A pH-responsive nucleic acid delivery composite material for radiosensitizing nasopharyngeal carcinoma, its preparation method and application

Abstract

This invention discloses a pH-responsive nucleic acid delivery composite material for radiosensitization in nasopharyngeal carcinoma, its preparation method, and its application, belonging to the field of biomedical technology. The composite material consists of a composite carrier LZMA formed by CoAl-LDH, ZIF-8, MIL-101(Fe), and AgNO3, further loaded with Antago-miR-205-5p to obtain the nucleic acid delivery composite material A205-LZMA. Compared with existing single-carrier systems, this invention adopts a composite design of "layered nanosheets + two types of functional nanoparticles + active ionic components + nucleic acid drugs." By first preparing CoAl-LDH, ZIF-8, and MIL-101(Fe), and then performing composite formation and nucleic acid loading, the entire process route is clear, the steps are well-defined, and it is easy to repeat. By introducing ZIF-8 into the composite system, this invention endows the material with responsive release characteristics adapted to the acidic tumor microenvironment, which is beneficial to improving the local release efficiency of nucleic acid drugs. This invention integrates CoAl-LDH, ZIF-8, MIL-101(Fe) and AgNO3 in the same material, enabling the material to take into account the multi-component functional basis of nucleic acid delivery, iron source introduction and active ion synergy.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, specifically relating to a pH-responsive nucleic acid delivery composite material for radiosensitizing nasopharyngeal carcinoma, its preparation method, and its application. Background Technology

[0002] Nasopharyngeal carcinoma (NPC) is a common malignant tumor in southern my country and Southeast Asia. Radiotherapy remains one of its core treatment methods, but radiotherapy tolerance is a significant cause of local recurrence, distant metastasis, and poor prognosis. Current research indicates that miR-205-5p is abnormally upregulated in NPC, particularly in radiotherapy-tolerant cells. Targeting this pathway is considered a feasible approach to improve radiosensitivity in NPC. However, Antago-miR-205-5p, as a nucleic acid drug, is easily degraded in body fluids, and the enrichment efficiency of naked nucleic acids at tumor sites is limited, restricting its practical application as a radiosensitization strategy. Currently, common nucleic acid delivery systems mainly include liposomes, polymer nanoparticles, and some inorganic nanocarriers. While these systems can improve nucleic acid delivery efficiency to some extent, most still primarily focus on a single loading function, making it difficult to simultaneously achieve stable protection, local release, and radiotherapy synergy. Specifically, they have the following drawbacks: 1. Naked Antago-miR-205-5p has poor stability in the serum environment and is easily degraded after entering the body, resulting in a low proportion of the effective payload reaching the tumor site.

[0003] 2. Existing nucleic acid delivery systems are mostly based on single loading and lack structural designs that can trigger release in the acidic microenvironment of tumors. The release location and timing are not ideal.

[0004] 3. Existing materials often only solve the delivery problem and cannot simultaneously provide the iron source, active ions or other synergistic modules required for radiotherapy sensitization, resulting in insufficient overall material functionality.

[0005] 4. In existing multi-component nanosystems, the proportions of each component are usually only used as experimental conditions, lacking clear definition of key ratios, resulting in insufficient reproducibility and synergistic stability in preparation. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a pH-responsive nucleic acid delivery composite material for radiosensitization in nasopharyngeal carcinoma, its preparation method, and its application. The composite material is formed by CoAl-LDH, ZIF-8, MIL-101(Fe), and AgNO3 to form a composite carrier LZMA, which is further loaded with Antago-miR-205-5p to obtain the nucleic acid delivery composite material A205-LZMA.

[0007] The technical solution of this invention mainly includes the following two aspects: firstly, the synthesis method of the composite material, namely the order of introduction, compounding method and preparation steps of CoAl-LDH, ZIF-8, MIL-101(Fe), AgNO3 and Antago-miR-205-5p; secondly, the key component ratio of the composite material, namely the compounding ratio between CoAl-LDH, ZIF-8, MIL-101(Fe) and AgNO3, and the loading ratio between Antago-miR-205-5p and the composite carrier.

[0008] In one aspect, the present invention provides a pH-responsive nucleic acid delivery composite material, the composite material comprising a layered host material CoAl-LDH, a first nanoparticle component ZIF-8, a second nanoparticle component MIL-101(Fe), an active ion source component AgNO3, and a nucleic acid drug Antago-miR-205-5p loaded in the composite carrier.

[0009] In this composite system, CoAl-LDH serves as the layered main framework, ZIF-8 and MIL-101(Fe) are introduced as nanoparticle components, AgNO3 is further introduced as a functional component, and Antago-miR-205-5p is loaded onto the aforementioned composite system, thereby forming the A205-LZMA composite material.

[0010] To address the first problem—that naked Antago-miR-205-5p has poor stability in the serum environment, is easily degraded after entering the body, and results in a low proportion of the effective payload reaching the tumor site—this invention does not employ the direct administration of naked nucleic acid drugs. Instead, it first constructs a composite carrier composed of CoAl-LDH, ZIF-8, MIL-101(Fe), and AgNO3, and then loads Antago-miR-205-5p into the composite carrier to form a nucleic acid drug-loaded composite material.

[0011] To address the second problem—that existing nucleic acid delivery systems primarily rely on single-load transport and lack structural designs capable of triggering release in the acidic tumor microenvironment, leading to unsatisfactory release locations and timing—this invention introduces ZIF-8 as an acid-responsive component into the composite system. ZIF-8 is not used alone as a carrier, but rather forms a hierarchical composite structure with CoAl-LDH and MIL-101(Fe), enabling the material to exhibit responsive release characteristics in the acidic tumor microenvironment. The ZIF-8 is composed of Zn²⁺. +The node and the deprotonated 2-methylimidazolium ligand are formed through Zn-N coordination. In a near-physiologically neutral environment, the ZIF-8 framework remains relatively stable; however, when the carrier enters a tumor-associated weakly acidic environment, especially after endocytosis into acidic compartments such as endosomes or lysosomes, the nitrogen-containing coordination sites of the 2-methylimidazolium ligand gradually undergo protonation, thus enhancing its coordination with Zn²⁺. + The coordination ability of Zn decreases, leading to the weakening and gradual breakage of the Zn-N coordination bonds, which in turn causes the ZIF-8 framework to disintegrate. This process removes the physical protection and diffusion restriction of the inner Antago-miR-205-5p complex by the ZIF-8 shell, promoting the release of nucleic acid drugs in the acidic intracellular environment, thereby achieving pH-responsive delivery. In the examples, the increased release rate observed at pH 5.5 can serve as experimental evidence for the above-mentioned acid-responsive disintegration and release mechanism.

