Drug-loading targeting nano-platform for targeted delivery of ZIF-8 drug-loading nano-particles by macrophages, preparation method and application of drug-loading targeting nano-platform
By targeting and delivering ZIF-8 drug-loaded nanoparticles to macrophages, and utilizing the EMS microenvironment and the chemotactic ability of macrophages, highly efficient targeted therapy of EMS lesions was achieved, reducing damage to normal tissues and solving the problems of high side effects and recurrence rates of existing treatment methods.
Patent Information
- Application Number
- CN202511226512.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-29
- Publication Date
- 2025-12-16
AI Technical Summary
Existing treatments for EMS, such as hormone drugs and surgical interventions, have problems such as large side effects and high recurrence rates. Furthermore, existing targeted therapies have broad target points, making it difficult to effectively target EMS lesions.
ZIF-8 drug-loaded nanoparticles were delivered via macrophage targeting. The drugs camel pine or levonorgestrel were loaded onto the ZIF-8 nanoparticle carriers, and the drugs were delivered to EMS lesions by taking advantage of the chemotactic ability of macrophages. The controlled release of the drugs was achieved by combining the acidic characteristics of the EMS microenvironment.
It improves the drug's targeting and uptake rate in EMS lesions, reduces damage to normal tissues, significantly inhibits ectopic endometrial cells, reduces drug toxicity and side effects, and has a simple preparation method with low environmental dependence and high stability.
Smart Images
Figure CN121130106A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology, specifically relating to a drug-loaded targeted nanoplatform for macrophage-targeted delivery of ZIF-8 drug-loaded nanoparticles, its preparation method, and its application. Background Technology
[0002] Endometriosis (EMS) is a common disease among women of reproductive age, characterized by the growth of endometrial tissue outside the uterine cavity, leading to symptoms such as chronic pelvic pain, dysmenorrhea, and infertility. EMS not only severely impacts patients' quality of life but also places a significant burden on social medical resources. Furthermore, the lesions are highly invasive and prone to recurrence.
[0003] Currently, treatment options for EMS mainly include hormonal medications and surgical intervention. Hormonal medications, such as oral contraceptives and gonadotropin-releasing hormone agonists (GnRH-a), can relieve symptoms, but long-term use can lead to side effects such as bone density loss and vasomotor dysregulation. As for surgical treatment, although it can directly remove the lesion, the recurrence rate is high, with approximately 75% of patients experiencing symptom recurrence within 2-9 years post-surgery. Furthermore, the surgery carries significant risks, potentially leading to complications such as pelvic adhesions and urinary system dysfunction.
[0004] In recent years, researchers have also focused on applying targeted therapy to the treatment of endometriosis. For example, in 2024, researchers used a mifepristone-coated fetal bovine serum albumin (FBO) targeted nanoplatform for endometriosis treatment, and in 2025, researchers used albumin-cerium oxide nanoparticles as a STAT3 inhibitor for endometriosis treatment. Although both methods utilize FBO or albumin respectively to enhance targeting, their treatment methods are still largely confined to hormone therapy; furthermore, they rely solely on protein targeting of the inflammatory environment, resulting in a relatively broad target. Therefore, developing novel targeted therapy strategies that incorporate the characteristics of the EMS microenvironment has become an urgent need for EMS treatment. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a drug-loaded targeted nanoplatform for macrophage-targeted delivery of ZIF-8 drug-loaded nanoparticles, its preparation method, and its application.
[0006] This invention targets the microenvironment of endometriosis by delivering drugs through macrophages coated with ZIF-8 drug-loaded nanoparticles, which effectively enhances the targeting of the delivery. Furthermore, the selected drugs are based on energy metabolism levels and have stronger killing specificity for M2 cells, which can effectively treat lesions while reducing damage to normal cells.
[0007] The first objective of this invention is to provide a drug-loaded targeted nanoplatform for macrophage-targeted delivery of ZIF-8 drug-loaded nanoparticles, comprising ZIF-8 drug-loaded nanoparticles and macrophages targeted and modified on the outermost layer of the ZIF-8 drug-loaded nanoparticles.
[0008] Preferably, the ZIF-8 drug-loaded nanoparticles include a ZIF-8 nanoparticle carrier and a drug loaded on the ZIF-8 nanoparticle carrier.
[0009] Preferably, the drug is harlotinib (HA) or levonorgestrel (LNG).
[0010] Preferably, the ZIF-8 nanoparticle carrier has a particle size of 91.28–295.31 nm, and the ZIF-8 drug-loaded nanoparticles have a particle size of 141.77–396.06 nm.
[0011] A second objective of this invention is to provide a method for preparing the above-mentioned macrophage-targeted delivery ZIF-8 drug-loaded nanoparticle platform, comprising the following steps:
[0012] Step 1: Zinc nitrate hexahydrate and 2-methylimidazole were used to synthesize zeolite-type imidazole salt framework 8-nanometer particles, namely ZIF-8 nanoparticle carriers.
[0013] The molar ratio of zinc nitrate hexahydrate to 2-methylimidazole is 1:8.2–11;
[0014] Step 2: Add drugs to ZIF-8 nanoparticle carriers to synthesize ZIF-8 drug-loaded nanoparticles;
[0015] The mass ratio of drug to ZIF-8 nanoparticle carrier is 2–4.2:1;
[0016] Step 3: Incubate macrophages with ZIF-8 drug-loaded nanoparticles for 2 hours, wash, transfer cells, and obtain a drug-loaded targeted nanoplatform.
[0017] Preferably, step 1 includes the following steps:
[0018] Step 11: Weigh zinc nitrate hexahydrate and 2-methylimidazole in a molar ratio of 1:8.2 to 11, and dissolve them in methanol solution to obtain zinc nitrate hexahydrate solution and 2-methylimidazole solution, respectively.
[0019] Step 12: While stirring, add zinc nitrate hexahydrate solution to 2-methylimidazole solution, and then stir at a constant speed for 3 hours under closed conditions to obtain a light white suspension.
[0020] Step 13: After centrifuging the light white suspension multiple times, retain the precipitate, dry it, and obtain the ZIF-8 nanoparticle carrier.
[0021] Preferably, step 2 includes the following steps:
[0022] Step 21: Weigh the drug and ZIF-8 nanoparticle carrier in a mass ratio of 2 to 4.2:1, and dissolve them in methanol solution to obtain ZIF-8 nanoparticle carrier suspension and drug solution respectively.
[0023] Step 22: While stirring, add the drug solution to the ZIF-8 nanoparticle carrier suspension, and then stir at a constant speed for 24 hours in a closed state to obtain a light white suspension.
[0024] Step 23: After centrifuging the light white suspension multiple times, retain the precipitate, dry it, and obtain ZIF-8 drug-loaded nanoparticles.
[0025] Preferably, in step 3, macrophages are extracted using the following method:
[0026] Step 31: Wash bone marrow macrophages with DMEM medium, centrifuge, and separate the cells into single-cell suspensions;
[0027] Step 32: Under conditions of 37℃ and 5% CO2, the single-cell suspension is inoculated onto the cell culture medium for culture. On the 3rd to 4th day of culture, half of the culture medium is replaced, and on the 5th day, all the culture medium is replaced. Macrophages can be obtained on the 7th day.
