Imaging probe based on radionuclide labeling, preparation method and application

Abstract

The invention relates to the field of nuclear medicine, in particular to an imaging probe based on radionuclide labeling and application. The imaging probe based on radionuclide labeling provided by the invention comprises lanthanide nanocrystals, an amphiphilic block copolymer self-assembled by the lanthanide nanocrystals, and radionuclide ions labeled on the nanocrystals, and the imaging probe simultaneously realizes time-gated intensity imaging, life imaging and SPECT (Single Photon Emission Computed Tomography) imaging. The imaging probe based on radionuclide labeling is provided for the first time, the radionuclide labeling rate as high as 99.6% is achieved, the imaging probe not only supports time gating intensity imaging with high signal-to-background ratio, but also supports life imaging and SPECT imaging, and in-vivo biological distribution and metabolism tracking of radioactive nanoparticles are achieved.

Description

Technical Field

[0001] The present invention relates to the field of nuclear medicine technology, and in particular to an imaging probe based on radionuclide labeling, a preparation method and an application thereof. Background Art

[0002] In recent years, the application of radioactive nanoparticles in nuclear medicine has been increasing, particularly in tumor treatment. Radioactive nanoparticles precisely kill tumor cells by locally releasing radioactive isotopes, thereby minimizing damage to surrounding normal tissue. This treatment method is known as brachytherapy. Brachytherapy involves placing radionuclides directly into diseased tissue or cavities. The high-energy rays or particles generated by their decay process are used to deliver short-term, high-dose irradiation to specific tumor tissue sites, minimizing radiation damage to surrounding normal tissue.

[0003] Despite the remarkable therapeutic effects of radioactive nanoparticles, their in vivo metabolism remains a pressing challenge. Accurately tracking the distribution, metabolic pathways, and bioavailability of nanoparticles is crucial for assessing therapeutic efficacy and safety. However, existing technologies primarily employ imaging techniques such as Micro PET / CT, Micro SPECT / CT, and Micro PET / MRI to investigate the in vivo distribution of free and labeled rare earth radionuclides. Gamma counters are used to investigate the distribution of radioactivity in heart, liver, spleen, lung, kidney, intestine, and tumor tissues, and to compare and analyze the in vivo metabolic rates of free and labeled rare earth radionuclides. However, single imaging modalities often have shortcomings, leading to the development of multimodal imaging techniques that combine the advantages of multiple imaging modalities to achieve better imaging results. For example, existing technologies combine fluorescence imaging and SPECT imaging to form dual-modality imaging. However, since this imaging method primarily performs imaging in the near-infrared region II and requires the use of excitation light for signal capture, significant interference from excitation light and autofluorescence can occur, resulting in poor imaging results. Particularly in the complex biological environment of the body, nonspecific fluorescence signals can severely impact the accuracy of imaging results. SPECT imaging can provide three-dimensional images of radioactivity distribution, making it suitable for tracking low-dose radioactive nanoparticles. However, SPECT imaging has relatively low spatial resolution and long imaging times, making it difficult to achieve dynamic monitoring with high temporal resolution.

[0004] Therefore, even in combined imaging, the problem of background signal interference from fluorescence imaging still exists, especially in the case of low-dose radioactive nanoparticles. The background signal may mask the target signal, leading to inaccurate imaging results. To ensure patient safety, the dose of injected radioactive nanoparticles is usually low, which reduces the signal-to-noise ratio of SPECT imaging and affects imaging quality. Summary of the Invention

[0005] In response to the above-mentioned shortcomings of the prior art, the present invention provides an imaging probe based on radionuclide labeling and its application. The imaging probe provided by the present invention can simultaneously realize time-gated intensity imaging, lifetime imaging and SPECT imaging. Single time-gated intensity imaging can effectively eliminate the interference of excitation light and autofluorescence, but it is easily affected by tissue scattering, so the accurate acquisition of in vivo luminescence signals is limited. In contrast, single lifetime imaging relies on luminescence lifetime as the imaging signal, which can extract information within biological tissues in the time domain. Lifetime imaging is independent of probe concentration, excitation conditions and signal changes caused by biological tissues, so it can be quantitatively imaged. However, lifetime imaging involves fitting multiple intensity images, and its imaging frame rate is usually low, making it difficult to capture rapid dynamic changes. In addition, it can only track in vivo in a two-dimensional plane. Single photon emission computed tomography (SPECT) imaging modality provides high-resolution imaging and radionuclide-specific tracking. The inventors unexpectedly discovered for the first time a trimodal imaging probe that can simultaneously display time-gated intensity imaging, lifetime imaging and SPECT imaging, providing a new approach for non-invasive in vivo imaging and long-term metabolic tracking of radionuclide nanoparticles.

[0006] The “time-gated intensity imaging” mentioned in the context of the present invention is to delay the excitation light pulse by a certain time (gating delay time, t delay ) and then open the signal acquisition window (gate width, Δt) to record only the light signal intensity within a specific time range after the delay. This method primarily uses the difference in lifetime between the imaging probe and background signals (such as short-lived autofluorescence) to isolate the target signal in the time dimension.