[0012] To address the third problem—that existing materials often only solve the delivery issue and cannot simultaneously provide the iron source, active ions, or other synergistic modules required for radiotherapy sensitization, resulting in insufficient overall material functionality—this invention introduces MIL-101(Fe) and AgNO3 into the same material system. MIL-101(Fe) serves as the iron-containing functional component, while AgNO3 acts as the active ion source component. After introducing AgNO3 into the composite hybrid material system, silver primarily functions as an Ag... + The ions bind to coordinating sites on the support surface, forming a stable anchor, thereby preventing free Ag from escaping. + Rapid diffusion or excessive accumulation in the blood or body fluids reduces the potential risk of cytotoxicity. Locally released Ag under acidic or reducing conditions in the tumor microenvironment... + It can participate in reactions such as ROS generation, enhanced lipid peroxidation, and DNA damage, thereby amplifying the cell damage effect under combined conditions of radiotherapy or ferroptosis, so that the material, in addition to nucleic acid delivery, also has the multi-component basis required for radiotherapy synergy.

[0013] To address the fourth problem, namely that the component ratios in existing multi-component nanosystems are often treated as experimental conditions and lack clear definition of key ratios, resulting in insufficient reproducibility and synergistic stability in preparation, this invention provides clear definition of the component ratios and nucleic acid loading ratios in the composite material.

[0014] In a preferred embodiment, the mass ratio of CoAl-LDH, ZIF-8, MIL-101(Fe) to AgNO3 in the composite material is 5:1:1:1.

[0015] Furthermore, the loading ratio of Antago-miR-205-5p to LZMA was screened by mass ratio, with a screening range of 1:5, 1:10, 1:20, 1:40, 1:60, 1:80 and 1:100, among which the preferred mass ratio was 1:60 (w / w).

[0016] In another aspect, the present invention also provides a method for preparing the pH-responsive nucleic acid delivery composite material, the method comprising: preparing CoAl-LDH nanosheets, ZIF-8 nanoparticles and MIL-101(Fe) nanoparticles respectively; then compounding CoAl-LDH, ZIF-8, MIL-101(Fe) with AgNO3 at a predetermined mass ratio to obtain LZMA; subsequently introducing Antago-miR-205-5p during the compounding process to obtain A205-LZMA.

[0017] Preferably, the composite process is carried out in the following order: first, a premixed system of CoAl-LDH, ZIF-8, and MIL-101(Fe) is formed; then, AgNO3 is introduced; and simultaneously, a predetermined amount of Antago-miR-205-5p is added during this composite step, thereby forming a composite material loaded with nucleic acid drugs. By limiting the order of introduction, the composite path, loading path, and preparation boundary of the material are more clearly defined.

[0018] The preparation of the CoAl-LDH nanosheets includes: taking 20.0 mL of a mixed metal nitrate solution, wherein the concentration of Co(NO3)2 is 0.032 M and the concentration of Al(NO3)3 is 0.018 M; simultaneously adding the mixed metal nitrate solution and 0.25 M NaOH solution to 20.0 mL of NaNO3 solution containing 23 vol% formamide, wherein the concentration of NaNO3 solution is 0.018 M; maintaining the pH of the system at 9.8–10.2 by adjusting the NaOH addition rate under magnetic stirring at 80 °C; after the metal salt solution is added, continuing to stir the reaction at 80 °C for 20 min, then rapidly cooling to room temperature, and obtaining the CoAl-LDH nanosheet dispersion system by centrifugation, washing, and redispersing. Under conditions of approximately pH 10, Co... 2+ And Al 3+ Synergistic hydrolysis and co-precipitation occur, forming positively charged layers composed of interconnected metal-oxygen octahedra. Formamide reduces electrostatic attraction and hydrogen bonding between adjacent layers by forming hydrogen bonds with hydroxyl groups on the layer surface and regulating the solvation environment of the reaction medium. This inhibits the vertical stacking and secondary aggregation of the layers, ensuring that the generated layered double hydroxides mainly maintain a low-stack or ultrathin nanosheet structure. The 20-minute period mainly corresponds to the limited growth and structure formation stage after nucleation, eliminating the need for the long hydrothermal aging process commonly used in the preparation of traditional bulk LDH.

[0019] The preparation of the ZIF-8 nanoparticles includes: weighing 734 mg of Zn(NO3)2·6H2O and 811 mg of 2-methylimidazole, and dissolving each in 50 mL of methanol; mixing the two solutions and stirring briefly, centrifuging to collect the white precipitate; washing three times with methanol and drying overnight in an oven at 60 °C to obtain ZIF-8 nanoparticles.

[0020] The preparation of the MIL-101(Fe) nanoparticles includes: dissolving 0.675 g of FeCl3·6H2O in 30 mL of N,N-dimethylformamide, then adding 0.206 g of terephthalic acid and dissolving it; transferring the resulting solution to a 100 mL polytetrafluoroethylene-lined reactor and reacting at 110 °C for 20 h; washing the product with methanol, then stirring it in fresh methanol for 2 days, centrifuging, washing it again, and drying it to obtain MIL-101(Fe) nanoparticles.

[0021] The preparation of LZMA includes: first, dispersing CoAl-LDH, ZIF-8 and MIL-101(Fe) in ethanol, and controlling the final composite ratio of CoAl-LDH:ZIF-8:MIL-101(Fe):AgNO3=5:1:1:1; after stirring magnetically at 500 rpm for 2 h at room temperature, adding AgNO3 and continuing to stir for 30 min; collecting the precipitate by centrifugation at 8000 rpm for 5 min, and freeze-drying to obtain the LZMA composite material.

[0022] The preparation of A205-LZMA includes: following the same compounding method as LZMA, CoAl-LDH, ZIF-8 and MIL-101(Fe) are first dispersed in ethanol and stirred. In this compounding step, AgNO3 and a predetermined amount of Antago-miR-205-5p are added simultaneously, and stirring is continued for 30 min. The precipitate is collected by centrifugation at 8000 rpm for 5 min, and then lyophilized to obtain A205-LZMA loaded with nucleic acid drugs.

