Exosome freeze-drying composition as well as preparation method and application thereof

Abstract

The invention discloses an exosome freeze-drying composition as well as a preparation method and application thereof, and relates to the technical field of biology, the exosome freeze-drying composition comprises the following components: 1 * 10 < 9 >-1 * 10 < 11 > Particle / bottle exosome, 1-20 mM of a buffering agent, 5-10% (w / v) of non-reducing sugar, 5-10% (w / v) of a filling agent, 5-20 mM of amino acid and a solvent; the preparation method of the exosome freeze-drying composition comprises the following steps: preparing liquid: dissolving the exosome, the buffer agent, the non-reducing sugar, the filling agent and the amino acid in the solvent, and uniformly mixing to a constant volume; freeze-drying: putting the prepared solution into a freeze-drying machine for vacuum freeze-drying; the vacuum freeze drying procedure comprises an annealing step; by exploring the components and the dosages, a freeze-drying process curve meeting laboratory type and pilot plant type vacuum freeze-drying is set, the production scale of experimental type and pilot plant type freeze-drying machines is met, and the problems of stability and integrity in the preservation process of exosome freeze-dried powder can be effectively solved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an exosome freeze-dried composition, its preparation method, and its application. Background Technology

[0002] In recent years, with the rapid development of biomedical technology, innovative products based on gene therapy, stem cell therapy, and immune cell therapy have provided unprecedented treatment options for intractable diseases such as cancer and autoimmune diseases. Against this backdrop, extracellular vesicles (EVs), as a natural intercellular communication medium and an excellent drug delivery carrier, have seen a continuous surge in research and development. Compared to artificially synthesized nanocarriers, EVs possess significant biological advantages: they exhibit low immunogenicity, excellent biocompatibility, lower tumorigenicity and embolism risks, and can naturally cross biological barriers. Furthermore, EVs themselves are rich in various bioactive substances such as proteins, lipids, and nucleic acids, making them not only suitable as carriers but also possessing enormous clinical application potential as innovative cell-free therapeutic agents.

[0003] Exosomes are a highly regarded subgroup of EVs, typically ranging in size from 30 to 150 nm and possessing a typical phospholipid bilayer structure. Exosome biogenesis originates from intracellular multivesicular bodies (MVBs), which invaginate the cell membrane and fuse with the plasma membrane before being released into the extracellular environment. As intercellular "messengers," exosomes are taken up by receptor cells through membrane fusion, receptor-mediated endocytosis, or direct signal transduction, releasing their carried miRNAs, mRNAs, enzymes, and signaling proteins, thus playing a crucial role in physiological and pathological processes such as anti-inflammation, immune regulation, tissue repair, and regeneration.

[0004] Despite the promising prospects of exosomes, their industrialization and clinical translation face severe challenges in long-term storage and stability. Currently, the standard preservation method recommended by the International Society for Extracellular Vesicles (ISEV) is to suspend exosomes in PBS buffer and store them at -80°C. However, existing technologies have significant drawbacks: (1) Loss of bioactivity: Long-term ultra-low temperature storage still cannot completely inhibit the physicochemical degradation of exosomes. Over time, the formation and recrystallization of ice crystals may puncture the phospholipid membrane, leading to exosome structural collapse, membrane fusion, aggregation and precipitation, as well as leakage or inactivation of the bioactive substances contained within. (2) Cold chain dependence and high cost: The storage conditions at -80°C place extremely high demands on cold chain logistics. Maintaining a constant ultra-low temperature is extremely difficult and costly during transportation and distribution from the production end to pharmacies, clinics, and even patients. Repeated freeze-thaw cycles will further accelerate the destruction of exosomes, severely limiting their popularization and application as drugs. Therefore, there is an urgent need in this field for a novel preservation method that can break free from cold chain dependence and effectively maintain the morphological integrity and biological activity of exosomes.

[0005] In recent years, lyophilization / freeze-drying technology has become increasingly important in the pharmaceutical industry, and has been widely used in the preparation of monoclonal antibodies, recombinant proteins, vaccines, and gene therapy products. This technology is based on the triple point principle of water. First, the water-containing material is rapidly frozen at low temperatures. Then, under vacuum conditions, the solid ice is directly sublimated into water vapor (primary drying). Finally, bound water is removed through desorption (secondary drying), thereby obtaining a dried product with low water content.

