Nintedanib-loaded composite nanoparticles for injection as well as preparation method and application of nintedanib-loaded composite nanoparticles

Abstract

The invention belongs to the technical field of biological medicines, and relates to a nintedanib-loaded composite nanoparticle for injection as well as a preparation method and application of the nintedanib-loaded composite nanoparticle. The composite nanoparticles take albumin as a main body skeleton, a nanostructure is formed through anti-solvent induced self-assembly, jellyfish collagen is embedded in the composite nanoparticles in a non-covalent manner, and meanwhile, an anti-fibrosis drug is entrapped. The preparation method does not depend on a chemical cross-linking agent, and self-assembly granulation is driven based on protein conformation transformation and non-covalent interaction between molecules; the composite nano-particles are good in nano-scale and dispersibility, the water dispersibility and the utilization rate of the indissolvable drug nintedanib are improved, and cell and animal experiment results prove that on the microscopic level, the composite nano-particles can remarkably inhibit TGF-beta1-induced fibroblasts from being excessively proliferated, migrated and converted into myofibroblasts; on the macroscopic level, excessive collagen deposition in the lungs of mice in a treatment group is obviously reduced, alveolar structure damage is relieved, and the fibrosis degree is obviously relieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to an injectable nintedanib-loaded composite nanoparticle, its preparation method, and its application. Background Technology

[0002] Pulmonary fibrosis (PF) is a chronic, progressive interstitial lung disease that primarily affects middle-aged and elderly men. Symptoms include cough and shortness of breath, and it is often accompanied by complications such as pulmonary hypertension, lung cancer, and ischemic heart disease, severely impacting patient survival. Its core pathological process is related to abnormal repair following alveolar epithelial damage. Fibroblasts are continuously activated and differentiate into myofibroblasts, producing and depositing excessive extracellular matrix (ECM), leading to fibrotic scarring of the lung structure, lung structural destruction, and loss of lung function.

[0003] Currently, only pirfenidone and nintedanib are approved by the U.S. Food and Drug Administration and the European Medicines Agency for the treatment of fibrosis (PF). Nintedanib, as a multi-target tyrosine kinase inhibitor, can simultaneously and potently block multiple key cellular signaling pathways driving the fibrosis process. However, existing nintedanib formulations are all oral soft capsules; no other formulations, such as inhalation or injection, have been approved for clinical use. Administration only via oral route presents challenges such as gastrointestinal adverse reactions and first-pass effects in the liver, resulting in low bioavailability and limiting its efficacy.

[0004] Therefore, developing novel drug delivery systems is crucial for improving the treatment efficacy of pulmonary fibrosis. Nanoparticle and microparticle formulations, with their excellent biocompatibility and physicochemical properties, show great potential in the field of drug delivery. Microvascular damage and permeability changes in fibrotic lungs, along with an increase in immune cells and fibroblasts, can lead to enhanced nanoparticle uptake, potentially increasing lung exposure. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes an injectable nintedanib-loaded composite nanoparticle, its preparation method, and its application.

[0006] The technical solution of this invention is as follows: A method for preparing injectable nintedanib-loaded composite nanoparticles, characterized by comprising the following steps: S1. Adjust the pH of the human serum albumin solution to alkaline, mix and stir the jellyfish collagen solution with the human serum albumin solution to obtain a mixed protein solution; S2. Nintedanib is dissolved in ethanol to prepare a nintedanib ethanol solution. Under continuous stirring, the nintedanib ethanol solution is added dropwise to the mixed protein solution described in step S1 to obtain a dispersion. S3. The dispersion obtained in step S1 is subjected to ultrasonic treatment to obtain a composite nanoparticle suspension. The composite nanoparticle suspension is centrifuged, washed, and then freeze-dried to obtain nydanib-loaded composite nanoparticles for injection.

[0007] Furthermore, the pH of the human serum albumin solution described in step S1 is 7.5-9.0; Furthermore, the technical parameters for stirring in step S1 are: stirring at 300-500 rpm for 30-60 minutes at a temperature of 25±1℃.

[0008] Further, the preparation method of the jellyfish collagen solution in step S1 is as follows: jellyfish tissue is desalted, cut into small pieces, soaked in NaOH solution for 20-30 hours, and then washed; the washed jellyfish tissue is added to glacial acetic acid solution and stirred at 4°C for 36-72 hours to obtain an extract; pepsin is added to the extract and reacted for 20-30 hours, centrifuged, and the supernatant is retained; the supernatant is precipitated by NaCl salting out, the precipitate is dissolved in acetic acid solution, dialyzed to remove salt, and freeze-dried to obtain jellyfish collagen; the jellyfish collagen is redissolved in deionized water to obtain a jellyfish collagen solution.

