Magnetic navigation mediated anal fistula drug sustained-release carbon dot gel and preparation method thereof

Abstract

The invention discloses a magnetic navigation mediated anal fistula drug sustained-release carbon dot gel and a preparation method thereof.The magnetic navigation mediated anal fistula drug sustained-release carbon dot gel comprises a carbon dot matrix, a magnetic navigation orientation module and an antibacterial modification component, embedded nanoscale ferroferric oxide Fe3O4 magnetic particles are dispersed in the carbon dot matrix to form a carbon dot-magnetic composite structure, and the carbon dot-magnetic composite structure is formed by the magnetic navigation orientation module and the antibacterial modification component; and the outer surface of the carbon dot-magnetic composite structure is coated with a drug sustained-release layer. Through magnetic navigation mediation, the carbon dot gel embedded with the ferroferric oxide magnetic particles can be directionally migrated to an anal fistula focus under the action of an alternating magnetic field and efficiently detained, the drug targeting efficiency is remarkably improved, compared with a traditional preparation, distribution of drugs in a non-focus area is reduced, and the whole body exposure risk is reduced; meanwhile, the drug sustained-release layer formed by sodium alginate and chitosan has pH responsiveness, long-acting release of 7-14 days can be achieved in a simulated intestinal juice environment, local drug concentration fluctuation within 48 hours is smaller than or equal to 10%, and effective treatment concentration can be continuously maintained.

Description

Technical Field

[0001] This application belongs to the field of anal fistula drug technology, specifically relating to a magnetic navigation-mediated sustained-release carbon dot gel for anal fistula drugs and its preparation method. Background Technology

[0002] Currently, traditional drug delivery methods for treating anal fistulas mainly include oral antibiotics, topical suppositories or ointments, and intra-fistula injection of ordinary gels. Oral medications need to reach the lesion through systemic blood circulation, posing a risk of systemic toxicity and resulting in low local drug concentrations. Topical suppositories and ointments are affected by the physiological environment of the rectum, and the drugs are easily lost with defecation, resulting in a short retention time (usually <6 hours), making it difficult to act on deep fistulas. Although ordinary injectable gels can be directly injected into the fistula, they lack targeting and cannot accurately remain in the core area of ​​the lesion.

[0003] Traditional techniques have significant drawbacks: First, they lack targeting, preventing drugs from reaching deep lesions and resulting in excessive drug exposure in non-lesion areas and insufficient concentration at the lesion site. Second, they lack sustained-release properties, with short drug release cycles (usually <24 hours), large fluctuations in local concentration, requiring frequent administration and leading to poor patient compliance. Third, they have limited antibacterial capabilities, lacking sustained antibacterial design and failing to inhibit the proliferation of complex bacterial flora within the fistula, easily leading to recurrent infections. Fourth, they lack biocompatibility and mechanical adaptability; some traditional gel materials are prone to causing local irritation and cannot adapt to the complex structure of the fistula to achieve stable retention. These shortcomings result in limited efficacy and significant side effects with traditional treatments, failing to meet the needs for precise and long-term treatment of anal fistulas. Summary of the Invention

[0004] This application provides a magnetic navigation-mediated sustained-release carbon dot gel for anal fistula and its preparation method, in order to solve the technical problems of frequent drug administration and unstable local concentration in traditional formulations.

[0005] To solve the above-mentioned technical problems, one technical solution adopted in this application is: a magnetic navigation-mediated sustained-release carbon dot gel for anal fistula drugs, comprising: a carbon dot matrix, a magnetic navigation orientation module, and an antibacterial modification component, wherein embedded nano-sized Fe3O4 magnetic particles are dispersed in the carbon dot matrix to form a carbon dot-magnetic composite structure, and the outer surface of the carbon dot-magnetic composite structure is coated with a sustained-release drug layer.

[0006] Furthermore, the carbon dot matrix uses biocompatible carbon dots as the core carrier, and the carbon dot surface is functionalized to contain active groups containing carboxyl or amino groups for loading drug molecules.

[0007] Furthermore, the embedded nano-sized Fe3O4 magnetic particles have a particle size range of 50-200 nm and are coated with polyethylene glycol (PEG) on their surface.

[0008] Furthermore, the drug sustained-release layer is a polymer cross-linked network wrapped around the surface of the carbon dot-magnetic composite structure, formed by the dynamic covalent cross-linking of sodium alginate and chitosan, with a cross-linking density of 0.5-2.0 mol / L.

[0009] Furthermore, the magnetic navigation orientation module is used to apply an alternating magnetic field externally, with a frequency of 10-50Hz and an intensity of 0.1-0.5T. Through the magnetic dipole interaction of the carbon dot-magnetic composite structure, the gel is driven to migrate and remain in the anal fistula lesion site in a directional manner.

[0010] Furthermore, the antibacterial modifying component includes silver ions (Ag). + Or zinc dioxide (ZnO) nanoparticles, with a mass percentage of 0.1-1.0%.

[0011] Another technical solution adopted in this application is: a method for preparing magnetically guided sustained-release carbon dot gel for anal fistula drugs, comprising:

[0012] S1. Carbon dot synthesis and functionalization: Carbon dots were prepared by reacting citric acid and urea at a mass ratio of 1:2.5 using a hydrothermal method at a temperature range of 160-200℃ for 4-10 hours. After acidification, carboxyl or amino groups were grafted onto the carbon dots via an EDC / NHS coupling reaction to obtain a surface-modified carbon dot matrix.

[0013] S2. Magnetic particle composite: Fe3O4 nanoparticles and polyethylene glycol are ultrasonically dispersed in an ethanol solution with an ultrasonic power range of 30-70W and an ultrasonic time range of 20-40 minutes. Then, they are mixed with carbon dots and formed a carbon dot-magnetic composite through electrostatic self-assembly.

