CN115920015B

CN115920015B - Sulfate crosslinked lysozyme hydrogel and preparation method thereof

Info

Publication number: CN115920015B
Application number: CN202310168742.4A
Authority: CN
Inventors: 蒋邦平; 沈星灿; 王瑷辉; 汪怡沁
Original assignee: Guangxi Normal University
Current assignee: Guangxi Normal University
Priority date: 2023-02-27
Filing date: 2023-02-27
Publication date: 2024-04-26
Anticipated expiration: 2043-02-27
Also published as: CN115920015A

Abstract

The invention discloses a preparation method of sulfate cross-linked lysozyme hydrogel, which comprises the steps of hydrolyzing lysozyme in an acidic environment with pH of 2.0 to obtain lysozyme starch fibrous sample; dialyzing with a dialysis bag to remove unreacted lysozyme and other impurities from the lysozyme amyloid; freeze-drying the lysozyme amyloid fibril with the impurities removed; and (3) preparing a lysozyme amyloid fibrous solution with the concentration of 20 mg/mL after drying, then adding each metal sulfate with the concentration of SO ₄ ^2‑ of 0.05 mmol/mL into the lysozyme amyloid fibrous solution, and uniformly mixing to obtain the serial sulfate crosslinked lysozyme fibrous hydrogel. On one hand, the lysozyme fiber hydrogel can effectively activate the antibacterial activity of the lysozyme fiber hydrogel under a pathogenic microenvironment, so that the effect of killing bacteria is achieved and bacterial drug resistance is not generated. On the other hand, various metal ions are involved in the regulation of cell metabolism and cytokines in wound healing, and play different roles in wound healing, thereby accelerating wound healing.

Description

Sulfate crosslinked lysozyme hydrogel and preparation method thereof

Technical Field

The invention belongs to the field of pharmaceutical preparations, and particularly relates to sulfate crosslinked lysozyme hydrogel, and a preparation method and application thereof.

Background

Antibiotics have been the primary drug for the treatment of bacterial infections since penicillin was found. However, with the wide clinical use of antibiotics, bacteria are rapidly gaining resistance. They evolved drug efflux pumps, permeation barriers, inactivating enzymes, targeting modification mechanisms, etc. to avoid the impact of antibiotics.

Studies have shown that biofilms provide a physical barrier to inhibit penetration of antibiotics and inactivate antibiotics by enzymatic breakdown or adsorption to protect pathogens from antibiotics and the host immune system. As a unique biocatalyst capable of hydrolyzing polysaccharides and peptidoglycans on bacterial cell walls, lysozyme (LYZ) can slow down bacterial resistance caused by antibiotic overdose, LYZ causes bacterial autolysis primarily by hydrolyzing peptidoglycans on cell walls and does not produce resistant bacteria, but the effective activation of lysozyme's antibacterial activity in pathogenic microenvironments remains a challenge. Fortunately, the antibacterial activity of lysozyme amyloid fibrils (LZF) appears to be superior to that of spheroplast lysozyme, which can be attributed to the reduction in positive charge and the increase in hydrophobic β -sheet assemblies, allowing sufficient physical contact with microorganisms to promote hydrolysis of peptidoglycans.

Currently, the main therapeutic strategies for biofilm removal are surgical debridement and antibiotic treatment. However, during surgical debridement, post-operative bacterial residues can lead to recurrence of the infection. Second, negatively charged biofilms not only block the entry of antibiotics into the biofilm, but also adsorb large amounts of positively charged antibiotics. Thus, antibiotic treatment of biofilm infections is facing a major challenge, and there is a strong need to develop innovative strategies for treating biofilm infections.

Disclosure of Invention

The process of wound healing is often driven by a variety of macro and micro nutrients, and the absence of any critical nutrients can lead to delays in healing of the damaged wound. Mineral elements including manganese, magnesium, iron, copper, zinc, and the like play a key role in cellular metabolism during wound healing.

In a physiological environment, zn ²⁺ can promote collagen deposition. Mg ²⁺ can effectively activate actin of fat cells under acidic condition, and accelerate healing at the initial stage of wound healing. Cu ²⁺ has also been shown to stimulate angiogenesis, accelerating the delivery of nutrients and oxygen during the wound healing process. In addition to the above metal ions, mn ²⁺,Fe³⁺,Al³⁺ and the like play an important role in promoting the wound healing process.

