CN110997635A

CN110997635A - AIE probe for identifying and killing cancer cells and gram-positive bacteria

Info

Publication number: CN110997635A
Application number: CN201880033098.7A
Authority: CN
Inventors: 唐本忠; 康苗苗; 顾星桂
Original assignee: Hong Kong University of Science and Technology HKUST
Current assignee: Hong Kong University of Science and Technology HKUST
Priority date: 2017-05-19
Filing date: 2018-05-18
Publication date: 2020-04-10
Anticipated expiration: 2038-05-18
Also published as: CN110997635B; WO2018210334A1

Abstract

The AIE molecule used as a diagnosis and treatment integrated reagent can be used for detecting cancer cells and gram-positive bacteria and killing photodynamic. The diagnosis and treatment integrated reagent is an organic small molecular compound with aggregation-induced emission property, and can generate active oxygen under the white light irradiation condition. The integrated diagnostic and therapeutic agent can be used for in-situ targeted visualization and/or prevention or inhibition of growth of cancer cells or gram-positive bacteria during a surgical procedure.

Description

AIE probe for identifying and killing cancer cells and gram-positive bacteria

Cross application

This application claims priority from us patent application 62/603, 131 filed by the inventors temporarily on 19/5/2017 and is incorporated by reference in its entirety.

Technical Field

The present application relates generally to the synthesis of a series of compounds with aggregation-induced emission properties and their use as fluorescent probes in selective imaging and killing of cancer cells and gram-positive bacteria.

Background

In recent years, the emergence of integrated diagnostic and therapeutic reagents has opened a new door for cancer research. The diagnosis and treatment integrated reagent can realize the integration of real-time diagnosis and in-situ treatment functions. Most cancers have small early stage tumor sizes, and thus early diagnosis of cancer is difficult. Therefore, a highly selective and sensitive diagnostic method is of great importance for the early diagnosis of cancer and the study of cancer metastasis.

Bacteria are closely related to various aspects of human life. Most of the bacteria are symbiotic with human beings and beneficial to human bodies. However, a small number of bacterial species cause infectious diseases and even are life threatening. For example, Escherichia coli (a gram-negative bacterium) is capable of producing vitamin B and vitamin K in the large intestine of a human body, and thus, a proper amount of Escherichia coli in the intestinal tract has a positive effect on maintaining normal physiological functions of the human body. Staphylococcus aureus, a gram-positive bacterium, is a pathogenic bacterium that produces endotoxin, resulting in acute gastroenteritis. Therefore, in the case of bacterial-related diseases, it is important to develop a simple and rapid method for distinguishing beneficial bacteria from pathogenic bacteria and selectively killing the pathogenic bacteria.

The living body is a comprehensive system in which cells and bacteria always coexist. For example, during surgery, which is a common method of removing tumors, infection often occurs and is defined as Surgical Site Infection (SSI). SSI has attracted public and investigator interest. Current methods for cancer diagnosis include ultrasound, X-ray, photoacoustic, computed tomography, positron emission tomography, and magnetic resonance imaging. On the other hand, the gram staining technique after direct smear method or isolated culture identification has been developed and widely used for bacterial detection. Compared with the traditional imaging technology, the fluorescence imaging has the advantages of high sensitivity, good accessibility, low cost, safety, reliability and the like. In addition, there are a considerable number of fluorescent imaging agents suitable for photoluminescence applications because they can undergo photophysical and photochemical reactions under illumination to generate toxic Reactive Oxygen Species (ROS) in situ, which are widely used in photodynamic therapy (PDT) of tumors. However, the most widely used materials for PDT, such as porphyrins and phenothiazines, have low photobleaching resistance due to their aggregation quenching (ACQ) effect and limited active oxygen generation. This results in low therapeutic efficiency, which severely hampers the practical use of these materials as therapeutic agents. Recently developed aggregation-induced emission (AIE) compounds have shown a phenomenon opposite to the ACQ effect. Luminescent agents with AIE properties (AIEgens) emit little light when dissolved in solution, but exhibit fluorescence enhancement in the aggregated state. This property allows AIEgens to operate stably at high concentrations or in the aggregate state, with high fluorescence properties and high photobleaching thresholds. In addition, some probes based on AIE properties also show highly efficient ROS generation in the aggregated state.

Therefore, there is a need for an AIE diagnosis and treatment integrated probe for identifying and killing cancer cells and gram-positive bacteria.

Disclosure of Invention

The invention relates to an AIE probe for diagnosis and treatment integration. The diagnosis and treatment integrated reagent can be used for selectively imaging and killing harmful cells. Harmful cells may include cancer cells and gram positive bacteria. To selectively identify and/or kill unwanted cells, treating a patient may reduce the therapeutic agent. The diagnosis and treatment integrated reagent can be administered in situ at a surgical operation site. The integrated diagnostic and therapeutic agent can selectively image cancer cells and/or gram-positive bacteria through AIE-mediated fluorescence. The integrated diagnostic and therapeutic agent can be used in photodynamic therapy to kill or impair the growth of unwanted cells by exposing the integrated diagnostic and therapeutic agent to white light. In this regard, the integrated diagnostic and therapeutic agent may be administered to a patient using fluorescence imaging to locate the site of a harmful cell in the patient. Once the site of the unwanted cells is identified, the site may be illuminated with white light, under which the compounds of the present invention may kill or inhibit the growth of the unwanted cells by releasing ROS.

In one embodiment, the AIE probe is a small molecule organic compound having a backbone structural formula selected from the group consisting of:

wherein R ', R "and R'" are each independently selected from the group consisting of: h, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-NCS, alkyl-N₃alkyl-NH₂；

X^-Independently selected from I^-、PF₆ ^-、BF₄ ^-、SbF₆ ^-、SbF₅ ^-、CH₃COO^-、CF₃COO^-、CO₃ ^2-、SO₄ ^2-、SO₃ ^2-、CF₃SO₂ ^-、TsO^-、ClO₄ ^-、F^-、Cl^-、Br^-、(F₃CSO₂)N^-、PO₄ ^3-One kind of (1).

In a further embodiment, the compound has a backbone structure selected from the group consisting of:

X^-Independently selected from I^-、PF₆ ^-、BF₄ ^-、SbF₆ ^-One kind of (1).

In one embodiment, the compound is one of the following structural formulas:

X^-independently selected from I^-、PF₆ ^-One kind of (1).

Drawings

Various embodiments will now be described in detail with reference to the accompanying drawings.

FIG. 1 shows the UV absorption spectrum of TPPCN in DCM (dichloromethane).

FIG. 2A shows TPPCN at various n-hexane contents (f)_HexaneVol%) in a mixed solvent of dichloromethane/n-hexane (v/v).

FIG. 2B is a graph showing the relative fluorescence intensity (I/I) of TPPCN in a mixed solvent of dichloromethane/n-hexane (v/v)₀) (ii) a Insertion diagram: fluorescence photographs of TPPCN before (0% n-hexane) and after (99% n-hexane) aggregation were taken under 365 nm-excited, hand-held UV light. Concentration: 10 mu M; excitation wavelength: 440 nm.

FIG. 3 shows the cell viability of HeLa cells after incubation with different concentrations of TPPCN.

FIG. 4A is a fluorescent photograph (wavelength of excitation TPPCN 400-440nM) of HeLa cells after 10 minutes of co-staining with TPPCN (5. mu.M) and a commercial mitochondrial Red fluorescent Probe (MTR,100 nM).

FIG. 4B is a fluorescent photograph (wavelength of excitation MTR is 510-550nM) of HeLa cells co-stained with TPPCN (5. mu.M) and a commercial mitochondrial red fluorescent probe (MTR,100nM) for 10 min.

Fig. 4C is a superimposed photograph of fig. 4A and 4B, with a pearson correlation coefficient Rr of 0.983 and a scale bar of 20 μm.

FIG. 4D is a bright field photograph of HeLa cells.

FIG. 5A is a graph showing the decrease in fluorescence intensity of HeLa cells after co-staining with TPPCN and MTR after multiple laser scans.

FIG. 5B is a photograph of HeLa cells after TPPCN (upper column) and MTR (lower column) staining respectively before and after 50 scanning times (13min) under laser confocal. The staining concentrations of TPPCN and MTR were 10. mu.M and 100nM, respectively; the wavelengths of the excited TPPCN and the excited MTR are respectively 405nm and 560 nm; the laser intensity for exciting TPPCN and MTR is 6 muW and 2.16 muW respectively; the scale bar is 20 μm.

FIG. 6 ROS release monitored by H2 DCF-DA: as the white light irradiation time was prolonged, the fluorescence intensity at 525nm measured in PBS of TPPCN, H2DCF-DA and the mixture thereof was changed. TPPCN, tetracycline and H2DCF-DA were at concentrations of 10. mu.M, 10. mu.M and 5. mu.M, respectively.

FIGS. 7A and 7B are superimposed images of the bright field and ROS indicator H2DCF-DA fluorescence field before and after irradiation of 10s under 405nm laser for HeLa cells co-stained with TPPCN (10. mu.M, staining 10min) and H2DCF-DA (10. mu.M, staining 60min), respectively; the excitation wavelength was 488nm, and the scale bar was 20 μm.

FIGS. 7C and 7D are superimposed images of bright field and ROS indicator H2DCF-DA fluorescence field before and after irradiation of HeLa cells stained with H2DCF-DA (10. mu.M) for 60min with 405nm laser for 10s, respectively; the excitation wavelength was 488nm, and the scale bar was 20 μm.