[0007] "Lifetime imaging," as used in the context of this invention, is a technique for generating images by measuring the average time it takes for a fluorescent molecule to return from an excited state to a ground state (i.e., the fluorescence lifetime, τ). Its core principle is to use fluorescence lifetime as contrast, rather than traditional fluorescence intensity. Unlike time-gated intensity imaging, which uses time gating to filter out short-lived background signals and only captures the intensity signal within a specific time window, lifetime imaging directly extracts lifetime values through full decay curve fitting or phase analysis, thereby analyzing in vivo parameter information.

[0008] The "SPECT imaging" mentioned in the context of the present invention generally refers to single-photon emission computed tomography (SPECT), which is a functional imaging method based on radionuclide tracer technology. It reconstructs three-dimensional tomographic images by detecting single gamma photons emitted by radioactive probes in the body, and is used to visualize dynamic processes such as biomolecule metabolism, blood flow distribution or target binding.

[0009] Although time-gated intensity imaging, lifetime imaging, and SPECT imaging have been reported separately in the prior art, there is no prior art report on combining them because this requires probes with suitable performance, which cannot be achieved by just selecting any probe. Time-gated intensity imaging and lifetime imaging rely on the luminescence lifetime of the luminescent substance. The lifetimes of different luminescent substances vary greatly, for example, Tm 3+ The luminescence lifetime of Er is in the millisecond order, while 3+ and Yb 3+ The luminescence lifetime is in the order of microseconds. If the luminescence lifetimes of the rare earth ions in the core and shell are inconsistent, it may cause signal interference in time-gated imaging. SPECT imaging relies on the gamma-ray emission of radionuclides. Although there are references in the prior art that nanomaterials can be labeled with radionuclides, different radionuclides (such as 177 Lu 3+ ) have different half-lives and energies, and appropriate radionuclides need to be selected to ensure imaging results.

[0010] In a first aspect of the present invention, an embodiment of the present invention provides an imaging probe based on radionuclide labeling, wherein the imaging probe includes lanthanide nanocrystals, amphiphilic block copolymers self-assembled from lanthanide nanocrystals, and radionuclide ions labeled on the nanocrystals, and the imaging probe simultaneously realizes time-gated intensity imaging, lifetime imaging and SPECT imaging.

[0011] Lanthanide nanocrystals have long luminescence lifetimes (typically in the microsecond to millisecond range) and narrow emission bands, making them suitable for long-duration imaging. Amphiphilic block copolymers can self-assemble into micelles in aqueous solution, forming stable nanostructures with the lanthanide nanocrystals. Radioactive nuclides are then labeled on the surface of lanthanide nanocrystals, enabling them to acquire radioactive images. These radionuclides have specific gamma-ray energies suitable for SPECT imaging. The long luminescence lifetime of lanthanide nanocrystals allows for time-gated imaging by selecting a specific time window (e.g., delaying signal acquisition by a few microseconds) to effectively reduce interference from short-lived background fluorescence and improve the signal-to-noise ratio (SBR). Furthermore, the high quantum yield of lanthanide nanocrystals enables sufficient signal intensity even at low concentrations, enhancing imaging sensitivity. This makes them suitable for the injection of low-dose radioactive nanoparticles. The gamma rays emitted by the radionuclide ions labeled on the lanthanide nanocrystals can be detected by the SPECT device, providing high-spatial-resolution three-dimensional images for assessing organ function and lesion location. Although other fluorescent materials (such as organic dyes, quantum dots, etc.) can also be used for time-gated intensity imaging and lifetime imaging, their fluorescence lifetime is usually short, the background interference is large, and the signal-to-noise ratio is not as good as that of lanthanide nanocrystals.

[0012] In some embodiments of the present invention, the valence state of the radioactive nuclide ion labeled on the nanocrystal is the same as the valence state of the lanthanide ion in the lanthanide nanocrystal. This is based on the fact that the radioactive nuclide ion is labeled by a simple cation exchange method. The exchange rate between ions with the same valence state is usually faster because their chemical behavior in solution is similar and they are more susceptible to exchange reactions. Ions with different valence states or with large differences in ionic radius can cause charge imbalance, which results in a longer labeling time for the radioactive nuclide ion and lower efficiency. In addition, the substitution position and coordination environment of ions with the same valence state in the nanocrystal are similar, making it easier to maintain the structural stability of the nanocrystal. Ions with similar ionic radius are more likely to enter the lattice structure of the nanocrystal and replace the original lanthanide ion. If the ionic radius differs greatly, lattice distortion may result, affecting the structural integrity and optical properties of the nanocrystal.

[0013] In some embodiments of the present invention, the crystalline phase of the lanthanide nanocrystals is a hexagonal phase, and the nanocrystals include NaL doped with at least one of the rare earth ions. n F4, at least one of the doped rare earth ions replaces the part L n 3+ location.

