Chemiluminescent afterglow nanoprobe based on magnetic regulation and control as well as preparation method and application of chemiluminescent afterglow nanoprobe

Abstract

The invention relates to the technical field of biomedical nanomaterials, and particularly discloses a chemiluminescence afterglow nanoprobe based on magnetic regulation and control as well as a preparation method and application of the chemiluminescence afterglow nanoprobe. The nanoprobe takes zinc-doped ferroferric oxide (ZnFe2O4, ZF) magnetic nanoparticles with enzyme-like catalytic activity as an inner core, a chemiluminescent molecule QM-CF with a singlet oxygen response characteristic is wrapped, and the nanoprobe is formed by packaging an amphiphilic material DSPE-PEG-2000. According to the nanoprobe, under the action of an alternating magnetic field, a ZF core can be catalyzed to generate singlet oxygen, so that QM-CF molecules are efficiently activated to generate chemical afterglow luminescence with extremely high intensity and long half-life period; and meanwhile, the ZF kernel endows excellent T2 weighted magnetic resonance imaging capability and magnetocaloric effect. According to the invention, remote and controllable activation and enhancement of chemical afterglow luminescence through an external magnetic field are realized, a multi-mode imaging function integrating magnetic resonance imaging, magnetic afterglow optical imaging and thermal imaging is successfully integrated, and a novel tool is provided for precise diagnosis and imaging guide treatment of solid tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanomaterials and molecular imaging technology, specifically relating to a magnetically modulated chemiluminescent afterglow nanoprobe, its preparation method, and its application. Background Technology

[0002] Malignant tumors, especially solid tumors, are major diseases that seriously threaten human health. The internal microenvironment of these lesions is often complex, with conditions such as hypoxia and acidity, posing significant challenges to accurate diagnosis and treatment. Medical imaging technology is a key tool for tumor diagnosis and efficacy evaluation. Currently, commonly used single-modal imaging techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and optical imaging, each have their own advantages and limitations. For example, MRI has excellent soft tissue resolution and is radiation-free, but its sensitivity is relatively low; optical imaging has high sensitivity, but its tissue penetration depth is limited, and it is subject to interference from the autofluorescence of biological tissues.

[0003] In recent years, multimodal imaging probes, by integrating the advantages of two or more imaging modalities, can provide complementary diagnostic information, thereby achieving a more comprehensive and accurate characterization of tumors, and have become a hot topic in cutting-edge research. Among them, the strategy of combining the depth penetration and high resolution of MRI with the high sensitivity of optical imaging has attracted much attention.

[0004] Afterglow luminescence is a special optical phenomenon that refers to the continued emission of light from a material after the excitation source has been removed. Afterglow luminescence imaging eliminates the need for real-time excitation light, completely avoiding interference from tissue autofluorescence and excitation light scattering, significantly improving the signal-to-noise ratio and imaging depth, making it ideal for high-contrast imaging of deep tissues. However, existing afterglow materials mostly rely on external excitation sources such as light and X-rays for "charging," and their excitation processes often have limited penetration capabilities, high equipment dependence, and cannot achieve real-time, controllable regulation of the afterglow luminescence process.

[0005] Magnetic nanomaterials, such as iron(III) oxide (Fe3O4), have been widely used as T2 contrast agents in MRI due to their excellent biocompatibility and unique magnetic properties. Furthermore, some magnetic materials (such as zinc-doped iron(III) oxide (ZnFe2O4, ZF)) exhibit peroxidase-like activity, capable of catalyzing chemical reactions of endogenous hydrogen peroxide and other substances under alternating magnetic field (AMF) stimulation, controllably generating reactive oxygen species (ROS), especially singlet oxygen. 1 O2). At the same time, magnetic nanoparticles under the action of alternating magnetic fields can also generate magnetocaloric effects, which can be used for thermal imaging or thermotherapy.

[0006] On the other hand, chemiluminescent molecules are a class of functional molecules that emit light through chemical reactions without the need for external light source excitation. Among them, some chemiluminescent molecules (such as QM-CF) have extremely high response sensitivity and specificity to singlet oxygen, and can produce afterglow emission with extremely high intensity and long half-life when excited by singlet oxygen.

