CN118286487A - Preparation method of multifunctional nanofiber membrane for promoting healing of complex infected wound - Google Patents
Preparation method of multifunctional nanofiber membrane for promoting healing of complex infected wound Download PDFInfo
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- CN118286487A CN118286487A CN202410386789.2A CN202410386789A CN118286487A CN 118286487 A CN118286487 A CN 118286487A CN 202410386789 A CN202410386789 A CN 202410386789A CN 118286487 A CN118286487 A CN 118286487A
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- nanofiber membrane
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- enzyme
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Abstract
The invention discloses a preparation method of a multifunctional nanofiber membrane for promoting healing of complex infected wounds, which belongs to the technical field of functional medical dressings, and the preparation method comprises the steps of taking ZIF-8 nanocrystals as precursors, etching and carbonizing to obtain hollow nano-enzyme HZC with good photo-thermal conversion capability, POD-like enzyme catalytic activity and drug carrying capability, loading berberine hydrochloride to obtain drug carrying carbon nano-enzyme HZCB, and preparing the multifunctional nanofiber membrane by an electrostatic manner comprising HZCB.
Description
Technical Field
The invention belongs to the technical field of functional medical dressings, and particularly relates to a preparation method of a multifunctional nanofiber membrane for promoting healing of complex infected wounds.
Background
Bacterial infection is one of the diseases that severely threatens public health, and has attracted considerable attention worldwide for recent decades. The limited antimicrobial capabilities and other drawbacks of conventional dressings have prevented their effective treatment of complex infected wounds. Electrospun fiber membranes are becoming increasingly popular due to their three-dimensional topology like extracellular matrix and large specific surface area for further biofunctionalization. The flexible preparation method can easily realize the loading or doping of various antibacterial materials, so that the antibacterial material becomes an ideal substrate for developing advanced wound dressing. Generally, the antimicrobial function of the fibers is achieved by introducing antibiotics during processing. In general, infected wounds always contain a variety of bacteria, the two most common of which are methicillin-resistant staphylococcus aureus (MRSA) and Pseudomonas Aeruginosa (PA). It is well known that they readily form biofilms and are thus insensitive to various antibiotics, reducing the efficacy of the antibiotics and thus preventing wound healing. In addition, cross infection of multiple bacteria can lead to bacterial quorum sensing, which in turn can lead to strong resistance to antibiotics. The risk of this type of resistant bacteria greatly limits the development of antibiotics. The long term abuse of antibiotics has also led to the evolution, increase and spread of drug resistant pathogens at a surprising rate. Thus, there is an urgent need to develop new antibacterial strategies and effective drugs to more effectively and safely combat complex bacterial infections.
At present, natural active ingredients of plant sources are widely studied in dermatological preparations due to their abundant biological activity and rare adverse reactions. Berberine hydrochloride is the hydrochloride of isoquinoline alkaloid and exists in various medicinal plants. A great deal of researches show that the berberine hydrochloride has a certain antibacterial effect on different bacteria. Berberine hydrochloride can interfere with the formation of a biofilm by inhibiting the formation of extracellular amyloid peptides in bacteria; disruption of bacterial biofilm integrity and permeability can also be achieved by binding to some proteins on the biofilm, disrupting the protein structure. In view of its excellent antibacterial activity, berberine hydrochloride can be an ideal substitute for antibiotics. However, its limited bioavailability and low water solubility limit its medical use. Thus, there is an urgent need to design new drug delivery strategies to meet their development needs for wound dressings.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of a multifunctional nanofiber membrane for promoting healing of complex infection wounds, and the invention constructs a near-infrared triggered three-mode synergistic antibacterial fiber membrane (PVA/HZCB) so as to effectively eliminate complex infection induced by MRSA and PA. The hollow carbon nano enzyme HZCB carrying berberine hydrochloride is prepared by taking ZIF-8 nanocubes as precursors and carrying out etching, pyrolysis and drug loading treatment. Compared with the solid nano structure, the multi-reflection light path in the inner cavity of the hollow structure is beneficial to improving the collection efficiency of the near infrared laser, and more reaction sites and contact interfaces can be exposed, so that the catalytic activity of the near infrared laser is improved. Under low intensity 808nm near infrared laser (0.3W/cm 2) irradiation, the PVA/HZCB fiber film can reach satisfactory PTT temperature. At the temperature, the POD-like enzyme activity and drug release are enhanced, a PTT-CDT-plant drug synergistic antibacterial system is formed, and the effect of 'one arrow three carving' is realized. The PCB nanofiber membrane has good biocompatibility, can effectively kill MRSA and PA in vitro and in vivo, reduces inflammation, and promotes healing of complex infected wounds.
