CN113332484B

CN113332484B - Preparation method of photo-thermal antibacterial nanofiber membrane

Info

Publication number: CN113332484B
Application number: CN202110502003.5A
Authority: CN
Inventors: 尹学琼; 田华; 李萌婷; 秦梓喻
Original assignee: Hainan University
Current assignee: Hainan University
Priority date: 2021-05-08
Filing date: 2021-05-08
Publication date: 2022-06-21
Anticipated expiration: 2041-05-08
Also published as: CN113332484A

Abstract

The invention discloses a preparation method of a photo-thermal antibacterial nanofiber membrane, which comprises the following steps: mixing chitosan and ethylenediamine, stirring, uniformly mixing, dropwise adding concentrated sulfuric acid, cooling to room temperature, adding water, stirring, precipitating with ethanol, centrifuging, filtering, rotary steaming, and oven drying to obtain N, S-CDs; adding chitosan into acetic acid solution, stirring to dissolve, adding HAuCl₄Heating the solution for reaction, cooling the solution to room temperature after the reaction, and filtering the reaction solution to obtain an AuNPs solution; mixing the N, S-CDs aqueous solution with the AuNPs solution, standing at room temperature in a dark place, dialyzing, and filtering to obtain an N, S-CDs @ AuNPs solution; mixing a polyvinyl alcohol aqueous solution with the N, S-CDs @ AuNPs solution, and stirring at room temperature to obtain an electrostatic spinning solution; at room temperature, electrostatic spinning is carried out by adopting electrostatic spinning solution, and the obtained photo-thermal antibacterial nanofiber membrane has excellent photo-thermal effect, excellent photo-thermal antibacterial effect and good biocompatibility.

Description

Preparation method of photo-thermal antibacterial nanofiber membrane

Technical Field

The invention belongs to the technical field of membrane materials, and particularly relates to a preparation method of a photo-thermal antibacterial nanofiber membrane.

Background

Bacteria have strong adaptability and reproductive capacity, and bacterial infection is always a problem in the field of medical health. Traditional antibiotic therapy treats disease by interfering with the normal metabolic processes of pathogenic bacteria. In recent years, the abuse of antibiotics makes the drug resistance of bacteria stronger and stronger, and some inherent limitations may exist, including high systemic cytotoxicity, poor solubility and poor dosage dependence, so that the development of multifunctional bacteriostatic materials, effective inhibition of bacteria and reduction of the drug resistance risk of bacteria is urgent.

Near Infrared (NIR) laser-induced photothermal therapy has been widely used as a powerful tool in the treatment of cancer and bacterial infections due to its noninvasive operation of antimicrobial dressings, good controllability, and high tissue penetration. The selection of a suitable photothermal agent that absorbs near infrared light and produces photothermal is critical for effective treatment or antimicrobial. Among various photothermal agents, gold-based nanomaterials are receiving much attention due to their surface plasmon effect and photothermal effect in the near infrared region. However, under long-term laser irradiation, due to the melting effect, the photothermal effect is reduced with the change of the morphology of some gold-based nanomaterials, and it is difficult to maintain efficient and stable heat generation. Therefore, there is a need to further functionalize the gold nano-material to protect it and improve its light stability and biocompatibility.

Therefore, the development of a method for preparing a photothermal antibacterial nanofiber membrane with excellent photothermal effect and good biocompatibility is a problem that needs to be solved by those skilled in the art.

Disclosure of Invention

In view of this, the invention provides a preparation method of a photothermal antibacterial nanofiber membrane with excellent photothermal effect and good biocompatibility.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of a photo-thermal antibacterial nanofiber membrane comprises the following steps:

(1) mixing chitosan and ethylenediamine, stirring, uniformly mixing, dropwise adding concentrated sulfuric acid, cooling to room temperature, adding water, stirring, precipitating with ethanol, centrifuging, filtering, rotary steaming, and oven drying to obtain N, S-CDs;

(2) adding chitosan into acetic acid solution, stirring to dissolve to obtain chitosan acetic acid solution, adding HAuCl₄Heating the solution for reaction, cooling to room temperature after the reaction is finished, and filtering the reaction solution to obtain an AuNPs solution;

(3) mixing the N, S-CDs aqueous solution with the AuNPs solution, standing at room temperature in a dark place, dialyzing, and filtering to obtain an N, S-CDs @ AuNPs solution;

(4) mixing a polyvinyl alcohol aqueous solution with the N, S-CDs @ AuNPs solution, and stirring at room temperature to obtain an electrostatic spinning solution;

(5) and (3) spinning the electrostatic spinning solution at room temperature by adopting an electrostatic spinning technology to obtain the photo-thermal antibacterial nanofiber membrane.

The invention has the beneficial effects that:

1. the method adopts natural biological macromolecular chitosan to reduce chloroauric acid, is green and environment-friendly, simple to operate and low in cost, and the prepared gold nanoparticles are high in stability and can be used for large-scale production.

