CN114404392A - A kind of preparation method and use of pH and heat-responsive CuAu nano-assembly - Google Patents

Abstract

The invention discloses a preparation method and application of a pH- and heat-responsive CuAu nanometer assembly, comprising the following steps: preparing oleylamine-coated copper-gold nanoparticles; preparing pH- and heat-responsive small molecules containing sulfhydryl groups; The amine-coated copper-gold nanoparticles were subjected to ligand exchange to obtain monodisperse copper-gold nanoparticles; pH- and thermal-responsive copper-gold nano-assemblies were prepared. The pH- and thermally responsive copper-gold assembly has good photothermal properties. On the basis of realizing photothermal resistance to biofilm, it can reduce the damage to normal tissues around the wound due to excessive heating; meanwhile, it can promote the deep penetration and release of copper ions, It acts as an antibacterial and promotes wound healing.

Description

A kind of preparation method and use of pH and heat-responsive CuAu nano-assembly

technical field

The invention relates to a preparation method and application of biomedical engineering materials, in particular to a preparation method and application of a pH- and heat-responsive CuAu nano-assembly.

Background technique

In recent years, the incidence of chronic wound healing complicated by diseases such as diabetes has continued to rise and has become a major global medical burden. When superinfection occurs in chronic wounds, treatment often fails because the presence of biofilm at the wound site and the release of endotoxins delay the healing process. Biofilm is a bacterial community with an extracellular polymer (EPS) matrix formed by the adhesion and proliferation of microorganisms. Difficulty of antibiotic treatment.

Unlike traditional chemotherapy, photothermal therapy (PTT) uses the heat generated by the material under the irradiation of a near-infrared laser light source of 650-950 nm to kill bacteria and destroy the biofilm structure, which is not easy to develop drug resistance. Inorganic nanomaterials such as silver, copper, zinc, etc. have antibacterial functions, but the current antibacterial materials have high toxicity and single function, and cannot promote wound healing while antibacterial. Copper-based nanomaterials have received extensive attention due to their inherent photothermal properties and their role in promoting wound healing. In addition, copper ions at the wound surface can enhance the antibacterial effect, promote wound healing and angiogenesis. However, in photothermal therapy, delocalized heat and uncontrollable temperature and ion release often cause enormous damage to healthy tissue. Therefore, there is an urgent clinical need to find a safe and effective treatment solution that can rapidly remove biofilm while promoting the healing of chronic wounds.

SUMMARY OF THE INVENTION

Purpose of the invention: The purpose of the present invention is to provide a preparation method of pH- and heat-responsive CuAu nano-assemblies with good biocompatibility and promoting wound healing.

The present invention is based on Edman degradation sequencing technology pH and heat-responsive CuAu nano-assembly, the nano-assembly is mainly composed of copper-gold nanoparticles, the surface modification compound 3 is a pH- and heat-responsive group, and one end has a thiol group compound 4 It is a hydrophilic functional group, and forms pH- and thermal-responsive CuAu nanoassemblies through the chemical reaction of the 3-terminal amino group of the compound and p-phenylenediisothiocyanate.

The present invention also provides the application of the nano-assembly.

Technical solution: the preparation method of pH and heat-responsive CuAu nano-assemblies of the present invention includes the following steps:

(1) preparation of oleylamine-coated copper-gold nanoparticles;

(2) Preparation of pH- and heat-responsive small molecules containing sulfhydryl groups;

(3) performing ligand exchange on the copper-gold nanoparticles coated with oleylamine to obtain monodisperse copper-gold nanoparticles;

(4) Preparation of pH- and heat-responsive copper-gold nanoassemblies.

Further, the synthesis of oleylamine-coated copper-gold nanoparticles: mixing copper acetylacetonate and oleylamine, heating up and feeding an inert gas, keeping the temperature for 30-60 minutes, taking the n-hexane solution of the gold nanoparticles of secondary growth and adding the reaction In the system, the oxygen and n-hexane are removed by inert gas, the temperature is raised, the reaction is continued and then lowered to room temperature, the nanoparticles are precipitated with a polar solvent, and the oleylamine-coated copper-gold nanoparticles are obtained after removing the supernatant.

