Enzyme-responsive inhalable microgel as well as preparation method and application thereof

Abstract

The invention provides inhalable microgel with enzyme responsiveness and a preparation method of the inhalable microgel, and belongs to the technical field of inhalation type preparations. Microgel constructed by a cross-linking agent with MMP2 enzyme responsiveness serves as a carrier, nintedanib and trastuzumab are jointly entrapped, enzyme-triggered targeted release of the medicine at the pulmonary fibrosis part is achieved through inhalation administration, and the curative effect is synergistically enhanced; the preparation method of the microgel comprises the following steps: preparing nintedanib lipidosome, and reducing disulfide bonds of trastuzumab; the preparation method comprises the following steps: preparing a nintedanib lipidosome and trastuzumab through a sulfydryl-maleimide reaction to obtain a solution containing free sulfydryl, connecting the nintedanib lipidosome and trastuzumab through the sulfydryl-maleimide reaction, mixing the nintedanib lipidosome solution connected with trastuzumab with a cross-linking agent prepared from CPVGLIDC and 4-arm polyethylene glycol maleimide according to a molar ratio of 1: 1, and preparing microgel through a water-in-oil emulsification method; and finally, carrying out gradient centrifugal screening to obtain microgels with three particle sizes. Nintedanib and trastuzumab synergistically enhance the anti-pulmonary fibrosis effect, and the effect is better than that of single-drug treatment.

Description

Technical Field

[0001] This invention belongs to the field of inhaled formulation technology, specifically relating to an enzyme-responsive inhalable microgel, its preparation method, and its application. Background Technology

[0002] Microgels are a class of nanoscale soft matter particles composed of a triple-crosslinked polymer network. They possess swelling properties and, compared to traditional hydrogels, are smaller in volume and have a larger specific surface area, making them widely used in drug delivery, tissue engineering, and smart materials. Microgels offer significant advantages as drug carriers for pulmonary delivery. Their network structure provides a suitable aerodynamic diameter for deposition deep within the lungs, and their swelling properties allow them to expand in the moist environment of the lungs, increasing their volume and preventing phagocytosis by pulmonary macrophages, thus achieving effective drug delivery.

[0003] Considering drug safety and biocompatibility, 4ARM-PEG-MAL and CPVGLIGC can be used as cross-linking agents to construct microgels. 4ARM-PEG-MAL is readily soluble in water and common organic solvents. Its structure has four branches, each ending in a maleimide (MAL) group, which can undergo Michael addition reactions with thiol groups to form stable covalent bonds. CPVGLIGC is a short peptide sequence composed of eight amino acids (Cys-Pro-Val-Gly-Leu-Ile-Gly-Cys). It has thiol groups on its cysteine ​​side chains at the beginning and end of the molecule, which can react with the maleimide groups in 4ARM-PEG-MAL to cross-link and form a network structure. Since MMP2 enzymes tend to cleave peptide bonds near hydrophobic amino acids, the Gly-Leu (GL) bond in the CPVGLIGC sequence is the main cleavage site for MMP2 enzymes, therefore CPVGLIGC exhibits MMP2 enzyme responsiveness. Since the activity and expression of MMP2 enzyme are abnormal in the pulmonary fibrosis site, affecting the metabolism of extracellular matrix components in the lungs, 4ARM-PEG-MAL and CPVGLIGC which are MMP2 enzyme responsive can be used as cross-linking agents to construct MMP2 enzyme responsive microgels.

[0004] Nintedanib is a triple angiokinase inhibitor that can reduce cell proliferation and migration and extracellular matrix formation, thereby slowing the progression of pulmonary fibrosis. It is used to treat pulmonary fibrosis and various lung diseases, and is mostly administered orally. However, it has significant toxic side effects, specifically nausea, vomiting, diarrhea, and elevated liver transaminase levels.

[0005] Trastuzumab is a drug that targets the human epidermal growth factor receptor 2 (HER2) gene. Studies have shown that blocking HER2 signaling can inhibit pulmonary fibrosis. Therefore, trastuzumab can also be used to treat pulmonary fibrosis. It is usually administered intravenously or subcutaneously. Patient compliance is poor and it has certain cardiotoxicity. The efficacy and safety need to be evaluated individually.

