CN112755924A - Preparation method of vinyl collagen microspheres - Google Patents
Preparation method of vinyl collagen microspheres Download PDFInfo
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- CN112755924A CN112755924A CN202110004777.5A CN202110004777A CN112755924A CN 112755924 A CN112755924 A CN 112755924A CN 202110004777 A CN202110004777 A CN 202110004777A CN 112755924 A CN112755924 A CN 112755924A
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 160
- 108010035532 Collagen Proteins 0.000 title claims abstract description 160
- 229920001436 collagen Polymers 0.000 title claims abstract description 160
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 title claims abstract description 110
- 229920002554 vinyl polymer Polymers 0.000 title claims abstract description 110
- 239000004005 microsphere Substances 0.000 title claims abstract description 77
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 29
- DCUFMVPCXCSVNP-UHFFFAOYSA-N methacrylic anhydride Chemical compound CC(=C)C(=O)OC(=O)C(C)=C DCUFMVPCXCSVNP-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000002699 waste material Substances 0.000 claims abstract description 20
- 238000012986 modification Methods 0.000 claims abstract description 9
- 230000004048 modification Effects 0.000 claims abstract description 9
- 239000007864 aqueous solution Substances 0.000 claims description 62
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 48
- 229940057995 liquid paraffin Drugs 0.000 claims description 40
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 33
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 32
- 238000003756 stirring Methods 0.000 claims description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- 238000006243 chemical reaction Methods 0.000 claims description 22
- 239000000839 emulsion Substances 0.000 claims description 17
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 16
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 16
- 239000007853 buffer solution Substances 0.000 claims description 16
- 238000004108 freeze drying Methods 0.000 claims description 16
- 238000010438 heat treatment Methods 0.000 claims description 16
- 238000002156 mixing Methods 0.000 claims description 16
- 239000008055 phosphate buffer solution Substances 0.000 claims description 16
- 239000004094 surface-active agent Substances 0.000 claims description 16
- 238000004132 cross linking Methods 0.000 claims description 10
- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical group CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 claims description 8
- 238000001816 cooling Methods 0.000 claims description 8
- 230000001804 emulsifying effect Effects 0.000 claims description 8
- 239000012535 impurity Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- 238000005063 solubilization Methods 0.000 claims 1
- 230000007928 solubilization Effects 0.000 claims 1
- 239000000126 substance Substances 0.000 abstract description 3
- 239000000758 substrate Substances 0.000 abstract 1
- 238000004090 dissolution Methods 0.000 description 7
- 239000010985 leather Substances 0.000 description 7
- 239000002861 polymer material Substances 0.000 description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 125000000524 functional group Chemical group 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 2
- 150000008064 anhydrides Chemical class 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229920005615 natural polymer Polymers 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 239000012434 nucleophilic reagent Substances 0.000 description 1
- 238000010534 nucleophilic substitution reaction Methods 0.000 description 1
- BHTJEPVNHUUIPV-UHFFFAOYSA-N pentanedial;hydrate Chemical compound O.O=CCCCC=O BHTJEPVNHUUIPV-UHFFFAOYSA-N 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000010526 radical polymerization reaction Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000002910 solid waste Substances 0.000 description 1
- 238000000371 solid-state nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
- C08H1/00—Macromolecular products derived from proteins
- C08H1/06—Macromolecular products derived from proteins derived from horn, hoofs, hair, skin or leather
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- Chemical Kinetics & Catalysis (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
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Abstract
The invention discloses a preparation method of a vinyl collagen microsphere, which is implemented according to the following steps: and step 1, performing double bond modification on the waste skin collagen to obtain the vinyl collagen. And 2, preparing the vinyl collagen microspheres. According to the method, the leather-making waste collagen is used as a substrate, is modified and modified by methacrylic anhydride, is further emulsified and crosslinked into balls, realizes resource utilization of the waste, and simultaneously grafts unsaturated double bonds on the microspheres, so that the chemical modifiability of the microspheres is improved.
Description
Technical Field
The invention relates to the technical field of natural high polymer materials, in particular to a preparation method of vinyl collagen microspheres.
