An exosome lyophilized composition and a method for preparing the same

Abstract

The application discloses an exosome freeze-drying composition and a preparation method thereof, relates to the field of pharmaceutical preparations, and comprises the following components in parts by mass: 1E9 particles exosome, 1-2 parts of PEG2000 modified beta-sitosterol, 2-4 parts of cholesterol, 20-30 parts of HSA, 30-40 parts of trehalose and 0.5-1.5 parts of poloxamer 188. The preparation method comprises the following steps: step one, preparation of a lipid film; step two, hydration of the liposome; step three, fusion with the exosome; step four, preparation of a pre-freeze-drying suspension; and step five, freeze-drying and sub-packaging. The formula and the preparation method can effectively protect the structural integrity of the exosome in the freeze-drying process and improve the stability of the exosome.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparations, and in particular to a lyophilized exosome composition. Background Technology

[0002] Exosomes are a type of extracellular vesicle (EV), and the smallest of them. EVs are extracellular bodies containing progenitor components, secreted by almost all cell types and originating from a lipid bilayer membrane structure formed during endocytosis. Based on their size, origin, and function, EVs can be classified into three types: exosomes (30-150 nm), ectosomes (100 nm-1 μm), and apoptotic bodies (50 nm-5 μm). Structurally, exosomes have a typical lipid bilayer membrane structure surrounding a water-soluble medium containing various biological molecules, including nucleic acids, peptides, lipids, and proteins. Currently, nearly 100,000 proteins and over 1,000 lipids have been recorded in exosomes. These components play important roles in intercellular and intracellular communication, including transmitting genetic information and regulating physiological functions. Exosomes are found in various biological fluids, including blood, urine, saliva, breast milk, amniotic fluid, synovium, cerebrospinal fluid, and tears. The function of exosomes depends on the cell type from which they originate, and they can participate in the body's immune response, antigen presentation, cell migration, cell differentiation, and tumor invasion.

[0003] Existing technologies employ composite preservatives such as combinations of platelet-rich factors, albumin, glycine, lysine, glycerol, gelatin hydrolysate, arginine, sucrose, sorbitol, and ceramides. However, these lyophilization preservatives cannot effectively guarantee the integrity and biological function of exosomes before and after lyophilization, and still have shortcomings.

[0004] The market needs an exosome lyophilization composition that can protect the stability of the exosome lipid bilayer membrane structure during low-temperature lyophilization, and this invention solves this problem. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an exosome freeze-dried composition. The formulation and preparation method of this invention can effectively protect the structural integrity of exosomes during the freeze-drying process and improve their stability.

[0006] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution: A lyophilized exosome composition comprising, by weight parts: 1E9 particles exosomes, 1-2 parts PEG2000 modified β-sitosterol, 2-4 parts cholesterol, 20-30 parts HSA, 30-40 parts trehalose, and 0.5-1.5 parts poloxamer 188.

[0007] The aforementioned exosome lyophilized composition is formulated as follows: 1E9 particles exosomes, 1.5 mg PEG2000 modified β-sitosterol, 3 mg cholesterol, 25.0 mg HSA, 34.5 mg trehalose, and 1.0 mg poloxamer 188.

[0008] The aforementioned method for preparing an exosome lyophilized composition includes the following steps: Step 1, preparation of lipid film: Dissolve the weighed PEG2000 modified β-sitosterol and cholesterol in chloroform, transfer the solution to a round-bottom flask, and use a rotary evaporator under reduced pressure to remove the organic solvent, so that the lipid forms a uniform film on the flask wall. Continue to vacuum dry the flask overnight to completely remove the residual organic solvent. Step 2, hydration of liposomes: Add an appropriate amount of preheated PBS buffer or purified water to the flask, heat and rotate for 30 minutes to hydrate the lipid film off the flask wall, forming a crude liposome suspension; use a liposome extruder to repeatedly extrude the suspension to finally obtain liposomes; Step 3, Fusion with exosomes: The prepared liposomes and 1E9 particles of exosomes are incubated together at 37°C for 1 day to allow the liposome membrane to fuse with the exosome membrane, forming an exosome-liposome complex suspension; Step 4, prepare the suspension before lyophilization: Dissolve the weighed HSA, trehalose, and poloxamer 188 in water for injection, and stir thoroughly until completely dissolved to obtain a protective agent solution. Gently and thoroughly mix the exosome-liposome complex suspension with the protective agent solution. Step 5: freeze-drying and packaging.

