Method for improving active oxygen generation capability of photosensitizer

Abstract

The invention provides a method for improving the active oxygen generation capacity of a photosensitizer. The problem that the active oxygen generation capacity of an existing photosensitizer is limited is solved. According to the invention, starting from a light source, a light path is changed, and a dual-wavelength excitation method is adopted to improve the active oxygen generation capability of the photosensitizer, so that the current situation that the generation of active oxygen of the photosensitizer is almost completely based on the absorption of the light source by the ground state is broken, and two exciting lights with different wavelengths are superposed together to act on the photosensitizer together; therefore, the ground state and the excited state of the photosensitizer are absorbed and superposed again, more active oxygen is generated, the utilization rate of the photosensitizer is effectively improved, the photodynamic antibacterial efficiency can be greatly improved, and practical application is further facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of photosensitizers, and particularly relates to a method for enhancing the ability of photosensitizers to generate reactive oxygen species. Background Art

[0002] Compared with traditional methods, photodynamic therapy has the advantages of high spatiotemporal selectivity, non-invasiveness, small long-term adverse reactions, and low drug resistance. Photodynamic therapy requires three basic elements: a photosensitizer, an excitation light, and molecular oxygen. Under light illumination, the photosensitizer molecules absorb photons of a specific wavelength, transition from the ground state to the excited state, and further interact with substrates such as molecular oxygen and water to undergo energy or electron transfer reactions to generate reactive oxygen species, which then play a killing role in photodynamic therapy. The process of photodynamic therapy highly depends on the reactive oxygen species production of the photosensitizer. Current research hotspots mainly focus on the design and synthesis of new photosensitizers with high reactive oxygen species production. This kind of design and development based on photosensitizers often requires a large amount of time and cost, and it takes a long verification cycle from design to actual clinical application. As an important part of photodynamic therapy, enhancing the reactive oxygen species production of existing commercial or clinically approved photosensitizers from the perspective of the light source can greatly shorten the R & D cycle and accelerate the clinical application of photosensitizers. For the same photosensitizer, its reactive oxygen species production is related to the energy of the light it absorbs. As the light intensity increases, the generation of reactive oxygen species increases. However, when the energy of the light source is too large, it will exceed the safe tolerance range of the human body and cause hazards such as burns. Obviously, simply enhancing the light intensity has limited improvement in the reactive oxygen species production of photosensitizers, which limits the utilization of photosensitizers in practical applications.

[0003] Therefore, it is necessary to explore a new method to enhance the ability of photosensitizers to generate reactive oxygen species. Summary of the Invention

[0004] The present invention proposes a method for enhancing the ability of photosensitizers to generate reactive oxygen species, solves the problem of limited ability of current photosensitizers to generate reactive oxygen species, and further improves the utilization rate of photosensitizers.

[0005] Concept of the present invention:

[0006] Aiming at the problem of limited ability of current photosensitizers to generate reactive oxygen species, in order to further enhance the reactive oxygen species production of photosensitizers while shortening the R & D cycle and reducing costs, the present invention continues to conduct in-depth exploration based on the optical properties of existing clinically approved or commercial photosensitizers from the perspective of the light source.

[0007] The excitation light source is an important part of photodynamic therapy. Under the irradiation of the excitation light, the photosensitizer changes from the ground state to the excited state. The excited state interacts with substrates such as molecular oxygen and water through energy transfer or electron transfer to generate reactive oxygen species, thereby exerting photodynamic antibacterial or therapeutic effects. However, the research team of this invention found that the current research on the process of reactive oxygen species generation by photosensitizers mainly focuses on the absorption of light sources by their ground states, without considering the absorption properties of their excited states, which limits the generation of reactive oxygen species by photosensitizers.

[0008] Ultrafast transient absorption spectroscopy can provide information on the excited state dynamics behavior on the femtosecond time scale. Therefore, this invention uses ultrafast transient absorption spectroscopy technology to analyze the basic optical properties and excited state dynamics behavior of existing clinically approved or commercial photosensitizers based on them, and further clarifies the role of the excited state absorption of photosensitizers in the generation of reactive oxygen species. It is proposed to start from the design of the light source, and by superimposing the second excitation wavelength, utilize the superposition effect of ground state absorption and excited state reabsorption to maximize the utilization rate of the photosensitizer and amplify its reactive oxygen species production.

