Suction type nano enzyme-polyvinyl alcohol gel particle based on microfluidic technology as well as preparation method and application of suction type nano enzyme-polyvinyl alcohol gel particle

Abstract

The invention discloses suction type nano enzyme-polyvinyl alcohol gel particles based on a microfluidic technology as well as a preparation method and application of the suction type nano enzyme-polyvinyl alcohol gel particles. According to the method, monodisperse oil-in-water liquid drops are prepared through a micro-fluidic technology, and the PVA gel particles loaded with the nano-enzyme are prepared by combining freeze-thaw cycle crosslinking and supercritical drying. The prepared particles have a unique structure of large geometric size and small aerodynamic size, the physical particle size (5-20 [mu] m) of the particles is beneficial for avoiding too fast removal of macrophages, and the aerodynamic diameter (1-5 [mu] m) of the particles is suitable for deep deposition in the lung due to the extremely low density of the particles. Nano-enzymes (such as FeMn (at) MOF) in the particles can be continuously released, active oxygen is effectively removed, inflammatory factors are reduced, and loaded sodium chloride is beneficial to enhancing mucous penetration. The particle system shows an excellent antioxidant and anti-inflammatory synergistic treatment effect in an acute lung injury model, and a new strategy is provided for inhalation type targeted drug delivery.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an inhaled nanoenzyme-polyvinyl alcohol gel microparticle based on microfluidic technology, its preparation method, and its application. Background Technology

[0002] Acute lung injury (ALI) is a severe inflammatory syndrome of the lungs induced by infection, trauma, etc. Its core pathology includes alveolar barrier disruption, reactive oxygen species (ROS) bursts, and cytokine storms. It easily develops into acute respiratory distress syndrome (ARDS) and has a high mortality rate. Currently used clinical drugs such as glucocorticoids and antioxidants have problems such as strong systemic side effects, poor lung targeting, low clearance efficiency, and the need for frequent dosing.

[0003] While inhaled drug delivery can directly reach the lungs, its efficacy is limited by particulate matter properties: excessively small particles are easily cleared by macrophages, while excessively large particles struggle to penetrate the mucus barrier; excessively large aerodynamic diameters deposit in the upper respiratory tract, while excessively small particles are exhaled, making it difficult for traditional formulations to achieve both. Biomicrogels, due to their high porosity and low density, can separate physical size from aerodynamic behavior, making them ideal carriers for deep lung deposition, but precise particle size control is required. Meanwhile, nanozymes (such as Au, Pt, and FeMn) possess various enzyme-mimicking activities and can effectively scavenge ROS and alleviate oxidative stress, but their ease of aggregation, lack of inflammatory regulation, and uncontrollable release behavior limit their application.

[0004] Therefore, there is an urgent need to develop an inhaled drug delivery system that can precisely control particle size and possesses both good lung deposition properties and antioxidant-anti-inflammatory functions to improve the treatment efficacy of ALI. Microfluidic technology provides a feasible approach for preparing monodisperse, parameter-tunable gel microparticles, which is expected to integrate the advantages of nanozymes and microgel carriers to achieve efficient and targeted ALI treatment. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing an inhalation-type nanoenzyme-polyvinyl alcohol gel microparticle based on microfluidic technology, its preparation method, and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect is to provide a method for preparing inhaled nanoenzyme-polyvinyl alcohol gel microparticles based on microfluidic technology, the method comprising the following steps: 1) Dissolve polyvinyl alcohol (PVA), sodium chloride (NaCl) and nanozyme in water in a certain proportion to form a dispersed phase, sterilize it through a filter membrane and set it aside for later use; mix Span80 and paraffin oil in a certain proportion, sterilize it and use it as the continuous phase; 2) The dispersed phase and the continuous phase are injected into a PDMS microfluidic chip respectively, and water-in-oil (W / O) droplets are prepared under controlled flow rate ratio conditions; 3) The obtained W / O droplets are subjected to a cyclic freeze-thaw process to achieve gelation; wherein the low-temperature freezing temperature is -40℃ to -10℃, the high-temperature thawing temperature is 20℃ to 40℃, and the number of cycles is 2 to 5. 4) The cross-linked gel droplets were washed successively with organic solvent and deionized water, and finally critical point dried in CO2 medium to obtain the final inhalation nanoenzyme-polyvinyl alcohol gel microparticles.

