A long-acting antibacterial hydrogel containing polyguanidine polymer and preparation method thereof

Abstract

The present invention discloses a long-acting antibacterial hydrogel containing a polyguanidine polymer and a preparation method, and belongs to the field of hydrogels. The present invention first performs aldehyde modification on hyaluronic acid (HA) to obtain aldehyded hyaluronic acid (OHA); then the aldehyded hyaluronic acid (OHA) and the amino group of a chitosan derivative / polyguanidine polymer undergo a Schiff base chemical cross-linking reaction to obtain a long-acting antibacterial hydrogel. The long-acting antibacterial hydrogel prepared by the present invention has good controllability and durability of release, does not affect its mechanical properties, and has a wide range of applications.

Description

Technical Field

[0001] The invention relates to a long-acting antibacterial hydrogel containing a polyguanidine macromolecular polymer and a preparation method thereof, and belongs to the field of antibacterial hydrogel dressings. Background Art

[0002] The skin is the largest organ in the human body, including the epidermis, dermis and subcutaneous tissue. In general, the main function of the skin is to inhibit the loss of water, electrolytes and nutrients in the body, and to a certain extent defend against external mechanical, chemical, biological and physical stimuli, while also preventing the invasion of bacteria and fungi on the skin surface. Normal skin will be damaged and traumatized by external factors, which will damage the structural integrity and protective function of the skin, lose its ability to protect the body, and lead to infection risks. Bacterial infection at the wound site will delay the healing process, leading to necrosis, sepsis, and even death (Wang J, Duan X, Zhong D, Zhang M, et al. International journal of biological macromolecules, 2023, 261 (1): 129064).

[0003] Wound dressings with good antibacterial properties can prevent wound infection. An ideal wound dressing should be able to absorb exudate from the wound surface while maintaining a moist environment in the wound to prevent the wound surface from drying out and becoming overly moist. At the same time, it isolates the influence of external factors such as dust and bacteria, and ultimately promotes wound healing. Antibacterial hydrogels have good biocompatibility, high water content, porous structure, suitable physical and chemical properties, and mechanical properties similar to those of extracellular matrix, making them show good application potential in wound hemostasis, tissue regeneration, and wound healing (Yang K, Han Q, Chen B, et al. International journal of nanomedicine, 2023, 20(5): 2217–2263).

[0004] At present, conventional antibacterial hydrogels are prepared by embedding antibacterial substances (such as metal nanoparticles, antibiotics, antimicrobial peptides, etc.) into or combining them with polymer networks. For example, by embedding metal nanoparticles (such as silver nanoparticles AgNPs) into the hydrogel network, the antibacterial properties of metal particles are used to achieve long-term antibacterial effects; antibiotics (such as ciprofloxacin, gentamicin, etc.) are combined with hydrogel matrices (such as carbomer) to achieve antibacterial effects by controlling the release of antibiotics; antimicrobial peptides (AMPs) are combined with hydrogels (S.Li et al.) , S. Dong, W. Xu, S. Tu, L. Yan, C. Zhao, J. Ding, X. Chen, Adv. Sci. 2018, 5, 1700527. https: / / doi.org / 10.1002 / advs.553); however, the antibacterial hydrogels prepared by these methods have poor controllability and durability in release, and will quickly release antibacterial agents in the early stage, and then lose antibacterial properties, thereby affecting wound healing and even causing wound infection, which is not conducive to wound repair. Moreover, improving antibacterial properties will affect the mechanical properties of hydrogels, thereby limiting the scope of application.

[0005] Therefore, how to obtain a hydrogel with sustained-release function and long-lasting antibacterial function has become a technical problem to be solved urgently in this field. Summary of the invention

[0006] [Technical issues]

[0007] The release controllability and durability of antimicrobial hydrogels are poor;

[0008] Improving antibacterial properties will affect the mechanical properties of hydrogels, thus limiting their application range.

[0009] [Technical solution]

[0010] In order to solve the above problems, the present invention first performs aldehyde modification on hyaluronic acid (HA) to obtain aldehyded hyaluronic acid (OHA); then the aldehyded hyaluronic acid (OHA) and the amino group of chitosan derivative / polyguanidine polymer undergo Schiff base chemical cross-linking reaction to obtain a long-acting antibacterial hydrogel. The long-acting antibacterial hydrogel prepared by the present invention has good controllability and durability of release, does not affect its mechanical properties, and has a wide range of applications.

