CN114561046B - Guanidine hyaluronic acid type antibacterial hydrogel and preparation method and application thereof - Google Patents
Guanidine hyaluronic acid type antibacterial hydrogel and preparation method and application thereof Download PDFInfo
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- CN114561046B CN114561046B CN202210188225.9A CN202210188225A CN114561046B CN 114561046 B CN114561046 B CN 114561046B CN 202210188225 A CN202210188225 A CN 202210188225A CN 114561046 B CN114561046 B CN 114561046B
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- hyaluronic acid
- gel
- reaction
- hydrogel
- antibacterial
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Classifications
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- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
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- A61L26/0061—Use of materials characterised by their function or physical properties
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- A—HUMAN NECESSITIES
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- A61L26/008—Hydrogels or hydrocolloids
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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Abstract
The invention relates to the field of antibacterial materials, in particular to a guanidino hyaluronic acid type antibacterial hydrogel, and a preparation method and application thereof. Through a series of reactions, alkyl guanidine with different chain lengths is successfully connected to a carboxyl end side chain of hyaluronic acid in a chemical bond mode, the antibacterial performance of the hyaluronic acid is endowed, and then the guanidine hyaluronic acid type antibacterial hydrogel is obtained through a ring-opening polymerization reaction by using a glycol diglycidyl ether cross-linking agent. The guanidine hyaluronic acid type antibacterial hydrogel can also effectively inhibit bacterial growth and prevent wound infection, is an excellent antibacterial gel auxiliary material, and has excellent antibacterial performance on gram-negative bacteria such as escherichia coli and gram-positive bacteria such as staphylococcus aureus; the cell compatibility is good; the reaction condition is easy to control, the cost is low, and the raw materials are easy to obtain; can be used for wound injury antibacterial dressing, and has good application prospect.
Description
Technical Field
The invention relates to the technical field of antibacterial materials, in particular to a guanidino hyaluronic acid type antibacterial hydrogel, a preparation method and application thereof.
Background
Skin wound injuries are common in production and life, such as burns and scalds, external force injuries, surgical traumas, electric injuries and the like. Wound healing is a very complex process, and is slow, during which bacterial infection and body fluid loss easily occur, endangering life health. Therefore, there is an urgent need for a wound dressing that functions to protect wounds, promote cell growth, and avoid bacterial infection during wound repair. Traditional wound dressing such as gauze and cotton wool have the defects that the open wound surface is difficult to attach, the wound surface cannot be kept moist, the wound surface is easy to infect, and the like, and the wound dressing is easy to adhere to the periphery of wound tissue, so that dehydration and secondary injury are caused. The hydrogel wound dressing is used as a novel medical dressing, has the advantages of softness, easiness in elasticity, transparency in permeation of oxygen and water, provision of wound healing environment and the like, and the hydrogel dressing based on natural biological polysaccharide has been widely paid attention to because of the advantages of good biocompatibility, easiness in degradation, abundant sources, difficulty in sensitization and the like.
Hyaluronic acid, a linear anionic acidic mucopolysaccharide with good biocompatibility, is widely distributed in the extracellular matrix of soft connective tissue of human and animals. Hyaluronic acid has good biocompatibility and degradability, is commonly used for researching the field of biological medicine, and is a natural macromolecular material with great potential. In recent years, the wound dressing is widely used for treating various wound injuries and repairing, and the application forms are mainly novel wound dressing such as hydrogel and the like. However, conventional hyaluronic acid gels, such as methacrylic acid hyaluronic acid hydrogel, have problems such as the need of various initiators upon crosslinking, and the susceptibility to introduction of highly cytotoxic impurities; the need to introduce additional functional groups for crosslinking, the crosslinking step is complex; moreover, the gel has the defects of weak mechanical strength, poor swelling performance and the like; there is also a slight disadvantage in combating bacterial infections.
Guanidino is a strongly basic group with high thermal and alkaline stability. The bond between the guanidine groups and the phosphate groups on the cell wall is much stronger than the bond between the amino groups and the phosphate groups when exposed to bacteria, resulting in a guanidine antibacterial agent with a higher antibacterial rate at lower human concentrations. Thus, the guanidine group can be introduced into the hyaluronic acid hydrogel structure to impart antibacterial properties thereto.
Therefore, how to develop a novel hyaluronic acid hydrogel wound dressing can ensure that the preparation method is simple and convenient, no exogenous high-cytotoxicity impurities are introduced, the mechanical property and the swelling property are excellent, and the novel hyaluronic acid hydrogel wound dressing also has high-efficiency long-term antibacterial property and is an urgent need of the market.
Disclosure of Invention
In order to solve the problems that the wound surface of the skin is extremely easy to be infected by bacteria and body fluid loss occurs, and the wound is difficult to heal due to inflammation. The invention provides a preparation method of a natural polysaccharide-based guanidyl hyaluronic acid type antibacterial hydrogel with a strong bactericidal and bacteriostatic effects. The preparation method of the guanidine hyaluronic acid type antibacterial hydrogel is simple and convenient, no exogenous high-cytotoxicity impurity is introduced, the mechanical strength is high, the guanidine hyaluronic acid type antibacterial hydrogel has broad-spectrum antibacterial capability, excellent swelling performance, high moisture retention, good biocompatibility and high safety, is not easy to generate drug resistance, can promote wound healing, and effectively overcomes the defects of the traditional hyaluronic acid hydrogel in application.
The invention adopts the following technical scheme:
the first object of the present invention is to provide a guanidino hyaluronic acid type antibacterial hydrogel prepared from guanidino hyaluronic acid (HA-RSG or HA-RBG) and a glycol diglycidyl ether crosslinking agent by ring-opening polymerization.
Further, the guanylated hyaluronic acid (HA-RSG or HA-RBG) is obtained by modifying alkyl monoguanidine or biguanide with different chain lengths through amidation reaction by carboxyl groups on Hyaluronic Acid (HA).
Further, the alkyl monoguanidine or biguanide with different chain lengths is obtained by substitution reaction of different alkyl amine side chains and a guanidyl donor.
