Injectable self-healing self-assembling herring sperm extract hydrogel, and preparation method and application thereof

Abstract

The application discloses an injectable self-healing self-assembling protamine extract hydrogel and a preparation method and application thereof, and belongs to the technical field of hydrogel materials and wound repair. The hydrogel is prepared by mixing protamine extract and a crosslinking agent solution; the crosslinking agent solution comprises a salt solution with a final concentration of 0.01-4.5 M, 0.01-0.1 M sodium citrate and 0.01-0.1 M sodium bicarbonate; the protamine extract is mixed with the crosslinking agent solution at different proportions (1%-50%, w / v%) and is stirred at 20-35 DEG C until complete gelation; and a series of hydrogels can be prepared after multiple freeze-thaw cycles. The hydrogel prepared by the application has good self-healing, injectable characteristics, blood compatibility and tissue regeneration capacity, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of hydrogel materials and wound repair, and relates to an injectable self-healing self-assembled milt extract hydrogel as well as a preparation method and application thereof. BACKGROUND

[0002] Skin wound repair is a multi-stage process involving the interaction of multiple cells and molecules, and under the regulation of cell behavior and dynamic remodeling of extracellular matrix (ECM), the damaged tissue is repaired and regenerated. Skin damage can trigger a series of physiological responses, including accelerated metabolism, protein and water loss, and endocrine and immune system imbalance, in addition to granulation tissue edema, wound infection, etc.

[0003] Therefore, research on how to repair skin wounds has broad application prospects. SUMMARY

[0004] The application aims to provide an injectable self-healing self-assembled milt extract hydrogel, and also provides a preparation method and application thereof, so as to make up for the deficiencies in the prior art.

[0005] In order to achieve the above application purposes, the specific technical scheme adopted by the application is as follows:

[0006] An injectable self-healing self-assembled milt extract hydrogel is prepared by mixing milt extract and a crosslinking agent solution.

[0007] Further, the crosslinking agent solution comprises a salt solution with a final concentration of 0.01-4.5M, 0.01-0.1M sodium citrate and 0.01-0.1M sodium bicarbonate. The salt solution comprises sodium chloride solution, potassium chloride solution, potassium chloride solution, calcium chloride solution, sodium acetate solution, etc.

[0008] Further, the crosslinking agent solution comprises a salt solution with a final concentration of 1-4.5M, 0.01-0.5M sodium citrate and 0.05-0.1M sodium bicarbonate.

[0009] Further, the preparation method of the milt extract comprises the following steps:

[0010] (1) washing milt raw materials;

[0011] (2) performing defatting treatment on the milt raw materials;

[0012] (3) homogenizing the defatted milt raw materials in extraction solution I;

[0013] (4) centrifuging the solution treated in step (3) to obtain a precipitate;

[0014] (5) The precipitate obtained in step (4) is added into 1-5 times volume of extraction solution II for homogenization to obtain a homogenate solution;

[0015] (6) The homogenate solution obtained in step (5) is centrifuged, and the supernatant is discarded after centrifugation to obtain a light yellow precipitate, i.e. the fish milt extract.

[0016] Further, the defatting agent used in step (2) is 75-100% pre-cooled ethanol, diethyl ether, petroleum ether, acetone, etc.

[0017] Further, the extraction solution I in step (3) is a mixture of 0.05-0.1 M salt solution and 0.01-0.05 M sodium citrate, and the pH of the extraction solution I is 8-9. The defatted fish milt raw material is added into the extraction solution for homogenization for 1.5 min. The salt solution includes sodium chloride solution, potassium chloride solution, sodium acetate solution, potassium acetate solution, sodium lactate solution, calcium chloride solution, sodium citrate solution, etc.

[0018] Further, in step (4), the homogenized solution is centrifuged at 4°C and 4000 rpm for 15 min, and the supernatant is discarded to obtain a precipitate close to the skin color.