Further, the synthesis of thiol-containing pH and thermally responsive small molecules: N-tert-butoxycarbonyl-amino acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide was sequentially added to anhydrous N,N-dimethylformamide solution for activation, and then cystamine was added to anhydrous N,N-dimethylformamide solution, and the resulting mixture was heated under nitrogen. Stir in medium, react, purify to obtain compound 1, take trifluoroacetic acid and anhydrous dichloromethane, add compound 1 to it, stir at room temperature to obtain compound 2, and then add compound 2 and dithiothreitol to methanol to obtain compound 3. The amino acids of the N-tert-butoxycarbonyl-amino acid include α-amino acids such as glycine, alanine, valine, leucine, and isoleucine, which have only one amino group and carboxyl group and no thiol group and no disulfide bond.

Further, ligand exchange is performed on the oleylamine-coated copper-gold nanoparticles: the oleylamine-coated copper-gold nanoparticles are dispersed in a good solvent, and compound 3 and compound 4 with a thiol group at one end are added to dissolve in proportion to react, After cooling, it was centrifuged, and deionized water was added to obtain monodisperse copper-gold nanoparticles (CuAu NPs).

Further, the preparation of pH and thermally responsive copper-gold nano-assemblies: dissolving p-phenylenediisothiocyanate in a good solvent, then adding monodisperse copper-gold nanoparticles and emulsifier, after ultrasonic emulsification, reducing pressure Evaporated to obtain pH- and thermally responsive copper-gold nanoassemblies (sCuAu NAs).

Further, in step (1), the particle size of the copper-gold nanoparticles coated with oleylamine is 6-8 nm, and the copper-gold ratio thereof is Cu:Au=0-5:1. In step (2), the amino acids of the N-tert-butoxycarbonyl-amino acid include glycine, alanine, valine, leucine and isoleucine, etc., have only one amino group and carboxyl group and have no sulfhydryl and disulfide groups. alpha-amino acids of the bond. In step (3), the particle size of the monodisperse copper-gold nanoparticles is 6-8 nm; compound 4 is a hydrophilic small molecule with a molecular weight of one end less than 1000 Da and having a sulfhydryl group. The good solvent is selected from one or more of chloroform, dichloromethane, cyclohexane and n-hexane; the emulsifier is selected from cetyltrimethylammonium bromide or sodium dodecyl sulfate; sCuAu NAs The hydrated particle size is between 100 and 200 nm. The pH- and heat-responsive copper-gold nano-assemblies are used in anti-drug-resistant bacteria or anti-bacterial biofilms or in the preparation of medicaments for treating wounds. The bacteria include Escherichia coli, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

The nanometer assembly is prepared into a solution of a certain concentration to become a medicine for treating wounds, and the medicine is sprayed on the surface of the wounds to perform antibacterial activities on the wounds and promote wound healing.

Further, the preparation method of the gold nanoparticles of secondary growth:

(1) Dissolve tetrachloroauric acid trihydrate in a mixed solution of oleylamine and 1,2,3,4-tetralin, pass in an inert gas, and keep the temperature at 2-5°C for 30-60 minutes to remove the reaction The water vapor and oxygen in the system are added with tetrabutylammonium bromide dissolved in oleylamine and 1,2,3,4-tetrahydronaphthalene. The solution changes from golden yellow to purple, and the reaction continues at 2～5℃. The nanoparticles were precipitated with a polar solvent, and the supernatant was removed to obtain gold nanoparticles, which were dissolved in n-hexane for use.

(2) Dissolving tetrachloroauric acid trihydrate in the mixed solution of oleylamine and octadecene, feeding in an inert gas, keeping the temperature for 30-60 minutes, adding the gold nanoparticles dissolved in n-hexane in step 1) , remove oxygen and n-hexane with an inert gas, continue the reaction and then drop to room temperature, precipitate nanoparticles with a polar solvent, and remove the supernatant to obtain secondary growth of gold nanoparticles, which are dissolved in n-hexane for use.

Preferably, in the step 1), the feed ratio of tetrachloroauric acid trihydrate, oleylamine, 1,2,3,4-tetralin and tetrabutylammonium bromide is 100～200mg: 5～10mL: 5～10mL: 45～90mg, the particle size of gold nanoparticles is 4～6nm.

Preferably, in the step 2), the feeding ratio of tetrachloroauric acid trihydrate, oleylamine, octadecene and gold nanoparticles is 50-100 mg: 3-6 mL: 3-6 mL: 25-50 mg, and the secondary growth The particle size of the gold nanoparticles is 6-8 nm.

Preferably, in the step 3), the feeding ratio of copper acetylacetonate, oleylamine and the gold nanoparticles for secondary growth is 20-40mg: 3-6mL: 15-30mg, and the synthesized oleylamine-coated copper-gold nanoparticles The copper-gold ratio is 0.8 to 1.2:l.

