A coumarin derivative-based afterglow luminescent nano probe, a preparation method and application thereof
By using coumarin derivative-based afterglow luminescent nanoprobes, the challenge of highly specific imaging of peroxynitrite ions in deep tissues has been solved, enabling accurate diagnosis and treatment efficacy evaluation for ulcerative colitis and Parkinson's disease.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HANGZHOU INSTITUTE OF MEDICAL SCIENCES CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2026-03-25
- Publication Date
- 2026-07-31
AI Technical Summary
Existing technologies struggle to achieve highly specific afterglow imaging of peroxynitrite ions in deep tissues such as the abdominal cavity or brain, and lack precise detection methods for ulcerative colitis and Parkinson's disease.
A coumarin derivative-based afterglow luminescence nanoprobe was designed. It is formed by the self-assembly of coumarin derivative and amphiphilic polymer, and combined with the afterglow substrate MP which is responsive to peroxynitrite ions, to realize the activation and imaging of afterglow luminescence signal.
It enables precise visualization of lesions and evaluation of treatment effects in mouse models of ulcerative colitis and Parkinson's disease, with high sensitivity and low background interference in afterglow imaging.
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Figure CN122483784A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biosensor technology, specifically relating to a coumarin derivative-based afterglow luminescent nanoprobe, its preparation method, and its application. Background Technology
[0002] Afterglow luminescence is an optical imaging modality that does not require real-time excitation. Afterglow molecules with afterglow luminescence properties can store light energy and slowly release photons, effectively eliminating interference from tissue autofluorescence. Compared with traditional fluorescence imaging, this technology has the advantages of low background interference, high signal-to-noise ratio, and deep tissue penetration. Afterglow materials are mainly divided into inorganic and organic materials, among which organic materials have attracted much attention due to their excellent biocompatibility and tunable molecular structure. Organic afterglow materials can achieve afterglow luminescence lasting from several minutes to several days, thus they can be used for longitudinal monitoring of lesion imaging, biomarker detection, and image-guided therapy. Existing organic afterglow molecules mainly include thiophene semiconductor polymers, dihydroporphyrin, methylene blue, BODIPY derivatives, trianthracene derivatives, and luminescent systems based on dioxane structures. Among them, derivatives based on trianthracene and dioxane have been proven to be used in the development of next-generation ultrasound or X-ray excitation luminescence technologies, highlighting the cornerstone role of afterglow materials in the development of advanced imaging systems. Therefore, designing novel organic afterglow molecules is crucial for promoting the development of luminescence technology and expanding bioimaging applications.
[0003] peroxynitrite ions (ONOO) - ) is a typical reactive oxygen species (ROS / NOx) generated in situ from the reaction of nitric oxide and superoxide anion in vivo, playing a crucial role in numerous physiological processes. - Overexpression of ONOO is closely related to cancer, inflammatory bowel disease, and neurodegenerative diseases. Accurate detection of ONOO is crucial. - It aids in disease diagnosis and treatment monitoring. However, ONOO - The extremely short in vivo half-life (approximately 20 milliseconds) and low steady-state concentration (nanomolar level) place higher demands on detection technologies. Afterglow luminescence imaging, with its advantages of being non-invasive, highly sensitive, and requiring no external excitation, has become an effective imaging tool for visualizing biomarkers. Currently, ONOO... - Studies on afterglow imaging have mostly focused on tumor models, while highly specific probes for complex physiological environments such as deep tissues, abdominal cavities, or the brain still need to be developed. Summary of the Invention
[0004] To address the shortcomings of existing technologies, the present invention aims to provide a coumarin derivative-based afterglow luminescent nanoprobe, its preparation method, and its application. This afterglow luminescent nanoprobe can be used for the detection and imaging of peroxynitrite ions. By utilizing the changes in the afterglow luminescence signal during this process, precise visualization of lesions and evaluation of treatment effects can be achieved in mice with ulcerative colitis and Parkinson's disease.
[0005] To achieve the above objectives, the present invention provides the following technical solution:
[0006] In a first aspect, the present invention provides a coumarin derivative-based afterglow luminescent nanoprobe, which is self-assembled from coumarin derivatives and amphiphilic polymers.
[0007] The coumarin derivatives comprise a coumarin skeleton, and rigid and flexible coumarin derivatives, including any one of the following structural formulas:
[0008] .
[0009] In a preferred embodiment, the coumarin derivative is coumarin 545 (Formula VI).
