ROS-responsive DNA light-operated hydrogel with AIE characteristics and application of ROS-responsive DNA light-operated hydrogel

Abstract

The invention belongs to the field of application of biological materials, and particularly relates to ROS-responsive DNA light-operated hydrogel with AIE characteristics and application of the ROS-responsive DNA light-operated hydrogel. The DNA hydrogel is prepared by carrying out polymerization reaction on AIE-linker and three DNA single chains, namely, Y-1a, Y-1b and Y-1c, the AIE-linker is obtained by connecting a linker chain with an amino group and a disulfide bond and an AIE fluorescent molecule with a carboxyl group through an amidation reaction. The multifunctional DNA light-operated hydrogel prepared by the invention breaks through the defects that the traditional hydrogel is single in function, long in preparation period, incapable of regulating and controlling the microenvironment of infected wounds and the like, and has the functions of rapid preparation, safety, high efficiency, responsive release, self-adaptability, cascade antibacterial property, angiogenesis promotion and immunity regulation. The multifunctional DNA light-operated hydrogel can cover infectious wound tissues, responsively releases wrapped ginseng exosomes and NO donors under the high ROS level of infectious wounds, plays a role under laser irradiation, and remarkably promotes healing of the infectious wounds.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a ROS-responsive DNA light-controlled hydrogel with AIE characteristics and applications thereof. Background Art

[0002] With the development of nanomedicine and nanotechnology, the development and application of nanodrug delivery systems (NDDS) have made significant progress. In order to achieve dynamic real-time visualization of nanocarriers and payloads, after studying various imaging tools, scientists tend to use fluorescent agents as imaging molecules. The fluorescence intensity of traditional fluorescent groups drops significantly when they aggregate in solution due to π-π stacking, which is called the "polyfluorescence quenching" (ACQ) effect. AIE luminescent materials can emit stronger fluorescence when forming aggregates. The main reason is that they have adjustable fluorescence properties. Therefore, combining AIE into NDDS can display the movement and behavior of the carrier or the entire NDDS. In addition, AIE with photodynamic therapy (PDT) further expands its application in disease diagnosis and treatment.

[0003] DNA hydrogel is a smart material with multifunctional physical and chemical properties constructed from DNA polymer chains. It mainly uses three strategies to form a three-dimensional network structure, including self-assembly of dendritic DNA, entanglement of ultra-long linear DNA segments, and hybridization with other materials. It has the advantages of sequence programmability, precise molecular recognition, easy functional modification, stimulus responsiveness, biocompatibility and biodegradability, laying a solid foundation for its application in tissue repair engineering. Currently, it has been applied in the fields of drug delivery, biosensor devices, tumor treatment, 3D cell culture and tissue repair.

[0004] At present, there is no report on the application of AIE luminescent materials in DNA hydrogels. Summary of the invention

[0005] The first object of the present invention is to provide a ROS-responsive DNA photo-controlled hydrogel with AIE characteristics, and the second object of the present invention is to provide applications of the DNA hydrogel.

[0006] The first object of the present invention is achieved as follows: a ROS-responsive DNA light-controlled hydrogel with AIE characteristics is formed by polymerization reaction of an AIE-linker and three DNA single strands: Y-1a, Y-1b, and Y-1c; The AIE-linker is obtained by connecting a linker chain having an amino group and a disulfide bond with an AIE fluorescent molecule having a carboxyl group through an amidation reaction; The Y-1a has a nucleotide sequence as shown in SEQ ID NO: 1; The Y-1b has a nucleotide sequence as shown in SEQ ID NO: 2; The Y-1c has a nucleotide sequence as shown in SEQ ID NO: 3; The linker chain has a nucleotide sequence as shown in SEQ ID NO:4.

[0007] The second object of the present invention is achieved by providing a multifunctional DNA hydrogel based on the ROS-responsive DNA photo-controlled hydrogel with AIE characteristics, which includes a composite nanodrug and a ROS-responsive DNA photo-controlled hydrogel with AIE characteristics loaded with the composite nanodrug.

