Milk exosome-based mouth ulcer sustained-release powder spray and method of preparing the same

Abstract

This invention relates to the fields of biomedical technology and pharmaceutical formulations, and discloses a sustained-release powder spray for oral ulcers based on milk exosomes and its preparation method. The spray uses milk exosomes as the active ingredient, combined with an extracellular matrix-simulated network composed of sodium hyaluronate and collagen, and the stabilizer trehalose. It is prepared into a dry powder with a particle size of 2-15 μm through a microdroplet freeze-drying process. During preparation, the components are mixed and formed into microdroplets using a microfluidic device, then instantaneously frozen in liquid nitrogen at -80°C, and finally freeze-dried to obtain the powder. This formulation can be stably stored for a long time at 2-8°C. When used, it is sprayed onto the ulcer surface using a quantitative spray device. Upon contact with moisture, the gel network is reconstructed in situ, achieving sustained release and protection of the exosomes. It has the advantages of convenient use, accurate dosage, and long-lasting healing promotion.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and pharmaceutical formulation, specifically relating to a powder formulation prepared using milk-derived exosomes as the active ingredient, combined with specific biological materials to form the extracellular matrix (ECM), and prepared using a special freeze-drying process. This formulation can be used for the local treatment of oral ulcers via a metered-dose spray device, and features sustained release, stability, and ease of use. Background Technology

[0002] Oral ulcers are a common oral mucosal disease, and their treatment usually focuses on anti-inflammatory, analgesic, and healing-promoting measures. However, conventional ointments or patches often have limited efficacy due to poor adhesion and short retention time in the moist and dynamic oral environment.

[0003] Exosomes, as naturally occurring nanoscale extracellular vesicles, have shown great potential in disease treatment and drug delivery in recent years. They possess low immunogenicity, good biocompatibility, and the ability to cross biological barriers, and can mediate intercellular communication, exhibiting inherent advantages in regulating inflammation and promoting tissue repair. Milk-derived exosomes, in particular, have attracted attention due to their wide availability and relatively low cost.

[0004] However, exosomes, especially their liquid formulations, suffer from stability issues and typically require storage and transportation under harsh conditions of -80°C, severely limiting their clinical applications. Although freeze-drying is a common method for improving the stability of biopharmaceuticals, traditional freeze-drying processes often first form a blocky solid, requiring physical grinding to obtain a powder. This process easily damages the integrity of the exosomes and any intricate structures that may exist in the formulation (such as the matrix constructed to achieve sustained-release effects), and results in uneven particle size of the final powder, affecting the accuracy of the dosage.

[0005] In addition, direct application of exosomes to oral ulcer lesions presents challenges: exosomes are easily cleared away by saliva, and various enzymes present in the oral environment may degrade the lipid bilayer of exosomes, reducing their efficacy.

[0006] Therefore, there is an urgent need in this field to develop a novel exosome formulation that can protect exosome activity, achieve long-lasting sustained release, improve storage stability, and act precisely and conveniently on the oral ulcer site. To this end, a sustained-release powder spray for oral ulcers based on milk exosomes and its preparation method have been proposed. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a sustained-release powder spray for oral ulcers based on milk exosomes and its preparation method, thereby resolving the problems in the prior art.

[0008] To achieve the above objectives, the present invention provides the following technical solution: Firstly, a sustained-release powder spray for oral ulcers based on milk exosomes, the powder spray comprising a formulation mixture containing the following components: Active ingredient: Milk exosomes; Matrix materials: sodium hyaluronate and collagen, used to construct an extracellular matrix-mimicking network loading the milk exosomes; Stabilizer: Trehalose; And, pharmaceutically acceptable buffer solutions; The spray is a dry powder obtained by microdroplet freeze-drying process, with a particle size distribution between 2μm and 15μm.

[0009] Preferably, the concentration of sodium hyaluronate in the formulation mixture is 1.0% to 2.5% (w / v), and the concentration of collagen in the formulation mixture is 0.5% to 2.0% (w / v).

[0010] Preferably, the concentration of trehalose in the formulation mixture is 2% to 10% (w / v).

[0011] Preferably, the formulation mixture further contains calcium chloride at a concentration of 0.5% to 2.0% (w / v).

[0012] Secondly, a method for preparing a sustained-release powder spray for oral ulcers based on milk exosomes, the method comprising the following steps: Step 1: Dissolve milk exosomes, sodium hyaluronate, collagen and trehalose in a buffer solution in proportion to form a homogeneous preparation mixture; Step 2: The formulation mixture is formed into microdroplets using a microfluidic device, and the microdroplets are introduced into an ultra-low temperature medium for instantaneous freezing to obtain frozen microspheres; Step 3: Freeze-dry the frozen microspheres to obtain a dried powder.

