Preparation method and application of thermo-sensitive collagen-chitosan composite gel

Abstract

The invention discloses a preparation method of a thermo-sensitive collagen-chitosan composite gel and an application of the thermo-sensitive collagen-chitosan composite gel. The preparation method of the thermo-sensitive collagen-chitosan composite gel comprises the following steps: dissolving hydroxybutyl chitosan and / or hydroxypropyl chitosan and recombinant I-type collagen in water at the temperature of 2 to 5 DEG C, so as to obtain the thermo-sensitive collagen-chitosan composite gel, the amino acid sequence of the recombinant I type collagen is an amino acid sequence obtained by repeating a short amino acid sequence as shown in SEQ ID No: 1 (GAPGAPGSQGAPGLQ) for 52 times. In the thermo-sensitive collagen-chitosan composite gel prepared by the preparation method disclosed by the invention, the recombinant I-type collagen is self-assembled to form collagen fibers which are more beneficial to aggregation and solidification of platelets and can act on a wound surface for a longer time, so that the thermo-sensitive collagen-chitosan composite gel has a better hemostatic effect and a better wound surface healing effect.

Description

Technical Field

[0001] This patent application belongs to the field of biomaterial technology, and specifically relates to a preparation method and application of a thermosensitive collagen-chitosan composite gel. Background Art

[0002] Wound healing includes a series of continuous, dynamic, complex and overlapping processes, including hemostasis, inflammatory response, proliferation and remodeling. Disordered wound healing can lead to a series of clinical problems, such as diabetic foot ulcers and hypertrophic scars. Millions of people suffer skin injuries every year due to physical and chemical factors or diseases, and subsequent wound infection and severe tissue necrosis endanger human life.

[0003] In recent years, wound dressings have become an indispensable element in clinical wound care due to their advantages such as low cost, ease of use, and significant effects. With the rapid development of materials science and technology, new medical wound dressings (hydrogels, electrospun fibers, films, sponges, etc.) have become a promising strategy in wound healing management. Ideal wound dressings should be non-toxic, degradable, biocompatible, have moderate mechanical strength, be easy to peel off from the wound surface, be antibacterial, promote healing, and be beneficial to reduce scar formation. To this end, researchers use different types of biomaterials to design new medical wound dressings with different functions.

[0004] Collagen is widely used in human wound dressings and tissue engineering products. When soft tissue is damaged, collagen can activate platelet response, promote platelet adhesion and aggregation at the damaged site, form clots, and improve hemostasis efficiency. At the same time, collagen promotes granulation tissue deposition and fibroblast proliferation, thereby accelerating wound healing. In addition, collagen also has good biocompatibility, biodegradability and low immunogenicity. Due to the weak mechanical strength of collagen, its mechanical properties cannot support soft tissue regeneration. It can form a highly ordered network structure through self-assembly in vitro, which is conducive to cell adhesion, proliferation, diffusion and migration. It has good biocompatibility, excellent mechanical properties, biodegradability and weak antigenicity, which provides unlimited possibilities for the application of this material.

[0005] Chitosan is a linear polymer compound composed of glucosamine and N-acetylglucosamine. It is the product of the deacetylation of the natural polymer chitin. Because chitosan has good biodegradability and biocompatibility, it has very wide applications in biology and pharmaceutical fields. Hydroxybutyl chitosan is a degradable, biocompatible, water-soluble chitosan derivative that can spontaneously form a reversible thermosensitive hydrogel at a certain concentration and temperature through intermolecular hydrogen bonding, avoiding the residual chemical crosslinking agent of traditional hydrogels.

[0006] Therefore, how to give full play to the respective advantages of collagen and chitosan and develop medical wound dressings with better hemostasis and wound healing effects is a technical problem faced by those skilled in the art. Summary of the invention

[0007] In view of the problems existing in the prior art, the object of the present invention is to provide a thermosensitive collagen-chitosan composite gel with better hemostatic effect, better wound healing effect and stronger adhesion.

[0008] The inventors conducted in-depth research to solve the above technical problems, and found that the recombinant type I collagen obtained by repeating the short amino acid sequence shown in SEQ ID No: 1 (GAPGAPGSQGAPGLQ) 52 times and mixed with hydroxybutyl chitosan and / or hydroxypropyl chitosan at low temperature (2-5°C) to prepare a thermosensitive collagen-chitosan composite gel showed better performance in hemostatic effect, wound repair and mechanical properties, thereby completing the present invention.

