Preparation method and application of composite drug-loaded microgel spheres with controlled drug release

Abstract

The present invention provides a method for preparing a composite drug-loaded microgel ball with controllable drug release and its application, wherein the preparation method comprises the following steps: preparing Gelma hydrogel; preparing AuBTO; preparing composite drug-loaded microgel balls. The composite drug-loaded microgel balls contain a scaffold Gelma seaweed ball, a therapeutic drug, verbascoside, a piezoelectric bactericidal preparation AuBTO, and an anti-inflammatory preparation PDA. Verascoside can regulate the balance of estrogen in women, effectively improve endometrial damage, and promote endometrial regeneration. AuBTO is a strong dielectric compound material with a high dielectric constant and low dielectric loss. By encapsulating the drug inside or modifying the surface of barium titanate nanoparticles, targeted release and delivery can be achieved to improve the effect and selectivity of the drug or gene. In addition, the metal nanoparticles can associate with the surface of bacteria, dissolve substances by releasing toxic ions that damage enzyme functions or DNA, or produce ROS that cause oxidative stress, thereby exerting antibacterial activity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for preparing composite drug-loaded microgel spheres capable of controlling drug release and application thereof. Background Art

[0002] The uterus is an important organ in the female reproductive system, playing a key role in nurturing the embryo and supporting the growth and development of the fetus. Endometrial damage refers to a disease caused by damage to the basal layer of the endometrium and endometrial repair disorders caused by various pathogenic factors. It can often lead to intrauterine adhesions, thin endometrium, and pelvic inflammatory disease. The clinical manifestations are symptoms such as menstrual disorders, lower abdominal pain, infertility, and recurrent miscarriage, which seriously affect women's physical and mental health. Uterine cavity operation, intrauterine infection, genetic factors, endometrial diseases, etc. are closely related to the occurrence of endometrial damage. Current methods for treating endometrial damage include drug therapy and surgical treatment. Drug treatment usually includes hormone therapy, such as oral contraceptives or other hormonal drugs, to regulate the menstrual cycle and relieve symptoms. Surgical treatment includes curettage and curettage, endometrial ablation, etc., which are used to remove abnormal endometrial tissue.

[0003] However, these treatments also have certain limitations and cannot meet the needs of clinical treatment. On the one hand, although drug therapy, such as large amounts of estrogen stimulation, can promote the regeneration and re-epithelialization of the endometrium, there are still certain problems, such as the lack of standard doses or regimens. On the other hand, drug therapy may cause side effects, such as hormone drugs may cause hormonal imbalance or other adverse reactions. Surgical treatment may be accompanied by surgical risks, and may not be effective for some endometrial injuries with more complex causes, and is ineffective for patients with severe intrauterine adhesions. Therefore, existing strategies are difficult to effectively repair the endometrium, and there is an urgent need to develop a treatment method to repair endometrial damage, avoid intrauterine adhesions and infections, and thus improve pregnancy outcomes. Summary of the invention

[0004] The purpose of the present invention is to solve the technical problem that the existing treatment methods are difficult to effectively repair the endometrium and there is an urgent need to develop a treatment method to repair endometrial damage. The present invention provides a composite drug-loaded microgel ball with controlled drug release. The composite drug-loaded microgel ball in the present invention contains a scaffold Gelma seaweed ball, a therapeutic drug verbascoside, a piezoelectric bactericidal preparation AuBTO, and an anti-inflammatory preparation PDA. Among them, verbascoside is the phenylethanol glycoside monomer compound with the highest content among the active ingredients of the genus Cistanche of the Orobanchaceae family. The 2020 edition of the Chinese Pharmacopoeia contains verbascoside as an indicator component for controlling the quality of Cistanche medicinal materials. In recent years, studies have found that Cistanche, known as the "desert ginseng", has the effect of plant hormones and can regulate the balance of estrogen in women. And clinical trials have shown that the famous old Chinese medicine prescription with Cistanche as the main medicine can rebuild the balance of the reproductive endocrine environment and improve the reproductive function indicators of rats. In this study, verbascoside can effectively improve endometrial damage and promote endometrial regeneration. AuBTO is a strong dielectric compound material with high dielectric constant and low dielectric loss. By encapsulating drugs inside barium titanate nanoparticles or modifying their surface, targeted release and delivery can be achieved to improve the effect and selectivity of drugs or genes. In addition, metal nanoparticles can associate with the surface of bacteria, dissolve substances by releasing toxic ions that damage enzyme function or DNA, or produce ROS that cause oxidative stress, thus exerting antibacterial activity. PDA is also considered to be an effective ROS scavenger in a variety of inflammatory diseases, which can effectively remove reactive oxygen species and normalize the antioxidant status.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for preparing composite drug-loaded microgel spheres with controllable drug release, characterized in that it comprises the following steps:

