A high light efficiency heterostructure rare earth nanometer fluorescence imaging agent and a preparation method and application thereof

Abstract

This invention discloses a high-efficiency heterostructure rare-earth nanofluorescent imaging agent, its preparation method, and its application. The rare-earth nanofluorescent imaging agent of this invention has a core-shell-shell structure, wherein the core of the core-shell-shell structure is a rare-earth ion luminescent layer core NaYbF4:Er. 3+ Ce 3+ ,Zn 2+ The rare earth ion luminescent layer core is sequentially coated with a rare earth ion sensitizer shell layer NaYbF4:Er 3+ Ce 3+ M 2+ The rare earth nano-fluorescent imaging agent of the present invention contains a heterogeneous inert protective shell MF2, wherein M is selected from at least one of Ca, Sr, and Ba. The rare earth nano-fluorescent imaging agent of the present invention can absorb excitation light in the ~980nm wavelength band and simultaneously generate emission light in the wavelength band from the visible region (400-700nm) to the near-infrared IIb region (1400-1700nm). It has advantages such as strong penetration into biological tissues and weak autofluorescence interference, which is beneficial for its application in deep tissue penetration in vivo real-time fluorescence imaging and tumor surgical navigation.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomedical imaging technology, and particularly relates to a heterostructured rare earth nanofluorescent imaging agent, its preparation method and application. Specifically, it relates to a high-efficiency MF2 heterostructure-coated core-shell-shell structured rare earth nanofluorescent imaging agent, its preparation method and its application in high-resolution rare earth upconversion luminescence and near-infrared fluorescence medical imaging in living organisms. Background Technology

[0002] Rare earth elements possess abundant electronic energy levels, capable of absorbing or emitting light waves from the ultraviolet to the visible and even near-infrared bands, giving them broad application prospects in the field of materials science. Due to their advantages such as broad fluorescence emission spectrum, good fluorescence stability, and low biotoxicity, rare earth nanofluorescent imaging agents show great application potential in fields such as medical detection, in vivo imaging, drug delivery, and real-time surgical navigation.

[0003] As a medical imaging technique, fluorescence imaging currently commonly uses emission bands in the visible light region (390–780 nm) and the near-infrared I region (750–900 nm). The emission bands in this region overlap with the emission bands of autofluorescence in biological tissues, resulting in problems such as high background, high scattering, and low penetration depth in the imaging results. In contrast, emission light in the near-infrared IIb region (NIR-IIb, 1500–1700 nm) not only distinguishes itself from tissue autofluorescence, avoiding high background and high scattering, but also has a deeper penetration depth, significantly improving the resolution of the imaging image. Among various developed NIR-IIb optical probes, rare-earth-doped nanomaterials possess advantages such as high photostability, tunable luminescence, narrow emission band, and long luminescence lifetime, leading to their widespread application in biomedicine. However, their low fluorescence quantum yield limits their development. Core-shell structure design can reduce surface quenching, lattice defects, and external interference, improving luminescence efficiency and enhancing material properties.

[0004] Rare earth nanomaterials can be classified into two types based on their core-shell structure: homogeneous and heterogeneous. Most existing research focuses on homogeneous core-shell structures, but their preparation often encounters the problem of cation mixing, leading to compromised structural integrity of the core nanoparticles and redistribution of optically active centers that should be confined within the core, thus reducing luminescence efficiency. In contrast, the growth of heterogeneous shells can form true core-shell structures with clear and thin interfaces, maintaining the integrity and chemical stability of the structural domains, effectively preventing unnecessary energy loss and surface quenching, thereby significantly improving quantum yield. However, achieving heterogeneous growth typically requires meticulous core-shell structure synthesis design, which not only increases the complexity of the synthesis steps but also reduces the yield, and the obtained core-shell materials often exhibit poor structural uniformity. Summary of the Invention

[0005] To overcome the shortcomings of the existing technology, the present invention provides the following technical solution:

[0006] A rare-earth nano-fluorescent imaging agent has a core-shell-shell structure, wherein the core of the core-shell-shell structure is a rare-earth ion luminescent layer core NaYbF4:Er 3+ Ce 3+ Zn 2+ The rare earth ion luminescent layer core is sequentially coated with a rare earth ion sensitizer shell layer NaYbF4:Er 3+ Ce 3+ M 2+ , and a heterogeneous inert protective shell MF2, wherein M is selected from at least one of Ca, Sr, and Ba.

[0007] In this invention, the introduction of two shells in the developer specifically involves a rare earth ion sensitizer shell and a heterogeneous inert protective shell, thus making it a heterogeneous rare earth nano-fluorescent developer.

[0008] The inventors discovered that when the rare earth ion sensitizer shell NaYbF4:Er 3+ Ce 3+ M 2+ Medium doped M 2+ This process facilitates the growth of the heterogeneous inert protective shell MF2, thereby obtaining a heterogeneous rare earth nano-fluorescent imaging agent with uniform particle size.

[0009] According to an embodiment of the present invention, M is preferably Ca. More preferably, the rare earth ion sensitizer shell is preferably NaYbF4:Er 3+ Ce 3+ Ca 2+ Furthermore, the heterogeneous inert protective shell is preferably CaF2.

[0010] According to an embodiment of the present invention, the rare earth ion luminescent layer core NaYbF4:Er 3+ Ce 3+ Zn 2+ In the middle, the first doped ion includes Er 3+ Ce 3+ and Zn 2+ The total molar concentration of the first doped ion is 1-10 mol%, exemplarily 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol%; furthermore, Er 3+ The doping concentration is 0-10 mol% (e.g., 1, 2, 3, 4, 5, 6, 7, 8 mol%); furthermore, Ce 3+ The doping concentration is 0-10 mol% (e.g., 1, 2, 3, 4, 5, 6, 7, 8 mol%); furthermore, Zn2+ The doping concentration is 0-10 mol% (e.g., 1, 2, 3, 4, 5, 6, 7, 8 mol%).