[0009] Further, the mass ratio of jellyfish collagen to human serum albumin in step S1 is 1:(3-5).

[0010] Further, in step S2, the mass ratio of albumin in the nintedanib:mixed protein solution is 1:(10-20).

[0011] Furthermore, the technical parameters for the dripping in step S2 are 10-20 drops / min, with each drop being 20 μL.

[0012] Furthermore, the technical parameters for the ultrasonic treatment in step S3 are: power of 200W, ultrasonic treatment for 5 seconds, interval for 10 seconds, and total duration of 3 minutes.

[0013] The nydanib-loaded composite nanoparticles for injection were prepared according to the preparation method described above.

[0014] Furthermore, the particle size of the injectable nydanib-loaded composite nanoparticles is 80-140 nm.

[0015] The application of the injectable nintedanib-loaded composite nanoparticles in the preparation of drugs for treating pulmonary fibrosis, wherein the pulmonary fibrosis is TGF-β1- or BLM-induced pulmonary fibrosis.

[0016] Compared with the prior art, the present invention has at least the following advantages: 1. This invention relates to injectable nintedanib-loaded composite nanoparticles and their preparation method. The composite nanoparticles use human serum albumin as the main framework, forming a nanostructure through antisolvent-induced self-assembly. Jellyfish collagen is non-covalently embedded within the nanostructure, and an anti-fibrotic drug is encapsulated therein. The preparation method does not rely on chemical cross-linking agents, but is mainly based on protein conformational changes and non-covalent intermolecular interactions driving self-assembly into nanoparticles. The composite nanoparticles prepared by this method exhibit good dispersibility at the nanoscale, improving the water dispersibility and bioavailability of the poorly soluble drug nintedanib, which is beneficial for pulmonary administration. Furthermore, they remain stable in the bloodstream (hemolysis rate <5%), making them suitable for intravenous injection and providing a new dosage form option for the treatment of pulmonary fibrosis.

[0017] 2. This invention also relates to the application of the aforementioned injectable nintedanib-loaded composite nanoparticles in the preparation of drugs for treating pulmonary fibrosis. Cellular and animal experimental results demonstrate that, at the microscopic level, the composite nanoparticles significantly inhibit TGF-β1-induced excessive proliferation, migration, and transformation into myofibroblasts; at the macroscopic level, excessive collagen deposition in the lungs of mice in the treatment group was significantly reduced, alveolar structural damage was alleviated, and the degree of fibrosis was significantly reduced. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0019] Figure 1 This is a SEM image of CHA@N prepared in Example 3.

[0020] Figure 2 The particle size distribution of CHA@N prepared in Example 3 is shown.

[0021] Figure 3 Biocompatibility evaluation of CHA@N prepared in Example 3; Figure 3 (a) Cell viability of A549 cells and NIH / 3T3 cells after 2 days of treatment with different concentrations of CHA@N; Figure 3 (b) is a graph showing hemolysis experiments with different concentrations of CHA@N; Figure 3 (c) is a statistical chart of hemolysis rate for different concentrations of CHA@N.

[0022] Figure 4 This is a statistical chart showing the results of inhibiting the excessive proliferation of NIH / 3T3 cells in each experimental group in Example 6.

[0023] Figure 5 The results of inhibiting the migration of NIH / 3T3 cells in each experimental group of Example 6 are shown.

[0024] Figure 6This is a stained image of lung tissue sections from different animal experimental groups in Example 7.

[0025] Figure 7 The immunoblots show the expression levels of α-SMA protein in different animal experimental groups in Example 7. Detailed Implementation

[0026] The present invention will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0027] This invention provides a general and / or specific description of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods; reagents or instruments used, unless otherwise specified, are commercially available conventional products prepared or used using conventional methods.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0029] Example 1: Extraction and Preparation of Jellyfish Collagen This embodiment provides a method for preparing high-purity, low-immunogenic jellyfish collagen, the specific steps of which are as follows.

[0030] S1. Pretreatment: Fresh jellyfish tissue was desalted, cut into pieces, and placed in 0.1 mol / L NaOH solution for 24 h at room temperature to remove non-collagenous proteins from the tissue.

