A polypeptide self-assembled hydrogel and a preparation method and application thereof

Abstract

The application discloses a kind of polypeptide self-assembly hydrogel and its preparation method and application.The application is obtained by dissolving sodium ascorbate in 0.1M phosphate buffer solution to obtain sodium ascorbate solution;Antibacterial peptide Jelleine-1 is dissolved in DMSO to be configured into 100mg / mL polypeptide stock solution;Sodium ascorbate solution is added to polypeptide stock solution, fully stirred and mixed, and placed into shape, to obtain polypeptide self-assembly hydrogel.The preparation method of the application is simple, the gelation speed is fast, the production cost is low, the obtained polypeptide self-assembly hydrogel has excellent antibacterial performance and antioxidant performance, and almost has no cytotoxicity and hemolytic toxicity, has good biocompatibility, can effectively inhibit bacterial infection in atopic dermatitis, reduce oxidative stress, thereby significantly relieving dermatitis symptoms.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanobiomedical materials, and particularly relates to a polypeptide self-assembled hydrogel and a preparation method and application thereof. BACKGROUND

[0002] Atopic dermatitis is a chronic, recurrent, inflammatory skin disease. Intense itching causes patients to constantly scratch their skin, causing epidermal dysfunction, and damaged skin is highly susceptible to pathogenic microbial infection. Chronicity causes patients to suffer greatly, and has a serious impact on patients' physiology, psychology and social life. Although current drug treatments such as glucocorticoids, antihistamines and antibiotic therapy have certain effects, they are often accompanied by multiple systemic side effects, and continuous use of antibiotics can increase the generation of drug-resistant bacteria, which makes atopic dermatitis still a great challenge in clinical practice. Therefore, it is urgent to develop safer and more effective treatment strategies to combat atopic dermatitis.

[0003] Hydrogel is a highly water-swollen three-dimensional network material, which has strong moisturizing properties and can effectively relieve dry and itchy symptoms, and has great application prospects in AD treatment. In recent years, hydrogels based on active peptides have attracted extensive research by scientists due to their rich biological functions. Among them, antibacterial peptides have great advantages in combating bacterial infections due to their unique membrane disruption and bactericidal mechanism, which is not prone to cause bacterial resistance.

[0004] The development of atopic dermatitis is a complex process, and increased pathological oxidative stress plays a key role in the development of AD. Abnormal accumulation of ROS will trigger a cascade of oxidative damage, leading to cell apoptosis and necrosis. In addition, excessive generation of ROS can activate the NF-κB cascade signal, promoting the secretion of pro-inflammatory factors, thereby aggravating the AD pathological process. Therefore, preparing a hydrogel with inherent antibacterial activity and antioxidant properties through a rapid and simple method may be an effective strategy to combat atopic dermatitis. SUMMARY

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present application is to provide a polypeptide self-assembled hydrogel and a preparation method and application thereof.

[0006] The present application is implemented in the following way: a preparation method of a polypeptide self-assembled hydrogel, the method comprising the following steps:

[0007] (1) dissolving sodium ascorbate in 0.1M phosphate buffer to obtain a sodium ascorbate solution;

[0008] (2) dissolving antibacterial peptide Jelleine-1 in DMSO to prepare a polypeptide stock solution of 100mg / mL;

[0009] (3) adding the sodium ascorbate solution into the polypeptide stock solution, fully stirring and mixing, and standing to form a polypeptide self-assembled hydrogel.

[0010] Preferably, in step (1), the pH of the phosphate buffer ranges from 6.5 to 7.5.

[0011] Preferably, in step (1), the concentration of sodium ascorbate in the sodium ascorbate solution ranges from 10 to 30 mg / mL.

[0012] Preferably, in step (3), the concentration of the antimicrobial peptide Jelleine-1 in the polypeptide self-assembled hydrogel ranges from 6 to 10 mg / mL.

[0013] The application further discloses the polypeptide self-assembled hydrogel prepared by the method.

[0014] The application further discloses application of the polypeptide self-assembled hydrogel in preparation of a therapeutic drug for atopic dermatitis.

[0015] The application overcomes the defects of the prior art, and provides a polypeptide self-assembled hydrogel, a preparation method and application thereof.