[0023] In a preferred embodiment, the specific amounts of CoAl-LDH, ZIF-8, MIL-101(Fe), AgNO3, and Antago-miR-205-5p can be scaled up or down proportionally according to the target preparation scale. The aforementioned four-component composite ratio of 5:1:1:1 and the nucleic acid loading ratio of 1:60 (w / w) are key parameters in the preferred embodiment.

[0024] In another aspect, the present invention also provides the application of the A205-LZMA in the preparation of nucleic acid delivery materials for radiosensitization in nasopharyngeal carcinoma radiotherapy. The application includes using the prepared A205-LZMA as a nucleic acid delivery composite material for radiosensitization in nasopharyngeal carcinoma radiotherapy.

[0025] Compared with existing single-carrier systems, this invention employs a composite design of "layered nanosheets + two types of functional nanoparticles + active ionic components + nucleic acid drugs," which has the following beneficial effects: This invention first prepares CoAl-LDH, ZIF-8 and MIL-101(Fe), and then performs complexation and nucleic acid loading, making the entire process route clear, the steps well-defined, and easy to repeat.

[0026] This invention introduces ZIF-8 into the composite system, enabling the material to possess responsive release characteristics adapted to the acidic microenvironment of tumors, which is beneficial to improving the local release efficiency of nucleic acid drugs.

[0027] This invention integrates CoAl-LDH, ZIF-8, MIL-101(Fe) and AgNO3 in the same material, enabling the material to take into account the multi-component functional basis of nucleic acid delivery, iron source introduction and active ion synergy.

[0028] This invention explicitly defines key parameters such as CoAl-LDH:ZIF-8:MIL-101(Fe):AgNO3=5:1:1:1 and Antago-miR-205-5p:LZMA=1:60 (w / w), which is beneficial to improving the consistency and repeatability of material preparation.

[0029] The A205-LZMA developed in this invention can be used as a nucleic acid delivery composite material for radiosensitizing nasopharyngeal carcinoma radiotherapy. Attached Figure Description

