A composite antibacterial nanomaterial, preparation method and application
By modifying amino and carboxyl monomers on the surface of gold nanoparticles, the problem of oxygen-dependent release of free radicals in low oxygen environments is solved, and targeted release and efficient antibacterial effects are achieved at bacterial infection sites.
Patent Information
- Application Number
- CN202310204787.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-03-06
- Publication Date
- 2025-07-08
- Estimated Expiration
- 2043-03-06
AI Technical Summary
Existing photodynamic therapies in treating bacterial infections rely on oxygen for free radical release, resulting in poor results in low oxygen environments and potential damage to normal tissues.
Using composite antibacterial nanomaterials, monomers with amino and carboxyl groups are modified by the surface of gold nanoparticles, and polyacrylamide as a linker, nanomaterials with targeting and reversible agglomeration at different pH values were prepared, and alkyl radicals were activated by near-infrared light.
It realizes the site release of free radicals at bacterial infection sites, avoids damage to normal tissues, expands the therapeutic effect in a hypoxic environment, and has good cell compatibility and antibacterial properties.
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Figure CN116159139B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biological antibacterial materials, and particularly relates to a composite antibacterial nanomaterial, a preparation method and an application thereof. Background Art
[0002] Bacterial infections seriously threaten human health. Worldwide, approximately 17 million victims die from bacterial infections every year, causing losses of up to $10 million, imposing a huge economic pressure on society. In the past few decades, the abuse of antibiotics has led to a large number of pathogens and unprecedented drug resistance. Traditional physical debridement is likely to cause damage to surrounding normal tissues or the spread of infection due to bacteria entering the bloodstream. In the context where traditional antibiotic treatments are ineffective and the development of new generation antibiotic technologies is difficult, costly, and has low returns, it is of great significance to find a safer and more effective new treatment method.
[0003] In response to this problem, many new solution strategies have been proposed currently, including chemotherapy using antimicrobial peptides, polysaccharides, quaternary ammonium salt compounds, cationic materials, antibacterial coatings, and antibacterial polymers, and non-chemical therapies such as photothermal therapy and photodynamic therapy have also been proposed. Among them, photodynamic therapy is a new technology developed in recent years for the diagnosis and treatment of bacterial infections, with obvious advantages such as a broad antibacterial spectrum, effectiveness against drug-resistant strains, good selectivity, low toxicity, and low adverse reactions. However, there are still many problems to be solved when photodynamic therapy (PDT) is applied to bacterial infections. Among them, the most critical problem is the non-localized release of free radicals, that is, controlling the release of free radicals at the site of bacterial infection without causing damage to normal tissues. In addition, usually, the ablation of tumors or bacteria by photodynamic therapy (PDT) is achieved by the reactive oxygen species (ROS) generated by photoexciting photosensitizers acting on bacteria. However, the generation of ROS highly depends on oxygen, greatly limiting its application in treating hypoxic bacterial infection wounds. And the latest research shows that photodynamic therapy (PDT) can act on tumors or bacteria through alkyl free radicals R·, thereby achieving the ablation of tumors or bacteria under anaerobic or hypoxic conditions, so as to achieve the purpose of treatment. Summary of the Invention
[0004] The purpose of the present invention is to provide a composite antibacterial nanomaterial, a preparation method and an application thereof, in order to obtain a nanomaterial with high reactivity centered on carbon (such as alkyl free radicals R·, where R can be alkyl, alkoxy, aldehyde group, etc.) that does not rely on oxygen and is only released at the site of bacterial infection.
[0005] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:
[0006] A composite antibacterial nanomaterial, including gold nanoparticles (GNPs), the surface of the gold nanoparticles (GNPs) is modified with a monomer containing amino and carboxyl groups, and the structural formula of the monomer containing amino and carboxyl groups is shown as follows:
[0007] Among them, the value of n is an integer between 20 and 40.
[0008] The present invention also provides a preparation method of the composite antibacterial nanomaterial of the present invention, including the following steps:
[0009] S1. Prepare gold nanoparticles (GNPs) by the citrate reduction method;
[0010] S2. Mix the gold nanoparticles (GNPs) with polyacryloyl alanine to obtain gold nanoparticles modified with polyacryloyl alanine (PAV);
[0011] S3. Under the action of a catalyst, covalently crosslink a compound containing amino groups at both ends onto the gold nanoparticles modified with polyacryloyl alanine (PAV) to obtain an intermediate product;
[0012] S4. Under the action of a catalyst, covalently crosslink 2,2'-azobis(2-methylpropionamidine) dihydrochloride onto the intermediate product to obtain the composite antibacterial nanomaterial.
