Construction of dha@zif-8@ha / alin and its application in hormone-induced femoral head necrosis

Abstract

This invention discloses the construction of DHA@ZIF-8@HA / ALN and its application in hormone-induced osteonecrosis of the femoral head, relating to the field of nanomedicine technology. The invention includes: dropping DHA@ZIF-8 nanoparticles into an HA / ALN complex, reacting at room temperature, and centrifuging to obtain DHA@ZIF-8@HA / ALN; and constructing a DHA@ZIF-8@HA / ALN nanodelivery system with dual bone-macrophage targeting and pH-responsive drug release characteristics by loading dihydroartemisinin onto a zeolite imidazole ester backbone-8 nanocarrier modified with hyaluronic acid and alendronate sodium. This significantly improves the water solubility and bioavailability of DHA, achieving precise enrichment and responsive release of the drug at the SONFH lesion site, providing a safe and efficient new targeted drug delivery strategy for early hip-preserving treatment of SONFH.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, specifically to the construction of DHA@ZIF-8@HA / ALN and its application in hormone-induced avascular necrosis of the femoral head. Background Technology

[0002] Steroid-induced osteonecrosis of the femoral head (SONFH) is a challenging problem in orthopedics, and its pathogenesis is closely related to the disruption of the local bone immune microenvironment in the femoral head caused by long-term use of glucocorticoids. Advanced SONFH can lead to femoral head collapse, which can only be treated with total hip replacement, imposing a heavy medical and economic burden on patients and society.

[0003] Dihydroartemisinin (DHA), a highly active derivative of artemisinin, possesses both anti-inflammatory and bone metabolism-regulating biological activities. It can improve femoral head bone mineral density and trabecular bone structure in SONFH model rats by inhibiting macrophage M1 polarization and downregulating inflammatory cytokine secretion, demonstrating promising application potential in SONFH treatment. Zeolitic imidazolate frameworks-8 (ZIF-8) are a novel metal-organic framework material. Formed by the coordination of zinc ions with 2-methylimidazolium, they possess a porous crystal structure with good biocompatibility, pH sensitivity, and drug encapsulation capabilities. Hyaluronic acid (HA) can specifically bind to the CD44 receptor on the surface of macrophages, and alendronate sodium (ALN) has a high affinity for hydroxyapatite in bone tissue. Both provide important ligands for the targeted modification of nanocarriers.

[0004] Current methods for DHA delivery primarily rely on conventional formulations or simple nanocarrier encapsulation. While these methods can improve water solubility to some extent, they lack a dual-targeting design for bone and macrophages targeting SONFH lesions, hindering precise drug accumulation and resulting in low bioavailability and high potential risks of systemic side effects. Therefore, this invention proposes the construction of DHA@ZIF-8@HA / ALN and its application in hormone-induced avascular necrosis of the femoral head. Summary of the Invention

[0005] The purpose of this invention is to provide the construction of DHA@ZIF-8@HA / ALN and its application in hormone-induced osteonecrosis of the femoral head, in order to solve the technical problem that the lack of bone-macrophage dual targeting capability of existing nanodelivery systems leads to their low bioavailability in SONFH treatment.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing DHA@ZIF-8@HA / ALN, comprising the following steps:

[0007] S1. Dissolve zinc nitrate hexahydrate in deionized water to form an aqueous solution of zinc nitrate, dissolve 2-methylimidazole in methanol to form a 2-methylimidazole solution, and dissolve DHA in dimethylformamide to form a DHA solution.

[0008] S2. Mix 2-methylimidazole solution with DHA solution, add zinc nitrate aqueous solution under stirring at room temperature, mix and react for 3-5 min, then centrifuge, wash with methanol, and freeze dry to obtain DHA@ZIF-8 nanoparticles.

[0009] S3. Dissolve hyaluronic acid in deionized water, add N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, then add alendronate sodium solution dropwise, mix and react, and then dialyze to obtain HA / ALN complex.

[0010] S4. The DHA@ZIF-8 nanoparticles obtained in step S2 are added to the HA / ALN complex obtained in step S3, and the reaction is carried out at room temperature. After centrifugation, DHA@ZIF-8@HA / ALN is obtained.

[0011] Furthermore, in step S2, the centrifugation speed is 12000 rpm and the time is 10 min.

[0012] Furthermore, the methanol washing in step S2 is performed three times.

[0013] Furthermore, the amidation reaction of hyaluronic acid with sodium alendronate in step S3 is carried out in the presence of N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0014] Furthermore, the hydrodynamic particle size of the DHA@ZIF-8@HA / ALN is 130–160 nm, and the Zeta potential is -15–-20 mV.