[0013] Preferably, the cryogenic medium is liquid nitrogen at -80°C.

[0014] Preferably, the droplet diameter formed in step b is 5 μm to 70 μm.

[0015] Thirdly, a drug delivery system includes a milk exosome-based sustained-release powder spray for oral ulcers as described in the first aspect, and a spraying device for containing and metering the dried powder.

[0016] Preferably, the powder spray undergoes in-situ hydration and forms a three-dimensional gel structure after contacting the moisture of the oral mucosa.

[0017] Fourthly, the use of a milk exosome-based sustained-release powder spray for oral ulcers, as described in the first aspect, or the use of the drug delivery system described in the third aspect, in the preparation of a medicament for treating oral ulcers.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention uses a composite matrix composed of sodium hyaluronate, collagen, and trehalose, and employs a microdroplet freeze-drying process to encapsulate milk exosomes in a dried extracellular matrix (ECM) simulated network. This allows the formulation to be stably preserved for a long time at 2-8°C, overcoming the limitation of liquid exosome formulations requiring deep freezing at -80°C, and improving the product's storage and transportation convenience and clinical applicability.

[0019] 2. This invention uses a constructed three-dimensional ECM network as a carrier. After contact with wound moisture, the network can reconstruct the gel structure in situ. On the one hand, it can physically block the degradation of enzymes in the oral environment and protect the integrity of exosomes. On the other hand, it can continuously and slowly release exosomes to the ulcer site through the sustained-release effect of the mesh, achieving long-term dual protection and treatment.

[0020] 3. This invention uses microfluidic technology to form microdroplets with controllable particle size from a mixture, followed by instantaneous freezing with liquid nitrogen and then freeze-drying to directly obtain a dry powder. This process avoids the physical damage to the ECM microstructure and exosomes caused by grinding after freeze-drying in traditional methods, ensuring a highly uniform final powder particle size and good batch-to-batch reproducibility, thus guaranteeing the accuracy of dosage and the consistency of product quality.

[0021] 4. This invention forms a ready-to-use drug delivery system by filling the prepared uniform powder into a quantitative spray device. When in use, the powder can be accurately and quantitatively sprayed onto the oral ulcer lesion without pre-reconstitution, achieving convenient and intelligent drug delivery that directly reaches the affected area and has controllable dosage, overcoming the shortcomings of traditional ointments such as poor adhesion and short retention time.

[0022] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures pointed out in the description, claims and drawings. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the preparation process of the present invention; Figure 2 A scanning electron microscope (SEM) image of the powder prepared in this invention; Figure 3This is a schematic diagram comparing the effects of traditional freeze-drying and grinding methods (left side) and the method of this invention (right side) on the microstructure. Figure 4 This is a schematic diagram illustrating the action of the powder spray of the present invention on oral ulcer wounds; Figure 5 This is a graph showing the cumulative release of exosomes in vitro according to the present invention. Figure 6 This is a graph showing the exosome activity retention rate of the structure of this invention under different storage conditions; Figure 7 This is a particle size distribution curve of the powder of the present invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Please see Figures 1-7 The present invention relates to a sustained-release powder spray for oral ulcers based on milk exosomes and its preparation method.

[0026] Example 1: Extraction of milk exosomes Freshly collected pasteurized milk samples were first centrifuged at 3000g for 30 minutes at 4°C to remove the fat layer and large particulate impurities. The supernatant was collected and centrifuged again at 10000g for 45 minutes at 4°C to further remove cell debris and larger vesicles. The supernatant was then filtered through a 0.22μm filter membrane. The filtrate was ultracentrifuged at 100000g for 70 minutes at 4°C, and the supernatant was discarded. The resulting precipitate was the crude milk exosome extract. The precipitate was gently resuspended in pre-cooled sterile phosphate-buffered saline (PBS, pH 7.4) to obtain a milk exosome suspension. Nanoparticle tracking analysis (NTA) was used to determine the particle size distribution and concentration, and Western blotting was used to detect exosome marker proteins (such as CD63 and TSG101) to verify their characteristics.