Preferably, the polar solvent is selected from one or more of isopropanol, ethanol and acetone.

Beneficial effect: Compared with the prior art, the present invention has the following advantages:

1. The copper-gold nanoparticles of the present invention can exist stably and are not easily oxidized. At the same time, the release of copper ions increased.

2. The CuAu NPs in the present invention have certain absorption characteristics in the region of 650-950 nm, and can achieve temperature increase under the stimulation of 660 nm laser. Furthermore, to achieve more efficient photothermal sterilization, sCuAu NAs were prepared for potential photothermal enhancement, and the photothermal conversion efficiency (η) was determined to be 66.32% according to the heating-cooling curve.

3. Due to the limited bactericidal effect of copper ions, the prepared sCuAuNAs are rapidly heated up by near-infrared light irradiation, which further promotes the dissociation of the assembly, and accelerates the release of copper ions when heated, which is synergistic with photothermal therapy.

4. In the acidic microenvironment of the wound biofilm infection site, sCuAu NAs can be disassembled responsively under laser irradiation, and when they play the role of photothermal anti-biofilm, they can avoid damage to surrounding normal tissues due to excessive temperature. At the same time, the release of copper ions increases and promotes wound healing.

Description of drawings

Fig. 1 is the transmission electron microscope picture of the copper-gold nanoparticles coated with oleylamine;

Fig. 2 is the X-ray powder diffraction pattern of oleylamine-coated copper-gold nanoparticles;

Fig. 3 is the nuclear magnetic resonance spectrum of compound 3;

Figure 4 is a transmission electron microscope image of CuAuNPs;

Figure 5 shows the TEM images of sCuAuNAs at pH=7.4, 37°C and pH=5.5, 50°C;

Figure 6 shows the particle size distribution of sCuAuNAs at pH=7.4, 37°C and pH=5.5, 50°C;

Figure 7 shows the heating-cooling curves and photothermal conversion efficiencies of CuAu NPs and sCuAu NAs;

Figure 8 shows the copper ion release curves of sCuAu NAs under different conditions;

Fig. 9 is the antibacterial curve of different concentrations of sCuAuNAs;

Figure 10 is the photothermal antibacterial image of CuAuNPs and sCuAuNAs against MRSA;

Figure 11 is the photothermal antibacterial image of CuAuNPs and sCuAuNAs on E. coli;

Fig. 12 is the stained image of live and dead bacteria in biofilms treated with CuAu NPs and sCuAu NAs;

Figure 13 is a graph showing the toxicity of sCuAuNA to HUVEC cells;

Figure 14 is the migration diagram of HUVEC cells after different treatments with sCuAuNAs;

Figure 15 is a wound healing diagram of MRSA infected diabetic wounds in mice;

Fig. 16 is a graph showing the measurement of wound size in MRSA-infected diabetic wound mice.

Detailed ways

Example 1: Preparation of oleylamine-coated copper-gold nanoparticles

(1) Preparation of gold nanoparticles: Dissolve 200 mg of tetrachloroauric acid trihydrate in a mixed solution of 10 mL of oleylamine and 10 mL of 1,2,3,4-tetrahydronaphthalene, pass in an inert gas, and at 2-5 Incubate for 30-60 minutes to remove water vapor and oxygen in the reaction system, add 90 mg of tetrabutylammonium bromide dissolved in 1 mL of oleylamine and 1 mL of 1,2,3,4-tetralin mixed solution, and the solution is composed of The golden yellow color turned purple. After the reaction was continued for 1 h at 2-5 °C, the nanoparticles were precipitated with acetone, centrifuged at 8000 rpm for 5 minutes, the supernatant was discarded, the precipitate was dissolved in n-hexane, and then centrifuged with ethanol to obtain gold nanoparticles.

(2) Preparation of secondary grown gold nanoparticles: Dissolve 100 mg of tetrachloroauric acid trihydrate in a mixed solution of 6 mL of oleylamine and 6 mL of octadecene, pass in inert gas, and keep at 80°C for 30-60 minutes , add the gold nanoparticles dissolved in n-hexane in step (1), pass inert gas to remove oxygen and n-hexane, continue to react for 2h and then drop to room temperature, precipitate the nanoparticles with isopropanol, centrifuge at 8000rpm for 5 minutes, discard The supernatant, the precipitate was dissolved in n-hexane, and then centrifuged with ethanol to obtain secondary growth gold nanoparticles.

