Application and preparation method of GW806742X in targeted treatment of inflammatory lung injury

Abstract

The present invention discloses the use of GW806742X in a drug for the targeted treatment of inflammatory lung injury and its preparation method. The invention encapsulates the small molecule inhibitor GW806742X into lipid particles to form a uniformly sized, stable liposome suspension. Through chemical bond interactions, antibodies or peptides targeting type II pneumocyte surface marker proteins are combined with the lipid nanoparticles, enhancing the targeting properties of the lipid nanoparticles, enabling them to target type II pneumocytes, thereby inhibiting necroptosis of alveolar epithelial cells and achieving the purpose of treating inflammatory lung injury in the airways.

Description

Technical Field

[0001] The present invention relates to the technical field of drugs for treating inflammatory lung injury, and in particular to an application of GW806742X in drugs for the targeted treatment of inflammatory lung injury and a preparation method thereof. Background Art

[0002] Acute lung injury (ALI) and the more severe acute respiratory distress syndrome (ARDS) refer to acute, progressively worsening diffuse inflammatory lung injury caused by various intrapulmonary or extrapulmonary factors, leading to respiratory insufficiency or respiratory failure. ARDS, the latter, is the result of an uncontrolled systemic inflammatory response in the lungs and is a severe manifestation of ALI. The main pathological features of ALI / ARDS are pulmonary inflammation and increased microvascular permeability, leading to pulmonary edema and hyaline membrane formation. This process may also include pulmonary interstitial fibrosis, resulting in decreased lung compliance, increased intrapulmonary shunt, and ventilation-perfusion imbalance, ultimately leading to clinical manifestations of respiratory distress and refractory hypoxemia. Current treatment remains primarily symptomatic and supportive, with key strategies including low tidal volume lung protection, prone positioning, and extracorporeal circulation. There are no globally available therapeutic agents targeting the inflammation. The airway epithelium is the first line of defense against inflammatory or environmental physical stimuli. The interaction between these stimuli and epithelial cells ultimately leads to cellular edema, inflammatory response, and airway remodeling. Type II alveolar cells (ATⅡ) are the "stem cells" of alveolar epithelial cells. They have multiple functions, proliferating into new ATⅡ cells and differentiating into other epithelial cells such as ATⅠ cells. They have the functions of synthesizing and secreting surfactant, transporting pulmonary water, and having immune functions. They play a very important role in maintaining the normal physiological state of the airways. Damage to alveolar epithelial cells is an important pathological feature of airway inflammatory diseases. Currently, there are no targeted drugs for the treatment of airway epithelial cell damage in acute respiratory distress syndrome worldwide.

[0003] During the pathological process of ARDS, activation of the necroptosis pathway in alveolar epithelial cells can lead to the release of proinflammatory signals, exacerbating the overall inflammatory response in ARDS and contributing to disease progression. Furthermore, cell death caused by necroptosis can exacerbate tissue damage and inflammation in ARDS. The release of intracellular substances by dying cells, and the immune response of immune cells to these substances, can further lead to tissue damage, inflammation, and the progression of ARDS. The multiple effects of proinflammatory signal release and exacerbated tissue damage highlight the important role of necroptosis in the progression and severity of ARDS.

[0004] GW806742X is a small molecule drug that can inhibit the membrane translocation process of mixed lineage kinase domain like protein (MLKL) by competing with ATP or ADP for the nucleic acid binding site of MLKL, thereby inhibiting cell necroptosis. However, its non-specific effects on cells and the large therapeutic dose required limit its use, so it has not been developed as a therapeutic drug. Summary of the Invention

[0005] The present invention aims to provide a method for preparing GW806742X for use in a drug for the targeted treatment of inflammatory lung injury. The small molecule inhibitor GW806742X screened by the present invention can be used in drugs to effectively alleviate and treat airway inflammatory lung injury.

[0006] The technical solution provided by the present invention is as follows: Application of GW806742X in drugs for the targeted treatment of inflammatory lung injury.

[0007] In the above application, the drug inhibits the necroptosis of alveolar epithelial cells by targeting type II alveolar cells, thereby achieving the purpose of treating airway inflammatory lung injury.

[0008] A method for preparing a drug for the targeted treatment of inflammatory lung injury comprises encapsulating GW806742X into lipid nanoparticles to form a liposome suspension, and then combining an antibody or peptide targeting a type II alveolar cell surface marker protein with the lipid nanoparticles in the liposome suspension to obtain a finished drug.

