Near-infrared luminescent cationic radical bacterial fluorescence imaging antibacterial agent and preparation and application thereof

Abstract

The application belongs to the technical field of medical materials, and discloses a near-infrared luminescent cationic radical bacterial fluorescence imaging antibacterial agent and a preparation and application thereof.The bacterial fluorescence imaging antibacterial agent is composed of a cationic radical compound of formula II and calabash [7] urea.The application further discloses a preparation method of the antibacterial agent.The cationic radical compound in the antibacterial agent is generated through in-situ reaction, does not need to be separated, has near-infrared luminescence, and has the advantages of high generation efficiency and good stability.The antibacterial agent can be directly used for bacterial fluorescence imaging, can effectively distinguish gram-positive bacteria and gram-negative bacteria, has good antibacterial effect on gram-positive bacteria, thereby realizing the integration of diagnosis and treatment of bacteria, and can realize drug screening on gram-negative bacteria.In addition, the antibacterial agent can be used as a fluorescent probe for detecting the interaction between bacteria and immune cells.

Description

Technical Field

[0001] This invention belongs to the technical field of medical materials, specifically relating to a bacterial fluorescence imaging antibacterial agent with near-infrared luminescent cationic free radicals, its preparation method, and its application. The bacterial fluorescence imaging antibacterial agent is used in bacterial fluorescence imaging and monitoring the interaction between bacteria and immune cells. Background Technology

[0002] Antibiotics play a crucial role in the treatment of modern bacterial infections. However, the overuse of antibiotics has led to the emergence of drug-resistant bacteria, causing serious diseases worldwide. Therefore, developing novel antimicrobial agents to achieve integrated diagnosis and treatment of bacteria is essential. Bacterial membranes are negatively charged; to accurately locate bacteria, antimicrobial agents must be further modified with targeting groups. Currently, a series of positively charged bacterial targeting agents have been developed, such as ammonium salts, antimicrobial peptides, and metal complexes. However, these integrated diagnostic and therapeutic agents typically involve cumbersome synthetic processes. In recent years, supramolecular host compounds have stabilized free radicals for photothermal therapy. However, no supramolecular host compound-stabilized free radicals serve as integrated diagnostic and therapeutic drugs for bacteria. This is because most cationic free radicals exhibit low fluorescence quantum yields. This is likely due to the low energy gap between the excited and ground states and the rapid non-radiative decay of the excitation energy. Summary of the Invention

[0003] To overcome the shortcomings and defects of existing technologies, the present invention aims to provide a near-infrared luminescent cationic free radical bacterial fluorescence imaging antibacterial agent and its preparation method. The reagent of the present invention is an integrated bacterial diagnostic and therapeutic agent, enabling integrated bacterial diagnosis and treatment.

[0004] Another object of the present invention is to provide applications of the aforementioned antibacterial agent. Specifically, the application of the cationic radical compound in integrated bacterial diagnostic and therapeutic reagents or drugs, wherein the integration refers to imaging and killing of Gram-positive bacteria. The antibacterial agent can also be used to monitor the process of phagocytosis of bacteria by immune cells in vivo and in vitro, and for drug screening against Gram-negative bacteria.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A near-infrared luminescent cationic free radical bacterial fluorescence imaging antibacterial reagent is composed of a formula II cationic free radical compound and cucurbita[7]urea;

[0007] Compound of Formula II:

[0008] Where R 1 It is hydrogen, substituted or unsubstituted aryl, or heteroaryl;

[0009] Where R2 R 3 R 4 R 5 Independently hydrogen, substituted or unsubstituted alkyl, halogen, alkyloxy, alkylamino, aryl, heteroaryl, aryloxy (Ar-O-), arylamino (Ar-NH-), arylthio (Ar-S-), heteroaryloxy, heteroarylamino, heteroarylthio.

[0010] R 1 In this context, the aryl group refers to a monocyclic or polycyclic aromatic group having 6-20 carbon atoms. Representative aryl groups include: phenyl, naphthyl, anthracene, and pyrene.

[0011] The substituted aryl group refers to the hydrogen on the aryl ring being replaced by one or more of alkoxy, amino, and carboxyl groups;

[0012] The heteroaryl group refers to a monocyclic or polycyclic heteroaryl group having 1-20 carbon atoms and 1-4 heteroatoms selected from N, S, and O. Representative heteroaryl groups include: pyrrole, pyridinyl, pyrimidinyl, imidazolyl, thiazolyl, indolyl, azanaphthyl, azaanthrayl, and azapyrene.

[0013] R 2 R 3 R 4 R 5 In this context, the alkyl group is C10. 1-30 Alkyl, alkyloxy group is C 1-30 Alkyloxy and alkylamino are C 1-30 Alkylamino, with an alkyl thio group of C 1-30 Alkyl thio;

[0014] The substituted alkyl group refers to the alkyl group in which hydrogen atoms are replaced by groups such as hydroxyl, methoxy, carboxyl, and halogen.

[0015] The aryl group refers to a monocyclic or polycyclic aromatic group having 6-20 carbon atoms. Representative aryl groups include phenyl, naphthyl, anthracene, and pyrene. The aryl groups in aryloxy, arylamino, and arylthio groups are each independently monocyclic or polycyclic aromatic groups having 6-20 carbon atoms. Representative aryl groups include phenyl, naphthyl, anthracene, and pyrene.

