A polyelectrolyte double network hydrogel for bacterial infection wound healing and a preparation method and application thereof

Abstract

The application discloses a polyelectrolyte double-network hydrogel for bacterial infection wound healing and a preparation method and application thereof, and constructs a polyelectrolyte double-network hydrogel by introducing a quaternary ammonium salt modified chitosan cationic polyelectrolyte into a propylene monomer-co-sulfonate anion copolymer, wherein the quaternary ammonium carboxymethyl chitosan cationic polyelectrolyte is used as a first network, and the poly(propylene monomer-co-sulfonate) anionic polymer is used as a second network; the two polymer networks with opposite charges strongly interact with each other, and the mechanical property, the adhesion property and the antibacterial property of the polyelectrolyte double-network hydrogel are synergistically improved.

Description

Technical Field

[0001] This invention belongs to the field of medical dressings, specifically relating to a polyelectrolyte dual-network hydrogel for healing bacterial infection wounds, its preparation method, and its application. Background Technology

[0002] Hydrogels are three-dimensional network gels formed through chemical and / or physical cross-linking. Due to their excellent biocompatibility, controllable physical properties, and natural drug-carrying structures, hydrogels have broad application prospects in fields such as biosensors, regenerative medicine, tissue engineering, and wound dressings. In wound healing applications, hydrogel dressings are infused with one or more functions according to the healing requirements of different wounds, such as good tissue adhesion, excellent mechanical properties, antibacterial activity, local immunomodulation, and antitumor properties. Currently, most clinical antibacterial wound dressings lack elasticity and adhesion; therefore, there is an urgent need to develop wound dressings with good mechanical, adhesive, and antibacterial properties, which remains a significant challenge. Summary of the Invention

[0003] To address the above problems, this invention provides a polyelectrolyte dual-network hydrogel for healing bacterial infection wounds, its preparation method, and its application. The polyelectrolyte dual-network hydrogel is constructed by introducing a quaternary ammonium salt-modified chitosan cationic polyelectrolyte into an anionic copolymer containing propylene monomer-co-sulfonate. The quaternized carboxymethyl chitosan (QCMCS) cationic polyelectrolyte forms the first network, and the poly(propylene monomer-co-sulfonate) anionic polymer forms the second network. The two polymer networks with opposite charges interact strongly, synergistically enhancing the mechanical, adhesive, and antibacterial properties of the polyelectrolyte dual-network hydrogel.

[0004] This invention is achieved through the following technical solution:

[0005] A method for preparing a polyelectrolyte dual-network hydrogel for healing bacterial infection wounds includes the following steps:

[0006] S1. Using carboxymethyl chitosan (CMCS) as raw material and quaternizing agent as modifier, N-quaternized carboxymethyl chitosan (QCMCS) is obtained by reacting the quaternizing agent with the amino groups on the carboxymethyl chitosan backbone.

[0007] S2. The N-quaternized carboxymethyl chitosan synthesized in step S1 is pre-dispersed in deionized water to obtain a dispersion. Then, a copolymer system containing propylene monomers and sulfonates is added to the dispersion. Under the action of crosslinking agent, initiator and coagulant, a polyelectrolyte double network hydrogel is obtained by free radical polymerization.

[0008] Further, in step S1, the mass ratio of the carboxymethyl chitosan to the quaternizing agent is 1:3-8.

[0009] Further, in step S1, the quaternizing agent is any one of 3-chloro-2-hydroxypropyltrimethylammonium chloride (Quat-188) and 2,3-epoxypropyltrimethylammonium (GTA).

[0010] Further, in step S1, the specific preparation process of the N-quaternized carboxymethyl chitosan is as follows: carboxymethyl chitosan is pre-dispersed in deionized water and fully dissolved, then a quaternization reagent is added, and after reacting at 60-90℃, N-quaternized carboxymethyl chitosan is obtained by dialysis and freeze-drying.

[0011] Chitosan is currently the only known cationic polysaccharide with good blood compatibility and low biotoxicity. However, chitosan has limited antibacterial activity and poor water solubility under weakly alkaline physiological conditions. This invention modifies chitosan with a quaternizing agent to obtain N-quaternized carboxymethyl chitosan, which is soluble in a wide pH range and has higher water solubility in acidic, neutral, and alkaline solutions. At the same time, the grafting of quaternary ammonium salt endows chitosan with more durable antibacterial properties, which can accelerate wound healing and achieve a dual enhancement of chitosan's water solubility and antibacterial properties.

