Near-infrared two-region excitation and emission lanthanide series nano fluorescent probe as well as preparation method and application of near-infrared two-region excitation and emission lanthanide series nano fluorescent probe

Abstract

The invention belongs to the technical field of nano biological materials, and particularly relates to a near-infrared two-region excitation and emission lanthanide nano fluorescent probe as well as a preparation method and application thereof. Lanthanide ions with near-infrared two-region absorption characteristics are used as sensitizers, nanocrystalline with a co-doped structure formed by the lanthanide ions and activator ions is used as a core, and a pure-phase matrix material shell layer which is the same as a core lattice is grown on the surface of a core particle in situ. The nano fluorescent probe can generate tunable near-infrared two-region fluorescence emission with the wavelength of 1000 to 3000 nm under the excitation of near-infrared light with the wavelength of more than 1000 nm. The near-infrared two-region excitation and emission characteristics of the nano fluorescent probe can provide higher tissue penetration depth and signal-to-noise ratio for in-vivo imaging. The nano fluorescent probe shows excellent performance in living body angiography, dynamic observation of intestinal tracts and multi-channel near-infrared two-region imaging, and has wide application prospects in the fields of biomedical detection, early diagnosis of diseases, surgical navigation and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to a near-infrared two-region excitation and emission lanthanide nanofluorescent probe, a preparation method and application thereof, and belongs to the technical field of nanobiomaterials. BACKGROUND

[0002] Optical imaging in biomedical research can observe the characteristics and states of certain regions in the body in real time, providing accurate and rich information about cell and tissue structures, thus helping to gain a deeper understanding of complex physiological and pathological processes in the body. However, the implementation of optical imaging in the body faces certain challenges, because the light scattering, light absorption and autofluorescence caused by the heterogeneity and complexity of biological tissues can reduce the resolution of optical imaging and limit its penetration depth. In recent years, the progress of near-infrared two-region (NIR-II, 1000-3000 nm) fluorescence imaging technology shows that longer wavelengths can greatly suppress photon scattering and reduce tissue autofluorescence, thus significantly improving the penetration depth and signal-to-noise ratio of in vivo optical imaging. Benefiting from the excellent performance of NIR-II fluorescence imaging in in vivo imaging, various NIR-II fluorophores and nanoprobes have been developed in recent years. Among them, lanthanide nanomaterials are considered as promising contrast agents for deep tissue imaging due to their rich narrow-band emission across the NIR-II region, as well as excellent optical stability and biocompatibility. However, at present, the effective excitation wavelength of lanthanide nanomaterials is still mainly limited below 1000 nm, such as the classic Nd 3+ sensitization system 808 nm excitation or Yb 3 + sensitization system 980 nm excitation, which hinders their further improvement in deep tissue imaging performance because of strong photon scattering. There have been some studies trying to use longer excitation wavelengths to excite lanthanide nanomaterials; however, due to the non-negligible absorption of water to long-wavelength photons, because water has obvious absorption peaks at 980 nm, 1200 nm, 1445 nm and 1920 nm, which limits the excitation efficiency of the material when used for in vivo imaging. Therefore, how to obtain an ideal NIR-II probe that balances the effects of tissue scattering and water absorption on the excitation wavelength is a technical problem that needs to be solved.

[0003] On the other hand, in vivo imaging, the imaging and detection of a single biomarker can not accurately reflect the whole picture of the physiological or pathological process in the organism. Therefore, it is particularly crucial to observe the characteristics and state of different biomarkers in multiple regions or specific regions of the organism in real time. Based on the above considerations, the main research content of the present application focuses on designing and developing a new type of lanthanide nanoprobes toolbox with NIR-II excitation and adjustable multi-emission characteristics to meet the needs of long-wavelength excitation, emission and further expansion of the number of imaging channels for NIR-II multi-imaging in deep tissues in vivo. SUMMARY

[0004] (I) Technical problems to be solved

[0005] In order to solve the above-mentioned problems of the prior art, the present application provides a near-infrared two-region excitation and emission lanthanide nanofluorescent probe, its preparation method and application, which provides a nanofluorescent probe whose excitation and fluorescence emission are located in the NIR-II window, and can be used for high-resolution in vivo imaging.

[0006] (II) Technical solutions

[0007] In order to achieve the above-mentioned purpose, the main technical solutions adopted by the present application include:

[0008] A near-infrared two-region excitation and emission lanthanide nanofluorescent probe, which is a core-shell composite structure.

[0009] The core layer is a nanocrystal of a matrix material co-doped with a sensitizer ion and an activator ion; the matrix material is any one selected from NaYF4, NaGdF4, LiYF4, CaF2, NaLuF4 and KYF4; the sensitizer ion is a lanthanide ion with NIR-II absorption characteristics, and the activator ion is one or more of praseodymium, dysprosium, samarium, holmium, erbium, thulium and ytterbium; wherein the sensitizer ion and the activator ion are the same or different;

[0010] The shell layer is a pure phase matrix material which is grown in situ on the surface of the core layer and has the same lattice as the matrix material of the core layer.

[0011] The lanthanide nanofluorescent probe as described above, preferably, the lanthanide ion in the sensitizer is one or more of praseodymium, dysprosium and samarium.

[0012] The lanthanide nanofluorescent probe as described above, preferably, the structural formula of the lanthanide nanofluorescent probe is β-NaYF4: x%X, y%Ln@NaYF4, X is any one or more of Ho, Pr, Sm and Dy, Ln is any one or more of Ho, Tm, Er, Yb, Pr, Dy and Sm, x is 0.1-0.7, and y is 0.05-0.5; β represents a hexagonal phase.

[0013] Further, the structure of the lanthanide nanofluorescent probe is as follows:

[0014] β-NaYF4:70%Ho @NaYF4, β-NaYF4:15%Ho, 40%Er@NaYF4, β-NaYF4:70%Ho, 20%Tm@NaYF4, β-NaYF4:10%Ho, 40%Yb@NaYF4.