2. The carbon dots and the gold nano-particles are self-assembled under the electrostatic action at room temperature, the carbon dots are coated on the surfaces of AuNPs to generate N, S-CDs @ AuNPs, and the method is simple to operate, mild in reaction conditions, low in preparation cost and environment-friendly. The N, S-CDs @ AuNPs photo-thermal conversion capability is stable, the photo-thermal conversion efficiency is high, the carbon dots can obviously improve the photo-thermal effect of the gold nano-material, the using amount of the gold nano-material can be reduced, the cost is saved, and the carbon dots can improve the biocompatibility of the gold nano-material.

3. The N, S-CDs @ AuNPs photo-thermal reagent in the photo-thermal antibacterial nanofiber membrane can be protected by polyvinyl alcohol, and materials such as the polyvinyl alcohol and the like have good biocompatibility, strong degradability, no toxicity, no harm and no pollution to the environment.

4. The photo-thermal antibacterial nanofiber membrane disclosed by the invention adopts carbon dots and gold nanoparticles to cooperate with a photo-thermal effect, can quickly obtain a higher temperature through infrared laser irradiation, and is stable in photo-thermal conversion capability and high in photo-thermal conversion efficiency, so that a good thermal effect sterilization effect is generated.

Further, in the step (1), the mass ratio of the chitosan to the ethylenediamine is 1: 10-20.

Further, in the step (1), 3-6mL of concentrated sulfuric acid is added to every 400mg of chitosan.

Adopt above-mentioned further beneficial effect: the method adopts the concentrated sulfuric acid and the ethylenediamine with the proportion for neutralization reaction, releases high-heat carbonized chitosan, does not need to provide additional heat source, and can reduce the cost of synthesizing carbon points.

Furthermore, in the step (1), the alcohol precipitation is performed by adding 200-300mL ethanol into 400mg chitosan, the centrifugation speed is 6000-9000rmp, the centrifugation time is 10-30min, and the rotary evaporation temperature is 50-70 ℃.

Further, in the step (1), the drying temperature is 50-80 ℃, and the drying time is 10-20 h.

Furthermore, in the step (1), the rotation speed of mixing and stirring the chitosan and the ethylenediamine is 50-100rpm, the stirring time is 10-20min, the concentration of the concentrated sulfuric acid is 80-98 wt%, the dropping speed is 0.5-1 drop/second, 15-25mL of water is added into every 400mg of chitosan, the stirring rotation speed is 50-100rpm, the stirring time is 6-10h, and a filter head with the aperture of 0.22 μm is adopted for the filtration.

Adopt above-mentioned further beneficial effect: according to the invention, through a large number of experiments, the accurate parameters in the step (1) are set, the reaction efficiency can be improved, chitosan is carbonized through a large amount of heat released by acid-base neutralization reaction to prepare carbon dots, the neutralization heat release of ethylenediamine and sulfuric acid is beneficial to nitrogen and sulfur hybridization on the surfaces of the carbon dots, the optical performance of the carbon dots is further improved, and the nitrogen, sulfur and oxygen groups can make the surfaces of the carbon dots carry negative charges. And (3) removing impurities in the carbon dots by ethanol precipitation to obtain purified carbon dots, so that the uniformity of the nano size of the carbon dots is ensured.

Further, in the step (2), the concentration of the chitosan acetic acid solution is 10-25mg/mL, and HAuCl is added into every 200mg of chitosan₄Solution 300-₄The concentration of the solution is 0.5-2 wt%.

Further, in the step (2), the solvent of the acetic acid solution is water, HAuCl₄The solvent of the solution is water.

Furthermore, in the step (2), a filter head with a pore size of 0.45 μm is used for the filtration.

Further, in the step (2), the heating reaction temperature is 85-100 ℃, and the heating reaction time is 1-2 h.

Adopt above-mentioned further beneficial effect: according to the invention, through a large number of experiments and reaction conditions optimization, the particle size of the obtained gold nanoparticles is kept in a nanometer size, the solution property is stable, agglomeration is not easy to occur, and subsequent functionalization operation is facilitated. Is favorable for self-assembly with carbon dots through electrostatic interaction.

Further, in the step (3), the concentration of the N, S-CDs aqueous solution is 5-20mg/mL, the concentration of the AuNPs solution is 5-15mg/mL, and the volume ratio of the N, S-CDs aqueous solution to the AuNPs solution is 0.5-2: 1.

Further, in the step (3), standing for 6-10h at room temperature in a dark place, and dialyzing for 30-40h by using a 500-Da dialysis bag.

Adopt above-mentioned further beneficial effect: the method is simple to operate, mild in reaction conditions, and capable of effectively combining N, S-CDs and AuNPs to generate stable N, S-CDs @ AuNPs, and the N, S-CDs and AuNPs are self-assembled through electrostatic interaction, and the particle size of the N, S-CDs is smaller to cover the surface of the AuNPs with a larger particle size, so that the biocompatibility is further improved, and the cytotoxicity is reduced. The carbon dots can obviously improve the photothermal effect of the gold nano material, reduce the using amount of the gold nano material and save the cost, and the N, S-CDs @ AuNPs photothermal conversion capability is stable, the photothermal conversion efficiency is high, thereby obviously improving the photothermal antibacterial effect.

