CN114796593A - Antibacterial peptide-hydrogel for medical dressing and preparation method and application thereof - Google Patents
Antibacterial peptide-hydrogel for medical dressing and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
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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
- A61L26/008—Hydrogels or hydrocolloids
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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
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0014—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
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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
- A61L26/0028—Polypeptides; Proteins; Degradation products thereof
- A61L26/0047—Specific proteins or polypeptides not covered by groups A61L26/0033 - A61L26/0042
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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
- A61L26/0066—Medicaments; Biocides
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
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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
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- A—HUMAN NECESSITIES
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- 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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Abstract
The invention discloses an antibacterial peptide-hydrogel for medical dressings, and a preparation method and application thereof. The LCST of the antibacterial peptide-hydrogel obtained by the invention is about 37.7 ℃, is close to the body temperature of a human body, has good inhibiting effect on staphylococcus aureus, and is characterized by SEM that the antibacterial peptide solution in the antibacterial peptide-hydrogel is distributed in pores of the hydrogel, has water storage property and is beneficial to slow drug release; DMA results show that the antibacterial peptide hydrogel has the maximum bearing stress of 0.038MPa, has certain mechanical property and certain thermal stability, and has early scarring due to the antibacterial peptide through skin irritation tests and wound healing tests, obviously higher overall wound healing capability, almost no irritation to the skin of a mouse, no secondary damage to a wound and good application value in the aspect of medical dressing.
Description
Technical Field
The invention relates to skin dressing in the medical field, in particular to antibacterial peptide-hydrogel for medical dressing and a preparation method and application thereof.
Background
In order to prevent wound infection, promote wound healing and reduce scar formation, wound dressings need to be used, and the wound dressings need to be easily stripped from healed wounds so as to avoid secondary trauma to the wounds. Compared with the traditional gauze dressing, the hydrogel dressing can lower the temperature of the surface of a wound, relieve pain, maintain a moist wound environment, absorb tissue exudates, and promote fibroblast proliferation and keratinocyte migration, and the characteristics are necessary for the complete epithelialization and healing of the wound; secondly, the hydrogel has a modulus comparable to that of human soft tissue, skin, etc., which reduces connection instability with wounds and increases comfort.
The antibacterial material used as the biomedical material can reduce the possibility of pollution of the material in the using process, reduce the replacement times and prolong the service life of the material. Therefore, antibacterial hydrogels have received much attention from researchers. Antimicrobial peptides (AMPs), also known as antimicrobial peptides, are small proteins or polypeptides with a net positive charge, which are widely present in multicellular organisms and are produced by encoding specific genes and inducing them by external conditions. Besides having broad spectrum of antibacterial, antifungal, viral, protozoan and ability to inhibit and kill tumor cells, etc., the antibacterial peptide is also excellent in heat stability and water solubility, and in addition, it has almost no toxic effect on normal cells of higher animals.
Hydrogels have received extensive attention in biomedical applications due to their high water content, good biocompatibility, and versatile functionalities. The current methods for preparing antibacterial hydrogel mainly comprise: (1) antibiotic, inorganic nano metal particles, organic antibacterial agent or semiconductor material with photocatalytic antibacterial effect and the like are loaded; (2) constructing an endogenous antibacterial hydrogel by adopting an antibacterial cationic electrolyte monomer; (3) and (3) constructing an endogenous antibacterial hydrogel and loading an antibacterial agent and the like. However, the preparation of the currently reported antibacterial hydrogel is often complex, and the application and popularization of the antibacterial hydrogel are greatly restricted. The hydrogel material with biological viscosity may have secondary wound on tissues in the replacement process, the introduction of the controllable adhesion-debonding performance can solve the problem, and the hydrogel material with a special structure such as tubular biomedical hydrogel plays an important role in cell culture and tissue engineering, but the preparation method is not mature at present and needs to be developed further.
Disclosure of Invention
Aiming at the technical current situation that the preparation of the reported antibacterial hydrogel is complex, the application and popularization of the antibacterial hydrogel are greatly restricted, and the preparation method of the tubular biomedical hydrogel with a special structure is immature at present, the invention provides the antibacterial peptide-hydrogel for the medical dressing and the preparation method and the application thereof. The antibacterial peptide-hydrogel provided by the invention comprises N-isopropyl acrylamide, ammonium persulfate, N-N methylene bisacrylamide, NNNN-tetramethyl ethylenediamine, an antibacterial peptide solution with the concentration of 0.3mg/mL, and the balance of deionized water. According to the invention, the LCST of the finally obtained antibacterial peptide-hydrogel is about 37.7 ℃ through raw material screening and preparation process optimization, the antibacterial peptide-hydrogel is close to the body temperature and has a good inhibiting effect on staphylococcus aureus, and SEM representation shows that the antibacterial peptide solution in the antibacterial peptide-hydrogel is distributed in pores of the hydrogel, has water storage property and is beneficial to slow drug release; DMA results show that the antibacterial peptide hydrogel has the maximum bearing stress of 0.038MPa, certain mechanical property and certain thermal stability, and is applied to hydrogel accessories on the surface of skin, so that the antibacterial peptide hydrogel accessories are antibacterial, excellent in water retention property and stable in mechanical and thermal stability, and provide a large amount of data support for the application of the antibacterial peptide hydrogel accessories in medical accessories.
