Biological coupling reagent initiated by near-infrared light as well as preparation method and application of biological coupling reagent

Abstract

The invention provides a biological coupling reagent initiated by near-infrared light as well as a preparation method and application of the biological coupling reagent. The biological coupling agent is prepared from the following raw material components: lanthanide series up-conversion luminescence nano particles and 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol grafted with tetrafluorophenyl azide, wherein the 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol is used as a carrier; the structural general formula of the biological coupling agent is as follows: NaGdF4: (50-x)% Yb, x% A (at) NaYF4: 20% Yb (at) NaGdF4: 10% Yb, 50% Nd (at) NaGdF4 (at) DSPE-PEG-PFPA, wherein x is more than or equal to 0.5 and less than or equal to 2, and A is selected from one of Tm, Ho and Er. The biological coupling reagent disclosed by the invention can be initiated by near-infrared light, can realize biological coupling between cells and a functional molecular material interface, between cells and between living biological tissues and a nano material, and has space-time controllability, high efficiency and universality.

Description

Technical Field

[0001] The present invention belongs to the field of biophysics cross technology, and in particular relates to a near-infrared light-triggered biocoupling reagent and a preparation method and application thereof. Background Art

[0002] Bioconjugation is the process of covalently linking natural biomolecules (such as carbohydrates, proteins, lipids, and nucleic acids) to functional molecules (such as drugs, dyes, polymers, or nanoparticles) through specific chemical reaction strategies. The conjugation of biomolecules to synthetic functional moieties is of great significance in biology and biomedicine, enabling diverse applications such as tracking and localization of biomolecules, biosensing, targeted drug delivery, and the development of new diagnostic and therapeutic tools. In recent decades, bioconjugation technologies have been continuously developed to improve the efficiency, biostability, and applicability of conjugation reactions for diverse applications, such as the azide-based Staudinger ligation, copper-catalyzed alkyne-azide cycloaddition (CuAAC), and copper-free click reactions.

[0003] Due to the dynamic nature of living systems, photoinduced bioconjugation strategies with high spatiotemporal precision, low trauma, and biocompatibility have profound implications in chemical biology. Photoresponsive crosslinkers can form covalent bonds with biomolecules at their binding sites upon light stimulation. Widely used photoresponsive crosslinkers include benzophenones, aryldiaziridines, aryl azides, photocycloadditions of diketones and vinyl ethers, and photocaged phosphines, which bioconjugate via free radical, electronic, or insertion reactions. Tetrafluorophenyl azide (PFPA) is one of the most popular photoaffinity agents due to its high stability, enhanced reaction yield, and kinetics. Upon photoactivation, it generates a highly reactive singlet tetrafluorophenylnitrene, which then undergoes 004007-H / NH insertion and C=C addition reactions, making it particularly suitable for conjugating complex biomacromolecules lacking characteristic functional groups.

[0004] However, the excitation wavelength of PFPA lies in the ultraviolet region, which has a shallow penetration depth. It is strongly absorbed and scattered by tissues, and can potentially damage living cells, significantly limiting its application in biological systems. Although the response wavelength of certain aryl azides can be extended to the visible red region (within 660 nm) by introducing photosensitizers or photocatalysts through electron transfer or energy transfer processes, this process requires ensuring that the aryl azide and these introducing agents have matching triplet energy levels, which limits further red shifts. Furthermore, the reactive oxygen species (ROS) generated by photosensitizers are incompatible with normal cells and tissues. Given the large number of CH / NH bonds present in the body, PFPA as a crosslinker can significantly improve the targeted delivery efficiency of nanomedicines into biological systems.

[0005] Lanthanide upconversion luminescent nanoparticles (UCNPs) can convert near-infrared excitation light into high-energy ultraviolet and visible light emissions, making them a promising candidate for expanding the excitation wavelength of photoinduced bioconjugation. Ultraviolet upconversion light emission in the 200-400 nm range can be used to trigger PFPA-based bioconjugation reactions.

[0006] Therefore, there is an urgent need to develop a longer wavelength photocontrollable bioconjugate with tunable spatiotemporal resolution and high biocompatibility to facilitate conjugation with various biomacromolecules in the body, thereby providing possibilities for a variety of applications such as tracking and positioning of biomolecules, biosensing, targeted drug delivery, and the invention of new diagnostic and therapeutic tools. Summary of the Invention

[0007] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a near-infrared light-triggered bioconjugation reagent and its preparation method and use, so as to solve the problem in the prior art that tetrafluorophenyl azide (PFPA) has an excitation wavelength in the ultraviolet region, which limits its application in biological systems.

[0008] To achieve the above-mentioned purpose and other related purposes, the present invention is achieved through the following technical solutions.

[0009] The first aspect of the present invention provides a near-infrared light-triggered biocoupling agent, wherein the raw material components of the biocoupling agent include lanthanide upconversion luminescent nanoparticles and tetrafluorophenyl azide-grafted 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol.

[0010] Preferably, the lanthanide upconversion luminescent nanoparticles are of a core-shell structure; the core layer is based on NaGdF4 as a crystal matrix and is doped with Yb and A elements, and based on the total molar number of Gd, Yb and A elements, the doping amount of the Yb element is (50-x)%, the doping amount of the A element is x%, and 0.5≤x≤2, and A is selected from one or more of Tm, Ho and Er; the core layer is marked as: NaGdF4: (50-x)% Yb, x% A; the shell layer is a three-level shell layer, which is the first shell layer, the second shell layer and the third shell layer from the inside to the outside; the first shell layer is based on NaYF4 as a crystal matrix and is doped with Yb element, and the Y and Yb elements are used as the base. The doping amount of the Yb element is (15-25)% based on the total molar number of Gd, Yb and Nd elements, and the first shell layer is marked as: NaYF4: (15-25)% Yb; the second shell layer uses NaGdF4 as a crystal matrix and is doped with Yb and Nd elements. Based on the total molar number of Gd, Yb and Nd elements, the doping amount of the Yb element is (10-15)%, and the doping amount of the Nd element is (45-55)%. The second shell layer is marked as: NaGdF4: (10-15)% Yb, (45-55)% Nd; the third shell layer is NaGdF4 crystal, and the third shell layer is marked as: NaGdF4.

[0011] More preferably, in the first shell layer, the doping amount of the Yb element is 20%.

[0012] More preferably, in the second shell layer, the doping amount of the Yb element is 10%, and the doping amount of the Nd element is 50%.

[0013] Preferably, the lanthanide upconversion luminescent nanoparticles carry oleic acid ligands, and the oleic acid ligands are used to achieve self-assembly of the lanthanide upconversion luminescent nanoparticles and tetrafluorophenyl azide-grafted 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol.

[0014] Preferably, the particle size of the biocoupling agent is 60 to 100 nm, such as 65 to 85 nm, 70 to 85 nm, or 70 to 90 nm.

[0015] Preferably, the crystal structure of the biocoupling agent is hexagonal.

[0016] Preferably, the crystal structure of the lanthanide up-conversion luminescent nanoparticles is hexagonal.

