Nanocomposite, intermediate thereof, preparation method, application, injection device and system thereof

Abstract

The invention discloses a nano-composite for near-infrared induced explosive ferroptosis and a preparation method and application thereof.The nano-composite is prepared from nano-particles loaded with a ferroptosis inducer and iron-doped dopamine and targeting ligands modified on the surfaces of the nano-particles, the nano-particles take manganese dioxide of a hollow mesoporous structure as a carrier, the ferroptosis inducer is loaded in the hollow mesopores, and the iron-doped dopamine is assembled on the surface of the carrier. Dual regulation and control of effective degradation of endogenous GSH and specific improvement of ROS level in tumor cells are realized through a specific nano composite material and a unique structure, and ferroptosis is increased in combination with a photothermal effect so as to inhibit tumor growth.

Description

[0001] This application is a divisional application of Chinese application filed on March 11, 2025, with application number 2025102855892 and entitled "Near-infrared induced explosive ferroptosis nanocomposite and its preparation method and application". Technical Field

[0002] This invention relates to the field of nanobiotechnology, specifically to near-infrared induced explosive ferroptosis nanocomposites, their preparation methods, and applications. Background Technology

[0003] Ferroptosis, a novel form of programmed cell death, is characterized by lipid peroxidation (LPO) triggered by the accumulation of iron-dependent reactive oxygen species (ROS). In recent years, this mechanism has been shown to be an effective strategy for cancer treatment and has received widespread attention in the field of anticancer research.

[0004] Existing research indicates that the execution mechanism of ferroptosis mainly relies on ROS-mediated phospholipid oxidation, thereby regulating cell death through the formation of oxidized phospholipid products. However, the tumor microenvironment (TME) contains the following key limiting factors:

[0005] 1. Insufficient endogenous ROS levels: The inherent ROS concentration in tumor cells is insufficient to induce the production of sufficient LPO, which significantly weakens the ferroptosis effect.

[0006] 2. Antioxidant defense mechanism: The glutathione (GSH) system, which is highly expressed in tumor cells, can clear ROS, maintain intracellular redox homeostasis and protect cells from external oxidation, thereby reducing the production of LPO.

[0007] The aforementioned mechanisms lead to technical bottlenecks in traditional ferroptosis induction strategies, such as low therapeutic efficiency and the ease with which tumor cells develop adaptive resistance.

[0008] In addition, some small molecule iron-lowering inducers exhibit poor pharmacokinetics and unsatisfactory GSH depletion when inducing iron lowering by directly or indirectly inhibiting or degrading glutathione peroxidase (GPX4), making them unsuitable for achieving precise and efficient regulatory results in antitumor therapy.

[0009] Therefore, developing complexes that can simultaneously achieve the effective degradation of endogenous GSH and the specific enhancement of ROS levels in tumor cells has become a key technological requirement for enhancing ferroptosis to fight tumors. Summary of the Invention

[0010] To address the aforementioned problems, the present invention aims to provide a near-infrared induced burst ferroptosis nanocomposite, its preparation method, and its application. Through specific nanocomposite materials and unique structures, it achieves dual regulation of effective degradation of endogenous GSH and specific enhancement of ROS levels in tumor cells, and combines photothermal effects to increase ferroptosis to inhibit tumor growth.

[0011] This invention provides a near-infrared induced burst ferroptosis nanocomposite, comprising nanoparticles loaded with a ferroptosis inducer and iron-doped dopamine, and a targeting ligand modified on the surface of the nanoparticles. The nanoparticles are supported by manganese dioxide with a hollow mesoporous structure. The ferroptosis inducer is loaded within the hollow mesopores, and the iron-doped dopamine is assembled on the surface of the support. The nanocomposite encapsulates the ferroptosis inducer RSL3 within the hollow mesopores of manganese dioxide HM-MnO2 nanoparticles, assembles iron-doped dopamine (Fe-PDA) on the HM-MnO2 surface, and modifies the nanoparticles with a cRGD tumor-targeting peptide. When the nanoparticles are targeted and delivered to the tumor site, the nanocomposite exhibits peroxidase (POD), oxidase (OXD), glutathione peroxidase (GPx), and NADH (Nicotinamide Adenine Dinucleotide). Hydride exhibits multi-enzyme activity similar to nicotinamide adenine dinucleotide oxidase (NOx), thereby generating reactive oxygen species (ROS) and depleting glutathione (GSH) to construct a tumor microenvironment (TME) suitable for ferroptosis. It simultaneously achieves a dual regulatory strategy of effectively degrading endogenous GSH and specifically enhancing intracellular ROS levels in tumor cells. Furthermore, under near-infrared irradiation at a specific wavelength, the Fe-PDA-mediated photothermal effect can accelerate the degradation of the nanocomposite and achieve a large release of RSL3. These cascade reactions, in synergistic photothermal effects, can induce a strong ferroptosis effect, significantly inhibiting tumor growth.

[0012] In some embodiments, the nanoparticles are hollow spherical structures with an average diameter of about 50-200 nm and a hydrodynamic diameter of 181±10.2 nm.

[0013] Nanomaterials with specific structures and sizes, such as the hollow mesoporous nanospheres in the examples, when used as drugs, not only enhance drug loading but also enable therapeutic drugs to be released in response to the tumor microenvironment (TME) to enhance iron ion apoptosis.

[0014] In some embodiments, the loading of the ferroptosis inducer in the nanoparticles is 50-70%.

[0015] In some embodiments, the specific surface area of ​​the manganese dioxide carrier is 20-60 m². 2 / g, with an average pore size of 2-10nm.

[0016] In some embodiments, the targeting ligand is a cyclic arginine-glycine-aspartic peptide.

[0017] This invention also provides a method for preparing a near-infrared induced explosive ferroptosis nanocomposite, comprising the following steps:

[0018] S1. Preparation of hollow mesoporous manganese dioxide nanoparticles;

[0019] S2. Hollow mesoporous manganese dioxide nanoparticles and ferrode death inducer were dispersed in methanol, ultrasonicated, stirred for 8-16 hours, and centrifuged to collect the first intermediate product.

[0020] S3. Disperse the first intermediate product in a weakly alkaline buffer solution, add dopamine and iron salt, and stir for 6-10 hours to obtain the second intermediate product.

[0021] S4. Add a crosslinking agent containing disulfide bonds to the second intermediate product and continue stirring for 20-28 hours;

[0022] S5. React the obtained product with the targeted ligand solution activated by the activator, and continue stirring for 12-24 hours;

[0023] S6. Centrifuge to collect the final product, wash and freeze-dry to obtain the nanocomposite.

[0024] In some embodiments, the hollow mesoporous manganese dioxide nanoparticles described in step S1 are prepared by a template method.

[0025] In some embodiments, the mass ratio of the hollow mesoporous manganese dioxide nanoparticles to the ferrodegeneration inducer in step S2 is (3.5-4.5):1, and the ultrasonic treatment time is 25-35 minutes.

[0026] In some embodiments, the buffer solution in step S3 is a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution with a pH of 8.3-8.7, and the iron salt is Fe(NO3)3·9H2O, with an addition amount of 0.05-0.15 mL per 10 mg of the first intermediate product.

[0027] In some embodiments, the disulfide-containing crosslinking agent in step S4 is NH2-SS-NH2 cystamine disulfide, and the amount added is 8-12 mg per 10 mg of the second intermediate product.

