CN110664781A - Preparation method of calcium alginate-loaded rhizoma drynariae microspheres with controllable particle size distribution - Google Patents
Preparation method of calcium alginate-loaded rhizoma drynariae microspheres with controllable particle size distribution Download PDFInfo
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- CN110664781A CN110664781A CN201911006485.4A CN201911006485A CN110664781A CN 110664781 A CN110664781 A CN 110664781A CN 201911006485 A CN201911006485 A CN 201911006485A CN 110664781 A CN110664781 A CN 110664781A
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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
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/11—Pteridophyta or Filicophyta (ferns)
- A61K36/12—Filicopsida or Pteridopsida
- A61K36/126—Drynaria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
Abstract
A controllable particle size distribution calcium alginate carries the preparation method of the rhizoma drynariae microballoons, use the conventional dropping method to make more than 95% of particle size distribute in 300-.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a preparation method of calcium alginate-loaded rhizoma drynariae microspheres with controllable particle size distribution.
Background
The drug-loaded microspheres are micro spherical entities formed by dissolving or dispersing drugs in high polymer materials, are spherical or spheroidal, have various advantages in practical application, have spherical shape, improve flowability, are beneficial to injection operation and realize targeting, are used for encapsulating the drugs in a carrier, are beneficial to maintaining the stability of the drugs, and can control the release dosage of the drugs by controlling the size and the material of the microspheres and other modes.
In the field of bone repair materials, a novel use method of drug-loaded microspheres is provided, and degradable drug-loaded polymer microspheres are added into the bone repair materials to prepare the composite bone repair materials containing the drug-loaded microspheres. The added microspheres have an entity occupying function, the microspheres in the bone repair material matrix can occupy the position as large as the size of the microspheres, and the porous bone repair material is formed by forming holes in situ after the microspheres are degraded by utilizing the characteristic that the microspheres can be degraded more quickly. As is known, the natural anatomical structure of the porous bone tissue is a porous structure with 300-; meanwhile, the composite bone repair material containing the drug-loaded microspheres can also realize controllable drug release performance by utilizing the drug release function of the drug-loaded microspheres and adjusting the types and the number of the microspheres and the like, thereby achieving the purposes of preventing infection and promoting repair. In addition, the microspheres are spherical, have good flowing property and good injectability, have small influence on the injectability when being added into injectable bone repair materials, and are beneficial to realizing clinical minimally invasive operation.
Because the microsphere is used in the bone repair field, the diameter of the microsphere particles added into the bone repair material is generally similar to the porous structure of bone tissues and is concentrated in the range of 300-. At present, a plurality of reports exist for compounding calcium alginate microspheres loaded with drugs and even live cells into bone repair materials, but the process for preparing the calcium alginate microspheres with the particle size of 300-800 mu m is only reported. The existing methods for preparing the calcium alginate microspheres with controllable size comprise an ultrasonic atomization method, a microemulsion method, an emulsion gel method and the like, the methods either need special equipment and have high manufacturing cost, or other auxiliary agents such as ethanol or liquid paraffin and the like need to be added, so that the risk of residual damage is high, and no special process for preparing the microspheres with narrow particle size exists in the existing preparation process.
Disclosure of Invention
In order to solve the problems, the invention provides a preparation method of calcium alginate-loaded rhizoma drynariae microspheres with controllable particle size distribution.
The technical scheme of the invention is as follows: a preparation method of calcium alginate-loaded rhizoma drynariae microspheres with controllable particle size distribution comprises the following steps:
step one, preparing 1.5wt% sodium alginate solution and 2wt% CaCl2Solution and 0.5M citric acid solution containing 1 wt% chitosan;
step two, putting the drynaria extract into a mortar, grinding for 7-13 minutes, and sieving to obtain drynaria powder;
step three, weighing the drynaria powder in the step two, adding the drynaria powder into 0.5-2wt% of sodium alginate solution, stirring the solution in a magnetic stirrer at the temperature of 60 ℃, and fully and uniformly mixing the solution to obtain drynaria-carrying sodium alginate suspension;
step four, dripping the rhizoma drynariae sodium alginate suspension into CaCl by adopting a syringe with a needle head specification of 32G and under a constant pressure of 70N-90N2In the solution, the formed microspheres are kept stand for crosslinking for 4 hours, the obtained drug-loaded microspheres are washed for 5 times by deionized water, and water is filtered out to obtain primary drug-loaded microspheres;
step five, soaking the primary drug-loaded microspheres in the step four in 0.5M citric acid solution for 12 hours;
and step six, washing the microspheres in the filtering step five by using a large amount of deionized water, pre-freezing the filtered microspheres in a refrigerator at the temperature of-80 ℃ for 24 hours, and freeze-drying the microspheres for 6 hours by using a freeze dryer to obtain the drug-loaded microspheres.
And further optimizing, wherein the sieving screen in the second step is 200 meshes.