[0006] In summary, the further clinical application of nintedanib and trastuzumab is limited. However, combining nintedanib and trastuzumab using a microgel as a carrier for pulmonary inhalation can reduce their toxic side effects, enhance targeting, and improve bioavailability. Furthermore, the abnormal MMP2 enzyme activity and expression in pulmonary fibrosis sites allows for MMP2 enzyme responsiveness studies. Therefore, inhalable microgels with MMP2 enzyme responsiveness can be constructed. Summary of the Invention

[0007] This invention addresses the problems of existing technologies by providing an enzyme-responsive inhalable microgel, its preparation method, and its applications.

[0008] To achieve the above objectives, the technical solution adopted in this application is as follows: An enzyme-responsive inhalable microgel includes a pharmaceutically active ingredient and a drug carrier, wherein the pharmaceutically active ingredient includes nintedanib and trastuzumab, and the drug carrier comprises a microgel.

[0009] A method for preparing an enzyme-responsive inhalable microgel, the specific steps of which are as follows: Step 1: Preparation of nintedanib liposomes: Nintedanib raw material, soybean lecithin, cholesterol, and DSPE-PEG2K-MAL were mixed and dissolved in anhydrous ethanol. The solution was then slowly and uniformly injected into a nitrogen-deoxygenated phosphate buffer solution. The mixture was heated and stirred at 40°C for 2 h to remove the ethanol. Subsequently, the mixture was dialyzed overnight using a 1 kD dialysis bag to obtain nintedanib liposomes. Step 2: Preparation of trastuzumab solution: Trastuzumab was reduced using TCEP reducing agent to open the disulfide bonds in trastuzumab. The mixture was incubated on a shaker for 4 h and then dialyzed overnight at 4°C. After dialysis, the TCEP reducing agent was removed using a 10 kD dialysis bag to obtain the trastuzumab solution. Step 3: Connecting nintedanib liposomes to trastuzumab: Take the above nintedanib liposomes and trastuzumab solutions, mix them evenly at a molar ratio of 1:1, react overnight at 4°C, and then dialyze with a 150 KD dialysis bag to obtain a nintedanib liposome solution connected with trastuzumab. Step 4: Preparation of microgels: CPVGLIGC and 4-arm polyethylene glycol maleimide were mixed at a molar ratio of 1:1 to prepare a crosslinking agent. Then, the above-mentioned nintedanib liposome solution linked with trastuzumab was mixed with the crosslinking agent. The mixture of nintedanib liposome solution linked with trastuzumab and crosslinking agent was slowly injected into mineral oil using a syringe. The mixture was heated and stirred at 37°C for 2 h to obtain the prepared microgels. Step 5: Sieving the microgels: Take out the mixture of the prepared microgels and mineral oil, add 2-3 times the total volume of the prepared microgels and mineral oil in phosphate buffer solution, and then centrifuge at 1000 rpm for 20 min to obtain the first oil supernatant, intermediate layer P1, and first PBS layer; take the first oil supernatant and add 2-3 times the volume of the first oil supernatant in phosphate buffer solution, and then centrifuge at 2000 rpm for 20 min to obtain the second oil supernatant, intermediate layer P2, and second PBS layer; take the second oil supernatant and add 2-3 times the volume of the second oil supernatant in phosphate buffer solution, and then centrifuge at 3000 rpm for 20 min to obtain the intermediate layer P3; then wash the obtained intermediate layer repeatedly with PBS solution 3 times to obtain microgels of three particle sizes: P1, P2, and P3.

[0010] Preferably, in step one, the molar ratio of nintedanib raw material to soybean lecithin is 1:10 to 1:5; and the pH of the phosphate buffer solution is 5-8.

[0011] Preferably, in step four, the mineral oil is a mineral oil containing 0.5% to 3% (V / V) of surfactant; the surfactant is one or more of Tween or Span.

[0012] Preferably, the Tween is Tween 80, and the hydrophilic-lipophilic balance value of Tween 80 is 15; the Span is Span 80, and the hydrophilic-lipophilic balance value of Span 80 is 4.3.

