A near-infrared second-region responsive nitrogen-doped carbon dots N-CDs and its preparation method and application

Abstract

This invention belongs to the field of nanophotothermal materials. To address the problems of poor fluorescence response and short fluorescence emission wavelength in the near-infrared region II (NIR II) of current carbon dot materials, a method for preparing and applying NIR II nitrogen-doped carbon dots (N-CDs) is provided. Citric acid and biuret are used as substrates, and N,N-dimethylformamide (DMF) is added for a solvothermal reaction. A red product is generated, which is then dispersed in a sodium hydroxide solution. Ultrasonic treatment and centrifugation yield a black precipitate, which is then washed sequentially with hydrochloric acid and deionized water to obtain black NIR II nitrogen-doped carbon dots (N-CDs). The fluorescence emission of the N-CDs exhibits a significant red shift, enhancing their photoconversion capability in the near-infrared region II (1060 nm). This technology offers a simple preparation process, low cost, and short reaction time. It possesses advantages such as a wide photoresponse range and high quantum efficiency, and is suitable for photothermal diagnosis and treatment, particularly for tumor cells, bioimaging, and catalytic antibacterial applications.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanophotothermal materials, and specifically relates to nitrogen-doped carbon dots (N-CDs) that respond in the second near-infrared region, as well as their preparation method and applications, especially in photothermal diagnosis and treatment, bioimaging, and catalytic antibacterial applications. Background Art

[0002] The morbidity and mortality rates of malignant tumors continue to rise, and cancer has become a serious threat to human health and life. In recent years, technologies such as photothermal therapy and fluorescence imaging have gained increasing attention due to their non-contact and non-ionizing properties, and are expected to find applications in cancer diagnosis and treatment.

[0003] Photothermal therapy (PTT) utilizes nanomaterials with photothermal conversion capabilities to convert light energy into heat under external illumination. This results in a localized increase in body temperature, destroying bioactive molecules within cells (such as protein denaturation and enzyme inactivation), and thereby killing cancer cells. In vivo bioluminescence imaging can directly and rapidly measure tumor growth, metastasis, and drug response in various cancer models. Its exceptional sensitivity allows detection of even tiny tumor lesions (as few as a few hundred cells), significantly improving sensitivity compared to traditional methods and making it ideally suited for quantitative analysis of tumor growth in vivo.

[0004] Near-infrared (NIR) light has been widely used as an advanced diagnostic and therapeutic tool in the medical field due to its non-invasive controllable properties, high resolution, and deep tissue penetration. It holds significant significance in the comprehensive diagnosis and treatment of NIR biological windows (NIR-I, 650-950 nm; NIR-II, 1000-1700 nm). In particular, NIR-II light, due to its excellent penetration, holds great promise for the treatment of deep-seated tumors.

[0005] Traditional photosensitive materials suffer from shortcomings such as short fluorescence emission wavelengths and low photothermal conversion efficiency. To redshift the fluorescence emission wavelength and improve photothermal conversion efficiency, researchers are exploring novel photothermal conversion materials using various approaches. Carbon dots (CDs), a novel carbon-based fluorescent nanomaterial with a size less than 10 nm, offer advantages such as environmental friendliness, excellent biocompatibility, tunable photoluminescence, high quantum yield, and unique physicochemical properties. However, most CD materials exhibit poor response in the near-infrared region II and exhibit fluorescence emission wavelengths below the near-infrared region, significantly limiting their application in the near-infrared region II and in tumor diagnosis and treatment. Summary of the Invention

[0006] To address the issues of poor response and short fluorescence emission wavelengths in the near-infrared region II (NIR II) of existing carbon dot materials, this invention provides nitrogen-doped carbon dots (N-CDs) that respond in the NIR II region, as well as their preparation and application. Citric acid is used as the carbon source, and nitrogen is doped to effectively improve the carbon dots' light conversion efficiency, enhance their NIR II response, and red-shift their fluorescence emission wavelength into the NIR II band.