[0009] The aforementioned preparation method includes the following freeze-drying steps: Pre-freezing: Temperature: -5℃, -45℃, -8℃, -45℃; Set time: 10min, 120min, 30min, 60min; Holding time: 30min, 60min, 90min, 10min; First drying: Temperature: -32℃; Set time: 90min; Holding time: 2700min; Vacuum degree: 0.15 mbar; Second drying: Temperature: 30℃, 30℃; Set time: 240℃, 1min; Holding time: 120℃, 240min; Vacuum degree: 0.20℃, 0.01 mbar.

[0010] The aforementioned exosome lyophilized composition is packaged as follows: the filling volume is 0.50 ml / bottle, and a 13 mm rubber stopper is added after filling.

[0011] The advantages of this invention are: Cholesterol is responsible for reinforcing the membrane structure, while PEG lipids are responsible for isolating interparticle interactions. When used in a suitable ratio, they can achieve a stable state that is both "rigid inside and flexible outside." Their mechanisms for membrane stability are complementary, providing both internal and external protection: preventing leakage internally and preventing sticking externally; preserving the membrane's shape while promoting resolubility. This invention utilizes a thin-film hydration method to prepare liposomes from PEG2000-modified β-sitosterol and cholesterol. After fusing with exosomes, these liposomes are mixed with a lyophilization protectant composed of HSA, trehalose, and poloxamer 188, and finally freeze-dried. This combination effectively protects the structural integrity of exosomes during the freeze-drying process and enhances their stability.

[0012] Definition of noun: HSA: Human serum albumin (HSA) is the main protein in human plasma, with a molecular weight of 66 kDa, composed of 585 amino acids, and accounting for approximately 60% of total plasma protein. During freezing and dehydration, the lipid bilayer membrane of exosomes is easily damaged. As a macromolecule, HSA can replace water molecules to form hydrogen bonds with the lipid membrane, maintaining membrane integrity and preventing fusion or rupture, thus protecting the membrane structure. The freeze-drying process may cause exosomes to irreversibly aggregate. HSA can separate exosome particles through steric hindrance, thereby improving the dispersibility and consistency of the reconstituted freeze-dried formulation. During freeze-drying, HSA can form an amorphous glassy matrix that encapsulates the exosomes. This state greatly restricts the molecular movement of exosomes, thereby slowing down their degradation and inactivation rates.

[0013] Trehalose is a non-reducing disaccharide that protects exosomes during freeze-drying through the following key mechanisms.

[0014] Glassy Embedding and Structural Fixation: Trehalose possesses a high glass transition temperature. During the drying stage, it forms a glass-like amorphous structure that tightly encapsulates exosomes. This "glassy" matrix significantly restricts the molecular movement of exosome membrane lipids and proteins, effectively preventing structural collapse and bioactive degradation. During freezing and dehydration, the "hydration layer" surrounding exosome membrane proteins is removed, potentially leading to protein denaturation. Trehalose's hydroxyl groups can form hydrogen bonds with the polar heads of membrane phospholipids and the hydrophilic groups of membrane proteins, effectively "replacing" lost water molecules and maintaining the natural structure and function of these biomolecules. During freezing, trehalose can influence the growth morphology and size distribution of ice crystals, making them smaller and more uniform, thereby reducing mechanical damage to the exosome membrane caused by large ice crystals.

[0015] Poloxamer 188 is a triblock copolymer and a nonionic surfactant. The ice-water interface during freeze-drying has high surface energy, which can cause severe interfacial stress on exosomes adsorbed there, leading to structural damage. As a surfactant, poloxamer 188 preferentially occupies these ice-water interfaces, "displacing" exosomes from this dangerous area, thus effectively preventing interface-induced protein denaturation and aggregation. In freeze-dried cakes, poloxamer 188 can act as a filler or framework agent, helping to form structurally intact and aesthetically pleasing freeze-dried cakes and preventing collapse. Poloxamer 188 alone is not an effective freeze-drying protectant; it must be used in combination with sugars such as trehalose to achieve optimal protective effects. Simultaneously, the presence of sugars (such as trehalose) can inhibit the crystallization of poloxamer 188 during freeze-drying, ensuring its continued and effective interfacial protection function.

