Exosome lyophilization protectant, exosome lyophilized formulation comprising the protectant, method for preparing the same, and use thereof in nebulized inhalation therapy

Abstract

The application is an exosome freeze-drying protective agent, an exosome freeze-drying preparation containing the protective agent, a preparation method thereof and application thereof in nebulization inhalation therapy. The freeze-drying protective agent contains trehalose, inulin, arginine and Tween 80, the concentration of the trehalose in the protective agent solution is 5%-7%, the concentration of the inulin in the protective agent solution is 1%-3%, the concentration of the arginine in the protective agent solution is 0.1%-0.2%, and the concentration of the Tween 80 in the protective agent solution is 0.05%-0.1%. The formula can effectively maintain the vesicle structure integrity and biological activity of the exosome in the process of exosome freeze-drying and subsequent storage and transportation, and significantly improve the long-term stability of the exosome.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an exosome lyophilization protectant, an exosome lyophilized formulation containing the protectant, its preparation method, and its application in nebulized inhalation therapy. Background Technology

[0002] Exosomes are extracellular vesicles with a diameter of approximately 30-150 nanometers, secreted by almost all cell types. They carry bioactive molecules such as specific proteins, lipids, and nucleic acids (e.g., mRNA, miRNA, lncRNA) from their source cells, playing a crucial "messenger" role in intercellular communication. Based on their natural biocompatibility, low immunogenicity, excellent targeted homing ability, and potential to cross biological barriers (such as the blood-brain barrier), exosomes have been considered highly promising next-generation drug delivery carriers and therapeutic agents, demonstrating significant clinical application value in regenerative medicine, tumor immunotherapy, neurological diseases, and inflammatory diseases.

[0003] However, the transition of exosomes from laboratory research to large-scale production and clinical application faces severe stability challenges. Exosomes are essentially vesicles encapsulated in a phospholipid bilayer, and long-term storage in an aqueous environment can easily lead to the following problems: vesicle aggregation, fusion, increased particle size, and even membrane rupture, resulting in leakage of contents; oxidation of membrane lipids; degradation or denaturation of proteins; and degradation of nucleic acids by nucleases.

[0004] Currently, research institutions and companies typically suspend exosomes in aqueous solutions such as phosphate buffer and preserve them at -80°C or liquid nitrogen. While this method can maintain activity in the short term, it has significant drawbacks: First, cryogenic storage equipment is expensive, energy-intensive, and dependent on uninterrupted power supplies, resulting in extremely high storage and transportation costs, severely hindering commercialization; second, repeated freeze-thaw cycles can cause irreversible damage to exosomes, while clinical use often requires multiple retrievals; finally, long-distance cold chain transportation in liquid form is risky and logistically complex, greatly limiting its accessibility as a standardized drug.

[0005] Freeze-drying (lyophilization) technology is considered an effective way to solve the long-term stability problem of biopharmaceuticals. This technology removes most of the water by freezing the sample at low temperatures and then directly sublimating the ice under vacuum, leaving the product in a solid form. Lyophilized formulations can be stably stored for a long time at 2-8°C or even room temperature, facilitating transportation and distribution, and can be directly reconstituted for use. However, exosomes, as intricately structured nanovesicles, undergo multiple stresses during freeze-drying: mechanical damage caused by ice crystal formation, chemical stress caused by drastic changes in salt content and pH in the concentrated phase, and damage to the membrane structure during the removal of bound water. Without protection, direct freeze-drying can lead to almost complete inactivation, aggregation, or disintegration of exosomes. Therefore, developing a highly efficient "lyophilization protectant" is crucial for the success of exosome freeze-drying technology.

