A liupao tea exosome freeze-drying preparation with low atomization loss rate and a preparation method and application thereof

Abstract

This invention discloses a lyophilized exosome formulation of Liubao tea with low nebulization loss rate, its preparation method, and its application, relating to the fields of biomedicine and inhalation formulation technology. The active ingredient is Liubao tea exosomes, and it also contains a pharmaceutically acceptable lyophilization protectant. The lyophilized formulation, after reconstitution, is adapted to a mesh nebulizer for nebulization inhalation. The Liubao tea exosomes are extracted by the following method: Liubao tea leaves are washed, rapidly frozen and ground in liquid nitrogen, homogenized with buffer solution, and allowed to stand at low temperature. Then, they are subjected to gradient centrifugation, filtration clarification, and purification and enrichment using a microfluidic chip system. The microfluidic chip system is the Huixin Bio EXODUS fully automated exosome extraction system, which relies on sonication and eddy current coupling physical fields to achieve selective separation of exosomes. This invention prepares Liubao tea exosomes into a lyophilized formulation specifically for nebulization inhalation, expanding the application direction of exosomes from fermented tea plants in the field of respiratory inhalation drug delivery.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and inhalation formulation technology, specifically to a freeze-dried exosome formulation of Liubao tea with low atomization loss rate, its preparation method, and its application. Background Technology

[0002] Plant-derived exosomes are natural nanoscale vesicle structures that can serve as drug delivery carriers, showing significant application potential in the treatment of respiratory diseases. Liubao tea, a microbially fermented tea, has exosomes rich in active substances such as theaflavins and polyphenols, possessing anti-inflammatory and antioxidant pharmacological activities, and showing promising development prospects in the intervention of respiratory diseases.

[0003] Currently, there are many shortcomings in the application and formulation technology of plant-derived exosomes, which seriously restrict their clinical and civilian translation:

[0004] Exosomes are prone to aggregation, sedimentation, and membrane fusion in liquid environments, and their carried active components, such as miRNAs, lipids, and functional proteins, degrade rapidly at room temperature. Existing products generally require storage and transportation under a low-temperature cold chain of -80℃ to -20℃, which significantly increases usage costs and operational complexity, becoming a major bottleneck for industry development.

[0005] Nebulized inhalation drug delivery places stringent requirements on formulation dispersibility, particle size uniformity, and shear resistance. When liquid exosomes are directly nebulized, the gas-liquid interface forces and fluid shear forces easily cause exosome vesicle rupture, leading to leakage of active ingredients and loss of function. In existing technologies, the activity loss rate of plant exosomes that have not undergone specific protection after nebulization can reach 30%–50%.

[0006] Currently available exosome lyophilized powders are mostly used in the fields of beauty and skincare, and oral administration, and have not been optimized for nebulized inhalation scenarios: the particle size cannot be restored to the original nanoscale after reconstitution; the nebulization shear force tolerance has not been verified; the aerosol particle size has not been tested in accordance with the inhalation formulation standards; and the lyophilization process parameters have not been customized and optimized in combination with the physicochemical properties of exosomes.

[0007] Exosomes from different plant sources exhibit significant differences in lipid membrane composition and structural stability. Existing research has largely focused on plants such as ginger, grape, and lemon, and there are no reports on the application of exosomes from fermented tea, such as Liubao tea, in freeze-dried formulations and nebulized inhalation.

[0008] Conventional exosome extraction methods, including ultracentrifugation, PEG precipitation, and chromatography, generally suffer from low recovery rates, poor product purity, cumbersome procedures, and difficulty in large-scale production. Furthermore, they can easily cause mechanical damage to the vesicle structure, making it impossible to guarantee the quality of the raw materials.

[0009] Therefore, we propose a freeze-dried preparation of Liubao tea exosomes with low atomization loss rate, its preparation method, and its application. Summary of the Invention

[0010] The purpose of this invention is to provide a freeze-dried exosome preparation of Liubao tea with low atomization loss rate, its preparation method and application, so as to solve the problems mentioned in the background art.

[0011] To achieve the above objectives, the present invention provides the following technical solution: a lyophilized preparation of Liubao tea exosomes with low atomization loss rate, wherein the active ingredient is Liubao tea exosomes, and also contains a pharmaceutically acceptable lyophilization protectant; the lyophilized preparation is reconstituted and adapted to a mesh nebulizer for nebulization inhalation.

