Near-infrared induced explosive ferroptosis nano-composite as well as preparation method and application 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

Technical Field

[0001] The present invention relates to the field of nano-biotechnology, and in particular to a near-infrared-induced explosive ferroptosis nanocomposite and a preparation method and application thereof. Background Art

[0002] Ferroptosis is a new type of programmed cell death. Its core feature is the lipid peroxidation (LPO) effect triggered by iron-dependent reactive oxygen species (ROS) accumulation. In recent years, this mechanism has been proven to be an effective tumor treatment strategy and has received widespread attention in the field of anti-cancer research.

[0003] Existing studies have shown that the execution mechanism of ferroptosis mainly depends on the ROS-mediated phospholipid oxidation process, which regulates cell death by forming oxidized phospholipid products. However, there are the following key limiting factors in the tumor microenvironment TME:

[0004] 1. Insufficient endogenous ROS levels: The inherent ROS concentration in tumor cells is difficult to induce sufficient LPO production, significantly weakening the ferroptosis effect;

[0005] 2. Antioxidant defense mechanism: The glutathione (GSH) system highly expressed in tumor cells will scavenge ROS, maintain intracellular redox homeostasis and protect cells from external oxidation, thereby reducing the production of LPO.

[0006] The above-mentioned mechanisms lead to technical bottlenecks in traditional ferroptosis induction strategies, such as low therapeutic efficiency and susceptibility of tumor cells to adaptive resistance.

[0007] In addition, some small molecule ferroptosis inducers, when inducing ferroptosis by directly or indirectly inhibiting or degrading glutathione peroxidase (GPX4), have poor pharmacokinetics and produce unsatisfactory GSH consumption, which makes them less suitable for achieving precise and efficient regulatory results in anti-tumor.

[0008] Therefore, the development of a dual-regulatory complex that can simultaneously achieve the effective degradation of endogenous GSH and the specific increase of ROS levels in tumor cells has become a key technical requirement for enhancing ferroptosis to fight tumors. Summary of the invention

[0009] To solve the above problems, the purpose of the present invention is to provide a near-infrared induced burst ferroptosis nanocomposite and its preparation method and application, which realizes the dual regulation of effective degradation of endogenous GSH and specific increase of ROS level in tumor cells through specific nanocomposite materials and unique structure, and combines photothermal effect to increase ferroptosis to inhibit tumor growth.

[0010] The embodiment of the present 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, wherein the nanoparticles use manganese dioxide with 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. The nanocomposite is obtained by encapsulating the ferroptosis inducer RSL3 in manganese dioxide HM-MnO with a hollow mesoporous structure. 2 The hollow mesopores of nanoparticles and the 2 Iron-doped dopamine (Fe-PDA) is assembled on the surface, and cRGD tumor targeting peptide is modified on the nanoparticles. When the nanoparticles are targeted and delivered to the tumor site, the nanocomplex exhibits peroxidase (POD), oxidase (OXD), glutathione peroxidase (GPx) and NADH (Nicotinamide Adenine Dinucleotide Hydride, nicotinamide adenine dinucleotide reduced state) oxidase (NOx)-like multi-enzyme activities, thereby generating reactive oxygen species (ROS) and depleting glutathione (GSH) to construct a tumor microenvironment TME suitable for ferroptosis, while achieving a dual regulatory strategy of effective degradation of endogenous GSH and specific enhancement of ROS levels in tumor cells. At the same time, under near-infrared irradiation of a specific wavelength, the photothermal effect mediated by Fe-PDA can accelerate the degradation of the nanocomplex and achieve a large amount of RSL3 release. These cascade reactions synergize with the photothermal effect to induce a strong ferroptosis effect, significantly inhibiting tumor growth.

[0011] 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.

[0012] When used as drugs, nanomaterials with specific structures and sizes, such as the hollow mesoporous nanospheres in the examples, not only enhance drug loading, but also enable the therapeutic drugs to be responsively released to the tumor microenvironment (TME) to enhance iron ion apoptosis.

[0013] In some embodiments, the loading amount of the ferroptosis inducing agent in the nanoparticles is 50-70%.

[0014] In some embodiments, the specific surface area of ​​the manganese dioxide carrier is 20-60m 2 / g, and the average pore size is 2-10nm.

[0015] In some embodiments, the targeting ligand is a cyclic arginine-glycine-aspartic acid peptide.

