A preparation method of rare earth up-conversion nanoparticles, products thereof and application in preparation of photopolymerization hydrogel and drug release

Abstract

The application discloses a preparation method of rare earth up-conversion nanoparticles, a product thereof and application of the product in preparation of photopolymerization hydrogel and drug release. The application first synthesizes core nanoparticles, then step-by-step synthesizes core-shell nanoparticles, optimizes the structure of UCNPs through design, establishes a double-wavelength orthogonal light-emitting system, makes the UCNPs emit visible green light under 808nm near-infrared excitation for assisting photopolymerization hydrogel, then makes the UCNPs emit ultraviolet light under 980nm excitation for cutting chemical bonds, and the drug is continuously and slowly released in the hydrogel wrapping. The power density and irradiation time length of the near-infrared light excited UCNPs are controlled through feedback regulation of gelation and drug release rate, and the application has wide application prospect in cancer treatment.

Description

Technical Field

[0001] This invention relates to a method for preparing rare earth upconversion nanoparticles and their products, as well as their applications in the preparation of photopolymer hydrogels and drug release, belonging to the field of biofunctional materials. Background Technology

[0002] Precise spatiotemporal delivery of oncology drugs is crucial for reducing drug side effects and enhancing therapeutic efficacy. Hydrogels, as an emerging drug carrier, can achieve sustained local drug delivery at the tumor site and have been widely used in cancer treatment in recent years. Compared with systemic chemotherapy, hydrogel drug carriers have lower toxicity, better biocompatibility and biodegradability, and can respond to environmental stimuli (such as light, heat, pH, and ultrasound) to achieve in-situ gelation and controlled drug release, thus greatly improving the convenience and efficiency of drug delivery.

[0003] Photopolymerization refers to a light-induced polymerization process. In a typical photopolymerization process, a photoinitiator absorbs incident light of a specific wavelength (ultraviolet or visible light) and generates an active substance, which then initiates a chain polymerization reaction of oligomers / comonomers to form a cross-linked or linear polymer structure. Photoresponsive drug delivery carriers prepared by combining photopolymerization with hydrogels possess excellent biocompatibility and abundant porous channels, allowing for the loading of photosensitizers or other therapeutic drugs for phototherapy. However, the vast majority of photopolymerization reactions require excitation by ultraviolet or visible light, which significantly limits their clinical application.

[0004] Compared to ultraviolet / visible light, near-infrared (NIR) light has greater penetrating power, lower phototoxicity, and lower thermal effects. NIR can excite upconversion nanoparticles (UCNPs) to emit ultraviolet / visible light; therefore, the fluorescence of UCNPs can be used to trigger the photoreaction of photosensitive compounds in vivo.

[0005] Upconversion luminescence is an anti-Stokes phenomenon, referring to the phenomenon where a material, when excited by low-energy light, transitions from its ground state to a metastable state I, absorbs another low-energy photon, transitions to a metastable state III, relaxes through internal energy conversion to a metastable state II, and finally returns to its ground state via radiative relaxation, producing nonlinear photon emission. It has been widely used in fields such as bioimaging, temperature sensing, and drug delivery. UCNPs represent a class of optical nanomaterials, generally composed of an optically inert inorganic matrix and optically active rare-earth (RE) trivalent ions. Currently, REs are commonly used... 3+ It is Yb 3+ Er 3+ and Tm 3+The former has a large absorption cross section in the NIR region (980nm) and is a relatively ideal sensitizer material. The latter two ions have a ladder-like energy level arrangement, which helps to absorb multiple photons to achieve upconversion emission. Co-doping the sensitizer and the activator can efficiently emit high-energy photons.

[0006] UCNPs have a series of advantages as therapeutic diagnostic agents: (1) The excitation wavelength of UCNPs is usually 808nm or 980nm, which is exactly in the "transparency window" (700-1000nm) of biological tissues, increasing the penetration depth; (2) When UCNPs are used as upconversion luminescent biomedical imaging probes, the endogenous and exogenous fluorophores in the organism cannot be excited by NIR radiation, so no background autofluorescence is generated, resulting in a better signal-to-noise ratio; (3) Due to Ln 3+ With similar radii and chemical properties, UCNPs can be controlled by doping with different types of Ln. 3+ Used for multimodal imaging without significantly affecting its fluorescence, such as Gd 3+ Used for MRI, Lu 3+ Used in CT, etc.; (4) The surface of UCNPs can be further modified, for example, by combining with organic active groups, biological macromolecules and prodrugs to make them functional, and has now become an ideal choice for therapeutic diagnostics and stimulus-responsive therapeutic diagnostics; (5) The advantages of narrow-band emission, long fluorescence lifetime, good anti-photobleaching stability and high photochemical stability make UCNPs widely used in in vivo and in vitro therapy.

[0007] Currently, common methods for synthesizing rare-earth-doped UCNPs include thermal decomposition, hydrothermal synthesis, solvothermal synthesis, sol-gel treatment, combustion synthesis, and coprecipitation. Because the nuclear fluorescence of UCNPs is prone to quenching, Zhang Fan et al. proposed a successive layer-by-layer (SLBL) epitaxial growth strategy. This involves continuously introducing a shell precursor solution to deposit uniform multi-shell layers on a hexagonal (β) or cubic (α) phase core. By adjusting the number of shell precursors, nanoparticles with a narrow size distribution (σ < 10%) can be obtained, and the shell thickness can be controlled from one monolayer (~0.36 nm) to more than 20 monolayers (~8 nm). However, due to energy loss during transitions such as cross-relaxation, current techniques mostly synthesize single-wavelength excited upconversion nanoparticles, and the luminescence efficiency and stability of the synthesized nanomaterials are relatively low. Summary of the Invention

[0008] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for preparing dual-wavelength orthogonal luminescent nanoparticles with high upconversion luminescence efficiency, as well as the products and applications thereof, to achieve upconversion emission of visible green light to assist photopolymerization of hydrogels under near-infrared excitation at 808nm, and upconversion emission of ultraviolet light to release drugs under ultraviolet light under 980nm excitation.

[0009] Technical Solution: To solve the above-mentioned technical problems, the present invention provides a method for preparing rare earth upconversion nanoparticles, comprising the following steps:

[0010] (1) GdCl3, YbCl3 and ErCl3 were mixed and oleic acid, 1-octadecene and methanol solution containing NaOH and NH4F were added to obtain core nanoparticle solution;

[0011] (2) Mix oleic acid, 1-octadecene and the core nanoparticle solution described in step (1), and alternately add Y-Yb-OA and Na-TFA-OA shell precursors to obtain CS1 core-shell nanoparticle solution;

[0012] (3) A solution of mixed oleic acid, 1-octadecene, and the CS1 core-shell nanoparticles described in step (2) was mixed, and Gd-Yb-Nd-OA and Na-TFA-OA shell precursors were added alternately to obtain CS1 core-shell nanoparticles. 1-2 Core-shell nanoparticle solution;

[0013] (4) A mixture of oleic acid, 1-octadecene and the CS described in step (3). 1-2 A solution of core-shell nanoparticles was prepared by alternately adding Y-OA and Na-TFA-OA shell precursors to obtain CS. 1-3 Core-shell nanoparticle solution;

[0014] (5) A mixture of oleic acid, 1-octadecene and the CS described in step (4). 1-3 A solution of core-shell nanoparticles was prepared by alternately adding Gd-Yb-Tm-OA and Na-TFA-OA shell precursors to obtain CS. 1-4 Core-shell nanoparticle solution;

[0015] (6) A mixture of oleic acid, 1-octadecene and the CS described in step (5). 1-4 A solution of core-shell nanoparticles was prepared by alternately adding Y-OA and Na-TFA-OA shell precursors to obtain rare earth upconversion nanoparticles.