[0015] Furthermore, the encapsulation rate of DHA in the DHA@ZIF-8@HA / ALN is above 85%, and the drug loading is above 10%.

[0016] Application of DHA@ZIF-8@HA / ALN in the preparation of drugs for treating hormone-induced avascular necrosis of the femoral head.

[0017] Compared with existing technologies, the construction of DHA@ZIF-8@HA / ALN and its application in hormone-induced avascular necrosis of the femoral head provided by this invention, by loading dihydroartemisinin into a zeolite imidazole ester backbone-8 nanocarrier modified with hyaluronic acid and alendronate sodium, a DHA@ZIF-8@HA / ALN nanodelivery system with dual bone-macrophage targeting and pH-responsive drug release characteristics was constructed. This significantly improved the water solubility and bioavailability of DHA, achieving precise enrichment and responsive release of the drug at the SONFH lesion site. At the same time, the modification of DHA effectively reduced the risk of hemolysis at high concentrations of ZIF-8-based carrier and greatly improved blood compatibility. Moreover, this nanosystem can significantly reverse the pathological damage of glucocorticoid-induced avascular necrosis of the femoral head and bring the bone microstructure close to the normal level, providing a safe and efficient new targeted drug delivery strategy for early hip-preserving treatment of SONFH. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0019] Figure 1 A diagram of a UV-Vis spectrophotometer provided in an embodiment of the present invention;

[0020] Figure 2 The transmission electron microscope scan image provided in the embodiment of the present invention;

[0021] Figure 3 Particle size distribution of DHA@ZIF-8@HA / ALN provided in this embodiment of the invention;

[0022] Figure 4 A pH response result diagram provided in an embodiment of the present invention;

[0023] Figure 5 Hemolysis level map provided for embodiments of the present invention;

[0024] Figure 6 Cell viability diagram provided for embodiments of the present invention;

[0025] Figure 7 Micro-CT scan images provided in embodiments of the present invention;

[0026] Figure 8 This is an image showing the HE staining results provided in an embodiment of the present invention. Detailed Implementation

[0027] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0028] Example 1:

[0029] Construction of DHA@ZIF-8@HA / ALN:

[0030] 1. Dissolve 75 mg zinc nitrate hexahydrate in 2.5 mL deionized water, 165 mg 2-methylimidazole in 4.5 mL methanol, and 10 mg DHA in 0.5 mL dimethylformamide. Stir at room temperature.

[0031] 2. Adding zinc nitrate aqueous solution to the 2-methylimidazole and DHA solution, the synthesis solution rapidly becomes turbid. After 3-5 min, DHA@ZIF-8 nanoparticles form. Centrifuging the solution for 10 min yields the DHA@ZIF-8 nanoparticles. The DHA@ZIF-8 nanoparticles are then washed three times with methanol to completely remove unreacted reagents. All supernatants are collected for measuring the encapsulation efficiency and drug loading of DHA. Finally, the nanoparticles are freeze-dried and stored at -20°C for further use.

[0032] 3. Hyaluronic acid (HA) was dissolved in deionized water, then N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added and stirred to activate the carboxyl groups. Subsequently, ALN (alendronate sodium) solution was added dropwise to the mixture, and the reaction continued for 24 h. The mixture was then dialyzed for 42 h using a dialysis bag to remove excess 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and free HA.

[0033] 4. Add the prepared DHA@ZIF-8 dropwise into HA / ALN and react at room temperature for 24 hours. After centrifugation, obtain DHA@ZIF-8@HA / ALN.

[0034] Example 2:

[0035] Please see Figure 1 The encapsulation efficiency and drug loading of DHA@ZIF-8@HA / ALN were determined using a UV-Vis spectrophotometer. The encapsulation efficiency and drug loading of the nanocarrier were evaluated by subtracting the residual DHA in the supernatant from the total feed amount.

[0036] The drug was dissolved to release DHA, and the drug loading efficiency of the nanocarrier was calculated based on the standard curve and UV-Vis spectrophotometry. The calculation formula is shown below:

[0037] Drug encapsulation efficiency = (Mass of drug in microspheres / Dosage amount) × 100%;

[0038] Drug loading rate = (mass of drug in microspheres / mass of microspheres weighed) × 100%;

[0039] The encapsulation efficiency of the material was calculated to be 85.3%, and the drug loading was 10.4%.

[0040] Example 3:

[0041] Please see Figure 2 The morphology of DHA@ZIF-8@HA / ALN was observed using transmission electron microscopy (TEM). DHA@ZIF-8@HA / ALN was prepared using the method described in Example 1, with DHA@ZIF-8 serving as a control. The TEM scans showed that the shape of DHA@ZIF-8 modified with HA and ALN changed from hexagonal to circular.