[0006] This process avoids hydrolysis in the presence of liquid water and denaturation caused by high temperatures, thus maximizing the preservation of the physicochemical properties and bioactivity of heat-sensitive bioproducts. Therefore, applying freeze-drying technology to the storage of exosomes is considered a promising technical strategy for solving their stability problems and achieving room-temperature storage and convenient transportation. By constructing a suitable freeze-drying protection system (such as introducing trehalose, sucrose, etc. to form a glassy matrix), the supporting role of water molecules on the exosome membrane structure can be effectively replaced, preventing membrane fusion and protein denaturation during the drying process. This allows for a significant restoration of the original physicochemical properties and functions of the exosomes after reconstitution. Therefore, there is a need to provide an exosome freeze-dried composition, its preparation method, and its applications to meet practical needs. Summary of the Invention

[0007] In view of this, the present invention addresses the deficiencies of the existing technology, and its main objective is to provide an exosome freeze-dried composition, its preparation method, and its application. By exploring different contents of exosomes, histidine, histidine hydrochloride, trehalose, mannitol, and arginine, a freeze-drying process curve is set to meet the requirements of laboratory and pilot-scale vacuum freeze-drying. The excipients used are safe and stable, and the freeze-drying process is guided by key temperature detection, meeting the production scale requirements of experimental and pilot-scale freeze dryers. This effectively solves the stability and integrity problems encountered during the storage of exosome freeze-dried powder.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: An exosome lyophilized composition comprising the following components: 1×10^9-1×10^11 particles / vial of exosomes, 1-20 mM of buffer, 5-10% (w / v) of non-reducing sugar, 5-10% (w / v) of filler, 5-20 mM of amino acids and water for injection.

[0009] As a preferred embodiment: the buffer is selected from one or both of histidine and histidine hydrochloride; the non-reducing sugar is trehalose; the filler is mannitol; the amino acid is arginine; and the solvent is selected from water for injection, phosphate buffer, or physiological saline.

[0010] As a preferred embodiment, the exosome lyophilized composition comprises the following components: 5×10^9-5×10^10 particles / vial of exosomes, 5-15 mM of histidine, 5-15 mM of histidine hydrochloride, 5%-7% (w / v) of trehalose, 8%-10% (w / v) of mannitol, 10-20 mM of arginine, and a solvent.

[0011] As a preferred embodiment: the solvent is water for injection; the exosomes are derived from stem cells, immune cells, plant cell tissues, and body fluids.

[0012] A method for preparing the exosome lyophilized composition includes the following steps: Solution preparation: Dissolve exosomes, buffers, non-reducing sugars, fillers, and amino acids in a solvent, mix well, and bring to a final volume. Freeze-drying: The solution obtained from the preparation is placed in a freeze dryer for vacuum freeze-drying; the vacuum freeze-drying process includes an annealing step.

[0013] As a preferred embodiment, the vacuum freeze-drying process specifically includes: S1. Pre-freezing: Cool to -45℃±5℃ in 0.5-1.5 h, and keep warm for 2-4 h; S2, Annealing: Heat to -15℃±3℃ within 1-3 hours, and hold for 2-5 hours; S3. Secondary pre-freezing: Cool down to -45℃±5℃ again within 1-3 hours and keep warm for 2-4 hours; S4. Sublimation drying (single drying): Heat to -30℃±5℃ within 0.5-1.5 h, control the vacuum degree at 10-30Pa, and dry for 60-80 h; S5. Desorption and drying (secondary drying): The temperature is raised to 25℃±5℃ in stages, and the vacuum degree is controlled at 0-30 Pa. Drying continues until the end.

[0014] As a preferred embodiment, the analytical drying in step S5 specifically includes: First stage: heating to 5℃±5℃ within 1-3 hours, controlling the vacuum degree at 10-30 Pa, and holding for 1-3 hours; Second stage: heating to 25℃±5℃ within 1-3 hours, controlling the vacuum degree at 10-30 Pa, and holding for 1-3 hours; Third stage: maintaining the temperature at 25℃±5℃, reducing the vacuum degree to the ultimate vacuum (0 Pa or close to 0 Pa), and holding for 1-3 hours.

[0015] As a preferred embodiment, the vacuum freeze-drying process specifically includes: S1. Pre-freezing: Cool to -42℃ in 1 hour and keep warm for 3 hours; S2, Annealing: Heat to -15℃ for 3 hours and hold for 4 hours; S3. Secondary pre-freezing: Cool down to -42℃ again after 2 hours and keep warm for 3 hours; S4. Sublimation drying (single drying): Heat to -30℃ for 1 hour, control the vacuum degree at 20 Pa, and dry for 70 hours; S5. Desorption and drying (secondary drying): Heat to 25 ℃ in stages, control the vacuum degree at 0-20 Pa, and dry until the end.

[0016] As a preferred embodiment, the analytical drying in step S5 specifically includes: the first stage: heating to 5°C for 2 hours, controlling the vacuum at 20 Pa, and holding for 2 hours; the second stage: heating to 25°C for 2 hours, controlling the vacuum at 20 Pa, and holding for 2 hours; the third stage: maintaining the temperature at 25°C, reducing the vacuum to the ultimate vacuum (0 Pa or close to 0 Pa), and holding for 2 hours.

