Poly (6-aminocaproic acid) / hyaluronic acid-based multifunctional composite hydrogel and its preparation method and application

Abstract

The present invention belongs to the technical field of multifunctional new materials, and relates to a poly-6-aminocaproic acid / hyaluronic acid-based multifunctional composite hydrogel and its preparation method and application. Hyaluronic acid modified with hydrazide, hyaluronic acid modified with aldehyde and poly-6-aminocaproic acid modified with dopamine are subjected to a pre-crosslinking reaction. After the reaction is completed, the reaction system is immersed in a strontium chloride aqueous solution to stabilize the structure. The dopamine-modified thiol-terminated poly-aminocaproic acid of the present invention dissociates H in an aqueous medium. + , and hydrazide-modified hyaluronic acid showed the ability to absorb free H + , showing the behavior of changing the pH value of the aqueous medium. The glycopeptide-based hydrogel prepared by the present invention has good mechanical properties, rapid gelation behavior and biocompatibility, and has excellent anti-inflammatory and antibacterial properties.

Description

Technical Field

[0001] The invention belongs to the technical field of multifunctional new materials and relates to a poly-6-aminocaproic acid / hyaluronic acid-based multifunctional composite hydrogel and a preparation method and application thereof. Background Art

[0002] The skin is the first line of defense for the human body against external invasion. Due to the neurological degeneration of blood vessels, diabetic patients are very likely to have ulcerated wounds in their distal skin tissues. Excessive expression of inflammation hinders the healing of skin tissues, causing skin trauma to lose its normal repair ability, and severe cases face the risk of amputation. In this case, clinical means can no longer repair skin wounds. Therefore, more and more new treatment methods have emerged, such as photothermal therapy, oxygen release, wound dressing therapy, and engineered stem cell-based therapies. Among them, hydrogel therapy has attracted widespread attention due to its structure similar to the extracellular matrix. Most hydrogel dressings achieve immune regulation of skin wounds by loading growth factors or living cells, but hydrogels loaded with growth factors or living cells are expensive and complicated to operate, which is not conducive to clinical use. Therefore, it is of great significance to construct a multifunctional hydrogel dressing with intrinsic immune regulation for the repair of diabetic distal ulcers.

[0003] Hyaluronic acid (HA) is a natural non-sulfated glycosaminoglycan, which is a non-toxic, biodegradable and biocompatible natural polymer. As the main component of the extracellular matrix, the diglucose carboxyaldehyde structure in HA gives it a high reactive oxygen species (ROS) scavenging performance, which plays a vital role in regulating inflammation; 6-aminocaproic acid is used as a hemostatic drug in clinical practice, but its short hydrophobic side chain at the end and the presence of a large number of carboxyl groups make it have excellent performance in regulating pH environment. The change in pH environment induces cells to undergo immunomodulation to obtain the effect of regulating inflammation and produces a large number of quaternary amine structures in the process of pH regulation to obtain antibacterial ability. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an anti-inflammatory and antibacterial poly-6-aminocaproic acid / hyaluronic acid-based multifunctional composite hydrogel in view of the deficiencies in the prior art.

[0005] The technical problem that the present invention also aims to solve is to provide a method for preparing the above-mentioned poly 6-aminocaproic acid / hyaluronic acid-based multifunctional composite hydrogel.

[0006] A further technical problem to be solved by the present invention is to provide an application of the above-mentioned poly 6-aminocaproic acid / hyaluronic acid-based multifunctional composite hydrogel.

[0007] Invention idea: In the present invention, a composite hydrogel with anti-inflammatory and antibacterial properties is prepared by mixing hyaluronic acid modified with adipic acid dihydrazide (HA-ADH), aldehyde-modified hyaluronic acid (OHA) and dopamine-terminated poly-6-aminocaproic acid (PADA). First, during the mixing process, Schiff base bonds are formed between the aldehyde groups and hydrazides in HA-ADH and OHA and associate to form a hydrogel at room temperature. Subsequently, under the action of strontium ions, a stable structure is further formed through the metal complexation reaction between the phenolic hydroxyl groups in PADA and the strontium ions. PADA with rich -COOH can release H under neutral conditions. + , H + The quaternary amine structure is captured by hydrazide and formed during the dynamic regulation process. This quaternary amine structure not only retains its antibacterial properties without affecting the biocompatibility of the hydrogel, but also synergistically regulates anti-inflammatory effects through hyaluronic acid with excellent biological functions and dynamic pH environment.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0009] The invention discloses a preparation method of a poly 6-aminocaproic acid / hyaluronic acid-based multifunctional composite hydrogel. The method comprises the following steps: mixing hydrazide-modified hyaluronic acid with a phosphate buffer to obtain a first prepolymerization liquid; mixing aldehyde-modified hyaluronic acid with a phosphate buffer to obtain a second prepolymerization liquid; mixing dopamine-terminated modified poly 6-aminocaproic acid with a phosphate buffer to obtain a third prepolymerization liquid; mixing the first prepolymerization liquid, the second prepolymerization liquid and the third prepolymerization liquid to carry out a pre-crosslinking reaction, and after the reaction is completed, immersing the reaction system in a strontium chloride aqueous solution to stabilize the structure, thereby obtaining the composite hydrogel.