[0010] The amphiphilic polymer is styrene-maleic anhydride copolymer (PSMA).
[0011] Secondly, the present invention also provides another afterglow luminescent nanoprobe based on coumarin derivatives, wherein the afterglow luminescent nanoprobe is self-assembled from coumarin derivatives, afterglow substrates, and amphiphilic polymers.
[0012] The structure of the afterglow substrate is shown below:
[0013] .
[0014] Among them, coumarin derivatives can generate singlet oxygen under light conditions; afterglow substrates contain peroxynitrite ions (ONOO). - It exhibits dual response characteristics of peroxynitrite ions and singlet oxygen; when peroxynitrite ions and singlet oxygen coexist, the afterglow emission signal of the afterglow substrate is activated, thereby achieving afterglow imaging.
[0015] In a preferred embodiment, the afterglow luminescent nanoprobe is based on a coumarin derivative and an afterglow substrate MP, with an amphiphilic polymer Pluronic F-127 as the surface modification layer.
[0016] Furthermore, the afterglow luminescent nanoprobe uses coumarin 545 and afterglow substrate MP as its core and the amphiphilic polymer Pluronic F-127 as its surface modification layer, and is named C545-MP NPs.
[0017] In a preferred embodiment, the afterglow luminescent nanoprobe has a uniform spherical structure.
[0018] Furthermore, the particle size of the afterglow luminescent nanoprobe is 1~1000 nanometers.
[0019] In a preferred embodiment, the emission wavelength range of the afterglow luminescent nanoprobe is 200~2000 nanometers.
[0020] Thirdly, the present invention also provides a method for preparing the afterglow luminescent nanoprobe, specifically as follows:
[0021] A dispersion of coumarin derivatives, afterglow substrate MP, and amphiphilic polymers was mixed and then added to water for ultrasonication to prepare afterglow luminescent nanoprobes based on coumarin derivatives.
[0022] Fourthly, the present invention also provides the application of the afterglow luminescent nanoprobe in the preparation of peroxynitrite ion detection reagent;
[0023] When peroxynitrite ions are present, the afterglow luminescent nanoprobe can emit light continuously after being excited by light.
[0024] In some specific implementations, the illumination time is 0s to 90s, and the illumination power ranges from 0.1 mW / cm². 2 ~100 mW / cm 2 .
[0025] In some specific implementations, the afterglow emission time ranges from 1 second to 24 hours; the luminous intensity of its long afterglow ranges from 10. 2 ~10 10 p / sec.
[0026] In some specific embodiments, the afterglow luminescent nanoprobe is mixed with a peroxynitrite ion solution, and under illumination, the change in the afterglow signal is used to detect and image the peroxynitrite ions.
[0027] The luminescence mechanism of the peroxynitrite ion-responsive afterglow luminescent nanoprobe prepared in this invention involves a synergistic process between peroxynitrite ions and photoactivation: First, peroxynitrite ions, acting as strong nucleophiles, attack electron-deficient boron atoms on the borate ester group of the afterglow substrate MP, initiating electron rearrangement and a rapid "de-cage" reaction to generate phenoxy anions. These phenoxy anions have a stronger electron-donating ability, making the afterglow substrate MP more easily oxidized by singlet oxygen. Subsequently, under light irradiation, the singlet oxygen generated by the afterglow initiator coumarin 545 oxidizes the methylene cyclobutane moiety of the afterglow substrate MP molecule, forming a dioxane intermediate. Subsequently, the O–O bond in the dioxane intermediate breaks, releasing energy to achieve afterglow luminescence.
[0028] The present invention has the following beneficial technical effects:
[0029] (1) This invention elucidates the afterglow mechanism of coumarin derivatives, and the afterglow emission efficiency is determined by the degree of molecular rigidity and the ability to generate singlet oxygen under photoexcitation.
[0030] (2) This invention designs a novel afterglow substrate MP that is responsive to peroxynitrite ions. The substrate uses borate ester as a selective responsive group and methylenecyclobutane as a singlet oxygen addition site. Subsequently, the MP molecule is co-assembled with coumarin 545, an afterglow initiator based on coumarin derivatives, to obtain afterglow luminescent nanoprobes C545-MP NPs.
[0031] (3) The present invention also provides the application of the coumarin derivative-based afterglow luminescent nanoprobe in in vivo imaging, which can accurately visualize lesions and evaluate treatment effects in mouse models of ulcerative colitis and Parkinson's disease. Attached Figure Description
[0032] Figure 1 This is a schematic diagram of the construction of the nanoparticles prepared in Example 1.