Furthermore, in the step (3), a filter head with a pore size of 0.45 μm is used for the filtration.

Further, in the step (4), the concentration of the polyvinyl alcohol aqueous solution is 10-20 wt%, and the volume ratio of the polyvinyl alcohol aqueous solution to the N, S-CDs @ AuNPs solution is 1: 0.3-1.8.

With the above further advantageous effects: according to the invention, a large number of experiments are carried out, the accurate raw material proportion is set, the spinning obtained by the electrostatic spinning technology is uniform in thickness and good in form, and the photo-thermal reagent can be effectively wrapped in the spinning, so that the photo-thermal reagent is protected.

Furthermore, in the step (4), the stirring time is 10-13h, and the stirring speed is 200-300 rpm.

Further, in the step (4), the preparation method of the polyvinyl alcohol aqueous solution comprises: polyvinyl alcohol was added to water and stirred at 90 ℃ until dissolved.

Further, in the step (5), the electrostatic spinning method comprises: at room temperature, a 20mL disposable syringe is used for absorbing 10mL of electrostatic spinning solution and is connected with a No. 6 needle, the syringe is fixed on a liquid transfer pump, a layer of tin foil paper is laid on a metal receiving plate for receiving spinning, the distance between the needle and the receiving plate is adjusted to be 15cm, the receiving plate is grounded, the needle is connected with a live wire, the high voltage is adjusted to be 20kV, and the output speed of the spinning solution is 0.5 mL/h.

Adopt above-mentioned further beneficial effect: according to the invention, through a large number of experiments, the raw material proportion is optimized, accurate electrostatic spinning parameters are set, the spinning obtained by the electrostatic spinning technology is uniform in thickness and good in form, and a photo-thermal reagent can be effectively dispersed in the spinning, so that the photo-thermal reagent is protected, and the biocompatibility is further improved.

Drawings

FIG. 1(a) Transmission Electron Microscopy (TEM) of N, S-CDs. The inset is a histogram of the particle size distribution of N, S-CDs; (b) high power transmission electron micrographs (HRTEM) of N, S-CDs; (c) transmission Electron Microscopy (TEM) of AuNPs. The inset is a histogram of the particle size distribution of AuNPs; (d) high power transmission electron microscopy (HRTEM) of AuNPs; (e) transmission Electron Microscopy (TEM) of N, S-CDs @ AuNPs; (f) high power transmission electron microscopy (HRTEM) of N, S-CDs @ AuNPs.

FIG. 2PVA nanofibers, AuNPs nanofibers and N, S-CDs @ AuNPs nanofibers (V)_PVA/V_{N，S-CDs@AuNPs}1:0.3,1:0.6,1:1,1:1.5,1:1.8) under Scanning Electron Microscopy (SEM).

FIG. 3 is an infrared image of PVA nanofiber membrane, AuNPs nanofiber membrane and N, S-CDs @ AuNPs nanofiber membrane under 808nm laser irradiation of different powers.

FIG. 4(a) temperatures of PVA nanofiber membrane, AuNPs nanofiber membrane and N, S-CDs @ AuNPs nanofiber membrane under 808nm laser irradiation of different powers; (b) heating curves of PVA, AuNPs and N, S-CDs @ AuNPs nanofiber membranes under 3W laser irradiation at 808 nm.

FIG. 5 is a photograph of bacterial colonies after 10min of no light treatment of Escherichia coli and Staphylococcus aureus and laser irradiation at 808nm of 3W.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Description of the drawings: in the invention, the AuNPs solution is gold nanometer solution, and the N, S-CDs aqueous solution is nitrogen and sulfur hybridized carbon dot aqueous solution.

Example 1

The preparation method of the photothermal antibacterial nanofiber membrane comprises the following steps:

(1) mixing and stirring chitosan and ethylenediamine at the stirring speed of 50rmp for 10min, uniformly mixing, dropwise adding concentrated sulfuric acid at the dropping speed of 0.5 drop/second and the concentration of the concentrated sulfuric acid of 80 wt%, cooling to room temperature, adding water, stirring at the stirring speed of 50rmp for 6h, then carrying out alcohol precipitation, wherein the alcohol precipitation is carried out by adding 200mL of ethanol into every 400mg of chitosan for alcohol precipitation, centrifuging at the centrifuging speed of 6000rmp for 10min, filtering by using a filter head with the aperture of 0.22 mu m, carrying out rotary evaporation at the rotary evaporation temperature of 50 ℃, drying at the drying temperature of 50 ℃ for 10h, and obtaining N, S-CDs; wherein the mass ratio of the chitosan to the ethylenediamine is 1:10, and 3mL of concentrated sulfuric acid and 15mL of water are added into every 400mg of chitosan.

(2) Adding chitosan into acetic acid solution, stirring to dissolve to obtain chitosan acetic acid solution, wherein the concentration of the chitosan acetic acid solution is 10mg/mL, the concentration of the acetic acid solution is 1 wt%, and then adding HAuCl₄Heating the solution to react, HAuCl₄The concentration of the solution is 0.5 wt%, the heating reaction temperature is 85 ℃, the heating reaction time is 1h, the solution is cooled to room temperature after the reaction is finished, and the reaction solution is filtered by a filter head with the aperture of 0.45 mu m to obtain an AuNPs solution; wherein HAuCl is added to every 200mg of chitosan ₄300 mu L of solution, the solvent of acetic acid solution is water and HAuCl₄The solvent of the solution is water.

