Photo-thermal antibacterial material and preparation method thereof

Abstract

The invention discloses a photo-thermal antibacterial material which is an oxidation-state product generated by carrying out oxidation reaction on 3, 3 ', 5, 5'-tetramethyl benzidine by using a novel nano material, a substance with a photo-thermal effect is generated through an oxidation reaction of 2, 3, 3 ', 5, 5'-tetramethyl benzidine, and the material has the advantages of being strong in photo-thermal effect, good in bacterium selectivity, high in near-infrared region absorption capacity, high in photo-thermal conversion efficiency, good in biocompatibility, low in cost and convenient to synthesize. Meanwhile, the hydrogel microspheres wrapped with the photo-thermal antibacterial material are prepared through a microfluidic technology, and the synergistic treatment prospect of slow-release photo-thermal antibacterial and anti-inflammatory is achieved.

Description

Technical Field

[0001] The invention relates to an antibacterial material, and in particular to a photothermal antibacterial material which converts light energy into heat energy for antibacterial and sterilization and a preparation method thereof. Background Art

[0002] Infections caused by drug-resistant bacteria have posed a serious threat to human health. At present, traditional antibiotics are a common strategy for treating diseases induced by pathogenic bacteria and have been successful to a certain extent. However, excessive use of antibiotics has greatly increased the risk of bacterial resistance and the emergence of superbugs, resulting in a significant reduction in treatment efficacy. Therefore, the development of new antibacterial strategies and antibacterial agents is imperative. Photothermal therapy (PTT) is considered to be a safe and effective antibacterial strategy that shows great promise in treating bacterial infections. Near-infrared light triggers the conversion of light energy into heat energy, which can cause irreversible damage to bacterial cells, causing leakage of cell contents by changing the permeability of the biofilm, thereby killing bacteria. At present, the photothermal materials under research mainly include: 1. Precious metal nanoparticles, which have strong NIR absorption and are easy to combine with biological molecules, but have poor selectivity for bacteria; 2. Carbon nanomaterials, which have a large photothermal conversion area, but poor absorption capacity in the near-infrared region; 3. Metal and non-metal compounds, which have the characteristics of low cost and convenient synthesis, but low photothermal conversion efficiency; 4. Polymer nanomaterials, which are easy to form antibacterial coatings or films, but have low photothermal conversion efficiency and poor biocompatibility, etc.

[0003] Therefore, a new type of photothermal antibacterial material is needed that can simultaneously solve the technical problems of existing photothermal antibacterial materials, such as poor bacterial selectivity, poor near-infrared absorption capacity, low photothermal conversion efficiency, and poor biocompatibility, while also having the characteristics of low cost and easy synthesis. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a photothermal antibacterial material and a preparation method thereof, which can simultaneously solve the technical problems of existing photothermal antibacterial materials such as poor bacterial selectivity, poor near-infrared absorption capacity, low photothermal conversion efficiency, and poor biocompatibility, while also having the characteristics of low cost and convenient synthesis.

[0005] A photothermal antibacterial material of the present invention is an oxidized product generated by an oxidation reaction of 3,3',5,5'-tetramethylbenzidine;

[0006] Furthermore, the 3,3',5,5'-tetramethylbenzidine is catalyzed by the modified novel nano-cerium oxide to generate an oxidized product;

[0007] Furthermore, the mass ratio of the 3,3',5,5'-tetramethylbenzidine to the nano-cerium oxide is 4-6:1. ;

[0008] Furthermore, the photothermal antibacterial material is a gel microsphere containing an oxidized product encapsulated in gel;

[0009] The method for preparing the photothermal antibacterial material of the present invention comprises the following steps:

[0010] S1, preparation of nano-cerium oxide;

[0011] S2, mixing a mixed solution of nano-cerium oxide and 3,3',5,5'-tetramethylbenzidine with a photoinitiator and a hydrogel material to obtain a hydrogel precursor mixed solution, and then using microfluidic technology to prepare the hydrogel precursor mixed solution into uniform-sized nano-hydrogel microspheres of photothermal material carriers;