[0013] Among them, polyacryloyl alanine (PAV) is the PAV disclosed in the literature "Surface-anchored poly(acryloyl-L(D)-valine) with enhanced chirality-selective effect on cellular uptake of gold nanoparticles".
[0014] Preferably, in S1, under the condition of 140-160 °C, reduce chloroauric acid with citrate and wash the product to obtain gold nanoparticles.
[0015] Preferably, the citrate is selected from trisodium citrate, potassium citrate or calcium citrate.
[0016] Preferably, in S2, under the condition of 25-35 °C, stir and mix the gold nanoparticles with polyacryloyl alanine for 0.5-1.5 h and wash the product to obtain gold nanoparticles modified with polyacryloyl alanine.
[0017] Preferably, the catalyst is a mixture of N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC);
[0018] The compound containing amino groups at both ends is selected from ethylenediamine, carbamide, p-phenylenediamine or diamino polyethylene glycol.
[0019] Preferably, in S3, under the action of a catalyst, the gold nanoparticles modified with polyacryloyl alanine are added to ethylenediamine, and the product is washed to obtain an intermediate product.
[0020] Among them, the temperature during the whole process of preparing the intermediate product needs to be maintained at about 30 °C, with a plus or minus not exceeding 0.5 °C.
[0021] Preferably, in S4, under the action of a catalyst, the intermediate product is added to azobis (N-hydroxyisobutyramidine) dihydrate, and the product is washed to obtain a composite antibacterial nanomaterial. Among them, azobis (N-hydroxyisobutyramidine) dihydrate is azobis (N-hydroxyisobutyramidine) dihydrate.
[0022] Among them, the temperature during the whole process of preparing the composite antibacterial nanomaterial in step S4 needs to be maintained at about 30 °C, with a plus or minus not exceeding 0.5 °C.
[0023] Preferably, in S1, the particle size of the prepared gold nanoparticles is 13-16 nm.
[0024] The present invention also provides an application of the composite antibacterial nanomaterial prepared by the preparation method of the present invention, and the use of the composite antibacterial nanomaterial as a medical material for photodynamic therapy of bacterial infections.
[0025] The beneficial effects of the present invention:
[0026] The composite antibacterial nanomaterial and the preparation method thereof of the present invention use gold nanoparticles (GNPs) as a carrier of an initiator and a heat source to promote the release of free radicals, and use polyacrylamide (PAV) with thiol and carboxyl groups as a linker to modify the surface of the gold nanoparticles with molecules with two weak electrolytes, amino and carboxyl groups, so as to obtain the composite antibacterial nanomaterial. The composite antibacterial nanomaterial can adjust the aggregation or dispersion performance of the gold nanoparticles under different pH conditions according to the degree of protonation or deprotonation of the amino and / or carboxyl groups, so that the composite antibacterial nanomaterial of the present invention has targeting to the bacterial infection site when treating bacterial infection, that is, the free radicals are released at a fixed point at the bacterial infection site by thermal decomposition. The composite antibacterial nanomaterial can release a large amount of alkyl free radicals under the irradiation of near-infrared light, which effectively solves the problem that the existing photodynamic therapy is highly dependent on oxygen, and expands the application of the composite antibacterial nanomaterial in the treatment of wounds infected with hypoxic bacteria. The composite antibacterial nanomaterial prepared by the method of the present invention has the advantages of uniform particle size, good cell compatibility and good antibacterial effect, and provides a promising nano-platform for efficient clinical bacterial infection treatment in the future. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 Flow chart for the synthesis of composite antibacterial nanomaterials;
[0028] Figure 2 TEM image of the gold nanoparticles prepared in Example 1;
[0029] Figure 3 TEM image of the gold nanoparticles modified with polyacrylamide prepared in Example 1;
[0030] Figure 4 TEM image of the intermediate product obtained in Example 1;
[0031] Figure 5 TEM image of the composite antibacterial nanomaterial prepared in Example 1;
[0032] Figure 6 This is a color change diagram of the composite antibacterial nanomaterial prepared in Example 1 in PBS buffer solutions with different pH values;
[0033] Figure 7 This is a transmission electron microscopy image of the composite antibacterial nanomaterial prepared in Example 1 in a PBS buffer solution at pH 7.0;
[0034] Figure 8TEM image of the composite antibacterial nanomaterial prepared in Example 1 in PBS buffer solution at pH 7.4;
[0035] Figure 9 TEM image of the composite antibacterial nanomaterial prepared in Example 1 in PBS buffer solution at pH 6.0;
[0036] Figure 10 TEM image of the composite antibacterial nanomaterial prepared in Example 1 in PBS buffer solution at pH 5.0;
[0037] Figure 11 TEM image of the composite antibacterial nanomaterial prepared in Example 1 in PBS buffer solution at pH 4.0;
[0038] Figure 12 UV-Vis-IR absorption spectra of the composite antibacterial nanomaterial prepared in Example 1 in PBS buffer solutions with different pH values;
[0039] Figure 13 ESR spectrum of the composite antibacterial nanomaterial prepared in Example 1;
[0040] Figure 14 Antibacterial result diagram of the composite antibacterial nanomaterial prepared in Example 1. Detailed implementation manners
[0041] The following will illustrate the implementation manners of the present invention with reference to the accompanying drawings and preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention, rather than for limiting the protection scope of the present invention.