[0016] The preparation method of the Y-Yb-OA shell precursor in step (2) includes the following steps: stirring and heating a mixture of YCl3, YbCl3, OA and ODE to obtain the Y-Yb-OA shell precursor.

[0017] The preparation method of the Na-TFA-OA shell precursor in step (2) includes the following steps: stirring and heating a mixture of Na-TFA and OA to obtain the Na-TFA-OA shell precursor.

[0018] The preparation method of the Gd-Yb-Nd-OA shell precursor in step (3) includes the following steps: magnetically stirring and heating a mixture of GdCl3, YbCl3, NdCl3, OA and ODE to obtain the Gd-Yb-Nd-OA shell precursor.

[0019] The present invention also provides rare earth upconversion nanoparticles prepared by the method described above.

[0020] This invention also provides a method for preparing photopolymerized hyaluronic acid hydrogel using the aforementioned rare-earth upconversion nanoparticles, comprising the following steps:

[0021] (1) Add excess N-(3-dimethylaminopropyl)-N'-ethylcarbodimethylimine hydrochloride and excess N-hydroxysuccinimide to a hyaluronic acid solution, then add 2-aminoethyl methacrylate hydrochloride. After thorough mixing, adjust the pH of the solution to a stable value of 4.7 and react overnight at room temperature. Adjust the pH of the solution to 7.0, dialyze, and dialyze with ethanol and deionized water respectively. Freeze dry to obtain the cotton-like flocculent product HA-AEME.

[0022] (2) Eosin Y, triethanolamine, monomer NVP and HA-AEMA described in step (1) are dissolved and mixed in PBS solution, and then the upconversion nanoparticles described in claim 1 are added and irradiated with 808nm near-infrared light to obtain photopolymerized hyaluronic acid hydrogel.

[0023] The present invention also provides a photopolymerized hyaluronic acid hydrogel prepared by the method described above.

[0024] The present invention also provides the application of the aforementioned rare earth upconversion nanoparticles in in vivo imaging.

[0025] The present invention also provides the application of the aforementioned photopolymerized hyaluronic acid hydrogel in in vivo imaging.

[0026] The present invention also provides the application of the aforementioned rare earth upconversion nanoparticles in the conversion of near-infrared light.

[0027] The rare earth upconversion nanoparticles convert near-infrared light into visible green light, blue light, or ultraviolet light.

[0028] The present invention also provides the application of the aforementioned rare earth upconversion nanoparticles in the preparation of drugs and / or diagnostic reagents for treating and / or detecting tumors.

[0029] The present invention also provides the use of the photopolymerized hyaluronic acid hydrogel in the preparation of drugs and / or diagnostic reagents for treating and / or detecting tumors.

[0030] This invention optimizes the SLBL method, designing and optimizing the upconversion nanoparticle structure as β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%@NaGdF4:Yb20% / Nd50%@NaYF4@NaGdF4:Yb80% / Tm1%@NaYF4. Then, UCNPs are modified with hyaluronic acid ligands, and the prodrug ONB-5-FU is covalently crosslinked. The schematic diagram of the synthesis is shown below. Figure 9 As shown, the resulting UCNPs-AEMA-ONB-5-FU complex exhibits good cell affinity, enabling precise tumor therapy by targeting cancer cells with CD44, a drug development target derived from tumor stem cells. By adjusting the type and order of the shell precursors, tunable doping sites (core doping and shell doping) can be achieved, thus improving the core-shell nanostructure in several aspects, including upconversion luminescence efficiency, stability, and multicolor luminescence.

[0031] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: 1. This invention designs and optimizes the structure of upconversion nanoparticles (UCNPs), establishes a dual-wavelength orthogonal luminescence system, and achieves upconversion emission of visible green light with a main wavelength of 545nm under near-infrared excitation at 808nm, and upconversion emission of blue light with a main wavelength of 460nm and ultraviolet light with a main wavelength of 365nm under near-infrared excitation at 980nm; 2. This invention applies green light to assist photopolymerization of hydrogels, blue light to in vivo imaging, and ultraviolet light to break chemical bonds to release anti-tumor drugs, solving the problem of low tissue penetration of ultraviolet-visible light; 3. The UCNPs synthesized in this invention effectively reduce energy loss caused by cross-relaxation, have high upconversion luminescence efficiency, and good uniformity and stability; 4. The UCNPs synthesized in this invention, after modification with hyaluronic acid ligands, have good biocompatibility and cell affinity, and can better target cancer cells by leveraging the drug development target CD44 of tumor stem cells to release drugs for precise tumor treatment. Attached Figure Description

[0032] Figure 1 Characterization of UCNPs: where a is the result of transmission electron microscopy (TEM); b is the upconversion emission spectrum of UCNPs excited by near-infrared light at 808 nm and 980 nm, respectively; c is a schematic diagram of the upconversion energy level transition principle;

[0033] Figure 2 Synthetic route and 1H NMR characterization of the hydrogel comonomer HA-AEMA;

[0034] Figure 3Hydrogels with low, medium, and high crosslinking densities formed under different irradiation times: a is the storage modulus G'; b and c are morphological comparisons of different hydrogels after swelling and freeze-drying.

[0035] Figure 4 The retention of hydrogels with low, medium, and high crosslinking densities formed at different illumination times in mice: a) fluorescence imaging; b) fluorescence quantitative curve;

[0036] Figure 5 The synthetic route and 1H NMR characterization of the prodrug ONB-5-FU were described.

[0037] Figure 6 In the figure, a is the standard curve of 5-FU; b and c are the high performance liquid chromatograms (HPLC) of ONB-5-FU and 5-FU.

[0038] Figure 7 In Figure a, the quantitative curve of the cumulative release of 5-FU from the ONB-5-FU aqueous solution under UV light is shown; in Figure b, the quantitative curve of the cumulative release of 5-FU from the hydrogel loaded with UCNPs-ONB-5-FU under 980nm near-infrared on-off excitation is shown.

[0039] Figure 8 The following are the results of the MTT assay: a) Survival rate of B16-F10 cells after 24 hours of co-incubation with different concentrations of ONB-5-FU and 5-FU; b) Survival rate of B16-F10 cells after 4 hours of co-incubation with 10 μMUCNPs-ONB-5-FU followed by 20 hours of incubation with 980nm near-infrared light for different durations; c) Survival rate of B16-F10 cells after 24 hours of co-incubation with different concentrations of UCNPs-ONB-5-FU; d) Survival rate of B16-F10 cells after 4 hours of co-incubation with different concentrations of UCNPs-ONB-5-FU followed by 50 seconds of incubation with 980nm near-infrared light for 20 hours; e) Survival rate of Hacat cells after 24 hours of co-incubation with different concentrations of 5-FU; f) ​​Survival rate of Hacat cells after 4 hours of co-incubation with different concentrations of UCNPs-ONB-5-FU followed by 50 seconds of incubation with 980nm near-infrared light for 20 hours.

[0040] Figure 9 A schematic diagram illustrating the invention mechanism for forming a UCNPs-AEMA-ONB-5-FU complex by surface modification of UCNPs;

[0041] Figure 10Upconversion emission spectra of β-NaGdF4:Yb20% / Tm0.5%@NaYF4:Yb20%(0.8mmol)@NaGdF4:Yb20% / Er2%(0.4mmol)@NaGdF4:Yb20% / Nd50%(0.6mmol)@NaYF4(1mmol) under near-infrared excitation at 980nm and 808nm, respectively.

[0042] Figure 11 The upconversion emission spectra of β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%(0.8mmol)@NaGdF4:Yb20% / Nd50%(0.4mmol)@NaYF4(1mmol)@NaGdF4:Yb20% / Tm0.5%(1mmol)@NaYF4(1mmol) are shown under near-infrared excitation at 980nm and 808nm, respectively.

