Preparation method and application of novel photodynamic antibacterial material Cu-Cy-PEG (at) Au NPs

Abstract

The invention discloses a preparation method of a novel photodynamic antibacterial material Cu-Cy-PEG (at) Au NPs, which comprises the following steps: 1) dissolving CuCl22H2O in deionized water, adding cysteamine hydrochloride and PEG 4000, and regulating the pH value to 7 by using sodium hydroxide; 2) after stirring reaction, heating to 100 DEG C in an oil bath to continue reaction, and then adding a chloroauric acid solution and trisodium citrate; 3, after centrifugation and washing, a product is dried in a vacuum furnace, and the Cu-Cy-PEG (at) Au NPs composite material is obtained.The nano-gold-loaded copper cysteamine (Cu-Cy-PEG (at) Au NPs) is prepared through an in-situ reduction method, has good photodynamic antibacterial performance and can be widely applied to the fields of clinical application, biochemical treatment and the like.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine / antibacterial new materials, and specifically discloses a preparation method and application of a novel photodynamic antibacterial material Cu-Cy-PEG@Au NPs. Background Art

[0002] Photodynamic antimicrobial chemotherapy (PACT) is a method that combines light irradiation and photosensitizers to produce reactive oxygen species (ROS) and kill pathogenic bacteria, replacing traditional antibiotics. PACT has many advantages over traditional antibiotics. PACT is cost-effective and has high biosafety in various applications. In PACT, various photosensitizers are used to convert incident light irradiation into oxygen free radicals. Copper cysteamine (Cu-Cy), as a new type of photosensitizer, can be activated not only by conventional UV light, but also by X-rays, microwaves, and ultrasound to produce singlet oxygen in deep tissues for infection. At the same time, Cu-Cy is very effective in killing Gram-positive bacteria because the peptidoglycan layer of Gram-positive bacteria is vulnerable to ROS attack. However, the antibacterial effect on Gram-negative bacteria is limited. Gold nanoparticles (Au NPs), with low cytotoxicity and good biocompatibility, have shown great potential in biomedical and advanced catalytic applications.

[0003] In order to further improve the antibacterial performance, researchers are also committed to developing new antibacterial materials with synergistic effects. For example, hydrogen peroxide (H2O2), as a commonly used oxidant, can destroy the cell structure of bacteria through oxidation, thereby achieving the purpose of sterilization. However, the antibacterial effect of H2O2 is greatly affected by its concentration and reaction conditions. It has the disadvantage of poor effect at low concentrations, and its use at high concentrations is relatively limited. Summary of the invention

[0004] In order to solve the above problems, the present invention discloses a preparation method and application of a novel photodynamic antibacterial material Cu-Cy-PEG@Au NPs.

[0005] The present invention includes the following technical solutions:

[0006] A method for preparing a novel photodynamic antibacterial material Cu-Cy-PEG@Au NPs comprises the following steps:

[0007] 1) Dissolve CuCl2·2H2O in deionized water, add cysteamine hydrochloride and PEG4000, and adjust the pH to 7 with sodium hydroxide;

[0008] 2) After stirring the reaction, heat the oil bath to 100°C to continue the reaction, and then add chloroauric acid solution and trisodium citrate;

[0009] 3) After centrifugation and washing, the product is dried in a vacuum oven to obtain a Cu-Cy-PEG@Au NPs composite material.

[0010] Furthermore, in the preparation method of the novel photodynamic antibacterial material Cu-Cy-PEG@Au NPs, the step 1) comprises the following specific steps:

[0011] Weigh a certain mass of CuCl2·2H2O, dissolve it in an appropriate amount of deionized water, stir magnetically at 25°C until dissolved, add a certain mass of cysteamine hydrochloride and PEG4000 under nitrogen protection, and adjust the pH value to 7 with 1M sodium hydroxide solution; wherein, the final concentration of CuCl2 is 0.03 mol / L, the final concentration of cysteamine hydrochloride is 0.06 mol / L, and the final concentration of PEG4000 is 0.8 mg / mL.

[0012] Furthermore, in the preparation method of the novel photodynamic antibacterial material Cu-Cy-PEG@Au NPs, the step 2) comprises the following specific steps:

[0013] Continue stirring and reacting for about 15 to 30 minutes, a dark purple substance appears on the wall of the flask, and then transfer to an oil bath; heat the oil bath to 100°C and react for 20 minutes; then add 1 / 1000 volume of 0.1 g / ml chloroauric acid solution and a certain mass of trisodium citrate to make the mass volume concentration of trisodium citrate reach about 0.1%.

[0014] Furthermore, in the preparation method of the novel photodynamic antibacterial material Cu-Cy-PEG@Au NPs, the step 3) comprises the following specific steps:

[0015] The mixture was centrifuged at 9000 rpm for 10 min, washed three times with a mixture of deionized water and ethanol in a volume ratio of 4:5, and then completely dried in a vacuum oven at 40 °C overnight to obtain a Cu-Cy-PEG@Au NPs composite material.