[0030] Figure 1 The results show the nucleic acid loading of A205-LZMA under different Antago-miR-205-5p:LZMA mass ratios in Example 1. Figure 2 Serum stability results of A205-LZMA prepared in Example 1; Figure 3 The release curve of A205-LZMA prepared in Example 1; Figure 4 The results show the effect of A205-LZMA prepared in Example 1 on the activity of nasopharyngeal carcinoma cells. Figure 4 A represents the cell viability assay results of C666-1R cells under different concentrations of LZMA or A205-LZMA treatment and with or without combined irradiation. Figure 4 B represents the cell viability test results of HONE1R cells under different concentrations of LZMA or A205-LZMA treatment and whether or not they were treated with combined irradiation. Figure 5The results show the intracellular miR-205-5p expression level detection after delivery of A205-LZMA prepared in Example 1. Figure 5 A represents the results of detecting the relative expression level of miR-205-5p in C666-1R cells. Figure 5 B represents the results of the relative expression level detection of miR-205-5p in HONE1R cells; Figure 6 The fluorescence microscopy results of A205-LZMA prepared in Example 1 are shown below. Figure 6 A shows the fluorescence microscopy results of Cy3-labeled nucleic acid delivery in C666-1R cells at 0 h and 24 h. Figure 6 B shows the fluorescence microscopy observation results of Cy3-labeled nucleic acid delivery in HONE1R cells at 0 h and 24 h; Figure 7 The flow cytometry uptake results of A205-LZMA prepared in Example 1 are shown below. Figure 7 A represents the flow cytometry results of Cy3-labeled nucleic acid delivery in C666-1R cells. Figure 7 B represents the flow cytometry results of Cy3-labeled nucleic acid delivery in HONE1R cells; Figure 8 Fe in nasopharyngeal carcinoma cells of each group in Example 2 2+ And GSH level test results, among which Figure 8 A represents Fe in C666-1R and HONE1R cells. 2+ Horizontal test results Figure 8 B represents the GSH level detection results in C666-1R and HONE1R cells; Figure 9 The results of lipid peroxidation level detection in each group of nasopharyngeal carcinoma cells in Example 2 are shown below. Figure 9 A is a flow cytometry image showing the lipid peroxidation level in C666-1R cells. Figure 9 B is a flow cytometry image showing the lipid peroxidation level in HONE1R cells. Figure 9 C represents the statistical results of lipid peroxidation levels in C666-1R cells. Figure 9 D represents the statistical results of lipid peroxidation levels in HONE1R cells; Figure 10 The results of the nasopharyngeal carcinoma cell death analysis in each group in Example 2 are shown below. Figure 10 Image A shows a flow cytometry image of C666-1R cells stained with Annexin. Figure 10 B is a flow cytometry image of HONE1R cells stained with Annexin. Figure 10 C represents the statistical results of the C666-1R cell mortality rate. Figure 10D represents the statistical results of HONE1R cell mortality; Figure 11 The results of EdU proliferation detection in each group of nasopharyngeal carcinoma cells in Example 2 are shown below. Figure 11 Image A shows the EdU flow cytometry analysis of C666-1R cells. Figure 11 B is a flow cytometry image of HONE1R cells analyzed using EdU. Figure 11 C represents the statistical result of the proportion of EdU-positive cells in C666-1R cells. Figure 11 D represents the statistical result of the proportion of EdU-positive cells in HONE1R cells; Figure 12 The results of DNA damage detection in nasopharyngeal carcinoma cells in each group in Example 2 are shown below. Figure 12 A shows representative images from the C666-1R and HONE1R cell comet experiments. Figure 12 B represents the statistical results of the comet tail length of C666-1R cells. Figure 12 C represents the statistical results of the comet tail length of HONE1R cells; Figure 13 The results of HUVEC cell tube formation experiments after treatment in each group in Example 2 are shown below. Figure 13 Image A shows a representative image of HUVEC cell tube formation under C666-1R and HONE1R cell-related treatments. Figure 13 B represents the statistical results of HUVEC cell tube formation rate under C666-1R cell-related treatment conditions. Figure 13 C represents the statistical results of HUVEC cell tube formation rate under HONE1R cell-related treatment conditions; Figure 14 This is a schematic diagram of the construction of each Hu-PBMC-PDX nasopharyngeal carcinoma humanized xenograft model in Example 2, along with the drug administration and sampling time points; Figure 15 and Figure 16 for Figure 14 Results of primary and metastatic tumor growth in each treatment group, including Figure 15 This is an overall observational image showing the location and growth of primary and metastatic tumors in mice of each treatment group. Figure 16 A represents the observation results of primary tumor samples taken from each treatment group. Figure 16 B represents the observation results of metastatic tumor samples taken from each treatment group; Figure 17 for Figure 14 Tumor volume growth curves of primary and metastatic tumors in each treatment group, among which Figure 17 A represents the curve showing the change in the volume of the primary tumor. Figure 17 B represents the curve showing the change in the volume of metastatic tumors; Figure 18 for Figure 14 Statistical results of final tumor weight of primary and metastatic tumors in each treatment group, among which... Figure 18 A represents the statistical results of the primary tumor weight. Figure 18 B represents the statistical results of metastatic tumor weight; Figure 19 , Figure 20 and Figure 21 for Figure 14 Histological examination results of primary tumors in each treatment group, among which Figure 19 Representative immunofluorescence images of γH2AX and CDKN1A in the primary tumor tissues of each treatment group are shown. Figure 19 The top three rows are γH2AX, DAPI and the merged graph, respectively; the bottom three rows are CDKN1A, DAPI and the merged graph, respectively. Figure 20 The images show the HE staining and Ki67 immunohistochemical staining results of primary tumor tissues in each treatment group. The top row shows representative HE staining images, and the bottom row shows representative Ki67 immunohistochemical staining images. Figure 21 A represents the statistical results of γH2AX fluorescence intensity. Figure 21 B represents the statistical results of CDKN1A fluorescence intensity. Figure 21 C represents the statistical results of Ki67 staining scores; Figure 22 for Figure 14 Immunohistochemical detection results of CRT and HMGB1 in the primary tumor tissues of each treatment group, among which Figure 22 The top row (A) shows representative immunohistochemical staining images of calreticulin (CRT). Figure 22 The bottom row of A shows representative immunohistochemical staining images of high-mobility group box 1 (HMGB1); Figure 22 B represents the statistical results of CRT semi-quantitative scoring. Figure 22 C represents the statistical results of the HMGB1 semi-quantitative scoring. Figure 23 for Figure 14 CD3 in metastatic tumor tissues of each treatment group + CD4 + Results of T-cell infiltration ratio detection, including Figure 23 A shows the CD3 / CD4 ratio detected by flow cytometry in metastatic tumor tissue from the control group. Figure 23 B shows the CD3 / CD4 ratio in the metastatic tumor tissue of the PD-1 monoclonal antibody group detected by flow cytometry. Figure 23 C shows the CD3 / CD4 ratio in metastatic tumor tissue from the A205-LZMA group using flow cytometry. Figure 23 D is a flow cytometry graph showing the CD3 / CD4 ratio in metastatic tumor tissue from the PD-1 monoclonal antibody + A205-LZMA group. Figure 23 E is CD3 + CD4 + Statistical results of T cell infiltration ratio; Figure 24 for Figure 14CD3 in metastatic tumor tissues of each treatment group + CD8 + Results of T-cell infiltration ratio detection, including Figure 24 A shows the CD3 / CD8 ratio detected by flow cytometry in metastatic tumor tissue from the control group. Figure 24 B is a flow cytometry image of CD3 / CD8 ratio in metastatic tumor tissue from the PD-1 monoclonal antibody group. Figure 24 C shows the CD3 / CD8 ratio in metastatic tumor tissue from the A205-LZMA group detected by flow cytometry. Figure 24 D is a flow cytometry result of CD3 / CD8 ratio in metastatic tumor tissue from the PD-1 monoclonal antibody + A205-LZMA group. Figure 24 E is CD3 + CD8 + Statistical results of T cell infiltration ratio; Figure 25 for Figure 14 Results of TNF-α level detection in metastatic tumor tissues of each treatment group; Figure 26 for Figure 14 Results of IFN-γ level detection in metastatic tumor tissues of each treatment group; Figure 27 for Figure 14 Results of IL-6 level detection in metastatic tumor tissues of each treatment group. Detailed Implementation

[0031] To explain in detail the technical content, structural features, objectives, and effects of the technical solution, the following description is provided in conjunction with specific embodiments and accompanying drawings.

[0032] Example 1

[0033] Preparation of A205-LZMA composite material This embodiment provides a method for preparing A205-LZMA composite material, which specifically includes the following steps.

[0034] Step 1: Preparation of CoAl-LDH nanosheets. Take 20.0 mL of a mixed metal nitrate solution, where the concentration of Co(NO3)2 is 0.032 M and the concentration of Al(NO3)3 is 0.018 M; simultaneously add this mixed metal nitrate solution and 0.25 M NaOH solution to 20.0 mL of NaNO3 solution containing 23 vol% formamide, where the NaNO3 solution concentration is 0.018 M; under magnetic stirring at 80℃, the pH of the system is maintained at 9.8–10.2 by adjusting the NaOH addition rate; after the metal salt solution is added, continue stirring at 80℃ for 20 min, then rapidly cool to room temperature, centrifuge, wash, and redisperse to obtain the CoAl-LDH nanosheet dispersion system. Under conditions of approximately pH 10, Co... 2+ And Al 3+ Synergistic hydrolysis and co-precipitation occur, forming positively charged layers composed of interconnected metal-oxygen octahedra. Formamide reduces electrostatic attraction and hydrogen bonding between adjacent layers by forming hydrogen bonds with hydroxyl groups on the layer surface and regulating the solvation environment of the reaction medium. This inhibits the vertical stacking and secondary aggregation of the layers, ensuring that the generated layered double hydroxides mainly maintain a low-stack or ultrathin nanosheet structure. The 20 min mainly corresponds to the limited growth and structure formation stage after nucleation, eliminating the need for the long hydrothermal aging process commonly used in the preparation of traditional bulk LDH.

