A nanocomposite photosensitizer for transepithelial ion introduction corneal collagen cross-linking and its preparation method and application
By co-doping nitrogen and sulfur with graphene quantum dots to modify riboflavin to form a nanocomposite photosensitizer, the problems of insufficient surface charge distribution and insufficient ROS in the matrix in the prior art were solved, and the efficacy of corneal cross-linking was significantly improved.
Patent Information
- Application Number
- CN202411865833.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-18
- Publication Date
- 2025-05-06
- Estimated Expiration
- 2044-12-18
AI Technical Summary
In the current transepithelial ion-introduction corneal cross-linking, the surface charge distribution of riboflavin and insufficient reactive oxygen free radicals in the matrix, resulting in poor efficacy.
Riboflavin was modified by nitrogen and sulfur co-doped graphene quantum dots, and nanocomposite photosensitizer was synthesized through esterification reaction to improve the transepithelial absorption rate of riboflavin and the ROS generation ability in the matrix.
The electric field response and permeability of riboflavin are improved, the generation efficiency of reactive oxygen free radicals in the matrix is enhanced, and the matrix riboflavin content similar to that of the gold standard group and slightly better biomechanical properties than the gold standard group are achieved.
Smart Images
Figure CN119326884B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biomedical materials, and in particular to a nano-composite photosensitizer used for trans-epithelial ion introduction in corneal collagen cross-linking surgery, and a preparation method and application thereof. Background Art
[0002] Keratoconus (KC) is a common, bilateral, asymmetric, progressive, multifactorial, and non-inflammatory corneal ectasia that can lead to visual impairment and even legal blindness. Conventional corneal collagen cross-linking (C-CXL) is considered the "gold standard" treatment for progressive keratoconus. The main steps include removing the corneal epithelium, dripping riboflavin 5′-phosphate sodium (RF) solution, and then using 370 nm ultraviolet light to induce cross-linking. However, epithelial removal can lead to some side effects, including impaired epithelial healing, infectious keratitis, corneal scarring, and low vision.
[0003] Iontophoresis-mediated transepithelial cross-linking with iontophoresis (I-CXL) is a new non-invasive technique that preserves the corneal epithelium. It applies a small electric current to cause negatively charged riboflavin sodium phosphate to penetrate the epithelial cell barrier and enter the stroma in a short time. Clinical studies have shown that I-CXL can slow the progression of KC in most patients and effectively improve topography and visual parameters. However, animal experiments have shown that the parameters such as riboflavin concentration in the stroma, the depth of the stroma demarcation line, biomechanical properties, and resistance to corneal collagenase digestion achieved by I-CXL are not as good as those of C-CXL. In addition, long-term clinical trials have shown that the progression rate of KC after I-CXL is higher than that of C-CXL. It is speculated that the above results are mainly due to the small distribution of riboflavin surface charge, which has limited response to ion introduction current, thereby affecting the transepithelial absorption rate of riboflavin. In addition, the lack of reactive oxygen species (ROS) in the corneal stroma also limits the efficacy of I-CXL.
[0004] Graphene quantum dots (GQDs) are zero-dimensional graphene nanostructured materials with lateral dimensions less than 10 nanometers. They exhibit excellent physical, chemical and biological properties derived from graphene structures and quantum dot edge effects. GQDs have high stability and good biosafety; have a wide absorption band including 370 nm used in corneal cross-linking; have a large specific surface area, strong π-π stacking interaction, and the ability to be functionally modified; have unique electron transfer properties; and have the ability to produce a family of reactive oxygen free radicals such as singlet oxygen, superoxide anions and hydroxyl radicals under photoexcitation. In short, all of the above characteristics make GQDs widely used in optoelectronics, catalysis, sensors, biomedicine and other fields. However, their application in corneal cross-linking has not been reported in the literature. Summary of the invention
[0005] In order to solve the technical defects of too little surface charge distribution of riboflavin and insufficient reactive oxygen free radicals in the matrix in transepithelial iontophoresis corneal cross-linking in the prior art, the present invention provides a nanocomposite photosensitizer for transepithelial iontophoresis corneal collagen cross-linking and a preparation method and application thereof. For the first time, nitrogen-sulfur co-doped graphene quantum dots are used to modify riboflavin to prepare a nanocomposite photosensitizer, which is applied to the I-CXL procedure, thereby providing new insights into the treatment of keratoconus.
