Hydrogel loaded with repair-promoting and antioxidant peptide as well as preparation method and application of hydrogel

Abstract

The invention discloses hydrogel loaded with repair-promoting and antioxidant peptides as well as a preparation method and application of the hydrogel, and belongs to the technical field of preparation of medical materials. According to the preparation method, firstly, polypeptide with the amino acid sequence of CEYWVKWCGEKGPAGERG is synthesized, and the polypeptide has antioxidant activity and cell proliferation promoting activity and has good structural stability and enzymatic degradation resistance; and loading the polypeptide into the hyaluronic acid hydrogel to obtain the hyaluronic acid hydrogel. The prepared hydrogel loaded with the repair-promoting and antioxidant peptide has excellent injectable performance and cornea spreadability, can enhance the retention of the polypeptide on the ocular surface, effectively solves the technical problem of high administration frequency of the existing preparation, has good cell and tissue compatibility, and has no adverse reaction in vivo.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical material preparation, and particularly relates to a hydrogel loaded with a repair-promoting and antioxidant peptide as well as a preparation method and application thereof. BACKGROUND

[0002] Dry eye disease (DED), also known as keratoconjunctival xerosis, is an ocular disease caused by tear film instability and ocular surface damage leading to abnormal tear quantity or quality. As a common multifactorial ocular disease in modern society, DED affects the quality of life of millions of people worldwide. DED mainly affects the cornea and conjunctiva, causing corneal epithelial damage, reducing the regularity and sensitivity of the corneal surface, and interfering with the refractive system, thereby causing blurred vision and severely affecting the daily life of patients. At present, the main drugs for treating DED include artificial tears, anti-inflammatory drugs and immunosuppressive agents. However, these treatment methods have their own limitations: artificial tears can only provide temporary symptomatic relief and cannot cure DED; steroid anti-inflammatory drugs are effective but can cause serious side effects such as glaucoma and cataracts; and immunosuppressive agents such as cyclosporine A require long-term use (3-6 months) to be effective. Therefore, it is particularly urgent to develop a safe and effective new DED therapy.

[0003] Recent studies have shown that oxidative stress damage caused by excessive production of reactive oxygen species (ROS) is an important upstream factor in the occurrence and development of DED. Preclinical studies and clinical data have shown a close link between ROS production, membrane damage related to lipid peroxidation, protein oxidation and inflammation. Direct exposure of the eye to ultraviolet light, drugs, air pollutants and excessive use of electronic devices can lead to accumulation of ROS on the ocular surface, triggering oxidative stress damage, manifested as decreased tear film stability, tear hyperosmolarity, corneal epithelial cell dysfunction, loss of goblet cells and increased inflammation, forming a vicious cycle. Based on this, scavenging excess ROS is considered a potential new strategy to improve the development of DED. Currently, some antioxidants such as Reproxalap (targeting reactive aldehydes) and the mitochondria-targeted antioxidant Visomitin (SKQ1) are being evaluated in clinical trials as new DED treatment drugs. However, all existing antioxidant eye drops are small molecule drugs that are consumed after reacting with ROS, and due to normal blinking action and tear metabolism, they have a very short residence time on the ocular surface. Hydrogels have many advantages, including their excellent hydrophilicity, transparency, biocompatibility and porous three-dimensional structure that supports cell activity and metabolite transport, and have been shown to extend the residence time of drugs on the ocular surface, thus representing a major choice for ocular drug delivery systems. SUMMARY

[0004] In view of the above prior art, the application discloses a hydrogel loaded with repair-promoting and antioxidant peptides, a preparation method and application thereof, so as to solve the technical problems of short residence time on the ocular surface and poor treatment effect of the antioxidant eye drops for treating DED in the prior art.

[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the application is to provide a hydrogel loaded with repair-promoting and antioxidant peptides, wherein the amino acid sequence of the repair-promoting and antioxidant peptides loaded by the hydrogel is shown as SEQ ID NO: 1.