[0010] Wherein, the concentration of potassium dihydrogen phosphate in the phosphate buffer is 1.5 mmol / L, the concentration of sodium dihydrogen phosphate in the buffer is 8 mmol / L, the concentration of NaCl in the buffer is 0.2 mol / L, the concentration of KCl in the buffer is 2.7 mmol / L, and the pH of the phosphate buffer is 7.4.

[0011] Specifically, the concentration of hydrazide-modified hyaluronic acid in the first prepolymer liquid is 0.5-3% g / mL; the concentration of aldehyde-modified hyaluronic acid in the second prepolymer liquid is 1-3% g / mL; the concentration of dopamine-terminated poly-6-aminocaproic acid in the third prepolymer liquid is 1-3% g / mL; the concentration of strontium chloride in the strontium chloride aqueous solution is 0.005%-0.05% g / mL, preferably 0.01% g / mL; the volume ratio of the first prepolymer liquid, the second prepolymer liquid and the third prepolymer liquid is 0.5-1:0.5-1:0.5-1.

[0012] Among them, the first prepolymer liquid, the second prepolymer liquid and the third prepolymer liquid are mixed to carry out a pre-crosslinking reaction. After the reaction is completed, a gelled hydrogel is obtained. Subsequently, the gelled hydrogel needs to be immersed in a strontium chloride aqueous solution to stabilize the structure. Therefore, the amount of strontium chloride aqueous solution is excessive and the gelled hydrogel needs to be immersed.

[0013] Specifically, the pre-crosslinking reaction has a reaction temperature of room temperature and a reaction time of 1 to 3 minutes, preferably 2 minutes; and the soaking has a soaking temperature of room temperature and a soaking time of 1 to 10 minutes.

[0014] Specifically, the preparation method of the hydrazide-modified hyaluronic acid is as follows: the preparation method of the hydrazide-modified hyaluronic acid is as follows: hyaluronic acid, a condensation agent and adipic acid dihydrazide are reacted to obtain hydrazide-modified hyaluronic acid, and the pH value of the reaction system is controlled between 7.5 and 7.8 within the first 48 hours of the total reaction time. After the total reaction time is 72 hours, the pH value of the reaction system is maintained at a neutral range of 7.35 to 7.45.

[0015] Preferably, the preparation method of the hydrazide-modified hyaluronic acid is as follows: dissolving hyaluronic acid in deionized water to obtain a mixed solution A; adding a condensing agent to the mixed solution A for activation to obtain a mixed solution B; adding adipic acid dihydrazide to the mixed solution B for a first reaction, controlling the pH value of the reaction system between 7.5 and 7.8 within the first 48 hours of the total reaction time, and maintaining the pH value of the reaction system at a neutral range of 7.35 to 7.45 after a total reaction time of 72 hours, dialyzing the reaction solution, and freeze-drying to obtain the product.

[0016] The pH value of the reaction system was controlled by adding 1 mol / L sodium hydroxide aqueous solution and 1 mol / L hydrochloric acid aqueous solution to the system to adjust the pH value.

[0017] Specifically, the condensing agent is 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; the concentration of hyaluronic acid in the mixed solution A is 5-20 mg / mL, preferably 10 mg / mL; the molar ratio of hyaluronic acid to 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.2-1.5:1.2-1.5, preferably 1:1.2:1.2; the molar ratio of carboxyl in hyaluronic acid to amino in adipic acid dihydrazide is 1:50-80; the activation temperature is room temperature and the activation time is 10-30 min; the first reaction has a reaction temperature of room temperature.

[0018] Among them, 1-hydroxybenzotriazole is abbreviated as HOBT, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is abbreviated as EDC.

[0019] Specifically, the preparation method of the aldehyde-modified hyaluronic acid is as follows: hyaluronic acid is reacted with sodium periodate and quenched to obtain the aldehyde-modified hyaluronic acid.