[0011] The first object of the present invention is to provide a method for preparing a long-acting antibacterial hydrogel containing a polyguanidine polymer, comprising the following steps:

[0012] (1) dispersing aldehyded hyaluronic acid (OHA) in water to obtain aqueous solution I;

[0013] (2) dispersing the polyguanidine polymer and the chitosan derivative in water to obtain aqueous solution II;

[0014] (3) The aqueous solution I and the aqueous solution II are mixed to carry out a Schiff base reaction to obtain a long-lasting antibacterial hydrogel.

[0015] In one embodiment of the present invention, the aldehyde-modified hyaluronic acid (OHA) in step (1) is prepared by a sodium periodate oxidation method, as follows:

[0016] Dissolving hyaluronic acid (HA) in water to obtain a hyaluronic acid (HA) aqueous solution; wherein the amount ratio of hyaluronic acid (HA) to water is 1 g: 10 mL;

[0017] Dissolve sodium periodate in water to obtain a sodium periodate aqueous solution; wherein the ratio of sodium periodate to water is 0.54 g:1 mL;

[0018] Slowly add sodium periodate aqueous solution dropwise to hyaluronic acid (HA) aqueous solution to react in the dark; after the reaction is completed, add ethylene glycol to quench the unreacted sodium periodate, dialyze, and freeze-dry to obtain aldehyded hyaluronic acid (OHA); wherein the reaction in the dark is carried out at 20-30°C (room temperature) for 1 hour, and the dosage ratio of sodium periodate to ethylene glycol is 0.54g:1mL.

[0019] In one embodiment of the present invention, the molecular weight of hyaluronic acid (HA) in step (1) is between 50 kDa and 5000 kDa.

[0020] In one embodiment of the present invention, the concentration of aldehyded hyaluronic acid (OHA) in the aqueous solution I of step (1) is 1-8% (w / v, g / mL).

[0021] In one embodiment of the present invention, the polyguanidine polymer in step (2) is one or both of polyhexamethylene biguanidine hydrochloride and polyhexamethylene monoguanidine hydrochloride.

[0022] In one embodiment of the present invention, the chitosan derivative in step (2) is one or more chitosans such as chitosan, carboxymethyl chitosan, hydroxypropyl chitosan, hydroxybutyl chitosan, etc.

[0023] In one embodiment of the present invention, the amino content of the chitosan derivative in step (2) is 0.5-15 mmol / g, more preferably 2-6 mmol / g.

[0024] In one embodiment of the present invention, the concentration of the polyguanidine polymer in the aqueous solution II of step (2) is 0.1-2% (w / v, g / mL), and the concentration of the chitosan derivative is 1-8% (w / v, g / mL); % is based on the ratio of the polyguanidine polymer or the chitosan derivative to water.

[0025] In one embodiment of the present invention, the volume ratio of aqueous solution I to aqueous solution II in step (3) is 1:10-10:1, preferably 1:3-3:1.

[0026] In one embodiment of the present invention, the Schiff base reaction in step (3) is carried out at 20-30° C. (normal temperature) for 4-10 min.

[0027] The second object of the present invention is the long-acting antibacterial hydrogel prepared by the method of the present invention.

[0028] The third object of the present invention is to use the long-acting antibacterial hydrogel of the present invention in drug delivery, food packaging or medical dressing.

[0029] The fourth object of the present invention is to provide a wound covering, which adopts the long-acting antibacterial hydrogel of the present invention.

[0030] A fifth object of the present invention is to provide a wound dressing, which uses the long-acting antibacterial hydrogel described in the present invention.

[0031] [Beneficial Effects]

[0032] (1) The long-acting antibacterial hydrogel prepared by the present invention can be directly applied and adhered to the skin, and can provide appropriate humidity and physical barriers.

[0033] (2) The long-acting antibacterial hydrogel prepared by the present invention is completely biodegradable, and the polysaccharide component is beneficial to skin repair.

[0034] (3) During the preparation process of the long-acting antibacterial hydrogel prepared by the present invention, a polyguanidine polymer antibacterial agent is introduced into the hydrogel through a Schiff base chemical cross-linking reaction, thereby effectively extending the antibacterial effect time of the hydrogel.

[0035] (4) The long-acting antibacterial hydrogel prepared by the present invention has good controllability and durability in release, does not affect its mechanical properties, and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a reaction mechanism diagram for preparing the long-acting antibacterial hydrogel of the present invention. DETAILED DESCRIPTION

[0037] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0038] Test method:

[0039] 1. Cytotoxicity test:

[0040] The sample was mixed with MES buffer at a mass ratio of 1:5 to extract the extract, and the cytotoxicity of the extract was characterized by the MTT method.

