CN117442779A - Preparation method and application of injectable polymer microsphere loaded and crosslinked with hyaluronic acid - Google Patents
Preparation method and application of injectable polymer microsphere loaded and crosslinked with hyaluronic acid Download PDFInfo
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- CN117442779A CN117442779A CN202311558546.4A CN202311558546A CN117442779A CN 117442779 A CN117442779 A CN 117442779A CN 202311558546 A CN202311558546 A CN 202311558546A CN 117442779 A CN117442779 A CN 117442779A
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- 239000004005 microsphere Substances 0.000 title claims abstract description 91
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 90
- 229920002674 hyaluronan Polymers 0.000 title claims abstract description 90
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- 229920000436 Poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide) Polymers 0.000 claims description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 2
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- 239000008103 glucose Substances 0.000 claims description 2
- WXZMFSXDPGVJKK-UHFFFAOYSA-N pentaerythritol Chemical compound OCC(CO)(CO)CO WXZMFSXDPGVJKK-UHFFFAOYSA-N 0.000 claims description 2
- 229920000762 poly(caprolactone)-poly(ethylene glycol)-poly(caprolactone) Polymers 0.000 claims description 2
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- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 claims 1
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- 230000015556 catabolic process Effects 0.000 abstract description 24
- 238000006731 degradation reaction Methods 0.000 abstract description 24
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- 238000001727 in vivo Methods 0.000 abstract description 6
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- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 28
- 238000002347 injection Methods 0.000 description 20
- 239000007924 injection Substances 0.000 description 20
- 238000004132 cross linking Methods 0.000 description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
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- 241000700159 Rattus Species 0.000 description 8
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 7
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 6
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
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- 229920000432 Polylactide-block-poly(ethylene glycol)-block-polylactide Polymers 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/58—Materials at least partially resorbable by the body
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
Abstract
The application provides a preparation method and application of injectable polymer microspheres loaded and crosslinked with hyaluronic acid, and belongs to the technical field of high molecular granulation treatment. Preparing hyaluronic acid solution, high polymer solution and surfactant solutions with two different concentrations respectively; adding hyaluronic acid solution into high-speed emulsified high-molecular polymer solution, and completely emulsifying to obtain a colostrum solution; adding the primary emulsion solution into a high-concentration solution of a surfactant, and emulsifying again to obtain a multiple emulsion solution; adding the double emulsion solution into a low-concentration solution of a surfactant, sequentially adding EDC and NHS to crosslink, centrifugally washing after reaction, and freeze-drying to obtain polymer microspheres loaded with and crosslinked with hyaluronic acid. The polymer microsphere loaded and crosslinked with hyaluronic acid prepared by the method has controllable microsphere size and degradation performance, can effectively promote regeneration of collagen in vivo, and has good application prospect in the field of medical injection.
Description
Technical Field
The application relates to a preparation method and application of injectable polymer microspheres loaded and crosslinked with hyaluronic acid, and belongs to the technical field of high molecular granulation treatment.
Background
With the development of society and the pursuit of beauty by people, the medical and aesthetic industries are vigorously developed, and higher requirements are put on injectable materials. Hyaluronic acid (hyaluronic acid) is a polysaccharide naturally existing in human tissues as a traditional medical and medical injectable material, has extremely high compatibility with the physiological environment of the human body, almost has no risk of causing allergy or rejection reaction, and can be gradually absorbed and metabolized by the human body after injection, thereby imparting adjustability and reversibility to the hyaluronic acid, and being capable of being adjusted or corrected according to personal needs in the medical and medical process. Meanwhile, the medical and aesthetic injectable material can also increase the volume and the hydration capacity of tissues, has the effects of improving the wrinkle condition, smoothing the skin surface and the like, and ensures that the skin looks younger and smoother and has a natural filling effect.
Although hyaluronic acid meets the consumer demand to some extent, there are some disadvantages. Briefly summarized, the following are broadly divided:
first, hyaluronic acid is gradually absorbed by the human body over time, and its filling effect is not permanent, so that regular supplementary injections are required to maintain the effect.
