An organic afterglow probe, a preparation method and application thereof

Abstract

The application belongs to the technical field of nanobiomaterials, and particularly relates to an organic afterglow probe and a preparation method and application thereof. The probe is a nanoparticle formed by self-assembly of a squarylium dye and a PEG liposome; and the specific preparation steps are as follows: (E)-4-2-(1-methyl-1H-pyrrole-2-yl)vinyl)pyridine and 3-N,N-(dibutylamino)phenyl, 4-hydroxy-3-cyclobutene-1,2-diketone are added into a mixed solvent of n-butanol and benzene, and the squarylium dye is obtained by purification after heating and refluxing; the prepared squarylium dye and PEG liposome are added into ultrapure water for self-assembly, and the organic long afterglow probe is obtained. The preparation method is simple, the conditions are mild, raw materials are cheap and environmentally friendly, the prepared probe has good water dispersibility, uniform particle size distribution, and excellent afterglow luminescence performance, high afterglow luminescence intensity, long half-life, and has clinical application potential.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomaterials technology, specifically relating to an organic afterglow probe, its preparation method, and its application. Background Technology

[0002] Optical imaging is a powerful tool for disease diagnosis and treatment monitoring. Compared with traditional imaging methods, optical imaging has advantages such as high imaging sensitivity, high spatiotemporal resolution, low imaging agent cost, and real-time imaging capability, and can sensitively and specifically detect biomarkers in vivo.

[0003] Fluorescence imaging requires real-time photoexcitation for fluorescence emission, and its imaging sensitivity and resolution are typically affected by low tissue penetration depth and high background noise caused by tissue autofluorescence. Chemiluminescence / bioluminescence imaging relies on chemical or enzymatic reactions to trigger the chemiluminescence of certain materials, encountering problems with limited available imaging substrates and relatively complex substrate synthesis routes. In contrast, afterglow imaging, as a promising imaging method utilizing persistent emission from chemical defects, has attracted considerable attention. It can reveal molecules or cells in living systems with ideal signal-to-background ratios and imaging sensitivity for several minutes after external light irradiation ceases. Afterglow emission is also repeatable after multiple light reflections because the afterglow substrate can act as an optical cell storing light energy upon illumination.

[0004] Afterglow materials can be inorganic or organic. Inorganic afterglow imaging agents typically contain rare earth metal ions, transition metal ions, or some main group metal ions with highly conductive structures. However, the use of inorganic materials in biomedical applications may be limited due to the potential leakage of toxic heavy metal ions and the limited targeting ability for specific molecules. Organic afterglow imaging agents exhibit high biocompatibility and molecular-level specificity because of the high structural diversity of organic afterglow matrices. The emission of organic afterglow materials can also be fine-tuned by doping with photosensitizers and introducing afterglow relay units into the afterglow imaging agent to enhance afterglow intensity, redshift the afterglow wavelength to the NIR window, and prolong the afterglow duration, making them ideal for in vivo imaging.

[0005] Molecular afterglow imaging is an emerging technology that enables visual monitoring in vivo with high sensitivity and signal-to-background ratio. Afterglow substrates can be combined with photosensitizers and afterglow relay units to construct multi-component afterglow imaging probes, making them ideal for in vivo bioimaging. However, the design and application of afterglow imaging probes still face many challenges, including limited tissue penetration depth, low singlet oxygen generation efficiency, and insufficient afterglow intensity and half-life.

[0006] Therefore, developing a new organic long-afterglow probe is of great significance. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a self-assembled organic long-persistence probe that addresses the shortcomings of the prior art. It can achieve long-persistence luminescence with a lifespan of several minutes, and has the advantages of high sensitivity, high resolution and real-time imaging, enabling it to specifically detect biomarkers.

