CN114404392B

CN114404392B - Preparation method and application of pH and thermal response type CuAu nano-assembly

Info

Publication number: CN114404392B
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: 2023-06-20
Anticipated expiration: 2042-03-02
Also published as: CN114246844A; CN114404392A

Abstract

The invention discloses a preparation method and application of a pH and thermal response CuAu nano-assembly, comprising the following steps: preparing copper gold nanoparticles coated with oleylamine; preparing a thiol-containing pH and thermally responsive small molecule; ligand exchange is carried out on the copper gold nanoparticles coated by the oleylamine to obtain monodisperse copper gold nanoparticles; preparing the pH and thermal response copper gold nano-assembly. The pH and thermal response type copper-gold assembly has good photo-thermal performance, and reduces damage to normal tissues around a wound due to overheating on the basis of realizing a photo-thermal anti-biological film; simultaneously promote the deep penetration and release of copper ions, and play roles of resisting bacteria and promoting wound healing.

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 CuAu nano-assembly.

Background

In recent years, the incidence of chronic wound healing accompanied by diseases such as diabetes has been increasing, and this has become a major medical burden worldwide. When chronic wounds develop overlapping infections, the treatment often fails due to the presence of biofilm at the wound site and release of endotoxin which delays the healing process. The biofilm is a bacterial community formed by microbial adhesion and proliferation and provided with Extracellular Polymer (EPS) matrix, and extracellular polysaccharide in the extracellular polymer is used as a physical barrier for resisting the attack of innate immune cells and the permeation of antibiotics, so that the difficulty of the traditional antibiotic treatment is greatly increased.

Unlike conventional chemotherapy, photothermal therapy (PTT) uses heat generated by a material under irradiation of a near infrared laser light source of 650-950nm to kill bacteria and destroy biofilm structure, and is not prone to drug resistance. Inorganic nano materials such as silver, copper, zinc and the like have antibacterial functions, but the existing antibacterial materials have higher toxicity and single function, and can not promote wound healing while resisting bacteria. Copper-based nanomaterials have received much attention due to their inherent photo-thermal properties and wound healing promoting effects. In addition, copper ions at the wound surface can improve the antibacterial effect and promote wound healing and angiogenesis. However, in photothermal therapy, delocalized heat and difficult-to-control temperatures and ion release often cause significant damage to healthy tissue. Thus, there is a great clinical need to find a safe and effective treatment regimen that can rapidly clear biofilm while promoting chronic wound healing.

Disclosure of Invention

The invention aims to: the invention aims to provide a preparation method of a pH and thermal response 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 Edman degradation sequencing technology, which takes copper gold nano-particles as a main body, wherein a surface modification compound 3 is a pH and thermal response type group, a compound 4 with a sulfhydryl group at one end is a hydrophilic functional group, and the pH and thermal response type CuAu nano-assembly is formed through the chemical reaction of the terminal amino group of the compound 3 and terephthalyl isothiocyanate.

The invention also provides application of the nano assembly.

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

(1) Preparing copper gold nanoparticles coated with oleylamine;

(2) Preparing a thiol-containing pH and thermally responsive small molecule;

(3) Ligand exchange is carried out on the copper gold nanoparticles coated by the oleylamine to obtain monodisperse copper gold nanoparticles;

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

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

Further, synthesis of thiol-containing pH and thermally responsive small molecules: n-tert-butoxycarbonyl-amino acid, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are sequentially added into an anhydrous N, N-dimethylformamide solution for activation, cystamine is added into the anhydrous N, N-dimethylformamide solution, the obtained mixture is stirred in nitrogen for reaction, purification is carried out to obtain a compound 1, trifluoroacetic acid and anhydrous dichloromethane are taken to add the compound 1 into the mixture, stirring is carried out at room temperature to obtain a compound 2, and then the compound 2 and dithiothreitol are added into methanol to obtain a compound 3. The amino acid of the N-t-butoxycarbonyl-amino acid includes glycine, alanine, valine, leucine, isoleucine and the like as alpha-amino acids having only one amino group and carboxyl group and no mercapto group and disulfide bond.

