CN109535447B - Thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel and preparation method thereof - Google Patents
Thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel and a preparation method thereof, and the preparation method comprises the steps of preparing collagen self-assembly hydrogel, crushing the collagen nanofiber hydrogel and synthesizing poly (isopropyl acrylamide) (PNIPAM) in situ, introducing a thermosensitive polymer, forming a semi-interpenetrating macromolecular network with the self-assembled collagen nanofiber in situ, and preparing the thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel, wherein the contraction-expansion behavior of the hydrogel has responsiveness to temperature (near the body temperature), so that the controllable release of a medicament is realized; the collagen hydrogel prepared by the invention induces collagen to form nano-fibers by using simulated physiological conditions, and then enables the collagen nano-fibers and PNIPAM to form a semi-interpenetrating gel network by using polymerization of NIPAM monomers, so that no additional chemical cross-linking agent is required, and potential cytotoxicity is reduced.
Description
Technical Field
The invention belongs to the technical field of collagen materials, and particularly relates to a thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel and a preparation method thereof.
Background
Collagen is the major structural protein of extracellular matrix, the most abundant protein in the organism, and it is widely present in the human and animal bodies. As a natural polymer material with a wide variety of sources, collagen has many excellent properties, such as high affinity with cells, hemagglutination with platelets, promotion of wound healing and creeping coverage of epidermal cells, biodegradability, and the like.
Under the simulated physiological condition, collagen molecules in the solution can be subjected to in-vitro self-assembly to form collagen nanofibers, and the collagen nanofiber network binds a large number of water molecules to form the collagen nanofiber hydrogel. The self-assembled collagen hydrogel has wide application in the fields of biomedical materials, tissue engineering and the like, and can be used as cell culture scaffold materials, cosmetic injection filling materials, drug slow-release carriers and the like.
Disclosure of Invention
The invention aims to provide a thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel and a preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel comprises the following steps:
(1) preparation of collagen self-assembled hydrogel: dissolving collagen sponge in 0.005-0.02mol/L acetic acid solution to obtain collagen solution with concentration of 5-15 mg/ml; adding the collagen solution, phosphate buffer solution, deionized water and sodium chloride into a glass container under an ice bath state, mixing and stirring until the mixture is transparent and clear to obtain a mixed solution A, then placing 5ml of the mixed solution A into a water bath at 37 ℃ for constant temperature for 30-90min, and obtaining collagen nanofiber hydrogel after collagen self-assembly is completed;
(2) crushing the collagen nanofiber hydrogel: fully crushing the collagen nanofiber hydrogel through a vortex mixer to obtain a flowable collagen nanofiber hydrogel, wherein the nanofiber structure of the collagen is not damaged;
(3) in-situ synthesis of polyisopropylacrylamide: dissolving N-isopropyl acrylamide, N, N-methylene bisacrylamide and potassium persulfate in 200 mu L of phosphate buffer solution, adding the mixed solution into the flowable collagen nanofiber hydrogel obtained in the step (2), uniformly mixing the flowable collagen nanofiber hydrogel by using a vortex mixer, placing the mixture in a water bath at the temperature of 20-26 ℃, dropwise adding 20 mu LN, N, N ', N' -tetramethyl ethylenediamine under the nitrogen atmosphere to initiate in-situ polymerization of an N-isopropyl acrylamide monomer, reacting for 5-10min to obtain collagen self-assembled hydrogel, and then soaking, cleaning and removing impurities to obtain the heat-sensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel.
Further, the phosphate buffer in step (1) and step (3) was 0.2mol/L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, and the pH was 8.0.
Further, the volume ratio of the collagen solution, the phosphate buffer solution and the deionized water in the mixed solution A is 5:2: 3.
Further, the concentration of sodium chloride in the mixed solution A is 80-100 mmol/L.
Further, the mass of the N-isopropylacrylamide in the step (3) is 0.02 to 0.1 g.
Further, in the step (3), the mass ratio of the N, N-methylene bisacrylamide to the N-isopropylacrylamide is (1-3): 20.
further, the mass of potassium persulfate in the step (3) was 0.02 g.