[0020] Preferably, the preparation method of the aldehyde-modified hyaluronic acid is as follows: dissolving hyaluronic acid in deionized water to obtain a mixed solution C; adding sodium periodate to the mixed solution C for a second reaction, quenching the reaction after the reaction is completed, dialyzing the reaction solution, and freeze-drying to obtain the aldehyde-modified hyaluronic acid.

[0021] Specifically, the molar ratio of the carboxylic acid ring to sodium periodate in the hyaluronic acid is 1:1-2.5, preferably 1:1.5; the second reaction is a light-proof reaction at room temperature for 4-8 hours; the quenching reaction is quenched by adding ethylene glycol to the reaction solution, and the mass volume ratio of hyaluronic acid to ethylene glycol is 1g:1-5mL.

[0022] In the mixed solution C, the amount of deionized water used is such that the hyaluronic acid in the mixed solution is dissolved and the viscosity of the mixed solution is moderate.

[0023] Specifically, the preparation method of the dopamine-terminated modified poly-6-aminocaproic acid comprises the following steps:

[0024] (1) reacting 6-aminocaproic acid, sodium hydroxide and acryloyl chloride to obtain 6-acryloylaminocaproic acid;

[0025] (2) reacting β-mercaptoethanol, ammonium persulfate, tetramethylethylenediamine, and sodium hydroxide with the 6-acryloylaminocaproic acid obtained in step (1) to obtain poly-6-aminocaproic acid;

[0026] (3) reacting 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, dopamine hydrochloride with the poly-6-aminocaproic acid obtained in step (2) to obtain.

[0027] Preferably, the preparation method of the dopamine-terminated modified poly-6-aminocaproic acid comprises the following steps:

[0028] (1) dissolving 6-aminocaproic acid and sodium hydroxide in deionized water to obtain a mixed solution D; dissolving acryloyl chloride in an organic solvent to obtain a mixed solution E; mixing the mixed solution D and the mixed solution E to perform a third reaction to obtain 6-acryloylaminocaproic acid;

[0029] (2) dissolving the 6-acryloylaminocaproic acid obtained in step (1) and sodium hydroxide in deionized water to obtain a mixed solution F; dissolving β-mercaptoethanol, ammonium persulfate and tetramethylethylenediamine in deionized water to obtain a mixed solution G; mixing the mixed solution F with the mixed solution G to perform a fourth reaction to obtain poly-6-aminocaproic acid;

[0030] (3) dissolving the poly-6-aminocaproic acid obtained in step (2) with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in deionized water to obtain a mixed solution H; dissolving dopamine hydrochloride in deionized water to obtain a mixed solution I; mixing the mixed solution H with the mixed solution I to carry out a fifth reaction, and after the reaction is completed, dialyzing the reaction solution and freeze-drying to obtain;

[0031] Step (2) and step (3) are carried out under the protection of inert gas.

[0032] Specifically, in step (1), the organic solvent is tetrahydrofuran; the concentration of 6-aminocaproic acid in the mixed solution D is 0.05-0.25 g / mL; the molar ratio of 6-aminocaproic acid, sodium hydroxide and acryloyl chloride is 1:1-1.2:1-1.2, preferably 1:1.1:1.1; and the reaction temperature of the third reaction is -4-4°C, and the reaction time is 8-12 h.

[0033] Wherein, in step (1), the amount of organic solvent in the mixed solution E is such that the acryloyl chloride solid in the mixed solution is dissolved and the viscosity of the mixed solution is moderate.

[0034] Specifically, in step (2), the molar ratio of 6-acryloylaminocaproic acid, sodium hydroxide, β-mercaptoethanol, ammonium persulfate and tetramethylethylenediamine is 50:50-60:0.1-2:0.1-1:0.1-1, preferably 50:50:1.185:0.41:1; and the fourth reaction has a reaction temperature of 22-27°C and a reaction time of 8-12h.

[0035] Wherein, in step (2), the amount of deionized water used is: enough to dissolve the solid in the mixed solution and to achieve a moderate viscosity of the mixed solution.

[0036] Specifically, in step (3), the molar ratio of poly 6-aminocaproic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and dopamine hydrochloride is 10:1-3:0.5-2:1-3, preferably 10:2:1:2; the fifth reaction is a light-proof reaction with a reaction temperature of 25-35°C and a reaction time of 8-12h.

[0037] Here, the abbreviation of N-hydroxysuccinimide is NHS.

[0038] Wherein, in step (3), the amount of deionized water used is: enough to dissolve the solid in the mixed solution and to achieve a moderate viscosity of the mixed solution.

[0039] The poly-6-aminocaproic acid / hyaluronic acid-based multifunctional composite hydrogel prepared by the above-mentioned preparation method is also within the protection scope of the present invention.