[0016] The present invention also provides a method for preparing a near-infrared-induced explosive ferroptosis nanocomposite, comprising the following steps:

[0017] S1. Preparation of hollow mesoporous manganese dioxide nanoparticles;

[0018] S2, dispersing the hollow mesoporous manganese dioxide nanoparticles and the ferroptosis inducer in methanol, stirring for 8-16 hours after ultrasonic treatment, and centrifuging to collect the first intermediate product;

[0019] S3, dispersing the first intermediate product in a weak alkaline buffer, adding dopamine and iron salt, and stirring for 6-10 hours to obtain a second intermediate product;

[0020] S4, adding a disulfide bond-containing cross-linking agent to the second intermediate product, and continuing stirring for 20-28 hours;

[0021] S5, reacting the obtained product with a targeting ligand solution activated by an activator, and continuing stirring for 12-24 hours;

[0022] S6. Collect the final product by centrifugation, wash it, and freeze-dry it to obtain the nanocomposite.

[0023] In some embodiments, the hollow mesoporous manganese dioxide nanoparticles in step S1 are prepared by a template method.

[0024] In some embodiments, the mass ratio of the hollow mesoporous manganese dioxide nanoparticles to the ferroptosis inducer in step S2 is (3.5-4.5):1, and the ultrasonic treatment time is 25-35 minutes.

[0025] In some embodiments, the buffer in step S3 is tris-hydrochloric acid buffer with a pH of 8.3-8.7, and the iron salt is Fe(NO 3 ) 3 9H 2 O, and its added amount is 0.05-0.15 mL per 10 mg of the first intermediate product.

[0026] In some embodiments, the disulfide bond-containing cross-linking agent in step S4 is NH 2 -SS-NH 2 Cystamine disulfide is added in an amount of 8-12 mg per 10 mg of the second intermediate product.

[0027] In some embodiments, the targeting ligand in step S5 is synthesized by a functionalized polyethylene glycol derivative, and the activator is a mixed solution of EDC and NHS.

[0028] In some embodiments, the washing in step S6 uses a mixture of ethanol and water in a volume ratio of (1:1)-(3:1), and the number of washing times is 2-4 times.

[0029] The method for preparing the near-infrared induced explosive ferroptosis nanocomposite provided in the embodiment of the present invention comprises firstly forming a hollow mesoporous HM-MnO 2 The ferroptosis inducer RSL3 is encapsulated in the nanoparticles and ultrasonically stirred to obtain a first intermediate product, then the first intermediate product is dispersed in a weak alkaline buffer, dopamine and iron salt are added and stirred to obtain a second intermediate product, then a disulfide bond-containing cross-linking agent is added to the second intermediate product and stirred, the obtained product is reacted with a targeting ligand solution activated by an activator and stirred, the final product is collected by centrifugation, washed, and freeze-dried to obtain a surface-assembled nanocomplex of iron-doped dopamine (Fe-PDA) and modified cRGD tumor targeting peptide. The preparation process is simple and controllable, and the drug loading capacity is high.

[0030] The present invention also provides application of the near-infrared responsive nanocomposite in preparing anti-tumor drugs.

[0031] In some embodiments, the tumor is head and neck squamous cell carcinoma.

[0032] The application of the near-infrared-induced explosive ferroptosis nanocomplex provided in the embodiments of the present invention induces explosive ferroptosis in tumor cells, especially oral squamous cell carcinoma (OSCC), by inhibiting glutathione and enhancing LOP lipid peroxidation, showing excellent tumor targeting and near-infrared enhanced degradation, and has beneficial effects in the application of anti-tumor drugs.

[0033] The near-infrared induced explosive ferroptosis nanocomplex provided in the embodiments of the present invention, as well as its preparation method and application, induces 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%. Secondly, the photothermal performance is significantly improved by surface modification of iron-doped dopamine (Fe-PDA). The finally synthesized cRGD-modified multifunctional MnO2-R@FePDA-cRGD nanoreactor exhibits excellent tumor targeting and near-infrared enhanced degradation. Both in vitro and in vivo experiments show that after the nanocomplex accumulates in the tumor site, it responds to the degradation of the tumor microenvironment under near-infrared irradiation, and explosively releases the carried RSL3 to induce ferroptosis. At the same time, the photothermal effect promotes the activity of multiple enzymes, catalyzes the generation of ROS and enhances LPO. By consuming GSH and oxidizing NADH, the GPX4 and FSP1 pathways are synergistically inhibited to achieve efficient tumor ferroptosis.