Second, hyaluronic acid, while rarely causing serious adverse reactions, may present some temporary discomfort during or after injection, such as swelling, redness, congestion, etc.
Third, hyaluronic acid filling is a medical practice that requires manipulation by a trained and experienced physician to ensure safety and effectiveness.
Thus, more research into novel medical and medical injectable materials is extended, including development of filling materials with longer effectiveness, more accurate injection technology, methods for enhancing safety and biocompatibility of materials, and the like. The polymer microsphere is used as a novel medical filling material, and has good biocompatibility and longer durability. However, the polymer microspheres studied at present are single polymer powder particles, and after filling, the polymer microspheres are incompatible with tissue at an injection site, have the problems of inflammation and the like; some documents use crosslinking and balling to achieve a simple coating effect, and neither degradation time nor injectability is ideal.
Disclosure of Invention
In view of the above, the present application provides a method for preparing a polymer microsphere capable of injecting and supporting and crosslinking hyaluronic acid, which uses the polymer microsphere to support hyaluronic acid, and the hyaluronic acid solution released by the microsphere after injection forms gel filling at the injection site, so that tissue filling can be rapidly realized, and meanwhile, the crosslinking structure of the polymer microsphere and the microsphere can also effectively slow down rapid loss of hyaluronic acid.
Specifically, the application is realized through the following scheme:
a method for preparing injectable polymer microspheres loaded and crosslinked with hyaluronic acid, comprising the following steps.
Step one, respectively preparing a Hyaluronic Acid (HA) solution, a high polymer solution and a surfactant solution with high and low different concentrations;
step two, adding the hyaluronic acid solution into the high-speed emulsified high-molecular polymer solution, and completely emulsifying to obtain a colostrum solution;
adding the primary emulsion solution into a high-concentration solution of a surfactant, and emulsifying again to obtain a multiple emulsion solution;
step four, adding the double emulsion solution into a low-concentration solution of a surfactant, and adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to crosslink;
and fifthly, after the crosslinking reaction is finished, centrifugally washing, and freeze-drying to obtain the polymer microsphere loaded and crosslinked with hyaluronic acid.
The preparation process adopts a two-step emulsification method to prepare multiple emulsion with a water-in-oil-in-water (W/O/W) structure, and the hyaluronic acid and the high polymer are subjected to cross-linking reaction under the combined action of EDC and NHS, so that the hyaluronic acid and the polymer microspheres are cross-linked and restrained, and meanwhile, the finished product obtained by freeze drying is also subjected to complete coating of the loaded hyaluronic acid in the polymer microspheres, the traditional common coating mode is broken, and the degradation time of the hyaluronic acid is greatly prolonged.
Further, as preferable:
the high molecular polymer is any one of carboxyl-terminated Polycaprolactone (PCL), polylactic acid (PLA), L-polylactic acid (PLLA), polyglycolic acid (PGA), polylactic acid-glycolic acid copolymer (PLGA), polyhydroxyfatty acid (PHA), PLGA-PEG, PLA-PEG, PLLA-PEG, PCL-PEG, PLGA-PEG-PLGA, PLA-PEG-PLGA, PLLA-PEG-PLGA, PCL-PEG-PLGA, PLGA-PEG-PLA, PLA-PEG-PLA, PLLA-PEG-PLA, PCL-PEG-PLA, PLGA-PEG-PLLA, PLLA-PEG-PLLA, PCL-PEG-PLLA, PLGA-PEG-PCL, PLLA-PEG-PCL, PCL-PEG-PCL. The carboxyl-terminated high polymer is used as a crosslinking carrier, and can be crosslinked with amine groups on the hyaluronic acid through EDC/NHS effect, so that the hyaluronic acid is fixed on the high polymer microsphere in a chemical bond mode.
The surfactant is one or more of glycerol, pentaerythritol, sucrose, glucose, sorbitol and polyvinyl alcohol.
In the first step:
the mass concentration of the hyaluronic acid solution is 10-20%.
The mass volume ratio of the concentration of the high molecular polymer solution is 5-15%.