Further, the molar ratio of the primary alcohol group of the guanylated hyaluronic acid (HA-RSG or HA-RBG) to the glycol diglycidyl ether crosslinking agent is 1 (1.0-2), and more preferably 1 (1.2-1.6).
The second object of the present invention is to provide a method for preparing a guanidine hyaluronic acid type antibacterial hydrogel, comprising the steps of:
(1) Amino alkylated hyaluronic acid (HA-R-NH) 2 ) Is prepared from the following steps: under the action of a coupling condensing agent, amidating reaction is carried out on hyaluronic acid and alkyl diamine, and the amino alkylated hyaluronic acid is obtained through sedimentation, dialysis and freeze-drying at room temperature overnight;
(2) Preparation of guanidino hyaluronic acid (HA-RSG or HA-RBG): at room temperature, catalyzing by N, N-Diisopropylethylamine (DIEA), carrying out substitution reaction on amino alkylated hyaluronic acid and a guanidyl donor, and carrying out sedimentation, dialysis and freeze-drying to obtain guanidyl hyaluronic acid, namely monoguanidine hyaluronic acid or biguanide hyaluronic acid;
(3) Preparation of guanidino hyaluronic acid type antibacterial hydrogel (HA-RG-Gel): dissolving the guanidyl hyaluronic acid in alkali liquor, regulating the pH of the system, adding a certain amount of glycol diglycidyl ether cross-linking agent, uniformly mixing, heating to obtain gel, washing the gel, and neutralizing the pH by adopting acid to obtain the guanidyl hyaluronic acid type antibacterial hydrogel.
Further, in the amidation reaction of the step (1), the molecular weight of the Hyaluronic Acid (HA) is 10-1000 k Da; the alkyl diamine is one of ethylenediamine, butanediamine, hexamethylenediamine and 1, 2-bis (2-aminoethoxy) ethane; the coupling condensing agent is dimethylaminopropyl carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS).
Further, in the amidation reaction of the step (1), the molar ratio of the carboxyl group on the hyaluronic acid to the alkyl diamine is 1 (1-2), more preferably 1 (1-1.2), and the reaction temperature is 25-80 ℃, more preferably 25-60 ℃. The reaction temperature is further optimized to be 25 ℃, and the reaction time is 10-25 h.
Further, in the substitution reaction of the step (2), the guanidino donor is 1H-pyrazole-1-carboxamidine hydrochloride or 1H-pyrazole-1-carboxamidine hydrochloride, and the molar ratio of the guanidino donor to the amino groups in the aminoalkylated hyaluronic acid is (1.5-2.5): 1, more preferably (1.8 to 2.2): 1, a step of; the mass of the catalyst N, N-Diisopropylethylamine (DIEA) is 5-10% of the amount of amino alkylated hyaluronic acid.
Further, the reaction system solvents in the step (1) and the step (2) are formamide and H 2 One of O.
Further, when the gel is prepared in the step (3), the concentration of the NaOH solution is 0.1-1M, and the pH of the system is adjusted to 6-10, and more preferably 7-8; the glycol diglycidyl ether cross-linking agent is one of Ethylene Glycol Diglycidyl Ether (EGDE), butanediol diglycidyl ether (BDDE) and polyethylene glycol diglycidyl ether (PEGDE); the reaction temperature is 40-80 ℃; 1 using ethanol and water: 1 washing the gel by the mixed solution, and neutralizing the pH of the gel to 6-8.
Further, the prepared guanidinium hyaluronic acid type antibacterial hydrogel is preserved by immersing in ultrapure water or PBS solution.
The third purpose of the invention is to provide the application of the guanidine hyaluronic acid type antibacterial hydrogel, which can be used as an antibacterial dressing for wound repair, such as wound caused by burns, scalds, external force injury, surgical trauma, electric injury and the like.
The invention has the beneficial effects that:
(1) The invention successfully introduces alkyl guanidine with different chain lengths into hyaluronic acid through a series of reactions, and endows the hyaluronic acid with antibacterial performance. Then using glycol diglycidyl ether cross-linking agents such as glycol diglycidyl ether (EGDE), butanediol diglycidyl ether (BDDE) and polyethylene glycol diglycidyl ether (PEGDE) to prepare the guanidino hyaluronic acid type antibacterial hydrogel, solving the problem of poor mechanical strength of the traditional hyaluronic acid hydrogel; compared with other hyaluronic acid hydrogels, such as methacrylic acid hyaluronic acid hydrogel, the method does not need to introduce additional functional groups for crosslinking, does not have initiator to participate in the reaction, and avoids introducing impurities with high cytotoxicity; moreover, the crosslinked network formed by the hydrogel is more hydrophilic than methacrylic hyaluronic acid hydrogel, and the crosslinked network shows higher swelling performance in aqueous solution; the mechanical properties are also greatly superior to those of the hyaluronic acid gel reported before.
(2) The guanidino hyaluronic acid type antibacterial hydrogel prepared by the invention is characterized in that guanidino and a hyaluronic acid main chain are connected in a covalent bond mode, the conventional guanidino compound and the hyaluronic acid are compounded in a physical method, such as a blending mode, for example, patent CN 113244437A and CN 101932301B, and the hydrogel is simple and convenient to prepare, but has the defects of uneven internal components, poor mechanical strength and the like.
(3) The guanidine hyaluronic acid type antibacterial hydrogel prepared by the invention enhances antibacterial performance through the synergistic effect of the monoguanidine or biguanide group of the glycosyl side chain and the alkyl chain, so that the guanidine hyaluronic acid type antibacterial hydrogel has excellent broad-spectrum antibacterial performance and shows excellent antibacterial performance on gram-negative bacteria such as escherichia coli and gram-positive bacteria such as staphylococcus aureus; the hydrogel reaction condition is easy to control, the cost is low, and the raw materials are easy to obtain; can be used for wound surface injury antibacterial dressing such as wound surface caused by burn and scald, external force injury, operation trauma, electric injury, etc., and has good reference significance for wound surface injury after repair.