[0041] 2. Antibacterial performance test:

[0042] Escherichia coli (10 4 cfu / mL) and Staphylococcus aureus (10 4 cfu / mL) as working bacterial solution, take 0.1mL of working bacterial solution to 5mL of test sample and mix well, take 1.0mL of mixed solution to 9mL of diluent at the test time, dilute in sequence, take 1mL to a sterile plate in parallel and then pour the culture medium, incubate at 35±2℃ for 48h to count the viable bacteria, and calculate the antibacterial rate of the sample according to the following formula:

[0043] X=(A–B) / A×100%

[0044] Wherein, X: inhibition rate, %; A: average colony count of control samples; : average colony count of tested samples.

[0045] Evaluation criteria:

[0046] If the antibacterial rate is ≥50-90%, the product has antibacterial effect; if the antibacterial rate is ≥90%, the product has a strong antibacterial effect.

[0047] 3. Long-term antibacterial test:

[0048] After rubbing the arm evenly, take a test sample (5cm*5cm) and stick it on the divided arm surface. Take the sample at intervals from 0h to 48h. Use a sterile cotton swab to take standardized samples at the arm patch. Then dilute and pour the parallel sampling liquid for culture. After culture at 37±2℃ for 24h, count and calculate the long-term antibacterial effect of the test sample.

[0049] 4. Mechanical properties test:

[0050] The 1 mm thick hydrogel was cut into 4*50 mm dumbbell-shaped tensile test samples and tested using a universal testing machine at a speed of 100 mm / min.

[0051] 5. Washing test:

[0052] Prepare 5 hydrogels of the same size, each weighing about 5 g, rinse them in water 3 times, and test the antibacterial effect after rinsing.

[0053] The raw materials used in the examples are:

[0054] Hyaluronic acid: molecular weight 40kDa-4000kDa, purchased from Bloomage Biotech Co., Ltd.;

[0055] Carboxymethyl chitosan; amino content 2-6 mmol / g, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0056] Hydroxypropyl chitosan: amino content of 2-6 mmol / g, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0057] Hydroxybutyl chitosan: amino content of 2-6 mmol / g, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0058] Polyhexamethylene biguanide hydrochloride: purity 98%, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.;

[0059] Aldehyde hyaluronic acid (OHA): prepared by sodium periodate oxidation method; details are as follows:

[0060] Dissolve 1 g of hyaluronic acid (HA) in 10 mL of deionized water to obtain a hyaluronic acid (HA) aqueous solution;

[0061] Dissolve 0.54 g of sodium periodate in 1 mL of deionized water to obtain a sodium periodate aqueous solution;

[0062] The sodium periodate aqueous solution was slowly added dropwise to the hyaluronic acid (HA) aqueous solution, and the reaction was carried out in the dark at 25°C (room temperature) for 1 hour. After the reaction was completed, 1 mL of ethylene glycol was added to quench the unreacted sodium periodate. The reaction solution was then dialyzed in deionized water for 3 days and freeze-dried to obtain aldehyded hyaluronic acid (OHA).

[0063] The w / v involved in the examples is g / mL, and % is the ratio of a substance to the solvent (water).

[0064] Example 1

[0065] A method for preparing a long-acting antibacterial hydrogel containing a polyguanidine polymer comprises the following steps:

[0066] (1) dispersing aldehyded hyaluronic acid (OHA) in water to obtain a 2% w / v aqueous solution I;

[0067] (2) dispersing polyhexamethylene biguanide hydrochloride and carboxymethyl chitosan in water to obtain aqueous solution II in which the concentration of polyhexamethylene biguanide hydrochloride is 0.1% w / v and the concentration of carboxymethyl chitosan is 2% w / v;

[0068] (3) Aqueous solution I and aqueous solution II were mixed in a volume ratio of 1:1, and allowed to stand at 25°C (room temperature) for 5 minutes to undergo a Schiff base reaction to obtain a long-lasting antibacterial hydrogel.

[0069] Example 2

[0070] A method for preparing a long-acting antibacterial hydrogel containing a polyguanidine polymer comprises the following steps:

[0071] (1) dispersing aldehyded hyaluronic acid (OHA) in water to obtain a 4% w / v aqueous solution I;

[0072] (2) dispersing polyhexamethylene biguanide hydrochloride and carboxymethyl chitosan in water to obtain aqueous solution II in which the concentration of polyhexamethylene biguanide hydrochloride is 0.1% w / v and the concentration of carboxymethyl chitosan is 4% w / v;

[0073] (3) Aqueous solution I and aqueous solution II were mixed in a volume ratio of 1:1, and allowed to stand at 25°C (room temperature) for 5 minutes to undergo a Schiff base reaction to obtain a long-lasting antibacterial hydrogel.