[0019] Further, the extraction solution II in step (5) is a mixture of 0.05-0.1 M salt solution and 0.01-0.05 M sodium citrate, and the pH of the extraction solution II is adjusted to 7-8. The precipitate obtained in step (4) is transferred into 1-5 times weight of pre-cooled extraction solution of the raw material for homogenization for 1.5 min. The salt solution includes sodium chloride solution, potassium chloride solution, sodium acetate solution, potassium acetate solution, sodium lactate solution, calcium chloride solution, sodium citrate solution, etc.

[0020] Further, in step (6), the homogenate solution is centrifuged at 4°C and 4000 rpm for 15 min, and the supernatant is discarded, and the operation is repeated for 3 times.

[0021] The preparation method of the hydrogel is that the fish milt extract and the crosslinking agent solution are mixed in different proportions (1%-50%, w / v%) and stirred at 20-35°C until complete gelation, and a series of hydrogels are prepared after multiple freeze-thaw cycles.

[0022] Further, the fish milt extract and the crosslinking agent solution are mixed in 5%-30% (w / v%) and stirred at 20-35°C until complete gelation.

[0023] The injectable self-healing self-assembled fish milt extract hydrogel is applied to the preparation of a wound healing promoting product.

[0024] Compared with the prior art, the advantages and beneficial results of the present application are that:

[0025] The application comprehensively utilizes fish extract and green crosslinking agent solution to construct an injectable self-healing self-assembled hydrogel which can promote wound healing. The fish extract and crosslinking agent solution are mixed for the first time to prepare an injectable self-assembled self-healing hydrogel which can promote wound healing. The hydrogel has good self-healing, injectable characteristics, blood compatibility and tissue regeneration capacity, and has wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Morphological analysis of fish extract hydrogel formed.

[0027] Figure 2 Morphological analysis of fish extract hydrogel formed after freeze-thaw cycle.

[0028] Figure 3 Rheological mechanics performance diagram of fish extract hydrogel prepared in different concentrations; wherein (a) is the G' and G" of fish extract hydrogel with a concentration of 20% in strain scanning, (b) is the G' and G" of fish extract hydrogel with a concentration of 10% in strain scanning, (c) is the G' and G" of fish extract hydrogel with a concentration of 5% in strain scanning.

[0029] Figure 4 Rheological analysis of fish extract hydrogel in different concentrations.

[0030] Figure 5 Self-healing property diagram of fish extract hydrogel prepared in different proportions; wherein (a) is the strain scanning of fish extract hydrogel with a concentration of 20% under alternating strain, (b) is the strain scanning of fish extract hydrogel with a concentration of 10% under alternating strain, (c) is the strain scanning of fish extract hydrogel with a concentration of 5% under alternating strain.

[0031] Figure 6 Injectability diagram of fish extract hydrogel prepared in different proportions.

[0032] Figure 7 Blood compatibility analysis of fish extract hydrogel.

[0033] Figure 8 Hemolysis rate analysis of red blood cells after incubation and culture with each group of samples.

[0034] Figure 9 Fish extract hydrogel promotes mouse wound healing effect diagram.

[0035] Figure 10 Wound healing trajectory diagram of each group of mice.

[0036] Figure 11The changes of the wound sizes of the mice in each group after treatment. DETAILED DESCRIPTION

[0037] The present application is further illustrated by the following examples without limiting the present application to the examples. The experimental methods in the following examples are selected according to the conventional methods and conditions, or according to the commercial instructions, if the specific conditions are not mentioned. The numerical values disclosed in the examples of the present application are approximate values, not definite values. All the values within the error range are included without being limited to the specific numerical values disclosed in the examples of the present application, if the error or experimental conditions permit.

[0038] The drugs and reagents involved in the examples are all the ordinary commercially available products, if not otherwise specified.