Further, ligand exchange is carried out on the copper gold nanoparticles coated by the oleylamine: dispersing the copper gold nanoparticles coated by the oleylamine in a good solvent, adding a compound 3 and a compound 4 with a sulfhydryl group at one end which are dissolved in proportion, reacting, cooling, centrifuging, and adding deionized water to obtain monodisperse copper gold nanoparticles (CuAu NPs).

Further, preparation of pH and thermally responsive copper gold nanoparticle assemblies: dissolving terephthalyl isothiocyanate in a good solvent, adding monodisperse copper gold nanoparticles and an emulsifier, performing ultrasonic emulsification, and performing reduced pressure evaporation to obtain a pH and thermal response copper gold nanoparticle assembly (sCuAu NAs).

Further, the particle size of the oleylamine coated copper gold nanoparticles in the step (1) is 6-8 nm, and the copper-gold ratio is Cu/Au=0-5:1. In the step (2), the amino acid of the N-t-butoxycarbonyl-amino acid includes an alpha-amino acid having only one amino group and carboxyl group and no mercapto group and disulfide bond, such as glycine, alanine, valine, leucine and isoleucine. In the step (3), the particle size of the monodisperse copper gold nanoparticles is 6-8 nm; compound 4 is a small hydrophilic molecule with one end having a molecular weight of less than 1000Da and a thiol group. The good solvent is one or more selected from chloroform, dichloromethane, cyclohexane and n-hexane; the emulsifier is selected from cetyl trimethyl ammonium bromide or sodium dodecyl sulfate; the hydration particle size of sCuAu NAs is 100-200 nm. The pH and thermal response copper-gold nano-assembly is applied to resisting drug-resistant bacteria or antibacterial biological films or preparing drugs for treating wound. The bacteria include Escherichia coli, pseudomonas aeruginosa, streptococcus pneumoniae, staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

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

Further, the preparation method of the secondarily-grown gold nanoparticles comprises the following steps:

(1) Dissolving tetrachloro-gold acid trihydrate in a mixed solution of oleylamine and 1,2,3, 4-tetrahydronaphthalene, introducing inert gas, preserving heat for 30-60 minutes at 2-5 ℃ to remove water vapor and oxygen in a reaction system, adding tetrabutylammonium bromide dissolved by the oleylamine and the 1,2,3, 4-tetrahydronaphthalene, changing the solution into purple from golden yellow, continuously reacting at 2-5 ℃, precipitating nano particles by a polar solvent, removing supernatant liquid to obtain gold nano particles, and dissolving the gold nano particles in n-hexane for standby.

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

Preferably, the feeding ratio of the trichlorogold 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 trichlorogold 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 the copper acetylacetonate, the oleylamine and the 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.

The beneficial effects are that: compared with the prior art, the invention has the following advantages:

1. the copper gold nanoparticles can exist stably, are not easy to oxidize, sCuAu NAs have good biocompatibility, exist stably under neutral or weak alkaline conditions, are assembled and dispersed under acidic and heating conditions, and meanwhile copper ion release is increased.

2. The CuAu NPs in the invention have certain absorption characteristics in the 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, sCuAu NAs was prepared to achieve potential photo-thermal enhancement, and the photo-thermal conversion efficiency (. Eta.) was determined to be 66.32% according to the heating-cooling curve.

3. Because the sterilizing effect of copper ions is limited, the prepared sCuAuNAs is rapidly heated by near infrared light irradiation, so that the assembly is promoted to be dissociated, the copper ions are released by heating in an accelerating way, and the sCuAuNAs and the photothermal therapy are synergistic for sterilization.

4. In the acidic microenvironment of the wound biofilm infection part, sCuAu NAs can be responsively disassembled under the irradiation of laser, and when the photothermal anti-biofilm function is exerted, the damage of the excessive temperature to surrounding normal tissues is avoided. And copper ions are released more, so that wound healing is promoted.