[0027] Example 2: Preparation of the formulation powder (basic formula) Weigh 1.5 g of sodium hyaluronate, 1.0 g of type I collagen, 3.0 g of trehalose, and 1.0 g of calcium chloride, dissolve them in 100 mL of sterile PBS (pH 7.4), stir until completely dissolved, and filter through a 0.22 μm filter membrane to obtain a matrix solution. Mix the milk exosome suspension obtained in Example 1 with the above matrix solution at a volume ratio of 1:10, and gently vortex to obtain a formulation mixture. Using a device equipped with a microfluidic chip with an inner diameter of 20 μm, pump the mixture at a stable flow rate to form uniform microdroplets at the outlet. Drop the microdroplets into a container filled with liquid nitrogen at -80°C for instantaneous freezing to form spherical frozen microspheres. Collect the frozen microspheres and transfer them to a freeze dryer for primary drying and desorption drying to finally obtain a white, free-flowing dry powder. The preparation process is as follows: Figure 1 The microfluidic-liquid nitrogen freeze-drying process from the mixture to the final powder is shown in the figure. The resulting powder was characterized: scanning electron microscope (SEM) images are shown below. Figure 2 As shown, the powder is spherical with a smooth surface and intact microstructure; the particle size distribution curve measured by the laser particle size analyzer is as follows. Figure 7 As shown, the curve peaks are narrow and mainly distributed within the range of 5μm±2μm, indicating that the powder particle size is uniform.

[0028] Example 3: Preparation of formulation powders with different formulations To illustrate the formulation range, the following three formulation mixtures with different compositions were prepared: Formula A: Sodium hyaluronate 1.0%, collagen 2.0%, trehalose 5.0%, calcium chloride 0.5%.

[0029] Formula B: Sodium hyaluronate 2.0%, collagen 0.5%, trehalose 2.0%, calcium chloride 2.0%.

[0030] Formula C: Sodium hyaluronate 2.5%, collagen 1.5%, trehalose 10.0%, no added calcium chloride.

[0031] All components were dissolved in PBS at w / v percentage. Equal volumes of milk exosome suspension were mixed with each formulation solution at a 1:10 ratio, and three powder formulations (labeled Powder-A, Powder-B, and Powder-C) were prepared using the same microfluidic parameters (chip inner diameter 30 μm) and liquid nitrogen freezing and lyophilization processes as in Example 2. Observations showed that all three were loose powders with good flowability, and while the particle size distribution differed slightly, it remained within the designed range of 2-15 μm. To further verify the advantages of the process of this invention, the microstructure of the powder obtained in Example 2 was compared with that of a sample prepared by the traditional freeze-drying and grinding method (where the mixture is directly freeze-dried into blocks and then ground and sieved). Figure 3As shown, the left side is an SEM image of the powder obtained by the traditional method, which shows a broken structure and inconsistent particle size; the right side is the powder obtained by the method of this invention, which has intact spherical shape and a dense surface. This comparison intuitively demonstrates the advanced nature of microdroplet freeze-drying technology in protecting microstructure.

[0032] Example 4: Stability Test The powder formulation prepared in Example 2 was aliquoted into sealed vials. One group was stored in a refrigerator at 4°C, and the other group was stored at room temperature (simulating ambient temperature) at 25°C. Samples were taken at 0, 3, 6, 12, 24, and 36 months of storage. Stability was evaluated using the following methods: 1) Appearance and resolubility: Observe the powder morphology and sprinkle it on a wet surface to observe its gel reconstruction ability; 2) Exosome integrity: The particle size and concentration of reconstituted exosomes were remeasured using NTA and compared with the initial values; 3) Bioactivity: The ability of reconstituted exosomes to promote the migration of human gingival epithelial cells was assessed using a cell migration assay (scratch test). The change in activity retention over time is shown in the curve. Figure 6 As shown, the results indicate that after 36 months of storage at 4°C, the powder appearance remained unchanged, and it could still rapidly hydrate to form a gel. The exosome recovery rate exceeded 85%, and the biological activity was not significantly different from the initial sample. After 24 months of storage at 25°C, all indicators remained good.

[0033] Example 5: In vitro release and enzyme protection experiment Simulated saliva environment: Prepare a buffer solution (pH 6.8) containing a certain concentration of lysozyme, amylase, etc. Take a certain amount of the powder from Example 2 and place it in a dialysis bag, immersing it in the simulated saliva release medium, and incubate at 37°C with constant shaking. The in vitro release curve is shown below. Figure 5 As shown, samples were taken at predetermined time points (e.g., 0.5, 1, 2, 4, 8, 12, 24 hours) and replenished with an equal volume of fresh medium. The cumulative release of the samples was detected using the BCA protein quantification method (for total exosome protein) or an ELISA assay for exosome-specific markers, and release curves were plotted. The results showed that exosomes exhibited a slow and continuous release over 24 hours, with a cumulative release rate exceeding 80%, indicating that the ECM network has excellent sustained-release properties. Simultaneously, a control group was set up: an equal volume of free milk exosomes was directly added to enzyme-containing simulated saliva and incubated at 37°C for the same time. Western blotting of exosome marker proteins was performed on both the experimental group (release medium after powder formulation) and the control group. The results showed that the exosome protein bands were clear in the experimental group, while the bands in the control group were significantly weakened or disappeared, demonstrating that the ECM gel effectively protected exosomes from degradation by oral enzymes.