[0039] Example 1

[0040] 1. A preparation method of fish sperm extract comprising the following steps:

[0041] (1) washing the fish sperm raw material with ultrapure water;

[0042] (2) adding an appropriate volume of pre-cooled 75% ethanol to the fish sperm raw material for degreasing, and placing in a 4°C refrigerator overnight;

[0043] The extraction solution I in the step (3) is a mixture of 0.01-1M salt solution and 0.01-0.1M sodium citrate, and the pH of the extraction solution I is 8-9. The degreased fish sperm raw material is homogenized in the extraction solution for 1.5 min.

[0044] The extraction solution II in the step (5) is a mixture of 0.01-1M salt solution and 0.01-0.1M sodium citrate, and the pH of the extraction solution II is adjusted to 7-8.

[0045] (3) homogenizing the degreased fish sperm raw material in the extraction solution I (a mixture of 0.05M salt solution and 0.05M sodium citrate) with pH of 9 for 1.5 min;

[0046] (4) centrifuging the solution treated in the step (3) at 4000 rpm for 15 min at 4°C, and discarding the supernatant;

[0047] (5) transferring the precipitate obtained in the step (4) to the extraction solution II (a mixture of 0.1M salt solution and 0.05M sodium citrate) with pH of 8, which is pre-cooled and 1-5 times the weight of the raw material, and homogenizing for 1.5 min;

[0048] (6) centrifuging the solution treated in the step (5) at 4000 rpm for 15 min at 4°C, and discarding the supernatant, and repeating the operation for 3 times to obtain a light yellow precipitate, which is the fish sperm extract.

[0049] 2. Preparation of crosslinking agent solution: mixture of sodium chloride with final concentration of 0.5-5 M and sodium citrate, sodium bicarbonate with final concentration of 0.01-0.1 M.

[0050] Example 2:

[0051] A method for preparing an injectable self-assembled self-healing fish mucus extract hydrogel, comprising the following steps:

[0052] 1 g of fish mucus extract prepared by Example 1 was weighed and added to 4 mL of crosslinking agent solution, and completely gelled after stirring at 25°C. After gelling, multiple freeze-thaw cycles were performed to obtain an injectable self-assembled self-healing fish mucus extract hydrogel for promoting wound healing.

[0053] Example 3

[0054] A method for preparing an injectable self-assembled self-healing fish mucus extract hydrogel, comprising the following steps:

[0055] 1 g of fish mucus extract prepared by Example 1 was weighed and added to 9 mL of crosslinking agent solution, and completely gelled after stirring at 25°C. After gelling, multiple freeze-thaw cycles were performed to obtain an injectable self-assembled self-healing fish mucus extract hydrogel for promoting wound healing.

[0056] Example 4

[0057] A method for preparing an injectable self-assembled self-healing fish mucus extract hydrogel, comprising the following steps:

[0058] 1 g of fish mucus extract prepared by Example 1 was weighed and added to 19 mL of crosslinking agent solution, and completely gelled after stirring at 25°C. After gelling, multiple freeze-thaw cycles were performed to obtain an injectable self-assembled self-healing fish mucus extract hydrogel for promoting wound healing.

[0059] Example 5

[0060] The fish mucus extract and crosslinking agent solution were mixed in different proportions to prepare the hydrogel, which was photographed and recorded. The changes in gelling ability and the properties of the gels formed were observed and analyzed. The gelling ability of the fish mucus extract hydrogel synthesized in Examples 2-4 was characterized by observation as shown in Figure 1 and Figure 2 . Figure 1 The state of gelling under stirring at 25°C, Figure 2 The state of the fish mucus extract hydrogel after freeze-thaw cycles. As can be seen from Figure 1 , Figure 2 , with the decrease of the concentration of fish mucus extract, the gelling ability of the hydrogel gradually changed from gel state to sol state, and the gelling ability showed a significant downward trend. After mold shaping and demolding to a flat plate, this phenomenon was more obvious.