The thermal sensitive collagen nanofiber hydrogel prepared by the preparation method has the shrinkage-expansion behavior with the responsiveness to temperature (near the body temperature of a human body), and can realize the controlled release of the drug.
The invention has the following remarkable advantages:
(1) compared with the existing collagen hydrogel, the hydrogel prepared by the technology has heat sensitivity, and can generate shrinkage-swelling reversible phase change under the stimulation effect of certain temperature. Particularly, under the action of body temperature (37 ℃) higher than LCST of PNIPAM, the gel slowly shrinks to slowly release the medicine, thereby achieving the purpose of slow release. The existing collagen hydrogel has weak sensitivity to temperature and has no temperature regulation and control characteristic; pure PNIPAM hydrogel releases almost no drug after initial burst.
(2) The collagen hydrogel prepared by the technology belongs to the nanofiber network hydrogel formed by self-assembly, has a bionic structure similar to that of in-vivo collagen fibers, and is beneficial to the application of the collagen hydrogel in the field of biomedical materials. Secondly, the collagen is induced to form the nano-fiber by using the simulated physiological condition, and then the collagen nano-fiber and the PNIPAM form a semi-interpenetrating gel network by using the polymerization of the NIPAM monomer, so that no additional chemical cross-linking agent is required to be added, and the potential cytotoxicity is reduced.
Drawings
FIG. 1 is a comparison graph of pure collagen nanofiber hydrogel and thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel of the present invention;
FIG. 2 is a network structure diagram of pure collagen nanofiber hydrogel and the thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel of the present invention;
fig. 3 is a graph comparing the release rates of the thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network hydrogel (PNIPAM/collagen), pure PNIPAM hydrogel and pure collagen hydrogel according to the present invention.
Detailed Description
In order to facilitate an understanding of the present invention, the following examples are provided to further illustrate the present invention, but are not intended to limit the scope of the present invention.
Example 1
(1) Preparation of collagen self-assembled hydrogel: dissolving collagen sponge in 0.005mol/L acetic acid solution to obtain collagen solution with concentration of 5 mg/ml; preparing 0.2mol/L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH8.0) as phosphate buffer solution; 5ml of a solution with a collagen concentration of 2.5mg/ml are prepared according to the following proportion:
collagen solution 2.5ml
Phosphate buffer 1ml
Deionized water 1.5ml
Sodium chloride is added to a concentration of 100mmol/L
The components are added into a flask in sequence under the ice bath state, and the solution is stirred to become transparent and clear. Then placing the mixture in a water bath with the temperature of 37 ℃ for constant temperature for 60 min. The collagen solution becomes collagen nanofiber hydrogel.
(2) Crushing the collagen nanofiber hydrogel: the collagen hydrogel was sufficiently crushed by applying vortex action with a vortex mixer, but the nanofiber structure of the collagen was not damaged.
(3) In-situ synthesis of polyisopropylacrylamide: preparing a mixed solution according to the following proportion:
NIPAM (N-isopropylacrylamide) 0.06g
BIS (N, N-methylenebisacrylamide) BIS/NIPAM mass ratio = 3:20
KPS (potassium persulfate) 0.02g
TEMED (N, N, N ', N' -tetramethylethylenediamine) 20. mu.L
The NIPAM, BIS and KPS were dissolved in phosphate buffer solution in advance. Adding the mixed solution into the crushed collagen nanofiber gel, completely mixing the crushed collagen nanofiber gel and the crushed collagen nanofiber gel by using a vortex mixer, then placing the mixture into a water bath at 20 ℃, dropwise adding TEMED under the nitrogen atmosphere, and uniformly mixing by using the vortex mixer to initiate NIPAM monomer to carry out in-situ polymerization. And continuing to react in the water bath at 20 ℃, purging with nitrogen for protection, changing the solution into transparent hydrogel again after a plurality of minutes, and continuing to react for 3 hours. Soaking in deionized water, and changing the solution for 3 times to allow water-soluble linear PNIPAM and other impurities not forming a network structure to escape, thereby obtaining the thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel.
