Injectable self-healing self-assembly milt extract hydrogel as well as preparation method and application thereof

Abstract

The invention discloses injectable self-healing self-assembly milt extract hydrogel as well as a preparation method and application thereof, and belongs to the technical field of hydrogel materials and wound repair. The hydrogel is prepared by mixing a milt extract and a cross-linking agent solution; the cross-linking agent solution comprises a salt solution with the final concentration of 0.01 to 4.5 M, sodium citrate with the final concentration of 0.01 to 0.1 M and sodium bicarbonate with the final concentration of 0.01 to 0.1 M; the preparation method comprises the following steps: mixing the milt extract and a cross-linking agent solution according to different proportions (1-50%, w / v%), stirring at 20-35 DEG C until the mixture is completely gelatinized, and performing multiple freezing and thawing cycles after gelatinizing to obtain a series of hydrogels. The hydrogel prepared by the invention has good self-healing and injectable characteristics, blood compatibility and tissue regeneration capacity, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogel materials and wound repair, and relates to an injectable self-healing self-assembling fish sperm extract hydrogel and a preparation method and application thereof. Background Art

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

[0003] Therefore, studying how to repair skin wounds has broad application prospects. Summary of the invention

[0004] The purpose of the invention is to provide an injectable self-healing self-assembling fish sperm extract hydrogel, and also provide a preparation method and application of the hydrogel to make up for the deficiencies of the prior art.

[0005] In order to achieve the above-mentioned invention object, the specific technical solution adopted by the present invention is:

[0006] The invention discloses an injectable self-healing and self-assembling fish sperm extract hydrogel, which is prepared by mixing the fish sperm extract and a cross-linking agent solution.

[0007] Furthermore, the cross-linking agent solution includes 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, wherein the salt solution includes sodium chloride solution, potassium chloride solution, potassium chloride solution, calcium chloride solution, sodium acetate solution and the like.

[0008] Furthermore, the cross-linking agent solution includes a final concentration of 1-4.5 M saline solution, 0.01-0.5 M sodium citrate and 0.05-0.1 M sodium bicarbonate.

[0009] Furthermore, the preparation method of the fish sperm extract comprises the following steps:

[0010] (1) Cleaning fish essence raw materials;

[0011] (2) defatting the fish essence raw material;

[0012] (3) adding the defatted fish essence raw material into the extracting solution I for homogenization;

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

[0014] (5) adding 1-5 times the volume of extracting solution II to the precipitate obtained in step (4) for homogenization to obtain a homogenate;

[0015] (6) Centrifuging the homogenate obtained in step (5), discarding the supernatant after centrifugation, and obtaining a light yellow precipitate, namely, the fish sperm extract.

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

[0017] Furthermore, the extracting solution I in step (3) is a mixture of 0.05-0.1M saline solution and 0.01-0.05M sodium citrate, the pH of the extracting solution I is 8-9, and the defatted fish essence raw material is put into the extracting solution for homogenization for 1.5 minutes. The saline 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] Furthermore, 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] Furthermore, the extracting solution II in step (5) is a mixture of 0.05-0.1M salt solution and 0.01-0.05M sodium citrate, and the pH of the extracting solution II is adjusted to 7-8; the precipitate obtained in step (4) is transferred to the extracting solution which is 1-5 times the weight of the raw material and pre-cooled and homogenized for 1.5 minutes. 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] Furthermore, in step (6): the homogenate is centrifuged at 4°C, 4000 rpm for 15 min, the supernatant is discarded, and the operation is repeated 3 times.

[0021] The preparation method of the hydrogel is as follows: fish sperm extract and crosslinking agent solution are mixed in different proportions (1% to 50%, w / v%), and stirred at 20 to 35° C. until completely gelled, and after gelling, multiple freeze-thaw cycles are performed to prepare a series of hydrogels.

[0022] Furthermore, 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.

[0023] Application of the injectable self-healing self-assembling fish sperm extract hydrogel in preparing products that promote wound healing.

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

[0025] The present invention comprehensively utilizes fish sperm extract and green crosslinker solution to construct an injectable self-healing self-assembling hydrogel that can promote wound healing. The fish sperm extract and the crosslinker solution are mixed for the first time to prepare an injectable self-assembling self-healing hydrogel that can promote wound healing. The hydrogel has good self-healing, injectable properties, blood compatibility and tissue regeneration ability, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0028] Figure 3 These are graphs of the rheological mechanical properties of the prepared fish sperm extract hydrogels with different concentrations; wherein (a) shows the G' and G", of the fish sperm extract hydrogel with a concentration of 20% during strain scanning, (b) shows the G' and G", of the fish sperm extract hydrogel with a concentration of 10% during strain scanning, and (c) shows the G' and G", of the fish sperm extract hydrogel with a concentration of 5% during strain scanning.