[0014] In some embodiments of the present invention, the radionuclide ions replace L in the nanocrystals. n 3+ location.

[0015] In some embodiments of the present invention, the radionuclide ions include 177 Lu 3+ 、 90 Y 3+ 、 153 Sm 3+ 、 166 Ho 3 + At least one of .

[0016] In some embodiments of the present invention, the amphiphilic block copolymer includes one of DSPE-PEG2000, polyethylene oxide-polypropylene oxide block copolymer (PEO-PPO-PEO), polyethylene oxide-polycaprolactone block copolymer (PEO-PCL), polyethylene oxide-polystyrene block copolymer (PEO-PS), polyethylene oxide-polyacrylic acid block copolymer (PEO-PAA), polyethylene oxide-polymethyl methacrylate block copolymer (PEO-PMMA), polyethylene oxide-polyacrylonitrile block copolymer (PEO-PAN), polyethylene oxide-polyacrylamide block copolymer (PEO-PAM), polyethylene oxide-polyglycerol adipate block copolymer (PEO-PGA), polyethylene oxide-polylactic acid block copolymer (PEO-PLA), and polyethylene oxide-polycaprolactone-polyethylene oxide block copolymer (PEO-PCL-PEO).

[0017] In some embodiments of the present invention, the radionuclide ions are labeled on the nanocrystals via cation exchange. This method utilizes the rapid diffusion and exchange properties of ions in solution, allowing for a shorter labeling time and improved efficiency. Furthermore, the procedure is relatively simple, requiring no complex instrumentation or equipment, and exhibits excellent reproducibility, ensuring consistent and reliable labeling results.

[0018] In some embodiments of the present invention, the particle size distribution coefficient of the lanthanide nanocrystals is no more than 10%.

[0019] In some embodiments of the present invention, the particle size distribution coefficient of the lanthanide nanocrystals is no greater than 5%.

[0020] In some embodiments of the present invention, the characteristic emission peak displayed by the lanthanide nanocrystals under the excitation of a light source corresponds to the characteristic luminescence spectrum of doped rare earth ions.

[0021] In some embodiments of the present invention, the ratio of doped rare earth ions in the lanthanide nanocrystals to the total of lanthanide ions and rare earth particles is in the range of 0.1% to 3%.

[0022] In some embodiments of the present invention, the ratio of doped rare earth ions in the lanthanide nanocrystals ranges from 0.5% to 1.5%.

[0023] In some embodiments of the present invention, the luminescence lifetime decay curve of the imaging probe is a single exponential decay characteristic.

[0024] In some embodiments of the present invention, the particle size of the lanthanide nanocrystals and the amphiphilic block polymer after self-assembly is in the range of 5 to 100 nm.

[0025] In some embodiments of the present invention, the particle size of the lanthanide nanocrystals and the amphiphilic block polymer after self-assembly is in the range of 10 to 50 nm.

[0026] In some embodiments of the present invention, the labeling rate of the radioactive rare earth ions on the lanthanide nanocrystals is not less than 95%.

[0027] In some embodiments of the present invention, the labeling rate of the radioactive rare earth ions on the lanthanide nanocrystals is not less than 99%.

[0028] In a second aspect of the present invention, a method for preparing an imaging probe based on radionuclide labeling is provided, comprising the following steps:

[0029] Step 1: synthesizing oleic acid-coated lanthanide nanocrystals doped with rare earth ions by a solvothermal method and dispersing the nanocrystals in cyclohexane;

[0030] Step 2: adding lanthanide nanocrystals doped with rare earth ions dispersed in cyclohexane to a saturated NOBF4-CH2Cl2 solution, collecting the precipitate, and dispersing the precipitate in methanol. The methanol-containing precipitate is added to a methanol solution containing DSPE-PEG2000, ultrasonically mixed, and the solvent is removed by rotary evaporation. The resulting nanoparticles are dispersed in deionized water.

[0031] Step 3: adding a salt solution containing radionuclide ions to the nanoparticles obtained above, performing ultrasonic centrifugation at room temperature to separate the precipitate, and washing to obtain the radionuclide-labeled imaging probe.

[0032] In some embodiments of the present invention, the specific steps of step 1 of the preparation method are as follows:

[0033] Step 10, adding the salt solution containing lanthanide ions and the salt solution containing rare earth ions to a three-necked flask containing oleic acid and octadecene, heating and stirring the resulting mixture under an inert gas atmosphere, and then cooling the solution;

[0034] Step 11. NH4F and NaOH dissolved in methanol are then added dropwise to the mixture, the mixture is heated to a first temperature and stirred, and the methanol is removed under an inert gas atmosphere, and then vacuumed. The mixture is then heated to a second temperature and stirred, and then naturally cooled to room temperature. The lanthanide nanocrystals doped with rare earth particles are collected, washed, and dispersed in cyclohexane.

[0035] In some embodiments of the present invention, the molar ratio of the salt solution containing lanthanide ions to the salt solution containing rare earth ions in step 10 is in the range of 97:3 to 999:1.

