阅读说明：本技术 基于吡啰红肟的近红外HClO荧光探针、制备及其应用 (Near-infrared HClO fluorescent probe based on pyrrazone oxime, and preparation and application thereof ) 是由 刘景� 王林芳 郭炜 于 2020-11-27 设计创作，主要内容包括：本发明开发了一种基于吡啰红肟的近红外HClO荧光探针(PyOX)、制备及应用,荧光探针的结构式如下：。本发明是将肟反应基团与吡啰红染料的9位相结合构建的探针,该探针对HClO显示出快速、灵敏的近红外荧光off-on响应,检出限为2.4 nM,并已成功应用于癌细胞/组织与正常细胞/组织的区分。(The invention discloses a near-infrared HClO fluorescent probe (PyOX) based on pyrrosine oxime, and preparation and application thereof, wherein the structural formula of the fluorescent probe is as follows: . The invention relates to a probe constructed by combining an oxime reaction group and 9-phase of a red-yellow-oil dye, which shows rapid and sensitive near-infrared fluorescence off-on response to HClO, has the detection limit of 2.4 nM, and is successfully applied to the differentiation of cancer cells/tissues and normal cells/tissues.)

1. A near-infrared HClO fluorescent probe based on pyrrosine oxime is characterized in that the structural formula of the fluorescent probe is as follows:

。

2. the method for preparing a fluorescent probe according to claim 1, characterized by comprising the steps of:

(1) dissolving xanthone in anhydrous tetrahydrofuran at 0 deg.C under nitrogen atmosphere, slowly adding THF solution of methyl magnesium bromide into the reaction solution, stirring at room temperature overnight, quenching with water, extracting with DCM, removing solvent under reduced pressure, dissolving the obtained product in acetonitrile and perchloric acid aqueous solution, stirring for 10 min, extracting with DCM again, and dissolving the organic phase with anhydrous Na₂SO₄Drying, filtering and evaporating to obtain a crude product, and purifying by column chromatography to obtain an intermediate 2;

(2) intermediate 2 and I obtained in step (1)₂Dissolving in CHCl₃Refluxing for 0.5 h, adding dimethyl sulfoxide into the solution, refluxing for 48 h, cooling to room temperature, and mixing with saturated Na₂S₂O₃Quenching, extracting with dichloromethane, and passing the organic phase over anhydrous Na₂SO₄Drying, filtering, evaporating and purifying by column chromatography to obtain an intermediate 3;

(3) the intermediate 3 and NH obtained in the step (2)₂And adding an OH & HCl and 4A molecular sieve into acetonitrile, stirring at room temperature for 6 h, filtering the reaction solution, washing with water, extracting with DCM, drying the organic phase, filtering, evaporating, and purifying by column chromatography to obtain the probe PyOX.

3. The method for preparing a fluorescent probe according to claim 2, wherein the structural formula of xanthone in step (1) is as follows:

。

4. the method for preparing a fluorescent probe according to claim 2, wherein the intermediate 2 has the following structural formula:

。

5. the method for preparing a fluorescent probe according to claim 2, wherein the intermediate 3 has the following structural formula:

。

6. the method for preparing a fluorescent probe according to any one of claims 2 to 5, characterized in that: the molar ratio of the xanthone compound to the methyl magnesium bromide in the step (1) is 1: 1.25; step (2) intermediates 2 and I₂In a molar ratio of 1:1, step (3) intermediate 3 and NH₂The molar ratio of OH to HCl is 1: 10.

7. Use of the fluorescent probe of claim 1 to detect HClO in the near infrared region with a minimum detection limit of 2.4 nM.

8. Use of the fluorescent probe of claim 1 to distinguish between cancer cells and normal cells, wherein: cancer cells are distinguished from normal cells by detecting intracellular ROS levels.

Technical Field

The invention relates to the field of fluorescent probes, and in particular relates to a near-infrared HClO fluorescent probe based on pyrrazone oxime, and preparation and application thereof.

Background

Differentiation of cancer from normal cells/tissues is critical for early diagnosis and treatment of cancer. Among the tumor imaging tools that have been developed so far, tumor imaging technologies based on fluorescent probes are receiving much attention because of their advantages of visualization, non-invasiveness, high sensitivity, no ionizing radiation, real-time imaging of living bodies, etc., and the most common design strategy of such fluorescent probes is to chemically couple fluorophores with ligands, including chemical molecules, polypeptides, proteins, antibodies, etc., which can specifically bind to surface markers over-expressed by cancer cells. Imaging differences between cancer and normal cells/tissues using such probes is helpful for individualized treatment of patients, but it remains difficult to use such differences to diagnose a broad spectrum of cancers due to genetic or phenotypic heterogeneity of cancer cells and tumors.