[0028] The third objective of this invention is to provide an application of the above-mentioned macrophage-targeted delivery ZIF-8 drug-loaded nanoparticle platform in the preparation of drugs for treating endometriosis.
[0029] Compared with the prior art, the advantages of this invention are as follows:
[0030] (1) This invention uses ZIF-8 nanoparticles combined with HA or LNG targeting energy metabolism levels as a carrier to target endometriosis cells with an acidic cellular microenvironment. In the acidic environment of the lesion, the drug is released in a controlled manner, and the effective uptake of the drug by endometriosis cells is enhanced, thereby effectively increasing the uptake rate and the drug concentration at the target site. This is beneficial to improving the efficacy of the drug against endometriosis, while reducing the distribution of the drug in normal tissues or cells and reducing the toxic side effects of the drug.
[0031] (2) This invention combines macrophages with ZIF-8 nanoparticle carriers. By utilizing the natural chemotactic ability of macrophages to actively migrate to the site of inflammation or lesion, the drug targeting of ZIF-8 nanoparticle carriers can be significantly improved.
[0032] (3) In vitro experiments showed that the drug-loaded macrophages had good migration ability and significant inhibitory effect on ectopic endometrial cells; in vivo experiments showed that the drug-loaded targeted nanoplatform provided by the present invention can effectively reduce the size, volume and weight of ectopic lesions and has good biocompatibility.
[0033] (4) The preparation method provided by the present invention is simple and has low dependence on environmental conditions (temperature and humidity). It can be carried out stably at room temperature (20–30℃) and in the normal humidity range (relative humidity 30–70%). There is no need to precisely control environmental parameters. Under uncontrolled conditions, the consistency and yield of the product can still be maintained. The yield is basically stable at about 93.4%, and the success rate is high. Attached Figure Description
[0034] Figure 1 This is a flowchart illustrating the preparation process of the drug-loaded targeted nanoplatform for macrophage-targeted delivery of ZIF-8 drug-loaded nanoparticles provided in Example 1 of the present invention.
[0035] Figure 2 This is a particle size distribution diagram of the ZIF-8 drug-loaded nanoparticles and ZIF-8 nanoparticle carrier in the embodiments of the present invention; wherein, A is the ZIF-8 nanoparticle carrier; B is the ZIF-8 drug-loaded nanoparticle (drug is HA); C is the ZIF-8 drug-loaded nanoparticle (drug is LNG).
[0036] Figure 3 These are SEM and TEM images of the ZIF-8 drug-loaded nanoparticles in the embodiments of the present invention at different scales.
[0037] Figure 4 The image shows the infrared spectrum of the ZIF-8 drug-loaded nanoparticles in this embodiment of the invention; where A represents the ZIF-8 drug-loaded nanoparticles (the drug is HA); and B represents the ZIF-8 drug-loaded nanoparticles (the drug is LNG).
[0038] Figure 5 The purity of BMDM cultured for 5 days using F4 / 80 and CD11b double staining was analyzed by flow cytometry in Example 1 of this invention.
[0039] Figure 6 The images are BMDM scanning confocal microscopy images of ZIF-8 drug-loaded nanoparticles after cell uptake; where AC represents ZIF-8 drug-loaded nanoparticles (drug is HA) at different magnifications; DF represents ZIF-8 drug-loaded nanoparticles (drug is LNG) at different magnifications.
[0040] Figure 7 Staining results of live / dead cells 2 hours after loading BMDM, HA@ZIF-8@BMDM and LNG@ZIF-8@BMDM (scale bar = 50 μm);
[0041] Figure 8 Figures show the Transwell experimental results for BMDM, HA@ZIF-8@BMDM, and LNG@ZIF-8@BMDM; where A is an optical microscope image stained with crystal violet; and B is the quantitative measurement result.
[0042] Figure 9 The image shows the expression of M1 / M2 macrophages in the peritoneal fluid of mice in the drug-loaded targeted nanoplatform provided in this embodiment of the invention and the untreated group; where A is untreated BMDM; B is HA@ZIF-8@BMDM; and C is LNG@ZIF-8@BMDM.
[0043] Figure 10 The images show a comparison of the autologous graft volume between the drug-loaded targeted nanoplatform provided in this embodiment of the invention and the untreated group of mice; where A represents the appearance comparison of the autologous graft volume; and B represents the quantitative measurement results.
[0044] Figure 11 The H&E staining results of the drug-loaded targeted nanoplatform and the major organs of untreated mice provided in the embodiments of the present invention are shown in the figure. Detailed Implementation
[0045] The following will be described in conjunction with embodiments of the present invention. Figures 1 to 11 The technical solutions in the embodiments of the present invention are clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0046] Example 1
[0047] This invention provides a drug-targeted nanoplatform for macrophage-targeted delivery of ZIF-8 drug-loaded nanoparticles, comprising ZIF-8 drug-loaded nanoparticles and macrophages targeted and modified on the outermost layer of the ZIF-8 drug-loaded nanoparticles.
[0048] In this embodiment of the invention, the ZIF-8 drug-loaded nanoparticles include a ZIF-8 nanoparticle carrier and a drug loaded on the ZIF-8 nanoparticle carrier.
[0049] In this embodiment of the invention, the drug is camel vesicant (HA).
[0050] In this embodiment of the invention, the particle size of the ZIF-8 nanoparticle carrier is 91.28–295.31 nm, and the particle size of the ZIF-8 drug-loaded nanoparticles is 141.77–396.06 nm.
[0051] This invention also provides a method for preparing the above-mentioned macrophage-targeted delivery ZIF-8 drug-loaded nanoparticle platform, specifically including the following steps:
[0052] Step 1: Synthesize zeolite-type imidazole salt framework 8-nanometer particles, namely ZIF-8 nanoparticle carriers, using zinc nitrate hexahydrate and 2-methylimidazole. This process includes the following steps:
[0053] Step 11: Weigh 4g of 2-methylimidazole powder and 1.45g of zinc nitrate hexahydrate crystals using a precision balance, and dissolve them in 40mL and 20mL of analytical grade methanol solution, respectively, to obtain zinc nitrate hexahydrate solution and 2-methylimidazole solution.
[0054] Step 12: Add the 2-methylimidazole solution to a flat-bottomed flask and place it on a magnetic stirrer at 550 rpm at room temperature. -1 Stir at a constant speed, and at the same time, add zinc nitrate hexahydrate solution slowly and steadily to 2-methylimidazole solution at a speed of 1 drop / s. Stir and mix thoroughly. Cover the mouth of the flask with multiple layers of sealing film to ensure that the methanol solution does not evaporate in large quantities and cause changes in the concentration of the solution system. After stirring at a constant speed for 3 hours, remove the sealing film and quickly dispense the resulting light white suspension into several 50 mL centrifuge tubes.
