheme is that this bag carries carrier is polyanion electrolyte
Doping calcium carbonate micron ball, a diameter of 1 ~ 5 μm, in cellular structures, recording aperture is 10 ~ 80nm.Carrier is capable of
Carrying basic protein bag, the surface being because doping polyanion electrolyte calcium carbonate micron ball exists stronger with basic protein
Electrostatic force, and its loose structure adds the specific surface area of micron ball, thus add carrier and the bag of basic protein is carried
Amount.
Arrange further be described polyanion electrolyte be kayexalate, polyacrylic acid, polymethyl
In acid, chondroitin sulfate, heparin sodium, hyaluronic acid, dextran sulfate, carboxymethyl cellulose, carrageenan, sodium alginate a kind of or
Multiple combination.
For realizing second object of the present invention, its technical scheme is the preparation method that a kind of basic protein bag carries carrier,
Comprise the following steps:
At 4 ~ 40 DEG C, by molten for calcium salt soln and the polyanion electrolyte that concentration is 0.1 ~ 20mg/ml that concentration is 0.1 ~ 1M
After liquid mixing, stirring and evenly mixing, when being stirred vigorously afterwards or be ultrasonic, rapidly join molten with the carbonate of calcium salt soln equimolar amounts
Liquid, stirs 30 ~ 60s, stands 10 ~ 30min, generates polyanion electrolyte doping calcium carbonate micron ball, calcium salt soln, poly-cloudy from
The volume ratio of sub-electrolyte solution and carbonate solution is (1 ~ 3): (0.5 ~ 1.5): (1.5 ~ 4.5), the polyanion that will generate
Electrolyte doping calcium carbonate micron ball pelleting centrifugation is collected, and then cleans and removes solution residual.
The calcium salt being described is set further and includes that calcium chloride or calcium nitrate, described carbonate include ammonium hydrogen carbonate, carbon
Acid sodium or potassium carbonate.
The polyanion electrolyte being described is set further and includes kayexalate, polyacrylic acid, poly-methyl-prop
In olefin(e) acid, chondroitin sulfate, heparin sodium, hyaluronic acid, dextran sulfate, carboxymethyl cellulose, carrageenan, sodium alginate one
Plant or multiple combination.
Arrange further be described in be stirred vigorously and refer to that mixing speed is at 400 ~ 1500 revs/min；And ultrasonic refer to frequency
At 20 ~ 60kHz.
For realizing third object of the present invention, its technical scheme is to take polyanion electrolyte doping calcium carbonate micron ball
Being placed in centrifuge tube, add the alkaline protein solution of 0.1 ~ 3mg/ml, hatch 15 ~ 90 minutes at 0 ~ 30 DEG C, centrifugal, lyophilizing is i.e.
Bag can be obtained and carry the calcium carbonate micron ball of basic protein.
The basic protein being described is set further and includes all albumen that isoelectric point, IP is more than 7.
It is an advantage of the current invention that:
(1) this carrier can realize carrying for the bag of all basic proteins, has the widest universality, doping polyanion electrolysis
There is stronger electrostatic force with basic protein in the surface of matter calcium carbonate micron ball, and its loose structure adds micron ball
Specific surface area, thus considerably increase the carrier bag carrying capacity to basic protein.
(2) this carrier is high for the bag carrying capacity of basic protein；
(3) the basic protein carrier that this bag support method is formed has good biocompatibility；
(4) this basic protein carrier is easily achieved the release of albumen, and is prone to wrap load further or modify；
(5) preparation of this carrier and the bag support method of basic protein are simple, and equipment cost is low, simple operation, it is adaptable to industrialization
Produce.
The basic protein bag of the application carries carrier and can ensure that basic protein has the bioactive native conformation of maintenance, tool
There is the universality and high efficiency that basic protein bag is carried.Preparation method and bag support method, technique is simple, and mild condition can be often
Warm water solution is carried out.Additionally, the decorative material kind that the method can use is many, bag carries the particle surface that basic protein obtains
With electrically, it is easy to do further layer assembly or modification etc. on surface, calcium carbonate bio-toxicity is low, it is easy under temperate condition
Remove.Therefore, the method can as albumen transmission template, be widely used in albumen and protein drug etc. bag carry, transmission and
The fields such as controlled release, have well research and application prospect in biological medicine Material Field.
Below in conjunction with specification drawings and specific embodiments, the present invention is described further.
Accompanying drawing explanation
The heparin sodium that Fig. 1 provides for the present invention modifies calcium carbonate micron ball；
It is 1.5mg/ml lysozyme that the 20mg heparin sodium that Fig. 2 provides for the present invention is modified under calcium carbonate difference pH 1ml concentration
Bag load situation.
Detailed description of the invention
Below by embodiment, the present invention is specifically described, is served only for the present invention being further described, no
It is understood that for limiting the scope of the present invention, the technician in this field can be according to the content of foregoing invention to the present invention
Make some nonessential improvement and adjustment.
Embodiment 1
The present embodiment relates to the calcium carbonate micron ball bag support method for chymotrypsin protein of a kind of heparin sodium that adulterates.