Drawings

FIG. 1 is a transmission electron microscope image of an oleylamine coated copper gold nanoparticle;

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

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

FIG. 4 is a transmission electron microscope image of CuAuNPs;

fig. 5 is a transmission electron micrograph of cuaunas at ph=7.4, 37 ℃ and ph=5.5, 50 ℃;

fig. 6 is a graph of particle size distribution of cuaunas at ph=7.4, 37 ℃ and ph=5.5, 50 ℃;

FIG. 7 is a graph showing the temperature rise-cooling curves and the photothermal conversion efficiency of CuAu NPs and sCuAu NAs;

FIG. 8 is a graph showing the copper ion release profile of sCuAu NAs under various conditions;

fig. 9 shows the bacteriostatic curves for different concentrations of cuaunas;

fig. 10 is a photothermal antimicrobial diagram of CuAuNPs and sgaunans against MRSA;

FIG. 11 is a photothermal antimicrobial diagram of CuAuNPs and sCuAuNAs versus E.coli;

FIG. 12 is a live-dead bacterial staining of biofilms after CuAu NPs and sCuAu NAs treatment;

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

FIG. 14 is a graph of HUVEC cell migration following various treatments of sCuAuNAs;

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

fig. 16 is a graph showing wound size measurement of MRSA infected diabetic mice.

Detailed Description

Example 1: preparation of copper gold nanoparticles coated with oleylamine

(1) Preparation of gold nanoparticles: 200mg of tetrachloroauric acid trihydrate is dissolved in a mixed solution of 10mL of oleylamine and 10mL of 1,2,3, 4-tetrahydronaphthalene, inert gas is introduced, the temperature is kept at 2-5 ℃ for 30-60 minutes to remove water vapor and oxygen in a reaction system, 90mg of tetrabutylammonium bromide dissolved in the mixed solution of 1mL of oleylamine and 1mL of 1,2,3, 4-tetrahydronaphthalene is added, the solution turns from golden yellow to purple, after the continuous reaction for 1h at 2-5 ℃, nano particles are precipitated by acetone, the supernatant is removed, the precipitate is dissolved in n-hexane and then the gold nano particles are obtained by centrifugation and precipitation by ethanol.

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

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

Example 2: preparation of copper gold nanoparticles coated with oleylamine

The synthesis was performed with reference to the preparation process of example l, except that the copper acetylacetonate added in the step (3) was changed to 0 to 200mg, and the oleylamine coated copper gold nanoparticles were also obtained. Wherein, the copper-gold ratio of the copper-gold nanoparticles coated by the oleylamine is Cu to Au=0-5 to 1.

Example 3: synthesis of thiol-containing pH and thermally responsive small molecules

N-Boc-glycine (525 mg,3 mmol), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (1719 mg,9 mmol) and N-hydroxysuccinimide (1035 mg,9 mmo1) were added sequentially to an anhydrous N, N-dimethylformamide solution (10 mL) and activated for half an hour. Cystamine (152.0 mg,1 mmol) was then added to an anhydrous N, N-dimethylformamide solution and the resulting mixture was stirred under nitrogen at room temperature for 24h. And (3) carrying out overnight reaction 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 points on the chromatography plate disappear. The solvent was removed by rotary evaporation, 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, the compound 1 was dissolved in 5mL of methylene chloride, 1mL of trifluoroacetic acid was added thereto, and the mixture was stirred at room temperature for 4 hours, and the solvent was removed by rotary evaporation to give the compound 2. Then, to 5mL of methanol, compound 2 and dithiothreitol (309 mg,2 mmol) were added, and the mixture was stirred at room temperature for 12 to 24 hours, the solvent was removed by rotary evaporation, the product was dissolved in water, extracted 3 times with ethyl acetate, dried, and purified by column chromatography (eluent acetonitrile: 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 of compound 3: ¹ H NMR(300MHz，DMSO-d ₆ )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 compound 3 is shown in fig. 3, and represents successful synthesis of small molecules with pH and thermal responsiveness.