[0014] S3. Drug loading and cross-linking: Metronidazole or clindamycin was dissolved in 0.01 mol / L phosphate buffer at pH 7.4, and drug loading was completed by ultrasonic-assisted diffusion and centrifugation. Sodium alginate and chitosan, both at a concentration of 2%, were dissolved in 0.1 mol / L NaCl solution to obtain a mixed solution. The pH of the mixed solution was adjusted to 6.5-7.0 with 1% calcium chloride solution to construct the cross-linking network.

[0015] S4. Construction of magnetic navigation structure: After the construction of the cross-linked network is completed, the magnetic navigation structure is constructed. The magnetic field parameters need to be adjusted according to the condition of the anal fistula lesion.

[0016] S5. Antibacterial modification: After the magnetic navigation structure is constructed, antibacterial modification is carried out, in which the corresponding antibacterial agent is determined according to the type of bacteria that infect;

[0017] S6. Mechanical property optimization: Adjust the crosslinking agent ratio and ultraviolet crosslinking according to the fistula condition to regulate the mechanical properties of the gel and obtain a sustained-release carbon dot gel for anal fistula drugs.

[0018] Furthermore, the carbon dot matrix has a particle size range of 50-100 nm, a surface carboxyl group density range of 1.2-2.0 mmol / g, an EDC of 0.2 mmol / mL, and an NHS of 0.1 mmol / mL.

[0019] Furthermore, the Fe3O4 nanoparticles had a particle size range of 50-200 nm, the surface-coated PEG had a molecular weight range of 1000-3000, and the mass ratio of carbon dots to Fe3O4 ranged from 1:1 to 1:3. The nanoparticles were allowed to self-assemble at 25 °C for 4 h to form a carbon dot-magnetic composite. The nanoparticles were gently stirred once every 30 minutes to avoid local sedimentation. After assembly, the nanoparticles were centrifuged at 5000 rpm for 5 minutes, the supernatant was discarded, and the precipitate was washed three times with deionized water to obtain the carbon dot-magnetic composite structure.

[0020] The beneficial effects of this application are as follows: Through magnetic navigation, the carbon dot gel embedded with ferric oxide magnetic particles can migrate directionally to the anal fistula lesion and remain efficiently under the action of an alternating magnetic field, significantly improving drug targeting efficiency. Compared with traditional formulations, it reduces the distribution of drugs in non-lesion areas and reduces the risk of systemic exposure. At the same time, the drug sustained-release layer formed by sodium alginate and chitosan has pH responsiveness and can achieve long-term release for 7-14 days in a simulated intestinal fluid environment. Moreover, the local drug concentration fluctuation is ≤10% within 48 hours, which can continuously maintain an effective therapeutic concentration and solve the problems of frequent administration and unstable local concentration of traditional formulations. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments.

[0022] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification.

[0023] This application provides a magnetically guided sustained-release carbon dot gel for anal fistula drugs, comprising: a carbon dot matrix, a magnetic navigation orientation module, and an antibacterial modification component, wherein embedded nano-sized Fe3O4 magnetic particles are dispersed in the carbon dot matrix to form a carbon dot-magnetic composite structure, and the carbon dot-magnetic composite structure is coated with a sustained-release drug layer.

[0024] The carbon dot matrix uses biocompatible carbon dots as the core carrier, with the carbon dot surface functionalized to contain carboxyl or amino groups for loading drug molecules. Embedded nano-sized Fe3O4 magnetic particles have a particle size range of 50-200 nm and are coated with polyethylene glycol (PEG). The drug-releasing layer is a polymer cross-linked network encapsulated on the surface of the carbon dot-magnetic composite structure, formed by dynamic covalent bonding of sodium alginate and chitosan, with a cross-linking density of 0.5-2.0 mol / L, ensuring pH-responsive drug release in simulated intestinal fluid. The magnetic navigation and orientation module applies an alternating magnetic field with a frequency of 10-50 Hz and an intensity of 0.1-0.5 T. Through the magnetic dipole interaction of the carbon dot-magnetic composite structure, it drives the gel to migrate and remain directionally at the anal fistula lesion site. The antibacterial modification component includes silver ions (Ag). + Alternatively, zinc dioxide (ZnO) nanoparticles, with a mass percentage of 0.1-1.0%, are used to inhibit local bacterial infection. By introducing a dynamic thiol-olefin click chemistry reaction, a three-dimensional reversible cross-linked network is constructed, allowing the gel to maintain shear-thinning properties at 37°C, facilitating rapid recovery of its original shape after injection. The carbon dot gel of this application achieves targeted drug delivery to deep tissues of anal fistulas via magnetic navigation, and under simulated physiological conditions, the drug release cycle is 7-14 days. Compared to traditional formulations, the targeting efficiency is improved, and the local drug concentration gradient is maintained for ≥48 hours.

[0025] This application also provides a method for preparing a magnetically guided sustained-release carbon dot gel for anal fistula drugs, comprising the following steps:

[0026] Step 1, Carbon dot synthesis and functionalization:

[0027] Carbon dots were prepared by reacting citric acid and urea at a mass ratio of 1:2.5 as precursors via a hydrothermal method at a temperature range of 160-200℃ for 4-10 hours. After acidification, carboxyl or amino groups were grafted onto the carbon dots via an EDC / NHS coupling reaction to obtain surface-modified carbon dots. The particle size of the carbon dots ranged from 50-100 nm, the surface carboxyl group density ranged from 1.2-2.0 mmol / g, the EDC concentration was 0.2 mmol / mL, and the NHS concentration was 0.1 mmol / mL.

[0028] Specifically, citric acid is analytical grade with a purity ≥99.5%. Urea is biological reagent grade with a purity ≥99%. Citric acid and urea are accurately weighed at a mass ratio of 1:2.5 and dissolved in deionized water, with a solid-liquid ratio of 1:10 (g / mL). The solution is magnetically stirred for 30 minutes until completely dissolved, forming a transparent solution.

[0029] The reaction temperature is divided into two stages: the first two hours are at 160-180℃ to promote carbon nucleation; the next two to eight hours are at 180-200℃ to control the carbon particle size and prevent carbon agglomeration due to prolonged reaction. The reaction vessel is a polytetrafluoroethylene hydrothermal reactor, and the cooling method is natural cooling to room temperature to avoid sudden cooling that could damage the carbon particle structure.