[0007] S1: Preparation of Gelma hydrogel;

[0008] S2: Preparation of AuBTO:

[0009] 20 mM BTO was mixed with 1.6 mL NH 3 · H 2 O in an alkaline environment with a pH value of 11;

[0010] Add 4 mL of tetrachloroauric acid hydrogen trihydrate (HAuCl4·3H2O) to the above solution, where Au 3+ Concentration 10mg mL -1 ;

[0011] The suspension was vigorously stirred at 80 °C for 1 h, washed, dried, and calcined at 300 °C for 1 h;

[0012] S3: Preparation of composite drug-loaded microgel spheres:

[0013] Gelma hydrogel, verbascoside, and AuBTO were mixed in pure water at 60°C and treated with ultrasound after dissolution;

[0014] The drug-loaded microgel beads are titrated using a microfluidic device; the dripping composite drug-loaded microgel beads are irradiated using an ultraviolet lamp;

[0015] Remove the oil phase: add the drug-loaded microgel spheres wrapped in liquid paraffin oil into pure water, stir and centrifuge at 3500g for 3 minutes, and collect the precipitate;

[0016] The oil phase was removed repeatedly to obtain clean drug-loaded microgel spheres;

[0017] The obtained drug-loaded microgel spheres were added into the PDA solution, stirred for reaction for 30 minutes, and then washed with pure water and freeze-dried to obtain composite drug-loaded microgel spheres.

[0018] Preferably, the specific steps of S1 are as follows:

[0019] 1) Add 10 g of Gelatin to 100 mL of DPBS or pure water, stir at 50°C for 3 h;

[0020] After Gelatin was completely dissolved, 6 mL of methacrylic anhydride (MA) was added dropwise into the bottle at 50 °C and stirred for 3 h;

[0021] The reacted solution was poured into a 50 mL centrifuge tube and centrifuged at 3500 g for 3 min to remove the unreacted MA;

[0022] Pour the supernatant into a large beaker and dilute with 200 mL of 40°C pure water;

[0023] The diluted solution was poured into a 12-14 kDa standard grade dialysis bag and dialyzed in heated 60°C pure water for 7 days. The pure water was changed twice a day for the first two days and at least once a day thereafter.

[0024] After dialysis, the solution was filtered with a 0.2 μm filter and pre-frozen at -20°C overnight;

[0025] The synthesized material was freeze-dried in a lyophilizer for 2-4 days, dried thoroughly and stored in a desiccator.

[0026] Preferably, S3 also includes the preparation of a microfluidic device, and the steps are as follows:

[0027] A glass microfluidic device was made by coaxially assembling and fixing glass tubes of different thicknesses on a glass slide;

[0028] The inner capillary is used to introduce a dispersed phase, which is a drug-loaded microgel solution, and the outer capillary is used to introduce a continuous phase, which is liquid paraffin oil containing 5% span.

[0029] Preferably, the titration method of the drug-loaded microgel spheres in S3 is as follows:

[0030] The drug-loaded microgel solution is cut into droplets by the liquid paraffin oil solution at the outlet of the inner tube. By adjusting the flow rate of the two-phase solution, the size of the droplets and the corresponding pore size can be controlled. As the dispersed phase velocity increases, the droplet diameter increases significantly, while the continuous phase velocity is inversely related to the droplet diameter.

[0031] Preferably, after the gelatin in S1 is completely dissolved, NaHCO3 or NaOH needs to be added dropwise to the stirred solution before adding MA to adjust the pH to between 7 and 8; in addition, after the dialysis of Gelma is completed, the pH also needs to be adjusted to about 7.2.