(3) preparation of oleylamine-coated copper-gold nanoparticles: 40mg copper acetylacetonate and 6mL oleylamine are placed in a four-necked flask, be warming up to 80° C. and pass into inert gas, be incubated for 30-60 minutes, take step (2) The n-hexane solution of gold nanoparticles grown in ) was added to the reaction system, and the oxygen and n-hexane were removed by inert gas, and then the temperature was raised to 210 ° C at a rate of 3 ° C/min, and the reaction was continued for 1 h. The particles were centrifuged at 8000 rpm for 5 minutes, the supernatant was discarded, the precipitate was dissolved in n-hexane, and washed three times repeatedly to obtain oleylamine-coated copper-gold nanoparticles. The copper-gold ratio of the oleylamine-coated copper-gold nanoparticles is about Cu:Au=1:1. The morphology of the oleylamine-coated copper-gold nanoparticles prepared in this example was characterized by transmission electron microscopy, as shown in FIG. 1 . Figure 2 shows the crystal form analysis of copper-gold nanoparticles by X-ray diffraction.

Example 2: Preparation of oleylamine-coated copper-gold nanoparticles

The synthesis is carried out with reference to the preparation process of Example 1, the difference is that the copper acetylacetonate added in the step (3) is changed to 0-200 mg, and the copper-gold nanoparticles coated with oleylamine are also obtained. The copper-gold ratios of the copper-gold nanoparticles coated with oleylamine are respectively Cu:Au=0-5:1.

Example 3: Synthesis of thiol-containing pH and thermoresponsive small molecules

N-tert-butoxycarbonyl-glycine (525 mg, 3 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1719 mg, 9 mmol) and N-hydroxysuccinimide The amine (1035 mg, 9 mmol) was sequentially added to anhydrous N,N-dimethylformamide solution (10 mL) and activated for half an hour. Cystamine (152.0 mg, 1 mmol) was then added to anhydrous N,N-dimethylformamide solution and the resulting mixture was stirred under nitrogen for 24 h at room temperature. The reaction was carried out overnight at room temperature under nitrogen protection, and the progress of the reaction was detected by thin-layer chromatography. The reaction was completed when the raw material point on the chromatography plate disappeared. The solvent was removed by rotary evaporation, the product was dissolved in dichloromethane, washed three times with water, dried and purified by column chromatography (eluent: dichloromethane:methanol=10:1) to obtain compound 1. Subsequently, compound 1 was dissolved in 5 mL of dichloromethane, 1 mL of trifluoroacetic acid was added thereto, stirred at room temperature for 4 h, and the solvent was removed by rotary evaporation to obtain compound 2. Then, compound 2 and dithiothreitol (309 mg, 2 mmol) were added to 5 mL of methanol, stirred at room temperature for 12 to 24 h, and the solvent was removed by rotary evaporation. The product was dissolved in water, extracted three times with ethyl acetate, dried and then subjected to column chromatography. method (eluent: acetonitrile: water = 10: 1) to obtain compound 3. After drying and purification, compound 3 was obtained. The synthetic route of the compound is as follows:

NMR data of compound 3: ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 1.31 (1H, s), 2.82-2.85 (2H, t), 3.50 (2H, s), 3.53-3.55 (2H, t) , 8.12 (2H, s), 8.63-8.66 (1H, t).

The NMR spectrum of compound 3 is shown in Fig. 3, indicating that the pH- and heat-responsive small molecules were successfully synthesized.

Example 4: Synthesis of thiol-containing pH and thermoresponsive small molecules

Synthesize with reference to the preparation technique of Example 3, the difference is that the amino acids in the described N-tert-butoxycarbonyl-glycine are respectively only possessed by alanine, valine, leucine and isoleucine etc. An alpha-amino acid substitution of amino and carboxyl groups without sulfhydryl and disulfide bonds.

Example 5: Preparation of CuAuNPs

1 mM oleylamine-coated copper-gold nanoparticles were dispersed in 10 mL of tetrahydrofuran, 1 mM of compound 3 dissolved in DMF and 1.5 mM of compound 4 whose one end was modified with sulfhydryl groups were added, reacted at 50 °C for 4 h, centrifuged at 3000 rpm for 3 minutes after cooling, and precipitated. After dissolving with DMF and then centrifuging twice to remove excess small molecules, the precipitate was dissolved with deionized water to obtain CuAu NPs. The morphology of CuAuNPs was characterized by transmission electron microscopy. The results are shown in Figure 4. The particle size is 6-8 nm, and they are dispersed in aqueous solution, showing the successful modification of thiol-containing small molecules.