[0006] In terms of drug delivery, nebulized inhalation can directly deliver drugs to the lesions (respiratory tract and lungs), increasing local drug concentration and reducing systemic exposure and side effects, showing significant advantages in the treatment of lung diseases. The use of exosomes in nebulized therapy has become a research hotspot. However, almost all current studies use liquid formulations composed of freshly prepared or cryopreserved and thawed liquid exosome suspensions for nebulization. This approach has inherent drawbacks: liquid formulations have a short stability window and must be used within a short period; ultra-low temperature freezers are required in clinical settings, making the process cumbersome; furthermore, liquid formulations may be affected by shear forces and gas-liquid interfaces during nebulization, and the loss of activity has not been fully assessed. Moreover, there is a lack of standardized solutions for transitioning from a stable solid at room temperature to a ready-to-use liquid state.

[0007] In summary, there is a significant gap and deficiency in existing technologies: on the one hand, exosome lyophilization protection technology is immature, lacking efficient and universally applicable protective agent formulations and stable processes; on the other hand, research on exosome nebulization therapy remains in the early stages of using unstable liquid formulations. Currently, there is no complete technical solution that combines efficient lyophilization protection, long-term stable storage, convenient reconstitution, and formulation characteristics suitable for nebulization inhalation. Therefore, developing a comprehensive solution specifically designed for exosomes, enabling them to maintain high activity after lyophilization and reconstitution and suitable for nebulization inhalation, is of urgent need and significant importance for promoting the practical clinical application of exosome therapeutic products. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a highly efficient and stable exosome lyophilization protectant that can maximally maintain the structural integrity and biological activity of exosomes during lyophilization and reconstitution. This formulation can be stably stored at room temperature for extended periods. Furthermore, this invention provides applications of the aforementioned exosome lyophilized formulation, particularly its reconstitution for nebulized inhalation therapy, thus resolving the issues of poor stability and inconvenience in clinical use of liquid exosome formulations.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a highly efficient and stable exosome freeze-drying protectant, the protectant comprising trehalose, inulin, arginine and Tween 80.

[0010] Preferably, the concentration of trehalose in the protective agent solution is 5%-7%, the concentration of inulin in the protective agent solution is 1%-3%, the concentration of arginine in the protective agent solution is 0.1%-0.2%, and the concentration of Tween 80 in the protective agent solution is 0.05%-0.1%.

[0011] Preferably, exosomes are extracted from the supernatant collected during the 2nd to 6th passages of human umbilical cord mesenchymal stem cells (hP-MSCs).

[0012] Preferably, exosomes are extracted by ultrafiltration.

[0013] This invention also provides an atomization application of exosome freeze-dried exosomes.

[0014] Preferably, the exosome lyophilized powder prepared according to this invention is reconstituted with sterile physiological saline or water for injection. The ratio of lyophilized powder to reconstitution solvent is 1:5-1:10 (g / mL). The mixture is gently inverted and allowed to stand for 5-10 min without ultrasonic treatment to obtain a uniformly dispersed exosome reconstituted solution. The exosome concentration after reconstitution is maintained at 2×10⁻⁶. 9 -5×10 9 The particle size was maintained at 30-150 nm, with a bioactivity recovery rate of ≥80% and no obvious aggregation.

[0015] Preferably, to further improve the nebulization effect of the exosome reconstituted solution and ensure no aggregation and high lung deposition rate during nebulization, the reconstituted exosome solution is optimized to prepare a nebulized formulation. The specific formulation is as follows: 0.05% Tween 80 is added to the reconstituted exosome solution, mixed thoroughly, and then aseptically filtered through a 0.22 μm filter membrane to remove impurities and any potentially present microparticles, obtaining the exosome nebulized solution. The pH value of this nebulized solution is controlled between 7.0 and 7.4, meeting the quality standards for inhaled formulations.

