A dual-channel near-infrared two-region rare earth nanoprobes, a preparation method and application thereof

Abstract

The application relates to a double-channel near-infrared two-region rare earth nanometer probe and a preparation method and application thereof. The double-channel near-infrared two-region rare earth nanometer probe comprises a probe A and a probe B; the probe A and the probe B are both three-layer structures, and sequentially comprise a core layer, a shell layer and a hydrophilic coating layer from inside to outside; in the probe A, the core layer material is NaYF4:Er, and the shell layer material is NaYF4; in the probe B, the core layer material is NaYbF4:Ce,Er,Zn, and the shell layer material is NaYF4. The probe has the characteristics of 808 nm / 940 nm orthogonal excitation and 1532 nm narrow-band emission, has the advantages of high light stability, rapid metabolism and high biocompatibility, and can realize deep tissue high signal-to-noise ratio in-vivo imaging.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomedical technology, specifically relating to a dual-channel near-infrared II rare-earth nanoprobe, its preparation method, and its application. Background Technology

[0002] Tumor vaccines, as a key means of activating the body's anti-tumor immunity, can activate specific immune responses by delivering antigens and adjuvants, and have become a highly promising tumor treatment strategy after immune checkpoint inhibitors. However, the clinical translation efficiency of tumor vaccines is limited, with core bottlenecks including low lymphatic transport efficiency, insufficient antigen presentation, and the inability to visualize the immune activation process. Furthermore, there is a lack of non-invasive, real-time, and dynamic methods for tracing vaccine delivery and evaluating treatment efficacy. Traditional assessment methods rely on tumor volume measurement and tissue sections, which are lagging, invasive, and unable to reflect the true early response to immunotherapy, hindering precision diagnosis and treatment.

[0003] Near-infrared II (NIR-II, 1000-1700 nm) fluorescence imaging, with its advantages of weak tissue scattering, deep penetration, low autofluorescence, and high spatiotemporal resolution, has become an ideal technique for non-invasive in vivo monitoring. Among them, the NIR-IIb (1500-1700 nm) window offers superior imaging quality and is suitable for tracing lymph nodes and deep tumors. Rare-earth core-shell nanoprobes, with their narrow emission peaks, strong photostability, tunable excitation wavelengths, and optimizable biocompatibility, are ideal materials for realizing vaccine lymph node tracing and apoptosis imaging. Current NIR-II probes and immunoimaging still have several key limitations: First, they are primarily single-channel imaging, which cannot simultaneously distinguish between vaccine distribution and apoptosis signals, easily causing signal crosstalk; second, lymphatic tracking resolution is insufficient, with traditional probes such as ICG showing weak signals in the NIR-II region, making it difficult to clearly display lymphatic vessels and sentinel lymph nodes; third, apoptosis monitoring lacks deep tissue-adapted probes, with traditional Annexin V fluorescent probes only suitable for in vitro detection, exhibiting poor penetration and low signal-to-noise ratio in vivo; fourth, nanomaterials are metabolized slowly in vivo and tend to accumulate in the liver and spleen, raising concerns about their long-term biosafety; and fifth, their diagnostic and therapeutic functions are limited, making it difficult to achieve integrated visualization and correlation assessment of vaccine delivery, lymphatic drainage, immune activation, and apoptosis.

[0004] Current technologies have not yet achieved a dual-channel independent excitation and interference-free NIR-II imaging system, making it impossible to simultaneously trace tumor vaccine lymphatic transport and quantitatively monitor treatment-induced tumor cell apoptosis at the in vivo level. Furthermore, the lack of rare-earth nanoplatforms that combine high brightness, high biocompatibility, rapid metabolism, and functionalization makes it difficult to meet the needs of dynamic monitoring throughout the entire process of tumor immunotherapy.

[0005] Therefore, constructing dual-channel NIR-II rare-earth nanoprobes that can be excited and differentiated, have independent signals, and are biosafe, to achieve integrated non-invasive imaging for precise lymph node tracing of tumor vaccines and real-time assessment of apoptosis induced by treatment, has become a key technical problem that urgently needs to be solved to improve the efficacy of tumor vaccines and promote the clinical translation of precision immunotherapy. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a dual-channel near-infrared II rare-earth nanoprobe, its preparation method, and its applications. This dual-channel near-infrared II rare-earth nanoprobe possesses advantages such as narrow-band emission of 1532 nm, high photostability, rapid metabolism, and high biocompatibility, enabling high signal-to-noise ratio in vivo imaging of deep tissues.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a dual-channel near-infrared II rare-earth nanoprobe, wherein the dual-channel near-infrared II rare-earth nanoprobe includes probe A and probe B; Both probe A and probe B have a three-layer structure, consisting of a core layer, a shell layer, and a hydrophilic coating layer from the inside out. In probe A, the core layer material is NaYF4:Er, and the shell material is NaYF4; In probe B, the core layer material is NaYbF4:Ce,Er,Zn, and the shell material is NaYF4.

