CN114617964A

CN114617964A - Enzyme-responsive photo-thermal nano material G @ CuS and preparation method thereof

Info

Publication number: CN114617964A
Application number: CN202210263044.8A
Authority: CN
Inventors: 王建浩; 韩倩倩; 惠泽轩; 周心霈; 邱琳; 崔朋飞; 王程; 胡华安子; 周舒文
Original assignee: Changzhou University
Current assignee: Changzhou University
Priority date: 2022-03-17
Filing date: 2022-03-17
Publication date: 2022-06-14

Abstract

The invention belongs to the technical field of nano materials, and particularly discloses an enzyme-responsive photo-thermal nano material G @ CuS and a preparation method thereof. The nano material consists of enzyme-responsive gelatin nano particles and copper sulfide nano dots. Gelatin is used as a nano-carrier, and a gelatin-coated copper sulfide nano-material (G @ CuS) is synthesized by self-assembly, and due to the existence of copper sulfide nano-dots, the material can be rapidly heated under the action of near-infrared illumination and release active oxygen, so that bacteria can be killed by the material in cooperation with photo-thermal photodynamic; the gelatin nano-carrier is introduced, so that the copper ions can be released through gelatinase response degradation to selectively kill the staphylococcus aureus. In addition, the G @ CuS has good biocompatibility and can promote the proliferation and migration of cells. The nano material disclosed by the invention is low in synthesis cost and less in operation, has excellent photo-thermal performance, stability and biocompatibility, and can provide a new solution strategy for treating bacterial infection.

Description

Enzyme-responsive photo-thermal nano material G @ CuS and preparation method thereof

Technical Field

The invention belongs to the technical field of nano materials, and particularly relates to an enzyme-responsive photo-thermal nano material G @ CuS and a preparation method thereof.

Background

The skin serves as a barrier connecting the body to the outside, and plays an essential role in maintaining biostability and protecting the human body from harmful substances and microorganisms. Once damaged, the skin undergoes a complex series of biological processes that require activation and coordination of various intracellular and intercellular pathways to accelerate and enhance healing. Although most skin defects heal in a short period of time, this regenerative property is compromised when bacterial infections, especially full-thickness infections, are encountered and wound-induced inflammation will not heal rapidly.

Antibiotics, the most widely used form of chemotherapy for bacterial infections, can effectively kill bacteria by reducing their ability to grow by either destroying their cellular integrity or inhibiting their essential growth and division processes (i.e., synthesis of cell walls, proteins and nucleic acids). However, due to their widespread use and misuse, bacteria produce a protective matrix (EPS) composed of polysaccharides, proteins and extracellular DNA, which acts as a shelter for the host immune system, hindering the diffusion and penetration of antibacterial agents and inducing the production of resistant pathogens. These drug-resistant pathogens cause fatal diseases such as soft tissue infection, pneumonia, bacteremia and the like. Therefore, it is crucial to find new strategies against drug-resistant bacteria and biofilm-associated infections.

At present, the light-based photothermal therapy has wide application prospects in the aspects of preventing wound infection and promoting wound healing. Photothermal therapy (PTT) can penetrate tissues and is less toxic and will completely eradicate bacteria without inducing drug resistance and damaging the body. More importantly, the PTT and other treatments such as Chemotherapy (CDT), photodynamic/catalytic treatment (PDT/PCT), immunotherapy, and sonodynamic treatment (SDT) can be used for reducing the laser density introduced into the PTT under the synergistic effect, so that unnecessary overheating damage to normal tissues is avoided. And metal-based nanomaterials, such as gold, silver, ruthenium, copper, and the like, are considered as attractive candidate antimicrobial nanomaterials due to excellent light-to-heat conversion efficiency. Nevertheless, it is a great hidden danger that ions released from the metal-based nano system are over-expressed in the lesion part, so it is important to develop a photothermal metal nano system with good biocompatibility.

Disclosure of Invention

The invention aims to provide an enzyme-responsive photo-thermal nano material G @ CuS which can rapidly ablate bacteria and promote wound healing and a preparation method thereof. The antibacterial nano material prepared by the invention can respond to the release of gelatinase, can effectively eradicate bacteria by cooperating with photothermal photodynamic therapy, and can promote the proliferation and migration of cells in vitro research. The antibacterial nano material is expected to become a replacement therapy in the aspect of wound healing of bacterial infection.