[0017] The use of the exosome lyophilized composition described above in the preparation of exosome drugs, biological skin care products or diagnostic reagents.

[0018] Compared with existing technologies, this invention has significant advantages and beneficial effects. Specifically, as can be seen from the above technical solution, by exploring different contents of exosomes, histidine, histidine hydrochloride, trehalose, mannitol, and arginine, a freeze-drying process curve suitable for laboratory and pilot-scale vacuum freeze-drying is established. The excipients used are safe and stable, and the freeze-drying process is guided by key temperature detection, meeting the production scale requirements of experimental and pilot-scale freeze dryers. This effectively solves the stability and integrity problems encountered during the storage of exosome freeze-dried powder. This exosome freeze-dried composition is suitable for storage... It has 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 appearance, residual water content, solubility, pH and osmotic pressure changes, endotoxins, microorganisms, exosome particle size and number, and electron microscopic structure of the exosome freeze-dried composition were evaluated to screen out the appropriate excipient ratio to obtain a good appearance. When reconstituted with water for injection, it dissolves rapidly and forms a clear and transparent liquid. The reconstituted exosomes have intact morphology, and the particle size and number do not change significantly. The pH and osmotic pressure are within a reasonable range.

[0019] To more clearly illustrate the structural features and effects of the present invention, a detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. Attached Figure Description

[0020] Figure 1 These are morphological images of the exosome freeze-dried compositions of Examples 1-5 and Comparative Examples 1-11 of the present invention. Figure 2 The graph shows the results of nanoparticle size tracking analysis (NTA) of the exosomes before and after freeze-drying and after 6 and 12 months of storage at room temperature in Examples 1-5 and Comparative Examples 1-11 of the present invention to detect the exosome particle retention rate. Figure 3 The graph shows the NTA detection results of exosome particle size before and after freeze-drying and after storage at room temperature for 6 and 12 months in Examples 1-5 and Comparative Examples 1-11 of the present invention. Figure 4 These are morphological images of exosomes before and after freeze-drying, and after being stored at room temperature for 6 and 12 months, as described in Examples 1-5 of the present invention, under transmission electron microscopy (TEM). Figure 5 The images show rat skin photographs and HE staining results from the skin irritation test of the lyophilized composition of Example 1 of this invention after reconstitution. Figure 6 The image shows the in vitro hemolytic activity test results of the lyophilized powder after reconstitution in Example 1 of this invention. Figure 7The results of HE staining in a rat repeated-dose toxicity test after reconstitution of the lyophilized powder in Example 1 of this invention are shown. Detailed Implementation

[0021] The present invention is as follows Figure 1 As shown in Figure 7, an exosome lyophilized composition comprises the following components: 1×10^9-1×10^11 particles / vial of exosomes, 1-20 mM of buffer, 5-10% (w / v) of non-reducing sugar, 5-10% (w / v) of filler, 5-20 mM of amino acids and solvent.

[0022] The buffer is selected from one or both of histidine and histidine hydrochloride; the non-reducing sugar is trehalose; the filler is mannitol; the amino acid is arginine; and the solvent is selected from water for injection, phosphate buffer, or physiological saline.

[0023] The exosome lyophilized composition comprises the following components: 5×10^9-5×10^10 particles / vial of exosomes, 5-15 mM of histidine, 5-15 mM of histidine hydrochloride, 5%-7% (w / v) of trehalose, 8%-10% (w / v) of mannitol, 10-20 mM of arginine, and water for injection.

[0024] The solvent is water for injection; the exosomes are derived from stem cells, immune cells, plant cell tissues, and body fluids.

[0025] A method for preparing a lyophilized exosome composition includes the following steps: Solution preparation: Dissolve exosomes, buffers, non-reducing sugars, fillers, and amino acids in a solvent, mix well, and bring to a final volume. Freeze-drying: The solution obtained from the preparation is placed in a freeze dryer for vacuum freeze-drying; the vacuum freeze-drying process includes an annealing step.

[0026] The specific process of vacuum freeze drying is as follows: S1. Pre-freezing: Cool to -45℃±5℃ in 0.5-1.5 h, and keep warm for 2-4 h; S2, Annealing: Heat to -15℃±3℃ within 1-3 hours, and hold for 2-5 hours; S3. Secondary pre-freezing: Cool down to -45℃±5℃ again within 1-3 hours and keep warm for 2-4 hours; S4. Sublimation drying (single drying): Heat to -30℃±5℃ within 0.5-1.5 h, control the vacuum degree at 10-30Pa, and dry for 60-80 h; S5. Desorption and drying (secondary drying): The temperature is raised to 25℃±5℃ in stages, and the vacuum degree is controlled at 0-30 Pa. Drying continues until the end.

