Room-temperature phosphorescent nanoprobe based on high-active oxygen phosphorescent molecules as well as preparation method and application of room-temperature phosphorescent nanoprobe

Abstract

The invention discloses a room-temperature phosphorescent nanoprobe based on high-active oxygen phosphorescent molecules as well as a preparation method and application of the room-temperature phosphorescent nanoprobe. According to the probe, phosphorescent molecules with high active oxygen generation efficiency are encapsulated in polymer nano-microspheres through an emulsion polymerization method, and the compact structure of the nano-microspheres can effectively inhibit the quenching effect of water molecules on phosphorescence through a physical barrier; and macroscopic phosphorescence emission in an air environment is successfully realized. The probe has a high signal-to-noise ratio, excellent tissue penetrability and biocompatibility, is suitable for the fields of tumor imaging, intraoperative navigation, lymphatic system imaging and the like, and has a good clinical application prospect.

Description

A room-temperature phosphorescent nanoprobe based on highly reactive oxygen phosphorescent molecules, its preparation method, and its application. Technical Field

[0001] This invention relates to the field of organic phosphorescent nanomaterials, and in particular to a room-temperature phosphorescent nanoprobe constructed by emulsion polymerization of organic phosphorescent molecules with high reactive oxygen generation capacity, as well as its preparation method and application. Background Technology

[0002] Organic room-temperature phosphorescent (RTP) materials have broad application prospects in fields such as bioimaging, information encryption, and sensing due to their long-lifetime luminescence properties. However, existing RTP materials are susceptible to quenching by oxygen and water molecules in aqueous environments, resulting in low luminescence efficiency and poor stability, making it difficult to meet the requirements of biomedical imaging for high signal-to-noise ratios and deep tissue penetration. Currently, two strategies based on crystal engineering and supramolecular self-assembly are considered effective ways to achieve aqueous RTP emission. However, these methods generally suffer from complex preparation processes, high crystal dependence, and poor host-guest compatibility, which limits their widespread application in practice. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a room-temperature phosphorescent nanoprobe based on highly reactive oxygen phosphorescent molecules, its preparation method, and its applications. The probe of this invention can achieve long-lifetime, visible phosphorescence emission in air and aqueous environments. Furthermore, the probe possesses deep tissue penetration capability, high signal-to-noise ratio, and good biocompatibility, making it suitable for applications in tumor imaging, surgical navigation, and lymphatic system imaging.

[0004] In a first aspect, the present invention provides a method for preparing room temperature phosphorescent nanoprobes based on highly active oxygen phosphorescent molecules, which is achieved through the following technical solution.

[0005] A method for preparing room temperature phosphorescent nanoprobes based on highly active oxygen phosphorescent molecules includes the following steps: S1. Mixing organic phosphorescent molecules, hydrophobic monomers, and emulsifiers with water in a molar ratio of 1:(200-400):(0.8-1.2) and ultrasonically forming a pre-emulsion; S2. Adding an initiator and carrying out an emulsion polymerization reaction under an inert atmosphere to obtain phosphorescent nanospheres.

[0006] By employing the above technical solution, organic phosphorescent molecules with high reactive oxygen species (ROS) generation capabilities are encapsulated within polymer nanospheres via emulsion polymerization, forming a nanoprobe structure internally loaded with phosphorescent molecules possessing high ROS generation capabilities. The prepared probe exhibits phosphorescence lifetime greater than 100 ms under air exposure and aqueous phase conditions; furthermore, this probe can generate singlet oxygen (SO4) under ultraviolet light excitation. 1 O2) creates a localized low-oxygen microenvironment, thereby enhancing phosphorescence emission.

[0007] Furthermore, the organophosphorescent molecule is selected from benzo[carbazole] derivatives; the benzo[carbazole] derivative is 7H-dibenzo[carbazole] or 7H-benzo[C]carbazole.

[0008] The structural formulas of 7H-dibenzo[c]carbazole (DBCz, CAS: 194-59-2) and 7H-benzo[c]carbazole (BCz, CAS: 205-25-4) are as follows: .

[0009] Furthermore, the hydrophobic monomer is selected from methyl methacrylate or benzyl methacrylate.