FIGS. 8A and 8B are photographs of mitochondrial morphological changes before and after 10s of light exposure; the excitation wavelength was 400-440nm and the scale bar was 15 μm.

FIGS. 8C and 8D are photographs of mitochondrial morphological changes before and 10s after TPPCN (10 μ M) staining for 10 min; the excitation wavelength was 400-440nm and the scale bar was 15 μm.

FIG. 9A is a confocal picture of HeLa cells and MDCK-II cells after staining with TPPCN (10. mu.M).

FIG. 9B is a confocal picture of HeLa cells and MDCK-II cells co-stained with TPPCN (10 μ M) and H2DCF-DA (10 μ M) after 10s pre-laser irradiation (405 nm). The wavelengths of excitation TPPCN and H2DCF-DA were 405nm and 488nm, respectively, and the scale bar was 20 μm.

FIG. 9C is a confocal picture of co-cultured HeLa cells and MDCK-II cells co-stained with TPPCN (10 μ M) and H2DCF-DA (10 μ M) after laser irradiation at 405nm for 10 s. The wavelengths of excitation TPPCN and H2DCF-DA were 405nm and 488nm, respectively, and the scale bar was 20 μm.

FIG. 10A is a photograph showing the superposition of bright field and fluorescence field after H2DCF-DA (10. mu.M) staining of HeLa cells and MDCK-II cells cultured in combination for 60 min. The excitation wavelength was 488nm, and the scale bar was 20 μm.

FIG. 10B is a photograph showing the superposition of bright field and fluorescence field after 10s of excitation irradiation at 405nm after H2DCF-DA (10. mu.M) staining for 60min for HeLa cells and MDCK-II cells cultured in mixture. The excitation wavelength was 488nm, and the scale bar was 20 μm.

FIG. 11A is a graph of the cell viability of HeLa cells incubated with different concentrations of TPPCN in the presence or absence of white light (power 36 mW).

Fig. 11B is a graph of the cell viability of MDCK-II cells incubated with different concentrations of TPPCN in the presence or absence of white light (power 36 mW).

FIG. 12A is a fluorescent photograph of HeLa cells and MDCK-II cells grown in mixed culture, which were stained with TPPCN (10. mu.M) for 40min, irradiated with a 36mW white light lamp for 30min, incubated 24h in the dark, and stained with propidium iodide (1.5. mu.M) for 15 min. The wavelength of the excitation TPPCN is 400-440nm, and the scale bar is 15 μm.

FIG. 12B is a fluorescent photograph of HeLa cells and MDCK-II cells, which were polycultured, stained with TPPCN (10. mu.M) for 40min, irradiated with a 36mW white light lamp for 30min, incubated 24h in the dark, and stained with propidium iodide (1.5. mu.M) for 15 min. The wavelength of the excitation PI is 510-540nm, and the scale bar is 15 μm.

Fig. 12C is a fluorescence overlay of fig. 12A and 12B.

FIG. 12D is a photograph of the mixotrophic HeLa cells and MDCK-II cells of FIGS. 12A and 12C in the light field.

FIG. 12E is a fluorescent photograph of HeLa cells and MDCK-II cells mixed cultured under a white light lamp with a power of 36mW, after being irradiated for 30min and incubated for 24h in the absence of light, and then stained with propidium iodide (1.5. mu.M) for 15 min. The wavelength of the excitation TPPCN is 400-440nm, and the scale bar is 15 μm.

FIG. 12F is a fluorescent photograph of HeLa cells and MDCK-II cells grown in mixed culture after being irradiated under a white light lamp of 36mW power for 30min, incubated for 24h in the dark, and stained with propidium iodide (1.5. mu.M) for 15 min. The wavelength of the excitation PI is 510-540nm, and the scale bar is 15 μm.

Fig. 12G is a fluorescence overlay of fig. 12E and 12F.

FIG. 12H is a photograph of the mixotrophic HeLa cells and MDCK-II cells of FIGS. 12E and 12G in the open field.

FIG. 13A is a confocal picture of TPPCN (5. mu.M) co-stained U87 cells with MTR (100nM) for 10 min. The wavelength of the excitation TPPCN was 405 nm.

FIG. 13B is a confocal picture of TPPCN (5. mu.M) and MTR (100nM) co-stained U87 cells for 10 min. The wavelength of the excitation MTR was 561 nm.

Fig. 13C is a superimposed confocal photograph of fig. 13A and 13B. The Pearson correlation coefficient Rr was 0.773, and the scale bar was 20 μm.

Fig. 13D is a graph of cell viability of U87 cells incubated with different concentrations of TPPCN in the presence or absence of white light (power 36 mW).

FIG. 14A is a superimposed image of the bright and fluorescent fields after staining U87 cells with TPPCN (20 μ M, staining for 10min) and H2DCF-DA (10M, staining for 60 min). Excitation wavelength 488nm, scale bar 20 μm.

FIG. 14B is a superimposed picture of the bright and fluorescent fields of TPPCN (20. mu.M, staining for 10min) and H2DCF-DA (10. mu.M, staining for 60min) stained U87 cells after laser irradiation at 405nm for 10 s; excitation wavelength 488nm, scale bar 20 μm.

FIG. 14C is a superimposed image of the bright and fluorescent fields after H2DCF-DA (10. mu.M) staining of U87 cells for 60 min; excitation wavelength 488nm, scale bar 20 μm.

FIG. 14D is a superimposed image of the bright and fluorescent fields of U87 cells after H2DCF-DA (10 μ M) staining for 60min after laser irradiation at 405nm for 10 s; excitation wavelength 488nm, scale bar 20 μm.

FIG. 15A is a confocal fluorescence image of Staphylococcus epidermidis (gram-positive bacteria) after incubation for 10min with TPPCN (10. mu.M); excitation wavelength 405nm, scale bar 10 μm.

FIG. 15B is a confocal bright field image of Staphylococcus epidermidis after incubation for 10min with TPPCN (10. mu.M); excitation wavelength 405nm, scale bar 10 μm.

FIG. 15C is a confocal fluorescence image of E.coli (gram-negative bacteria) after incubation with TPPCN (10 μ M) for 10 min; excitation wavelength 405nm, scale bar 10 μm.

FIG. 15D is a confocal bright field picture of E.coli (gram-negative bacteria) after incubation with TPPCN (10. mu.M) for 10 min; excitation wavelength 405nm, scale bar 10 μm.

FIG. 15E is a confocal fluorescence image of Staphylococcus epidermidis and Escherichia coli polycultured after incubation with TPPCN (10 μ M) for 10 min; excitation wavelength 405nm, scale bar 10 μm.

FIG. 15F is a confocal brightfield image of polycultured Staphylococcus epidermidis and Escherichia coli after incubation for 10min with TPPCN (10. mu.M); excitation wavelength 405nm, scale bar 10 μm.

FIG. 16A is a photograph of Staphylococcus epidermidis in bright field after incubation for 10min without TPPCN (10. mu.M), irradiation for 20min under a white light lamp, and staining for 15min with PI (1.5. mu.M); the excitation wavelength is 510-550nm, and the scale bar is 15 μm.

FIG. 16B is a photograph of a fluorescence field of Staphylococcus epidermidis not incubated with TPPCN (10. mu.M) for 10min, irradiated under a white light lamp for 20min, and stained with PI (1.5. mu.M) for 15 min; the excitation wavelength is 510-550nm, and the scale bar is 15 μm.

FIG. 16C is an SEM picture of Staphylococcus epidermidis after 1h exposure to natural light without TPPCN (10 μ M) incubation for 10 min; the scale bar is 500 nm.

FIG. 16D is a photograph of Staphylococcus epidermidis after incubation for 10min with TPPCN (10. mu.M), exposed to white light for 20min, and stained with PI (1.5. mu.M) for 15min in bright field; the excitation wavelength is 510-550nm, and the scale bar is 15 μm.

FIG. 16E is a fluorescent photograph of Staphylococcus epidermidis after incubation for 10min with TPPCN (10 μ M), irradiated for 20min with a white light lamp, and stained with PI (1.5 μ M) for 15 min; the excitation wavelength is 510-550nm, and the scale bar is 15 μm.

FIG. 16F is an SEM picture of Staphylococcus epidermidis after being incubated for 10min with TPPCN (10 μ M) and after being exposed for 1h in natural light; the scale bar is 500 nm.

FIG. 17A is a photograph of E.coli cells after incubation for 10min without TPPCN (10. mu.M), after exposure to light under a white light lamp for 20min and staining for 15min with PI (1.5. mu.M); the excitation wavelength is 510-550nm, and the scale bar is 15 μm.

FIG. 17B is a photograph of a fluorescence field of E.coli after incubation for 10min without TPPCN (10. mu.M), irradiated under a white light lamp for 20min, and stained with PI (1.5. mu.M) for 15 min; the excitation wavelength is 510-550nm, and the scale bar is 15 μm.

FIG. 17C is a photograph of E.coli cells incubated with TPPCN (10. mu.M) for 10min, irradiated with white light for 20min, and stained with PI (1.5. mu.M) for 15min in bright field; the excitation wavelength is 510-550nm, and the scale bar is 15 μm.

FIG. 17D is a fluorescent photograph of E.coli incubated with TPPCN (10. mu.M) for 10min, irradiated with white light for 20min, and stained with PI (1.5. mu.M) for 15 min; the excitation wavelength is 510-550nm, and the scale bar is 15 μm.