[0008] Probe A consists of a NaYF4:Er core encapsulating a NaYF4 inert shell with a coupled hydrophilic coating, exhibiting luminescence upon excitation at 808 nm. Probe B consists of a NaYbF4:Ce,Er,Zn core encapsulating a NaYF4 inert shell with a coupled hydrophilic coating, exhibiting luminescence upon excitation at 940 nm. Both probes A and B show narrow emission peaks at 1532 nm, retain over 95% fluorescence after 3 hours of continuous irradiation, and remain stable in physiological media for 30 days without aggregation or quenching.

[0009] The energy level structures and luminescence mechanisms of probe A and probe B are as follows: Figure 1 As shown.

[0010] For probe A, Er 3+ As a direct absorption center, it is excited at 808 nm by... 4 I 15 / 2 Leap to 4 I 9 / 2 The energy level, which then relaxes to nonradiative levels. 4 I 13 / 2 It reaches the energy level and returns to the ground state through radiative transition, emitting a near-infrared signal of approximately 1532 nm.

[0011] For probe B, Yb is used. 3+As a sensitizer, it exhibits strong absorption in the 940 nm wavelength range. During excitation, Yb 3+ After absorbing external photons, it transfers energy to Er. 3+ This causes it to change from the ground state ( 4 I 15 / 2 ) transition to the excited state ( 4 I 11 / 2 ). Subsequently, Er 3+ Transitioning to via a nonradiative relaxation process 4 I 13 / 2 Energy level, and ultimately through 4 I 13 / 2 → 4 I 15 / 2 The radiative transition produces an NIR-II emission signal of approximately 1532 nm.

[0012] This invention constructs a dual-channel NIR-II rare-earth nanoprobe system with orthogonal excitation at 808 nm / 940 nm and narrow-band emission at 1532 nm. It exhibits high photostability, no signal interference or crosstalk between the two channels, rapid metabolism, and good biocompatibility, enabling high signal-to-noise ratio in vivo imaging of deep tissues.

[0013] This probe can be further coupled with OVA antigen and CpG B adjuvant to obtain a tumor vaccine tracer probe, or coupled with a targeting ligand that can specifically recognize apoptotic cell surface markers to obtain an apoptosis-targeting probe, thereby enabling dual probes to simultaneously trace vaccine lymphatic transport and monitor tumor apoptosis.

[0014] Preferably, in the core layer material of probe A, the molar ratio of Y to Er is 1:3-3:1 (e.g., it can be 1:3, 2:3, 1:1, 2:1, 3:1, etc.), and preferably 1:1.

[0015] Preferably, in the core layer material of probe B, the molar ratio of Yb, Ce and Er is (94-98):(1-3):(1-3), and more preferably 96:2:2.

[0016] Among them, the specific point values ​​in 94-98 can be 94, 95, 96, 97, 98, etc., and the specific point values ​​in 1-3 can be 1, 1.5, 2, 2.5, 3, etc.

[0017] Preferably, the raw materials for preparing the hydrophilic coatings of probe A and probe B both include PMH, 8Arm-PEG-NH2, PAA and m-PEG-NH2.

[0018] Preferably, the number-average molecular weight of PMH is 30-50 kDa; the molecular weight of 8Arm-PEG-NH2 is 30-50 kDa; the molecular weight of PAA is 1-3 kDa; and the molecular weight of m-PEG-NH2 is 3-5 kDa.

[0019] Among them, specific point values ​​in the 30-50 kDa range can be 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, etc.; specific point values ​​in the 1-3 kDa range can be 1 kDa, 1.5 kDa, 2 kDa, 2.5 kDa, 3 kDa, etc.; and specific point values ​​in the 3-5 kDa range can be 3 kDa, 3.5 kDa, 4 kDa, 4.5 kDa, 5 kDa, etc.

[0020] Preferably, the hydrophilic coatings of probe A and probe B both include a PMH adsorption layer, an 8Arm-PEG-NH2 and PAA crosslinking layer, and an m-PEG-NH2 layer from the inside out.

[0021] The structural schematic diagrams of probe A and probe B of this invention are shown below. Figure 2 As shown, oil-phase nanoparticles were transformed into water-soluble nanoprobes by layer-by-layer cross-linking coating (PMH adsorption, 8Arm-PEG-NH2 cross-linking, PAA network construction, and m-PEG-NH2 end modification) on the surface of core-shell structured nanoparticles.

[0022] In a second aspect, the present invention provides a method for preparing a dual-channel near-infrared II rare-earth nanoprobe as described in the first aspect, the method comprising: The cores of probe A and probe B were obtained by thermal decomposition, and a shell material was wrapped around the core by epitaxial growth. Then, probe A and probe B were obtained by crosslinking with the raw materials for preparing the hydrophilic coating.

