Multifunctional nano-platform, preparation method and application thereof

Abstract

This invention belongs to the field of biomedicine and nanomedicine, specifically relating to a multifunctional nanoplatform, its preparation method, and its applications. The multifunctional nanoplatform comprises gastrodin-iron ion coordinated nanoparticles and an immune checkpoint inhibitor loaded onto the nanoparticles. The multifunctional nanoplatform is obtained by the coordination self-assembly of gastrodin and iron ions to form a nanostructure, with the immune checkpoint inhibitor loaded during the assembly process. The nanoplatform exhibits good stability and biocompatibility, and can be used in the treatment and imaging of glioblastoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanomedicine technology, specifically relating to a multifunctional nanoplatform and its preparation method and application, particularly to a multifunctional nanoplatform loaded with an immune checkpoint blocker based on the coordination self-assembly of gastrodin and iron ions, and its application in the treatment and imaging of glioblastoma. Background Technology

[0002] Glioblastoma (GBM) is one of the most malignant and aggressive primary brain tumors of the central nervous system, characterized by rapid growth, high recurrence rate, and poor prognosis. Currently, clinical treatment for glioblastoma mainly includes a combination of surgical resection, radiotherapy, and chemotherapy. However, due to the highly aggressive and heterogeneous nature of glioblastoma, existing treatments are insufficient to completely eradicate tumor cells, and most patients experience tumor recurrence after treatment, resulting in limited overall treatment efficacy.

[0003] In recent years, immunotherapy has become an important research direction in cancer treatment, achieving good therapeutic effects in various solid tumors. Among them, immune checkpoint blockade therapy, represented by programmed death receptor 1 (PD-1) / programmed death ligand 1 (PD-L1), can enhance the body's anti-tumor immune response by restoring T-cell immune activity. However, the therapeutic effect of immune checkpoint blockers in glioblastoma remains significantly limited.

[0004] On the one hand, the presence of the blood-brain barrier (BBB) ​​severely restricts the entry of therapeutic drugs into brain tissue. Tight junctions form between the endothelial cells of brain microvessels, and various efflux transport proteins exist in brain tissue, making it difficult for most antibody drugs and macromolecular drugs to effectively penetrate the blood-brain barrier, thereby reducing the efficiency of drug accumulation at brain tumor sites.

[0005] On the other hand, glioblastoma exhibits a distinctly immunosuppressive tumor microenvironment. Tumor tissue typically contains a large number of immunosuppressive cells and factors, leading to reduced T cell activity and insufficient tumor immune response, thereby further weakening the effectiveness of immune checkpoint blockade therapy. Therefore, relying solely on a single immunotherapy modality is unlikely to achieve ideal therapeutic results.

[0006] Furthermore, recent studies have revealed that ferroptosis, a novel programmed cell death mechanism dependent on iron ions and accompanied by lipid peroxidation, can kill tumor cells through reactive oxygen species generation and lipid peroxidation, demonstrating potential applications in cancer treatment. However, existing ferroptosis induction systems typically suffer from insufficient brain targeting capabilities, limited biocompatibility, and difficulty in effectively synergizing with immunotherapy, thus limiting their further application in glioblastoma treatment.

[0007] Therefore, developing a multifunctional nanoplatform that can effectively penetrate the blood-brain barrier and simultaneously induce ferroptosis and block immune checkpoints is of great significance for improving the treatment efficacy of glioblastoma. Summary of the Invention

[0008] In view of this, the purpose of the present invention is to provide a multifunctional nanoplatform for targeted immunotherapy of glioblastoma, its preparation method and application, so as to solve the problems in existing glioblastoma treatments, such as the inability of drugs to effectively penetrate the blood-brain barrier, insufficient delivery efficiency at the tumor site, limited anti-tumor effect of single treatment methods, and difficulty in effectively improving the tumor immunosuppressive microenvironment.

[0009] To achieve the above objectives, the present invention provides the following technical solution: In one embodiment of the present invention, a multifunctional nanoplatform is provided, the multifunctional nanoplatform comprising gastrodin-iron ion coordination nanoparticles and an immune checkpoint blocker loaded on the gastrodin-iron ion coordination nanoparticles.

[0010] Furthermore, the immune checkpoint inhibitor is selected from one or more of anti-PD-L1 antibody, anti-PD-1 antibody, and anti-CTLA-4 antibody.

[0011] Furthermore, the molar ratio of gastrodin to iron ions is 1:2 to 1:10.

[0012] Preferably, the molar ratio of gastrodin to iron ions is 1:5.

[0013] Furthermore, the mass ratio of the gastrodin-iron ion coordination nanoparticles to the immune checkpoint blocker is 2:1 to 20:1.

[0014] Preferably, the mass ratio of the gastrodin-iron ion coordination nanoparticles to the immune checkpoint blocker is 6:1.

[0015] In one embodiment of the present invention, a method for preparing the multifunctional nanoplatform is provided, comprising the following steps: Gastrodin solution was mixed with iron salt solution to form gastrodin-iron ion coordination nanoparticles through coordination self-assembly. An immune checkpoint inhibitor is added during the coordination self-assembly process, thereby loading the immune checkpoint inhibitor onto the gastrodin-iron ion coordination nanoparticles to obtain the multifunctional nanoplatform.

[0016] Furthermore, the iron salt is ferric chloride.

[0017] Alternatively, the mixing is carried out under vortex conditions, which include a vortex time of 1-2 minutes and a rotation speed of 3000 rpm.

[0018] In one embodiment of the present invention, the use of the multifunctional nanoplatform or the multifunctional nanoplatform prepared by the method is provided in the preparation of a medicament for treating glioblastoma.

[0019] In one embodiment of the present invention, the application of the multifunctional nanoplatform or the multifunctional nanoplatform prepared by the method is provided in the preparation of a drug for glioblastoma imaging.

[0020] Based on the above technical solution, the multifunctional nanoplatform provided by this invention forms a nanostructure by coordinating gastrodin with iron ions, and loads an immune checkpoint inhibitor into the nanostructure, thereby integrating brain-targeted delivery, ferroptosis induction, and immune checkpoint blockade functions into a single nanoplatform. This nanoplatform is simple to prepare under mild conditions, exhibits good stability, biocompatibility, and potential for glioblastoma treatment and imaging applications, and can provide a new technical solution for the synergistic treatment of glioblastoma.

[0021] Compared with the prior art, the present invention has at least the following beneficial effects: First, the present invention uses gastrodin as a component unit of the nanoplatform. Gastrodin can participate in the coordination self-assembly of iron ions as an organic ligand, and can also exert its brain-targeting related properties, which helps to improve the ability of the nanoplatform to penetrate the blood-brain barrier and promote its accumulation in brain tumor sites.

[0022] Second, this invention utilizes iron ions to construct a nanoplatform, enabling the nanoplatform to promote the generation of reactive oxygen species and induce ferroptosis in tumor cells within the tumor microenvironment, thereby achieving direct killing of glioblastoma cells.

[0023] Third, by loading an immune checkpoint inhibitor, the present invention enables the nanoplatform to block tumor immune escape pathways, improve the level of anti-tumor immune response, and thus enhance the therapeutic effect of glioblastoma.

[0024] Fourth, this invention integrates brain-targeted delivery, ferroptosis induction, and immune checkpoint blockade into the same nanoplatform, which helps to achieve synergistic effects among multiple therapeutic mechanisms and improves the limited efficacy of existing single-treatment methods for glioblastoma.

[0025] Fifth, the multifunctional nanoplatform provided by this invention is prepared by coordination self-assembly, without the need for complex chemical modification steps, and has the advantages of simple operation, mild reaction conditions, green preparation process and easy scale-up preparation.

[0026] Sixth, the multifunctional nanoplatform provided by this invention has good colloidal stability and biocompatibility, and did not cause significant toxic damage to major organs under experimental drug administration conditions, demonstrating good safety and potential for further clinical translation.

[0027] Seventh, the multifunctional nanoplatform provided by this invention can also be used for glioblastoma imaging. By loading fluorescent molecules, it is possible to monitor the in vivo distribution of the nanoplatform and the enrichment of brain tumors, providing a technical basis for the integrated diagnosis and treatment of glioblastoma. Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the preparation process and mechanism of action of the multifunctional nanoplatform of the present invention.

[0029] Figure 2 This is a particle size distribution diagram of GAS-Fe NPs and GAS-Fe@αPD-L1 NPs in Example 1 of the present invention.

[0030] Figure 3 The images are transmission electron microscope images of GAS-Fe NPs and GAS-Fe@αPD-L1 NPs in Example 1 of this invention.

[0031] Figure 4 This is an EDS elemental surface scan of GAS-Fe@αPD-L1 NPs in Embodiment 1 of the present invention.

[0032] Figure 5 The UV-Vis absorption spectra of GAS, Fe3+, GAS-Fe NPs and GAS-Fe@αPD-L1 NPs in Example 1 of this invention are shown.