[0007] Further, in step 1), the mass ratio of polyvinyl alcohol to NaCl is 1:1-5:1, and the mass ratio of polyvinyl alcohol to nanozyme is 2:1-10:1; the nanozyme is one of Au nanoparticles, Pt nanoparticles, FeMn nanoparticles, FeMn@MOF nanoparticles, and CuMn nanoparticles.

[0008] Furthermore, in step 1), the volume ratio of Span80 to paraffin oil is 1:1 to 1:20.

[0009] Further, in step 2), the flow rate ratio of the continuous phase to the dispersed phase is 10:1-2:1; the flow rate of the continuous phase is 10-500 μm / s.

[0010] Furthermore, in step 3), the low-temperature freezing time is 1h-8h; the high-temperature melting time is 0.5h-5h.

[0011] Furthermore, in step 4), the organic solvent used for washing is selected from one or two of petroleum ether, ethanol, methanol, acetonitrile, ethyl acetate, and dimethyl sulfoxide.

[0012] Further, in step 4), the critical point drying process is as follows: cooling for 5-20 minutes → filling with liquid for 5-15 minutes → draining with liquid for 2-20 minutes → secondary filling with liquid for 4-10 minutes → critical point treatment for 5-20 minutes → depressurization for 5-20 minutes → exhausting with gas for 2-20 minutes.

[0013] The second aspect is to provide an inhaled nanoenzyme-polyvinyl alcohol gel microparticle based on microfluidic technology, wherein the inhaled nanoenzyme-polyvinyl alcohol gel microparticle is prepared by the above-mentioned preparation method.

[0014] Furthermore, the physical particle size of the inhaled nanoenzyme-polyvinyl alcohol gel microparticles is 5-20 μm, and the aerodynamic diameter of the inhaled nanoenzyme-polyvinyl alcohol gel microparticles is 1-5 μm.

[0015] The third aspect is to provide the application of the above-mentioned microfluidic-based inhaled nanoenzyme-polyvinyl alcohol gel microparticles in the preparation of drugs for treating acute lung injury.

[0016] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: This invention utilizes microfluidic chip technology to prepare well-dispersed oil-in-water (W / O) droplet precursors by precisely controlling parameters such as two-phase flow rates and chip size. Combined with subsequent freeze-thaw crosslinking and supercritical drying processes, polyvinyl alcohol aerogel microparticles with regular geometry and controllable particle size distribution can be obtained. Furthermore, this invention constructs a three-dimensional PVA network with high porosity through freeze-thaw crosslinking and supercritical drying. Nanozymes are effectively embedded and immobilized within this network, forming a sustained-release system. In vitro release experiments of FeMn@MOF nanozyme-PVA gel microparticles show that this system can achieve sustained release of nanozymes for up to 72 hours, potentially prolonging drug retention time in the lungs, reducing dosing frequency, and overcoming the drawback of naked nanozymes being easily and rapidly cleared.

[0017] The gel microparticles prepared by this invention possess unique characteristics of "large geometric size and small aerodynamic size". Their physical particle size (5-20 μm) helps to avoid being cleared too quickly by alveolar macrophages, while their extremely low material density allows their median mass aerodynamic diameter (MMAD) to be controlled within the ideal range of 1-5 μm. This ensures that the microparticles can be efficiently deposited in the lesion area deep in the lungs, achieving a balance between "effective deposition" and "long-term retention" in lung delivery.