[0017] Preferably, the lanthanide upconversion luminescent nanoparticles have a particle size of 40 to 70 nanometers, such as 45 to 65 nanometers, or 45 to 62 nanometers. If the particle size is too large, the bioconjugation agent cannot be stably dispersed in the aqueous solution. If the particle size is too small, the upconversion luminescence intensity is low and the bioconjugation efficiency is low.

[0018] Preferably, the particle size of the core layer of the lanthanide upconversion luminescent nanoparticles is 20 to 35 nanometers.

[0019] Preferably, based on the molar amount of lanthanide chloride, the amount of oleic acid added is: 5-15 mL of oleic acid for every 1 mmol of lanthanide chloride. For example, 7-13 mL of oleic acid, 8-12 mL of oleic acid, and 9-11 mL of oleic acid can be added for every 1 mmol of lanthanide chloride.

[0020] Preferably, the grafting yield of the tetrafluorophenyl azide-grafted 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol is 40% to 75%. For example, it may be 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, or 65% to 75%. Only within this grafting yield range can the prepared biocoupling agent be uniformly dispersed in water while achieving high coupling efficiency.

[0021] Preferably, the mass ratio of the lanthanide upconversion luminescent nanoparticles to the tetrafluorophenyl azide-grafted 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol is 1:1-2. For example, the mass ratio may be 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, or 1:2. If the mass of the lanthanide upconversion luminescent nanoparticles is too high, the nanoparticles will not be dispersed in the aqueous solution. If the mass of the lanthanide upconversion luminescent nanoparticles is too low, the yield of the effective bioconjugate will be low.

[0022] More preferably, the general structural formula of the bioconjugate is: NaGdF4:(50-x)%Yb,x%A@NaYF4:20%Yb@NaGdF4:10%Yb,50%Nd@NaGdF4@DSPE-PEG-PFPA; wherein 0.5≤x≤2, and A is selected from one of Tm, Ho and Er.

[0023] The second aspect of the present invention provides a method for synthesizing the near-infrared light-triggered biocoupling agent as described above, wherein the biocoupling agent is formed by self-assembly of lanthanide upconversion luminescent nanoparticles and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol grafted with tetrafluorophenyl azide.

[0024] Preferably, the temperature during the self-assembly process is 20-40° C., for example, 20-35° C., 20-30° C., or 20-25° C. Under these temperature conditions, the oleic acid ligands on the surface of the nanoparticles and the 1,2-distearoyl groups in the tetrafluorophenyl azide-grafted 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol achieve assembly through hydrophobic interactions.

[0025] Preferably, the self-assembly process further comprises a first reaction medium, and the first reaction medium is one or more of dichloromethane, chloroform and toluene.

[0026] Preferably, a first impurity removal is performed before the self-assembly. More preferably, the first impurity removal is performed by rotary evaporation to remove the first reaction medium and other organic impurities in the reaction system.

[0027] Preferably, the synthesis method of the tetrafluorophenyl azide-grafted 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol is as follows: N-hydroxysuccinimide-functionalized tetrafluorophenyl azide (PFPA-NHS) and 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol-amine (DSPE-PEG-NH2) are subjected to an amidation reaction to synthesize the tetrafluorophenyl azide-grafted 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol.

[0028] The choice of DSPE-PEG-NH2 is not arbitrary. In this application, the oleic acid ligand on the nanoparticle surface interacts hydrophobically with the 1,2-distearoyl group in the tetrafluorophenyl azide-grafted 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol, ultimately achieving self-assembly. Furthermore, DSPE-PEG-NH2 exhibits enhanced biosafety, making it more suitable for a wide range of subsequent applications.

[0029] Preferably, the amount of the first reaction medium added is 1-5 mL per 10 mg of lanthanide upconversion luminescent nanoparticles, such as 1-3 mL, 1-2.5 mL, or 2-2.5 mL.

[0030] Preferably, the molar ratio of the N-hydroxysuccinimide-functionalized tetrafluorophenyl azide to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-amine is 1:1 to 1.5, such as 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, and more preferably 1:1 to 1.1.

[0031] Preferably, the amidation reaction further comprises a catalyst. More preferably, the catalyst comprises one or both of triethylamine and pyridine.

[0032] Preferably, the amidation reaction further comprises a second reaction medium, and the second reaction medium is selected from one or more of dichloromethane, toluene and tetrahydrofuran.

[0033] Preferably, after the amidation reaction is completed, a second impurity removal is performed. More preferably, the second impurity removal is performed by rotary evaporation, that is, rotary evaporation is used to remove the reaction medium and other organic impurities in the reaction system.

[0034] Preferably, the catalyst comprises one or both of triethylamine and pyridine.

[0035] Preferably, the molar ratio of the N-hydroxysuccinimide-functionalized tetrafluorophenyl azide to the catalyst is 1:1 to 1.5, such as 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, and more preferably 1:1 to 1.1.

[0036] Preferably, based on the molar amount of N-hydroxysuccinimide-functionalized tetrafluorophenyl azide, the amount of the second reaction medium added is 1-2 mL per 0.011 mol of N-hydroxysuccinimide-functionalized tetrafluorophenyl azide.

[0037] A third aspect of the present invention provides an application as described above, comprising any one of the following:

[0038] 1) As a coupling agent;

[0039] 2) as a signaling molecule;

[0040] 3) As a drug delivery vehicle.

[0041] Preferably, the application is achieved based on the biocoupling agent undergoing insertion reaction of carbon-hydrogen bonds and nitrogen-hydrogen bonds under near-infrared light to form covalent crosslinks.

[0042] Preferably, the biocoupling agent is used for coupling biomolecules with synthetic functional molecules.

[0043] Preferably, the biocoupling agent is used for coupling biomolecules to biomolecules.

[0044] Preferably, the biomolecules include one or more of carbohydrates, proteins, lipid molecules and nucleic acids.

[0045] Preferably, the functional molecules include one or both of drugs and dyes.

[0046] Preferably, the functional molecule comprises a polymer.

[0047] Preferably, the functional molecules include nanoparticles.

[0048] Preferably, the wavelength of the near-infrared light is 780-2500 nm, such as 780-1100 nm, 780-808 nm, 808-1100 nm, or 808-2500 nm.

[0049] The present invention creatively obtains a biocoupling agent by compounding lanthanide upconversion luminescent nanoparticles with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol grafted with tetrafluorophenyl azide. This solves the problem in the prior art that tetrafluorophenyl azide (PFPA) has an excitation wavelength in the ultraviolet region, which limits its application in biological systems.

[0050] The biocoupling agent provided by the present invention can undergo insertion reactions on carbon-hydrogen bonds and nitrogen-hydrogen bonds under near-infrared light to form covalent crosslinks, thereby achieving biocoupling between the interface of cells and functional molecular materials, cells and cells, and living biological tissues and nanomaterials.

[0051] The beneficial effects of the present invention are:

[0052] 1) The technical solution of the present invention can extend the response wavelength of light-induced biocoupling to the near-infrared region (e.g., 780-2500 nm), has a high penetration depth in biological tissues, and can achieve in vivo biocoupling in deep tissues and living organisms in a non-invasive manner.