Example 4: synthesis of thiol-containing pH and thermally responsive small molecules

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

Example 5: preparation of CuAuNPs

Dispersing 1mM of oleylamine coated copper gold nanoparticles in 10mL of tetrahydrofuran, adding 1mM of compound 3 and 1.5mM of compound 4 with one end being mercapto-modified, reacting for 4 hours at 50 ℃, centrifuging at 3000rpm for 3 minutes after cooling, dissolving the precipitate with DMF, centrifuging for 2 times again, removing redundant small molecules, and dissolving the precipitate with deionized water to obtain CuAu NPs. The CuAu NPs are subjected to morphology characterization by a transmission electron microscope, and 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 the successful modification of small molecules containing sulfhydryl groups is shown.

Example 6: preparation of sCuAuNAs

Terephthaloyl isothiocyanate (5 mM) was dissolved in chloroform (1 mL), then CuAu NPs (1 mM,10 mL) and cetyltrimethylammonium bromide as an emulsifier were added, and ultrasonic emulsification was performed, chloroform was volatilized after stirring overnight at room temperature, excess terephthaloyl isothiocyanate was removed by centrifugation at 500rpm for 3 minutes, and excess cetyltrimethylammonium bromide was removed by dialysis with a 1000Da dialysis bag to obtain sCuAu NAs.

The particle size distribution of sCuAu NAs at different temperatures and different pH values was analyzed by dynamic light scattering, and the results are shown in Table 1. The morphology of the resulting socuau NAs was characterized by transmission electron microscopy at ph=7.4, 37 ℃ and ph=5.5, 50 ℃ as shown in fig. 5, and the particle size distribution of the socuau NAs at ph=7.4, 37 ℃ and ph=5.5, 50 ℃ was analyzed by dynamic light scattering as shown in fig. 6. The results prove that the sCuAu NAs has good pH and thermal response, and is completely disassembled under the condition of pH=5.5 and 50 ℃.

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

Example 7: preparation of sCuAuNAs

The synthesis was performed with reference to the preparation process of example 6, except that the emulsifier added was sodium dodecyl sulfate.

Example 8: photothermal performance test of sCuAu NAs

CuAu NPs (prepared according to example 5) and CuAu NAs (prepared according to example 6) were diluted with deionized water to prepare a solution of 15 μg/mL. After the two solutions are irradiated by a 660nm near infrared light emitter for 5 minutes, the laser is turned off, the solutions are naturally cooled to room temperature, and the real-time temperatures of different solutions are recorded by an infrared thermal imager. The heating-cooling curves of CuAu NPs and sCuAu NAs are 1.0W/cm ² Obtained. The photothermal conversion efficiency (η) is calculated by the following formula:

equation 1

Equation 2

Equation 3

Q ₀ ＝hS(T _max，water -T _surr )

τ _s The value of (2) is calculated from the linear regression curve in the cooling curve, m _d And C _d The mass (1 g) and heat capacity (4.2J/(g.K)) of the solution are expressed, respectively, so that the value of hS can be obtained, and then Q is calculated by the formula 3 ₀ Represents the background energy input in the absence of MPDA nanoparticles. Wherein T is _max，water And T _surr Representing the steady state maximum temperature of water and the ambient room temperature, respectively. Thus, in determining hS and Q ₀ After the value, the light-heat conversion efficiency can be calculated according to formula 1. T (T) _max Maximum temperature, I and A, indicating solution stability ₆₆₀ The laser power (1.0W) and the absorbance of the nanoparticle at 660nm are shown, respectively. As a result, as shown in FIG. 7, the photothermal conversion efficiency of sCuAu NAs was 66.32% higher than that of CuAu NPs (46.65%).

Example 9: responsive release of copper ions

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

R _t ＝C _t ×50/2×100％

wherein R is _t Copper release rate at response time point, C _t The copper concentration in the solution was taken out at this time point in units of: mg/mL.