[0040] Specifically, in the composite hydrogel, the final concentration of hydrazide-modified hyaluronic acid is 0.3% to 1% g / mL, the final concentration of aldehyde-modified hyaluronic acid is 0.3% to 1% g / mL, and the final concentration of dopamine-terminated poly-6-aminocaproic acid is 0.3% to 1% g / mL.

[0041] The use of the above-mentioned poly 6-aminocaproic acid / hyaluronic acid-based multifunctional composite hydrogel in the preparation of anti-inflammatory and antibacterial medical materials is also within the protection scope of the present invention.

[0042] Beneficial effects:

[0043] (1) The composite hydrogel of the present invention has a fast gelation time and good mechanical properties and cell compatibility.

[0044] (2) Hydrogels in the prior art achieve antibacterial effects by loading antibacterial drugs or nanoparticles, but the potential biological toxicity of such loaded drugs is not conducive to their application. The composite hydrogel described in the present invention can dynamically generate quaternary amine structures during use, greatly improving the biocompatibility of the hydrogel without losing the antibacterial effect.

[0045] (3) The present invention uses hyaluronic acid as the matrix material and introduces poly 6-aminocaproic acid. As the -COOH at the end of the poly 6-aminocaproic acid and the -NH2 at the end of the hyaluronic acid are protonated and deprotonated, a quaternary amine structure is dynamically generated.

[0046] (4) The present invention uses hyaluronic acid as the raw material for hydrogel. The structure of glucose dicarboxaldehyde in hyaluronic acid promotes the scavenging effect of hydrogel on ROS, and the raw material has been commercialized. Therefore, its selection and the establishment, promotion and promotion of such gel method in regenerative medicine are of great value. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0048] Figure 1 These are the H-NMR spectra of hyaluronic acid modified with adipic acid dihydrazide and hyaluronic acid modified with aldehyde.

[0049] Figure 2 This is the NMR spectrum of dopamine-terminated poly (6-aminocaproic acid).

[0050] Figure 3 This is the modulus-frequency curve of the composite hydrogel.

[0051] Figure 4 Image showing the antibacterial effect of the composite hydrogel.

[0052] Figure 5 The images show the inhibition of TNF-α secretion by composite hydrogel.

[0053] Figure 6 The images show the inhibition of IL-6 secretion by composite hydrogel.

[0054] Figure 7 The images show the inhibition of IL-1β secretion by composite hydrogel.

[0055] Figure 8 This is the FTIR infrared spectrum of aldehyde-modified hyaluronic acid. DETAILED DESCRIPTION

[0056] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0057] The phosphate buffer (PBS) used in the embodiments of the present invention has a potassium dihydrogen phosphate concentration of 1.5 mmol / L, a sodium dihydrogen phosphate concentration of 8 mmol / L, a NaCl concentration of 0.2 mol / L, a KCl concentration of 2.7 mmol / L, and a pH of 7.4 in the phosphate buffer.

[0058] Example 1: Preparation of hyaluronic acid modified with adipic acid dihydrazide

[0059] (1) Weigh 4 g of hyaluronic acid (HA, Mw: 1100 kDa) and dissolve it in 400 mL of deionized water until it is fully dissolved to form a mixed solution A (the concentration of hyaluronic acid in the mixed solution A is 10 mg / mL). Add 1.62 g of HOBT and 2.3 g of EDC to the mixed solution A and activate it at room temperature for 30 min to obtain a mixed solution B. Add 43.55 g of adipic acid dihydrazide (ADH) to the mixed solution B and react at room temperature. During the reaction, add 1 mol / L of sodium hydroxide aqueous solution and 1 mol / L of hydrochloric acid aqueous solution to the reaction solution to adjust the pH of the reaction solution. The pH value of the reaction system is controlled between 7.5 and 7.8 within the first 48 h of the total reaction time. After the total reaction time of 72 h, the pH value of the reaction system is maintained at a neutral pH of 7.35 to 7.45. The reaction solution is placed in a dialysis bag (8000 to 14000 Da) and dialyzed for 3 days. The water is changed every 4 h in the first two days and three times a day in the next few days. The dialyzed product was placed in a freeze dryer for freeze drying, and a white sponge-like sample was finally obtained, namely, adipic acid dihydrazide-modified hyaluronic acid (HA-ADH), which was stored in a desiccator for later use.

[0060] (2) 10.00 mg HA and 10.00 mg HA-ADH were dissolved in D2O (1000 μL) and the samples were recorded by NMR spectrometer. 1 H NMR spectrum.

