A stem cell exosome composite gel dressing and a preparation method thereof

Abstract

The application belongs to the technical field of medical dressing preparation, and particularly relates to a stem cell exosome composite gel dressing and a preparation method thereof. The preparation method of the composite gel dressing comprises the following steps: S1. adding exosomes, sophorolipids and diammonium glycyrrhizinate into water, stirring, and preparing an exosome mixture; S2. adding a silk fibroin solution into the exosome mixture in step S1, stirring, and preparing a mixed solution; and S3. adding sodium alginate oxide into the mixed solution in step S2, and stirring. The preparation method has mild conditions, is simple and convenient to operate, and has good repeatability. The composite gel dressing can accelerate wound healing and improve healing quality.

Description

Technical Field

[0001] This invention belongs to the field of medical dressing preparation technology, specifically relating to a stem cell exosome composite gel dressing and its preparation method. Background Technology

[0002] In recent years, stem cell-derived exosomes have shown great promise as a core carrier for cell-free therapy strategies. Exosomes are nanoscale vesicles secreted by cells, carrying bioactive substances such as proteins, nucleic acids, and lipids from the mother cell. They can precisely regulate key biological processes such as proliferation, migration, differentiation, and angiogenesis of recipient cells through intercellular communication. Mesenchymal stem cell exosomes, due to their powerful anti-inflammatory, pro-angiogenic, and tissue repair functions, and by avoiding the tumorigenicity and immune rejection risks of direct cell transplantation, are considered highly promising "next-generation" therapeutic biological agents. Loading them into wound dressings holds promise for achieving active wound repair and functional reconstruction.

[0003] However, successfully transforming exosomes into stable and effective medical products still faces significant technical bottlenecks. The core challenge lies in the poor stability and susceptibility to inactivation of exosomes. Exosomes themselves have a phospholipid bilayer structure, making them extremely sensitive to the external environment. They easily aggregate and degrade at room temperature, and their contained bioactive molecules are prone to leakage. In traditional physical mixing or simple encapsulation processes, the mechanical shear forces, pH changes, organic solvent exposure, and lyophilization-reconstitution operations involved in the preparation process can easily damage the integrity of their vesicle structure, leading to loss of function and severely restricting their clinical translation and application.

[0004] To address these challenges, researchers have attempted to integrate exosomes into various hydrogel carriers. Hydrogels provide a moist healing environment and allow for controlled drug release. However, conventional gelation processes (such as ionic crosslinking and free radical polymerization) often involve harsh conditions, which also affect exosome activity. Furthermore, existing systems primarily focus on simple "loading," lacking strategies for the continuous stabilization and protection of exosomes during long-term storage, resulting in short product shelf lives and uncontrollable biological effects. Therefore, how to construct an intelligent gel dressing that can protect exosome activity, achieve long-term sustained release, and possess multiple functions such as antibacterial and anti-inflammatory properties under mild conditions is a key scientific problem that urgently needs to be solved in this field.

[0005] Against this backdrop, this study aims to develop a novel stem cell exosome composite gel dressing. This dressing utilizes carefully selected biocompatible natural materials as the matrix and innovatively incorporates a composite bioprotective agent system, striving to maximize the preservation of the original activity and function of exosomes in a mild preparation environment, providing a highly efficient and safe new strategy for the treatment of refractory wounds in clinical practice. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, the first objective of this invention is to provide a method for preparing a stem cell exosome composite gel dressing, which has a simple process.

[0007] The second objective of this invention is to provide a stem cell exosome composite gel dressing that can accelerate wound healing and improve healing quality.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing a stem cell exosome composite gel dressing includes the following steps: S1. Take exosomes, sophorolipids and diammonium glycyrrhizate, add them to water, stir, and prepare an exosome mixture; S2. Add the silk fibroin solution to the exosome mixture from step S1, stir, and obtain a mixed solution; S3. Add sodium alginate oxide to the mixed solution from step S2, stir, and it is ready.

[0009] This invention creatively introduces sophorolipid and diammonium glycyrrhizate as a composite protective agent. The two work synergistically to form an effective protection for the lipid bilayer of exosomes during preparation and storage, significantly reducing the damage to their structure caused by mechanical shearing and environmental stress, and ensuring the integrity and functionality of the bioactive components of exosomes.

[0010] In addition, the present invention constructs a hydrogel network by cross-linking silk fibroin and sodium oxidized alginate through a mild Schiff base reaction. This not only provides a stable three-dimensional support carrier for exosomes, enabling their controllable and continuous release, but also the gel matrix itself has good biocompatibility, moisture permeability, and biomimetic extracellular matrix properties, which can provide an ideal microenvironment for wound healing.

