A method for preparing and applying a glutathione-specific responsive dye-sensitized rare-earth upconversion nanoassemblies

Abstract

This invention discloses a method for preparing and applying a dye-sensitized rare-earth upconversion nanoassemblies that are specifically responsive to glutathione (DAG), belonging to the fields of bionanomaterials and medical technology. The invention prepares the nanoassemblies through the following steps: first, core-shell-shell structured rare-earth upconversion nanoparticles NaYF4:Yb are synthesized. 3+ ,Tm 3+ @NaYF4:Nd 3+ @NaYF4, after modification with citric acid, reacts with tetracarboxyphenylporphyrin iron and ZrOCl2•8H₂O via a solvothermal reaction to form UCM, which is then loaded with the glutathione-responsive dye Cy-GSH and finally modified with hyaluronic acid to obtain Cy-UCMA. This nanoassembly, triggered by high glutathione expression in the tumor microenvironment, converts Cy-GSH to Cy-SG, enhancing upconversion luminescence through non-radiative energy transfer, thereby improving the fluorescence resonance energy transfer efficiency with porphyrin iron, significantly increasing singlet oxygen production, efficiently inducing immunogenic cell death, and activating anti-tumor immune responses. This invention achieves tumor-specific photodynamic immunotherapy, effectively inhibiting in situ tumor growth and distant metastasis, and has good biosafety and clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of bionanomaterials and medical technology, specifically relating to a dye-sensitized rare earth upconversion nanoassembly that is specifically responsive to glutathione and its preparation method and its application in photodynamic immunotherapy. Background Technology

[0002] Tumor immunotherapy represents a major breakthrough in the treatment of malignant tumors. However, its clinical efficacy is often limited by the immunosuppressive microenvironment caused by the low immunogenicity of tumor cells. Transforming immune "cold" tumors into "hot" tumors, thereby effectively activating the anti-tumor immune response, is crucial for the success of tumor immunotherapy.

[0003] Immunogenic cell death (ICD) is a crucial bridge connecting tumor cell death and anti-tumor immune responses. Increased ICD levels promote the release of damage-associated molecular patterns (DAMPs), including the extravasation of calreticulin to the cell membrane, increased adenosine triphosphate (ATP) secretion, and the release of high-mobility group box 1 (HMGB1) from the nucleus. These signaling molecules recruit antigen-presenting cells (such as dendritic cells), promote their maturation and migration to draining lymph nodes, thereby activating naive T cells, differentiating them into cytotoxic T lymphocytes (CTLs), and ultimately achieving specific killing of tumor cells.

[0004] Photodynamic immunotherapy mediated by rare-earth upconversion nanocomposites is effective because it can generate singlet oxygen under near-infrared light excitation. 1 Near-infrared light (NIIR) has attracted much attention for inducing ICD (intracytoplasmic Degeneration) with oxygen (O2). Compared with traditional photodynamic therapy excited by ultraviolet or visible light, NIIR light has deeper tissue penetration and less damage to biological tissues. In upconversion nanocomposites, upconversion nanoparticles (UCNPs) can convert near-infrared light into visible or ultraviolet light, and then transfer the energy to the loaded photosensitizer molecules (such as porphyrins) through the fluorescence resonance energy transfer (FRET) mechanism, thereby efficiently generating... 1 O2.

[0005] However, the efficacy of existing rare-earth upconversion photodynamic therapy systems is mainly limited by two key factors. First, the inherently low upconversion luminescence quantum yield of rare-earth upconversion nanoparticles limits their energy transfer efficiency to photosensitizers, leading to... 1Insufficient O2 generation capacity. To address this issue, researchers have attempted to sensitize UCNPs with organic dyes as antenna molecules. By broadening the light absorption range and non-radiatively transferring energy to the UCNPs, the upconversion luminescence efficiency can be improved. For example, studies have reported upconversion nanoprobes sensitized with cyanine dyes. However, these dye sensitization strategies are mostly static sensitization, meaning the dye remains constantly activated, lacking a selective response to the tumor microenvironment.

[0006] Secondly, glutathione (GSH), highly expressed in the tumor microenvironment, is a potent antioxidant, with concentrations reaching 2-10 mM, far exceeding those in normal tissues. GSH can effectively quench the antioxidants produced during photodynamic therapy. 1 O2 significantly weakens the induction effect of ICD. Although researchers have developed GSH-responsive nanoplatforms, such as using GSH to trigger the disintegration of nanostructures or activate therapeutic functions, these systems typically only use GSH as a depleting target to reduce its antioxidant capacity, failing to translate high GSH expression into a positive regulatory signal that enhances photodynamic therapy efficacy.