[0028] In some embodiments, the targeting ligand in step S5 is synthesized from a functionalized polyethylene glycol derivative, and the activator is a mixed solution of EDC and NHS.

[0029] In some embodiments, the washing in step S6 uses a mixture of ethanol and water in a volume ratio of (1:1) to (3:1), and the washing is performed 2 to 4 times.

[0030] The near-infrared induced burst ferroptosis nanocomposite preparation method provided in this invention involves first encapsulating the ferroptosis inducer RSL3 in hollow mesoporous HM-MnO2 nanoparticles and ultrasonically stirring to obtain a first intermediate product. Then, the first intermediate product is dispersed in a weakly alkaline buffer solution, and dopamine and iron salt are added and stirred to obtain a second intermediate product. Next, a crosslinking agent containing disulfide bonds is added to the second intermediate product and stirred. The resulting product is reacted with a target ligand solution activated by an activator and stirred. After centrifugation, the final product is collected, washed, and freeze-dried to obtain a nanocomposite with surface-assembled iron-doped dopamine (Fe-PDA) and modified cRGD tumor-targeting peptide. The preparation process is simple and controllable, and the drug loading is high.

[0031] This invention also provides the application of the above-mentioned near-infrared responsive nanocomposite in the preparation of antitumor drugs.

[0032] In some embodiments, the tumor is squamous cell carcinoma of the head and neck.

[0033] The near-infrared induced explosive ferroptosis nanocomposite provided in this invention induces explosive ferroptosis in tumor cells, especially oral squamous cell carcinoma (OSCC), by inhibiting glutathione and enhancing LOP lipid peroxidation. It exhibits excellent tumor targeting and near-infrared enhanced degradation, and has beneficial effects in the application of anti-tumor drugs.

[0034] The near-infrared induced explosive ferroptosis nanocomposite, its preparation method, and its application provided in this invention embodiment induce explosive ferroptosis in oral squamous cell carcinoma (OSCC) by inhibiting glutathione (GSH) and enhancing LOP lipid peroxidation. First, the prepared hollow MnO2 nanozyme has a high drug loading capacity, achieving a drug loading rate of 67%. Second, by modifying the surface with iron-doped dopamine (Fe-PDA), the photothermal properties are significantly improved. Finally, the synthesized cRGD-modified multifunctional MnO2-R@FePDA-cRGD nanoreactor exhibits excellent tumor targeting and near-infrared enhanced degradation. In vitro / in vivo experiments show that after the nanocomposite accumulates at the tumor site, it responds to the degradation of the tumor microenvironment under near-infrared irradiation, explosively releasing the carried RSL3 to induce ferroptosis. At the same time, the photothermal effect promotes multi-enzyme activity, catalyzes ROS generation and enhances LPO, and achieves efficient tumor ferroptosis by consuming GSH and oxidizing NADH, synergistically inhibiting the GPX4 and FSP1 pathways.

[0035] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0036] Figure 1 Characterization of MNO2R@FEPDAC in Examples 1-3 is shown, where (a) shows a transmission electron microscope (TEM) image of HM-MnO2, (b) shows the nitrogen adsorption-desorption isotherm and pore size distribution curve of HM-MnO2, (c) shows a TEM image of MNO2R@FEPDAC and (d) shows an EDS elemental distribution map with a resolution of 50 nm, (e) shows a comparison of the XRD patterns of MnO2 nanoparticles and MNO2R@FEPDAC, (f) shows the full-spectrum XPS spectrum, (g) shows the corresponding Mn 2p and (h) shows the high-resolution XPS spectrum of Fe 2p, and (i) shows the Fourier transform infrared spectrum.

[0037] Figure 2 The photothermal properties of MNO2R@FEPDAC in Examples 4-5 are shown, where (a) shows the photothermal properties at 1 W / cm². 2 The temperature change curves under near-infrared irradiation, (b) show the photothermal stability of MNO2R@FEPDAC (100 μg / mL) during five switching cycles, and (c) show the photothermal conversion efficiency: the red line represents 1 W / cm². 2 Photothermal effect after 10 minutes of laser irradiation; violet line shows the time constant (τs) measured during the cooling phase; (d) shows the UV-Vis absorption spectra of free HM-MnO2, RSL3, and MNO2R@FEPDAC; (e) shows the effect with and without near-infrared irradiation (1W / cm²) under simulated tumor microenvironment conditions (pH = 5.4 containing 10 mM GSH and 100 μM H2O2). 2 The RSL3 release behavior of MNO2R@FEPDAC after 10 minutes of continuous exposure is shown in (f), which shows the absorbance of oxTMB at 652 nm after different treatments, indicating peroxidase-like activity (n=3, **p<0.01). (g) shows the transmission electron microscopy image of MNO2R@FEPDAC after 1 hour of incubation at pH 7.4 with 10 mM GSH. (h) shows the transmission electron microscopy image after 1 hour of incubation under simulated tumor microenvironment conditions. (i) shows the transmission electron microscopy image after 10 minutes of near-infrared irradiation under simulated tumor microenvironment conditions.

[0038] Figure 3The enzymatic reaction kinetics of MNO2R@FEPDAC in Examples 6-10 are shown. (a) shows peroxidase-like activity with H2O2 as the substrate; (b) shows oxidase-like activity with TMB as the substrate; (c) shows the Michaelis-Menten equation curves for glutathione peroxidase-like activity; and (d) shows the Michaelis-Menten equation curves for nitric oxide synthase-like activity. (e) shows a schematic diagram of the mechanism of action of the multi-enzyme mimicry activity; (f) shows the generation of hydroxyl radicals (·OH) and (g) superoxide anion radicals (O2·-) detected by electron spin resonance spectroscopy; and (h) shows the absorbance changes at 652 nm after each experimental group reacted with 100 μM H2O2 and 2 mM TMB for 5 minutes.

[0039] Figure 4 Examples 11-14 illustrate the in vitro antitumor activity and mechanism of action of MNO2R@FEPDAC on HN6 cells. Specifically, (a) shows a confocal laser scanning microscopy (CLSM) image of IR-780-labeled MNO2R@FEPDAC after 6 hours of treatment; (b) shows the relative cell viability after 24 hours of treatment with different concentrations of free HM-MnO2 (n=6); (c) shows the relative cell viability of different treatment groups (n=6); (d) shows fluorescent staining images of live cells (green) / dead cells (red); (e) shows intracellular reactive oxygen species (ROS, green) / nuclear (blue) fluorescence images; (f) shows the Liperfluo probe fluorescence imaging of lipid peroxide (LPO) content; (g) shows the MDA content of HN6 cells detected by the kit; (h) shows the GSH content; (j) shows the NADH content (n=6); (i) shows bio-transmission electron microscopy (Bio-TEM) images of HN6 cells with different pretreatments; and (k) shows Western spectroscopy images. Blot analysis was performed on the protein expression levels of GPX4, FSP1, FTH1, and FDX1 after 24 hours of different treatments. The groups were: i. control group, ii. near-infrared group, iii. MNO2R@FEPDAC group, and iv. MNO2R@FEPDAC + near-infrared group. Near-infrared parameters: 808nm laser, 0.5W / cm². 2 Irradiation for 5 minutes, *p<0.05, ***p<0.001.