[0006] The technical solution adopted by the present invention is: a nano-composite photosensitizer for trans-epithelial ion introduction corneal collagen cross-linking surgery, wherein the nano-composite photosensitizer is nitrogen-sulfur co-doped graphene quantum dot-modified riboflavin synthesized by esterification reaction of nitrogen-sulfur co-doped graphene quantum dots and riboflavin.
[0007] The particle size distribution of the nitrogen-sulfur co-doped graphene quantum dots modified riboflavin is 8.0±1.0 nm; the nitrogen-sulfur co-doped graphene quantum dots modified riboflavin has a wide excitation band of 332-585 nm; the Zeta potential of the nanocomposite photosensitizer solution used for transepithelial ion introduction corneal collagen cross-linking surgery is -20.1 mV.
[0008] The encapsulation rate of riboflavin in the nanocomposite photosensitizer is about 76.7%, and the drug loading rate is about 79.1%.
[0009] The nanocomposite photosensitizer has an electron transfer phenomenon; the nanocomposite photosensitizer of nitrogen-sulfur co-doped graphene quantum dots modified with riboflavin has excellent ability to generate active oxygen free radicals by light excitation; and the nanocomposite photosensitizer of nitrogen-sulfur co-doped graphene quantum dots modified with riboflavin has excellent biological safety.
[0010] A method for preparing a nanocomposite photosensitizer for transepithelial ion introduction in corneal collagen cross-linking, the preparation method comprising the following steps:
[0011] S1. At room temperature, weigh riboflavin sodium phosphate, dehydrating agent 1-ethyl-(3-dimethylaminopropyl)
[0012] Dissolve the imine hydrochloride and catalyst 4-dimethylaminopyridine in acidic PBS, stir until dissolved, and set aside;
[0013] S2, adding nitrogen-sulfur co-doped graphene quantum dots in a concentration range of 3 to 15 mg / mL to the solution obtained in step S1, and stirring to mix evenly;
[0014] S3, transfer the mixed solution to a brown bottle and stir magnetically at 500 rpm at room temperature for 48 to 72 h;
[0015] S4. The resulting solution after the reaction is dialyzed and purified in double distilled water to obtain a nitrogen-sulfur co-doped graphene quantum dot-modified riboflavin nanocomposite photosensitizer (NS-GQDs-RF) solution.
[0016] Preferably, the acidic PBS is a 1 M phosphate buffer solution with a pH of 4 to 6.
[0017] Preferably, in step S1, the mass ratio of riboflavin sodium phosphate, dehydrating agent 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and catalyst 4-dimethylaminopyridine is 8:5.6:2.8 to 8:5.6:1.
[0018] Preferably, in step S2, the volume ratio of nitrogen-sulfur co-doped graphene quantum dots to acidic PBS as a solvent is 1:10 to 2:10.
[0019] Preferably, the dialysis in step S4 uses a cellulose dialysis bag with a molecular weight cutoff of 1000 Da, and the dialysis time is 8 to 12 h.
[0020] Preferably, the nitrogen-sulfur co-doped graphene quantum dots in step S2 are prepared by the following steps:
[0021] Anhydrous citric acid and reduced glutathione were weighed and added into double distilled water in a mass ratio of 5:1.5-5:1. After ultrasonic mixing and dissolution, the mixture was transferred into a reactor and reacted at 180-240°C for 6-16 h. The nitrogen-sulfur co-doped graphene quantum dots (NS-GQDs) solution was obtained after ultrafiltration and dialysis purification.
[0022] Preferably, the ultrafiltration uses a 50 nm filter membrane; the dialysis uses a cellulose dialysis bag with a molecular weight cutoff of 500 to 1000 Da, and the dialysis time is 12 to 48 h.
[0023] The particle size distribution of the nitrogen-sulfur co-doped graphene quantum dots is 2.7±0.7 nm; the ultraviolet absorption peak of the nitrogen-sulfur co-doped graphene quantum dots is at 335 nm; and the Zeta potential of the nitrogen-sulfur co-doped graphene quantum dot solution is -11.5 mV.
[0024] Application of a nanocomposite photosensitizer in the preparation of an iontophoresis-mediated transepithelial corneal cross-linking agent for treating keratoconus.
