CN114404392A

CN114404392A - Preparation method and application of pH and thermal response type CuAu nano assembly

Info

Publication number: CN114404392A
Application number: CN202210201542.XA
Authority: CN
Inventors: 孙晓莲; 赵桂桢; 吴晓静
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-12-28
Filing date: 2022-03-02
Publication date: 2022-04-29
Anticipated expiration: 2042-03-02
Also published as: CN114246844A; CN114404392B

Abstract

The invention discloses a preparation method and application of a pH and thermal response type CuAu nano assembly, which comprises the following steps: preparing oleylamine coated copper gold nanoparticles; preparing pH and thermal response type micromolecules containing sulfydryl; ligand exchange is carried out on the copper-gold nano particles coated by oleylamine to obtain monodisperse copper-gold nano particles; preparing the pH and thermal response type copper-gold nano assembly. The pH and thermal response type copper-gold assembly has good photo-thermal performance, and reduces the damage to normal tissues around a wound due to overheating on the basis of realizing the photo-thermal anti-biofilm; meanwhile, the deep penetration and release of copper ions are promoted, and the effects of resisting bacteria and promoting wound healing are achieved.

Description

Preparation method and application of pH and thermal response type 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 thermal response type CuAu nano assembly.

Background

In recent years, the incidence of chronic wound healing complicated by diseases such as diabetes has been increasing, and this has become a major medical burden worldwide. When chronic wounds present superinfection, treatment often fails due to the presence of biofilm at the wound site and the release of endotoxin slowing the healing process. Biofilms are bacterial communities with Extracellular Polymeric Substance (EPS) matrixes formed by adhesion and proliferation of microorganisms, and extracellular polysaccharides in the EPS matrixes serve as physical barriers to resist attack of innate immune cells and penetration of antibiotics, so that the difficulty of traditional antibiotic treatment is greatly increased.

Different from traditional chemotherapy, photothermal therapy (PTT) utilizes the heat generated by the material under the irradiation of a near-infrared laser light source of 650-950nm to kill bacteria and destroy the biofilm structure, and is not easy to generate drug resistance. Inorganic nano materials such as silver, copper, zinc and the like have an antibacterial function, but the existing antibacterial materials have high toxicity and single function and cannot promote wound healing while resisting bacteria. Copper-based nano materials are receiving wide attention due to the inherent photo-thermal property and the effect of promoting wound healing. In addition, the copper ions on the wound surface can improve the antibacterial effect and promote the wound surface healing and the blood vessel regeneration. However, in photothermal therapy, delocalized heat and difficult to control temperature and ion release often cause great damage to healthy tissue. Therefore, there is an urgent clinical need to find a safe and effective treatment scheme that can rapidly remove biofilms while promoting healing of chronic wounds.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a preparation method of a pH and thermal response type CuAu nano assembly which has good biocompatibility and can promote wound healing.

The invention relates to a pH and thermal response type CuAu nano assembly based on an Edman degradation sequencing technology, which takes copper-gold nano particles as a main body, a surface modification compound 3 as a pH and thermal response type group, a compound 4 with a sulfydryl at one end as a hydrophilic functional group, and the pH and thermal response type CuAu nano assembly is formed through the chemical reaction of an amino group at the tail end of the compound 3 and p-phenylene isothiocyanate.

The invention also provides an application of the nano assembly.

The technical scheme is as follows: the preparation method of the pH and thermal response type CuAu nano assembly comprises the following steps:

(1) preparing oleylamine coated copper gold nanoparticles;

(2) preparing pH and thermal response type micromolecules containing sulfydryl;

(3) ligand exchange is carried out on the copper-gold nano particles coated by oleylamine to obtain monodisperse copper-gold nano particles;

(4) preparing the pH and thermal response type copper-gold nano assembly.

Further, synthesis of oleylamine coated copper gold nanoparticles: mixing copper acetylacetonate and oleylamine, heating, introducing inert gas, keeping the temperature for 30-60 minutes, adding the n-hexane solution of the gold nanoparticles growing secondarily into a reaction system, introducing the inert gas to remove oxygen and n-hexane, heating, continuously reacting, cooling to room temperature, precipitating the nanoparticles by using a polar solvent, and removing supernatant to obtain the oleylamine coated copper-gold nanoparticles.

