A bimodal imaging nanoprobe material and a preparation method and application thereof

Abstract

This invention discloses a dual-modal imaging nanoprobe, its preparation method, and its application, belonging to the field of biomedical engineering and nanotechnology. The dual-modal imaging nanoprobe comprises lanthanide-doped upconversion nanocrystals, NAD(P)H sensitive molecular probes loaded on their surface, and ONOO... ‑ Sensitive molecular probes and glycosylated encapsulation layers enable the detection of intracellular energy metabolism marker NAD(P)H and M1 macrophage polarization-related nitrosation stress marker ONOO through near-infrared light-excited upconversion luminescence and multispectral photoacoustic dual-modal imaging. ‑ Simultaneous, highly sensitive, and highly specific detection. This invention can dynamically and non-invasively screen and evaluate the efficacy of treatment regimens combining calorie restriction mimics and immunotherapy drugs from two dimensions: energy metabolism and M1 phenotype levels, and predict treatment outcomes at an early stage, providing a powerful visualization tool for the precision development of cancer combination immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering and nanotechnology, specifically relating to a dual-modal imaging nanoprobe material, its preparation method, and its application. Background Technology

[0002] Tumor-associated macrophages (TAMs) play a crucial role in tumor progression and treatment resistance, and polarizing them from the tumor-promoting M2 phenotype to the tumor-killing M1 phenotype is an important strategy in cancer immunotherapy. Caloric restriction or its mimics (CRMs, such as 2-deoxy-D-glucose, 2-DG) can inhibit energy metabolism in cancer cells and may enhance the efficacy of immunotherapy. CR combination therapy is a treatment strategy that combines TAM-targeted immunotherapies (such as the TLR7 / 8 agonist R848 or the CSF-1R inhibitor PLX3397 / BLZ945) with energy metabolism inhibitors (CRMs, such as 2-DG). However, developing effective calorie restriction (CR) combined with immunotherapy faces several challenges, including: (i) a lack of visualization tools to simultaneously analyze the complex information generated by immunotherapy and energy metabolism during treatment; (ii) difficulty in understanding how energy metabolism inhibition regulates TAM-mediated immune responses; (iii) insufficient understanding of how novel CR combination therapies affect TAM phenotypes; and (iv) an unclear relationship between the dose ratio of immunotherapies to CRM and treatment efficacy.

[0003] Currently, traditional optical imaging techniques used for treatment assessment suffer from drawbacks such as invasiveness, discontinuity, lag, and limited tissue penetration depth. Although photoacoustic imaging and upconversion luminescence imaging each have their advantages, a tool that can achieve internal signal cross-referencing and simultaneously provide highly sensitive, spatiotemporally accurate dynamic imaging of energy metabolism and M1 phenotype-related nitrosation stress is still lacking.

[0004] Therefore, there is an urgent need in this field to develop a novel nanoprobe and imaging strategy that can perform non-invasive, real-time, and dynamic dual-dimensional screening and efficacy evaluation of CR combined with tumor immunotherapy from the two dimensions of energy metabolism and M1 phenotype level. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for simultaneously, with high sensitivity and high specificity, detecting intracellular nicotinamide adenine dinucleotide (NAD(P)H) and peroxynitrite (ONOO). -This invention discloses a near-infrared light-modulated multispectral upconversion-photoacoustic dual-modal imaging nanoprobe (named MEI-1) and its application in dual-dimensional screening of calorie restriction combined with cancer immunotherapy. Specifically, this invention provides a near-infrared light-modulated multispectral upconversion-photoacoustic dual-modal imaging nanoprobe and its application in simultaneously and dynamically monitoring the energy metabolism reprogramming and nitrosation stress levels of tumor-associated macrophages, particularly for screening and evaluating the efficacy of calorie restriction combined with cancer immunotherapy.

[0006] To address the aforementioned technical problems, this invention discloses a dual-modal imaging nanoprobe material, its preparation method, and its application. The specific technical solution is as follows: In a first aspect, the present invention provides a method for preparing a dual-modal imaging nanoprobe material, comprising the following steps: (1) Prepare glycosylated upconversion nanocrystals, wherein the upconversion nanocrystals are NaGdF4:Yb,Er,Tm@NaGdF4, which can generate multiple upconversion emission peaks under 980 nm near-infrared light excitation, especially the emission peaks at 660 nm and 800 nm; (2) The molecular probe and the glycosylated upconversion nanocrystals are mixed in a first solvent to prepare the dual-modality imaging nanoprobe material; wherein the molecular probe includes NADH / NADPH sensitive molecular probes and ONOO. - Sensitive molecular probe. In some embodiments of the present invention, the first solvent includes ethanol.

[0007] In step (1), the upconversion nanocrystals NaGdF4: Yb, Er, Tm@NaGdF4 are prepared by thermal decomposition synthesis, including the following steps: (a) The rare earth salt precursor is dissolved in methanol and mixed with oleic acid and 1-octadecene. The mixture is reacted at 150-160°C for 1-2 h to obtain a rare earth-oleic acid precursor complex. The rare earth salt precursor includes a combination of Gd(OAc)3·4H2O, Y(OAc)3·4H2O, Er(OAc)3·4H2O and Tm(OAc)3·4H2O, with a mixing molar ratio of 77.6:20:1.8:0.6. (b) Gd(OAc)3·4H2O was dissolved in methanol, mixed with oleic acid and 1-octadecene, and reacted at 150~160 °C for 1~2 h to obtain a yellow solution; (c) The rare earth-oleic acid precursor complex and the yellow solution are mixed with a methanol solution containing NaOH and NH4F for 20-40 min, and then kept at 300-310 °C for 1-2 hours to obtain the upconversion nanocrystals.

[0008] In some embodiments of the present invention, the preparation of the upconversion nanocrystals NaGdF4:Yb, Er, Tm@NaGdF4 includes the following steps: (a) The rare earth salt precursor is dissolved in methanol and mixed with oleic acid and 1-octadecene. The mixture is heated to 150°C and held for 1 h under an inert gas atmosphere to remove moisture and methanol, forming a homogeneous rare earth-oleic acid precursor complex. The amount of methanol used is calculated as 10 mL of methanol per mmol of rare earth salt precursor, the amount of oleic acid used is calculated as 7 mL of oleic acid per mmol of rare earth salt precursor, and the amount of 1-octadecene used is calculated as 15 mL of 1-octadecene per mmol of rare earth salt precursor.

