Gadolinium-based nanocomposite and application thereof in preparation of medicine for reversing NSCLC radiation resistance

Abstract

The invention is applicable to the technical field of biological medicine, and provides a gadolinium-based nano-composite and application thereof in preparation of a medicine for reversing NSCLC radiation resistance, and a preparation method of the gadolinium-based nano-composite comprises the following steps: synthesizing NaYF4: Yb / Er; synthesizing NaYF4: Yb / Er (at) NaGdF4; synthesizing NaYF4: Yb / Er (at) NaGdF4 (at) mSiO2; 3-aminopropyltrimethoxysilane is added into a NaYF4: Yb / Er (at) NaGdF4 (at) mSiO2 aqueous solution, stirring is performed overnight, washing is performed, shSPARC plasmids are added, and a reaction is performed at the room temperature. The gadolinium-based nanocomposite prepared by the invention has both gene intervention and radiation sensitization functions, is good in biocompatibility, and can regulate the ferroptosis sensitivity of radiation-resistant tumors in a targeted manner, improve the ferroptosis inhibition effect, reverse the radiation resistance of resistant cells and improve the radiotherapy effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a gadolinium-based nanocomposite and its application in the preparation of a drug for reversing the radiation resistance of NSCLC. Background Art

[0002] Lung cancer is a malignant tumor. Non-small cell lung cancer (NSCLC) is one of the main pathological types of lung cancer and poses a great threat to public health. As an important means of lung cancer treatment, radiotherapy is applied in all stages of NSCLC treatment. However, no matter which radiotherapy technique is adopted, the emergence of tumor cell radiation resistance inevitably leads to the failure of radiotherapy and local recurrence. Simply increasing the radiation dose not only fails to improve the survival benefit, but may also exacerbate the adverse reactions and lead to poor prognosis. Therefore, in-depth exploration of the radiation resistance mechanism and finding targets for reversing the radiation resistance of lung cancer have become important concerns for reversing the radiation resistance of NSCLC.

[0003] Recently, ferroptosis, a mode of cell death triggered by excessive production of reactive oxygen species (ROS) by mitochondria and excessive accumulation of iron-dependent lipid peroxides, has become a research hotspot. It can be inhibited by antioxidants and is closely related to oxidative stress, and finally shows obvious mitochondrial morphological changes, accumulation of iron and lipid peroxidation, and changes in the expression levels of some key ferroptosis regulators such as glutathione peroxidase-4 (GPX4). As a natural anti-tumor method, ferroptosis is closely related to radiation resistance. It can improve the sensitivity of tumor radiotherapy through multiple mechanisms such as enhancing the cell oxidative stress pathway, increasing radiation-induced DNA damage, regulating the tumor microenvironment, and activating the immune synergy effect. Therefore, in-depth exploration of the regulatory mechanism of ferroptosis in radiation-resistant cells and targeted regulation of ferroptosis-related pathways are expected to provide new theoretical bases and clinical application directions for overcoming tumor radiation resistance.

[0004] In recent years, as a tumor-targeted therapy carrier, nanomaterials have been widely used in various fields including chemodynamic therapy, photodynamic therapy, and photothermal therapy. High-Z rare earth nanomaterials represented by gadolinium (Gd) have been found to be used as radiotherapy sensitizers, which can effectively amplify the local radiation dose without causing additional radioactive toxicity to the surrounding normal tissues. Developing nanomaterials with radiation sensitization benefits, making them targeted, safe, and efficient, and having an anti-cancer effect on the targeted unit and only existing in specific types of tumor cells to improve the radiation resistance of NSCLC and have a stronger tumor killing effect have important scientific significance and clinical value. Summary of the Invention

[0005] The purpose of the embodiment of the present invention is to provide a preparation method of gadolinium-based nanocomposites, aiming to solve the problems proposed in the above-mentioned background technology.

[0006] The embodiment of the present invention is implemented as follows. The preparation method of gadolinium-based nanocomposites includes the following steps: Synthesize NaYF4:Yb / Er; Use NaYF4:Yb / Er to synthesize NaYF4:Yb / Er@NaGdF4; Use NaYF4:Yb / Er@NaGdF4 to synthesize NaYF4:Yb / Er@NaGdF4@mSiO2; Synthesize gadolinium-based nanocomposites: Add 3-aminopropyltrimethoxysilane to the aqueous solution of NaYF4:Yb / Er@NaGdF4@mSiO2 and stir overnight. Wash off the unreacted 3-aminopropyltrimethoxysilane, and then add shSPARC plasmid and react at room temperature. The nucleotide sequence of the shSPARC plasmid is as shown in SEQ ID NO:1. The loading ratio of the aqueous solution of NaYF4:Yb / Er@NaGdF4@mSiO2 to the shSPARC plasmid is mg / mL:μg = 1:5.

[0007] Preferably, the step of synthesizing NaYF4:Yb / Er specifically includes: Add oleic acid and octadecene to YCl3·6H2O, YbCl3·6H2O and ErCl3·6H2O and stir. Heat up in an argon environment, repeatedly evacuate until no bubbles, continue to heat up, then cool down to room temperature. Add a methanol solution containing NaOH and NH4F and gradually heat up, repeatedly evacuate, and then carry out the reaction under programmed temperature rise.

[0008] Preferably, the step of using NaYF4:Yb / Er to synthesize NaYF4:Yb / Er@NaGdF4 specifically includes: Add Gd(CF3COO)3 and CF3COONa to oleic acid and octadecene, react after heating, cool down to room temperature and then add NaYF4:Yb / Er. Heat up in an argon environment, repeatedly evacuate and then carry out the reaction under programmed temperature rise.