[0007] In summary, current technologies still lack a solution that can simultaneously address the low upconversion luminous efficiency and GSH consumption of UCNPs. 1 Photodynamic therapy systems that address both O2 and GSH issues, particularly lacking a "dual-lock" response mechanism capable of transforming the detrimental factor of GSH into an enhancing signal, are inadequate for achieving selective activation and efficient production. 1 The combined effect of O2 and anti-tumor immune response. Summary of the Invention

[0008] To address the shortcomings of existing rare-earth upconversion photodynamic therapy systems, such as low upconversion luminescence efficiency and glutathione (GSH) consumption of singlet oxygen in the tumor microenvironment... 1 To address the problem of poor induction of immunogenic cell death (ICD) caused by O2, this invention provides a dye-sensitized rare earth upconversion nanoassembly that is specifically responsive to glutathione, its preparation method, and its application.

[0009] To address the aforementioned technical problems, the present invention provides the following technical solutions.

[0010] A method for preparing a dye-sensitized rare-earth upconversion nanoassemblies that are specifically responsive to glutathione (D&G) includes the following steps: (1) Preparation of rare earth upconversion nanoparticles NaYF4:Yb with core-shell-shell structure 3+ ,Tm 3+ @NaYF4:Nd 3+@NaYF4: Rare earth upconversion nanoparticles were obtained by thermal decomposition in oleic acid and octadecene solvent, using rare earth trifluoroacetate as a precursor, under argon protection, heating to 110-130℃ and holding for 30-60 minutes, and then heating to 300-320℃ and holding for 30-60 minutes. (2) Modify the rare earth upconversion nanoparticles obtained in step (1) with citric acid: Disperse the rare earth upconversion nanoparticles in chloroform and stir with a citric acid water / ethanol mixed solution at room temperature for 24-72 hours to obtain citric acid modified rare earth upconversion nanoparticles. (3) The citric acid-modified rare earth upconversion nanoparticles obtained in step (2) are dispersed with tetracarboxyphenylporphyrin iron, ZrOCl2•8H2O and benzoic acid in a mixed solvent of ethanol and N,N-dimethylformamide in a volume ratio of 1:1, and reacted at 80-100℃ for 4-6 hours to obtain rare earth upconversion composite nanomaterial UCM; wherein the mass ratio of the citric acid-modified rare earth upconversion nanoparticles, tetracarboxyphenylporphyrin iron, ZrOCl2•8H2O and benzoic acid is 1:1:3:28; (4) Disperse the UCM obtained in step (3) and DSPE-MPEG-1000 in chloroform at a mass ratio of 1:1, stir for 6-12 hours, remove the chloroform and disperse in buffer solution to obtain PEG-modified UCM. (5) Dissolve the glutathione-specific responsive dye Cy-GSH in dimethyl sulfoxide, mix Cy-GSH with PEG-modified UCM at a mass ratio of 1:1 to 1:5, and incubate in buffer for 6-12 hours to obtain dye-sensitized rare earth upconversion nanoassemblies Cy-UCM. (6) Hyaluronic acid and Cy-UCM obtained in step (5) are mixed in water at a mass ratio of 2:1 to 4:1. The mixture is stirred for 6-12 hours in the dark, centrifuged and washed to obtain the dye-sensitized rare earth upconversion nanoassembly Cy-UCMA that is specifically responsive to glutathione.

[0011] The core chemical composition of the rare earth upconversion nanoparticles mentioned in step (1) above is NaYF4:20%Yb. 3+ 0.5%Tm 3+ The chemical composition of the middle shell is NaYF4:20%Nd. 3+ The outer shell is undoped NaYF4.

[0012] Furthermore, as a preferred technical solution for the preparation method of the present invention: The heating rate in step (1) to raise the temperature to 300-320℃ is 15-25℃ / min.

[0013] In step (2), the volume ratio of water to anhydrous ethanol in the citric acid / ethanol mixed solution is 3:2, and the concentration of citric acid is 20 mg / mL.

[0014] The reaction temperature in step (3) is 90°C and the reaction time is 5 hours.

[0015] The mass ratio of Cy-GSH to PEG-modified UCM in step (5) is 1:1.

