CN117797258A - Preparation method and application of photo-thermal synergistic chemical kinetics nano gel for resisting bacterial biofilm infection - Google Patents
Preparation method and application of photo-thermal synergistic chemical kinetics nano gel for resisting bacterial biofilm infection Download PDFInfo
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- CN117797258A CN117797258A CN202311845320.2A CN202311845320A CN117797258A CN 117797258 A CN117797258 A CN 117797258A CN 202311845320 A CN202311845320 A CN 202311845320A CN 117797258 A CN117797258 A CN 117797258A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 12
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- 239000001263 FEMA 3042 Substances 0.000 claims abstract description 31
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- BLUAFEHZUWYNDE-NNWCWBAJSA-N artemisinin Chemical class C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2OC(=O)[C@@H]4C BLUAFEHZUWYNDE-NNWCWBAJSA-N 0.000 claims abstract description 22
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims abstract description 19
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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
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
-
- 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
- A61K33/26—Iron; Compounds thereof
-
- 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
- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
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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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- General Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Communicable Diseases (AREA)
- Inorganic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a preparation method and application of a nano gel for resisting bacterial biofilm infection by photo-thermal synergistic chemical kinetics, wherein the nano gel is prepared by loading natural polyphenol Tannic Acid (TA) on natural substance Hyaluronic Acid (HA) in a covalent bond combination mode, and simultaneously, forming a metal-polyphenol network structure (MPN) by utilizing TA and Fe2+ self-assembly, and packaging artemisinin analogues (Aas). The components of the nanogel are synergistic, wherein MPN formed by TA and Fe2+ has good photo-thermal performance, and under the irradiation of near infrared light, the photo-energy can be converted into heat energy for photo-thermal treatment, so that the nanogel has good anti-biofilm effect. HA is a natural polysaccharide which can be degraded by the action of hyaluronidase produced by bacteria, facilitating the release of artemisinin species. The invention realizes the effect of combining photo-thermal and chemical kinetics to resist the biological membrane, and the nanogel has good biocompatibility and high biological safety, and is very suitable for treating the biological membrane infection.
Description
Technical Field
The invention belongs to the technical field of antibacterial materials, and in particular relates to a nanogel for treating biomembrane infection by photo-thermal synergistic chemical kinetics, and a preparation method and application thereof.
Background
Research in the U.S. centers for disease control and prevention has shown that about 65% -80% of human bacterial infections are associated with bacterial biofilms. Bacterial biofilms are three-dimensional microflora formed by extracellular polymeric substances secreted by bacteria and surrounding the bacteria, the bacteria themselves, and the like. Compared with planktonic bacteria without forming a biological film, the tolerance of the biological film bacteria to antibiotics is 10-1000 times that of planktonic bacteria, and the biological film bacteria have stronger resistance to complex environments, and can reduce or invalidate the drug effect of antibiotics and antibacterial products/disinfectants. Therefore, it is urgent to explore a highly effective anti-biofilm means. It has been found that bacterial biofilm infections are often accompanied by microenvironmental disruption and abnormal physiological signals, such as a decrease in pH. Once a biofilm is formed at the bacterial infection site, the production and excessive accumulation of anaerobic glycolytic acid metabolites will result, ultimately, in a localized acidic microenvironment of the bacterial biofilm. Therefore, the pH difference between the bacterial biofilm infection site and normal tissues can be used as a trigger signal to develop a corresponding antibacterial drug delivery platform.
Photothermal therapy (PTT) is a therapeutic method in which a photothermal conversion agent absorbs near infrared light, and then converts the absorbed light energy into heat energy by using the photothermal effect, and kills bacteria and eliminates biofilms by thermal injury. The green sterilization effect which is rapid and efficient, is not easy to resist medicine, is noninvasive and plays a role locally is attracting a great deal of attention. Many nanomaterials with photothermal effects are used in photothermal antimicrobial applications. Wherein the metal-polyphenol network (MPN) is formed by rapid self-assembly of polyphenol compounds and metal ions in aqueous solution through coordination chelation. Coordination of metal ions in MPN with polyphenols allows MPN to have a relatively broad absorption band in the near infrared region. Therefore, the MPN has higher photo-thermal conversion efficiency and good photo-thermal stability, and can be used as a photo-thermal conversion material for photo-thermal treatment. In addition, MPN has pH responsiveness and will decompose in the acidic microenvironment of the bacterial biofilm. Therefore, MPN can be used as a gate of drug release to control the release of drugs at the bacterial biofilm site.
However, PTT alone is used to sterilize the local high temperatures that it generates may destroy the undesirable inflammation caused by surrounding healthy tissue, and the therapeutic efficiency of single photothermal therapy is limited. In recent years, chemical kinetics (CDT) has been widely studied for its excellent antibacterial properties. CDT is produced by using Fe 2+ Or other metal ions with H 2 O 2 Fenton or Fenton-like reactions occur to produce highly toxic ∙ OH, which induces bacterial death by destroying DNA, inactivating proteins, and causing peroxidation of cell membrane lipids. The method is only carried out in a slightly acidic environment of the bacterial biomembrane, does not need exogenous light radiation, and ensures the safety of normal tissues to a certain extent. PTT in combination with CDT may achieve synergistic therapeutic effects.
