CN118812877B

CN118812877B - A long-acting antibacterial hydrogel containing polyguanidine polymer and preparation method thereof

Info

Publication number: CN118812877B
Application number: CN202410776500.8A
Authority: CN
Inventors: 袁海; 宋正财; 毕伟华; 黄碧峰; 何佳佳
Original assignee: Hongzhi Biotechnology Zhejiang Co ltd
Current assignee: Hongzhi Biotechnology Zhejiang Co ltd
Priority date: 2024-06-17
Filing date: 2024-06-17
Publication date: 2025-02-14
Anticipated expiration: 2044-06-17
Also published as: CN118812877A

Abstract

The invention discloses a long-acting antibacterial hydrogel containing polyguanidine high molecular polymers and a preparation method thereof, and belongs to the field of hydrogels. The invention firstly carries out aldehyde modification on Hyaluronic Acid (HA) to obtain aldehyde hyaluronic acid (OHA), and then carries out Schiff base chemical crosslinking reaction on the aldehyde hyaluronic acid (OHA) and amino groups of chitosan derivatives/polyguanidine polymers to obtain the long-acting antibacterial hydrogel. The long-acting antibacterial hydrogel prepared by the invention has good release controllability and durability, does not influence the mechanical properties, and has wide application range.

Description

Long-acting antibacterial hydrogel containing polyguanidine high molecular polymer and preparation method thereof

Technical Field

The invention relates to a long-acting antibacterial hydrogel containing polyguanidine high molecular polymers and a preparation method thereof, belonging to the field of antibacterial hydrogel dressing.

Background

Skin is the largest organ of the human body and includes epidermis, dermis, and subcutaneous tissue. In general, the main function of skin is to inhibit the loss of water, electrolytes and nutrients in the body, and to protect against external mechanical, chemical, biological and physical stimuli to some extent, while also preventing bacterial, fungal invasion etc. at the skin surface. Normal skin is damaged and wounded under the influence of external factors, the structural integrity and the protection function of the skin are damaged, the protection capability of the body is lost, and the infection risk is caused. Bacterial infection at the wound site delays the healing process, leading to necrosis, sepsis and even death (Wang J,Duan X,Zhong D,Zhang M,et al.Internationaljournal ofbiological macromolecules,2023,261(1):129064).

The wound dressing with good antibacterial property can avoid wound infection. An ideal wound dressing should be able to absorb exudates from the wound surface while maintaining a moist environment at the wound site, avoiding drying and excessive moisture of the wound surface. And meanwhile, the influence of external factors such as dust, bacteria and the like is isolated, and the wound healing is finally promoted. The antibacterial hydrogel has good biocompatibility, high water content, porous structure, proper physicochemical properties and mechanical properties similar to extracellular matrix, so that the antibacterial hydrogel has good application potential in the aspects of wound hemostasis, tissue regeneration and wound healing (Yang K, han Q, chen B, et al International journal ofnanomedicine,2023,20 (5): 2217-2263).

At present, the conventional antibacterial hydrogel is prepared by embedding or combining antibacterial substances (such as metal nano particles, antibiotics, antibacterial peptides and the like) with a high polymer network, for example, the antibacterial properties of the metal nano particles (such as silver nano particles AgNPs) are utilized to realize long-acting antibacterial effect by embedding the metal nano particles into the hydrogel network, the antibiotics (such as ciprofloxacin, gentamicin and the like) are combined with a hydrogel matrix (such as carbomer) to realize antibacterial effect by controlling the release of the antibiotics, and the antibacterial peptide (AMPs) is combined with the hydrogel to realize antibacterial effect by controlling (S.Li,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);, but the antibacterial hydrogel prepared by the methods has poor release controllability and durability, can quickly release the antibacterial agent at early stage, lose antibacterial performance after that, thereby influencing wound healing, even causing wound infection and being unfavorable for wound repair. Moreover, increasing the antimicrobial properties affects the mechanical properties of the hydrogels, which limits the range of applications.

Therefore, how to obtain the hydrogel with the slow release function and the long-acting antibacterial function becomes a technical problem to be solved in the field.

Disclosure of Invention

[ Technical problem ]

The release controllability and durability of the antibacterial hydrogel are poor;

increasing the antimicrobial properties affects the mechanical properties of the hydrogels, which limits the range of applications.

Technical scheme

In order to solve the problems, the invention firstly carries out hydroformylation modification on Hyaluronic Acid (HA) to obtain aldehyde hyaluronic acid (OHA), and then carries out Schiff base chemical crosslinking reaction on the aldehyde hyaluronic acid (OHA) and amino groups of chitosan derivatives/polyguanidine polymers to obtain the long-acting antibacterial hydrogel. The long-acting antibacterial hydrogel prepared by the invention has good release controllability and durability, does not influence the mechanical properties, and has wide application range.