[0009] The above-mentioned preparation method, the preparation method of the liposome suspension includes dialysis method, thin film hydration method, reverse evaporation method, solvent injection method, surfactant removal method and microfluidic method.

[0010] The aforementioned preparation method, the process of preparing the liposome suspension by dialysis is to dissolve the lipid nanoparticles and GW806742X in methanol at a mass ratio of 15-25:1 and shake to mix, then place them in a dialysis bag, and perform rotary dialysis with PBS at 1-5°C for 20-26 hours. During the rotary dialysis, the external phase is replaced with fresh PBS buffer solution several times. After the rotary dialysis is completed, the liposome suspension in the dialysis bag is removed and stored on ice.

[0011] The aforementioned preparation method, the process of preparing the liposome suspension by dialysis is to dissolve DSPE-PEG 2000, DSPE-PEG 2000-MAL and GW806742X in methanol according to a mass ratio of 16:4:1 and shake to mix, then place it in a dialysis bag, and perform rotary dialysis with PBS at 4°C for 24 hours. The external phase is replaced with fresh PBS buffer solution at 2, 4, 6, 8 and 12 hours. After the rotary dialysis is completed, the liposome suspension in the dialysis bag is removed and stored on ice.

[0012] In the aforementioned preparation method, the antibody against the surface marker protein of type II alveolar cells is an SPC antibody.

[0013] The aforementioned preparation method combines the SPC antibody with lipid nanoparticles in the liposome suspension by first dissolving the SPC antibody powder in HEPES buffer to obtain an antibody dilution solution; adding the liposome suspension to a glucose solution so that the final product contains 5% glucose; then mixing the antibody dilution solution with the glucose-containing liposome suspension at a molar ratio of 1:2 between the antibody and DSPE-PEG2000-MAL, and placing the mixture on a 3D suspension instrument at 4°C. After 6 hours, the sample is collected and placed in a dialysis bag, and rotary dialyzed with PBS at 4°C for 24 hours. The external phase is replaced with fresh buffer solution at 2, 4, 6, 8 and 12 hours. After the rotary dialysis, the collected sample is placed in an ultrafiltration tube with a pore size of 3KDa for concentration and purification to obtain a lipid micelle suspension.

[0014] In the above-mentioned preparation method, the conditions for the concentration and purification are a rotation speed of 3000 rpm, a temperature of 4° C., and a time of 30 min.

[0015] In the above-mentioned preparation method, the sample is tested after concentration and purification: the lipid micelle suspension obtained by concentration and purification is diluted 1:100, and the zeta potential, hydration diameter and drug concentration are measured to calculate the drug loading and encapsulation efficiency. The drug product is successfully prepared if the hydrated diameter is measured to be between 80-200nm, the dispersion coefficient is less than 0.1, and the encapsulation efficiency reaches more than 60%.

[0016] Compared with existing technologies, the present invention has screened out a small molecule inhibitor, GW806742X, that targets alveolar epithelial cell necroptosis, which can effectively alleviate inflammatory lung injury. This invention encapsulates the small molecule inhibitor GW806742X into lipid particles to form a uniformly sized, stable liposome suspension. Through chemical interactions, antibodies or peptides targeting type II pneumocyte surface marker proteins are combined with the lipid nanoparticles, enhancing the targeting properties of the lipid nanoparticles, enabling them to target type II pneumocytes, thereby inhibiting necroptosis of alveolar epithelial cells and achieving the purpose of treating airway inflammatory lung injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 GSEA analysis of bulk RNA sequencing data of lung tissue;

[0018] Figure 2 It is a heat map after gene correlation analysis based on the number of fragments per kilobase of exon model per million mapped fragments (FPKM);

[0019] Figure 3 This is the protein expression level and statistical graph of RIPK1 / p-RIPK1, a key factor in lung necroptosis, detected by Western blot;

[0020] Figure 4 This is the protein expression level and statistical graph of RIPK3 / p-RIPK3, a key factor in lung necroptosis, detected by Western blot;

[0021] Figure 5 The protein expression level and statistical graph of MLKL / p-MLKL, a key factor in lung necroptosis, were detected by Western blot;

[0022] Figure 6 This is the result of HE pathological staining of lung tissue;