[0034] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The characterization of MNO2R@FEPDAC in Examples 1-3 is shown, wherein (a) shows the transmission electron microscope image of HM-MnO2, (b) shows the nitrogen adsorption-desorption isotherm and pore size distribution curve of HM-MnO2, (c) shows the transmission electron microscope image of MNO2R@FEPDAC and (d) shows the EDS element distribution map with a resolution of 50nm, (e) shows the XRD spectrum comparison between MnO2 nanoparticles and MNO2R@FEPDAC, (f) shows the full spectrum XPS spectrum and (g) shows the corresponding Mn 2p and (h) shows the high-resolution XPS spectrum of Fe2p, and (i) shows the Fourier transform infrared spectrum.

[0036] Figure 2 The photothermal properties of MNO2R@FEPDAC in Examples 4-5 are shown, where (a) shows the photothermal properties of MNO2R@FEPDAC at 1 W / cm 2 Temperature variation curve under near-infrared irradiation. (b) shows the photothermal stability of MNO2R@FEPDAC (100 μg / mL) during five switching cycles. (c) shows the photothermal conversion efficiency: the red line indicates 1 W / cm 2 Photothermal effect of laser irradiation for 10 min; the purple line shows the time constant (τs) measured during the cooling phase, (d) shows the UV-visible absorption spectra of free HM-MnO2, RSL3, and MNO2R@FEPDAC, and (e) shows the UV-visible absorption spectra of free HM-MnO2, RSL3, and MNO2R@FEPDAC under simulated tumor microenvironment conditions (pH = 5.4 containing 10 mM GSH and 100 μM H2O2) with and without near-infrared irradiation (1 W / cm 2 (f) 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 incubation for 1 hour at pH 7.4 containing 10 mM GSH, (h) shows the transmission electron microscopy image after incubation for 1 hour under conditions of simulated tumor microenvironment, and (i) shows the transmission electron microscopy image after near-infrared irradiation for 10 minutes under conditions of simulated tumor microenvironment.

[0037] Figure 3 The enzymatic reaction kinetics of MNO2R@FEPDAC in Examples 6-10 are shown. (a) shows peroxidase-like activity, where the substrate is H 2 O 2, (b) shows the oxidase-like activity, with TMB at the bottom, (c) shows the Michaelis-Menten curves of glutathione peroxidase-like activity and (d) shows the nitric oxide synthase-like activity. (e) shows the schematic diagram of the mechanism of multi-enzyme mimicking 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 differences between each experimental group and 100μM H 2 O 2 And the absorbance change at 652 nm after 5 minutes of reaction with 2 mM TMB.

[0038] Figure 4 The in vitro antitumor activity and mechanism of action of MNO2R@FEPDAC on HN6 cells in Examples 11-14 are shown. Among them, (a) shows the confocal laser scanning microscopy (CLSM) image of IR-780-labeled MNO2R@FEPDAC after 6 hours of treatment, (b) shows the relative survival rate of cells after 24 hours of treatment with different concentrations of free HM-MnO2, n=6, (c) shows the relative survival rate of cells in different treatment groups, n=6, (d) shows the live cell (green) / dead cell (red) fluorescence staining image, (e) shows the intracellular reactive oxygen species (ROS, green) / nucleus (blue) fluorescence image, (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 the biological transmission electron microscopy (Bio-TEM) images of HN6 cells with different pretreatments, and (k) shows the Western Blot analysis was performed to detect the expression levels of GPX4, FSP1, FTH1 and FDX1 proteins after 24 hours of different treatments, including ⅰ. control group, ⅱ. near-infrared group, ⅲ. MNO2R@FEPDAC group, ⅳ. MNO2R@FEPDAC+near-infrared group. Near-infrared parameters: 808 nm laser, 0.5 W / cm 2 Irradiation for 5 minutes, *p<0.05, ***p<0.001.