Background art
China is a big leather producing country, a large number of export trades exist every year, and a series of problems can be brought while the economy is increased, such as: environmental pollution, resource waste, etc. These problems restrict the development of the industry, so the resource utilization of leather waste is very important. More than 80% of leather solid waste is reported to be collagen, and collagen with different molecular weights and structures can be extracted by processing defective leather materials, leftover materials and the like generated in the leather making process so as to adapt to different requirements. The collagen extracted from the tanning waste can reduce the pollution to the environment, can provide new raw materials for other industries, and achieves the purposes of recycling the waste and changing waste into valuable.
The polymer microsphere has the special characteristics of shape specificity (spherical shape), good embedding property, small size, large specific surface area, strong adsorbability, surface enrichment of functional groups and the like, so that the polymer microsphere is widely applied to multiple fields such as biomedicine, coating and food and becomes one of the hotspots of current scientific research. There are many carrier materials for preparing microspheres, which are mainly classified into natural polymer materials and synthetic polymer materials. The natural polymer material is a common raw material for preparing the microspheres due to good biocompatibility and biodegradability and no toxicity and harm. Collagen is one of common materials for preparing microspheres as a natural high polymer material which is cheap, easily available, wide in source, environment-friendly, degradable, non-toxic and contains a large number of active groups (amino groups, hydroxyl groups and carboxyl groups) on a molecular chain, and collagen microspheres prepared from the collagen are widely applied to the aspects of medicine, food, dye treatment and the like.
The vinyl collagen microspheres are prepared by modifying collagen through methacrylic anhydride and performing an emulsion crosslinking method, not only have the properties of the collagen microspheres, but also have better reaction activity because unsaturated double bonds are grafted on the microspheres due to the reaction with the methacrylic anhydride, so that the microspheres can perform free radical polymerization reaction with various substances with sulfydryl more conveniently and can be self-crosslinked.
Disclosure of Invention
The invention aims to provide a preparation method of vinyl collagen microspheres, which not only realizes resource utilization of leather waste collagen, but also grafts unsaturated double bonds onto the microspheres and increases the chemical modifiability of the microspheres.
The technical scheme adopted by the invention is that the preparation method of the vinyl collagen microspheres is implemented according to the following steps;
step 1, performing double bond modification on waste skin collagen to obtain vinyl collagen;
and 2, preparing the vinyl collagen microspheres.
The present invention is also characterized in that,
the specific implementation process of the step 1 is as follows:
adding a certain amount of collagen and phosphoric acid buffer solution into a conical flask, then placing the conical flask into a constant-temperature magnetic stirrer at 70 ℃, heating and stirring until the collagen is completely dissolved, then cooling the temperature of the constant-temperature magnetic stirrer to 50 ℃, adding a certain amount of methacrylic anhydride into the conical flask for reaction for 2 hours after 20 minutes, wherein the addition amount of the methacrylic anhydride is 3/500 of the volume of the phosphoric acid buffer solution, dialyzing for 24 hours, freeze-drying for 12-24 hours, and placing the conical flask into a dryer for later use;
wherein 10ml of phosphate buffer solution is required for 1g of collagen.
The specific implementation process of the step 2 is as follows:
preparing a vinyl collagen aqueous solution with the mass fraction of 15-40%; mixing liquid paraffin and a surfactant according to a volume ratio of 50: 1, adding the mixture into a three-neck flask, mixing, placing the three-neck flask into a water bath, heating the water bath to 60 ℃ at the rotating speed of 400-1200 rpm/min, slowly dripping the vinyl collagen aqueous solution into liquid paraffin after 10min, wherein the volume ratio of the vinyl collagen aqueous solution to the liquid paraffin is 1: 4-8; emulsifying for 20min, quickly transferring to an ice bath at 0-10 ℃, adding a glutaraldehyde aqueous solution for crosslinking for 2h after 15min, wherein the addition amount of the glutaraldehyde aqueous solution is 0.4-1% of the volume of the liquid paraffin; adding isopropanol into the emulsion, wherein the adding amount of the isopropanol is 1/10 of the volume of the liquid paraffin, stirring for 10min, stopping the reaction, centrifugally separating the emulsion to obtain vinyl collagen microspheres, washing the vinyl collagen microspheres for 3 times respectively by using the isopropanol, acetone and water to remove impurities, and freeze-drying to obtain pure vinyl collagen microspheres;
wherein the mass fraction of the glutaraldehyde in the glutaraldehyde aqueous solution is 25%.
