A composite implant containing agarose of different molecular weights, its preparation method and application

Abstract

The application provides a composite implant containing different molecular weight agarose and a preparation method and application thereof, and belongs to the technical field of biomedical materials. The composite implant comprises two or more than two combinations 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. The application combines medical grade agarose with different molecular weights, so that the composite material has good injectability, moderate gel strength and controllable degradation performance. The prepared composite implant containing different molecular weight agarose has good antioxidant and anti-wrinkle effects, and has a wide application prospect in the field of skin anti-aging.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular to a composite implant containing agarose with different molecular weights and a preparation method and application thereof. BACKGROUND

[0002] With age, people's skin gradually shows signs of aging, including thinning of the dermis, decreased elasticity, and deepening of wrinkles. Endogenous aging is regulated by genes and is accompanied by degradation of extracellular matrix components; while exogenous aging is mainly induced by ultraviolet radiation and oxidative stress, leading to collagen degradation and loss of elastin. Existing anti-aging treatments, such as laser therapy, injection of fillers and topical antioxidants, can improve skin aging to some extent, but still have problems such as limited efficacy, insufficient durability and potential side effects.

[0003] Agarose is a natural linear polysaccharide extracted from red algae, and the basic structural unit is β-D-galactose and 3,6-anhydrous-α-L-galactose, which has excellent biocompatibility and non-immunogenicity. Agarose has temperature-sensitive gelation, which can melt or solidify into a gel with temperature change. In the gel state, the molecular chains exist in the form of double helix, and the double helix aggregates 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 cell growth and proliferation, and agarose also has good biocompatibility and non-immunogenicity, etc. Based on its unique properties, agarose has wide application potential in the field of dermatology.

[0004] However, the application of traditional high molecular weight agarose in skin repair is limited by its high mechanical strength, which leads to injection difficulty and limited bioactivity, affecting its feasibility in clinical application. Therefore, it is urgent to develop an agarose multifunctional implant with good injectability, appropriate gel strength, controllable degradation performance and excellent bioactivity, to meet the needs of tissue repair and regenerative medicine and other application scenarios. SUMMARY

[0005] In view of the shortcomings of the prior art, the present application provides a composite implant containing agarose with different molecular weights and a preparation method and application thereof. By compounding medical-grade agarose with different molecular weights, the composite material is endowed with good injectability, moderate gel strength and controllable degradation performance, and also has good antioxidant and anti-wrinkle effects, providing an innovative biomaterial choice for skin anti-aging treatment.

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

[0007] In a first aspect, the present application provides a composite implant containing different molecular weight agarose, comprising: two or more combinations 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] The high molecular weight medical grade agarose has a molecular weight greater than 100,000;

[0009] The medium-high molecular weight medical grade agarose has a molecular weight of 30,000-100,000;

[0010] The medium molecular weight medical grade agarose has a molecular weight of 3,000-30,000;

[0011] The low molecular weight medical grade agarose has a molecular weight less than 3,000.

[0012] In view of the problems of injection difficulty caused by high mechanical strength and limited bioactivity of traditional high molecular weight agarose in skin repair, the present application combines agarose with different molecular weights and uses specific proportions and preparation methods to prepare a composite gel implant with excellent rheological properties and injectability, while having the mechanical support of high molecular weight agarose and the permeability and bioactivity of low molecular weight agarose. Through the synergistic effect of agarose with different molecular weights, better comprehensive performance and repair effect are achieved, making the composite implant of the present application an ideal biological material for the new generation of anti-aging treatment. In addition, the raw materials of the composite implant of the present application only include agarose with different molecular weights and water, which has fewer types of raw materials, high biocompatibility, reduces the risk of adverse reactions caused by the interaction between materials, and reduces allergic or rejection reactions caused by multiple material combinations. Moreover, the production process of the present application is simpler and is conducive to industrial scale production.

[0013] Further, 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 / 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 agarose with different molecular weights in the composite implant is 0.5%-10%.

