Milk exosome composite hydrogel system as well as preparation method and application thereof

Abstract

The invention provides milk exosome composite hydrogel as well as a preparation method and application thereof, and belongs to the technical field of medical materials. The milk exosome composite hydrogel provided by the invention is prepared from milk exosome, hyaluronic acid, epsilon-polylysine, 3-aminophenylboronic acid and tannic acid. The exosome is extracted from milk of a non-stem cell source; the molecular weight of the hyaluronic acid is 200 to 400 KDa. The invention provides a hydrogel carrier capable of loading a large amount of milk exosomes, and the hydrogel carrier can continuously release a large amount of milk exosomes and can give full play to the anti-oxidation and inflammation inhibition effects of the milk exosomes. In the aspect of wound healing, the milk exosome and the hydrogel carrier have a synergistic effect, so that the healing effect can be remarkably improved, and the healing period is shortened.

Description

[Technical field]

[0001] The present invention relates to the technical field of medical materials, and in particular to a milk exosome composite hydrogel system and a preparation method and application thereof. [Background technology]

[0002] Exosomes are endogenous vesicles with a diameter of about 30 to 150 nm. They are one of the extracellular vesicles with multiple biological activities and intercellular communication functions. Because they inherit the characteristics of parental cells, they can improve and accelerate wound healing through inflammation regulation, immune protection, angiogenesis, cell proliferation and migration, and collagen remodeling imbalance regulation. As a new cell-free treatment strategy, it has attracted widespread attention from researchers and has broad application prospects. However, exosomes can only exist in the body for 3 hours and will be lost with physiological circulation and metabolism, thereby losing their effect. This limits its application. To solve this problem, researchers use constructed carriers to coat them to achieve the effect of sustained release drug delivery. For example, some people load exosomes derived from umbilical cord mesenchymal stem cells onto hydrogels for liver regeneration after liver resection (CN202410215305.8, 2024.02.27). However, most exosomes are derived from stem cells, which leads to high production costs and is not suitable for large-scale preparation, so that the load of exosomes is very small. Milk exosomes are derived from non-stem cell sources. They are not only inexpensive to prepare, but also can accelerate wound healing, with a healing effect similar to that of stem cell-derived exosomes. Therefore, we constructed a hydrogel carrier that can load a large amount of milk exosomes, so that it can continuously release a large amount of milk exosomes and give full play to the antioxidant and anti-inflammatory effects of milk exosomes. Milk exosomes and hydrogel carriers synergistically heal wounds, which can significantly improve the healing effect and shorten the healing cycle. [Summary of the invention]

[0003] The object of the present invention is to provide a milk exosome composite hydrogel and a preparation method and application thereof. The milk exosome composite hydrogel provided by the present invention can continuously release a large amount of milk exosomes, significantly reduce the inflammatory response of the wound, promote the formation of blood vessels and granulation tissue, thereby shortening the healing cycle and healing the wound faster.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for preparing a milk exosome composite hydrogel, comprising the following steps:

[0006] (1) Hyaluronic acid is oxidized with sodium periodate, and after the reaction is completed, it is dialyzed and freeze-dried to obtain oxidized hyaluronic acid.

[0007] (2) 3-aminophenylboronic acid is dissolved in methanol to prepare a solution, and tannic acid is dissolved in phosphate buffered saline (PBS) to prepare a solution of a certain concentration, and finally the two are mixed to prepare a phenylboronic acid-tannic acid solution.

[0008] (3) The oxidized hyaluronic acid, ε-polylysine and milk exosomes in step (1) are dissolved in PBS to prepare solutions. These solutions are then mixed with the phenylboronic acid-tannic acid solution in step (2) in different proportions at room temperature to form a hydrogel in a short time.

[0009] Preferably, the optimal molar ratio between the 3-aminophenylboronic acid group and tannic acid in step (2) is 1:0.1; and the weight percentage of the phenylboronic acid-tannic acid solution is 2.4wt%.

[0010] Preferably, in step (3), the molar ratio of the oxidized hyaluronic acid to ε-polylysine is 1:1; the weight percentage of the oxidized hyaluronic acid solution is 5wt%; and the weight percentage of the ε-polylysine solution is 3.9wt%.

[0011] Preferably, in step (3), the oxidized hyaluronic acid solution, the ε-polylysine solution and the phenylboronic acid-tannic acid solution are mixed in a volume ratio of 2:1:1; and the final concentration of the exosomes is 0.5 mg / mL.

[0012] Preferably, the pH environment of the reaction in step (3) is 7.5, and the gel time is about 10 seconds.

[0013] The invention provides a milk exosome composite hydrogel prepared according to the method.

[0014] The present invention also provides application of the milk exosome composite hydrogel in the field of promoting wound healing.

[0015] The milk exosome composite hydrogel provided by the present invention has good biocompatibility, and has the characteristics of antibacterial, antioxidative, self-healing and injectable. The milk exosome composite hydrogel is used for wound healing, which can continuously release a large amount of milk exosomes and synergize with the carrier to significantly reduce the inflammatory response of the wound, promote the formation of blood vessels and granulation tissue, thereby shortening the healing cycle and healing the wound faster.

Brief Description of the Drawings

[0016] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification for explanation. The accompanying drawings are as follows:

[0017] Figure 1 This is a physical picture of the successful preparation verification of the hydrogel of the method for preparing milk exosome composite hydrogel of the present invention.

[0018] Figure 2 This is a self-healing injectable physical picture of the preparation method of a milk exosome composite hydrogel of the present invention.

