A targeted nano-drug for treating radiation-resistant non-small cell lung cancer and a preparation method and application thereof

Abstract

The application discloses a kind of targeted nanomedicine for treating radiation-resistant NSCLC and its preparation method and application, and belongs to the medical technical field, wherein the preparation method of targeted nanomedicine includes the following steps: using NaGdF4 as core, mesoporous silica as carrier, NaGdF4 is coated, and radio-sensitizing nanomaterial is obtained;Radio-sensitizing nanomaterial is co-incubated with PD-1 protein, and the targeted nanomedicine for treating radiation-resistant NSCLC is obtained.The application regulates radiation-resistant NSCLC tumor immune microenvironment by constructing radiosensitizing nano platform and loading PD-1 protein, controls the proliferation, migration and invasion of radiation-resistant NSCLC cells, successfully reverses the radioresistance of NSCLC, and significantly enhances the response of resistant cells to radiotherapy.The method provides valuable insights for developing targeted nanotherapeutic drugs to effectively treat NSCLC.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of medicines, in particular to a targeted nano-drug for treating radiation-resistant NSCLC and a preparation method and application thereof. BACKGROUND

[0002] Lung cancer is the leading cause of cancer morbidity and mortality worldwide, and non-small cell lung cancer (NSCLC) is the most common histological subtype, accounting for about 85% of all new lung cancer cases. According to existing evidence, about 77% of lung cancer patients have indications for radiotherapy. However, the effect of radiotherapy in the treatment of NSCLC is often severely limited due to radiation resistance. Radiation resistance refers to the ability of cancer cells to survive and continue to proliferate after being subjected to X-ray ionizing radiation. This phenomenon not only significantly weakens the effect of radiotherapy, but also greatly increases the risk of tumor recurrence and metastasis. Therefore, it is of great significance to deeply explore the molecular mechanism of radiation resistance, and it is particularly urgent to develop innovative treatment strategies that can reverse radiation resistance.

[0003] The expression of immune checkpoints is one of the key factors affecting tumor occurrence and progression. Among them, PD-1 (programmed cell death protein-1) and its ligand PD-L1 (programmed death ligand 1) are important members of the immune checkpoint family, which participate in the regulation of the interaction between immune cells and tumor cells. In the tumor microenvironment, tumor cells can inhibit the activation of T cells by high expression of PD-L1 binding to PD-1 on the surface of T cells, thus achieving immune escape. This mechanism enables tumor cells to continuously proliferate, spread and invade surrounding tissues. Studies have shown that in radiation-resistant non-small cell lung cancer, PD-L1 expression is significantly up-regulated. This phenomenon is due to the activation of related signaling pathways induced by DNA damage during radiotherapy, which promotes tumor cells to up-regulate PD-L1 as an immune escape mechanism. At the same time, the activation of interferon signaling pathways and the remodeling of tumor microenvironment caused by radiotherapy further strengthen this effect. The high expression of PD-L1 not only enhances the immune escape ability of tumor cells, but also forms a positive feedback loop with radioresistance, which together leads to local control failure and immune therapy resistance after radiotherapy, and constitutes an important mechanism basis for the poor treatment effect of such patients.

[0004] For radiation-resistant NSCLC, the current treatment regimen mainly revolves around two cores of optimizing radiotherapy technology and combined systemic drugs. In terms of radiotherapy itself, the use of precise technologies such as stereotactic body radiotherapy (SBRT) can give a high dose in a short time, effectively overcoming part of the resistance and shortening the course of treatment, but its main drawback is that it has a higher risk of hemorrhage and damage to tumors located in the central region, and the tolerance dose of normal tissues limits its unlimited increase. In terms of combination therapy, concurrent chemoradiotherapy is the standard regimen, which has the advantage of sensitizing radiotherapy and controlling systemic micrometastases, but at the cost of significantly additive toxicities such as radiation esophagitis and pneumonia, and poor patient tolerance; the biggest drawback of combined targeted therapy (for EGFR / ALK genes, etc.) is that it will dramatically increase the risk of life-threatening radiation pneumonitis, and is only suitable for specific mutant populations. The combination of immunotherapy and radiotherapy can destroy tumor cells, release antigens, activate systemic immune responses, and change the tumor microenvironment, but it will also significantly increase the risk and complexity of immune pneumonitis, and the efficacy is uncertain, lacking reliable predictive markers. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of a targeted nanodrug for treating radiation-resistant NSCLC to solve the problems raised in the above background art.

