Visible light activated organic afterglow Janus hydrogel and preparation and application thereof

Abstract

This invention provides a visible light-activated organic afterglow Janus hydrogel, its preparation, and its application. The compound has structures shown in I-VI, and upon visible light excitation in an aqueous environment, it produces afterglow luminescence and continuously generates reactive oxygen species. This invention covalently grafts this compound onto an outer hydrogel network formed by copolymerizing chondroitin sulfate with methacrylamide and N,N-dimethylacrylamide, and combines it with a visible light-curable liquid inner layer precursor to construct a Janus bilayer hydrogel system. In use, the inner layer precursor is sprayed or injected onto the wound, and after applying the outer hydrogel, a single visible light irradiation simultaneously triggers in-situ curing of the inner layer and functional activation of the outer layer. This hydrogel combines afterglow imaging and long-lasting reactive oxygen species release, enabling real-time visual monitoring and continuous therapeutic intervention during treatment. It is suitable for clinical scenarios such as skin infection, postoperative adhesion prevention, prevention of postoperative tumor recurrence, repair of deep tissue defects, and treatment of adjacent important blood vessels where surgical resection is not possible.

Description

Technical Field

[0001] This application relates to the field of biomedical polymer materials technology, and more specifically, to a visible light-activated organic afterglow Janus hydrogel and its preparation and application. Background Technology

[0002] Visible light-activated organic afterglow materials are a class of functional materials that continue to emit light and generate reactive oxygen species even after photoexcitation has ceased. Compared to traditional ultraviolet or X-ray excitation systems, visible light activation offers higher biosafety and demonstrates unique advantages in biomedical imaging and phototherapy. However, existing organic afterglow materials suffer from the following technical limitations: they mostly rely on multi-component assembly systems, with single-component molecules being extremely rare; they are prone to luminescence quenching in aqueous physiological environments, making stable in vivo application difficult; and they are typically applied as nanoparticle suspensions, making it impossible to form mechanically strong three-dimensional structures in situ, hindering the achievement of targeted physical isolation and mechanical support in complex wounds such as penetrating defects and postoperative cavities.

[0003] Currently, organic afterglow materials mainly rely on multi-component assembly systems, with truly single-component molecules being extremely rare. Most existing organic afterglow molecules are prone to luminescence quenching in aqueous physiological environments, severely limiting their medical applications in living organisms. Existing visible light-activated organic afterglow materials are typically applied in the form of nanoparticle suspensions, lacking the ability to form three-dimensional structures with sufficient mechanical strength in situ, making it difficult to achieve targeted, long-lasting physical isolation and mechanical support in complex wounds (such as penetrating defects, near-vascular areas, and postoperative cavities).

[0004] On the other hand, in-situ hydrogel formation technology can achieve sealing and repair of irregular wounds. Among them, Janus bilayer hydrogel, by constructing an asymmetric structure, can meet the needs of both mechanical support and tissue repair. However, existing in-situ gelation systems (such as enzyme-initiated systems) rely on specific endogenous triggers, and the hydrogel itself does not have imaging monitoring capabilities, making it difficult to achieve real-time visualization feedback of the treatment process and long-term therapeutic intervention.

