CN113384743A - Preparation method of temperature-sensitive dressing with tissue repair promoting and antibacterial functions - Google Patents
Preparation method of temperature-sensitive dressing with tissue repair promoting and antibacterial functions Download PDFInfo
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- 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
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/008—Hydrogels or hydrocolloids
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- A—HUMAN NECESSITIES
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- 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
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0052—Mixtures of macromolecular compounds
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- 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
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
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- 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
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/0066—Medicaments; Biocides
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- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/0085—Porous materials, e.g. foams or sponges
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- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
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- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
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- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/602—Type of release, e.g. controlled, sustained, slow
Abstract
The invention discloses a preparation method of a temperature-sensitive dressing with the functions of promoting tissue repair and resisting bacteria, which comprises the following steps: step S1: grafting RADA16 and Amps to form self-assembly peptide, dissolving the self-assembly peptide into 2-20mg/mL self-assembly polypeptide aqueous solution by using double distilled water to form the self-assembly peptide; step S2: dissolving poly N-isopropyl acrylamide in HCl-Tris buffer solution, and stirring with a magnetic stirrer to obtain poly N-isopropyl acrylamide solution; step S3: stirring and mixing the polypeptide solution subjected to self-assembly with the poly N-isopropylacrylamide solution by using a magnetic stirrer to obtain a final solution-hydrogel; step S4: the repair peptide is added into the final solution to obtain the product. The method realizes the preparation of the reversible hydrogel with temperature responsiveness by a physical crosslinking method between the nano structure obtained by self-assembly of the self-assembly polypeptide and the poly-N-isopropylacrylamide, mixes the soluble repair peptide to realize the loading and controlled release of the drug, and is simple and easy to implement.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a preparation method of a temperature-sensitive dressing with the functions of promoting tissue repair and resisting bacteria.
Background
The biological intelligent material is a material which can be functionalized by changing parameters such as temperature, pH value or magnetic field. Among them, the intelligent hydrogel is similar to biological tissues, has good biocompatibility, can be widely applied to the biomedical field as a tissue engineering scaffold, a drug sustained release carrier and the like, and is therefore of great interest in recent years.
The temperature-sensitive hydrogel can form in-situ hydrogel at the phase transition temperature, namely, when the temperature is lower than the phase transition temperature, the system is in a sol state; above the phase transition temperature, the system undergoes phase transition and changes to a gel state. Because the drug is in a solution state at low temperature, the drug can be well loaded, the temperature is increased to form gel after injection (such as subcutaneous injection) or skin smearing, the embedded drug can be diffused to human tissues by self to realize effective and controllable release, and the characteristic makes the drug have new progress in the aspects of drug delivery, tissue engineering, cell membrane engineering and the like.
The self-assembly peptide forms a plurality of peptide chains into nano structures with different shapes through non-covalent bond action, such as assemblies with different structures, such as nano particles, nano tubes, nano fibers, nano rods, nano capsules or gel, and the like, has good biocompatibility due to the amino acid as a basic composition unit, has different functions by modifying functional short peptide sequences on the peptide chains, can provide surface groups or hydrophobic internal cavities for combining and loading drug molecules, is an excellent carrier for loading drugs, but does not have temperature-sensitive property.
The phase transition temperature of the PNIPAM hydrogel of the temperature-sensitive copolymer poly N-isopropylacrylamide (PNIPAM) is about 32 ℃, the temperature-sensitive characteristic of the PNIPAM hydrogel is mainly generated by a monomer material NIPAM (N-isopropylacrylamide) forming the PNIPAM hydrogel, and the NIPAM molecules simultaneously have hydrophilic amido bond (-CONH-) and hydrophobic isopropyl (-CH (CH)3)2). When the temperature is low, the polymer moleculeThe hydrophilic groups in the PNIPAM are dominant, and the hydrophilic amide groups and water molecules form hydrogen bonds to form a compact hydrated shell structure, so that the PNIPAM polymer is in an extended 'coil' state and is soluble at the moment. When the temperature rises to reach the LCST, the hydrogen bonding action is weakened, the hydrophobic action between polymers is strengthened due to the existence of isopropyl, and water is released from the polymers to form compact 'spheres'. In summary, PNIPAM produces a linear to spherical structural transition upon stimulation by temperature.