[0016] The heteroaryl group refers to a monocyclic or polycyclic heteroaryl group having 1-20 carbon atoms and 1-4 heteroatoms selected from N, S, and O. Representative heteroaryl groups include: pyrrole, pyridinyl, pyrimidinyl, imidazolyl, thiazolyl, indolyl, azanaphthyl, azaanthrayl, and azapyrene. The heteroaryl group in the heteroaryloxy, heteroarylamino, and heteroarylthio groups is independently a monocyclic or polycyclic heteroaryl group having 1-20 carbon atoms and 1-4 heteroatoms selected from N, S, and O. Representative heteroaryl groups include: pyrrole, pyridinyl, pyrimidinyl, imidazolyl, thiazolyl, indolyl, azanaphthyl, azaanthrayl, and azapyrene.

[0017] R 1 Preferably, the phenyl or alkoxy-substituted phenyl group (the alkoxy group is preferably C10). 1-5 Alkyloxy group); R 2 R 5 Alkyl groups are preferred on their own (the alkyl group is preferably C10). 1-5 Alkyl); R 3 R 4 The preferred form is hydrogen-containing or substituted alkyl group (preferably hydroxyl-substituted alkyl group, especially C-substituted alkyl group). 1-5 alkyl).

[0018] The near-infrared luminescent cationic radical bacterial fluorescence imaging antibacterial agent is specifically obtained by oxidizing the compound of formula I under aerobic conditions with cucurbit[7]urea to obtain a complex composed of the cationic radical compound of formula II and cucurbit[7]urea, namely the cationic radical bacterial fluorescence imaging antibacterial agent.

[0019] The compound of formula I is:

[0020]

[0021] R 1 R 2 R 3 R 4 R 5 As defined in Equation II above.

[0022] The molar ratio of CB[7] to compound I is 1:(2-4).

[0023] The near-infrared luminescent cationic radical compound (Formula II) is obtained by in-situ oxidation of Formula I compound with cucurbita[7]urea in oxygen. Specifically, it is obtained by oxidation in an aqueous solution.

[0024] The preparation method of the near-infrared luminescent cationic free radical bacterial fluorescence imaging antibacterial agent includes the following steps: mixing the compound of formula I and cucurbit[7]urea in water, and subjecting the compound of formula I to an oxidation reaction under aerobic conditions to obtain a complex composed of the cationic free radical compound of formula II and cucurbit[7]urea, namely the cationic free radical bacterial fluorescence imaging antibacterial agent.

[0025] Equation for oxidation reaction:

[0026]

[0027] The water is ultrapure water;

[0028] The aforementioned aerobic conditions refer to the reaction under the action of an oxidant, where the oxidant is oxygen or an atmosphere containing oxygen (such as air).

[0029] When the reaction takes place under the action of an oxidant, it can be carried out under light or without light.

[0030] When the oxidant is oxygen or air, oxidation can be carried out under light or without light.

[0031] When light shines on an object, the oxidation reaction is called photo-oxidation. Photo-oxidation refers to oxidation that occurs under conditions of oxygenation.

[0032] The illumination includes sunlight and ultraviolet light.

[0033] The concentration of the compound of formula I in water is 1 μM to 1000 mM; the molar ratio of CB[7] to the compound of formula I is 1:(2 to 4).

[0034] The reaction time is 1 to 30 minutes.

[0035] The cationic free radical bacterial fluorescence imaging antibacterial reagent of the present invention is used as a bacterial fluorescence imaging agent for bacterial staining; in particular, it is a fluorescence imaging agent for Gram-positive bacteria.

[0036] The cationic free radical bacterial fluorescence imaging antibacterial agent of the present invention is used to distinguish between Gram-positive and Gram-negative bacteria. Gram-positive bacteria are stained and emit red fluorescence, while Gram-negative bacteria are not stained. The Gram-positive bacteria are Staphylococcus aureus and methicillin-resistant Staphylococcus aureus; the Gram-negative bacteria are Escherichia coli and Pseudomonas aeruginosa.

[0037] The cationic free radical bacterial fluorescence imaging antibacterial agent of the present invention can be used as a reagent to distinguish between Gram-positive and Gram-negative bacteria.

[0038] The cationic free radical bacterial fluorescence imaging antibacterial reagent of the present invention is used for drug screening against Gram-negative bacteria; the Gram-negative bacteria are Escherichia coli and Pseudomonas aeruginosa.

[0039] The cationic free radical bacterial fluorescence imaging antibacterial agent of the present invention is used to detect the interaction between Gram-positive bacteria and immune cells.