[0007] Currently, there are no reports of combining the enzymatic catalytic properties, MRI function, and magnetocaloric effect of magnetic nanomaterials with the singlet oxygen-responsive afterglow properties of chemiluminescent molecules to generate afterglow luminescence (magnetoafterglow) through alternating magnetic fields, thereby achieving multimodal imaging using MRI / magnetoafterglow / magneocaloric imaging. Developing such "magnetoafterglow" nanoprobes is of great significance for promoting the precision diagnosis and integrated treatment of solid tumors. Summary of the Invention

[0008] Technical problem: The purpose of this invention is to overcome the shortcomings of the prior art and provide a magnetically controlled chemiluminescent afterglow nanoprobe QM-CF@ZF NPs.

[0009] Technical solution:

[0010] A magnetically controlled chemiluminescent afterglow nanoprobe, characterized in that it comprises: a magnetic core, which is zinc-doped iron tetroxide nanoparticles (ZF); a chemiluminescent layer encapsulating the magnetic core, which contains QM-CF chemiluminescent molecules; and an amphiphilic polymer shell encapsulating the magnetic core and the chemiluminescent layer, which is composed of DSPE-PEG-2000.

[0011] The structure of QM-CF is shown below:

[0012]

[0013] The zinc-doped iron oxide nanoparticles were prepared according to the following steps: 5 mmol FeCl3·6H2O and 2.5 mmol ZnCl2 were dissolved in 40 mL of ethylene glycol solution, and then 3.6 g sodium acetate, MW=4000, and 1.0 g PEG were added; the solution was stirred for 30 minutes, and the reaction was carried out at 200 °C for 8 hours; the black product was washed, dried, and dispersed in ultrapure water to obtain zinc-doped iron oxide nanoparticles.

[0014] The nanoprobe is characterized in that the half-life of the afterglow emission generated after the QM-CF chemiluminescent molecule is activated is 2.8 hours.

[0015] The nanoprobe is characterized in that the average hydrated particle size of the nanoprobe is 150-180 nm.

[0016] The method for preparing the magnetically responsive afterglow nanoprobe includes the following steps:

[0017] DSPE-PEG-2000 was mixed with QM-CF solution and ZnFe2O4 solution. The mixed solution was sonicated, centrifuged, washed with water multiple times, and then redispersed in deionized water for later use, to obtain chemiluminescent afterglow nanoprobes QM-CF@ZF NPs.

[0018] A magnetically modulated chemiluminescent afterglow nanoprobe, characterized in that it comprises: a magnetic core, which is zinc-doped iron tetroxide nanoparticles (ZF); a chemiluminescent layer encapsulating the magnetic core, which contains QM-CF chemiluminescent molecules; and an amphiphilic polymer shell encapsulating the magnetic core and the chemiluminescent layer, which is composed of DSPE-PEG-2000.

[0019] This invention provides a method for preparing the above-mentioned magnetically responsive afterglow nanoprobe, comprising the following steps:

[0020] (1) Zinc-doped iron oxide nanoparticles were synthesized by hydrothermal method and QM-CF chemiluminescent molecules were synthesized by literature method;

[0021] (2) After the zinc-doped iron tetroxide nanoparticles and QM-CF molecules obtained in step (1) are mixed evenly, they are added to the DSPE-PEG-2000 aqueous solution.

[0022] (3) Sonicate the mixed solution obtained in step (2) at 20% working intensity in an ultrasonic cell disruptor for 10 minutes;

[0023] (4) Purify the dispersion obtained in step (3) to remove unencapsulated QM-CF molecules.

[0024] (5) Centrifuge the solution obtained in step (4) and wash it with water several times, then redisperse it in deionized water for later use; put a portion into a freeze dryer to freeze and form a product precipitate for later use.

[0025] This invention provides the application of the above-mentioned magnetic afterglow nanoprobe in the preparation of diagnostic reagents for multimodal imaging of solid tumors.

