Composite implant containing agarose with different molecular weights as well as preparation method and application of composite implant

Abstract

The invention provides a composite implant containing agarose with different molecular weights as well as a preparation method and application of the composite implant, and belongs to the technical field of biomedical materials. The composite implant comprises two or a combination of more than two of high-molecular-weight medical-grade agarose, medium-high-molecular-weight medical-grade agarose, medium-molecular-weight medical-grade agarose and low-molecular-weight medical-grade agarose. According to the invention, medical grade agarose with different molecular weights is compounded, so that the composite material is endowed with good injectability, moderate gel strength and controllable degradation performance, and the prepared composite implant containing agarose with different molecular weights shows good anti-oxidation and anti-wrinkle effects; wide application prospects are realized in the field of skin anti-aging.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly relates to a composite implant containing agarose with different molecular weights, a preparation method thereof, and an application thereof. Background Art

[0002] As people age, their skin gradually exhibits aging characteristics, including thinning of the dermis, decreased elasticity, and deepening of wrinkles. Endogenous aging is regulated by genes and is accompanied by the degradation of extracellular matrix components; while exogenous aging is mainly induced by ultraviolet radiation and oxidative stress, resulting in the degradation of collagen and the loss of elastin. Existing anti-aging treatment methods, such as laser treatment, injection fillers, and topical antioxidants, although they can improve skin aging to a certain extent, still have problems such as limited efficacy, insufficient persistence, and potential side effects.

[0003] Agarose is a natural linear polysaccharide extracted from red algae. The basic structural unit is β-D-galactose and 3,6-anhydro-α-L-galactose. It has excellent biocompatibility and non-immunogenicity. Agarose has thermosensitive gel-forming properties and can melt or solidify into a gel with temperature changes. In the gel state, the molecular chains exist in a double helix form, and the double helices aggregate to form a three-dimensional network structure of agarose gel. The agarose hydrogel structure is similar to the extracellular matrix, which can create a good environment for the growth and proliferation of cells. Moreover, agarose also has properties such as good biocompatibility and non-immunogenicity. Based on its unique properties, agarose has broad application potential in the field of skin medicine.

[0004] However, the application of traditional high-molecular-weight agarose in skin repair is restricted by the injection difficulty caused by its high mechanical strength and limited biological activity, which affects its feasibility in clinical applications. Therefore, there is an urgent need to develop an agarose multifunctional implant with advantages such as good injectability, appropriate gel strength, controllable degradation performance, and excellent biological activity to meet the requirements of application scenarios such as tissue repair and regenerative medicine. Summary of the Invention

[0005] Aiming at the above deficiencies of the prior art, the present invention provides a composite implant containing agarose with different molecular weights, a preparation method thereof, and an application thereof. By compounding medical-grade agarose with different molecular weights, the composite material is given good injectability, moderate gel strength, and controllable degradation performance. At the same time, it also has good antioxidant and anti-wrinkle effects, providing an innovative biological material choice for skin anti-aging treatment.

[0006] To achieve the above object, the specific technical solutions of the present invention are as follows:

[0007] In a first aspect, the present invention provides a composite implant containing agarose of different molecular weights, comprising: a combination of two or more of high-molecular-weight medical-grade agarose, medium-high-molecular-weight medical-grade agarose, medium-molecular-weight medical-grade agarose, and low-molecular-weight medical-grade agarose;

[0008] wherein, the molecular weight of the high-molecular-weight medical-grade agarose is greater than 100,000;

[0009] the molecular weight of the medium-high-molecular-weight medical-grade agarose is 30,000 - 100,000;

[0010] the molecular weight of the medium-molecular-weight medical-grade agarose is 3,000 - 30,000;

[0011] the molecular weight of the low-molecular-weight medical-grade agarose is less than 3,000.

[0012] Aiming at the problems that the application of traditional high-molecular-weight agarose in skin repair is limited by the injection difficulty caused by its high mechanical strength and limited biological activity, the present invention prepares a composite gel implant with excellent rheological properties and injectability by compounding agarose of different molecular weights and adopting a specific ratio and preparation method. At the same time, it combines the mechanical support of high-molecular-weight agarose with the permeability and biological activity of low-molecular-weight agarose. Through the mutual cooperation and synergistic effect of agarose of different molecular weights, better comprehensive performance and repair effect are achieved, making the composite implant of the present invention an ideal biomaterial for the new generation of anti-aging treatment. In addition, the raw materials of the composite implant of the present invention only include agarose of different molecular weights and water, with few raw material types and high biocompatibility, reducing the risk of adverse reactions caused by the interaction between materials and reducing allergic or rejection reactions caused by the combination of multiple materials; moreover, the production process of the present invention is simpler, which is conducive to industrial scale production.

[0013] Further, the combination of agarose of different molecular weights includes: high / medium-high-molecular-weight medical-grade agarose, high / medium-molecular-weight medical-grade agarose, high / low-molecular-weight medical-grade agarose, high / medium-high / medium-molecular-weight medical-grade agarose, high / medium-high / low-molecular-weight medical-grade agarose, high / medium / low-molecular-weight medical-grade agarose, high / medium-high / medium / low-molecular-weight medical-grade agarose, medium-high / medium-molecular-weight medical-grade agarose, medium-high / low-molecular-weight medical-grade agarose, medium-high / medium / low-molecular-weight medical-grade agarose, or medium / low-molecular-weight medical-grade agarose.

[0014] Further, the total mass percentage concentration of the agarose of different molecular weights in the composite implant is 0.5% - 10%.