[0011] According to an embodiment of the present invention, the rare earth ion luminescent layer core NaYbF4:Er 3+ Ce 3+ Zn 2+ The thickness is 0-30nm excluding 0, preferably 2-20nm, for example 2, 5, 10, 15 or 20nm.

[0012] According to an embodiment of the present invention, the rare earth ion sensitizer shell NaYbF4:Er 3+ Ce 3+ M 2+ In the middle, the second doped ion includes Er 3+ Ce 3+ and M 2+ (M is preferably Ca), the total molar concentration of the second doped ion is 1-10 mol%, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol%; furthermore, Er 3+ The doping concentration is 0-10 mol% (e.g., 1, 2, 3, 4, 5, 6, 7, 8 mol%); furthermore, Ce 3+ The doping concentration is 0-10 mol% (e.g., 1, 2, 3, 4, 5, 6, 7, 8 mol%); furthermore, M 2+ The doping concentration is 0-10 mol% (e.g., 1, 2, 3, 4, 5, 6, 7, 8 mol%).

[0013] According to an embodiment of the present invention, the rare earth ion sensitizer shell NaYbF4:Er 3+ Ce 3+ M 2+ The thickness is 0-20nm excluding 0, preferably 0.1-8nm, for example 0.5, 1, 2, 3, 4, 5, 6, 7, 8nm.

[0014] According to an embodiment of the present invention, the thickness of the heterogeneous inert protective shell MF2 is 0-20 nm and does not include 0, preferably 0.5-10 nm, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nm.

[0015] According to an embodiment of the present invention, the particle size of the rare earth nanofluorescent imaging agent is 5-80 nm, preferably 10-50 nm, for example 15, 20, 25, 30, 35, 40, 45 nm.

[0016] According to an embodiment of the present invention, the rare earth nano-fluorescent imaging agent is a crystal, preferably having a cubic phase structure.

[0017] According to an embodiment of the present invention, the rare earth nanofluorescent imaging agent is optionally further loaded with a modified ligand, the modified ligand being loaded on the surface of the heterogeneous inert protective shell.

[0018] According to an embodiment of the present invention, the modified ligand is selected from water-soluble ligands, preferably at least one of water-soluble ligands without tumor-targeting function, biomacromolecules with targeting function, or biosmall molecules.

[0019] According to an embodiment of the present invention, the water-soluble ligand without tumor-targeting function is selected from at least one of methoxy polyethylene glycol alendronate (MPEG-ALE), polyacrylic acid (PAA), and distearate phosphatidylethanolamine-polyethylene glycol (DSPE-PEG).

[0020] According to an embodiment of the present invention, the biomacromolecule with targeting function is selected from peptides and / or antibodies, such as phospholipid polyethylene glycol penetrant peptide (DSPE-PEG2000-iRGD).

[0021] According to an embodiment of the present invention, the biomolecule is selected from at least one of protein kinase inhibitors, poly(ADP-ribose) polymerase (PARP) inhibitors, HDAC inhibitors, proteasome inhibitors, and Hedgehog pathway inhibitors, for example, folic acid.

[0022] According to an exemplary embodiment of the present invention, the surface load of the heterogeneous inert protective shell layer is DSPE-PEG-2000 and DSPE-PEG2000-iRGD, and the mass ratio of DSPE-PEG-2000 and DSPE-PEG2000-iRGD is (5~10):2, exemplarily 5:2, 6:2, 7:2, 8:2, 9:2 or 10:2.

[0023] According to an embodiment of the present invention, the rare earth nano-fluorescent imaging agent has essentially the following properties: Figure 1 The transmission electron microscope image shown in the middle left figure.

[0024] According to an embodiment of the present invention, the rare earth nano-fluorescent imaging agent has essentially the following properties: Figure 2 The element distribution diagram shown.

[0025] According to an embodiment of the present invention, the rare earth nano-fluorescent imaging agent has essentially the following properties: Figure 3 The diagram shows the energy transfer of rare earth ions.

[0026] According to an embodiment of the present invention, the rare-earth nano-fluorescent imaging agent, when excited by 980 nm excitation light, exhibits upconversion luminescence in the visible light region and luminescence in the near-infrared IIb region, with the luminescence spectrum essentially as follows: Figure 4 As shown.

[0027] According to an embodiment of the present invention, when the rare earth nano-fluorescent imaging agent is loaded with modified ligands, its hydrated particle size is 10-500 nm, for example 20-200 nm, and exemplaryly 50, 80, 100, 120, 140, 160, 180 nm, specifically as follows: Figure 5 As shown.

[0028] The present invention also provides a method for preparing the above-mentioned rare earth nano-fluorescent imaging agent, wherein the preparation method preferably employs at least one of the following methods: high-temperature co-precipitation method, high-temperature thermal decomposition method, hydrothermal method, or sol-gel method.

[0029] According to an exemplary embodiment of the present invention, the high-temperature pyrolysis method includes the following steps:

[0030] S1. Preparation of rare earth ion luminescent layer core NaYbF4:Er 3+ Ce 3+ Zn 2+ ;

[0031] S2, in the rare earth ion luminescent layer core NaYbF4:Er 3+ Ce 3+ Zn 2+ On the surface, a rare earth ion sensitizer shell NaYbF4:Er was prepared by shell epitaxial growth method. 3+ Ce 3+ M 2+ The core-shell structure NaYbF4:Er was obtained. 3+ Ce 3+ Zn 2+ @NaYbF4:Er 3+ Ce 3+ M 2+ ;

[0032] S3, the rare earth ion sensitizer shell layer NaYbF4:Er with a core-shell structure from step S2. 3+ Ce 3+ Zn 2+ On the surface of NaYbF4:Er, a heterogeneous inert protective shell MF2 was prepared to obtain a core-shell-shell structure. 3+ Ce 3+ Zn 2+ @NaYbF4:Er 3+ Ce 3+ M 2+ @MF2 is the rare earth nano fluorescent imaging agent.

