A ternary complex coacervate and a method of making the same

Abstract

The application relates to the field of medical materials and discloses a ternary composite coacervate and a preparation method thereof, which comprises the following steps: dissolving a first component, a second component and a third component in deionized water respectively to prepare a first component aqueous solution, a second component aqueous solution and a third component aqueous solution; blending the first component aqueous solution, the second component aqueous solution and the third component aqueous solution; standing and stratifying or centrifugal treatment stratifying, collecting the ternary composite coacervate in the lower layer, repeating the process 5-10 times until the ternary composite coacervate is clear and transparent. The preparation method of the ternary composite coacervate is simple, the balance between the infiltration and integrity of the ternary composite coacervate at a wound defect is ensured through polysaccharide molecular weight control and the addition of a third component polyphenol substance, the ternary composite coacervate can be directly applied to an irregular wound, and the ternary composite coacervate has excellent wound healing promotion activity.

Description

Technical Field

[0001] This application relates to the field of medical materials, and mainly to a ternary composite aggregate and its preparation method. Background Technology

[0002] Full-thickness skin defects, characterized by slow regeneration and infection risks during treatment, pose a significant challenge in clinical practice. Hydrogels, with their high water content network and tunable physicochemical properties, are among the most ideal wound dressings because they maintain a moist microenvironment for wound healing, absorb exudate, allow gas permeation, and isolate the wound from adverse environments. However, traditional hydrogels are generally formed through chemical cross-linking, requiring complex chemical reaction processes that may introduce cytotoxic cross-linking agents. Furthermore, hydrogels are prone to detachment from the wound site during joint movement. Therefore, there is an urgent need to develop wound dressings that are simple to prepare, highly adaptable to different shapes, biocompatible, and have adhesion suitable for complex wounds.

[0003] Polyelectrolyte aggregates refer to the mixture of polyionic solutions with opposite charges, where liquid-liquid phase separation is driven by non-covalent interactions, transforming the charged polymer from a soluble dispersion to a water-insoluble aggregate. These non-covalent interactions include electrostatic interactions, hydrogen bonds, and hydrophobic interactions. Aggregates possess independent physicochemical properties such as high polymer density, low interfacial tension, water immiscibility, adhesion, and self-healing ability. They can diffuse across tissue surfaces by repelling interfacial water, making them excellent candidate materials for wound healing. Current research often uses combinations of macromolecules and small molecules to obtain polyelectrolyte aggregates rather than solid precipitates, such as polyacrylic acid and chitosan oligosaccharide (CN116725946A), and polydiallyl dimethylammonium chloride and adenine triphosphate (CN116655066A). However, in wound healing applications, these often suffer from low strength and excessive fluidity, hindering their retention on wound surfaces. Furthermore, the bioactivity of existing polyelectrolyte aggregates is limited, necessitating the combination with other bioactive molecules to expand their application in wound repair.

[0004] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this application is to provide a ternary composite aggregate and its preparation method, which aims to solve the problems of low strength, excessive fluidity and poor biological activity of existing polyelectrolyte aggregates.

[0006] The technical solution of this application is as follows:

[0007] A method for preparing a ternary composite condensate, comprising the following steps:

[0008] The first component, the second component, and the third component were dissolved in deionized water to prepare aqueous solutions of the first component, the second component, and the third component, respectively.

[0009] The first component aqueous solution, the second component aqueous solution, and the third component aqueous solution are blended together;

[0010] Allow the mixture to stand and separate into layers or centrifuge it to separate into layers. Collect the lower layer of the ternary composite aggregate. Repeat this process 5-10 times until the ternary composite aggregate is clear and transparent.

[0011] The first component is one of chitosan quaternary ammonium salt, chitosan hydrochloride, chitosan acetate, chitosan lactate, and carboxymethyl chitosan;

[0012] The second component is one of sodium hyaluronate and gum arabic;

[0013] The third component is a polyphenolic substance.

[0014] This application uses a spontaneous liquid-liquid phase separation process in aqueous solution to prepare ternary composite aggregates. The preparation method is simple. The addition of the third component, polyphenolic substances, ensures the balance between the infiltration and integrity of the ternary composite aggregates at the wound defect site. It can be directly applied to irregular wounds and has excellent wound healing activity.

[0015] The method for preparing the ternary composite aggregate, wherein the first component is chitosan quaternary ammonium salt, the second component is sodium hyaluronate, and the third component is propyl gallate.

