A chlorella extract-loaded hyaluronic acid hydrogel, a preparation method thereof, and application thereof

Abstract

The application provides a chlorella extract-loaded hyaluronic acid hydrogel, a preparation method and application thereof, and belongs to the field of biomedical materials.The chlorella extract-loaded hyaluronic acid hydrogel provided by the application comprises: a hyaluronic acid hydrogel comprising hyaluronic acid and polyethylene glycol; and a chlorella vulgaris extract comprising water-extracted chlorella vulgaris extract and polyethylene glycol-extracted chlorella vulgaris extract, wherein the chlorella vulgaris extract is dispersed in the hyaluronic acid hydrogel.The hyaluronic acid hydrogel has good biocompatibility and excellent antioxidant, anti-inflammatory and antibacterial properties, can significantly accelerate the healing of a diabetic wound surface, can be applied to the preparation of a diabetic wound injury treatment drug, and can also be used to prepare an antioxidant preparation, an anti-inflammatory preparation or an antibacterial preparation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomaterials, in particular to a chlorella extract-loaded hyaluronic acid hydrogel, a preparation method and application thereof. BACKGROUND

[0002] Diabetic ulcers are one of the serious complications of diabetes, and are plaguing a large number of type I or type II diabetes patients worldwide, and can further lead to a risk of serious infection and amputation, and are one of chronic refractory ulcers. Compared with ordinary ulcer and other wound injuries, diabetic patients often have skin nutrition disorders due to microvascular and peripheral neuropathy, resulting in skin damage, secondary infection, and poor wound repair ability, thereby causing more difficult-to-heal ulcers. Therefore, the methods for treating ordinary wound healing are not suitable for treating diabetic ulcers, such as surgical debridement and high-pressure wound irrigation. Hospital-based studies have shown that the mortality rate of patients with diabetic foot ulcers is about twice that of patients with diabetes without foot ulcers. Therefore, chronic wound care has become a major healthcare problem. There are many drugs on the current market for treating diabetic wound healing (such as collagenase ointment, gentamicin, and concentrated povidone-iodine solution), but the effects are not very significant.

[0003] A hydrogel is a reticular polymer sweller containing a large amount of water composed of non-water-soluble polymers, has good water absorption, and has a dual function of providing water to a wound and absorbing exudate when the hydrogel is in contact with the wound, and is a new type of medical dressing commonly used in clinical practice. Hyaluronic acid, as a macromolecular polysaccharide widely existing in living organisms, has good biocompatibility, is easy to produce on a large scale, and has functions such as regulating osmotic pressure, maintaining tissue morphology, lubrication, and buffering stress, and is applied in the biomedical field. At present, there are gels prepared by using hyaluronic acid as a matrix, but the construction often needs to modify the hyaluronic acid by using a chemical crosslinking agent to make it gelatinized, so as to obtain gels with various mechanical properties and controllable degradation time. However, these crosslinking agents all have certain toxic chemical substances, and the application of the crosslinking agents to wound injuries will have a certain impact on the treatment. Or, a small molecule substance is used to modify the hyaluronic acid to form a gel, but the small molecule substance needs to be dialyzed in deionized water for 5-7 days in the synthesis process to completely remove potential small molecule impurities, which limits the industrialized production and application of the hyaluronic acid-based hydrogel. Therefore, it is extremely urgent to develop a hyaluronic acid-based hydrogel that is simple to prepare, green to synthesize, has good mechanical properties, and can treat diabetic ulcers. SUMMARY

[0004] The present application is carried out to solve the above problems, and aims to provide a chlorella extract-loaded hyaluronic acid hydrogel, a preparation method and application thereof.

[0005] The present application provides a chlorella extract-loaded hyaluronic acid hydrogel, having the characteristics of comprising: a hyaluronic acid hydrogel comprising hyaluronic acid and polyethylene glycol; and a chlorella extract comprising a water-extracted chlorella extract and a polyethylene glycol-extracted chlorella extract, wherein the chlorella extract is dispersed in the hyaluronic acid hydrogel.