[0016] The Cy-GSH is prepared through the following steps: (a) 2,3,3-trimethyl-3H-indole and 5-bromopentanoic acid were dissolved in toluene at a molar ratio of 1:1.2 to 1:1.6, refluxed under nitrogen protection for 8-16 hours, and purified by silica gel column chromatography to obtain compound 1; (b) Compound 1 and 2-chloro-1-formyl-3-(hydroxymethylene)cyclohex-1-ene were dissolved in a mixed solvent of n-butanol / benzene at a molar ratio of 1.5:1 to 2.5:1 and refluxed for 4 to 8 hours. The mixture was then purified by silica gel column chromatography to obtain compound 2. (c) Under an argon atmosphere, sodium hydride and 4-hydroxy-4'-nitroazobenzene were reacted in DMF at a molar ratio of 1.2:1 to 1.8:1. After stirring for 5 to 15 minutes, a DMF solution of compound 2 was added dropwise, wherein the molar ratio of compound 2 to 4-hydroxy-4'-nitroazobenzene was 0.8:1 to 1.2:1. The reaction was carried out at room temperature for 8 to 16 hours. The mixture was purified by silica gel column chromatography to obtain Cy-GSH.

[0017] The mass ratio of hyaluronic acid to Cy-UCM in step (6) is 2:1.

[0018] The structure of the double-locked glutathione-specific responsive dye-sensitized rare-earth upconversion nanoassemblies prepared by the above method includes: Rare earth upconversion nanoparticles, wherein the rare earth upconversion nanoparticles have a core-shell-shell structure, consisting of NaYF4:Yb in sequence. 3+ ,Tm 3+ Core layer, NaYF4:Nd 3+ Shell and NaYF4 outer shell; A metal-organic framework layer is coated on the surface of the rare earth on-conversion nanoparticles. The metal-organic framework layer is composed of tetracarboxyphenylporphyrin iron and zirconium clusters, and the metal-organic framework layer is loaded with glutathione-specific responsive dye Cy-GSH. And a hyaluronic acid targeting layer modified on the outer surface of the metal-organic framework layer.

[0019] The mechanism of action of the nanoassembly of this invention is as follows: The dye Cy-GSH itself does not exhibit sensitization of rare earth upconversion nanoparticles, but when it enters the tumor microenvironment, it specifically reacts with overexpressed glutathione to generate Cy-SG molecules. As a sensitizer for rare earth upconversion nanoparticles, Cy-SG molecules can efficiently absorb 808 nm near-infrared light and transfer energy to UCNPs via nonradiative energy transfer (NRET), significantly enhancing their upconversion luminescence. The enhanced upconversion luminescence is further transferred to tetracarboxyphenylporphyrin iron in the metal-organic framework layer via fluorescence resonance energy transfer (FRET), thereby maximizing the generation of singlet oxygen.

[0020] Compared with existing technologies, this invention has the following beneficial effects: This invention achieves tumor-specific activation photodynamic therapy through a "dual-lock" response mechanism (i.e., high expression of glutathione in the tumor microenvironment triggers the conversion of Cy-GSH to Cy-SG, which in turn enhances upconversion luminescence and improves FRET efficiency via NRET), significantly reducing toxic side effects on normal tissues. Simultaneously, this mechanism can increase the upconversion luminescence intensity several times, significantly improving singlet oxygen production in the presence of glutathione, effectively overcoming the quenching effect of glutathione on singlet oxygen. The enhanced singlet oxygen production can efficiently induce immunogenic cell death in tumor cells, promote calreticulin eversion, and release ATP and HMGB1, thereby recruiting and maturing dendritic cells, activating cytotoxic T lymphocytes, and transforming immune "cold" tumors into "hot" tumors. Furthermore, hyaluronic acid modification enables active targeting of the CD44 receptor on tumor cells, combined with the good water solubility and long-term circulation capability conferred by PEGylation modification, giving the nanoassemblies excellent tumor enrichment efficiency and biocompatibility in vivo. Animal experiments show that the nanoassemblies of this invention exhibit excellent tumor accumulation efficiency and biocompatibility at 808... Irradiation with nm near-infrared light can significantly inhibit the growth of subcutaneous liver cancer tumors in mice and effectively control distant lung metastases, demonstrating a powerful anti-tumor immunotherapy effect. Moreover, the preparation method is mild and the parameters are controllable, showing good reproducibility and potential for large-scale production. Attached Figure Description

[0021] Figure 1 This is a synthetic route diagram of Cy-GSH obtained in Example 1 of the present invention.

[0022] Figure 2 The image shows the hydrogen nuclear magnetic resonance spectrum of Cy-GSH obtained in Example 1 of this invention.

[0023] Figure 3 The image shows the carbon NMR spectrum of Cy-GSH obtained in Example 1 of this invention.

[0024] Figure 4 This is the mass spectrum of Cy-GSH obtained in Example 1 of the present invention.