[0032] Preferably, the final concentration of Gelma in S1 is 10%, the concentration of AuBTO is 500 μg / mL, and the concentration of verbascoside is 200 μg / mL.

[0033] Preferably, the diameter of the inner capillary is 300 mm, the diameter of the outer capillary is 900 mm, and the ratio of the flow rate of the outer phase to the flow rate of the inner phase is 1:10.

[0034] Preferably, the preparation method of the PDA solution in S3 is as follows: dopamine DA (0.5 mg / mL) is dissolved in an alkaline solution of 1 M Tris-HCl pH=8.0 and stirred at 60° C. for 24 hours to prepare a PDA solution.

[0035] The present application also provides a composite drug-loaded microgel sphere with controllable drug release, which is prepared using the above-mentioned preparation method.

[0036] The present application also provides the use of the composite drug-loaded microgel spheres with controllable drug release prepared by the above-mentioned preparation method in the preparation of drugs for preventing and treating endometrial damage.

[0037] Compared with the prior art, this application has the following beneficial effects:

[0038] 1. The verbascoside in the composite drug-loaded microgel spheres with controlled drug release prepared in the present application has estrogenic effect and can effectively improve endometrial damage. In addition, under the piezoelectric effect of BTO, ultrasound can slowly control the release of verbascoside;

[0039] 2. In the composite drug-loaded microgel spheres with controlled drug release prepared in the present application, Au ions have high bactericidal activity and low toxic side effects, which can provide more effective and safe treatment options;

[0040] 3. The PDA solution used in the composite drug-loaded microgel spheres with controlled drug release prepared in the present application is an effective ROS scavenger in a variety of inflammatory diseases, has antioxidant effects, and can help alleviate or reduce inflammatory responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a material characterization diagram of the composite drug-loaded microgel spheres in one embodiment of the present invention;

[0042] Figure 2 This is a diagram showing the biocompatibility and anti-inflammatory effect of the composite drug-loaded microgel spheres in one embodiment of the present invention;

[0043] Figure 3 This is a diagram showing the drug release effect of the composite drug-loaded microgel spheres in one embodiment of the present invention;

[0044] Figure 4 This is a diagram showing the bactericidal effect of the composite drug-loaded microgel spheres in one embodiment of the present invention;

[0045] Specifically, Figure 1 This is a material characterization diagram of the composite drug-loaded microgel spheres in an embodiment of the present invention. Figure 1 A: Scanning electron micrograph of composite drug-loaded microgel spheres. Figure 1 B, C: PDA molecules attached to the surface of composite drug-loaded microgel spheres. Figure 1 DI: Elemental analysis of composite drug-loaded microgel spheres (O; Au; Ba; Ti).

[0046] Figure 2 Graph showing biocompatibility and anti-inflammatory effects in an embodiment of the present invention. Figure 2 A: Fluorescence images of cell life and death under the intervention of culture media with different components. Figure 2 B: ROS fluorescence image under the intervention of culture medium with different components.

[0047] Figure 2 C: Cell survival rate under the intervention of culture medium with different components. Figure 2 D, E: Expression of inflammatory factors TNF-a and IL-1β in cell supernatants under the intervention of different composition culture media

[0048] Figure 3 This is a drug release diagram of verbascoside from the composite drug-loaded microgel spheres in an embodiment of the present invention.

[0049] Figure 4 It is a diagram of the sterilization effect in an embodiment of the present invention. Figure 4A: Escherichia coli and Staphylococcus aureus agar plate renderings. Figure 4 B: Scanning electron microscope images of Escherichia coli and Staphylococcus aureus silicon wafer. Figure 4 C: OD value levels of LB culture medium of Enterobacteriaceae and Staphylococcus aureus treated with different concentrations of AuBTO. DETAILED DESCRIPTION

[0050] The present invention is further described in detail below in conjunction with specific embodiments.

[0051] A method for preparing composite drug-loaded microgel spheres with controllable drug release comprises the following steps:

[0052] S1: Preparation of Gelma hydrogel;

[0053] In one embodiment, the specific preparation method of Gelma is as follows:

[0054] 1) Add 10 g of Gelatin into 100 mL of DPBS (or pure water) at 50°C and stir for 3 h.

