Conductive antibacterial composite hydrogel and preparation method and application thereof
阅读说明：本技术 一种导电抗菌复合水凝胶及其制备方法、应用 (Conductive antibacterial composite hydrogel and preparation method and application thereof ) 是由 姜英男 匡玉兰 闫茹月 赵新宇 田腾辉 张哲� 王继凤 肖利智 孙天霞 朱迪夫 于 2021-11-02 设计创作，主要内容包括：本发明提供了一种复合水凝胶,所述复合水凝胶包括聚乙烯醇、明胶、导电聚苯胺和银纳米粒子；所述聚乙烯醇、明胶和导电聚苯胺形成交联的聚合物网络结构；所述银纳米粒子复合在所述聚合物网络结构中。本发明以PVA与明胶作为复合水凝胶的主体基质,以植酸作为交联剂引入导电聚苯胺,赋予水凝胶体系导电性能,同时还形成了物理交联的3D聚合物网络结构,再结合银纳米粒子,提高水凝胶敷料的抗菌性能。该材料兼具良好的力学性能、导电性能、生物容性与抑菌性,而且即使对感染细菌一段时间,感染程度较重的伤口,仍然具有较好的促进伤口愈合效果,包括促进血管、皮肤细胞、毛囊等的生成的效果,是一种极具应用潜力的医用水凝胶敷料。(The invention provides a composite hydrogel which comprises polyvinyl alcohol, gelatin, conductive polyaniline and silver nanoparticles; the polyvinyl alcohol, the gelatin and the conductive polyaniline form a cross-linked polymer network structure; the silver nanoparticles are complexed in the polymer network structure. According to the invention, PVA and gelatin are used as main matrixes of the composite hydrogel, phytic acid is used as a cross-linking agent to introduce the conductive polyaniline, so that the hydrogel system is endowed with conductive performance, a physically cross-linked 3D polymer network structure is formed, and the antibacterial performance of the hydrogel dressing is improved by combining with silver nanoparticles. The material has good mechanical property, conductivity, biocompatibility and bacteriostasis, and still has good effect of promoting wound healing even for wounds with serious infection degree to infectious bacteria for a period of time, including the effect of promoting the generation of blood vessels, skin cells, hair follicles and the like, thus being a medical hydrogel dressing with great application potential.)
1. The composite hydrogel is characterized by comprising polyvinyl alcohol, gelatin, conductive polyaniline and silver nanoparticles;
the polyvinyl alcohol, the gelatin and the conductive polyaniline form a cross-linked polymer network structure;
the silver nanoparticles are complexed in the polymer network structure.
2. The composite hydrogel according to claim 1, wherein the composite hydrogel comprises polyvinyl alcohol and gelatin as main matrices;
the conductive polyaniline comprises phytic acid crosslinked polyaniline;
the particle size of the silver nanoparticles is 10-50 nm;
the crosslinked polymer network structure comprises a physically crosslinked 3D polymer network structure;
the composite hydrogel comprises a composite hydrogel dressing.
3. The composite hydrogel according to claim 1, wherein the polymer network structure is an interpenetrating network structure in which three polymers, namely polyvinyl alcohol, gelatin and conductive polyaniline, are interpenetrated;
the molecular weight of the polyvinyl alcohol is 120000-1500000 Da;
the molecular weight of the gelatin is 50000-100000 Da;
the molecular weight of the conductive polyaniline is 600-800 Da;
the composite hydrogel includes an electrically conductive composite hydrogel dressing.
4. The composite hydrogel according to claim 1, wherein the crosslinked polymer network structure is formed by compounding a phytic acid crosslinked polyaniline network with polyvinyl alcohol and gelatin as main skeletons;
the polymer network structure has a coral-shaped and/or dendritic network structure appearance;
the aperture of the composite hydrogel is 1-50 mu m;
the porosity of the composite hydrogel is 50-99%;
the specific surface area of the composite hydrogel is 4-8 m2/g；
The composite hydrogel comprises a bacteriostatic composite hydrogel dressing.
5. The composite hydrogel according to claim 1, which comprises, in mass fraction:
the composite hydrogel comprises a medical composite hydrogel dressing.
6. The preparation method of the composite hydrogel is characterized by comprising the following steps:
1) mixing and dissolving polyvinyl alcohol, gelatin and water to obtain a mixed solution;
2) mixing the mixed solution obtained in the step, phytic acid, aniline, ammonium persulfate and water again, and freezing and removing impurities to obtain conductive hydrogel;
3) and (3) soaking the conductive hydrogel obtained in the step (a) in water, and then placing the conductive hydrogel in a silver nanoparticle solution to obtain the composite hydrogel.
7. The preparation method according to claim 6, wherein the temperature of the mixed dissolution is 80-90 ℃;
the mass ratio of the polyvinyl alcohol to the gelatin is (0.05-2): 1;
the mass ratio of the phytic acid to the aniline is (1-5): 1;
the mass ratio of the ammonium persulfate to the aniline is (0.2-5): 1.
8. the preparation method according to claim 6, wherein one or more of the phytic acid, aniline and ammonium persulfate is added to the system in the form of a solution;
the mass concentration of the phytic acid solution is 45-55 wt%;
the mass concentration of the aniline solution is 99.0-99.9%;
the concentration of the ammonium persulfate solution is 1-1.5M.
9. The method of claim 6, wherein the freezing temperature is-20 to-30 ℃;
the freezing time is 10-24 h;
the impurity removal mode comprises soaking in water to remove impurities;
the placing time is 48-72 h;
the mass ratio of the silver nanoparticles to the polyvinyl alcohol is (0.001-0.01): 1;
the concentration of the silver nanoparticle solution is 0.05-0.5M.
10. Use of the composite hydrogel according to any one of claims 1 to 5 or the composite hydrogel prepared by the preparation method according to any one of claims 6 to 9 in medical materials.
Technical Field
The invention belongs to the technical field of medical dressings, relates to a composite hydrogel and a preparation method and application thereof, and particularly relates to a medical conductive antibacterial composite hydrogel and a preparation method and application thereof.
Background
Wound infection generally refers to a more severe inflammatory reaction after a longer time (more than 24 h) of invasion of the skin lesion by microorganisms. The clinical manifestations are local red and swollen, pain, pus exudation, peculiar smell and the like, and the severe can cause systemic infection and even threaten the life of people. Therefore, the clinical application significance of curing the infected wound and accelerating the healing of the infected wound is high. Traditional dressings such as gauze, bandage and the like have good air permeability, but obviously cannot play a role in bacteriostasis treatment on infected wounds. And in the treatment process, wound adhesion is easy to occur to cause secondary injury, the wound healing period is prolonged, and even larger scars are generated. In recent years, various novel antibacterial dressings such as films, foams, alginates and the like are developed according to the clinical requirements of wound infection healing. However, these materials also have the disadvantages of poor exudate absorption, poor wound surface adherence, poor air permeation. Therefore, the research on the medical dressing which is light, thin and portable, can effectively resist bacteria and promote the healing of infected wounds has important research and application significance.
