Exosome sustained-release preparation and application thereof in treatment of tendon injury

Abstract

The application discloses an exosome sustained-release preparation and application thereof in treatment of tendon injury, and the sustained-release preparation takes a silk fibroin frozen sponge as a carrier, and the carrier is loaded with exosomes. The application provides the silk fibroin frozen sponge as the carrier of the exosomes, and the silk fibroin frozen sponge has good biocompatibility and biodegradability; the sustained release of the exosomes is realized by controlling a protein fiber network structure of the silk fibroin. The application utilizes the silk fibroin frozen sponge to release the exosomes in situ at a tendon injury site, so that a better tendon repair effect is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to an exosome sustained-release preparation and its application in treating tendon injuries. Background Art

[0002] In recent years, the application of exosomes, a bioactive substance, in tendon injury repair has garnered widespread attention. Exosomes are bioactive substances secreted by cells. They possess specific biological activities and can play a variety of roles in the body, including cell communication, cell repair, and immune regulation. In the treatment of tendon injuries, exosomes can promote tenocyte proliferation and differentiation, stimulate collagen synthesis, and inhibit inflammatory responses.

[0003] However, drug delivery via exosomes presents several challenges. For one thing, exosomes are complex and cannot be directly delivered using traditional drug delivery methods. Furthermore, direct injection of exosomes into the body is prone to metabolic clearance and difficulty localizing to damaged tendons. Therefore, a specialized exosome carrier is required that can sustain sustained release of exosomes, produce no side effects during internalization, and exhibit a slow degradation rate.

[0004] Prior art reports have reported the encapsulation of exosomes into peptide hydrogels for delivery. However, after soaking the peptide hydrogel in PBS solution, the exosomes diffuse and release at an initial release rate within the first few days, with the release rate exceeding that of tendon injury repair. Therefore, the effectiveness of this approach is limited.

[0005] Exosomes are encapsulated into polypeptide hydrogels for delivery. However, after being soaked in a PBS solution, the polypeptide hydrogel diffuses and releases exosomes at an initial release rate within the first few days, and the amount released is faster than tendon damage repair. Therefore, the effectiveness of this solution is limited. The purpose of the present invention is to solve the problems that cannot be solved by the existing technology. A new strategy based on silk fibroin freezing sponge is proposed, which can continuously release exosomes for a longer period of time, thereby achieving better tendon repair effects. This technology achieves precise control and delivery of exosomes by designing new biomaterials and therapeutic drugs and utilizing the self-assembly properties of natural protein motifs. Therefore, the present invention aims to improve the delivery efficiency and efficacy of exosomes, thereby solving the problems existing in the existing technology. Summary of the Invention

[0006] Based on the above reasons, the present invention aims to solve the problems of exosomes as a complex bioactive substance in drug delivery, especially in the application of tendon injury repair. The existing exosome delivery strategy is usually to passively encapsulate it into a polypeptide hydrogel, but in the first few days, the exosomes will be diffused and released at the initial release rate, and the release amount is faster than the tendon injury repair. Therefore, the present invention proposes a new strategy, that is, to use silk fibroin frozen sponge to continuously release exosomes over a long period of time, thereby achieving better tendon repair effect. Specifically, in order to achieve the purpose of the present invention, the present invention intends to adopt the following technical solutions:

[0007] One aspect of the present invention relates to an exosome sustained-release preparation, characterized in that the sustained-release preparation uses a silk fibroin frozen sponge as a carrier, and the carrier is loaded with exosomes.

[0008] In a preferred embodiment of the present invention, peaks appear at 11.9°, 15.7°, 19.8° and 24.5° in the X-ray diffraction spectrum of the sustained-release preparation.

[0009] In a preferred embodiment of the present invention, the Fourier transform infrared spectrum of the sustained-release preparation appears at 1658 cm -1 Main peak.

[0010] In a preferred embodiment of the present invention, the silk fibroin is silk I.

[0011] Another aspect of the present invention relates to a method for preparing the above-mentioned exosome sustained-release preparation, the method comprising:

[0012] (1) Mixing the silk fibroin solution and the exosome solution;

[0013] (2) The mixed solution was frozen at -4°C for more than 24 hours. After thawing, a solid sponge loaded with exosomes was obtained and soaked in PBS.

[0014] Another aspect of the present invention relates to the use of the above-mentioned exosome sustained-release preparation in the preparation of a tendon repair preparation.