[0055] 2) After Gelatin is completely dissolved, add 6 mL of methacrylic anhydride (MA) dropwise into the bottle at 50°C and stir for 3 h;

[0056] 3) The reacted solution was poured into a 50 mL centrifuge tube and centrifuged at 3500 g for 3 min to remove the unreacted MA;

[0057] 4) Pour the supernatant into a large beaker and dilute with 200 mL of 40°C pure water;

[0058] 5) The diluted solution was poured into a 12-14 kDa standard grade dialysis bag and dialyzed in heated 60°C pure water for 7 days. The pure water was changed twice a day for the first two days and at least once a day thereafter;

[0059] 6) After dialysis, filter with a 0.2 μm filter and pre-freeze at -20°C overnight;

[0060] 7) Freeze-dry the synthesized material in a freeze dryer for 2-4 days, dry thoroughly and store in a desiccator.

[0061] S2: Preparation of AuBTO;

[0062] 1) Mix 20 mM BTO with 1.6 mL NH3·H2O in an alkaline environment with a pH value of 11.

[0063] 2) Add 4 mL of tetrachloroauric acid trihydrate (HAuCl4·3H2O) to the above solution, where Au 3+ Concentration 10mg mL -1 .

[0064] 3) The suspension was vigorously stirred at 80°C for 1 hour, washed, dried, and calcined at 300°C for 1 hour.

[0065] S3: Preparation of composite drug-loaded microgel spheres.

[0066] In one embodiment, the composite drug-loaded microgel spheres are prepared by mixing using microfluidic chip technology, and the polydopamine layer is wrapped around the outside of the drug-loaded microgel spheres.

[0067] In one embodiment, the method for preparing the composite drug-loaded microgel spheres comprises the following steps: preparing a drug-loaded microgel solution, preparing a microfluidic device, titrating the drug-loaded microgel spheres, solidifying the drug-loaded microgel spheres, removing the oil phase, and preparing the drug-loaded microgel spheres.

[0068] The specific steps are as follows:

[0069] 1) Preparation of drug-loaded microgel solution: Gelma hydrogel, verbascoside, and AuBTO were mixed in pure water at 60°C, dissolved, and then ultrasonically treated for 10 min to promote full dissolution and mixing of the solution;

[0070] 2) Preparation of microfluidic device: Glass tubes of different thicknesses were coaxially assembled and fixed on a glass slide to make a glass microfluidic device. The inner capillary was used to introduce the dispersed phase (i.e., drug-loaded microgel solution), and the outer capillary was used to introduce the continuous phase (i.e., liquid paraffin oil containing 5% span).

[0071] 3) Titration of drug-loaded microgel spheres: The drug-loaded microgel solution is cut into droplets by the liquid paraffin oil solution at the outlet of the inner tube. By adjusting the flow rate of the two-phase solution, the size of the droplets and the corresponding pore size can be controlled. With the increase of the dispersed phase velocity, the droplet diameter increases significantly, while the continuous phase velocity is inversely related to the droplet diameter.

[0072] 4) Curing of drug-loaded microgel spheres: The dripped composite drug-loaded microgel spheres are immediately irradiated with ultraviolet light to accelerate the curing speed.

[0073] 5) Removing the oil phase: Add the drug-loaded microgel spheres wrapped in liquid paraffin oil into pure water, stir, centrifuge at 3500g for 3 min, and collect the precipitate;

[0074] 6) Repeat step 5 about 10 times to obtain clean drug-loaded microgel spheres;

[0075] 7) Preparation of composite drug-loaded microgel spheres: Add the drug-loaded microgel spheres to the PDA solution, stir and react for 30 minutes, then wash with pure water and freeze-dry to obtain composite drug-loaded microgel spheres.

[0076] The present application also provides a composite drug-loaded microgel sphere with controllable drug release, and the composite drug-loaded microgel sphere is prepared using the above-mentioned preparation method.

[0077] In addition, the present application also provides an application of composite drug-loaded microgel spheres with controllable drug release in the preparation of drugs for preventing and treating endometrial damage.