[0010] Furthermore, the emulsifier is selected from F127 or hexadecyltrimethylammonium bromide.

[0011] Furthermore, in step S1, the concentrations of each component in the aqueous phase are: 0.8-1.2 mM for organic phosphorescent molecules, 240-360 mM for hydrophobic monomers, and 0.8-1.2 mM for emulsifiers.

[0012] Furthermore, the amount of initiator added is 0.45 wt%-0.9 wt% of the total monomer mass; the initiator is selected from potassium persulfate or ammonium persulfate.

[0013] Specifically, the concentration of the initiator in the aqueous phase is 7.0 mM-8.0 mM.

[0014] Furthermore, the inert atmosphere is nitrogen or argon.

[0015] Furthermore, the emulsion polymerization reaction conditions are: temperature 70-80℃, oil bath reaction for 2-4 hours.

[0016] Secondly, the present invention provides a room-temperature phosphorescent nanoprobe based on highly active oxygen phosphorescent molecules, which is achieved through the following technical solution.

[0017] A room-temperature phosphorescent nanoprobe based on highly reactive oxygen phosphorescent molecules prepared by the above preparation method.

[0018] In an air environment, phosphorescent molecules in the probe of this invention generate ROS under ultraviolet light excitation, consuming triplet oxygen. 3O2 creates a low-oxygen microenvironment inside the nanospheres; the polymer nanosphere structure effectively isolates the quenching effect of water molecules on phosphorescence and inhibits non-radiative transitions of triplet excitons; ultimately achieving long-lifetime phosphorescence emission (lifetime > 100 ms, up to 356.1 ms) in air and aqueous environments; and possessing high quantum yield (Φ > 10%) and high signal-to-noise ratio (SBR > 500, up to 556 in mouse subcutaneous imaging), it can penetrate 8 mm of chicken breast tissue, has low cytotoxicity, and is suitable for in vivo imaging.

[0019] Thirdly, the present invention provides an application of a room-temperature phosphorescent nanoprobe based on highly active oxygen phosphorescent molecules, which is achieved through the following technical solution.

[0020] Application of the above-mentioned room-temperature phosphorescent nanoprobe based on highly reactive oxygen phosphorescent molecules in the fields of bioimaging, intraoperative navigation, tumor labeling, and lymphatic imaging.

[0021] Specifically, in vivo imaging includes high-contrast phosphorescence imaging of subcutaneous tissue, lymph nodes, and tumor sites.

[0022] Specifically, intraoperative navigation involves marking tumor boundaries in real time using phosphorescent signals during tumor resection surgery.

[0023] Specifically, time-resolved imaging utilizes long-lifetime phosphorescent signals, combined with time-gating technology, to effectively filter out biological autofluorescence interference.

[0024] This application has the following beneficial effects.

[0025] (1) This invention combines ROS-generated phosphorescent molecules with emulsion polymerization technology to prepare phosphorescent nanoprobes that achieve long lifespan in air atmosphere and aqueous environment; (2) The phosphorescent nanoprobes of this invention can be widely used in imaging, navigation, treatment, anti-counterfeiting and other fields, and have good transformation prospects; (3) The preparation method of this invention is highly versatile and applicable to a variety of highly active oxygen phosphorescent molecules and polymer systems, breaking through the limitations of crystal or host-guest matching; (4) The preparation method of this invention is simple and scalable, and the emulsion polymerization process is mature and suitable for large-scale production. Attached Figure Description