Disclosure of Invention
The invention aims to provide a preparation method of a temperature-sensitive dressing with the functions of promoting tissue repair and resisting bacteria, so as to achieve the aim.
In order to achieve the purpose, the invention provides the following technical scheme:
a preparation method of a temperature-sensitive dressing with the functions of promoting tissue repair and resisting bacteria comprises the following steps:
step S1: grafting RADA16 (short peptide hydrogel) and Amps to form self-assembled peptide, dissolving the self-assembled peptide with double distilled water to prepare 2-20mg/mL self-assembled polypeptide aqueous solution, carrying out ultrasonic treatment for 15-30min under the ice bath condition, and then placing the self-assembled peptide for 12-48h at the temperature of 4 ℃ to complete self-assembly of the self-assembled peptide to form the self-assembled peptide with the antibacterial function;
step S2: dissolving poly-N-isopropylacrylamide in HCl-Tris (Tris (hydroxymethyl) aminomethane hydrochloride) buffer solution with the pH =7.4, stirring by using a magnetic stirrer at the stirring speed of 200 rpm/min at the temperature of 4 ℃ for 1-4 hours to obtain poly-N-isopropylacrylamide solution, and standing to fully crosslink the poly-N-isopropylacrylamide solution;
step S3: stirring and mixing the polypeptide solution after the self-assembly and the poly N-isopropylacrylamide solution at room temperature and 4 ℃ by using a magnetic stirrer according to the following compounding conditions, wherein the stirring speed is 200-400rpm/min, and the stirring time is 2-5h, so as to obtain a final solution-hydrogel;
step S4: the repair peptide is added into the final solution to obtain the product.
On the basis of the technical scheme, the invention also provides the following optional technical scheme:
in one alternative: the compounding condition in the step S3 is that the concentration of the polypeptide in the final solution is greater than the self-assembly critical concentration of the polypeptide and the polypeptide is uniformly dispersed, and the concentration of the poly-N-isopropylacrylamide in the final solution is 5.0-40 mg/mL.
In one alternative: and the standing time of the poly N-isopropylacrylamide solution in the step S2 is 48-72 h.
In one alternative: in the step S1, the ultrasonic power of the self-assembly polypeptide aqueous solution in the ultrasonic treatment is 480W.
In one alternative: the operation step of adding the repair peptide into the final solution-hydrogel in the step S4 comprises the steps of stirring and mixing at the temperature of 4 ℃ by using a magnetic stirrer, wherein the stirring speed is 200-400rpm/min, the stirring time is 1-4 hours, standing for 3-5 hours at the temperature of 4 ℃ after stirring, fully and uniformly mixing the drugs in the hydrogel solution to obtain a drug-loaded mixed solution, and heating the mixed solution to the temperature of more than 32 ℃ to form the temperature-sensitive dressing for promoting tissue repair and antibiosis.
In one alternative: the repair peptide is one or more of MGFE peptide, IGF-1, GHRP6, VEGF and recombinant type III collagen.
In one alternative: the concentration range of the repair peptide is a concentration effective for cells or organisms.
The temperature-responsive reversible hydrogel dressing prepared by the preparation method of the temperature-sensitive dressing with the functions of promoting tissue repair and resisting bacteria is applied to tissue repair.