Example 6: Preparation of sCuAuNAs

P-phenylenediisothiocyanate (5mM) was dissolved in chloroform (1mL), then CuAu NPs (1mM, 10mL) and emulsifier cetyltrimethylammonium bromide were added, sonicated, and stirred overnight at room temperature. Chloroform was evaporated, and the excess p-phenylenediisothiocyanate was removed by centrifugation at 500 rpm for 3 minutes, and the excess cetyltrimethylammonium bromide was removed by dialysis with a 1000 Da dialysis bag to obtain sCuAu NAs.

The particle size distribution of sCuAu NAs at different temperatures and pH conditions was analyzed by dynamic light scattering, and the results are shown in Table 1. The obtained sCuAu NAs were characterized by transmission electron microscopy at pH=7.4, 37°C and pH=5.5, 50°C. The results are shown in Fig. 5. Dynamic light scattering analysis was used at pH=7.4, 37°C and pH=7.4. The particle size distribution of sCuAu NAs at pH=5.5 and 50 °C is shown in Figure 6. The above results all prove that the sCuAu NAs have good pH and thermal responsiveness, and are completely disassembled at pH=5.5 and 50 °C.

Table 1. Particle size variation of sCuAu NAs at different temperatures and pH conditions

Example 7: Preparation of sCuAuNAs

Synthesize with reference to the preparation process of Example 6, the difference is that the added emulsifier is sodium lauryl sulfate.

Example 8: Photothermal performance test of sCuAu NAs

CuAu NPs (prepared according to Example 5) and sCuAu NAs (prepared according to Example 6) were diluted with deionized water to prepare a solution of 15 μg/mL. After irradiating the above two solutions with a 660 nm near-infrared light emitter for 5 minutes, the laser was turned off, the solutions were cooled to room temperature naturally, and an infrared thermal imager was used to record the real-time temperature of different solutions. The heating-cooling curves of CuAu NPs and sCuAu NAs were obtained at 1.0 W/ ^cm2 . The photothermal conversion efficiency (η) is calculated by the following formula:

Formula 1

Formula 2

Formula 3

Q ₀ =hS(T _{max, water} -T _surr )

The value of τ _s is calculated from the linear regression curve in the cooling curve, m _d and C _d represent the mass (1g) and heat capacity (4.2J/(g K)) of the solution, respectively, so that the value of hS can be obtained , and then, Q ₀ is calculated by Equation 3, representing the background energy input in the absence of MPDA nanoparticles. where _{Tmax, water} and _Tsurr represent the steady state maximum temperature of water and ambient room temperature, respectively. Therefore, after the hS and Q0 values are determined, the photothermal conversion efficiency can be calculated according to Equation ₁ . _Tmax represents the maximum temperature at which the solution is stable, and I and A ₆₆₀ represent the laser power (1.0 W) and the absorbance of the nanoparticles at 660 nm, respectively. The results are shown in Fig. 7, the photothermal conversion efficiency of sCuAu NAs is 66.32% higher than that of CuAu NPs (46.65%).

Example 9: Responsive release of copper ions

Accurately measure 1 mL of sCuAu NAs (prepared according to Example 6) (2 mg/mL) into a dialysis bag with Mw=1000, and immerse the dialysis bag in 50 mL of pH 7.4 or pH 5.5 buffer solution at 37°C and 50°C, At the corresponding time point, 1 mL of buffer solution was taken out and 1 mL of blank buffer solution of phase acidity was added. After sampling, the copper content in each sample was measured by inductively coupled plasma chromatography, and then the copper release amount was calculated. Calculated as follows:

R _t =C _t ×50/2×100%

where R _t is the copper release rate at the response time point, and C _t is the copper concentration in the solution taken out at this time point, in mg/mL.

Figure 8 was then plotted by release rate versus time. It can be observed that there is almost no copper release from sCuAuNAs at 37 °C, pH 7.4, while the copper release rate can exceed 10% in buffer solution at pH 5.5 at 50 °C. The copper release rates of sCuAu NAs in buffer solutions at pH 5.5 or 50 °C were all lower than 5%. This proves that the synthesized sCuAu NAs can remain stable in a neutral environment, while more copper ions are released due to disassembly under acidic and heated conditions.