[0015] Even further, the total mass percentage concentration of the agarose of different molecular weights in the composite implant is 1.0% - 3.7%.

[0016] Furthermore, in the combined high / medium molecular weight medical-grade agarose, the mass percentage concentrations of the high and medium molecular weight medical-grade agarose in the composite implant are (0.7% - 1.5%):(0.5% - 1.6%);

[0017] In the combined high / medium molecular weight medical-grade agarose, the mass percentage concentrations of the high and medium molecular weight medical-grade agarose in the composite implant are (0.7% - 1.5%):(0.5% - 1.6%);

[0018] In the combined high / low molecular weight medical-grade agarose, the mass percentage concentrations of the high and low molecular weight medical-grade agarose in the composite implant are (0.7% - 1.5%):(0.5% - 1.6%);

[0019] In the combined high / medium-high / medium molecular weight medical-grade agarose, the mass percentage concentrations of the high, medium-high, and medium molecular weight medical-grade agarose in the composite implant are (0.5% - 1.2%):(0.4% - 1.3%):(0.5% - 1.2%);

[0020] In the combined high / medium-high / low molecular weight medical-grade agarose, the mass percentage concentrations of the high, medium-high, and low molecular weight medical-grade agarose in the composite implant are (0.5% - 1.2%):(0.4% - 1.3%):(0.5% - 1.2%);

[0021] In the combined high / medium / low molecular weight medical-grade agarose, the mass percentage concentrations of the high, medium, and low molecular weight medical-grade agarose in the composite implant are (0.5% - 1.2%):(0.4% - 1.3%):(0.5% - 1.2%);

[0022] In the combined high / medium-high / medium / low molecular weight medical-grade agarose, the mass percentage concentrations of the high, medium-high, medium, and low molecular weight medical-grade agarose in the composite implant are (0.4% - 0.7%):(0.3% - 0.7%):(0.2% - 0.8%):(0.1% - 0.9%);

[0023] In the combined medium-high / medium molecular weight medical-grade agarose, the mass percentage concentrations of the medium-high and medium molecular weight medical-grade agarose in the composite implant are (0.7% - 1.5%):(0.5% - 1.6%);

[0024] In the combined medium-high / low molecular weight medical-grade agarose, the mass percentage concentrations of the medium-high and low molecular weight medical-grade agarose in the composite implant are (0.7% - 1.5%):(0.5% - 1.6%);

[0025] In the combination of high / middle / low molecular weight medical grade agarose, the mass percentage concentrations of high-middle, middle, and low molecular weight medical grade agarose in the composite implant are (0.5% - 1.2%):(0.4% - 1.3%):(0.5% - 1.2%);

[0026] In the combination of middle / low molecular weight medical grade agarose, the mass percentage concentrations of middle and low molecular weight medical grade agarose in the composite implant are (0.7% - 1.5%):(0.5% - 1.6%).

[0027] Furthermore, the combination is high / middle-high / middle / low molecular weight medical grade agarose.

[0028] Furthermore, in the combination of high / middle-high / middle / low molecular weight medical grade agarose, the mass percentage concentration of high molecular weight medical grade agarose in the composite implant is 0.625%; the mass percentage concentration of middle-high molecular weight medical grade agarose in the composite implant is 0.625%; the mass percentage concentration of middle molecular weight medical grade agarose in the composite implant is 0.625%; the mass percentage concentration of low molecular weight medical grade agarose in the composite implant is 0.625%.

[0029] In a second aspect, the present invention provides a method for preparing the composite implant containing agarose with different molecular weights, comprising the following steps:

[0030] Mix medical grade agarose with different molecular weights with water and heat to 90 - 120 °C until completely dissolved to obtain a clear and transparent solution; cool the solution to 10 - 40 °C and let it stand for reaction to obtain a composite implant containing agarose with different molecular weights.

[0031] Further, the dissolution temperature is 95 - 105 °C, and the reaction temperature is 25 - 37 °C.

[0032] In a third aspect, the present invention provides the application of the composite implant containing agarose with different molecular weights in the field of skin anti-aging.

[0033] Compared with the prior art, the advantages of the present invention are as follows:

[0034] (1) The composite implant containing agarose with different molecular weights of the present invention has excellent rheological properties and injectability, and at the same time combines the mechanical support of high molecular weight agarose and the permeability and biological activity of low molecular weight agarose, achieving better comprehensive performance and repair effect, making the composite implant of the present invention an ideal biomaterial for a new generation of anti-aging treatment;

[0035] (2) Compared with traditional high-molecular-weight agarose implants, the composite implants containing agarose with different molecular weights in the present invention have reduced mechanical strength and viscosity, enhancing their injectability and effectively solving the problem of uneven injection force of traditional high-molecular-weight agarose implants;

[0036] (3) The composite implants containing agarose with different molecular weights in the present invention have antioxidant and anti-wrinkle effects and have significant application potential in the field of skin anti-aging;

[0037] (4) The raw materials of the composite implants in the present invention only include agarose with different molecular weights and water. The types of raw materials are few, and the biocompatibility is high. At the same time, the risk of adverse reactions caused by the interaction between materials is reduced, as well as the allergic or rejection reactions caused by the combination of multiple materials are reduced; moreover, the production process of the present invention is simpler, which is conducive to industrial scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a physical diagram of the composite implant containing agarose with different molecular weights in the present invention;

[0039] Figure 2 is the gel strength of the composite implant containing agarose with different molecular weights in the present invention;