[0009] (b) Gd(OAc)3·4H2O is dissolved in methanol, mixed with oleic acid and 1-octadecene, and reacted at 150 °C for 1-2 h to obtain a yellow solution; in some embodiments of the present invention, the amount of Gd(OAc)3·4H2O is the same as that in step (a), the amount of methanol is calculated as 5 mL of methanol per mmol of rare earth salt precursor in step (a), and the amount of oleic acid and 1-octadecene is the same as that in step (a).

[0010] (c) The rare earth-oleic acid precursor complex and the yellow solution are mixed with a methanol solution containing NaOH and NH4F for 30 min. This step introduces fluoride and sodium ions necessary for the formation of the NaGdF4 lattice. Providing sufficient fluoride and sodium ions in this step is crucial for the formation of NaGdF4. The mixture is then slowly heated to 100°C to remove low-boiling-point solvents such as methanol, and then rapidly heated to 305°C and held for 1.5 hours. This is the most critical step for nanocrystal nucleation and growth, determining the crystallinity, size, and phase of the nanocrystals. The most important aspect of this step is controlling the reaction temperature and time. After the reaction system is naturally cooled to room temperature, ethanol is added to precipitate the nanocrystals. The precipitates are obtained by centrifugation and repeated washing with ethanol. In some embodiments of the present invention, the amount of NaOH used is 2.5 mmol of NaOH per mmol of the rare earth acid precursor in step (a), and the amount of NH4F used is 4 mmol of NH4F per mmol of the rare earth acid precursor in step (a).

[0011] In some embodiments of the present invention, in step (c), before mixing the rare earth-oleic acid precursor complex and the yellow solution, the rare earth-oleic acid precursor complex is treated to obtain core nanoparticles before being mixed with the yellow solution. The core nanoparticles are prepared by the following method: the rare earth-oleic acid precursor complex is mixed with a methanol solution containing NaOH and NH4F for 30 min, the mixture is slowly heated to 100 °C to remove low-boiling-point solvents such as methanol, and then the temperature is rapidly raised to 305 °C and held for 1.5 hours to obtain the core nanoparticles.

[0012] In step (1), the glycosylation modification is a polyethyleneimine-polyethylene glycol-mannose copolymer modification.

[0013] In step (1), the glycosylated upconversion nanocrystals (PEI-PEG-Man-LUNCs) are prepared as follows: Upconversion nanocrystals are mixed with a polyethyleneimine solution to obtain polyethyleneimine-modified upconversion nanocrystals; then, the polyethyleneimine-modified upconversion nanocrystals and mannose-polyethylene glycol-carboxylic acid are mixed in a second solvent to obtain glycosylated upconversion nanocrystals. The mannose-polyethylene glycol-carboxylic acid (Man-PEG-COOH) is prepared as follows: Amino-polyethylene glycol-carboxylic acid and D-mannopyranoyl isothiocyanate are covalently coupled. The second solvent is an EtOH / H2O (1:1 v / v) solution; in some embodiments of the present invention, the second solvent is an EtOH / H2O (1:1 v / v) solution containing EDC and NHS.

[0014] In step (2), the NADH / NADPH sensitive molecular probe includes Glu-RB, whose structural formula is shown in Formula I; the ONOO - The sensitive molecular probe includes iNOS-CY, whose structural formula is shown in Formula II. The Glu-RB comprises a hybrid rhodamine spironolactam bridged with 3-substituted methylquinoline and a polyene. In the presence of NAD(P)H, its quinoline group undergoes a specific hydrogen transfer reaction, transforming into a dihydropyridine structure and initiating ring-opening of the spironolactam, resulting in enhanced photoacoustic signal at 710 nm, while simultaneously weakening the upconversion luminescence signal at 660 nm through fluorescence resonance energy transfer. The iNOS-CY comprises a polyacetylene linker. In the presence of ONOO⁻, its polyacetylene linker undergoes irreversible oxidative cleavage, leading to weakened photoacoustic signal at 800 nm, while simultaneously restoring the upconversion luminescence signal at 800 nm (and resulting in enhanced upconversion luminescence signal near 800 nm).

[0015] Formula I, Formula II.

[0016] In some embodiments of the present invention, in step (2), the molecular probe is a molecular probe with a terminal carboxyl group.

[0017] In step (2), the NADH / NADPH sensitive molecular probe and ONOO - The mass ratio of the sensitive molecular probe is 3 to 5:1, preferably 4:1.

[0018] In step (2), the amount of glycosylated upconversion nanocrystals added is based on the mass of the polyethyleneimine-modified upconversion nanocrystals (PEI-LUNCs), with each mg of glycosylated upconversion nanocrystals mixed with 1-2 mg of molecular probe. In some embodiments of the present invention, the amount of glycosylated upconversion nanocrystals added is based on the mass of the PEI-LUNCs, with each mg of glycosylated upconversion nanocrystals mixed with 1.5 mg of molecular probe.

[0019] Secondly, the present invention provides a dual-modal imaging nanoprobe material prepared by the preparation method described in the first aspect. In some embodiments of the present invention, the dual-modal imaging nanoprobe material includes a core, a molecular probe loaded on the core, and an encapsulation layer; the core is upconversion nanocrystals NaGdF4:Yb, Er, Tm@NaGdF4, and the molecular probe is an NAD(P)H sensitive probe (Glu-RB) and an ONOO. - The sensitive probe (iNOS-CY) has an encapsulation layer that encapsulates the core and molecular probe, preferably a glycosylated graft copolymer, specifically polyethyleneimine-polyethylene glycol-mannose. This encapsulation layer not only improves the water solubility and stability of the nanoprobe and prevents the molecular probe from dissociating, but its exposed mannose residues can also specifically bind to the mannose receptors overexpressed on the TAM surface, enhancing the uptake of the nanoprobe by the TAM.

[0020] Thirdly, the present invention provides the dual-modal imaging nanoprobe material described in the second aspect for simultaneous in vivo or in vitro imaging of NADH / NADPH and ONOO. - Applications in [the context of the text].