[0031] S2. Acid-enzyme extraction: Discard the alkaline solution, wash the precipitate repeatedly with deionized water until neutral, then add 0.5 mol / L glacial acetic acid solution at a material-to-liquid ratio of 1:10 w / v, and extract magnetically at 4°C for 48 h. Then add 0.1 wt% of the substrate mass of pepsin to the system, maintain the acidic conditions and continue stirring for 24 h. The pepsin can improve collagen solubility and reduce its immunogenicity.

[0032] S3. Purification and Drying: After the reaction, centrifuge at 10,000 rpm for 30 min to remove undissolved residue. Add NaCl to the supernatant to achieve a final concentration of 0.9 mol / L for salting out. Let stand overnight to precipitate collagen. Centrifuge to collect the precipitate, redissolve it in 0.1 mol / L acetic acid solution, place it in a dialysis bag, and dialyze it in deionized water to remove salt. Freeze-dry the dialyzed solution to obtain jellyfish collagen, and store it at -20℃ for later use.

[0033] Example 2: Preparation of Jellyfish Collagen-Albumin Composite Nanoparticles (CHA) This embodiment prepares drug-free protein composite nanoparticles, and the preparation method is shown below.

[0034] S1. Preparation of protein solution: Take 1g of jellyfish collagen prepared in Example 1, dissolve it in deionized water, and stir until fully dissolved to prepare a 1% (w / v) jellyfish collagen solution; take human serum albumin (HSA), add deionized water to prepare a 1% (w / v) HSA solution; adjust the pH of the HSA solution to 8.0 with NaOH / HCl, stir at room temperature for 2 hours to allow hydration and expansion, filter through a filter membrane to remove insoluble matter / aggregates, and obtain a clear HSA solution for later use. The pH of the HSA solution described in this invention can be adjusted between 7.5 and 9.0.

[0035] S2. Mixing and self-assembly: Take the clarified HSA solution obtained in step S1 and mix it with the jellyfish collagen solution, wherein the mass ratio of jellyfish collagen to albumin is 1:4; gently stir at 300 rpm for 30 min at room temperature to avoid generating bubbles, so that the two proteins form a homogeneous mixed protein solution in the aqueous phase.

[0036] S3. Antisolvent-induced particle formation: Under stirring at 1000 rpm, anhydrous ethanol was slowly added dropwise to the mixed protein solution from step S2 using a syringe pump. The ethanol dropping rate was controlled at 20 drops / min, with each drop being 20 μL. The final volume ratio of ethanol to the mixed protein solution was 15:1. After the addition of ethanol, the conformation of HSA molecules changed and they co-assembled with jellyfish collagen. After the ethanol addition was complete, stirring was continued for 2 hours. The dispersion system was then subjected to ultrasonic treatment at a power of 200W, with a 3-second working time followed by a 10-second interval, for a total of 3 minutes, to improve particle dispersibility and prevent aggregation.

[0037] S4. Solidification and collection: The suspension after sonication in S3 was centrifuged at 12,000 rpm for 10 min, the supernatant was discarded, the precipitate was resuspended in deionized water and washed three times by centrifugation to remove residual ethanol; finally, the precipitate was freeze-dried to obtain jellyfish collagen-albumin composite nanoparticles (CHA).

[0038] Example 3: Preparation of Nydanibue jellyfish collagen-albumin composite nanoparticles (CHA@N) In this embodiment, the drug nintedanib is encapsulated in composite nanoparticles by antisolvent coprecipitation. The preparation method of CHA@N in this embodiment is the same as in Embodiment 2, except for step S3, and the preparation method is as follows.

[0039] S1. Preparation of protein solution: Take 1g of jellyfish collagen prepared in Example 1, dissolve it in deionized water, and stir until fully dissolved to prepare a 1% (w / v) jellyfish collagen solution; take human serum albumin (HSA), add deionized water to prepare a 1% (w / v) HSA solution; adjust the pH of the HSA solution to 8.0 with NaOH / HCl, stir at room temperature for 2 hours to allow hydration and expansion, filter through a filter membrane to remove insoluble matter / aggregates, and obtain a clear HSA solution for later use. The pH of the HSA solution described in this invention can be adjusted between 7.5 and 9.0.

[0040] S2. Mixing and Self-Assembly: The clarified HSA solution obtained in step S1 is mixed with the jellyfish collagen solution, wherein the mass ratio of jellyfish collagen to albumin is 1:4; the mixture is gently stirred at 300 rpm for 30 min at room temperature to avoid generating bubbles, so that the two proteins form a homogeneous mixed protein solution in the aqueous phase. The mass ratio of jellyfish collagen to albumin in this invention can be adaptively adjusted within the range of 1:(3-5).