(1), by after calcium chloride solution that 2ml concentration is 0.5M and heparin sodium mixing that 1ml concentration is 6mg/ml, stirring 1 ~
5min, afterwards under 900 revs/min, rapidly joins the sodium carbonate liquor that 3ml concentration is 0.33M, stirs 35 ~ 40s, stand
15min, collects centrifugal for the precipitation of calcium carbonate containing heparin sodium generated, washes 3 times with water.The carbonic acid containing heparin sodium of preparation
Calcium mean particle dia is about 2 ~ 5 μm.
(2), taking the most above-mentioned calcium carbonate microparticle containing heparin sodium and be placed in the centrifuge tube of 1.5ml, adding 1ml concentration is
The chymotrypsin protein solution (pH=7.2) of 1mg/ml, hatches 30min at 4 DEG C, centrifugal, and lyophilizing i.e. can obtain bag and carry pancreas curdled milk egg
The calcium carbonate microparticle of white heparin sodium, albumen bag load rate is up to more than 95%.
Embodiment 2
The present embodiment relates to the calcium carbonate micron ball bag support method for lysozyme of a kind of heparin sodium that adulterates.
(1), by after calcium chloride solution that 2ml concentration is 0.5M and heparin sodium mixing that 1ml concentration is 6mg/ml, stirring 1 ~
5min, afterwards under 1000 revs/min, rapidly joins the sodium carbonate liquor that 3ml concentration is 0.33M, stirs 35 ~ 40s, stand
25min, collects centrifugal for the precipitation of calcium carbonate containing heparin sodium generated, washes 3 times with water.The carbonic acid of the doping heparin sodium of preparation
Calcium micron ball diameter is about 2 ~ 5 μm, sees Fig. 1.
(2), the calcium carbonate micron ball that takes appropriate above-mentioned doping heparin sodium be placed in the centrifuge tube of 1.5ml, add 1ml concentration
For the lysozyme soln (pH=7.2) of 1mg/ml, hatching 60min at 10 DEG C, centrifugal, lyophilizing i.e. can obtain bag and carry lysozyme
The calcium carbonate micron ball of doping heparin sodium, albumen bag load rate is up to more than 92%..
Embodiment 3
The present embodiment relates to the calcium carbonate micron ball bag support method for lysozyme of a kind of doped sulfuric acid glucosan.
(1), by after calcium chloride solution that 2ml concentration is 0.5M and dextran sulfate mixing that 1ml concentration is 6mg/ml, stir
Mix 1 ~ 5min, afterwards under 1200 revs/min, rapidly join the sodium carbonate liquor that 3ml concentration is 0.33M, stir 35 ~ 40s, quiet
Put 20min, collect centrifugal for the precipitation of calcium carbonate of the doped sulfuric acid glucosan generated, wash 3 times with water.The doped sulfuric acid Portugal of preparation
The calcium carbonate microparticle diameter of polysaccharide is about 2 ~ 5 μm.
(2), the calcium carbonate microparticle that takes appropriate above-mentioned doped sulfuric acid glucosan be placed in the centrifuge tube of 1.5ml, add 1ml dense
Degree is the lysozyme soln (pH=7.2) of 1mg/ml, hatches 90min at 25 DEG C, centrifugal, and lyophilizing i.e. can obtain bag and carry lysozyme
The calcium carbonate micron ball of doped sulfuric acid glucosan, albumen bag load rate is up to more than 94%.
Embodiment 4
The present embodiment relates to the calcium carbonate micron ball bag support method for lysozyme of a kind of heparin sodium that adulterates.
(1), by after calcium chloride solution that 2ml concentration is 0.5M and heparin sodium mixing that 1ml concentration is 6mg/ml, stirring 1 ~
5min, afterwards under 1200 revs/min, rapidly joins the sodium carbonate liquor that 3ml concentration is 0.33M, stirs 35 ~ 40s, stand
15min, collects centrifugal for the precipitation of calcium carbonate of the doping heparin sodium generated, washes 3 times with water.The carbonic acid of the doping heparin sodium of preparation
Calcium mean particle dia is about 2 ~ 5 μm.
(2), the calcium carbonate microparticle that takes appropriate above-mentioned doping heparin sodium be placed in the centrifuge tube of 1.5ml, adding 1ml concentration is
The lysozyme soln (pH=7.0) of 2mg/ml, hatches 30min at 25 DEG C, centrifugal, and lyophilizing i.e. can obtain bag and carry mixing of lysozyme
The calcium carbonate micron ball of miscellaneous heparin sodium, albumen bag load rate is up to more than 70%, as shown in Figure 2.
Embodiment 5
The present embodiment relates to the calcium carbonate micron ball bag support method for lysozyme of a kind of heparin sodium that adulterates.
(1), by after calcium chloride solution that 2ml concentration is 0.5M and heparin sodium mixing that 1ml concentration is 6mg/ml, stirring 1 ~
5min, afterwards under 1200 revs/min, rapidly joins the sodium carbonate liquor that 3ml concentration is 0.33M, stirs 35 ~ 40s, stand
15min, collects centrifugal for the precipitation of calcium carbonate of the doping heparin sodium generated, washes 3 times with water.The carbonic acid of the doping heparin sodium of preparation
Calcium mean particle dia is about 2 ~ 5 μm.