[0005] In summary, while existing visible light-activated organic afterglow materials offer the advantage of long-lasting, image-guided therapy, they lack the ability to form mechanically adaptable hydrogel structures in situ. Conversely, existing in-situ gelling Janus hydrogel systems, while providing robust sealing and mechanical support for moist tissues, lack afterglow imaging and long-lasting therapeutic capabilities. Especially in clinical scenarios such as skin infections, inoperable lesions near blood vessels, postoperative adhesion prevention, inhibition of tumor recurrence and metastasis, and repair of deep tissue defects, there is an urgent need for a hydrogel dressing that can generate real-time afterglow imaging and long-lasting therapeutic effects under visible light activation, and can also form a mechanically adaptable Janus bilayer structure through in-situ spraying. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a visible light-activated organic afterglow Janus hydrogel, its preparation, and its application. Addressing the dual technical challenges of existing organic afterglow materials relying on multi-component assembly, easy quenching in aqueous phases, inability to form mechanically adaptable three-dimensional structures in situ, and the lack of imaging guidance and long-term therapeutic functions in existing in-situ gelled Janus hydrogels, this invention designs and synthesizes a single-molecule afterglow probe with a well-defined structure that can efficiently generate afterglow luminescence and continuously produce reactive oxygen species under visible light excitation. This probe is covalently grafted onto an outer hydrogel network formed by the copolymerization of chondroitin sulfate methacrylamide and N,N-dimethylacrylamide. Simultaneously, a liquid composition containing a visible light initiator, monomer, and crosslinking agent is formulated as an inner hydrogel precursor. During application, the inner precursor is simply sprayed or injected onto the wound, followed by the application of the outer hydrogel. A single visible light irradiation simultaneously triggers the in-situ solidification of the inner precursor and the functional activation of the outer afterglow molecules, ultimately forming a Janus bilayer hydrogel with both a mechanical support layer and a tissue-adhesive layer. This hydrogel combines background-free afterglow imaging with long-lasting reactive oxygen species release, enabling real-time visual monitoring and continuous treatment intervention during the treatment process. It is suitable for complex clinical scenarios such as skin infections, inoperable lesions near blood vessels, postoperative adhesion prevention, prevention of tumor recurrence and metastasis, and repair of deep tissue defects, achieving seamless integration from material application to tissue repair.

[0007] In a first aspect, the present invention provides a visible light-activated organic afterglow small molecule compound, said compound having any of the structures shown in general formulas I to VI: Ⅰ: II: III: , IV: V: VI: .

[0008] Most existing organic afterglow materials rely on multi-component assembly systems, which are complex in composition, cumbersome in preparation, and have poor batch stability. The compound of this invention is a single molecule with a well-defined structure, which can achieve afterglow luminescence and reactive oxygen generation under visible light excitation without multi-component assembly, simplifying the material system and improving reproducibility and ease of application.

[0009] Secondly, the present invention provides a method for preparing a visible light-activated Janus bilayer hydrogel, characterized by comprising the following steps: Chondroitin sulfate methacrylamide and N,N-dimethylacrylamide are copolymerized to form a hydrogel network, and the organic afterglow small molecule compound of claim 1 is attached to the hydrogel network to obtain an outer hydrogel. A liquid composition containing a visible light initiator, a polymerizable monomer, and a crosslinking agent was prepared to obtain an inner layer hydrogel precursor; The outer hydrogel and the inner hydrogel precursor are stacked together to obtain the Janus bilayer hydrogel.

[0010] In existing technologies, the application of organic afterglow materials typically requires a separate photoactivation step, and the in-situ molding of hydrogels also requires independent photo- or chemical triggering. This invention requires only a single visible light irradiation to simultaneously trigger the in-situ solidification of the inner hydrogel precursor and the functional activation of the organic afterglow molecules in the outer hydrogel. This not only simplifies the operation process but also achieves simultaneous completion of structural molding and functional attribution.

[0011] Preferably, the molar ratio of chondroitin sulfate methacrylamide to N,N-dimethylacrylamide is 1:1 to 1:10.

[0012] Preferably, the organic afterglow small molecule compound is covalently linked to the hydrogel network in the following manner: the organic afterglow small molecule compound is first prepared into nano micelles, then the compound in the nano micelles is activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and then the activated compound is mixed and reacted with the hydrogel network to achieve covalent grafting.

[0013] Preferably, the preparation steps of the nanomicelles are as follows: dissolving the compound and DSPE-PEG-NH2 together in an organic solvent, adding the organic phase dropwise to water to form micelles, removing the organic solvent, and obtaining a nanomicelle solution by dialysis and filtration.

[0014] Preferably, the visible light initiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, the polymerizable monomer is polyethylene glycol diacrylate or acrylamide, and the crosslinking agent is N,N-methylenebisacrylamide.

[0015] Preferably, the process also includes a post-processing step: applying visible light to the Janus bilayer hydrogel to solidify the inner hydrogel precursor in situ, while simultaneously activating the compound to produce afterglow luminescence and reactive oxygen species; the visible light irradiation uses an LED light source with a wavelength of 420~700 nm.