In order to achieve the above purpose, the present invention provides the following technical solutions:
One of the technical schemes of the invention is as follows:
A method for preparing a multifunctional nanofiber membrane for promoting healing of complex infected wounds, which comprises the following steps:
a. dispersing ZIF-8 in absolute ethanol, adding tannic acid solution, stirring uniformly, centrifuging, and drying to obtain HZIF-8;
b. Calcining HZIF-8, and cooling to obtain hollow carbon nano-enzyme HZC;
c. Dispersing the hollow carbon nano enzyme HZC in methanol, adding berberine hydrochloride, stirring uniformly, centrifuging, and drying to obtain drug-loaded carbon nano enzyme HZCB;
d. adding the drug-loaded carbon nano-enzyme HZCB into polyvinyl alcohol to prepare spinning solution, carrying out electrostatic spinning and drying to obtain the multifunctional nanofiber membrane for promoting healing of complex infected wounds.
The invention synthesizes a multifunctional nanofiber membrane for promoting healing of complex infected wounds, the hollow carbon nano-enzyme HZC can wrap guest molecules, and the porous carbon material hollow carbon nano-enzyme HZC derived from a Metal Organic Framework (MOF) has the advantages of high specific surface area, high capacity, high encapsulation and good biocompatibility, and has application potential in biomedical application, drug delivery and treatment. Due to its surface groups and unique electronic structure, MOF-derived porous carbon material hollow carbon nanoenzymes HZC have a variety of enzymatic activities that can enable chemical kinetic therapy (CDT). In addition, the hollow carbon nano enzyme HZC has strong light absorption in a near infrared region, is favorable for realizing near infrared light irradiation induced photo-thermal therapy (PTT), can be designed into an intelligent medicine carrying system triggered by near infrared laser, and improves the medicine delivery efficiency. According to the invention, berberine hydrochloride is loaded on MOF-derived hollow carbon nano enzyme HZC, and a PTT-CDT-plant drug three-mode synergistic antibacterial platform is constructed, so that the limited effect of a single antibacterial strategy or side effects and metabolism burden possibly brought by large-dose use are overcome, and the three-mode synergistic antibacterial platform is hardly reported in the treatment of complex wound infection.
Further, the preparation method of ZIF-8 comprises the following steps: dissolving 2-methylimidazole in 100mL of deionized water, adding 4mL of CTAB (cetyltrimethylammonium bromide) solution, dissolving zinc nitrate hexahydrate in 100mL of deionized water, slowly adding the solution into the solution, stirring to obtain a milky solution, and centrifugally drying the milky solution to obtain ZIF-8.
Further, in the preparation method of ZIF-8, the addition amount of 2-methylimidazole is 8.8-10.8g, the concentration of CTAB solution is 0.005-0.01M, and the addition amount of zinc nitrate hexahydrate is 0.5-0.7g.
Further, in the preparation method of ZIF-8, the stirring time is 1-3h, and the stirring speed is 600-800r/min;
The centrifugation times are 1-3 times, the drying temperature is 40-60 ℃, and the drying time is 6-8 hours.
Further, in the step a, ZIF-8 is added in an amount of 80-100mg and the concentration of the tannic acid solution is 10-20g/L.
Further, in the step a, the stirring time is 1-5min, and the stirring rotating speed is 600-800r/min;
The centrifugation times are 1-3 times, the drying temperature is 40-60 ℃, and the drying time is 6-8 hours.
Further, in the step b, the calcining temperature of HZIF-8 is 800-1000 ℃ and the heat preservation time is 1-3h.
Further, in the step c, the mass ratio of the hollow carbon nano enzyme HZC to the berberine hydrochloride is (1:2) - (1:4).
Further, in the step d, the adding amount of the drug-loaded carbon nano-enzyme HZCB in the spinning solution is 150-250 mug/mL, and the adding amount of the polyvinyl alcohol in the spinning solution is 60-80mg/mL.