[0043] Figure 12 The upconversion emission spectra of β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%(0.8mmol)@NaGdF4:Yb20% / Nd50%(0.6mmol)@NaYF4(1mmol)@NaGdF4:Yb80% / Tm1%(1mmol)@NaYF4(1mmol) are shown under near-infrared excitation at 980nm and 808nm, respectively.

[0044] Figure 13 The upconversion emission spectra of β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%(0.8mmol)@NaGdF4:Yb20% / Nd50%(0.6mmol)@NaYF4(1mmol)@NaGdF4:Yb80% / Tm1%(3mmol)@NaYF4(2mmol) are shown under near-infrared excitation at 980nm and 808nm, respectively.

[0045] Figure 14 The upconversion emission spectra of β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%(0.8mmol)@NaGdF4:Yb20% / Nd50%(0.6mmol)@NaYF4(1mmol)@NaGdF4:Yb80% / Tm1%(1.6mmol)@NaYF4(1mmol) are shown under near-infrared excitation at 980nm and 808nm, respectively. Detailed Implementation

[0046] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0047] Example 1: Design, Synthesis and Characterization of UCNPs

[0048] 1. Raw materials

[0049] Anhydrous gadolinium(III) chloride (GdCl3, 99.99%), anhydrous yttrium(III) chloride (YCl3, 99.9%), anhydrous ytterbium(III) chloride (YbCl3, 99.9%), anhydrous neodymium(III) chloride (NdCl3, 99.9%), anhydrous erbium(III) chloride (ErCl3, 99.9%), anhydrous thulium(III) chloride (TmCl3, 99.9%), sodium trifluoroacetate (Na-TFA, 98%), 1-octadecene (ODE, 90%), and oleic acid (OA, 90%) were purchased from [unspecified sources]. Sigma-Aldrich; sodium hydroxide (NaOH, 96%) and ammonium fluoride (NH4F, 96%) were purchased from Aladdin Reagent Co., Ltd. (Shanghai); anhydrous ethanol (99%) and cyclohexane (99.7%) were purchased from Sinopharm Chemical Reagent Co., Ltd.; N,N-dimethylformamide (DMF, 99%) was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd.; nitrosamine tetrafluoroborate (BF4NO, 95%) was purchased from Shanghai Maclean Biotechnology Co., Ltd.; hyaluronic acid (HA, 55kDa) was purchased from Bloomage Biotechnology Co., Ltd. All chemicals were used as is without further purification.

[0050] 2. Design and Synthesis of UCNPs

[0051] (1) Preparation of shell precursor

[0052] Y-OA main shell precursor: A mixture of YCl3 (5.0 mmol), OA (10.0 mL) and ODE (15.0 mL) was placed into a 100 mL round-bottom three-necked flask and heated under vacuum at 140 °C with magnetic stirring for 30 min to remove residual water and oxygen, resulting in a colorless and transparent Y-OA shell precursor solution (0.2 M).

[0053] Y-Yb-OA, Gd-Yb-Nd-OA, and Gd-Yb-Tm-OA dopant shell precursors: The synthesis of Y-Yb-OA (0.20 M), Gd-Yb-Nd (0.05 M), and Gd-Yb-Tm (0.20 M) is the same as that of the Y-OA host shell precursor: the rare earth chlorides of Y-Yb-OA are replaced with YCl3 (4.0 mmol) and YbCl3 (1.0 mmol) instead of 5.0 mmol YCl3, and the rare earth chlorides of Gd-Yb-Nd-OA (0.05 M) are replaced with GdCl3 (0.375 mmol), YbCl3 (0.25 mmol), and NdCl3 (0.625 mmol) instead of 5.0 mmol. The rare earth chlorides of YCl3 and Gd-Yb-Tm-OA (0.2M) are prepared by replacing 5.0 mmol of YCl3 with YCl3 (0.95 mmol), YbCl (4.0 mmol), and TmCl3 (0.05 mmol), while other raw materials and amounts remain the same.

[0054] Na-TFA-OA shell precursor: A mixture of Na-TFA (8.0 mmol) and OA (10 mL) was placed in a 100 mL round-bottom three-necked flask and heated under vacuum at 120 °C with magnetic stirring for 30 min to remove residual water and oxygen, yielding a colorless and transparent Na-TFA-OA shell precursor solution (0.8 M). The synthesis of the Na-TFA-OA shell precursor (0.2 M) was carried out in the same manner, except that the amount of Na-TFA was reduced to 2.0 mmol.

[0055] (2) Synthesis of UCNPs core (β-NaGdF4:Yb20% / Er2%)

[0056] In a 100 mL round-bottom three-necked flask, 0.78 mmol GdCl3, 0.2 mmol YbCl3, and 0.02 mmol ErCl3 rare earth raw materials were first weighed out, followed by the addition of 6 mL OA and 15 mL ODE as high-boiling-point solvents. The flask was then placed in a temperature-programmed environment and heated to 140 °C at a rate of 10 °C / min under vacuum and magnetic stirring, and held for 1 hour to remove water and oxygen. The resulting mixed solution was cooled to 50 °C, and 10 mL of methanol containing 2.5 mmol NaOH and 4 mmol NH4F was added. The mixture was held at 50 °C for 30 minutes, then heated to 80 °C, and the methanol was removed under vacuum and held for 30 minutes. High-purity nitrogen was then introduced as a protective gas, and the temperature was increased to 285 °C at a rate of 15 °C / min, and the reaction was held for 100 minutes. Magnetic stirring was maintained throughout the preparation process. After the reaction was complete, the mixture was cooled to room temperature, 20 mL of ethanol was added to precipitate the precipitate, and the mixture was centrifuged at 8500 r / min for 5 minutes. Then, it was washed twice with anhydrous ethanol to obtain pure core nanoparticles β-NaGdF4:Yb20% / Er2% (1 mmol). Finally, the nanoparticles were dispersed in 2 mL of cyclohexane and stored for later use.

[0057] (3) Synthesis of β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20% (CS1)

[0058] A single-pot continuous layer-by-layer (SLBL) growth strategy was employed. In a 100 mL round-bottom three-necked flask, 4.0 mL of OA, 6.0 mL of ODE, and 0.5 mL (0.25 mmol) of purified cyclohexane solution of β-NaGdF4:Yb20% / Er2% core nanoparticles were added. The flask was then placed in a temperature-programmed environment and heated to 100 °C at a rate of 10 °C / min under vacuum and magnetic stirring, with evacuation for 30 minutes to remove cyclohexane and residual air. Subsequently, nitrogen protection was switched, and the temperature was increased to 280 °C at a rate of 15 °C / min. After temperature stabilization, 1 mL of Y-Yb-OA (0.2 M) and 0.5 mL of Na-TFA-OA (0.8 M) shell precursor were added dropwise at a rate of 0.1 mL / min, alternating between the two. This process was repeated four times, with each addition held for 30 minutes to allow the shell precursor to fully coat the core nanoparticles. Finally, the obtained β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%(CS1) core-shell nanoparticles were precipitated and washed twice with ethanol, and then dispersed in 2 mL of cyclohexane for later use.

