CN116898793B - Lysozyme hydrogel for diabetic foot ulcers and preparation method thereof - Google Patents
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/32—Manganese; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
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- Health & Medical Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
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- Obesity (AREA)
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- Immunology (AREA)
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- Medicinal Preparation (AREA)
Abstract
The invention belongs to the field of pharmaceutical preparations, and discloses a lysozyme hydrogel for diabetic foot ulcers, which is prepared by dissolving lysozyme in secondary water, and reacting under water bath and acidic conditions to obtain a lysozyme amyloid fibrous solution; dialyzing with dialysis bag to remove unreacted lysozyme and other impurities; freezing and drying again for standby; drying, dissolving lysozyme amyloid and genipin in secondary water, and performing water bath reaction to obtain blue liquid, namely LZF/GP; adding LZF/GP into MnSO 4 for crosslinking to obtain blue gel, namely LZF/GP-MnSO 4 hydrogel. In the lysozyme hydrogel, manganese ions are used for enhancing cGSA sensitivity to extracellular DNA, sting signals are activated to promote inflammatory reaction to clear residual bacteria and destroy biological membranes, and GP plays roles in resisting inflammation and reducing blood sugar, so that dynamic pro-inflammatory and anti-inflammatory balance is achieved, and healing of diabetic foot ulcers is promoted.
Description
Technical Field
The invention belongs to the field of pharmaceutical preparations, and particularly relates to lysozyme hydrogel for diabetic foot ulcers and a preparation method thereof.
Background
With the advent of the worldwide aging society, the treatment of Diabetic Foot Ulcers (DFUs) is increasingly under pressure. Of which about 25% of diabetic patients develop diabetic foot ulcers throughout their lives, and 40% of foot ulcers relapse after treatment, diabetic foot ulcers have become one of the most common and serious complications of diabetes. Local hyperglycemia induces chronic inflammation and bacterial invasion, which makes it more difficult to cure diabetic foot ulcers using existing chemotherapy.
Reducing the inflammatory response of DFUs may help it heal, but may compromise the antimicrobial activity of local innate immunity, leading to persistent biofilm-related infections. In contrast, proper inflammation is essential in wound repair, and by activation of antibacterial pro-inflammatory responses, inflammation is promoted to clear pathogenic bacteria, cell debris and damaged matrix. However, overactivated pro-inflammatory reactions may lead to high oxidative stress, impeding wound healing.
Currently, the primary treatment strategy for diabetic foot ulcers is surgical debridement, removal of the diseased limb, and combination antibiotic treatment. The fundamental approach to radical treatment is to start from enhancing physical quality and enhancing immunity. Interferon gene stimulators (Sting) have become a promising target for immunotherapy of cancer, viral and bacterial infections as another class of independent innate immune risk sensors. Thus, immunotherapy of diabetic foot ulcers caused by drug-resistant bacteria is facing a great challenge, and there is an urgent need to explore innovative strategies for immunotherapy of foot ulcers.
The ability to sense microbial pathogens by recognizing their nucleic acids has been a key feature of mammalian cell innate immunity for the past two decades. In addition, interferon gene Stimulators (STING) have become a promising target for cancer immunotherapy as another class of independent innate immune risk sensors. Mn 2+ is the only metal ion reported in the prior art that increases the sensitivity of cyclic GMP-AMP synthase (cGAS) to extracellular DNA, promotes Sting activation and is widely used in the treatment of tumor, viral and bacterial infections. How to achieve anti-inflammatory effects of diabetic foot ulcers after Sting activates the inflammatory response is a challenge.
Genipin (GP) is reported to have anti-inflammatory, down-regulating insulin, protecting liver, nerves, and other biological properties. In addition, genipin is a naturally derived cross-linking agent that is a water-soluble bifunctional cross-linking agent that reacts rapidly with amine-containing molecules (e.g., proteins) to produce a dark blue substance and is therefore also used in photothermal therapy.