[0013] An enzyme-responsive inhalable microgel for use in the preparation of enzyme-responsive pulmonary inhalation drug delivery.

[0014] Compared with the prior art, the advantages and positive effects of the present invention are as follows: This application discloses an enzyme-responsive inhalable microgel, its preparation method, and its application. A microgel constructed with a cross-linking agent responsive to the MMP2 enzyme is used as a carrier to jointly encapsulate nintedanib and trastuzumab. Inhalation administration enables enzyme-triggered targeted release of the drug at the site of pulmonary fibrosis, synergistically enhancing therapeutic efficacy. The microgel preparation steps include: preparation of nintedanib liposomes; reduction of the disulfide bonds of trastuzumab to obtain a solution containing free thiol groups; linking nintedanib liposomes and trastuzumab via a thiol-maleimide reaction; mixing the nintedanib liposome solution linked to trastuzumab with a cross-linking agent prepared by CPVGLIGC and 4-arm polyethylene glycol maleimide in a 1:1 molar ratio; and preparing the microgel via a water-in-oil emulsion method. Finally, microgels of three particle sizes are obtained by gradient centrifugation and sieving. The synergistic enhancement of anti-pulmonary fibrosis effects by nintedanib and trastuzumab is superior to monotherapy. The microgels were prepared using the specific enzyme-responsive peptide crosslinking agent CPVGLIGC and 4ARM-PEG-MAL, and a microgel network structure was constructed by water-in-oil emulsification. This design allows the microgels to specifically dissociate and release drugs in the pathological microenvironment of pulmonary fibrosis where MMP2 enzymes are enriched, thereby effectively avoiding non-specific phagocytosis and clearance by pulmonary macrophage microgels, and significantly improving drug retention and targeted therapeutic effects in the lungs. This invention encapsulates nintedanib and trastuzumab in a liposomal microgel and uses inhalation administration to improve drug targeting and bioavailability, providing a reference for the clinical treatment of pulmonary fibrosis; moreover, the microgel can significantly inhibit the progression of pulmonary fibrosis and has high safety. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This image shows the effect of nintedanib liposomes conjugated with trastuzumab (TRA). Figure 2 This is a microgel particle size diagram under a fluorescence microscope; Figure 3 TEM images of the microgel before and after incubation with MMP2 enzyme; Figure 4 This is a fluorescence microscopy image showing the fluorescence distribution of liposome microgels containing coumarin 6 (C6) before and after incubation with MMP2 enzyme. Figure 5This image shows the effect of microgel samples of different particle sizes on preventing macrophage phagocytosis under a fluorescence microscope. Figure 6 Figures showing the hemolytic phenomenon of nintedanib in red blood cell suspension at different concentrations; Figure 7 Figures showing the results of the cell scratch experiment for each experimental group; Figure 8 The figures show the Transwell experimental results for each experimental group. Detailed Implementation

[0017] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0018] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification.

[0019] This application discloses an enzyme-responsive inhalable microgel comprising a pharmaceutical active ingredient and a drug carrier, wherein the pharmaceutical active ingredient comprises nintedanib and trastuzumab, and the drug carrier comprises a microgel.