[0040] The application of the cationic free radical bacterial fluorescence imaging antibacterial agent of the present invention in antibacterial applications is used to inhibit and kill Gram-positive bacteria; the Gram-positive bacteria are Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0041] This invention has the following excellent effects:

[0042] The cationic free radical compound of this invention is generated through an in-situ reaction without separation, and possesses advantages such as near-infrared luminescence, high generation efficiency, and good stability, making it directly applicable to bacterial fluorescence imaging. It can effectively distinguish between Gram-positive and Gram-negative bacteria, while exhibiting good antibacterial effects against Gram-positive bacteria, thus achieving integrated diagnosis and treatment of bacteria; it can also facilitate drug screening for Gram-negative bacteria. Furthermore, this cationic free radical can serve as a fluorescent probe for detecting the interaction between bacteria and immune cells. Attached Figure Description

[0043] Figure 1 I-1 is oxidized in situ to cationic free radical compound I-1 under the action of CB[7] and air. ·+ Absorption and fluorescence emission spectra: (A) Absorption spectrum; (B) Fluorescence emission spectrum, λ ex =600nm;

[0044] Figure 2 II-1 was oxidized in situ to cationic free radical compound II-1 under the action of CB[7] and air. ·+ Absorption and fluorescence emission spectra: (A) Absorption spectrum; (B) Fluorescence emission spectrum, λ ex =600nm;

[0045] Figure 3 III-1 was oxidized in situ to cationic radical compound III-1 under the action of CB[7] and air. ·+ Absorption and fluorescence emission spectra: (A) Absorption spectrum; (B) Fluorescence emission spectrum, λ ex =600nm;

[0046] Figure 4 For I-1 ·+ -CB[7] Fluorescence spectra and confocal images after incubation with Gram-negative and Gram-positive bacteria: (A) Gram-positive Staphylococcus aureus and (B) Gram-negative Escherichia coli after incubation with ethanol and without ethanol treatment. ·+Fluorescence spectrum after incubation with -CB[7](100μM), λ ex =600nm; using I-1 ·+ -CB[7] Confocal images of (C) Gram-positive Staphylococcus aureus and (D) Gram-negative Escherichia coli incubated for 2 hours;

[0047] Figure 5 I-1 at different concentrations ·+ -CB[7] Bar chart of zeta potential changes after bacterial incubation: using I-1 ·+ Zeta potentials of Staphylococcus aureus and Escherichia coli after incubation with CB[7] (0, 100, 200, 300 μM) for 2 h;

[0048] Figure 6 For different concentrations of I-1 ·+ Morphological characterization of Staphylococcus aureus after incubation with CB[7]: The culture was prepared by incubation with I-1 at concentrations of 0, 100, and 400 μM. ·+ -CB[7] SEM and TEM images of Staphylococcus aureus after treatment;

[0049] Figure 7 For I-1 ·+ -CB[7] Confocal images after incubation with Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa: (A) Gram-positive Staphylococcus aureus and (B) Gram-negative Pseudomonas aeruginosa I-1 ·+ -CB[7](100μM) confocal imaging image after 2 hours of incubation for 2 hours;

[0050] Figure 8 For I-1 ·+ -CB[7] confocal images after incubation for 2 hours with a mixed culture of Gram-negative and Gram-positive bacteria: (A) Staphylococcus aureus and Escherichia coli, (B) Staphylococcus aureus and Pseudomonas aeruginosa with I-1 ·+ -CB[7](100μM) confocal imaging image after 2 hours of incubation for 2 hours;

[0051] Figure 9 Different concentrations of I-1, CB[7] and I-1 ·+ - CB[7] bar chart and agar plate plot of antibacterial activity against Gram-positive bacteria - Staphylococcus aureus: (A) I-1, CB[7] and I-1 ·+ -CB[7] concentration-dependent antibacterial activity against Staphylococcus aureus; using different concentrations of (B)I-1, CB[7] and I-1 ·+ -CB[7] Image of Staphylococcus aureus agar plates treated with CB;

[0052] Figure 10 Different concentrations of I-1, CB[7], and I-1 ·+ - CB[7] bar chart and agar plate plot of antibacterial activity against Gram-negative bacteria - Escherichia coli: (A) I-1, CB[7] and I-1 ·+ -CB[7] concentration-dependent antibacterial activity against Escherichia coli; using different concentrations of (B)I-1, CB[7] and I-1 ·+ - CB[7] image of E. coli agar plates treated with E. coli;

[0053] Figure 11 I-1 at 100 μM or 400 μM ·+ -CB[7] after incubation with Staphylococcus aureus and subsequent treatment with SYTOXGreen or FITC-glucan: confocal images with different concentrations of I-1 ·+ -CB[7] and confocal images after (A) SYTOX Green treatment or (B) FITC-dextran treatment;

[0054] Figure 12 After treatment with different drugs, I-1 ·+ -CB[7] staining effect confocal image: treated with vancomycin and colistin for 3 hours respectively, and then in PBS with 100 μM I-1 ·+ CLSM image of E. coli stained with CB[7] for 2 hours;

[0055] Figure 13 For I-1 ·+ -CB[7] after incubation with Staphylococcus aureus, confocal images and flow cytometry were measured at different times: (A) Staphylococcus aureus after I-1 ·+ -CB[7] incubation, confocal images at different time points after washing with PBS; (B) and I-1 ·+ -CB[7] Staphylococcus aureus incubated with PBS was washed with PBS and the corresponding flow cytometry results at different time points;

[0056] Figure 14 For I-1 ·+ -CB[7] was incubated with Staphylococcus aureus and then infected with macrophages. Confocal images were measured at different time points.

[0057] Figure 15 For I-1 ·+ -CB[7] Confocal images of zebrafish infected with Staphylococcus aureus after incubation: (A) shows the process of neutrophils phagocytizing bacteria at different time points; (B) shows the process of neutrophils phagocytizing bacteria at different time points. Detailed Implementation

[0058] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0059] The compounds provided in Examples 1-3 of the present invention have good antibacterial effects against Gram-positive bacteria and selective fluorescence imaging, which can effectively distinguish between Gram-positive and Gram-negative bacteria; they can also be used for drug screening of Gram-negative bacteria.