[0016] Thirdly, the present invention provides a Janus bilayer hydrogel.

[0017] Fourthly, the present invention provides the application of Janus bilayer hydrogel in the preparation of long-acting therapeutic drugs or medical devices for imaging guidance.

[0018] In summary, the present invention has at least one of the following beneficial technical effects: 1. This invention provides a single-component, aqueously stable visible-light-activated organic afterglow molecule, overcoming the shortcomings of existing materials that rely on multi-component assembly and aqueous-phase quenching. The organic afterglow molecule compound of this invention has a well-defined single-molecule structure and can efficiently generate afterglow luminescence and continuously produce reactive oxygen species under visible-light excitation without multi-component assembly. This molecule maintains excellent afterglow performance in an aqueous environment, with an afterglow duration of nearly 14 hours, and can be cycled for more than 8 times without significant performance decay, overcoming the technical bottleneck of traditional organic afterglow molecules being easily quenched in aqueous physiological environments.

[0019] 2. This invention covalently grafts organic afterglow molecules onto an outer hydrogel network and combines them with a visible-light-curable inner precursor to form a Janus bilayer system. After application, only one visible-light irradiation is required to simultaneously trigger in-situ curing of the inner precursor and functional activation of the outer afterglow molecules, forming a Janus hydrogel that serves as both a mechanical support layer and a tissue-adhesive layer. This hydrogel not only firmly adheres to irregular wound surfaces and provides mechanical support against dynamic stress, but also continues to generate afterglow luminescence and reactive oxygen species after light irradiation ceases, achieving simultaneous structural formation and functional activation.

[0020] 3. The Janus bilayer hydrogel of this invention, upon activation by visible light, produces afterglow luminescence without background fluorescence interference, enabling real-time visual monitoring of the reactive oxygen species (ROS) generation status in the treatment area and providing imaging feedback for precise control of phototherapy parameters. Simultaneously, the continuously released ROS from the hydrogel can effectively eliminate pathogens, induce tumor cell apoptosis, or regulate the immune microenvironment. This invention is applicable to complex clinical scenarios such as skin and soft tissue infections, lesions near blood vessels that are inoperable, postoperative adhesion prevention, prevention of postoperative tumor recurrence and metastasis, and repair of deep tissue defects. It achieves seamless integration from material application to tissue repair, avoiding the difficulties of sutures, frequent dressing changes, secondary trauma, and postoperative recurrence in traditional treatments. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The synthetic route diagram of anthraquinone-2-formyl chloride provided in the embodiments of this application is shown; Figure 2A synthetic route diagram of compound I (a melamine derivative) provided in the embodiments of this application is shown; Figure 3 The mass spectrum (MS) of compound I provided in the embodiments of this application is shown; Figure 4 The present application illustrates compound I provided in the embodiments of this application. 1 H NMR spectrum; Figure 5 The present application illustrates compound I provided in the embodiments of this application. 13 C NMR spectrum; Figure 6 A synthetic route diagram of compound II (pyrimidine derivative) provided in the embodiments of this application is shown; Figure 7 The mass spectrum (MS) of compound II provided in the embodiments of this application is shown; Figure 8 The embodiments of compound II provided in this application are shown. 