[0009] To achieve the above object, the solution provided by this invention is as follows:

[0010] A method for enhancing the ability of a photosensitizer to generate reactive oxygen species, which is characterized in that it includes the following steps:

[0011] 1) Analyze and select the ground state absorption wavelength of the photosensitizer according to the basic optical properties of the photosensitizer and the wavelength range of the light source used in daily life (such as: the light source used clinically); analyze and select the excited state reabsorption wavelength of the photosensitizer according to the excited state dynamics behavior of the photosensitizer;

[0012] The photosensitizer is a photosensitizer with excited state absorption properties; those skilled in the art can screen out photosensitizers with excited state absorption properties by respectively characterizing the basic optical properties and excited state dynamics behavior of the photosensitizer;

[0013] 2) Select the first excitation light source for ground state absorption and the second excitation light source for excited state reabsorption according to the wavelength bands where the ground state absorption wavelength and excited state reabsorption wavelength selected in step 1) are located and the safety threshold of the application object (such as: the human safety threshold);

[0014] 3) Use the first excitation light source and the second excitation light source selected in step 2) to construct a dual-wavelength excitation system, and irradiate the photosensitizer with two beams of excitation light simultaneously to enhance the ability of the photosensitizer to generate reactive oxygen species.

[0015] Furthermore, the basic optical properties of the photosensitizer refer to the maximum absorption wavelength and the maximum emission wavelength of the photosensitizer, which can be characterized by ultraviolet-visible absorption spectroscopy and fluorescence emission spectroscopy;

[0016] The excited-state kinetic behavior of the photosensitizer refers to the excited-state absorption signal of the photosensitizer, which can be characterized by detecting the changes in the excited state through femtosecond transient absorption spectroscopy.

[0017] Further, in step 3), the dual-wavelength excitation system includes a first excitation light source, a second excitation light source, and an optical transmission unit; wherein, the optical transmission unit includes M1, M2, M3, M4, and M5 connected according to the designed optical path by a Y-shaped optical fiber;

[0018] The light beam emitted by the first excitation light source irradiates the photosensitizer through the optical path formed by M1, M2, and M3;

[0019] The light beam emitted by the second excitation light source is superimposed on the light beam emitted by the first excitation light source and irradiates the photosensitizer through the optical path formed by M4, M5, and M3;

[0020] Among them, M1, M2, M4, and M5 are all total reflection mirrors; M3 is a harmonic beam splitter.

[0021] Further, in step 2), the first excitation light source and the second excitation light source are laser or xenon lamp light sources.

[0022] Further, the photosensitizer is rose bengal, hematoporphyrin monomethyl ether, or zinc phthalocyanine.

[0023] In addition, the present invention also provides a dual-wavelength excitation system for enhancing the ability of a photosensitizer to generate reactive oxygen species, which is characterized in that it includes a first excitation light source for ground-state absorption, a second excitation light source for excited-state reabsorption, and an optical transmission unit;

[0024] Among them, the first excitation light source is selected according to the following principle:

[0025] First, analyze and select the ground-state absorption wavelength of the photosensitizer according to the basic optical properties of the photosensitizer and the wavelength range of the light sources commonly used in daily life, and then screen out the first excitation light source according to the wavelength band where the ground-state absorption wavelength is located and the safety threshold of the application object;

[0026] The second excitation light source is selected according to the following principle:

[0027] First, analyze and select the excited-state reabsorption wavelength of the photosensitizer according to the excited-state kinetic behavior of the photosensitizer, and then screen out the second excitation light source according to the wavelength band where the excited-state reabsorption wavelength is located and the safety threshold of the application object;

[0028] The optical transmission unit includes M1, M2, M3, M4, and M5;

[0029] The light beam emitted by the first excitation light source irradiates the photosensitizer through the optical path formed by M1, M2, and M3; the light beam emitted by the second excitation light source is superimposed on the light beam emitted by the first excitation light source through the optical path formed by M4, M5, and M3 and irradiates the photosensitizer.

[0030] Among them, M1, M2, M4, and M5 are all total reflection mirrors; M3 is a harmonic beam splitter.