[0033] Figure 6 The Fourier transform infrared spectra of GAS and GAS-Fe NPs in Example 1 of this invention are shown.

[0034] Figure 7 This is a stability evaluation diagram of GAS-Fe NPs in different media in Example 2 of the present invention.

[0035] Figure 8This is a graph showing the long-term storage stability evaluation of GAS-Fe NPs in Example 2 of the present invention.

[0036] Figure 9 This is a diagram illustrating the establishment and verification of the organ-on-a-chip blood-brain barrier model in Embodiment 3 of the present invention.

[0037] Figure 10 This is a graph showing the cumulative permeation changes of different treatment groups in the blood-brain barrier model in Example 4 of the present invention.

[0038] Figure 11 This is a Western blot analysis of Glut1 protein expression under different treatment conditions in Example 5 of the present invention.

[0039] Figure 12 This is a diagram showing the uptake of Cy5-labeled GAS-Fe@αPD-L1 NPs in U251 cells in Example 6 of this invention.

[0040] Figure 13 This is a graph showing the cytotoxicity of GAS-Fe NPs and GAS-Fe@αPD-L1 NPs on U251 and GL261 cells in Example 7 of this invention.

[0041] Figure 14 This is a graph showing the ROS levels in U251 cells after different treatments in Example 8 of the present invention.

[0042] Figure 15 This is a graph showing the detection level of intracellular lipid peroxidation in U251 cells after different treatments in Example 8 of the present invention.

[0043] Figure 16 This is a graph showing the detection of Fe2+ levels in U251 cells after different treatments in Example 8 of the present invention.

[0044] Figure 17 This is a graph showing the expression of GPX4 protein in U251 cells after different treatments in Example 9 of the present invention.

[0045] Figure 18 This is a graph showing the detection of ATP release from U251 cells after different treatments in Example 10 of the present invention.

[0046] Figure 19 This is a graph showing the detection of HMGB1 release levels in U251 cells after different treatments in Example 10 of the present invention.

[0047] Figure 20 This is a detection image of the CRT membrane surface exposure of U251 cells after different treatments in Example 10 of the present invention.

[0048] Figure 21This is a graph showing the detection of PD-L1 mRNA expression in GL261 cells after different treatments in Example 11 of the present invention.

[0049] Figure 22 This is a graph showing the expression of PD-L1 protein in GL261 cells after different treatments in Example 11 of the present invention.

[0050] Figure 23 This is an in vivo fluorescence imaging image of Cy5-labeled nanoparticles in an in situ U251 glioma mouse, as shown in Example 12 of this invention.

[0051] Figure 24 This is an in vitro fluorescence imaging image of the main organs and brain tissue in Example 12 of the present invention.

[0052] Figure 25 This is a graph showing the in vivo antitumor efficacy evaluation of the GL261 and U251 models in Embodiment 13 of the present invention.

[0053] Figure 26 This is an immunohistochemical staining image of tumor tissue in Example 14 of the present invention.

[0054] Figure 27 This is a graph showing the detection of serum cytokine levels in Example 14 of the present invention.

[0055] Figure 28 This is a H&E staining image of the main organs in Example 15 of the present invention.

[0056] Figure 29 The image shows the results of hematological index analysis and hemolysis experiment in Example 15 of this invention. Detailed Implementation

[0057] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can understand and implement the present invention. It should be understood that the following embodiments are only preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention.

[0058] Without departing from the concept of this invention, those skilled in the art can make appropriate adjustments or equivalent substitutions to the types of raw materials, proportion ranges, preparation conditions, administration methods, detection methods, and application scenarios according to actual application needs; such adjustments or substitutions, as long as they do not depart from the essential content of this invention, should fall within the protection scope of this invention.

[0059] Unless otherwise specified, the reagents, materials, cell lines, animal models, and detection methods used in the embodiments of this invention can all be obtained using commercially available products or conventional experimental methods in the art. The specific parameters involved in the embodiments are used to illustrate the feasibility of this invention and do not constitute a limitation on the scope of protection of this invention.

[0060] Example 1: Preparation and characterization of the multifunctional nanoplatform GAS-Fe@αPD-L1 NPs (corresponding to...) Figures 1-6 ) This embodiment provides a method for preparing a multifunctional nanoplatform GAS-Fe@αPD-L1 NPs based on the coordination self-assembly of gastrodin (GAS) and iron ions, and characterizes and analyzes its particle size, morphology, elemental composition and coordination structure.

[0061] (1) Preparation of GAS-Fe NPs 50 mg of gastrodin (GAS) was dissolved in 10 mL of ultrapure water to obtain a gastrodin solution; 25 mg of FeCl3·6H2O was dissolved in 10 mL of ultrapure water to obtain an iron salt solution.

[0062] At room temperature, the gastrodin solution and the iron salt solution were mixed at a molar ratio of gastrodin to iron ions of 1:5, and vortexed at 3000 rpm for 1-2 min to form gastrodin-iron ion coordination nanoparticles (GAS-Fe NPs) through coordination self-assembly reaction.

[0063] In this embodiment, the phenolic hydroxyl groups in GAS coordinate with Fe3+, thereby driving the self-assembly of the nanostructure to form stable nanoparticles.

[0064] Furthermore, this embodiment uses an aqueous system to prepare the nanoplatform, which does not require the introduction of organic solvents or additional surface modifiers, and has the advantages of simple preparation process, mild conditions and good biocompatibility.

[0065] (2) Preparation of GAS-Fe@αPD-L1 NPs The αPD-L1 antibody was added to the gastrodin solution and mixed thoroughly at room temperature.

[0066] Furthermore, an iron salt solution was added to the above mixture under vortex conditions, causing gastrodin to coordinate with iron ions and self-assemble, and simultaneously encapsulating the αPD-L1 antibody in the formed nanostructure, thereby obtaining a multifunctional nanoplatform GAS-Fe@αPD-L1 NPs loaded with an immune checkpoint blocker.

[0067] In this embodiment, the concentration of the αPD-L1 antibody is 1 mg / mL.

[0068] Furthermore, the mass ratio of the GAS-Fe NPs to the αPD-L1 antibody is 6:1.

[0069] Furthermore, the reaction conditions are room temperature and vortexing at 3000 rpm for 1-2 min.

[0070] Figure 1 A schematic diagram illustrating the preparation process and mechanism of action of the multifunctional nanoplatform GAS-Fe@αPD-L1 NPs of the present invention is shown.

[0071] (3) Particle size and zeta potential detection The hydration size and zeta potential of GAS-Fe NPs and GAS-Fe@αPD-L1 NPs were detected using dynamic light scattering (DLS).

[0072] Test results as follows Figure 2 As shown.

[0073] The results showed that the average hydrated particle size of GAS-Fe NPs was approximately 60 nm, and the Zeta potential was -20.7 ± 2.5 mV. After loading with αPD-L1 antibody, the average particle size of GAS-Fe@αPD-L1 NPs increased to approximately 170 nm, and the Zeta potential changed to -21.9 ± 0.8 mV.

[0074] The above results indicate that the αPD-L1 antibody was successfully loaded into the nanoplatform, and the resulting nanoparticles exhibit good dispersibility and colloidal stability.

[0075] (4) Transmission electron microscopy (TEM) characterization The microstructure and dispersion of the nanoplatform were observed using transmission electron microscopy (TEM).

[0076] The results are as follows Figure 3 As shown.

[0077] The results showed that both GAS-Fe NPs and GAS-Fe@αPD-L1 NPs exhibited relatively uniform spindle-shaped nanostructures with good particle dispersion and no obvious aggregation.

[0078] Furthermore, the overall size of the nanoparticles increased after loading with αPD-L1 antibody, consistent with the DLS detection results.

[0079] (5) EDS elemental surface scanning analysis Elemental surface scan analysis of GAS-Fe@αPD-L1 NPs was performed using energy dispersive X-ray spectroscopy (EDS).

[0080] The results are as follows Figure 4 As shown.

[0081] EDS results showed that Fe, O, N and C elements were uniformly distributed throughout the nanoparticles.

[0082] Among them, Fe element comes from iron ions, O and C elements mainly come from gastrodin molecules, and N element mainly comes from αPD-L1 antibody.

[0083] The above results indicate that iron ions, gastrodin, and αPD-L1 antibody were successfully assembled together in the same nanoplatform.

[0084] (6) Ultraviolet-Visible Absorption Spectroscopy Analysis GAS, Fe3+, GAS-Fe NPs and GAS-Fe@αPD-L1 NPs were detected and analyzed using ultraviolet-visible absorption spectroscopy (UV-Vis).

[0085] The results are as follows Figure 5 As shown.

[0086] The results showed that after GAS coordinated with Fe3+, its characteristic absorption peaks underwent a significant red shift, indicating that an effective coordination reaction occurred between the phenolic hydroxyl groups in GAS and Fe3+.

