Antioxidant and antibacterial temperature control hydrogel as well as preparation and application thereof

Abstract

The invention relates to the technical field of polymer composite materials, in particular to antioxidant and antibacterial temperature control hydrogel as well as preparation and application thereof. According to the temperature-control hydrogel disclosed by the invention, a Ce-MOF material is used as a carrier, and F127 is used as a temperature-control material; wherein active ingredients are loaded in pores of the Ce-MOF material, and the Ce-MOF loaded with the active ingredients is wrapped by the F127. The temperature control hydrogel provided by the invention has good oxidation resistance, antibacterial ability and biocompatibility, is subjected to gelation transformation at the human body temperature of 37 DEG C, and has good temperature control performance.

Description

Technical Field

[0001] The present invention relates to the technical field of polymer composite materials, and in particular to an antioxidant and antibacterial temperature-controlled hydrogel and its preparation and application. Background Art

[0002] Skin tissue is the largest organ of the human body and an irreplaceable barrier for maintaining the normal functions of the body, and also plays an important role in protecting the human body from external injuries. However, the modern people's long-term habit of staying up late will affect the metabolism of the skin and reduce the skin's self-repair ability. At the same time, excessive cleaning or using strong irritating cleaning products will also damage the skin barrier function and form sensitive skin. This brings continuous pain and burden to patients, and there is also a risk of further infection and deterioration. Therefore, it is imperative to explore a material that is easy to obtain, has good biocompatibility and antibacterial properties.

[0003] In recent years, flavonoids and polyphenolic compounds such as quercetin, catechin, dihydromyricetin or procyanidin have received extensive attention in the fields of health, nutrition, medicine, etc. due to their antioxidant, anti-inflammatory, antibacterial, anti-allergic and other properties, but their poor bioavailability has also become a difficult point.

[0004] Therefore, it is crucial to provide a technical solution that can solve the above technical problems. Summary of the Invention

[0005] In order to solve the above problems, the purpose of the present invention is to provide an antioxidant and antibacterial temperature-controlled hydrogel and its preparation and application. The temperature-controlled hydrogel provided by the present invention has good antioxidant properties, antibacterial ability and biocompatibility, and undergoes a gelation transition at the human body temperature of 37°C, and has good temperature control performance.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] The first purpose of the present invention is to provide an antioxidant and antibacterial temperature-controlled hydrogel, wherein the temperature-controlled hydrogel uses a Ce-MOF material as a carrier and F127 as a temperature control material;

[0008] Among them, the pores of the Ce-MOF material are loaded with active ingredients;

[0009] F127 wraps the Ce-MOF loaded with active ingredients.

[0010] In an embodiment of the present invention, the active ingredient is selected from one of flavonoid active ingredients, polysaccharide active ingredients or phenols, active ingredients, alkaloids, terpenoids, organic sulfides, saponins, carotenoids, phytosterols, phytoestrogens or phytic acid;

[0011] Preferably, the active ingredient is a flavonoid active ingredient;

[0012] More preferably, the active ingredient is dihydromyricetin.

[0013] In one embodiment of the present invention, the Ce-MOF material is prepared by the following method:

[0014] Mix the terephthalic acid solution and the ammonium cerium nitrate solution, and then place them in an oil bath for reaction. After the reaction is completed, post-treatment is carried out to obtain the Ce-MOF material.

[0015] In one embodiment of the present invention, the volume ratio of terephthalic acid to the ammonium cerium nitrate solution is 1-3:1;

[0016] During the reaction, the temperature is 80-99 °C and the time is 0.5-1.5 h;

[0017] Preferably, during the reaction, the temperature is 99 °C and the time is 1 h.

[0018] In one embodiment of the present invention, the terephthalic acid solution is a mixed solution of terephthalic acid and DMF;

[0019] The concentration of the terephthalic acid solution is 0.7 wt% - 2 wt%;

[0020] Preferably, the concentration of the terephthalic acid solution is 1.4 wt%;

[0021] The ammonium cerium nitrate solution is a mixed solution of ammonium cerium nitrate and deionized water;

[0022] The concentration of the ammonium cerium nitrate solution is 0.6 wt% - 2.95 wt%;

[0023] Preferably, the concentration of the ammonium cerium nitrate solution is 1.47 wt%.

[0024] In one embodiment of the present invention, the post-treatment is centrifugation and washing in sequence.