[0035] Step 2: Preparation of ZIF-8 nanoparticles. 734 mg (2.47 mmol) of Zn(NO3)2·6H2O and 811 mg (9.87 mmol) of 2-methylimidazole were weighed and dissolved separately in 50 mL of methanol. The two solutions were mixed and stirred briefly, and the white precipitate was collected by centrifugation. After washing three times with methanol, the precipitate was dried overnight in a 60℃ oven to obtain ZIF-8 nanoparticles.

[0036] Step 3: Preparation of MIL-101(Fe) nanoparticles. 0.675 g (2.45 mmol) of FeCl3·6H2O was dissolved in 30 mL of N,N-dimethylformamide, followed by the addition and dissolution of 0.206 g (1.24 mmol) of terephthalic acid. The resulting solution was transferred to a 100 mL polytetrafluoroethylene-lined reactor and reacted at 110 °C for 20 h. The product was washed with methanol, then stirred in fresh methanol for 2 days, centrifuged, washed again, and dried to obtain MIL-101(Fe) nanoparticles.

[0037] Step 4: Preparation of LZMA composite material. CoAl-LDH, ZIF-8, and MIL-101(Fe) were first dispersed in ethanol, and the final composite ratio of CoAl-LDH:ZIF-8:MIL-101(Fe):AgNO3 was controlled at 5:1:1:1. After stirring magnetically at 500 rpm for 2 h at room temperature, AgNO3 was added and stirring was continued for 30 min. The precipitate was collected by centrifugation at 8000 rpm for 5 min and then freeze-dried to obtain LZMA composite material.

[0038] Step 5: Preparation of A205-LZMA. Following the same procedure as in Step 4, CoAl-LDH, ZIF-8, and MIL-101(Fe) were first dispersed in ethanol and magnetically stirred at 500 rpm for 2 h at room temperature. Then, AgNO3 and a predetermined amount of Antago-miR-205-5p were added simultaneously, and stirring was continued for 30 min. The precipitate was collected by centrifugation at 8000 rpm for 5 min and lyophilized to obtain A205-LZMA.

[0039] Step 6: Nucleic acid loading ratio control. Antago-miR-205-5p and LZMA were screened at mass ratios of 1:5, 1:10, 1:20, 1:40, 1:60, 1:80, and 1:100 to determine the optimal loading ratio of nucleic acid drugs in A205-LZMA.

[0040] Figure 1 Nucleic acid loading results of A205-LZMA under different Antago-miR-205-5p:LZMA mass ratios were presented. 20 nmol of Antago-miR-205-5p was mixed with LZMA at different mass ratios of 1:5, 1:10, 1:20, 1:40, 1:60, 1:80, and 1:100 (w / w), and incubated at room temperature for 30 min. Nucleic acid loading was then detected by 2% agarose gel electrophoresis. The results showed that when Antago-miR-205-5p was present alone, obvious free nucleic acid migration bands were observed. With increasing LZMA concentration, the migration bands of free Antago-miR-205-5p gradually weakened, and the complex formed by the nucleic acid and LZMA mainly remained near the sample wells, indicating that LZMA can effectively bind and load Antago-miR-205-5p. When the mass ratio of Antago-miR-205-5p to LZMA is 1:60, the migration bands of free Antago-miR-205-5p basically disappear, indicating that Antago-miR-205-5p has been effectively loaded at this ratio. Therefore, the preferred mass ratio of Antago-miR-205-5p to LZMA is 1:60 (w / w).

[0041] exist Figure 1 Based on the determined optimal nucleic acid loading ratio, subsequent assays for serum stability, acid-responsive release, cell viability, gene silencing effect, and cell uptake were performed using A205-LZMA prepared with Antago-miR-205-5p and LZMA at a mass ratio of 1:60 (w / w). The results are as follows: Figures 2 to 7 As shown: Figure 2 The serum stability results of A205-LZMA are presented. Free Antago-miR-205-5p solution and A205-LZMA solution were co-incubated with a system containing 50% fetal bovine serum at 37°C, and samples were taken at predetermined time points. Nucleic acid stability was detected by 2% agarose gel electrophoresis. The results showed that, compared with free Antago-miR-205-5p, the nucleic acid loaded with A205-LZMA maintained better stability within 24 h, indicating that A205-LZMA can reduce nucleic acid degradation in the serum environment.

[0042] Figure 3 The release curves of A205-LZMA are shown. Cy3-labeled Antago-miR-205-5p was loaded into A205-LZMA and placed in PBS buffer at pH 7.4 and pH 5.5, respectively, and incubated with shaking at 37°C. The concentration of A205-LZMA was 1 mg / mL. The supernatant was collected by centrifugation at different time points, and its fluorescence intensity was measured to characterize nucleic acid release. The results showed that the release rate of A205-LZMA was faster under acidic conditions than under neutral conditions, indicating that A205-LZMA has acid-responsive release characteristics.

[0043] Figure 4 The results show the effect of A205-LZMA on the activity of nasopharyngeal carcinoma cells, among which... Figure 4 A represents the cell viability assay results of C666-1R cells under different concentrations of LZMA or A205-LZMA treatment and with or without combined irradiation. Figure 4 B shows the cell viability of HONE1R cells under different concentrations of LZMA or A205-LZMA treatment and with or without combined irradiation. Radiotherapy-resistant nasopharyngeal carcinoma cells C666-1R and HONE1R were treated with 1×10⁻⁶ LZMA. 4 Cells were seeded per well in 96-well plates and treated with 0, 25, 50, and 100 μg / mL LZMA or A205-LZMA for 24 h. Cell viability was assessed using the CCK-8 assay, and the synergistic effect was evaluated in conjunction with radiotherapy. Results showed that cell viability decreased after the combination of nanomaterials and radiotherapy, with the A205-LZMA group exhibiting the most significant inhibitory effect, where cell viability remained below 25%.

[0044] Figure 5The results show the intracellular miR-205-5p expression level after A205-LZMA delivery. Figure 5 A represents the results of detecting the relative expression level of miR-205-5p in C666-1R cells. Figure 5 B shows the results of the relative expression level detection of miR-205-5p in HONE1R cells. After treating C666-1R and HONE1R cells with A205-LZMA or liposome-delivered Antago-miR-205-5p, the intracellular expression level of miR-205-5p was detected by RT-qPCR. The A205-LZMA treatment concentration was 50 μg / mL. The results showed that the expression level of miR-205-5p decreased more significantly in the A205-LZMA group, indicating that A205-LZMA has a better gene silencing effect.