A hydrogel is a 3D polymer network that swells in water and can swell in water and retain a significant amount of water. Unlike other dressings, the hydrogel as a wound dressing can provide a wet environment for the wound to heal more easily, accelerate granulation and angiogenesis, and promote autolysis of necrotic tissue. And the exudate which is easy to cause wound infection not to be healed can be absorbed highly. Due to the excellent characteristics, the composite medical hydrogel dressing has better application and development prospects in clinic, and particularly has unique advantages in the field of biomedical sensing due to the characteristics of stretchability, wearability and drug loading along with the rapid development of artificial intelligence and micro robots. Thus, there is a further need in the art for a medical hydrogel dressing that combines the properties of these dressings. Meanwhile, although the existing hydrogel dressing can provide a wet environment which is beneficial to wound recovery, most of hydrogels do not have a bacteriostatic function, but bacteria can be adhered and grow and reproduce, so that the existing hydrogel dressing is not beneficial to wound healing and even incapable of healing infectious wounds. Some hydrogel materials added with antibiotics have the problems of antibiotic abuse and continuous increase of drug-resistant bacteria types. More importantly, in clinical practice, the wound is not treated in time, and serious infection and suppuration even cause life danger. In the study of antimicrobial hydrogel dressings, where the environment in which the experiment is typically conducted is clean (typically SPF grade), researchers have focused only on promoting healing of full-thickness skin lesions. Or a simple bacteria coating experiment is carried out, the bacteriostatic dressing is immediately applied to the wound surface after bacteria coating, and the wound infection degree is very low. The research and application of the bacteriostatic hydrogel application do not relate to the simulation and treatment of serious wound infection, which is one of the blind points of research in the industry.
Therefore, how to find a more suitable composite hydrogel material not only has various comprehensive properties, but also can solve the existing defects in the aspect of bacteriostasis, and meanwhile, has a good effect on severe wound infection, and has become one of the focuses of great concern of a plurality of researchers with prospective prospects in the field.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a composite hydrogel, a preparation method and an application thereof, and in particular, to a medical conductive antibacterial composite hydrogel, a preparation method and an application thereof. The invention synthesizes the novel medical hydrogel with good mechanical property, conductivity, biocompatibility and bacteriostasis, and even for wounds with serious infection degree for a period of time of infected bacteria, the prepared hydrogel still has good effect of promoting wound healing, including the effect of promoting the generation of blood vessels, skin cells, hair follicles and the like, and is a medical hydrogel dressing with great application potential.
The invention provides a composite hydrogel which comprises polyvinyl alcohol, gelatin, conductive polyaniline and silver nanoparticles;
the polyvinyl alcohol, the gelatin and the conductive polyaniline form a cross-linked polymer network structure;
the silver nanoparticles are complexed in the polymer network structure.
Preferably, the composite hydrogel takes polyvinyl alcohol and gelatin as main matrix;
the conductive polyaniline comprises phytic acid crosslinked polyaniline;
the particle size of the silver nanoparticles is 10-50 nm;
the crosslinked polymer network structure comprises a physically crosslinked 3D polymer network structure;
the composite hydrogel comprises a composite hydrogel dressing.
Preferably, the polymer network structure is an interpenetrating network structure formed by interpenetrating three polymers, namely polyvinyl alcohol, gelatin and conductive polyaniline;
the molecular weight of the polyvinyl alcohol is 120000-1500000 Da;
the molecular weight of the gelatin is 50000-100000 Da;
the molecular weight of the conductive polyaniline is 600-800 Da;
the composite hydrogel includes an electrically conductive composite hydrogel dressing.
Preferably, the crosslinked polymer network structure is formed by compounding a phytic acid crosslinked polyaniline network with polyvinyl alcohol and gelatin as main skeleton;
the polymer network structure has a coral-shaped and/or dendritic network structure appearance;
the aperture of the composite hydrogel is 1-50 mu m;
the porosity of the composite hydrogel is 50-99%;
the specific surface area of the composite hydrogel is 4-8 m2/g；
The composite hydrogel comprises a bacteriostatic composite hydrogel dressing.
Preferably, the composite hydrogel comprises the following components in percentage by mass:
the composite hydrogel comprises a medical composite hydrogel dressing.
The invention provides a preparation method of composite hydrogel, which comprises the following steps:
1) mixing and dissolving polyvinyl alcohol, gelatin and water to obtain a mixed solution;
2) mixing the mixed solution obtained in the step, phytic acid, aniline, ammonium persulfate and water again, and freezing and removing impurities to obtain conductive hydrogel;
3) and (3) soaking the conductive hydrogel obtained in the step (a) in water, and then placing the conductive hydrogel in a silver nanoparticle solution to obtain the composite hydrogel.
Preferably, the temperature of the mixing and dissolving is 80-90 ℃;
the mass ratio of the polyvinyl alcohol to the gelatin is (0.05-2): 1;
the mass ratio of the phytic acid to the aniline is (1-5): 1;
the mass ratio of the ammonium persulfate to the aniline is (0.2-5): 1.
preferably, one or more of the phytic acid, the aniline and the ammonium persulfate are added into the system in the form of solution;
the mass concentration of the phytic acid solution is 45-55 wt%;
the mass concentration of the aniline solution is 99.0-99.9%;
the concentration of the ammonium persulfate solution is 1-1.5M.
Preferably, the freezing temperature is-20 to-30 ℃;
the freezing time is 10-24 h;
the impurity removal mode comprises soaking in water to remove impurities;
the placing time is 48-72 h;
the mass ratio of the silver nanoparticles to the polyvinyl alcohol is (0.001-0.01): 1;
the concentration of the silver nanoparticle solution is 0.05-0.5M.
The invention also provides application of the composite hydrogel in any one of the technical schemes or the composite hydrogel prepared by the preparation method in any one of the technical schemes in medical materials.
The invention provides a composite hydrogel which comprises polyvinyl alcohol, gelatin, conductive polyaniline and silver nanoparticles; the polyvinyl alcohol, the gelatin and the conductive polyaniline form a cross-linked polymer network structure; the silver nanoparticles are complexed in the polymer network structure. Compared with the prior art, the invention aims at the problems that the existing hydrogel medical material has single function, most of the hydrogel medical material does not have bacteriostatic function, antibiotics are added, and the variety of drug-resistant bacteria is increased continuously, and the simulation and treatment of serious wound infection are lacked in the research direction.
The invention creatively obtains a composite hydrogel material with specific composition and structure, which is composed of polyvinyl alcohol, gelatin, conductive polyaniline and silver nanoparticles and forms the hydrogel material with specific structure. The invention takes PVA and gelatin as main bodies, takes phytic acid as a cross-linking agent, introduces conductive polyaniline for cross-linking, forms a polymer network structure of the cross-linking of the PVA and the gelatin, and then loads silver nanoparticles to obtain the novel medical hydrogel with good mechanical property, conductive property, biocompatibility and antibacterial property. Polyvinyl alcohol (PVA) has good lubricity, elasticity and shock absorption capacity, excellent mechanical properties and good performance when being compounded with other materials. Gelatin (Gelatin) contains a large number of active functional groups such as amino groups, carboxyl groups, hydroxyl groups and the like, has high water absorption, low antigenicity, good biocompatibility, biodegradability, reversible gel formation property along with temperature change and the like, but the hydrogel taking the Gelatin as a main body has poor mechanical property and thermal stability, so that the hydrogel taking the Gelatin as a main body is compounded with polyvinyl alcohol to prepare hydrogel with interpenetrating networks, double networks and nano composite structures, has better mechanical property, particularly adopts polyaniline with good conductivity, is further modified by phytic acid, has simple synthesis or modification process, and is easier to compound with other hydrogel materials, so that the composite hydrogel material prepared by the invention has advantages in the aspects of bioelectricity sensing and electrochemical detection, and the hydrogel dressing containing the conductive material is beneficial to the transmission of electric signals, the skin can be better attached; the composite material has the characteristics of mechanical stretchability, sensitivity and self-healing and sensing of skin model, and the doped polyaniline also has an antibacterial effect and can prevent microorganisms such as bacteria from breeding on the surface of the material, so that the composite hydrogel provided by the invention has both electric conductivity and antibacterial performance and good skin bonding effect. In addition, the silver nanoparticles are used as an antibacterial material, can change the permeability of the cell membrane of bacteria, release silver ions to damage the DNA of the bacteria and reduce the activity of dehydrogenase, have strong sterilization property, and hardly increase drug-resistant bacteria, thereby avoiding the abuse problem of antibiotics.