[0040] Figure 5 The in vivo antitumor activity of MNO2R@FEPDAC in the HN6 tumor-bearing mouse model as shown in Examples 15-16 is illustrated. (a) shows whole-body optical in vivo imaging at different time points after administration of IR-780-labeled MNO2R@FEPDAC and in vitro fluorescence imaging 48 hours after injection. (b) shows the in vivo antitumor activity at 808 nm and 0.5 W / cm² at 48 hours after injection. 2Infrared thermographic image of the tumor site under near-infrared laser irradiation, (c) shows Figure 5 (b) shows the temperature change curve of the tumor region; (d) shows the tumor growth curves of different experimental groups over 14 days (n=5); (e) shows macroscopic photographs of tumor samples from each group after treatment; (f) shows the tumor weight statistics (n=5); (g) shows H&E staining, TUNEL cell apoptosis detection, and Ki67 proliferation marker immunofluorescence staining of tumor tissues; (g) shows the immunofluorescence staining results of FTH1, GPX4, and FDX1 proteins. Scale bar: 100 micrometers. ***p<0.001.

[0041] Figure 6 The in vivo biodegradation and biosafety of MNO2R@FEPDAC in Example 17 are shown, where (a) shows the changes in manganese (Mn) and iron (Fe) concentrations in various organs at different time points after injection. (b) shows the analysis of blood biochemical indicators 14 days after different treatments.

[0042] Figure 7 The photothermal conversion efficiency of MnO2@PDA was shown.

[0043] Figure 8 The image shown is a scanning electron microscope (SEM) image of HM-MnO2 nanoparticles from Example 3.

[0044] Figure 9 The results of dynamic light scattering (DLS) tests on the MNO2R@FEPDAC of Example 3 are shown.

[0045] Figure 10 The results of the elemental surface scan in the material obtained by energy-dispersive X-ray spectroscopy (EDS) in Example 3 are shown, with a resolution of 100 nm.

[0046] Figure 11 The results of the d-stability experiment of de MNO2R@FEPDAC in physiological media over 7 days in Example 3 are shown.

[0047] Figure 12 The UV-Vis-NIR spectrophotometric absorption spectrum of the MNO2R@FEPDAC of Example 4 is shown, in which a polysiloxane phosphate composite material (PSPc) is used.

[0048] Figure 13 The different power ratings (0.5 W / cm²) of the MNO2R@FEPDAC in Example 4 are shown. 2 1.0W / cm 2 1.5W / cm 2 The heating results under ( ).

[0049] Figure 14 The concentration standard curve of RSL3 in Example 5 is shown.

[0050] Figure 15 The results of TMB oxidation capacity of Fe-PDA and MNO2R@FEPDAC in Example 6 are shown.

[0051] Figure 16 The results of the concentration gradient experiments of the oxidase-like enzyme (OXD) of Example 6 with different concentrations of TMB as substrate are shown.

[0052] Figure 17 The results of the peroxidase-like (POD) activity experiment of Example 6 with different concentrations of H2O2 as substrates are shown.

[0053] Figure 18 The absorbance characteristics of 5,5-dithiobis(2-nitrobenzoic acid) DTNB of Example 7 with varying incubation times are shown to indicate GSH consumption of MNO2R@FEPDAC.

[0054] Figure 19 The results of the Michaelis-Menten kinetic parameters of the Fe-PDA nanozyme of Example 7, which mimics glutathione peroxidase (GPx), catalyze glutathione (GSH).

[0055] Figure 20 The UV-Vis absorbance of different concentrations of MNO2R@FEPDAC in Example 8 is shown to demonstrate the NADH oxidation capacity of MNO2R@FEPDAC.

[0056] Figure 21 The results of the Michaelis-Menten kinetic parameters of NADH catalyzed by the Fe-PDA nanozyme mimicking nitrite oxidase (NOx-like) of Example 8 are shown.

[0057] Figure 22 The results show the differences in mouse body weight in the control group, near-infrared light group, MNO2R@FEPDAC group, and MNO2R@FEPDAC+near-infrared light group of Example 16.

[0058] Figure 23 The results of control H&E (hematoxylin & eosin) staining of the major organs in Example 17 are shown to confirm the biocompatibility of MNO2R@FEPDAC. Detailed Implementation

[0059] The following will clearly and completely describe the concept and technical effects of this application in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are all within the scope of protection of this application.

[0060] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0061] For details regarding the reagents and their manufacturers used in the following examples, please refer to Table 1:

[0062] Table 1. Main Reagent Names and Manufacturers

[0063]

[0064]

[0065] For details regarding the instrument models and manufacturers used in the following embodiments, please refer to Table 2:

[0066] Table 2. Main Instrument Models and Manufacturers

[0067]

[0068] Example 1: Preparation method of hollow mesoporous manganese dioxide HM-MnO2 nanoparticles

[0069] This invention provides a method for preparing hollow mesoporous manganese dioxide nanoparticles, comprising the following steps:

[0070] (1) Synthesis of solid silica nanoparticles (SiO2): In this embodiment, solid silica nanoparticles (SiO2) are synthesized by reducing TEOS (tetraethyl orthosilicate) in ammonia water. By adjusting the reaction parameters, including temperature, concentration, stirring speed, etc., silica nanoparticles with an adjustable particle size in the range of 30-500nm can be obtained. In this embodiment, by adjusting the ammonia water concentration, silica nanoparticles with a particle size in the range of 50-200nm are obtained.

[0071] (2) KMnO4 was then added under ultrasonic conditions. After reacting for 6 hours, the suspension was centrifuged at 14,000 rpm to collect the precipitate.

[0072] (3) The obtained mesoporous MnO2 coated SiO2 was then dispersed in a Na2CO3 aqueous solution at 60℃ and stirred overnight to remove the SiO2 template, and finally hollow mesoporous MnO2 (HM-MnO2) nanoparticles were obtained.

[0073] Example 2: Preparation method of MnO2-RSL3@FePDA-cRGD nanocomposite

[0074] This invention provides a method for preparing MnO2-RSL3@FePDA-cRGD nanocomposites, comprising the following steps:

[0075] (1) Solid silica nanoparticles (SiO2) are synthesized by existing methods, such as template method, using PS microspheres as templates, depositing SiO2 and then removing the template to obtain a hollow porous structure.

[0076] (2) KMnO4 was then added under ultrasonic conditions. After reacting for 6 hours, the suspension was centrifuged at 14,000 rpm to collect the precipitate.

[0077] (3) The obtained mesoporous MnO2 coated SiO2 was then dispersed in a Na2CO3 aqueous solution at 60℃ and left to stand overnight to finally obtain hollow mesoporous MnO2 (HM-MnO2) nanoparticles.

[0078] (4) Disperse 20 mg HM-MnO2 in 10 mL of methanol, then add 5 mg RSL3.

[0079] (5) Sonicate the mixture for 30 minutes and stir continuously overnight.

[0080] (6) The product was collected by centrifugation at 8000 rpm, washed three times with PBS buffer, and labeled as MnO2-RSL3.

[0081] (7) Then 10 mg of MnO2-RSL3 was dispersed in 10 mL of Tris-HCl buffer (pH 8.5), and 1 mg of dopamine and 0.1 mL of Fe(NO3)3·9H2O solution were added.

[0082] (8) After stirring at room temperature for 8 hours, add 10 mg of NH2-SS-NH2 and continue stirring for 24 hours.

[0083] (9) The obtained material was mixed with COOH-PEG activated by EDC / NHS. 2000 The -cRGD solution was reacted and stirred overnight.