FIG. 18A is a photograph of a mixed culture of Staphylococcus epidermidis and Escherichia coli after incubation for 10min without TPPCN (10. mu.M), irradiation for 20min under a white light lamp, and staining for 15min with PI (1.5. mu.M); the excitation wavelength is 510-550nm, and the scale bar is 30 μm.

FIG. 18B is a photograph of a fluorescence field of a polycultured Staphylococcus epidermidis and Escherichia coli after incubation for 10min without TPPCN (10. mu.M), irradiation for 20min under a white light lamp, and staining for 15min with PI (1.5. mu.M); the excitation wavelength is 510-550nm, and the scale bar is 30 μm.

FIG. 18C is a photograph of a mixed culture of Staphylococcus epidermidis and Escherichia coli after incubation for 10min with TPPCN (10 μ M), irradiated for 20min under a white light lamp, and stained with PI (1.5 μ M) for 15min in a bright field; the excitation wavelength is 510-550nm, and the exemplary scale is 30 μm.

FIG. 18D is a fluorescent field photograph of a polycultured Staphylococcus epidermidis and Escherichia coli after incubation for 10min with TPPCN (10 μ M), irradiated for 20min under a white light lamp, and stained with PI (1.5 μ M) for 15 min; the excitation wavelength is 510-550nm, and the scale bar is 30 μm.

FIGS. 19A and 19B are optical density change pictures of Staphylococcus epidermidis after incubation with 1 ‰ DMSO and 10 μ M TPPCN for different periods of time, respectively.

FIGS. 20A and 20B are graphs showing the change in optical density of Staphylococcus epidermidis after incubation with TPPCN for various periods of time.

Fig. 20C and 20D are pictures of the killing effect of TPPCN dissolved in 1 ‰ DMSO at different concentrations after incubation with staphylococcus epidermidis for 4h, respectively.

FIG. 21A is a photograph of a plate of Staphylococcus epidermidis not incubated with TPPCN after 0.5h without illumination with a 36mW power white light lamp.

FIG. 21B is a photograph of a plate of Staphylococcus epidermidis not incubated with TPPCN after 0.5h of irradiation with a 36mW power white light lamp.

FIG. 21C is a photograph of Staphylococcus epidermidis after incubation for 10min with TPPCN (10. mu.M), after irradiation for 0.5h under a white light lamp with a power of 36 mW.

FIG. 21D is a photograph of plates of E.coli not incubated with TPPCN after 0.5h without illumination with a 36mW power white light lamp.

FIG. 21E is a photograph of plates of E.coli not incubated with TPPCN after 0.5h irradiation with a 36mW power white light lamp.

FIG. 21F is a photograph of a plate of E.coli after incubation with TPPCN (10. mu.M) for 10min, irradiated with a 36mW white light for 0.5 h.

FIG. 22A is a photograph of a plate after 0.5h irradiation without a 36mW power white light lamp of Staphylococcus epidermidis incubated with no TPPCN.

FIG. 22B is a photograph of Staphylococcus epidermidis after incubation for 30min with TPPCN (10. mu.M), after irradiation for 0.5h without a 36mW power white light lamp.

FIG. 22C is a photograph of Staphylococcus epidermidis after incubation for 30min with TPPCN (10. mu.M), after irradiation for 0.5h under a white light lamp with a power of 36 mW.

FIG. 22D is a photograph of plates after 0.5h of irradiation without TPPCN incubated E.coli under a white light lamp of 36mW power.

FIG. 22E is a photograph of E.coli plates incubated for 30min with TPPCN (10. mu.M) after 0.5h irradiation without a 36mW power white light lamp.

FIG. 22F is a photograph of a plate of E.coli incubated for 30min with TPPCN (10. mu.M) and irradiated for 0.5h under a white light lamp of 36mW power.

FIG. 23A is a confocal fluorescence image of Staphylococcus epidermidis, Hela cells and MDCK-II cells after incubation for 10min with TPPCN (10. mu.M); the excitation wavelength was 405nm and the scale bar was 15 μm.

FIG. 23B is a confocal brightfield image of a polycultured Staphylococcus epidermidis, Hela cells and MDCK-II cells incubated with TPPCN (10. mu.M) for 10 min; the excitation wavelength was 405nm and the scale bar was 15 μm.

FIG. 23C is a confocal fluorescence field and bright field overlay image of a co-cultured Staphylococcus epidermidis, Hela cells and MDCK-II cells after incubation for 10min with TPPCN (10 μ M); the excitation wavelength was 405nm and the scale bar was 15 μm.

FIG. 24A is a confocal fluorescence image of Staphylococcus epidermidis and Hela cells after incubation for 10min with TPPCN (10 μ M), irradiation for 60min with white light, and staining for 15min with PI (1.5 μ M); the wavelength of the excited TPPCN is 400-440nm, and the scale bar is 15 μm.

FIG. 24B is a confocal fluorescence image of Staphylococcus epidermidis and Hela cells after incubation for 10min with TPPCN (10 μ M), irradiation for 60min with white light, and staining for 15min with PI (1.5 μ M); the wavelength of the excited PI was 510-540nm, and the scale bar was 15 μm.

FIG. 24C is a confocal brightfield image of a mixture of Staphylococcus epidermidis and Hela cells incubated with TPPCN (10 μ M) for 10min, irradiated with white light for 60min, and stained with PI (1.5 μ M) for 15 min; the wavelength of the excited PI was 510-540nm, and the scale bar was 15 μm.

FIG. 25 shows the UV absorption spectrum of TPE-CP in methylene chloride.

FIG. 26A TPE-CP with different n-hexane content (f)_HexaneVol%) of a dichloromethane/n-hexane (v/v) mixed solvent; TPE-CP concentration is 10 μ M, excitation wavelength is 475 nm.

FIG. 26B relative fluorescence intensity (I/I) of TPE-CP in mixed solvent of dichloromethane/n-hexane (v/v)₀) (ii) a TPE-CP concentration is 10 μ M, excitation wavelength is 475 nm.

FIG. 27 shows the cell viability of HeLa cells after incubation with different concentrations of TPE-CP.

FIG. 28A is a confocal picture of HeLa cells co-stained with TPE-CP (10. mu.M, 30min stain) and BODIPY 493/503 (1. mu.g/ml, 15min stain) (488 nm wavelength for excitation of TPE-CP).

FIG. 28B is a confocal picture of TPE-CP (10. mu.M, stained for 30min) and BODIPY 493/503 (1. mu.g/ml, stained for 15min) co-stained HeLa cells (wavelength of excited BODIPY 493/503 at 488 nm).

Fig. 28C is a superimposed photograph of fig. 28A and 28B, with a pearson correlation coefficient Rr of 0.94 and a scale bar of 20 μm.

FIG. 29A is a graph showing the decrease in fluorescence intensity of HeLa cells after co-staining with TPE-CP and BODIPY 493/503 after multiple laser scans.

FIG. 29B is a photograph of HeLa cells stained with TPE-CP (top) and BODIPY 493/503 (bottom) respectively, scanned 50 times (about 13min) in confocal laser light before (left) and after (right) scans; the staining concentrations of TPE-CP and BODIPY 493/503 were 20. mu.M and 1. mu.g/mL, respectively; the excitation wavelength is 488 nm; the laser intensity is 0.55 muW respectively; the scale bar is 20 μm.

FIG. 30A is a graph showing the change in fluorescence intensity at 525nm measured in PBS for TPE-CP, tetracycline, ROS indicator H2DCF-DA, a mixture of TPE-CP and H2DCF-DA, and a mixture of tetracycline and H2DCF-DA as white light exposure time is extended; the concentrations of TPE-CP, tetracycline and H2DCF-DA were 10. mu.M, 10. mu.M and 5. mu.M, respectively.

FIG. 30B is a superimposed image of the bright and fluorescent fields of HeLa cells stained with TPE-CP (10. mu.M, 30min stain) and H2DCF-DA (10. mu.M, 60min stain); the excitation wavelength was 488nm, and the scale bar was 20 μm.

FIG. 30C is a superimposed picture of the bright field and ROS indicator H2DCF-DA fluorescence field after irradiation of 30s under 405nm laser for HeLa cells stained with TPE-CP (10 μ M, 30min stain) and H2DCF-DA (10 μ M, 60min stain); the excitation wavelength was 488nm, and the scale bar was 20 μm.

FIG. 30D is a photograph of a superposition of the bright and fluorescent fields of HeLa cells stained with H2DCF-DA (10. mu.M) for 60 min; the excitation wavelength was 488nm, and the scale bar was 20 μm.

FIG. 30E is a photograph showing the superposition of the bright field and the fluorescence field of HeLa cells stained with H2DCF-DA (10. mu.M) for 60min after laser irradiation at 405nm for 30 s; the excitation wavelength was 488nm, and the scale bar was 20 μm.

FIG. 31A is a confocal bright field image of E.coli incubated with TPE-CP (10. mu.M) for 30 min; the excitation wavelength is 400-440nm, and the scale bar is 15 μm.

FIG. 31B is a confocal fluorescence image of E.coli incubated with TPE-CP (10. mu.M) for 30 min; the excitation wavelength is 400-440nm, and the scale bar is 15 μm.