[0060] Example 1: Compound I-1 in aqueous solution ·+ In-situ generation

[0061]

[0062] When compound I-1 (2,5-dimethyl-3-hydroxymethylene-1-phenyl-1H-pyrrole) (300 μM) was added to an aqueous solution of cucurbita[7]urea (100 μM), the pyrrole compound I-1 was rapidly converted into the corresponding cationic free radical compound I-1. ·+ It further forms a stable complex I-1 with cucurbita[7]urea. ·+ -CB[7] can be monitored in situ through ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy. The results are as follows Figure 1 As shown, (A) is the absorption spectrum; (B) is the fluorescence emission spectrum.

[0063] Example 2: Compound II-1 in aqueous solution ·+ In-situ generation

[0064]

[0065] When compound II-1 (1-(4-methoxyphenyl)-2,5-dimethyl-3-hydroxymethylene-1H-pyrrole) (300 μM) was added to an aqueous solution of cucurbita[7]urea (100 μM), the pyrrole compound II-1 was rapidly converted into the corresponding cationic free radical compound II-1. ·+ It further forms a stable complex II-1 with cucurbita[7]urea. ·+ -CB[7]. Its formation process can be monitored in situ by ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy. The results are as follows: Figure 2 As shown, (A) is the absorption spectrum; (B) is the fluorescence emission spectrum.

[0066] Example 3: Compound III-1 in aqueous solution ·+ In-situ generation

[0067]

[0068] When compound III-1 (2,5-diethyl-3-hydroxymethylene-1H-pyrrole) (300 μM) was added to an aqueous solution of cucurbita[7]urea (100 μM), the pyrrole compound III-1 was rapidly converted into the corresponding cationic free radical compound III-1. ·+ It further forms a stable complex III-1 with cucurbita[7]urea. ·+ -CB[7]. Its formation process can be monitored in situ by ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy. The results are as follows: Figure 3 As shown, (A) is the absorption spectrum; (B) is the fluorescence emission spectrum.

[0069] Example 4:

[0070] Applications of fluorescence imaging in bacteria (see) Figures 4-5 ):

[0071] (1) Bacterial culture

[0072] A colony of *Staphylococcus aureus* (Gram-positive bacterium, *S. aureus*) was transferred to NB medium and incubated for 8 hours at 37°C and 180 rpm on a shaker. The bacteria were obtained by centrifugation (8000 rpm, 3 min) and washing (PBS, 10 mM, pH 7.4) three times. Finally, the bacteria were resuspended in PBS, and the optical density at 600 nm was 1.0 (OD0.0). 600 =1.0) bacterial culture. When culturing Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli, the culture medium needs to be replaced with LB medium.

[0073] (3) Fluorescence imaging experiment of bacteria

[0074] Imaging of live bacteria: in bacterial suspensions (OD) 600 Dilute with phosphate-buffered saline (PBS, 10 mM, pH 7.4) to 1.0, then add 10 μL of 10 mM I-1. ·+ -CB[7] solution, making I-1 ·+ -CB[7] was incubated at a concentration of 100 μM. After mixing, the bacterial suspension was placed on a shaker and incubated at 37°C for 2 h. Then the bacterial suspension was centrifuged (8000 rpm, 3 min) and washed twice with PBS. Finally, 50 μL of PBS was added to resuspend the bacterial suspension. 2 μL was dropped onto a glass slide, covered with a coverslip, and finally fluorescence imaging was performed. For I-1 ·+ -CB[7], with an excitation wavelength of 633nm and an emission wavelength of 650-750nm.

[0075] Imaging of dead bacteria: First, centrifuge 200 μL of bacterial suspension (8000 rpm, 3 min), discard the supernatant, then resuspend in 1 mL of 75% EtOH and incubate at 37°C for 30 min on a shaker. Centrifuge again (8000 rpm, 3 min) and wash twice with phosphate-buffered saline (PBS, 10 mM, pH = 7.4). Finally, resuspend in PBS to obtain the bacterial suspension. Then add 10 μL of 10 mM I-1. ·+ -CB[7] solution, making I-1 ·+ The final concentration of -CB[7] was 100 μM. After mixing evenly, the bacterial suspension was placed on a shaker and incubated at 37°C for 2 h. The bacterial suspension was centrifuged (8000 rpm, 3 min) and washed twice with phosphate-buffered saline (PBS, 10 mM, pH = 7.4) to obtain 50 μL of bacterial suspension. 2 μL was dropped onto a glass slide, covered with a coverslip, and finally subjected to fluorescence imaging using a ZEISS 880. For I-1 ·+ -CB[7], with an excitation wavelength of 633nm and an emission wavelength of 650-750nm.

[0076] Figure 4 For I-1 ·+ -CB[7] Fluorescence spectra and confocal images after incubation with Gram-negative and Gram-positive bacteria: (A) Gram-positive Staphylococcus aureus and (B) Gram-negative Escherichia coli after incubation with ethanol and without ethanol treatment. ·+ Fluorescence spectrum after incubation with -CB[7](100μM), λ ex =600nm; using I-1 ·+ -CB[7] Confocal images of (C) Gram-positive Staphylococcus aureus and (D) Gram-negative Escherichia coli incubated for 2 hours.