Example 10: Inhibitory effect of sCuAu NAs on planktonic bacteria

Take methicillin-resistant Staphylococcus aureus (MRSA) in logarithmic growth phase, dilute the bacterial solution with fresh liquid medium to OD ₆₀₀ =0.01, take sCuAu NAs (prepared according to Example 6), and dilute with normal saline to Different concentrations were added to the bacterial solution and mixed, so that the final concentrations were 0 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL and 40 μg/mL, 100 μL per well, and cultured in a constant temperature shaking incubator at 37 °C. After 1 hour of incubation, irradiate the solution with or without 660nm laser (10 minutes, 1.0W/cm2), continue to incubate for 18 hours, measure its absorbance at 600nm on a microplate reader, and determine the bacterial survival rate, the results are shown in Figure 9 As shown, when the concentration is 20 μg/mL and 40 μg/mL, sCuAu NAs plus laser irradiation basically kills more than 95% of planktonic bacteria, and without laser, sCuAu NAs have no obvious killing ability to bacteria at 20 μg/mL and 40 μg/mL The survival rate was about 60%, so the experimental concentration of sCuAu NAs was 20 μg/mL.

Example 11: Photothermal sterilization of planktonic bacteria by CuAu NPs and sCuAu NAs

Take the methicillin-resistant Staphylococcus aureus in the logarithmic growth phase, dilute the bacterial solution with fresh liquid medium to OD ₆₀₀ =0.01, take CuAuNPs (prepared according to Example 5) and sCuAuNAs (prepared according to Example 6), Dilute the medium to 20 μg/mL, 100 μL per well, and culture in a constant temperature shaking incubator at 37°C. After 1 hour of incubation, the solution was irradiated with or without a 660nm laser (10 minutes, 1.0W/cm2), and the incubation was continued until 12 hours. The absorbance at 600nm was measured on a microplate reader, and the bacterial survival rate was determined by optical density. The results are shown in Fig. 10. Both CuAuNPs and sCuAuNAs can kill about 35% of planktonic bacteria without laser irradiation, while sCuAu NAs in laser irradiation group can kill 99% of bacteria, and CuAu NPs can only kill about 60% of bacteria. , which fully shows that the improvement of photothermal efficiency is beneficial to improve the photothermal antibacterial effect, and sCuAu NAs have superior photothermal antibacterial effect.

Example 12: Photothermal sterilization of planktonic bacteria by CuAu NPs and sCuAu NAs

The experiment was carried out with reference to the protocol of Example 11, except that the bacterium used was Escherichia coli (E.coli). The bacterial viability was determined by densitometry, and the results are shown in Figure 11. This shows that the photothermal treatment of sCuAu NAs has a broad-spectrum antibacterial effect.

In order to prove the broad-spectrum antibacterial effect of sCuAu NAs photothermal therapy, the bacteria used also include antibiotic resistance of Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, Salmonella, Diphtheria, Bacillus anthracis, and Escherichia coli. The bacterial survival rate after treatment was determined by optical density method to verify that sCuAu NAs have good photothermal treatment effect.

Example 13: Killing and Inhibitory Effect of sCuAu NAs on Bacterial Biofilms

Take the methicillin-resistant Staphylococcus aureus in logarithmic growth phase, dilute the bacterial solution with fresh liquid medium to OD ₆₀₀ = 0.05, inoculate in a 96-well plate, 200 μL per well, and keep it in a constant temperature incubator at 37 °C. Incubate for 48 hours and replace with fresh medium every 24 hours. After bacteria form a biofilm, the culture medium is discarded, and the biofilm is rinsed with physiological saline to wash away planktonic bacteria and bacteria desorbed from the biofilm. Take CuAu NPs (prepared according to Example 5) and sCuAu NAs (prepared according to Example 6), diluted with fresh medium to 20 μg/mL, added to biofilm, 200 μL per well, and cultured in a constant temperature incubator at 37 °C. . Meanwhile, blank medium was used as control group. After 1 hour of incubation, the solution was irradiated with or without 660nm laser (10 minutes, 1.0W/cm2), and the incubation was continued until 12 hours. The biofilm was thoroughly washed and replaced with propidium iodide (PI, 54.9μM) and SYTO 9 (5μM). ) of normal saline. After incubation at 37°C for 20 minutes, the fluorescent dye was washed away and the bacterial biofilm was observed under a fluorescence microscope. PI dyes stain dead bacteria, while SYTO 9 stains both live and dead bacteria. When the biofilm is intact, PI cannot penetrate the bacterial cell wall, and live bacteria are stained green by SYTO 9. The results are shown in Fig. 12. More than 99% of the normal saline group is green fluorescence. Without laser, CuAu NPs and sCuAu NAs can only destroy about 20% of the biofilm. However, after the laser irradiation group was treated with sCuAu NAs, the bacteria in the biofilm Almost all were stained by PI, showing red fluorescence, and the activity was significantly reduced. CuAu NPs could only destroy about 50% of the biofilm, showing that sCuAu NAs had a significant photothermal antibacterial biofilm effect.

