Dihydroartemisinin-loaded pH-responsive MOF (Metal Organic Framework) drug loading system as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of drug loading systems, and particularly discloses a dihydroartemisinin-loaded pH response type MOF drug loading system and a preparation method and application thereof, and the preparation method comprises the following steps: preparing MIL-101-NH2; preparing HA-FA; preparing MD; and preparing MD-coated HF. According to the dihydroartemisinin-loaded pH response type MOF drug loading system as well as the preparation method and the application thereof, the drug loading system has good pH responsiveness, biocompatibility, antibacterial property and wound healing promoting capability; the preparation method is stable and controllable in process and easy for industrial production; the healing of a wound infected by the staphylococcus aureus is promoted.

Description

Technical Field

[0001] This invention relates to the field of drug delivery system technology, and in particular to a pH-responsive MOF drug delivery system loaded with dihydroartemisinin, its preparation method and application. Background Technology

[0002] Dihydroartemisinin (DHA) is an important derivative of artemisinin, possessing a variety of pharmacological activities including broad-spectrum antibacterial, anti-inflammatory, and antitumor effects, showing particular potential in combating bacterial infections. However, DHA suffers from poor water solubility, low bioavailability, a short half-life in vivo, and is easily released at non-target sites, leading to reduced efficacy and potential toxic side effects, thus limiting its clinical application.

[0003] Metal-organic frameworks (MOFs) are porous materials with a periodic network structure formed by coordination bonds between metal ions or metal clusters and organic ligands. They possess advantages such as large specific surface area, tunable pore size, structural diversity, and good biocompatibility, attracting widespread attention in the field of drug delivery. Among them, MIL-101-NH2, as a typical MOF material, not only has a large specific surface area and pore volume, but its surface amino groups can also bind to drug molecules through hydrogen bonding, electrostatic interactions, etc., achieving efficient drug loading. At the same time, MIL-101-NH2 undergoes structural dissociation in an acidic environment, enabling pH-responsive drug release, making it suitable for drug delivery in tumor microenvironments or bacterial infection sites (which are usually acidic).

[0004] Hyaluronic acid (HA) is a natural polysaccharide with good biocompatibility, biodegradability, and targeting properties (it can bind to CD44 receptors on the surface of cells at tumor or inflammatory sites). Folic acid (FA) can specifically bind to folate receptors highly expressed on the surface of tumor cells or bacteria, achieving active targeted delivery. Combining HA and FA to form an HA-FA complex for encapsulating MOF drug delivery systems not only improves the stability and biocompatibility of the drug delivery system but also enhances its targeting properties, reduces drug distribution in normal tissues, and improves efficacy.

[0005] Currently, there are no reports on pH-responsive drug delivery systems that use MIL-101-NH2 as a carrier to load DHA and combine it with HA-FA for targeted modification. Therefore, developing a pH-responsive MIL-101-NH2 / HA-FA drug delivery system loaded with DHA to solve the DHA delivery challenge is of significant research importance and clinical application value. Summary of the Invention

[0006] The purpose of this invention is to provide a pH-responsive MOF drug delivery system loaded with dihydroartemisinin, its preparation method, and its application. The drug delivery system has good pH responsiveness, biocompatibility, antibacterial properties, and wound healing promotion ability. The preparation method is stable, controllable, and easy to industrialize. It promotes the healing of wounds infected with Staphylococcus aureus.

[0007] To achieve the above objectives, the present invention provides a method for preparing a pH-responsive MOF drug delivery system loaded with dihydroartemisinin, comprising the following steps: Step 1, Preparation of MIL-101-NH2: 2-Aminoterephthalic acid and FeCl3•6H2O were placed in a polytetrafluoroethylene liner, N,N-dimethylformamide was added, and the mixture was magnetically stirred until completely dissolved. The mixture was then transferred to a high-pressure reactor and reacted at 120°C for 20 hours. After cooling, the mixture was centrifuged, washed, and vacuum dried to obtain MIL-101-NH2. Step 2, Preparation of HA-FA: Folic acid was dissolved in dimethyl sulfoxide and sonicated to dissolve. N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide were added, and the mixture was activated by reacting in a water bath at 30°C for 5 hours. The precipitate was removed by centrifugation. Hyaluronic acid was dissolved in formamide and stirred at 40°C to dissolve. The activated folic acid solution was added dropwise, and the mixture was reacted in the dark for 48 hours. The mixture was then centrifuged, dialyzed, and freeze-dried to obtain HA-FA. Step 3, Preparation of MD: Dissolve MIL-101-NH2 and dihydroartemisinin from Step 1 in anhydrous ethanol and sonicate. After mixing, place the mixture in a parallel reactor and stir magnetically in the dark under the conditions of drug loading time of 24 h, drug loading temperature of 34 °C, drug loading ratio of 0.63:1 and rotation speed of 300 rpm. After reaction, centrifuge and wash to obtain MD. Step 4, Preparation of MD@HF: Dissolve the MD from Step 3 and the HA-FA from Step 2 in ultrapure water and sonicate. Add the MD solution to the HA-FA solution and stir at room temperature for 36 hours in the dark. Centrifuge, wash, and vacuum dry to obtain the MD@HF drug delivery system.

[0008] Preferably, in step 1, the amount of 2-aminoterephthalic acid is 360 mg, the amount of FeCl3•6H2O is 1 g, and the amount of N,N-dimethylformamide is 40 mL; the centrifugation speed is 10000 rpm, and the centrifugation time is 5 min; the washing process involves washing three times each with N,N-dimethylformamide and anhydrous ethanol.

[0009] Preferably, in step 2, the dosage of folic acid is 50 mg, the dosage of dimethyl sulfoxide is 3 mL, the dosage of N,N'-dicyclohexylcarbodiimide is 1 g, the dosage of N-hydroxysuccinimide is 288 mg, the dosage of hyaluronic acid is 100 mg, and the dosage of formamide is 10 mL; the dialysis process uses ultrapure water for dialysis for 48 h, and the water is changed every 6 h.

