Double-crosslinking personalized hydrogel for promoting wound healing and preparation process thereof

Abstract

The invention discloses double-crosslinking personalized hydrogel for promoting wound healing and a preparation process thereof, and relates to the technical field of biomedical materials. The hydrogel comprises sodium alginate, modified chitosan, calcium carbonate, glucolactone, acrylamide, methylene diacrylamide, ammonium persulfate and tetramethylethylenediamine, and the hydrogel can also carry drugs in the preparation process, including but not limited to drugs with the functions of diminishing inflammation, resisting bacteria, stopping bleeding and the like. The hydrogel is prepared in a double-crosslinking mode of physical crosslinking and chemical crosslinking. The hydrogel provided by the invention has the advantages of high biocompatibility, adjustable viscosity, easy replacement, adjustable water absorbability and temperature responsiveness, has a loose porous structure, has better water content, cell proliferation and migration performance, swelling performance, drug sustained release performance and the like, can effectively promote wound healing, and has a good application prospect. The method has potential application prospects in skin wound treatment and repair.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly relates to a double-crosslinked personalized hydrogel for promoting wound healing, its preparation process and application. Background Art

[0002] With the intensification of the global population aging trend and the frequent occurrence of various traumatic injury events, the treatment demand for skin wounds is increasing day by day, which puts higher requirements on the existing treatment methods. Skin wounds include incisions, burns, abrasions, ulcers, etc. In terms of surgical incisions, more than 300 million surgical operations are performed worldwide every year, and more than one-third of them lead to the formation of hypertrophic scars or keloids; in terms of diabetic foot ulcers (DFUs), about 30% of global diabetic patients suffer from DFUs, and more than one-third of the diabetic patient care costs are used to treat DFU-related problems, which greatly increases the physical and economic burden on diabetic patients; in terms of burns, 7-12 million people need treatment due to burns every year, and the scars and chronic wounds left after burns bring long-term physical and psychological burdens to the injured; in terms of traumatic wounds, traumatic wounds caused by accidental injuries such as traffic accidents and falls are an important cause of skin damage, and the number of people injured in traffic accidents worldwide exceeds 50 million every year. Skin wounds are a global health problem, which not only affects the quality of life of individuals, but also brings a heavy burden to the medical system.

[0003] Wound healing is a dynamic and complex tissue regeneration and growth process, including four stages: coagulation and hemostasis, inflammation, proliferation, and remodeling stages. Different from acute wounds, the healing of chronic wounds or difficult-to-heal wounds such as ulcers usually stagnates in the early inflammatory stage after injury and does not reach the proliferation and subsequent healing. The damage to the normal wound healing process and the resulting formation of difficult-to-heal wounds are the most common reasons for the decline in the quality of life of patients.

[0004] In this context, it is particularly important to develop new wound dressings with the advantages of efficiently promoting healing, reducing the risk of infection, and improving the quality of life of patients. The new wound dressings based on hydrogels show great potential in this field due to their unique physical and chemical properties, but many hydrogels in the prior art are prone to cause secondary damage to the wound surface during the dressing change process. Therefore, in-depth research on the design of new wound dressings based on hydrogels and their application and effects in the treatment of skin wounds can not only promote the development of related technologies, but also provide strong support for clinical practice, thereby improving the prognosis of patients and enhancing the quality of life. Summary of the Invention

[0005] The purpose of the present invention is to provide a double-crosslinked personalized hydrogel for promoting wound healing and its preparation process to help improve wound trauma repair and improve the prognosis of the wound surface of patients.

[0006] To achieve the above object, the present invention provides a double-crosslinked personalized hydrogel for promoting wound healing, which hydrogel comprises sodium alginate, modified chitosan, calcium carbonate, glucono delta-lactone, acrylamide, methylene bisacrylamide, ammonium persulfate, and tetramethylethylenediamine, wherein the modified chitosan is 90% chitosan + 10% chitosan quaternary ammonium salt.

[0007] Preferably, the above hydrogel further contains drugs including but not limited to those with anti-inflammatory, antibacterial, and hemostatic functions, such as shikonin, etc., and the drugs can be loaded during the preparation process.

[0008] The thickness of the hydrogel provided by the present invention is 0.5 - 10 mm, and this hydrogel can be applied to the repair and treatment of skin wounds.

[0009] The present invention also provides a preparation process of the hydrogel, which comprises the following steps: (1) Dissolve sodium alginate, modified chitosan, and calcium carbonate in water in sequence, and stir to obtain a uniform precursor solution; (2) Take acrylamide and add it to the above precursor solution, and keep it warm and static continuously to form a pre-gel; (3) Prepare aqueous solutions of methylene bisacrylamide and ammonium persulfate respectively, and add them dropwise to the obtained pre-gel in sequence. After stirring evenly, keep it warm continuously to obtain a pre-mixed gel; (4) Add tetramethylethylenediamine and glucono delta-lactone to the obtained pre-mixed gel, stir evenly, and then pour it into a mold for cross-linking reaction; (5) After the cross-linking reaction is completed, let it cool to room temperature to obtain the hydrogel.

[0010] Preferably, in step (1) of the above preparation process, the final concentrations of sodium alginate, modified chitosan, and calcium carbonate in the hydrogel are 20, 10, and 2.73 mg / mL respectively.

[0011] Preferably, in step (2) of the above preparation process, the final concentration of acrylamide in the hydrogel is 100 mg / mL; the temperature for keeping warm continuously is 60 °C.

[0012] Preferably, in step (3) of the above preparation process, the final concentration of methylene bisacrylamide in the hydrogel is 0.05 mg / mL, the final concentration of ammonium persulfate in the hydrogel is 0.5 mg / mL, and the temperature for keeping warm continuously is 60 °C.

[0013] Preferably, in the above preparation process, the volume ratio of the dosage of tetramethylethylenediamine in the hydrogel is 13.333 μL / 10 mL, the final concentration of glucono delta-lactone in the hydrogel is 14.2 μg / mL, and the cross-linking reaction conditions are: reacting at 60 °C for 1 hour.

[0014] The present invention has the following advantages: The hydrogel provided by the present invention is prepared by a dual cross-linking method of physical cross-linking and chemical cross-linking. Among the physically cross-linked high molecular polymers, sodium alginate (SA) is a natural biopolymer. When combined with modified chitosan, it has unique advantages such as environmental friendliness, low cost, sustainability, and biodegradability. The addition of the chemically cross-linked high molecular polymer polyacrylamide (PAM) significantly improves the mechanical properties of the hydrogel, making the hydrogel more uniform and dense. The present invention provides a preparation method of a hydrogel with high biocompatibility, adjustable viscosity and easy replacement, adjustable water absorption, and temperature responsiveness. This hydrogel can be prepared into corresponding hydrogels according to the size and shape of the wound; the water absorption capacity can be adjusted according to the amount of wound exudate, so as to achieve personalized treatment. It is further confirmed that this hydrogel has a loose porous structure, and has good water content, cell proliferation and migration performance, water absorption performance, drug slow release and other properties, and can effectively promote wound healing. Description of the Drawings

[0015] Figure 1 SEM image of the hydrogel in Example 1 of the present invention.

[0016] Figure 2 Results of X-ray energy spectrum analysis (EDS) of the hydrogel in Example 1 of the present invention.

[0017] Figure 3 SEM image of the hydrogel in Example 2 of the present invention.

[0018] Figure 4 Results of X-ray energy spectrum analysis (EDS) of the hydrogel in Example 2 of the present invention.

[0019] Figure 5 SEM image of the hydrogel in Example 3 of the present invention.

[0020] Figure 6 Results of X-ray energy spectrum analysis (EDS) of the hydrogel in Example 3 of the present invention.