[0006] The present application also provides a method for preparing a chlorella extract-loaded hyaluronic acid hydrogel, having the characteristics of comprising the following steps: step S1, extracting Chlorella protothecoides in extractant A and extractant B respectively, and after the extraction reaction is completed, taking the supernatant to obtain extract A and extract B respectively; and step S2, dissolving hyaluronic acid in extract A, then adding extract B, and obtaining a chlorella extract-loaded hyaluronic acid hydrogel after the cross-linking reaction is completed, wherein the extractant A is a solvent capable of dissolving a hyaluronic acid solution, and the extractant B is polyethylene glycol.

[0007] In the method for preparing a chlorella extract-loaded hyaluronic acid hydrogel provided by the present application, the extractant A in step S1 can also be PBS buffer or water.

[0008] In the method for preparing a chlorella extract-loaded hyaluronic acid hydrogel provided by the present application, the mass-volume ratio of Chlorella protothecoides to extractant A in step S1 can also be (1-1.5) g: 10 mL,

[0009] The mass-volume ratio of Chlorella protothecoides to extractant B can also be (1-1.5) g: 25 mL.

[0010] In the method for preparing a chlorella extract-loaded hyaluronic acid hydrogel provided by the present application, the extraction temperature in step S1 can also be 60-100℃.

[0011] In the method for preparing a chlorella extract-loaded hyaluronic acid hydrogel provided by the present application, the mass ratio of hyaluronic acid to extract B in step S2 can also be 1: (3-7), and the volume ratio of extract A to extract B can also be 1: (2-4).

[0012] The present application also provides a chlorella extract-loaded hyaluronic acid hydrogel for use in diabetic wound injury.

[0013] In the use of a chlorella extract-loaded hyaluronic acid hydrogel in diabetic wound injury provided by the present application, the diabetic wound injury can also be diabetic ulcer.

[0014] The application further provides application of the chlorella extract-loaded hyaluronic acid hydrogel in a drug for treating a diabetic wound injury.

[0015] The application further provides application of the chlorella extract-loaded hyaluronic acid hydrogel in preparation of an antioxidant preparation, an anti-inflammatory preparation or an antibacterial preparation.

[0016] Effects of the application

[0017] The chlorella extract-loaded hyaluronic acid hydrogel provided by the application has excellent anti-inflammatory, antioxidant and antibacterial properties and can significantly accelerate healing of a diabetic wound.

[0018] In the preparation method of the chlorella extract-loaded hyaluronic acid hydrogel provided by the application, polyethylene glycol and an extractant A (a solvent capable of dissolving a hyaluronic acid solution) are used to extract protein core chlorella without an external crosslinking agent, and the protein core chlorella is combined with hyaluronic acid to prepare a hydrogel for promoting repair of a diabetic wound injury through multiple hydrogen bond crosslinking. Through a preliminary experiment, it is found that hyaluronic acid hydrogel directly loaded with protein core chlorella without extraction and hyaluronic acid hydrogel loaded with a chlorella extract obtained by water extraction have unsatisfactory antioxidant and anti-inflammatory effects in repair of a diabetic wound injury, and compared with the water extraction method, the polyethylene glycol used in the application can extract some hydrophobic components in chlorella, has a more significant effect on promoting wound injury, and has a dual effect of promoting crosslinking.

[0019] In addition, the chlorella extract-loaded hyaluronic acid hydrogel of the application can improve biocompatibility in repair of a diabetic wound, reduce pro-inflammatory factors CD86, TNF-alpha and IL1-beta, increase anti-inflammatory factors CD206, IL-10 and TGF-alpha, achieve an anti-inflammatory effect, can be applied to preparation of a drug for treating a diabetic wound injury, and can be used to prepare an antioxidant preparation, an anti-inflammatory preparation or an antibacterial preparation.

