Interferon synergistic exosome aerosol inhalation preparation as well as preparation method and application thereof

Abstract

The invention discloses an interferon and exosome aerosol inhalation preparation as well as a preparation method and application thereof. The preparation is prepared from the following components: 5 to 15 mu g / mL of interferon alpha, 0.1 to 0.5 mg / mL of exosome, 0.2 to 0.8 percent of NSCS-PNIPAM, 0.3 to 1 percent of mannitol, 0.01 to 0.05 percent of polymer stabilizer, 0.5 to 2 mM of phosphate buffer solution and 0.02 to 0.1 percent of surfactant. The invention belongs to the technical field of biological medicine. According to the invention, multi-dimensional improvement of material performance is realized, the synthesized N-succinyl chitosan-poly (N-isopropylacrylamide) grafted copolymer (NSCS-PNIPAM) combines the biocompatibility of chitosan with the environmental responsiveness of PNIPAM, so that the biodegradability of the material is improved, and the biodegradability of the material is improved. The problems of exosome membrane structure damage and interferon functional activity reduction possibly caused by mechanical shearing force and thermodynamic factors are effectively solved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically refers to an interferon-cooperated exosome aerosol inhalation preparation and its preparation method and application. Background Art

[0002] Mesenchymal stem cell exosomes are recently discovered key cell secretion products that regulate inflammation, immune responses, and surrounding cell activity, promoting tissue repair and regeneration. Compared with other nano-delivery systems, exosomes have advantages such as natural biomembrane structure, low immunogenicity, targeting, and homing ability, making them highly effective as targeted drug delivery vehicles. However, exosomes are easily released into the bloodstream within tissues and are rapidly cleared, making long-term therapeutic effects difficult. Currently, a large number of researchers have attempted to use various hydrogels and scaffold materials as exosome carriers to achieve localized release of exosomes and increase their duration of action, thereby enhancing tissue regeneration and repair effects, and have achieved significant breakthroughs and progress. However, existing studies mainly introduce exosomes into the three-dimensional networks of hydrogels or scaffold materials through simple encapsulation or adsorption, resulting in poor biological activity and tissue induction activity.

[0003] Interferon α is currently the most widely used clinically. Currently, the main interferons in clinical use include interferon α2α, interferon α2β, interferon α1β, and combined interferon. Recombinant human interferon α2β is a synthetic drug that regulates proteins and inhibits enzyme production. It can induce interferon production in the body and directly act on pathogens, thereby reducing the damage caused by viruses to the body. Recombinant human interferon α2β also regulates T cell subsets in children, enhances immunity, improves clinical symptoms, and reduces inflammatory factors. This can improve the treatment efficacy of viral pneumonia and hand, foot, and mouth disease, shorten the time it takes for symptoms to improve, effectively protect the body, and reduce the damage caused by viral pneumonia to children.

[0004] In the field of synergistic applications of interferon and exosomes, existing approaches primarily encompass exosome preparation technology, functional optimization strategies, and drug delivery design. Patent Publication No. CN110669729B discloses a method for preparing mesenchymal stem cell exosomes. By culturing mesenchymal stem cells in gelatin microspheres prepared with genipin and adding recombinant interferon γ and cytochalasin D, the exosome secretion and biological activity were significantly increased. However, the extraction and purification steps of the exosomes in this technology are complex, potentially leading to high production costs and technical barriers. Furthermore, this approach focuses on large-scale exosome production and does not address specific dosage form design or administration routes, which poses certain limitations for clinical translation. Patent Publication No. CN104274828B proposes an application scheme for the inhibition of hepatitis B and C viruses using exosomes secreted by IFN-α-treated cells. Studies have shown that these exosomes can be internalized by hepatocytes and effectively inhibit viral replication. However, the application of the exosomes in this technology is limited to injection or topical administration. The lack of aerosol inhalation formulations designed for specific locations such as the respiratory tract makes it difficult to meet the requirements for rapid onset of action or targeted delivery. At the same time, this program's functional optimization of exosomes mainly focuses on antiviral activity, with less consideration given to the comprehensive improvement of immune regulatory function.

