NIR-IIb / c rare earth nanoparticle fluorescent probe as well as preparation method and application thereof

Abstract

The invention provides an NIR-IIb / c rare earth nanoparticle fluorescent probe as well as a preparation method and application thereof. The fluorescent probe sequentially comprises a core, an active shell and an inert shell from inside to outside, wherein the core comprises Tm < 3 + > ions and Er < 3 + > ions, and the active shell comprises Er < 3 + > ions, Yb < 3 + > ions and Ce < 3 + > ions. The fluorescent probe provided by the invention has an efficient energy transfer system, not only has bright luminescence in an NIR-IIb wave band (1500-1700 nm), but also has luminous intensity increased by dozens of times in an NIR-IIc wave band (1700-1900 nm) compared with that of a traditional probe. The hydrophilic modification method of the probe is also improved, and the biocompatibility of the probe is improved by a multi-layer polymer cross coating method, so that the probe still has good luminescence property in an organism, and the penetration depth and resolution of biological tissues during imaging are improved.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescent probes, and in particular to a NIR-IIb / c rare earth nanoparticle fluorescent probe and a preparation method and application thereof. Background Art

[0002] In vivo optical imaging is widely used in research in biology, medicine and other fields due to its high sensitivity, good biosafety, dynamic observation and non-invasive analysis. However, when light propagates in tissues, it will be interfered by interface reflection, scattering, absorption and autofluorescence, which seriously limit the penetration depth and spatial resolution of imaging. As the wavelength increases to the near-infrared region II after 1000 nm, the autofluorescence and scattering of biological tissues have decreased significantly, and the absorption of water is also at a low level. The near-infrared region II is divided into several sub-regions suitable for in vivo biological imaging, such as NIR-IIa (1300-1400 nm), NIR-IIb (1500-1700 nm), and NIR-IIc (1700-1900 nm). In recent years, NIR-IIb (1500-1700 nm) imaging has been widely used in multi-channel functional imaging, surgical navigation, tumor microenvironment monitoring and other fields due to its low absorption and scattering effects, no autofluorescence interference, high resolution and signal-to-noise ratio, and high tissue penetration depth. Compared with the NIR-IIb window, the absorption of water in the NIR-IIc window is basically the same, but the wavelength increases and the influence of interference factors such as autofluorescence decreases. Therefore, the imaging depth and resolution of NIR-IIc are higher than those of NIR-IIb. InGaAs near-infrared cameras have the characteristics of high frame rate, high resolution, low noise and high stability, and are currently a very commonly used biological imaging detection device. The spectral response range of traditional InGaAs cameras is 900-1700 nm, which cannot detect the NIR-IIc band. In recent years, researchers have optimized the camera to extend its working wavelength to 1900 nm, which meets the requirements of window biological imaging for equipment, but the camera response performance decreases after the detection wavelength is extended, which puts higher requirements on the performance of the probe.

[0003] Currently, fluorescent probes that can be used for NIR-IIc biological imaging mainly include semiconductor quantum dots and rare earth nanoparticles. Rare earth nanoparticles have obvious advantages in biological imaging applications due to their large Stokes shift, narrow emission half-peak width, low toxicity, long luminescence lifetime, good stability, and strong controllability. However, the current NIR-IIc rare earth nanoparticle probes have shortcomings and deficiencies such as small quantity, low brightness, and poor stability. Therefore, developing new NIR-IIc rare earth nanoparticle probes with better performance and optimizing the hydrophilic modification method to improve the biocompatibility of the probe are crucial to exploring deep tissue visualization imaging, disease diagnosis, and treatment.

[0004] The transitions between the energy levels of the 4f electron orbits of rare earth ions are large in quantum number, so there are many transitions between energy levels, which can form spectra in all bands from ultraviolet light to infrared light. 3+ ion 3 F4→ 3 The H6 energy level transition will produce luminescence around 1800 nm, which is exactly in the NIR-IIc window. Therefore, Tm 3+ ions as the luminescent center of the probe material to prepare NIR-IIc rare earth nanoparticle fluorescent probe. 3+ Ions are commonly used as sensitizers for rare earth nanoparticle probes, but Yb 3+ Ions and Tm 3+ The energy transfer efficiency of ions is low, Tm 3+ The luminescence intensity of ions near 1800 nm is weak and cannot achieve ideal imaging effects. Therefore, an efficient sensitization system is established to enhance the Tm 3+ The problem that needs to be solved urgently is to improve the down-conversion efficiency of ions, thereby increasing the luminescence intensity near 1800 nm so that the probe can be better applied to NIR-IIc imaging. Most of the existing rare earth nanoparticle probes are single-emission, that is, there is only one main emission peak in a certain band. If the probe is improved and can emit two or more emission peaks in different bands at the same time, the scope of application and application performance of the probe will be greatly improved. Therefore, the field urgently needs to focus on the research and development of multi-band rare earth nanoparticle fluorescent probes with excellent performance.

[0005] In addition, the biocompatibility and stability of nanomaterials directly affect their application scenarios and effects in organisms, as well as their safety of use. Currently, there is a lack of stable and reliable methods and technologies for modifying the surface hydrophilicity of inorganic nanoparticles, which is another difficult problem that needs to be solved in this field. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a NIR-IIb / c rare earth nanoparticle fluorescent probe and a preparation method and application thereof.

[0007] The novel NIR-IIb / c rare earth nanoparticle fluorescent probe provided by the present invention is mainly used in the field of NIR-IIb / c biological imaging. The more mature NIR-IIb biological imaging in the near-infrared biological field is NIR-IIb biological imaging. Due to its low absorption and scattering effects, no spontaneous fluorescence interference, high resolution and signal-to-noise ratio, and high tissue penetration depth, it is widely used in multi-channel functional imaging, surgical navigation, tumor microenvironment monitoring, and tumor treatment effect monitoring. However, relative to the NIR-IIb window, the absorption intensity of water in the NIR-IIc window is basically the same, but the wavelength is increased, and the influence of interference factors such as spontaneous fluorescence is reduced. Therefore, the imaging depth and resolution of NIR-IIc are higher than those of NIR-IIb, and it has more advantages in deep biological imaging, so it has broad application prospects in the fields of visualization imaging of deep tissues, disease monitoring, mechanism research, and drug development. The efficient energy transfer system designed by the present invention enables the probe to have good luminescence performance and improve the penetration depth and resolution of biological tissues. The NIR-IIb / c rare earth nanoparticle fluorescent probe has a dual-sensitized core-shell structure, which improves the energy transfer efficiency and promotes the Er in the core-shell 3+ The energy migration of ions further improves the efficiency of energy transfer across the shell. The efficient energy transfer system enables the probe to emit bright light not only in NIR-IIb, but also in NIR-IIc, where the intensity is increased by dozens of times compared to traditional probes.