[0025] The beneficial effects of the present invention are as follows: (1) The present invention provides a nanocomposite photosensitizer for transepithelial ion introduction corneal collagen cross-linking, wherein the prepared nitrogen-sulfur co-doped graphene quantum dots have the characteristics of small particle size, negative potential, high electron transfer efficiency, high light excitation ROS generation efficiency, etc., and are excellent nanocarriers of riboflavin sodium phosphate. The nanocomposite photosensitizer prepared by modifying riboflavin with nitrogen-sulfur co-doped graphene quantum dots has high electric field responsiveness, high corneal riboflavin penetration ability, high ROS generation efficiency and good biocompatibility.
[0026] (2) The present invention also provides a method for preparing a nanocomposite photosensitizer for transepithelial ion introduction into corneal collagen cross-linking, that is, firstly synthesizing nitrogen-sulfur co-doped graphene quantum dots by a simple hydrothermal method, and then synthesizing a nanocomposite photosensitizer of nitrogen-sulfur co-doped graphene quantum dots modified with riboflavin by a one-step esterification reaction. The method is simple to operate, environmentally friendly and safe, with low cost, and is conducive to industrialization and entering the clinical trial stage.
[0027] (3) The present invention also pioneered the use of a nanocomposite photosensitizer of nitrogen-sulfur co-doped graphene quantum dots modified with riboflavin for transepithelial ion introduction corneal collagen cross-linking. The experimental results showed that the use of this composite photosensitizer can achieve a matrix riboflavin content similar to that of the gold standard group for epithelial removal; achieve slightly better biomechanical properties than the gold standard group; and achieve the superiority of epithelial preservation and shorter penetration time compared to the gold standard procedure. This research result will surely provide new insights into the treatment of keratoconus. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 Schematic diagram of the synthesis process of the nitrogen-sulfur co-doped graphene quantum dots modified riboflavin nanocomposite photosensitizer (NS-GQDs-RF) prepared by the present invention.
[0029] Figure 2 Schematic diagram of the process and mechanism of applying the nitrogen-sulfur co-doped graphene quantum dots modified riboflavin nanocomposite photosensitizer (NS-GQDs-RF) prepared by the present invention to transepithelial ion introduction corneal collagen cross-linking.
[0030] Figure 3Are transmission electron microscopy characterization images, where A is a transmission electron microscopy representative image of NS-GQDs, and B is a transmission electron microscopy representative image of NS-GQDs-RF.
[0031] Figure 4 A is the UV-visible absorption spectra of riboflavin sodium phosphate (RF), NS-GQDs and NS-GQDs-RF, and B is the excitation spectra of riboflavin sodium phosphate (RF), NS-GQDs and NS-GQDs-RF at 525 nm.
[0032] Figure 5 A is the Zeta potential of riboflavin sodium phosphate (RF), NS-GQDs and NS-GQDs-RF in double distilled water (pH 7, n=3), and B is the difference in peak binding energy between NS-GQDs-RF and RF and NS-GQDs.
[0033] Figure 6 is the glutathione depletion rate of NS-GQDs-RF and RF under UV-A irradiation (n=6).
[0034] Figure 7 The results of evaluating the cell viability of human corneal epithelial cells (HCECs) and rabbit corneal stromal cells (RCSCs) after treatment with different concentrations of NS-GQDs-RF for 24 h using the CCK-8 kit (n=4).
[0035] Figure 8 The results of HE staining of different treatment groups at 2 and 4 weeks after surgery.
[0036] Fig. 9 The following are the evaluation diagrams of in vivo riboflavin penetration in different penetration treatment groups, where A is a laser confocal 2.5D image and B is a slit lamp image under cobalt blue light.
[0037] Fig.10 Figure 2 shows the biomechanical properties of the cornea in different treatment groups (n=5), where A is the statistical diagram of Young's modulus under different strains, and B is the stress-strain curve. DETAILED DESCRIPTION
[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0039] The raw materials in the examples were obtained from reagent companies such as Aladdin, McLean, and Shanghai Yuanye Biotechnology Co., Ltd. Human corneal epithelial cells (HCECs) were purchased from the American Type Culture Collection (ATCC, USA), and rabbit primary corneal stromal cells were purchased from Synbio (Shanghai) Biotechnology Co., Ltd.