According to the invention, PVA and gelatin are used as main matrixes of the composite hydrogel, phytic acid is used as a cross-linking agent to introduce conductive polyaniline, so that the hydrogel system is endowed with conductivity, a physically cross-linked 3D polymer network structure is formed, the hydrogel dressing has a typical interpenetrating network and multi-network cross-linked structure, and silver nanoparticles are combined, so that the antibacterial performance of the hydrogel dressing is improved, the hydrogel dressing has a good treatment effect on infected wounds, and finally, a novel hydrogel material which has good mechanical properties and antibacterial effect and is attached to the skin is obtained. The conductive medical hydrogel dressing prepared by the material has good mechanical property, conductivity, biocompatibility and bacteriostasis, and still has good effect of promoting wound healing even for wounds with serious infection degree after a period of time of infectious bacteria, including the effect of promoting the generation of blood vessels, skin cells, hair follicles and the like, thus being a medical hydrogel dressing with great application potential.
Experimental results show that the novel system hydrogel prepared by the invention has good mechanical property, biocompatibility, conductivity and wound bacteriostasis effect, is particularly suitable for animal models with severe wound infection, still shows good bacteriostasis effect on infected wounds infected with bacteria for 48 hours, and is a novel medical hydrogel application capable of being used for severe wound infection.
Drawings
FIG. 1 is a TEM image of silver nanoparticles prepared according to the present invention;
FIG. 2 is a graph showing mechanical property tests and physical deformation of Ag NPs/CPH prepared with different amounts (5 wt%, 7.5 wt%, 10 wt%, 12.5 wt%) of PVA of different activities prepared according to the present invention;
FIG. 3 is a graph showing the conductivity test of AgNPs/CPHs prepared by the present invention;
FIG. 4 is SEM scanning electron micrographs of Ag NPs/CPH of different gelatin reaction contents prepared by the invention and the effect curves on storage modulus and loss modulus;
FIG. 5 is a photograph showing the state detection of the hydrogel prepared according to the present invention;
FIG. 6 is a graph of five time-dependent (0-72 h) volume swell curves for five sets of CPH and Ag NPs/CPH samples prepared in accordance with the present invention;
FIG. 7 is an XPS spectrum of Ag NPs/CPH prepared according to the present invention;
FIG. 8 is a photograph of an experiment on e-coli and S.aureus resistance of a hydrogel plate and a corresponding diameter calculation chart;
FIG. 9 shows lb Medium, CPH and Ag NPs loaded with different Ag NpsLiquid OD of Co-culture of CPHAg NCs and e-coli600A measured data graph of values (0-48 h);
FIG. 10 is a liquid OD obtained by co-culturing lb medium, CPH and Ag NPs/CPHAg NCs loaded with different Ag NCs with S.aureus600A measured data graph of values (0-48 h);
FIG. 11 shows the cell survival rate of HaCat cells measured by CCK8 method after the HaCat cells are co-cultured in PBS and hydrogel soak solutions of different concentrations for 48 h;
FIG. 12 shows the cell viability of LO2 cells measured by CCK8 after the cells are co-cultured in PBS and hydrogel soaking solutions of different concentrations for 48 h;
FIG. 13 shows the cell survival rate of 293T cells after being co-cultured in PBS and hydrogel soak solutions of different concentrations for 48h, which is measured by the CCK8 method;
FIG. 14 is a photograph of wounds from Ag NPs/CPH, CPH and PBS treated mice on days 1, 3, 7 and 14;
FIG. 15 is a graph showing statistical calculations of the remaining wound area over time for each group of mice corresponding to FIG. 14;
FIG. 16 is a H & E stained section of skin tissue near the wound of gauze (control), CPH and Ag NPs/CPH treated mice and corresponding counts of inflammatory cells on different days (scale: 50 μm);
FIG. 17 is a CD31 stained section of skin tissue near the wound and corresponding blood vessel counts in skin tissue of gauze (control), CPH and Ag NPs/CPH treated mice on different days (scale: 50 μm);
FIG. 18 is a Munsen stained section and corresponding percentage of collagen deposition near the wound of gauze (control), CPH and Ag NPs/CPH treated mice on different days (scale: 50 μm);
figure 19 is a simplified schematic illustration of a hydrogel material prepared in accordance with the present invention and a medical hydrogel dressing for severe wound infection.
Detailed Description
For a further understanding of the invention, preferred embodiments of the invention are described below in conjunction with the examples, but it should be understood that these descriptions are included merely to further illustrate the features and advantages of the invention and are not intended to limit the invention to the claims.
All of the starting materials of the present invention, without particular limitation as to their source, may be purchased commercially or prepared according to conventional methods well known to those skilled in the art.
All the raw materials of the invention are not particularly limited in purity, and the invention preferably adopts the purity commonly used in the field of analytical purity or medical hydrogel accessories.
All the raw materials, the marks and the acronyms thereof belong to the conventional marks and acronyms in the field, each mark and acronym is clear and definite in the field of related application, and the raw materials can be purchased from the market or prepared by a conventional method by the technical staff in the field according to the marks, the acronyms and the corresponding application.
The invention provides a composite hydrogel which comprises polyvinyl alcohol, gelatin, conductive polyaniline and silver nanoparticles;
the polyvinyl alcohol, the gelatin and the conductive polyaniline form a cross-linked polymer network structure;
the silver nanoparticles are complexed in the polymer network structure. Specifically, the silver nanoparticles are preferably supported on an interpenetrating network structure.
In the present invention, the composite hydrogel preferably comprises polyvinyl alcohol and gelatin as main matrix.
In the present invention, the conductive polyaniline preferably comprises phytic acid crosslinked polyaniline.
In the invention, the particle size of the silver nanoparticles is preferably 10-50 nm, more preferably 15-45 nm, more preferably 20-40 nm, and more preferably 25-35 nm.
In the present invention, the crosslinked polymer network structure preferably comprises a physically crosslinked 3D polymer network structure.
In the invention, the polymer network structure is preferably an interpenetrating network structure formed by interpenetrating three polymers of polyvinyl alcohol, gelatin and conductive polyaniline.
In the present invention, the crosslinked polymer network structure is preferably formed by compounding a phytic acid crosslinked polyaniline network with polyvinyl alcohol and gelatin as main skeletons.
In the present invention, the polymer network preferably has a coral-like and/or dendritic network morphology, and more preferably has a coral-like or dendritic network morphology.
In the present invention, the composite hydrogel comprises, by mass:
in the present invention, the composite hydrogel preferably comprises a medical composite hydrogel dressing.
In the present invention, the polyvinyl alcohol is preferably added in an amount of 0.5 to 20 parts by weight, more preferably 4 to 16 parts by weight, and still more preferably 8 to 12 parts by weight.
In the present invention, the gelatin is preferably added in an amount of 0.5 to 10 parts by weight, more preferably 2 to 8 parts by weight, and still more preferably 4 to 6 parts by weight.
In the invention, the addition amount of the conductive polyaniline is preferably 5 to 20 parts by weight, more preferably 8 to 17 parts by weight, and more preferably 11 to 14 parts by weight.
In the present invention, the amount of the silver nanoparticles added is preferably 0.01 to 0.1 part by weight, more preferably 0.03 to 0.08 part by weight, and more preferably 0.05 to 0.06 part by weight.