[0059] (4)β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%@NaGdF4:Yb20% / Nd50% (CS 1-2 Synthesis of

[0060] The preparation method is similar to that of the UCNPs described above. In a 100 mL round-bottom three-necked flask, 4.0 mL of OA, 6.0 mL of ODE, and 2 mL of purified cyclohexane solution containing β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20% (CS1, 0.25 mmol) core-shell nanoparticles were added. The flask was then placed in a temperature-programmed device and heated to 100 °C at a rate of 10 °C / min under vacuum and magnetic stirring, with evacuation for 30 minutes to remove cyclohexane and residual air. Subsequently, nitrogen protection was switched, and the temperature was increased to 280 °C at a rate of 15 °C / min. After the temperature stabilized, 1 mL of Gd-Yb-Nd-OA (0.05 M) and 0.5 mL of Na-TFA-OA (0.2 M) shell precursor were added alternately at a rate of 0.1 mL / min using a dropwise method, with 20 cycles. After each injection, the solution was held for 15 minutes. (Because Nd readily forms heterogeneous nucleations, the concentration of the Gd-Yb-Nd-OA shell precursor solution was reduced from 0.2 M to 0.05 M, and the corresponding concentration of Na-TFA-OA was reduced from 0.8 M to 0.2 M, increasing the number of cycles to 20 to ensure that the shell precursor was uniformly coated on the core-shell nanoparticles.) Finally, the obtained β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%@NaGdF4:Yb20% / Nd50% (CS) was processed. 1-2 The core-shell nanoparticles were precipitated and washed twice with ethanol, then dispersed in 2 mL of cyclohexane for later use.

[0061] (5)β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%@NaGdF4:Yb20% / Nd50%@NaYF4(CS 1-3 Synthesis of

[0062] The preparation method is similar to that of the UCNPs described above. In a 100 mL round-bottom three-necked flask, add 4.0 mL of OA, 6.0 mL of ODE, and 2 mL of purified β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%@NaGdF4:Yb20% / Nd50% (CS) 1-2A cyclohexane solution of 0.25 mmol core-shell nanoparticles was prepared. A three-necked flask was then placed in a temperature-programmed flask and heated to 100 °C at a rate of 10 °C / min under vacuum and magnetic stirring, with evacuation for 30 minutes to remove cyclohexane and residual air. Subsequently, nitrogen protection was switched, and the temperature was increased to 280 °C at a rate of 15 °C / min. After the temperature stabilized, 1 mL of Y-OA (0.2 M) and 0.5 mL of Na-TFA-OA (0.8 M) shell precursor were added dropwise at a rate of 0.1 mL / min, alternating between the two. This process was repeated 5 times, with each addition held for 30 minutes to allow the shell precursor to fully coat the nanoparticles. Finally, the resulting β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%@NaGdF4:Yb20% / Nd50%@NaYF4(CS) 1-3 The core-shell nanoparticles were precipitated and washed twice with ethanol, then dispersed in 2 mL of cyclohexane for later use.

[0063] (6)β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%@NaGdF4:Yb20% / Nd50%@NaYF4@NaGdF4:Yb80% / Tm1% (CS 1-4 Synthesis of

[0064] The preparation method is similar to that of the UCNPs described above. In a 100 mL round-bottom three-necked flask, add 4.0 mL of OA, 6.0 mL of ODE, and 2 mL of purified β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%@NaGdF4:Yb20% / Nd50%@NaYF4(CS) 1-3 A solution of cyclohexane containing 0.25 mmol of nanoparticles was prepared. A three-necked flask was then placed in a temperature-programmed flask and heated to 100 °C at a rate of 10 °C / min under vacuum and magnetic stirring, with the temperature evacuated for 30 minutes to remove cyclohexane and residual air. Subsequently, nitrogen protection was switched, and the temperature was increased to 280 °C at a rate of 15 °C / min. After the temperature stabilized, 1 mL of Gd-Yb-Tm-OA (0.2 M) and 0.5 mL of Na-TFA-OA (0.8 M) shell precursor were added dropwise at a rate of 0.1 mL / min, alternating between the two. This process was repeated 10 times, with each addition held for 30 minutes to allow the shell precursor to fully coat the nanoparticles. Finally, the obtained β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%@NaGdF4:Yb20% / Nd50%@NaYF4@NaGdF4:Yb80% / Tm1% (CS) 1-4 The core-shell nanoparticles were precipitated and washed twice with ethanol, then dispersed in 2 mL of cyclohexane for later use.

[0065] (7)β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%@NaGdF4:Yb20% / Nd50%@NaYF4@NaGdF4:Yb80% / Tm1%@NaYF4 (CS 1-5 Synthesis of

[0066] The preparation method is similar to that of the UCNPs described above. In a 100 mL round-bottom three-necked flask, add 4.0 mL of OA, 6.0 mL of ODE, and 2 mL of purified β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%@NaYF4@NaGdF4:Yb80% / Tm1% (CS) 1-4 A cyclohexane solution of 0.25 mmol of core-shell nanoparticles was prepared. A three-necked flask was then placed in a temperature-programmed flask and heated to 100 °C at a rate of 10 °C / min under vacuum and magnetic stirring, with evacuation for 30 minutes to remove cyclohexane and residual air. Subsequently, nitrogen protection was switched, and the temperature was increased to 280 °C at a rate of 15 °C / min. After the temperature stabilized, 1 mL of Y-OA (0.2 M) and 0.5 mL of Na-TFA-OA (0.8 M) shell precursor were added dropwise at a rate of 0.1 mL / min, alternating between the two. This process was repeated five times, with each addition held for 30 minutes to allow the shell precursor to fully coat the core-shell nanoparticles. Finally, the final product β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%@NaGdF4:Yb20% / Nd50%@NaYF4@NaGdF4:Yb80% / Tm1%@NaYF4(CS) was obtained. 1-5 The core-shell nanoparticles were precipitated and washed twice with ethanol, then dispersed in 2 mL of cyclohexane.

[0067] (8)UCNPs(CS 1-5 Ligand modification to aqueous phase

[0068] The OA-terminated UCNPs (2.0 mL, 0.25 mmol) prepared above were diluted 5-fold with cyclohexane and added to 6.0 mL of DMF along with BF4NO (233.6 mg, 2 mmol). The mixture was sonicated for 10 minutes to remove the surface OA ligands. After standing for 5 minutes, the nanoparticles separated into layers at the bottom of the DMF. The upper cyclohexane layer was removed, and 10 mL of ethanol was added. The mixture was centrifuged at 10,000 rpm for 10 minutes, the supernatant was discarded, and the nanoparticles were washed twice with ethanol to obtain BF4 ligand-modified water-soluble nanoparticles. These nanoparticles were dissolved in 5 mL of DMF and added to 5 mL of water along with 400 mg of hyaluronic acid. The mixture was stirred vigorously for 2 hours to achieve ligand conversion. Then, 10 mL of ethanol was added to precipitate the nanoparticles. The mixture was centrifuged at 15,000 rpm for 15 minutes, the supernatant was discarded, and the nanoparticles were washed twice with ethanol to obtain hyaluronic acid ligand-modified water-soluble nanoparticles with better biocompatibility. These nanoparticles were then lyophilized and stored.

[0069] 2. Characterization of UCNPs

[0070] The synthesized UCNPs (β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%@NaGdF4:Yb20% / Nd50%@NaYF4@NaGdF4:Yb80% / Tm1%@NaYF4) were scanned using transmission electron microscopy (TEM). Figure 1 As shown in Figure a, gadolinium (Gd, Z=64) is much brighter under TEM because its atomic number is much larger than that of yttrium (Y, Z=39). By using different matrix elements to achieve alternating bright and dark layers in UCNPs, it is shown that different shells were successfully grown on the core nanoparticles, and the core was completely covered. The TEM image shows that the UCNPs have good size uniformity, with an average particle size of about 63 nm, which is consistent with the particle size results measured by dynamic light scattering particle size potentiometer (DLS).

