CN106177924A - The preparation method of nanoscale lactoferrin chitosan particle - Google Patents
The preparation method of nanoscale lactoferrin chitosan particle Download PDFInfo
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- CN106177924A CN106177924A CN201610560376.7A CN201610560376A CN106177924A CN 106177924 A CN106177924 A CN 106177924A CN 201610560376 A CN201610560376 A CN 201610560376A CN 106177924 A CN106177924 A CN 106177924A
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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
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
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- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
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- C08K3/32—Phosphorus-containing compounds
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- C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
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- C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
- C08J2305/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K3/00—Use of inorganic substances as compounding ingredients
- C08K3/32—Phosphorus-containing compounds
- C08K2003/321—Phosphates
- C08K2003/324—Alkali metal phosphate
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Abstract
The preparation method of nanoscale lactoferrin chitosan particle, relates to the preparation method of a kind of lactoferrin chitosan particle.The particle diameter being intended to solve existing lactoferrin chitosan particle is relatively big, envelop rate and the low problem of carrying drug ratio.Method: one, preparation chitosan solution, lysozyme mixed liquor and lactoferrin solution；Two, in chitosan solution, add lysozyme mixed liquor, concussion, boil, obtain modified chitosan solution；Three, lactoferrin solution is mixed with modified chitosan solution, stirring, add sodium tripolyphosphate solution stirring；Four, centrifuging and taking precipitation, is lactoferrin chitosan nanoparticles.Oligo-chitosan prepared by the method is conducive to body to absorb, and has higher biological activity, is easier to expose and cross-link with phosphate radical compared to long-chain chitosan molecule protonated amino simultaneously, thus strengthens the combination degree of medicine-carried system.Envelop rate is up to 30%, and carrying drug ratio is up to 26%.Preparation for oral lactoferrin medicine.
Description
Technical field
The present invention relates to the preparation method of a kind of lactoferrin chitosan particle.
Background technology
Lactoferrin is the protide medicine with extensive biological function, is directly administered by oral lactoferrin
Though convenient, protein medicaments needs to overcome a large amount of obstacle from digestive system entrance blood circulation, and chitosan is as load
Protein medicaments carrier is while keeping self slow release characteristic, it is also possible to protect environmentally sensitive protein medicaments from fall
Solve, thus be the desirable combination of medicine carrying microgranule.
Preparing the microgranule of lactoferrin chitosan at present, its size is micron number magnitude, simultaneously preparation method such as breast
Changing cross-linking method, there is certain toxicity, is not suitable for directly acting on human body in required cross-linking agent such as glutaraldehyde etc.；Additionally, prepare
Medicine carrying granule character can be affected by factors such as chitosan raw material relevant parameter and reaction conditions.
Summary of the invention
The present invention is to solve that the particle diameter of existing lactoferrin chitosan particle is relatively big, for micron order, envelop rate and medicine carrying
The problem that rate is low, it is provided that the preparation method of nanoscale lactoferrin chitosan particle.
The present invention prepares lactoferrin chitosan nanoparticles by ionic cross-linking, and the method uses tripolyphosphate
Sodium, as cross-linking agent, carries out ionic gel inductionization to chitosan, in the range of specific pH, and the protonated amino of chitosan and three
The phosphate radical of polyphosphate sodium interacts, and forms pharmaceutical carrier parcel lactoferrin.
The preparation method of nanoscale lactoferrin chitosan particle of the present invention, sequentially includes the following steps:
One, agent treated:
Dissolving the chitosan in glacial acetic acid, adjustment pH is 4-5, and prepared concentration is that the chitosan of 0.6-0.8mg/mL is molten
Liquid；
9-11mg lysozyme is added 1ml sterilized water or 10mmol/L Tris Cl (pH8.0) solution, obtains lysozyme
Mixed liquor；
Lactoferrin is dissolved in 0.1mol/L sodium chloride solution, and concussion mixing, prepared concentration is the newborn ferrum egg of 9-11mg/mL
White solution；
Two, chitin modified:
In chitosan solution, add appropriate lysozyme mixed liquor, shake 3-4 hour in 38-42 DEG C 75-85 rev/min,
Being oligochitosan by long-chain degradation of chitosan, then heated and boiled 3-5 minute makes lysozyme inactivate, and obtains modified chitosan molten
Liquid；
Three, crosslinking:
Appropriate lactoferrin solution is mixed with modified chitosan solution, makes every 50ml chitosan solution contain
Lactoferrin 5mg-10mg, continuing magnetic force stirs, is added dropwise over 0.5-0.6mg/mL sodium tripolyphosphate solution simultaneously and carries out ion
Crosslinking, continuing magnetic force stirs 30-40 minute, and solution entirety presents blue-opalescent；
Four, separate:
By step 3 cross-link after solution under the conditions of 5-15 DEG C 15000-25000 rev/min centrifugal 0.5-1 hour,
Take precipitation, be lactoferrin chitosan nanoparticles.