[0009] Preferably, the step of using NaYF4:Yb / Er@NaGdF4 to synthesize NaYF4:Yb / Er@NaGdF4@mSiO2 specifically includes: Dissolve CTAB, add NaYF4:Yb / Er@NaGdF4 and disperse it by stirring overnight. Then add water, NaOH and ethanol, heat in a water bath. After the temperature is balanced, slowly add tetraethyl orthosilicate solution to react. Wash and then add an ethanol solution containing NH4NO3, and add anhydrous ethanol to make up the volume. React in a water bath, and then fully wash the precipitate.

[0010] Another object of the embodiments of the present invention is to provide a gadolinium-based nanocomposite prepared by the above preparation method.

[0011] Another object of the embodiments of the present invention is to provide an application of a gadolinium-based nanocomposite in the preparation of a drug for reversing the radiation resistance of NSCLC.

[0012] Preferably, the gadolinium-based nanocomposite targets the release of shSPARC plasmid in an acidic tumor microenvironment, and the shSPARC plasmid affects the ferroptosis process by dynamically regulating the expression of GPX4 / SLC7A11 / PTGS2.

[0013] The gadolinium-based nanocomposite provided by the embodiments of the present invention has both gene intervention and radiation sensitization functions, good biocompatibility, targets and regulates the ferroptosis sensitivity of radiation-resistant tumors, improves the ferroptosis inhibition effect, integrates physical energy deposition and gene-targeted therapy, breaks through the limitations of a single sensitization strategy, realizes radiotherapy sensitization from physical sensitization and gene regulation, reverses the radiation resistance of resistant cells, and improves the radiotherapy effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Clonogenic formation results and analysis of A549 and A549-RR provided in Example 1 of the present invention (n = 3, **** p < 0.0001); Figure 2 Fe levels after irradiation of A549 and A549-RR provided in Example 2 of the present invention (n = 3, *** 2+ 0.001, **** p< 0.0001); p< 0.0001); Figure 3 Expression levels of ferroptosis-related proteins after irradiation of A549 and A549-RR provided in Example 3 of the present invention; Figure 4 Differential gene statistics of A549, A549-RR, and A549-RR+RSL3 groups provided in Example 4 of the present invention; Figure 5 Volcano plot of differential genes of A549 and A549-RR groups provided in Example 4 of the present invention; Figure 6 Volcano plot of differential gene groups of A549-RR and A549-RR+RSL3 provided in Example 4 of the present invention; Figure 7 Venn diagram of differential genes between A549 and A549-RR and differential genes after treatment of A549-RR and RSL3 provided in Example 4 of the present invention; Figure 8Heatmap showing the correlation between the differential genes of A549 and A549-RR provided in Example 4 of the present invention and after A549-RR and RSL3 treatment (group1: A549 group; group2: A549-RR group; group3: A549-RR + RSL3 group); Figure 9 SPARC mRNA expression levels of A549, A549-RR and A549-RR provided in Example 5 of the present invention (n = 3, **** p< 0.0001); Figure 10 SPARC protein expression levels during the establishment of radiation resistance in A549-RR provided in Example 5 of the present invention; Figure 11 SPARC immunohistochemical results and related lung cancer patient survival analysis curves in normal lung tissues, lung adenocarcinoma and squamous cell carcinoma tissues in the TCGA database provided in Example 5 of the present invention; Figure 12 Protein expression changes after knocking down SPARC in A549-RR provided in Example 6 of the present invention (A-NC and H-NC: A549 transfected with empty plasmid group, A-S1 / 2 / 3: A549 transfected with shSPARC-1 / 2 / 3 group); Figure 13 Fluorescence imaging of SPARC in A549-RR cells provided in Example 6 of the present invention (nucleus stained blue with DAPI; cytoskeleton stained red with F-actin; SPARC protein stained green, scale bar = 10 μm); Figure 14 Effect of SPARC knockdown on the colony formation ability of A549-RR provided in Example 6 of the present invention (n = 3, *** p< 0.001, **** p< 0.0001); Figure 15 Colony formation results and analysis after knocking down SPARC and treating with Fer1 in A549-RR provided in Example 6 of the present invention (n = 3, * p< 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001); Figure 16 Levels of ferroptosis-related proteins after knocking down SPARC and treating with Fer1 in A549-RR provided in Example 6 of the present invention; Figure 17 Agarose gel electrophoresis detection of the ability of DNSP (1 mg / mL) to load shSPARC provided in Example 7 of the present invention; Figure 18 For Example 7 of the present invention, agarose horizontal electrophoresis was used to detect the release of shSPARC from DNSP under different pH conditions; Figure 19 Appearance characterization results provided for Example 7 of the present invention (a is the transmission electron microscope image of NaYF4:Yb / Er, b is the transmission electron microscope image of NaYF4:Yb / Er@NaGdF4, c is the transmission electron microscope image of DNS, d is the transmission electron microscope image of DNSP (scale bar = 5 nm)); Figure 20 Transmission electron microscope elemental mapping of DNS provided for Example 7 of the present invention (scale bar = 5 nm; Figure 21 Analysis of the line scan results of the transmission electron microscope of DNS provided for Example 7 of the present invention; Figure 22 Results of the synthesis quality and performance characteristics of DNSP provided for Example 7 of the present invention (A is the XRD results of each component of DNS, B is the FTIR spectrum of each component of DNSP, C is the hydrodynamic particle size distribution of DNSP, D is the zeta potential of DNS, DNS-NH2 and DNSP); Figure 23 Near-infrared imaging and fluorescence intensity under 980 nm laser emission after intratumoral injection of DNSP provided for Example 7 of the present invention; Figure 24 Cell uptake of FITC-labeled DNSP provided for Example 7 of the present invention (scale bar = 200 μm; Figure 25 Uptake of DNSP by A549-RR cells in a two-dimensional system and A549-RR tumor spheres in a three-dimensional system provided for Example 7 of the present invention (blue DAPI: cell nucleus; red: lysosome probe; green: FITC-labeled DNSP; (left) scale bar = 10 μm (right) scale bar = 40 μm); Figure 26 Immunofluorescence detection of SPARC protein expression after transfection of A549-RR with DNSP compared to commercial lipo6000 (scale bar = 100 μm); Figure 27 Colony formation ability after transfection of A549-RR with DNSP compared to commercial lipo6000 provided for Example 7 of the present invention (n = 3, * p< 0.05, **** p< 0.0001); Figure 28Photographs of different groups for treating A549-RR cell xenografts in SCID mice provided in Example 7 of the present invention (G1: Control, G2: DNS, G3: DNSP, G4: X-ray, G5: DNS + X-ray, G6: DNSP + X-ray); Figure 29 Change in the total tumor volume of each group during the treatment of A549-RR cell-bearing SCID mice provided in Example 7 of the present invention (n = 3, * p< 0.05, *** p< 0.001, **** p< 0.0001); Figure 30 Change in the tumor volume of each group of A549-RR cell-bearing SCID mice during the treatment provided in Example 7 of the present invention; Figure 31 Tumor mass at 14 days after irradiation of A549-RR cell-bearing SCID mice provided in Example 7 of the present invention (n = 3, ** p< 0.01, *** p< 0.001, **** p< 0.0001); Figure 32 Change in body weight of different groups of A549-RR cell-bearing SCID mice provided in Example 7 of the present invention; Figure 33 HE staining results of various organs of A549-RR cell-bearing SCID mice provided in Example 7 of the present invention (scale bar = 100 μm); Figure 34 Expression levels of ferroptosis-related proteins and SPARC protein in different groups for treating A549-RR cell xenografts in SCID mice provided in Example 7 of the present invention; Figure 35 TUNEL fluorescence staining results of different groups for treating A549-RR cell xenografts in SCID mice provided in Example 7 of the present invention (DAPI: blue fluorescence; TUNEL: red fluorescence, scale bar = 100 μm) (n = 3, *** p< 0.001, **** p< 0.0001); Figure 36H&E staining results of different groups of tissues of A549-RR cell xenografts in SCID mice and immunohistochemical expressions of SPARC, GPX4, and PTGS2 proteins in different groups of tissues of A549-RR cell xenografts in SCID mice, and TUNEL fluorescence staining results (DAPI: blue fluorescence; TUNEL: red fluorescence; scale bar = 100 μm) provided in Example 7 of the present invention (n = 3, * p< 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001). Detailed implementation manners