[0025] The second object of the present invention is to provide a preparation method of an antioxidant and antibacterial temperature-controlled hydrogel, comprising the following steps:

[0026] (S1) Add dihydromyricetin to the Ce-MOF material and mix well to obtain a hydrogel precursor;

[0027] (S2) Use the F127 solution to coat and stir the hydrogel precursor prepared in step (S1) to obtain an antioxidant and antibacterial temperature-controlled hydrogel: TCHY hydrogel.

[0028] In one embodiment of the present invention, in step (S1), the mass ratio of dihydromyricetin to the Ce-MOF material is 1:0.8 to 1.2; preferably, the mass ratio of dihydromyricetin to the Ce-MOF material is 1:1;

[0029] During the mixing process, the time is 20 to 28 h; preferably, the time is 24 h.

[0030] In one embodiment of the present invention, the concentration of dihydromyricetin is 300 to 700 μg / mL; preferably, the concentration of dihydromyricetin is 700 μg / mL.

[0031] In one embodiment of the present invention, in step (S2), the mass ratio of the F127 solution to the hydrogel precursor is 1000:5 to 9; preferably, the mass ratio of the F127 solution to the hydrogel precursor is 1000:7;

[0032] The coating stirring condition is an ice bath.

[0033] In one embodiment of the present invention, the concentration of the F127 solution is 18 wt% to 24 wt%; preferably, the concentration of the F127 solution is 20 wt%.

[0034] In one embodiment of the present invention, ultrasonic degassing treatment is performed after stirring and coating.

[0035] The third object of the present invention is to provide an application of an antioxidant and antibacterial temperature-controlled hydrogel in the preparation of functional products, or, in the medical field.

[0036] In one embodiment of the present invention, the functional product is a product having lubricating and / or moisturizing and / or thickening and / or repairing functions;

[0037] The antioxidant and antibacterial temperature-controlled hydrogel is used for drug delivery, as a wound dressing, and for lubricating medical devices.

[0038] In one embodiment of the present invention, the product is a product having both antioxidant and antibacterial functions.

[0039] The temperature-controlled hydrogel of the present invention can be further applied to the research of sensitive skin, and at the same time, its potential for promoting wound healing can also be explored.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The temperature-controlled hydrogel provided by the present invention can obtain a stable combination of two polymers with different properties and functions, thereby achieving property complementarity between components. Among them, the Ce-MOF material has porous characteristics, and there is little research on its drug-loading ability. It can improve the bioavailability of active ingredients and has a temperature control effect. It undergoes a gelation transition at 37°C and has strong competitiveness in the application of cosmetics. In addition, dihydromyricetin used in the present invention is a general term for a large class of flavonol compounds widely present in plants and has the functions of strong antioxidant and free radical scavenging. The present invention combines the two to provide an antioxidant and antibacterial temperature-controlled hydrogel and its preparation method and application. Description of the Drawings

[0042] Figure 1 It is a state diagram of different concentrations of F127 at 4°C or 37°C;

[0043] Figure 2 It is a state diagram of F127 and TCHY700 at 4°C or 37°C;

[0044] Figure 3 It is a DPPH scavenging ability diagram of different samples;

[0045] Figure 4 It is an ABTS scavenging ability diagram of different samples;

[0046] Figure 5 It is a total reducing power determination diagram of different samples;

[0047] Figure 6 It is a cytotoxicity effect diagram of different samples;

[0048] Figure 7 It is an antibacterial performance determination diagram of different samples;

[0049] Figure 8 It is an Escherichia coli inhibition rate determination diagram of different samples;

[0050] Figure 9 It is an antibacterial performance determination diagram of different samples;

[0051] Figure 10 It is a Staphylococcus aureus inhibition rate determination diagram of different samples. Detailed Embodiments

[0052] The present invention will be described in detail below with reference to the drawings and specific embodiments.

[0053] In the following embodiments, unless otherwise specified, the reagents used are all commercially available reagents, and the detection means and methods used are all conventional detection means and methods in the art.

[0054] Example 1

[0055] Dissolve F127 in deionized water and mix well to obtain F127 solutions with concentrations of 16 wt%, 18 wt%, 20 wt%, 22 wt%, and 24 wt% respectively; observe the states of F127 solutions with different concentrations at low temperatures and the gelation changes at 37 °C; the results are as Figure 1 shown. Through Figure 1 it can be found that at 4 °C, F127 solutions with different concentrations are all in a liquid state. When the temperature rises to 37 °C, F127 solutions with a concentration of 20 wt% or more are in a gel state. Subsequently, further exploration is carried out at this concentration (20 wt%).