[0053] 2) The biocoupling agent provided by the present invention is triggered by light and has spatiotemporal controllability. It can also achieve biocoupling between cells and functional molecular material interfaces, cells and cells, and living biological tissues and nanomaterials.

[0054] 3) Since organisms are rich in CH / NH bonds, the insertion cross-linking reaction between the biocoupling agent of the present invention and biological molecules is highly efficient and universal. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Shown is the structure and application diagram of the near-infrared light-triggered spatiotemporal controllable bioconjugate LINC.

[0056] Figure 2 Shown is a transmission electron microscope image of NaGdF4:49% Yb, 1% Tm (Tm-C) during the preparation process of Example 1 of this application.

[0057] Figure 3 Shown is a transmission electron microscope image of NaGdF4:49% Yb, 1% Tm@NaYF4:20% Yb (Tm-CS) during the preparation process of Example 1 of this application.

[0058] Figure 4 Shown is a transmission electron microscope image of NaGdF4:49% Yb, 1% Tm@NaYF4:20% Yb@NaGdF4:10% Yb, 50% Nd (Tm-CSS) during the preparation process of Example 1 of this application.

[0059] Figure 5 Shown is a transmission electron microscope image of NaGdF4:49% Yb, 1% Tm@NaYF4:20% Yb@NaGdF4:10% Yb, 50% Nd@NaGdF4 (Tm-CSSS) prepared in Example 1 of this application.

[0060] Figure 6 Shown is the X-ray powder diffraction analysis pattern of Tm-CSSS and its intermediates prepared in Example 1 of the present application.

[0061] Figure 7 Shown is a transmission electron microscope structural characterization imaging image of the Ho-CSSS prepared in Example 2 of this application.

[0062] Figure 8 Shown is the transmission electron microscope structure characterization imaging image of Er-CSSS prepared in Example 3 of this application.

[0063] Figure 9 Shown is the Fourier transform infrared spectrum of PFPA-DSPE-PEG prepared in Example 4 of this application.

[0064] Figure 10 Shown are Fourier transform infrared (FTIR) spectra of LINC-Tm and hydrophobic nanomaterial Tm-CSSS prepared in Example 5 of the present application.

[0065] Figure 11 Shown are the ultraviolet absorption spectra of LINC-Tm and hydrophobic nanomaterial Tm-CSSS prepared in Example 5 of the present application.

[0066] Figure 12 Shown is the particle size distribution diagram of LINC-Tm prepared in Example 5 of this application in PBS buffer.

[0067] Figure 13 Shown is the UV-visible emission spectrum of LINC-Tm prepared in Example 5 of the present application when excited at 808 nm.

[0068] Figure 14 Shown is the UV-visible emission spectrum of LINC-Ho prepared in Example 6 of the present application when excited at 808 nm.

[0069] Figure 15Shown is the UV-visible emission spectrum of LINC-Er prepared in Example 7 of the present application when excited at 808 nm.

[0070] Figure 16 Shown are the ultraviolet absorption spectra of the LINC-Tm and dextran mixed aqueous solution prepared in Example 5 of the present application under 808 nm excitation at different times.

[0071] Figure 17 Shown are the ultraviolet absorption spectra of the LINC-Ho and dextran mixed aqueous solution prepared in Example 6 of the present application under 808 nm excitation at different times.

[0072] Figure 18 Shown are the ultraviolet absorption spectra of the LINC-Er and dextran mixed aqueous solution prepared in Example 7 of the present application under 808 nm excitation at different times.

[0073] Figure 19 Shown are the ultraviolet absorption spectra of a mixed aqueous solution containing only DSPE-PEG-PFPA and dextran in Comparative Example 1 of the present application under different excitation times at 808 nm.

[0074] Figure 20 Shown is the N1s spectrum in the X-ray photoelectron spectrum of the silicon wafers that have undergone different treatments in Example 14 of the present application.

[0075] Figure 21 Shown is a near-infrared second-third zone imaging analysis diagram of a silicon wafer that has undergone different treatments in Example 14 of this application.

[0076] Figure 22 The figure shows the results of the methylthiazolyl tetrazolium (MTT) assay of LINC in mouse breast cancer 4T1 cells in Example 15 of the present application.

[0077] Figure 23 Shown is a quantitative analysis graph of the number of cells on agarose culture dishes treated under different conditions for 30 minutes in Example 16 of this application.

[0078] Figure 24 Shown is a laser confocal micrograph of 4T1 cells in Example 17 of the present application after incubation with 400 μg / mL of LINC at 37° C. and 5% CO 2 for 2 h.

[0079] Figure 25 Shown are the confocal microscopy results of 4T1 cells treated with different conditions for 30 minutes in Example 17 of this application.

[0080] Figure 26 Shown are images of hematoxylin and eosin (H&E) stained tissue sections (heart, liver, spleen, lung, and kidney) of different groups of mice in Example 18 of the present application after 30 days.

[0081] Figure 27 Shown are near-infrared imaging images of mice after injection of LINC or UCNP in Example 19 of the present application.

[0082] Figure 28 Shown are near-infrared zone 2 imaging images of mice treated under different conditions in Example 19 of this application.

[0083] Figure 29 Shown is a statistical graph of body temperature changes of mice after being treated under different conditions in Example 19 of this application. DETAILED DESCRIPTION

[0084] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0085] Before further describing the specific embodiments of the present invention, it should be understood that the scope of the present invention is not limited to the specific embodiments described below. It should also be understood that the terminology used in the examples is intended to describe specific embodiments and is not intended to limit the scope of the present invention. The experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers.

[0086] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0087] The present application does not specifically limit the method for synthesizing lanthanide upconversion luminescent nanoparticles, as long as lanthanide upconversion luminescent nanoparticles with oleic acid ligands and the general structural formula of NaGdF4: (50-x)% Yb, x% A@NaYF4: 20% Yb@NaGdF4: 10% Yb, 50% Nd@NaGdF4 (wherein 0.5≤x≤2, A is selected from one of Tm, Ho and Er) can be obtained.

[0088] Example 1

[0089] This embodiment provides a specific lanthanide upconversion nanoparticle (Tm-CSSS) with Gd / Tm as the luminescence center and a preparation method thereof.

[0090] The nanoparticles in this embodiment have a 4-layer multi-level core-shell structure, and the structural formula is: NaGdF4:49% Yb, 1% Tm@NaYF4:20% Yb@NaGdF4:10% Yb, 50% Nd@NaGdF4. The specific synthesis steps are as follows:

[0091] 1) Synthesis of Core NaGdF4:49% Yb, 1% Tm (Tm-C) 1-1) 0.5 mmol GdCl3, 0.49 mmol YbCl3, and 0.01 mmol TmCl3 were mixed with 10 ml oleic acid (OA) and 10 ml 1-octadecene in a three-necked flask, heated to 125°C and maintained for 40 min to obtain a clear solution.

[0092] 1-2) After the clear solution obtained in step 1-1) was cooled to 40°C, 11 ml of a methanol solution containing 3.4 mmol of NH4F and 2.5 mmol of NaOH was added to the clear solution. The methanol was removed by degassing at 80°C for 40 minutes, and then heated at 300°C for 1.5 hours in an argon atmosphere.