[0010] Preferably, in step 2, the grafting rate of FA in HA-FA is 17.69%.

[0011] Preferably, in step 3, the amount of MIL-101-NH2 used is 10 mg, and the amount of anhydrous ethanol used is 5 mL; the amount of dihydroartemisinin used is 10 mg, and the amount of anhydrous ethanol used is 2 mL; the centrifugation speed is 10000 rpm, the centrifugation time is 5 min, and the product is washed twice with anhydrous ethanol.

[0012] Preferably, in step 3, the drug loading rate of MD is 46.41±0.03%, and the encapsulation rate is 86.61±0.11%.

[0013] Preferably, in step 4, the amount of MD is 20 mg, the amount of HA-FA is 4 mg, and the amount of ultrapure water is 20 mL; the centrifugation speed is 10000 rpm, the centrifugation time is 5 min, and the product is washed three times with ultrapure water.

[0014] Preferably, the drug loading rate of the MD@HF drug delivery system is 29.00±0.08%, and the MD@HF drug delivery system is pH responsive, with a dihydroartemisinin release rate of 56.72±1.40% under acidic conditions of pH 5.0 and a dihydroartemisinin release rate of 29.05±3.88% under neutral conditions of pH 7.4.

[0015] The present invention also provides a pH-responsive MOF drug delivery system loaded with dihydroartemisinin, which is prepared by the above-described preparation method.

[0016] This invention also provides the application of a pH-responsive MOF drug delivery system loaded with dihydroartemisinin, used in the preparation of antibacterial drugs that inhibit the growth of Staphylococcus aureus.

[0017] The advantages and beneficial effects of the pH-responsive MOF drug delivery system loaded with dihydroartemisinin, its preparation method, and its application in this invention are as follows: 1. The drug delivery system of the present invention uses MIL-101-NH2 as a carrier, and utilizes its porous structure and pH sensitivity to achieve efficient loading and pH-responsive release of DHA. It releases the drug at acidic infection sites and slowly releases it in normal physiological environments, thereby improving the drug's targeting and bioavailability.

[0018] 2. This invention employs HA-FA coating of MD, which not only improves the stability and biocompatibility of the drug delivery system but also endows it with a certain degree of active targeting, enhancing drug accumulation at the infection site and reducing drug damage to normal tissues. The drug delivery system exhibits excellent antibacterial properties and wound-healing capabilities, particularly showing significant efficacy in treating wounds infected with Staphylococcus aureus, and has broad application prospects in the biomedical field. 3. The preparation method of the present invention has clear, stable and controllable process parameters, good repeatability, and is easy to industrialize.

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0020] Figure 1 This is a superimposed 1H NMR spectrum of HA, FA, and HA-FA of the present invention; Figure 2 These are the infrared spectra of HA, FA, and HA-FA of the present invention; Figure 3 This is a characterization of the nanoparticles prepared in this invention, wherein A is a TEM image of MIL-101-NH2, B is a TEM image of MD; C is a particle size distribution chart of MIL-101-NH2, and D is a particle size distribution chart of MD. Figure 4 This is a characterization of the nanoparticles prepared in this invention, where A is a SEM image of MIL-101-NH2 and B is a SEM image of MD. Figure 5 This is a hydrodynamic dimension diagram of MIL-101-NH2, MD, and MD@HF of the present invention; Figure 6 This is a Zeta potential diagram of MIL-101-NH2, MD, and MD@HF of the present invention; Figure 7 This is a stability test of the MD@HF of the present invention, where A is the particle size change over seven days and B is the PDI stability (n=3). Figure 8 In the figure, A represents the TGA curves of MIL-101-NH2, MD, and MD@HF, and B represents the DTG curves. Figure 9 These are the XRD patterns of MIL-101-NH2, MD, and MD@HF of this invention; Figure 10 This is the EDS diagram of MIL-101-NH2 of the present invention, where A is a SEM image, B is an energy dispersive spectroscopy (EDS) diagram, C is a C element distribution diagram, D is a N element distribution diagram, E is an O element distribution diagram, and F is a Fe element distribution diagram. Figure 11 These are the infrared spectra of different samples from this invention; Figure 12 These are the EPR diagrams of MIL-101-NH2, MD, and MD@HF of the present invention; Figure 13 This is the XPS total spectrum of MIL-101-NH2, MD, and MD@HF of this invention; Figure 14In the image, A is the high-resolution 1s spectrum of C, B is the high-resolution 1s spectrum of N, C is the high-resolution 1s spectrum of O, and D is the high-resolution 2p spectrum of Fe. Figure 15 The N2 adsorption-desorption isotherms and average pore size distribution of MIL-101-NH2, MD and MD@HF of this invention are shown. Figure 16 This invention presents DHA release curves at pH values ​​of 5.0 and 7.4, where A represents MD and B represents MD@HF. Figure 17 This is the hemolysis result of the present invention, wherein A is MIL-101-NH2, B is MD, and C is MD@HF; Figure 18 The present invention describes the cytotoxicity of different concentrations of MIL-101-NH2, MD, and MD@HF on HaCaT cells, wherein A is MIL-101-NH2, B is MD, and C is MD@HF; Figure 19 (a) shows an infected rat model, (b) shows a schematic diagram of the wound area, and (c) shows a statistical graph of changes in the wound area. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0022] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0023] The following examples are not intended to limit the invention, but are only for illustration. Unless otherwise specified, the experimental methods used in the following examples are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0024] Example 1 A method for preparing a pH-responsive MOF (metal-organic framework) drug delivery system loaded with dihydroartemisinin includes the following steps: Step 1, Preparation of MIL-101-NH2: 360 mg of 2-aminoterephthalic acid and 1 g of FeCl3•6H2O were placed in a polytetrafluoroethylene (PTFE) liner, and 40 mL of N,N-dimethylformamide (DMF) was added. The mixture was magnetically stirred until the solid was completely dissolved. The PTFE liner was then transferred to a high-pressure reactor and reacted at 120 °C for 20 h. After the reaction, the mixture was cooled to room temperature, and the resulting solution was centrifuged at 10,000 rpm for 5 min. The precipitate was collected and washed three times sequentially with DMF and anhydrous ethanol to remove unreacted raw materials and impurities. Finally, the washed precipitate was dried in a vacuum drying oven to obtain MIL-101-NH2 powder.