Further, in the step d, during electrostatic spinning, the voltage is 13-15KV, the distance between a receiver and a nozzle is 10-15cm, and the injection speed of the spinning solution is 0.2-0.4mL/h.
Further, in the step d, the drying temperature is 40-60 ℃ and the drying time is 6-8h.
The second technical scheme of the invention is as follows:
The multifunctional nanofiber membrane for promoting healing of complex infection wounds is prepared by the preparation method.
The third technical scheme of the invention:
The multifunctional nanofiber membrane is applied to the preparation of medicines for promoting healing of complex infected wounds.
Compared with the prior art, the invention has the following advantages and technical effects:
(1) According to the invention, ZIF-8 nanocrystals are used as precursors, and hollow nano-enzyme HZC with good photo-thermal conversion capability, POD-like enzyme catalytic activity and drug carrying capability is obtained after etching and carbonization, and then berberine hydrochloride is carried to obtain the drug carrying carbon nano-enzyme HZCB. In addition, in order to achieve the aim of local application to wounds, PVA with good biocompatibility and high safety is used as a carrier, HZCB with a therapeutic effect is wrapped in the PVA, PVA/HZCB fibers are obtained through electrostatic spinning, and the therapeutic system provides a comfortable microenvironment for cell proliferation, angiogenesis, granulation tissue formation and epithelial regeneration.
(2) The preparation process of the multifunctional nanofiber membrane has the advantages of simplicity, low cost and short process period. The relative specific surface area of the hollow carbon-based nano enzyme HZC in the PVA/HZCB nanofiber component is remarkably increased compared with that of the precursor HZIF-8, the pore volume and the specific surface area of the HZC are respectively 0.650cm 3/g and 529.9m 2/g, and the pore volume and the specific surface area of uncarbonized HZIF-8 are only 0.246cm 3/g and 101.9m 2/g. By virtue of a larger specific surface area and an ordered mesoporous structure, the HZC can provide more active sites, is favorable for being combined with a substrate more effectively, improves the catalytic activity of enzyme, can effectively load berberine hydrochloride, and has the load rate of 42.3+/-2.2%.
(3) The multifunctional nanofiber membrane prepared by the invention has good photo-thermal performance, and can exert photo-thermal effect only by low-power laser irradiation (0.3W/cm 2), so that skin is not easy to burn, energy is saved, and meanwhile, the inflammatory damage to skin during high-temperature treatment in the traditional photo-thermal therapy is avoided. Meanwhile, the low-temperature heat effect achieved under the irradiation of low-intensity NIR enhances the catalytic activity of POD nano-enzyme and the release of berberine hydrochloride, improves the killing ability to bacteria, and provides synergistic effect. The combination of these different antibacterial mechanisms overcomes the metabolic burden of the single mode high dose of the PCB nanofibers and realizes the rapid and efficient sterilization at low dose (HZCB is preferably added in the spinning solution at 250 mug/mL).
(4) The multifunctional nanofiber membrane prepared by the invention has good biocompatibility and high safety. The in vitro hemolysis experiment and the cell living and dying staining experiment prove that the dye has good biocompatibility and can not cause damage to the cell activity or the skin of the mice.