[0039] Figure 5 The in vivo antitumor activity of MNO2R@FEPDAC in Examples 15-16 in the HN6 tumor-bearing mouse model is shown, wherein (a) shows the whole-body optical in vivo imaging of IR-780-labeled MNO2R@FEPDAC at different time points after administration and the in vitro fluorescence imaging 48 hours after injection, and (b) shows the fluorescence intensity of IR-780-labeled MNO2R@FEPDAC at 808 nm and 0.5 W / cm 2 Infrared thermal imaging of the tumor site under near-infrared laser irradiation, (c) shows Figure 5Temperature change curve of the tumor area in b, (d) shows the tumor growth curve of different experimental groups within 14 days, n=5, (e) shows the macroscopic photos of tumor samples in each group after treatment, (f) shows the statistical results of tumor weight, n=5, (g) shows H&E staining, TUNEL cell apoptosis detection and Ki67 proliferation marker immunofluorescence staining of tumor tissue, () shows the immunofluorescence staining results of FTH1, GPX4 and FDX1 proteins, scale bar is 100 μm. ***p<0.001.

[0040] Figure 6 The in vivo biodegradation and biosafety of MNO2R@FEPDAC in Example 17 are shown, wherein (a) shows the concentration change curves of manganese (Mn) and iron (Fe) in various organs at different time points after injection. (b) shows the analysis of blood biochemical indicators after 14 days of intervention with different treatments.

[0041] Figure 7 The photothermal conversion efficiency of MnO2@PDA is shown.

[0042] Figure 8 The HM-MnO of Example 3 is shown 2 Scanning electron microscope (SEM) image of nanoparticles.

[0043] Fig. 9 The dynamic light scattering (DLS) test results of MNO2R@FEPDAC of Example 3 are shown.

[0044] Fig.10 The surface scan result of the elements in the material obtained by the energy dispersive X-ray spectroscopy (EDS) technique in Example 3 is shown, wherein the resolution is 100 nm.

[0045] Fig.11 The stability test results of the MNO2R@FEPDAC of Example 3 in physiological medium for 7 days are shown.

[0046] Fig.12 The ultraviolet-visible-near-infrared spectrophotometric absorption spectrum (UV-Vis-NIR) of MNO2R@FEPDAC of Example 4 is shown, in which a polysiloxane phosphate composite material (PSPc) is used.

[0047] Fig.13 The different power (0.5 W / cm 2 ,1.0W / cm 2 , 1.5W / cm 2 ) under heating results.

[0048] Fig.14A concentration standard curve of RSL3 of Example 5 is shown.

[0049] Fig.15 The TMB oxidation ability results of Fe-PDA and MNO2R@FEPDAC of Example 6 are shown.

[0050] Fig.16 The results of the concentration gradient experiment of the oxidase-like enzyme (OXD) of Example 6 with different concentrations of TMB as substrate are shown.

[0051] Fig.17 The peroxidase-like (POD) activity of Example 6 was shown to be increased at different concentrations of H 2 O 2 The results of the substrate concentration gradient experiment.

[0052] Fig.18 The absorbance characteristic results of 5,5-dithiobis(2-nitrobenzoic acid) DTNB of Example 7 with different incubation time extensions are shown to indicate the GSH consumption of MNO2R@FEPDAC.

[0053] Fig.19 The Michaelis-Menten kinetic parameter results of the Fe-PDA nanozyme in Example 7 simulating glutathione peroxidase (GPx) catalyzing glutathione (GSH) are shown.

[0054] Fig. 20 The UV-visible absorbance of different concentrations of MNO2R@FEPDAC of Example 8 is shown to indicate the NADH oxidation ability of MNO2R@FEPDAC.

[0055] Fig.21 The Michaelis-Menten kinetic parameter results of the Fe-PDA nanozyme in Example 8 simulating nitrite oxidase (NOx-like) catalyzing NADH are shown.

[0056] Fig. 22 The results of the difference in mouse body weight among the control group, near-infrared light group, MNO2R@FEPDAC group and MNO2R@FEPDAC+near-infrared light group of Example 16 are shown.

[0057] Fig.23 Control H&E (hematoxylin & eosin) staining results of the main organs of Example 17 are shown to confirm the biocompatibility of MNO2R@FEPDAC. DETAILED DESCRIPTION

[0058] The following will clearly and completely describe the concept of the present application and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of them. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present application.