[0008] The beneficial effects of the present invention are: (1) The present invention uses a palindromic complementary DNA sequence as a linker, avoiding the double linker design and use of previous DNA hydrogels, and reducing the subsequent preparation cost of DNA hydrogels.

[0009] (2) The ROS-responsive DNA photo-controlled hydrogel with AIE characteristics provided by the present invention has high biocompatibility and adaptability, and the preparation method is simple and fast.

[0010] (3) The multifunctional DNA light-controlled hydrogel provided by the present invention can adapt to and respond to different disease environments by changing the responsive modules in the DNA sequence (for example, the tumor microenvironment is usually more acidic than normal tissue, and the pH-responsive sequence added to the DNA sequence can be used to release drugs in the tumor environment). At the same time, it can also change the type of plant-derived exosomes loaded to produce different therapeutic effects, thereby broadening the application field of DNA hydrogels.

[0011] (4) The multifunctional DNA light-controlled hydrogel for infectious wounds prepared by the present invention can cover the infected wound tissue and quickly release the encapsulated exosomes and NO donors under the high ROS level of the infected wound. Under 680 nm laser irradiation, AIE with PDT effect generates reactive oxygen species, further oxidizes NO donors, and cascades to generate NO with antibacterial activity, which significantly promotes the healing of infected wounds ( Figure 5 ). The multifunctional DNA hydrogel for infectious wounds provided by the present invention overcomes the shortcomings of traditional hydrogels such as single function, long preparation cycle, and inability to regulate the microenvironment of infected wounds, and has the functions of rapid preparation, safety and efficiency, responsive release, self-adaptation, cascade antibacterial properties, angiogenesis promotion, and immune regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1Graph showing the antibacterial activity of the multifunctional DNA light-controlled hydrogel of the present invention; wherein A is a plate coating graph after different treatments; B is a graph showing the corresponding bacterial survival rate; C is a staining graph showing live and dead bacteria after different treatments (G1: PBS; G2: L-arginine; G3: ginseng exosomes; G4: blank AIE-DNA hydrogel; G5: blank AIE-DNA hydrogel + laser; G6: drug-loaded AIE-DNA hydrogel; G7: drug-loaded AIE-DNA hydrogel + laser); Figure 2 Figure 1 is a graph showing the biosafety test results of blank AIE-DNA hydrogels; A is the cell viability of HaCat and Raw 264.7 cells after being treated with blank AIE-DNA hydrogels at different concentrations; B is the live and dead cell staining of HaCat and Raw 264.7 cells after being treated with blank AIE-DNA hydrogels (100 μM); C is the hemolysis of red blood cells after being treated with blank AIE-DNA hydrogels at different concentrations; Figure 3 The figure shows the therapeutic effect of the multifunctional DNA light-controlled hydrogel on the mouse wound infection model; A is the wound area of ​​each group during the treatment; B is the wound healing rate of each group after treatment; C is the HE and Masson staining of the wound tissue of each group after treatment (G1: PBS; G2: L-arginine; G3: ginseng exosomes; G4: blank AIE-DNA hydrogel; G5: blank AIE-DNA hydrogel + laser; G6: drug-loaded AIE-DNA hydrogel; G7: drug-loaded AIE-DNA hydrogel + laser); Figure 4 Figure 1 is a graph showing the results of in vivo safety testing of multifunctional DNA light-controlled hydrogels; A is a graph showing the HE staining results of mouse organs in the Control group, blank AIE-DNA hydrogel group, and drug-loaded AIE-DNA hydrogel group; B is a graph showing the ALT, AST, CREA, and UA levels in the serum of mice in the Control group, blank AIE-DNA hydrogel group, and drug-loaded AIE-DNA hydrogel group; C is a graph showing the results of routine blood tests of mice in the Control group, blank AIE-DNA hydrogel group, and drug-loaded AIE-DNA hydrogel group; Figure 5 This is a diagram showing the mechanism of action of the multifunctional DNA light-controlled hydrogel of the present invention on wounds infected with Staphylococcus aureus. DETAILED DESCRIPTION

[0013] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the protection scope of the present invention.