[0016] This invention also provides a method for nebulized drug delivery using a medical compression nebulizer. The prepared exosome nebulization solution is added to the nebulizer, and the nebulization parameters are adjusted to control the diameter of the nebulized particles to 1-5 μm. Nebulized particles within this size range can pass smoothly through the respiratory tract and reach the alveoli and small bronchi. The lung deposition rate is 10 times that of intravenous injection, significantly improving the lung delivery efficiency of exosomes.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The freeze-drying protectant formulation of this invention is safe and efficient, and can effectively protect the structural integrity and biological activity of exosomes during the freeze-drying process, enabling long-term stable storage of exosomes at room temperature (room temperature > 6 months). The activity recovery rate after reconstitution is ≥ 80%, meaning that the activity is still 80% after reconstitution. There is no significant change in particle size and no aggregation phenomenon, which completely solves the technical problems of traditional liquid exosomes that rely on cold chain, are prone to inactivation, and are inconvenient to store and transport.

[0018] The exosome lyophilized powder prepared by this invention is small in volume, lightweight, and stable in properties. It requires no cold chain transportation and can be stored and transported at room temperature, completely eliminating reliance on cold chains and significantly reducing drug distribution costs. Furthermore, the lyophilized powder is easy to reconstitute, requiring no specialized precision equipment; it can be rapidly reconstituted and administered using only conventional medical solvents. The operation is not technically demanding, meeting both the standardized clinical medication needs of hospitals and the needs of patients undergoing home nebulization therapy (especially for lung diseases), significantly improving the convenience of medication and patient adherence to long-term treatment. Compared to existing exosome liquid formulations, intravenous administration methods, and conventional nebulized formulations, this invention offers multiple technological advantages in terms of exosome lyophilized powder dosage form, nebulization performance, preparation process, and clinical application system. It effectively solves industry pain points such as stringent storage and transportation conditions, poor targeting, low bioavailability, difficulty in mass production, and lack of standardized application solutions, demonstrating significant inventiveness, clinical value, and industrialization potential.

[0019] This invention allows for direct nebulization therapy using lyophilized exosome powder. The exosome nebulization preparation has high nebulization efficiency, uniform atomized particles, and high lung deposition rate. The nebulization delivery method is non-invasive and highly targeted, and can directly act on lung lesions. Compared with intravenous injection, it significantly improves bioavailability, has fewer systemic side effects, and is well tolerated by patients.

[0020] The preparation process of this invention is simple, controllable, and low-cost. All components are conventional pharmaceutical raw materials, making large-scale industrial production easily achievable and meeting the industrialization needs of biopharmaceutical preparations. Furthermore, it clarifies the specific application scheme for nebulized therapy, providing feasible technical support for the clinical promotion of exosomes in the treatment of respiratory diseases, and possesses broad clinical application prospects and market value. Existing technologies only detect and analyze changes in the physicochemical properties after lyophilization, without exploring changes in the functional activity of the samples after lyophilization, which is a major shortcoming in current research in this field.

[0021] Currently, intravenous administration is commonly used in clinical practice for lung diseases as a systemic drug delivery method. The drug is dispersed throughout the body via the bloodstream, resulting in low drug accumulation and poor bioavailability in lung lesions. Furthermore, the drug tends to accumulate in the liver and kidneys, leading to systemic toxicity. Conventional nebulizer formulations on the market generally suffer from uneven particle size, low nebulization efficiency, and limited lung deposition. The nebulizer formulation of this invention boasts high nebulization efficiency and uniform, stable droplet particles. It can directly target lung lesions via non-invasive inhalation, bypassing systemic circulation, exhibiting strong targeting and significantly improving local drug concentration and bioavailability in the lungs. Simultaneously, local nebulization effectively avoids systemic adverse reactions, eliminates puncture damage, is suitable for a wide range of patients, and demonstrates significantly better patient tolerance and safety compared to traditional intravenous administration.