[0033] Figure 2 The image shows the afterglow emission pattern of the nanoparticles prepared in Example 1.
[0034] Figure 3 The image shows a TEM image of the C545 NPs prepared in Example 3.
[0035] Figure 4 The image shows the afterglow emission of C545 NPs prepared in Example 3 under different light power excitation.
[0036] Figure 5 The image shows the afterglow emission of C545 NPs prepared in Example 3 under different illumination times.
[0037] Figure 6 The image shows the persistent afterglow emission of the C545 NPs prepared in Example 3 after photoexcitation.
[0038] Figure 7 The afterglow emission band diagram of the C545 NPs prepared in Example 3.
[0039] Figure 8 This is a schematic diagram of the construction of the nanoprobe prepared in Example 4.
[0040] Figure 9 The image shows the afterglow emission pattern of C545-MP NPs prepared in Example 4 for detecting peroxynitrite ions.
[0041] Figure 10 The afterglow emission band diagram of C545-MP NPs prepared in Example 4.
[0042] Figure 11 This is a post-luminescence imaging image of the abdomen of mice with ulcerative colitis in Example 5 after injection of C545-MP NPs.
[0043] Figure 12 This is an afterglow luminescence imaging image of the brain of a Parkinson's mouse after injection of C545-MP NPs in Example 6. Detailed Implementation
[0044] Unless otherwise specified, the experimental methods described in the following examples are conventional methods, and the reagents and materials described are commercially available unless otherwise specified.
[0045] The technical solution of the present invention will be further illustrated below through specific experimental methods.
[0046] Example 1: Preparation of Nanoparticles with Afterglow Luminescence Properties
[0047] This embodiment is based on the synthesis of coumarin molecules with afterglow luminescence nanoparticles. The specific general synthesis method is as follows:
[0048] The synthesis was performed directly using a nano-coprecipitation method. First, 1 mL of THF stock solution containing coumarin derivatives of formulas 1-IV (25 μg) with afterglow luminescence properties and styrene-maleic anhydride copolymer (PSMA) (2.5 mg) was prepared. 9 mL of H2O was placed in a serum bottle, and the prepared tetrahydrofuran solution was quickly injected into the water and sonicated for 8 minutes. After removing THF by rotary evaporation, the solution was centrifuged (4000 rpm, 5 minutes), washed with deionized water, concentrated, and stored in the dark.
[0049] The construction of nanoparticles with afterglow luminescence properties in this embodiment is as follows: Figure 1 As shown.
[0050] Example 2: Property Testing of Nanoparticles with Afterglow Emission
[0051] The afterglow emission signal of the nanoparticles with afterglow emission properties obtained in Example 1 was collected using a small animal imaging instrument.
[0052] from Figure 2 It can be seen that, at the same mass concentration, the afterglow luminescence ability of coumarin 545-based nanoparticles (C545 NPs) is the strongest.
[0053] Example 3: Synthesis and Property Verification of C545 NPs
[0054] This embodiment is based on the study of the properties of C545 NPs, and the specific synthesis steps are as in Example 1.
[0055] Figure 3 The TEM image of C545 NPs obtained in Example 3 is shown below. Figure 3 It can be seen that the synthesized particles have a diameter of approximately 30 nanometers.
[0056] (1) Study on photoactivated luminescence properties of C545 NPs: The obtained C545 NPs were excited under different light power or different light exposure time, and then the afterglow luminescence imaging was tested by a small animal imaging instrument.
[0057] from Figure 4 and Figure 5 It can be seen that the afterglow luminescence intensity of C545 NPs increases with the extension of illumination time and with the increase of illumination power.
[0058] (2) Study on the luminescence time of C545 NPs: The obtained C545 NPs were illuminated for 30 seconds, and afterglow luminescence images were collected at different time points after the illumination stopped using a small animal imaging device.
[0059] from Figure 6 It can be seen that the emission signal of C545 NPs can still be collected 8 minutes after the photoexcitation stops.
[0060] (3) Study on the emission band of C545 NPs: The obtained C545 NPs were illuminated for 30 seconds, and then afterglow emission images were collected by small animal imaging device in different bands (510~570 nm, 570~650 nm, 690~700 nm, 800~820 nm).
[0061] from Figure 7 It can be seen that the afterglow emission of C545 NPs is mainly concentrated in the 550~650 nm range.
