Exosome-loaded electroactive hydrogel dressing and preparation method thereof

Abstract

The invention relates to a gel biological material, in particular to an exosome-loaded electroactive hydrogel dressing and a preparation method thereof. The preparation method comprises the following steps: adding pyrrole into a phytic acid aqueous solution, stirring for reaction, mixing with an ammonium persulfate solution to promote gelation, and removing the residual reaction reagent to obtain conductive hydrogel; and dropwise adding exosome extracted from bone marrow mesenchymal stem cells to the surface of the conductive hydrogel to enable the exosome to freely permeate, thereby obtaining the exosome-loaded electroactive hydrogel dressing. The gel has biocompatibility, is soft in texture, rich in moisture and strong in adhesion, and has self-healing property. As a highly conductive thin film, the thin film is attached to injured nerves of a diabetes model, is automatically wrapped in a tubular structure with matched size, and forms a stable and intimate electric bridge with electrical protogenic nervous tissues to promote axonal regeneration and remyelination.

Description

Technical Field

[0001] The present invention relates to an exosome-loaded hydrogel material, and more specifically, to an exosome-loaded electroactive hydrogel dressing. The present invention also relates to a method for preparing the exosome-loaded electroactive hydrogel dressing. Background Art

[0002] Conductive electroactive materials have a wide range of applications in the medical field. Common electroactive materials include polypyrrole (PPy), polyaniline (PAni), polythiophene (PTh), etc. These materials are also often used in the manufacture of artificial nerve catheters. However, the catheter materials made of these materials have a rigid structure, lack biocompatibility with the body's nerve tissue, and are sticky and inelastic. These characteristics of traditional catheters usually require further trimming of residual nerve fibers and suturing the catheter to the distal end. Surgical interventions, including sutures or insertion of catheters to bridge the gap, are ineffective for diabetic nerve damage. This is because diabetic patients have extremely slow regeneration of damaged nerves (about 1mm / day) due to complex neurophysiological defects, accompanied by severe demyelination. In addition, invasive interventions and implantation of mismatched implants may lead to additional trauma and complications, such as scarring, increased inflammation, and progressive WD. Therefore, biomaterials used to treat diabetic nerve crush injuries should be soft, adhesive, biocompatible, and able to be inserted through non-invasive procedures. Summary of the invention

[0003] In view of the above technical problems, the purpose of the present invention is to provide an exosome-loaded biocompatible conductive hydrogel (ECH) dressing with excellent mechanical properties, electrical activity and the ability to promote nerve repair, so as to achieve local neuromodulation and treat peripheral nerve damage in diabetic patients, and provide a method for preparing the exosome-loaded electroactive hydrogel dressing.

[0004] The purpose of the present invention is achieved through the following technical solutions: A method for preparing an exosome-loaded electroactive hydrogel dressing comprises the following steps: (1) adding pyrrole to a phytic acid aqueous solution and stirring the mixture to react, mixing the mixture with an ammonium persulfate solution to promote gelation, and removing the remaining reaction reagents to obtain a conductive hydrogel; (2) Exosomes extracted from bone marrow mesenchymal stem cells are dropped onto the surface of the conductive hydrogel to allow them to penetrate freely, thereby obtaining an exosome-loaded electroactive hydrogel dressing.

[0005] Furthermore, the mass percentage concentration of phytic acid in the phytic acid aqueous solution in step (1) is 0.15%-0.6%.

[0006] Furthermore, the dosage of the pyrrole is 1-1.5 mmol pyrrole / mL phytic acid solution.

[0007] Furthermore, the gelation is promoted at a temperature of 4°C.

[0008] Furthermore, in the step (2), after the bone marrow mesenchymal stem cells are cultured in a culture medium, an in vitro culture medium free of peripheral fetal bovine serum is prepared for further BMSCs culture; after the culture, the exosomes are obtained by centrifugation.

[0009] Furthermore, the bone marrow mesenchymal stem cells were cultured at 37° C. and 5% CO 2 .