[0029] Figure 4 Rheological analysis of fish sperm extract hydrogels with different concentrations.

[0030] Figure 5 The figures are graphs showing the self-healing properties of fish sperm extract hydrogels prepared with different ratios; wherein (a) is a strain scan of a 20% fish sperm extract hydrogel under alternating strain, (b) is a strain scan of a 10% fish sperm extract hydrogel under alternating strain, and (c) is a strain scan of a 5% fish sperm extract hydrogel under alternating strain.

[0031] Figure 6 This is a diagram showing the injectability of fish sperm extract hydrogels prepared with different ratios.

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

[0033] Figure 8 To analyze the hemolysis rate of red blood cells after incubation with each group of samples.

[0034] Fig. 9 This is a diagram showing the effect of fish sperm extract hydrogel in promoting wound healing in mice.

[0035] Fig.10 Figure 2 shows the wound healing trajectory of mice in each group.

[0036] Fig.11The graph shows the changes in wound size of mice in each group after treatment. DETAILED DESCRIPTION

[0037] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples described. The experimental methods in the following examples that do not specify specific conditions are selected according to conventional methods and conditions, or according to the product specifications. The numerical values ​​disclosed in the examples of the present invention are approximate values, not definite values. When errors or experimental conditions permit, all values ​​within the error range may be included without being limited to the specific numerical values ​​disclosed in the examples of the present invention.

[0038] Unless otherwise specified, the drugs and reagents mentioned in the examples are all common commercially available products.

[0039] Embodiment 1:

[0040] 1. A method for preparing a fish sperm extract comprises the following steps:

[0041] (1) Using ultrapure water to clean the fish essence raw material;

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

[0043] The extracting solution I in step (3) is a mixture of 0.01-1M saline solution and 0.01-0.1M sodium citrate, the pH of the extracting solution I is 8-9, and the defatted fish essence raw material is put into the extracting solution for homogenization for 1.5 minutes.

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

[0045] (3) The defatted fish essence raw material was homogenized in extraction solution I (a mixture of 0.05 M saline solution and 0.05 M sodium citrate) with a pH of 9 for 1.5 min;

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

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

[0048] (6) The solution treated in step (5) was centrifuged at 4000 rpm for 15 min at 4°C, and the supernatant was discarded. The operation was repeated three times to obtain a light yellow precipitate, which was the fish sperm extract.

[0049] 2. Prepare the crosslinker solution: a mixture of 0.5-5 M sodium chloride, 0.01-0.1 M sodium citrate, and 0.01-0.1 M sodium bicarbonate at a final concentration.

[0050] Embodiment 2:

[0051] A method for preparing an injectable self-assembling and self-healing fish sperm extract hydrogel comprises the following steps:

[0052] Weigh 1 g of the fish sperm extract prepared in Example 1, add 4 mL of the cross-linking agent solution, place at 25° C. and stir until completely gelled. After gelling, perform multiple freeze-thaw cycles to obtain an injectable self-healing self-assembling hydrogel that promotes wound healing.

[0053] Example 3

[0054] A method for preparing an injectable self-assembling and self-healing fish sperm extract hydrogel comprises the following steps:

[0055] Weigh 1 g of the fish sperm extract prepared in Example 1, add 9 mL of the cross-linking agent solution, place at 25° C. and stir until completely gelled. After gelling, perform multiple freeze-thaw cycles to obtain an injectable self-healing self-assembling hydrogel that promotes wound healing.

[0056] Example 4

[0057] A method for preparing an injectable self-assembling and self-healing fish sperm extract hydrogel comprises the following steps:

[0058] 1 g of the fish sperm extract prepared in Example 1 was weighed, and 19 mL of the cross-linking agent solution was added, and the mixture was stirred at 25° C. to completely gel. After gelation, multiple freeze-thaw cycles were performed to obtain an injectable self-healing self-assembling hydrogel that promotes wound healing.

[0059] Example 5

[0060] The hydrogels prepared by mixing the fish sperm extract and the crosslinking agent solution in different proportions were photographed and recorded, and the changes in the gelling ability and the properties of the resulting gels were observed and analyzed. The gelling ability of the fish sperm extract hydrogels synthesized in Examples 2-4 was characterized by observation. Figure 1 and Figure 2 shown. Figure 1 It is the state of being stirred into gel at 25℃. Figure 2 The state of the fish sperm extract hydrogel after freeze-thaw cycles. Figure 1 , Figure 2 It can be seen that as the concentration of fish sperm extract decreases, the gelling ability of the hydrogel gradually changes from the gel state to the sol state, and the gelling ability shows a significant downward trend. This phenomenon is more obvious when the mold is finalized and then demolded to a flat plate.