[0042] Example 4:

[0043] Please see Figure 3 The particle size of DHA@ZIF-8@HA / ALN was detected using a nanoparticle tracking analyzer (NTA, Malvern NanoSight). DHA@ZIF-8@HA / ALN was prepared using the method described in Example 1, with DHA@ZIF-8 serving as a control. The hydrodynamic particle size distribution of DHA@ZIF-8 showed an average particle size intensity distribution of 140.1 nm and a PDI of 0.069, indicating uniform particle size distribution. The average zeta potential was -18.3 mV, indicating that the negatively charged HA coating on the surface of DHA@ZIF-8@HA / ALN caused a zeta potential reversal.

[0044] Example 5:

[0045] Please see Figure 4 pH-responsive release of DHA@ZIF-8@HA / ALN.

[0046] Using the DHA@ZIF-8@HA / ALN provided in Example 1 as a sample, its release rate was tested. The pH-responsive release experiment results showed that the DHA@ZIF-8@HA / ALN system exhibited a significant acceleration in drug release under strongly acidic conditions (pH 1-2), with a release rate significantly higher than under neutral and weakly alkaline conditions. As the pH increased (pH 3-10), the release rate rapidly decreased and remained at a low level, indicating that the carrier structure was stable within this pH range. Under strongly alkaline conditions (pH 11-13), the release rate increased again, but the overall release amplitude was lower compared to the acidic environment. The overall curves showed a typical "V-shaped" or "U-shaped" trend, consistent with the expected pH-responsive release characteristics, especially with faster release in acidic environments, indicating that this carrier system has good acid-responsive drug release capability.

[0047] Example 6:

[0048] Please see Figure 5 Hemolysis experiment of DHA@ZIF-8@HA / ALN.

[0049] DHA@ZIF-8@HA / ALN was prepared using the method described in Example 1, with ZIF-8@HA / ALN serving as a control. Hemolysis experiments showed that both ZIF-8@HA / ALN and DHA@ZIF-8@HA / ALN exhibited low hemolysis rates (<10%) within the concentration range of 40-640 ng / kg, but there were significant differences in their biocompatibility. The unmodified ZIF-8@HA / ALN showed a significantly increased hemolysis rate at high concentrations of 320 ng / kg and 640 ng / kg, exhibiting a concentration-dependent hemolysis trend, indicating that the material may have some impact on erythrocyte membrane stability at high concentrations.

[0050] In contrast, DHA@ZIF-8@HA / ALN maintained stable low hemolysis levels across all tested concentrations. Even at high concentrations of 320 ng / kg and 640 ng / kg, its hemolysis rate did not increase significantly, demonstrating superior blood compatibility. This indicates that DHA modification significantly improves the biocompatibility of the material system, effectively suppressing the potential hemolysis risk at high concentrations and broadening the safe concentration range for use. Therefore, DHA@ZIF-8@HA / ALN not only inherits the low hemolysis characteristics of ZIF-8@HA / ALN but also further enhances its blood safety through DHA modification, providing strong support for its application as a safe and efficient anti-inflammatory delivery system in subsequent in vivo studies.

[0051] Example 7:

[0052] Please see Figure 6 Cell compatibility assays of DHA@ZIF-8@HA / ALN with RAW264.7 cells. In vitro cytotoxicity assays were performed using the CCK-8 assay to evaluate the effects of different drugs on cell viability. DHA-free ZIF-8@HA / ALN was prepared using the method provided in Example 1, with DHA and DHA@ZIF serving as controls.

[0053] The results showed that free DHA exhibited significant concentration- and time-dependent cytotoxicity. After 12 hours of treatment, low concentrations could slightly promote cell proliferation. When the concentration was ≥5 μg / mL, cell viability decreased significantly with increasing concentration. When the concentration was greater than 50 μg / mL, cell viability was less than 20%. After 24 hours of treatment, the toxicity was further enhanced. When the concentration was greater than 0.5 μg / mL, significant cell viability inhibition was observed. Compared to free DHA, the cytotoxicity of each nanoparticle was significantly reduced: the ZIF-8@HA / ALN blank carrier maintained more than 70% cell viability at a concentration of 75 μg / mL; DHA@ZIF-8 maintained cell viability of more than 70% at concentrations below 75 μg / mL, with a safe dose much higher than that of free DHA at the same concentration; DHA@ZIF-8@HA / ALN had no significant effect on cell viability at treatment times of 12 h and 24 h and concentrations below 20 μg / mL, with significant inhibition only at high concentrations. This confirms that the nanodelivery system can effectively reduce the cytotoxicity of DHA and has excellent in vitro biosafety, providing a safe basis for subsequent in vivo applications.