[0005] Secondly, during the aerosolization process of nebulized inhalation preparations, mechanical shear and thermodynamic factors may damage the exosome membrane structure and reduce the functional activity of interferon, which poses a challenge to the stability and efficacy of the preparation. Traditional methods for addressing the synergistic effect of exosomes and interferon often fail to fully balance the relationship between preparation stability and functionality, thus affecting their effectiveness and scope of application in clinical applications. Summary of the Invention

[0006] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides an interferon-cooperated exosome aerosol inhalation preparation and its preparation method and application, which effectively solves the problem that exosome preparations on the current market may cause damage to the exosome membrane structure and decreased interferon functional activity due to mechanical shear force and thermodynamic factors.

[0007] The present invention provides an interferon-synergistic exosome aerosol inhalation preparation, which comprises the following components: 5-15 μg / mL interferon α, 0.1-0.5 mg / mL exosomes, 0.2-0.8% NSCS-PNIPAM, 0.3-1% mannitol, 0.01-0.05% polymer stabilizer, 0.5-2 mM phosphate buffer and 0.02-0.1% surfactant.

[0008] Preferably, the formulation consists of the following components: 10 μg / mL interferon α, 0.3 mg / mL exosomes, 0.5% NSCS-PNIPAM, 0.5% mannitol, 0.03% polymer stabilizer, 1 mM phosphate buffer, and 0.05% surfactant.

[0009] Interferon alpha (IFN-α) in this invention is an important protein cytokine with significant effects in antiviral, immunomodulatory, and tumor growth inhibition. By binding to exosomes, the delivery efficiency of interferon alpha is improved, while its stability is protected during the aerosolization process.

[0010] Exosomes, as used in this study, are nanoscale vesicles secreted by cells, typically mesenchymal stem cells (MSCs). They contain bioactive substances such as proteins, lipids, and nucleic acids, and can be used as drug carriers for targeted delivery. The membrane structure of exosomes gives them excellent biocompatibility and low immunogenicity, making them suitable for aerosol inhalation.

[0011] Among them, mannitol is a commonly used osmotic pressure regulator and lyoprotectant, which plays a role in stabilizing exosomes and interferon α in the preparation, while reducing the impact of water evaporation on particle dispersibility during the nebulization process; phosphate buffer: used to adjust the pH value of the preparation to the range of 6.8-7.4 to ensure that interferon α and exosomes maintain functional activity in a suitable environment.

[0012] Preferably, the polymer stabilizer can be polyvinylpyrrolidone (PVP), which is not specifically limited in the present invention. It can prevent the aggregation of exosomes during the aerosolization process through the steric hindrance effect, thereby maintaining their dispersion and uniformity.

[0013] Preferably, the surfactant is generally selected from Tween-80, which is not limited in the present invention and can reduce the surface tension of the liquid, improve the particle distribution characteristics during the atomization process, and reduce the deposition resistance of aerosol particles on the respiratory mucosa.

[0014] As a further preferred embodiment of the present invention, the method for preparing the aerosol inhalation preparation comprises the following steps:

[0015] (1) Preparation of NSCS-PNIPAM (N-succinylchitosan-poly(N-isopropylacrylamide) graft copolymer);

[0016] (2) Take 1×10 10 Exosomes and IFN-α are mixed in electroporation buffer, and interferon IFN-α is loaded onto the exosomes by electroporation;

[0017] (3) adding NSCS-PNIPAM to the above mixture, mixing and fixing by photocrosslinking;

[0018] (4) adding mannitol, polymer stabilizer and surfactant in sequence;

[0019] (4) The resulting mixture was processed by a high-pressure homogenizer, with the pressure controlled at 200-400 bar and the temperature at 20-25° C., and the process was circulated 3-5 times;

[0020] (5) The microemulsion is freeze-dried to obtain a powdered preparation.

[0021] The preparation method of NSCS-PNIPAM includes the following steps: dissolving 2g of chitosan in 100mL of 2% acetic acid solution, adding 5mL of succinic anhydride dropwise, reacting under magnetic stirring for 6h, dialysis purification and freeze-drying to obtain N-succinyl chitosan, dissolving NSCS and NIPAM monomers in deoxygenated PBS, adding cystamine crosslinker and photoinitiator, and ultrafiltration and concentration under 365nm ultraviolet radiation under nitrogen protection to obtain the grafted product.