Compared with the prior art, the invention has the following beneficial effects:
1. the self-assembly polypeptide RA-Amps is derived from the synthesis of amino acid, and the formed three-dimensional scaffold has good histocompatibility and bioactivity. The functional motif RRWRVIVKW is modified, so that the bacteriostatic property of the functional motif RRWRVIVKW can be improved; according to the method, the preparation of the reversible hydrogel with temperature responsiveness is realized by a physical crosslinking method between the nano structure obtained by self-assembly of the self-assembly polypeptide and the poly-N-isopropylacrylamide, the soluble repair peptide is mixed in, the loading and controlled release of the drug are realized, and the method is simple and easy to implement;
2. the temperature response type reversible hydrogel can provide a hydrophobic environment or surface groups for the interaction of medicines, thereby realizing the high-efficiency loading of the medicines, having temperature sensitivity and being capable of controlling the release of the medicines;
3. the phase transition temperature of the temperature-responsive reversible hydrogel is near body temperature, the temperature can be raised to form the hydrogel through the temperature of a human body, and the temperature reduction and solution recovery can be simply and conveniently realized through physical temperature reduction modes such as ice compress and the like, so that secondary damage caused when dressing is replaced can be reduced.
Drawings
FIG. 1 is a graph showing the results of experiments on the bacteriostatic properties of RA-Amps against Staphylococcus aureus and Escherichia coli provided in the examples of the present invention.
FIG. 2 shows the gel time variation of temperature-sensitive hydrogels formed by combining PNIPAM and RA-Amps at different ratios provided by the embodiment of the present invention.
FIG. 3 is a photograph of PNI/RA-Amps temperature-sensitive hydrogel provided in the examples of the present invention in different states at 25 ℃ and 37 ℃.
FIG. 4 shows the rheological performance results of temperature-sensitive hydrogels formed by combining PNIPAM and RA-Amps at different ratios provided by the examples of the present invention.
FIG. 5 is a microscopic SEM image of a PNI/RA-Amps temperature-sensitive hydrogel provided in the examples of the present invention.
FIG. 6 is a graph showing the time-dependent release of MGF E from PNI/RA-Amps temperature-sensitive hydrogels provided in the examples of the present invention at 37 ℃.
FIG. 7 shows the results of the HFF cytotoxicity test of PNI/RA-Amps temperature-sensitive hydrogel provided in the examples of the present invention.
FIG. 8 shows the effect of PNI/RA-Amps/E drug-loaded temperature-sensitive hydrogel provided in the present invention on wound healing.
Detailed Description
The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention. Any obvious modifications or variations can be made to the present invention without departing from the spirit or scope of the present invention.
Example 1
The embodiment provides a preparation method of a temperature-sensitive dressing with the functions of promoting tissue repair and resisting bacteria, which essentially screens out the optimal proportion of a temperature-sensitive copolymer and a self-assembled peptide solution to form a stable hydrogel structure at 37 ℃, and specifically comprises the following steps:
s1: grafting RADA16 (short peptide hydrogel) and Amps to form Ac-RADARADARADARADA- (Acp) -RRWRVIVKW self-assembly peptide, dissolving the peptide by double distilled water to prepare a self-assembly polypeptide aqueous solution of 2mg/mL, performing ultrasonic treatment for 30min under an ice bath condition, performing ultrasonic treatment, and standing for 12h at 4 ℃ to complete self-assembly of the self-assembly peptide to form the self-assembly peptide with an antibacterial function;
the self-assembly polypeptide RA-Amps is prepared in the step: the polypeptide sequence Ac-RADARADARADARADA- (Acp) -RRWRVIVKW, and the polypeptide is modified by functional motif RRWRVIVKW. The self-assembly polypeptide is self-assembled into a nanofiber structure, and the formed three-dimensional scaffold has good histocompatibility and bioactivity. And a functional motif RRWRVIVKW is added for modifying so that the peptide fiber has bacteriostatic property. The concentration of the polypeptide in the final solution is a concentration that is greater than its critical concentration for self-assembly while maintaining uniform dispersion.