Further, in step 2, the volume ratio of chitosan solution and lysozyme mixed liquor is 1000:1.
Further, in step 3, the rate of addition of sodium tripolyphosphate solution is 1 drop/sec.
Further, modified in step 3 chitosan solution is (4-5) with the volume ratio of sodium tripolyphosphate solution: 1.
Beneficial effects of the present invention:
The present invention utilizes lysozyme can hydrolyze β-this feature of Isosorbide-5-Nitrae glycosidic bond, and be hydrolyzed modification to chitosan, modified
Rear oligochitosan is mainly 2-7 and gathers, and molecular weight substantially 3000-10000D, oligo-chitosan is conducive to body to absorb, and has higher
Biological activity, is easier to expose and cross-link with phosphate radical compared to long-chain chitosan molecule protonated amino simultaneously, thus strengthens
The combination degree of medicine-carried system, preparing chitosan lactoferrin nanometer medicine-carried system for ionic cross-linking provides reference.
Present invention preparation isolated nano level lactoferrin chitosan particle, the method utilization has no side effect
Sodium tripolyphosphate carries out ion induction gelation to chitosan, utilizes the protonated amino of positively charged and electronegative phosphate radical phase
Attract mutually, medicine is wrapped to form drug-carrying nanometer particle by cross-linking process.Nanoscale lactoferrin chitosan prepared by this method is micro-
Grain has higher envelop rate and carrying drug ratio.The microgranule natural air drying obtained after being centrifuged, is measured by Coomassie Brilliant Blue
Protein concentration in clear liquid, in conjunction with the amount of input protein drug, the envelop rate of estimation protein drug and carrying drug ratio, measurement result
For: envelop rate is 20%-30%, and carrying drug ratio is 9%-26%.
The inventive method is easy and simple to handle, it is adaptable to the preparation of oral lactoferrin medicine.
Accompanying drawing explanation
Fig. 1 is the SEM figure of the lactoferrin chitosan nanoparticles of embodiment 1 preparation.
Detailed description of the invention
Technical solution of the present invention is not limited to act detailed description of the invention set forth below, also includes between each detailed description of the invention
Combination in any.
Detailed description of the invention one: the preparation method of present embodiment nanoscale lactoferrin chitosan particle, by following step
Suddenly carry out:
One, agent treated:
Dissolving the chitosan in glacial acetic acid, adjustment pH is 4-5, and prepared concentration is that the chitosan of 0.6-0.8mg/mL is molten
Liquid；
9-11mg lysozyme is added 1ml sterilized water or Tris Cl (pH8.0) solution of 1ml 10mmol/L, obtains molten
Bacterium enzyme mixation；
Lactoferrin is dissolved in 0.1mol/L sodium chloride solution, and concussion mixing, prepared concentration is the newborn ferrum egg of 9-11mg/mL
White solution；
Two, chitin modified:
In chitosan solution, add appropriate lysozyme mixed liquor, shake 3-4 hour in 38-42 DEG C 75-85 rev/min,
Being oligochitosan by long-chain degradation of chitosan, then heated and boiled 3-5 minute makes lysozyme inactivate, and obtains modified chitosan molten
Liquid；
Three, crosslinking:
Appropriate lactoferrin solution is mixed with modified chitosan solution, makes every 50ml chitosan solution contain
Lactoferrin 5mg-10mg, continuing magnetic force stirs, is added dropwise over 0.5-0.6mg/mL sodium tripolyphosphate solution simultaneously and carries out ion
Crosslinking, continuing magnetic force stirs 30-40 minute, and solution entirety presents blue-opalescent；
Four, separate:
By step 3 cross-link after solution under the conditions of 5-15 DEG C 15000-25000 rev/min centrifugal 0.5-1 hour,
Take precipitation, be lactoferrin chitosan nanoparticles.