[0061] The mechanical properties of the fish sperm extract hydrogel were characterized by a rotational rheometer. The rheological tests were performed on the rheometer platform. The self-healing properties of the fish sperm extract hydrogel obtained in Examples 2-4 were tested using an Anton Paar rheometer. The strain sweep test was performed with a strain variation range of 0.01% to 1000% and a constant angular frequency of 1 rad / s. The results are shown in Figure 2. Figure 3 As shown. Figure 3 It can be seen that the three hydrogels with different ratios all have obvious platform areas at small strains (Strain < 1%), proving that the network structure of the gel is not destroyed under small strains. As the strain increases further, the gel loss modulus has an upward period and then decreases. The storage modulus and loss modulus successively intersect, and the G' and G" curves disintegrate the gel after the intersection. When the concentration is 20%, the strain corresponding to the intersection of the storage modulus (G') and loss modulus (G") curves of the fish sperm extract hydrogel is 56.5%; when the concentration is 10%, the strain corresponding to the intersection of the storage modulus (G') and loss modulus (G") curves of the fish sperm extract hydrogel is 91.3%; when the concentration is 5%, the strain corresponding to the intersection of the storage modulus (G') and loss modulus (G") curves of the fish sperm extract hydrogel is 148%. Figure 3 The results showed that the fish sperm extract hydrogels cross-linked at different concentrations all had good toughness.

[0062] The fish sperm extract hydrogels obtained in Examples 2-4 were tested using an Anton Paar rheometer. The hydrogels were subjected to a frequency sweep in the range of 0.01 to 100 rad / s under a shear strain of γ = 1%. The results were recorded as the storage modulus (G') and loss modulus (G") of the viscoelastic properties. The results are shown in Table 1. Figure 4 As shown. Figure 4 It can be seen that within the scanning frequency range of 0.01 to 100 rad / s, the hydrogel's G' is greater than G", indicating that it is in a gel state, exhibiting solid-state properties and good stability. Moreover, the gel's G' is relatively stable and does not depend on changes in frequency. In addition, the viscoelasticity of the gel gradually increases with the addition of fish sperm extract, indicating that as the amount of fish sperm extract added increases, the proportion of weakly alkaline solution in the hydrogel also decreases, affecting the cross-linking density and increasing G'.

[0063] The mechanical properties of the fish sperm extract hydrogel were characterized by a rotational rheometer, and the rheological test was performed on the rheometer platform. The fish sperm extract hydrogel obtained in Examples 2-4 was subjected to self-healing tests using an Anton Paar rheometer. It started with a small strain (γ = 1%) and then changed to a large strain (the parameters of the large strain setting are applicable to the measurement of fish sperm extract hydrogels with different ratios). The results are shown in Figure 2. Figure 5 .Depend on Figure 5It can be seen that when the concentration is 20%, the fish extract hydrogel still quickly recovers from sol state to normal hydrogel state after continuous and alternating multiple times (1% to 70%); when the concentration is 10%, the fish extract hydrogel quickly recovers from sol state to normal hydrogel state after continuous and alternating multiple times (1% to 120%); when the concentration is 5%, the fish extract hydrogel still quickly recovers from sol state to normal hydrogel state after continuous and alternating multiple times (1% to 200%). The collapse and recovery behavior of the hydrogel structure can be repeated multiple times, indicating that the fish extract hydrogel with different proportions has a repeatable self-healing ability.

[0064] The mechanical properties of the fish extract hydrogel were characterized by a rotational rheometer, and the rheological test was performed on a rheometer platform. The fish extract hydrogel obtained in Examples 2-4 was tested for injectability using an Anton Paar rheometer, and the shear rate varied from 0.1 to 100 s -1 . The results are shown in Figure 6 , and it can be seen that the viscosity of the fish extract with different proportions decreases rapidly with the increase of the shear rate, at which time the gel enters a relatively viscous and flowable state, and can be injected out through a syringe. Figure 6 The experimental results of shear thinning experiment verify the micro shear thinning of the fish extract hydrogel from the perspective of rheology, and prove the injectability of the fish extract hydrogel.