[0022] Current research on exosome therapy for lung diseases largely remains at the level of basic laboratory research, merely verifying biological activity. It lacks mature and replicable formulation preparation processes and standardized clinical dosing protocols, making it difficult for ordinary technicians to implement and translate into clinical practice. This invention constructs a complete technical system covering raw material formulation, preparation, room temperature storage and transportation, and clinical and home nebulization dosing. The system is clear, highly operable, and stably replicable, effectively filling the gap in the industrialization and clinical application of exosome nebulization therapy for respiratory diseases. It provides reliable technical support for the clinical promotion of exosome-based targeted non-invasive lung therapy, possessing outstanding innovation, practicality, and broad market application prospects. Attached Figure Description

[0023] Figure 1 These are comparative images showing the appearance of exosome lyophilized powders from Examples 1-3, Comparative Examples 1-3, and Comparative Example 5 of the present invention. Figure 2 This is a comparison chart of the particle size results of lyophilized exosomes from Examples 1–3, Comparative Examples 1–3, Comparative Examples 5, and Comparative Example 4. Figure 3 The results of the average particle size (Number Mean), peak Z-average, and dispersity (PDI) of the lyophilized exosomes of Examples 1–3, Comparative Examples 1–3 and 5, and Comparative Example 4 are as follows: Figure 4 The images show the electron microscope images of the lyophilized exosomes of Examples 1-3, Comparative Examples 1-3 and Comparative Examples 5, and the reconstituted exosomes of Comparative Example 4. Figure 5 The figures show the Western blot results of exosome marker proteins CD9, CD63 and CD81 after reconstitution of exosomes in Examples 1-3 and Comparative Examples 1-5 of this invention. Figure 6 This is a schematic diagram of nebulizer therapy; Figure 7 This is an in vivo distribution imaging and efficiency quantification result of exosomes after reconstitution and intravenous injection or nebulization in Example 1 of the present invention; Figure 8 HE staining results and statistical graphs of lyophilized exosomes of Examples 1-3, Comparative Examples 1-3, Comparative Examples 5 and Comparative Example 4, and exosome nebulization treatment of radiation-induced lung injury in mice. Figure 9 Masson staining results and statistical graphs of lyophilized exosomes in Examples 1–3, Comparative Examples 1–3, Comparative Examples 5 and Comparative Example 4, and exosome nebulization treatment of radiation-induced lung injury in mice. Figure 10 This is a graph showing the changes in the recovery rate of the lyophilized exosome preparations of Examples 1-3, Comparative Examples 1-3, and Comparative Examples 5 after different storage times. Figure 11 DSC results for trehalose and inulin; Figure 12 X-ray diffraction results for trehalose and inulin; Figure 13 Infrared spectra of trehalose and inulin. Detailed Implementation

[0024] The following examples will further illustrate the specific steps and features of the invention. These examples are for illustrative purposes only and are not intended to limit the invention. Unless otherwise specified, the methods used in this invention are conventional methods in the art. Unless otherwise specified, all reagents and materials involved in this invention are commercially available, including inulin (4kDa).

[0025] Examples 1-3: Table 1: Components and dosages of the freeze-dried compositions of Examples 1-3 and Comparative Examples 1-3

[0026] Based on the component dosage design in Table 1, Examples 1-3 and Comparative Examples 1-3 dissolved each excipient component in phosphate buffer, added exosomes to each formulation to form a lyophilized exosome composition liquid, and dispensed into EP tubes, each containing 1 mL of liquid and 5 × 10⁻⁶ ppm. 9 Particles are exosomes.

[0027] Comparative Examples 4 and 5: According to the formulation design in Table 1, Comparative Examples 4 and 5 were freshly extracted and undried exosomes and undried exosomes without protective agents, respectively. The exosomes were dissolved in phosphate-buffered saline (PBS) buffer and aliquoted into EP tubes, each containing 1 mL of liquid and 5 × 10⁻⁶ ppm.9 Particles are exosomes.