[0074] Example 3

[0075] A method for preparing a long-acting antibacterial hydrogel containing a polyguanidine polymer comprises the following steps:

[0076] (1) dispersing aldehyded hyaluronic acid (OHA) in water to obtain a 4% w / v aqueous solution I;

[0077] (2) dispersing polyhexamethylene biguanide hydrochloride and carboxymethyl chitosan in water to obtain aqueous solution II in which the concentration of polyhexamethylene biguanide hydrochloride is 0.5% w / v and the concentration of carboxymethyl chitosan is 4% w / v;

[0078] (3) Aqueous solution I and aqueous solution II were mixed in a volume ratio of 1:1, and allowed to stand at 25°C (room temperature) for 5 minutes to undergo a Schiff base reaction to obtain a long-lasting antibacterial hydrogel.

[0079] Example 4

[0080] A method for preparing a long-acting antibacterial hydrogel containing a polyguanidine polymer comprises the following steps:

[0081] (1) dispersing aldehyded hyaluronic acid (OHA) in water to obtain a 4% w / v aqueous solution I;

[0082] (2) dispersing polyhexamethylene biguanide hydrochloride and carboxymethyl chitosan in water to obtain aqueous solution II in which the concentration of polyhexamethylene biguanide hydrochloride is 2% w / v and the concentration of carboxymethyl chitosan is 4% w / v;

[0083] (3) Aqueous solution I and aqueous solution II were mixed in a volume ratio of 1:1, and allowed to stand at 25°C (room temperature) for 5 minutes to undergo a Schiff base reaction to obtain a long-lasting antibacterial hydrogel.

[0084] Example 5

[0085] The carboxymethyl chitosan in step (2) of Example 3 was adjusted to hydroxypropyl chitosan, and the other steps were kept consistent with Example 3 to obtain a long-acting antibacterial hydrogel.

[0086] Example 6

[0087] The carboxymethyl chitosan in step (2) of Example 3 was adjusted to hydroxybutyl chitosan, and the other parts were kept consistent with Example 3 to obtain a long-acting antibacterial hydrogel.

[0088] Comparative Example 1

[0089] The polyhexamethylene biguanide hydrochloride in step (2) of Example 3 was omitted, and the other steps were the same as those of Example 3 to obtain a hydrogel.

[0090] Comparative Example 2

[0091] The polyhexamethylene biguanide hydrochloride in step (2) of Example 3 was adjusted to dodecyltrimethylammonium chloride, and the rest was kept the same as Example 3 to obtain a hydrogel.

[0092] Comparative Example 3

[0093] The carboxymethyl chitosan in step (2) of Example 3 was omitted, and the other steps were the same as those of Example 3 to obtain a hydrogel.

[0094] Comparative Example 4

[0095] A method for preparing an antibacterial hydrogel comprises the following steps:

[0096] Aldehydated hyaluronic acid (OHA), polyhexamethylene biguanide hydrochloride, and carboxymethyl chitosan were dispersed in water and allowed to stand at 25°C (normal temperature) for 5 minutes for Schiff base reaction to obtain a hydrogel;

[0097] The concentration of polyhexamethylene biguanide hydrochloride was 0.2% w / v, the concentration of carboxymethyl chitosan was 2% w / v, and the concentration of aldehyded hyaluronic acid (OHA) was 2% w / v.

[0098] Comparative Example 5

[0099] A method for preparing an antibacterial hydrogel comprises the following steps:

[0100] Aldehydated hyaluronic acid (OHA) and carboxymethyl chitosan were dispersed in PBS buffer and allowed to stand at 25°C (room temperature) for 5 min for Schiff base reaction to obtain a hydrogel;

[0101] The concentration of carboxymethyl chitosan is 2% w / v, and the concentration of aldehyded hyaluronic acid (OHA) is 2% w / v.

[0102] Comparative Example 6

[0103] The carboxymethyl chitosan in step (2) of Example 3 was adjusted to chitosan, and the other steps were kept consistent with Example 3 to obtain a hydrogel.

[0104] The obtained hydrogel was subjected to performance testing, and the test results are as follows:

[0105] (1) Cytotoxicity:

[0106] After testing, the hydrogels prepared in Examples 1-6 and Comparative Examples 1-6 were non-cytotoxic.

[0107] (2) Antibacterial properties:

[0108] Table 1 shows the antibacterial performance test results. It can be seen from Table 1 that Examples 1-6 all have good antibacterial performance, and the antibacterial performance of the hydrogel without adding an antibacterial agent is poor.