[0027] The specific steps of the drying process in step S5 are as follows: First stage: within 1-3 hours, the temperature is raised to 5℃±5℃, the vacuum degree is controlled at 10-30 Pa, and the temperature is maintained for 1-3 hours; Second stage: within 1-3 hours, the temperature is raised to 25℃±5℃, the vacuum degree is 10-30 Pa, and the temperature is maintained for 1-3 hours; Third stage: the temperature is maintained at 25℃±5℃, the vacuum degree is reduced to the ultimate vacuum (0 Pa or close to 0 Pa), and the temperature is maintained for 1-3 hours.

[0028] The specific process of vacuum freeze drying is as follows: S1. Pre-freezing: Cool to -42℃ in 1 hour and keep warm for 3 hours; S2, Annealing: Heat to -15℃ for 3 hours and hold for 4 hours; S3. Secondary pre-freezing: Cool down to -42℃ again after 2 hours and keep warm for 3 hours; S4. Sublimation drying (single drying): Heat to -30℃ for 1 hour, control the vacuum degree at 20 Pa, and dry for 70 hours; S5. Desorption and drying (secondary drying): Heat to 25 ℃ in stages, control the vacuum degree at 0-20 Pa, and dry until the end.

[0029] The specific steps of the drying process in step S5 are as follows: First stage: heat up to 5°C for 2 hours, control the vacuum at 20 Pa, and hold for 2 hours; Second stage: heat up to 25°C for 2 hours, control the vacuum at 20 Pa, and hold for 2 hours; Third stage: maintain the temperature at 25°C, reduce the vacuum to the ultimate vacuum (0 Pa or close to 0 Pa), and hold for 2 hours.

[0030] The use of an exosome lyophilized composition in the preparation of exosome drugs, biological skin care products or diagnostic reagents.

[0031] Histidine / histidine hydrochloride plays multiple roles in lyophilized formulations, including pH buffering, protein conformational stabilization, antioxidation, and viscosity reduction. As a buffer, histidine's imidazole side chain with a pKa of approximately 6.0 provides excellent buffering capacity within a pH range of 5.5-7.0, effectively maintaining the pH stability of the solution before lyophilization and preventing protein denaturation due to pH fluctuations. Simultaneously, histidine binds to protein molecules through non-covalent interactions (such as hydrogen bonds), stabilizing their conformation, particularly inhibiting hydrolysis of antibody hinge regions and reducing aggregation tendency. Histidine hydrochloride plays a crucial role in the lyophilization process; its high solubility helps form an amorphous glassy matrix, encapsulating and isolating protein molecules and preventing crystallization damage.

[0032] Trehalose, as a non-reducing sugar, protects proteins through two main mechanisms: First, its unique spatial structure makes it less prone to forming intramolecular hydrogen bonds, and more inclined to form intermolecular hydrogen bonds with water molecules. This "preferential hydration" characteristic reduces direct competition between proteins and water molecules, lowering the risk of protein aggregation. Second, during freeze-drying, trehalose forms an amorphous glassy matrix, which, through its high viscosity (10... 12 Pa·s Restricting the movement of protein molecules slows down conformational transitions and relaxation, thereby maintaining the structure and function of the molecules.

[0033] Mannitol, as a crystalline filler and scaffold forming agent, effectively prevents powder collapse and remelting during freeze-drying by forming a stable crystalline matrix, ensuring the formulation possesses good appearance, mechanical strength, and rapid reconstitution characteristics. Its high eutectic temperature and crystallization tendency can synergistically work with amorphous protectants to optimize freeze-drying process efficiency and maintain the long-term physical stability of the active ingredient. Studies have shown that when mannitol is appropriately combined with other excipients such as trehalose, it can provide good structural support without affecting the glassy protective function of trehalose, achieving an optimal balance between physical form and functionality.

[0034] Arginine plays a major role in lyophilized formulations by reducing viscosity, stabilizing, and protecting the protein during lyophilization. As a basic amino acid (positively charged), arginine can reduce the viscosity of high-concentration protein solutions and improve lyophilization efficiency by weakening electrostatic repulsion and hydrophobic interactions between proteins. During lyophilization, arginine forms an amorphous glassy matrix, maintaining the protein hydration layer through preferential hydration and preventing conformational changes caused by dehydration.