[0056] Example 2

[0057] This example provides a method for preparing a hydrogel, which specifically includes the following steps:

[0058] (S1) Dissolve 0.708 g of terephthalic acid in 48 mL of DMF to obtain a terephthalic acid solution;

[0059] Dissolve 2.23 g of ammonium cerium nitrate in 16 mL of deionized water to obtain an ammonium cerium nitrate solution;

[0060] Mix the above-prepared terephthalic acid solution and ammonium cerium nitrate solution evenly and then stir to obtain a mixture;

[0061] Place the obtained mixture in an oil bath pot, react at 99 °C for 1 h, collect the product, and then perform centrifugation (8000 r / min, 10 min) and washing treatment (wash three times with deionized water) in sequence, and dry until the weight remains unchanged to obtain a Ce-MOF material (which can be abbreviated as Ce-MOF in the following figures);

[0062] (S2) Dissolve dihydromyricetin and the Ce-MOF material prepared in step (S1) in water and mix well to obtain a first hydrogel precursor (the final concentration of dihydromyricetin is 600 μg / mL, and the final concentration of the Ce-MOF material is 700 μg / mL);

[0063] Dissolve dihydromyricetin and the Ce-MOF material prepared in step (S1) (mass ratio 1:1) in water and mix well to obtain a second hydrogel precursor (the final concentration of dihydromyricetin is 700 μg / mL, and the final concentration of the Ce-MOF material is 700 μg / mL);

[0064] (S3) Add 1 g of F127 powder to 5 mL of the first hydrogel precursor and mix well to obtain a hydrogel: TCHY600;

[0065] Add 1 g of F127 powder to 5 mL of the second hydrogel precursor and mix well to obtain a hydrogel: TCHY700 (asFigure 2 As shown in the figure, marked as TCHY in the figure.

[0066] (1) Determination of antioxidant property:

[0067] 1) Determination of DPPH radical scavenging rate:

[0068] Weigh 2 mg of DPPH powder and dissolve it in 40 mL of ethanol solution with a volume concentration of 95%. Ultrasonic for 15 min to make it evenly mixed to obtain DPPH solution; take 2 mL of DPPH solution and measure its absorbance at 517 nm, and adjust the absorbance of DPPH solution to be between 1.2 and 1.3.

[0069] Disperse 700 μg of DMY (final concentration), 700 μg of Ce-MOF material, 1 g of F127, and 1 g of TCHY700 in 1 mL of ethanol solution (concentration 10% w / v) respectively to obtain DMY700 dispersion, Ce-MOF dispersion, F127 dispersion, and hydrogel dispersion; mix the DMY700 dispersion, Ce-MOF dispersion, F127 dispersion, and hydrogel dispersion with DPPH solution in equal volume to obtain mixtures. Incubate the mixtures in the dark for 0 h, 0.5 h, 1 h, 1.5 h, 2 h, and 2.5 h respectively, and measure the optical density value of the mixtures at 517 nm with an enzyme-labeled instrument; the DPPH radical scavenging rate is calculated by the following formula:

[0070]

[0071] In the formula: A1 is the absorbance of the blank (0 h), and A0 is the absorbance of the hydrogel sample (0.5 h, 1 h, 1.5 h, 2 h, 2.5 h).

[0072] The results are as Figure 3 shown, through Figure 3 It can be seen that: with the increase of time, dihydromyricetin in the hydrogel is slowly released, and the DPPH scavenging rate increases with the increase of the concentration of the released dihydromyricetin. The scavenging rate can reach up to 79.90% at 1 h, and the scavenging rate tends to be stable at subsequent times. The above results indicate that the hydrogel prepared in this example has certain antioxidant effects.

[0073] 2) Determination of ABTS radical scavenging rate:

[0074] Mix 5 mL of 7.4 mmol / L ABTS stock solution with 88 μL of 2.6 mmol / L K2S2O8 and let it stand for 14 hours to obtain ABTS solution; take 0.4 mL of ABTS solution and dilute the ABTS solution with PBS solution until the absorbance of the ABTS solution at 734 nm is 0.7 ± 0.02 (measured at room temperature) to obtain ABTS working solution.

[0075] Take 2 mL of ABTS working solution respectively, and mix it with 600 μg DMY, 700 μg DMY, 700 μg Ce-MOF material, 0.5 g F127, 0.5 g TCHY600, and 0.5 g TCHY700 respectively, and let it stand at room temperature in the dark for 10 min. At room temperature, measure the absorbance at a wavelength of 734 nm in parallel 3 times. The scavenging ability of each sample for ABTS free radicals (ABTS free radical scavenging rate) is calculated by the following formula:

[0076]

[0077] In the formula: A0 is the absorbance of adding only ABTS working solution without adding the sample; A1 is the absorbance of adding the sample and ABTS.