[0028] Application Example 1: Extraction of Exosomes 1000 mL of serum-free culture supernatant from human umbilical cord mesenchymal stem cells was collected and centrifuged using differential centrifugation to remove cells and cell debris (500 g × 10 min, to remove red blood cells and large debris; 2000 g × 20 min, to remove cell debris). Exosomes were then obtained by ultracentrifugation at 130,000 g for 2 h. The exosomes were washed three times with PBS buffer and the concentration was adjusted to 5 × 10⁻⁶. 10 Particles / mL, total protein concentration up to 3 mg / mL, for later use.

[0029] Application Example 2: Preparation of exosome lyophilized powder Take 20 mL of the above exosome concentrate, add exosome lyophilization protectant, gently invert and mix, and let stand at room temperature for 15 min; dispense the mixture into EP tubes, 1 mL per tube, and place them in a lyophilizer for lyophilization according to the following process: pre-freeze at -50℃ for 3 h, dry once at -20℃ under 15 Pa vacuum for 15 h, and dry again at 22℃ under 8 Pa vacuum for 8 h to obtain a white and loose exosome lyophilized powder; store under sterile conditions at room temperature and away from light.

[0030] Application Example 3: Nebulized Treatment of Exosomes Take one tube of exosome lyophilized powder prepared in Application Example 2, add 1 mL of sterile physiological saline, gently invert and mix, let stand for 8 min to obtain exosome reconstituted solution; The lyophilized powder corresponding to the protective agent in Example 1, after reconstitution, showed an exosome concentration of 4 × 10⁻⁶. 9 The particle size ranges from 30 to 150 nm, with particles per mL, and no obvious aggregation.

[0031] Add 0.05% Tween 80 to the above reconstituted solution, mix thoroughly, and then sterilely filter using a 0.22 μm filter membrane to obtain an exosome nebulizer with a pH of 7.3.

[0032] Turn on the mouse-specific compression nebulizer 30 minutes in advance, adjust the equipment parameters, set the gas flow rate to 6L / min, the nebulization pressure to 200 kPa, and the nebulization time to 15 minutes. Confirm that the diameter of the atomized particles is 1-5 μm (of which 2-5 μm particles account for ≥75%), check that the tubing connections are tight and there are no leaks, that the induction chamber is clean and free of impurities, and that the filter is working properly to avoid affecting the nebulization efficiency.

[0033] Five mice were placed in each nebulization induction chamber in sequence (to avoid crowding and affecting the inhalation effect). The chamber door was closed, and the mice were allowed to adapt to the environment for 5 minutes to reduce stress response. The mice were observed during nebulization. If any abnormalities such as restlessness or difficulty breathing occurred, the ventilation of the induction chamber was adjusted in time to ensure that the mice could breathe normally.

[0034] Add the prepared corresponding nebulizing solutions (exosome nebulizing solution and blank nebulizing solution) into the nebulizer, start the nebulization pump, and begin nebulization drug delivery. Strictly control the nebulization time to 15 minutes. During the nebulization process, utilize the Venturi effect of the nebulizer to form uniform aerosol droplets, which enter the mouse respiratory tract with the airflow, ensuring that the droplets can directly reach the alveolar area of ​​the mouse and increase the lung deposition rate.

[0035] After nebulization, turn off the nebulizer pump and keep the mouse in the induction chamber for 5 minutes. After the residual nebulizer droplets in the chamber have completely dissipated, remove the mouse. After each nebulization, clean the instrument with sterile saline, air dry, and then autoclave to avoid cross-contamination. Replace the filter regularly after long-term use to ensure stable equipment performance.

[0036] Before each day's nebulization treatment, weigh the mice, observe their mental state, diet, water intake and respiration, and record any abnormal reactions (such as hair loss, diarrhea, difficulty breathing, etc.). If any abnormalities occur, record them in time and analyze the cause. If necessary, remove the mouse.

[0037] Effect test 1. Appearance of exosome lyophilized powder like Figure 1 The appearance of the exosome lyophilized powders in Examples 1-3, Comparative Examples 1-3, and Comparative Examples 5 is shown. In Examples 1-3 and Comparative Examples 1-3, the exosome lyophilized powders exhibit a complete cake-like structure with uniform texture and high mechanical strength. In Comparative Example 5, only PBS was used as the preservation solution, and no protective agent was added. The lyophilized powder showed melting and a granular appearance.