[0022] Preferably, the addition ratio of the cystamine crosslinker is 0.1 mol%, and the addition ratio of the photoinitiator is 0.05%.

[0023] The cystamine crosslinker in the present invention is cystamine dihydrochloride (CAS No.: 56-17-7), and the photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) (CAS No.: 106797-53-1).

[0024] In addition, the present invention also provides an application method of an interferon-exosome nebulized inhalation preparation, characterized in that the method comprises the following steps:

[0025] Step 1: After reconstitution, the preparation is loaded into the nebulizer and the nebulization parameters are set to an air flow rate of 5-10 L / min and a nebulization temperature of 20-25°C;

[0026] Step 2: The patient inhales aerosol particles through the mouth and nose, and the diameter of the atomized particles is controlled within the range of 1-5μm;

[0027] Step 3: Inhale 1-2 times a day, each inhalation time is 10-15 minutes, and use continuously for 7-14 days.

[0028] Preferably, in step 1, the atomizer is an ultrasonic atomizer with a frequency range of 1.7-2.5 MHz and an amplitude of 30-50 μm.

[0029] Preferably, in step 2, the particle size distribution of the aerosol particles is measured by a dynamic light scattering instrument to ensure that more than 90% of the particles have a diameter within the range of 1-5 μm.

[0030] In addition, the present invention also provides the use of interferon-assisted exosome aerosol inhalation preparation in the preparation of antiviral drugs.

[0031] In addition, the present invention also provides an application method of interferon-assisted exosome aerosol inhalation preparation in the preparation of immunomodulatory therapeutic products.

[0032] The technical solution adopted by the present invention is as follows: The beneficial effects achieved by the present invention using the above structure are as follows:

[0033] (1) The present invention achieves a multi-dimensional improvement in material performance through a unique molecular design and three-dimensional protection mechanism. The synthesized N-succinyl chitosan-poly (N-isopropylacrylamide) graft copolymer (NSCS-PNIPAM) combines the biocompatibility of chitosan with the environmental responsiveness of PNIPAM, effectively solving the problem that mechanical shear force and thermodynamic factors may cause damage to the exosome membrane structure and decrease the functional activity of interferon.

[0034] (2) PNIPAM undergoes a conformational transition from β-sheet to α-helix under shear force, efficiently absorbing external kinetic energy by stretching, providing strong mechanical protection for the material;

[0035] (3) Based on the LCST characteristics of PNIPAM at 34°C, a thermally induced hydration layer (phase change enthalpy ΔH = 38 J / g) is rapidly formed under high temperature conditions, significantly reducing the film temperature and avoiding high temperature damage to the material and the encapsulated substances;

[0036] (4) Introducing a dynamic disulfide bond cross-linking system and using cystamine cross-linker to construct a material that can reconstruct the membrane structure according to the GSH concentration in a ROS environment, effectively improving the stability and adaptability of the material in complex physiological environments;

[0037] This invention expands the application potential of exosomes in biomedicine, tissue engineering and other fields, and has significant innovation and practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a morphological diagram of exosomes of the present invention;

[0039] Figure 2 Zeta potential diagram;

[0040] Figure 3 Virus titers in mouse lung tissues.

[0041] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0043] The present invention provides an interferon-synergistic exosome aerosol inhalation preparation, which comprises the following components: 5-15 μg / mL interferon α, 0.1-0.5 mg / mL exosomes, 0.2-0.8% NSCS-PNIPAM, 0.3-1% mannitol, 0.01-0.05% polymer stabilizer, 0.5-2 mM phosphate buffer and 0.02-0.1% surfactant.

[0044] The formulation consisted of the following components: 10 μg / mL interferon α, 0.3 mg / mL exosomes, 0.5% NSCS-PNIPAM, 0.5% mannitol, 0.03% polymer stabilizer, 1 mM phosphate buffer, and 0.05% surfactant.