[0008] In the first aspect, the NIR-IIb / c rare earth nanoparticle fluorescent probe provided by the present invention comprises a core, an active shell and an inert shell from the inside to the outside; wherein the core comprises Tm 3+ Ions and Er 3+ ion, the active shell comprising Er 3+ Ion, Yb 3+ Ions and Ce 3+ ion.

[0009] The NIR-IIb / c rare earth nanoparticle fluorescent probe provided by the present invention is a dual-sensitized core-shell structure with Tm 3+ Ions are luminescent centers, Er 3+ ions serve as both luminescence centers and sensitization centers, Yb 3+ Ions improve the energy transfer efficiency as sensitization centers, while Ce 3+ Ions and Er 3+ Ion cross relaxation promotes the core-shell Er 3+ The energy migration of ions further improves the efficiency of energy transfer across the shell. The efficient energy transfer system enables the probe to emit bright light not only in NIR-IIb, but also in NIR-IIc, where the intensity is increased by dozens of times compared to traditional probes.

[0010] Preferably, the inert shell comprises Y 3+ ion.

[0011] Preferably, the general structural formula of the fluorescent probe for NIR-IIb / c bioimaging is: A x F4:Tm y @AYb m F4:Er n ,Ce p @AYF4 Type 1

[0012] Among them, x+y=1, m+n+p=1, A is Na + Ion or K + ion.

[0013] Preferably, x is 80% to 99.99%, preferably 88% to 98%, more preferably 92%; y is 0.01 to 20%, preferably 2% to 12%, more preferably 8%; m is 38% to 100%, preferably 60% to 97%, more preferably 84%; n is 0 to 50%, preferably 2% to 30%, more preferably 10%; p is 0 to 12%, preferably 1% to 10%, more preferably 6%; A is Na + Ion or K + Ion, preferably Na + ion.

[0014] The NIR-IIb / c rare earth nanoparticle fluorescent probe provided by the present invention realizes efficient energy transfer and excellent biological imaging effect by optimizing ion combination and structural design, significantly improves energy transfer efficiency by adjusting energy transfer system and energy transfer mode, and especially improves the luminous intensity of probe in NIR-IIc, significantly improves the penetration depth and resolution of imaging. The inventors have found that by adjusting the ratio of x, y, m, n, p, the energy transfer efficiency of the probe from outside to inside is significantly improved, and the preferred parameters can significantly improve the luminous intensity of the probe in NIR-IIc, which is dozens of times higher than the brightness of the traditional NIR-IIc probe. The probe treated with hydrophilic treatment on the basis thereof can significantly improve the penetration depth and imaging resolution in biological tissues, reaching sub-centimeter penetration depth and micron resolution, so that the imaging effect of deeper tissues is clearer. At the same time, the stability of the probe in the organism is enhanced, further improving the in vivo imaging effect, and has good application prospects in many fields such as biomedical imaging, drug monitoring and disease diagnosis.

[0015] In a second aspect, the present invention provides a method for preparing a NIR-IIb / c rare earth nanoparticle fluorescent probe, comprising sequentially preparing a 3+ Ions and Er 3+ The nucleus of the ion contains Er 3+Ion, Yb 3+ Ions and Ce 3+ The active shell of the ion and the Y 3+ Preferably, the process comprises the following steps: 1) Mix the mixed powder of Tm(CH3COO)3 and Er(CH3COO)3 with oleic acid and octadecene, heat under an inert atmosphere, cool and add methanol solution of NH4F and NaOH, evacuate and heat under an inert atmosphere, continue to heat and heat, cool, add ethanol, centrifuge and wash, and disperse in cyclohexane to obtain a first dispersion.

[0016] 2) Add oleic acid and octadecene to the mixed powder of CF3COONa, Er(CF3COO)3, Ce(CF3COO)3 and Yb(CF3COO)3, add the first dispersion, heat and evacuate, continue to heat and heat, add ethanol after cooling, centrifuge and wash, and disperse in cyclohexane to obtain a second dispersion.

[0017] 3) Add oleic acid and octadecene to the mixed powder of CF3COONa and Y(CF3COO)3, add the second dispersion, heat and evacuate, continue heating, add ethanol after cooling, centrifuge and wash, and disperse in cyclohexane to obtain NIR-IIb / c rare earth nanoparticle fluorescent probe. In the present invention, nanoparticles of suitable size are obtained by regulating the ratio of drug reagents and the temperature and time of the reaction, and hydrophilic modification can be better performed while ensuring high luminous efficiency.

[0018] Preferably, in step 1), the molar ratio of Tm(CH3COO)3 and Er(CH3COO)3 is 0.02-0.12:0.88-0.98; and / or, the mass ratio of oleic acid and octadecene is 4-7:4-8; and / or, the mass ratio of NH4F and NaOH is 65-85:100-120; and / or, heating at 145-155°C for 25-35 min under an inert atmosphere, cooling to 70-80°C and adding methanol solutions of NH4F and NaOH, evacuating and heating at 290-310°C for 50-70 min under an inert atmosphere, and continuing to heat to 305-310°C and heating for 15-25 min.

[0019] Preferably, in step 2), the molar ratio of CF3COONa, Er(CF3COO)3, Ce(CF3COO)3 and Yb(CF3COO)3 is 0.75-1.35:0.02-0.3:0.01-0.1:0.6-0.97; and / or, the mass ratio of oleic acid and octadecene is 3-6:8-11; and / or, stirring and vacuuming at 110-130 °C for 15-45 min, heating to 280-300 °C for 60-90 min, and continuing to heat to 305-320 °C for 10-30 min.