[0045] Figure 6 These are fluorescence microscopy results of the A205-LZMA, among which... Figure 6 A shows the fluorescence microscopy results of Cy3-labeled nucleic acid delivery in C666-1R cells at 0 h and 24 h. Figure 6 B shows the fluorescence microscopy observation results of Cy3-labeled nucleic acids in HONE1R cells at 0 h and 24 h after delivery. Cy3-labeled nucleic acids were delivered to C666-1R and HONE1R cells using either A205-LZMA or Antago-miR-205-5p / lipo3000. The cells were fixed with 4% paraformaldehyde and stained with Hoechst 33342 at 0 h and 24 h, respectively. Intracellular fluorescence distribution was observed using fluorescence microscopy. The results showed that the A205-LZMA group exhibited stronger red fluorescence signals, mainly distributed in the cytoplasm.

[0046] Figure 7 The results of flow cytometry uptake of A205-LZMA are shown, among which Figure 7 A represents the flow cytometry results of Cy3-labeled nucleic acid delivery in C666-1R cells. Figure 7 B shows the flow cytometry results of Cy3-labeled nucleic acid delivery in HONE1R cells. The cells treated as described above were analyzed by flow cytometry, with the A205-LZMA treatment concentration at 50 μg / mL. The results showed that the uptake efficiency of the A205-LZMA group was higher than that of the control group.

[0047] In conclusion, Figure 1 This indicates that LZMA can effectively load Antago-miR-205-5p, and the preferred loading ratio of Antago-miR-205-5p:LZMA is determined to be 1:60 (w / w). Figure 2-7Further evidence indicates that A205-LZMA prepared at this ratio exhibits good serum stability, acid-responsive release capacity, and high cellular uptake and delivery efficiency. In summary, Figure 2-7 This indicates that the A205-LZMA composite material has high nucleic acid loading capacity, good serum stability, acid-responsive release capacity, and high cell uptake and delivery efficiency.

[0048] Example 2

[0049] Application and efficacy verification of A205-LZMA in radiosensitization for nasopharyngeal carcinoma. This embodiment provides the application method and effect verification of A205-LZMA composite material in radiosensitization of nasopharyngeal carcinoma.

[0050] First, three basic nanomaterials, CoAl-LDH, ZIF-8, and MIL-101(Fe), were prepared according to the method in Example 1. Then, they were composited at a mass ratio of CoAl-LDH:ZIF-8:MIL-101(Fe):AgNO3 = 5:1:1:1. Next, Antago-miR-205-5p was introduced during the composite process, and the ratio of Antago-miR-205-5p:LZMA was controlled to be 1:60 (w / w) to obtain A205-LZMA.

[0051] Finally, the obtained A205-LZMA was used as a nucleic acid delivery composite material for radiosensitization in nasopharyngeal carcinoma radiotherapy, with a corresponding control group set up. The results are as follows: Figures 8 to 27 As shown: Figure 8 Fe in each group of nasopharyngeal carcinoma cells 2+ And GSH level test results, among which Figure 8 A represents Fe in C666-1R and HONE1R cells. 2+ Horizontal test results Figure 8 B shows the GSH level detection results in C666-1R and HONE1R cells. Radiotherapy-resistant nasopharyngeal carcinoma cells C666-1R and HONE1R were selected and treated with Antago-NC, Antago-miR-205-5p, LZMA, and A205-LZMA, respectively. A ferroptosis inhibitor Ferr-1 intervention group was also included. The Ferr-1 concentration was 1 μM. After treatment, each group received 4 Gy irradiation. Intracellular Fe was detected using iron ion detection kits and glutathione detection kits. 2+ And GSH levels. Results showed that, compared with the Antago-NC group, Antago-miR-205-5p group, and LZMA group, the A205-LZMA group had lower intracellular Fe content. 2+ As levels rise, GSH levels decrease.

[0052] Figure 9 The results show the lipid peroxidation levels of nasopharyngeal carcinoma cells in each group. Figure 9 A is a flow cytometry image showing the lipid peroxidation level in C666-1R cells. Figure 9 B is a flow cytometry image showing the lipid peroxidation level in HONE1R cells. Figure 9 C represents the statistical results of lipid peroxidation levels in C666-1R cells. Figure 9 D represents the statistical results of lipid peroxidation levels in HONE1R cells. Cells were treated with Antago-NC, Antago-miR-205-5p, LZMA, and A205-LZMA, respectively, and the lipid peroxidation levels in each group were detected by C11-BODIPY flow cytometry. The results showed that lipid peroxidation levels were increased in the A205-LZMA group; these changes were reversed to varying degrees after the addition of Ferr-1.

[0053] Figure 10 The results of the nasopharyngeal carcinoma cell death analysis for each group are as follows: Figure 10 Image A shows a flow cytometry image of C666-1R cells stained with Annexin. Figure 10 B is a flow cytometry image of HONE1R cells stained with Annexin. Figure 10 C represents the statistical results of the C666-1R cell mortality rate. Figure 10 D represents the statistical results of HONE1R cell death rate. Cells were treated with Antago-NC, Antago-miR-205-5p, LZMA, and A205-LZMA, respectively. Flow cytometry and cytotoxicity assays were used to evaluate cell death in each group. Results showed that the cell death rate increased in the A205-LZMA group after irradiation with 4 Gy.

[0054] Figure 11 The results of EdU proliferation detection for nasopharyngeal carcinoma cells in each group are shown below. Figure 11 Image A shows the EdU flow cytometry analysis of C666-1R cells. Figure 11 B is a flow cytometry image of HONE1R cells analyzed using EdU. Figure 11 C represents the statistical result of the proportion of EdU-positive cells in C666-1R cells. Figure 11 D represents the statistical result of the proportion of EdU-positive cells in HONE1R cells. Cells were treated with Antago-NC, Antago-miR-205-5p, LZMA, and A205-LZMA, respectively, and cell proliferation was detected by EdU flow cytometry. The results showed that cell proliferation was decreased in the A205-LZMA group.

[0055] Figure 12 The results of DNA damage detection in each group of nasopharyngeal carcinoma cells are as follows: Figure 12A shows representative images from the C666-1R and HONE1R cell comet experiments. Figure 12 B represents the statistical results of the comet tail length of C666-1R cells. Figure 12 C represents the statistical results of the comet tail length of HONE1R cells. Cells were treated with Antago-NC, Antago-miR-205-5p, LZMA, and A205-LZMA, respectively, and the degree of DNA damage in each group was detected using the comet assay. The results showed that the degree of DNA damage was increased in the A205-LZMA group.