[0015] In order to make the objectives, technical solutions, and advantages of the present invention more clear and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0016] The following describes the specific implementation of the present invention in detail with reference to specific embodiments.

[0017] Example 1: Construction of a radiation-resistant cell model and verification of radiation sensitivity: Cell culture: The human lung cancer cell line A549 (gifted by the research group of Jin Shunzi in the Radiation Biology Laboratory of the School of Public Health, Jilin University) was used and maintained in DMEM high-glucose medium containing 10% premium fetal bovine serum and 2% penicillin-streptomycin solution. All cells were cultured in a cell incubator at 37°C and 5% CO2 concentration to ensure the best growth conditions; Cell treatment: For cell digestion, when the cells reached approximately 90% confluence in the culture dish, the old culture medium was discarded, and after washing 2-3 times with PBS, 0.25% trypsin (containing EDTA) was added for digestion; when the cells became round and in a flowing sand-like state, complete medium was added to terminate digestion, and the cells were blown into a single-cell suspension, centrifuged, and resuspended; for cell passage, an appropriate amount of cell suspension was added to a new culture dish for continued culture; for cell resuscitation, the cells stored in a -80°C refrigerator / liquid nitrogen were thawed in a 37°C water bath. After the cryopreservation solution was fully dissolved, it was mixed at a ratio of 1:10 (cryopreservation solution: complete medium), centrifuged at 1000 rpm for 5 min, and the cells were resuspended with an appropriate amount of medium and added to a new culture dish for continued culture; for cryopreservation, the cells to be treated were digested with trypsin, centrifuged to discard the supernatant, resuspended with a commercial cryopreservation solution, and aliquoted into cryopreservation tubes. The types of cells and the cryopreservation time were marked and promptly stored in a -80°C refrigerator. Important cell lines for seed preservation were transferred from the -80°C refrigerator to a liquid nitrogen tank the next day for long-term preservation; Irradiation conditions: Using the method of hypofractionated large-dose irradiation, A549 cells were irradiated 5 times at a dose rate of 1 Gy / min (total dose 30 Gy, single dose 6 Gy) to construct a radiation-resistant model A549-RR. After irradiation, at least 4 passages were performed to complete the post-irradiation stabilization culture. After ensuring the stability of cell phenotype, subsequent functional verification was carried out; Radiosensitivity verification: The colony formation ability of the radiation-resistant cell model at 0, 2, 4, 6, and 8 Gy was detected by the colony formation assay. The results are as Figure 1 shown. With the increase of irradiation dose, the colony formation ability of each group of cells decreased in a dose-dependent manner. In the same dose irradiation group, the colony formation rate of A549-RR cells was significantly better than that of parental cells. The results indicate that the ability of radiation-resistant cells to repair sublethal damage is significantly stronger and the radiosensitivity is reduced.