[0020] Preferably, in step 3), the molar ratio of CF3COONa to Y(CF3COO)3 is 0.75-1.35:0.75-1.35; and / or, the mass ratio of oleic acid to octadecene is 3-6:8-11; and / or, stirring and vacuuming at 110-130°C for 15-45 min, heating to 280-300°C for 60-90 min, and continuing to heat to 305-320°C for 10-30 min.

[0021] In the present invention, by optimizing the reagent ratio, reaction time, temperature, etc. in each step, adjusting the size of the core and the thickness of the active shell and the inert shell, the size of the fluorescent probe is regulated, the quenching effect is reduced, the luminescence efficiency and stability of the fluorescent probe can be improved, and the energy transfer path and transfer method can be regulated, the utilization of multiple luminescence centers is optimized, and the imaging effect is improved.

[0022] In a third aspect, the present invention provides a method for hydrophilizing a NIR-IIb / c rare earth nanoparticle fluorescent probe, wherein the NIR-IIb / c rare earth nanoparticle fluorescent probe is the NIR-IIb / c rare earth nanoparticle fluorescent probe or the NIR-IIb / c rare earth nanoparticle fluorescent probe obtained by the preparation method. The NIR-IIb / c fluorescent nanoprobe provided by the present invention improves the hydrophilicity and biocompatibility of the probe by selecting the selection of the reagents, optimizing the reagent ratio, and adopting multilayer polymer cross-coupling.

[0023] Preferably, the hydrophilic modification method comprises the following steps: 1) polymaleic anhydride-1-hexadecene (PMAH), chloroform and the NIR-IIb / c rare earth nanoparticle fluorescent probe are mixed and stirred until the liquid evaporates; 4-dimethylaminopyridine (DMAP) and water are added, ultrasonic dispersion and centrifugal treatment are performed, and the obtained particles are dispersed in a 4-morpholineethanesulfonic acid hydrate solution (MES solution) to obtain a first solution.

[0024] 2) Eight-arm polyethylene glycol amine (8Arm-PEG-NH2) and a cross-linking agent are dissolved in a MES solution and added to the first solution for reaction, and Tris base (Tris-base) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) are added. After the reaction, centrifugation and dialysis are performed, and then ultrafiltration tube centrifugation is performed, and the obtained particles are dispersed in the MES solution to obtain a second solution.

[0025] 3) dissolving polyacrylic acid (PAA) and EDC in MES solution and adding them to the second solution, centrifuging after the reaction, and then performing ultrafiltration tube centrifugation to disperse the obtained particles in MES solution to obtain a third solution.

[0026] 4) dissolving methoxypolyethylene glycol amine (mPEG-NH2), 8Arm-PEG-NH2 and a cross-linking agent in a MES solution and adding them to the third solution for reaction, adding Tris-base and EDC, centrifuging after the reaction, and then performing ultrafiltration tube centrifugation, dispersing the obtained particles in a MES solution, and obtaining a hydrophilically modified NIR-IIb / c rare earth nanoparticle fluorescent probe.

[0027] Preferably, in step 1), the mass volume ratio of the PMAH, chloroform, NIR-IIb / c rare earth nanoparticle fluorescent probe, and DMAP is 70-90 mg: 2-8 mL: 300-500 μL: 70-150 mg; and / or, the rotation speed of the centrifugal treatment is 12000-14000 rpm, and the treatment time is 1.5-3 h; and / or, the pH of the MES solution is 8-11.

[0028] Preferably, in step 2), the mass volume ratio of the 8Arm-PEG-NH2, the cross-linking agent, the first solution, Tris-base and EDC is 10-14 mg: 6-10 mg: 4-6 mL: 30-50 mg: 15-25 mg; and / or, the cross-linking agent is EDC; and / or, the reaction time is 2-4 h; and / or, centrifuged at 4000-5000 rpm for 20-40 min, then dialyzed in a 280-350 k dialysis bag for 10-14 h, and then centrifuged at 4000-5000 rpm in an 80-120 k ultrafiltration tube for 20-40 min.

[0029] Preferably, in step 3), the mass volume ratio of PAA, EDC and the second solution is 2-5 mg: 6-10 mg: 4-6 mL, and the reaction time is 1-2 h; centrifugation is performed at 4000-5000 rpm for 20-40 min, and then centrifugation is performed at 4000-5000 rpm for 20-40 min in an 80-120 k ultrafiltration tube.

[0030] Preferably, in step 4), the mass volume ratio of mPEG-NH2, 8Arm-PEG-NH2, cross-linking agent, third solution, Tris-base and EDC is 3~5 mg: 0.5~1 mg: 6~10 mg: 4~6 mL: 15~25 mg: 8~15 mg; and / or, the cross-linking agent is EDC; and / or, centrifuged at 4000~5000 rpm for 20~40 min, and then centrifuged at 4000~5000 rpm in a 80~120 k ultrafiltration tube for 20~40 min.

[0031] In the present invention, by optimizing the hydrophilic modification method, the amphiphilic polymer polymaleic anhydride-1-hexadecene is selected to transfer the rare earth nanoparticle fluorescent probe in the oil phase to the aqueous phase. The most advanced hydrophilic modification method for nanoparticles currently uses polymaleic anhydride-1-octadecene, compared with which the combination of polymaleic anhydride-1-hexadecene and nanoparticles is more stable. In order to make the surface polymer wrapping more dense and compact, a multilayer polymer cross-coupling method under the action of chemical bonds is used to form a firm polymer network on the surface of the nanoparticles, and the overall performance is enhanced by optimizing parameters such as drug ratio and reaction time, ensuring that the synthesized nanoprobe has higher biocompatibility and fluorescence characteristics, thereby improving the application effect in biological imaging.

[0032] In a fourth aspect, the present invention provides a hydrophilically modified NIR-IIb / c rare earth nanoparticle fluorescent probe, which is obtained by the above-mentioned hydrophilic modification method of the NIR-IIb / c rare earth nanoparticle fluorescent probe. Preferably, the NIR-IIb / c rare earth nanoparticle fluorescent probe includes at least one layer of hydrophilic polymer, and the number of layers is, for example, an integer between 2 and 8.