[0084] (10) Finally, wash three times with an ethanol / water mixture, freeze-dry and store at 4°C.

[0085] Example 3 Characterization and Performance Testing

[0086] The HM-MnO2 nanoparticles of Example 1 were tested as follows:

[0087] Please participate Figure 1 Transmission electron microscopy (TEM) of a and Figure 8 Scanning electron microscope (SEM) images confirmed that the material formed a hollow mesoporous structure with a clear surface morphology.

[0088] Brunauer-Emmett-Teller (BET) tests showed that the specific surface area of ​​the HM-MnO2 nanoparticles in Example 1 was 33.013 m². 2 / g, with an average pore size of 3.5nm, indicating that it has high specific surface area and mesoporous properties. Figure 1 b).

[0089] These properties make it a highly efficient nanocarrier.

[0090] The MnO2-RSL3@FePDA-cRGD nanocomposite of Example 2 was tested as follows:

[0091] like Figure 1 As shown in c, the final MNO2-RSL3@FePDA-cRGD nanocomposite (hereinafter referred to as MNO2R@FEPDAC) obtained in Example 2 maintains a hollow spherical structure with an average diameter of about 150 nm.

[0092] Dynamic light scattering (DLS) measurements showed its hydrodynamic diameter to be 181 ± 10.2 nm (see...). Figure 9 ).

[0093] Energy-dispersive X-ray spectroscopy (EDS) surface scan results indicate that the material is rich in Mn and Fe elements, and also contains C, N, O, S, and Cl elements (see [link to EDS]). Figure 1 d and Figure 10 ).

[0094] X-ray diffraction (XRD) analysis showed that the characteristic diffraction peaks at 36.7° and 66.3° belonged to MnO2 crystals, while broadened peaks were present in the 20-30° range, corresponding to the presence of polydopamine (PDA). Figure 1 e).

[0095] In-depth analysis of the elemental composition and chemical state of materials using X-ray photoelectron spectroscopy (XPS) Figure 1 f).

[0096] The binding energy peaks at 652.5 eV and 640.8 eV correspond to the Mn 2p1 / 2 and Mn 2p3 / 2 orbitals, respectively, and are assigned to Mn orbitals through peak fitting.4+ 2p1 / 2 and Mn 4+ 2p3 / 2 confirms that Mn is predominantly in the +4 oxidation state. Figure 1 g).

[0097] Similarly, the fitting peaks at 723.5 eV and 716.7 eV in the Fe 2p1 / 2 spectrum correspond to Fe, respectively. 3+ with Fe 2+ The 712.2 eV and 710.0 eV peaks in the Fe 2p3 / 2 spectrum further confirm that Fe 3+ / Fe 2+ coexist( Figure 1 h).

[0098] Fourier transform infrared spectroscopy (FT-IR) verified the successful modification of the material surface with PDA and cRGD. Figure 1 i).

[0099] Stability tests show (see) Figure 11 MNO2R@FEPDAC maintained excellent stability in physiological media for 7 days, with no decrease in UV-Vis absorption.

[0100] In summary, the experimental data confirm that the nanoreactor was successfully constructed and possesses good stability.

[0101] Example 4: MNO2R@FEPDAC Photothermal Effect Test

[0102] MNO2R@FEPDAC was dispersed in PBS solutions with concentrations of 0, 25, 50, 75, and 100 μg / mL, using a 1 W / cm² concentration. 2 Irradiate with an 808nm laser at a power density for 10 minutes.

[0103] Temperature changes were monitored using a Fluke Ti27 infrared thermal imager (USA). Additionally, MNO2R@FEPDAC solutions with concentrations of 100 μg / mL were subjected to thermal stresses of 0.5, 1.0, and 1.5 W / cm². 2 The system was irradiated with a power density, and the temperature change curves of the heating process and subsequent natural cooling stage were fully recorded.

[0104] The photothermal conversion efficiency (η) is calculated using the following formulas 1-4.

[0105] η=(hS(T max -T surr )-Q dis ) / I(1–10 -A (1)

[0106] hS=∑mCp / τS (2)

[0107] τS =-t / lnθ (3)

[0108] θ=(TT surr ) / (T max -T surr (4)

[0109] In the formula, h is the heat transfer coefficient, S is the container area, τS represents the system heat transfer time constant, m is the system mass (1 gram), Cp (4.2 joules / (g·℃)) is the specific heat capacity of water, and τS = 408.1 seconds is taken from... Figure 2 c.

[0110] hS is calculated using formula (2) (hS = 1 × 4.2 / 408.1 = 10.29 mW / ℃), Qdis is independently measured to be 74.84 mW, Tmax is the equilibrium temperature, Tsurr is the ambient temperature, and I = 1.0 W / cm². 2 A represents the absorbance at a wavelength of 808 nanometers (A808 = 0.425).

[0111] Therefore, the conversion efficiency η is calculated as follows:

[0112] η={[10.29×(48.0-24.3)-74.84] / [1000×(1-10 -0 · 425 )]}×100%=39.1%.

[0113] Specifically, its absorption spectrum was analyzed using a UV-Vis-NIR spectrophotometer.

[0114] like Figure 12 As shown, MNO2R@FEPDAC exhibits significant absorption characteristics in the near-infrared region. The absorption of nanoparticle suspensions at different concentrations under an 808 nm laser (1.0 W / cm²) was monitored using an infrared thermal imager. 2 The temperature change after 10 minutes of irradiation was evaluated to assess its photothermal performance.

[0115] like Figure 2 As shown in Figure a, compared with the PBS control group, the MNO2R@FEPDAC solution exhibits a concentration-dependent temperature increase, indicating its ability to efficiently convert laser energy into heat energy. Furthermore, the photothermal properties of this material show a power density-dependent trend (see Figure a). Figure 13 ).

[0116] It is worth noting that no temperature decay was observed after five cycles of heating and cooling. Figure 2 b) This fully demonstrates its excellent photothermal stability.

[0117] like Figure 2c shows that at 1.0 W / cm 2 Under irradiation conditions, the photothermal conversion efficiency of MNO2R@FEPDAC reaches 39.1%, which is comparable to that of other technologies. Figure 7 The photothermal conversion efficiency of MnO2@PDA is 24.03%, which provides sufficient energy support for improving the catalytic performance and degradation ability of MNO2R@FEPDAC.

[0118] Example 5: Controlled release behavior of MNO2R@FEPDAC and near-infrared response degradation characteristics of tumor microenvironment (TME).

[0119] MNO2R@FEPDAC was incubated for 1 hour in phosphate-buffered saline (PBS) at pH 5.5 or 7.4 with or without glutathione (GSH) and hydrogen peroxide (H2O2). The release kinetics of RSL3 under different conditions were quantitatively analyzed by UV-Vis spectrophotometry. The samples were then characterized by transmission electron microscopy (TEM).

[0120] like Figure 2 As shown in Figure d, the characteristic absorption peak at 280 nm confirms the successful loading of RSL3 in MNO2R@FEPDAC, based on the concentration standard curve of RSL3 ( Figure 14 The calculated drug loading was 67%.

[0121] Under simulated TME conditions (pH = 5.4, containing 10 mM GSH and 100 μM H2O2), MNO2R@FEPDAC exhibited a slow RSL3 release profile, while near-infrared (NIR) irradiation triggered a rapid and broad drug release. Figure 2 e).