[0012] Further, in step S2, the mass-to-volume ratio of propylene monomer to dispersion is 0.2-0.4 g / ml; taking propylene monomer as a reference, the mass percentage of N-quaternized carboxymethyl chitosan is 15-20%, the mass percentage of sulfonate is 1-3%, the mass percentage of crosslinking agent is 0.05-0.1%, the mass percentage of initiator is 0.1-0.3%, and the mass percentage of coagulant is 0.001-0.005%.

[0013] Further, in step S2, the propylene monomer is any one of an acrylamide or an acrylate monomer. Acrylamide may be, but is not limited to, N-hydroxyethylacrylamide or acrylamide. Acrylate monomers include, but are not limited to, methyl acrylate, ethyl acrylate, methyl 2-methacrylate, and ethyl 2-methacrylate.

[0014] Further, in step S2, the sulfonate is any one of styrene sulfonate, alkyl sulfonate, and aminosulfonate.

[0015] Further, in step S2, the crosslinking agent is N,N-methylenebisacrylamide.

[0016] Further, in step S2, the initiator is any one of potassium sulfate, ammonium persulfate, azobisisobutyronitrile, azodimethylpentanonitrile, sodium azodicyanoacrylate, and azomidine salts (such as azo N-substituted amidine propane hydrochloride).

[0017] Furthermore, in step S2, the coagulant is tetramethylethylenediamine (TEMED).

[0018] Furthermore, in step S2, the free radical polymerization is carried out at a temperature of 60-90°C for a time of 0.5-2 hours.

[0019] This invention first modifies carboxymethyl chitosan with a quaternizing agent to obtain N-quaternized carboxymethyl chitosan cationic polyelectrolyte. Then, it is introduced into an anionic copolymer system containing propylene monomers and sulfonates, and a polyelectrolyte dual-network hydrogel is constructed via free radical polymerization. In this hydrogel, the quaternized carboxymethyl chitosan (QCMCS) cationic polyelectrolyte forms the first network, and the poly(propylene monomer-co-sulfonate) anionic polymer forms the second network. Based on the hydrogen bonds between polysaccharide molecular chains and the electrostatic adsorption between the polyelectrolytes, the two polymer networks with opposite charges interact strongly, synergistically enhancing the mechanical, adhesive, and antibacterial properties of the polyelectrolyte dual-network hydrogel.

[0020] The present invention also provides a polyelectrolyte dual-network hydrogel for healing bacterial infection wounds prepared by the above preparation method.

[0021] The present invention also provides the application of the above-mentioned polyelectrolyte dual-network hydrogel for healing bacterial infection wounds, using it to prepare dressings for bacterial infection wounds.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The polyelectrolyte dual-network hydrogel of the present invention introduces N-quaternized carboxymethyl chitosan (QCMCS) and sulfonate as cationic electrolyte and anionic electrolyte, respectively, into the hydrogel system to prepare a polyelectrolyte dual-network hydrogel for healing bacterial infection wounds, thus broadening its potential application value in wound dressings.

[0024] (2) The polyelectrolyte dual-network hydrogel of the present invention is first modified with a quaternizing agent to obtain N-quaternized carboxymethyl chitosan, which is soluble in a wide pH range and has higher water solubility in acidic, neutral and alkaline solutions. At the same time, the grafting of quaternary ammonium salt endows chitosan with more durable antibacterial properties, which can accelerate wound healing and achieve a dual enhancement of chitosan's water solubility and antibacterial properties. Furthermore, due to the presence of sulfonic acid groups, the outer membrane stress of microbial cells is increased, thereby achieving the purpose of killing microorganisms. At the same time, sulfonates can establish strong connections with substances of opposite charge, and enhance the mechanical and adhesive properties of the hydrogel network through electrostatic adsorption.

[0025] (3) The polyelectrolyte dual-network hydrogel of the present invention uses N-quaternized carboxymethyl chitosan (QCMCS) cationic polyelectrolyte as the first network and poly(propylene monomer-co-sulfonate) anionic polymer as the second network. The two have opposite charges. Based on the hydrogen bonds between polysaccharide molecular chains and the electrostatic adsorption between polyelectrolytes, the continuous and uniform network of the hydrogel is conducive to the construction of the hydrogel network. At the same time, the polyelectrolytes can promote wound healing by establishing an intercellular communication network.