[0077] Both fluorescence spectroscopy and confocal imaging results can illustrate I-1 ·+ -CB[7] can stain live and dead Staphylococcus aureus, but cannot stain live Gram-negative bacteria - Escherichia coli.

[0078] (4) Bacterial surface potential test:

[0079] 200 μL of OD 600 The bacterial suspension with a pH of 1.0 was diluted in 800 μL of PBS solution, and then 10 μL of 10 mM I-1 was added. ·+ -CB[7] solution, making I-1 ·+-CB[7] was incubated at a concentration of 100 μM. After mixing thoroughly, the bacterial suspension was placed on a shaker and incubated at 37°C for 2 h. Then, the bacterial suspension was centrifuged (8000 rpm, 3 min), washed 3 times with PBS, and finally resuspended in 1 mL of PBS solution. The control group did not add I-1. ·+ -CB[7] incubation, other steps are the same. Zeta potential of bacteria is measured using Zeta sizer NanoZSE. The test method for S. aureus and E. coli is the same. The results show that I-1 ·+ -CB[7] probes are positively charged and can interact electrostatically with Staphylococcus aureus bacteria that are negatively charged on their surface, resulting in a significant increase in the Zeta potential, while I-1 ·+ -CB[7] cannot stain E. coli with negative surface charge, so the Zeta potential remains basically unchanged.

[0080] Figure 5 I-1 at different concentrations ·+ -CB[7] Bar chart of zeta potential changes after bacterial incubation: using I-1 ·+ Zeta potentials of Staphylococcus aureus and Escherichia coli after incubation with CB[7](0, 100, 200, 300 μM) for 2 h.

[0081] Example 5

[0082] I-1 ·+ -CB[7] Morphological characterization of Gram-positive bacteria ( Figure 6 ):

[0083] (1) SEM characterization

[0084] Dilute the bacterial suspension with PBS, then add 10-40 μL of 10 mM I-1. ·+ -CB[7] solution, making I-1 ·+ -CB[7] was incubated at concentrations of 100 and 400 μM. After mixing evenly, the mixture was incubated on a shaker at 37°C for 2 h. The bacterial suspension was then centrifuged (8000 rpm, 3 min) and washed twice with PBS. The bacteria were then fixed with 4% paraformaldehyde and placed at 4°C. After 12 h, the suspension was centrifuged (8000 rpm, 3 min), washed three times with PBS, and dehydrated for 30 min with a gradient of different concentrations of alcohol solution. The silicon wafer was then immersed in anhydrous ethanol and sonicated for 30 min. The bacterial suspension was then dropped onto the silicon wafer and allowed to dry naturally. After sputtering the sample with gold for 90 seconds, it was photographed using a high-resolution field emission scanning electron microscope.

[0085] (2) TEM characterization

[0086] Dilute the bacterial suspension with PBS, then add 10-40 μL of 10 mM I-1. ·+ -CB[7] solution, making I-1 ·+ -CB[7] was incubated at concentrations of 100 and 400 μM. After mixing evenly, the mixture was incubated on a shaker at 37°C for 2 h. The bacterial suspension was then centrifuged (8000 rpm, 3 min) and washed twice with PBS. The bacteria were then fixed with 4% paraformaldehyde and placed at 4°C. After 12 h, the suspension was centrifuged (8000 rpm, 3 min), washed three times with PBS, and dehydrated for 30 min with a gradient of different concentrations of alcohol solution. The silicon wafer was then immersed in anhydrous ethanol and sonicated for 30 min. The bacterial suspension was then dropped onto a copper plate and photographed using a transmission electron microscope.

[0087] Electron microscopy can further confirm I-1. ·+ -CB[7] can disrupt the membrane structure of Gram-positive bacteria, Staphylococcus aureus. Without I-1 ·+ -CB[7] treatment of Staphylococcus aureus membrane surface is smooth; after 100μM treatment, the bacterial membrane begins to become uneven; after 400μM treatment, contents will flow out between the bacteria and the bacterial membrane is destroyed.

[0088] Figure 6 For different concentrations of I-1 ·+ Morphological characterization of Staphylococcus aureus after incubation with CB[7]: The culture was prepared by incubation with I-1 at concentrations of 0, 100, and 400 μM. ·+ SEM and TEM images of Staphylococcus aureus after CB[7] treatment.

[0089] Example 6

[0090] I-1 ·+ -CB[7] selective staining for Gram-positive and Gram-negative bacteria ( Figures 7-8 ):

[0091] (1) Universality verification

[0092] Study I-1 using laser confocal microscopy ·+ -CB[7] probe fluorescence imaging ability against Gram-positive bacteria - methicillin-resistant Staphylococcus aureus and Gram-negative bacteria - Pseudomonas aeruginosa. Take 2 μL and add it to a glass slide, cover with a coverslip, and finally perform fluorescence imaging. For I-1 ·+ -CB[7], with an excitation wavelength of 633nm and an emission wavelength of 650-750nm.

