CN116549716A - Hydrogel dressing with bacteria in-situ detection and photoresponsive antibacterial function, and preparation method and application thereof - Google Patents
Hydrogel dressing with bacteria in-situ detection and photoresponsive antibacterial function, and preparation method and application thereof Download PDFInfo
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- CN116549716A CN116549716A CN202310474611.9A CN202310474611A CN116549716A CN 116549716 A CN116549716 A CN 116549716A CN 202310474611 A CN202310474611 A CN 202310474611A CN 116549716 A CN116549716 A CN 116549716A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/008—Hydrogels or hydrocolloids
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
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- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
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- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0004—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing inorganic materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0023—Polysaccharides
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- A—HUMAN NECESSITIES
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- A61L26/0061—Use of materials characterised by their function or physical properties
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- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Materials Engineering (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Inorganic Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Dispersion Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Dermatology (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention discloses a hydrogel dressing with bacteria in-situ detection and photoresponsive antibacterial function, and a preparation method and application thereof. The invention takes carboxymethyl chitosan and oxidized sodium alginate as raw materials and loads fluorescent substrate 4-methyl umbrella type ketone-beta-D-glucuronide and antibacterial agent UCNPs@TiO 2 Nanoparticles, forming a hydrogel dressing by a schiff base reaction. When bacteria exist at the wound, the hydrogel dressing can emit blue fluorescence under the excitation of ultraviolet light, and the infection degree can be identified and judged by naked eyes or a smart phone, so that the hydrogel dressing is practicalIn situ and rapid detection of bacteria at the wound site. Meanwhile, the hydrogel dressing has excellent photodynamic antibacterial performance under the irradiation of near infrared light, and a large amount of ROS can be generated in a short time to kill bacteria, so that the light-controlled antibacterial function is realized. In addition, the hydrogel dressing has good biocompatibility and can promote wound healing. The invention has important application value in the aspects of in-situ detection of wound pathogenic bacteria, prevention of infection and on-demand treatment.
Description
Technical Field
The invention belongs to the field of biomedical engineering, and particularly relates to a hydrogel dressing with bacteria in-situ detection and photoresponsive antibacterial function, and a preparation method and application thereof.
Background
Bacterial infection of wounds is prone to sepsis, toxemia, and may even be life threatening. Traditional methods for detecting bacterial infection of wounds mainly comprise tissue biopsy, curettage, wound swabs and the like, and all have the main defects of pain, invasive operation and the like. At the same time, these methods require long sample analysis times, which can easily lead to increased infection or delayed treatment. Although the novel bacteria detection means based on PCR and GC-MS technology effectively shortens the detection time and has high accuracy, the detection means still needs several hours to process and detect the sample, and the sample needs to be sampled after the wound dressing is removed. And frequent dressing changes may result in secondary trauma to the wound bed, increased pain and additional costs for patient care. Furthermore, both PCR and GC-MS detection methods require expensive instrumentation and specialized operators. Therefore, how to quickly, timely and conveniently detect wound bacteria is a key to avoiding wound infection occurrence and deterioration and guiding clinical medication.
The construction of intelligent dressings for in situ detection of wound infection and promotion of wound healing by integrating flexible electronic sensors is a leading study in the current biomedical engineering field. The intelligent dressing mainly takes the change of the pH value or the temperature of the wound as an infection detection index. However, detectable pH or temperature changes often occur after infection formation and wound pH changes are affected by a variety of factors, thus making it difficult to accurately assess infection by pH magnitude. In addition, temperature variations are difficult to avoid interference from external environmental factors. Thus, the method is applicable to a variety of applications. Developing a smart dressing that is non-invasive, non-interfering, reliable and suitable for early detection of infection is a challenge in biomedical engineering and clinical medicine fields.
In wound infection treatment, antibiotics or antibiotic-loaded antimicrobial dressings are mainly used to treat wounds. However, the side effects of the overused antibiotics on the human body cannot be ignored. Meanwhile, the biomembrane formed by infection can resist the antibacterial action of antibiotics. Secondly, the lack of a controlled and triggered release means results in an antimicrobial dressing that will also release the antiseptic agent continuously in the absence of bacteria from the wound, thereby creating cytotoxicity, increasing costs and delaying wound healing, even leading to the emergence of drug resistant strains. The application of the intelligent dressing not only can realize the real-time acquisition of wound surface information, but also has more outstanding clinical significance in exerting antibacterial performance as required. Smart dressings with rapid and excellent antimicrobial activity triggered by external stimuli have become alternatives to antibiotics without involvement of drugs or antimicrobial agents, providing more positive and effective guidance to clinicians in the clinical practice of wound treatment. Therefore, developing an intelligent dressing which can realize in-situ bacteria detection and bacteria inhibition at the same time of wounds has important application value and social significance.