[0087] Furthermore, GAS-Fe@αPD-L1 NPs also retained the corresponding coordination characteristic absorption peaks, indicating that the nanoplatform structure remained stable after loading with αPD-L1 antibody.

[0088] (7) Fourier transform infrared spectroscopy analysis GAS and GAS-Fe NPs were analyzed using Fourier Transform Infrared Spectroscopy (FTIR).

[0089] The results are as follows Figure 6 As shown.

[0090] The results showed that, compared with free GAS, the characteristic absorption peaks associated with hydroxyl groups in GAS-Fe NPs underwent a significant shift.

[0091] The above results further demonstrate that the hydroxyl groups in GAS react with Fe. 3+ Coordination bonds were formed between them, thus successfully constructing a gastrodin-iron ion coordination nanoplatform.

[0092] In summary, this embodiment successfully constructed a multifunctional nanoplatform, GAS-Fe@αPD-L1 NPs, based on the coordination self-assembly of gastrodin and iron ions. This nanoplatform exhibits uniform particle size, structural stability, good dispersibility, and the ability to load immune checkpoint inhibitors, laying the foundation for subsequent blood-brain barrier penetration, ferroptosis induction, and synergistic immunotherapy for glioblastoma.

[0093] Example 2: Stability assessment of the nanoplatform (corresponding to) Figures 7-8 ) This embodiment evaluates the colloidal stability and long-term storage stability of the GAS-Fe NPs prepared in Example 1 to verify the stability of the nanoplatform under different physiological environmental conditions.

[0094] (1) Stability evaluation in different physiological media The GAS-Fe NPs prepared in Example 1 were dispersed in the following different media: 1) Physiological saline; 2) PBS buffer (pH 7.4); 3) Milli-Q ultrapure water; 4) Cell culture medium containing 10% fetal bovine serum (FBS).

[0095] Furthermore, the different dispersion systems were incubated at 4°C, and samples were taken on day 1, day 3 and day 7.

[0096] Dynamic light scattering (DLS) was used to detect the changes in the hydration particle size of each group of nanoparticles in order to evaluate their colloidal stability.

[0097] Test results as follows Figure 7 As shown.

[0098] The results showed that after incubation in the above-mentioned different media for 1 day, 3 days and 7 days, the average particle size of GAS-Fe NPs did not change significantly, and no obvious precipitation or particle aggregation was observed.

[0099] The above results indicate that the GAS-Fe NPs constructed in this invention have good colloidal stability in different physiologically relevant environments.

[0100] Furthermore, the fact that it can maintain a stable dispersion in serum-containing culture medium indicates that the nanoplatform has good anti-protein adsorption and anti-aggregation capabilities, which is beneficial for subsequent in vivo circulation and biomedical applications.

[0101] (2) Long-term storage stability evaluation Furthermore, the GAS-Fe NPs prepared in Example 1 were stored at 4°C in the dark for 30 days.

[0102] During storage, the changes in particle size, polydispersity index (PDI), and zeta potential of the nanoparticles were periodically monitored to evaluate their long-term storage stability.

[0103] Test results as follows Figure 8 As shown.

[0104] The results showed that after storage at 4℃ for 30 days, the average particle size, PDI, and Zeta potential of GAS-Fe NPs did not fluctuate significantly.

[0105] The nanoparticles consistently maintained a low PDI value, indicating that the particle distribution was uniform and no significant aggregation occurred.

[0106] Furthermore, the Zeta potential remains in a stable negative charge state, indicating that the surface structure of the nanoparticles is stable.

[0107] The above results indicate that the GAS-Fe NPs provided by this invention have good long-term storage stability and can maintain stable physicochemical properties over a relatively long period of time.

[0108] In summary, the results of this embodiment demonstrate that the GAS-Fe NPs provided by this invention not only exhibit good colloidal stability in a variety of physiologically relevant media, but also maintain a stable structure under long-term storage conditions, which is beneficial for their subsequent formulation development, transportation, storage, and clinical translation applications.

[0109] Example 3: Establishment and Validation of an Organ-on-a-Chip Blood-Brain Barrier Model (corresponding to) Figure 9 ) In this embodiment, a dual-channel microfluidic organ-on-a-chip was used to construct an in vitro blood-brain barrier (BBB) ​​model, and the integrity and barrier function of the established model were verified.

[0110] (1) Construction of an organ-on-a-chip blood-brain barrier model This embodiment uses a dual-channel organ-on-a-chip as a platform for constructing the blood-brain barrier model.

[0111] The organ-on-a-chip includes an upper channel and a lower channel, which are separated by a semi-permeable membrane to simulate the barrier structure between brain microvessels and brain tissue.

[0112] Furthermore, the upper channel is used to simulate the cerebral blood vessel side, and the lower channel is used to simulate the brain parenchyma side.

[0113] First, human brain microvascular endothelial cells HCMEC / D3 were seeded into the upper channel.

[0114] In this embodiment, the HCMEC / D3 cell seeding density was 1 × 10⁻⁶. 6 cells / mL.

[0115] Furthermore, astrocytes (ASTs) were seeded into the lower channel.

[0116] In this embodiment, the ASTs cell seeding density is 5 × 10⁻⁶. 5 cells / mL.

[0117] After cell seeding, the organ-on-a-chip was placed in a 37°C, 5% CO2 incubator for static culture for 24 h to promote cell adhesion and the formation of a stable cell layer.

[0118] Furthermore, after static culture is completed, the organ-on-a-chip is connected to a microfluidic perfusion system.

[0119] We used EBM-2 medium to continuously perfuse culture at a flow rate of 2 μL / min for 24 h to simulate the dynamic fluid environment in brain microvessels.

[0120] In this embodiment, microfluidic perfusion culture can promote the formation of tight junctions in brain microvascular endothelial cells and improve the physiological relevance of the blood-brain barrier model.

[0121] (2) Validation of the integrity of the blood-brain barrier model To verify the integrity and barrier function of the constructed blood-brain barrier model, this embodiment uses FITC-glucan of different molecular weights for permeation experiments.

[0122] Specifically, FITC-glucan with molecular weights of 10 kDa, 40 kDa, and 70 kDa were used as fluorescent tracer molecules and added to the channels on the organ-on-a-chip.

[0123] Samples were collected from the lower channel at predetermined time points, and the permeation of FITC-glucan was analyzed using a fluorescence detection method.

[0124] Furthermore, the apparent permeability coefficient (Papp) of FITC-glucan with different molecular weights was calculated based on the changes in fluorescence intensity.

[0125] Test results as follows Figure 9 As shown.

[0126] The results showed that as the molecular weight of FITC-glucan increased, its apparent permeability coefficient gradually decreased.

[0127] Among them, 10 kDa FITC-glucan has a relatively high permeability, while the permeability of 40 kDa and 70 kDa FITC-glucan is significantly inhibited.

[0128] The above results indicate that the blood-brain barrier model constructed in this embodiment exhibits typical molecular weight-dependent barrier characteristics, which can effectively restrict the transport of macromolecules across the barrier.

[0129] Furthermore, this indicates that a relatively complete tight junction structure has been formed between the endothelial cells of brain microvessels.

[0130] In summary, this embodiment successfully established an in vitro blood-brain barrier model based on organ-on-a-chip. The established model exhibits good barrier integrity and physiological relevance, and can be used for subsequent evaluation of the blood-brain barrier penetration capability of nanoplatforms and research on brain-targeted delivery mechanisms.

[0131] Example 4: Evaluation of the blood-brain barrier penetration ability of the nanoplatform (corresponding to) Figure 10 ) This embodiment uses the organ-on-a-chip blood-brain barrier model constructed in Example 3 to evaluate the blood-brain barrier penetration ability of GAS-FeNPs and GAS-Fe@αPD-L1 NPs provided in this invention.

[0132] (1) Experimental grouping The completed and validated organ-on-a-chip blood-brain barrier models were randomly divided into the following treatment groups: 1) PBS control group; 2) Free GAS group; 3) GAS-Fe NPs group; 4) GAS-Fe@αPD-L1 NPs group.

[0133] In this embodiment, the free GAS group, the GAS-Fe NPs group, and the GAS-Fe@αPD-L1 NPs group were all calculated with a GAS concentration of 10 μg / mL.

[0134] Furthermore, the samples from each group were added to the channels on the organ-on-a-chip for perfusion processing.

[0135] (2) Blood-brain barrier penetration test Culture medium samples were collected from the organ-on-a-chip channel at 0 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h and 24 h after drug administration.

[0136] The changes in fluorescence intensity in the samples at each time point were measured using a fluorescence detection method.

[0137] Furthermore, the cumulative permeation and apparent permeability coefficient (Papp) of the nanoplatforms in different treatment groups were calculated based on the changes in fluorescence intensity to evaluate their blood-brain barrier penetration ability.

[0138] Test results as follows Figure 10 As shown.

[0139] (3) Analysis of experimental results The results showed that no significant changes in fluorescence signal were observed in the PBS control group, indicating that the blood-brain barrier model was structurally stable and there was no significant leakage.