FIG. 31C is a confocal brightfield image of Staphylococcus epidermidis incubated with TPE-CP (10 μ M) for 30 min; the excitation wavelength is 400-440nm, and the scale bar is 15 μm.

FIG. 31D is a confocal fluorescence image of Staphylococcus epidermidis after incubation for 30min with TPE-CP (10 μ M); the excitation wavelength is 400-440nm, and the scale bar is 15 μm.

FIGS. 32A and 32B are optical density change pictures of Staphylococcus epidermidis after incubation with 1 ‰ DMSO and 10 μ M TPE-CP for different periods of time, respectively.

FIG. 33A is a graph of the change in optical density of TPE-CP at various concentrations and after incubation with Staphylococcus epidermidis for various periods of time.

FIG. 33B is a graph of the change in optical density of TPE-CP at different concentrations and after incubation with Staphylococcus epidermidis for different periods of time.

Fig. 33C is a picture of the killing effect of TPE-CP dissolved in 1 ‰ DMSO at different concentrations after incubation with staphylococcus epidermidis for 4 h.

Fig. 33D is a picture of the killing effect of TPE-CP dissolved in 1 ‰ DMSO at different concentrations after incubation with staphylococcus epidermidis for 4 h.

FIG. 34A is a photograph of Staphylococcus epidermidis not incubated with TPE-CP, after plates were not irradiated for 0.5h with a 36mW power white light lamp.

FIG. 34B is a photograph of a plate of Staphylococcus epidermidis not incubated with TPE-CP after 0.5h of irradiation with a 36mW white light lamp.

FIG. 34C is a photograph of a plate of Staphylococcus epidermidis after incubation with TPE-CP (10. mu.M) for 30min without white light illumination.

FIG. 34D is a photograph of Staphylococcus epidermidis after incubation for 30min with TPE-CP (10. mu.M), after 0.5h irradiation under a 36mW white light lamp.

FIG. 34E is a photograph of E.coli not incubated with TPE-CP, plates not irradiated for 0.5h with a 36mW power white light lamp.

FIG. 34F is a photograph of a plate after 0.5h of E.coli without TPE-CP incubation under a 36mW power white light lamp.

FIG. 34G is a photograph of a plate without white light irradiation after incubation of E.coli with TPE-CP (10. mu.M) for 30 min.

FIG. 34H is a photograph of E.coli incubated with TPE-CP (10. mu.M) for 30min, after 0.5H irradiation with a 36mW white light lamp.

FIG. 35A is an SEM picture of Staphylococcus epidermidis after exposure to natural light for 1h without incubation for 10min with TPE-CP (10. mu.M).

FIG. 35B is an SEM image of Staphylococcus epidermidis after incubation with TPE-CP (10 μ M) for 10min after exposure to natural light for 1 h.

Detailed Description

The following definitions are provided to understand the present application and to construct the appended patent claims.

Definition of

It should be understood that the drawings described above or below are for illustration purposes only. The drawings are not necessarily to scale, emphasis generally being placed upon illustrating the principles of the technology contemplated in this disclosure. The drawings are not intended to limit the scope of the present invention in any way.

Throughout this application, where a composition is described as "having," "including," or "containing" a particular component, or where the process is described as "having," "including," or "containing" a particular process step. It is contemplated that a composition of the present teachings can also consist essentially of, or consist of, the recited components, and that a process of the present teachings can also consist essentially of, or consist of, the recited process steps.

In the present application, where an element or component is referred to as being included in and/or selected from a list of recited elements or components, it is to be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components. Moreover, it should be understood that elements and/or features of the compositions, devices, or methods described herein may be combined in various ways, whether explicit or implicit, without departing from the spirit and scope of the application.

The use of the terms "comprising," "including," "having," or "with" is generally to be construed as open-ended and non-limiting unless otherwise specifically stated.

The use of the singular herein includes the plural (and vice versa) unless specifically stated otherwise. In addition, where the term "about" is used before a quantitative value, the application also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ± 10% variation from the nominal value, unless otherwise specified or inferred.

It should be understood that the order of steps or order of performing certain actions is immaterial so long as the application remains operable. Further, two or more steps or actions may be performed simultaneously.

As used herein, "aromatic heterocyclic ring" refers to an aromatic monocyclic or fused ring system containing at least one hetero atom such as oxygen (O), nitrogen (N), sulfur (S), silicon (Si), selenium (Se) in addition to carbon atoms. Polyheterocyclic groups include groups in which two or more heterocyclic rings are fused together, groups in which a single heterocyclic ring is fused to one or more carbocyclic rings, groups in which a single heterocyclic ring is fused to a non-aromatic carbocyclic ring or groups in which a single heterocyclic ring is fused to a non-aromatic hybrid alkyl ring. Typically, an aromatic heterocyclic group will contain from 5 to 22 ring atoms, including from 1 to 5 heteroatoms (e.g., a 5-20 membered heterocyclic group). The heteroaromatic group is attached to a heteroatom or carbon atom of a particular chemical structure to form a stable chemical structure, and typically the heteroaromatic group does not contain an O-O bond, an S-S bond, or an S-O bond, however, one or more of the N, S atoms in the heteroaromatic group can be oxidized, such as pyridine nitroxide, thiophene sulfoxide, thiophene sulfide, sulfur dioxide. The aromatic heterocyclic system of the five-membered or six-membered monocyclic ring and the five-membered and six-membered bicyclic ring is shown as the following figure:

wherein T represents O, S, NH, N-alkyl, N-aryl, N- (alkylaryl) (e.g., N-phenyl), bishydrosilyl, alkylhydrosilyl, bialkylsilyl, aralkylhydrosilyl, diaralkyl silyl, or alkyl aralkylsilyl. Heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, isothiazolyl, thiazolyl, thiadiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, indolyl, isoindolyl, benzofuranyl, benzimidazolyl, benzothiazolyl, benzisothiazolyl, benzisoxazolyl, benzoxazolyl, cinnolinyl, 1 hydro-indolyl, 2 hydro-indolyl, indolizinyl, isobenzofuranyl, naphthyridinyl, phthalazinyl, pteridinyl, purinyl, oxazolopyridyl, thiazolopyridyl, imidazopyridinyl, furopyridinyl, thienopyrimidinyl, pyridoazinyl, pyridopyridazinyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl. Heteroaromatic groups also include, but are not limited to, 4,5,6, 7-tetrahydroindolyl, tetrahydroquinolinyl, benzothiophenylpyridinyl, and benzofuranpyridinyl.

Here, "halogen" means F, Cl, Br, I.

As used herein, "alkyl" refers to a saturated hydrocarbon straight or branched substituent containing from 1 to 40 carbon atoms, including but not limited to methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (n-butyl, z '-butyl, sec-butyl, tert-butyl), pentyl (n-pentyl, z' -pentyl, -pentyl), hexyl, wherein the alkyl chain containing from 1 to 6 carbon atoms is referred to as a short chain alkyl, such as methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, sec-butyl, tert-butyl). In the examples, the alkyl chain may be substituted as described herein. One alkyl chain may not be substituted by another alkyl, alkenyl or alkynyl group.

As used herein, "alkenyl" refers to straight or branched chain substituents containing from 2 to 40(C2-40) or 2 to 20(C2-20) carbons, wherein one or more C ═ C bonds, including but not limited to ethenyl, propenyl, butenyl, pentadienyl, hexenyl, butadienyl, pentadienyl, hexadiene. One or more of the C ═ C double bonds present therein may be in an intermediate position (e.g., 2-butene) or in a terminal position (e.g., 1-butene). In embodiments, the alkenyl groups referred to may be replaced by alkenyl chains as described above.

As used herein, "fused ring" or "fused ring moiety" refers to a polycyclic ring formed by two or more carbocyclic or heterocyclic rings sharing a ring edge, wherein at least one of the rings is aromatic (either carbocyclic or heterocyclic). These polycyclic systems are highly conjugated and in embodiments, fused ring groups may be replaced as described herein.

Here, "heteroatom" means any element other than C, H, such as N, O, Si, S, P, Se.

As used herein, "aryl" refers to a hydrocarbon aromatic ring containing 6 to 24 carbon atoms (e.g., C6-24 aryl), either a single aromatic ring or a polycyclic ring formed by two or more carbocyclic rings sharing a common ring edge, wherein at least one of the carbocyclic rings is aromatic. In some embodiments, a polycyclic aromatic group can contain 8 to 24 carbon atoms, and any aromatic group can be covalently bonded to the chemical structure at a suitable site. Aromatic groups include, but are not limited to, carbocyclic aromatic rings such as phenyl, 1-naphthyl (bicyclic), 2-naphthyl (bicyclic), anthracenyl (tricyclic), phenanthrenyl (tricyclic), fused pentacenyl (pentacyclic). Aromatic groups also include carbocyclic aromatic rings attached to one or more cyclic alkyl or cyclic heteroalkyl groups, such as phenyl derivatives of cyclopentane (e.g., indanyl of a 5, 6-bicyclic cycloalkyl/aryl system), phenyl derivatives of cyclohexane (e.g., tetrahydronaphthyl of a 6, 6-bicyclic cycloalkyl/aryl system), phenyl derivatives of imidazoline (e.g., benzimidazolinyl of a 5, 6-bicyclic cycloheteroalkyl/aryl system), and phenyl derivatives of pyran (e.g., benzopyranyl of a 6, 6-bicyclic cycloheteroalkyl/aryl system). Aromatic groups also include benzodioxazolyl, benzodifuran, chroman, indoline and similar structures. In some embodiments, aryl groups may be replaced with aryl groups as mentioned herein. In the examples, "haloalkyl" means an aryl group containing one or more halogen substituents including perhalogenated aryl (e.g., -C6F 5). In the examples, a bisaryl group refers to an aryl group containing an aromatic ring substituent, and any aromatic group in the bisaromatic ring may be replaced with an aryl group as described herein.