[0042] It should be noted that the diagrams provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner. Therefore, only the components related to the present invention are shown in the diagrams, rather than being drawn according to the number, shape, and size of the components in actual implementation. The type, quantity, and ratio of each component in actual implementation can be arbitrarily changed, and the component layout type may also be more complex.
[0043] The synthesis process of the composite antibacterial nanomaterial of the present invention is as Figure 1 shown, Figure 1 in which, represents polyacryloyl alanine, and its structural formula is represents ethylenediamine, and its structural formula is Denotes azodicarbonyl-hydroxyisobutylamidine, and its structural formula is
[0044] Example 1
[0045] As Figure 1 shown, a preparation method of a composite antibacterial nanomaterial includes the following steps:
[0046] S1. Reducing chloroauric acid with trisodium citrate to prepare gold nanoparticles, specifically as follows: Take 600 mL of pure water in a 1000 mL two-necked flask, heat it to 153 °C, then add 6 mL of an aqueous chloroauric acid solution with a mass fraction of 1%, keep it at 153 °C for 10 min, under the state of violent boiling and uniform stirring, add dropwise 6 mL of an aqueous trisodium citrate solution with a mass fraction of 3%, after the solution turns wine red, continue heating and stirring for 15 min, and then centrifuge and wash the product 3 times with ultrapure water to make gold nanoparticles (GNPs) with a particle size of about 15 nm;
[0047] S2. Modifying polyacryloyl amino acid on the surface of gold nanoparticles, specifically as follows: Using ultrapure water to prepare a gold nanoparticle solution with a concentration of 0.38 mg·mL -1 from the gold nanoparticles (GNPs) prepared in S1; Using methanol to prepare a polyacryloyl amino acid solution with a concentration of 10 mg·mL -1 ;
[0048] Take 1 mL of the gold nanoparticle solution in a 1.5 mL centrifuge tube, then add 50 μL of the polyacryloyl amino acid solution, mix and stir at 30 °C for 1 h, and centrifuge and wash the product 3 times with ultrapure water to obtain gold nanoparticles modified with polyacryloyl amino acid (GNPs-PVA);
[0049] S3. Prepare an intermediate product, specifically as follows: Using ultrapure water to prepare an N-hydroxysuccinimide (NHS) solution with a concentration of 20 mg·mL -1 ; Using ultrapure water to prepare a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) solution with a concentration of 20 mg·mL -1 ; Using ultrapure water to prepare an ethylenediamine aqueous solution with a concentration of 88.9 mg·mL -1 ;
[0050] Using ultrapure water to prepare the gold nanoparticles modified with polyacryloyl amino acid (GNPs-PVA) obtained in S2 into a gold concentration of 0.38 mg·mL -1The gold nanoparticle solution modified with polyacryloyl amino acid, take 15 μL of N-hydroxysuccinimide solution and 25 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution and add them to the gold nanoparticle solution modified with polyacryloyl amino acid (Au 0.38 mg·mL -1 ), stir vigorously at 30 °C for 0.5 h, then inject 34 μL of ethylenediamine aqueous solution and mix and stir for 1 h, and then centrifuge and wash the product with ultrapure water 3 times to obtain the intermediate product (GNPs-PVA-ED);
[0051] S4. Prepare the composite antibacterial nanomaterial as follows: Dissolve azodicarbonyl-hydroxyisobutylamidine in ultrapure water to form an azodicarbonyl-hydroxyisobutylamidine solution with a concentration of 10 mg·mL -1 ;
[0052] Use ultrapure water to prepare the intermediate product (GNPs-PVA-ED) prepared in S3 into an intermediate product solution with a gold concentration of 0.38 mg·mL -1 , take 7.5 μL of the N-hydroxysuccinimide solution prepared in S3 and 10 μL of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution prepared in S3 and add them to the intermediate product solution, stir vigorously at 30 °C for 0.5 h, then add 50 μL of the azodicarbonyl-hydroxyisobutylamidine solution, stir vigorously at 30 °C for 2 h, and centrifuge and wash the product with ultrapure water 3 times to obtain the composite antibacterial nanomaterial (GNPs-PVA-ED-VA057), where the value of n in the composite antibacterial nanomaterial is 30.