[0026] In summary, this polyelectrolyte dual-network hydrogel has the characteristics of stable coverage, small displacement, long antibacterial time, and rapid wound healing. Under the synergistic effect of each component, it is endowed with good mechanical properties, adhesion properties and antibacterial properties. Attached Figure Description

[0027] Figure 1 Fourier transform infrared spectra of carboxymethyl chitosan (bottom) and N-quaternized carboxymethyl chitosan (top).

[0028] Figure 2 The tensile stress-strain curves (right) and the corresponding elastic modulus and toughness curves (left) of the polyelectrolyte dual-network hydrogels prepared in Examples 1-2 and Comparative Examples 1-2 under external force are shown.

[0029] Figure 3 This is a schematic diagram of the skin tissue adhesion of the polyelectrolyte dual-network hydrogel prepared in Example 1.

[0030] Figure 4 The graph shows the antibacterial properties of the polyelectrolyte dual-network hydrogels prepared in Examples 1-2 and Comparative Examples 1-2 after co-culturing with bacteria for 24 hours.

[0031] Figure 5 This is a representative healing diagram of a wound infected with Staphylococcus aureus. Detailed Implementation

[0032] The following examples further illustrate specific implementations of the present invention, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described below are those that can be implemented or understood by those skilled in the art by referring to existing technology. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0033] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0034] Example 1

[0035] This example provides a polyelectrolyte dual-network hydrogel for treating bacterial infected wounds and its preparation method, specifically including the following steps:

[0036] (1) Preparation of N-quaternized carboxymethyl chitosan (QCMCS):

[0037] 3.28 g of CMCS was pre-dispersed in 100 mL of deionized water and fully dissolved. Then, 15.1 g of GTA was added, and the mixture was stirred at 80 °C for 1 h. After the reaction was completed, the mixture was dialyzed in a dialysis tube with a molecular weight cutoff of 8000–12000 Da for 48 h. Finally, the mixture was freeze-dried under vacuum to remove excess water, yielding a yellow powder, QCMCS.

[0038] (2) Preparation of polyelectrolyte dual-network hydrogel:

[0039] 0.21 g of QCMCS was poured into a flask containing 5 mL of deionized water and stirred to form a uniform dispersion. Then, acrylamide monomer AM (1.4 g), sodium styrene sulfonate SS (0.03 g), crosslinking agent N,N'-methylenebisacrylamide (0.0010 g), and initiator ammonium persulfate APS (0.0028 g) were added to the flask respectively. Then, 5 μL of coagulant tetramethylethylenediamine (TEMED) was added to obtain the precursor solution. Finally, the precursor solution was heated at 70 °C for 1 h to initiate polymerization and obtain a polyelectrolyte double network hydrogel, denoted as PAAm:SS:QCMCS. 15 QCMCS accounts for 15% of the mass percentage of acrylamide monomer AM.

[0040] Example 2

[0041] Same as Example 1, except that: in step (2), the amount of QCMCS added is 0.28g, and the resulting sample is denoted as PAAm:SS:QCMCS. 20 QCMCS accounts for 20% of the mass percentage of acrylamide monomer AM.

[0042] Comparative Example 1

[0043] Same as Example 1, except that: in step (2), the amount of QCMCS added is 0.07g, and the sample obtained is denoted as PAAm:SS:QCMCS5, wherein QCMCS accounts for 5% of the mass percentage of acrylamide monomer AM.

[0044] Comparative Example 2

[0045] Same as Example 1, except that: in step (2), the amount of QCMCS added is 0.14g, and the resulting sample is denoted as PAAm:SS:QCMCS. 10 QCMCS accounts for 10% of the mass percentage of acrylamide monomer AM.

[0046] Table 1. Experimental results of polyelectrolyte dual-network hydrogels prepared in Examples 1-2 and Comparative Examples 1-2.