[0014] The present invention discloses a ROS-responsive DNA light-controlled hydrogel with AIE characteristics, which is formed by polymerization reaction of an AIE-linker and three DNA single strands: Y-1a, Y-1b, and Y-1c; The AIE-linker is obtained by connecting a linker chain having an amino group and a disulfide bond with an AIE fluorescent molecule having a carboxyl group through an amidation reaction; The Y-1a has a nucleotide sequence as shown in SEQ ID NO: 1; The Y-1b has a nucleotide sequence as shown in SEQ ID NO: 2; The Y-1c has a nucleotide sequence as shown in SEQ ID NO: 3; The linker chain has a nucleotide sequence as shown in SEQ ID NO:4.

[0015] The AIE fluorescent molecule is a molecule TPA-COOH having a triphenylamine (TPA) structure and having a carboxyl group.

[0016] The preparation method of the AIE-linker is as follows: TPA-COOH was mixed with EDC and NHS in a molar ratio of 1:10:10 and stirred at room temperature for 2 h to activate the carboxyl group of TPA-COOH. Then the linker solution was added dropwise and the reaction was continued for 24 h. The reaction mixture was dialyzed for 2 d and freeze-dried.

[0017] The molar ratio of Y-1a, Y-1b, Y-1c and AIE-linker is 2:2:2:1.

[0018] The present invention also provides a multifunctional DNA light-controlled hydrogel based on the ROS-responsive DNA light-controlled hydrogel with AIE characteristics, which comprises a composite nano-drug and a ROS-responsive DNA light-controlled hydrogel with AIE characteristics loaded with the composite nano-drug.

[0019] The composite nanomedicine comprises plant-derived exosomes and an NO donor.

[0020] The multifunctional DNA light-controlled hydrogel is used for preparing a medicine for treating infectious wounds; and the exosomes are ginseng exosomes.

[0021] The preparation method of ginseng exosomes is achieved by the following steps: 1) Wash and dry the ginseng, add PBS solution and stir evenly, filter with gauze to obtain ginseng liquid; perform gradient centrifugation on the ginseng liquid to obtain ginseng crude extract; perform ultra-high speed centrifugation on the ginseng crude extract, discard the supernatant, resuspend the precipitate with PBS solution to obtain ginseng crude exosomes; 2) After ultracentrifugation of crude ginseng exosomes with sucrose solutions of different concentrations, the supernatant was discarded and the precipitate was resuspended with PBS solution. After ultracentrifugation again, the supernatant was discarded and the precipitate was resuspended with PBS solution to obtain ginseng exosomes with an average particle size of 134.22-156.5 nm, which were packaged and stored in a -80℃ refrigerator.

[0022] The NO donor is L-arginine.

[0023] The target multifunctional DNA hydrogel was obtained by mixing ROS-responsive DNA photocontrollable hydrogel with AIE characteristics, exosomes and NO donors. Example 1 Extraction and purification of ginseng exosomes 1. Extraction of ginseng exosomes: After washing the ginseng, add an appropriate amount of PBS with a pH of 7.4, put it in a blender to make a homogenate, filter, centrifuge at 3000×g for 30 min, take the supernatant; centrifuge at 10000×g for 1 h, take the supernatant; centrifuge at 100,000×g for 2 h, discard the supernatant, resuspend the precipitate with PBS with a pH of 7.4, and vortex thoroughly to obtain crude ginseng exosomes.

[0024] 2. Purification of ginseng exosomes: Prepare 1 mol / L and 2 mol / L sucrose solutions, place the resuspended ginseng crude exosomes in a centrifuge tube, and then add 1 mol / L sucrose solution and 2 mol / L sucrose solution through the sample layer. Centrifuge at 100,000×g for 2 h, gently aspirate the exosome layer, discard the remaining solution containing sucrose, add PBS to the obtained exosomes to the whole tube, centrifuge at 100,000×g for 2 h, discard the supernatant, resuspend the precipitate with PBS, obtain purified ginseng exosomes, and determine the protein concentration with a BCA kit. Take an appropriate amount of ginseng exosomes for the experiment, and store the remaining ginseng exosomes at -80℃ for subsequent experiments.