[0140] Furthermore, a certain degree of cross-barrier penetration was observed in the free GAS group at each time point, indicating that GAS itself has a certain ability to penetrate the blood-brain barrier.

[0141] Compared to the free GAS group, the cumulative permeation of the GAS-Fe NPs group was significantly higher at all time points, and its 24-hour cumulative permeation was significantly higher than that of the free GAS group.

[0142] Furthermore, the GAS-Fe@αPD-L1 NPs group also exhibited high trans-blood-brain barrier transport capacity.

[0143] The above results indicate that the gastrodin-iron ion coordination nanoplatform constructed in this invention can effectively improve the blood-brain barrier penetration efficiency of the drug system.

[0144] Further analysis revealed that both the GAS-Fe NPs group and the GAS-Fe@αPD-L1 NPs group exhibited significant time-dependent transbarrier transport characteristics, with their cumulative permeation gradually increasing over time.

[0145] In this embodiment, the reason why the nanoplatform enhances the blood-brain barrier penetration ability may be related to the promoting effect of gastrodin on the brain-targeted transport process.

[0146] Meanwhile, nanostructures can improve the stability of drug systems and help prolong their effective circulation time in blood-brain barrier models, thereby further improving cross-barrier transport efficiency.

[0147] In summary, the results of this embodiment demonstrate that the GAS-Fe NPs and GAS-Fe@αPD-L1 NPs provided by this invention have good blood-brain barrier penetration capabilities, which can effectively improve the drug delivery efficiency to brain tissue and provide a foundation for subsequent glioblastoma brain-targeted therapy.

[0148] Example 5: Study on the regulatory mechanism of Glut1 expression (corresponding) Figure 11 ) This embodiment investigates the regulatory effect of the nanoplatform provided by the present invention on Glut1 expression by detecting changes in the expression of glucose transporter 1 (Glut1) in brain microvascular endothelial cells, and further analyzes its relationship with the blood-brain barrier penetration ability.

[0149] (1) Cell culture and experimental grouping Human brain microvascular endothelial cells (HCMEC / D3) were seeded into 6-well plates and cultured at 37°C in a 5% CO2 incubator until the cells adhered stably.

[0150] Furthermore, the cells were randomly divided into the following treatment groups: 1) PBS control group; 2) Free GAS group; 3) Free GAS + Phloretin (PHL) group; 4) GAS-Fe NPs group; 5) GAS-Fe NPs + phloretin (PHL) group.

[0151] In this embodiment, phloretin (PHL) was used as a Glut1 inhibitor to verify the mechanism of action of Glut1 in the brain-targeted delivery process of the nanoplatform.

[0152] Further, each group of cells was treated for 24 hours before subsequent testing was performed.

[0153] (2) Western blot detection After processing, cells from each group were collected and total protein was extracted.

[0154] After quantifying the protein concentration using the BCA method, an equal amount of protein was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane.

[0155] Furthermore, the corresponding protein bands were incubated with Glut1 primary antibody and then detected by colorimetric assay with the corresponding secondary antibody.

[0156] Finally, Western blot was used to analyze the expression level of Glut1 protein under different treatment conditions.

[0157] Test results as follows Figure 11 As shown.

[0158] (3) Analysis of experimental results The results showed that the expression level of Glut1 protein was significantly increased in the free GAS group compared with the PBS control group.

[0159] Furthermore, the upregulation of Glut1 expression induced by free GAS was significantly inhibited after the addition of the Glut1 inhibitor phloretin (PHL).

[0160] The above results indicate that GAS can promote the expression of Glut1 protein in brain microvascular endothelial cells.

[0161] Furthermore, the GAS-Fe NPs treatment group also significantly increased the expression level of Glut1 protein.

[0162] Compared to the free GAS group, the expression level of Glut1 was further enhanced in the GAS-Fe NPs group.

[0163] In addition, although the expression of Glut1 in the GAS-Fe NPs group decreased after the addition of PHL, it still remained at a relatively high level.

[0164] The above results demonstrate that the GAS-Fe NPs constructed in this invention not only retain the ability of GAS to regulate Glut1 expression, but also maintain good brain-targeting related biological functions after nanostructuring.

[0165] In this embodiment, the upregulation of Glut1, an important glucose transporter in brain microvascular endothelial cells, helps promote the transport of the nanoplatform across the blood-brain barrier.

[0166] Therefore, the nanoplatform provided by this invention may partially rely on the Glut1-mediated pathway to achieve brain-targeted delivery.

[0167] In summary, the results of this embodiment demonstrate that the GAS-Fe NPs provided by this invention can effectively upregulate the expression of Glut1 protein in brain microvascular endothelial cells, providing a mechanistic basis for enhancing its blood-brain barrier penetration ability and brain-targeted delivery capability.

[0168] Example 6: Cell uptake experiment (corresponding) Figure 12 ) In this embodiment, the uptake of Cy5-labeled GAS-Fe@αPD-L1 NPs in glioblastoma cells was observed using confocal fluorescence microscopy to evaluate the endocytic capacity of the nanoplatform provided by this invention.

[0169] (1) Cell culture Human glioblastoma cells U251 were seeded into 4-well confocal culture dishes and cultured in a 37°C, 5% CO2 incubator.

[0170] In this embodiment, the U251 cell seeding density was 6 × 10⁻⁶. 3 cells / pores.

[0171] After the cells have adhered stably, subsequent experiments will be conducted.

[0172] (2) Preparation of Cy5-labeled nanoplatforms GAS-Fe@αPD-L1 NPs were fluorescently labeled using Cy5 fluorescent dye.

[0173] Furthermore, Cy5-labeled GAS-Fe@αPD-L1 NPs were added to the cell culture system and co-incubated with U251 cells.

[0174] In this embodiment, the treatment concentration of Cy5-labeled GAS-Fe@αPD-L1 NPs was 10 μg / mL.

[0175] (3) Cell uptake experiment Incubation time points of 1 h, 2 h, 4 h and 6 h were set respectively.

[0176] After the corresponding time points, the culture medium was discarded, and the cells were washed with PBS buffer to remove nanoparticles that had not been taken up by the cells.

[0177] Furthermore, DAPI was used to stain the cell nuclei.

[0178] After staining, the distribution of Cy5-labeled nanoplatforms in cells was observed using a confocal laser scanning microscope.

[0179] Test results as follows Figure 12 As shown.

[0180] (4) Analysis of experimental results The results showed that a small amount of red fluorescence signal could be observed in the cells after 1 h of incubation, indicating that Cy5-labeled GAS-Fe@αPD-L1 NPs had begun to be taken up by U251 cells.

[0181] Furthermore, as the incubation time was extended, the intensity of red fluorescence within the cells gradually increased.

[0182] Between 2 and 4 hours, the fluorescence signal in the cytoplasm region increased significantly, suggesting that the nanoplatform continued to penetrate into the cell.

[0183] After 6 hours of incubation, the intracellular red fluorescence reached a high level and was mainly distributed in punctate patterns in the cytoplasm.

[0184] The above results indicate that the GAS-Fe@αPD-L1 NPs provided by this invention can be effectively taken up by glioblastoma cells and exhibit significant time-dependent endocytosis characteristics.

[0185] Furthermore, the dotted distribution of the nanoplatform in the cytoplasm suggests that it may enter the cell via endocytosis.

[0186] In summary, the results of this embodiment demonstrate that the GAS-Fe@αPD-L1 NPs provided by this invention have good cellular uptake capabilities and can effectively enter the interior of glioblastoma cells, providing a foundation for subsequent ferroptosis induction and synergistic immunotherapy.

[0187] Example 7: Cytotoxicity Experiment (corresponding) Figure 13 ) In this embodiment, the CCK-8 assay was used to evaluate the cytotoxicity of GAS-Fe NPs and GAS-Fe@αPD-L1 NPs provided by this invention against glioblastoma cells, in order to verify the killing ability of the nanoplatform against tumor cells.

[0188] (1) Cell culture Human glioblastoma cells U251 and mouse glioma cells GL261 were seeded into 96-well cell culture plates, respectively.

[0189] In this embodiment, the cell seeding density was 6000 cells / well, and 100 μL of culture medium was added to each well.

[0190] After inoculation, the cells were placed in a 37°C, 5% CO2 incubator and cultured overnight to allow the cells to adhere fully to the incubator wall.

[0191] (2) Drug treatment After the cells adhered and stabilized, different concentrations of GAS-Fe NPs and GAS-Fe@αPD-L1 NPs were added for treatment.

[0192] In this embodiment, the concentrations treated by the nanoplatform are as follows: 0 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL and 200 μg / mL.

[0193] Furthermore, the cells in each group were cultured for another 24 hours.

[0194] (3) CCK-8 detection After drug treatment, add 10 μL of CCK-8 detection reagent to each well.

[0195] Furthermore, the cells were incubated for another 1 hour.