[0065] Example 6

[0066] The fish extract hydrogel prepared in Examples 2-4 was subjected to blood compatibility test: the fish extract hydrogel extract was obtained by extraction method; physiological saline was used as negative control; deionized water was used as positive control. The red blood cell diluent and the material extract were mixed according to the corresponding proportion, and then the centrifuge tube was placed in a water bath oven for warm bath. After the warm bath, the OD value was measured at 540 nm by an enzyme marker.

[0067] The results are shown in Figure 7 and Figure 8 , and it can be seen that the supernatant color of the fish extract hydrogel prepared in Examples 2-4 is completely consistent with the hemolysis rate shown in Figure 7 : the results of the hydrogel group are similar to those of the negative control group, and the supernatant is almost colorless, while the supernatant of the positive control group is bright red due to the rupture of red blood cells. The hemolysis rate measured by Figure 8 indicates that the hemolysis rate of the fish extract with different concentrations on blood cells is lower than 5% of the evaluation standard, indicating that the dressing has low hemolysis effect on red blood cells, which meets the relevant standards of biological materials.

[0068] Example 7

[0069] The fish extract hydrogel prepared in Example 2-4 was subjected to wound healing test: a skin wound damage model was established for studying the effect of fish extract hydrogel dressing on wound healing. First, the mice were anesthetized, the back of the mice was depilated using a depilatory agent, and then a 5mm diameter circular full-thickness defect wound was made on the back using a punch, and then all the wound model mice were randomly divided into 4 groups (12 mice in each group), wherein the control group mice were smeared with 40 μL green crosslinking agent solution on the wound, and the experimental group mice were respectively administered fish extract hydrogel (10%, w / v%) and fish extract hydrogel (20%, w / v%) on the wound, and the positive control group mice were administered 40 μL human epidermal growth factor gel on the wound. The 3M medical adhesive tape was used to fix the hydrogel on the mice in each group, and the wound was changed daily. After treatment, the wound healing of the skin of the mice in each group was recorded by real-time imaging during the wound healing period of 0-14d. The wound images were analyzed by ImageJ software analysis system, and the size of the wound was measured and calculated. The size of the wound was calculated according to the following formula:

[0070]

[0071] wherein W0 represents the initial wound area, W n represents the wound area at different time points.

[0072] The test results are shown in Figure 9 , Figure 10 , Figure 11 Figure 9 It is shown that during the healing period of 2, 4, 8, 14 days, the wound healing speed is improved to a certain extent after using the hydrogel dressing. Among them, the high-concentration fish extract hydrogel group has the most obvious promoting effect on wound healing, and the wound healing effect is slightly better than that of the mice using epidermal growth factor gel. On the 14th day, it can be seen that the high-concentration fish extract hydrogel group wound has almost completely healed, and the low-concentration fish extract hydrogel has a significant decrease in wound area compared with the control group. The experimental results show that the fish extract hydrogel can promote wound healing, and the healing speed is obviously related to the concentration of fish extract. Figure 10 The wound healing trajectory of the mice is shown in Figure 9 ​The wound healing area can be more intuitively reflected. At the same time, the wound healing area of each group of mice was investigated: on the 2nd day of treatment, no new skin tissue reconstruction was observed in the wound tissue of each group of mice, and on the 4th day of treatment, the wound of each group of mice began to heal, and the formation of new skin tissue was observed. Among them, the fish sperm extract hydrogel with a concentration of 20% has the most obvious healing effect, and the size of the wound is reduced to 40.1%. The fish sperm extract hydrogel with a concentration of 10% has a significant effect compared with the control group, but the effect is slightly worse than that of the positive control group, and the size of the wound is reduced to 52.9%, while the size of the wound of the positive control group of mice smeared with epidermal growth gel is 42.3%; on the 8th day of treatment, compared with the control group and the low-concentration fish sperm extract hydrogel group, the high-concentration fish sperm extract hydrogel group has a significantly reduced wound size, and the wound size is reduced to 29.5%; after 14 days of treatment, the wound healing rate of the high-concentration fish sperm extract hydrogel group is 95.6%, and the wound is basically completely healed, which represents the best healing effect, while the average wound healing rates of the control group, the positive control group and the low-concentration fish sperm extract hydrogel group are only 71.5%, 93.2% and 84.8%, respectively. Compared with the rest of the groups, the wound tissue of the mice still has different degrees of damage, and during the entire healing period, the high-concentration fish sperm extract hydrogel shows the best treatment effect, and the low-concentration fish sperm extract hydrogel group has a better healing effect than the control group, but the effect is slightly worse than that of the positive control group.