[0016] In the application, the antimicrobial peptide Jelleine-1 is a natural antimicrobial peptide isolated from bee royal jelly, has broad-spectrum antibacterial activity, and has the following structural formula:

[0017]

[0018] In addition, ascorbic acid is a simple low-molecular-weight polyhydroxyl compound, is an essential vitamin for human body, and is a natural antioxidant, and has the following structural formula:

[0019]

[0020] Self-assembly refers to a process in which basic structural units spontaneously organize into an ordered structure under the driving of non-covalent interaction. The application finds that the antimicrobial peptide Jelleine-1 can self-assemble into a hydrogel in a sodium ascorbate phosphate solution through non-covalent interaction, and does not need to add any crosslinking agent.

[0021] In the process of atopic dermatitis, the secondary infection caused by microbial colonization and the oxidative stress-inflammation cascade amplification effect are important factors to aggravate its symptoms, and traditional drugs have systemic side effects, single action mechanism and other defects. Based on this, the local treatment strategy of "antibacterial-antioxidant dual functional synergy" is proposed, the natural source of antibacterial peptide Jelleine-1 and natural antioxidant sodium ascorbate are selected as the basic component unit, and the hydrogel is self-assembled under physiological conditions by using non-covalent interaction, which is easily injected into the skin lesion through simple injection, and the antibacterial peptide Jelleine-1 and VcNa are continuously released to simultaneously play the antibacterial and antioxidant functions, reduce the pathogenic microorganisms and inhibit the oxidative stress reaction, and then relieve the inflammatory reaction.

[0022] Since the hydrogel prepared by the application does not need any chemical crosslinking agent, the toxicity problem caused by the traditional crosslinking agent can be avoided. The design based on all-natural ingredients endows the hydrogel with excellent biocompatibility and degradability. The hydrogel not only regulates the oxidative stress-inflammation cascade reaction, but also reduces the colonization of pathogens, and improves the skin lesions of dermatitis from multiple aspects.

[0023] Compared with the defects and deficiencies of the prior art, the application has the following beneficial effects:

[0024] (1) The preparation method of the application is simple, the gelation speed is fast, and no crosslinking agent is needed, the required compounds are easy to obtain, and the production cost is low.

[0025] (2) The polypeptide self-assembled hydrogel has excellent antibacterial performance and antioxidant performance, and almost no cytotoxicity and hemolytic toxicity, and has good biocompatibility.

[0026] (3) The polypeptide self-assembled hydrogel can effectively inhibit bacterial infection in atopic dermatitis and reduce oxidative stress, thereby significantly relieving the symptoms of dermatitis. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is the microstructure of the polypeptide self-assembled hydrogel.

[0028] Figure 2 It is the hemolytic toxicity test result of the hydrogel; wherein, Figure 2 a is a representative picture of red blood cells after incubation with the hydrogel, Figure 2 b is the hemolysis rate statistics.

[0029] Figure 3 It is the cytotoxicity test result of the hydrogel; wherein, Figure 3 a is the cell survival rate of L929 cells and hydrogel leachate respectively incubated for 24h (left), 48h (middle) and 72h (right). Figure 3b is the live / dead staining image of L929 cells incubated with PBS and hydrogel leachate for 24h, 48h, 72h, respectively.

[0030] Figure 4 are the results of the antibacterial performance test of the hydrogel; wherein, Figure 4 a and Figure 4 c are the representative images of S. aureus and E. coli incubated with J-1-VcNa hydrogel at different time points; Figure 4 b and Figure 4 d are the colony quantification analysis of S. aureus and E. coli incubated with J-1-VcNa hydrogel at different time points, respectively; Figure 4 e is the representative picture of the inhibition zone; Figure 4 f is the inhibition zone diameter of J-1-VcNa hydrogel against S. aureus, methicillin-resistant S. aureus (MRSA), E. coli, and extended-spectrum beta-lactamase-producing enterobacteriaceae (ESBLs-E. coli).

[0031] Figure 5 are the results of the antioxidant performance test of the hydrogel; wherein, Figure 5 a is the scavenging activity of the hydrogel on DPPH free radicals; Figure 5 b is the scavenging activity of the hydrogel on ABTS free radicals.