[0053] Example 2
[0054] As Figure 1 shown, a preparation method of a composite antibacterial nanomaterial includes the following steps:
[0055] S1. Reduce chloroauric acid with trisodium citrate to prepare gold nanoparticles as follows: Take 600 mL of pure water in a 1000 mL two-necked flask, heat to 160 °C, then add 5.5 mL of 1% chloroauric acid aqueous solution, continuously react at 160 °C for 8 min, under the state of violent boiling and uniform stirring, dropwise add 6 mL of 3% trisodium citrate aqueous solution, continue to heat and stir the reaction for 15 min after the solution turns wine red, and then centrifuge and wash the product with ultrapure water 3 times to make gold nanoparticles (GNPs) with a particle size of about 15 nm;
[0056] S2. Modify polyacryloyl amino acid on the surface of gold nanoparticles as follows: Use ultrapure water to prepare the gold nanoparticles (GNPs) prepared in S1 into 0.38 mg·mL -1Gold nanoparticle solution; polyacryloyl alanine was dissolved in methanol to prepare a 10 mg·mL -1 polyacryloyl alanine solution;
[0057] Take 1 mL of the gold nanoparticle solution into a 1.5 mL centrifuge tube, then add 60 μL of the polyacryloyl alanine solution, mix and stir at 30 °C for 1 h, and centrifuge and wash the product 3 times with ultrapure water to obtain gold nanoparticles modified with polyacryloyl alanine (GNPs-PVA);
[0058] S3. Prepare the intermediate product as follows: Dissolve N-hydroxysuccinimide (NHS) in ultrapure water to prepare a 20 mg·mL -1 N-hydroxysuccinimide solution; Dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in ultrapure water to prepare a 20 mg·mL -1 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution; Dissolve ethylenediamine in ultrapure water to prepare an 88.9 mg·mL -1 ethylenediamine aqueous solution;
[0059] Dissolve the gold nanoparticles modified with polyacryloyl alanine (GNPs-PVA) prepared in S2 in ultrapure water to prepare a gold nanoparticle solution with a gold concentration of 0.38 mg·mL -1 Take 15 μL of the N-hydroxysuccinimide solution and 25 μL of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution and add them to the gold nanoparticle solution modified with polyacryloyl alanine (Au 0.38 mg·mL -1 ), stir vigorously at 30 °C for 0.5 h, then inject 34 μL of the ethylenediamine aqueous solution and mix and stir for 1 h, and centrifuge and wash the product 3 times with ultrapure water to obtain the intermediate product (GNPs-PVA-ED);
[0060] S4. Prepare the composite antibacterial nanomaterial as follows: Dissolve azodicarbonyl diamino-hydroxyisobutyramidine in ultrapure water to prepare a 10 mg·mL -1 azodicarbonyl diamino-hydroxyisobutyramidine solution;
[0061] Dissolve the intermediate product (GNPs-PVA-ED) prepared in S3 in ultrapure water to prepare a gold nanoparticle solution with a gold concentration of 0.38 mg·mL -1Intermediate product solution; Take 7.5 μL of the N-hydroxysuccinimide solution prepared in S3 and 10 μL of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution prepared in S3 and add them to the intermediate product solution. Stir vigorously at 30 °C for 0.5 h, then add 50 μL of the azodiisobutyramidine dihydrochloride solution, stir vigorously at 30 °C for 2 h, and centrifuge and wash the product 3 times with ultrapure water to obtain the composite antibacterial nanomaterial (GNPs-PVA-ED-VA057). Among them, the value of n in the composite antibacterial nanomaterial is 28.
[0062] Example 3
[0063] As Figure 1 shown, a preparation method of a composite antibacterial nanomaterial includes the following steps:
[0064] S1. Reduce chloroauric acid with trisodium citrate to prepare gold nanoparticles, specifically as follows: Take 600 mL of pure water in a 1000 mL two-necked flask, heat to 153 °C, then add 6 mL of a 1% aqueous solution of chloroauric acid, continuously react at 153 °C for 10 min, under the state of violent boiling and uniform stirring, dropwise add 6 mL of a 3% aqueous solution of trisodium citrate. After the solution turns wine red, continuously react for 15 min, and then centrifuge and wash the product 3 times with ultrapure water to make gold nanoparticles (GNPs) with a particle size of about 15 nm.