Disclosure of Invention
In view of the above, the invention aims to provide a hydrogel dressing with bacteria in-situ detection and photoresponsive antibacterial function, and a preparation method and application thereof. The hydrogel dressing consists of a hydrogel matrix, 4-methyl umbrella-type ketone-beta-D-glucuronide and a photosensitive antibacterial agent UCNPs@TiO 2 The preparation method comprises the steps of taking chitosan and sodium alginate as raw materials of the hydrogel matrix, and respectively preparing carboxymethyl chitosan and oxidized sodium alginate through carboxymethylation and oxidation reaction. The 4-methyl umbrella-type ketone-beta-D-glucuronide of the hydrogel dressing can be hydrolyzed by special enzyme beta-GUS secreted by bacteria to generate 4-methyl umbrella-type ketone, and the hydroxyl in the 4-methyl umbrella-type ketone is dissociated and then generates blue fluorescence under the excitation of ultraviolet light area, so that the visual detection of wound bacteria can be realized. In addition, UCNPs@TiO in hydrogel dressing 2 Nanoparticles in near infrared lightAnd under the irradiation, reactive Oxygen Species (ROS) are generated to kill bacteria, so that the in-situ detection and photoresponsive antibacterial functions of the composite hydrogel are realized.
The preparation method of the hydrogel dressing with the functions of in-situ detection of bacteria and light-responsive antibacterial comprises the following steps:
(1) Taking carboxymethyl chitosan solution with the concentration of 1-6wt% as hydrogel precursor solution 1; adding 4-methyl umbrella type ketone-beta-D-glucuronide and UCNPs@TiO into 1-10wt% oxidized sodium alginate solution 2 Nanoparticles to obtain hydrogel precursor solution 2;
(2) And uniformly mixing the hydrogel precursor solution 1 and the hydrogel precursor solution 2 according to the volume ratio of 1:1, and stirring at room temperature to a gel state to obtain the hydrogel dressing with the functions of in-situ bacteria detection and photoresponsive antibacterial.
Wherein the hydrogel dressing contains 0.1-10 mM 4-methyl umbrella type ketone-beta-D-glucuronide and 10-1000 mug/mL UCNPs@TiO 2 And (3) nanoparticles.
The preparation method of the carboxymethyl chitosan comprises the following steps: dissolving chitosan in a NaOH solution with the mass fraction of 50%, uniformly stirring, then placing in a refrigerator with the temperature of 0-4 ℃ for freezing for 24 hours, thawing, filtering to remove alkali liquor, and obtaining alkalized chitosan; dispersing the alkalized chitosan in isopropanol, adding chloroacetic acid, reacting for 5 hours at 60 ℃, filtering after the reaction is finished, and washing, dialyzing and freeze-drying a solid phase to obtain carboxymethyl chitosan; the chitosan: the proportion of NaOH solution is 5-20 g:100mL, the alkalized chitosan: isopropyl alcohol: the ratio of chloroacetic acid is 10-20 g:200mL: 15-30 g.
The preparation method of the oxidized sodium alginate comprises the following steps: dissolving sodium alginate in ultrapure water, adding sodium periodate, stirring at room temperature in a dark place for reaction for 6 hours, then adding ethylene glycol, and continuously stirring for 3 hours to terminate the reaction, thus obtaining oxidized sodium alginate mixture solution; dialyzing, freeze-drying the oxidized sodium alginate mixture solution to obtain oxidized sodium alginate; the sodium alginate comprises the following components: ultrapure water: sodium periodate: the proportion of the glycol is 3-6 g:300mL: 5-10 g:5mL.
Wherein the UCNPs@TiO 2 The preparation method of the nano-particles comprises the following steps: dissolving yttrium acetate, ytterbium acetate and thulium acetate in mixed solution of oleic acid and 1-octadecene, heating to 150deg.C, maintaining for 30min to dissolve solid completely, cooling to room temperature, adding NaOH and NH 4 F, heating the methanol solution to 60 ℃ for heat preservation treatment for 30min to discharge methanol, heating to 320 ℃ under the protection of nitrogen for reaction for 1h, cooling to 60 ℃ after the reaction is finished, adding absolute ethyl alcohol, centrifuging, and collecting precipitate to obtain UCNPs; dissolving UCNPs in absolute ethyl alcohol, adding polyvinylpyrrolidone in a stirring state, and continuously stirring at room temperature for 1h to obtain UCNPs suspension; uniformly mixing tetrabutyl titanate and absolute ethyl alcohol to obtain a tetrabutyl titanate ethanol mixed solution, dropwise adding UCNPs suspension into the tetrabutyl titanate ethanol mixed solution, stirring for 2 hours, adding absolute ethyl alcohol, reacting for 6 hours at 180 ℃, cooling to room temperature, centrifuging, collecting precipitate, and washing to obtain UCNPs@TiO 2 A nanoparticle; the yttrium acetate: ytterbium acetate: the molar ratio of the thulium acetate is 0.2-1: 0.1 to 0.5:0.001 to 0.005, the NaOH: NH (NH) 4 F has a mole ratio of 1-5: 2 to 10.
A hydrogel dressing prepared by the above preparation method.
The application of the hydrogel dressing is any one of the following 1) to 3):
1) Use in the visual detection of bacteria;
2) Use in the preparation of a formulation for the visual detection of bacteria;
3) Use in the preparation of a wound dressing having antibacterial properties.