[0026] Preferably, the multimodal imaging includes magnetic resonance imaging, afterglow luminescence imaging, and thermal imaging.

[0027] Preferably, the application includes: activating the nanoprobe to generate singlet oxygen by an external alternating magnetic field, thereby exciting QM-CF molecules to produce afterglow luminescence; performing T2-weighted magnetic resonance imaging using the magnetic core of the nanoprobe; and performing thermal imaging using the magnetocaloric effect of the nanoprobe under an alternating magnetic field.

[0028] principle:

[0029] This invention uses QM-CF@ZF NPs as a nanoprobe, which utilizes the alternating magnetic field of zinc-doped iron oxide nanoparticles (ZF NPs) to remotely activate them to generate singlet oxygen, thereby triggering the internal QM-CF chemiluminescent molecules to generate a strong afterglow signal. It also has T2-weighted MRI contrast imaging and magnetocaloric imaging functions, realizing integrated multimodal imaging.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0031] 1. Innovation: The novel concept of "magnetic afterglow" is proposed for the first time, realizing the remote and controllable activation and enhancement of afterglow luminescence through a non-invasive, deep-penetrating alternating magnetic field, providing a new paradigm for optical imaging of deep tissues.

[0032] 2. Multimodal integration: A single nanoprobe integrates three functions: magnetic resonance imaging (T2 contrast imaging), afterglow optical imaging, and magnetocaloric imaging. This overcomes the limitations of a single imaging mode, enables information complementarity, and greatly improves the accuracy and reliability of tumor diagnosis.

[0033] 3. Excellent performance: The selected ZF material has a high singlet oxygen yield, excellent matching with QM-CF molecules, and produces high afterglow intensity (up to 100 times or more), long half-life (about 2.8 hours), wide imaging window, and extremely high signal-to-noise ratio.

[0034] 4. Controllability and safety: Imaging Activation Mode (AMF) is non-radioactive and can be started and stopped at any time, enabling real-time control of the imaging process and potentially higher safety.

[0035] 5. Strong scalability and potential for integrated diagnosis and treatment: The magnetic afterglow nanoprobe platform provided by this invention has excellent modifiability. Based on this platform, QM-CF molecules or PEG chains can be further functionalized, for example, by introducing chemical bonds that specifically respond to the tumor microenvironment, thereby achieving a more precise and intelligent response to tumors. In particular, "responsive magnetic afterglow nanoprobes" can be constructed by modifying the nanoprobes to respond to nitroreductase (NTR). The hypoxic microenvironment of solid tumors specifically upregulates NTR expression. Upon entering tumor tissue, the linker arm of this responsive probe is cleaved by high levels of NTR enzymes, thereby: enhancing tumor targeting and retention effect (EPR effect); achieving specific activation and enrichment of the probe within the tumor, further improving the signal-to-noise ratio; real-time monitoring of tumor hypoxia: NTR response efficiency is positively correlated with the degree of hypoxia, therefore the activation intensity and dynamics of the afterglow signal can serve as a direct indicator for monitoring tumor hypoxia levels; providing feedback and efficacy assessment for treatment: after interventions such as radiotherapy or hypoxia-activated chemotherapy, changes in the afterglow signal can be monitored to non-invasively and in real-time assess treatment effects (e.g., whether hypoxia has been relieved), providing a powerful tool for achieving precise "integrated diagnosis and treatment." Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the assembly and structure of QM-CF@ZF NPs prepared in Example 1 of the present invention;

[0037] Figure 2 The images show the ESR detection spectrum (A) and singlet oxygen production graph (B) of the ZF nanomaterials generated under an alternating magnetic field in Example 2 of this invention.

[0038] Figure 3 The following are characterization images of QM-CF@ZF NPs in Example 3 of this invention: A. Transmission electron microscope (TEM) image; B. Scanning electron microscope (SEM) image; C. Dynamic light scattering (DLS) particle size distribution map; DX-ray diffraction (XRD) pattern; E. Elemental analysis results of QM-CF@ZF NPs;

[0039] Figure 4 These are the afterglow decay curves and half-life fitting diagrams of QM-CF@ZF NPs and QM-CF@MF NPs in Embodiment 4 of the present invention.