[0032] Figure 6 are the results of the anti-inflammatory performance test of the hydrogel; wherein, Figure 6 a, 6b and 6c are the expression levels of inflammatory factors IL-1β, IL-6 and TNF-α in RAW264.7 cells after treatment with the hydrogel, respectively.

[0033] Figure 7 are the results of the therapeutic effect test of the hydrogel in atopic dermatitis, wherein, Figure 7 a is the representative photo of the dorsal skin of mice in each treatment group on day 1, day 7 and day 11; Figure 7 b is the H&E staining of the dorsal skin tissue of mice in different groups after 11 days of treatment; Figure 7 c is the DHE and DAPI staining of the dorsal skin tissue of mice in different treatment groups after 11 days of treatment; Figure 7 d is the dermatitis score of mice in different treatment groups within 11 days; Figure 7 e is the epidermis thickness of different treatment groups calculated from the H&E staining results; Figure 7 f is the MRSA load in the dorsal skin tissue of mice in different groups after 4 days of treatment. DETAILED DESCRIPTION

[0034] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and not to limit the present application.

[0035] Example 1

[0036] (1) Sodium ascorbate was dissolved in 0.1M, pH 7.0 phosphate buffer (PB, made of sodium dihydrogen phosphate and disodium hydrogen phosphate) to obtain a sodium ascorbate solution with a concentration of 20mg / mL;

[0037] (2) Antimicrobial peptide Jelleine-1 (purchased from Nanjing Jiepeibio Technology Co., Ltd.) was dissolved in DMSO to prepare a polypeptide stock solution with a concentration of 100mg / mL;

[0038] (3) 920μL of the sodium ascorbate solution was added to the polypeptide stock solution, fully stirred and mixed, and shaped by standing to obtain a polypeptide self-assembled hydrogel with a concentration of 8mg / mL of antimicrobial peptide Jelleine-1.

[0039] The microstructure of the polypeptide self-assembled hydrogel was observed by FE-SEM, as shown in FIG. 1, the formation of nanofiber bundles can be observed. Figure 1

[0040] Example 2

[0041] (1) Sodium ascorbate was dissolved in 0.1M, pH 6.5 phosphate buffer (PB, made of sodium dihydrogen phosphate and disodium hydrogen phosphate) to obtain a sodium ascorbate solution with a concentration of 10mg / mL;

[0042] (2) Antimicrobial peptide Jelleine-1 (purchased from Nanjing Jiepeibio Technology Co., Ltd.) was dissolved in DMSO to prepare a polypeptide stock solution with a concentration of 100mg / mL;

[0043] (3) 940μL of the sodium ascorbate solution was added to the polypeptide stock solution, fully stirred and mixed, and shaped by standing to obtain a polypeptide self-assembled hydrogel with a concentration of 6mg / mL of antimicrobial peptide Jelleine-1.

[0044] The microstructure of the polypeptide self-assembled hydrogel was observed by FE-SEM, and the formation of nanofiber bundles can be observed.

[0045] Example 3

[0046] ​(1) Dissolve sodium ascorbate in 0.1M, pH 7.5 phosphate buffer (PB, made of sodium dihydrogen phosphate and disodium hydrogen phosphate) to obtain a sodium ascorbate solution with a concentration of 30mg / mL;

[0047] (2) Dissolve antibacterial peptide Jelleine-1 (purchased from Nanjing Jiepeibio Technology Co., Ltd.) in DMSO to prepare a polypeptide stock solution with a concentration of 100mg / mL;

[0048] (3) Add 900μL of the sodium ascorbate solution to the polypeptide stock solution, mix thoroughly, and stand to form a polypeptide self-assembled hydrogel with a concentration of 10mg / mL of antibacterial peptide Jelleine-1.

[0049] FE-SEM was used to observe the microstructure of the polypeptide self-assembled hydrogel, and the formation of nanofiber bundles was observed.

[0050] Application Example

[0051] The polypeptide self-assembled hydrogel prepared in Example 1 was selected for the following implementation operation.