As used herein, "integrated diagnostic and therapeutic agent" refers to an organic material having both diagnostic and therapeutic functions.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently described subject matter belongs.

Where a range of values is provided (e.g., a concentration range, a percentage range, or a ratio range), it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit and any other stated or intervening value in that stated range is encompassed within the subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and these embodiments are also encompassed within the described subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges that do not include either or both of those included limits are also included in the subject matter described.

Throughout this application, various embodiments are described using the language "comprising". However, those skilled in the art will understand that in some particular instances, embodiments may alternatively be described using language "consisting essentially of.

For a better understanding of the present teachings and in no way limiting the scope of the present application, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Diagnosis and treatment integrated reagent

The present subject matter relates to Aggregation Induced Emission (AIE) fluorescent probes useful as integrated diagnostic and therapeutic agents. The diagnosis and treatment integrated reagent can be used for selectively imaging and killing harmful cells. Harmful cells may include cancer cells and gram-positive pathogenic bacteria. The integrated diagnostic and therapeutic agent can be administered to a patient undergoing a surgical procedure to selectively identify and/or kill unwanted cells. The diagnosis and treatment integrated reagent can be administered in situ at a surgical operation site. The integrated diagnostic and therapeutic agent can selectively image cancer cells and/or gram positive bacteria through AIE mediated fluorescence. The diagnosis and treatment integrated reagent is used for photodynamic therapy to kill harmful cells or inhibit the growth of the harmful cells.

In one embodiment, the aggregation-inducing luminescent probe of the present invention is a small molecule organic compound having a backbone structure selected from the group consisting of:

In a further embodiment, the compound has a backbone structural formula selected from the group consisting of:

In one embodiment, the compound is one of the following structural formulas:

X^-independently selected from I^-、PF₆ ^-One kind of (1).

An exemplary reaction scheme for preparing TPPCN compounds is as follows:

an exemplary reaction scheme for preparing TPE-CP compounds is as follows:

selectively recognizing harmful cells and inhibiting cell growth

The compounds of the invention may be administered to a patient as a contrast agent for use in locating unwanted cells in the patient's body by fluorescence imaging techniques. For example, the compounds may be used in situ in a patient undergoing surgery. This in situ administration can be used to detect residual cancer cells during tumor ablation surgery, or to detect the presence of potentially harmful (gram positive) bacteria in or near the surgical site. In another embodiment, the compound may be administered by intravenous injection.

As detailed herein, imaging studies demonstrate that this compound can be used as an effective probe for the selective identification of cancer cells and gram-positive bacteria. Once the harmful cells are identified, the compound can generate active oxygen under the white light irradiation condition, and the harmful cells are selectively killed. In one embodiment, the unwanted cells may be cancer cells and/or gram positive bacteria. Photodynamic therapy of unwanted cells with these compounds can induce apoptosis or growth inhibition.

Since the compounds of the present invention are completely organic, these compounds exhibit good biocompatibility and no detectable toxicity. The compounds have ultra-high light stability and good photodynamic performance, and are expected to be used for in-situ diagnosis and photodynamic therapy of diseases.

The invention is illustrated by the following examples.

Examples

Materials and instruments

Minimal Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Fetal Bovine Serum (FBS), penicillin and streptomycin, Phosphate Buffered Saline (PBS), mitochondrial Red fluorescent Probe: (

Red FM) and BODIPY 493/503 were purchased from Invitrogen. LB agar and LB broth were purchased from USB co, Propidium Iodide (PI) and H2DCF-DA from Sigma-Aldrich and used without further purification. Tetrahydrofuran (THF) was purified by distillation in benzophenone benzyl ester before use. Other reagents used in this work, such as dimethyl sulfoxide, potassium chloride and sodium chloride were purchased from Sigma-Aldrich and used without further purification. Milli-Q water is prepared fromMilli-Q Plus System (Millipore corporation, USA). All chemicals used to synthesize TPPCN and TPE-CP were purchased from Sigma-Aldrich.

Characterization of

Using CD₂Cl₂As deuterated solvent, determination was made on a Bruker ARX 400 nuclear magnetic spectrometer¹H and¹³c nuclear magnetic spectrum. High resolution mass spectra were recorded on a Finnigan MAT TSQ 7000 mass spectrometer system operating in time-of-flight mass spectrometry mode. Ultraviolet absorption spectra were taken on a Milton Ray spectral 3000 array spectrophotometer. The steady state fluorescence spectra were recorded on a Perkin elmer ls 55 spectrometer. Fluorescence images were collected on an Olympus BX41 fluorescence microscope. Confocal laser scanning microscope images were collected on a Zeiss laser scanning confocal microscope (LSM7 DUO) and analyzed using ZEN 2009 software (Carl Zeiss). Particle size was measured on a Zeta potential analyzer (Brookhaven, ZETAPLUS). Observations of bacterial morphology were studied using a scanning electron microscope (JSM-6390 (JEOL)).

Cell culture

For cell culture, HeLa cells were placed in a humidity incubator (37 ℃, 5% CO)₂Atmosphere) containing 10% FBS and antibiotics (100 units/mL penicillin and 100. mu.g/mL streptomycin). MDCK-II and U87 cells were placed in a humidified incubator (37 ℃, 5% CO)₂Atmosphere) with 10% FBS and antibiotics (100 units/mL penicillin and 100. mu.g/mL streptomycin).

Biocompatibility

For cellular biocompatibility assessed by MTT, cells were seeded in 96-well plates at a density of 5000 cells per well. After overnight incubation, the medium in each well was replaced with 100 μ L of fresh medium containing different concentrations of TPPCN and TPE-CP. Volume of DMSO contained in the solution is less than 0.2%. After 24 hours of treatment, 10. mu.L of MTT solution (5 mg/mL in PBS) was added to each well. After 4 hours of incubation, the MTT-containing solution was gently removed and replaced with 100 μ L DMSO. After shaking, the cells were passed through a microplate reader (Perkin-Elmer Victor 3)^TM) The absorbance at 490nm was recorded for each well. Each experiment was performed in parallel in 6 wells. Background of blank was subtracted and control group was thinnedThe cell activity was set to 1.

Cytotoxicity

For cytotoxicity assessed by MTT, HeLa, MDCK-II or U87 cells were seeded in 96-well plates at a density of 5000 cells per well. After overnight incubation, the medium in each well was replaced with 100 μ L of fresh medium containing different TPPCN concentrations. Volume of DMSO contained in the solution is less than 0.2%. After 8 hours of incubation, three plates containing HeLa, MDCK-II and U87 cells were exposed to white light (36mW) for 30min, and three other plates with cells were kept in the dark as controls.

Cell culture

For cellular imaging, a coverslip or plasma treated 25mm round coverslip was mounted on the bottom of a 35mm dish with a viewing window and cells were cultured overnight in the 35mm dish. Viable cells were incubated with a specific concentration of a certain dye for a certain time (2. mu.L of stock solution in DMSO solution was added to 2mL of cell culture medium, DMSO volume was less than 0.1%). Dye-labeled cells were fixed and imaged under a fluorescent microscope (BX41 microscope). Conditions are as follows: for TPPCN and TPE-CP, the excitation filter is 400-440nm, the dichroic mirror is 455nm, and the emission filter is 465nm long pass; for the mitochondrion red fluorescent probe MTR, the excitation filter is 510-550nm, the dichroic mirror is 570nm, and the emission filter is 590nm long pass; for BODIPY, the excitation filter is 460-490nm, the dichroic mirror is 505nm, and the emission filter is 515nm long pass.

Light stability

Dye-labeled HeLa cells were imaged by confocal microscopy (Zeiss LSM7 DUO) using ZEN 2009 software (Carl Zeiss). Conditions are as follows: for TPPCN, the excitation wavelength was 405 nm; for a mitochondrial red fluorescent probe MTR, the excitation wavelength is 560 nm; the laser power was unified to 6 μ W (TPPCN) and 2.16 μ W (MTR). For TPE-CP and BODIPY 493/503, the excitation wavelength is 489 nm; the laser power was set to 0.55. mu.W.

Bacterial culture, imaging and killing

Single bacterial colonies on solid agar plates were transferred to 5mL of the corresponding liquid medium and cultured at 37 ℃ for 10 hours (culture)Base: LB for e.coli and staphylococcus epidermidis). By measuring the Optical Density (OD) at 600nm₆₀₀) Determining the bacterial concentration, then 1X 10⁹Bacteria of Colony Forming Units (CFU) were transferred to 1.5mL EP tubes. After centrifugation (13000rpm, 3 minutes), the upper layer of the culture was discarded, and the lower layer was washed twice with PBS. After removal of the supernatant, the remaining bacteria were resuspended in PBS to obtain 1mL of OD₆₀₀1.0 (about 10)⁸CFU mL^-1) The bacterial suspension of (1). To this bacterial suspension was added the compound and incubated for a certain period of time.