[0015] The present invention utilizes a silk fibroin frozen sponge as a carrier of exosomes. During the process of being released from the frozen solvent crystals, high-concentration silk fibroin macromolecules entangle and enhance cross-linking in a limited space, thereby forming a protein network structure. The sustained release of exosomes is achieved through the protein fiber network structure of silk fibroin. The microporous structure of the silk fibroin frozen sponge can provide sufficient space for the exosomes to be encapsulated and stably present therein. Unlike traditional polypeptide hydrogels, the silk fibroin frozen sponge has a slower degradation rate and can continuously release exosomes for a longer period of time, thereby achieving better tendon repair effects. In addition, as a natural protein, silk fibroin has good biocompatibility and biodegradability, which can avoid adverse effects on the body.

[0016] Beneficial effects

[0017] 1. The present invention provides a silk fibroin frozen sponge as a carrier of exosomes, which has good biocompatibility and biodegradability, and achieves the sustained release of exosomes by controlling the protein fiber network structure of silk fibroin.

[0018] 2. The present invention utilizes silk fibroin frozen sponge to slowly release exosomes in situ at the site of tendon injury, achieving better tendon repair effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 X-ray diffraction spectra of silk fibroin solution and exosome-loaded silk fibroin frozen sponge showed a broad peak around 20° in the SF solution, indicating an amorphous structure. After freeze-self-assembly, peaks appeared at 11.9°, 15.7°, 19.8°, and 24.5°, corresponding to typical silk I spacings of 0.74, 0.56, 0.45, and 0.36 nm, confirming self-assembly into a frozen sponge structure.

[0020] Figure 2 : Fourier transform infrared spectroscopy test results of the silk fibroin frozen sponge loaded with exosomes. The peak after freezing self-assembly is at 1628cm -1 It represents the formation of sponge-like structure.

[0021] Figure 3 : SEM images of SF sponge Under scanning electron microscopy, SF sponges with different concentrations can self-assemble to form sponge-like structures.

[0022] Figure 4 : Degradation curve of SF sponge. The experimental results show that SF sponge can be degraded under the action of protease.

[0023] Figure 5: SF sponge sustained release of fluorescently labeled exosomes (the left leg of the rat was injected with fluorescently labeled exosomes directly into the joint cavity, and the right leg was injected with SF sponge sustained release of exosomes) showed that SF sponge can release exosomes in situ for a long time in vivo.

[0024] Figure 6 The tendon repair effect of SF sponge with sustained release of exosomes was verified using a rat patellar tendon defect model. The defect was filled with SF sponges of appropriate size containing stem cell-derived exosomes, and samples were taken after 1, 2, and 4 weeks to observe the repair effect. Compared with the blank group, the defect area was filled with scar tissue and the surface was uneven at 4 weeks. In the SF+exosome group, the defect area was filled with new white tendon tissue at 4 weeks.

[0025] Figure 7 HE staining was used to observe the tendon repair effect. It was found that in the early stage of repair, inflammatory cells were infiltrated in the blank group and the SF sponge group, while the number of inflammatory cells in the SF sponge + exosome group was relatively small.

[0026] Figure 8 : In vivo repair effect, Sirius red staining; green represents type I collagen, red represents type III collagen. At 4 weeks, the SF sponge + exosome group had mature type III collagen deposition. In comparison, the blank group and the SF sponge group had more type I collagen and less type III collagen deposition at 4 weeks. DETAILED DESCRIPTION

[0027] To further understand the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0028] Unless otherwise specified, all reagents involved in the embodiments of the present invention are commercially available products and can be purchased through commercial channels.

[0029] Example 1

[0030] Preparation of silk fibroin solution:

[0031] 1. Take about 15g of silk cocoons and cut them into pieces (about 1 x 0.3 cm);

[0032] 2. Take 4.24g of anhydrous sodium carbonate, dissolve it in 2L of deionized water, and heat to boil;

[0033] 3. Pour the cocoons into water and boil for 30 minutes to remove the sericin;

[0034] 4. Remove the remaining white sponge, rinse it with deionized water several times, and then dry it thoroughly at 37°C overnight;

[0035] 5. Calculate the required amount of lithium bromide based on a silk fibroin:lithium bromide mass ratio of 1:4. Calculate the total volume of lithium bromide solution required based on a 9.3 mol / L ratio. Slowly add lithium bromide powder to 70% of the total volume of deionized water while stirring. Once completely dissolved, pour the powder into the graduated cylinder and fill the remaining volume.

[0036] 6. Squeeze the silk fibroin sponge fully into a 100 mL beaker, slowly pour the lithium bromide solution into the beaker to completely immerse the silk fibroin sponge, seal the beaker, and heat and stir at 60 degrees for 4 hours to completely dissolve it, obtaining a yellow viscous solution.

[0037] 7. Pour the solution into a dialysis bag with a molecular weight cut-off of 7k for dialysis treatment (fill with about 25% of the volume), change the water 1-2 times a day, and change the water 5 times in total;

[0038] 8. Pour the protein solution into a large beaker, divide it into centrifuge tubes, and centrifuge at 9000 rpm at 4°C for 20 minutes. Remove the supernatant and centrifuge again for 20 minutes.