[0023] Preferably, the preparation method of probe A includes: (1) Erbium acetate, oleic acid, 1-octadecene, sodium hydroxide dissolved in alcohol solution and ammonium fluoride are mixed and reacted at 280-350℃ for 0.5-3 h under argon protection to obtain the core NaYF4:Er of probe A; (2) Sodium trifluoroacetate, yttrium trifluoroacetate, core NaYF4:Er, oleic acid and 1-octadecene are mixed and reacted at 280-350℃ for 0.5-3 h under argon protection to grow a NaYF4 shell on the core NaYF4:Er, and nanoparticles A (abbreviated as pErNPs) are obtained. (3) Nanoparticle A is mixed with PMH to obtain PMH-encapsulated nanoparticle A, which is then sequentially coupled with 8Arm-PEG-NH2, PAA and m-PEG-NH2 to obtain probe A (abbreviated as pEr-p). 3 ).

[0024] Preferably, the alcohol solution in step (1) includes a methanol solution.

[0025] Preferably, the preparation method of probe B includes: (I) Ytterbium acetate, cerium acetate, erbium acetate, zinc acetate, oleic acid, 1-octadecene, sodium hydroxide dissolved in alcohol solution and ammonium fluoride are mixed and reacted at 280-350℃ for 0.5-3 h under argon protection to obtain the core NaYbF4:Ce,Er,Zn of probe B; (II) Sodium trifluoroacetate, yttrium trifluoroacetate, core NaYbF4:Ce,Er,Zn, oleic acid, and 1-octadecene are mixed and reacted at 280-350℃ for 0.5-3 h under argon protection to epitaxially grow a NaYF4 shell on the core NaYbF4:Ce,Er,Zn, to obtain nanoparticles B (abbreviated as ErNPs). (III) Nanoparticle B is mixed with PMH to obtain PMH-encapsulated nanoparticle B, which is then sequentially coupled with 8Arm-PEG-NH2, PAA and m-PEG-NH2 to obtain probe B (abbreviated as Er-p). 3 ).

[0026] Among them, the specific point values ​​in the 280-350℃ range can be 280℃, 290℃, 300℃, 310℃, 320℃, 330℃, 340℃, 350℃, etc., and the specific point values ​​in the 0.5-3 h range can be 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, etc.

[0027] Preferably, the alcohol solution in step (I) comprises a methanol solution.

[0028] Thirdly, the present invention provides a tumor vaccine tracer probe, which is obtained by conjugating antigen OVA to probe A in the dual-channel near-infrared II rare earth nanoprobe described in the first aspect and loading adjuvant CpG B.

[0029] The structural diagram of the tumor vaccine tracer probe (abbreviated as pEr-OVA-CpG B) is shown below. Figure 3 As shown. It uses probe A (pEr-p 3 Using OVA antigen and electrostatically loaded CpG B adjuvant as a carrier, lymph node tracing and immunotherapy are integrated. Under 808 nm laser excitation, the ratio of fluorescence signal intensity of lymph nodes to normal tissue (iLN / NT) at emission wavelength of 1532 nm can reach 25.5, indicating that lymph node tracing can be well achieved at the same time as immunotherapy.

[0030] Fourthly, the present invention provides an apoptosis-targeting probe, wherein the apoptosis-targeting probe is obtained by coupling probe B of the dual-channel near-infrared II rare earth nanoprobe described in the first aspect with a phosphatidylserine-specific recognition protein. Preferably, the phosphatidylserine-specific recognition protein includes Annexin A5 protein.

[0031] The structural diagram of the apoptosis-targeting probe (abbreviated as Er-Annexin A5) is shown below. Figure 4 As shown. It uses probe B (Er-p) 3 Using a carrier, Annexin A5 protein is covalently coupled to the probe, which can specifically recognize phosphatidylserine (PS) in apoptotic cells. Under 940 nm laser excitation, at an emission wavelength of 1532 nm, the ratio of fluorescence signal intensity (T / NT) between tumor tissue and normal tissue can reach 56, indicating that the probe can be successfully used to assess tumor apoptosis, thereby achieving early assessment and accurate feedback of treatment efficacy.

[0032] Fifthly, the present invention provides a dual-channel near-infrared two-zone imaging platform, wherein the dual-channel near-infrared two-zone imaging platform includes the tumor vaccine tracer probe described in the third aspect and the apoptosis-targeting probe described in the fourth aspect.

[0033] Sixthly, the present invention provides the application of the dual-channel near-infrared II rare-earth nanoprobe described in the first aspect, the tumor vaccine tracer probe described in the third aspect, the apoptosis-targeting probe described in the fourth aspect, or the dual-channel near-infrared II imaging platform described in the fifth aspect in the preparation of reagents or kits, wherein the reagents or kits are used for at least one of the following purposes: (a) Used for monitoring vaccine lymphatic drainage and / or lymph node enrichment; (b) Used to evaluate the response to tumor immunotherapy.

[0034] Compared with the prior art, the present invention has the following beneficial effects: The dual-channel near-infrared II rare-earth nanoprobe provided by this invention has the characteristics of 808 nm / 940 nm orthogonal excitation and 1532 nm narrowband emission, and has advantages such as high photostability, rapid metabolism and high biocompatibility, which can realize high signal-to-noise ratio in vivo imaging of deep tissues. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the energy level structure and luminescence mechanism of the dual-channel near-infrared second-region rare-earth nanoprobe of the present invention.