[0061] 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 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 had 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 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 a rising period and then decreases. The storage modulus and loss modulus have intersections successively, 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 hydrogel obtained in Examples 2-4 was tested using an Anton Paar rheometer. The hydrogel was subjected to a frequency sweep in the frequency range of 0.01 to 100 rad / s under the condition of shear deformation γ = 1%, and the results were recorded as the data values ​​of the storage modulus (G') and loss modulus (G") of viscoelasticity. The results are shown in FIG. Figure 4 As shown. Figure 4 It can be seen that the hydrogel has a G' greater than G" in the scanning frequency range of 0.01 to 100 rad / s, and is in a gel state, showing solid-state properties and good stability. Moreover, the G' of the gel is relatively stable and does not depend on the change of frequency. In addition, the viscoelasticity of the gel gradually increases with the increase of fish sperm extract, indicating that with the increase in the amount of fish sperm extract added, 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 tested for self-healing 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 suitable for the measurement of fish sperm extract hydrogels with different ratios). The results are shown in Figure 5 .Depend on Figure 5It can be seen that when the concentration is 20%, the fish sperm extract hydrogel can still quickly recover from the sol state to the normal hydrogel state after multiple consecutive alternations (1% to 70%); when the concentration is 10%, the fish sperm extract hydrogel can still quickly recover from the sol state to the normal hydrogel state after multiple consecutive alternations (1% to 120%); when the concentration is 5%, the fish sperm extract hydrogel can still quickly recover from the sol state to the normal hydrogel state after multiple consecutive alternations (1% to 200%). The collapse and recovery behavior of the hydrogel structure can be repeated alternately many times, indicating that the fish sperm extract hydrogels with different ratios have repeatable and efficient self-healing ability.

[0064] 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 injectability of the fish sperm extract hydrogel obtained in Examples 2-4 was tested using an Anton Paar rheometer, with a shear rate range of 0.1 to 100 s -1 The results are as follows Figure 6 As shown by Figure 6 It can be seen that the viscosity of fish sperm extracts with different ratios decreases rapidly with the increase of shear rate. At this time, the gel enters a relatively viscous and flowable state and can be injected through a syringe. The experimental results of the shear thinning experiment verified the microscopic shear thinning of the fish sperm extract hydrogel from the perspective of rheology and proved the injectability of the fish sperm extract hydrogel.

[0065] Example 6

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

[0067] The results are as follows Figure 7 and Figure 8 As shown, the color of the supernatant of the fish sperm extract hydrogel prepared in Example 2-4 was observed by macroscopic observation. Figure 7 The hemolysis rates shown were exactly the same: the results of the hydrogel group were similar to those of the negative control group, with the supernatant being almost colorless, while the supernatant of the positive control group was bright red due to the rupture of red blood cells. Figure 8 The results of the measured hemolysis rate show that the hemolysis rate of fish sperm extract at different concentrations on blood cells is lower than the evaluation standard of 5%, indicating that the dressing has a low effect on the hemolysis of red blood cells and meets the relevant standards for biomaterials.

[0068] Example 7

[0069] The fish sperm extract hydrogel prepared in Example 2-4 was subjected to wound healing test: a skin wound injury model was established to study the effect of fish sperm extract hydrogel dressing on wound healing. First, after the mice were anesthetized, a depilatory agent was used to remove hair from the back of the mice, and then a circular full-thickness defect wound with a diameter of 5 mm was made on the back with a puncher. Then all the trauma model mice were randomly divided into 4 groups (12 in each group), among which the control group mice were smeared with 40 μL of green crosslinker solution on their wounds, and the wounds of the experimental group mice were respectively administered with fish sperm extract hydrogel (10%, w / v%) and fish sperm extract hydrogel (20%, w / v%), and the positive control group mice were administered with 40 μL of human epidermal growth factor gel on their wounds. The hydrogel was fixed to each group of mice with 3M medical tape, and the wounds were changed every day. After treatment, the healing of the skin wounds of each group of model mice was recorded by real-time imaging during the wound healing period of 0 to 14 days. The wound tissue images were analyzed by the ImageJ software analysis system, and the wound size was measured and statistically calculated. The wound size ratio was calculated according to the following formula:

[0070]