[0030] A 0.1 mol / L hydrochloric acid solution (analytical grade) was used. The pH of the carbon dot solution was adjusted to 2.0-3.0 with this hydrochloric acid solution, and the solution was magnetically stirred for 1 hour. After centrifugation at 8000 rpm for 10 minutes, the supernatant was collected to remove unreacted precursors.

[0031] EDC is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, with a purity ≥98%. NHS is N-hydroxysuccinimide, with a purity ≥98%. EDC and NHS are dissolved in carbon dot solution at a ratio of 0.2 mmol / mL:0.1 mmol / mL and reacted at 25°C in the dark for 2 hours to graft carboxyl or amino groups. Grafted carboxyl groups are used for loading basic drugs, and grafted amino groups are used for loading acidic drugs. The modified carbon dots require dialysis. The dialysis bag has a molecular weight cutoff of 3500 Da, and the dialysis time is 48 hours, with deionized water changed every 6 hours. This is to ensure the removal of free EDC and NHS.

[0032] Step 2, magnetic particle composite:

[0033] Fe3O4 nanoparticles and polyethylene glycol (PEG2000) were ultrasonically dispersed in an ethanol solution with an ultrasonic power range of 30-70W and an ultrasonic time range of 20-40 minutes. Subsequently, they were mixed with carbon dots and formed a carbon dot-magnetic composite through electrostatic self-assembly. The Fe3O4 nanoparticles had a particle size range of 50-200 nm, the PEG coating had a molecular weight range of 1000-3000, and the mass ratio of carbon dots to Fe3O4 ranged from 1:1 to 1:3. The nanoparticles were allowed to self-assemble at 25°C for 4 hours to form the carbon dot-magnetic composite, with gentle stirring every 30 minutes to avoid localized sedimentation. After assembly, the nanoparticles were centrifuged at 5000 rpm for 5 minutes, the supernatant was discarded, and the precipitate was washed three times with deionized water.

[0034] PEG with a molecular weight of 1000 is used for superficial lesions, and PEG with a molecular weight of 3000 is used for deep lesions. The purity is ≥99%. The mass ratio of PEG to Fe3O4 is 1:5. The ultrasonic dispersion parameters are 30-50W (50-100nm particles) for 20-30 minutes and 50-70W (100-200nm particles) for 30-40 minutes, ensuring that the particle dispersion index (PDI) is ≤0.2.

[0035] Step 3, Drug Loading and Crosslinking:

[0036] The medications used are commonly used antibiotics for anal fistulas, including metronidazole and clindamycin. The concentration of metronidazole is 5 mg / mL, and the concentration of clindamycin is 2 mg / mL. These medications are dissolved in phosphate-buffered saline (PBS) at pH 7.4. The PBS concentration is 0.01 mol / L, containing 0.138 mol / L NaCl and 0.0027 mol / L KCl. The PBS needs to be sterilized at 121°C for 20 minutes.

[0037] The mass ratio of drug to carbon dots varies depending on the type of drug. Metronidazole has a mass ratio of 1:5 to 1:8, as it is relatively water-soluble. Clindamycin has a mass ratio of 1:8 to 1:10, as it is relatively lipid-soluble.

[0038] The conditions for ultrasound-assisted diffusion are as follows: It is performed at 25℃, with an ultrasound power of 20-30W for metronidazole and 30-40W for clindamycin, for a duration of 10-15 minutes. This ultrasound time is controlled to avoid drug degradation. After loading, centrifuge at 3000 rpm for 5 minutes. The precipitate is then collected to determine the loading efficiency, which should be ≥85%.

[0039] After drug loading, a cross-linked network was constructed. The sodium alginate used was of low molecular weight, with a viscosity between 200-300 mPa·s. The degree of deacetylation of chitosan was ≥90%. Two mass ratios of sodium alginate to chitosan were chosen: 1:1 and 1:3. A 1:1 mass ratio resulted in a softer gel, suitable for mucosal sensitive areas. A 1:3 mass ratio resulted in a harder gel, suitable for fibrotic lesions. Both sodium alginate and chitosan were 2% (w / v) and dissolved in a 0.1 mol / L NaCl solution.

[0040] The pH of the above mixed solution was adjusted to 6.5-7.0 with a 1% (w / v) calcium chloride solution to promote the cross-linking of dynamic covalent bonds. The cross-linking time varied depending on the mass ratio of sodium alginate to chitosan. At a mass ratio of 1:1, the cross-linking time was 10-20 minutes. At a mass ratio of 1:3, the cross-linking time was 20-30 minutes. It is necessary to ensure that the swelling rate of the gel in simulated intestinal fluid at pH 7.4 remains stable at 150-180%. Excessive swelling rate can cause the gel to rupture, while insufficient swelling rate will result in slow drug release.

[0041] Step 4: Construction of the magnetic navigation structure:

[0042] After constructing the cross-linked network, the magnetic navigation structure is built, and the magnetic field parameters need to be adjusted according to the condition of the anal fistula lesion. The alternating magnetic field frequency varies depending on the depth of the lesion. For superficial anal fistulas with a lesion depth <3cm, the corresponding alternating magnetic field frequency is 10-30Hz; for deep anal fistulas with a lesion depth in the range of 3-5cm, the corresponding alternating magnetic field frequency is 30-50Hz. A higher frequency is chosen to enhance the penetration of the magnetic field.

[0043] The magnetic field strength is set in different stages. In the initial positioning stage, the magnetic field strength is 0.1-0.3T to avoid excessive magnetic field impact. In the retention stage, the magnetic field strength is 0.3-0.5T to enhance the adhesion of the gel to the lesion site. Simultaneously, the magnetic field uniformity error must be ≤±5%.

[0044] The magnetic mold design has specific parameter requirements. The NdFeB permanent magnet array used is grade N52, with a remanence between 1.48-1.52T and a size of 5cm × 5cm. The permanent magnets are arranged in a "dense at the center and sparse at the edges" pattern, with the central part corresponding to the core of the lesion. The spacing between adjacent permanent magnets is 0.5cm.