Fig. 8 is then obtained by plotting the release rate against time. It was observed that the sCuAuNAs showed substantially no copper release at 37℃at pH 7.4, whereas the copper release rate in buffer at 50℃at pH 5.5 could exceed 10%. The copper release rate of sCuAu NAs in buffer solution with pH of 5.5 or 50 ℃ is lower than 5%. This demonstrates that the above-described synthetic sCuAu NAs can remain stable in a neutral environment, while releasing more copper ions due to disassembly under acidic and heated conditions.

Example 10: inhibition of sCuAu NAs killing planktonic bacteria

Taking methicillin-resistant staphylococcus aureus (MRSA) in logarithmic phase, diluting bacterial liquid to OD with fresh liquid culture medium ₆₀₀ sCuAu NAs (prepared according to example 6) were diluted to different concentrations with physiological saline, added to the bacterial liquid 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 cultured in a shaking incubator at 37℃with 100. Mu.L per well. After incubation for 1 hour, the solution was irradiated with or without 660nm laser (10 min, 1.0W/cm 2) and incubation was continued for 18 hours, the absorbance at 600nm was measured on a microplate reader, and the bacterial viability was determined, 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 more than 95% of planktonic bacteria, and without laser sCuAu NAs was killed at 20. Mu.g/mL and 40. Mu.g/mLThe survival rates are about 60% without obvious difference, so that the concentration of sCuAu NAs is 20 mug/mL later in experiment.

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

Taking methicillin-resistant staphylococcus aureus in logarithmic phase, diluting bacterial liquid to OD with fresh liquid culture medium ₆₀₀ =0.01 CuAuNPs (prepared according to example 5) and cuaunas (prepared according to example 6) were taken, diluted to 20 μg/mL with medium, 100 μl per well, and incubated in a shaking incubator at 37 ℃. After incubation for 1 hour, the solution was irradiated with or without 660nm laser (10 minutes, 1.0W/cm 2), incubation was continued for 12 hours, absorbance at 600nm was measured on a microplate reader, and bacterial viability was determined by densitometry, as shown in fig. 10, and both CuAuNPs and cuaunas were able to kill about 35% of planktonic bacteria without adding laser, whereas the laser irradiation group CuAu NAs was able to kill 99% of bacteria, cuAu NPs was able to kill only about 60% of bacteria, which sufficiently showed that improvement in photothermal efficiency was favorable for improvement of photothermal antibacterial effect, and CuAu NAs had superior photothermal antibacterial effect.

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

Experiments were performed with reference to the protocol of example 11, except that the bacteria used were E.coli (E.coli). Bacterial viability was determined by densitometry and the results are shown in figure 11. This shows that photothermal treatment of sCuAu NAs has a broad-spectrum antibacterial effect.

In order to prove the broad-spectrum antibacterial effect of the sCuAu NAs photothermal treatment, the adopted 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 an optical density method to verify that the sCuAu NAs has good photothermal treatment effect.

Example 13: inhibition of bacterial biofilm killing by sCuAu NAs

Taking methicillin-resistant staphylococcus aureus in logarithmic phase, diluting bacterial liquid to OD with fresh liquid culture medium ₆₀₀ =0.05, inoculated into 96-well plates, 200 μl per well, and cultured in a constant temperature incubator at 37 ℃ for 48 hours with fresh culture medium replacement every 24 hours. After the bacteria form a biofilm, the culture solution is discarded, and the biofilm is rinsed with physiological saline to wash away planktonic 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 biofilm, 200. Mu.L per well and incubated in a 37℃incubator. Meanwhile, a blank medium was used as a control group. After 1 hour incubation, the solution was irradiated with or without 660nm laser (10 min, 1.0W/cm 2) and incubation was continued for 12 hours to thoroughly wash the biofilm, replacing the physiological saline containing propidium iodide (PI, 54.9. Mu.M) and SYTO 9 (5. Mu.M). After incubation at 37 ℃ for 20 minutes, the fluorescent dye was washed away and the bacterial biofilm was observed under a fluorescent microscope. PI dye can stain dead bacteria, while SYTO 9 can stain live bacteria and dead bacteria at the same time. When the biofilm is intact, PI cannot penetrate the bacterial cell wall, whereas viable bacteria are stained green by SYTO 9. As shown in FIG. 12, more than 99% of physiological saline groups are green fluorescent, cuAu NPs and sCuAu NAs only destroy about 20% of biofilms when no laser is added, and bacteria in the biofilms are basically fully dyed by PI after the laser irradiation groups are treated by sCuAu NAs, red fluorescent is displayed, the activity is obviously reduced, and the CuAu NPs only destroy about 50% of biofilms, so that the sCuAu NAs has obvious photo-thermal antibacterial biofilm effect.