Further, synthesis of pH and thermo-responsive small molecules containing sulfhydryl groups: adding N-tert-butyloxycarbonyl-amino acid, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide into an anhydrous N, N-dimethylformamide solution in sequence for activation, then adding cystamine into the anhydrous N, N-dimethylformamide solution, stirring the obtained mixture in nitrogen for reaction, purifying to obtain a compound 1, adding the compound 1 into trifluoroacetic acid and anhydrous dichloromethane, stirring at room temperature to obtain a compound 2, and then adding the compound 2 and dithiothreitol into methanol to obtain a compound 3. The amino acids of the N-t-butoxycarbonyl-amino acid include glycine, alanine, valine, leucine, isoleucine and the like α -amino acids having only one amino group and carboxyl group and no thiol group and disulfide bond.

Further, ligand exchange was performed on the oleylamine coated copper gold nanoparticles: dispersing the copper-gold nanoparticles coated with oleylamine in a good solvent, adding a compound 3 dissolved in proportion and a compound 4 with a mercapto group at one end, reacting, cooling, centrifuging, and adding deionized water to obtain monodisperse copper-gold nanoparticles (CuAu NPs).

Further, preparation of pH and thermal response type copper-gold nano-assembly: dissolving terephthalocyanurate in a good solvent, adding monodisperse copper-gold nanoparticles and an emulsifier, ultrasonically emulsifying, and evaporating under reduced pressure to obtain a pH and thermal response type copper-gold nano assembly (sCuAu NAs).

Further, the particle size of the copper-gold nanoparticles coated with oleylamine in the step (1) is 6-8 nm, and the ratio of copper to gold is 0-5: 1. In the step (2), the amino acids of the N-t-butoxycarbonyl-amino acid include glycine, alanine, valine, leucine, isoleucine and the like, which are α -amino acids having only one amino group and carboxyl group and having no thiol group and disulfide bond. In the step (3), the particle size of the monodisperse CuAu nanoparticles is 6-8 nm; the compound 4 is a hydrophilic small molecule with a sulfhydryl group at one end and the molecular weight is less than 1000 Da. The good solvent is selected from one or more of chloroform, dichloromethane, cyclohexane and n-hexane; the emulsifier is selected from cetyl trimethyl ammonium bromide or lauryl sodium sulfate; the hydrated particle size of the sCuAu NAs is 100-200 nm. The pH and thermal response type copper-gold nano assembly is applied to drug-resistant bacteria or antibacterial biofilm or preparation of a drug for treating wound. The bacteria include Escherichia coli, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

The nano assembly is prepared into a solution with a certain concentration to become a medicine for treating the wound, and the medicine is sprayed on the surface of the wound to resist bacteria and promote the wound healing.

Further, the preparation method of the gold nanoparticles with secondary growth comprises the following steps:

(1) dissolving tetrachloroaurate trihydrate into a mixed solution of oleylamine and 1, 2, 3, 4-tetrahydronaphthalene, introducing inert gas, keeping the temperature at 2-5 ℃ for 30-60 minutes to remove water vapor and oxygen in a reaction system, adding tetrabutylammonium bromide dissolved by oleylamine and 1, 2, 3, 4-tetrahydronaphthalene, continuously reacting at 2-5 ℃, precipitating nanoparticles by using a polar solvent, removing a supernatant to obtain gold nanoparticles, and dissolving the gold nanoparticles in n-hexane for later use.

(2) Dissolving tetrachloroauric acid trihydrate in a mixed solution of oleylamine and octadecene, introducing inert gas, preserving heat for 30-60 minutes, adding the gold nanoparticles dissolved in n-hexane in the step 1), introducing the inert gas to remove oxygen and the n-hexane, continuously reacting, cooling to room temperature, precipitating the nanoparticles by using a polar solvent, removing supernatant liquid to obtain secondarily grown gold nanoparticles, and dissolving the secondarily grown gold nanoparticles in the n-hexane for later use.

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

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

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

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

Has the advantages that: compared with the prior art, the invention has the following advantages:

1. the copper-gold nanoparticles can exist stably and are not easy to oxidize, the sCuAu NAs has good biocompatibility and exists stably under a neutral or weakly alkaline condition, and the sCuAu NAs can be assembled and dispersed under an acidic and heating condition, and the release of copper ions is increased.

2. The CuAu NPs have certain absorption characteristics in a 650-950nm area, and can realize temperature rise under the stimulation of 660nm laser. In addition, in order to achieve more efficient photo-thermal sterilization, the sccuau NAs was prepared to achieve a potential photo-thermal enhancement effect, and the photo-thermal conversion efficiency (η) was determined to be 66.32% according to the heating-cooling curve.