[0036] Figure 2 This is a schematic diagram of the structure of the dual-channel near-infrared second-region rare-earth nanoprobe of the present invention.

[0037] Figure 3 This is a schematic diagram of the structure of the tumor vaccine tracer probe of the present invention.

[0038] Figure 4 This is a schematic diagram of the apoptosis-targeting probe of the present invention.

[0039] Figure 5 This is a transmission electron microscope (TEM) image of the pErNPs of this invention and their particle size distribution.

[0040] Figure 6 This is a transmission electron microscope (TEM) image of the ErNPs of this invention, showing their morphology and particle size distribution.

[0041] Figure 7 This invention is pEr-p 3 With Er-p 3 Dual-channel excitation fluorescence imaging.

[0042] Figure 8 This invention is pEr-p 3 With Er-p 3 Histopathological sections of major organs of the treated mouse.

[0043] Figure 9 This is a dynamic imaging image of lymphatic transport and tumor enrichment of the tumor vaccine tracer probe in mice; where a is the imaging result at different times, b is a comparison of fluorescence intensity of lymph node tissue, tumor tissue and normal tissue at different times, and c is a fluorescence imaging image of mice at different angles 6 hours after the first injection of the tumor vaccine tracer probe.

[0044] Figure 10 This is a comparison chart of the anti-tumor efficacy evaluation and tumor growth curve of the tumor vaccine tracer probe of this invention.

[0045] Figure 11 This invention presents a dual-channel imaging strategy for monitoring and quantitatively analyzing the dynamic changes in tumor apoptosis.

[0046] Figure 12 This is a superimposed image of the dual-channel imaging results under the dual-channel imaging strategy of this invention. Detailed Implementation

[0047] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0048] The PMH mentioned in the following specific implementation method is from Sigma-Aldrich. The 8Arm-PEG-NH2 involved in the following specific embodiments is sourced from Xiamen Sinobond Biotechnology Co., Ltd. The PAA used in the following specific implementation is from Sigma-Aldrich. The m-PEG-NH2 involved in the following specific embodiments is from Laysan-Bio.

[0049] Example 1 This embodiment provides a dual-channel NIR-II rare-earth fluorescent nanoprobe, prepared by the following method: (1) Synthesis of precursors yttrium trifluoroacetate (CF3COO)3Y and ytterbium trifluoroacetate (CF3COO)3Yb.

[0050] (1.1) Precursor yttrium trifluoroacetate (CF3COO)3Y: Weigh 3 g of Y₂O₃ and transfer it to a round-bottom flask. Add 15 g of CF₃COOH and react at 90 °C for 1 h. Add 15 mL of deionized water and react for another 2 h. After the reaction is complete, a colorless and transparent solution is obtained. (If the solution is not clear, it may be because (CF₃COO)₃Y is not easily soluble in CF₃COOH. You can add another 5 mL of deionized water to dissolve (CF₃COO)₃Y). Fix the round-bottom flask on a rotating shaft and set the temperature of the rotary evaporator to 60 °C to remove trifluoroacetic acid and water. Place the sample in a 60 °C drying oven overnight. Collect the dried (CF₃COO)₃Y, grind it into powder using a mortar and pestle under an infrared heating lamp, place it in a glass bottle, and store it in a 60 °C vacuum drying oven (to prevent water absorption) to obtain the precursor yttrium trifluoroacetate.

[0051] (1.2) Precursor ytterbium trifluoroacetate (CF3COO)3Yb: The precursor ytterbium trifluoroacetate was synthesized according to step (1.1). The difference between step (1.1) and step (1.1) is that 3 g of Y2O3 was replaced with 3 g of Yb2O3, and the reaction time with CF3COOH at 90℃ was adjusted from 1 h to 12 h. All other steps were the same as in step (1.1). The precursor ytterbium trifluoroacetate was obtained.

[0052] (2) Preparation of probe core-shell structured nanoparticles.

[0053] (2.1) Synthesis of NaYF4:Er@NaYF4 nanoparticles in probe A: (2.1.1) Synthesis of NaYF4:Er (kernel layer): A mixture of (CH3COO)3Er (0.258 g, 0.619 mmol), oleic acid (OA, 4.25 g, 15 mmol), and 1-octadecene (ODE, 9.47 g, 37.5 mmol) was added to a 50 mL double-necked flask. The solution was heated to 150 °C under argon atmosphere and stirred for 30 min, then cooled to 50 °C. When the solution temperature dropped to 50 °C, NaOH (75 mg, 1.875 mmol) and NH4F (111 mg, 2.999 mmol) dissolved in anhydrous methanol (8 mL) were added to the above solution, and the mixture was purged with argon atmosphere at 50 °C for 1.5 h to remove methanol. Next, the solution was heated to 100°C under argon protection in a closed system. After vacuuming for 10 min, it was heated to 300°C and reacted at 300°C for 1 h. Then, it was heated to 305°C and reacted for 19 min. After the reaction was completed, it was allowed to cool naturally to room temperature. The reaction mixture was collected into a 50 mL centrifuge tube, ethanol was added, and the mixture was centrifuged for 30 min. The mixture was washed with ethanol and finally dispersed in 3 mL of cyclohexane for the next step of shell coating.