[0016] The electrostatic complexation of chitosan quaternary ammonium salt and sodium hyaluronate constructs a physical cross-linked network of aggregates with certain fluidity. It has excellent infiltration properties in irregular wounds and can completely fill defects. The third component, propyl gallate, due to its pyrogallol structure, can interact with polysaccharides through hydrophobic interactions, hydrogen bonds, and electrostatic interactions to form a ternary composite aggregate, which can enhance the strength of the binary aggregate and help maintain its integrity at the wound site.

[0017] The method for preparing the ternary composite aggregate, wherein the mass concentration of the first component aqueous solution is 4-10%, and the mass concentration of the second component aqueous solution is 4-10%.

[0018] The method for preparing the ternary composite aggregate, wherein the molecular weight of the chitosan quaternary ammonium salt is 50-250 kDa, and the molecular weight of the sodium hyaluronate is 10-100 kDa.

[0019] In the method for preparing the ternary composite aggregate, the mass concentration of the aqueous solution of the third component is 0.1-2%.

[0020] The method for preparing the ternary composite condensate includes dissolving the third component in deionized water and then subjecting it to a heat treatment at 50-70°C.

[0021] In the method for preparing the ternary composite condensate, the volume ratio of the first component aqueous solution to the second component aqueous solution is 1:4 to 4:1.

[0022] In the method for preparing the ternary composite aggregate, the amount of the third component aqueous solution added is 10% to 80% of the total volume of the first component aqueous solution and the first component aqueous solution.

[0023] The method for preparing the ternary composite condensate, wherein the centrifugation process is centrifugation at 6000-8000 rpm for 5-20 min.

[0024] A ternary composite condensate, wherein the ternary composite condensate is prepared by the preparation method of the ternary composite condensate as described above.

[0025] Beneficial effects: The preparation method of the ternary composite aggregate provided in this application is simple. By controlling the molecular weight of polysaccharides and adding polyphenolic substances as a third component, the balance between the infiltration and integrity of the ternary composite aggregate at the wound defect site is ensured. It can be directly applied to irregular wounds and has excellent wound healing activity. Attached Figure Description

[0026] Figure 1 These are scanning electron microscope (SEM) images of the ternary composite aggregate of Example 2 and the binary aggregate of Comparative Example 1 in this application.

[0027] Figure 2 This is a comparison of the oscillation scan curves of the ternary composite condensate of Example 2 and the binary condensate of Comparative Example 1 in this application.

[0028] Figure 3 The images show a comparison of wound healing between the blank control group, the binary aggregate applied to Comparative Example 1, the ternary composite aggregate applied to Example 1, and the ternary composite aggregate applied to Example 2 in this application.

[0029] Figure 4 Comparative images of HE staining of wounds treated with the binary aggregate of Comparative Example 1, the ternary composite aggregate of Example 1, and the ternary composite aggregate of Example 2 in this application. Detailed Implementation

[0030] This application provides a ternary composite condensate and its preparation method. To make the objectives, technical solutions, and effects of this application clearer and more explicit, the following provides a more detailed description. It should be understood that the specific embodiments described herein are merely illustrative of this application and are not intended to limit this application.

[0031] This application utilizes a spontaneous liquid-liquid phase separation process in aqueous solution to prepare a ternary composite aggregate. The electrostatic complexation of chitosan quaternary ammonium salt and sodium hyaluronate constructs a physical cross-linked network within the aggregate, exhibiting a certain degree of fluidity and excellent wetting properties at irregular wound sites, capable of completely filling defects. The third component, propyl gallate, due to its pyrogallol structure, interacts with the polysaccharide through hydrophobic interactions, hydrogen bonds, and electrostatic interactions, forming a ternary composite aggregate that enhances the strength of the binary aggregate, thus helping to maintain its integrity at the wound site. Furthermore, this ternary composite aggregate not only provides physical protection to the wound site but also integrates the excellent bioactivity of chitosan quaternary ammonium salt and propyl gallate, effectively promoting the repair of full-thickness wound defects.

[0032] Specifically, the method for preparing the ternary composite condensate provided in this application includes the following steps:

[0033] (1) Preparation of aqueous solutions of chitosan quaternary ammonium salt and sodium hyaluronate:

[0034] Chitosan quaternary ammonium salt and sodium hyaluronate were dissolved in deionized water to prepare aqueous solutions of chitosan quaternary ammonium salt and sodium hyaluronate with a mass concentration of 4-10%.