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

the enzyme-responsive photo-thermal nano material G @ CuS provided by the invention consists of gelatinase-responsive nano particles GeL and photo-thermal agent copper sulfide nano dots.

Wherein, the copper sulfide nano dots (CuS NDs) are prepared by an auxiliary coprecipitation method, the absorption wave band is between 980 and 1100nm, the particle size is 10nm, and the potential is-7.4 mV.

The prepared enzyme-responsive photo-thermal nano material G @ CuS has a hydrated particle size of 145nm and a potential of +20.4mV, and has excellent photo-thermal performance and biocompatibility.

The enzyme-responsive photo-thermal nano material G @ CuS has the advantages of low synthesis cost, simplicity in operation and stable property.

The preparation method comprises the following steps:

dissolving the type A gelatin in deionized water at 40 ℃, and stirring at the speed of 500r/min for 30 min. To the solution was added an equal volume of acetone solution and allowed to stand at room temperature for 1 h. The supernatant was discarded, the precipitate was dissolved with deionized water and, after complete dissolution, 8mg/mL of CuS NDs solution was added dropwise to the solution. The pH was adjusted to 2.5(1M HCl), and acetone solution (2mL/min) was added slowly to the above solution and stirred for 1 h. Dripping a cross-linking agent (50% w glutaraldehyde aqueous solution), stirring at room temperature for 16h, removing acetone and glutaraldehyde solution by rotary evaporation, and centrifuging for 10min at 8000r/min by a high-speed centrifuge. The non-crosslinked gelatin and non-embedded CuS NPS were removed, filtered through a water system (0.22 μm) filter and stored in a refrigerator at 4 ℃ until use. The nano material obtained by the method is called G @ CuS for short.

The enzyme-responsive photo-thermal nano material G @ CuS can be independently subjected to response degradation in a microenvironment where staphylococcus aureus exists, copper ions are released to perform chemical sterilization, and photo-thermal sterilization can be performed under the near-infrared effect.

Compared with the prior art, the invention has the following beneficial effects:

(1) the antibacterial photo-thermal nano material obtained by the invention can respond to microenvironment release, has excellent functions of promoting tissue regeneration and cell adhesion and can avoid potential safety hazards caused by free metal nanoparticles.

(2) The antibacterial photo-thermal nano material has simple preparation process and low cost, and can be popularized to large-scale production.

(3) The antibacterial photo-thermal nano material inherits the photo-thermal effect of copper sulfide, and can rapidly realize the ablation of bacteria under the excitation of near infrared light in cooperation with photo-thermal photodynamic power, so that the bacteria are damaged and die.

Description of the drawings:

FIG. 1 is a particle size distribution diagram of CuS NDs and a Transmission Electron Microscope (TEM) image;

FIG. 2 is a UV-VIS absorption spectrum of GeL, CuS, G @ CuS;

FIG. 3 is a graph of the particle size distribution of GeL, CuS, G @ CuS;

FIG. 4 is a graph of potential changes of GeL, CuS, G @ CuS;

FIG. 5 is an X-ray powder diffractometer (XRD) of GeL, CuS, G @ CuS;

FIG. 6 is a Fourier transform infrared (FT-IR) spectrum of GeL, CuS, G @ CuS;

FIG. 7 is a photo-thermal temperature rise graph of different concentrations G @ CuS;

FIG. 8 is a photo-thermal heating diagram for different power G @ CuS;

FIG. 9 is a thermal imager temperature map of GeL, CuS, G @ CuS;

FIG. 10 is a comparative graph of CuS, G @ CuS temperature increase and decrease;

FIG. 11 is a graph of the production of reactive oxygen species under near infrared illumination by CuS, G @ CuS;

FIG. 12 is a graph of the bactericidal effect of different concentrations of CuS and G @ CuS on S.aureus;

fig. 13 is a graph of the bactericidal effect of different concentrations of CuS and G @ CuS on e.coil;

fig. 14 is a photo-thermal antibacterial plot against s.aureus/e.coil;

FIG. 15 is a graph of the effect of G @ CuS on the S.aureus/E.coil growth curve;

FIG. 16 is a plot of Live/Dead staining before and after the effect on S.aureus/E.coil.