[0018] This invention combines the antioxidant function of nanozymes with the physical drug loading and sustained-release properties of PVA gel carriers. Experiments show that loaded nanozymes (such as FeMn@MOF) can effectively scavenge ROS and reduce inflammatory factor levels in vitro. In a rat model of LPS-induced acute lung injury, inhalation of this microparticle formulation significantly reduced ROS and inflammatory factor (such as IL-6 and IL-1β) levels in bronchoalveolar lavage fluid, confirming its good synergistic antioxidant and anti-inflammatory therapeutic effects. The NaCl loaded in the microparticles, after dissolving in the lungs, can form a local hypertonic environment, theoretically helping to absorb water from the mucus and temporarily reduce mucus viscosity, thereby potentially promoting microparticle diffusion and drug release. Experimental results show that, compared to the drug-loaded system without NaCl, NaCl release can increase drug penetration efficiency by 3.2 times in a dense mucus environment. This provides a potential physical aid mechanism for enhancing drug delivery to lesions within a dense mucus barrier. Attached Figure Description

[0019] Figure 1 The image shows a scanning electron microscope (SEM) image of the Au nanozyme-PVA gel microparticles prepared in Example 1.

[0020] Figure 2 The aerodynamic particle size distribution diagrams are shown for the nanozyme-PVA gel microparticles prepared in Examples 1-4.

[0021] Figure 3 The image shows a scanning electron microscope (SEM) image of the FeMn nanozyme-PVA gel microparticles prepared in Example 2.

[0022] Figure 4 The image shows a scanning electron microscope (SEM) image of the FeMn@MOF nanozyme-PVA gel microparticles prepared in Example 3.

[0023] Figure 5 The image shows a scanning electron microscope (SEM) image of the CuMn nanozyme-PVA gel microparticles prepared in Example 4.

[0024] Figure 6 The in vitro nanozyme release curve of the FeMn@MOF nanozyme-PVA gel microparticles prepared in Example 3 in simulated lung fluid.

[0025] Figure 7 The particle deposition evaluation results are for the Au nanozyme-PVA gel particles prepared in Example 1.

[0026] Figure 8 The image shows the in vitro ROS scavenging effect of the FeMn@MOF nanozyme-PVA gel microparticles prepared in Example 3.

[0027] Figure 9 The scavenging effect of FeMn@MOF nanozyme-PVA gel microparticles prepared in Example 3 on inflammatory factors IL-6 and IL-1β.

[0028] Figure 10 The results of the in vitro mucus penetration experiment using the FeMn@MOF-PVA gel microparticles prepared in Example 3 are shown.

[0029] Figure 11 The image shows a comparison of the fluorescence intensity of ROS in bronchoalveolar lavage fluid (BALF) of rats treated with CuMn nanozyme-PVA gel microparticles prepared in Example 4.

[0030] Figure 12 The bar chart shows the levels of inflammatory factors IL-6 and IL-1β in BALF after treating ALI rats with CuMn nanozyme-PVA gel microparticles prepared in Example 4. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0032] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions or as recommended by the manufacturer. Unless otherwise stated, all reagents and materials used in the following examples were commercially available.

[0033] Example 1 This embodiment provides a method for preparing inhaled Au nanozyme-polyvinyl alcohol gel microparticles based on microfluidic technology, the method comprising: (1) Add 1.0g PVA powder, 1.0g NaCl and 0.2g Au nanoparticles to 10mL of sterile deionized water, filter and sterilize through a 0.22μm filter to obtain the dispersed phase. Add 1mL Span 80 solution to 10mL paraffin oil, filter and sterilize through a 0.22μm filter to obtain the continuous phase.

[0034] (2) The dispersed phase and the continuous phase obtained in step (1) are injected into the PDMS microfluidic chip respectively, and the cross flow cut size is selected as 10 μm. The flow rate of the continuous phase is 100 μm / s and the flow rate of the dispersed phase is 50 μm / s (flow rate ratio 2:1). W / O droplets are formed by the shearing of the two phases, and the outflowing liquid is collected at the outlet of the chip tail end.

[0035] (3) Freeze the W / O droplets obtained in step (2) at -40°C for 1 hour, then melt them at 40°C for 1 hour, repeating the cycle twice to form a gel structure.