(4) The guanidino hyaluronic acid type antibacterial hydrogel prepared by the invention is used as a novel wound dressing, is convenient for the treatment of wound surface wounds, can promote the repair and healing of inflammatory wound positions, and avoids bacterial infection. The invention adopts bioactive macromolecular hyaluronic acid as a framework, and can accelerate wound healing. After the prepared guanidino hyaluronic acid type antibacterial hydrogel is co-cultured with L-929 mouse fibroblasts, the cell survival rate is higher, and even exceeds that of a control group, so that the antibacterial hydrogel has excellent cell compatibility and can promote cell proliferation.
(6) The main raw material adopted by the invention is natural polysaccharide hyaluronic acid, and the method has the advantages of wide source, abundant reserves, easily obtained raw materials, lower cost, simple operation, mild condition, easy control, suitability for industrial production and wide application prospect.
Drawings
FIG. 1 is a schematic diagram of the synthetic route of the guanidine hyaluronic acid-type antibacterial hydrogel of the present invention.
FIG. 2 is a schematic structural view of the guanidine hyaluronic acid-type antibacterial hydrogel of the present invention. Wherein HA represents a long chain hyaluronic acid, RG represents an alkyl guanidine side chain, and EO represents a glycol diglycidyl ether chain.
FIG. 3 is a view of the ethylamine hyaluronic acid (HA-E-NH) of the present invention 2 ) The influence factor of the Grafting Ratio (GR), FIG. 3A shows the change in the molar ratio of Ethylenediamine (EDA) to HA carboxyl groups versus HA-E-NH at a reaction time of 12h 2 The effect of GR of (2); FIG. 3B shows the change in reaction time versus HA-E-NH when the molar ratio of EDA to HA carboxyl groups is 1 2 GR, is determined.
FIG. 4 shows Hyaluronic Acid (HA) and ethambutol hyaluronic acid (HA-E-NH) in examples 1 and 2 2 ) Nuclear magnetic resonance hydrogen spectra of ethanamine monoguanidine hyaluronic acid (HA-ESG) and ethanamine biguanide hyaluronic acid (HA-EBG).
FIG. 5 shows nuclear magnetic resonance carbon spectra of ethanamine monoguanidine hyaluronic acid (HA-ESG) and ethanamine biguanide hyaluronic acid (HA-EBG) in examples 1 and 2.
FIG. 6 is a graph showing swelling properties of guanidinium hyaluronic acid type antibacterial hydrogels (HA-ESG-Gel and HA-EBG-Gel) in examples 1 and 2.
FIG. 7 is a rheological property curve of the guanidinium hyaluronic acid type antibacterial hydrogels (HA-ESG-Gel and HA-EBG-Gel) of example 1 and example 2.
FIG. 8 is a graph showing the antibacterial effect of the guanidinium hyaluronic acid-type antibacterial hydrogels (HA-ESG-Gel and HA-EBG-Gel) of examples 1 and 2.
FIG. 9 is a graph showing cytotoxicity of guanidinium hyaluronan-type antibacterial hydrogels (HA-ESG-Gel and HA-EBG-Gel) of example 1 and example 2 against mouse fibroblast L-929.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments.
The synthetic route of the guanidine hyaluronic acid type antibacterial hydrogel is schematically shown in fig. 1. The design idea is as follows: in order to introduce guanidine groups into the hyaluronic acid chain, it is required that the side chains have primary amino groups, and the active groups on the side chains of the hyaluronic acid have only carboxyl groups for modification, so that the primary amino groups can be introduced into the hyaluronic acid chain through amide condensation reaction of alkyl diamine and carboxyl groups. It is subsequently crosslinked to form a hydrogel, which is more useful as a wound dressing.
Reaction mechanism:
amidation, i.e., the reaction of carboxyl groups with amine groups, requires the addition of a condensing agent to facilitate the reaction. The reaction principle is that carboxyl is activated and then reacts with amine to obtain amide, EDC is used as an amidation condensing agent, and NHS is used as an acylation catalyst to convert the amide into corresponding active ester or active amide because the intermediate generated in the first stage of the reaction is unstable, otherwise, the amide is easy to form urea.
When the hydrogel is crosslinked, ring-opening polymerization reaction is carried out on the double-end ethylene oxide group in the ethylene glycol diglycidyl ether and the primary alcohol group of the hyaluronic acid side chain to form a hydrogel network.
Example 1: preparation method of ethylamine monoguanidine hyaluronic acid Gel (HA-ESG-Gel)
Step S1: ethylamine hyaluronic acid (HA-E-NH) 2 ) Is synthesized by the following steps:
at room temperature, 4g of sodium hyaluronate (12 k Da HA, wherein the amount of carboxyl groups is 10 mmol) was dissolved in 40ml of deionized water and stirred well; 600mg of ethylenediamine (10 mmol) was dissolved in 10ml of pure water and added to the aforementioned HA solution. Adjusting the pH value of the reaction mixture to 7.5 by using 1M HCl solution, and stirring for later use; 2.304g of EDC (12 mmol) and 1.380g of NHS (12 mmol) were each dissolved in 10ml of pure water and mixed homogeneously. The mixed solution of HA and ethylenediamine with the pH value adjusted is slowly added into the mixed aqueous solution of EDC and NHS in a dropwise manner, and after the mixed aqueous solution is stirred uniformly under a magnetic stirrer, the pH value of a reaction system is kept at about 7.5 by using a 1M NaOH solution, and the reaction is carried out for 24 hours at room temperature. After the reaction was completed, the reaction solution was settled in absolute ethanol for 8 hours, the precipitate was washed with absolute ethanol for 2 times, the precipitate was redissolved with deionized water, and then dialyzed using a dialysis bag (mwco=3500), with ultrapure water being replaced every 8 hours, and dialyzed for 72 hours. Freeze-drying the dialyzed sample to obtain HA-E-NH 2 ;
Step S2: synthesis of ethylamine monoguanidine hyaluronic acid (HA-ESG):
at room temperature, 2g of HA-E-NH was first added 2 (the amount of amino groups was 2.4 mmol) was dissolved in 10ml of deionized water, and the solution was stirred with heating to complete dissolution. Then 0.5ml of N, N-Diisopropylethylamine (DIEA) was slowly added dropwise thereto. Then 703mg of 1H-pyrazole-1-carboxamidine hydrochloride (4.8 mmol) is slowly added in portions, the mixture is stirred uniformly to be completely dissolved, the mixture is stirred at 30 ℃ for 48 hours, after the reaction is finished, the reaction solution is slowly dripped into 12 times of absolute ethyl alcohol, and white precipitation is generatedSedimentation is carried out for 8 hours at 4 ℃. The supernatant was then discarded, the lower suspension was centrifuged at 7500rpm for 10min, the supernatant was discarded, and the pellet was washed 2 times with absolute ethanol. After washing, re-dissolving the precipitate by using deionized water, and then deep dialyzing the precipitate in deionized water for 72 hours by using a resin dialysis bag with the molecular weight of 3500, wherein the deionized water is replaced every 8 hours; and freeze-drying the dialyzed sample to obtain the HA-ESG.