[0033] In a fifth aspect, the present invention provides the use of the NIR-IIb / c rare earth nanoparticle fluorescent probe, the NIR-IIb / c rare earth nanoparticle fluorescent probe obtained by the method, or the hydrophilically modified NIR-IIb / c rare earth nanoparticle fluorescent probe obtained by the method in NIR-IIb / c biological imaging.

[0034] According to the present invention, the energy transfer of some NIR-IIb / c rare earth nanoparticle fluorescent probes is to excite Tm with 800 nm laser. 3+ arrive 3 H4 energy level, and then the energy relaxes to 3 The F4 level emits light at 1600-2100 nm, with a small amount of Er doped in it. 3+ions as auxiliary transition channels. However, this energy transfer path has low efficiency and weak luminescence intensity, and cannot fully reflect the advantages of NIR-IIc imaging in biological applications. The NIR-IIb / c rare earth nanoparticle fluorescent probe of the present invention is excited by 980 nm laser, and Yb 3+ Ions and Er 3+ The ions also serve as sensitizers to form a dual sensitization system, and the energy transfer route from outside to inside is Yb→Er→Tm. This energy transfer method significantly improves the energy transfer efficiency, and the luminescence intensity of the probe in NIR-IIc is significantly improved. The energy transfer route of some NIR-IIb / c rare earth nanoparticle fluorescent probes is Yb→Er→Tm, and the energy migration route is not introduced, so the luminescence efficiency is still insufficient. At the same time, the probe material has not been hydrophilically modified for actual application in biological imaging. The NIR-IIb / c rare earth nanoparticle fluorescent probe of the present invention is excited by a 980 nm laser, and Yb 3+ Ions and Er 3 + The ions also serve as sensitizers to form a dual sensitization system. In addition to the energy transfer route of Yb→Er→Tm, the energy transfer route of core-shell energy migration Yb→Er→Er→Tm is also introduced. Er is doped in the middle shell. 3+ Ions and Ce 3+ ions, assisting energy cross relaxation to 4 I 13 / 2 energy level, from which energy can be transferred directly to Tm 3+ Ionic 3 F4 energy level can also migrate to the nucleus 3+ The same energy level of the ion is then transferred to Tm 3+ Ionic 3 F4 energy level luminescence. This energy transfer mode further improves the energy transfer efficiency, and the luminescence intensity of the probe in NIR-IIc is significantly improved. In addition, the hydrophilic modification method of the NIR-IIb / c fluorescent nanoprobe provided by the present invention has good dispersibility in water and good biocompatibility after hydrophilic modification, and can realize NIR-IIb / c window living biological imaging, thereby improving the penetration depth of biological tissues and the resolution of imaging.

[0035] The beneficial effects of the present invention are at least that: the probe provided by the present invention has a Tm 3+ Ions are luminescent centers, Er 3+ ions serve as both luminescence centers and sensitization centers, Yb 3+ Ions improve the energy transfer efficiency as sensitization centers, while Ce 3+ Ions and Er 3+ Ion cross relaxation promotes the core-shell Er 3+The energy migration of ions further improves the efficiency of energy transfer across the shell. The efficient energy transfer system enables the probe to not only emit bright light in NIR-IIb, but also improves the luminescence intensity of NIR-IIc by dozens of times compared with traditional probes. The present invention provides a method for preparing NIR-IIb / c fluorescent nanoprobes and a method for hydrophilic modification, in which the amphiphilic polymer is replaced by polymaleic anhydride-1-octadecene with polymaleic anhydride-1-hexadecene; this technical upgrade and optimization makes its combination with nanoparticles more stable, and after hydrophilic modification, the probe has good dispersibility in water and good biocompatibility, and can realize NIR-IIb / c window in vivo biological imaging. Compared with NIR-IIb imaging, NIR-IIc imaging improves the penetration depth of biological tissues and the resolution of imaging in biological in vivo imaging, thereby supporting the visualization imaging of deep tissues, which is of great significance for monitoring, mechanism research, drug development, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 Schematic diagram of the structure and energy transfer of the NIR-IIb / c rare earth nanoparticle fluorescent probe provided in an embodiment of the present invention.

[0038] Figure 2 (a) TEM image and (b) STEM and EDX images of the NIR-IIb / c rare earth nanoparticle fluorescent probe provided in an embodiment of the present invention.

[0039] Figure 3 This is the XRD diffraction pattern of the NIR-IIb / c rare earth nanoparticle fluorescent probe provided in an embodiment of the present invention.

[0040] Figure 4 This is an absorption spectrum of the NIR-IIb / c rare earth nanoparticle fluorescent probe provided in an embodiment of the present invention.

[0041] Figure 5 This is a fluorescence lifetime diagram of the NIR-IIb / c rare earth nanoparticle fluorescent probe provided in an embodiment of the present invention.

[0042] Figure 6 The invention provides a single-factor regulation of the optimal ion doping concentration of the NIR-IIb / c rare earth nanoparticle fluorescent probe in different sensitization systems.

[0043] Figure 7 The invention provides a single-factor regulation of the optimal ion doping concentration of the NIR-IIb / c rare earth nanoparticle fluorescent probe in different sensitization systems and core-shell structures.

[0044] Figure 8 The invention provides a single-factor regulation of the optimal ion doping concentration of the NIR-IIb / c rare earth nanoparticle fluorescent probe in different energy migration systems.

[0045] Fig. 9 This is an emission spectrum diagram of the NIR-IIb / c rare earth nanoparticle fluorescent probe during the regulation process provided by an embodiment of the present invention.

[0046] Fig.10 This is a transmission electron micrograph of the NIR-IIb / c rare earth nanoparticle fluorescent probe after hydrophilic modification provided in an embodiment of the present invention.

[0047] Fig.11 This is an emission spectrum diagram of the NIR-IIb / c rare earth nanoparticle fluorescent probe after hydrophilic modification provided in an embodiment of the present invention.