[0025] Step 2, Preparation of HA-FA: Weigh 50 mg of folic acid (FA) and dissolve it in 3 mL of dimethyl sulfoxide (DMSO), then sonicate until completely dissolved. Add 1 g of N,N'-dicyclohexylcarbodiimide (DCC) and 288 mg of N-hydroxysuccinimide (NHS) to the above solution, and place the mixed solution in a 30°C water bath for 5 h to activate the carboxyl group of FA. After the reaction is complete, centrifuge the solution at 10000 rpm for 5 min to remove the precipitate formed by the reaction, and collect the supernatant (activated FA solution) for later use.

[0026] Separately, 100 mg of HA was dissolved in 10 mL of formamide and stirred on a magnetic stirrer at 40 °C until the HA was completely dissolved. Then, the activated FA solution was added dropwise to the HA solution, and the reaction was carried out under light-protected conditions for 48 h. After the reaction was complete, the mixture was centrifuged to remove the precipitate, and the supernatant was collected and placed in a dialysis bag. Dialysis was performed with ultrapure water for 48 h (changing the ultrapure water every 6 h) to remove unreacted small molecules. After dialysis, the solution in the dialysis bag was freeze-dried to obtain HA-FA solid powder. The grafting rate of FA in HA-FA was 17.69%.

[0027] Step 3, Preparation of MD: Weigh 10 mg MIL-101-NH2 and dissolve it in 5 mL of anhydrous ethanol, and dissolve 10 mg dihydroartemisinin (DHA) in 2 mL of anhydrous ethanol. Both solutions are sonicated to ensure complete dissolution. The two solutions are then mixed and transferred to a parallel reactor. Under conditions of drug loading time of 24 h, drug loading temperature of 34 °C, drug-to-load ratio of 0.63:1, and rotation speed of 300 rpm, the mixture is magnetically stirred in the dark. After the reaction is complete, the mixture is centrifuged at 10,000 rpm for 5 min, the precipitate is collected, and washed twice with anhydrous ethanol to remove free DHA, yielding MD solid powder. The drug loading rate of MD is 46.41 ± 0.03%, and the encapsulation efficiency is 86.61 ± 0.11%.

[0028] Step 4, Preparation of MD@HF: Weigh 20 mg MD and 4 mg HA-FA, dissolve them separately in 20 mL of ultrapure water, and sonicate until completely dissolved. Slowly add the MD solution to the HA-FA solution, and stir at room temperature in the dark for 36 h to ensure HA-FA fully coats the MD surface. After the reaction is complete, centrifuge the mixed solution at 10000 rpm for 5 min, collect the precipitate, wash three times with ultrapure water to remove uncoated HA-FA. Finally, dry the precipitate in a vacuum drying oven to obtain the MD@HF drug delivery system. The drug loading rate of the MD@HF drug delivery system is 29.00 ± 0.08%. The MD@HF drug delivery system is pH responsive; the dihydroartemisinin release rate is 56.72 ± 1.40% under acidic conditions (pH 5.0) and 29.05 ± 3.88% under neutral conditions (pH 7.4).

[0029] Example 2 Physicochemical characterization of pH-responsive MOF (metal-organic framework) drug delivery system loaded with dihydroartemisinin.

[0030] Using deuterated hydrochloric acid and heavy water (DCl 20% in D2O) as solvents, the structural features of HA, FA, and HA-FA were characterized by 1H-NMR. The obtained data were imported into the software MestReNova for analysis and processing of 1H-NMR data and plotting.

[0031] The functional group changes of HA, FA, HA-FA, DHA, MIL-101-NH2, MD, and MD@HF were analyzed by Fourier transform infrared spectroscopy (FT-IR).

[0032] The morphology of MIL-101-NH2, MD, and MD@HF was analyzed by transmission electron microscopy (TEM).

[0033] The morphology of MIL-101-NH2, MD, and MD@HF was analyzed by scanning electron microscopy (SEM).

[0034] The particle size, zeta potential, and polydispersity index (PDI) of samples MIL-101-NH2, MD, and MD@HF were measured by dynamic light scattering (DLS) to determine whether the MD@HF nanoparticles were successfully synthesized.

[0035] The changes in the crystal structure of MIL-101-NH2, MD, and MD@HF were determined by X-ray diffraction (XRD).

[0036] The surface elemental composition of MIL-101-NH2 was determined by X-ray photoelectron spectroscopy (XPS).

[0037] The thermal stability of MIL-101-NH2, MD, and MD@HF was obtained by thermogravimetric analysis (TGA).

[0038] The elemental composition of MIL-101-NH2 was analyzed by energy-dispersive X-ray spectroscopy.

[0039] The ability of MIL-101-NH2 to promote ROS generation was investigated by electron paramagnetic resonance spectroscopy (EPR).

[0040] The ultraviolet spectra of MIL-101-NH2, MD, and MD@HF were obtained using a UV-Vis spectrophotometer.