[0008] To address the aforementioned technical problems, this invention discloses an organic afterglow probe, wherein the probe is a nanoparticle formed by the self-assembly of squaric acid cyanine dye and PEG liposomes;

[0009] The structural formula of the squaric acid cyanine dye is as follows:

[0010] This invention also discloses a method for preparing the above-mentioned organic long-afterglow probe, the specific steps of which are as follows:

[0011] Step 1: Add (E)-4-2-(1-methyl-1H-pyrrolo-2-yl)vinyl)pyridine and 3-N,N-(dibutylamino)phenyl,4-hydroxy-3-cyclobutene-1,2-dione to a mixed solvent of n-butanol and benzene, heat under reflux and then purify to obtain the squaric acid cyanine dye;

[0012] Step 2: Add the squaric acid cyanine dye and PEG liposomes obtained in Step 1 to ultrapure water for self-assembly to obtain the organic long afterglow probe.

[0013] The mass ratio of (E)-4-2-(1-methyl-1H-pyrrolo-2-yl)vinyl)pyridine to 3-N,N-(dibutylamino)phenyl,4-hydroxy-3-cyclobutene-1,2-dione is (0.4-0.8):1.

[0014] Preferably, the mass ratio of (E)-4-2-(1-methyl-1H-pyrrolo-2-yl)vinyl)pyridine to 3-N,N-(dibutylamino)phenyl,4-hydroxy-3-cyclobutene-1,2-dione is 0.6:1.

[0015] The volume ratio of n-butanol to benzene in the mixed solvent is 1:1.

[0016] Wherein, the total mass of (E)-4-2-(1-methyl-1H-pyrrolo-2-yl)vinyl)pyridine and 3-N,N-(dibutylamino)phenyl,4-hydroxy-3-cyclobutene-1,2-dione and the mass-volume ratio of the mixed solvent are (5-7) mg:1 mL;

[0017] Preferably, the mass-to-volume ratio of the total mass of (E)-4-2-(1-methyl-1H-pyrrolo-2-yl)vinyl)pyridine and 3-N,N-(dibutylamino)phenyl,4-hydroxy-3-cyclobutene-1,2-dione to the mixed solvent is 6 mg:1 mL.

[0018] The specific conditions for the heating and reflux are: reflux at 80-100°C for 8-12 hours;

[0019] Preferably, the heating and reflux is performed under the following conditions: reflux at 90°C for 10 hours.

[0020] The specific purification steps are as follows: after heating and reflux, most of the solvent is removed by rotary evaporation of the reaction solution, petroleum ether is added and the crude product is precipitated, filtered and separated, and then the crude product is dissolved in chloroform and purified by column chromatography to obtain squaric acid cyanine dye.

[0021] The PEG liposomes are DSPE-PEG, and the molecular weight of DSPE-PEG is 2000.

[0022] The mass ratio of the squaric acid cyanine dye to DSPE-PEG is 1:(3-5);

[0023] Preferably, the mass ratio of the squaricine dye to DSPE-PEG is 1:4.

[0024] The self-assembly is specifically performed under the following conditions: stirring at room temperature for 1 to 3 hours.

[0025] Preferably, the self-assembly is performed under the following conditions: stirring at room temperature for 2 hours.

[0026] Furthermore, the application of the aforementioned organic long-afterglow probe in bioimaging is also within the scope of protection of this invention.

[0027] Specifically, in some embodiments of the present invention, self-assembled organic long-persistent-light probes were prepared by the above-described preparation method. By testing the ultraviolet absorption spectrum, fluorescence emission spectrum, in vitro reactive oxygen species (ROS) generation level, in vitro singlet oxygen generation level, and afterglow imaging of the organic long-persistent-light probes, it was shown that the organic long-persistent-light probes provided by the present invention have good in vitro ROS generation and in vitro singlet oxygen generation levels, and possess long-persistent-light luminescence performance, demonstrating the application prospects of the organic long-persistent-light probes in bioimaging.

[0028] Beneficial effects:

[0029] The method for preparing the self-assembled organic long-afterglow probe described in this invention is simple, the conditions are mild, the raw materials are inexpensive and environmentally friendly, and the prepared probe has good singlet oxygen generation ability.

[0030] The self-assembled organic long afterglow probe of the present invention has good water dispersibility, uniform size, and good morphology.