[0040] Example 1 Preparation method and performance characterization of nitrogen-sulfur co-doped graphene quantum dots modified riboflavin nanocomposite photosensitizer (NS-GQDs-RF)
[0041] This embodiment provides a NS-GQDs-RF nanocomposite photosensitizer, and its preparation method is as follows Figure 1 As shown, the specific steps include:
[0042] (1) Preparation of nitrogen-sulfur co-doped graphene quantum dots (NS-GQDs) by a simple hydrothermal method
[0043] 5 g of anhydrous citric acid and 1.5 g of reduced glutathione were dissolved in 30 mL of double distilled water and subjected to ultrasonic dissolution. The resulting transparent solution was then transferred to a 100 mL polytetrafluoroethylene-lined autoclave and reacted at 180 °C in an oven for 6 h. After the reaction, the autoclave was allowed to cool naturally to room temperature. The resulting solution was filtered through a 50 nm filter membrane to remove larger particles, and then dialyzed for 24 h using a cellulose dialysis bag with a molecular weight cutoff of 500 to 1000 Da to obtain a purified NS-GQDs solution.
[0044] (2) Preparation of nitrogen-sulfur co-doped graphene quantum dots modified riboflavin nanocomposite photosensitizer (NS-GQDs-RF) by one-step esterification reaction
[0045] 40 mg of riboflavin 5′-sodium phosphate, 28 mg of dehydrating agent 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 7 mg of catalyst 4-dimethylaminopyridine (DMAP) were dissolved in 10 mL of PBS (1 M, pH 5), stirred until dissolved, and then 1 mL of NS-GQDs solution (10 mg / mL) was added and mixed evenly. The resulting mixed solution was then transferred to a brown bottle and stirred at room temperature with magnetic stirring at 500 rpm for 48 h. Then, the purified NS-GQDs-RF nanocomposite photosensitizer solution was obtained by dialyzing in double distilled water with a cellulose dialysis bag with a molecular weight cutoff of 1000 Da for 8 h.
[0046] The mechanism of the NS-GQDs-RF nanocomposite photosensitizer prepared in this example is shown in the figure below: Figure 2 As shown; transmission electron microscopy characterization diagram as shown Figure 3 As shown; UV-visible absorption spectrum and excitation spectrum are shown Figure 4 As shown; Zeta potential diagram and peak binding energy difference diagram are shown Figure 5 As shown; the glutathione depletion rate results are shown in Figure 6 As shown; the cell survival rate results tested using the CCK-8 method are shown in Figure 7 shown.
[0047] Example 2 Application of Nitrogen-Sulfur Co-doped Graphene Quantum Dots Modified Riboflavin Nanocomposite Photosensitizer (NS-GQDs-RF) in Transepithelial Iontophoresis Corneal Collagen Cross-linking
[0048] The NS-GQDs-RF nanocomposite photosensitizer prepared in Example 1 was applied to transepithelial iontophoresis corneal cross-linking to verify its effect on corneal riboflavin penetration and biomechanical properties. The specific process is as follows:
[0049] Four-month-old male New Zealand white rabbits were randomly divided into four groups. The NS-GQDs-RF (ion) group was infiltrated with NS-GQDs-RF (0.1% RF) composite photosensitizer solution for 10 min (e.g. Figure 2 The RF (ion) group used 0.1% RF solution for ion introduction for 10 min, and the RF (epi-off) group (the traditional gold standard group) used 0.1% RF dissolved in 20% dextran T500 for 30 min after corneal epithelium removal, followed by standard ultraviolet irradiation mode at 370 nm 3 mw / cm 2 Irradiate under UV light for 30 min. Blank group is a blank control group without any treatment. One batch of rabbits was observed for riboflavin penetration by slit lamp under cobalt blue light immediately after infiltration, and then OCT-embedded ice-cut samples were taken for confocal microscopy photography. Another batch of rabbits were taken for paraffin sectioning and HE staining at the 2nd and 4th week after cross-linking. The last batch of rabbits were taken immediately after cross-linking in each group with 1mm sclera for uniaxial tensile test to evaluate the biomechanical properties of different treatment groups.
[0050] Experimental Results
[0051] Figure 3 The transmission electron microscopy images confirm that the NS-GQDs prepared in Example 1 are composed of small-sized, monodispersed nanoparticles with an average particle size of 2.7±0.7 nm; and the particle size of the NS-GQDs-RF prepared in Example 1 is increased, with an average particle size of 8.0±1.0 nm.
[0052] Figure 4The UV-visible absorption spectrum of A shows that NS-GQDs-RF has a fusion wave of RF and NS-GQDs, and the UV absorption peak of NS-GQDs-RF shows an obvious blue shift compared with NS-GQDs. Figure 4 B shows that when monitored at a wavelength of 525 nm, RF exhibits a wide ultraviolet excitation band of 301 to 594 nm; when loaded with NS-GQDs, the shape and range of the excitation spectrum curve of the NS-GQDs-RF composite photosensitizer changed.