In step 1, the pH of the phosphate buffer solution was 7.4.
In step 2, the surfactant is Span 80.
In the step 2, when preparing the vinyl collagen aqueous solution, the vinyl collagen aqueous solution needs to be heated to 60 ℃ under the stirring state for dissolution assistance.
The invention has the beneficial effects that: the method of the invention utilizes the leather waste collagen as the raw material to carry out the reaction, thereby not only changing waste into valuable and improving the economic value, but also opening up a new way for recycling the waste. Meanwhile, double bond functional groups are introduced to the collagen microspheres, so that the types of the functional groups on the molecular chains of the microspheres are enriched, and the repairability of the microspheres is enhanced.
Drawings
FIG. 1 is an SEM photograph of vinyl collagen microspheres prepared in example 1;
FIG. 2 is a graph showing a distribution of particle sizes of the vinyl collagen microspheres prepared in example 1;
FIG. 3 is a schematic diagram of the chemical reaction process involved in step 1 of the process of the present invention;
FIG. 4 is a schematic diagram of the chemical reaction process involved in step 2 of the method of the present invention;
FIG. 5 shows collagen microspheres and vinyl collagen microspheres1H solid-state NMR contrast map.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
A preparation method of vinyl collagen microspheres is implemented according to the following steps;
step 1, performing double bond modification on waste skin collagen to obtain vinyl collagen; the specific implementation process of the step 1 is as follows:
adding a certain amount of collagen and phosphoric acid buffer solution into a conical flask, then placing the conical flask into a constant-temperature magnetic stirrer at 70 ℃, heating and stirring until the collagen is completely dissolved, then cooling the temperature of the constant-temperature magnetic stirrer to 50 ℃, adding a certain amount of methacrylic anhydride into the conical flask for reaction for 2 hours after 20 minutes, wherein the addition amount of the methacrylic anhydride is 3/500 of the volume of the phosphoric acid buffer solution, dialyzing for 24 hours, freeze-drying for 12-24 hours, and placing the conical flask into a dryer for later use;
wherein 10ml of phosphate buffer solution is required for 1g of collagen.
In step 1, the pH of the phosphate buffer solution was 7.4.
And 2, preparing the vinyl collagen microspheres.
The specific implementation process of the step 2 is as follows:
preparing a vinyl collagen aqueous solution with the mass fraction of 15-40%; mixing liquid paraffin and a surfactant according to a volume ratio of 50: 1, adding the mixture into a three-neck flask, mixing, placing the three-neck flask into a water bath, heating the water bath to 60 ℃ at the rotating speed of 400-1200 rpm/min, slowly dripping the vinyl collagen aqueous solution into liquid paraffin after 10min, wherein the volume ratio of the vinyl collagen aqueous solution to the liquid paraffin is 1: 4-8; emulsifying for 20min, quickly transferring to an ice bath at 0-10 ℃, adding a glutaraldehyde aqueous solution for crosslinking for 2h after 15min, wherein the addition amount of the glutaraldehyde aqueous solution is 0.4-1% of the volume of the liquid paraffin; adding isopropanol into the emulsion, wherein the adding amount of the isopropanol is 1/10 of the volume of the liquid paraffin, stirring for 10min, stopping the reaction, centrifugally separating the emulsion to obtain vinyl collagen microspheres, washing the vinyl collagen microspheres for 3 times respectively by using the isopropanol, acetone and water to remove impurities, and freeze-drying to obtain pure vinyl collagen microspheres;
wherein the mass fraction of the glutaraldehyde in the glutaraldehyde aqueous solution is 25%.
In step 2, the surfactant is Span 80.
In the step 2, when preparing the vinyl collagen aqueous solution, the vinyl collagen aqueous solution needs to be heated to 60 ℃ under the stirring state for dissolution assistance.