The invention has the remarkable advantages that:
1) The hydrogel dressing is prepared by loading the fluorescent substrate with the polymer material with good biocompatibility, and can realize in-situ detection of the hydrogel on wound bacteria under ultraviolet irradiation (preferably 365 nm).
2) The hydrogel dressing has excellent light response antibacterial performance, and can generate ROS to cause oxidative damage of bacteria under the irradiation of near infrared light (preferably 980 nm), so that the light control response antibacterial performance of the hydrogel is realized.
3) The hydrogel dressing can be used for wound healing and detection and treatment of wound infection.
Drawings
Fig. 1: UCNPs@TiO 2 ROS production occurs.
Fig. 2: and (5) observing microscopic morphology of the section of the hydrogel.
Fig. 3: swelling properties of hydrogels.
Fig. 4: degradation rate of hydrogel.
Fig. 5: rheological properties of hydrogels.
Fig. 6: EDX elemental analysis (for CMCS/OSA/MUG/UCNPs@TiO2 hydrogels).
Fig. 7: transmittance and visibility of CMCS/OSA/MUG/UCNPs@TiO2 hydrogels.
Fig. 8: and (3) respectively imaging the composite hydrogel under normal light and ultraviolet irradiation.
Fig. 9: and detecting a result image by the smart phone.
Fig. 10: images of hydrogels at different types of wounds under normal light and ultraviolet light, respectively.
Fig. 11: coated plate images of two bacteria under light conditions with different hydrogels.
Fig. 12: different groups of wound healing rate change graphs.
Fig. 13: h & E staining of each organ of the rat after treatment with hydrogel and light.
Detailed Description
In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.
Example 1
Preparation of carboxymethyl chitosan (CMCS): dissolving 10g of chitosan in 40mL of NaOH solution with the mass fraction of 50%, uniformly stirring, then placing in a refrigerator with the temperature of 4 ℃ for freezing for 24 hours, thawing, filtering to remove alkali liquor, and obtaining alkalized chitosan; dispersing 10g of alkalized chitosan in 200mL of isopropanol, adding 15g of chloroacetic acid, reacting for 5 hours at 60 ℃, filtering after the reaction is finished, washing a solid phase with absolute ethyl alcohol for 3 times and 5 minutes each time, then redissolving with 200mL of ultrapure water, putting into a dialysis bag with the molecular weight cutoff of 8000Da, dialyzing with deionized water as dialysis solution for 72 hours, collecting liquid in the dialysis bag, transferring the liquid into a polytetrafluoroethylene plate, pre-freezing for 5 hours in a refrigerator at-80 ℃, and freeze-drying for 72 hours at-50 ℃ under the vacuum degree of 1-20 Pa to obtain CMCS.
Preparation of Oxidized Sodium Alginate (OSA): dissolving 10g of sodium alginate in 300mL of ultrapure water, adding 5g of sodium periodate, stirring at room temperature in a dark place for reaction for 6 hours, then adding 5mL of ethylene glycol, and continuously stirring for 3 hours to terminate the reaction, thus obtaining oxidized sodium alginate mixture solution; transferring the oxidized sodium alginate mixture solution into a dialysis bag with the molecular weight cutoff of 8000Da, dialyzing for 72 hours by taking deionized water as dialyzate, collecting liquid in the dialysis bag, transferring into a polytetrafluoroethylene plate, pre-freezing for 5 hours in a refrigerator at the temperature of minus 80 ℃, and then freeze-drying for 72 hours at the temperature of minus 50 ℃ under the vacuum degree of 1-20 Pa to obtain OSA.
Preparation of UCNPs nanoparticles: dissolving 0.26g yttrium acetate, 0.084g ytterbium acetate and 0.005g thulium acetate in a mixture of 6mL oleic acid and 15mL 1-octadecene, heating to 150deg.C, maintaining for 30min to dissolve the solid completely, cooling to room temperature, adding 10mL solution of NaOH (0.1 g) and NH 4 F (0.148 g) methanol solution is heated to 60 ℃ and is subjected to heat preservation treatment for 30min to discharge methanol, then the methanol solution is heated to 320 ℃ under the protection of nitrogen to react for 1h, after the reaction is finished, the methanol solution is cooled to 60 ℃, 25mL of absolute ethanol is added, the ethanol solution is centrifuged for 3 times at 10000r/min for 5min each time, and UCNPs are obtained by collecting sediment.
UCNPs@TiO 2 Preparation of nanoparticles: dissolving 0.01g UCNPs in 10mL absolute ethyl alcohol, adding 0.1g polyvinylpyrrolidone (PVP) under stirring, and continuously stirring at room temperature for 1h to obtain UCNPs suspension; uniformly mixing 0.02mL of tetrabutyl titanate with 5mL of absolute ethyl alcohol to obtain a tetrabutyl titanate ethanol mixed solution; dropwise adding 10mL UCNPs suspension into 5mL tetrabutyl titanate ethanol mixed solution, stirring for 2h, adding 15mL absolute ethanol, reacting for 6h at 180 ℃ in an autoclave with a polytetrafluoroethylene lining, cooling to room temperature, centrifuging for 3 times at 10000r/min for 5min each time, collecting precipitate, washing for 3 times with absolute ethanol for 5min each time, and obtaining UCNPs@TiO 2 And (3) nanoparticles.
