Hydrogel beneficial to wound healing and preparation method thereof

Abstract

The invention relates to hydrogel beneficial to wound healing and a preparation method thereof. The hydrogel comprises chitosan and polyamino acid, and the mass ratio of the chitosan to the polyamino acid is (5: 1)-(1: 2); the mass of the mixed solution accounts for 70%-98% of the total mass. According to the technical scheme, the PRP / chitosan / polyamino acid three-network hydrogel is formed, the infectious wound inflammation microenvironment can be doubly adjusted, and the hydrogel has good mechanical performance and self-healing performance and is suitable for being applied to wounds. Most importantly, the hydrogel not only can inhibit bacteria and shorten the inflammation period, but also can supplement repair factors and accelerate the repair period.

Description

Technical Field

[0001] The invention belongs to the medical field, and particularly relates to a hydrogel that helps wound healing and a preparation method thereof. Background Art

[0002] Wound healing can be roughly divided into four stages: hemostasis, inflammation, proliferation and remodeling. If the wound is infected by bacteria, it will lead to the extension of the inflammatory stage, aggravate the inflammatory response, and destroy the balance of the immune environment of the wound tissue; it will also lead to insufficient growth factors in the repair stage, resulting in insufficient angiogenesis, reduced collagen synthesis, delayed epithelialization, and ultimately insufficient wound proliferation and remodeling.

[0003] Therefore, antibacterial, regulating inflammatory state and promoting tissue regeneration are the key to the treatment of infected wounds.

[0004] Traditional wound treatment methods mainly involve wound debridement and dressing, and the use of sterile gauze bandages, and the use of antibiotics to control infection when necessary. However, the frequent use of bactericidal solutions (such as hydrogen peroxide) can cause significant damage to tissues, destroy granulation tissue, inhibit re-epithelialization, and be detrimental to the later repair of wounds. In addition, the problem of increased bacterial resistance caused by the widespread use of antibiotics in recent years has also become increasingly serious.

[0005] In order to deal with the above problems, in recent years, bio-derived gel materials such as chitosan, mussel protein, alginate, etc. have been widely used in wound healing research due to their good biocompatibility and good antibacterial properties. However, the use of antibacterial gel alone is not enough to deal with the entire process of infectious wound repair. There are also some composite hydrogels under research, but there are problems in the design, such as poor antibacterial effect, addition of antibiotics, lack of immune regulation, or insufficient wound repair ability, which cannot play a role in the entire process of wound repair.

[0006] In summary, the existing technology cannot both effectively fight bacteria and promote wound healing. Summary of the invention

[0007] The purpose of the present invention is to solve the problem that the medical materials in the prior art cannot both effectively fight bacteria on wounds and promote wound healing.

[0008] To achieve the above object, the present invention provides a network hydrogel that helps wound healing, comprising:

[0009] Chitosan,

[0010] Polyamino acid, the mass ratio of chitosan to polyamino acid is 5:1 to 1:2;

[0011] Mixed liquid, the mass of the mixed liquid accounts for 70% to 98% of the total mass.

[0012] Furthermore, the chitosan is N-acylated chitosan, chitosan hydrochloride, N-carboxymethyl chitosan, O-carboxymethyl chitosan, O-acylated chitosan, carboxylated chitosan, N-alkylated chitosan, O-alkylated chitosan, quaternized chitosan, N-carboxy chitosan or O-carboxy chitosan.

[0013] Furthermore, the polyamino acid is polyleucine, polyaspartic acid, polylysine, polythreonine, polycysteine ​​or polyglutamic acid.

[0014] Furthermore, the mixed solution is pure water, ultrapure water, heavy water, organic acid, inorganic acid, organic base, inorganic base or cell buffer.

[0015] Furthermore, the mass ratio of chitosan to polyamino acid is 3:1.

[0016] Furthermore, the mass of the mixed liquid accounts for 88% of the total mass.

[0017] Furthermore, it also includes:

[0018] The volume ratio of PRP, PRP to double network hydrogel was 1:10 to 2:1.

[0019] Furthermore, it also includes:

[0020] The volume ratio of PRP to double network hydrogel was 1:3.