Step S3: synthesis of ethylamine monoguanidine hyaluronic acid Gel (HA-ESG-Gel):
first 1g of HA-ESG (primary alcohol group 2 mmol) was dissolved in 4mL of 1M NaOH solution, and the pH of the solution was adjusted to 6 to 10, N 2 Protecting, stirring at 4deg.C overnight to ensure that HA-ESG is fully dissolved; then 522mg of ethanol solution of ethylene glycol diglycidyl ether (3 mmol,0.5mL of ethanol) was added, and the mixed solution was stirred at 60℃for 15 minutes; the gel obtained was put into an excess of distilled water/ethanol (volume ratio 1:1), and neutralized with 0.5M hydrochloric acid, and then the gel was washed with distilled water/ethanol (1:1 v/v) several times; finally, washing with distilled water instead, and freeze-drying to obtain HA-ESG-Gel.
Example 2: preparation of ethambutol biguanide hyaluronic acid Gel (HA-EBG-Gel)
Step S1: ethylamine hyaluronic acid (HA-E-NH) 2 ) Is synthesized by the following steps:
10g of sodium hyaluronate (260 kDa HA, 25mmol of carboxyl) was dissolved in 100ml of formamide at room temperature, stirred well, 1.8g of ethylenediamine (30 mmol) was dissolved in 10ml of pure water and added dropwise to the HA solution; adjusting the pH value of the reaction mixture to 7.5 by using 1M HCl solution, and stirring for later use; 4.518g of DCC (30 mmol) and 3.467g of NHS (30 mmol) are weighed and dissolved in 20ml of formamide respectively and mixed uniformly; slowly dripping the mixed solution of HA and ethylenediamine with the pH value regulated into the mixed aqueous solution of DCC and NHS, uniformly stirring under a magnetic stirrer, and then keeping the pH value of a reaction system at about 7.5 by using a 1M NaOH solution for reaction for 12 hours at 40 ℃; after the reaction, the reaction solution was settled in absolute ethanol for 8 hours, the precipitate was washed with absolute ethanol for 2 times, re-dissolved with deionized water, and then dialyzed with a dialysis bag (mwco=3500), with replacement every 8 hoursUltrapure water was dialyzed for 72 hours. Spray drying the dialyzed sample to obtain HA-E-NH 2 。
Step S2: synthesis of ethambutol hyaluronic acid (HA-EBG):
at room temperature, 8g of ethanamine hyaluronic acid (HA-E-NH) 2 The amino group was dissolved in an amount of 10 mmol) in 50ml of formamide, and stirred to dissolve completely. 2ml of N, N-Diisopropylethylamine (DIEA) was then slowly added dropwise thereto. 2.92g of 1H-pyrazole-1-dicarboxamidine hydrochloride (20 mmol) was weighed and added slowly to the reaction system in portions, stirred well to dissolve completely, and the mixture was stirred at 45℃for 24H. After the reaction was completed, the reaction solution was slowly dropped into 8 volumes of absolute ethanol, and white precipitate was generated and was settled at 4℃for 8 hours. The supernatant was then discarded, the lower suspension was centrifuged at 7500rpm for 10min, the supernatant was discarded, and the pellet was washed 2 times with absolute ethanol. After washing, the precipitate was redissolved with deionized water and then ultrafiltered. And freeze-drying the ultrafiltered sample to obtain the HA-EBG.
Step S3: synthesis of ethambutol hyaluronic acid Gel (HA-EBG-Gel):
firstly, 3g of HA-EBG (the amount of primary alcohol group is 6 mmol) is dissolved in 12mL of 1M NaOH solution, the pH of the solution is adjusted to 8-10, N 2 Protection, stirring overnight at 4℃ensured adequate dissolution of HA-EBG. Then, 1.566g of an ethanol solution (9 mmol,2mL of ethanol) of ethylene glycol diglycidyl ether was added, and the mixed solution was stirred at 50℃for 25 minutes. The gel obtained was put into an excess of distilled water/ethanol (volume ratio 1:1) and neutralized with 0.5M hydrochloric acid, and then the gel was washed with distilled water/ethanol (1:1 v/v) a plurality of times. Finally, washing with distilled water instead, and freeze-drying to obtain HA-EBG-Gel.
FIG. 4 shows Hyaluronic Acid (HA) and ethambutol hyaluronic acid (HA-E-NH) in examples 1 and 2 2 ) Nuclear magnetic resonance hydrogen spectra of ethanamine monoguanidine hyaluronic acid (HA-ESG) and ethanamine biguanide hyaluronic acid (HA-EBG).
In the presence of hyaluronic acid (HA-E-NH) 2 ) In the spectra, delta=2.8-3.2 ppm is the resonance peak of methylene hydrogen on ethylamine side chain, the appearance of the peak indicates successful reaction initiationAlkyl amine is added; in the ethylamine monoguanidine hyaluronic acid (HA-ESG) and ethylamine biguanide hyaluronic acid (HA-EBG) spectra, the characteristic peak of alkyl hydrogen at δ=2.8 to 3.2ppm disappeared, indicating successful introduction of guanidine group, the primary amino group on the side chain was fully monoguanidine or biguanidized, resulting in an increase in chemical shift of alkyl hydrogen near guanidine group on the alkyl side chain, and disappearance of the original characteristic peak.