[0012] Furthermore, the exosomes from Liubao tea leaves are extracted by the following method: Liubao tea leaves are washed, quick-frozen and ground in liquid nitrogen, homogenized in buffer solution, allowed to stand at low temperature, then subjected to gradient centrifugation, filtered and clarified, and finally purified and enriched by a microfluidic chip system.

[0013] Furthermore, the microfluidic chip system is Huixin Bio's EXODUS fully automated exosome extraction system, which relies on sonication and eddy current coupling physical fields to achieve selective separation of exosomes.

[0014] Furthermore, the freeze-drying protectant is composed of trehalose, mannitol, and resistant dextrin in a mass ratio of 2:1:1; the ratio of the total mass of the freeze-drying protectant to the total protein mass of the exosomes of Liubao tea is 10:1.

[0015] Furthermore, the freeze-drying protectant is dissolved in purified water to prepare a mother liquor with a final concentration of 2% (w / v).

[0016] Furthermore, the freeze-drying process for preparing the freeze-dried formulation is as follows: pre-freezing at -80℃ until the sample is completely frozen, and then continuously drying for 72 hours under conditions where the cold trap temperature is below -40℃ and the system vacuum degree is below 300Pa.

[0017] Furthermore, after the freeze-dried preparation is reconstituted, it is atomized by a mesh atomizer. The relative loss rate of Liubao tea exosome particle concentration before and after atomization is ≤3.125%; the exosome particle size after reconstitution is 70~110nm.

[0018] Furthermore, the mesh nebulizer is a Yuwell M102 medical nebulizer with a median atomized particle size of approximately 3.7 μm and aerosol particles with a diameter of less than 5 μm accounting for ≥60%.

[0019] A method for preparing a lyophilized exosome formulation of Liubao tea with low atomization loss rate, comprising the following steps:

[0020] (1) Extraction of exosomes from Liubao tea leaves;

[0021] (2) Mix the obtained Liubao tea exosomes with the freeze-drying protectant mother liquor evenly and dispense them into freeze-drying containers;

[0022] (3) Place the dispensed samples in an environment of -80℃ to pre-freeze until completely frozen;

[0023] (4) Place the pre-frozen sample in a freeze dryer, control the cold trap temperature < -40℃ and the vacuum degree < 300Pa, dry for 72h, and the finished freeze-dried preparation is obtained by discharging the material.

[0024] The application of the described lyophilized preparation of Liubao tea exosomes for nebulization in the preparation of nebulized inhalation drugs for treating respiratory diseases.

[0025] This invention provides a lyophilized exosome preparation of Liubao tea with low atomization loss rate, its preparation method and application, which has the following beneficial effects: Liubao tea exosomes are prepared into a lyophilized preparation for atomization inhalation, opening up the application direction of exosomes of fermented tea plants in the field of respiratory inhalation drug delivery.

[0026] Exosomes were extracted using sonophoresis-vortex coupled microfluidics technology, which has a gentle physical effect and preserves the complete vesicle structure of exosomes to the greatest extent, thus greatly improving the quality of raw materials.

[0027] Through customized freeze-drying processes and compound protective agents, the formulation can be stored stably at room temperature for a long time, completely eliminating the need for low-temperature cold chains and significantly reducing storage, transportation, and usage costs.

[0028] After atomization by a mesh nebulizer, the exosome concentration loss rate is only 3.125%, which is far lower than the 30% to 50% activity loss of traditional liquid formulations, solving the industry problem of exosomes being easily broken and losing activity during atomization. Attached Figure Description

[0029] Figure 1 This is a particle size distribution diagram of the quality control standard spheres of this invention;

[0030] Figure 2 This is a graph showing the particle size distribution and concentration detection of the Liubao tea exosome sample LY-1 in this invention.

[0031] Figure 3 This is a graph showing the particle size distribution and concentration detection of the Liubao tea exosome sample LY-2 in this invention;

[0032] Figure 4 This is a summary chart of the detection results of exosome concentration in Liubao tea leaves according to the present invention;

[0033] Figure 5 The Zeta potential distribution diagram of the exosomes from Liubao tea in this invention shows that the Zeta potential is approximately -20 mV;

[0034] Figure 6This is a transmission electron microscope (TEM) image of the exosomes from Liubao tea leaves in this invention.

[0035] Figure 7 This is a graph showing the particle size distribution and concentration of exosome samples before atomization in this invention.