S2: dissolving poly-N-isopropylacrylamide (PNIPAM) in HCl-Tris (Tris (hydroxymethyl) aminomethane hydrochloride) buffer solution with the pH =7.4, stirring by using a magnetic stirrer, wherein the stirring speed is 200-400rpm/min at the temperature of 4 ℃, and the stirring time is 2-5 hours to obtain poly-N-isopropylacrylamide solution with the concentration of 40mg/mL, and standing the obtained sample to fully crosslink the sample;
in the step, the dissolving speed of the poly-N-isopropylacrylamide is low, and the poly-N-isopropylacrylamide needs to be fully dissolved; and in dissolving poly-N-isopropylacrylamide, at 4 ℃; the poly-N-isopropylacrylamide has temperature-sensitive characteristic, namely, the poly-N-isopropylacrylamide is in a solution state at room temperature (20-25 ℃) and forms a gel structure at the temperature of more than 32 ℃.
S3: and (3) stirring and mixing the self-assembled polypeptide solution and the poly N-isopropylacrylamide solution at room temperature and 4 ℃ by using a magnetic stirrer according to the following compounding conditions, wherein the stirring speed is 200-400rpm/min, and the stirring time is 3h to obtain the final solution.
In the step, the preparation process of the integral temperature-sensitive hydrogel is carried out at the temperature of 4 ℃. The preparation of the temperature response type reversible hydrogel is realized between the self-assembly polypeptide and the PINPAM by a physical crosslinking method.
The concentration of the final solution is adjusted so that the final solution is in a solution state at 20 to 25 ℃ and in a hydrogel state at a temperature of more than 32 ℃.
In the step, the final solution is heated to a temperature of over 32 ℃, the solution loses fluidity, hydrogel can be formed, and the fluidity is recovered at a temperature of between 20 and 25 ℃.
The invention provides the application of the temperature response type reversible hydrogel in preparing an intelligent hydrogel dressing, wherein the mechanical property of a system is improved and the viscoelasticity of the hydrogel is increased due to the addition of the self-assembly peptide.
The invention provides an application of the temperature response type reversible hydrogel in loading repair peptide, which comprises the following steps:
s4: adding the repair peptide into the final solution, stirring by a magnetic stirrer at the stirring speed of 200-400rpm/min at the stirring speed of 1-4 hours, standing at 4 ℃ for 3 hours after stirring to fully and uniformly mix the drug in the hydrogel solution to obtain a drug-loaded mixed solution, and heating the mixed solution to above 32 ℃ to form the drug-loaded temperature-sensitive dressing for promoting tissue repair and antibiosis. And (3) putting the temperature-sensitive dressing loaded with the repair peptide into a buffer solution at 37 ℃ to perform drug release control.
As shown in fig. 3, which is a schematic diagram of gel formation of PNI/RA-Amps hydrogel, the PNI/RA-Amps composite hydrogel is in a transparent flowing solution state at room temperature, and is in a white gel state at body temperature, which indicates that the addition of the self-assembly peptide does not affect the macro-morphology of the composite hydrogel.
In yet another embodiment, the present invention provides the use of the temperature responsive reversible hydrogels.
In the embodiment, as the temperature-sensitive hydrogel, the process of 'sol-gel' can enable the hydrogel to easily enter the human body in a minimally invasive manner; the low viscosity of the sol phase can easily flow to fill cavities, defects or surgical wounds, so that the hydrogel can perfectly adapt to boundaries with irregular shapes;
the invention further provides application of the temperature-responsive reversible hydrogel dressing prepared by the preparation method of the temperature-sensitive dressing with the functions of promoting tissue repair and resisting bacteria in tissue repair
As shown in fig. 2, the phase transition time of the hydrogel as a liquid dressing is also one of the factors that must be considered.
The phase transition time of the composite hydrogel is changed by adding the self-assembly peptide, the phase transition time changes gradually after being reduced along with the increase of the concentration of the self-assembly peptide, and the shortest gel forming time of the composite hydrogel at 37 ℃ is (23 +/-1) s under the proportion of PNI/RA-Amps 3.
As shown in fig. 4, the hydrogel used as a wound dressing needs to have good mechanical properties, and not be easily damaged on the surface of a wound, so as to provide a good environment for wound healing.