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

[0016] Further, in the combined high / medium high molecular weight medical grade agarose, the mass percentage concentration of high, medium high molecular weight medical grade agarose in the composite implant is (0.7%~1.5%): (0.5%~1.6%);

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

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

[0019] In the combined high / medium high / medium molecular weight medical grade agarose, the mass percentage concentration of high, medium high, medium molecular weight medical grade agarose in the composite implant is (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 concentration of high, medium high, low molecular weight medical grade agarose in the composite implant is (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 concentration of 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%);

[0022] In the combined high / medium high / medium / low molecular weight medical grade agarose, the mass percentage concentration of high, medium high, medium, 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%);

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

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

[0025] 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 (0.625%): (0.625%): (0.625%): (0.625%).

[0026] In the combination of medium / low molecular weight medical grade agarose, the mass percentage concentration of medium and low molecular weight medical grade agarose in the composite implant is (0.7%~1.5%): (0.5%~1.6%).

[0027] Further, the combination is high / medium high / medium / low molecular weight medical grade agarose.

[0028] Further, in the combination of high / medium high / medium / 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 medium high molecular weight medical grade agarose in the composite implant is 0.625%; the mass percentage concentration of medium molecular weight medical grade agarose in the composite implant is 0.625%; and 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 application provides a preparation method of the composite implant containing different molecular weight agarose, comprising the following steps:

[0030] Mixing medical grade agarose with different molecular weights with water, and heating to 90~120 ℃ to completely dissolve, to obtain a clear and transparent solution; cooling the solution to 10~40 ℃, and standing for reaction to obtain the composite implant containing different molecular weight agarose.

[0031] Further, the dissolution temperature is 95~105 ℃, and the reaction temperature is 25~37 ℃.

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

[0033] Compared with the prior art, the present application has the following advantages:

[0034] (1) The composite implant containing different molecular weight agarose has excellent rheological properties and injectability, and at the same time has the mechanical support of high molecular weight agarose and the permeability and biological activity of low molecular weight agarose, so as to realize better comprehensive performance and repair effect, so that the composite implant becomes an ideal biological material for new generation anti-aging treatment.

[0035] (2) Compared with the traditional high molecular weight agarose implant, the complex implant containing different molecular weight agaroses has reduced mechanical strength and viscosity, and enhanced injectability, effectively solving the problem of uneven injection force of the traditional high molecular weight agarose implant;

[0036] (3) The complex implant containing different molecular weight agaroses has antioxidant and anti-wrinkle effects, and has significant application potential in the field of skin anti-aging;

[0037] (4) The complex implant only includes different molecular weight agaroses and water as raw materials, has less types of raw materials, high biocompatibility, reduces the risk of adverse reactions caused by the interaction between materials, and reduces allergic or rejection reactions caused by multi-material combination; and the production process of the complex implant is simpler, which is conducive to industrial scale production. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is a physical map of the complex implant containing different molecular weight agaroses of the present application;

[0039] Figure 2 is the gel strength of the complex implant containing different molecular weight agaroses of the present application;

[0040] Figure 3 is the viscosity of the complex implant containing different molecular weight agaroses of the present application;

[0041] Figure 4 is the injectability of the complex implant containing different molecular weight agaroses of the present application;

[0042] Figure 5 is the degradability of the complex implant containing different molecular weight agaroses of the present application;

[0043] Figure 6 is the antioxidant capacity of the complex implant containing different molecular weight agaroses of the present application;

[0044] Figure 7 is the zebrafish anti-wrinkle effect of the complex implant containing different molecular weight agaroses of the present application. DETAILED DESCRIPTION

[0045] The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0046] The present application provides a complex implant containing different molecular weight agarose, comprising: two or more combinations 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] The high molecular weight medical grade agarose has a molecular weight greater than 100,000;

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

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

[0050] The low molecular weight medical grade agarose has a molecular weight less than 3,000.

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

[0052] In some examples, the combination of different molecular weight agarose 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.