[0052] 1. Hemolytic toxicity test of hydrogel

[0053] Centrifuge rat blood at 1500rpm for 10 minutes to obtain red blood cells, and prepare an 8%(v / v) red blood cell suspension after washing twice with PBS. Mix 500μL of hydrogel with an equal volume of red blood cell suspension, use 0.1% Triton X-100 as a positive control and PBS as a negative control, centrifuge at 1500rpm for 10 minutes after incubation at 37℃ for 2 hours, and take 100μL of supernatant in a 96-well plate, and measure the absorbance at 540nm using an enzyme marker.

[0054]

[0055] Wherein, As is the absorbance value of the hydrogel, At is the absorbance value of Triton, and Ap is the absorbance value of PBS.

[0056] The experimental results are shown in Figure 2 After incubation of the hydrogel and red blood cells for 1h, almost no hemolysis occurred Figure 2 a), and the hemolysis rate was only 2.8% Figure 2 b), indicating that the hydrogel has good blood compatibility.

[0057] 2. Cytotoxicity test of hydrogel

[0058] MTT method and live / dead cell staining test were used to test the cytotoxicity of the hydrogel.

[0059] First, a certain amount of hydrogel was prepared and soaked in DMEM at 37°C for 24 h to obtain different concentrations of hydrogel extracts. L929 cells (8 x 10 4 / mL) were inoculated in a 96-well plate at 100 μL / well and incubated overnight in an incubator to allow the cells to adhere. Different concentrations of hydrogel extracts were incubated with L929 cells for 24, 48, and 72 h, with three parallel control wells, followed by the addition of 10 μL of MTT solution per well and continued incubation for 4 h. The supernatant was discarded, 150 μL of DMSO was added to each well to fully dissolve the formazan, and the absorbance at 570 nm was measured using a multifunctional enzyme label instrument (FlexStation 3, Molecular Devices, USA) to calculate the cell survival rate.

[0060] For live / dead staining, L929 cells (8 x 10 4 / mL) were first inoculated in a 96-well plate at 100 μL / well and incubated overnight in an incubator to allow the cells to adhere. Then, hydrogel extracts were added for continued culture for 24, 48, and 72 h. After incubation, the supernatant was discarded, and Calcein-AM (5 μM, 30 min) and PI (50 μM, 15 min) dyes were added for staining. Finally, the samples were observed under a fluorescence microscope and photographed.

[0061] The experimental results are shown in Figure 3 After L929 cells were treated with different concentrations of hydrogel extracts for 24, 48, and 72 h, they showed the same proliferation trend as the control group, with a cell survival rate of >80% ( Figure 3 a). The results of live / dead staining also showed that the hydrogel had almost no cytotoxicity and did not affect the basic morphology and proliferation of the cells ( Figure 3 b).

[0062] 3. Test of the antibacterial performance of the hydrogel

[0063] The antibacterial ability of the hydrogel was detected by measuring the number of colonies of S. aureus and E. coli after incubation with the hydrogel for a certain period of time.

[0064] First, 400 μL of hydrogel was placed in a 1.5 mL centrifuge tube, and then 400 μL of S. aureus or E. coli with a concentration of 1 x 10 5CFU / mL bacterial suspension was placed on top of the hydrogel and incubated on a shaker at 120 rpm and 37°C. After incubation for 0 h, 6 h, 12 h, and 24 h, the bacterial suspension was taken from the top of the gel, diluted appropriately, and evenly spread on solid culture medium. Finally, it was incubated in a constant temperature and humidity incubator (temperature: 37°C; humidity: 60%) for 18 h. Bacterial growth was observed, colonies were counted, and the bactericidal ability of the hydrogel was determined. The results were photographed and recorded. Simultaneously, the antibacterial activity of the hydrogel was determined using the inhibition zone method. First, 100 μL of 1×10⁻⁶ CFU / mL bacterial suspension was added to the hydrogel. 7 The bacterial suspension of CFU / mL was evenly spread on MH agar plates, and 6 mm holes were punched in the plates. Hydrogel was added into the holes, and the plates were incubated at 37°C for 18–24 h. The size of the inhibition zone was measured and photographed.