[0078] The following is an explanation of the above content in combination with specific verification experiments:

[0079] Example 1: Evaluation of the controlled drug release performance of composite drug-loaded microgel spheres:

[0080] Experimental methods:

[0081] 200 mg of drug-loaded microspheres were accurately weighed and placed in 1 mL of PBS. One group was subjected to ultrasound with a 30 MHz ultrasound probe, and the other group was left to stand normally. 100 μL of supernatant was regularly drawn at four time points: 0, 5, 10, and 15 min. The drug concentration was determined by UV spectrophotometer, and the in vitro drug release curve was drawn. According to the literature, 332 nm was selected as the detection wavelength of verbascoside.

[0082] Experimental results:

[0083] See also Figure 2 , under the assistance of ultrasound, the composite drug-loaded microgel spheres have good controlled drug release performance.

[0084] Example 2: Verification of biocompatibility and anti-inflammatory effect of composite drug-loaded microgel spheres:

[0085] Experimental methods:

[0086] 1. Detect cell viability using CCK8 method

[0087] Soak 100 mg of seaweed balls with different compositions of Gelma, Gelma@Acteoside, Gelma@Acteoside@AuBTO, and Gelma@Acteoside@AuBTO@PDA in 10 mL of complete culture medium, place in a cell culture incubator at 37°C for 24 hours, take the supernatant, and filter it with a 0.2 μm filter to obtain culture medium with different compositions.

[0088] The cells were plated at 1.0 × 10 9 The cells were inoculated in a 96-well plate at a density of 1.50 μg / mL. After incubation overnight, the cells were co-cultured with culture media of different compositions for 24 hours, and then 10 μL of CCK-8 was added to each well and incubated at 37°C for half an hour. Finally, the absorbance at 450 nm was measured to calculate the cell activity.

[0089] 2. Detect cell death and viability using immunofluorescence

[0090] The cells were co-cultured with the above culture medium for 24 hours, the culture medium was discarded, and the cells were washed once with PBS. An appropriate volume of CalceinAM / PI detection working solution was added and incubated at 37°C in the dark for 30 minutes. After the incubation, the staining effect was observed under a fluorescence microscope (CalceinAM of live cells is green fluorescence; PI of dead cells is red fluorescence).

[0091] 3. Reactive oxygen detection kit quantitatively detects intracellular reactive oxygen levels under different treatments

[0092] Prepare cell suspension and count cells; according to the appropriate number of cells to be plated (about 1×10 4 ), inoculate the cell suspension (1mL / well) in a six-well plate, and place the culture plate in an incubator for 4 hours; LPS stimulation was performed on the positive control wells, and the culture medium with different components was added to the intervention wells, and the culture plate was incubated in an incubator for 24 hours; DCFH-DA was diluted with serum-free culture medium at 1:1000 to a final concentration of 10 micromol / L; the cell culture medium was removed, and 1mL of the diluted DCFH-DA was added to each well to fully cover the cells. Incubate in a 37℃ cell culture incubator for 20 minutes; wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA that did not enter the cells. Take pictures under a fluorescence microscope.

[0093] 4. ELISA detection of intracellular inflammatory factor levels stimulated by LPS under different culture medium treatments

[0094] The cells were plated at 1.0 × 10 9 The cells were inoculated in a 96-well plate at a density of 1.50 μg / mL. After one night of incubation, the cells were co-cultured with culture media with different components for 24 hours, the supernatant was aspirated, and the levels of inflammatory factors (TNF-a, IL-1β) in the cell supernatant were detected by ELISA kit.

[0095] Experimental results:

[0096] See also Figure 2 A, C, the composite drug-loaded microgel spheres have good biocompatibility and can maintain high cell activity when co-cultured with cells;

[0097] like Figure 2 As shown in B, the composite drug-loaded microgel spheres have good reactive oxygen scavenging ability;

[0098] In addition, please refer to Figures D and E. The composite drug-loaded microgel spheres have a good effect in clearing inflammation, and the levels of inflammatory factors in the supernatant of the intervened cells are reduced.