[0023] Figure 7 This is a schematic diagram of the preparation method of a drug for the targeted treatment of inflammatory lung injury;

[0024] Figure 8 are transmission electron microscopy images of lipid nanomicelles without antibody binding (M-GW) and lipid nanomicelles with antibody binding (SPC-M-GW);

[0025] Figure 9 is the statistical graph of hydration diameter of M-GW and SPC-M-GW;

[0026] Figure 10 It is a statistical diagram of the potential of M-GW and SPC-M-GW;

[0027] Figure 11 Fluorescence microscopy images of mouse lung tissue showing DAPI (nuclei), SPC (alveolar type II epithelial cells) antibodies, and Cy5 staining (CY5 fluorescent material coated with DSPE-PEG lipids modified with SPC antibodies);

[0028] Figure 12 This is the HE staining image of lung tissue of the disease model;

[0029] Figure 13 It is a scoring chart for the degree of lung damage. DETAILED DESCRIPTION

[0030] The present invention is described in further detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention, not for limiting the scope of the invention. The examples provided below can be used as a guide for further improvement by those of ordinary skill in the art and do not constitute a limitation of the present invention in any way. The experimental methods in the following examples, unless otherwise specified, are conventional methods and are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0031] Example: Application of GW806742X in targeted therapy for inflammatory lung injury.

[0032] 1. Demonstrate the existence of necroptosis in airway epithelium in inflammatory lung injury:

[0033] 1.1. Animal Model Construction: Healthy male C57BL / 6 wild-type mice aged 6-8 weeks were anesthetized with an intraperitoneal injection of sodium pentobarbital (0.2 ml / mouse, 1% pentobarbital). After anesthesia, the mice were kept upright (with the tip of the nose aligned with the spine) and the tongue extended out of the mouth. 50 μl of LPS dilution was aspirated using a 100 μl pipette and LPS (Lipopolysaccharide, derived from Pseudomonas aeruginosa, 10 mg / kg, 50 μl) was instilled into the airways from the mouth along the base of the tongue. After instillation, the mice were gently shaken upright to allow LPS to diffuse within the airways while maintaining airway patency to induce acute lung injury. A control group of mice was treated with the same volume of phosphate-buffered saline (PBS) intratracheally using the same technique. After model construction, mice were treated at different time points according to the experimental requirements and samples of lung tissue, serum, and bronchoalveolar lavage fluid were collected for storage or further experiments.

[0034] 1.2. 24 hours after LPS airway instillation, mice were killed by intraperitoneal overdose of anesthetic (0.5 ml / mouse, 1% pentobarbital). After disinfection with 75% alcohol, the heart and lungs were fully exposed.

[0035] 1.3. Collect blood from the heart using a 1 ml sterile syringe, centrifuge at 3000 g, 4°C, for 15 min, collect the supernatant, store at -80°C, and detect inflammatory factors.

[0036] 1.4. Free the lung lobes and ligate the remaining lobes. Use sterile curved forceps to free the airways. Use tissue scissors to create a small "V"-shaped incision beneath the thyroid cartilage. Insert a BALF needle (pre-treated 20ml syringe needle) through the incision into the airway and secure with cotton thread. Withdraw 0.4ml of PBS using a 1ml syringe and slowly perfuse it into the lung tissue through the BALF needle. Then withdraw the PBS and collect the recovered fluid in a 1.5ml EP tube. Repeat this process three times, for a total of 1.2ml per mouse. Recover 1ml of alveolar lavage fluid and store at -80°C for analysis of inflammatory factors.

[0037] 1.5. Cut the lung tissue ligated in step 1.5, separate the lobes, and place them in cryopreservation tubes. Store at -80°C for detection of inflammatory factors or transcriptome sequencing.

[0038] 1.6. Free the lung lobes as described above, ligate the remaining lobes, draw 0.6 ml of 4% formaldehyde with a 1 ml syringe, and slowly perfuse into the lung lobes through a BALF needle. When the lung lobes are observed to expand, ligate the trachea, remove the BALF needle, and carefully remove the formaldehyde-filled lung lobes. Immerse them in 3 ml of 4% formaldehyde and store at 4°C. Embed them in paraffin, slice them at 3 μm, and stain them with immunofluorescence and HE.