Example 14: cytotoxicity assay of 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 were grown to 80% confluency, three duplicate wells per concentration, with blank medium as control. After 24h incubation, the culture medium containing the nanoparticles was removed, and pre-formulated MTT solution (5 mg/mL) was added to each well, 150. Mu.L per well was placed in the incubator for further incubation for 4h; the MTT solution was carefully removed, 150. Mu.L of dimethyl sulfoxide (DMSO) was added to each well, shaking was performed for 3 minutes, the absorbance OD value of each well at 492nm was measured by an ELISA reader, and the average OD value of three wells was used as the OD value of the target sample to calculate the cell viability:

cell viability = sample _OD Blank group _OD ×100％

As shown in FIG. 13, sCuAu NAs was slightly toxic at a concentration of 40. Mu.g/mL and substantially non-toxic at 20. Mu.g/mL and below, demonstrating that biosafety was ensured at an antimicrobial concentration of 20. Mu.g/mL.

Example 15: cell migration experiments of sCuAu NAs

First, sCuAu NAs (prepared as in example 6) and sCuAu NAs (pH 5.5, 10 minutes) irradiated with 660nm laser 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 complete, cell streaks were performed with a 200. Mu.L gun head, and cell debris was washed off with blank medium. Then 800. Mu.L of the above-prepared solution was added to each well, wherein the Control group was a blank group, the sCuAu NAs group was a solution containing sCuAu NAs added at 20. Mu.g/mL, and the sCuAu NAs+Laser group was a solution containing sCuAu NAs added at 20. Mu.g/mL (after photo-thermal treatment for 10 minutes). The results of migration of HUVEC cells from each group after the addition of the above-mentioned scratches to the cells in the solution culture are shown in FIG. 14. As shown in FIG. 14, after the HUVEC cells are scratched for 36 hours, the Control group has only a small amount of cell migration, while the sCuAu NAs+Laser group has obvious cell migration, which is about 30% higher than that of the blank group, and the sCuAu NAs group also has the capability of promoting cell migration, but the effect is not obvious in the sCuAu NAs+Laser group, which indicates that copper ions can promote cell migration. This means that the sCuAuNAs can not only realize photothermal antibacterial effect, but also promote wound healing.

Example 16 in vivo wound healing experiments with sCuAu NAs

1. Experimental part: selecting 22-25g male BALB/c mice, and performing intraperitoneal injection of STZ molding to perform type I diabetes molding; preparing wound surface on back of diabetic mouse with 8mm punch with concentration of 1×10 ⁸ The diabetic mouse wound surface with serious infection of drug-resistant bacteria is prepared after 20 mu L of the methoxy-resistant staphylococcus aureus bacterial liquid of CFU/mL and 48 hours. Grouping the mice successfully molded: 100 mu L of physiological Saline, called Saline group, is administered to the wound surface of a diabetic mouse infected with MRSA, and is also the present inventionControl group of experiment. mu.L of the solution containing sCuAu NAs prepared as above at 20. Mu.g/mL was administered to the wound surface of MRSA-infected diabetic mice, which was designated as sCuAu NAs group. After 100. Mu.L of the sCuAuNAss-containing solution prepared as above was applied to the wound surface of MRSA-infected diabetic mice for 1 hour, 660nm Laser (10 minutes, 1.0W/cm 2) was irradiated, which was designated as sCuAu NAs+Laser group.