The invention provides an antibacterial peptide-hydrogel for medical dressings, which comprises, by weight, 300 parts of N-isopropylacrylamide 200-containing material, 8-10 parts of ammonium persulfate, 10-15 parts of N-N methylene bisacrylamide, 3-5 parts of NNN-tetramethylethylenediamine, 50-150 parts of an antibacterial peptide solution with the concentration of 0.3mg/mL, and the balance of deionized water.
The invention further provides the antibacterial peptide-hydrogel for the medical dressing, which comprises 230 parts of N-isopropyl acrylamide, 9 parts of ammonium persulfate, 12 parts of N-N methylene bisacrylamide, 4 parts of NNNN-tetramethyl ethylenediamine, 100 parts of an antibacterial peptide solution with the concentration of 0.3mg/mL and the balance of deionized water in parts by weight.
Meanwhile, the invention provides a preparation method of the antibacterial peptide-hydrogel for the medical dressing, and the antibacterial peptide-hydrogel for the medical dressing is obtained through the following test steps:
(1) NIPAM monomer is accurately weighed, the solid is crushed and poured into a clean beaker, a certain amount of MBAA and APS are weighed and poured into the beaker, a certain amount of deionized water is added, and a glass rod is stirred at a constant speed at room temperature until white crystals are completely dissolved.
(2) Adding TEMED into the solution obtained in the step (1), placing the solution in an ice box, continuously stirring for 1.0min, injecting the liquid in the beaker into a gel making plate by using a liquid transfer gun, and standing and reacting for 6-12h at 4 ℃ to obtain the P (NIPAM) hydrogel.
(3) Soaking the P (NIPAM) hydrogel obtained in the step (2) in deionized water for 6-18h, changing water every 6h, soaking in hot water at 60-80 ℃ until the gel is swelled to be balanced, taking out when the gel is whitish and has shrunk volume, and airing in a fume hood.
(4) And (3) adding a certain amount of antibacterial peptide solution with the concentration of 0.3mg/mL into the hydrogel obtained in the step (3), and obtaining the antibacterial peptide-hydrogel after the gel is swelled to be balanced.
In the preparation method of the antibacterial peptide-hydrogel for the medical dressing, the standing reaction time in the step (2) is 8 hours.
In the preparation method of the antibacterial peptide-hydrogel for the medical dressing, the soaking time of deionized water in the step (3) is 12 hours, and the temperature of hot water is 70 ℃.
The invention provides application of an antibacterial peptide-hydrogel for medical dressings in the medical dressings.
Through the technical scheme, the invention achieves the following technical effects:
(1) the antibacterial peptide-hydrogel for medical dressings provided by the invention has wide raw material sources and a simple preparation method, and the P (NIPAM) hydrogel added with the antibacterial peptide is transparent when being gelatinized at 4 ℃, and the gel formed at normal temperature is white. The antibacterial peptide-P (NIPAM) hydrogel has good antibacterial activity.
(2) According to the invention, the LCST of the antibacterial peptide-hydrogel finally obtained through raw material screening and preparation process optimization is about 37.7 ℃, the antibacterial peptide-hydrogel is close to the body temperature of a human body and has a good inhibiting effect on staphylococcus aureus, and SEM (scanning electron microscope) representation shows that the antibacterial peptide-hydrogel has a loose and porous structure, can well store an antibacterial peptide solution, slowly releases a drug along with the rise of the environmental temperature, and is beneficial to slow drug release.
(3) The antibacterial peptide-hydrogel provided by the invention has the maximum bearing stress of 0.038MPa, certain mechanical property and certain thermal stability, wherein the C, H, N content is 52.38%, 9.44% and 11.43%, and the mass loss process of the antibacterial peptide-P (NIPAM) hydrogel is divided into 3 stages: the hydrogel loses internal bound water at about 100 ℃; the mass loss at 150 ℃ to 360 ℃ may be the cleavage and decomposition of poly-N-isopropylacrylamide.
Drawings
FIG. 1 is a diagram showing the bacteriostatic activity of the antibacterial peptide and the antibacterial peptide hydrogel of the present invention; FIG. 1 a: 1-5 antibacterial peptide bacteriostasis experiment (Oxford cup method); FIG. 1 b: 1, P (NIPAM) hydrogel (soaking treatment), 2, P (NIPAM) hydrogel (non-soaking treatment), 3, antimicrobial peptide-P (NIPAM) hydrogel (gelling at 4 ℃), and 4, antimicrobial peptide-P (NIPAM) hydrogel (gelling at normal temperature).
FIG. 2 is an SEM image of a P (NIPAM) hydrogel and an antimicrobial peptide-P (NIPAM) hydrogel of the present invention; wherein FIG. 2a is a 5000 magnification of the P (NIPAM) hydrogel; FIG. 2b is a 10000 times magnification of P (NIPAM) hydrogel; FIG. 2c is a 5000-fold magnification of an antimicrobial peptide-P (NIPAM) hydrogel; FIG. 2d is a 10000-fold magnification of the antimicrobial peptide-P (NIPAM) hydrogel.