Further optimizing, the rhizoma drynariae sodium alginate suspension liquid in the fourth step is dripped in the CaCl distance2The horizontal distance of the solution liquid surface is 40cm, and the vertical distance is 30 cm.
Further optimized, the dosage of the 0.5M citric acid solution in the step five is preferably just submerging the microspheres.
The invention has the beneficial effects that:
the drug-loaded calcium alginate microspheres with the required particle size of 300-.
Drawings
FIG. 1 is a topography inspection chart of example 1
FIG. 2 is a profile inspection chart of example 2
FIG. 3 is a topography test chart of example 3
FIG. 4 is an analytical particle size plot for example 1
FIG. 5 is an analytical particle size plot for example 2
FIG. 6 is an analytical particle size plot for example 3
FIG. 7 is a cytotoxicity assay of examples 1-3 and control.
Detailed Description
The specific implementation mode of the invention is as follows:
example 1
A preparation method of calcium alginate-loaded rhizoma drynariae microspheres with controllable particle size distribution comprises the following steps:
respectively preparing 1.5wt% sodium alginate solution and 2wt% CaCl2Putting the drynaria extract into a mortar, grinding for 10 minutes, sieving by a 200-mesh sieve to obtain drynaria powder, weighing 0.5wt% of drynaria powder according to the mass fraction, adding the drynaria powder into a sodium alginate solution, placing the solution into a magnetic stirrer at the temperature of 60 ℃, stirring for 30 minutes, and fully and uniformly mixing to obtain a drynaria-carrying sodium alginate suspension; dripping rhizoma Drynariae-loaded sodium alginate suspension into CaCl with syringe needle specification of 32G at constant pressure of 70N2In the solution, the needle of the syringe is far from CaCl during the dripping process2The liquid level of the solution is horizontally spaced by 40cm and vertically spaced by 30cm, the formed microspheres are kept stand and crosslinked for 4h, the obtained drug-loaded microspheres are washed by deionized water for 5 times to obtain primary drug-loaded microspheres, 0.5M citric acid solution is added to immerse the primary drug-loaded microspheres for 12 h, a large amount of deionized water is used for washing and filtering after being taken out, the filtered microspheres are placed in a refrigerator with the temperature of-80 ℃ for pre-freezing for 24 h, and then a freeze drier is used for freeze drying for 6h, so that the drug-loaded microspheres with the particle size mainly distributed in the range of 300-800 mu M and mostly distributed in the range of 300-600 mu M are obtained.
Example 2
A preparation method of calcium alginate-loaded rhizoma drynariae microspheres with controllable particle size distribution comprises the following steps:
respectively preparing 2wt% sodium alginate solution and 6 wt% CaCl2Adding rhizoma Drynariae extract into 0.5M citric acid solution containing 1 wt% chitosan, grinding in mortar for 10 min, sieving with 200 mesh sieve,weighing 1.5wt% of rhizoma drynariae powder according to the mass fraction, adding the rhizoma drynariae powder into the sodium alginate solution, placing the mixture into a magnetic stirrer at the temperature of 60 ℃, stirring for 30 minutes, and fully and uniformly mixing to obtain a rhizoma drynariae-carrying sodium alginate suspension; dripping rhizoma Drynariae-loaded sodium alginate suspension into CaCl with syringe needle specification of 32G at constant pressure of 80N2In the solution, the needle of the syringe is far from CaCl during the dripping process2The liquid level of the solution is horizontally spaced by 40cm and vertically spaced by 30cm, the formed microspheres are kept stand and crosslinked for 4h, the obtained drug-loaded microspheres are washed by deionized water for 5 times to obtain primary drug-loaded microspheres, 0.5M citric acid solution is added to immerse the primary drug-loaded microspheres for 12 h, a large amount of deionized water is used for washing and filtering after being taken out, the filtered microspheres are placed in a refrigerator with the temperature of-80 ℃ for pre-freezing for 24 h, and then a freeze drier is used for freeze drying for 6h, so that the drug-loaded microspheres with the particle size mainly distributed in the range of 300-800 mu M and mostly distributed in the range of 300-600 mu M are obtained.
Example 3
A preparation method of calcium alginate-loaded rhizoma drynariae microspheres with controllable particle size distribution comprises the following steps:
respectively preparing 2.5wt% sodium alginate solution and 3 wt% CaCl2Putting the drynaria extract into a mortar, grinding for 10 minutes, sieving by a 200-mesh sieve to obtain drynaria powder, weighing 2wt% of drynaria powder according to the mass fraction, adding the drynaria powder into a sodium alginate solution, stirring for 30 minutes in a magnetic stirrer at the temperature of 60 ℃, and fully and uniformly mixing to obtain a drynaria-carrying sodium alginate suspension; dripping rhizoma Drynariae-loaded sodium alginate suspension into CaCl with syringe needle specification of 32G at constant pressure of 90N2In the solution, the needle of the syringe is far from CaCl during the dripping process2Keeping the solution level at a horizontal distance of 40cm and a vertical distance of 30cm, standing and crosslinking the formed microspheres for 4h, washing the obtained drug-loaded microspheres with deionized water for 5 times to obtain primary drug-loaded microspheres, adding 0.5M citric acid solution to immerse the primary drug-loaded microspheres for 12 h, taking out, washing and filtering with a large amount of deionized water, pre-freezing the filtered microspheres in a refrigerator at the temperature of-80 ℃ for 24 h, and freeze-drying with a freeze dryer for 6h to obtain the microspheres with the main particle sizeDrug-loaded microspheres distributed in the range of 300-800 μm, wherein the drug-loaded microspheres are distributed in the range of 300-600 μm.