[0040] Figure 3 is the viscosity of the composite implant containing agarose with different molecular weights in the present invention;

[0041] Figure 4 is the injectability of the composite implant containing agarose with different molecular weights in the present invention;

[0042] Figure 5 is the degradability of the composite implant containing agarose with different molecular weights in the present invention;

[0043] Figure 6 is the antioxidant capacity of the composite implant containing agarose with different molecular weights in the present invention;

[0044] Figure 7 is the anti-wrinkle effect of zebrafish of the composite implant containing agarose with different molecular weights in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0045] The technical solutions of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0046] The present invention provides a composite implant containing agarose with different molecular weights, comprising: a combination of two or more of high molecular weight medical grade agarose, medium-high molecular weight medical grade agarose, medium molecular weight medical grade agarose, and low molecular weight medical grade agarose;

[0047] Among them, the molecular weight of the high molecular weight medical grade agarose is greater than 100,000;

[0048] The molecular weight of the medium-high molecular weight medical grade agarose is 30,000 - 100,000;

[0049] The molecular weight of the medium molecular weight medical grade agarose is 3,000 - 30,000;

[0050] The molecular weight of the low molecular weight medical grade agarose is less than 3,000.

[0051] In some examples, the total mass percentage concentration of the agarose with different molecular weights in the composite implant is 0.5% - 10%.

[0052] In some examples, the combinations of the agarose with different molecular weights include: high / medium-high molecular weight medical grade agarose, high / medium molecular weight medical grade agarose, high / low molecular weight medical grade agarose, high / medium-high / medium molecular weight medical grade agarose, high / medium-high / low molecular weight medical grade agarose, high / medium / low molecular weight medical grade agarose, high / medium-high / medium / low molecular weight medical grade agarose, medium-high / medium molecular weight medical grade agarose, medium-high / low molecular weight medical grade agarose, medium-high / medium / low molecular weight medical grade agarose, or medium / low molecular weight medical grade agarose.

[0053] In some examples, in the combined high / middle molecular weight medical-grade agarose, the mass percentage concentrations of the high and middle molecular weight medical-grade agarose in the composite implant are (0.7% - 1.5%):(0.5% - 1.6%); in the combined high / middle molecular weight medical-grade agarose, the mass percentage concentrations of the high and middle molecular weight medical-grade agarose in the composite implant are (0.7% - 1.5%):(0.5% - 1.6%); in the combined high / low molecular weight medical-grade agarose, the mass percentage concentrations of the high and low molecular weight medical-grade agarose in the composite implant are (0.7% - 1.5%):(0.5% - 1.6%); in the combined high / middle-high / middle molecular weight medical-grade agarose, the mass percentage concentrations of the high, middle-high, and middle molecular weight medical-grade agarose in the composite implant are (0.5% - 1.2%):(0.4% - 1.3%):(0.5% - 1.2%); in the combined high / middle-high / low molecular weight medical-grade agarose, the mass percentage concentrations of the high, middle-high, and low molecular weight medical-grade agarose in the composite implant are (0.5% - 1.2%):(0.4% - 1.3%):(0.5% - 1.2%); in the combined high / middle / low molecular weight medical-grade agarose, the mass percentage concentrations of the high, middle, and low molecular weight medical-grade agarose in the composite implant are (0.5% - 1.2%):(0.4% - 1.3%):(0.5% - 1.2%); in the combined high / middle-high / middle / low molecular weight medical-grade agarose, the mass percentage concentrations of the high, middle-high, middle, and low molecular weight medical-grade agarose in the composite implant are (0.4% - 0.7%):(0.3% - 0.7%):(0.2% - 0.8%):(0.1% - 0.9%); in the combined middle-high / middle molecular weight medical-grade agarose, the mass percentage concentrations of the middle-high and middle molecular weight medical-grade agarose in the composite implant are (0.7% - 1.5%):(0.5% - 1.6%); in the combined middle-high / low molecular weight medical-grade agarose, the mass percentage concentrations of the middle-high and low molecular weight medical-grade agarose in the composite implant are (0.7% - 1.5%):(0.5% - 1.6%); in the combined middle-high / middle / low molecular weight medical-grade agarose, the mass percentage concentrations of the middle-high, middle, and low molecular weight medical-grade agarose in the composite implant are (0.5% - 1.2%):(0.4% - 1.3%):(0.5% - 1.2%); in the combined middle / low molecular weight medical-grade agarose, the mass percentage concentrations of the middle and low molecular weight medical-grade agarose in the composite implant are (0.7% - 1.5%):(0.5% - 1.6%).

[0054] The preparation method of the composite implant containing agarose with different molecular weights includes the following steps:

[0055] Mix medical-grade agarose with different molecular weights with ultrapure water and heat to 90-120 °C until completely dissolved to obtain a clear and transparent solution; cool the solution to 10-40 °C, let it stand for reaction, and perform aseptic packaging after forming a gel to obtain a composite implant containing agarose with different molecular weights.

[0056] In the following specific examples, unless otherwise specified, the molecular weight of the high-molecular-weight medical-grade agarose is 106,000; the molecular weight of the medium-high-molecular-weight medical-grade agarose is 67,000; the molecular weight of the medium-molecular-weight medical-grade agarose is 6493; the molecular weight of the low-molecular-weight medical-grade agarose is 2000.