The mechanical properties of the hydrogel at 37 ℃ are characterized by a rheometer in the experiment. PNIPAM alone solution at 37 deg.C, G' > G ", indicates that PNIPAM alone exhibits some stiffness at 37 deg.C, but the resulting hydrogel is less elastic. Similarly, the defect of weak elasticity of the hydrogel cannot be changed by adding short-chain Amps antibacterial peptide into the system, but after the self-assembly peptide RA-Amps is added, G '> 10G' among groups indicates that after the self-assembly peptide is added, the composite hydrogel has good rigidity, and the system forms solid hydrogel. And the concentration of the self-assembly peptide in the system is in positive correlation with G 'and G', which shows that the addition of the self-assembly peptide can obviously adjust the viscoelasticity of the system and is beneficial to forming a stable hydrogel structure.
Example 2
The embodiment provides a preparation method of a temperature-sensitive dressing with the functions of promoting tissue repair and resisting bacteria, which comprises the following steps:
s1: grafting RADA16 and Amps to form Ac-RADARADARADARADA- (Acp) -RRWRVIVKW (RA-Amps) self-assembly peptide, dissolving the peptide by using double distilled water to prepare a 20mg/mL self-assembly polypeptide aqueous solution, performing ultrasonic treatment for 30min under an ice bath condition, performing ultrasonic treatment, and standing for 48h at 4 ℃ to complete self-assembly of the self-assembly peptide to form the self-assembly peptide with an antibacterial function;
s2: dissolving poly-N-isopropylacrylamide (PNIPAM) in HCl-Tris buffer solution with the pH =7.4, stirring by using a magnetic stirrer, and stirring at the stirring speed of 300rpm/min for 4 hours at the temperature of 4 ℃ to obtain poly-N-isopropylacrylamide solution with the concentration of 30mg/mL, standing the obtained sample, and fully crosslinking the sample;
s3: and (3) stirring and mixing the polypeptide solution after the self-assembly and the poly N-isopropylacrylamide solution at room temperature and 4 ℃ by using a magnetic stirrer according to the following compounding conditions, wherein the stirring speed is 400rpm/min, and the stirring time is 3h to obtain a final solution.
The concentration of the final solution is adjusted so that the final solution is in a solution state at 20 to 25 ℃ and in a hydrogel state at a temperature of more than 32 ℃.
S4: adding the repair peptide into the final solution, stirring by a magnetic stirrer at the stirring speed of 200-400rpm/min for 4 hours at the temperature of 4 ℃, standing for 5 hours at the temperature of 4 ℃ after stirring to fully and uniformly mix the drug in the hydrogel solution to obtain a drug-loaded mixed solution, and heating the mixed solution to above 32 ℃ to form the drug-loaded temperature-responsive reversible hydrogel capable of promoting tissue repair and antibiosis. The repair peptide may include MGF E peptide, IGF-1, GHRP6, VEGF and other repair peptides capable of promoting cell proliferation, cell migration and other effects, and the concentration range of the repair peptides is effective cell or body concentration. In this embodiment, due to the presence of the fiber structure of the self-assembled polypeptide, a binding site is provided for the repair peptide, and loading and release control of different drugs can be easily achieved.
As shown in figure 6, the slow release curve of the drug-loaded PNI/RA-Amps/E composite hydrogel shows burst release, sustained release and slow release, and mainly shows slow initial release at 12h, and the release rate reaches (65 +/-2.5)%. The in-situ hydrogel loaded with the repair peptide can form hydrogel at a wound and locally release a drug for a long time, so that the administration frequency is reduced, the drug targeting efficiency is improved, and the wound healing rate is accelerated.
The invention provides application of the temperature-sensitive dressing with the functions of promoting tissue repair and resisting bacteria in an embodiment. In the embodiment, the gel has a high-water-content material, and the loose and porous three-dimensional network structure is favorable for promoting absorption of wound exudate, keeping the wound moist and continuously and locally delivering loaded drugs from the interior of the gel.