About 2. mu.L of the stained bacterial solution was transferred onto a glass slide, and then covered with a cover slip for imaging. Coli and staphylococcus epidermidis were imaged under a fluorescence Microscope (BX41 Microscope) with the following settings: for TPPCN and TPE-CP, excitation filter 400-; for PI, excitation filter 510-.

For PI staining experiments, after incubation with TPPCN (10 μ M) for 10 minutes, the bacteria were exposed to white light illumination for 20 minutes while the control group was placed in the dark. Then, PI was added to the experimental group and the control group at a final concentration of 1.5 μ M, followed by incubation in the dark for another 15 minutes. Then, the bacteria were imaged under a fluorescence microscope, specifically set as follows: the excitation filter is 510-550nm, the dichroic mirror is 570nm, and the emission filter is 590nm long pass.

For the light induced toxicity experiments, 1 × 10⁸CFU bacteria were dispersed in 1mL PBS. After incubation with TPPCN (10 μ M) for 10 minutes or TPE-CP (10 μ M) for 30 minutes, the solution was centrifuged (13000rpm, 3 minutes), then the supernatant was removed and washed 2 times with PBS before adding PBS for resuspension, exposing the experimental groups to white light for the designed time while the control group was left in the dark. The viability of the bacteria was then assessed by plate counting.

For antimicrobial performance studies, the microorganism concentration was diluted to OD₆₀₀Close to 0.2. TPPCN with different predetermined concentrations was added to each experimental group. And adding the same amount of the same to the control groupPure DMSO. At designed time intervals, by measuring OD₆₀₀The concentration of the microorganism is determined. The test was repeated three times to ensure reliability.

For SEM studies, the concentration of Staphylococcus epidermidis was diluted to OD₆₀₀Close to 0.2. Bacteria were incubated with TPPCN (10 μ M) for 4 hours and irradiated with white light for 1 hour, then dried, and SEM images were collected. Untreated bacteria were also imaged under SEM for comparison.

Example 1

Synthesis of TPPCN and TPE-CP

4- (1- (4- (2, 2-dicyano-1-styryl) phenyl-2, 2-bis (4-methoxystyryl) -1-methylpyridine hexafluorophosphate (TPPCN,6)

Mixing TiCl₄(1mL,9.0mmol) was added gradually to dry THF (50mL) containing zinc powder (1.17g,18.0mmol) at-78 deg.C to form a reaction mixture. The reaction was refluxed for 2 hours and 4,4' -dimethoxybenzophenone (1.090g,4.5mmol) and compound 2(0.786g,3mmol) were added to the reaction mixture mixed in dry THF (20 ml). The reaction mixture was refluxed for an additional 5 hours. After the reaction was completed, the solvent was removed with compressed air, and the residue was extracted with DCM, and anhydrous Na was used₂SO₄And (5) drying. The crude product was purified on silica gel column using DCM as eluent. Compound 3 was obtained as a yellow solid in 50% yield.

n-BuLi (0.6mL,1.2mmol,2.0M n-hexane) was added slowly dropwise to a solution of compound 3(0.471g,1.0mmol) in dry THF (20mL) at-78 deg.C under nitrogen. After stirring at this temperature for 2 hours, N-dimethylbenzamide (0.179g,1.2mmol) was added slowly and stirring was continued at-78 ℃ for 1 hour. The mixture was then warmed to room temperature (22 ℃) and quenched with 10% aqueous HCl (10mL) and stirred for 30 minutes. The aqueous phase was separated, washed with DCM (30ml) and anhydrous MgSO₄Drying and vacuum evaporating. The crude product is reacted with TiCl₄And pyridine under reflux to give a mixture, which was purified on a silica gel column using DCM eluent to give compound 5 as a pale yellow solid (0.382g, 70%).

To a solution of compound 5(0.50mmol) in toluene was added methyl iodide (5mmol CH)₃I) Forming a reaction mixture. The reaction mixture was heated and stirred at 110 ℃ under nitrogen for 4 hours. The precipitate was collected and washed with toluene. And (3) recrystallizing the crude product by adopting a mixed solvent of n-hexane and dichloromethane. The pure product was dissolved in acetone and subjected to counter ion exchange with potassium hexafluorophosphate to give orange-yellow TPPCN (yield 90%).¹H-NMR(400MHz；CD₂Cl₂)δH 8.15(d,2H),7.61(t,1H),7.52(t,2H),7.45-7.42(m,4H),7.31(d,2H),7.16(d,2H),7.03(d,2H),6.94(d,2H),6.83(d,2H),6.70(d,2H),4.22(s,3H),3.81(s,3H),3.76(s,3H)ppm；¹³C-NMR(400MHz；CD₂Cl₂) δ C174.0,161.4,161.0,153.1,146.0,143.5,135.8,135.4,133.3,133.2,132.7,132.6,132.0,131.5,130.9,130.4,129.7,128.9,114.4,113.9,113.8,113.5,81.7,55.4,55.3,47.7 ppm; MALDI-MS (C) to calculate TPPCN cation₃₈H₃₀N₃O₂ ⁺) 560.2333, found 560.2355.

4- (1- (4- (2, 2-bis (4-methoxyphenyl) -1-phenyl) -2, 2-bisiminofuranyl) -1-methylhexafluorophosphorus Pyridine acid (TPE-CP,12)

The overall synthesis of TPE-CP was similar to that of TPPCN described above, and then a solution of compound 11(0.50mmol) in toluene was added to methyl iodide (5mmol CH)₃I) In (b), a reaction mixture is formed. The reaction mixture was heated and stirred at 110 ℃ under nitrogen for 4 hours. The precipitate was collected and washed with toluene, and the crude product was recrystallized using a mixed solvent of n-hexane and dichloromethane. The pure product was dissolved in acetone and counter-ion exchanged with potassium hexafluorophosphate to give a deep red TPE-CP (yield 90%).¹H-NMR(400MHz；CD₂Cl₂)δH 8.76(d,2H),7.99(d,2H),7.27(d,2H),7.20-7.14(m,5H),7.06-7.03(m,2H),6.95-6.91(m,4H),6.70-6.63(m,4H),4.51(s,3H),3.75(s,3H),3.73(s,3H)ppm；¹³C-NMR(400MHz；CD₂Cl₂) δ C171.2,158.9,158.6,152.6,150.4,145.9,145.8,143.1,137.7,137.4,135.5,135.4,132.7,132.6,132.5,132.2,131.8,131.4,131.3,130.0,129.9,129.8,129.0,128.0,127.5,126.8,126.6,113.4,113.3,113.2,113.0, 84.9,55.1,55.0,49.4 ppm; MALDI-MS (C) to calculate TPPCN cation₃₈H₃₀N₃O₂ ⁺) 560.2333, found 560.2321.

Example 2

Photophysical properties of TPPCN

As shown in fig. 1, the uv absorption spectrum of TPPCN in Dichloromethane (DCM) solution has a maximum absorption peak at 440nm in the visible range, which is less damaging to biological systems than uv light. The fluorescence spectra of the solution and the aggregation state show that the TPPCN has typical AIE characteristics, as shown in FIGS. 2A-2B, in a mixed system of n-hexane and dichloromethane, when the volume fraction of 2 n-hexane is 0-70%, the TPPCN hardly emits fluorescence, and when the volume fraction of n-hexane is increased to 80%, the fluorescence gradually appears, and the peak value is 606 nm. After further increasing the n-hexane volume fraction to 90%, the peak fluorescence intensity was about 217 times higher than that in pure DCM solution. Under the irradiation of 365nm light source of a hand-held ultraviolet lamp, bright yellow fluorescence can be observed. This significant difference in fluorescence intensity was probably due to the formation of TPPCN aggregates induced by n-hexane as a poor solvent for TPPCN. When the Taiji fraction of n-hexane increased from 90% to 99%, a decrease in fluorescence intensity occurred, which was probably caused by precipitation of TPPCN and adhesion to the inner wall of the cuvette. These precipitates can be observed by the naked eye under irradiation of an ultraviolet lamp. The Quantum Yields (QY) of TPPCN were 0% and 15.2% in pure DCM and DCM/n-hexane mixture (1: 99, v/v), respectively. The fluorescence spectrum and quantum yield result show that TPPCN has AIE property.

Example 3

Biocompatibility of TPPCN

The cytotoxicity of TPPCN on HeLa cells was examined using the 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyltetrazolium bromide (MTT) method. When TPPCN concentration was increased to 10 μ M, cell viability was not significantly affected (fig. 3), indicating that TPPCN has good biocompatibility for HeLa cells over the tested concentration range.

Example 4

Mitochondrial targeting of TPPCN

The ability of TPPCN to image mitochondria in HeLa cells in a targeted manner was further investigated. HeLa cells were incubated with TPPCN and the commercial mitochondrial Red dye MitoTracker Red FM (MTR) for 10 minutes. As shown in fig. 4A-4D, TPPCN selectively accumulates in the cell mitochondria and emits intense blue-green fluorescence, which can coincide well with the red fluorescence of MTR. The Pearson correlation coefficient (Rr; from +1 to-1) was 0.983, indicating that TPPCN is highly specific for mitochondria.

Example 5

Photostability of TPPCN

The light stability of TPPCN was further examined by confocal laser microscopy scanning. As shown in fig. 5A-5B, TPPCN fluorescence was clearly observed in mitochondria after 50 scans (13min), with more than 95% signal retained. In contrast, after 25 scans, the fluorescence of MTR had quenched by more than half, and after 50 scans, the red fluorescence of MTR almost completely disappeared. It is clear that TPPCN has more excellent photostability than MTR.