[0035] The excipients in the freeze-dried formulation of this invention form a multiple protective mechanism through synergistic effects: histidine and trehalose together form an amorphous glassy matrix, inhibiting protein aggregation through hydrogen bonding and steric hindrance, respectively. Arginine and histidine reduce the surface charge attraction of proteins through charge complementarity, further inhibiting aggregation; both also reduce the viscosity of the protein solution, optimizing freeze-drying efficiency. Mannitol and trehalose complement each other through physical morphology to form a stable freeze-dried structure: mannitol crystals provide mechanical support, while the amorphous form of trehalose provides a protective matrix; their reasonable ratio balances structural strength and protective effect, ensuring smooth freeze-drying and the formation of a uniform and stable powder cake.

[0036] This application explores different amounts of exosomes, histidine, histidine hydrochloride, trehalose, mannitol, arginine, and solvents. After vacuum freeze-drying, the morphology, residual water content, solubility, pH and osmotic pressure changes, endotoxin content, microbial exosome particle size and number, and electron microscopic structure of the exosome lyophilized composition are evaluated. Suitable excipient ratios are selected to obtain a good appearance. When reconstituted with water for injection, it dissolves rapidly to form a clear and transparent liquid. The reconstituted exosomes maintain their morphology, and their particle size and number do not change significantly. The pH and osmotic pressure remain within a reasonable range. Furthermore, the safety of the preferred formulation is evaluated through multi-dimensional safety analysis of the exosome lyophilized preparation, including rat skin irritation tests, active anaphylaxis tests, hemolytic tests, and repeated-dose toxicity tests.

[0037] The freeze-drying process conditions set in this invention are suitable for laboratory and pilot-scale vacuum freeze dryers, and can operate at half or full load. This invention, through a scientifically sound technical solution, develops an exosome freeze-dried composition, its preparation method, and its applications. The excipients used are safe and stable, and the freeze-drying process is guided by key temperature detection, meeting the requirements for half or full load production scale in experimental and pilot-scale freeze dryers. It effectively solves the stability and integrity issues encountered during the storage of exosome freeze-dried powder. This exosome freeze-dried composition is suitable for storage over 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.

[0038] Examples 1-5: Table 1: Components and dosages of the lyophilized compositions of Examples 1-5 and Comparative Examples 1-11

[0039] Based on the component dosage design in Table 1, Examples 1-5 used water for injection to dissolve each excipient component, added exosomes to each formulation to form an exosome lyophilized liquid composition, and dispensed it into vials, each vial containing 2 mL of liquid and 1*10^10 exosome particles.

[0040] Comparative Examples 1-11: According to the formulation design in Table 1, Comparative Examples 1-6 and 7-11 dissolved each excipient component in PBS and physiological saline, respectively. Exosomes were added to each formulation to form a lyophilized exosome composition liquid, which was then dispensed into vials, each containing 2 mL of liquid and 1*10^10 exosome particles. Comparative Example 1 was an exosome solution preserved only in PBS.

[0041] Table 2: Freeze-drying process procedure settings

[0042] The exosome lyophilized composition of the above prescription was lyophilized according to the vacuum freeze-drying process in Table 2, including the following steps: S1. Pre-freezing: After placing the vial containing the exosome lyophilized composition into the lyophilizer, cool it to -42°C within 1 hour and keep it frozen for 3 hours. S2, Annealing: Raise the freezing temperature to -15°C within 3 hours and keep it frozen for 4 hours; S3, Secondary Pre-freezing: Lower the freezing temperature to -42℃ within 2 hours and keep it frozen for 3 hours; S4. Sublimation drying (single drying): The temperature is raised to -30℃ within 1 h, the vacuum degree is maintained at 20 Pa, and the sublimation drying is continued under these conditions for 70 h. S5. Desorption and drying (secondary drying): Within 2 hours, the temperature is raised to 5°C, the vacuum degree is maintained at 20 Pa, and desorption and drying are continued under these conditions for 2 hours; then, within 2 hours, the temperature is raised to 25°C, the vacuum degree is maintained at 20 Pa, and desorption and drying are continued under these conditions for 2 hours; finally, at 25°C, the vacuum degree is reduced to 0 Pa (or close to 0 Pa), and desorption and drying are continued under these conditions for 2 hours.

[0043] Effect test 1. Appearance of the exosome lyophilized composition like Figure 1 The appearance of the exosome lyophilized compositions in Examples 1-5 and Comparative Examples 1-11 is shown below. In Examples 1-5, when the exosome lyophilized compositions were prepared with water for injection, the exosome lyophilized powder exhibited a complete cake-like structure with uniform texture and high mechanical strength. In Comparative Example 1, only PBS was used as the preservation solution without adding any protective agent, and the lyophilized powder showed melting and a granular appearance. In Comparative Examples 2-6 and 7-11, PBS and physiological saline were used to prepare the exosome lyophilized compositions, respectively, and the exosome lyophilized powder showed texture separation and bottom collapse.