[0059] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0060] In the following examples, the detailed information of the reagents used and the manufacturers are shown in Table 1:

[0061] Table 1 Main reagent names and manufacturers

[0062]

[0063]

[0064] In the following examples, the detailed information of the instrument models and manufacturers used is shown in Table 2:

[0065] Table 2 Main instrument models and manufacturers

[0066]

[0067] Example 1 Hollow Mesoporous Manganese Dioxide HM-MnO 2 Method for preparing nanoparticles

[0068] The present invention provides a method for preparing hollow mesoporous manganese dioxide nanoparticles, comprising the following steps:

[0069] (1) Synthesis of solid silica nanoparticles (SiO 2 ), in this embodiment, TEOS (tetraethyl orthosilicate) is reduced in ammonia water to form solid silicon dioxide nanoparticles (SiO 2 ), by adjusting the reaction parameters, including temperature, concentration, stirring speed, etc., silica nanoparticles with adjustable particle size in the range of 30-500nm can be obtained. In this embodiment, silica nanoparticles with a particle size in the range of 50-200nm are obtained by adjusting the concentration of ammonia water.

[0070] (2) Then KMnO was added under ultrasonic conditions. 4After 6 hours of reaction, the suspension was centrifuged at 14,000 rpm to collect the precipitate.

[0071] (3) Obtained mesoporous MnO 2 Coated SiO 2 Then dispersed in 60℃ Na 2 CO 3 The aqueous solution was stirred overnight to remove SiO 2 template, and finally obtain hollow mesoporous MnO 2 (HM-MnO 2 ) nanoparticles.

[0072] Example 2MnO 2 - Preparation method of RSL3@FePDA-cRGD nanocomposite

[0073] The embodiment of the present invention provides a MnO 2 -The preparation method of RSL3@FePDA-cRGD nanocomposite comprises the following steps:

[0074] (1) Synthesize solid silica nanoparticles (SiO 2 ), for example, PS microspheres can be used as templates to deposit SiO 2 The template is then removed to obtain a hollow porous structure.

[0075] (2) Then KMnO was added under ultrasonic conditions. 4 After 6 hours of reaction, the suspension was centrifuged at 14,000 rpm to collect the precipitate.

[0076] (3) Obtained mesoporous MnO 2 Coated SiO 2 Then dispersed in 60℃ Na 2 CO 3 After standing in the aqueous solution overnight, hollow mesoporous MnO 2 (HM-MnO 2 ) nanoparticles.

[0077] (4) 20 mg HM-MnO 2 Dispersed in 10 ml of methanol, followed by the addition of 5 mg of RSL3.

[0078] (5) The mixture was sonicated for 30 minutes and stirred overnight.

[0079] (6) The product was collected by centrifugation at 8000 rpm, washed three times with PBS buffer, and labeled as MnO 2 -RSL3.

[0080] (7) Then 10 mg MnO2 -RSL3 was dispersed in 10 ml Tris-HCl buffer (pH 8.5), and 1 mg dopamine and 0.1 ml Fe(NO 3 ) 3 9H 2 O solution.

[0081] (8) After stirring at room temperature for 8 hours, 10 mg NH 2 -SS-NH 2 Stirring was continued for 24 hours.

[0082] (9) The obtained material was mixed with COOH-PEG activated by EDC / NHS 2000 -cRGD solution was reacted and stirred overnight.

[0083] (10) The resulting product was washed three times with an ethanol / water mixed solution, freeze-dried, and stored at 4°C.

[0084] Example 3 Characterization and performance testing

[0085] For the HM-MnO of Example 1 2 Nanoparticles were tested as follows:

[0086] Please attend Figure 1 a transmission electron microscope (TEM) and Figure 8 Scanning electron microscope (SEM) images of the material confirmed that the material formed a hollow mesoporous structure with a clear surface morphology.

[0087] The Brunauer-Emmett-Teller (BET) test showed that the specific surface area of ​​the HM-MnO2 nanoparticles of Example 1 was 33.013 m 2 / g, with an average pore size of 3.5nm, indicating that it has high specific surface area and mesoporous properties ( Figure 1 b).

[0088] These properties make it a highly efficient nanocarrier.

[0089] For the MnO of Example 2 2 -RSL3@FePDA-cRGD nanocomposite was tested as follows:

[0090] like Figure 1 c shows the final MNO obtained in Example 2. 2 -RSL3@FePDA-cRGD nanocomposite (hereafter referred to as MNO2R@FEPDAC) maintained a hollow spherical structure with an average diameter of approximately 150 nm.

[0091] Dynamic light scattering (DLS) tests showed that its hydrodynamic diameter was 181±10.2nm (see Fig. 9 ).

[0092] Energy dispersive X-ray spectroscopy (EDS) surface scanning results show that the material is rich in Mn and Fe elements, and coexists with C, N, O, S, and Cl elements (see Figure 1 d and Fig.10 ).