[0065] S2. Modify polyacryloyl amino acid on the surface of gold nanoparticles, specifically as follows: Use ultrapure water to prepare a gold nanoparticle solution of 0.38 mg·mL -1 with the gold nanoparticles prepared in S1; Use methanol to prepare a polyacryloyl amino acid solution of 10 mg·mL -1 of polyacryloyl amino acid;
[0066] Take 1 mL of the gold nanoparticle solution in a 1.5 mL centrifuge tube, then add 50 μL of the polyacryloyl amino acid solution, mix and stir at 30 °C for 1 h, and centrifuge and wash the product 3 times with ultrapure water to obtain gold nanoparticles modified with polyacryloyl amino acid (GNPs-PVA).
[0067] S3. Prepare an intermediate product, specifically as follows: Use ultrapure water to prepare an N-hydroxysuccinimide solution of 20 mg·mL -1 of N-hydroxysuccinimide; Use ultrapure water to prepare a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution of 20 mg·mL -1 of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; Use ultrapure water to prepare ethylenediamine into 88.9 mg·mL-1 An aqueous solution of ethylenediamine;
[0068] Using ultrapure water, the gold nanoparticles modified with polyacryloyl alanine (GNPs-PVA) prepared in S2 were formulated into a gold nanoparticle solution with a gold concentration of 0.38 mg·mL -1 of the gold nanoparticle solution modified with polyacryloyl alanine; 20 μL of N-hydroxysuccinimide solution and 20 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution were added to the gold nanoparticle solution modified with polyacryloyl alanine (Au 0.38 mg·mL -1 ), and the mixture was vigorously stirred at 30 °C for 1 h, then 40 μL of the ethylenediamine aqueous solution was injected and stirred for 1 h, and the product was centrifugally washed 3 times with ultrapure water to obtain an intermediate product (GNPs-PVA-ED);
[0069] S4. Preparation of the composite antibacterial nanomaterial, specifically as follows: Using ultrapure water, azodicarbonyl-hydroxyisobutylamidine was formulated into a solution with a concentration of 10 mg·mL -1 of the azodicarbonyl-hydroxyisobutylamidine solution;
[0070] Using ultrapure water, the intermediate product (GNPs-PVA-ED) prepared in S3 was formulated into an intermediate product solution with a gold concentration of 0.38 mg·mL -1 ; 7.5 μL of the N-hydroxysuccinimide solution prepared in S3 and 10 μL of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution prepared in S3 were added to the intermediate product solution, and the mixture was vigorously stirred at 30 °C for 0.5 h, then 50 μL of the azodicarbonyl-hydroxyisobutylamidine solution was added, and the mixture was vigorously stirred at 30 °C for 2 h. The product was centrifugally washed 3 times with ultrapure water to obtain the composite antibacterial nanomaterial (GNPs-PVA-ED-VA057), where the value of n in the composite antibacterial nanomaterial is 32.
[0071] Example 4
[0072] As Figure 1 shown, a preparation method of a composite antibacterial nanomaterial, comprising the following steps:
[0073] S1. Prepare gold nanoparticles by reducing chloroauric acid with trisodium citrate as follows: Take 600 mL of pure water in a 1000 mL two-necked flask, heat it to 153 °C, then add 6 mL of 1% (by mass) chloroauric acid aqueous solution. React continuously at 153 °C for 10 min. While boiling violently and stirring evenly, add 6 mL of 3% (by mass) trisodium citrate aqueous solution dropwise. After the solution turns wine red, react continuously for 15 min. Then wash the product by centrifugation with ultrapure water three times to prepare gold nanoparticles (GNPs) with a particle size of about 15 nm.
[0074] S2. Modify polyacrylamide on the surface of gold nanoparticles as follows: Use ultrapure water to prepare a gold nanoparticle solution with a concentration of 0.38 mg·mL -1 from the gold nanoparticles (GNPs) prepared in S1; Use methanol to prepare a polyacrylamide solution with a concentration of 10 mg·mL -1 .
[0075] Take 1 mL of the gold nanoparticle solution in a 1.5 mL centrifuge tube, then add 50 μL of the polyacrylamide solution, mix and stir at 30 °C for 1 h, and wash the product by centrifugation with ultrapure water three times to obtain gold nanoparticles modified with polyacrylamide (GNPs-PVA).
[0076] S3. Prepare an intermediate product as follows: Use ultrapure water to prepare a N-hydroxysuccinimide (NHS) solution with a concentration of 20 mg·mL -1 ; Use ultrapure water to prepare a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) solution with a concentration of 20 mg·mL -1 ; Use ultrapure water to prepare an ethylenediamine aqueous solution with a concentration of 88.9 mg·mL -1 .