[0033] According to an embodiment of the present invention, when preparing the rare earth nanofluorescent material, the molar ratio of M in the heterogeneous inert protective shell to Yb in the rare earth ion sensitizer shell is not greater than 4:1, for example (4-0.1):1, or (4-0.01):1, and exemplary ratios are 3:1, 2:1, 1:1, and 0.1:1.

[0034] According to an embodiment of the present invention, step S1, the preparation of the rare earth ion luminescent layer core specifically includes the following steps: adding erbium trifluoroacetate, ytterbium trifluoroacetate, cerium trifluoroacetate, zinc trifluoroacetate, and sodium trifluoroacetate to a first solvent, heating to 250-350°C in an inert atmosphere, and reacting for 10-120 minutes to obtain the rare earth ion luminescent layer core NaYbF4:Er 3+ Ce 3+ Zn 2 + .

[0035] According to an embodiment of the present invention, in step S1, the molar ratio of sodium in sodium trifluoroacetate, ytterbium in ytterbium trifluoroacetate, erbium in erbium trifluoroacetate, cerium in cerium trifluoroacetate, and zinc in zinc trifluoroacetate is 1:(0-1):(0-1):(0-1):(0-1) and is not 0, for example, 1:0.20:0.05:0.02:0.01.

[0036] According to an embodiment of the present invention, the first solvent comprises at least one of oleic acid, oleylamine and octadecene, preferably a mixture of oleic acid, oleylamine and octadecene, for example, by molar amount, the first solvent comprises 1 part oleic acid, (0.5-1) parts oleylamine and (0.5-1) parts octadecene.

[0037] According to an embodiment of the present invention, step S1 further includes the following step before heating: heating under vacuum until sodium trifluoroacetate, ytterbium trifluoroacetate, erbium trifluoroacetate, cerium trifluoroacetate, and zinc trifluoroacetate dissolve in a solvent.

[0038] According to an embodiment of the present invention, after the reaction, step S1 further includes the following steps: allowing the reacted solution to cool naturally, precipitating, washing and dispersing.

[0039] According to an embodiment of the present invention, in step S2, the shell epitaxial growth method includes the following steps: growing the rare earth ion luminescent layer core NaYbF4:Er from step S1... 3+ Ce 3+ Zn 2+Erbium trifluoroacetate, ytterbium trifluoroacetate, cerium trifluoroacetate, a trifluoroacetate salt of M (e.g., calcium trifluoroacetate), and sodium trifluoroacetate are added to a second solvent and mixed. The mixture is then heated to 250-350°C in an inert atmosphere and reacted for 10-120 minutes to obtain the rare earth ion sensitizer shell NaYbF4:Er 3+ Ce 3+ M 2+ The core-shell structure NaYbF4:Er was obtained. 3+ Ce 3+ Zn 2+ @NaYbF4:Er 3+ Ce 3+ M 2+ .

[0040] According to an embodiment of the present invention, in step S2, the molar ratio of sodium:ytterbium:erbium:cerium:M in the trifluoroacetate erbium, trifluoroacetate ytterbium, trifluoroacetate cerium, M trifluoroacetate salt and sodium trifluoroacetate is 1:(0-1):(0-1):(0-1):(0-1), for example 1:0.01:0.01:0.01:0.01.

[0041] According to an embodiment of the present invention, step S3 specifically includes the following steps: first, the core-shell structure NaYbF4:Er 3+ Ce 3+ Zn 2+ @NaYbF4:Er 3+ Ce 3+ M 2+ The trifluoroacetate salt of M (such as calcium trifluoroacetate) is added to the second solvent and mixed. The mixture is heated to 250-350℃ in an inert atmosphere and reacted for 10-120 minutes to obtain a heterogeneous inert protective shell MF2, thus obtaining the rare earth nano fluorescent imaging agent, which has a heterogeneous structure.

[0042] According to an embodiment of the present invention, the second solvent comprises at least one of oleic acid and octadecene, preferably a mixture of oleic acid and octadecene, for example, by molar amount, the second solvent comprises 1 part oleic acid and (0.5-1.5) parts octadecene.

[0043] According to an embodiment of the present invention, the preparation method further includes: after preparing the heterogeneous inert protective shell MF2 in step S3, optionally performing the following step: loading a modified ligand onto the surface of the heterogeneous inert protective shell. Preferably, the modified ligand has the definition described above.

[0044] According to an embodiment of the present invention, loading modified ligands onto the surface of a heterogeneous inert protective shell specifically includes: mixing the core-shell-shell structured rare-earth nanofluorescent imaging agent and the modified ligand in an organic solvent, stirring at a temperature below 40°C for 1-24 hours, and removing the organic solvent to obtain the rare-earth nanofluorescent imaging agent loaded with the modified ligand. Preferably, the organic solvent is selected from chloroform and dichloromethane. Preferably, the removal of the organic solvent can be carried out using methods known in the art, such as rotary evaporation; furthermore, after removing the organic solvent, ultrapure water can be added for washing.

[0045] The present invention also provides a developer comprising the rare earth nano-fluorescent developer as described above, wherein the surface of the heterogeneous inert protective shell of the rare earth nano-fluorescent developer is loaded with modified ligands, and the modified ligands have the meanings described above.

[0046] According to an embodiment of the present invention, the developer is capable of emitting light in the visible green region (around 540 nm) and the visible red region (around 650 nm) using a 980 nm laser as a light source.

[0047] According to an embodiment of the present invention, the developer is capable of emitting near-infrared light in the near-infrared IIb region to around 1530nm using a 980nm laser as a light source.

[0048] The present invention also provides the above-mentioned rare earth nano fluorescent imaging agents and / or the use of the imaging agents in fluorescence imaging, such as for medical imaging.