Preparation of CMCS/OSA/MUG/UCNPs@TiO 2 Hydrogel: carboxymethyl chitosan was buffered with PBS (0.1 m, ph=7.2)Preparing a solution with the concentration of 4.8wt% to obtain a hydrogel precursor solution 1; oxidized sodium alginate was prepared as a 7.2wt% solution in PBS buffer (0.1M, pH=7.2), to which 7mg of 4-methyl umbrella-type ketone-beta-D-glucuronide (MUG) and 2mg of UCNPs@TiO were added 2 Obtaining hydrogel precursor solution 2; the hydrogel precursor solution 1 and the hydrogel precursor solution 2 are mixed according to a volume ratio of 1:1 are evenly mixed and stirred at room temperature to be in a gel state to obtain CMCS/OSA/MUG/UCNPs@TiO 2 A hydrogel.
Preparation of CMCS/OSA hydrogels: carboxymethyl chitosan was formulated as a 4.8wt% solution with PBS buffer (0.1 m, ph=7.2) to give hydrogel precursor solution 1; preparing oxidized sodium alginate into 7.2wt% solution by using PBS buffer solution (0.1M, pH=7.2) to obtain hydrogel precursor solution 2; the hydrogel precursor solution 1 and the hydrogel precursor solution 2 are mixed according to a volume ratio of 1:1, uniformly mixing, and stirring to gel state at room temperature to obtain the CMCS/OSA hydrogel.
Preparation of CMCS/OSA/MUG hydrogels: carboxymethyl chitosan was formulated as a 4.8wt% solution with PBS buffer (0.1 m, ph=7.2) to give hydrogel precursor solution 1; oxidized sodium alginate was prepared as a 7.2wt% solution in PBS buffer (0.1 m, ph=7.2), to which 7mg of 4-methyl umbrella-type ketone-beta-D-glucuronide (MUG) was added to obtain hydrogel precursor solution 2; the hydrogel precursor solution 1 and the hydrogel precursor solution 2 are mixed according to a volume ratio of 1:1, uniformly mixing, and stirring to a gel state at room temperature to obtain the CMCS/OSA/MUG hydrogel.
Preparation of CMCS/OSA/UCNPs@TiO 2 : carboxymethyl chitosan was formulated as a 4.8wt% solution with PBS buffer (0.1 m, ph=7.2) to give hydrogel precursor solution 1; oxidized sodium alginate was prepared as a 7.2wt% solution in PBS buffer (0.1M, pH=7.2), to which 2mg UCNPs@TiO was added 2 Obtaining hydrogel precursor solution 2; the hydrogel precursor solution 1 and the hydrogel precursor solution 2 are mixed according to a volume ratio of 1:1 are evenly mixed and stirred to gel state at room temperature to obtain CMCS/OSA/UCNPs@TiO 2 A hydrogel.
Characterization of the hydrogels of this example:
in vitro experiments using DPBF as probe for detecting ROS production, 980nm near-red was studiedUCNPs@TiO under external light irradiation 2 In the case of ROS production, the results are shown in fig. 1A, where the absorption value of DPBF at 410nm decreases with increasing illumination time, indicating ROS production. In addition, UCNPs@TiO is treated under 365nm ultraviolet light condition 2 As shown in fig. 1B, the absorption value of DPBF gradually decreases with increasing irradiation time under 365nm ultraviolet irradiation, which demonstrates ROS generation, and ROS generation speed is about the same as 980nm near infrared irradiation, but in view of radiation hazard of ultraviolet rays to human skin and excellent penetration of near infrared light.
Microscopic morphology observation is carried out on the section of the hydrogel under a scanning electron microscope. As shown in fig. 2, each hydrogel has a continuous and stable three-dimensional network structure, and a plurality of microporous structures are more uniformly distributed in the interior of each hydrogel; CMCS/OSA, CMCS/OSA/MUG, CMCS/OSA/UCNPs@TiO 2 、CMCS/OSA/MUG/UCNPs@TiO 2 The average pore size of the hydrogels was 283.2 μm, 265.5 μm, 270.8 μm and 263.5 μm, UCNPs@TiO, respectively 2 The addition of nanoparticles did not have a significant effect on the pore size of the hydrogels.
The swelling rate of the hydrogels in PBS buffer (ph=7.4) was tested at room temperature. As shown in FIG. 3, each hydrogel showed a certain swelling property because CMCS and OSA each contain a large amount of hydrophilic groups such as-OH, -NH 2 and-COOH, which are capable of allowing more water molecules to enter the hydrogel network; CMCS/OSA, CMCS/OSA/MUG, CMCS/OSA/UCNPs@TiO 2 And CMCS/OSA/MUG/UCNPs@TiO 2 The maximum swelling ratios of the hydrogels were 161.3%, 166.4%, 171.7% and 175.9%, UCNPs@TiO, respectively 2 The presence of the nanoparticles does not significantly affect the swelling properties of the hydrogels, UCNPs@TiO 2 Hydrogels still exhibit good water absorption in the presence of nanoparticles.