3. Because the sterilization effect of copper ions is limited, the prepared sCuAuNAs is rapidly heated through near infrared light irradiation, so that the assembly is promoted to be dissociated, the copper ions are released in an accelerated manner by heating, and the prepared sCuAuNAs and the photothermal therapy cooperate to realize antibiosis.

4. In an acidic microenvironment of a wound biofilm infection part, the sCuAu NAs can be responsively disassembled under laser irradiation, and when the photothermal anti-biofilm effect is exerted, damage of excessive temperature to surrounding normal tissues is avoided. And simultaneously, the release of copper ions is increased, and the wound healing is promoted.

Drawings

FIG. 1 is a transmission electron microscope image of oleylamine coated bronze nanoparticles;

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

FIG. 3 is a nuclear magnetic resonance spectrum of Compound 3;

FIG. 4 is a transmission electron micrograph of CuAuNPs;

fig. 5 is a transmission electron micrograph of sccaunas at pH 7.4, 37 ℃ and pH 5.5, 50 ℃;

fig. 6 is a graph of the particle size distribution of sccaunas at pH 7.4, 37 ℃ and pH 5.5, 50 ℃;

FIG. 7 shows the temperature rise-cooling curves and photothermal conversion efficiency of CuAu NPs and sCuAu NAs;

FIG. 8 is a copper ion release profile of sCuAu NAs under different conditions;

FIG. 9 is a bacteriostasis curve for different concentrations of sCuAuNAs;

FIG. 10 is a photothermal antimicrobial graph of CuAuNPs and sCuAuNAs against MRSA;

fig. 11 is a photothermal antimicrobial graph of CuAuNPs and cuaunas against e.coli;

FIG. 12 is a graph of staining of biofilm by viable bacteria after treatment with CuAu NPs and sCuAu NAs;

FIG. 13 is a graph showing the results of toxicity of sCuAuNA on HUVEC cells;

FIG. 14 is a graph of HUVEC cell migration after different treatments of sCuAuNAs;

FIG. 15 is a graph of wound healing in MRSA infected diabetic mice;

fig. 16 is a graph for measuring the size of the wound surface of MRSA-infected diabetic wound mice.

Detailed Description

Example 1: preparation of oleylamine coated copper gold nanoparticles

(1) Preparing gold nanoparticles: dissolving 200mg of tetrachloroaurate trihydrate into a mixed solution of 10mL of oleylamine and 10mL of 1, 2, 3, 4-tetrahydronaphthalene, introducing inert gas, keeping the temperature at 2-5 ℃ for 30-60 minutes to remove water vapor and oxygen in a reaction system, adding 90mg of tetrabutylammonium bromide dissolved in a mixed solution of 1mL of oleylamine and 1mL of 1, 2, 3, 4-tetrahydronaphthalene, changing the solution from golden yellow to purple, continuously reacting at 2-5 ℃ for 1 hour, precipitating nanoparticles by using acetone, centrifuging at 8000rpm for 5 minutes, discarding the supernatant, dissolving the precipitate in n-hexane, and centrifuging and precipitating by using ethanol to obtain the gold nanoparticles.

(2) Preparing secondary growth gold nanoparticles: dissolving 100mg of tetrachloroaurate trihydrate in a mixed solution of 6mL of oleylamine and 6mL of octadecene, introducing inert gas, preserving heat at 80 ℃ for 30-60 minutes, adding the gold nanoparticles dissolved in n-hexane in the step (1), introducing the inert gas to remove oxygen and the n-hexane, continuously reacting for 2 hours, cooling to room temperature, precipitating the nanoparticles with isopropanol, centrifuging at 8000rpm for 5 minutes, discarding supernatant, dissolving the precipitate in the n-hexane, and centrifuging and precipitating with ethanol to obtain secondarily grown gold nanoparticles.

(3) Preparing oleylamine coated copper gold nanoparticles: placing 40mg of copper acetylacetonate and 6mL of oleylamine in a four-necked bottle, heating to 80 ℃, introducing inert gas, preserving heat for 30-60 minutes, adding the n-hexane solution of the gold nanoparticles secondarily grown in the step (2) into a reaction system, introducing the inert gas to remove oxygen and the n-hexane, heating to 210 ℃ at the speed of 3 ℃/minute, continuously reacting for 1 hour, cooling to room temperature, precipitating the nanoparticles with ethanol, centrifuging at 8000rpm for 5 minutes, discarding the supernatant, dissolving the precipitate in the n-hexane, and repeatedly cleaning for three times to obtain the oleylamine-coated copper-gold nanoparticles. Wherein, the copper-gold ratio of the copper-gold nano-particles coated by the oleylamine is about 1: 1 relative to 1: Au. The appearance of the oleylamine coated cu — au nanoparticles prepared in this example was characterized by a transmission electron microscope, as shown in fig. 1. Fig. 2 is a crystal analysis of cu-au nanoparticles by X-ray diffraction.