[0093] 1-3) After the solution from step 1-2) was cooled to room temperature, it was centrifuged at 12,500 rpm for 15 minutes to obtain the core NaGdF4:49% Yb, 1% Tm (Tm-C). The precipitate was washed three times with a 1:1 volume ratio of ethanol to cyclohexane, and the Tm-C nanoparticles were then dispersed in 3 mL of 1-octadecene (ODE).

[0094] The morphology of NaGdF4:49% Yb, 1% Tm prepared in this step is as follows Figure 2 As shown by Figure 2 It can be seen that the morphology of Tm-C is spheres with an average particle size of 28 nm.

[0095] 2) Synthesis of NaGdF4:49%Yb,1%Tm@NaYF4:20%Yb(Tm-CS)

[0096] The Tm-C obtained in step 1) is used as the seed of the core-shell structure and is coated with a shell layer, wherein the molar ratio of the core to the shell is 1:1.

[0097] 2-1) Add 0.4 mmol YCl3, 0.1 mmol YbCl3 (0.5 mmol ReCl3) to 10 mL oleic acid (OA) and 7 mL 1-octadecene (ODE) solution, mix well, degas at 60°C to remove moisture and oxygen from the reaction system, and then heat to 125°C for 40 min to obtain a clear solution.

[0098] 2-2) After the clear solution obtained in step 2-1) was cooled to 40°C, 0.5 mmol of Tm-C prepared in step 1) and 8 ml of a methanol solution containing 1.7 mmol of NH4F and 1.25 mmol of NaOH were added to the clear solution. After degassing at 80°C for 40 minutes to remove the methanol, the solution was heated at 300°C for 1.5 hours in an argon atmosphere.

[0099] 2-3) After the solution in step 2-2) was cooled to room temperature, it was centrifuged at 12,500 rpm for 15 minutes to obtain the NaGdF4:49% Yb, 1% Tm@NaYF4:20% Yb (Tm-CS). The precipitate was washed three times with a 1:1 volume ratio of ethanol and cyclohexane, and the Tm-CS nanoparticles were then dispersed in 3 mL of 1-octadecene (ODE).

[0100] The morphology of NaGdF4:49%Yb,1%Tm@NaYF4:20%Yb prepared in this step is as follows Figure 3 As shown by Figure 3 It can be seen that the average particle size of Tm-CS is 32 nm and it has a hexagonal crystal structure.

[0101] 3) Synthesis of NaGdF4:49%Yb,1%Tm@NaYF4:20%Yb@NaGdF4:10%Yb,50%Nd (Tm-CSS)

[0102] Continue to coat the Tm-CS obtained in step 2), wherein the molar ratio of Tm-CS to Tm-CSS is 1:1

[0103] 3-1) 0.2 mmol GdCl3, 0.05 mmol YbCl3, and 0.25 mmol NdCl3 (0.5 mmol ReCl3) were added to 10 mL oleic acid (OA) and 7 mL 1-octadecene (ODE) solution, mixed well, degassed at 60°C to remove moisture and oxygen from the reaction system, and then heated to 125°C for 40 min to obtain a clear solution.

[0104] 2-2) After the clear solution obtained in step 2-1) was cooled to 40°C, 0.5 mmol of Tm-CS and 8 ml of a methanol solution containing 1.7 mmol of NH4F and 1.25 mmol of NaOH were added to the clear solution. After degassing at 80°C for 40 minutes to remove the methanol, the solution was heated at 300°C for 1.5 hours in an argon atmosphere.

[0105] 2-3) After the solution in step 2-2) was cooled to room temperature, it was centrifuged at 12,500 rpm for 15 minutes to obtain the NaGdF4:49% Yb, 1% Tm@NaYF4:20% Yb@NaGdF4:10% Yb, 50% Nd (Tm-CSS). The precipitate was washed three times with a 1:1 solution of ethanol and cyclohexane by volume, and the Tm-CSS nanoparticles were then dispersed in 3 mL of 1-octadecene (ODE).

[0106] The morphology of NaGdF4:49%Yb,1%Tm@NaYF4:20%Yb@NaGdF4:10%Yb,50%Nd(Tm-CSS) prepared in this step is as follows Figure 4 As shown by Figure 4 It can be seen that the average particle size of Tm-CSS is 45 nm and it has a hexagonal crystal structure.

[0107] 4) Synthesis of NaGdF4: 49% Yb, 1% Tm@NaYF4: 20% Yb @ NaGdF4: 10% Yb, 50% Nd @ NaGdF4 (Tm-CSSS)

[0108] Continue to coat the Tm-CSS obtained in step 3), wherein the molar ratio of Tm-CSS to Tm-CSSS is 1:1

[0109] 4-1) 0.5 mmol of GdCl3 was added to 10 mL of oleic acid (OA) and 7 mL of 1-octadecene (ODE) solution, mixed thoroughly, degassed at 60°C to remove moisture and oxygen from the reaction system, and then heated to 125°C for 40 min to obtain a clear solution.

[0110] 4-2) After the clear solution obtained in step 3-1) was cooled to 40°C, 0.5 mmol of Tm-CSS and 8 ml of a methanol solution containing 1.7 mmol of NH4F and 1.25 mmol of NaOH were added to the clear solution. After degassing at 80°C for 40 minutes to remove the methanol, the solution was heated at 300°C for 1.5 hours in an argon atmosphere.

[0111] 4-3) After the solution from step 3-2) was cooled to room temperature, it was centrifuged at 12,500 rpm for 15 minutes to obtain the NaGdF4:49% Yb, 1% Tm@NaYF4:20% Yb@NaGdF4:10% Yb, 50% Nd@NaGdF4 (Tm-CSSS). The precipitate was washed three times with a 1:1 solution of ethanol and cyclohexane by volume, and the Tm-CSSS was then dispersed in 5 mL of cyclohexane.

[0112] The morphology of NaGdF4:49%Yb,1%Tm@NaYF4:20%Yb@NaGdF4:10%Yb,50%Nd@NaGdF4(Tm-CSSS) prepared in this step is as follows Figure 5 As shown by Figure 5 It can be seen that the average particle size of Tm-CSSS is 54 nm and it has a hexagonal crystal structure.

[0113] Figure 6 The X-ray powder diffraction analysis pattern of Tm-CSSS and its intermediates prepared in this example is shown. Figure 6 It can be seen that Tm-CSSS and its intermediates exhibit X-ray diffraction peaks of hexagonal NaYbF4 and NaGdF4, indicating that Tm-CSSS and its intermediates are hexagonal, proving that Tm-CSSS was prepared in this example.

[0114] Example 2

[0115] This embodiment provides a specific lanthanide upconversion nanoparticle (Ho-CSSS) with Gd / Ho as the luminescence center and a preparation method thereof.