[0057] Example 1 A composite implant containing agarose with different molecular weights

[0058] 1. Preparation of a composite implant containing high-molecular-weight / medium-high-molecular-weight medical-grade agarose

[0059] Weigh 125 mg of high-molecular-weight medical-grade agarose powder and 125 mg of medium-high-molecular-weight medical-grade agarose powder respectively, dissolve them in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved to obtain a clear and transparent solution. Then, cool the obtained solution to 37 °C, let it stand for reaction, and obtain a composite implant containing high / medium-high-molecular-weight medical-grade agarose.

[0060] 2. Preparation of a composite implant containing high-molecular-weight / medium-molecular-weight medical-grade agarose

[0061] Weigh 125 mg of high-molecular-weight medical-grade agarose powder and 125 mg of medium-molecular-weight medical-grade agarose powder respectively, dissolve them in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved to obtain a clear and transparent solution. Then, cool the obtained solution to 37 °C, let it stand for reaction, and obtain a composite implant containing high / medium-molecular-weight medical-grade agarose.

[0062] 3. Preparation of a composite implant containing high-molecular-weight / low-molecular-weight medical-grade agarose

[0063] Weigh 125 mg of high-molecular-weight medical-grade agarose powder and 125 mg of low-molecular-weight medical-grade agarose powder respectively, dissolve them in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved to obtain a clear and transparent solution. Then, cool the obtained solution to 37 °C, let it stand for reaction, and obtain a composite implant containing high / low-molecular-weight medical-grade agarose.

[0064] 4. Preparation of a composite implant containing high-molecular-weight / medium-high-molecular-weight / medium-molecular-weight medical-grade agarose

[0065] Weigh 83.3 mg of high-molecular-weight medical-grade agarose powder, 83.3 mg of medium-high-molecular-weight medical-grade agarose powder, and 83.3 mg of medium-molecular-weight medical-grade agarose powder respectively, dissolve them in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved to obtain a clear and transparent solution. Then, cool the obtained solution to 37 °C, let it stand for reaction, and obtain a composite implant containing high / medium-high / medium-molecular-weight medical-grade agarose.

[0066] 5. Preparation of Composite Implant Containing High-Molecular-Weight / Medium-High-Molecular-Weight / Low-Molecular-Weight Medical-Grade Agarose

[0067] Weigh 83.3 mg of high-molecular-weight medical-grade agarose powder, 83.3 mg of medium-high-molecular-weight medical-grade agarose powder, and 83.3 mg of low-molecular-weight medical-grade agarose powder respectively, dissolve them in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved to obtain a clear and transparent solution. Then, cool the obtained solution to 37 °C, let it stand for reaction, and obtain a composite implant containing high / medium / low-molecular-weight medical-grade agarose.

[0068] 6. Preparation of Composite Implant Containing High-Molecular-Weight / Medium-Molecular-Weight / Low-Molecular-Weight Medical-Grade Agarose

[0069] Weigh 83.3 mg of high-molecular-weight medical-grade agarose powder, 83.3 mg of medium-molecular-weight medical-grade agarose powder, and 83.3 mg of low-molecular-weight medical-grade agarose powder respectively, dissolve them in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved to obtain a clear and transparent solution. Then, cool the obtained solution to 37 °C, let it stand for reaction, and obtain a composite implant containing high / medium / low-molecular-weight medical-grade agarose.

[0070] 7. Preparation of Composite Implant Containing High-Molecular-Weight / Medium-High-Molecular-Weight / Medium-Molecular-Weight / Low-Molecular-Weight Medical-Grade Agarose

[0071] Weigh 62.5 mg of high-molecular-weight medical-grade agarose powder, 62.5 mg of medium-high-molecular-weight medical-grade agarose powder, 62.5 mg of medium-molecular-weight medical-grade agarose powder, and 62.5 mg of low-molecular-weight medical-grade agarose powder respectively, dissolve them in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved to obtain a clear and transparent solution. Then, cool the obtained solution to 37 °C, let it stand for reaction, and obtain a composite implant containing high / medium-high / medium / low-molecular-weight medical-grade agarose.

[0072] 8. Preparation of Composite Implant Containing Medium-High-Molecular-Weight / Medium-Molecular-Weight Medical-Grade Agarose

[0073] Weigh 125 mg of medium-high molecular weight medical-grade agarose powder and 125 mg of medium molecular weight medical-grade agarose powder separately, dissolve them in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved to obtain a clear and transparent solution. Then, cool the obtained solution to 37 °C and let it stand for reaction to obtain a composite implant containing medium-high molecular weight / medium molecular weight medical-grade agarose.

[0074] 9. Preparation of a composite implant containing medium-high molecular weight / low molecular weight medical-grade agarose

[0075] Weigh 125 mg of medium-high molecular weight medical-grade agarose powder and 125 mg of low molecular weight medical-grade agarose powder separately, dissolve them in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved to obtain a clear and transparent solution. Then, cool the obtained solution to 37 °C and let it stand for reaction to obtain a composite implant containing medium-high molecular weight / low molecular weight medical-grade agarose.

[0076] 10. Preparation of a composite implant containing medium molecular weight / low molecular weight medical-grade agarose

[0077] Weigh 125 mg of medium molecular weight medical-grade agarose powder and 125 mg of low molecular weight medical-grade agarose powder separately, dissolve them in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved to obtain a clear and transparent solution. Then, cool the obtained solution to 37 °C and let it stand for reaction to obtain a composite implant containing medium molecular weight / low molecular weight medical-grade agarose.