[0010] Compared with the prior art, the present invention has the following beneficial effects: The exosome-loaded electroactive hydrogel dressing obtained by the preparation method of the present invention is composed of phytic acid, pyrrole, ammonium persulfate and exosomes, has biocompatibility, soft texture, rich in water, strong adhesion, and self-healing. As a highly conductive film, it adheres to the damaged nerves in the diabetic model and is automatically wrapped in a tubular structure of matching size, forming a stable and intimate bridge with the electrogenic nerve tissue, promoting axon regeneration and remyelination. In addition, exosomes can promote the repair of damaged peripheral nerves and regulate immune responses. Therefore, the exosome-loaded biocompatible electroactive hydrogel dressing can work together in many aspects to repair damaged peripheral nerves. DETAILED DESCRIPTION

[0011] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods, but does not constitute any limitation to the present invention. Example

[0012] Phytic acid (PA) was dissolved in deionized water to obtain a PA solution with a mass percentage concentration of 0.6%. 0.5 mmol pyrrole (py, 99%, Aldrich, St. Louis, USA) was added to 0.6 mL PA solution and then stirred to react to obtain solution I. 0.316 g ammonium persulfate (APS) powder was dissolved in 0.6 mL water to prepare solution II at 4°C. Solutions I and II were gently mixed at 4°C to promote gelation. The hydrogel was incubated in distilled water (DI) overnight at 4°C to remove any remaining reaction reagents to obtain a biocompatible conductive hydrogel (ECH).

[0013] Bone marrow mesenchymal stem cells were obtained by flushing the bone marrow cavity from the bilateral humerus and femur of 1-2 week old female SD rats. The obtained bone marrow mesenchymal stem cells were cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO) containing 10% fetal bovine serum (FBS, GIBCO) at 37°C and 5% CO2.

[0014] RAW264.7 cells were purchased from ATCC Cell Bank and cultured in 1640 medium (GIBCO) at 37°C and 5% CO 2 Culture under the conditions of 2.2.

[0015] First, at 4°C, the culture medium was centrifuged at 120,000 g (Optima-90 K, Beckman Coulter) for 14 h to prepare the in vitro free peripheral fetal bovine serum for further BMSCs culture. After 48 h of culture, the BMSCs culture medium was collected and then subjected to multiple steps of centrifugation, including 2000 g for 20 min, 10,000 g for 30 min, and finally 100,000 g for 90 min to separate the exosomes. The exosomes were added dropwise to the surface of the conductive hydrogel to allow them to freely penetrate into the hydrogel.

[0016] The obtained exosome-loaded electroactive hydrogel dressing has matching tissue mechanical properties, with an experimentally measured Young's modulus of about 1000 pa and an electroactivity of 3.6*10-4 S to achieve local neuromodulation. The exosome-loaded electroactive hydrogel dressing is composed of phytic acid, pyrrole, ammonium persulfate and exosomes, and is biocompatible, soft, rich in water, highly adhesive and self-healing. As a highly conductive film, it adheres to the damaged nerves in the diabetic model and is automatically wrapped in a tubular structure of matching size, forming a stable and intimate bridge with the electrogenic nerve tissue, promoting axonal regeneration and remyelination. In addition, exosomes can promote the repair of damaged peripheral nerves and regulate immune responses. Therefore, exosome-loaded electroactive hydrogel dressings can work together in multiple aspects to repair damaged peripheral nerves.

[0017] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. A person skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features may be replaced by equivalents. These modifications or replacements should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing an exosome-loaded electroactive hydrogel dressing, characterized in that: The following steps are involved: (1) adding pyrrole to a phytic acid aqueous solution and stirring the mixture to react, mixing the mixture with an ammonium persulfate solution to promote gelation, and removing the remaining reaction reagents to obtain a conductive hydrogel; (2) Exosomes extracted from bone marrow mesenchymal stem cells are dropped onto the surface of the conductive hydrogel to allow them to penetrate freely, thereby obtaining an exosome-loaded conductive hydrogel dressing.

2. The method for preparing the exosome-loaded electroactive hydrogel dressing according to claim 1, characterized in that: The mass percentage concentration of phytic acid in the phytic acid aqueous solution in step (1) is 0.15%-0.6%.

3. The method for preparing the exosome-loaded electroactive hydrogel dressing according to claim 1, characterized in that: The dosage of the pyrrole is 1-1.5 mmol pyrrole / mL phytic acid solution.

4. The method for preparing the exosome-loaded electroactive hydrogel dressing according to claim 3, characterized in that: The gel acceleration was carried out at a temperature of 4°C.

5. The method for preparing the exosome-loaded electroactive hydrogel dressing according to claim 1, characterized in that: In the step (2), bone marrow mesenchymal stem cells are cultured in a culture medium to prepare in vitro peripheral fetal bovine serum-free culture for further BMSCs culture; after culture, exosomes are obtained by centrifugation.

6. The method for preparing the exosome-loaded electroactive hydrogel dressing according to claim 5, characterized in that: The bone marrow mesenchymal stem cells were cultured at 37° C. and 5% CO 2 .

7. An exosome-loaded electroactive hydrogel dressing obtained by the preparation method of claim 1.