A photothermal-photodynamic antibacterial probe and a preparation method and application thereof

Abstract

The application discloses a kind of photothermal-photodynamic synergistic antibacterial multifunctional probe and its preparation method and application, and is related to biomedical antibacterial field.The antibacterial probe includes gold-silver alloy hollow nanoparticles and photosensitizer modified on the surface of the gold-silver alloy hollow nanoparticles, and the photosensitizer for photodynamic antibacterial effect is chlorin e6 (Ce6).The antibacterial probe in the application has simple synthesis steps, excellent photothermal properties and photodynamic effect, has a significant effect on antibacterial growth, and has good biocompatibility, and can be used for the skin surface of animal tissue to carry out antibacterial effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical optical antibacterial, specifically to a photothermal-photodynamic antibacterial probe, its preparation method, and its application. Background Technology

[0002] Bacterial infections are one of the most serious threats to human health, imposing a huge medical and economic burden on society. If bacterial infections are not diagnosed and treated in time, they can become chronic and develop into sepsis, multiple organ failure, and other conditions that severely impact a patient's survival.

[0003] Currently, antibiotic therapy remains the primary method for treating bacterial infections in clinical practice. However, due to the emergence and evolution of bacterial resistance, the situation regarding the treatment of bacterial infections is becoming increasingly challenging, both now and in the future. This compels clinicians and researchers to focus on developing new, highly effective treatment methods that are less likely to induce resistance.

[0004] In recent years, the emergence of novel treatment methods such as photothermal therapy (PTT) and photodynamic therapy (PDT) based on nanomaterials has provided new directions for antibacterial treatment. PTT eliminates bacteria by inducing local overheating through non-invasive light irradiation, thereby disrupting the integrity of pathogens. It has the characteristics of spatiotemporal controllability and is less likely to induce drug resistance. However, the high temperature generated by the high-power laser irradiation used in PTT may damage normal tissues. PDT uses photosensitizers to generate singlet oxygen under laser irradiation, which has a killing effect on cells. However, the antibacterial effect of photosensitizers alone in PDT is still insufficient. Summary of the Invention

[0005] This invention provides a photothermal-photodynamic antibacterial probe, its preparation method, and its application, aiming to solve the problems existing in the above-mentioned background technology, and to construct an antibacterial probe that has both photothermal and photodynamic effects. This can avoid the damage to normal tissues caused by high-power lasers generated by photothermal treatment alone, and can also enhance the antibacterial effect of photodynamic therapy alone.

[0006] To achieve the above-mentioned technical objectives, the present invention mainly adopts the following technical solutions:

[0007] This invention discloses a photothermal-photodynamic antibacterial probe, characterized in that the surface of the gold-silver alloy hollow nanoparticles is modified with functionalized polyethylene glycol (HS-PEG-NH2). The probe comprises gold-silver alloy hollow nanoparticles and a photosensitizer, dihydroporphyrin E6 (Ce6), modified on the surface of the gold-silver alloy hollow nanoparticles. The photosensitizer is cross-linked with the surface of the gold-silver alloy hollow nanoparticles via the functionalized polyethylene glycol.

[0008] The present invention also provides a method for preparing the above-mentioned photothermal-photodynamic antibacterial probe, comprising the following steps:

[0009] S1: Silver nanoparticles were synthesized by reducing silver chloride with ascorbic acid under alkaline conditions, and gold-silver alloy hollow nanoparticles were synthesized by chloroauric acid substitution.

[0010] S2: Tris(2-carboxyethyl)phosphine was added to the gold-silver alloy hollow nanoparticles in S1, followed by the addition of mercapto-polyethylene glycol-amine groups, and the mixture was stirred evenly overnight.

[0011] S3: Dihydroporphyrin e6 and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were mixed evenly in dimethyl sulfoxide medium and reacted at 25°C for 30 minutes. Then, N-hydroxysuccinimide was added and the reaction was continued for 90 minutes.

[0012] S4: Add the activated carboxyl group of dihydroporphyrin e6 from S3 to S2, mix thoroughly and shake for 24 hours, centrifuge and resuspend to obtain the photothermal-photodynamic antibacterial probe.

[0013] Another object of the present invention is to provide the application of the above-mentioned photothermal-photodynamic antibacterial probe in antibacterial therapy.

[0014] Preferably, the antibacterial treatment is an in vitro antibacterial effect or an antibacterial effect on the skin surface of an animal model.