[0026] Figure 1 shows photographs of the polymer emulsion of the present invention under 365 nm ultraviolet light irradiation and after irradiation (nitrogen atmosphere); Figure 2 shows the delayed spectrum of DBCz@PMMA of the present invention under nitrogen atmosphere; Figure 3 shows the time-resolved decay curve of DBCz@PMMA of the present invention under nitrogen atmosphere; Figure 4 shows the delayed spectrum of BCz@PMMA of the present invention under nitrogen atmosphere; Figure 5 shows the time-resolved decay curve of BCz@PMMA of the present invention under nitrogen atmosphere; Figure 6 shows the hydrodynamic diameter of DBCz@PMMA nanospheres measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM) images of the present invention; Figure 7 shows afterglow photographs of the polymer emulsion of the present invention under oxygen atmosphere, without ultraviolet light pre-excitation, during 365 nm ultraviolet light irradiation, and after irradiation; Figure 8 shows afterglow photographs of the polymer emulsion of the present invention under oxygen atmosphere, after 2 minutes of ultraviolet light pre-excitation, during 365 nm ultraviolet light irradiation, and after irradiation; Figure 9 shows the DBCz@PMMA of the present invention. Figure 10 shows the time-resolved attenuation curve of DBCz@PMMA in air atmosphere; Figure 11 shows the time-resolved spectrum of BCz@PMMA in air atmosphere; Figure 12 shows the time-resolved attenuation curve of BCz@PMMA in air atmosphere; Figure 13 shows the PL spectrum change of DCFDA under ultraviolet irradiation in the presence of DBCz@PMMA nanospheres; Figure 14 shows the PL spectrum change of DCFDA under ultraviolet irradiation in the presence of CE6@PMMA nanospheres; Figure 15 shows the slope of the PL emission peak intensity of DCFDA at 525 nm over time; Figure 16 shows the time-dependent phosphorescence images of various NPs obtained by pre-irradiation with ultraviolet lamp at 37 °C for 2 minutes using an IVIS instrument in bioluminescence mode (Ex: 365 nm); Figure 17 shows the phosphorescence images of DBCz@PMMA nanospheres and BCz@PMMA nanospheres covered with chicken tissue of different thicknesses after 2 minutes of ultraviolet pre-irradiation (Ex: 365 nm). Figure 18 shows the afterglow imaging obtained by injecting DBCz@PMMA nanospheres and BCz@PMMA nanospheres subcutaneously into mice according to the present invention; Figure 19 shows the tumor-targeting imaging of DBCz@PMMA nanoparticles according to the present invention; Figure 20 shows the tissue section of the tumor tissue according to the present invention; Figure 21 shows the lymphatic system imaging of the present invention using DBCz@PMMA nanospheres; Figure 22 is a schematic diagram of the experimental principle of the present invention. Detailed Implementation

[0027] The invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise specified, the experimental methods used in this invention are conventional methods, and the experimental equipment, materials, reagents, etc. used can all be purchased from relevant material sales companies.

[0028] Example 1: Preparation of DBCz@PMMA Nanoprobes 1. Raw material preparation: Phosphorescent molecule: DBCz (7H-dibenzocarbazole), with high ROS generation ability; Monomer: Methyl methacrylate (MMA); Emulsifier: F127 (Pluronic® F-127); Initiator: Potassium persulfate (KPS); Solvent: Deionized water.

[0029] 2. Preparation steps: (1) Add 1 mg DBCz and 480 μL MMA to an EP tube and sonicate for 5 minutes to fully dissolve the small molecule in MMA; (2) Add 240 μL of the above solution, 1 mL F127 (25 mg / mL) and 4 mL deionized water to a reaction flask and sonicate for 30 minutes to form a uniform pre-emulsion; (3) Add 1 mL KPS (2 mg / mL) and perform three freeze-pump-thaw deoxygenation under a nitrogen atmosphere; place the reaction system in a 75°C oil bath and stir magnetically for 4 hours. Cool to room temperature, collect the emulsion, and obtain DBCz@PMMA.

[0030] Example 2: Preparation of BCz@PMMA nanoprobe (1) Add 1 mg BCz and 480 μL MMA to an EP tube and sonicate for 5 minutes to fully dissolve the small molecule in MMA; (2) Add 240 μL of the above solution, 1 mL F127 (25 mg / mL) and 4 mL deionized water to a reaction flask and sonicate for 30 minutes to form a uniform pre-emulsion; (3) Add 1 mL KPS (2 mg / mL) and perform three freeze-pump-thaw deoxygenation under a nitrogen atmosphere; place the reaction system in a 75°C oil bath and stir magnetically for 4 hours. Cool to room temperature and collect the emulsion to obtain BCz@PMMA.