FIG. 5 shows nuclear magnetic resonance carbon spectra of ethanamine monoguanidine hyaluronic acid (HA-ESG) and ethanamine biguanide hyaluronic acid (HA-EBG) in examples 1 and 2.
The presence of a single or double peak at δ=156 to 162ppm also demonstrates successful introduction of guanidinium groups.
Comparative example 1: synthesis of HA-ESG-Gel
Referring to the method of example 1, the only difference is that the amount of alkali is increased so that pH > 10, specific step S3:
1g of HA-ESG (primary alcohol group 2 mmol) was first dissolved in 10mL of 1M NaOH solution, N 2 Protecting, stirring at 4deg.C overnight to ensure that HA-ESG is fully dissolved; then 522mg of ethanol solution of ethylene glycol diglycidyl ether (3 mmol,0.5mL of ethanol) was added, and the mixed solution was stirred at 60℃for 15 minutes; the gel obtained was put into an excess of distilled water/ethanol (volume ratio 1:1), and neutralized with 0.5M hydrochloric acid, and then the gel was washed with distilled water/ethanol (1:1 v/v) several times; finally, washing with distilled water instead, and freeze-drying to obtain HA-ESG-Gel.
As a result, it was found that when the amount of alkali was too large, the gel color was yellow, the washing was not completely colorless, the appearance was not beautiful when used and the visualization was lowered when used as a wound dressing, and the wound condition was not observed at any time.
Comparative example 2: synthesis of HA-ESG-Gel
The procedure of example 1 is followed except that the amount of crosslinking agent is reduced so that the molar ratio of primary alcohol groups on the guanidinated hyaluronic acid (HA-RSG or HA-RBG) to glycol diglycidyl ether crosslinking agent is 1:0.8, specific step S3:
1g of HA-ESG (primary alcohol group 2 mmol) was first dissolved in 4mL of 1M NaOH solution and the solution was adjustedpH is 6-10, N 2 Protecting, stirring at 4deg.C overnight to ensure that HA-ESG is fully dissolved; then 278mg of ethanol solution of ethylene glycol diglycidyl ether (1.6 mmol,0.5ml ethanol) was added, and the mixed solution was stirred at 60℃for 15 minutes; the gel obtained was put into an excess of distilled water/ethanol (volume ratio 1:1), and neutralized with 0.5M hydrochloric acid, and then the gel was washed with distilled water/ethanol (1:1 v/v) several times; finally, washing is carried out with distilled water instead, followed by freeze-drying.
As a result, it was found that the gel morphology was broken after the amount of the crosslinking agent was reduced.
Comparative example 3: synthesis of HA-ESG-Gel:
referring to the method of example 1, the only difference is that the reaction time of the synthetic gel is reduced, specific step S3:
1g of HA-ESG (primary alcohol group 2 mmol) was first dissolved in 4mL of 1M NaOH solution to prepare a 20wt% solution, N 2 Protecting, stirring at 4deg.C overnight to ensure that HA-ESG is fully dissolved; then 522mg of ethanol solution of ethylene glycol diglycidyl ether (3 mmol,0.5mL of ethanol) was added, and the mixed solution was stirred at 60℃for 5 minutes; the gel obtained was put into an excess of distilled water/ethanol (volume ratio 1:1), and neutralized with 0.5M hydrochloric acid, and then the gel was washed with distilled water/ethanol (1:1 v/v) several times; finally, washing is carried out with distilled water instead, followed by freeze-drying.
As a result, it was found that the reaction system was still fluid, and the gel was not formed after the reaction time was too short.
Example 3: effect of molar ratio of Ethylenediamine (EDA) to HA on guanidine graft Rate
A preparation method of ethylamine monoguanidine hyaluronic acid (HA-ESG) comprises the steps of setting the reaction time to be 12h, and adjusting the EDA dosage in the step S1 to enable the molar ratio of EDA to HA carboxyl to be 0.4-2.0, specifically:
step S1: 1.2g of hyaluronic acid (HA, 10K Da, carboxyl group in an amount of 3 mmol) was completely dissolved in 10mL of ultrapure water to obtain an aqueous HA solution; 680mg of dimethylaminopropyl-ethyl-carbodiimide hydrochloride (EDC. HCl,3.6 mmol) and 480mg of N-hydroxysuccinimide (NHS, 3.6 mmol) were then added in portions to the aqueous HA solution, frequentlyStirring for 1h; then slowly dripping 72-360 mg of ethylenediamine (1.2-6 mmol) into the mixture, and stirring the mixture at 25 ℃ for reaction for 12 hours; after the reaction, the reaction solution is settled in absolute ethyl alcohol, the sediment is washed by absolute ethyl alcohol for 2 times, and then the hyaluronic acid-ethylenediamine (HA-E-NH) is obtained after filtration, washing and freeze drying 2 ) Is designated as HA-E-NH 2 The change chart of the Grafting Ratio (GR) of X is shown in figure 3A, and GR is calculated to be 20% -55%.
Step S2: 1g of HA-E-NH was first added at room temperature 2 (the amount of amino groups was about 1.2 mmol) was dissolved in 10ml of deionized water, and the solution was stirred with heating to complete dissolution. Then 0.5ml of N, N-Diisopropylethylamine (DIEA) was slowly added dropwise thereto. Then 352mg of 1H-pyrazole-1-carboxamidine hydrochloride (2.4 mmol) was slowly added in portions, stirred uniformly to dissolve completely, the mixture was stirred at 30℃for 48H, after the reaction was completed, the reaction solution was slowly added dropwise to 12 volumes of absolute ethanol, white precipitate was generated, and the mixture was settled at 4℃for 8H. The supernatant was then discarded, the lower suspension was centrifuged at 7500rpm for 10min, the supernatant was discarded, and the pellet was washed 2 times with absolute ethanol. After washing, re-dissolving the precipitate by using deionized water, and then deep dialyzing the precipitate in deionized water for 72 hours by using a resin dialysis bag with the molecular weight of 3500, wherein the deionized water is replaced every 8 hours; and freeze-drying the dialyzed sample to obtain the HA-ESG, wherein the grafting rate of the guanidine groups is the same as that of GR in S1.