Fig. 17 is a graph of s.aureus biofilm inhibition experiments;

fig. 18 is a graph of e.coil biofilm inhibition assay;

FIG. 19 is a graph showing measurement of hemolysis rate;

FIG. 20 is a cytotoxicity plot of CuS against L929/HUVEC;

FIG. 21 is a graph of cytotoxicity of G @ CuS on L929/HUVEC;

FIG. 22 is a graph of cell proliferation of G @ CuS vs L929;

FIG. 23 is a cell scratch graph of G @ CuS versus L929;

fig. 24 is a graph of G @ CuS versus a wound model of s.aureus infected mice;

FIG. 25 is a graph of the change in body weight of mice;

FIG. 26 is a drawing of a wound tissue coating of an animal.

Detailed Description

The present invention is described in detail below with reference to examples, but these examples are only for illustrative purposes and should not be construed as limiting the practice of the present invention.

Example 1

1. Preparation of enzyme-responsive photo-thermal nano material G @ CuS

1) Synthesis of copper sulfide nanodots (CuS NDs)

The copper sulfide nanodots are synthesized by an auxiliary precipitation method, wherein copper chloride dihydrate provides copper ions, PVP is used as a dispersoid, sodium sulfide nonahydrate provides sulfur ions, nucleation and growth of crystals are carried out under the action of high temperature, and finally the uniformly dispersed nanodots are formed. The specific operation method comprises the following steps: to 5mL of deionized water was added 21mg of CuCl₂·2H₂O and 60mg of polyvinylpyrrolidone PVP (m.w.24000), and stirred to obtain a clear solution. Then, 30mg of Na was added during stirring₂S·9H₂And (O). The solution was heated to 90 ℃ and stirred for 30min to obtain a greenish black nanodispersion. Dialyzing in deionized water for 24h to obtain copper sulfide nanodots (CuS NDs), filtering the synthesized copper sulfide nanodots with a water system (0.22 μm) filter membrane, and storing in a refrigerator at 4 deg.C for later use.

2) Preparation of G @ CuS

The invention finally prepares the G @ CuS nano solution with better dispersity and stability by adjusting the dosage for several times, and the implementation process is as follows:

a. gelatin type 1.0g A was dissolved in 20mL of deionized water at 40 ℃ and stirred at 500r/min for 30 min. An equal volume of acetone solution was added to the above solution and allowed to stand at room temperature for 1 h. The supernatant was discarded and the pellet was dissolved in 9mL of deionized water and to this solution 8mg/mL of 4.5mL CuS NDs solution was added dropwise until dissolution was complete. The pH was adjusted to 2.5(1M HCl), and 50mL (2mL/min) of acetone was added slowly to the above solution and stirred for 1 h. 75 μ L of crosslinker (50% w glutaraldehyde in water) was added dropwise and stirred at room temperature for 16 h.

b.0.55g of type A gelatin is dissolved in 15mL of deionized water at 40 ℃ and stirred at 500r/min for 30 min. To the solution was added an equal volume of acetone solution and allowed to stand at room temperature for 1 h. The supernatant was discarded and the pellet was dissolved in 7mL of deionized water and to this solution 8mg/mL of 4.5mL of CuS NDs solution was added dropwise until dissolution was complete. The pH was adjusted to 2.5(1M HCl), and 40mL (2mL/min) of acetone was added slowly to the above solution and stirred for 1 h. 50 μ L of crosslinker (50% w glutaraldehyde in water) was added dropwise and stirred at room temperature for 16 h.

c. Gelatin type 0.55g A was dissolved in 15mL of deionized water at 40 ℃ and stirred at 500r/min for 30 min. An equal volume of acetone solution was added to the above solution and allowed to stand at room temperature for 1 h. The supernatant was discarded and the pellet was dissolved in 7mL of deionized water and 8mg/mL of 3mL CuS NDs solution was added dropwise to the solution until dissolution was complete. The pH was adjusted to 2.5(1M HCl), and 30mL (2mL/min) of acetone was added slowly to the above solution and stirred for 1 h. 50 mu L of cross-linking agent (50% w glutaraldehyde aqueous solution) is dripped, stirred for 16h at room temperature, and then the acetone and glutaraldehyde solution are removed by rotary evaporation, and centrifuged for 10min by a high-speed centrifuge 8000 r/min. The non-crosslinked gelatin and non-embedded CuS NDs were removed, filtered through a water-based (0.22 μm) filter, and stored in a refrigerator at 4 ℃ until use. The nano material obtained by the method is called G @ CuS for short.