[0065] The results are as follows Figure 4 As shown, the hydrogel exhibits broad-spectrum antibacterial activity and rapid bactericidal ability, killing Staphylococcus aureus and Escherichia coli in 4 hours and 2 hours, respectively. Figure 4 a~ Figure 4 d), and all of them have certain antibacterial activity against drug-resistant bacteria. Figure 4 e~ Figure 4 f).

[0066] 4. Antioxidant performance test of hydrogel

[0067] (1) DPPH· free radical scavenging experiment

[0068] Add 100 μL of DPPH ethanol solution (1 mg / mL) to the hydrogel, then add 1 mL of anhydrous ethanol and react in the dark for 1 h. Use N-acetylcysteine ​​as a positive control, measure the absorbance at 517 nm, and calculate the clearance rate (%).

[0069] (2) ABTS+· free radical scavenging experiment

[0070] An equal volume of ABTS solution (7 mmol / L) and potassium persulfate (2.45 mmol / L) was mixed and reacted in the dark for 12 h, then diluted to an absorbance of 0.70 ± 0.02 at 734 nm. The hydrogel was then reacted with 1.5 mL of ABTS. + The solution was reacted in the dark for 20 minutes, with N-acetylcysteine ​​as a positive control. The absorbance at 734 nm was measured using an ELISA reader to calculate the ABTS clearance rate.

[0071] The results are as follows Figure 5 As shown, the hydrogel exhibited 80% scavenging activity against both DPPH· and ABTS+· free radicals, demonstrating excellent antioxidant capacity.

[0072] 5. Anti-inflammatory properties test of hydrogel

[0073] An inflammation model was constructed using RAW 264.7 mouse mononuclear macrophage leukemia cells. The specific steps are as follows: RAW 264.7 cells were cultured at a rate of 1×10⁻⁶ cells / cells. 6 Cells were seeded at a density of 1 cell per well and co-cultured with 30 μL of hydrogel extract. Simultaneously, 100 ng / mL LPS was added to induce a systemic inflammatory microenvironment, and stimulation was continued for 24 h. The control group received only an equal volume of culture medium. Cell suspensions were collected, RNA was extracted and reverse transcribed into cDNA, and the expression levels of inflammatory factors IL-1β, IL-6, and TNF-α were detected using qPCR.

[0074] GAPDH was used as the internal reference gene, and the primer sequences are as follows:

[0075] GADPH-F: 5'-TGTGTCCGTCGTGGATCTGA-3';

[0076] GADPH-R: 5'-TTGCTGTTGAAGTCGCAG GAG-3';

[0077] IL-1β-F: 5'-TGGTGTGTGACGTTCCCATT-3';

[0078] IL-1β-R: 5'-TGTCGTTGCTTGGT TCTCCT-3';

[0079] IL-6-F: 5'-CCACTTCACAAGTCGGAGGCTTA-3';

[0080] IL-6-R: 5'-TGCAAGTGCATCATCGTTGTTC-3';

[0081] TNF-α-F: 5'-ACTCCAGGCGGTGCCTATGT-3';

[0082] TNF-α-R: 5'-GTGAG GGTCTGGGCCATAGAA-3'.

[0083] Experimental results are as follows Figure 6 As shown, compared with the control group, the expression levels of IL-1β, IL-6, and TNF-α in cells were significantly increased after LPS treatment, indicating that the inflammation model was successfully established. After hydrogel treatment, the expression levels of the three were significantly reduced, indicating that hydrogel can effectively control the expression of inflammatory factors.

[0084] 6. Therapeutic efficacy test of hydrogel in atopic dermatitis

[0085] Kunming male mice weighing 20±2 g were selected, the back hair was removed, and dinitrochlorobenzene (DNCB) was dissolved in a mixed solvent of acetone and olive oil (acetone: olive oil = 1:3) to induce dermatitis on the back skin. In the first two weeks, the mice were smeared with 150 μL of 0.2% DNCB on the back every day for primary sensitization. From the third week, the mice were smeared with 150 μL of 1% DNCB on the back every day for seven consecutive days for secondary sensitization.