[0036] (4) The gel particles obtained in step (3) are washed sequentially with petroleum ether, ethyl acetate, and distilled water. The water-dispersed gel microspheres are replaced with anhydrous ethanol using a dialysis bag. The ethanol-dispersed gel microspheres are placed in the sample chamber of a supercritical dryer and subjected to supercritical drying in CO2 medium according to the procedure of "cooling for 5 min → filling for 10 min → draining for 20 min → filling again for 5 min → critical point treatment for 10 min → depressurization for 10 min → exhausting for 15 min". The dried powder is collected, which is Au nanozyme-PVA gel microparticles.

[0037] Characterization results: The obtained particles are as follows Figure 1 As shown, SEM reveals that it is spherical with abundant wrinkles and pores on its surface. Random statistical analysis was performed using ImageJ software. Figure 2 The number-average physical diameter was (12.8 ± 2.1) μm. Its mass median aerodynamic diameter (MMAD) was determined to be (2.8 ± 0.3) μm using the Anderson cascade impactor.

[0038] Example 2 This embodiment provides a method for preparing inhaled FeMn nanoenzymes-polyvinyl alcohol gel microparticles based on microfluidic technology, the method comprising: (1) Add 2.0g PVA powder, 1.0g NaCl and 0.5g FeMn nanoparticles to 10mL of sterile deionized water, filter and sterilize through a 0.22μm filter to obtain the mobile phase. Add 1mL Lpan 80 solution to 15mL paraffin oil, filter and sterilize through a 0.22μm filter to obtain the continuous phase.

[0039] (2) Inject the dispersed phase and continuous phase obtained in step (1) into the PDMS microfluidic chip respectively, and select a cross flow slit size of 5 μm. The flow rate of the continuous phase is 300 μm / s, and the flow rate of the dispersed phase is 60 μm / s (flow rate ratio 5:1). Collect W / O droplets.

[0040] (3) The W / O droplets obtained in step (2) are frozen at -10℃ for 3 hours, then thawed at 20℃ for 2 hours, and cross-linked three times to form a gel structure; (4) The gel particles obtained in step (3) were washed sequentially with ethanol, ethyl acetate, and distilled water.

[0041] The solution was replaced with ethanol and subjected to supercritical drying (program: cooling for 10 min → filling for 15 min → draining for 20 min → filling again for 10 min → critical point treatment for 15 min → depressurization for 20 min → exhaust for 20 min) to obtain FeMn nanozyme-PVA gel microparticles.

[0042] Characterization results: The obtained particles are as follows Figure 3 As shown, SEM revealed that the particles were regularly shaped spheres with a number-average physical particle size of (8.2±1.5) μm and a MMAD of (3.5±0.4) μm.

[0043] Example 3: This embodiment provides a method for preparing inhaled FeMn@MOF nanoenzymes-polyvinyl alcohol gel microparticles based on microfluidic technology, the method comprising: (1) Add 2.0g PVA powder, 1.0g NaCl and 1.0g FeMn@MOF nanoparticles to 10mL of sterile deionized water, filter and sterilize to obtain the dispersed phase. Add 1mL Span 80 to 15mL paraffin oil, filter and sterilize to obtain the continuous phase.

[0044] (2) Inject the two phases into the PDMS chip with a cut size of 20 μm. The continuous phase flow rate is 100 μm / s and the dispersed phase flow rate is 50 μm / s (flow rate ratio 2:1). Collect the W / O droplets.

[0045] (3) The droplets were frozen at -20℃ for 1 hour and thawed at 30℃ for 2 hours, and the cycle was repeated 3 times.

[0046] (4) Wash with petroleum ether, ethyl acetate and distilled water in sequence, replace with ethanol and perform supercritical drying (the procedure is the same as in Example 2) to obtain FeMn@MOF nanozyme-PVA gel microparticles.

[0047] Characterization results: Figure 4 SEM results showed that the obtained microparticles were spherical porous structures loaded with nanozymes. The number-average physical diameter of the obtained microparticles was (18.5±2.8) μm, and the MMAD was (4.2±0.5) μm.

[0048] Example 4 This embodiment provides a method for preparing inhaled CuMn nanoenzymes-polyvinyl alcohol gel microparticles based on microfluidic technology, the method comprising: (1) Add 3.0g PVA powder, 1.5g NaCl and 0.6g CuMn nanozyme to 10mL of sterile deionized water, filter and sterilize to obtain the dispersed phase. Mix 1mL Span80 with 15mL paraffin oil and sterilize to obtain the continuous phase.