As shown in FIG. 5, the hydrogel system is a three-dimensional porous structure, which facilitates the exchange of gas and tissue fluid during the healing process of skin wounds, and the surface of the hydrogel is smooth, which facilitates the optimal microenvironment for vascularization and cell colonization when the hydrogel is embedded in living tissue to promote tissue healing.
Example 3
The embodiment provides a preparation method of a biological material with the functions of promoting tissue repair and resisting bacteria, which comprises the following steps:
s1: grafting RADA16 and Amps to form Ac-RADARADARADARADA- (Acp) -RRWRVIVKW (RA-Amps) self-assembly peptide, dissolving the peptide by using double distilled water to prepare a 10mg/mL self-assembly polypeptide aqueous solution, performing ultrasonic treatment for 30min under an ice bath condition, performing ultrasonic treatment, and standing for 24h at 4 ℃ to complete self-assembly of the self-assembly peptide to form the self-assembly peptide with an antibacterial function;
s2: dissolving poly-N-isopropylacrylamide (PNIPAM) in HCl-Tris buffer solution with pH =7.4, stirring with a magnetic stirrer at the stirring speed of 300rpm/min at the temperature of 4 ℃ for 4 hours to obtain poly-N-isopropylacrylamide solution with the concentration of 10mg/mL, and standing the obtained sample to fully crosslink the sample. The single self-assembly peptide solution and the PNIPAM solution at the concentration still have fluidity after being heated, and cannot form stable hydrogel;
s3: and (3) stirring and mixing the polypeptide solution after the self-assembly and the poly N-isopropylacrylamide solution at room temperature and 4 ℃ by using a magnetic stirrer according to the following compounding conditions, wherein the stirring speed is 400rpm/min, and the stirring time is 2h to obtain a final solution.
The concentration of the final solution is adjusted so that the final solution is in a solution state at 20 to 25 ℃ and in a hydrogel state at a temperature of more than 32 ℃.
S4: adding the repair peptide into the final solution, stirring by a magnetic stirrer at the stirring speed of 200-400rpm/min for 3 hours at 4 ℃, standing for 5 hours after stirring to fully and uniformly mix the drug in the hydrogel solution to obtain a drug-loaded mixed solution, and heating the mixed solution to above 32 ℃ to form the drug-loaded temperature-responsive reversible hydrogel capable of promoting tissue repair and antibiosis.
Experiment one
The antibacterial ability of the polypeptide nanofibers coupled with antibacterial peptides improved by the above example 1 was evaluated by using staphylococcus aureus (s. aureus) and escherichia coli (e.coli) in vitro culture experiments. The specific operation method comprises the following steps:
(1) respectively placing RA-Amps and Amps samples with different concentrations of 0.29 muM, 2.90 muM, 29.00 muM, 74.00 muM and 148.00 muM prepared in example 1 into a 48-well plate, and taking a phosphate buffered saline (PBS, pH = 7.4) as a control group;
(2) 10 μ L of 10 was added dropwise to each well6CFU/mL Staphylococcus aureus;
(3) culturing in 37 deg.C incubator for 6h, inoculating 100 μ L diluted bacterial liquid on LB solid culture medium, culturing in 37 deg.C incubator for 12h, and observing the bactericidal effect of each material, with the result shown in FIG. 1B;
(5) the bacteriostatic analysis on escherichia coli was the same as the analysis on staphylococcus aureus, except that the staphylococcus aureus added to the wells was replaced with escherichia coli, and the results are shown in fig. 1A.
Fig. 1 is a graph showing the experimental results of the photothermal bacteriostatic properties of the polypeptide nanofibers coupled with antibacterial peptides prepared in example 1 on escherichia coli (e.coli) and staphylococcus aureus (s.aureus): (A) optical photographs of the bactericidal effect of each group of samples on escherichia coli; (B) optical photographs of the bactericidal effect on staphylococcus aureus for each set of samples; wherein PBS is a blank control group; the experimental groups were RA-Amps and Amps at different concentrations.
