Organic afterglow probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nano biological materials, and particularly relates to an organic afterglow probe as well as a preparation method and application thereof. The probe is nano-particles formed by self-assembly of squarylium cyanine dye and PEG (polyethylene glycol) lipidosome; the preparation method comprises the following specific preparation steps: adding (E)-4-2-(1-methyl-1H-pyrrole-2-yl) vinyl) pyridine, 3-N, N-(dibutylamino) phenyl and 4-hydroxy-3-cyclobutene-1, 2-diketone into a mixed solvent of n-butyl alcohol and benzene, heating and refluxing, and purifying to obtain the squarylium cyanine dye, and adding the prepared squarylium cyanine dye and PEG liposome into ultrapure water for self-assembly to obtain the organic long afterglow probe. The preparation method is simple, conditions are mild, raw materials are cheap and environmentally friendly, and the prepared probe has good water dispersibility, uniform particle size distribution, excellent afterglow luminescence performance, high afterglow luminescence intensity, long half-life period and clinical application potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanobiomaterials, and particularly relates to an organic afterglow probe, a preparation method thereof, and an application thereof. Background Art

[0002] Optical imaging is a powerful tool for disease diagnosis and treatment monitoring. Compared with traditional imaging methods, optical imaging has the advantages of high imaging sensitivity, high spatio-temporal resolution, low cost of imaging agents, and real-time imaging ability, and can sensitively and specifically detect biomarkers in vivo.

[0003] Fluorescence imaging requires real-time light excitation for fluorescence emission, and its imaging sensitivity and resolution are usually 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 / bioluminescence of certain materials, and it encounters problems such as limited available imaging substrates and relatively complex substrate synthesis routes. On the contrary, afterglow imaging, as a promising imaging method that utilizes persistent luminescence emitted from chemical defects, has currently attracted great attention. It persists for several minutes after stopping external light irradiation and reveals molecules or cells in a living system with an ideal signal-to-background ratio and imaging sensitivity. The afterglow emission is also repeatable after multiple light reflections because the afterglow substrate can act as an optical battery that stores light energy during light irradiation.

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

[0005] Molecular afterglow imaging is a new emerging technology that enables visual monitoring in vivo with high sensitivity and signal-to-background ratio. The afterglow substrate can be combined with photosensitizers and afterglow relay units to construct multi-component afterglow imaging probes, making them ideally suitable 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, insufficient afterglow intensity and half-life, etc.

[0006] Therefore, it is of great significance to develop a new organic long afterglow probe. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a self-assembled organic long afterglow probe in view of the deficiencies of the prior art. It can achieve long afterglow luminescence, and its lifetime lasts for several minutes. It has the advantages of high sensitivity, high resolution and real-time imaging, and can specifically detect biomarkers.

[0008] To solve the above technical problem, the present invention discloses an organic afterglow probe, and the probe is a nanoparticle formed by the self-assembly of squarylium dye and PEG liposome;

[0009] Among them, the structural formula of the squarylium dye is:

[0010] The present invention also discloses a preparation method of the above-mentioned organic long afterglow probe, and the specific steps are as follows:

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

[0012] Step 2: Self-assemble the squarylium dye prepared in Step 1 and PEG liposome in ultrapure water to obtain the organic long afterglow probe.

[0013] Among them, the mass ratio of (E)-4-2-(1-methyl-1H-pyrrol-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-pyrrol-2-yl)vinyl)pyridine to 3-N,N-(dibutylamino)phenyl, 4-hydroxy-3-cyclobutene-1,2-dione is 0.6:1.

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

[0016] Among them, the total mass of (E)-4-2-(1-methyl-1H-pyrrol-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 is (5-7) mg:1 mL;

[0017] Preferably, the total mass of (E)-4-2-(1-methyl-1H-pyrrol-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 is 6 mg: 1 mL.

[0018] Among them, for the heating under reflux, the specific conditions are: refluxing for 8 - 12 h at 80 - 100 °C;

[0019] Preferably, for the heating under reflux, the specific conditions are refluxing for 10 h at 90 °C.

[0020] Among them, the specific steps for purification are: after the heating under reflux is completed, the reaction solution is rotary evaporated to remove most of the solvent, petroleum ether is added and precipitation gives the crude product, after filtration separation, the crude product is then dissolved in chloroform, and the squarylium dye is obtained through column chromatography separation and purification.