[0016] The synergistic stabilizing effect of cholesterol and PEG2000-β-sitosterol in lipid membrane modification stems from their complementary roles in molecular mechanisms: cholesterol is responsible for reinforcing the membrane structure, while PEG lipids are responsible for isolating interparticle interactions. When used in a suitable ratio, they can achieve a stable state that is both "rigid inside and flexible outside."

[0017] Cholesterol is a derivative of cyclopentanoperhydrophenanthrene. Its chemical formula is C63-C62 ... 27 H 46 O, a white or pale yellow crystal; the rigid steroidal ring of cholesterol inserts into the hydrophobic region of phospholipids (or exosome membranes), reducing membrane fluidity and increasing bilayer thickness above the phase transition temperature, preventing membrane fusion and rupture; below the phase transition temperature, it inhibits crystallization, avoiding phase separation and leakage of contents. It promotes the formation of a "liquid ordered phase": at appropriate ratios (usually molar ratio ≤50%), cholesterol and saturated phospholipids form a dense, ordered liquid phase, endowing the membrane with high mechanical strength and low permeability, significantly reducing vesicle rupture during the freeze-drying-reconstitution process.

[0018] β-Sitosterol is an organic compound with the molecular formula C0. 29 H 50 O is a white crystalline solid. PEG2000-β-sitosterol is coupled to PEG2000 via succinic anhydride as a linker, forming an ester bond structure. The specific synthesis was completed by Aivitol (Shanghai) Pharmaceutical. β-sitosterol can be firmly embedded in the lipid bilayer (less prone to detachment compared to DSPE anchors), extending the hydrophilic PEG2000 long chain to the particle surface. The PEG chain forms a dense hydration layer ("conformation cloud") on the surface. When two particles approach each other, the PEG chain compresses, generating repulsive osmotic pressure, preventing irreversible aggregation during lyophilization concentration and reconstitution. Detailed Implementation

[0019] The present invention will be described in detail below with reference to specific embodiments.

[0020] Example 1: The formulation is as follows: 1E9 particles exosomes, 1mg PEG2000 modified β-sitosterol, 2mg cholesterol, 20mg HSA, 30mg trehalose, and 0.5mg poloxamer 188.

[0021] Example 2: The formulation is as follows: 1E9 particles exosomes, 1.5 mg PEG2000 modified β-sitosterol, 3 mg cholesterol, 25.0 mg HSA, 34.5 mg trehalose, and 1.0 mg poloxamer 188.

[0022] Example 3: The formulation is as follows: 1E9 particles exosomes, 2 mg PEG2000 modified β-sitosterol, 4 mg cholesterol, 30 mg HSA, 40 mg trehalose, and 1.5 mg poloxamer 188.

[0023] Comparative Example 1: The formulation consisted of 1E9 particles exosomes, 3 mg cholesterol, 25.0 mg HSA, 34.5 mg trehalose, and 1.0 mg poloxamer 188. It lacked the PEG2000-modified β-sitosterol found in Example 2.

[0024] Comparative Example 2: The formulation consisted of 1E9 particles exosomes, 1.5 mg PEG2000-modified β-sitosterol, 25.0 mg HSA, 34.5 mg trehalose, and 1.0 mg poloxamer 188. This formulation lacked cholesterol compared to Example 2.

[0025] Comparative Example 3: The formulation consisted of 1E9 particles exosomes, 1.5 mg β-sitosterol, 3 mg cholesterol, 25.0 mg HSA, 34.5 mg trehalose, and 1.0 mg poloxamer 188. The difference from Example 2 was that the β-sitosterol was not modified with PEG.

[0026] Comparative Example 4: The formulation consisted of 1E9 particles exosomes, 1.5 mg of PEG2000-modified β-sitosterol, 25.0 mg of HSA, 34.5 mg of trehalose, and 1.0 mg of poloxamer 188. Compared to Example 2, it lacked PEG2000-modified β-sitosterol and cholesterol.