TABLE 1

a.b. embodiment implementation, resulting in more aggregation, supernatant dilution, particle size measurement with malvern particle sizer, triplicate determinations for each sample, and average. The results are shown in Table 1, and the particle diameter and dispersibility obtained were inferior.

c. The nano material obtained by the scheme has good dispersibility and stability.

The invention prepares the nano material G @ CuS by the scheme c for subsequent characterization and in vivo and in vitro experiments.

The synthesis method of the gelatin nanoparticles (GeL NPs) is similar, and only the copper sulfide nanodot solution is not needed to be added.

2. Characterization of enzyme-responsive photo-thermal nano material G @ CuS

1) Determination of hydrated particle size and appearance of CuS

Filtering the synthesized copper sulfide nanodots through a water system (0.22 mu m) filter membrane, diluting by 10 times, measuring each sample in parallel for three times, taking an average value, and measuring the distribution of hydrated particle size and appearance by using a Malvern particle size analyzer and a transmission electron microscope. The results are shown in FIG. 1: the copper sulfide nanodot dispersion is good, and the particle size distribution is about 10 nm.

2) Ultraviolet-visible absorption spectrum, hydrated particle size and Zeta potential measurement of GeL, CuS and G @ CuS

CuS and G @ CuS are diluted into aqueous solutions with equal concentration, the aqueous solutions are respectively filtered through filter membranes (0.22 mu m), the equal volume is taken for measurement, each sample is measured in parallel for three times, an average value is taken, and an ultraviolet-visible spectrophotometer and a Malvern particle size analyzer are used for measuring the maximum absorption wavelength, the particle size and the Zeta potential change of the sample. Gelatin nanoparticles were used as a control sample in this procedure. The results are shown in FIGS. 2, 3 and 4. Fig. 2 shows that the maximum absorption wavelength of copper sulfide nano-dots is about 980nm, and after gelatin coating, red shift occurs and ultraviolet absorption changes to a certain extent. FIG. 3 shows that the particle size increases in turn during the embedding process, and the potential of FIG. 4 is reversed from that of the negative potential (-7.4mV) of the copper sulfide nano-dots after embedding and is lower than that of gelatin, and a part of the charge is neutralized. The above results preliminarily indicate that the synthesis of G @ CuS is successful.

3) X-ray powder diffractometer spectrogram and Fourier infrared spectrogram determination of GeL, CuS and G @ CuS

Freeze-drying the sample, grinding the sample into powder, and taking a proper amount of the sample powder for XRD and Fourier infrared scanning. As can be seen from fig. 5, the copper sulfide has a certain crystal structure, which is mainly represented by an X-ray diffraction (XRD) pattern consistent with that of the CuS standard hexagonal phase (JCPDS 06-0464), indicating that CuS (2 θ diffraction peaks near 29.277 °, 32.852 °, 47.941 ° correspond to the lattice planes of (102), (103), (110) copper) is successfully synthesized. After gelatin embedding, a dispersion peak is shown, which may be related to the coating of gelatin and the smaller particle size of CuS in the composite. Meanwhile, the FT-IR spectrum (figure 6) analyzes the chemical composition of G @ CuS at 3308cm^-1And 2936cm^-1The characteristic peak is mainly O-H stretching vibration and C-H asymmetric stretching vibration, and is 1659cm^-1The characteristic peak is mainly an N-H stretching vibration peak and has the same stretching vibration characteristic peak as that of the free gelatin nanoparticles. The above results indicate the successful synthesis of G @ CuS.