[0031] The self-assembled organic long-afterglow probe described in this invention has excellent afterglow luminescence performance, with strong afterglow luminescence intensity and long half-life, and has potential for clinical application. Attached Figure Description

[0032] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0033] Figure 1 The high-resolution mass spectra of the squaric acid cyanine dye (Sq) prepared in the embodiments of the present invention are used for molecular weight characterization.

[0034] Figure 2 Transmission electron microscopy characterization of the self-assembled organic long afterglow probe in this embodiment of the invention;

[0035] Figure 3 The images show the UV absorption and fluorescence emission spectra of the self-assembled organic long-afterglow probe in this embodiment of the invention, where A is the UV absorption spectrum and B is the fluorescence emission spectrum.

[0036] Figure 4 This is a characterization of the in vitro reactive oxygen species (ROS) generation level of the self-assembled organic long-afterglow probe in this embodiment of the invention, where A is the in vitro ROS generation level of the ultrapure water group and B is the in vitro ROS generation level of the self-assembled organic long-afterglow probe group.

[0037] Figure 5 This is a characterization of the in vitro singlet oxygen generation level of the self-assembled organic long afterglow probe in this embodiment of the invention;

[0038] Figure 6 This invention provides an example of afterglow imaging of a self-assembled organic long afterglow probe at different concentrations.

[0039] Figure 7 This is an example of afterglow imaging of a self-assembled organic long afterglow probe under different illumination durations in an embodiment of the present invention;

[0040] Figure 8 This invention provides an example of afterglow imaging using a self-assembled organic long afterglow probe at different oxygen contents.

[0041] Figure 9 The half-life of the self-assembled organic long-afterglow probe in this embodiment of the invention is characterized. Detailed Implementation

[0042] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0043] Compound 1 in the following examples is (E)-4-2-(1-methyl-1H-pyrrolo-2-yl)vinyl)pyridine, and the specific preparation steps are as follows:

[0044] (1) Weigh 1.08g of sodium hydride and dissolve it in 20mL of ultra-dry DMF, then add 2.79g of 4-methylpyridine and heat under reflux at 60℃ for 2h.

[0045] (2) Weigh 3.27g of N-methylpyrrole-2-carboxaldehyde, add it slowly dropwise with a syringe, and heat under reflux at 150℃ for 8h;

[0046] (3) Cool the reaction mixture, pour it into crushed ice, stir at room temperature for 1 hour, filter to collect the solid product, and dry to obtain compound 1.

[0047] Compound 2 in the following examples is 3-N,N-(dibutylamino)phenyl,4-hydroxy-3-cyclobutene-1,2-dione, and the specific preparation steps are as follows:

[0048] (1) Weigh 5.58g aniline, 19.08g sodium carbonate, 20.55g n-butyl bromide and 200mg iodine and dissolve them in 80mL n-butanol and reflux at 100℃ for 24h;

[0049] (2) Cool the reaction mixture, remove the solvent by rotary evaporation, then suspend the mixture in water and extract with dichloromethane;

[0050] (3) Collect the organic layer, wash it with brine, and then dry it with anhydrous sodium sulfate.

[0051] (4) After column chromatography separation and purification, the eluent ratio was: ethyl acetate: n-hexane = 1:20, to obtain the product N,N-dibutylaniline;

[0052] (5) Weigh 2g of squaric acid and dissolve it in 50mL of benzene, then add 4mL of thionyl chloride, stir at room temperature for 24h, then add 4.2g of N,N-dibutylaniline, and heat under reflux at 80℃ for 6h.

[0053] (6) After the reaction mixture is cooled, it is poured into 500 mL of ice water, extracted with dichloromethane, the organic layer is collected, and the solvent is removed by rotary evaporation.

[0054] (7) Dissolve the residue in a mixed solution of 20 mL acetic acid, 1 mL hydrochloric acid and 20 mL water, and heat under reflux at 100 °C for 2 h;

[0055] (8) Cool the reaction mixture, separate the solid product by filtration, wash with ether and dry to give compound 2.