[0049] According to an embodiment of the present invention, the medical imaging includes real-time fluorescence imaging and / or surgical navigation.

[0050] Beneficial effects

[0051] (1) The heterostructure rare earth nanofluorescent imaging agent with core-shell-shell structure of the present invention provides a template for the layer-by-layer epitaxial growth method through the synthesized rare earth ion luminescent core, which ensures the cubic phase of the nanoparticles. The heterostructure shell coating has stronger luminescence than the homostructure shell coating, thereby enabling the heterostructure fluorescent nanofluorescent imaging agent of the present invention to have high-brightness rare earth visible upconversion luminescence and NIR-IIb fluorescence emission.

[0052] (2) At the same time, unlike the existing rare earth-based core-shell structures which all rely on homogeneous tetrafluororare earth sodium core and shell, the outermost layer of the nano fluorescent imaging agent of the present invention is a completely inert MF2 that can maintain the structural and chemical integrity of the two structural domains, while effectively preventing unnecessary energy migration and surface quenching, thereby significantly improving the quantum yield.

[0053] (3) The rare earth nanofluorescent imaging agent with core-shell structure of the present invention, after surface modification with water-soluble ligands without tumor targeting effect or antibodies, small biological molecules and large biological molecules with tumor targeting effect, improves biocompatibility and biosafety, and can have a targeting effect on cancer cells, and has good application prospects in the field of biological in vivo imaging.

[0054] (4) The core-shell-shell heterostructure rare earth nanofluorescent imaging agent of the present invention can absorb excitation light in the ~980nm band and generate emission light in the band from 400-700nm in the visible region to 1400-1700nm in the near-infrared IIb region. It has the characteristics of high quantum yield and long emission lifetime, which provides more possibilities for the biomedical application of this rare earth nanofluorescent medical imaging agent. For example, it has upconversion fluorescence in the visible region with a wavelength of 400-700nm, which is beneficial for the application of visualization of cell microscopic confocal imaging and upconversion luminescence imaging of living organisms. The strong luminescence in the near-infrared IIb region with a wavelength of 1400-1700nm has the advantages of strong biological tissue penetration and weak autofluorescence interference, which is beneficial for its application in deep tissue penetration biological living real-time fluorescence imaging and tumor surgery navigation, etc., and can obtain universal high-resolution, high signal-to-noise ratio vascular medical images. Attached Figure Description

[0055] Figure 1 These are transmission electron microscope images of the rare earth nano-fluorescent imaging agents prepared in Example 1 and Comparative Example 2 of the present invention.

[0056] Figure 2 The elemental distribution diagram of the rare earth nano-fluorescent imaging agent prepared in Example 1 of this invention is shown.

[0057] Figure 3 This is a schematic diagram of rare earth ion energy transfer in the rare earth nano-fluorescent imaging agent prepared in Example 1 of the present invention.

[0058] Figure 4 The rare earth nano-fluorescent imaging agents of Comparative Example 1 and Example 1 of the present invention exhibit upconversion luminescence in the visible light region and luminescence in the near-infrared IIb region under 980 nm excitation, respectively.

[0059] Figure 5 The images shown are transmission electron microscope (TEM) images and dynamic light scattering particle size distribution diagrams of the surface-modified rare-earth nano-fluorescent imaging agent in Example 2 of this invention.

[0060] Figure 6 The 24-hour cytotoxicity of the surface-modified rare-earth nano-fluorescent imaging agent in Example 2 of this invention;

[0061] Figure 7These are medical imaging images of upconversion luminescence in cells excited by a 980nm laser in Application Example 1 of this invention.

[0062] Figure 8 This is an example of an in vivo fluorescence medical imaging image, a microscopic medical imaging image, and the corresponding minimum imaging resolution in the near-infrared IIb region (~1530nm) of the present invention, as shown in Application Example 2. Detailed Implementation

[0063] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of 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 content of the present invention are covered within the scope of protection intended by the present invention.

[0064] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0065] Comparative Example 1:

[0066] Preparation of NaYbF4:Er 3+ Ce 3+ Zn 2+ @NaYbF4:Er 3+ Ce 3+ Ca 2+ @NaYF4 core-shell-shell structure contrast nanofluorescent imaging agent can be prepared by methods known in the art for preparing nanomaterials, such as hot injection, high-temperature coprecipitation, high-temperature thermal decomposition, hydrothermal method, or sol-gel method.

[0067] This comparative example uses a high-temperature thermal decomposition method, which specifically includes the following steps:

[0068] S101. Weigh 0.1360 g sodium trifluoroacetate, 0.4962 g ytterbium trifluoroacetate, 0.0051 g erbium trifluoroacetate, 0.0032 g cerium trifluoroacetate, and 0.0029 g zinc trifluoroacetate into a three-necked flask at room temperature. Add 3.17 mL oleic acid, 3.29 mL oleylamine, and 6.40 mL octadecene as a mixed solvent. Heat under vacuum until the sodium trifluoroacetate and gadolinium trifluoroacetate salts dissolve. Continue heating to 310 °C under an inert atmosphere. After reacting for 1 hour, allow to cool naturally to room temperature, precipitate, and wash to obtain α-phase NaYbF4:Er 3+ Ce 3+ Zn 2+ Nanoparticles.

[0069] S102. The α-phase NaYbF4:Er prepared in step S101 is... 3+ Ce 3+ Zn2+ Add the following to a mixed solvent of 6.4 mL oleic acid and 6.4 mL octadecene: 0.1360 g sodium trifluoroacetate, 0.4962 g ytterbium trifluoroacetate, 0.0051 g erbium trifluoroacetate, 0.0032 g cerium trifluoroacetate, and 0.0027 g calcium trifluoroacetate. Heat under vacuum until the trifluoroacetates dissolve, then continue heating to 310 °C under an inert atmosphere. After reacting for 1 hour, allow to cool naturally to room temperature, precipitate, and wash to obtain the desired product.