[0021] Figure 7 Preparation shapes of the hydrogels prepared in Examples 1-3 in different specifications of molds.

[0022] Figure 8 Preparation shapes of the hydrogels prepared in Examples 4-9 in a 24-well plate.

[0023] Figure 9 SEM image of the hydrogel in Example 4 of the present invention.

[0024] Figure 10 SEM image of the hydrogel in Example 5 of the present invention.

[0025] Figure 11 SEM image of the hydrogel in Example 6 of the present invention.

[0026] Figure 12 SEM image of the hydrogel in Example 7 of the present invention.

[0027] Figure 13 SEM image of the hydrogel in Example 8 of the present invention.

[0028] Figure 14 SEM image of the hydrogel in Example 9 of the present invention.

[0029] Figure 15 Analysis results of the water content of different groups of hydrogels prepared in Examples 1-3.

[0030] Figure 16 Determination results of the swelling properties of different groups of hydrogels prepared in Examples 1-3 after freeze-drying and under normal conditions.

[0031] Figure 17 Determination results of the adhesion properties of different groups of hydrogels prepared in Examples 1-3.

[0032] Figure 18 Determination results of the compression stress-strain curves and compression moduli of different groups of hydrogels prepared in Examples 1-3.

[0033] Figure 19 Cell viability results after incubating mouse epithelial fibroblasts L929 cells with different hydrogel extracts prepared in Examples 1-3.

[0034] Figure 20 Determination results of live-dead staining after incubating L929 cells with different hydrogel solutions (10 mg / mL) prepared in Examples 1-3 for 48 h.

[0035] Figure 21 Cell viability results after incubating mouse embryonic fibroblasts NIH3T3 cells with different hydrogel extracts prepared in Examples 1-3.

[0036] Figure 22 Determination results of live-dead staining after incubating NIH3T3 cells with different hydrogel solutions (10 mg / mL) prepared in Examples 1-3 for 48 h.

[0037] Figure 23 Cell migration of different groups of hydrogel extracts prepared in Examples 1-3 co-cultured with L929 cells for different times.

[0038] Figure 24Quantitative analysis results of cell migration of different groups of hydrogel extracts prepared in Examples 1-3 co-cultured with L929 cells for different times.

[0039] Figure 25 Cell migration of different groups of hydrogel extracts prepared in Examples 1-3 co-cultured with NIH3T3 cells for different times.

[0040] Figure 26 Quantitative analysis results of cell migration of different groups of hydrogel extracts prepared in Examples 1-3 co-cultured with NIH3T3 cells for different times.

[0041] Figure 27 Drug release curves of different groups of hydrogels loaded with shikonin prepared in Examples 7-9.

[0042] Figure 28 Repair effect of the hydrogel prepared in Example 2 on mouse skin wound repair for 12 days.

[0043] Figure 29 Quantitative analysis results of wound healing of the hydrogel prepared in Example 2 on mouse skin wound repair for 12 days.

[0044] Figure 30 HE staining of the wound of the hydrogel prepared in Example 2 on mouse skin wound repair for 12 days.

[0045] Figure 31 Masson trichrome staining of the wound of the hydrogel prepared in Example 2 on mouse skin wound repair for 12 days.

[0046] Figure 32 Comparison results of the antibacterial effects of the hydrogels prepared before and after chitosan modification. Detailed implementation manners

[0047] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0048] Note: The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the technologies or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0049] The present invention provides a hydrogel, which is obtained by physically crosslinking a macromolecular compound of sodium alginate and chitosan, calcium carbonate and glucono delta-lactone, and chemically crosslinking a macromolecular compound of acrylamide, methylene bisacrylamide, ammonium persulfate and tetramethylethylenediamine.

[0050] Among them, the chitosan is modified by replacing 100% chitosan with 90% chitosan + 10% chitosan quaternary ammonium salt. The chitosan quaternary ammonium salt is obtained by introducing a quaternary ammonium group (such as hydroxypropyltrimethylammonium chloride) onto the chitosan molecular chain through chemical modification, so that a permanently positively charged quaternary ammonium group is connected to the C2 position or amino group of each sugar residue, and the structure is more stable.

[0051] The antibacterial effects of the hydrogels prepared before and after the modification of chitosan were compared and detected, and Escherichia coli and Staphylococcus aureus were selected for verification. By inoculating the two strains of bacteria in LB liquid medium and different treatments and co-culturing for 24 hours, the control group was added with sterile up water, the chitosan group was added with the unmodified hydrogel in the medium, and the chitosan quaternary ammonium salt group was added with the modified hydrogel in the medium. The comparison of antibacterial effects is shown in Figure 32 As shown, where A is the growth of bacteria on the plate and B is the quantitative result of the antibacterial rate, and the control is. It can be seen that after replacing chitosan with modified chitosan, its antibacterial effect is significantly improved. Based on this, a hydrogel with better antibacterial effect and its preparation method are further verified and optimized as follows:

[0052] Example 1 This example provides a hydrogel, which is obtained by physically crosslinking a macromolecular compound of sodium alginate and modified chitosan, calcium carbonate and glucono delta-lactone, and chemically crosslinking a macromolecular compound of acrylamide with a mass-volume fraction of 7.5%, methylene bisacrylamide, ammonium persulfate and tetramethylethylenediamine. The thickness of the obtained hydrogel is 0.5 - 10.0 mm.

[0053] Among them, the preparation method of the obtained hydrogel is as follows: First, weigh 200.0 mg of sodium alginate, 100.0 mg of modified chitosan, and 27.3 mg of calcium carbonate powder at room temperature and dissolve them successively in 9 mL of deionized water. Stir vigorously to obtain a homogeneous precursor solution. The stirring method is magnetic stirring at a speed of 800 r / min for 0.5 - 1 h. The heating method is water bath heating at a temperature of 45°C. Then, weigh 750.0 mg of acrylamide and add it to the above mixture, and continue to heat the precursor solution in a water bath at a temperature of 60°C, and keep it warm and static to form a pre-gel. Then, weigh 0.5 mg of methylene bisacrylamide and dissolve it in 0.5 mL of deionized water, weigh 5 mg of ammonium persulfate and dissolve it in 0.5 mL of deionized water. The mixing method is vortex oscillation at a speed of 800 r / min for 5 - 10 min, and it is prepared at room temperature. Then, add the methylene bisacrylamide aqueous solution and the ammonium persulfate aqueous solution drop by drop to the pre-gel in turn. Under water bath heating at 60°C, after stirring evenly, add 10.0 μL of tetramethylethylenediamine and 142.0 μg of glucono-δ-lactone, stir for 30 s, then pour it into a square glass mold (length = 10 cm, width = 8 cm, depth = 0.5 - 10 mm) or a round 12-, 24-, 48-well plate, and carry out a thermal initiation cross-linking reaction at 60°C for 1 hour. Let the cross-linked pre-gel stand at room temperature for 24 hours to obtain a hydrogel.

[0054] In the preparation method provided by the present invention, the cross-linking of sodium alginate and calcium ions is physical cross-linking. Adding glucono-δ-lactone as a cross-linking agent can control the release rate of calcium ions, thereby regulating the gel formation time. At the same time, acrylamide, methylene bisacrylamide, ammonium persulfate, and tetramethylethylenediamine form a polyacrylamide polymer gel through a chemical cross-linking reaction.

[0055] Physical cross-linking and chemical cross-linking are classifications of fixed hydrogel gelation methods. The hydrogel provided by the present invention mainly uses two cross-linking methods together and cross-links separately. During the reaction, glucono-δ-lactone and tetramethylethylenediamine are cross-linking agents for physical cross-linking and chemical cross-linking respectively. Both cross-linking methods require heating, but since gelation occurs quickly after addition, therefore, the cross-linking agent is selected to be added last, and the hydrogel provided by the present invention is obtained through thermal initiation cross-linking.