(3) Mixing the N, S-CDs aqueous solution with the AuNPs aqueous solution, wherein the concentration of the N, S-CDs aqueous solution is 5 mg/mL; the concentration of the AuNPs solution is 5mg/mL, the AuNPs solution is kept stand for 6h in a dark place at room temperature, then is dialyzed for 30h by adopting a 500-plus-one 1000Da dialysis bag, and is filtered by adopting a filter head with the aperture of 0.45 mu m to obtain an N, S-CDs @ AuNPs solution; wherein the volume ratio of the N, S-CDs aqueous solution to the AuNPs solution is 0.5: 1.

(4) Adding polyvinyl alcohol into water, stirring at 90 ℃ until the polyvinyl alcohol is dissolved to obtain a polyvinyl alcohol aqueous solution with the concentration of 10 wt%, mixing the polyvinyl alcohol aqueous solution with the N, S-CDs @ AuNPs solution, stirring at room temperature for 10 hours at the stirring speed of 200rmp to obtain an electrostatic spinning solution; wherein the volume ratio of the polyvinyl alcohol aqueous solution to the N, S-CDs @ AuNPs solution is 1: 0.3.

(5) At room temperature, a 20mL disposable syringe is used for absorbing 10mL of electrostatic spinning solution and is connected with a No. 6 needle, the syringe is fixed on a liquid transfer pump, a layer of tin foil paper is laid on a metal receiving plate for receiving spinning, the distance between the needle and the receiving plate is adjusted to be 15cm, the receiving plate is grounded, the needle is connected with a live wire, the high voltage is adjusted to be 20kV, the output speed of the spinning solution is 0.5mL/h, and the photothermal antibacterial nanofiber membrane is obtained.

Example 2

(1) mixing and stirring chitosan and ethylenediamine at the stirring speed of 80rmp for 15min, uniformly mixing, dropwise adding concentrated sulfuric acid at the dropping speed of 1 drop/second and the concentration of the concentrated sulfuric acid of 90 wt%, cooling to room temperature, adding water, stirring at the stirring speed of 80rmp for 7h, then carrying out alcohol precipitation, wherein the alcohol precipitation is carried out by adding 250mL of ethanol into every 400mg of chitosan, carrying out alcohol precipitation, centrifuging at the centrifuging speed of 8000rmp for 20min, filtering by using a filter with the aperture of 0.22 mu m, carrying out rotary evaporation at the rotary evaporation temperature of 60 ℃, drying at the drying temperature of 70 ℃ for 15h, and obtaining N, S-CDs; wherein the mass ratio of the chitosan to the ethylenediamine is 1:15, and 5mL of concentrated sulfuric acid and 20mL of water are added into every 400mg of chitosan.

(2) Adding chitosan into acetic acid solution, stirring to dissolve to obtain chitosan acetic acid solutionThe concentration of the solution was 20mg/mL and the concentration of the acetic acid solution was 1.5 wt%, followed by the addition of HAuCl₄Heating the solution to react, HAuCl₄The concentration of the solution is 1 wt%, the heating reaction temperature is 90 ℃, the heating reaction time is 1.5h, the solution is cooled to room temperature after the reaction is finished, and the reaction solution is filtered by a filter head with the aperture of 0.45 mu m to obtain an AuNPs solution; wherein HAuCl is added to every 200mg of chitosan₄500 μ L of solution, acetic acid solution solvent is water, HAuCl₄The solvent of the solution is water.

(3) Mixing an N, S-CDs aqueous solution with an AuNPs solution, wherein the concentration of the N, S-CDs aqueous solution is 10mg/mL, the concentration of the AuNPs solution is 10mg/mL, standing for 8h at room temperature in a dark place, dialyzing for 35h by using a 500-plus-1000 Da dialysis bag, and filtering by using a filter head with the aperture of 0.45 mu m to obtain an N, S-CDs @ AuNPs solution; wherein the volume ratio of the N, S-CDs aqueous solution to the AuNPs solution is 1.5: 1.

(4) Adding polyvinyl alcohol into water, stirring at 90 ℃ until the polyvinyl alcohol is dissolved to obtain a polyvinyl alcohol aqueous solution with the concentration of 15 wt%, mixing the polyvinyl alcohol aqueous solution with the N, S-CDs @ AuNPs solution, stirring at room temperature for 12 hours, and stirring at the rotating speed of 250rmp to obtain an electrostatic spinning solution; wherein the volume ratio of the polyvinyl alcohol aqueous solution to the N, S-CDs @ AuNPs solution is 1: 1.2.