[0071] UCNPs were excited using 808 nm and 980 nm exciters, respectively, and emission spectrum data were collected using a spectrometer. The results are as follows: Figure 1 b. With the core doped with the rare-earth element erbium (Er), the outer shell of ytterbium (Yb) effectively improves the inner-layer luminescence efficiency. Neodymium (Nd) can be excited under 808nm near-infrared light, transferring energy inward to Yb and Er, achieving upconversion emission of visible green light with a dominant wavelength around 545nm. The outer thulium (Tm) layer can be excited by 980nm near-infrared light under Yb sensitization, upconverting and emitting blue and ultraviolet light with dominant wavelengths around 460nm and 365nm, respectively. The middle shell and the outermost inert element yttrium (Y) effectively prevent cross-relaxation and surface quenching, improving the upconversion luminescence efficiency. Figure 1 As shown in c, Nd 3+ After absorbing 808nm photons, 4 I9 / 2 ground state transition to 4 F 5 / 2 Excited state, energy transfer makes Yb 3+ Depend on 4 F 7 / 2 ground state transition to 4 F 5 / 2 In the excited state, energy is further transferred to Er 3+ , so that it is from 4 I 15 / 2 ground state transition to 4 S 3 / 2 The excited state returns to the ground state and emits a 545nm photon; Yb 3+ After absorbing 980nm photons, 4 F 7 / 2 ground state transition to 4 F 5 / 2 Excited state, energy transferred to Tm 3+ , so that it is from 3 H6 ground state transition to 1 D2 and 1 G4 excited state, return to 3 After F4 and the ground state, it emits photons at 365nm and 460nm.

[0072] Example 2: Application of UCNPs in the synthesis of photopolymerized hyaluronic acid hydrogels and in vivo imaging

[0073] 1. Raw materials

[0074] Hyaluronic acid (HA, 55 kDa) was purchased from Bloomage Biotechnology Co., Ltd.; 2-aminoethyl methacrylate hydrochloride (AEMA·HCl), N-(3-dimethylaminopropyl)-N'-ethylcarbodimethylimine hydrochloride (EDC, 98%), N-hydroxysuccinimide (NHS, 98%), and triethanolamine (TEOA, 99%) were purchased from Aladdin Reagent Co., Ltd. (Shanghai); N-vinylpyrrolidone (NVP, 99%) and Acid Red 87 (Eosin Y) were purchased from Shanghai Maclean Biotechnology Co., Ltd.; BALB / c nude mice were obtained from Nanjing Annokang Biotechnology Co., Ltd. All chemicals were used as is without further purification.

[0075] 2. Synthesis and characterization of HA-AEMA

[0076] AEMA was covalently attached to the HA side chain via an EDC / NHS coupling reaction. HA (4.000 g, 10.40 mmol) was added to 400 mL of ultrapure water and stirred to dissolve. Then, carboxyl activator EDC (2.192 g, 11.44 mmol, 10% excess) and amino acid protectant NHS (1.328 g, 11.44 mmol, 10% excess) were added to the solution to fully activate the carboxyl groups on the HA side chain. An equimolar amount of AEMA (1.720 g, 10.40 mmol) was then added, and the mixture was thoroughly mixed. The pH of the solution was adjusted to a stable value of 4.7, and the reaction was allowed to proceed overnight at room temperature. The next day, the solution was adjusted to pH 7.0, transferred to a dialysis bag (MWCO = 3000 Da), and dialyzed against 0.1% NaCl for two days, followed by dialyzing against 75% ethanol and deionized water for one day each. The product was then frozen at -80°C and freeze-dried for 7 days to obtain 3.518 g of cotton-like flocculent product (HA-AEME), with a yield of 61.5%. It was stored at -20°C for later use. The synthetic route is as follows: Figure 2 As shown in a.

[0077] Using nuclear magnetic resonance hydrogen spectroscopy (NMR) 1 The HA-AEMA structure was confirmed by 1H NMR (400MHz, D2O), and the results are as follows: Figure 2 As shown in b, by calculating the ratio of methyl hydrogen on AEMA to methyl hydrogen in the HA skeleton, the degree of substitution of AEMA on the HA skeleton (the number of AEMA molecules contained in every 100 HA repeating units) is 33%.

[0078] 3. Synthesis and characterization of photopolymerized hyaluronic acid hydrogel

[0079] Eosin Y (2 μL, 25 mg / mL), which can be excited under 545 nm green light, was selected as a photosensitizer, and triethanolamine (5 μL) was selected as a photoinitiator. The copolymerization of monomers NVP (10 μL) and HA-AEMA (50 mg) increased the crosslinking speed and the mechanical strength of the hydrogel. The solvent was 500 μL of PBS solution containing UCNPs (2 mg). The UCNPs were used to convert 808 nm near-infrared laser light into green light to assist in the formation of photopolymerization hydrogel.

[0080] Hydrogels with different mechanical properties can be obtained by controlling the laser irradiation time. Table 1 shows the performance parameters of hydrogels with low, medium, and high crosslinking densities obtained by irradiation with 808nm near-infrared light for 5, 10, and 15 minutes, respectively. The mass swelling ratio, volume swelling ratio, crosslinking density, and pore size of the hydrogels formed by irradiation for 10 and 15 minutes are approximately equal. Figure 3 As shown, the storage modulus of the two is not much different, and the volume after water absorption and swelling is basically the same as the morphology after freeze-drying, indicating that the hydrogel has basically stabilized after irradiation for 10 minutes.

[0081] Table 1 Properties of hydrogels with different crosslinking densities

[0082]

[0083] 4. In vivo imaging experiment of photopolymerized hyaluronic acid hydrogel

[0084] Weigh HA-AEMA (250 mg) and dissolve it in 2.5 mL of PBS buffer solution (0.2 mg / mL). Add comonomer NVP (50 μL), photosensitizer Eosin Y (10 μL, 25 mg / mL), photoinitiator triethanolamine (25 μL), and light conversion material UCNPs (10 mg). Mix well to prepare the hydrogel precursor solution.

[0085] The retention of photopolymerizable hydrogels with different crosslinking densities in vivo was studied using BALB / c nude mice. Eighteen male nude mice weighing approximately 20g were randomly divided into three groups (A, B, and C) with six mice in each group. Each mouse was subcutaneously injected with 100μL of hydrogel precursor solution in its back. Immediately after injection, the mice were irradiated with an 808nm near-infrared laser. Group A mice were irradiated for 5 minutes, Group B mice for 10 minutes, and Group C mice for 15 minutes. The 808nm near-infrared light was converted into green light using UCNPs to assist in the formation of the photopolymerizable hydrogel.

[0086] Fluorescence signal changes in mice were analyzed using a chemiluminescence imaging system at 0.5h, 2h, 4h, 24h, 3 days, 6 days, 9 days, 12 days, 15 days, and 18 days. The excitation light was a 980nm near-infrared laser, the emission wavelength was 460nm, and the exposure time was 10ms. Finally, the fluorescence intensity at the injection site of the hydrogel was quantified using ImageJ software. All animal procedures were performed in accordance with the National Institutes of Health (NIH) guidelines for laboratory animal care and use, and were approved by the Animal Ethics Committee of China Pharmaceutical University. Figure 4 As shown, the longer the laser irradiation-assisted gelation time, the more stable the hydrogel is in vivo and the longer the retention time. Group C was basically completely metabolized after about 18 days in vivo, showing good biocompatibility.

[0087] Example 3: Application of UCNPs in drug release and tumor therapy

[0088] 1. Raw materials

[0089] Isenhanol, sodium borohydride (NaBH4), anhydrous sodium sulfate, potassium carbonate, ethyl acetate, acetyl chloride, triphenylphosphine (PPh3), carbon tetrabromide (CBr4), 5-fluoro-2,4-bis((trimethylsilyl)oxy)pyrimidine, and 5-fluorouracil (5-FU) were purchased from Shanghai Haohong Biomedical Technology Co., Ltd.; nitric acid (HNO3) and anhydrous methanol (MeOH, 99.5%) were purchased from Sinopharm Chemical Reagent Co., Ltd.; dichloromethane (DCM, 99.5%) and acetonitrile (CH3CN, 99.9%) were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd.; mouse melanoma cell lines (B16-F10) and human immortalized epidermal cells (Hacat) were purchased from ATCC (USA); fetal bovine serum (FBS), RPMI-1640 cell culture medium, DMEM medium, and trypsin were purchased from Gibco (New York, USA). All chemicals were used as is without further purification.