[0031] According to the basic optical properties and excited state kinetic characteristics of commercially / clinically approved photosensitizers, the present invention screens the first excitation light source (ground state absorption) and the second excitation light source (excited state reabsorption), and through a dual-wavelength excitation method for enhancing the ability of photosensitizers to generate reactive oxygen species, maximizes the utilization of photosensitizers. In the process of photodynamic antibacterial (common examples include: food preservation or antibacterial on the surface of objects by spraying or coating materials containing photosensitizers and then irradiating with light) or photodynamic therapy, by using the above method, the two excitation lights are made to irradiate the photosensitizer simultaneously by changing the optical path, so that the ground state absorption and excited state reabsorption of the photosensitizer are superimposed, generating more reactive oxygen species, thereby improving the utilization rate of the photosensitizer.

[0032] The advantages of the present invention are as follows:

[0033] 1. Starting from the light source, the present invention changes the optical path and uses a dual-wavelength excitation method to enhance the ability of photosensitizers to generate reactive oxygen species, breaking the current situation that the generation of reactive oxygen species by photosensitizers is almost entirely based on the absorption of the light source in the ground state. The two excitation lights with different wavelengths are superimposed and act on the photosensitizer together, so that the ground state and excited state reabsorption of the photosensitizer are superimposed, generating more reactive oxygen species, effectively improving the utilization rate of the photosensitizer, greatly enhancing the photodynamic antibacterial efficiency, and being beneficial to practical applications.

[0034] 2. The present invention does not need to design and develop new photosensitizers through chemical synthesis or modify photosensitizers by means of nanoengineering, etc. Instead, based on the optical properties of existing clinically approved or commercial photosensitizers, it makes full use of their excited state characteristics, can largely shorten the R & D time, shorten the clinical conversion cycle, accelerate clinical application, reduce costs, and can select light sources with different wavelength ranges according to the photophysical and chemical properties of the selected photosensitizer, greatly improving the use effects of different photosensitizers, and has universality. Description of the Drawings

[0035] Figure 1 It is a schematic diagram of the optical path of a dual-wavelength excitation light source;

[0036] Figure 2 It is the ultraviolet absorption spectrum and fluorescence emission spectrum of RB;

[0037] Figure 3 It is the ultraviolet absorption spectrum and fluorescence emission spectrum of HMPE;

[0038] Figure 4 It is the femtosecond transient absorption spectrum diagram of RB;

[0039] Figure 5 It is the femtosecond transient absorption spectrum diagram of HMPE;

[0040] Figure 6 It is the comparison diagram of reactive oxygen species generated by RB after light irradiation by combining a single - wavelength xenon lamp with a 550nm band - pass filter, a 1064nm laser and a xenon light source loaded with a 550nm band - pass filter;

[0041] Figure 7 It is the comparison diagram of reactive oxygen species generated by HMPE after light irradiation by combining a single - wavelength xenon lamp with a 520nm band - pass filter, a 1064nm laser and a xenon light source loaded with a 520nm band - pass filter;

[0042] Figure 8 It is the image of colonies on the agar plate;

[0043] Figure 9 It is the survival rate diagram of MRSA treated with PBS, single - wavelength excited RB and dual - wavelength excited RB;

[0044] Figure 10 It is the detection of ROS generation in MRSA under dual - wavelength;

[0045] Figure 11 It is the fluorescence image of live / dead stained bacteria;

[0046] Figure 12 It is the SEM image of MRSA;

[0047] Figure 13 It is the comparison diagram of the cytotoxicity of photosensitizers with different concentrations;

[0048] Figure 14 It is the picture of the wound infected with MRSA;

[0049] Figure 15 It is the wound area during the wound healing process;

[0050] Figure 16 It is the H&E staining comparison diagram of the heart, liver, spleen, lung and kidney of mice in different groups. Detailed implementation mode

[0051] The following further elaborates on the content of the present invention in combination with the attached drawings and specific embodiments. Unless otherwise emphasized, the chemical reagents and solvents used in the embodiments are all commercially available analytical pure. The photosensitizers used for testing are commercially available rose bengal (RB) and hematoporphyrin monomethyl ether (HMPE).