Example 2: preparation of oleylamine coated copper gold nanoparticles

The preparation process of example l is referred to for synthesis, except that 0-200 mg of copper acetylacetonate added in the step (3) is changed to obtain oleylamine coated copper gold nanoparticles. The copper-gold ratio of the oleylamine coated copper-gold nanoparticles is 0-5: 1 to 0-5: 1.

Example 3: synthesis of sulfhydryl-containing pH and thermal response type small molecule

N-tert-Butoxycarbonyl-glycine (525mg, 3mmol), 1-ethyl- (3-dimethylaminopropyl) carbodiimides hydrochloride (1719mg, 9mmol) and N-hydroxysuccinimide (1035mg, 9mmol 1) were sequentially added to an anhydrous N, N-dimethylformamide solution (10mL) and activated for half an hour. Cystamine (152.0mg, 1mmol) was then added to the anhydrous N, N-dimethylformamide solution and the resulting mixture was stirred under nitrogen at room temperature for 24 h. Reacting overnight at room temperature under the protection of nitrogen, detecting the reaction progress by using a thin layer chromatography, and finishing the reaction when the raw material point on the chromatography plate disappears. The solvent was removed by rotary evaporation and the product was dissolved in dichloromethane, washed 3 times with water, dried and purified by column chromatography (eluent dichloromethane: methanol 10: 1) to give compound 1. Subsequently, compound 1 was dissolved in 5mL of dichloromethane, 1mL of trifluoroacetic acid was added thereto, stirred at room temperature for 4 hours, and the solvent was removed by rotary evaporation to give compound 2. Then, compound 2 and dithiothreitol (309mg, 2mmol) are added into 5mL of methanol, stirring is carried out for 12-24 h at room temperature, the solvent is removed by rotary evaporation, the product is dissolved in water, extraction is carried out for 3 times by ethyl acetate, and purification is carried out by column chromatography after drying (eluent is acetonitrile: water is 10: 1), so as to obtain compound 3. Drying and purifying to obtain the compound 3. The synthetic route of the compound is as follows:

nuclear magnetic data for compound 3:¹H NMR(300MHz，DMSO-d₆)δ1.31(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 nuclear magnetic resonance spectrum of the compound 3 is shown in fig. 3, which shows that the pH and the thermal response micromolecules are successfully synthesized.

Example 4: synthesis of sulfhydryl-containing pH and thermal response type small molecule

The synthesis was performed in accordance with the preparation process of example 3, except that the amino acids in N-t-butoxycarbonyl-glycine were replaced with alpha-amino acids having only one amino group and carboxyl group and no thiol group and disulfide bond, such as alanine, valine, leucine, and isoleucine, respectively.

Example 5: preparation of CuAuNPs

1mM oleylamine coated copper gold nanoparticles are dispersed in 10mL tetrahydrofuran, 1mM compound 3 dissolved in DMF and 1.5mM compound 4 with one end modified by sulfydryl are added, the mixture reacts for 4h at 50 ℃, the mixture is cooled and centrifuged at 3000rpm for 3 min, the precipitate is dissolved in DMF and then centrifuged for 2 times, redundant micromolecules are removed, and the precipitate is dissolved in deionized water to obtain CuAu NPs. And (3) carrying out appearance characterization on the CuAu NPs by using a transmission electron microscope, wherein the result is shown in figure 4, the particle size is 6-8 nm, and the CuAu NPs are dispersed in an aqueous solution, so that successful modification of the sulfydryl-containing micromolecules is shown.

Example 6: preparation of sCuAuNAs

Dissolving terephthalocyanuric acid diisocyanate (5mM) in chloroform (1mL), adding CuAu NPs (1mM, 10mL) and cetyl trimethylammonium bromide as an emulsifier, sonicating, stirring overnight at room temperature, then volatilizing the chloroform, centrifuging at 500rpm for 3 minutes to remove excess terephthalocyanuric acid isothiocyanate, dialyzing against a 1000Da dialysis bag to remove excess cetyl trimethylammonium bromide, and obtaining sCuAu NAs.