[0078] 11. Preparation of a composite implant containing medium-high molecular weight / medium molecular weight / low molecular weight medical-grade agarose

[0079] Weigh 83.3 mg of medium-high molecular weight medical-grade agarose powder, 83.3 mg of medium molecular weight medical-grade agarose powder, and 83.3 mg of low molecular weight medical-grade agarose powder separately, dissolve them in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved to obtain a clear and transparent solution. Then, cool the obtained solution to 37 °C and let it stand for reaction to obtain a composite implant containing medium-high molecular weight / medium molecular weight / low molecular weight medical-grade agarose.

[0080] Example 2 A composite implant containing agarose with different molecular weights

[0081] Weigh 70 mg of high molecular weight medical grade agarose powder, 55 mg of medium-high molecular weight medical grade agarose powder, 80 mg of medium molecular weight medical grade agarose powder, and 45 mg of low molecular weight medical grade agarose powder respectively, dissolve them in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved to obtain a clear and transparent solution. Then, cool the obtained solution to 37 °C and let it stand for reaction to obtain a composite implant containing high molecular weight / medium-high molecular weight / medium molecular weight / low molecular weight medical grade agarose.

[0082] Example 3 Preparation of a composite implant containing agarose with different molecular weights

[0083] Weigh 50 mg of high molecular weight medical grade agarose powder, 60 mg of medium-high molecular weight medical grade agarose powder, 75 mg of medium molecular weight medical grade agarose powder, and 65 mg of low molecular weight medical grade agarose powder respectively, dissolve them in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved to obtain a clear and transparent solution. Then, cool the obtained solution to 37 °C and let it stand for reaction to obtain a composite implant containing high molecular weight / medium-high molecular weight / medium molecular weight / low molecular weight medical grade agarose.

[0084] Example 4 Preparation of a composite implant containing agarose with different molecular weights

[0085] Weigh 150 mg of high molecular weight medical grade agarose powder, 150 mg of medium-high molecular weight medical grade agarose powder, 150 mg of medium molecular weight medical grade agarose powder, and 150 mg of low molecular weight medical grade agarose powder respectively, dissolve them in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved to obtain a clear and transparent solution. Then, cool the obtained solution to 37 °C and let it stand for reaction to obtain a composite implant containing high molecular weight / medium-high molecular weight / medium molecular weight / low molecular weight medical grade agarose.

[0086] Example 5 Preparation of a composite implant containing agarose with different molecular weights

[0087] Weigh 270 mg of high molecular weight medical grade agarose powder, 280 mg of medium-high molecular weight medical grade agarose powder, 280 mg of medium molecular weight medical grade agarose powder, and 270 mg of low molecular weight medical grade agarose powder respectively, dissolve them in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved to obtain a clear and transparent solution. Then, cool the obtained solution to 37 °C and let it stand for reaction to obtain a composite implant containing high molecular weight / medium-high molecular weight / medium molecular weight / low molecular weight medical grade agarose.

[0088] Comparative Example 1 Preparation of a traditional high molecular weight agarose implant

[0089] Weigh 250 mg of high-molecular-weight medical-grade agarose powder, dissolve it in 10 mL of ultrapure water, and heat it to 100 °C until completely dissolved to obtain a clear and transparent solution. Then, cool the obtained solution to 37 °C and let it stand for reaction to obtain an implant containing high-molecular-weight medical-grade agarose.

[0090] Perform physicochemical property tests on the implants obtained in Example 1 and Comparative Example 1.

[0091] 1. Gel-forming property of the implant

[0092] Take 1 mL of the agarose mixed solution with different molecular weights in Example 1 and 1 mL of the traditional high-molecular-weight agarose solution in Comparative Example 1 respectively, transfer them to a transparent vial while it is hot, and let it stand at room temperature; take another 1 mL of the implant sample and place it in the lid of a centrifuge tube, and also let it stand at room temperature. The physical appearance of the implant is as Figure 1 shown. The results show that both the composite implant containing agarose with different molecular weights of the present invention and the traditional high-molecular-weight agarose implant can form hydrogels.

[0093] 2. Gel strength test of the implant

[0094] Prepare gel blocks with a uniform thickness of 1 cm × 1 cm × 2 mm from the agarose mixed solution with different molecular weights in Example 1 and 1 mL of the traditional high-molecular-weight agarose solution in Comparative Example 1 respectively, place this gel block on the sample stage of a rheometer, fix the shear strain (1%) of the rheometer, and test G'(Pa) and G"(Pa) of the gel as the angular frequency changes. The test results of G'(Pa) and G"(Pa) of the hydrogels containing agarose with different molecular weights and the hydrogel of traditional high-molecular-weight agarose are as Figure 2 shown. The results show that the gel strength is in the order of: high > high / medium-high > high / medium > high / low > high / medium-high / medium > high / medium-high / low > high / medium / low > high / medium-high / medium / low > medium-high / medium > medium-high / low > medium-high / medium / low > medium / low; the gel strength of the combined gel without high-molecular-weight is lower than that of the combined gel containing high-molecular-weight, and the smallest gel strength in the combined gels containing high-molecular-weight is high / medium-high / medium / low.