[0011] Furthermore, the mass ratio of exosomes, sophorolipids, diammonium glycyrrhizate, and water in step S1 is 1:(0.1-0.2):(0.03-0.1):(100-110).

[0012] Furthermore, in step S2, the volume ratio of the silk fibroin solution and the exosome mixture is 1:(1-1.2); the concentration of the silk fibroin solution is 0.03-0.05 g / mL; and the stirring time is 30-40 min.

[0013] Furthermore, in step S3, the amount of sodium alginate added is 1-2 wt% of the mixed solution; and the stirring time is 1-2 h.

[0014] Furthermore, the preparation method of the oxidized sodium alginate is as follows: Add sodium periodate aqueous solution to sodium alginate ethanol solution and react in the dark for 18-24 hours.

[0015] Furthermore, the mass ratio of sodium alginate to sodium periodate is 1:(1-6); the mass concentration of the sodium periodate aqueous solution is 20wt%; and the concentration of the sodium alginate ethanol solution is 0.2g / mL.

[0016] Furthermore, the exosomes are umbilical cord mesenchymal stem cell exosomes.

[0017] Furthermore, the method for preparing the umbilical cord mesenchymal stem cell exosomes is as follows: Umbilical cord mesenchymal stem cells are seeded into a culture medium, cultured, and the supernatant is collected and centrifuged in a gradient.

[0018] Furthermore, the seeding density of the umbilical cord mesenchymal stem cells is (1-3) × 10⁻⁶. 5 The number of cells / mL; the umbilical cord mesenchymal stem cells are P2-P4 generation umbilical cord mesenchymal stem cells; the culture is carried out at 37℃, 5% CO2, and 1% O2 for 48 hours.

[0019] A stem cell exosome composite gel dressing is prepared using the above-described preparation method.

[0020] The beneficial technical effects of this invention are as follows: 1. This invention discloses a method for preparing a stem cell exosome composite gel dressing. The preparation method is mild, simple to operate, and has good repeatability.

[0021] 2. This invention innovatively uses sophorolipid and diammonium glycyrrhizate as a composite protective agent, which effectively solves the key problem of poor stability and easy inactivation of exosomes in gel loading.

[0022] 3. The composite hydrogel dressing prepared by this invention is based on a mild cross-linking system of silk fibroin and sodium oxidized alginate. It can stably load and slowly release active exosomes, and at the same time synergistically exert multiple effects of antibacterial, anti-inflammatory and tissue regeneration promotion, thereby significantly accelerating wound healing, improving healing quality, and facilitating storage and transportation. It has broad application prospects in the field of medical dressings. Attached Figure Description

[0023] Figure 1 This is a morphological diagram of the P3 generation umbilical cord mesenchymal stem cells obtained in this invention. Detailed Implementation

[0024] The following is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all reagents or instruments used are conventional products obtained through commercial channels.

[0025] The molecular weight of the sodium alginate of this invention is 25 kDa.

[0026] The exosomes of the present invention were prepared by the following method: Clean 1mm 3 Umbilical cord tissue of various sizes was placed in a cell culture flask, and α-MEM complete medium containing 10% FBS was added. The flask was then placed in a hypoxic environment (37℃, 5% CO2, 1% O2, and the remainder being nitrogen) and cultured. The medium was changed every 3 days. When fibroblasts crawled out from around the tissue block and fused in large areas, the supernatant and tissue block were removed. The tissue was then digested with 0.25% trypsin-EDTA and passaged to obtain P3 generation umbilical cord mesenchymal stem cells.

[0027] With 2×10 5 P3 generation umbilical cord mesenchymal stem cells were seeded at a density of cells / mL to a height of 75cm. 2 Cell culture flasks were filled with serum-free α-MEM medium and cultured in a hypoxic environment (37℃, 5% CO2, 1% O2, and the remainder nitrogen) for 72 hours. The culture medium was collected and centrifuged at 300×g for 10 minutes to obtain the umbilical cord mesenchymal stem cell culture supernatant. Gradient centrifugation was performed according to the following settings: centrifugation at 2000×g for 30 minutes, centrifugation at 10000×g for 30 minutes, and centrifugation at 120000×g for 90 minutes. The precipitate obtained was the umbilical cord mesenchymal stem cell exosomes.