Detailed description of the invention two: present embodiment is unlike detailed description of the invention one: preparing concentration in step one is
The chitosan solution of 0.7mg/mL.Other is identical with detailed description of the invention one.
Detailed description of the invention three: present embodiment is unlike detailed description of the invention one or two: prepare dense in step one
Degree is the lactoferrin solution of 10mg/mL.Other is identical with detailed description of the invention one or two.
Detailed description of the invention four: present embodiment is unlike one of detailed description of the invention one to three: step 2 mesochite
Polysaccharide solution is 1000:1 with the volume ratio of lysozyme mixed liquor.Other is identical with one of detailed description of the invention one to three.
Detailed description of the invention five: present embodiment is unlike one of detailed description of the invention one to four: in step 2 in
40 DEG C 80 revs/min shake 3 hours.Other is identical with one of detailed description of the invention one to four.
Detailed description of the invention six: present embodiment is unlike one of detailed description of the invention one to five: make in step 3
Every 50ml chitosan solution contains lactoferrin 6mg-9mg.Other is identical with one of detailed description of the invention one to five.
Detailed description of the invention seven: present embodiment is unlike one of detailed description of the invention one to five: make in step 3
Every 50ml chitosan solution contains lactoferrin 7mg-8mg.Other is identical with one of detailed description of the invention one to five.
Detailed description of the invention eight: present embodiment is unlike one of detailed description of the invention one to seven: in step 3 three
The rate of addition of polyphosphate sodium solution is 1 drop/sec.Other is identical with one of detailed description of the invention one to seven.
Detailed description of the invention nine: present embodiment is unlike one of detailed description of the invention one to eight: change in step 3
Chitosan solution after property is (4-5) with the volume ratio of sodium tripolyphosphate solution: 1.Other with detailed description of the invention one to eight it
One is identical.
Detailed description of the invention ten: present embodiment is unlike one of detailed description of the invention one to nine: in step 3
It is centrifuged under the conditions of 10 DEG C.Other is identical with one of detailed description of the invention one to nine.
Detailed description of the invention 11: present embodiment is unlike one of detailed description of the invention one to ten: in step 3
Centrifugal rotational speed is 20000 revs/min.Other with detailed description of the invention one to ten together.
Detailed description of the invention 12: present embodiment is unlike one of detailed description of the invention one to ten one: step 3
Middle centrifugation time is 0.7-0.8 hour.Other is identical with one of detailed description of the invention one to ten one.
For checking beneficial effects of the present invention, carry out following experiment:
Embodiment 1
The preparation method of the present embodiment nanoscale lactoferrin chitosan particle, sequentially includes the following steps:
One, agent treated:
Dissolving the chitosan in glacial acetic acid, adjusting pH is 4.5, and prepared concentration is the chitosan solution of 0.7mg/mL；
10mg lysozyme adds 1ml sterilized water, obtains lysozyme mixed liquor；
Lactoferrin is dissolved in 0.1mol/L sodium chloride solution, and concussion mixing, prepared concentration is that the lactoferrin of 10mg/mL is molten
Liquid；
Two, chitin modified:
Adding appropriate lysozyme mixed liquor to chitosan solution, chitosan solution with the volume ratio of lysozyme mixed liquor is
1000:1,40 DEG C 80 revs/min shake 3 hours, are oligochitosan by long-chain degradation of chitosan, and then heated and boiled makes molten in 5 minutes
Bacterium enzyme inactivates, and obtains modified chitosan solution；
Three, crosslinking:
Appropriate lactoferrin solution is mixed with modified chitosan solution, makes every 50ml chitosan solution contain
Lactoferrin 5mg, continuing magnetic force stirs, is added dropwise over 0.5mg/mL sodium tripolyphosphate solution simultaneously and carries out ionomer, modified
After the volume ratio of chitosan solution and sodium tripolyphosphate solution be 4.5:1, continuing magnetic force stirs 30 minutes, and solution entirety presents
Blue-opalescent；Wherein the rate of addition of sodium tripolyphosphate solution is 1 drop/sec.
