CN116617155A - Hyaluronic acid hydrogel carrying chlorella extract, preparation method and application thereof - Google Patents
Hyaluronic acid hydrogel carrying chlorella extract, preparation method and application thereof Download PDFInfo
- Publication number
- CN116617155A CN116617155A CN202310236838.XA CN202310236838A CN116617155A CN 116617155 A CN116617155 A CN 116617155A CN 202310236838 A CN202310236838 A CN 202310236838A CN 116617155 A CN116617155 A CN 116617155A
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- China
- Prior art keywords
- hyaluronic acid
- extract
- chlorella
- acid hydrogel
- hydrogel
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- Pending
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/02—Algae
- A61K36/05—Chlorophycota or chlorophyta (green algae), e.g. Chlorella
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/02—Local antiseptics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Biotechnology (AREA)
- Dermatology (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Microbiology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Inorganic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Mycology (AREA)
- Medical Informatics (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Botany (AREA)
- Alternative & Traditional Medicine (AREA)
- Medicinal Preparation (AREA)
Abstract
The application provides a hyaluronic acid hydrogel carrying chlorella extract, a preparation method and application thereof, belonging to the field of biomedical materials. The hyaluronic acid hydrogel carrying chlorella extract provided by the application comprises the following components: hyaluronic acid hydrogel, comprising hyaluronic acid and polyethylene glycol; and the chlorella pyrenoidosa extract comprises a chlorella pyrenoidosa extract obtained by water extraction and a chlorella pyrenoidosa extract obtained by polyethylene glycol extraction, wherein the chlorella pyrenoidosa extract is dispersed in hyaluronic acid hydrogel. The hyaluronic acid hydrogel has good biocompatibility and excellent antioxidation, anti-inflammatory and antibacterial properties, can remarkably accelerate the healing of diabetic wounds, can be applied to the preparation of drugs for treating the damage of the diabetic wounds, and can also be used for preparing antioxidation preparations, anti-inflammatory preparations or antibacterial preparations.
Description
Technical Field
The application relates to the field of biomedical materials, in particular to a hyaluronic acid hydrogel carrying chlorella extract, a preparation method and application thereof.
Background
Diabetic ulcers are one of the serious complications of diabetes mellitus, which is afflicting a large number of type I or type II diabetics worldwide, and may further lead to the risk of serious infection and amputation, one of the chronic refractory ulcers. Compared with common ulcer and other wound injuries, diabetic patients often cause skin nutritional disorder, skin damage and secondary infection due to microvascular and peripheral neuropathy, and the wound surface has poor repairing capability, so that ulcer which is more difficult to heal is caused, and therefore, the method for treating common wound healing is not suitable for treating diabetic ulcer, 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 diabetic patients without foot ulcers. Chronic wound care has therefore become a major healthcare problem. There are many drugs (such as collagenase ointment, gentamicin, concentrated povidone iodine solution) for treating diabetic wound healing in the current market, but the effect is not obvious.
The hydrogel is a reticular polymer swelling body containing a large amount of water and composed of water-insoluble polymers, has good water absorption, can be repeatedly hydrated when contacting with a wound surface, has the dual functions of providing water for the wound surface and absorbing seepage, and is a novel medical dressing commonly used clinically at present. Hyaluronic acid is widely used as macromolecular polysaccharide in organisms, is easy for large-scale production due to good biocompatibility, has the functions of regulating osmotic pressure, maintaining tissue morphology, lubricating, buffering stress and the like, and is applied to the biomedical field. At present, the gel prepared by taking hyaluronic acid as a matrix is available, but the hyaluronic acid is often required to be modified by a chemical cross-linking agent during construction to be gelled to obtain the gel with various mechanical properties and controllable degradation time, and the cross-linking agents all have chemical substances with certain toxic components, so that the chemical cross-linking agents have certain influence on treatment when applied to wound surface injury; or the hyaluronic acid is modified by a small molecular substance to form gel, but the hyaluronic acid needs to be dialyzed in deionized water for 5-7 days in the synthesis process to completely remove potential small molecular impurities, which limits the industrial production and application of the hyaluronic acid-based hydrogel. Therefore, development of a hyaluronic acid-based hydrogel which is simple to prepare, green to synthesize and has good mechanical properties and can treat diabetic ulcers has become very urgent.