The hydrogel was tested for degradation rate in PBS buffer (ph=7.4) at room temperature. As shown in fig. 4, the weight of each hydrogel gradually decreases with the increase of time; after 10 days of degradation in PBS buffer, CMCS/OSA/MUG, CMCS/OSA/UCNPs@TiO 2 And CMCS/OSA/MUG/UCNPs@TiO 2 The degradation rates of the hydrogel are 62.1%, 58.1%, 52.8% and 50.5%, respectively, which shows that each hydrogel dressing has good in-vitro degradability; compared with CMCS/OSA hydrogel, UCNPs@TiO is doped 2 The degradation rate of the hydrogel of the nanoparticle is relatively low due to ucnps@tio 2 The nanoparticles improve the mechanical properties and stability of the hydrogels.
Detection of CMCS/OSA, CMCS/OSA/MUG, CMCS/OSA/UCNPs@TiO, respectively, using a rotarheometer 2 And CMCS/OSA/MUG/UCNPs@TiO 2 Rheological properties of hydrogels. As shown in fig. 5, each hydrogel had excellent rheological properties, and as the angular frequency was increased, the storage modulus of the hydrogel was higher than the energy consumption modulus, indicating that each hydrogel remained in a complete gel form; wherein UCNPs@TiO is doped 2 The hydrogels of the nanoparticles clearly show a higher storage modulus (G') mainly due to UCNPs@TiO 2 The addition of (3) improves the mechanical strength and crosslink density of the hydrogel.
For CMCS/OSA/MUG/UCNPs@TiO 2 EDX elemental analysis was performed on the hydrogels to verify UCNPs@TiO 2 Loading of nanoparticles on hydrogels. As shown in FIG. 6, UCNPs@TiO in hydrogels 2 The absorption peaks appear in the four trace elements F, Y, yb, tm and Ti, which indicates UCNPs@TiO 2 Successfully loaded inside the hydrogel.
CMCS/OSA/MUG/UCNPs@TiO using an ultraviolet-visible spectrophotometer pair 2 The transmittance of the hydrogels was tested. As shown in fig. 7, the hydrogel has a high light transmittance of 80.1% in the visible light range of 400 to 800 nm; in addition, each color of the 12-color ring can still be clearly observed through the hydrogel, which indicates that the hydrogel has good visibility and is favorable for detecting wound pathogenic bacteria.
Example 2
Preparation of carboxymethyl chitosan (CMCS): dissolving 10g of chitosan in 40mL of NaOH solution with the mass fraction of 50%, uniformly stirring, then placing in a refrigerator with the temperature of 4 ℃ for freezing for 24 hours, thawing, filtering to remove alkali liquor, and obtaining alkalized chitosan; dispersing 10g of alkalized chitosan in 200mL of isopropanol, adding 15g of chloroacetic acid, reacting for 5 hours at 60 ℃, filtering after the reaction is finished, washing a solid phase with absolute ethyl alcohol for 3 times and 5 minutes each time, then redissolving with 200mL of ultrapure water, putting into a dialysis bag with the molecular weight cutoff of 8000Da, dialyzing with the ultrapure water as a dialysis solution for 72 hours, collecting liquid in the dialysis bag, transferring the liquid into a polytetrafluoroethylene plate, pre-freezing for 5 hours in a refrigerator at-80 ℃, and freeze-drying for 72 hours at-50 ℃ under the vacuum degree of 1-20 Pa to obtain the CMCS.
Preparation of Oxidized Sodium Alginate (OSA): dissolving 10g of sodium alginate in 300mL of ultrapure water, adding 5g of sodium periodate, stirring at room temperature in a dark place for reaction for 6 hours, then adding 5mL of ethylene glycol, and continuously stirring for 3 hours to terminate the reaction, thus obtaining oxidized sodium alginate mixture solution; transferring the oxidized sodium alginate mixture solution into a dialysis bag with the molecular weight cutoff of 8000Da, dialyzing for 72 hours by taking ultrapure water as dialyzate, collecting liquid in the dialysis bag, transferring into a polytetrafluoroethylene plate, pre-freezing for 5 hours in a refrigerator at the temperature of minus 80 ℃, and then freeze-drying for 72 hours at the temperature of minus 50 ℃ under the vacuum degree of 1-20 Pa to obtain OSA.
Preparation of UCNPs nanoparticles: dissolving 0.26g yttrium acetate, 0.084g ytterbium acetate and 0.005g thulium acetate in a mixture of 6mL oleic acid and 15mL 1-octadecene, heating to 150deg.C, maintaining for 30min to dissolve the solid completely, cooling to room temperature, adding 10mL solution of NaOH (0.1 g) and NH 4 F (0.148 g) methanol solution is heated to 60 ℃ and is subjected to heat preservation treatment for 30min to discharge methanol, then the methanol solution is heated to 320 ℃ under the protection of nitrogen to react for 1h, after the reaction is finished, the methanol solution is cooled to 60 ℃, 25mL of absolute ethanol is added, the ethanol solution is centrifuged for 3 times at 10000r/min for 5min each time, and UCNPs are obtained by collecting sediment.