[0049] (2) Inject the two phases into the PDMS chip with a cut size of 15 μm. The continuous phase flow rate is 450 μm / s and the dispersed phase flow rate is 50 μm / s (flow rate ratio 9:1). Collect the W / O droplets.

[0050] (3) The droplets were frozen at -38℃ for 2 hours and thawed at 22℃ for 4 hours, and the cycle was repeated 5 times.

[0051] (4) Wash with acetonitrile, methanol and distilled water in sequence, replace with ethanol and carry out supercritical drying (program: cooling for 18 min → filling for 8 min → draining for 18 min → filling for 4 min → critical point treatment for 18 min → depressurizing for 18 min → exhausting for 18 min) to obtain CuMn nanozyme-PVA gel microparticles.

[0052] Characterization results: Figure 5 The SEM results showed that the obtained particles were regular spheres with a number-average geometric diameter of (6.5±1.2) μm and a MMAD of (1.8±0.2) μm.

[0053] Verification Example 1 The four nanozyme-polyvinyl alcohol gel microparticles (hereinafter referred to as "microparticles") prepared in Examples 1-4 were systematically characterized in terms of their physical properties to confirm their morphology, size and aerodynamic characteristics.

[0054] 1. Morphological and geometrical analysis Observation was performed using scanning electron microscopy. SEM images of the particles in Examples 1-4 are shown below. Figure 1 , Figures 3-5 The geometric diameter of its particles is in the range of 5-20 μm.

[0055] 2. Aerodynamic particle size determination The Anderson cascade impactor was used for determination. Test conditions: flow rate 28.3 L / min, relative humidity <15%. Approximately 20 mg of dried particles were placed in the dry powder inhaler for aerodynamic classification. The median mass aerodynamic diameter (MMAD) was calculated. Results are as follows: Figure 2 As shown, the MMAD of all the microparticles in the examples were within the ideal lung deposition range of 1-5 μm, and the ratio of geometric particle size to MMAD (i.e., shape factor) was greater than 2, confirming their low-density, porous aerogel properties.

[0056] 3. Drug release curve Taking the FeMn@MOF nanozyme-PVA gel microparticles from Example 3 as an example, a precise amount of microparticles was weighed and dispersed in pH 7.4 phosphate buffer (simulating lung fluid) containing 0.1% Tween 80, and placed in a shaker at 37°C (100 rpm). Samples were taken at predetermined time points (0.5, 1, 2, 4, 8, 12, 24, 48, 72 h), and an equal volume of fresh medium was added. After centrifugation, the Fe / Mn ion concentration in the supernatant was determined by inductively coupled plasma mass spectrometry, and the cumulative release rate was calculated. The results are as follows: Figure 6 As shown, the microparticle exhibits a continuous and slow release characteristic over 72 hours. Initially (0-8h), there is a certain burst release (approximately 25%), followed by a gradual release. The cumulative release rate over 72 hours is approximately 78%, which is consistent with the characteristics of a sustained-release formulation.

[0057] Verification Example 2 The Anderson sampler was used to simulate the deposition patterns in the human lungs to determine the potential deposition locations of aerosol microparticles after nebulization and inhalation, using Au nanozyme-PVA gel microspheres as an example. Figure 7 As shown, the particles can be effectively distributed in the terminal respiratory tract and alveoli of S4 level and above through physical deposition mechanisms, and Au nanozyme-PVA gel microspheres can be effectively deposited in the respiratory area deep in lung tissue.

[0058] Verification Example 3 1. The FeMn@MOF nanozyme-PVA gel microparticles prepared in Example 3 were subjected to in vitro ROS scavenging and inflammatory factor scavenging experiments. In vitro ROS scavenging assay: RAW 264.7 cells were cultured in six-well confocal plates (2 × 10⁶ cells per well). 5Cells were pretreated with LPS for 4 hours and then incubated with gel particles of varying concentrations for 24 hours. ROS in the cells were detected on CLSM (Leica, STELLARIS) using a fluorescent probe (DCFH-DA). Furthermore, the fluorescence intensity of ROS was detected via the FITC channel using flow cytometry (BD, CytoFLEXLX).