(5) The multifunctional nanofiber membrane prepared by the invention can promote angiogenesis, collagen deposition and granulation tissue formation, effectively treat complex wound infection caused by various bacteria, and accelerate the wound healing process.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
FIG. 1 is a representation of a hollow carbon nanoenzyme HZC, a hollow drug-loaded carbon nanoenzyme HZCB and berberine hydrochloride in example 1, wherein A is an SEM image of the drug-loaded carbon nanoenzyme HZCB, B is a particle size distribution diagram of the drug-loaded carbon nanoenzyme HZCB, C is an N 2 adsorption-desorption isotherm of the hollow carbon nanoenzyme HZC and HZIF-8, D is pore volume of the hollow carbon nanoenzyme HZC and HZIF-8, and E is an FT-IR spectrum image of the hollow drug-loaded carbon nanoenzyme HZCB and berberine hydrochloride;
FIG. 2 is a representation of PVA/HZCB fibers from example 1, wherein A is an SEM image and fiber diameter profile of the PVA/HZCB fibers, B is a time-temperature profile of the PVA/HZCB fibers under low intensity NIR illumination of 0.3W/cm 2, and the control is a blank nanofiber;
FIG. 3 is a photo-thermal enhancement POD enzyme activity characterization of the nanomaterial in example 1, wherein A is the absorption spectrum of HZCB and PVA/HZCB nanofibers at the same HZC concentration, and B is the absorption spectrum of the reaction system with or without NIR laser irradiation and a digital picture;
FIG. 4 is a graph showing the light responsive drug release characteristics of PVA/HZCB fibers of example 1;
FIG. 5 is an in vitro antimicrobial property of PVA/HZCB fibers of example 1, wherein A is a digital photograph of colony genus of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa after different groups of treatments, and B is a corresponding bacteriostatic rate;
FIG. 6 is a graph showing the in vitro safety test results of PVA/HZCB fibers in example 1, wherein A is the cell viability of HaCaT cells of the PVA/HZCB fiber group and the control group without any treatment, B is the fluorescent live dead staining pattern of the PVA/HZCB fiber group and the control group with no treatment, and C is the hemolysis rate of the PVA/HZCB fibers;
FIG. 7 is a graph showing the results of the PVA/HZCB in vivo complex infection wound healing and biosafety test in example 1, wherein A is a digital photograph of the complex infection wound healing of mice in the PVA/HZCB fiber group and the control group without any treatment, B is the wound healing rate of the PVA/HZCB fiber group and the control group without any treatment on days 4, 7 and 10, and C is the colony count of wound tissue in the PVA/HZCB fiber group and the control group without any treatment;
FIG. 8A is the histological features of PVA/HZCB fiber wound healing in example 1, B is the inflammatory level of PVA/HZCB fiber wound healing in example 1, and C is the expression level of the healing factor of PVA/HZCB fiber wound healing in example 1.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The room temperature in the examples of the present invention was 25.+ -. 2 ℃ unless otherwise specified.
The raw materials used in the examples of the present invention are all commercially available.
The technical scheme of the invention is further described by the following examples.
Example 1
A. 10.8g of 2-methylimidazole is dissolved in 100mL of deionized water, and 4mL of a 0.01M CTAB solution is added to obtain a mixed solution; dissolving 0.7g of zinc nitrate hexahydrate in 100mL of deionized water, slowly adding the solution into the solution, stirring the solution at a speed of 800r/min for 3 hours to obtain a milky white solution, centrifuging the milky white solution for 3 times, and drying the milky white solution at 60 ℃ for 8 hours to obtain ZIF-8 powder;
b. C, ultrasonically dispersing 100mg of ZIF-8 powder obtained in the step a in 25mL of absolute ethyl alcohol, then adding 25mL of 10g/L tannic acid solution, stirring at a speed of 800r/min for 5min to obtain a mixed solution, centrifuging the mixed solution for 3 times, and drying at 60 ℃ for 8h to obtain hollow nanocrystals HZIF-8;
c. Placing HZIF-8 obtained in the step b into a tube furnace, heating to 800 ℃ in argon at a heating rate of 5 ℃/min, preserving heat for 3 hours, and naturally cooling to room temperature to obtain ZIF-8 derived empty drug carbon nano-enzyme HZC;
d. Dispersing 10mg of HZC obtained in the step c in 10mL of methanol by ultrasonic, adding 40mg of berberine hydrochloride, stirring for 24 hours, centrifuging for 3 times, and drying at 60 ℃ for 8 hours to obtain drug-loaded carbon nano-enzyme HZCB;
e. 8g of polyvinyl alcohol (PVA) is dissolved in 100mL of deionized water to obtain 8wt% PVA spinning solution; adding 25mg HZCB into the PVA spinning solution, and uniformly stirring to obtain a spinning solution of PVA/HZCB fibers; carrying out electrostatic spinning on the spinning solution of the PVA/HZCB fiber, wherein the parameters of the electrostatic spinning are as follows: the high-voltage power supply is set to 15KV, the distance between the nozzle and the receiver is 15cm, and the solution propelling speed is 0.4mL/h. After the setting is finished, the electrostatic spinning process is started, after the electrostatic spinning, the product is dried at 60 ℃ for 8 hours, and finally PVA/HZCB fiber is obtained, namely the multifunctional nanofiber membrane for promoting the healing of complex infection wounds.