[0041] 1. In vitro release experiment The in vitro release of DHA from MD and MD@HF was detected by dynamic dialysis. 5 mg of MD and MD@HF were dispersed separately in 2 mL of PBS solution (pH 7.4, pH 5.0) containing 15% DMSO, and ultrasonically dispersed using an ultrasonic cleaner before being transferred to dialysis bags. The dialysis bags were then placed into centrifuge tubes containing 20 mL of release medium, and the tubes were placed in a THZ-92B air bath constant temperature shaker and gently shaken (150 rpm / min) at 37°C in the dark. At appropriate time intervals (1, 2, 4, 6, 8, 10, 12, 24, 36, 48 h), 2 mL of release medium was collected and 2 mL of fresh PBS solution (pH 7.4, pH 5.0) was added to maintain a constant release volume. The collected release medium was mixed with 2% NaOH and incubated in a 50°C water bath for 30 min. Subsequently, the absorbance at 290 nm was measured under UV-Vis. Based on the standard curve of DHA in PBS solutions (pH 7.4, pH 5.0), the release rate of DHA was calculated using the following formula: ; Total DHA released from the release medium; The volume of sample drawn each time is 2 mL; Volume of release medium, 20 mL; : No. i The concentration of DHA in the release medium during the first sampling, mg / mL; DHA mass, mg; Number of samplings; : The concentration of DHA in the release medium during the nth (last) sampling.

[0042] 2. Hemolysis test Positive control group: 1 mL of physiological saline containing 1% Triton X-100 + 20 μL of erythrocyte suspension; Negative control group: 1 mL of physiological saline + 20 μL of erythrocyte suspension; each concentration gradient constitutes one group, and five parallel experiments are set up. 1 mL of working solution from each group is added to 20 μL of erythrocyte suspension. All samples are incubated at 37℃ for 1 h, then centrifuged at 1500 rpm for 10 min. 100 μL of supernatant is transferred to a 96-well plate, and the absorbance at 540 nm is measured using a microplate reader (images are collected after sample centrifugation). The hemolysis rate is calculated using the following formula: ; In the formula Represents the absorbance of the sample. The absorbance represents the positive control group. The absorbance represents the negative control group.

[0043] 3. Experimental Results (1) Characterization of HA-FA 1 1 H-NMR analysis: The superimposed proton NMR spectra of FA, HA, and HA-FA are shown below. Figure 1 In the 1H NMR spectrum of FA, the peaks shifted around 11 and 12 ppm represent the protons of NH atoms on FA. In the HA-FA overlay spectrum, the peak position of this structure is 10.78 ppm. Coupling with HA increases the electron cloud density, causing the chemical shift to move to a higher field. The multiplets with a chemical shift around 3.7 ppm represent the proton peaks of hydrogen atoms on the FA structure. The nucleophilicity of HA causes them to shift to a lower field, indicating successful grafting of HA and FA. The d-peak with a shift of δ=8.3 ppm represents a proton of hydrogen atom on the N-containing heterocycle of FA, with an integral value of 1. Compared with the integral value of 6 (two proton peaks) of HA at δ=4.48 ppm, the grafting rate of FA on HA is estimated to be approximately 33%. That is, there are 33 FAs per 1000 HA structural units.

[0044] Analysis of Fourier transform infrared spectroscopy: like Figure 2 As shown, in the infrared spectrum of the HA-FA complex, at 3450 cm⁻¹... -1 There is a broad peak at 1741 cm⁻¹, which may be attributed to an overlap of the characteristic absorption peaks of amino and hydroxyl groups. Compared with HA, its intensity is relatively lower, possibly because some hydroxyl groups in HA are combined with the carboxyl groups in FA, resulting in a relative weakening of the hydroxyl intensity. -1 The peak at 1656 cm⁻¹ is attributed to the stretching vibration of the C=O bond formed by the reaction of HA and FA to form an ester bond. -1 The peak at [value missing] is attributed to the stretching vibration of the C=O group of the carboxyl group; this is a strong absorption peak of amides. It is located in the 1200-1000 cm⁻¹ range.-1 Absorption peaks were also observed within the range, which may originate from the stretching vibrations of COC and C-OH in hyaluronic acid. These results indicate that the HA-FA polymer was successfully synthesized.

[0045] (2) TEM analysis The morphology of MIL-101-NH2 and MD was observed using TEM, and their particle size was statistically analyzed using NanoMeasurer 1.2 software. The results are as follows: Figure 3 As shown. Figure 3 China A and Figure 3 Figure B shows the TEM images of MIL-101-NH2, which exhibit a typical and complete octahedral structure with relatively uniform particle size. The statistical results of MIL-101-NH2 obtained using NanoMeasurer 1.2 are shown in Figure C of Figure 3, with an average particle size of 250.0000 ± 79.4244 nm. The statistical results of MD obtained using NanoMeasurer 1.2 are shown in Figure C of Figure 3. Figure 3 As shown in Figure D, its average particle size is 288.4158±17.3955nm, similar to the size of MIL-101-NH2, indicating that the loading of DHA has little effect on the structure of the material.

[0046] (3) SEM analysis Figure 4 China A and Figure 4 Image B shows the SEM images of MIL-101-NH2 and MD, respectively. It can be seen that DHA loading on MIL-101-NH2 results in a relatively rough surface. However, its octahedral structure remains unchanged, and the particle size is relatively uniform. This indicates that DHA loading has little impact on the morphology and structure of the nanoparticles.

[0047] (4) DLS particle size analysis of MIL-101-NH2, MD and MD@HF like Figure 5As shown, the hydrodynamic average particle sizes of MIL-101-NH2, MD, and MD@HF in ultrapure water are 271.0133±1.0309 nm, 305.3833±0.7528 nm, and 367.7667±0.6054 nm, respectively. The particle size results obtained by DLS are larger than those obtained by TEM. This is because hydrodynamic particle size is measured in a liquid medium using methods such as dynamic light scattering. Nanoparticles adsorb surrounding water molecules in the solution, forming a solvation layer, which leads to an inflated hydrodynamic particle size. In addition, in solution, nanoparticles aggregate due to interactions such as surface charge and van der Waals forces. DLS may measure the size of the aggregates, resulting in an inflated particle size. The PDI values ​​of MIL-101-NH2, MD, and MD@HF in ultrapure water were 0.0180±0.0121, 0.0583±0.0050, and 0.3150±0.0102, respectively, indicating that the prepared nanoparticles had good dispersibility in water.