[0056] Freeze the prepared hydrogel at -20°C overnight. The solidified hydrogel is quenched with liquid nitrogen to expose the cross-section, and then vacuum freeze-dried for more than 48 hours. After drying, the sample is fixed on a conductive adhesive, with the cross-section of the hydrogel facing up. After sputtering with gold, a scanning electron microscope (SEM) is used to test the surface of the prepared hydrogel, and the SEM image of the hydrogel in Example 1 is as Figure 1As shown, where A is at low magnification (scale: 100 μm) and B is at high magnification (scale: 20 μm). It can be seen that the hydrogel has a loose porous structure with a pore size of 74.69 ± 17.42 μm and a porosity of 76.13%. In the SEM images of the hydrogel taken, a high-magnification image area is selected for X-ray energy spectrum analysis. The X-ray energy spectrum analysis (EDS) results of the hydrogel are as Figure 2 shown. It can be seen that the distribution of each element in the hydrogel is relatively uniform, and the hydrogel forms a stable and uniform gel. It can be seen that the proportions of the four elements C, O, N, and Na are the largest, which is consistent with the proportions of the raw materials for preparing the hydrogel.

[0057] Example 2 This example provides a hydrogel, which is obtained by physically cross-linking sodium alginate, a high-molecular compound of modified chitosan, calcium carbonate, and glucono-δ-lactone, and chemically cross-linking a high-molecular compound of 10% acrylamide, methylene bisacrylamide, ammonium persulfate, and tetramethylethylenediamine by mass / volume fraction. The thickness of the hydrogel is 0.5 - 10.0 mm.

[0058] Among them, the preparation method of the obtained hydrogel is as follows: First, at room temperature, 200.0 mg of sodium alginate, 100.0 mg of modified chitosan, and 27.3 mg of calcium carbonate powder are successively dissolved in 8.666 mL of deionized water, and vigorously stirred to obtain a uniform precursor solution. The stirring method is magnetic stirring with a rotation speed of 800 r / min for 0.5 - 1 h. The heating method is water bath heating at a temperature of 45°C. Then, 1000.0 mg of acrylamide is weighed and added to the above mixture, and the precursor solution is continuously heated in a water bath at a temperature of 60°C and kept warm and static to form a pre-gel. Then, 0.667 mg of methylene bisacrylamide is dissolved in 0.667 mL of deionized water, and 6.667 mg of ammonium persulfate is dissolved in 0.667 mL of deionized water. The mixing method is vortex oscillation with a rotation speed of 800 r / min for 5 - 10 min, and it is prepared under normal temperature conditions. Then, the methylene bisacrylamide aqueous solution and the ammonium persulfate aqueous solution are successively added dropwise to the pre-gel. Under water bath heating at 60°C, after stirring evenly, 13.333 μL of tetramethylethylenediamine and 142.0 μg of glucono-δ-lactone are added, and after stirring for 30 s, it is poured into a square glass mold (length = 10 cm, width = 8 cm, depth = 0.5 - 10 mm) or a round 12-, 24-, or 48-well plate, and a thermal initiation cross-linking reaction is carried out at 60°C for 1 hour. The cross-linked pre-gel is placed at normal temperature for 24 hours to obtain the hydrogel.

[0059] The prepared hydrogel was frozen at -20 °C overnight. The solidified hydrogel was quenched with liquid nitrogen to expose the cross-section, and then vacuum freeze-dried for more than 48 hours. After drying, the sample was fixed on a conductive adhesive with the cross-section of the hydrogel facing upward. After sputtering with gold, the surface of the prepared hydrogel was tested by scanning electron microscopy (SEM), and the SEM image of the hydrogel in Example 2 was obtained as Figure 3 shown. Among them, A is at low magnification (scale bar: 100 μm), and B is at high magnification (scale bar: 20 μm). It can be seen that the hydrogel obtained in this example has a loose porous structure, with a pore size of 62.70 ± 13.12 μm and a porosity of 78.17%. An X-ray energy spectrum analysis was performed on a high-magnification image area selected from the SEM image of the hydrogel. The X-ray energy spectrum analysis (EDS) results of the hydrogel are as Figure 4 shown. It can be seen that the distribution of each element in the hydrogel is relatively uniform, the gelation of the hydrogel is stable and uniform, and the proportions of the four elements C, O, N, and Na are the largest, which is consistent with the proportion of the raw materials for preparing the hydrogel.

[0060] Example 3 This example provides a hydrogel, which is obtained by physical cross-linking of sodium alginate, a high-molecular compound of modified chitosan, calcium carbonate, and glucono-delta-lactone, and chemical cross-linking of a high-molecular compound of 15% acrylamide, methylene bisacrylamide, ammonium persulfate, and tetramethylethylenediamine in terms of mass-volume fraction. The thickness of the hydrogel is 0.5 - 10.0 mm.

[0061] Among them, the preparation method of the obtained hydrogel is as follows: First, weigh 200.0 mg of sodium alginate, 100.0 mg of modified chitosan, and 27.3 mg of calcium carbonate powder at room temperature and dissolve them in 8 mL of deionized water in turn. Stir vigorously to obtain a uniform precursor solution. The stirring method is magnetic stirring, with a rotation speed of 800 r / min, and stir for 0.5 - 1 h. The heating method is water bath heating, with a temperature of 45 °C. Then, weigh 1500.0 mg of acrylamide and add it to the above mixture, and continue to heat the precursor solution in a water bath at a temperature of 60 °C, and keep it warm and static to form a pre-gel. Then, weigh 1.0 mg of methylene bisacrylamide and dissolve it in 1.0 mL of deionized water, weigh 10.0 mg of ammonium persulfate and dissolve it in 1.0 mL of deionized water. The mixing method is vortex oscillation, with a rotation speed of 800 r / min, and stir for 5 - 10 min, prepared at room temperature. Then, add the methylene bisacrylamide aqueous solution and ammonium persulfate aqueous solution drop by drop to the pre-gel in turn. Under 60 °C water bath heating, after stirring evenly, add 20 μL of tetramethylethylenediamine and 142.0 μg of glucono-delta-lactone, stir for 30 s and then pour it into a square glass mold (length = 10 cm, width = 8 cm, depth = 0.5 - 10 mm) or round 12-, 24-, 48-well plates, and carry out a 1-hour thermal initiation cross-linking reaction at 60 °C. Place the cross-linked pre-gel at room temperature for 24 hours to obtain a hydrogel.

[0062] Freeze the prepared hydrogel at -20 °C overnight. The solidified hydrogel is quenched with liquid nitrogen to expose the cross-section, and then vacuum freeze-dried for more than 48 hours. After drying, the sample is fixed on a conductive adhesive, with the cross-section of the hydrogel facing up. After sputtering with gold, the surface of the prepared hydrogel is tested by scanning electron microscopy (SEM) to obtain the SEM image of the hydrogel in Example 3 as Figure 5 shown. Among them, A is at low magnification (scale bar: 100 μm), and B is at high magnification (scale bar: 20 μm). It can be seen that the hydrogel has a loose porous structure, with a pore size of 56.54 ± 8.25 μm and a porosity of 79.93%. Select a high-magnification image area in the SEM image of the hydrogel taken for X-ray energy spectrum analysis. The X-ray energy spectrum analysis (EDS) result of the hydrogel is as Figure 6 shown. It can be seen that the distribution of each element in the hydrogel is relatively uniform, and the gelation of the hydrogel is stable and uniform. Among them, the proportions of the four elements C, O, N, and Na are the largest, which is in line with the proportion of the raw materials for preparing the hydrogel.