[0021] Compared with the prior art, this technical solution has the following advantages:

[0022] This technical solution forms a PRP / chitosan / polyamino acid three-network hydrogel, which can dually regulate the inflammatory microenvironment of the infectious wound. Chitosan can be used as an activator of PRP to make it self-gelling, without the need for calcium chloride and bovine thrombin. The formation of the hydrogel containing three components is based on intramolecular amide bonds, intermolecular amide bonds and Schiff base bonds, which makes the hydrogel have good mechanical properties and self-healing properties, suitable for application on the wound surface. The most important thing is that this hydrogel can not only inhibit bacteria and shorten the inflammatory period, but also supplement repair factors and accelerate the repair period.

[0023] The present invention also provides a method for preparing the above network hydrogel, comprising:

[0024] Taking chitosan, polyamino acid, and a mixed solution and mixing them in proportion, wherein the mass ratio of chitosan to polyamino acid is 5:1 to 1:2, and the mass of the mixed solution accounts for 70% to 98% of the total mass;

[0025] Through catalysis, heating or high-speed stirring, the three react to form a network hydrogel;

[0026] Sterilize by heat or irradiation.

[0027] Furthermore, the preparation method of the network hydrogel also includes:

[0028] Add PRP and mix well by stirring or pipetting. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of a double network hydrogel in the first embodiment of the present invention;

[0030] Figure 2 This is a schematic diagram of a three-network hydrogel in the first embodiment of the present invention;

[0031] Figure 3 This is a schematic diagram of a method for preparing a triple-network hydrogel in a second embodiment of the present invention;

[0032] Figure 4 This is a schematic diagram of a method for preparing a triple-network hydrogel in a second embodiment of the present invention;

[0033] Figure 5 This is an experimental diagram showing that the three-network hydrogel in the first embodiment of the present invention has suitable pH value, is injectable, has strong adhesion, can absorb water, retain water and self-degrades;

[0034] Figure 6 This is an experimental diagram for characterizing the composition and structure of the triple-network hydrogel material in the first embodiment of the present invention;

[0035] Figure 7 This is an experimental diagram showing that the network hydrogel can accelerate wound healing in the first embodiment of the present invention;

[0036] Figure 8 This is an experimental diagram showing the excellent antibacterial performance of the network hydrogel in the first embodiment of the present invention.

[0037] (Note: The structures shown in the drawings are only for the purpose of illustrating the features of the present invention and are not intended to be based on the structures shown in the drawings.) DETAILED DESCRIPTION

[0038] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0039] In the prior art, although chitosan exhibits good antibacterial properties, can inhibit bacterial proliferation, reduce the inflammatory response caused by bacterial reproduction, and thus shorten the inflammatory period, it cannot replenish sufficient repair factors during the repair period, so its ability to activate repair cells, promote the polarization of macrophages M1 to M2, and promote local vascularization and re-epithelialization of the wound surface is relatively weak. That is, although chitosan can control infection, inhibit inflammation, and shorten the inflammatory period, it cannot accelerate the repair process of infectious wounds during the repair period. Secondly, due to its weak adhesion, chitosan is easy to fall off when applied to the wound.

[0040] Embodiment 1

[0041] This embodiment provides a network hydrogel that helps wound healing, including: chitosan.

[0042] The chitosan is N-acylated chitosan, chitosan hydrochloride, N-carboxymethyl chitosan, O-carboxymethyl chitosan, O-acylated chitosan, carboxylated chitosan, N-alkylated chitosan, O-alkylated chitosan, quaternized chitosan, N-carboxy chitosan or O-carboxy chitosan.

[0043] Among them, CMCS, namely carboxymethyl chitosan, was found to have better effect through experiments.

[0044] Modified dressings based on chitosan can enhance the antibacterial effect and promote the healing of infected wounds. Later, as a water-soluble derivative of chitosan, chitosan has antibacterial, antifungal, antioxidant and antitumor biological activities due to the addition of carboxymethyl groups, making it more suitable for wound healing.