[0055] Step 13: Balance the light white suspension in several centrifuge tubes and place them in a centrifuge. Centrifuge at 5500 rpm for 5 minutes, then discard the supernatant. Add an equal volume of methanol solution as before centrifugation. Invert the centrifuge tubes (or shake the machine) to resuspend the milky white ZIF-8 precipitate. Centrifuge at 8500 rpm for 5 minutes. Discard the supernatant. Drill several vent holes in the centrifuge tube caps and place the samples in a 60℃ oven to dry overnight (6-8 hours). After overnight drying, pour out the ZIF-8 powder and grind it into a fine powder using an agate mortar. Seal the powder and store it in a bottle at room temperature to obtain the ZIF-8 nanoparticle carrier, i.e., ZIF-8.
[0056] Step 2: Camelamine (HA) is added to the ZIF-8 nanoparticle carrier to synthesize ZIF-8 drug-loaded nanoparticles, which specifically includes the following steps:
[0057] Step 21: Accurately weigh 30 mg of ZIF-8 nanoparticles and 72 mg of camel thorn alkaloid (HA) using an analytical balance, and dissolve them in 10 ml of methanol analytical grade solution to obtain ZIF-8 nanoparticle carrier suspension and HA solution.
[0058] Step 22: Pour the ZIF-8 nanoparticle carrier suspension into a flask and place it on a magnetic stirrer at 550 r·min at room temperature. -1Stir the mixture at a constant speed while simultaneously adding the HA solution dropwise at a rate of 1 drop / s, ensuring thorough mixing. Seal the flask with sealing film. After stirring at a constant speed for 24 hours, remove the sealing film and quickly aliquot the resulting light white suspension into 50 mL centrifuge tubes.
[0059] Step 23: Balance the light white suspension in several centrifuge tubes and centrifuge at 5500 rpm for 5 minutes, then discard the supernatant. Add an equal volume of methanol solution as before centrifugation, invert the centrifuge tubes (or shake the machine) to resuspend the milky white ZIF-8 precipitate, and centrifuge at 8500 rpm for 5 minutes. Repeat the above steps until the supernatant is clear, then discard the supernatant. Dissolve the supernatant in 1 ml of methanol, transfer it to a 10 ml centrifuge tube, seal and dry. Drill several vent holes in the centrifuge tube cap and place the sample in a 60°C oven to dry, then let it stand overnight (6-8 hours). The next day, pour out the powder, grind it into a fine powder using an agate mortar and pestle, seal it in a bottle, and store it at room temperature to obtain ZIF-8 drug-loaded nanoparticles, i.e., HA@ZIF-8. In this example, the mass of the ZIF-8 drug-loaded nanoparticles is approximately 95.27 mg, and the yield is approximately 94%.
[0060] Step 3: Incubate macrophages with ZIF-8 drug-loaded nanoparticles for 1 hour, wash, transfer cells to obtain the drug-loaded targeted nanoplatform, namely HA@ZIF-8@BMDM;
[0061] In step 3, macrophages were extracted using the following method:
[0062] Step 31: Use a 26G puncture needle to wash bone marrow macrophages with DMEM culture medium, centrifuge (1500 rpm, 6 min) and separate the cells into a single-cell suspension;
[0063] Step 32: Under conditions of 37℃ and 5% CO2, single-cell suspensions were seeded into 10cm culture dishes containing 10mL of cell culture medium. Half of the medium was replaced on day 3, and all the medium was replaced on day 5. Macrophages (BMDM) were obtained on day 7. The differentiation status of BMMs was detected by FITC-F4 / 80 and PE-CD11b double staining flow cytometry. The results are as follows: Figure 5 As shown, through Figure 5 The results showed that over 98% of the cells were CD11b+F4 / 80+, confirming that the cell purity was sufficient for experimental use.
[0064] In this embodiment, the cell culture medium consists of 15% FBS, 1% antibiotics, 40% L929 cell conditioned medium (L929-CM), and 44% DMEM medium. The L929 cell conditioned medium (L929-CM) is prepared by culturing L929 cells to a confluence state, continuing the culture for one week, and then centrifuging, filtering, and collecting the supernatant.
[0065] In this embodiment, bone marrow macrophages were prepared by euthanizing 8-12 week old C57BL / 6 mice and then separating their femurs and tibias.
[0066] In this embodiment, step S3 specifically includes the following steps:
[0067] Macrophage BMDMs were seeded in 12-well plates (8*10). 5 Cells were placed on a plate (1 cell / well) and then incubated with cell culture medium containing HA@ZIF-8 (HA@ZIF-8 concentration 6 mg / ml) for 1 h. Untaken particles were gently washed away with PBS solution, repeated three times. Cells were gently scraped off with a cell scraper, and the cells were transferred to centrifuge tubes with 5 ml of cell culture medium and frozen at -40°C to obtain HA@ZIF-8@BMDM.
[0068] Example 2
[0069] This invention provides a drug-targeted nanoplatform for macrophage-targeted delivery of ZIF-8 drug-loaded nanoparticles, comprising ZIF-8 drug-loaded nanoparticles and macrophages targeted and modified on the outermost layer of the ZIF-8 drug-loaded nanoparticles.
[0070] In this embodiment of the invention, the ZIF-8 drug-loaded nanoparticles include a ZIF-8 nanoparticle carrier and a drug loaded on the ZIF-8 nanoparticle carrier.
[0071] In this embodiment of the invention, the drug is camel vesicant (HA).
[0072] In this embodiment of the invention, the particle size of the ZIF-8 nanoparticle carrier is 91.28–295.31 nm, and the particle size of the ZIF-8 drug-loaded nanoparticles is 141.77–396.06 nm.
[0073] This invention also provides a method for preparing the above-mentioned macrophage-targeted delivery ZIF-8 drug-loaded nanoparticle platform, specifically including the following steps:
[0074] Step 1: Synthesize zeolite-type imidazole salt framework 8-nanometer particles, namely ZIF-8 nanoparticle carriers, using zinc nitrate hexahydrate and 2-methylimidazole. This process includes the following steps:
[0075] Step 11: Weigh 4g of 2-methylimidazole powder and 1.68g of zinc nitrate hexahydrate crystals using a precision balance, and dissolve them in 40mL and 20mL of analytical grade methanol solution, respectively, to obtain zinc nitrate hexahydrate solution and 2-methylimidazole solution.
[0076] Step 12: Add the 2-methylimidazole solution to a flat-bottomed flask and stir it at a constant speed of 550 r·min⁻¹ on a magnetic stirrer at room temperature. At the same time, add the zinc nitrate hexahydrate solution slowly and steadily to the 2-methylimidazole solution at a speed of 1 drop / s. Stir thoroughly and cover the mouth of the flask with multiple layers of sealing film to ensure that the methanol solution does not evaporate in large quantities and cause changes in the concentration of the solution system. After stirring at a constant speed for 3 hours, remove the sealing film and quickly dispense the resulting light white suspension into several 50 mL centrifuge tubes.
[0077] Step 13: Balance the light white suspension in several centrifuge tubes and place them in a centrifuge. Centrifuge at 5500 rpm for 5 minutes, then discard the supernatant. Add an equal volume of methanol solution as before centrifugation. Invert the centrifuge tubes (or shake the machine) to resuspend the milky white ZIF-8 precipitate. Centrifuge at 8500 rpm for 5 minutes. Discard the supernatant. Drill several vent holes in the centrifuge tube caps and place the samples in a 60℃ oven to dry overnight (6-8 hours). After overnight drying, pour out the ZIF-8 powder and grind it into a fine powder using an agate mortar. Seal the powder and store it in a bottle at room temperature to obtain the ZIF-8 nanoparticle carrier, i.e., ZIF-8.