[0059] In vitro inflammatory factor clearance assay: The levels of IL-6 and IL-1β in the cell supernatant were detected according to the ELISA kit instructions. 50 μL of the assay solution was added to each well of the coated plate and incubated at 37°C for 2 h. After washing five times with washing buffer, 100 μL of enzyme-labeled secondary antibody dilution buffer (goat anti-mouse IgG-HRP) was added to each well, and the plate was incubated at 37°C for 1 h. After washing five times with washing buffer, 100 μL of TMB substrate solution was added to each well. After developing the color at room temperature in the dark for 10 min, 30 μL of stop solution was added to each well. The color turned yellow, and the absorbance at 450 nm and 630 nm was measured using a microplate reader, and the results were recorded.

[0060] Figure 8 The results of the in vitro ROS scavenging experiment show that the ROS scavenging rate of FeMn@MOF nanozyme-PVA gel microparticles decreased by 81.7±3.8% compared with the LPS model group (p<0.01). Figure 9 The results showed that after treatment with FeMn@MOF nanozyme-PVA gel microparticles, the inflammatory factors IL-6 and IL-1β in the supernatant decreased by 74.9±4.6% (p<0.01) and 74.9±4.6% (p<0.01).

[0061] 2. The prepared FeMn@MOF nanozyme-PVA gel microparticles were subjected to in vitro mucus penetration experiments. The simulated lung mucus was prepared according to the literature: mucin (porcine gastric mucin, Sigma Type II) 50 mg / mL; DNA (salmon sperm DNA, Sigma) 5 mg / mL; NaCl 5 mg / mL; Tween 80 0.1% (w / v). The experimental procedure was as follows: 1.5 mL of PBS was added to the lower chamber of a Transwell chamber (Corning, 3 μm pore size); 200 μL of simulated mucus (thickness ≈ 100 μm) was spread in the upper chamber and allowed to stand for equilibration for 30 min; FITC-labeled FeMn@MOF nanozyme-PVA gel microparticles (5 mg) were dispersed in 20 μL of PBS and evenly spread on the surface of the mucus layer; incubation was performed at 37±0.5℃ and 5% CO2 for 2 h; the solution from the lower chamber was collected, and the fluorescence intensity was measured using a fluorescence spectrophotometer (Hitachi F-7000, Ex / Em=490 / 520 nm). Calculate the transmittance = (lower chamber fluorescence intensity / total fluorescence intensity) × 100%; total fluorescence intensity = fluorescence value of particles completely dissolved in 1% SDS solution.

[0062] Figure 10 The results of in vitro mucus penetration experiments using the prepared inhaled FeMn@MOF-PVA gel microparticles are presented. Calculations of the penetration rate show that, compared to the drug-loaded system without NaCl (FeMn@MOF), NaCl release from the FeMn@MOF-PVA gel microparticles can increase drug penetration efficiency by 3.2 times in a dense mucus environment.

[0063] Verification Example 4 The CuMn nanozyme-PVA gel microparticles prepared in Example 4 were used in a rat experiment to treat LPS-induced acute lung injury. The model was established using male SD rats (200±20g, from Speford Biotechnology) randomly divided into three groups (n=6): 1) Control group: 1 mL / kg of physiological saline was administered intratracheally; 2) LPS model group: 5 mg / kg of LPS (E. coli O55:B5) was administered intratracheally; 3) Treatment group: LPS + microparticle suspension (20 mg / kg, prepared with physiological saline) was administered (n=6). 72 h after administration, the rats were anesthetized with sodium pentobarbital via intraperitoneal injection, and bronchoalveolar lavage fluid (BALF, 5 mL PBS lavage 3 times) was collected after tracheal intubation. The supernatant was collected after centrifugation of the BALF, and the DCFH-DA probe was added. The reaction was carried out at 37℃ in the dark for 30 min, and detected using a fluorescence microplate reader (Ex / Em=488 / 525nm). The ROS equivalent was calculated using the H2O2 standard curve. BALF supernatant was detected using a rat IL-6 / IL-1β ELISA kit, strictly following the instructions.