The high concentration of the surfactant is 3-7%, and the low concentration is 0.2-0.8%.
In the second step, the emulsifying speed is 5000-8000 rpm, and the emulsifying time is 2-5 min.
In the third step, the emulsifying speed is 2000-5000 rpm, and the emulsifying time is 2-4 h.
Compared with the traditional medical filling material of hyaluronic acid, the preparation method introduces the polymer microsphere material with controllable degradation rate to encapsulate the hyaluronic acid, and simultaneously fixes the hyaluronic acid on the polymer microsphere bracket in a crosslinking mode for controlling the release behavior of the hyaluronic acid. The polymer microsphere loaded with and crosslinked with hyaluronic acid prepared by the method has controllable microsphere size and degradation performance, and can effectively promote regeneration of collagen in vivo.
The polymer microsphere prepared by the scheme is used as an injectable medical filler, and the filler comprises the polymer microsphere and a phosphate buffer solution, wherein the pH value of the phosphate buffer solution is 5.5-7.5. When filling, the polymer microsphere is directly contacted with the injection site instead of the traditional hyaluronic acid, and the hyaluronic acid only provides early degradation and assists the biological combination of the high polymer and the injection site, so that the injectability of the high polymer reaches an ideal state.
Compared with the prior art, the application has the following beneficial effects:
1. the preparation method of the polymer composite microsphere provided by the invention is simple, the size and the distribution of the composite microsphere can be effectively controlled by a double-emulsion method, meanwhile, the slow release of the loaded and crosslinked hyaluronic acid can be realized, and the porous structure of the microsphere after the release can effectively induce the filling of extracellular matrixes.
2. The polymer composite microsphere loaded and crosslinked with hyaluronic acid prepared by the invention is simple to use in the medical injection filling process, and the hyaluronic acid loaded and crosslinked in the microsphere can serve as a continuous phase in the mixing process of the composite microsphere and the phosphate buffer solution, so that the microsphere particles can be effectively and rapidly dispersed to obtain an injectable suspension, and meanwhile, the problems of layering, agglomeration and the like of the suspension can be effectively solved.
3. The raw materials involved in the polymer composite microsphere for loading and crosslinking the hyaluronic acid are degradable materials, the biocompatibility is good, compared with the traditional hyaluronic acid filler and the common polymer microsphere, the polymer composite microsphere has a longer filling effect, and the loaded hyaluronic acid can also effectively induce the generation of collagen at an injection site.
Drawings
FIG. 1 is a microstructure of a polymer microsphere prepared according to the present application;
FIG. 2 is a graph of the external degradation rate of polymer microspheres prepared according to the present application;
FIG. 3 is a graph showing the in vivo degradation rate after injection of polymer microspheres prepared according to the present application;
FIG. 4 shows the collagen content of the polymer microsphere injection site prepared according to the present application.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The following describes the invention in detail with reference to examples.
Example 1
The preparation method of the polymer microsphere capable of loading and crosslinking hyaluronic acid in an injection manner comprises the following steps:
1) Colostrum stage: 0.1g of HA (molecular weight 800,000) is weighed and dispersed in 0.6mL of deionized water, and stirred at room temperature until the HA is completely dissolved, so as to obtain an HA solution; 1.0g of PLGA is weighed and dispersed in 10mL of dichloromethane, and the mixture is stirred at room temperature until the PLGA is completely dissolved, so as to obtain PLGA solution; the HA solution was added to the PLGA solution and emulsified at 6000rpm for 3min to give a colostrum solution.
2) And (3) re-emulsifying: 4g of polyvinyl alcohol is weighed and dispersed in 100mL of ultrapure water, stirred at 90 ℃ until the polyvinyl alcohol is completely dissolved, a high-concentration surfactant solution is obtained, the colostrum solution obtained in the step 1) is poured into the high-concentration surfactant solution, and emulsification is carried out for 3min at a speed of 3000rpm, so as to obtain a multiple emulsion solution.