[0044] 2. Residual water content and solubility of the exosome lyophilized composition Table 3: Residual water content and dissolution time of exosome lyophilized compositions

[0045] As shown in Table 3, the residual water content and dissolution time with water for injection of Examples 1-5 and Comparative Examples 1-11 are as follows: the residual water content of Examples 1-5 is ≤3.00%, while only some samples of Comparative Examples 1-11 have a residual water content ≤3.00%; Examples 1-5 and Comparative Examples 1-11 can all be rapidly dissolved in water for injection.

[0046] 3. Changes in pH and osmotic pressure before and after lyophilization of the exosome lyophilized composition. Table 4: Changes in pH and osmotic pressure of the exosome lyophilized composition before and after lyophilization

[0047] Table 4 shows the changes in pH and osmotic pressure before and after freeze-drying in Examples 1-5 and Comparative Examples 1-11. The pH changes before and after freeze-drying in Examples 1-5 were all within the normal range, while the pH of Comparative Examples 1-11 showed more severe drift. The osmotic pressure before and after freeze-drying in Examples 1-5 was less than 500 mOsmol / kg, while the osmotic pressure before and after freeze-drying in Comparative Examples 2-11 exceeded 700 mOsmol / kg. Hypertonic solutions may cause cell damage, affect drug distribution, and may cause pain to patients during use, thereby affecting patient compliance.

[0048] 4. Results of endotoxin and microbial detection before and after lyophilization of exosome lyophilized compositions Table 5: Results of endotoxin and microbial detection of exosome lyophilized compositions before and after lyophilization

[0049] As shown in Table 5, the endotoxin and microbial detection results of Examples 1-5 and Comparative Examples 1-11 before and after freeze-drying were all <0.083 EU / mL, which met the requirements of the Chinese Pharmacopoeia. In addition, the microbial culture tests of Examples 1-5 and Comparative Examples 1-11 before and after freeze-drying were all negative.

[0050] 5. Retention rate of exosome lyophilized composition after reconstitution at 0 days, 6 days, and 12 months. like Figure 2The retention rate of exosomes was determined by nanoparticle size tracking analysis (NTA) before and after lyophilization and after 6 and 12 months of storage at room temperature in Examples 1-5 and Comparative Examples 1-11. The results showed that, compared with the exosome compositions of Examples 1-5 before lyophilization, the number of exosome particles did not change significantly after dissolving the lyophilized exosome compositions with water for injection on day 0, 6, and 12 months after lyophilization. However, the number of particles in the lyophilized exosome compositions of Comparative Examples 1-11 showed significant degradation after dissolution.

[0051] 6. Particle size changes of the exosome lyophilized composition at 0 days, 6 months and 12 months after reconstitution like Figure 3 The results of NTA analysis of exosome particle size in Examples 1-5 and Comparative Examples 1-11 before and after lyophilization, and after 6 and 12 months of storage at room temperature, are shown. The results indicate that, compared to the exosome compositions of Examples 1-5 before lyophilization, the particle size of the exosomes did not change significantly after dissolving the lyophilized exosome compositions with water for injection on day 0, 6, and 12 months after lyophilization; however, the particle size of the exosome lyophilized compositions of Comparative Examples 1-11 increased significantly after dissolution.

[0052] 7. Electron microscopy morphology of exosome lyophilized compositions at 0 days, 6 months and 12 months after reconstitution like Figure 4 The images show the morphological structures of exosomes before and after lyophilization and after one month of storage at room temperature in Examples 1-5. The results indicate that, compared to the exosome compositions of Examples 1-5 before lyophilization, the exosomes retained their intact morphology and exhibited a double-membrane structure, resembling a saucer, after being dissolved with water for injection on day 0, 6, and 12 months after lyophilization. In contrast, no vesicle structures were observed under electron microscopy after reconstitution of the exosome lyophilized compositions of Comparative Examples 1-11.

[0053] 8. Skin irritation test and HE staining in rats after reconstitution of the exosome lyophilized composition of Example 1 like Figure 5The results of skin irritation test and HE staining of rats after reconstitution of the exosome lyophilized composition of Example 1 are shown. Twelve SD rats were randomly divided into three groups: (1) blank control group: the back of the rats was shaved and no operation was performed; (2) water for injection group: the back of the rats was shaved and 0.1 mL of water for injection was injected and applied to the upper and lower backs respectively; (3) exosome lyophilized composition group: the back of the rats was shaved and 0.1 mL of the reconstituted exosome lyophilized composition was injected and applied to the upper and lower backs respectively. The results showed that, compared with the blank control group and the water for injection group, the reconstituted exosome lyophilized composition of Example 1 did not cause any inflammatory reaction or allergic reaction when applied to the skin of rats after application and injection. HE staining results showed that no pathological changes occurred in the skin tissue of rats after application and injection. These results prove that the formulation of Example 1 and the exosome lyophilized composition produced after vacuum freeze-drying do not have skin irritation.