The particle size distribution of the sCuAu NAs was analyzed by dynamic light scattering at different temperatures and different pH, and the results are shown in Table 1. The morphology of the resulting cuau NAs was characterized by a transmission electron microscope at pH 7.4, 37 ℃ and pH 5.5, 50 ℃ as shown in fig. 5, and the particle size distribution of the cuau NAs was analyzed by dynamic light scattering at pH 7.4, 37 ℃ and pH 5.5, 50 ℃ as shown in fig. 6. The above results all prove that the sCuAu NAs has good pH and thermal responsiveness, and is completely disassembled under the conditions of pH 5.5 and 50 ℃.

TABLE 1 variation of sCuAu NAs particle size at different temperatures and different pH

Example 7: preparation of sCuAuNAs

The synthesis was performed according to the preparation process of example 6, except that the emulsifier added was sodium lauryl sulfate.

Example 8: photothermal performance test of sCuAu NAs

CuAu NPs (prepared as in example 5) and sCuAu NAs (prepared as in example 6) were diluted with deionized water to make up a 15. mu.g/mL solution. And (3) irradiating the two solutions for 5 minutes by a 660nm near-infrared light emitter, closing the laser, naturally cooling the solutions to room temperature, and recording the real-time temperatures of the different solutions by using an infrared thermal imager. The heating-cooling curves of CuAu NPs and sCuAu NAs are at 1.0W/cm²And (4) obtaining the product. The photothermal conversion efficiency (η) is calculated by the following formula:

equation 1

Equation 2

Equation 3

Q₀＝hS(T_max，water-T_surr)

τ_sIs calculated from a linear regression curve in the cooling curve, m_dAnd C_dThe mass (1g) and heat capacity (4.2J/(g. K)) of the solution were respectively expressed, and thus the value of hS was obtained, and then Q was calculated by the formula 3₀Representing the background energy input in the absence of MPDA nanoparticles. Wherein T is_max，waterAnd T_surrRespectively representing the steady state maximum temperature of water and the ambient room temperature. Thus, in determining hS and Q₀After the value, the photothermal conversion efficiency can be calculated according to equation 1. T is_maxThe maximum temperature at which the solution is stable, I and A₆₆₀Respectively, the laser power (1.0W) and the absorbance of the nanoparticles at 660 nm. As a result, as shown in FIG. 7, the photothermal conversion efficiency of sCuAu NAs was improved by 66.32% as compared with CuAu NPs (46.65%).

Example 9: responsive release of copper ions

1mL of sCuAu NAs (prepared as in example 6) (2mg/mL) was measured accurately and placed in a dialysis bag with Mw 1000, the bag was immersed in 50mL of a buffer solution at 37 ℃ and 50 ℃ at pH 7.4 or pH 5.5, and at the corresponding time point 1mL of the buffer solution was removed and supplemented with 1mL of a blank buffer solution of the phase acidity. And after sampling, measuring the copper content in each sample by using inductively coupled plasma chromatography, and further calculating the copper release amount. The calculation formula is as follows:

R_t＝C_t×50/2×100％

wherein R is_tIs the copper release rate at the response time point, C_tIs the copper concentration in the solution taken at this time point in units of: mg/mL.

Subsequently, by plotting the release rate against time, fig. 8 was obtained. It can be observed that the sCuAuNAs has substantially no copper release at 37 ℃ at pH 7.4, whereas the copper release rate in a buffer solution at 50 ℃ at pH 5.5 can exceed 10%. The copper release rate of the sCuAu NAs in the buffer solution with the pH value of 5.5 or 50 ℃ is lower than 5 percent. This demonstrates 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: killing and inhibiting effect of sCuAu NAs on planktonic bacteria

Taking methicillin-resistant staphylococcus aureus (MRSA) in logarithmic growth phase, and diluting the bacterial liquid to OD (OD) by using a fresh liquid culture medium₆₀₀0.01, sCuAu NAs (prepared according to example 6) was diluted with physiological saline to different concentrations, added to the bacterial suspension and mixed so that the final concentrations were 0. mu.g/mL, 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL and 40. mu.g/mL, respectively, and 100. mu.L per well was cultured in a constant temperature shaking incubator at 37 ℃. After 1 hour of incubation, the solution was irradiated with or without 660nm laser (10 minutes, 1.0W/cm2), incubation was continued for 18 hours, and the absorbance at 600nm was measured on a microplate reader to determine the survival rate of the bacteria, as shown in FIG. 9, at concentrations of 20. mu.g/mL and 40. mu.g/mL, sCuAu NAs was irradiated with laser to substantially kill 95% or more of the planktonic bacteria, and when no laser was applied, sCuAu NAs had no significant difference in the killing power against bacteria at 20. mu.g/mL and 40. mu.g/mL, and the survival rate was about 60%, so that the experimental concentration of sCuAu NAs was 20. mu.g/mL thereafter.