[0012] Further, in step S1, solid cerium nitrate hexahydrate, sodium fluoride, boric acid and sodium phosphate solution are mixed and reacted at a temperature of 160-180° C., and then the mixture is separated and dried to obtain powdered nano cerium oxide particles;

[0013] Furthermore, the molar ratio of the cerium nitrate hexahydrate solid, sodium fluoride, boric acid, and sodium phosphate solution is 1:3:1:0.01;

[0014] Further, in step S2, the aqueous solution A formed by the nano-cerium oxide particles and 3,3',5,5'-tetramethylbenzidine is mixed with the gel material containing the photoinitiator, and micro-droplets are generated through the microchannel of the droplet microfluidic chip, and then cured by ultraviolet irradiation to obtain hydrogel microspheres;

[0015] Further, in step S2, the method for preparing the aqueous phase solution comprises the following steps: uniformly mixing nano-cerium oxide particles, 3,3',5,5'-tetramethylbenzidine, water, a photoinitiator, and a gel material to obtain a hydrogel precursor mixed solution;

[0016] Further, in step S2, the photoinitiator is any one of lithium phenyl-2,4,6-trimethylbenzoylphosphonate, phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, and 2,4,6(trimethylbenzoyl)diphenylphosphine oxide, corresponding to different hydrogel materials; the gelling material is any one of methacrylated carboxymethyl cellulose, methacrylated gelatin, methacrylated sodium hyaluronate, methacrylated sodium alginate, methacrylated chitosan, methacrylated carboxymethyl chitosan, methacrylated chondroitin sulfate, methacrylated gellan gum, acrylated poloxamer, and acrylated RGD peptide.

[0017] The beneficial effects of the present invention are as follows: the photothermal antibacterial material and preparation method thereof of the present invention, 3,3',5,5'-tetramethylbenzidine generates a substance with photothermal effect through oxidation reaction, has a strong photothermal effect, good selectivity for bacteria, strong absorption capacity in the near-infrared region, high photothermal conversion efficiency, good biocompatibility, and has the characteristics of low cost and convenient synthesis. The photothermal antibacterial material wrapped by hydrogel microspheres has a sustained release effect, and has the application prospects of photothermal antibacterial and anti-inflammatory in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0019] Figure 1 This is an antibacterial effect diagram of the photothermal antibacterial material of the present invention;

[0020] Figure 2 This is a photothermal cycle effect diagram of the photothermal antibacterial material of the present invention;

[0021] Figure 3 This is a photothermal heating effect diagram of the photothermal antibacterial material of the present invention. DETAILED DESCRIPTION

[0022] Embodiment 1

[0023] The method for preparing the photothermal antibacterial material of this embodiment comprises the following steps:

[0024] Preparation of nano-cerium oxide: Using the hydrothermal method, hexahydrate cerium nitrate solid, sodium fluoride, and boric acid are dissolved in sodium phosphate solution and stirred, and the materials are added according to the molar ratio of 1:3:1:0.01; the reactants are transferred to a reactor lined with polytetrafluoroethylene and reacted at 170°C for 13 hours; after the reaction, the mixture is transferred to a centrifuge tube and centrifuged on a centrifuge. After the reaction, the supernatant is poured out, water is added, and stirred with glass for 2min-5min, and centrifuged again. Repeat the centrifugation to complete the water washing, and then change to an organic solvent for repeated washing. Dry in a 50°C constant temperature oven for 4h to obtain powdered nano-cerium oxide.