Artemisinin and its derivatives are most well known for their potent antimalarial effect. With the continuous and intensive research on artemisinin and its derivatives, it is found that artemisinin and its derivatives not only can resist malaria, but also have the functions of resisting tumor, inflammation, bacteria and the like. The treatment mechanism is similar to Fenton reaction: at the position ofFe 2+ Or other metal ions, the peroxy bridge in the artemisinin derivative structure can be broken, and a large amount of active oxygen with high toxicity is generated to kill cells or bacteria.
Disclosure of Invention
The invention aims to provide a preparation method and application of a nano gel for resisting bacterial biofilm infection by photo-thermal synergistic chemical kinetics, and aims to overcome the defects that local high temperature generated by photo-thermal treatment alone used for sterilization can damage surrounding healthy tissues to cause bad inflammation, single photo-thermal treatment duration is short, treatment efficiency is limited and the like. The nanogel loads natural polyphenol Tannic Acid (TA) on natural Hyaluronic Acid (HA) in a covalent bond mode, and simultaneously utilizes TA and Fe 2+ Self-assembling to form metal-polyphenol network structure (MPN), and packaging artemisinin analogue (Aas) to obtain HA-Ta-Aas-Fe 2+ . TA and Fe in the nano gel 2+ The formed MPN can convert light energy into heat energy for photothermal treatment, and has good anti-biofilm effect. In addition, under the acidic microenvironment of the bacterial biofilm, TA-Fe with MPN structure 2+ Will decompose into free TA and Fe 2+ And release artemisinin substances, fe 2+ Can be combined with hydrogen peroxide (H) 2 O 2 ) Fenton reaction occurs to generate high toxicity ∙ OH to kill bacteria in the biological film, and chemical kinetics treatment effect is exerted. At the same time Fe 2+ Can react with artemisinin substances to generate carbon free radical for sterilization. In addition, fe generated by the reaction 3+ Can be reduced to Fe again under the action of TA 2+ The therapeutic effect is continuously exerted. HA is a natural polysaccharide which can be degraded by the action of hyaluronidase produced by bacteria, facilitating the release of artemisinin species. The components of the nano gel cooperate together, so that the anti-biofilm effect of the nano gel can be effectively improved, the biocompatibility is good, and the biological safety is high.
The aim of the invention can be achieved by the following technical scheme:
photo-thermal synergistic chemistryThe nanometer gel is prepared by loading natural polyphenol tannic acid TA on natural hyaluronic acid HA in a covalent bond mode, and simultaneously utilizing TA and Fe 2+ Self-assembling to form MPN with metal-polyphenol network structure, and packaging the artemisinin analogue Aas.
As a further scheme of the invention: the arteannuin substance comprises dihydroarteannuin DHA, arteannuin ARN, artesunate ART, artemether ARM, etc.
As a further scheme of the invention: the method specifically comprises the following steps:
s1, dissolving 20.0g,11.8mmol TA in 50mL DMF, adding 3.4g,14.2mmol 3-boc-bromopropylamine and 20.3g,147.0mmol K2CO3, heating to 60℃under nitrogen and stirring 6h with a magnetic stirrer, cooling to room temperature, adding 100mL deionized water, acidifying the solution with 50mL 6M HCl, extracting three times with 100mL ethyl acetate, washing the separated ethyl acetate layer twice with 100mL deionized water, anhydrous Na 2 SO 4 Drying for 12 hours, then removing ethyl acetate by rotary evaporation, adding 50mL absolute ethyl alcohol for re-dissolving, dialyzing the absolute ethyl alcohol for two days, removing the absolute ethyl alcohol by rotary evaporation, and pumping the solution by a suction filtration bottle to obtain white solid TA-Boc-NH 2 ；
S2, taking 10g of TA-Boc-NH 2 Dissolving in 100mL of absolute ethyl alcohol, introducing HCl vapor generated by the reaction of concentrated sulfuric acid and NaCl into the solution for 6h, then continuously stirring at room temperature for 24 hours, removing the absolute ethyl alcohol by rotary evaporation after the reaction is finished, adding 50mL of deionized water for redissolution and dialyzing for two days, and freeze-drying to obtain light yellow amino tannic acid solid ATA;
s3, 10-20 Wan, 300mg,0.74mmol HA was added to 40mL deionized water and stirred at room temperature for 30 minutes, followed by 285.19mg, 0.292 mol/L, 5mL EDC.HCl in aqueous solution and 5mL, 171.22mg, 0.292 mol/L, NHS in aqueous solution stirred at room temperature for 12 hours. Then under the protection of nitrogen, adding 10mL, 267mg,0.015mol/L and ATA dissolved in DMSO, stirring at room temperature for reaction for 24h, dialyzing with deionized water of 350 Da for 1-2 days, freeze-drying to obtain HA-TA, and preserving at-20 ℃ for later use;
s4, 30mg of HA-TDissolving A in 20mL of deionized water, regulating the solution to be slightly alkaline pH to 8 by using 5mg/mL of Tris base, transferring into a three-necked flask, introducing nitrogen, stirring at room temperature for 30min, and slowly adding 2mL of Aas solution, namely DMSO solution, and dissolving 12.5 mg/mL; then 20mL of 0.1% FeCl was added dropwise at a rate of 40mL/h using a syringe pump 2 •4H 2 O, continuing stirring and reacting for 2h; finally, deionized water is used for washing the supernatant liquid to be colorless, and the HA-TA-Aas-Fe is prepared 2+ The prepared HA-TA-Aas-Fe 2 + The nanogel is dispersed in an aqueous solution and stored at 4 ℃.