[0038] 2. Particle size of exosome lyophilized products like Figure 2 The results shown are the particle size results of lyophilized exosomes from Examples 1–3, Comparative Examples 1–3, Comparative Examples 5, and Comparative Example 4, combined with… Figure 2 and Figure 3In Example 1, the average particle size, peak value, and PDI of exosomes were close to those of exosomes in Comparative Example 4. In Examples 2-3 and Comparative Examples 1-3, the average particle size, peak value, and PDI of exosomes were significantly increased compared to those of exosomes in Comparative Example 4. In Comparative Example 5, the average particle size and peak value of exosomes were decreased compared to those of exosomes in Comparative Example 4, while the PDI was significantly increased.

[0039] 3. The state of exosomes under electron microscopy after reconstitution. like Figure 4 The images show the electron microscope (EM) images of the reconstituted exosomes of Examples 1–3, Comparative Examples 1–3, Comparative Examples 5, and Comparative Example 4. In Examples 1 and Comparative Example 4, the exosomes retained their intact morphology and exhibited a double-membrane structure, resembling a tea saucer. Examples 2–3 and Comparative Examples 1–3 also exhibited a double-membrane structure, resembling a tea saucer, but the membrane structure was ruptured. In Comparative Example 2, no vesicle structure could be observed under an E microscope after the reconstituted exosome lyophilized powder was dissolved.

[0040] 4. Detection of exosome marker proteins like Figure 5 As shown, Western blot was used to detect the expression of typical exosome marker proteins after rehydration of exosomes in Examples 1–3 and Comparative Examples 1–5. The results showed that CD9, CD63, and CD81 protein bands were detected in all examples and comparative examples, indicating that the lyophilized and rehydrated samples retained exosome-related molecular markers. In particular, the CD9, CD63, and CD81 bands in Example 1 were clear, and the overall expression was similar to that of the fresh exosome group in Comparative Example 4, suggesting that the protective agent in Example 1 can effectively maintain the molecular characteristics of exosomes during the lyophilization and rehydration process. These results further demonstrate that the protective agent of this invention can not only maintain the vesicle morphology of exosomes but also preserve the detectability of their typical exosome marker proteins.

[0041] 5. In vivo delivery efficiency of exosome reconstituted and nebulized drug administration like Figure 6 As shown, this invention reconstitutes lyophilized exosomes into an exosome nebulizer solution, which is then administered via nebulization using a medical compression nebulizer. During nebulization, the exosome nebulizer solution forms aerosol droplets under the influence of airflow, which are then carried into a mouse nebulization induction chamber, allowing the mice to inhale the exosome nebulizer solution through spontaneous respiration. This administration method is simple, non-invasive, and can directly deliver the reconstituted exosomes to the respiratory tract and lungs.

[0042] like Figure 7As shown, the in vivo delivery efficiency of exosomes after lyophilization and reconstitution in Example 1 was evaluated. Compared with intravenous administration of exosomes in Example 1, exosomes administered via nebulization in Example 1 showed a stronger imaging signal in the lung region, suggesting that nebulized inhalation can improve the local delivery efficiency of exosomes in the respiratory tract and lungs. Further quantitative results showed that the signal intensity of the nebulized administration group in Example 1 was significantly higher than that of the intravenous injection group and close to that of the nebulized administration group in Comparative Example 4, indicating that the exosomes treated with the protective agent of the present invention can still maintain good nebulized delivery capability after lyophilization and reconstitution. The above results indicate that the protective agent of Example 1 can not only maintain the stability of exosomes after lyophilization and reconstitution but also ensure their suitability for subsequent nebulized inhalation administration.

