Polylipoic acid-based zwitterionic hydrogel as well as preparation method and application thereof

Abstract

The invention discloses a polylipoic acid-based zwitter-ion hydrogel and a preparation method and application thereof, and relates to the technical field of hydrogel materials.The preparation method comprises the steps that sodium ethoxide and lipoic acid powder are dissolved in absolute ethyl alcohol respectively, and a sodium ethoxide solution and a lipoic acid solution are obtained; dropwise adding the sodium ethoxide solution into the lipoic acid solution to form uniform mixed slurry; centrifuging and drying the mixed slurry to obtain yellow sodium lipoate powder; the preparation method comprises the following steps: adding lipoic acid and sodium lipoate powder into deionized water to form a lipoic acid and sodium lipoate mixed dispersion liquid, and then adding zwitter-ion powder under continuous stirring for reaction to obtain polylipoic acid-based zwitter-ion hydrogel; according to the polylipoic acid-based zwitterionic hydrogel as well as the preparation method and the application thereof, the polylipoic acid-based zwitterionic hydrogel is endowed with anti-pollution performance through zwitterions, the structure of the polylipoic acid-based zwitterionic hydrogel is more stable due to the hydrogen-bond interaction of lipoic acid and sodium lipoate and is similar to a natural protein structure, and the melting temperature is suitable for in-vivo application.

Description

Technical Field

[0001] The invention relates to hydrogel material technology, and in particular to a polylipoic acid-based zwitterionic hydrogel and a preparation method and application thereof. Background Art

[0002] Polylipoic acid-based zwitterionic hydrogel is a new type of biomedical material with unique structure and excellent properties. It can be used in drug delivery carriers, biosensor dressings and medical dressings.

[0003] A Chinese invention patent with publication number CN118388801A discloses a self-aggregation-induced polymerized lipoic acid-based zwitterionic hydrogel and a preparation method thereof. The self-aggregation-induced polymerized lipoic acid-based zwitterionic hydrogel is prepared by activating lipoic acid into a lipoic acid-based intermediate containing an amide bond and a tertiary amino group, and then zwitterionizing the lipoic acid-based zwitterionic monomer by reacting β-propiolactone or 1,3-propane sultone to obtain a lipoic acid-based zwitterionic monomer, which is then prepared into solutions of different concentrations. The superhydrophilic effect of the zwitterions in the solution promotes the hydrophobic aggregation of dithiolane, resulting in an increase in its local concentration, thereby achieving concentration-induced autonomous ring-opening polymerization. Finally, lipoic acid-based hyaluronic acid is added to prepare the lipoic acid-based zwitterionic hydrogel by self-aggregation-induced polymerization.

[0004] The existing lipoic acid-based zwitterionic hydrogel modifies the carboxy betaine group of the zwitterionic group to the carboxyl group on the side chain of lipoic acid, and then synthesizes a polymerizable zwitterionic polymer by thermally initiated ring-opening polymerization (ROP) using the acylation reaction of lipoic acid with N,N-dimethylethylenediamine and the ring-opening reaction of β-propiolactone. This technology still has the disadvantages of complex operation and the introduction of too many substances, making it difficult to be widely used in clinical practice. Summary of the invention

[0005] The purpose of the present invention is to provide a polylipoic acid-based zwitterionic hydrogel and a preparation method and application thereof, so as to solve the problem that the prior art has the disadvantages of complicated operation and introduction of too many substances, making it difficult to be applied in clinical practice.

[0006] In order to achieve the above object, the present invention provides the following technical solution: a method for preparing a polylipoic acid-based zwitterionic hydrogel, comprising:

[0007] S1. Dissolving sodium ethoxide and thioctic acid powder in anhydrous ethanol respectively to obtain sodium ethoxide solution and thioctic acid solution;

[0008] S2, adding the sodium ethoxide solution dropwise to the lipoic acid solution, and stirring the mixed solution at room temperature to form a uniform mixed slurry;

[0009] S3, then centrifuging the mixed slurry and drying under vacuum to obtain yellow sodium thioctic acid powder, and storing the yellow sodium thioctic acid powder in a refrigerator;

[0010] S4, adding thioctic acid and sodium thioctic acid powder into deionized water to form a mixed dispersion of thioctic acid and sodium thioctic acid;

[0011] S5, heating and stirring the mixed dispersion of lipoic acid and sodium lipoate until it is molten, then adding zwitterion powder to react under continuous stirring, and naturally cooling to room temperature after the reaction is completed to obtain polylipoic acid-based zwitterion hydrogel.