[0196] After incubation, the absorbance value (OD450) of each well was measured at a wavelength of 450 nm using an ELISA reader.

[0197] Furthermore, cell viability in different treatment groups was calculated based on OD450 values.

[0198] Test results as follows Figure 13 As shown.

[0199] (4) Analysis of experimental results The results showed that as the concentrations of GAS-Fe NPs and GAS-Fe@αPD-L1 NPs increased, the cell viability of both U251 and GL261 cells gradually decreased.

[0200] Furthermore, both nanoplatforms exhibited significant concentration-dependent cytotoxicity characteristics.

[0201] At lower concentrations, cell viability changes were relatively small; however, as the concentration of the nanoplatform increased further, tumor cell viability decreased significantly.

[0202] Further analysis revealed that the GAS-Fe@αPD-L1 NPs group exhibited a strong overall cytotoxic effect.

[0203] The above results indicate that the nanoplatform provided by this invention can effectively inhibit the proliferation of glioblastoma cells and induce tumor cell death.

[0204] In this embodiment, the cytotoxicity of the nanoplatform may be related to iron ion-induced reactive oxygen species generation and ferroptosis.

[0205] Meanwhile, loading immune checkpoint inhibitors helps to further enhance the anti-tumor therapeutic potential of the nanoplatform.

[0206] In summary, the results of this embodiment demonstrate that the GAS-Fe NPs and GAS-Fe@αPD-L1 NPs provided by this invention exhibit good in vitro antitumor activity against U251 and GL261 glioma cells, providing a foundation for subsequent research on ferroptosis mechanisms and in vivo antitumor experiments.

[0207] Example 8: Verification of the ferroptosis mechanism (corresponding to) Figures 14-16 ) This embodiment verifies the ability of the nanoplatform provided by the present invention to induce ferroptosis in tumor cells by detecting intracellular reactive oxygen species (ROS), lipid peroxidation (LPO), and Fe2+ levels.

[0208] (1) Cell culture and drug treatment U251 glioblastoma cells were seeded in confocal culture dishes and cultured at 37°C in a 5% CO2 incubator until the cells adhered stably.

[0209] Furthermore, the cells were randomly divided into the following treatment groups: 1) PBS control group; 2) GAS-Fe NPs group; 3) GAS-Fe@αPD-L1 NPs group.

[0210] In this embodiment, the treatment concentration for both the GAS-Fe NPs group and the GAS-Fe@αPD-L1 NPs group was 60 μg / mL.

[0211] Further, each group of cells was treated for 24 hours before subsequent testing was performed.

[0212] (2) Detection of intracellular ROS levels After drug treatment, ROS fluorescent probe DCF was added to each group of cells for incubation.

[0213] Furthermore, after washing the cells with PBS buffer, the changes in intracellular fluorescence signals were observed using a confocal laser scanning microscope.

[0214] Test results as follows Figure 14 As shown.

[0215] The results showed that, compared with the PBS control group, both the GAS-Fe NPs group and the GAS-Fe@αPD-L1 NPs group exhibited significantly enhanced green fluorescence signals.

[0216] The above results indicate that the nanoplatform provided by this invention can significantly improve intracellular ROS levels.

[0217] In this embodiment, the increased ROS level indicates that iron ions in the nanoplatform can promote oxidative stress.

[0218] (3) Detection of lipid peroxidation level Furthermore, the intracellular lipid peroxidation level in each group was detected using the Liperfluo fluorescent probe.

[0219] After drug treatment, the Liperfluo probe was added to the cell culture system for incubation.

[0220] Subsequently, confocal laser scanning microscopy was used to observe changes in intracellular fluorescence.

[0221] Test results as follows Figure 15 As shown.

[0222] The results showed that, compared with the PBS control group, both the GAS-Fe NPs group and the GAS-Fe@αPD-L1 NPs group exhibited significantly enhanced fluorescence signals.

[0223] The above results demonstrate that the nanoplatform provided by this invention can significantly induce intracellular lipid peroxidation.

[0224] Furthermore, elevated lipid peroxidation levels are one of the key characteristics of ferroptosis.

[0225] (4) Detection of intracellular Fe2+ levels Furthermore, the intracellular Fe2+ level was detected using the FerroOrange fluorescent probe.

[0226] After drug treatment, the FerroOrange probe was added to the cell culture system for incubation.

[0227] Subsequently, confocal laser scanning microscopy was used to observe intracellular Fe2+-related fluorescence signals.

[0228] Test results as follows Figure 16 As shown.

[0229] The results showed that, compared with the PBS control group, both the GAS-Fe NPs group and the GAS-Fe@αPD-L1 NPs group exhibited significantly enhanced orange fluorescence signals.

[0230] The above results indicate that the nanoplatform provided by this invention can increase intracellular Fe in tumor cells. 2+ level.

[0231] In this embodiment, intracellular Fe 2+ Increased levels help promote the Fenton reaction, thereby further inducing ROS generation and lipid peroxidation.

[0232] (5) Analysis of experimental results Comprehensive ROS, lipid peroxidation and Fe 2+ The results of the level detection show that both GAS-Fe NPs and GAS-Fe@αPD-L1 NPs provided by this invention can significantly increase the intracellular Fe content in tumor cells. 2+ This increases the level of ROS and further induces ROS accumulation and lipid peroxidation.

[0233] The above results indicate that the nanoplatform provided by this invention can effectively induce ferroptosis in glioblastoma cells.

[0234] Furthermore, GAS-Fe@αPD-L1 NPs can load immune checkpoint inhibitors while maintaining ferroptosis induction capabilities, providing a basis for the synergistic effect of subsequent ferroptosis and immunotherapy.

[0235] In summary, the results of this embodiment demonstrate that the nanoplatform provided by the present invention has good ferroptosis induction ability and can kill glioblastoma cells by promoting Fe2+ accumulation, reactive oxygen species generation, and lipid peroxidation.

[0236] ## Example 9: Detection of GPX4 protein expression (corresponding to) Figure 17 ) This embodiment further verifies the molecular mechanism of ferroptosis induced by the nanoplatform provided by the present invention by detecting the expression level of glutathione peroxidase 4 (GPX4) protein.

[0237] GPX4 is a key antioxidant enzyme in the regulation of ferroptosis. Its downregulation usually indicates a decrease in the cell's ability to resist lipid peroxidation and is closely related to the occurrence of ferroptosis.

[0238] (1) Cell culture and drug treatment U251 glioblastoma cells were seeded into cell culture dishes and cultured at 37°C in a 5% CO2 incubator until the cells adhered stably.

[0239] Furthermore, the cells were randomly divided into the following treatment groups: 1) PBS control group; 2) GAS-Fe NPs group; 3) GAS-Fe@αPD-L1 NPs group.

[0240] In this embodiment, the treatment concentration for both the GAS-Fe NPs group and the GAS-Fe@αPD-L1 NPs group was 60 μg / mL.

[0241] Furthermore, each group of cells was continuously treated for 24 hours before subsequent testing was performed.

[0242] (2) Extraction of cell proteins After drug treatment, the culture medium was discarded, and the cells were washed with pre-cooled PBS buffer.

[0243] Further, cell lysis buffer was added to lyse the cells, and total protein was collected.

[0244] The protein concentration of each group of samples was determined using the BCA protein quantification kit to ensure that the sample loading amount of each group was consistent in subsequent tests.

[0245] (3) Western blot detection Equal amounts of protein samples were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane.

[0246] Furthermore, the target protein was incubated and detected using GPX4 primary antibody.

[0247] Vinculin was used as an internal reference protein for calibration.

[0248] After incubation, the corresponding secondary antibody was added for colorimetric detection, and protein band analysis was performed using a chemiluminescence imaging system.

[0249] Test results as follows Figure 17 As shown.

[0250] (4) Analysis of experimental results The results showed that, compared with the PBS control group, the expression level of GPX4 protein in both the GAS-Fe NPs group and the GAS-Fe@αPD-L1 NPs group was significantly reduced.

[0251] Furthermore, the GAS-Fe@αPD-L1 NPs group also maintained a significant GPX4 inhibitory effect.

[0252] The above results indicate that the nanoplatform provided by this invention can effectively inhibit the expression of GPX4 protein in tumor cells.

[0253] In this embodiment, GPX4, as an important antioxidant enzyme for scavenging lipid peroxides, will lead to a further increase in the level of cellular lipid peroxidation when its expression is downregulated, thereby promoting ferroptosis.

[0254] Combining ROS, lipid peroxidation, and Fe from Example 8 2+ The test results further demonstrate that the nanoplatform provided by this invention can effectively induce ferroptosis in glioblastoma cells by increasing oxidative stress levels and inhibiting the cellular antioxidant defense system.

[0255] In summary, the results of this embodiment show that the GAS-Fe NPs and GAS-Fe@αPD-L1 NPs provided by this invention can significantly downregulate GPX4 protein expression, further verifying their good ferroptosis induction ability.