[0077] Use ultrapure water to prepare a gold nanoparticle solution modified with polyacrylamide (GNPs-PVA) with a gold concentration of 0.38 mg·mL -1 from the gold nanoparticles modified with polyacrylamide prepared in S2; Take 15 μL of the N-hydroxysuccinimide solution and 25 μL of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution and add them to the gold nanoparticle solution modified with polyacrylamide (Au 0.38 mg·mL -1 ). Stir vigorously at 30 °C for 0.5 h, then inject 34 μL of the ethylenediamine aqueous solution and mix and stir for 1 h. Then wash the product by centrifugation with ultrapure water three times to obtain the intermediate product (GNPs-PVA-ED).
[0078] S4. Prepare the composite antibacterial nanomaterial as follows: Dissolve azodicarbonamide-hydroxyisobutylamidine in ultrapure water to form an azodicarbonamide-hydroxyisobutylamidine solution with a concentration of 10 mg·mL -1 .
[0079] Dissolve the intermediate product (GNPs-PVA-ED) obtained in S3 in ultrapure water to form an intermediate product solution with a gold concentration of 0.38 mg·mL -1 . Take 15 μL of the N-hydroxysuccinimide solution obtained in S3 and 15 μL of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution obtained in S3 and add them to the intermediate product solution. Stir vigorously at 30 °C for 1 h, then add 70 μL of the azodicarbonamide-hydroxyisobutylamidine solution, and stir vigorously at 30 °C for 3 h. Centrifuge and wash the product 3 times with ultrapure water to obtain the composite antibacterial nanomaterial (GNPs-PVA-ED-VA057), where the value of n in the composite antibacterial nanomaterial is 30.
[0080] Detection and analysis
[0081] 1) Use a transmission electron microscope to analyze the morphologies of the gold nanoparticles, gold nanoparticles modified with polyacryloyl amino acid, the intermediate product, and the composite antibacterial nanomaterial prepared in Example 1. The results are as Figures 2 to 5 shown.
[0082] From the Figures 2 to 5 observation and analysis, it can be seen that the van der Waals force and hydrogen bond force between the composite antibacterial nanomaterials far exceed the hydration electrostatic and hydration repulsion, resulting in tight aggregation. Under neutral conditions, the amino and carboxyl groups are deprotonated, and the absolute value of the surface potential of the composite antibacterial nanomaterials increases. The strong electrostatic and hydration repulsion between the composite antibacterial nanomaterials disperses the composite antibacterial nanomaterials.
[0083] 2) Place the composite antibacterial nanomaterial prepared in Example 1 in PBS buffer solutions with different pH values to compare the sensitivity of the composite antibacterial nanomaterial to pH values. The color changes of the composite antibacterial nanomaterial in PBS buffer solutions with different pH values are as Figure 6 shown, and the transmission electron microscope images of the composite antibacterial nanomaterial in PBS buffer solutions with different pH values are as Figures 7 to 11 shown.
[0084] From the Figure 6It can be observed and analyzed that the composite antibacterial nanomaterial (i.e., pH-sensitive gold nanoparticles) has a significant color difference in PBS buffer solutions with different pH values. When the pH value is 7.4 and 7.0, the composite antibacterial nanomaterial has good dispersion performance and the color is wine red. As the pH value decreases to 6.0, the composite antibacterial nanomaterial gradually agglomerates and the color changes to dark purple. As the pH value decreases to 5.0, the agglomeration of the composite antibacterial nanomaterial deepens and the color changes to grayish purple. As the pH value decreases to 4.0, the agglomeration of the composite antibacterial nanomaterial further intensifies and the color changes to gray. This proves that the pH value affects the dispersion of the composite antibacterial nanomaterial, and as the pH value decreases, the agglomeration property is better. The pH of the wound surface infected by bacteria is generally acidic (about between 5 and 6.5), so that the composite antibacterial nanomaterial prepared by the present invention has targeting to the bacterial infection site and effectively avoids damage to normal tissues.
[0085] From Figures 7 to 11 It can be observed and analyzed that the agglomeration degree of the composite antibacterial nanomaterial varies greatly in environments with different pH values, and obvious agglomeration behavior is shown as the pH value decreases, and the aggregation state gradually changes from a loose state to a tight state. This is because when the pH value is 7.4 and 7.0, the amino and carboxyl groups on the surface of the composite antibacterial nanomaterial are in a deprotonated state, and the large negative charge on the surface makes the particles have strong electrostatic and hydration repulsion forces, and its dispersion performance is good. When the pH is 6.0, due to the partial protonation of the amino and carboxyl groups, the absolute value of the surface potential of the composite antibacterial nanomaterial decreases, the van der Waals and hydrogen bond attractions increase, and the electrostatic and hydration repulsion forces weaken, showing a loose aggregation state. When the pH decreases to 5.0 and 4.0, due to the increased degree of protonation of the amino and carboxyl groups, the van der Waals attraction and hydrogen bond attraction on the surface of the composite antibacterial nanomaterial far exceed the hydration electrostatic and hydration repulsion effects and tightly aggregate.