[0021] Fourthly, the present invention provides the use of the dual-modal imaging nanoprobe material described in the second aspect in the preparation of reagents for screening or evaluating the efficacy of calorie-limiting combined immunotherapy for cancer, the use including: (1) Using the nanoprobe, the energy metabolism level (reflected by NAD(P)H signal) and M1 macrophage polarization level (reflected by ONOO) in the tumor region or immune cells can be monitored simultaneously by multispectral photoacoustic imaging and / or upconversion luminescence imaging. - (Signal reflection).

[0022] (2) Based on the monitored NAD(P)H signal and ONOO - Changes in signals and ratios (such as R-PAP / PAE or R-ULP / ULE) can be used to screen effective combination therapies for complete remission (CR) and immunotherapy, assess treatment efficacy, predict treatment prognosis, or guide patient stratification.

[0023] (3) The calorie restriction combined with cancer immunotherapy regimen includes calorie restriction mimics (such as 2-DG) and immunotherapy drugs, including but not limited to Toll-like receptor 7 / 8 agonists (such as R848) or colony-stimulating factor 1 receptor inhibitors (such as PLX3397, BLZ945).

[0024] Beneficial effects: This invention creatively designs and constructs a highly integrated multifunctional nanoplatform (MEI-1) and applies it to solve the cutting-edge biomedical challenge of dynamic, non-invasive, in vivo screening-based calorie-restricted combined immunotherapy. This integration is not a simple assembly; its technical difficulty and advantages are significant. First, combining upconversion nanocrystals with molecular probes to construct the MEI-1 probe for in vivo dual-modal imaging requires complex, multi-step processes, each requiring precise control of reaction conditions to ensure successful execution. Furthermore, the construction process is not a simple mixing but rather the creation of a highly integrated multifunctional nanoplatform. The technical challenge lies in ensuring that the functional independence and synergy of each component are maintained after integration, achieving dual-modal, dual-channel signal decoupling. Overcoming these technical difficulties brings breakthrough advantages that cannot be achieved by a single technology. Traditional methods can only detect signals in a relatively single dimension, making it difficult to dynamically correlate two aspects simultaneously at the same in vivo site. The MEI-1 platform of this invention possesses innovative dynamic, non-invasive, in vivo, dual-dimensional assessment capabilities, enabling real-time monitoring of dynamic changes in tumor microenvironment energy metabolism and immune status during treatment. Furthermore, the two signals corroborate each other, enabling rapid prediction and comparison of the long-term effects of multiple combination therapy regimens in the early stages of treatment based on objective imaging indicators, thereby significantly reducing the cycle and cost of preclinical drug screening. Specific beneficial effects are as follows: (1) Dual-channel, dual-modal imaging: MEI-1 nanoparticles for the first time integrate NAD(P)H and ONOO -The detection is integrated into a nanoplatform, and the photoacoustic signals at different wavelengths (710 nm and 800 nm) are decoupled to achieve simultaneous imaging at both the energy metabolism and M1 phenotype levels. The photoacoustic imaging and upconversion luminescence imaging modalities can be internally cross-validated, improving the accuracy and reliability of the detection and enabling synchronous, non-interfering monitoring of macrophage polarization and energy metabolism in the tumor microenvironment. (2) High sensitivity and specificity: The probe is sensitive to NAD(P)H and ONOO - The detection limits reach the nanomolar level and have little interference with other reactive oxygen species / nitrogen species, enabling specific detection under complex physiological environments; (3) Excellent penetration depth and spatial resolution: Existing detection methods are generally in vitro detection methods, requiring post-sampling processing and unable to perform dynamic monitoring. Furthermore, traditional optical imaging methods have shallow tissue penetration and are easily affected by autofluorescence interference. This invention combines the deep tissue penetration capability (centimeter level) and micrometer-level spatial resolution of mPAT with the background-free fluorescence interference characteristics of upconversion luminescence, making it suitable for in vivo imaging of subcutaneous solid tumors and achieving high-resolution, three-dimensional imaging of deep tumors. It can achieve non-invasive, real-time, longitudinal monitoring of the dynamic changes of the tumor microenvironment during treatment, greatly improving the efficiency and information content of preclinical research. (4) Dynamic screening and prognosis prediction: Existing technologies for evaluating the efficacy of combination therapy usually rely on endpoint indicators such as tumor volume and survival time, lacking real-time and quantitative process monitoring. The imaging strategy provided by this invention can non-invasively and dynamically monitor the dynamic correlation between TAM energy metabolism and polarization during CR combined immunotherapy, and use the imaging signal ratio (such as R-PA~P~ / PA~E~) to predict tumor volume changes and treatment outcomes in the early stage, so that the efficacy of different drug combinations can be compared intuitively and quantitatively, providing guidance for personalized medicine and precision medicine. The method shown in this invention can not only determine the effectiveness of combination therapy, but also screen out the optimal CR and immunotherapy combination scheme through this value, realizing a leap from qualitative to quantitative. (5) Promote therapy development: This technology provides a powerful visualization tool for the rapid screening and evaluation of novel CR combined with immunotherapy, and is expected to accelerate the clinical translation process of "repurposing old drugs". Attached Figure Description

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0026] Figure 1 This is the synthetic route diagram for the molecular probe Glu-RB, where 1, 2, and 3 represent intermediate 1, intermediate 2, and intermediate 3, respectively.

[0027] Figure 2 The diagram shows the synthetic route of the molecular probe iNOS-CY, where 4 and 5 represent compounds 4 and 5, respectively.

[0028] Figure 3 This is a schematic diagram of the synthetic route for the nanoprobe MEI-1 (LUNCs@PEI-PEG-Man / Glu-RB / iNOS-CY).

[0029] Figure 4 The image shows the in vitro optical sensing performance evaluation of MEI-1 nanoprobes. (a) compares the UV-Vis absorption spectra; (b) shows the PA signal intensity versus NADH and ONOO. - (c) shows the normalized PAf / PAi intensities of MEI-1 at 710 and 800 nm, where: ONOO - : peroxynitrite, HOCl: hypochlorous acid, H2O2: hydrogen peroxide, NO: nitric oxide, HNO: nitroglycerin, O2·-: superoxide anion, OH·: hydroxyl radical, NAD(P)H: nicotinamide adenine dinucleotide (phosphate) (reduced form), Cys: cysteine, GSH: glutathione, AS: ascorbic acid, DTT: dithiothreitol, Glu: glucose, Pyr: pyruvate, ATP: adenosine triphosphate; (d) shows the PA and UL signals of MEI-1 in tissues.