1 H NMR spectrum; Figure 9 The embodiments of compound II provided in this application are shown. 13 C NMR spectrum; Figure 10 A synthetic route diagram of compound III (a heptaazine derivative) provided in the embodiments of this application is shown; Figure 11 The mass spectrum (MS) of compound III provided in the embodiments of this application is shown; Figure 12 The embodiments of compound III provided in this application are shown. 1 H NMR spectrum; Figure 13 A synthetic route diagram for compound IV (a triphenylamine derivative) provided in the embodiments of this application is shown; Figure 14 The mass spectrum (MS) of compound IV provided in the embodiments of this application is shown; Figure 15 The embodiments of compound IV provided in this application are shown. 1 H NMR spectrum; Figure 16 A synthetic route diagram for compound V (a tetraphenylethylene derivative) provided in the embodiments of this application is shown; Figure 17 The mass spectrum (MS) of compound V provided in the embodiments of this application is shown; Figure 18 The embodiments of compound V provided in this application are shown. 1 H NMR spectrum; Figure 19A synthetic route diagram of compound VI (porphyrin derivative) provided in the embodiments of this application is shown; Figure 20 The mass spectrum (MS) of compound VI provided in the embodiments of this application is shown; Figure 21 The embodiments of compound VI provided in this application are shown. 1 H NMR spectrum; Figure 22 This illustration shows a schematic diagram of the synthetic route for forming micelles by encapsulating small organic afterglow molecules in DSPE-PEG-NH2, as provided in an embodiment of this application. Figure 23 The image shows a transmission electron microscope (TEM) image of nanomicelles provided in an embodiment of this application; Figure 24 The afterglow emission spectra of nanomicelles under different wavelength excitations provided in the embodiments of this application are shown. Figure 25 The following diagrams show the afterglow emission intensity of nanomicelles under different wavelength excitations provided in the embodiments of this application. Figure 26 The diagram shows the afterglow duration of the nanomicelles provided in the embodiments of this application; Figure 27 The image shows the afterglow luminescence intensity after eight cycles of activation of the nanomicelles provided in this application embodiment; Figure 28 This paper illustrates the effect of different treatment groups on the fluorescence intensity of reactive oxygen species in cells, as provided in the embodiments of this application. Figure 29 The figure showing the effect of different treatment groups under normoxic and hypoxic conditions on the apoptosis rate of 4T1 cells provided in the embodiments of this application is illustrated. Figure 30 The diagram illustrates the effects of different treatment groups provided in the embodiments of this application on DNA damage and mitochondrial membrane potential in 4T1 cells. Figure 31 The figure shows the effect of different treatment groups provided in the embodiments of this application on the tumor volume growth curve of 4T1 tumor-bearing mice; Figure 32 The graphs showing the changes in body weight of mice in different treatment groups provided in the embodiments of this application are shown. Figure 33 The diagram shows the fit effect of the Janus bilayer hydrogel provided in the embodiment of this application during joint flexion; Figure 34 The image shows the adhesion effect of Janus double-layer hydrogel covering a postoperative wound according to an embodiment of this application; Figure 35 The diagram shows the antibacterial effect of the Janus bilayer hydrogel provided in the embodiments of this application against Escherichia coli and Staphylococcus aureus. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention. Furthermore, all other embodiments obtained by those skilled in the art without inventive effort are within the protection scope of the present invention.