[0078] The results are as Figure 4 shown. It can be seen through Figure 4 that the drug loading of dihydromyricetin has little effect on the ABTS scavenging of the hydrogel, and the ABTS scavenging rate of Ce-MOF and F127 is relatively low. It can be known that the scavenging ability of the hydrogel for ABTS mainly comes from DMY, and can reach up to 99.26%. The above results show that the hydrogel prepared in this example has a certain antioxidant effect.

[0079] 3) Determination of ferric ion reducing antioxidant power:

[0080] Take 700 μg DMY, 700 μg Ce-MOF, 0.2 g F127, and 0.2 g TCHY700 respectively, add 0.5 mL of phosphate buffer solution (pH = 7.2) and 0.5 mL of 1 wt% potassium ferricyanide solution respectively. After incubating in a water bath at 50 °C for 20 min, terminate the reaction in an ice bath. Add 0.5 mL of 10% (w / v) trichloroacetic acid solution to each portion, and centrifuge at 3000 r·min -1 for 10 min. Take the supernatant, divide it into two portions, a and b. Add 1.7 mL of distilled water and 0.17 mL of 0.1% (w / v) ferric chloride solution to portion b, let it stand for 10 min, and measure the absorbance of b at a wavelength of 70 nm as (A1). Portion a of the sample is the control group, and its absorbance is (A2);

[0081] In the blank group (A0), 0.2 g of the sample is replaced with 0.2 g of deionized water, and the other conditions are the same as those of portion a of the sample. Each group of experiments is parallel 3 times, and the experimental results are averaged. The formula for calculating the total reducing power is as follows. The formula for the moisture retention rate is as follows:

[0082] A0 = (A1 - A2 - A3) × 100%

[0083] Where: A1 is the absorbance of the sample; A2 is the absorbance of the control; A3 is the absorbance of the blank group.

[0084] The results are as Figure 5 shown. It can be seen from Figure 5 that the highest clearance rate of TCHY700 (labeled as TCHY in the figure) can reach 59.35%. The above results indicate that the hydrogel prepared in this example has a certain antioxidant effect.

[0085] (2) Cytotoxicity assay:

[0086] The cytocompatibility test was used to evaluate cytotoxicity by the CCK-8 assay. 20 wt% F127 and TCHY700 were sterilized under ultraviolet light for 30 minutes respectively, and then 1 g of sterile F127 and 1 g of sterile TCHY700 were immersed in 1 mL of DMEM medium to obtain the initial leaching solution (1000 mg / mL), and they were leached for 24 h.

[0087] HACAT cells were seeded into 96-well plates at an initial density of 20,000 cells / well (cultured with DMEM medium, where DMEM was supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin); then the HACAT cells were placed in an incubator at 37 °C and 5% CO2 for 24 hours.

[0088] The initial leaching solution (1000 mg / mL) was diluted to 500 μg / mL, 1000 μg / mL, 1500 μg / mL, 2000 μg / mL, and the diluted solutions with different concentrations were added to the 96-well plates and incubated for 24 hours.

[0089] The wells without any sample leaching solution were set as the control group.

[0090] After removing the supernatant and cell washing steps, CCK-8 and DMEM were mixed at a volume ratio of 1:10 and incubated at 37 °C for 15 min. The absorbance was measured at 450 nm with an enzyme-linked immunosorbent assay reader. The relative cell viability was calculated using the following formula:

[0091]

[0092] Where A T is the absorbance of the well containing the hydrogel sample, A B is the absorbance of the blank (only CCK8 in the well of the 96-well plate), and A C is the absorbance of the control. The average value of three measurements was calculated for each group of samples.

[0093] The results are as Figure 6 shown. Through Figure 6It can be seen that the cell viability of the temperature control materials F127 and TCHY700 (labeled as TCHY in the figure) is 96.71% at 2000 μg / mL, and there is basically no cytotoxicity, indicating good biocompatibility.