[0061] (3) In the NMR spectra of hyaluronic acid (HA) and HA-ADH ( Figure 1 ) it can be seen that after ADH grafting, two new signal peaks will appear at about δ=1.65ppm(b) and 2.39ppm(a), which are the NMR peaks of the methylene group on adipic acid dihydrazide, proving that hyaluronic acid has been successfully adipic acid dihydrazide-modified.

[0062] Example 2: Preparation of aldehyde-modified hyaluronic acid

[0063] (1) Weigh 3 g of hyaluronic acid (HA, Mw: 1100 kDa) and dissolve it in 300 mL of deionized water until it is fully dissolved to form a mixed solution C; add 2.53 g of sodium periodate (NaIO4) to the mixed solution C and react at room temperature in the dark for 6 h; after the reaction is completed, add 15 mL of ethylene glycol to the reaction solution to quench the reaction, and then place the reaction solution in a dialysis bag (8000-14000 Da) for 3 days, changing the water every 4 h for the first two days and changing the water three times a day for the next few days. The dialyzed product is placed in a freeze dryer and freeze-dried to finally obtain a white sponge-like sample, i.e., aldehyde-modified hyaluronic acid (OHA), which is stored in a desiccator in the dark.

[0064] (2) The infrared characterization of OHA was carried out by potassium bromide tableting. An appropriate amount of the product was ground into fine powder and pressed into tablets using a Thermo Scientific Nicolet iS5 500-4000 cm -1 The chemical structure of the obtained product was confirmed by FT-IR analysis. The infrared spectrum of OHA is shown in Figure 8 As shown, 1725cm -1 The vibration peak of C=O double bond appeared at , indicating the successful grafting of aldehyde group. 1 H NMR analysis confirmed the chemical structure of the obtained product. The NMR spectrum of OHA is shown in Figure 1 As shown, the triplet peak (a, b and c peaks) around 5.0 ppm indicated the successful grafting of aldehyde groups, indicating the successful preparation of aldehyde-modified hyaluronic acid.

[0065] Example 3: Preparation of dopamine-terminated poly-6-aminocaproic acid

[0066] (1) Weigh 13.17 g of 6-aminocaproic acid (AA) and 4.4 g of sodium hydroxide (NaOH) and place them in an eggplant-shaped flask. Add 80 mL of deionized water in an ice bath at 0°C and stir thoroughly to obtain a mixed solution D. Weigh 10 g of acryloyl chloride (AC) and dissolve it in 15 mL of tetrahydrofuran to obtain a mixed solution E. The mixed solution E was added to a 50 mL constant pressure dropping funnel, and slowly added dropwise to the mixed solution D. The mixture was reacted at 0°C for 8 h. After the reaction was completed, 50 mL of ethyl acetate was added to the reaction solution to extract the reaction solution. The mixture was allowed to stand in a separatory funnel until layers were separated. The organic phase was collected. The pH of the aqueous phase was adjusted to 2-3 with 1 M hydrochloric acid. The aqueous phase was extracted with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate overnight, filtered with a sand core funnel, and the organic phase obtained by filtration was rotary evaporated until it became viscous. The mixture was purified by column chromatography using a petroleum ether / ethyl acetate system to obtain a white powder 6-acrylamidohexanoic acid (AACA). The AACA was dried in a vacuum drying oven overnight, and then stored in a desiccator for later use.

[0067] (2) Weigh 2 g of AACA and put it into a two-necked flask, evacuate for 30 min, and pass nitrogen for 5 min. Repeat this step twice, add 15 mL of deionized water solution containing 0.432 g of sodium hydroxide, and stir well to obtain mixed solution F; weigh 0.02 g of β-mercaptoethanol, 0.02 g of ammonium persulfate, and 0.025 g of tetramethylethylenediamine (TEMED) and dissolve them in 1 mL of deionized water, and stir well to obtain mixed solution G. Add mixed solution G to mixed solution F and react at 25°C for 12 h. After the reaction, dialyze the reaction solution. Three days later, put the dialyzed product into a freeze dryer and freeze-dry it to finally obtain a white sponge-like product (PACA), i.e., poly-6-aminocaproic acid, which is stored in a desiccator away from light.

[0068] (3) Weigh 0.5 g of PACA and put it into a two-necked flask, evacuate for 30 min, and pass nitrogen for 5 min. Repeat this step twice, add 50 mL of deionized water solution containing 0.1035 g of EDC and 0.031 g of NHS, and stir well to obtain mixed solution H; weigh 0.1024 g of dopamine hydrochloride and dissolve it in 1 mL of deionized water, and stir well to obtain mixed solution I. Add mixed solution I to mixed solution H and react at 25°C in the dark for 12 h. After the reaction, dialyze the reaction solution. Three days later, put the dialyzed product into a freeze dryer and freeze-dry it to finally obtain a white sponge-like product (PADA), i.e., dopamine-terminated poly-6-aminocaproic acid, which is stored in a desiccator in the dark.