[0015] The antibacterial effect of bacteria in vitro includes the following steps:

[0016] Bacteria were co-incubated with a photothermal-photodynamic antibacterial probe and then irradiated with 808nm and 660nm lasers to produce an antibacterial effect.

[0017] The animal model-level antibacterial activity includes the following steps:

[0018] (1) Construct an animal skin infection model;

[0019] (2) Incubate the infected skin wound with photothermal-photodynamic antibacterial probe solution;

[0020] (3) The antibacterial effect was produced by irradiation with 808nm and 660nm lasers and the antibacterial effect was evaluated by plate coating of skin samples taken from the wound.

[0021] The bacteria described in this invention are Staphylococcus aureus ATCC25923 and Escherichia coli ATCC25922.

[0022] The present invention has the following beneficial effects:

[0023] This invention discloses a photothermal-photodynamic antibacterial probe, its preparation method, and its application, relating to the field of biomedical antibacterial agents. The antibacterial probe comprises hollow gold-silver alloy nanoparticles and a photosensitizer modified on the surface of the hollow gold-silver alloy nanoparticles. The photosensitizer for photodynamic antibacterial activity is dihydroporphyrin E6. The photothermal-photodynamic antibacterial probe of this invention has a simple synthesis procedure, excellent photothermal properties and photodynamic activity, significant effects in inhibiting bacterial growth, good biocompatibility, and can be used on the skin surface of animal tissues for antibacterial activity. Attached Figure Description

[0024] Figure 1 The flowchart and structural schematic diagram of the photothermal-photodynamic antibacterial probe prepared according to the present invention are shown.

[0025] Figure 2 Transmission electron microscopy and elemental analysis characterization of the photothermal-photodynamic antibacterial probe prepared in this invention.

[0026] Figure 3 The particle size distribution of Ag@Au, Ag@Au-PEG, and Ag@Au-PEG-Ce6 (photothermal-photodynamic antibacterial probes) prepared in this invention is shown in the figure.

[0027] Figure 4 Zeta potential diagrams of Ag@Au, Ag@Au-PEG, and Ag@Au-PEG-Ce6 (photothermal-photodynamic antibacterial probes) prepared in this invention.

[0028] Figure 5 The photothermal stability diagram of the photothermal-photodynamic antibacterial probe prepared in this invention is shown.

[0029] Figure 6 This is a diagram showing the singlet oxygen generation of the photothermal-photodynamic antibacterial probe prepared in this invention.

[0030] Figure 7 The image shows the fluorescence staining results of Staphylococcus aureus and Escherichia coli after incubation and light treatment with the photothermal-photodynamic antibacterial probe prepared in this invention. The scale bar in the image is 10 μm.

[0031] Figure 8 The image shows the results of antibacterial treatment of skin infection wounds using the photothermal-photodynamic antibacterial probe prepared in this invention. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0033] Example 1: Synthesis of Silver Nanospheres

[0034] Weigh 85 mg of PVP and add it to 20 mL of ultrapure water, stirring to dissolve it on a magnetic stirrer. Add 85 mg of silver nitrate, and after dissolving, add 200 μL of 5 M NaCl solution. Stir for 15 minutes in the dark to obtain silver chloride colloid. Separately, weigh 0.528 g of ascorbic acid and add it to 60 mL of ultrapure water, followed by 6.9 mL of 0.5 M sodium hydroxide solution. Add the above silver chloride colloid (7.5 mL) dropwise while stirring evenly with a magnetic stirrer. Stir for 2 hours in the dark to prepare silver nanospheres.

[0035] Example 2: Synthesis of Gold-Silver Alloy Hollow Nanoparticles

[0036] Take 12.5 mL of the above-mentioned silver nanospheres, wash them several times with ultrapure water, and concentrate them to 2 mL. Quickly dissolve 50 mg PVP in 48 mL of ultrapure water, add 2 mL of concentrated silver nanoparticles, and heat. After the solution boils, adjust the temperature to maintain a gentle boil for 10 minutes. Add chloroauric acid solution (0.4 mM, 0.75 mL / h) dropwise using a high-precision syringe pump. Stop adding chloroauric acid when the maximum absorption wavelength of the solution reaches 750 nm after adding 22 mL, and continue heating and stirring for 5 minutes before stopping heating. After cooling in an ice bath, add 7 mL of ammonia solution and continue stirring for half an hour. Then wash three times with ultrapure water, and finally suspend the nanoparticle precipitate in 12.5 mL of ultrapure water to prepare Ag@Au (gold-silver alloy hollow nanoparticles).