[0045] The angle between the magnetic field direction and the injection direction needs to be determined according to the fistula type. For straight fistulas, the angle is 45°±5°; for curved fistulas, the angle is between 35° and 45°. The angle needs to be adjusted according to the fistula's direction, and can be preset using CT images.

[0046] Step 5, Antibacterial Modification:

[0047] After the magnetic navigation structure is constructed, antibacterial modification is performed. The selection of the antibacterial agent depends on the type of infecting bacteria. For infections primarily caused by Gram-negative bacteria, such as those caused by Escherichia coli, Ag is selected. + Perform antibacterial treatment. The specific procedure is as follows: Use a 0.2-0.5 mg / mL AgNO3 solution dissolved in deionized water. Immerse the gel in this solution for 2 hours, ensuring the Ag... + It can be uniformly adsorbed onto the gel.

[0048] For infections primarily caused by Gram-positive bacteria, such as Staphylococcus aureus, ZnO is selected for antibacterial treatment. The ZnO nanoparticles used have a particle size between 20-50 nm, a purity ≥99.5%, and a mass percentage of 0.5-1.0% in the gel. Before use, the ZnO nanoparticles are first ultrasonically dispersed at a power of 40 W for 15 minutes, and then mixed with the gel.

[0049] Ag +The reduction process follows strict operating procedures. The 10mM NaBH4 solution used must be freshly prepared and used immediately, with a volume ratio of 1:5 between the solution and the gel. The reaction is carried out at a constant temperature of 37°C for 20-30 minutes. This temperature is controlled to prevent drug inactivation due to high temperatures. After the reduction reaction is complete, the gel is washed three times with PBS to remove free NaBH4.

[0050] Ag + The loading was detected by ICP-MS using an Agilent 7900 instrument. Prior to detection, the gel underwent microwave digestion using a nitric acid-hydrogen peroxide system. To ensure the Ag content was within acceptable limits... + The loading rate should be between 0.3-0.6 wt%. Excessive loading rate can easily lead to cytotoxicity.

[0051] Step 6: Optimization of mechanical properties

[0052] After completing the antibacterial modification, mechanical property optimization is required. Among them, the crosslinking agent ratio and ultraviolet light crosslinking are key operations. Their role is to regulate the mechanical properties of the gel so that the gel can be smoothly injected into the anal fistula lesion site and maintain structural stability after reaching the lesion, thus meeting the mechanical requirements during the treatment process.

[0053] The cross-linking agent ratio needs to be adjusted according to the fistula condition. The cysteine ​​and acrylic acid used should have a purity ≥99%. When injection is needed through a thin fistula (diameter <5mm), the molar ratio of cysteine ​​to acrylic acid is 1:1.2-1:1.5. This ratio is to reduce the gel viscosity, facilitating passage through the thin fistula. When the lesion space is larger, the molar ratio of cysteine ​​to acrylic acid is 1:1-1:1.2. This ratio is to enhance the structural stability of the gel. The total concentration of cysteine ​​and acrylic acid should be controlled at 1% (w / v). Dissolve them in deionized water and add them to the gel, then magnetically stir for 10 minutes until uniformly mixed.

[0054] The parameters for UV crosslinking are set as follows: UV power 30-50 mW / cm², wavelength 365 nm. This wavelength is chosen to avoid drug degradation caused by short wavelengths below 360 nm. The irradiation time varies depending on the ratio of cysteine ​​to acrylic acid. When the molar ratio is 1:1.5, the irradiation time is 3-5 minutes. When the molar ratio is 1:1, the irradiation time is 5-7 minutes. It is necessary to ensure the elastic modulus of the gel. The pressure was stable at 500-800 Pa. The elastic modulus was tested using a TAAR2000 rheometer at a frequency of 1 Hz.

[0055] elastic modulus of gel The density of the cross-linked network is determined by the dynamic cross-linking network, and this network density is directly related to the molar ratio of cysteine ​​(containing thiol groups, SH) and acrylic acid (containing olefin groups, C=C) and the duration of UV irradiation. Therefore, a qualitative correlation is constructed based on the process parameters:

[0056] The elastic modulus of the gel. For cross-linked network density, The molar ratio of crosslinking agents, For the duration of ultraviolet radiation, when Decreasing the proportion of acrylic acid increases the amount of olefin groups, which in turn increases the number of crosslinking points. Increase, therefore Follow Decrease and increase.

[0057] when Extended exposure time allows for more complete UV-induced thiol-olefin click reactions. Increase, therefore Follow It extends and increases in size.

[0058] The target range (500-800 Pa) is controlled by adjusting... and make It is within a specific range, that is:

[0059] ;

[0060] Step 7, Sterilization and Packaging:

[0061] The finished gel was treated with gamma rays for sterilization at a dose range of 20-30 kGy and a dose rate range of 5-10 kGy / min. The drug activity retention rate of the gel after sterilization was ≥90%. The sterilized gel was then packaged in a sterile syringe and stored at a refrigerated temperature range of 2-8℃.

[0062] This method solves the problems of low drug delivery efficiency and excessively high local concentration in existing technologies by constructing carbon dot-magnetic composite structures, magnetic response directional alignment and dynamic cross-linking networks in a stepwise manner. Moreover, the preparation process does not require organic solvents and is environmentally friendly.

[0063] Example 1:

[0064] To achieve precise drug delivery to the anal fistula lesion site, this protocol utilizes a magnetically responsive carbon dot gel, with its movement trajectory guided and controlled by an external magnetic field. Embedded Fe3O4 magnetic particles within the gel enable directional migration and targeted retention under magnetic field influence. However, the efficiency of magnetic navigation largely depends on the gel's response speed and controllability under different magnetic field intensities, thus affecting its positioning accuracy and therapeutic effect in complex physiological environments. Therefore, it is necessary to systematically evaluate the gel's navigation performance under different magnetic field intensities to quantify the relationship between its movement speed and magnetic field strength, providing a theoretical basis and experimental support for optimizing magnetic navigation parameters. Based on this, experiments were conducted to determine the relationship between the navigation speed and magnetic field strength of the magnetically responsive carbon dot gel.