[0006] To achieve the above-mentioned purpose, the embodiments of the present application provide the following technical solutions:

[0007] A preparation method of a targeted nanodrug for treating radiation-resistant NSCLC, comprising the following steps:

[0008] Coating NaGdF4 with mesoporous silica as a carrier to obtain a radiosensitizing nanomaterial;

[0009] Co-incubating the radiosensitizing nanomaterial with PD-1 protein to obtain a targeted nanodrug for treating radiation-resistant NSCLC.

[0010] Further, the step of coating NaGdF4 with mesoporous silica as a carrier to obtain a radiosensitizing nanomaterial specifically comprises:

[0011] Synthesizing NaGdF4 nanocrystals in an oil phase;

[0012] Hydrophilic treatment of the NaGdF4 nanocrystals;

[0013] Coating the hydrophilically treated NaGdF4 nanocrystals with a mesoporous silica shell layer to obtain a radiosensitizing nanomaterial.

[0014] Further, the step of synthesizing NaGdF4 nanocrystals in an oil phase specifically comprises:

[0015] Gd(CF3COO)3, CF3COONa, YCl3.6H2O, YbCl3.6H2O and ErCl3.6H2O were placed in a mixed solution containing oleic acid and octadecene, and reacted by heating to 140-160°C under a protective atmosphere to obtain a reaction solution;

[0016] After the reaction solution was cooled, it was heated to 90-110°C under a protective atmosphere, and then heated to 290-310°C after repeatedly vacuumizing, and then reacted by adding a methanol solution containing NaOH and NH4F to obtain NaGdF4nanocrystals.

[0017] Further, the step of hydrophilic treatment of the NaGdF4nanocrystals specifically includes:

[0018] The NaGdF4nanocrystals were dispersed in cyclohexane to obtain a cyclohexane solution of NaGdF4;

[0019] The cyclohexane solution of NaGdF4was mixed with a tetramethylammonium hydroxide aqueous solution and subjected to ultrasonic treatment, at which time the NaGdF4gradually transferred from the upper organic phase to the lower aqueous phase, the two phases were centrifuged and separated, and the aqueous phase containing the hydrophilic NaGdF4was collected;

[0020] The NaGdF4nanocrystals in the aqueous phase were collected by centrifugation to obtain the hydrophilic treated NaGdF4nanocrystals.

[0021] Further, the step of coating the hydrophilic treated NaGdF4nanocrystals with a mesoporous silica shell to obtain a radiosensitizing nanomaterial specifically includes:

[0022] The hydrophilic treated NaGdF4nanocrystals were dispersed in ultrapure water containing cetyltrimethylammonium bromide, and ultrasonic treatment was performed to form a uniform dispersion;

[0023] The dispersion was heated to 60-80°C under stirring, and then a tetraethyl orthosilicate solution was added dropwise to react to obtain a precipitate;

[0024] The precipitate was washed and dispersed in ethanol, and then an ethanol solution containing NH4NO3was added and reacted in a 50-70°C water bath to obtain a radiosensitizing nanomaterial.

[0025] Further, the temperature of the co-incubation is 3-5°C.

[0026] Further, the mass ratio of the radiosensitizing nanomaterial to the PD-1 protein is 1:(1-10).