3. Photo-thermal property research of enzyme-responsive photo-thermal nano material G @ CuS

150. mu.L of G @ CuS aqueous solutions (1.25, 1.0, 0.75, 0.5, 0.25mg/mL) having different concentrations were placed in a 2mL centrifuge tube and irradiated with a near-infrared laser (808nm, 1.8W/cm) at a distance of 2cm from the liquid surface²) And 5 min. The treatment methods with different powers are consistent, and the irradiation with different power densities is carried out at the concentration of 0.75 mg/mL. Using a 808nm laser (1.8W/cm)²) Irradiating 150 mu L of G @ CuS and CuS samples (the concentration is 1.25mg/mL) for 5min, naturally cooling for 5min, continuously irradiating the nano material for 5min by using a 808nm laser with the same power model, monitoring the temperature change by a thermal imager, and repeating for 5-6 times. Fig. 7 and 8 show that the photothermal effect of G @ CuS is concentration-dependent and power density-dependent, i.e., the photothermal effect is better as the concentration and power density are increased. Meanwhile, FIGS. 9 and 10 show that the same concentration (1.25mg/mL) and the same time (5min) and the same power density (1.8W/cm)²) The photo-thermal effects of G @ CuS and free CuS under near-infrared irradiation are equivalent, and the photo-thermal stability of the G @ CuS and the free CuS is relatively good.

Exploration on generation of catalytic active oxygen under near-infrared illumination of G @ CuS

Absorbance values of DPBF with singlet oxygen ((II))¹O₂) Is reduced by oxidation and is suitable for detection¹O₂The resulting probe. DPBF was dissolved in acetonitrile at a final concentration of 500. mu.M and then mixed with 100. mu.L of CuS, G @ CuS, water and gelatin nanoparticle solution (as a control) and added to a 96-well plate (120. mu.L/well). The well plate was vibrated away from light to ensure uniform mixing and the UV spectra (300-800nm) of the different samples were determined by a microplate reader. As a result, as shown in fig. 11, the synthesized CuS and G @ CuS can generate singlet oxygen under the stimulation of near infrared light, and destroy the conjugated structure of DPBF, so that the absorption thereof is reduced, which can provide a basis for photodynamic therapy.

5. In vitro antibacterial experiment of G @ CuS

1) Near-infrared photothermal G @ CuS bacteria coating experiment is not carried out

Sterile water is used for respectively preparing CuS and G @ CuS solutions with the concentration of 2.0, 1.5, 1 and 0.5mg/mL, and 10 is taken⁸CFU/mL S.aureus bacterial suspension 100. mu.L was incubated with 100. mu.L CuS and G @ CuS solutions of different concentrations for 1h (actual working concentrations of G @ CuS were about 1, 0.75, 0.5, 0.25 mg/mL). Here, 100. mu.L of PBS and GeL NPs solution were used as negative controls, and after completion of incubation, 100. mu.L of the diluted solution was applied to a plate, and the plate was placed in a biochemical incubator (37 ℃ C.) and incubated overnight, and the number of colonies growing on the TSA plate was counted and each sample was assayed in triplicate. Wherein the Escherichia coli is treated in accordance with the above-mentioned treatment steps. The results in figure 12 show that G @ CuS demonstrates comparable antibacterial activity to free copper sulfide, since gelatin degrades in response to gelatinase secreted by staphylococcus aureus, releasing copper ions to induce bacterial death and achieving a mortality rate of about 50% for staphylococcus aureus at concentrations of 0.5-0.75 mg/mL. Unlike the results for S.aureus, FIG. 13, the effect of G @ CuS on E.coli is not as good as that of free copper sulfide, since E.coli does not secrete gelatinase. The above results indicate that the nanosystems have release-responsive properties.

2) Near-infrared photo-thermal G @ CuS bacteria coating experiment

100 μ L of PBS, CuS and G @ CuS in water and 100 μ L of Staphylococcus aureus were mixed, respectively (10)⁸CFU/mL), near infrared illumination was performed for 5min using a 808nm laser. And (5) finishing the incubation for 1h after the illumination. After incubation, the plate was diluted 2 ten thousand times, 100. mu.L of the plate was plated and incubated overnight in a biochemical incubator (37 ℃), and the number of colonies growing on the TSA plate was counted and each sample was assayed in triplicate. The experiment was divided into 6 groups in total: PBS, PBS + IR, CuS + IR, G @ CuS group, G @ CuS + IR group. (Note: working concentration 0.75mg/mL) the results are shown in FIG. 14.