Therefore, researchers have focused on the abnormal metabolism of tumors, namely aerobic glycolysis (also known as the Warburg effect), and this particular metabolic pattern of cancer cells creates a cancer cell-specific microenvironment, independent of the type of cancer, such as decreased extracellular pH, increased intracellular Glutathione (GSH) and Reactive Oxygen Species (ROS), and the like. However, since there is not much difference in extracellular pH and intracellular GSH levels between cancer cells and normal cells (pH 6.5 and 7.4, respectively; glutathione is in mM grade), there are very few examples of achieving differentiation by this strategy to date.

In contrast, the concentration of ROS in cancer cells, including O₂ ^-、H₂O₂、OH•、ONOO^-And ClO^-Etc., approximately 10-fold higher than normal cells, and therefore, the ROS-sensitive probe should have a higher sensitivity to cancer cells than to normal cells/tissues. In fact, this property of cancer cells has been widely applied in ROS-responsive drug delivery systems relevant for cancer therapy. Although a large number of ROS fluorescent probes have been reported in the last decade, only a few ROS fluorescent probes have been able to achieve the effect of distinguishing between cancerous and normal cells/tissues. One possible reason for this is due to insufficient probe sensitivity, which makes it difficult for most probes to respond to background ROS in cancer cells; another reason may be that the poor tissue penetration of visible light compared to near infrared light (650-900 nm), due to the absorption and emission wavelengths of most ROS probes in the visible region, severely limits the imaging applications of such probes in tissue or living bodies. Therefore, development of ROS fluorescence capable of simultaneously overcoming the above disadvantagesThe probe has important significance for diagnosing broad-spectrum tumors.

Disclosure of Invention

The invention provides a near-infrared HClO fluorescent probe based on pyrrosine oxime, and preparation and application thereof. These excellent properties have led to their success in practical applications for differentiating between cancer cells and normal cells/tissues.

The technical scheme for realizing the invention is as follows:

a near-infrared HClO fluorescent probe (PyOX) based on red oxime of pyranthralfate has the following structural formula:

。

the preparation method of the fluorescent probe comprises the following steps:

(1) dissolving xanthone in anhydrous tetrahydrofuran at 0 deg.C under nitrogen atmosphere, slowly adding THF solution of methyl magnesium bromide into the reaction solution, stirring at room temperature overnight, quenching with water, extracting with DCM, removing solvent under reduced pressure, dissolving the obtained product in water solution of acetonitrile and perchloric acid, stirring for 10 min, extracting with DCM again, and dissolving the organic phase with anhydrous Na₂SO₄Drying, filtering and evaporating to obtain a crude product, and purifying by column chromatography to obtain an intermediate 2;

(2) intermediate 2 and I obtained in step (1)₂Dissolving in CHCl₃Refluxing for 0.5 h, adding dimethyl sulfoxide into the solution, refluxing for 48 h, cooling to room temperature, and mixing with saturated Na₂S₂O₃Quenching, extracting with dichloromethane, and passing the organic phase over anhydrous Na₂SO₄Drying, filtering, evaporating and purifying by column chromatography to obtain an intermediate 3;

(3) the intermediate 3 and NH obtained in the step (2)₂Adding OH & HCl and 4A molecular sieve into acetonitrile, stirring at room temperature for 6 h, filtering the reaction solution, washing with water, extracting with DCM, drying the organic phase, filtering, evaporating, and purifying by column chromatography to obtain the final productNeedle PyOX.

The structural formula of the xanthone in the step (1) is as follows:

。

the structural formula of the intermediate 2 is as follows:

。

the structural formula of the intermediate 3 is as follows:

。

the molar ratio of xanthone to methyl magnesium bromide in the step (1) is 1: 1.25; step (2) intermediates 2 and I₂Is 1:1, and the molar ratio of the intermediate 3 to the hydroxylamine hydrochloride in the step (3) is 1: 10.

The fluorescent probe is applied to detecting HClO in a near infrared region, and the lowest detection limit is 2.4 nM.

The synthesis of the probe and the sensing mechanism of the probe on HClO are shown as follows:

the fluorescent probe is applied to distinguishing cancer cells from normal cells, and distinguishing cancer cells from normal cells by detecting the level of intracellular ROS.