The first object of the invention is to provide a method for preparing a long-acting antibacterial hydrogel containing polyguanidine high molecular polymer, which comprises the following steps:

(1) Dispersing aldehyde hyaluronic acid (OHA) in water to obtain an aqueous solution I;

(2) Dispersing a polyguanidine high molecular polymer and a chitosan derivative in water to obtain an aqueous solution II;

(3) And mixing the aqueous solution I with the aqueous solution II, and performing Schiff base reaction to obtain the long-acting antibacterial hydrogel.

In one embodiment of the present invention, the aldehyde-modified hyaluronic acid (OHA) of step (1) is prepared by sodium periodate oxidation, specifically as follows:

Dissolving Hyaluronic Acid (HA) in water to obtain aqueous solution of Hyaluronic Acid (HA), wherein the dosage ratio of Hyaluronic Acid (HA) to water is 1 g/10 mL;

dissolving sodium periodate in water to obtain a sodium periodate aqueous solution, wherein the dosage ratio of the sodium periodate to the water is 0.54 g/1 mL;

Slowly dripping sodium periodate aqueous solution into Hyaluronic Acid (HA) aqueous solution for light-proof reaction, adding glycol to quench unreacted sodium periodate after the reaction is finished, dialyzing, freeze-drying to obtain aldehyde hyaluronic acid (OHA), wherein the light-proof reaction is carried out for 1h at 20-30 ℃ at room temperature, and the dosage ratio of sodium periodate to glycol is 0.54g to 1mL.

In one embodiment of the invention, the Hyaluronic Acid (HA) in step (1) HAs a molecular weight of between 50kDa and 5000kDa.

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

In one embodiment of the present invention, the polyguanidine-based polymer in the step (2) is one or two of polyhexamethylene biguanidine hydrochloride and polyhexamethylene monoguanidine hydrochloride.

In one embodiment of the present invention, the chitosan derivative in the step (2) is one or more of chitosan, carboxymethyl chitosan, hydroxypropyl chitosan, hydroxybutyl chitosan, and the like.

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

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

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

In one embodiment of the present invention, the Schiff base reaction in step (3) is a 20-30 ℃ (normal temperature) reaction for 4-10min.

The second purpose of the invention is to prepare the long-acting antibacterial hydrogel by the method.

The third object of the invention is the application of the long-acting antibacterial hydrogel in drug delivery, food packaging or medical dressing.

A fourth object of the present invention is to provide a wound dressing employing the long-acting antibacterial hydrogel of the present invention.

A fifth object of the present invention is to provide a wound dressing employing the long-acting antibacterial hydrogel of the present invention.

[ Advantageous effects ]

(1) The long-acting antibacterial hydrogel prepared by the invention can be directly smeared and adhered on skin, and can provide proper humidity and physical barrier.

(2) The long-acting antibacterial hydrogel prepared by the invention can be completely biodegraded, and polysaccharide components are beneficial to skin repair.

(3) The long-acting antibacterial hydrogel prepared by the invention introduces the polyguanidine polymer antibacterial agent into the hydrogel through Schiff base chemical crosslinking reaction in the preparation process, so that the antibacterial action time of the hydrogel is effectively prolonged.

(4) The long-acting antibacterial hydrogel prepared by the invention has good release controllability and durability, does not influence the mechanical properties, and has wide application range.

Drawings

FIG. 1 is a diagram of the reaction mechanism of the preparation of the long-acting antibacterial hydrogel.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for better illustration of the invention, and should not be construed as limiting the invention.

The testing method comprises the following steps:

1. Cytotoxicity test:

the samples and MES buffer are extracted into leaching solution according to the mass ratio of 1:5, and the cytotoxicity of the leaching solution is characterized by an MTT method.

2. Antibacterial performance test:

E.coli (10 ⁴ cfu/mL) and staphylococcus aureus (10 ⁴ cfu/mL) are used as working bacteria liquid, 0.1mL of working bacteria liquid is taken and evenly mixed with 5mL of test sample, 1.0mL of mixed liquid is taken to 9mL of diluent until the test time, gradient dilution is carried out sequentially, 1mL of mixed liquid is taken to a sterile plate in parallel, then a culture medium is poured, and the culture is carried out for 48 hours at 35+/-2 ℃ to be used as viable bacteria count, and the bacteriostasis rate of the sample is calculated according to the following formula:

X=(A–B)/A×100%

Wherein, X is the bacteriostasis rate,% A is the average colony number of the control sample, and the average colony number of the tested sample.