Disclosure of Invention
The present application has been made to solve the above problems, and an object of the present application is to provide a hyaluronic acid hydrogel carrying chlorella extract, a preparation method and an application thereof.
The present application provides a hyaluronic acid hydrogel carrying chlorella extract, having such characteristics as comprising: hyaluronic acid hydrogel, comprising hyaluronic acid and polyethylene glycol; and the chlorella pyrenoidosa extract comprises a chlorella pyrenoidosa extract obtained by water extraction and a chlorella pyrenoidosa extract obtained by polyethylene glycol extraction, wherein the chlorella pyrenoidosa extract is dispersed in hyaluronic acid hydrogel.
The application also provides a preparation method of the hyaluronic acid hydrogel carrying chlorella extract, which has the characteristics that the preparation method comprises the following steps: step S1, respectively placing chlorella pyrenoidosa in an extracting agent A and an extracting agent B for extraction, and taking supernatant after the extraction reaction is finished to respectively obtain an extracting solution A and an extracting solution B; and S2, dissolving hyaluronic acid in the extracting solution A, then adding the extracting solution B, and obtaining the hyaluronic acid hydrogel of the chlorella pyrenoidosa extract after the crosslinking reaction is finished, wherein the extracting agent A is a solvent capable of dissolving the hyaluronic acid solution, and the extracting agent B is polyethylene glycol.
The preparation method of the hyaluronic acid hydrogel carrying chlorella extract provided by the application can also have the following characteristics: in step S1, the extractant a is PBS buffer or water.
The preparation method of the hyaluronic acid hydrogel carrying chlorella extract provided by the application can also have the following characteristics: wherein in the step S1, the mass volume ratio of the chlorella pyrenoidosa to the extractant A is (1-1.5) g to 10mL,
the mass volume ratio of the chlorella pyrenoidosa to the extractant B is (1-1.5) g/25 mL.
The preparation method of the hyaluronic acid hydrogel carrying chlorella extract provided by the application can also have the following characteristics: wherein, in the step S1, the extraction temperature is 60-100 ℃.
The preparation method of the hyaluronic acid hydrogel carrying chlorella extract provided by the application can also have the following characteristics: in the step S2, the mass ratio of the hyaluronic acid to the extracting solution B is 1 (3-7), and the volume ratio of the extracting solution A to the extracting solution B is 1 (2-4).
The application also provides application of the hyaluronic acid hydrogel carrying chlorella extract in wound injury of diabetes.
In the application of the chlorella extract-carrying hyaluronic acid hydrogel in the wound injury of diabetes, the chlorella extract-carrying hyaluronic acid hydrogel can also have the following characteristics: wherein the wound injury of diabetes is diabetic ulcer.
The application also provides an application of the chlorella extract-loaded hyaluronic acid hydrogel in a medicament for treating diabetic wound injury.
The application also provides application of the chlorella extract-carrying hyaluronic acid hydrogel in preparation of an antioxidant preparation, an anti-inflammatory preparation or an antibacterial preparation.
Effects and effects of the application
The hyaluronic acid hydrogel carrying chlorella extract provided by the application has excellent anti-inflammatory, antioxidant and antibacterial properties, and can remarkably accelerate the healing of diabetic wounds.