3) Preparing polymer composite microspheres: 1.6g of polyvinyl alcohol is weighed and dispersed in 400mL of deionized water, stirred at 90 ℃ until the polyvinyl alcohol is completely dissolved, a low-concentration surfactant solution is obtained, the pH value of the solution is adjusted to 5.5 by using concentrated hydrochloric acid, then the double emulsion solution obtained in the step 2) is poured into the low-concentration surfactant solution with the pH value of 5.5, then 0.2g of EDC and 0.5g of NHS solid powder are sequentially added, stirring is carried out for 3 hours at 800rpm at room temperature, after complete crosslinking and dichloromethane volatilization, centrifugation is carried out for 10 minutes at 5000rpm, centrifugal washing is carried out for 5 times by using deionized water under the same conditions, and polymer microsphere powder loaded with and crosslinked hyaluronic acid is obtained through freeze drying.
Example 2
This embodiment is identical to the arrangement of embodiment 1, except that: in the step 1), emulsifying for 3min at a speed of 5000rpm to obtain a colostrum solution; in step 2), the emulsion was carried out at 2500rpm for 3 minutes to obtain a multiple emulsion solution.
Example 3
This embodiment is identical to the arrangement of embodiment 1, except that: in the step 1), emulsifying for 3min at 7000rpm to obtain a colostrum solution; in step 2), the emulsion was carried out at 4000rpm for 3min to obtain a multiple emulsion solution.
Example 4
The preparation method of the polymer microsphere in the embodiment comprises the following steps:
1) 4g of polyvinyl alcohol is weighed and dispersed in 100mL of ultrapure water, stirred at 90 ℃ until the polyvinyl alcohol is completely dissolved to obtain a high-concentration surfactant solution, PLGA solution (1.0 g of PLGA is weighed and dispersed in 10mL of dichloromethane, and stirred at room temperature until the PLGA solution is completely dissolved) is poured into the high-concentration surfactant solution, and the mixture is emulsified for 3min at a speed of 3000rpm to obtain a multiple emulsion solution.
2) 1.6g of polyvinyl alcohol is weighed and dispersed in 400mL of deionized water, stirred at 90 ℃ until the polyvinyl alcohol is completely dissolved, a low-concentration surfactant solution is obtained, the pH value of the solution is adjusted to 5.5 by using concentrated hydrochloric acid, then the re-emulsion solution obtained in the step 1) is poured into the low-concentration surfactant solution with the pH value of 5.5, stirred at 800rpm for 3 hours at room temperature, after complete crosslinking and volatilization of methylene dichloride, the solution is centrifuged at 5000rpm for 10 minutes, and the solution is centrifuged and washed for 5 times by using deionized water under the same conditions, and the polymer microsphere powder loaded with and crosslinked hyaluronic acid is obtained through freeze drying.
Example 5
1) Colostrum stage: 0.1g of HA (molecular weight 800,000) is weighed and dispersed in 0.6mL of deionized water, and stirred at room temperature until the HA is completely dissolved, so as to obtain an HA solution; 1.0g of PLGA is weighed and dispersed in 10mL of dichloromethane, and the mixture is stirred at room temperature until the PLGA is completely dissolved, so as to obtain PLGA solution; the HA solution was added to the PLGA solution and emulsified at 6000rpm for 3min to give a colostrum solution.
2) And (3) re-emulsifying: 4g of polyvinyl alcohol is weighed and dispersed in 100mL of ultrapure water, stirred at 90 ℃ until the polyvinyl alcohol is completely dissolved, a high-concentration surfactant solution is obtained, the colostrum solution obtained in the step 1) is poured into the high-concentration surfactant solution, and emulsification is carried out for 3min at a speed of 3000rpm, so as to obtain a multiple emulsion solution.
3) Preparing polymer composite microspheres: 1.6g of polyvinyl alcohol is weighed and dispersed in 400mL of deionized water, stirred at 90 ℃ until the polyvinyl alcohol is completely dissolved, a low-concentration surfactant solution is obtained, the pH value of the solution is adjusted to 5.5 by using concentrated hydrochloric acid, then the re-emulsion solution obtained in the step 2) is poured into the low-concentration surfactant solution with the pH value of 5.5, stirred at 800rpm for 3 hours at room temperature, after complete crosslinking and volatilization of methylene dichloride, the solution is centrifuged at 5000rpm for 10 minutes, and the solution is centrifuged and washed for 5 times by using deionized water under the same conditions, and the polymer microsphere powder loaded with and crosslinked hyaluronic acid is obtained through freeze drying.