[0037] Example 3: Functionalized Polyethylene Glycol-Amine Group Combination

[0038] 1 mL of gold-silver alloy hollow nanoparticles were mixed with tris(2-carboxyethyl)phosphine (2 × 10⁻⁶). -4 M, 30 μL), followed by the addition of mercapto-polyethylene glycol-amine (10 -4 Mix M, 30 μL) thoroughly and stir overnight at room temperature;

[0039] Example 4: Activation of the carboxyl group of dihydroporphyrin E6

[0040] Take 0.036 mg of dihydroporphyrin E6 and 0.288 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and mix them evenly by ultrasonication in dimethyl sulfoxide medium. After reacting at 25°C for 30 minutes, add 0.144 mg of N-hydroxysuccinimide and continue the reaction for 90 minutes.

[0041] Example 5: Synthesis of Ag@Au-PEG-Ce6 (Photothermal-Photodynamic Antibacterial Probe)

[0042] Add the carboxyl-activated dihydroporphyrin e6 from Example 4 to Example 3, mix thoroughly and shake for 24 hours, centrifuge and resuspend to obtain the photothermal-photodynamic antibacterial probe.

[0043] Example 6: Photothermal stability test of photothermal-photodynamic antibacterial probe

[0044] The 1 nM photothermal-photodynamic antibacterial probe from Example 5 was irradiated with an 808 nm laser for 5 minutes, stopped for 15 minutes, and repeated for a total of 5 times. The temperature change was recorded every 30 seconds using an infrared thermal imager.

[0045] The effect is as follows Figure 5 After irradiation with an 808nm laser for 5 minutes, the temperature of the probe solution can reach above 55℃, and after 5 photothermal cycles, the probe solution can still reach this temperature, indicating that the probe solution has excellent photothermal performance and excellent photothermal stability.

[0046] Example 7: Test of the singlet oxygen production capacity of the photothermal-photodynamic antibacterial probe

[0047] The photothermal-photodynamic antibacterial probe solution from Example 5 was added to a quartz cup and irradiated with a 660 nm wavelength laser. Fluorescence intensity was measured using an SOSG fluorescent probe (final concentration 1 μM) at minutes 1, 2, 5, 10, 15, and 20, indicating the generation of singlet oxygen. (E) x 488nm, E m (500-650nm)

[0048] The effect is as follows Figure 6 The photothermal-photodynamic antibacterial probe solution showed a continuous increase in SOSG fluorescence intensity upon excitation light irradiation, indicating the continuous generation of singlet oxygen and suggesting that photodynamic action can be used for antibacterial purposes.

[0049] Example 8: The photothermal-photodynamic antibacterial probe showed antibacterial effects after incubation with Staphylococcus aureus and Escherichia coli and light treatment, as indicated by fluorescent staining.

[0050] Collect freshly cultured bacteria (Staphylococcus aureus, Escherichia coli) in the logarithmic growth phase, centrifuge to collect the bacterial pellet, wash with sterile physiological saline and resuspend to a concentration of 10. 8 CFU / mL. Divided into four groups: PBS group, PBS + Laser (808nm 0.8W / cm) group, etc. 2 5 minutes + 660nm 0.2W / cm 2 5-minute group, probe group, probe + laser (808nm 0.8W / cm) 2 5 minutes + 660nm 0.2W / cm 2For the 5-minute group, 200 μL of bacterial suspension was added to 50 μL of PBS or a photothermal-photodynamic antibacterial probe, followed by laser irradiation or without irradiation. After different treatments, Syto 9 and PI from the kit were added to appropriate dye concentrations, incubated for 10 minutes, centrifuged, resuspended in 10 μL of sterile physiological saline, and dropped onto a glass slide for observation under a confocal fluorescence microscope.

[0051] The effect is as follows Figure 7 The results showed similar outcomes in both the Staphylococcus aureus and Escherichia coli groups. With PBS alone and PBS plus light irradiation, almost no dead bacteria were observed using the live-dead bacterial dye (red). In the group using the photothermal-photodynamic probe alone without laser irradiation, the bacteria were almost all in a green, viable state. Only when the photothermal-photodynamic probe was used in conjunction with laser irradiation were bacterial deaths clearly observed (red).