[0006] The application further discloses a preparation method of the hydrogel loaded with repair-promoting and antioxidant peptides. S1: synthesizing a polypeptide, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1; S2: synthesizing a hyaluronic acid hydrogel by using sodium hyaluronate and 1,4-butanediol diglycidyl ether (BDDE) as raw materials; S3: mixing the polypeptide and the hyaluronic acid hydrogel uniformly to obtain the hydrogel loaded with repair-promoting and antioxidant peptides.

[0007] On the basis of the above technical scheme, the application can be further improved as follows.

[0008] Further, the molecular weight of the sodium hyaluronate is 18,000,000-25,000,000 Da, and the mass ratio of the sodium hyaluronate with a molecular weight of 18,000,000-25,000,000 Da to 1,4-butanediol diglycidyl ether is 6-10:0.2-0.8.

[0009] Further, the dosage ratio of the polypeptide to the hyaluronic acid hydrogel is 25-2,000 μg:1 mL.

[0010] The application further discloses application of the above hydrogel loaded with repair-promoting and antioxidant peptides in treatment of dry eye.

[0011] The application has the following beneficial effects: 1、The CYCLO-17 peptide synthesized in the application has antioxidant activity and cell proliferation promoting activity, and has good structural stability and enzyme degradation resistance; the amino acid sequence is CEYWVKWCGEKGPAGERG; the two cysteines (Cys1 and Cys8) in the sequence are the first and eighth positions, and form an intramolecular disulfide bond (Cys1-Cys8) under oxidation conditions, forming a small ring structure (8-membered ring). The CYCLO-17 structure with disulfide bond has stronger structural stability and is more resistant to protease degradation. The active fragment (GEKGPAGERG) contains two active sites (GEK and GER), which are derived from the high cell adhesion function region of human type III collagen; the antioxidant fragment (EYWVKW) has free radical scavenging ability, which can reduce oxidative stress, and Lys and Arg interfere with the NF-κB pathway or inhibit matrix metalloproteinase.

[0012] 2、The hydrogel loaded with the repair-promoting and antioxidant peptide in the application has a simple preparation process, has superior injectability and corneal spreading property, can enhance the retention of the polypeptide on the ocular surface, and effectively solves the technical problem of high drug administration frequency of existing preparations.

[0013] 3、The hydrogel loaded with the repair-promoting and antioxidant peptide in the application has low residual crosslinking agent after application, has good cell and tissue compatibility, and has no adverse reactions in vivo. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Figure 1 is the effect of different concentrations of CYCLO-17 peptide on the survival rate of HCE cells in the experimental example; Figure 2 Figure 2 is the DPPH scavenging capacity of different concentrations of CYCLO-17 peptide in the experimental example; Figure 3 Figure 3 is the corneal surface spreading property of hydrogels with different moduli in the experimental example; Figure 4 Figure 4 is a slit lamp cobalt blue staining image of the cornea of a mouse after different treatments in the experimental example; Figure 5 Figure 5 is H&E staining of the cornea of a mouse after different treatments in the experimental example. DETAILED DESCRIPTION

[0015] The specific embodiments of the application will be described in detail below with reference to the examples.

[0016] Example 1 A hydrogel loaded with a repair-promoting and antioxidant peptide, the preparation method thereof comprises the following steps: ①Using a polypeptide synthesizer to synthesize polypeptides by solid phase synthesis Take 0.4 g Rink Amide MBHA Resin into the synthesis column, add 20 mL DMF, stir at room temperature for 30 min, and make the resin fully swollen; add amino acids one by one from C to N, and the synthesis sequence is Gly 17 → Arg 16 → Glu 15 → Gly 14 → Ala 13 → Pro 12 → Gly 11 → Lys 10 → Glu 9 → Cys 8 → Trp 7 → Lys 6 → Val 5 → Trp 4 → Tyr 3 → Glu 2 → Cys 1 ; finally, cut with trifluoroacetic acid, deionized water and triisopropylsilane solution, precipitate with ice ether, freeze-dry to obtain the target polypeptide, and obtain the antioxidant peptide (labeled as CYCLO-17 peptide) after lyophilization.