[0065] By simulating the clinical anal fistula lesion environment through in vitro 3D printing of anal fistula models, the directional migration ability, lesion targeting and retention efficiency of magnetic navigation-mediated carbon dot gel under the action of alternating magnetic field were verified, demonstrating the role of the "magnetic navigation module" in improving the accuracy of drug delivery.

[0066] Based on the type of anal fistula (superficial / deep, straight / curved) and the magnetic field parameters, the experiments were divided into the following groups, with each group undergoing three repeated trials:

[0067] Model type grouping:

[0068] Superficial straight fistula group: simulates anal fistula with lesion depth <3cm and straight fistula course. The 3D printed model has a lumen diameter of 5mm, a length of 2cm, and a course angle of 0° (straight).

[0069] Superficial curved fistula group: simulates anal fistula with lesion depth <3cm and curved fistula course. The 3D printed model has a lumen diameter of 5mm, a length of 2cm, and a course angle of 30° (arc).

[0070] Deep straight fistula group: simulates anal fistula with a lesion depth of 3-5cm and a straight fistula course. The 3D printed model has a lumen diameter of 4mm, a length of 4cm, and a course angle of 0° (straight).

[0071] Deep curved fistula group: simulates anal fistula with a lesion depth of 3-5cm and a curved fistula course. The 3D printed model has a lumen diameter of 4mm, a length of 4cm, and a course angle of 45° (arc).

[0072] Magnetic field parameters are grouped (each group corresponds to the model type mentioned above, with a magnetic field / no magnetic field control).

[0073] Experimental group (with magnetic field): The alternating magnetic field specified in the plan is applied, with the following parameters:

[0074] Superficial anal fistula (straight / curved fistula): frequency 10-30Hz, intensity 0.1-0.3T (initial location) → 0.3-0.5T (retention).

[0075] Deep anal fistula (straight / curved fistula): frequency 30-50Hz, intensity 0.1-0.3T (initial location) → 0.3-0.5T (retention).

[0076] Control group (no magnetic field): Except for the absence of an alternating magnetic field, all other conditions (model type, gel injection volume, etc.) were exactly the same as those of the experimental group.

[0077] Experimental steps:

[0078] Experimental Step 1, Preparation of Simulated Intestinal Fluid: Prepare according to the pH 7.4 phosphate buffer formula in the protocol (0.01 mol / L PBS, containing 0.138 mol / L NaCl and 0.0027 mol / L KCl), sterilize at 121℃ for 20 minutes, cool to 37℃, and then inject into the lumen of the 3D model.

[0079] Experimental step 2, gel injection: Use a 1mL sterile syringe to draw 0.2mL of fluorescently labeled gel and slowly inject it along the model inlet, avoiding the generation of air bubbles.

[0080] Experimental step 3, magnetic field application: The experimental group immediately started the alternating magnetic field generator after injection. The magnetic field direction was set according to the plan. The angle between the straight fistula and the injection direction was 45°±5°, and the angle between the curved fistula and the injection direction was 35°-45°. The magnetic field was applied for 30 minutes, including 10 minutes for initial positioning and 20 minutes for retention. The control group was placed in the same environment without magnetic field application.

[0081] Experimental step 4, real-time monitoring: The model was scanned in real time using a fluorescence imager (once every 5 minutes) to record the positional changes of the gel in the lumen and save the image data.

[0082] The results of each group of three repeated experiments are taken as the mean ± standard deviation.

[0083] The t-test was used to compare the migration speed, arrival time, and retention rate between the experimental group and the control group. P < 0.05 was considered statistically significant.

[0084] The above experiments clearly demonstrate the targeting and migration capabilities of the gel in different types of anal fistula models under the influence of a magnetic field, directly verifying the actual effect of the magnetic navigation module in improving the targeting efficiency of lesions.

[0085] Table 1 shows the comparison of the targeting migration ability of the gel in different types of anal fistula models in Example 1.

[0086] Model type Group Magnetic field parameters (frequency / intensity) Migration speed ( / min) Time to reach the lesion (in) Retention rate (%) Difference from the control group (P value) Superficial straight fistula (<3cm, 0°) experimental group 10-30Hz / 0.1-0.5T 2.5±0.3 8.2±1.1 85.6±4.2 <0.01 Superficial straight fistula (<3cm, 0°) Control group (no magnetic field) - 0.8±0.2 22.5±3.3 32.1±5.7 Superficial curved fistula (<3cm, 30°) experimental group 10-30Hz / 0.1-0.5T 2.1±0.2 10.5±1.5 78.3±3.8 <0.01 Superficial curved fistula (<3cm, 30°) Control group (no magnetic field) - 0.6±0.1 28.7±4.1 25.4±4.3 - Deep straight fistula (3-5cm, 0°) experimental group 30-50Hz / 0.1-0.5T 1.8±0.2 15.3±2.2 72.5±4.5 <0.01 Deep straight fistula (3-5cm, 0°) Control group (no magnetic field) - 0.5±0.1 35.6±5.2 18.7±3.6 Deep bend (3-5cm, 45°) experimental group 30-50Hz 1.5±0. 18.8±2 65.2±3 <0.01 Deep curved fistula (3-5cm, 45°) Control group (no magnetic field) - 0.4±0.1 42.3±6.1 12.5±2.8 -

[0087] The experimental results in Table 1 show that, in this technical solution, the experimental group with an alternating magnetic field applied, corresponding to the superficial straight fistula, superficial curved fistula, deep straight fistula, and deep curved fistula models, are significantly better than the control group without a magnetic field in terms of migration speed, time to reach the lesion, and retention rate.