[0099] Example 3: Verification of the bactericidal effect of composite drug-loaded microgel spheres:

[0100] Experimental methods:

[0101] 1. Plate count method

[0102] The bactericidal effect of AuBTO was evaluated by plate coating experiment, using Escherichia coli and Staphylococcus aureus as typical models of Gram-negative bacteria and Gram-positive bacteria, respectively. The activated Staphylococcus aureus and Escherichia coli were mixed with LB medium and diluted to 1×10 6 CFU mL -1 The bacterial solution sample was mixed with 1×10 6 CFU mL -1 The bacteria were incubated together for 20 minutes, and 50 μL of the bacterial turbid solution was spread onto the LB medium in the culture dish using a triangular glass spreading rod. The culture dish was incubated at 37°C for 24 hours, and the culture dish was photographed and recorded. At the same time, a bacterial culture dish without AuBTO solution was used as a control group, and the same experimental operation was performed.

[0103] 2. ELISA

[0104] To further study the antibacterial ability, AuBTO with different concentrations was placed in 4 mL of 400 μL bacterial suspension (1×10 6 CFU mL -1 ) in a sterile LB medium solution and shake the mixture in a constant temperature shaker at 37°C. After 6 hours, 200 μL of the suspension was collected and the absorbance (OD) was recorded at 570 nm by a microplate reader. The higher the OD value, the more turbid the bacterial solution.

[0105] 3. Silicon wafer scanning electron microscope detection method

[0106] The silicon wafer was placed on a 96-well plate, and 200 μL of the intervention Escherichia coli and Staphylococcus aureus solution was drawn into the 96-well plate, centrifuged (3000-4000 rpm), the supernatant was removed, and an appropriate amount of PBS was added to wash three times. The bacteria were gently suspended, centrifuged, and the supernatant was removed. 2.5% glutaraldehyde was used for fixation for 3 hours, centrifuged, washed three times with PBS, and the supernatant was removed. Dehydration was performed on the sample in a gradient of 30%, 50%, 70%, 80%, and 90% ethanol aqueous solution, each step was 15 minutes, centrifuged, and dehydrated in anhydrous ethanol for 15 minutes, twice, and the supernatant was removed. The sample was then placed in a mixed solution of ethanol and tert-butanol at a ratio of 1:1 for 15 minutes, the supernatant was removed, and then the alcohol was replaced with pure tert-butanol twice, each time for 15 minutes, and the supernatant was removed. The 96-well plate containing the sample was frozen at -80°C, and then placed in a freeze dryer for freeze drying. After sufficient drying, the silicon wafer was gold-plated and observed under an electron microscope.

[0107] See also Figure 4 A, C, compared with the control group, the survival rate of Staphylococcus aureus and Escherichia coli treated with AuBTO decreased, which showed a certain bactericidal ability, and the bactericidal effect showed a trend of increasing concentration;

[0108] See also Figure 4 B, Compared with the control group, the Staphylococcus aureus and Escherichia coli treated with AuBTO were attached, broken and shrunk by AuBTO wrapping.

[0109] The above verification experiments have proved that the composite drug-loaded microgel spheres provided in the present application have potent bactericidal and anti-inflammatory effects, and have the effect of controlling drug release, thereby treating endometrial damage.

[0110] In summary, the present application provides a composite drug-loaded microgel ball with controlled drug release, which contains a scaffold Gelma seaweed ball, a therapeutic drug, verbascoside, a piezoelectric bactericidal preparation AuBTO, and an anti-inflammatory preparation PDA. Among them, verbascoside has the effect of plant hormones, can regulate the balance of estrogen in women, can effectively improve endometrial damage, and promote endometrial regeneration. AuBTO is a strong dielectric compound material with a high dielectric constant and low dielectric loss. The drug is wrapped inside or surface-modified with barium titanate nanoparticles, which can achieve targeted release and delivery to improve the effect and selectivity of drugs or genes. In addition, metal nanoparticles can be associated with the surface of bacteria, dissolve substances by releasing toxic ions that damage enzyme function or DNA, or produce ROS that cause oxidative stress, and play an antibacterial activity. PDA, as an effective ROS scavenger in a variety of inflammatory diseases, can effectively remove reactive oxygen and normalize the antioxidant state. In summary, the composite drug-loaded microgel ball provided in the present application has the effects of controlling drug release, sterilization and anti-inflammatory.