[0031] Preparation method of Ce6@PMMA in Comparative Example 1 (1) Add 1 mg Ce6 (dihydroporphyrin E6) and 480 μL MMA to an EP tube and sonicate for 5 minutes to fully dissolve the small molecule in MMA; (2) Add 240 μL of the above solution, 1 mL F127 (25 mg / mL) and 4 mL deionized water to a reaction flask and sonicate for 30 minutes to form a uniform pre-emulsion; (3) Add 1 mL KPS (2 mg / mL) and perform three freeze-pump-thaw deoxygenation under a nitrogen atmosphere; place the reaction system in a 75°C oil bath and stir magnetically for 4 hours. Cool to room temperature and collect the emulsion to obtain Ce6@PMMA.

[0032] Experimental Example 1: Photophysical Performance Testing of Nanoprobes 1. Performance of Phosphorescent Probes in Nitrogen Atmosphere: As shown in Figure 1-6, under 365 nm ultraviolet light excitation, the nanoprobes exhibited significant yellow-green phosphorescence. The afterglow time in a nitrogen atmosphere was 8-10 s. The delayed emission peak of DBCz@PMMA in a nitrogen atmosphere was 560 nm, and its emission intensity was 1.3 × 10⁻⁶. 4 The lifetime under nitrogen atmosphere is 647 ms, and the quantum yield is 11.17%. The delayed emission peak of BCz@PMMA under nitrogen atmosphere is at 530 nm, with an intensity of 0.8 × 10⁻⁶. 4 The lifetime under a nitrogen atmosphere is 880 ms. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) of DBCz@PMMA indicate an average particle size of 52.9 nm.

[0033] 2. Performance of the phosphorescent probe in air atmosphere: As shown in Figure 7-12, the nanoprobe does not emit phosphorescence when there is no pre-excitation under 365 nm ultraviolet light; after 2 minutes of pre-excitation under 365 nm ultraviolet light, the nanoprobe exhibits obvious yellow-green phosphorescence in air; the phosphorescence lifetime is 356 ms; after excitation is stopped, the afterglow is visible to the naked eye for more than 5 seconds.

[0034] 3. ROS generation capability verification: DCFDA was used as an indicator to detect the ability of the nanoprobe to generate ROS under 365 nm UV irradiation; the ability of the nanoprobe to generate reactive oxygen species under 365 nm UV irradiation was determined according to the following steps: (1) Prepare a 1.0 mM DCFDA stock solution with anhydrous DMSO; (2) Take 20 μL of the stock solution, add 180 μL of 0.010 M NaOH aqueous solution, mix well, and hydrolyze in the dark for 5 min to obtain 2.0 × 10 -4 M DCFDA working solution; (3) Mix 1800 μL of the sample to be tested with 200 μL of the DCFDA working solution to make the final concentration of DCFDA 2.0 × 10 -5 M; (4) Irradiate the mixture with 365 nm ultraviolet light, immediately record the 500-600 nm excitation spectrum, and monitor the fluorescence intensity change with an emission wavelength of 488 nm.

[0035] As shown in Figures 13-15, compared with the control group (Ce6@PMMA), the fluorescence enhancement factor induced by DBCz@PMMA was as high as 17, indicating that it has excellent ROS generation performance and enhances phosphorescence emission in the local hypoxic environment of nanospheres when pre-excited by a 365 nm UV lamp.

[0036] Experimental Example 2: Bioimaging Performance Test In Vitro Tissue Penetration Experiment: (1) Using a wavelength of 365 ± 5 nm and a power density of 10-20 mW / cm² -2 (1) Use an ultraviolet light source to vertically irradiate the polymer emulsion for 120 ± 10 s; (2) Sequentially cover the well plate with chicken breast slices of thickness 0 mm, 1 mm, 2 mm, 3 mm, 4 mm and 5 mm; (3) Immediately use the IVIS imaging system to collect signals, excite the filter at 420-440 nm, emit the filter at 560-600 nm, and expose for 1-60 s.