[0048] (5) Zeta potential analysis of MIL-101-NH2, MD and MD@HF Figure 6 The figures show the Zeta potentials of MIL-101-NH2, MD, and MD@HF. MIL-101-NH2 exhibits a positive charge. With DHA loading, the charge of the nanoparticles decreases from 27.0567±1.0735 mV to 11.7633±0.6054 mV, indicating successful DHA loading. MD shows a significant negative charge after HA-FA modification, decreasing to -8.4700±0.3636 mV, indicating that negatively charged HA-FA is successfully coated on the surface of MD.

[0049] (6) Stability test of MD@HF like Figure 7 China A and Figure 7 As shown in Figure B, the stability of MD@HF in water was investigated. Dynamic light scattering (DLS) results showed that the size of MD@HF remained around 380 nm, and the polydispersity index (PDI) was around 0.4, indicating good stability.

[0050] (7) Thermogravimetric analysis (TGA) like Figure 8 China A and Figure 8As shown in Figure B, the three samples MIL-101-NH2, MD, and MD@HF all experienced a decrease in mass from room temperature to approximately 100°C. This may be due to the evaporation of moisture from the surface or some pores of the MOF material, or the evaporation of residual organic solvents (DMF, ethanol) from the synthesis or cleaning process. Further mass reduction in the 110-180°C range is likely due to the evaporation of strongly adsorbed interlayer water and organic solvents (DMF, ethanol) in the pores. During this stage, the mass of MIL-101-NH2, MD, and MD@HF decreased by 3.94%, 2.59%, and 2.98%, respectively. MIL-101-NH2 experienced the greatest mass loss, indicating that it contained more interlayer water and residual organic solvents.

[0051] Within the temperature range of 180-300℃, the mass losses of MIL-101-NH2, MD, and MD@HF were 9.2%, 9.50%, and 15.96%, respectively, possibly due to the partial removal of structured hydroxyl groups. However, the overall mass change was small, indicating good thermal stability under the experimental temperature conditions. The relatively large mass loss of MD@HF is likely due to the thermal decomposition of some side chains or the main chain of HA-FA.

[0052] Significant mass loss occurs within the 350-500℃ range, which may be due to the decomposition of the organic ligands in the material and the presence of some ferric iron in the form of oxides, leading to the destruction of the material structure and resulting in mass loss.

[0053] Within the temperature range of 500-700℃, the high temperature completely destroys and decomposes the material structure. By comparing the weight loss percentage of MD and MD@HF at 700℃, it can be shown that the modification amount of HA-FA coated on the MD surface is 3.898%.

[0054] (8) X-ray diffraction spectroscopy (XRD) analysis like Figure 9 As shown, MIL-101-NH2 exhibited characteristic diffraction peaks at 2θ = 8.4°, 8.9°, 10.5°, 16.3°, and 18.8°. These strong and sharp peaks indicate good crystallinity and purity, demonstrating the successful synthesis of MIL-101-NH2. The characteristic peaks of the DHA-loaded and HA-FA-coated MIL-101-NH2 were almost identical to those of MIL-101-NH2, with only a decrease in the intensity of some peaks. This indicates that neither the DHA loading nor the HA-FA coating on the surface of MIL-101-NH2 affected its crystal structure.

[0055] (9) Energy-dispersive X-ray spectroscopy (EDS) analysis EDS can detect the overall elemental composition and distribution of MIL-101-NH2. For example... Figure 10 As shown in A, B, C, D, E, and F, MIL-101-NH2 mainly contains four elements: C, N, O, and Fe. Among them, C is the most abundant element, with a wt% content of 78.58%, followed by O, with a wt% content of 16.83%, and Fe is present in a wt% content of 4.59%.

[0056] (10) Fourier transform infrared spectroscopy (FT-IR) analysis like Figure 11 As shown, in the infrared spectrum of MIL-101-NH2, 1575 cm⁻¹ -1 The absorption peak at 1381 cm⁻¹ is attributed to the vibration of the C=O bond in the carboxylate. -1 The absorption peak at 1257 cm⁻¹ is attributed to the vibration of the C-C bond on the aromatic ring of the carboxylate. -1 and 3353cm -1 The absorption peaks at 800-1200 cm⁻¹ are attributed to the stretching vibrations of the CN and NH bonds in 2-aminoterephthalic acid, indicating the successful synthesis of MIL-101-NH₂. Furthermore, these absorption peaks are also observed in the infrared spectra of MD and MD@HF, suggesting that drug loading did not alter the material's structure. In the infrared spectra of MD and MD@HF, the peaks in the 800-1200 cm⁻¹ range are also significant. -1 The characteristic absorption peak of DHA is located at 2940 cm⁻¹. This peak is attributed to the vibration of the OO and CO bonds in the peroxide (COOC) in DHA. In the infrared spectrum of DHA, this peak is at 2940 cm⁻¹. -1 and 2850cm -1 The peak at 3450 cm⁻¹ corresponds to the stretching vibrations of the methyl and ethyl groups in DHA, which are also reflected in the IR spectrum of MD. These results indicate that DHA has been successfully loaded onto MIL-101-NH₂. HA-FA at 3450 cm⁻¹... -1 There is an overlapping peak at 1656 cm⁻¹, formed by the superposition of absorption peaks of amino and hydroxyl groups. -1 There is a peak generated by the C=O stretching vibration of the carboxyl group, which is also present on MD@HF, indicating that HA-FA has been successfully coated on the surface of MD without changing the original structure of the material.