[0016] Furthermore, the preparation method of the above-mentioned novel photodynamic antibacterial material Cu-Cy-PEG@Au NPs also includes step 4) testing the catalytic synergistic photothermal bactericidal performance of Cu-Cy-PEG@Au NPs: mixing the material with bacterial solution, H2O2 and PBS solution, and irradiating with 880nm near-infrared laser.

[0017] The invention also discloses a novel photodynamic antibacterial drug / material Cu-Cy-PEG@Au NPs, which is prepared by the above method.

[0018] The present invention also discloses the use of the novel photodynamic antibacterial drug / material Cu-Cy-PEG@Au NPs in preparing materials / drugs for improving the antibacterial ability of H2O2.

[0019] The invention also discloses a composite antibacterial material / drug, which contains the above-mentioned photodynamic antibacterial material Cu-Cy-PEG@Au NPs and H2O2.

[0020] The present invention also discloses an antibacterial method, which comprises simultaneously applying antibacterial materials Cu-Cy-PEG@Au NPs and H2O2 to a material to be treated under near-infrared light conditions.

[0021] Furthermore, in the above antibacterial method, the bacteria is Escherichia coli or Staphylococcus aureus.

[0022] Compared with the prior art, the present invention has the following outstanding beneficial effects:

[0023] In order to improve the antibacterial ability of Cu-Cy, the present invention synthesized a hybrid of cysteamine copper nanosheets and gold nanoclusters (AuNPs), and proved its excellent antibacterial ability. The present invention prepared nano-gold-loaded copper cysteamine (Cu-Cy-PEG@Au NPs), which has good antibacterial activity against Gram-negative bacteria and Gram-positive bacteria, has significant bactericidal properties, and has a clearing effect on stubborn bacterial biofilms. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Transmission electron microscopy image of Cu-Cy-PEG@Au NPs;

[0025] Figure 2 is the UV absorption spectrum of Cu-Cy-PEG@Au NPs;

[0026] Figure 3 Comparison of the bactericidal effect of Cu-Cy-PEG@Au NPs. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Example 1

[0029] Preparation Example

[0030] 1) Weigh a certain amount of CuCl2·2H2O, dissolve it in an appropriate amount of deionized water, and stir it with a magnetic stirrer at 25°C until it dissolves. Add a certain amount of cysteamine hydrochloride and PEG4000 under nitrogen protection, and adjust the pH value to 7 with 1M sodium hydroxide solution. Among them, the final concentration of CuCl2 is 0.03mol / L, the final concentration of cysteamine hydrochloride is 0.06mol / L, and the concentration of PEG4000 is 0.8mg / mL;

[0031] 2) Continue stirring and reacting for about 15 to 30 minutes, and a dark purple substance will appear on the wall of the flask. Then transfer it to an oil bath. Heat the oil bath to 100°C and react for 20 minutes. Then add 0.1g / ml chloroauric acid solution and a certain amount of trisodium citrate to make the mass volume concentration of trisodium citrate reach about 0.1%;

[0032] 3) centrifuging at 9000 rpm for 10 min, washing three times with a mixture of deionized water and ethanol in a volume ratio of 4:5, and then completely drying the obtained product in a vacuum oven at 40° C. overnight to obtain a Cu-Cy-PEG@Au NPs composite material.

[0033] Example 2

[0034] The Cu-Cy-PEG@Au NPs composite material prepared in Example 1 was examined by electron microscopy. Figure 1 The figure shows the successful synthesis of the Cu-Cy-PEG@Au NPs composite material, whose basic morphology is rectangular nanosheets, with gold nanoparticles loaded on the surface of Cu-Cy. However, there are still a small amount of free gold nanoparticles, which may be caused by the shedding caused by ultrasound during the sample preparation process.

[0035] The Cu-Cy-PEG@Au NPs composite material prepared in Example 1 was tested by ultraviolet absorption spectrum. Figure 2 As shown, 365 nm is the representative absorption peak of Cu-Cy. The absorption peak at 825 nm indicates that Cu-Cy-PEG@Au NPs can effectively absorb near-infrared light.

[0036] Example 3

[0037] In this example, we tested the antibacterial ability and antibacterial use of Cu-Cy-PEG@Au NPs composite materials. We selected Escherichia coli (E. coli) as a representative of Gram-negative bacteria and Staphylococcus aureus (S. aureus) as a representative of Gram-positive bacteria, and conducted an in vitro catalytic antibacterial performance test of Cu-Cy-PEG@Au NPs. The bacteria used were the above-mentioned bacteria diluted to OD600 = 0.1. The antibacterial performance of Cu-Cy-PEG@Au NPs catalyzed H2O2, and the bacterial survival rate after treatment with different materials was measured by the plate count method. Three parallel experiments were performed for each sample.