[0036] Figure 8 This is a graph showing the particle size distribution and concentration detection of exosome samples after atomization according to the present invention. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to specific embodiments. Those skilled in the art should understand that the following embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0038] Example 1: Extraction of exosomes from Liubao tea leaves: 100 g of fresh tender Liubao tea leaves were pretreated and purified according to steps 2.1.1-2.1.4, yielding approximately 5 mL of exosome suspension. NanoFCM analysis showed a particle concentration of 1.09 × 10¹¹ particles / mL and a peak particle size of 85.6 nm. Transmission electron microscopy (TEM) observations showed (… Figure 6 The exosomes of Liubao tea exhibit a typical saucer-shaped vesicle structure, with complete morphology and uniform particle size.

[0039] Example 2: Preparation of Liubao Tea Exosome Lyophilized Formulation: The Liubao tea exosome suspension prepared in Example 1 was taken, and a lyophilization protectant stock solution was added at a ratio of 10:1 (total mass of protectant to total exosome protein). The lyophilization protectant stock solution was prepared by mixing trehalose, mannitol, and resistant dextrin at a mass ratio of 2:1:1, with a final concentration of 2% (w / v).

[0040] After mixing, the mixture was dispensed into freeze-drying containers and pre-frozen overnight at -80℃. Freeze-drying was performed using a freeze dryer manufactured by Guangzhou Huoyuan Medical Equipment Co., Ltd., with the following parameters: cold trap temperature < -40℃ (actual measurement -41.9℃), vacuum degree < 300 Pa (actual measurement 0.100 Pa), and a single drying cycle of 72 hours.

[0041] After freeze-drying, the lyophilized powder for Liubao tea exosome nebulization inhalation was obtained. Testing showed that after reconstitution, the particle size was 74.1 ± 6.5 nm (particle size retention rate 99.6%), the Zeta potential was -20.3 ± 3.6 mV, and the appearance was a white, loose, blocky substance. The reconstitution time was approximately 11 seconds.

[0042] Example 3: Compatibility verification of nebulized drug delivery: One vial of the lyophilized formulation prepared in Example 2 was taken, and a reconstitution solution (0.9% physiological saline) was added. After gentle shaking to dissolve, the solution was poured into the drug cup of the Yuwell M102 nebulizer. Nebulization was performed according to standard operating procedures, and samples before and after nebulization were collected for NanoFCM analysis.

[0043] Results: The particle concentration before atomization was 1.60 × 10⁻⁶. 10 The particle density was 1.55 × 10⁻⁶ particles / mL, with an average particle size of 74.4 nm; the particle concentration after atomization was 1.55 × 10⁻⁶. 10 The particle size distribution was 74.1 nm, with a relative loss rate of approximately 3.125%. The particle size was essentially uniform.

[0044] Example 4: Room temperature stability test: The lyophilized formulation prepared in Example 2 was stored at 25℃±2℃. Samples were taken at preset time points (0, 7, 14, 30, 60, and 90 days), and the particle size, particle concentration, and zeta potential were measured after reconstitution.

[0045] 0 74.1 100% 100% -20.3 7 74.3 99.7% 98.5% -20.1 14 74.7 99.1% 97.2% -19.8 30 75.3 98.4% 95.6% -19.5 60 75.8 97.8% 93.8% -19.1 90 76.2 97.2% 92.1% -18.7

[0046] The results showed that the formulation of the present invention, after being stored at room temperature (25°C) for 90 days, maintained a particle size retention rate of >97% and a particle concentration retention rate of >92% after reconstitution, and the absolute value of the Zeta potential was always greater than 18 mV, demonstrating good room temperature stability.

[0047] Comparative Example 1: Comparison of different exosome extraction methods: Under the condition that the raw materials, pretreatment, and subsequent formulation processes are completely identical, exosomes from Liubao tea were extracted using the microfluidic method of this invention and the traditional ultracentrifugation method, respectively, and the performance indicators were compared:

[0048] experimental group The microfluidic method of this application ≥90% ≥99% 74.4±6.5 3.14% control group Ultracentrifugation 30%-50% 60%-80% 98.6±12.3 28.6%

[0049] It is evident that the extraction method of this invention has a much higher recovery rate and purity than traditional methods, and causes less damage to the exosome structure, resulting in significantly improved atomization tolerance.