Claims

1. A method for preparing composite drug-loaded microgel spheres with controlled drug release, characterized in that: The following steps are involved: S1: Preparation of Gelma hydrogel; S2: Preparation of AuBTO: 1) 20 mM BTO and 1.6 mL NH 3 · H 2 O were mixed in an alkaline environment with a pH value of 11 to form a mixed solution; 2) Add 4 mL of tetrachloroauric acid trihydrate (HAuCl4·3H2O) to the above mixed solution, where Au 3+ Concentration 10mg mL -1 ; 3) the suspension was vigorously stirred at 80°C for 1 hour, washed, dried, and calcined at 300°C for 1 hour; S3: Preparation of composite drug-loaded microgel spheres: Gelma hydrogel, verbascoside, and AuBTO were mixed in pure water at 60°C and treated with ultrasound after dissolution; The drug-loaded microgel beads are titrated using a microfluidic device; the dripping composite drug-loaded microgel beads are irradiated using an ultraviolet lamp; The preparation steps of the microfluidic device are as follows: A glass microfluidic device was made by coaxially assembling and fixing glass tubes of different thicknesses on a glass slide; The inner capillary is used to introduce the dispersed phase, which is a drug-loaded microgel solution, and the outer capillary is used to introduce the continuous phase, which is liquid paraffin oil containing 5% span; the inner capillary diameter is 300 mm, and the outer capillary diameter is 900 mm; the ratio of the outer phase flow rate to the inner phase flow rate is 1:10; The titration method of the drug-loaded microgel spheres is as follows: The drug-loaded microgel solution was cut into droplets by the liquid paraffin oil solution at the outlet of the inner tube. The droplet size and the corresponding pore size can be controlled by adjusting the flow rate of the two-phase solution. With the increase of the dispersed phase velocity, the droplet diameter increased significantly, while the continuous phase velocity was inversely related to the droplet diameter. Remove the oil phase: add the drug-loaded microgel spheres wrapped in liquid paraffin oil into pure water, stir and centrifuge at 3500g for 3 minutes, and collect the precipitate; The oil phase was removed repeatedly to obtain clean drug-loaded microgel spheres; The obtained drug-loaded microgel spheres were added into the PDA solution, stirred for reaction for 30 minutes, and then washed with pure water and freeze-dried to obtain composite drug-loaded microgel spheres.

2. The method for preparing a composite drug-loaded microgel sphere with controlled drug release according to claim 1, characterized in that: The specific steps of S1 are as follows: 1) Add 10 g of Gelatin to 100 mL of DPBS or pure water, stir at 50°C for 3 h; 2) After Gelatin is completely dissolved, add 6 mL of methacrylic anhydride (MA) dropwise into the bottle at 50°C and stir for 3 h; 3) The reacted solution was poured into a 50 mL centrifuge tube and centrifuged at 3500 g for 3 min to remove the unreacted MA; 4) Pour the supernatant into a large beaker and dilute with 200 mL of 40°C pure water; 5) The diluted solution was poured into a 12-14 kDa standard grade dialysis bag and dialyzed in heated 60°C pure water for 7 days. The pure water was changed twice a day for the first two days and at least once a day thereafter; 6) After dialysis, filter with a 0.2 μm filter and pre-freeze at -20°C overnight; 7) Freeze-dry the synthesized material in a freeze dryer for 2-4 days, dry thoroughly and store in a desiccator.

3. The method for preparing a composite drug-loaded microgel sphere with controlled drug release according to claim 1, characterized in that: After the gelatin in S1 is completely dissolved, NaHCO3 or NaOH needs to be added dropwise to the stirred solution before adding MA to adjust the pH to between 7 and 8; in addition, after the dialysis of Gelma is completed, the pH also needs to be adjusted to about 7.

2.

4. The method for preparing a composite drug-loaded microgel sphere with controlled drug release according to claim 1, characterized in that: The final concentration of Gelma in the S1 is 10%, the concentration of AuBTO is 500 μg / mL, and the concentration of verbascoside is 200 μg / mL.

5. The method for preparing a composite drug-loaded microgel sphere with controlled drug release according to claim 1, characterized in that: The preparation method of the PDA solution in S3 is as follows: 0.5 mg / mL dopamine DA is dissolved in an alkaline solution of 1 M Tris-HCl pH=8.0 and stirred at 60° C. for 24 hours to prepare a PDA solution.

6. A composite drug-loaded microgel sphere with controlled drug release, characterized in that: The method is used to prepare the product according to any one of claims 1 to 5.

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