[0018] Example 2. Analysis of ferroptosis metabolic characteristics of radiation-resistant cells: The core mechanism of ferroptosis is the catalysis of cell membrane structure by intracellular ferrous ions, leading to lipid peroxidation. In this example of the present invention, a colorimetric method was used to detect the intracellular Fe 2+ content. The results are as Figure 2 shown. The basal Fe 2+ content of A549-RR cells was 0.15 ± 0.06 nmol / 10 6 cells, which was decreased by 62.5% compared with parental cells A549 (0.40 ± 0.06 nmol / 10 6 cells, p < 0.001); 48 hours after 6 Gy irradiation, the ability of resistant cells to accumulate Fe 2+ was significantly limited. A549-RR (0.42 ± 0.02 nmol / 10 6 cells) was decreased by 46.2% compared with A549 (0.78 ± 0.03 nmol / 10 6 cells) (p < 0.0001), suggesting that radiation-resistant cells resist the ferroptosis process by inhibiting iron accumulation.

[0019] Example 3. Analysis of changes in key ferroptosis proteins in radiation-resistant cells: PTGS2, as a key catalytic enzyme in the lipid peroxidation process, its up-regulation can promote the ferroptosis process; while SLC7A11 (regulating cysteine uptake) and GPX4 (reducing lipid peroxides) inhibit the occurrence of ferroptosis through synergistic effects. To explore the changes in ferroptosis-related proteins in radiation-resistant cells, in this example of the present invention, Western Blot was used to detect the changes in the expression of PTGS2, SLC7A11, and GPX4 proteins in cells. The results are as Figure 3As shown, under the unirradiated state, the expression intensities of GPX4 and SLC7A11 proteins in radiation-resistant cells (A549-RR) were significantly higher than those in parental cells, while the basal expression level of PTGS2 was relatively low; 48 hours after 6 Gy irradiation, the expression of GPX4 and SLC7A11 proteins in parental cells (A549) showed a downward trend, and the expression of PTGS2 showed an upward trend, suggesting the activation of radiation-induced ferroptosis; after irradiation, the downregulation amplitude of GPX4 / SLC7A11 in resistant cells was smaller than that in parental cells, and the upregulation degree of PTGS2 was limited, indicating that it has stronger antioxidant defense ability. The above protein expression trends were highly consistent with the ferroptosis resistance phenotype of radiation-resistant cells, suggesting that the continuous activation of the antioxidant defense system may be the key mechanism for its acquisition of radiation resistance.

[0020] Example 4. Screening and verification of ferroptosis-sensitive targets in radiation-resistant cells: RNA-seq (Illumina NovaSeq 6000, sequencing depth 30M reads / sample) was used to perform transcriptome sequencing analysis on three groups of cells: A549, A549-RR, and A549-RR + RSL3 (ferroptosis inducer) (1.2 μM, 48 h). Differentially expressed genes were screened based on the DESeq2 algorithm (v1.34.0), and the screening criteria were false discovery rate (FDR) < 0.05 and |log2FC| > 1. The results are as Figure 4 、 5 shown. The volcano plot of differential genes between the A549-RR and parental A549 groups showed a total of 236 differential genes (76 upregulated and 160 downregulated); as Figure 6 shown, treatment with RSL3 induced 1856 differential genes in A549-RR (800 upregulated and 1056 downregulated); To find genes that are commonly involved in radiation resistance in the two groups of A549, A549-RR, and A549-RR, A549-RR + RSL3, Venn diagram analysis was used, and the results are as Figure 7 shown. It was found through analysis that 92 genes were commonly involved in the basal resistance group (A549 and A549-RR) and the RSL3 stress treatment group (A549-RR and A549-RR + RSL3), suggesting that these genes may be involved in the core regulatory network of radiation resistance; further through Pearson correlation heat map analysis, as Figure 8 shown, 11 of these genes were highly expressed in resistant cells and were significantly downregulated after treatment with RSL3.

[0021] Example 5. Verification of SPARC expression and clinical correlation analysis: Based on the screening results of transcriptome data, in the embodiments of the present invention, the qPCR method was used to verify candidate genes with consistent expression trends in parental cells and the RSL3 treatment group. The results are as follows Figure 9 shown. In A549-RR, the expression of SPARC was consistent with the trend of second-generation sequencing. The expression of SPARC mRNA in A549-RR increased by 1.5-fold (p < 0.0001). After RSL3 treatment, the expression level of SPARC decreased by 1.3-fold respectively (p < 0.0001). Further, Western Blot was used to detect the expression level of SPARC protein. The results are as follows Figure 10 shown. The SPARC protein was upregulated in a dose-dependent manner during the establishment of radiation resistance, suggesting that it may be a potential target for reversing radiotherapy resistance; Using the immunohistochemistry data of pathological tissues in the TCGA database for analysis, the results are as follows Figure 11 shown. Compared with normal lung tissues, the expression of SPARC was upregulated in lung adenocarcinoma and squamous cell carcinoma tissues. The survival analysis of GEPIA2 further confirmed that high expression of SPARC in NSCLC was closely related to the poor prognosis of NSCLC patients (HR = 1.5, p = 0.0065). This suggests that SPARC is a potential clinical prognostic marker for NSCLC. Based on these results, it is speculated that SPARC may reduce the radiation sensitivity of resistant cells by inhibiting the ferroptosis pathway.