According to the preparation method of the chlorella extract-carrying hyaluronic acid hydrogel, polyethylene glycol and an extracting agent A (a solvent capable of dissolving hyaluronic acid solution) are adopted for the first time under the condition of no external cross-linking agent, and are combined with hyaluronic acid, so that hydrogel for promoting repair of diabetic wound injury is prepared through multiple hydrogen bond cross-linking. Meanwhile, through preliminary experiments, the hyaluronic acid hydrogel of the chlorella pyrenoidosa and the hyaluronic acid hydrogel of the chlorella pyrenoidosa extract extracted by water are directly loaded without extraction operation, and the effects of antioxidation and inflammation inhibition are not ideal in repairing diabetic wound surface injury, and compared with a water extraction method, the polyethylene glycol adopted by the application can extract hydrophobic components in some chlorella pyrenoidosa, has more obvious effect on promoting wound surface injury, and has the double effects of promoting cross-linking.
In addition, the hyaluronic acid hydrogel carrying chlorella extract can improve biocompatibility in repairing diabetic wounds, reduce pro-inflammatory factors CD86, TNF-alpha and IL 1-beta, improve anti-inflammatory factors CD206, IL-10 and TGF-alpha, realize anti-inflammatory effects, can be applied to preparing drugs for treating diabetic wounds and can also be used for preparing antioxidant preparations, anti-inflammatory preparations or antibacterial preparations.
In addition, the gel provided by the application has the following advantages when being used for treating diabetic wounds: (1) The hydrogel dressing loaded with chlorella extract is environment-friendly and pollution-free, and has good biocompatibility; (2) The hydrogel dressing loaded with chlorella extract has strong bioadhesion effect and good bioadhesion effect on diabetic wounds; (3) The hydrogel dressing loaded with chlorella extract has good antioxidant, anti-inflammatory and antibacterial effects, and has good therapeutic effects on the generation of diabetes wound surface ROS and the generated inflammation and bacterial infection; (4) The hydrogel dressing loaded with the chlorella extract ensures the sterility of the diabetic wound surface and provides nutrition for the diabetic wound surface, the hydrogel dressing loaded with the chlorella extract is a dressing rich in nutrition, and the chlorella pyrenoidosa contains various nutrients, so that the repair of the diabetic wound surface can be promoted; (5) The hydrogel loaded with chlorella extract is convenient to use, is in gel form immediately after being covered on the surface of a wound, forms a physical protective layer, and is beneficial to the repair of the wound surface; (6) The hydrogel dressing loaded with chlorella extract is convenient to store and transport.
Drawings
Fig. 1 is a graph showing the results of gel formation detection of the dressing prepared in example 1 and comparative example 1.
Fig. 2 is an SEM image of the hydrogel dressing prepared in example 1.
FIG. 3 is a graph showing the tensile test results of the hydrogel dressing prepared in example 1.
Fig. 4 is a graph of compression test results of the hydrogel dressing prepared in example 1.
Fig. 5 is a graph of the biocompatibility test and anti-inflammatory and antioxidant effects of the hydrogel dressing. A) Different concentrations of CPHA treated the viability of NIH-3T3 fibroblasts. B) NIH-3T3 fibroblasts were treated with the same concentration of H in the absence or presence of CPHA 2 O 2 Cell viability after treatment. C) CPHA scavenges superoxide radicals through SOD mimic activity. D) Effect of CPHA on LPS-activated RAW264.7 cell pro-inflammatory (CD 86) gene expression. E) CP (control program)Effect of HA on LPS-activated RAW264.7 cell pro-inflammatory (IL-1 β) gene expression. F) Effect of CPHA on LPS-activated RAW264.7 cell pro-inflammatory (TNF- α) gene expression. G) Effect of CPHA on LPS-activated RAW264.7 cell anti-inflammatory (CD 206) gene expression. H) Effect of CPHA on LPS-activated RAW264.7 cell anti-inflammatory (IL-10) gene expression. I) Effect of CPHA on expression of anti-inflammatory (TGF- β) genes in LPS-activated RAW264.7 cells.
Figure 6 is an in vitro antimicrobial property of a hydrogel dressing. CPHA completely inhibited the growth of Staphylococcus aureus and Escherichia coli.