Example 14: Cytotoxicity assay of sCuAu NAs

HUVEC cells were seeded in 96-well plates and cultured overnight. When the cells grew to 80% confluence, sCuAuNAs (1.25, 2.5, 5, 10, 20, 40 μg/mL) were added in three replicate wells for each concentration, and blank medium was used as a control. After culturing for 24 h, remove the medium containing nanoparticles, add pre-prepared MTT solution (5 mg/mL) to each well, and continue to incubate 150 μL per well in an incubator for 4 h; 150 μL dimethyl sulfoxide (DMSO), shaken for 3 minutes, use a microplate reader to detect the absorbance OD value of each well at 492 nm, take the average OD value of the three duplicate wells as the OD value of the target sample, and calculate the cell viability. :

Cell viability = sample _OD / blank control _OD × 100%

As shown in Figure 13, when the concentration of sCuAu NAs is 40 μg/mL, it is slightly toxic, and 20 μg/mL and below are basically non-toxic, indicating that the biosafety can be guaranteed when the antibacterial concentration is 20 μg/mL.

Example 15: Cell Migration Experiment of sCuAu NAs

Two solutions were first prepared by diluting sCuAu NAs (prepared according to Example 6) and 660 nm laser irradiated sCuAu NAs (pH 5.5, 10 min) in basal medium (DMEM) containing 1% serum. The HUVEC cells were cultured in a 12-well plate for 24 hours. When the cells were completely confluent, a 200 μL pipette tip was used to scratch the cells, and blank medium was used to wash away cell debris. Afterwards, 800 μL of the above-configured solution was added to each well. Among them, the Control group was a blank group without any drug treatment, the sCuAu NAs group was added with 20 μg/mL solution containing sCuAu NAs, and the sCuAu NAs+Laser group was added with 20 μg/mL Solution containing sCuAu NAs (photothermal for 10 min). The above solution was added to culture the scratched HUVEC cells for 36 h, and the cell migration results of each group are shown in Figure 14. As can be seen from Figure 14, after 36 hours of scratching HUVEC cells, only a small amount of cells migrated in the Control group, while the cells in the sCuAu NAs+Laser group migrated significantly, an increase of about 30% compared with the blank group. The cells in the sCuAu NAs group also had the ability to promote cell migration. However, the effect was not as obvious as that in the sCuAu NAs+Laser group, indicating that copper ions can promote cell migration. This means that sCuAuNAs can not only achieve photothermal antibacterial, but also promote wound healing.

Example 16 In vivo wound healing experiments of sCuAu NAs

1. Experimental part: 22-25g male BALB/c mice were selected, and STZ was injected intraperitoneally to make a model of type I diabetes; an 8mm*8mm hole punch was used on the back of the diabetic mice to prepare wounds, and the concentration of the bacterial solution was 1. 20 μL of 1×10 ⁸ CFU/mL methicillin-resistant Staphylococcus aureus solution was used to prepare wounds of diabetic mice severely infected with drug-resistant bacteria after 48 hours. Grouping of successful model mice: 100 μL of normal saline was given to the wounds of MRSA-infected diabetic mice, called Saline group, which was also the control group in this experiment. 100 μL of the 20 μg/mL sCuAu NAs-containing solution prepared above was administered to the wounds of MRSA-infected diabetic mice, referred to as the sCuAu NAs group. The wounds of diabetic mice infected with MRSA were given 100 μL of the 20 μg/mL sCuAuNAss-containing solution prepared above and incubated for 1 h, and then irradiated with 660 nm laser (10 minutes, 1.0 W/cm2), which was called the sCuAu NAs+Laser group.

2. Photographs of wound healing: Take pictures of the wounds at 0, 2, 4, 6, 8, and 10 days after drug administration in the above-mentioned mice, as shown in Figure 15. Figure 15 shows the wound healing of MRSA infected diabetic wounds in mice Figure 15 shows that after 10 days of treatment, the wound area of the sCuAu NAs alone group was reduced, but the wound was not healed, indicating that the effect of repair alone was not good; it is worth mentioning that the sCuAu NAs+ The Laser group combined the dual effects of photothermal antibacterial and copper ions to promote wound healing, and played a synergistic effect. The wounds were basically healed, and the repair of MRSA-infected diabetic wounds achieved optimal results.

