Exosome-loaded heat-sensitive reversible adhesive gel and preparation method thereof

Abstract

The invention relates to a gel biological material, in particular to exosome-loaded heat-sensitive reversible adhesive gel and a preparation method thereof. According to the preparation method, periodate is used as an oxidizing agent to oxidize and specifically crack vicinal diol in chondroitin sulfate to form a dialdehyde derivative, and each r-ethylene glycol group consumes one molecule of periodate; carrying out self-crosslinking on the hydroformylated chondroitin sulfate and gelatin in the presence of borax to form heat-sensitive reversible adhesive gel; and dropwise adding the exosome to the surface of the heat-sensitive reversible adhesive gel to enable the exosome to freely permeate, so as to obtain the exosome-loaded heat-sensitive reversible adhesive gel. The gel has relatively high biocompatibility and relatively strong adhesion strength, and can regulate and control the immune response of an organism.

Description

Technical Field

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

[0002] Surgical adhesives and sealants have a wide range of applications in surgery. There are a limited number of artificial and natural surgical adhesives and sealants in clinical use. For example, natural fibrin glue sealants have been developed for tissue sealing, wound closure, and hemostasis. Although these fibrin-based glues are biocompatible, they have weak adhesion strength, high cost, and a risk of viral infection. Synthetic-based adhesives, especially the commonly used cyanoacrylates, have strong adhesion strength to tissues; however, they have low biocompatibility and biodegradability, and the degradation products are highly toxic, causing excessive inflammatory responses in the host. Summary of the invention

[0003] In view of the above technical problems, the object of the present invention is to provide an exosome-loaded thermosensitive reversible adhesion gel with high biocompatibility, strong adhesion strength and the ability to regulate the body's immune response, and to provide a method for preparing the exosome-loaded thermosensitive reversible adhesion gel.

[0004] The purpose of the present invention is achieved through the following technical solutions: A method for preparing an exosome-loaded thermosensitive reversible adhesion gel comprises the following steps: (1) Periodate acts as an oxidant to specifically cleave the vicinal diol in chondroitin sulfate to form a dialdehyde derivative. Each r-ethylene glycol group consumes one molecule of periodate. The aldehyde-modified chondroitin sulfate self-crosslinks with gelatin in the presence of borax to form a thermosensitive reversible adhesive gel. (2) Exosomes are dropped onto the surface of the thermosensitive reversible adhesion gel to allow them to penetrate freely, thereby obtaining the exosome-loaded thermosensitive reversible adhesion gel.

[0005] Furthermore, the periodate described in step (1) is sodium periodate.

[0006] Furthermore, the preparation method of the thermosensitive reversible adhesive gel is as follows: dissolving chondroitin sulfate in distilled water, adding sodium periodate solution under light-proof conditions, and stirring vigorously; after the oxidation reaction is completed, adding borax and stirring for further reaction for 20-30 minutes. Dialyzing the reaction solution for 48 hours, freezing the dialysate and drying it to obtain oxidized chondroitin sulfate; preparing the oxidized chondroitin sulfate into an oxidized chondroitin sulfate solution and quickly mixing it with a gelatin solution, and continuing the reaction for 5-6 hours to obtain the gelatin ...

[0007] Furthermore, the chondroitin sulfate solution is added to the sodium periodate solution at a speed of 5-6 drops / second.

[0008] Furthermore, the method for drying the dialysate after freezing is freeze drying, and the drying time is 6-8 days.

[0009] Furthermore, the reaction temperature of the oxidized chondroitin sulfate solution and the gelatin solution is 37°C.

[0010] Compared with the prior art, the present invention has the following beneficial effects: 1. The preparation method of the exosome-loaded thermosensitive reversible adhesion gel of the present invention does not add any toxic cross-linking agent, has good biosafety, and has mild preparation conditions.

[0011] 2. The exosome-loaded thermosensitive reversible adhesion gel obtained by the preparation method of the present invention has high biocompatibility and strong adhesion strength. The adhesion strength measured by the pig skin lap shear test is about 37 kPa. It can also regulate the body's immune response and regulate the host's immune response after implantation, so that the body can reduce inflammatory response or even anti-inflammatory. DETAILED DESCRIPTION

[0012] 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

[0013] Chondroitin sulfate was dissolved in 90 ml of distilled water and added to 20 ml of an aqueous solution containing 1.93 g of sodium periodate at a rate of 6 drops / second under light-proof conditions, while stirring vigorously. After the oxidation reaction was completed, 5 ml of ethylene glycol was added and stirred for 30 min, and then 1.93 g of borax was added and stirred for further 30 min. The reaction solution was dialyzed with a dialysis bag at room temperature for 48 h, and then the dialysate was frozen and freeze-dried for 7 days to obtain a white cotton-like substance, which was oxidized chondroitin sulfate. 1 g of oxidized chondroitin sulfate was dissolved in 10 mL of PBS solution to prepare an oxidized chondroitin sulfate solution with a concentration of 10 w / v%. 6 g of gelatin was dissolved in 20 mL of PBS solution at 60°C, and then 0.5 mL of each solution was taken in a 2 m PE tube and mixed quickly, and then placed in a 37°C oven for continuous reaction for 5 h to obtain a thermosensitive reversible adhesion gel.