UCNPs@TiO 2 Preparation of nanoparticles: dissolving 0.01g UCNPs in 10mL absolute ethyl alcohol, adding 0.1g polyvinylpyrrolidone (PVP) under stirring, and continuously stirring at room temperature for 1h to obtain UCNPs suspension; uniformly mixing 0.02mL of tetrabutyl titanate with 5mL of absolute ethyl alcohol to obtain a tetrabutyl titanate ethanol mixed solution; dropwise adding 10mL UCNPs suspension into 5mL tetrabutyl titanate ethanol mixed solution, stirring for 2h, adding 15mL absolute ethanol, reacting for 6h at 180 ℃ in an autoclave with a polytetrafluoroethylene lining, cooling to room temperature, centrifuging for 3 times at 10000r/min for 5min each time, collecting precipitate, washing for 3 times with absolute ethanol for 5min each time, and obtaining UCNPs@TiO 2 And (3) nanoparticles.
Preparation of CMCS/OSA/MUG/UCNPs@TiO 2 Hydrogel: preparing carboxymethyl chitosan into a solution with 4wt% by using ultrapure water to obtain a hydrogel precursor solution 1; the oxidized sodium alginate was prepared into an 8wt% solution with ultra pure water, to which 7mg of 4-methyl umbrella type ketone-beta-D-glucuronide (MUG) and 2mg of UCNPs@TiO were added 2 Obtaining hydrogel precursor solution 2; the hydrogel precursor solution 1 and the hydrogel precursor solution 2 are mixed according to a volume ratio of 1:1 are evenly mixed and stirred at room temperature to be in a gel state to obtain CMCS/OSA/MUG/UCNPs@TiO 2 A hydrogel.
Characterization of the hydrogels of this example:
respectively selecting five strains of escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumoniae and enterococcus faecalis and CMCS/OSA/MUG/UCNPs@TiO 2 Co-culturing the hydrogel for 3 hours, and setting the bacterial concentration to be 10 5 CFU/mL, the duration of the change in blue fluorescence intensity in the hydrogel was measured under 365nm ultraviolet light, and the average time consumption of the PCR detection method commonly used in clinic was selected as a control (Table 1). CMCS/OSA/MUG/UCNPs@TiO 2 The blue fluorescence phenomenon of the hydrogel under normal light conditions and 365nm ultraviolet light irradiation after the hydrogel is contacted and cultured with Escherichia coli for 3 hours is shown in FIG. 8, the left side is the hydrogel without 4-methyl umbrella-type ketone-beta-D-glucuronide, and the result shows that under the irradiation of an ultraviolet lamp, CMCS/OSA/MUG/UCNPs@TiO is shown 2 The fluorescence phenomenon of the hydrogel is obvious, and can be used as a judging basis for distinguishing bacterial infection.
TABLE 1 determination of the detection duration of different bacteria
Colony forming unit was taken to be 10 3 Is added into 200 mu L CMCS/OSA/MUG/UCNPs@TiO by liquid drops 2 In the hydrogel, after co-culturing for 3 hours, 980nm near infrared light is applied for 15 minutes, the hydrogel is photographed at fixed time nodes respectively, and the fluorescence intensity value of the hydrogel is calculated. By not applying lightPositive control group, negative control group without bacteria added. The results are shown in Table 2, CMCS/OSA/MUG/UCNPs@TiO 2 The hydrogel has good bacterial detection and photoresponsive antibacterial performance in vitro.
TABLE 2 fluorescence intensity Change after Co-culture of hydrogels with bacteria
A circular wound with the diameter of 1cm is constructed on the back of a rat, 20 mu L of mixed bacterial liquid of escherichia coli and staphylococcus aureus is dripped, and then CMCS/OSA/MUG/UCNPs@TiO is coated once on days 1, 3, 5 and 7 2 The hydrogel was photographed 3 hours after the application of the hydrogel to the wound and the fluorescence intensity was calculated, and the sterilization treatment was performed using 980nm near infrared light to irradiate the hydrogel for 15 minutes, using SD rat wound without bacteria as a control group. The results are shown in Table 3, CMCS/OSA/MUG/UCNPs@TiO 2 The hydrogel has excellent bacteria detection and response antibacterial capability in the wound healing process.
TABLE 3 variation of fluorescence intensity of hydrogels during wound healing
Colony forming unit was taken to be 10 3 Is added into 200 mu L CMCS/OSA/MUG/UCNPs@TiO by liquid drops 2 In the hydrogel, smart phones are used for photographing the hydrogel at different time nodes respectively, and a mobile phone interface image for judging the infection degree is used by using a mobile phone detection program Pathogenic Detection which is developed by the applicant. As shown in fig. 9, the smart phone can accurately detect the fluorescence intensity change of the hydrogel, and the applicability of the hydrogel dressing is enhanced by detecting the fluorescence intensity change in cooperation with the smart phone.