[0063] Example 4 This example provides a hydrogel, which can carry drugs with functions including but not limited to anti-inflammatory, antibacterial, hemostatic, etc., such as shikonin. This hydrogel is obtained by physically cross-linking sodium alginate, a polymer compound of modified chitosan, calcium carbonate, and glucono-δ-lactone, and chemically cross-linking a polymer compound of acrylamide, methylene bisacrylamide, ammonium persulfate, and tetramethylethylenediamine with a mass-volume fraction of 7.5%. Add 10% ethanol solution of 1 mg / mL shikonin standard product, and the obtained hydrogel has a thickness of 0.5 - 10.0 mm.

[0064] Among them, the preparation method of the obtained hydrogel is as follows: First, take 100 mL of absolute ethanol, add 900 mL of distilled water, and mix evenly to obtain a 10% ethanol solution. Weigh 10 mg of shikonin and dissolve it in 10 mL of 10% ethanol solution to prepare a 1 mg / mL shikonin solution. Then, at room temperature, weigh 200.0 mg of sodium alginate, 100.0 mg of modified chitosan, and 27.3 mg of calcium carbonate powder and dissolve them in 9 mL of shikonin solution in turn, and stir vigorously to obtain a uniform precursor solution. The stirring method is magnetic stirring with a rotation speed of 800 r / min, and the stirring time is 0.5 - 1 h. The heating method is water bath heating at a temperature of 45 °C. Then, weigh 750.0 mg of acrylamide and add it to the above mixture, and continue to heat the precursor solution in a water bath at a temperature of 60 °C, and keep it warm and static to form a pre-gel. Then, weigh 0.5 mg of methylene bisacrylamide and dissolve it in 0.5 mL of shikonin solution, weigh 5 mg of ammonium persulfate and dissolve it in 0.5 mL of shikonin solution, and the mixing method is vortex oscillation with a rotation speed of 800 r / min, and the stirring time is 5 - 10 min, which is prepared under normal temperature conditions. Then, add the methylene bisacrylamide solution and ammonium persulfate solution drop by drop to the pre-gel in turn. Under water bath heating at 60 °C, after stirring evenly, add 10.0 μL of tetramethylethylenediamine and 142.0 μg of glucono-δ-lactone, stir for 30 s, and then pour it into a square glass mold (length = 10 cm, width = 8 cm, depth = 0.5 - 10 mm) or a round 12-, 24-, 48-well plate, and carry out a thermal initiation cross-linking reaction at 60 °C for 1 hour. Let the cross-linked pre-gel stand at room temperature for 24 hours to obtain the hydrogel.

[0065] Example 5 This example provides a hydrogel, which can carry drugs with functions including but not limited to anti-inflammatory, antibacterial, hemostatic, etc., such as shikonin. This hydrogel is obtained by physically cross-linking sodium alginate, a polymer compound of modified chitosan, calcium carbonate, and glucono-δ-lactone, and chemically cross-linking a polymer compound of acrylamide, methylene bisacrylamide, ammonium persulfate, and tetramethylethylenediamine with a mass-volume fraction of 10%. Add 10% ethanol solution of 1 mg / mL shikonin standard product, and the obtained hydrogel has a thickness of 0.5 - 10.0 mm.

[0066] Among them, the preparation method of the obtained hydrogel is as follows: First, take 100 mL of absolute ethanol, add 900 mL of distilled water, and mix evenly to obtain a 10% ethanol solution. Weigh 10 mg of shikonin and dissolve it in 10 mL of the 10% ethanol solution to prepare a 1 mg / mL shikonin solution. Then, at room temperature, weigh 200.0 mg of sodium alginate, 100.0 mg of modified chitosan, and 27.3 mg of calcium carbonate powder and dissolve them successively in 8.666 mL of the shikonin solution, and stir vigorously to obtain a uniform precursor solution. The stirring method is magnetic stirring, with a rotation speed of 800 r / min, and stir for 0.5 - 1 h. The heating method is water bath heating, with a temperature of 45 °C. Then, weigh 1000.0 mg of acrylamide and add it to the above mixture, and continue to heat the precursor solution in a water bath at a temperature of 60 °C, and keep it warm and static to form a pre-gel. Then, weigh 0.667 mg of methylene bisacrylamide and dissolve it in 0.667 mL of the shikonin solution, weigh 6.667 mg of ammonium persulfate and dissolve it in 0.667 mL of the shikonin solution. The mixing method is vortex oscillation, with a rotation speed of 800 r / min, and the stirring time is 5 - 10 min, prepared under normal temperature conditions. Then, add the methylene bisacrylamide solution and the ammonium persulfate solution drop by drop to the pre-gel in sequence. Under 60 °C water bath heating, after stirring evenly, add 13.333 μL of tetramethylethylenediamine and 142.0 μg of glucono-δ-lactone, stir for 30 s, and then pour it into a square glass mold (length = 10 cm, width = 8 cm, depth = 0.5 - 10 mm) or a round 12-, 24-, or 48-well plate, and carry out a 1-hour thermal initiation cross-linking reaction at 60 °C. Let the cross-linked pre-gel stand at room temperature for 24 hours to obtain the hydrogel.

[0067] Example 6 This example provides a hydrogel, which can carry drugs including but not limited to those with functions such as anti-inflammatory, antibacterial, and hemostatic effects, such as shikonin. This hydrogel is obtained by physically cross-linking high-molecular compounds of sodium alginate and modified chitosan, and calcium carbonate and glucono-δ-lactone, and chemically cross-linking high-molecular compounds of 15% acrylamide, methylene bisacrylamide, ammonium persulfate, and tetramethylethylenediamine. Add a 10% ethanol solution of 1 mg / mL shikonin standard product, and the thickness of the obtained hydrogel is 0.5 - 10.0 mm.

[0068] Among them, the preparation method of the obtained hydrogel is as follows: First, take 100 mL of absolute ethanol and add 900 mL of distilled water. Mix them evenly to obtain a 10% ethanol solution. Weigh 10 mg of shikonin and dissolve it in 10 mL of the 10% ethanol solution to prepare a 1 mg / mL shikonin solution. Then, at room temperature, weigh 200.0 mg of sodium alginate, 100.0 mg of modified chitosan, and 27.3 mg of calcium carbonate powder and dissolve them successively in 8 mL of the shikonin solution. Stir vigorously to obtain a homogeneous precursor solution. The stirring method is magnetic stirring with a rotation speed of 800 r / min for 0.5 - 1 h. The heating method is water bath heating at a temperature of 45 °C. Then, weigh 1500.0 mg of acrylamide and add it to the above mixture, and continue to heat the precursor solution in a water bath at a temperature of 60 °C. Keep it warm and static to form a pre-gel. Then, weigh 1.0 mg of methylene bisacrylamide and dissolve it in 1.0 mL of the shikonin solution, weigh 10.0 mg of ammonium persulfate and dissolve it in 1.0 mL of the shikonin solution. The mixing method is vortex oscillation with a rotation speed of 800 r / min for 5 - 10 min at room temperature. Then, add the methylene bisacrylamide solution and the ammonium persulfate solution drop by drop to the pre-gel successively. Under water bath heating at 60 °C, after stirring evenly, add 20 μL of tetramethylethylenediamine and 142.0 μg of glucono-δ-lactone. Stir for 30 s and then pour it into a square glass mold (length = 10 cm, width = 8 cm, depth = 0.5 - 10 mm) or round 12-, 24-, 48-well plates, and carry out a thermal initiation cross-linking reaction at 60 °C for 1 hour. Let the cross-linked pre-gel stand at room temperature for 24 hours to obtain the hydrogel.