[0090] 2. Design, synthesis and characterization of prodrugs

[0091] (1) Synthesis of compound 1:

[0092] according to Figure 5 According to the synthetic route shown in a, 10 mL of HNO3 was added to a 250 mL round-bottom flask, and the mixture was cooled to -10 °C in an ice-salt bath. Isovalin (3.20 g, 21.05 mmol) was added in 5 portions, and the mixture was stirred at room temperature for 2 hours. Then, the reaction solution was slowly added dropwise to 30.0 mL of ice water, filtered, and dried to give compound 1 (3.97 g yellow solid) with a yield of 78%.

[0093] (2) Synthesis of compound 2:

[0094] Compound 1 (3.97 g, 16.40 mmol) was dissolved in 10.0 mL of anhydrous methanol, cooled to 0 °C in an ice bath, and sodium borohydride (1.41 g, 32.80 mmol) was slowly added. The mixture was stirred at room temperature for 3 hours, and then the solvent was removed by rotary evaporation. The compound was dissolved in water, and the pH was adjusted to 2–3 with hydrochloric acid. The mixture was extracted with ethyl acetate, and the combined organic layers were washed with saturated brine. Finally, the mixture was dried over anhydrous sodium sulfate to give compound 2 (3.80 g brown solid) in 95% yield.

[0095] (3) Synthesis of compound 3:

[0096] Compound 2 (3.80 g, 15.58 mmol) and acetyl chloride (0.88 mL, 12.46 mmol) were dissolved in 20.0 mL of dichloromethane, stirred at room temperature for 12 hours, concentrated, and purified by column chromatography to give compound 3 (1.69 g white solid) in 38% yield.

[0097] (4) Synthesis of compound 4:

[0098] Compound 3 (1.69 g, 5.92 mmol), triphenylphosphine (3.10 g, 11.84 mmol), and carbon tetrabromide (3.87 g, 11.84 mmol) were dissolved in 15.0 mL of dichloromethane, stirred at room temperature for 12 hours, concentrated, and purified by column chromatography to give compound 4 (906 mg white solid) in a yield of 44%.

[0099] (5) Synthesis of ONB-5-FU:

[0100] Compound 4 (906 mg, 2.60 mmol) and an equimolar amount of 5-fluoro-2,4-bis((trimethylsilyl)oxy)pyrimidine (564 mg, 2.60 mmol) were placed in a 15 mL sealed tube. The tube was evacuated and purged with nitrogen three times. 4.0 mL of acetonitrile was added, and the mixture was reacted at 85 °C in the dark for 72 hours. The mixture was then transferred to room temperature, and 4.0 mL of methanol and potassium carbonate (358 mg, 2.60 mmol) were added. The reaction was carried out for 1.5 hours, concentrated, and purified by column chromatography to obtain ONB-5-FU (278 mg, yellow solid), with a yield of 30%. The results were analyzed using 1H NMR spectroscopy (1H NMR spectroscopy). 1 The structure was confirmed by H NMR (300 MHz, DMSO-d6), and the results are as follows: Figure 5 As shown in b, the δ values ​​are 11.82 (d, J = 5.0 Hz, 1H), 7.52 (d, J = 7.0 Hz, 1H), 7.45 (s, 1H), 4.88 (s, 2H), and 3.74 (s, 3H). The 1H NMR spectrum results are consistent with the theoretical structure.

[0101] 3. In vitro drug release and qualitative and quantitative analysis

[0102] Table 2 Mobile phase conditions for high performance liquid chromatography (HPLC)

[0103]

[0104] (1) Determination of the 5-FU standard curve

[0105] Accurately weigh 10 mg of 5-FU technical powder, dissolve it in 0.01 M PBS buffer, and dilute to 100 mL to obtain a stock solution. Accurately transfer 3 mL of the stock solution to dilute to 10 mL to obtain a 5-FU solution with a concentration of 30 μg / mL. Prepare 5-FU solutions with concentrations of 25, 20, 15, 10, and 5 μg / mL sequentially using the same method. Measure the absorption peak of the 5-FU solution at 256 nm using high-performance liquid chromatography (HPLC) at room temperature (Wukong K2025, C18 column). The mobile phase conditions are shown in Table 2. Plot a standard curve for 5-FU with sample concentration as the x-axis and the corresponding absorption peak area as the y-axis. Fit a linear regression equation; the results are shown in Table 2. Figure 6As shown in Figure a, it can be seen that the HPLC peak area exhibits a good linear relationship with the drug concentration, R 2 >0.999.

[0106] (2) In vitro drug release of ONB-5-FU under ultraviolet light irradiation

[0107] 5.0 mg ONB-5-FU was dissolved in 25 mL PBS buffer solution to prepare a prodrug stock solution (0.2 mg / mL). 2 mL of the prodrug stock solution was transferred to a release vial and irradiated under a UV lamp to convert ONB-5-FU into 5-FU, thus preparing the release solution. 200 μL of the release solution was collected at 0, 20, 40, 60, 80, 100, 120, 140, 160, and 180 minutes, with fresh PBS buffer solution added simultaneously to maintain a constant volume. Each experiment was performed in triplicate. Finally, the concentration of 5-FU in the release solution was quantitatively analyzed by HPLC. The mobile phase gradient is shown in Table 2, and the qualitative results are as follows: Figure 6 b, c, The retention time of ONB-5-FU is 13 min, and the retention time of 5-FU is 3 min, such as Figure 7 a represents the cumulative drug release curve. After 3 hours, ONB-5-FU was almost completely converted to 5-FU, with a release rate of 98%.

[0108] (3) Photopolymerized hydrogels in near-infrared in vitro drug release

[0109] Weigh HA-AEMA (50 mg) and dissolve it in 500 μL of PBS buffer solution containing ONB-5-FU (0.2 mg / mL). Add comonomer NVP (10 μL), photosensitizer Eosin Y (2 μL, 25 mg / mL), photoinitiator triethanolamine (5 μL), and light conversion material UCNPs (2 mg). Irradiate with 808 nm near-infrared laser for 10 minutes to convert near-infrared light to green light to assist in the formation of photopolymerization hydrogel.

[0110] Then, 1 mL of PBS release medium was added, and the sample was irradiated with a 980 nm near-infrared laser. Using UCNPs, the near-infrared light was upconverted to ultraviolet light, acting on the o-nitrobenzyl site of the prodrug ONB-5-FU, cleaving the chemical bond and releasing the antitumor drug 5-FU. Irradiation was performed for 10 minutes on (ON) and then stopped for 10 minutes, repeated 6 times. Every 10 minutes, 200 μL of the release solution was collected, and fresh release medium was added simultaneously to maintain a constant volume. Each experiment was performed in triplicate. Figure 7 As shown in b, due to the sustained-release effect of the hydrogel, the drug concentration also increases during the OFF activation process, but the release rate is significantly slower than that in the ON activation state, at 3.5 W / cm². 2 At the excitation power density, approximately 18% of the drug is released in 2 hours.