[0069] (3) Dissolve 10.00 mg AA, 10.00 mg PACA and 10.00 mg PADA in CDCl3, D2O and D2O (1000 μL) respectively. Record the samples by NMR spectrometer. 1 H NMR spectrum.

[0070] from Figure 2 It can be seen that in the NMR spectrum of AACA, multiple new signal peaks appear around δ=5.9-6.5ppm, which are the NMR peaks of -CH2- on propylene. In the NMR spectrum of PADA, δ=6-7ppm is the NMR peak of the benzene ring on dopamine, which proves the successful synthesis of PADA.

[0071] Example 4

[0072] (1) The HA-ADH prepared in Example 1 was dissolved in PBS to obtain a first prepolymer solution (the concentration of HA-ADH was 2% g / mL); the OHA prepared in Example 2 was dissolved in PBS to obtain a second prepolymer solution (the concentration of OHA was 2% g / mL); the first prepolymer solution and the second prepolymer solution were mixed in a volume ratio of 1:1 until gelation was obtained to obtain an OHA / HA-ADH hydrogel, wherein the final concentration of HA-ADH in the hydrogel was 1% g / mL, and the final concentration of OHA was 1% g / mL.

[0073] (2) The PADA and SrCl2 prepared in Example 3 were dissolved in PBS and allowed to stand until gelation occurred to obtain a PADA / Sr hydrogel. The concentration of PADA in the hydrogel was 1% g / mL, and the concentration of strontium chloride was 0.01% g / mL.

[0074] (3) The HA-ADH prepared in Example 1 was dissolved in PBS to prepare a prepolymer solution containing HA-ADH, and a first prepolymer solution was obtained, wherein the concentration of HA-ADH in the first prepolymer solution was 3% g / mL; the OHA prepared in Example 2 was dissolved in PBS to prepare a prepolymer solution containing OHA, and a second prepolymer solution was obtained, wherein the concentration of OHA in the second prepolymer solution was 3% g / mL. The PADA prepared in Example 3 was dissolved in PBS to prepare a prepolymer solution containing PADA, and a third prepolymer solution was obtained, wherein the concentration of PADA in the third prepolymer solution was 3% g / mL. The above prepolymer solutions were mixed in a volume ratio of 1:1:1 and added into a glass bottle for pre-crosslinking reaction. The reaction temperature was room temperature and the crosslinking was allowed to proceed for 2 minutes. The crosslinked hydrogel was then placed in an aqueous solution of strontium chloride containing 0.01% g / mL Sr ions and soaked at room temperature for 5 minutes. After soaking, the hydrogel was taken out and washed with PBS to obtain a PADA / OHA / HA-ADH hydrogel. The final concentration of HA-ADH in the PADA / OHA / HA-ADH hydrogel was 1% g / mL, the final concentration of OHA was 1% g / mL, and the final concentration of PADA was 1% g / mL.

[0075] (4) The compression performance of the hydrogel was tested using a rheometer with an upper clamp of 20 mm. 500 μL of hydrogel was injected into a 2 mL glass bottle and added to the parallel plates after gelation. The sample was compressed at 10.0 μm / s and tested at 37°C with a strain of 0.1% in the frequency range of 0.1 to 100 Hz to obtain the modulus-frequency curve. Figure 3 The modulus-frequency curve of the hydrogel (G' represents the ability of the material to store elastic deformation energy, and G" represents the energy lost by the material under irreversible deformation), which can reflect the mechanical properties of the hydrogel; among them, PADA / OHA / HA-ADH hydrogel exhibits a higher storage modulus, which may be because the introduction of the double network increases the cross-linking density of the hydrogel, thereby improving the mechanical properties of the hydrogel.

[0076] Example 5

[0077] (1) The OHA / HA-ADH, PADA / Sr, and PADA / OHA / HA-ADH hydrogels prepared in Example 4 were applied to this example.

[0078] (2) Control group: PBS and bacteria were co-cultured in the well plate for 12 h, and the supernatant was directly centrifuged and discarded. After adding 10 times the amount of normal saline and blowing evenly, 10 times, 100 times, 1000 times, 10000 times, and 100000 times of diluted liquid were taken, and then 10uL of each multiple of three parallel samples was dropped onto the agar plate and cultured for 12 hours.