[0069] Example 7 This example provides a hydrogel, which can carry drugs including but not limited to those with functions such as anti-inflammatory, antibacterial, and hemostatic, such as shikonin. The hydrogel is obtained by physically cross-linking high molecular compounds of sodium alginate and modified chitosan with calcium carbonate and glucono-δ-lactone, and chemically cross-linking high molecular compounds of 7.5% acrylamide, methylene bisacrylamide, ammonium persulfate, and tetramethylethylenediamine. After freeze-drying, it is soaked in 10 mL of a 10% ethanol solution of 1 mg / mL shikonin standard to obtain a normally moist hydrogel, and the thickness of the obtained hydrogel is 0.5 - 10.0 mm.

[0070] Among them, the preparation method of the obtained hydrogel is as follows: First, take 100 mL of absolute ethanol and add 900 mL of distilled water. Mix them evenly to obtain a 10% ethanol solution. Weigh 10 mg of shikonin and dissolve it in 10 mL of the 10% ethanol solution to prepare a 1 mg / mL shikonin solution. Then, at room temperature, weigh 200.0 mg of sodium alginate, 100.0 mg of modified chitosan, and 27.3 mg of calcium carbonate powder and dissolve them in 9 mL of deionized water in sequence. Stir vigorously to obtain a uniform precursor solution. The stirring method is magnetic stirring with a rotation speed of 800 r / min for 0.5 - 1 h. The heating method is water bath heating at a temperature of 45 °C. Then, weigh 750.0 mg of acrylamide and add it to the above mixture, and continue to heat the precursor solution in a water bath at a temperature of 60 °C. Keep it warm and static to form a pre-gel. Then, weigh 0.5 mg of methylene bisacrylamide and dissolve it in 0.5 mL of deionized water, weigh 5 mg of ammonium persulfate and dissolve it in 0.5 mL of deionized water. The mixing method is vortex oscillation with a rotation speed of 800 r / min for 5 - 10 min at room temperature. Then, add the methylene bisacrylamide aqueous solution and ammonium persulfate aqueous solution drop by drop to the pre-gel in sequence. Under 60 °C water bath heating, after stirring evenly, add 10.0 μL of tetramethylethylenediamine and 142.0 μg of glucono-δ-lactone, stir for 30 s, and then pour it into a square glass mold (length = 10 cm, width = 8 cm, depth = 0.5 - 10 mm) or round 12-, 24-, 48-well plates, and carry out a 1-hour thermal initiation cross-linking reaction at 60 °C. Let the cross-linked pre-gel stand at room temperature for 24 hours to obtain a hydrogel. Then, freeze the prepared hydrogel at -20 °C overnight, vacuum freeze-dry the solidified hydrogel for more than 48 hours, and soak it in 10 mL of 1 mg / mL shikonin standard 10% ethanol solution to make it fully absorb the shikonin solution to obtain a normally moist hydrogel for subsequent use.

[0071] Example 8 This example provides a hydrogel, which can carry drugs including but not limited to those with functions such as anti-inflammatory, antibacterial, and hemostatic, such as shikonin. This hydrogel is obtained by physically cross-linking high molecular compounds of sodium alginate and modified chitosan and calcium carbonate and glucono-δ-lactone, and chemically cross-linking high molecular compounds of acrylamide, methylene bisacrylamide, ammonium persulfate, and tetramethylethylenediamine with a mass-volume fraction of 10%. After freeze-drying, it is soaked in 10 mL of 1 mg / mL shikonin standard 10% ethanol solution to obtain a normally moist hydrogel, and the thickness of the obtained hydrogel is 0.5 - 10.0 mm.

[0072] Among them, the preparation method of the obtained hydrogel is as follows: First, take 100 mL of absolute ethanol and add 900 mL of distilled water. Mix them evenly to obtain a 10% ethanol solution. Weigh 10 mg of shikonin and dissolve it in 10 mL of the 10% ethanol solution to prepare a 1 mg / mL shikonin solution. Then, at room temperature, weigh 200.0 mg of sodium alginate, 100.0 mg of modified chitosan, and 27.3 mg of calcium carbonate powder and dissolve them successively in 8.666 mL of deionized water. Stir vigorously to obtain a homogeneous precursor solution. The stirring method is magnetic stirring at a speed of 800 r / min for 0.5 - 1 h. The heating method is water bath heating at a temperature of 45°C. Then, weigh 1000.0 mg of acrylamide and add it to the above mixture, and continue to heat the precursor solution in a water bath at a temperature of 60°C. Keep it warm and static to form a pre-gel. Then, weigh 0.667 mg of methylene bisacrylamide and dissolve it in 0.667 mL of deionized water, weigh 6.667 mg of ammonium persulfate and dissolve it in 0.667 mL of deionized water. The mixing method is vortex oscillation at a speed of 800 r / min for 5 - 10 min, and it is prepared at room temperature. Then, add the methylene bisacrylamide aqueous solution and the ammonium persulfate aqueous solution drop by drop into the pre-gel in turn. Under 60°C water bath heating, after stirring evenly, add 13.333 μL of tetramethylethylenediamine and 142.0 μg of glucono-δ-lactone, stir for 30 s, and then pour it into a square glass mold (length = 10 cm, width = 8 cm, depth = 0.5 - 10 mm) or round 12-, 24-, 48-well plates, and carry out a 1-hour thermal initiation cross-linking reaction at 60°C. Let the cross-linked pre-gel stand at room temperature for 24 hours to obtain a hydrogel. Then, freeze the prepared hydrogel at -20°C overnight, and vacuum freeze-dry the solidified hydrogel for more than 48 hours. Immerse it in 10 mL of 1 mg / mL shikonin standard 10% ethanol solution to make it fully absorb the shikonin solution to obtain a normally moist hydrogel for subsequent use.

[0073] Example 9 This example provides a hydrogel, which can carry drugs including but not limited to those with functions such as anti-inflammatory, antibacterial, and hemostatic, such as shikonin. This hydrogel is obtained by physically cross-linking high-molecular compounds of sodium alginate and modified chitosan with calcium carbonate and glucono-δ-lactone, and chemically cross-linking high-molecular compounds of 15% acrylamide, methylene bisacrylamide, ammonium persulfate, and tetramethylethylenediamine. After freeze-drying, it is immersed in 10 mL of 1 mg / mL shikonin standard 10% ethanol solution to obtain a normally moist hydrogel, and the thickness of the obtained hydrogel is 0.5 - 10.0 mm.

[0074] Among them, the preparation method of the obtained hydrogel is as follows: First, take 100 mL of absolute ethanol, add 900 mL of distilled water, and mix evenly to obtain a 10% ethanol solution. Weigh 10 mg of shikonin and dissolve it in 10 mL of the 10% ethanol solution to prepare a 1 mg / mL shikonin solution. Then, at room temperature, weigh 200.0 mg of sodium alginate, 100.0 mg of modified chitosan, and 27.3 mg of calcium carbonate powder and dissolve them successively in 8 mL of deionized water. Stir vigorously to obtain a uniform precursor solution. The stirring method is magnetic stirring, with a rotation speed of 800 r / min and stirring for 0.5 - 1 h. The heating method is water bath heating at a temperature of 45 °C. Then, weigh 1500.0 mg of acrylamide and add it to the above mixture, and continue to heat the precursor solution in a water bath at a temperature of 60 °C. Keep it warm and static to form a pre-gel. Then, weigh 1.0 mg of methylene bisacrylamide and dissolve it in 1.0 mL of deionized water, weigh 10.0 mg of ammonium persulfate and dissolve it in 1.0 mL of deionized water. The mixing method is vortex oscillation with a rotation speed of 800 r / min and stirring for 5 - 10 min, prepared under normal temperature conditions. Then, add the methylene bisacrylamide aqueous solution and ammonium persulfate aqueous solution dropwise into the pre-gel in turn. Under 60 °C water bath heating, after stirring evenly, add 20 μL of tetramethylethylenediamine and 142.0 μg of glucono-δ-lactone, stir for 30 s, and then pour it into a square glass mold (length = 10 cm, width = 8 cm, depth = 0.5 - 10 mm) or a round 12-, 24-, 48-well plate, and carry out a 1-hour thermal initiation cross-linking reaction at 60 °C. Place the cross-linked pre-gel at room temperature for 24 hours to obtain a hydrogel. Then, freeze the prepared hydrogel (-20 °C) overnight, and vacuum freeze-dry the solidified hydrogel for more than 48 hours. Immerse it in 10 mL of a 1 mg / mL shikonin standard 10% ethanol solution to make it fully absorb the shikonin solution to obtain a normally moist hydrogel for subsequent use.