[0017] ②Synthesis of hyaluronic acid hydrogel Slowly add 6 g of sodium hyaluronate (molecular weight 2.2 million) to 100 mL of 0.1 M sodium hydroxide solution to obtain solution A; then take 0.2 g of BDDE and dissolve it in anhydrous ethanol (3 mL), then slowly drop it into solution A, and continue to slowly stir (200 r / min) for 6 h; then the reaction liquid is loaded into a MWCO 12-14 kDa dialysis bag, dialyzed in deionized water for 72 h, and the water is changed every 6 h during the dialysis, and finally balanced in 0.9 wt% NaCl solution for 12 h; collect the gel in the bag, and after sterilization with an autoclave (121℃, 21 min), an injectable cross-linked hyaluronic acid hydrogel is obtained.

[0018] ③Mix 1 mL of hydrogel with 100 μg of CYCLO-17 peptide on a clean bench, and repeatedly push and squeeze with a two-milliliter syringe without a needle for 3-5 times, mix uniformly, and then place in a 4℃ refrigerator for use.

[0019] Example 2 A water gel loaded with repair-promoting and antioxidant peptides, the preparation method comprising the following steps: ①Solid-phase synthesis of polypeptide using polypeptide synthesizer Take 0.4 g Rink Amide MBHA Resin into the synthesis column, add 20 mL DMF, stir at room temperature for 30 min, and make the resin fully swollen; add amino acids one by one from C to N, and the synthesis sequence is Gly 17 → Arg 16 → Glu 15 → Gly 14 → Ala 13 → Pro 12 → Gly 11 → Lys 10 → Glu 9 → Cys 8 → Trp 7 → Lys 6 → Val 5 → Trp 4 → Tyr 3 → Glu 2 → Cys 1 ; finally, cut with trifluoroacetic acid, deionized water and triisopropylsilane solution, precipitate with ice ether, freeze-dry to obtain the target polypeptide, and obtain the antioxidant peptide (labeled as CYCLO-17 peptide) after freeze-drying.

[0020] ②Synthesis of hyaluronic acid hydrogel Slowly add 6 g of sodium hyaluronate (molecular weight 2.2 million) to 100 mL of 0.1 M sodium hydroxide solution to obtain solution A; then take 0.5 g of BDDE and dissolve it in anhydrous ethanol (3 mL), then slowly drop it into solution A, and continue to slowly stir (200 r / min) for 6 h; then the reaction liquid is loaded into a MWCO 12-14 kDa dialysis bag, dialyzed in deionized water for 72 h, and the water is changed every 6 h during the dialysis, and finally balanced in 0.9 wt% NaCl solution for 12 h; collect the gel in the bag, and after sterilization with an autoclave (121℃, 21 min), an injectable cross-linked hyaluronic acid hydrogel is obtained.

[0021] ③Mix 1 mL of hydrogel with 100 μg of CYCLO-17 peptide on a clean bench, and repeatedly push and squeeze with a two-milliliter syringe without a needle for 3-5 times, mix uniformly, and then place in a 4℃ refrigerator for use.

[0022] Example 3 A water gel loaded with repair-promoting and antioxidant peptides, the preparation method comprising the following steps: ①Solid-phase synthesis of polypeptide using polypeptide synthesizer Take 0.4 g Rink Amide MBHA Resin into the synthesis column, add 20 mL DMF, stir at room temperature for 30 min, and make the resin fully swollen; add amino acids one by one from C to N, and the synthesis sequence is Gly 17 → Arg 16 → Glu 15 → Gly 14 → Ala 13 → Pro 12 → Gly 11 → Lys 10 → Glu 9 → Cys 8 → Trp 7 → Lys 6 → Val 5 → Trp 4 → Tyr 3 → Glu 2 → Cys 1 ; finally, cut with trifluoroacetic acid, deionized water and triisopropylsilane solution, precipitate with ice ether, freeze-dry to obtain the target polypeptide, and obtain the antioxidant peptide (labeled as CYCLO-17 peptide) after freeze-drying.