[0122] In addition, we investigated the effect of NIR irradiation on the catalytic ability of MNO2R@FEPDAC by evaluating the peroxidase (POD) activity of MNO2R@FEPDAC with and without NIR irradiation.

[0123] After NIR treatment, the absorbance of oxTMB at 652 nm was significantly enhanced. Figure 2 f) indicates that the photothermal effect of MNO2R@FEPDAC not only promotes rapid drug release but also significantly enhances catalytic performance, providing sufficient energy for inducing explosive ferroptosis.

[0124] The NIR-responsive drug release mechanism of MNO2R@FEPDAC was further investigated by observing its morphological changes. Under physiological conditions of pH 7.4, MNO2R@FEPDAC exhibited excellent structural stability. Figure 2 g).

[0125] However, after incubation for 1 hour under simulated TME conditions, the nanoparticle structure exhibited partial degradation. Figure 2 In contrast, under simulated TME conditions, MNO2R@FEPDAC underwent complete degradation after 10 minutes of NIR irradiation (h). Figure 2 i).

[0126] This dual-response characteristic demonstrates that MNO2R@FEPDAC possesses both physiological stability and the ability to achieve precise drug release and catalytic enhancement through photothermal triggering within the tumor microenvironment, providing technical support for spatiotemporally controllable ferroptosis therapy.

[0127] Example 6: Multienzyme mimicry activity characterization test of MNO2R@FEPDAC

[0128] This embodiment provides a multi-enzyme simulation test of the MNO2R@FEPDAC nanoreactor. Peroxidase-like (POD) activity was detected using 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate in the presence of hydrogen peroxide (H2O2). The absorbance change of oxidized TMB (oxTMB) at a wavelength of 652 nm was monitored at specific reaction time points using a UV-Vis spectrophotometer. Relevant kinetic parameters were calculated based on the Michaelis-Menten equation and its two reciprocals (as shown in Equation (5) below).

[0129]

[0130] In formula (5), V0 and Vmax represent the initial reaction rate and the maximum reaction rate, respectively, [S] represents the TMB substrate concentration, and Km is the Michaelis constant (i.e., the substrate concentration corresponding to the reaction rate reaching half of the maximum rate).

[0131] For the standard assay of oxidase-like (OXD) activity, the kinetic characteristics of the MNO2R@FEPDAC catalyst were evaluated by setting different concentrations of TMB substrate (0.1-8 mmol / L) in acidic buffer solutions. The absorbance values ​​of each group at 652 nm were recorded using a UV-Vis spectrophotometer.

[0132] To evaluate nitric oxide synthase (NOx)-like activity, solutions containing different concentrations of MNO2R@FEPDAC (0, 5, 10, 50, 100, 150, and 200 μg / mL) were incubated with 2 mmol / L NADH and 100 μmol / L H2O2 at room temperature for 30 minutes, and the absorbance was recorded using a UV-Vis spectrophotometer. Subsequently, the 200 μg / mL MNO2R@FEPDAC solution was dispersed in pH 4.5 buffer containing different concentrations of NADH (0.05–2 mmol / L) and reacted for 10 minutes, and the absorbance of the supernatant of each sample was measured at 340 nm.

[0133] Glutathione peroxidase (GPx) activity was assessed using DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) as a substrate. Specifically, 100 μg / mL MNO2R@FEPDAC solution was mixed with 0.5 mmol / L glutathione (GSH) solution. At a specified time point, 0.5 mL of the mixture was added to 2.5 mL of DTNB solution, thoroughly mixed, and centrifuged at 14,000 rpm for 5 minutes. The supernatant was collected for absorbance measurement. Separately, 200 μg / mL MNO2R@FEPDAC was dispersed in pH 4.5 buffer containing different concentrations of GSH (0.1–3 mmol / L), reacted with DTNB for 10 minutes, and the absorbance at 412 nm was measured using a UV-Vis spectrophotometer.

[0134] The peroxidase-like (POD) activity assessment used 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate, which generates a blue product (oxTMB) with a characteristic absorption peak at 652 nm. Figure 15 As shown, both Fe-PDA and MNO2R@FEPDAC exhibit TMB oxidation ability under acidic conditions.

[0135] In a concentration gradient experiment using H₂O₂ as the substrate, the kinetic parameters were determined by fitting curves to the Michaelis equation.

[0136] The Michaelis constant Km of MNO2R@FEPDAC is 0.011 mM (millomoles / liter), and the maximum reaction rate Vmax is 2.136 × 10⁻⁷ M·s⁻¹. In contrast, Fe-PDA has a Km value of 0.259 mM and a Vmax value of 3.56 × 10⁻⁹ M s⁻¹, which are two orders of magnitude lower than those of MNO2R@FEPDAC (see...). Figure 3 a and Figure 16 ).

[0137] Oxidase-like (OXD) activity was assessed using a TMB substrate concentration gradient assay. Steady-state analysis showed:

[0138] Km=0.059mM, Vmax=3.453×10 of MNO2R@FEPDAC -7 M·s -1 Compared to Fe-PDA, it exhibits a significantly improved catalytic efficiency (see...). Figure 3 b and Figure 17 ).

[0139] The above data confirm that MNO2R@FEPDAC exhibits significant advantages in both substrate affinity and catalytic activity. Its lower Km value indicates a stronger binding ability to substrates such as H2O2, while its higher Vmax value reflects a more efficient catalytic conversion.

[0140] Example 7: Glutathione (GSH) Consumption Capacity Test of MNO2R@FEPDAC

[0141] The residual GSH level of MNO2R@FEPDAC after incubation with excess GSH for different times was quantitatively analyzed using 5,5-dithiobis(2-nitrobenzoic acid) (DTNB).

[0142] like Figure 18 As shown, the characteristic absorbance of DTNB at 412 nm decreased significantly with increasing incubation time, indicating that MNO2R@FEPDAC can effectively consume GSH.

[0143] Furthermore, under simulated tumor microenvironment (TME) conditions, MNO2R@FEPDAC exhibited Michaelis kinetics in substrate concentration-dependent experiments with GSH.

[0144] The measured Km and Vmax values ​​were 0.24 mM and 1.657 × 10⁻⁶, respectively. -9 M·s -1 ( Figure 3 c) significantly higher than the corresponding values ​​for Fe-PDA (1.52 mM and 0.213 × 10⁻⁶). -9 M·s -1 ,See Figure 19 ).

[0145] Example 8: Assay of NOx-like nitrite oxidase activity of MNO2R@FEPDAC

[0146] Ferrocyte inhibitor protein (FSP1), an NADH-dependent coenzyme Q oxidoreductase, exerts its anti-ferroptosis effect by inhibiting lipid peroxidation (LPO) by reducing ubiquinone (CoQ10) to ubiquinol (CoQ10H2).

[0147] In this process, NADH provides the necessary reducing power for FSP1 to perform its antioxidant function. Therefore, NADH is oxidized to NAD.+ This is an effective strategy to enhance ferroptosis.

[0148] To evaluate the nitrite oxidase (NOx-like) activity of MNO2R@FEPDAC, it was incubated with 2 mM NADH at pH 4.5 and 37°C for 30 minutes.

[0149] like Figure 20 As shown, the UV-Vis spectrum reveals that the absorbance at 340 nm gradually decreases with increasing MNO2R@FEPDAC concentration, indicating its high NADH oxidation capacity.