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

Taking methicillin-resistant staphylococcus aureus in logarithmic growth phase, and diluting the bacterial liquid to OD by using a fresh liquid culture medium₆₀₀CuAuNPs (prepared according to example 5) and scaunas (prepared according to example 6) were taken, diluted to 20 μ g/mL with medium, 100 μ L per well, and cultured in a 37 ℃ incubator with shaking at a constant temperature. After 1 hour of incubation, the solution was irradiated with or without 660nm laser (10 minutes, 1.0W/cm2), incubation was continued for 12 hours, absorbance at 600nm was measured on a microplate reader, and the survival rate of the bacteria was determined by densitometry, with the results shown in FIG. 10, CuAuNPs and sCuAuNAs both kill about 35% of planktonic bacteria without laser, whereas the laser-irradiated group sCuAu NAs kills 99% of bacteria and CuAu NPs kills only about 60% of bacteria, which fully shows that the improvement of photothermal efficiency is favorable for improving photothermal and anti-viral propertiesThe bacteria effect, sCuAu NAs has excellent photo-thermal antibacterial effect.

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

An experiment was performed with reference to the protocol of example 11, except that the bacterium used was escherichia coli (e. The survival rate of bacteria was determined by densitometry and the results are shown in figure 11. This shows that photothermal therapy of cuau NAs has broad spectrum antibacterial effects.

In order to prove the broad-spectrum antibacterial effect of the sCuAu NAs photo-thermal treatment, the used bacteria also comprise antibiotic resistant strains of pseudomonas aeruginosa, streptococcus pneumoniae, staphylococcus aureus, salmonella, diphtheria bacillus, bacillus anthracis and escherichia coli, and the survival rate of the treated bacteria is respectively determined by a densitometry to verify that the sCuAu NAs has good photo-thermal treatment effect.

Example 13: killing inhibition of bacterial biofilms by sCuAu NAs

Taking methicillin-resistant staphylococcus aureus in logarithmic growth phase, and diluting the bacterial liquid to OD by using a fresh liquid culture medium₆₀₀The cells were inoculated into a 96-well plate at 200. mu.L/well, and incubated at 37 ℃ for 48 hours while leaving the plate static in a constant temperature incubator, and the culture medium was replaced with fresh medium every 24 hours. After the biofilm formed by the bacteria, the culture solution was discarded, and the biofilm was rinsed with physiological saline to wash away floating bacteria and bacteria desorbed from the biofilm. CuAu NPs (prepared according to example 5) and sCuAu NAs (prepared according to example 6) were diluted to 20. mu.g/mL with fresh medium, added to the biofilm at 200. mu.L per well, and incubated at 37 ℃ in a constant temperature incubator. Meanwhile, blank medium was used as a control group. After 1 hour of incubation, the solution was irradiated with or without 660nm laser (10 min, 1.0W/cm2), incubation was continued for 12 hours, the biofilm was thoroughly washed, and the physiological saline containing propidium iodide (PI, 54.9. mu.M) and SYTO 9 (5. mu.M) was replaced. After incubation at 37 ℃ for 20 minutes, the fluorochrome was washed off and the bacterial biofilm was observed under a fluorescent microscope. PI dye can stain dead bacteria, SYTO 9 can stain live bacteria and dead bacteria simultaneously. When the biofilm is intact, PI cannot penetrate the bacterial cell wall, while the viable bacteria are coveredSYTO 9 was stained green. As shown in FIG. 12, 99% or more of the saline group showed green fluorescence, and CuAu NPs and sCuAu NAs only destroyed about 20% of the biofilm without laser, while the laser-irradiated group treated with sCuAu NAs showed substantially all of the bacteria in the biofilm stained with PI, exhibited red fluorescence, and significantly reduced activity, and CuAu NPs only destroyed about 50% of the biofilm, indicating that sCuAu NAs had a significant photothermal antibacterial biofilm effect.