[0012] Furthermore, the weight ratio of the sodium ethoxide and anhydrous ethanol in S1 is 1:4-5; the weight ratio of the lipoic acid and anhydrous ethanol in S1 is 1:0.5-2.

[0013] Furthermore, the stirring time of S2 is 10 to 15 hours.

[0014] Furthermore, the temperature of the refrigerator in S2 is -20°C.

[0015] Furthermore, the molar ratio of lipoic acid and sodium lipoate added to the deionized water in S4 is 2:1, and the mass of the deionized water in S4 is 2.5-3 times the total mass of lipoic acid and sodium lipoate.

[0016] Furthermore, in S5, the heating and stirring are performed until the mixture is in a molten state, and the heating temperature is 80°C.

[0017] Furthermore, the zwitterionic powder in S5 is betaine sulfonate methacrylate.

[0018] A polylipoic acid-based zwitterionic hydrogel is prepared by a polylipoic acid-based zwitterionic hydrogel preparation method.

[0019] The invention discloses an application of the polylipoic acid-based zwitterionic hydrogel prepared by the preparation method in the preparation of antifouling coatings, drug delivery carriers, biosensor dressings and medical dressings.

[0020] Application of polylipoic acid-based zwitterionic hydrogel in the preparation of antifouling coatings, drug delivery carriers, biosensor dressings and medical dressings.

[0021] Compared with the prior art, the present invention provides a polylipoic acid-based zwitterion hydrogel and a preparation method and application thereof. The super-hydrophilicity of zwitterions is utilized to improve the inherent hydrophobicity of the existing lipoic acid polymer itself, and the hydrophilicity of the polymer is significantly enhanced, thereby further increasing the biocompatibility of the polymer hydrogel. In addition, in an aqueous environment, a large number of water molecules attracted by zwitterions through electrostatic attraction form a hydration layer on the surface of the material, which weakens the interaction between pollutants and the material body, thereby giving the material anti-fouling ability. The polymer has the effect of resisting protein adsorption and bacterial adhesion, has a good resistance to the adhesion of Gram-positive and Gram-negative bacteria, and does not adhere to platelets on its surface, and has good blood compatibility. At the same time, the special structure of zwitterions will not interfere with the synergistic effect of peptide chains and side groups, can maintain the normal conformation of proteins, and can still effectively promote the functional repair of bone tissue at the defect under the condition of reducing the amount of bone morphogenetic protein-2 loading. In addition, the structure of zwitterion polymers is similar to that of natural proteins, and attracts mineralization precursor ions by imitating natural proteins, thereby inducing biomineralization.

[0022] By utilizing the characteristics of lipoic acid polymers that can degrade and release lipoic acid through reverse cyclization depolymerization triggered by dynamic disulfide bonds and terminal carboxyl reactions, polylipoic acid-based zwitterionic hydrogels have the excellent properties of both. By utilizing the multiple hydrogen bond interactions between lipoic acid and sodium lipoate, the polymer is stabilized and the melting temperature is suitable for use in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0024] Figure 1 A schematic diagram of the overall preparation method provided by an embodiment of the present invention;

[0025] Figure 2 A graph showing the water absorption rate of the polylipoic acid-based zwitterionic hydrogel provided in an embodiment of the present invention;

[0026] Figure 3 A graph showing the biocompatibility test results of the polylipoic acid-based zwitterionic hydrogel provided in an embodiment of the present invention;

[0027] Figure 4 This is a graph showing the antibacterial performance test results of the polylipoic acid-based zwitterionic hydrogel provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0029] Embodiment 1:

[0030] See also Figure 1 , a method for preparing a polylipoic acid-based zwitterionic hydrogel, comprising:

[0031] S1. Dissolving sodium ethoxide and lipoic acid powder in anhydrous ethanol respectively to obtain sodium ethoxide solution and lipoic acid solution; the weight ratio of sodium ethoxide to anhydrous ethanol is 1:4-5; the weight ratio of lipoic acid to anhydrous ethanol is 1:0.5-2;

[0032] S2. Add the sodium ethoxide solution dropwise to the lipoic acid solution, and stir the mixed solution at room temperature to form a uniform mixed slurry; the stirring time is 10 to 15 hours;

[0033] S3, then centrifuging the mixed slurry and drying it under vacuum to obtain yellow sodium lipoate powder, and storing the yellow sodium lipoate powder in a refrigerator; the temperature of the refrigerator is -20°C;

[0034] S4, adding thioctic acid and sodium thioctic acid powder to deionized water to form a mixed dispersion of thioctic acid and sodium thioctic acid; the molar ratio of thioctic acid and sodium thioctic acid added to the deionized water is 2:1, and the mass of the deionized water is 2.5-3 times the total mass of thioctic acid and sodium thioctic acid;

[0035] S5. Heat and stir the mixed dispersion of lipoic acid and sodium lipoate until it is molten, then add zwitterion powder under continuous stirring to react, and naturally cool to room temperature after the reaction to obtain a polylipoic acid / zwitterion composite hydrogel; heat and stir until it is molten, the heating temperature is 80° C.; the zwitterion powder is sulfobetaine methacrylate.

[0036] The specific implementation method is as follows: S1, dissolving sodium ethoxide and lipoic acid powder in anhydrous ethanol respectively, and obtaining sodium ethoxide solution and lipoic acid solution after they are completely dissolved, wherein the weight ratio of sodium ethoxide to anhydrous ethanol is 1:4-5; the weight ratio of lipoic acid to anhydrous ethanol is 1:0.5-2;

[0037] S2, adding the completely dissolved sodium ethoxide solution dropwise to the completely dissolved lipoic acid solution, and stirring the mixed solution at room temperature for 12 hours until a uniform slurry is formed;

[0038] S3. The slurry was then centrifuged and dried under vacuum to obtain yellow sodium lipoate (LANa) powder, and the yellow sodium lipoate powder was stored in a refrigerator at -20°C.

[0039] S4. Add lipoic acid (LA) and sodium lipoate powder into a vial containing deionized water at a molar ratio of 2:1 to form a mixed dispersion of lipoic acid and sodium lipoate (LA / LANa), wherein the mass fraction of deionized water in the mixed dispersion is 72.5%.

[0040] S5. The mixed dispersion of lipoic acid and sodium lipoate (LA / LANa) is heated and stirred at 80°C until it is molten, and then sulfobetaine methacrylate (SBMA) powder is added under continuous stirring, and the mixture is stirred and reacted at 80°C for 5 minutes. After the reaction is completed, the mixture is naturally cooled to room temperature to obtain a polylipoic acid-based zwitterionic hydrogel.

[0041] Embodiment 2:

[0042] This embodiment provides a technical solution based on the first embodiment: a polylipoic acid-based zwitterionic hydrogel, which is prepared by a method for preparing a polylipoic acid-based zwitterionic hydrogel.

[0043] The polylipoic acid-based zwitterion hydrogel utilizes the super-hydrophilicity of zwitterions to improve the inherent hydrophobicity of existing lipoic acid polymers, significantly enhancing the hydrophilicity of the polymer, thereby further increasing the biocompatibility of the polymer hydrogel; and in an aqueous environment, zwitterions form a hydration layer on the surface of the material through electrostatic attraction of a large number of water molecules, weakening the interaction between pollutants and the material itself, thereby giving the material anti-fouling ability, and can be used in the preparation of anti-fouling coatings.