[0035] The molecular weight of chitosan quaternary ammonium salt is 50-250 kDa, and the molecular weight of sodium hyaluronate is 10-100 kDa. These molecular weight ranges are used to ensure a high initial solution concentration and to guarantee the preparation of polyelectrolyte aggregates with suitable mechanical properties. If the molecular weight exceeds this range, the two aggregates will form fine, fragmented gel blocks when combined; if the molecular weight is below this range, the resulting complex will have excessively high fluidity.

[0036] In this application, the concentration of the aqueous solution of chitosan quaternary ammonium salt and sodium hyaluronate is limited to 4-10%. If it is less than 4%, the yield will be low and the separation time will be increased. If it is more than 10%, the initial solution concentration will be too high and the viscosity will be too high, which will easily cause uneven mixing.

[0037] In this application, the first component can be chitosan quaternary ammonium salt, or one of chitosan hydrochloride, chitosan acetate, chitosan lactate, or carboxymethyl chitosan. The second component can be sodium hyaluronate or gum arabic.

[0038] (2) Preparation of propyl gallate aqueous solution:

[0039] Propyl gallate was dissolved in deionized water to prepare an aqueous solution of propyl gallate with a mass concentration of 0.1-2%.

[0040] Propyl gallate is a commonly used food additive and pharmaceutical excipient with excellent biocompatibility. It can not only serve as a physical cross-linking point for binary aggregates, thereby enhancing the mechanical properties of aggregates and ensuring a balance between wettability and integrity, but also act as a bioactive component to endow aggregates with antioxidant and anti-inflammatory properties.

[0041] In this application, the mass concentration of the aqueous propyl gallate solution is limited to 0.1-2% because the effect only occurs when the concentration of the aqueous propyl gallate solution exceeds 0.1%, but the solubility of propyl gallate is only up to 2%. Therefore, the concentration of the aqueous propyl gallate solution is limited to 0.1-2%.

[0042] In this application, when dissolving propyl gallate in deionized water, a heating treatment of 50-70°C is performed to promote the dissolution of aqueous propyl gallate.

[0043] In this application, the third component, in addition to propyl gallate, can theoretically also include polyphenolic substances such as tannic acid, catechin, 3,4-dihydroxyphenylpropionic acid, caffeic acid, and quercetin.

[0044] (3) Mix the chitosan quaternary ammonium salt aqueous solution, sodium hyaluronate aqueous solution, and propyl gallate aqueous solution in a certain proportion and stir thoroughly.

[0045] The volume ratio of chitosan quaternary ammonium salt aqueous solution to sodium hyaluronate aqueous solution is 1:4 to 4:1. The amount of propyl gallate aqueous solution added can be 10% to 80% of the total volume of chitosan quaternary ammonium salt aqueous solution and sodium hyaluronate aqueous solution. Adding more than 10% can ensure a significant effect, while adding less than 80% is because there is an upper limit to the enhancement effect, and adding more will not have a better effect.

[0046] (4) Allow the mixture to stand and separate into layers or centrifuge it to separate into layers. Collect the lower layer of ternary composite aggregates and repeat this process 5-10 times until the ternary composite aggregates are clear and transparent and reach equilibrium.

[0047] In this application, centrifugation is preferred for phase separation because there is an internal network rearrangement process after the condensate phases separate, during which excess water is removed. Centrifugation can accelerate this water removal process, resulting in higher preparation efficiency. Furthermore, the centrifugation phase separation process involves centrifugation at 6000-8000 rpm for 5-20 minutes.

[0048] The method for preparing ternary composite condensates provided in this application has the following advantages:

[0049] (1) The preparation method provided in this application is simple and mild. Ternary composite aggregates can be prepared through a spontaneous liquid-liquid phase separation process, which effectively ensures the biocompatibility of the product and the process is easy to scale up.

[0050] (2) Because there are a large number of pyrogallol structures in the propyl gallate molecule, it can interact with polysaccharides through hydrophobic interactions, hydrogen bonds and electrostatic interactions, and act as a physical cross-linking point, which can enhance the mechanical strength of binary aggregates.

[0051] (3) The mechanical properties of the ternary composite aggregate prepared in this application can ensure the balance between infiltration and integrity at the wound defect site. It has strong tissue adhesion and can be directly applied to the wound surface to effectively protect the wound. At the same time, the antibacterial properties of chitosan quaternary ammonium salt and the antioxidant and anti-inflammatory activities of propyl gallate make the ternary composite aggregate have excellent wound healing activity.