Example 6

Reactive Oxygen Species (ROS) generating effects of TPPCN

More interestingly, TPPCN can generate ROS as a photosensitizer. Since TPPCN has strong absorption in the visible region, white light is used as an excitation light source. H2DCF-DA is selected as an ROS indicator, and can emit fluorescence with the wavelength of about 530nm in the presence of ROS. In the presence of TPPCN, the fluorescence of H2DCF-DA gradually increased with the increase of the irradiation time (FIG. 6). After 30 minutes of white light irradiation, the fluorescence intensity of H2DCF-DA was 500 times the initial value. However, no such change was observed in the TPPCN or H2DCF-DA only groups. TPPCN is much more efficient in ROS production than the commonly used photosensitizer, tetracycline. In addition, the invention also researches the ROS generation efficiency of TPPCN in cells. As shown in FIGS. 7A-7D, almost no fluorescence from H2DCF-DA was detected in the cells before 405nm laser irradiation; after 10 seconds of laser irradiation at 405nm, green fluorescence of H2DCF-DA was observed. For cells incubated with H2DCF-DA only, the green fluorescence was weak before and after 405nm laser irradiation. These results above indicate that TPPCN can generate ROS in solution and in cells as a photosensitizer.

Excess ROS are harmful to the cell. As shown in fig. 8A-D, after 10 seconds of light exposure, the mitochondrial morphology of the TPPCN-stained HeLa cells changed from long tubular to small and dispersed fragments. In contrast, mitochondria in control cells remained in good condition, indicating that ROS produced by TPPCN can cause mitochondrial damage and cell death.

Example 7

Selective imaging and killing of cancer cells by TPPCN

TPPCN selectively stains cancer cells but not normal cells. Cancerous HeLa cells and normal MDCK-II cells were incubated with TPPCN, respectively, under the same conditions. As shown in fig. 9A, intense blue-green fluorescence was observed in mitochondria of HeLa cells. In contrast, little fluorescence was observed in MDCK-II cells. This result is sufficient to confirm the following assumptions: the differentiating effect on cancer cells and normal cells comes from the difference in Mitochondrial Membrane Potential (MMP) between cancer cells and normal cells and the different strength of electrostatic interaction of TPPCN with mitochondria.

To demonstrate the ability of TPPCN to selectively kill cancer cells, the present inventors investigated the intracellular ROS production efficiency of TPPCN in co-culture systems. Little green fluorescence of H2DCF-DA was detected in the cells before 405nm laser irradiation (FIG. 9B). After 10 seconds of laser irradiation at 405nm, green fluorescence of H2DCF-DA was observed in HeLa cells. However, little green fluorescence was observed in MDCK-II cells (FIG. 9C). For cells incubated with H2DCF-DA only, the green fluorescence was weak before and after 405nm laser irradiation (FIGS. 10A-10B). The results indicate that TPPCN is able to selectively produce ROS in cancer cells in a co-culture system of cancer cells with normal cells.

The MTT assay was used to examine the cell viability of HeLa cells and MDCK-II cells under different treatment conditions. For HeLa cells, white light irradiation hardly affected cell viability in the absence of TPPCN. However, for TPPCN stained cells, cell viability gradually decreased with increasing TPPCN concentration under white light irradiation conditions (fig. 11A). Notably, cell viability was nearly unchanged in TPPCN-stained cells in the absence of light. TPPCN was almost non-toxic to MDCK-II cells when its concentration reached 12.5. mu.M under white light irradiation (FIG. 11B). In contrast, the survival rate of HeLa cells decreased by nearly 80% under the same conditions. The result shows that TPPCN can effectively kill cancer cells under the irradiation of white light, but does not kill normal cells, and the TPPCN can selectively target mitochondria in the cancer cells and induce the death of the mitochondria through photodynamic therapy and has good potential as a diagnosis and treatment integrated reagent.

To further test the toxicity of TPPCN to cancer cells in the co-culture system, Propidium Iodide (PI) staining was applied. PI is a cell membrane impermeable dye that can only enter dead or late apoptotic cells for staining. As shown in FIGS. 12A to 12H, only blue-green fluorescence of TPPCN was observed in HeLa cells in the co-culture system of HeLa cells and MDCK-II cells. Under white light illumination, red fluorescence of PI was observed in all HeLa cells stained with TPPCN, indicating that these cells were effectively killed by ROS production by TPPCN. The red fluorescence of PI could not be observed in unstained cells treated with pure white light, demonstrating that TPPCN plays an important role in inducing cell death.

The present invention incorporates another cancer cell line (U87, commonly used in neuroscience) for demonstrating the prevalence of TPPCN for selective imaging and killing of cancer cell mitochondria. As shown in fig. 13A-13D and fig. 14A-14D, TPPCN can specifically target mitochondria of U87 cells and can selectively generate ROS as a photosensitizer in U87 cells.

Example 8

Selective imaging and antibacterial of TPPCN on gram-positive bacteria

Similar to the difference in Mitochondrial Membrane Potential (MMP) between cancer cells and normal cells, there is a difference in the membrane structure between gram-positive and gram-negative bacteria due to the negative charge on the surface of the bacteria. Therefore, TPPCN was applied to bacteria differential imaging. Representative of the biologically safe non-pathogenic bacteria staphylococcus epidermidis (gram positive bacteria) and escherichia coli (gram negative bacteria) were selected, and as shown in fig. 15A-15B, after incubation with TPPCN (10 μ M) for 10 minutes, the spherical gram positive bacteria staphylococcus epidermidis fluoresced in blue-green color, while the rod-shaped gram negative bacteria escherichia coli fluoresced almost no fluorescence (fig. 15C-15D), indicating that TPPCN selectively stained gram positive bacteria but not gram negative bacteria. These results were also confirmed in the co-culture system of gram-positive and gram-negative bacteria (FIGS. 15E-15F).

The killing effect of TPPCN on gram positive and gram negative bacteria was assessed by Propidium Iodide (PI) staining. Staphylococcus epidermidis was first incubated with TPPCN for 10 minutes, then irradiated with white light for 20 minutes, and then stained with PI. As shown in fig. 16D-16F, bright red fluorescence emission of PI was clearly observed in the TPPCN-stained cells, and almost no red color from PI was observed in the control group without TPPCN staining (fig. 16A-16B). Since PI can only penetrate bacteria with damaged membrane structure, the above results demonstrate that TPPCN can kill staphylococcus epidermidis under white light irradiation. Similarly, gram-negative E.coli was treated in the same manner, and the experimental group treated with TPPCN and the control group treated with no TPPCN showed no red fluorescence of PI (FIGS. 17A to 17D). The antibacterial action and mechanism of TPPCN on Staphylococcus epidermidis are further studied by Scanning Electron Microscopy (SEM). SEM was used to observe the integrity of the bacterial cell wall under the combined effect of TPPCN and white light irradiation. In the control group without TPPCN treatment and with only light exposure, the staphylococcus epidermidis was morphologically regular with clear borders and smooth surface (fig. 16C). There are well-defined boundaries between overlapping bacteria, indicating the presence of an intact cell wall in Staphylococcus epidermidis. After TPPCN and white light irradiation co-treatment, the cell wall of Staphylococcus epidermidis shrinks, divides and the shape of the bacteria changes significantly (FIG. 16F). The results show that TPPCN can kill bacteria by destroying the cell wall of staphylococcus epidermidis under the condition of white light irradiation, and has almost no killing effect on escherichia coli. This result was also confirmed in the co-culture system of gram-positive and gram-negative bacteria (FIGS. 18A-18D), further indicating that TPPCN is capable of selective killing of gram-positive bacteria.

Based on the killing effect of TPPCN on Staphylococcus epidermidis, the Optical Density (OD) of bacteria incubated with TPPCN is measured₆₀₀) The antibacterial ability of TPPCN was further investigated. As shown in FIGS. 19A-19B, TPPCN showed inhibitory effect on the proliferation of Staphylococcus epidermidis. The time-dependent and concentration-dependent changes in the density of Staphylococcus epidermidis incubated with TPPCN are shown in FIGS. 20A-20B. FIGS. 20C-20D are graphs showing the change in bacterial survival and bactericidal efficacy of TPPCN with increasing TPPCN concentration, showing the IC of TPPCN in inhibiting the growth of Staphylococcus epidermidis₅₀The value was about 3.068. mu.M. This result indicates that TPPCN can be a potential drug against gram-positive bacteria.

Example 9

Potential of TPPCN (AIE fluorogram) as diagnosis and treatment integrated reagent

The use of TPPCN in complex systems including cells and bacteria was further investigated. As shown in FIGS. 23A-23C, TPPCN selectively stained cancer cells HeLa and gram-positive Staphylococcus epidermidis, but not normal cells MDCK-II. ROS generated by TPPCN can selectively kill HeLa cells and staphylococcus epidermidis after white light irradiation (fig. 24A-24C). The above results indicate that TPPCN is capable of selectively imaging cancer cells and gram positive bacteria and generating ROS under light conditions, thereby killing cancer cells and gram positive bacteria. These properties are of great value, particularly in the search for TPPCN as a diagnostic and therapeutic integration reagent.