[0116] The nanoparticles in this embodiment have a 4-layer multi-level core-shell structure, and the structural formula is: NaGdF4:49% Yb, 2% Ho@NaYF4:20% Yb@NaGdF4:10% Yb, 50% Nd@NaGdF4. The specific synthesis steps are as follows:

[0117] 1) Synthesis of core NaGdF4:49% Yb, 2% Ho (Ho-C)

[0118] 1-1) Mix 0.49 mmol GdCl3, 0.49 mmol YbCl3, and 0.02 mmol HoCl3 with 10 ml oleic acid (OA) and 10 ml 1-octadecene in a three-necked flask, heat to 125°C and hold for 40 min to obtain a clear solution.

[0119] 1-2) is exactly the same as in Example 1.

[0120] 1-3) are exactly the same as in Example 1.

[0121] 2) Synthesis of NaGdF4:49%Yb,2%Ho@NaYF4:20%Yb(Ho-CS)

[0122] Exactly the same as Example 1.

[0123] 3) The synthesis of NaGdF4:49%Yb,2%Ho@NaYF4:20%Yb@NaGdF4:10%Yb,50%Nd(Ho-CSS) is exactly the same as that in Example 1.

[0124] 3) Synthesis of NaGdF4:49%Yb,2%Ho@NaYF4:20%Yb@NaGdF4:10%Yb,50%Nd@NaGdF4 (Ho-CSSS)

[0125] Exactly the same as Example 1.

[0126] Figure 7 a is a transmission electron micrograph of Ho-CSSS prepared in this example; Figure 7 b is a high-resolution transmission electron microscope image. Figure 7 c is a high-angle annular dark field image of a transmission electron microscope.

[0127] Figure 7 d~h show the elemental maps of Ho-CSSS energy dispersive X-ray spectroscopy (EDS), where Figure 7 d is the distribution of Gd element; Figure 7 e is the distribution of Nd element; Figure 7 f is the distribution of Y elements; Figure 7 g is the distribution of Yb element; Figure 7 h is the merge diagram of the distribution of Gd, Nd, Y and Yb elements.

[0128] Depend on Figure 7 It can be seen that the Ho-CSSS prepared in this embodiment has an average particle size of 62 nm and a hexagonal crystal structure.

[0129] Example 3

[0130] This embodiment provides a specific lanthanide upconversion nanoparticle (Er-CSSS) with Gd / Er as the luminescence center and a preparation method thereof.

[0131] The nanoparticles in this embodiment have a 4-layer multi-level core-shell structure, and the structural formula is: NaGdF4:49% Yb, 1.5% Er@NaYF4:20% Yb@NaGdF4:10% Yb, 50% Nd@NaGdF4. The specific synthesis steps are as follows:

[0132] 1) Synthesis of NaGdF4:49% Yb,1.5% Er (Er-C)

[0133] 1-1) Mix 0.495 mmol GdCl3, 0.49 mmol YbCl3, and 0.015 mmol ErCl3 with 10 ml oleic acid (OA) and 10 ml 1-octadecene in a three-necked flask, heat to 125°C and hold for 40 min to obtain a clear solution.

[0134] 1-2) is exactly the same as in Example 1.

[0135] 1-3) are exactly the same as in Example 1.

[0136] 2) Synthesis of NaGdF4:49%Yb,2%Ho@NaYF4:20%Yb(Er-CS)

[0137] Exactly the same as Example 1.

[0138] 3) The synthesis of NaGdF4:49%Yb,2%Ho@NaYF4:20%Yb@NaGdF4:10%Yb,50%Nd(Er-CSS) is exactly the same as that in Example 1.

[0139] 4) Synthesis of NaGdF4:49%Yb,2%Ho@NaYF4:20%Yb@NaGdF4:10%Yb,50%Nd@NaGdF4(Er-CSSS)

[0140] Exactly the same as Example 1.

[0141] Figure 8 a is a transmission electron micrograph of Er-CSSS prepared in this example; Figure 8 b is a high-resolution transmission electron microscope image. Figure 8 c is a high-angle annular dark field image of a transmission electron microscope.

[0142] Figure 8 d~h show the energy dispersive X-ray spectroscopy (EDS) elemental maps of Er-CSSS, where Figure 8 d is the distribution of Gd element; Figure 8 e is the distribution of Nd element; Figure 8 f is the distribution of Y elements; Figure 8 g is the distribution of Yb element; Figure 8 h is the merge diagram of the distribution of Gd, Nd, Y and Yb elements.

[0143] Depend on Figure 8 It can be seen that the Er-CSSS prepared in this embodiment has an average particle size of 45 nm and a hexagonal crystal structure.

[0144] Example 4

[0145] This embodiment provides a specific tetrafluorophenyl azide-grafted distearoylphosphatidylacetamide-polyethylene glycol (DSPE-PEG-PFPA) and its synthesis method.

[0146] 0.055 mmol of N-hydroxysuccinimide-functionalized tetrafluorophenyl azide (PFPA-NHS) was mixed with 5 ml of CHCl solution. Then, 0.05 mmol of the amphiphilic ligand 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-amine (DSPE-PEG-NH2) and 0.055 mmol of triethylamine were added. The mixture was stirred at room temperature for 8 hours. The CHCl and triethylamine were removed by rotary evaporation to obtain the DSPE-PEG-PFPA grafting product with a yield of 66%.

[0147] The Fourier transform infrared (FTIR) spectra of DSPE-PEG-PFPA and its synthetic raw materials PFPA-NHS and DSPE-PEG-NH2 are as follows Figure 9 As shown. Figure 9 It can be seen that azide (N3) is at 2134 cm -1 The stretching vibration of C=O in ester is at 1740cm -1 and amide at 1648 cm -1 stretching vibration, which indicates the formation of PFPA-DSPE-PEG.

[0148] Example 5

[0149] This example provides a specific bioconjugate (LINC-Tm; Tm-CSSS@DSPE-PEG-PFPA) and its preparation method.

[0150] The bioconjugate in this embodiment is prepared by combining DSPE-PEG-PFPA with lanthanide upconversion luminescent nanoparticles with Gd / Tm as the luminescent center, and the specific steps are as follows:

[0151] 30 mg of the DSPE-PEG-PFPA prepared in Example 4 was mixed evenly with 5 mL of a CH2Cl2 solution, and 20 mg of the cyclohexane-dispersed Tm-CSSS prepared in Example 1 was added. Organic matter was removed from the system by rotary evaporation. The DSPE-PEG-PFPA then self-assembled on the surface of the Tm-CSSS with oleic acid ligands at 25°C. After self-assembly, 1 mL of phosphate buffered saline (PBS) was added for ultrasonic dispersion to obtain the LINC-Tm.

[0152] Figure 10 Shown are the Fourier transform infrared (FTIR) spectra of LINC-Tm and the hydrophobic nanomaterial Tm-CSSS. Figure 11 Shown are the UV absorption spectra of LINC-Tm and hydrophobic nanomaterial Tm-CSSS. Figure 12 Shown is the particle size distribution of LINC-Tm in PBS buffer.

[0153] Depend on Figure 10 It can be seen that in the Fourier transform infrared spectrum of LINC-Tm, N3 is at 2133 cm -1 Stretching vibration occurs at Figure 11 It can be seen that the characteristic absorption of tetrafluorophenyl azide in the 256 nm range can be detected in the UV-visible absorption spectrum of LINC-Tm, which confirms the successful assembly of DSPE-PEG-PFPA on the Tm-CSSS surface to obtain LINC-Tm.