[0020] In addition, the gel of the present invention has the following advantages when used to treat diabetic wounds: (1) The hydrogel dressing loaded with Chlorella extract is green, environmentally friendly and pollution-free, and has good biocompatibility; (2) The hydrogel dressing loaded with Chlorella extract has strong bioadhesion and has a good bioadhesion effect on diabetic wounds; (3) The hydrogel dressing loaded with Chlorella extract has good antioxidant, anti-inflammatory and antibacterial effects, and has a good therapeutic effect on the generation of ROS and the resulting inflammation and bacterial infection in diabetic wounds; (4) The hydrogel dressing loaded with Chlorella extract ensures the sterility of diabetic wounds while providing them with nutrition. The hydrogel dressing loaded with Chlorella extract is a nutrient-rich dressing. Chlorella proteinensis contains a variety of nutrients that can promote the repair of diabetic wounds; (5) The hydrogel loaded with Chlorella extract is easy to use. After being covered on the wound surface, it immediately becomes gel-like and forms a physical protective layer, which is beneficial to the repair of the wound; (6) The hydrogel dressing loaded with Chlorella extract is convenient to store and transport. Attached Figure Description

[0021] Figure 1 The image shows the gelation test results of the dressings prepared in Example 1 and Comparative Example 1.

[0022] Figure 2 SEM image of the hydrogel dressing prepared in Example 1.

[0023] Figure 3 The figure shows the tensile test results of the hydrogel dressing prepared in Example 1.

[0024] Figure 4 The image shows the compression test results of the hydrogel dressing prepared in Example 1.

[0025] Figure 5Figures for biocompatibility test and anti-inflammatory and antioxidant effects of the hydrogel dressing. A) Viability of NIH-3 T3 fibroblasts treated with different concentrations of CPHA. B) Cell viability of NIH-3 T3 fibroblasts treated with the same concentration of H2O2 without or with CPHA. C) Superoxide radical scavenging by CPHA through SOD mimetic activity. D) Effect of CPHA on pro-inflammatory (CD86) gene expression of LPS-activated RAW264.7 cells. E) Effect of CPHA on pro-inflammatory (IL-1β) gene expression of LPS-activated RAW264.7 cells. F) Effect of CPHA on pro-inflammatory (TNF-α) gene expression of LPS-activated RAW264.7 cells. G) Effect of CPHA on anti-inflammatory (CD206) gene expression of LPS-activated RAW264.7 cells. H) Effect of CPHA on anti-inflammatory (IL-10) gene expression of LPS-activated RAW264.7 cells. I) Effect of CPHA on anti-inflammatory (TGF-β) gene expression of LPS-activated RAW264.7 cells.

[0026] Figure 6 Figures for in vitro antibacterial performance of the hydrogel dressing. CPHA completely inhibited the growth of Staphylococcus aureus and Escherichia coli.

[0027] Figure 7 Figures for wound healing promotion of the hydrogel dressing in diabetic mice. A) Wound photos of control group, PHA, CPGel, and CPHA mice at different time periods; B) Wound closure area of each group. DETAILED DESCRIPTION

[0028] In order to make the technical means, creative features, purposes and effects achieved by the present application easy to understand, the following embodiments will be specifically described in combination with the drawings.

[0029] The material involved in the present application is as follows:

[0030] The fibroblast NIH-3T3 and RAW 264.7 macrophage cells of the present application are purchased from Shanghai Zhiyan Biotechnology Co., Ltd. Hydrogen peroxide (H2O2) is purchased from Shanghai Mokang Biotechnology Co., Ltd.

[0031] The equipment used in the present application is purchased from the United States Molecular Devices Company; the nanodrop 2000 ultramicro nucleic acid protein detector is purchased from the United States Thermo Company.

[0032] This study was approved by the Animal Ethics Committee of Mudanjiang Medical College, and all animal care, feeding and killing procedures were performed in accordance with the national animal experiment regulations "Regulations on the Management of Laboratory Animals (Revised in 2017)".

[0033] All data results of the present application are expressed as mean ± standard deviation. Measurement of statistical significance adopts one-way ANOVA, and multiple comparisons adopt LSD-t test and indicative control. If the data does not pass the normality test, Tamhane's T2 test is adopted. The judgment standard of difference with statistical significance is P value <0.05.

[0034] <Embodiment 1>

[0035] The present embodiment provides a chlorella extract-loaded hyaluronic acid hydrogel and a preparation method thereof.

[0036] The preparation method of the chlorella extract-loaded hyaluronic acid hydrogel comprises the following steps:

[0037] Step S1, 1.25g of Chlorella vulgaris was weighed and dissolved in 10mL of PBS buffer and 25mL of polyethylene glycol 200 respectively, and reacted at 80℃ for 4h; then centrifuged at 5000 rpm for 5 minutes, and the supernatant was collected to obtain PBS extract and polyethylene glycol extract respectively.