Example 6
The preparation method of the polymer microsphere capable of loading and crosslinking hyaluronic acid in an injection manner comprises the following steps:
1) Colostrum stage: 0.1g of HA (molecular weight 800,000) is weighed and dispersed in 0.6mL of deionized water, and stirred at room temperature until the HA is completely dissolved, so as to obtain an HA solution; 1.0g of PGA is weighed and dispersed in 10mL of dichloromethane, and the mixture is stirred at room temperature until the PGA is completely dissolved to obtain PLGA solution; adding the HA solution into the PGA solution, and emulsifying at 6000rpm for 3min to obtain a colostrum solution.
2) And (3) re-emulsifying: 4g of polyvinyl alcohol is weighed and dispersed in 100mL of ultrapure water, stirred at 90 ℃ until the polyvinyl alcohol is completely dissolved, a high-concentration surfactant solution is obtained, the colostrum solution obtained in the step 1) is poured into the high-concentration surfactant solution, and emulsification is carried out for 3min at a speed of 3000rpm, so as to obtain a multiple emulsion solution.
3) Preparing polymer composite microspheres: 1.6g of polyvinyl alcohol is weighed and dispersed in 400mL of deionized water, stirred at 90 ℃ until the polyvinyl alcohol is completely dissolved, a low-concentration surfactant solution is obtained, the pH value of the solution is adjusted to 5.5 by using concentrated hydrochloric acid, then the double emulsion solution obtained in the step 2) is poured into the low-concentration surfactant solution with the pH value of 5.5, then 0.2g of EDC and 0.5g of NHS solid powder are sequentially added, stirring is carried out for 3 hours at 800rpm at room temperature, after complete crosslinking and dichloromethane volatilization, centrifugation is carried out for 10 minutes at 5000rpm, centrifugal washing is carried out for 5 times by using deionized water under the same conditions, and polymer microsphere powder loaded with and crosslinked hyaluronic acid is obtained through freeze drying.
Example 7
The preparation method of the polymer microsphere capable of loading and crosslinking hyaluronic acid in an injection manner comprises the following steps:
1) Colostrum stage: 0.1g of HA (molecular weight 800,000) is weighed and dispersed in 0.6mL of deionized water, and stirred at room temperature until the HA is completely dissolved, so as to obtain an HA solution; 1.0g of PLA is weighed and dispersed in 10mL of dichloromethane, and the mixture is stirred at room temperature until the mixture is completely dissolved to obtain PLGA solution; adding the HA solution into the PLA solution, and emulsifying for 3min at 6000rpm to obtain a colostrum solution.
2) And (3) re-emulsifying: 4g of polyvinyl alcohol is weighed and dispersed in 100mL of ultrapure water, stirred at 90 ℃ until the polyvinyl alcohol is completely dissolved, a high-concentration surfactant solution is obtained, the colostrum solution obtained in the step 1) is poured into the high-concentration surfactant solution, and emulsification is carried out for 3min at a speed of 3000rpm, so as to obtain a multiple emulsion solution.
3) Preparing polymer composite microspheres: 1.6g of polyvinyl alcohol is weighed and dispersed in 400mL of deionized water, stirred at 90 ℃ until the polyvinyl alcohol is completely dissolved, a low-concentration surfactant solution is obtained, the pH value of the solution is adjusted to 5.5 by using concentrated hydrochloric acid, then the double emulsion solution obtained in the step 2) is poured into the low-concentration surfactant solution with the pH value of 5.5, then 0.2g of EDC and 0.5g of NHS solid powder are sequentially added, stirring is carried out for 3 hours at 800rpm at room temperature, after complete crosslinking and dichloromethane volatilization, centrifugation is carried out for 10 minutes at 5000rpm, centrifugal washing is carried out for 5 times by using deionized water under the same conditions, and polymer microsphere powder loaded with and crosslinked hyaluronic acid is obtained through freeze drying.