Example 10

Photophysical Properties of TPE-CP

As shown in FIG. 25, in the UV absorption spectrum of TPE-CP in Dichloromethane (DCM), the maximum absorption peak of TPPCN is 475nm, which is in the visible range, and the visible light is more friendly to the biological system than the UV light. The fluorescence spectra of the solution and the aggregation state show that the TPE-CP has typical AIE characteristics, as shown in FIGS. 26A-26B, the fluorescence of the TPE-CP in a pure dichloromethane solution is weak, and the fluorescence is gradually enhanced along with the increase of the volume fraction of poor solvent n-hexane. The Quantum Yields (QY) of TPPCN were 0% and 6.7% in pure DCM and the DCM/n-hexane mixture (1: 99, v/v), respectively. The fluorescence spectrum and quantum yield result show that TPPCN has AIE property.

Example 11

Biocompatibility of TPE-CP

The cytotoxicity of TPPCN on HeLa cells was examined using the 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyltetrazolium bromide (MTT) method. When the TPE-CP concentration was as high as 50. mu.M, the cell viability was still not significantly affected after 24h incubation (FIG. 27), indicating that TPE-CP has good biocompatibility for HeLa cells.

Example 12

Lipid droplet targeting of TPE-CP

The TPE-CP ability to target lipid droplets in HeLa cells was further investigated. HeLa cells were incubated with TPE-CP and the commercial lipid droplet green dye BODIPY 493/503 (1. mu.g/mL) for 15 minutes. As shown in fig. 28A-28C, TPE-CP selectively accumulated in the cell lipid droplets and emitted intense orange-red fluorescence, which coincided well with the green fluorescence of BODIPY 493/503. The Pearson correlation coefficient (Rr; from +1 to-1) was 0.94, indicating that TPE-CP is highly specific for lipid droplets.

Example 13

Light stability of TPE-CP

And further examining the light stability of the TPE-CP through scanning of a laser confocal microscope. As shown in fig. 29A-29B, fluorescence of TPE-CP was still clearly observed in the lipid droplets after 50 scans, with over 80% of the signal retained. In contrast, more than 40% of the green fluorescence of BODIPY 493/503 was quenched after 50 scans. Clearly, TPE-CP has superior light stability to BODIPY 493/503.

Example 14

Reactive Oxygen Species (ROS) generating effects of TPE-CP

TPE-CP can also act as a photosensitizer to generate ROS. Since TPE-CP absorbs strongly in the visible region, white light is used as the excitation light source. H2DCF-DA is selected as an ROS indicator, and can emit fluorescence with the wavelength of about 530nm in the presence of ROS. In the presence of TPE-CP, the fluorescence of H2DCF-DA gradually increased with the increase of the irradiation time (FIG. 30A). After 10 minutes of white light irradiation, the fluorescence intensity of H2DCF-DA was 670 times the initial value. However, no such change was observed in the TPE-CP or H2DCF-DA only groups. In addition, the invention also researches the ROS generation efficiency of TPE-CP in cells. Little fluorescence from H2DCF-DA was detected in the cells before 405nm laser irradiation (FIG. 30B); after 10 seconds of laser irradiation at 405nm, green fluorescence of H2DCF-DA was observed (FIG. 30C). For cells incubated with H2DCF-DA only, the green fluorescence was weak before and after 405nm laser irradiation (FIGS. 30D-30E). These results above indicate that TPE-CP can generate ROS in solution and cells as a photosensitizer.

Example 15

Selective imaging and antibacterial of TPE-CP on gram-positive bacteria

Similar to the difference in Mitochondrial Membrane Potential (MMP) between cancer cells and normal cells, there is a difference in the membrane structure between gram-positive and gram-negative bacteria due to the negative charge on the surface of the bacteria. Therefore, TPE-CP was applied for bacteria differential imaging. As representative of the biologically safe non-pathogenic bacteria staphylococcus epidermidis (gram positive bacteria) and escherichia coli (gram negative bacteria) were selected, as shown in fig. 31A-31D, after incubation with TPE-CP (10 μ M) for 30 minutes, the spherical gram positive bacteria staphylococcus epidermidis fluoresced in blue-green color, while the rod-shaped gram negative bacteria escherichia coli fluoresced almost no fluorescence, indicating that TPPCN selectively stained gram positive bacteria but not gram negative bacteria. These results were also confirmed in a co-culture system of gram-positive and gram-negative bacteria.

Based on the selective imaging of Staphylococcus epidermidis by TPE-CP and the ROS production capability of TPE-CP, the antibacterial capability of TPPCN is further discussed. As shown in FIGS. 32A-32B, TPE-CP showed inhibitory effect on the proliferation of Staphylococcus epidermidis. The time-dependent and concentration-dependent changes in the density of Staphylococcus epidermidis incubated with TPE-CP are shown in FIGS. 33A-33B. FIGS. 33C-33D show the changes in bacterial survival and TPE-CP bactericidal efficacy with increasing TPE-CP concentration, showing TPE-CP inhibitionIC for Staphylococcus epidermidis growth₅₀The value was about 3.144. mu.M. The results indicate that TPE-CP can be used as a potential drug against gram-positive bacteria.

FIGS. 34A-34H are images of agar plates used to quantify the killing effect of TPE-CP on Staphylococcus epidermidis and Escherichia coli. In the control group without any treatment, the bacteria grew healthily on the solid medium. The single illumination treatment can reduce the colony number of the staphylococcus epidermidis to a certain extent, but can not obviously influence the survival rate of the escherichia coli; TPE-CP treatment alone was effective at killing staphylococcus epidermidis (fig. 34C) without significant effect on e.coli survival (fig. 34E); the TPE-CP and the light act together to effectively kill the staphylococcus epidermidis, as shown in FIG. 34C, the colony growth of the staphylococcus epidermidis hardly occurs on the agar plate, and the TPE-CP has a high antibacterial effect on the staphylococcus epidermidis under the light condition (FIG. 34C). In contrast, TPE-CP and light combined had little antibacterial effect on E.coli colonies (FIG. 34H). These results demonstrate that TPE-CP alone kills gram-positive bacteria and that light exposure provides TPE-CP with a more potent antimicrobial effect.

The integrity of the bacterial cell wall was observed by Scanning Electron Microscopy (SEM) under the combined effect of TPE-CP and white light irradiation. In the control group without TPE-CP treatment and with only light, the Staphylococcus epidermidis was morphologically regular with clear boundaries and smooth surface (FIG. 35A). There are well-defined boundaries between overlapping bacteria, indicating the presence of an intact cell wall in Staphylococcus epidermidis. After the TPE-CP and white light irradiation co-treatment, the cell wall of Staphylococcus epidermidis shrinks, divides and the shape of the bacteria changes significantly (FIG. 35B). The results show that the TPE-CP can generate an antibacterial effect by destroying the cell wall of the staphylococcus epidermidis under the white light irradiation condition, and almost has no antibacterial effect on escherichia coli.

Having thus described the subject matter, it will be apparent that the same may be modified or varied in many ways. Such modifications and variations are not to be regarded as a departure from the spirit and scope of the present subject matter, and all such modifications and variations are intended to be included within the scope of the following claims.

Claims

1. An AIE fluorescent probe comprising a compound having a backbone structure selected from the group consisting of:

2. The AIE fluorescent probe of claim 1, wherein the compound comprises a backbone structural formula selected from the group consisting of:

X^-independently selected from I^-、PF₆ ^-One kind of (1).

3. The AIE fluorescent probe of claim 1, wherein the probe exhibits mitochondrial selective staining.

4. The AIE fluorescent probe of claim 1, wherein the probe exhibits lipid droplet specific staining.

5. The AIE fluorescent probe of claim 1, wherein the probe exhibits cancer cell specific staining.

6. The AIE fluorescent probe of claim 5, wherein the cancer cell is a HeLa cell.

7. The AIE fluorescent probe of claim 1, wherein the probe exhibits gram positive specific staining.

8. The AIE fluorescent probe of claim 7, wherein the gram-positive bacterium is Staphylococcus epidermidis.

9. The AIE fluorescent probe of claim 1, wherein the probe is capable of generating reactive oxygen species under light conditions.

10. The AIE fluorescent probe of claim 1, wherein the probe is a photosensitizer.

11. The AIE fluorescent probe of claim 1, wherein the probe is useful for cancer cell imaging and killing.

12. The AIE fluorescent probe of claim 1, wherein the probe is useful for gram-positive bacteria imaging and for antimicrobial applications.

13. An AIE fluorescent probe comprising a compound having a backbone structure selected from the group consisting of:

14. The AIE fluorescent probe of claim 13, wherein the compound comprises a backbone structural formula selected from the group consisting of:

X^-independently selected from I^-、PF₆ ^-One kind of (1).

15. A method of locating unwanted cells in a patient, comprising: administering to a patient an AIE probe as defined in claim 1; and localizing the unwanted cells using fluorescence imaging.

16. The method of claim 15, wherein the unwanted cells are cancer cells.

17. The method of claim 15, wherein the unwanted cells are gram positive bacteria.

18. The method of claim 15, wherein the AIE fluorescent probe is used at a surgical site.

19. The method of claim 15, wherein the amount of harmful cells is quantified based on fluorescence intensity.

20. A method of terminating or inhibiting the growth of unwanted cells in a patient comprising: administering to a patient the AIE fluorescent probe of claim 1; locating harmful cells by a fluorescence imaging method; and in the presence of the fluorescent probe, illuminating the position where the harmful cell is located, so as to prevent or inhibit the growth of the harmful cell.