[0048] Fig.12 This is a DLS graph of the NIR-IIb / c rare earth nanoparticle fluorescent probe after hydrophilic modification provided in an embodiment of the present invention.

[0049] Fig.13 NIR-IIb / c rat lymphatic vessel imaging provided by an embodiment of the present invention.

[0050] Fig.14 NIR-IIc dual-channel imaging of mouse intestinal vascular provided by an embodiment of the present invention.

[0051] Fig.15 This is a schematic diagram of the luminescence intensity of Tm-based rare earth nanoparticles provided as a comparative example of the present invention. DETAILED DESCRIPTION

[0052] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods. If no specific techniques or conditions are specified in the examples, they are all conventional methods or the techniques or conditions described in the literature in this field, or according to the product instructions. If the manufacturers of the reagents and instruments are not specified, they are all conventional products that can be purchased through regular channels.

[0054] The present invention will be further described below with reference to the embodiments.

[0055] Example 1

[0056] The synthesis method of the NIR-IIb / c rare earth nanoparticle fluorescent probe provided by the present invention is as follows.

[0057] The mixed powder (258.3 mg) prepared according to the stoichiometric ratio of Tm(CH3COO)3: Er(CH3COO)3=0.08:0.92 was added to the mixed solution of oleic acid (4.25 g) and octadecene (9.47 g), and heated at 150 °C for 30 min under argon protection. When cooled to 75 °C, 111 mg NH4F and 75 mg NaOH corresponding methanol solution (8 mL) were added and mixed. After vacuuming for 10 min, the mixture was heated to 300 °C under argon protection and maintained for 1 h, and then continued to heat to 305 °C and maintained for 20 min before stopping heating. After cooling to room temperature, ethanol was added and centrifuged for washing twice, and then dispersed in 3 mL cyclohexane to obtain dispersion 1.

[0058] The mixed powder (654.47 mg) prepared according to the stoichiometric ratio of CF3COONa: Er(CF3COO)3: Ce(CF3COO)3: Yb(CF3COO)3 = 1: 0.1: 0.06: 0.84 was added to the mixed solution of oleic acid (5.15 g) and octadecene (5.66 g), and the above dispersion 1 was added, and the mixture was stirred and vacuumed at 120 °C for 30 min, heated to 300 °C and maintained for 75 min, and then continued to heat to 305 °C and maintained for 20 min before stopping heating. After cooling to room temperature, ethanol was added and centrifuged for washing twice, and then dispersed in 3 mL of cyclohexane to obtain dispersion 2.

[0059] The mixed powder (564 mg) prepared according to the stoichiometric ratio of CF3COONa:Y(CF3COO)3=1:1 was added to the mixed solution of oleic acid (5.15 g) and octadecene (5.66 g), and the above dispersion 2 was added, and the mixture was stirred and vacuumed at 120°C for 30 min, heated to 300°C and maintained for 75 min, and then continued to heat to 305°C and maintained for 20 min before stopping heating. After cooling to room temperature, ethanol was added and centrifuged and washed twice, and the mixture was dispersed in 3 mL of cyclohexane to obtain the NIR-IIb / c rare earth nanoparticle fluorescent probe solution.

[0060] The NIR-IIb / c rare earth nanoparticle fluorescent probe provided in this embodiment has a structural formula of NaEr 0.92 F4:Tm 0.08 @NaYb 0.84 F4:Er 0.1 ,Ce 0.06 @NaYF4.

[0061] Figure 1 The schematic diagram of the structure and energy transfer of NIR-IIb / c rare earth nanoparticle fluorescent probe is shown in Figure 1 As shown, the 980 nm laser excites the Yb in the active shell 3+ Ions and Er 3+ Ion, Yb 3+ The energy of the ion is then transferred to the Er in the active shell. 3+ Ions. Excited Er 3+ In addition to emitting light in NIR-IIb, the ions also transfer energy directly to the Tm 3+ Ions emit light, and on the other hand they pass through Ce 3+ After cross relaxation, the ions transfer energy to Er in the nucleus through migration. 3+ ions, which are then transferred to Tm 3+ Ion luminescence. The present invention also tested the NIR-IIb / c rare earth nanoparticle fluorescent probe prepared in Example 1, and the test results are as follows. Figure 2 (a) TEM image and (b) STEM and EDX images of NIR-IIb / c rare earth nanoparticle fluorescent probe. Figure 2 As shown, the size of rare earth nanoparticles is 20-40 nm, and the structure is hexagonal; the innermost layer is Tm 3+ Ions and Er 3+ ions, the middle layer is Yb 3+ Ion, Er 3+ Ions and Ce 3+ ions, the outermost layer is Y 3+ ions, which conform to the structural design of the present invention. Figure 3-5They are the XRD diffraction pattern, absorption spectrum and fluorescence lifetime diagram of NIR-IIb / c rare earth nanoparticle fluorescent probe, such as Figure 3 As shown, XRD data further verified the hexagonal phase structure of the NIR-IIb / c rare earth nanoparticle fluorescent probe; Figure 4 As shown in Figure 2, the absorption peaks of the NIR-IIb / c rare earth nanoparticle fluorescent probe are approximately at 650 nm, 808 nm, and 980 nm; Figure 5 As shown, the lifetime of the NIR-IIb / c rare earth nanoparticle fluorescent probe is about 2.15 ms when excited by 980 nm laser.