[0025] Preparation of photothermal antibacterial materials:

[0026] Step A: dispersing a plurality of nano-cerium oxide particles with a particle size of 20 nm, 3,3',5,5'-tetramethylbenzidine and a photoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), in a methacrylated carboxymethyl cellulose solution, and dispersing them uniformly by ultrasonic treatment to obtain a mixed colloidal solution; the mass ratio of the 3,3',5,5'-tetramethylbenzidine to the nano-cerium oxide is 4:1;

[0027] Step B: using the mixed colloidal solution obtained in step A to prepare a water-in-oil emulsion by a microfluidic method, and then irradiating the water-in-oil emulsion under an ultraviolet lamp with a wavelength of 311 nm, so that the mixed colloidal solution is solidified into nano-cerium oxide / hydrogel composite microspheres;

[0028] Step C: using an organic solvent to clean the nano-cerium oxide / hydrogel composite microspheres cured in step B, thereby obtaining clean cerium nanoparticles / hydrogel composite microspheres.

[0029] Embodiment 2

[0030] The method for preparing the photothermal antibacterial material of this embodiment comprises the following steps:

[0031] Preparation of nano-cerium oxide: Using the hydrothermal method, hexahydrate cerium nitrate solid, sodium fluoride, and boric acid are dissolved in sodium phosphate solution and stirred, and the materials are added according to the molar ratio of 1:3:1:0.01; the reactants are transferred to a reactor lined with polytetrafluoroethylene and reacted at 160°C for 13.5 hours; after the reaction, the mixture is transferred to a centrifuge tube and centrifuged on a centrifuge. After the reaction, the supernatant is poured out, water is added, and stirred with glass for 2min-5min, and centrifuged again. Repeat the centrifugation to complete the water washing, and then change to an organic solvent for repeated washing. Dry in a constant temperature oven at 45°C for 5.5 hours to obtain powdered nano-cerium oxide.

[0032] Preparation of photothermal antibacterial materials:

[0033] Step A: Dispersing multiple nano-cerium oxides with a particle size of 100 nm, 3,3',5,5'-tetramethylbenzidine and a photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) in a methacrylated gelatin solution, and dispersing them evenly by ultrasonic treatment to obtain a mixed colloidal solution; the mass ratio of the 3,3',5,5'-tetramethylbenzidine to the nano-cerium oxide is 6:1.

[0034] Step B: using the mixed solution obtained in step A to prepare a water-in-oil emulsion by a microfluidic method, and then irradiating the water-in-oil emulsion under an ultraviolet lamp with a wavelength of 330 nm, so that the mixed colloidal solution in the water-in-oil emulsion is solidified into cerium nanoparticles / hydrogel composite microspheres;

[0035] Step C: using an organic solvent to clean the nano-cerium oxide / hydrogel composite microspheres cured in step B, thereby obtaining clean nano-cerium oxide / hydrogel composite microspheres.

[0036] Embodiment 3

[0037] The method for preparing the photothermal antibacterial material of this embodiment comprises the following steps:

[0038] Preparation of nano-cerium oxide: Using the hydrothermal method, solid cerium nitrate hexahydrate, sodium fluoride, and boric acid are dissolved in sodium phosphate solution and stirred, and the materials are added according to the molar ratio of 1:3:1:0.01; the reactants are transferred to a reactor lined with polytetrafluoroethylene and reacted at 180°C for 11 hours; after the reaction, the mixture is transferred to a centrifuge tube and centrifuged on a centrifuge. After the reaction, the supernatant is poured out, water is added, and stirred with glass for 2min-5min, and centrifuged again. Repeat the centrifugation to complete the water washing, and then change to an organic solvent for repeated washing. Dry in a constant temperature oven at 55°C for 4h to obtain powdered nano-cerium oxide.

[0039] Preparation of antibacterial materials:

[0040] Step A: dispersing a plurality of nano-cerium oxides with a particle size of 60 nm, 3,3',5,5'-tetramethylbenzidine and a photoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), in a methacrylated gelatin liquid to obtain a mixed colloidal solution; the mass ratio of the 3,3',5,5'-tetramethylbenzidine to the nano-cerium oxide is 5.1:1;

[0041] Step B: preparing a water-in-oil emulsion by a microfluidic method from the mixed colloidal solution obtained in step A, and then irradiating the water-in-oil emulsion under an ultraviolet lamp with a wavelength of 300 nm, so that the mixed colloidal solution in the water-in-oil emulsion is solidified into nano-cerium oxide / hydrogel composite microspheres;

[0042] Step C: using an organic solvent to clean the cerium nanoparticles / hydrogel composite microspheres cured in step B, thereby obtaining clean nano-cerium oxide / hydrogel composite microspheres.