[0150] The measured values ​​of Km and Vmax were 0.15 mM and 1.455 × 10⁻⁶, respectively. -6 M·s -1 It is significantly better than the corresponding value of Fe-PDA (see Figure 3 d and Figure 21 ).

[0151] These results highlight the superior multi-enzyme biomimetic catalytic properties of MNO2R@FEPDAC, including peroxidase-like (POD-), oxidase-like (OXD-), glutathione peroxidase-like (GPx-), and nitrite oxidase-like (NOx-like) activities. Figure 3 e), demonstrating its great potential in inducing ferroptosis in tumor cells.

[0152] Example 9: Catalytic potential test of MNO2R@FEPDAC in regulating free radicals

[0153] The generation of hydroxyl radicals (·OH) and superoxide radicals (O2·-) was detected by electron spin resonance (ESR) spectroscopy combined with the radical spin trapping agent 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO).

[0154] like Figure 3 As shown in f, even without the addition of H2O2, a characteristic ·OH signal peak with an intensity ratio of approximately 1:2:2:1 can still be observed in the MNO2R@FEPDAC solution at pH 5.4. Upon the addition of H2O2, the ·OH signal is significantly enhanced, indicating that this material possesses intrinsic oxidase-like (OXD-like) activity under acidic conditions and can catalyze the decomposition of H2O2 to generate a large amount of ·OH.

[0155] Similarly, under the same conditions without the addition of H2O2, a weak O2·- signal can be detected, while the O2·- signal intensity increases sharply in the presence of H2O2. Figure 3 g).

[0156] These findings suggest that MNO2R@FEPDAC has significant catalytic potential in regulating ·OH and O2·- free radicals, indicating its broad application prospects in the field of tumor therapy.

[0157] Example 10: Synergistic effect test of near-infrared irradiation and MNO2R@FEPDAC

[0158] This embodiment provides a test of the enhancing effect of near-infrared (NIR) irradiation on the rate of Fenton / Fenton-like reactions and the generation of reactive oxygen species (ROS).

[0159] Specifically:

[0160] At pH 5.4, the appearance of a characteristic absorption peak at 652 nm after introducing MNO2R@FEPDAC into H2O2 solution confirmed the formation of ROS. Figure 3 h).

[0161] The intensity of this characteristic peak increased significantly after NIR irradiation. Experimental results indicate that near-infrared radiation can accelerate the Fenton / Fenton-like reaction rate and increase the ROS yield.

[0162] Therefore, the synergistic effect of near-infrared irradiation and MNO2R@FEPDAC provides a powerful strategy for amplifying the ferroptosis effect in tumor cells.

[0163] Example 11: Assay of the ability of MNO2R@FEPDAC cRGD tumor-targeting peptides to internalize into tumor cells

[0164] 5×10 4 Each HN6 cell was seeded into a confocal culture dish and cultured overnight.

[0165] After incubating with 20 μg / mL IR780-labeled MNO2R@FEPDAC for 6 hours, the cells were washed with PBS and stained with Hoechst 33342 for 10 minutes.

[0166] Confocal images were acquired using a Leica Stellaris 5 microscope from Germany.

[0167] This embodiment evaluated the ferroptosis-mediated killing efficiency of MNO2R@FEPDAC against oral squamous cell carcinoma (OSCC) cells. Efficient endocytosis of nanomaterials is a key step in achieving tumor cell clearance.

[0168] To visualize the cellular uptake process, the internalization behavior of IR-780-labeled MNO2R@FEPDAC in HN6 cells was observed using confocal laser scanning microscopy (CLSM).

[0169] After 6 hours of incubation, dense red fluorescent signals were visible around the cell nucleus. Figure 4 a) indicates that the cRGD tumor-targeting peptide modified on the surface of the nanomaterial can effectively promote its efficient internalization by tumor cells.

[0170] This embodiment also first evaluated the potential cytotoxicity of free HM-MnO2. After treatment with 100 μg / mL HM-MnO2 for 24 hours, the cell viability remained above 90%. Figure 4 b) This fully demonstrates its good biocompatibility.

[0171] Example 12: Near-infrared Response Cell Killing Rate Test of MNO2R@FEPDAC

[0172] HN6 cells were distributed at a rate of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 1,000 cells per well in 96-well plates and incubated overnight.

[0173] The following day, cells were exposed to different concentrations of MNO2R@FEPDAC (0, 10, 25, 50, 100 μg / mL) for 24 hours to assess cytotoxicity.

[0174] In the antitumor experiment of this embodiment, the cells were first co-incubated with medium containing 40 μg / mL MNO2R@FEPDAC for 6 hours, then replaced with fresh medium, and a power density of 0.5 W / cm² was used. 2 Irradiate with an 808nm laser for 5 minutes.

[0175] After culturing for another 24 hours, cell viability was detected using a CCK-8 assay kit.

[0176] In addition, under the same treatment conditions, cells were stained at 37°C in the dark for 15 minutes using 1 μg / mL calcein-AM / PI double staining reagent, and finally, fluorescent images of live / dead cells were captured using an Olympus IX71 fluorescence microscope from Japan.

[0177] Compared with the control group, the group receiving only near-infrared (NIR) irradiation (0.5 W / cm²) 2 Cell viability remained close to 100% even after 5 minutes. Figure 4 c) indicates that the near-infrared radiation phototoxicity under this parameter is extremely low.

[0178] When treated with 40 μg / mL MNO2R@FEPDAC, cell viability decreased by 31.8%, indicating a moderate ferroptosis effect.

[0179] After being exposed to near-infrared irradiation, the cell killing rate reached as high as 95.2%, which is presumably due to the photothermal activation of ferroptosis.

[0180] To visually verify the treatment effect, a live / dead cell staining experiment was conducted using calcein-AM (green fluorescent labeling of live cells) and propidium iodide (red fluorescent labeling of dead cells).

[0181] The results showed that cells in the MNO2R@FEPDAC combined with near-infrared irradiation group exhibited complete red fluorescence. Figure 4 d), which is highly consistent with cell survival data.

[0182] This embodiment demonstrates that the synergistic combination of photothermal effect and ferroptosis mechanism offers promise for the application of antitumor drugs, especially drugs for the treatment of oral squamous cell carcinoma (OSCC).

[0183] Example 13: Multi-enzyme catalytic activity and photothermal response RSL3 release characteristics of MNO2R@FEPDAC under tumor microenvironment (TME) conditions.

[0184] This embodiment includes:

[0185] 1. Detection of intracellular reactive oxygen species (ROS) and lipid peroxides (LPO):

[0186] HN6 cells were administered at a rate of 1×10 4 Inoculated at a density of cells / well in 96-well plates and incubated for 24 hours, then replaced with fresh medium (pH = 6.5) containing 100 μM H2O2 and 40 μg / mL MNO2R@FEPDAC solution.

[0187] Cells in the near-infrared treatment group were subjected to a power density of 0.5 W / cm². 2 Cells were irradiated with an 808nm laser for 5 minutes and then cultured overnight. Next, the cells were incubated with DCFH-DA (ROS probe) and Liperfluo (LPO probe) for 30 minutes each, washed three times with PBS, and observed under a fluorescence microscope.

[0188] 2. Detection of cellular malondialdehyde (MDA), glutathione (GSH), and reduced coenzyme I (NADH) levels:

[0189] Commercially available reagent kits were used to quantitatively detect the levels of MDA, GSH, and NADH.