[0015] The NIR-II excitation and emission lanthanide nanofluorescent probe β-NaYF4:70%Ho @NaYF4, β-NaYF4:15%Ho, 40%Er@NaYF4, β-NaYF4:70%Ho, 20%Tm@NaYF4, β-NaYF4:10%Ho, 40%Yb@NaYF4 provided by the application has main fluorescence emission wavelengths of 2030 nm, 1525 nm, 1250 nm and 980 nm respectively, and the excitation wavelength used is preferably 1140-1160 nm, preferably 1150 nm.

[0016] An injectable imaging contrast agent, comprising: the lanthanide nanofluorescent probe as described above is hydrophilized and stability modified by DSPE-PEG2000-CH3, and is dispersed in sterile water.

[0017] Further, the preparation method of the imaging contrast agent is as follows:

[0018] The oil phase solution of the lanthanide nanofluorescent probe is mixed with chloroform and the oil phase solution of DSPE-PEG2000-CH3, the solvent is evaporated to obtain a transparent film, the film is dried, preheated water is added, ultrasonic treatment is performed, and then ultracentrifugation is performed, the supernatant is discarded, and the precipitate is resuspended in sterile water to obtain the imaging contrast agent.

[0019] A preparation method of a near-infrared two-region excitation and emission lanthanide nanofluorescent probe, comprising the following steps:

[0020] S1, a mixture of YCl3·6H2O, HoCl3·6H2O and LnCl3·6H2O is dissolved in a mixed solvent of oleic acid and octadecene, vacuum heating treatment is performed to remove water and oxygen, and a transparent solution is obtained;

[0021] S2, a methanol solution containing NaOH and NH4F is added to the transparent solution; after reaction, the methanol and water are completely removed by heating under vacuum;

[0022] S3, after continuous heating, the reaction is continuously stirred under argon protection, and finally cooled to room temperature; the nanoparticles are obtained by precipitation with excess anhydrous ethanol, washed by centrifugation, and added with cyclohexane to obtain a nanoparticle suspension;

[0023] S4, mixing the nanoparticle suspension with a shell precursor and a solvent, heating under vacuum to completely remove residual water, oxygen and cyclohexane, continuing to heat for high-temperature reaction, and cooling to obtain a nanofluorescent probe β-NaYF4:Ho, Ln@NaYF4.

[0024] Preferably, in step S1, the molar ratio of YCl3·6H2O, HoCl3·6H2O and LnCl3·6H2O is 1-x-y:x:y, wherein x is 0.1-0.7 and y is 0-0.5; the volume ratio of oleic acid and octadecene is 2:5, and the amount of mixed solvent added is 20-25 L:1 mol relative to the total molar amount of YCl3·6H2O, HoCl3·6H2O and LnCl3·6H2O.

[0025] The heating temperature is 130-150°C, and the duration of continuous stirring is 30-60 min.

[0026] Further, y is 0.05-0.5.

[0027] Preferably, in step S2, the final concentrations of NaOH and NH4F in the methanol solution are 0.3-0.5 mol / L and 0.3-0.8 mol / L; the volume ratio of methanol solution to mixed solvent is 21:8, and the temperature is raised to 100-115°C. Further, the final concentrations of NaOH and NH4F in the methanol solution are 0.3125 mol / L and 0.5 mol / L; the temperature is raised to 110°C. Further, the reaction temperature is 50°C, and the reaction time is 30 min.

[0028] Preferably, in step S3, the temperature is continuously raised to 280-320°C, and the stirring reaction time is 30-70 min; the centrifugation rate is 4000-5000 rpm / min, and the centrifugation time is 5-10 min.

[0029] Preferably, in step S4, the shell precursor is yttrium trifluoroacetate and sodium trifluoroacetate in an equimolar ratio, and the solvent is oleic acid and octadecene in an equal volume ratio; the molar ratio of nanoparticle suspension to shell precursor is 1:4, and the volume-molar ratio of solvent to shell precursor is 1 L:2 mol; heating under vacuum to 120-130°C; the high-temperature reaction temperature is 280-320°C, and the time is 30-70 min.

[0030] Use of the lanthanide nanofluorescent probe as described above or the lanthanide nanofluorescent probe obtained by the preparation method as described above in preparation of an imaging contrast agent. Specifically, the imaging contrast agent is used for in vivo blood vessel imaging, intestinal tract dynamic observation, NIR-II multi-channel in vivo imaging.

[0031] In a preferred embodiment, the preparation method of the imaging contrast agent is mixing the lanthanide nanofluorescent probe solution with chloroform and DSPE-PEG2000-CH3, forming a transparent film after evaporation of chloroform, adding preheated water after drying, centrifuging after ultrasonic treatment, and obtaining water-phase nanoparticles by resuspending the precipitate in water.

[0032] The lanthanide nanofluorescent probe prepared by the method has a deeper tissue penetration performance on the in vivo than the common lanthanide nanoprobes with an excitation wavelength of 808 nm or 980 nm.

[0033] (III) Beneficial effects

[0034] The beneficial effects of the present application are:

[0035] The lanthanide nanofluorescent probe provided by the present application can produce tunable near-infrared two-region fluorescence emission of 1000-3000 nm under the excitation of near-infrared light greater than 1000 nm. The near-infrared two-region excitation and emission characteristics of the nanofluorescent probe can provide higher tissue penetration depth and signal-to-noise ratio for in vivo imaging. The nanocrystal exhibits excellent performance in in vivo blood vessel imaging, intestinal tract dynamic observation, and multi-channel near-infrared two-region imaging, and has a wide application prospect in the fields of biomedical detection, early disease diagnosis, and surgical navigation.