Based on the above, the invention provides a sulfate cross-linked lysozyme hydrogel, and a preparation method and application thereof.

The invention relates to a preparation method of sulfate cross-linked lysozyme hydrogel, which comprises the following steps:

S1, dissolving lysozyme in secondary water, regulating the pH value of the solution to 2.0 by using 0.1M HCl, and hydrolyzing in an oil bath at 80 ℃ to obtain lysozyme amyloid;

S2, dialyzing the obtained lysozyme amyloid fibrous sample in secondary water by using a dialysis bag to remove unreacted lysozyme and other impurities;

S3, freeze-drying the lysozyme amyloid fibrous sample with the impurities removed for later use;

S4, after drying, dissolving a proper amount of lysozyme amyloid in secondary water, preparing a lysozyme amyloid solution with the concentration of 20 mg/mL, then adding each metal sulfate with the SO 42-concentration of 0.05 mmol/mL into the lysozyme amyloid solution, and uniformly mixing to obtain the serial sulfate crosslinked lysozyme fiber (LZF) hydrogel.

Further, the molecular weight of the dialysis bag of S2 is 8000-14000Da.

Further, each metal sulfate in S4 is Na₂SO₄、K₂SO₄、ZnSO₄、Fe₂(SO₄)₃、CuSO₄、MgSO₄、MnSO₄ or Al ₂(SO₄)₃, and the solidification condition is that sulfate is used as a cross-linking agent instead of metal ions;

the dosage ratio of the lysozyme amyloid fibrous solution to the metal sulfate is 9:1.

The lysozyme fiber hydrogel prepared by the preparation method can be used for preparing medicines for promoting wound healing.

In the treatment of bacterial wound infection, the lysozyme fiber hydrogel can effectively activate the antibacterial activity of the lysozyme fiber hydrogel under pathogenic microenvironment, so that the effect of killing bacteria is achieved and bacterial drug resistance is not generated. On the other hand, various metal ions are involved in the regulation of cell metabolism and cytokines in wound healing, and play different roles in wound healing, thereby accelerating wound healing.

Drawings

FIG. 1 shows the setting effect of the series LZF prepared in the examples with sodium and potassium salts as cross-linking agents;

FIG. 2 shows the setting effect of the series LZF prepared in the examples with chloride, nitrate and sulfate as cross-linking agents;

FIG. 3 is a graph of dynamic time and frequency scans of a series of sulfate LZF gels prepared in the examples;

Antibacterial Activity of series sulfate Cross-Linked LZF gels

FIG. 4 is a photograph of the in vitro antimicrobial activity of a series of sulfate crosslinked LZF hydrogels prepared in the examples against E.coli and MRSA and corresponding colony statistics;

FIG. 4 (A) is a photograph of the in vitro antimicrobial activity of a series of sulfate-crosslinked LZF hydrogels against E.

FIG. 4 (B) is colony statistics of a series of sulfate crosslinked LZF hydrogels versus E.coli;

FIG. 4 (C) is a photograph of the in vitro antimicrobial activity of a series of sulfate crosslinked LZF hydrogels against MRSA;

FIG. 4 (D) is colony statistics for series of sulfate crosslinked LZF hydrogels versus MRSA;

Fig. 5a time-dependent ion release profile of sulfate crosslinked LZF hydrogels prepared in the examples.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but is not limited thereto.

Examples

Preparation of sulfate-crosslinked lysozyme hydrogel:

1) Preparation of lysozyme amyloid fibrils (LZF)

Dissolving 4 g lysozyme (egg white lysozyme, purchased from source leaf biotechnology) in 100 mL secondary water, adjusting pH of the solution to pH=2 with 0.1M HCl, and hydrolyzing 24h in 80 deg.C oil bath; after cooling to room temperature, loading into dialysis bag with molecular weight of 8000-14000Da, and dialyzing for 3 days to remove unreacted lysozyme and other impurities; removing impurities, and freeze-drying to obtain lysozyme amyloid (LZF);

2) Preparation of a series of LZF hydrogels

A proper amount of dried LZF was dissolved in secondary water to prepare a LZF solution with a concentration of 20 mg/mL, and then each metal salt with an anion concentration of 50 mL of 0.05 mmol/mL was added to the 450 mL LZF solution, as a result, as shown in FIG. 1, under the condition that the anion concentrations were the same, LZF could not form a gel under the crosslinking of Cl ^- and NO ^3-, and divalent SO ₄ ^2- and CO ₃ ^2- could form a gel. Therefore, the setting effect of the series sulfate as a crosslinker for LZF was further studied, and as shown in fig. 2, LZF was able to form gel with the series sulfate as the crosslinker, while LZF was unable to form hydrogel with the series chloride and nitrate as the crosslinker, and sulfate was selected as the crosslinker.