[0027] The exosomes listed above are PBMC-derived Treg exosomes, provided by Celsin. Exosomes can also be CBMC-derived Treg exosomes; there are no restrictions.

[0028] Sample preparation method: The preparation method includes the following steps: Step 1, preparation of lipid film: Dissolve the weighed PEG2000 modified β-sitosterol and cholesterol in chloroform, transfer the solution to a round-bottom flask, and use a rotary evaporator under reduced pressure to remove the organic solvent, so that the lipid forms a uniform film on the flask wall. Continue to vacuum dry the flask overnight to completely remove the residual organic solvent. Step 2, hydration of liposomes: Add an appropriate amount of preheated PBS buffer or purified water to the flask, heat and rotate for 30 minutes to hydrate the lipid film off the flask wall, forming a crude liposome suspension; use a liposome extruder to repeatedly extrude the suspension to finally obtain liposomes; Step 3, Fusion with exosomes: The prepared liposomes and 1E9 particles of exosomes are incubated together at 37°C for 1 day to allow the liposome membrane to fuse with the exosome membrane, forming an exosome-liposome complex suspension; Step 4, prepare the suspension before lyophilization: Dissolve the weighed HSA, trehalose, and poloxamer 188 in water for injection, and stir thoroughly until completely dissolved to obtain a protective agent solution. Gently and thoroughly mix the exosome-liposome complex suspension with the protective agent solution. Step 5, freeze drying: The freeze drying steps are as follows: Pre-freezing: Temperature: -5℃, -45℃, -8℃, -45℃; Set time: 10min, 120min, 30min, 60min; Hold time: 30min, 60min, 90min, 10min; First drying: Temperature: -32℃; Set time: 90min; Hold time: 2700min; Vacuum degree: 0.15 mbar; Second drying: Temperature: 30℃, 30℃; Set time: 240℃, 1min; Hold time: 120℃, 240min; Vacuum degree: 0.20℃, 0.01 mbar.

[0029] Samples 1-3 and comparative samples 1-3 were obtained using the formulations of Examples 1-3 and Comparative Examples 1-3, and the methods described above. Sample 4 was prepared by dissolving weighed HSA, trehalose, and poloxamer 188 in water for injection, stirring thoroughly until completely dissolved to obtain a protective agent solution. The exosome suspension of 1E9 particles incubated for one day was then incorporated into the protective agent solution, and the same freeze-drying process as in step five was used to obtain comparative sample 4.

[0030] Experiment 1: Stability Test 1.1 Test conditions and sampling time points: Samples were taken and tested at 0, 4 and 8 weeks after the lyophilized powder was placed at 40°C ± 2°C / 75% ± 5% RH, and the experimental data were compared. 1.2 Detection Indicators and Methods

[0031] 1.3 Comparing the appearance of the samples at week 0 and week 4, both samples 1-3 and control samples 1-4 were clear and had uniform particle size.

[0032] 1.4 Table 1 shows the changes in test results and appearance after reconstitution of the lyophilized powder at 40°C ± 2°C / 75% ± 5% RH for 0 weeks and 8 weeks respectively: Table 1

[0033] Analysis of experimental results: Comparative sample 4 (naked exosomes): The NTA recovery rate and biomarker signal were lowest after lyophilization, proving that lyophilization protectants alone cannot completely maintain exosome membrane integrity. Comparative sample 2 (cholesterol-deficient): High leakage rate and abnormally reduced particle size indicate that the membrane's mechanical strength is insufficient in the absence of cholesterol, and ice crystal stress leads to membrane rupture and leakage of contents. Cholesterol is an essential component for maintaining membrane structural rigidity.

[0034] Comparative Sample 1 (without PEG and β-sitosterol): Severe aggregation and a surge in PDI occurred after reconstitution. This demonstrates that while cholesterol reinforces the membrane, the lack of a hydrophilic protective layer on the surface leads to particle fusion and precipitation during freeze-drying and concentration. Comparative Sample 3 (unmodified β-sitosterol): The degree of aggregation is between that of Comparative Sample 1 and Sample 2, indicating that β-sitosterol lacking the long PEG chain cannot provide sufficient steric hindrance, resulting in significantly lower stability compared to the PEG-modified product. Conclusion: The steric hindrance provided by the long PEG chain is a key factor in preventing particle aggregation during freeze-drying and reconstitution.