Example 2
A. 9.8g of 2-methylimidazole was dissolved in 100mL of deionized water, and 4mL of a 0.0075M CTAB solution was added to obtain a mixed solution; dissolving 0.6g of zinc nitrate hexahydrate in 100mL of deionized water, slowly adding the solution into the solution, stirring the solution at a speed of 700r/min for 2 hours to obtain a milky white solution, centrifuging the milky white solution for 2 times, and drying the milky white solution at 50 ℃ for 7 hours to obtain ZIF-8 powder;
b. Dispersing 90mg ZIF-8 powder obtained in the step a in 25mL of absolute ethyl alcohol by ultrasonic, then adding 25mL of 15g/L tannic acid solution, stirring at the speed of 700r/min for 2.5min to obtain a mixed solution, centrifuging the mixed solution for 2 times, and drying at 50 ℃ for 7h to obtain hollow nanocrystals HZIF-8;
c. Placing HZIF-8 obtained in the step b into a tube furnace, heating to 900 ℃ in argon at a heating rate of 5 ℃/min, preserving heat for 2 hours, and naturally cooling to room temperature to obtain ZIF-8 derived empty drug carbon nano-enzyme HZC;
d. C, dispersing 10mg of HZC obtained in the step c in 10mL of methanol by ultrasonic, adding 30mg of berberine hydrochloride, stirring for 24 hours, centrifuging for 3 times, and drying at 50 ℃ for 7 hours to obtain drug-loaded carbon nano-enzyme HZCB;
e. 7g of PVA was dissolved in 100mL of deionized water to give a 7wt% PVA spinning solution; adding 20mg HZCB into the PVA spinning solution, and uniformly stirring to obtain a spinning solution of PVA/HZCB fibers; carrying out electrostatic spinning on the spinning solution of the PVA/HZCB fiber, wherein the parameters of the electrostatic spinning are as follows: the high-voltage power supply is set to be 14KV, the distance between the nozzle and the receiver is 12.5cm, and the solution advancing speed is 0.3mL/h. After the setting is finished, the electrostatic spinning process is started, after the electrostatic spinning, the product is dried for 7 hours at 50 ℃, and finally PVA/HZCB fiber is obtained, namely the multifunctional nanofiber membrane for promoting the healing of complex infection wounds.
Example 3
A. 8.8g of 2-methylimidazole was dissolved in 100mL of deionized water, and 4mL of a 0.005M CTAB solution was added to obtain a solution; dissolving 0.5g of zinc nitrate hexahydrate in 100mL of deionized water, slowly adding the solution into the solution, stirring the solution at a speed of 600r/min for 1h to obtain a milky white solution, centrifuging the milky white solution for 1 time, and drying the milky white solution at 40 ℃ for 6h to obtain ZIF-8 powder;
b. Dispersing 80mg ZIF-8 powder obtained in the step a in 25mL of absolute ethyl alcohol by ultrasonic, then adding 25mL of 20g/L tannic acid solution, stirring at 600r/min for 1min to obtain a mixed solution, centrifuging the mixed solution for 1 time, and drying at 40 ℃ for 6h to obtain hollow nanocrystals HZIF-8;
c. Placing HZIF-8 obtained in the step b into a tube furnace, heating to 1000 ℃ in argon at a heating rate of 5 ℃/min, preserving heat for 1h, and naturally cooling to room temperature to obtain ZIF-8 derived empty drug carbon nano-enzyme HZC;
d. dispersing 10mg of HZC obtained in the step c in 10mL of methanol by ultrasonic, adding 20mg of berberine hydrochloride, stirring for 24h, centrifuging for 1 time, and drying at 40 ℃ for 6h to obtain drug-loaded carbon nano-enzyme HZCB;
e. 6g of PVA was dissolved in 100mL of deionized water to give a 6wt% PVA spinning solution; adding 15mg HZCB into the PVA spinning solution, and uniformly stirring to obtain a spinning solution of PVA/HZCB fibers; carrying out electrostatic spinning on the spinning solution of the PVA/HZCB fiber, wherein the parameters of the electrostatic spinning are as follows: the high-voltage power supply is set to 13KV, the distance between the nozzle and the receiver is 10cm, and the solution advancing speed is 0.2mL/h. After the setting is finished, the electrostatic spinning process is started, after the electrostatic spinning, the product is dried for 6 hours at 40 ℃, and finally PVA/HZCB fiber is obtained, namely the multifunctional nanofiber membrane for promoting the healing of complex infection wounds.