[0020] A method for preparing an enzyme-responsive inhalable microgel, the specific steps of which are as follows: Step 1: Preparation of nintedanib liposomes: Nintedanib raw material, soybean lecithin, cholesterol, and DSPE-PEG2K-MAL were mixed and dissolved in anhydrous ethanol. The solution was then slowly and uniformly injected into nitrogen-deoxygenated phosphate-buffered saline (PBS). The mixture was heated and stirred at 40°C for 2 h to remove the ethanol. Subsequently, the mixture was dialyzed overnight using a 1 kD dialysis bag to obtain nintedanib liposomes. The molar ratio of nintedanib raw material to soybean lecithin was 1:10 to 1:5. The pH of the phosphate-buffered saline solution was 5-8. Step 2: Preparation of trastuzumab solution: The disulfide bonds of trastuzumab are reduced using TCEP reducing agent; during this process, the disulfide bonds of trastuzumab are reduced to thiol groups by TCEP; incubate on a shaker for 4 h, and dialyze overnight at 4°C; the overnight dialysis process is carried out in the dark; after dialysis, a 10 kD dialysis bag is used to remove the TCEP reducing agent to obtain the trastuzumab solution; Step 3: Ligation of nintedanib liposomes with trastuzumab: Take the above nintedanib liposomes and trastuzumab solutions, mix them evenly at a molar ratio of 1:1, react overnight at 4°C, and dialyze with a 150 KD dialysis bag to obtain a nintedanib liposome solution conjugated with trastuzumab; wherein, the thiol groups of the trastuzumab solution react with the maleimide of DSPE-PEG2K-MAL in the nintedanib liposomes, thereby linking the nintedanib liposomes with trastuzumab; Step 4: Preparation of microgels: A crosslinking agent was prepared by mixing CPVGLIGC and 4-arm polyethylene glycol maleimide (4ARM-PEG-MAL) at a molar ratio of 1:1. The nintedanib liposome solution linked to trastuzumab was then mixed with the crosslinking agent. Using a syringe, the mixture of the nintedanib liposome solution linked to trastuzumab and the crosslinking agent was slowly injected into mineral oil. The mixture was heated and stirred at 37°C for 2 hours to obtain the prepared microgels. In Step 4, the mineral oil contained 0.5%–3% (V / V) of a surfactant. The surfactant was one or more of Tween or Span. The Tween was Tween 80 with a hydrophilic-lipophilic balance (HLB) value of 15. The Span was Span 80 with a HLB value of 4.3. Step 5: Sieving the microgels: Take out the mixture of the prepared microgels and mineral oil, add 2-3 times the total volume of the prepared microgels and mineral oil in phosphate buffer solution, and then centrifuge at 1000 rpm for 20 min to obtain the first oil supernatant, intermediate layer P1, and first PBS layer; take the first oil supernatant and add 2-3 times the volume of the first oil supernatant in phosphate buffer solution, and then centrifuge at 2000 rpm for 20 min to obtain the second oil supernatant, intermediate layer P2, and second PBS layer; take the second oil supernatant and add 2-3 times the volume of the second oil supernatant in phosphate buffer solution, and then centrifuge at 3000 rpm for 20 min to obtain the intermediate layer P3; then wash the obtained intermediate layer repeatedly with PBS solution 3 times to obtain microgels of three particle sizes: P1, P2, and P3.