Disclosure of Invention
Aiming at the anti-inflammatory problem of diabetic foot ulcers, the invention provides lysozyme fiber-like hydrogel and a preparation method thereof.
The invention discloses a preparation method of lysozyme hydrogel for diabetic foot ulcer, which comprises the following steps:
S1, dissolving lysozyme in secondary water, and then reacting under water bath and acidic conditions to obtain a lysozyme amyloid fibril (LZF) solution;
S2, dialyzing the lysozyme amyloid fibrous solution obtained in the S1 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, dissolving lysozyme amyloid and Genipin (GP) in secondary water after drying, and carrying out water bath reaction to obtain blue liquid, namely LZF/GP;
s5, adding the LZF/GP obtained in the S4 into MnSO 4 for crosslinking to obtain blue gel, namely the lysozyme hydrogel LZF/GP-MnSO 4 for diabetic foot ulcers.
Further, the water bath reaction temperature of S1 is 70-80 ℃, the acidic condition is pH 2.0-4.0, and the dosage of secondary water is suitable for dissolving lysozyme.
Further, the molecular weight of the dialysis bag of S2 is 8000-14000Da.
Further, the mass ratio of the lysozyme amyloid to genipin in the S4 is: 20-200 mg, 0.113-11.3 mg, water bath reaction temperature is 30-60 ℃, and the dosage of secondary water is suitable for dissolving the raw materials which participate in the reaction.
Further, the volume ratio of the LZF/GP to the MnSO 4 is 9:1, and the concentration of the MnSO 4 is 0.01-0.2 mmol/mL.
Further, when the concentration of the lysozyme amyloid is 20 mg/mL or more, the LZF/GP-MnSO 4 hydrogel can be obtained.
Hydrogels are extremely hydrophilic three-dimensional network-like structures that provide physical isolation and create a moist environment. The LZF/GP-MnSO 4 hydrogel prepared by the method can be used for solving the problem of diabetic foot ulcer caused by drug-resistant bacterial infection. During the treatment of diabetic foot ulcers, lysozyme fiber-Like (LZF) has unique antibacterial activity and photothermal energy to effectively kill pathogenic bacteria, and in addition, manganese ions can increase the sensitivity of cyclic GMP-AMP synthase (cGAS) to extracellular DNA, promote the activation of interferon gene stimulator Sting and promote inflammatory reaction to clear residual bacteria and destroy biological membranes. And GP has the functions of anti-inflammation and reducing blood sugar, so that the dynamic pro-inflammatory and anti-inflammatory balance is achieved, and the healing of diabetic foot ulcers is promoted. The LZF/GP-MnSO 4 hydrogel can be widely used for treating tumors, viruses and bacterial infections.
Drawings
FIG. 1 shows the setting effects of LZF-MnSO 4 hydrogels and LZF/GP-MnSO 4 hydrogels prepared in the examples at different concentrations of LZF;
FIG. 2 is a graph of dynamic time and frequency scans of a series of sulfate LZF gels prepared in the examples;
Fig. 2 (a) is a dynamic time scan, and fig. 2 (b) is a dynamic frequency scan;
FIG. 3 is an ultraviolet absorption diagram of LZF-MnSO 4 hydrogel and LZF/GP-MnSO 4 hydrogel prepared in the examples;
FIG. 4 is a graph showing the antibacterial activity of LZF-MnSO 4 hydrogels and LZF/GP-MnSO 4 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 lysozyme hydrogel for diabetic foot ulcers:
(1) Preparation of lysozyme amyloid (LZF):
Dissolving 4 g lysozyme (egg white lysozyme, purchased from the biotechnology of the source leaf) in 200 mL secondary water, and then carrying out hydrolysis reaction under the acidic condition of pH=2 in a water bath at 80 ℃ for 24 h; 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 LZF/GP-MnSO 4 hydrogel:
Dissolving dried LZF (20-80 mg) and 1.13 mg Genipin (GP) in 900 uL secondary water, and then carrying out water bath reaction at 45 ℃ for 12 h to obtain blue liquid (LZF/GP);
Adding LZF/GP into 100 uL MnSO 4 (0.05 mmol/mL) for crosslinking to obtain blue gel, namely the LZF/GP-MnSO 4 hydrogel.