[0053] In some examples, in the combination high / medium-high molecular weight medical grade agarose, the mass percentage concentration of high, medium-high molecular weight medical grade agarose in the composite implant is (0.7%~1.5%): (0.5%~1.6%); in the combination high / medium molecular weight medical grade agarose, the mass percentage concentration of high, medium molecular weight medical grade agarose in the composite implant is (0.7%~1.5%): (0.5%~1.6%); in the combination high / low molecular weight medical grade agarose, the mass percentage concentration of high, low molecular weight medical grade agarose in the composite implant is (0.7%~1.5%): (0.5%~1.6%); in the combination high / medium-high / medium molecular weight medical grade agarose, the mass percentage concentration of high, medium-high, medium molecular weight medical grade agarose in the composite implant is (0.5%~1.2%): (0.4%~1.3%): (0.5%~1.2%); in the combination high / medium-high / low molecular weight medical grade agarose, the mass percentage concentration of high, medium-high, 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 combination high / medium / low molecular weight medical grade agarose, the mass percentage concentration of 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 combination high / medium-high / medium / low molecular weight medical grade agarose, the mass percentage concentration of high, medium-high, medium, 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 combination medium-high / medium molecular weight medical grade agarose, the mass percentage concentration of medium-high, medium molecular weight medical grade agarose in the composite implant is (0.7%~1.5%): (0.5%~1.6%); in the combination medium-high / low molecular weight medical grade agarose, the mass percentage concentration of medium-high, low molecular weight medical grade agarose in the composite implant is (0.7%~1.5%): (0.5%~1.6%); in the combination medium-high / medium / low molecular weight medical grade agarose, 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 combination medium / low molecular weight medical grade agarose, the mass percentage concentration of medium, low molecular weight medical grade agarose in the composite implant is (0.7%~1.5%): (0.5%~1.6%).

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

[0055] The medical grade agarose with different molecular weights is mixed with ultrapure water and heated to 90-120℃ to completely dissolve, to obtain a clear and transparent solution; the solution is cooled to 10-40℃, and left to react, and after the gel is formed, sterile packaging is performed, 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; and 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 / medium-high molecular weight medical grade agarose

[0059] 125 mg of high molecular weight medical grade agarose powder and 125 mg of medium-high molecular weight medical grade agarose powder are weighed, respectively, dissolved in 10 mL of ultrapure water, and heated to 100℃ until completely dissolved, to obtain a clear and transparent solution. Then, the obtained solution is cooled to 37℃, left to react, and a composite implant containing high / medium-high molecular weight medical grade agarose is obtained.

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

[0061] 125 mg of high molecular weight medical grade agarose powder and 125 mg of medium molecular weight medical grade agarose powder are weighed, respectively, dissolved in 10 mL of ultrapure water, and heated to 100℃ until completely dissolved, to obtain a clear and transparent solution. Then, the obtained solution is cooled to 37℃, left to react, and a composite implant containing high / medium molecular weight medical grade agarose is obtained.

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

[0063] 125 mg of high molecular weight medical grade agarose powder and 125 mg of low molecular weight medical grade agarose powder are weighed, respectively, dissolved in 10 mL of ultrapure water, and heated to 100℃ until completely dissolved, to obtain a clear and transparent solution. Then, the obtained solution is cooled to 37℃, left to react, and a composite implant containing high / low molecular weight medical grade agarose is obtained.

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

[0065] Take 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 in 10 mL of ultrapure water, and heat to 100 ℃ until completely dissolved, to obtain a clear transparent solution. Then, the obtained solution is cooled to 37 ℃, and left to react to obtain a composite implant containing high / medium high / medium molecular weight medical grade agarose.

[0066] 5. Preparation of a composite implant containing high molecular weight / medium high molecular weight / low molecular weight medical grade agarose

[0067] Take 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 in 10 mL of ultrapure water, and heat to 100 ℃ until completely dissolved, to obtain a clear transparent solution. Then, the obtained solution is cooled to 37 ℃, and left to react to obtain a composite implant containing high / medium / low molecular weight medical grade agarose.

[0068] 6. Preparation of a composite implant containing high molecular weight / medium molecular weight / low molecular weight medical grade agarose

[0069] Take 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 in 10 mL of ultrapure water, and heat to 100 ℃ until completely dissolved, to obtain a clear transparent solution. Then, the obtained solution is cooled to 37 ℃, and left to react to obtain a composite implant containing high / medium / low molecular weight medical grade agarose.

[0070] 7. Preparation of a composite implant containing high molecular weight / medium high molecular weight / medium molecular weight / low molecular weight medical grade agarose

[0071] Take 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 in 10 mL of ultrapure water, and heat to 100 ℃ until completely dissolved, to obtain a clear transparent solution. Then, the obtained solution is cooled to 37 ℃, and left to react to obtain a composite implant containing high molecular weight / medium high molecular weight / medium molecular weight / low molecular weight medical grade agarose.