[0094] (3) Antibacterial performance determination

[0095] The antibacterial performance of the hydrogel was quantitatively evaluated by the shake flask method. Weigh 700 μg of DMY, 700 μg of Ce-MOF, 1 g of F127, and 1 g of TCHY700 (as samples) respectively, and sterilize them with ultraviolet light. Two kinds of bacteria (Escherichia coli (labeled as E.coli in the figure) and Staphylococcus aureus) were cultured in nutrient broth for 24 h, with a shaking speed of 130 revolutions per minute and a temperature of 37 °C. Dilute the bacterial growth with sterile PBS solution to make the test bacterial density range from 1×10 8 ~5×10 8 CFU / mL. Subsequently, mix the above samples (700 μg of DMY, 700 μg of Ce-MOF, 1 g of F127, 1 g of TCHY700) with 1 mL of bacterial suspension respectively, and culture them in an oscillator at a speed of 150 r / min at 24 ± 1 °C for 18 - 24 hours. The control group was cultured under the same conditions (without adding samples). After that, take out the cultured mixed solution and perform gradient dilution with PBS (10 1 、10 2 、10 3 、10 4 、10 5 and 10 6 times). Drop 20 μL of each gradient dilution solution into a sterile petri dish containing solidified agar medium, and incubate it at 37 °C for 24 h, and count the colony forming units (CFU). Each group was repeated three times, and the antibacterial performance test of the sample was calculated according to the following equation:

[0096]

[0097] Among them, A b is the number of surviving bacteria in the control group, and A S is the number of surviving bacteria in the sample group.

[0098] The results are as Figures 7 - 10 shown, through Figures 7 - 10It can be seen that the three components in TCHY700 (all marked as TCHY in the figure) all have a certain inhibitory effect on Escherichia coli. Due to insufficient time, DMY was not completely released, so the antibacterial effect of the finished hydrogel TCHY700 is relatively poor compared to DMY, but its highest inhibitory rate against Escherichia coli can reach 63.24%. Staphylococcus aureus, as a representative of Gram-positive bacteria, has stronger resistance compared to Gram-negative bacteria. The highest inhibitory rate of the finished hydrogel TCHY700 against Staphylococcus aureus can reach 32.23%. The above results indicate that the hydrogel prepared in the above examples has certain antibacterial properties.

[0099] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those familiar with the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention based on the interpretation of the present invention should be within the protection scope of the present invention.

Claims

1. An antioxidant and antibacterial temperature-controlled hydrogel, characterized in that, The temperature-controlled hydrogel uses Ce-MOF material as a carrier and F127 as a temperature-controlled material; Among them, the pores of the Ce-MOF material are loaded with active ingredients; F127 wraps the Ce-MOF loaded with active ingredients.

2. The thermosensitive hydrogel with antioxidant and antibacterial properties according to claim 1, wherein The active ingredient is selected from one of flavonoid active ingredients, polysaccharide active ingredients, polyphenol active ingredients, alkaloids, terpenoids, organic sulfides, saponins, carotenoids, phytosterols, phytoestrogens or phytic acid components.

3. The thermosensitive hydrogel with antioxidant and antibacterial properties according to claim 1, characterized in that, The active ingredient is dihydromyricetin.

4. The temperature-controlled hydrogel with antioxidant and antibacterial properties according to claim 1, characterized in that, The Ce-MOF material is prepared by the following method: Mix the terephthalic acid solution and the ammonium cerium nitrate solution, place them in an oil bath and react. After the reaction, post-treatment is carried out to obtain the Ce-MOF material.

5. An antioxidant and antibacterial temperature-controlled hydrogel according to claim 4, characterized in that, The volume ratio of terephthalic acid to ammonium cerium nitrate solution is 1-3:1; During the reaction, the temperature is 80-99 °C and the time is 0.5-1.5 h.

6. An antioxidant and antibacterial temperature-controlled hydrogel according to claim 4, characterized in that, The post-treatment is to carry out centrifugation and washing treatment in sequence.

7. A method for preparing the antioxidant and antibacterial temperature-controlled hydrogel according to any one of claims 1 to 6, characterized in that, It includes the following steps: (S1) Add dihydromyricetin to the Ce-MOF material and mix well to obtain a hydrogel precursor; (S2) Use the F127 solution to perform coating and stirring treatment on the hydrogel precursor prepared in step (S1) to obtain an antioxidant and antibacterial temperature-controlled hydrogel: TCHY hydrogel.

8. The preparation method of an antioxidant and antibacterial temperature-controlled hydrogel according to claim 1, characterized in that, In step (S1), the mass ratio of dihydromyricetin to the Ce-MOF material is 1:0.8-1.2; During the mixing process, the time is 20-28 h.

9. The preparation method of an antioxidant and antibacterial temperature-controlled hydrogel according to claim 1, characterized in that, In step (S2), the mass ratio of the F127 solution to the hydrogel precursor is 1000:5-9; The coating and stirring condition is an ice bath.

10. Use of an antioxidant and antibacterial temperature-controlled hydrogel as described in any one of claims 1-6 in the preparation of functional products, or, use in the medical field.

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