The principle of using PyOX probes to distinguish cancer cells from normal cells/tissues is shown:

the invention has the beneficial effects that: the probe constructed by combining the oxime reaction group and the 9-phase of the pyrromone red fluorophore shows rapid and obvious fluorescence off-on response to HClO in a near infrared region, has extremely high sensitivity, and has been successfully applied to the differentiation of cancer cells/tissues and normal cells/tissues.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a drawing of intermediate 2 in the example¹H NMR chart;

FIG. 2 is a drawing of intermediate 2 in the example¹³C NMR chart;

FIG. 3 is a HRMS plot of intermediate 2 in the examples;

FIG. 4 is a drawing of intermediate 3 in example¹H NMR chart;

FIG. 5 is a drawing of intermediate 3 in example¹³C NMR chart;

FIG. 6 is a HRMS plot of intermediate 3 in the examples;

FIG. 7 is PyOX¹H NMR chart;

FIG. 8 is PyOX¹³C NMR；

FIG. 9 is a HRMS map of PyOX;

FIG. 10(A, B) is a spectrum of absorption and emission spectra of PyOX (2 μ M) after ClO-treatment and inset in B is the change in fluorescence intensity at 680 nm of PyOX (2 μ M) after 2 equivalents of ClO-treatment; c is the change of PyOX (2 μ M) fluorescence spectrum with increasing ClO-dosage; d is PyOX (2 μ M) via different substances (including 2 μ M ClO)^-And ONOO^-(ii) a 20 μ M of O₂ ^·, ^·OH, ¹O₂, H₂O₂And NO; 200 μ M Cys, Hcy, GSH, SH-and HSO₃ ^-(ii) a 200 μ M HCO₃ ^-, AcO^-, NO₃ ^-, NO₂ ^-, Cl^-, Cu²⁺ , Ca²⁺ , Fe²⁺ , Fe³⁺And Zn²⁺) After treatmentA change in fluorescence spectrum of (a);

FIG. 11(A) is a photograph of fluorescent images of normal and cancer cells after pre-treatment with PyOX (2. mu.M) for 20 minutes; (B) quantitative data for (a); (C) fluorescence imaging of mixed cultured A549 and RAW264.7 cells after PyOX (2 μ M) treatment for 20 minutes; the collection wavelength was 650-750nm (λ ex = 633 nm);

FIG. 12 is a fluorescence image of RAW264.7 cells under different conditions. (i) RAW264.7 cells treated with PyOX (2. mu.M) only, (ii-iv) RAW264.7 cells pretreated with PyOX (2. mu.M) and then 2. mu.M ClO^-SIN-1 and H₂O₂Treated RAW264.7 cells; (v) RAW264.7 cells previously incubated with LPS (1 mg/mL)/IFN-. gamma. (50 ng/mL) for 12 h, followed by PyOX (2. mu.M); (vi) the inhibitors ABAH (300. mu.M), LPS (1. mu.g/mL)/IFN-^γCultured (50 ng/mL) for 12 h, then PyOX (2. mu.M) -treated RAW264.7 cells, harvested at a wavelength of 650-750nm (. lamda. ex = 633 nm);

FIG. 13 (A) in vivo imaging of tumor-bearing mice injected with PyOX (20 μ M, 50 μ L) via tail vein or intratumorally; (B) graph of fluorescence versus time for HepG2 tumor-bearing mice injected tail vein with PyOX (20. mu.M, 50. mu.L); adopting an excitation filter of 630 nm and an emission filter of 700 nm;

FIG. 14 is a photograph of an image of exposed tumor and surrounding normal tissue of MCF-7 tumor-bearing mice in vivo after 5 minutes of PyOX (20 μ M) spray. (a) A bright image; (b) a fluorescence image; (c) fluorescence images after PyOX spray; (d) fluorescence images after tumor resection.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

Examples

The preparation method of the near-infrared HClO fluorescent probe based on the red oxime of prasuzumi comprises the following steps:

(1) dissolving xanthone (1.01 g, 3 mmol) in anhydrous tetrahydrofuran (30 mL) at 0 ℃ under nitrogen, slowly adding a THF solution of methylmagnesium bromide (3.75 mmol) to the reaction solution, stirring at room temperature overnight, quenching with water (50 mL), extracting with DCM (3X 50 mL) after the reaction is finished, removing the solvent under reduced pressure, redissolving the obtained product in an aqueous solution of acetonitrile and perchloric acid, stirring for 10 minutes, extracting with DCM (3X 50 mL) again, and using anhydrous Na as an organic phase₂SO₄Drying, filtration and evaporation gave the crude product which was purified by column chromatography to give intermediate 2 (1.05 g, 80%).