[0088] Specifically, the experimental group exhibited a migration speed of 1.5-2.5 mm / min, a arrival time at the lesion of 8.2-18.8 min, and a retention rate of 65.2%-85.6%. In contrast, the control group had a migration speed of only 0.4-0.8 mm / min, a arrival time of 22.5-42.3 min, and a retention rate of only 12.5%-32.1%. All differences between the experimental and control groups were statistically significant (P < 0.01). This result directly validates the effectiveness of the magnetic navigation module in the protocol, demonstrating that alternating magnetic fields can drive the carbon dot gel to migrate directionally to the anal fistula lesion and achieve efficient retention, significantly improving targeted delivery efficiency.

[0089] Example 2: In this example, the pH-responsive sustained-release performance and local concentration maintenance ability of the magnetically guided anal fistula drug-releasing carbon dot gel were verified through in vitro drug release kinetics and in vivo drug concentration maintenance tests, including:

[0090] The study verified whether the gel could achieve a drug release cycle of 7-14 days in a simulated physiological environment (pH 7.4), with local drug concentration fluctuation ≤10% within 48 hours, demonstrating the advantage of "long-acting sustained release".

[0091] The release behavior of the gel was verified to differ in different pH environments (pH 6.0, pH 7.4, pH 8.0), demonstrating its "pH-responsive release" characteristic (adapting to pH changes in the anal fistula lesion and surrounding tissues).

[0092] To verify whether the gel can maintain a local drug concentration gradient at the anal fistula lesion site for ≥48 hours in an animal model, and whether the systemic drug exposure is lower than that of traditional formulations.

[0093] Table 2 shows the comparison of the release behavior of gels under different pH environments.

[0094] Grouping type Types of drugs Environmental pH Control group setup Number of repetitions Experimental group 1 Metronidazole 6.0 - 3 times Experimental group 2 Metronidazole 7.4 Traditional metronidazole solution (pH 7.4) 3 times Experimental group 3 Metronidazole 8.0 - 3 times Experimental group 4 clindamycin 6.0 - 3 times Experimental group 5 clindamycin 7.4 Traditional clindamycin solution (pH 7.4) 3 times Experimental group 6 clindamycin 8.0 - 3 times

[0095] In the experimental group, all drugs were loaded with drug-eluting gels, respectively loaded with metronidazole or clindamycin; the control group was a traditional drug solution without gel carrier, set only at pH 7.4 to compare the sustained-release effect; pH 6.0 simulated the acidic environment of the anal fistula inflammation area, pH 7.4 simulated the normal intestinal fluid environment, and pH 8.0 simulated the abnormal alkaline environment of intestinal fluid.

[0096] Table 3 shows the comparison of different types of gels in mouse experiments.

[0097] Grouping type Types of drugs Formulation type animal models Number of animals per group Experimental group (gel group) Metronidazole Magnetic navigation sustained-release carbon dot gel anal fistula model rats 6 Control group (traditional group) Metronidazole Regular metronidazole gel (commercially available) anal fistula model rats 6 Experimental group (gel group) clindamycin Magnetic navigation sustained-release carbon dot gel anal fistula model rats 6 Control group (traditional group) clindamycin Regular clindamycin gel (commercially available) anal fistula model rats 6

[0098] Specific experimental steps:

[0099] Experimental Step 1: Prepare simulation solutions, including 0.1 mol / L citrate-sodium citrate buffer (pH 6.0), 0.01 mol / L phosphate buffer (pH 7.4, containing 0.138 mol / L NaCl and 0.0027 mol / L KCl), and 0.05 mol / L Tris-HCl buffer (pH 8.0). All buffers were sterilized at 121°C for 20 minutes and then cooled to 37°C for later use. Prepare carbon dot gels loaded with metronidazole (5 mg / mL) or clindamycin (2 mg / mL) according to the protocol. Take 3 portions of each group, each 0.5 g, and weigh them accurately. Prepare metronidazole solution (5 mg / mL) and clindamycin solution (2 mg / mL) with the same initial drug concentration as the gel, and dissolve them in pH 7.4 PBS as control solutions. Prepare dialysis bags (metronidazole corresponds to a molecular weight cutoff of 3500 Da, and clindamycin corresponds to 1000 Da), pre-treat them by boiling them in deionized water for 10 minutes, and then drain them. Meanwhile, SPF-grade SD rats (weighing 250-300g) were selected, and an anal fistula model (fistula depth approximately 2cm) was surgically constructed after inducing inflammation by injecting carrageenan around the anus. The formation of the fistula was confirmed by imaging 3 days postoperatively.

[0100] In experimental step 2, the experimental group placed 0.5g of drug-loaded gel into a dialysis bag and sealed it. The bag was then placed in a 50mL centrifuge tube, and 30mL of simulated solution corresponding to the pH was added. The mixture was incubated in a constant temperature shaker (100rpm) at 37℃. The control group placed 3mL of drug solution (consistent with the total amount of drug in the gel) into a dialysis bag and incubated it in the same manner under pH 7.4 conditions. At time points of 1h, 6h, 12h, 24h, 48h, 7d, and 14d, 2mL of the release solution was taken from each centrifuge tube, and 2mL of fresh simulated solution was added to maintain a constant volume. The released solution was then refrigerated at 4℃ for testing.

[0101] Experimental Step 3: In vitro concentration detection and data analysis. High-performance liquid chromatography (HPLC) was used to detect the drug concentration in the release solution. Metronidazole was detected using a C18 column (250 mm × 4.6 mm), with a mobile phase of methanol-water (30:70), a flow rate of 1.0 mL / min, and a detection wavelength of 320 nm. Clindamycin was detected using a C18 column (250 mm × 4.6 mm), with a mobile phase of acetonitrile-0.1% phosphoric acid (25:75), a flow rate of 1.0 mL / min, and a detection wavelength of 220 nm. 10nm; Calculate the cumulative release rate (total drug released at each time point / initial drug loading of gel × 100%) and plot the release curve to verify whether the cumulative release rate of the pH7.4 group reaches more than 80% after 14 days. Calculate whether the drug concentration fluctuation value within 48 hours ((highest concentration - lowest concentration) / average concentration × 100%) of this group is ≤10%. Compare the differences in the 24-hour cumulative release rate of the same drug in the pH6.0, pH7.4, and pH8.0 groups to verify pH responsiveness (difference between the pH7.4 group and other groups ≥20%).