[0093] X-ray diffraction (XRD) analysis showed that the characteristic diffraction peaks at 36.7° and 66.3° belonged to MnO2 crystals, and there was a broadened peak in the range of 20-30°, corresponding to the presence of polydopamine (PDA) ( Figure 1 e).

[0094] X-ray photoelectron spectroscopy (XPS) is used to deeply analyze the elemental composition and chemical state of the material. Figure 1 f).

[0095] The binding energy peaks at 652.5 eV and 640.8 eV correspond to Mn 2p1 / 2 and Mn 2p3 / 2 orbitals, respectively, and are assigned to Mn by peak fitting. 4+ 2p1 / 2 and Mn 4+ 2p3 / 2, confirming that Mn is mainly in the +4 oxidation state ( Figure 1 g).

[0096] Similarly, the fitted peaks at 723.5 eV and 716.7 eV in the Fe 2p1 / 2 spectrum correspond to Fe 3+ with Fe 2+ The peaks of Fe 2p3 / 2 spectrum at 712.2eV and 710.0eV further confirm that Fe 3+ / Fe 2+ coexist( Figure 1 h).

[0097] Fourier transform infrared spectroscopy (FT-IR) confirmed the successful modification of PDA and cRGD on the material surface. Figure 1 i).

[0098] Stability experiments showed (see Fig.11 ), MNO2R@FEPDAC maintained excellent stability in physiological medium for 7 days, and no attenuation of UV-visible absorption was observed.

[0099] In summary, the experimental data confirmed that the nanoreactor was successfully constructed and had good stability.

[0100] Example 4 Photothermal effect test of MNO2R@FEPDAC

[0101] MNO2R@FEPDAC was dispersed in PBS solutions with concentrations of 0, 25, 50, 75, and 100 μg / mL and heated at 1 W / cm 2 The laser was irradiated at 808 nm for 10 min.

[0102] Fluke Ti27 infrared thermal imager (USA) was used to monitor the temperature change. In addition, 0.5, 1.0 and 1.5 W / cm 2 The power density was irradiated, and the temperature change curves of the heating process and the subsequent natural cooling stage were fully recorded.

[0103] The photothermal conversion efficiency (η) is calculated using the following formula 1-4.

[0104] η=(hS(T max -T surr )-Q dis ) / I(1–10 -A ) (1)

[0105] hS=∑mCp / τS (2)

[0106] τ S =-t / lnθ (3)

[0107] θ=(TT surr ) / (T max -T surr ) (4)

[0108] 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 / (gram·℃)) is the specific heat capacity of water, and τS = 408.1 seconds is taken from Figure 2 c.

[0109] hS is calculated by formula (2) (hS = 1 × 4.2 / 408.1 = 10.29 mW / °C), Qdis is independently measured as 74.84 mW, Tmax is the equilibrium temperature, Tsurr is the ambient temperature, and I = 1.0 W / cm 2 , A represents the absorbance at a wavelength of 808 nanometers (A808=0.425).

[0110] Therefore, the conversion efficiency η is calculated as follows:

[0111] η={[10.29×(48.0-24.3)-74.84] / [1000×(1-10 -0.425 )]}×100%=39.1%.

[0112] Specifically, an ultraviolet-visible-near infrared spectrophotometer is used to analyze its absorption spectrum.

[0113] like Fig.12 As shown in the figure, MNO2R@FEPDAC exhibits significant absorption characteristics in the near-infrared region. The absorption characteristics of nanoparticle suspensions with different concentrations were monitored by infrared thermal imaging at 808 nm laser (1.0 W / cm 2 ) to evaluate its photothermal performance.

[0114] like Figure 2 As shown in a, compared with the PBS control group, the MNO2R@FEPDAC solution showed a concentration-dependent temperature increase, indicating that it has the ability to efficiently convert laser energy into thermal energy. In addition, the photothermal performance of the material showed a power density-dependent trend (see Fig.13 ).

[0115] It is worth noting that no temperature decay was observed after five heating and cooling cycles ( Figure 2 b), fully demonstrating its excellent light and heat stability.

[0116] like Figure 2 c shows that at 1.0W / cm 2 Under irradiation conditions, the photothermal conversion efficiency of MNO2R@FEPDAC reached 39.1%. Figure 7 As mentioned above, the photothermal conversion efficiency of MnO2@PDA is 24.03%. This characteristic can provide sufficient energy support for improving the catalytic performance and enhancing the degradation ability of MNO2R@FEPDAC.