Examples LZF-MnSO 4 hydrogels and LZF/GP-MnSO 4 hydrogels of different concentrations of LZF were further prepared and their gel forming effects were investigated. LZF concentration in the hydrogel is 5 mg/mL, 10 mg/mL, 20 mg/mL, 40 mg/mL and 80 mg/mL respectively, and when the LZF concentration is greater than or equal to 20 mg/mL as shown in FIG. 1, the LZF-MnSO 4 hydrogel and the LZF/GP-MnSO 4 hydrogel can be obtained.
Referring to FIG. 2, the examples also explored the rheological properties of the series LZF-MnSO 4 hydrogel and LZF/GP-MnSO 4 hydrogel, as shown in FIG. 2 (a), from dynamic time scans, both LZF-MnSO 4 hydrogel and LZF/GP-MnSO 4 hydrogel were able to rapidly set (G ¢ > G); as shown in fig. 2 (b), the dynamic frequency scanning chart shows that the gel structure is maintained all the time in the dynamic frequency scanning interval, which also shows that the series of sulfate crosslinked LZF hydrogels have the characteristic of rapid solidification and meet the basic requirements of wound dressing.
Examples the UV absorption of LZF-MnSO 4 hydrogels and LZF/GP-MnSO 4 hydrogels were further investigated by UV spectrophotometers and the results are shown in FIG. 3. LZF, after reaction with GP, presents a new UV absorption band at 600 nm, which provides the basis for the LZF/GP-MnSO 4 hydrogels to kill pathogenic bacteria by photothermal means.
Examples the antimicrobial activity of LZF-MnSO 4 hydrogels and LZF/GP-MnSO 4 hydrogels was further investigated by selecting methicillin-resistant Lin Putao cocci (MRSA) and pseudomonas aeruginosa (p. Aerosa) as antimicrobial models, as shown in fig. 4, LZF/GP-MnSO 4 hydrogels were able to kill 98.7% of MRSA and 96.0% of p. Aerosa under 650 nm laser irradiation.
As a wound dressing, the hydrogel has excellent tissue adhesion capability, so that the LZF/GP-MnSO 4 hydrogel can be directly applied to the surface of a wound in a smearing way to treat diabetic foot ulcers caused by drug-resistant bacterial infection.
Claims (5)
1. A method for preparing lysozyme hydrogel for diabetic foot ulcers, characterized in that the method comprises the following steps:
s1, dissolving lysozyme in secondary water, and then reacting in a water bath under an acidic condition to obtain a lysozyme amyloid fibrous solution;
the water bath reaction temperature is 70-80 ℃, and the acidic condition is pH 2.0-4.0;
S2, dialyzing the lysozyme amyloid fibrous solution obtained in the S1 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, dissolving lysozyme amyloid and genipin in secondary water after drying, and carrying out water bath reaction to obtain blue liquid, namely LZF/GP;
the concentration of the lysozyme amyloid fibrous solution is more than or equal to 20 mg/mL;
s5, adding the LZF/GP obtained in the S4 into MnSO 4 for crosslinking to obtain blue gel, namely the lysozyme hydrogel LZF/GP-MnSO 4 for diabetic foot ulcers.
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: the mass ratio of the lysozyme amyloid to the genipin is as follows: 20-200 mg, 0.113-11.3 mg, and the water bath reaction temperature is 30-60 ℃.
4. The method for preparing lysozyme hydrogel according to claim 1, characterized in that: the volume ratio of LZF/GP to MnSO 4 is 9:1, and the concentration of MnSO 4 is 0.01-0.2 mmol/mL.
5. A lysozyme hydrogel for diabetic foot ulcers prepared by the method of any one of claims 1 to 4.
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| CA1221046A (en) * | 1984-05-30 | 1987-04-28 | Lorne S. Reid | Method for separating lysozyme from egg-white |
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