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

[0072] The test results are as follows Fig. 9 , Fig.10 , Fig.11 shown. Fig. 9 It was shown that within the healing period of 2, 4, 8, and 14 days, the speed of wound healing was improved to a certain extent after using the hydrogel dressing. Among them, the high-concentration fish sperm extract hydrogel group had the most obvious promoting effect on wound healing, and the wound healing effect was slightly better than that of the mice using the epidermal growth factor gel group. On the 14th day, it can be clearly seen that the wounds in the high-concentration fish sperm extract hydrogel group have almost completely healed, and the wound area of ​​the low-concentration fish sperm extract hydrogel has also been significantly reduced compared to the control group. The experimental results show that fish sperm extract hydrogel can promote wound healing, and the healing speed is significantly correlated with the concentration of fish sperm extract. Fig.10 The wound healing trajectory of mice is based on Fig. 9The wound healing area was outlined, which can more intuitively reflect the size of the wound healing area. At the same time, the wound healing area of ​​each group of mice was investigated: on the second day of treatment, no reconstruction of new skin tissue was observed in the wound tissue of each group of mice. After the fourth day of treatment, the wounds of each group of mice began to heal, and the formation of new skin tissue was observed. Among them, the healing effect of the fish sperm extract hydrogel with a concentration of 20% was the most obvious, and its wound size was reduced to 40.1%. The healing effect of the 10% fish sperm extract hydrogel was more obvious than that of the control group, but slightly worse than that of the positive control group. Its wound size was reduced to 52.9%, while the wound size of the positive control group mice coated with epidermal growth gel was 42.3%; on the 8th day of treatment, the wound size of the high-concentration fish sperm extract hydrogel group was significantly reduced compared with the control group and the low-concentration fish sperm extract hydrogel group, and its wound size had dropped to 29.5%; after 14 days of treatment, the wound healing rate of the high-concentration fish sperm extract hydrogel group was 95.6%, and the wound was basically healed completely, reflecting 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 were only 71.5%, 93.2% and 84.8% respectively. In comparison, the wound tissues of the other groups of mice still showed varying degrees of damage. During the entire healing cycle, the high-concentration fish sperm extract hydrogel showed the best therapeutic effect, while the healing effect of the low-concentration fish sperm extract hydrogel group was stronger than that of the control group, but slightly worse than that of the positive control group.

[0073] The above description is only a preferred embodiment of the present invention and does not constitute a limitation on the scope of the claims. Other substantially equivalent substitutes that can be thought of by those skilled in the art are all within the protection scope of the present invention.

Claims

1. An injectable self-healing self-assembling fish sperm extract hydrogel, characterized in that: The hydrogel is prepared by mixing fish sperm extract and cross-linking agent solution.

2. The injectable self-healing self-assembling fish sperm extract hydrogel according to claim 1, characterized in that: The cross-linking agent solution includes a final concentration of 0.01-4.5 M saline solution, 0.01-0.1 M sodium citrate, and 0.01-0.1 M sodium bicarbonate.

3. The injectable self-healing self-assembling fish sperm extract hydrogel according to claim 1, characterized in that: The preparation method of the fish sperm extract comprises the following steps: Cleaning fish essence raw materials; Degreasing the fish essence raw materials; Put the defatted fish essence raw material into the extracting solution I for homogenization; Centrifuging the solution treated in step (3) to obtain a precipitate; The precipitate obtained in step (4) is added with 1-5 times the volume of extracting solution II for homogenization to obtain a homogenate; The homogenate obtained in step (5) is centrifuged, and the supernatant is discarded to obtain a light yellow precipitate, namely, the fish sperm extract.

4. The injectable self-healing self-assembling fish sperm extract hydrogel according to claim 1, characterized in that: The extracting solution I in step (3) is a mixture of 0.05-0.1M saline solution and 0.01-0.05M sodium citrate, 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.

5. The injectable self-healing 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.

6. The injectable self-healing self-assembling fish sperm extract hydrogel according to claim 1, characterized in that: The extracting solution II in step (5) is a mixture of 0.05-0.1M salt solution and 0.01-0.05M sodium citrate, and the pH of the extracting solution II is adjusted to 7-8; the precipitate obtained in step (4) is transferred to the pre-cooled extracting solution with a weight of 1-5 times that of the raw material for homogenization.

7. The injectable self-healing self-assembling fish sperm extract hydrogel according to claim 1, characterized in that: In the 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.

8. 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: fish sperm extract and cross-linking agent solution are mixed at 1%~50%, w / v%, and stirred at 20~35°C until completely gelled. After gelation, multiple freeze-thaw cycles are performed to prepare a series of hydrogels.

9. The preparation method according to claim 8, characterized in that: The fish sperm extract and the cross-linking agent solution are mixed at 5% to 30% (w / v%) and stirred at 20 to 35°C until they are completely gelled.

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

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