Example 3

(1) mixing and stirring chitosan and ethylenediamine at the stirring speed of 100rmp for 20min, uniformly mixing, dropwise adding concentrated sulfuric acid at the dropping speed of 1 drop/second and the concentration of the concentrated sulfuric acid of 98 wt%, cooling to room temperature, adding water, stirring at the stirring speed of 100rmp for 10h, then carrying out alcohol precipitation, wherein the alcohol precipitation is carried out by adding 300mL of ethanol into every 400mg of chitosan, carrying out alcohol precipitation, centrifuging at the centrifuging speed of 9000rmp for 30min, filtering by using a filter head with the aperture of 0.22 mu m, carrying out rotary evaporation at the rotary evaporation temperature of 70 ℃, drying at the drying temperature of 80 ℃ for 20h, and obtaining N, S-CDs; wherein the mass ratio of the chitosan to the ethylenediamine is 1:20, and 6mL of concentrated sulfuric acid and 25mL of water are added into every 400mg of chitosan.

(2) Adding chitosan into acetic acid solution, stirring to dissolve to obtain chitosan acetic acid solution, wherein the concentration of the chitosan acetic acid solution is 25mg/mL, the concentration of the acetic acid solution is 2 wt%, and then adding HAuCl₄Heating the solution to react, HAuCl₄The concentration of the solution is 2 wt%, the heating reaction temperature is 100 ℃, the heating reaction time is 2 hours, the solution is cooled to room temperature after the reaction is finished, and the reaction solution is filtered by a filter head with the aperture of 0.45 mu m to obtain an AuNPs solution; wherein HAuCl is added to every 200mg of chitosan₄600 μ L of solution, acetic acid solution solvent is water, HAuCl₄The solvent of the solution is water.

(3) Mixing an N, S-CDs aqueous solution with an AuNPs solution, wherein the concentration of the N, S-CDs aqueous solution is 20mg/mL, the concentration of the AuNPs solution is 15mg/mL, standing for 10h at room temperature in a dark place, dialyzing for 40h by using a 500-plus-1000 Da dialysis bag, and filtering by using a filter head with the aperture of 0.45 mu m to obtain an N, S-CDs @ AuNPs solution; wherein the volume ratio of the N, S-CDs aqueous solution to the AuNPs solution is 2: 1.

(4) Adding polyvinyl alcohol into water, stirring at 90 ℃ until the polyvinyl alcohol is dissolved to obtain a polyvinyl alcohol aqueous solution with the concentration of 20 wt%, mixing the polyvinyl alcohol aqueous solution with the N, S-CDs @ AuNPs solution, stirring at room temperature for 13 hours at the stirring speed of 300rmp to obtain an electrostatic spinning solution; wherein the volume ratio of the polyvinyl alcohol aqueous solution to the N, S-CDs @ AuNPs solution is 1: 1.8.

Example 4

(1) mixing chitosan and ethylenediamine, stirring at the rotation speed of 80rpm for 15min, uniformly mixing, dropwise adding concentrated sulfuric acid at the dropping speed of 0.5 drop/S and the concentration of the concentrated sulfuric acid of 98 wt%, cooling to room temperature, adding water, stirring at the stirring rotation speed of 80rpm for 8h, carrying out alcohol precipitation, adding 250mL of ethanol into every 400mg of chitosan for alcohol precipitation, centrifuging at the centrifugation speed of 8000rpm for 20min, filtering by using a filter with the aperture of 0.22 mu m, carrying out rotary evaporation at the rotary evaporation temperature of 60 ℃, drying at the drying temperature of 60 ℃ for 12h, and obtaining N, S-CDs; wherein the mass ratio of the chitosan to the ethylenediamine is 1:15, 4mL of concentrated sulfuric acid and 20mL of water are added into every 400mg of chitosan.

(2) Adding chitosan into acetic acid solution, stirring to dissolve to obtain chitosan acetic acid solution, wherein the concentration of the chitosan acetic acid solution is 20mg/mL, the concentration of the acetic acid solution is 1 wt%, and then adding HAuCl₄Heating the solution to react, HAuCl₄The concentration of the solution is 1 wt%, the heating reaction temperature is 95 ℃, the heating reaction time is 1h, the solution is cooled to room temperature after the reaction is finished, and the reaction solution is filtered by a filter head with the aperture of 0.45 mu m to obtain an AuNPs solution; wherein HAuCl is added to every 200mg of chitosan₄The solution is 500 μ L, and the solvent of acetic acid solution is water, HAuCl₄The solvent of the solution is water.

(3) Mixing an N, S-CDs aqueous solution with an AuNPs solution, wherein the concentration of the N, S-CDs aqueous solution is 5mg/mL, the concentration of the AuNPs solution is 10mg/mL, standing for 8h at room temperature in a dark place, dialyzing for 36h by using a 500-plus-1000 Da dialysis bag, and filtering by using a filter head with the aperture of 0.45 mu m to obtain an N, S-CDs @ AuNPs solution; wherein the volume ratio of the N, S-CDs aqueous solution to the AuNPs solution is 1.5: 1.

(4) Adding polyvinyl alcohol into water, stirring at 90 ℃ until the polyvinyl alcohol is dissolved to obtain a polyvinyl alcohol aqueous solution with the concentration of 20 wt%, mixing the polyvinyl alcohol aqueous solution with the N, S-CDs @ AuNPs solution, stirring at room temperature for 12 hours, and stirring at the rotation speed of 280rpm to obtain an electrostatic spinning solution; wherein the volume ratio of the polyvinyl alcohol aqueous solution to the N, S-CDs @ AuNPs solution is 1: 1.5.