[0154] Depend on Figure 12 It can be seen that the average particle size of the LINC-Tm prepared in this application is about 78.5 nm and is well dispersed in the aqueous phase.

[0155] Example 6

[0156] This example provides a specific bioconjugate (LINC-Ho; Ho-CSSS@DSPE-PEG-PFPA) and its preparation method.

[0157] The bioconjugate in this example is obtained by combining lanthanide upconversion luminescent nanoparticles with Gd / Ho as the luminescent center and DSPE-PEG-PFPA. The specific steps are the same as those in Example 5, except that the Tm-CSSS used is replaced by the Ho-CSSS obtained in Example 2. The remaining steps are exactly the same.

[0158] Example 7

[0159] This embodiment provides a specific bioconjugate (LINC-Er; Er-CSSS@DSPE-PEG-PFPA) and its preparation method.

[0160] The bioconjugate in this example is obtained by combining lanthanide upconversion luminescent nanoparticles with Gd / Er as the luminescent center with DSPE-PEG-PFPA. The specific steps are the same as those in Example 5, except that the Tm-CSSS used is replaced by the Er-CSSS obtained in Example 3. The remaining steps are exactly the same.

[0161] Example 8

[0162] In this example, 808 nm was used as an excitation source to observe whether the LINC-Tm prepared in Example 5 could convert near-red light into ultraviolet light to verify the feasibility of LINC-Tm as a bioconjugating agent. The specific steps were as follows:

[0163] LINC-Tm was dispersed in PBS buffer to prepare a 20 mg / mL solution. 1 mL was placed in a micro-cuvette and UV-visible emission spectra were collected under 808 nm excitation. The specific results are as follows: Figure 13 shown.

[0164] Depend on Figure 13 It can be seen that LINC-Tm exhibits multiple strong emission bands in the ultraviolet band, which indicates that the LINC-Tm prepared in the present application can convert near-red light into ultraviolet light.

[0165] Example 9

[0166] Same as Example 8, except that LINC-Tm in Example 8 was replaced by LINC-Ho prepared in Example 6.

[0167] Specific results such as Figure 14 As shown by Figure 14 It can be seen that LINC-Ho exhibits multiple strong emission bands in the ultraviolet band, which indicates that the LINC-Ho prepared in this application can convert near-red light into ultraviolet light.

[0168] Example 10

[0169] Same as Example 8, except that LINC-Tm in Example 8 was replaced by LINC-Er prepared in Example 7.

[0170] Specific results such as Figure 15 As shown by Figure 15 It can be seen that LINC-Er exhibits multiple strong emission bands in the ultraviolet band, which indicates that the LINC-Er prepared in this application can convert near-red light into ultraviolet light.

[0171] Example 11

[0172] In this example, 808 nm was used as an excitation source to test the changes in ultraviolet light absorption of the mixed solution of LINC-Tm and dextran prepared in Example 5 at different excitation times, thereby verifying whether LINC-Tm can be coupled with dextran (functional molecule). The specific steps are as follows:

[0173] The LINC-Tm prepared in Example 5 was dispersed in water to prepare a 3 mg / mL solution. 3 mL was placed in a standard cuvette, and 60 mg of dextran was added. The solution was mixed thoroughly. The cuvette was irradiated at 808 nm for 0 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, and 180 minutes. After the solution in the cuvette returned to room temperature, the UV absorption spectrum was collected at each excitation time period. The results are shown in Figure 2. Figure 16 shown.

[0174] Depend on Figure 16 It can be seen that with the increase of 808 nm laser irradiation time, the absorption peak of the azide group at 257 nm gradually decreased, which indicates that the azide group in the bioconjugate LINC-Tm underwent an insertion reaction and produced cross-linking with the dextran molecules.

[0175] Example 12

[0176] Same as Example 11, except that LINC-Tm in Example 11 was replaced by LINC-Ho prepared in Example 6, the specific results are as follows Figure 17 shown.

[0177] Depend on Figure 17 It can be seen that with the increase of 808 nm laser irradiation time, the absorption peak of the azide group at 257 nm gradually decreased, which indicates that the azide group in the bioconjugate LINC-Ho underwent an insertion reaction and produced cross-linking with the dextran molecules.

[0178] Example 13

[0179] Same as Example 11, except that LINC-Tm in Example 11 was replaced by LINC-Er obtained in Example 7, the specific results are as follows Figure 18 shown.

[0180] Depend on Figure 18 It can be seen that with the increase of 808 nm laser irradiation time, the absorption peak of the azide group at 257 nm gradually decreased, which indicates that the azide group in the bioconjugate LINC-Er underwent an insertion reaction and produced cross-linking with the dextran molecules.

[0181] Comparative Example 1

[0182] The applicant also used a mixed solution containing only DSPE-PEG-PFPA and dextran as a control, and used 808 nm as the excitation source to test the changes in its absorption of ultraviolet light at different excitation times. The specific operation steps were the same as those in Example 11, except that the LINC-Tm in Example 11 was replaced by DSPE-PEG-PFPA. The specific results are shown in Figure 11. Figure 19 shown.

[0183] Depend on Figure 19 It can be seen that the absorption spectrum hardly changes after 808 nm laser irradiation for 0 minutes, 5 minutes, 30 minutes, and 60 minutes, eliminating the interference of thermal effects on the azide insertion reaction.

[0184] The above experiments show that the LINC prepared in the present application can be coupled with dextran (functional molecule) under 808 nm laser irradiation conditions.

[0185] Example 14

[0186] In this example, LINC-Tm was used as a bioconjugating agent to investigate whether it could undergo a bioconjugation reaction with bovine serum albumin (BSA) under near-infrared light (808 nm).

[0187] The specific steps are as follows: LINC-Tm aqueous solution is added dropwise onto a single crystal silicon wafer modified with bovine serum albumin (BSA@Si) and irradiated with 808nm laser for 1 hour. The X-ray photoelectron spectroscopy (XPS) results are shown in Figure 2. Figure 20 As shown, the results of near-infrared second-third zone imaging analysis are as follows Figure 21 shown.

[0188] Depend on Figure 20 It can be seen that compared with the substrate without 808 nm irradiation, the N1s peak corresponding to the azide structure at 401 eV and 404 eV disappeared after 808 nm irradiation, which proves that under 808 nm irradiation, LINC-Tm undergoes an azide insertion reaction, that is, LINC-Tm is bioconjugated with BSA.

[0189] exist Figure 21 In the figure, the blank column indicates LINC-Tm was added to an unmodified silicon wafer and excited at 808 nm for 1 h; LINC indicates LINC-Tm was added to a silicon wafer modified with BSA; and LINC+808 indicates LINC-Tm was added to a silicon wafer modified with BSA and excited at 808 nm for 1 h. Error bars define the standard deviation (SD) (n = 3).

[0190] Depend on Figure 21 It can be seen that compared with the silicon wafers marked as blank or LINC, the silicon wafer marked as LINC+808, that is, the silicon wafer treated with LINC-Tm and irradiated with 808nm, showed stronger emission intensity in the near-infrared region, indicating that LINC-Tm can undergo coupling reaction with BSA under 808nm laser irradiation conditions.