[0021] Among them, the PEG liposome is DSPE-PEG; the molecular weight of DSPE-PEG is 2000.

[0022] Among them, the mass ratio of the squarylium dye to DSPE-PEG is 1: (3 - 5);

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

[0024] Among them, for the self-assembly, the specific conditions are: stirring at room temperature for 1 - 3 h;

[0025] Preferably, for the self-assembly, the specific conditions are: stirring at room temperature for 2 h.

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

[0027] Specifically, in some embodiments of the present invention, a self-assembled organic long afterglow probe is prepared by the above preparation method. By testing the ultraviolet absorption spectrum, fluorescence emission spectrum, in vitro reactive oxygen species generation level, in vitro singlet oxygen generation level and afterglow imaging of the organic long afterglow probe, it is shown that the organic long afterglow probe provided by the present invention has good in vitro reactive oxygen species generation level and in vitro singlet oxygen generation level, and has long afterglow luminescence performance, demonstrating the application prospect of the organic long afterglow probe in bioimaging.

[0028] Beneficial effects:

[0029] The preparation method of the self-assembled organic long afterglow probe of the present invention is simple, the conditions are mild, the raw materials are cheap 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 of the present invention has excellent afterglow luminescence performance, with strong afterglow luminescence intensity, long half-life, and potential for clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The following further specific description of the present invention will be made in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0033] Figure 1 It is the molecular weight characterization of the high-resolution mass spectrum of the squarylium dye (Sq) prepared in the embodiment of the present invention;

[0034] Figure 2 It is the transmission electron microscope characterization of the self-assembled organic long afterglow probe in the embodiment of the present invention;

[0035] Figure 3 It is the ultraviolet absorption and fluorescence emission spectra of the self-assembled organic long afterglow probe in the embodiment of the present invention, where A is the ultraviolet absorption spectrum and B is the fluorescence emission spectrum;

[0036] Figure 4 It is the characterization of the in vitro reactive oxygen species generation level of the self-assembled organic long afterglow probe in the embodiment of the present invention, where A is the in vitro reactive oxygen species generation level of the ultrapure water group and B is the in vitro reactive oxygen species generation level of the self-assembled organic long afterglow probe group;

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

[0038] Figure 6 It is the afterglow imaging of the self-assembled organic long afterglow probe in the embodiment of the present invention at different concentrations;

[0039] Figure 7 It is the afterglow imaging of the self-assembled organic long afterglow probe in the embodiment of the present invention at different light illumination durations;

[0040] Figure 8 It is the afterglow imaging of the self-assembled organic long afterglow probe in the embodiment of the present invention at different oxygen contents;

[0041] Figure 9 It is the half-life characterization of the self-assembled organic long afterglow probe in the embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0042] In the following examples, the experimental methods are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial sources unless otherwise specified.

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

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

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

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

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

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

[0049] (2) Cool the reaction mixture, rotary evaporate to remove the solvent, 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) Purify by column chromatography, and the eluent ratio is: ethyl acetate: n-hexane = 1:20 to obtain the product N,N-dibutylaniline;

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

[0053] (6) After the reaction mixture is cooled, pour it into 500 mL of ice water, extract with dichloromethane, collect the organic layer, and rotary evaporate to remove the solvent;

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

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

[0056] Example 1: Preparation of Self-Assembled Organic Long Afterglow Probes

[0057] The synthesis route of squarylium dye (Sq) in this example 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-pyrrol-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, and then add 40 mL of n-butanol and 40 mL of benzene respectively. Heat under reflux at 90 °C for 10 h;

[0061] (2) Rotary evaporate to remove most of the solvent, add 50 mL of petroleum ether to the reaction flask, and the crude product will precipitate at the bottom of the flask;

[0062] (3) Filter the solid product by suction, add it to chloroform, and separate and purify it by column chromatography. The eluent ratio is: dichloromethane: methanol = 10:1 to obtain the product squarylium dye;

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

[0064] Figure 1 is the mass spectrum of squarylium dye, which is obtained by dissolving it in dichloromethane and testing on a high-resolution mass spectrometer. The test results show that the molecular weight is 468.2632, which is consistent with the actual molecular weight.