[0007] The present invention is achieved by the following technical solution: a nitrogen-doped carbon dot N-CDs responsive to the second near-infrared region, wherein citric acid and biuret are used as substrates, N,N-dimethylformamide (DMF) is added to carry out a solvent thermal reaction, a red product is generated and then dispersed in a sodium hydroxide solution, ultrasonically treated and centrifuged to obtain a black precipitate, which is washed with hydrochloric acid and deionized water in sequence to obtain black nitrogen-doped carbon dot N-CDs responsive to the second near-infrared region.

[0008] The method for preparing the nitrogen-doped carbon dots N-CDs that respond to the near-infrared second region comprises the following specific steps:

[0009] (1) Preparation of N-CDs: 2 g of citric acid and 4.5 g of biuret were completely dissolved in 30-40 mL of DMF to form a clear colorless solution. The mixed solution was transferred to a reactor and heated to 180 °C at a rate of 10 °C / min for hydrothermal reaction for 4 h. The mixture was then cooled to room temperature. The red product obtained by the hydrothermal reaction was dispersed in 50 mg / mL of sodium hydroxide solution. The mixture was ultrasonically treated at 80 W for 5 min and centrifuged at 12,000 rpm for 10 min to obtain a black precipitate, which was N-CDs.

[0010] (2) Purification of N-CDs: The obtained black precipitate was washed with 1 mol / L hydrochloric acid and deionized water in sequence, and freeze-dried to obtain a black powder; the freeze-drying temperature was controlled at -40 °C and the drying time was 24-72 h.

[0011] Furthermore, the amount of DMF used was 40 mL.

[0012] Furthermore, the specific purification method in step (2) is as follows: the obtained black precipitate is dissolved with 1 mol / L hydrochloric acid, and the solution is centrifuged at 8000-10000 rpm for 10 min to collect the precipitate; then the precipitate is dissolved with deionized water, and the solution is centrifuged at 8000-10000 rpm for 10 min to collect the black precipitate, after the precipitate is dissolved with hydrochloric acid once, the precipitate is dissolved again with deionized water three times; finally, the sample is placed in a vacuum freeze dryer and freeze-dried for 24-72 h to obtain the purified N-CDs powder.

[0013] The present invention also provides the use of the nitrogen-doped carbon dots N-CDs responsive to the second near-infrared region or the nitrogen-doped carbon dots N-CDs responsive to the second near-infrared region obtained by the preparation method as a photosensitizer in antibacterial sterilization. The target bacteria of the antibacterial sterilization is Staphylococcus aureus; the concentration of the N-CDs aqueous solution is 0.2-1.0 mg / L, and the initial concentration of Staphylococcus aureus is 1×10 5 CFU / mL; 1060 nm and 808 nm light sources for 10 min.

[0014] The present invention also provides the use of the nitrogen-doped carbon dots N-CDs responsive to the second near-infrared region or the nitrogen-doped carbon dots N-CDs responsive to the second near-infrared region obtained by the preparation method as a photosensitizer in eliminating tumors. The tumor cells are human prostate cancer cells DU145, and the concentration of the N-CDs is 1.0 mg / mL; 1.0 W / cm 2 , 808 nm or 1060 nm laser irradiation for 10 min.

[0015] The present invention also provides the use of the nitrogen-doped carbon dots (N-CDs) responsive to the second near-infrared region, or the nitrogen-doped carbon dots (N-CDs) responsive to the second near-infrared region obtained using the preparation method, as a photosensitizer in fluorescence imaging. The N-CDs concentration is 0.5 mg / kg, and the fluorescence intensity is measured after 1 hour of treatment.

[0016] The nitrogen-doped carbon dots N-CDs responsive to the second near-infrared region prepared by the present invention are particularly suitable for use as photosensitizers for killing bacteria, eliminating tumors and fluorescence imaging in the field of photothermal diagnosis and treatment, and have a wide range of applications.