The invention is described below in principle:
step 1, methacrylic anhydride modifies collagen to obtain vinyl collagen
In the step 1, the preparation principle of the vinyl collagen is that amino on a molecular chain of the collagen and anhydride of methacrylic anhydride perform nucleophilic substitution reaction, the amino is used as a nucleophilic reagent to attack carbon in one carboxyl of the anhydride, and then the other carboxyl leaves to form an amide bond. The chemical reaction is shown in FIG. 3;
in the step 2, the principle of crosslinking the vinyl collagen into the vinyl collagen microspheres in the emulsification process is that amino groups on molecular chains of the collagen and aldehyde groups of glutaraldehyde undergo aldehyde-amine condensation to generate amide bonds. Glutaraldehyde acts as a "bridge" linking the vinyl collagen chains, as shown in figure 4.
Compared with collagen microspheres, the vinyl collagen microspheres are modified with double bonds, and signal peaks of unsaturated bonds just appear at 5.4-6 ppm, which proves that double bonds exist on the microspheres, as shown in fig. 5.
Example 1
A preparation method of vinyl collagen microspheres is implemented according to the following steps;
step 1, performing double bond modification on waste skin collagen to obtain vinyl collagen; the specific implementation process of the step 1 is as follows:
adding a certain amount of collagen and phosphoric acid buffer solution into a conical flask, then placing the conical flask into a constant-temperature magnetic stirrer at 70 ℃, heating and stirring until the collagen is completely dissolved, then cooling the temperature of the constant-temperature magnetic stirrer to 50 ℃, adding a certain amount of methacrylic anhydride into the conical flask for reaction for 2 hours after 20 minutes, wherein the addition amount of the methacrylic anhydride is 3/500 of the volume of the phosphoric acid buffer solution, dialyzing for 24 hours, freeze-drying for 12 hours, and placing the conical flask into a dryer for later use;
wherein 10ml of phosphate buffer solution is required for 1g of collagen.
In step 1, the pH of the phosphate buffer solution was 7.4.
And 2, preparing the vinyl collagen microspheres.
The specific implementation process of the step 2 is as follows:
preparing a vinyl collagen aqueous solution with the mass fraction of 15%; mixing liquid paraffin and a surfactant according to a volume ratio of 50: 1, adding the mixture into a three-neck flask, mixing, placing the three-neck flask into a water bath, heating the water bath to 60 ℃ at the rotating speed of 800rpm/min, slowly dripping the vinyl collagen aqueous solution into liquid paraffin after 10min, wherein the volume ratio of the vinyl collagen aqueous solution to the liquid paraffin is 1: 5; emulsifying for 20min, quickly transferring to an ice bath at 10 ℃, adding glutaraldehyde aqueous solution for crosslinking for 2h after 15min, wherein the addition amount of the glutaraldehyde aqueous solution is 0.8% of the volume of the liquid paraffin; adding isopropanol into the emulsion, wherein the adding amount of the isopropanol is 1/10 of the volume of the liquid paraffin, stirring for 10min, stopping the reaction, centrifugally separating the emulsion to obtain vinyl collagen microspheres, washing the vinyl collagen microspheres for 3 times respectively by using the isopropanol, acetone and water to remove impurities, and freeze-drying to obtain pure vinyl collagen microspheres;
wherein the mass fraction of the glutaraldehyde in the glutaraldehyde aqueous solution is 25%.
In step 2, the surfactant is Span 80.
In the step 2, when preparing the vinyl collagen aqueous solution, the vinyl collagen aqueous solution needs to be heated to 60 ℃ under the stirring state for dissolution assistance.
As can be seen from FIG. 1, the vinyl collagen microspheres prepared by the method of the present invention have regular morphology and smooth surface.
As can be seen from FIG. 2, the average particle size of the vinyl collagen microspheres prepared by the method of the present invention is 21.204 μm, and the average particle size is mostly distributed between 20 μm and 40 μm.
Example 2
A preparation method of vinyl collagen microspheres is implemented according to the following steps;
step 1, performing double bond modification on waste skin collagen to obtain vinyl collagen; the specific implementation process of the step 1 is as follows:
adding a certain amount of collagen and phosphoric acid buffer solution into a conical flask, then placing the conical flask into a constant-temperature magnetic stirrer at 70 ℃, heating and stirring until the collagen is completely dissolved, then cooling the temperature of the constant-temperature magnetic stirrer to 50 ℃, adding a certain amount of methacrylic anhydride into the conical flask for reaction for 2 hours after 20 minutes, wherein the addition amount of the methacrylic anhydride is 3/500 of the volume of the phosphoric acid buffer solution, dialyzing for 24 hours, freeze-drying for 12 hours, and placing the conical flask into a dryer for later use;
wherein 10ml of phosphate buffer solution is required for 1g of collagen.