[0078] Step 2: Camelin is added to the ZIF-8 nanoparticle carrier to synthesize ZIF-8 drug-loaded nanoparticles, which specifically includes the following steps:
[0079] Step 21: Accurately weigh 30 mg of ZIF-8 nanoparticles and 72 mg of camel thorn alkaloid (HA) using an analytical balance, and dissolve them in 10 ml of methanol analytical grade solution to obtain ZIF-8 nanoparticle carrier suspension and HA solution.
[0080] Step 22: Pour the ZIF-8 nanoparticle carrier suspension into a flask and stir it at a constant speed of 550 r·min⁻¹ on a magnetic stirrer at room temperature. Simultaneously, add the HA solution dropwise into the flask at a rate of 1 drop / s, and mix thoroughly. Seal the flask with sealing film. After stirring at a constant speed for 24 hours, remove the sealing film and quickly aliquot the resulting light white suspension into 50 mL centrifuge tubes.
[0081] Step 23: Balance the light white suspension in several centrifuge tubes and centrifuge at 5500 rpm for 5 minutes, then discard the supernatant. Add an equal volume of methanol solution as before centrifugation, invert the centrifuge tubes (or shake the machine) to resuspend the milky white ZIF-8 precipitate, then centrifuge at 8500 rpm for 5 minutes. Repeat the above steps until the supernatant is clear, then discard the supernatant. Dissolve the supernatant in 1 ml of methanol, transfer it to a 10 ml centrifuge tube, seal and dry. Drill several vent holes in the centrifuge tube cap and place the sample in a 60℃ oven to dry overnight (6-8 hours). The next day, pour out the powder, grind it into a fine powder using an agate mortar and pestle, seal it in a bottle, and store it at room temperature to obtain ZIF-8 drug-loaded nanoparticles, i.e., HA@ZIF-8.
[0082] Step 3: Incubate macrophages with ZIF-8 drug-loaded nanoparticles for 1 hour, wash, transfer cells to obtain the drug-loaded targeted nanoplatform, namely HA@ZIF-8@BMDM;
[0083] In step 3, macrophages were extracted using the following method:
[0084] Step 31: Use a 26G puncture needle to wash bone marrow macrophages with DMEM culture medium, centrifuge (1500 rpm, 6 min) and separate the cells into a single-cell suspension;
[0085] Step 32: Under conditions of 37℃ and 5% CO2, the single-cell suspension was inoculated into a 10cm culture dish containing 10mL of cell culture medium. On the 3rd day, half of the culture medium was replaced, and on the 5th day, all of the culture medium was replaced. Macrophage BMDMs could be obtained on the 7th day.
[0086] In this embodiment, the cell culture medium consists of 15% FBS, 1% antibiotics, 40% L929 cell conditioned medium (L929-CM), and 44% DMEM medium. The L929 cell conditioned medium (L929-CM) is prepared by culturing L929 cells to a confluence state, continuing the culture for one week, and then centrifuging, filtering, and collecting the supernatant.
[0087] In this embodiment, bone marrow macrophages were prepared by euthanizing 8-12 week old C57BL / 6 mice and then separating their femurs and tibias.
[0088] In this embodiment, step S3 specifically includes the following steps:
[0089] Macrophage BMDM cells were seeded into 12-well plates (8*10⁵ cells / well) and then incubated with cell culture medium containing HA@ZIF-8 (HA@ZIF-8 concentration 6 mg / ml) for 1 h. Untaken particles were gently washed away with PBS solution, repeated three times. Cells were gently scraped off with a cell scraper, and the cells were transferred to centrifuge tubes with 5 ml of cell culture medium and frozen at -40°C to obtain HA@ZIF-8@BMDM.
[0090] Example 3
[0091] This invention provides a drug-targeted nanoplatform for macrophage-targeted delivery of ZIF-8 drug-loaded nanoparticles, comprising ZIF-8 drug-loaded nanoparticles and macrophages targeted and modified on the outermost layer of the ZIF-8 drug-loaded nanoparticles.
[0092] In this embodiment of the invention, the ZIF-8 drug-loaded nanoparticles include a ZIF-8 nanoparticle carrier and a drug loaded on the ZIF-8 nanoparticle carrier.
[0093] In this embodiment of the invention, the drug is camel vesicant (HA).
[0094] In this embodiment of the invention, the particle size of the ZIF-8 nanoparticle carrier is 91.28–295.31 nm, and the particle size of the ZIF-8 drug-loaded nanoparticles is 141.77–396.06 nm.
[0095] This invention also provides a method for preparing the above-mentioned macrophage-targeted delivery ZIF-8 drug-loaded nanoparticle platform, specifically including the following steps:
[0096] Step 1: Synthesize zeolite-type imidazole salt framework 8-nanometer particles, namely ZIF-8 nanoparticle carriers, using zinc nitrate hexahydrate and 2-methylimidazole. This process includes the following steps:
[0097] Step 11: Weigh 4g of 2-methylimidazole powder and 1.32g of zinc nitrate hexahydrate crystals using a precision balance, and dissolve them in 40mL and 20mL of analytical grade methanol solution, respectively, to obtain zinc nitrate hexahydrate solution and 2-methylimidazole solution.
[0098] Step 12: Add the 2-methylimidazole solution to a flat-bottomed flask and stir it at a constant speed of 550 r·min⁻¹ on a magnetic stirrer at room temperature. At the same time, add the zinc nitrate hexahydrate solution slowly and steadily to the 2-methylimidazole solution at a speed of 1 drop / s. Stir thoroughly and cover the mouth of the flask with multiple layers of sealing film to ensure that the methanol solution does not evaporate in large quantities and cause changes in the concentration of the solution system. After stirring at a constant speed for 3 hours, remove the sealing film and quickly dispense the resulting light white suspension into several 50 mL centrifuge tubes.
[0099] Step 13: Balance the light white suspension in several centrifuge tubes and place them in a centrifuge. Centrifuge at 5500 rpm for 5 minutes, then discard the supernatant. Add an equal volume of methanol solution as before centrifugation. Invert the centrifuge tubes (or shake the machine) to resuspend the milky white ZIF-8 precipitate. Centrifuge at 8500 rpm for 5 minutes. Discard the supernatant. Drill several vent holes in the centrifuge tube caps and place the samples in a 60℃ oven to dry overnight (6-8 hours). After overnight drying, pour out the ZIF-8 powder and grind it into a fine powder using an agate mortar. Seal the powder and store it in a bottle at room temperature to obtain the ZIF-8 nanoparticle carrier, i.e., ZIF-8.