[0021] In addition, nintedanib raw material was purchased from Belka Pharmaceutical Co., Ltd., soybean lecithin from Xi'an Ruixi Biotechnology, cholesterol from SIGMA, DSPE-PEG2K-MAL and 4ARM-PEG-MAL from Xi'an Ruixi Biotechnology, TCEP from ALDAICH, trastuzumab from Shanghai Zhangjiang Biotechnology Co., Ltd., CPVGLIGC from Wuhan Xidan Port Biotechnology, and SPAN80 and TWEEN80 from Tianjin Damao Chemical Reagent Factory. Example 1 describes a method for preparing an enzyme-responsive inhalable microgel, the specific steps of which are as follows: Step 1: Preparation of nintedanib liposomes: 3.02 mg of nintedanib active pharmaceutical ingredient (API) was mixed with 4.83 mg of cholesterol, 18.97 mg of soy lecithin, and 2.09 mg of DSPE-PEG2K-MAL in a 1.5 mL EP tube, followed by the addition of 0.5 mL of anhydrous ethanol for dissolution. Nitrogen-deoxygenated phosphate-buffered saline (PBS) solution was added to a glass bottle. The prepared solution was then slowly and uniformly injected into the PBS, and the mixture was heated and stirred at 40°C for 2 h to evaporate the ethanol. After evaporation, the mixture was dialyzed overnight at 4°C using a 1 kDa dialysis bag and PBS as the dialysate to obtain nintedanib liposomes. The molar ratio of nintedanib API to soy lecithin was 1:10; the pH of the phosphate-buffered saline solution was 7.4. Step 2: Preparation of trastuzumab solution: Dissolve 2.00 mg of trastuzumab powder in 0.5 mL of PB solution at pH 7.1; reduce trastuzumab with 3.12 mg of TCEP reducing agent to open the disulfide bonds in trastuzumab; incubate on a shaker in the dark for 4 h, and dialyze overnight at 4°C; the overnight dialysis process should be carried out in the dark; after dialysis, remove the TCEP reducing agent using a 10 kD dialysis bag to obtain the trastuzumab solution; during this process, the disulfide bonds of trastuzumab are reduced to thiol groups by TCEP; Step 3: Ligation of nintedanib liposomes with trastuzumab: Take the above nintedanib liposomes and trastuzumab solutions, mix them evenly at a molar ratio of 1:1, react overnight at 4°C, and dialyze with a 150 KD dialysis bag to obtain a nintedanib liposome solution conjugated with trastuzumab; wherein, the thiol groups of the trastuzumab solution react with the maleimide of DSPE-PEG2K-MAL in the nintedanib liposomes, thereby linking the nintedanib liposomes with trastuzumab; Step 4: Preparation of microgels: A crosslinking agent was prepared by mixing CPVGLIGC and 4-arm polyethylene glycol maleimide (4ARM-PEG-MAL) at a molar ratio of 1:1. The nintedanib liposome solution linked to trastuzumab was then mixed with the crosslinking agent. Using a syringe, the mixture of the nintedanib liposome solution linked to trastuzumab and the crosslinking agent was slowly injected into mineral oil. The mixture was heated and stirred at 37°C for 2 hours to obtain the prepared microgels. In Step 4, the mineral oil contained 1% (V / V) of surfactant; the surfactant was one or more of Tween or Span; the Tween was Tween 80 with a hydrophilic-lipophilic balance (HLB) value of 15; the Span was Span 80 with a HLB value of 4.3. Step 5: Sieving the microgels: Take out the mixture of the prepared microgels and mineral oil, add 2-3 times the total volume of the prepared microgels and mineral oil in phosphate buffer solution, and then centrifuge at 1000 rpm for 20 min to obtain the first oil supernatant, intermediate layer P1, and first PBS layer; take the first oil supernatant and add about 2-3 times the volume of the first oil supernatant in phosphate buffer solution, and then centrifuge at 2000 rpm for 20 min to obtain the second oil supernatant, intermediate layer P2, and second PBS layer; take the second oil supernatant and add 2-3 times the volume of the second oil supernatant in phosphate buffer solution, and then centrifuge at 3000 rpm for 20 min to obtain the intermediate layer P3; then wash the obtained intermediate layer repeatedly with PBS solution 3 times to obtain microgels of three particle sizes: P1, P2, and P3.

[0022] Application of an enzyme-responsive inhalable microgel in the preparation of enzyme-responsive pulmonary inhalation drug delivery: Experiment 1: Two portions of the nintedanib lipids obtained in Step 1 were used to determine the drug encapsulation efficiency. The specific procedures are as follows: First, the nintedanib liposomes were demulsified: 0.1 mL of nintedanib liposomes was added to 9.9 mL of methanol, and the mixture was ultrasonically cleaned for 10 min to demulsify and release the encapsulated drug. The filtrate was then filtered through a 0.22 μm microporous membrane to remove insoluble impurities and ensure purity. The resulting filtrate was then analyzed by HPLC to determine the peak area, content, and encapsulation efficiency of nintedanib. The results are shown in Table 1 below.

[0023] Table 1 Peak area under nidanib gradient concentration

[0024] Experiment 2: Testing the binding effect of nintedanib liposomes with trastuzumab. The specific procedures are as follows: The conjugation effect of nintedanib liposomes with trastuzumab was detected by SDS-polyacrylamide gel electrophoresis. The procedure was performed according to the following steps: leak detection, gel preparation, sample loading, electrophoresis, staining, destaining, and development. The results are as follows. Figure 1 As shown, compared with trastuzumab (TRA), nintedanib liposome trastuzumab microgel showed a significant upshift of the band, indicating that TRA can be attached to nintedanib liposomes.

[0025] Experiment 3: Determination of microgel particle size. The specific procedures are as follows: Appropriate samples of the microgels with particle sizes P1, P2, and P3 prepared in Example 1 were placed on glass slides and observed using an inverted fluorescence microscope and image processing system. The particle size was measured, and the results are as follows. Figure 2As shown, the microgels observed under a microscope are mostly spherical in shape. The particle size of the large-diameter (P1) microgels is mostly between 10-20 μm, the particle size of the medium-diameter (P2) microgels is mostly between 5-8 μm, and the particle size of the small-diameter (P3) microgels is mostly between 4-6 μm. The large, medium, and small-diameter microgels have a high degree of differentiation in particle size.