Evaluation criteria:

the antibacterial rate is more than or equal to 50-90%, the product has antibacterial effect, the antibacterial rate is more than or equal to 90%, and the product has strong antibacterial effect.

3. Long-acting bacteriostasis test:

And uniformly rubbing the arm, then taking test sample pieces (5 cm and 5 cm) and pasting the test sample pieces on the surface of the divided arm, respectively taking the sample pieces from each interval time period of 0 to 48 hours, simultaneously taking out standard samples at the sample pasting position of the arm by using a sterile cotton swab, diluting, pouring parallel sampling liquid for culture, and counting and calculating the long-acting antibacterial effect of the test sample after culturing for 24 hours at 37+/-2 ℃.

4. Mechanical property test:

The 1mm thick hydrogel was cut into 4 x 50mm dumbbell shaped tensile test specimens and tested using a universal tester at a speed of 100 mm/min.

5. Washing test:

5 pieces of hydrogel with the same size are prepared, each piece of hydrogel is about 5g, the hydrogel is put into water to be washed 3 times, and the antibacterial effect after washing is tested.

The raw materials used in the examples:

hyaluronic acid with molecular weight of 40kDa-4000kDa, purchased from Kaschin Biotech Co., ltd;

Carboxymethyl chitosan with amino content of 2-6mmol/g, purchased from Shanghai Ala Biochemical technology Co., ltd;

hydroxypropyl chitosan with amino content of 2-6mmol/g is purchased from Shanghai Ala Biochemical technology Co., ltd;

hydroxybutyl chitosan, with an amino content of 2-6mmol/g, purchased from Shanghai Ala Biochemical technologies Co., ltd;

polyhexamethylene biguanide hydrochloride with a purity of 98% and purchased from Shanghai Meilin Biochemical technologies Co., ltd;

the aldehyde hyaluronic acid (OHA) is prepared by sodium periodate oxidation method, and concretely comprises the following steps:

1g of Hyaluronic Acid (HA) was dissolved in 10mL of deionized water to obtain an aqueous Hyaluronic Acid (HA) solution;

Dissolving 0.54g of sodium periodate in 1mL of deionized water to obtain a sodium periodate aqueous solution;

Slowly dripping sodium periodate aqueous solution into Hyaluronic Acid (HA) aqueous solution, reacting for 1h at 25 ℃ in darkness (normal temperature), adding 1mL of ethylene glycol after the reaction is finished to quench unreacted sodium periodate, dialyzing the reaction solution in deionized water for 3 days, and freeze-drying to obtain aldehyde hyaluronic acid (OHA).

The w/v referred to in the examples is g/mL,% is the ratio of a substance to a solvent (water).

Example 1

A method for preparing a long-acting antibacterial hydrogel containing polyguanidine high molecular polymers comprises the following steps:

(1) Dispersing aldehyde hyaluronic acid (OHA) in water to obtain an aqueous solution I with the concentration of 2% w/v;

(2) Dispersing polyhexamethylene biguanide hydrochloride and carboxymethyl chitosan in water to obtain an aqueous solution II with the concentration of polyhexamethylene biguanide hydrochloride being 0.1% w/v and the concentration of carboxymethyl chitosan being 2% w/v;

(3) Mixing the aqueous solution I and the aqueous solution II according to a volume ratio of 1:1, and standing at 25 ℃ for 5min to obtain the long-acting antibacterial hydrogel.

Example 2

(1) Dispersing aldehyde hyaluronic acid (OHA) in water to obtain an aqueous solution I with the concentration of 4% w/v;

(2) Dispersing polyhexamethylene biguanide hydrochloride and carboxymethyl chitosan in water to obtain an aqueous solution II with the concentration of polyhexamethylene biguanide hydrochloride being 0.1% w/v and the concentration of carboxymethyl chitosan being 4% w/v;

Example 3

(2) Dispersing polyhexamethylene biguanide hydrochloride and carboxymethyl chitosan in water to obtain an aqueous solution II with the concentration of polyhexamethylene biguanide hydrochloride being 0.5% w/v and the concentration of carboxymethyl chitosan being 4% w/v;

Example 4

(2) Dispersing polyhexamethylene biguanide hydrochloride and carboxymethyl chitosan in water to obtain an aqueous solution II with the concentration of polyhexamethylene biguanide hydrochloride being 2% w/v and the concentration of carboxymethyl chitosan being 4% w/v;

Example 5

And (3) adjusting the carboxymethyl chitosan in the step (2) of the example 3 to be hydroxypropyl chitosan, and keeping the same with the example 3 to obtain the long-acting antibacterial hydrogel.