Anaesthetizing the rat by intraperitoneal injection, constructing a circular full-thickness skin defect wound (non-infected wound) model with the diameter of 10mm after dehairing the back of the rat, and then dripping 20 mu L of 10 at the wound 7 CFU/mL mixed bacterial liquid of escherichia coli and staphylococcus aureus to construct an infection wound model, and CMCS/OSA/MUG/UCNPs@TiO 2 After the hydrogel is placed at the full-thickness skin infection and non-infection wounds of the rats for 15min, the blue fluorescence phenomenon of the hydrogel under the normal light condition and 365nm ultraviolet light irradiation respectively, the result is shown in figure 10, the left side infection type wound emits obvious blue fluorescence under the ultraviolet light irradiation due to the existence of bacteria, and the right side conventional wound does not emit fluorescence, which indicates that CMCS/OSA/MUG/UCNPs@TiO 2 Photo-control detection performance of hydrogels.
Hydrogel group: drop 50. Mu.L of bacterial suspension into CMCS/OSA/MUG/UCNPs@TiO 2 A hydrogel surface; hydrogel + light group: drop 50. Mu.L of bacterial suspension into CMCS/OSA/MUG/UCNPs@TiO 2 The hydrogel surface was irradiated with 980nm near infrared light for 15min; gentamicin group: mu.L of the bacterial suspension was added dropwise to a 0.5mM gentamicin solution; illumination group: mu.L of the bacterial suspension was irradiated with 980nm near infrared light for 15min. Culturing at 37 ℃ for 2 hours at constant temperature, cleaning bacteria by using PBS buffer solution, re-suspending to obtain bacterial suspension, taking 50 mu L of the bacterial suspension, coating the bacterial suspension on a flat plate, incubating the bacterial suspension in a constant temperature incubator for 24 hours, counting colonies appearing in a culture dish, and calculating bacterial survival rates under different conditions. The results are shown in Table 4 and FIG. 11, in which the hydrogel+light group showed little bacterial survival, while the hydrogel alone had no bacterial killing effect, indicating CMCS/OSA/MUG/UCNPs @ TiO 2 The hydrogel has good photoresponsive antibacterial capability.
TABLE 4 statistics of antibacterial Rate of hydrogels under different conditions
The E.coli and Staphylococcus aureus suspensions were added to the well plates for 48h to form complete biofilms, which were then grouped for different treatments: no light group: placing CMCS/OSA/MUG/UCNPs@TiO2 hydrogel on the surface of a biological membrane for 15min; hydrogel + light group: CMCS/OSA/MUG/UCNPs@TiO 2 Placing the hydrogel on the surface of a biological film and irradiating with 980nm near infrared light for 15min; gentamicin group: a solution of 0.5mM gentamicin was added dropwise to the surface of the biofilm for 15min. After the end of the treatment 100. Mu.L of the biofilm solution was pipetted and the absorbance at 590nm was measured. The results are shown in Table 5, where the biofilm clearance rate was significantly higher in the hydrogel + light group than in the no light group and gentamicin group.
TABLE 5 biofilm removal Capacity
The healing promotion effect of the composite hydrogel on the wound is studied by using SD male rats, firstly, anaesthetizing the rats by intraperitoneal injection, constructing a circular full-layer skin defect wound model with the diameter of 10mm after dehairing the backs of the rats, and dripping 20 mu L of 10 at the wound 7 The mixed bacterial solution of CFU/mL escherichia coli and staphylococcus aureus is used for constructing an infected wound model, then wounds are treated according to different groups, and the wound model is fixed by using an indwelling needle patch, and dressing is replaced for rats every 3 days until the wounds are completely healed. The wound healing calculation formula is: wound closure rate (%) = (a) 0 -A t )/A 0 X 100%, where A 0 Represents the area of the wound on day 0; a is that t Represents the wound area on the t-th day. The specific grouping is as follows: control group: no treatment is performed; hydrogel alone group: coating CMCS/OSA/MUG/UCNPs@TiO 2 A hydrogel; hydrogel + light group: coating CMCS/OSA/MUG/UCNPs@TiO 2 Hydrogel was irradiated with 980nm near infrared light for 15min; commercial dressing sets: medical antibacterial dressing (acasin-Ag) coated with Aikexin nano silver + ). The experimental results are shown in fig. 12 and table 6, and the wound closure rate of the hydrogel + light group is significantly increased compared to the commercial dressing group, mainly because the excellent biocompatibility of carboxymethyl chitosan and oxidized sodium alginate promotes the growth and proliferation of cells in the wound healing process, and accelerates the healing of the wound. At the wound is completeH for each organ of rat after healing&E staining, the results are shown in FIG. 13, and the rat organs are normal in morphology, clear in structure and free of obvious pathological damage, indicating CMCS/OSA/MUG/UCNPs@TiO 2 The hydrogel has excellent biocompatibility and can be used for the treatment and repair of wounds.