[0036] In some embodiments of the present invention, the temperature range of heating the mixture obtained in step 10 under an inert gas atmosphere is 100-150°C.

[0037] In some embodiments of the present invention, the temperature range of the solution cooling in step 10 is 45-80°C.

[0038] In some embodiments of the present invention, the temperature range of the solution cooling in step 10 is 55-65°C.

[0039] In some embodiments of the present invention, the molar ratio of NH4F to NaOH in step 11 is in the range of 1:5 to 1:8.

[0040] In some embodiments of the present invention, the first temperature range in which the mixture is heated in step 11 is the same as the temperature range in which the mixture is heated under an inert gas atmosphere in step 10.

[0041] In some embodiments of the present invention, the second temperature range of heating the mixture in step 11 is 280-320°C.

[0042] In some embodiments of the present invention, the time for stirring the mixture under the second temperature heating in step 11 is 40 to 150 minutes.

[0043] In some embodiments of the present invention, the radioactivity of the solution containing radionuclide ions in step 3 ranges from 14 to 56 MBq.

[0044] In a third aspect of the present invention, there is provided a use of an imaging probe based on radionuclide labeling, wherein the use includes at least one of the following uses:

[0045] Monitor changes during tumor treatment;

[0046] Study the distribution of radionuclide-labeled drugs in the body;

[0047] Assess the functional status of the liver and kidneys.

[0048] The radionuclide-labeled imaging probe provided by the present invention has the following technical effects:

[0049] The imaging probe is prepared using simple solvothermal and cation exchange methods. It features a simple structure, a relatively easy preparation process, and low cost. Furthermore, the resulting imaging probe supports time-gated intensity imaging, lifetime imaging, and SPECT imaging simultaneously, achieving high signal-to-background ratio (SBR) of up to 2300 without background interference, enabling precise three-dimensional in vivo tracking of nanocrystals. This non-invasive, multifunctional imaging effect is consistent with the organ distribution patterns observed through gamma counting analysis, thus enabling the practical implementation of metabolic tracking applications of radionuclide nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The present invention is shown to be prepared by cation exchange method 177 Schematic diagram of Lu-labeled lanthanide nanocrystals;

[0051] Figure 2 Calculated SBR ratios in time-gated intensity imaging in mice according to an embodiment of the present invention are shown;

[0052] Figure 3 Showing the time-gated intensity imaging diagram and SBR value in mice in the prior art;

[0053] Figure 4 The signal distribution of the embodiment of the present invention through time-gated intensity imaging and lifetime imaging is shown;

[0054] Figure 5 The time-gated intensity imaging and lifetime imaging images of the embodiment of the present invention are shown at different excitation power densities, nanocrystal concentrations and penetration depths;

[0055] Figure 6 Showing an embodiment of the present invention Figure 6 The 3D reconstruction results of SPECT / CT imaging of M1 over a three-day period and the metabolic pathways of the nanocrystals presented in the embodiment of the present invention are shown;

[0056] Figure 7 The time-gated imaging, lifetime imaging and SPECT imaging results of the embodiment of the present invention are shown;

[0057] Figure 8 An enlarged view showing the gated intensity imaging and lifetime imaging results of Example M1 of the present invention after 24 hours. DETAILED DESCRIPTION

[0058] The following will further describe the radionuclide-labeled composite material for time-series multidimensional imaging and its applications in conjunction with specific examples. It should be understood that the following examples are intended only to illustrate and explain the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above-mentioned disclosure are encompassed within the scope of protection intended by the present invention.

[0059] It should be understood that the various steps described in the method embodiments of the present disclosure can be performed sequentially and / or in parallel. In addition, the method embodiments may include additional steps and / or omit the steps shown. The scope of the present disclosure is not limited in this respect.

[0060] As used herein, the term "including" and its variations are open-ended, i.e., "including but not limited to." The term "based on" means "based, at least in part, on." The term "one embodiment" means "at least one embodiment," the term "another embodiment" means "at least one additional embodiment," and the term "some embodiments" means "at least some embodiments." Other terms are defined in the following description.

[0061] Non-invasive in vivo imaging and tracking technologies play a vital role in modern medical and biological research. They not only provide key information for early diagnosis of diseases, but also provide intuitive visualization methods for evaluating the efficacy of disease treatment and studying complex physiological and pathological processes in organisms. However, with the deepening of scientific research, traditional single imaging modes such as optical imaging or single-photon emission computed tomography (SPECT) imaging have gradually revealed their limitations and are unable to meet the current demand for high-resolution, high-sensitivity, and multifunctional imaging. Therefore, overcoming the shortcomings of single modes and achieving more comprehensive and accurate imaging of the internal structure and function of organisms has become an important issue.