[0026] Experiment 4: The morphology of the microgels before and after MMP2 enzyme incubation was examined using transmission electron microscopy (TEM). The specific procedures are as follows: Take 80 μL of the prepared medium-sized (P2) microgel, add 20 μL of MMP2 enzyme with a concentration of 10 μg / mL, and incubate at 37℃ for 4 h in an incubator to obtain the incubated medium-sized microgel sample. Medium-sized microgel samples before and after incubation were placed in a TEM, allowing the electron beam to penetrate the samples. Because different parts of the sample scatter electrons differently, images of varying brightness are formed on the imaging plate, enabling observation of the internal structure of the microgels. The specific structure is shown below. Figure 3 As shown, the medium-sized microgel sample before incubation is spherical with a darker color in the center; after incubation, some particles of the medium-sized microgel sample become flattened and the color in the center becomes lighter. A clear network structure can be observed inside, indicating that the drug encapsulated inside the microgel has been released after the action of MMP2 enzyme, indicating that the microgel has MMP2 enzyme responsiveness.

[0027] Experiment 5: Comparison of particle size of medium-sized microgel samples before and after incubation with MMP2 enzyme. The specific procedures are as follows: Take 80 μL of C6-labeled medium-sized microgel sample, add 20 μL of MMP2 enzyme at a concentration of 10 μg / mL, and incubate at 37℃ for 4 h in an incubator; Medium-sized microgel samples labeled with C6 before and after incubation were observed under a fluorescence microscope to compare their particle size dispersion. Since the MMP2 enzyme can react with the MMP2 enzyme-responsive peptide in CPVGLIGC, releasing the drug, the particle size after incubation was more dispersed than before incubation. Details are as follows: Figure 4 As shown, before incubation, the medium-sized microgel samples labeled with C6 exhibited spherical shapes of varying sizes, with fluorescent labeling only present inside the particles. After incubation, the range of fluorescent labeling in the medium-sized microgel samples labeled with C6 expanded, indicating that the drug inside the microgel particles was released and became more dispersed, suggesting that the microgel exhibits MMP2 enzyme responsiveness.

[0028] Experiment 6: Macrophage phagocytosis experiment of microgel samples with different particle sizes (P1, P2, P3). The specific operation is as follows: RAW264.7 cells were used for the experiment. First, the frozen RAW264.7 cells were rapidly thawed and seeded in medium containing 10% FBS. They were cultured at 37°C with 5% CO2 until adherence. The RAW264.7 cells were then seeded at a density of 40% in 24-well plates. Subsequently, the cells were cultured in medium containing 5% FBS for 12 hours to starve them and prevent cell proliferation. Two hours before the experiment, the culture medium was replaced with one containing 10% FBS to restore cell viability. Three microgel samples (P1, P2, P3) with different particle sizes containing C6 were diluted to the same concentration with the culture medium and then added to the RAW264.7 cell culture system and gently mixed. They were incubated for 0.5 h, 1 h, and 2 h, respectively. During incubation, care was taken to avoid light to prevent fluorescence quenching. After incubation, cells were washed with cold PBS to stop phagocytosis. The culture plate was placed on ice for 5 min to reduce cell membrane fluidity. Cells were then gently washed three times with pre-cooled PBS at 4°C to remove unphagocytosed microgels. Cells were then fixed with 4% paraformaldehyde and washed with PBS. After fixation, cells were stained with 10 μg / mL DAPI, washed with PBS, and then infiltrated with PBS. The purpose of infiltrating cells with PBS is to prevent cell death. The phagocytosis of microgels of different particle sizes by RAW cells at different time points was observed under a fluorescence microscope for the above samples. The results are as follows: Figure 5 As shown, it can be observed that at the same time point, RAW cells take up microgels of different particle sizes. RAW cells with added small-particle-size microgels have stronger fluorescence signals, while those with large-particle-size microgels have the weakest. Therefore, large-particle-size microgels are most effective in preventing macrophage phagocytosis and achieving effective drug delivery.