[0086] 3) The composite antibacterial nanomaterial prepared in Example 1 was placed in PBS buffer solutions with different pH values for ultraviolet-visible-infrared absorption spectroscopy analysis, and the results are as Figure 12 shown.
[0087] Figure 12 It includes the ultraviolet-visible-infrared absorption spectra of the composite antibacterial nanomaterial placed in PBS buffer solutions with pH values of 7.4 and 5.0 and the ultraviolet-visible-infrared absorption spectrum after adjusting the pH from 5.0 to 7.4.
[0088] From Figure 12Analysis shows that the aggregation of the composite antibacterial nanomaterials results in a gradual red shift of their SPR peaks and a gradual increase in the peak intensity at 808 nm, thus proving that the composite antibacterial nanomaterials have better photothermal conversion efficiency in the aggregated state. In addition, the pH-responsive aggregation of the composite antibacterial nanomaterials is reversible. After the composite antibacterial nanomaterials are used to treat wound infections and aggregate in the body, since the pH value in the body becomes 7.4 under normal physiological conditions, the composite antibacterial nanomaterials can be redispersed and quickly excreted from the body, effectively avoiding the damage caused to the human body by the long-term retention of the aggregated composite antibacterial nanomaterials in the body after treatment.
[0089] 4) Free radical generation test
[0090] The composite antibacterial nanomaterials prepared in Example 1 were placed in an acidic environment (PBS pH 5.0) for free radical generation evaluation. Electron spin resonance (ESR) technology was used to select a free radical scavenger (POBN) to detect free radical generation, and the results are as Figure 13 shown.
[0091] Figure 13 The figure shows the ESR spectrogram of the composite antibacterial nanomaterials in PBS solution (pH 5.0) under near-infrared light (808 nm) irradiation as the time extends.
[0092] From Figure 13 analysis, it can be seen that the generation amount of alkyl free radicals gradually increases with the increase of irradiation time. The signal of alkyl free radicals could hardly be detected within the first 2 minutes, but increased rapidly within the subsequent 5 minutes, indicating that with the increase of irradiation time, alkyl free radicals gradually decomposed from the composite antibacterial nanomaterials, and with the extension of time, the signal of alkyl free radicals gradually strengthened, indicating that the amount of decomposed alkyl free radicals gradually increased. This proves that a large amount of alkyl free radicals can be generated from the composite antibacterial nanomaterials under the action of heat.
[0093] 5) In vitro antibacterial test
[0094] The in vitro antibacterial performance of the composite antibacterial nanomaterials prepared in Example 1 was evaluated as follows: The composite antibacterial nanomaterials (0.1 mg / mL) were dissolved in PBS 7.4 aqueous solution to obtain a composite antibacterial solution. Then, 100 μL of equal amounts of the composite antibacterial solution and PBS 7.4 aqueous solution were respectively mixed with a methicillin-resistant Staphylococcus aureus (MRSA) biofilm. Under near-infrared light (808 nm) irradiation, a fluorescence microscope was used to observe the bacterial death situation, and the results are as Figure 14 shown.
[0095] Figure 14 In the figure, Figure 14-A1 is the result diagram after taking 100 μL of the mixture of PBS 7.4 aqueous solution and methicillin-resistant Staphylococcus aureus (MRSA) biofilm, placing it in a 96-well plate, and irradiating it with 808 near-infrared light for 10 minutes; Figure 14 -B1 is the result diagram after taking 100 μL of the mixture of the composite antibacterial solution and methicillin-resistant Staphylococcus aureus (MRSA) biofilm, placing it in a 96-well plate, and irradiating it with 808 near-infrared light for 10 minutes; Figure 14 -A2 is the result diagram of bacterial staining after mixing PBS 7.4 aqueous solution and methicillin-resistant Staphylococcus aureus (MRSA) biofilm; Figure 14 -B2 is the result diagram of bacterial staining after mixing the composite antibacterial solution and methicillin-resistant Staphylococcus aureus (MRSA) biofilm, where Figure 14 -The green in -A2 represents live bacteria, Figure 14 -The red in -B2 represents dead bacteria.