The photo-thermal synergistic chemical kinetics antibacterial biofilm infection nanogel has a synergistic killing effect on bacterial biofilms.
The invention has the beneficial effects that: the components of the nanogel act synergistically, wherein TA and Fe 2+ The formed MPN has good photo-thermal performance, can convert the photo-energy into heat energy for photo-thermal treatment under the irradiation of near infrared light, and has good anti-biofilm effect. In addition, MPN has pH responsiveness, and TA-Fe with MPN structure under acidic microenvironment of bacterial biofilm 2+ Will decompose into free TA and Fe 2+ And release artemisinin analogues, fe 2+ Can be combined with hydrogen peroxide (H) 2 O 2 ) Fenton reaction occurs to generate high toxicity ∙ OH to kill bacteria in the biological film, and chemical kinetics treatment effect is exerted. At the same time Fe 2+ Can react with artemisinin analogues to generate carbon radical sterilization. In addition, fe generated by the reaction 3+ Can be reduced to Fe again under the action of TA 2+ The therapeutic effect is continuously exerted. HA is a natural polysaccharide which can be degraded by the action of hyaluronidase produced by bacteria, facilitating the release of artemisinin species. The invention realizes the effect of combining photo-thermal and chemical kinetics to resist the biological membrane, and the nanogel has good biocompatibility and high biological safety, and is very suitable for treating the biological membrane infection.
Drawings
The present invention is further described below with reference to the accompanying drawings for the convenience of understanding by those skilled in the art.
Fig. 1 is a schematic diagram of a preparation process and bacteriostasis of a nanogel for treating biofilm infection by photo-thermal synergistic chemical kinetics according to the invention.
FIG. 2 shows HA-TA-DHA-Fe prepared according to example 1 of the present invention 2+ Transmission electron microscopy of nanogels. (lack of drawings)
FIG. 3 shows HA-TA-DHA-Fe prepared according to example 1 of the present invention 2+ DLS diagram of nanogel.
FIG. 4 shows the HA-TA-DHA-Fe of different concentrations prepared in example 1 of the present invention 2+ Photo-thermal heating profile under 808nm near infrared light irradiation.
FIG. 5 shows the HA-TA-DHA-Fe of different concentrations prepared in example 1 of the present invention 2+ Fenton reaction Activity assay.
Fig. 6 is a graph and a bar graph showing biofilm effect coatings against staphylococcus aureus (s. Aureus) and escherichia coli (e. Coli) under different conditions for different nanogels (HTF, HTDF) prepared in example 1 and comparative example 1 of the present invention.
FIG. 7 is a confocal plot of live-dead staining of the biofilm effects of different nanogels (HTF, HTDF) prepared in example 1 and comparative example 1 of the invention against Staphylococcus aureus (S. Aureus) and Escherichia coli (E. Coli) under different conditions.
FIG. 8 is an in vivo image of HTF-ICG obtained by converting DHA as a drug carried in HTDF prepared in example 1 of the present invention into ICG as a fluorescent reagent.
Fig. 9 is a graph of animal wound after treatment of the different nanogels (HTF, HTDF) prepared in example 1 and comparative example 1 of the invention under different conditions. b. Day 6 animal wound-dressing pictures. c. Skin tissue HE staining on day 6. d. Animal wound bacterial survival bar graph at day 6.
Description of the embodiments
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
As shown in figures 1-5, the preparation method of the nano gel for resisting bacterial biofilm infection by photo-thermal synergistic chemical kinetics comprises the steps of loading natural polyphenol tannic acid TA on natural hyaluronic acid HA in a covalent bond manner, and simultaneously utilizing TA and Fe 2+ Self-assembling to form MPN with metal-polyphenol network structure, and packaging the artemisinin analogue Aas.
As a further scheme of the invention: the arteannuin substance comprises dihydroarteannuin DHA, arteannuin ARN, artesunate ART, artemether ARM, etc.