[0029] Experiment 7: To investigate the safety of the prepared liposome microgels, an in vitro hemolysis test was conducted on the prepared microgel samples. The specific procedures are as follows: Blood was collected from the eyeball of a mouse to obtain an appropriate amount of venous blood. The blood was centrifuged at 2000 rpm for 10 min to remove the upper plasma layer. The lower blood cells were resuspended in cooled physiological saline and centrifuged at 2000 rpm for 10 min. This process was repeated until the upper solution became clear to obtain red blood cells. An appropriate amount of physiological saline was added to prepare a red blood cell suspension. The prepared red blood cell suspension was divided into several portions, and small-particle microgels were added to prepare different drug concentrations. The concentrations of nintedanib were 4.94 μg / mL, 6.08 μg / mL, 7.60 μg / mL, and 12.54 μg / mL; the concentrations of trastuzumab were 7.96 μg / mL, 9.80 μg / mL, 12.25 μg / mL, and 20.21 μg / mL. A positive control group was prepared by replacing physiological saline with ultrapure water, and a negative control group was prepared by not adding any drugs to the red blood cell suspension. All samples were incubated at 37℃ for 1 h. The results are as follows: Figure 6 As shown, the drug did not exhibit hemolysis within the above concentration range, indicating that the formulation has good safety.

[0030] Experiment 8, to detect the efficacy of nintedanib combined with trastuzumab in the treatment of pulmonary fibrosis, performed a cell scratch assay on nintedanib, trastuzumab, and the combination of the two, respectively. The specific procedures are as follows: BEAS-2B cells were fed at a rate of 2 × 10⁻⁶ 5 Cells were seeded at a density of 1 / well in 6-well plates and cultured at 37°C and 5% CO2 until 90% confluence. A 200 μL sterile pipette tip was used to make a vertical scratch on the wells. After washing with PBS to remove detached cells, the cells were divided into a blank control group (untreated), a model group (containing 5 ng / mL TGF-β), and a model group (TGF-β1-stimulated with nintedanib (200 nM) liposomes, trastuzumab microgel, and nintedanib liposomes coupled with trastuzumab microgel, respectively). Each group had 3 replicates. All groups were incubated in medium containing 10% FBS at 37°C for 48 h. The scratched areas were photographed under an inverted microscope at 0 h and 48 h. The results are shown below. Figure 7 As shown.

[0031] Both nintedanib and trastuzumab, when used alone, can inhibit the migration of BEAS-2B cells, and the inhibitory effect is even better when used in combination. Therefore, it is believed that the combined use of nintedanib and trastuzumab is more effective in treating pulmonary fibrosis. Thus, this enzyme-responsive inhalable microgel can be used in the preparation of enzyme-responsive pulmonary inhalation drug delivery.

[0032] Experiment Nine involves conducting Transwell experiments on the experimental groups from Experiment Eight. The specific procedures are as follows: Each experimental group included a control group without treatment, a model group containing 5 ng / mL TGF-β, a first-drug single-drug group with 200 nM nintedanib added to the upper chamber of the Transwell, a second-drug single-drug group with 1.2 μg / mL trastuzumab microgel added to the lower chamber of the Transwell, and a combination-drug group with simultaneous addition of nintedanib liposomes and trastuzumab to link the two. Each group had 3 replicates. BEAS-2B cells were cultured at 2 × 10⁻⁶ cells / well. 5 Cells were seeded at a density of 200 μL serum-free medium in the upper chamber of a Transwell; 600 μL of medium containing 10% FBS was added to the lower chamber of the Transwell, with an additional 5 ng / mL TGF-β added to the model group, the first single-drug group, the second single-drug group, and the combination drug group. After incubation at 37℃ and 5% CO2 for 24 h, the culture medium was discarded, and the cells were fixed in 4% paraformaldehyde for 30 min, stained with 0.1% crystal violet for 20 min, and gently washed with PBS to remove unmigrated cells. The cells were then photographed and analyzed under an inverted microscope. The results are as follows: Figure 8 As shown.