[0014] 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 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.

[0015] RAW264.7 cells were purchased from ATCC Cell Bank and cultured in 1640 medium (GIBCO) at 37°C and 5% CO2 2.2.

[0016] First, at 4 ° C, centrifuge at 120000 g ultracentrifugation (Optima-90 K, Beckman Coulter) for 14 h to prepare in vitro peripheral fetal bovine serum for further BMSCs culture. After 48 h of culture, the BMSCs culture medium was collected and then subjected to multi-step centrifugation, 2000 g centrifugation for 20 min, 10000 g centrifugation for 30 min, and finally 100000 g centrifugation for 90 min to separate exosomes. The exosomes were added dropwise to the surface of the thermosensitive reversible adhesive gel to allow it to freely penetrate into the hydrogel, thereby obtaining an exosome-loaded thermosensitive reversible adhesive gel. The exosome-loaded thermosensitive reversible adhesive gel obtained in the example was subjected to a pigskin lap shear test, and the adhesion strength was measured to be 37Kpa.

[0017] Exosome-loaded thermosensitive reversible adhesive gel can have high biocompatibility, strong adhesion strength, and can regulate the body's immune response, regulate the host's immune response after implantation, and reduce the body's inflammatory response or even anti-inflammatory. The hydrogel is self-healing, injectable, and remodelable, so it is easy to use in surgical operations through syringes and other delivery systems frequently used in interventional procedures. The hydrogel has good biocompatibility and degradability. The outstanding adhesion and hemostasis ability of the hydrogel helps to effectively treat fatal liver bleeding wounds and immediately seal injured uterine tissue without sutures or staples. In addition, exosomes can promote the repair of damaged tissues and regulate immune responses without causing excessive immune responses in the body.

[0018] 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 thermosensitive reversible adhesion gel, characterized in that: The following steps are involved: (1) Periodate acts as an oxidant to specifically cleave the vicinal diol in chondroitin sulfate to form a dialdehyde derivative. Each r-ethylene glycol group consumes one molecule of periodate. The aldehyde-modified chondroitin sulfate self-crosslinks with gelatin in the presence of borax to form a thermosensitive reversible adhesive gel. (2) Exosomes are dropped onto the surface of the thermosensitive reversible adhesion gel to allow them to penetrate freely, thereby obtaining the exosome-loaded thermosensitive reversible adhesion gel.

2. The method for preparing the exosome-loaded thermosensitive reversible adhesion gel according to claim 1, characterized in that: The periodate described in step (1) is sodium periodate.

3. The method for preparing the exosome-loaded thermosensitive reversible adhesion gel according to claim 1, characterized in that: The preparation method of the thermosensitive reversible adhesive gel is as follows: dissolving chondroitin sulfate in distilled water, adding sodium periodate solution under light-proof conditions, and stirring vigorously at the same time; after the oxidation reaction is completed, adding borax and stirring for further reaction for 20-30 minutes. The reaction solution is dialyzed for 48 hours, and the dialysate is frozen and dried to obtain oxidized chondroitin sulfate; the oxidized chondroitin sulfate is prepared into an oxidized chondroitin sulfate solution and quickly mixed with a gelatin solution, and the reaction is continued for 5-6 hours to obtain the gelatin ...

4. The method for preparing the exosome-loaded thermosensitive reversible adhesion gel according to claim 3, characterized in that: The chondroitin sulfate solution was added to the sodium periodate solution at a rate of 5-6 drops / second.

5. The method for preparing the exosome-loaded thermosensitive reversible adhesion gel according to claim 3, characterized in that: The method for drying the dialysate after freezing is freeze drying, and the drying time is 6-8 days.

6. The method for preparing the exosome-loaded thermosensitive reversible adhesion gel according to claim 5, characterized in that: The reaction temperature of the oxidized chondroitin sulfate solution and the gelatin solution was 37°C.

7. An exosome-loaded thermosensitive reversible adhesion gel obtained by the preparation method of claim 1.