[0062] Example 4: Synthesis and Property Verification of C545-MP NPs
[0063] This embodiment is based on the synthesis and property study of C545-MP NPs. The specific synthesis method is as follows:
[0064] (1) Preparation of C545-MP NPs nanoparticles:
[0065] Nanoparticles based on C545-MP NPs were synthesized directly using a nano-coprecipitation method: First, 1 mL of THF stock solution containing coumarin 545 (100 μg), MP (100 μg) and the amphiphilic polymer Pluronic F-127 (2.5 mg) was prepared. 9 mL of H2O was placed in a serum bottle, and the prepared tetrahydrofuran solution was quickly injected into the water and sonicated for 8 minutes. After removing THF by rotary evaporation, the mixture was centrifuged (4000 rpm, 5 minutes), washed with deionized water, concentrated, and stored in the dark.
[0066] This embodiment describes the construction of a coumarin derivative-based afterglow luminescent nanoprobe as follows: Figure 8 As shown.
[0067] (2) Verification of the ability of C545-MP NPs to detect peroxynitrite ions by afterglow imaging: The obtained C545-MP NPs were mixed with peroxynitrite ion solutions of different concentrations (e.g., 0, 1, 2, 4, 8, 16 μM), and then illuminated for 60 seconds. After that, the afterglow emission imaging of C545-MP NPs was tested using a small animal imaging instrument.
[0068] Figure 9 Afterglow images of C545-MP NPs detecting different concentrations of peroxynitrite ions were obtained. Figure 9 It can be seen that the afterglow emission signal of C545-MP NPs increases with the increase of peroxynitrite ion concentration.
[0069] (3) Study on the emission band of C545-MP NPs: The obtained C545-MP NPs were mixed with peroxynitrite ion solution (16 μM) and irradiated for 30 seconds. After that, the afterglow emission images were collected by small animal imaging instrument in different bands (510~570 nm, 570~650 nm, 690~700 nm, 800~820 nm).
[0070] from Figure 10 It can be seen that the afterglow emission of C545-MP NPs is mainly concentrated in the 550~650 nm range.
[0071] Example 5: C545-MP NPs achieve accurate diagnosis and treatment efficacy evaluation of ulcerative colitis using afterglow luminescence imaging.
[0072] To establish an ulcerative colitis (UC) model, female BALB / c mice were randomly divided into four groups (n=3 per group): Group 1 (control group): Mice drank purified water for 6 days. Group 2 (DSS-induced UC model): Mice were treated with sodium dextran sulfate (DSS, 3% (w / v), orally) for 6 days. Group 3 (BR treatment): Mice were treated with DSS (3% (w / v), orally) for 6 days. From day 4 to day 6, bilirubin (BR, 3 mg / mL, 200 µL / mouse) was injected intraperitoneally. Group 4 (5-ASA treatment): Mice were treated with DSS (3% (w / v), orally) for 6 days. From day 4 to day 6, 5-aminosalicylic acid (5-ASA, 3 mg / mL, 200 µL / mouse) was injected intraperitoneally. On day 8, mice were anesthetized with 2% isoflurane oxygen and injected intraperitoneally with C545-MP NPs. Five minutes after injection, the mice abdomen was exposed to light (30 mW / cm²). 2 Immediately after 5 seconds, an image is taken using a small animal imaging device.
[0073] Figure 11 The image obtained in Example 5 is from... Figure 11 It can be seen that the afterglow intensity of the colon region of mice in the DSS-treated group was 2.14 times that of the control group. After treatment with BR and 5-ASA drugs, the afterglow signal in the colon region of mice in the DSS+BR treatment group and the DSS+5-ASA treatment group was lower than that in the DSS-treated group. This indicates that the afterglow emission of C545-MP NPs can be detected by ONOO. - To achieve the diagnosis and treatment efficacy evaluation of ulcerative colitis.
[0074] Example 6: C545-MP NPs enable early diagnosis and treatment efficacy assessment of Parkinson's disease using afterglow luminescence imaging.