[0043] 6. HE staining of mice with radiation-induced lung injury treated by nebulization after exosome reconstitution like Figure 8 The figures show the HE staining results and statistical graphs of mice treated with exosome nebulization for radiation-induced lung injury in Examples 1-3, Comparative Examples 1-3, Comparative Examples 5, and Comparative Example 4. The effects of exosome nebulization treatment in Examples 1 and Comparative Example 4 were similar. However, after exosome nebulization treatment in Examples 2-3, Comparative Examples 1-3, and Comparative Example 5, pathological changes still occurred in the lung tissue, indicating relatively poor treatment efficacy.

[0044] 7. Masson staining of mice with radiation-induced lung injury treated by nebulization after exosome reconstitution. like Figure 9 The figures show Masson staining results and fibrosis area percentage statistics for mice treated with exosome nebulization in Examples 1-3, Comparative Examples 1-3, Comparative Example 5, and Comparative Example 4. The effects of exosome nebulization treatment in Examples 1 and 4 were close to those in the normal group. However, pathological changes were still observed in the lung tissue after exosome nebulization treatment in Examples 2-3, Comparative Examples 1-3, and Comparative Example 5.

[0045] Example 1 showed comparable therapeutic effects to fresh exosomes.

[0046] 8. Evaluation of the storage stability of exosome lyophilized formulations like Figure 10As shown, the exosome activity recovery rates of Examples 1–3 and Comparative Examples 1–5 were tested at different storage times. The results showed that the exosome activity recovery rates of all groups decreased to varying degrees with prolonged storage time. However, the Example 1 group maintained a high recovery rate at 0.5, 1, 3, and 6 months, with a significantly lower decrease than the other formulation groups and the comparative examples. Even at 6 months, the Example 1 group maintained a high exosome recovery level, while the recovery rates of Examples 2–3 and Comparative Examples 1–5 decreased more significantly. These results indicate that the trehalose, inulin, arginine, and Tween 80 composite protection system described in Example 1 can effectively reduce exosome loss during storage and maintain the long-term stability of the lyophilized formulation.

[0047] 9. The reason why trehalose and inulin act as protective agents like Figure 11 , Figure 12 , Figure 13 The figures show the DSC, X-ray diffraction (XRD) patterns, and infrared (IR) spectra of trehalose and inulin, respectively. Trehalose has a glass transition temperature of 117℃, while inulin has a glass transition temperature of 78℃, enabling them to construct a stable glassy matrix that provides excellent protection. The XRD pattern shows that inulin has no obvious crystallization peaks, while trehalose does, which may affect the exosome membrane during freeze-drying. The IR spectra show that both trehalose and inulin can form hydrogen bonds with the exosome membrane, stabilizing exosomes during freeze-drying.

[0048] 10. Selection of Protective Agent Group Based on the above results regarding appearance, particle size distribution, transmission electron microscopy morphology, exosome marker protein expression, material characterization, nebulization delivery efficiency, lung injury treatment effect, and storage stability, the results indicate that the size and particle size of the exosomes after lyophilization and reconstitution in Example 1 are similar to those in Comparative Example 4, and the treatment effect is comparable, making it the most suitable example.

[0049] The key point of this invention is to effectively solve the stability and integrity problems encountered in the preservation of exosome lyophilized powder by exploring different contents of trehalose, inulin, arginine, and Tween 80. This exosome lyophilized composition is suitable for storage in a wide temperature range and can significantly extend the shelf life of exosomes, providing reliable technical support for the long-term preservation and application of exosomes. The exosome lyophilized powder can be stably stored for 6 months at room temperature (25°C), relative humidity (50%), and in the dark. After reconstitution, the activity recovery rate is ≥80%, the particle size does not change significantly, and there is no aggregation. This completely solves the technical problems of traditional liquid exosomes, such as reliance on cold chain, easy inactivation, and inconvenient storage and transportation.