[0075] The present invention provides two different drug addition methods, which can be applicable to adding different drugs to the hydrogel. Specifically, if a drug will react with other components during the direct addition to the hydrogel gelation process (refer to the preparation methods of Examples 4, 5, and 6), the drug addition method of Examples 7, 8, and 9 can be used, which can effectively reduce the risk of the change in drug efficacy caused by the reaction between the drug and the hydrogel.

[0076] Experimental Example 1 Performance Test and Result Analysis of Hydrogel 1. Hydrogel Preparation Shape Test: Test the shapes that can be prepared from the hydrogels prepared in Examples 1 - 3 above, and obtain different hydrogel preparation shapes as Figure 7As shown, it can be seen that the prepared hydrogel can form a gel in any mold, including a square glass mold (length = 10 cm, width = 8 cm, depth = 0.5 - 10 mm) or a round 12-, 24-, or 48-well plate, and can also be arbitrarily cut according to the needs of the wound to achieve personalized treatment.

[0077] The prepared hydrogel products in Examples 4 - 9, their preparation shapes in a 24-well plate are as Figure 8 shown, and their SEM images of the hydrogel are respectively successively as Figure 9 , 10 , 11, 12, 13, 14 shown, where Figures 9 - 14 in A is at low magnification (scale bar: 100 μm), B is at high magnification (scale bar: 30 μm). It can be seen that this hydrogel also has a loose porous structure. Because it contains shikonin, the hydrogel becomes purplish red, and the gelation of the hydrogel remains stable and uniform.

[0078] This hydrogel has two drug-loading methods, which can be selected according to the different properties of the drugs. In Examples 4 - 6, the drugs are directly added during the gelation process to jointly prepare the hydrogel; in Examples 7 - 9, after the hydrogel is gelled, it is vacuum freeze-dried, and then, by virtue of its large swelling ratio, a certain amount of liquid medicine is absorbed to restore the normal moist state of the hydrogel for use.

[0079] 2. Testing of water content and swelling properties of the hydrogel: (1) Water content: To test the water content of the hydrogels prepared in Examples 1 - 3, first, hydrogels with fixed shapes and sizes are prepared through the above-mentioned examples. Each sample has 3 parallel samples. Weigh their mass (M0) and freeze (-20 °C) overnight, then vacuum freeze-dry them for more than 48 hours, and weigh the mass of the hydrogel after freeze-drying (M1). The calculation formula for the water content of the hydrogel is: water content (%) = (M0 - M1) / M0 * 100%. The analysis results of the water content of different groups of hydrogels are as Figure 15 shown. For each group (n = 3), where * is P < 0.05, ** is P < 0.01, *** is P < 0.001. It can be seen that the water content rate of the 7.5% acrylamide group is 97.07%; the 10% group is 95.87%; the 15% group is 84.91%. The water content of each group remains at a relatively high level, which is beneficial to maintaining the moist environment of the wound.

[0080] (2) Swelling properties: Test the swelling properties of the hydrogels prepared in Test Examples 1-3. Keep the hydrogels in a vacuum freeze-dried or wet state. Weigh appropriate amounts of different hydrogels and place them in different centrifuge tubes. Each sample has 3 parallel samples. Record the initial mass of the hydrogel as M0. Immerse the hydrogels in 0.01M PBS buffer (pH = 7.2 - 7.4). Take out 3 replicated hydrogels at time points of 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 hours. Blot the water on the surface of the hydrogels with absorbent paper, and then weigh their swollen weights (M t ). The swelling rate calculation formula of the hydrogel is: Swelling rate (%) = (M t - M0) / M0 * 100%. Obtain the swelling properties of different groups of hydrogels after freeze-drying and under normal conditions. The measurement results are as shown in Figure 16 . Among them, for each group (n = 3), Figure 16 in which A is for testing the hydrogel after freeze-drying, and B is for directly testing the normal hydrogel. It can be seen that the hydrogels can all swell by more than 100% within 15 min after freeze-drying, and reach swelling equilibrium after 12 h. The swelling rates of each group of hydrogels all exceed 2500%. The final swelling rate of the 7.5% group is 2704.93%; the final swelling rate of the 10% group is 3081.48%; the final swelling rate of the 15% group is 3252.91%. The swelling rate of thousands of times of the hydrogel after freeze-drying endows the hydrogel with the property of adjustable water absorption. When directly testing the swelling properties of the normal hydrogel, it can all swell by more than 70% within 30 min, and more than 100% within 1 h. The 7.5% group reaches swelling equilibrium in about 3 h, and the final swelling rate is 152.75%; the 10% group reaches swelling equilibrium in about 6 h, and the final swelling rate is 253.23%; the 15% group reaches swelling equilibrium in about 10 h, and the final swelling rate is 364.26%. The swelling rate of hundreds of times of the normal wet hydrogel also meets the requirement of absorbing general wound exudate.

[0081] By comparison, the swelling rate of the normal hydrogel can already meet the clinical requirement of absorbing wound exudate. The extremely large swelling rate of the freeze-dried hydrogel provides a sufficient adjustable range for its high drug loading and absorption of wound exudate, as shown in Examples 7 - 9. Therefore, this property also endows the hydrogel with adjustable water absorption, and this characteristic is more in line with the variability during the wound healing process in clinical use, and can modify the treatment plan in a timely manner according to the actual situation such as wound exudate to achieve personalized treatment.

[0082] 3. Hydrogel adhesion property test: Test the adhesion performance of the hydrogels prepared in Test Examples 1-3. Prepare fresh porcine skin of 10 mm * 60 mm, remove the hair and excess fat layer on the surface of the porcine skin, soak it in absolute ethanol for 30 minutes, and then let it stand at room temperature to volatilize. Apply different hydrogels between two pieces of porcine skin. There are 3 parallel samples for each sample. The contact surface between the porcine skins is about 10 mm * 10 mm. Press the porcine skin coated with the hydrogel under a heavy object and store it at room temperature for more than 2 hours to achieve adhesion. Use a universal testing machine to test through a tensile experiment. The tensile speed is 1 mm / min, and the load sensor is 1000 N. The adhesion of different groups of hydrogels is obtained as Figure 17 shown. Among them, A is a schematic diagram of the hydrogel adhering to the surfaces of different materials; B is the adhesion strength of each group of hydrogels (n = 3 for each group). It can be seen that the hydrogel can adhere tightly to various materials such as plastic ( Figure 17 a in A), glass (b), wood (c), foam (e), paper (f), stainless steel (g), etc., and can also adhere to internal organs such as the mouse heart (d) and intestine (h). Among them, the adhesion strength of the 7.5% group is 120.19 KPa; the adhesion strength of the 10% group is 216.85 KPa; the adhesion strength of the 15% group is 362.60 KPa. The hydrogel can firmly adhere to the wound surface through strong adhesion, control wound bleeding, prevent bacterial infection, and promote wound healing.