[0043] In the above embodiment, in order to improve the uniformity of mixing, a microfluidic device can be used for mixing: the device includes a "Y"-shaped microfluidic chip with a serpentine mixing channel, wherein the chip is provided with a first liquid inlet channel, a second liquid inlet channel and a mixing channel; the mixing channel includes an inlet section and a main section connected to each other; the outlet of the first liquid inlet channel is connected to the first channel wall of the inlet section, and the outlet of the second liquid inlet channel is connected to the second channel wall of the inlet section; the main section forms continuous undulations; taking the undulation direction of the main section as the height direction, the first channel wall and the second channel wall are at different heights.

[0044] A "Y"-shaped microfluidic chip with a serpentine mixing channel contains two injection ports and one outlet, forming a "Y" microchannel structure consisting of two branch channels converging and connecting to a main channel. The two branch channels form two precursor solution injection ports, and the end of the main channel forms a product outlet. The total length of the serpentine mixing channel is not less than 4 cm.

[0045] A constant pressure injection pump is used to inject the mixture of nano-cerium oxide solution, TMB and hydrogel microsphere precursor solution into the branch microchannel of the "Y"-shaped microfluidic chip through the injection port to prepare the hydrogel microsphere precursor solution mixture of the microfluidic chip. The total flow rate of the solution is set, and the flow rate ratio of the nano-cerium oxide solution to TMB and the hydrogel microsphere precursor solution is adjusted. The product is collected at the outlet of the main microchannel of the "Y"-shaped microfluidic chip. The product is the prepared hydrogel microspheres.

[0046] The photothermal antibacterial material prepared in Example 1 was subjected to the following tests.

[0047] 1. Antibacterial performance test:

[0048] 1) Use autoclaved double distilled water to prepare 10 mg / mL nano-cerium oxide and 3,3',5,5'-tetramethylbenzidine to obtain a mixed solution A. Take 1×10 8 0.9mL of CFU / mL Staphylococcus aureus solution was mixed with 100μL of sample and incubated at 37℃ for 12 hours. The control group was not treated. The experimental group was irradiated with 808nm laser for 10min or 15min, diluted 1000 times, 10μL was taken to spread on the plate, and then placed in a biochemical incubator at 37℃ for 15 hours, and the number of colonies on the plate was observed. Common bacteria Escherichia coli and Staphylococcus aureus were selected and cultured in liquid LB medium.

[0049] The results are as follows Figure 1 As shown: After 10 minutes of 808nm laser irradiation, the colonies of Staphylococcus aureus and Escherichia coli were significantly reduced, but the number of colonies was still large, indicating that the photothermal antibacterial effect of mixed solution A was not optimal; after 15 minutes of 808nm laser irradiation, the colonies of Staphylococcus aureus and Escherichia coli were significantly reduced and the number of colonies decreased significantly, indicating that the photothermal antibacterial effect of mixed solution A was good after 15 minutes. This is because after laser irradiation, mixed solution A generates a large amount of heat, the temperature of the microorganisms rises, causing cell damage and death, and the antibacterial effect is better.

[0050] 2. Compatibility evaluation of mixed solution A:

[0051] After the red blood cells were diluted with PBS, they were mixed with different concentrations of mixed solution A, and the mixed solution was incubated at 37°C for 4 hours. Then the mixed solution was centrifuged at 12000rpm for 5 minutes at 4°C. 100μL of the supernatant was aspirated and added to a 96-well plate. The absorbance of the supernatant at 492nm was measured with an enzyme marker to calculate the hemolysis rate. Sterile deionized water was used as the positive control in this experiment, and PBS was used as the negative control. Mixed solution A has good blood compatibility within a concentration of 200μg / mL.