[0041] S3. Antisolvent-induced granulation: Nintedanib was dissolved in 120 mL of anhydrous ethanol to obtain a nintedanib ethanol solution, with a nintedanib to albumin mass ratio of 1:20. Under stirring at 1000 rpm, the nintedanib ethanol solution was slowly added dropwise to the mixed protein solution from step S2 using a syringe pump at a rate of 15 drops / min, with each drop being 20 μL. After the ethanol was added, the conformation of HSA molecules changed, and they co-assembled with jellyfish collagen. After the nintedanib ethanol solution was completely added, stirring continued for 2 hours. The dispersion system was then sonicated to obtain a suspension. The sonication was performed at 200 W for 3 seconds with a 10-second interval, for a total of 3 minutes, to improve particle dispersibility and prevent aggregation. The mass ratio of nintedanib to albumin in the mixed protein solution of this invention can be adaptively adjusted within the range of 1:(10-20).

[0042] S4. Solidification and Collection: The suspension after S3 sonication was centrifuged at 12000 rpm for 10 min, the supernatant was discarded, the precipitate was resuspended in deionized water and washed three times by centrifugation to remove residual ethanol to avoid toxicity; finally, the precipitate was freeze-dried to obtain nydandelion collagen-albumin composite nanoparticles (CHA@N).

[0043] Example 4: Characterization of the physicochemical properties of CHA@N This embodiment characterizes the morphology and particle size distribution of CHA@N prepared in Example 3.

[0044] The results of scanning electron microscopy (SEM) observations are as follows: Figure 1 As shown in the figure, the prepared CHA@N exhibits a distinct nanosphere structure with uniform size and good dispersibility.

[0045] Dynamic light scattering (DLS) particle size determination: CHA@N was tested using a dynamic light scattering instrument, and the results are as follows: Figure 2 As shown in the figure, the particle size of CHA@N is 80-140 nm, and the average particle size is 107 nm.

[0046] In summary, the CHA@N particles prepared by the method described in this invention have small particle size and good dispersibility, which is beneficial for pulmonary drug delivery.

[0047] Example 5: CHA@N Biocompatibility Evaluation Cytotoxicity assay: A549 and NIH / 3T3 cells were cultured in cell culture dishes. When the confluence reached 70-80%, trypsin was added for digestion, and the cells were collected by centrifugation. The cells were then seeded into 96-well plates at a density of 5 × 10⁶ cells per well. 3 Cells were incubated for 24 hours. After incubation, different concentrations of CHA@N were co-cultured with the cells for 48 hours. Then, CCK-8 reagent was added, and cell metabolic activity was quantified by detecting the absorbance (OD value) at 450 nm. Cell viability was calculated to assess toxicity. CCK-8 results are shown below. Figure 3 As shown in (a).

[0048] Hemolytic activity: Whole blood was obtained from healthy BALB / c mice via venous collection and collected in anticoagulant tubes containing EDTA or sodium citrate. After thorough shaking, the blood cells were obtained by centrifugation at 3000 rpm for 15 min. Protein composite nanoparticles of different concentrations were prepared, and 1 mL of each nanoparticle solution, 1 mL of ultrapure water, and 1 mL of PBS solution were mixed with 20 μL of blood cells. The mixtures were incubated at 37°C for 4 h, followed by centrifugation at 3000 rpm for 15 min. The samples were then arranged horizontally and photographed. Figure 3 As shown in (b). The supernatant was then collected by centrifugation, and the absorbance resulting from hemoglobin release was measured at 545 nm using a spectrophotometer. The absorbance was compared with the OD values ​​of the negative control (physiological saline) and the positive control (deionized water). Figure 3 As shown in (c), the hemolysis rate was calculated to evaluate the destructive effect of the material on red blood cells.

[0049] Figure 3 The results showed that the protein-composite nanoparticles exhibited good cell compatibility and no significant cytotoxicity within the tested concentration range. The supernatants of all experimental groups and the negative control group (PBS) were clear with no significant heme release; the positive control group (H2O) showed virtually no intact red blood cell deposition at the bottom, and almost all red blood cells ruptured. Meanwhile... Figure 3(c) The hemolysis rate in all experimental and negative control groups shown was below 5%, which indicates that CHA@N has good blood compatibility, is stable in the blood circulation, and is suitable for intravenous injection.