(2), the calcium carbonate microparticle that takes appropriate above-mentioned doping heparin sodium be placed in the centrifuge tube of 1.5ml, adding 1ml concentration is
The lysozyme soln (pH=11.5) of 1.5mg/ml, hatches 30min at 25 DEG C, centrifugal, and lyophilizing i.e. can obtain bag and carry lysozyme
The calcium carbonate micron ball of doping heparin sodium, albumen bag load rate is up to more than 98%, as shown in Figure 2.
Claims (8)
1. a basic protein bag carries carrier, it is characterised in that: it is that polyanion electrolyte doping calcium carbonate is micro-that this bag carries carrier
Rice ball, diameter is 1 ~ 5 μm, and stereoscan photograph shows that its microsphere Cross Section Morphology is cellular structures, records aperture
It is 10 ~ 80nm.
The bag of a kind of basic protein the most according to claim 1 carries carrier, it is characterised in that: described polyanion electrolysis
Matter be kayexalate, polyacrylic acid, polymethylacrylic acid, chondroitin sulfate, heparin sodium, hyaluronic acid, sulphuric acid Portugal gather
One or more combinations in sugar, carboxymethyl cellulose, carrageenan, sodium alginate.
3. the preparation method of a basic protein bag load carrier, it is characterised in that: comprise the following steps:
At 4 ~ 40 DEG C, by molten for calcium salt soln and the polyanion electrolyte that concentration is 0.1 ~ 20mg/ml that concentration is 0.1 ~ 1M
After liquid mixing, stirring and evenly mixing, when being stirred vigorously afterwards or be ultrasonic, rapidly join molten with the carbonate of calcium salt soln equimolar amounts
Liquid, stirs 30 ~ 60s, stands 10 ~ 30min, generates polyanion electrolyte doping calcium carbonate micron ball, calcium salt soln, poly-cloudy from
The volume ratio of sub-electrolyte solution and carbonate solution is (1 ~ 3): (0.5 ~ 1.5): (1.5 ~ 4.5), the polyanion that will generate
Electrolyte doping calcium carbonate micron ball pelleting centrifugation is collected, and then cleans and removes solution residual.
A kind of basic protein bag the most according to claim 3 carries the preparation method of carrier, it is characterised in that: described calcium salt
Including calcium chloride or calcium nitrate, described carbonate includes ammonium hydrogen carbonate, sodium carbonate or potassium carbonate.
A kind of basic protein bag the most according to claim 3 carries the preparation method of carrier, it is characterised in that: described poly-the moon
Ionic electrolytes includes kayexalate, polyacrylic acid, polymethylacrylic acid, chondroitin sulfate, heparin sodium, hyalomitome
One or more combinations in acid, dextran sulfate, carboxymethyl cellulose, carrageenan, sodium alginate.
A kind of basic protein bag the most according to claim 3 carries the preparation method of carrier, it is characterised in that: described is violent
Stirring refers to that mixing speed is at 400 ~ 1500 revs/min；And ultrasonic refer to that frequency is at 20 ~ 60kHz.
7. a basic protein bag support method for carrier, its feature is carried based on the basic protein bag one of claim 1-2 Suo Shu
It is: taking polyanion electrolyte doping calcium carbonate micron ball and be placed in centrifuge tube, the basic protein adding 0.1 ~ 3mg/ml is molten
Liquid, hatches 15 ~ 90 minutes at 0 ~ 30 DEG C, centrifugal, and lyophilizing i.e. can obtain bag and carry the calcium carbonate micron ball of basic protein.
The bag support method of basic protein the most according to claim 7, it is characterised in that: described basic protein includes waiting electricity
The point all albumen more than 7.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108014727A (en) * | 2017-12-13 | 2018-05-11 | 温州生物材料与工程研究所 | The method that one step absorption method prepares cationic polyelectrolyte microcapsules |
| CN108451924A (en) * | 2017-12-13 | 2018-08-28 | 温州生物材料与工程研究所 | The method that one step absorption method prepares protein microcapsules |
| CN110756181A (en) * | 2019-10-25 | 2020-02-07 | 西安医学院 | Magnetic adsorbent with surface modified polyacrylic acid and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108014727A (en) * | 2017-12-13 | 2018-05-11 | 温州生物材料与工程研究所 | The method that one step absorption method prepares cationic polyelectrolyte microcapsules |
| CN108451924A (en) * | 2017-12-13 | 2018-08-28 | 温州生物材料与工程研究所 | The method that one step absorption method prepares protein microcapsules |
| CN108451924B (en) * | 2017-12-13 | 2020-03-27 | 温州生物材料与工程研究所 | Method for preparing protein microcapsule by one-step adsorption method |
| CN110756181A (en) * | 2019-10-25 | 2020-02-07 | 西安医学院 | Magnetic adsorbent with surface modified polyacrylic acid and preparation method thereof |
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Application publication date: 20160817 |