[0023] ②Synthesis of hyaluronic acid hydrogel Slowly add 6 g of sodium hyaluronate (molecular weight 2.2 million) to 100 mL of 0.1 M sodium hydroxide solution to obtain solution A; then take 0.8 g of BDDE and dissolve it in anhydrous ethanol (3 mL), then slowly drop it into solution A, and continue to slowly stir (200 r / min) for 6 h; then the reaction liquid is loaded into a MWCO 12-14 kDa dialysis bag, dialyzed in deionized water for 72 h, and the water is changed every 6 h during the dialysis, and finally balanced in 0.9 wt% NaCl solution for 12 h; collect the gel in the bag, and after sterilization with an autoclave (121℃, 21 min), an injectable cross-linked hyaluronic acid hydrogel is obtained.

[0024] ③Mix 1 mL of hydrogel with 1000 μg of CYCLO-17 peptide on a clean bench, and repeatedly push and squeeze with a two-milliliter syringe without a needle for 3-5 times, mix uniformly, and then obtain, and finally place in a 4℃ refrigerator for use.

[0025] Example 4 A water gel loaded with repair-promoting and antioxidant peptides, the preparation method comprising the following steps: ①Solid-phase synthesis of polypeptide using polypeptide synthesizer Take 0.4 g Rink Amide MBHA Resin into the synthesis column, add 20 mL DMF, stir at room temperature for 30 min, and make the resin fully swollen; add amino acids one by one from C to N, and the synthesis sequence is Gly 17 → Arg 16 → Glu 15 → Gly 14 → Ala 13 → Pro 12 → Gly 11 → Lys 10 → Glu 9 → Cys 8 → Trp 7 → Lys 6 → Val 5 → Trp 4 → Tyr 3 → Glu 2 → Cys 1 ; finally, cut with trifluoroacetic acid, deionized water and triisopropylsilane solution, precipitate with ice ether, freeze-dry to obtain the target polypeptide, and obtain the antioxidant peptide (labeled as CYCLO-17 peptide) after freeze-drying.

[0026] ② Synthesis of hyaluronic acid hydrogel Slowly add 10 g of sodium hyaluronate (molecular weight 2.2 million) to 100 mL of 0.1 M sodium hydroxide solution to obtain solution A; then take 0.2 g of BDDE and dissolve it in anhydrous ethanol (5 mL), then slowly drop it into solution A, and continue to slowly stir (400 r / min) for 12 h; then the reaction liquid is loaded into a MWCO 12-14 kDa dialysis bag, dialyzed in deionized water for 48 h, and the water is changed every 8 h during the dialysis, and finally balanced in 0.9 wt% NaCl solution for 12 h; collect the gel in the bag, and after sterilization with an autoclave (121℃, 21 min), an injectable cross-linked hyaluronic acid hydrogel is obtained.

[0027] ③Mix 1 mL of hydrogel with 25 μg of CYCLO-17 peptide on a clean bench, and repeatedly push and squeeze with a two-milliliter syringe without a needle for 3-5 times, mix uniformly, and then place in a 4℃ refrigerator for standby.