FIG. 3 is a graph comparing the results of absorbance and DSC of the antimicrobial peptide-hydrogel of the present invention; wherein FIG. 3A is the absorbance of the antimicrobial peptide hydrogel; FIG. 3B is a DSC with a P (NIPAM) hydrogel; b is antibacterial peptide-P (NIPAM) hydrogel.
FIG. 4 is a comparison graph of the P (NIPAM) hydrogel and antibacterial peptide-P (NIPAM) hydrogel element analysis of the present invention, wherein 1 is antibacterial peptide Cecropin-XJ element analysis; 2, PNIPAM hydrogel element analysis; 3. the analysis is the element analysis of the antibacterial peptide-P (NIPAM) hydrogel.
FIG. 5 is an infrared spectrum of the P (NIPAM) hydrogel and the antimicrobial peptide-P (NIPAM) hydrogel of the present invention; a is P (NIPAM) hydrogel, and b is antibacterial peptide-P (NIPAM) hydrogel.
FIG. 6 is a DMA diagram of the antimicrobial peptide-P (NIPAM) hydrogel of the present invention, wherein a in A is a stress-strain relationship diagram; in B, a is a temperature-strain curve, and B is a temperature-length curve.
FIG. 7 is an enthusiasm analysis chart of the antimicrobial peptide-P (NIPAM) hydrogel of the present invention, Mass is a Mass loss curve; DTG is the mass loss rate curve.
FIG. 8 is a graph of skin condition of the invention after multiple skin irritation tests with antimicrobial peptide-P (NIPAM) hydrogel treatment and other treatments at different times, wherein 1 is a graph of skin condition of the test group after 24h treatment, 2 is a graph of skin condition of the test group after 48h treatment, and 3 is a graph of skin condition of the test group after 72h treatment; the skin condition of the positive control group after 24h treatment is shown in figure 4, the skin condition of the positive control group after 48h treatment is shown in figure 5, and the skin condition of the positive control group after 72h treatment is shown in figure 6.
FIG. 9 is a comparison of wound healing with antimicrobial peptide-P (NIPAM) hydrogel treatment of the present invention; wherein, A is the general view of the skin fault wound surface model, a is an experimental group, and b is a control group; b is a test group and a positive control group, a is a test group, and B is a positive control group; panel C shows the test group and the negative control group, a the test group and b the negative control group.
FIG. 10 is a graph comparing the healing results of different treatment wounds at different times; wherein A is a 5d test group and a negative control group, a is a test group, and b is a negative control group; b is the 10d test group and the negative control group, a is the test group, B is the negative control group; c is the 5d test group and the positive control group, a is the test group, b is the positive control group; d is the 10D test group and the positive control group, a is the test group, and b is the positive control group.
Detailed Description
The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.
All materials, practices and instrumentation assays selected for use in the present invention are well known in the art and are not intended to limit the practice of the invention, and other reagents and equipment well known in the art may be suitable for use in the practice of the following embodiments of the invention.
The staphylococcus aureus selected by the invention is a conventional strain, is a common publicly known strain, can be purchased and obtained by a person skilled in the art through public channels, and can be obtained by adopting common reports in the field as culture conditions and culture media.
Example 1: antibacterial peptide-hydrogel for medical dressing
The embodiment provides an antibacterial peptide-hydrogel for medical dressings, which comprises, by weight, 300 parts of N-isopropylacrylamide 200, 8-10 parts of ammonium persulfate, 10-15 parts of N-N methylene bisacrylamide, 3-5 parts of NNN-tetramethylethylenediamine, 50-150 parts of an antibacterial peptide solution with a concentration of 0.3mg/mL, and the balance of deionized water.
Example 2: antibacterial peptide-hydrogel for medical dressing
This example provides an antimicrobial peptide-hydrogel for medical dressing based on example 1, which comprises, by weight, 230g of N-isopropylacrylamide, 9g of ammonium persulfate, 12g of N-methylenebisacrylamide, 4mL of NNNN-tetramethylethylenediamine, 100mL of an antimicrobial peptide solution with a concentration of 0.3mg/mL, and the balance deionized water.
Example 3: antibacterial peptide-hydrogel for medical dressing
This example provides an antimicrobial peptide-hydrogel for medical dressing based on example 1, which comprises 200g of N-isopropylacrylamide, 8g of ammonium persulfate, 10g of N-methylenebisacrylamide, 3mL of NNN-tetramethylethylenediamine, 50mL of antimicrobial peptide solution with concentration of 0.3mg/mL, and the balance of deionized water in parts by weight.
Example 4: antibacterial peptide-hydrogel for medical dressing
This example provides an antimicrobial peptide-hydrogel for medical dressing based on example 1, which comprises, by weight, 300g of N-isopropylacrylamide, 10g of ammonium persulfate, 15g of N-methylenebisacrylamide, 5mL of NNNN-tetramethylethylenediamine, 150mL of an antimicrobial peptide solution with a concentration of 0.3mg/mL, and the balance deionized water.