[0056] Figure 13 The results of HUVEC cell tube formation experiments after treatment in each group are shown below. Figure 13 Image A shows a representative image of HUVEC cell tube formation under C666-1R and HONE1R cell-related treatments. Figure 13 B represents the statistical results of HUVEC cell tube formation rate under C666-1R cell-related treatment conditions. Figure 13 C represents the statistical results of HUVEC cell tube formation rate under HONE1R cell-related treatment conditions. Nasopharyngeal carcinoma cell cultures were treated with Antago-NC, Antago-miR-205-5p, LZMA, and A205-LZMA, respectively. The supernatant from each group's nasopharyngeal carcinoma cell culture was collected and used to treat HUVEC cells for tube formation experiments. The results showed that the A205-LZMA group had the most significant inhibitory effect on HUVEC cell tube formation; the addition of Ferr-1 weakened this effect to varying degrees.

[0057] In conclusion, Figure 8-13 This indicates that A205-LZMA can promote ferroptosis in nasopharyngeal carcinoma cells under radiotherapy conditions and enhance the cell damage and killing effect caused by radiotherapy, thereby increasing the sensitivity of nasopharyngeal carcinoma cells to radiotherapy.

[0058] Figure 14 This diagram illustrates the construction of the Hu-PBMC-PDX nasopharyngeal carcinoma humanized xenograft model and shows the drug administration and sampling time points. First, 1×10⁻⁶… 7 Personal PBMCs were injected into 6-week-old NSG mice via the tail vein. One week later, the proportion of human CD45-positive cells in peripheral blood was measured. When the proportion of human CD45-positive cells was greater than 1%, a nasopharyngeal carcinoma PDX xenograft model was established bilaterally in mice, with the left tumor serving as the primary tumor and the right tumor as the metastatic tumor. When the tumor volume reached approximately 100 mm... 3 Drug administration and irradiation were initiated at the following times: 4 Gy irradiation was administered to the side of the primary tumor every 5 days; PD-1 monoclonal antibody was administered by intraperitoneal injection every 5 days at a dose of 180 μg; A205-LZMA was administered by intravenous injection every 5 days; samples were collected 1 week after the last irradiation.

[0059] Figure 15 and Figure 16 for Figure 14 Results of primary and metastatic tumor growth in each treatment group, including Figure 15 This is an overall observational image showing the location and growth of primary and metastatic tumors in mice of each treatment group. Figure 16 A represents the observation results of primary tumor samples taken from each treatment group. Figure 16 B shows the observation results of metastatic tumor samples taken from each treatment group. Tumor growth was observed in mice after 35 days of treatment. Results showed that, in addition to radiotherapy for the primary tumor, both PD-1 monoclonal antibody and A205-LZMA alone inhibited the growth of both primary and metastatic tumors. The combination of PD-1 monoclonal antibody and A205-LZMA showed a more significant inhibitory effect on both primary and metastatic tumors.

[0060] Figure 17 for Figure 14 Tumor volume growth curves of primary and metastatic tumors in each treatment group, among which Figure 17 A represents the curve showing the change in the volume of the primary tumor. Figure 17 B represents the curve showing the change in the volume of metastatic tumors; Figure 18 for Figure 14 Statistical results of final tumor weight of primary and metastatic tumors in each treatment group, among which... Figure 18 A represents the statistical results of the primary tumor weight. Figure 18 B represents the statistical results of metastatic tumor weight. The results showed that the combination of PD-1 monoclonal antibody and A205-LZMA resulted in slower tumor volume growth and a decrease in final tumor weight.

[0061] Figure 19 , Figure 20 and Figure 21 for Figure 14 Histological examination results of primary tumors in each treatment group, among which Figure 19 Representative immunofluorescence images of γH2AX and CDKN1A in the primary tumor tissues of each treatment group are shown. Figure 19 The top three rows are γH2AX, DAPI and the merged graph, respectively; the bottom three rows are CDKN1A, DAPI and the merged graph, respectively. Figure 20 The images show the HE staining and Ki67 immunohistochemical staining results of primary tumor tissues in each treatment group. The top row shows representative HE staining images, and the bottom row shows representative Ki67 immunohistochemical staining images. Figure 21 A represents the statistical results of γH2AX fluorescence intensity. Figure 21 B represents the statistical results of CDKN1A fluorescence intensity. Figure 21C represents the statistical results of Ki67 staining scores. The cumulative irradiation dose to the primary tumor side was 16 Gy. Immunofluorescence was used to detect γH2AX and CDKN1A, HE staining was used to detect cell necrosis or apoptosis, and IHC was used to detect Ki67 expression. The results showed that in the combined treatment group, γH2AX and CDKN1A signals were enhanced, Ki67 expression was decreased, and cell necrosis or apoptosis was more pronounced in the primary tumor tissue.

[0062] In conclusion, Figure 14-21 This indicates that A205-LZMA can synergize with radiotherapy and PD-1 monoclonal antibody immunotherapy, thereby improving the overall treatment effect of nasopharyngeal carcinoma.

[0063] Figure 22 for Figure 14 Immunohistochemical detection results of CRT and HMGB1 in the primary tumor tissues of each treatment group, among which Figure 22 A shows representative immunohistochemical staining images of CRT and HMGB1 in the primary tumor tissues of each treatment group. Figure 22 The top row (A) shows representative immunohistochemical staining images of calreticulin (CRT). Figure 22 The bottom row of A shows representative immunohistochemical staining images of high-mobility group box 1 (HMGB1); Figure 22 B represents the statistical results of CRT semi-quantitative scoring. Figure 22 C represents the statistical results of the HMGB1 semi-quantitative score. After treatment, primary tumor tissue was collected, and immunohistochemistry was used to detect CRT and HMGB1 expression. Semi-quantitative scoring was performed based on the proportion and intensity of staining-positive cells. Results showed that the expression levels of CRT and HMGB1 were increased in the group treated with PD-1 monoclonal antibody in combination with A205-LZMA.