In the present invention, the composite hydrogel preferably includes a composite hydrogel dressing. Preferably, the composite hydrogel preferably comprises an electrically conductive composite hydrogel dressing. Preferably, the composite hydrogel preferably comprises a bacteriostatic composite hydrogel dressing.
In the invention, the pore diameter of the composite hydrogel is preferably 1-50 μm, more preferably 10-40 μm, and more preferably 20-30 μm.
In the present invention, the porosity of the composite hydrogel is preferably 50% to 99%, more preferably 60% to 90%, and more preferably 70% to 80%.
In the invention, the specific surface area of the composite hydrogel is preferably 4-8 m2A more preferable range is 4.5 to 7.5 m/g2A concentration of 5 to 7m2A more preferable range is 5.5 to 6.5 m/g2/g。
In the invention, the molecular weight of the polyvinyl alcohol is preferably 120000-1500000 Da, more preferably 125000-1450000 Da, and more preferably 130000-1400000 Da.
In the invention, the molecular weight of the conductive polyaniline is preferably 600-800 Da, more preferably 640-760 Da, and more preferably 680-720 Da.
The invention also provides a preparation method of the composite hydrogel, which comprises the following steps:
1) mixing and dissolving polyvinyl alcohol, gelatin and water to obtain a mixed solution;
2) mixing the mixed solution obtained in the step, phytic acid, aniline, ammonium persulfate and water again, and freezing and removing impurities to obtain conductive hydrogel;
3) and (3) soaking the conductive hydrogel obtained in the step (a) in water, and then placing the conductive hydrogel in a silver nanoparticle solution to obtain the composite hydrogel.
According to the invention, firstly, polyvinyl alcohol, gelatin and water are mixed and dissolved to obtain a mixed solution.
In the invention, the mixing and dissolving temperature is preferably 80-90 ℃, more preferably 82-88 ℃, and more preferably 84-86 ℃.
In the invention, the mass ratio of the polyvinyl alcohol to the gelatin is preferably (0.05-2): 1, more preferably (0.4 to 1.6): 1, more preferably (0.8 to 1.2): 1.
the mixed solution obtained in the step, phytic acid, aniline, ammonium persulfate and water are mixed again, and the conductive hydrogel is obtained after freezing and impurity removal.
In the invention, the mass ratio of the phytic acid to the aniline is preferably (1-5): 1, more preferably (1.5 to 4.5): 1, more preferably (2-4): 1, more preferably (2.5 to 3.5): 1.
in the invention, the mass ratio of the ammonium persulfate to the aniline is preferably (0.2-5): 1, more preferably (1.2 to 4): 1, more preferably (2.2 to 3): 1.
in the present invention, one or more of the phytic acid, aniline and ammonium persulfate is preferably added to the system in the form of a solution.
In the present invention, the mass concentration of the phytic acid solution is preferably 45 wt% to 55 wt%, more preferably 47 wt% to 53 wt%, and more preferably 49 wt% to 51 wt%.
In the present invention, the mass concentration of the aniline solution is preferably 99.0% to 99.9%, more preferably 99.2% to 99.7%, and still more preferably 99.4% to 99.5%.
In the invention, the concentration of the ammonium persulfate solution is preferably 1-1.5M, more preferably 1.1-1.4M, and more preferably 1.2-1.3M.
In the present invention, the temperature of the freezing is preferably-20 to-30 ℃, more preferably-22 to-28 ℃, and still more preferably-24 to-26 ℃.
In the invention, the freezing time is preferably 10-24 h, more preferably 13-21 h, and more preferably 16-19 h.
In the present invention, the removing of impurities preferably comprises soaking in water to remove impurities.
Finally, soaking the conductive hydrogel obtained in the steps in water, and then placing the conductive hydrogel in a silver nanoparticle solution to obtain the composite hydrogel.
In the invention, the time for placing is preferably 48-72 h, more preferably 53-67 h, and more preferably 58-62 h.
In the invention, the mass ratio of the silver nanoparticles to the polyvinyl alcohol is preferably (0.001-0.01): 1, more preferably (0.003 to 0.008): 1, more preferably (0.005 to 0.006): 1.
in the present invention, the concentration of the silver nanoparticle solution is preferably 0.05 to 0.5M, more preferably 0.15 to 0.4M, and more preferably 0.25 to 0.3M.
The invention takes PVA and gelatin as main frameworks, is compounded with a phytic acid cross-linked polyaniline network, and then is loaded with silver nanoparticles by using an immersion method, so as to synthesize a novel hydrogel patch which can be used for promoting the rapid healing of infected wounds. Through tensile property experiments, scanning electron microscope tests and electrical property investigation, the optimal reaction concentration of PVA, gelatin and aniline is determined, and after the silver nanoparticle aqueous solution is fully soaked, the skin-attached conductive medical hydrogel dressing Ag NPs/CPH is obtained. Then, the biological toxicity and the antibacterial property of the Ag NPs/CPH are inspected, and the result shows that the obtained Ag NPs/CPH has good mechanical property, abundant internal pores, good conductivity, effective antibacterial property and lower biological toxicity.
The invention also provides application of the composite hydrogel in any one of the technical schemes or the composite hydrogel prepared by the preparation method in any one of the technical schemes in medical materials.
In the present invention, the medical material preferably comprises a medical dressing, more preferably a medical hydrogel dressing, more preferably a bacteriostatic medical hydrogel dressing, more preferably a conductive medical hydrogel dressing. The invention can be used for medical hydrogel application for severe wound infection.
In the biological experiment, the invention also designs an animal wound inflammation model with longer time (20 h) for infecting bacteria and heavier infection degree. And a series of biological experiments show the application potential of the prepared antibacterial hydrogel dressing in treating human infected and inflamed wounds. The work establishes a new research idea and reference basis for the design and application of the composite medical hydrogel dressing in the future.
Referring to fig. 19, fig. 19 is a simplified schematic illustration of a hydrogel material prepared in accordance with the present invention and a medical hydrogel application for severe wound infection.
The invention provides a medical conductive antibacterial composite hydrogel and a preparation method and application thereof. The composite hydrogel material with specific composition and structure is composed of polyvinyl alcohol, gelatin, conductive polyaniline and silver nanoparticles, and the hydrogel material with specific structure is formed. The invention takes PVA and gelatin as main bodies, takes phytic acid as a cross-linking agent, introduces conductive polyaniline for cross-linking, forms a polymer network structure of the cross-linking of the PVA and the gelatin, and then loads silver nanoparticles to obtain the novel medical hydrogel with good mechanical property, conductive property, biocompatibility and antibacterial property. Polyvinyl alcohol (PVA) has good lubricity, elasticity and shock absorption capacity, excellent mechanical properties and good performance when being compounded with other materials. Gelatin (Gelatin) contains a large number of active functional groups such as amino groups, carboxyl groups, hydroxyl groups and the like, has high water absorption, low antigenicity, good biocompatibility, biodegradability, reversible gel formation property along with temperature change and the like, but the hydrogel taking the Gelatin as a main body has poor mechanical property and thermal stability, so that the hydrogel taking the Gelatin as a main body is compounded with polyvinyl alcohol to prepare hydrogel with interpenetrating networks, double networks and nano composite structures, has better mechanical property, particularly adopts polyaniline with good conductivity, is further modified by phytic acid, has simple synthesis or modification process, and is easier to compound with other hydrogel materials, so that the composite hydrogel material prepared by the invention has advantages in the aspects of bioelectricity sensing and electrochemical detection, and the hydrogel dressing containing the conductive material is beneficial to the transmission of electric signals, the skin can be better attached; the composite material has the characteristics of mechanical stretchability, sensitivity and self-healing and sensing of skin model, and the doped polyaniline also has an antibacterial effect and can prevent microorganisms such as bacteria from breeding on the surface of the material, so that the composite hydrogel provided by the invention has both electric conductivity and antibacterial performance and good skin bonding effect. In addition, the silver nanoparticles are used as an antibacterial material, can change the permeability of the cell membrane of bacteria, release silver ions to damage the DNA of the bacteria and reduce the activity of dehydrogenase, have strong sterilization property, and hardly increase drug-resistant bacteria, thereby avoiding the abuse problem of antibiotics.