[0045] The preparation method of the aerosol inhalation preparation comprises the following steps:

[0046] (1) Preparation of NSCS-PNIPAM;

[0047] (2) Take 1×10 10 Exosomes and IFN-α are mixed in electroporation buffer, and interferon IFN-α is loaded onto the exosomes by electroporation;

[0048] (3) adding NSCS-PNIPAM to the above mixture, mixing and fixing by photocrosslinking;

[0049] (4) adding mannitol, polymer stabilizer and surfactant in sequence;

[0050] (4) The resulting mixture was processed by a high-pressure homogenizer, with the pressure controlled at 200-400 bar and the temperature at 20-25° C., and the process was circulated 3-5 times;

[0051] (5) The microemulsion is freeze-dried to obtain a powdered preparation.

[0052] The preparation method of NSCS-PNIPAM comprises the following steps: dissolving 2 g of chitosan (degree of deacetylation ≥ 95%) in 100 mL of 2% acetic acid solution, adding 5 mL of succinic anhydride dropwise (in an ice bath at 0°C), reacting with magnetic stirring (500 rpm) for 6 h, dialysis purification (MWCO 8 kDa), and then freeze-drying to obtain N-succinyl chitosan (NSCS), dissolving NSCS and NIPAM monomers (molar ratio 1:50) in deoxygenated PBS, adding 0.1 mol% of cystamine crosslinker and 0.05% of photoinitiator (I2959), and irradiating with 365 nm ultraviolet light (10 mW / cm2) under nitrogen protection. 2 ×15 min) and concentrated by ultrafiltration (100 kDa MWCO) to obtain the grafted product.

[0053] A method for applying an interferon-exosome nebulized inhalation preparation, comprising the following steps:

[0054] Step 1: reconstitute the preparation prepared according to any one of claims 1 to 5 and load it into a nebulizer, setting the nebulization parameters to an air flow rate of 5-10 L / min and a nebulization temperature of 20-25°C;

[0055] Step 2: The patient inhales aerosol particles through the mouth and nose, and the diameter of the atomized particles is controlled within the range of 1-5μm;

[0056] Step 3: Inhale 1-2 times a day, each inhalation time is 10-15 minutes, and use continuously for 7-14 days.

[0057] In step 1, the atomizer uses an ultrasonic atomization device with a frequency range of 1.7-2.5 MHz and an amplitude of 30-50 μm.

[0058] In step 2, the particle size distribution of the aerosol particles is measured by a dynamic light scattering instrument to ensure that more than 90% of the particles have a diameter within the range of 1-5 μm.

[0059] The interferon-exosome nebulized inhalation preparation is used in the preparation of antiviral drugs.

[0060] The application method of the interferon-cooperative exosome aerosol inhalation preparation is used in the preparation of immunomodulatory therapeutic products.

[0061] Example 1

[0062] The present invention provides an interferon-cooperated exosome aerosol inhalation preparation.

[0063] The formulation consisted of the following components: 10 μg / mL interferon α, 0.3 mg / mL exosomes, 0.5% NSCS-PNIPAM, 0.5% mannitol, 0.03% polymer stabilizer, 1 mM phosphate buffer, and 0.05% surfactant.

[0064] The preparation method of the aerosol inhalation preparation comprises the following steps:

[0065] (1) Preparation of NSCS-PNIPAM;

[0066] (2) Take 1×10 10 Exosomes and IFN-α are mixed in electroporation buffer, and interferon IFN-α is loaded onto the exosomes by electroporation;

[0067] (3) NSCS-PNIPAM was added to the above mixture, mixed and fixed by photocrosslinking (exosome coating);

[0068] (4) adding mannitol, polymer stabilizer and surfactant in sequence;

[0069] (4) The resulting mixture was processed by a high-pressure homogenizer, with the pressure controlled at 200-400 bar and the temperature at 20-25° C., and the process was circulated 3-5 times;

[0070] (5) The microemulsion is freeze-dried to obtain a powdered preparation.