[0050] Existing exosome biological agents often suffer from complex formulations, specialized excipients, stringent preparation processes, and poor mass production stability, limiting their application to small-scale laboratory trials and hindering industrialization. This invention, however, utilizes only conventional pharmaceutical raw materials and excipients listed in the pharmacopoeia, ensuring a safe and compliant formulation, low cost, and readily available raw materials. The overall preparation process is simple, standardized, and features controllable parameters and good reproducibility, enabling standardized automated production. This effectively overcomes the technical bottlenecks of traditional exosome preparations, such as difficulty in large-scale mass production and high industrialization costs, fully meeting the needs of industrialized biopharmaceutical production and market promotion.

[0051] This invention further describes the reconstitution of exosomes after lyophilization using the protective agent in phosphate buffer to prepare a formulation suitable for nebulized administration, which is then applied to the nebulized inhalation therapy for lung diseases. This application method leverages the convenience and high local concentration advantages of nebulized inhalation to significantly improve the delivery efficiency and therapeutic effect of exosomes in treatment, providing a new solution for the non-invasive therapeutic application of exosomes.

[0052] Any aspects not covered in this invention are applicable to existing technologies.

Claims

1. An exosome freeze-drying protectant, characterized in that, It includes the following ingredients: trehalose, inulin, arginine, and Tween 80.

2. The protective agent according to claim 1, characterized in that, The concentration of trehalose in the protective agent solution is 5%-7% (w / v), the concentration of inulin in the protective agent solution is 1%-3% (w / v), the concentration of arginine in the protective agent solution is 0.1%-0.2% (w / v), and the concentration of Tween 80 in the protective agent solution is 0.05%-0.1% (w / v).

3. The protective agent according to claim 2, characterized in that, The concentration of trehalose in the protective agent solution is 6% (w / v), the concentration of inulin in the protective agent solution is 2% (w / v), the concentration of arginine in the protective agent solution is 0.1%-0.2% (w / v), and the concentration of Tween 80 in the protective agent solution is 0.05%-0.1% (w / v).

4. The protective agent according to claim 1, characterized in that, The inulin has a molecular weight of 3.5 to 4.5 kilodaltons; the protective agent also includes a buffer system, which is a phosphate buffer solution with a pH of 6.8 to 7.

8.

5. A lyophilized exosome formulation, characterized in that, It comprises exosomes and the protective agent as described in any one of claims 1-4.

6. A method for preparing the exosome lyophilized formulation according to claim 5, characterized in that, Includes the following steps: S1: The purified exosomes are mixed with the protective agent according to any one of claims 1-4 to obtain a mixture; S2: Pre-freeze the mixture; S3: Freeze-dry the pre-frozen sample to obtain the exosome lyophilized formulation.

7. The preparation method according to claim 6, characterized in that, The pre-freezing process is as follows: pre-freezing at -50℃ for 3 hours; the freeze-drying process is as follows: drying at -20℃ for 15 hours under a vacuum of 15Pa, followed by drying at 22℃ for 8 hours under a vacuum of 8Pa.

8. The use of an exosome lyophilized formulation according to claim 6 or an exosome lyophilized formulation obtained by the preparation method according to claim 6 or 7 in the preparation of a nebulized inhalation drug.

9. The application according to claim 8, characterized in that, The nebulization therapy involves directly delivering a solution formed by reconstituted exosome lyophilized preparation to the respiratory tract or lungs via a nebulizer inhalation device. 0.05% Tween 80 is added to the reconstituted exosome solution, and after thorough mixing, it is aseptically filtered using a 0.22 μm filter membrane to remove impurities and any possible small particles, thus obtaining the exosome nebulization solution.

10. The application according to claim 7, characterized in that, The exosome lyophilized formulation is reconstituted using sterile water for injection or physiological saline and is used for nebulized treatment of radiation-induced lung injury. The reconstituted exosome activity recovery rate is not less than 80%, the particle size is 30–150 nm, and the exosome marker proteins CD9, CD63, and CD81 can be detected.

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