[0024] Specific experimental steps or conditions are not specified in the embodiments; they can be performed according to the conventional experimental steps or conditions described in the prior art. Reagents and other instruments used, unless otherwise specified, are all commercially available conventional reagent products. Furthermore, the accompanying drawings are merely illustrative diagrams of the embodiments of the present invention and are not necessarily drawn to scale. The same reference numerals in the drawings denote the same or similar parts, and therefore, repeated descriptions of them will be omitted. Some block diagrams shown in the drawings are functional entities and do not necessarily correspond to physically or logically independent entities.

[0025] Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of this specification.

[0026] In the description of this invention, it should be understood that the use of terms such as "first" and "second" to define components is merely for the purpose of distinguishing the corresponding components. Unless otherwise stated, the above terms have no special meaning and therefore should not be construed as limiting the scope of protection of this invention.

[0027] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0028] To enable those skilled in the art to better understand this application, the following examples provide a detailed description of a visible light-activated organic afterglow Janus hydrogel, its preparation, and its application.

[0029] Example Example 1: Preparation of visible light-activated small organic afterglow compounds In a dry 250 mL round-bottom flask, 30.0 g (0.12 mol) of anthraquinone-2-carboxylic acid was added and cooled in an ice bath at 0°C, strictly avoiding moisture. 2.0 mL of N,N-dimethylformamide (DMF) was added as a catalyst. 100.0 mL of thionyl chloride (SOCl2) was slowly added, and stirring continued at 0°C for 30 minutes. The mixture was slowly heated to reflux temperature (82°C) and reacted for 8 hours until the system became a clear, transparent solution. After the reaction was complete, excess thionyl chloride was removed by vacuum distillation. The remaining solid was recrystallized from approximately 80 mL of toluene, filtered, washed with cold toluene, and dried to give approximately 27.0 g of anthraquinone-2-formyl chloride, with a yield of approximately 83%. The synthetic route is as follows: Figure 1 As shown, Take approximately 30 g of the above-mentioned crude anthraquinone-2-formyl chloride and dissolve it in 50 mL of anhydrous N-methylpyrrolidone (NMP) for later use. In a dry 500 mL three-necked flask, add 4.0 g (31.7 mmol) of dried melamine and 150 mL of anhydrous NMP, and stir to disperse the solid. Add 20.0 mL (14.5 g, 143.5 mmol) of anhydrous triethylamine as an acid-binding agent. Under nitrogen protection and ice bath cooling (maintaining the internal temperature not exceeding 10°C), slowly add the previously prepared anthraquinone-2-formyl chloride NMP solution. During the addition, the system gradually changes from a suspension to a dark solution, followed by the gradual precipitation of solids. After the addition is complete, remove the ice bath and slowly raise the temperature to 80–90°C, continuing the reaction with stirring at this temperature for 12–16 hours. Thin-layer chromatography (TLC) is used to monitor the consumption of melamine amino groups during the reaction.

[0030] After the reaction was complete, the mixture was cooled to room temperature. 200 mL of deionized water was slowly added to the reaction solution to quench the reaction, resulting in the precipitation of a large amount of yellow to brown solid. The mixture was filtered, and the filter cake was washed successively with 100 mL of 5% sodium bicarbonate solution, 100 mL of deionized water (twice), and 50 mL of anhydrous ethanol. The obtained solid was dried in a vacuum drying oven at 80°C to constant weight, yielding approximately 23.5 g of the target compound I, with a yield of approximately 90% (based on melamine). The synthetic route is as follows: Figure 2 As shown.

[0031] The obtained compound I was subjected to MS, 1 H NMR and 13 The C NMR characterization results are as follows: Figures 3-5 As shown, its chemical structure has been confirmed.

[0032] Following the same reaction steps, amounts, and conditions as described above, compounds II, III, IV, V, and VI were obtained by replacing the intermediate melamine with equimolar amounts of pyrimidine, heptaazine, triphenylamine, tetraphenylethylene, and porphyrin, respectively. The purification methods for each compound were the same as described above.

[0033] The synthetic route of compound II is as follows: Figure 6 As shown, via MS, 1 H NMR and 13 The C NMR characterization results are as follows: Figures 7-9 As shown, its chemical structure has been confirmed.

[0034] The synthetic route of compound III is as follows: Figure 10 As shown, via MS, 1 H NM characterization results are as follows Figure 11 , 12 As shown, its chemical structure has been confirmed.

[0035] The synthetic route of compound IV is as follows: Figure 13 As shown, via MS, 1 H NMR such as Figure 14 , 15 As shown, its chemical structure has been confirmed.

[0036] The synthetic route of compound V is as follows: Figure 16 As shown, via MS, 1 The H NMR characterization results are as follows: Figure 17 , 18 As shown, its chemical structure has been confirmed.

[0037] The synthetic route of compound VI is as follows: Figure 19 As shown, via MS, 1 The H NMR characterization results are as follows: Figure 20 , 21 As shown, its chemical structure has been confirmed.

[0038] This embodiment successfully synthesized a class of visible light-activated organic afterglow small molecule compounds I–VI. Anthraquinone-2-carboxylic acid was used as the starting material, and anthraquinone-2-formyl chloride was prepared via acylation reaction. This chloride was then reacted with melamine, pyrimidine, heptaazine, triphenylamine, tetraphenylethylene, and porphyrin, respectively, to obtain the target compounds I–VI. The structures of each compound were determined by MS, 1 H NMR and 13 The results were confirmed by C10 NMR characterization. This method is simple to operate and has a high yield, providing a functional core material for the subsequent preparation of Janus bilayer hydrogels.