[0071] The specific experimental steps for loading interferon IFN-α into exosomes are as follows: take 1×10 10 Exosomes were mixed with IFN-α (final concentration 100 μg / mL) in electroporation buffer (10 mM HEPES, 250 mM sucrose, pH 7.4). The cells were transferred to a 4 mm electroporation cuvette using the following parameters: voltage 150 V, capacitance 950 μF, and three pulses. The cells were incubated at 37°C for 30 minutes to restore membrane integrity, and free IFN-α was removed by ultrafiltration and centrifugation (100 kDa MWCO).

[0072] The preparation method of NSCS-PNIPAM includes the following steps: dissolving 2g of chitosan in 100mL of 2% acetic acid solution, adding 5mL of succinic anhydride dropwise, reacting under magnetic stirring for 6h, dialysis purification and freeze-drying to obtain N-succinyl chitosan, dissolving NSCS and NIPAM monomers in deoxygenated PBS, adding cystamine crosslinker and photoinitiator, and ultrafiltration and concentration under 365nm ultraviolet radiation under nitrogen protection to obtain the grafted product.

[0073] In this example, the specific experimental method for exosome coating was as follows: exosome pretreatment: ultracentrifugation purification (100,000 g × 2 h), resuspended in 4 ° C pre-cooled PBS (containing 1 mM EDTA and then gradient cooling: 25 ° C to 15 ° C (-1 ° C / min) to 10 ° C (maintained for 30 min); complex formation: NSCS-PNIPAM solution (final concentration 0.5% w / v) was added dropwise, vortex mixing (2000 rpm × 30 s, interval 5 min × 3 times); photocrosslinking fixation: 365 nm UV LED array irradiation (5 mW / cm 2 The viscosity of the solution was monitored in real time (45±5 cP). Purification: ultrafiltration centrifugation (300 kDa MWCO, 4°C×15,000 g×30 min), and sterile filtration (0.22 μm PVDF membrane).

[0074] In addition, the experimental steps for exosome extraction and purification are as follows:

[0075] Step 1: Cell culture and conditioned medium collection

[0076] hUC-MSCs were seeded in 15 cm culture dishes (density 1×10 6 cells / dish) and cultured in serum-free medium for 48 h.

[0077] The conditioned medium was collected, centrifuged at 10,000 g for 30 min at 4°C to remove cell debris, and filtered through a 0.22 μm filter.

[0078] Step 2: Ultracentrifugation purification;

[0079] Transfer the filtrate to an ultracentrifuge tube and centrifuge at 110,000 g for 70 min at 4°C. Discard the supernatant and gently resuspend the pellet in pre-chilled PBS (pH 7.4). Determine the protein concentration by BCA assay (target 1-2 mg / mL). Filter through a 0.22 μm filter and store in aliquots at -80°C (containing 5% trehalose).

[0080] Also provided is a method for applying an interferon-exosome nebulized inhalation preparation, the method comprising the following steps:

[0081] Step 1: reconstitute the preparation prepared according to any one of claims 1 to 5 and load it into a nebulizer, setting the nebulization parameters to an air flow rate of 5-10 L / min and a nebulization temperature of 20-25°C;

[0082] Step 2: The patient inhales aerosol particles through the mouth and nose, and the diameter of the atomized particles is controlled within the range of 1-5μm;

[0083] Step 3: Inhale 1-2 times a day, each inhalation time is 10-15 minutes, and use continuously for 7-14 days.

[0084] In step 1, the atomizer uses an ultrasonic atomization device with a frequency range of 1.7-2.5 MHz and an amplitude of 30-50 μm.

[0085] In step 2, the particle size distribution of the aerosol particles is measured by a dynamic light scattering instrument to ensure that more than 90% of the particles have a diameter within the range of 1-5 μm.

[0086] Also provided is the use of the interferon-assisted exosome aerosol inhalation preparation in the preparation of antiviral drugs, and the use of the interferon-assisted exosome aerosol inhalation preparation in the preparation of immunomodulatory therapeutic products.

[0087] Example 2:

[0088] The formulation includes the following components: 5 μg / mL interferon α, 0.1 mg / mL exosomes, 0.2% NSCS-PNIPAM, 0.3% mannitol, 0.01% polymer stabilizer, 0.5 mM phosphate buffer, and 0.02% surfactant.

[0089] Other preparation methods are shown in Example 1.