[0025] Figure 5 This is a schematic diagram of the synthesis process of the dye-sensitized rare earth upconversion nanoassemblies (Cy-UCMA) that are specifically responsive to glutathione and prepared in Example 1 of the present invention.

[0026] Figure 6 This is a transmission electron microscope (TEM) image of the Cy-UCMA nanoassembly prepared in Example 1 of the present invention.

[0027] Figure 7 The image shows the X-ray diffraction (XRD) pattern of the Cy-UCMA nanoassembly prepared in Example 1 of this invention.

[0028] Figure 8 The images show the upconversion luminescence spectra of the Cy-UCMA nanoassembly reacted with glutathione (GSH) for different times in Example 2 of this invention.

[0029] Figure 9 This is a test diagram (DPBF absorption change curve) of the Cy-UCMA nanoassembly generated by 808 nm laser irradiation in the presence of GSH in Example 2 of the present invention.

[0030] Figure 10 This is a fluorescence detection image of intracellular singlet oxygen generation after co-incubation of Cy-UCMA nanoassemblies with liver cancer cells (HepG2) in Example 3 of the present invention and irradiation with an 808nm laser.

[0031] Figure 11 This is a cell viability test (CCK-8 assay) of Cy-UCMA nanoassemblies co-incubated with liver cancer cells (HepG2) in Example 3 of the present invention, followed by irradiation with an 808nm laser.

[0032] Figure 12 This is a monitoring graph showing the tumor volume changes during the treatment of a mouse subcutaneous liver cancer tumor model using Cy-UCMA nanoassemblies in Example 4 of this invention.

[0033] Figure 13 This is a graph showing the monitoring of mouse weight changes during the treatment of a mouse subcutaneous liver cancer tumor model using Cy-UCMA nanoassemblies in Example 4 of this invention.

[0034] Figure 14 This is a flow cytometry analysis of the proportion of cytotoxic T lymphocytes infiltrating the tumor tissue after treating a mouse subcutaneous liver cancer model with Cy-UCMA nanoassemblies in Example 4 of this invention. Detailed Implementation

[0035] To further illustrate the present invention, a detailed description is provided below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the methods described in the embodiments are conventional methods; unless otherwise specified, the reagents are commercially available analytical grade reagents.

[0036] Example 1: Preparation of Cy-UCMA nanoassemblies This embodiment provides a method for preparing a dye-sensitized rare-earth upconversion nanoassemblies (Cy-UCMA) that are specifically responsive to glutathione (DAG), comprising the following steps: 1. Synthesis of the glutathione-specific responsive dye Cy-GSH (1) Synthesis of compound 1 2,3,3-Trimethyl-3H-indole (2 g, 12.5 mmol) and 5-bromopentanoic acid (3.28 g, 18.2 mmol) were dissolved in toluene and refluxed overnight under nitrogen protection. After the reaction was complete, the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to give compound 1.

[0037] (2) Synthesis of compound 2 Compound 1 (3.6 g, 11.5 mmol) and 2-chloro-1-formyl-3-(hydroxymethylene)cyclohexyl-1-ene (1.0 g, 5.75 mmol) were dissolved in 100 mL of a mixed solvent of n-butanol / benzene (10:1, v / v). The mixture was refluxed for 6 hours to give a dark green solution. After the reaction was complete, the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to give compound 2.

[0038] (3) Synthesis of Cy-GSH Under an argon atmosphere, sodium hydride (62 mg, 2.6 mmol) was slowly added to a 3 mL solution of DMF containing 0.42 g, 1.73 mmol. After stirring the mixture for 10 minutes, a DMF solution of compound 2 (1.465 g, 1.73 mmol) was slowly added dropwise. The resulting reaction mixture was stirred for 12 hours at room temperature. After the reaction was complete, the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography using dichloromethane / methanol (10:1, v / v) as eluent to give a deep blue solid, Cy-GSH. The structure and characterization of the product are as follows. Figure 1-4 As shown.