[0052] The dual-wavelength excitation system can superimpose ground state absorption and excited state absorption. The optical path diagram is shown in the figure Figure 1 As shown, the first excitation light source, the second excitation light source, and the optical transmission unit composed of M1, M2, M3, M4, and M5 connected by a Y-shaped optical fiber, in the present invention, the first excitation light source and the second excitation light source can select laser or xenon light sources with different wavelength ranges according to the type of photosensitizer selected. The selection principle of the first excitation light source is: first analyze and select the ground state absorption wavelength of the photosensitizer according to the basic optical properties of the photosensitizer and the wavelength range of the light source used in daily life, and then select the first excitation light source according to the band where the ground state absorption wavelength is located and the application object using the safety threshold; the selection principle of the second excitation light source is: first analyze and select the excited state reabsorption wavelength of the photosensitizer according to the excited state kinetic behavior of the photosensitizer, and then select the second excitation light source according to the band where the excited state reabsorption wavelength is located and the application object using the safety threshold.

[0053] 1. Basic optical testing

[0054] The specific details of the experiment are as follows:

[0055] 1. UV absorption spectrum test experiment of RB and HMPE

[0056] Use deionized water as the solvent to prepare RB and HMPE solutions with a mass concentration of 1 μg / mL. After RB and HMPE are fully dissolved, use a pipette to draw 2 mL of the above solution and add it to a four-way quartz cuvette for later use.

[0057] A Hitachi UH5300 UV spectrophotometer was used to perform baseline calibration on a four-way quartz cuvette with deionized water added. After the above operation was completed, a cuvette containing the sample to be tested was placed and its absorption spectrum in the range of 300 nm to 1100 nm was measured.

[0058] 2. Fluorescence emission spectrum test experiment of RB and HMPE

[0059] Use deionized water as solvent to prepare RB and HMPE solutions with a mass concentration of 1 μg / mL. After RB and HMPE are fully dissolved, use a pipette to draw 2 mL of the above solution and add it to a four-way quartz cuvette for later use. The fluorescence spectrometer measures the fluorescence emission spectrum of the sample solution to be tested. The excitation wavelengths are 580nm and 540nm, the slit is 5×5mm, the excitation voltage is 650V, and the measurement ranges are 590 to 900nm and 550 to 800nm ​​respectively.

[0060] like Figure 2 , Figure 3As shown in the UV absorption spectra and fluorescence emission spectra of RB and HMPE, the maximum absorption wavelength of RB is 550 nm, and the maximum emission wavelength is 567 nm. The main absorption of HMPE is around 390 nm, and it has a relatively wide absorption in the wavelength range of 500 - 600 nm, with a maximum emission wavelength of 612 nm.

[0061] II. Femtosecond transient absorption spectroscopy experiment (fs - ta)

[0062] The fs - ta spectrum was obtained using an amplified Ti:sapphire laser system that produces a fundamental output of 800 nm, with a pulse width of 120 fs and a repetition rate of 1 kHz (Solstice Ace, Newport Spectra - Physics). The average power provided by this laser system is 7 W, which is split into two beams (7:3) for pumping and probing respectively. The pump beam ranges from 240 nm to 2600 nm and is generated using an optical parametric amplifier (TOPAS, Light Conversion).

[0063] The sample was pumped at 550 nm by a femtosecond Spectra - Physics Spitre regenerative amplifier (120 fs, 1000 Hz). The probe pulse was generated by focusing a small portion of the fundamental 800 - nm laser pulse on a thin CaF 2 plate, ranging from 400 nm to 1600 nm.

[0064] As Figure 4 、 Figure 5 shown, according to the spectra, there is a relatively wide excited - state absorption signal (ΔA > 0) in the second near - infrared region (1000 - 1700 nm) near 1064 nm, indicating that RB and HMPE have certain potential to improve the efficiency of reactive oxygen species generation through the mechanism of ground - state absorption superimposed with excited - state re - absorption.