[0050] Example 6: Evaluation of In vitro inhibition of fibroblast activation and migration Inhibition of cell proliferation: NIH / 3T3 cells were cultured in cell culture dishes. When the confluence reached 70-80%, they were digested with trypsin, centrifuged to collect the cells, and then seeded into 96-well plates at 3000 cells per well, with 200 μL of DMEM complete medium added. The seeded 96-well plates were placed in a cell culture incubator to allow the cells to adhere and grow overnight. After 24 h of culture, the medium was replaced with serum-free medium and starved for 24 h, followed by stimulation with TGF-β1 (10 ng / ml) for 48 h to induce excessive proliferation and activation of fibroblasts. The experimental groups were: normal control group (Control group), TGF-β1 group, TGF-β1+CHA group, and TGF-β1+CHA@N group. At the corresponding time points, remove the corresponding well plates, aspirate the liquid from the 96-well plates using a 200 μL pipette, wash once with 100 μL PBS buffer, add 100 μL LMEM basal medium and 10 μL CCK8 solution to each well, and then incubate the 96-well plates back in the cell culture incubator for 2-4 hours, observing the color change. When the color turns golden yellow, place the 96-well plates in a spectrophotometer and measure the OD value at 450 nm. For result analysis, subtract the control well or OD value from the OD value of each well, and take the average of the OD values ​​of all parallel wells. Cell viability % = (OD of drug-treated cells - OD of blank cells) / (OD of control cells - OD of blank cells) × 100%. Plot the time line on the x-axis and the OD value on the y-axis, and compare the growth rate of the curves between different groups to reflect the differences in cell proliferation capacity. Figure 4 The results showed that TGF-β1+CHA@N drug-loaded protein composite nanoparticles could effectively inhibit the proliferation of NIH / 3T3 cells.

[0051] Inhibition of cell migration: After the same treatment, NIH / 3T3 cells were seeded in six-well plates and cultured until 100% confluence was achieved. Then, a straight incision was made along the midline of the cell layer using a sterile 200 μL pipette tip, followed by a slight rinse with 1 mL of PBS buffer to remove cell debris. Cells in the six-well plates were photographed using a microscope. Grouping was the same as above. The specific intervention method involved pretreatment with the drug for 3 hours, followed by stimulation with TGF-β1 (10 ng / ml) for 24 hours. After stimulation, microscopic images were taken again. ImageJ was used to calculate the area of ​​cell-free regions in the six-well plates. By comparing the reduction in cell area after corresponding stimulation in different cell groups, the differences in cell migration ability could be reflected. Figure 5 The results showed that TGF-β1+CHA@N drug-loaded protein composite nanoparticles could effectively inhibit the migration of NIH / 3T3 cells.

[0052] Example 7: In vivo efficacy evaluation of anti-pulmonary fibrosis drugs The experimental groups were set up as a control group (Control), a model group (BLM), a carrier treatment group (BLM+CHA), and a drug-loaded protein composite nanoparticle treatment group (BLM+CHA@N). Eight male C57BL / 6 mice were used in each group. Mice were anesthetized with isoflurane and intratracheally instilled with 0.2 mL of bleomycin (BLM) at a concentration of 3 mg / kg to establish a pulmonary fibrosis model. The control group received an equal volume of saline. Treatment began 7 days later. The carrier treatment group and the drug-loaded protein composite nanoparticle treatment group received a tail vein injection of 2 mg / kg of the drug every two days (CHA and CHA@N were reconstituted with 0.9% saline), while the model group received an equal volume of saline. The experiment ended on day 28. Lung tissue sections were collected for hematoxylin-eosin (H&E) staining and Masson's staining to observe the pulmonary fibrosis status.

[0053] like Figure 6 As shown, drug-loaded protein composite nanoparticles can effectively inhibit lung fibrosis in mice. Compared with the model group, the CHA@N treatment group can alleviate BLM-induced fibrosis to a certain extent, reduce collagen deposition, preserve some alveolar structure, and reduce the area of ​​fibrosis.

[0054] Alpha-smooth muscle actin (α-SMA) is a core biomarker in the pathological process of pulmonary fibrosis. In normal lung tissue, α-SMA is expressed in large quantities during the fibrotic process. The expression level of α-SMA directly indicates the number of myofibroblasts transforming and the intensity of fibrotic activity, and is a key indicator for assessing disease severity and verifying the effectiveness of anti-fibrotic therapies in inhibiting this core process. Figure 7As shown, compared to the model group, CHA@N, after intravenous injection, can target lung lesions, effectively release nintedanib, inhibit the expression level of α-SMA, and suppress the progression of fibrosis.