3) Growth Curve determination of G @ CuS

To investigate the effect of G @ CuS nanoparticles on the growth of two bacteria, overnight cultured s⁵CFU/mL, into 96-well plates, respectivelyAdding 100 mu L of bacterial liquid, and respectively adding 100 mu L of G @ CuS nano material with the concentration of 1.5mg/mL (the actual working concentration is 0.75mg/mL), and dividing into 8 groups: s.aureus group, s.aureus + IR (laser irradiation) group, s.aureus + G @ CuS + IR group, e.coli + IR group, e.coli + G @ CuS + IR group), and each group was used as a parallel sample. Using a 808nm laser (1.8W/cm)²) The light group was irradiated for 5min, and the control group was prepared without laser irradiation and with PBS solution. And detecting and recording the absorbance of each hole at 600nm every other hour by using a microplate reader, continuously monitoring for 12 hours, and drawing and observing the change of the growth curves of the bacteria in the experimental group and the control group according to the measured data. The results are shown in FIG. 15.

As shown in fig. 14 and 15, consistent with the hypothesis and the plating experiment, the G @ CuS has a better effect of inhibiting the growth of staphylococcus aureus when no near-infrared light is applied, and has a better effect of inhibiting both bacteria when light is applied.

4) Staining for live and dead bacteria

The influence of the photothermal nano system G @ CuS on the viability of staphylococcus aureus and escherichia coli before and after photothermal is respectively researched by a live/dead bacterium staining method. 100 mu L G @ CuS was added to 100 mu L of 10⁸Incubating in S.aureus at CFU/mL for 30min, and irradiating with near infrared laser (808nm, 1.8W/cm)²) The bacteria were irradiated for 10 min. (PBS, PBS + IR, CuS + IR, G @ CuS five groups as controls) six groups of samples were centrifuged at 5000rpm for 10min, the supernatant was discarded, the precipitate was stained with 30. mu.L live/dead reagent, blown down evenly, vortexed, kept in the dark for 20min, and finally 20. mu.L of sample was dropped onto a slide glass and observed with an inverted fluorescence microscope. According to the manufacturer's instructions, live bacterial cells were stained with SYTO 9 dye (green), while dead bacterial cells were labeled with propidium iodide dye (red) due to cell wall and membrane damage. As a result, as shown in fig. 16, the red fluorescence emitted was the most under the irradiation of near infrared light, whereas the PBS group showed almost only green light despite the addition of the near infrared irradiation. This shows that the nano system can perform photothermal and photodynamic synergistic effects and has excellent photothermal antibacterial effects.

5) G @ CuS inhibits biofilm formation

Flat-bottom 96-well plates were selected and 100 μ L of s.aureus strain in logarithmic growth phase was added to selected 12 wells and divided into 4 groups: PBS control group, PBS + NIR, G @ CuS group, G @ CuS + NIR (808nm laser irradiation) group, final volume of each group was 200. mu.L and actual working concentration of G @ CuS was 0.75mg/mL, and 3 replicates for each group were set up. The illumination group used a 808nm laser (1.8W/cm)²) Irradiating for 5 min. And (3) placing the 96-well plate in a biochemical incubator at 37 ℃ for incubation for 48h, after the incubation is finished, slowly sucking out the upper culture solution by using a liquid transfer gun, washing the wells for 2-3 times by using PBS (phosphate buffer solution), air-drying for 10min, and adding 100 mu L of crystal violet solution with the concentration of 1% into each well for dyeing for 20 min. After dyeing is finished, slowly and gently sucking away the crystal violet solution along the wall by using a pipette gun, washing the selected holes for 3 times by using sterile PBS (phosphate buffer solution), drying in air for 10min, adding 200 mu L of 80% ethanol into each experimental hole, placing the 96-hole plate on a constant-temperature shaking table, oscillating until the crystal violet is completely dissolved, detecting the absorbance of each hole at 590nm by using an enzyme-labeling instrument, taking an average value and recording data. (Note: working concentration 0.75mg/mL) the results are shown in FIGS. 17 and 18, with the absorbance values for the G @ CuS + NIR group being around 0.2-0.4, approximately one fifth of the absorbance value for the PBS control group, which is clearly effective in inhibiting biofilm formation in both bacteria.