[0100] Step 2: Camelin is added to the ZIF-8 nanoparticle carrier to synthesize ZIF-8 drug-loaded nanoparticles, which specifically includes the following steps:
[0101] Step 21: Accurately weigh 30 mg of ZIF-8 nanoparticles and 72 mg of camel thorn alkaloid (HA) using an analytical balance, and dissolve them in 10 ml of methanol analytical grade solution to obtain ZIF-8 nanoparticle carrier suspension and HA solution.
[0102] Step 22: Pour the ZIF-8 nanoparticle carrier suspension into a flask and stir it at a constant speed of 550 r·min⁻¹ on a magnetic stirrer at room temperature. Simultaneously, add the HA solution dropwise into the flask at a rate of 1 drop / s, and mix thoroughly. Seal the flask with sealing film. After stirring at a constant speed for 24 hours, remove the sealing film and quickly aliquot the resulting light white suspension into 50 mL centrifuge tubes.
[0103] Step 23: Balance the light white suspension in several centrifuge tubes and centrifuge at 5500 rpm for 5 minutes, then discard the supernatant. Add an equal volume of methanol solution as before centrifugation, invert the centrifuge tubes (or shake the machine) to resuspend the milky white ZIF-8 precipitate, then centrifuge at 8500 rpm for 5 minutes. Repeat the above steps until the supernatant is clear, then discard the supernatant. Dissolve the supernatant in 1 ml of methanol, transfer it to a 10 ml centrifuge tube, seal and dry. Drill several vent holes in the centrifuge tube cap and place the sample in a 60℃ oven to dry overnight (6-8 hours). The next day, pour out the powder, grind it into a fine powder using an agate mortar and pestle, seal it in a bottle, and store it at room temperature to obtain ZIF-8 drug-loaded nanoparticles, i.e., HA@ZIF-8.
[0104] Step 3: Incubate macrophages with ZIF-8 drug-loaded nanoparticles for 1 hour, wash, transfer cells to obtain the drug-loaded targeted nanoplatform, namely HA@ZIF-8@BMDM;
[0105] In step 3, macrophages were extracted using the following method:
[0106] Step 31: Use a 26G puncture needle to wash bone marrow macrophages with DMEM culture medium, centrifuge (1500 rpm, 6 min) and separate the cells into a single-cell suspension;
[0107] Step 32: Under conditions of 37℃ and 5% CO2, the single-cell suspension was inoculated into a 10cm culture dish containing 10mL of cell culture medium. On the 3rd day, half of the culture medium was replaced, and on the 5th day, all of the culture medium was replaced. Macrophage BMDMs could be obtained on the 7th day.
[0108] In this embodiment, the cell culture medium consists of 15% FBS, 1% antibiotics, 40% L929 cell conditioned medium (L929-CM), and 44% DMEM medium. The L929 cell conditioned medium (L929-CM) is prepared by culturing L929 cells to a confluence state, continuing the culture for one week, and then centrifuging, filtering, and collecting the supernatant.
[0109] In this embodiment, bone marrow macrophages were prepared by euthanizing 8-12 week old C57BL / 6 mice and then separating their femurs and tibias.
[0110] In this embodiment, step S3 specifically includes the following steps:
[0111] Macrophage BMDM cells were seeded into 12-well plates (8*10⁵ cells / well) and then incubated with cell culture medium containing HA@ZIF-8 (HA@ZIF-8 concentration 6 mg / ml) for 1 h. Untaken particles were gently washed away with PBS solution, repeated three times. Cells were gently scraped off with a cell scraper, and the cells were transferred to centrifuge tubes with 5 ml of cell culture medium and frozen at -40°C to obtain HA@ZIF-8@BMDM.
[0112] Example 4
[0113] This invention provides a drug-targeted nanoplatform for macrophage-targeted delivery of ZIF-8 drug-loaded nanoparticles, comprising ZIF-8 drug-loaded nanoparticles and macrophages targeted and modified on the outermost layer of the ZIF-8 drug-loaded nanoparticles.
[0114] In this embodiment of the invention, the ZIF-8 drug-loaded nanoparticles include a ZIF-8 nanoparticle carrier and a drug loaded on the ZIF-8 nanoparticle carrier.
[0115] In this embodiment of the invention, the drug is camel alkaloid.
[0116] In this embodiment of the invention, the particle size of the ZIF-8 nanoparticle carrier is 91.28–295.31 nm, and the particle size of the ZIF-8 drug-loaded nanoparticles is 141.77–396.06 nm.
[0117] This invention also provides a method for preparing the above-mentioned macrophage-targeted delivery ZIF-8 drug-loaded nanoparticle platform, specifically including the following steps:
[0118] Step 1: Synthesize zeolite-type imidazole salt framework 8-nanometer particles, namely ZIF-8 nanoparticle carriers, using zinc nitrate hexahydrate and 2-methylimidazole. This process includes the following steps:
[0119] Step 11: Weigh 4g of 2-methylimidazole powder and 1.45g of zinc nitrate hexahydrate crystals using a precision balance, and dissolve them in 40mL and 20mL of analytical grade methanol solution, respectively, to obtain zinc nitrate hexahydrate solution and 2-methylimidazole solution.
[0120] Step 12: Add the 2-methylimidazole solution to a flat-bottomed flask and stir it at a constant speed of 550 r·min⁻¹ on a magnetic stirrer at room temperature. At the same time, add the zinc nitrate hexahydrate solution slowly and steadily to the 2-methylimidazole solution at a speed of 1 drop / s. Stir thoroughly and cover the mouth of the flask with multiple layers of sealing film to ensure that the methanol solution does not evaporate in large quantities and cause changes in the concentration of the solution system. After stirring at a constant speed for 3 hours, remove the sealing film and quickly dispense the resulting light white suspension into several 50 mL centrifuge tubes.
[0121] Step 13: Balance the light white suspension in several centrifuge tubes and place them in a centrifuge. Centrifuge at 5500 rpm for 5 minutes, then discard the supernatant. Add an equal volume of methanol solution as before centrifugation. Invert the centrifuge tubes (or shake the machine) to resuspend the milky white ZIF-8 precipitate. Centrifuge at 8500 rpm for 5 minutes. Discard the supernatant. Drill several vent holes in the centrifuge tube caps and place the samples in a 60℃ oven to dry overnight (6-8 hours). After overnight drying, pour out the ZIF-8 powder and grind it into a fine powder using an agate mortar. Seal the powder and store it in a bottle at room temperature to obtain the ZIF-8 nanoparticle carrier, i.e., ZIF-8.
[0122] Step 2: Camelin is added to the ZIF-8 nanoparticle carrier to synthesize ZIF-8 drug-loaded nanoparticles, which specifically includes the following steps:
[0123] Step 21: Accurately weigh 30 mg of ZIF-8 nanoparticles and 126 mg of camel thorn alkaloid (HA) using an analytical balance, and dissolve them in 10 ml of methanol analytical grade solution to obtain ZIF-8 nanoparticle carrier suspension and HA solution.
[0124] Step 22: Pour the ZIF-8 nanoparticle carrier suspension into a flask and stir it at a constant speed of 550 r·min⁻¹ on a magnetic stirrer at room temperature. Simultaneously, add the HA solution dropwise into the flask at a rate of 1 drop / s, and mix thoroughly. Seal the flask with sealing film. After stirring at a constant speed for 24 hours, remove the sealing film and quickly aliquot the resulting light white suspension into 50 mL centrifuge tubes.