Step S3: firstly, 1g of HA-ESG (the amount of primary alcohol groups is 2 mmol) is dissolved into 4mL of 1M NaOH solution, the pH of the solution is regulated to 6-10, N2 is used for protection, and stirring is carried out at 4 ℃ overnight to ensure that the HA-ESG is fully dissolved; then 522mg of ethanol solution of ethylene glycol diglycidyl ether (3 mmol,0.5mL of ethanol) was added, and the mixed solution was stirred at 60℃for 15 minutes; the gel obtained was put into an excess of distilled water/ethanol (volume ratio 1:1), and neutralized with 0.5M hydrochloric acid, and then the gel was washed with distilled water/ethanol (1:1 v/v) several times; finally, washing with distilled water instead, and freeze-drying to obtain HA-ESG-Gel.
The analysis in conjunction with fig. 3A yields:
in order to achieve a guanidine group grafting ratio of 45% or more, the molar ratio of EDA to HA carboxyl groups is preferably 1.0 to 2.0.
In order to achieve a grafting ratio of 50% or more and to achieve cost savings, it is preferable that the molar ratio of EDA to HA carboxyl groups is 1.0 to 1.2.
Example 4: effect of amidation reaction time on guanidine graft ratio
A preparation method of ethylamine monoguanidine hyaluronic acid (HA-ESG) comprises the following steps of adjusting reaction time in the step S1 to be 4-24 hours, specifically:
step S1: 1.2g of hyaluronic acid (HA, 10K Da, carboxyl group in an amount of 3 mmol) was completely dissolved in 10mL of ultrapure water to obtain an aqueous HA solution; 680mg of dimethylaminopropyl-ethyl-carbodiimide hydrochloride (EDC. HCl,3.6 mmol) and 480mg of N-hydroxysuccinimide (NHS, 3.6 mmol) were added in portions to the aqueous HA solution and stirred at ambient temperature for 1h; then slowly dripping 72-360 mg of ethylenediamine (3 mmol) into the mixture, and stirring the mixture at 25 ℃ for reaction for 4-24 hours; after the reaction, the reaction solution is settled in absolute ethyl alcohol, the sediment is washed by absolute ethyl alcohol for 2 times, and then the hyaluronic acid-ethylenediamine (HA-E-NH) is obtained after filtration, washing and freeze drying 2 ) Is designated as HA-E-NH 2 The change chart of the Grafting Ratio (GR) of X is shown in FIG. 3B, and GR is calculated to be 10% -50%.
Step S2: 1g of HA-E-NH was first added at room temperature 2 (the amount of amino groups was about 1.2 mmol) was dissolved in 10ml of deionized water, and the solution was stirred with heating to complete dissolution. Then 0.5ml of N, N-Diisopropylethylamine (DIEA) was slowly added dropwise thereto. Then 352mg of 1H-pyrazole-1-carboxamidine hydrochloride (2.4 mmol) was slowly added in portions, stirred uniformly to dissolve completely, the mixture was stirred at 30℃for 48H, after the reaction was completed, the reaction solution was slowly added dropwise to 12 volumes of absolute ethanol, white precipitate was generated, and the mixture was settled at 4℃for 8H. The supernatant was then discarded, the lower suspension was centrifuged at 7500rpm for 10min, the supernatant was discarded, and the pellet was washed 2 times with absolute ethanol. After washing, re-dissolving the precipitate by using deionized water, and then deep dialyzing the precipitate in deionized water for 72 hours by using a resin dialysis bag with the molecular weight of 3500, wherein the deionized water is replaced every 8 hours; and freeze-drying the dialyzed sample to obtain the HA-ESG, wherein the grafting rate of the guanidine groups is the same as that of GR in S1.
Step S3: firstly, 1g of HA-ESG (the amount of primary alcohol groups is 2 mmol) is dissolved into 4mL of 1M NaOH solution, the pH of the solution is regulated to 6-10, N2 is used for protection, and stirring is carried out at 4 ℃ overnight to ensure that the HA-ESG is fully dissolved; then 522mg of ethanol solution of ethylene glycol diglycidyl ether (3 mmol,0.5mL of ethanol) was added, and the mixed solution was stirred at 60℃for 15 minutes; the gel obtained was put into an excess of distilled water/ethanol (volume ratio 1:1), and neutralized with 0.5M hydrochloric acid, and then the gel was washed with distilled water/ethanol (1:1 v/v) several times; finally, washing with distilled water instead, and freeze-drying to obtain HA-ESG-Gel.
When the molar ratio of Ethylenediamine (EDA) to HA carboxyl groups is 1, the change in reaction time is relative to HA-E-NH 2 As shown in FIG. 3.B, the effect of GR on HA-E-NH was increased with the increase of the reaction time 2 The GR of (C) also increases rapidly, and when the reaction time reaches 12h, the GR reaches the maximum value, and the GR decreases slightly with the subsequent increase of the reaction time. Preferably, the amidation reaction time is 12 to 24 hours, in which case GR is 50% or more.
Example 5: swelling Performance test of guanidine-modified hyaluronic acid antibacterial gel
Cylindrical samples of guanidino-modified hyaluronic acid antibacterial gel (prepared by using a 48-well plate as a mold) were placed in pure water and PBS solution (0.1 m, ph=7.4), respectively, and tested in an incubator at 37 ℃. It was taken out of the pure water and PBS solution at regular intervals, and the surface water was removed with filter paper. The initial body weight (W) 0 ) And body weight after swelling for different periods of time (W t ) The Swelling ratio (Swelling ratio) was calculated.
Swelling ratio=W t /W 0
The equilibrium swelling ratio of hydrogels is an important factor in characterizing the hydrogel structure. The hydrogel has swelling property because of a large number of crosslinking points in the hydrogel, and can perform molecular chain segment movement, so that water molecules can fill the hydrogel.