Example 5: antibacterial peptide-hydrogel for medical dressing
This example provides an antimicrobial peptide-hydrogel for medical dressing based on example 1, which comprises, by weight, 250g of N-isopropylacrylamide, 9g of ammonium persulfate, 13g of N-methylenebisacrylamide, 4mL of NNNN-tetramethylethylenediamine, 120mL of an antimicrobial peptide solution with a concentration of 0.3mg/mL, and the balance deionized water.
Example 6: preparation of antibacterial peptide-hydrogel for medical dressing
This example provides a method for preparing an antimicrobial peptide-hydrogel for medical dressing based on examples 1 to 5, and the method specifically includes the following steps:
(1) accurately weighing NIPAM monomer by an electronic analytical balance, grinding the solid and pouring into a clean beaker, weighing a certain amount of MBAA and APS and pouring into the beaker, adding a certain amount of deionized water, and stirring at a constant speed by a glass rod at room temperature until white crystals are completely dissolved.
(2) Adding TEMED into the solution obtained in the step (1), placing the solution in an ice box, continuously stirring for 1.0min, injecting the liquid in the beaker into a gel making plate by using a liquid transfer gun, and standing and reacting for 6-12h at 4 ℃ to obtain the P (NIPAM) hydrogel.
(3) Soaking the P (NIPAM) hydrogel obtained in the step (2) in deionized water for 6-18h, changing water every 6h, soaking in hot water at 60-80 ℃ until the gel is swelled to be balanced, taking out when the gel is whitish and has shrunk volume, and airing in a fume hood.
(4) And (4) adding a certain amount of antibacterial peptide solution with the concentration of 0.3mg/mL into the hydrogel obtained in the step (3), and obtaining the antibacterial peptide-hydrogel after the gel is swelled to be balanced.
Example 7: preparation of antibacterial peptide-hydrogel for medical dressing
This example provides a method for preparing an antimicrobial peptide-hydrogel for medical dressing based on examples 1 to 6, wherein the reaction time of standing at 4 ℃ is 8 hours, the soaking time of deionized water is 12 hours, and the temperature of hot water in hot water soaking is 70 ℃.
Example 8: preparation of antibacterial peptide-hydrogel for medical dressing
This example provides a method for preparing an antimicrobial peptide-hydrogel for medical dressing based on examples 1 to 6, wherein the reaction time of standing at 4 ℃ is 6 hours, the soaking time of deionized water is 6 hours, and the temperature of hot water in hot water soaking is 60 ℃.
Example 9: preparation of antibacterial peptide-hydrogel for medical dressing
This example provides a method for preparing an antimicrobial peptide-hydrogel for medical dressing based on examples 1 to 6, wherein the reaction time of standing at 4 ℃ is 12 hours, the soaking time of deionized water is 18 hours, and the temperature of hot water in hot water soaking is 80 ℃.
Example 10: preparation of antibacterial peptide-hydrogel for medical dressing
This example provides a method for preparing an antimicrobial peptide-hydrogel for medical dressing based on examples 1 to 6, wherein the reaction time of standing at 4 ℃ is 10 hours, the soaking time of deionized water is 10 hours, and the temperature of hot water in hot water soaking is 70 ℃.
Example 11: performance characterization of antimicrobial peptide-hydrogels for medical dressings
(1) Detection of bacteriostatic activity
And (3) oxford cup method detection: the method influences the growth and reproduction of staphylococcus aureus by inhibiting and killing the staphylococcus aureus, so that bacterial colonies form an antibacterial zone, and the strength of the antibacterial ability of the additive is determined by measuring the size of the antibacterial zone. The sterilized LB medium was heated to completely melt and poured into petri dishes at 15mL per dish (lower layer) to allow it to solidify. The melted LB medium was cooled to about 50 deg.C, Staphylococcus aureus was added, and 5mL of the mixed medium was added to the solidified medium to be solidified (upper layer). Directly and vertically placing an oxford cup on the surface of a culture medium in a sterile operation, slightly pressurizing to ensure that the oxford cup is in contact with the culture medium without a gap, adding an antibacterial peptide solution into the oxford cup, wherein the oxford cup can be generally filled with 240 mu L of liquid. After the culture medium is filled, the culture medium is placed at 36 ℃ for culturing for 16-18h with the front side facing upwards, and the size of the inhibition zone is measured by observing the result.
A patch method: soaking the antibacterial peptide-hydrogel subjected to RO to absorb the antibacterial peptide to swell and adsorb, cutting into a wafer with the diameter of 8mm, melting an LB solid culture medium in an ultra-clean workbench, cooling to about 50 ℃, adding staphylococcus aureus, slowly pouring into a culture dish, and waiting for the solidification. Gently clamping the disc by using forceps, sticking the disc on a staphylococcus aureus solid culture medium, putting the disc into a constant-temperature incubator at 36 ℃ for culturing for 12 hours, observing the result, and measuring the size of the inhibition zone.
The test adopts an oxford cup method and a patch method to detect the antibacterial activity of the antibacterial peptide-hydrogel prepared by the invention, and the test result is shown in figure 1.