Screening experiment for optimal concentration of chitosan acetic acid solution and optimal volume ratio of N, S-CDs aqueous solution to AuNPs solution

Reducing HAuCl by using chitosan acetic acid solutions of 1mg/mL, 10mg/mL and 20mg/mL respectively₄AuNPs nanoparticles of different sizes were prepared and the other steps were performed according to step (2) of example 4, and the results are shown in Table 1. AuNPs prepared from 1mg/mL, 10mg/mL and 20mg/mL chitosan acetic acid solutions have the sizes of 101.20nm,37.78nm and 72.95nm respectively, and the potentials of 36.6mV, 23.3mV and 33.3mV respectively. The potential of N, S-CDs was-7.84 mV. Therefore, AuNPs and N, S-CDs prepared by the method can self-assemble through electrostatic interaction. Considering that AuNPs need to be combined with carbon dots in the next step and the size is increased, for application in subsequent biological systems, the combination of small-sized AuNPs prepared by using 10mg/mL and 20mg/mL chitosan acetic acid solution and the carbon dots generates the change of the size and the potential of N, S-CDs @ AuNPs nanoparticles, so that optimization is continued.

TABLE 1 preparation of AuNPs from chitosan acetic acid solutions of different concentrations

AuNPs solutions prepared by respectively adopting 10mg/mL of N, S-CDs aqueous solution and 10mg/mL and 20mg/mL of chitosan acetic acid solution are reacted according to the volume ratio of 0.5:1,1:1,1.5:1 and 2:1, other steps are operated according to the step (3) in the example 4, and as shown in the table 2, the positive potential of the obtained N, S-CDs @ AuNPs nano particles is gradually reduced and the size is gradually increased along with the increase of the volume of the N, S-CDs. This is because N, S-CDs are negatively charged and AuNPs are positively charged, and when they are combined, the positive and negative charges partially cancel each other. AuNPs are gathered on the surface of AuNPs by inducing N, S-CDs, so that the size of the N, S-CDs @ AuNPs is increased. Since the size of the generated N, S-CDs @ AuNPs is relatively stable after the AuNPs prepared from the chitosan acetic acid solution of 20mg/mL react with the N, S-CDs, and the particle size is always kept within 105nm, the AuNPs prepared from the chitosan acetic acid solution of 20mg/mL are used for carrying out the next experiment.

TABLE 2 potentials and particle sizes of N, S-CDs @ AuNPs

AuNPs prepared from 10mg/mL of N, S-CDs aqueous solution and 20mg/mL of chitosan acetic acid solution are uniformly mixed according to the volume ratio of 0.5:1,1:1,1.5:1 and 2:1 respectively, and then react to generate N, S-CDs @ AuNPs nanocomposite, and the N, S-CDs @ AuNPs nanocomposite is subjected to Dynamic Light Scattering (DLS) characterization, wherein DLS results show that as the adding amount of N, S-CDs is increased from 0.5mL to 2mL, the size of N, S-CDs @ AuNPs is increased from 69.13nm to 103.70nm, N, S-CDs can cover the surface of AuNPs, and the N, S-CDs @ AuNPs are aggregated, so that the size is increased. As is clear from tables 1 and 2, the potential of AuNPs is 33.3mV, the potential of N, S-CDs is-7.84 mV, and the potential of N, S-CDs @ AuNPs (1.5:1) is 27.6mV, with the significant change in potential due to the charge-charge interaction between the two oppositely charged nanoparticles, so that the positively charged AuNPs electrostatically interact with the negatively charged N, S-CDs, allowing self-assembly. Comprehensively considering, the optimal volume ratio of N, S-CDs to AuNPs is 1.5: 1.

Secondly, in order to better observe whether the carbon dots are successfully synthesized and the size and surface morphology of the carbon dots, a Transmission Electron Microscope (TEM) characterization is carried out on the carbon dots, FIG. 1 is a TEM image of example 4, FIG. 1(a) is a TEM image of the carbon dots, the carbon dots are good in dispersity and are spherical, the particle size ranges from 1.76 nm to 6.69nm, and the average particle size is 3.68nm, and HRTEMimage in FIG. 1(b) can show that the carbon dots present amorphous carbon which has clear lattice fringes and shows a lattice spacing of 0.201nm, and the amorphous carbon belongs to the diffraction plane of the graphitic carbon (100). Fig. 1(c) is a TEM image of AuNPs, in which AuNPs nanoparticles are shown to have a regular spherical shape and an average particle diameter of 10.02nm, and the AuNPs are shown to be in a monodisperse state. FIG. 1(d) is a HRTEM image of AuNPs, which was measured to show that AuNPs have a lattice spacing of 0.235nm, which matches the (111) crystal plane of metallic gold. FIG. 1(e) is a TEM image of N, S-CDs @ AuNPs, and it can be seen that N, S-CDs @ AuNPs are aggregated and tend to aggregate in a chain form due to the attraction between N, S-CDs and AuNPs. FIG. 1(f) HRTEM image shows that AuNPs attract each other and are combined, and the surface of AuNPs forms a thin carbon shell layer, which shows the successful synthesis of N, S-CDs @ AuNPs.