[0057] (11) Electron paramagnetic resonance (EPR) spectroscopy analysis Using DMPO as a trapping agent, the hydroxyl radicals (·OH) of MIL-101-NH2, MD, and MD@HF were detected. The EPR test results are as follows: Figure 12As shown, the EPR spectra of MIL-101-NH2, MD, and MD@HF exhibit a characteristic signal peak of 1:2:2:1, which is caused by DMPO-OH, indicating the type of ROS. MIL-101-NH2, MD, and MD@HF can all generate ·OH in the presence of H2O2, with the ability to generate free radicals increasing sequentially. The iron nodes (Fe) in MIL-101-NH2... 3+ It may be partially reduced to Fe by H2O2. 2+ And generate ·OH through the Fenton reaction. The porous structure of MIL-101-NH2 loaded with DHA may increase the local concentration of DHA and H2O2, thereby promoting reaction efficiency. The enhanced free radical generation ability of MD may be due to the peroxy-bridged bond (-OO-) of DHA in Fe... 2+ Catalytic decomposition produces additional ·OH. Simultaneously, DHA can act as a reducing agent to promote Fe... 3+ →Fe 2+ The cycling of MD@HF maintains the persistence of the Fenton reaction, resulting in a significantly stronger ·OH signal compared to MIL-101-NH2. The further increase in ·OH production in MD@HF may be attributed to: the HA-FA coating potentially delaying H2O2 diffusion and prolonging the reaction time; simultaneously, the low molecular weight HA fragments or carboxyl groups can promote iron-mediated ·OH formation, and the carboxyl and amino groups of FA can stabilize Fe ions, optimizing Fe... 2 + / Fe 3+ This ratio improves Fenton's efficiency.

[0058] (12) X-ray photoelectron spectroscopy (XPS) analysis Table 1 shows the percentages of Fe 2p, C 1s, O 1s, and N 1s in XPS.

[0059] The elemental composition and electron binding energy changes of MIL-101-NH2, MD, and MD@HF were characterized and analyzed by X-ray photoelectron spectroscopy (XPS), which can be used to measure the elements and chemical bonds present in a depth range of 10 nm on the sample surface. (Full scan spectrum) Figure 13 The results show that all three materials contain the four elements C, N, O, and Fe, indicating that the materials were successfully prepared. Figure 14 As shown in Figure A, MD@HF exhibits three peaks: 284.80 eV, 286.66 eV, and 288.69 eV, corresponding to C-C, CO, and C=O bonds, respectively. Compared to MIL-101-NH2 and MD, the peaks for CO and C=O bonds in MD@HF are significantly enhanced, attributed to the CO bond on the HA polysaccharide chain and the amide bond NC=O in the FA chain, respectively. The XPS spectrum of N 1s is shown below. Figure 14As shown in Figure B, the binding energies of MD@HF at 399.09 eV and 400.00 eV correspond to the -NH2 bond and CN bond, respectively. The decrease in binding energy at 400.00 eV may be due to DHA and HA-FA shading. The XPS spectrum of MD@HF at O ​​1s is shown below. Figure 14 As shown in Figure C, the binding energy at 532.42 eV is enhanced, possibly due to the increased OC=O bonds caused by the -COOH group of FA. The binding energies at 531.26 eV and 530.92 eV correspond to the oxygen in 2-aminoterephthalic acid and the oxygen in Fe-O, respectively. Figure 14 As shown in Figure D, the Fe 2p spectrum consists of three peaks. The binding energies at 725.67 eV, 723.63 eV, and 710.88 eV in MD@HF correspond to Fe 2p peaks, respectively. 1 / 2 Fe2p 3 / 2 satellite peaks and Fe 2p 3 / 2 This indicates that Fe in the sample 3+ The existence of.

[0060] (13) N2 adsorption-desorption analysis To characterize the specific surface area, pore structure, and pore size distribution of the materials as nano-formulations, BET tests were performed on MIL-101-NH2, MD, and MD@HF. BET testing can reveal the textural properties of the materials; their high specific surface area and abundant pore structure are beneficial for improving drug loading capacity, providing a structural basis for their excellent performance. As shown in Table 2, the specific surface area of ​​MIL-101-NH2 is 107.342 m² / g. 2 / g, with a relatively large pore volume of 0.472cm. 3 / g, which is consistent with its large pore size. MD has the largest specific surface area, reaching 369.830 m². 2 / g, pore volume is 0.505cm³ 3 The concentration of 0.164 cm² / g indicates that it possesses good porous properties. After HA-FA modification, its specific surface area decreased to 0.164 cm² / g. 3 / g, pore volume decreased to 0.164cm 3 The / g indicates successful HA-FA coating. However, with increasing HA-FA loading, it occupies a portion of the surface and pore space of the MD, reducing the effective surface and space available for nitrogen molecule adsorption. Furthermore, the average pore size of MIL-101-NH2 and the MD is too large, potentially leading to drug leakage after loading. HA-FA coating effectively prevents premature drug release.

[0061] Table 2 BET surface properties of MIL-101-NH2, MD, and MD@HF

[0062] like Figure 15 As shown, the nitrogen adsorption-desorption isotherms of MIL-101-NH2, MD, and MD@HF exhibit Type IV isotherm characteristics. They rise sharply in the low relative pressure region (P / P0), indicating the presence of numerous micropores. In the medium-to-high relative pressure region, significant capillary condensation accompanied by distinct hysteresis loops occurs, confirming the existence of mesoporous structures, which are predominantly mesoporous in all three materials. Furthermore, the hysteresis loops of MIL-101-NH2 close at higher relative pressures, and its adsorption capacity continues to increase in the high-pressure region, a typical characteristic of the MIL-101 series materials with large pore sizes and cage-like structures. The hysteresis loops of MD and MD@HF are relatively narrow and symmetrical, indicating a more uniform pore size distribution.