[0030] Figure 5 The results of MEI-1 assessment of the relationship between macrophage energy metabolism and polarization are shown in the figure. Among them, (a) is the upconversion luminescence and photoacoustic image after MEI-1 treatment, (b) is the oxygen consumption rate and extracellular acidification rate under different polarization conditions, (c) is the photoacoustic image after MEI-1 treatment, and (d) is the UL (660 and 800 nm) intensity and normalized PA (R-PA, 710 and 800 nm) intensity after treatment.

[0031] Figure 6 A diagram illustrating the MEI-1 evaluation of the CR combined with R848 immunotherapy regimen.

[0032] Figure 7 Image of UL for MEI-1 evaluation of CR combined with R848 immunotherapy.

[0033] Figure 8 A diagram illustrating a combined immunotherapy regimen for dynamic screening of CR in tumor-bearing mice.

[0034] Figure 9 The results of UL imaging during TLR7 / 8 targeted therapy.

[0035] Figure 10 The results of UL imaging during CSF-1R targeted therapy. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation of the invention in any way.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0038] In the following examples, unless otherwise specified, the PBS buffer is 10 mM, pH 7.4.

[0039] Example 1 Synthesis of core-shell structured upconversion nanocrystals Core-shell structured NaGdF4 upconversion nanocrystals (LUNCs) were synthesized via thermal decomposition: 20% Yb, 1.8% Er, 0.6% Tm@NaGdF4. The specific method is as follows: (1) Preparation of core nanoparticles (NaGdF4: Yb / Er / Tm): Gd(OAc)3·4H2O (0.776 mmol), Y(OAc)3·4H2O (0.20 mmol), Er(OAc)3·4H2O (0.018 mmol) and Tm(OAc)3·4H2O (CAS No. 15280-58-7, 0.006 mmol) were added to a 50 mL flask and dissolved in 10 mL of methanol by sonication to form a transparent solution. After removing the methanol by slow heating to 100 °C for 10 minutes, 7 mL of oleic acid and 15 mL of 1-octadecene were added, and the mixture was heated at 150 °C for 60 minutes under an inert gas (argon) to form a homogeneous solution, which is a rare earth-oleic acid precursor complex. After the solution cooled to room temperature, 10 mL of methanol solution containing NaOH (2.5 mmol) and NH4F (4 mmol) was added. The mixture was stirred at room temperature for 30 minutes to obtain a colloidal dispersion. The temperature was slowly increased to 100 °C and held for 10 minutes to remove the methanol. Subsequently, the temperature was increased to 305 °C under argon protection and reacted for 1.5 hours. After natural cooling, ethanol was added to precipitate the core particles. After centrifugation and washing with ethanol three times, the core nanoparticles of the product were redispersed in 10 mL of cyclohexane.

[0040] (2) Synthesis of core-shell structured LUNCs (NaGdF4: 20% Yb, 1.8% Er, 0.6% Tm@NaGdF4): Gd(OAc)3·4H2O (0.776 mmol) dissolved in 5 mL methanol was added to a 50 mL flask containing 7 mL oleic acid and 15 mL 1-octadecene. The mixture was slowly heated to 100 °C under an argon atmosphere to remove the methanol, and then heated to 150 °C for 60 minutes to form a homogeneous yellow solution. 10 mL of cyclohexane-dispersed core nanoparticles synthesized in step (1) and 10 mL of methanol solution containing NaOH (2.5 mmol) and NH4F (4 mmol) were added to the homogeneous yellow solution, and the mixture was stirred at room temperature for 30 minutes. The resulting dispersion was slowly heated to 100 °C and held for 10 minutes to remove low-boiling-point solvents, and then heated to 305 °C for 1.5 hours under argon protection. After the solution cooled naturally, the obtained nanoparticles were precipitated with ethanol. After centrifugation and washing with ethanol multiple times, the core-shell structured LUNCs were obtained. Finally, the obtained core-shell structured LUNCs (still containing oleic acid ligands) were redispersed in 10 mL of cyclohexane for subsequent experiments.

[0041] Example 2 Synthesis of molecular probe Glu-RB Starting with 3-aldehyde quinoline and a rhodamine derivative (intermediate 2), intermediate 3 was generated via a condensation reaction. Subsequently, intermediate 3 underwent a quaternization reaction with iodomethane to yield the final product Glu-RB. The synthetic route is as follows: Figure 1 As shown. The specific synthesis steps are as follows: (1) Synthesis of 9-(2-carboxyphenyl)-6-(dimethylamino)-1,2,3,4-tetrahydrozanton perchlorate (rhodamine derivative, intermediate 2): 3-hydroxy-N,N-dimethylaniline (100 mmol) and isobenzofuran-1,3-dione (14.8 g, 100 mmol) were refluxed in 50 mL toluene for 24 hours. The solvent was removed under reduced pressure, and 50 mL of 35% NaOH (w / w) aqueous solution was added. The mixture was then heated to 90 °C and held for 12 hours. The resulting mixture was cooled to room temperature, acidified with 20 mL of 1 M HCl, and allowed to stand at room temperature for 2 hours. The suspension was filtered through a 0.45 μm filter and dried to give intermediate 1 (26.8 g). Cyclohexanone (0.29 g, 3 mmol) was added dropwise to 10 mL of concentrated H2SO4 under ice bath conditions. Intermediate 1 (3 mmol) was then slowly added while stirring vigorously, and the mixture was heated at 90 °C for 3 hours. After cooling, the mixture was poured onto ice and 2 mL of 70% v / v perchloric acid was added. The resulting precipitate was filtered, washed three times with cold water, and air-dried to give the rhodamine derivative (intermediate 2), which could be used directly for subsequent condensation reactions without purification.

[0042] (2) Synthesis of 9-(2-carboxyphenyl)-6-(dimethylamino)-4-(quinoline-3-ylmethylene)-1,2,3,4-tetrahydrozanton perchlorate (intermediate 3): Intermediate 2 (rhodamine derivative, 10 mmol) prepared in step (1) and 3-aldehyde quinoline (10 mmol) were dissolved in 50 mL of acetic acid and then heated at 90 °C for 8 hours under N2 atmosphere. After removing acetic anhydride under reduced pressure, the crude product was washed with ethyl acetate and purified by rapid chromatography (DCM / EtOAc, 49:1) as the eluent to obtain a purple solid, which was intermediate 3 (3.23 g, yield: 55%).