[0064] Figure 11 Immunofluorescence images (DCFH-DA) of ROS in BALF collected after treating LPS-induced ALI rats with the prepared inhaled CuMn nanozyme-PVA gel microparticles showed a significant decrease in ROS in the treatment group compared to the LPS model group. Bar charts of IL-6 and IL-1β levels are shown below. Figure 12 As shown, IL-6 decreased by 83.8±4.6% (p<0.01), and IL-1β decreased by 80.3±5.5% (p<0.01).

[0065] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing inhalable nanoscale enzyme-polyvinyl alcohol gel microparticles based on microfluidic technology, characterized by, The method includes the following steps: 1) Dissolve polyvinyl alcohol (PVA), sodium chloride (NaCl) and nanozyme in water in a certain proportion to form a dispersed phase, sterilize it through a filter membrane and set it aside for later use; mix Span80 and paraffin oil in a certain proportion, sterilize it and use it as the continuous phase; 2) The dispersed phase and the continuous phase are injected into a PDMS microfluidic chip respectively, and water-in-oil (W / O) droplets are prepared under controlled flow rate ratio conditions; 3) The obtained W / O droplets are subjected to a cyclic freeze-thaw process to achieve gelation; wherein the low-temperature freezing temperature is -40℃ to -10℃, the high-temperature thawing temperature is 20℃ to 40℃, and the number of cycles is 2 to 5. 4) The cross-linked gel droplets were washed successively with organic solvent and deionized water, and finally critical point dried in CO2 medium to obtain the final inhalation nanoenzyme-polyvinyl alcohol gel microparticles.

2. The production method according to claim 1, characterized by, In step 1), the mass ratio of polyvinyl alcohol to NaCl is 1:1-5:1, and the mass ratio of polyvinyl alcohol to nanozyme is 2:1-10:1; the nanozyme is one of Au nanoparticles, Pt nanoparticles, FeMn nanoparticles, FeMn@MOF nanoparticles, and CuMn nanoparticles.

3. The preparation method according to claim 1, characterized in that, In step 1), the volume ratio of Span80 to paraffin oil is 1:1 to 1:

20.

4. The preparation method according to claim 1, characterized in that, In step 2), the flow rate ratio of the continuous phase to the dispersed phase is 10:1-2:1; the flow rate of the continuous phase is 10-500 μm / s.

5. The preparation method according to claim 1, characterized in that, In step 3), the low-temperature freezing time is 1h-8h; the high-temperature melting time is 0.5h-5h.

6. The preparation method according to claim 1, characterized in that, In step 4), the organic solvent used for washing is selected from one or two of petroleum ether, ethanol, methanol, acetonitrile, ethyl acetate, and dimethyl sulfoxide.

7. The preparation method according to claim 1, characterized in that, In step 4), the critical point drying process is as follows: cooling for 5-20 minutes → filling with liquid for 5-15 minutes → draining liquid for 2-20 minutes → secondary filling with liquid for 4-10 minutes → critical point treatment for 5-20 minutes → depressurization for 5-20 minutes → exhausting liquid for 2-20 minutes.

8. An inhaled nanoenzyme-polyvinyl alcohol gel microparticle based on microfluidic technology, characterized in that, The inhaled nanoenzyme-polyvinyl alcohol gel microparticles are prepared by the preparation method according to any one of claims 1-7.

9. The microfluidic-based inhaled nanoenzyme-polyvinyl alcohol gel microparticles according to claim 8, characterized in that, The physical particle size of the inhaled nanoenzyme-polyvinyl alcohol gel microparticles is 5-20 μm, and the aerodynamic diameter of the inhaled nanoenzyme-polyvinyl alcohol gel microparticles is 1-5 μm.

10. The application of the microfluidic-based inhaled nanoenzyme-polyvinyl alcohol gel microparticles as described in claim 8 or 9 in the preparation of drugs for treating acute lung injury.

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