[0028] Example 5 A water gel loaded with repair-promoting and antioxidant peptides, the preparation method comprising the following steps: ① Use a polypeptide synthesizer to synthesize a polypeptide by solid phase synthesis Take 0.4 g Rink Amide MBHA Resin into the synthesis column, add 20 mL DMF, stir at room temperature for 30 min, and make the resin fully swollen; add amino acids one by one from C to N, and the synthesis sequence is Gly 17 → Arg 16 → Glu 15 → Gly 14 → Ala 13 → Pro 12 → Gly 11 → Lys 10 → Glu 9 → Cys 8 → Trp 7 → Lys 6 → Val 5 → Trp 4 → Tyr 3 → Glu 2 → Cys 1 ; finally, cut with trifluoroacetic acid, deionized water and triisopropylsilane solution, precipitate with ice ether, freeze-dry to obtain the target polypeptide, and obtain the antioxidant peptide (marked as CYCLO-17 peptide) after freeze-drying.

[0029] ② Synthesis of hyaluronic acid hydrogel Slowly add 8 g of sodium hyaluronate (molecular weight 2.2 million) to 100 mL of 0.1 M sodium hydroxide solution to obtain solution A; then take 0.2 g of BDDE and dissolve it in anhydrous ethanol (2 mL), then slowly drop it into solution A, and continue to slowly stir (300 r / min) for 8 h; then the reaction liquid is loaded into a MWCO 12-14 kDa dialysis bag, dialyzed in deionized water for 64 h, and the water is changed every 6 h during the dialysis, and finally balanced in 0.9 wt% NaCl solution for 12 h; collect the gel in the bag, and after sterilization with an autoclave (121℃, 21 min), an injectable cross-linked hyaluronic acid hydrogel is obtained.

[0030] ③Mix 1 mL of hydrogel with 2000 μg of CYCLO-17 peptide on a clean bench, and repeatedly push and squeeze with a two-milliliter syringe without a needle for 3-5 times, mix uniformly, and then place in a 4℃ refrigerator for use.

[0031] Experimental example I. CYCLO-17 peptide activity detection ① CYCLO-17 peptide promotes cell proliferation activity The corneal epithelial cells (density 5×10 4Cells / mL) were inoculated into 96-well plates (i.e. about 5000 cells per well), incubated at 37℃ in a 5% CO2 incubator for 24 h to allow the cells to adhere and recover. Serial concentrations of CYCLO-17 polypeptide were prepared (diluted with complete culture medium, 12.5, 25, 50, 100 μg / mL); the old culture medium was discarded, and 100 μL of fresh complete culture medium + corresponding concentration of polypeptide was added to each well, negative control wells (only medium), blank wells (no cells), and incubated for 48 h. The culture medium was carefully aspirated from each well, washed once with PBS, and 100 μL of fresh complete culture medium + 10 μL of CCK-8 reagent (i.e. CCK-8: medium = 1:10) was added to each well. The 96-well plate was placed in an incubator (37℃, 5% CO2) for 3 h, and the absorbance of each well was measured at 450 nm using a microplate reader. The results are shown in Figure 1 As can be seen from the figure, CYCLO-17 peptide has a significant proliferative effect on corneal epithelial cells, and when the polypeptide concentration is 50 μg / mL, the cell survival rate is more than 120% compared with the control group.

[0032] ② Antioxidant activity of CYCLO-17 peptide An equal volume of 0.1 mM DPPH solution was mixed with different concentrations of CYCLO-17 peptide solution (0, 25, 50, 100 μg / mL), gently shaken after sampling (to avoid bubbles), wrapped with aluminum foil or placed in a dark box, and allowed to react at room temperature (25℃) for 30 min. After the reaction was completed, the absorbance was measured at 517 nm using a microplate reader. The results are shown in Figure 2 As can be seen from the figure, the free radical scavenging ability of CYCLO-17 is proportional to its concentration, and when the concentration is 100 μg / mL, the scavenging rate is more than 80%, indicating its good free radical scavenging activity.