[0052] This application also provides a ternary composite agglomerate, which is prepared using the above-described method. The polyphenolic substance propyl gallate acts as a small-molecule cross-linking agent in the agglomerate network, enhancing the mechanical properties of the chitosan quaternary ammonium salt / sodium hyaluronate agglomerate. The antibacterial properties of chitosan quaternary ammonium salt and the antioxidant and anti-inflammatory effects of propyl gallate give the ternary composite agglomerate excellent wound-healing activity. The ternary composite agglomerate of this application has high polymer concentration, good tissue adhesion, suitable viscoelasticity and integrity, and can be directly applied to irregular wounds, significantly promoting the wound healing process.

[0053] The present application will be further described below through specific embodiments.

[0054] Example 1:

[0055] Chitosan quaternary ammonium salt (100 kDa) and sodium hyaluronate (50 kDa) were dissolved separately in deionized water to a concentration of 5% (w / w). Propyl gallate was dissolved in deionized water and heated at 60°C to a concentration of 0.5% (w / w). Equal volumes of the three aqueous solutions were mixed and thoroughly stirred. The mixture was centrifuged at 8000 rpm for 10 min, and the lower ternary composite aggregate was collected. This process was repeated 8 times until the ternary composite aggregate was clear and transparent.

[0056] Example 2:

[0057] Chitosan quaternary ammonium salt (100 kDa) and sodium hyaluronate (50 kDa) were dissolved separately in deionized water to a concentration of 5% by mass. Propyl gallate was dissolved in deionized water and heated at 60 °C to a concentration of 1% by mass. Equal volumes of the three aqueous solutions were mixed and stirred thoroughly. The mixture was centrifuged at 8000 rpm for 10 min, and the lower ternary composite aggregate was collected. This process was repeated 6 times until the ternary composite aggregate was clear and transparent.

[0058] Example 3:

[0059] Chitosan quaternary ammonium salt (50 kDa) and sodium hyaluronate (100 kDa) were dissolved separately in deionized water to a concentration of 5% (w / w). Propyl gallate was dissolved in deionized water and heated at 60 °C to a concentration of 0.5% (w / w). 40 mL of chitosan quaternary ammonium salt aqueous solution, 20 mL of sodium hyaluronate aqueous solution, and 30 mL of propyl gallate aqueous solution were mixed and stirred thoroughly. The mixture was centrifuged at 8000 rpm for 10 min, and the lower ternary composite aggregate was collected. This process was repeated 8 times until the ternary composite aggregate was clear and transparent.

[0060] Example 4:

[0061] Chitosan quaternary ammonium salt (50 kDa) and sodium hyaluronate (100 kDa) were dissolved separately in deionized water to a concentration of 5% (w / w). Propyl gallate was dissolved in deionized water and heated at 60 °C to a concentration of 0.5% (w / w). 20 mL of the chitosan quaternary ammonium salt aqueous solution, 40 mL of the sodium hyaluronate aqueous solution, and 30 mL of the propyl gallate aqueous solution were mixed and stirred thoroughly. The mixture was centrifuged at 8000 rpm for 10 min, and the lower ternary composite aggregate was collected. This process was repeated 6 times until the ternary composite aggregate was clear and transparent.

[0062] Comparative Example 1:

[0063] Chitosan quaternary ammonium salt (100 kDa) and sodium hyaluronate (50 kDa) were dissolved separately in deionized water, each with a mass concentration of 5%. Equal volumes of the two aqueous solutions were mixed and stirred thoroughly. The mixture was centrifuged at 8000 rpm for 10 min, and the lower binary aggregate was collected. This process was repeated 10 times until the binary aggregate became clear and transparent.

[0064] The solid content of the ternary composite condensates prepared in Examples 1 and 2 and the binary condensate prepared in Comparative Example 1 were tested. The solid content of the ternary composite condensate in Example 1 was 18.3±0.3%, the solid content of the ternary composite condensate in Example 2 was 19.5±0.4%, and the solid content of Comparative Example 1 was 15.7±0.3%. The changes in solid content show that propyl gallate participated in the formation of the condensate and further concentrated it.

[0065] Scanning electron microscopy was performed on the ternary composite condensate prepared in Example 2 and the binary condensate prepared in Comparative Example 1. The results are as follows: Figure 1 As shown, a is a scanning electron microscope (SEM) image of the binary condensate of Comparative Example 1, and b is a scanning electron microscope (SEM) image of the ternary composite condensate of Example 2. Figure 1 It can be seen that the addition of propyl gallate increases the degree of cross-linking of the polymer network, reduces the water content, and increases the wall thickness.