[0050] Comparative Example 2: Comparison of different freeze-drying protectant formulations. With the exosome raw material and freeze-drying process unchanged, multiple groups of protectant formulations were set up for control experiments. The results are as follows:

[0051] Control group 1 No protective agent 156.8±18.2 52.3% 52.6% —— Control group 2 Trehalose only (2%) 98.5±8.6 82.1% 11.2% 45 Control group 3 Trehalose + Mannitol (2:1) 86.3±7.2 89.5% 6.8% 32 Control group 4 Trehalose + resistant dextrin (2:1) 92.4±9.1 86.2% 8.3% 18 experimental group Trehalose + Mannitol + Resistant Dextrin (2:1:1) 74.1±6.3 99.6% 3.125% 11

[0052] Experiments have shown that the combination of trehalose, mannitol, and resistant dextrin produces a synergistic effect, providing the best protection for exosomes in Liubao tea, while significantly shortening the reconstitution time.

[0053] It should be noted that, in this document, the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0054] This article uses specific examples to illustrate the principles and implementation methods of the present invention. The above examples are only for the purpose of helping to understand the method and core ideas of the present invention. The above descriptions are only preferred embodiments of the present invention. It should be noted that due to the limitations of textual expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of the present invention, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of the present invention.

Claims

1. A freeze-dried preparation of Liubao tea exosomes with low atomization loss rate, characterized in that, The active ingredient is exosomes from Liubao tea leaves, and it also contains a pharmaceutically acceptable freeze-drying protectant; the freeze-dried preparation is reconstituted and adapted to a mesh nebulizer for nebulization inhalation.

2. The lyophilized exosome preparation of Liubao tea with low atomization loss rate according to claim 1, characterized in that, The exosomes from Liubao tea leaves were extracted by the following method: Liubao tea leaves were washed, quick-frozen and ground in liquid nitrogen, homogenized in buffer solution, allowed to stand at low temperature, then subjected to gradient centrifugation, filtered and clarified, and finally purified and enriched by a microfluidic chip system.

3. The lyophilized exosome preparation of Liubao tea with low atomization loss rate according to claim 1, characterized in that, The microfluidic chip system is Huixin Bio's EXODUS fully automated exosome extraction system, which relies on sonication and eddy current coupling physical field to achieve selective separation of exosomes.

4. The lyophilized preparation of Liubao tea exosomes with low atomization loss rate according to claim 3, characterized in that, The freeze-drying protectant is composed of trehalose, mannitol, and resistant dextrin in a mass ratio of 2:1:1; the ratio of the total mass of the freeze-drying protectant to the total protein mass of the exosomes of Liubao tea is 10:

1.

5. The lyophilized preparation of Liubao tea exosomes with low atomization loss rate according to claim 4, characterized in that, The freeze-drying protectant was dissolved in purified water to prepare a mother liquor with a final concentration of 2% (w / v).

6. The lyophilized preparation of Liubao tea exosomes with low atomization loss rate according to claim 4, characterized in that, The freeze-drying process for preparing the freeze-dried formulation is as follows: pre-freezing at -80℃ until the sample is completely frozen, and then continuously drying for 72 hours under conditions where the cold trap temperature is below -40℃ and the system vacuum degree is below 300Pa.

7. The lyophilized exosome preparation of Liubao tea with low atomization loss rate according to claim 6, characterized in that, After the freeze-dried preparation is reconstituted, it is atomized by a mesh atomizer. The relative loss rate of Liubao tea exosome particle concentration before and after atomization is ≤3.125%; the exosome particle size after reconstitution is 70~110nm.

8. The lyophilized exosome preparation of Liubao tea with low atomization loss rate according to claim 7, characterized in that, The mesh nebulizer is a Yuwell M102 medical nebulizer with a median atomized particle size of approximately 3.7 μm and aerosol particles with a diameter of less than 5 μm accounting for ≥60%.

9. A method for preparing a lyophilized Liubao tea exosome formulation with low atomization loss rate, used to prepare the lyophilized Liubao tea exosome formulation with low atomization loss rate as described in claim 8, characterized in that, Includes the following steps: (1) Extraction of exosomes from Liubao tea leaves; (2) Mix the obtained Liubao tea exosomes with the freeze-drying protectant mother liquor evenly and dispense them into freeze-drying containers; (3) Place the dispensed samples in an environment of -80℃ to pre-freeze until completely frozen; (4) Place the pre-frozen sample in a freeze dryer, control the cold trap temperature < -40℃ and the vacuum degree < 300Pa, dry for 72h, and the finished freeze-dried preparation is obtained by discharging the material.

10. The use of the lyophilized formulation of Liubao tea exosome nebulization inhalation according to any one of claims 1-8 in the preparation of nebulized inhalation drugs for treating respiratory diseases.

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