As a further scheme of the invention: the method specifically comprises the following steps:
s1, dissolving 20.0g,11.8mmol TA in 50mL DMF, adding 3.4g,14.2mmol 3-boc-bromopropylamine and 20.3g,147.0mmol K2CO3, heating to 60℃under nitrogen and stirring 6h with a magnetic stirrer, cooling to room temperature, adding 100mL deionized water, acidifying the solution with 50mL 6M HCl, extracting three times with 100mL ethyl acetate, washing the separated ethyl acetate layer twice with 100mL deionized water, anhydrous Na 2 SO 4 Drying for 12 hours, then removing ethyl acetate by rotary evaporation, adding 50mL absolute ethyl alcohol for re-dissolving, dialyzing the absolute ethyl alcohol for two days, removing the absolute ethyl alcohol by rotary evaporation, and pumping the solution by a suction filtration bottle to obtain white solid TA-Boc-NH 2 ；
S2, taking 10g of TA-Boc-NH 2 Dissolving in 100mL of absolute ethyl alcohol, introducing HCl vapor generated by the reaction of concentrated sulfuric acid and NaCl into the solution for 6h, then continuously stirring at room temperature for 24 hours, removing the absolute ethyl alcohol by rotary evaporation after the reaction is finished, adding 50mL of deionized water for redissolution and dialyzing for two days, and freeze-drying to obtain light yellow amino tannic acid solid ATA;
s3, 10-20 Wan, 300mg,0.74mmol HA was added to 40mL deionized water and stirred at room temperature for 30 minutes, followed by 285.19mg, 0.292 mol/L, 5mL EDC.HCl in aqueous solution and 5mL, 171.22mg, 0.292 mol/L, NHS in aqueous solution stirred at room temperature for 12 hours. Then under the protection of nitrogen, adding 10mL, 267mg,0.015mol/L and ATA dissolved in DMSO, stirring at room temperature for reaction for 24h, dialyzing with deionized water of 350 Da for 1-2 days, freeze-drying to obtain HA-TA, and preserving at-20 ℃ for later use;
s4, dissolving 30mg of HA-TA in 20mL of deionized water, regulating the solution to be slightly alkaline pH to 8 by using 5mg/mL of Tris base, then transferring the solution to a three-necked flask, introducing nitrogen, stirring at room temperature for 30min, and slowly adding 2mL of Aas solution, namely DMSO, to dissolve, wherein 12.5 mg/mL; then 20mL of 0.1% FeCl was added dropwise at a rate of 40mL/h using a syringe pump 2 •4H 2 O, continuing stirring and reacting for 2h; finally, deionized water is used for washing the supernatant liquid to be colorless, and the HA-TA-Aas-Fe is prepared 2+ The prepared HA-TA-Aas-Fe 2 + Dispersing the nano gel in an aqueous solution, and preserving at 4 ℃;
the photo-thermal synergistic chemical kinetics antibacterial biofilm infection nanogel has a synergistic killing effect on bacterial biofilms.
Example 1: nanogel HA-TA-DHA-Fe for treating biomembrane infection by photo-thermal synergistic chemical kinetics 2+
The nanogel HA-TA-DHA-Fe with photo-thermal synergistic chemical kinetics for treating biomembrane infection 2+ The preparation method specifically comprises the following steps:
s1 TA (20.0 g,11.8 mmol) was dissolved in 50mL DMF and 3-boc-bromopropylamine (3.4 g,14.2 mmol) and K2CO3 (20.3 g,147.0 mmol) were added, heated to 60℃under nitrogen and stirred with a magnetic stirrer for 6h. After cooling to room temperature, 100mL of deionized water was added, the solution was acidified with 50mL of HCl (6M) and extracted three times with 100mL of ethyl acetate, the separated ethyl acetate layer was washed twice with 100mL of deionized water, dried over anhydrous Na2SO4 for 12 hours, then the ethyl acetate was removed by rotary evaporation, re-dissolved with 50mL of absolute ethanol, dialyzed for two days and then removed by rotary evaporation, and the white solid TA-Boc-NH2 was obtained by suction filtration bottle suction.
S2, taking TA-Boc-NH 2 (10g) Dissolving in 100mL of absolute ethanol, introducing HCl vapor generated by reacting concentrated sulfuric acid with NaCl into the solution for 6h, stirring at room temperature for 24h, removing absolute ethanol by rotary evaporation after the reaction, adding 50mL of deionized water for redissolution and dialyzing for two daysFreeze-drying to obtain yellowish amino tannic acid solid ATA.
S3, adding HA (10-20 ten thousand, 300mg,0.74 mmol) into 40mL of deionized water, stirring for 30 minutes at room temperature, then adding 5mLEDC.HCl (285.19 mg, 0.292 mol/L, aqueous solution) and 5mLNHS (171.22 mg, 0.292 mol/L, aqueous solution), stirring at room temperature for reaction for 12 hours, then adding 10mLATA (267 mg,0.015mol/L, DMSO solution) under the protection of nitrogen, stirring at room temperature for reaction for 24 hours, dialyzing for 1-2 days (3500 Da) with deionized water, freeze-drying to obtain HA-TA, and storing at-20 ℃ for later use;
s4, dissolving 30mg of HA-TA in 20mL of deionized water, regulating the solution to be slightly alkaline (pH to 8) by using Trisbase (5 mg/mL), transferring the solution into a three-necked flask, introducing nitrogen, stirring at room temperature for 30min, and slowly adding 2mL of an HA solution (DMSO solution, 12.5 mg/mL); then, 20mL of the Fecl is dripped into the mixture at the speed of 40mL/h by using a syringe pump 2 •4H 2 O (0.1%), stirring and reacting for 2h, washing with deionized water to obtain colorless supernatant, and making into HA-TA-DHA-Fe 2+ The prepared HA-TA-DHA-Fe 2+ The nanogel is dispersed in an aqueous solution and stored at 4 ℃.