[0086] The mice successfully modeled were randomly divided into three groups, and were treated with normal saline (100 μL), hydrogel (100 μL), and mometasone furoate cream, respectively, each group of 12, and at the same time, randomly divided into MRSA-infected and non-infected groups, each group of 6. The MRSA-infected group was infected with MRSA on the back skin of the mice one day before treatment, and then the treatment was started. The treatment group was treated with J-1-VcNa hydrogel / mometasone furoate, and the non-treatment group was treated with normal saline, once a day for 3 consecutive days.

[0087] After the treatment, the skin tissue infected with MRSA was taken for grinding and plating, the skin bacterial load was counted, and the photographs were recorded. The non-infected MRSA group was treated for 11 consecutive days, and during the treatment, 1% DNCB was used for re-sensitization every 3 days. On the 1st, 7th, and 11th day of treatment, the four typical symptoms of dermatitis, erythema, scar / dryness, edema / abrasion, and erosion were scored according to the severity, each symptom was scored as 0-3 points (0: none, 1: mild, 2: moderate, 3: severe), after the treatment, all mice were sacrificed, the back skin of the mice was collected and fixed with 4% formaldehyde for paraffin section, then H&E staining and histological examination were performed. In order to measure the ROS in the cells of the dermatitis tissue, DHE staining was performed. Briefly, fresh specimens were taken from different groups for frozen section, incubated with DHE (5 mM) and DAPI at 37°C in the dark for 20 minutes. Fluorescence imaging was performed using CLSM. ImageJ software was used for semi-quantitative calculation of ROS intensity: DHE / DAPI ratio (% of positive area) = DHE fluorescent positive area / DAPI fluorescent positive area x 100.

[0088] The experimental results are shown in Figure 7 At the beginning of the treatment, the back skin of the mice in each group showed severe epidermal dryness, chapping, and erythema, and the average dermatitis score reached 9 points. After 11 days of treatment, compared with the non-treatment group, the skin pathological symptoms of the hydrogel group and the positive control group were significantly reduced, showing a healthy and rosy state Figure 7 a), the dermatitis score was reduced to 2.0±0.31 and 1.5±0.31 Figure 7 d), which was significantly lower than that of the non-treatment group (4.5±0.49). As Figure 7As shown in b and 7e, after 11 days of treatment, the epidermis thickness of untreated mice was 8 times of that of normal mice, while the hydrogel group and the positive control group recovered to thinner epidermis, only 1 / 3 of the untreated group. The ROS content in the skin of mice after treatment was detected by DHE staining, as shown in Figure 7 As shown in c, the dorsal skin of normal mice showed almost no red fluorescence after DHE staining, while the skin of untreated mice showed a large amount of red fluorescence, and the red fluorescence was significantly reduced after hydrogel treatment, while the red fluorescence intensity of the positive control drug mometasone furoate group did not show obvious reduction, which indicated that mometasone furoate did not have antioxidant capacity. We also evaluated the antibacterial effect of hydrogel in the treatment of AD, as shown in Figure 7 As shown in f, the colony load in the skin tissue of AD mice infected with MRSA was significantly reduced after 3 days of hydrogel treatment, reducing the risk of skin infection by pathogenic bacteria, while mometasone furoate did not show antibacterial effect.

[0089] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of a polypeptide self-assembled hydrogel in the preparation of a therapeutic drug for atopic dermatitis, wherein the polypeptide self-assembled hydrogel is prepared by the following steps: (1) dissolving sodium ascorbate in 0.1M phosphate buffer to obtain a sodium ascorbate solution; (2) dissolving antibacterial peptide Jelleine-1 in DMSO to prepare a polypeptide stock solution with a concentration of 100 mg / mL; (3) adding the sodium ascorbate solution to the polypeptide stock solution, thoroughly mixing, and standing to form a polypeptide self-assembled hydrogel.

2. Use according to claim 1, wherein In step (1), the pH of the phosphate buffer is in the range of 6.5-7.

5.

3. The use according to claim 1, wherein In step (1), the concentration of sodium ascorbate in the sodium ascorbate solution is 10-30 mg / mL.

4. The use according to claim 1, wherein In step (3), the concentration of antibacterial peptide Jelleine-1 in the polypeptide self-assembled hydrogel is 6-10 mg / mL.

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