[0102] Step 4 of the experiment involved in vivo drug administration and sample collection. In the experimental group, each rat with anal fistula was injected with 0.2 mL of drug-loaded gel (metronidazole gel containing 1 mg of drug and clindamycin gel containing 0.4 mg of drug) through the fistula entrance. The control group was injected with an equal amount of ordinary gel containing the same amount of drug. At 6 h, 24 h, 48 h, and 72 h after drug administration, 3 rats from each group were randomly selected and sacrificed. Tissue (approximately 0.5 g) from the anal fistula lesion was dissected and homogenized with 1 mL of pH 7.4 PBS. The tissue was then centrifuged at 4°C (12000 rpm, 10 min) to obtain the supernatant. At the same time, 2 mL of blood was collected from the abdominal aorta, allowed to stand for 30 min, and then centrifuged (3000 rpm, 10 min) to obtain the plasma.

[0103] Experimental Step 5: In vivo concentration detection and data analysis

[0104] The drug concentration in the lesion tissue supernatant and plasma was detected using the same HPLC method as in in vitro experiments. The ratio of drug concentration in lesion tissue to drug concentration in plasma was calculated to verify whether the ratio was maintained at >10 for ≥48h. The lesion tissue concentration (experimental group should be more than 30% higher than control group) and plasma concentration (experimental group should be more than 40% lower than control group) at 48h were compared between the experimental group and the control group to verify the gel's targeting properties and low systemic toxicity.

[0105] Table 4 compares the cumulative release rates of metronidazole gel and clindamycin gel over time.

[0106] Types of drugs Group / Environment Cumulative release rate over 1 hour (%) 24-hour cumulative release rate (%) 48-hour concentration fluctuation (%) 14-day cumulative release rate (%) Metronidazole pH 6.0 (Experimental group) 28.5±2.1 52.3±3.2 - 85.6±4.5 Metronidazole pH 7.4 (Experimental Group) 15.2±1.8 30.1±2.5 8.7±1.2 88.3±3.8 Metronidazole pH 8.0 (Experimental group) 42.6±2.7 68.5±3.6 - 90.2±4.1 Metronidazole pH 7.4 (control group) 65.3±3.5 92.1±2.8 25.6±2.3 99.5±1.2 clindamycin pH 6.0 (Experimental group) 25.3±1.9 48.7±2.9 - 82.4±3.6 clindamycin pH 7.4 (Experimental Group) 2.5±1.5 26.8±2.2 7.5±1.0 86.7±3.2 clindamycin pH 8.0 (Experimental group) 38.7±2.4 63.2±3.1 - 89.5±3.7 clindamycin pH 7.4 (control group) 60.2±3.2 89.7±2.5 22.3±1.8 98.8±1.5 Metronidazole Experiment (gel) - - - - Metronidazole Comparison (normal) - - - - clindamycin Experiment (gel) - - - - clindamycin Comparison (normal) - - - - Types of drugs Group / Environment Cumulative release rate over 1 hour (%) 24-hour cumulative release rate (%) 48-hour concentration fluctuation (%) 14-day cumulative release rate (%)

[0107] Table 5 compares the concentration ratios of metronidazole gel and clindamycin gel in lesion tissue and plasma over time.

[0108] Types of drugs Group / Environment Lesion tissue / plasma concentration ratio (48h) Lesion concentration over 48 hours (μg / g) 48-hour plasma concentration (μg / mL) Metronidazole pH 6.0 (Experimental group) - - - Metronidazole pH 7.4 (Experimental Group) - - - Metronidazole pH 8.0 (Experimental group) - - - Metronidazole pH 7.4 (control group) - - - clindamycin pH 6.0 (Experimental group) - - - clindamycin pH 7.4 (Experimental Group) - - - clindamycin pH 8.0 (Experimental group) - - - clindamycin pH 7.4 (control group) - - - Metronidazole Experimental group (gel) 15.6±1.8 48.5±3.2 3.1±0.4 Metronidazole Control group (normal) 4.2±0.6 32.8±2.7 7.8±0.9 clindamycin Experimental group (gel) 14.3±1.5 35.2±2.8 2.5±0.3 clindamycin Control group (normal) 3.8±0.5 24.7±2.1 6.5±0.7 Types of drugs Group / Environment Lesion tissue / plasma concentration ratio (48h) Lesion concentration over 48 hours (μg / g) 48-hour plasma concentration (μg / mL)

[0109] As shown in Table 4, in the in vitro release experiment, the cumulative release rate of the experimental group in the pH 7.4 environment reached more than 85% in 14 days, and the concentration fluctuation in 48 hours was ≤10%, which met the design of "7-14 day release cycle and local concentration stability"; and the release rate of the pH 7.4 group in 24 hours was significantly lower than that of the pH 6.0 and pH 8.0 groups (difference ≥20%), which verified the pH responsiveness.

[0110] The control group (traditional solution) released too quickly, with large concentration fluctuations over 48 hours, and could not achieve long-term sustained release.

[0111] As shown in Table 5, in the in vivo experiment, the lesion tissue / plasma concentration ratio of the experimental group was >10 and maintained for ≥48h. The lesion concentration at 48h was more than 30% higher than that of the control group, and the plasma concentration was more than 40% lower than that of the control group, demonstrating the advantages of maintaining local concentration gradient and low systemic toxicity.

[0112] The main technical advantages of this application lie in the synergistic effect of targeted delivery and long-lasting sustained release. Through magnetic navigation, carbon dot gels embedded with magnetite magnetic particles can migrate directionally to the anal fistula lesion and remain efficiently under the influence of an alternating magnetic field, significantly improving drug targeting efficiency. Compared to traditional formulations, this reduces drug distribution in non-lesion areas and lowers the risk of systemic exposure. Simultaneously, the drug-releasing layer formed by sodium alginate and chitosan is pH-responsive, achieving long-lasting release for 7-14 days in a simulated intestinal fluid environment, with local drug concentration fluctuations ≤10% within 48 hours, maintaining an effective therapeutic concentration and solving the problems of frequent administration and unstable local concentrations associated with traditional formulations.