Example 2: nanogel HA-TA-ARN-Fe for treating biomembrane infection by photo-thermal synergistic chemical kinetics 2+
The nanogel (HA-TA-DHA-Fe) with photo-thermal synergistic chemical kinetics for treating biomembrane infection 2+ ) The preparation method specifically comprises the following steps:
s1 TA (20.0 g,11.8 mmol) was dissolved in 50mL DMF and 3-boc-bromopropylamine (3.4 g,14.2 mmol) and K were added 2 CO 3 (20.3 g,147.0 mmol) was heated to 60℃under nitrogen and stirred with a magnetic stirrer for 6h, after cooling to room temperature, 100mL of deionized water was added, the solution was acidified with 50mL of HCl (6M) and extracted three times with 100mL of ethyl acetate, the ethyl acetate layer separated was washed twice with 100mL of deionized water, anhydrous Na 2 SO 4 Drying for 12 hours. Then removing ethyl acetate by rotary evaporation, adding 50mL of absolute ethyl alcohol for redissolution, dialyzing with absolute ethyl alcohol for two days, removing the absolute ethyl alcohol by rotary evaporation, and pumping with a suction filtration bottle to obtain white solid TA-Boc-NH2;
s2, taking TA-Boc-NH 2 (10g) Dissolving solutionIntroducing HCl vapor generated by the reaction of concentrated sulfuric acid and NaCl into the solution for 6 hours in 100mL of absolute ethyl alcohol, then continuously stirring for 24 hours at room temperature, removing the absolute ethyl alcohol by rotary evaporation after the reaction is finished, adding 50mL of deionized water for redissolution and dialyzing for two days, and freeze-drying to obtain pale yellow amino tannic acid solid ATA;
s3, adding HA (10-20 ten thousand, 300mg,0.74 mmol) into 40mL of deionized water, stirring for 30 minutes at room temperature, then adding 5mLEDC.HCl (285.19 mg, 0.292 mol/L, aqueous solution) and 5mLNHS (171.22 mg, 0.292 mol/L, aqueous solution), stirring for reaction for 12 hours at room temperature, then adding 10mLATA (267 mg,0.015mol/L, DMSO solution) under the protection of nitrogen, stirring for reaction for 24 hours at room temperature, dialyzing for 1-2 days (3500 Da) by using deionized water, freeze-drying to obtain HA-TA, and storing at-20 ℃ for later use.
S4, dissolving 30mg of HA-TA in 20mL of deionized water, regulating the solution to be slightly alkaline (pH to 8) by using Trisbase (5 mg/mL), transferring the solution into a three-necked flask, introducing nitrogen, stirring at room temperature for 30min, slowly adding 2mLARN solution (DMSO solution, 12.5 mg/mL), and then dropwise adding 20mLFECl at a speed of 40mL/h by using a syringe pump 2 •4H 2 O (0.1%) is continuously stirred for 2 hours, finally deionized water is used for washing the supernatant liquid to be colorless, and the HA-TA-ARN-Fe is prepared 2+ . The prepared HA-TA-ARN-Fe 2+ The nanogel is dispersed in an aqueous solution and stored at 4 ℃.
Example 3: nanogel HA-TA-ART-Fe for treating biomembrane infection by photo-thermal synergistic chemical kinetics 2+
The nanogel HA-TA-DHA-Fe with photo-thermal synergistic chemical kinetics for treating biomembrane infection 2+ The preparation method specifically comprises the following steps:
s1 TA (20.0 g,11.8 mmol) was dissolved in 50mL DMF and 3-boc-bromopropylamine (3.4 g,14.2 mmol) and K were added 2 CO 3 (20.3 g,147.0 mmol) was heated to 60℃under nitrogen and stirred with a magnetic stirrer for 6h, after cooling to room temperature, 100mL of deionized water was added, the solution was acidified with 50mL of HCl (6M) and extracted three times with 100mL of ethyl acetate, the ethyl acetate layer separated was washed twice with 100mL of deionized water, anhydrous Na 2 SO 4 Drying for 12 hours, followed by rotary evaporationRemoving ethyl acetate, adding 50mL of absolute ethyl alcohol for redissolution, dialyzing with absolute ethyl alcohol for two days, removing absolute ethyl alcohol by rotary evaporation, and pumping with a suction filtration bottle to obtain white solid TA-Boc-NH 2 ；
S2, taking TA-Boc-NH 2 (10g) Dissolving in 100mL of absolute ethyl alcohol, introducing HCl vapor generated by the reaction of concentrated sulfuric acid and NaCl into the solution for 6h, then continuously stirring at room temperature for 24h, removing the absolute ethyl alcohol by rotary evaporation after the reaction is finished, adding 50mL of deionized water for redissolution and dialyzing for two days, and freeze-drying to obtain pale yellow amino tannic acid solid ATA;
s3, adding HA (10-20 ten thousand, 300mg,0.74 mmol) into 40mL of deionized water, stirring for 30 minutes at room temperature, then adding 5mLEDC.HCl (285.19 mg, 0.292 mol/L, aqueous solution) and 5mLNHS (171.22 mg, 0.292 mol/L, aqueous solution), stirring at room temperature for reaction for 12 hours, then adding 10mLATA (267 mg,0.015mol/L, DMSO solution) under the protection of nitrogen, stirring at room temperature for reaction for 24 hours, dialyzing for 1-2 days (3500 Da) with deionized water, freeze-drying to obtain HA-TA, and storing at-20 ℃ for later use;
s4, dissolving 30mg of HA-TA in 20mL of deionized water, adjusting the solution to be slightly alkaline (pH to 8) by using Trisbase (5 mg/mL), transferring to a three-necked flask, introducing nitrogen, and stirring at room temperature for 30min. 2mLART solution (DMSO solution, 12.5 mg/mL) was slowly added. Then, 20mL of the Fecl is dripped into the mixture at the speed of 40mL/h by using a syringe pump 2 •4H 2 O (0.1 percent), continuously stirring and reacting for 2 hours, finally washing the supernatant liquid with deionized water to be colorless, and preparing the HA-TA-ART-Fe 2+ The prepared HA-TA-ART-Fe 2+ The nanogel is dispersed in an aqueous solution and stored at 4 ℃.