[0093] (2) Bacterial co-incubation

[0094] Submit I-1 ·+-CB[7] was incubated with a mixture of Gram-negative bacteria—Pseudomonas aeruginosa or Escherichia coli and Gram-positive bacteria—Staphylococcus aureus to verify whether selective imaging of Gram-positive bacteria was possible. 2 μL was added to a glass slide, covered with a coverslip, and finally fluorescence imaging was performed. For I-1 ·+ -CB[7], with an excitation wavelength of 633nm and an emission wavelength of 650-750nm.

[0095] Figure 7 For I-1 ·+ -CB[7] Confocal images after incubation with Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa: (A) Gram-positive Staphylococcus aureus and (B) Gram-negative Pseudomonas aeruginosa I-1 ·+ Confocal imaging image after 2 hours of incubation with CB[7](100μM).

[0096] Figure 8 For I-1 ·+ -CB[7] confocal images after incubation for 2 hours with a mixed culture of Gram-negative and Gram-positive bacteria: (A) Staphylococcus aureus and Escherichia coli, (B) Staphylococcus aureus and Pseudomonas aeruginosa with I-1 ·+ Confocal imaging image after 2 hours of incubation with CB[7](100μM).

[0097] In the mixed bacterial culture, I-1 ·+ -CB[7] can specifically illuminate Gram-positive bacteria, showing red fluorescence emission, while Gram-negative bacteria do not emit fluorescence. This proves that the molecule has the ability to selectively stain Gram-positive bacteria.

[0098] Example 7

[0099] Antibacterial test ( Figures 9-10 )

[0100] The antibacterial properties were characterized using the plate count method. Concentrations of ~10... 6 CFU mL -1 Bacterial suspensions were reacted with different concentrations of I-1 ·+ -CB[7], I-1, and CB[7] were mixed thoroughly and then incubated on a shaker at 37°C for 2 hours. The bacterial mixture was then diluted to 10... 4 CFU mL -1 Take 10 μL and drop it onto an agar plate, then spread it evenly using a bacterial spreader. Finally, record the bacterial colony count. The test method for *S. aureus* is the same as that for *E. coli*. I-1 ·+ -CB[7] shows stronger antibacterial properties against Staphylococcus aureus with increasing concentration. Therefore, I-1 generates cationic free radicals I-1.·+ Subsequently, its antibacterial properties against Staphylococcus aureus were enhanced. In comparison, I-1... ·+ -CB[7] showed no significant difference in antibacterial properties against Escherichia coli with increasing concentration. This indicates that I-1 ·+ -CB[7] does not have antibacterial properties against Escherichia coli.

[0101] Figure 9 Different concentrations of I-1, CB[7] and I-1 ·+ - CB[7] bar chart and agar plate plot of antibacterial activity against Gram-positive bacteria - Staphylococcus aureus: (A) I-1, CB[7] and I-1 ·+ -CB[7] concentration-dependent antibacterial activity against Staphylococcus aureus; using different concentrations of (B)I-1, CB[7] and I-1 ·+ -CB[7] Photograph of Staphylococcus aureus agar plates treated with CB[7].

[0102] Figure 10 Different concentrations of I-1, CB[7], and I-1 ·+ - CB[7] bar chart and agar plate plot of antibacterial activity against Gram-negative bacteria - Escherichia coli: (A) I-1, CB[7] and I-1 ·+ -CB[7] concentration-dependent antibacterial activity against Escherichia coli; using different concentrations of (B)I-1, CB[7] and I-1 ·+ - CB[7] Agar plate of Escherichia coli treated with CB.

[0103] Example 8

[0104] Antibacterial mechanism experiment ( Figure 11 )

[0105] In bacterial suspension (OD) 600 Dilute the solution with phosphate-buffered saline (PBS, 10 mM, pH 7.4) to 1.0, then add 10 and 40 μL of 10 mM I-1 solution respectively. ·+ -CB[7] solution, making I-1 ·+-CB[7] incubation concentration is 100, 400 μM. After mixing evenly, place the bacterial suspension on a shaker and incubate at 37℃ for 2 h. Centrifuge the bacterial suspension (8000 rpm, 3 min), wash twice with PBS, then add SYTOX Green (5 μM) and incubate for 10 min or FITC-modified dextran (FITC-dextran, 500 μg / mL) and incubate for 30 min. Centrifuge the bacterial suspension (8000 rpm, 3 min), wash twice with PBS. Finally, add 50 μL of PBS to resuspend the bacterial suspension. Take 2 μL and drop it onto a glass slide, cover with a coverslip, and finally perform fluorescence imaging. For I-1 ·+ -CB[7], excitation wavelength is 633nm, emission wavelength is 650-750nm, for SYTOX Green and FITC-dextran, excitation wavelength is 488nm, emission wavelength is 490-550nm. The nucleic acid of bacteria incubated at high concentrations was stained with a small amount of SYTOX Green and FITC-dextran, indicating that the high concentration of I-1 ·+ -CB[7] can cause damage to the Staphylococcus aureus membrane, leading to nucleic acid efflux.