[0009] That is, the present invention includes:

[0010] 1. A method for preparing a thermosensitive collagen-chitosan composite gel, comprising: dissolving hydroxybutyl chitosan and / or hydroxypropyl chitosan and recombinant type I collagen in water at 2 to 5° C. to obtain the thermosensitive collagen-chitosan composite gel; the amino acid sequence of the recombinant type I collagen is an amino acid sequence obtained by repeating the short amino acid sequence shown in SEQ ID No: 1 (GAPGAPGSQGAPGLQ) 52 times.

[0011] 2. The preparation method according to item 1, wherein the final concentration of the hydroxybutyl chitosan and / or hydroxypropyl chitosan is 1-10 wt %, preferably 1-6 wt %, more preferably 2-5 wt %.

[0012] 3. The preparation method according to item 1, wherein the final concentration of the recombinant type I collagen is 0.01-5% by weight, preferably 0.1-1% by weight.

[0013] 4. The preparation method according to item 1, wherein the weight ratio of hydroxybutyl chitosan and / or hydroxypropyl chitosan to recombinant collagen is 1:1-60:1, preferably 2:1-40:1, more preferably 2:1-20:1.

[0014] 5. The preparation method according to item 1, which does not include a cross-linking step.

[0015] 6. The preparation method according to item 1, further comprising packaging the obtained thermosensitive collagen-chitosan composite gel into syringes.

[0016] 7. The preparation method according to item 1 is carried out at an ambient temperature of 2 to 5°C.

[0017] 8. Thermosensitive collagen-chitosan composite gel prepared by the preparation method described in any one of items 1 to 8.

[0018] The thermosensitive collagen-chitosan composite gel is preferably stored under low temperature conditions (eg, 0-15° C., 0-10° C., 2-5° C.), and the thermosensitive collagen-chitosan composite gel is in a liquid state when stored under low temperature conditions.

[0019] 9. Use of the thermosensitive collagen-chitosan composite gel according to item 8 in the preparation of medical wound dressings.

[0020] The medical wound dressing can be preferably used for repairing irregular, closed or narrow soft tissue or bone tissue. In the field of wound healing, the medical wound dressing can be widely used in different types of wounds such as oral cavity, nasal cavity, traumatic wounds, surgical incisions after suture, abrasion wounds, diabetic wounds and infected wounds, and can quickly fill the wound space and form a physical barrier, effectively prevent blood loss, and show the characteristics of rapid hemostasis, promotion of wound healing, effective avoidance of wound infection, inhibition of excessive inflammation and relief of wound pain.

[0021] Effects of the Invention

[0022] The thermosensitive collagen-chitosan composite gel of the present invention has good hemostatic effect and wound healing effect. The reason may be that the recombinant type I collagen can self-assemble and aggregate to form collagen fibers under low temperature conditions (see Chinese patent application publication CN118718121A), and due to factors such as charge, hydroxybutyl chitosan and / or hydroxypropyl chitosan can interact with the collagen self-assembly, produce certain constraints to the collagen self-assembly, and it is not easy to dissociate when it returns to normal temperature. Compared with single collagen, collagen fibers are more conducive to the aggregation and coagulation of platelets, so hemostatic effect is better. Compared with single collagen, collagen fibers are not easy to degrade, and can act on the wound surface for a longer time, so the wound healing effect is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a graph showing the hemostatic effects of Example 1 and Comparative Example 1.

[0024] Figure 2 It is a diagram showing the wound healing effects of Example 1 and Comparative Example 1. DETAILED DESCRIPTION

[0025] The present application is further described below in conjunction with examples. It should be understood that the examples are only used to further describe and illustrate the present application and are not intended to limit the present invention.

[0026] The recombinant type I collagen (i.e., the HLC protein referred to in the above Chinese patent application publication) was prepared according to Example 2 of Chinese patent application publication CN115947828A. Hydroxybutyl chitosan was prepared according to the method reported in the literature (Li Jingjing. Preparation of hydroxybutyl chitosan and study on its hydrogel sensitivity (temperature / pH) and biocompatibility [D]. Ocean University of China, 2011.), and hydroxypropyl chitosan is a commercially available product.

[0027] Example 1 Preparation of thermosensitive wound protection hemostatic repair gel 1

[0028] Take 10g of hydroxybutyl chitosan, 0.5g of recombinant type I collagen, and 5g of glycerol, add them into 500ml of purified water precooled to 2-5°C, and dissolve them completely at ambient temperature 2-5°C to obtain a thermosensitive wound protection hemostatic repair gel 1, which is divided into syringes and stored in a low temperature environment.