[0070] NaYbF4:Er 3+ Ce 3+ Zn 2+ @NaYbF4:Er 3+ Ce 3+ Ca 2+ Nanoparticles with a particle size of approximately 11 nm.

[0071] S103. The NaYbF4:Er prepared in step S102 3+ Ce 3+ Zn 2+ @NaYbF4:Er 3+ Ce 3+ Ca 2+ Add 0.2561 g of ytterbium trifluoroacetate to a mixed solvent of 6.4 mL oleic acid and 6.4 mL octadecene; then add 0.2561 g of ytterbium trifluoroacetate; heat under vacuum until the calcium trifluoroacetate dissolves, then continue heating to 310 °C under an inert atmosphere, react for 1 hour, and allow to cool naturally to room temperature. The precipitate is then washed to obtain...

[0072] NaYbF4:Er 3+ Ce 3+ Zn 2+ @NaYbF4:Er 3+ Ce 3+ Ca 2+ @NaYF4 nanocrystals, with a particle size of approximately 13 nm, are the core-shell-shell structure contrast fluorescent nano-imaging agents prepared in this comparative example.

[0073] Example 1:

[0074] Preparation of core-shell-shell structured nanofluorescent imaging agents

[0075] NaYbF4:Er 3+ Ce 3+ Zn 2+ @NaYbF4:Er 3+ Ce 3+ Ca 2+@CaF2 can be prepared using methods known in the art for preparing nanomaterials, such as hot injection, high-temperature co-precipitation, high-temperature thermal decomposition, hydrothermal method, or sol-gel method. This embodiment uses the high-temperature thermal decomposition method, and the preparation method specifically includes the following steps:

[0076] S201. Preparation of the rare-earth ion-emitting core: Weigh 0.1360 g sodium trifluoroacetate, 0.4962 g ytterbium trifluoroacetate, 0.0051 g erbium trifluoroacetate, 0.0032 g cerium trifluoroacetate, and 0.0029 g zinc trifluoroacetate into a three-necked flask at room temperature. Add 3.17 mL oleic acid, 3.29 mL oleylamine, and 6.40 mL octadecene as a mixed solvent. Heat under vacuum until the sodium trifluoroacetate and gadolinium trifluoroacetate salts dissolve. Continue heating to 310 °C under an inert atmosphere. After reacting for 1 hour, allow to cool naturally to room temperature, precipitate, and wash to obtain the α-phase NaYbF4:Er 3+ Ce 3+ ,Zn 2+ The nanoparticles are rare-earth ion luminescent cores; the molar content of doped ions in these rare-earth ion luminescent cores is NaYbF4:1%Er. 3+ 1% Ce 3+ 1% Zn 2+ .

[0077] S202. Preparation of rare earth ion sensitizer shell: The α phase prepared in step S101...

[0078] NaYbF4:Er 3+ Ce 3+ ,Zn 2+ Add the following to a mixed solvent of 6.4 mL oleic acid and 6.4 mL octadecene: sodium trifluoroacetate, 0.4962 g ytterbium trifluoroacetate, 0.0051 g erbium trifluoroacetate, 0.0032 g cerium trifluoroacetate, and 0.0027 g calcium trifluoroacetate. Heat under vacuum until the trifluoroacetates dissolve, then continue heating to 310 °C under an inert atmosphere. After reacting for 1 hour, allow to cool naturally to room temperature, precipitate, and wash. Obtain the rare earth ion sensitizer shell NaYbF4:Er on the surface of the rare earth ion luminescent core via epitaxial growth. 3+ Ce 3+ Ca 2+ denoted as NaYbF4:Er 3+ Ce 3+ ,Zn 2+ @NaYbF4:Er 3+ Ce 3+ Ca 2+ Nanoparticles with a particle size of approximately 11 nm; in the shell of this rare earth ion sensitizer, the molar content of doped ions is NaYbF4:1%Er 3+1% Ce 3+ 1% Ca 2+ .

[0079] S203. Preparation of heterogeneous inert protective layer: The layer prepared in step S202...

[0080] NaYbF4:Er 3+ Ce 3+ Zn 2+ @NaYbF4:Er 3+ Ce 3+ Ca 2+ Nanocrystals were added to a mixed solvent of 6.4 mL oleic acid and 6.4 mL octadecene, followed by 0.1331 g of calcium trifluoroacetate. The mixture was heated under vacuum until the calcium trifluoroacetate dissolved, then heated to 310 °C under an inert atmosphere. After reacting for 1 hour, the mixture was allowed to cool naturally to room temperature, precipitate was formed, and washed to obtain the desired product.

[0081] NaYbF4:Er 3+ Ce 3+ Zn 2+ @NaYbF4:Er 3+ Ce 3+ Ca 2+ @CaF2 nanocrystals, with a particle size of approximately 13 nm, are the core-shell-shell structured nanofluorescent imaging agents of this embodiment.

[0082] Comparative Example 2:

[0083] Preparation of NaYbF4:Er 3+ Ce 3+ Zn 2+ @NaYbF4:Er 3+ Ce 3+ The core-shell-shell structure of @CaF2 was compared with that of a nano-fluorescent imaging agent. The preparation method was basically the same as in Example 1, except that calcium trifluoroacetate was not added when preparing the rare earth ion sensitizer shell in step S202; the remaining conditions and steps were the same as in Example 1, resulting in NaYbF4:Er 3+ Ce 3+ Zn 2+ @NaYbF4:Er 3+ Ce 3 + @CaF2 nanocrystals are the core-shell-shell structured nanofluorescent imaging agents used in this comparative example.