[0095] 3. Shear viscosity test of the implant

[0096] Prepare gel blocks with a uniform thickness of 1 cm × 1 cm × 2 mm from the agarose mixed solution with different molecular weights in Example 1 and 1 mL of the traditional high-molecular-weight agarose solution in Comparative Example 1 respectively, place this gel block on the sample stage of a rheometer, and at a shear rate of 0.01 s -1 ~100 s -1Peristaltic scanning was performed below to obtain the shear viscosity of the gel. The test results of the shear viscosity of the hydrogels containing agarose with different molecular weights and the hydrogels of traditional high molecular weight agarose are as Figure 3 shown. The shear viscosities are in the order of: high > high / medium-high > high / medium > high / low > high / medium-high / medium > high / medium-high / low > high / medium / low > high / medium-high / medium / low > medium-high / medium > medium-high / low > medium-high / medium / low > medium / low; the viscosity of the combination without high molecular weight is lower than that of the combination containing high molecular weight, and the combination with the smallest viscosity among the combinations containing high molecular weight is high / medium-high / medium / low.

[0097] 4. Injectability test of the implant

[0098] The agarose mixed solutions with different molecular weights in Example 1 and 1 mL of the traditional high molecular weight agarose solution in Comparative Example 1 were respectively filled into 1 mL syringe needles while they were still hot, cooled overnight at room temperature, and then the sample was tested for needle penetration using a needle passing instrument. During the test, the syringe needle was installed, the plunger was pushed at a constant speed, and the sample in the syringe needle was extruded through the needle tip to obtain the extrusion force curve.

[0099] The test results of the injectability of the hydrogels containing agarose with different molecular weights and the hydrogels of traditional high molecular weight agarose are as Figure 4 shown. The maximum extrusion forces are in the order of: high > high / medium-high > high / medium > high / low > high / medium-high / medium > high / medium-high / low > high / medium / low > high / medium-high / medium / low > medium-high / medium > medium-high / low > medium-high / medium / low > medium / low; the injectability of the combination without high molecular weight is better than that of the combination containing high molecular weight, and the combination with the best injectability among the combinations containing high molecular weight is high / medium-high / medium / low.

[0100] 5. Degradability test of the implant

[0101] Appropriate amounts (W0) of the freeze-dried samples of the implant in Example 1 and the traditional high molecular weight agarose implant in Comparative Example 1 were respectively weighed and placed in centrifuge tubes, and soaked in 1.0 M hydrochloric acid solution at 37 °C to characterize the degradation behavior of the hydrogel. Samples were taken at regular intervals, the supernatant was removed after centrifugation, and the precipitate was freeze-dried and weighed (W t ). The acid degradation rate was calculated according to the following formula: Weight loss (%) = (W0 - W t ) / W0 × 100%. The degradation results are shown in Figure 5 .

[0102] The results showed that on the 7th day, the degradation rate of the implant containing high molecular weight agarose in Comparative Example 1 was 34%; in Example 1, the degradation rate of the agarose implant containing a high / mid-high combination was 50%, the degradation rate of the agarose implant containing a high / mid combination was 67%, the degradation rate of the agarose implant containing a high / low combination was 67%, the degradation rate of the agarose implant containing a high / mid-high / mid combination was 66%, the degradation rate of the agarose implant containing a high / mid-high / low combination was 66%, the degradation rate of the agarose implant containing a high / mid / low combination was 78%, the degradation rate of the agarose implant containing a high / mid-high / mid / low combination was 75%, the degradation rate of the agarose implant containing a mid-high / mid combination was 83%, the degradation rate of the agarose implant containing a mid-high / low combination was 83%, the degradation rate of the agarose implant containing a mid / low combination was 100%, and the degradation rate of the agarose implant containing a mid-high / mid / low combination was 88%. The overall trend indicated that the degradation rate of the combination without high molecular weight was higher than that of the combination containing high molecular weight.

[0103] 6. Antioxidant Capacity Test of Implants

[0104] The ABTS method was used to evaluate the in vitro antioxidant capacity of the implants. The stable ABTS radical solution was blue-green and had a maximum absorption peak at 734 nm. When the radicals were scavenged by the implant samples, the number of radicals decreased, the color of the solution gradually became lighter, and the absorbance at 734 nm decreased accordingly. Therefore, the ability of the sample to scavenge ABTS radicals could be judged by measuring the change in absorbance.

[0105] The experimental procedure was as follows: A 7.4 mmol / L ABTS stock solution and a 2.6 mmol / L potassium persulfate (K2S2O8) solution were prepared and mixed in a 1:1 ratio, and left to stand in the dark for 12 h to generate the ABTS radical solution. Before the experiment, the solution was diluted with ultrapure water to an absorbance of 0.70 ± 0.02 and used as the working solution. After the implant sample solution was prepared at different concentrations, it was measured in a 96-well plate. 20 µL of the implant sample solution and 180 µL of the working solution were added to each well, and after reacting at room temperature in the dark for 3 - 5 min, the absorbance was measured at 734 nm using a microplate reader. The scavenging rate was calculated according to the following formula: Scavenging rate (%) = [(A0 - A) / A0] × 100, where A0 was the absorbance of the working solution and A was the absorbance after mixing the implant sample with the working solution. The test results are as Figure 6 shown.