[0256] Example 10: Immunogenic cell death detection (corresponding to) Figures 18-20 ) This embodiment evaluates the ability of the nanoplatform provided by the present invention to induce immunogenic cell death by detecting immunogenic cell death (ICD) related indicators such as ATP release, HMGB1 release, and exposure of calreticulin (CRT) membrane surface.

[0257] (1) Cell culture and experimental grouping U251 glioblastoma cells were seeded into 6-well cell culture plates and cultured at 37°C in a 5% CO2 incubator until the cells adhered stably.

[0258] Furthermore, the cells were randomly divided into the following treatment groups: 1) PBS control group; 2) Free GAS group; 3) GAS-Fe NPs group.

[0259] In this embodiment, the treatment concentration for both the free GAS group and the GAS-Fe NPs group was 60 μg / mL.

[0260] Further, each group of cells was treated for 24 hours before subsequent testing was performed.

[0261] (2) ATP release detection After drug treatment, the cell culture supernatant of each group was collected.

[0262] The level of extracellular ATP release was detected using an ATP assay kit.

[0263] Furthermore, the intensity of the corresponding fluorescence or luminescence signal was measured using an ELISA reader, and the amount of ATP released was calculated based on the standard curve.

[0264] Test results as follows Figure 18 As shown.

[0265] The results showed that, compared with the PBS control group, the extracellular ATP release in the GAS-Fe NPs group was significantly increased.

[0266] Furthermore, the change in ATP release was relatively small in the free GAS group.

[0267] The above results indicate that the GAS-Fe NPs provided by this invention can promote the release of ATP from tumor cells.

[0268] In this embodiment, ATP, as one of the important damage-associated molecular patterns (DAMPs), helps promote the recruitment of immune cells and immune activation through its release.

[0269] (3) HMGB1 release detection Further, the cell culture supernatant from each group was collected.

[0270] Extracellular HMGB1 levels were detected using an HMGB1 ELISA kit.

[0271] Test results as follows Figure 19 As shown.

[0272] The results showed that, compared with the PBS control group, the HMGB1 release level in the GAS-Fe NPs group was significantly increased.

[0273] The above results demonstrate that the nanoplatform provided by this invention can promote the release of HMGB1.

[0274] In this embodiment, HMGB1 release is one of the key characteristics of immunogenic cell death, which can further promote antigen presentation and immune system activation.

[0275] (4) CRT membrane surface exposure detection Furthermore, CRT membrane surface exposure was detected in cells from different treatment groups.

[0276] After drug treatment, the cells were immunofluorescence stained with CRT antibody.

[0277] Furthermore, the cell nuclei were counterstained with DAPI, and the changes in CRT fluorescence signal were observed using a confocal laser scanning microscope.

[0278] Test results as follows Figure 20 As shown.

[0279] The results showed that, compared with the PBS control group, the CRT fluorescence signal on the cell membrane surface of the GAS-Fe NPs group was significantly enhanced.

[0280] The above results indicate that the nanoplatform provided by this invention can promote the translocation of CRT from the intracellular space to the cell membrane surface.

[0281] In this embodiment, exposure of the CRT membrane surface helps promote the recognition and phagocytosis of tumor cells by dendritic cells, thereby enhancing the anti-tumor immune response.

[0282] (5) Analysis of experimental results The combined results of ATP release, HMGB1 release, and CRT membrane surface exposure detection show that the GAS-Fe NPs provided by this invention can significantly induce immunogenic cell death in glioblastoma cells.

[0283] Furthermore, the nanoplatform provided by this invention can not only directly kill tumor cells through ferroptosis, but also promote the release of DAMPs, thereby enhancing tumor-associated antigen exposure and immune system activation.

[0284] The above results indicate that the nanoplatform provided by this invention has good potential for immune activation, laying the foundation for synergistic effects with subsequent immune checkpoint blockade therapy.

[0285] In summary, the results of this embodiment demonstrate that the GAS-Fe NPs provided by this invention can effectively induce immunogenic cell death and promote the release of various immune-related signals, which is beneficial for enhancing the anti-tumor immune response in glioblastoma.

[0286] Example 11: Study on PD-L1 expression regulation (corresponding) Figures 21-22 ) This embodiment investigates the regulatory effect of the nanoplatform provided by the present invention on immune checkpoint-related molecules in tumor cells by detecting PD-L1 mRNA and protein expression levels.

[0287] (1) Cell culture and experimental grouping Mouse glioma cells GL261 were seeded into cell culture dishes and cultured at 37°C in a 5% CO2 incubator until the cells adhered stably.

[0288] Furthermore, the cells were randomly divided into the following treatment groups: PBS control group; 2) Free GAS group; 3) GAS-Fe NPs group; 4) GAS-Fe@αPD-L1 NPs group.

[0289] In this embodiment, the drug concentration in each treatment group was 60 μg / mL.

[0290] Furthermore, each group of cells was continuously treated for 24 hours before subsequent testing was performed.

[0291] (2) Detection of PD-L1 mRNA expression After drug treatment, total RNA was extracted from cells in each group using the TRIzol method.

[0292] Furthermore, the RNA was reverse transcribed to obtain cDNA, and the expression level of PD-L1 mRNA was detected by real-time quantitative PCR (qPCR).

[0293] Test results as follows Figure 21 As shown.

[0294] The results showed that, compared with the PBS control group, the expression of PD-L1 mRNA in each treatment group changed to some extent.

[0295] In some treatment groups, the level of PD-L1 mRNA showed an upregulation trend.

[0296] The above results indicate that the nanoplatform provided by this invention can influence PD-L1-related transcription processes in tumor cells.

[0297] (3) Detection of PD-L1 protein expression Furthermore, the expression level of PD-L1 protein in different treatment groups was detected by Western blot.

[0298] Specifically, after drug treatment, total protein was extracted from cells in each group, and protein quantification was performed using the BCA method.

[0299] SDS-PAGE electrophoresis was then performed to separate the components, which were then transferred to a PVDF membrane.

[0300] Furthermore, the cells were incubated with PD-L1 primary antibody and then incubated with the corresponding secondary antibody for colorimetric detection.

[0301] Finally, the expression of PD-L1 protein was analyzed using a chemiluminescence imaging system.

[0302] Test results as follows Figure 22 As shown.

[0303] The results showed that, compared with the PBS control group, the expression of PD-L1 protein was significantly reduced in the free GAS group, the GAS-Fe NPs group, and the GAS-Fe@αPD-L1 NPs group.

[0304] Furthermore, the GAS-Fe@αPD-L1 NPs group showed a more significant inhibitory effect on PD-L1 protein.

[0305] (4) Analysis of experimental results The combined results of qPCR and Western blot analysis show that the nanoplatform provided by this invention regulates PD-L1 at different levels, with some differences between the mRNA and protein levels.

[0306] Furthermore, although PD-L1 mRNA expression showed an upregulation trend in some treatment groups, PD-L1 protein expression still decreased significantly.

[0307] The above results indicate that the regulation of PD-L1 by the nanoplatform provided by this invention may mainly occur at the post-translational level or during protein degradation.

[0308] In this embodiment, GAS-Fe@αPD-L1 NPs can not only achieve immune checkpoint blockade by loading αPD-L1 antibody, but also reduce the expression level of PD-L1 protein in tumor cells, thereby further weakening the tumor's immune escape ability.

[0309] Furthermore, the nanoplatform provided by this invention, through the combination of ferroptosis induction and immune checkpoint regulation, helps to enhance the efficacy of anti-tumor immunotherapy.

[0310] In summary, the results of this embodiment demonstrate that the GAS-Fe NPs and GAS-Fe@αPD-L1 NPs provided by this invention can effectively regulate PD-L1 expression in tumor cells and have good immunomodulatory potential, providing a foundation for subsequent in vivo immunotherapy research.

[0311] Example 12: In vivo biodistribution experiment (corresponding to) Figures 23-24 ) This embodiment establishes an in situ glioblastoma animal model and uses in vivo fluorescence imaging technology to evaluate the distribution of the nanoplatform provided by this invention in vivo and its ability to target and enrich brain tumors.

[0312] (1) Establishment of an in situ glioma model A nude mouse model of glioblastoma in situ was established using U251 cells.

[0313] Specifically, U251 cells in the logarithmic growth phase were digested, collected, and prepared into a cell suspension.

[0314] Furthermore, under anesthesia, U251 cells were injected into the brain tissue of nude mice via stereotactic injection to establish an orthotopic glioma model.

[0315] After the model was established, the experimental animals were fed in a routine manner, and the tumor growth was observed.

[0316] (2) Preparation of Cy5-labeled nanoplatform GAS-Fe NPs and GAS-Fe@αPD-L1 NPs were fluorescently labeled using Cy5 fluorescent dye.

[0317] Furthermore, the successful labeling of Cy5 in the nanoplatform was confirmed by detecting the fluorescence signal.