[0125] Step 23: Balance the light white suspension in several centrifuge tubes and centrifuge at 5500 rpm for 5 minutes, then discard the supernatant. Add an equal volume of methanol solution as before centrifugation, invert the centrifuge tubes (or shake the machine) to resuspend the milky white ZIF-8 precipitate, then centrifuge at 8500 rpm for 5 minutes. Repeat the above steps until the supernatant is clear, then discard the supernatant. Dissolve the supernatant in 1 ml of methanol, transfer it to a 10 ml centrifuge tube, seal and dry. Drill several vent holes in the centrifuge tube cap and place the sample in a 60℃ oven to dry overnight (6-8 hours). The next day, pour out the powder, grind it into a fine powder using an agate mortar and pestle, seal it in a bottle, and store it at room temperature to obtain ZIF-8 drug-loaded nanoparticles, i.e., HA@ZIF-8.
[0126] Step 3: Incubate macrophages with ZIF-8 drug-loaded nanoparticles for 1 hour, wash, transfer cells to obtain the drug-loaded targeted nanoplatform, namely HA@ZIF-8@BMDM;
[0127] In step 3, macrophages were extracted using the following method:
[0128] Step 31: Use a 26G puncture needle to wash bone marrow macrophages with DMEM culture medium, centrifuge (1500 rpm, 6 min) and separate the cells into a single-cell suspension;
[0129] Step 32: Under conditions of 37℃ and 5% CO2, single-cell suspensions were seeded into 10cm culture dishes containing 10mL of cell culture medium. Half of the medium was replaced on day 3, and all the medium was replaced on day 5. Macrophage BMDMs were obtained on day 7. The differentiation status of BMMs was detected by FITC-F4 / 80 and PE-CD11b double staining flow cytometry. The results are as follows: Figure 8 As shown, through Figure 8 The results showed that over 98% of the cells were CD11b+F4 / 80+, confirming that the cell purity was sufficient for experimental use.
[0130] In this embodiment, the cell culture medium consists of 15% FBS, 1% antibiotics, 40% L929 cell conditioned medium (L929-CM), and 44% DMEM medium. The L929 cell conditioned medium (L929-CM) is prepared by culturing L929 cells to a confluence state, continuing the culture for one week, and then centrifuging, filtering, and collecting the supernatant.
[0131] In this embodiment, bone marrow macrophages were prepared by euthanizing 8-12 week old C57BL / 6 mice and then separating their femurs and tibias.
[0132] In this embodiment, step S3 specifically includes the following steps:
[0133] Macrophage BMDMs were seeded into 12-well plates (8 x 10⁵ cells / well) and then incubated with cell culture medium containing HA@ZIF-8 (HA@ZIF-8 concentration 6 mg / ml) for 1 h. Untaken particles were gently washed away with PBS solution, repeated three times. Cells were gently scraped off with a cell scraper, and the cells were transferred to centrifuge tubes with 5 ml of cell culture medium and frozen at -40°C to obtain HA@ZIF-8@BMDMs.
[0134] Example 5
[0135] This invention provides a drug-targeted nanoplatform for macrophage-targeted delivery of ZIF-8 drug-loaded nanoparticles, comprising ZIF-8 drug-loaded nanoparticles and macrophages targeted and modified on the outermost layer of the ZIF-8 drug-loaded nanoparticles.
[0136] In this embodiment of the invention, the ZIF-8 drug-loaded nanoparticles include a ZIF-8 nanoparticle carrier and a drug loaded on the ZIF-8 nanoparticle carrier.
[0137] In this embodiment of the invention, the drug is camel alkaloid.
[0138] In this embodiment of the invention, the particle size of the ZIF-8 nanoparticle carrier is 91.28–295.31 nm, and the particle size of the ZIF-8 drug-loaded nanoparticles is 141.77–396.06 nm.
[0139] This invention also provides a method for preparing the above-mentioned macrophage-targeted delivery ZIF-8 drug-loaded nanoparticle platform, specifically including the following steps:
[0140] Step 1: Synthesize zeolite-type imidazole salt framework 8-nanometer particles, namely ZIF-8 nanoparticle carriers, using zinc nitrate hexahydrate and 2-methylimidazole. This process includes the following steps:
[0141] Step 11: Weigh 4g of 2-methylimidazole powder and 1.45g of zinc nitrate hexahydrate crystals using a precision balance, and dissolve them in 40mL and 20mL of analytical grade methanol solution, respectively, to obtain zinc nitrate hexahydrate solution and 2-methylimidazole solution.
[0142] Step 12: Add the 2-methylimidazole solution to a flat-bottomed flask and stir it at a constant speed of 550 r·min⁻¹ on a magnetic stirrer at room temperature. At the same time, add the zinc nitrate hexahydrate solution slowly and steadily to the 2-methylimidazole solution at a speed of 1 drop / s. Stir thoroughly and cover the mouth of the flask with multiple layers of sealing film to ensure that the methanol solution does not evaporate in large quantities and cause changes in the concentration of the solution system. After stirring at a constant speed for 3 hours, remove the sealing film and quickly dispense the resulting light white suspension into several 50 mL centrifuge tubes.
[0143] Step 13: Balance the light white suspension in several centrifuge tubes and place them in a centrifuge. Centrifuge at 5500 rpm for 5 minutes, then discard the supernatant. Add an equal volume of methanol solution as before centrifugation. Invert the centrifuge tubes (or shake the machine) to resuspend the milky white ZIF-8 precipitate. Centrifuge at 8500 rpm for 5 minutes. Discard the supernatant. Drill several vent holes in the centrifuge tube caps and place the samples in a 60℃ oven to dry overnight (6-8 hours). After overnight drying, pour out the ZIF-8 powder and grind it into a fine powder using an agate mortar. Seal the powder and store it in a bottle at room temperature to obtain the ZIF-8 nanoparticle carrier, i.e., ZIF-8.
[0144] Step 2: Camelin is added to the ZIF-8 nanoparticle carrier to synthesize ZIF-8 drug-loaded nanoparticles, which specifically includes the following steps:
[0145] Step 21: Accurately weigh 30 mg of ZIF-8 nanoparticles and 105 mg of camel thorn alkaloid (HA) using an analytical balance, and dissolve them in 10 ml of methanol analytical grade solution to obtain ZIF-8 nanoparticle carrier suspension and HA solution.
[0146] Step 22: Pour the ZIF-8 nanoparticle carrier suspension into a flask and stir it at a constant speed of 550 r·min⁻¹ on a magnetic stirrer at room temperature. Simultaneously, add the HA solution dropwise into the flask at a rate of 1 drop / s, and mix thoroughly. Seal the flask with sealing film. After stirring at a constant speed for 24 hours, remove the sealing film and quickly aliquot the resulting light white suspension into 50 mL centrifuge tubes.