[0079] Hydrogel group: A circular hydrogel sample was made with a mold. The hydrogel and bacteria were co-cultured in a well plate for 12 hours. A 10-fold amount of normal saline was added, and the sample was vortexed for 2 minutes after breaking. The liquid was then aspirated for later use. The sample was diluted to 10, 100, 1000, 10,000, and 100,000 times of the original solution in sequence. Then, 10uL of each multiple was aspirated and 3 parallel samples in each group were dropped on an agar plate for 12 hours.

[0080] from Figure 4 It can be seen that compared with the control group, the PADA / OHA / HA-ADH composite hydrogel showed obvious bacterial inhibition effect, which was due to the synergistic antibacterial effect of quaternary amine and aldehyde groups in the acidic environment. OHA / HA-ADH and PADA / Sr hydrogels showed different colony inhibition effects, inhibiting bacterial proliferation through aldehyde groups and acidic microenvironments respectively. In addition, due to the double-layer cell membrane structure of Escherichia coli, acidic means have no effect on it, and it can only be sterilized through groups. Among them, Figure 4 The PADA group in the gel is PADA / Sr hydrogel.

[0081] Example 6

[0082] (1) The OHA / HA-ADH, PADA / Sr, and PADA / OHA / HA-ADH hydrogels prepared in Example 4 were applied to this example.

[0083] (2) Macrophages in the logarithmic growth phase were cultured by pipetting and centrifugation, with 2×10 4 The number of cells was added to a 24-well plate and cultured until adhered. A medium containing LPS (5ug / mL) was added to induce the cells for 24 hours. After the hydrogel was added and cultured for 24 hours, the hydrogel was removed, the macrophages were blown and centrifuged, and the supernatant was taken to obtain solution L, which was placed in a -20°C refrigerator for later use.

[0084] Among them, the Norml group (negative control group) and the LPS group (positive control group) did not contain hydrogel; the Normal group: co-culture of culture medium and macrophages; the LPS group: co-culture of culture medium containing LPS (lipopolysaccharide) and macrophages.

[0085] (3) Test solution L for TNF-α, IL-1β, and IL-6 related proteins using an Elisa kit. Set the microplate reader to measure the OD value of each group in the well plate at 450 nm. The blank wells are used as background plates to deduct the corresponding OD values. Figure 5 , Figure 6 , Figure 7 As shown, each group of hydrogels showed a reducing effect on inflammatory factors, among which PADA / OHA / HA-ADH hydrogel had the best anti-inflammatory effect.

[0086] Among them, Figure 5 , Figure 6 and Figure 7 In the figure, PADA / HA-ADH / / OHA hydrogel is PADA / OHA / HA-ADH hydrogel; and PADA group is PADA / Sr hydrogel.

[0087] The present invention provides a poly-6-aminocaproic acid / hyaluronic acid-based multifunctional composite hydrogel and its preparation method and application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing a poly (6-aminocaproic acid) / hyaluronic acid-based multifunctional composite hydrogel, characterized in that: The hydrazide-modified hyaluronic acid is mixed with a phosphate buffer to obtain a first prepolymer solution; the aldehyde-modified hyaluronic acid is mixed with a phosphate buffer to obtain a second prepolymer solution; the dopamine-terminated poly-6-aminocaproic acid is mixed with a phosphate buffer to obtain a third prepolymer solution; the first prepolymer solution, the second prepolymer solution and the third prepolymer solution are mixed to perform a pre-crosslinking reaction, and after the reaction is completed, the reaction system is immersed in a strontium chloride aqueous solution to stabilize the structure, thereby obtaining a composite hydrogel; The preparation method of the hydrazide-modified hyaluronic acid is as follows: dissolving hyaluronic acid in deionized water to obtain a mixed solution A; adding a condensing agent to the mixed solution A for activation to obtain a mixed solution B; adding adipic acid dihydrazide to the mixed solution B for a first reaction, controlling the pH value of the reaction system between 7.5 and 7.8 within the first 48 hours of the total reaction time, and maintaining the pH value of the reaction system at a neutral range of 7.35 to 7.45 after the total reaction time of 72 hours, dialyzing the reaction solution, and freeze-drying to obtain the hyaluronic acid; The preparation method of the aldehyde-modified hyaluronic acid is as follows: dissolving hyaluronic acid in deionized water to obtain a mixed solution C; adding sodium periodate to the mixed solution C for a second reaction, quenching the reaction after the reaction is completed, dialyzing the reaction solution, and freeze-drying to obtain the aldehyde-modified hyaluronic acid; The preparation method of the dopamine-terminated modified poly-6-aminocaproic acid comprises the following steps: (1) dissolving 6-aminocaproic acid and sodium hydroxide in deionized water to obtain a mixed solution D; dissolving acryloyl chloride in an organic solvent to obtain a mixed solution E; mixing the mixed solution D and the mixed solution E to perform a third reaction to obtain 6-acryloylaminocaproic acid; (2) dissolving the 6-acryloylaminocaproic acid obtained in step (1) and sodium hydroxide in deionized water to obtain a mixed solution F; dissolving β-mercaptoethanol, ammonium persulfate and tetramethylethylenediamine in deionized water to obtain a mixed solution G; mixing the mixed solution F with the mixed solution G to perform a fourth reaction to obtain poly-6-aminocaproic acid; (3) dissolving the poly-6-aminocaproic acid obtained in step (2) with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in deionized water to obtain a mixed solution H; dissolving dopamine hydrochloride in deionized water to obtain a mixed solution I; mixing the mixed solution H with the mixed solution I to carry out a fifth reaction, and after the reaction is completed, dialyzing the reaction solution and freeze-drying to obtain; Step (2) and step (3) are carried out under the protection of inert gas.