[0025] Example 2 Rapid synthesis of ROS-responsive DNA photo-controlled hydrogels with AIE characteristics 1. Synthesis of ROS-responsive DNA single strands with AIE characteristics (i.e., AIE-linker) The DNA single strands used in the experiment were purchased from Sangon Biotechnology Co., Ltd. The linker with an amino group and a disulfide bond was connected to TPA-COOH through an amidation reaction to form a ROS-responsive DNA single strand with AIE characteristics.

[0026] The specific synthesis method of AIE-linker is as follows: TPA-COOH was mixed with EDC and NHS in a molar ratio of 1:10:10 and stirred at room temperature for 2 h to activate the carboxyl group of TPA-COOH. The linker solution was added dropwise and the reaction was continued for 24 h. After the reaction was completed, the reaction mixture was dialyzed for 2 d and lyophilized.

[0027] 2. Annealing assembly of Y-1a, Y-1b, Y-1c and AIE-linker Dissolve the purchased Y-1a, Y-1b, Y-1c and the prepared AIE-linker in sterilized dd H2O to prepare a high-concentration stock solution. Keep a certain proportion of Y-1a, Y-1b, Y-1c and AIE linker at 95 °C for 3 min. Mix Y-1a, Y-1b, Y-1c and AIE linker, add a certain proportion of Tri-HCl & Mg 2+ Buffer solution was used to prepare blank AIE-DNA hydrogel at room temperature.

[0028] The base sequence of the single-stranded DNA used in the present invention is shown in Table 1.

[0029] Table 1 Base sequences of the DNA single strands used DNA single strand Base sequence Y-1a tcc-att-ccc-agg-ttg-gat-ccg-cat-gac-att-cgc-cgt-aag Y-1b tcc-att-ccc-agg-tct-tac-ggc-gaa-tga-ccg-aat-cag-cct Y-1c cc-att-ccc-agg-tag-gct-gat-tcg-gtt-cat-gcg-gat-cca Linker acc-tgg-gaa-tgg-aat-ac / his-sh / aac-gtt-aac-gtt-gta-t Example 3 Preparation of drug-loaded multifunctional DNA hydrogel 1. Load NO donor and ginseng exosomes into DNA hydrogel.

[0030] NO donor and ginseng exosomes were added to the blank AIE-characterized ROS-responsive DNA hydrogel and vortexed to obtain the drug-loaded AIE-DNA hydrogel.

[0031] 2. The ratio of NO donor-ginseng exosomes-DNA hydrogel is shown in Table 2.

[0032] Table 2 Ratios of Y-1a, Y-1b, Y-1c, AIE-linker, NO donor and ginseng exosomes Material concentration Y-1a 100 μM Y-1b 100 μM Y-1c 100 μM AIE-linker 50 μM NO Donor 150 μM Ginseng Exosomes 5 μg / mL Example 4 In vitro multifunctional DNA light-controlled hydrogel experiment 1. In vitro antibacterial activity test Staphylococcus aureus was selected as the bacterial species to evaluate the antibacterial properties of the multifunctional DNA light-controlled hydrogel. This experiment was divided into 7 groups, namely G1-G7. Group G1 (control group): PBS; Group G2: L-arginine (150 μM); Group G3: ginseng exosomes (5 μg / mL); Group G4: blank AIE-DNA hydrogel (AIE-DNA hydrogel: 100 μM); Group G5: blank AIE-DNA hydrogel (AIE-DNA hydrogel: 100 μM) + laser; Group G6: drug-loaded AIE-DNA hydrogel (L-arginine: 150 μM, ginseng exosomes: 5 μg / mL, AIE-DNA hydrogel: 100 μM); Group G7: drug-loaded AIE-DNA hydrogel (L-arginine: 150 μM, ginseng exosomes: 5 μg / mL, AIE-DNA hydrogel: 100 μM) + laser. Staphylococcus aureus was first cultured at 37°C and diluted to 10 7 CFU / mL, and equal volumes of PBS, L-arginine, ginseng exosomes, blank AIE-DNA hydrogel, and drug-loaded AIE-DNA hydrogel were added to the diluted bacterial suspension and cultured for 16 h. Among them, Staphylococcus aureus cultured in groups G5 and G7 were irradiated with 680 nm laser for 15 min. 100 μL of bacterial suspension was taken from each group and evenly spread on the agar plate and placed in an inverted bacterial incubator for culture. After 24 h, the colonies were counted and photographed. The bacterial survival rate of each group was calculated by the following formula: The bacterial samples were fixed with 2.5% glutaraldehyde fixative (for electron microscopy) at 4°C for 6 h. Then, they were dehydrated with ethanol (15 min) in sequence, and the ethanol concentration was reduced in sequence. After dehydration, the samples were centrifuged and the bacteria were resuspended with tert-butyl alcohol. The resuspended bacteria were dropped on a silicon wafer and freeze-dried, then sprayed with gold and observed under a scanning electron microscope. Bacterial live or dead staining was used to evaluate the bacterial survival rate of the multifunctional DNA hydrogel, and the untreated samples were used as controls. DMPO and propidium iodide (PI) were used as dyes, and after staining for 30 minutes, the samples were placed under a fluorescence microscope for observation.