[0106] The results showed that when the concentration of the implant sample solution was 0.67 mg / mL, the ABTS radical scavenging rate of the high-molecular-weight agarose implant in Comparative Example 1 was 26.12%; in Example 1, the ABTS radical scavenging rate of the agarose implant containing a high / medium-high combination was 28%, the ABTS radical scavenging rate of the agarose implant containing a high / medium combination was 32.39%, the ABTS radical scavenging rate of the agarose implant containing a high / low combination was 38.97%, the ABTS radical scavenging rate of the agarose implant containing a high / medium-high / medium combination was 31.53%, the ABTS radical scavenging rate of the agarose implant containing a high / medium-high / low combination was 35.95%, the ABTS radical scavenging rate of the agarose implant containing a high / medium / low combination was 38.81%, the ABTS radical scavenging rate of the agarose implant containing a high / medium-high / medium / low combination was 38.91%, the ABTS radical scavenging rate of the agarose implant containing a medium-high / medium combination was 34.16%, the ABTS radical scavenging rate of the agarose implant containing a medium-high / low combination was 40.79%, the ABTS radical scavenging rate of the agarose implant containing a medium / low combination was 45.12%, and the ABTS radical scavenging rate of the agarose implant containing a medium-high / medium / low combination was 39.98%. The overall trend indicated that the combination containing low-molecular-weight agarose had stronger antioxidant ability.

[0107] 7. Anti-wrinkle Ability Test of Implants on Zebrafish

[0108] Ultraviolet irradiation can cause changes in collagen, elastin, etc. in the skin structure of zebrafish, resulting in phenomena such as fin shrinkage and deformation. After adding the test substance, by analyzing the change in the area of the caudal fin, the degree of protection of the test substance against caudal fin deformation of zebrafish is analyzed, so as to evaluate its anti-wrinkle efficacy. The test zebrafish were divided into three groups: a normal control group, a model control group, and a test article group. The normal control group was not treated with anything; the model control group was only irradiated with ultraviolet light; the test article group was quantitatively added with the test sample (the concentration of the test article was 0.25%) while being irradiated with ultraviolet light. Incubate for 2 h, and perform 3 times of ultraviolet light irradiation under the condition of an irradiance of 2.0 - 2.5 Mw / cm 2 , and each irradiation time was 15 min, with an interval of 30 min. Continue to incubate in the dark in a biochemical incubator at (28.5 °C ± 1.0 °C) for 22 h and then observe. After the incubation ended, the zebrafish were photographed, and the anti-wrinkle efficacy of the test article was evaluated by the area of the caudal fin of the zebrafish in the selected quantitative region.

[0109] The test samples included: the implant in Example 1 and the traditional high-molecular-weight agarose implant in Comparative Example 1 with a concentration diluted to 0.25%. The test results are as Figure 7 shown. The results showed that the agarose implant with a high / medium-high / medium / low combination had the best anti-wrinkle effect.

[0110] 8. Screening of the Optimal Combinations of Agarose with Different Molecular Weights in the Implant

[0111] For different combinations, the optimal scheme was screened by evaluating their gel-forming ability, gel strength, viscosity, injectability, degradability, antioxidant property, and anti-wrinkle property. A scoring system was used to evaluate various properties of the implant. The evaluation criterion for gel-forming ability was that if it could form a gel, it was rated 1 point, and if it could not, it was rated 0 point. Excessively high gel strength would affect the injection performance, while too low strength would be difficult to provide effective support; therefore, the evaluation criterion for gel strength was set as follows: when the angular frequency ω = 1.27 rad / s, if the storage modulus (G') > 45,000 Pa, it was rated 0 point, if G' < 15,000 Pa, it was rated 0 point, and if 15,000 Pa < G' < 45,000 Pa, it was rated 1 point. Excessively high viscosity would reduce the injection performance, while too low viscosity might cause the implant to displace at the implantation site; therefore, the evaluation criterion for viscosity was set as follows: when the shear rate = 0.407 s -1 -1, if the viscosity > 30,00000 mPa·s, it was rated 0 point, if the viscosity < 4,00000 mPa·s, it was rated 0 point, and if 4,00000 mPa·s < viscosity < 30,00000 mPa·s, it was rated 1 point. The smaller the thrust required for injection, the better the injectability of the implant; therefore, the higher the thrust, the lower the score, the highest thrust was rated 0 point, and as the thrust decreased, the score increased successively (each combination was scored with the maximum value). Too fast degradation rate would affect the persistence of the implant; therefore, the degradation rate on the 7th day was used as the evaluation criterion, the higher the degradation rate, the lower the score, the highest degradation rate was rated 0 point, and as the degradation rate decreased, the score increased successively. The better the antioxidant and anti-wrinkle effects, the better the anti-aging effect, the best effect was rated 11 points, and the scores decreased successively. The scoring results are shown in Table 1.

[0112] Table 1: Comprehensive Evaluation of Agarose Implants Prepared in Example 1 and Comparative Example 1

[0113]

[0114] The comprehensive evaluation results showed that the composite implant containing high-molecular-weight / middle-high-molecular-weight / middle-molecular-weight / low-molecular-weight agarose showed excellent performance in terms of gel-forming ability, gel strength, viscosity, injectability, degradability, antioxidant property, and anti-wrinkle property, with the highest score, and was determined as the optimal implant combination for anti-aging treatment.

[0115] Verified by the experiments of the inventors, the composite implants prepared in Examples 2 - 6 also showed good injectability, antioxidant, and anti-wrinkle effects, and could be applied to the field of skin anti-aging.

[0116] In summary, the present invention provides a composite implant containing agarose with different molecular weights and a preparation method thereof. By combining medical-grade agarose with high molecular weight (>100,000), medium-high molecular weight (30,000-100,000), medium molecular weight (3,000-30,000), and low molecular weight (<3,000) in a specific ratio, a composite agarose implant with excellent performance is prepared. For different combinations, the optimal scheme, namely the high molecular weight / medium-high molecular weight / medium molecular weight / low molecular weight composite agarose implant, is screened by evaluating its gel-forming property, rheological property, injectability, degradability, antioxidant property, and anti-wrinkle property. The composite implant containing agarose with different molecular weights prepared by the present invention has low mechanical strength and viscosity and excellent injectability. The composite implant containing agarose with different molecular weights exhibits good antioxidant and anti-wrinkle effects and has broad application prospects in the field of skin anti-aging.