[0038] Step S2, hyaluronic acid was weighed and dissolved in the PBS extract, and then added to the polyethylene glycol extract, and mixed at 60℃. The mixed solution was injected into a mold to obtain a chlorella extract-loaded hyaluronic acid hydrogel, denoted as CPHA. The volume ratio of the PBS extract to the polyethylene glycol extract is 1:2, and the mass ratio of the hyaluronic acid to the polyethylene glycol extract is 1:3.

[0039] <Embodiment 2>

[0040] I. The chlorella extract-loaded hyaluronic acid hydrogel (hereinafter referred to as hydrogel) prepared in Example 1 was subjected to the following detection:

[0041] (1) Gelation test: the hydrogel prepared in Example 1 was placed at the bottom of the sample bottle, and the control was the hyaluronic acid solution in Comparative Example 1, and the results are shown in Figure 1 As shown in Comparative Example 1 (left), the hyaluronic acid solution flowed naturally from the bottom to the bottle cap under the action of gravity, while the sample of Example 1 (right) still remained at the bottom, indicating that the hyaluronic acid, polyethylene glycol and chlorella extract could self-assemble into a hydrogel without an external crosslinking agent, and had good adhesion.

[0042] (2) Hydrogel SEM test: The hydrogel prepared in Example 1 was freeze-dried in a freeze dryer after being washed with water, and its structure was observed by SEM after being sprayed with gold. The results are shown in Figure 2: The hydrogel showed a typical honeycomb structure. Figure 2

[0043] (3) Tensile and compression tests were measured using a universal tensile testing machine. In the tensile test, the hydrogel was in the shape of a dumbbell, with a narrow width of 4 mm, a narrow length of 20 mm, a width of 12 mm, and a total length of 70 mm. The speed was set to 50 mm min-1. The hydrogel sample for compression test was 10 mm in diameter and 20 mm in height. The results are shown in Figures 3 and 4: The tensile strength of the hydrogel prepared in Example 1 reached 0.485 MPa, and the compression strength reached 15.07 MPa. Figure 3 4

[0044] II. Biological experiments

[0045] Materials involved in the following experiments:

[0046] A. PHA group: Hyaluronic acid was dissolved in PBS buffer, then polyethylene glycol 200 was added, mixed at 60°C, and injected into the mold to obtain a hydrogel dressing, which was recorded as PHA. The volume ratio of PBS buffer to polyethylene glycol 200 was 1:2, and the mass ratio of hyaluronic acid to polyethylene glycol was 1:3.

[0047] B. CPGel group: 1.25 g of Chlorella pyrenoidosa was dissolved in 10 mL of PBS buffer and 25 mL of polyethylene glycol 200, respectively, and reacted at 80°C for 4 h; then centrifuged at 5000 rpm for 5 minutes, and the supernatant was collected to obtain PBS extract and polyethylene glycol extract, respectively. Gelatin was weighed and dissolved in the PBS extract, then added to the polyethylene glycol extract, mixed at 60°C, and the mixed solution was injected into the mold to obtain a Chlorella pyrenoidosa hydrogel, which was recorded as CPGel. The volume ratio of PBS extract to polyethylene glycol extract was 1:2, and the mass ratio of gelatin to polyethylene glycol extract was 1:3.

[0048] C. HA group: pure hyaluronic acid gel.

[0049] D. CPHA group: the hydrogel prepared in Example 1.

[0050] (1) Biocompatibility of hydrogel

[0051] MTT:

[0052] ​​​Mouse fibroblasts (NIH-3T3) were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin and incubated at 37 °C in a CO2 incubator for 24 h. To determine the in vitro cytotoxicity of CPHA, NIH-3T3 cells were seeded in 96-well tissue culture plates (5000 cells / well). After cell attachment, PHA, CPGel, CPHA hydrogel were added to the cells. After 24 h incubation, the cells were washed with fresh culture medium containing MTT reagent and incubated at 37 °C for 4 h. Finally, the absorbance of the formazan crystals dissolved in DMSO was determined at 490 nm wavelength using a microplate reader. Cell survival rate was then calculated according to the absorbance Figure 5 A.