1H NMR (600 MHz, CDCl3) δ 7.96 (d, J = 9.6 Hz, 2H), 7.03 (d, J = 9.6 Hz, 2H), 6.70 (s, 2H), 3.63 (q, J1 = 7.2 Hz, J2 = 6.6 Hz, 8H), 2.88 (s, 3H), 1.34 (t, J = 6.6 Hz, 12H); 13C NMR (150 MHz, CDCl3) δ 152.39, 140.597, 123.53, 123.06, 115.71, 115.02, 66.78, 49.03, 21.55; ESI-MS [M+H]+: calcd for 337.2274, Found 337.2276.

(2) Intermediate 2 (1.5 g, 3.43 mmol) from step (1) and I₂(0.87 g, 3.44 mmol) was dissolved in CHCl₃Refluxing for 0.5 h, adding dimethyl sulfoxide (8 mL) into the solution, refluxing for 48 h, cooling to room temperature, and mixing with saturated Na₂S₂O₃Quench (50 mL), extract with dichloromethane (3X 50 mL), and pass the organic phase over anhydrous Na₂SO₄Drying, filtration and evaporation, column chromatography purification gave intermediate 3 (0.81 g, 52%);

¹H NMR (600 MHz, CDCl₃) δ 10.98 (s, 1H), 8.20 (d, J= 9.6 Hz, 2H), 7.12 (d, J= 9.6 Hz, 2H), 6.90 (s, 2H), 3.70 (q, J ₁ = 7.2 Hz, J ₂ = 6.6 Hz, 8H), 1.38 (t, J= 6.6 Hz, 12H); ¹³C NMR (150 MHz, CDCl₃) δ 191.35, 158.42, 155.45, 140.42, 129.47, 115.69, 111.87, 97.55, 46.43, 12.69; ESI-MS [M+H]⁺: calcd for 351.2067, Found 351.2068.

(3) the intermediate 3 (4) obtained in the step (2)5 mg, 0.1 mmol）、NH₂OH HCl (69 mg, 1 mmol) and molecular 4 a sieve (5 particles) were added to acetonitrile (10 mL), stirred at room temperature for 6 h, the reaction was filtered, washed with water, extracted with DCM (3 × 50 mL), the organic phase was dried, filtered and evaporated to give probe PyOX (25 mg, 55%).

1H NMR (600 MHz, DMSO-d6) δ 13.01 (s, 1H), 9.10 (s, 1H), 8.26(d , J = 9.6 Hz, 2H), 7.23 (d, J = 9.6 Hz, 2H), 6.91 (s, 2H), 3.68 (d, J = 6.6, 8H), 1.23 (t, J = 6.6 Hz, 12H); 13C NMR (150 MHz, DMSO-d6) δ 157.76, 155.35, 144.60, 144.17, 131.49, 114.98, 111.72, 96.60, 45.76, 12.98; ESI-MS [M+H]+: calcd for 366.2176, Found 366.2180.

Performance testing

1. Solution preparation

The probe PyOX was made up to 2 mM stock with acetonitrile and subsequently diluted to the corresponding concentration with 20 mM PBS (pH 7.4).

Hypochlorite solution (ClO)^-) Prepared by dilution of commercial NaClO solution in deionized water, the concentration of which was determined by measuring the absorption of the solution at 292 nm (ClO)^-Molar extinction coefficient in deionized water of 350M^-1 cm^-1）。

Peroxynitrite solution (ONOO)^-) Prepared according to literature reports (R.M. Up, W.A. Pryor, Synthesis of peroxinitrite in a two-phase system using isocamyl nitrate and hydrogen peroxide,Anal. Biochem1996, 236, 242-249.), whose concentration was determined by measuring the absorption of the solution at 302 nm (ONOO)^-The molar extinction coefficient of the solution in 0.1M NaOH was 1670M^-1 cm^-1)。

Hydrogen peroxide solution (H)₂O₂) By commercialization of H₂O₂The solution was prepared by dilution in deionized water.

Cell culture and fluorescence imaging

All cell lines were purchased from GeneFull biotechnology limited (china).