[0019] Figure 3 This is a diagram showing the animal test results of a method for preparing a milk exosome composite hydrogel of the present invention. [Specific implementation method]

[0020] The present invention will be further described and illustrated by specific embodiments and accompanying drawings below:

[0021] (1) Preparation of oxidized hyaluronic acid: First, weigh 2 g of hyaluronic acid (200-400 KDa) and dissolve it in 200 ml of deionized water to prepare a solution; then weigh 1.13 g of sodium periodate at a molar ratio of 1:1 to hyaluronic acid, dissolve it in 15 ml of deionized water, add it dropwise to the hyaluronic acid solution, and stir it for 4 hours in a dark environment at a speed of 600 rpm. Then add 1 ml of ethylene glycol to quench the unreacted sodium periodate, and stir it for another hour. Finally, the obtained product is dialyzed with a dialysis bag (8000-14000 Da) for 3 days, and then freeze-dried to finally obtain oxidized hyaluronic acid.

[0022] (2) Preparation of phenylboronic acid-tannic acid: Weigh 0.1 g of 3-aminophenylboronic acid, dissolve it in 2.75 ml of methanol, and stir at room temperature until it is completely dissolved. Then, according to the molar ratio between 3-aminophenylboronic acid group and tannic acid (1:0.05, 1:0.075, 1:0.1, 1:0.125), weigh 0.0155 g, 0.0233 g, 0.031 g, and 0.0388 g of tannic acid, respectively, dissolve them in 2.75 ml of PBS to prepare a solution. Then, mix the two at room temperature to prepare a phenylboronic acid-tannic acid solution, which is stored for later use.

[0023] (3) Preparation of hydrogel: 0.55 g of oxidized hyaluronic acid and 0.213 g of ε-polylysine were weighed according to the ratio of aldehyde group: amino group = 2:1. The oxidized hyaluronic acid was dissolved in 11 mL of PBS to prepare a 5 wt% solution; the ε-polylysine was dissolved in 5.5 mL of PBS to prepare a 3.9 wt% solution. Then, ε-polylysine, phenylboronic acid-tannic acid and milk exosomes were mixed together at room temperature under a pH value of 7.5. The final concentration of milk exosomes was 0.5 mg / mL. The oxidized hyaluronic acid solution was then added according to the volume ratio of oxidized hyaluronic acid: ε-polylysine: phenylboronic acid-tannic acid = 2:1:1. The hydrogel was formed in a short time.

[0024] Hydrogel formation and confirmation: References Figure 1 , if no obvious flow of the mixed solution is observed during the inversion of the container, the formation of the hydrogel is confirmed.

[0025] refer to Figure 2 , the hydrogel is self-healing and injectable.

[0026] Specific experimental test results are as follows Figure 3 , showing the changes in wounds after one of the hydrogels was used in an animal experiment 14 days later.

[0027] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the present invention.

Claims

1. A milk exosome composite hydrogel, characterized in that: The invention comprises milk exosomes, hyaluronic acid, ε-polylysine, 3-aminophenylboronic acid and tannic acid. The exosomes are exosomes extracted from milk of non-stem cell source; and the molecular weight of the hyaluronic acid is 200-400KDa.

2. The method for preparing the milk exosome composite hydrogel according to claim 1, characterized in that: The following steps are involved: (1) Hyaluronic acid is oxidized with sodium periodate, and after the reaction is completed, it is dialyzed and freeze-dried to obtain oxidized hyaluronic acid. (2) 3-aminophenylboronic acid is dissolved in methanol to prepare a solution, and tannic acid is dissolved in phosphate buffered saline (PBS) to prepare a solution of a certain concentration, and finally the two are mixed to prepare a phenylboronic acid-tannic acid solution. (3) The oxidized hyaluronic acid, ε-polylysine and milk exosomes in step (1) are dissolved in PBS to prepare solutions. These solutions are then mixed with the phenylboronic acid-tannic acid solution in step (2) in different proportions at room temperature to form a hydrogel in a short time.

3. The method for preparing the milk exosome composite hydrogel according to claim 2, characterized in that: The optimal molar ratio between the 3-aminophenylboronic acid group and tannic acid in step (2) is 1:0.1; the weight percentage of the phenylboronic acid-tannic acid solution is 2.4wt%.

4. The method for preparing the milk exosome composite hydrogel according to claim 2, characterized in that: In step (3), the molar ratio of the oxidized hyaluronic acid to ε-polylysine is 1:1; the weight percentage of the oxidized hyaluronic acid solution is 5wt%; and the weight percentage of the ε-polylysine solution is 3.9wt%.

5. The method for preparing the milk exosome composite hydrogel according to claim 2, characterized in that: In step (3), the oxidized hyaluronic acid solution, the ε-polylysine solution and the phenylboronic acid-tannic acid solution are mixed in a volume ratio of 2:1:1; the final concentration of the exosomes is 0.5 mg / mL.

6. The method for preparing the exosome composite hydrogel according to claim 2, characterized in that: The pH environment of the reaction in step (3) is 7.5, and the gel time is about 10 seconds.

7. The method for preparing the milk exosome composite hydrogel according to any one of claims 2 to 6, characterized in that: The hydrogel is cross-linked by dynamically reversible Schiff base bonds and has the characteristics of self-healing and injectability, which can cope with the healing of irregular wounds. Moreover, the hydrogel can serve as a carrier to load a large amount of milk exosomes and can continuously release a large amount of milk exosomes, giving full play to the antioxidant and anti-inflammatory effects of milk exosomes, significantly improving the healing effect and shortening the healing cycle.

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