[0191] Example 15

[0192] This example investigates the in vitro biocompatibility of LINC-Tm prepared in Example 5. The specific steps are as follows:

[0193] (1) Mouse breast cancer 4T1 cells were seeded into 96-well cell culture plates at 1×10 4 cells per well and cultured at 37°C and 5% CO 2 for 24 h.

[0194] (2) DMEM culture medium containing different concentrations of LINC (0, 50, 100, 200, 300, 400, and 500 μg / mL) was added to the wells, and 4T1 cells were incubated at 37°C and 5% CO2 for 48 h.

[0195] (3) Methylthiazolyl tetrazolium (MTT) (20 μL; 5 mg / mL) was added to each well, and the assay plate was incubated at 37° C. and 5% CO 2 for another 4 hours.

[0196] (4) Measure the optical density of each well using an enzyme-labeled instrument.

[0197] The cell survival rate (%) of the control group was taken as 100%, and the cell survival rate (%) was calculated by the ratio of the absorbance values ​​of the methylthiazolyl tetrazolium product formazan produced in the treatment group and the control group. The specific results are as follows: Figure 22 shown.

[0198] Depend on Figure 22 It can be seen that LINC did not show obvious toxicity at an incubation dose of up to 500 μg / mL, wherein the cell viability was still maintained above 85%, which indicates that the LINC prepared in Example 5 of the present application has good biocompatibility in vitro.

[0199] Example 16

[0200] In this example, LINC-Tm obtained in Example 5 was used as a biocoupling agent to explore whether it could be used to couple cells with agarose molecules. The specific steps were as follows:

[0201] After coating the agarose substrate on the confocal culture dish, 1 mL of LINC-Tm aqueous solution (concentration of 20 mg / mL) was added to the agarose surface and allowed to stand overnight. The excess liquid was then aspirated and the dish was rinsed three times with PBS. 4T1 cells (1×10 5 / ), treated under different conditions for 30 minutes, and then washed three times with PBS. The number of cells in the cell culture dish under different conditions was observed under a confocal microscope. The specific results are as follows Figure 23 shown.

[0202] Specifically, blank means: 4T1 cells were not treated in any way; LINC means: LINC-Tm was added to the agarose substrate; 808 means: 4T1 cells on the agarose substrate were irradiated with 808 nm laser; LINC+808 means: the agarose substrate with LINC-Tm was irradiated with 808 nm laser for 30 min.

[0203] Depend on Figure 23 It can be seen that the number of cells in the LINC+808 group is 29 times that of the blank group, which indicates that under near-infrared light irradiation, the agarose substrate can couple with cells through LINC-Tm, thereby enhancing the adhesion of cells on the substrate.

[0204] Example 17

[0205] In this example, LINC-Tm prepared in Example 5 was used as a bioconjugating agent to investigate its ability to perform bioconjugation between cells in vitro. The specific steps were as follows:

[0206] Adherent 4T1 cells were incubated with Roswell Park Memorial Institute medium (RPMI 1640) containing 400 μg / mL LINC-Tm at 37°C and 5% CO2 for 2 h, washed three times with PBS, and observed under a laser confocal microscope.

[0207] Depend on Figure 24 It can be seen that the upconversion luminescence signal (400-500 nm) of LINC-Tm was detected on the cell surface, indicating that LINC-Tm was successfully labeled on the cell membrane surface.

[0208] The 4T1 cells were digested and treated under different conditions for 30 minutes, then observed using a confocal laser scanning microscope. Specifically, "Blank" indicates untreated cells; "LINC" indicates cells surface-labeled with LINC-Tm but without 808nm excitation; "808" indicates cells unlabeled with LINC-Tm irradiated with 808nm for 30 minutes; and "LINC+808" indicates cells labeled with LINC-Tm irradiated with 808nm for 30 minutes.

[0209] Figure 25 Shown are confocal microscopy images of 4T1 cells treated with different conditions for 30 min. Figure 25 It can be seen that the LINC+808 group showed obvious cell clusters. Z-axis confocal scanning of cells stained with cell membrane dye revealed that compared with the Blank, LINC, and 808 groups, the LINC+808 group showed a clustered distribution. This shows that the LINC-Tm prepared in Example 5 of this application can couple cells to cells in vitro.

[0210] Example 18

[0211] This example investigates the in vivo biocompatibility of the LINC-Tm prepared in Example 5. The specific steps are as follows:

[0212] 200 μL (concentration of 20 mg / mL) of LINC-Tm and Tm-CSSS@DSPE-PEG (UCNP) without PFPA grafting were injected intravenously into mice, and the main organs (heart, liver, spleen, lung and kidney) of mice in different groups were analyzed by hematoxylin and eosin staining 30 days later. The specific results are as follows: Figure 26 shown.

[0213] Depend on Figure 26It can be seen that no organ abnormalities or damage were observed in either the group injected with LINC-Tm alone or the group injected with LINC-Tm and excited at 808nm for 1h (LINC+808 / UCNP+808), indicating that the LINC-Tm prepared in this application and irradiation with 808nm laser do not cause significant toxicity to living animals.

[0214] Example 19

[0215] In this example, the LINC-Tm prepared in Example 5 was used as a bioconjugating agent to conduct an in vivo bioconjugation experiment between LINC-Tm and tumor areas controlled by near-infrared light. The specific steps are as follows:

[0216] 200 μL LINC-Tm (20 mg / mL) and 200 μL UCNP (20 mg / mL) dispersed in PBS buffer were injected into the mice through the tail vein, and then the mice were immediately imaged by near-infrared optical imaging. Figure 27 It can be seen that luminescent signals can be observed in the blood vessels of mice, indicating that LINC-Tm has good blood circulation. Figure 27 The area marked by the yellow box in the middle is the tumor site in the mouse.

[0217] Mice injected with LINC-Tm and UCNP were irradiated at 808 nm for 1 h, and near-infrared optical imaging of mice under different conditions was observed, and the body temperature of the mice was measured.

[0218] Figure 28 Shown are near-infrared optical images of mice under different conditions; Figure 29 Shown are temperature statistics of mice under different conditions. Specifically, LINC represents the accumulation of LINC-Tm in mice after injection, UCNP+808 represents the near-infrared (NIR) zone II imaging of mice injected with UCNPs and excited at 808 nm for 1 hour, and LINC+808 represents the near-infrared (NIR) zone II imaging of mice injected with LINC-Tm and excited at 808 nm for 1 hour.

[0219] Depend on Figure 28 It can be seen that the tumor area of ​​the LINC+808 group mice had stronger luminescence signals than the LINC group and UCNP+808 group. Figure 28 The yellow circle in the upper right corner is the tumor area, indicating that the LINC-Tm prepared in this application is controlled by near-infrared light and can be coupled with organisms in the tumor area.

[0220] Depend on Figure 29 It can be seen that no obvious increase in mouse body temperature was found under 808nm irradiation, which ruled out the influence of thermal effect on the coupling reaction.