[0054] (2.1.2) Synthesis of NaYF4:Er@NaYF4 (core-shell structure): At room temperature, CF3COONa (0.136 g, 1 mmol), (CF3COO)3Y (0.428 g, 1 mmol), and 3 mL of the core nanoparticles prepared in step (2.1.1) were added to a mixture of oleic acid (5.66 g, 20 mmol) and 1-octadecene (ODE, 5.15 g, 20 mmol). The solution was first evacuated for 20 min, then heated to 120 °C under vacuum, and evacuated for another 30 min to remove moisture and oxygen. Then, under an argon atmosphere, the solution was heated to 300 °C for 75 min, and then heated to 305 °C for 19 min. After the reaction was completed, the solution was allowed to cool naturally to room temperature, the reaction solution was collected, excess ethanol was poured into the solution, the obtained nanoparticles were centrifuged at 4400 r / min for 30 min, washed with ethanol, and then dispersed in 3 mL of cyclohexane.

[0055] At room temperature, CF3COONa (0.136 g, 1 mmol), (CF3COO)3Y (0.428 g, 1 mmol) and 3 mL of the solution prepared in the previous step were added to a mixture of oleic acid (5.66 g, 20 mmol) and 1-octadecene (5.15 g, 20 mmol). The above steps were repeated once to obtain core-shell structured oil-phase nanoparticles NaYF4:Er@NaYF4 (abbreviated as pErNPs).

[0056] (2.2) Synthesis of NaYbF4:Ce,Er,Zn@NaYF4 nanoparticles in probe B: (2.2.1) Synthesis of NaYbF4:Ce,Er,Zn (core layer): A mixture of (CH3COO)3Yb (0.252 g, 0.72 mmol), (CH3COO)3Ce (0.0048 g, 0.015 mmol), (CH3COO)3Er (0.0052 g, 0.0125 mmol), (CH3COO)2Zn (0.0138 g, 0.075 mmol), oleic acid (4.25 g, 15 mmol), and 1-octadecene (9.47 g, 37.5 mmol) was added to a 50 mL double-necked round-bottom flask. The solution was heated to 150 °C under argon protection, stirred for 30 min, and then cooled to 50 °C. When the solution temperature dropped to 50°C, NaOH (75 mg, 1.875 mmol) and NH4F (111 mg, 2.999 mol) dissolved in anhydrous methanol (8 mL) were added to the above solution. The mixture was kept under argon atmosphere and purged at 50°C for 1.5 h to remove methanol. Next, the solution was heated to 100°C under argon protection in a closed system, evacuated for 10 min, and then heated to 306°C. The solution was reacted at 306°C for 1 h, then heated to 311°C for 19 min. After the reaction, it was allowed to cool naturally. After cooling to room temperature, the reaction mixture was collected in a 50 mL centrifuge tube, ethanol was added, and the mixture was centrifuged for 30 min. The mixture was washed with ethanol and finally dispersed in 3 mL of cyclohexane for the next shell coating step.

[0057] (2.2.2) Synthesis of NaYbF4:Ce,Er,Zn@NaYF4 (core-shell structure): At room temperature, CF3COONa (0.136 g, 1 mmol), (CF3COO)3Y (0.214 g, 0.5 mmol), (CF3COO)3Yb (0.255 g, 0.5 mmol), and 3 mL of the core nanoparticles prepared in step (2.2.1) were added to a mixture of oleic acid (5.66 g, 20 mmol) and 1-octadecene (5.15 g, 20 mmol). The solution was first evacuated for 20 min, then heated to 120 °C under vacuum, and evacuated for another 30 min to remove moisture and oxygen. Then, under an argon atmosphere, the solution was heated to 306 °C for 75 min, and then heated to 311 °C for 19 min. After the reaction was completed, the solution was allowed to cool naturally to room temperature, the reaction solution was collected, excess ethanol was poured into the solution, the resulting nanoparticles were centrifuged at 4400 r / min for 30 min, washed with ethanol, and then dispersed in 3 mL of cyclohexane.

[0058] At room temperature, CF3COONa (0.136 g, 1 mmol), (CF3COO)3Y (0.428 g, 1 mmol), and 3 mL of the solution prepared in the previous step were added to a mixture of oleic acid (5.66 g, 20 mmol) and 1-octadecene (5.15 g, 20 mmol). The above steps were repeated once to obtain core-shell structured oil-phase nanoparticles NaYbF4:Ce,Er,Zn@NaYF4 (abbreviated as ErNPs).