[0027] Further, the mass ratio of the radiosensitizing nanomaterial to the PD-1 protein is 1:5.

[0028] Another object of the present application is to provide a targeted nanomedicine for treating radiation-resistant NSCLC prepared by the above preparation method.

[0029] Another object of the present application is to provide the use of the above targeted nanomedicine in the preparation of a drug for combined radiotherapy of radiation-resistant NSCLC.

[0030] The preparation method of the targeted nanomedicine for treating radiation-resistant NSCLC provided by the present application regulates the immune microenvironment of radiation-resistant NSCLC tumor by constructing a precise targeting strategy and a radiosensitizing nano-platform with good biological safety and loading PD-1 protein, controls the proliferation, migration and invasion of radiation-resistant NSCLC cells, successfully reverses the radioresistance of NSCLC, and significantly enhances the response of resistant cells to radiotherapy. This method provides valuable insights for the development of targeted nanotherapeutic drugs for the effective treatment of NSCLC, and provides a better treatment option for patients with radiation-resistant NSCLC. Specifically, the present application uses mesoporous silica as a carrier, core-encapsulates high-atomic-number elements (Gd) to form a radiosensitizing nano material, and covalently connects PD-1 protein on the surface of the radiosensitizing nano material through chemical modification, for specific targeting of tumor cells expressing PD-L1. The targeted nanomedicine is used in combination with radiotherapy, which not only enhances local energy deposition, induces strong oxidative stress, ultimately leading to irreparable DNA damage and cell death, but also remodels the tumor immune microenvironment, increases T cell infiltration in tumor tissue, and inhibits the growth of unirradiated metastatic lesions. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Middle: a is a schematic diagram of PD-1 protein amplification; b is a transmission electron micrograph of PD-1@NP; c is a zeta potential change diagram of NaGdF4 (left), NP (middle), and PD-1@NP (right); d is the hydration particle size of NaGdF4 (left) and PD-1@NP (right); e is the NP loading PD-1 protein content shown by agarose gel electrophoresis; f is the release of PD-1 at different pH.

[0032] Figure 2 Middle: a is a schematic diagram of resistant cell construction; b is the change in cell viability of A549 and A549 RR cells at different times after radiation treatment; c is the apoptosis of A549 and A549 RR cells after radiation treatment; d is the colony formation ability of A549 and A549 RR cells after 6Gy radiation treatment; e is the scratch healing ability of A549 and A549 RR cells after 6Gy radiation treatment; f is the ROS production of A549 and A549 RR cells after 6Gy radiation treatment.

[0033] Figure 3Fig. 6: PD-1@NP treatment inhibits the growth of A549 RR cells in vitro. a, Uptake of PD-1@NP by A549 RR cells; b, Cell viability of A549 RR cells treated with different concentrations of NP; c, Cell viability of A549 RR cells treated with different concentrations of NP after irradiation; d, Colony formation ability of A549 RR cells treated with different methods; e, Immunofluorescence of DNA damage of A549 RR cells treated with different methods; f-i, Secretion of various important immune factors (TNF-a, CXCL10, IL-6 and INF-b) of A549 RR cells treated with different methods.

[0034] Figure 4 Fig. 7: PD-1@NP treatment inhibits the growth of A549 RR cells in vivo. a, Changes in body weight of mice in different treatment groups; b, Changes in tumor volume of mice in different treatment groups; c, HE staining of important organs of mice in different treatment groups; d, Changes in CD4+T cell content in the spleen of mice in different treatment groups; e, Changes in CD80+and CD86+T cell content in the lymph nodes of mice in different treatment groups. + CD8 + + CD86 + DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0036] As a multifunctional drug delivery system, nano-platform (NP) has the advantages of small size, large specific surface area and strong targeting ability, which can significantly improve the bioavailability and therapeutic effect of drugs. In particular, by modifying specific molecules, NP can achieve precise targeting of tumor cells, thereby further improving the specificity and effectiveness of treatment. In the embodiments of the present application, a PD-1 modified nano-drug (PD-1@NP) is developed, which can specifically target tumor cells expressing PD-L1, restore the activation of T cells to enhance the overall effect of cancer treatment, and provide a new synergistic treatment strategy for NSCLC. The embodiments of the present application aim to systematically evaluate the therapeutic potential of PD-1@NP in NSCLC and reveal its molecular mechanism through in vitro and in vivo experiments, thereby providing a new theoretical basis for clinical treatment.