FIG. 6 is a graph showing swelling properties of guanidinium hyaluronic acid type antibacterial hydrogels (HA-ESG-Gel and HA-EBG-Gel) in examples 1 and 2. As can be seen from the graph, when the treatment time reaches 36h, the HA-ESG-Gel and the HA-EBG-Gel basically reach the swelling balance in water, and the swelling rates of the HA-ESG-Gel and the HA-EBG-Gel in water are 24.3 and 23.8 respectively; when the treatment time reached 24h, the HA-ESG-Gel and HA-EBG-Gel reached a swelling equilibrium in water with PBS solution (0.1 m, ph=7.4) as medium, and the swelling rates in water were 20.5 and 20.2, respectively. Compared with other hyaluronic acid hydrogel products, such as methacrylic acid hydrogel in CN 106488772B or other biological guanidyl biological polysaccharide hydrogels, such as guanidyl chitosan hydrogel in CN 112980003A, the guanidyl hyaluronic acid type antibacterial gel has better swelling performance, can absorb wound tissue fluid such as blood water in time when being used as a wound dressing, ensures the wetting around a wound, reduces the risk of microbial infection caused by wound exudate accumulation, and is beneficial to wound healing recovery.
Example 6: rheological property test of guanidine-modified hyaluronic acid antibacterial gel
A cylindrical sample (prepared by taking a 48-pore plate as a die) of the guanidino modified hyaluronic acid antibacterial gel is placed on a base plate of a rheometer, the diameter of a cone plane is 7mm, and the distance between the cone plane and the base plate is kept at 1 mm. Dynamic strain scanning is carried out at room temperature within a certain time range, the linear viscoelasticity range of the hydrogel is determined, and the dynamic storage modulus (G ') and loss modulus (G') change curves are recorded.
FIG. 7 is a rheological property curve of the guanidinium hyaluronic acid type antibacterial hydrogels (HA-ESG-Gel and HA-EBG-Gel) of example 1 and example 2. From the figure, the storage modulus (elastic modulus) G 'of the HA-ESG-Gel and the HA-EBG-Gel are always larger than the loss modulus (viscous modulus) G', which indicates that the two guanidyl hyaluronic acid type antibacterial gels can maintain Gel state at 100-10000Pa, ensure the shape memory capability of the hydrogel in application, have extremely excellent mechanical properties, and can better maintain the wrapping capability on wounds. For example, the pure hyaluronic acid hydrogel in CN108853569a or the methacrylic acid hyaluronic acid hydrogel in CN 106488772B is easy to break under a larger pressure and cannot maintain the gel state, and the guanidino hyaluronic acid hydrogel provided by the invention effectively solves the problem.
Example 7: in vitro antibacterial performance test of guanidine-modified hyaluronic acid antibacterial gel
Diluting cultured Staphylococcus aureus (S.aureus) and Escherichia coli (E.coli) with LB liquid medium, and measuring absorbance (OD) of the bacterial liquid at wavelength of 630nm with a microplate reader 630 ) The bacterial liquid was diluted to an absorbance of about 0.1. Taking 10mL of bacterial liquid, respectively adding into sterilized test tubes, leaving one test tube without sample as control, respectively adding 0.1g of different guanidino hyaluronic acid hydrogel into the rest test tubes, setting three groups of parallel experiments, plugging a plug on a bottle mouth, placing into a constant-temperature oscillator, culturing at constant temperature of 37 ℃ and 200rpm, respectively sampling at 0h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 6h, 8h, 12h and 24h, and measuring OD of the bacterial liquid by an enzyme-labeling instrument 630 。
FIG. 8 is a graph showing the antibacterial effect of the guanidinium hyaluronic acid-type antibacterial hydrogels (HA-ESG-Gel and HA-EBG-Gel) of examples 1 and 2. As can be seen from the graph, the antibacterial performance of the HA-ESG-Gel and the HA-EBG-Gel can be maintained for at least 24 hours, and the OD630 of the bacterial liquid is obviously reduced within 2 hours. The guanidine hyaluronic acid type antibacterial hydrogel gel has extremely strong antibacterial performance and long retention time. When used as wound dressing, can effectively inhibit bacterial infection of wound. The invention provides the antibacterial property of the hyaluronic acid hydrogel by modifying the hyaluronic acid with alkyl guanidine, and the antibacterial capability of the hyaluronic acid hydrogel is very excellent and stronger than that of other guanidine hydrogels, such as guanidine chitosan hydrogel in CN 112980003A.
Example 8: cytotoxicity test of guanidino hyaluronic acid hydrogel
The radiation sterilized HA-ESG-Gel and HA-EBG-Gel hydrogels were soaked in DMEM medium (10% fetal bovine serum, 100IU/mL penicillin, 100. Mu.g/mL streptomycin) for 1 day to obtain a leaching solution, and diluted 10-fold. 100. Mu.L of L-929 mouse fibroblast suspension was inoculated into 96-cell well plates, and 100. Mu.L of each of the two diluted extracts was added thereto at 37℃and 5%CO 2 The culture medium is changed every two days. Cell culture medium was aspirated at 24h, 48h, 72h of incubation, and 10% CCK-8 (CCK 8/DMEM) solution containing 100. Mu.L was added to each well. The well plate was returned to the cell incubator for continuous culture for 1 hour, the CCK-8 reaction solution of the well plate was transferred to another 96-cell well plate, 100. Mu.L of the reaction solution was added to each well, and the absorbance (OD value) was immediately measured at 492nm wavelength of the microplate reader. The group without added material is set as a blank group. Cell viability was calculated as follows:
cell viability (%) = (experimental OD value-negative control OD value)/(blank OD value-negative control OD value) ×100%.
FIG. 9 is a graph showing cytotoxicity of guanidinium hyaluronan-type antibacterial hydrogels (HA-ESG-Gel and HA-EBG-Gel) of example 1 and example 2 against mouse fibroblast L-929. From the graph, the cell viability of the HA-ESG-Gel and the HA-EBG-Gel is high in 72 hours of culture, and even exceeds that of a control group, so that the antibacterial hydrogel HAs excellent cell compatibility and can promote cell proliferation. In acting as a wound dressing, it can promote wound healing, and compared with other non-hyaluronic acid hydrogels, such as alginate hydrogels in CN 112980003A, there is no obvious effect of promoting cell proliferation.