FIG. 7 is a graph of hydrogel dressing promoting wound healing in diabetic mice A) control group, photographs of the wound of PHA, CPGel, CPHA mice at different time periods; the method comprises the steps of carrying out a first treatment on the surface of the B) Wound closure area for each group.
Detailed Description
In order to make the technical means, creation characteristics, achievement purposes and effects of the application easy to understand, the following examples are specifically described with reference to the accompanying drawings for carrying chlorella extract hyaluronic acid hydrogel, preparation method and application thereof.
The application relates to a material as follows:
the fibroblasts NIH-3T3 and RAW264.7 macrophages of the application were purchased from Shanghai Enzymogen Biotechnology Inc. Hydrogen peroxide (H) 2 O 2 ) Purchased from Shanghai Kangshen Biotech Co., ltd;
the equipment enzyme label instrument used by the application is purchased from Molecular Devices company in the United states; ultramicro nucleic acid protein assay (nanodrop 2000) was purchased from Thermo corporation of the united states.
The study was approved by the ethical committee of the university of Paeonia suffruticosa, and all animal care, feeding and sacrifice procedures were performed in accordance with the national animal regulations of the subject animal administration (revised 2017).
All data results of the present application are expressed as mean ± standard deviation. Statistically significant measurements were performed using one-way ANOVA (one-way ANOVA), multiple comparisons were performed using LSD-t test with an indicative control, and if the data did not pass the normalization test, tamhane's T test was performed. P value <0.05 is a statistically significant criterion for variance.
Example 1 ]
The embodiment provides a hyaluronic acid hydrogel carrying chlorella extract and a preparation method thereof.
The preparation method of the hyaluronic acid hydrogel carrying chlorella extract comprises the following steps:
step S1, respectively weighing 1.25g of chlorella pyrenoidosa, respectively dissolving the chlorella pyrenoidosa in 10mL of PBS buffer solution and 25mL of polyethylene glycol 200, and reacting for 4 hours at 80 ℃; then, the mixture was centrifuged at 5000rpm for 5 minutes, and the supernatant was collected to obtain PBS extract and polyethylene glycol extract, respectively.
And S2, weighing hyaluronic acid, dissolving in PBS (phosphate buffer solution), adding the solution into polyethylene glycol extract, mixing at 60 ℃, and injecting the mixed solution into a mold to obtain hyaluronic acid hydrogel carrying chlorella extract, which is marked as CPHA. Wherein, the volume ratio of PBS extract to polyethylene glycol extract is 1:2, and the mass ratio of hyaluronic acid to polyethylene glycol extract is 1:3.
Example 2 ]
1. The following tests were carried out on the chlorella extract-carrying hyaluronic acid hydrogel (hereinafter referred to as hydrogel) prepared in example 1:
(1) Gel test: the hydrogel prepared in example 1 was placed at the bottom of a sample bottle, and the control was hyaluronic acid solution in comparative example 1, and the results are shown in fig. 1: the hyaluronic acid solution in comparative example 1 (left) naturally flows from the bottom to the bottle cap under the action of gravity, while the sample in example 1 (right) remains at the bottom, which shows that hyaluronic acid, polyethylene glycol and chlorella pyrenoidosa extract can self-assemble into hydrogel without an external cross-linking agent, and has good adhesion performance.
(2) Hydrogel SEM test: the hydrogel prepared in example 1 was sufficiently washed with water and lyophilized in a freeze dryer, and after surface metal spraying, the structural morphology was observed by SEM, and the result is shown in fig. 2: hydrogels exhibit a typical cellular structure.
(3) The tensile test and the pressure test are measured by using a universal tensile machine, wherein the hydrogel is dumbbell-shaped and has a narrow width of 4mm during the tensile testThe narrow length is 20mm, the width is 12mm, the total length is 70mm, and the speed is set to be 50mm & min -1 The method comprises the steps of carrying out a first treatment on the surface of the Whereas the hydrogel sample used for the compression test had a diameter of 10 mm and a height of 20 mm. The results are shown in fig. 3 and 4: the tensile strength of the hydrogel prepared in example 1 reached 0.485MPa and the compressive strength reached 15.07MPa.