2. Photographing and recording wound healing: taking photos of wound surfaces of the mice at 0, 2, 4, 6, 8 and 10 days after the mice are treated, as shown in fig. 15, fig. 15 is a graph of wound surface healing of the mice infected by MRSA and diabetes, and as can be seen from fig. 15, after the mice are treated for 10 days, the wound surface area of the single sCuAu NAs group is reduced, but the wound is not healed yet, which indicates that the single repairing effect is poor; it is worth mentioning that the sCuAu NAs+Laser group combines the dual effects of photothermal antibiosis and copper ion promotion of wound healing, plays a synergistic effect, and has the advantages of basically healing wounds and optimally repairing the wound surface of MRSA infected diabetes.

3. Wound size measurement: the wound sizes were measured on

mice

0, 2, 4, 6, 8, and 10 days after the above drug administration treatment using vernier calipers, as shown in fig. 16. Fig. 16 is a graph showing the wound size measurement of MRSA-infected diabetic mice, and as can be seen from fig. 16, the wound measurement data is substantially identical to the wound healing graph of fig. 15, and the cuau NAs+laser group can heal the wound in a short time.

Claims

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

(1) Preparing copper gold nanoparticles coated with oleylamine;

(2) Preparing a thiol-containing pH and thermally responsive small molecule;

(4) Preparing a pH and thermal response copper gold nano assembly;

synthesis of the oleylamine coated copper gold nanoparticles: mixing copper acetylacetonate and oleylamine, heating, introducing inert gas, preserving heat for 30-60 minutes, adding n-hexane solution of gold nanoparticles growing for the second time into a reaction system, introducing inert gas to remove oxygen and n-hexane, heating, continuously reacting, cooling to room temperature, precipitating the nanoparticles with a polar solvent, and removing supernatant to obtain the oleylamine coated copper gold nanoparticles;

the synthesis of the thiol-containing pH and thermally responsive small molecule: sequentially adding N-tert-butoxycarbonyl-amino acid, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide into an anhydrous N, N-dimethylformamide solution 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 trifluoroacetic acid and anhydrous dichloromethane into the compound 1, 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 is carried out on the copper gold nanoparticles coated by the oleylamine: dispersing the copper gold nanoparticles coated by the oleylamine in a good solvent, adding a compound 3 and a compound 4 with a sulfhydryl group at one end which are dissolved in proportion, reacting, cooling, centrifuging, and adding deionized water to obtain monodisperse copper gold nanoparticles;

preparation of the pH and thermally responsive copper gold nanoparticle assembly: dissolving terephthalyl isothiocyanate in a good solvent, adding monodisperse copper gold nanoparticles and an emulsifier, performing ultrasonic emulsification, and performing reduced pressure evaporation to obtain sCuAu NAs;

the structural formula of the compound 3 is

；

Compound 4 is a hydrophilic small molecule with a molecular weight of less than 1000Da and a sulfhydryl group at one end.

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

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

4. The method for preparing the pH and thermal response CuAu nano-assembly according to claim 1, wherein: 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 sodium dodecyl sulfate; the hydration particle size of sCuAu NAs is 100-200 nm.

5. The use of the pH and thermally responsive CuAu nano-assemblies prepared by the method of any one of claims 1-4 in the preparation of antibacterial biofilms or in the preparation of medicaments for the treatment of wound wounds.

6. The use of the pH and thermally responsive CuAu nano-assembly prepared by the method of any one of claims 1-4 in the preparation of a drug resistant bacterial biofilm resistant drug.

7. The use according to claim 5, wherein the bacteria comprises escherichia coli, pseudomonas aeruginosa, streptococcus pneumoniae, staphylococcus aureus, salmonella, diphtheria bacillus, bacillus anthracis.

8. The use according to claim 6, wherein the bacteria comprise an antibiotic resistant strain of escherichia coli or methicillin-resistant staphylococcus aureus.