As can be seen from the data in the attached figure 1, the antimicrobial activity detection is carried out on the antimicrobial peptide solution by adopting an oxford cup, and the activity is good as shown in figure 1-a; different treatments of p (NIPAM) hydrogel, as shown in fig. 1-b (1) and 2, the hydrogel without the antibacterial peptide has bacteriostatic effect before soaking, and does not have antibacterial effect after soaking, because the NIPAM monomer has certain toxicity, and the monomer is basically eliminated after soaking. The hydrogel containing antibacterial peptide P (NIPAM) is transparent at 4 deg.C, and white at room temperature, as shown in fig. 1-b (3) and (4). The antibacterial peptide-P (NIPAM) hydrogel has good antibacterial activity. This shows that the NIPAM monomer treated by the method of the present invention has no toxicity, and the antibacterial activity of the finally obtained antibacterial peptide-hydrogel is obviously enhanced compared with that of the antibacterial peptide monomer.
(2) Characterization of microscopic features
In this test, the hydrogel samples which have reached equilibrium swelling are placed in a freeze dryer at-40 ℃ for 24 hours in vacuum freeze-drying. The obtained sample is subjected to freezing and brittle fracture in liquid nitrogen, then gold spraying is carried out to prepare a sample, then the sample is placed in a VEGA3SBU scanning electron microscope, observation is carried out under the condition of 10kV acceleration voltage, the micro-morphology of the hydrogel and the antimicrobial peptide hydrogel is observed under different magnification factors, the influence of the hydrogel structure on the performance of the antimicrobial peptide is inspected, and the specific result is shown in attached figure 2.
As shown in fig. 2, fig. 2(a) is a porous network structure obtained by amplifying the internal space structure of p (NIPAM) hydrogel 5000 times, and fig. 2(b) is a result of amplifying the circle part of fig. 2(a) 10000 times, which is observed to show that the hydrogel is smooth and flat; fig. 2(c) is a result of amplifying the antimicrobial peptide-p (nipam) hydrogel to 5000 times, and a plurality of pores are inlaid in the surface of the originally smooth mesh structure, and fig. 2(d) is a result of amplifying the circled portion of fig. 2(c) to 10000 times, and it can be observed that the diameters of the pores are about 0.1 μm to 2 μm, the distribution is dense, and no special rule exists. The antibacterial peptide hydrogel prepared by the invention has a loose and porous structure, provides a foundation for storing the antibacterial peptide solution, and can slowly release the antibacterial peptide solution along with the rise of the environmental temperature.
(3) Determination of the lower Critical transition temperature LCST
In order to explore the lower critical transition temperature LCST of the antibacterial peptide-hydrogel, the antibacterial peptide hydrogel is subjected to DSC analysis in a 200F3 differential scanning calorimeter; cutting the antibacterial peptide hydrogel sample into a cuvette width, placing the cuvette into which RO is added, adding an RO group as a control, and detecting the light transmittance of the hydrogel in a 756PC ultraviolet spectrophotometer at different temperatures, wherein the specific result is shown in figure 3.
As can be seen from the results in the attached figure 3, the antibacterial peptide hydrogel prepared by the invention has the advantages that the light transmittance is sharply reduced at about 33 ℃, and the antibacterial peptide hydrogel is almost opaque at 35 ℃, and is closer to the DSC result of the antibacterial peptide-hydrogel in the attached figure 3B; in FIG. 3B, a is P (NIPAM) hydrogel endothermic peak range of 34.4 deg.C-41.8 deg.C, peak value of 38.2 deg.C; b is antibacterial peptide-P (NIPAM) hydrogel endothermic peak range of 33.4-41.3 ℃ and peak value of 37.7 ℃, which is slightly lower than that of P (NIPAM) hydrogel. The LCST of the antibacterial peptide hydrogel prepared by the invention is 37.7 ℃, and the LCST of the hydrogel can be reduced by the NIPAM monomer content and the antibacterial peptide solution.
(4) Elemental analysis
To analyze the elemental composition of the antimicrobial peptide-hydrogel and further confirm the successful modification of the active groups, an EL cube elemental analyzer was used to perform elemental analysis on the lyophilized antimicrobial peptide-hydrogel powder samples, and a set of repeat results was established for each sample to obtain an average, with the specific results shown in fig. 4.
As shown in the result of elemental analysis in FIG. 4, the antimicrobial peptides C, H, N content is 33.96%, 6.97%, 6.57%, the P (NIPAM) hydrogel C, H, N content is 53.58%, 9.69%, 11.07%, the antimicrobial peptide-P (NIPAM) hydrogel C, H, N content is 52.38%, 9.44%, 11.43%, the content is relatively compromised, the doping of the antimicrobial peptides makes the complex gel element content lower than that of P (NIPAM), and the content of N element is relatively high, probably because the antimicrobial peptides contain more amino groups than P (NIPAM). It follows that antimicrobial peptides have been successfully modified in p (NIPAM) hydrogels.