All cells were cultured in an incubator containing 5% carbon dioxide at 37 ℃ in which Raw264.7 cells and Cos-7 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100U/mL penicillin G sodium and 100. mu.g/mL streptomycin; a549 cells, HepG2 cells, T98G cells, BEAS-2B cells and HUCEC cells were cultured in DMEM (high-sugar) medium containing 10% fetal bovine serum, 100U/mL penicillin G sodium and 100. mu.g/mL streptomycin; before cell imaging experiments, cells were placed on a 30 mm glass-bottom cell culture dish in advance, allowed to stand for 12 hours until the cells were attached to the wall, washed 3 times with Phosphate Buffered Saline (PBS), and then subjected to fluorescence imaging using a Ceiss LMS 710 confocal microscope, and collected at a wavelength of 650-750nm (λ ex = 633 nm).

3. Cytotoxicity

The cytotoxicity of the probe PyOX was investigated using a CCK-8 cell proliferation assay. Briefly, adherently grown A549 cells were first digested into a cell suspension at 5.0X 10 cells per well³The density of individual cells was seeded in 96-well plates and cells were attached by incubation overnight with 100 μ L of DMEM medium. Then adding a PyOX stock solution (2 mM) into the culture medium, keeping the final concentration at 0-10 mu M, repeating the control group and the test group for 6 times, and continuing to incubate the cells; after culturing the cells for 24 h, discarding the old medium, washing with PBS 2 times, replacing the fresh medium containing 10% CCK-8, incubating for 0.5 h, and culturing withiMark ^TM MicroplateAbsorbance ReaderThe absorbance at 450 nm was measured.

4. Subcellular localization

Before the experiment, the cells are placed on a 30 mm glass-bottom cell culture dish, the cell culture dish is kept stand for 12 hours until the cells adhere to the wall, and the cells are washed by PBS for 3 times; cells were stained with PyOX (2. mu.M) and LysoTracker Green DND-26 (50 nM, 10 min)/MitoTracker Green FM (0.2. mu.M, 10 min), respectively, in PBS buffer at 37 ℃ and fluorescence-imaged after 3 washes in PBS. The collection wavelength for PyOX was 650-750nm (λ ex = 633 nm) and for LysoTracker Green DND-26/MitoTracker Green FM was 500-600 nm (λ ex = 488 nm).

HClO imaging in living cells

In experiments with the addition of HClO to image cells, Raw264.7 cells were first pretreated with PyOX (2. mu.M) for 20 min and then with 2. mu.M ClO, respectively^-SIN-1 (commercial ONOO)^-Donor) and H₂O₂Treating for 20 minutes; in experiments to image endogenous HClO in cells, Raw264.7 cells were treated with LPS (1. mu.g/mL)/IFN-^γ(50 ng/mL) pretreatment for 12 h, followed by PyOX (2. mu.M) treatment for 20 min, three PBS washes, and fluorescence imaging; in inhibition experiments, cells were treated with the MPO-specific inhibitors ABAH (300. mu.M), LPS (1. mu.g/mL)/IFN-^γ(50 ng/mL) for 12 h, then PyOX (2. mu.M) for 20 min, PBS washing three times, fluorescence imaging.

Tumor imaging of tumor-bearing murine models with PyOX

All animal experiments were performed according to the relevant laws and guidelines promulgated by the ethical committee of the university of shanxi. BALB/c male nude mice (6-8 weeks old) were purchased from Experimental animals technology, Inc., Viton, Beijing.

A549 cells, HepG2 cells or MCF-7 cells (1X 10)⁶Individual cells) were injected subcutaneously into the left axilla (or left leg) of nude mice, and after 15 days of inoculation, tumor-bearing mice were injected with PyOX via tail vein or intratumorally. Living animal imaging was performed in a Bruker multimode in vivo imaging system, with an excitation filter of 630 nm and an emission filter of 700 nm selected.

Image guided ablation using PyOX

MCF-7 tumor-bearing mice are dissected to expose tumor and normal tissues, PyOX (2 mu M) is sprayed on the exposed tumor and the surrounding normal tissues for 5 minutes for imaging; the tumor was removed and imaged again. Living animal imaging was performed in a Bruker multimode in vivo imaging system, with an excitation filter of 630 nm and an emission filter of 700 nm selected.