[0039] 9. The silk fibroin solution was divided into 15 ml centrifuge tubes, 8 ml per tube, frozen, lyophilized and dehydrated, and stored at room temperature;

[0040] 10. Dissolve the unextruded freeze-dried silk fibroin solid in 1*PBS according to the required concentration (for 5%, multiply the protein amount by 20). Shake by hand for a few minutes until dissolved. Centrifuge at 4000 rpm for 3 minutes, remove the clear liquid in the middle, and sterilize under UV for 30 minutes (using a UV lamp in a clean bench).

[0041] 11. Add excess L-glutamic acid / aspartic acid to the silk fibroin solution, adjust the pH value of the solution to 4, and let it stand for 30 minutes to 1 hour.

[0042] 12. Centrifuge at 1000 rpm for 3 min, remove the supernatant silk fibroin solution, and mix it with the exosome solution;

[0043] 13. Freeze the mixed solution at -6°C for 24 hours. After thawing, a solid sponge loaded with exosomes is obtained. Soak it in PBS for long-term storage.

[0044] XRD (X-ray diffraction spectrum, Figure 1) The test results showed that the silk fibroin solution exhibited a broad peak at 20°. The traditional silk fibroin sponge (ESF) exhibited a sharp peak at 20.6°, and shoulder peaks at 8.7° and 24°, indicating the emergence of silk II structure. A weak peak also appeared at 19.8°, indicating a mixture of silk I and silk II structures. The exosome-loaded silk fibroin frozen sponge (CSF) showed peaks at 11.9°, 15.7°, 19.8°, and 24.5°, which corresponded to the silk I structure.

[0045] FTIR (Fourier transform infrared spectroscopy, Figure 2 ) The test results show that the spectrum of the exosome-loaded silk fibroin frozen sponge is at 1658 cm -1 and 1534cm -1 The main peak is at 1628cm, indicating that the structure of the protein has changed from random segments to α-helices. -1 The peak at indicates the presence of β-sheet conformation.

[0046] The above test results show that as the freezing storage treatment time in step 13 increases, the 1628cm -1 The peak at 1658cm -1 After 24 hours of freezing, the silk fibroin solution self-assembled into a sponge, and the main peak shifted to 1658 cm -1 .

[0047] Figure 3 : Place the prepared silk fibroin frozen sponge in liquid nitrogen and freeze for 5 minutes. Transfer the frozen solid silk fibroin sponge to a freeze dryer and freeze-dry at -50°C in vacuum for 24-48 hours to completely remove moisture. Take a cross-section of the frozen sponge, plate gold on the surface to be observed, and observe its morphology under a high-resolution field emission scanning electron microscope.

[0048] Exosome release curve and in vivo sustained release of silk fibroin frozen sponge: To quantify the release of exosomes from the scaffold, exosomes were fluorescently labeled with PKH67. Exosome gradient experiments were used to draw a standard curve between exosome concentration and fluorescence intensity, with the maximum concentration of the curve set at 1×10 7 / uL. Silk fibroin-coated exosomes and fibrin glue-coated exosomes contain 1×10 6Scaffolds were prepared in advance. Scaffolds were cut to 5 × 5 × 0.3 mm. A 0.4 U / mL pepsin K solution was used as the working solution for the enzymatic degradation curve, and PBS was used as the working solution for the hydrolysis curve. Scaffolds were immersed in 0.5 mL of the solution. On days 1, 2, 3, 4, 5, 6, 8, and 10, 0.2 mL of the supernatant was collected and neutralized with an equal volume of 1% BSA solution. Fresh, equal volumes of the working solution were replenished. After 10 days, the remaining scaffolds were digested with 4 U / mL pepsin K solution. Exosomes released from the scaffolds were quantified using a microplate reader (excitation: 485 nm; emission: 600 nm), and the corresponding concentrations were calculated using a standard curve. The cumulative exosome release was expressed as the ratio of daily exosome release to the total payload. To measure the degradation rate of the scaffolds, the same volume of scaffolds was lyophilized and weighed, then immersed in 0.5 mL of the degradation solution. On days 1, 2, 3, 4, 5, 6, 8, and 10, the residual scaffolds were removed and rinsed three times with PBS, lyophilized, and weighed. The results were expressed as the ratio of the residual mass to the initial mass.

[0049] The experimental results are as follows Figure 4 As shown, the experimental results show that SF sponge can be degraded under the action of proteases.