[0039] 1.7. Perform transcriptome sequencing analysis (RNA-seq) on the lung tissue in the cryopreserved tube.

[0040] 1.8. 20 mg of lung tissue / animal was cryopreserved in a tube. RIPA (200 μl / sample) containing phosphatase and protease inhibitors was added. The tissue was magnetically ground at 7500 rpm for 30 sec three times. The tissue was lysed on ice for 30 min and then centrifuged at 12000 rpm at 4°C for 15 min. The supernatant was collected and normalized by BCA protein quantification. 5x Loading Buffer was added. The tissue was vortexed to mix and incubated in a metal bath at 100°C for 10 min. The supernatant was centrifuged at 4°C for 10 min. The supernatant was collected for electrophoresis (western blot).

[0041] Figure 1 GSEA analysis of bulk RNA sequencing data of lung tissue showed that the necroptosis pathway was significantly activated in the disease group model. Figure 2 Heatmap of gene correlation analysis based on exon model fragments per kilobase per million mapped fragments (FPKM). Figure 3 This is the protein expression level and statistical graph of RIPK1 / p-RIPK1, a key factor in lung tissue necroptosis, detected by Western blot. Figure 4 This is the protein expression level and statistical graph of RIPK3 / p-RIPK3, a key factor in lung tissue necroptosis, detected by Western blot. Figure 5The following is a statistical chart of the protein expression levels of MLKL / p-MLKL, a key factor in lung necroptosis, detected by Western blot, where *P<0.05, ***P<0.001. Figures 1 to 5 The results can confirm the presence of necroptosis of alveolar epithelial cells in inflammatory lung injury.

[0042] 2. GW806742X treats inflammatory lung injury disease models;

[0043] 2.1. The method and steps for constructing the animal model are the same as those in 1.

[0044] 2.2 Dissolution of GW806742X: Take 1 mg of GW806742X powder, naturally warm to room temperature, add 100 μl of sterile DMSO, and vortex to dissolve to a concentration of 10 mg / ml. Take out the corresponding volume of drug stock solution required for the administration of 2 mg / kg, 1 mg / kg, and 0.5 mg / kg, add it to PBS (pH = 7.2-7.4) and vortex to mix.

[0045] 2.3. GW806742X treatment: LPS airway instillation was used to construct an inflammatory lung injury model. The method was the same as before. After 2-4 hours, GW806742X drugs fully dissolved in PBS and DMSO were locally administered by airway instillation at doses of 2 mg / kg, 1 mg / kg, and 0.5 mg / kg. After 24 hours, alveolar lavage fluid, serum, and lung tissue were collected for testing (the steps are the same as in 1). Figure 6 This is the result of HE pathological staining of lung tissue. Figure 6 It can be seen that at a dose of 2 mg / kg, it can significantly inhibit the accumulation of airway inflammatory cells, relieve alveolar congestion and edema, and improve alveolar destruction. This result confirms that GW806742X can significantly inhibit inflammatory lung injury at a dose of 2 mg / kg.

[0046] 3. Preparation method of drugs for targeted treatment of inflammatory lung injury, the principle is as follows Figure 7 As shown, Figure 7 SPC-M-GW (SPC-Micelle-GW806742X) in the figure represents the drug, SPC indicates that it can target the surface protein SPC of alveolar type II epithelial cells, M indicates that it is coated with DSPE-PEG2000 lipids to form micelles, and GW indicates that the content is GW806742X. The specific process is as follows:

[0047] 3.1. Weigh appropriate amounts of DSPE-PEG 2000, DSPE-PEG 2000-MAL, and GW806742X in a mass ratio of 16:4:1, shake well, and dissolve in methanol (1 mg GW806742X in 1 ml methanol).

[0048] 3.2. Place the sample in a dialysis bag (MWCO 100-500) and dialyze against PBS (pH 7.5) at 4°C for 24 hours. Replace the external phase with fresh PBS buffer solution at 2, 4, 6, 8, and 12 hours.

[0049] 3.3. After 24 hours, remove the liposome suspension from the dialysis bag and store it on ice;

[0050] 3.4. Dissolve SPC antibody (Surfactant Associated Protein C) powder in HEPES buffer (1 M, pH 7.5);

[0051] 3.5. Add the liposome suspension to an appropriate amount of 50% glucose solution so that the final product contains 5% glucose;

[0052] 3.6. Mix the antibody dilution with the glucose-containing liposome suspension at a molar ratio of 1:2 between antibody and DSPE-PEG2000-MAL and place on a 3D suspension instrument at 4°C.