[0063] (14) In vitro release experiment like Figure 16 China A and Figure 16 As shown in Figure B, both MD and MD@HF release DHA under different pH conditions. DHA release essentially reaches equilibrium by 12 hours. MD releases DHA rapidly under acidic conditions (pH 5.0), with a cumulative release rate of (77.36 ± 4.96)% over 48 hours, while release is slower under neutral conditions (pH 7.4), with a cumulative release rate of (42.17 ± 4.14)% over 48 hours. MD@HF releases DHA at 56.72 ± 1.40% under acidic conditions (pH 5.0), but only 29.05 ± 3.88% under neutral conditions (pH 7.4). The higher DHA release rates in acidic environments compared to neutral environments indicate that DHA release in MD and MD@HF is pH-responsive. Because the MIL-101 framework is unstable in acidic environments, its lattice may partially dissociate or deform, causing the pores to open or expand, thus promoting DHA release. Compared to MD, MD@HF exhibits a lower DHA release rate under the same conditions, and its release is particularly slow at pH 7.4. This is because HA-FA acts as a protective layer on the outside of the nanoparticles. The carboxyl groups of HA undergo protonation, causing the molecular chains to coil or change conformation. This change, to some extent, hinders the direct contact between acidic substances and the surface of the nanoparticles, delaying the change in the nanoparticle structure. Consequently, MD@HF possesses a sustained-release function, preventing premature release of DHA during blood circulation.

[0064] Example 3 Antibacterial properties and wound healing promotion of pH-responsive MOF drug delivery system loaded with dihydroartemisinin 1. Cytotoxicity test The effects of MIL-101-NH2, MD, and MD@HF on HaCaT cell viability were detected using the CCK-8 assay. HaCaT cells were cultured at 5 × 10⁶ cells / year.3 Cells were seeded at a density of 1 / 2 well in 96-well plates and cultured at 37°C for 24 h. Then, the old medium was discarded, and 100 mL of DMEM medium containing different concentrations (0, 6, 12, 25, 50, 100, and 200 μg / mL) of MIL-101-NH2, MD, and MD@HF were added, and the cells were incubated for 24 h. The old medium was then discarded, and the cells were washed twice with PBS. 100 mL of fresh medium containing 10 mL of CCK-8 was added to each well, and the cells were incubated at 37°C for 2 h. The absorbance at 450 nm was then measured for each well. Cell viability was calculated using the formula.

[0065] ; : Absorbance of the experimental well; : Absorbance of blank well; : Absorbance of the control well.

[0066] 2. Experimental Results 1. Hemolysis test Hemolysis testing is the most effective and classic method for evaluating the biocompatibility of carriers or nanoparticles. When the hemolysis rate of a material is less than 5%, it meets the safety standards stipulated by national standards, indicating that the material is safe for in vivo application and can be used as a biomedical material. Figure 17 Figures A, B, and C show the hemolysis results of MIL-101-NH2, MD, and MD@HF, respectively. The hemolysis rates of both the blank carrier and the drug-loaded nanoparticles were below the national standard of 5% within the concentration range of 12-400 μg / mL. Even at a higher concentration of 400 μg / mL, the hemolysis rates were below 3.1%, indicating that MIL-101-NH2, MD, and MD@HF have good blood compatibility and can be used as biomedical materials for in vivo drug loading and delivery.

[0067] 2. Cytotoxicity The cytotoxicity of different concentrations of MIL-101-NH2, MD, and MD@HF on human immortalized keratinocytes (HaCaT cells) was evaluated using the CCK-8 assay. The results are as follows: Figure 18 As shown in Figures A, B, and C: With increasing concentration, MIL-101-NH2, MD, and MD@HF all exhibited certain toxicity to HaCaT cells. At the maximum concentration of 100 μg / mL, the cell viability of MIL-101-NH2, MD, and MD@HF remained above 80%, indicating that both the metal-organic framework MIL-101-NH2 and the drug-loaded nanoformulation possess good biocompatibility and low toxicity.

[0068] To evaluate the therapeutic effect of nanoparticles in promoting in vivo wound healing, a subcutaneous wound model of Staphylococcus aureus infection was established. First, a 10 mm diameter wound was constructed on the back of male SD rats, and 100 μL of Staphylococcus aureus solution (1×10⁻⁶) was injected. 6 CFUml -1 To establish an infected wound model, infected SD rats were randomly divided into seven groups: (I) Control(-), (II) S. aureus, (III) Control(+), (IV) DHA, (V) MIL-101-NH2, (VI) MD, and (VII) MD@HF. Figure 19 In vivo experiments were conducted using the procedure in (a), and photographs of rat wounds were recorded at different times. Figure 19 Figure (b) shows a schematic diagram of wound closure at different time points in different drug administration groups. It can be seen that the Control(-) and S. aureus groups exhibited severe local infection symptoms during treatment, with large areas still unhealed by day 14. This indicates that SD rats have very limited resistance to Staphylococcus aureus infection, making wound healing difficult. In contrast, due to the mild antibacterial ability of CDT, the abscesses in the other five experimental groups showed slight relief, and the treatment effect on infected wounds was significantly better than that in the Control(-) and S. aureus groups. Furthermore, the addition of DHA improved the therapeutic effect of CDT. The scar area decreased rapidly. This demonstrates that DHA, MIL-101-NH2, MD, and MD@HF all possess certain antibacterial properties. The quantitative statistical analysis of the wound size over time is as follows: Figure 19 As shown in (c), by day 14, approximately 18.7% and 28.7% of the wound area in the Control(-) and S. aureus groups, respectively, remained unhealed; MIL-101-NH2 showed limited wound healing ability, with approximately 7.6% of the wounds remaining unhealed; the wound area in the MD@HF treatment group was only approximately 5.9% or less. This directly demonstrates that MD@HF has a better ability to promote wound healing than MIL-101-NH2, showcasing the advantages of material design in promoting in vivo wound healing.