As can be seen from FIG. 1, when the bactericidal effect of both bacteria on live bacteria was investigated by the plate coating method, the numbers of colonies obtained by plate coating of both bacteria were significantly reduced as the concentrations of RA-Amps and Amps were increased. After 12h of culture, the sterilization effect is obvious (P is less than 0.01) when the E.coli and the S.aureus processed by the RA-Amps are compared with those processed by the Amps under the concentration of 2.9 mu M and 29 mu M, and the sterilization rate of the E.coli and the S.aureus processed by the RADA16-Amps and the Amps reaches 100% when the concentration reaches 148 mu M.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Experiment 2
The cytotoxicity of the PNI/RA-Amps composite hydrogel improved by the above example 1 was evaluated by using Human Foreskin Fibroblast (HFF) in vitro culture experiment.
(1) Taking HFF cells in exponential growth phase, digesting with pancreatin, and adjusting cell concentration to 2 × 104cells/mL were seeded into 96-well plates, and then 100. mu.L of the mixed solution was added to the 96-well plates and incubated at 37 ℃ under 5% (v/v) CO 2.
(2) Discarding the culture medium in the 96-well plate, adding DMEM culture medium containing 0.0625, 0.125, 0.25, 0.5 and 1mg/mL composite hydrogel, setting 3 multiple wells for each concentration, and culturing the 96-well plate in a 5% CO2 incubator at 37 ℃ for 24 h;
(3) the medium in the 96-well plate was discarded, and 10% CCK-8 solution was added to the medium instead of the original medium and incubated for 45min in a 37 ℃ cell incubator. The absorbance at 450nm was read with a microplate reader and the procedure was repeated three times.
The influence of PNI/RA-Amps composite hydrogel on the relative proliferation rate of cells is tested by using CCK-8 in the figure 7, HFF cells are used as model cells in the experiment, and leaching solutions with different concentrations are selected to be co-cultured with the HFF cells for 24h, 48h and 72h to judge the cytotoxicity of the PNI/RA-Amps composite hydrogel. Along with the increase of the culture time, the number of cells cultured by the composite hydrogel leaching liquor with different concentrations is increased, and the increase rate is higher than 80%, which indicates that the biocompatibility of the material is good. It can be seen from the composite hydrogel leaching solutions with different concentrations that the cell proliferation rate is relatively reduced with the increase of the concentration, and it is possible that the composite hydrogel leaching solutions with high concentration cause the obstruction of cell metabolites, thereby reducing the cell activity, but the cell relative proliferation rates are all more than 80%, that is, in the leaching solutions with high concentration, the cells can still grow and proliferate normally. The composite hydrogel can effectively promote the adhesion and proliferation of cells on the first day compared with the third day. From the results, the PNI/RA-Amps composite hydrogel has good biological safety, and the composite hydrogel has no cytotoxicity.
Experiment three
The effect of the PNI/RA-Amps/E composite hydrogel improved in the above example 1 on the healing of the skin wound of a rat was evaluated by using an animal skin wound model. The specific operation method comprises the following steps:
(1) before the experiment, newly purchased rats need to be raised in an SPF rat room for one week to adapt to the environment, free drinking water is provided for the rats, quantitative rat food is fed, the raising environment temperature is maintained at 24-26 ℃, illumination is performed alternately day and night, and the experiment can be performed without adverse reaction.
(2) One day before the experiment, rats are marked in groups, and 15 rats are divided into three groups;
the Sutai anesthetic Zoletil 50 is injected into the abdominal cavity for anesthesia, injection is carried out according to the instruction strictly, and the necessary drug analgesia is carried out on the rat after the operation for not less than 3 days. The skin of the back was wiped clean with iodophors (0.2% w/v).
(3) The dorsal skin was then operated to remove four full thickness skin plaques (1 cm diameter) and the model was completed. The wounds selected on the back of each animal were set as control (treated by injection of normal saline), hydrogel group, drug-loaded hydrogel group and commercial control group, respectively, and changed at fixed time each day.