[0083] In actual applications, the problem of difficult replacement of the hydrogel is likely to cause secondary damage to the wound surface. Therefore, developing a hydrogel with adjustable adhesion and easy replacement is more in line with actual clinical applications, which can reduce the pain and discomfort caused by frequent dressing changes for patients. The adhesion performance of this hydrogel is tested again after soaking in water for 5 seconds. The adhesion strength of the 7.5% group is 68.59 KPa; the adhesion strength of the 10% group is 115.96 KPa; the adhesion strength of the 15% group is 296.17 KPa. The adhesion performance of each group of hydrogels decreases after soaking in water. Therefore, this property also endows the hydrogel with adjustable adhesion, which is more conducive to painless replacement of the dressing during clinical use.

[0084] 4. Test the mechanical properties of the hydrogel: Test the mechanical properties of the hydrogels prepared in Test Examples 1-3. Use the compression mode of a dynamic mechanical analyzer. Place an appropriate amount of circular hydrogel in the center of the fixture. There are 3 parallel samples for each sample. Set the compression parameter to 3 mm / min. The compression stress-strain curves and their compression modulus of different groups of hydrogels are obtained as shown in Figure 18As shown, where A is a schematic diagram of the hydrogel compression process; B is the stress-strain curve of each group of hydrogels; C is the compression modulus of each group of hydrogels (n = 3). It can be seen that the compression modulus of the 7.5% group is 212.81 Pa; the compression modulus of the 10% group is 320.22 Pa; the compression modulus of the 15% group is 440.33 Pa. Each group of hydrogels has good softness and elasticity, can adapt to a variety of irregular shapes, and has a certain plasticity, capable of forming a variety of different structures to meet different application requirements. At the same time, when the hydrogel is subjected to pressure or force, it will deform and can buffer external impacts, having high damping performance; when the external pressure is removed, its shape can quickly return to its original state.

[0085] 5. Hydrogel cell proliferation and toxicity performance test: Take 10 mg of the hydrogel and place it in a centrifuge tube. Each sample has 3 parallel samples. Add 1 mL of 0.01 M PBS buffer (pH = 7.2 - 7.4), and incubate it in a shaker at 37 °C and 50 rpm for 24 hours. After filtration and sterilization, prepare the hydrogel extract for storage and standby. Inoculate cells in the logarithmic growth phase at an appropriate density in a 96-well plate. After overnight culture until the cells adhere, add the hydrogel extract to test the gel cell proliferation and toxicity performance.

[0086] (1) Determination of cell viability of mouse epithelial fibroblasts L929 cells after incubation in the hydrogel (10 mg / mL) extract for different times: Test the cell proliferation and toxicity performance of the hydrogels prepared in Test Examples 1 - 3. Add hydrogel extracts with different concentrations to different wells and co-culture them with mouse epithelial fibroblasts L929 cells. Add CCK8 reagent and incubate in the dark for 2 hours at 24 hours and 48 hours. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at a wavelength of 450 nm. Use a CCK8 kit to test the cell viability of mouse epithelial fibroblasts L929 cells after incubation with different hydrogel extracts. The results are as Figure 19 shown, where Figure 19 A is for incubation for 24 h and B is for incubation for 48 h. It can be seen that the survival rate of each group remains above 90%, and with the increase in the concentration of the hydrogel extract or the increase in the co-incubation time, the ability to promote cell proliferation also increases to a certain extent. After incubating L929 cells with different hydrogel solutions (10 mg / mL) for 48 h, perform live-dead staining tests. Live cells show green and dead cells show red. The results are as Figure 20 shown. It can be seen that the proportion of dead cells (red) in different hydrogel solutions is relatively small, and the proportion of live cells (green) is relatively large, which is consistent with the CCK8 quantitative test results.

[0087] (2)Determination of the cell viability of mouse embryonic fibroblast NIH3T3 cells after incubation in the extract of the hydrogel (10 mg / mL) for different times: To test the cell proliferation and toxicity properties of the hydrogels prepared in Test Examples 1-3, different concentrations of the hydrogel extract were added to different wells and co-cultured with mouse embryonic fibroblast NIH3T3 cells. CCK8 reagent was added at 24 hours and 48 hours and incubated in the dark for 2 hours. The absorbance at a wavelength of 450 nm was measured using a microplate reader. The CCK8 kit was used to test the cell viability of mouse embryonic fibroblast NIH3T3 cells after incubation with different hydrogel extracts. The results are as Figure 21 shown, where Figure 21 A is for incubation for 24 h and B is for incubation for 48 h. It can be seen that the survival rate of each group remained above 80%. Moreover, with the increase in the concentration of the hydrogel extract or the increase in the co-incubation time, the ability to promote cell proliferation also increased to a certain extent. After incubating NIH3T3 cells with different hydrogel solutions (10 mg / mL) for 48 h, a live-dead staining test was performed. Live cells were shown in green and dead cells were shown in red. The results are as Figure 22 shown. It can be seen that the proportion of dead cells (red) in different hydrogel solutions was relatively small, and the proportion of live cells (green) was relatively large, which was consistent with the CCK8 quantitative test results.

[0088] By testing the cell viability of two types of cells, NIH3T3 cells and L929 cells, after incubation with different hydrogel extracts, the results all showed that the sodium alginate-modified chitosan-polyacrylamide composite hydrogel had good biocompatibility, could promote cell proliferation, and was beneficial to promoting wound healing.

[0089] 6. Test on the cell migration performance of the hydrogel: At least three horizontal lines were drawn with a marker pen along the ruler at the bottom of each well of the well plate as marking lines. The cells were cultured until they reached about 80-90% confluence, and the cell density was about 5x10 5 cells / well. A scratch was made in the center of the cell monolayer well plate with a 200 μL yellow sterile pipette tip, so that the scratch intersected with the marking lines, forming several intersection points as fixed detection points to simulate a wound. The culture dish was gently washed with sterile PBS buffer to remove floating cells, and fresh serum-free medium was added. Different hydrogel extracts could be added according to the experimental needs. Each sample had 3 parallel samples. The immediate image (0 hour) after scratching was observed and photographed using a microscope. The culture dish was placed back into the 37 °C, 5% CO2 incubator. The culture dish was taken out after culturing for 12 and 24 hours, and the images of cell migration were observed and photographed. Image analysis software (such as ImageJ) was used to measure the width of the scratch or the number of migrated cells at different time points. The cell migration rate was calculated and statistical analysis was performed.

[0090] The cell migration performance of the hydrogels prepared in Test Examples 1-3 was tested. The cell migration of different groups of hydrogel extracts co-cultured with L929 cells for different times is shown in Figure 23 as follows. Quantitative analysis of cell migration was performed on each group, and the results are shown in Figure 24 as follows (n = 3). The results indicate that the hydrogel extracts of different groups all promoted the migration of L929 cells, and the promotion increased with the increase in the concentration of polyacrylamide.

[0091] The cell migration performance of the hydrogels prepared in Test Examples 1-3 was tested. The cell migration of different groups of hydrogel extracts co-cultured with NIH3T3 cells for different times is shown in Figure 25 as follows. Quantitative analysis of cell migration was performed on each group, and the results are shown in Figure 26 as follows (n = 3). The results indicate that the hydrogel extracts of different groups all promoted the migration of NIH3T3 cells, and the promotion increased with the increase in the concentration of polyacrylamide.