2. Biological experiments
The materials involved in the following experiments:
A. PHA group: dissolving hyaluronic acid in PBS buffer solution, adding polyethylene glycol 200, mixing at 60deg.C, and injecting into a mold to obtain hydrogel dressing, denoted PHA. Wherein the volume ratio of the PBS buffer solution to the polyethylene glycol 200 is 1:2, and the mass ratio of the hyaluronic acid to the polyethylene glycol is 1:3.
B. CPGel group: 1.25g of Chlorella pyrenoidosa is weighed and dissolved in 10mL of PBS buffer solution and 25mL of polyethylene glycol 200 respectively, and reacted for 4 hours at 80 ℃; then, the mixture was centrifuged at 5000rpm for 5 minutes, and the supernatant was collected to obtain PBS extract and polyethylene glycol extract, respectively. Weighing gelatin, dissolving in PBS extract, adding into polyethylene glycol extract, mixing at 60deg.C, and injecting the mixed solution into a mold to obtain Chlorella hydrogel, which is denoted CPGel. Wherein, the volume ratio of PBS extract to polyethylene glycol extract is 1:2, and the mass ratio of gelatin to polyethylene glycol extract is 1:3.
C. HA group: pure hyaluronic acid gel.
D. CPHA group: the hydrogel prepared in example 1.
(1) Biocompatibility of hydrogels
MTT：
Mouse fibroblasts (NIH-3T 3) were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin and at 37C CO 2 Incubation was performed in an incubator for 24 hours. To determine the in vitro cytotoxicity of CPHA, NIH-3T3 cells were seeded into 96-well tissue culture plates (5000 cells/well). After cell attachment, PHA, CPGel, CPHA hydrogel was added to the cells. After 24h of incubation, the cells were washed with fresh medium containing MTT reagent at 37 ℃ and incubated for 4h. Finally, the dissolution in DMSO was determined with a microplate reader at a wavelength of 490nmAbsorbance of the resolved formazan crystals. Cell viability was then calculated from the absorbance and the calculation is shown in figure 5A.
Fig. 5B is a graph showing the results of the MTT assay for cell viability, with no significant difference between the cell viability of the different treatments and the different concentrations compared to the control. Therefore, the hydrogel has good biocompatibility and no cytotoxicity.
(2) Cytoprotective effect
To verify the cytoprotective effect of CPHA, NIH-3T3 cells were incubated with hydrogen peroxide to mimic the ROS environment. NIH-3T3 cells were grown in 96-well plates and treated with PHA, CPGel, CPHA hydrogel, respectively. After 24h incubation, the cells were washed and cell viability was determined by MTT assay.
As shown in fig. 5B, with H 2 O 2 The increase in concentration significantly reduces cell viability. The addition of PHA, CPGel, CPHA hydrogel improves cell viability.
(3) Peroxide scavenging activity of CPHA
The ROS scavenging activity of CPHA hydrogels was analyzed using SOD assay kits according to the manufacturer's instructions. With or without the addition of hydrogel or Chlorella extract (200 μg mL) -1 ). In this experiment, superoxide anions generated by the xanthine/xanthine oxidase reaction system reacted with the WST-1 reagent to produce yellow Fu Ma Zan product, which was measured by absorbance at 450 nm. The absorbance change is proportional to the superoxide concentration. The superoxide scavenging activity was calculated as follows (formula (3)):
superoxide scavenging activity (%) = (control-sample)/control×100% (3)
As shown in FIG. 5C, with the addition of PHA, CPGel and CPHA hydrogels, the superoxide scavenging activity gradually increased in sequence, indicating that the hydrogels of the present application can enhance peroxide scavenging.