Example 6

And (3) adjusting the carboxymethyl chitosan in the step (2) of the example 3 to be hydroxybutyl chitosan, and keeping the same with the example 3 to obtain the long-acting antibacterial hydrogel.

Comparative example 1

The polyhexamethylene biguanide hydrochloride in step (2) of example 3 was omitted, and the other was kept the same as in example 3 to obtain a hydrogel.

Comparative example 2

The polyhexamethylene biguanide hydrochloride in step (2) of example 3 was adjusted to dodecyltrimethylammonium chloride, and the other conditions were kept the same as those of example 3 to obtain a hydrogel.

Comparative example 3

The carboxymethyl chitosan in step (2) of example 3 was omitted, and the other conditions were the same as in example 3, to obtain a hydrogel.

Comparative example 4

A method of preparing an antimicrobial hydrogel comprising the steps of:

Dispersing aldehyde hyaluronic acid (OHA), polyhexamethylene biguanide hydrochloride and carboxymethyl chitosan in water, standing at 25 ℃ for Schiff base reaction for 5min to obtain hydrogel;

Wherein the concentration of polyhexamethylene biguanide hydrochloride is 0.2% w/v, the concentration of carboxymethyl chitosan is 2% w/v, and the concentration of hydroformylation hyaluronic acid (OHA) is 2% w/v.

Comparative example 5

A method of preparing an antimicrobial hydrogel comprising the steps of:

dispersing aldehyde hyaluronic acid (OHA) and carboxymethyl chitosan in PBS buffer solution, and standing at 25 ℃ for Schiff base reaction for 5min to obtain hydrogel;

Wherein the concentration of carboxymethyl chitosan is 2% w/v and the concentration of aldehyde hyaluronic acid (OHA) is 2% w/v.

Comparative example 6

The carboxymethyl chitosan in the step (2) of the example 3 was adjusted to chitosan, and the other materials were kept the same as the example 3 to obtain a hydrogel.

The performance of the obtained hydrogel was tested as follows:

(1) Cytotoxicity:

The hydrogels prepared in examples 1-6 and comparative examples 1-6 were tested to be non-cytotoxic.

(2) Antibacterial performance:

Table 1 shows the results of the antibacterial performance test, and it can be seen from Table 1 that examples 1-6 all have good antibacterial performance, and the antibacterial performance of the hydrogel without the antibacterial agent is poor.

TABLE 1 antibacterial Rate of hydrogels

(3) Long-acting bacteriostasis:

Table 2 shows the results of the long-acting performance test, and from Table 2, it can be seen that examples 1-6 have good long-acting antibacterial performance, and examples 3-6 have no microbial growth after 48 hours.

TABLE 2 Long-acting antibacterial Properties of hydrogels

Note that "-" represents no microbial growth, "" represents a bacterial colony count of <10 "" represents a bacterial colony count of <20 "" represents a bacterial colony count of greater than or equal to 20 or colony agglomeration.

(4) Mechanical properties:

table 3 shows the mechanical properties, and from Table 3, examples 2-6 and comparative examples 1,2, 3 and 5 have good mechanical properties, the solid content in example 1 is low, the tensile strength and elongation are reduced, the carboxymethyl chitosan in comparative example 3 is absent, the prepared hydrogel has almost no tensile strength, schiff base reaction starts to occur when the raw material in comparative example 4 is dissolved, so that the gel is uneven, and the mechanical properties are poor.

TABLE 3 mechanical Properties of hydrogels

(5) Washing test

Table 4 shows that examples 1-6 have higher antibacterial rate after washing, and comparative examples 1-6 have a clear decrease in antibacterial rate after washing, indicating that the antibacterial active ingredients in the hydrogel are washed away at this time.

TABLE 4 antibacterial Rate of hydrogel after Water washing

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for preparing a long-acting antibacterial hydrogel containing a polyguanidine polymer, characterized in that it comprises the following steps:

(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 the aldehyde-modified hyaluronic acid (OHA) in step (1) is prepared by sodium periodate oxidation method.

4. The method according to claim 3, characterized in that the preparation method of aldehyded hyaluronic acid OHA in step (1) is:

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 50 kDa-5000 kDa.

6. The method according to claim 1, characterized in that the volume ratio of aqueous solution I to aqueous solution II in step (3) 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 patch, characterized in that it uses the long-acting antibacterial hydrogel according to claim 7.

10. A wound dressing, characterized in that it uses the long-acting antibacterial hydrogel according to claim 7.