[0022] Example 6. Analyze the regulatory effect of SPARC knockdown on the ferroptosis level of radiation-resistant cells: 1. Construction and verification of the SPARC knockdown cell model: To clarify the role of SPARC in radiation-resistant cells, in the embodiments of the present invention, 3 shRNAs (shSPARC-1 / 2 / 3) targeting human SPARC were designed, and their nucleotide sequences are shown in SEQ ID NO: 1-3. A stable SPARC knockdown cell line was constructed by lentivirus infection. As Figure 12 shown, the Western Blot results showed that the best knockdown effect was achieved by shSPARC-1. Therefore, shSPARC-1 was selected for subsequent functional studies; as Figure 13 shown, immunofluorescence was used to detect the expression of SPARC protein. In the control group, SPARC was mainly localized in the cytoplasm (green fluorescence) and co-localized with F-actin (red fluorescence) to form an orange-yellow signal. After transfection with shSPARC-1, the green fluorescence of A549-RR cells was significantly weakened, indicating that the expression of SPARC was effectively inhibited; 2. Effect of SPARC knockdown on radiation sensitivity: To evaluate the regulatory effect of SPARC on radiosensitivity, a colony formation assay was used to analyze the effect of SPARC knockdown on the survival ability of radiation-resistant cells. The results are as followsFigure 14 As shown, with the increase of irradiation dose, the cell colony formation ability weakens. Under the same dose of irradiation, compared with NC, knocking down SPARC results in a weaker cell colony formation ability of A549-RR cells. This indicates that low expression of SPARC enhances the radiation sensitivity of A549-RR cells; 3. Sensitization of radiotherapy by knocking down SPARC through the ferroptosis pathway: To clarify whether the radiosensitization effect induced by knocking down SPARC depends on the ferroptosis pathway, the ferroptosis inhibitor Ferrostatin-1 (Fer1, 2 μM) was used for treatment, and the results are as follows Figure 15 shown. Pretreatment with Fer1 can significantly restore the inhibitory effect of knocking down SPARC on the colony formation of radiation-resistant cells. The colony formation rate of the A549-RR group increased from 17.4 ± 3.8% to 28.0 ± 3.2% ( p <0.01). This result confirms that knocking down SPARC significantly enhances radiation sensitivity by activating the ferroptosis pathway, and its radiosensitization effect is ferroptosis-dependent; 4. Effects of knocking down SPARC on ferroptosis-related proteins: To clarify the effects of knocking down SPARC on ferroptosis-related proteins in radiation-resistant cells, Western Blot immunoblotting was used to detect the protein expression levels of SPARC, PTGS2, SLC7A11, and GPX4 in cells, and the results are as follows Figure 16 shown. Under basal conditions, the protein expression intensities of GPX4 and SLC7A11 in the SPARC knockdown group A549-RR (A-S1) were significantly lower than those in the negative control group. After irradiation, this down-regulation trend was more obvious. On the contrary, the pro-ferroptosis protein PTGS2 showed an up-regulated basal expression in the SPARC knockdown group, and its expression intensity further increased after irradiation. Treatment with the ferroptosis inhibitor Fer1 can partially reverse the above protein expression trends, indicating that SPARC affects the ferroptosis process by dynamically regulating the expression of GPX4 / SLC7A11 / PTGS2. This result shows that knocking down SPARC weakens the antioxidant defense of radiation-resistant cells and increases the level of cellular ferroptosis by enhancing the level of lipid peroxidation; Example 7. Construction of gadolinium-based nanocomposites (DNSP) and analysis of their radiosensitization effects: 1. Construction and characterization of DNSP: To overcome radiation resistance during NSCLC radiation, the present invention's example designed and constructed a gadolinium-based core-shell rare earth upconversion nanoradiosensitizer (DNSP), and the specific steps are as follows: Synthesis of NaYF4:Yb / Er: Weigh 0.78 mM YCl3·6H2O, 0.2 mM YbCl3·6H2O, and 0.02 mM ErCl3·6H2O into a three-necked flask, add 6 mL of oleic acid and 15 mL of octadecene, stir at 800 rpm, heat to 100 °C under an argon atmosphere, repeatedly evacuate until no bubbles are present, continue heating to 160 °C, cool to room temperature after 30 min, add a methanol solution containing NaOH and NH4F and gradually heat to 100 °C, repeatedly evacuate, and program the temperature to 300 °C for reaction for 1 h. After the reaction, wash with acetone and cyclohexane 3 - 5 times and disperse in 10 mL of cyclohexane for standby (as Reagent 1); Synthesis of NaYF4:Yb / Er@NaGdF4: Take 0.144 mM Gd(CF3COO)3 and 0.32 mM CF3COONa and place them in a three-necked flask containing 10 mL of oleic acid and 10 mL of octadecene, heat to 120 °C for reaction for 30 min, cool to room temperature, add 9 mL of Reagent 1, heat to 100 °C under an argon atmosphere, repeatedly evacuate, and then program the temperature to 290 °C for reaction for 1 h. After the reaction, wash with acetone and cyclohexane 3 - 5 times and disperse in 10 mL of cyclohexane for standby (as Reagent 2); Synthesis of NaYF4:Yb / Er@NaGdF4@mSiO2 (DNS): Dissolve 66 mg of CTAB in 20 mL of distilled water at 60 °C, add 4 mL of Reagent 2, disperse by ultrasonic probe for 30 min, and then stir overnight at 800 rpm. The next day, add 20 mL of distilled water, 150 μL of 2M NaOH, and 3 mL of ethanol to 10 mL of the above mixture, heat in a water bath at 70 °C. After the temperature is balanced, slowly add the tetraethyl orthosilicate solution. After reaction for 2 h, wash the precipitate thoroughly with ethanol and distilled water and disperse in 10 mL of ethanol for the next step of reaction. Add an ethanol solution containing NH4NO3 to the above washed solution and add anhydrous ethanol to make up to 50 mL, react in a water bath at 60 °C for 2 h, wash the precipitate thoroughly with ethanol and distilled water to obtain DNS and disperse in 10 mL for use; Synthesis of DNSP: Add 2% 3-aminopropyltrimethoxysilane (APTMS) to the DNS aqueous solution and stir overnight to modify -NH2. Wash the unreacted APTMS thoroughly with ethanol / distilled water. Then react the modified DNS with the shSPARC plasmid at room temperature for 2 h. To optimize the loading ratio of shSPARC and DNS, use agarose gel electrophoresis to detect the loading stability when 1 mg / mL DNS loads different masses of shSPARC. The results are as Figure 17As shown, 1 mg / mL DNS can completely load 5 μg shSPAR (without free bands). When the ratio is greater than this, plasmid leakage occurs due to oversaturation of the loading amount (with free bands), indicating that the loading ratio of 1:5 (DNS:shSPARC) is the best. Therefore, the DNSP with this loading ratio is used for subsequent experiments. To verify the pH-responsive ability of DNSP, as Figure 18 shown, the release rate of shSPARC from DNSP in an acidic environment (pH 5.5) is higher than that in a neutral condition (pH 7.4), suggesting that DNSP can target the release of shSPARC in an acidic tumor microenvironment; In the examples of the present invention, core-shell coated NaYF4:Yb / Er@NaGdF4 is selected as the basic structure instead of pure NaGdF4. This choice is based on the fact that the physical and chemical properties of NaGdF4 itself show severe aggregation, resulting in poor dispersibility and increasing the difficulty of coating mesoporous silica. To solve this problem, NaYF 4: Yb / Er is introduced as a supporting structure to improve the size stability and dispersibility of the overall upconversion nanomaterials; Characterization: First, the morphology of each shell layer of the nanomaterials was observed by transmission electron microscopy (FEI Tecnai G2 S-Twin 200 kV equipment), and the results are as Figure 19 shown in a. The core layer NaYF4:Yb / Er presents hexagonal phase nanocrystals, as Figure 19 shown in b. The radiation sensitization layer NaGdF4 is uniformly coated, forming an obvious core-shell structure, as Figure 19 shown in c. The carrier layer is DNS coated with mesoporous silica, presenting a uniformly dispersed structure, as Figure 19 shown in d. After loading shSPARC, the mesopores of DNSP become denser, indicating successful loading of shSPARC; Figure 20 For the element mapping of DNS by transmission electron microscopy, it can be seen from the figure that Y, Yb, and Er are enriched in the core region of DNS, Gd is uniformly distributed in the core-shell layer, and the Si signal is distributed throughout the domain, as Figure 21 shown. Y, Yb, and Er reach peaks in the core region, the Gd peak in the shell layer shows a double peak, and the Si peak shows a wide peak. The elemental gradient distribution at the interface confirms the core-shell structure. This design realizes the integration of triple functions through the core-shell strategy: the NaYF4:Yb / Er core provides upconversion luminescence properties; the NaGdF4 shell enhances the radiosensitization effect of radiotherapy; the mesoporous SiO2 carrier realizes efficient loading of shSPARG; To verify the synthesis quality and performance characteristics of DNSP, the crystal phase compositions of NaYF4:Yb / Er, NaYF4:Yb / Er@NaGdF4, and DNS were detected by X-ray diffraction (XRD) (VG ESCALAB MK II electron energy spectrometer), as Figure 22As shown in Figure A, the diffraction peaks of DNSP are highly consistent with the standard card PDF#16_0334 of hexagonal phase NaYF4, confirming the successful construction of the core-shell structure and the absence of impurity phases. At the same time, Fourier transform infrared spectroscopy (FTIR) was used for detection. As Figure 22 shown in Figure B, DNSP has characteristic absorption peaks at 1090 cm -1 , 960 cm -1 and 2920 cm -1 , indicating that the mesoporous silica coating is complete and the APTMS modification is successful. As Figure 22 shown in Figure C, dynamic light scattering (DLS) analysis shows that the hydrodynamic diameter of DNSP is 170.3 ± 5.7 nm, and the polydispersity index (PDI) is 0.12, confirming that the material has good monodispersity. As Figure 22 shown in Figure D, DNS-NH2 was obtained by modifying with APTMS, which increased the Zeta potential of DNSP from -32.1 mV to +18.6 mV. This potential reversal helps shSPARC to be efficiently loaded through electrostatic adsorption. These characterization data confirm the successful preparation of DNSP, and its physicochemical parameters meet the requirements of subsequent experiments.