[0147] Step 23: Balance the light white suspension in several centrifuge tubes and centrifuge at 5500 rpm for 5 minutes, then discard the supernatant. Add an equal volume of methanol solution as before centrifugation, invert the centrifuge tubes (or shake the machine) to resuspend the milky white ZIF-8 precipitate, then centrifuge at 8500 rpm for 5 minutes. Repeat the above steps until the supernatant is clear, then discard the supernatant. Dissolve the supernatant in 1 ml of methanol, transfer it to a 10 ml centrifuge tube, seal and dry. Drill several vent holes in the centrifuge tube cap and place the sample in a 60℃ oven to dry overnight (6-8 hours). The next day, pour out the powder, grind it into a fine powder using an agate mortar and pestle, seal it in a bottle, and store it at room temperature to obtain ZIF-8 drug-loaded nanoparticles, i.e., HA@ZIF-8.
[0148] Step 3: Incubate macrophages with ZIF-8 drug-loaded nanoparticles for 1 hour, wash, transfer cells to obtain the drug-loaded targeted nanoplatform, namely HA@ZIF-8@BMDM;
[0149] In step 3, macrophages were extracted using the following method:
[0150] Step 31: Use a 26G puncture needle to wash bone marrow macrophages with DMEM culture medium, centrifuge (1500 rpm, 6 min) and separate the cells into a single-cell suspension;
[0151] Step 32: Under conditions of 37℃ and 5% CO2, single-cell suspensions were seeded into 10cm culture dishes containing 10mL of cell culture medium. Half of the medium was replaced on day 3, and all the medium was replaced on day 5. Macrophage BMDMs were obtained on day 7. The differentiation status of BMMs was detected by FITC-F4 / 80 and PE-CD11b double staining flow cytometry. The results are as follows: Figure 8 As shown, through Figure 8 The results showed that over 98% of the cells were CD11b+F4 / 80+, confirming that the cell purity was sufficient for experimental use.
[0152] In this embodiment, the cell culture medium consists of 15% FBS, 1% antibiotics, 40% L929 cell conditioned medium (L929-CM), and 44% DMEM medium. The L929 cell conditioned medium (L929-CM) is prepared by culturing L929 cells to a confluence state, continuing the culture for one week, and then centrifuging, filtering, and collecting the supernatant.
[0153] In this embodiment, bone marrow macrophages were prepared by euthanizing 8-12 week old C57BL / 6 mice and then separating their femurs and tibias.
[0154] In this embodiment, step S3 specifically includes the following steps:
[0155] Macrophage BMDMs were seeded into 12-well plates (8 x 10⁵ cells / well) and then incubated with cell culture medium containing HA@ZIF-8 (HA@ZIF-8 concentration 6 mg / ml) for 1 h. Untaken particles were gently washed away with PBS solution, repeated three times. Cells were gently scraped off with a cell scraper, and the cells were transferred to centrifuge tubes with 5 ml of cell culture medium and frozen at -40°C to obtain HA@ZIF-8@BMDMs.
[0156] Example 6
[0157] This invention provides a drug-targeted nanoplatform for macrophage-targeted delivery of ZIF-8 drug-loaded nanoparticles, comprising ZIF-8 drug-loaded nanoparticles and macrophages targeted and modified on the outermost layer of the ZIF-8 drug-loaded nanoparticles. The preparation method of the drug-targeted nanoplatform for macrophage-targeted delivery of ZIF-8 drug-loaded nanoparticles provided in this invention is the same as in Example 1, except that the drug used in this invention is levonorgestrel (LNG), and the drug-targeted nanoplatform for macrophage-targeted delivery of ZIF-8 drug-loaded nanoparticles obtained in this invention is denoted as LNG@ZIF-8@BMDM.
[0158] Comparative Example 1
[0159] This comparative example provides a drug-loaded targeted nanoplatform for macrophage-targeted delivery of ZIF-8 drug-loaded nanoparticles, comprising ZIF-8 drug-loaded nanoparticles and macrophages targeted and modified on the outermost layer of the ZIF-8 drug-loaded nanoparticles. The preparation method of the macrophage-targeted targeted nanoplatform for ZIF-8 drug-loaded nanoparticle delivery provided in this comparative example is the same as in Example 1, except that the molar ratio of zinc nitrate hexahydrate to 2-methylimidazole is 1:19. The mass of the ZIF-8 drug-loaded nanoparticles in this comparative example is approximately 43.88 mg, with a yield of 43%, which is significantly lower than the yield of the ZIF-8 drug-loaded nanoparticles prepared in Example 1 of this invention. This indicates that the molar ratio between zinc nitrate hexahydrate and 2-methylimidazole in the example is specifically set; if it is not within the set range of this invention, the yield of ZIF-8 drug-loaded nanoparticles will be lower.
[0160] Since the therapeutic effects of the drug-loaded targeted nanoplatforms provided in Examples 1-5 of this invention are basically the same, the therapeutic effects of the drug-loaded targeted nanoplatforms synthesized in Examples 1 and 6 of this invention will be studied below.
[0161] I. Experimental Design
[0162] 1. The prepared ZIF-8 nanoparticle carrier and ZIF-8 drug-loaded nanoparticles loaded with HA and LNG respectively were characterized.
[0163] The concentration of nanoparticles was detected by inductively coupled plasma optical emission spectrometry (ICP-OES). The size and morphology of the obtained ZIF-8, HA@ZIF-8, and LNG@ZIF-8 nanoparticles were characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The functional groups of HA@ZIF-8 and LNG@ZIF-8 were characterized by measuring zeta potential and by Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). The purity of BMDM cultured for 5 days with F4 / 80 and CD11b double staining was detected by flow cytometry. The results of BMDM uptake of HA@ZIF-8 and LNG@ZIF-8 for 2 hours were observed by scanning confocal microscopy. The cell state after BMDM was co-incubated with HA@ZIF-8 and LNG@ZIF-8 for 2 hours was observed by live / dead cell staining experiments. This invention conducted a Transwell experiment to test the migration ability of BMDM, HA@ZIF-8@BMDM, and LNG@ZIF-8@BMDM treated with ectopic endometrial stromal cells (EESCs), and quantitatively assessed the number of migrating cells in different treatment groups.
[0164] 2. The effect of the drug-loaded targeted nanoplatform prepared in this invention on endometriosis.
[0165] Simulating the primate menstrual cycle, this invention administered cyclical estrogen and progesterone to mice, and then established an endometriosis model in mice by implanting autologous peritoneal lesions. The model mice were injected intravenously with HA@ZIF-8@BMDM and LNG@ZIF-8@BMDM, respectively. Twenty-four hours after injection, the transplanted tumor was removed, and its volume was measured. The heart, liver, and kidneys of the mice were also removed for HE staining. To further clarify the treatment mechanism of endometriosis, the peritoneal lavage fluid from the model mice was collected, and the levels of CD86 and CD206 were detected by flow cytometry to determine the M1 ratio.
[0166] II. Experimental Results
[0167] pass Figure 2 As can be seen, the average particle size of ZIF-8 prepared in the embodiments of the present invention is approximately 130.5 nm, the average particle size of HA@ZIF-8 is approximately 276.6 nm, and the average particle size of LNG@ZIF-8 is approximately 251.5 nm. These results indicate that the size of the nanoparticles increases after drug loading, demonstrating that the drug can be successfully loaded into the nanoparticles.