[0053] Figure 5 The Zeta potential of A shows that the Zeta potential of NS-GQDs-RF is -20.1 mV, which is more negative than that of NS-GQDs (-11.5 mV) and RF (-6.3 mV). Figure 5 The peak binding energy difference diagram of B shows that compared with NS-GQDs, the peak binding energies of C 1s and S 2p in NS-GQDs-RF increased by 0.82 eV and 0.16 eV, respectively, which indicates that NS-GQDs have a tendency to lose electrons. On the contrary, compared with RF, the peak binding energies of Na 1s and P 2p in NS-GQDs-RF decreased by 0.19 eV and 0.27 eV, respectively, indicating that RF has a tendency to gain electrons. The above results all illustrate the existence of electron transfer in the NS-GQDs-RF composite system, that is, transfer from NS-GQDs to RF, thereby increasing its response efficiency to the ion introduction electric field force.
[0054] from Figure 6 It can be seen that under ultraviolet A irradiation, the glutathione loss rate of NS-GQDs-RF is significantly increased compared with RF, indicating that the loading of NS-GQDs will enhance the efficiency of active oxygen free radical generation of NS-GQDs-RF composite photosensitizer.
[0055] Figure 7 The CCK-8 result graph shows that different concentrations of NS-GQDs-RF (0-2 mg / mL RF) were co-incubated with human corneal epithelial cells (HCECs) and rabbit corneal epithelial cells (RCSCs) for 24 h, and the survival rates of both cells exceeded 90%, indicating that NS-GQDs-RF has good cell biocompatibility.
[0056] Figure 8The HE staining results of different treatment groups at 2 and 4 weeks after surgery showed that the corneal structure of the NS-GQDs-RF (ion) group was as clear as that of the Blank group. There was no significant change in the thickness of each layer of the cornea in the NS-GQDs-RF (ion) group; the corneal epithelial cells and stromal cells were evenly distributed without obvious damage, and the number of inflammatory cells did not increase significantly. The above results indicate that NS-GQDs-RF has good in vivo biocompatibility.
[0057] Fig. 9 The laser confocal 2.5D image of A shows that the green fluorescence signal of the NS-GQDs-RF (ion) group increased significantly compared with the RF (ion) group, and even reached a level comparable to that of RF (epi-off). Fig. 9 The slit lamp image under cobalt blue light in B shows the same trend. The above results indicate that the loading of NS-GQDs increases the penetration efficiency of RF in the NS-GQDs-RF composite system.
[0058] from Fig.10 It can be seen that when the strain is 8% and 10%, the Young's modulus and stress of NS-GQDs-RF are significantly increased compared with the RF (ion) and Blank groups, and slightly greater than the gold standard RF (epi-off) group, indicating that the application of the NS-GQDs-RF composite photosensitizer synthesized in Example 1 in transepithelial ion introduction corneal cross-linking can produce the best biomechanical enhancement effect.
[0059] In summary, the present invention provides a nanocomposite photosensitizer for transepithelial ion introduction corneal collagen cross-linking and its preparation method and application. First, nitrogen-sulfur co-doped graphene quantum dots are synthesized by a simple hydrothermal method, and then a nanocomposite photosensitizer of nitrogen-sulfur co-doped graphene quantum dots modified with riboflavin is synthesized by a one-step esterification reaction. The method is simple to operate, environmentally friendly and safe, and has low cost, which is conducive to industrialization and entering the clinical trial stage. The prepared nanocomposite photosensitizer of nitrogen-sulfur co-doped graphene quantum dots modified with riboflavin has high electric field responsiveness, high corneal riboflavin penetration ability, high reactive oxygen free radical generation efficiency and good biocompatibility. It is applied to transepithelial ion introduction corneal collagen cross-linking, which can achieve a matrix riboflavin content similar to that of the gold standard group of epithelial removal; achieve slightly better biomechanical properties than the gold standard group; and achieve the superiority of retaining the epithelium and a shorter penetration time compared with the gold standard procedure. The present invention has good application prospects and will provide assistance for the treatment of keratoconus.
[0060] Technical personnel should note that although the present invention has been explained through the above specific implementation methods, the inventive concept of the present invention is not limited thereto. Any improvement or variation based on the inventive concept belongs to the protection scope of this patent right.