[0059] (3) Encapsulation with a hydrophilic coating: (3.1) Cross-linked polymer network coating encapsulates NaYF4:Er@NaYF4: PMH (80 mg, 30–50 kDa) was dissolved in 5 mL of chloroform and placed in a 10 mL glass bottle. Then, pErNPs (32 mg, ~400 µL) dispersed in cyclohexane were added. The solution was stirred for 3 h and then left uncapped at room temperature overnight. The solution was then dried in a 60 °C oven for 3 h to remove excess organic solvent. DMAP (80 mg) dissolved in 6 mL of aqueous solution was added to the dried glass bottle. The mixture was stirred and then sonicated until NaYF4:Er@NaYF4 was completely dispersed. The solution was centrifuged twice at 14000 r / min to remove excess PMH and DMAP. The precipitate was resuspended in 3 mL of MES solution (pH=11). Then, 8Arm-PEG-NH2 (12 mg) and EDC (8 mg) dissolved in 3 mL of MES solution were added. The solution was shaken and reacted for 3 h. Tris-base (40 mg) and EDC (20 mg) dissolved in MES solution were added to block excess -COOH derived from PMH. The solution was reacted for another 3 h, then centrifuged at 4400 r / min for 30 min to remove potentially large flocs. The supernatant was dialyzed with running water for 12 h (300 kDa dialysis bag) to completely remove excess 8Arm-PEG-NH2 and deactivate EDC. The solution was washed four times with an ultrafiltration centrifuge tube (100 kDa), and the concentrated reaction solution was resuspended and dispersed in 3 mL of MES solution. PAA (4 mg) and EDC (8 mg) dissolved in 3 mL of MES solution were added to the above solution. The solution was shaken for 1 h. The longer reaction time would produce a large amount of flocculent material. The solution was then centrifuged at 4400 r / min for 30 min to remove potentially large flocculent material. The supernatant was washed four times with an ultrafiltration centrifuge tube (100 kDa) to remove excess PAA, and then dispersed in 3 mL of MES solution. To this solution, 4 mg of mPEG-NH2, 0.8 mg of 8Arm-PEG-NH2, and 8 mg of EDC dissolved in 3 mL of MES solution were added, and the mixture was reacted on a shaker at 150 r / min for 3 h. Tris base (20 mg) and EDC (10 mg) were then added to the solution to block excess -COOH groups derived from PAA. The solution was shaken for another 3 h and then centrifuged at 4400 r / min for 30 min to remove potentially large flocs. The supernatant was washed four times through a centrifugal filter (100 kDa) to remove excess m-PEG-NH2. The final product was NaYF4:Er@NaYF4 (abbreviated as pEr-p) with a cross-linked polymer network coating. 3 (~16 mg), dispersed in 1.6 mL PBS buffer and stored at 4°C for long-term preservation.

[0060] (3.2) Cross-linked polymer network coating encapsulates NaYbF4:Ce,Er,Zn@NaYF4: Refer to step (3.1) pErNPs-p 3 The preparation of the cross-linked polymer network coating is different from step (3.1) in that pErNPs (32 mg, ~400 µL) is replaced with ErNPs (32 mg, ~400 µL), while the rest of the steps are the same as in step (3.1); NaYbF4:Ce,Er,Zn@NaYF4 (abbreviated as Er-p) is obtained. 3 ).

[0061] Verification Example 1 (1) Probe structure characterization: The transmission electron microscopy morphology and particle size distribution of pErNPs and ErNPs obtained in step (2) of Example 1 are as follows: Figure 5 and Figure 6 As shown, the probe exhibits a uniform core-shell structure, with pErNPs having a particle size of approximately 23.86 nm and ErNPs having a particle size of approximately 16.83 nm.

[0062] (2) Probe imaging verification: The pEr-p obtained in step (3) of Example 1 was excited using 808 nm and 940 nm lasers. 3 Probes and Er-p 3 The probe was used, and fluorescence imaging images under wide field of view were collected using an InGaAs camera. The results are as follows: Figure 7 As shown, only pEr-p under 808 nm excitation 3 Emission, only Er-p under 940 nm excitation 3 It exhibits excellent light emission and dual-channel separation performance.

[0063] (3) Security verification: C57BL / 6 mice in the experimental group were injected with pEr-p via the tail vein. 3 probe or Er-p 3 The probe (dissolved in physiological saline, injection dose 2 mg / 200 μL per mouse); control C57BL / 6 mice were injected with an equal volume of physiological saline via the tail vein; on day 30 post-administration, mice in both groups were euthanized and dissected, and the main target organs (liver, spleen, kidney, heart, and lungs) were collected for HE staining. Results are as follows: Figure 8 As shown, organ pathological sections confirmed pEr-p 3 Probes and Er-p 3 The probe has good biocompatibility and causes no tissue damage.

[0064] Example 2 Construction of tumor vaccine tracer probes and apoptosis-targeting probes.