[0039] 2. Preparation of core-shell-shell structured rare earth upconversion nanoparticles (1) Preparation of NaYF4: 20%Yb 3+ 0.5%Tm 3+ Nanoparticles: 1 mmol of Ln(CF3COO)3 (Ln being Y, Yb, and Tm, with a molar ratio of Y:Yb:Tm = 0.795:0.20:0.05), 6 mL of oleic acid, and 12 mL of octadecene were added to a 50 mL three-necked round-bottom flask. The flask was placed in a magnetically stirred heating mantle, and argon gas was continuously introduced to remove oxygen from the reaction system. After 30 minutes of argon gas introduction, the temperature of the reaction system was slowly raised to 120°C (heating rate of 5°C / min) and held for 40 minutes until the solid was completely dissolved. Then, the temperature was rapidly raised to 310°C (heating rate of 20°C / min) and held for 40 minutes. After cooling to room temperature (argon gas was continuously introduced during heating, holding, and cooling), the reaction was allowed to cool naturally to room temperature. Anhydrous ethanol was added, and the precipitated product was collected by centrifugation and washed three times with a mixed solution of cyclohexane and ethanol to obtain NaYF4:20% Yb with uniform morphology. 3+ 0.5%Tm 3+ Nanoparticles were dispersed in 10 mL of cyclohexane to obtain a cyclohexane dispersion. (2) Preparation of core-shell structured NaYF4: 20% Yb 3+ 0.5%Tm 3+ @NaYF4,20%Nd 3+ Rare earth upconversion nanoparticles: Take 5 mL of cyclohexane dispersion in a three-necked round-bottom flask, add 0.8 mmol Y(CF3COO)3, 1 mmol CF3COONa and 0.2 mmol Nd(CF3COO)3, then add 6 mL oleic acid and 12 mL octadecene. Continuously purge with argon gas to remove oxygen from the reaction system. After purging with argon gas for 30 minutes, raise the temperature to 120 °C (heating rate 5 °C / min) and hold for 40 minutes to remove cyclohexane and low-boiling-point solvents from the system. Under continuous argon gas protection, rapidly raise the temperature to 310 °C and hold for 40 minutes (heating rate 20 °C / min). Allow to cool naturally to room temperature, add 20 mL anhydrous ethanol, and after the reaction product precipitates, collect the reaction product by centrifugation. Wash three times with a mixed solution of cyclohexane and anhydrous ethanol, disperse in 10 mL of cyclohexane for later use, and obtain core-shell structured rare earth upconversion nanoparticles (abbreviated as: NaYF4: 20%Yb). 0.5%Tm@NaYF4, 20%Nd).

[0040] (3) Preparation of core-shell structured NaYF4: 20% Yb 3+ 0.5%Tm 3+ @NaYF4,20%Nd 3+ @ NaYF4 rare earth upconversion nanoparticles: Take 5 mL of cyclohexane dispersion in a three-necked round-bottom flask, add 0.5 mmol Y(CF3COO)3 and 1 mmol CF3COONa, then add 6 mL oleic acid and 12 mL octadecene. Continuously purge with argon gas to remove oxygen from the reaction system. After purging with argon gas for 30 minutes, raise the temperature to 120 °C (heating rate 5 °C / min) and hold for 40 minutes to remove cyclohexane and low-boiling solvents from the system. Under continuous argon gas protection, rapidly raise the temperature to 310 °C and hold for 40 minutes (heating rate 20 °C / min). Allow to cool naturally to room temperature, add 20 mL of anhydrous ethanol. After the reaction product precipitates, collect the reaction product by centrifugation, wash three times with a mixed solution of cyclohexane and anhydrous ethanol, and dry under vacuum at 60 °C to obtain core-shell structured rare earth upconversion nanoparticles (abbreviation: NaYF4: 20%Yb, 0.5%Tm@NaYF4, 20%Nd@NaYF4).

[0041] 3. Preparation of citric acid-modified rare earth upconversion nanoparticles (CA-UCNP) Add 20 mg of NaYF4: 20%Yb, 0.5%Tm@NaYF4, 20%Nd@NaYF4 to 5 mL of chloroform, and disperse evenly by ultrasonication to obtain the first solution; Dissolve 200 mg of citric acid in 10 mL of a mixed solvent, with the mixed solvent being in a volume ratio of V... 水 V 无水乙醇 A second solution is prepared by mixing water and anhydrous ethanol in a ratio of 3:2. The first and second solutions were mixed and stirred at room temperature for 48 hours. The precipitate was collected by centrifugation and washed with deionized water to obtain citric acid-modified rare earth upconversion nanoparticles (CA-UCNP).

[0042] 4. Preparation of rare earth upconversion composite nanomaterials (UCM) 20 mg CA-UCNP, 20 mg tetracarboxyphenylporphyrin iron (TCPP-Fe), 60 mg ZrOCl2·8H2O and 560 mg benzoic acid were dispersed in 20 mL of ethanol / N,N-dimethylformamide (volume ratio 1:1) mixed solvent and reacted at 90 °C for 5 hours. The product was collected by centrifugation and washed with ethanol to obtain UCM.