[0056] Example 1: Preparation of self-assembled organic long-afterglow probe

[0057] The synthetic route for succinic acid cyanine dye (Sq) in this embodiment is as follows:

[0058]

[0059] The preparation process of the self-assembled organic long-afterglow probe is as follows:

[0060] (1) Weigh 184 mg of (E)-4-2-(1-methyl-1H-pyrrolo-2-yl)vinyl)pyridine and 301 mg of 3-N,N-(dibutylamino)phenyl,4-hydroxy-3-cyclobutene-1,2-dione, add them to a 250 mL round-bottom flask, then add 40 mL of n-butanol and 40 mL of benzene respectively, and heat under reflux at 90 °C for 10 h;

[0061] (2) Remove most of the solvent by rotary evaporation, add 50 mL of petroleum ether to the reaction flask, and the crude product will precipitate and settle at the bottom of the flask.

[0062] (3) The solid product was separated by vacuum filtration, added to chloroform, and purified by column chromatography. The eluent ratio was: dichloromethane: methanol = 10:1 to obtain the product, succinic acid dye.

[0063] (4) Weigh 1 mg of squaric acid cyanine dye and 4 mg of DSPE-PEG (2000), add them to 10 mL of ultrapure water, stir at room temperature for 2 h, and prepare the above self-assembled organic long afterglow probe.

[0064] Figure 1 The mass spectrum of squaric acid cyanine dye was obtained by dissolving it in dichloromethane and then measuring it on a high-resolution mass spectrometer. The test results showed a molecular weight of 468.2632, which is consistent with the actual molecular weight.

[0065] Figure 2 This is a transmission electron microscope image of a self-assembled organic long-afterglow probe. The test sample was an aqueous solution of the organic long-afterglow probe with a concentration of 10 μg / mL. Figure 2 As can be seen, the self-assembled organic long afterglow probe prepared in this embodiment is capsule-shaped, uniform in size, and has a good morphology.

[0066] Example 2: Spectral Testing of Self-Assembled Organic Long Afterglow Probe

[0067] Combination Figure 3 This paper introduces the basic spectroscopic characterization of self-assembled organic long-afterglow probes, and the specific test steps are as follows:

[0068] The self-assembled organic long-afterglow probe was prepared into aqueous solutions with concentrations of 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL, respectively. 3 mL of each solution was added to a quartz cuvette, and its UV absorption spectrum was measured using a UV-Vis spectrophotometer. The test results (…) Figure 3 A) shows that its ultraviolet absorption peak is located at 670 nm, and the ultraviolet absorption intensity increases with increasing concentration gradient; then, samples with concentrations of 20 μg / mL and 40 μg / mL were added to quartz cuvettes, and their fluorescence emission spectra were measured on a fluorescence spectrophotometer. The test results ( Figure 3 B) shows that its fluorescence emission peak is located at 680 nm, and the fluorescence emission intensity increases with increasing concentration gradient.

[0069] Example 3: Test of Reactive Oxygen Species (ROS) Generation Level

[0070] Combination Figure 4 and Figure 5 This paper introduces the in vitro singlet oxygen generation level of a self-assembled organic long-afterglow probe. The specific test steps are as follows:

[0071] (1) The self-assembled organic long-afterglow probe was prepared into an aqueous solution with a concentration of 20 μg / mL. 3 mL of the solution was added to a quartz cuvette, followed by the addition of 10 μL of 10 μM singlet oxygen indicator ABDA. The UV absorption intensity was measured under light-protected conditions, and then the solution was concentratedly irradiated with a xenon lamp with a power density of 0.7 W / cm². 2 The illumination duration was 1 minute, for a total of 5 minutes. The UV absorption intensity was measured every 1 minute of illumination, for a total of 5 tests. The entire test was conducted in the dark. To eliminate the influence of the singlet oxygen indicator ABDA itself, 10 μL of 10 μM singlet oxygen indicator ABDA was added to 3 mL of ultrapure water for the same test, while keeping other conditions unchanged.