Four, separate:
In solution after step 3 is cross-linked under the conditions of 10 DEG C 20000 revs/min centrifugal 1 hour, take precipitation, be breast
Ferritin chitosan nanoparticles.
The SEM scanning result of microgranule prepared by the present embodiment is as shown in Figure 1, it can be seen that this method has obtained nano level
Lactoferrin chitosan particle.
The microgranule natural air drying obtained after being centrifuged, measures the protein concentration in supernatant, knot by Coomassie Brilliant Blue
Closing the amount putting into protein drug, the envelop rate of estimation protein drug and carrying drug ratio, measurement result is: envelop rate is 30%, medicine carrying
Rate is 26%.
This method utilizes lysozyme can hydrolyze β-this feature of Isosorbide-5-Nitrae glycosidic bond, and be hydrolyzed modification to chitosan, modified
Rear oligochitosan is mainly 2-7 and gathers, and molecular weight is 3000-10000D, and mean molecule quantity is 5000-5500D, and oligo-chitosan is favourable
Absorb in body, have a higher biological activity, be easier to expose compared to long-chain chitosan molecule protonated amino simultaneously and with
Phosphate radical cross-links, thus strengthens the combination degree of medicine-carried system, prepares chitosan lactoferrin nano drug-carrying for ionic cross-linking
System provides reference.
Claims (10)
1. the preparation method of nanoscale lactoferrin chitosan particle, it is characterised in that the method sequentially includes the following steps:
One, agent treated:
Dissolving the chitosan in glacial acetic acid, adjustment pH is 4-5, and prepared concentration is the chitosan solution of 0.6-0.8mg/mL；
9-11mg lysozyme is added 1ml sterilized water or the Tris Cl solution of 1ml 10mmol/L, obtains lysozyme mixed liquor；
Lactoferrin is dissolved in 0.1mol/L sodium chloride solution, and concussion mixing, prepared concentration is that the lactoferrin of 9-11mg/mL is molten
Liquid；
Two, chitin modified:
In chitosan solution, add appropriate lysozyme mixed liquor, shake 3-4 hour in 38-42 DEG C 75-85 rev/min, will be long
Chain degradation of chitosan is oligochitosan, and then heated and boiled 3-5 minute makes lysozyme inactivate, and obtains modified chitosan solution；
Three, crosslinking:
Appropriate lactoferrin solution is mixed with modified chitosan solution, makes every 50ml chitosan solution contain breast ferrum
Albumen 5mg-10mg, continuing magnetic force stirs, is added dropwise over 0.5-0.6mg/mL sodium tripolyphosphate solution simultaneously and carries out ionomer,
Continuing magnetic force stirs 30-40 minute, and solution entirety presents blue-opalescent；
Four, separate:
By step 3 cross-link after solution under the conditions of 5-15 DEG C 15000-25000 rev/min centrifugal 0.5-1 hour, it is heavy to take
Form sediment, be lactoferrin chitosan nanoparticles.
The preparation method of nanoscale lactoferrin chitosan particle the most according to claim 1, it is characterised in that step one
In prepare concentration be the chitosan solution of 0.7mg/mL.
The preparation method of nanoscale lactoferrin chitosan particle the most according to claim 1, it is characterised in that step one
In prepare concentration be the lactoferrin solution of 10mg/mL.
The preparation method of nanoscale lactoferrin chitosan particle the most according to claim 1, it is characterised in that step 2
Middle chitosan solution is 1000:1 with the volume ratio of lysozyme mixed liquor.
The preparation method of nanoscale lactoferrin chitosan particle the most according to claim 1, it is characterised in that step 2
In in 40 DEG C 80 revs/min shake 3 hours.
The preparation method of nanoscale lactoferrin chitosan particle the most according to claim 1, it is characterised in that step 3
In make every 50ml chitosan solution contain lactoferrin 6mg-9mg.
The preparation method of nanoscale lactoferrin chitosan particle the most according to claim 1, it is characterised in that step 3
The rate of addition of middle sodium tripolyphosphate solution is 1 drop/sec.
The preparation method of nanoscale lactoferrin chitosan particle the most according to claim 1, it is characterised in that step 3
In the volume ratio of modified chitosan solution and sodium tripolyphosphate solution be (4-5): 1.