[0052] 3. Near infrared absorption capacity:

[0053] Evaluation of the photothermal conversion effect of mixed solution A: Mixed solution A was prepared into a 200 μg / mL solution with PBS at pH = 5 and incubated for 6 h. 2 The dispersion was irradiated with NIR and the temperature of the system was detected with a FLIR infrared thermal imager. The temperature value was recorded every 30 seconds until the temperature of the system stopped increasing. Under NIR irradiation, the temperature of the solution did not change significantly when the concentration of mixed solution A was 0 (PBS). After irradiation for 300 seconds, the temperature increased by about 10°C, while 200μg / mL mixed solution A could increase the solution temperature by about 50°C within 300 seconds. It can be seen that mixed solution A has a photothermal conversion effect, which can convert NIR light energy into heat, and can be used as a photothermal conversion agent for photothermal antibacterial.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A photothermal antibacterial material, characterized in that: The photothermal antibacterial material is an oxidized product generated by an oxidation reaction of 3,3',5,5'-tetramethylbenzidine.

2. The photothermal antibacterial material according to claim 1, characterized in that: The 3,3',5,5'-tetramethylbenzidine is catalyzed by the modified novel nano-cerium oxide to generate an oxidized product.

3. The photothermal antibacterial material according to claim 2, characterized in that: The mass ratio of the 3,3',5,5'-tetramethylbenzidine to nano-cerium oxide is 4-6:

1.

4. The photothermal antibacterial material according to claim 2, characterized in that: The photothermal antibacterial material is gel microspheres containing oxidized products wrapped in gel.

5. The method for preparing the photothermal antibacterial material according to claim 1, characterized in that: The following steps are involved: S1, preparation of nano-cerium oxide; S2, mixing a mixture of nano-cerium oxide and 3,3',5,5'-tetramethylbenzidine with a photoinitiator and a hydrogel material to obtain a hydrogel precursor mixed solution, and then using microfluidic technology to make the hydrogel precursor mixed solution into uniform-sized nano-hydrogel microspheres of photothermal material carriers.

6. The method for preparing the photothermal antibacterial material according to claim 5, characterized in that: In step S1, solid cerium nitrate hexahydrate, sodium fluoride, boric acid and sodium phosphate solution are mixed and reacted at a temperature of 160-180° C., and then the mixture is separated and dried to obtain powdered nano cerium oxide particles.

7. The method for preparing the photothermal antibacterial material according to claim 6, characterized in that: The molar ratio of the hexahydrate cerium nitrate solid, sodium fluoride, boric acid and sodium phosphate solution is 1:3:1:0.

01.

8. The method for preparing the photothermal antibacterial material according to claim 5, characterized in that: In step S2, the aqueous solution formed by nano-cerium oxide particles and 3,3',5,5'-tetramethylbenzidine is mixed with a gel material containing a photoinitiator, and microdroplets are generated through microchannels of a droplet microfluidic chip, which are then cured by ultraviolet irradiation to obtain hydrogel microspheres.

9. The method for preparing the photothermal antibacterial material according to claim 8, characterized in that: In step S2, the method for preparing the aqueous phase solution comprises the following steps: mixing nano-cerium oxide particles, 3,3',5,5'-tetramethylbenzidine, water, a photoinitiator, and a gel material uniformly to obtain a hydrogel precursor mixed solution.

10. The method for preparing the photothermal antibacterial material according to claim 9, characterized in that: In step S2, the photoinitiator is any one of lithium phenyl-2,4,6-trimethylbenzoylphosphonate, phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, and 2,4,6(trimethylbenzoyl)diphenylphosphine oxide, corresponding to different hydrogel materials; the gelling material is any one of methacrylated carboxymethyl cellulose, methacrylated gelatin, methacrylated sodium hyaluronate, methacrylated sodium alginate, methacrylated chitosan, methacrylated carboxymethyl chitosan, methacrylated chondroitin sulfate, methacrylated gellan gum, acrylated poloxamer, and acrylated RGD peptide.

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