[0064] Figure 23 and Figure 24 for Figure 14 CD4 in metastatic tumor tissues of each treatment group + T cells and CD8 + Results of T-cell infiltration ratio detection, including Figure 23 AD represents, in order, the CD3 / CD4 ratio in metastatic tumor tissues of the control group, PD-1 monoclonal antibody group, A205-LZMA group, and PD-1 monoclonal antibody + A205-LZMA group, as shown in the flow cytometry images. Figure 23 E is CD3 + CD4 + Statistical results of T cell infiltration ratio; Figure 24 AD represents the CD3 / CD8 ratio in metastatic tumor tissues from the control group, PD-1 monoclonal antibody group, A205-LZMA group, and PD-1 monoclonal antibody + A205-LZMA group, in that order. Figure 24 E is CD3 + CD8 +Statistical results of T-cell infiltration ratio. Metastatic tumor tissue was collected after treatment, and CD4 count was detected by flow cytometry. + T cells and CD8 + The results showed that the proportion of T cell infiltration was higher in the group using PD-1 monoclonal antibody in combination with A205-LZMA. + T cells and CD8 + The proportion of T-cell infiltration increases.

[0065] Figure 25-27 They are respectively Figure 14 The levels of TNF-α, IFN-γ, and IL-6 in metastatic tumor tissues from each treatment group were measured using ELISA. Results showed that the levels of TNF-α, IFN-γ, and IL-6 were elevated in the group treated with PD-1 monoclonal antibody in combination with A205-LZMA.

[0066] In conclusion, Figure 22-27 This indicates that A205-LZMA can enhance the release of immunogenic cell death-related signals, promote T cell infiltration, and increase the expression of immune-related cytokines during synergistic radiotherapy and immunotherapy, thereby activating the anti-tumor immune response.

[0067] In summary, this embodiment demonstrates that the A205-LZMA composite material can not only deliver Antago-miR-205-5p and achieve acid-responsive release, but also promote ferroptosis in nasopharyngeal carcinoma cells in vitro, increase the sensitivity of nasopharyngeal carcinoma cells to radiotherapy, and form a synergistic effect with radiotherapy and PD-1 monoclonal antibody immunotherapy in vivo, showing good prospects for anti-tumor applications.

[0068] Although the above embodiments have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the above descriptions are merely embodiments of the present invention and do not limit the scope of patent protection of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A pH-responsive nucleic acid delivery composite material for radiosensitizing nasopharyngeal carcinoma radiotherapy, characterized in that, The nucleic acid delivery composite material is obtained by forming a composite carrier from CoAl-LDH, ZIF-8, MIL-101(Fe) and AgNO3, and further loading Antago-miR-205-5p onto the composite carrier.

2. The nucleic acid delivery composite material according to claim 1, characterized in that, The mass ratio of CoAl-LDH, ZIF-8, MIL-101(Fe) to AgNO3 in the nucleic acid delivery composite material is 5:1:1:

1.

3. The nucleic acid delivery composite material according to claim 1, characterized in that, The loading ratio of Antago-miR-205-5p to the composite carrier is 1:60 by mass.

4. A method for preparing the nucleic acid delivery composite material according to any one of claims 1 to 3, characterized in that, include: CoAl-LDH nanosheets, ZIF-8 nanoparticles, and MIL-101(Fe) nanoparticles were prepared separately; then, the CoAl-LDH nanosheets, ZIF-8 nanoparticles, and MIL-101(Fe) nanoparticles were composited with AgNO3; subsequently, Antago-miR-205-5p was introduced during the composite process to obtain a nucleic acid delivery composite material.

5. The preparation method according to claim 4, characterized in that, The compounding process is carried out in the following order: first, a premixed system of CoAl-LDH, ZIF-8 and MIL-101(Fe) is formed, then AgNO3 is introduced, and a predetermined amount of Antago-miR-205-5p is added at the same time as AgNO3, thereby obtaining the nucleic acid delivery composite material.

6. The preparation method according to claim 4, characterized in that, The preparation of the CoAl-LDH nanosheets includes: taking 20.0 mL of a mixed metal nitrate solution, wherein the concentration of Co(NO3)2 is 0.032 M and the concentration of Al(NO3)3 is 0.018 M; simultaneously adding the mixed metal nitrate solution and 0.25 M NaOH solution to 20.0 mL of NaNO3 solution containing 23 vol% formamide, wherein the concentration of NaNO3 solution is 0.018 M; maintaining the pH of the system at 9.8–10.2 by adjusting the NaOH addition rate under magnetic stirring at 80 °C; after the metal salt solution is added, continuing to stir the reaction at 80 °C for 20 min, then rapidly cooling to room temperature, and obtaining the CoAl-LDH nanosheet dispersion system by centrifugation, washing and redispersing.

7. The preparation method according to claim 4, characterized in that, The preparation of the ZIF-8 nanoparticles includes: weighing 734 mg of Zn(NO3)2·6HO and 811 mg of 2-methylimidazole, and dissolving each in 50 mL of methanol; mixing the two solutions and stirring briefly, centrifuging to collect the white precipitate; washing three times with methanol and drying overnight in an oven at 60 °C to obtain ZIF-8 nanoparticles.

8. The preparation method according to claim 4, characterized in that, The preparation of the MIL-101(Fe) nanoparticles includes: dissolving 0.675 g of FeCl3·6H2O in 30 mL of N,N-dimethylformamide, then adding 0.206 g of terephthalic acid and dissolving it; transferring the resulting solution to a 100 mL polytetrafluoroethylene-lined reactor and reacting at 110 °C for 20 h; washing the product with methanol, then stirring it in fresh methanol for 2 days, centrifuging, washing it again, and drying it to obtain MIL-101(Fe) nanoparticles.

9. The preparation method according to claim 4, characterized in that, The preparation of the nucleic acid delivery composite material includes: first, dispersing CoAl-LDH nanosheets, ZIF-8 nanoparticles and MIL-101(Fe) nanoparticles in ethanol and stirring, then simultaneously adding AgNO3 and Antago-miR-205-5p, and continuing to stir for 30 min; collecting the precipitate by centrifugation at 8000 rpm for 5 min, and then freeze-drying to obtain the nucleic acid delivery composite material.

10. The use of the nucleic acid delivery composite material according to any one of claims 1 to 3 in the preparation of a medicament for radiosensitizing nasopharyngeal carcinoma.

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