[0190] Specifically, HN6 cells were seeded in 96-well plates and pre-cultured for 24 hours, and then co-cultured for 24 hours with different treatment groups (PBS group, near-infrared group, MNO2R@FEPDAC group, and MNO2R@FEPDAC+near-infrared combined group).

[0191] After culture, cells were collected by digestion and centrifugation, and quantitative analysis of each indicator was performed using the corresponding kit.

[0192] Specifically, detection using the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe revealed that cells treated with MNO2R@F1, EPDAC, and near-infrared spectroscopy (NIR) under simulated TME conditions produced a large amount of ROS (reactive oxygen species). Figure 4 e).

[0193] The production of lipid peroxides (LPO), a key trigger for ferroptosis, was visualized using fluorescence imaging. The MNO2R@FEPDAC+NIR group showed the most significant intracellular LPO levels. Figure 4 f).

[0194] In addition, the formation of LPO was verified by detecting malondialdehyde (MDA), a decomposition product of LPO.

[0195] like Figure 4 As shown in g, near-infrared irradiation alone has no effect on MDA levels, while MNO2R@FEPDAC treatment significantly increases MDA levels, and the index further increases after combined near-infrared treatment.

[0196] Glutathione (GSH) depletion is also crucial for enhancing ferroptosis.

[0197] Specifically, the relative GSH level in HN6 cells treated with MNO2R@FEPDAC+NIR decreased by 75%, significantly lower than that in other experimental groups. Figure 4 This indicates that the nanomaterial possesses excellent glutathione peroxidase (GPx) biomimicry and can effectively consume intracellular GSH.

[0198] Previous studies have reported that cancer cells rely on high levels of reduced nicotinamide adenine dinucleotide (NADH) to promote GSH synthesis, maintain cell homeostasis, and resist ferroptosis.

[0199] Therefore, we evaluated the biomimicry of NADH oxidase (NOx) in HN6 cells. Figure 4 j) The results showed that the NADH level in the MNO2R@FEPDAC+NIR group was significantly reduced, confirming that the nanomaterial can inhibit intracellular GSH production by consuming NADH.

[0200] These data indicate that the strong multi-enzyme activity of MNO2R@FEPDAC can significantly enhance the ferroptosis effect in tumor cells.

[0201] Example 14: Intracellular ferroptosis effect of MNO2R@FEPDAC cells

[0202] Ferrapoptosis is known to induce oxidative damage to the inner mitochondrial membrane and cause significant morphological changes.

[0203] Transmission electron microscopy (TEM) images showed that HN6 cells treated with MNO2R@FEPDAC (with or without near-infrared irradiation) exhibited ferroptosis-related mitochondrial dysfunction features, including reduced mitochondrial volume, increased membrane density, decreased or absent cristae, and balloon-like morphology. Figure 4 i).

[0204] To further investigate the effects of ferroptosis, we used Western blot to detect the expression levels of intracellular ferroptosis-related proteins.

[0205] Specifically, this embodiment uses a Western blot experiment, and the steps are as follows:

[0206] 1. Cells were lysed on ice using radioimmunoprecipitation analysis buffer to obtain protein extracts.

[0207] 2. Protein concentration was quantified using the BCA method (dioctanoic acid method).

[0208] 3. Equal amounts of protein were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane with a pore size of 0.45 micrometers.

[0209] 4. The membrane was blocked with bovine serum albumin (BSA) and then incubated with primary antibody overnight at 4°C.

[0210] 5. After the washing step, the membrane was incubated with horseradish peroxidase-labeled secondary antibody at room temperature for 1 hour.

[0211] 6. Protein bands were visualized using a chemiluminescence imaging system (Bio-Rad, Singapore), and the band intensity was normalized to the β-actin level.

[0212] like Figure 4 As shown in k, the expression of glutathione peroxidase 4 (GPX4), a key antagonist of ferroptosis, was significantly downregulated, which is consistent with the decrease in intracellular GSH levels.

[0213] Ferrocyte inhibitory protein (FSP1) plays a crucial role in scavenging LPO radicals by converting coenzyme Q10 (CoQ10(H)) to CoQ10H2. However, the oxidation of NAD(P)H to NAD(P)+ disrupts the biological function of FSP1. The biomimetic NADH oxidase (NOx) of MNO2R@FEPDAC effectively inhibits the ferroptosis-resistant FSP1 protein.

[0214] In addition, MNO2R@FEPDAC treatment also suppressed the expression of other ferroptosis-related proteins such as ferritin heavy chain 1 (FTH1) and ferroredoxin 1 (FDX1).

[0215] In summary, MNO2R@FEPDAC triggers a strong intracellular ferroptosis effect through multiple mechanisms, including efficient intracellular uptake, multi-enzyme biomimicry, photothermal responsive ROS storm, lipid metabolism disruption, and GPX4 / FSP1 pathway regulation, thereby significantly enhancing ferroptosis in tumor cells.

[0216] Example 15: Evaluation of MNO2R@FEPDAC's in vivo tumor targeting and anti-tumor capabilities

[0217] The in vivo biodistribution and photothermal effect experiment in this embodiment specifically includes:

[0218] 1. Tumor-bearing mice were administered 10 mg / kg of IR780-labeled MNO2R@FEPDAC via intravenous injection.

[0219] 2. In vivo fluorescence images were acquired at set time points (3, 6, 12, 24 and 48 hours) using the Caliper IVIS Spectrum imaging system (PerkinElmer, USA).

[0220] 3. Ex vivo fluorescence imaging of the heart, liver, spleen, lungs, kidneys and tumors was performed 48 hours after injection.

[0221] 4. Mice were anesthetized 48 hours after injection, and the tumor site was treated with an 808nm laser at 0.5W / cm². 2 Irradiate for 5 minutes.

[0222] 5. Use an infrared thermal imager to monitor temperature changes at the tumor site.

[0223] Specifically, based on the above experimental methods, the tumor targeting ability of MNO2R@FEPDAC was studied using the HN6 tumor-bearing mouse model. After intravenous injection of IR-780-labeled MNO2R@FEPDAC, its biodistribution characteristics over time were monitored using an in vivo fluorescence imaging system (IVIS).

[0224] like Figure 5 As shown in Figure a, the fluorescence signal at the tumor site gradually increased over time, reaching a peak at 48 hours post-injection and beginning to decay after 72 hours. Ex vivo fluorescence imaging at 48 hours confirmed the effective accumulation of MNO2R@FEPDAC in tumor tissue. This targeted accumulation may be attributed simultaneously to the size-dependent enhanced penetration and retention (EPR) effect and the active targeting capability mediated by cRGD.

[0225] By monitoring 0.5W / cm 2 The in vivo photothermal properties of MNO2R@FEPDAC were assessed by evaluating tumor site temperature changes under near-infrared irradiation. Thermal imaging showed that the tumor area temperature gradually increased to a steady state of 48.2℃ within 5 minutes. Figure 5 b) and c) confirm that the nanomaterial has excellent photothermal effect in vivo.

[0226] Therefore, MNO2R@FEPDAC shows significant application potential in amplifying reactive oxygen species generation and promoting ferroptosis.

[0227] Example 16: In vivo antitumor effect of MNO2R@FEPDAC

[0228] To confirm the in vivo therapeutic effect of MNO2R@FEPDAC, we evaluated its anti-tumor activity in an HN6 tumor-bearing mouse model.