[0065] III. Dual - wavelength excitation test of RB reactive oxygen species

[0066] First, according to the results of the absorption spectrum and femtosecond transient absorption spectrum, the excitation light sources were selected. For RB, the xenon light source equipped with a 550 - nm band - pass filter was used as the first excitation light source, and the 1064 - nm laser was used as the second excitation light source. For HMPE, the xenon light source equipped with a 520 - nm band - pass filter was used as the first excitation light source, and the 1064 - nm laser was used as the second excitation light source. Then, according to the dual - wavelength excitation method provided by the present invention, the generation of reactive oxygen species of RB and HMPE was tested, using SOSG (singlet oxygen sensor green probe, for detecting 1 O 2 ), APF (aminophenyl fluorescence probe, for detecting ·OH), DHE (dihydroethidium probe, for detecting O 2 -·) and DHR 123 (dihydrorhodamine probe, used for detecting O 2 - ·).

[0067] As Figure 6 shown, under dual-wavelength excitation, compared with single-wavelength excitation (550 nm), for RB, 1 O 2 production increased by 71%, ·OH decreased by 17%, and O 2 - · production increased by 10%. As Figure 7 shown, under dual-wavelength excitation, compared with single-wavelength excitation, for HMPE, ·OH production increased by 81%, and O 2 - · production decreased by 2%, 1 O 2 production decreased by 2%. Under dual-wavelength excitation, the generation of reactive oxygen species in RB and HMPE was generally enhanced. This enhancement means that the excited-state reabsorption effect introduced by the second excitation wavelength can effectively participate in the generation of reactive oxygen species, greatly improving the production efficiency of reactive oxygen species.

[0068] IV. In vitro antibacterial experiment

[0069] 1. Bacterial culture:

[0070] Culture MRSA (USA 300) in LB liquid medium at 4 °C and centrifuge at 5000 rpm for 3 minutes to collect bacteria. Measure the turbidity of the bacteria at 600 nm using an ultraviolet spectrophotometer, and adjust the bacterial solution concentration to 10 8 CFU mL -1 for subsequent experiments.

[0071] 2. Plate counting method:

[0072] Add the adjusted-concentration MRSA bacterial solution (10 8 CFU mL -1 , 100 μL) to 900 μL of PBS containing RB to make the final concentration 0.1 μg / mL, and then incubate at 37 °C for 4 h. After incubation, for the dual-wavelength excitation group, irradiate with 550 nm + 1064 nm (550 nm: 20 mW / cm 2 , 1064 nm: 0.8 W / cm 2 ) for 15 min. For the single-wavelength excitation group, irradiate with 550 nm (20 mW / cm 2 ) or 1064 nm (0.8 W / cm 2 ) for 15 min. After irradiation, continue to incubate for 3 h. After incubating for 3 h, dilute the MRSA bacterial solution with PBS buffer to 10 5 CFU mL-1 Subsequently, 5 μL of the bacterial suspension was taken and serially diluted 10-fold in a 96-well plate, spotted onto plates and counted. Another 50 μL of the bacterial solution was added to an LB solid culture dish and incubated for 24 hours. The number of colony-forming units (CFUs) on the RB-treated plates was divided by the number of CFUs in the PBS control to determine the survival rate after colony formation. As Figure 8 and Figure 9 shown, when excited with a single wavelength of 1064 nm, there was almost no bactericidal effect. When excited with a single wavelength of 550 nm, the antibacterial rate reached 83.54%. When excited with a dual wavelength of 550 nm + 1064 nm, the antibacterial rate was 99.99%.

[0073] 3. Reactive oxygen species (ROS) generation test:

[0074] At 37 °C, the fluorescent probe (DCFH-DA) was added to MRSA (10 8 CFU mL -1 ) and placed in an incubator for 30 min. RB was added to the bacterial solution, and the concentration of RB was adjusted to 0.1 μg / mL, and incubation was continued for 1 h. Then the suspension was irradiated at 550 nm + 1064 nm for 15 min. 10 μL of the pre-prepared mixed suspension was added to a confocal dish, and the cover glass was fixed. The fluorescence intensity of the DCFH solution at 500 to 540 nm was recorded at an excitation wavelength of 488 nm. As Figure 10 shown, clear green fluorescence could be observed in MRSA bacteria, indicating that RB has good ROS generation ability under dual-wavelength excitation.

[0075] 4. Bacterial live / dead staining assay:

[0076] The MRSA bacterial solution was treated by the plate counting method, the bacteria were collected by centrifugation, washed twice with 9% NaCl, resuspended with 9% NaCl (1 mL) containing the bacterial live / dead stain, and stained for 20 minutes. 10 μL of the bacterial solution was added to the bottom of a confocal dish, the cover glass was fixed, and the live / dead images of the bacteria were captured using a CLSM (Nikon C2+ & N-SIM E). As Figure 11 shown, RB only produced bright red fluorescence under dual-wavelength excitation, indicating its superior bactericidal effect.