[0054] 9. Active hypersensitivity test in guinea pigs after reconstitution of the exosome lyophilized composition of Example 1 Nine adult white guinea pigs were randomly divided into three groups: (1) Negative control group: during the sensitization phase, 0.5 mL of water for injection was administered intraperitoneally every other day for a total of three times; during the challenge phase, 1 mL of water for injection was administered intravenously 14 days after the last sensitization. (2) Positive control group: during the sensitization phase, 0.5 mL of 1% bovine serum albumin was administered intraperitoneally every other day for a total of three times; during the challenge phase, 1 mL of 1% bovine serum albumin was administered intravenously 14 days after the last sensitization. (3) Exosome lyophilized composition group: during the sensitization phase, 0.5 mL of the reconstituted solution of the lyophilized composition of Example 1 was administered intraperitoneally every other day for a total of three times; during the challenge phase, 1 mL of the reconstituted solution of the lyophilized composition of Example 1 was administered intravenously 14 days after the last sensitization. The guinea pigs' reactions were observed immediately after the challenge, lasting from 30 minutes to 3 hours, and symptoms such as restlessness, piloerection, trembling, sneezing, difficulty breathing, convulsions, and death were recorded. The severity of the allergic reaction was determined according to the severity of symptoms in Table 6. Table 7 shows the results of the guinea pig active allergy test. No abnormal reactions were observed in any guinea pigs during clinical observation before challenge. After intravenous injection of samples from each group, no allergic reaction symptoms were observed in the three animals in the negative control group, indicating a negative allergic reaction. In the positive control group, three animals died, indicating a strongly positive to extremely strongly positive allergic reaction. In the case of the three animals intravenously injected with the reconstituted exosome lyophilized composition from Example 1, no allergic reaction symptoms were observed, indicating a negative allergic reaction.

[0055] Table 6: Evaluation Table of Active Hypersensitivity in Guinea Pigs

[0056] Table 7: Results of the active hypersensitivity test in guinea pigs

[0057] 10. In vitro hemolytic test of the reconstituted exosome lyophilized composition of Example 1 like Figure 6 The results of the in vitro hemolytic activity test of the lyophilized powder of Example 1 after reconstitution are shown. Blood from healthy rabbits was used to prepare a 2% erythrocyte suspension. Six centrifuge tubes were used, and the reconstituted liquid of the lyophilized composition of Example 1 (tubes 5 and 6), physiological saline (tubes 7 and 8), and water for injection (tubes 9 and 10) were added sequentially. The tubes were placed in a 37°C incubator. Incubation was performed at different time points: 0 min, 15 min, 30 min, 45 min, 1 h, 2 h, and 3 h. After incubation, the tubes were shaken and centrifuged. Visual observation and recording of hemolysis were performed, and photographs were taken. Figure 6 The test results showed that the solution in the water for injection test tube was clear red with no cell residue at the bottom, indicating hemolysis. However, in the test tubes of the reconstituted lyophilized composition of Example 1 and the physiological saline, all red blood cells sank to the bottom, and the supernatant was colorless and clear, indicating that no hemolysis occurred.

[0058] 11. Repeated-dose toxicity test in rats after reconstitution of the exosome lyophilized composition of Example 1 like Figure 7 The results of HE staining in the repeated-dose toxicity test of the lyophilized powder reconstituted in Example 1 are shown. Six SD rats were randomly divided into two groups for a 28-day repeated-dose toxicity test (with a 14-day recovery period). The animal groups were: (1) Blank control group: 0.5 mL of water for injection was injected intravenously, and the drug was repeated for 14 days, once a day; (2) Reconstituted lyophilized composition of Example 1 group: 0.5 mL of the reconstituted lyophilized composition of Example 1 was injected intravenously, and the drug was repeated for 14 days, once a day. After the 28-day observation period, tissue samples from the heart, liver, spleen, lungs, and kidneys of the rats were collected and HE stained for analysis. Figure 7 The results showed that, compared with the blank control group, repeated administration of the lyophilized composition of Example 1 to rats after reconstitution did not cause pathological changes in tissues, and there was no significant difference.