[0065] (1) Preparation of tumor vaccine tracer probes: The pEr-p prepared in Example 1 3 (~2 mg), OVA (100 µg), EDC (1.5 mg), and MES solution (pH=11, 500 μL) were mixed to make pEr-p 3 The pEr-OVA solution was covalently coupled with OVA and, after 3 h, washed and dispersed in 100 µL of 1×PBS buffer via ultrafiltration centrifuge tube to obtain the pEr-OVA solution. The prepared pEr-OVA solution was mixed with CpG B (5 µg) and incubated at 25°C for 0.5 h to electrostatically load CpG B adjuvant, thus obtaining the pEr-OVA-CpG B tumor vaccine tracer probe for subcutaneous injection at the tail root.

[0066] (2) Preparation of apoptosis-targeting probes: The Er-p prepared in Example 1 3 (~2 mg ErNPs), Annexin A5 (24 µg) and MES solution (pH=11, 500 μL) were mixed and reacted. After 3 h, the mixture was washed in an ultrafiltration centrifuge tube and then dispersed in 200 µL 1×PBS buffer to obtain the Er-Annexin A5 apoptosis-targeting probe for tail vein injection.

[0067] Test Example 1 Validation of the NIR-II dual-channel imaging system: (1) Experimental method: (1.1) Establishment of E.G7-OVA tumor-bearing mouse model: 5×10 6 E.G7-OVA cells dispersed in complete culture medium were subcutaneously injected into the left hind limb of C57BL / 6 mice to establish an E.G7-OVA tumor-bearing mouse model. Tumor size was measured periodically after injection, and the tumor was considered closed when it reached 50 mm². 3 This is used for subsequent experiments.

[0068] (1.2) Trial grouping and intervention method: On day 0, tumor-bearing mice in the experimental group were subcutaneously injected with pEr-OVA-CpG B tumor vaccine tracer probe (5 mg / kg) at the base of their tails to induce a tumor-specific immune response; on day 3, Er-Annexin A5 apoptosis-targeting probe (1.2 mg / kg) was injected via the tail vein to allow it to enter the tumor through blood circulation; and on day 7, the same dose of pEr-OVA-CpG B tumor vaccine tracer probe was injected subcutaneously at the base of the tails again for booster immunization.

[0069] The control group of tumor-bearing mice were injected with the same volume of physiological saline on day 0, day 3, and day 7, respectively.

[0070] (1.3) Constructing a dual-channel live-body imaging strategy: The excitation wavelength of 808 nm is used for tumor vaccine tracing, and the excitation wavelength of 940 nm is used for apoptosis imaging. The emission window is >1500 nm.

[0071] (2) Test results: (2.1) Lymphatic transport and tumor accumulation of nanovaccines in mice: Imaging results after subcutaneous injection of pEr-OVA-CpG B tumor vaccine tracer probe at the tail base are as follows: Figure 9 As shown, a) is the imaging results at different times after the first injection of the tumor vaccine tracer probe, b) is the comparison of fluorescence intensity of lymph node tissue, tumor tissue and normal tissue at different times after the first injection of the tumor vaccine tracer probe (where iLN represents lymph node tissue, NT represents normal tissue and T represents tumor tissue), and c) is the fluorescence imaging of mice at different angles 6 h after the first injection of the tumor vaccine tracer probe.

[0072] It can be seen that after the tumor vaccine tracer probe is injected, it drains from the tail root along the lymphatic vessels to the inguinal lymph nodes (the enrichment of the nano-vaccine in the lymph nodes reaches its peak 6 hours after injection), and is enriched at the tumor site, with clear lymphatic tracer.

[0073] The tumor size in mice after subcutaneous injection of pEr-OVA-CpG B tumor vaccine tracer probe at the base of the tail is as follows: Figure 10 As shown, after injection of pEr-OVA-CpG B, the tumor volume in mice gradually decreased and rapidly regressed after 6 days; while the tumors in the control group continued to grow. This indicates that the vaccine, after accumulation at the tumor site, has a good therapeutic effect on tumors.

[0074] (2.2) Monitoring tumor apoptosis using a channel imaging system: Tumor apoptosis was observed at different time points after injection of pEr-OVA-CpG B and Er-Annexin A5 under 940 nm excitation light. Results are as follows: Figure 11 As shown, the number of apoptotic cells in the tumor site of the experimental group mice gradually increased. The Er-Annexin A5 apoptosis-targeting probe can specifically recognize phosphatidylserine in apoptotic cells. The apoptosis signal T / NT (the ratio of fluorescence signal intensity of tumor tissue to normal tissue) was the highest, about 56, which was highly positively correlated with the treatment effect.

[0075] Dual-channel fluorescence imaging was performed 24 h after injection of the Er-Annexin A5 apoptosis-targeting probe (i.e., day 4), and the results are as follows: Figure 12As shown in the image, the 808 nm excitation wavelength channel reveals that the tumor vaccine tracer probes are mainly concentrated in the reservoir formed at the tail root injection site and the tumor region, indicating that the tumor vaccine tracer probes successfully targeted the tumor. In the 940 nm excitation wavelength channel, the destination of the apoptotic cell-targeting probes can be observed; the Er-Annexin A5 apoptosis-targeting probes are abundantly aggregated at the tumor site, indicating the presence of a large number of apoptotic cells in the tumors of vaccine-treated mice. These results demonstrate the successful construction of this NIR-II dual-channel imaging strategy, enabling simultaneous visualization and monitoring of vaccine distribution and tumor cell apoptosis in the same live mouse.