[0111] (4) In vitro antitumor experiment

[0112] First, the biocompatibility of the prodrug ONB-5-FU and the drug 5-FU was studied. B16-F10 mouse melanoma cells were cultured in 1640 medium containing 10% fetal bovine serum (FBS). The cell suspension was seeded into 96-well plates at a density of 100 μL per well (3000 cells / well). After culturing for 24 h at 37℃, 5% CO2, the medium was discarded, and 200 μL of ONB-5-FU and 5-FU solutions at concentrations of 0.625, 1.25, 2.50, 5.00, and 10.0 μM were added, with five replicates for each concentration. An equal volume of 1640 medium served as a control. Cells were incubated for 24 h after treatment. After incubation, 20 μL of MTT reagent was added to each well, and the cells were incubated at 37°C in the dark for 24 h. The supernatant was then discarded, and 200 μL of DMSO was added to each well to dissolve the formazan generated by the reduction of live cells. Finally, the absorbance of each well at 492 nm was measured using a microplate reader, and the corresponding cell viability was calculated using Origin software. Each experiment was performed in triplicate. Figure 8 a represents the survival of B16-F10 cells. It can be seen that the prodrug ONB-5-FU has weaker toxicity, while the original drug 5-FU is more toxic. Cell survival rate decreases with increasing 5-FU concentration, indicating that 5-FU has an inhibitory effect on cancer cells and can effectively kill tumor cells. IC50 50 It is 2.5 μM.

[0113] Subsequently, the inhibitory effect of 5-FU generated from a mixed solution of ONB-5-FU (10.0 μM) and UCNPs (200 μg / mL) (UCNPs-ONB-5-FU in the figure) on B16-F10 cells under 980 nm near-infrared light irradiation for different durations was compared. Measurements were performed using the method described above, with each group of cells irradiated with a 980 nm laser (2.5 W / cm²) 4 h after the addition of the sample solution. 2 The cells were irradiated for 0, 30, 35, 40, 45, and 50 seconds respectively, with a pure cell group irradiated at the same time gradient without the mixed solution as a control. The cells were incubated for a total of 24 hours. The experimental results are as follows: Figure 8 As shown in b, it can be seen that the damage to pure cells within 50 seconds of laser irradiation is relatively weak. However, in the experimental group, the 980nm near-infrared light is converted into ultraviolet light by UCNPs, which converts the prodrug ONB-5-FU into the anti-tumor drug 5-FU. As the irradiation time increases, the survival rate of tumor cells decreases, and the cell survival rate is only about 40% after 50 seconds of irradiation.

[0114] The inhibitory effect of 5-FU generated from mixed solutions of UCNPs-ONB-5-FU at different concentrations on B16-F10 cells after irradiation with 980 nm near-infrared light for 50 seconds was then compared. The assay was performed using the method described above. Each group of cells was treated with sample solutions (mixed solutions of UCNPs-ONB-5-FU) at concentration gradients of 0, 0.625, 1.25, 2.50, 5.00, and 10.0 μM, respectively, for 4 hours, followed by irradiation with a 980 nm laser (2.5 W / cm²). 2 Irradiate for 50 seconds, and use a control group with the same concentration gradient but no light as a reference, for a total incubation of 24 hours. Experimental results are as follows: Figure 8 As shown in c and d, it can be seen that UCNPs-ONB-5-FU has good biocompatibility. However, after 50 seconds of laser irradiation, as the concentration of UCNPs increases, the upconversion emission of ultraviolet light intensifies, causing more ONB-5-FU to be converted into the antitumor drug 5-FU, resulting in decreased cell viability and IC50. 50 It is 10 μM.

[0115] Furthermore, the effects of 5-FU and 980 nm near-infrared light irradiation on human immortalized epidermal cells (Hacat) were investigated. Measurements were performed using the methods described above, with each group of cells receiving 5-FU solutions at concentration gradients of 0, 0.625, 1.25, 2.50, 5.00, and 10.0 μM. The experimental results are as follows: Figure 8 As shown in Figure e, 5-FU exhibits low toxicity to Hacat cells, indicating it is a relatively selective antitumor drug. Different concentration gradients of UCNPs-ONB-5-FU also showed minimal damage to Hacat cells under 50-second illumination. Figure 8 f) demonstrates that 980nm near-infrared light causes less damage to normal cells and can be safely used for in vivo imaging and drug delivery therapy in a short period of time.

[0116] Comparative Example 1

[0117] β-NaGdF4:Yb20% / Tm0.5%@NaYF4:Yb20%(0.8mmol)@NaGdF4:Yb20% / Er2%(0.4mmol)@NaGdF4:Yb20% / Nd50%(0.6mmol)@NaYF4(1mmol) were prepared according to the method of Example 1, except that: the 0.02mmol ErCl3 rare earth raw material in step (2) of Example 1 was replaced with 0.005mmol TmCl3, and the amount of GdCl3 was changed to 0.795mmol; the 1mL Gd-Yb-Nd-OA(0.05M) and 0.5mL Na-TFA-OA(0.2M) shell precursor in step (4) were replaced with 1mL Gd-Yb20%-Er2%-OA(0.2M) and 0.5mL Na-TFA-OA (0.8M) was added dropwise twice, alternatingly (the amount of rare earth shell precursor used was 0.2M*2mL=0.4mmol); the 1mL Y-OA (0.2M) and 0.5mL Na-TFA-OA (0.8M) shell precursor in step (5) were replaced with 1mL Gd-Yb20%-Nd50%-OA (0.05M) and 0.5mL Na-TFA-OA (0.2M), added dropwise 12 times (the amount of rare earth shell precursor used was 0.05M*12mL=0.6mmol); step (6) was removed.

[0118] UCNPs were excited using 808 nm and 980 nm exciters, respectively, and emission spectrum data were collected using a spectrometer. The results are as follows: Figure 10 As shown in the figure. The results show that the inner layer of Nd contains both Er and Tm luminescent elements. The two near-infrared excitation beams are not selective, and blue, green and ultraviolet light are emitted under excitation at 980 nm and 808 nm.

[0119] Comparative Example 2

[0120] β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%(0.8mmol)@NaGdF4:Yb20% / Nd50%(0.4mmol)@NaYF4(1mmol)@NaGdF4:Yb20% / Tm0.5%(1mmol)@NaYF4(1mmol) were prepared according to the method of Example 1, except that: the amount of 1 mL Gd-Yb-Nd-OA (0.05M) and 0.5 mL Na-TFA-OA (0.2M) shell precursor used in step (4) of Example 1 was reduced to 8 alternating drops (the amount of rare earth shell precursor used was 0.05M*8mL=0.4mmol); the amount of 1 mL in step (6) was reduced to 8 alternating drops. The Gd-Yb-Tm-OA(0.2M) shell precursor was replaced with Gd-Yb20%-Tm0.5%-OA(0.2M), and the dosage was changed to 5 drops (the dosage of rare earth shell precursor is 0.2M*5mL=1mmol).

[0121] UCNPs were excited using 808 nm and 980 nm exciters, respectively, and emission spectrum data were collected using a spectrometer. The results are as follows: Figure 11 As shown in the figure. The results indicate that blue and ultraviolet light are weak under 980nm near-infrared excitation.

[0122] Comparative Example 3

[0123] β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%(0.8mmol)@NaGdF4:Yb20% / Nd50%(0.6mmol)@NaYF4(1mmol)@NaGdF4:Yb80% / Tm1%(1mmol)@NaYF4(1mmol) were prepared according to the method of Example 1, except that: the amount of 1 mL Gd-Yb-Nd-OA (0.05M) and 0.5 mL Na-TFA-OA (0.2M) shell precursor in step (4) of Example 1 was changed to be added alternately 12 times (the amount of rare earth shell precursor is 0.05M*12mL=0.6mmol); the amount of 1 mL Gd-Yb-Tm-OA (0.2M) and 0.5 mL in step (6) was changed to be added alternately 12 times (the amount of rare earth shell precursor is 0.05M*12mL=0.6mmol); The amount of Na-TFA-OA (0.8M) shell precursor was reduced to 5 alternating drops (the amount of rare earth shell precursor used was 0.2M * 5mL = 1mmol).