[0038] The typical method includes: mixing 100 μL Cu-Cy-PEG@Au NPs solution with 20 μL bacterial solution, adding 20 μL H2O2 solution and 60 μL PBS solution, and using a power density of 3 W cm -2 The final concentration of the materials in the above reaction was 100 μg / mL, and the final concentration of H2O2 was 1 mM.

[0039] The remaining single-factor experiments used the single-factor concentrations and treatment times described above.

[0040] like Figure 3 As shown in the figure, whether it is for Gram-negative bacteria or Gram-positive bacteria, Cu-Cy-PEG@Au NPs has the best bactericidal effect in the presence of hydrogen peroxide and near-infrared light conditions, that is, it has a significant photodynamic antibacterial effect.

[0041] As can be seen from the above examples, the present invention synthesized a hybrid of cysteamine copper nanosheets and gold nanoclusters (Au NPs), and demonstrated its excellent antibacterial ability. The present invention prepared nano-gold-loaded copper cysteamine (Cu-Cy-PEG@AuNPs), which has good antibacterial activity against Gram-negative and Gram-positive bacteria, has significant bactericidal properties, and has a clearing effect on stubborn bacterial biofilms.

[0042] The above are only a few preferred embodiments of the present invention, which are described in a more specific and detailed manner, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing a novel photodynamic antibacterial material Cu-Cy-PEG@Au NPs, characterized in that: The following steps are involved: 1) Dissolve CuCl2•2H2O in deionized water, add cysteamine hydrochloride and PEG4000, and adjust the pH to 7 with sodium hydroxide; 2) After stirring the reaction, heat the oil bath to 100°C to continue the reaction, and then add chloroauric acid solution and trisodium citrate; 3) After centrifugation and washing, the product is dried in a vacuum oven to obtain a Cu-Cy-PEG@Au NPs composite material.

2. The method for preparing a novel photodynamic antibacterial material Cu-Cy-PEG@Au NPs according to claim 1, characterized in that: The step 1) includes the following specific steps: Weigh a certain mass of CuCl2·2H2O, dissolve it in an appropriate amount of deionized water, stir magnetically at 25°C until dissolved, add a certain mass of cysteamine hydrochloride and PEG4000 under nitrogen protection, and adjust the pH to 7 with 1M sodium hydroxide solution; wherein, the final concentration of CuCl2 is 0.03 mol / L, the final concentration of cysteamine hydrochloride is 0.06 mol / L, and the final concentration of PEG4000 is 0.8 mg / mL.

3. The method for preparing a novel photodynamic antibacterial material Cu-Cy-PEG@Au NPs according to claim 1, characterized in that: The step 2) includes the following specific steps: Continue stirring the reaction for about 15 to 30 minutes. A dark purple substance will appear on the wall of the flask. Then transfer it to an oil bath. Heat the oil bath to 100 °C and react for 20 min. Then add 1 / 1000 volume of 0.1 g / ml chloroauric acid solution and a certain mass of trisodium citrate to make the mass volume concentration of trisodium citrate reach about 0.1%.

4. The method for preparing a novel photodynamic antibacterial material Cu-Cy-PEG@Au NPs according to claim 1, characterized in that: The step 3) includes the following specific steps: After centrifugation at 9000 rpm for 10 min, the mixture was washed three times with a mixture of deionized water and ethanol in a volume ratio of 4:5, and then the obtained product was completely dried in a vacuum oven at 40 °C overnight to obtain a Cu-Cy-PEG@Au NPs composite material.

5. The method for preparing a novel photodynamic antibacterial material Cu-Cy-PEG@Au NPs according to claim 1, characterized in that: It also includes step 4) testing the catalytic synergistic photothermal bactericidal performance of Cu-Cy-PEG@Au NPs: the material is mixed with bacterial solution, H2O2 and PBS solution, and irradiated with 880 nm near-infrared laser.

6. A new type of photodynamic antibacterial drug / material Cu-Cy-PEG@Au NPs, characterized in that: The method is prepared by any one of claims 1 to 5.

7. Use of the novel photodynamic antibacterial material Cu-Cy-PEG@Au NPs as claimed in claim 6 in the preparation of materials / drugs for enhancing the antibacterial ability of H2O2.

8. A composite antibacterial material / drug, characterized in that: Contains the photodynamic antibacterial material Cu-Cy-PEG@Au NPs as claimed in claim 6 and H2O2.

9. An antibacterial method, characterized in that: The method comprises simultaneously applying the antibacterial material Cu-Cy-PEG@Au NPs and H2O2 to the material to be treated under near-infrared light conditions.

10. The antibacterial method according to claim 9, characterized in that: The bacteria is Escherichia coli or Staphylococcus aureus.

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