The effect of hydrogels on the expression of pro-inflammatory genes (CD 86, IL-1. Beta. And TNF-. Alpha.) and anti-inflammatory genes (CD 206, IL-10 and TGF-. Beta.) in LPS-activated RAW264.7 cells is shown in FIGS. 5D-I. As shown in fig. 5D-F, the expression of pro-inflammatory cytokines (CD 86, IL-1 β and TNF- α) was significantly increased in macrophages after LPS stimulation, however, the expression of these pro-inflammatory cytokines was reduced after LPS-treated RAW264.7 cells were incubated with PHA hydrogel, CPGel and CPHA hydrogel, especially in the CPHA hydrogel group, where the pro-inflammatory cytokines were significantly lower than in the CPGel hydrogel group, indicating that the inhibition of pro-inflammatory cytokine expression by CPHA hydrogel treatment was best. As shown in FIGS. 5G-I, the expression of the anti-inflammatory genes (CD 206, IL-10 and TGF-. Beta.) was decreased after LPS stimulation, and the expression of the anti-inflammatory genes was improved after each hydrogel group treatment, but only CPHA hydrogel treatment showed the highest expression and higher than that of the blank group. These results indicate that CPHA hydrogels can act as anti-inflammatory mediators.
(5) Antibacterial ability test of CPHA
OD value for measuring bacterial concentration
The concentration of bacteria was measured using an enzyme-labeled instrument and formulated to 10 7 CFU, add 1.5ml bacterial liquid, 1.5ml hydrogel into 5ml EP tube, put into constant temperature shaking table (220 rpm) of 37 ℃; incubation was performed for 24h, and the od values (3 replicates per group) were measured using a microplate reader (600) every 3 hours
As shown in fig. 6, the OD value was measured as PHA, CPGel, CPHA hydrogel group without bacteria, HA group without bacteria, and the difference between the HA group and the control group was not significant. Therefore, the hydrogel without PHA, CPGel, CPHA has no antibacterial function, and the PHA, CPGel, CPHA hydrogel has a remarkable antibacterial effect.
(6) In vivo experiments in diabetic mice
Male ICR mice of 8 weeks of age were intraperitoneally injected with 100mg/kg Streptozotocin (STZ) after the end of fasting, followed by 50mg/kg for 3 consecutive days to induce a model of type I diabetes. Mice with blood glucose levels exceeding 16.7mM are considered diabetic. A total of 20 diabetic mice were used. The mice were then randomly divided into 4 groups, control group, PHA, CPGel, CPHA hydrogel group, respectively. Before surgery, 20 diabetic mice were anesthetized with the respiratory anesthetic isoparaffin. The hair on the back of each mouse was shaved and then the back was rubbed with 70% ethanol. 2 full-skin defect wound surfaces with the diameter of 6mm are respectively arranged on the back area of the mouse along the two sides of the spine, the mouse is covered by wet gauze after being subjected to local injection, the surgical suture is fixed for four weeks, and the control group is injectedEqual amount of PBS.
After the wounds were treated separately in the manner described above, the wounds were covered with wet gauze and secured around with surgical sutures. Standard rule for wound size and image processing software
(Imagej) was measured and analyzed at fixed time intervals and the size of the wound was photographed. Representative images of diabetic wound healing in mice are shown in fig. 7A, and fig. 7B shows wound healing rates at various time points.
First, as can be seen from the observation of the control group in fig. 7, the mice have a certain self-healing capacity; secondly, the area of the wound surface of the mice in the PHA, CPGel, CPHA hydrogel group at different times is smaller than that of the mice in the control group, which shows that the PHA, CPGel, CPHA hydrogel dressing has obvious healing promotion effect on the diabetic wound surface of the mice. CPHA hydrogel dressing has the best promoting effect on the healing aspect of the diabetic wound surface.