[0062] Although combining multiple imaging techniques is theoretically attractive, it faces numerous challenges in practical application. First, the implementation of multiple imaging techniques is often accompanied by high background noise. This is because different imaging modalities have different sensitivities to environmental factors. For example, optical imaging is susceptible to the influence of tissue autofluorescence, while SPECT imaging can increase background noise due to the nonspecific distribution of radiolabeled materials. This background noise not only degrades image quality but also affects the accuracy and reliability of the final data, thus limiting the application scope of combining multiple imaging techniques. Second, the heterogeneity of biological tissue is another factor that cannot be ignored. Biological tissue is composed of a variety of cells and matrices with different properties, and these components have different absorption, scattering, and reflection properties for light. Light scattering and reflection are particularly severe when imaging deep tissue. This physical effect can lead to attenuation and distortion of the imaging signal, thereby affecting imaging depth and resolution. Furthermore, the background autofluorescence of biological tissue can significantly reduce the signal-to-background ratio (SBR), making it difficult to distinguish the target signal from the background. This problem is particularly prominent when imaging in vivo. Furthermore, the composite materials currently used for multimodal imaging are relatively complex in structure, which not only increases the difficulty and cost of material preparation. At the same time, existing imaging technologies mostly focus on near-infrared II (NIR-II) imaging. Although NIR-II imaging has good tissue penetration and low light scattering, it requires the excitation light source to be continuously on to collect signals. This continuous illumination not only causes thermal damage to biological tissues, but can also cause phenomena such as photobleaching, further affecting imaging quality and accuracy of the results.

[0063] In view of the above-mentioned shortcomings, an embodiment of the present invention provides an imaging probe based on radionuclide labeling, which integrates the advantages of time-gated intensity imaging, lifetime imaging and SPECT imaging, and truly achieves the interference of no background fluorescence in application. It breaks through the limitation of the original time-resolved imaging two-dimensional signal being limited by tissue overlap, provides three-dimensional visualization through tomographic reconstruction, and supports intensity imaging and lifetime imaging at the same time. Therefore, this imaging probe can be used for long-term non-invasive metabolic tracking of nanoparticles, such as radioactive nanoparticles, in vivo in practical applications.

[0064] The following are some specific embodiments:

[0065] Example 1

[0066] This example demonstrates the synthesis process of NaYF4:1%Tm nanoparticles, as follows:

[0067] YCl3 (0.99 mmol) and TmCl3 (0.01 mmol) were added to a three-necked flask containing oleic acid (6 mL) and octadecene (15 mL). The mixture was heated to 120°C under an argon (Ar) atmosphere and magnetic stirring was maintained until a clear solution was formed. The clear solution was then cooled to 60°C, kept clear and transparent, and NH4F (0.4 mmol, 148.2 mg) and NaOH (2.5 mmol, 100 mg) dissolved in methanol (10 mL) were subsequently added dropwise to the mixture. Afterwards, the mixture was heated to 120°C and maintained under an argon atmosphere for 30 minutes to remove the methanol, and then vacuumed for 10 minutes. The mixture was then heated to 300°C and magnetic stirring was maintained for 60 minutes. Subsequently, the flask was cooled naturally to room temperature. The nanoparticles were collected by centrifugation (12000 rpm, 10 minutes), washed three times with a cyclohexane / ethanol solution, and finally dispersed in cyclohexane (10 mL).

[0068] In this example, oleic acid-coated NaYF4:1% Tm nanocrystals were synthesized by a solvent thermal method and dispersed in cyclohexane. Transmission electron microscopy (TEM) observations showed that these nanocrystals had a uniform morphology with an average diameter of 20.7±0.8nm. High-resolution transmission electron microscopy (HR-TEM) and selected area electron diffraction (SAED) confirmed their hexagonal (β-) crystalline phase. Under 980nm excitation, the luminescence spectrum showed a characteristic emission peak at 800nm, corresponding to Tm 3+ The transient photoluminescence spectrum measured the emission decay curve at 800 nm (excitation wavelength: 785 nm), which was well fitted to a single exponential decay characteristic, and the luminescence lifetime in cyclohexane was determined to be 735 μs.

[0069] Example 2

[0070] This example demonstrates the synthesis of NaYF4:1%Tm@PEG nanocrystals, as follows:

[0071] First, NaYF4:1% Tm (10 mg) dispersed in cyclohexane was added to 3 mL of saturated NOBF4-CH2Cl2 solution to remove the oleic acid ligands on the surface. A white precipitate was observed. After shaking for 2 minutes, centrifugation (11000 rpm, 10 minutes) was performed to obtain a precipitate. 2 mL of methanol was added to disperse the ligand-free nanoparticles for further use. In a flask, DSPE-PEG2000 (15 mg) was added to 4 mL of methanol. Then 1 mL of ligand-free nanoparticles was added. Ultrasonic treatment was performed for 1 minute to ensure that the mixture was evenly dispersed. The solvent was removed using a rotary evaporator at a pressure of 100 mbar. The resulting nanoparticles were then redispersed in 1 mL of deionized water, centrifuged (12000 rpm) and washed three times with deionized water. Finally, the nanocomposite material was dispersed in 1 mL of deionized water.