[0028] Example 1 This embodiment provides a method for preparing a stem cell exosome composite gel dressing, including the following steps: S1. Using exosomes, sophorolipids, diammonium glycyrrhizate, and water in a mass ratio of 1:0.15:0.07:105, add exosomes, sophorolipids, and diammonium glycyrrhizate to water and stir to mix evenly to obtain an exosome mixture. S2. Add 0.04 g / mL silk fibroin solution to the exosome mixture in step S1 at a volume ratio of 1:1.1, and stir at room temperature for 30-40 min to obtain the mixed solution; S3. Add sodium alginate oxide to the mixed solution in step S2 at a concentration of 1.5 wt% of the mixed solution, adjust the pH to 8, and stir at room temperature for 1.5 h.

[0029] The preparation method of oxidized sodium alginate is as follows: with the mass ratio of sodium alginate to sodium periodate being 1:3, 20wt% sodium periodate aqueous solution is added to 0.2g / mL sodium alginate ethanol solution, reacted in the dark for 20h, purified, and dried to obtain oxidized sodium alginate.

[0030] This embodiment also provides a stem cell exosome composite gel dressing, which is prepared using the above-described preparation method.

[0031] Example 2 This embodiment provides a method for preparing a stem cell exosome composite gel dressing, including the following steps: S1. Using exosomes, sophorolipids, diammonium glycyrrhizate, and water in a mass ratio of 1:0.1:0.03:110, add exosomes, sophorolipids, and diammonium glycyrrhizate to water, stir and mix evenly to obtain an exosome mixture; S2. Add 0.03 g / mL silk fibroin solution to the exosome mixture in step S1 at a volume ratio of 1:1, and stir at room temperature for 30 min to obtain the mixed solution; S3. Add sodium alginate oxide to the mixed solution in step S2 at a concentration of 1 wt% of the mixed solution, adjust the pH to 8, and stir at room temperature for 1 hour.

[0032] The preparation method of oxidized sodium alginate is as follows: with the mass ratio of sodium alginate and sodium periodate being 1:1, 20wt% sodium periodate aqueous solution is added to 0.2g / mL sodium alginate ethanol solution, reacted in the dark for 18h, purified, and dried to obtain oxidized sodium alginate.

[0033] This embodiment also provides a stem cell exosome composite gel dressing, which is prepared using the above-described preparation method.

[0034] Example 3 This embodiment provides a method for preparing a stem cell exosome composite gel dressing, including the following steps: S1. Using exosomes, sophorolipids, diammonium glycyrrhizate, and water in a mass ratio of 1:0.2:0.03:100, add the exosomes, sophorolipids, and diammonium glycyrrhizate to water and stir to mix evenly to obtain an exosome mixture. S2. Add 0.05 g / mL silk fibroin solution to the exosome mixture in step S1 at a volume ratio of 1:1.2, and stir at room temperature for 40 min to obtain the mixed solution; S3. Add sodium alginate oxide to the mixed solution in step S2 at a concentration of 2 wt%, adjust the pH to 8, and stir at room temperature for 2 hours.

[0035] The preparation method of oxidized sodium alginate is as follows: with the mass ratio of sodium alginate to sodium periodate being 1:6, 20wt% sodium periodate aqueous solution is added to 0.2g / mL sodium alginate ethanol solution, reacted in the dark for 24h, purified, and dried to obtain oxidized sodium alginate.

[0036] This embodiment also provides a stem cell exosome composite gel dressing, which is prepared using the above-described preparation method.

[0037] Comparative Example 1 Based on Example 1, sophorolipids were omitted to form Comparative Example 1.

[0038] Comparative Example 2 Based on Example 1, glycyrrhizic acid diammonium was omitted to form Comparative Example 2.

[0039] Comparative Example 3 Based on Example 1, glycyrrhizic acid diammonium was omitted, and the amount of sophorolipid was increased to be consistent with the total amount of glycyrrhizic acid diammonium and sophorolipid in Example 1, forming Comparative Example 3.

[0040] Experimental Example 1 The morphology of the P3 generation umbilical cord mesenchymal stem cells obtained by this invention was observed, and the results are shown in [reference needed]. Figure 1 .

[0041] Figure 1 This is a morphological image of the P3 generation umbilical cord mesenchymal stem cells obtained in this invention. From... Figure 1 It can be seen that the cells are short spindle-shaped and uniform in size, which are typical morphological characteristics of umbilical cord mesenchymal stem cells.

[0042] Experimental Example 2 The exosome mixture obtained in the examples or comparative examples was mixed with PBS buffer at a volume ratio of 1:5 and stored at 4°C for 7 days. Then, the characteristic proteins CD63 and CD9 were detected by the mouse CD63 molecule (CD63) ELISA kit and the mouse exosome CD9 detection kit, respectively. The fluorescence intensity data (mean values) obtained are shown in Table 1.