[0055] The technical principle of CHA@N preparation described in this invention is as follows: HSA solution serves as the framework material for nanoparticles. When the pH of HSA is adjusted to be alkaline and far from the isoelectric point, the protein carries a negative charge, maintaining an extended molecular state to prepare for subsequent self-assembly. When an ethanol solution containing the hydrophobic drug nintedanib is slowly introduced into the HSA aqueous solution as an antisolvent, the dielectric constant of the system decreases, the protein solvation layer is weakened, and the HSA molecules undergo conformational rearrangement, exposing hydrophobic regions. Subsequently, driven by hydrophobic interactions, hydrogen bonding, and electrostatic interactions, nanoscale aggregates are formed, manifested as the solution changing from clear to turbid and eventually forming stable, dispersed nanoparticles. When HSA aggregates under ethanol induction, jellyfish collagen adsorbs / accompanies the hydrophilic regions on the particle surface or inside the particle through hydrogen bonding, electrostatic interactions, and physical entanglement, forming composite particles in a one-step process, thereby enhancing surface hydrophilicity and cell compatibility. The composite nanoparticles gradually degrade and release the loaded drug in the lung tissue microenvironment, effectively slowing down the proliferation, migration and transformation of fibroblasts into activated myofibroblasts, thereby inhibiting the excessive secretion and deposition of extracellular matrix and delaying the progression of pulmonary fibrosis.

[0056] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for preparing injectable nintedanib-loaded composite nanoparticles, characterized in that, Includes the following steps: S1. Adjust the pH of the human serum albumin solution to alkaline, mix and stir the jellyfish collagen solution with the human serum albumin solution to obtain a mixed protein solution; S2. Nintedanib is dissolved in ethanol to prepare a nintedanib ethanol solution. Under continuous stirring, the nintedanib ethanol solution is added dropwise to the mixed protein solution described in step S1 to obtain a dispersion. S3. The dispersion obtained in step S1 is subjected to ultrasonic treatment to obtain a composite nanoparticle suspension. The composite nanoparticle suspension is centrifuged, washed, and then freeze-dried to obtain nydanib-loaded composite nanoparticles for injection.

2. The preparation method according to claim 1, characterized in that, The pH of the human serum albumin solution in step S1 is 7.5-9.0; the stirring parameters in step S1 are: stirring at 300-500 rpm for 30-60 min at a temperature of 25±1℃.

3. The preparation method according to claim 1, characterized in that, The specific method for preparing the jellyfish collagen solution in step S1 is as follows: jellyfish tissue is desalted, cut into small pieces, soaked in NaOH solution for 20-30 hours, and then washed; the washed jellyfish tissue is added to glacial acetic acid solution and stirred at 4°C for 36-72 hours to obtain an extract; pepsin is added to the extract and reacted for 20-30 hours, centrifuged, and the supernatant is retained; the supernatant is precipitated by NaCl salting out, the precipitate is dissolved in acetic acid solution, dialyzed to remove salt, and freeze-dried to obtain jellyfish collagen; the jellyfish collagen is redissolved in deionized water to obtain the jellyfish collagen solution.

4. The preparation method according to claim 1, characterized in that, The mass ratio of jellyfish collagen to human serum albumin in step S1 is 1:(3-5).

5. The preparation method according to claim 1, characterized in that, In step S2, the mass ratio of albumin in the nintedanib mixed protein solution is 1:(10-20).

6. The preparation method according to claim 1, characterized in that, The technical parameters for the dripping in step S2 are 10-20 drops / min, with each drop being 20 μL.

7. The preparation method according to claim 1, characterized in that, The technical parameters for the ultrasonic treatment in step S3 are: power of 200W, ultrasonic treatment for 5 seconds, interval for 10 seconds, and total duration of 3 minutes.

8. Injectable nydanib-loaded composite nanoparticles prepared by the preparation method according to any one of claims 1-7.

9. The injectable nintedanib-loaded composite nanoparticles according to claim 8, characterized in that, The particle size of the injectable nydanib-loaded composite nanoparticles is 80-140 nm.

10. The use of the injectable nintedanib-loaded composite nanoparticles according to claim 8 in the preparation of drugs for treating pulmonary fibrosis, characterized in that, The pulmonary fibrosis is TGF-β1- or BLM-induced pulmonary fibrosis.

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