[0069] Therefore, this invention employs the aforementioned pH-responsive MOF drug delivery system loaded with dihydroartemisinin, its preparation method, and its application. The drug delivery system uses MIL-101-NH2 as a carrier, utilizing its porous structure and pH sensitivity to achieve efficient loading and pH-responsive release of DHA, thus improving drug targeting and bioavailability. Encapsulating MD with HA-FA not only improves the stability and biocompatibility of the drug delivery system but also endows it with a certain degree of active targeting, enhancing drug accumulation at the infection site and reducing damage to normal tissues. The preparation method has well-defined, stable, and controllable process parameters, good reproducibility, and is easy for industrial production. This drug delivery system exhibits good antibacterial properties and wound-healing capabilities, especially showing significant efficacy in treating wounds infected with Staphylococcus aureus, and has broad application prospects in the biomedical field.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a pH-responsive MOF drug delivery system loaded with dihydroartemisinin, characterized in that, Includes the following steps: Step 1, Preparation of MIL-101-NH2: 2-Aminoterephthalic acid and FeCl3•6H2O were placed in a polytetrafluoroethylene liner, N,N-dimethylformamide was added, and the mixture was magnetically stirred until completely dissolved. The mixture was then transferred to a high-pressure reactor and reacted at 120°C for 20 hours. After cooling, the mixture was centrifuged, washed, and vacuum dried to obtain MIL-101-NH2. Step 2, Preparation of HA-FA: Folic acid was dissolved in dimethyl sulfoxide and sonicated to dissolve. N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide were added, and the mixture was activated by reacting in a water bath at 30°C for 5 hours. The precipitate was removed by centrifugation. Hyaluronic acid was dissolved in formamide and stirred at 40°C to dissolve. The activated folic acid solution was added dropwise, and the mixture was reacted in the dark for 48 hours. The mixture was then centrifuged, dialyzed, and freeze-dried to obtain HA-FA. Step 3, Preparation of MD: Dissolve MIL-101-NH2 and dihydroartemisinin from Step 1 in anhydrous ethanol and sonicate. After mixing, place the mixture in a parallel reactor and stir magnetically in the dark under the conditions of drug loading time of 24 h, drug loading temperature of 34 °C, drug loading ratio of 0.63:1 and rotation speed of 300 rpm. After reaction, centrifuge and wash to obtain MD. Step 4, Preparation of MD@HF: Dissolve the MD from Step 3 and the HA-FA from Step 2 in ultrapure water and sonicate. Add the MD solution to the HA-FA solution and stir at room temperature for 36 hours in the dark. Centrifuge, wash, and vacuum dry to obtain the MD@HF drug delivery system.

2. The method for preparing the pH-responsive MOF drug delivery system loaded with dihydroartemisinin according to claim 1, characterized in that: In step 1, the amount of 2-aminoterephthalic acid used is 360 mg, the amount of FeCl3•6H2O used is 1 g, and the amount of N,N-dimethylformamide used is 40 mL; the centrifugation speed is 10000 rpm, and the centrifugation time is 5 min; the washing process involves washing three times each with N,N-dimethylformamide and anhydrous ethanol.

3. The method for preparing the pH-responsive MOF drug delivery system loaded with dihydroartemisinin according to claim 1, characterized in that: In step 2, the dosage of folic acid is 50 mg, the dosage of dimethyl sulfoxide is 3 mL, the dosage of N,N'-dicyclohexylcarbodiimide is 1 g, the dosage of N-hydroxysuccinimide is 288 mg, the dosage of hyaluronic acid is 100 mg, and the dosage of formamide is 10 mL. Ultrapure water is used for dialysis for 48 hours, and the water is changed every 6 hours.

4. The method for preparing the pH-responsive MOF drug delivery system loaded with dihydroartemisinin according to claim 1, characterized in that: In step 2, the grafting rate of FA in HA-FA is 17.69%.

5. The method for preparing the pH-responsive MOF drug delivery system loaded with dihydroartemisinin according to claim 1, characterized in that: In step 3, the amount of MIL-101-NH2 used is 10 mg, and the amount of anhydrous ethanol used is 5 mL; the amount of dihydroartemisinin used is 10 mg, and the amount of anhydrous ethanol used is 2 mL; the centrifugation speed is 10000 rpm, the centrifugation time is 5 min, and the product is washed twice with anhydrous ethanol.

6. The method for preparing the pH-responsive MOF drug delivery system loaded with dihydroartemisinin according to claim 1, characterized in that: In step 3, the drug loading rate of MD was 46.41±0.03%, and the encapsulation rate was 86.61±0.11%.

7. The method for preparing the pH-responsive MOF drug delivery system loaded with dihydroartemisinin according to claim 1, characterized in that: In step 4, the amount of MD used is 20 mg, the amount of HA-FA used is 4 mg, and the amount of ultrapure water used is 20 mL; the centrifugation speed is 10000 rpm, the centrifugation time is 5 min, and the product is washed three times with ultrapure water.

8. The method for preparing the pH-responsive MOF drug delivery system loaded with dihydroartemisinin according to claim 1, characterized in that: The drug loading rate of the MD@HF drug delivery system was 29.00±0.08%. The MD@HF drug delivery system is pH responsive, with a dihydroartemisinin release rate of 56.72±1.40% under acidic conditions at pH 5.0 and 29.05±3.88% under neutral conditions at pH 7.

4.

9. A pH-responsive MOF drug delivery system loaded with dihydroartemisinin, characterized in that: It is prepared by the preparation method described in any one of claims 1-8.

10. The application of the pH-responsive MOF drug delivery system loaded with dihydroartemisinin according to claim 9, characterized in that: It is used in the preparation of antibacterial drugs that inhibit the growth of Staphylococcus aureus.

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