[0029] Example 2 Preparation of Thermosensitive Wound Hemostatic Repair Gel 2

[0030] Thermosensitive wound protection and hemostatic repair gel 2 was prepared as in Example 1, except that 40 g of hydroxybutyl chitosan and 20 g of recombinant type I collagen were used.

[0031] Example 3 Preparation of Thermosensitive Wound Hemostatic Repair Gel 3

[0032] Thermosensitive wound protection and hemostatic repair gel 3 was prepared as in Example 1, except that 10 g of hydroxypropyl chitosan and 0.2 g of recombinant type I collagen were used.

[0033] Comparative Example 1 Comparison of the preparation of thermosensitive wound protection hemostatic repair gel 1

[0034] A comparative thermosensitive wound protection hemostatic repair gel 1 was prepared as in Example 1, except that the purified water was at 10-12° C. and all operations were performed at 10-12° C. At 10-12° C., hydroxybutyl chitosan is soluble in water, but the recombinant type I collagen cannot self-assemble.

[0035] Comparative Example 2 Comparative Preparation of Thermosensitive Wound Hemostatic Repair Gel 2

[0036] Use commercially available thermosensitive hydroxybutyl chitosan wound dressing.

[0037] Comparative Example 3 Preparation of Thermosensitive Wound Hemostasis Repair Gel 3

[0038] A comparative thermosensitive wound protection hemostatic repair gel 3 was prepared as in Example 1, except that:

[0039] Poly (N-isopropylacrylamide) was used as the thermosensitive polymer instead of hydroxybutyl chitosan.

[0040] Comparative Example 4 Comparison of the preparation of thermosensitive wound protection hemostatic repair gel 4

[0041] A comparative thermosensitive wound protection hemostatic repair gel 4 was prepared as in Comparative Example 3, except that:

[0042] The recombinant type I collagen was not added.

[0043] Comparative Example 5 Comparative Preparation of Thermosensitive Wound Hemostatic Repair Gel 5

[0044] Comparative thermosensitive wound protection hemostatic repair gel 5 was prepared as in Comparative Example 3, except that the purified water was 10-12° C. and all operations were performed at 10-12° C. At 10-12° C., N-isopropylacrylamide is soluble in water, but the recombinant type I collagen cannot self-assemble.

[0045] The thermosensitive wound protection hemostatic repair gels obtained in Examples 1-3 and Comparative Examples 1-4 were taken out from the low-temperature storage environment, and hemostasis and wound healing tests were performed at room temperature to observe the hemostatic effect and wound healing effect of each thermosensitive wound protection hemostatic repair gel. The results are shown in Table 1:

[0046] Table 1 Preparation conditions and related indicators of thermosensitive wound protection and hemostatic repair gel

[0047]

Claims

1. A method for preparing a thermosensitive collagen-chitosan composite gel, comprising: Hydroxybutyl chitosan and / or hydroxypropyl chitosan and recombinant type I collagen are dissolved in water at 2 to 5° C. to obtain the thermosensitive collagen-chitosan composite gel; the amino acid sequence of the recombinant type I collagen is an amino acid sequence obtained by repeating the short amino acid sequence shown in SEQ ID No: 1 (GAPGAPGSQGAPGLQ) 52 times.

2. The preparation method according to claim 1, wherein The final concentration of the hydroxybutyl chitosan and / or hydroxypropyl chitosan is 1-10% by weight, preferably 1-6% by weight, more preferably 2-5% by weight.

3. The preparation method according to claim 1, wherein The final concentration of the recombinant type I collagen is 0.01-5% by weight, preferably 0.1-1% by weight.

4. The preparation method according to item 1, wherein: The weight ratio of the hydroxybutyl chitosan and / or hydroxypropyl chitosan to the recombinant collagen is 1:1-60:1, preferably 2:1-40:1, more preferably 2:1-20:

1. The preparation method according to claim 1 , wherein no cross-linking step is included. The preparation method according to claim 1 , further comprising dispensing the obtained thermosensitive collagen-chitosan composite gel into syringes. The preparation method according to claim 1 , which is carried out at an ambient temperature of 2 to 5° C. .

8. Thermosensitive collagen-chitosan composite gel prepared by the preparation method according to any one of claims 1 to 8; The thermosensitive collagen-chitosan composite gel is preferably stored under low temperature conditions (eg, 0-15° C., 0-10° C., 2-5° C.), and the thermosensitive collagen-chitosan composite gel is in a liquid state when stored under low temperature conditions.

9. Use of the thermosensitive collagen-chitosan composite gel according to claim 8 in the preparation of medical wound dressings.

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