[0096] From Figure 14 -A1 and Figure 14 -B1, through comparative analysis, it can be seen that after mixing the composite antibacterial solution and methicillin-resistant Staphylococcus aureus (MRSA) biofilm, the surface structure of methicillin-resistant Staphylococcus aureus (MRSA) biofilm can be significantly damaged after light treatment, while PBS 7.4 aqueous solution has almost no effect on methicillin-resistant Staphylococcus aureus (MRSA) biofilm; From Figure 14 -A2 and Figure 14 -B2, through comparative analysis, it can be seen that the bacteria mixed with the composite antibacterial solution are almost all killed, thus proving that the composite antibacterial nanomaterial has excellent bactericidal effects.
[0097] In summary, for the composite antibacterial nanomaterial and its preparation method of the present invention, gold nanoparticles (GNPs) are used as the carrier of the initiator and the heat source for promoting the release of free radicals, and polyacryloyl amino acid (PAV) with sulfhydryl and carboxyl groups is used as the linker to modify the molecules with two weak electrolytes of amino and carboxyl groups on the surface of the gold nanoparticles, so as to obtain the composite antibacterial nanomaterial. The composite antibacterial nanomaterial can adjust the aggregation or dispersion performance of the composite antibacterial nanomaterial under different pH conditions according to the degree of protonation or deprotonation of amino and / or carboxyl groups. Therefore, when the composite antibacterial nanomaterial of the present invention is used to treat bacterial infections, it has targeting to the bacterial infection site, that is, free radicals are released at the bacterial infection site, effectively avoiding damage to normal tissues. At the same time, the composite antibacterial nanomaterial of the present invention can release a large amount of free radicals under the irradiation of near-infrared light, effectively solving the problem that the existing photodynamic therapy highly depends on oxygen, expanding the application of the composite antibacterial nanomaterial in the treatment of hypoxic bacterial infection wounds. Moreover, the composite antibacterial nanomaterial prepared by the method of the present invention has the advantages of uniform particle size, good cell compatibility and excellent antibacterial effect, providing a promising nanoplatform for future efficient clinical treatment of bacterial infections.
[0098] The above embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention.
Claims
1. A composite antibacterial nanomaterial, characterized in that, It includes gold nanoparticles, and the surfaces of the gold nanoparticles are modified with monomers containing amino groups and carboxyl groups. The structural formula of the composite antibacterial nanomaterial containing amino groups and carboxyl groups is shown as follows: ， Among them, n is an integer between 20 and 40.
2. The preparation method of the composite antibacterial nanomaterial according to claim 1, characterized in that, It includes the following steps: S1. Prepare gold nanoparticles by the citrate reduction method; S2. Mix the gold nanoparticles with polyacryloyl alanine to obtain gold nanoparticles modified with polyacryloyl alanine; S3. Under the action of a catalyst, make a compound with amino groups at both ends covalently crosslinked on the gold nanoparticles modified with polyacryloyl alanine to obtain an intermediate product; S4. Under the action of a catalyst, make 2,2'-azobis(2-methylpropionamidine) dihydrochloride covalently crosslinked on the intermediate product to obtain a composite antibacterial nanomaterial.
3. The preparation method of the composite antibacterial nanomaterial according to claim 2, characterized in that, In S1, under the condition of 140 - 160 °C, reduce chloroauric acid with citrate and wash the product to prepare gold nanoparticles.
4. The preparation method of the composite antibacterial nanomaterial according to claim 2, characterized in that, The citrate is selected from trisodium citrate or potassium citrate.
5. The preparation method of the composite antibacterial nanomaterial according to claim 2, characterized in that, In S2, under the condition of 25 - 35 °C, stir and mix the gold nanoparticles with polyacryloyl alanine for 0.5 - 1.5 h and wash the product to obtain gold nanoparticles modified with polyacryloyl alanine.
6. The preparation method of the composite antibacterial nanomaterial according to claim 2, wherein, The catalyst is a mixture of N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; The compound with amino groups at both ends is selected from ethylenediamine.
7. The preparation method of the composite antibacterial nanomaterial according to claim 6, characterized in that, In S3, under the action of a catalyst, add the gold nanoparticles modified with polyacryloyl alanine to ethylenediamine and wash the product to prepare an intermediate product.
8. The preparation method of the composite antibacterial nanomaterial according to claim 6, wherein, In S4, under the action of a catalyst, add the intermediate product to 2,2'-azobis(2-methylpropionamidine) dihydrochloride and wash the product to prepare a composite antibacterial nanomaterial.
9. The preparation method of the composite antibacterial nanomaterial according to claim 3, characterized in that, In S1, the prepared gold nanoparticles have a particle size of 13 - 16 nm.
10. Use of the composite antibacterial nanomaterial prepared by the preparation method according to any one of claims 2 to 9, characterized in that, Use of the composite antibacterial nanomaterial in the preparation of medical materials for photodynamic therapy.