[0037] ​​Specifically, the embodiment of the present application combines radiosensitizing nanomaterials with PD-1 protein. The surface of the nanomaterials is modified by PD-1 to specifically recognize the tumor cell surface antigen ligand PD-L1, realize accurate guidance, and maximize the enrichment of radiosensitizing nanomaterials in the tumor, greatly enhancing the killing efficiency of radiotherapy on tumor cells, especially triggering stronger and more extensive immunogenic cell death, thereby enhancing the killing of radiotherapy on tumors. At the same time, reverse the immune suppression microenvironment, increase the number of T cells, and eliminate immune suppression cells, reducing the risk of radiation damage to surrounding healthy lung tissue, esophagus, etc., providing a possibility for solving the core toxicity problem of combined therapy. The combination of radiosensitizing nanomaterials and PD-1 protein constructs a powerful precise sensitization-strong activation-system attack treatment system. It not only optimizes the shortcomings of traditional treatment at every link, but also through the synergy of multiple mechanisms, can achieve deep and persistent control of radiation-resistant non-small cell lung cancer.

[0038] Embodiment 1: The embodiment provides a preparation method of a targeted nanodrug for treating radiation-resistant NSCLC, comprising the following steps:

[0039] S1, taking NaGdF4 as the core and mesoporous silica (mSiO2) as the carrier, coating NaGdF4 to obtain radiosensitizing nanomaterials (NP); specifically as follows:

[0040] Gd(CF3COO)3, 44 mg of CF3COONa, 210 mg of YCl3.6H2O, 80 mg of YbCl3.6H2O and 10 mg of ErCl3.6H2O were placed in a three-necked flask containing 10 mL of oleic acid and 10 mL of octadecene. The mixture was slowly heated to 150°C under argon and maintained for 30 min. Then the mixture was cooled to room temperature and rapidly heated to 100°C under argon. The mixture was repeatedly vacuumed and programmed to 300°C. 5 mL of a methanol solution containing NaOH and NH4F was rapidly added and reacted for 1 h. After the reaction was completed, the obtained product NaGdF4nanocrystals were washed with acetone and cyclohexane for 3-5 times, and dispersed in 10 mL of cyclohexane for standby. Since the NaGdF4nanocrystals synthesized by the oil phase method are hydrophobic, they need to be surface modified to be hydrophilic to facilitate subsequent packaging. Specifically, 10 mL of 0.1M tetramethylammonium hydroxide aqueous solution was prepared and added to the above 10 mL cyclohexane solution containing NaGdF4nanocrystals, and ultrasonic treatment was performed for 30 min. At this time, NaGdF4gradually transferred from the upper organic phase to the lower aqueous phase, indicating that the hydrophilization was successful. The two phases were separated by low-speed centrifugation, and the aqueous phase containing hydrophilic NaGdF4was collected. The NaGdF4nanocrystals in the aqueous phase were collected by high-speed centrifugation (12000 rpm, 10 min), and washed with ultrapure water for 2-3 times. The above hydrophilic NaGdF4nanocrystals were dispersed in 80 mL of ultrapure water containing 0.1 g of cetyltrimethylammonium bromide, and ultrasonic treatment was performed to form a uniform dispersion. Under strong stirring (1000 rpm), the dispersion system was heated to 70°C, and 1 mL of tetraethyl orthosilicate solution was added dropwise, and the reaction was continued for 2 h. The precipitate was washed with ethanol and distilled water and dispersed in 10 mL of ethanol, then an ethanol solution containing NH4NO3was added, and anhydrous ethanol was added to 50 mL, and reacted at 60°C water bath for 2 h. After the reaction was completed, the precipitate was washed with ethanol and distilled water, and the radiosensitizing nanomaterial NaGdF4@mSiO2(NP) was obtained. The mSiO2shell not only improves the biocompatibility of the material, but also provides an effective carrier for the subsequent loading of PD-1 protein; at the same time, Gd metal is a high atomic number metal, which can enhance local energy deposition and induce strong oxidative stress, ultimately leading to irreparable DNA damage and cell death.