[0053] 3.7. After 6 hours of rotational incubation, collect the sample, place it in a dialysis bag (MWCO 1000K), and remove excess SPC antibodies by dialysis or ultrafiltration.

[0054] 3.8. Place at 4°C for 24 hours and dialyze against PBS (pH 7.5). Replace the external phase with fresh buffer solution at 2, 4, 6, 8, and 12 hours.

[0055] 3.9. The collected sample was placed in an ultrafiltration tube with a pore size of 3 kDa for concentration and purification (3000 rpm, 4°C, 30 min) to obtain a lipid micelle suspension.

[0056] 3.10. The collected lipid micelle suspension was diluted 1:100 (the diluent was PBS, and the zeta potential was negative at this time). The zeta potential, hydration diameter, and drug concentration were measured to calculate the drug loading and encapsulation efficiency. The preparation was successful if the hydrated diameter was between 80-200 nm, the dispersion coefficient was less than 0.1, and the encapsulation efficiency was greater than 60%.

[0057] In this example, dialysis was used to prepare the liposome suspension. Other known methods for preparing liposome suspensions include thin film hydration, reverse evaporation, solvent injection, surfactant removal, and microfluidics, all of which can replace step 3.2 of this product. The SPC antibody used in this example can be replaced with a peptide targeting the SPC protein or other similar antibodies or peptides targeting alveolar type II epithelial cell surface marker proteins. The DSPE-PEG2000 used in this example can be replaced with similar nationally approved lipid molecules.

[0058] Figure 8 The transmission electron microscopy images show lipid nanomicelles without antibody binding (M-GW) and lipid nanomicelles with antibody binding (SPC-M-GW). The translucent substance outside the particles in the right image is the SPC antibody. Figure 9 and Figure 10 These are statistical diagrams of the hydration diameter and potential of M-GW and SPC-M-GW, respectively. The vertical axis represents the diameter of the micelle particles and the size of the charge on the micelle surface, respectively.

[0059] 4. Targeted verification of finished drug products:

[0060] 4.1. Use CY5 powder (pink fluorescence) instead of GW806742X as feed material, and use the above method to prepare targeted lipid nanomedicines with CY5 fluorescence labeling in a light-proof environment.

[0061] 4.2. Calculate the required airway delivery volume of the GW806742X nanodrug targeting alveolar type II epithelial cells to achieve a dose of 2 mg / kg, 1 mg / kg, and 0.5 mg / kg. Based on the calculated results, prepare a 50 μl diluted solution of the same volume of fluorescent targeted nanodrug and administer it to the airways of healthy mice in a dark environment.

[0062] 4.3. 24 hours after instillation, lung tissue was collected from mice in the dark (same steps as in step 1), paraffin-fixed, and sectioned into 3 μm slices. The sections were fluorescently stained with SPC antibody (green) and DAPI (blue), and photographed using a fluorescence microscope. It was observed that the pink fluorescent particles in the field of view were mostly concentrated around cells labeled with green fluorescence, indicating that the targeted nanoparticles were present at high concentrations around alveolar epithelial II cells.

[0063] Figure 11Fluorescence microscopy images of mouse lung tissue showing DAPI (cell nucleus), SPC (alveolar type II epithelial cells) antibodies and Cy5 staining (CY5 fluorescent material coated with DSPE-PEG lipids modified with SPC antibodies). It can be seen that SPC and Cy5 are co-localized in alveolar type II epithelial cells, indicating that the fluorescent lipid micelles targeting alveolar type II epithelial cells are fused with alveolar type II epithelial cells.

[0064] 5. GW806742X nanomedicine targeting alveolar type II epithelial cells in the treatment of inflammatory lung injury disease models;

[0065] 5.1. The steps for constructing the animal model are the same as those described in 1 above.

[0066] 5.2. Targeted drug therapy: LPS was instilled into the airway to construct an inflammatory lung injury model (the method is the same as above). After 2-4 hours, the prepared GW806742X nanodrug targeting airway epithelial ATII cells was locally administered by airway instillation at a dose of 2 mg / kg, 1 mg / kg, and 0.5 mg / kg. After 24 hours, alveolar lavage fluid, serum, and lung tissue were collected for testing (the steps are the same as described in (1)).