According to the invention, PVA and gelatin are used as main matrixes of the composite hydrogel, phytic acid is used as a cross-linking agent to introduce conductive polyaniline, so that the hydrogel system is endowed with conductivity, a physically cross-linked 3D polymer network structure is formed, the hydrogel dressing has a typical interpenetrating network and multi-network cross-linked structure, and silver nanoparticles are combined, so that the antibacterial performance of the hydrogel dressing is improved, the hydrogel dressing has a good treatment effect on infected wounds, and finally, a novel hydrogel material which has good mechanical properties and antibacterial effect and is attached to the skin is obtained. The conductive medical hydrogel dressing prepared by the material has good mechanical property, conductivity, biocompatibility and bacteriostasis, and still has good effect of promoting wound healing even for wounds with serious infection degree after a period of time of infectious bacteria, including the effect of promoting the generation of blood vessels, skin cells, hair follicles and the like, thus being a medical hydrogel dressing with great application potential.
Experimental results show that the novel system hydrogel prepared by the invention has good mechanical property, biocompatibility, conductivity and wound bacteriostasis effect, is particularly suitable for animal models with severe wound infection, still shows good bacteriostasis effect on infected wounds infected with bacteria for 48 hours, and is a novel medical hydrogel application capable of being used for severe wound infection.
For further illustration of the present invention, the following will describe in detail a composite hydrogel and its preparation method and application in conjunction with the following examples, but it should be understood that these examples are implemented on the premise of the technical solution of the present invention, and the detailed embodiments and specific procedures are given, only for further illustration of the features and advantages of the present invention, not for limitation of the claims of the present invention, and the scope of protection of the present invention is not limited to the following examples.
Materials and chemicals
Aniline(AN,≈99.5％),phytic acid(PA,≈50wt％,Mw≈660.04)was purchased from Aladdin,polyvinylpyrrolidone(PVP,Mw≈40,000),PVA(≈99％hydrolyzed,Mw≈130，000),and gelatin(～300g Bloom)were purchased from Sigma–Aldrich.
silver nitrate(AgNO3,≈99.8％),and sodium borohydride(NaBH4,≈96％)were purchased from Sinopharm Chemical Reagent CO.,Ltd.(Shanghai,P.R.China).,
Ammonium persulfate(APS,≈98.5％),hydrogen peroxide(H2O230 wt%), sodium citrate (. apprxeq.99.0%) wee associated from Beijing Chemical Works (Beijing, P.R.China)
Materials for bacteria and cell experiments LB medium, e-coli, Staphylococcus aureus from ATCC, Phosphor Buffer (PBS) from, CCK-8 Reagent test kit from, HACAT
Instrument for measuring the position of a moving object
Prepare hydrogel samples for infrared spectroscopy,SEM,and TEM tests.X-ray photoelectron spectroscopy(XPS)were measured on a Thermo ESCALAB 250.The radiation source is monochromatic Al Ka(hv＝1486.6eV).The passing energy is 20eV.C1s(284.6eV)is used for calibration.The vacuum degree is better than 2x10-6Pa.The FTIR spectra of the synthetic raw materials and samples were characterized on Fourier transform infrared spectroscopy BRUKER VECTOR33.Scanning electron microscope(SEM)images were obtained using JEOL FESEM6700F electron microscope with a primary electron energy of 3kV.Transmission electron microscopy(TEM)images of the Ag NPs were obtained using a Tecnai GI F20 U-TWIN.
Example 1
Preparation of Ag NPs
First, a prepared silver nitrate solution (0.1 x 10)-3M, 97mL) was stirred vigorously at room temperature, and then 1mL of sodium citrate solution (180 x 10) was added in sequence-3M), 1mL PVP solution (4.2 x 10)-3M)，240μL H2O2Solution (30 wt%) and 800. mu.L of NaBH was added4Solution (100 x 10)-3M). After a few minutes, the solution rapidly finishes the color conversion from colorless solution, the final color is dark blue, and the stirring lasts for more than 2 hours. Finally, stable 0.1 x 10 with antibacterial effect is obtained-3M/L silver nanoparticle solution is pasted for standby, and the nanoparticle form can be observed through a transmission electron microscope.
The silver nanoparticles prepared according to the invention were characterized.
Referring to fig. 1, fig. 1 is a TEM electron micrograph of silver nanoparticles prepared according to the present invention.
Preparation of CPH
0.25g of PVA and 0.075g of gelatin are taken, 4.675mL of purified water are added, and the mixture is stirred vigorously at 90 ℃ until complete dissolution. The resulting mixed solution was transferred to a magnetic stirrer, and 1mL of phytic acid solution (PA,. apprxeq.50 wt%), 600. mu.L of aniline solution (AN,. apprxeq.99.5%), 1mL of APS solution (1.25M) were added in that order, and the solution turned from colorless to a dark green color near black. Put into a refrigerator at-20 ℃ overnight. Taking out the mixture the next day, soaking the mixture in ultrapure water, removing reactant monomers and impurities, replacing the ultrapure water every 8h, and repeating the process for 6 times. Thus obtaining the conductive hydrogel CPH.
Preparation of Ag NPs/CPH
And (3) soaking the hydrogel soaked in the distilled water into the silver nanoparticle solution, taking out after 24 hours, filling the hydrogel in a glass container, and sealing the hydrogel by using a sealing film to obtain the antibacterial hydrogel with a good antibacterial effect. And storing at 4 ℃ in dark.
Example 2
The Ag NPs/CPH prepared by the invention is subjected to performance detection.
In the synthesis process of Ag NPs/CPH, PVA is a main base material, and the influence of PVA reaction content on the mechanical properties of the prepared Ag NPs/CPH is firstly examined.
Within a certain reaction concentration range (2.5-10 wt%), of PVA in the Ag NPs/CPH can prepare hydrogel.
The degree of compression of the hydrogels was similar when a certain pressure was applied to the surface of the hydrogels prepared with different reaction concentrations of PVA. After the pressure was removed, the hydrogel recovered to its original state with little change in its macroscopic state.
The mechanical properties of hydrogel with different PVA concentrations are tested by using a universal material machine for testing the tensile properties of the hydrogel.
Referring to FIG. 2, FIG. 2 is a graph showing mechanical property tests and physical deformation of Ag NPs/CPH prepared with different amounts (5 wt%, 7.5 wt%, 10 wt%, 12.5 wt%) of PVA of different activities prepared according to the present invention. Wherein A is tensile elastic modulus; b is tensile strength; c is tensile strain at break.
A is an elastic stretching mode; b is tensile strength; c is strain at break; d is a storage (G +) and loss (G +) modality; e is a compression mode; f is a photograph of the compression set effect of pre-made Ag NPs/CPH with different PVA concentrations at a certain pressure.