[0041] S2, 1 μg of the above radiosensitizing nanomaterial NP was mixed with the amplified PD-1 protein (as shown in a of Figure 1 ) at different mass ratios (1:0, 1:1, 1:5, 1:10, 1:20) and co-incubated at 4°C on a shaking table to obtain a targeted nanodrug PD-1@NP for treating radiation-resistant NSCLC.

[0042] Example 2: Transmission electron microscopy (as shown in Figure 1As shown in b), observation of the PD-1@NP prepared in Example 1 (NP to PD-1 protein mass ratio of 1:5) confirmed that the synthesized nanoparticles were of uniform size and regular morphology. Zeta potential (as shown in b) Figure 1 (as shown in c) and dynamic light scattering (such as Figure 1 The results (as shown in d) further validate the successful construction of PD-1@NP.

[0043] To optimize the loading conditions of PD-1 protein on NP, this embodiment of the invention uses agarose gel electrophoresis to measure the loading of 1 mg·mL⁻¹ PD-1 protein on NP. -1 The stability of NP-loaded with different masses of PD-1 protein was evaluated. The results showed (e.g.) Figure 1 As shown in Figure e), when the mass ratio of NP to PD-1 is 1:5, the protein is completely bound, and no free bands appear. However, when the ratio exceeds this threshold, significant protein leakage occurs, indicating that the loading has reached saturation. Therefore, this optimal loading ratio (NP:PD-1=1:5) was used to prepare PD-1@NP for subsequent experiments. Furthermore, to evaluate the responsive release behavior of PD-1@NP in the tumor microenvironment, the embodiments of this invention investigated its PD-1 protein release kinetics under different pH conditions, such as... Figure 1 As shown in f, the release rate of PD-1 under acidic conditions (pH=5.5) is significantly higher than that under neutral conditions (pH=7.4), indicating that the NP nanoplatform has good tumor microenvironment responsiveness and can specifically release PD-1 protein at the tumor site, providing an effective delivery basis for targeted immunotherapy.

[0044] Example 3: Using A549 non-small cell lung cancer cells as a model, a radiation-resistant cell line was constructed using multiple low-dose X-ray irradiation methods. Specifically, cells in the logarithmic growth phase were subjected to five separate 6 Gy X-ray irradiations (e.g.,...). Figure 2 (As shown in a). Cell viability was detected by CCK-8 assay (e.g., ...). Figure 2 (as shown in b), flow cytometry analysis of apoptosis (e.g.) Figure 2 (as shown in c) and clonogenic experiments (such as Figure 2 As shown in d), under the same radiation dose, the survival fraction of the screened cells was significantly higher than that of the parent cells, indicating that their radiation resistance was significantly enhanced. Therefore, they were named A549 RR cells (radiation-resistant A549 cells).

[0045] Further cell scratch assays revealed that A549 RR cells exhibited stronger migration and healing capabilities after irradiation (e.g., Figure 2 (As shown in e). Furthermore, the cells also exhibited significantly enhanced antioxidant stress resistance after irradiation (e.g., Figure 2The above results show that the A549 RR cell line with stable radiation resistance phenotype is successfully established by the embodiment of the application, which provides a reliable experimental model for subsequent research, and on this basis, the following experiments are carried out.