[0043] 5. PEG modification and Cy-GSH loading 20 mg UCM and 20 mg DSPE-MPEG-1000 were dispersed in chloroform and stirred overnight. The chloroform was slowly evaporated under a nitrogen atmosphere, and the residue was dispersed in ultrapure water. After centrifugation and washing three times, the PEG-modified UCM was collected and resuspended in HEPES buffer (pH 7.2). Cy-GSH was dissolved in DMSO and slowly added to the dispersion at a Cy-GSH to PEG-modified UCM mass ratio of 1:1. The mixture was incubated overnight on a shaker, centrifuged, and washed to obtain Cy-UCM.

[0044] 6. Hyaluronic acid (HA) modification Dissolve 10 mg HA in 1 mL DMSO, mix with 5 mg Cy-UCM in 5 mL water, and stir overnight in the dark. Centrifuge and wash with ultrapure water to obtain the target product Cy-UCMA.

[0045] The obtained Cy-UCMA was characterized by morphology observation using transmission electron microscopy (TEM). Figure 6 X-ray diffraction (XRD) analysis of crystal structure Figure 7 The results showed that a uniform morphology and a size of approximately 100 nm nanoassemblies were successfully prepared.

[0046] Example 2: In vitro GSH responsiveness and singlet oxygen generation assay of Cy-UCMA 1. GSH Responsive Upconversion Emission Test Cy-UCMA dispersion (1 mg / mL) was added to PBS buffer (pH 6.5) containing 10 mM GSH. After reacting for 0, 0.5, 1, 2, and 4 hours, the upconversion emission spectra (808 nm excitation) were measured using a fluorescence spectrometer. The results are as follows: Figure 8 As shown, the upconversion luminescence intensity gradually increases with increasing reaction time, proving that Cy-UCMA has a specific response to GSH and that the generation of Cy-SG significantly sensitizes the luminescence of UCNPs.

[0047] 2. Singlet oxygen generation test 1,3-Diphenylisobenzofuran (DPBF) was used as a singlet oxygen probe. A PBS solution (pH 6.5) containing 100 μg / mL Cy-UCMA, 10 mM GSH, and 50 μM H₂O₂ was added to a quartz cuvette, followed by the addition of DPBF (final concentration 50 μM). The cuvette was then irradiated with an 808 nm laser (power density 0.5 W / cm²). 2 The UV-Vis absorption spectrum was measured at regular intervals to monitor the decrease in the characteristic absorption of DPBF at 420 nm. The results are as follows: Figure 9As shown, DPBF absorption decreases significantly with increasing illumination time, indicating that Cy-UCMA can efficiently generate DPBF in the presence of GSH. 1 O2.

[0048] Example 3: Cellular-level photodynamic activity and cytotoxicity assays of Cy-UCMA 1. Intracellular singlet oxygen detection Human hepatocellular carcinoma cells (HepG2) were seeded into 24-well plates and cultured for 12 hours. Cy-UCMA and unsensitized UCMA (as a control) were then added to a final concentration of 100 μg / mL, and incubated for 4 hours. The plates were washed three times with PBS, and then irradiated with a singlet oxygen-specific probe (SOSG) (5 μM) using an 808 nm laser (0.5 W / cm²). 2 (5 minutes). Observe and photograph under a fluorescence microscope. Results are as follows. Figure 10 As shown, the Cy-UCMA group exhibited strong green fluorescence (SOSG emitted light after oxidation), while the UCMA group showed very weak fluorescence, indicating that Cy-UCMA can respond to GSH and efficiently generate singlet oxygen in cells.

[0049] 2. Cytotoxicity assay (CCK-8 assay) HepG2 cells were seeded in 96-well plates and cultured overnight. Different concentrations (0, 10, 25, 50, 100, 200 μg / mL) of Cy-UCMA and UCMA were added and incubated for 4 hours. Cells were washed three times with PBS, added with fresh culture medium, and irradiated with an 808 nm laser (0.5 W / cm²). 2 (5 minutes). After culturing for another 24 hours, CCK-8 reagent was added to each well, and the cells were incubated for 2 hours. The absorbance at 450 nm was measured. Cell viability was calculated. Results are as follows. Figure 11 As shown, Cy-UCMA has a significant killing effect on HepG2 cells under light conditions, with cell survival rate below 20% at a concentration of 100 μg / mL; while the UCMA group still has a cell survival rate above 80% under the same conditions, proving that dye sensitization significantly enhances the photodynamic therapy effect.