[0044] In addition, the polylipoic acid-based zwitterionic hydrogel has the effects of resisting protein adsorption and bacterial adhesion, and has a good resistance to the adhesion of Gram-positive and Gram-negative bacteria. Its surface does not adhere to platelets and has good blood compatibility. At the same time, the special structure of the zwitterion will not interfere with the synergistic effect of the peptide chain-side group, and can maintain the normal conformation of the protein. Under the condition of reducing the loading amount of bone morphogenetic protein-2 (BMP-2), it can still effectively promote the functional repair of bone tissue in the defect. And because the zwitterionic polymer is similar to the natural protein structure, it can attract mineralization precursor ions by imitating the natural protein and induce biomineralization.

[0045] By utilizing the characteristics of lipoic acid polymers that can degrade and release lipoic acid through reverse cyclization depolymerization triggered by dynamic disulfide bonds and terminal carboxyl reactions, the polylipoic acid-based zwitterionic hydrogel has the excellent properties of both. By utilizing the multiple hydrogen bond interactions between lipoic acid and sodium lipoate, the polylipoic acid-based zwitterionic hydrogel is stabilized and has a melting temperature suitable for in vivo use, and can be used to prepare drug delivery carriers, biosensor dressings and medical dressings.

[0046] Embodiment three:

[0047] See also Figure 2 This embodiment provides a technical solution based on the first or second embodiment: measuring the water absorption rate of the polylipoic acid-based zwitterionic hydrogel.

[0048] The water absorption rate of polylipoic acid-based zwitterionic hydrogel was measured by weight method. The polylipoic acid-based zwitterionic hydrogel was divided into four parallel groups, numbered A-1, A-2, A-3 and A-4; the purchased commercial hydrogel was used as the control group, numbered NC; the polylipoic acid-based zwitterionic hydrogel and the purchased commercial hydrogel were evenly applied to the ABS plate, and the coated plate was obtained after curing, and the coated plate was weighed and the weight Mi was recorded;

[0049] Soak the coated plate in deionized water for 24 hours. After soaking, take out the hydrogel sample and gently absorb the water on the surface of the hydrogel with filter paper to ensure that there is no excess water attached to the surface. Immediately weigh the mass Mt of the hydrogel sample after water absorption and calculate the water absorption rate (W). The calculation formula of the water absorption rate (W) is as follows:

[0050] W=Mt-Mi / Mi-M0×100%.

[0051] Among them, M0 is the mass of the ABS board;

[0052] Mi is the unimmersed mass of the coated plate;

[0053] Mt is the mass of the coated board after immersion in deionized water for 24 h.

[0054] See also Figure 2 , the X-axis represents different experimental groups, including A-1, A-2, A-3, A-4 and the control group NC; the Y-axis represents the percentage (%) of water absorption.

[0055] The water absorption rate of group A-1 was 295.24%, which was the highest among all experimental groups; the water absorption rate of group A-2 was 284.49%, slightly lower than that of group A-1, but still relatively high; the water absorption rate of group A-3 was 257.77%, lower than that of groups A-1 and A-2, but still showed good water absorption performance; the water absorption rate of group A-4 was 268.76%, which was between groups A-3 and NC; the water absorption rate of group NC (control group) was 166.76%, which was the lowest among all groups.

[0056] As can be seen from the figure, the water absorption rate of polylipoic acid-based zwitterionic hydrogel is generally higher than that of the control group (NC), which indicates that polylipoic acid-based zwitterionic hydrogel has good water absorption performance. The determination of this water absorption performance is very important for evaluating the performance of hydrogels in practical applications, especially in applications that require high water absorption, such as tissue engineering, drug delivery systems or wound dressings.

[0057] Embodiment 4:

[0058] See also Figure 3 This embodiment provides a technical solution based on the first or second embodiment: determining the biocompatibility of polylipoic acid-based zwitterionic hydrogel.

[0059] Mouse fibroblasts (L929) were selected as the experimental subjects. This cell line has the characteristics of stable growth, easy culture, and sensitive reaction to a variety of biomaterials, and is widely used in cytotoxicity experiments. Before the experiment, L929 cells were revived from liquid nitrogen, inoculated in DMEM medium containing 10% fetal bovine serum, and pre-cultured in an incubator at 37°C and 5% CO2. After the cells grew to the logarithmic growth phase, subsequent experimental operations were carried out.