[0046] Example 4: In order to evaluate the uptake of A549 RR cells to PD-1@NP, the embodiment of the application carries out cell uptake experiment by using FITC-labeled PD-1@NP. As shown in FIG. 4a, with the extension of time from 0 h to 8 h, the green fluorescence signal in the cells gradually increases, and the Merge image further shows that PD-1@NP accumulates in the cells in a time-dependent manner, indicating that the cells have effective and sustained uptake ability to the nanoparticles. Figure 3 Figure 3 Figure 3 Figure 3 Figure 3 Figure 3 ​​​​​TNF-a and IL-6 levels in the control and NP groups were not significantly different under non-irradiation condition; while the contents of TNF-a, CXCL10, IL-6 and IFN-b in the PD-1@NP group were significantly increased, suggesting that the introduction of PD-1 could activate the expression of some immune-related factors under non-irradiation condition. After X-ray irradiation, the expression levels of the above immune factors in the NP group were significantly higher than those in the control and non-irradiated NP groups, indicating that radiation combined with NP treatment could further induce the release of immune factors, suggesting that the degree of cellular immune damage was exacerbated. Notably, the secretion levels of TNF-a and IL-6 in the PD-1@NP group were particularly prominent after irradiation, indicating that PD-1@NP could effectively promote the release of pro-inflammatory factors under the synergistic effect of irradiation. In summary, PD-1@NP can activate and amplify the systemic immune response in the tumor microenvironment by enhancing the secretion of multiple key immune factors in A549 RR cells, providing a potential immune regulation mechanism for reversing radioresistance.

[0047] Example 5: To evaluate the in vivo anti-tumor effect of PD-1@NP, the present embodiment constructed an A549-RR radioresistant tumor model in severe combined immunodeficiency (SCID) mice, which focused on the radioresistance mechanism urgently needed to be solved in the treatment of non-small cell lung cancer (NSCLC). The research results showed that PD-1@NP exhibited significant therapeutic potential in combination with radiotherapy. In the non-irradiation group, PD-1@NP treatment had a certain inhibitory effect on drug-resistant tumor growth, suggesting that it could effectively regulate tumor proliferation through PD-1-mediated targeting (such as Figure 4 Figures 2a and 2b). After combined radiotherapy, the therapeutic effect was more significant: the simple radiotherapy group showed a decrease in tumor volume at the initial stage, but rapidly relapsed after the 5th day; in contrast, the PD-1@NP combined radiotherapy group not only enhanced the initial therapeutic effect, but also effectively inhibited the regrowth of tumors, indicating that this strategy could significantly improve the intensity and persistence of radiotherapy treatment response. In addition, hematoxylin-eosin staining histological analysis of major organs showed that none of the treatment groups caused obvious tissue damage (such as Figure 4 Figure 2c), suggesting that PD-1@NP had good biological safety at an effective dose. Immunophenotyping analysis further revealed its potential mechanism of action. After irradiation, the proportion of CD4 + CD8 + T cells in the spleen of the PD-1@NP group was significantly higher than that in other groups (such as Figure 4 Figure 2d), suggesting that this treatment might promote the generation or recruitment of this T cell subset; at the same time, the proportion of CD4 + CD8 + cells (dendritic cell maturation markers) in the lymph nodes of this group also increased significantly (such as Figure 4PD-1@NP combined with radiotherapy can enhance the activation and immune function of antigen-presenting cells. In summary, the examples of the present application demonstrate that PD-1@NP can effectively reverse the radioresistance phenotype of NSCLC by enhancing the PD-1 targeting and immune regulation function, and inhibit the progression of tumor in vivo. This strategy not only widens the radiotherapy treatment window of NSCLC, but also provides a new targeted treatment path for overcoming intrinsic drug resistance of tumor.