[0040] Figure 5 This is a graph showing the afterglow luminescence intensity under different AMF parameters (A), different AMF treatment times (B), and different NP concentrations (C) in Example 5 of the present invention.

[0041] Figure 6These are the multimodal imaging images of QM-CF@ZF NPs in Embodiment 6 of the present invention (AB magnetic afterglow imaging results and semi-quantitative afterglow emission curves; CD magnetocaloric imaging and its temperature curves; EF magnetic resonance imaging and its imaging intensity curves). Detailed Implementation

[0042] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0043] Example 1: Preparation of QM-CF@ZF NPs

[0044] like Figure 1 As shown:

[0045] (1) Synthesis of ZF NPs: A modified hydrothermal method was used for synthesis. Specifically, FeCl3·6H2O (1.35 g, 5 mmol) and ZnCl2 (0.34 g, 2.5 mmol) were dissolved in ethylene glycol solution (40 mL), followed by the addition of sodium acetate (3.6 g) and PEG (MW = 4000, 1.0 g). The solution was stirred for 30 minutes and then transferred to a stainless steel high-pressure Teflon tube (100 mL). The reaction was carried out at 200 °C for 8 hours. The black product was repeatedly washed with ethanol, dried at 60 °C for 6 hours, and finally dispersed in ultrapure water to obtain a dispersion of zinc-doped iron tetroxide nanoparticles, abbreviated as ZF NPs.

[0046] (2) Synthesis of QM-CF: (Synthesized according to the patent method [China Pharmaceutical University. An enzyme-catalyzed chemiluminescent probe based on sulfatase and its preparation method and application: 202311520881.5 [P]. 2024-02-20]);

[0047]

[0048] (3) Dissolution of DSPE-PEG-2000: Dissolve 5 mg of DSPE-PEG-2000 in 5 mL of ultrapure water to obtain a DSPE-PEG-2000 solution of 1 mg / mL.

[0049] (4) Assembly of magnetic afterglow nanoprobes QM-CF@ZF NPs: QM-CF was dissolved in anhydrous DMSO solution (concentration 1 mg / mL). 200 μL of QM-CF solution (1 mg / mL) was slowly added dropwise to 2 mL of ZF (200 μg / mL) aqueous solution. Then, 200 μL of DSPE-PEG-2000 solution was added to the mixed solution. The resulting mixture was sonicated for 5 min at 20% working intensity (i.e., sonicated for 3 s, with a 5 s interval) in an ultrasonic cell disruptor. The entire operation was performed in the dark. After the system was naturally cooled to room temperature, it was stirred for 2 hours. The resulting solution was centrifuged and washed several times with water, then redispersed in deionized water and stored at 4℃ for later use. A portion was placed in a freeze dryer to freeze and form a product precipitate for later use.

[0050] Example 2: Determination of the ROS generation ability of ZF magnetic material

[0051] Electron spin resonance (ESR) spectroscopy was used with TEMP as the trapping agent to detect ZF after treatment in an alternating magnetic field (AMF, 300 kHz, 10 A) for 5 minutes. 1 O2 signal. Results are as follows: Figure 2 As shown in Figure A, ZF exhibits a strong characteristic signal of singlet oxygen. Further quantitative detection of singlet oxygen production was performed using DPBF ultraviolet absorption spectrometry. Specifically, DPBF (10 μM) solution was added to the ZF solution (50 μg / mL), and after treatment with AMF (with the same parameters) for different times, the absorbance change at 415 nm was measured. The results are as follows: Figure 2 As shown in Figure B, the characteristic absorption peak of DPBF in ZF decreases significantly, and its singlet oxygen yield is calculated to be 0.62, significantly higher than that of methylene blue MB. Surface ZF exhibits excellent yield under the action of AMF. 1 The O2 capability provides preliminary proof that QM-CF@ZF NPs possess magnetic afterglow luminescence capability.