[0072] 8. Preparation of a composite implant containing medium high molecular weight / medium molecular weight medical grade agarose

[0073] Take 125 mg of medium high molecular weight medical grade agarose powder and 125 mg of medium molecular weight medical grade agarose powder, respectively, dissolve in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved, to obtain a clear transparent solution. Then, the obtained solution is cooled to 37 °C, and left to react at rest 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] Take 125 mg of medium high molecular weight medical grade agarose powder and 125 mg of low molecular weight medical grade agarose powder, respectively, dissolve in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved, to obtain a clear transparent solution. Then, the obtained solution is cooled to 37 °C, and left to react at rest 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] Take 125 mg of medium high molecular weight medical grade agarose powder and 125 mg of low molecular weight medical grade agarose powder, respectively, dissolve in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved, to obtain a clear transparent solution. Then, the obtained solution is cooled to 37 °C, and left to react at rest to obtain a composite implant containing medium high 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] Take 125 mg of medium high molecular weight medical grade agarose powder and 125 mg of low molecular weight medical grade agarose powder, respectively, dissolve in 10 mL of ultrapure water, and heat to 100 °C until completely dissolved, to obtain a clear transparent solution. Then, the obtained solution is cooled to 37 °C, and left to react at rest to obtain a composite implant containing medium high molecular weight / low molecular weight medical grade agarose.

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

[0081] Take 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 in 10 mL of ultrapure water, and heat to 100 ℃ until completely dissolved, to obtain a clear transparent solution. Then, the obtained solution is cooled to 37℃, and left to react 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 A composite implant containing agarose of different molecular weights

[0083] Take 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 in 10 mL of ultrapure water, and heat to 100 ℃ until completely dissolved, to obtain a clear transparent solution. Then, the obtained solution is cooled to 37℃, and left to react 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 A composite implant containing agarose of different molecular weights

[0085] Take 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 in 10 mL of ultrapure water, and heat to 100 ℃ until completely dissolved, to obtain a clear transparent solution. Then, the obtained solution is cooled to 37℃, and left to react 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 A composite implant containing agarose of different molecular weights

[0087] Take 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 in 10 mL of ultrapure water, and heat to 100 ℃ until completely dissolved, to obtain a clear transparent solution. Then, the obtained solution is cooled to 37℃, and left to react 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 conventional high molecular weight agarose implant

[0089] Take 250 mg of high molecular weight medical grade agarose powder, dissolve in 10 mL of ultrapure water, and heat to 100°C until completely dissolved to obtain a clear transparent solution. Then, the obtained solution is cooled to 37°C, and left to react to obtain an implant containing high molecular weight medical grade agarose.

[0090] The implant obtained in Example 1 and Comparative Example 1 is subjected to physical and chemical performance testing

[0091] 1. Gelation of the implant

[0092] 1 mL of the agarose mixed solution of different molecular weights in Example 1 and 1 mL of the traditional high molecular weight agarose solution in Comparative Example 1 are respectively transferred into transparent vials while hot and left to stand at room temperature; 1 mL of the implant sample is placed in a centrifuge tube cap and also left to stand at room temperature. The actual implant is as shown in Figure 1 The results show that the composite implant containing different molecular weight agarose of the present application and the traditional high molecular weight agarose implant can both form hydrogel.

[0093] 2. Gel strength test of the implant

[0094] The agarose mixed solution of different molecular weights in Example 1 and 1 mL of the traditional high molecular weight agarose solution in Comparative Example 1 are respectively prepared into gel blocks with uniform thickness of 1 cm x 1 cm x 2 mm, and the gel blocks are placed on the sample table of a rheometer, and the shear strain (1%) of the rheometer is fixed to test the G'(Pa) and G"(Pa) of the gel with the change of angular frequency. The G'(Pa) and G"(Pa) test results of the hydrogel containing different molecular weight agarose and the traditional high molecular weight agarose hydrogel are as shown in Figure 2 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 combination without high molecular weight is lower than that of the combination with high molecular weight, and the minimum gel strength of the combination with high molecular weight is high / medium high / medium / low.