[0076] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0077] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0078] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A dual-channel near-infrared II rare-earth nanoprobe, characterized in that, The dual-channel near-infrared II rare-earth nanoprobe includes probe A and probe B; Both probe A and probe B have a three-layer structure, consisting of a core layer, a shell layer, and a hydrophilic coating layer from the inside out. In probe A, the core layer material is NaYF4:Er, and the shell material is NaYF4; In probe B, the core layer material is NaYbF4:Ce,Er,Zn, and the shell material is NaYF4.

2. The dual-channel near-infrared II rare-earth nanoprobe according to claim 1, characterized in that, In the core layer material of probe A, the molar ratio of Y to Er is 1:3-3:1; Preferably, the molar ratio of Yb, Ce and Er in the core layer material of probe B is (94-98):(1-3):(1-3).

3. The dual-channel near-infrared II rare-earth nanoprobe according to claim 1 or 2, characterized in that, The raw materials for preparing the hydrophilic coatings of probes A and B both include PMH, 8Arm-PEG-NH2, PAA and m-PEG-NH2; Preferably, the hydrophilic coatings of probe A and probe B both include a PMH adsorption layer, an 8Arm-PEG-NH2 and PAA crosslinking layer, and an m-PEG-NH2 layer from the inside out.

4. A method for preparing a dual-channel near-infrared II rare-earth nanoprobe as described in any one of claims 1-3, characterized in that, The preparation method includes: The cores of probe A and probe B were obtained by thermal decomposition, and a shell material was wrapped around the core by epitaxial growth. Then, probe A and probe B were obtained by crosslinking with the raw materials for preparing the hydrophilic coating.

5. The preparation method according to claim 4, characterized in that, The preparation methods for probe A include: (1) Erbium acetate, oleic acid, 1-octadecene, sodium hydroxide dissolved in alcohol solution and ammonium fluoride are mixed and reacted at 280-350℃ for 0.5-3 h under argon protection to obtain the core NaYF4:Er of probe A; (2) Sodium trifluoroacetate, yttrium trifluoroacetate, core NaYF4:Er, oleic acid and 1-octadecene are mixed and reacted at 280-350℃ for 0.5-3 h under argon protection to grow a NaYF-4 shell on the core NaYF4:Er to obtain nanoparticle A; (3) Nanoparticle A is mixed with PMH to obtain PMH-encapsulated nanoparticle A, and then 8Arm-PEG-NH2, PAA and m-PEG-NH2 are coupled sequentially to obtain probe A.

6. The preparation method according to claim 4 or 5, characterized in that, The preparation methods for probe B include: (I) Ytterbium acetate, cerium acetate, erbium acetate, zinc acetate, oleic acid, 1-octadecene, sodium hydroxide dissolved in alcohol solution and ammonium fluoride are mixed and reacted at 280-350℃ for 0.5-3 h under argon protection to obtain the core NaYbF4:Ce,Er,Zn of probe B; (II) Sodium trifluoroacetate, yttrium trifluoroacetate, core NaYbF4:Ce,Er,Zn, oleic acid, and 1-octadecene are mixed and reacted at 280-350℃ for 0.5-3 h under argon protection to epitaxially grow a NaYF4 shell on the core NaYbF4:Ce,Er,Zn, thus obtaining nanoparticles B; (III) Nanoparticle B is mixed with PMH to obtain PMH-encapsulated nanoparticle B, which is then sequentially coupled with 8Arm-PEG-NH2, PAA and m-PEG-NH2 to obtain probe B.

7. A tumor vaccine tracer probe, characterized in that, The tumor vaccine tracer probe is obtained by conjugating probe A of the dual-channel near-infrared II rare earth nanoprobe according to any one of claims 1-4 with antigen OVA and loading adjuvant CpG B.

8. An apoptosis-targeting probe, characterized in that, The apoptosis-targeting probe is obtained by coupling probe B of the dual-channel near-infrared II rare-earth nanoprobe according to any one of claims 1-4 with a phosphatidylserine-specific recognition protein. Preferably, the phosphatidylserine-specific recognition protein includes Annexin A5 protein.

9. A dual-channel near-infrared two-zone imaging platform, characterized in that, The dual-channel near-infrared two-zone imaging platform includes the tumor vaccine tracer probe of claim 7 and the apoptosis-targeting probe of claim 8.

10. The application of the dual-channel near-infrared II rare-earth nanoprobe of any one of claims 1-4, the tumor vaccine tracer probe of claim 7, the apoptosis-targeting probe of claim 8, or the dual-channel near-infrared II imaging platform of claim 9 in the preparation of reagents or kits, wherein the reagents or kits are used for at least one of the following purposes: (a) Used for monitoring vaccine lymphatic drainage and / or lymph node enrichment; (b) Used to evaluate the response to tumor immunotherapy.

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