[0039] Example 2: Preparation of Organic Afterglow Small Molecule Nanomicelles and Their Visible Light Excitation Properties Weigh 5-10 mg of DSPE-PEG-NH2 (Mw = 2000 Da) and 1-2 mg of the organic afterglow small molecule compound prepared in Example 1, and dissolve them together in 12 mL of tetrahydrofuran (THF). Dissolve by vortexing or sonication to obtain a homogeneous organic phase solution. Under sonication, slowly add the organic phase solution dropwise to 9-20 mL of ultrapure water (volume ratio THF:H2O = 1:9-1:20), at a dropping rate of about 1 drop / 5-10 seconds. After the addition is complete, continue sonication for 1-5 minutes to obtain a translucent micelle solution with a pale blue opalescence.

[0040] The micelle solution was transferred to a round-bottom flask and subjected to rotary evaporation at 25–30°C under reduced pressure to remove THF for approximately 10–20 minutes. The evaporated micelle solution was then transferred to a dialysis bag (molecular weight cutoff MWCO 3,500 or 8,000–14,000 Da) and dialyzed with ultrapure water for 12–24 hours (changing the dialysis medium 2–3 times during this period) to further remove residual organic solvents and unencapsulated drug. After dialysis, the micelle solution in the bag was collected and filtered through a 0.22 μm or 0.45 μm microporous membrane to obtain the drug-loaded DSPE-PEG-NH2 micelle solution. This micelle solution can be stored at 4°C or freeze-dried for solid storage.

[0041] The synthetic route for forming micelles by encapsulating small organic afterglow molecules in DSPE-PEG-NH2 is as follows: Figure 22 As shown. The transmission electron microscope (TEM) image of the obtained nanomicelles is shown below. Figure 23 As shown, the micelles are spherical with a uniform particle size distribution.

[0042] The above-mentioned nanomicelle solution was subjected to performance testing under visible light excitation. The test results are as follows: Afterglow emission spectrum: In aqueous nanomicelles, upon visible light excitation, the compound exhibits afterglow emission in the near-infrared I (NIR-I) region. The afterglow emission spectra under different wavelengths of excitation are shown below. Figure 24 As shown, the corresponding emission intensity is as follows Figure 25 As shown.

[0043] Afterglow duration as Figure 26 As shown, the afterglow emission lasts for nearly 14 hours, exhibiting exceptionally long afterglow performance. Figure 27 As shown, after 8 cycles of activation, the afterglow luminescence intensity remained unchanged, indicating that the nanomicelles have good re-activation performance.

[0044] The above results show that the nanomicelles prepared in this embodiment maintain excellent afterglow luminescence performance in an aqueous environment and have good cycling stability.

[0045] Example 3 Evaluation of the in vitro and in vivo antitumor effects of organic afterglow small molecule compound I Triple-negative breast cancer cells (4T1 cells) were collected and divided into four groups: a blank control group, a compound I-only group (no light exposure), a light-exposed group (visible light exposure only), and a compound I + light-exposed group. The compound I + light-exposed group was given compound I first, followed by visible light irradiation for 30 seconds. After irradiation, intracellular reactive oxygen species (ROS) levels were detected using a reactive oxygen species fluorescent probe.

[0046] The results are as follows Figure 28 As shown, compared with the blank control group, the simple I group and the light irradiation group, the fluorescence intensity of reactive oxygen species in cells of the compound I + light irradiation group was significantly enhanced, indicating that compound I can efficiently generate reactive oxygen species after activation by visible light.

[0047] Four T1 cells were collected and divided into four groups (as above), and cultured under normoxic (5% O2) and hypoxic (1% O2) conditions, respectively. The compound I+ light-illuminated group was irradiated with visible light for 30 seconds and then cultured for another 24 hours. The apoptosis rate was detected by flow cytometry. Results are as follows: Figure 29 As shown, compound I+ light irradiation group showed significant apoptosis induction under both normoxic and hypoxic environments, indicating that compound I can still effectively kill tumor cells in hypoxic tumor environments.

[0048] Four T1 cells were collected, grouped, and treated as above. After irradiation, DNA damage was detected by immunofluorescence staining, and mitochondrial membrane potential was detected by JC-1 staining (red fluorescence indicates normal membrane potential, and green fluorescence indicates loss of membrane potential). The results are as follows. Figure 30 As shown, cells in the compound I+ light-irradiated group exhibited significant DNA damage (strong green fluorescence) and a decrease in mitochondrial membrane potential (enhanced green fluorescence and weakened red fluorescence), indicating that visible light-activated compound Ia can induce severe organelle damage in tumor cells.