Example 2
(1) Preparation of collagen self-assembled hydrogel: the collagen sponge is dissolved in 0.02mol/L acetic acid solution, and the concentration is 10 mg/ml. A0.2 mol/L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 8.0) was prepared. 5ml of a solution with a collagen concentration of 5mg/ml is prepared according to the following proportion:
collagen 2.5ml
Phosphate buffer 1ml
Deionized water 1.5ml
Sodium chloride was added to a concentration of 90mmol/L
The components are added into a small flask under the ice bath state, and the solution is stirred to be transparent and clear. Then placing the mixture in a water bath with the temperature of 37 ℃ for 30 min. At the moment, the collagen self-assembles, and the collagen solution becomes the collagen nanofiber hydrogel.
(2) Crushing the collagen nanofiber hydrogel: the collagen hydrogel is swirled by a swirl mixer to fully break the gel into flowable collagen nanofiber gel, and the nanofiber structure of the collagen is not damaged.
(3) In-situ synthesis of polyisopropylacrylamide: preparing a mixed solution according to the following proportion:
NIPAM (N-isopropylacrylamide) 0.02g
BIS (N, N-methylenebisacrylamide) BIS/NIPAM mass ratio =1:10
KPS (potassium persulfate) 0.02g
TEMED (N, N, N ', N' -tetramethylethylenediamine) 20. mu.L
The NIPAM, BIS and KPS were dissolved in phosphate buffer solution in advance. Adding the mixed solution into the crushed collagen nanofiber gel, completely mixing the crushed collagen nanofiber gel and the crushed collagen nanofiber gel by using a vortex mixer, placing the mixture into a water bath at 26 ℃, dropwise adding TEMED under the nitrogen atmosphere, and uniformly mixing by using the vortex mixer to initiate NIPAM monomer to carry out in-situ polymerization. And continuing to react in the water bath at 26 ℃, purging with nitrogen for protection, changing the solution into transparent hydrogel again after a plurality of minutes, and continuing to react for 1 hour. Soaking in deionized water, and changing the solution for 6 times to allow water-soluble linear PNIPAM and other impurities not forming a network structure to escape, thereby obtaining the thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel.
Example 3
(1) Preparation of collagen self-assembled hydrogel: the collagen sponge is dissolved in 0.01mol/L acetic acid solution, and the concentration is 15 mg/ml. A0.2 mol/L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 8.0) was prepared. 5ml of a solution with a collagen concentration of 7.5mg/ml are prepared according to the following proportion:
collagen 2.5ml
Phosphate buffer 1ml
Deionized water 1.5ml
Sodium chloride was added to a concentration of 80mmol/L
The components are added into a small sesame seed cake in an ice bath state, and the solution is stirred to be transparent and clear. Then placing the mixture in a water bath with the temperature of 37 ℃ for constant temperature for 90 min. At the moment, the collagen self-assembles, and the collagen solution becomes the collagen nanofiber hydrogel.
(2) Crushing the collagen nanofiber hydrogel: the collagen hydrogel is swirled by a swirl mixer to fully break the gel into flowable collagen nanofiber gel, and the nanofiber structure of the collagen is not damaged.
(3) In-situ synthesis of polyisopropylacrylamide: preparing a mixed solution according to the following proportion:
NIPAM (N-isopropylacrylamide) 0.10g
BIS (N, N-methylenebisacrylamide) BIS/NIPAM mass ratio =1:20
KPS (potassium persulfate) 0.02g
TEMED (N, N, N ', N' -tetramethylethylenediamine) 20. mu.L
The NIPAM, BIS and KPS were dissolved in phosphate buffer solution in advance. Adding the mixed solution into the crushed collagen nanofiber gel, completely mixing the crushed collagen nanofiber gel and the crushed collagen nanofiber gel by using a vortex mixer, then placing the mixture into a water bath at 23 ℃, dropwise adding TEMED under the nitrogen atmosphere, and uniformly mixing by using the vortex mixer to initiate NIPAM monomer to carry out in-situ polymerization. And continuing to react in the water bath at the temperature of 23 ℃, purging with nitrogen for protection, changing the solution into transparent hydrogel again after a plurality of minutes, and continuing to react for 2 hours. Soaking in deionized water, and changing the solution for 4 times to allow water-soluble linear PNIPAM and other impurities not forming a network structure to escape, thereby obtaining the thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel.