[0023] 2. Biodistribution and cellular uptake of DNS: 2.1 Experimental animals and treatments: The experimental animals were purchased from Beijing Huafukang Biotechnology Co., Ltd., 3-4-week-old SCID mice. All experimental animals were raised and handled completely in accordance with the "Jilin University Guide for the Care and Use of Laboratory Animals" and were approved by the Animal Experiment Welfare Ethics Committee of the School of Public Health of Jilin University (SY: 2024-07-001). Irradiation conditions: Dose rate of 2 Gy / min, total dose of 20 Gy, source-skin distance of 60 cm. The parts of the mice outside the irradiation field were covered with lead plates. The mice were divided into 6 groups (8 mice in each group). Establishment of the resistant NSCLC tumor model: A cell suspension of 1×10 7 A549-RR cells was subcutaneously inoculated into the right hind leg of the mice; in the treatment group, the cells were replaced with A549-RR cells stably infected with shSPARC lentivirus. One week after tumor implantation, the subcutaneous tumor formation was observed every 3 days. The tumor volume formula is: (length × width 2 2) / 2; when the tumors of each group of mice grew to 100 mm 2Perform intratumoral injection / irradiation operations on the left and right; for the DNSP treatment group, 12 h before irradiation of the mice, intratumorally inject DNSP (100 μL / mouse, 50 mg / mL) into the A549-RR tumor-bearing mice, and irradiate the mice after anesthesia the next day; the body weight and tumor size of the experimental mice were recorded every 2 days, the physical condition of the mice was observed, the mice were euthanized uniformly on the 14th day after irradiation, and samples such as various organs and tumor tissues were taken, the tumor mass was weighed, and the tissues were fixed or frozen according to the experimental needs; 2.2 In vivo distribution characteristics of DNSP: Use a 980 nm laser source to excite the intratumoral injection site. As Figure 23 shown, the fluorescence intensity of DNSP reached its peak 12 h after intratumoral injection and remained until 24 h, indicating its good retention at the tumor site; 2.3 DNS cell uptake ability: Evaluate the uptake function of DNS by FITC fluorescence labeling tracer method. As Figure 24 shown, DNS is taken up by A549-RR cells through endocytosis, and as the incubation time prolongs, more fluorescence is taken up by the cells, and the intracellular uptake reaches the highest level at 12 h.