[0106] Figure 11 I-1 at 100 μM or 400 μM ·+ -CB[7] after incubation with Staphylococcus aureus and subsequent treatment with SYTOXGreen or FITC-glucan: confocal images with different concentrations of I-1 ·+ -CB[7] and confocal images after (A) SYTOX Green treatment or (B) FITC-dextran treatment;

[0107] Example 9

[0108] Drug sensitivity test ( Figure 12 )

[0109] I-1 ·+ -CB[7] Antimicrobial susceptibility imaging experiment on Escherichia coli: Take 200 μL OD 600 The bacterial suspension with a concentration of 1.0 was diluted to 800 μL of PBS solution. Then, colistin sulfate and vancomycin (final concentration 5 μM) were added to the bacterial solution, and the mixture was incubated on a shaker at 37°C for 3 h. The bacterial suspension was then centrifuged (8000 rpm, 3 min) and washed three times with PBS. Next, 10 μL of 10 mM I-1 was added. ·+ -CB[7] solution, making I-1 ·+-CB[7] was incubated at a concentration of 100 μM. After mixing thoroughly, it was incubated on a shaker at 37°C for 3 h. Then the bacterial suspension was centrifuged (8000 rpm, 3 min), washed 3 times with PBS, and 50 μL of PBS solution was added to resuspend the bacteria. Finally, 2 μL of the bacterial suspension was dropped onto a glass slide, covered with a coverslip, and the bacteria were fluorescently imaged on a ZEISS 880. For I-1 ·+ -CB[7], with an excitation wavelength of 633nm and an emission wavelength of 650-750nm. Almost all E. coli treated with colistin were treated with I-1 ·+ -CB[7] lit up, and only a very small portion of the vancomycin-treated bacteria emitted red light. This indicates that I-1 ·+ -CB[7] can be used to test the effectiveness of drugs against Gram-negative bacteria.

[0110] Figure 12 After treatment with different drugs, I-1 ·+ -CB[7] staining effect confocal image: treated with vancomycin and colistin for 3 hours respectively, and then in PBS with 100 μM I-1 ·+ CLSM image of E. coli stained with CB[7] for 2 hours.

[0111] Example 10

[0112] Applications of monitoring the interaction between immune cells and bacteria Figure 13-15 )

[0113] (1)I-1 ·+ -CB[7] staining bacteria's retention capacity

[0114] Fluorescence imaging experiment: Take 200 μL of OD 600 The bacterial suspension with a concentration of 1.0 was diluted in 800 μL of PBS solution, and then 10 μL of 10 mM I-1 was added. ·+ -CB[7] solution, making I-1 ·+ -CB[7] was incubated at a concentration of 100 μM. After mixing, the mixture was incubated at 37°C for 2 h on a shaker. The bacterial suspension was centrifuged (8000 rpm, 3 min), washed twice with PBS, and then resuspended in 50 μL of PBS solution. Then, at different time points (0, 2.0, 3.0 and 5.0 h), 2 μL of bacterial suspension was dropped onto a glass slide and covered with a coverslip. Finally, fluorescence imaging was performed on a ZEISS 880. For I-1 ·+ -CB[7], with an excitation wavelength of 633nm and an emission wavelength of 650-750nm.

[0115] Flow cytometry: Take 200 μL of OD 600The bacterial suspension with a concentration of 1.0 was diluted in 800 μL of PBS solution, and then 10 μL of 10 mM I-1 was added. ·+ -CB[7] solution, making I-1 ·+ The incubation concentration of -CB[7] was 100 μM. After mixing evenly, the mixture was incubated at 37 °C for 2 h on a shaker. The bacterial suspension was then centrifuged (8000 rpm, 3 min), washed twice with PBS, and then resuspended in 1 mL of PBS solution. Finally, the fluorescence intensity of the bacteria was detected by flow cytometry at different time points (2.0, 4.0, 8.0 and 12.0 h).

[0116] I-1 ·+ -CB[7] treatment of Staphylococcus aureus did not show significant changes in fluorescence intensity within several hours. This indicates that I-1 ·+ -CB[7] did not dissociate on Staphylococcus aureus and has good chemical stability.

[0117] (2) In vitro experiments

[0118] The RAW 264.7 cells used in this experiment were purchased from the National Laboratory Cell Resource Sharing Platform. The complete culture medium for RAW 264.7 cells consisted of 10% FBS, 1% penicillin and streptomycin, and DMEM. Cells were cultured in a humidified cell culture incubator at 37°C with 5% CO2. Cells were passaged at 80% confluence and seeded at a density of 1 × 10⁶ cells / mL in confocal dishes. 5 The culture medium was incubated at 37°C and 5% CO2 for 24 hours. Then, the culture medium was changed to fresh medium. ·+ -CB[7]-treated Staphylococcus aureus was added to a confocal dish at a ratio of bacteria:cells = 8:1. One confocal dish was used directly for laser confocal microscopy to observe the interaction between macrophages and Staphylococcus aureus without any post-treatment. In the other confocal dish, after incubation for 90 min, the culture medium was removed and the sample was washed three times with PBS to remove Staphylococcus aureus outside the macrophages.

[0119] (3) In vivo experiments

[0120] Preparation of bacterial suspension: 200 μL of bacterial suspension (OD) 600 =1.0) was added to PBS (800 μL) solution for dilution, and then 10 μL of 10 mM I-1 was added. ·+ -CB[7] solution, making I-1 ·+The final concentration of -CB[7] was 100 μM. After mixing evenly, the mixture was incubated at 37 °C for 2 h. The bacterial suspension was then centrifuged (8000 rpm, 3 min), the supernatant was discarded, and the suspension was washed twice with phosphate-buffered saline (PBS, 10 mM, pH = 7.4) to obtain 200 μL of bacterial suspension.