[0318] (3) In vivo fluorescence imaging Nude mice with U251 gliomas in situ were randomly divided into groups and given Cy5-labeled nanoplatforms via tail vein injection.

[0319] In this embodiment, the dosage of both the GAS-Fe NPs group and the GAS-Fe@αPD-L1 NPs group was 10 mg / kg.

[0320] Furthermore, in vivo fluorescence imaging of small animals was performed at 0 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h and 24 h after drug administration.

[0321] Test results as follows Figure 23 As shown.

[0322] (4) Analysis of experimental results The results showed that both Cy5-labeled nanoplatforms were able to gradually accumulate in vivo after drug administration and reach a high fluorescence signal level at about 6 hours.

[0323] Furthermore, compared with the GAS-Fe NPs group, the GAS-Fe@αPD-L1 NPs group showed a stronger fluorescence signal in the brain tumor region.

[0324] The above results indicate that the nanoplatform provided by this invention has good brain tumor targeting and enrichment capabilities.

[0325] Furthermore, GAS-Fe@αPD-L1 NPs showed superior enrichment in brain tissue compared to GAS-Fe NPs without immune checkpoint inhibitor loading.

[0326] In this embodiment, the better brain tumor enrichment capacity of the nanoplatform may be related to the brain-targeted delivery mediated by GAS and the blood-brain barrier penetration ability enhanced by nanoparticles.

[0327] (5) Fluorescence imaging analysis of ex vivo organs Furthermore, the experimental animals were sacrificed 24 hours after drug administration, and their major organs and brain tissue were collected for in vitro fluorescence imaging analysis.

[0328] In this embodiment, the major organ includes: Heart, liver, spleen, lungs, and kidneys.

[0329] Test results as follows Figure 24 As shown.

[0330] The results showed that the nanoplatforms in each group were distributed to a certain extent in the liver and spleen, indicating that the nanoparticles can be metabolized through the reticuloendothelial system.

[0331] Furthermore, in brain tissue, the fluorescence signal of the GAS-Fe@αPD-L1 NPs group was significantly higher than that of the GAS-Fe NPs group and the free Cy5 group.

[0332] The above results further demonstrate that the nanoplatform provided by this invention can effectively penetrate the blood-brain barrier and achieve enrichment at the site of brain tumors.

[0333] (6) Summary of experimental results In summary, the results of this embodiment demonstrate that the GAS-Fe NPs and GAS-Fe@αPD-L1 NPs provided by this invention have good in vivo brain-targeted delivery capabilities.

[0334] Furthermore, GAS-Fe@αPD-L1 NPs can effectively penetrate the blood-brain barrier and achieve high enrichment at the glioblastoma site, providing a foundation for subsequent in vivo anti-tumor therapy and brain tumor imaging applications.

[0335] Example 13: Evaluation of in vivo antitumor efficacy (corresponding to) Figure 25 ) This embodiment evaluates the in vivo antitumor therapeutic effects of GAS-Fe NPs and GAS-Fe@αPD-L1 NPs provided by this invention by establishing an in situ glioma animal model.

[0336] (1) Establishment of an in situ glioma model Orthotopic glioma models were established using U251 cells and GL261 cells, respectively.

[0337] The U251 model was used to evaluate the therapeutic effect of the nanoplatform in a human glioblastoma model; the GL261 model was used to evaluate the antitumor immunotherapy effect of the nanoplatform in an immune-intact mouse model.

[0338] Specifically, U251 cells or GL261 cells in the logarithmic growth phase were prepared into cell suspensions.

[0339] Furthermore, under anesthesia, cells were seeded into the brain tissue of experimental animals using stereotactic injection.

[0340] After the model was established, the experimental animals were fed in a routine manner, and the tumor growth was observed.

[0341] (2) Experimental grouping and dosing regimen After the in situ tumor model was established, the experimental animals were randomly divided into the following treatment groups: 1) PBS control group; 2) GAS-Fe NPs group; 3) GAS-Fe@αPD-L1 NPs group.

[0342] In this embodiment, all treatment groups were administered the medication via tail vein injection.

[0343] Furthermore, the dosage for both the GAS-Fe NPs group and the GAS-Fe@αPD-L1 NPs group was 10 mg / kg.

[0344] In some implementations, the drug is administered once every 2 days for a total of 3 consecutive administrations.

[0345] (3) In vivo tumor growth monitoring For the U251 orthotopic glioma model, a small animal in vivo fluorescence imaging system was used to monitor tumor growth.

[0346] Furthermore, changes in fluorescence signals in the brains of each group of animals were detected at different time points.

[0347] For the GL261 orthotopic glioma model, a bioluminescence imaging system was used to monitor tumor growth.

[0348] Test results as follows Figure 25 As shown.

[0349] (4) Analysis of experimental results The results showed that the tumor fluorescence signal in the PBS control group continued to increase, indicating that the tumor was growing rapidly.

[0350] In contrast, tumor growth was somewhat inhibited in the GAS-Fe NPs group, and the tumor fluorescence signal was significantly lower than that in the PBS control group.

[0351] Furthermore, the GAS-Fe@αPD-L1 NPs group exhibited the most significant anti-tumor effect, with its tumor fluorescence signal being significantly lower than that of the PBS control group and the GAS-Fe NPs group.

[0352] The above results indicate that the GAS-Fe@αPD-L1 NPs provided by this invention can effectively inhibit the growth of glioblastoma.

[0353] Further analysis revealed that, in the GL261 immune integrity model, the GAS-Fe@αPD-L1 NPs group also showed significantly better anti-tumor therapeutic effects than the GAS-Fe NPs group.

[0354] The above results indicate that loading immune checkpoint inhibitors onto the nanoplatform can further enhance the anti-tumor immunotherapy effect.

[0355] In this embodiment, the excellent in vivo antitumor effect of GAS-Fe@αPD-L1 NPs may be due to the following reasons: 1) GAS-mediated brain-targeted delivery improves the enrichment efficiency of nanoplatforms at brain tumor sites; 2) Iron ions induce ferroptosis, thereby directly killing tumor cells; 3) αPD-L1 antibodies block tumor immune escape and enhance anti-tumor immune responses; 4) Ferroplasmosis-induced immunogenic cell death further enhances the efficacy of immunotherapy.

[0356] (5) Summary of experimental results In summary, the results of this embodiment demonstrate that the GAS-Fe@αPD-L1 NPs provided by this invention have good in vivo anti-glioblastoma therapeutic effects.

[0357] Furthermore, this nanoplatform can effectively inhibit glioblastoma through the synergistic effects of brain-targeted delivery, ferroptosis induction, and immune checkpoint blockade, providing a new technical solution for the treatment of glioblastoma.

[0358] Example 14: Immunohistochemistry and cytokine detection (corresponding) Figures 26-27 ) This embodiment evaluates the immunomodulatory effect of the nanoplatform provided by the present invention in vivo by immunohistochemical staining of tumor tissue and detection of serum cytokines.

[0359] (1) Handling of laboratory animals Subsequent experiments were conducted using the GL261 orthotopic glioma mouse model established in Example 13.

[0360] After the drug treatment was completed, the experimental animals in each group were euthanized, and brain tumor tissue and blood samples were collected.

[0361] Furthermore, tumor tissue was used for immunohistochemical staining analysis; blood samples were centrifuged and serum was collected for cytokine level detection.

[0362] (2) Immunohistochemical staining The collected brain tumor tissue was fixed with 4% paraformaldehyde and then embedded in paraffin.

[0363] Furthermore, the paraffin tissue sections were dewaxed, hydrated, and subjected to antigen retrieval treatment.

[0364] Subsequently, the tissue sections were subjected to immunohistochemical staining using CD8 antibody, CD4 antibody, and PD-L1 antibody, respectively.

[0365] After staining, the infiltration of immune cells and the expression of PD-L1 in tumor tissues of different treatment groups were observed under a microscope.

[0366] Test results as follows Figure 26 As shown.

[0367] (3) Analysis of immunohistochemical results The results showed that, compared with the PBS control group, the infiltration of CD8+ T cells and CD4+ T cells in tumor tissues of the GAS-Fe NPs group was increased.

[0368] Furthermore, the infiltration levels of CD8+ T cells and CD4+ T cells were significantly enhanced in the GAS-Fe@αPD-L1 NPs group.

[0369] The above results indicate that the nanoplatform provided by this invention can promote the recruitment and infiltration of immune cells in tumor tissues.

[0370] Further analysis revealed that, compared with the PBS control group, the expression of PD-L1 in tumor tissues of the GAS-Fe@αPD-L1 NPs group was significantly reduced.

[0371] The above results demonstrate that the nanoplatform provided by this invention can effectively reduce tumor immune escape and enhance the level of local tumor immune activation.

[0372] (4) Serum cytokine detection Furthermore, the serum levels of IL-2, IFN-γ, and TNF-α in each group of experimental animals were detected using an ELISA kit.

[0373] Specifically, serum samples from each group were added to the corresponding ELISA test plate, and the reaction and color development were performed according to the kit instructions.