[0050] Example 4: Evaluation of the in vivo antitumor immunotherapy efficacy of Cy-UCMA 1. Establishment of animal models Female BALB / c mice aged 6-8 weeks were injected subcutaneously into the right hind limb with mouse hepatocellular carcinoma H22 cells (1×10⁻⁶). 6 (One tumor per individual) to establish a subcutaneous tumor model. The tumor volume was increased to approximately 100 mm. 3The mice were randomly divided into three groups (5 mice per group) for about 7 days: (1) Control group (injected with an equal amount of PBS); (2) UCMA + light irradiation group (injected with unsensitized rare earth upconversion nanoassemblies UCMA and irradiated); (3) Cy-UCMA + light irradiation group (injected with Cy-UCMA prepared in this invention and irradiated).

[0051] 2. Drug administration and phototherapy regimen UCMA and Cy-UCMA were administered via tail vein injection at a dose of 20 mg / kg (based on nanoassemblies). Twenty-four hours after administration, the tumor sites of mice in the light-treated group were irradiated with an 808 nm laser (power density 0.3 W / cm², irradiation time 5 minutes). Administration was repeated every two days for a total of three doses, with light exposure 24 hours after each administration. Mouse weight and tumor volume were recorded every two days.

[0052] 3. Treatment efficacy The results are as follows Figure 12 As shown, the tumor volume in the control group increased rapidly; the tumor growth in the UCMA+ light irradiation group was inhibited to some extent, but the effect was limited; while the tumor volume in the Cy-UCMA+ light irradiation group was significantly reduced, and the tumor inhibition rate was significantly higher than that in the UCMA+ light irradiation group. Figure 13 As shown, there was no significant difference in the body weight of mice in each group during the treatment period, indicating that both nanoassemblies have good biocompatibility.

[0053] 4. Immune cell infiltration analysis Mice were sacrificed after treatment, tumor tissue was dissected, and single-cell suspensions were prepared. Flow cytometry was used to detect CD8+ in tumor-infiltrating lymphocytes (TILs). + The proportion of T cells. Results as follows: Figure 14 As shown, CD8+ in tumor tissue of the Cy-UCMA+ light-irradiated group + The T cell infiltration rate was significantly higher than that of other groups, indicating that the nanoassembly can effectively activate adaptive anti-tumor immune responses.

[0054] In summary, the dual-locked glutathione-specific responsive dye-sensitized rare earth upconversion nanoassemblies (Cy-UCMA) provided by this invention exhibit excellent GSH-responsive upconversion luminescence enhancement, efficient singlet oxygen generation, and significant photodynamic killing ability of tumor cells both in vivo and in vitro. They can also effectively induce immunogenic cell death, activate anti-tumor immunity, inhibit in situ tumor growth and distant metastasis, while having good biosafety.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a dye-sensitized rare-earth upconversion nanoassembly that is specifically responsive to glutathione, characterized in that, Includes the following steps: (1) Preparation of rare earth upconversion nanoparticles NaYF4:Yb with core-shell-shell structure 3+ ,Tm 3+ @NaYF4:Nd 3+ @NaYF4: Rare earth upconversion nanoparticles were obtained by thermal decomposition in oleic acid and octadecene solvent, using rare earth trifluoroacetate as a precursor, under argon protection, heating to 110-130℃ and holding for 30-60 minutes, and then heating to 300-320℃ and holding for 30-60 minutes. (2) Modify the rare earth upconversion nanoparticles obtained in step (1) with citric acid: Disperse the rare earth upconversion nanoparticles in chloroform and stir with a citric acid water / ethanol mixed solution at room temperature for 24-72 hours to obtain citric acid modified rare earth upconversion nanoparticles. (3) The citric acid-modified rare earth upconversion nanoparticles obtained in step (2) are dispersed with tetracarboxyphenylporphyrin iron, ZrOCl2•8H2O and benzoic acid in a mixed solvent of ethanol and N,N-dimethylformamide in a volume ratio of 1:1, and reacted at 80-100℃ for 4-6 hours to obtain rare earth upconversion composite nanomaterial UCM; wherein the mass ratio of the citric acid-modified rare earth upconversion nanoparticles, tetracarboxyphenylporphyrin iron, ZrOCl2•8H2O and benzoic acid is 1:1:3:28; (4) Disperse the UCM obtained in step (3) and DSPE-MPEG-1000 in chloroform at a mass ratio of 1:1, stir for 6-12 hours, remove the chloroform and disperse in buffer solution to obtain PEG-modified UCM. (5) Dissolve the glutathione-specific responsive dye Cy-GSH in dimethyl sulfoxide, mix Cy-GSH with PEG-modified UCM at a mass ratio of 1:1 to 1:5, and incubate in buffer for 6-12 hours to obtain dye-sensitized rare earth upconversion nanoassemblies Cy-UCM. (6) Hyaluronic acid and Cy-UCM obtained in step (5) are mixed in water at a mass ratio of 2:1 to 4:

1. The mixture is stirred for 6-12 hours in the dark, centrifuged and washed to obtain the dye-sensitized rare earth upconversion nanoassembly Cy-UCMA that is specifically responsive to glutathione.