Comparative example 1: nanogel HA-TA-Fe for photothermal treatment of biofilm infection 2+
The nanometer gel (HA-TA-Fe) for photothermal treatment of biomembrane infection 2+ ) The preparation method specifically comprises the following steps:
s1 TA (20.0 g,11.8 mmol) was dissolved in 50mL DMF and 3-boc-bromopropylamine (3.4 g,14.2 mmol) and K were added 2 CO 3 (20.3 g,147.0 mmol) was heated to 60℃under nitrogen and stirred with a magnetic stirrer for 6h, cooled to room temperature, 100mL of deionized water was added and the solution was acidified with 50mL of HCl (6M)Extracting with 100mL ethyl acetate three times, washing the separated ethyl acetate layer with 100mL deionized water twice, drying with anhydrous Na2SO4 for 12 hours, then spin-evaporating to remove ethyl acetate, adding 50mL absolute ethanol for redissolution, dialyzing with absolute ethanol for two days, spin-evaporating to remove absolute ethanol, and pumping with a suction filtration bottle to obtain white solid TA-Boc-NH 2 。
S2, dissolving TA-Boc-NH2 (10 g) in 100mL of absolute ethyl alcohol, introducing HCl vapor generated by the reaction of concentrated sulfuric acid and NaCl into the solution for 6 hours, then continuously stirring at room temperature for 24 hours, removing the absolute ethyl alcohol by rotary evaporation after the reaction is finished, adding 50mL of deionized water for redissolution and dialysis for two days, and freeze-drying to obtain pale yellow amino tannic acid solid ATA;
s3, adding HA (10-20 ten thousand, 300mg,0.74 mmol) into 40mL of deionized water, stirring for 30 minutes at room temperature, then adding 5mLEDC.HCl (285.19 mg, 0.292 mol/L, aqueous solution) and 5mLNHS (171.22 mg, 0.292 mol/L, aqueous solution), stirring for reaction at room temperature for 12 hours, then adding 10mLATA (267 mg,0.015mol/L, DMSO solution) under the protection of nitrogen, stirring for reaction at room temperature for 24 hours, dialyzing for 1-2 days (3500 Da) by using deionized water, and freeze-drying to obtain HA-TA. And storing at-20deg.C for use;
s4, dissolving 30mg of HA-TA in 20mL of deionized water, adjusting the solution to be slightly alkaline (pH to 8) by using Trisbase (5 mg/mL), transferring to a three-necked flask, introducing nitrogen, and stirring at room temperature for 30min. Then, 20mL of the Fecl is dripped into the mixture at the speed of 40mL/h by using a syringe pump 2 •4H 2 O (0.1%), stirring and reacting for 2 hr, washing with deionized water to obtain colorless supernatant, and making into HA-TA-Fe 2+ . The prepared HA-TA-Fe 2+ Dispersing the nano gel in an aqueous solution, and preserving at 4 ℃;
FIG. 2 shows HA-TA-DHA-Fe prepared according to example 1 of the present invention 2+ A transmission electron microscope image of the nano gel; as can be seen from the figure, the nanogel is uniformly dispersed and has a particle size of about 50-60nm.
FIG. 3 is a DLS map of HTDF nanogel prepared in example 1 of the invention. As can be seen from the graph, the hydrated particle size of the HA-TA-Fe2+ (HTF) nanogel is about 265nm, and the hydrated particle size of the HA-TA-DHA-Fe2+ (HTDF) nanogel is about 277nm.
Fig. 4 is a photo-thermal temperature rise graph of HTDF nanogels of different concentrations prepared in example 1 of the present invention under irradiation of 808 and nm near infrared light. HTDF nanogel suspensions of different concentrations (0, 100, 400, 600, 700, 800. Mu.g/mL) were irradiated with 808nm laser light at a power of 1.5W/cm2, as shown, the rising temperature gradually increased with increasing laser irradiation time for each concentration nanogel, and the greater the concentration of the nanogel, the higher the rising temperature. When the concentration of the HTDF nanogel suspension is 800 mug/mL, the temperature can be increased by 39.1 ℃ after 600 s is irradiated by near infrared laser. The HTDF nanogel has excellent light-heat conversion performance.
FIG. 5 shows the HTDF Fenton reaction activity experiments of different concentrations prepared in example 1 of the present invention. We measured the resulting change in active oxygen concentration indirectly by detecting the change in absorbance of methylene blue using an ultraviolet-visible spectrophotometer. To HTDF solutions (1 mL) of different concentrations (0, 100, 200, 400, 600 and 700. Mu.g/mL) were added PBS solution (pH 5.5, 880. Mu.L), methylene blue solution (4 mM, 20. Mu.L) and H2O2 solution (100. Mu.M, 100. Mu.L), respectively, and after incubation for 10 minutes, the mixture was centrifuged (5000 rpm,3 min), the supernatant was removed, and the spectrum was scanned with an ultraviolet-visible spectrophotometer and the absorption curve was recorded. The methylene blue solution had a distinct absorption peak at 664 nm, and as can be seen in the spectrum, the absorbance of the supernatant at 664 nm gradually decreased with increasing concentration of HTDF solution, indicating that the HTDF reacted with H2O 2. In addition, the concentration of active oxygen produced increases gradually as the concentration of HTDF increases.