Examples the rheological properties of sulfate crosslinked LZF hydrogels were further explored:

The serial LZF hydrogels prepared by 0.3 mL are taken on a sample carrying table of a rotary rheometer, and the setting time and mechanical properties of the serial LZF hydrogels are respectively explored by utilizing dynamic time scanning and dynamic frequency scanning. Wherein the time interval is 0-600 s, and the frequency interval is 1-100 rad/s. As shown in FIG. 3, the series sulfate crosslinked LZF gel can rapidly gel (G ¢ > G), and the gel structure is always maintained in the dynamic frequency scanning interval, which also shows that the series sulfate crosslinked LZF hydrogel has the characteristic of rapid gel formation, and meets the basic requirements of wound dressing.

Examples the antibacterial effect of sulfate crosslinked LZF hydrogels was further explored:

the antibacterial activity of a series of sulfate-crosslinked LZF gels was explored by selecting escherichia coli (e.coli) and methicillin-resistant Lin Putao cocci (MRSA) as antibacterial models, as shown in fig. 4, fig. 4 is an in vitro antibacterial activity photograph of a series of sulfate-crosslinked LZF hydrogels prepared in the examples on e.coli (fig. 4A) and MRSA (fig. 4C) and corresponding e.coli (fig. 4B) and MRSA (fig. 4D) colony statistics; as can be seen from fig. 4, sulfate-crosslinked LZF hydrogels all exhibited respective unique antimicrobial activities.

Examples the ion release profile of sulfate crosslinked LZF hydrogels was further explored:

The ICP-Ms test was performed by taking 1 mL each sulfate crosslinked LZF hydrogel in 39 mL deionized water, standing at 37 ℃ and taking 1 mL each of 0.5 h, 1 h, 2h, 4h, 6 h each of aqueous solutions (taking out 1 ml aqueous solution and adding 1 mL deionized water again to maintain the solution volume at 40 mL). The results are shown in fig. 5, where monovalent sodium and potassium ions release most rapidly after 12 h, while trivalent iron and aluminum ions release most slowly, divalent metal ions.

Claims

1. A method for preparing a sulfate-crosslinked lysozyme hydrogel, comprising the steps of:

s1, dissolving lysozyme in secondary water, regulating the pH value of a solution to 2.0 by using 0.1M HCl, and hydrolyzing in an oil bath at 80 ℃ to obtain lysozyme amyloid fibers;

S2, dialyzing the obtained lysozyme amyloid fibers in secondary water by using a dialysis bag to remove unreacted lysozyme and other impurities;

s3, freeze-drying the lysozyme amyloid fibers with the impurities removed for later use;

S4, after drying, dissolving a proper amount of lysozyme amyloid fiber in secondary water, preparing a lysozyme amyloid fiber solution with the concentration of 20 mg/mL, then adding each metal sulfate with the concentration of SO ₄ ^2- of 0.05 mmol/mL into the lysozyme amyloid fiber solution, and uniformly mixing to obtain the serial sulfate crosslinked lysozyme fiber hydrogel.

2. The method for preparing lysozyme hydrogel according to claim 1, characterized in that: the molecular weight of the dialysis bag is 8000-14000Da.

3. The method for preparing lysozyme hydrogel according to claim 1, characterized in that: s4, each metal sulfate is Na₂SO₄、K₂SO₄、ZnSO₄、Fe₂(SO₄)₃、CuSO₄、MgSO₄、MnSO₄ or Al ₂(SO₄)₃;

The dosage ratio of the lysozyme amyloid fibrous solution to each metal sulfate is 9:1.

4. Use of a lysozyme fiber hydrogel prepared by the method of any one of claims 1 to 3, characterized in that: the lysozyme fiber hydrogel is applied to the preparation of medicines for promoting wound healing.