According to the experimental period, all wounds show a gradual healing trend, and the healing rates of all experimental groups are inconsistent. Compared with the control group, the samples of each group can shrink the wound well on the 5 th day, and the wound is basically epithelialized and has small scars on the 9 th day. On day 13, epithelialization of the drug-loaded composite hydrogel group was substantially complete, close to healing, and the composite hydrogel group was the second time.
The above description is only for the specific embodiments of the present disclosure, but the scope of the present disclosure is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present disclosure, and all the changes or substitutions should be covered within the scope of the present disclosure. Therefore, the protection scope of the present disclosure shall be subject to the protection scope of the claims.
Claims (8)
1. A preparation method of a temperature-sensitive dressing with the functions of promoting tissue repair and resisting bacteria is characterized by comprising the following steps:
step S1: grafting RADA16 and Amps to form self-assembled peptide, dissolving the self-assembled peptide by using double distilled water to prepare 2-20mg/mL self-assembled polypeptide aqueous solution, carrying out ultrasonic treatment for 15-30min under the ice bath condition, and then placing the self-assembled peptide for 12-48h at the temperature of 4 ℃ to complete self-assembly of the self-assembled peptide to form the self-assembled peptide with the antibacterial function;
step S2: dissolving poly N-isopropylacrylamide in HCl-Tris buffer solution with the pH =7.4, stirring by using a magnetic stirrer, stirring at the speed of 200-400rpm/min at the temperature of 4 ℃ for 1-4 hours to obtain poly N-isopropylacrylamide solution, and standing;
step S3: stirring and mixing the self-assembled polypeptide solution and the poly N-isopropylacrylamide solution at 4 ℃ by using a magnetic stirrer according to the following compounding conditions, wherein the stirring speed is 200-400rpm/min, and the stirring time is 2-5h, so as to obtain a final solution;
step S4: and adding the repair peptide into the final solution to obtain the temperature-responsive reversible hydrogel dressing.
2. The method for preparing a temperature-sensitive dressing with effects of promoting tissue repair and resisting bacteria according to claim 1, wherein the compounding conditions in step S3 are that the concentration of the polypeptide in the final solution is greater than its self-assembly critical concentration while maintaining a uniformly dispersed concentration, and the concentration of the poly N-isopropylacrylamide in the final solution is 5.0-40 mg/mL.
3. The method for preparing a temperature-sensitive dressing with the functions of promoting tissue repair and resisting bacteria according to claim 1, wherein the poly-N-isopropylacrylamide solution is kept standing at 4 ℃ for 48-72h in the step S2.
4. The method for preparing a temperature-sensitive dressing with the functions of promoting tissue repair and resisting bacteria according to claim 1, wherein the ultrasonic power of the self-assembly polypeptide aqueous solution in the ultrasonic treatment in the step S1 is 480W.
5. The method for preparing a temperature-sensitive dressing with effects of promoting tissue repair and resisting bacteria as claimed in claim 1, wherein the step of adding repair peptide into the final solution in step S4 comprises the steps of stirring and mixing at 4 ℃ by a magnetic stirrer, wherein the stirring speed is 200-400rpm/min, the stirring time is 1-4 hours, standing for 3-5 hours at 4 ℃ after stirring to fully mix the drug in the hydrogel solution to obtain a drug-loaded mixed solution, and heating the mixed solution to above 32 ℃ to form the temperature-sensitive dressing with effects of promoting tissue repair and resisting bacteria.
6. The preparation method of the temperature-sensitive dressing with the functions of promoting tissue repair and resisting bacteria according to claim 1, wherein the repair peptide is one or more of MGFE peptide, IGF-1, GHRP6, VEGF and recombinant type III collagen.
7. The method for preparing a temperature-sensitive dressing having the effects of promoting tissue repair and resisting bacteria according to claim 6, wherein the concentration range of the repair peptide is a concentration effective for cells or organisms.
8. Use of the temperature-responsive reversible hydrogel dressing prepared by the method for preparing a temperature-sensitive dressing for promoting tissue repair and antisepsis according to any one of claims 1-6 in tissue repair.
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