Example 14: cytotoxicity assays for sCuAu NAs

HUVEC cells were seeded in 96-well plates and cultured overnight. sCuAu NAs (1.25, 2.5, 5, 10, 20, 40. mu.g/mL) were added when the cells grew to 80% confluence, in triplicate per concentration, in blank medium as a control. After 24h of culture, removing the culture medium containing the nanoparticles, adding a pre-prepared MTT solution (5mg/mL) into each well, and continuously placing 150 mu L of each well in an incubator for incubation for 4 h; the MTT solution was carefully removed, 150. mu.L of dimethyl sulfoxide (DMSO) was added to each well, the wells were shaken well for 3 minutes, the OD value of the absorbance at 492nm of each well was measured using a microplate reader, and the cell viability was calculated by taking the average OD value of three duplicate wells as the OD value of the target sample:

cell viability ═ sample_ODBlank control group_OD×100％

As shown in FIG. 13, the sCuAu NAs is slightly toxic at a concentration of 40. mu.g/mL, and is substantially non-toxic at concentrations of 20. mu.g/mL or less, indicating that biological safety can be ensured at an antibacterial concentration of 20. mu.g/mL.

Example 15: cell migration assay for sCuAu NAs

First, sCuAu NAs (prepared according to example 6) and sCuAu NAs irradiated with 660nm laser (pH 5.5, 10 minutes) were diluted in a basal medium (DMEM) containing 1% serum to prepare two solutions. HUVEC cells were cultured in 12-well plates for 24h, and when cell growth was completely confluent, cells were scored using a 200. mu.L tip and cell debris was washed off with blank medium. Then, 800 μ L of the prepared solution was added to each well, wherein Control group was blank group and no drug treatment was added, sCuAu NAs group was added with 20 μ g/mL solution containing sCuAu NAs, and sCuAu NAs + Laser group was added with 20 μ g/mL solution containing sCuAu NAs (subjected to photo-thermal treatment for 10 minutes). The results of cell migration of each group are shown in FIG. 14, after 36h of addition of the above-mentioned streaked HUVEC cells. As can be seen from FIG. 14, after the HUVEC cells are scratched for 36h, the Control group only has a small amount of cell migration, while the cell migration of the sCuAu NAs + Laser group is obvious and increased by about 30% compared with the blank group, and the cells of the sCuAu NAs group also have the cell migration promoting ability, but the effect is not obvious as the sCuAu NAs + Laser group, which indicates that the copper ions can promote the cell migration. This means that sCuAuNAs not only can realize photothermal antibiosis, but also can promote wound healing.

Example 16 in vivo wound healing experiments with sCuAu NAs

1. Experimental part: selecting 22-25g male BALB/c mice, and carrying out intraperitoneal injection STZ molding to carry out type I diabetes molding; preparing wound surface of diabetic mouse with 8 mm-8 mm perforator, and dripping bacteria solution with concentration of 1 × 10⁸20 mu L of CFU/mL of MRSA bacterial liquid, and preparing the wound surface of the diabetic mouse seriously infected by the drug-resistant bacteria after 48 hours. Grouping mice successfully modeled: the wound surface of a diabetic mouse infected with MRSA was given 100 μ L of physiological Saline, which was called a Saline group and was also a control group in this experiment. The wound surface of a diabetic mouse infected with MRSA was administered 100. mu.L of the above-prepared 20. mu.g/mL sCuAu NAs-containing solution, which was referred to as sCuAu NAs group. After 100. mu.L of the 20. mu.g/mL sCuAuNAss-containing solution prepared above was incubated for 1h on the wound surface of a diabetic mouse infected with MRSA, it was irradiated with a 660nm Laser (10 minutes, 1.0W/cm2), and the resulting wound surface was designated as sCuAu NAs + Laser group.

2. Taking a picture of wound healing: wound surface photographing records are carried out 0, 2, 4, 6, 8 and 10 days after the mice are subjected to administration treatment, as shown in fig. 15, fig. 15 is a wound surface healing graph of mice with diabetes infected by MRSA, and as can be seen from fig. 15, after 10 days of treatment, the wound area of the single sCuAu NAs group is reduced, but the wound is not healed, which indicates that the single repairing effect is not good enough; it is worth mentioning that the sCuAu NAs + Laser group combines the dual effects of photo-thermal antibiosis and copper ion promotion of wound healing, the synergistic effect is exerted, the wound is basically healed, and the MRSA infection diabetic wound surface is repaired to obtain the optimal effect.