FIG. 2: the pure collagen water gel network formed by self-assembly under the simulated physiological condition consists of nano fibers, and the average diameter is about 100nm magnitude; the semi-interpenetrating thermosensitive collagen hydrogel network is formed by hybridizing and interspersing collagen nanofibers and PNIPAM macromolecules, the collagen nanofibers and the PNIPAM macromolecules have interface combination effect, and the shapes of the nanofiber and the macromolecule components can be distinguished.
FIG. 3: releasing conditions are as follows: 37 ℃, pH = 7.2. The semi-interpenetrating thermosensitive collagen nanofiber hydrogel (PNIPAM/collagen) disclosed by the invention has an obvious slow-release effect and a high cumulative release rate. The following properties are embodied: under the body temperature environment (37 ℃) higher than LCST of PNIPAM, the hybrid hydrogel slowly shrinks to slowly release the medicine, thereby achieving the purpose of slow release. In addition, the pure PNIPAM hydrogel only has small initial burst release and no slow release action, and the cumulative release rate is extremely low because the gel pore channels are almost completely blocked at the temperature. The pure collagen hydrogel has insufficient sensitivity to body temperature (the release curves at 37 ℃ and 25 ℃ are highly coincident), the release behavior of the pure collagen hydrogel can not be effectively regulated and controlled by temperature, and the cumulative release rate is not high.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (6)
1. A preparation method of thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel is characterized by comprising the following steps: the method comprises the following steps:
(1) preparation of collagen self-assembled hydrogel: dissolving collagen sponge in 0.005-0.02mol/L acetic acid solution to obtain collagen solution with concentration of 5-15 mg/ml; adding the collagen solution, phosphate buffer solution, deionized water and sodium chloride into a glass container under an ice bath state, mixing and stirring until the mixture is transparent and clear to obtain a mixed solution A, then placing 5ml of the mixed solution A into a water bath at 37 ℃ for constant temperature for 30-90min, and obtaining collagen nanofiber hydrogel after collagen self-assembly is completed;
(2) crushing the collagen nanofiber hydrogel: fully crushing the collagen nanofiber hydrogel through a vortex mixer to obtain flowable collagen nanofiber hydrogel;
(3) in-situ synthesis of polyisopropylacrylamide: dissolving N-isopropylacrylamide (NIPAM), N, N-methylenebisacrylamide and potassium persulfate in 200 mu L of phosphate buffer solution, adding the mixed solution into the flowable collagen nanofiber hydrogel obtained in the step (2), uniformly mixing the flowable collagen nanofiber hydrogel with a vortex mixer, placing the mixture in a water bath at the temperature of 20-26 ℃, dropwise adding 20 mu L N, N, N ', N' -tetramethylethylenediamine under the nitrogen atmosphere to initiate NIPAM to carry out in-situ polymerization, reacting for 5-10min to obtain collagen self-assembled hydrogel, and then soaking, cleaning and removing impurities to obtain the thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network hydrogel;
the volume ratio of the collagen solution, the phosphate buffer solution and the deionized water in the mixed solution A is 5:2: 3; in the step (3), the mass ratio of the N, N-methylene bisacrylamide to the N-isopropyl acrylamide is (1-3): 20.
2. the method for preparing the thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel as claimed in claim 1, wherein the method comprises the steps of: the phosphate buffer solution in the step (1) and the step (3) is 0.2mol/L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, and the pH value is 8.0.
3. The method for preparing the thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel as claimed in claim 1, wherein the method comprises the steps of: the concentration of sodium chloride in the mixed solution A is 80-100 mmol/L.
4. The method for preparing the thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel as claimed in claim 1, wherein the method comprises the steps of: the mass of the N-isopropyl acrylamide in the step (3) is 0.02-0.1 g.
5. The method for preparing the thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel as claimed in claim 1, wherein the method comprises the steps of: the mass of the potassium persulfate in the step (3) was 0.02 g.
6. The heat-sensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel prepared by the preparation method of any one of claims 1-5.
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