As can be seen from A, B, C, D, E and F in FIG. 2, as the reaction content of PVA increases (5 wt%, 7.5 wt%, 10 wt%, 12.5 wt%), the tensile modulus of elasticity of the hydrogel gradually increases, and the tensile strength and elongation at break also regularly increase. The lower the PVA content, the softer the hydrogel. This shows that the prepared hydrogel has a certain tensile strength, which can ensure that the material is not easily damaged in processing and use. When the PVA content is 2.5% or less, the hydrogel prepared is liable to be broken, probably because the gel formation is not facilitated by the small matrix content; the hydrogel prepared when the PVA content is 10% or more is too fast in curing and gelling speed in the preparation process, so that the system is inhomogeneous, and the hydrogel is too high in rigidity and not easy to be subjected to addition forming. Clearly, the softer the hydrogel, the easier it will conform to the skin and the less the skin wound will be squeezed and irritated during use.
In order to make the material better applied to the field of bioelectricity sensing, the invention prepares hydrogel with different aniline reaction amount (400 muL, 600 muL, 800 muL). When the reaction amount of aniline was 200. mu.L, the hydrogel had poor conductivity. When the reaction amount of aniline was increased to 1000. mu.L, the hydrogel formability was poor and the hydrogel was easily broken. The circuit is connected with a 1.5V power supply for supplying power, and the two leads are pressed on the surface of the prepared hydrogel by an insulating glass plate.
Referring to FIG. 3, FIG. 3 is a graph showing the conductivity of AgNPs/CPHs prepared by the present invention.
As shown in FIG. 3, AgNPs/CPHs (active volume of 600 μ L) is connected to the circuit to make the bulb emit light, and the bulb emits bright light when the polyaniline reaction content is 400-800 μ L. The hydrogel prepared by the method has good conductivity and can be used as a conductive medical dressing for skin attachment.
The dielectric constant detection result shows that when the aniline has different reaction contents (400 mu L, 600 mu L and 800 mu L), the dielectric constants obtained by using a dielectric impedance instrument are 1.46, 1.25 and 1.11 respectively.
As another host substrate for CPH, gelatin consists of a large number of triplex helices, usually dense in structure. Too large a reactive amount of gelatin may make the prepared gel more dense, resulting in low internal porosity, which is detrimental to the large loading and gradual release of Ag NCs.
Therefore, on the basis of determining the reaction amount of the main material PVA and the conductive material AN, the influence of the reaction amount of the water-absorbing material gelatin on the structure of the prepared CPH is detected.
Referring to fig. 4, fig. 4 is a SEM scanning electron micrograph of Ag NPs/CPH of different gelatin reaction contents prepared according to the present invention and a curve of the effect on storage modulus and loss modulus. Wherein, (a)0.5 wt%, (b)1 wt%, (c)1.5 wt%, (d)2 wt%, (e)2.5 wt%, and (f)3 wt%.
As can be seen from the internal morphology structure diagram of the hydrogel with different gelatin reaction amounts in FIG. 4, when the gelatin reaction amount is 0.5 wt% (FIG. 4-a), the interior of the prepared hydrogel has a loose and macroporous structure, which results in poor mechanical properties, low porosity and low specific surface area. As the reaction amount of gelatin gradually increased (1 wt% and 1.5 wt%, FIG. 4-b and FIG. 4-c), the inside of the hydrogel became dense, the pore diameter became smaller, and the specific surface area was higher. And at higher magnification (upper right corner of fig. 4-c) the coral-like, dendritic 3D network structure typical of polyaniline can be clearly seen. While the reaction amount of gelatin continued to increase (2 wt%, 2.5 wt%, 3 wt%, FIG. 4-d, FIG. 4-e and FIG. 4f), it can be seen that since the reaction amount of gelatin participated in was too high, the hydrogel was dense inside and no longer had a large number of pores for supporting Ag NCs and preserving fluid. Therefore, when a reaction amount of 1.5 wt% gelatin is used to prepare hydrogel, it provides optimum porosity, specific surface area and water absorption. And changes in storage modulus and loss modulus with gelatin concentration increasing from 0.5 wt% to 3 wt%.
The hydrogel prepared by the invention is subjected to state detection.
Referring to FIG. 5, FIG. 5 is a photograph showing a state detection of the hydrogel prepared according to the present invention. Wherein, B is Ag NPs/CPH solution which is not frozen and thawed, and is solid gel which does not flow at room temperature after aniline in-situ polymerization and freezing and thawing; C. pouring the mixed solution added with APS (ammonium persulfate) into moulds with different shapes before freezing, and taking out after freezing and thawing.
The invention adopts 5 Wt% of PVA reaction amount, 1.5 Wt% of gelatin reaction amount and 12 Wt% of aniline reaction amount, and the reaction amount is frozen and thawed (10 h). As can be seen in FIG. 5, the hydrogel solution transformed from a liquid phase to a stable solid gel, which was not flowable when tilted. And before the sol is frozen, the sol can be poured into molds with various shapes, and after freezing and thawing, the sol is taken out from the molds, so that the hydrogel CPHs with the required size and shape can be obtained.
The detection result shows that the hydrogel prepared by the invention has good elasticity, and can still maintain good mechanical property after the external force action disappears even if the hydrogel is stretched, compressed or sheared by certain external force. These excellent mechanical properties facilitate their storage, transportation or cutting for practical use.
And (3) carrying out swelling performance detection on the hydrogel material prepared by the invention.
The swelling properties of hydrogels play an important role in wound healing. An ideal inflammatory wound dressing should be able to absorb a large amount of inflammatory wound exudate and maintain a moist environment for the wound, accelerate granulation and angiogenesis, and promote autolysis of necrotic tissue. The five prepared groups of 10 CPH and Ag NPs/CPH samples were dried at 40 ℃ for 24 h.
Referring to FIG. 6, FIG. 6 is a graph showing five time-dependent (0-72 h) volume swelling curves for five sets of CPH and Ag NPs/CPH samples prepared according to the present invention. Wherein A is a volume swelling curve diagram; b is the statistical result curve of A.
FIG. 6 is a swelling curve of 10 samples examined under an excessive moisture environment, wherein the volume of the samples changes with time (0-72 h), and it can be seen from the swelling curve that the dried samples continuously and rapidly absorb moisture to swell within-6 h. And in 6-12 h, the water absorption rate of the sample is reduced and still has a relatively high water absorption speed. The swelling ratio of the hydrogel is basically unchanged for a long time (72h) after 12h, and the imbibed liquid can be stored in the internal pores for a long time, so that the hydrogel has good stability. And the water absorption expansibility of CPH and Ag NPs/CPH samples is not greatly different, namely the soaking load of the Ag NCs has low influence on the structure of the CPH.
Therefore, it can be concluded that when Ag NPs/CPH is used as a patch, the loaded Ag NCs exchange with wound exudate through concentration difference in aqueous solution, act on the wound surface, and effectively inhibit bacteria. The wound exudate is not easy to seep again after being absorbed into the gel, and causes secondary infection to the wound.
The hydrogel prepared by the invention is characterized by infrared spectrum and XPS.
The lyophilized sample of hydrogel was tested by infrared spectroscopy and found to be at 3422cm-1The position peak is derived from C-H, N-H and 2906cm contained in gelatin and aniline-1The peak-OH of (A) is from gelatin. 1648, 1567 belong to C ═ O and are derived from gelatin. 1490, 1427, 1292, 1079 represents-CH from PVA. 823cm-1The para-disubstituted benzene ring is from polyaniline.
Referring to FIG. 7, FIG. 7 is an XPS spectrum of Ag NPs/CPH prepared according to the present invention. Wherein C is XPS spectrum full scan spectrum of Ag NPs/CPH; d and E are high resolution XPS spectra of C1s and Ag 3D.