Test results

1. Water solubility and light stability

Since the probe has good solubility in water (up to 60. mu.M), pure PBS buffer system was selected for the in vitro test system. The change in absorption spectra before and after reaction of PyOX (2. mu.M) with HClO was first investigated in PBS (20 mM, pH 7.4). As shown in FIG. 10A, the maximum absorption peak of PyOX in PBS (20 mM, pH 7.4) is 581 nm, which is significantly higher than that of classical pyrrosia leaf dyePeak length due to the increased Intramolecular Charge Transfer (ICT) process of the excited state of the dye by the electron withdrawing action of the oxime; as shown in FIG. 9, ClO was added to PyOX solution^-Thereafter, the maximum absorption peak of the probe was red-shifted to 649 nm, and the HRMS data confirmed that the reaction produced PyCNO (m/z = 365.173).

Under the same conditions, the change in fluorescence spectrum before and after reaction of PyOX (2. mu.M) with HClO was further investigated. As shown in fig. 10B, PyOX showed little fluorescence in PBS due to non-radiative transition processes resulting from C = N isomerization; however, addition of ClO to PyOX solution^-Thereafter, significant fluorescence enhancement was caused in the near infrared region, the maximum emission peak was located at 680 nm, and the reaction could be completed within 10 s. The fluorescence titration experiment shows that ClO^-The concentration of (A) was well linear with the fluorescence intensity at 680 nM, with a limit of detection as low as 2.4 nM (FIG. 10C).

In addition, PyOX to ClO^-The selectivity of (a) was higher than other ROS/RNS and biologically relevant cations, anions and biological thiols (fig. 10D).

Cell culture and cell imaging

As shown in fig. 11A, images of PyOX-treated cancer cells, including a549 cells, HepG2 cells, and T98G cells, after 20 minutes, all cells had bright red fluorescence; whereas, images of normal cells treated with PyOX, including BEAS-2B cells, HUVEC cells and COS-7 cells, showed faint fluorescence in all cells after 20 minutes. Quantitative fluorescence analysis showed that the fluorescence enhancement of cancer cells was about 3-fold higher than that of normal cells (FIG. 11B), indicating that PyOX has the potential to distinguish cancer cells from normal cells based on the difference in intracellular ROS levels.

In the cancer cell and normal cell co-culture experiments, a549 cells and RAW264.7 cells were co-cultured in the same confocal dish for 24 hours, treated with PyOX for 20 minutes after cell attachment, and then imaged under a Confocal Laser Scanning Microscope (CLSM). As shown in fig. 11C, a549 cells (green arrows) showed a distinct red fluorescence signal, whereas normal raw264.7 cells showed only a weak fluorescence (white arrows), which indicates that PyOX has a certain potential in vivo tumor diagnosis.

Selective imaging and cytotoxicity of HClO in cells

PyOX imaging ability on HClO was examined in RAW264.7 cells. As shown in FIG. 12, when the RAW264.7 cells incubated with PyOX in advance were excited with laser light of 633 nm, the red fluorescence was almost negligible; when the RAW264.7 cells previously incubated with PyOX were incubated with several representative ROS, only HClO induced the cells to emit bright red fluorescence; when RAW264.7 cells were previously induced by LPS for 12 h and incubation with PyOX continued, bright red fluorescence was observed. These results indicate that PyOX has good cell membrane permeability and can selectively image both endogenous and exogenous HClO in cells.

In addition, CCK-8 cell proliferation experiments showed that PyOX has lower cytotoxicity in the concentration range of 0-10 μm, and the cell survival rate is more than 85%.

4. Tumor imaging

PyOX (20 μ M, 50 μ L) was subcutaneously injected into the tumor region and normal tissue region of a549 tumor-bearing mice, HepG2 tumor-bearing mice, and MCF7 tumor-bearing mice, respectively, and subsequently imaged under a small animal in vivo imaging system. As shown in fig. 13A, the tumor region showed a significant fluorescence signal, while the normal tissue region showed a weak fluorescence signal; in addition, PyOX (20 μ M, 50 μ L) was injected tail vein into HepG2 tumor-bearing mice, and only the tumor area showed significant fluorescence signal, which was maximal at 10 min and gradually disappeared due to metabolism after 30 min (fig. 11B). These results indicate that PyOX can selectively image tumor sites in tumor-bearing mice due to higher ROS levels in the background of cancer cells than in normal cells.

Image guided ablation using PyOX

As shown in fig. 14, PyOX sprayed onto exposed tumor and surrounding normal tissues, only the tumor area showed bright fluorescence signal, while the surrounding normal tissues showed negligible fluorescence; after complete tumor resection of the mice, little fluorescence was seen in the remaining tissue. The results show that PyOX has good application prospect in surgical tumor resection.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.