(5) FIR Infrared Spectroscopy
Mixing and grinding a freeze-dried antibacterial peptide hydrogel polymer sample and spectral-grade KBr powder, tabletting, measuring on a Frontier infrared spectrometer, and judging functional groups through analysis, wherein the test result is shown in figure 5.
As can be seen from the results of FIG. 5, 3440.82cm -1 The left and right are the stretching vibration peak of the N-H bond, 2973cm -1 The left and right peaks are due to C-H stretching vibration; 1641.26cm -1 Is amide I band (meaning C ═ O stretch/hydrogen bond combined with C — N stretch, C — CN deformation); 1546.59cm -1 Is the amide II band (caused by bending vibration of the N-H group and stretching vibration of the C-N group); at 1382cm -1 And 1460cm -1 The left and right double peaks are of equal strength, and are two-CH of isopropyl 3 Absorption peak 1170cm -1 The left and right peaks are due to ether linkages; 1382.54cm -1 And 1460.37cm -1 The left and right double peaks are of equal strength, and are two-CH of isopropyl 3 An absorption peak; therefore, the antibacterial peptide is successfully crosslinked to P (NIPAM), and the result shows that the target product, namely the antibacterial peptide-P (NIPAM) hydrogel, is successfully prepared.
(6) Dynamic thermomechanical analysis DMA analysis
And taking an antibacterial peptide hydrogel sample, and performing mechanical dynamic analysis in a DMAQ800 dynamic thermal mechanical analyzer. The test condition is that the constant temperature is 30 ℃, and the relation between the compression strain (%) and the stress (MPa) is tested; the temperature dependence of the strain (%) at constant stress is in the range of 25 ℃ to 50 ℃ and the results are shown in FIG. 6.
As shown in the results of FIG. 6, in FIG. 6A, the strain of the hydrogel decreases with increasing stress at 30 ℃ and the fracture-type sudden drop of the hydrogel occurs at about 0.04 MPa; in FIG. 6(B), the hydrogel is under constant stress of 0.038MPa, and the strain and length change with the slow increase of ambient temperature, and at 40 ℃, the strain and length of the hydrogel suddenly drop, indicating that the inside of the hydrogel may be changed from a uniform structure to a non-uniform collapsed structure, thereby causing the colloid to break.
(7) Thermogravimetric analysis
In order to investigate the thermal stability and decomposition process at high temperature of the antimicrobial peptide hydrogel, the lyophilized antimicrobial peptide hydrogel was ground into fine powder and analyzed for thermal stability under an STA-409PC comprehensive thermal analyzer, and the specific results are shown in FIG. 7.
As can be seen from the results in the attached FIG. 7, the mass loss process of the antibacterial peptide-P (NIPAM) hydrogel prepared by the invention is divided into 3 stages: the mass loss is caused by the weight loss of the bound water in the hydrogel at about 100 ℃; the mass loss from 150 ℃ to 360 ℃ may be caused by the cleavage and decomposition of poly-N-isopropylacrylamide.
By combining the data analysis, the temperature-sensitive antibacterial peptide-hydrogel is successfully prepared by mixing the antibacterial peptide and the P (NIPAM) polymer. The main reason for the phase transition of hydrogels is that the inside of the gel changes from a coil structure to a spherical structure when the temperature rises. When the temperature is lower than the LCST, the hydrophilic gel absorbs the antibacterial peptide after removing the NIPAM monomer and stores the antibacterial peptide, and when the temperature is higher than the LCST, the antibacterial peptide is slowly released along with the change of the hydrogel structure, and the experimental result shows that the LCST of the antibacterial peptide hydrogel is about 37.7 ℃, is close to the body temperature of a human body and has good inhibitory effect on staphylococcus aureus, so that the hydrogel dressing is suitable for being used as the hydrogel dressing on the surface of the skin of the human body. SEM results show that the addition of the antibacterial peptide enables the original loose and porous structure of the hydrogel to have water storage performance, and the antibacterial peptide solution is distributed in pores, so that slow drug release is facilitated. The results of element analysis and infrared spectroscopy both show that the antimicrobial peptide has been successfully embedded in the P (NIPAM) hydrogel. DMA analysis shows that the maximum bearing stress of the antibacterial peptide hydrogel is 0.038MPa, the antibacterial peptide hydrogel breaks due to the change of the internal structure at about 40 ℃, and the antibacterial peptide hydrogel has certain mechanical properties when used as a medical dressing. The thermogravimetric analysis result shows that the antibacterial peptide hydrogel has certain thermal stability. The function and practicability of the antibacterial peptide-hydrogel are verified through experiments, so that the antibacterial peptide-hydrogel can be used as a medical dressing to be applied to actual life.
Example 12: application test of antibacterial peptide-hydrogel for medical dressing
In this example, an application verification test was performed on the antimicrobial peptide-hydrogel for medical dressing provided by the present invention based on the tests provided in examples 1 to 11, and the skin irritation and the effect on wound healing were examined.
1. Skin irritation test
The experimental biocompatibility procedure followed the GB/T16886.10-2017 procedure. The animal welfare requirement conforms to GB/T16886.2-2011-part 2 of biological evaluation of medical instruments: animal welfare requirements; acute skin irritation studies were performed according to the current version of the guidelines of GB/T16886.10-2017.