[0084] Test Example 1

[0085] Figure 1The left-middle image shows a transmission electron microscope (TEM) image of the core-shell-shell structured rare-earth nanofluorescent imaging agent prepared in Example 1. As can be seen from the image, the nanomaterial prepared in this example exhibits a distinct core-shell structure and a particle size of approximately 13 nm. In contrast, Figure 1 The middle right figure shows the nano-imaging agent prepared in Comparative Example 2. Its particle size is uneven and its purity is not high, making it unsuitable as a drug.

[0086] See Figure 2 The image shown is a HAADF-STEM image (high-angle annular dark-field scanning transmission electron microscope) of the rare earth nanofluorescent imaging agent with a core-shell-shell structure prepared in Example 1, and the elemental distribution diagrams of Na, Er, Ce, Yb, and Ca.

[0087] See Figure 3 The diagram shows the energy transfer pathway of the core-shell-shell structured rare-earth nanofluorescent imaging agent prepared in Example 1, where Yb 3+ Ions act as sensitizers, filling Yb by pumping electrons. 3+ of 2 F 5 / 2 To harvest 980nm photons. Highly efficient Yb 3+ →Er 3+ Energy transfer ensures Er 3+ stimulated to the middle 4 I 11 / 2 Energy level. An intermediate state with a sufficiently long lifetime during upconversion emission. 4 I 11 / 2 Er 3+ The ions were further excited to higher levels 2 H 11 / 2 and 4 S 3 / 2 The energy level then undergoes upconversion emission. For NIR-IIb emission, the short lifetime... 4 I 11 / 2 Excited-state lifetimes will allow for rapid nonradiative decay to 4 I 13 / 2 Horizontal, then near-infrared emission at 1550 nm. Via Ce 3+ Doping promotes Er 3+4 I 11 / 2 → 4 I 13 / 2 nonradiative relaxation, Er 3+4 I 13 / 2 Significant energy level filling suppresses upconversion and enhances downconversion emission, resulting in enhanced 1550nm luminescence of nanoparticles.

[0088] Test Example 2

[0089] See Figure 4 NaYbF4:Er prepared as Comparative Example 13+ Ce 3+ Zn 2+ @

[0090] NaYbF4:Er 3+ Ce 3+ Ca 2+ @NaYF4 nanofluorescent imaging agent, comparative example 2NaYbF4:Er 3+ Ce 3+ Zn 2+ @NaYbF4:Er 3+ Ce 3+ A comparison of the core-shell-shell structure of @CaF2 with that of the nanofluorescent imaging agent and NaYbF4:Er prepared in Example 1. 3+ Ce 3+ Zn 2+ @NaYbF4:Er 3+ Ce 3+ Ca 2+ The CaF2 nano-fluorescent imaging agent exhibits upconversion luminescence in the visible light region (400-700 nm) and luminescence in the near-infrared region (IIb region) (1500-1700 nm) under 980 nm excitation; [The text abruptly ends here, likely due to an incomplete translation or missing information.] Figure 4 It can be seen that the fluorescence intensity of the nanocrystals prepared in this embodiment is significantly higher than that of Comparative Example 1 and Comparative Example 2.

[0091] Example 2:

[0092] The preparation method of surface-modified rare-earth nano-fluorescent imaging agent is as follows:

[0093] Weigh out the core-shell-shell structured rare earth nanofluorescent imaging agent prepared in Example 1, and add 8 times the mass of the rare earth nanofluorescent imaging agent, namely distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000, purchased from Shanghai Pengshuo Biotechnology Co., Ltd.), and 2 times the mass of the nanofluorescent imaging agent, namely phospholipid polyethylene glycol penetrating peptide (DSPE-PEG2000-iRGD, purchased from Xi'an Ruixi Biotechnology Co., Ltd.), to a globular flask. Add 30 mL of chloroform as a solvent, mix ultrasonically, and stir overnight at room temperature. Remove the remaining chloroform from the flask by rotary evaporation, add ultrapure water, disperse, and centrifuge to obtain the precipitate, which is the surface-modified rare earth nanofluorescent imaging agent.

[0094] NaYbF4:Er 3+ Ce 3+ Zn 2+ @NaYbF4:Er 3+ Ce 3+ Ca 2+ @CaF2@DSPE-PEG2000-iRGD dispersed the precipitate in physiological saline.

[0095] See Figure 5 The figures show a transmission electron microscope (TEM) image of the surface-modified rare-earth nanofluorescent imaging agent prepared in this embodiment in physiological saline and a dynamic light scattering particle size distribution over two weeks. As can be seen from the figures, the surface-modified rare-earth nanofluorescent imaging agent is uniformly dispersed in physiological saline, with a hydrated particle size of approximately 120 nm, and no aggregation occurred within 14 days. This indicates that when surface-modifying the rare-earth nanofluorescent imaging agent, the simultaneous addition of DSPE-PEG2000-iRGD and DSPE-PEG2000 not only enhances the targeting ability of the imaging agent but also its water solubility.

[0096] See Figure 6 The image shows the biosafety test of the surface-modified rare-earth nanofluorescent imaging agent prepared in this embodiment. The specific test method is as follows:

[0097] Based on 24-hour cytotoxicity, different concentrations (0 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL) of surface-modified rare-earth nano-fluorescent imaging agents were co-incubated with different cell types (human gastric cancer cells MGC803, human lung cancer cells A549, human glioma cells U87, and human pancreatic cancer cells PANC-1, all purchased from Fenghui Biotechnology). The effects of the rare-earth nano-fluorescent imaging agents on the growth inhibition of different cell types were compared using the CCK8 colorimetric assay (a commercially available CCK8 detection kit).

[0098] Different cell lines were seeded at a density of 4000 cells / well in 96-well plates. After 48 h of seeding, the rare earth nano-fluorescent imaging agent prepared in Example 1 was diluted to 0, 0.25, 0.5, 1, 2, 5, 10, and 20 μM, respectively, and 100 μL was added to each well, with 4 replicates per group. After incubation at 37°C for 48 h, 10 μL of CCK8 was added to each well and incubated in the dark for 1 h. The OD value at 450 nm was measured using a multi-functional microplate reader, and the viability was calculated.