TABLE 6 wound closure statistics
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (10)
1. A preparation method of a hydrogel dressing with bacteria in-situ detection and photoresponsive antibacterial functions is characterized by comprising the following steps: the method comprises the following steps:
(1) Taking carboxymethyl chitosan solution with the concentration of 1-6wt% as hydrogel precursor solution 1; adding 4-methyl umbrella type ketone-beta-D-glucuronide and UCNPs@TiO into an oxidized sodium alginate solution with the concentration of 1-10wt% 2 Nanoparticles to obtain hydrogel precursor solution 2;
(2) Hydrogel precursor solution 1 and hydrogel precursor solution 2 were mixed according to 1:1, mixing uniformly in volume ratio, and stirring to gel state at room temperature to obtain the hydrogel dressing with bacteria in-situ detection and photoresponsive antibacterial effect.
2. The method of manufacturing according to claim 1, characterized in that: the hydrogel dressing contains 0.1-10 mM 4-methyl umbrella type ketone-beta-D-glucuronide and 10-1000 mug/mL UCNPs@TiO 2 And (3) nanoparticles.
3. The method of manufacturing according to claim 1, characterized in that: the preparation method of the carboxymethyl chitosan comprises the following steps: dissolving chitosan in a NaOH solution with the mass fraction of 50%, uniformly stirring, then, freezing in a refrigerator with the temperature of 0-4 ℃ for 24 hours, thawing, and filtering to remove alkali liquor to obtain alkalized chitosan; dispersing the alkalized chitosan in isopropanol, adding chloroacetic acid, reacting for 5 hours at 60 ℃, filtering after the reaction is finished, and washing, dialyzing and freeze-drying a solid phase to obtain carboxymethyl chitosan.
4. A method of preparation according to claim 3, characterized in that: the chitosan: the proportion of NaOH solution is 5-20: g:100mL, the alkalized chitosan: isopropyl alcohol: the proportion of chloroacetic acid is 10-20 g:200mL: 15-30 g.
5. The method of manufacturing according to claim 1, characterized in that: the preparation method of the oxidized sodium alginate comprises the following steps: dissolving sodium alginate in ultrapure water, adding sodium periodate, stirring at room temperature in a dark place for reaction for 6 hours, then adding ethylene glycol, and continuously stirring for 3 hours to terminate the reaction, thus obtaining oxidized sodium alginate mixture solution; and (5) dialyzing and freeze-drying the oxidized sodium alginate mixture solution to obtain the oxidized sodium alginate.
6. The method of manufacturing according to claim 5, wherein: the sodium alginate comprises the following components: ultrapure water: sodium periodate: the proportion of the ethylene glycol is 3-6 g:300mL: 5-10 g:5mL.
7. The method of manufacturing according to claim 1, characterized in that: the UCNPs@TiO 2 The preparation method of the nano-particles comprises the following steps:
dissolving yttrium acetate, ytterbium acetate and thulium acetate in mixed solution of oleic acid and 1-octadecene, heating to 150deg.C, maintaining for 30min to dissolve solid completely, cooling to room temperature, adding NaOH and NH 4 F, heating the methanol solution to 60 ℃ for heat preservation treatment for 30min to discharge methanol, heating to 320 ℃ under the protection of nitrogen for reaction for 1h, cooling to 60 ℃ after the reaction is finished, adding absolute ethyl alcohol, centrifuging, and collecting precipitate to obtain UCNPs; dissolving UCNPs in absolute ethyl alcohol, adding polyvinylpyrrolidone in a stirring state, and continuously stirring at room temperature for 1h to obtain UCNPs suspension;
uniformly mixing tetrabutyl titanate and absolute ethyl alcohol to obtain a tetrabutyl titanate ethanol mixed solution, dropwise adding UCNPs suspension into the tetrabutyl titanate ethanol mixed solution, stirring for 2 hours, adding absolute ethyl alcohol, reacting for 6 hours at 180 ℃, cooling to room temperature, centrifuging, collecting precipitate, and washing to obtain UCNPs@TiO2 nano particles.
8. The method of manufacturing according to claim 7, wherein: the yttrium acetate: ytterbium acetate: the molar ratio of the thulium acetate is 0.2-1: 0.1 to 0.5: 0.001-0.005, naOH: NH (NH) 4 F has a molar ratio of 1-5: 2-10.
9. A hydrogel dressing made by the method of making of claim 1.
10. Use of a hydrogel dressing according to claim 9, wherein: is any one of the following 1) to 3):
1) Use in the visual detection of bacteria;
2) Use in the preparation of a formulation for the visual detection of bacteria;
3) Use in the preparation of a wound dressing having antibacterial properties.
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| CN202310474611.9A CN116549716A (en) | 2023-04-28 | 2023-04-28 | Hydrogel dressing with bacteria in-situ detection and photoresponsive antibacterial function, and preparation method and application thereof |
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| CN202310474611.9A CN116549716A (en) | 2023-04-28 | 2023-04-28 | Hydrogel dressing with bacteria in-situ detection and photoresponsive antibacterial function, and preparation method and application thereof |
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