In step 1, the pH of the phosphate buffer solution was 7.4.
And 2, preparing the vinyl collagen microspheres.
The specific implementation process of the step 2 is as follows:
preparing a vinyl collagen aqueous solution with the mass fraction of 15%; mixing liquid paraffin and a surfactant according to a volume ratio of 50: 1, adding the mixture into a three-neck flask, mixing, placing the three-neck flask into a water bath, heating the water bath to 60 ℃ at the rotating speed of 400rpm/min, slowly dripping the vinyl collagen aqueous solution into liquid paraffin after 10min, wherein the volume ratio of the vinyl collagen aqueous solution to the liquid paraffin is 1: 4; emulsifying for 20min, quickly transferring to an ice bath at 0 ℃, adding glutaraldehyde aqueous solution for crosslinking for 2h after 15min, wherein the addition amount of the glutaraldehyde aqueous solution is 0.4% of the volume of the liquid paraffin; adding isopropanol into the emulsion, wherein the adding amount of the isopropanol is 1/10 of the volume of the liquid paraffin, stirring for 10min, stopping the reaction, centrifugally separating the emulsion to obtain vinyl collagen microspheres, washing the vinyl collagen microspheres for 3 times respectively by using the isopropanol, acetone and water to remove impurities, and freeze-drying to obtain pure vinyl collagen microspheres;
wherein the mass fraction of the glutaraldehyde in the glutaraldehyde aqueous solution is 25%.
In step 2, the surfactant is Span 80.
In the step 2, when preparing the vinyl collagen aqueous solution, the vinyl collagen aqueous solution needs to be heated to 60 ℃ under the stirring state for dissolution assistance.
Example 3
A preparation method of vinyl collagen microspheres is implemented according to the following steps;
step 1, performing double bond modification on waste skin collagen to obtain vinyl collagen; the specific implementation process of the step 1 is as follows:
adding a certain amount of collagen and phosphoric acid buffer solution into a conical flask, then placing the conical flask into a constant-temperature magnetic stirrer at 70 ℃, heating and stirring until the collagen is completely dissolved, then cooling the temperature of the constant-temperature magnetic stirrer to 50 ℃, adding a certain amount of methacrylic anhydride into the conical flask for reaction for 2 hours after 20 minutes, wherein the addition amount of the methacrylic anhydride is 3/500 of the volume of the phosphoric acid buffer solution, dialyzing for 24 hours, freeze-drying for 24 hours, and placing the conical flask into a dryer for later use;
wherein 10ml of phosphate buffer solution is required for 1g of collagen.
In step 1, the pH of the phosphate buffer solution was 7.4.
And 2, preparing the vinyl collagen microspheres.
The specific implementation process of the step 2 is as follows:
preparing a vinyl collagen aqueous solution with the mass fraction of 40%; mixing liquid paraffin and a surfactant according to a volume ratio of 50: 1, adding the mixture into a three-neck flask, mixing, placing the three-neck flask into a water bath, heating the water bath to 60 ℃ at the rotating speed of 1200rpm/min, slowly dripping the vinyl collagen aqueous solution into liquid paraffin after 10min, wherein the volume ratio of the vinyl collagen aqueous solution to the liquid paraffin is 1: 8; emulsifying for 20min, quickly transferring to an ice bath at 10 ℃, adding glutaraldehyde aqueous solution for crosslinking for 2h after 15min, wherein the addition amount of the glutaraldehyde aqueous solution is 1% of the volume of the liquid paraffin; adding isopropanol into the emulsion, wherein the adding amount of the isopropanol is 1/10 of the volume of the liquid paraffin, stirring for 10min, stopping the reaction, centrifugally separating the emulsion to obtain vinyl collagen microspheres, washing the vinyl collagen microspheres for 3 times respectively by using the isopropanol, acetone and water to remove impurities, and freeze-drying to obtain pure vinyl collagen microspheres;
wherein the mass fraction of the glutaraldehyde in the glutaraldehyde aqueous solution is 25%.