[0043] Table 1 As can be seen from the fluorescence intensity data in Table 1, compared with Comparative Examples 1-3, the exosome mixture obtained in Example 1 exhibited the strongest activity of its characteristic proteins CD63 and CD9 after preservation. The experimental conclusion is that the combination of sophorolipid and diammonium glycyrrhizate as exosome protectants can effectively preserve the activity of exosomes.

[0044] Experimental Example 3 Fifty male mice weighing 20-22g were acclimatized for 7 days. They were then randomly divided into the following groups, with 10 mice in each group: Examples 1-3 and Comparative Examples 1-2. A skin defect model with a diameter of 10mm was created on the back of each mouse in each group. The wound was treated with the corresponding dressing, and the dressing was changed once a day. The area of ​​the remaining wound was recorded each day. The results are shown in Table 2.

[0045] Table 2 As can be seen from the residual wound area data in Table 2, compared with Comparative Examples 1-3, the composite gel dressing obtained in Example 1 can promote faster wound healing and improve the quality of wound healing.

[0046] As shown in Tables 1-2, this invention, through the innovative use of sophorolipid and diammonium glycyrrhizate as composite protective agents, effectively solves the key problem of exosome inactivation in gel loading, and successfully constructs a multifunctional composite gel dressing. This dressing, based on a mild cross-linking system of silk fibroin and oxidized sodium alginate, can stably load and sustainably release active exosomes, while synergistically exerting multiple effects of antibacterial, anti-inflammatory, and tissue regeneration promotion. This significantly accelerates wound healing, improves healing quality, and facilitates storage and transportation, showing broad application prospects in the field of medical dressings.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A method for preparing a stem cell exosome composite gel dressing, characterized in that, Includes the following steps: S1. Take exosomes, sophorolipids and diammonium glycyrrhizate, add them to water, stir, and prepare an exosome mixture; S2. Add the silk fibroin solution to the exosome mixture from step S1, stir, and obtain a mixed solution; S3. Add sodium alginate oxide to the mixed solution from step S2, stir, and it is ready.

2. The method for preparing the stem cell exosome composite gel dressing according to claim 1, characterized in that, The mass ratio of exosomes, sophorolipids, diammonium glycyrrhizate, and water in step S1 is 1:(0.1-0.2):(0.03-0.1):(100-110).

3. The method for preparing the stem cell exosome composite gel dressing according to claim 1, characterized in that, In step S2, the volume ratio of the silk fibroin solution and the exosome mixture is 1:(1-1.2); the concentration of the silk fibroin solution is 0.03-0.05 g / mL; and the stirring time is 30-40 min.

4. The method for preparing the stem cell exosome composite gel dressing according to claim 1, characterized in that, In step S3, the amount of sodium alginate added is 1-2 wt% of the mixed solution; the stirring time is 1-2 h.

5. The method for preparing the stem cell exosome composite gel dressing according to claim 4, characterized in that, The method for preparing the oxidized sodium alginate is as follows: Add sodium periodate aqueous solution to sodium alginate ethanol solution and react in the dark for 18-24 hours.

6. The method for preparing the stem cell exosome composite gel dressing according to claim 5, characterized in that, The mass ratio of sodium alginate to sodium periodate is 1:(1-6); the mass concentration of the sodium periodate aqueous solution is 20wt%; and the concentration of the sodium alginate ethanol solution is 0.2g / mL.

7. The method for preparing the stem cell exosome composite gel dressing according to claim 2, characterized in that, The exosomes are umbilical cord mesenchymal stem cell exosomes.

8. The method for preparing the stem cell exosome composite gel dressing according to claim 7, characterized in that, The method for preparing the umbilical cord mesenchymal stem cell exosomes is as follows: Umbilical cord mesenchymal stem cells are seeded into a culture medium, cultured, and the supernatant is collected and centrifuged in a gradient.

9. The method for preparing the stem cell exosome composite gel dressing according to claim 8, characterized in that, The seeding density of the umbilical cord mesenchymal stem cells was (1-3) × 10⁻⁶. 5 The number of cells / mL; the umbilical cord mesenchymal stem cells are P2-P4 generation umbilical cord mesenchymal stem cells; the culture is carried out at 37℃, 5% CO2, and 1% O2 for 48 hours.

10. A stem cell exosome composite gel dressing, characterized in that, It is prepared by the preparation method according to any one of claims 1-9.

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