FIG. 2 shows the PVA nanofibers, AuNPs nanofibers, a series of N, S-CDs @ AuNPs nanofibers (V) in example 4_PVA/V_{N,S-CDs@AuNPs}SEM pictures of 1:0.3,1:0.6,1:1,1:1.5,1: 1.8). As can be seen from the figure, all the nanofibers prepared had good fiber morphology, and the fiber surface was smooth. At V_PVA/V_{N,S-CDs@AuNPs}<1:1, uniform fiber thickness, no synaptic structure, at V_PVA/V_{N,S-CDs@AuNPs}At the ratio of 1:1 or more, the fiber shows synapse structures, but all the synapses can be spun to form good fiber morphology, and synapses are increased along with the increase of the addition amount of N, S-CDs @ AuNPs, which is caused by the fact that the synapses are wrapped by nanoparticles.

Third, performance test

1. Test of photothermal Effect

The nano-spinning film in the embodiment 4 is cut into a circular membrane with the diameter of 6mm by a perforating machine, the circular membrane is irradiated by 808nm near-infrared laser with the power of 1.5-3W for 5min, a laser probe is 2cm away from the surface of the film, and the temperature change of the nano-spinning film within a certain time is recorded. The results are shown in FIGS. 3 and 4.

Fig. 3 is an infrared imaging diagram of the nanofiber membrane, and fig. 4(a) is the temperature of the nanofiber membrane under 808nm laser irradiation of different powers for 5 min. Under the irradiation of laser light with the wavelength of 1.5W and 808nm, the PVA nanofiber membrane, the AuNPs nanofiber membrane and the N, S-CDs @ AuNPs nanofiber membrane (V)_PVA/V_{N,S-CDs@AuNPs}Temperature of 1:0.3,1:0.6,1:1,1:1.5,1:1.8) was raised to 40 ℃,42 ℃,42 ℃,46 ℃,52 ℃,50 ℃,51 ℃ respectively; at 2.0W, 80Under the irradiation of 8nm laser, the temperature of the materials is respectively raised to 41 ℃,44 ℃,45 ℃,50 ℃,54 ℃,56 ℃ and 60 ℃; under the laser irradiation of 2.5W and 808nm, the temperature of the materials is respectively raised to 42 ℃,47 ℃,48 ℃,52 ℃,57 ℃,59 ℃ and 67 ℃; the temperature of the above materials was raised to 43 deg.C, 50 deg.C, 56 deg.C, 61 deg.C, 64 deg.C, and 72 deg.C, respectively, under the irradiation of laser light of 808nm at 3.0W. These results indicate that the photothermal effect of the nanofiber membrane increases with increasing laser power; and under the condition of the same content of AuNPs (AuNPs nanofiber membrane and V)_PVA/V_{N,S-CDs@AuNPs}The photo-thermal conversion capacity of the N, S-CDs @ AuNPs nanofiber membrane is obviously higher than that of the AuNPs nanofiber membrane; the photo-thermal conversion capability of the N, S-CDs @ AuNPs nanofiber membrane is enhanced along with the increase of the content of the N, S-CDs @ AuNPs. FIG. 4(b) shows the temperature rise curves of PVA, AuNPs and N, S-CDs @ AuNPs nanofiber membranes under 3W laser irradiation at 808nm, and it can be seen from the graphs that the temperature rise rate of PVA is 16 ℃ for min^-1Temperature rise rate of AuNPs 19.5 ℃ min^-1N, S-CDs @ AuNPs nanofiber membrane (V)_PVA/V_{N,S-CDs@AuNPs}Heating rates of 1:0.3,1:0.6,1:1,1:1.5,1:1.8) were 26 ℃ min, respectively^-1,38℃min^-1,46℃min^-1,54℃min^-1,68℃min^-1. The results all show that the addition of the carbon dots remarkably improves the photothermal conversion efficiency of the AuNPs.

2. Test for antibacterial application

Taking PVA, AuNPs and a series of N, S-CDs @ AuNPs nanofibers (V) prepared in example 4_PVA/V_{N,S-CDs@AuNPs}1:0.3,1:0.6,1:1,1:1.5,1:1.8) the membrane was placed in a small well of a 96-well plate, 40. mu.L of the bacterial solution was added to each well, and the mixture was incubated at 37 ℃ for 2 hours in an incubator. After incubation, 40 μ L of physiological saline was added to each well, and the solution was blown up with a pipette gun to mix the solution uniformly and diluted to 100 times. And (3) an illumination group: irradiation with 3.0W, 808nm NIR laser for 10 min. Control group, not illuminated. And (3) uniformly coating 50 mu L of the solution on an agar culture medium, finally incubating the sample in a thermostat at 37 ℃ for 12 hours, counting by adopting a colony counting method, and calculating the survival rate of bacteria. The results are shown in FIG. 5.