2. The preparation method according to claim 1, characterized in that, The core chemical composition of the rare earth upconversion nanoparticles mentioned in step (1) is NaYF4:20%Yb. 3+ 0.5%Tm 3+ The chemical composition of the middle shell is NaYF4:20%Nd. 3+ The outer shell is undoped NaYF4.

3. The preparation method according to claim 1, characterized in that, The heating rate in step (1) to raise the temperature to 300-320℃ is 15-25℃ / min; In step (2), the volume ratio of water to anhydrous ethanol in the citric acid / ethanol mixed solution is 3:2, and the concentration of citric acid is 20 mg / mL. The reaction temperature in step (3) is 90°C and the reaction time is 5 hours; The mass ratio of Cy-GSH to PEG-modified UCM in step (5) is 1:1; The mass ratio of hyaluronic acid to Cy-UCM in step (6) is 2:

1.

4. The preparation method according to claim 1, characterized in that, The glutathione-specific responsive dye Cy-GSH described in step (5) is prepared through the following steps: (a) 2,3,3-trimethyl-3H-indole and 5-bromopentanoic acid were dissolved in toluene at a molar ratio of 1:1.2 to 1:1.6, refluxed under nitrogen protection for 8-16 hours, and purified by silica gel column chromatography to obtain compound 1; (b) Compound 1 and 2-chloro-1-formyl-3-hydroxymethylene-1-cyclohexene were dissolved in a mixed solvent of n-butanol / benzene at a molar ratio of 1.5:1 to 2.5:1 and refluxed for 4 to 8 hours. The mixture was then purified by silica gel column chromatography to obtain compound 2. (c) Under an argon atmosphere, sodium hydride and 4-hydroxy-4'-nitroazobenzene were reacted in DMF at a molar ratio of 1.2:1 to 1.8:

1. After stirring for 5 to 15 minutes, a DMF solution of compound 2 was added dropwise, wherein the molar ratio of compound 2 to 4-hydroxy-4'-nitroazobenzene was 0.8:1 to 1.2:

1. The reaction was carried out at room temperature for 8 to 16 hours. The mixture was purified by silica gel column chromatography to obtain Cy-GSH.

5. A glutathione-specific responsive dye-sensitized rare-earth upconversion nanoassemblies prepared by the preparation method according to any one of claims 1-4.

6. The dye-sensitized rare-earth upconversion nanoassembly according to claim 5, characterized in that, The nanoassembly includes: Rare earth upconversion nanoparticles, wherein the rare earth upconversion nanoparticles have a core-shell-shell structure, consisting of NaYF4:Yb in sequence. 3 + ,Tm 3+ Core layer, NaYF4:Nd 3+ Shell and NaYF4 outer shell; A metal-organic framework layer is coated on the surface of the rare earth on-conversion nanoparticles. The metal-organic framework layer is composed of tetracarboxyphenylporphyrin iron and zirconium clusters, and the metal-organic framework layer is loaded with glutathione-specific responsive dye Cy-GSH. And a hyaluronic acid targeting layer modified on the outer surface of the metal-organic framework layer; The dye Cy-GSH reacts with glutathione overexpressed in the tumor microenvironment to generate Cy-SG molecules. The Cy-SG molecules act as sensitizers for the rare earth upconversion nanoparticles, enhancing the upconversion luminescence of the rare earth upconversion nanoparticles through a non-radiative energy transfer mechanism. This, in turn, enhances the fluorescence resonance energy transfer between the rare earth upconversion nanoparticles and the tetracarboxyphenylporphyrin iron, thereby increasing the singlet oxygen yield.

7. The application of the dual-locked glutathione-specific responsive dye-sensitized rare-earth upconversion nanoassemblies according to claim 6 in the preparation of photodynamic immunotherapy drugs for treating tumors.

8. The application according to claim 7, characterized in that, The tumor is a solid tumor that overexpresses glutathione, including liver cancer cells.

9. The application according to claim 7, characterized in that, The photodynamic immunotherapy drug is administered via intravenous injection, followed by irradiation of the tumor site with 808 nm near-infrared light at a power density of 0.2-0.5 W / cm². 2 The irradiation time is 3-10 minutes; the photodynamic immunotherapy drug induces immunogenic cell death of tumor cells, activates adaptive anti-tumor immune response, and inhibits the growth of in situ tumors and / or distant metastases.

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