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

[0066] Example 2: Spectral Test of Self-Assembled Organic Long Afterglow Probes

[0067] Combined with Figure 3 , the basic spectral characterization of the self-assembled organic long afterglow probe is introduced. The specific test steps are as follows:

[0068] The self-assembled organic long afterglow probes were respectively configured into aqueous solutions with concentrations of 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL. 3 mL of each solution was taken and added to a quartz cuvette, and its ultraviolet absorption spectrum was measured on an ultraviolet-visible spectrophotometer. The test results ( Figure 3 A) showed that the position of its ultraviolet absorption peak was at 670 nm, and with the increase of the concentration gradient, the ultraviolet absorption intensity also increased in a gradient manner; then, 3 mL of the samples with concentrations of 20 μg / mL and 40 μg / mL were respectively taken and added to a quartz cuvette, and their fluorescence emission spectra were measured on a fluorescence spectrophotometer. The test results ( Figure 3 B) showed that the position of its fluorescence emission peak was at 680 nm, and with the increase of the concentration gradient, the fluorescence emission intensity also increased in a gradient manner.

[0069] Example 3: Measurement of the generation level of reactive oxygen species (ROS)

[0070] Combined with Figure 4 and Figure 5 , the in vitro singlet oxygen generation level of the self-assembled organic long afterglow probe is introduced. The specific test steps are as follows:

[0071] (1) The self-assembled organic long afterglow probe was configured into an aqueous solution with a concentration of 20 μg / mL. 3 mL of the solution was taken and added to a quartz cuvette, and then 10 μL of the singlet oxygen indicator ABDA with a concentration of 10 μM was added. The ultraviolet absorption intensity was measured under dark conditions, and then it was irradiated intensively with a xenon lamp. The power density of the xenon lamp was 0.7 W / cm 2 , the light irradiation duration was 1 min, for a total of 5 min. The ultraviolet absorption intensity was measured once every 1 min for a total of 5 times, and the whole test was carried out under dark conditions. To exclude the influence of the singlet oxygen indicator ABDA itself, under the condition that other conditions remained unchanged, 10 μL of the singlet oxygen indicator ABDA with a concentration of 10 μM was added to 3 mL of ultrapure water for the same test.

[0072] The 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) showed that in the presence of the self-assembled organic long afterglow probe, with the increase of the light irradiation time, Figure 4 the ultraviolet absorption intensity at 400 nm in B decreased, while Figure 4 the ultraviolet absorption intensity of the ultrapure water group in A completely overlapped at different times and showed no downward trend. This was because the singlet oxygen generated by the self-assembled afterglow probe during light irradiation reacted with ABDA, resulting in a decrease in the concentration of ABDA and a decrease in the absorption intensity of ABDA at 400 nm, indicating that the self-assembled organic long afterglow probe could generate singlet oxygen well.

[0073] (2) The self-assembled organic long afterglow probe was configured into an aqueous solution with a concentration of 20 μg / mL. 3 mL was taken and added into a quartz cuvette. Subsequently, 10 μL of the singlet oxygen indicator SOSG with a concentration of 500 μM was added. The fluorescence emission intensity was measured under dark conditions. Then, it was irradiated intensively with a xenon lamp. The power density of the xenon lamp was 0.7 W / cm 2 , and the illumination duration was 1 min, for a total of 5 min. The fluorescence emission intensity was measured once every 1 min for a total of 5 times. The whole measurement process was carried out under dark conditions.

[0074] The test results ( Figure 5 ) showed that in the presence of the self-assembled organic long afterglow probe, as the illumination time increased, the fluorescence emission intensity at 520 nm increased. This was because singlet oxygen generated by the self-assembled afterglow probe during illumination interacted with SOSG to form SOSG-EP, resulting in the activation of fluorescence output, indicating that the self-assembled organic long afterglow probe could generate singlet oxygen well, and the fluorescence intensity increased uniformly with the increase of illumination time. It can be seen that the self-assembled afterglow probe could continuously generate singlet oxygen under continuous illumination conditions, indicating its good stability.

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

[0076] Combined with Figures 6 to 9 , the afterglow performance of the self-assembled organic long afterglow probe was introduced. The specific test steps were as follows:

[0077] (1) The self-assembled organic long afterglow probe was respectively configured into aqueous solutions with concentrations of 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL. 3 mL was taken from each solution and added into centrifuge tubes. They were irradiated intensively with a xenon lamp. The power density of the xenon lamp was 0.7 W / cm 2 , and the illumination time was 1 min. After the illumination ended, they were quickly placed into a small animal in vivo imaging system (IVIS LUMINA K Series Ⅲ). The exposure time was 60 s, the binning was 8, the aperture size was 1, and the filter was turned on to measure the afterglow intensity under different concentration conditions.