3. Measurement of wound size: The size of the wound was measured on the mice at 0, 2, 4, 6, 8, and 10 days after the above administration and treatment with a vernier caliper, as shown in FIG. 16 . Figure 16 is the measurement chart of the wound size of MRSA infected diabetic wounds in mice. It can be seen from Figure 16 that the wound measurement data is basically the same as that of the wound healing chart in Figure 15, and the sCuAu NAs+Laser group can heal the wound in a short time.

Claims (10)

1. a preparation method of pH and heat-responsive CuAu nano-assembly, is characterized in that: comprise the steps: (1) preparation of oleylamine-coated copper-gold nanoparticles; (2) Preparation of pH- and heat-responsive small molecules containing sulfhydryl groups; (3) performing ligand exchange on the copper-gold nanoparticles coated with oleylamine to obtain monodisperse copper-gold nanoparticles; (4) Preparation of pH- and heat-responsive copper-gold nanoassemblies. 2. the preparation method of pH according to claim 1 and thermal response type CuAu nano-assembly, it is characterized in that: the synthesis of the copper-gold nanoparticle of oleylamine coating: copper acetylacetonate and oleylamine mix, heat up and pass into Inert gas, keep the temperature for 30-60 minutes, take the n-hexane solution of the gold nanoparticles for secondary growth and add it to the reaction system, pass the inert gas to remove oxygen and n-hexane, heat up, continue the reaction and then drop to room temperature, and precipitate the nanoparticles with a polar solvent , after removing the supernatant, oleylamine-coated copper-gold nanoparticles were obtained. 3. the preparation method of pH and heat-responsive CuAu nano-assembly according to claim 1, is characterized in that: the synthesis of thiol-containing pH and heat-responsive small molecules: N-tert-butoxycarbonyl-amino acid, 1 -Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were sequentially added to anhydrous N,N-dimethylformamide solution for activation, and then the Cystamine was added to anhydrous N,N-dimethylformamide solution, the resulting mixture was stirred under nitrogen, reacted, and purified to obtain compound 1. Compound 1 was added to trifluoroacetic acid and anhydrous dichloromethane. Stir to obtain compound 2, and then add compound 2 and dithiothreitol to methanol to obtain compound 3. 4. the preparation method of pH and heat-responsive CuAu nano-assembly according to claim 1, is characterized in that: carry out ligand exchange to the copper-gold nanoparticles coated with oleylamine: the copper-gold nanoparticles coated with oleylamine Disperse in a good solvent, add compound 3 dissolved in proportion and compound 4 with a thiol group at one end, react, cool, centrifuge, and add deionized water to obtain monodispersed copper-gold nanoparticles. 5. The preparation method of pH and heat-responsive CuAu nano-assemblies according to claim 1, characterized in that: the preparation of pH and heat-responsive copper-gold nano-assemblies: dissolved in p-phenylenediisothiocyanate In the good solvent, monodispersed copper-gold nanoparticles and an emulsifier were added, and after ultrasonic emulsification, sCuAu NAs were obtained by evaporation under reduced pressure. 6. The preparation method of pH and heat-responsive CuAu nano-assembly according to claim 1, characterized in that: in step (1), the particle size of the copper-gold nanoparticles coated with oleylamine is 6-8 nm, and the copper-gold nanoparticles have a particle size of 6-8 nm. The ratio is Cu:Au=0-5:1. 7. The preparation method of pH and heat-responsive CuAu nano-assembly according to claim 1 or 4, characterized in that: in step (3), the particle size of the monodispersed copper-gold nanoparticles is 6-8 nm; The solvent is selected from chloroform or tetrahydrofuran; compound 4 is a small hydrophilic molecule with a mercapto group at one end whose molecular weight is less than 1000 Da. 8. The preparation method of pH and heat-responsive CuAu nano-assemblies according to claim 5, wherein the good solvent is selected from the group consisting of chloroform, dichloromethane, cyclohexane and n-hexane or more; the emulsifier is selected from cetyltrimethylammonium bromide or sodium dodecyl sulfate; the hydrated particle size of the sCuAu NAs is 100-200 nm. 9. Application of the pH- and heat-responsive CuAu nanoassembly of any one of claims 1 to 8 in anti-drug-resistant bacteria or anti-bacterial biofilms or in the preparation of medicaments for treating wounds. 10. The application according to claim 9, wherein the bacteria comprise antibiotic-resistant strains of Escherichia coli, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, Salmonella, Bacillus diphtheriae, Bacillus anthracis, Escherichia coli Or methicillin-resistant Staphylococcus aureus.

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