[0073] The above only describes the preferred embodiments of the present application and does not limit the scope of the claims. Other alternatives that can be thought of by those skilled in the art are within the scope of the present application.

Claims

1. An injectable self-healing and self-assembling fish sperm extract hydrogel, characterized in that: The hydrogel is prepared by mixing a fish sperm extract and a cross-linking agent solution; the cross-linking agent solution includes a salt solution with a final concentration of 0.01-4.5M, 0.01-0.1M sodium citrate, and 0.01-0.1M sodium bicarbonate, and the salt solution includes a sodium chloride solution, a potassium chloride solution, a calcium chloride solution, or a sodium acetate solution. The preparation method of the fish sperm extract includes the following steps: (1) Cleaning fish essence raw materials; (2) Degreasing the fish essence raw materials; (3) The defatted fish essence raw material is put into the extracting solution I for homogenization; the extracting solution I is a mixture of 0.05-0.1M saline solution and 0.01-0.05M sodium citrate, the saline solution is sodium chloride solution, potassium chloride solution, sodium acetate solution, potassium acetate solution, sodium lactate solution or calcium chloride solution, and the pH of the extracting solution I is 8-9. The defatted fish essence raw material is put into the extracting solution for homogenization; (4) centrifuging the solution treated in step (3) to obtain a precipitate; (5) adding 1-5 times the volume of extracting solution II to the precipitate obtained in step (4) for homogenization to obtain a homogenate; the extracting solution II is a mixture of 0.05-0.1M saline solution and 0.01-0.05M sodium citrate, the saline solution is sodium chloride solution, potassium chloride solution, sodium acetate solution, potassium acetate solution, sodium lactate solution or calcium chloride solution, and adjusting the pH of the extracting solution II to 7-8; transferring the precipitate obtained in step (4) to 1-5 times the weight of the raw material pre-cooled extracting solution for homogenization; (6) The homogenate obtained in step (5) is centrifuged, and the supernatant is discarded to obtain a light yellow precipitate, which is the fish sperm extract.

2. The injectable self-healing and self-assembling fish sperm extract hydrogel according to claim 1, characterized in that: In the step (4), the homogenized solution is centrifuged at 4°C and 4000 rpm, and the supernatant is discarded to obtain a precipitate close to the skin color.

3. The injectable self-healing and self-assembling fish sperm extract hydrogel according to claim 1, characterized in that: In step (6): the homogenate is placed at 4°C and centrifuged at 4000 rpm, the supernatant is discarded, and the process is repeated several times.

4. The method for preparing the injectable self-healing and self-assembling fish sperm extract hydrogel according to claim 1, characterized in that: The preparation method is as follows: fish sperm extract is mixed with a cross-linker solution at 1% to 50% (w / v%), and stirred at 20 to 35°C until complete gelation. After gelation, multiple freeze-thaw cycles are performed to prepare a series of hydrogels.

5. The preparation method according to claim 4, wherein The fish sperm extract is mixed with the cross-linking agent solution at 5% to 30% (w / v%) and stirred at 20 to 35°C until it is completely gelled.

6. Use of the injectable self-healing and self-assembling fish sperm extract hydrogel according to claim 1 in the preparation of products promoting wound healing.

Images