[0052] In summary, the photothermal-photodynamic probe described in this invention has excellent antibacterial effects.

[0053] Example 9: Antibacterial effect of photothermal-photodynamic probes in animal models

[0054] Establishment of an animal skin infection model: On day 0, BALB / c mice (18-22g) were randomly divided into a blank control group, a PBS group, and an Ag@Au group (808nm 0.8W / cm²). 2 5-minute group, Ce6 (660nm 0.2W / cm) 2 5-minute group, Ag@Au-Ce6 group, Ag@Au-Ce6 (808nm 0.8W / cm) 2 5 minutes + 660nm 0.2W / cm 2 (5 minutes) group (n=3). Mice were anesthetized with sodium pentobarbital solution, and hair was removed from the right hindquarters of the back. A circular wound with a diameter of 8 mm was constructed using a sterilized punch. Except for the control group, 50 μL of bacterial suspension (2×10^7 CFU / mL) was added to each of the other groups.

[0055] On days 1 and 3, according to the above groupings, PBS, Ag@Au, Ce6, and Ag@Au-Ce6 were added to the wounds for laser or no laser irradiation, respectively. The wound size was photographed and recorded on days 0, 2, 4, 6, and 8. On day 8, skin samples were taken from the wounds, thoroughly mixed in sterile saline, and plated.

[0056] Depend on Figure 8The wound healing in the mouse group treated with photothermal-photodynamic antibacterial therapy was only slightly worse than the control group, better than the treatment-only group, and significantly better than the PBS group without any treatment. Agar smears of skin samples from the wounds also showed that bacterial numbers were effectively suppressed in the photothermal-photodynamic antibacterial therapy group, thereby promoting wound healing.

[0057] In the photothermal-photodynamic antibacterial probes prepared in the above embodiments, the photothermal properties of the synthesized Ag@Au structure, combined with the photodynamic effect through the polyethylene glycol-linked photosensitizer, generate singlet oxygen to kill bacteria through photothermal-photodynamic killing. This avoids the damage to normal tissues caused by high-power lasers generated by photothermal therapy alone, while enhancing the antibacterial effect of photodynamic therapy alone.

[0058] The above description is only a part of the examples of the present invention, and not all of them. The detailed description of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected examples. All other examples obtained by those skilled in the art based on the examples of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. Application of photothermal-photodynamic antibacterial probe in the preparation of antibacterial therapeutic agents, wherein the photothermal-photodynamic antibacterial probe comprises gold-silver alloy hollow nanoparticles and a photosensitizer modified on the surface of the gold-silver alloy hollow nanoparticles, wherein the photosensitizer is dihydroporphyrin e6, i.e. Ce6; The surface of the gold-silver alloy hollow nanoparticles is modified with functionalized polyethylene glycol HS-PEG-NH2; The photosensitizer is cross-linked with the surface of gold and silver alloy hollow nanoparticles via functionalized polyethylene glycol. The preparation method of the photothermal-photodynamic antibacterial probe includes the following steps: S1: Silver nanospheres were synthesized by reducing silver chloride with ascorbic acid under alkaline conditions as a substrate, and gold-silver alloy hollow nanoparticles were synthesized by substitution with chloroauric acid. S2: Tris(2-carboxyethyl)phosphine was added to the gold-silver alloy hollow nanoparticles in S1, followed by the addition of mercapto-polyethylene glycol-amine groups, and the mixture was stirred evenly overnight. S3: Ce6 and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were mixed evenly in dimethyl sulfoxide medium and reacted at 25°C for 30 minutes. Then, N-hydroxysuccinimide was added and the reaction was continued for 90 minutes. S4: Add the Ce6 with activated carboxyl group from S3 to the solution after S2, mix thoroughly and shake for 24 hours, centrifuge and resuspend to obtain the photothermal-photodynamic antibacterial probe.

2. The application according to claim 1, characterized in that: The antibacterial treatment involves inhibiting bacteria in vitro.

3. Use according to claim 2, characterized in that, The in vitro antibacterial process includes the following steps: co-incubating free bacteria with a photothermal-photodynamic antibacterial probe and then irradiating them with 808nm and 660nm lasers to produce an antibacterial effect.

4. Use according to claim 3, characterized in that: The bacteria are Staphylococcus aureus ATCC25923 and Escherichia coli ATCC25922.

Images