Claims (7)
1. A method for preparing a guanidino hyaluronic acid type antibacterial hydrogel, comprising the steps of:
(1) Preparation of aminoalkylated hyaluronic acid: under the action of a coupling condensing agent, amidating reaction is carried out on hyaluronic acid and alkyl diamine, and the amino alkylated hyaluronic acid is obtained through sedimentation, dialysis and freeze-drying at room temperature overnight;
(2) Preparation of guanidinated hyaluronic acid: at room temperature, catalyzing N, N-diisopropylethylamine, carrying out substitution reaction on amino alkylated hyaluronic acid and a guanidino donor, and carrying out sedimentation, dialysis and freeze-drying to obtain guanidino hyaluronic acid-monoguanidine hyaluronic acid or biguanide hyaluronic acid;
(3) Preparation of guanidino hyaluronic acid type antibacterial hydrogel: dissolving guanylated hyaluronic acid in a proper amount of NaOH solution, regulating the pH of the system to 6-10, adding a glycol diglycidyl ether cross-linking agent, uniformly mixing, heating to obtain gel, washing the gel, and neutralizing the pH with acid to obtain the guanylated hyaluronic acid type antibacterial hydrogel;
wherein, the mol ratio of the primary alcohol group of the guanidyl hyaluronic acid to the glycol diglycidyl ether cross-linking agent is 1: (1.0-2);
in the amidation reaction in the step (1), the molar ratio of carboxyl on the hyaluronic acid to the alkyl diamine is 1 (1-1.2);
the reaction time in the amidation reaction of the step (1) is 12h;
the glycol diglycidyl ether cross-linking agent is ethylene glycol diglycidyl ether;
the heating time in the step (3) is 15 minutes, and the heating temperature is 60 ℃.
2. The method according to claim 1, wherein in the amidation reaction in the step (1), the reaction temperature is 25℃and the reaction time is 12 h.
3. The method of claim 2, wherein the NaOH solution concentration is 0.1-1 m during the gel preparation in step (3).
4. The method according to claim 2, wherein in the substitution reaction of step (2), the guanidino donor is 1H-pyrazole-1-carboxamidine hydrochloride or 1H-pyrazole-1-carboxamidine hydrochloride; the molar amount ratio of the guanidyl donor to the amino groups in the amino alkylated hyaluronic acid is (1.5-2.5): 1, a step of; the mass of the N, N-diisopropylethylamine is 5-10% of the mass of the amino alkylated hyaluronic acid.
5. The method according to claim 2, wherein in step (3) 1 of ethanol and water is used: 1. and washing the gel by the mixed solution, and neutralizing the pH of the gel to be 6-8.
6. The method according to claim 2, wherein the prepared guanidinium hyaluronic acid-type antibacterial hydrogel is preserved by immersing in ultrapure water or PBS solution.
7. Use of the guanidino hyaluronic acid type antibacterial hydrogel prepared by the method of any of claims 1-6 in the preparation of wound adjuvant.
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6831172B1 (en) * | 1998-11-11 | 2004-12-14 | Farmila-Thea Farmaceutici S.P.A. | Cross-linked hyaluronic acids and medical uses thereof |
CN102675651A (en) * | 2012-04-20 | 2012-09-19 | 常州华联保健敷料有限公司 | Preparation method of chitosan hydrogel for antiseptic dressing |
CN102690373A (en) * | 2012-06-21 | 2012-09-26 | 南京大学 | Preparation method of guanidinylated chitosan |
WO2017141204A2 (en) * | 2016-02-17 | 2017-08-24 | Chindera Kantaraja | Biguanide grafted / modified biopolymers as drug delivery vehicles |
CN108853569A (en) * | 2018-06-27 | 2018-11-23 | 海南大学 | A kind of covalent cross-linking hyaluronic acid aeroge and its hydrogel and preparation method |
CN109535430A (en) * | 2017-09-22 | 2019-03-29 | 天津大学 | A kind of amphipathic derivatives and preparation method thereof of olelated chitosan oligosaccharide guanidine grafting polyethylene imine |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11389539B2 (en) * | 2016-05-11 | 2022-07-19 | Chugai Seiyaku Kabushiki Kaisha | Hyaluronic acid derivatives into which cationic and hydrophobic groups are introduced |
-
2022
- 2022-02-28 CN CN202210188225.9A patent/CN114561046B/en active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6831172B1 (en) * | 1998-11-11 | 2004-12-14 | Farmila-Thea Farmaceutici S.P.A. | Cross-linked hyaluronic acids and medical uses thereof |
CN102675651A (en) * | 2012-04-20 | 2012-09-19 | 常州华联保健敷料有限公司 | Preparation method of chitosan hydrogel for antiseptic dressing |
CN102690373A (en) * | 2012-06-21 | 2012-09-26 | 南京大学 | Preparation method of guanidinylated chitosan |
WO2017141204A2 (en) * | 2016-02-17 | 2017-08-24 | Chindera Kantaraja | Biguanide grafted / modified biopolymers as drug delivery vehicles |
CN109535430A (en) * | 2017-09-22 | 2019-03-29 | 天津大学 | A kind of amphipathic derivatives and preparation method thereof of olelated chitosan oligosaccharide guanidine grafting polyethylene imine |
CN108853569A (en) * | 2018-06-27 | 2018-11-23 | 海南大学 | A kind of covalent cross-linking hyaluronic acid aeroge and its hydrogel and preparation method |
Non-Patent Citations (3)
Title |
---|
刘瑞雪等."透明质酸".《高分子材料》.河南大学出版社,2018, * |
姚日生."HA".《药用高分子材料 第2版》.化学工业出版社,2008, * |
徐开俊等."水凝胶".《物理化学 第3版》.中国医药科技出版社,2019, * |
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