[0095] 3. Shear viscosity test of the implant

[0096] The agarose mixed solution of different molecular weights in Example 1 and 1 mL of the traditional high molecular weight agarose solution in Comparative Example 1 are respectively prepared into gel blocks with uniform thickness of 1 cm x 1 cm x 2 mm, and the gel blocks are placed on the sample table of a rheometer, and the shear rate is set to 0.01 s -1 ~1 00 s -1The shear viscosity of the hydrogels containing different molecular weight agarose and the traditional high molecular weight agarose hydrogel was tested by performing a peristaltic scan. The results are shown in Table 2. Figure 3 The shear viscosity 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 viscosity of the hydrogel without the high molecular weight combination is lower than that of the hydrogel with the high molecular weight combination, and the viscosity of the hydrogel with the high / medium high / medium / low combination is the lowest.

[0097] 4. Test of injection performance of the implant

[0098] The hot mixed solution of agarose 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 a 1 mL injection needle, cooled at room temperature overnight, and then the needle passability of the sample was tested using a needle passability tester. During the test, the injection needle was installed, the core rod was pushed at a constant speed, and the sample in the injection needle was pushed out through the needle head to obtain a pushing force curve.

[0099] The injection performance test results of the hydrogels containing different molecular weight agarose and the traditional high molecular weight agarose hydrogel are shown in Table 3. Figure 4 The maximum pushing force 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 injection performance of the hydrogel without the high molecular weight combination is better than that of the hydrogel with the high molecular weight combination, and the injection performance of the hydrogel with the high / medium high / medium / low combination is the best.

[0100] 5. Test of degradability of the implant

[0101] The appropriate amount of the lyophilized sample (W0) of the implant in Example 1 and the traditional high molecular weight agarose implant in Comparative Example 1 was respectively weighed and placed in a centrifugal tube, and then soaked in a 1.0 M hydrochloric acid solution at 37 ℃ to characterize the degradation behavior of the hydrogel. The sample was taken every certain time, the supernatant was removed after centrifugation, and the precipitate was lyophilized 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 Table 4. Figure 5 .

[0102] The results show that the degradation rate of the high molecular weight agarose-containing implant of Comparative Example 1 was 34% at day 7; in Example 1, the degradation rate of the high / medium high combination-containing agarose implant was 50%, the high / medium combination-containing agarose implant was 67%, the high / low combination-containing agarose implant was 67%, the high / medium high / medium combination-containing agarose implant was 66%, the high / medium high / low combination-containing agarose implant was 66%, the high / medium / low combination-containing agarose implant was 78%, the high / medium high / medium / low combination-containing agarose implant was 75%, the medium high / medium combination-containing agarose implant was 83%, the medium high / low combination-containing agarose implant was 83%, the medium / low combination-containing agarose implant was 100%, and the medium high / medium / low combination-containing agarose implant was 88%. The overall trend indicates that the degradation rate of the combination without high molecular weight is higher than that of the combination with high molecular weight.

[0103] 6. Test of antioxidant capacity of implants

[0104] The ABTS method was used to evaluate the in vitro antioxidant capacity of the implants. The stable ABTS free radical solution is blue-green and has a maximum absorption peak at 734 nm. When the free radicals are scavenged by the implant sample, the number of free radicals decreases, the color of the solution gradually lightens, and the absorbance at 734 nm decreases accordingly. Therefore, the ability of the sample to scavenge ABTS free radicals can be determined by measuring the change in absorbance.

[0105] The experimental procedure was as follows: a 7.4 mmol / L ABTS stock solution was prepared with a 2.6 mmol / L potassium peroxydisulfate (K2S2O8) solution, mixed at a ratio of 1:1, and placed in the dark for 12 h to generate an ABTS free radical solution. Before the experiment, the solution was diluted with ultrapure water to an absorbance of 0.70 ± 0.02 as a working solution. The implant sample solution was prepared at different concentrations and 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 the absorbance was measured at 734 nm using a microplate reader after incubation at room temperature in the dark for 3-5 min. The scavenging rate was calculated according to the following formula: Scavenging rate (%) = [(A0-A) / A0] x 100, where A0 is the absorbance of the working solution and A is the absorbance of the implant sample mixed with the working solution. The test results are shown in Table 2. Figure 6