Hemolysis assay of G @ CuS

Erythrocytes were collected after centrifugation (300 rpm) of fresh female mouse blood for 5min, and after centrifugation, washed 3 times with sterile PBS (0.1M, pH 7.4). The purified erythrocytes were then diluted with PBS to a concentration of 20%. Then 20. mu.L of the aspirated erythrocytes were incubated with different concentrations of G @ CuS in a centrifuge tube in an incubator (37 ℃). PBS and 1% triton solution were negative and positive controls, respectively. To eliminate the interference of G @ CuS on blood absorption, PBS was mixed with G @ CuS and the absorbance was measured as follows. The hemolysis rate is calculated as follows:

hemolysis rate (%) - (A)_s-A_i-A_n)/(A_p-A_n)

Wherein A is_s、A_i、A_n、A_pThe absorbance values of the G @ CuS, the negative control and the positive control of the sample to be detected and the same concentration are respectively.

The hemolysis results are shown in FIG. 19 (the inset shows hemolysis), and the hemolysis rate is only about 3% and less than 5% when the concentration of G @ CuS is as high as (1.0 mg/mL). At the in vivo experimental concentration (0.75mg/mL), the hemolysis rate is about 1.5%, which indicates that the biocompatibility of the G @ CuS nano system is better. It is noted that the free CuS with the same concentration gradient generates aggregation when subjected to a hemolysis experiment, which indicates that gelatin can improve the dispersibility of CuS and effectively reduce the risk of toxicity to tissues caused by aggregation of CuS.

Cytotoxicity assay of G @ CuS

L929 and HUVEC are used as experimental materials, and the cytotoxicity of the CuS and G @ CuS nano system is detected by adopting an MTT method. First, L929 and HUVEC were subjected to procedures such as recovery, fluid exchange, and passaging, and experiments were performed when the cell viability was good. In the actual experiment, L929 and HUVEC cells were seeded in a 96-well microplate (10 wells each)⁴Individual cells) overnight and treated with different concentrations (0, 0.25, 0.5, 0.75, 1.0mg/mL) of CuS, G @ CuS nanosystems, respectively. The non-seeded pores of the process were filled with sterile PBS. After incubation for 24h and 48h respectively, MTT detection is carried out, and the absorbance of L929 and HUVEC cells at 490nm is measured by a microplate reader to evaluate the cytotoxicity of CuS and G @ CuS nano systems, so as to research the activity of the cells. The results are shown in fig. 20, 21 and 22, the survival rates of two cells of G @ CuS at the in vivo experimental concentration (0.75mg/mL) are respectively about 85% and 95%, and both are higher than 80%, and meanwhile, after the G @ CuS nano system is incubated with the L929 cells for 48 hours, the G @ CuS nano system shows good cell proliferation and differentiation capacity, while copper sulfide at the same concentration generates certain cytotoxicity to the cells. These results indicate that the G @ CuS nanosystem has good biocompatibility.

Cell scratch test of G @ CuS

After incubation of L929 cells in 6-well plates (50 ten thousand cells per well) for 12h, G @ CuS samples were added with fresh medium as a blank. After reaching approximately 90% confluence, the cell layer was scratched with a scratch-specific tip to simulate an incision wound. After co-culturing for 0, 12h, 24h and 48h, the scratched area of the cells is observed by using a fluorescence inverted microscope to photograph and track the closure condition. The results are shown in FIG. 23. It can be seen from the figure that the scratch area of the G @ CuS sample group is significantly narrowed compared to the control group after culturing for 48h with L929 cells, which indicates that it effectively promotes cell migration and facilitates cell differentiation.

Study of G @ CuS for Staphylococcus aureus infection

1) Establishing a staphylococcus aureus skin infection model

The modeling method comprises the following steps: shaving in Balb/c mice to expose the skin of the back overnight, and constructing an oval full-thickness wound with a perforator on the back the next day, with a major axis of about 8mm and a minor axis of about 6 mm; then, 20. mu.L of Staphylococcus aureus solution (10. mu.L) was dropped on the wound⁸CFU/mL) overnight, two infections were performed to ensure successful infection.