[0043] (3) Intermediate 3 (1 mmol) was dissolved in 15 mL of anhydrous acetonitrile, and then CH3I (8 mmol) was added dropwise. The solution was refluxed at 90 °C for 48 hours. After removing the solvent under reduced pressure, the crude product was dissolved in 2 mL of methanol and purified by rapid chromatography (CH2Cl2 / MeOH, 10:1) as the eluent to obtain the target probe Glu-RB as a purple solid (444 mg, yield: 61%).

[0044] Example 3 Synthesis of the molecular probe iNOS-CY iNOS-CY was obtained by reacting the cyanine dye derivative Cy-Cl with 3-mercaptopropionic acid to introduce a carboxyl terminus. The synthetic route is as follows: Figure 2 As shown. The specific synthesis steps are as follows: (1) Synthesis of compound 4: 2,3,3-trimethylindoline (10.0 mmol) and iodoethane (10.0 mmol) were dissolved in acetonitrile (10 mL) under an argon atmosphere and heated under reflux for 48 hours. After cooling, the mixture was treated with diethyl ether (50 mL) for about 24 hours, and then the solvent was removed by decanting. The final product was repeatedly dissolved in a small amount of ethanol, followed by precipitation with diethyl ether. Compound 4 was obtained by vacuum filtration in a yield of 79% (3.57 g).

[0045] (2) Synthesis of compound 5: At 0°C, a solution of phosphorus oxychloride (37.0 mL) in dichloromethane (40 mL) was added dropwise to N,N-dimethylformamide (DMF, 40.0 mL), and the reaction mixture was stirred for 30 minutes. The resulting solution was added to cyclohexanone (10 g), and the mixture was refluxed for 4 hours. After the mixture was cooled to room temperature, it was treated with 300 mL of ice water for about 24 hours, resulting in the precipitation of a large amount of pale yellow solid. The product was filtered and washed with water, and the crude product was purified by recrystallization from acetone to obtain a yellow crystalline solid compound 5 (10.75 g, yield: 62%).

[0046] (3) Synthesis of Cy-Cl: Compound 4 (5.0 mmol), compound 5 (2.0 mmol), and NaAc (0.8 mmol) were dissolved in 15 mL of acetic anhydride, refluxed for 24 hours, and then cooled to room temperature. The resulting solution was green and concentrated under reduced pressure. The residue was first eluted with DCM to remove nonpolar impurities, and then purified with a DCM solution of 5% v / v methanol. The product was further washed twice with 10 mL of diethyl ether to give a bright red solid Cy-Cl (458 mg, yield: 36%).

[0047] (4) Synthesis of probe iNOS-CY: Cy-Cl (0.5 mmol), 3-mercaptopropionic acid (0.6 mmol), and triethylamine (0.6 mmol) were mixed in 5 mL of DMF solution and stirred overnight at room temperature. The mixture was then added dropwise to diethyl ether and filtered to obtain a crude product with a green precipitate. The crude product was washed with ethyl acetate and purified by rapid chromatography (DCM:MeOH, 10:0-10:1) as the eluent to obtain the target probe iNOS-CY as a golden yellow solid (276 mg, yield: 78%).

[0048] Example 4 Preparation of PEI-LUNCs The synthesis steps of PEI-LUNCs are as follows: Figure 3 As shown, 50 mg of the core-shell structured LUNCs with oleic acid ligands prepared in Example 1 were added to 3 mL of ethanol and then dispersed in 10 mL of acidic aqueous solution (pH=4) adjusted with 0.1 M HCl. The oleic acid ligands were removed by sonication for 10 minutes, and the ligand-free LUNCs were obtained by centrifugation. The LUNCs were washed three times each with ethanol and deionized water and redispersed in 5 mL of deionized water. 100 μL of branched polyethyleneimine solution (PEI, 20 mg / mL, molecular weight 25 kDa) was added dropwise to the ligand-free LUNCs dispersion while sonicating to aid redispersion. Sonication was then continued for 15 minutes at room temperature, followed by stirring for 12 hours. The LUNCs were washed three times with deionized water to remove excess polyethyleneimine, yielding the product PEI-LUNCs, which were then redispersed in 5 mL of water.

[0049] Example 5 Preparation of Glu-RB and iNOS-CY modified upconversion nanoprobe MEI-1 The synthetic route of the nanoprobe MEI-1 (LUNCs@PEI-PEG-Man / Glu-RB / iNOS-CY) is as follows: Figure 3 As shown, the specific steps include the following: Synthesis of polymer mannose-polyethylene glycol-carboxylic acid (Man-PEG-COOH): Man-PEG-COOH was prepared by direct covalent coupling of amino-polyethylene glycol-carboxylic acid (NH2-PEG-COOH, Mw = 2 kDa) with D-mannose pyranocyanate (CAS No. 96345-79-8). The synthesis steps are as follows: NH2-PEG-COOH (100 mg) and D-mannose pyranocyanate (0.06 mmol) were dissolved in dimethyl sulfoxide (5 mL), stirred at 25 °C for 24 hours, and the resulting solution was evaporated to dryness to obtain Man-PEG-COOH, which was then redispersed in 6 mL of water.

[0050] EDC (0.5 mmol) and NHS (0.5 mmol) were dissolved in 5 mL of EtOH / H2O (1:1 v / v) solution and added to a 50 mL flask containing Man-PEG-COOH (100 mg). After stirring at room temperature for 30 minutes, the solution was slowly added dropwise to 2 mL of aqueous PEI-LUNCs (10 mg / mL). The mixture was sonicated for 30 minutes and then stirred overnight. Free polymer was removed by centrifugation, and the nanoparticles (PEI-PEG-Man-LUNCs) were collected by centrifugation after washing with 5 mL of ethanol. The nanoparticles were then sonicated and redispersed in 2 mL of PBS solution (10 mg / mL based on PEI-LUNCs). -1 )middle.