[0221] In summary, PFPA-functionalized LINC-Tm as a near-infrared light-triggered bioconjugate agent effectively promoted its labeling in the tumor area, realizing the temporal and spatial controllability of bioconjugate agents in living systems, and proving that near-infrared light-triggered bioconjugate agents can effectively promote the targeting of tumor sites by cross-linking with biological interfaces in vivo.

[0222] In this application, the experimental methods involved are:

[0223] 1) X-ray diffraction analysis

[0224] The prepared sample was subjected to X-ray diffraction analysis using a Bruker D2 X-ray diffraction analyzer to obtain an X-ray diffraction analysis spectrum.

[0225] 2) X-ray photoelectron spectroscopy analysis

[0226] The prepared sample was subjected to X-ray photoelectron spectroscopy analysis using a K-alpha X-ray photoelectron spectrometer to obtain an X-ray photoelectron spectroscopy analysis spectrum.

[0227] 3) Fourier transform infrared (FTIR) method

[0228] The prepared samples were analyzed using a VERTEX 70 Fourier transform infrared spectrometer to obtain Fourier transform infrared (FTIR) spectra.

[0229] 4) Ultraviolet absorption spectroscopy

[0230] The prepared samples were analyzed using a Shimadzu 3000 ultraviolet absorption spectrometer to obtain ultraviolet absorption spectra.

[0231] 5) Particle size distribution

[0232] The sample was dispersed in deionized water and analyzed using a laser particle size analyzer to obtain a particle size distribution diagram.

[0233] 6) Near-infrared second-third zone imaging analysis

[0234] The sample was dispersed in physiological saline and analyzed using a NIR vana 640 near-infrared camera to obtain near-infrared optical imaging images.

[0235] 7) Hematoxylin and eosin staining analysis

[0236] The tissue section samples were stained with hematoxylin and Yin Hong dye respectively, then washed with deionized water, and the tissue structure was observed after sealing.

[0237] In summary, the biocoupling agent provided by the present invention can be triggered by near-infrared light and has a high penetration depth in biological tissues, thereby achieving in vivo biocoupling in deep tissues and living organisms; the biocoupling agent has good biocompatibility and can achieve coupling of biomolecules and functional molecules in vivo and in vitro, and it has high spatiotemporal controllability, high efficiency and universal applicability.

[0238] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A near-infrared light-induced biocoupling agent, characterized in that: The raw material components of the biocoupling agent include lanthanide up-conversion luminescent nanoparticles and tetrafluorophenyl azide-grafted 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol.

2. The biocoupling agent according to claim 1, characterized in that The lanthanide upconversion luminescent nanoparticles are of a core-shell structure; The core layer uses NaGdF4 as a crystal matrix and is doped with Yb and A elements, wherein the doping amount of the Yb element is (50-x)% based on the total molar number of the Gd, Yb and A elements, and the doping amount of the A element is x%; and 0.5≤x≤2, and A is selected from one or more of Tm, Ho and Er; The shell is a three-level shell, from the inside to the outside, it is the first shell, the second shell and the third shell; The first shell layer uses NaYF4 as a crystal matrix and is doped with Yb element, and the doping amount of the Yb element is (15-25)% based on the total molar number of Y and Yb elements; The second shell layer uses NaGdF4 as a crystal matrix and is doped with Yb and Nd elements. Based on the total molar number of Gd, Yb and Nd elements, the doping amount of the Yb element is (10-15)%, and the doping amount of the Nd element is (45-55)%. The third shell is NaGdF4 crystal; And / or, the lanthanide upconversion luminescent nanoparticles carry oleic acid ligands.

3. The biocoupling agent according to claim 1, characterized in that The average particle size of the biocoupling agent is 60 to 100 nanometers; And / or, the crystal structure of the biocoupling agent is hexagonal; And / or, the crystal structure of the lanthanide up-conversion luminescent nanoparticles is hexagonal; And / or, the particle size of the lanthanide upconversion luminescent nanoparticles is 40 to 70 nanometers; And / or, the particle size of the core layer of the lanthanide upconversion luminescent nanoparticles is 20 to 35 nanometers; and / or, based on the molar amount of lanthanide chloride, 5 to 15 mL of oleic acid is added to 1 mmol of lanthanide chloride; and / or, the grafting rate of the tetrafluorophenyl azide-grafted 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol is 40% to 75%; And / or, the mass ratio of the lanthanide upconversion luminescent nanoparticles to the tetrafluorophenyl azide-grafted 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol is 1:1-2.

4. A method for synthesizing a near-infrared light-triggered biocoupling agent as claimed in claims 1 to 3, characterized in that: Lanthanide upconversion luminescent nanoparticles and tetrafluorophenyl azide-grafted 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol are self-assembled to form the biocoupling agent.

5. The synthesis method according to claim 4, characterized in that The temperature during the self-assembly process is 20-40°C; And / or, the self-assembly process further comprises a first reaction medium, wherein the first reaction medium is one or more of dichloromethane, chloroform and toluene; and / or, before the self-assembly, performing a first impurity removal; And / or, the synthesis method of the tetrafluorophenyl azide-grafted 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol is: N-hydroxysuccinimide-functionalized tetrafluorophenyl azide and 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol-amine are subjected to an amidation reaction to synthesize the tetrafluorophenyl azide-grafted 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol.

6. The synthesis method according to claim 5, characterized in that The amount of the first reaction medium added is 1 to 5 mL per 10 mg of lanthanide upconversion luminescent nanoparticles; and / or, the molar ratio of the N-hydroxysuccinimide-functionalized tetrafluorophenyl azide to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-amine is 1:1 to 1.5; And / or, the amidation reaction further uses a catalyst; And / or, the amidation reaction further comprises a second reaction medium, wherein the second reaction medium is selected from one or more of dichloromethane, toluene and tetrahydrofuran; And / or, after the amidation reaction is completed, a second impurity removal is performed.

7. The synthesis method according to claim 6, characterized in that The catalyst includes one or two of triethylamine and pyridine; and / or, the molar ratio of the N-hydroxysuccinimide-functionalized tetrafluorophenyl azide to the catalyst is 1:1 to 1.5; And / or, the amount of the second reaction medium added is 1 to 2 mL per 0.011 mol of N-hydroxysuccinimide-functionalized tetrafluorophenyl azide.

8. A use of the biocoupling agent according to any one of claims 1 to 3, comprising any one of the following: 1) As a coupling agent; 2) as a signaling molecule; 3) As a drug delivery vehicle.

9. The application according to claim 8, characterized in that: The application is achieved based on the biocoupling agent inserting carbon-hydrogen bonds and nitrogen-hydrogen bonds under near-infrared light to form covalent crosslinks; And / or, the biocoupling agent is used for coupling biomolecules with functional molecules; And / or, the biocoupling agent is used for coupling biomolecules to biomolecules.

10. The use according to claim 9, characterized in that The biomolecules include carbohydrates, proteins, one or more of lipid molecules and nucleic acids; And / or, the functional molecules include one or both of drugs and dyes; and / or, the functional molecule comprises a polymer; And / or, the functional molecules include nanoparticles.

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