[0062] Example 2

[0063] The NIR-IIb / c rare earth nanoparticle fluorescent probe was prepared in the same manner as in Example 1, with the only difference being that the sensitization system, core-shell structure and element doping ratio were serially regulated to improve the energy transfer efficiency of the probe, thereby improving its luminescence performance. Figure 6 To improve the energy transfer efficiency by adjusting the optimal ion doping concentration in different sensitization systems. Figure 6 As shown in a, Tm 3+ ions are the luminescence center, Yb 3+ ions as sensitizers, and the outer layers are inert layers to prepare fluorescent probes. Experiments have found that when Tm 3+ When the ion doping concentration is 8%, the NIR-IIc fluorescence intensity of the probe is the highest under 980 nm laser excitation. Figure 6 As shown in b, Tm 3+ Ions and Er 3+ ions are the luminescence center, and Er 3+ Ions also act as sensitizers, and the outer layer is an inert layer to prepare fluorescent probes. The experiment found that the concentration of Tm is still 8%. 3+ When ion doped, the NIR-IIc fluorescence intensity of the probe is the highest under 980 nm laser excitation. Figure 7 To optimize the energy transfer efficiency of the probe by regulating the sensitization system and core-shell structure. Figure 7 As shown, Tm 3+ Ions and Er 3+ ions are the luminescent center and the doping ratio is the same as the optimal ratio after optimization, divided by Er 3+ ions as sensitizers, adding Yb 3+ The ion is the active shell of the sensitizer, and the outermost layer is covered with an inert shell. Under the secondary structure, the concentration of Yb in the active shell is 75%. 3+ When ion doped, the NIR-IIc fluorescence intensity of the probe is the highest under 980 nm laser excitation, while the NIR-IIb intensity is also the strongest. The fluorescence intensity change trends of the two windows are the same, indicating that the energy transfer between the two is related. Figure 8From the perspective of regulating energy migration, the optimal ion doping concentration is optimized to improve energy transfer efficiency. Figure 8 As shown in a, Tm 3+ Ions and Er 3+ ions are the luminescent center and the doping ratio is the same as the optimal ratio after optimization. The active shell is doped with the sensitizer Yb 3+ Ions and Er 3+ ions, the outermost layer is an inert layer to prepare fluorescent probes. The experiment found that when Er 3+ When the ion doping concentration is 10%, the NIR-IIc fluorescence intensity of the probe is the highest under 980 nm laser excitation. Figure 8 As shown in b, Tm 3+ Ions and Er 3+ ions as the luminescent center and the doping ratio is the same as the optimal ratio after optimization. 3+ Ions and Er 3+ Ce doped on the active shell layer with ions as sensitizer 3+ ions, the outermost layer is an inert layer to prepare fluorescent probes. Experiments have found that when Ce 3+ When the ion doping concentration is 6%, the NIR-IIc fluorescence intensity of the probe is the highest under 980 nm laser excitation. Fig. 9 This is the emission spectrum after a series of adjustments to the probe sensitization system, core-shell structure, and element doping ratio, such as Fig. 9 As shown, the NIR-IIb / c rare earth nanoparticle fluorescent probe has three emission peaks, at 1550 nm, 1680 nm and 1860 nm, among which the luminescence at 1860 nm is about 43.5 times higher than the fluorescence intensity of traditional probes.

[0064] Example 3

[0065] The hydrophilic modification method of the NIR-IIb / c rare earth nanoparticle fluorescent probe provided in the embodiment of the present invention is as follows.

[0066] 1) Add 80 mg of PMAH to a glass bottle and then add 4 mL of chloroform and stir. Add 400 μL of the NIR-IIb / c rare earth nanoparticle fluorescent probe solution prepared in Example 1 and stir until the liquid evaporates completely. Add 120 mg of DMAP and 6 mL of ultrapure water to a glass bottle and disperse by ultrasonic. Centrifuge twice at 13000 rpm for 1.5 h. After centrifugation, disperse the particles into MES solution (pH=8.5).

[0067] 2) 12 mg 8Arm-PEG-NH2(40 k) and 8 mg EDC were dissolved in 500 μL MES solution and added to the above solution (5 mL in total) obtained in step 1) and shaken for 3 h. 40 mg Tris-base and 20 mg EDC were added to terminate the reaction. After centrifugation at 4500 rpm for 30 min, the solution was added to a 300 k dialysis bag and dialyzed for 12 h. After the dialysis, the liquid was added to a 100 k ultrafiltration tube and centrifuged at 4500 rpm for 30 min to disperse the solid particles into the MES solution again.

[0068] 3) 4 mg PAA and 8 mg EDC were dissolved in 500 μL MES solution and added to the above solution (5 mL in total) obtained in step 2) and shaken for 1 h. After the reaction, centrifuge at 4500 rpm for 30 min, and then centrifuge at 4500 rpm for 30 min using a 100 k ultrafiltration tube. Disperse the particles with ultrapure water and continue centrifugation for 3 times, and then disperse the solid particles in MES solution again.

[0069] 4) 4 mg mPEG-NH2 (5 k), 0.8 mg 8Arm-PEG-NH2 (40 k) and 8 mg EDC were dissolved in 500 μL MES solution and added to the above solution (5 mL in total) obtained in step 3) and shaken for 3 h. 20 mg Tris-base and 10 mg EDC were added to terminate the reaction. After centrifugation at 4500 rpm for 30 min, the liquid was added to a 100 k ultrafiltration tube and centrifuged at 4500 rpm for 30 min. The particles were dispersed with ultrapure water and centrifuged twice. The solid particles were dispersed again in MES solution to obtain the hydrophilic modified NIR-IIb / c rare earth nanoparticle fluorescent probe.

[0070] Fig.10 This is a transmission electron microscopy image of the NIR-IIb / c rare earth nanoparticle fluorescent probe after hydrophilic modification. Fig.10 As shown, the rare earth nanoparticles are wrapped with a transparent polymer coating. After hydrophilic modification, the probe has good dispersibility in water and its biocompatibility is significantly improved. Fig.11 This is the emission spectrum of the NIR-IIb / c rare earth nanoparticle fluorescent probe after hydrophilic modification. Fig.11 As shown in the figure, the emission peak of the NIR-IIb / c rare earth nanoparticle fluorescent probe changed after hydrophilic modification. Before hydrophilic modification, the emission peak intensity at 1550 nm became weaker and red-shifted by about 20 nm to 1570 nm; before hydrophilic modification, the emission peak intensity at 1860 nm became stronger and blue-shifted by about 110 nm to 1750 nm, which may be related to the influence of water absorption. Fig.12 This is the DLS graph of the NIR-IIb / c rare earth nanoparticle fluorescent probe after hydrophilic modification. Fig.12 As shown, the NIR-IIb / c rare earth nanoparticle fluorescent probe has good stability in PBS after hydrophilic modification.

[0071] Example 4

[0072] The embodiment of the present invention provides a NIR-IIb / c rat lymphatic vessel imaging method, and the specific steps are as follows.