[0017] This invention incorporates nitrogen into carbon dots (CDs), successfully red-shifting the fluorescence emission wavelength of the CDs to the near-infrared range and enhancing their light conversion capability in the second near-infrared region (1060 nm). The fluorescent photothermal material N-CDs provided by this invention has a simple preparation process, low cost, and short preparation time. The fluorescent photothermal material N-CDs provided by this invention has advantages such as a wide photoresponse range and high quantum efficiency, and can be applied in photothermal diagnosis and treatment, particularly in photothermal therapy, antibacterial treatment, and fluorescence imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Transmission electron microscopy images of nitrogen-doped fluorescent photothermal materials N-CDs; Figure: A is the TEM image of N-CDs; B is the particle size distribution of N-CDs;

[0019] Figure 2The photothermal capacity curve of nitrogen-doped fluorescent photothermal material N-CDs under near-infrared light (1060 nm / 808 nm) is shown in Figure 1. A is the temperature rise curve of 1.0 mg / mL N-CDs solution under 808 nm laser irradiation with different powers over time; B is 1.0 W / cm 2 The temperature rise curves of N-CDs solutions with different concentrations under 808 nm laser irradiation over time; C is the temperature rise curves of 1.0 mg / mL N-CDs solution under 1060 nm laser irradiation with different powers over time; D is 1.0 W / cm 2 Temperature rise curves of N-CDs solutions with different concentrations under 1060nm laser irradiation over time;

[0020] Figure 3 Figure 2 shows the ability of nitrogen-doped fluorescent photothermal materials N-CDs to kill Staphylococcus aureus under near-infrared light (1060 nm / 808 nm). In the figure: A is the bactericidal ability of 1.0 mg / mL N-CDs solution under 808 nm or 1060 nm laser irradiation at different powers; B is 1.0 W / cm 2 The bactericidal ability of N-CDs solutions with different concentrations under 808nm or 1060nm laser irradiation;

[0021] Figure 4 This is a cytotoxicity test diagram of nitrogen-doped fluorescent photothermal material N-CDs under near-infrared light (1060 nm / 808 nm);

[0022] Figure 5 Live and dead cell staining of nitrogen-doped fluorescent photothermal material N-CDs under near-infrared light (1060 nm / 808 nm);

[0023] Figure 6 In vivo fluorescence imaging of nitrogen-doped fluorescent photothermal materials N-CDs;

[0024] Figure 7 This is a graph showing the in vivo anti-tumor ability of nitrogen-doped fluorescent photothermal materials N-CDs under near-infrared light (1060 nm / 808 nm) irradiation. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0027] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0028] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0029] Example 1: A method for preparing near-infrared-responsive nitrogen-doped carbon dots (N-CDs) comprises using citric acid and biuret as raw materials, adding DMF to conduct a solvothermal reaction, dispersing the resulting red product in a sodium hydroxide solution, and then sonicating and centrifuging to obtain a black precipitate. The precipitate is then washed sequentially with hydrochloric acid and deionized water to obtain black nitrogen-doped carbon dots (N-CDs). The specific steps are as follows:

[0030] (1) Preparation of N-CDs: Weigh 2 g of citric acid and 4.5 g of biuret and dissolve them in 40 mL of DMF. After they are fully dissolved, a clear colorless solution is formed. The solution is transferred to a 100 mL reactor and the temperature of the hydrothermal oven is increased at a rate of 10 °C / min. The solvent thermal reaction is carried out at 180 °C for 4 h. The reaction is cooled to room temperature. The black precipitate generated by the hydrothermal reaction is collected and dissolved in 50 mg / mL sodium hydroxide solution. The mixture is ultrasonicated for 5 min and centrifuged at 8000-12000 rpm for 10 min to obtain the black precipitate.

[0031] (2) The obtained black precipitate was dissolved with 1 mol / L hydrochloric acid, and the solution was centrifuged at 8000-10000 rpm for 10 min to collect the black precipitate; then, the black precipitate was dissolved with deionized water, and the solution was centrifuged at 8000-10000 rpm for 10 min to collect the black precipitate, and the process was repeated three times; the sample was placed in a vacuum freeze dryer at -40 °C and freeze-dried for 24-72 h to obtain purified N-CDs powder.