[0099] Survival rate calculation formula: Survival rate = [(As-Ab) / (Ac-Ab)] × 100%.

[0100] Depend on Figure 6 It can be seen that when the concentration of the surface-modified rare earth nano-fluorescent imaging agent reaches 100ug / mL, the cell survival rate is still higher than 75% after co-incubation for 24 hours. This indicates that the surface-modified rare earth nano-fluorescent imaging agent prepared in this embodiment has low biotoxicity and can be used for confocal cell microscopy and small animal in vivo imaging experiments.

[0101] Application Example 1:

[0102] Surface-modified rare-earth nanofluorescent imaging agents are used for visualizing cell microscopic confocal imaging.

[0103] The rare-earth nanofluorescent imaging agent coated with surface-modified ligands prepared in Example 2 was applied to cell imaging: 6*10 cells were seeded in a confocal dish (containing 1 mL of 1640 complete culture medium). 4 Human gastric cancer cells (MGC803) were incorporated into the confocal dish after overnight adhesion, followed by the addition of surface-modified rare-earth nanofluorescent imaging agents to achieve a final concentration of 200 μg / mL. After co-incubation at 37°C for 6 hours, excess nanoparticles were removed using PBS / NaCl aqueous solution, followed by fixation with paraformaldehyde fixative. The nuclei were stained with DAPI dye (4',6-diamidinyl-2-phenylindole dye), and the cells were then excited by a 980 nm laser. Figure 7 As shown, upconversion luminescence medical imaging of green light emission (~540nm) and red light emission (~650nm) in cells can be observed simultaneously under a confocal fluorescence microscope.

[0104] Application Example 2:

[0105] Surface-modified rare-earth nanofluorescent imaging agents applied to in vivo fluorescence imaging in the NIR-IIb region

[0106] Animal experiments were approved by the Laboratory Animal Ethics Committee of Fujian Medical University. BALB / c nude mice (female, 4 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed in the animal facility of the New Drug Safety Evaluation Center of Fujian Medical University.

[0107] The surface-modified rare-earth nanofluorescent imaging agent prepared in Example 2 was injected into BALB / cnude nude mice via the tail vein (injection dose of 150 mg / kg), and the NIR-IIb region fluorescence signal in the nude mice was obtained by a near-infrared imaging system.

[0108] See Figure 8 The images show in vivo fluorescence medical imaging, microscopic medical imaging, and corresponding imaging resolution in the NIR-IIb region (~1530nm). As can be seen from the images, the microscopic imaging system can clearly distinguish the arteries and veins of the thigh and the surrounding vascular branches when magnifying the abdominal venous plexus and the femoral artery and vein of the mouse. In the images, the edges of the small blood vessels in the abdomen are sharp, and the resolution can reach 25μm.

[0109] Therefore, when rare earth nanofluorescent imaging agents are coated with proteins or small molecules that can specifically bind to tumor cells, the rare earth nanofluorescent imaging agents can accumulate on the surface of tumor cells, and when used for in vivo imaging, clear images of tumor tissue can be obtained.

[0110] The exemplary embodiments of the present invention have been described above. However, the scope of protection of this application is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A rare-earth nano-fluorescent imaging agent, characterized in that, The rare earth nano-fluorescent imaging agent has a core-shell-shell structure, wherein the core of the core-shell-shell structure is a rare earth ion luminescent layer core NaYbF4:Er 3+ Ce 3+ Zn 2+ The rare earth ion luminescent layer core is sequentially coated with a rare earth ion sensitizer shell layer NaYbF4:Er 3+ Ce 3+ M 2+ , and a heterogeneous inert protective shell MF2, wherein M is selected from at least one of Ca, Sr, and Ba.

2. The rare earth nano-fluorescent imaging agent according to claim 1, characterized in that, M is Ca; the rare earth ion sensitizer shell is NaYbF4:Er 3+ Ce 3+ Ca 2+ The heterogeneous inert protective shell is CaF2.

3. The rare earth nano-fluorescent imaging agent according to claim 1, characterized in that, The rare earth ion luminescent layer core NaYbF4:Er 3+ Ce 3+ Zn 2+ In the middle, the first doped ion includes Er 3+ Ce 3+ and Zn 2+ The total molar concentration of the first doped ion is 1-10 mol%% The rare earth ion luminescent layer core NaYbF4:Er 3+ Ce 3+ Zn 2+ The thickness is 0-30 nm and does not include 0; The rare earth ion sensitizer shell NaYbF4:Er 3+ Ce 3+ M 2+ In the middle, the second doped ion includes Er 3+ Ce 3+ and M 2+ The total molar concentration of the second doped ions is 1-10 mol%% The rare earth ion sensitizer shell NaYbF4:Er 3+ Ce 3+ M 2+ The thickness is 0-20 nm and does not include 0; The thickness of the heterogeneous inert protective shell MF2 is 0-20 nm, excluding 0.

4. The rare earth nano-fluorescent imaging agent according to claim 1, characterized in that, In the first doped ion, Er 3+ The doping concentration is 1-10 mol%, Ce 3+ The doping concentration of Zn is 1-10 mol%. 2+ The doping concentration is 1-10 mol%; In the second doped ion, Er 3+ The doping concentration is 1-10 mol%, Ce 3+ The doping concentration is 1-10 mol%, M 2+ The doping concentration is 1-10 mol%.

5. The rare earth nano-fluorescent imaging agent according to claim 1, characterized in that, The particle size of the rare earth nano-fluorescent imaging agent is 5-80 nm. The rare earth nano-fluorescent imaging agent is a crystal.

6. The rare earth nano-fluorescent imaging agent according to claim 1, characterized in that, The rare earth nano-fluorescent imaging agent is also loaded with modified ligands, which are loaded on the surface of the heterogeneous inert protective shell. The modified ligand is selected from water-soluble ligands; The water-soluble ligand is at least one of a water-soluble ligand without tumor-targeting function, a biomacromolecule with targeting function, or a biosmall molecule.