[0033] Both nintedanib and trastuzumab, when used alone, can inhibit the migration of BEAS-2B cells. However, the combination of the two is more effective in inhibiting the migration of BEAS-2B cells than either of them alone, indicating that nintedanib combined with trastuzumab microgel can enhance the therapeutic effect on pulmonary fibrosis.

[0034] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments that can be applied to other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. An enzyme-responsive inhalable microgel, characterized in that, It includes a pharmaceutical active ingredient and a drug carrier, wherein the pharmaceutical active ingredient includes nintedanib and trastuzumab, and the drug carrier includes a microgel.

2. The method for preparing an enzyme-responsive inhalable microgel according to claim 1, characterized in that, The specific steps are as follows: Step 1: Preparation of nintedanib liposomes: Nintedanib raw material, soybean lecithin, cholesterol, and DSPE-PEG2K-MAL were mixed and dissolved in anhydrous ethanol. The solution was then slowly and uniformly injected into a nitrogen-deoxygenated phosphate buffer solution. The mixture was heated and stirred at 40°C for 2 h to remove the ethanol. Subsequently, the mixture was dialyzed overnight using a 1 kD dialysis bag to obtain nintedanib liposomes. Step 2: Preparation of trastuzumab solution: Trastuzumab was reduced using TCEP reducing agent to open the disulfide bonds in trastuzumab. The mixture was incubated on a shaker for 4 h and dialyzed overnight at 4°C. After dialysis, the TCEP reducing agent was removed using a 10 kD dialysis bag to obtain the trastuzumab solution. Step 3: Connecting nintedanib liposomes to trastuzumab: Take the above nintedanib liposomes and trastuzumab solutions, mix them evenly at a molar ratio of 1:1, react overnight at 4°C, and then dialyze with a 150 KD dialysis bag to obtain a nintedanib liposome solution connected with trastuzumab. Step 4: Preparation of microgels: CPVGLIGC and 4-arm polyethylene glycol maleimide were mixed at a molar ratio of 1:1 to prepare a crosslinking agent. Then, the above-mentioned nintedanib liposome solution linked with trastuzumab was mixed with the crosslinking agent. The mixture of nintedanib liposome solution linked with trastuzumab and crosslinking agent was slowly injected into mineral oil using a syringe. The mixture was heated and stirred at 37°C for 2 h to obtain the prepared microgels. Step 5: Sieving the microgels: Take out the mixture of the prepared microgels and mineral oil, add 2-3 times the total volume of the prepared microgels and mineral oil in phosphate buffer solution, and then centrifuge at 1000 rpm for 20 min to obtain the first oil supernatant, intermediate layer P1, and first PBS layer; take the first oil supernatant and add 2-3 times the volume of the first oil supernatant in phosphate buffer solution, and then centrifuge at 2000 rpm for 20 min to obtain the second oil supernatant, intermediate layer P2, and second PBS layer; take the second oil supernatant and add 2-3 times the volume of the second oil supernatant in phosphate buffer solution, and then centrifuge at 3000 rpm for 20 min to obtain the intermediate layer P3; then wash the obtained intermediate layer repeatedly with PBS solution 3 times to obtain microgels of three particle sizes: P1, P2, and P3.

3. The method for preparing an enzyme-responsive inhalable microgel according to claim 1, characterized in that, In step one, the nintedanib raw material and soybean lecithin are mixed in a molar ratio of 1:10 to 1:5; the pH of the phosphate buffer solution is 5-8.

4. The method for preparing an enzyme-responsive inhalable microgel according to claim 1, characterized in that, In step four, the mineral oil is a mineral oil containing 0.5% to 3% (V / V) surfactant; the surfactant is one or more of Tween or Span.

5. The method for preparing an enzyme-responsive inhalable microgel according to claim 4, characterized in that, The Tween is Tween 80, and the hydrophilic-lipophilic balance value of Tween 80 is 15; the Span is Span 80, and the hydrophilic-lipophilic balance value of Span 80 is 4.

3.

6. The application of an enzyme-responsive inhalable microgel in the preparation of an enzyme-responsive pulmonary inhalation drug delivery system, wherein the microgel is applied to the enzyme-responsive inhalable microgel of claim 1.

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