Taking example 1 as an example, a test was performed:
FIG. 1 is a representation of hollow carbon nanoenzyme HZC, hollow drug-loaded carbon nanoenzyme HZCB and berberine hydrochloride in example 1, wherein A is an SEM image of drug-loaded carbon nanoenzyme HZCB, B is a particle size distribution diagram of drug-loaded carbon nanoenzyme HZCB, C is an N 2 adsorption-desorption isotherm of hollow carbon nanoenzymes HZC and HZIF-8, D is pore volume of hollow carbon nanoenzymes HZC and HZIF-8, and E is an FT-IR spectrum image of hollow drug-loaded carbon nanoenzyme HZCB and berberine hydrochloride. As shown in fig. 1a and B, the apparent hollow internal structure of HZCB, which helps to better carry the drug, is clearly seen from SEM images, HZCB having a diameter around 100 nm. As shown in FIGS. 1C and D, the HZC after carbonization has a significant difference in pore volume and specific surface area compared to HZIF-8 before carbonization. The pore volume and specific surface area of HZC were 0.650cm 3/g and 529.9m 2/g, respectively, whereas the pore volume and specific surface area of uncarbonized HZIF-8 were only 0.246cm 3/g and 101.9m 2/g. HZC can provide more active sites by virtue of a larger specific surface area and an ordered mesoporous structure, is favorable for more effectively combining with a substrate, improves the catalytic activity of enzyme, and provides a foundation for drug loading. The successful loading of BBH on HZCB was also confirmed by the clear absorption peak at 345nm in the FT-IR spectrum as shown in FIG. 1E. According to the standard curve, the loading of BBH on HZCB is calculated to be 42.3+/-2.2%.
FIG. 2 is a characterization of PVA/HZCB fibers in example 1, where A is the SEM image and diameter profile of the PVA/HZCB fibers, B is the photo-thermal properties of the PVA/HZCB fibers, and the control is blank nanofibers. As shown in FIG. 2A, the PVA/HZCB fibers all have a good network porous structure, the fiber diameter is about 195nm, and the distribution is uniform. This indicates that HZCB addition did not affect the morphology of the nanofibers. Under near infrared laser irradiation, the photo-thermal properties of PVA/HZCB fibers were evaluated. As shown in FIG. 2B, the blank nanofibers did not show a significant temperature change under 0.3W/cm 2 NIR laser irradiation, whereas the PVA/HZCB fiber temperature reached 45.8℃in 5 minutes.
FIG. 3 is a photo-thermal enhancement POD enzyme activity characterization of the nanomaterial in example 1, wherein A is the absorption spectrum of HZCB and PVA/HZCB nanofibers at the same HZC concentration, and B is the absorption spectrum of the reaction system with or without NIR laser irradiation and digital pictures. The POD-like enzyme catalytic properties of HZC, HZCB and PCB nanofiber membranes were determined in PBS (ph=4.0, 10 mM) according to the chromogenic reaction for oxidizing TMB with the aid of H 2O2, all reactions were performed at 37 ℃, the reaction time was 5min, and the uv-visible absorbance spectra were recorded. Wherein, the concentration of H 2O2 is 25mM, the concentration of TMB is 1mM, the concentration of HZC is 40. Mu.g/mL, and the total reaction system is 400. Mu.L. For peroxidase-like activity under NIR laser irradiation, the reaction system was irradiated with NIR laser under the same conditions for 5min and ultraviolet absorption spectrum was recorded. As can be seen from fig. 3a, the catalytic activity of HZCB loaded with the drug was similar to that of the unloaded drug at the same HZC content, indicating that the drug loading did not affect the catalytic activity. Furthermore, at the same HZC content, the PVA/HZCB fibers also showed comparable catalytic activity, indicating that the PVA/HZCB fibers can be used as an effective peroxidase mimic. The intensity of the absorption peak at 652nm under NIR radiation was higher than that of the unirradiated system (B in FIG. 3), indicating that the catalytic activity of HZC can be enhanced by photothermal means. This phenomenon may be due to the increased temperature of the reaction system by the NIR laser irradiation.
FIG. 4 shows the light responsive drug release characteristics of PVA/HZCB fibers of example 1. As shown in FIG. 4, the PVA/HZCB fiber reached 77.7.+ -. 1.6% cumulative drug release after 3 replicates of 0.3W/cm 2 NIR laser irradiation, significantly higher than the non-irradiated control group (37.7.+ -. 0.7%). This demonstrates that PVA/HZCB fibers enable responsive release of drugs by NIR laser.