[0054] Example 8:

[0055] Please see Figure 7-8 The effect of DHA@ZIF-8@HA / ALN on SONFH in RAW264.7 rats

[0056] 1. Micro-CT

[0057] Micro-CT scanning and 3D reconstruction results showed that after one month of continuous injection of methylprednisolone, the SONFH model group showed obvious femoral head necrosis. Compared with the SONFH model group, the free DHA intervention group showed a significant reduction in the range of cystic changes in the femoral head and a significant improvement in trabecular bone fracture, confirming that DHA can effectively alleviate the pathological damage of glucocorticoid-induced femoral head necrosis. Compared with the free DHA group, the continuity and integrity of the trabecular bone in the femoral head of the DHA@ZIF-8 nano-drug-loaded group were further improved, and the repair effect of the necrotic area was more significant. The improvement effect of the DHA@ZIF-8@HA / ALN dual-target nano-delivery system intervention group was the most outstanding. The trabecular bone in the femoral head of the rats was arranged regularly and the structure was continuous. The cystic changes basically disappeared, and the bone microstructure was close to the level of the femoral head of normal rats, which fully verified that the dual-target nano-delivery system can significantly enhance the therapeutic efficacy of DHA on SONFH. Figure 7 ).

[0058] 2. HE staining results

[0059] HE staining results showed that: in the SONFH group, the trabeculae of the femoral head of rats were significantly thinned and fractured, with a large number of hollow lacunae visible within the trabeculae, and the number of adipocytes in the medullary cavity was significantly increased, with extensive adipocyte infiltration; in the DHA group, the fracture, thickness, and number of trabeculae in the femoral head of rats were improved, the number of hollow lacunae was relatively reduced, and the adipocyte infiltration in the medullary cavity was somewhat alleviated; in the DHA@ZIF-8 group, the continuity and morphology of the trabeculae of the femoral head of rats were further improved, the number of hollow lacunae was significantly reduced, and the degree of adipocyte infiltration in the medullary cavity was significantly reduced; in the DHA@ZIF-8@HA / ALN group, the trabeculae of the femoral head of rats were intact and regularly arranged, the number of hollow lacunae was extremely small, the adipocyte infiltration in the medullary cavity was basically eliminated, and the histological morphology was close to that of normal rat femoral head.

[0060] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for preparing DHA@ZIF-8@HA / ALN, characterized in that, Includes the following steps: S1. Dissolve zinc nitrate hexahydrate in deionized water to form an aqueous solution of zinc nitrate, dissolve 2-methylimidazole in methanol to form a 2-methylimidazole solution, and dissolve DHA in dimethylformamide to form a DHA solution. S2. Mix 2-methylimidazole solution with DHA solution, add zinc nitrate aqueous solution under stirring at room temperature, mix and react for 3-5 min, then centrifuge, wash with methanol, and freeze dry to obtain DHA@ZIF-8 nanoparticles. S3. Dissolve hyaluronic acid in deionized water, add N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, then add alendronate sodium solution dropwise, mix and react, and then dialyze to obtain HA / ALN complex. S4. The DHA@ZIF-8 nanoparticles obtained in step S2 are added to the HA / ALN complex obtained in step S3, and the reaction is carried out at room temperature. After centrifugation, DHA@ZIF-8@HA / ALN is obtained.

2. The preparation method of DHA@ZIF-8@HA / ALN according to claim 1, characterized in that, In step S2, the centrifugation speed is 12000 rpm and the time is 10 min.

3. The preparation method of DHA@ZIF-8@HA / ALN according to claim 1, characterized in that, The methanol washing in step S2 is performed three times.

4. The preparation method of DHA@ZIF-8@HA / ALN according to claim 1, characterized in that, The amidation reaction of hyaluronic acid with sodium alendronate in step S3 is carried out in the presence of N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

5. The preparation method of DHA@ZIF-8@HA / ALN according to claim 1, characterized in that, The hydrodynamic particle size of the DHA@ZIF-8@HA / ALN is 130–160 nm, and the zeta potential is -15–-20 mV.

6. The preparation method of DHA@ZIF-8@HA / ALN according to claim 1, characterized in that, The encapsulation rate of DHA in the DHA@ZIF-8@HA / ALN is over 85%, and the drug loading is over 10%.

7. The use of DHA@ZIF-8@HA / ALN prepared by any one of claims 1-6 in the preparation of a drug for treating hormone-induced avascular necrosis of the femoral head.

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