[0117] Example 5 Controlled release behavior of MNO2R@FEPDAC and tumor microenvironment (TME) near-infrared responsive degradation characteristics test.

[0118] MNO2R@FEPDAC was placed in the presence or absence of glutathione (GSH) and hydrogen peroxide (H 2 O 2 ) in phosphate buffered saline (PBS) at pH 5.5 or 7.4 for 1 h. The release kinetics of RSL3 under different conditions were quantitatively analyzed by ultraviolet-visible spectrophotometry (UV-Vis), and the samples were then characterized by transmission electron microscopy (TEM).

[0119] like Figure 2 As shown in d, the characteristic absorption peak at 280 nm confirms the successful loading of RSL3 in MNO2R@FEPDAC. According to the concentration standard curve of RSL3 ( Fig.14 ) calculated that the drug loading was 67%.

[0120] In the simulated TME condition (pH = 5.4, containing 10 mM GSH and 100 μM H 2 O 2 ), MNO2R@FEPDAC showed a slow RSL3 release profile, while near-infrared (NIR) irradiation triggered a rapid and extensive drug release ( Figure 2 e).

[0121] Furthermore, we investigated the effect of NIR irradiation on its catalytic ability by evaluating the peroxidase-like (POD) activity of MNO2R@FEPDAC with and without NIR irradiation.

[0122] After NIR treatment, the absorbance of oxTMB at 652 nm was significantly enhanced ( Figure 2 f), indicating that the photothermal effect of MNO2R@FEPDAC not only promotes the rapid release of drugs, but also significantly improves the catalytic performance, providing sufficient energy for inducing explosive ferroptosis.

[0123] The NIR-responsive drug release mechanism of MNO2R@FEPDAC was further studied by observing the morphological changes of MNO2R@FEPDAC. Under physiological conditions of pH 7.4, MNO2R@FEPDAC exhibited excellent structural stability ( Figure 2 g).

[0124] However, after 1 h of incubation in simulated TME conditions, the nanoparticle structure showed partial degradation ( Figure 2 h). In contrast, MNO2R@FEPDAC was completely degraded after 10 min of NIR irradiation under simulated TME conditions ( Figure 2 i).

[0125] This dual response characteristic proves that MNO2R@FEPDAC is both stable in the physiological environment and can achieve precise drug release and catalytic enhancement in the tumor microenvironment through photothermal triggering, providing technical support for spatiotemporally controllable ferroptosis therapy.

[0126] Example 6 Characterization test of multi-enzyme mimic activity of MNO2R@FEPDAC

[0127] This example provides a test of the multi-enzyme mimicking properties of the MNO2R@FEPDAC nanoreactor in the presence of hydrogen peroxide (H 2 O 2) was used as a substrate to detect the peroxidase (POD) activity in the presence of 3,3',5,5'-tetramethylbenzidine (TMB). The absorbance change of oxidized TMB (oxTMB) at a wavelength of 652 nm was monitored using a UV-visible spectrophotometer at a specific reaction time point. Based on the Michaelis-Menten equation and its double reciprocal equation (as shown in the following formula (5)), the relevant kinetic parameters were calculated.

[0128]

[0129] In formula (5), V 0 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).

[0130] The kinetic properties of the MNO2R@FEPDAC catalyst were evaluated by setting different concentrations of TMB substrate (0.1-8 mmol / L) in an acidic buffer solution for the standard test of oxidase-like (OXD) activity. The absorbance values ​​of each group at a wavelength of 652 nm were recorded using a UV-visible spectrophotometer.

[0131] To evaluate the nitric oxide synthase (NOx) activity, solutions containing different concentrations of MNO2R@FEPDAC (0, 5, 10, 50, 100, 150, and 200 μg / mL) were mixed with 2 mmol / L NADH and 100 μmol / L H 2 O 2 After incubation at room temperature for 30 minutes, the absorbance was recorded using a UV-visible spectrophotometer. Subsequently, 200 μg / mL MNO2R@FEPDAC solution was dispersed in a pH 4.5 buffer containing different concentrations of NADH (0.05-2 mmol / L) and reacted for 10 minutes. The absorbance of the supernatant of each sample was measured at 340 nm.