[0036] The lanthanide nanofluorescent probe prepared by the present application and the common lanthanide nanoprobes have the non-crosstalk excitation characteristics, and are suitable for orthogonal excitation in vivo multi-channel imaging. The lanthanide nanofluorescent probe prepared by the present application uses 1150 nm excitation light, which has a more excellent tissue penetration ability than the 808 nm or 980 nm excitation light used by the traditional lanthanide nanoprobes, and is suitable for in vivo imaging. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 Structure diagram of the lanthanide nanofluorescent probe for NIR-II excitation and emission;

[0038] Figure 2 Absorption spectrum diagram of Ho 3+

[0039] Figure 3 ​Fluorescence emission spectrum of the NIR-II excitation and emission lanthanide nanofluorescent probe 1, 2, 3, 4 prepared in the present application;

[0040] Figure 4 Transmission electron micrograph of the NIR-II excitation and emission lanthanide nanofluorescent probe 1, 2, 3, 4 prepared in the present application;

[0041] Figure 5 Attenuation of 1150 nm excitation light and common lanthanide nanoprobes using 808 nm and 980 nm excitation light in simulated tissues (1 wt% fat emulsion) of different thicknesses;

[0042] Figure 6 NIR-II excitation and emission lanthanide nanofluorescent probe 2 prepared in the present application for in vivo whole-body imaging;

[0043] Figure 7 NIR-II excitation and emission lanthanide nanofluorescent probes 2, 3 prepared in the present application for dynamic intestinal peristalsis observation;

[0044] Figure 8 NIR-II excitation and emission lanthanide nanofluorescent probes 2, 3, 4 prepared in the present application for in vivo multiple imaging;

[0045] Figure 9 In vivo brain vascular imaging experiment picture results;

[0046] Figure 10 Fluorescence intensity comparison results of in vivo brain vascular imaging experiment;

[0047] Figure 11 In vivo intestinal imaging experiment picture results;

[0048] Figure 12 Fluorescence intensity comparison results of in vivo intestinal imaging experiment. DETAILED DESCRIPTION

[0049] In order to solve the above problems, the present application designs and develops a new lanthanide nanoprobes toolbox, the core of the technical scheme of which comprises: by selecting a lanthanide ion with long-wavelength absorption characteristics as a sensitizer in a lanthanide nanomaterial, a nanocrystal with a co-doped structure of the sensitizer ion and an activator ion is formed as a core, and a shell layer of a pure phase matrix material with the same lattice as the core is grown in situ on the surface of the core particle. The lanthanide nanofluorescent probe can produce tunable fluorescence emission in the range of 1000-3000 nm under near-infrared light excitation greater than 1000 nm. The NIR-II excitation and emission characteristics of the nanofluorescent probe can provide higher tissue penetration depth and signal-to-noise ratio for in vivo imaging. The lanthanide nanofluorescent probe exhibits excellent performance in in vivo vascular imaging, intestinal dynamic observation and multi-channel NIR-II imaging, providing a powerful multifunctional NIR-II imaging platform for life science and medical research, and is expected to promote the application of in vivo optical imaging in the fields of precise diagnosis and drug research.

[0050] The present application provides a lanthanide nanofluorescent probe with NIR-II excitation and emission, which is a core-shell composite structure; the core layer is a nanocrystal of a matrix material co-doped with a sensitizer ion and an activator ion; the shell layer is a pure phase matrix material grown in situ on the surface of the core layer and has the same lattice as the core layer. Specifically, any one of NaYF4, NaGdF4, LiYF4, CaF2, NaLuF4, and KYF4 is used as a matrix material, doped with a lanthanide ion (one or more of praseodymium, dysprosium, samarium, and holmium) with NIR-II absorption characteristics as a sensitizer, and an activator ion (one or more of praseodymium, dysprosium, samarium, holmium, erbium, thulium, and ytterbium) to form a nanocrystal with a co-doped structure as a core, and a pure phase matrix material shell layer with the same lattice as the core is grown in situ on the surface of the core particle. The doping concentration of the sensitizer ion is 10%-70%, and the doping concentration of the activator ion is 5%-50%. By changing the types and concentrations of the sensitizer and the activator, the NIR-II absorption and emission wavelengths of the probe can be adjusted. Taking NaYF4 as the matrix material as an example, the structural formula is β-NaYF4:x%X,y%Ln@NaYF4, where X represents any one or more of praseodymium (Pr), dysprosium (Dy), and holmium (Ho), and Ln represents any one or more of praseodymium (Pr), dysprosium (Dy), samarium (Sm), erbium (Er), thulium (Tm), and ytterbium (Yb), and the structural diagram is as shown in Figure 1

[0051] Further, preferably, X is Ho, and Ln is Tm, Er, and Yb.

[0052] ​It is found through a large number of experimental researches that the doping concentration of the sensitizing agent is preferably 10% to 70%, which can absorb near-infrared excitation light and can transfer the absorbed photon energy to the activator ions. When the doping concentration of the sensitizing agent is less than 10%, the absorbed photon energy is too small, and finally the luminescence is weak; if it is higher than 70%, it will strongly cause the cross relaxation of the energy between ions, so that the energy is consumed, and finally the luminescence is quenched. Therefore, the doping concentration of the sensitizing agent is preferably 10% to 70%. The doping concentration of the activator ions is preferably 5% to 50%, which is used as a luminescent ion to absorb the energy transferred from the sensitizing agent to produce 1000-3000 nm band luminescence. When the doping concentration of the activator ions is less than 5%, the number of luminescent ions is small, resulting in low luminescent efficiency; if it is higher than 50%, it will also strongly cause the cross relaxation of the energy between ions, so that the energy is consumed, and finally the luminescence is quenched. Therefore, the doping concentration of the activator ions is preferably 5% to 50%.