[0032] The N-CDs prepared in Example 1 were examined by electron microscopy. Figure 1 As shown, it can be observed that the N-CDs sample is uniformly dispersed and has a uniform particle size distribution.

[0033] Experimental Example 1: Photothermal Capacity Test of N-CDs: The photothermal conversion capacity of N-CDs was evaluated under 1060 nm and 808 nm laser irradiation. The specific steps were as follows: the light sources were 1060 nm and 808 nm lasers, respectively, with a laser diameter of 10 mm and a power of 1.0 W / cm 2 The N-CDs samples were prepared into aqueous solutions with concentrations of 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1.0 mg / mL, and 200 μL was taken into a 96-well plate. The temperature of the samples was recorded at 2 min, 4 min, 6 min, 8 min, and 10 min. The experimental results are shown in Figure 2. Figure 2 As shown in the figure, the results show that after irradiation at 1060 nm and 808 nm for 10 min, the sample temperature can reach a maximum of 59 ℃ and 71 ℃, respectively.

[0034] Experimental Example 2: N-CDs sterilization activity test: Staphylococcus aureus was selected as the sterilization target, and 1060 nm and 808 nm lasers were selected as light sources, with a light intensity of 1.0 W / cm 2 N-CDs solutions (0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, 1.0 mg / L) were mixed with the initial concentration of about 1×10 5 After mixing CFU / mL of Staphylococcus aureus evenly, place it in a sterile reactor, turn on the light source for 10 minutes, dilute it with sterile PBS solution and evenly spread it on the agar medium, place it in an incubator at 37℃ for 24 hours, and count the number of colonies to determine the number of viable bacteria. The results of the light sterilization experiment are as follows Figure 4 As shown in the figure, the sterilization rates of Staphylococcus aureus can reach up to 95% and 90% after irradiation at 1060 nm and 808 nm for 10 min, respectively.

[0035] Experimental Example 3: Cytotoxicity test of N-CDs: DU145 cells were selected as the experimental subjects of this experiment, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) was used as a stain to evaluate the cytotoxicity of N-CDs. 1060 nm and 808 nm lasers were selected as light sources, respectively, with a light intensity of 1.0 W / cm 2 PBS and different concentrations of N-CDs (0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL) were added to DU145 cells (1×10 4The cells were then gently rinsed with PBS to remove any unabsorbed sample and incubated for a further 24 h.

[0036] In the phototoxicity experiment group, the light source was turned on for 10 min. After laser irradiation, the cells were further incubated for 24 h and each well was stained with MTT stain. After 4 h, the supernatant of each well was aspirated, DMSO was added, and cell survival was detected at a wavelength of 492 nm.

[0037] The results of cytotoxicity experiments were as follows Figure 5 As shown in the figure, the results showed that under dark conditions, after 10 min of illumination, DU145 cells still maintained a high cell viability of more than 80% after incubation with N-CDs for 24 h, indicating that the toxicity of N-CDs to cells was negligible and had good biocompatibility.

[0038] Illumination without N-CDs did not significantly damage DU145 cells. In contrast, N-CDs samples at varying concentrations exhibited a potent killing effect on DU145 tumor cells under 1060 nm and 808 nm near-infrared light. This was attributed to the photothermal effect and efficient reactive oxygen species (ROS) generation capacity of N-CDs, which were positively correlated with sample concentration. This resulted in significantly increased cell damage with increasing N-CDs concentration co-incubated with DU145 cells. When the sample concentration was increased to 1 mg / mL, the cell viability of DU145 cells co-incubated with N-CDs decreased to approximately 31% and 41% under 1060 nm and 808 nm NIR light, respectively. These results demonstrate that N-CDs exhibit a significant killing effect on tumor cells under near-infrared light, confirming their excellent photothermal conversion efficiency and suggesting that N-CDs have great potential for photothermal therapy of tumors.