7. The rare earth nano-fluorescent imaging agent according to claim 1, characterized in that, The water-soluble ligand without tumor-targeting function is selected from at least one of methoxy polyethylene glycol alendronate, polyacrylic acid, and distearate phosphatidylethanolamine-polyethylene glycol; The targeted biomolecules are selected from peptides and / or antibodies; The biomolecule is selected from at least one of protein kinase inhibitors, poly(ADP-ribose) polymerase inhibitors, HDAC inhibitors, proteasome inhibitors, and Hedgehog pathway inhibitors.

8. The rare earth nano-fluorescent imaging agent according to claim 1, characterized in that, The rare earth nano-fluorescent imaging agent emits upconversion light in the visible region and emits light in the near-infrared IIb region when excited by 980 nm excitation light. When the rare earth nanofluorescent imaging agent is loaded with modified ligands, its hydrated particle size is 10-500 nm.

9. The method for preparing the rare earth nano-fluorescent imaging agent according to any one of claims 1-8, characterized in that, The preparation method employs at least one of the following methods to prepare the rare earth nano fluorescent imaging agent: hot injection, high-temperature co-precipitation, high-temperature thermal decomposition, hydrothermal method, or sol-gel method.

10. The preparation method according to claim 9, characterized in that, The high-temperature pyrolysis method includes the following steps: S1. Preparation of rare earth ion luminescent layer core NaYbF4:Er 3+ Ce 3+ Zn 2+ ; S2, in the rare earth ion luminescent layer core NaYbF4:Er 3+ Ce 3+ Zn 2+ On the surface, a rare earth ion sensitizer shell NaYbF4:Er was prepared by shell epitaxial growth method. 3+ Ce 3+ M 2+ The core-shell structure NaYbF4:Er was obtained. 3+ Ce 3+ Zn 2+ @ NaYbF4:Er 3+ Ce 3+ M 2+ ; S3, the rare earth ion sensitizer shell layer NaYbF4:Er with a core-shell structure from step S2. 3+ Ce 3+ Zn 2+ On the surface of NaYbF4:Er, a heterogeneous inert protective shell MF2 was prepared to obtain a core-shell-shell structure. 3+ Ce 3+ Zn 2+ @ NaYbF4:Er 3+ Ce 3+ M 2+ @MF2 is the rare earth nano fluorescent imaging agent.

11. The preparation method according to claim 10, characterized in that, In step S1, the preparation of the rare earth ion luminescent layer core specifically includes the following steps: erbium trifluoroacetate, ytterbium trifluoroacetate, cerium trifluoroacetate, zinc trifluoroacetate, and sodium trifluoroacetate are added to the first solvent, and the mixture is heated to 250-350 °C in an inert atmosphere and reacted for 10-120 minutes to obtain the rare earth ion luminescent layer core NaYbF4:Er 3+ Ce 3+ Zn 2+ ; In step S1, the molar ratio of sodium in sodium trifluoroacetate, ytterbium in ytterbium trifluoroacetate, erbium in erbium trifluoroacetate, cerium in cerium trifluoroacetate, and zinc in zinc trifluoroacetate is 1:0-1:0-1:0-1:0-1 and is not 0; The first solvent includes at least one of oleic acid, oleylamine, and octadecene.

12. The preparation method according to claim 10, characterized in that, In step S2, the shell epitaxial growth method includes the following steps: growing the rare earth ion luminescent layer core NaYbF4:Er from step S1... 3+ Ce 3+ Zn 2+ Erbium trifluoroacetate, ytterbium trifluoroacetate, cerium trifluoroacetate, trifluoroacetate salt of M, and sodium trifluoroacetate are added to a second solvent and mixed. The mixture is then heated to 250-350 °C in an inert atmosphere and reacted for 10-120 minutes to obtain the rare earth ion sensitizer shell NaYbF4:Er 3+ Ce 3+ M 2+ The core-shell structure NaYbF4:Er was obtained. 3+ Ce 3+ Zn 2+ @ NaYbF4:Er 3+ Ce 3+ M 2+ ; In step S2, the molar ratio of sodium:ytterbium:erbium:cerium:M in erbium trifluoroacetate, ytterbium trifluoroacetate, cerium trifluoroacetate, the trifluoroacetate salt of M, and sodium trifluoroacetate is 1:0-1:0-1:0-1:0-1; Step S3 specifically includes the following steps: First, the core-shell structure NaYbF4:Er 3+ Ce 3+ Zn 2+ @ NaYbF4:Er 3+ Ce 3+ M 2 + The trifluoroacetate of M is added to the second solvent and mixed. The mixture is heated to 250-350 °C in an inert atmosphere and reacted for 10-120 minutes to obtain a heterogeneous inert protective shell MF2, thus obtaining the rare earth nano fluorescent imaging agent, which has a heterogeneous structure. The second solvent includes at least one of oleic acid and octadecene.

13. The preparation method according to claim 10, characterized in that, The preparation method further includes: after preparing the heterogeneous inert protective shell MF2 in step S3, optionally performing the following step: loading modified ligands on the surface of the heterogeneous inert protective shell; The surface loading of modified ligands on a heterogeneous inert protective shell specifically includes: mixing the core-shell-shell structure and the modified ligand in an organic solvent, stirring at a temperature below 40°C for 1-24 hours, and then removing the organic solvent.

14. A developer comprising the rare earth nanofluorescent developer according to any one of claims 1-8, wherein the rare earth nanofluorescent developer has a surface-loaded modified ligand in a heterogeneous inert protective shell.

15. The use of the rare earth nanofluorescent imaging agent according to any one of claims 1-8 and / or the imaging agent according to claim 14 in fluorescence imaging.

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