[0073] 100 μL of the hydrophilic modified NIR-IIb / c rare earth nanoparticle fluorescent probe (25 mg / mL) prepared by the method of Example 3 was injected into the tail root and foot pad of Wister rats, respectively. NIR-IIb and NIR-IIc were imaged separately by replacing filters of different bands under 980 nm laser excitation to observe the fluorescence signals of the lymphatic vessels connecting the tail root to the inguinal lymph nodes and the lymphatic vessels connecting the foot pad to the popliteal lymph nodes. Compared with NIR-IIb, the resolution and imaging depth of NIR-IIc imaging have been significantly improved. Fig.13 For NIR-IIb / c rat lymphatic imaging, Fig.13 As shown in the figure, the hydrophilic modified NIR-IIb / c rare earth nanoparticle fluorescent probe was injected into the tail root and foot pad of Wister rats, and NIR-IIb and NIR-IIc were imaged separately by replacing filters of different bands to observe the fluorescence signals of the lymphatic vessels connecting the tail root to the inguinal lymph nodes and the lymphatic vessels connecting the foot pad to the popliteal lymph nodes. Compared with NIR-IIb, the resolution and imaging depth of NIR-IIc imaging have been significantly improved.

[0074] Example 5

[0075] The embodiment of the present invention provides a NIR-IIc mouse intestinal imaging method, and the specific steps are as follows.

[0076] 100 μL of the hydrophilic modified NIR-IIb / c rare earth nanoparticle fluorescent probe (25 mg / mL) prepared by the method of Example 3 was added to the drinking water of mice for drinking. 100 μL of PbS QDs (5 mg / mL) was injected into the mice through the tail vein. 2 ) and 860 nm (325 mW / cm 2) laser for dual-channel imaging to observe the overall peristalsis of the intestine. Then, 1 g of agar was added to 10 mL of water and stirred, heated in a microwave oven for 1 minute, and when it cooled to 60 °C, 3 mL of hydrophilic modified NIR-IIb / c rare earth nanoparticle fluorescent probe (25 mg / mL) was added and stirred evenly, then poured into a glass dish to cool. After the agar solidified, it was cut into pieces of about 1 mm 3 The granules were placed at room temperature for 24 h to dehydrate and dry the granules, reduce their volume, and increase their hardness. The granules were mixed with mouse feed and fed. 100 μL PbS QDs (5 mg / mL) were injected into mice through the tail vein. The mice were illuminated by a 980 nm laser (247 mW / cm 2 ) and 860 nm (325 mW / cm 2 ) laser for dual-channel imaging to observe the movement of solid digestive matter in the intestine as it moves through the intestine. Fig.14 Dual-channel imaging of intestinal vascular in NIR-IIc mice. Fig.14 As shown, the hydrophilically modified NIR-IIb / c rare earth nanoparticle fluorescent probes were mixed into the drinking water and agar of mice respectively, and PbS QDs were injected into the mice through the tail vein, presenting the movement of digested matter in the intestine in the form of liquid and solid.

[0077] Comparative Example 1

[0078] This comparative example provides a tetragonal Tm-based rare earth nanoparticle LiTmF4:0.2%Er@LiYF4, with a luminescent wavelength of 1600-2100 nm and excited by 808 nm laser. The luminescent intensity of the nanoparticle is compared with that of the NIR-IIb / c rare earth nanoparticle fluorescent probe prepared in the above embodiment of the present invention in NIR-IIc. Fig.15 As shown, it is found that at the same power density (105 mW / cm 2 ) Under 980 nm and 808 nm lasers, respectively, the intensity of the probe of the present invention is about 5.82 times stronger.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A NIR-IIb / c rare earth nanoparticle fluorescent probe, characterized in that: The NIR-IIb / c rare earth nanoparticle fluorescent probe comprises a core, an active shell and an inert shell from the inside to the outside; wherein the core comprises Tm 3+ Ions and Er 3+ ion, the active shell comprising Er 3+ Ion, Yb 3+ Ions and Ce 3+ ion.

2. The NIR-IIb / c rare earth nanoparticle fluorescent probe according to claim 1, characterized in that: The inert shell comprises Y 3+ ion; Preferably, the general structural formula of the fluorescent probe for NIR-IIb / c bioimaging is: AEr x F4:Tm y @AYb m F4:Er n ,Ce p @AYF4 Type 1 Among them, x+y=1, m+n+p=1, A is Na + Ion or K + ion.

3. The NIR-IIb / c rare earth nanoparticle fluorescent probe according to claim 2, characterized in that: x is 80% to 99.99%, preferably 88% to 98%, more preferably 92%; y is 0.01 to 20%, preferably 2% to 12%, more preferably 8%; m is 38% to 100%, preferably 60% to 97%, more preferably 84%; n is 0 to 50%, preferably 2% to 30%, more preferably 10%; p is 0 to 12%, preferably 1% to 10%, more preferably 6%; A is Na + Ion or K + Ion, preferably Na + ion.

4. A method for preparing a NIR-IIb / c rare earth nanoparticle fluorescent probe, characterized in that: comprising sequentially preparing 3+ Ions and Er 3+ The nucleus of the ion contains Er 3+ Ion, Yb 3+ Ions and Ce 3+ The active shell of the ion and the Y 3+ An inert shell of an ion; preferably, comprising the steps of: 1) Mixing a mixed powder of Tm(CH3COO)3 and Er(CH3COO)3 with oleic acid and octadecene, heating under an inert atmosphere, cooling and adding a methanol solution of NH4F and NaOH, evacuating and heating under an inert atmosphere, continuing to heat and heat, cooling, adding ethanol, centrifugally washing and dispersing in cyclohexane to obtain a first dispersion; 2) Add oleic acid and octadecene to the mixed powder of CF3COONa, Er(CF3COO)3, Ce(CF3COO)3 and Yb(CF3COO)3, add the first dispersion, heat and evacuate, continue heating, cool, add ethanol, centrifuge and wash, and disperse in cyclohexane to obtain a second dispersion; 3) Add oleic acid and octadecene to the mixed powder of CF3COONa and Y(CF3COO)3, add the second dispersion, heat and evacuate, continue heating, add ethanol after cooling, centrifuge and wash, and disperse in cyclohexane to obtain NIR-IIb / c rare earth nanoparticle fluorescent probe.