[0024] 3 Analysis of the transfection ability of DNSP and its effect on radiation-related ferroptosis: To evaluate the cellular uptake and delivery efficacy of DNSP, co-localization analysis was performed using FITC labeling tracer technology combined with a lysosome-specific fluorescent probe (LysoTracker Red). As Figure 25 shown, 12 h after DNSP treatment, DNSP showed uniformly distributed green fluorescence signals in adherent A549-RR cells; in the three-dimensional tumor spheroid model, the penetration depth of DNSP reached 120 ± 12 microns, indicating that DNSP can be efficiently taken up by A549-RR cells and penetrate deep into the tumor; further, the expression of SPARC protein was quantified by immunofluorescence. As Figure 26 shown, the fluorescence expression trend of SPARC in the DNSP transfection group was not significantly different from that in the commercial lipo6000 transfection group, but was significantly lower than that in the untreated group. After 6 Gy irradiation, the SPARC fluorescence expression level in the DNSP treatment group was lower than that in the simple irradiation group, confirming that it can effectively inhibit radiation-induced overexpression of SPARC. The above data indicate that DNSP achieves targeted gene silencing by efficiently delivering shSPARC, providing a molecular basis for radiation sensitization.

[0025] 4 Analysis of the effect of DNSP on colony formation: To evaluate the inhibitory effect of DNSP on the colony formation ability of radiation-resistant cells, a colony formation assay system was used to analyze the survival characteristics of A549-RR cells in different treatment groups. As Figure 27As shown, the combination of DNSP and 6 Gy irradiation significantly reduced the colony formation rate of A549-RR cells to 3.3% ± 0.8%, which was lower than that of the commercial transfection reagent lipo6000 group (12.0% ± 1.0%) (p < 0.05), indicating that DNSP enhanced the colony inhibition ability of irradiation against resistant cells.

[0026] 5. Tumor suppression effect of DNSP combined with radiotherapy: In the in vivo animal experiments, the radiation doses used were all verified as normal tolerance doses through previous experiments. In the examples of the present invention, SCID mice were used to construct an A549-RR cell xenograft tumor model to evaluate the radiotherapy effects of DNS and DNSP on radiation-resistant non-small cell lung cancer. As Figure 28 shown, the DNSP combined radiotherapy group (G6) showed the strongest tumor growth inhibitory effect, and its effect was significantly better than that of the single radiotherapy group (G4) and the DNS combined radiotherapy group (G5); the tumor growth curve was plotted. As Figure 29 and 30 shown, 14 days after radiotherapy, the tumor volume of group G6 (122.9 ± 20.8 mm 3 ³) was smaller than that of the single radiotherapy group G4 (1001.7 ± 121.7 mm 3 ³) ( p <0.0001), and was significantly better than the DNS combined radiotherapy group (group G5, 364 ± 51.9 mm 3 , p <0.0001); as Figure 31 shown, at the end of the experiment when sacrificed, the wet weight of the tumor in group G6 was 0.12 ± 0.05 g, which was lower than that of group G4 (0.79 ± 0.12 g) (p < 0.01). The above results indicate that DNSP has excellent radiation sensitization characteristics and greatly improves the radiotherapy effect of radiation-resistant non-small cell lung cancer.

[0027] 6. Biosafety evaluation of DNSP: To evaluate the systemic toxicity of DNSP, the body weight changes of tumor-bearing mice and the pathological status of major organs were monitored. As Figure 32 shown, there was no significant difference in body weight between the DNS and DNSP treatment groups (G3, G6) and the control group (G1). Body weight analysis showed that there was no significant difference between the DNSP treatment group (G6: 22.38 ± 2.3 g), the DNS treatment group (G3: 22.37 ± 1.9 g), and the blank control group (G1: 21.20 ± 1.2 g) ( p > 0.05), suggesting that DNSP did not cause obvious systemic toxic reactions; further through H&E staining pathological analysis, as Figure 33As shown, the organizational structures of major organs such as the heart, liver, spleen, lungs, and kidneys of the mice in the DNSP treatment group were intact, and no pathological damage was observed, confirming its excellent biocompatibility.