[0117] The above specific embodiments have described the implementation of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple variations all belong to the protection scope of the present invention.

Claims

1. A composite implant containing agarose of different molecular weights, characterized in that: include: A combination of two or more of high molecular weight medical grade agarose, medium-high molecular weight medical grade agarose, medium molecular weight medical grade agarose, and low molecular weight medical grade agarose; Wherein, the molecular weight of the high molecular weight medical grade agarose is greater than 100,000; The molecular weight of the medium-high molecular weight medical-grade agarose is 30,000 to 100,000; The molecular weight of the medium molecular weight medical grade agarose is 3000-30,000; The molecular weight of the low molecular weight medical grade agarose is less than 3000.

2. A composite implant containing agarose of different molecular weights according to claim 1, characterized in that: The combination of agarose with different molecular weights includes: high / medium-high molecular weight medical grade agarose, high / medium molecular weight medical grade agarose, high / low molecular weight medical grade agarose, high / medium-high / medium molecular weight medical grade agarose, high / medium-high / low molecular weight medical grade agarose, high / medium-high / low molecular weight medical grade agarose, high / medium-high / medium / low molecular weight medical grade agarose, high / medium-high / medium / low molecular weight medical grade agarose, medium-high / medium molecular weight medical grade agarose, medium-high / low molecular weight medical grade agarose, medium-high / medium / low molecular weight medical grade agarose or medium / low molecular weight medical grade agarose.

3. A composite implant containing agarose of different molecular weights according to claim 2, characterized in that: The total mass percentage concentration of the agarose with different molecular weights in the composite implant is 0.5% to 10%.

4. A composite implant containing agarose of different molecular weights according to claim 3, characterized in that: The total mass percentage concentration of the agarose with different molecular weights in the composite implant is 1.0% to 3.7%.

5. A composite implant containing agarose of different molecular weights according to claim 4, characterized in that: In the combined high / medium-high molecular weight medical grade agarose, the mass percentage concentration of high and medium-high molecular weight medical grade agarose in the composite implant is (0.7%~1.5%): (0.5%~1.6%); In the combined high / medium molecular weight medical grade agarose, the mass percentage concentration of high and medium molecular weight medical grade agarose in the composite implant is (0.7%~1.5%): (0.5%~1.6%); In the combined high / low molecular weight medical grade agarose, the mass percentage concentration of high and low molecular weight medical grade agarose in the composite implant is (0.7%~1.5%): (0.5%~1.6%); In the combined high / medium-high / medium molecular weight medical grade agarose, the mass percentage concentrations of high, medium-high and medium molecular weight medical grade agarose in the composite implant are (0.5%~1.2%): (0.4%~1.3%): (0.5%~1.2%); In the combined high / medium-high / low molecular weight medical grade agarose, the mass percentage concentrations of high, medium-high and low molecular weight medical grade agarose in the composite implant are (0.5%~1.2%): (0.4%~1.3%): (0.5%~1.2%); In the combined high / medium / low molecular weight medical grade agarose, the mass percentage concentrations of high, medium and low molecular weight medical grade agarose in the composite implant are (0.5%~1.2%): (0.4%~1.3%): (0.5%~1.2%); In the combination of high / medium-high / medium / low molecular weight medical grade agarose, the mass percentage concentration of high, medium-high, medium and low molecular weight medical grade agarose in the composite implant is (0.4%~0.7%): (0.3%~0.7%): (0.2%~0.8%): (0.1%~0.9%); In the high / medium molecular weight medical grade agarose in the combination, the mass percentage concentration of medium-high and medium molecular weight medical grade agarose in the composite implant is (0.7%~1.5%): (0.5%~1.6%); In the combination of high / low molecular weight medical grade agarose, the mass percentage concentration of high and low molecular weight medical grade agarose in the composite implant is (0.7%~1.5%): (0.5%~1.6%); In the high / medium / low molecular weight medical grade agarose in the combination, the mass percentage concentration of medium-high, medium-low molecular weight medical grade agarose in the composite implant is (0.5%~1.2%): (0.4%~1.3%): (0.5%~1.2%); In the combined medium / low molecular weight medical grade agarose, the mass percentage concentration of the medium and low molecular weight medical grade agarose in the composite implant is (0.7%~1.5%): (0.5%~1.6%).

6. A composite implant containing agarose of different molecular weights according to claim 2, characterized in that: The combination is high / medium high / medium / low molecular weight medical grade agarose.

7. The method for preparing the composite implant containing agarose of different molecular weights according to any one of claims 1 to 6, characterized in that: The following steps are involved: Medical grade agarose of different molecular weights is mixed with water and heated to 90-120°C for complete dissolution to obtain a solution; the solution is cooled to 10-40°C and allowed to stand for reaction to obtain a composite implant containing agarose of different molecular weights.

8. The method for preparing the composite implant containing agarose of different molecular weights according to claim 7, characterized in that: The dissolution temperature is 95-105°C, and the reaction temperature is 25-37°C.

9. Use of the composite implant containing agarose of different molecular weights as claimed in any one of claims 1 to 6 in the field of skin anti-aging.

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