[0072] In this example, the NaYF4:1%Tm nanoparticles prepared in Example 1 were modified by self-assembly with DSPE-PEG2000 to prepare water-dispersible NaYF4:1%Tm@PEG nanocrystals. These PEGylated nanocrystals exhibited excellent dispersibility in aqueous solution and maintained a consistent morphology. The hydrated particle size measured by dynamic light scattering (DLS) was 34.8nm. In water, the luminescence decay followed a single exponential pattern, and the luminescence lifetime was shortened to 375μs. This reduction in luminescence lifetime was mainly attributed to the quenching effect caused by the frequency doubling of the hydroxyl vibration in water.

[0073] Example 3

[0074] This example shows NaYF4:1%Tm, 177 The synthesis of Lu@PEG nanocrystals is as follows:

[0075] 28MBq 177 LuCl3 solution (containing 3.83×10 -11 Moore's Lu 3+ ) was added to 10 mg of NaYF4:1%Tm@PEG solid. Ultrasonic treatment was performed at room temperature for 2 minutes, followed by centrifugation (12000 rpm, 10 minutes) to separate the precipitate. The precipitate was washed three times with PBS and finally dispersed in 1 mL of deionized water to obtain NaYF4:1%Tm, 177 Lu@PEG nanocrystals.

[0076] In this example, the NaYF4:1%Tm@PEG nanocrystals prepared in Example 2 were subjected to radionuclide 177 Lu 3+ Marking, its principle see Figure 1 , the prepared NaYF4:1%Tm,177 The Lu@PEG nanocrystals achieved a labeling efficiency of 99.6%. This efficient and rapid radiolabeling method also ensured the high stability of the labeled nanocrystals. The labeled nanocrystals maintained extremely high stability for two weeks in phosphate-buffered saline (PBS) and 0.5% human serum albumin (HSA).

[0077] Example 4

[0078] This embodiment provides a time-resolved in vivo imaging system independently built in a laboratory.

[0079] Another reason why time-gated intensity imaging, lifetime imaging, and SPECT imaging have not been reported in existing technologies is that they require extremely high detector temporal resolution, typically requiring picosecond or nanosecond response times. SPECT imaging, on the other hand, requires detectors with high spatial resolution and high sensitivity, and existing detectors struggle to meet these diverse requirements.

[0080] The in vivo resolution imaging system in this embodiment includes the following main components: an IsCMOS camera (2DSPC-GaAs-U), a lens assembly (f=50mm), a digital delay generator (STC 810), a TTL-modulated 785nm continuous wave laser, an 800 / 10nm bandpass filter, and an in vivo imaging box. Specifically, the digital delay generator controls the laser pulse width, repetition rate, and the relative delay between the laser and the camera. The digital delay generator generates a TTL signal to activate the laser for a period of time and then turns it off. After a preset delay time, ensuring that the laser is completely turned off, the photocathode is activated. The digital delay generator controls the gate width. At this stage, the emission signal is captured by the CMOS, while the laser signal is blocked due to the photocathode being turned off, thus achieving time-gated imaging. Signal acquisition is achieved by opening the photocathode multiple times during a single CMOS exposure. By adjusting the delay time gate, the emission signal can be captured at different time points, ultimately generating an emission decay curve.

[0081] The time-resolved in vivo imaging system provided in this embodiment has higher sensitivity, enabling the combination of time-gated intensity imaging, lifetime imaging, and SPECT imaging to track the accumulation and metabolic pathways of nanoparticles in vivo.

[0082] Example 5

[0083] This embodiment provides an application of the time-resolved in vivo imaging system provided in Example 4 to the time-gated intensity imaging of the embodiment of the present invention.

[0084] First, a 10% Intralipid solution was diluted tenfold to create a 1% fat emulsion to simulate tissue scattering, as its scattering properties are similar to those of tissue. A quartz tube containing nanocrystals was encapsulated for Phantom imaging studies. The tube was placed beneath a culture dish and covered with 1% fat emulsion at varying thicknesses. The first image in the sequence captured during lifetime imaging was selected as the time-gated intensity image.

[0085] The results show that, see Figure 5 , it was observed that the fluorescence intensity also decreased linearly with decreasing excitation light power density, demonstrating the system's strong correlation and accuracy in intensity measurement and temporal control, indicating that the fluorescence of NaYF4:1%Tm,177Lu nanocrystals follows a single-photon process. Furthermore, the fluorescence intensity also decreased with decreasing concentration, but still maintained a strong linear correlation, demonstrating the stability and reliability of the laboratory-built imaging system. This linear relationship indicates that the system can quantitatively reflect concentration-dependent changes in fluorescence intensity, which is critical for applications requiring precise signal quantification.

[0086] Example 6

[0087] This embodiment provides an application of the time-resolved in vivo imaging system provided in Example 4 in the lifespan imaging of an embodiment of the present invention.

[0088] Referring to the experimental process of Example 5, the lifetime imaging image is obtained by processing multiple time-gated intensity images at different delay times. The luminescence lifetime of each pixel can be obtained by fitting the decay of each pixel with a single exponential function.