[0059] The key design focus of this invention is to establish freeze-drying process curves suitable for laboratory and pilot-scale vacuum freeze-drying by exploring different contents of exosomes, histidine, histidine hydrochloride, trehalose, mannitol, and arginine. The excipients used are safe and stable, and the freeze-drying process is guided by key temperature monitoring, meeting the production scale requirements of experimental and pilot-scale freeze dryers. This effectively solves the stability and integrity issues encountered during the storage of exosome freeze-dried powder. This exosome freeze-dried composition is suitable for storage over 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 invention evaluates parameters such as the appearance, residual water content, solubility, pH and osmotic pressure changes, endotoxins, microorganisms, exosome particle size and number, and electron microscopy structure of the exosome freeze-dried composition, screening out suitable excipient ratios to obtain a good appearance. When reconstituted with water for injection, it dissolves rapidly to form a clear and transparent liquid, and the reconstituted exosomes retain their intact morphology, with no significant changes in particle size and number, and the pH and osmotic pressure remain within a reasonable range.

[0060] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A lyophilized exosome composition, characterized in that: It includes the following components: 1×10^9-1×10^11 particles / bottle of exosomes, 1-20 mM of buffer, 5-10% (w / v) of non-reducing sugar, 5-10% (w / v) of filler, 5-20 mM of amino acids and solvent.

2. The exosome lyophilized composition according to claim 1, characterized in that: The buffer is selected from one or both of histidine and histidine hydrochloride; the non-reducing sugar is trehalose; the filler is mannitol; the amino acid is arginine; and the solvent is selected from water for injection, phosphate buffer, or physiological saline.

3. The exosome lyophilized composition according to claim 2, characterized in that: It includes the following components: 5×10^9-5×10^10 particles / vial of exosomes, 5-15 mM of histidine, 5-15 mM of histidine hydrochloride, 5%-7% (w / v) of trehalose, 8%-10% (w / v) of mannitol, 10-20 mM of arginine, and water for injection.

4. The exosome lyophilized composition according to claim 2, characterized in that: The solvent is water for injection; the exosomes are derived from stem cells, immune cells, plant cell tissues, and body fluids.

5. A method for preparing an exosome lyophilized composition as described in any one of claims 1-4, characterized in that: Includes the following steps: Solution preparation: Dissolve exosomes, buffers, non-reducing sugars, fillers, and amino acids in a solvent, mix well, and bring to a final volume. Freeze-drying: The solution obtained from the preparation is placed in a freeze dryer for vacuum freeze-drying; the vacuum freeze-drying process includes an annealing step.

6. The preparation method according to claim 5, characterized in that: The vacuum freeze-drying process is specifically as follows: S1. Pre-freezing: Cool to -45℃±5℃ in 0.5-1.5 h, and keep warm for 2-4 h; S2, Annealing: Heat to -15℃±3℃ within 1-3 hours, and hold for 2-5 hours; S3. Secondary pre-freezing: Cool down to -45℃±5℃ again within 1-3 hours and keep warm for 2-4 hours; S4. Sublimation drying: Heat to -30℃±5℃ within 0.5-1.5 h, control the vacuum degree at 10-30 Pa, and dry for 60-80 h; S5. Desorption and drying: Heat the temperature in stages to 25℃±5℃, control the vacuum degree at 0-30 Pa, and continue drying until the end.

7. The preparation method according to claim 6, characterized in that: The desorption and drying process in step S5 specifically includes: the first stage: heating to 5℃±5℃ within 1-3 hours, controlling the vacuum at 10-30 Pa, and holding for 1-3 hours; the second stage: heating to 25℃±5℃ within 1-3 hours, controlling the vacuum at 10-30 Pa, and holding for 1-3 hours; the third stage: maintaining the temperature at 25℃±5℃, reducing the vacuum to the ultimate vacuum, and holding for 1-3 hours.

8. The preparation method according to claim 6, characterized in that: The vacuum freeze-drying process is specifically as follows: S1. Pre-freezing: Cool to -42℃ in 1 hour and keep warm for 3 hours; S2, Annealing: Heat to -15℃ for 3 hours and hold for 4 hours; S3. Secondary pre-freezing: Cool down to -42℃ again after 2 hours and keep warm for 3 hours; S4. Sublimation drying: Heat to -30℃ in 1 h, control the vacuum degree at 20 Pa, and dry for 70 h; S5. Desorption and drying: Heat to 25 ℃ in stages, control the vacuum degree at 0-20 Pa, and dry until the end.

9. The preparation method according to claim 8, characterized in that: The analytical drying process in step S5 specifically includes: the first stage: heating to 5°C for 2 hours, controlling the vacuum at 20 Pa, and holding for 2 hours; the second stage: heating to 25°C for 2 hours, controlling the vacuum at 20 Pa, and holding for 2 hours; the third stage: maintaining the temperature at 25°C, reducing the vacuum to the ultimate vacuum, and holding for 2 hours.

10. The use of an exosome lyophilized composition as described in any one of claims 1-4 in the preparation of exosome pharmaceuticals, biological skin care products, or diagnostic reagents.

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