The evaluation of the intensity of the applied irritability and the evaluation criteria are shown in tables 1-2, and the evaluation was made according to the skin irritation response evaluation criteria. The average reaction score (total score of erythema formation + total score of edema formation)/total number of animals was used to determine the skin irritation intensity.
(1) Test protocol
Single contact test: the method comprises the steps of carrying out short hair (5 cm x 6 cm) on the two sides of the spine and the lateral wing area of 6 healthy and skin-damage-free adult mice in advance for 4 h-24 h, establishing an antibacterial peptide hydrogel test group, a deionized water blank control group and a 1% sodium dodecyl sulfate positive control group, and repeating 3 groups in each group. The method comprises the steps of cutting each group of hydrogel into the size of 1cm & lt 1 & gt cm & lt), attaching the hydrogel to the back spine of a mouse which is shorn in advance, fixing the hydrogel for 12 hours by using medical gauze and adhesive tapes, and feeding the hydrogel separately. The contact site condition was recorded at (1. + -. 0.1) h, (24. + -. 2) h, (48. + -.2) h and (72. + -.2) h after application, respectively, and the results are shown in Table 3.
Table 1: skin irritation response scoring criteria
Table 2: evaluation criteria for skin irritation intensity
| Type of reaction | Average score |
| Has no |
0～0.5 |
| Mild degree of | 0.5～1.9 |
| Of |
2～4.9 |
| |
5～8 |
The contact test was repeated: grouping and skin treatment methods same as single contact experiment, applying 1 time daily, fixing with gauze and medical adhesive tape for 6 hr, and continuing for 3 days. Before daily application, visual observation and recording whether the application part has irritation phenomena such as edema and erythema are recorded and scored. The contact site condition was recorded at (1. + -. 0.1) h, (24. + -. 2) h, (48. + -.2) h and (72. + -.2) h after application, respectively, and the results are shown in Table 4 and FIG. 8.
(2) Analysis of test results
Single contact test: as can be seen from the data in Table 3, 24 hours after the dressing is removed, the applied part of an individual test group has very slight erythema, the average maximum stimulation is 0.33, and the whole is nonirritating; the positive control group has obvious stimulation; the blank control group exhibited no irritation.
Table 3: single application irritation score
Multiple skin irritations: as can be seen from the data of the attached table 4 and the attached figure 8, the skin of the mouse is slightly stimulated by multiple times of continuous administration, less clear erythema appears in a test group within 24 hours, the highest stimulation is averagely 0.83, and the mild stimulation is achieved; the average stimulation score of the positive control group in 48h and 72h is 3.00 continuously, and the positive control group has moderate stimulation; the blank group individually showed very mild erythema, with the highest stimulation averaging 0.33 and overall no irritation.
Table 4: repeated application irritation score
The antibacterial peptide is a small molecular protein, and whether the antibacterial peptide can cause skin irritation and anaphylactic reaction is not reported. In order to ensure the biocompatibility of the antibacterial peptide PNIPAM hydrogel, the experimental procedure completely follows the current version procedure of GB/T16886.10-2017. The animal welfare requirement conforms to GB/T16886.2-2011-biological evaluation of medical instruments-part 2: animal welfare requirements. The test group showed very slight erythema within 24 hours of the initial hydrogel application period, but disappeared and returned to the initial state within 72 hours without skin irritation such as edema, hyperemia, effusion, ulceration and pigmentation. Overall, the skin irritation of the test group was significantly less than that of the positive control group. Research results show that the antibacterial peptide hydrogel provided by the invention has good safety, almost has no irritation to mouse skin, and provides reference basis for practical safe use.
2. Wound healing test
In order to evaluate the wound healing capacity and biocompatibility of the dressing, 6 healthy guinea pigs with the size of 8 weeks are selected as study objects in the test and are respectively bred for 1 week in an adaptive and independent mode. During the feeding period, guinea pigs were fed with standard feed and were fed freely with water at room temperature (22 + -2) ° c, well ventilated, and the time of light irradiation was adjusted according to the normal circadian rhythm. All guinea pigs were kept under the same conditions without adverse effects.
(1) Test protocol
Guinea pigs are randomly marked as No. 1-6, and the antibacterial peptide-hydrogel provided by the invention is set to be applied as a test group, the Yunnan white drug powder woundplast is applied as a positive control group, and the PNIPAM hydrogel is set as a negative control group. Two test zones were placed on the back of each guinea pig and the test groups were determined to be: numbers 1-3 (test group + negative control); numbers 4-6 (test group + positive control). 4-24 h before the test, the guinea pig was depilated and skin-prepared on the back of the operation area using an electric shaver and depilatory cream. All experimental devices were subjected to uv sterilization prior to surgery.