[0060] First, 5×10 3 Mouse fibroblasts (L929) were inoculated on a 96-well plate to ensure that the cells were evenly distributed in each well. The 96-well plate was placed in an incubator at 37°C and 5% CO2 for 24 hours to allow the cells to fully adhere to the wall and grow to form a good monolayer of cells. This step is the key to ensuring the accuracy of the experimental results, because a good cell adhesion state can ensure the normal growth and metabolism of the cells in subsequent experiments, thereby accurately reflecting the effect of the hydrogel on them.

[0061] After 24 hours of culture, different concentrations of polylipoic acid-based zwitterionic hydrogel solutions were added to the 96-well plate. The concentration gradient of the hydrogel solution was set reasonably to fully cover the concentration range that may affect the cells. At the same time, a control group without polylipoic acid-based zwitterionic hydrogel was set up, and only an equal amount of culture medium was added. The 96-well plate was placed in an incubator at 37°C and 5% CO2 for another 24 hours to allow the cells to fully contact the hydrogel and observe the long-term effects of the hydrogel on cell growth and metabolism.

[0062] After the incubation time is over, gently aspirate the old culture medium in the 96-well plate, taking care to avoid damaging the cells. Subsequently, 100 μL of fresh culture medium and 20 μL of MTT (5 mg / mL) solution are added to each well. MTT is a reagent that can be reduced to purple crystals by succinate dehydrogenase in the mitochondria of living cells, and the amount of its reduction product is positively correlated with the number of living cells. The 96-well plate was placed in an incubator at 37 ° C and 5% CO2 for 4 hours to allow MTT to fully react. After the reaction is completed, the MTT solution is removed and an appropriate amount of DMSO is added to each well to dissolve the purple crystals at the bottom of the well plate. Finally, the absorbance of each well is measured at 490 nm using a microplate reader. The size of the absorbance value reflects the metabolic activity of the cells, and thus the toxic effect of polylipoic acid-based zwitterionic hydrogel on cells can be evaluated.

[0063] For specific test results, please refer to Figure 3 , Figure 3 The middle X-axis represents the concentration of polylipoic acid-based zwitterionic hydrogel in micromolar (μm), including six concentration gradients of 0, 20, 50, 100, 200, and 500 μm;

[0064] The Y axis represents the percentage (%) of cell activity, which was determined by MTT assay and reflects the proliferation and survival of cells.

[0065] The cell activity of the control group (0 μm) was close to 100%, which was used as a baseline value, indicating that the cell activity was normal in the absence of the polylipoic acid-based zwitterionic hydrogel.

[0066] In the 20μm, 50μm, 100μm, and 200μm groups, the cell activity decreased slightly with the increase in the concentration of polylipoic acid-based zwitterionic hydrogel, but remained above 80% overall, indicating that these concentrations of hydrogels had little effect on cell activity.

[0067] The cell activity of the 500μm group decreased compared with the other groups, but the effect was not significant.

[0068] As can be seen from the figure, the polylipoic acid-based zwitterionic hydrogel has little effect on the activity of mouse fibroblasts (L929) at a concentration of 0-500 μM, and the cell activity remains at a high level, indicating that the hydrogel has good biocompatibility at these concentrations.

[0069] Embodiment five:

[0070] See also Figure 4 This embodiment provides a technical solution based on the first or second embodiment: testing the antibacterial property of polylipoic acid-based zwitterionic hydrogel.

[0071] Escherichia coli and Staphylococcus aureus were selected as the test bacteria. These two bacteria are extremely common in clinical infections, representing two major categories of Gram-negative bacteria and Gram-positive bacteria, respectively, and are widely representative. Escherichia coli often causes diseases such as intestinal infections and urinary tract infections, while Staphylococcus aureus is an important pathogen that causes a variety of serious infections such as skin infections, respiratory infections, and sepsis. By studying the antibacterial activity of these two bacteria, the antibacterial spectrum of polylipoic acid-based zwitterionic hydrogel can be comprehensively evaluated.

[0072] Two typical pathogens, Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive), were used as test strains, and the antibacterial activity test was carried out using the mixed culture oscillation method.