[0033] II. Performance characterization of hyaluronic acid hydrogel ① Modulus determination The hyaluronic acid hydrogel synthesized in step ② of Examples 1-3 was measured at 25℃ using a rotational rheometer, about 1000 μL was taken with a pipette and dropped in the center of the measurement plate, the cone plate was slowly lowered to ensure uniform extrusion of the sample and avoid air bubbles, and the excess sample at the edges was gently removed with a spatula. The strain (1%) and the scanning angular frequency (ω) were fixed at 0.1-100 rad / s, and the G' value (unit: Pa or kPa) corresponding to 10 rad / s was selected. The results are shown in Table 1, which shows that the modulus of the hyaluronic acid hydrogel is proportional to the content of the crosslinking agent BDDE, and is between 38.74 and 212.58 Pa.

[0034] Table 1

[0035] ②Ocular surface spreading test: 0.1 mg / mL of fluorescein sodium was mixed with the hyaluronic acid hydrogel synthesized in step ② of Example 1-3, respectively, and added dropwise to the surface of the rabbit eye. As shown in Figure 3 , the modulus of the hydrogel increases from left to right, and the results show that the low modulus hydrogel in Example 1 has the best spreading, the best comfort and the best ocular surface retention.

[0036] III. In vivo effect evaluation of the hydrogel loaded with repair-promoting and antioxidant peptides A mouse dry eye model was successfully constructed by adding 5 μL of 0.2% benzalkonium chloride solution to both eyes of the mouse twice a day for 2 weeks. Benzalkonium chloride is a commonly used drug for inducing DED with acute corneal epithelial damage, excessive accumulation of ROS and corneal inflammation. The successful construction of the dry eye model was verified by corneal fluorescein staining. As shown in Figure 4 , compared with the control group and the blank hydrogel group, the hydrogel loaded with peptides showed a faster healing rate. In addition, the hydrogel with a concentration of 100 μg / mL had better therapeutic effect than the commercialized sea drop eye drops.

[0037] HE staining showed that the corneal epithelial cell layers of normal mice were clear under the microscope, and the cell morphology from the basal layer to the surface layer was normal. As can be seen from Figure 5 , the visible corneal epithelial cell layers of the control group and the blank gel group were disordered, the interfilament arrangement was disordered, the surface layer cells were significantly reduced, and vacuoles and nuclear fragmentation cells were visible; the corneal epithelium of the mouse treated with the hydrogel with a concentration of 100 μg / mL was complete, there was no obvious inflammatory reaction, and the stromal fibers were arranged in order without obvious edema, further proving its good therapeutic effect.

[0038] Although the specific embodiments of the present application are described in detail in combination with the examples, it should not be understood as limiting the protection scope of the present patent. Various modifications and variations made by those skilled in the art within the scope described in the claims are still within the protection scope of the present patent.

Claims

1. A hydrogel loaded with a pro-repair and antioxidant peptide, characterized in that, The amino acid sequence of the repair-promoting and antioxidant peptide loaded hydrogel is shown as SEQ ID NO:

1.

2. The method of claim 1, wherein the method of preparing the hydrogel loaded with the pro-repair and antioxidant peptide is characterized by, The method comprises the following steps: S1: synthesizing a polypeptide; the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1; S2: synthesizing a hyaluronic acid hydrogel from sodium hyaluronate and 1,4-butanediol diglycidyl ether; S3: mixing the polypeptide and the hyaluronic acid hydrogel uniformly to obtain the repair-promoting and antioxidant peptide loaded hydrogel.

3. The method for preparing a hydrogel loaded with repair-promoting and antioxidant peptides according to claim 2, characterized in that, The molecular weight of the sodium hyaluronate is 18,000,000-25,000,000 Da, and the mass ratio of the sodium hyaluronate to the 1,4-butanediol diglycidyl ether is 6-10:0.2-0.

8.

4. The method for preparing a hydrogel loaded with repair-promoting and antioxidant peptides according to claim 2, characterized in that, The usage ratio of the polypeptide to the hyaluronic acid hydrogel is 25-2,000 μg:1 mL.

5. Use of a hydrogel loaded with a pro-repair and antioxidant peptide, characterized in that: The repair-promoting and antioxidant peptide loaded hydrogel of claim 1 is used for treating dry eye.

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