Fig. 7C is an XPS spectrum of the prepared hydrogel surface elements (C, N, O, Ag, B) characterized and analyzed using an X-ray photoelectron spectrometer (XPS). The binding energies of C1s were at 287.7, 285.3 and 284.7eV, showing that the samples contained functional groups of-COOH, C ═ O/C-N and C ═ C/C-C (fig. 7D). The binding energy of 373.2 and 367.1eV indicated that the sample contained Ag 3d, confirming that the nano-silver had been successfully loaded into the hydrogel (fig. 7E). As can be seen from the figure, the Ag peak obtained by XPS scanning is weaker, presumably due to the fact that the nano-silver particles in the sample are mainly present in the pores inside the hydrogel, and less attached to the hydrogel surface in the dry state.
Example 2
Bacteriostatic experiment of Ag NPs/CPH
Uniformly spreading diluted bacterial liquid of two bacteria common to infected wound, Escherichia coli (Abbrevian e-coli) and Staphylococcus aureus (Abbrevian s. aureus), on agar plate, and spreading the diluted bacterial liquid carrying 0, 0.1 x 10-3M/L(1Ag)，0.2ⅹ10-3M/L(2Ag)，0.3ⅹ10-3M/L(3Ag)，0.4ⅹ10-3400 μ L of hydrogel sheet of M/L (4Ag) silver nanoparticle concentration was placed on the plate and refrigerated at 4 ℃ for one hour. Then, the cells were placed upside down and cultured at 37 ℃ for 12 hours. And measuring the size of the inhibition zone.
Detection of nano silver particles in Ag NPs/CPH in body fluidThe antibacterial effect is achieved by taking 10mL of blank culture medium (denuded as lb) and diluted bacterial liquid (e-coli and S.aureus) as a control group, and setting the concentration of CPH and soaked silver nanoparticles to be 0.1 x 10-3M/L(denoted 1Ag)，0.2ⅹ10-3M/L(denoted 2Ag)，0.3ⅹ10-3M/L(denoted 3Ag)，0.4ⅹ10-3M/L (condensed 4Ag) 1200. mu.L of Ag NPs/CPH was added to the medium tube of the diluted bacteria solution at equal concentration, and OD values were measured at 1h, 2h, 3h, 4h, 5h, 6h, 12h, 24h and 48h, respectively.
And (3) performing a hydrogel flat antibacterial experiment and measuring the OD value of the hydrogel co-culture bacterial liquid, and detecting the antibacterial performance of the Ag NPs/CPH.
The prepared Ag NCs solution (0.1 x 10)-3M/L, denoted as 1Ag) are respectively prepared into 0.2 x 10 concentrated solutions of different Ag NCs by a rotary evaporation mode-3M/L(denoted as 2Ag)，0.3ⅹ10-3M/L (dented as 3Ag) and 0.4 x 10-3M/L (condensed as 4Ag), and compounding the two CPHs respectively.
Referring to FIG. 8, FIG. 8 is a photograph of an experiment on hydrogel plates against e-coli and S.aureus and a corresponding diameter calculation chart. Wherein A is S.aureus, B is e-coli, i represents group 1Ag, ii represents group 2Ag, iii represents group 3Ag, iv represents group 4 Ag; (C) and (D) is a calculated diameter showing the experimental inhibition zone of the corresponding antimicrobial plate.
As shown in FIG. 8, Ag NPs/CPH soaked in silver nanoparticle aqueous solutions with different concentrations can release bacteriostatic Ag NCs, and effectively inhibit the propagation of bacteria e-coli and S. The size of the zone of inhibition on the plate increases with increasing concentration of the soaked Ag NCs solution. In addition, the CPH which is not soaked in the Ag NCs solution also has a certain inhibition zone, which is possibly endowed by the antibacterial property of polyaniline, but the area of the inhibition zone is obviously smaller than that of the Ag NPs/CPH.
Referring to FIG. 9, FIG. 9 is a liquid OD of co-culture of lb medium, CPH and Ag NPs/CPHAg NCs loaded with different Ag Nps with e-coli600The measured data of the values are shown in the graph (0-48 h).
Referring to FIG. 10, FIG. 10 is a liquid OD obtained by co-culturing lb medium, CPH and Ag NPs/CPHAg NCs loaded with different Ag NCs with S.aureus600Number of measurements of valueAccording to the graph (0-48 h).
FIG. 9(for e-coli) and FIG. 10(for S.aureus) are graphs showing the measurement of OD values of the coculture liquid. In FIG. 9, the black line (the last line from top to bottom) is used as lb medium of the control group, and the OD value thereof hardly changes with time. The red line (first line from top to bottom) is the OD measured for the diluted e-coli solution, respectively. And (3) rapidly increasing the OD value of the e-coli solution within 6-10 h, and keeping the log phase of bacterial growth. And (5) slowing the rise speed of the OD value for 10-24 h, and keeping the OD value in a stable period of bacterial growth. After 24h, the OD was substantially unchanged and was in the late phase of bacterial growth.
The gray line (second line from top to bottom) is a plot of OD data measured for CPH and e-coli cultured under the same conditions. The four different blue lines (the third line to the sixth line from top to bottom) are respectively an OD data graph of the Ag NPs/CPHAg NCs which are measured by the culture with the e-coli under the same condition after being soaked in different Ag NCs solutions. As shown in the figure, the OD value of the bacterial liquid decreased with the increase in the Ag NCs loading amount. Compared with e-col, the prepared Ag NCs load system has obvious bacteriostatic effect. Consistent with the results of fig. 8. The results of fig. 10 are substantially identical to those of fig. 9.
The experimental results show that the silver nanoparticle-loaded hydrogel has a good inhibition effect on common Escherichia coli and staphylococcus aureus infected by wounds.
Cytotoxicity assay of Ag NPs/CPH
A5 g aliquot of Ag NPs/CPH was placed in a centrifuge tube containing 30mL of PBS. The incubator was immersed at 37 ℃ for 24 h. After the Ag NPs/CPH is taken out, the concentration of the soaking solution is measured and diluted to obtain a series of Ag NPs/CPH soaking solutions with gradient concentrations (10 mu g/mL, 20 mu g/mL, 30 mu g/mL, 50 mu g/mL, 60 mu g/mL, 80 mu g/mL, 100 mu g/mL, 150 mu g/mL). By adopting a CCK-8 method, the toxic effect of the antibacterial hydrogel soaking solution on HaCat cells is investigated under the conditions of different concentrations.
HaCat cells are human immortalized keratinocytes which undergo self-transformation, have biological properties similar to those of normal keratinocytes, and are therefore suitable for various cell studies for examining skin toxicity.
And (3) detecting the influence of the Ag NPs/CPH on human skin cells, and performing toxicity experiments on the soak solution of the Ag NPs/CPH by adopting a CCK-8 method aiming at HaCat cells, LO2 cells and 293T cells.
Referring to FIG. 11, FIG. 11 shows the cell viability of HaCat cells measured by CCK8 after incubation in PBS and different concentrations of hydrogel soaking solution for 48 h.
Referring to fig. 12, fig. 12 shows the cell viability of LO2 cells measured by CCK8 after culturing in PBS and different concentrations of hydrogel soaking solution for 48 h.
Referring to FIG. 13, FIG. 13 shows the cell viability of 293T cells measured by CCK8 after incubation in PBS and different concentrations of hydrogel soaking solution for 48 h.