[0049] A 4T1 tumor-bearing nude mouse model was established, and the tumor volume was increased to approximately 100 mm. 3 Mice were randomly divided into four groups of five each: a blank control group, a compound I-only group (intratumoral injection of compound I, no light exposure), a light-exposed group (visible light exposure only), and a compound I + light-exposed group (intratumoral injection of compound I followed by visible light exposure). Visible light exposure was performed using an LED light source (wavelength 420–700 nm) for a cumulative exposure time of 1 minute. Tumor volume was measured periodically after treatment to assess the antitumor effect. Results are as follows: Figure 31 As shown, tumor growth was significantly inhibited in the compound I+ light-irradiated group, and its anti-tumor activity was the strongest among all groups.

[0050] During treatment, the body weight of mice in each group was measured regularly. Results are as follows: Figure 32As shown, the body weight of mice in each treatment group did not decrease significantly, and there was no statistically significant difference compared with the blank control group, indicating that compound I and visible light irradiation had no significant systemic toxicity to mice and had good biosafety.

[0051] Example 4: Preparation and performance evaluation of visible light-activated Janus bilayer hydrogels Chondroitin sulfate methacrylamide (CS-MA) and N,N-dimethylacrylamide (DMAA) were dissolved in deionized water at a molar ratio of 1:1 to 1:10. A photoinitiator was added, and the mixture was thoroughly injected into a mold. The mixture was then copolymerized under UV or visible light irradiation to form a hydrogel network. The nanomicelle solution prepared in Example 2 (i.e., DSPE-PEG-NH2 micelles of organic afterglow small molecule compounds I to VI) was taken, and EDC and NHS were added to activate the carboxyl groups on the micelle surface. The activated micelle solution was then soaked and mixed with the outer hydrogel, and the mixture was stirred at room temperature for 12 to 24 hours to covalently graft the organic afterglow small molecules onto the hydrogel network via amide bonds. After the reaction, the mixture was thoroughly washed with deionized water to remove ungrafted micelles, yielding the outer hydrogel loaded with organic afterglow small molecules.

[0052] A visible light initiator, polymerizable monomer, and crosslinking agent are dissolved in a phosphate buffer solution to prepare a liquid composition, thus obtaining the inner hydrogel precursor. This precursor is a flowable liquid at room temperature, facilitating injection or spraying.

[0053] For animal skin wounds or postoperative wounds, the inner hydrogel precursor is first evenly applied to the wound surface by spraying or injection. Then, the outer hydrogel is applied on top of the inner precursor and gently pressed to ensure a tight fit. Subsequently, the bilayer structure is irradiated with visible light using an LED light source (wavelength 420~700 nm) for 5 minutes.

[0054] During irradiation, visible light simultaneously triggers two processes: the photoinitiator in the inner hydrogel precursor is excited to generate free radicals, initiating the polymerization of monomers and crosslinking agents, forming an inner hydrogel with good tissue adhesion in situ; the covalently grafted organic afterglow molecules in the outer hydrogel absorb light energy, forming an inner peroxide intermediate, which stores energy for subsequent afterglow luminescence and reactive oxygen species release. After irradiation, a hydrogel dressing with a Janus bilayer structure is obtained.

[0055] The Janus bilayer hydrogel prepared by the above method was applied to the skin surface of the joint, and a bending test was performed after the gel cured. The results are as follows: Figure 33 As shown, when the joint is bent to nearly 90°, the double-layer hydrogel still maintains a complete fit without falling off or cracking, demonstrating excellent flexibility and adhesion.

[0056] A full-thickness skin defect was created on the back of mice, and Janus bilayer hydrogel was formed in situ using the above method. Postoperative observation showed that the hydrogel adhered firmly to the wound, tightly covered the defect area, and seamlessly integrated with the surrounding normal skin. Results are as follows: Figure 34 As shown, this hydrogel can be used for effective closure and physical isolation of postoperative wounds.