5. The method for preparing the NIR-IIb / c fluorescent nanoprobe according to claim 4, characterized in that: In step 1), the molar ratio of Tm(CH3COO)3 and Er(CH3COO)3 is 0.02-0.12:0.88-0.98; and / or, the mass ratio of oleic acid and octadecene is 4-7:4-8; and / or, the mass ratio of NH4F and NaOH is 65-85:100-120; and / or, heating at 145-155°C for 25-35 min under an inert atmosphere, cooling to 70-80°C and adding methanol solutions of NH4F and NaOH, evacuating and heating at 290-310°C for 50-70 min under an inert atmosphere, and continuing to heat to 305-310°C and heating for 15-25 min; and / or, in step 2), the molar ratio of CF3COONa, Er(CF3COO)3, Ce(CF3COO)3 and Yb(CF3COO)3 is 0.75-1.35:0.02-0.3:0.01-0.1:0.6-0.97; and / or, the mass ratio of oleic acid and octadecene is 3-6:8-11; and / or, stirring and vacuuming at 110-130°C for 15-45 min, heating to 280-300°C for 60-90 min, and continuing to heat to 305-320°C for 10-30 min; and / or, in step 3), the molar ratio of CF3COONa to Y(CF3COO)3 is 0.75-1.35:0.75-1.35; and / or, the mass ratio of oleic acid to octadecene is 3-6:8-11; and / or, stirring and vacuuming at 110-130°C for 15-45 min, heating to 280-300°C for 60-90 min, and continuing to heat to 305-320°C for 10-30 min.

6. A method for hydrophilizing a NIR-IIb / c rare earth nanoparticle fluorescent probe, characterized in that: The NIR-IIb / c rare earth nanoparticle fluorescent probe is the NIR-IIb / c rare earth nanoparticle fluorescent probe described in any one of claims 1 to 3 or the NIR-IIb / c rare earth nanoparticle fluorescent probe obtained by the preparation method described in claim 4 or 5.

7. The hydrophilic modification method according to claim 6, characterized in that: The following steps are involved: 1) mixing polymaleic anhydride-1-hexadecene, chloroform and the NIR-IIb / c rare earth nanoparticle fluorescent probe, and stirring until the liquid evaporates; adding 4-dimethylaminopyridine and water, performing ultrasonic dispersion and centrifugal treatment, and dispersing the obtained particles into a 4-morpholineethanesulfonic acid hydrate solution to obtain a first solution; 2) dissolving the eight-arm polyethylene glycol amine and the cross-linking agent in a MES solution and adding them to the first solution for reaction, adding Tris base and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, centrifuging and dialysis after the reaction, and then centrifuging in an ultrafiltration tube, dispersing the obtained particles in a MES solution to obtain a second solution; 3) dissolving polyacrylic acid and EDC in MES solution and adding them to the second solution, centrifuging after the reaction, and then centrifuging in an ultrafiltration tube, dispersing the obtained particles in MES solution to obtain a third solution; 4) Dissolving methoxypolyethylene glycol amine, 8Arm-PEG-NH2 and a cross-linking agent in a MES solution and adding them to the third solution for reaction, adding Tris-base and EDC, centrifuging after the reaction, and then performing ultrafiltration tube centrifugation, dispersing the obtained particles in a MES solution, and obtaining a hydrophilically modified NIR-IIb / c rare earth nanoparticle fluorescent probe.

8. The hydrophilic modification method according to claim 7, characterized in that: In step 1), the mass volume ratio of the PMAH, chloroform, NIR-IIb / c rare earth nanoparticle fluorescent probe, and DMAP is 70-90 mg: 2-8 mL: 300-500 μL: 70-150 mg; and / or, the speed of the centrifugal treatment is 12000-14000 rpm, and the treatment time is 1.5-3 h; and / or, the pH of the MES solution is 8-11; And / or, in step 2), the mass volume ratio of the 8Arm-PEG-NH2, the cross-linking agent, the first solution, Tris-base and EDC is 10-14 mg: 6-10 mg: 4-6 mL: 30-50 mg: 15-25 mg; and / or, the cross-linking agent is EDC; and / or, the reaction time is 2-4 h; and / or, centrifugation at 4000-5000 rpm for 20-40 min, then adding a 280-350 k dialysis bag for dialysis for 10-14 h, and then centrifuging at 4000-5000 rpm for 20-40 min in an 80-120 k ultrafiltration tube; And / or, in step 3), the mass volume ratio of PAA, EDC and the second solution is 2-5 mg: 6-10 mg: 4-6 mL, and the reaction time is 1-2 h; centrifugation is performed at 4000-5000 rpm for 20-40 min, and then centrifugation is performed at 4000-5000 rpm for 20-40 min in an 80-120 k ultrafiltration tube; And / or, in step 4), the mass volume ratio of mPEG-NH2, 8Arm-PEG-NH2, cross-linking agent, third solution, Tris-base and EDC is 3~5 mg: 0.5~1 mg: 6~10 mg: 4~6 mL: 15~25 mg: 8~15 mg; and / or, the cross-linking agent is EDC; and / or, centrifugation at 4000~5000 rpm for 20~40 min, and then centrifugation at 4000~5000 rpm in a 80~120 k ultrafiltration tube for 20~40 min.

9. A hydrophilically modified NIR-IIb / c rare earth nanoparticle fluorescent probe, characterized in that: The method is obtained by the hydrophilic modification method of the NIR-IIb / c rare earth nanoparticle fluorescent probe according to claim 7 or 8.

10. Use of the NIR-IIb / c rare earth nanoparticle fluorescent probe described in any one of claims 1 to 3, or the NIR-IIb / c rare earth nanoparticle fluorescent probe obtained by the method described in any one of claims 4 to 6, or the hydrophilically modified NIR-IIb / c rare earth nanoparticle fluorescent probe obtained by the method described in claim 7 or 8 in NIR-IIb / c biological imaging.

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