[0033] 2. In vitro cytotoxicity and hemocompatibility testing CCK8 and live-dead cell staining were used to detect the biocompatibility of blank AIE-DNA hydrogel. A certain concentration of hydrogel was added to the culture medium to prepare a hydrogel extract. Raw cells were inoculated on a 96-well plate and different concentrations of extracts were added and cultured for 24 h. CCK8 was added to the 96-well plate and the cells were cultured for 1 h. The absorbance at 490 nm was measured using an ELISA reader. The cell survival rate of each group was calculated by the formula: The hemocompatibility of blank AIE-DNA hydrogel was tested by hemolysis test. Fresh blood was centrifuged, the supernatant was discarded, and the cell pellet was resuspended in PBS (pH 7.4) to prepare a 2% red blood cell suspension. The red blood cell suspension with Triton X-100 was used as a positive control, and the red blood cell suspension with PBS (pH 7.4) was used as a negative control. The red blood cell suspension was incubated with different concentrations of hydrogel extract at 37°C for 4 h. The absorbance was measured at 541 nm. The hemolysis rate of each group of samples was calculated: Experimental results analysis: Figure 1 It can be seen from the LB plates of each group that the number of Staphylococcus aureus colonies in the G5 group is less than that in the G1-G4 group and the G6 group. Among them, the colony distribution in the G7 group is the least. Through the statistical analysis of colony counting, the colony survival rate of the G5 group was 31.18±11.82%, and the colony survival rate of the G7 group was 3.66±1.71%. Through the bacterial Live / Dead staining experiment, it can be seen that compared with other groups, the G5 group and the G7 group showed obvious red fluorescence, while the G7 group showed significant red fluorescence and weak green fluorescence, indicating that the G7 group had the best antibacterial effect.

[0034] The CCK-8 assay was used to evaluate the cytotoxicity of blank AIE-DNA hydrogels to HaCat and Raw264.7 cells. Figure 2 As shown, different concentrations of hydrogel extracts were co-incubated with HaCat and Raw264.7 cells for 24 h, and the cell viability was maintained above 95%, with no significant difference compared with the control group (0 μM). From the live-dead cell staining experiment, it can be seen that almost all cells in the control group and the blank AIE-DNA hydrogel group showed green fluorescence, indicating that the blank AIE-DNA hydrogel extract did not affect the activity of the cells after incubation for 2 days. The hemocompatibility of the blank AIE-DNA hydrogel was evaluated by hemolysis experiment. Significant color differences could be observed between the blank AIE-DNA hydrogel group and the positive control (Triton X-100), and the red blood cells treated with different concentrations of blank AIE-DNA hydrogels showed only slight hemolysis (<4%).