[0052] Example 3: Characterization of QM-CF@ZF NPs

[0053] The QM-CF@ZF NPs prepared in Example 1 were characterized. The microstructure of the prepared QM-CF@ZF NPs was characterized using transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM). (TEM images are shown below.) Figure 3 (A and 3B) show that the nanoparticles have a complete structure, uniform particle size distribution, and nanoscale structure. DLS detection ( Figure 3 C) shows that its hydrated particle size is approximately 145.1 nm, indicating good dispersibility. XRD pattern ( Figure 3 D) The structure matches that of ZnFe2O4, confirming the successful synthesis of QM-CF@ZFNPs. Scanning electron microscopy and elemental analysis were performed on the QM-CF@ZF NPs, such as... Figure 3 As shown in Figure E, the elements of QM-CF are uniformly distributed on the ZF surface, further demonstrating the successful synthesis of QM-CF@ZF NPs.

[0054] Example 4: Evaluation of Magnetic Afterglow Performance and Determination of Half-Life

[0055] The QM-CF@ZF NPs (concentration 100 μg / mL) prepared in Example 1 were dispersed in PBS and treated with AMF (300 kHz, 10 A) for 5 minutes. Immediately afterward, the samples were placed in a small animal in vivo imaging system, and chemiluminescence signals were acquired at different time points, with a monitoring range of 0-270 min. Afterglow intensity-time curves were plotted. Figure 4 The afterglow half-life of QM-CF@ZF NPs was calculated to be approximately 2.85 hours, demonstrating that the ZF-core magnetic afterglow nanoprobes possess ultra-long afterglow luminescence properties. The model was then fitted using an exponential decay model.

[0056] Example 5: Factors Affecting Magnetic Afterglow Performance

[0057] The effects of different factors on the intensity of magnetic afterglow luminescence were systematically investigated. QM-CF@ZF NPs (100 μg / mL) were dispersed in PBS, and the following experiments were conducted: (A) At a fixed exposure time (5 min), the magnetic afterglow intensity was examined after exposure to different AMF (alternating magnetic field) powers (0, 2, 5, 8, 10, 12 A); (B) At a fixed AMF power (10 A), the magnetic afterglow intensity was examined after exposure to different AMF times (0, 1, 2, 5, 8 min); (C) At a fixed AMF parameter (10 A, 5 min), the magnetic afterglow intensity was examined at different NP concentrations (0, 10, 20, 50, 100 μg / mL). The results are as follows: Figure 5 As shown, the magnetic afterglow intensity trend increases with the increase of AMF power, interaction time and NPs concentration. The optimal interaction time of the AMF was determined to be 5 min and the optimal AMF power was 10 A. This also indicates that the magnetic afterglow emission process is controllable and adjustable, providing a basis for parameter optimization under different imaging requirements.

[0058] Example 6: Verification of Multimodal Imaging Capability

[0059] To verify the multimodal imaging capability of QM-CF@ZF NPs, magnetic resonance imaging of QM-CF@ZF NPs was performed. Figure 6 A) Experiments in magnetothermal imaging and in vivo afterglow imaging ( Figure 6B); First, 200 μL of QM-CF@ZF NPs solution (100 μg / mL) was directly injected into the tumor of a 4T1 cell breast cancer subcutaneous tumor. The mouse was then placed in a coil with an alternating magnetic field (10 A, 5 min) and immediately placed in a small animal imaging system for magnetic afterglow imaging at different time points (1-110 min). Figure 6 C and D) 200 μL QM-CF@ZF NPs solution (100 μg / mL) was subjected to AMF (10A, 10 min). The solution temperature and magnetocaloric imaging were recorded continuously for 10 min with the AMF on, and then the solution temperature and magnetocaloric imaging were recorded continuously for 10 min with the AMF off. Figure 6 E and F) Different concentrations of QM-CF@ZF NPs solutions were placed in the magnetic resonance imaging chamber to record their T2 imaging. The results are as follows: Figure 6 As shown, QM-CF@ZF NPs simultaneously possess in vivo long-term afterglow imaging, tumor-targeted magnetothermal imaging, and high-efficiency T2-weighted magnetic resonance imaging capabilities: afterglow imaging enables long-term tracking of tumors at the molecular level, magnetothermal imaging can monitor temperature changes in the tumor region under magnetic stimulation, and MRI can accurately locate the anatomical position of the tumor; the three synergistically validate the multimodal imaging potential of NPs, providing technical support for the integrated diagnosis of solid tumors such as breast cancer, including "localization-qualification-dynamic monitoring".