Fig. 6 is a graph and a bar graph showing biofilm effect coatings against staphylococcus aureus (s. Aureus) and escherichia coli (e. Coli) under different conditions for different nanogels (HTF, HTDF) prepared in example 1 and comparative example 1 of the present invention. After we co-incubated different materials (HTF, HTDF) with bacteria, four different treatment conditions (H2O 2 (-) NIR (-), H2O2 (+) NIR (-), H2O2 (-) NIR (+) and H2O2 (+) NIR (+)) were applied, the bacteria viability was evaluated by plate coating experiments and bar graphs, and PBS was used as the material control. Where NIR (-) means dark conditions and NIR (+) means irradiation with 808nm laser (power 1.5W/cm 2) for 10 minutes. H2O2 (-) indicates that no H2O2 was added. H2O2 (+) means H2O2 (100. Mu.M) was added. From the graph, the antimicrobial effect of the HTF and HTDF nanogels is obviously different under different treatment conditions, wherein the survival rate of the S.aureus and E.colli of the H2O2 (+) NIR (+) group S.aureus and E.colli after treatment by NIR and H2O2 is the lowest. For HTF nanogels, the survival rates of the H2O2 (+) NIR (+) groups s.aureus and e.coli after NIR and H2O2 treatment were 11.6% and 16.3%, respectively. For HTDF nanogels, H2O2 (+) NIR (+) group s. Aureus and e. Coll survivors were 1.3% and 1.1%, respectively. Shows that the HTDF nano gel has better antibacterial effect under the synergistic effect of photothermal therapy and chemical kinetics therapy.
FIG. 7 is a confocal plot of live-dead staining of the biofilm effects of different nanogels (HTF, HTDF) prepared in example 1 and comparative example 1 of the invention against Staphylococcus aureus (S. Aureus) and Escherichia coli (E. Coli) under different conditions. Bacteria at a concentration of 1 mL at 2X 108 CFU/mL were added to the confocal dish and incubated 48 h on a shaker at 37℃to allow bacterial biofilm to develop at the bottom of the dish. After washing away planktonic bacteria with physiological saline, PBS, HTF (700. Mu.g/mL) and HTDF (700. Mu.g/mL) nanogel suspensions were added, respectively, with PBS group as control group. Then, treatments were given under different conditions (H2O 2 (-) NIR (-), H2O2 (+) NIR (-), H2O2 (-) NIR (+) and H2O2 (+) NIR (+) respectively) and incubated on a shaking table at 37℃for 12H. After washing the differently treated biofilms with physiological saline, live bacteria were labeled with a green fluorescent dye SYTO-9 and dead bacteria were labeled with a red Propidium Iodide (PI) dye under dark conditions. After incubation for 30 minutes in the dark at 37 ℃, the dye that did not enter the bacteria was washed out with physiological saline. And then adding 2.5% glutaraldehyde solution for fixation, and finally observing the fluorescence imaging effect of the live bacteria under a laser confocal microscope. As shown in the graph, the bacteria in the PBS group only carry green fluorescence under different treatment conditions, which indicates that the bacteria can survive well in the presence of only NIR and H2O2 treatment conditions, and the external conditions have little influence on the bacteria. However, when we were given HTF and HTDF nanogels, the different condition treatment groups all showed red fluorescence, and the red fluorescence intensity of the H2O2 (+) NIR (+) group was stronger than that of the laser irradiation treatment group alone (H2O 2 (-) NIR (+)) or the H2O2 addition treatment group alone (H2O 2 (+) NIR (-)). In addition, the red fluorescence intensity of the HTDF nanogel H2O2 (+) NIR (+) group was stronger than that of the HTF nanogel H2O2 (+) NIR (+) group. The results showed the most death of the H2O2 (+) NIR (+) group of bacteria, and also demonstrated that HTDF nanogels can synergistically kill bacteria through PTT/CDT.
FIG. 8 is an in vivo image of HTF-ICG obtained by converting DHA as a drug carried in HTDF prepared in example 1 of the present invention into ICG as a fluorescent reagent. We assessed their targeting ability by detecting the fluorescence intensity of the infected site in mice using a small animal in vivo imager. Firstly, 50 mu L staphylococcus aureus and 5 mu L staphylococcus aureus are subcutaneously injected to the position of the right leg of the mouse, and after 48 hours, the biomembrane molding is successful. Injecting 100 [ mu ] L, 50 [ mu ] g/mL of ICG and 100 [ mu ] L of HTF-ICG (ICG: 50 [ mu ] g/mL) into a mouse with a built biological membrane in a tail vein injection mode, and imaging the mouse with a living animal imager at 0, 4, 8, 12, 24, 48, 72 and 96 hours; at 96 hours, all mice were sacrificed, and their heart, liver, spleen, lung and kidney and skin tissues at the infected site were dissected and imaged again to observe the fluorescence intensity at the infected site. From the above graph, the fluorescence intensity of the infection site of the HIF-ICG group gradually increased with the lapse of time, while the fluorescence intensity of the free ICG group reached the highest intensity at 4 hours, and then decreased until 12 hours, and the fluorescence was substantially absent, indicating that HTF-ICG was able to target the infection site of the biofilm and accumulated at the site for a long period of time.