[0078] Figure 6 was the afterglow imaging of aqueous solutions of self-assembled organic long afterglow probes with different concentrations. It can be seen from the figure that the afterglow intensity of the self-assembled organic long afterglow probe increased with the increase of concentration.

[0079] (2) The self-assembled organic long afterglow probe was configured into an aqueous solution with a concentration of 50 μg / mL. 3 mL was taken and added into a centrifuge tube. It was irradiated intensively with a xenon lamp. The power density of the xenon lamp was 0.7 W / cm 2, with the light exposure times being 1 min, 2 min, 3 min, and 4 min respectively. After the light exposure, quickly place it into a small animal in vivo imaging system (IVIS LUMINA K Series Ⅲ), with the exposure time being 60 s, binning being 8, the aperture size being 1, and the filter being turned on to test the afterglow intensity at different light exposure times.

[0080] Figure 7 The afterglow imaging of the self-assembled organic long afterglow probe aqueous solution at 50 μg / mL under different light exposure times. It can be seen from the figure that the afterglow intensity of the self-assembled organic long afterglow probe increases with the increase of the light exposure time.

[0081] (3) Prepare an aqueous solution of the self-assembled organic long afterglow probe with a concentration of 50 μg / mL. Take 3 mL and add it to centrifuge tubes to prepare 4 samples. Sample 1 is the control group, and sample 2 is continuously purged with Ar for 10 min. 2 and sample 3 is continuously purged with N 2 and sample 4 is continuously purged with O 2 , then irradiate it intensively with a xenon lamp. The power density of the xenon lamp is 0.7 W / cm 2 , the light exposure time is 1 min. After the light exposure, quickly place it into a small animal in vivo imaging system (IVIS LUMINA K Series Ⅲ), with the exposure time being 60 s, binning being 8, the aperture size being 1, and the filter being turned on to test the afterglow intensity at different oxygen contents.

[0082] Figure 8 The afterglow imaging of the self-assembled organic long afterglow probe aqueous solution at 50 μg / mL under different oxygen contents. It can be seen from the figure that the afterglow intensity of the self-assembled organic long afterglow probe increases with the increase of the oxygen content.

[0083] (4) Prepare an aqueous solution of the self-assembled organic long afterglow probe with a concentration of 50 μg / mL. Take 3 mL and add it to a centrifuge tube, and irradiate it intensively with a xenon lamp. The power density of the xenon lamp is 0.7 W / cm 2 , the light exposure time is 1 min. After the light exposure, quickly place it into a small animal in vivo imaging system (IVIS LUMINA K Series Ⅲ), with the exposure time being 60 s, binning being 8, the aperture size being 1, and the filter being turned on. Test it 4 times continuously. Since the exposure time is 60 s, the imaging interval is 1 min each time to test the half-life of its afterglow.

[0084] Figure 9 The half-life diagram of the afterglow of the self-assembled organic long afterglow probe aqueous solution at 50 μg / mL after 1 min of light exposure. It can be seen from the figure that it still emits light continuously 4 min after the light exposure stops, indicating that the self-assembled afterglow probe has excellent afterglow luminescence performance.

[0085] The present invention provides an idea for an organic afterglow probe, its preparation method and application. There are many methods and ways to specifically implement this technical solution. The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

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

2. The method for preparing the organic afterglow probe according to claim 1, characterized in that: The specific steps are: Step 1: adding (E)-4-(2-(1-methyl-1H-pyrrol-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, heating under reflux and purifying to obtain the square acid cyanine dye; Step 2: Add the squaryl cyanine dye and PEG liposomes prepared in step 1 into 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 the (E)-4-(2-(1-methyl-1H-pyrrol-2-yl)vinyl)pyridine and 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 mass volume ratio of the total mass of the (E)-4-2-(1-methyl-1H-pyrrol-2-yl)vinyl)pyridine and 3-N,N-(dibutylamino)phenyl, 4-hydroxy-3-cyclobutene-1,2-dione to the mixed solvent is (5-7) mg:1 mL.

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

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

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

9. The preparation method according to claim 2, characterized in that: The specific conditions of the self-assembly are: stirring at room temperature for 1 to 3 hours.

10. Use of the organic afterglow probe according to claim 1 in biological imaging.

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