[0090] Example 3:

[0091] The formulation includes the following components: 15 μg / mL interferon α, 0.5 mg / mL exosomes, 0.8% NSCS-PNIPAM, 1% mannitol, 0.05% polymer stabilizer, 2 mM phosphate buffer, and 0.1% surfactant.

[0092] Other preparation methods are shown in Example 1.

[0093] Comparative Example 1

[0094] Chitosan was used instead of NSCS-PNIPAM treatment.

[0095] Test Example 1

[0096] Exosome membrane integrity and zeta potential detection

[0097] The aerosol inhalation preparations prepared in Example 1 and Comparative Example 1 of the present invention were used as test materials. 100 mg of each lyophilized preparation was reconstituted with 1 mL of PBS (pH 7.4) and ultrasonically shaken for 30 seconds until completely dissolved. Exosome morphology was observed using a negative staining method (2% phosphotungstic acid, pH 6.8) and scored according to the membrane integrity scoring criteria. Dynamic light scattering (DLS) was used to measure zeta potential. The membrane integrity scoring criteria were: 5: complete spherical; 3: slightly concave; 1: membrane rupture.

[0098] Result analysis: Figure 1As shown, 95% of the exosomes in the Example 1 group were scored as Grade 5 and 5% were scored as Grade 3, while 60% of the exosomes in the Comparative Example 1 group were scored as Grade 3 and 40% were scored as Grade 1. Figure 2 As shown, the Zeta potential of Example 1 group is higher than that of Comparative Example 1 group, and the surface charge of Example 1 group is relatively stable.

[0099] Test Example 2

[0100] Exosome integrity and IFN-α enzyme activity

[0101] The reconstituted preparations of Example 1 and Comparative Example 1 (5 mL each) were added to the PARILC Sprint nebulizer, connected to an air compressor, set the air flow rate to 6 L / min, and the nebulization temperature to 25°C. The outlet of the nebulizer was connected to an Andersen Cascade Impactor, which was divided into 8 stages to collect particles of different sizes, including Stage 2: a particle size of 3.3-4.7 μm (simulating alveolar deposition area); Stage 4: a particle size of 1.1-2.1 μm (simulating bronchial area). The nebulization lasted for 15 minutes, and 1 mL of PBS was pre-added to each impactor plate. The recovered liquid of all levels was collected, mixed evenly, and 200 μL was taken to the sample cell of the dynamic light scattering instrument. The integrity of the exosomes was compared using the Nanosight NS300 counting method; the IFN-α enzyme activity retention rate was detected using the ELISA method (Human IFNalpha ELISA Kit).

[0102] Table 1 Integrity of exosomes and IFN-α enzyme activity

[0103] Detection indicators Example 1 group Comparative Example 1 Complete exosome concentration / particles / ml <![CDATA[4.2×10 9 ]]> <![CDATA[2.9×10 9 ]]> IFN-α activity retention rate 89.5％ 68.3％

[0104] Analysis of results: As shown in Table 1, the exosome breakage rate in Example 1 was lower than that in Comparative Example 1, indicating that NSCS-PNIPAM reduced membrane rupture caused by atomization shear through mechanical protection. The IFN-α activity retention rate in Example 1 was significantly higher (P < 0.01), confirming that the thermoresponsive hydration layer of PNIPAM can prevent high temperature from damaging protein activity.

[0105] Test Example 3

[0106] Shear stress response performance evaluation

[0107] The distance between the rotor and stator of the Couette rheometer was adjusted to 1 mm, 5 mL of each of the reconstituted preparations of Example 1 and Comparative Example 1 were added, and the shear rates were set to 0 s -1 (static control), 10 3 s -1 (low shear), 10 4 s -1(High shear), each rate was maintained for 5 minutes; samples were taken immediately after each shear for subsequent detection. 100 μL of the sheared sample was added with Annexin V-FITC (1 μL) and PI (1 μL), incubated in the dark for 15 minutes, and 400 μL of binding buffer was added. The early stage of apoptosis (Annexin V + / PI-) and necrotic cells (Annexin V ＞ / PI + ) ratio, membrane integrity rate = 1-(early apoptosis rate + necrosis rate); the activity retention rate was calculated using ELISA (Human IFNalpha ELISA Kit) with the uncut sample as the control.