[0037] As shown in Figures 16-17, after pre-excitation of the nanoprobe, the afterglow of the image obtained by IVIS exceeded 400 seconds; after pre-excitation of the nanoprobe, imaging was performed on chicken breast tissue of different thicknesses, and the signal could penetrate 8 mm of tissue; when the nanoprobe was injected subcutaneously into the back of a mouse and immediately excited at 365 nm for 2 minutes, the phosphorescence signal recorded by the IVIS imaging system was as high as 556; Experimental Example 3: Tumor Targeting Imaging and Intraoperative Navigation 1. Tumor Enrichment: 200 μL of DBCz@PMMA nanoprobe was injected into a 4T1 breast cancer mouse model via the tail vein; 24 hours later, obvious phosphorescence signal was visible at the tumor site, with a signal-to-noise ratio of 63.99; indicating that the nanoprobe achieves passive tumor targeting through the EPR effect (Figure 18).

[0038] 2. Intraoperative navigation: During tumor resection surgery, the resection is performed based on the boundaries of the phosphorescent signal; after the resection is completed, the phosphorescent signal disappears, confirming that the tumor tissue has been completely removed; postoperative H&E staining of the tissue sections verifies that the resected area is tumor tissue (Figure 19-20).

[0039] Example 5: Lymphatic system imaging. 200 μL of DBCz@PMMA nanoprobe was injected into the footpad of a mouse, and lymphatic drainage was observed after excitation. The phosphorescent signal migrated along the lymphatic vessels and eventually accumulated in the inguinal lymph node (Figure 21). The signal-to-noise ratio reached 117, which is suitable for sentinel lymph node localization and metastasis monitoring.

[0040] The embodiments described herein are preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape, and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing room-temperature phosphorescent nanoprobes based on highly reactive oxygen phosphorescent molecules, characterized in that: Includes the following steps: S1. Mix organic phosphorescent molecules, hydrophobic monomers, and emulsifiers with water in a molar ratio of 1:(200-400):(0.8-1.2) and sonicate to form a pre-emulsion; S2. Add an initiator and carry out emulsion polymerization under an inert atmosphere to obtain phosphorescent nanospheres.

2. The method for preparing a room-temperature phosphorescent nanoprobe based on highly reactive oxygen phosphorescent molecules according to claim 1, characterized in that: The organic phosphorescent molecule is selected from benzo[carbazole] derivatives; the benzo[carbazole] derivative is 7H-dibenzo[carbazole] or 7H-benzo[C]carbazole.

3. The method for preparing a room-temperature phosphorescent nanoprobe based on highly reactive oxygen phosphorescent molecules according to claim 1, characterized in that: The hydrophobic monomer is selected from methyl methacrylate or benzyl methacrylate.

4. The method for preparing a room-temperature phosphorescent nanoprobe based on highly reactive oxygen phosphorescent molecules according to claim 1, characterized in that: The emulsifier is selected from F127 or hexadecyltrimethylammonium bromide.

5. The method for preparing a room-temperature phosphorescent nanoprobe based on highly reactive oxygen phosphorescent molecules according to claim 1, characterized in that: In step S1, the concentrations of each component in the aqueous phase are: organic phosphorescent molecules 0.8-1.2 mM, hydrophobic monomers 240-360 mM, and emulsifiers 0.8-1.2 mM.

6. The method for preparing a room-temperature phosphorescent nanoprobe based on highly reactive oxygen phosphorescent molecules according to claim 1, characterized in that: The amount of initiator added is 0.45 wt%-0.9 wt% of the total monomer mass; the initiator is potassium persulfate or ammonium persulfate.

7. The method for preparing a room-temperature phosphorescent nanoprobe based on highly reactive oxygen phosphorescent molecules according to claim 1, characterized in that: The inert atmosphere is nitrogen or argon.

8. The method for preparing a room-temperature phosphorescent nanoprobe based on highly reactive oxygen phosphorescent molecules according to claim 1, characterized in that: The emulsion polymerization reaction conditions are: temperature 70-80℃, oil bath reaction for 2-4 hours.

9. A room-temperature phosphorescent nanoprobe based on highly reactive oxygen phosphorescent molecules prepared by any of the preparation methods described in claims 1-8.

10. The application of the room-temperature phosphorescent nanoprobe based on highly reactive oxygen phosphorescent molecules as described in claim 9 in the fields of bioimaging, intraoperative navigation, tumor labeling, and lymphatic imaging.

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