[0039] Experimental Example 4: N-CDs live-dead cell staining experiment: DU145 cells were selected as the experimental subjects of this experiment, and 1060 nm and 808 nm lasers were selected as light sources, respectively, with a light intensity of 1.0 W / cm 2 DU145 cells were seeded into 24-well plates (5 × 10 4 The cells were cultured for 24 hours at 4 °C (100 μg / well). The culture medium was then replaced with fresh medium containing N-CDs solution (1 mg / mL) and incubated for 4 hours to allow for cellular uptake. The cells were then washed with PBS and incubated with fresh medium. The cells were illuminated for 10 minutes. A control group was cultured in the dark for 10 minutes. Calcein-AM / PI staining was then performed for 30 minutes before imaging with confocal laser scanning.

[0040] Live-dead cell staining results Figure 6 As shown in the results, cells incubated with PBS in the control group exhibited a strong green fluorescence signal in the absence of light, indicating good cell survival. Furthermore, cells in the PBS group also displayed strong green fluorescence when irradiated with near-infrared light at 1060 nm and 808 nm, indicating that illumination alone does not induce any actual damage to tumor cells. Meanwhile, DU145 cells incubated with N-CDs in the absence of light exhibited bright green fluorescence, indicating good cell health under these conditions and negligible cytotoxicity from N-CDs, further demonstrating the excellent biocompatibility of N-CDs.

[0041] In contrast, when N-CDs were combined with near-infrared light, they effectively induced a significant degree of cell death, with the vast majority of cells in the field of view being dyed red, again demonstrating the enormous potential of N-CDs for photothermal therapy of tumors.

[0042] Experimental Example 5: In Vivo Fluorescence Imaging of N-CDs: The feasibility of in vivo fluorescence imaging of N-CDs in tumor-bearing mice was investigated using the IVIS Spectral Imaging System (IVIS Lumina III; Perki Elmer, Waltham, MA, USA). DU145 tumor-bearing mice were selected as experimental subjects. PBS and N-CDs (0.5 mg / kg) were intratumorally injected into the tumor-bearing mice. In vivo fluorescence imaging was performed using the IVIS Lumina III Mouse Imaging System 1, 2, 4, 8, and 24 hours after injection. After 24 hours, the mice were euthanized, and the fluorescence intensity of major organs and tumors was recorded.

[0043] In vivo fluorescence imaging results Figure 7 As shown in the figure, after sample injection, a strong fluorescence signal from N-CDs was observed in the tumor area. The fluorescence intensity increased with injection time and reached its peak 1 hour after injection, indicating that cellular uptake of N-CDs in the tumor area reached its peak. Thereafter, the intensity steadily decreased over time. Eight hours after injection, the fluorescence intensity in the tumor site significantly weakened, and after 24 hours, the fluorescence in the tumor site required a high background intensity to be observed.

[0044] Experimental Example 6: Application of N-CDs photothermal diagnosis and treatment: DU145 (human prostate cancer cell) tumor-bearing mice were divided into 6 groups (three mice in each group) for in vivo anti-tumor experiments. The treatments were (1) PBS, (2) N-CDs, (3) 1060 nm light irradiation, (4) 808 nm light irradiation, (5) N-CDs + 1060 nm, and (6) N-CDs + 808 nm. After intratumoral injection of PBS or N-CDs (1.0 mg / mL, 200 μL), the mice were treated with 808 nm or 1060 nm (1.0 W / cm 2 ) laser irradiation for 10 minutes, and the temperature of the tumor site was monitored using a photothermal imager. The weight of the mice was recorded every two days, and the tumor volume of each group of mice was measured and calculated as follows: V = (width 2 × length) / 2. After 14 days of treatment, the mice were sacrificed and the tumors and major organs (heart, liver, spleen, lung, and kidney) were removed for H&E staining. Figure 7 As shown, the results showed that the tumor was completely ablated under 808 nm light irradiation, and the tumor volume was greatly reduced under 1060 nm irradiation.