[0067] Figure 12 HE staining of lung tissue from disease models was performed to evaluate inflammatory damage in the lung tissue of mice treated with the disease group, GW (GW806742X), M-GW (lipid-coated GW806742X to form micelles), or SPC-M-GW (a specific targeted drug targeting alveolar type II epithelial cells). The figure shows that the alveolar cavity of the lung tissue in the disease group was filled with inflammatory cells, the alveoli collapsed, the interstitial congestion and edema were evident, and the alveolar structure was significantly damaged. There was no improvement in inflammatory damage in the lung tissue of mice treated with 1 mg / kg of GW806742X or 1 mg / kg of lipid-coated GW806742X to form micelles (M-GW). In the lung tissue of mice treated with 1 mg / kg of lipid-coated GW806742X to form micelles (SPC-M-GW), which specifically targets the SPC protein, inflammatory cells were significantly reduced, interstitial congestion and edema were alleviated, and the alveolar structure was intact. Figure 13 The degree of lung injury was scored. Targeted drugs (SPC-M-GW) could significantly alleviate inflammatory damage, with **P<0.01, ***P<0.001.

[0068] In summary, the present invention has screened out a small molecule inhibitor, GW806742X, for necroptosis of alveolar epithelial cells, which can effectively alleviate inflammatory lung injury. The present invention encapsulates the small molecule inhibitor GW806742X into lipid particles to form a liposome suspension of uniform size and stable system. Through the interaction between chemical bonds, antibodies or peptides targeting type II alveolar cell surface marker proteins are combined with lipid nanoparticles, increasing the targeting properties of the lipid nanoparticles, enabling them to target type II alveolar cells, thereby inhibiting necroptosis of alveolar epithelial cells and achieving the purpose of treating airway inflammatory lung injury.

[0069] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention.

Claims

1. Use of GW806742X in the preparation of a drug for the targeted treatment of inflammatory lung injury, characterized in that: The preparation of the targeted treatment drug for inflammatory lung injury is to encapsulate GW806742X into lipid nanoparticles to form a liposome suspension, and then combine the antibody against the surface marker protein of type II alveolar cells with the lipid nanoparticles in the liposome suspension to obtain a finished drug. The liposome suspension was prepared by dissolving DSPE-PEG 2000, DSPE-PEG 2000-MAL, and GW806742X in methanol at a mass ratio of 16:4:1, shaking, and then placing the mixture in a dialysis bag. The mixture was dialyzed against PBS at 4°C for 24 h, with the external phase replaced with fresh PBS buffer at 2, 4, 6, 8, and 12 h. After the rotary dialysis, the liposome suspension in the dialysis bag was removed and stored on ice. The drug targets type II alveolar cells, inhibits necroptosis of alveolar epithelial cells, and achieves the purpose of treating airway inflammatory lung injury. The antibody against the surface marker protein of type II pneumocytes is an SPC antibody.

2. The use according to claim 1, characterized in that: The method for combining SPC antibodies with lipid nanoparticles in a liposome suspension comprises first dissolving SPC antibody powder in HEPES buffer to obtain an antibody dilution solution; adding the liposome suspension to a glucose solution so that the final product contains 5% glucose; then mixing the antibody dilution solution with the glucose-containing liposome suspension at a molar ratio of 1:2 between the antibody and DSPE-PEG2000-MAL, and placing the mixture on a 3D suspension instrument at 4°C. After 6 hours, a sample is collected and placed in a dialysis bag, and rotary dialyzed against PBS at 4°C for 24 hours. The external phase is replaced with fresh buffer solution at 2, 4, 6, 8, and 12 hours. After the rotary dialysis, the collected sample is placed in an ultrafiltration tube with a pore size of 3 KDa for concentration and purification to obtain a lipid micelle suspension; The conditions for the concentration and purification are a rotation speed of 3000 rpm, a temperature of 4° C., and a time of 30 min; The concentrated and purified lipid micelle suspension was diluted 1:100, and the zeta potential, hydration diameter, and drug concentration were measured to calculate the drug loading and encapsulation efficiency. The hydration diameter was measured to be between 80-200 nm, the dispersion coefficient was <0.1, and the encapsulation efficiency reached more than 60%, indicating that the finished drug product was successfully prepared.

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