The antibacterial performance of the PVA/HZCB fiber three-mode cooperative treatment system in vitro is evaluated by adopting an agar plate method system by using two troublesome common wound related strains (methicillin-resistant staphylococcus aureus (MRSA) and (pseudomonas aeruginosa) PA). FIG. 5 shows the in vitro antimicrobial properties of PVA/HZCB fibers of example 1. As shown in FIGS. 5A and B, when PVA/HZCB fiber is treated together with low concentration H 2O2 (100. Mu.M) and low intensity NIR (0.3W/cm 2), bacteria almost completely die, and the antibacterial rates of MRSA and PA reach 98.7+ -0.8% and 98.6+ -0.7%, respectively, which shows excellent antibacterial effects. This powerful antimicrobial efficacy can be attributed to a variety of antimicrobial modes: partial warming generated under NIR laser irradiation can kill partial bacteria (PTT efficacy), and the temperature rise can accelerate the release of berberine hydrochloride encapsulated in HZCB, thereby enhancing the sterilization effect (drug treatment efficacy); meanwhile, the catalytic activity of POD-like enzyme of HZCB is improved by heat generated under NIR laser irradiation, H 2O2 is more effectively converted into toxic substances OH, and the strong oxidization property of the POD-like enzyme can quickly destroy bacterial cell membranes and intracellular substances, so that bacteria die (chemical kinetics efficacy). The three-synergy sterilization system reduces the dosage of medicines and the intensity of NIR laser, greatly reduces the toxic and side effects on organisms, and achieves satisfactory antibacterial effect.
The toxicity of nanofibers was examined by MTT method using HaCaT cells as the subject, and FIG. 6 shows the in vitro safety test results of PVA/HZCB fibers in example 1. As shown in fig. 6a, PVA/HZCB fibers exhibited negligible cytotoxicity to HaCaT cells, with cell viability exceeding 90%, no significant difference from the normal cells of the control group without any treatment. This was further demonstrated by the cell viability/death double staining experiments, where the treated cells were morphologically normal and at a higher density (B in fig. 6). These results indicate that PVA/HZCB fibers have good cell compatibility. The material was then tested for hemolysis, and the results are shown in figure 6C for hemolysis less than 5%. Materials with a hemolysis ratio of less than 5% are generally considered to have good blood compatibility. Compared with the positive and negative control samples, the PVA/HZCB fiber of the present invention does not cause hemolysis.
A complex infection wound model is created on the skin surface of a mouse, the in-vivo sterilization effect of PVA/HZCB fibers is further studied, a circular wound surface with the diameter of 1cm is constructed on the back of all female ICR mice, and a wound is infected by high-concentration MRSA and PA mixed bacterial liquid, so that the skin complex infection model is established. The wound healing was recorded by photographing the wound site of the mice on a specific treatment date, and fig. 7 is the results of the PVA/HZCB in-vivo complex infection wound healing and biosafety test in the fiber of example 1. As shown in fig. 7a and B, the wound areas of the groups are similar at the beginning of wound creation. The fourth day after surgery, the wound area of the treatment group was significantly reduced. The PVA/HZCB fiber treatment group has a wound healing rate of 58.2+/-2.5% under the condition of applying low-concentration H 2O2 (100 mu M) and low-intensity NIR (0.3W/cm 2), and has no phenomena of redness, swelling and inflammation. On the 10 th day after operation, the wound of PVA/HZCB fiber +H 2O2 +NIR group is almost completely healed, and the healing rate is as high as 95.8+/-1.9%. Furthermore, the in vivo antibacterial activity of PVA/HZCB fibers was further assessed by plate counting. As shown in FIG. 7C, the results were comparable to in vitro antimicrobial, with the PVA/HZCB fiber+H 2O2 +NIR group being the most effective, and a large number of colonies were also present in the control group. These results demonstrate that the mild photothermal effect produced at low light intensity not only has no adverse effect, but also enhances the POD mimic enzyme performance and BBH release of PVA/HZCB fibers, effectively fights against complex bacterial infection, and promotes healing of complex infected wounds.