[0045] As a bioactive material, chitosan has good antibacterial properties, can inhibit bacterial proliferation, reduce inflammatory reactions caused by bacterial reproduction, and thus shorten the inflammatory period. However, because it cannot replenish sufficient repair factors during the repair period, its ability to activate repair cells, promote polarization of macrophages from M1 to M2, and promote local vascularization and re-epithelialization of wounds is relatively weak. That is, although chitosan can control infection, inhibit inflammation, and shorten the inflammatory period, it cannot accelerate the repair process of infected wounds during the repair period.

[0046] Chitosan is easy to fall off when applied on a wound due to its weak adhesion. Therefore, the network hydrogel further comprises polyamino acid, and the mass ratio of chitosan to polyamino acid is 5:1 to 1:2, and the best is 3:1. The polyamino acid is polyleucine, polyaspartic acid, polylysine, polythreonine, polycysteine ​​or polyglutamic acid.

[0047] Among them, γ-PGA, or polyglutamic acid, was found to have better effects through experiments.

[0048] Super-hydrophilic polyamino acid can drain moisture from the wound surface and has strong adhesion properties, which enhances the surface adhesion of the gel and makes it less likely to fall off.

[0049] In order to allow chitosan and polyamino acid to repeatedly contact and form a gel, a mixed solution is required, the mass of the mixed solution accounts for 70% to 98% of the total mass, and the best is 88%. The mixed solution can be pure water, ultrapure water, heavy water, organic acid, inorganic acid, organic base, inorganic base or cell buffer.

[0050] refer to Figure 1 The network hydrogel finally formed is a double network hydrogel, which has good antibacterial properties, can inhibit bacterial proliferation, reduce the inflammatory response caused by bacterial reproduction, and thus shorten the inflammatory period; and has strong adhesion, can cover the outside of the wound without falling off, does not require other fixatives, and has good mechanical properties, making it suitable for application on the wound surface.

[0051] The optimal double network hydrogel synthesized by CMCS and γ-PGA is referred to as Cγ. The relevant characterization information can be found in Figure 5-Figure 8 .

[0052] Embodiment 2

[0053] However, the double network hydrogel described in the first embodiment cannot replenish sufficient repair factors during the repair period, so its ability to activate repair cells, promote the polarization of M1 macrophages to M2 macrophages, and promote local vascularization and re-epithelialization of the wound surface is relatively weak.

[0054] In order to ensure the antibacterial properties of the above-mentioned double-network hydrogel, shorten the inflammatory period, further supplement related repair factors, accelerate wound repair, and achieve dual inflammatory microenvironment regulation, we added platelet-rich plasma (PRP) to the hydrogel. The volume ratio of PRP to double-network hydrogel is 1:10 to 2:1, and the optimal is 1:3.

[0055] PRP is an autologous growth factor library rich in a variety of growth factors. Under the action of activators, PRP is activated to form a platelet-rich gel. Platelets release high concentrations of growth factors through degranulation, which plays an important role in promoting wound healing. The combined action of these factors can stimulate the physiological response process of cells during the repair period of the wound, such as inducing the migration, proliferation, differentiation and stabilization of endothelial cells in new blood vessels to accelerate the revascularization and collagen deposition of damaged tissues; inducing the migration and proliferation of fibroblasts to repair damaged connective tissues, restore damaged extracellular matrix, and accelerate skin wound repair. PRP contains a large number of repair factors, which can significantly improve the inflammatory microenvironment of the wound during the repair period, including promoting macrophage M1 / M2 polarization, cell recruitment, tissue vascularization, collagen synthesis, and wound epithelialization, thereby promoting wound repair.

[0056] refer to Figure 2 , and finally form a PRP / chitosan / polyamino acid three-network hydrogel, which can dually regulate the inflammatory microenvironment of the infected wound. Chitosan can be used as an activator of PRP to make it self-gelling, without the need for calcium chloride and bovine thrombin. The formation of the hydrogel containing three components is based on intramolecular amide bonds, intermolecular amide bonds and Schiff base bonds, which makes the hydrogel have good mechanical properties and self-healing properties, suitable for application on the wound surface. The most important thing is that this hydrogel can not only inhibit bacteria and shorten the inflammatory period, but also supplement repair factors and accelerate the repair period.