[0092] By testing the cell migration rates of two types of cells, NIH3T3 cells and L929 cells, after incubation with different hydrogel extracts, the results all show that the sodium alginate-modified chitosan-polyacrylamide composite hydrogel can promote cell migration, which is beneficial to promoting wound healing.

[0093] 7. Test on the drug sustained-release performance of the hydrogel: The drug sustained-release performance of the hydrogels prepared in Test Examples 7-9 was tested. 10 mg of shikonin standard was weighed and dissolved in 1 mL of 10% ethanol solution by ultrasonic to obtain a shikonin stock solution. Then, the stock solution was diluted with UP water (ultrapure water) to a solution with a concentration of 0.1-1 mg / mL. The absorbance y of shikonin solutions with different concentrations x at 520 nm was measured respectively, and the standard curve of shikonin was calculated by linear regression. After adding an equal amount of shikonin to different hydrogels and forming gels, an appropriate amount of hydrogel was placed in a 50 mL centrifuge tube. Each sample had 3 parallel samples. 15 mL of UP water was added, and the mixture was incubated in a shaker at 37 °C and 50 rpm. 100 μL of supernatant was collected at different time points, and then 100 μL of UP water was added. The absorbance y of the supernatant at 520 nm at different time points was measured using an enzyme-linked immunosorbent assay (ELISA) reader. According to the standard curve, the shikonin concentration in different supernatants could be calculated, and the drug release curves of shikonin-loaded hydrogels of different groups were obtained. The results are shown in Figure 27 as follows. Among them, A is the standard curve of shikonin drug release; B is the shikonin drug release curve of different groups of hydrogels at 12 h (n = 3). It can be seen that the drug release of the 7.5% hydrogel group was relatively fast in the early stage, and 60% was released in 1 h. The drug release of the 10% and 15% hydrogel groups was relatively slow, realizing the continuous and stable release of the drug, thereby improving the therapeutic effect, reducing the number of drug administrations and drug side effects. The final drug release rate of the 10% hydrogel was greater than that of the 15% hydrogel group.

[0094] 8. Hydrogel animal wound healing test: By comparing the usage status of hydrogels with different ratios in Examples 1, 2, and 3 on the wound, it was found that the hydrogel in the 10% acrylamide group had moderate adhesion performance, neither too low to adhere to the wound nor too high to be difficult to replace; its mechanical properties were moderate, neither too hard nor too soft; its swelling properties were moderate, neither too high to absorb too much water required by normal growing cell tissues near the wound nor too low to incompletely absorb wound exudate; it had drug slow release and finally released the most. Based on this, the hydrogel in the 10% acrylamide group was selected for animal experiment verification.

[0095] To test the wound repair performance of the hydrogel prepared in Example 2, female mice about 6 weeks old were used. After being adaptively fed for one week to get familiar with the environment, the experiment was carried out when their states were stable. During the experiment, the mice were anesthetized with an isoflurane gas anesthesia machine. First, the hair on the backs of the mice was removed. After 12 hours, a full-thickness circular wound with a diameter of 10 mm was modeled at the depilated area. In the experimental group, the wound was covered with the 10% group hydrogel for treatment and wrapped with sterile gauze. In the control group, only bandaging was performed. During the whole treatment process, the hydrogel dressing was changed every 2 - 3 days, and the wound surface of the mice was photographed every 2 days for recording. The repair effects of different groups of hydrogels on the skin wound of mice for 12 days (measured and statistically analyzed every two days) are shown in Figure 28 as shown, and the quantitative analysis of the wound healing situation is shown in Figure 29 as shown. The results showed that compared with the control group that only used normal saline to clean and gauze bandage, the 10% hydrogel group showed better wound healing effect, with a significant difference from the control group.

[0096] On the 12th day of wound healing, the mice were euthanized, and the skin wound tissues were collected for further analysis. Part of the wound surfaces of the two groups were taken for pathological analysis. HE staining and Masson trichrome staining were performed on them, and the results are shown in Figure 30 (H&E staining) and 31 (Masson trichrome staining) respectively. Among them, Figure 30 and 31 in A - B are the control group, A is low magnification (scale bar: 100 μm), B is high magnification (scale bar: 10 μm); C - D are the 10% hydrogel group, C is low magnification (scale bar: 100 μm), D is high magnification (scale bar: 10 μm). It can be seen that the wound regeneration rate of the 10% hydrogel treatment group is higher, the wound regeneration tissue is thicker, and there is more granulation tissue. The wound tissue of the blank group only has a thin layer of granulation tissue, indicating that the wound treated with the hydrogel regenerates faster. In addition, the wound treated with 10% hydrogel shows a higher proportion of collagen in the MTS image, which indicates that this hydrogel indeed has the potential to accelerate the wound healing process. In summary, this hydrogel shows good wound healing ability and great potential for its further clinical application.

[0097] In summary, the present invention provides an adhesiveness and water absorption adjustable double-crosslinked hydrogel and a preparation method thereof. The obtained hydrogel can be used for treating skin wounds, effectively improving the technical problem of secondary damage to the wound caused by hydrogel replacement, better conforming to the dynamic variability of wound exudate in practical applications, and promoting skin wound healing.

[0098] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be regarded as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and substitutions of the present invention will be obvious. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. A double-crosslinked personalized hydrogel for promoting wound healing, characterized in that, The hydrogel contains sodium alginate, modified chitosan, calcium carbonate, glucono delta-lactone, acrylamide, methylene bisacrylamide, ammonium persulfate, and tetramethylethylenediamine, wherein the modified chitosan is 90% chitosan + 10% chitosan quaternary ammonium salt.

2. The hydrogel according to claim 1, wherein The hydrogel further contains drugs including but not limited to those with anti-inflammatory, antibacterial, and hemostatic functions.

3. The hydrogel according to claim 2, wherein The drug can be selected from shikonin.

4. The hydrogel according to any one of claims 1 to 3, characterized in that, The thickness of the hydrogel is 0.5 - 10 mm.

5. Use of the hydrogel according to any one of claims 1 - 4 in the treatment of skin wound repair.

6. The preparation process of the hydrogel according to claim 1, characterized in that, Comprising the following steps: (6) Dissolve sodium alginate, modified chitosan, and calcium carbonate in water in sequence, and stir to obtain a uniform precursor solution; (7) Add acrylamide to the precursor solution, and keep it warm and static to form a pre-gel; (8) Prepare aqueous solutions of methylene bisacrylamide and ammonium persulfate respectively, and add them drop by drop to the pre-gel in sequence. After stirring evenly, keep it warm to obtain a premixed gel; (9) Add tetramethylethylenediamine and glucono delta-lactone to the premixed gel, stir evenly, and then pour it into a mold for cross-linking reaction; (10) After the cross-linking reaction is completed, let it cool to room temperature to obtain the hydrogel.

7. The preparation process according to claim 6, characterized in that, The final concentrations of sodium alginate, modified chitosan, and calcium carbonate in the hydrogel in step (1) are 20, 10, and 2.73 mg / mL respectively.

8. The preparation process according to claim 6, characterized in that, The final concentration of acrylamide in the hydrogel in step (2) is 100 mg / mL; the temperature for keeping warm is 60 °C.

9. The preparation process according to claim 6, characterized in that, The final concentration of methylene bisacrylamide in the hydrogel in step (3) is 0.05 mg / mL, and the final concentration of ammonium persulfate in the hydrogel is 0.5 mg / mL. The temperature for keeping warm is 60 °C.

10. The preparation process according to any one of claims 6-9, characterized in that, The volume ratio of the amount of tetramethylethylenediamine in the hydrogel is 13.333 μL / 10 mL, the final concentration of glucono delta-lactone in the hydrogel is 14.2 μg / mL, and the conditions for the cross-linking reaction are: reacting at 60 °C for 1 hour.

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