[0108] Table 2 Membrane integrity rate and IFN-α activity retention rate

[0109]

[0110] Result analysis: As shown in Table 2, under static control, low shear rate and high shear rate, the membrane integrity rate of Example 1 group was higher than that of Comparative Example 1 group, and the IFN-α activity retention rate of Example 1 group was also higher than that of Comparative Example 1 group.

[0111] Test Example 4

[0112] Biosafety Verification

[0113] The samples prepared in Example 1 were subjected to biosafety tests, including cytotoxicity test and hemolysis test.

[0114] Cytotoxicity test (CCK-8 method):

[0115] (1) Cell culture: L929 fibroblasts were cultured at 5×10 3 cells / well were seeded in 96-well plates and cultured at 37°C, 5% CO2 for 24 h;

[0116] (2) Sample treatment: dilute the preparation solution 10-fold with complete culture medium (extract solution) and add it to the well plate (100 μL per well), with 3 replicate wells;

[0117] (3) Control settings: positive control (0.1% TritonX-100), negative control (complete culture medium);

[0118] (4) Detection: After 24 h of incubation, 10 μL of CCK-8 solution was added to each well and incubated for 2 h. The absorbance at 450 nm was read using a microplate reader.

[0119] (5) Calculation: relative proliferation rate (RG) = (OD value of sample group / OD value of negative control group) × 100%.

[0120] Hemolysis test:

[0121] (1) Preparation of red blood cell suspension: Fresh rabbit blood was collected, washed three times with normal saline, and centrifuged (2000 g for 5 minutes) to prepare a 2% (v / v) red blood cell suspension;

[0122] (2) Sample treatment: The preparation solution (distilled water for positive control and normal saline for negative control) was mixed with the red blood cell suspension at a volume ratio of 1:1 and incubated at 37°C for 30 min;

[0123] (3) Centrifugation and detection: Centrifuge at 1000 g for 5 minutes, take the supernatant and measure the absorbance at 540 nm. Hemolysis rate = (sample absorbance - negative control absorbance) / (positive control absorbance - negative control absorbance) × 100%.

[0124] Result analysis: CCK-8 assay showed that the relative proliferation rate (RG) of Example 1 group was 94.3% (conforming to medical grade requirements); hemolysis rate was 2.1% (<5% qualified).

[0125] Test Example 5

[0126] Antiviral and immunomodulatory effects in animal models

[0127] Twenty 6-week-old Balb / c mice were randomly divided into two groups, each with 10 mice, and recorded as Example 1 group (nebulized inhalation of Example 1 preparation) and Comparative Example 1 group (nebulized inhalation of Comparative Example 1 preparation). All mice were infected with the nasal cavity for 10 4 The mice were treated with PFU influenza virus A / PR / 8 / 34 (H1N1) starting on the first day after infection for 7 consecutive days. Mice were killed on the eighth day, and lung tissues were weighed and homogenized in 1 mL of TRIzol. Total RNA was extracted and reverse transcribed into cDNA. PCR amplification was performed using primers specific for the viral NP gene. The viral copy number was detected by fluorescent quantitative PCR, and the results were expressed as copies / mg of lung tissue. Mouse serum was collected and cytokine levels were detected according to the instructions of Mouse IFN-γ ELISA Kit and Mouse IL-6 ELISA Kit.

[0128] Result analysis: Figure 3 As shown, the viral titer in the lung tissue of the Example 1 group was much lower than that of the Comparative Example 1 group, indicating that the NSCS-PNIPAM-containing formulation can effectively inhibit respiratory viral replication through aerosol inhalation, confirming its antiviral efficacy. As shown in Table 3, the IFN-γ level in the Example 1 group increased by 65%, indicating that the formulation can enhance Th1 cellular immune response and improve antiviral immunity; the IL-6 level in the Example 1 group decreased by 42%, indicating that the formulation can alleviate lung inflammation and reduce the risk of excessive immune damage.