[0051] Subsequently, the PEI-PEG-Man-LUNCs prepared above were added to 5 mL of ethanol containing Glu-RB (24 mg) and iNOS-CY (6 mg), and the mixture was sonicated for 10 minutes and stirred at room temperature for 12 hours to form a homogeneous solution. The resulting solution was centrifuged, and the MEI-1 nanoprobe was washed with deionized water. The prepared MEI-1 could be redispersed by sonication in PBS to form a homogeneous blue solution, which was then stored at 4°C.

[0052] Example 6: Evaluation of the in vitro optical sensing performance of MEI-1 nanomaterials 1. Regarding NAD(P)H and ONOO - responsiveness In this example, the optical response of MEI-1 was tested in PBS buffer (pH 7.4), as detailed below: UV-Vis absorption and upconversion luminescence: After adding 150 μM NAD(P)H and 0.05 mg / mL MEI-1, the absorption spectrum showed a red shift, with a significant enhancement in absorption at 710 nm, as shown in the figure. Figure 4In (a), the upconversion luminescence (UL) emission intensity at 660 nm is weakened due to LRET. Adding 30 μM ONOO... - After adding 0.05 mg / mL MEI-1, the absorption at 800 nm decreased, while the upconversion emission intensity at 800 nm recovered. Upconversion emission lifetime tests further confirmed the existence of the LRET process: with the addition of NAD(P)H, the upconversion emission lifetime at 660 nm decreased from 369.2 μs to 211.2 μs; with the addition of ONOO... - Subsequently, the upconversion luminescence lifetime at 800 nm was extended from 185.4 μs to 260.9 μs.

[0053] Photoacoustic signal (PA): MEI-1 exhibits excellent photoacoustic sensing performance, and the photoacoustic signal is detected using multispectral photoacoustic tomography (mPAT). With increasing NAD(P)H concentration, the PA signal at 710 nm (PA ~ 710 ~) linearly increases, and the detection limit (LOD) is as low as approximately 466 nM. With ONOO - With increasing concentration, the PA signal at 800 nm (PA ~ 800 ~) decreases linearly, with a LOD of approximately 184 nM. Figure 4 (b) The PA spectrum clearly shows this change.

[0054] 2. Selectivity and Penetration Selectivity: MEI-1 for NAD(P)H and ONOO - It exhibits high selectivity. In the presence of various reactive oxygen species (ROS / RNS) and energy metabolites, it only exhibits high selectivity in ONOO. - When present with its degradation product HOCl (which can oxidize iNOS-CY), the PA~800~ channel shows a significant response, and the interference of other substances on the dual-channel PA signal is less than 10%, such as Figure 4 (c) The normalized PAF / PAi intensity values ​​for all substances are expressed in terms of NADH and ONOO. - The induced PAf / PAi ratio was normalized using it as a reference. That is, the response signals of other substances were compared with those of NADH (710 nm channel) and ONOO. - The response signals (800 nm channel) were compared to evaluate the selectivity of MEI-1 for different substances.

[0055] Tissue Penetration: The imaging capability of MEI-1 was evaluated by simulating the in vivo environment using chicken breast tissue of varying thicknesses. Specifically, the MEI-1 nanoprobe (concentration 0.05 mg / mL) was placed in a container. Chicken breast was sliced ​​into uniformly thick slices. By stacking different numbers of slices, a series of different tissue thicknesses were constructed above the probe sample, such as 0 mm, 2 mm, 4 mm, 6 mm, 8 mm, 10 mm, 15 mm, 20 mm, and 25 mm. Ultrasonic coupling gel (for PA imaging) or optically transparent media (for UL imaging) were used between the tissue slices and the sample container, and between the tissue slices and the imaging detector, to ensure effective signal transmission and reduce interface reflection loss.

[0056] The results showed that although both UL and PA signals attenuated with increasing tissue thickness, mPAT imaging exhibited significantly better signal-to-background ratio (SBR) and tissue penetration depth (up to 2.2 cm) than UL imaging. Figure 4 (d) For example, at a tissue depth of 10 mm, the SBR of PA~710~ and PA~800~ are 3.4 times and 2.0 times that of UL~660~ and UL~800~, respectively.

[0057] Example 7: Assessment of macrophage energy metabolism and polarization using MEI-1 1. Imaging under different glucose supply and CRM conditions RAW264.7 macrophages were seeded at 2×10^3 cells / well in 35 mm glass-bottomed culture dishes, and different glucose concentrations (5, 25, 35 mM) and 2-deoxy-D-glucose (2-DG, 1 mM) were added. After culturing for 24 h, 0.1 mg / mL MEI-1 was added and incubated for 45 min before UL / mPAT imaging was performed.

[0058] The results showed that within the glucose range of 5-25 mM, the PA~710~ signal increased with increasing glucose concentration, but the PA~800~ signal did not change significantly, indicating that M1 polarization did not occur in the cells. However, after treatment with high glucose (35 mM) or 2-DG, the PA~710~ signal changed significantly (increased with high glucose, inhibited with 2-DG), while the PA~800~ signal decreased.

[0059] like Figure 5 As shown in (a), within the 5-25 mM glucose range, the UL~660~ signal weakens with increasing glucose concentration, which, together with the enhancement of the PA~710~ signal, confirms the increase in NAD(P)H levels from glycolysis; while the UL~800~ signal remains stable, consistent with the lack of change in the PA~800~ signal, indicating the absence of ONOO related to M1 polarization. -In the presence of high glucose (35 mM) or 2-DG, the UL~660~ signal further weakened or showed a recovery trend, corresponding to excessive activation and inhibition of energy metabolism, respectively; while the UL~800~ signal was significantly enhanced, which, together with the weakening of the PA~800~ signal, confirms the occurrence of nitrosation stress. Drastic changes in energy metabolism (high glucose or deprivation) can promote M1 polarization and nitrosation stress.

[0060] 2. Two-dimensional imaging under different polarization states RAW264.7 macrophages were polarized into three phenotypes: M0 (resting, Control), M1 (LPS / IFN-γ induced), and M2 (IL-4 induced). Specifically, M0 cells were cultured in standard medium (without additional inducers) for 24 hours; M1 cells were cultured in standard medium supplemented with LPS (1 μg / mL) and IFN-γ (50 ng / mL) for 24 hours; and M2 cells were cultured in standard medium supplemented with IL-4 (20 ng / mL) for 24 hours. The resulting cells were then incubated with 0.05 mg / mL MEI-1 for 45 min before UL / mPAT imaging.