[0374] The absorbance of each well was then measured using an ELISA reader, and the corresponding cytokine concentrations were calculated based on the standard curve.

[0375] Test results as follows Figure 27 As shown.

[0376] (5) Analysis of cytokine detection results The results showed that, compared with the PBS control group, the serum levels of IL-2, IFN-γ and TNF-α in the GAS-Fe NPs group were significantly increased.

[0377] Furthermore, the levels of the aforementioned cytokines were significantly increased in the GAS-Fe@αPD-L1 NPs group.

[0378] The above results indicate that the nanoplatform provided by this invention can effectively enhance the body's anti-tumor immune response.

[0379] In this embodiment, IL-2, IFN-γ, and TNF-α are important anti-tumor immune-related cytokines, and their elevated levels indicate that the body's immune system has been effectively activated.

[0380] Furthermore, combined with the results of CD8+ T cell and CD4+ T cell infiltration, it can be demonstrated that the nanoplatform provided by the present invention can improve the immunosuppressive microenvironment of glioblastoma.

[0381] (6) Summary of experimental results In summary, the results of this embodiment show that the GAS-Fe@αPD-L1 NPs provided by the present invention can not only directly inhibit tumor growth, but also promote the infiltration of immune cells in tumor tissue and enhance the body's anti-tumor immune response.

[0382] Furthermore, the nanoplatform provided by this invention helps to enhance the immunotherapy effect of glioblastoma through the synergistic effect of ferroptosis induction and immune checkpoint blockade.

[0383] Example 15: Biosafety Evaluation (corresponding to) Figures 28-29 ) This embodiment evaluates the biosafety of the nanoplatform provided by the present invention through pathological analysis of major organ tissues, detection of hematological indicators, and hemolysis experiments.

[0384] (1) Handling of laboratory animals The experimental animals used in Example 13 were used for subsequent safety evaluation.

[0385] After completing the drug treatment, the experimental animals were euthanized at the end of the experiment, and samples of major organs and blood were collected.

[0386] In this embodiment, the major organ includes: Heart, liver, spleen, lungs, and kidneys.

[0387] Furthermore, major organs were used for histopathological analysis; blood samples were used for hematological marker detection and hemolysis experiments.

[0388] (2) H&E staining analysis of major organs The collected major organs were fixed with 4% paraformaldehyde and then embedded in paraffin.

[0389] Further, after dewaxing and hydrating the paraffin tissue sections, they were stained with hematoxylin-eosin (H&E).

[0390] After staining, the morphological changes of major organs and tissues in different treatment groups were observed using an optical microscope.

[0391] Test results as follows Figure 28 As shown.

[0392] (3) Analysis of H&E staining results The results showed that, compared with the PBS control group, no obvious pathological changes such as tissue damage, inflammatory infiltration, cell necrosis or structural abnormalities were observed in the major organs and tissues of the GAS-Fe NPs group and the GAS-Fe@αPD-L1 NPs group.

[0393] Furthermore, the structures of the heart, liver, spleen, lungs, and kidneys in each group remained intact.

[0394] The above results indicate that the nanoplatform provided by this invention did not cause significant toxic damage to major organs under experimental drug administration conditions.

[0395] (4) Hematological index testing Furthermore, whole blood samples were collected from the experimental animals for hematological testing.

[0396] In this embodiment, the detection indicators include: White blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), platelet count (PLT), and related biochemical indicators.

[0397] Test results as follows Figure 29 As shown.

[0398] The results showed that, compared with the PBS control group, no significant abnormalities were observed in any hematological parameters in the GAS-Fe NPs group and the GAS-Fe@αPD-L1 NPs group.

[0399] The above results demonstrate that the nanoplatform provided by this invention has good blood compatibility and in vivo biosafety.

[0400] (5) Hemolysis test Furthermore, an in vitro hemolysis experiment was used to evaluate the effect of the nanoplatform on erythrocytes.

[0401] Specifically, fresh anticoagulated blood is collected, washed with PBS, and a red blood cell suspension is obtained.

[0402] Subsequently, different concentrations of GAS-Fe NPs and GAS-Fe@αPD-L1 NPs were added and incubated at 37°C for a certain period of time.

[0403] Furthermore, PBS was used as a negative control and deionized water as a positive control.

[0404] After incubation, the supernatant was collected by centrifugation, and the absorbance at 540 nm was measured using an ELISA reader to calculate the hemolysis rate.

[0405] Test results as follows Figure 29 As shown.

[0406] (6) Analysis of hemolysis test results The results showed that, compared with the positive control group, no obvious hemolysis was observed in either the GAS-Fe NPs group or the GAS-Fe@αPD-L1 NPs group.

[0407] Furthermore, within the experimental concentration range, the hemolysis rate of each group of nanoplatforms remained at a low level.

[0408] The above results indicate that the nanoplatform provided by this invention has good blood compatibility and is unlikely to cause significant damage to red blood cells.

[0409] (7) Summary of experimental results In summary, the results of this embodiment demonstrate that the GAS-Fe NPs and GAS-Fe@αPD-L1 NPs provided by this invention have good biocompatibility under experimental drug administration conditions.

[0410] Furthermore, the nanoplatform did not cause significant toxic damage to major organs, and also exhibited good blood compatibility and low hemolytic properties.

[0411] Therefore, the nanoplatform provided by this invention has good potential for biomedical applications and clinical translation prospects.

[0412] In summary, this invention provides a multifunctional nanoplatform based on the coordination self-assembly of gastrodin and iron ions, its preparation method, and its applications. This nanoplatform can synergistically integrate brain-targeted delivery, ferroptosis induction, and immune checkpoint blockade functions, thereby improving the therapeutic effect of glioblastoma. Experimental results show that the nanoplatform provided by this invention possesses good stability, blood-brain barrier penetration ability, tumor cell uptake capacity, anti-tumor activity, and biosafety, demonstrating promising application prospects in the treatment and imaging of glioblastoma.

[0413] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that various modifications, substitutions or equivalent transformations can be made to the technical solutions of the present invention without departing from the spirit and substance of the present invention, and such modifications, substitutions or equivalent transformations should all fall within the scope of protection defined by the claims of the present invention.

[0414] Furthermore, the various technical features in the specific embodiments described in this specification can be combined in any appropriate manner without contradicting each other, and the resulting technical solutions should all be considered as part of the disclosure of this invention.

[0415] The terms “including,” “comprising,” and “having” used in this specification are open-ended expressions and do not exclude the existence of other components, steps, or method units not explicitly listed.

[0416] It will be understood by those skilled in the art that the above embodiments are merely preferred embodiments of the present invention, used to illustrate the technical principles of the present invention, and not intended to limit the scope of protection of the present invention. The scope of protection of the present invention should be determined by the scope defined in the appended claims.

Claims

1. A multifunctional nanoplatform, characterized in that, The multifunctional nanoplatform comprises gastrodin-iron ion coordination nanoparticles and an immune checkpoint blocker loaded on the gastrodin-iron ion coordination nanoparticles.

2. The multifunctional nanoplatform of claim 1, wherein, The immune checkpoint inhibitor is selected from one or more of anti-PD-L1 antibody, anti-PD-1 antibody, and anti-CTLA-4 antibody. 3.The multifunctional nano-platform according to claim 1 or 2, wherein, The molar ratio of gastrodin to iron ions is 1:2 to 1:

10.

4. The multifunctional nanoplatform of claim 3, wherein, The molar ratio of gastrodin to iron ions is 1:

5.

5. The multifunctional nanoplatform according to any one of claims 1 to 4, wherein, The mass ratio of the gastrodin-iron ion coordination nanoparticles to the immune checkpoint blocker is 2:1 to 20:

1.

6. The multifunctional nanoplatform of claim 5, wherein, The mass ratio of the gastrodin-iron ion coordination nanoparticles to the immune checkpoint blocker is 6:

1.

7. A method of preparing the multifunctional nanoplatform of any one of claims 1 to 6, characterized in that, Includes the following steps: Gastrodin solution was mixed with iron salt solution to form gastrodin-iron ion coordination nanoparticles through coordination self-assembly. An immune checkpoint inhibitor is added during the coordination self-assembly process, thereby loading the immune checkpoint inhibitor onto the gastrodin-iron ion coordination nanoparticles to obtain the multifunctional nanoplatform.

8. The method of claim 7, wherein, The iron salt is ferric chloride; and / or, The mixing is carried out under vortex conditions, which include a vortex time of 1-2 minutes and a rotation speed of 3000 rpm.

9. The use of the multifunctional nanoplatform of any one of claims 1 to 6, or the multifunctional nanoplatform prepared by the method of claim 7 or 8, in the preparation of a medicament for treating glioblastoma.

10. The use of the multifunctional nanoplatform of any one of claims 1 to 6, or the multifunctional nanoplatform prepared by the method of claim 7 or 8, in the preparation of a medicament for glioblastoma imaging.

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