[0066] Take 1g of each of the binary condensate prepared in Comparative Example 1 and the ternary composite condensate prepared in Example 1 as samples, and place them in the center of the sample stage of the rheometer for testing. Use a 35mm plate, select amplitude scanning mode, strain range of 0.1~200%, and test temperature of 25℃. Figure 2 This is a comparison of the oscillation scan curves of the binary condensate prepared in Comparative Example 1 and the ternary composite condensate prepared in Example 1. Figure 2 It can be seen that the addition of a small amount of propyl gallate can increase the storage modulus of the aggregate from 580 Pa to 970 Pa, indicating that propyl gallate can act as a physical crosslinking point through hydrophobic interactions, hydrogen bonds and electrostatic interactions, thereby enhancing the mechanical properties of the aggregate.

[0067] A full-thickness wound defect model was created on the back of rats, with a wound diameter of 12 mm. The ternary composite aggregates prepared in Examples 1 and 2, and the binary aggregate prepared in Comparative Example 1, were directly applied to the wound. The wound healing was observed and recorded on days 1, 4, 8, 12, and 16. A blank control group was also established, where physiological saline was applied. Figure 3 The images show a comparison of wound healing between the blank control group, the binary aggregate of Comparative Example 1, the ternary composite aggregate of Example 1, and the ternary composite aggregate of Example 2. In Figure a, image a compares the wound healing results, and in Figure b, image b compares the changes in wound area. Figure 3 This indicates that the ternary composite aggregate can significantly promote the wound healing process, and its wound healing activity is further enhanced with the increase of propyl gallate content.

[0068] Sixteen days after modeling, rats were sacrificed, and newly formed granulation tissue from the same wound site was collected. The specimens were fixed with paraformaldehyde fixative, stained with hematoxylin and eosin (HE), and observed for pathological tissue. Figure 4 This is a comparison of HE staining of the wounds in Comparative Example 1, Example 1, and Example 2. Figure 4 It can be seen that the thickness of granulation tissue in Comparative Example 1, Example 1 and Example 2 is significantly greater than that in the blank control group, indicating that the aggregates can promote wound healing. Furthermore, the granulation tissue in Example 2 shows certain skin appendages, indicating that the addition of propyl gallate can accelerate the wound healing process and improve the quality of wound healing.

[0069] It should be understood that the application of this application is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of this application.

Claims

1. A method for preparing a ternary complex coacervate, characterized by, The method comprises the following steps: dissolving the first component, the second component and the third component in deionized water respectively to prepare a first component aqueous solution, a second component aqueous solution and a third component aqueous solution; blending the first component aqueous solution, the second component aqueous solution and the third component aqueous solution, and forming ternary composite coacervates through hydrophobic interaction, hydrogen bonding and electrostatic interaction among the first component, the second component and the third component; standing for stratification or centrifugal treatment for stratification, collecting the lower layer ternary composite coacervates, repeating the process 5-10 times until the ternary composite coacervates are clear and transparent; the mass concentration of the third component aqueous solution is 0.1-2%; the amount of the third component aqueous solution added is 10%-80% of the total volume of the first component aqueous solution and the second component aqueous solution; the first component is chitosan quaternary ammonium salt, the second component is sodium hyaluronate, and the third component is propyl gallate; the mass concentration of the first component aqueous solution is 4-10%, and the mass concentration of the second component aqueous solution is 4-10%; the volume ratio of the first component aqueous solution to the second component aqueous solution is 1:4-4:

1.

2. The method for preparing a ternary complex coacervate according to claim 1, wherein the molecular weight of the chitosan quaternary ammonium salt is 50-250 kDa, and the molecular weight of the sodium hyaluronate is 10-100 kDa.

3. The method of claim 1, wherein the ternary complex coacervate is formed by mixing the polyelectrolyte, the polyelectrolyte-binding agent, and the polyelectrolyte-binding agent-binding agent. when dissolving the third component in deionized water, heating treatment at 50-70℃ is performed.

4. The method for preparing ternary complex coacervates according to claim 1, wherein, the centrifugal treatment process is centrifugation at 6000-8000 rpm for 5-20 min.

5. A ternary complex coacervate, characterized in that, the ternary composite coacervates are prepared by the preparation method of the ternary composite coacervates according to any one of claims 1-4.

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