Thus, the present application demonstrates that the effective anti-inflammatory effect of CPHA hydrogels in vivo can reduce macrophage burden in infected wound models and prevent the secretion of pro-inflammatory cytokines and increase the secretion of anti-inflammatory cytokines. Overall, the anti-inflammatory activity of CPHA hydrogels may help to prevent prolonged inflammatory phases and promote effective wound healing.
Comparative example 1 ]
Unlike example 1, comparative example 1 was not added with polyethylene glycol 200. Hyaluronic acid does not gel in PBS buffer, but remains in solution. That is to say,
the preparation method of the hyaluronic acid solution in comparative example 1 comprises the following steps:
step SA, weighing 1.25g of chlorella pyrenoidosa, dissolving the chlorella pyrenoidosa in 10mLPBS buffer solution, and reacting for 4 hours at 80 ℃; then, the mixture was centrifuged at 5000rpm for 5 minutes, and the supernatant was collected to obtain a PBS extract.
Step SB, weighing hyaluronic acid, dissolving in PBS extract, mixing at 60 ℃, and injecting the mixed solution into a mold to obtain hyaluronic acid solution, which is marked as CPHA. Wherein the mass ratio of hyaluronic acid to PBS extract was the same as that of example 1.
The above embodiments are preferred examples of the present application, and are not intended to limit the scope of the present application. Other variations and modifications of the present application will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present application.
Claims (10)
1. A hyaluronic acid hydrogel carrying chlorella extract, comprising:
hyaluronic acid hydrogel, comprising hyaluronic acid and polyethylene glycol; and
the chlorella pyrenoidosa extract comprises chlorella pyrenoidosa extract obtained by water extraction and chlorella pyrenoidosa extract obtained by polyethylene glycol extraction,
wherein the Chlorella pyrenoidosa extract is dispersed in the hyaluronic acid hydrogel.
2. A method for preparing the chlorella extract-carrying hyaluronic acid hydrogel according to claim 1, comprising the steps of:
step S1, respectively placing chlorella pyrenoidosa in an extracting agent A and an extracting agent B for extraction, and taking supernatant after the extraction reaction is finished to respectively obtain an extracting solution A and an extracting solution B;
step S2, dissolving hyaluronic acid in the extracting solution A, then adding the extracting solution B, obtaining hyaluronic acid hydrogel of the chlorella pyrenoidosa extract after the crosslinking reaction is finished,
wherein the extractant A is a solvent capable of dissolving hyaluronic acid solution, and the extractant B is polyethylene glycol.
3. The method for preparing a hyaluronic acid hydrogel carrying chlorella extract according to claim 2, wherein:
in step S1, the extractant a is PBS buffer or water.
4. The method for preparing a hyaluronic acid hydrogel carrying chlorella extract according to claim 2, wherein:
wherein in the step S1, the mass volume ratio of the chlorella pyrenoidosa to the extractant A is (1-1.5) g to 10mL,
the mass volume ratio of the chlorella pyrenoidosa to the extractant B is (1-1.5) g to 25mL.
5. The method for preparing a hyaluronic acid hydrogel carrying chlorella extract according to claim 2, wherein:
in the step S1, the extraction temperature is 60-100 ℃.
6. The method for preparing a hyaluronic acid hydrogel carrying chlorella extract according to claim 2, wherein:
wherein in the step S2, the mass ratio of the hyaluronic acid to the extracting solution B is 1 (3-7),
the volume ratio of the extracting solution A to the extracting solution B is 1 (2-4).
7. Use of the chlorella extract-carrying hyaluronic acid hydrogel according to claim 1 in wound lesions of diabetes.
8. The use according to claim 7, characterized in that:
wherein the diabetic wound injury is a diabetic ulcer.
9. The use of the chlorella extract-carrying hyaluronic acid hydrogel according to claim 1 in a medicament for treating diabetic wound injury.
10. Use of a chlorella extract-carrying hyaluronic acid hydrogel according to claim 1 for the preparation of an antioxidant, anti-inflammatory or antibacterial agent.
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