CN114246844A

CN114246844A - Preparation method and application of pH and thermal response type CuAu nano assembly

Info

Publication number: CN114246844A
Application number: CN202111628189.5A
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: 2021-12-28
Publication date: 2022-03-29
Also published as: CN114404392A; 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

Chronic healing of wounds poses an increasing public health problem and creates a significant medical and economic burden. The presence of bacteria at the wound site and the release of endotoxin delays the healing process, thereby increasing the risk of morbidity and mortality. Studies have shown that biofilms are present in more than 90% of chronic wounds and can induce excessive inflammation, leading to long-term release of inflammatory cytokines and activation of immune complexes, thereby impairing skin wound healing. MRSA is a common biofilm-forming bacterium identified from chronic wounds, and MRSA confined in biofilms is protected by extracellular polymeric substances secreted by itself, and conventional methods are difficult to work. Therefore, multimodal antibacterial drugs that synergistically remove biofilms, accelerating the wound healing process, are urgent requirements for effective wound care.

Among the various materials resistant to wound infection, copper-based nanomaterials such as copper sulfide (CuS) and copper oxide (CuO) have received extensive attention in recent years. These nanomaterials have inherent photothermal properties and can generate macroscopic heat to destroy bacterial biofilms under light irradiation. Meanwhile, the released copper ions can further reduce bacterial infection, stimulate angiogenesis and promote wound healing. However, due to the complex distribution of nanomaterials, it is difficult to precisely control photothermal temperature in vivo, and excessive heating may damage surrounding tissues, prolonging healing time. However, the damage to the metal steady state from uncontrolled copper ion release can be toxic. Achieving thermal management and desired copper ion release accurately at the site of infection remains a challenge.

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 (namely sCuAu NAs) which has good biocompatibility and can promote wound healing.

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 with good biocompatibility and capability of promoting wound healing 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. Synthesizing sulfydryl-containing pH and thermal response type micromolecules: adding N-tert-butyloxycarbonyl-glycine, 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. Ligand exchange of oleylamine coated copper gold nanoparticles: dispersing 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 (Mono-CuAu NPs). 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, 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 by the oleylamine in the step (1) is 6-8nm, and the ratio of copper to gold is Cu: au is 0.3-3: 1. 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 group at one end and the molecular weight is less than 1000 Da. In the step (4), 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: 1.

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 Mono-CuAu NPs have certain absorption characteristics in a 650-950 nm 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 sCuAu NAs 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 antibacterial effect is cooperated with the photothermal therapy.

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 Mono-CuAu NPs;

fig. 5 is a transmission electron micrograph of cuau NAs at pH 7.4, 37 ℃ and pH5.5, 50 ℃;

fig. 6 is a graph of the particle size distribution of cuau NAs at pH 7.4, 37 ℃ and pH5.5, 50 ℃;

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

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

FIG. 9 is a graph of the inhibition curves for different concentrations of sCuAu NAs;

FIG. 10 is a photothermal antimicrobial graph of Mono-CuAu NPs and sCuAu NAs;

FIG. 11 is a graph of viable bacteria staining of biofilms after treatment with Mono-CuAu NPs and sCuAu NAs;

FIG. 12 is a graph showing the results of toxicity of sCuAu NA on HUVEC cells;

FIG. 13 is a graph of HUVEC cell migration after different treatments of sCuAu NAs;

FIG. 14 is a graph of wound healing in MRSA infected diabetic mice;

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

fig. 16 is a schematic diagram of nanoparticle synthesis and mechanism of action: (A) schematic diagram of the assembly and disassembly process of sCuAu NAs; (B) schematic diagram of an in vivo mechanism of sCuAu NAs mediated photothermal antibiosis and wound healing promotion.

Detailed Description

Example 1: preparation of oleylamine coated copper gold nanoparticles

(1) 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) 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) 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.

And performing the appearance characterization of the prepared oleylamine coated copper gold nanoparticles by using a transmission electron microscope, as shown in figure 1. Fig. 2 is a crystal analysis of cu-au nanoparticles by X-ray diffraction.