[0057] PRP / chitosan / polyamino acid triple network hydrogel has the following advantages:

[0058] 1. Because it contains chitosan gel network, it can effectively inhibit bacterial colonization and growth, thereby promoting the transformation of neutrophil N1 to N2 in the early stage, shortening the early inflammation clearance period, and achieving the first level of inflammatory microenvironment regulation;

[0059] 2. Due to the morphology and cross-linking of the PRP / chitosan / polyamino acid tri-network hydrogel, the α particles in the PRP are relatively stable, and the growth factors can be slowly released, thus improving the utilization efficiency of the growth factors. This can then gradually promote the polarization of macrophages from M1 to M2, the recruitment of peripheral cells, tissue vascularization, collagen formation, and wound epithelialization under the premise that the infection is controlled, thereby greatly improving the efficiency of promoting the healing of infectious wounds and achieving the second level of inflammatory microenvironment regulation;

[0060] 3. The mechanical properties are further enhanced, and its strength and elasticity can meet the requirements of making dressings that are not easy to break, have certain ductility and high adhesion, and have self-healing properties;

[0061] 4. This hydrogel can be synthesized in one step by the double-barreled syringe method, in which the chitosan component can self-activate PRP to form a platelet-rich gel (PRG) gel network without the need for calcium chloride and exogenous bovine thrombin, thus avoiding coagulation disorders caused by the formation of coagulation factor V and XI antibodies.

[0062] In the PRP / chitosan / polyamino acid triple network hydrogel, the growth factors are simultaneously wrapped by the triple network hydrogel formed by the combination of fibrin, chitosan and polyamino acid, which greatly slows down the release of the factors and solves the problem of too fast release of PRP factors. At the same time, dual inflammatory microenvironment regulation is also expected to be achieved.

[0063] PRP / chitosan / polyamino acid triple-network hydrogel is a high-performance gel dressing that can dually regulate the inflammatory microenvironment of infectious wounds and has the characteristics of self-gelation, self-healing, and high adhesion. It has high clinical application value.

[0064] Due to the excellent properties of Cγ, we referred to the triple-network hydrogel obtained by mixing PRP and Cγ as CγR.

[0065] Experiment 1

[0066] refer to Figure 5 , we preliminarily characterized the PRP / chitosan / polyamino acid triple-network hydrogel and found that it has excellent mechanical properties, can form a self-gel, is injectable, and has high adhesion.

[0067] The PRP / chitosan / polyamino acid tri-network hydrogel can form its own gel, is injectable and highly adhesive. The description of each part in the figure is as follows:

[0068] A. Investigation of pH values ​​of Cγ, PRP, Cγ gel and CγR gel obtained by mixing PRP in different proportions;

[0069] B. Injectability of CγR gel (the ratio of CγR gel shown in the figure is Cγ:PRP=3:1);

[0070] C. Apparent adhesion of CγR gel. Photographs of CγR gel adhered to mouse skin, muscle, tendon, fat, human skin, ceramic pestle, plastic EP tube, and glass coverslip;

[0071] D. Schematic diagram of lap shear test;

[0072] E. Adhesion strength of hydrogels of CγR gel, Cγ gel, and PRP gel;

[0073] F. Adhesion strength of CγR gels with different γ-PGA concentrations;

[0074] G. Swelling rate of CγR gel, Cγ gel, and PRP gel;

[0075] H. Water retention rate of CγR gel, Cγ gel, and PRP gel;

[0076] I. Degradation rates of CγR gel, Cγ gel, and PRP gel.

[0077] Experiment 2

[0078] refer to Figure 6 , the PRP / chitosan / polyamino acid triple network hydrogel was characterized.

[0079] A. Infrared spectra of chitosan, polyamino acid and double network hydrogel formed by the two;

[0080] B. The pH value of the double network hydrogel formed by chitosan and polyamino acid at different concentrations and ratios is between 6.4 and 6.9, which is consistent with the weakly acidic environment of the skin, and weak acidity has an antibacterial effect;

[0081] C. Electron micrographs of three types of gels. From left to right: chitosan / polyamino acid double network hydrogel, PRP gel, PRP / chitosan / polyamino acid triple network hydrogel;

[0082] D. X-ray (EDS) spectrum analysis of three types of gels. From left to right: chitosan / polyamino acid double network hydrogel, PRP gel, PRP / chitosan / polyamino acid triple network hydrogel.