[0028] Effect of DNSP combined with radiotherapy on the ferroptosis pathway: To evaluate the regulatory effect of DNSP on radiation-induced ferroptosis in vivo, total proteins were extracted from A549-RR xenograft tumor tissues, and Western Blot immunoblotting was used to detect the expression levels of ferroptosis-related proteins in tumor tissues, such as Figure 34 As shown, the expression level of SPARC in the DNSP treatment groups (G3 and G6) was lower than that in the control group (G1 group). After radiotherapy, the expression of SPARC in the control group increased significantly, while the DNSP treatment groups still maintained low expression after radiotherapy. The expression of PTGS2 was significantly upregulated in the radiotherapy combined with DNSP treatment group (G6 group), while the expressions of SLC7A11 and GPX4 were the lowest, and the expression trend was stronger than that in the DNS treatment group (G5), suggesting that DNSP enhanced radiation-induced ferroptosis and improved the radiotherapy effect of radiation-resistant tumors; To evaluate the effect of irradiation on the DNA damage ability of tumor tissues after DNSP treatment, fluorescence staining of the collected tumor tissues was performed using the TUNEL method, as Figure 35 shown. The positive apoptosis expression rate in the DNSP combined with radiotherapy group (G6 group) reached 60.3 ± 2.4%, which was 93.3% higher than that in the simple radiotherapy group (G4: 31.2% ± 2.6%) ( p <0.0001), and 35.5% higher than that in the DNS combined with radiotherapy group (G5: 44.5% ± 4.3%) ( p <0.001); at the same time, H&E staining analysis of tumor tissues was performed, as Figure 36 shown. H&E staining showed the characteristics of lung adenocarcinoma of A549-RR cell implanted tumors. Extensive necrosis and foam cell infiltration were observed in the DNSP combined with radiotherapy group, suggesting that the synergistic radiation sensitization effect of DNSP caused the greatest damage to tumors. Immunohistochemistry was used to detect SPARC, GPX4, and PTGS2 proteins in tumors. The positive rate of SPARC decreased to 20.2 ± 3.3% in the G6 group (78.4 ± 5.7% in the G4 group, p < 0.0001), and the positive rate of GPX4 decreased synchronously to 10.7 ± 2.8% (27.2 ± 4.9% in the G4 group, p < 0.001), which was highly consistent with the Western Blot results. The above results indicate that DNSP enhances ferroptosis related to SPARC gene knockout, can effectively reverse the radiation resistance mechanism of radiation-resistant NSCLC, and improve the radiotherapy effect.

[0029] While retaining the radiation sensitization characteristics of DNS, the gadolinium-based nanocomposite DNSP prepared in the embodiments of the present invention can improve the ferroptosis inhibition effect by inhibiting the abnormal activation of SPARC in radiation-resistant cells, thereby reversing the radiation resistance of resistant cells.

[0030] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a gadolinium-based nanocomposite, characterized in that, Comprising the following steps: Synthesize NaYF4:Yb / Er; Use NaYF4:Yb / Er to synthesize NaYF4:Yb / Er@NaGdF4; Use NaYF4:Yb / Er@NaGdF4 to synthesize NaYF4:Yb / Er@NaGdF4@mSiO2; Synthesize gadolinium-based nanocomposite: Add 3-aminopropyltrimethoxysilane to the aqueous solution of NaYF4:Yb / Er@NaGdF4@mSiO2 and stir overnight, wash away the unreacted 3-aminopropyltrimethoxysilane, then add shSPARC plasmid and react at room temperature. The nucleotide sequence of the shSPARC plasmid is as shown in SEQ ID NO:

1. The loading ratio of the aqueous solution of NaYF4:Yb / Er@NaGdF4@mSiO2 to the shSPARC plasmid is mg / mL:μg = 1:

5.

2. The gadolinium-based nanocomposite according to claim 1, wherein The step of synthesizing NaYF4:Yb / Er specifically includes: Add oleic acid and octadecene to YCl3·6H2O, YbCl3·6H2O and ErCl3·6H2O and stir, heat up under an argon atmosphere, repeatedly evacuate until there are no bubbles, continue to heat up, then cool down to room temperature, add a methanol solution containing NaOH and NH4F and gradually heat up, repeatedly evacuate, and then carry out the reaction under programmed temperature rise.

3. The gadolinium-based nanocomposite according to claim 1, wherein The step of using NaYF4:Yb / Er to synthesize NaYF4:Yb / Er@NaGdF4 specifically includes: Add Gd(CF3COO)3 and CF3COONa to oleic acid and octadecene, react after heating, cool down to room temperature and then add NaYF4:Yb / Er, heat up under an argon atmosphere, repeatedly evacuate and then carry out the reaction under programmed temperature rise.

4. The gadolinium-based nanocomposite according to claim 1, wherein The step of using NaYF4:Yb / Er@NaGdF4 to synthesize NaYF4:Yb / Er@NaGdF4@mSiO2 specifically includes: Dissolve CTAB, add NaYF4:Yb / Er@NaGdF4 and disperse it, stir overnight, then add water, NaOH and ethanol, heat in a water bath, slowly add tetraethyl orthosilicate solution for reaction after the temperature is balanced, wash, add an ethanol solution containing NH4NO3, and add anhydrous ethanol to make up the volume, react in a water bath, and then wash the precipitate thoroughly.

5. Gadolinium-based nanocomposites, characterized in that, It is prepared by using the preparation method described in any one of claims 1-4.

6. Use of a gadolinium-based nanocomposite as described in claim 5 in the preparation of a drug for reversing the radiation resistance of NSCLC.

7. The application according to claim 6, characterized in that, The gadolinium-based nanocomposite targets and releases the shSPARC plasmid in an acidic tumor microenvironment, and the shSPARC plasmid affects the ferroptosis process by dynamically regulating the expression of GPX4 / SLC7A11 / PTGS2.

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