[0053] The application provides a synthesis method of a NIR-II excitation and emission lanthanide nanofluorescent probe, which can synthesize nanoparticles with uniform size through step-by-step synthesis of a core-shell structure. The specific steps are as follows:

[0054] (1) Synthesis of β-NaYF4:x%Ho, y%Ln (Ln=any one or more of Er, Tm, Yb, Pr, Dy, Sm):

[0055] First, a mixture of YCl3·6H2O, HoCl3·6H2O and LnCl3·6H2O with a total molar amount of 1 mole (molar ratio (1-x-y):x:y, wherein x is 0.1 to 0.7, and y is 0 to 0.5) is added to a three-necked flask containing a mixed solvent of oleic acid and octadecene (v / v = 2:5). Then the mixture is heated to 130-150℃ under vacuum until no bubbles are generated in the solution, and stirring is continued at this temperature for 50 minutes to completely remove water and oxygen; after the obtained transparent solution is cooled to room temperature, a methanol solution containing NaOH and NH4F is added. The mixture is continuously stirred at 50℃ for 30 minutes. Then it is heated to 100-115℃ under vacuum to completely remove methanol and water. Then it is heated to 280-320℃ at a rate of 11-15℃ / min, and the reaction is continuously stirred for 70-90 minutes under argon protection, and finally it is naturally cooled to room temperature. The nanoparticles are obtained by precipitation with excess anhydrous ethanol, centrifuged at 4000-5000 rpm / min for 5-10 minutes, washed with ethanol three times, and then dispersed in cyclohexane for standby.

[0056] (2) Synthesis of β-NaYF4:x%Ho, y%Ln@NaYF4 (Ln=Er, Tm, Yb, Pr, Dy, Sm):

[0057] The nanoparticle suspension (dispersed in cyclohexane) obtained in step (1) was added to a three-necked flask, which also included shell precursors (yttrium trifluoroacetate (C6F9O6Y) and sodium trifluoroacetate (C2F3NaO2)) and solvents (a mixture of oleic acid and octadecene). The mixture was heated to 120-130 °C under vacuum to completely remove residual water, oxygen and cyclohexane, then heated to 280-320 °C at a heating rate of 11 °C per minute, and stirred for 30-70 min under the protection of argon flow, and finally cooled to room temperature. The nanoparticles were obtained by precipitation with excess anhydrous ethanol, and dispersed in cyclohexane for use after centrifugation and ethanol washing three times.

[0058] In order to better explain the present application, so as to be understood, the present application is described in detail below by specific embodiments in combination with the accompanying drawings.

[0059] Example 1

[0060] Ho 3+ The preparation method of the single-doped NIR-II excitation and emission lanthanide nanofluorescent probe is as follows:

[0061] (1) First, a mixture of 0.3 millimoles of YCl3·6H2O and 0.7 millimoles of HoCl3·6H2O was added to a three-necked flask containing 21 mL of a mixed solvent of oleic acid and octadecene at a v / v ratio of 2:5; then the mixture was heated to 150 °C under vacuum until no bubbles were generated in the solution, and the stirring was continued at this temperature for 50 minutes to completely remove water and oxygen. After the obtained transparent solution was cooled to room temperature, 8 mL of a methanol solution containing 2.5 millimoles of NaOH and 4 millimoles of NH4F was added; the mixture was heated to 50 °C and continuously stirred for 30 minutes. Subsequently, it was heated to 110 °C under vacuum to completely remove methanol and water. Then it was heated to 300 °C at a rate of 11 °C / min, and continuously stirred for 90 minutes under the protection of argon, and finally naturally cooled to room temperature. The nanoparticles were obtained by precipitation with excess anhydrous ethanol (30 mL), and the obtained product β-NaYF4:70%Ho was dispersed in 10 mL of cyclohexane for use after centrifugation at 4500 rpm / min for 10 minutes and ethanol washing three times.

[0062] (2) 10 mL of the nanoparticle suspension (1 mmol, dispersed in cyclohexane) obtained in step (1) above was added to a three-necked flask, which also contained shell precursors (2 mmol of yttrium trifluoroacetate and 2 mmol of sodium trifluoroacetate) and solvents (10 mL of oleic acid and 10 mL of octadecene) to obtain a mixture. The mixture was heated to 130°C under vacuum to completely remove residual water, oxygen and cyclohexane, then heated to 300°C at a heating rate of 11°C per minute, stirred for 70 minutes under the protection of argon flow, and finally cooled to room temperature. The nanoparticles were obtained by precipitation with excess anhydrous ethanol (30 mL), centrifuged at 4500 rpm / min for 10 minutes, washed with ethanol three times, and then the obtained product β-NaYF4:70%Ho@NaYF4 was dispersed in 10 mL of cyclohexane for standby.

[0063] YCl3·6H2O was dissolved in heavy water to obtain a Ho 3+ ion solution with a concentration of 0.2 M, and the absorption spectrum of the solution was measured using a Lambda 750 S ultraviolet-visible-near infrared spectrophotometer of PerkinElmer, USA (the cuvette used was a four-side light-transmitting quartz glass cuvette with an optical path of 10 mm). The measured absorption spectrum is shown in Figure 2 According to the intensity of absorbance, the excitation wavelength of the present application is preferably 1150 nm.

[0064] The product β-NaYF4:70%Ho@NaYF4 obtained above is referred to as probe 1, and the fluorescence emission spectrum of probe 1 under 1150 nm excitation is shown in Figure 3 The emission peak is located at 2030 nm.