[0109] Table 1 Antibacterial rate of hydrogel

[0110]

[0111] (3) Long-term antibacterial effect:

[0112] Table 2 shows the long-term performance test results. It can be seen from Table 2 that Examples 1-6 have good long-term antibacterial performance, and Examples 3-6 have no microbial growth after 48 hours.

[0113] Table 2 Long-term antibacterial properties of hydrogels

[0114]

[0115] Note: “-” represents no microbial growth; “*” represents bacterial colony count <10; “**” represents bacterial colony count <20; “***” represents bacterial colony count ≥20 or colony agglomeration.

[0116] (4) Mechanical properties:

[0117] Table 3 shows the mechanical property test results. It can be seen from Table 3 that Examples 2-6 and Comparative Examples 1, 2, 3, and 5 have good mechanical properties; the solid content in Example 1 is low, and its tensile strength and elongation are both reduced; Comparative Example 3 lacks carboxymethyl chitosan, and the prepared hydrogel has almost no tensile strength; in Comparative Example 4, the Schiff base reaction begins to occur when the raw materials are dissolved, resulting in uneven gelation, resulting in poor mechanical properties.

[0118] Table 3 Mechanical properties of hydrogels

[0119]

[0120] (5) Water washing test

[0121] Table 4 shows the antibacterial test of the hydrogel after washing. It can be seen that Examples 1-6 have a high antibacterial rate after washing, while the antibacterial rate of Comparative Examples 1-6 decreases significantly after washing, indicating that the antibacterial active ingredients in the hydrogel are washed away.

[0122] Table 4 Antibacterial rate of hydrogel after washing

[0123]

[0124]

[0125] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing a long-acting antibacterial hydrogel containing a polyguanidine polymer, characterized in that: The steps include: (1) dispersing aldehyde-modified hyaluronic acid (OHA) in water to obtain aqueous solution I; wherein the concentration of aldehyde-modified hyaluronic acid (OHA) in aqueous solution I is 1-8% w / v; (2) dispersing a polyguanidine polymer and a chitosan derivative in water to obtain an aqueous solution II; wherein the polyguanidine polymer is one or both of polyhexamethylene biguanidine hydrochloride and polyhexamethylene monoguanidine hydrochloride; and the chitosan derivative is one or more of carboxymethyl chitosan, hydroxypropyl chitosan, and hydroxybutyl chitosan; and the concentration of the polyguanidine polymer in the aqueous solution II is 0.1-2% w / v, and the concentration of the chitosan derivative is 1-8% w / v; (3) mixing aqueous solution I and aqueous solution II, and performing Schiff base reaction to obtain a long-lasting antibacterial hydrogel; The volume ratio of aqueous solution I to aqueous solution II is 1:10-10:1; the Schiff base reaction is carried out at 20-30°C for 4-10 minutes.

2. The method according to claim 1, characterized in that The amino content of the chitosan derivative in step (2) is 0.5-15 mmol / g.

3. The method according to claim 1, characterized in that In step (1), the aldehyde-modified hyaluronic acid (OHA) is prepared by a sodium periodate oxidation method.

4. The method according to claim 3, characterized in that The preparation method of aldehyde-modified hyaluronic acid OHA in step (1) is as follows: Dissolving hyaluronic acid HA in water to obtain a hyaluronic acid HA aqueous solution; wherein the dosage ratio of hyaluronic acid HA to water is 1 g: 10 mL; Dissolve sodium periodate in water to obtain a sodium periodate aqueous solution; wherein the ratio of sodium periodate to water is 0.54 g:1 mL; Slowly add sodium periodate aqueous solution to hyaluronic acid HA aqueous solution and react in the dark; after the reaction is completed, add ethylene glycol to quench the unreacted sodium periodate, dialyze, and freeze-dry to obtain aldehyded hyaluronic acid OHA; the reaction in the dark is carried out at 20-30°C for 1 hour, and the dosage ratio of sodium periodate to ethylene glycol is 0.54g:1mL.

5. The method according to claim 4, characterized in that The molecular weight of hyaluronic acid HA is between 50kDa and 5000kDa.

6. The method according to claim 1, characterized in that In step (3), the volume ratio of aqueous solution I to aqueous solution II is 1:3-3:

1.

7. The long-acting antibacterial hydrogel prepared by the method according to any one of claims 1 to 6.

8. Use of the long-acting antibacterial hydrogel according to claim 7 in the preparation of drug delivery materials, food packaging materials or medical dressings.

9. A wound dressing, characterized in that: The long-acting antibacterial hydrogel according to claim 7 is used.

10. A wound dressing, characterized in that: The long-acting antibacterial hydrogel according to claim 7 is used.

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