[0229] Specifically, NOD-SCID mice were subcutaneously injected with 1×10⁻⁶ solution suspended in 100 μL of PBS. 6 HN6 cells. When the tumor volume reaches approximately 200 mm. 3 Mice were randomly divided into four groups (n=5 per group): control group, near-infrared light group, MNO2R@FEPDAC group and MNO2R@FEPDAC+near-infrared light group.

[0230] On day 0, mice were given intravenous injections of PBS (100 μL) or MNO2R@FEPDAC (10 μL) containing 10 mg / kg.

[0231] Forty-eight hours later, 808nm laser irradiation (0.5W / cm²) was applied to the tumor sites of the designated groups. 2 (Continued for 5 minutes). Weight was recorded every other day during the experiment, and tumor volume was measured every 2 days using calipers.

[0232] Mice were sacrificed on day 14, and tumors and major organs (heart, liver, spleen, lung, and kidney) were collected and fixed in 4% paraformaldehyde for histopathological and immunohistochemical analysis.

[0233] The formula for calculating tumor volume is V = major diameter × minor diameter. 2 / 2.

[0234] Based on the above experiments, the tumor suppression effect was evaluated by continuously measuring the tumor volume after different treatment regimens.

[0235] In the near-infrared (NIR) treatment group, an 808nm laser (0.5W / cm²) was used 48 hours after injection. 2 Irradiate the tumor for 5 minutes.

[0236] like Figure 5 As shown in d, MNO2R@FEPDAC effectively inhibits tumor growth by inducing ferroptosis through its multi-enzyme-mimicking activity in the tumor microenvironment (TME).

[0237] In stark contrast, the MNO2R@FEPDAC combined with NIR group showed complete inhibition or even ablation of tumor growth. Figure 5 e).

[0238] At the end of the treatment, the tumor weight in the combination therapy group was significantly lower than that in the other groups. Figure 5 f). Pathological analysis of tumor tissue, including hematoxylin-eosin (H&E) staining, TUNEL staining, and Ki67 staining, further confirmed the remarkable antitumor effect of MNO2R@FEPDAC combined with NIR. Figure 5 g).

[0239] No significant difference in mouse body weight was observed across the groups throughout the experiment, demonstrating the good biocompatibility of the nanomaterial (see...). Figure 22 ).

[0240] Example 17: In vivo biodegradation and biosafety of MNO2R@FEPDAC.

[0241] The in vivo clearance rate and biosafety assessment experiments in this embodiment include:

[0242] Major organs were collected on days 0, 1, 3, 7 and 14 following intravenous administration of MNO2R@FEPDAC (10 mg / kg).

[0243] After the organs were digested with nitric acid, the iron and manganese contents were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) to assess their biodegradability.

[0244] On day 14, a 0.6 mL blood sample was collected via the orbital venous plexus, placed in a heparinized test tube, and centrifuged at 3000 rpm for 15 minutes at 4°C to obtain serum.

[0245] Blood biochemistry tests related to liver and kidney function were performed by Wuhan Xavier Biotechnology Co., Ltd. (China).

[0246] Major organs such as the heart, liver, lungs, spleen, and kidneys were collected at different time points after intravenous injection.

[0247] The biodegradability of MNO2R@FEPDAC was characterized by quantitative analysis of manganese (Mn) and iron (Fe) concentrations using inductively coupled plasma optical emission spectrometry (ICP-OES).

[0248] like Figure 6 As shown in Figure a, the Mn and Fe contents increased significantly on the first day after injection, but gradually decreased from the 7th day. By the 14th day, the elemental levels returned to the baseline state before injection, indicating that the nanomaterial can be effectively cleared during the treatment cycle.

[0249] Furthermore, blood biochemical analyses were performed on day 14 post-treatment to assess liver function (including ALT, AST, ALP, ALB, and T-BIL, representing alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, albumin, and total bilirubin, respectively) and kidney function (UA, BUN, and CR, corresponding to uric acid, blood urea nitrogen, and creatinine, respectively). The levels of these biomarkers were within the normal range in all groups, indicating that no abnormalities in liver or kidney function were observed in any group. Figure 6 b).

[0250] Furthermore, H&E staining results of major organs showed that, compared with the control group, no significant tissue damage was observed in the MNO2R@FEPDAC injection group (see...). Figure 23 ).

[0251] These results confirm the excellent biocompatibility and degradability of MNO2R@FEPDAC.

[0252] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A nanocomposite intermediate, characterized in that, include: Using manganese dioxide with a hollow mesoporous structure as a carrier, iron death inducer is loaded in the hollow mesopores of the carrier, and iron-doped dopamine is assembled on the surface of the carrier; The iron-doped dopamine imparts a near-infrared photothermal effect to the intermediate, enabling it to respond to the tumor microenvironment and accelerate degradation under near-infrared irradiation, thereby releasing the ferroptosis inducer explosively.

2. The nanocomposite intermediate as described in claim 1, characterized in that, The loading of the ferroptosis inducer is 50-70%.

3. A nanocomposite, characterized in that, include: The nanocomposite intermediate as described in claim 1 or 2; as well as A targeting ligand attached to the surface of the intermediate; The nanocomposite exhibits multi-enzyme activity in the tumor microenvironment, generating reactive oxygen species and consuming glutathione. Through the synergistic effect of the explosive release of the ferroptosis inducer, the accumulation of reactive oxygen species, and the depletion of glutathione, it induces explosive ferroptosis in tumor cells.

4. The nanocomposite according to claim 3, characterized in that, The targeting ligand is a cyclic arginine-glycine-aspartic acid peptide.

5. The nanocomposite as described in claim 3 or 4, characterized in that, The multi-enzyme activity includes at least one of peroxidase-like activity, oxidase-like activity, glutathione peroxidase-like activity, and NADH oxidase-like activity.

6. A method for preparing the nanocomposite intermediate as described in claim 1 or 2, characterized in that, Includes the following steps: Hollow mesoporous manganese dioxide nanoparticles are provided; The hollow mesoporous manganese dioxide nanoparticles were mixed with an iron death inducer in a solvent, so that the iron death inducer was loaded into the hollow mesopores of the nanoparticles to obtain a first intermediate product. Under weakly alkaline conditions, the first intermediate product is reacted with dopamine and iron salt to assemble an iron-doped dopamine layer on its surface, thus obtaining the nanocomposite intermediate.

7. A method for preparing nanocomposites as described in any one of claims 3-5, characterized in that, Includes the following steps: The nanocomposite intermediate as described in claim 1 or 2 is crosslinked using a crosslinking agent containing disulfide bonds; Targeting ligands are attached to the cross-linked product.

8. Use of the nanocomposite as described in any one of claims 3-5 in the preparation of a medicament for treating oral squamous cell carcinoma.

9. An injection device, characterized in that, include: Therapeutic effective amount of the nanocomposite as described in any one of claims 3-5.

10. A system for inducing tumor ferroptosis, characterized in that, include: The injection device of claim 9 is used to deliver the nanocomposite to a tumor site; Near-infrared equipment is used to irradiate the tumor site; The near-infrared photothermal effect provided by the near-infrared device is configured to trigger the explosive degradation of the nanocomposite delivered by the injection device at the tumor site through the photothermal effect, thereby synergistically inducing ferroptosis in tumor cells.

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