Microstructure test:
the lyophilized microspheres of example 1, example 2 and example 3 were blown onto a conductive tape, and then ion-sputter coated for 30s to obtain the size and appearance structure of the microspheres, and the results are shown in fig. 1 (a is example 2, b is example 1, and c is example 3): under the same conditions, the particle sizes of the microspheres obtained by different emulsification speeds are different, and the higher the emulsification speed is, the smaller the microsphere size is.
In vitro degradation experiments
The polymer microspheres obtained by freeze-drying in example 1, example 6 and example 7 were weighed 0.1g and put into a phosphoric acid buffer having a pH of 5.5In the solution, 5 parallel samples are arranged in each group, the microspheres are centrifugally freeze-dried at 1 week, 2 weeks, 4 weeks and 8 weeks respectively after degradation under the condition of 37 ℃, and the weight m is tested xy Where x is 1, 6 and 7, respectively, representing example numbers, and y is 1, 2, 4 and 8, respectively, representing sampling times. The degradation rate of the composite microspheres was calculated according to the following formula, and the data are shown in table 1.
Table 1: in vitro degradation test data
As can be seen from table 1 and fig. 2: the degradation rates of the microspheres prepared from the three different polymers are obviously different, and compared with the example 1, in the example 6, the degradation rate is faster because the high polymer PLGA is replaced by PGA; whereas the high molecular polymer PLGA in example 7 was replaced with PLA, the degradation rate was slower.
In vivo degradation test:
weighing 0.1g of polymer microspheres obtained in the freeze-drying of the embodiment 1, the embodiment 4 and the embodiment 5, placing into a dialysis bag, implanting into subcutaneous tissue at the back of SD rats, subcutaneously implanting 20 SD rats into each group of samples, randomly selecting 5 SD rats at 1 week, 2 weeks, 4 weeks and 8 weeks respectively, taking out the implanted dialysis bag filled with the microspheres, washing the microspheres with normal saline, centrifuging, drying, weighing, and marking as m xy Where x is 1, 4 and 5, respectively, representing example numbers, and y is 1, 2, 4 and 8, respectively, representing sampling times. The degradation rate of the composite microspheres was calculated according to the following formula, and the data are shown in table 2.
Table 2: in vivo degradation test data
As can be seen from table 2 and fig. 3: the three types of microspheres have certain degradability, and compared with the scheme of adding hyaluronic acid in the embodiment 4, the addition of hyaluronic acid in the embodiment 1 is beneficial to the degradation of the microspheres in the early stage, and the degradation rate of the microspheres in the later stage is equal to that of the common polymer microspheres; in example 5, the pre-hyaluronic acid was released in a large amount by dissolution at the injection site, and the degradation rate at week 1 was 5% or so, and the degradation rate at week 8 was increased to 18% or so.
The method for applying the polymer microsphere prepared in the embodiment to the injectable medical filler comprises the following steps: weighing 0.1g of polymer microsphere powder, dispersing into 10mL of phosphoric acid buffer solution with pH value of 7.2 (microsphere concentration is 0.2 g/mL), packaging, and ultraviolet sterilizing to obtain the final product for injection.
Taking SD rats as an example, the collagen content of the injection site was tested to demonstrate the effect of the application.
Injection site collagen content test:
the injectable fillers of examples 1, 2 and 5 were injected into subcutaneous back tissue of SD rats, 20 SD rats were subcutaneously implanted in each group of samples, 5 SD rats were randomly selected at week 4, week 8 and week 12, and subcutaneous tissues regenerated at 5mm x 5mm implantation sites were removed, and collagen content was measured, and specific data are shown in table 3.
Table 3: collagen content test data at injection site
As can be seen from the combination of table 3 and fig. 4: the local collagen content of SD rats of example 1 and example 5 is greater than that of example 4. The composite microsphere has more obvious effect of promoting the regeneration of collagen compared with the common polymer microsphere under the condition that the hyaluronic acid exists.