[0065] Example 2

[0066] Ho 3+ , Er 3+ co-doped NIR-II excitation and emission lanthanide nanofluorescent probe preparation method, the specific steps are as follows:

[0067] (1) First, a mixture of 0.45 mmol of YCl3-6H2O, 0.15 mmol of HoCl3-6H2O and 0.4 mmol of ErCl3-6H2O in a ratio of 1:0.33:1 was added to a three-necked flask containing 21 mL (v / v = 2:5) of a mixed solvent of oleic acid and octadecene. Then the mixture was heated to 150°C under vacuum until the solution was free of bubbles, and continued to be stirred at this temperature for 50 minutes to completely remove water and oxygen; after the resulting transparent solution was cooled to room temperature, 8 mL of a methanol solution containing 2.5 mmol of NaOH and 4 mmol of NH4F was added. The mixture was continuously stirred at 50°C for 30 minutes; then it was heated to 110°C under vacuum to completely remove methanol and water. Subsequently, it was heated to 300°C at a rate of 11°C / min, and continuously stirred at 300°C for 90 minutes under argon protection, and finally naturally cooled to room temperature. The nanoparticles were obtained by precipitation with excess anhydrous ethanol (30 mL), centrifuged at 4500 rpm / min for 10 minutes, and then dispersed in 10 mL of cyclohexane for use.

[0068] (2) 10 mL of the nanoparticle suspension obtained in step (1) (1 mmol, dispersed in cyclohexane) was added to a three-necked flask, and 2 mmol of yttrium trifluoroacetate and 2 mmol of sodium trifluoroacetate were added as shell precursors, and 10 mL of oleic acid and 10 mL of octadecene were added as solvents. The mixture was heated to 130°C under vacuum to completely remove residual water, oxygen and cyclohexane, and then heated to 300°C at a rate of 11°C / min, and continuously stirred at 300°C for 65 minutes under argon protection, and finally cooled to room temperature. The nanoparticles were obtained by precipitation with excess anhydrous ethanol (30 mL), centrifuged at 4500 rpm / min for 10 minutes, and then dispersed in 10 mL of cyclohexane for use. The resulting product β-NaYF4:15%Ho, 40%Er@NaYF4 is referred to as Probe 2, and its fluorescence emission spectrum under 1150 nm excitation is shown in FIG. 1B, with an emission peak at 1525 nm. Figure 3

[0069] Example 3

[0070] Ho 3+ , Tm 3+ The preparation method of the co-doped NIR-II excitation and emission lanthanide nanofluorescent probe is as follows:

[0071] ​(1) First, a mixture of 0.1 mmol of YCl3-6H2O, 0.7 mmol of HoCl3-6H2O and 0.2 mmol of TmCl3-6H2O was added to a three-necked flask containing 21 mL (v / v = 2:5) of a mixed solvent of oleic acid and octadecene. Then the mixture was heated to 150°C under vacuum until the solution was free of bubbles, and the temperature was maintained at 150°C for 50 minutes to completely remove water and oxygen. After the resulting transparent solution was cooled to room temperature, 8 mL of a methanol solution containing 2.5 mmol of NaOH and 4 mmol of NH4F was added; the mixture was heated to 50°C and stirred at 50°C for 30 minutes. Subsequently, the temperature was raised to 110°C under vacuum to completely remove methanol and water. Then the temperature was raised to 300°C at a rate of 11°C / min, and the reaction was stirred at 300°C for 90 minutes under argon protection, and finally cooled to room temperature naturally. The nanoparticles were obtained by precipitation with excess anhydrous ethanol (30 mL), centrifuged at 4500 rpm / min for 10 minutes, and the resulting product β-NaYF4:70%Ho, 20%Tm was dispersed in 10 mL of cyclohexane for later use.

[0072] (2) 10 mL of the nanoparticle suspension obtained in step (1) (1 mmol, dispersed in cyclohexane) was added to a three-necked flask, and the flask was additionally provided with shell precursors (2 mmol of yttrium trifluoroacetate and 2 mmol of sodium trifluoroacetate) and solvents (10 mL of oleic acid and 10 mL of octadecene). The above mixture was heated to 130°C under vacuum to completely remove residual water, oxygen and cyclohexane, and then heated to 300°C at a rate of 11°C / min, stirred at 300°C for 65 minutes under argon flow, and finally cooled to room temperature. The nanoparticles were obtained by precipitation with excess anhydrous ethanol, centrifuged at 4500 rpm / min for 10 minutes, and the resulting product β-NaYF4:70%Ho, 20%Tm@NaYF4 was dispersed in 10 mL of cyclohexane for later use. The resulting product β-NaYF4:70%Ho, 20%Tm@NaYF4 was denoted as Probe 3, and its fluorescence emission spectrum under 1150 nm excitation is shown in FIG. 1B, with an emission peak at 1250 nm. Figure 3

[0073] Example 4

[0074] Ho 3+ , Yb 3+ co-doped NIR-II excitation and emission lanthanide nanofluorescent probes were prepared according to the following specific steps:

[0075] ​(1) First, a mixture of 0.5 mmol of YCl3-6H2O, 0.1 mmol of HoCl3-6H2O and 0.4 mmol of YbCl3-6H2O was added to a three-neck flask containing 21 mL (v / v = 2:5) of a mixed solvent of oleic acid and octadecene. Then the mixture was heated to 130°C under vacuum until the solution was free of bubbles, and the temperature was kept at this value for 40 minutes to completely remove water and oxygen. After the resulting transparent solution was cooled to room temperature, 8 mL of a methanol solution containing 2.5 mmol of NaOH and 4 mmol of NH4F was added. The mixture was continuously stirred at 50°C for 30 minutes. Subsequently, the temperature was raised to 110°C under vacuum to completely remove methanol and water. Then the temperature was raised to 300°C at a rate of 11°C / min, and the reaction was continuously stirred for 90 minutes under argon protection, and finally naturally cooled to room temperature. The nanoparticles were obtained by precipitation with excess anhydrous ethanol (30 mL), centrifuged at 4500 rpm / min for 10 minutes, and the resulting product β-NaYF4:10%Ho, 40%Yb was dispersed in 10 mL of cyclohexane for later use.