[0089] Results see Figure 4 and Figure 5 Despite changes in concentration, the fluorescence lifetime remains constant, confirming that lifetime imaging is inherently independent of the fluorescent probe's concentration. This is particularly advantageous in biological imaging, as changes in probe distribution or local accumulation, which typically affect intensity-based imaging, are less affected by lifetime measurements, making it a more robust and quantitative imaging modality.

[0090] Example 7

[0091] This embodiment provides a method for evaluating the performance of lifespan imaging under physiologically relevant conditions based on the time-resolved in vivo imaging system provided in Example 4.

[0092] In this example, a 1% intravenous fat emulsion solution was used to simulate in vivo tissue scattering, and the performance of intensity- and lifetime-based imaging was compared. Water-dispersible nanocrystals were encapsulated in small quartz tubes and covered with fat emulsion layers of varying thicknesses to simulate increased optical scattering. The results are consistent with previous observations that the strong scattering effect of the intravenous fat emulsion leads to a significant decrease in fluorescence intensity from intensity imaging. This decrease is not solely attributable to the thickness itself, but also suggests that multiple scattering events both attenuate the excitation light reaching the nanocrystals and scatter the emitted fluorescence, reducing signal intensity. Furthermore, scattering-induced intensity fluctuations can introduce artifacts in quantitative imaging, further complicating signal interpretation. However, lifetime imaging maintained remarkable stability across various penetration depths. The measured lifetime remained nearly constant regardless of the degree of scattering, further demonstrating that the lifetime signal is unaffected by variations in light scattering and absorption. This finding is crucial for in vivo imaging applications, where tissue heterogeneity and depth-dependent light attenuation often compromise the accuracy of intensity-based imaging. By leveraging the inherent stability of fluorescence lifetime measurements, lanthanide nanocrystals can be used for deep tissue imaging with minimal background interference, achieving precise, quantitative, and background-free imaging suitable for biomedical applications.

[0093] Example 8

[0094] This embodiment provides an application of time-gated intensity imaging, lifetime imaging, and SPECT imaging in analyzing the metabolic pathways of nanocrystals and observing their biodistribution. Figure 7 .

[0095] Nanocrystals were injected intravenously into mice. Time-resolved imaging can completely suppress interfering signals such as excitation light and autofluorescence, and time-gated intensity images were obtained. The results are shown in the figure. Figure 2 , achieving a signal-to-noise ratio of up to 2300. This high sensitivity enables detailed tracking of the accumulation and metabolic pathways of nanoparticles in the body. Figure 3 a. The signal-to-background ratio (SBR) of non-time-resolved imaging is 1.84, 2.20, and 2.91, while the SBR of time-resolved imaging after improvement in this report is 200. Figure 3 a, Figure 3The imaging results of b are obviously clearer in outline, and both accuracy and contrast are improved, which reduces the effect of tissue autofluorescence interference, thereby improving the accuracy and contrast of near-infrared II optical imaging. However, in the embodiment of the present invention, by utilizing the time-resolved in vivo imaging system independently built by the laboratory and the screening of imaging probes, its signal-to-background ratio is as high as 2300. It is known to those skilled in the art that SBR (Signal-to-Background Ratio) is a very important parameter in imaging technology, which is used to evaluate the quality and reliability of images. The definition of SBR is the ratio of signal intensity to background noise intensity. Specifically, the higher the SBR, the greater the intensity of the signal relative to the background noise, and the better the quality and clarity of the image. In imaging technologies such as SPECT and CT, by increasing SBR, high-quality images can be obtained at lower radiation doses, thereby reducing radiation exposure to patients. Therefore, this is a significant advancement in imaging technology, further enabling large-scale application of tracking the accumulation and metabolic pathways of nanoparticles in the body.

[0096] Furthermore, time-gated intensity imaging of the whole body of the mouse was performed one hour after injection, see Figure 8 The results showed that the nanocrystals mainly accumulated in the liver, and lifetime imaging further confirmed that the observed fluorescence originated from the injected NaYF4:1%Tm. 177 Lu@PEG nanocrystals were detected because the measured fluorescence lifetimes were identical to those measured in in vitro experiments. On the second day (24 hours), signals were detected in the liver and spleen of mouse 1 (M1), whereas no spleen signal was observed in mice M2 and M3. This was likely due to spatial overlap between liver and spleen signals, making it difficult to distinguish spleen-specific signals using two-dimensional imaging techniques. Notably, fluorescence signals were detected in the lungs on the third day. However, the weak signal intensity made it difficult to effectively extract lifetime information, possibly due to low nanoparticle concentration or increased scattering in lung tissue. In addition, fluorescence intensity imaging provided an estimate of nanoparticle clearance, as the overall intensity in the liver gradually decreased over time. However, due to variations in imaging settings, penetration depth, and physiological differences between mice between days, intensity measurements alone are insufficient to accurately quantify nanoparticle uptake. In contrast, no significant changes in fluorescence lifetime were observed using lifetime imaging, further confirming its robustness for tracking nanoparticles over long periods of time. Three days after injection, mice were euthanized, and excised liver and spleen tissues were subjected to ex vivo optical imaging. The results showed that although no obvious spleen signals (M2 and M3) were observed in in vivo imaging, nanocrystals were indeed present in the liver and spleen.