[0045] Example 2: A method for preparing near-infrared responsive nitrogen-doped carbon dots N-CDs, wherein the amount of DMF used is 30 mL, and the rest of the method is the same as that described in Example 1.

[0046] Example 3: A method for preparing near-infrared responsive nitrogen-doped carbon dots N-CDs, wherein the amount of DMF used is 35 mL, and the rest of the method is the same as that described in Example 1.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nitrogen-doped carbon dot (N-CD) with near-infrared (NIR) response in the second region, characterized by: Citric acid and biuret were used as substrates, and N,N-dimethylformamide (DMF) was added for a solvothermal reaction. A red product was generated and then dispersed in a sodium hydroxide solution. After ultrasonic treatment and centrifugation, a black precipitate was obtained. The black precipitate was washed with hydrochloric acid and deionized water in sequence to obtain black nitrogen-doped carbon dots N-CDs that responded in the second near-infrared region.

2. A method for preparing the nitrogen-doped carbon dots (N-CDs) with near-infrared second-region response according to claim 1, characterized in that: The specific steps are as follows: (1) Preparation of N-CDs: 2 g of citric acid and 4.5 g of biuret were completely dissolved in 30-40 mL of DMF to form a clear colorless solution. The mixed solution was transferred to a reactor and heated to 180 °C at a rate of 10 °C / min for a hydrothermal reaction for 4 h. The mixture was then cooled to room temperature. The red product obtained by the hydrothermal reaction was dispersed in a 50 mg / mL sodium hydroxide solution. The mixture was ultrasonically treated at 80 W for 5 min and centrifuged at 8000-12000 rpm for 10 min to obtain a black precipitate, which was N-CDs. (2) Purification of N-CDs: The obtained black precipitate was washed with 1 mol / L hydrochloric acid and deionized water in sequence, and freeze-dried to obtain a black powder; the freeze-drying temperature was controlled at -40 °C and the drying time was 24-72 h.

3. The preparation method according to claim 2, wherein: The amount of DMF used was 40 mL.

4. The preparation method according to claim 2, wherein: The specific purification method in step (2) is as follows: the obtained black precipitate is dissolved with 1 mol / L hydrochloric acid, and the solution is centrifuged at 8000-10000 rpm for 10 min to collect the precipitate; then the precipitate is dissolved with deionized water, and the solution is centrifuged at 8000-10000 rpm for 10 min to collect the black precipitate, after the precipitate is dissolved with hydrochloric acid once, the precipitate is dissolved again with deionized water three times; finally, the sample is placed in a vacuum freeze dryer and freeze-dried for 24-72 h to obtain the purified N-CDs powder.

5. Use of the nitrogen-doped carbon dots N-CDs responsive to the second near-infrared region according to claim 1 or the nitrogen-doped carbon dots N-CDs responsive to the second near-infrared region obtained by the preparation method according to any one of claims 2 to 4 as a photosensitizer in the preparation of antibacterial and bactericidal drugs.

6. The use according to claim 5, characterized in that: The target bacteria of the antibacterial sterilization is Staphylococcus aureus; the concentration of the N-CDs aqueous solution is 0.2-1.0 mg / L, and the initial concentration of Staphylococcus aureus is 1×10 5 CFU / mL; 1060 nm and 808 nm light sources for 10 min.

7. Use of the nitrogen-doped carbon dots (N-CDs) responsive to the second near-infrared region according to claim 1 or the nitrogen-doped carbon dots (N-CDs) responsive to the second near-infrared region obtained by the preparation method according to any one of claims 2 to 4 as a photosensitizer in the preparation of a drug for eliminating human prostate cancer cells DU145.

8. The use according to claim 7, characterized in that: The concentration of the N-CDs was 1.0 mg / mL; 1.0 W / cm 2 , 808 nm or 1060 nm laser irradiation for 10 min.

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