[0076] (2) 10 mL of the nanoparticle suspension (1 mmol, dispersed in cyclohexane) obtained in step (1) was added to a three-neck flask, which also contained shell precursors (2 mmol of yttrium trifluoroacetate and 2 mmol of sodium trifluoroacetate) and solvents (10 mL of oleic acid and 10 mL of octadecene). The mixture was heated to 130°C under vacuum to completely remove residual water, oxygen and cyclohexane, and then heated to 300°C at a rate of 11°C / min, stirred for 70 minutes under argon flow, and finally cooled to room temperature. The nanoparticles were obtained by precipitation with excess anhydrous ethanol (30 mL), centrifuged at 4500 rpm / min for 10 minutes, and the resulting product β-NaYF4:10%Ho, 40%Yb@NaYF4 was dispersed in 10 mL of cyclohexane for later use. The resulting product β-NaYF4:10%Ho, 40%Yb@NaYF4 is referred to as probe 4, and its fluorescence emission spectrum under 1150 nm excitation is shown in FIG. 1, with an emission peak at 980 nm. Figure 3

[0077] The above-mentioned probes of Examples 1-4 were respectively subjected to transmission electron microscopy detection, and the results are shown in FIG. 2, indicating that the prepared probes are at the nanoscale of 10-50 nm. Figure 4

[0078] Example 5

[0079] ​​To more accurately test the tissue penetration ability of different excitation lights, 1 wt% fat emulsion was used in this embodiment to simulate living tissue, and the tissue penetration ability of excitation light under different tissue thicknesses can be effectively compared. Test process: the decay of 1150 nm excitation light of the NIR-II excitation and emission lanthanide nanofluorescent probe prepared by the application and other classic lanthanide nanomaterial excitation light 808 nm and 980 nm in different thicknesses of simulated tissue (1 wt% fat emulsion) was measured by using an optical power meter. Using 1 wt% fat emulsion to simulate living tissue, when the thickness of the tissue is 1 mm, 2 mm, 3 mm, 4 mm and 5 mm, respectively, the optical power under the excitation light wavelength of 808 nm, 980 nm and 1150 nm is measured by using an optical power meter. The normalized results are shown in Figure 5 As the thickness of the tissue increases, the decay rate of the 1150 nm excitation light is slower, which indicates that its tissue penetration effect is obviously better than that of the 808 nm and 980 nm excitation wavelengths.

[0080] Example 6

[0081] The specific steps of surface hydrophilic modification of the lanthanide nanofluorescent probe for in vivo imaging are as follows:

[0082] 600 μL of chloroform dispersion of nanoprobes (containing 0.05 millimoles of nanoprobes) was mixed with 7 mL of chloroform and 6 mL of DSPE-PEG2000-CH3 solution (25 mg / mL, dissolved in chloroform) in a 100 mL pear-shaped flask. Under the condition of heating in a 39°C water bath, the chloroform in the flask was rapidly evaporated by a rotary evaporator until a transparent film was formed at the bottom of the flask. Then, the flask was dried at room temperature under vacuum for 60 min. Then, the flask was placed in a 100°C oven for further drying for 5 min, then 4 mL of deionized water preheated to 100°C was added to the flask, and ultrasonic treatment was carried out at 100°C to promote the dispersion of the film. Finally, the ultrasonic colloidal dispersion was transferred to an ultracentrifuge tube and centrifuged at 20000 rpm for 25 min at 10°C, the supernatant was discarded, and the precipitate was resuspended in 1 mL of deionized water to obtain the surface hydrophilic modified nanoprobes. It should be noted that all the aqueous nanoparticles need to be filtered through a 0.22 μm syringe filter before being used for in vivo imaging. The fluorescent probes of Examples 2-4 above can be made into surface hydrophilic modified lanthanide nanofluorescent probes 2, 3 and 4 by this method.

[0083] Application Example 1:

[0084] High-resolution imaging of whole-body blood vessels in mice was performed using NIR-II excitation and emission lanthanide nanofluorescent probe 2 with surface hydrophilic modification prepared according to Example 6. The specific steps are as follows:

[0085] First, the Balb / c mice (purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.) were anesthetized by intraperitoneal injection of anesthetic (2% avertin solution, v / v, injected at a dose of 10 μL / g according to the weight of the mice), and the body hair was carefully shaved. Subsequently, 200 μL of fluorescent probe 2 obtained in Example 6 (0.015 mmol) was injected into the mouse body through the tail vein. Under 1150 nm laser excitation, the whole-body blood vessels of the mouse were imaged using an InGaAs CCD camera, and the exposure time of each image was 4 seconds; the filter set used was 1319LP + 1500LP. Among them, the images of the abdominal blood vessels and the back blood vessels of the mouse are shown in FIGS. 1A and 1B, respectively. It can be clearly seen from the figures that the blood vessels are clearly imaged. Figure 6

[0086] Application Example 2:

[0087] High-resolution dynamic observation of the mouse intestine was performed using NIR-II excitation and emission lanthanide nanofluorescent probes 2 and 3 with surface hydrophilic modification prepared according to Example 6, respectively. The specific steps are as follows:

[0088] In order to visualize the dynamic peristalsis of the mouse intestine, the mouse was anesthetized and 350 μL of surface hydrophilic modified fluorescent probe 2 or 3 (0.018 mmol) obtained in Example 6 was perfused through the gavage needle. Under 1150 nm laser excitation, the emission signal of β-NaYF4:70%Ho, 20%Tm@NaYF4 was collected using an InGaAs CCD camera, with a range of 1270-1330 nm (filter set: 1250LP + 1300BP) and an exposure time of 50 milliseconds; the emission signal of β-NaYF4:15%Ho, 40%Er@NaYF4 was collected using an InGaAs CCD camera, with a range of 1500-1700 nm (filter set: 1319LP + 1500LP) and an exposure time of 100 ms. The video frame rate for observing the peristalsis of the mouse intestine using β-NaYF4:70%Ho, 20%Tm@NaYF4 and β-NaYF4:15%Ho, 40%Er@NaYF4 was 10.6 fps and 6.92 fps, respectively (see FIGS. 2A and 2B). Figure 7 The results show that both probes 2 and 3 can clearly observe the peristalsis of the intestine, but each has its own advantages and disadvantages. Probe 2 has less light scattering and is clearer, while probe 3 has a higher video frame rate and can more quickly monitor the peristalsis process of the intestine.