[0124] UCNPs were excited using 808 nm and 980 nm exciters, respectively, and emission spectrum data were collected using a spectrometer. The results are as follows: Figure 12 As shown in the figure. The results show that increasing the amount of Gd-Yb-Nd-OA shell precursor and increasing the proportion of Yb and Tm in the Gd-Yb-Tm-OA shell precursor enhances the blue, green and ultraviolet light, but the green light is stronger under 980nm near-infrared excitation, while the blue and ultraviolet light are still weak.

[0125] Comparative Example 4

[0126] β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%(0.8mmol)@NaGdF4:Yb20% / Nd50%(0.6mmol)@NaYF4(1mmol)@NaGdF4:Yb80% / Tm1%(3mmol)@NaYF4(2mmol) were prepared according to the method of Example 1. The difference is that, compared with Example 1, the amount of 1 mL Gd-Yb-Nd-OA (0.05M) and 0.5 mL Na-TFA-OA (0.2M) shell precursor in step (4) was changed to be added alternately 12 times (the amount of rare earth shell precursor is 0.05M*12mL=0.6mmol); the amount of 1 mL Gd-Yb-Tm-OA (0.2M) and 0.5 mL in step (6) was changed to be added alternately 12 times. The amount of Na-TFA-OA (0.8M) shell precursor is increased to 15 alternating drops (the amount of rare earth shell precursor is 0.2M*15mL=3mmol); the amount of 1mL Y-OA (0.2M) and 0.5mL Na-TFA-OA (0.8M) shell precursor in step (7) is increased to 10 alternating drops (the amount of rare earth shell precursor is 0.2M*10mL=2mmol).

[0127] UCNPs were excited using 808 nm and 980 nm exciters, respectively, and emission spectrum data were collected using a spectrometer. The results are as follows: Figure 13 As shown in the figure. The results indicate that excessive use of Gd-Yb-Tm-OA and Y-OA shell precursors resulted in excessively strong blue and ultraviolet light under 980nm near-infrared excitation, and weaker green light under 808nm excitation.

[0128] Comparative Example 5

[0129] β-NaGdF4:Yb20% / Er2%@NaYF4:Yb20%(0.8mmol)@NaGdF4:Yb20% / Nd50%(0.6mmol)@NaYF4(1mmol)@NaGdF4:Yb80% / Tm1%(1.6mmol)@NaYF4(1mmol) were prepared according to the method of Example 1, except that: the amount of 1 mL Gd-Yb-Nd-OA (0.05M) and 0.5 mL Na-TFA-OA (0.2M) shell precursor in step (4) of Example 1 was changed to be added alternately 12 times (the amount of rare earth shell precursor is 0.05M*12mL=0.6mmol); the amount of 1 mL Gd-Yb-Tm-OA (0.2M) and 0.5 mL in step (6) was changed to be added alternately 12 times (the amount of rare earth shell precursor is 0.05M*12mL=0.6mmol); The amount of Na-TFA-OA (0.8M) shell precursor was reduced to 8 alternating drops (the amount of rare earth shell precursor used was 0.2M * 8mL = 1.6mmol).

[0130] UCNPs were excited using 808 nm and 980 nm exciters, respectively, and emission spectrum data were collected using a spectrometer. The results are as follows: Figure 14 As shown in the figure. The results show that even with reduced amounts of Gd-Yb-Tm-OA and Y-OA shell precursors, the green light emitted under 808nm excitation is still relatively weak.

Claims

1. A method for preparing rare earth upconversion nanoparticles, characterized in that, Includes the following steps: (1) GdCl3, YbCl3 and ErCl3 were mixed and oleic acid, 1-octadecene and methanol solution containing NaOH and NH4F were added to obtain core nanoparticle solution; (2) Mix oleic acid, 1-octadecene and the core nanoparticle solution described in step (1), and alternately add Y-Yb-OA and Na-TFA-OA shell precursors to obtain CS1 core-shell nanoparticle solution; (3) Mix oleic acid, 1-octadecene, and the CS1 core-shell nanoparticle solution described in step (2), and alternately add Gd-Yb-Nd-OA and Na-TFA-OA shell precursors to obtain CS1 core-shell nanoparticle solution. 1-2 Core-shell nanoparticle solution; (4) A mixture of oleic acid, 1-octadecene and the CS described in step (3). 1-2 A solution of core-shell nanoparticles was prepared by alternately adding Y-OA and Na-TFA-OA shell precursors to obtain CS. 1-3 Core-shell nanoparticle solution; (5) A mixture of oleic acid, 1-octadecene and the CS described in step (4). 1-3 A solution of core-shell nanoparticles was prepared by alternately adding Gd-Yb-Tm-OA and Na-TFA-OA shell precursors to obtain CS. 1-4 Core-shell nanoparticle solution; (6) A mixture of oleic acid, 1-octadecene and the CS described in step (5) 1-4 A solution of core-shell nanoparticles was alternately supplemented with Y-OA and Na-TFA-OA shell precursors to obtain rare earth upconversion nanoparticles; the structure of the rare earth upconversion nanoparticles was β-NaGdF4:Yb20% / Er2% @ NaYF4:Yb20% @ NaGdF4:Yb20% / Nd50% @ NaYF4 @ NaGdF4:Yb80% / Tm1% @ NaYF4.

2. The method according to claim 1, characterized in that, The preparation method of the Y-Yb-OA shell precursor in step (2) includes the following steps: stirring and heating a mixture of YCl3, YbCl3, oleic acid and 1-octadecene to obtain the Y-Yb-OA shell precursor.

3. The method according to claim 1, characterized in that, The preparation method of the Na-TFA-OA shell precursor in step (2) includes the following steps: stirring and heating a mixture of Na-TFA and oleic acid to obtain the Na-TFA-OA shell precursor.

4. The method according to claim 1, characterized in that, The preparation method of the Gd-Yb-Nd-OA shell precursor in step (3) includes the following steps: magnetically stirring and heating a mixture of GdCl3, YbCl3, NdCl3, oleic acid and 1-octadecene to obtain the Gd-Yb-Nd-OA shell precursor.

5. A rare earth upconversion nanoparticle prepared by the method of claim 1.

6. A method for preparing photopolymerized hyaluronic acid hydrogel using the rare earth upconversion nanoparticles of claim 5, characterized in that, Includes the following steps: (1) Add excess N-(3-dimethylaminopropyl)-N'-ethylcarbodimethylimine hydrochloride and excess N-hydroxysuccinimide to hyaluronic acid solution, then add 2-aminoethyl methacrylate hydrochloride. After mixing thoroughly, adjust the pH of the solution to be stable in acidic conditions and react overnight at room temperature. Adjust the solution to neutral, dialyze, dialyze with ethanol and deionized water respectively, freeze dry, and finally obtain cotton flocculent product HA-AEME. (2) Eosin Y, triethanolamine, monomer NVP and HA-AEMA described in step (1) are dissolved and mixed in PBS solution, and rare earth upconversion nanoparticles as described in claim 5 are added and then irradiated with near-infrared light to obtain photopolymerized hyaluronic acid hydrogel.

7. A photopolymerized hyaluronic acid hydrogel prepared by the method of claim 6.

8. The application of the rare earth upconversion nanoparticles of claim 5 and / or the photopolymerized hyaluronic acid hydrogel of claim 7 in the preparation of drugs for in vivo imaging.

9. The application of the rare earth upconversion nanoparticles according to claim 5 in the preparation of drugs that convert near-infrared light.

10. The application of the photopolymerized hyaluronic acid hydrogel according to claim 7 in the preparation of a drug for treating tumors, characterized in that, The photopolymerized hyaluronic acid hydrogel is cross-linked with the prodrug ONB-5-FU via covalent interaction; the structural formula of ONB-5-FU is as follows: .

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