2) In vivo treatment of staphylococcus aureus infected wounds

To explore the treatment of the bacterial infected wound surface by G @ CuS, the experimental mice that were modeled were divided into 5 groups, including G @ CuS, G @ CuS + IR (808nm IR, 1.8W/cm)²5min), CuS, GeL and PBS, 3 replicates per group. Each mouse was individually housed to allow free access to water and diet to eliminate the remaining experimental interferences. The process is used for 5 times of administration, the illumination group uses a fixing plate to fix the mouse for each administration, and the whole near-infrared process uses a thermal imager to monitor the temperature, so that the temperature is maintained at about 46 ℃ to prevent the temperature from failing to meet the treatment requirement or damaging normal tissues too much. Finally, in order to monitor the change of the infection wound surface and the change of the physical sign of the mouse in the treatment process, a wound surface picture is taken every day and the weight of the mouse is weighed. On the ninth day of treatment, the tissue sites were removed after sterilization with medical scissors, each group of tissues was stored with an equal volume of sterile PBS for plating experiments, the plaques were placed in an incubator at 37 ℃ for overnight culture, and the number of colonies was counted. Wound healing results, weight change during treatment and plating results are shown in figures 24, 25 and 26.

As can be seen, the healing effect of G @ CuS + IR is the best. On the third day of treatment, the wound sites of the CuS, G @ CuS + IR groups gradually contracted, while the PBS group, GeL group did not change much, indicating that the PBS group, GeL group had no antibacterial activity at the early stage of wound healing, resulting in slow wound healing. It is worth noting that the two groups of G @ CuS and G @ CuS + IR have antibacterial activity due to the antibacterial activity of the two groups and the wound shrinkage is obvious due to the moisture retention of gelatin, and the near infrared light can effectively kill bacteria through photo-thermal and photodynamic light, so that the healing effect of the G @ CuS + IR is most obvious, the healing effect is most obvious in the group of G @ CuS + IR on the ninth day, and obvious scar scabbing is hardly seen compared with the rest groups. In the process, the weight change of each group of mice is not obvious, which indicates that the prepared material has low toxicity. Meanwhile, the plate coating results show that bacteria do not exist in the G @ CuS + NIR group, which indicates that the bacteria are eradicated, and the rest groups still have more bacteria in wound surface tissues, and the results indicate that the G @ CuS photothermal nano system can effectively eliminate the bacteria in infected wounds and promote wound healing under the near-infrared photothermal effect.

In light of the foregoing description of the preferred embodiment of the present invention, it is to be understood that various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the contents of the specification, and must be determined according to the scope of the claims.

Claims

1. The enzyme-responsive photo-thermal nano material G @ CuS is characterized by consisting of responsive drug-loaded nano particles GeL NPs and photo-thermal agents CuS NDs.

2. The enzyme-responsive photo-thermal nanomaterial G @ CuS of claim 1, wherein the CuS NDs maximum absorption band of the enzyme-responsive photo-thermal nanomaterial G @ CuS is between 980 and 1100nm, the hydrated particle size is 10nm, and the potential is-7.4 mV.

3. The enzyme-responsive photothermal nanomaterial G @ CuS of claim 1, wherein the CuS NDs in the enzyme-responsive photothermal nanomaterial G @ CuS are produced by assisted co-precipitation.

4. The enzyme-responsive photo-thermal nanomaterial G @ CuS of claim 1, wherein the maximum absorption band of the enzyme-responsive photo-thermal nanomaterial G @ CuS is between 980 and 1100nm, the hydrated particle size is 145nm, and the potential is +20.4 mV.

5. The enzyme-responsive photothermal nanomaterial G @ CuS of claim 1, wherein the enzyme-responsive photothermal nanomaterial G @ CuS is used as an antibacterial material for the treatment of bacterial infection of a wound.

6. The preparation method of the enzyme-responsive photothermal nanomaterial G @ CuS of claim 1, wherein the preparation method comprises: and (3) dropwise adding copper sulfide nanodots in the self-assembly process of the gelatin, adjusting the pH to 2.5, adding acetone for desolventizing, crosslinking by using glutaraldehyde, and purifying and removing a solvent from the obtained nano system by rotary evaporation and diafiltration.