[0053] Figure 5 A is the MTT method for detecting cell survival rate results figure, the cell survival rate of different treatment and different concentration treatment and the control group were no significant difference. Thus, the hydrogel biocompatibility is good, no cytotoxicity.

[0054] (2) Cell protection

[0055] To verify the cell protection of CPHA, NIH-3T3 cells were incubated with hydrogen peroxide to simulate the ROS environment. NIH-3T3 cells were grown in 96-well plates and treated with PHA, CPGel, CPHA hydrogel, respectively. After 24 h incubation, the cells were washed and cell viability was determined by MTT method.

[0056] As shown in Figure 5 B, with the increase of H2O2 concentration, the cell viability decreased significantly. The addition of PHA, CPGel, CPHA hydrogel improved the cell viability.

[0057] (3) Peroxide scavenging activity of CPHA

[0058] The ROS scavenging activity of CPHA hydrogel was analyzed using a SOD assay kit according to the manufacturer's instructions. Hydrogel or Chlorella extract (200 μg mL-1) was added or not. In this experiment, the superoxide anion produced by the xanthine / xanthine oxidase reaction system reacted with WST-1 reagent to generate a yellow formazan product, which could be measured by absorbance at 450 nm. The change in absorbance was proportional to the concentration of superoxide. The formula for calculating superoxide scavenging activity is as follows (equation (3)):

[0059] Superoxide scavenging activity (%) = (control - sample) / control x 100% (3)

[0060] As shown in Figure 5As shown in Figure C, the superoxide scavenging activity gradually increases with the addition of PHA, CPGel, and CPHA hydrogels, indicating that the hydrogel of this invention can improve the scavenging of peroxides.

[0061] The effects of hydrogel on the expression of pro-inflammatory genes (CD86, IL-1β, and TNF-α) and anti-inflammatory genes (CD206, IL-10, and TGF-β) in LPS-activated RAW264.7 cells are shown in [the table below]. Figure 5 DI. As shown in Figures 5D-F, LPS stimulation significantly increased the expression of pro-inflammatory cytokines (CD86, IL-1β, and TNF-α) in macrophages. However, after incubation with PHA hydrogel, CPGel, and CPHA hydrogel in LPS-treated RAW264.7 cells, the expression of these pro-inflammatory cytokines decreased, especially in the CPHA hydrogel group, where the expression of pro-inflammatory cytokines was significantly lower than in the CPGel hydrogel group, indicating that CPHA hydrogel treatment had the best inhibitory effect on the expression of pro-inflammatory cytokines. Figure 5 As shown in the GI, LPS stimulation reduced the expression of anti-inflammatory genes (CD206, IL-10, and TGF-β). Although the expression of anti-inflammatory genes increased after treatment with each hydrogel group, only CPHA hydrogel treatment showed the highest expression of anti-inflammatory genes, which was higher than that of the blank control group. These results indicate that CPHA hydrogel can serve as an anti-inflammatory mediator.

[0062] (5) Antibacterial ability test of CPHA

[0063] OD value measures bacterial concentration

[0064] The bacterial concentration was detected using an ELISA reader. The bacterial solution was prepared to 10⁷ CFU. 1.5 ml of bacterial solution and 1.5 ml of hydrogel were added to a 5 ml EP tube and placed in a constant temperature shaker at 37°C (220 rpm). The tube was incubated for 24 hours. The od value was detected every 3 hours using an ELISA reader (600) (each group had 3 replicate samples).

[0065] like Figure 6 As shown in the figure, the OD value measurement results showed that there were no bacteria in the PHA, CPGel, and CPHA hydrogel groups, while bacteria were present in the HA group, and the difference between the groups and the control group was not significant. This indicates that the hydrogels without PHA, CPGel, and CPHA do not have antibacterial function, while the hydrogels with PHA, CPGel, and CPHA have a significant antibacterial effect.