[0047]

[0048] Figure 1 Fourier transform infrared (FTIR) spectral analysis was performed on carboxymethyl chitosan (CMCS) and N-quaternized carboxymethyl chitosan (QCMCS) prepared in Example 1. Comparison of the FTIR spectra of the two substances showed that QCMCS had a peak value of 1606.25 cm⁻¹. -1 and 1405.44cm -1 There are two similar characteristic absorption peaks at 1058.29 cm⁻¹. In QCMCS, this is the peak at 1058.29 cm⁻¹. -1 The characteristic peaks are attributed to the CO stretching of the secondary hydroxyl groups in the cyclic alcohol. The results indicate that the secondary hydroxyl groups are unaffected during the modification of carboxymethyl chitosan. Due to the stretching vibrations of -OH and -NH, the FTIR spectrum in CMCS peaks at 3448.64 cm⁻¹. -1 A broad peak appears nearby. The results indicate that the introduction of GTA caused an amino reaction and hydrogen bond breaking, resulting in the peak at 3448.64 cm⁻¹ in the CMCS FTIR spectrum. -1 The peak direction shifted by 3438.22 cm. -1 3448.64cm -1 The offset is 3448.64cm. -1 In the infrared spectrum, 1477 cm⁻¹ -1 The new absorption peak at the methyl group is attributed to the vibrational peak of the trimethyl quaternary ammonium ion in the methyl group, indicating that the modification of carboxymethyl chitosan was successful.

[0049] Figure 2 (Right) shows the tensile stress-strain curves of the polyelectrolyte dual-network hydrogels prepared in Examples 1-2 and Comparative Examples 1-2 under external force. Figure 2 (Left) is the corresponding elastic modulus and toughness curve. Figure 2It can be seen that, compared with Comparative Examples 1-2, the tensile strength of the polyelectrolyte dual-network hydrogel is significantly improved when the amount of QCMCS accounts for 15-20% of the acrylamide monomer mass. This is because more cross-linking points are formed in the polyelectrolyte dual-network hydrogel through hydrogen bonding, electrostatic adsorption, and a denser porous network, resulting in a tensile stress of 718-770 kPa and a tensile strain of 1100-1106%. The first network is a tightly cross-linked rigid polyelectrolyte network, while the second network is a loosely cross-linked flexible acrylamide network. When force is applied to the polyelectrolyte dual-network hydrogel, the polyelectrolyte network structure provides sacrificial bonds for the hydrogel system, playing a role in energy dissipation and external stress dispersion. QCMCS and amino groups are electrostatically attracted to the sulfonic acid groups of SS, while the π-π accumulation between benzene rings in sodium styrene sulfonate also increases the mechanical properties of the hydrogel. Therefore, based on the synergistic effect of the polyelectrolyte dual-network hydrogel, this hydrogel exhibits excellent mechanical properties.

[0050] Figure 3 This is a schematic diagram illustrating the skin tissue adhesion of the polyelectrolyte dual-network hydrogel prepared in Example 1. Using pig skin as a model tissue, an overlap shear test was conducted on the hydrogel (20mm*20mm) using a universal tensile testing machine at a speed of 50mm / min. Table 1 shows that when the amount of QCMCS accounts for 15% of the acrylamide monomer mass percentage, the adhesive strength of the polyelectrolyte dual-network hydrogel reaches 6.2kPa, exhibiting strong adhesion under air conditions, thus meeting the practical application requirements of the hydrogel in biomedical dressings. Simultaneously, from... Figure 3 It is known that the polyelectrolyte hydrogel exhibits stable adhesion, stretches with tissue movement, achieves stretching on various dynamic skin surfaces, and can maintain autonomous adhesion to human skin for a long time.

[0051] Figure 4 This is a graph showing the antibacterial properties of the polyelectrolyte dual-network hydrogels prepared in Examples 1-2 and Comparative Examples 1-2 after co-culturing with bacteria for 24 hours. The polyelectrolyte dual-network hydrogels were then placed on Staphylococcus aureus (10... 5 CFU / mL, 4mL) and Escherichia coli (10 CFU / mL, 4mL) 5 When QCMCS was co-cultured with a solution of CFU / mL and 4mL for 24h, the antibacterial effect was not obvious when the mass percentage of QCMCS in acrylamide monomer AM was 5% and 10%. However, when the mass percentage of QCMCS in acrylamide monomer AM was increased to 15-20%, the antibacterial effect was significantly enhanced, and it showed good antibacterial activity against Staphylococcus aureus and Escherichia coli models.

[0052] Figure 5 Representative healing diagram of a Staphylococcus aureus-infected wound. (Based on QCMCS) 15The / P(AAm-co-SS) hydrogel was used in the experimental group (top), while the untreated group (bottom) was used in the control group. Photos of the wound contraction area taken at different times (every two days) during the wound healing process are shown, revealing a significant difference between the experimental and control groups. Suppurative lesions were visible in the control group, indicating slower wound healing. No suppurative reaction was observed in the experimental group after treatment, and QCMCS was observed on day 14. 15 / P(AAm-co-SS) hydrogel leaves almost no residual wound. The results indicate that QCMCS... 15 / P(AAm-co-SS) hydrogel has a significant therapeutic effect on full-thickness wounds infected with Staphylococcus aureus.