As shown in FIG. 11, HaCat cells showed substantially no inhibitory effect and even a tendency to proliferate at 48h after coculture with low concentration hydrogel soak (10. mu.g/mL, 20. mu.g/mL, 30. mu.g/mL) compared to the control (PBS, hydrogel soak concentration 0. mu.g/mL). This indicates that for normal wounds, the Ag NPs/CPH patch is used in a small amount, is substantially non-toxic to skin cells, and may promote skin cell growth and promote skin healing in small amounts. After co-culture with medium concentration hydrogel soak (50. mu.g/mL, 60. mu.g/mL, 80. mu.g/mL, 100. mu.g/mL), cells were slightly inhibited (< 10%); after co-culture with high concentration hydrogel soak (150 μ g/mL), cells were inhibited to a lesser extent (< 20%) and were very cytotoxic. It is demonstrated that the application of Ag NPs/CPH in large quantities to severe wounds (e.g., inflammatory suppuration) hardly adversely affects wound healing.
To further examine the in vivo safety of Ag NPs/CPH, CCK-8 experiments were performed on LO2 and 293T cells at different concentrations (5, 10, 15, 20, 30, 50, 100, 150 and 200. mu.g/mL). The results are shown in FIGS. 12 and 13, where each concentration of Ag NPs/CPH soak did not significantly inhibit LO2 and 293T cells, and there was even slight proliferation.
Therefore, the Ag NPs/CPH hydrogel prepared by the method has good antibacterial property and cell safety, and can be used as a promising medical wound dressing.
Animal infection wound healing experiments
Selecting male ICR mice to be grouped into gauze group, CPH group and Ag NPs/CPH group, carrying out wound modeling on the backs of the mice, measuring the sizes of wounds, and then coating staphylococcus aureus bacterial liquid with equal concentration on the wounds of each mouse to cause wound infection. After eating normally for 48 hours, red swelling of the wound with a small amount of exudate around it was observed, and then applied as a group. Wound area was measured and photographed on a time basis of 1d, 3d, 4d, 7d and 14 d. Mice were sacrificed and skin tissue near the wound was stored in paraformaldehyde and stored in a refrigerator at 4 ℃. After 14 days, the tissue sections were uniformly embedded and stained, and the inflammation and recovery of the tissue around the wound were observed under a 100 x microscope.
Referring to FIG. 14, FIG. 14 is a photograph of wounds from Ag NPs/CPH, CPH and PBS treated mice at days 1, 3, 7 and 14.
Referring to fig. 15, fig. 15 is a graph of statistical calculations of the wound remaining area over time for each group of mice corresponding to fig. 14.
The effect of the Ag NPs/CPH dressing on the infected wound of the mouse is shown in FIG. 14, the gauze dressing is a Control group, and the CPH group and the Ag NPs/CPH dressing group are used as experimental groups. On the first day, the wounds (diameter-1 cm) of three groups of mice are clear in edge, no blood seepage and liquid seepage exist, and no obvious infection sign exists in a short time after S.aureus liquid is uniformly smeared; in the experiment of the next day, the wounds of the three groups of mice are infected and suppurated, have obvious peculiar smell and are determined to be established as a severe infection model. Respectively giving corresponding dressing (diameter-1.2 cm) to each group of mice to treat wounds; in the seventh day of the experiment, gauze pieces adhered (Figure S) and thick scabs were present near the wound in the gauze group mice, and the mice experienced pain when changing dressings. The wounds of the mice in the CPH group and the Ag NPs/CPH group are better recovered, the drug change is not bonded, the struggle of the mice is less, and the secondary injury to the wounds of the mice is not caused. The residual area of the wound Ag NPs/CPH group is minimum; on the fourteenth day, the gauze group mice still had large residual wound area, whereas the CPH group and Ag NPs/CPH group mice had wounds that were barely visible under the neonatal hair mask. After shaving, wounds with better recovery and smaller residual area were visible. Among them, the Ag NPs/CPH group had the smallest wound residual area. The Ag NPs/CPH has obvious advantages of bacteriostasis, infection resistance and healing promotion when being used for treating severely infected wounds compared with the traditional dressing (such as gauze).
FIG. 15 is a statistical calculation chart of the change in the remaining wound area over time for the respective groups of mice. The wound shape is slightly changed on the next day due to normal infection and contraction of the wound and the influence of pulling of dressing change, but the area is still more than 90 percent; the wound area of the mice of each group is obviously different from the seventh day of the experiment, the wound area of the control group is still more than 60 percent, and the wound area of the CPH group is slightly less than 60 percent. The area of the wound of the Ag NPs/CPH group is close to 40 percent, and the wound recovery effect is obvious and best; from day 14 onwards, the wound area of the control group was still close to 40%. The CPH group (wound area-20%) and Ag NPs/CPH group mice had close wound healing. The Ag NPs/CPH group wound has the best recovery effect, and the wound area is close to 10%. Overall, the Ag NPs/CPH group healed best, and the CPH group was also better than the control, consistent with the results in fig. 12.
Referring to FIG. 16, FIG. 16 is a H & E stained section of skin tissue near the wound of gauze (control), CPH and Ag NPs/CPH treated mice and corresponding counts of inflammatory cells on different days (scale: 50 μm). Wherein, after (A) 2, 7 and 14d (scale: 50 μm), H & E staining of skin tissue was performed near the wounds of mice treated with gauze (control group), CPH and Ag NPs/CPH. (B) Corresponding counts of inflammatory cells.
FIG. 16 is a H & E stained section of mouse skin tissue near a wound observed under a microscope at 2d, 7d, and 14 d. As can be seen, when a severe infection wound model of three groups of mice is established on the third day, the inflammatory infiltration level of the three groups of mice is high. After different dressing treatments, on the seventh day, the number of inflammatory cells in the Control group in the stained section was still large, and the infiltration levels of inflammatory cells in the CPH group and the Ag NPs/CPH group were smaller than those in the Control group. Both the CPH group and the Ag NPs/CPH group suppressed the development of inflammation. But the CPH group still has a certain amount of inflammatory infiltration, the infiltration level of inflammatory cells in the Ag NPs/CPH group is the lowest, and the wound inflammation is better controlled; on day fourteen, the inflammatory phase in three groups of mice was over and the stained sections showed recovery of all wounds. The Ag NPs/CPH group had the highest numbers of hair follicle tissues and fibroblasts. This is probably due to the moist environment provided by the bacteriostatic hydrogel dressing which promotes wound healing at levels exceeding those of the control and CPH groups.
Referring to FIG. 17, FIG. 17 is a CD31 stained section of skin tissue near the wound of gauze (control), CPH and Ag NPs/CPH treated mice and corresponding blood vessel counts in the skin tissue on different days (scale: 50 μm). Wherein (C) is gauze (control), CD31 staining of 2, 7 and 14d of skin tissue near the wound of mice after CPH and Ag NPs/CPH treatment; (D) corresponding blood vessel counts in the skin tissue.
Referring to FIG. 18, FIG. 18 is a graph of the Munsen stained sections near the wound and the corresponding percentage of collagen deposition for gauze (control), CPH and Ag NPs/CPH treated mice on different days (scale: 50 μm). Wherein (E) is the Masson staining in the vicinity of the mouse wound after 2, 7 and 14d treatment with gauze (control), CPH and Ag NPs/CPH and (F) is the corresponding percentage of collagen deposition in the graph.
The above detailed description of the present invention provides a medical conductive antibacterial composite hydrogel and its preparation method and application, and the principle and embodiments of the present invention are explained in the present application by using specific examples, and the above description of the examples is only for helping to understand the method of the present invention and its core idea, including the best mode, and also for enabling anyone skilled in the art to practice the present invention, including making and using any device or system, and implementing any method in combination. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The scope of the invention is defined by the claims and may include other embodiments that occur to those skilled in the art. Such other embodiments are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.