[0121] (2) Needle preparation: Use tweezers to break off the needle tip at a point where the outer diameter of the needle is approximately 20 μm, using the scale in the eyepiece of the optical microscope. Avoid using needles with excessively large openings, as they can affect embryo survival.

[0122] (3) Bacterial injection: On the 3rd day after fertilization, the embryos were first anesthetized in a 100 mm culture dish containing 0.02% (w / v) 3-aminobenzoic acid. Embryos expressing green fluorescent protein were then selected using a stereofluorescence microscope. The embryos were transferred to agarose plates covered with E3 medium containing 0.02% (w / v) 3-aminobenzoic acid. Approximately 2 nL of Staphylococcus aureus suspension was injected subepithelially into the somatic epithelium of the embryo using a pneumatic microinjector. For zebrafish centrifuges or macrophages: excitation wavelength was 488 nm, emission wavelength was 500-550 nm; for I-1... ·+ -CB[7], with an excitation wavelength of 633nm and an emission wavelength of 650-750nm.

[0123] In vitro and in vivo experiments have demonstrated that it is possible to monitor I-1. ·+ -CB[7] treatment of Staphylococcus aureus is phagocytosed by immune cells.

[0124] Figure 13 For I-1 ·+ -CB[7] after incubation with Staphylococcus aureus, confocal images and flow cytometry were measured at different times: (A) Staphylococcus aureus after I-1 ·+ -CB[7] incubation, confocal images at different time points after washing with PBS; (B) and I-1 ·+ -CB[7] Flow cytometry analysis results of Staphylococcus aureus incubated with PBS at different time points.

[0125] Figure 14 For I-1 ·+ -CB[7] was incubated with Staphylococcus aureus and then infected with macrophages. Confocal images were obtained at different time points.

[0126] Figure 15 For I-1 ·+ -CB[7] Confocal images of zebrafish infected with Staphylococcus aureus after incubation: (A) shows the process of neutrophils phagocytizing bacteria at different time points; (B) shows the process of neutrophils phagocytizing bacteria at different time points.

Claims

1. A cationic free radical bacterial fluorescence imaging antibacterial agent with near-infrared luminescence, characterized in that: it is Composed of a cationic free radical compound of formula II and cucurbit[7]urea; Formula II: ; Where R 1 A phenyl or alkoxy-substituted phenyl group; R 2 R 5 Alkyl group alone; R 3 R 4 Alkyl groups that are hydrogen-rich or substituted; R 1 The alkoxy group mentioned is C 1-5 Alkyloxy; R 2 R 5 The alkyl group mentioned is C 1-5 Alkyl; R 3 R 4 The substituted alkyl group is a C that has been substituted with a hydroxyl group. 1-5 alkyl; The antibacterial agent refers to an antibacterial agent that inhibits and kills Gram-positive bacteria; The near-infrared luminescent cationic free radical bacterial fluorescence imaging antibacterial agent is specifically obtained by oxidizing the compound of formula I under aerobic conditions with cucurbit[7]urea to obtain a complex composed of the cationic free radical compound of formula II and cucurbit[7]urea, namely the cationic free radical bacterial fluorescence imaging antibacterial agent. The compound of formula I is: ； R 1 R 2 R 3 R 4 R 5 As defined in Equation II.

2. The preparation method of the near-infrared luminescent cationic free radical bacterial fluorescence imaging antibacterial agent according to claim 1, characterized in that: Includes the following steps: The compound of formula I and cucurbit[7]urea are mixed in water, and the compound of formula I is oxidized under aerobic conditions to obtain a complex composed of the cationic free radical compound of formula II and cucurbit[7]urea, namely the cationic free radical bacterial fluorescence imaging antibacterial agent. The molar ratio of the cucurbit[7]urea to the compound of formula I is 1:(2~4).

3. The application of the near-infrared luminescent cationic free radical bacterial fluorescence imaging antibacterial agent according to claim 1, characterized in that: The near-infrared luminescent cationic free radical bacterial fluorescence imaging antibacterial agent is used to prepare an antibacterial agent that inhibits and kills Gram-positive bacteria, wherein the Gram-positive bacteria is Staphylococcus aureus.

4. The application of the near-infrared luminescent cationic free radical bacterial fluorescence imaging antibacterial agent according to claim 1 for non-diagnostic and therapeutic purposes in bacterial fluorescence imaging, characterized in that: The near-infrared luminescent cationic free radical bacterial fluorescence imaging antibacterial agent is used to differentiate between Gram-positive and Gram-negative bacteria. Gram-positive bacteria are stained and emit red fluorescence, while Gram-negative bacteria are not stained; or it can be used for drug screening targeting Gram-negative bacteria.

5. The application according to claim 4, characterized in that: The Gram-positive bacteria are Staphylococcus aureus; the Gram-negative bacteria are Escherichia coli and Pseudomonas aeruginosa.

6. The application of the near-infrared luminescent cationic free radical bacterial fluorescence imaging antibacterial agent according to claim 1 for non-diagnostic and therapeutic purposes, characterized in that: The near-infrared luminescent cationic radical bacterial fluorescence imaging antibacterial agent is used as a fluorescent probe to detect the interaction between bacteria and immune cells.

7. The application according to claim 6, characterized in that: The bacteria in question are Gram-positive.

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