[0168] pass Figure 3It can be seen that both HA@ZIF-8 and LNG@ZIF-8 are spherical nanoparticles, with the particle size of HA@ZIF-8 ranging from 38.18 to 296.92 nm and the particle size of LNG@ZIF-8 ranging from 36.10 to 282.13 nm.
[0169] pass Figure 4 As can be seen, the infrared spectra of HA@ZIF-8 and LNG@ZIF-8 show characteristic peaks of eugenol and levonorgestrel, respectively, indicating that the loading of eugenol and levonorgestrel was successful.
[0170] pass Figure 5 The results showed that the proportion of CD11b+F4 / 80+ cells was higher than 98.5%, indicating that the cultured BMDMs had high purity and were suitable for subsequent experiments.
[0171] pass Figure 6 The results showed that cells were indeed able to take up HA@ZIF-8 and LNG@ZIF-8, and the results were relatively stable.
[0172] pass Figure 7 The results showed that almost all HA@ZIF-8@BMDM and LNG@ZIF-8@BMDM remained active after 2 hours, indicating that the nanoparticles had low toxicity to the carrier cells in the short term, providing valuable time for subsequent lesion tropism.
[0173] pass Figure 8 The results in A show that both HA@ZIF-8@BMDM and LNG@ZIF-8@BMDM effectively migrate to eesc, although the number of LNG@ZIF-8@BMDM is slightly lower than that of HA@ZIF-8@BMDM. Figure 8 Quantitative analysis of B cells revealed that both HA@ZIF-8@BMDM and LNG@ZIF-8@BMDM exhibited strong tumor tropism, with approximately 406 and 353 LNG@ZIF-8@BMDM cells migrating to eesc cells per field, respectively, compared to only 88 cells per field in untreated BMDM. This indicates that the drug-loaded macrophage nanoplatform demonstrates superior tumor tropism compared to unmodified BMDM.
[0174] pass Figure 9 The results showed that, compared with untreated BMDM, the proportion of M1 macrophages in HA@ZIF-8@BMDM and LNG@ZIF-8@BMDM was significantly increased, indicating that both HA@ZIF-8@BMDM and LNG@ZIF-8@BMDM can promote macrophage polarization towards M1.
[0175] pass Figure 10The results showed that the lesion volume in the treatment group was significantly reduced, indicating that HA@ZIF-8@BMDM and LNG@ZIF-8@BMDM have comparable therapeutic effects.
[0176] pass Figure 11 The results showed no abnormalities in histological examination, indicating that HA@ZIF-8@BMDM and LNG@ZIF-8@BMDM have low toxicity.
[0177] In summary, this invention uses ZIF-8 nanoparticles combined with HA or LNG targeting energy metabolism levels as a carrier to target endometriosis cells in an acidic cellular microenvironment. This allows for controlled drug release within the acidic environment of the lesion, enhancing the effective uptake of the drug by endometriosis cells, thereby effectively increasing the uptake rate and drug concentration at the target site. This is beneficial for improving the efficacy of the drug in treating endometriosis, while reducing the distribution of the drug in normal tissues or cells and lowering the toxic side effects of the drug.
[0178] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims
1. A drug-loaded targeted nano-platform for targeted delivery of ZIF-8 drug-loaded nanoparticles to macrophages, characterized in that, The macrophage-targeted drug-loaded ZIF-8 nanoparticle comprises a ZIF-8 nanoparticle carrier and a drug loaded on the ZIF-8 nanoparticle carrier. 2.The drug-loaded targeting nano-platform of macrophage-targeted delivery of ZIF-8 drug-loaded nanoparticles according to claim 1, characterized in that, The drug-loaded ZIF-8 nanoparticle comprises a ZIF-8 nanoparticle carrier and a drug loaded on the ZIF-8 nanoparticle carrier. 3.The drug-loaded targeting nano-platform of macrophage-targeted delivery of ZIF-8 drug-loaded nanoparticles according to claim 2, characterized in that, The drug is harmine or levonorgestrel. 4.The drug-loaded targeting nano-platform of macrophage-targeted delivery of ZIF-8 drug-loaded nanoparticles according to claim 2, characterized in that, The particle size of the ZIF-8 nanoparticle carrier is 91.28-295.31 nm, and the particle size of the drug-loaded ZIF-8 nanoparticle is 141.77-396.06 nm.
5. The method of claim 1-4, wherein the method of preparing the drug-loaded targeting nano-platform of macrophage-targeted delivery of ZIF-8 drug-loaded nanoparticles is characterized in that, The method comprises the following steps: Step 1: synthesizing a ZIF-8 nanoparticle carrier by using zinc nitrate hexahydrate and 2-methylimidazole; The molar ratio of zinc nitrate hexahydrate to 2-methylimidazole is 1:8.2-11; Step 2: synthesizing a drug-loaded ZIF-8 nanoparticle by adding a drug into the ZIF-8 nanoparticle carrier; The mass ratio of the drug to the ZIF-8 nanoparticle carrier is 2-4.2:1; Step 3: incubating macrophages with the drug-loaded ZIF-8 nanoparticle for 1 h, washing, transferring cells, and obtaining a drug-loaded targeted nanoparticle platform.
6. The production method according to claim 5, wherein Step 1 comprises the following steps: Step 11: weighing zinc nitrate hexahydrate and 2-methylimidazole according to a molar ratio of 1:8.2-11 respectively, and dissolving them in a methanol solution to obtain a zinc nitrate hexahydrate solution and a 2-methylimidazole solution; Step 12: under stirring, adding the zinc nitrate hexahydrate solution into the 2-methylimidazole solution, and then stirring at a constant speed for 3 h in a closed state to obtain a light white suspension; Step 13: after multiple centrifugation operations on the light white suspension, retaining the precipitate, and drying to obtain the ZIF-8 nanoparticle carrier.
7. The preparation method according to claim 5, characterized in that, Step 2 comprises the following steps: Step 21: weighing the drug and the ZIF-8 nanoparticle carrier according to a mass ratio of 2-4.2:1 respectively, and dissolving them in a methanol solution to obtain a ZIF-8 nanoparticle carrier suspension and a drug solution; Step 22: under stirring, adding the drug solution into the ZIF-8 nanoparticle carrier suspension, and then stirring at a constant speed for 24 h in a closed state to obtain a light white suspension; Step 23: after multiple centrifugation operations on the light white suspension, retaining the precipitate, and drying to obtain the drug-loaded ZIF-8 nanoparticle.
8. The preparation method according to claim 5, characterized in that, In step 3, the macrophages are obtained by the following method: Step 31: rinsing the bone marrow macrophages by using a DMEM culture medium, centrifuging, and separating the cells into a single-cell suspension; Step 32: under the condition of 37℃ and 5% CO2, inoculating the single-cell suspension on a cell culture solution for culture, replacing half of the culture medium on the 3rd-4th day of culture, replacing all the culture medium on the 5th day, and obtaining the macrophages on the 7th day.
9. A drug-loaded targeted nanoparticle platform for delivering a macrophage-targeted drug-loaded ZIF-8 nanoparticle in the preparation of a drug for treating endometriosis.