[0072] Test results ( Figure 4 (where A is the absorption intensity of the ultrapure water group and B is the absorption intensity of the self-assembled organic long-afterglow probe group) This indicates that, in the presence of the self-assembled organic long-afterglow probe, with the increase of illumination time, Figure 4 The ultraviolet absorption intensity at 400 nm in B decreases, while Figure 4 The ultraviolet absorption intensities of the ultrapure water group A all overlapped at different times and showed no decreasing trend. This is because the singlet oxygen generated by the self-assembled afterglow probe reacted with ABDA during illumination, resulting in a decrease in the concentration of ABDA and a decrease in the absorption intensity of ABDA at 400 nm. This indicates that the self-assembled organic long afterglow probe can generate singlet oxygen very well.

[0073] (2) The self-assembled organic long-afterglow probe was prepared into an aqueous solution with a concentration of 20 μg / mL. 3 mL of this solution was added to a quartz cuvette, followed by the addition of 10 μL of 500 μM singlet oxygen indicator SOSG. The fluorescence emission intensity was measured under light-shielded conditions, and then the solution was concentratedly irradiated with a xenon lamp with a power density of 0.7 W / cm². 2 The illumination duration was 1 minute, for a total of 5 minutes. The fluorescence emission intensity was tested once every 1 minute of illumination, for a total of 5 tests. The test was conducted in the dark throughout.

[0074] Test results ( Figure 5 The results show that, in the presence of the self-assembled organic long-afterglow probe, the fluorescence emission intensity at 520 nm increases with the increase of illumination time. This is because the singlet oxygen generated by the self-assembled afterglow probe interacts with SOSG to form SOSG-EP, which leads to the activation of fluorescence output. This indicates that the self-assembled organic long-afterglow probe can generate singlet oxygen well, and the fluorescence intensity increases uniformly with the increase of illumination time. It can be seen that the self-assembled afterglow probe can continuously generate singlet oxygen under continuous illumination conditions, indicating that it has good stability.

[0075] Example 4: Afterglow imaging of a self-assembled organic long-afterglow probe

[0076] Combination Figures 6-9 This paper introduces the afterglow performance of a self-assembled organic long afterglow probe, and the specific test steps are as follows:

[0077] (1) The self-assembled organic long-afterglow probe was prepared into aqueous solutions with concentrations of 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL, respectively. 3 mL of each solution was added to centrifuge tubes, and the tubes were irradiated with a xenon lamp at a power density of 0.7 W / cm². 2 The illumination time was 1 minute. After the illumination ended, the animal was quickly placed into the small animal live imaging system (IVIS LUMINA K Series Ⅲ), with an exposure time of 60 seconds, a binning of 8, an aperture of 1, and the filter was opened to test the afterglow intensity under different concentration conditions.

[0078] Figure 6 The image shows afterglow imaging of aqueous solutions of self-assembled organic long-afterglow probes at different concentrations. As can be seen from the figure, the afterglow intensity of the self-assembled organic long-afterglow probes increases with increasing concentration.

[0079] (2) Prepare a 50 μg / mL aqueous solution of the self-assembled organic long-afterglow probe. Add 3 mL of the solution to a centrifuge tube and irradiate it with a xenon lamp with a power density of 0.7 W / cm³. 2The illumination times were 1 min, 2 min, 3 min, and 4 min. After the illumination ended, the animal was quickly placed into the small animal live imaging system (IVIS LUMINA K Series Ⅲ), with an exposure time of 60 s, a binning of 8, an aperture of 1, and the filter was opened to test the afterglow intensity at different illumination times.

[0080] Figure 7 The image shows the afterglow of a 50 μg / mL self-assembled organic long-afterglow probe aqueous solution under different illumination times. As can be seen from the figure, the afterglow intensity of the self-assembled organic long-afterglow probe increases with the increase of illumination time.