[0035] Example 5 Establishment of Mouse Wound Model 1. Wound healing effect C57 male mice were selected to establish an infectious wound model. After the mice were anesthetized with isoflurane, the back hair was shaved and the back skin was cleaned with 75% alcohol solution. A skin sampler (6 mm in diameter) was used to create a full-thickness skin excision wound on the back of the mice. Staphylococcus aureus suspension (1 μL, 1×10 8CFU / mL) were collected and housed in cages after surgery and divided into 7 groups, namely G1-G7. Group G1 (control group): PBS; Group G2: L-arginine (150μM); Group G3: ginseng exosomes (5μg / mL); Group G4: blank AIE-DNA hydrogel (AIE-DNA hydrogel: 100μM); Group G5: blank AIE-DNA hydrogel (AIE-DNA hydrogel: 100μM) + laser; Group G6: drug-loaded AIE-DNA hydrogel (L-arginine: 150μM, ginseng exosomes: 5μg / mL, AIE-DNA hydrogel: 100μM); Group G7: drug-loaded AIE-DNA hydrogel (L-arginine: 150μM, ginseng exosomes: 5μg / mL, AIE-DNA hydrogel: 100μM) + laser. The dosage of each group was 10 μL / animal, and the control group used the same volume of PBS. Among them, the G5 and G7 groups were irradiated with 680 nm laser for 10 min. The drugs in each group were dripped into the wound, and the wound site was kept clean. The drugs were administered on the 1st, 3rd, and 5th day after the model was established, and the wound healing was observed by taking photos on the 2nd, 4th, 6th, and 8th day. The wound area of ​​each group was calculated using Image J software, and the wound healing rate of each group was calculated using the following method: 2. HE and Masson staining After 8 days of injury, skin wound samples of mice in each group were taken and immersed in 4% paraformaldehyde fixative. The skin wound tissues of mice were made into paraffin sections. After HE and Masson staining, they were observed and photographed under an inverted fluorescence microscope.

[0036] 3. In vivo safety testing Safety tests included organ HE staining (for detecting organ toxicity), serum biochemical tests (for detecting liver and kidney toxicity) and whole blood routine tests (for detecting blood safety in vivo). Healthy C57 mice were divided into three groups, namely Contol group, A-hydrogel group (blank AIE-DNA hydrogel group) and LGAH group (drug-loaded AIE-DNA hydrogel group). 10 μL of PBS, blank AIE-DNA hydrogel (AIE-DNA hydrogel: 100 μM) and drug-loaded AIE-DNA hydrogel (L-arginine: 150 μM, ginseng exosomes: 5 μg / mL, AIE-DNA hydrogel: 100 μM) were injected subcutaneously. The organs of mice in each group were immersed in 4% paraformaldehyde fixative to prepare paraffin sections. HE staining was performed. The fresh blood of mice was placed at 4°C overnight, centrifuged at 3000 rpm for 5 min, the supernatant was taken, placed on ice, and biochemical tests were performed on the machine. Add the extracted fresh blood of mice into the anticoagulant tube, mix well and place on ice, and then perform routine blood test on the machine.

[0037] Experimental results analysis: Figure 3 It can be seen that there is no significant difference between the G1 group and the G2 group, and the wound healing rates are approximately 65.57% and 63.99%, respectively. Compared with the G1 group and the G2 group, the other groups have better wound healing effects, among which the G7 group has the best wound healing effect. On the 8th day, the wounds infected with Staphylococcus aureus in the G7 group were almost completely healed, and the wound healing rate was as high as 92.16%. From the results of HE staining, the degree of wound healing in each group was different. Among them, the wound healing effect of the G7 group was the best, and the length and depth of the wound and the area of ​​inflammatory cell infiltration were the smallest. Masson staining is a classic collagen fiber staining method, which is mainly used to distinguish collagen fibers from muscle fibers. After staining, the muscle fibers of the tissue are red and the collagen fibers are blue. The blue spots in the wound tissue of the G7 group were the deepest and the widest, indicating that there were a large number of collagen fibers in the tissue of the G7 group. The results show that the G7 group has the best ability to promote the healing of infectious wounds.

[0038] The biosafety of blank AIE-DNA hydrogel and drug-loaded AIE-DNA hydrogel was evaluated by H&E staining of the main organs of mice, blood routine and serum biochemical analysis. Figure 4 shown.