Fig. 9 is a graph of animal wound after treatment of the different nanogels (HTF, HTDF) prepared in example 1 and comparative example 1 of the invention under different conditions. b. Day 6 animal wound-dressing pictures. c. Skin tissue HE staining on day 6. d. Animal wound bacterial survival bar graph at day 6. We assessed the antibacterial activity in mice by observing their healing, plating the mice with bacterial fluid, and HE staining the mice' skin. Firstly, 50 mu L staphylococcus aureus and 5 mu L staphylococcus aureus are subcutaneously injected to the upper right leg of the mouse, and after 48 hours, the biomembrane molding is successful. Mice that were successfully modeled were randomly divided into six groups and given different conditioning treatments: control, control + NIR, HTF, HTF + NIR, HTDF, HTDF +NIR 100. Mu.L, pH5.5 PBS, HTF (3.5 mg/mL) and HTDF (3.5 mg/mL) were injected into mice by tail vein injection on day 0 and day 3, respectively, and all mice of the NIR group were irradiated with 808nm laser light of 0.75W cm-2 on days 1, 2, 4 and 5 for 10 min, during which time the temperature change at the infected site was recorded with a near infrared imager. In addition, daily recordings of all mice' weight changes and healing at the site of infection are also required. On the 6 th day, 10 mu L of bacterial liquid is taken from the infected part of the mice to be coated, and the results of the coating are counted; on day 8, all mice were sacrificed and skin tissue at the affected site was HE stained. From the results of plating and the survival rate bar graph, the htdf+nir group has almost no bacterial growth, the sterilization rate is almost hundred percent, and from the HE staining results, the number of inflammatory cells is greatly reduced, and the inflammatory cell infiltration is also greatly reduced, which indicates that the HTDF has good in vivo antibacterial effect.
The preferred embodiments of the invention disclosed above are intended only to assist in the explanation of the invention. The preferred embodiments are not exhaustive or to limit the invention to the precise form disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
Claims (4)
1. A preparation method of a nano gel for resisting bacterial biofilm infection by photo-thermal synergistic chemical kinetics is characterized in that the nano gel loads natural polyphenol tannic acid TA on natural hyaluronic acid HA in a covalent bond mode, and simultaneously utilizes TA and Fe 2+ Self-assembling to form MPN with metal-polyphenol network structure, and packaging the artemisinin analogue Aas.
2. The method for preparing the nano gel for resisting bacterial biofilm infection by photo-thermal synergistic chemical kinetics according to claim 1, wherein the artemisinin substances comprise dihydroartemisinin DHA, artemisinin ARN, artesunate ART, artemether ARM and the like.
3. The method for preparing the nanogel for resisting bacterial biofilm infection by photo-thermal synergistic chemical kinetics according to claim 1, which is characterized by comprising the following steps:
s1, dissolving 20.0g,11.8mmol TA in 50mL DMF, adding 3.4g,14.2mmol 3-boc-bromopropylamine and 20.3g,147.0mmol K2CO3, heating to 60℃under nitrogen and stirring 6h with a magnetic stirrer, cooling to room temperature, adding 100mL deionized water, acidifying the solution with 50mL 6M HCl, extracting three times with 100mL ethyl acetate, washing the separated ethyl acetate layer twice with 100mL deionized water, anhydrous Na 2 SO 4 Drying for 12 hours, then removing ethyl acetate by rotary evaporation, adding 50mL absolute ethyl alcohol for re-dissolving, dialyzing the absolute ethyl alcohol for two days, removing the absolute ethyl alcohol by rotary evaporation, and pumping the solution by a suction filtration bottle to obtain white solid TA-Boc-NH 2 ；
S2, taking 10g of TA-Boc-NH 2 Dissolving in 100mL of absolute ethyl alcohol, introducing HCl vapor generated by the reaction of concentrated sulfuric acid and NaCl into the solution for 6h, then continuously stirring at room temperature for 24 hours, removing the absolute ethyl alcohol by rotary evaporation after the reaction is finished, adding 50mL of deionized water for redissolution and dialyzing for two days, and freeze-drying to obtain light yellow amino tannic acid solid ATA;
s3, 10-20 Wan, 300mg,0.74mmol HA was added to 40mL deionized water and stirred at room temperature for 30 minutes, followed by 285.19mg, 0.292 mol/L, 5mL EDC.HCl in aqueous solution and 5mL, 171.22mg, 0.292 mol/L, NHS in aqueous solution stirred at room temperature for 12 hours. Then under the protection of nitrogen, adding 10mL, 267mg,0.015mol/L and ATA dissolved in DMSO, stirring at room temperature for reaction for 24h, dialyzing with deionized water of 350 Da for 1-2 days, freeze-drying to obtain HA-TA, and preserving at-20 ℃ for later use;
s4, dissolving 30mg of HA-TA in 20mL of deionized water, regulating the solution to be slightly alkaline pH to 8 by using 5mg/mL of Tris base, transferring to a three-necked flask, and introducing nitrogenStirring at room temperature for 30min, slowly adding 2mL of Aas solution, namely DMSO solution, and 12.5. 12.5 mg/mL; then 20mL of 0.1% FeCl was added dropwise at a rate of 40mL/h using a syringe pump 2 •4H 2 O, continuing stirring and reacting for 2h; finally, deionized water is used for washing the supernatant liquid to be colorless, and the HA-TA-Aas-Fe is prepared 2+ The prepared HA-TA-Aas-Fe 2+ The nanogel is dispersed in an aqueous solution and stored at 4 ℃.
4. The use of a photo-thermal synergistic chemical kinetics antibacterial biofilm infection nanogel according to claim 1, wherein the photo-thermal synergistic chemical kinetics antibacterial biofilm infection nanogel has a synergistic killing effect on bacterial biofilms.
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