[0035] The particle size variation of samples 1-3 was significantly lower than that of other control samples. Recovery: The NTA recovery of samples 1-3 was significantly higher than that of control samples. Leakage inhibition: Samples 1-3 were able to simultaneously meet the requirements of "low leakage rate (membrane integrity)" and "low PDI (no aggregation)".

[0036] Experimental data show that PEG2000-modified β-sitosterol and cholesterol have a significant synergistic stabilizing effect in this lyophilization system. Cholesterol prevents membrane rupture caused by lyophilization stress by enhancing the rigidity of the lipid bilayer; PEG2000-modified β-sitosterol prevents irreversible aggregation between particles through steric hindrance. Both are indispensable and together achieve the uniformity of exosome lyophilized powder particle size, structural integrity, and high recovery rate after reconstitution.

[0037] Experiment 2: Moisture content of exosome lyophilized powder tested using samples from Example 2.

[0038] This indicates that freeze drying has removed most of the free water and some of the bound water, meeting the requirements for freeze dryers.

[0039] This invention utilizes a thin-film hydration method to prepare liposomes from PEG2000-modified β-sitosterol and cholesterol. These liposomes are then fused with exosomes and mixed with a lyophilization protectant composed of HSA, trehalose, and poloxamer 188 before final freeze-drying. This combination effectively protects the structural integrity of exosomes during freeze-drying and enhances their stability; simultaneously, the protectant formulation of HSA, trehalose, and poloxamer 188 also contributes to exosome stability.

[0040] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A lyophilized exosome composition, characterized in that, The formula, by weight, includes: 1 E9 particles exosomes, 1-2 parts PEG2000 modified β-sitosterol, 2-4 parts cholesterol, 20-30 parts HSA, 30-40 parts trehalose, and 0.5-1.5 parts poloxamer 188.

2. The exosome lyophilized composition according to claim 1, characterized in that, The formula is: 1E9 particles exosomes, 1.5 mg PEG2000 modified β-sitosterol, 3 mg cholesterol, 25.0 mg HSA, 34.5 mg trehalose, and 1.0 mg poloxamer 188.

3. The method for preparing an exosome lyophilized composition as described in claim 1, characterized in that, Includes the following steps: Step 1, preparation of lipid film: Dissolve the weighed PEG2000 modified β-sitosterol and cholesterol in chloroform, transfer the solution to a round-bottom flask, and use a rotary evaporator under reduced pressure to remove the organic solvent, so that the lipid forms a uniform film on the flask wall. Continue to vacuum dry the flask overnight to completely remove the residual organic solvent. Step 2, hydration of liposomes: Add an appropriate amount of preheated PBS buffer or purified water to the flask, heat and rotate for 30 minutes to hydrate the lipid film off the flask wall, forming a crude liposome suspension; use a liposome extruder to repeatedly extrude the suspension to finally obtain liposomes; Step 3, Fusion with exosomes: The prepared liposomes and 1E9 particles of exosomes are incubated together at 37°C for 1 day to allow the liposome membrane to fuse with the exosome membrane, forming an exosome-liposome complex suspension; Step 4, prepare the suspension before lyophilization: Dissolve the weighed HSA, trehalose, and poloxamer 188 in water for injection, and stir thoroughly until completely dissolved to obtain a protective agent solution. Gently and thoroughly mix the exosome-liposome complex suspension with the protective agent solution. Step 5: freeze-drying and packaging.

4. The preparation method according to claim 3, characterized in that, The freeze-drying steps are as follows: Pre-freezing: temperature: -5℃, -45℃, -8℃, -45℃; set time: 10min, 120min, 30min, 60min; holding time: 30min, 60min, 90min, 10min; First drying: temperature: -32℃; set time: 90min; holding time: 2700min; vacuum degree: 0.15mbar; Second drying: temperature: 30℃, 30℃; set time: 240℃, 1min; holding time: 120℃, 240min; vacuum degree: 0.20℃, 0.01mbar.

5. The preparation method according to claim 3, characterized in that, The dispensing steps are as follows: the filling volume is 0.50ml / bottle, and a 13mm rubber stopper is added after filling.

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