2. The preparation method according to claim 1, characterized in that: The concentration of hydrazide-modified hyaluronic acid in the first prepolymer solution is 0.5-3% g / mL; the concentration of aldehyde-modified hyaluronic acid in the second prepolymer solution is 1-3% g / mL; the concentration of dopamine-terminated poly-6-aminocaproic acid in the third prepolymer solution is 1-3% g / mL; the concentration of strontium chloride in the strontium chloride aqueous solution is 0.005%-0.05% g / mL; the volume ratio of the first prepolymer solution, the second prepolymer solution and the third prepolymer solution is 0.5-1:0.5-1:0.5-1.

3. The preparation method according to claim 1, characterized in that: The pre-crosslinking reaction has a reaction temperature of room temperature and a reaction time of 1 to 3 minutes; the soaking has a soaking temperature of room temperature and a soaking time of 1 to 10 minutes.

4. The preparation method according to claim 1, characterized in that: The condensing agents are 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; the concentration of hyaluronic acid in the mixed solution A is 5-20 mg / mL; the molar ratio of the hyaluronic acid to 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.2-1.5:1.2-1.5; the molar ratio of the carboxyl group in the hyaluronic acid to the amino group in the adipic acid dihydrazide is 1:50-80; the activation temperature is room temperature and the activation time is 10-30 min; the reaction temperature of the first reaction is room temperature.

5. The preparation method according to claim 1, characterized in that: The molar ratio of the carboxylic acid ring to sodium periodate in the hyaluronic acid is 1:1~2.5; the second reaction is a light-proof reaction at room temperature for 4~8 hours; the quenching reaction is quenched by adding ethylene glycol to the reaction solution, and the mass volume ratio of hyaluronic acid to ethylene glycol is 1g:1~5mL.

6. The preparation method according to claim 1, characterized in that: In step (1), the organic solvent is tetrahydrofuran; the concentration of 6-aminocaproic acid in the mixed solution D is 0.05-0.25 g / mL; the molar ratio of 6-aminocaproic acid, sodium hydroxide and acryloyl chloride is 1:1-1.2:1-1.2; and the reaction temperature of the third reaction is -4-4°C and the reaction time is 8-12 h.

7. The preparation method according to claim 1, characterized in that: In step (2), the molar ratio of 6-acryloylaminocaproic acid, sodium hydroxide, β-mercaptoethanol, ammonium persulfate and tetramethylethylenediamine is 50:50-60:0.1-2:0.1-1:0.1-1; and the fourth reaction has a reaction temperature of 22-27°C and a reaction time of 8-12 h.

8. The preparation method according to claim 1, characterized in that: In step (3), the molar ratio of poly 6-aminocaproic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and dopamine hydrochloride is 10:1-3:0.5-2:1-3; the fifth reaction is a light-proof reaction at a temperature of 25-35°C and a reaction time of 8-12 h.

9. The poly (6-aminocaproic acid) / hyaluronic acid-based multifunctional composite hydrogel prepared by the preparation method according to any one of claims 1 to 8.

10. The poly (6-aminocaproic acid) / hyaluronic acid-based multifunctional composite hydrogel according to claim 9, characterized in that: In the composite hydrogel, the final concentration of hydrazide-modified hyaluronic acid is 0.3%-1% g / mL, the final concentration of aldehyde-modified hyaluronic acid is 0.3%-1% g / mL, and the final concentration of dopamine-terminated poly-6-aminocaproic acid is 0.3%-1% g / mL.

11. Use of the poly (6-aminocaproic acid) / hyaluronic acid-based multifunctional composite hydrogel according to claim 9 in the preparation of anti-inflammatory and antibacterial medical materials.

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