In the application of the injectable medical filler, the preparation method introduces the polymer microsphere material with controllable degradation rate to encapsulate the hyaluronic acid, and further adopts a crosslinking mode to fix the hyaluronic acid on the polymer microsphere bracket in order to control the release behavior of the hyaluronic acid. The prepared polymer microsphere loaded with and crosslinked with hyaluronic acid has controllable microsphere size and degradation performance, can effectively promote regeneration of collagen in vivo, and has good application prospect in the field of medical injection.
The foregoing examples merely illustrate specific embodiments of the invention, which are described in greater detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (10)
1. A method for preparing injectable polymer microspheres loaded and crosslinked with hyaluronic acid, which is characterized by comprising the following steps:
step one, preparing a hyaluronic acid solution, a high-molecular polymer solution and surfactant solutions with high and low different concentrations respectively;
step two, adding the hyaluronic acid solution into the high-speed emulsified high-molecular polymer solution, and completely emulsifying to obtain a colostrum solution;
adding the primary emulsion solution into a high-concentration solution of a surfactant, and emulsifying again to obtain a multiple emulsion solution;
and fourthly, adding the re-emulsion solution into a low-concentration solution of the surfactant, sequentially adding EDC and NHS to crosslink, centrifugally washing after the reaction is finished, and freeze-drying to obtain the polymer microsphere loaded and crosslinked with hyaluronic acid.
2. The method for preparing injectable hyaluronic acid-loaded polymer microspheres according to claim 1, wherein the method comprises the steps of: the high molecular polymer is any one of carboxyl-terminated PCL, PLA, PGA, PLLA, PLGA, PHA, PLGA-PEG, PLA-PEG, PLLA-PEG, PCL-PEG, PLGA-PEG-PLGA, PLA-PEG-PLGA, PLLA-PEG-PLGA, PCL-PEG-PLGA, PLGA-PEG-PLA, PLLA-PEG-PLA, PLGA-PEG-PLLA, PLA-PEG-PLLA, PLLA-PEG-PLLA, PCL-PEG-PLLA, PLGA-PEG-PCL, PLA-PEG-PCL, PLLA-PEG-PCL, PCL-PEG-PCL.
3. The method for preparing injectable hyaluronic acid-loaded polymer microspheres according to claim 1, wherein the method comprises the steps of: the surfactant is one or more of glycerol, pentaerythritol, sucrose, glucose, sorbitol and polyvinyl alcohol.
4. The method for preparing injectable hyaluronic acid-loaded polymer microspheres according to claim 1, wherein the method comprises the steps of: the mass volume ratio of the concentration of the high polymer solution is 5-15%.
5. The method for preparing injectable hyaluronic acid-loaded polymer microspheres according to claim 1, wherein the method comprises the steps of: the mass concentration of the hyaluronic acid solution is 10-20%.
6. The method for preparing injectable hyaluronic acid-loaded polymer microspheres according to claim 1, wherein the method comprises the steps of: the two different concentrations of the surfactant are respectively 3-7% and 0.2-0.8%.
7. The method for preparing injectable polymer microspheres loaded with and crosslinked with hyaluronic acid according to any one of claims 1-6, wherein the method is characterized in that: in the second step, the emulsification speed is 5000-8000 rpm, and the emulsification time is 2-5 min.
8. The method for preparing injectable polymer microspheres loaded with and crosslinked with hyaluronic acid according to any one of claims 1-6, wherein the method is characterized in that: in the third step, the emulsifying speed is 2000-5000 rpm, and the emulsifying time is 2-4 h.
9. A method of using the injectable hyaluronic acid-loaded and crosslinked polymeric microspheres prepared by the method of claim 1 for injectable medical fillers.
10. A method for injectable use of polymeric microspheres loaded with and cross-linked with hyaluronic acid for injectable medical fillers prepared according to claim 9, characterized in that: the filler comprises polymer microspheres and phosphate buffer solution with the pH of 5.5-7.5.
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