[0113] This application enhances the safety and efficacy of treatment through antibacterial modification and biocompatibility design. The silver ion or zinc oxide nanoparticles doped in the gel effectively inhibit local bacterial infection. The three-dimensional reversible cross-linked network constructed by dynamic thiol-olefin click chemistry gives the gel shear-thinning properties, facilitating rapid recovery of its original shape after injection and adapting to the complex physiological environment of anal fistula. The preparation process requires no organic solvents, and all components meet biosafety requirements. After gamma-ray sterilization, the drug activity retention rate is ≥90%, providing a safe and reliable foundation for clinical application and comprehensively improving the precision, long-term effectiveness, and safety of anal fistula treatment.

[0114] The above description is merely an embodiment of this application and does not limit the patent scope of this application. Any equivalent structural or procedural transformations made using the content of this application's specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of this application.

Claims

1. A magnetically guided sustained-release carbon dot gel for anal fistula medication, characterized in that, include: The composition includes a carbon dot matrix, a magnetic navigation orientation module, and an antibacterial modification component. Embedded nano-sized Fe3O4 magnetic particles are dispersed in the carbon dot matrix to form a carbon dot-magnetic composite structure, and the carbon dot-magnetic composite structure is coated with a drug sustained-release layer.

2. The magnetically guided sustained-release carbon dot gel for anal fistula medication according to claim 1, characterized in that, The carbon dot matrix uses biocompatible carbon dots as the core carrier, and the carbon dot surface is functionalized with active groups containing carboxyl or amino groups for loading drug molecules.

3. The magnetically guided sustained-release carbon dot gel for anal fistula medication according to claim 1, characterized in that, The embedded nano-sized Fe3O4 magnetic particles have a particle size range of 50-200 nm and are coated with polyethylene glycol (PEG) on their surface.

4. The magnetically guided sustained-release carbon dot gel for anal fistula medication according to claim 1, characterized in that, The drug sustained-release layer is a polymer cross-linked network wrapped around the surface of the carbon dot-magnetic composite structure. It is formed by the dynamic covalent cross-linking of sodium alginate and chitosan, with a cross-linking density of 0.5-2.0 mol / L.

5. The magnetically guided sustained-release carbon dot gel for anal fistula medication according to claim 1, characterized in that, The magnetic navigation orientation module is used to apply an alternating magnetic field externally, with a frequency of 10-50Hz and an intensity of 0.1-0.5T. Through the magnetic dipole interaction of the carbon dot-magnetic composite structure, the gel is driven to migrate and remain in the anal fistula lesion site.

6. The magnetically guided sustained-release carbon dot gel for anal fistula medication according to claim 1, characterized in that, The antibacterial modifying component includes silver ions (Ag). + Or zinc dioxide (ZnO) nanoparticles, with a mass percentage of 0.1-1.0%.

7. A method for preparing a magnetically guided sustained-release carbon dot gel for anal fistula drugs, characterized in that, include: S1. Carbon dot synthesis and functionalization: Citric acid and urea at a mass ratio of 1:2.5 are used as precursors and reacted by hydrothermal method at a temperature range of 160-200℃ for a reaction time of 4-10 hours to prepare carbon dots; after acidification treatment, carboxyl or amino groups are grafted onto the carbon dots by EDC / NHS coupling reaction to obtain the surface-modified carbon dot matrix. S2. Magnetic particle composite: Fe3O4 nanoparticles and polyethylene glycol are ultrasonically dispersed in an ethanol solution with an ultrasonic power range of 30-70W and an ultrasonic time range of 20-40 minutes. Then, they are mixed with carbon dots and formed the carbon dot-magnetic composite through electrostatic self-assembly. S3. Drug loading and cross-linking: Metronidazole or clindamycin was dissolved in phosphate buffer solution at pH 7.4 with a concentration of 0.01 mol / L, and drug loading was completed by ultrasonic-assisted diffusion and centrifugation. Sodium alginate and chitosan, both at a concentration of 2%, were dissolved in 0.1 mol / L NaCl solution to obtain a mixed solution. The pH of the mixed solution was adjusted to 6.5-7.0 with 1% calcium chloride solution to construct the cross-linking network. S4. Construction of magnetic navigation structure: After the construction of the cross-linked network is completed, the magnetic navigation structure is constructed. The magnetic field parameters need to be adjusted according to the condition of the anal fistula lesion. S5. Antibacterial modification: After the magnetic navigation structure is constructed, antibacterial modification is carried out, in which the corresponding antibacterial agent is determined according to the type of bacteria that infect; S6. Mechanical property optimization: Adjust the crosslinking agent ratio and ultraviolet crosslinking according to the fistula condition to regulate the mechanical properties of the gel and obtain the sustained-release carbon dot gel for anal fistula.

8. The preparation method according to claim 7, characterized in that, The carbon dot matrix has a particle size range of 50-100 nm, a surface carboxyl density range of 1.2-2.0 mmol / g, an EDC of 0.2 mmol / mL, and an NHS of 0.1 mmol / mL.

9. The preparation method according to claim 7, characterized in that, The Fe3O4 nanoparticles have a particle size range of 50-200 nm, the PEG coating on the surface has a molecular weight range of 1000-3000, and the mass ratio of carbon dots to Fe3O4 ranges from 1:1 to 1:

3. They are allowed to self-assemble at 25°C for 4 hours to form a carbon dot-magnetic composite. During this time, the mixture is gently stirred once every 30 minutes to avoid local sedimentation. After assembly, the mixture is centrifuged at 5000 rpm for 5 minutes, the supernatant is discarded, and the precipitate is washed three times with deionized water to obtain the carbon dot-magnetic composite structure.