[0077] 5. SEM imaging:

[0078] The MRSA bacterial solution was processed according to the plate counting method, centrifuged, and the bacteria were collected, and then fixed with paraformaldehyde overnight. Next, the bacteria were washed twice with PBS, and then dehydrated successively with 20%, 40%, 60%, 80%, 90% and 100% ethanol for 10 minutes for the first three concentrations and 30 minutes for the last three concentrations. Finally, the samples were treated with metal spraying and observed by scanning electron microscopy (ZEISS Gemini SEM 300). As Figure 12 shown, the bacterial morphology in the PBS group remained intact, while treatment with RB excited at 550 nm or dual wavelengths caused varying degrees of shrinkage and damage, and the damage in the dual-wavelength group was more obvious. Compared with single-wavelength excitation, dual-wavelength excitation of RB had a stronger antibacterial effect and could rapidly kill bacteria.

[0079] 6. Cell culture:

[0080] Mouse embryonic fibroblasts (NIH 3T3) were cultured in high-glucose DMEM conventional medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C and 5% CO 2 .

[0081] 7. Cell compatibility test:

[0082] NIH 3T3 cells were seeded in 96-well plates at a density of 6×10 3 cells / well and incubated at 37 °C and 5% CO 2 for 24 h. Then, the cells were incubated with fresh medium (100 μL) containing different concentrations of RB (RB concentrations were 0.01, 0.05, 0.1, 0.15, 0.2 μg / mL) for 14 h. Finally, the medium was removed, and the cells were incubated with CCK-8 solution (100 μL / well, V / V = 1:9) for 30 min, and the absorbance of the cells at 450 nm was measured using a multi-functional microplate detector (TECAN Spark). As Figure 13 shown, even at a concentration as high as 0.2 μg / mL, RB did not show cytotoxicity.

[0083] V. In vivo antibacterial experiment:

[0084] To verify the antibacterial effect of this method, mice were selected as experimental materials. The selected photosensitizer was dropped on the wounds of the mice, and the wounds of the mice were treated using single-wavelength and dual-wavelength excitation methods respectively. The wound healing process was continuously monitored by taking photos, measuring the area of wound infection, and the changes in the body weight of infected mice during the treatment period.

[0085] All animal experiment procedures were conducted in accordance with the Regulations on the Administration of Laboratory Animals in China and were approved by the Animal Ethics Committee of Northwestern Polytechnical University. All mice were randomly divided into 6 groups: PBS, PBS + 550 + 1064, RB, RB + 1064, RB + 550, RB + 550 + 1064. Twelve hours after bacterial infection, 10 μL of PBS and 10 μL of RB (0.1 mg / mL) were dropped into the wound. The dual-wavelength excitation group was irradiated with 550 nm + 1064 nm (550 nm: 20 mW / cm 2 , 1064 nm: 0.8 W / cm 2 ) on the wound surface for 10 min, and the single-wavelength excitation group was irradiated with 550 nm (20 mW / cm 2 ) or 1064 nm (0.8 W / cm 2 ) for 10 min. After that, the wound healing process was continuously monitored by taking pictures and measuring the area of wound infection and the body weight of infected mice during the entire treatment period (9 days). As Figure 14 shown, the RB + 550 + 1064 group started to heal on the 3rd day of treatment, and the infected wound surface was close to healing on the 9th day. As Figure 15 shown, after 9 days of treatment, the wound areas of the RB + 550 group, RB + 1064 group, and RB + 550 + 1064 group decreased to 33.84%, 49.76%, and 7.09% respectively, indicating that dual-wavelength excitation exhibited good photodynamic antibacterial effects. The wound healing of mice in the dual-wavelength excitation group was significantly better than that of other groups.