3. Measuring the size of the wound surface: the wound size was measured with a vernier caliper in mice at 0, 2, 4, 6, 8, 10 days after the above dosing treatment, as shown in fig. 16. Fig. 16 is a graph for measuring the size of the wound surface of a mouse with MRSA infected diabetic wound surface, and it can be seen from fig. 16 that the wound surface measurement data is basically consistent with the wound surface healing graph result of fig. 15, and the cuau NAs + Laser group can heal the wound surface in a shorter time.

Claims

1. A preparation method of a pH and thermal response type CuAu nano assembly is characterized by comprising the following steps: the method comprises the following steps:

(1) preparing oleylamine coated copper gold nanoparticles;

(2) preparing pH and thermal response type micromolecules containing sulfydryl;

(4) preparing the pH and thermal response type copper-gold nano assembly.

2. The method of preparing a pH and thermal responsive CuAu nano-assembly according to claim i, wherein: synthesizing oleylamine coated copper gold nanoparticles: mixing copper acetylacetonate and oleylamine, heating, introducing inert gas, keeping the temperature for 30-60 minutes, adding the n-hexane solution of the gold nanoparticles growing secondarily into a reaction system, introducing the inert gas to remove oxygen and n-hexane, heating, continuously reacting, cooling to room temperature, precipitating the nanoparticles by using a polar solvent, and removing supernatant to obtain the oleylamine coated copper-gold nanoparticles.

3. The method of preparing a pH and thermal response type CuAu nano-assembly according to claim 1, wherein: synthesizing sulfydryl-containing pH and thermal response type micromolecules: adding N-tert-butyloxycarbonyl-amino acid, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide into an anhydrous N, N-dimethylformamide solution in sequence for activation, then adding cystamine into the anhydrous N, N-dimethylformamide solution, stirring the obtained mixture in nitrogen for reaction, purifying to obtain a compound 1, adding the compound 1 into trifluoroacetic acid and anhydrous dichloromethane, stirring at room temperature to obtain a compound 2, and then adding the compound 2 and dithiothreitol into methanol to obtain a compound 3.

4. The method of preparing a pH and thermal response type CuAu nano-assembly according to claim 1, wherein: ligand exchange of oleylamine coated copper gold nanoparticles: dispersing the copper-gold nanoparticles coated with oleylamine in a good solvent, adding a compound 3 dissolved in proportion and a compound 4 with a mercapto group at one end, reacting, cooling, centrifuging, and adding deionized water to obtain the monodisperse copper-gold nanoparticles.

5. The method of preparing a pH and thermal response type CuAu nano-assembly according to claim 1, wherein: preparing a pH and thermal response type copper-gold nano assembly: dissolving terephthalocyanurate in a good solvent, adding monodisperse copper-gold nanoparticles and an emulsifier, carrying out ultrasonic emulsification, and carrying out reduced pressure evaporation to obtain sCuAu NAs.

6. The method of preparing a pH and thermal response type CuAu nano-assembly according to claim 1, wherein: the particle size of the copper-gold nanoparticles coated by the oleylamine in the step (1) is 6-8 nm, and the ratio of copper to gold is 0-5: 1.

7. The method for preparing a pH and thermal response type CuAu nano-assembly according to claim 1 or 4, wherein: in the step (3), the particle size of the monodisperse CuAu nanoparticles is 6-8 nm; the good solvent is selected from trichloromethane or tetrahydrofuran; the compound 4 is a hydrophilic small molecule with a sulfhydryl at one end and the molecular weight of the hydrophilic small molecule is less than 1000 Da.

8. The method of preparing a pH and thermal responsive CuAu nano-assembly according to claim 5, wherein: the good solvent is selected from one or more of trichloromethane, dichloromethane, cyclohexane and n-hexane; the emulsifier is selected from cetyl trimethyl ammonium bromide or lauryl sodium sulfate; the hydrated particle size of the sCuAu NAs is 100-200 nm.

9. Use of a pH and thermal responsive CuAu nano-assembly according to any one of claims 1 to 8 in a drug resistant bacteria or antibacterial biofilm or in the preparation of a medicament for the treatment of a wound.

10. The use of claim 9, wherein the bacteria comprise escherichia coli, pseudomonas aeruginosa, streptococcus pneumoniae, staphylococcus aureus, salmonella, diphtheria, bacillus anthracis, antibiotic-resistant strains of escherichia coli, or methicillin-resistant staphylococcus aureus.