[0048] The above is based on the ideal embodiment of the present application. Through the above description, relevant personnel can make various changes and modifications without deviating from the technical idea of the present application. The technical scope of the present application is not limited to the contents of the specification.

Claims

1. A method for preparing a targeted nanodrug for treating radiation-resistant NSCLC, characterized in that, Includes the following steps: Using NaGdF4 as the core and mesoporous silica as the carrier, NaGdF4 was coated to obtain radiosensitizing nanomaterials. By co-incubating radiosensitizing nanomaterials with PD-1 protein, targeted nanomedicines for the treatment of radiation-resistant NSCLC were obtained. The preparation steps of the radiosensitizing nanomaterial include: NaGdF4 nanocrystals were synthesized in the oil phase. Hydrophilization treatment of NaGdF4 nanocrystals; A radiosensitizing nanomaterial was obtained by coating a mesoporous silica shell onto hydrophilicated NaGdF4 nanocrystals. The method for coating a mesoporous silica shell onto hydrophilically treated NaGdF4 nanocrystals includes: The hydrophilized NaGdF4 nanocrystals were dispersed in ultrapure water containing hexadecyltrimethylammonium bromide and ultrasonically treated to form a uniform dispersion. The dispersion was heated to 60-80℃ with stirring, and then tetraethyl orthosilicate solution was added dropwise to react and obtain a precipitate. The precipitate was washed and dispersed in ethanol, and then an ethanol solution containing NH4NO3 was added. The mixture was reacted in a water bath at 50-70℃ to obtain radiosensitizing nanomaterials.

2. The method for preparing the targeted nano-drug for treating radiation-resistant NSCLC according to claim 1, characterized in that, The steps for synthesizing NaGdF4 nanocrystals in the oil phase specifically include: Gd(CF3COO)3, CF3COONa, YCl3·6H2O, YbCl3·6H2O and ErCl3·6H2O were placed in a mixture containing oleic acid and octadecene, and heated to 140-160℃ under a protective atmosphere to react and obtain a reaction solution. After cooling the above reaction solution, the temperature was raised to 90-110℃ under a protective atmosphere. After repeated vacuuming, the temperature was raised to 290-310℃. Then, a methanol solution containing NaOH and NH4F was added to react and obtain NaGdF4 nanocrystals.

3. The method for preparing the targeted nano-drug for treating radiation-resistant NSCLC according to claim 1, characterized in that, The steps for hydrophilization treatment of NaGdF4 nanocrystals specifically include: NaGdF4 nanocrystals were dispersed in cyclohexane to obtain a cyclohexane solution of NaGdF4. A cyclohexane solution of NaGdF4 was mixed with an aqueous solution of tetramethylammonium hydroxide and subjected to ultrasonic treatment. During this process, NaGdF4 gradually transferred from the upper organic phase to the lower aqueous phase. The two phases were separated by centrifugation, and the aqueous phase containing hydrophilic NaGdF4 was collected. NaGdF4 nanocrystals were obtained by centrifugation to collect the aqueous phase, and then hydrophilized NaGdF4 nanocrystals were obtained.

4. The method for preparing the targeted nano-drug for treating radiation-resistant NSCLC according to claim 1, characterized in that, The co-incubation temperature is 3-5℃.

5. The method for preparing the targeted nano-drug for treating radiation-resistant NSCLC according to claim 1, wherein, The mass ratio of the radiosensitizing nanomaterial to PD-1 protein is 1:(1-10).

6. The method for preparing the targeted nano-drug for treating radioresistant NSCLC according to claim 5, characterized in that, The mass ratio of the radiosensitizing nanomaterial to PD-1 protein is 1:

5.

7. A targeted nanomedicine for treating radiation-resistant NSCLC prepared by the method described in any one of claims 1-6.

8. The use of the targeted nanomedicine as described in claim 7 in the preparation of a medicament for combined radiotherapy of radiation-resistant NSCLC.

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