Carrying out morphology detection, particle size analysis and cytotoxicity detection on the drug-loaded microspheres prepared in the embodiments 1 to 3, wherein the morphology detection is carried out by carrying out ultrasonic dispersion on the prepared microspheres, taking a picture under a light mirror and observing the balling condition, as shown in fig. 1, fig. 2 and fig. 3, the visible materials are all spherical, complete and full, no fragment is observed, and the material is proved to be solid; analysis particle size analysis method comprises the steps of carrying out ultrasonic dispersion on the prepared microspheres, taking pictures under an optical lens, respectively selecting 5 different typical view fields, using image processing software, counting the number of the microspheres with different particle sizes, and analyzing particle size analysis particle; cytotoxicity assay cytotoxicity was assayed by using the MTT assay in the national standard GB/T16886.5-2017 using a leach solution of the material using MG63 cells and an initial seeding density of 3000 cells/well in a 96-well plate, as shown in fig. 7, and the absorbance values of examples 1-3 were all higher than or equal to the blank control without any material added, indicating that the material was not cytotoxic.
The foregoing illustrates and describes the principal features, utilities, and principles of the invention, as well as advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made in the invention without departing from the spirit and scope of the invention. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (4)
1. A preparation method of calcium alginate-loaded rhizoma drynariae microspheres with controllable particle size distribution is characterized by comprising the following steps:
step oneRespectively preparing 1.5-2.5wt% sodium alginate solution and 2-6 wt% CaCl2Solution and 0.5M citric acid solution containing 1 wt% chitosan;
step two, putting the drynaria extract into a mortar, grinding for 7-13 minutes, and sieving to obtain drynaria powder;
step three, weighing the drynaria powder in the step two, adding the drynaria powder into 0.5-2wt% of sodium alginate solution, stirring the solution in a magnetic stirrer at the temperature of 60 ℃, and fully and uniformly mixing the solution to obtain drynaria-carrying sodium alginate suspension;
step four, dripping the rhizoma drynariae sodium alginate suspension into CaCl by adopting a syringe with a 32G needle specification and under the constant pressure of 70-90N2In the solution, the formed microspheres are kept stand for crosslinking for 4 hours, the obtained drug-loaded microspheres are washed for 5 times by deionized water, and water is filtered out to obtain primary drug-loaded microspheres;
step five, soaking the primary drug-loaded microspheres in the step four in 0.5M citric acid solution for 12 hours;
and step six, washing the microspheres in the filtering step five by using a large amount of deionized water, pre-freezing the filtered microspheres in a refrigerator at the temperature of-80 ℃ for 24 hours, and freeze-drying the microspheres for 6 hours by using a freeze dryer to obtain the drug-loaded microspheres.
2. The method for preparing calcium alginate-loaded rhizoma drynariae microspheres with controllable particle size distribution as claimed in claim 1, wherein the mesh of the screen in the second step is 200 meshes.
3. The method for preparing calcium alginate-loaded rhizoma drynariae microspheres with controllable particle size distribution as claimed in claim 1, wherein the needle tip of the suspension liquid of rhizoma drynariae sodium alginate in step four is placed in the CaCl space when the suspension liquid of rhizoma drynariae sodium alginate is added2The horizontal distance of the solution liquid surface is 40cm, and the vertical distance is 30 cm.
4. The controllable particle size distribution calcium alginate-loaded rhizoma drynariae micro powder of claim 1
The preparation method of the ball is characterized in that the dosage of the 0.5M citric acid solution in the step five is just submerging the microspheres.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113827767A (en) * | 2021-09-13 | 2021-12-24 | 西北大学 | Preparation method of novel microgel bone powder for repairing postoperative tissue of osteoma |
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| US20130039990A1 (en) * | 2010-04-30 | 2013-02-14 | University Of Maryland, Baltimore | Injectable, load-bearing cell/microbead/calcium phosphate bone paste for bone tissue engineering |
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| CN113827767B (en) * | 2021-09-13 | 2022-05-17 | 西北大学 | Preparation method of novel microgel bone powder for repairing postoperative tissue of osteoma |
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Application publication date: 20200110 |