[0061] Energy metabolism: The PA710 signal intensity in M1 macrophages was 1.4 times that in M0 cells, indicating enhanced glycolysis and NAD(P)H production. Figure 5 As shown in (b), Seahorse energy metabolism analysis confirmed that M1 cells had a higher extracellular acidification rate (ECAR, an indicator of glycolysis) and a lower oxygen consumption rate (OCR, an indicator of oxidative phosphorylation).

[0062] Nitrification stress: such as Figure 5 As shown in (c), the PA800 signal intensity of M1 macrophages was significantly lower than that of M0 and M2 cells, indicating that they produced a large amount of ONOO. - .

[0063] Inhibitor validation: The M1 macrophages obtained above were used with ONOO - Treatment with the scavenger mercaptoethylguanidine (MEG, 100 μM) or the energy metabolism inhibitor 2-DG (1 mM) resulted in the reversal of the expected directions of both the dual-channel PA and UL signals of MEI-1, demonstrating the specificity and reliability of its sensing mechanism. Figure 5 As shown in (d) in the figure.

[0064] Example 8: Evaluation of CR combined with R848 immunotherapy regimen using MEI-1 Immunotherapy was simulated using the TLR7 / 8 agonist R848 (resiquimod) and combined with 2-DG to simulate CR combination therapy. Figure 6 ).

[0065] Dual-modal imaging: Macrophages were treated with 4.5 µg / mL R848 alone (at approximately 2 × 10⁻⁶). 3 Seeding at a density of 10 cells / well in 35 mm glass-bottomed culture dishes for 48 hours resulted in enhanced PA~710~ signal and decreased PA~800~ signal, exhibiting typical M1 metabolic characteristics. Upconversion luminescence (UL) imaging results showed that, as Figure 7 As shown: In the energy metabolism channel, the decrease in UL~660~ signal corresponds to the increase in PA~710~ signal; in the M1 polarization / nitrosation stress channel, the increase in UL~800~ signal corresponds to the decrease in PA~800~ signal. After the combination of 2-DG (1 mM2-DG was added to the culture medium 30 minutes before the start of R848 treatment, i.e., cells were pre-incubated with 2-DG for 30 minutes before being stimulated by R848 for 48 hours), the PA~710~ signal decreased (energy metabolism was inhibited), but the PA~800~ signal only slightly increased, indicating that strong M1 polarization and killing ability (nitrosation stress) were still maintained under energy-limited conditions. At this time, the UL imaging results showed that the intervention of 2-DG inhibited glycolysis and affected the production of ONOO-. 2-DG and R484 can maintain strong M1 polarization and killing ability under energy-limited conditions through synergistic effects.

[0066] Flow cytometry validation: Flow cytometry analysis showed that R848 effectively polarized macrophages to the M1 phenotype (F4 / 80). + / iNOS + The efficiency was slightly lower than that of the classic LPS / IFN-γ, but much higher than that of the control group and the IL-4 group. In the presence of 2-DG, the proportion of M1 population induced by R848 was even higher than that of LPS / IFN-γ, which is consistent with the relatively high M1 phenotypic signal in the CR-R848 group in the imaging results.

[0067] Energy metabolism analysis: The Seahorse experiment showed that R848 induced enhanced glycolysis and inhibition of OXPHOS. Combined treatment with 2-DG significantly inhibited glycolysis but partially reversed the inhibition of mitochondrial OXPHOS by R848, suggesting metabolic reprogramming.

[0068] Example 9: Dynamic screening of CR combined with immunotherapy in tumor-bearing mice 4T1 mouse breast cancer cells (5×10) 6 (Number of tumor cells) were suspended in culture medium and subcutaneously injected into the backs of BALB / c nude mice (female, 6 weeks old, 18-20 grams). After 5-7 days of feeding, nodules could be palpated. The major and minor axes could be measured with calipers, and the volume was calculated. Tumor volumes reaching 1000 mm² were considered normal. 3 At that time, the 4T1 tumor-bearing mouse model was constructed.

[0069] In a 4T1 tumor-bearing mouse model, the efficacy of MEI-1 in in vivo screening for complete remission (CR) combined with immunotherapy (immunotherapy drugs including the TLR7 / 8 agonist R848 or the CSF-1R inhibitors PLX3397 and BLZ945) was evaluated. Figure 8 The aforementioned CR combined with immunotherapy specifically involves mice receiving multiple rounds of immunotherapy drugs and / or 2-DG injections during treatment, with the injection frequency adjusted so that the mouse tumor volume reaches 1000 mm². 3 Day 0 was designated as the treatment group, with five intratumoral injections administered on days 0, 2, 4, 6, and 8. MEI-1 imaging was performed on day 6 during treatment. All drugs were dissolved in 10 mM, pH 7.4 PBS buffer before injection. The injection details for each group of mice are as follows: Control group: Tumor-bearing mice received intratumoral injections of the same volume and frequency of PBS (10 mM, pH 7.4) as the treatment group; LPS / IFN-γ group: Each injection consisted of 100 μL of LPS at a concentration of 1 μg / mL and IFN-γ at a concentration of 50 ng / mL; R848 group: Each injection consisted of 100 μL of R484 at a concentration of 0.25 mg / mL; PLX3397 group: 100 μL of PLX3397 at a concentration of 12.5 mg / mL was injected each time; BLZ945 group: 100 μL of BLZ945 at a concentration of 50 mg / mL was injected each time; CR-LPS / IFN-γ group: each injection consisted of 50 μL of 250 mg / mL 2-DG and 50 μL of 2 μg / mL LPS and 100 ng / mL IFN-γ; CR-R848 group: Each injection consisted of 50 μL of 250 mg / mL 2-DG and 50 μL of 0.5 mg / mL R484; CR-PLX3397 group: Each injection consisted of 50 μL of 250 mg / mL 2-DG and 50 μL of 25 mg / mL PLX3397; CR-BLZ945 group: Each injection consisted of 50 μL of 250 mg / mL 2-DG and 50 μL of 100 mg / mL BLZ945.