[0075] To establish a Parkinson's disease model, male C57BL / 6 mice were randomly divided into three groups (n=3 per group) and treated as follows: Group 1 (wild-type): Mice were intraperitoneally injected with PBS for 7 consecutive days. Group 2 (MPTP-induced PD model): Mice were intraperitoneally injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 15 mg / kg / day) for 7 consecutive days. Group 3 (L-DOPA treatment): Mice received MPTP treatment for 7 days. On days 6 and 7, mice were additionally injected with levodopa (L-DOPA, 5 mg / kg / day). On day 8, mice were anesthetized with 2% isoflurane oxygen, and C545-MP NPs (500 µg / mL, 200 µL) were intravenously injected, followed by brain irradiation (23 mW / cm²). 2 Immediately after 30 seconds, use a small animal imaging device to take an image.
[0076] Figure 12The image obtained in Example 6 is from... Figure 12 It can be seen that the afterglow signal in the brain region of the MPTP group was the strongest, 1.45 times higher than that of the wild-type group. After L-DOPA treatment, the afterglow luminescence induced by MPTP in the brain region of PD mice was weakened. This indicates that C545-MPNPs are a reliable and sensitive afterglow luminescence probe that can be used to monitor ONOO in the brain of PD mice. - level.
[0077] This invention discovers that coumarin derivatives exhibit inherent afterglow emission after photoexcitation is stopped. The afterglow emission efficiency of coumarin is determined by both its molecular rigidity and its ability to generate singlet oxygen under photoexcitation. This is achieved by combining coumarin with ONOO containing borate esters and methylene cyclobutane units. - The binding of sensitive molecules can transform the afterglow luminescence of coumarin into imaging nanoprobes for disease responses. The coumarin backbone acts as a photoinitiator, while the MP moiety serves as an ONOO (on-off) mechanism. - The substrate and signal relay element for the response. The dual conditions of chemical activation and photoexcitation effectively suppress background interference and achieve a high signal-to-background ratio, enabling precise visualization of lesions and evaluation of treatment efficacy in mouse models of ulcerative colitis and Parkinson's disease.
[0078] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the embodiments and descriptions provided above, but is merely illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the scope of protection of the present invention.
Claims
1. A coumarin derivative-based afterglow luminescent nanoprobe, characterized in that, It is self-assembled from coumarin derivatives and amphiphilic polymers; The coumarin derivative includes any one of the following structural formulas: 。 2. The coumarin derivative-based afterglow luminescent nanoprobe according to claim 1, characterized in that, The coumarin derivative is coumarin 545 (Formula VI). The amphiphilic polymer is styrene-maleic anhydride copolymer (PSMA).
3. The coumarin derivative-based afterglow luminescent nanoprobe according to claim 1, characterized in that, The afterglow luminescent nanoprobe is self-assembled from coumarin derivatives, afterglow substrates, and amphiphilic polymers; The structure of the afterglow substrate is shown below: 。 4. The coumarin derivative-based afterglow luminescent nanoprobe according to claim 3, characterized in that, The afterglow luminescent nanoprobe has a coumarin derivative and afterglow substrate MP as its core, and an amphiphilic polymer Pluronic F-127 as its surface modification layer.
5. A coumarin derivative-based afterglow luminescent nanoprobe according to any one of claims 1 to 4, characterized in that, The afterglow luminescent nanoprobe has a uniform spherical structure; the particle size of the afterglow luminescent nanoprobe is 1~1000 nanometers.
6. A coumarin derivative-based afterglow luminescent nanoprobe according to any one of claims 1 to 4, characterized in that, The emission wavelength range of the afterglow luminescent nanoprobe is 200~2000 nanometers.
7. The method for preparing the afterglow luminescent nanoprobe according to claim 3 or 4, characterized in that, Specifically: A dispersion of coumarin derivatives, afterglow substrate MP, and amphiphilic polymers was mixed and then added to water for ultrasonication to prepare afterglow luminescent nanoprobes based on coumarin derivatives.
8. The application of the afterglow luminescent nanoprobe according to any one of claims 1 to 4 in the preparation of peroxynitrite ion detection reagent, characterized in that: When peroxynitrite ions are present, the afterglow luminescent nanoprobe can emit light continuously after being excited by light.
9. The application of the afterglow luminescent nanoprobe according to claim 8 in the preparation of peroxynitrite ion detection reagent, characterized in that, The illumination duration is 0s to 90s, and the illumination power ranges from 0.1 mW / cm². 2 ~100 mW / cm 2 .
10. The application of the afterglow luminescent nanoprobe according to claim 8 in the preparation of peroxynitrite ion detection reagent, characterized in that, The afterglow duration ranges from 1 second to 24 hours; the intensity of its long afterglow ranges from 10. 2 ~10 10 p / sec.