[0089] Application Example 3:

[0090] ​Three-channel in vivo imaging of mice was achieved using the NIR-II excitation and emission lanthanide nanofluorescent probes 2, 3, 4 with surface hydrophilic modification prepared according to Example 6. The specific steps are as follows:

[0091] To achieve three-channel in vivo imaging, 300 μL of the NIR-II excitation and emission lanthanide nanofluorescent probe 3 (β-NaYF4: 70% Ho, 20% Tm @ NaYF4, 0.015 mmol) with surface hydrophilic modification prepared according to Example 6 was first injected into the mouse body through a gastric tube. Three and a half hours later, 400 μL of the NIR-II excitation and emission lanthanide nanofluorescent probe 4 (β-NaYF4: 10% Ho, 40% Yb @ NaYF4, 0.02 mmol) with surface hydrophilic modification prepared according to Example 6 was again perfused through the gastric tube. Half an hour later, 200 μL of the NIR-II excitation and emission lanthanide nanofluorescent probe 2 (β-NaYF4: 15% Ho, 40% Er @ NaYF4, 0.015 mmol) with surface hydrophilic modification prepared according to Example 6 was injected intravenously through the tail vein. Under 1150 nm laser excitation (100 mW·cm -2 ), three-channel in vivo imaging was collected using an InGaAs CCD camera equipped with a 2-inch lens. The signal collection range of the Yb channel (probe 4) was 1000-1050 nm (filter set: 1000LP+1050SP+1100SP), the exposure time was 4 seconds; the signal collection range of the Tm channel (probe 3) was 1270-1330 nm (filter set: 1250LP+1300BP), the exposure time was 400 milliseconds; the signal collection range of the Er channel (probe 2) was 1500-1700 nm (filter set: 1319LP+1500LP), the exposure time was 4 seconds. The three-channel imaging results are shown in Figure 8 As can be seen from the imaging results of Figure 8 , the Yb channel clearly lights up the small intestine of the mouse, the Tm channel clearly lights up the large intestine of the mouse, the Er channel clearly lights up the liver, spleen and whole body vascular network of the mouse. From the picture of the composite three channels, the imaging profile is very clear, and there is almost no signal crosstalk. These results show that the new 1150 nm excitation lanthanide nanofluorescent material developed by the present application is a very promising candidate material for NIR-II in vivo multi-channel imaging.

[0092] Experimental Example 1

[0093] The in vivo brain vascular imaging experiment was used to evaluate the tissue penetration ability of the excitation light at 1150 nm used by the NIR-II excitation and emission lanthanide nanofluorescent probe prepared by the present application, compared with the common probe excitation light at 808 nm and 980 nm.

[0094] The specific steps are as follows:

[0095] First, the Balb / c mice were anesthetized by intraperitoneal injection of anesthetic (2% Avertin solution, v / v, injected at a dose of 10 μL / g according to the weight of the mice), and the body hair was carefully shaved. Subsequently, 200 μL of the surface hydrophilic modified fluorescent probe 2 (0.015 mmol) obtained by the method of Example 6 was injected into the mouse body through the tail vein. The brain blood vessels of the mouse were imaged using an InGaAs CCD camera under laser excitation at 808 nm, 980 nm and 1150 nm, respectively, and the exposure time was 4 seconds. The filter set used was 1319LP+1500LP. It can be seen from the pictures of the brain blood vessels excited by the three excitation lights that the fluorescence intensity of the brain blood vessels excited by the 1150 nm excitation light is stronger, which is significantly higher than the common fluorescence intensity, and is 2.37 and 1.69 times higher than the fluorescence intensity excited by the traditional 808 nm and 980 nm excitation lights, respectively, indicating that the excitation light (1150 nm) used by the NIR-II excitation and emission lanthanide nanofluorescent probe prepared by the present application has a deeper tissue penetration performance in vivo than the 808 nm and 980 nm excitation lights commonly used by lanthanide nanoprobes. Figure 9 Figure 10 It can be seen from the pictures of the brain blood vessels excited by the three excitation lights that the fluorescence intensity of the brain blood vessels excited by the 1150 nm excitation light is stronger, which is significantly higher than the common fluorescence intensity, and is 2.37 and 1.69 times higher than the fluorescence intensity excited by the traditional 808 nm and 980 nm excitation lights, respectively, indicating that the excitation light (1150 nm) used by the NIR-II excitation and emission lanthanide nanofluorescent probe prepared by the present application has a deeper tissue penetration performance in vivo than the 808 nm and 980 nm excitation lights commonly used by lanthanide nanoprobes.

[0096] Experimental Example 2

[0097] The tissue penetration ability of the excitation light at 1150 nm used by the NIR-II excitation and emission lanthanide nanofluorescent probe prepared by the present application was evaluated by in vivo intestinal imaging experiments, which had an advantage over the common probe excitation light at 808 nm and 980 nm.

[0098] First, the Balb / c mice were anesthetized by intraperitoneal injection of anesthetic (2% Avertin solution, v / v, injected at a dose of 10 μL / g according to the weight of the mice), and the body hair was carefully shaved. Subsequently, 200 μL of the surface hydrophilic modified fluorescent probe 2 (0.01 mmol) obtained by the method of Example 6 was perfused through the stomach tube. The intestine was imaged using an InGaAs CCD camera equipped with a 2-inch lens under laser excitation at 808 nm, 980 nm and 1150 nm, and the exposure time was 400 milliseconds. The results are shown in Figure 11 . The results show that the brightness of the intestine under 1150 nm excitation is about 1.2 times that of 980 nm excitation, but is only slightly higher than that of 808 nm excitation (see Figure 12 ). This difference may be due to the trade-off between water absorption and light scattering during intestinal imaging.