[0086] In vivo biosafety assessment: To evaluate the biosafety of RB, all infected mice were sacrificed at the end of the treatment (the 10th day), and blood was collected from healthy mice as a control for further analysis. Routine blood tests and liver function indicators (ALT, AST, ALP), kidney function indicators (UA, BUN, CREA) were analyzed, and the main organs (skin, liver, kidney, spleen, lung, heart) of infected mice in each group and the skin tissue of the wound were stained with hematoxylin and eosin (H&E), and then imaged and observed with an optical microscope (Thermo Fisher EVOS FL Auto 2). As Figure 16 shown, the wound surface of the RB + 550 + 1064 group healed well, and no obvious inflammatory cells were seen, further demonstrating that dual-wavelength excitation has good antibacterial effects.

[0087] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of various equivalent modifications or substitutions, and these modifications or substitutions should all be covered within the protection scope of the present invention.

Claims

1. A method for improving the active oxygen generation capacity of a photosensitizer, characterized in that: The following steps are involved: 1) Analyze and select the ground state absorption wavelength of the photosensitizer based on the basic optical properties of the photosensitizer and the wavelength range of the light source used in daily life; Analyze and select the excited state reabsorption wavelength of the photosensitizer based on the excited state kinetic behavior of the photosensitizer; The photosensitizer is a photosensitizer with excited state absorption properties; 2) screening the first excitation light source for ground state absorption and the second excitation light source for excited state reabsorption using a safety threshold according to the wavelength bands of the ground state absorption wavelength and the excited state reabsorption wavelength selected in step 1) and the application object; 3) constructing a dual-wavelength excitation system using the first excitation light source and the second excitation light source selected in step 2), so that the two excitation lights simultaneously irradiate the photosensitizer to enhance the photosensitizer's ability to generate active oxygen.

2. The method for improving the active oxygen generation capacity of a photosensitizer according to claim 1, characterized in that: In step 1), the basic optical properties of the photosensitizer refer to the maximum absorption wavelength and the maximum emission wavelength of the photosensitizer; The excited state kinetic behavior of the photosensitizer refers to the excited state absorption signal of the photosensitizer.

3. The method for improving the active oxygen generation capacity of a photosensitizer according to claim 1 or 2, characterized in that: In step 3), the dual-wavelength excitation system includes a first excitation light source, a second excitation light source and a light transmission unit; wherein the light transmission unit includes M1, M2, M3, M4 and M5; The light beam emitted by the first excitation light source irradiates the photosensitizer through the light path formed by M1, M2, and M3; The light beam emitted by the second excitation light source passes through the optical path formed by M4, M5, and M3 and is superimposed on the light beam emitted by the first excitation light source to irradiate the photosensitizer; Among them, M1, M2, M4 and M5 are all total reflection mirrors; M3 is a harmonic beam splitter.

4. The method for improving the active oxygen generation capacity of a photosensitizer according to claim 3, characterized in that: In step 2), the first excitation light source and the second excitation light source are both laser or xenon lamp light sources.

5. The method for improving the active oxygen generation capacity of a photosensitizer according to claim 1, characterized in that: The photosensitizer is Bengal rose red, hemoporfin or zinc phthalocyanine.

6. A dual-wavelength excitation system for improving the active oxygen generation capacity of a photosensitizer, characterized in that: It includes a first excitation light source for ground state absorption, a second excitation light source for excited state reabsorption, and a light transmission unit; The first excitation light source is selected according to the following principles: First, the ground state absorption wavelength of the photosensitizer is analyzed and selected according to the basic optical properties of the photosensitizer and the wavelength range of the light source used in daily life, and then the first excitation light source is screened out using a safety threshold according to the wavelength band of the ground state absorption wavelength and the application object; The second excitation light source is selected according to the following principles: First, the excited state reabsorption wavelength of the photosensitizer is selected according to the excited state kinetic behavior of the photosensitizer, and then the second excitation light source is selected according to the wavelength band of the excited state reabsorption wavelength and the application object using a safety threshold; The optical transmission unit includes M1, M2, M3, M4 and M5; The light beam emitted by the first excitation light source irradiates the photosensitizer through the light path formed by M1, M2, and M3; The light beam emitted by the second excitation light source passes through the optical path formed by M4, M5, and M3 and is superimposed on the light beam emitted by the first excitation light source to irradiate the photosensitizer; Among them, M1, M2, M4 and M5 are all total reflection mirrors; M3 is a harmonic beam splitter.

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