The preparation method of nanoscale lactoferrin chitosan particle the most according to claim 1, it is characterised in that step 3
In centrifugal under the conditions of 10 DEG C.
The preparation method of nanoscale lactoferrin chitosan particle the most according to claim 1, it is characterised in that step 3
Middle centrifugal rotational speed is 20000 revs/min.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109735525A (en) * | 2018-12-28 | 2019-05-10 | 浙江工业大学 | A kind of preparation method of cross-linking enzyme aggressiveness |
| CN110960512A (en) * | 2019-10-28 | 2020-04-07 | 武汉弘跃医药科技有限公司 | Amino acid-chitosan nano drug-loading system, preparation method and application thereof |
| CN111420067A (en) * | 2020-03-09 | 2020-07-17 | 西南交通大学 | Composite microsphere nano-carrier and preparation method and application thereof |
| CN111529546A (en) * | 2020-05-08 | 2020-08-14 | 深圳市大美康桥生物科技有限公司 | Iron-abstracting agent compound with therapeutic effect and preparation method thereof |
| CN112493317A (en) * | 2020-11-26 | 2021-03-16 | 拉格比(广东)健康科技有限公司 | Composition containing lactoferrin and preparation method thereof |
| CN113368037A (en) * | 2021-06-15 | 2021-09-10 | 北京农学院 | Bovine lactoferrin peptide nanoparticle-loaded chitosan temperature-sensitive hydrogel and preparation method and application thereof |
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| CN113875751A (en) * | 2021-08-17 | 2022-01-04 | 广东省科学院测试分析研究所(中国广州分析测试中心) | Preparation method and application of gold nanocluster-loaded antibacterial lactoferrin fiber aggregate |
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| CN109735525A (en) * | 2018-12-28 | 2019-05-10 | 浙江工业大学 | A kind of preparation method of cross-linking enzyme aggressiveness |
| CN110960512A (en) * | 2019-10-28 | 2020-04-07 | 武汉弘跃医药科技有限公司 | Amino acid-chitosan nano drug-loading system, preparation method and application thereof |
| CN110960512B (en) * | 2019-10-28 | 2022-03-22 | 武汉弘跃医药科技有限公司 | Amino acid-chitosan nano drug-loading system, preparation method and application thereof |
| CN111420067A (en) * | 2020-03-09 | 2020-07-17 | 西南交通大学 | Composite microsphere nano-carrier and preparation method and application thereof |
| CN111529546A (en) * | 2020-05-08 | 2020-08-14 | 深圳市大美康桥生物科技有限公司 | Iron-abstracting agent compound with therapeutic effect and preparation method thereof |
| CN112493317A (en) * | 2020-11-26 | 2021-03-16 | 拉格比(广东)健康科技有限公司 | Composition containing lactoferrin and preparation method thereof |
| CN113368037A (en) * | 2021-06-15 | 2021-09-10 | 北京农学院 | Bovine lactoferrin peptide nanoparticle-loaded chitosan temperature-sensitive hydrogel and preparation method and application thereof |
| CN113384687A (en) * | 2021-06-15 | 2021-09-14 | 北京农学院 | Bovine lactoferrin peptide-loaded chitosan nanoparticle, preparation method thereof and freeze-dried powder |
| CN113875751A (en) * | 2021-08-17 | 2022-01-04 | 广东省科学院测试分析研究所(中国广州分析测试中心) | Preparation method and application of gold nanocluster-loaded antibacterial lactoferrin fiber aggregate |
| CN113875751B (en) * | 2021-08-17 | 2022-06-07 | 广东省科学院测试分析研究所(中国广州分析测试中心) | Preparation method and application of gold nanocluster-loaded antibacterial lactoferrin fiber aggregate |
| CN116036054A (en) * | 2023-03-07 | 2023-05-02 | 广州见华医学科技有限公司 | Lactoferrin patch and application thereof in preparation of postoperative rehabilitation drugs for tumor patients |
| CN116036054B (en) * | 2023-03-07 | 2024-04-26 | 湖北嫦娥生物股份有限公司 | Lactoferrin patch and application thereof in preparation of postoperative rehabilitation drugs for tumor patients |
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Application publication date: 20161207 |