[0034] Example 6: Verification of effectiveness at the cellular level The experiment was conducted using human gingival epithelial cell lines.

[0035] Anti-inflammatory experiment: An inflammation model was established by stimulating cells with lipopolysaccharide (LPS). Cells were treated with culture medium containing free exosomes and culture medium reconstituted with the powder from Example 2. Cell supernatant was collected after 24 hours, and the levels of inflammatory factors (such as IL-6 and TNF-α) were detected by ELISA. The results showed that the levels of inflammatory factors in the powder treatment group were significantly lower than those in the model group and the free exosome group.

[0036] Healing promotion experiment: Scratches were created on cell monolayers, and cells were cultured in culture media containing free exosomes and in culture media containing reconstituted powder from Example 2 (the powder was pre-reconstituted with a small amount of culture media to form a gel, and the supernatant was used or appropriately diluted before use). The scratch area was observed and measured under a microscope at 0, 12, and 24 hours. The results showed that the cell migration rate and scratch closure rate of the powder treatment group were significantly higher than those of the blank control group and the free exosome group, proving that it can effectively promote epithelial repair.

[0037] Example 7: Assembly and Use of Powder Spray System The dried powder prepared in Example 2 was quantitatively filled into the storage hopper of a specially designed pressurized metered powder sprayer under aseptic drying conditions. A schematic diagram of the spray's action on oral ulcer lesions is shown below. Figure 4 As shown, during use, the patient aims the nozzle at the ulcer in the mouth and presses once, and the device sprays out a set dose of powder (e.g., about 5 mg). Upon contact with moisture on the mucous membrane, the powder hydrates in situ within seconds, forming a transparent, adhesive gel film that tightly covers the ulcer surface. This film can last for several hours, during which time it slowly releases exosomes to exert its effects, without affecting normal speech or drinking.

Claims

1. A sustained-release powder spray for oral ulcers based on milk exosomes, characterized in that, This powder spray consists of a formulation mixture containing the following components: Active ingredient: Milk exosomes; Matrix materials: sodium hyaluronate and collagen, used to construct an extracellular matrix-mimicking network loading the milk exosomes; Stabilizer: Trehalose; And, pharmaceutically acceptable buffer solutions; The spray is a dry powder obtained by microdroplet freeze-drying process, with a particle size distribution between 2μm and 15μm.

2. The oral ulcer sustained-release powder spray based on milk exosomes according to claim 1, characterized in that, The concentration of sodium hyaluronate in the formulation mixture is 1.0% to 2.5% (w / v), and the concentration of collagen in the formulation mixture is 0.5% to 2.0% (w / v).

3. The oral ulcer sustained-release powder spray based on milk exosomes according to claim 1, characterized in that, The concentration of trehalose in the formulation mixture is 2% to 10% (w / v).

4. The oral ulcer sustained-release powder spray based on milk exosomes according to claim 1, characterized in that, The formulation mixture also contains calcium chloride, which has a concentration of 0.5% to 2.0% (w / v) in the mixture.

5. A method for preparing a sustained-release powder spray for oral ulcers based on milk exosomes, characterized in that, This method is used to prepare a sustained-release powder spray for oral ulcers based on milk exosomes as described in any one of claims 1-4, and the method includes the following steps: Step 1: Dissolve milk exosomes, sodium hyaluronate, collagen and trehalose in a buffer solution in proportion to form a homogeneous preparation mixture; Step 2: The formulation mixture is formed into microdroplets using a microfluidic device, and the microdroplets are introduced into an ultra-low temperature medium for instantaneous freezing to obtain frozen microspheres; Step 3: Freeze-dry the frozen microspheres to obtain a dried powder.

6. The method for preparing a sustained-release powder spray for oral ulcers based on milk exosomes according to claim 5, characterized in that, The cryogenic medium is liquid nitrogen at -80°C.

7. The method for preparing a sustained-release powder spray for oral ulcers based on milk exosomes according to claim 5, characterized in that, The droplets formed in step b have a diameter of 5 μm to 70 μm.

8. A drug delivery system, characterized in that, Includes a milk exosome-based sustained-release powder spray for oral ulcers as described in any one of claims 1-4, and a spraying device for containing and metering the dried powder.

9. A drug delivery system according to claim 8, characterized in that, Upon contact with moisture in the oral mucosa, the powder spray undergoes in-situ hydration to form a three-dimensional gel structure.

10. Use of a milk exosome-based sustained-release powder spray for oral ulcers, as described in any one of claims 1-4, or the drug delivery system described in any one of claims 8-9, in the preparation of a medicament for treating oral ulcers.

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