[0106] ​The results show that when the concentration of the implant sample solution is 0.67 mg / mL, the ABTS free radical scavenging rate of the high molecular weight agarose implant of Comparative Example 1 is 26.12%; in Example 1, the ABTS free radical scavenging rate of the high / medium high combination agarose implant is 28%, the ABTS free radical scavenging rate of the high / medium combination agarose implant is 32.39%, the ABTS free radical scavenging rate of the high / low combination agarose implant is 38.97%, the ABTS free radical scavenging rate of the high / medium high / medium combination agarose implant is 31.53%, the ABTS free radical scavenging rate of the high / medium high / low combination agarose implant is 35.95%, the ABTS free radical scavenging rate of the high / medium / low combination agarose implant is 38.81%, the ABTS free radical scavenging rate of the high / medium high / medium / low combination agarose implant is 38.91%, the ABTS free radical scavenging rate of the medium high / medium combination agarose implant is 34.16%, the ABTS free radical scavenging rate of the medium high / low combination agarose implant is 40.79%, the ABTS free radical scavenging rate of the medium / low combination agarose implant is 45.12%, and the ABTS free radical scavenging rate of the medium high / medium / low combination agarose implant is 39.98%. The overall trend indicates that the combination containing low molecular weight agarose has stronger antioxidant capacity.

[0107] 7. Zebrafish anti-wrinkle ability test of implants

[0108] UV irradiation can cause changes in collagen, elastin and other components in the skin structure of zebrafish, causing the fish fin to wrinkle, deform and other phenomena. After adding the test substance, the degree of protection of the test substance against deformation of the zebrafish tail fin is analyzed by changes in the area of the tail fin, so as to evaluate its anti-wrinkle efficacy. The test zebrafish are divided into three groups: normal control group, model control group and test product group. The normal control group is not treated; the model control group is only subjected to UV irradiation; the test product group is quantitatively added with the test sample (test product concentration is 0.25%) while being subjected to UV irradiation. Incubate for 2 h, and irradiate with UV light for 3 times under the condition of irradiance of 2.0~2.5 Mw / cm 2 The observation is carried out after 22 h of incubation in the dark in a biochemical incubator (28.5 ℃±1.0 ℃). After incubation, the zebrafish are photographed, and the area of the zebrafish tail fin in the selected quantitative region is evaluated to evaluate the anti-wrinkle efficacy of the test product.

[0109] The test samples include: the implant of Example 1 and the traditional high molecular weight agarose implant of Comparative Example 1 diluted to a concentration of 0.25%. The test results are shown in Table 2. Figure 7 The results show that the high / medium high / medium / low combination agarose implant has the best anti-wrinkle effect.

[0110] 8. Screening of optimal combination of different molecular weight agarose in implant

[0111] The optimal scheme was screened by evaluating the gelation, gel strength, viscosity, injectability, degradability, antioxidant and anti-wrinkle properties of different combinations. The scores were used to evaluate the various properties of the implant. The evaluation standard for gelation was that 1 point was given to the gelation and 0 point was given to the non-gelation. The gel strength was too high to affect the injection performance, and the strength was too low to provide effective support; therefore, the evaluation standard for the gel strength was set as follows: when the angular frequency ω = 1.27 rad / s, the storage modulus (G') > 45,000 Pa was given 0 point, G' < 15,000 Pa was given 0 point, and 15,000 Pa < G' < 45,000 Pa was given 1 point. The viscosity was too high to reduce the injection performance, and the viscosity was too low to cause the implant to be displaced at the implant site; therefore, the evaluation standard for the viscosity was set as follows: when the shear rate = 0.407 s -1 , the viscosity > 30,00000 mPa·s was given 0 point, the viscosity < 4,00000 mPa·s was given 0 point, and 4,00000 mPa·s < viscosity < 30,00000 mPa·s was given 1 point. The smaller the injection force required, the better the injectability of the implant; therefore, the higher the injection force, the lower the score, and the highest injection force was given 0 point, and the score was increased in turn as the injection force decreased. The faster the degradation rate, the lower the score, and the highest degradation rate was given 0 point, and the score was increased in turn as the degradation rate decreased. The better the antioxidant and anti-wrinkle effects, the better the anti-aging effect, and the best effect was given 11 points, and the score was decreased in turn. The score results are shown in Table 1.