As can be seen from fig. 5, in the control group, the reaction mixture was purified by PVA, AuNPs,forming an obvious number of bacterial colonies on a solid culture medium treated by the bacterial colonies of the N, S-CDs @ AuNPs nanofiber membrane, irradiating for 10min (laser irradiation group) by 3W 808nm laser, and then forming the N, S-CDs @ AuNPs nanofiber membrane (V)_PVA/V_{N,S-CDs@AuNPs}1:0.3,1:0.6,1:1,1:1.5,1:1.8) has obvious killing effect on escherichia coli, bacterial colonies are obviously reduced, and N, S-CDs @ AuNPs nanofiber membrane (V)_PVA/V_{N,S-CDs@AuNPs}1:1.5,1:1.8) was irradiated with laser light, and the survival rate of escherichia coli was 0. After the membrane is irradiated by 3W 808nm laser for 10min, the AuNPs, N, S-CDs @ AuNPs nano fiber membrane has obvious killing effect on staphylococcus aureus, bacterial colonies are obviously reduced, and the N, S-CDs @ AuNPs nano fiber membrane (V)_PVA/V_{N,S-CDs@AuNPs}1:1.5,1:1.8) was irradiated with laser light, and the survival rate of staphylococcus aureus was 0. In conclusion, the N, S-CDs @ AuNPs nanofiber membrane has excellent photothermal antibacterial effect, and the photothermal antibacterial efficiency of the membrane is superior to that of AuNPs. V_PVA/V_{N,S-CDs@AuNPs}From 1:1.5, the N, S-CDs @ AuNPs nanofiber membrane has 100% of sterilization rate on two bacteria. Experiments prove that the N, S-CDs @ AuNPs nanofiber membrane has a strong antibacterial effect under the irradiation of near-infrared laser. In conclusion, the N, S-CDs @ AuNPs nanofiber membrane can be used as a strong antibacterial membrane, has an excellent antibacterial effect under near infrared light irradiation, and the carbon dots can enhance the photothermal antibacterial effect of AuNPs.

The description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The preparation method of the photothermal antibacterial nanofiber membrane is characterized by comprising the following steps of:

(3) mixing an N, S-CDs aqueous solution with an AuNPs solution, wherein in the self-assembly process of the N, S-CDs and the AuNPs through electrostatic interaction, the particle size of the N, S-CDs is smaller and covers the surface of the AuNPs with larger particle size, standing in a dark place at room temperature, then dialyzing, and filtering to obtain an N, S-CDs @ AuNPs solution;

2. The method for preparing a photothermal antibacterial nanofiber membrane according to claim 1, wherein in step (1), the mass ratio of chitosan to ethylenediamine is 1: 10-20.

3. The method for preparing a photothermal antibacterial nanofiber membrane according to claim 1, wherein in step (1), concentrated sulfuric acid is added in an amount of 3-6mL per 400mg of chitosan.

4. The method for preparing a photothermal antibacterial nanofiber membrane according to claim 1, wherein in the step (1), the drying temperature is 50-80 ℃ and the drying time is 10-20 h.

5. The method for preparing a photothermal antimicrobial nanofiber membrane according to claim 1, wherein in step (2), the chitosan acetic acid solution has a concentration of 10-25mg/mL, and HAuCl is added to every 200mg of chitosan₄Solution 300-₄The concentration of the solution is 0.5-2 wt%.

6. The method for preparing a photothermal antibacterial nanofiber membrane according to claim 1, wherein in the step (2), the heating reaction temperature is 85-100 ℃ and the heating reaction time is 1-2 h.

7. The method for preparing the photothermal antibacterial nanofiber membrane according to claim 1, wherein in step (3), the concentration of the N, S-CDs aqueous solution is 5-20mg/mL, the concentration of the AuNPs solution is 5-15mg/mL, and the volume ratio of the N, S-CDs aqueous solution to the AuNPs solution is 0.5-2: 1.

8. The method for preparing a photothermal antibacterial nanofiber membrane according to claim 1, wherein in step (3), the membrane is kept standing at room temperature for 6-10h in the dark, and the dialysis is performed for 30-40h by using a 500-Da dialysis bag.

9. The method for preparing a photothermal antibacterial nanofiber membrane according to claim 1, wherein in step (4), the concentration of the aqueous polyvinyl alcohol solution is 10 to 20 wt%, and the volume ratio of the aqueous polyvinyl alcohol solution to the N, S-CDs @ AuNPs solution is 1:0.3 to 1.8.

10. The method for preparing a photothermal antibacterial nanofiber membrane according to claim 1, wherein in the step (5), the electrostatic spinning method comprises: at room temperature, a 20mL disposable syringe is used for absorbing 10mL of electrostatic spinning solution and is connected with a No. 6 needle, the syringe is fixed on a liquid transfer pump, a layer of tin foil paper is laid on a metal receiving plate for receiving spinning, the distance between the needle and the receiving plate is adjusted to be 15cm, the receiving plate is grounded, the needle is connected with a live wire, the high voltage is adjusted to be 20kV, and the output speed of the spinning solution is 0.5 mL/h.