[0081] (3) The self-assembled organic long-afterglow probe was prepared into an aqueous solution with a concentration of 50 μg / mL. 3 mL of each solution was added to centrifuge tubes to prepare four samples: Sample 1 served as the control group; Sample 2 was continuously irradiated with Ar2 for 10 min; Sample 3 was continuously irradiated with N2 for 10 min; and Sample 4 was continuously irradiated with O2 for 10 min. The samples were then concentratedly irradiated with a xenon lamp with a power density of 0.7 W / cm². 2 The illumination time was 1 minute. After the illumination ended, the animal was quickly placed into the small animal live imaging system (IVIS LUMINA K Series Ⅲ). The exposure time was 60 seconds, the binning was 8, the aperture was 1, the filter was opened, and the afterglow intensity under different oxygen contents was tested.

[0082] Figure 8 The image shows the afterglow imaging of a 50 μg / mL self-assembled organic long-afterglow probe aqueous solution under different oxygen contents. As can be seen from the figure, the afterglow intensity of the self-assembled organic long-afterglow probe increases with increasing oxygen content.

[0083] (4) Prepare a 50 μg / mL aqueous solution of the self-assembled organic long-afterglow probe. Add 3 mL of the solution to a centrifuge tube and irradiate it with a xenon lamp with a power density of 0.7 W / cm³. 2 The illumination time was 1 minute. After the illumination ended, the animal was quickly placed into the small animal live imaging system (IVIS LUMINA K Series Ⅲ). The exposure time was 60 seconds, the binning was 8, the aperture was 1, the filter was opened, and the test was performed 4 times without interruption. Since the exposure time was 60 seconds, the interval between each imaging was 1 minute. The half-life of the afterglow was tested.

[0084] Figure 9 The figure shows the half-life of the afterglow of a 50 μg / mL self-assembled organic long afterglow probe aqueous solution after 1 min of illumination. As can be seen from the figure, it continues to emit light 4 min after the illumination is stopped, indicating that the self-assembled afterglow probe has excellent afterglow luminescence performance.

[0085] This invention provides an organic afterglow probe, its preparation method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. An organic afterglow probe, characterized in that, The probe is a nanoparticle formed by the self-assembly of squaric acid cyanine dye and PEG liposomes. The structural formula of the squaric acid cyanine dye is as follows:

2. The method for preparing the organic afterglow probe as described in claim 1, characterized in that, The specific steps are as follows: Step 1: Add (E)-4-2-(1-methyl-1H-pyrrolo-2-yl)vinyl)pyridine and 3-N,N-(dibutylamino)phenyl,4-hydroxy-3-cyclobutene-1,2-dione to a mixed solvent of n-butanol and benzene, heat under reflux and then purify to obtain the squaric acid cyanine dye; Step 2: Add the squaric acid cyanine dye and PEG liposomes obtained in Step 1 to ultrapure water for self-assembly to obtain the organic long afterglow probe.

3. The preparation method according to claim 2, characterized in that, The mass ratio of (E)-4-2-(1-methyl-1H-pyrrolo-2-yl)vinyl)pyridine to 3-N,N-(dibutylamino)phenyl,4-hydroxy-3-cyclobutene-1,2-dione is (0.4-0.8):

1.

4. The preparation method according to claim 2, characterized in that, The volume ratio of n-butanol to benzene in the mixed solvent is 1:

1.

5. The preparation method according to claim 3 or 4, characterized in that, The total mass of (E)-4-2-(1-methyl-1H-pyrrolo-2-yl)vinyl)pyridine and 3-N,N-(dibutylamino)phenyl,4-hydroxy-3-cyclobutene-1,2-dione and the mass-volume ratio of the mixed solvent are (5-7) mg:1 mL.

6. The preparation method according to claim 2, characterized in that, The specific conditions for the heating and reflux are: reflux at 80-100°C for 8-12 hours.

7. The preparation method according to claim 2, characterized in that, The PEG liposomes are DSPE-PEG; the molecular weight of DSPE-PEG is 2000.

8. The preparation method according to claim 2, characterized in that, The mass ratio of the squaric acid cyanine dye to DSPE-PEG is 1:(3-5).

9. The preparation method according to claim 2, characterized in that, The self-assembly is specifically performed under the following conditions: stirring at room temperature for 1–3 hours.

10. The application of the organic afterglow probe as described in claim 1 in bioimaging.

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