[0053] Comparative Example 3

[0054] Same as Example 1, except that SS is not added in step (2).

[0055] The hydrogel has a fracture stress of 579 kPa and an elongation at break of 801%. The addition of SS can effectively improve its mechanical properties.

[0056] Comparative Example 4

[0057] Same as Example 1, except that: no monomer AM was added in step (2).

[0058] The hydrogel has a fracture stress of 417 kPa and a fracture elongation of 622%. The addition of monomer AM endows the hydrogel with a double network structure, which enhances its mechanical properties.

[0059] Comparative Example 5

[0060] Same as Example 1, except that QCMCS was not added in step (2), and the hydrogel has no antibacterial properties.

[0061] Comparative Example 6

[0062] Same as Example 1, except that QCMCS is replaced with CMCS in step (2). The hydrogel has a bactericidal rate of 78.34% against Staphylococcus aureus and a bactericidal rate of 65.19% against Escherichia coli.

[0063] Table 2 shows the experimental results of the hydrogels prepared in Comparative Examples 3-6.

[0064]

Claims

1. A method for the preparation of a polyelectrolyte double network hydrogel for the healing of bacterial infected wounds, characterized in that, The method comprises the following steps: S1. Using carboxymethyl chitosan as raw material and quaternary ammonium reagent as modifier, the quaternary ammonium reagent reacts with the amino group on the carboxymethyl chitosan skeleton to obtain N-quaternary ammonium carboxymethyl chitosan; S2. The N-quaternary ammonium carboxymethyl chitosan synthesized in step S1 is dispersed in deionized water to obtain a dispersion liquid, and a copolymerization system containing an acryl monomer and a styrene sulfonate is added to the dispersion liquid, and under the action of a crosslinking agent, an initiator and a coagulant, a polyelectrolyte double network hydrogel is prepared by free radical polymerization; The mass percentage of the N-quaternary ammonium carboxymethyl chitosan is 15-20% and the mass percentage of the sulfonate is 1-3% with the acryl monomer as a reference.

2. The production method according to claim 1, characterized by, In step S1, the mass ratio of the carboxymethyl chitosan to the quaternary ammonium reagent is 1:3-8.

3. The production method according to claim 1, characterized by, In step S1, the quaternary ammonium reagent is any one of 3-chloro-2-hydroxypropyl trimethyl ammonium chloride and 2,3-epoxypropyl trimethyl ammonium.

4. The method of claim 1, wherein, In step S1, the specific preparation process of the N-quaternary ammonium carboxymethyl chitosan is as follows: the carboxymethyl chitosan is dispersed in deionized water and fully dissolved, then the quaternary ammonium reagent is added, and after reaction at 60-90℃, the N-quaternary ammonium carboxymethyl chitosan is prepared by dialysis and freeze-drying.

5. The preparation method according to claim 1, characterized in that, In step S2, the mass-volume ratio of the acryl monomer to the dispersion liquid is 0.2-0.4 g / ml; the mass percentage of the crosslinking agent is 0.05-0.1%, the mass percentage of the initiator is 0.1-0.3%, and the mass percentage of the coagulant is 0.001-0.005% with the acryl monomer as a reference.

6. The method of any one of claims 1-5, wherein, In step S2, the acryl monomer is any one of an acrylamide monomer or an acrylate monomer.

7. The method of any one of claims 1-5, wherein, In step S2, the crosslinking agent is N,N-methylene bisacrylamide; The initiator is any one of potassium sulfate, ammonium persulfate, azobisisobutyronitrile, azobisdimethylvaleronitrile, sodium azobis cyano acrylate, and azamidine salt; The coagulant is tetramethyl ethylenediamine.

8. The method of any one of claims 1-5, wherein, In step S2, the temperature of the free radical polymerization is 60-90℃ and the time is 0.5-2h.

9. The polyelectrolyte double network hydrogel for bacterial infection wound healing prepared by the preparation method of any one of claims 1-8.

10. The use of the polyelectrolyte double network hydrogel for the healing of bacterial infected wounds according to claim 9, characterized in that, It is used for preparing a bacterial infection wound dressing.

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