[0073] The diluted mixed bacterial suspension and polylipoic acid-based zwitterionic hydrogel were cultured in a constant temperature shaking incubator (maintained at 37° C.) for 12 h; the control group did not add polylipoic acid-based zwitterionic hydrogel.

[0074] Then 200 μL of the culture solution was evenly spread on the solid nutrient agar medium, and the growth of bacteria was recorded.

[0075] See also Figure 4 In the antibacterial activity experiment, it was observed that after the bacterial suspension of Escherichia coli and Staphylococcus aureus was co-cultured with polylipoic acid-based zwitterionic hydrogel, almost no colonies grew on the plate surface, indicating that the hydrogel had a significant antibacterial effect on these two bacteria. In contrast, a large number of Escherichia coli and Staphylococcus aureus colonies appeared on the surface of the control plate cultured under the same conditions, further confirming the excellent antibacterial activity of polylipoic acid-based zwitterionic hydrogel.

[0076] The preparation method of the polylipoic acid-based zwitterionic hydrogel is simple, and gelation can be achieved without the intervention of special external conditions. The hydrogel is degradable and has excellent biocompatibility, which makes it have potential application prospects in the biomedical field.

[0077] The above description is only by way of illustration of certain exemplary embodiments of the present invention. It is undoubted that those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a polylipoic acid-based zwitterionic hydrogel, characterized in that: include: S1. Dissolving sodium ethoxide and thioctic acid powder in anhydrous ethanol respectively to obtain sodium ethoxide solution and thioctic acid solution; S2, adding the sodium ethoxide solution dropwise to the lipoic acid solution, and stirring the mixed solution at room temperature to form a uniform mixed slurry; S3, then centrifuging the mixed slurry and drying under vacuum to obtain yellow sodium thioctic acid powder, and storing the yellow sodium thioctic acid powder in a refrigerator; S4, adding thioctic acid and sodium thioctic acid powder into deionized water to form a mixed dispersion of thioctic acid and sodium thioctic acid; S5, heating and stirring the mixed dispersion of lipoic acid and sodium lipoate until it is molten, then adding zwitterion powder under continuous stirring to react, and naturally cooling to room temperature after the reaction to obtain polylipoic acid-based zwitterion hydrogel.

2. The method for preparing a polylipoic acid-based zwitterionic hydrogel according to claim 1, wherein: The weight ratio of sodium ethoxide to anhydrous ethanol in S1 is 1:4-5; the weight ratio of lipoic acid to anhydrous ethanol in S1 is 1:0.5-2.

3. The method for preparing a polylipoic acid-based zwitterionic hydrogel according to claim 1, characterized in that: The stirring time of S2 is 10 to 15 hours.

4. The method for preparing a polylipoic acid-based zwitterionic hydrogel according to claim 1, characterized in that: The temperature of the refrigerator in S2 is -20°C.

5. The method for preparing a polylipoic acid-based zwitterionic hydrogel according to claim 1, characterized in that: The molar ratio of lipoic acid and sodium lipoate added to the deionized water in S4 is 2:1, and the mass of the deionized water in S4 is 2.5-3 times the total mass of lipoic acid and sodium lipoate.

6. The method for preparing a polylipoic acid-based zwitterionic hydrogel according to claim 1, characterized in that: S5 is described as heating and stirring until it is in a molten state, and the heating temperature is 80°C.

7. The method for preparing a polylipoic acid-based zwitterionic hydrogel according to claim 1, characterized in that: S5 The zwitterionic powder is sulfobetaine methacrylate.

8. A polylipoic acid-based zwitterionic hydrogel, characterized in that: The polylipoic acid-based zwitterionic hydrogel is prepared by the preparation method of a polylipoic acid-based zwitterionic hydrogel according to any one of claims 1 to 7.

9. Use of the polylipoic acid-based zwitterionic hydrogel prepared by the preparation method according to any one of claims 1 to 7 in the preparation of antifouling coatings, drug delivery carriers, biosensor dressings and medical dressings.

10. Use of the polylipoic acid-based zwitterionic hydrogel according to claim 8 in the preparation of antifouling coatings, drug delivery carriers, biosensor dressings and medical dressings.

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