[0097] In previous time-gated intensity imaging and lifetime imaging modes, the signals from the liver and spleen overlapped, making it difficult to clearly observe the metabolic behavior of nanocrystals. Further use of SPECT / CT imaging to monitor the long-term biodistribution of nanocrystals in vivo is discussed in

[15] . Figure 6 . SPECT / CT is capable of high spatial resolution deep tissue radionuclide imaging, providing complementary insights into the pharmacokinetics of nanoparticles beyond the limitations of optical imaging. The results showed that the nanocrystals initially accumulated in the liver and then gradually moved to the spleen over time. Two hours after injection, obvious signals were observed in all three mice, mainly accumulating in the liver, with only weak signals in the spleen. By the second day (24 hours), the liver signal began to weaken, while spleen uptake increased significantly. By the third day (48 hours), spleen accumulation gradually increased, while the liver signal continued to weaken. This pattern was highly consistent with the results obtained by optical imaging. These findings indicate that SPECT / CT imaging provides high-fidelity, long-term in vivo tracking of nanoparticles, effectively solving the spatial resolution limitations of optical imaging and making biodistribution analysis more accurate.

[0098] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An imaging probe based on radionuclide labeling, characterized in that: The imaging probe comprises lanthanide nanocrystals, amphiphilic block copolymers self-assembled from the lanthanide nanocrystals, and radioactive nuclide ions labeled on the lanthanide nanocrystals, and the imaging probe simultaneously realizes time-gated intensity imaging, lifetime imaging and SPECT imaging.

2. The imaging probe according to claim 1, wherein The imaging probe includes the radionuclide ion labeled on the lanthanide nanocrystal, and the valence state of the radionuclide ion is the same as the valence state of the lanthanide ion in the lanthanide nanocrystal.

3. The imaging probe according to claim 1, wherein The lanthanide nanocrystals include NaL doped with at least one rare earth ion n F4, the doped rare earth ions replace part L n 3+ location.

4. The imaging probe according to claim 2, wherein: The radioactive nuclide ions replace L in the nanocrystals n 3 + location.

5. The imaging probe according to claim 4, characterized in that The radionuclide ions include 177 Lu 3+ 、 90 Y 3 + 、 153 Sm 3+ 、 166 Ho 3+ At least one of .

6. The imaging probe according to claim 1, wherein The amphiphilic block copolymer includes one of DSPE-PEG2000, polyethylene oxide-polypropylene oxide block copolymer (PEO-PPO-PEO), polyethylene oxide-polycaprolactone block copolymer (PEO-PCL), polyethylene oxide-polystyrene block copolymer (PEO-PS), polyethylene oxide-polyacrylic acid block copolymer (PEO-PAA), polyethylene oxide-polymethyl methacrylate block copolymer (PEO-PMMA), polyethylene oxide-polyacrylonitrile block copolymer (PEO-PAN), polyethylene oxide-polyacrylamide block copolymer (PEO-PAM), polyethylene oxide-polyglycerol adipate block copolymer (PEO-PGA), polyethylene oxide-polylactic acid block copolymer (PEO-PLA), and polyethylene oxide-polycaprolactone-polyethylene oxide block copolymer (PEO-PCL-PEO).

7. The imaging probe according to claim 1, wherein The characteristic emission peak displayed by the lanthanide nanocrystals under the excitation of a light source corresponds to the characteristic luminescence spectrum of doped rare earth ions.

8. The imaging probe according to claim 2, wherein: The ratio of the doped rare earth ions in the lanthanide nanocrystals to the total of the lanthanide ions and the rare earth particles is in the range of 0.1% to 3%.

9. A method for preparing an imaging probe based on radionuclide labeling, characterized in that: Here are the steps: Step 1: synthesizing oleic acid-coated lanthanide nanocrystals doped with rare earth ions by a solvothermal method and dispersing the nanocrystals in cyclohexane; Step 2: adding lanthanide nanocrystals doped with rare earth ions dispersed in cyclohexane to a saturated NOBF4-CH2Cl2 solution, collecting the precipitate, and dispersing the precipitate in methanol. The methanol-containing precipitate is added to a methanol solution containing DSPE-PEG2000, ultrasonically mixed, and the solvent is removed by rotary evaporation. The resulting nanoparticles are dispersed in deionized water. Step 3: adding a salt solution containing radionuclide ions to the nanoparticles obtained above, performing ultrasonic centrifugation at room temperature to separate the precipitate, and washing to obtain the radionuclide-labeled imaging probe.

10. An application of an imaging probe based on radionuclide labeling, characterized in that: These include: Monitor changes during tumor treatment; Study the distribution of radionuclide-labeled drugs in the body; Assess the functional status of the liver and kidneys.

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