[0057] The inhibitory effect of Janus bilayer hydrogel on common surgical infections was evaluated using the inhibition zone method or colony counting method. The bilayer hydrogel was applied to agar plates coated with *Escherichia coli* or *Staphylococcus aureus*, activated by visible light irradiation for 5 minutes, and then incubated at 37°C for 24 hours. Results are as follows: Figure 35 As shown, a distinct inhibition zone appeared around the hydrogel, and the number of bacterial colonies was significantly reduced compared with the control group, indicating that the hydrogel has good antibacterial activity against both Gram-negative and Gram-positive bacteria.

[0058] In summary, this invention successfully synthesized six single-component visible-light-activated organic afterglow small molecule compounds I–VI and prepared their aqueous nanomicelles. These micelles exhibit near-infrared afterglow emission under visible light excitation, continuous afterglow luminescence for nearly 14 hours, and stable performance capable of being activated 8 times. In vitro and in vivo experiments confirmed that compound I, combined with visible light irradiation, can induce tumor cell apoptosis, cause DNA damage, and decrease mitochondrial membrane potential under normoxic and hypoxic conditions, significantly inhibiting tumor growth in tumor-bearing mice without systemic toxicity. The Janus bilayer hydrogel constructed based on the above molecules can be in-situ solidified and activated upon a single visible light irradiation, exhibiting excellent tissue adhesion, flexibility, and antibacterial activity. The system of this invention integrates imaging guidance with long-term therapeutic effects, providing a feasible material solution for various complex clinical scenarios.

[0059] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0060] Although preferred embodiments of the present application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present application.

[0061] Finally, it should be noted that in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes said element.

[0062] The above provides a detailed description of a visible light-activated organic afterglow Janus hydrogel, its preparation, and its application. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A visible light-activated organic afterglow small molecule compound, characterized in that, The compound has any of the structures shown in general formulas I to VI: Ⅰ： 、Ⅱ： 、Ⅲ： 、 Ⅳ： 、Ⅴ： 、Ⅵ： 。 2. A method for preparing a visible light-activated Janus bilayer hydrogel, characterized in that, Includes the following steps: Chondroitin sulfate methacrylamide and N,N-dimethylacrylamide are copolymerized to form a hydrogel network, and the organic afterglow small molecule compound of claim 1 is attached to the hydrogel network to obtain an outer hydrogel. A liquid composition containing a visible light initiator, a polymerizable monomer, and a crosslinking agent was prepared to obtain an inner layer hydrogel precursor; The outer hydrogel and the inner hydrogel precursor are stacked together to obtain the Janus bilayer hydrogel.

3. The preparation method according to claim 2, characterized in that, The molar ratio of chondroitin sulfate methyl methacrylate to N,N-dimethylacrylamide is 1:1 to 1:

10.

4. The preparation method according to claim 2, characterized in that, The organic afterglow small molecule compound is covalently linked to the hydrogel network in the following manner: the organic afterglow small molecule compound is first prepared into nano micelles, then the compound in the nano micelles is activated with EDC and NHS, and then the activated compound is mixed and reacted with the hydrogel network to achieve covalent grafting.

5. The preparation method according to claim 4, characterized in that, The preparation steps of the nanomicelles are as follows: the compound and DSPE-PEG-NH2 are dissolved together in an organic solvent, the organic phase is added dropwise to water to form micelles, the organic solvent is removed, and the nanomicelle solution is obtained by dialysis and filtration.

6. The preparation method according to claim 2, characterized in that, The visible light initiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, the polymerizable monomer is polyethylene glycol diacrylate or acrylamide, and the crosslinking agent is N,N-methylenebisacrylamide.

7. The preparation method according to claim 2, characterized in that, It also includes a post-processing step: applying visible light to the Janus bilayer hydrogel to solidify the inner hydrogel precursor in situ, while activating the compound to produce afterglow luminescence and active oxygen; the visible light irradiation uses an LED light source with a wavelength of 420~700 nm.

8. Janus bilayer hydrogel prepared by the preparation method according to any one of claims 2 to 7.

9. The use of the compound as described in claim 1 or the Janus bilayer hydrogel as described in claim 8 in the preparation of long-acting therapeutic drugs or medical devices for imaging guidance.

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