[0083] Experiment 3

[0084] refer to Figure 7 This is a general diagram of promoting the healing of Staphylococcus aureus infected wounds through PRP / chitosan / polyamino acid triple network hydrogel. A full-thickness skin defect infection model was constructed using SD rats and Staphylococcus aureus. From top to bottom, the four groups are no intervention, using rat autologous PRP gel, using chitosan / polyamino acid double network hydrogel, and using PRP / chitosan / polyamino acid triple network hydrogel. It can be seen that the use of chitosan / polyamino acid double network hydrogel and PRP / chitosan / polyamino acid triple network hydrogel can effectively accelerate healing.

[0085] Experiment 4

[0086] refer to Figure 8 , agarose gel was used as the base, and equal amounts of PBS, PRP, PRP / chitosan 1 / 3, PRP / chitosan 1 / 4, PRP / chitosan 1 / 5, and PRP / chitosan 1 / 6 were added to each group. The results showed that PRP gel had a weak ability to inhibit the growth of Staphylococcus aureus, and the amount of bacteria was reduced to a certain extent compared with the control group, while no Staphylococcus aureus grew on chitosan gel, and almost no bacteria grew on 1 / 6 mixed PRP / chitosan double network hydrogel, indicating that it has excellent antibacterial properties.

[0087] Embodiment 3

[0088] refer to Figure 3 and Figure 4 This embodiment provides a method for preparing the network hydrogel described in the first embodiment, comprising:

[0089] Taking chitosan, polyamino acid, and a mixed solution and mixing them in proportion, wherein the mass ratio of chitosan to polyamino acid is 5:1 to 1:2, and the mass of the mixed solution accounts for 70% to 98% of the total mass;

[0090] Through catalysis, heating or high-speed stirring, the three react to form a network hydrogel;

[0091] Sterilize by heat or irradiation.

[0092] Finally, add the PRP and mix well by stirring or by pipetting.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A network hydrogel that helps wound healing, characterized in that: include: Chitosan; Polyamino acid, the mass ratio of chitosan to polyamino acid is 5:1 to 1:2; Mixed liquid, the mass of the mixed liquid accounts for 70% to 98% of the total mass.

2. The network hydrogel according to claim 1, characterized in that The chitosan is N-acylated chitosan, chitosan hydrochloride, N-carboxymethyl chitosan, O-carboxymethyl chitosan, O-acylated chitosan, carboxylated chitosan, N-alkylated chitosan, O-alkylated chitosan, quaternized chitosan, N-carboxy chitosan or O-carboxy chitosan.

3. The network hydrogel according to claim 1, characterized in that The polyamino acid is polyleucine, polyaspartic acid, polylysine, polythreonine, polycysteine ​​or polyglutamic acid.

4. The network hydrogel according to claim 1, characterized in that The mixed solution is pure water, ultrapure water, heavy water, organic acid, inorganic acid, organic base, inorganic base or cell buffer.

5. The network hydrogel according to claim 1, characterized in that: The mass ratio of chitosan to polyamino acid is 3:

1.

6. The network hydrogel according to claim 1, characterized in that The mass of the mixed liquid accounts for 88% of the total mass.

7. The network hydrogel according to claim 1, characterized in that Also includes: The volume ratio of PRP, PRP to double network hydrogel was 1:10 to 2:

1.

8. The network hydrogel according to claim 7, characterized in that: Also includes: The volume ratio of PRP to double network hydrogel was 1:

3.

9. A method for preparing the network hydrogel according to claim 1, characterized in that: include: Taking chitosan, polyamino acid, and a mixed solution and mixing them in proportion, wherein the mass ratio of chitosan to polyamino acid is 5:1 to 1:2, and the mass of the mixed solution accounts for 70% to 98% of the total mass; Through catalysis, heating or high-speed stirring, the three react to form a network hydrogel; Sterilize by heat or irradiation.

10. The preparation method according to claim 9, characterized in that: Also includes: Add PRP and mix well by stirring or pipetting.

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