[0070] Imaging procedure: mPAT imaging was performed 24 h after intravenous injection (iv) of 200 μL of 5 mg / mL MEI-1, or UL imaging was performed 24 h after intratumoral injection (it) of 25 μL of 5 mg / mL MEI-1, to simultaneously monitor intratumoral energy metabolism (PA ~710~ / UL ~660~) and M1 polarization level (PA ~800~ / UL ~800~).

[0071] TLR7 / 8 targeted therapy screening: Compared with the control group, LPS / IFN-γ or R848 monotherapy both resulted in enhanced PA710 signal and decreased PA800 signal in the tumor region. The M1 polarization to energy metabolism ratio (R-PAP / PAE) was 1.48 and 1.43 in the LPS / IFN-γ group and the R848 group, respectively. However, after combination with 2-DG (CR-LPS / IFN-γ, CR-R848), although the absolute value of the dual-channel PA signal decreased, the R-PAP / PAE values ​​increased to 1.73 and 2.11 in the CR-LPS / IFN-γ group and the CR-R848 group, respectively, indicating a relatively higher M1 polarization level achieved under the background of overall low energy metabolism. UL imaging results showed good internal cross-reference with mPAT imaging, such as... Figure 9 As shown.

[0072] CSF-1R targeted therapy screening: PLX3397 monotherapy effectively reduced tumor PA800 signal, while BLZ945 showed a weaker effect. When combined with 2-DG (CR-PLX3397, CR-BLZ945), the R-PAP / PAE values ​​of both were significantly higher than those of monotherapy, at 1.59 and 1.46 respectively, and corresponding changes were observed in UL imaging, such as... Figure 10 The results demonstrate that CR can enhance the efficacy of CSF-1R inhibitors. MEI-1 successfully differentiated the effects of drugs targeting different targets and their CR combination regimens.

[0073] The R-PAP / PAE ratio is defined as: R-PAP / PAE = [PAE(Control) × PAP(Control)] / [PAE(CR) × PAP(CR)]; where PAE refers to the PA710 signal, which reflects energy metabolism within the tumor by detecting NAD(P)H; PAP refers to PA800, which reflects M1 polarization level by detecting ONOO⁻. This ratio reflects the intensity of M1 polarization relative to energy metabolism. A higher ratio indicates a higher M1 polarization level per unit energy metabolism level, which can more effectively promote macrophage polarization towards the anti-tumor M1 type and inhibit tumor glycolysis, predicting better anti-tumor effects. The method described in this invention monitors the dynamic changes of two key biomarkers, NAD(P)H and ONOO⁻, in the tumor microenvironment in real time, in situ, and quantitatively, and derives the comprehensive quantitative index R-PAP / PAE, providing a powerful tool for high-throughput screening and optimization of drug combinations for CR combined with immunotherapy at the in vivo level. By comparing the values ​​of this value in different treatment groups, it is possible to intuitively and quantitatively determine which drug combination is more effective in disrupting tumor energy metabolism and the immune microenvironment.

[0074] This invention provides a dual-modal imaging nanoprobe material, its preparation method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing a dual-modal imaging nanoprobe material, characterized in that, Includes the following steps: (1) Preparation of glycosylated upconversion nanocrystals, wherein the upconversion nanocrystals are NaGdF4: Yb, Er, Tm@NaGdF4; (2) The molecular probe and the glycosylated upconversion nanocrystals are mixed in a first solvent to prepare the dual-modality imaging nanoprobe material; wherein the molecular probe includes NADH / NADPH sensitive molecular probes and ONOO. - Sensitive molecular probes.

2. The preparation method according to claim 1, characterized in that, In step (1), the upconversion nanocrystals NaGdF4:Yb, Er, Tm@NaGdF4 are prepared by thermal decomposition synthesis, including the following steps: (a) The rare earth salt precursor is dissolved in methanol, mixed with oleic acid and 1-octadecene, and reacted at 150-160 °C for 1-2 h to obtain a rare earth-oleic acid precursor complex. The rare earth salt precursor includes a combination of Gd(OAc)3·4H2O, Y(OAc)3·4H2O, Er(OAc)3·4H2O and Tm(OAc)3·4H2O, with a mixing molar ratio of 77.6:20:1.8:0.

6. (b) Gd(OAc)3·4H2O was dissolved in methanol, mixed with oleic acid and 1-octadecene, and reacted at 150~160 °C for 1~2 h to obtain a yellow solution; (c) The rare earth-oleic acid precursor complex and the yellow solution are mixed with a methanol solution containing NaOH and NH4F for 20-40 min, and then kept at 300-310 °C for 1-2 hours to obtain the upconversion nanocrystals.

3. The preparation method according to claim 1, characterized in that, In step (1), the glycosylation modification is a polyethyleneimine-polyethylene glycol-mannose copolymer modification.

4. The preparation method according to claim 3, characterized in that, In step (1), the glycosylated upconversion nanocrystals are prepared by the following method: the upconversion nanocrystals are mixed with a polyethyleneimine solution to obtain polyethyleneimine-modified upconversion nanocrystals; then the polyethyleneimine-modified upconversion nanocrystals and mannose-polyethylene glycol-carboxylic acid are mixed in a second solvent to obtain glycosylated upconversion nanocrystals.

5. The preparation method according to claim 1, characterized in that, In step (2), the NADH / NADPH sensitive molecular probe includes Glu-RB, the structural formula of which is shown in Formula I; the ONOO - Sensitive molecular probes include iNOS-CY, whose structural formula is shown in Formula II. Equation I, Formula II.

6. The preparation method according to claim 1, characterized in that, In step (2), the NADH / NADPH sensitive molecular probe is combined with ONOO - The mass ratio of the sensitive molecular probe is 3~5:

1.

7. The preparation method according to claim 4, characterized in that, In step (2), the amount of glycosylated upconversion nanocrystals added is based on the mass of the polyethyleneimine-modified upconversion nanocrystals, with each mg of glycosylated upconversion nanocrystals mixed with 1-2 mg of molecular probe.

8. The dual-modal imaging nanoprobe material prepared by the preparation method according to any one of claims 1 to 7.

9. The dual-modal imaging nanoprobe material of claim 8 can simultaneously image NADH / NADPH and ONOO in vivo or in vitro. - Applications in [the field].

10. The use of the dual-modal imaging nanoprobe material of claim 8 in the preparation of reagents for screening or evaluating the efficacy of caloric restriction combined with immunotherapy for cancer.

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