[0061] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments and experimental examples. Any technical solution that follows the concept of the present invention is included in the protection scope of the present invention. It should be emphasized that for ordinary technicians in this technical field, without departing from the purpose and scope of the present invention, any modification or equivalent replacement should be regarded as part of the protection scope of the present invention.
Claims
1. Application of a nanocomposite photosensitizer solution in the preparation of an iontophoresis-mediated transepithelial corneal cross-linking agent for treating keratoconus, characterized in that: The final concentration of riboflavin in the nanocomposite photosensitizer solution is 0.1%. The nanocomposite photosensitizer solution is nitrogen-sulfur co-doped graphene quantum dot-modified riboflavin synthesized by esterification reaction of nitrogen-sulfur co-doped graphene quantum dots and riboflavin. The nanocomposite photosensitizer solution is prepared by the following steps: S1. At room temperature, weigh riboflavin sodium phosphate, dehydrating agent 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and catalyst 4-dimethylaminopyridine, dissolve them in acidic PBS, stir until dissolved, and set aside; S2, adding a nitrogen-sulfur co-doped graphene quantum dot solution with a concentration range of 3 to 15 mg / mL to the solution obtained in step S1, and stirring to mix evenly; S3, transfer the mixed solution to a brown bottle and stir magnetically at 500 rpm at room temperature for 48 to 72 h; S4. The solution obtained after the reaction is dialyzed and purified in double distilled water to obtain a nanocomposite photosensitizer solution of nitrogen-sulfur co-doped graphene quantum dots modified with riboflavin.
2. The use according to claim 1, characterized in that: The particle size distribution of the nitrogen-sulfur co-doped graphene quantum dots modified riboflavin is 8.0±1.0 nm; the nitrogen-sulfur co-doped graphene quantum dots modified riboflavin has a wide excitation band of 332-585 nm; and the Zeta potential of the nanocomposite photosensitizer solution is -20.1 mV.
3. A method for preparing the nanocomposite photosensitizer solution according to claim 1, characterized in that: The preparation method comprises the following steps: S1. At room temperature, weigh riboflavin sodium phosphate, dehydrating agent 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and catalyst 4-dimethylaminopyridine, dissolve them in acidic PBS, stir until dissolved, and set aside; S2, adding a nitrogen-sulfur co-doped graphene quantum dot solution with a concentration range of 3 to 15 mg / mL to the solution obtained in step S1, and stirring to mix evenly; S3, transfer the mixed solution to a brown bottle and stir magnetically at 500 rpm at room temperature for 48 to 72 h; S4. The solution obtained after the reaction is dialyzed and purified in double distilled water to obtain a nanocomposite photosensitizer solution of nitrogen-sulfur co-doped graphene quantum dots modified with riboflavin.
4. The preparation method according to claim 3, characterized in that The acidic PBS is a 1 M phosphate buffer solution with a pH of 4 to 6.
5. The preparation method according to claim 3, characterized in that: In the step S1, the mass ratio of riboflavin sodium phosphate, dehydrating agent 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and catalyst 4-dimethylaminopyridine is 8:5.6:2.8 to 8:5.6:
1.
6. The preparation method according to claim 3, characterized in that: In step S2, the volume ratio of the nitrogen-sulfur co-doped graphene quantum dot solution to the acidic PBS as a solvent is 1:10 to 2:
10.
7. The preparation method according to claim 3, characterized in that The dialysis in step S4 uses a cellulose dialysis bag with a molecular weight cutoff of 1000 Da, and the dialysis time is 8 to 12 hours.
8. The preparation method according to claim 3, characterized in that: The nitrogen-sulfur co-doped graphene quantum dot solution in step S2 is prepared by the following steps: Anhydrous citric acid and reduced glutathione were weighed and added into double distilled water in a mass ratio of 5:1.5 to 5:
1. After ultrasonic mixing and dissolution, the mixture was transferred into a reactor and reacted at 180 to 240°C for 6 to 16 hours. The mixture was then purified by ultrafiltration and dialysis to obtain a nitrogen-sulfur co-doped graphene quantum dot solution.
9. The preparation method according to claim 8, characterized in that The ultrafiltration uses a 50 nm filter membrane; the dialysis uses a cellulose dialysis bag with a molecular weight cutoff of 500 to 1000 Da, and the dialysis time is 12 to 48 hours.