Example 2: 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) were added to an anhydrous N, N-dimethylformamide solution (10mL) in this order and activated for half an hour. Then adding cystamine (152.0mg and 1 mmol) into an anhydrous N, N-dimethylformamide solution, stirring the obtained mixture in nitrogen for 24h at room temperature, reacting at room temperature overnight under the condition of nitrogen protection, detecting the reaction progress by using a thin layer chromatography, removing the solvent by rotary evaporation when the raw material point on a chromatography plate disappears, namely the reaction is finished, dissolving the product in dichloromethane, washing with water for 3 times, purifying by using a column chromatography after drying (an eluent is dichloromethane: methanol is 10: 1) to obtain a compound 1, then dissolving the compound 1 in 5mL dichloromethane, adding 1mL trifluoroacetic acid into the dichloromethane, stirring at room temperature for 4h, removing the solvent by rotary evaporation to obtain a compound 2, then adding the compound 2 and dithiothreitol (309mg and 2mmol) into 5mL methanol, stirring at room temperature for 12-24 h, removing the solvent by rotary evaporation, dissolving the product in water, extracting with ethyl acetate for 3 times, drying, and purifying by column chromatography (eluent is B)

Nitrile: water 10: 1) to give 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 3: preparation of Mono-CuAu NPs

Dispersing 10mg of oleylamine coated copper gold nanoparticles in 1mL of tetrahydrofuran, adding 5mg of compound 3 and 9mg of compound 4 dissolved in DMF, reacting for 4h at 50 ℃, cooling, centrifuging at 3000rpm for 3 min, dissolving precipitate in DMF, centrifuging for 2 times, removing redundant small molecules, and dissolving precipitate in deionized water to obtain Mono-CuAu NPs. The Mono-CuAu NPs are characterized by the appearance by a transmission electron microscope, and the result is shown in figure 4, the particle size is 6-8nm, the particles are dispersed in an aqueous solution, and successful modification of the sulfydryl-containing micromolecules is shown.

Example 4: preparation of sCuAu NAs

Terephthalic isothiocyanate (18.3mg) was dissolved in chloroform (100uL), and after adding Mono-CuAu NPs (5mg, 1mL) and cetyltrimethylammonium bromide (4mg), ultrasonication was carried out, the chloroform was volatilized after overnight stirring at room temperature, and the excess terephthalic isothiocyanate was removed by centrifugation at 500rpm for 3 minutes, and the excess cetyltrimethylammonium bromide was removed by dialysis in a dialysis bag of 1000Da to give 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 pH5.5, 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 pH5.5, 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 pH5.5 and 50 ℃.

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

Example 5: photothermal performance test of sCuAu NAs

Mono-CuAu NPs (prepared as in example 4) and sCuAu NAs (prepared as in example 5) 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 Mono-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 Mono-CuAu NPs (46.65%).

Example 6: responsive release of copper ions

1mL of sCuAu NAs (prepared as in example 5) (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 pH5.5, 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 sCuAu NAs has substantially no copper release at 37 deg.C, pH 7.4, while the copper release rate in the buffer solution at 50 deg.C, pH5.5 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 7: killing and inhibiting effect of sCuAu NAs on planktonic drug-resistant bacteria

Taking staphylococcus aureus (with drug resistance to partial antibiotics) in logarithmic growth phase, and diluting the bacteria liquid to OD by using fresh liquid culture medium₆₀₀0.01, sCuAu NAs (prepared according to example 5) 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 incubation, with or without a 660nm laser (10 min, 1.0W/cm)²) The solution is irradiated and incubated for 18 hours, the absorbance at 600nm is measured on a microplate reader, the survival rate of the bacteria is measured, and the result is shown in figure 9, when the concentration is 20 mu g/mL and 40 mu g/mL, the sCuAu NAs basically kills more than 95% of the planktonic bacteria by adding laser irradiation, and when the laser is not added, the killing capacity of the sCuAu NAs to the bacteria is not obviously different from that of the sCuAu NAs at 20 mu g/mL and 40 mu g/mL, and the survival rate is about 60%, so the experimental concentration of the sCuAu NAs is 20 mu g/mL.