[0099] ​In summary, through the in vivo brain blood vessel system and deep tissue organ (intestinal tract) imaging experiments, it is fully verified that the excitation light (1150nm) used by the NIR-II excitation and emission lanthanide series nano fluorescent probe prepared by the present application has a deeper tissue penetration performance than the common 808nm and 980nm excitation light used by the lanthanide series nano probe.

[0100] The above is only a preferred embodiment of the present application, and is not intended to limit the present application in other forms. Any person skilled in the art can modify or change the above disclosed technical content into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application without departing from the technical solution content of the present application still belongs to the protection scope of the technical solution of the present application.

Claims

1. A near-infrared two-region excitation and emission lanthanide nanofluorescent probe, characterized in that, It is a core-shell composite structure; The core layer is a matrix material nanocrystal co-doped with a sensitizer ion and an activator ion; the matrix material is any one selected from NaYF4, NaGdF4, LiYF4, CaF2, NaLuF4, KYF4; the sensitizer ion is a lanthanide ion with NIR-II absorption characteristics, and the activator ion is one or more of praseodymium, dysprosium, samarium, holmium, erbium, thulium, and ytterbium; wherein the sensitizer ion and the activator ion are the same or different; The shell layer is a pure-phase matrix material grown in situ on the surface of the core layer and has the same lattice as the matrix material of the core layer.

2. The lanthanide nanofluorescent probe according to claim 1, wherein, The lanthanide ion in the sensitizer is one or more of praseodymium, dysprosium, and samarium.

3. The lanthanide nanofluorescent probe according to claim 2, wherein, The structural formula of the lanthanide nanofluorescent probe is β-NaYF4: x%X, y%Ln@NaYF4, X is any one or more of Ho, Pr, Sm, and Dy, Ln is any one or more of Ho, Tm, Er, Yb, Pr, Dy, and Sm, x is 0.1-0.7, and y is 0.05-0.5; β represents a hexagonal phase.

4. The lanthanide nanofluorescent probe of claim 1, wherein, The structural formula of the lanthanide nanofluorescent probe is any one of the following: β-NaYF4:70%Ho@NaYF4, β-NaYF4:15%Ho,40%Er@NaYF4, β-NaYF4:70%Ho,20%Tm@NaYF4, β-NaYF4:10%Ho,40%Yb@NaYF4.

5. An injectable imaging contrast agent, characterized in that, It comprises: dispersing the lanthanide nanofluorescent probe in any one of claims 1-4 in sterile water after hydrophilization and stability modification by DSPE-PEG2000-CH3.

6. The injectable imaging contrast agent of claim 5, wherein, The preparation method is as follows: Mix the oil phase solution of the lanthanide nanofluorescent probe with chloroform and the oil phase solution of DSPE-PEG2000-CH3, evaporate the solvent to obtain a transparent film, dry the film, add preheated water, ultrasonic, perform ultracentrifugation, discard the supernatant, resuspend the precipitate in sterile water, and obtain the imaging contrast agent.

7. A method for preparing a near-infrared two-region excitation and emission lanthanide nanofluorescent probe, characterized in that, It comprises the following steps: S1, mix YCl3·6H2O, HoCl3·6H2O and LnCl3·6H2O mixture, dissolve with a mixed solvent of oleic acid and octadecene, remove water and oxygen by vacuum heating treatment, and obtain a transparent solution; S2, add a methanol solution containing NaOH and NH4F to the transparent solution; after the reaction, subsequently heat under vacuum conditions to completely remove methanol and water; S3, continue to heat and stir under argon protection after cooling to room temperature; obtain nanoparticles by precipitation with excess anhydrous ethanol, wash after centrifugation, and add cyclohexane to obtain a nanoparticle suspension; S4, mix the nanoparticle suspension with a shell precursor and a solvent, heat under vacuum conditions to completely remove residual water, oxygen and cyclohexane, continue to heat for high-temperature reaction, and cool to obtain the nanofluorescent probe β-NaYF4:Ho,Ln@NaYF4.

8. The production method according to claim 7, wherein In step S1, the molar ratio of YCl3·6H2O, HoCl3·6H2O and LnCl3·6H2O is 1-x-y:x:y, wherein x is 0.1-0.7, y is 0-0.5; the volume ratio of oleic acid and octadecene is 2:5, and the amount of mixed solvent is 20-25 L:1 mol of the total molar amount of YCl3·6H2O, HoCl3·6H2O and LnCl3·6H2O; The heating temperature is 130-150℃, and the duration of continuous stirring is 30-60 min.

9. The production method according to claim 7, wherein In step S2, the final concentration of NaOH and NH4F in the methanol solution is 0.3-0.5 mol / L and 0.3-0.8 mol / L; the volume ratio of methanol solution to mixed solvent is 21:8, and the temperature is raised to 100-115℃; In step S4, the shell precursor is yttrium trifluoroacetate and sodium trifluoroacetate in an equimolar ratio, and the solvent is oleic acid and octadecene in an equal volume ratio; the molar ratio of nanoparticle suspension to shell precursor is 1:4, and the volume to molar ratio of solvent to shell precursor is 1 L:2 mol; vacuum is applied and the temperature is raised to 120-130℃; the high-temperature reaction temperature is 280-320℃, and the time is 30-70 min.

10. The lanthanide series nanofluorescent probe of any one of claims 1-3 or the lanthanide series nanofluorescent probe obtained by the preparation method of any one of claims 7-9 is applied to the preparation of an imaging contrast agent; the application includes blood vessel imaging, intestinal tract dynamic observation, and NIR-II multi-channel in vivo imaging.

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