Claims

1. A magnetic regulation based chemiluminescence afterglow nanoprobes, characterized in that, It comprises a magnetic core, a chemiluminescence layer wrapped outside the magnetic core, and an amphiphilic polymer shell encapsulating the magnetic core and the chemiluminescence layer; wherein the magnetic core is composed of zinc-doped ferroferric oxide nanoparticles; the chemiluminescence layer is composed of QM-CF chemiluminescence molecules; and the amphiphilic polymer shell is composed of DSPE-PEG-2000. The structural formula of QM-CF is as follows:

2. The nanoprobe of claim 1, wherein, The zinc-doped ferroferric oxide nanoparticles are prepared by the following steps: 5 mmol FeCl3·6H2O and 2.5 mmol ZnCl2 are dissolved in 40 mL of ethylene glycol solution, then 3.6 g of sodium acetate and 1.0 g of PEG with MW = 4000 are added; the solution is stirred for 30 minutes, and the reaction is continued at 200℃ for 8 hours. The black product is washed, dried, and dispersed in ultrapure water to obtain zinc-doped ferroferric oxide nanoparticles.

3. The nanoprobe of claim 1, wherein, The average hydrated particle size of the nanoprobe is 150-180 nm; and the half-life of the afterglow light generated by the activated QM-CF chemiluminescence molecules is 2.8 hours.

4. The preparation method of the magnetic response-based afterglow nanoprobe according to any one of claims 1-3, comprising the following steps: The DSPE-PEG-2000 is mixed with the QM-CF solution and the zinc-doped ferroferric oxide nanoparticle dispersion, the mixed solution is ultrasonicated, the obtained solution is centrifuged and washed with water for multiple times, and then is redispersed in deionized water for standby, to obtain the nanoprobe.

5. The method of claim 4, wherein, The following steps are taken to achieve the above object (1) 5 mmol FeCl3·6H2O and 2.5 mmol ZnCl2 are dissolved in 40 mL of ethylene glycol solution, then 3.6 g of sodium acetate and 1.0 g of PEG with MW = 4000 are added; the solution is stirred for 30 minutes, and the reaction is continued at 200℃ for 8 hours; The black product is washed, dried, and dispersed in ultrapure water to obtain a zinc-doped ferroferric oxide nanoparticle dispersion; (2) The zinc-doped ferroferric oxide nanoparticles obtained in step (1) and the QM-CF molecules obtained in step (2) are mixed uniformly according to a mass molar ratio of 2:1, and then are added to a DSPE-PEG-2000 aqueous solution; (3) The mixed solution obtained in step (2) is ultrasonicated under the following conditions: ultrasonic time of 3 s, interval of 5 s, and 10 minutes; (4) The dispersion obtained in step (3) is purified to remove the unencapsulated QM-CF molecules; (5) The solution obtained in step (4) is centrifuged and washed with water for multiple times, and then is redispersed in deionized water for standby.

6. The method of claim 5, wherein, In step (1), the zinc-doped ferroferric oxide nanoparticles and the QM-CF are connected by the amphiphilic polymer DSPE-PEG-2000 through a nanoscale co-precipitation method.

7. The application of the magnetic regulation-based chemiluminescence afterglow nanoprobe according to any one of claims 1-3 in the preparation of a diagnostic preparation.

8. Use according to claim 7, characterized in that, The diagnostic preparation is used for imaging of solid tumors.

9. Use according to claim 7, characterized in that, The imaging is multi-modal imaging.

10. Use according to claim 9, characterized in that, The multi-modal imaging includes magnetic resonance imaging, magnetic afterglow imaging, and magnetic thermal imaging.

Images