Histological features of wound healing were assessed by H & E and Masson trichromatography, the results of which are shown in fig. 8. As can be seen from fig. 8a, on the 10 th day after operation, a large amount of inflammatory cells still infiltrate and severely damage the epidermis at the wound of all control groups, which indicates that the natural healing condition is poor under the complex bacterial infection condition, the PVA/HZCB fiber+h 2O2 +nir group shows the best healing condition, the complete epidermis structure is formed, and more newly-grown skin appendages can be observed. The Masson trichromatic staining results showed that the collagen deposition was significantly higher for the PVA/HZCB nanofiber + H 2O2 + NIR group than for the other groups.
To explore the intrinsic driving force for wound healing, the level of inflammation and the expression of healing factors in infected wound tissue was assessed by immunohistochemistry (B, C in fig. 8). The results of immunohistochemistry for IL-1β showed that the PVA/HZCB fiber + H 2O2 + NIR group had the lowest level of inflammation, probably due to other bacteria that effectively cleared the wound site, thereby reducing inflammatory factor expression. At the same time, the expression of PVA/HZCB fiber +H2t 2O2 +NIR VEGF-A is obviously higher compared with that of the control group. These results indicate that PVA/HZCB nanofiber membranes in combination with H 2O2 and NIR lasers can reduce inflammation by inhibiting bacterial infection, promote capillary vessel formation, and thus effectively promote wound healing.
The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.
Claims (10)
1. A method for preparing a multifunctional nanofiber membrane for promoting healing of complex infected wounds, which is characterized by comprising the following steps of:
a. dispersing ZIF-8 in absolute ethanol, adding tannic acid solution, stirring uniformly, centrifuging, and drying to obtain HZIF-8;
b. Calcining HZIF-8, and cooling to obtain hollow carbon nano-enzyme HZC;
c. Dispersing the hollow carbon nano enzyme HZC in methanol, adding berberine hydrochloride, stirring uniformly, centrifuging, and drying to obtain drug-loaded carbon nano enzyme HZCB;
d. adding the drug-loaded carbon nano-enzyme HZCB into polyvinyl alcohol to prepare spinning solution, carrying out electrostatic spinning and drying to obtain the multifunctional nanofiber membrane for promoting healing of complex infected wounds.
2. The method for preparing a multifunctional nanofiber membrane for promoting healing of complex infected wound according to claim 1, wherein the ZIF-8 is added in an amount of 80-100mg and the concentration of tannic acid solution is 10-20g/L in step a.
3. The method for preparing a multifunctional nanofiber membrane for promoting healing of complex infected wounds according to claim 1, wherein in the step a, the stirring time is 1-5min, and the stirring speed is 600-800r/min;
The centrifugation times are 1-3 times, the drying temperature is 40-60 ℃, and the drying time is 6-8 hours.
4. The method for preparing a multifunctional nanofiber membrane for promoting healing of complex infected wound according to claim 1, wherein in the step b, the calcination temperature of HZIF-8 is 800-1000 ℃ and the heat preservation time is 1-3h.
5. The method for preparing a multifunctional nanofiber membrane for promoting healing of complex infected wound according to claim 1, wherein in the step c, the mass ratio of the hollow carbon nano enzyme HZC to berberine hydrochloride is (1:2) - (1:4).
6. The method for preparing a multifunctional nanofiber membrane for promoting healing of complex infected wounds according to claim 1, wherein in the step d, the adding amount of drug-loaded carbon nano-enzyme HZCB in the spinning solution is 150-250 mug/mL, and the adding amount of polyvinyl alcohol in the spinning solution is 60-80mg/mL.
7. The method for preparing a multifunctional nanofiber membrane for promoting healing of complex infected wounds according to claim 1, wherein in the step d, the voltage is 13-15KV during electrostatic spinning, the distance between a receiver and a nozzle is 10-15cm, and the injection speed of spinning solution is 0.2-0.4mL/h.
8. The method for preparing a multifunctional nanofiber membrane for promoting healing of complex infected wound according to claim 1, wherein in the step d, the drying temperature is 40-60 ℃ and the drying time is 6-8h.
9. A multifunctional nanofiber membrane for promoting healing of complex infected wounds, characterized by being prepared according to the method of any one of claims 1-8.
10. Use of the multifunctional nanofiber membrane of claim 9 in the preparation of a medicament for promoting healing of complex infected wounds.
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