[0129] Table 3 Immune factor level detection

[0130] Group IFN-γ level / pg / ml IL-6 level / pg / ml Example 1 group 185±22 45±8 Comparative Example 1 112±15 78±11

[0131] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

[0132] The present invention and its embodiments are described above. This description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if a person skilled in the art is inspired by this and, without departing from the purpose of the present invention, designs structures and embodiments similar to this technical solution without inventiveness, they shall fall within the scope of protection of the present invention.

Claims

1. An interferon-exosome nebulized inhalation preparation, characterized by: The formulation includes the following components: 5-15 μg / mL interferon α, 0.1-0.5 mg / mL exosomes, 0.2-0.8% NSCS-PNIPAM, 0.3-1% mannitol, 0.01-0.05% polymer stabilizer, 0.5-2 mM phosphate buffer, and 0.02-0.1% surfactant.

2. The interferon-exosome nebulized inhalation preparation according to claim 1, characterized in that: The formulation consisted of the following components: 10 μg / mL interferon α, 0.3 mg / mL exosomes, 0.5% NSCS-PNIPAM, 0.5% mannitol, 0.03% polymer stabilizer, 1 mM phosphate buffer, and 0.05% surfactant.

3. The method for preparing the interferon-exosome nebulized inhalation preparation according to claim 1 or 2, characterized in that: The preparation method of the aerosol inhalation preparation comprises the following steps: (1) Preparation of NSCS-PNIPAM; (2) Take 1×10 10 Exosomes and IFN-α are mixed in electroporation buffer, and interferon IFN-α is loaded onto the exosomes by electroporation; (3) adding NSCS-PNIPAM to the above mixture, mixing and fixing by photocrosslinking; (4) adding mannitol, polymer stabilizer and surfactant in sequence; (4) The resulting mixture was processed by a high-pressure homogenizer, with the pressure controlled at 200-400 bar and the temperature at 20-25° C., and the process was circulated 3-5 times; (5) The microemulsion is freeze-dried to obtain a powdered preparation.

4. The method for preparing the interferon-exosome nebulized inhalation preparation according to claim 3, characterized in that: The preparation method of NSCS-PNIPAM comprises the following steps: dissolving chitosan in a 2% acetic acid solution, adding succinic anhydride dropwise, reacting under magnetic stirring for 6 hours, dialysis purification, and freeze-drying to obtain N-succinyl chitosan, dissolving NSCS and NIPAM monomers in deoxygenated PBS, adding a cystamine crosslinker and a photoinitiator, and ultrafiltration and concentration under nitrogen protection at 365nm ultraviolet radiation to obtain a grafted product.

5. The method for preparing the interferon-exosome nebulized inhalation preparation according to claim 4, characterized in that: The addition ratio of the cystamine cross-linking agent was 0.1 mol%, and the addition ratio of the photoinitiator was 0.05%.

6. A method for applying an interferon-exosome nebulized inhalation preparation, characterized in that: The method comprises the following steps: Step 1: reconstitute the preparation prepared according to any one of claims 1 to 5 and load it into a nebulizer, setting the nebulization parameters to an air flow rate of 5-10 L / min and a nebulization temperature of 20-25°C; Step 2: The patient inhales aerosol particles through the mouth and nose, and the diameter of the atomized particles is controlled within the range of 1-5μm; Step 3: Inhale 1-2 times a day, each inhalation time is 10-15 minutes, and use continuously for 7-14 days.

7. The method for using the interferon-exosome nebulized inhalation preparation according to claim 6, characterized in that: In step 1, the atomizer uses an ultrasonic atomization device with a frequency range of 1.7-2.5 MHz and an amplitude of 30-50 μm.

8. The method for using the interferon-cooperated exosome aerosol inhalation preparation according to claim 6, characterized in that: In step 2, the particle size distribution of the aerosol particles is measured by a dynamic light scattering instrument to ensure that more than 90% of the particles have a diameter within the range of 1-5 μm.

9. Use of the interferon-exosome nebulized inhalation preparation according to any one of claims 1 to 5 in the preparation of antiviral drugs.

10. Use of the interferon-synergistic exosome aerosol inhalation preparation according to claim 6 in the preparation of an immunomodulatory therapeutic product.

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