[0112] Table 1: Comprehensive evaluation of agarose implant prepared in Example 1 and Comparative Example 1

[0113]

[0114] The comprehensive evaluation results show that the composite implant containing high molecular weight / medium-high molecular weight / medium molecular weight / low molecular weight agarose performs excellent in the gelation, gel strength, viscosity, injectability, degradability, antioxidant and anti-wrinkle properties, and has the highest score, and is determined as the optimal implant combination for anti-aging treatment.

[0115] The composite implant prepared in Examples 2 to 6 also performs good injectability and antioxidant and anti-wrinkle effects, and can be applied to the field of skin anti-aging, which is verified by the inventors.

[0116] In summary, the present application provides a complex implant containing different molecular weight agarose and a preparation method thereof, wherein 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) is combined in a specific ratio to prepare a complex agarose implant with excellent performance. For different combinations, the optimal scheme is screened by evaluating the gel forming property, rheological property, injectability, degradability, antioxidant property and wrinkle resistance, i.e. a high molecular weight / medium-high molecular weight / medium molecular weight / low molecular weight complex agarose implant. The complex implant containing different molecular weight agarose prepared by the present application has low mechanical strength and viscosity, and has excellent injectability. The complex implant containing different molecular weight agarose shows good antioxidant and wrinkle resistance, and has a wide application prospect in the field of skin anti-aging.

[0117] The above detailed description of the embodiments of the present application, but the present application is not limited to the specific details of the above-described embodiments. Within the scope of the claims and technical concepts of the present application, various simple modifications and changes can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A composite implant comprising different molecular weight agarose, characterized in that, Comprise: two or more combinations of high molecular weight medical grade agarose, medium-high molecular weight medical grade agarose, medium molecular weight medical grade agarose, low molecular weight medical grade agarose; the combination is specifically: High / medium molecular weight medical grade agarose, the mass percentage concentration of the high, medium molecular weight medical grade agarose in the composite implant is (0.7%~1.5%): (0.5%~1.6%); or, High / low molecular weight medical grade agarose, the mass percentage concentration of the high, low molecular weight medical grade agarose in the composite implant is (0.7%~1.5%): (0.5%~1.6%); or, High / medium-high / medium molecular weight medical grade agarose, the mass percentage concentration of the high, medium-high, medium molecular weight medical grade agarose in the composite implant is (0.5%~1.2%): (0.4%~1.3%): (0.5%~1.2%); or, High / medium-high / low molecular weight medical grade agarose, the mass percentage concentration of the high, medium-high, low molecular weight medical grade agarose in the composite implant is (0.5%~1.2%): (0.4%~1.3%): (0.5%~1.2%); or, High / medium / low molecular weight medical grade agarose, the mass percentage concentration of the 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%); or, High / medium-high / medium / low molecular weight medical grade agarose, the mass percentage concentration of the high, medium-high, medium, 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%); or, Medium-high / medium molecular weight medical grade agarose, the mass percentage concentration of the medium-high, medium molecular weight medical grade agarose in the composite implant is (0.7%~1.5%): (0.5%~1.6%); or, Medium-high / low molecular weight medical grade agarose, the mass percentage concentration of the medium-high, low molecular weight medical grade agarose in the composite implant is (0.7%~1.5%): (0.5%~1.6%); 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 30,000~100,000; The molecular weight of the medium molecular weight medical grade agarose is 3,000~30,000; The molecular weight of the low molecular weight medical grade agarose is less than 3,000; The preparation method of the composite implant containing different molecular weight agarose comprises the following steps: Mix medical grade agarose with different molecular weights with water, heat to 90~120 ℃ to completely dissolve, and obtain a solution; cool the solution to 10~40 ℃, and stand to react to obtain a composite implant containing different molecular weight agarose.

2. The complex implant agent containing different molecular weight agarose according to claim 1, characterized in that, The combination is high / medium-high / medium / low molecular weight medical grade agarose.

3. The complex implant containing agarose of different molecular weight according to claim 1, wherein the agarose is agarose of 200,000 to 300,000 Da. The dissolution temperature is 95~105 ℃, and the reaction temperature is 25~37 ℃.

4. Use of the complex implant containing different molecular weight agarose according to any one of claims 1 to 3 for the preparation of a skin anti-aging material.

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