[0039] from Figure 4 It can be seen that after treatment with different hydrogels, the HE staining of the main organs of mice showed no obvious histological damage and no obvious immune cell infiltration. The results of routine blood examination showed that there were no abnormalities in the parameters of white blood cells, red blood cells and other blood cells. The biochemical indicators related to liver and kidney function (AST, ALT, UA, CREA) in the serum of each group of mice were detected. The four indicators of the control group and the hydrogel group were all within the reference range of normal mice, indicating that the blank AIE-DNA hydrogel and the drug-loaded AIE-DNA hydrogel have good biosafety.

Claims

1. A ROS-responsive DNA photo-controlled hydrogel with AIE characteristics, characterized in that: It is formed by the polymerization reaction of AIE-linker and three DNA single strands: Y-1a, Y-1b, and Y-1c; The AIE-linker is obtained by connecting a linker chain having an amino group and a disulfide bond with an AIE fluorescent molecule having a carboxyl group through an amidation reaction; The Y-1a has a nucleotide sequence as shown in SEQ ID NO: 1; The Y-1b has a nucleotide sequence as shown in SEQ ID NO: 2; The Y-1c has a nucleotide sequence as shown in SEQ ID NO: 3; The linker chain has a nucleotide sequence as shown in SEQ ID NO:

4.

2. The ROS-responsive DNA photo-controlled hydrogel with AIE characteristics according to claim 1, characterized in that: The molar ratio of Y-1a, Y-1b, Y-1c and AIE-linker is 2:2:2:

1.

3. The ROS-responsive DNA photo-controlled hydrogel with AIE characteristics according to claim 1, characterized in that: The AIE fluorescent molecule is a molecule TPA-COOH having a triphenylamine structure and a carboxyl group.

4. The ROS-responsive DNA photo-controlled hydrogel with AIE characteristics according to claim 3, characterized in that: The preparation method of AIE-linker is as follows: TPA-COOH was mixed with EDC and NHS in a molar ratio of 1:10:10 and stirred at room temperature for 2 h to activate the carboxyl group of TPA-COOH; The linker solution was then added dropwise and the reaction was continued for 24 hours. The reaction mixture was dialyzed for 2 days and then freeze-dried.

5. The multifunctional DNA photo-controlled hydrogel based on the ROS-responsive DNA photo-controlled hydrogel with AIE characteristics according to claim 1, characterized in that: The invention relates to a ROS-responsive DNA light-controlled hydrogel including a composite nano-drug and AIE characteristics loaded with the composite nano-drug.

6. The multifunctional DNA light-controlled hydrogel according to claim 5, characterized in that: The composite nanomedicine comprises plant-derived exosomes and an NO donor.

7. The multifunctional DNA light-controlled hydrogel according to claim 6, characterized in that: The multifunctional DNA light-controlled hydrogel is used for preparing a medicine for treating infectious wounds; and the exosomes are ginseng exosomes.

8. The multifunctional DNA light-controlled hydrogel according to claim 7, characterized in that: The preparation method of ginseng exosomes is achieved by the following steps: 1) Wash and dry the ginseng, add PBS solution and stir evenly, filter with gauze to obtain ginseng liquid; perform gradient centrifugation on the ginseng liquid to obtain ginseng crude extract; perform ultra-high speed centrifugation on the ginseng crude extract, discard the supernatant, resuspend the precipitate with PBS solution to obtain ginseng crude exosomes; 2) After ultra-high-speed centrifugation of crude ginseng exosomes with sucrose solutions of different concentrations, the supernatant was discarded and the precipitate was resuspended with PBS solution; after ultra-high-speed centrifugation again, the supernatant was discarded and the precipitate was resuspended with PBS solution to obtain ginseng exosomes with an average particle size of 134.22-156.5 nm, which were packaged and stored in a -80°C refrigerator.

9. The multifunctional DNA light-controlled hydrogel according to claim 6, characterized in that: The NO donor is L-arginine.

10. The method for preparing the multifunctional DNA light-controlled hydrogel according to claim 5, characterized in that: The ROS-responsive DNA photocontrollable hydrogel with AIE characteristics, exosomes and NO donor were mixed to obtain the target multifunctional DNA photocontrollable hydrogel.

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