In step 2, the surfactant is Span 80.
In the step 2, when preparing the vinyl collagen aqueous solution, the vinyl collagen aqueous solution needs to be heated to 60 ℃ under the stirring state for dissolution assistance.
Example 4
A preparation method of vinyl collagen microspheres is implemented according to the following steps;
step 1, performing double bond modification on waste skin collagen to obtain vinyl collagen; the specific implementation process of the step 1 is as follows:
adding a certain amount of collagen and phosphoric acid buffer solution into a conical flask, then placing the conical flask into a constant-temperature magnetic stirrer at 70 ℃, heating and stirring until the collagen is completely dissolved, then cooling the temperature of the constant-temperature magnetic stirrer to 50 ℃, adding a certain amount of methacrylic anhydride into the conical flask for reaction for 2 hours after 20 minutes, wherein the addition amount of the methacrylic anhydride is 3/500 of the volume of the phosphoric acid buffer solution, dialyzing for 24 hours, freeze-drying for 20 hours, and placing the conical flask into a dryer for later use;
wherein 10ml of phosphate buffer solution is required for 1g of collagen.
In step 1, the pH of the phosphate buffer solution was 7.4.
And 2, preparing the vinyl collagen microspheres.
The specific implementation process of the step 2 is as follows:
preparing a vinyl collagen aqueous solution with the mass fraction of 20%; mixing liquid paraffin and a surfactant according to a volume ratio of 50: 1, adding the mixture into a three-neck flask, mixing, placing the three-neck flask into a water bath, heating the water bath to 60 ℃ at the rotating speed of 800rpm/min, slowly dripping the vinyl collagen aqueous solution into liquid paraffin after 10min, wherein the volume ratio of the vinyl collagen aqueous solution to the liquid paraffin is 1: 5; emulsifying for 20min, quickly transferring to an ice bath at 0-10 ℃, adding a glutaraldehyde aqueous solution for crosslinking for 2h after 15min, wherein the addition amount of the glutaraldehyde aqueous solution is 0.5% of the volume of the liquid paraffin; adding isopropanol into the emulsion, wherein the adding amount of the isopropanol is 1/10 of the volume of the liquid paraffin, stirring for 10min, stopping the reaction, centrifugally separating the emulsion to obtain vinyl collagen microspheres, washing the vinyl collagen microspheres for 3 times respectively by using the isopropanol, acetone and water to remove impurities, and freeze-drying to obtain pure vinyl collagen microspheres;
wherein the mass fraction of the glutaraldehyde in the glutaraldehyde water solution is 25%.
In step 2, the surfactant is Span 80.
In the step 2, when preparing the vinyl collagen aqueous solution, the vinyl collagen aqueous solution needs to be heated to 60 ℃ under the stirring state for dissolution assistance.
Example 5
A preparation method of vinyl collagen microspheres is implemented according to the following steps;
step 1, performing double bond modification on waste skin collagen to obtain vinyl collagen; the specific implementation process of the step 1 is as follows:
adding a certain amount of collagen and phosphoric acid buffer solution into a conical flask, then placing the conical flask into a constant-temperature magnetic stirrer at 70 ℃, heating and stirring until the collagen is completely dissolved, then cooling the temperature of the constant-temperature magnetic stirrer to 50 ℃, adding a certain amount of methacrylic anhydride into the conical flask for reaction for 2 hours after 20 minutes, wherein the addition amount of the methacrylic anhydride is 3/500 of the volume of the phosphoric acid buffer solution, dialyzing for 24 hours, freeze-drying for 15 hours, and placing the conical flask into a dryer for later use;
wherein 10ml of phosphate buffer solution is required for 1g of collagen.
In step 1, the pH of the phosphate buffer solution was 7.4.
And 2, preparing the vinyl collagen microspheres.