[0050] Exosomes were labeled with DiR dye, and silk fibroin material encapsulating fluorescently labeled exosomes was implanted into the right knee joint of SD rats. Fluorescently labeled exosome solution was directly injected into the left knee joint. The in situ residence of fluorescently labeled exosomes in vivo was observed using a small animal in vivo imaging device at 1 day, 3 days, 1 week, 2 weeks, and 4 weeks. The experimental results are as follows: Figure 5 As shown, it can be seen that the silk fibroin frozen sponge can retain exosomes in situ for more than 4 weeks.

[0051] A 1 cm long and 2 mm wide defect was created in the patellar tendon of SD rats. The blank group received no treatment, the SF sponge group was filled with a silk fibroin sponge appropriate to the defect area, and the SF sponge + exosomes group was filled with a silk fibroin sponge loaded with exosomes. Samples were collected at 1, 2, and 4 weeks to observe the repair effects.

[0052] Figure 6 :Observing the macroscopic appearance of the rat patellar tendon defect model at different time points, it can be seen that there is almost no repair in the blank group, and the defect area is empty; in the SF sponge group, residual sponge tissue can be seen filling the defect area at 1, 2, and 4 weeks; in the SF+exosome group, new blood vessels can be seen at 1 week, and new tissue can be seen filling the defect at 2 and 4 weeks.

[0053] Figure 7HE staining of the rat patellar tendon defect model at different time points showed that inflammatory cells gathered in the blank group, mostly scar tissue. Residual sponge material could be seen in the SF sponge group, and inflammatory cells infiltrated around the material. At 4 weeks, most of the sponge material was degraded, the inflammation was alleviated, and scar tissue was surrounding it. In the SF sponge + exosome group, the inflammatory cell infiltration around the material was significantly reduced at 1 week compared with the SF sponge group. At 2 and 4 weeks, the new collagen tissue was neatly arranged compared with the other two groups.

[0054] Figure 8 : Observe the Sirius red staining of the rat patellar tendon defect model at different time points. Type I collagen is orange or red, and new collagen (type III collagen) is green. Mature tendon tissue is mainly composed of type I collagen. It can be seen that the blank group and SF sponge group are mainly type III collagen, while the SF sponge + silk fibroin group can generate neatly arranged type I collagen similar to mature tendon tissue at 4 weeks.

[0055] The preferred embodiments of the present invention are described above, but they are not intended to limit the present invention. Those skilled in the art may make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims

1. Application of an exosome sustained-release preparation in the preparation of a tendon repair preparation, characterized in that: The preparation method of the exosome sustained-release preparation includes: (1) Take 15g of silkworm cocoons and cut them into pieces; (2) Take 4.24 g of anhydrous sodium carbonate, dissolve it in 2 L of deionized water, and heat to boil; (3) Pour the cocoons into water and boil for 30 minutes to remove the sericin; (4) Remove the remaining white sponge, rinse it with deionized water several times, and then dry it thoroughly at 37°C overnight; (5) Calculate the required mass of lithium bromide based on the mass ratio of silk fibroin to lithium bromide = 1:

4. Calculate the total volume of lithium bromide solution required based on the ratio of 9.3 mol / L. Take 70% of the total volume of deionized water and slowly add lithium bromide powder while stirring. After it is completely dissolved, pour it into the graduated cylinder and make up the remaining volume. (6) Squeeze the silk fibroin sponge fully into a 100 mL beaker, slowly pour the lithium bromide solution into the beaker, completely immerse the silk fibroin sponge, seal it, and heat and stir at 60 degrees for 4 hours to completely dissolve it, obtaining a yellow viscous solution; (7) Pour the solution into a dialysis bag with a molecular weight cut-off of 7k for dialysis treatment. Change the water 1-2 times a day, and change the water 5 times in total; (8) Pour the protein solution into a large beaker, divide it into centrifuge tubes, and centrifuge at 9000 rpm at 4°C for 20 min. Remove the supernatant and centrifuge again for 20 min. (9) The silk fibroin solution was divided into 15 mL centrifuge tubes, with 8 mL in each tube. After freezing, lyophilization and dehydration were performed and stored at room temperature. (10) Dissolve the freeze-dried silk fibroin solid that has not been squeezed and stressed in 1× PBS according to the required concentration. Shake it by hand for a few minutes until it dissolves. Centrifuge it at 4000 rpm for 3 minutes, remove the clear liquid in the middle, and sterilize it with UV for 30 minutes. (11) Add excess L-glutamic acid / aspartic acid to the silk fibroin solution, adjust the pH value of the solution to 4, and let it stand for 30 minutes to 1 hour; (12) Centrifuge at 1000 rpm for 3 min, remove the supernatant silk fibroin solution, and mix it with the exosome solution; (13) The mixed solution was frozen at -6°C for 24 hours. After thawing, a solid sponge loaded with exosomes was obtained, which was soaked in PBS and could be stored for a long time.

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