The guinea pig is anesthetized by ether, when the anesthesia effect is satisfied, a fault skin wound surface model (1cm multiplied by 1cm and 3mm) is manufactured on two sides of the spinal column, after hemostasis is performed by pressing and alcohol disinfection is performed, hydrogel is cut into the size of (2cm multiplied by 2 cm) and is pasted on a wound, disinfected white gauze is used for external use to be sewn at four corners and is wrapped by medical adhesive tape for winding and fixing, the wound healing condition is observed and recorded every day, and photographing is performed. And finally, comparing with a control group to comprehensively evaluate the biocompatibility and the wound healing efficacy of the P (NIPAM-C) hydrogel, performing hemostasis and disinfection treatment on the wound after molding, observing the wound healing condition every day and taking a picture after the hydrogel is applied for the 0 th day, and comparing and observing and analyzing the wound healing result by taking 5d and 10d, wherein the specific result is shown in the attached figures 9-10.
(2) Analysis of test results
As can be seen from the results shown in FIGS. 9-10, the test groups were wet hydrogels compared to the negative control group, but the scar formation was earlier due to the antibacterial peptide contained therein, and the healing capacity of the whole wound surface was significantly higher; compared with a positive control, the wound healing conditions of the test group are basically in the same stage, the wound healing capacity to the skin wound surface is slightly strong, and the antibacterial peptide hydrogel is safer considering that the application part of the band-aid is easy to suppuration and the wound is easy to be secondarily damaged during dressing change.
In conclusion, through skin irritation tests and wound healing tests, the skin irritation of the antibacterial peptide-hydrogel provided by the invention is obviously less than that of a positive control group, the scar is earlier because of containing the antibacterial peptide, the whole wound healing capability is obviously higher, the antibacterial peptide-hydrogel hardly has an irritation effect on the skin of a mouse, secondary injury can not be caused to the wound, a reference basis is provided for safe use of the antibacterial peptide-hydrogel, and the antibacterial peptide-hydrogel has a good application value in the aspect of medical dressings
The above examples are merely illustrative for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications can be made while remaining within the scope of the present invention.
Claims (6)
1. The antibacterial peptide-hydrogel for the medical dressing is characterized by comprising, by weight, 300 parts of N-isopropylacrylamide 200-5 parts, 8-10 parts of ammonium persulfate, 10-15 parts of N-N methylene bisacrylamide, 3-5 parts of NNNN-tetramethylethylenediamine, 50-150 parts of an antibacterial peptide solution with the concentration of 0.3mg/mL, and the balance of deionized water.
2. The antimicrobial peptide-hydrogel for medical dressings according to claim 1, which comprises 230 parts by weight of N-isopropylacrylamide, 9 parts by weight of ammonium persulfate, 12 parts by weight of N-methylenebisacrylamide, 4 parts by weight of NNNN-tetramethylethylenediamine, 100 parts by weight of an antimicrobial peptide solution having a concentration of 0.3mg/mL, and the balance of deionized water.
3. The preparation method of the antibacterial peptide-hydrogel for the medical dressing is characterized in that the antibacterial peptide-hydrogel for the medical dressing is obtained through the following test steps:
(1) accurately weighing NIPAM monomer, grinding the solid and pouring into a clean beaker, weighing a certain amount of MBAA and APS and pouring into the beaker, adding a certain amount of deionized water, and stirring at a constant speed by using a glass rod at room temperature until white crystals are completely dissolved;
(2) adding TEMED into the solution obtained in the step (1), placing the solution in an ice box, continuously stirring for 1.0min, injecting the liquid in a beaker into a gel making plate by using a liquid transfer gun, and standing and reacting for 6-12h at 4 ℃ to obtain P (NIPAM) hydrogel;
(3) soaking the P (NIPAM) hydrogel obtained in the step (2) in deionized water for 6-18h, changing water every 6h, soaking in hot water at 60-80 ℃ until the gel is swelled to be balanced, taking out when the gel is whitish and has shrunk volume, and airing in a fume hood;
(4) and (3) adding a certain amount of antibacterial peptide solution with the concentration of 0.3mg/mL into the hydrogel obtained in the step (3), and obtaining the antibacterial peptide-hydrogel after the gel is swelled to be balanced.
4. The method for preparing the antibacterial peptide-hydrogel for medical dressings according to claim 3, wherein the standing reaction time in the step (2) is 8 hours.
5. The method for preparing the antibacterial peptide-hydrogel for medical dressings according to claim 3, wherein the soaking time of the deionized water in the step (3) is 12 hours, and the temperature of the hot water is 70 ℃.
6. The use of the antimicrobial peptide-hydrogel for medical dressings according to claim 1 in medical dressings.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103948962A (en) * | 2014-05-09 | 2014-07-30 | 天津工业大学 | Method for preparing growth-factor bound thermo-sensitive hydrogel biocarrier |
| CN114040785A (en) * | 2019-05-02 | 2022-02-11 | 国家健康科学研究所 | Hyaluronic acid hydrogels with extended antimicrobial activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103948962A (en) * | 2014-05-09 | 2014-07-30 | 天津工业大学 | Method for preparing growth-factor bound thermo-sensitive hydrogel biocarrier |
| CN114040785A (en) * | 2019-05-02 | 2022-02-11 | 国家健康科学研究所 | Hyaluronic acid hydrogels with extended antimicrobial activity |
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