The specific implementation process of the step 2 is as follows:
preparing a vinyl collagen aqueous solution with the mass fraction of 20%; mixing liquid paraffin and a surfactant according to a volume ratio of 50: 1, adding the mixture into a three-neck flask, mixing, placing the three-neck flask into a water bath, heating the water bath to 60 ℃ at the rotating speed of 800rpm/min, slowly dripping the vinyl collagen aqueous solution into liquid paraffin after 10min, wherein the volume ratio of the vinyl collagen aqueous solution to the liquid paraffin is 1: 5; emulsifying for 20min, quickly transferring to an ice bath at 6 ℃, adding glutaraldehyde aqueous solution for crosslinking for 2h after 15min, wherein the addition amount of the glutaraldehyde aqueous solution is 0.7% of the volume of the liquid paraffin; adding isopropanol into the emulsion, wherein the adding amount of the isopropanol is 1/10 of the volume of the liquid paraffin, stirring for 10min, stopping the reaction, centrifugally separating the emulsion to obtain vinyl collagen microspheres, washing the vinyl collagen microspheres for 3 times respectively by using the isopropanol, acetone and water to remove impurities, and freeze-drying to obtain pure vinyl collagen microspheres;
wherein the mass fraction of the glutaraldehyde in the glutaraldehyde aqueous solution is 25%.
In step 2, the surfactant is Span 80.
In the step 2, when preparing the vinyl collagen aqueous solution, the vinyl collagen aqueous solution needs to be heated to 60 ℃ under the stirring state for dissolution assistance.
Claims (6)
1. The preparation method of the vinyl collagen microspheres is characterized by comprising the following steps of;
step 1, performing double bond modification on waste skin collagen to obtain vinyl collagen;
and 2, preparing the vinyl collagen microspheres.
2. The method for preparing vinyl collagen microspheres according to claim 1, wherein the specific implementation process of step 1 is as follows:
adding a certain amount of collagen and phosphoric acid buffer solution into a conical flask, then placing the conical flask into a constant-temperature magnetic stirrer at 70 ℃, heating and stirring until the collagen is completely dissolved, then cooling the temperature of the constant-temperature magnetic stirrer to 50 ℃, adding a certain amount of methacrylic anhydride into the conical flask for reaction for 2 hours after 20 minutes, wherein the addition amount of the methacrylic anhydride is 3/500 of the volume of the phosphoric acid buffer solution, dialyzing for 24 hours, freeze-drying for 12-24 hours, and placing the conical flask into a dryer for later use;
wherein 10ml of phosphate buffer solution is required for 1g of collagen.
3. The method for preparing vinyl collagen microspheres according to claim 1, wherein the step 2 is carried out by:
preparing a vinyl collagen aqueous solution with the mass fraction of 15-40%; mixing liquid paraffin and a surfactant according to a volume ratio of 50: 1, adding the mixture into a three-neck flask, mixing, placing the three-neck flask into a water bath, heating the water bath to 60 ℃ at the rotating speed of 400-1200 rpm/min, slowly dripping the vinyl collagen aqueous solution into liquid paraffin after 10min, wherein the volume ratio of the vinyl collagen aqueous solution to the liquid paraffin is 1: 4-8; emulsifying for 20min, quickly transferring to an ice bath at 0-10 ℃, adding a glutaraldehyde aqueous solution for crosslinking for 2h after 15min, wherein the addition amount of the glutaraldehyde aqueous solution is 0.4-1% of the volume of the liquid paraffin; adding isopropanol into the emulsion, wherein the adding amount of the isopropanol is 1/10 of the volume of the liquid paraffin, stirring for 10min, stopping the reaction, centrifugally separating the emulsion to obtain vinyl collagen microspheres, washing the vinyl collagen microspheres for 3 times respectively by using the isopropanol, acetone and water to remove impurities, and freeze-drying to obtain pure vinyl collagen microspheres;
wherein the mass fraction of the glutaraldehyde in the glutaraldehyde aqueous solution is 25%.
4. The method for preparing vinyl collagen microspheres according to claim 2, wherein the pH of the phosphate buffer solution in step 1 is 7.4.
5. The method for preparing vinyl collagen microspheres according to claim 2, wherein in step 2, the surfactant is Span 80.
6. The method of claim 2, wherein the aqueous solution of collagen vinyl is heated to 60 degrees centigrade for solubilization under stirring in step 2.
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