[0066] (6) In vivo experiments in diabetic mice

[0067] 8-week-old male ICR mice were injected intraperitoneally with 100 mg / kg streptozotocin (STZ) at the end of fasting, and then 50 mg / kg continuously for 3 days to induce a type I diabetes model. Mice with blood glucose levels exceeding 16.7 mM were considered diabetic. A total of 20 diabetic mice were used. The mice were then randomly divided into 4 groups, namely the control group, the PHA group, the CPGel group, and the CPHA hydrogel group. Before the operation, 20 diabetic mice were anesthetized with the respiratory anesthetic isoflurane. The hair on the back of each mouse was shaved, and then the back was wiped with 70% ethanol. Two 6-mm-diameter full-skin defect wound surfaces were made on both sides of the spine in the back area of the mouse, and the mouse was locally injected and covered with a wet gauze, and the operation suture was fixed for four weeks, wherein the control group was injected with an equal amount of PBS.

[0068] After treating the wounds in the above manner, the wounds were covered with a wet gauze and fixed with surgical sutures around the perimeter. The size of the wounds was measured and analyzed at fixed time intervals using standard rulers and image processing software (Imagej), and the size of the wounds was photographed. Representative images of the healing of the diabetic wounds of the mice are shown in Figure 7 A, Figure 7 B is the wound healing rate at different time points.

[0069] First, it can be seen from the control group of Figure 7 that the mice have a certain self-healing ability; second, the area of the wounds of the mice in the PHA, CPGel, and CPHA hydrogel groups at different times is smaller than that of the control group, indicating that the PHA, CPGel, and CPHA hydrogel dressings have obvious effects on promoting the healing of diabetic wounds in mice. The CPHA hydrogel dressing has the best effect on promoting the healing of diabetic wounds.

[0070] Therefore, the present application demonstrates that the in vivo effective anti-inflammatory effect of the CPHA hydrogel can reduce the macrophage burden in the infected wound model and prevent the secretion of pro-inflammatory cytokines and increase the secretion of anti-inflammatory cytokines. In general, the anti-inflammatory activity of the CPHA hydrogel can help prevent prolonged inflammation and promote effective wound healing.

[0071] <Comparative Example 1>

[0072] Unlike Example 1, Comparative Example 1 does not add polyethylene glycol 200. Hyaluronic acid does not form a gel in PBS buffer and remains in a solution state. That is,

[0073] The preparation method of the hyaluronic acid solution in Comparative Example 1 includes the following steps:

[0074] Step SA, 1.25 g of Chlorella pyrenoidosa was weighed and dissolved in 10 mL of PBS buffer solution, and reacted at 80°C for 4 h; then centrifuged at 5000 rpm for 5 min, and the supernatant was collected to obtain a PBS extract.

[0075] Step SB, hyaluronic acid was weighed and dissolved in the PBS extract, and mixed at 60°C to obtain a hyaluronic acid solution, denoted as CPHA. The mass ratio of hyaluronic acid to PBS extract was the same as that in Example 1.

[0076] The above embodiments are preferred cases of the present application and are not intended to limit the protection scope of the present application. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. Use of a Chlorella extract-loaded hyaluronic acid hydrogel in the preparation of a medicament for diabetic wound injury, characterized in that: the preparation method of the Chlorella extract-loaded hyaluronic acid hydrogel comprises the following steps: Step S1, Chlorella protococcus is placed in extractant A and extractant B respectively for extraction, after the extraction reaction is completed, the supernatant is taken, and extract A and extract B are obtained respectively; Step S2, hyaluronic acid is dissolved in the extract A, then the extract B is added, and a Chlorella extract-loaded hyaluronic acid hydrogel is obtained after the crosslinking reaction is completed, Wherein, the extractant A is PBS buffer or water, and the extractant B is polyethylene glycol, In step S1, the mass-volume ratio of the Chlorella protococcus to the extractant A is (1-1.5) g: 10 mL, and the mass-volume ratio of the Chlorella protococcus to the extractant B is (1-1.5) g: 25 mL, and the extraction temperature is 60-100℃, In step S2, the mass ratio of the hyaluronic acid to the extract B is 1: (3-7), and the volume ratio of the extract A to the extract B is 1: (2-4).

2. The use of the Chlorella extract-loaded hyaluronic acid hydrogel according to claim 1 in the preparation of a medicament for diabetic wound injury, characterized in that: The diabetic wound injury is diabetic ulcer. wherein ​

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