Example 8: photo-thermal sterilization effect of Mono-CuAu NPs and sCuAu NAs on planktonic drug-resistant bacteria

Taking staphylococcus aureus (with drug resistance to partial antibiotics) in logarithmic growth phase, and diluting the bacteria liquid to OD by using fresh liquid culture medium₆₀₀Mono-CuAu NPs (prepared as in example 4) and sCuAu NAs (prepared as in example 5) were diluted to 20. mu.g/mL in medium and 100. mu.L per well, and cultured in a 37 ℃ incubator with shaking at constant temperature. After 1 hour incubation, with or without a 660nm laser (10 min, 1.0W/cm)²) The solution is irradiated and continuously incubated for 12 hours, the absorbance at 600nm of the solution is measured on a microplate reader, the survival rate of bacteria is determined, and the result is shown in figure 10, Mono-CuAu NPs and sCuAu NAs can kill about 35% of planktonic bacteria without laser, sCuAu NAs of the laser irradiation group can kill 99% of bacteria, Mono-CuAu NPs can only kill about 60% of bacteria, so that the improvement of the photo-thermal efficiency is favorable for improving the photo-thermal antibacterial effect, and the sCuAu NAs have the excellent photo-thermal antibacterial effect.

Example 9: killing and inhibiting effect of sCuAu NAs on drug-resistant bacteria biofilm

Staphylococcus aureus (resistant to some antibiotics) in the logarithmic growth phase was collected, diluted to an OD600 of 0.05 with a fresh liquid medium, and inoculated into a 96-well plate at 200. mu.L per well. The culture was allowed to stand in a constant temperature incubator at 37 ℃ for 48 hours, and the culture was replaced with fresh culture 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. Mono-CuAu NPs (prepared as in example 4) and sCuAu NAs (prepared as in example 5) were diluted to 20. mu.g/mL with fresh medium, added to the biofilm in an amount of 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 incubation, with or without a 660nm laser (10 min, 1.0W/cm)²) The solution was irradiated and incubation continued for up to 12 hours, the biofilm was thoroughly washed and replaced with normal saline containing propidium iodide (PI, 54.9. mu.M) and SYTO 9 (5. mu.M). 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 was intact, PI was unable to penetrate the bacterial cell wall, while live bacteria were stained green by SYTO 9. As shown in FIG. 11, 99% or more of the saline group showed green fluorescence, and Mono-CuAu NPs and sCuAu NAs only destroyed about 20% of the biofilm without laser, while the laser-irradiated group was treated with sCuAu NAs, and then the biofilm had almost all the bacteria stained with PI, which showed red fluorescence, and the activity was significantly reduced, while Mono-CuAu NPs only destroyed about 50% of the biofilm, indicating that sCuAu NAs had a significant photothermal antibacterial biofilm effect.

Example 10: 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. 12, 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 11: cell migration assay for sCuAu NAs

First, sCuAu NAs (prepared according to example 5) and sCuAu NAs irradiated with 660nm laser (pH5.5, 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. 13 after 36h of addition of the above-mentioned streaked HUVEC cells. As can be seen from FIG. 13, 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 the sCuAu NAs not only can realize photothermal antibiosis, but also can promote wound healing.

EXAMPLE 12 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 8mm × 8mm perforator, and dripping methoxyl-resistant golden yellow grape with bacterial liquid concentration of 1 × 108CFU/mL20 mu L of coccus (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 sCuAu NAss-containing solution prepared above was applied to the wound surface of a diabetic mouse infected with MRSA for 1h, a 660nm laser (10 minutes, 1.0W/cm)²) Irradiation, 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 the administration treatment, as shown in fig. 14, fig. 14 is a wound surface healing graph of mice with diabetes infected by MRSA, and as can be seen from fig. 14, after the treatment for 10 days, 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 on mice at 0, 2, 4, 6, 8, 10 days after the above dosing treatment, as shown in fig. 15. Fig. 15 is a graph for measuring the size of the wound of a mouse with MRSA infected diabetic wound, and it can be seen from fig. 15 that the wound measurement data is substantially the same as the wound healing graph of fig. 14, and the cuau NAs + Laser group can heal the wound 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 response type CuAu nano-assembly according to claim 1, 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-glycine, 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 6-8nm, and the ratio of copper to gold is 0.3-3: 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 group at one end and the molecular weight 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, or methicillin-resistant staphylococcus aureus.