[0132] Glutathione peroxidase (GPx) activity was evaluated 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 DTNB solution. After thorough mixing, the mixture was centrifuged at 14,000 rpm for 5 minutes, and the supernatant was collected for absorbance measurement. In addition, 200 μg / mL MNO2R@FEPDAC was dispersed in a pH 4.5 buffer containing different concentrations of GSH (0.1-3 mmol / L). After reacting with DTNB for 10 minutes, the absorbance at 412 nm was measured using a UV-Vis spectrophotometer.

[0133] Among them, the peroxidase-like (POD) activity assessment uses 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate, and the reaction will generate a blue product (oxTMB) with a characteristic absorption peak of 652 nanometers. Fig.15 As shown, both Fe-PDA and MNO2R@FEPDAC exhibit TMB oxidation ability under acidic conditions.

[0134] In H 2 O 2 In the concentration gradient experiment of the substrate, the kinetic parameters were determined by fitting the curve with the Michaelis-Menten equation:

[0135] The Michaelis constant Km of MNO2R@FEPDAC is 0.011mM (millimol / L), and the maximum reaction rate Vmax is 2.136×10-7M·s-1. The Km value of Fe-PDA is 0.259mM, and the Vmax value is 3.56×10-9M s-1, which is two orders of magnitude lower than that of MNO2R@FEPDAC (see Figure 3 a and Fig.16 ).

[0136] The activity of OXD was evaluated by TMB substrate concentration gradient experiment. Steady-state analysis showed:

[0137] Km=0.059mM, Vmax=3.453×10 of MNO2R@FEPDAC -7 M.s -1 , showing significantly improved catalytic efficiency compared to Fe-PDA (see Figure 3 b and Fig.17 ).

[0138] The above data confirm that MNO2R@FEPDAC exhibits significant advantages in terms of substrate affinity and catalytic activity. Its lower Km value indicates that it has a better affinity for H 2 O 2 The higher Vmax value reflects the more efficient catalytic conversion efficiency.

[0139] Example 7 Glutathione (GSH) consumption capacity test of MNO2R@FEPDAC

[0140] 5,5-Dithiobis(2-nitrobenzoic acid) (DTNB) was used to quantitatively analyze the residual GSH levels after incubation of MNO2R@FEPDAC with excess GSH for different time periods.

[0141] like Fig.18As shown, the characteristic absorbance of DTNB at 412 nm decreased significantly with the extension of incubation time, indicating that MNO2R@FEPDAC can effectively consume GSH.

[0142] In addition, under simulated tumor microenvironment (TME) conditions, MNO2R@FEPDAC exhibited Michaelis-Menten kinetic characteristics in substrate concentration-dependent experiments with GSH.

[0143] The Km and Vmax values ​​obtained were 0.24 mM and 1.657 × 10 -9 M.s -1 ( Figure 3 c), which is significantly higher than the corresponding values ​​of Fe-PDA (1.52 mM and 0.213×10 -9 M.s -1 ,See Fig.19 ).

[0144] Example 8 Test of Nitrite Oxidase-like (NOx-like) Activity of MNO2R@FEPDAC

[0145] Ferroptosis suppressor protein 1 (FSP1) acts as an NADH-dependent coenzyme Q oxidoreductase, which reduces ubiquinone (CoQ10) to ubiquinol (CoQ10H 2 ) to inhibit lipid peroxidation (LPO), thereby exerting an anti-ferroptosis effect.

[0146] In this process, NADH provides the necessary reducing power for FSP1 to perform its antioxidant function. + is an effective strategy to enhance ferroptosis.

[0147] To evaluate the NOx-like activity of MNO2R@FEPDAC, it was incubated with 2 mM NADH at pH 4.5 and 37 °C for 30 min.

[0148] like Fig. 20 As shown, the UV-visible spectrum showed that the absorbance at 340 nm gradually decreased with the increase of MNO2R@FEPDAC concentration, indicating that it has efficient NADH oxidation ability.

[0149] The measured Km and Vmax values ​​were 0.15 mM and 1.455 × 10 -6 M.s -1 , which is significantly better than the corresponding value of Fe-PDA (see Figure 3 d and Fig.21 ).

[0150] These results highlight the excellent 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 a burst of ferroptosis in tumor cells.

[0151] Example 9 Testing of the catalytic potential of MNO2R@FEPDAC in regulating free radicals

[0152] Electron spin resonance (ESR) spectroscopy combined with the free radical spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was used to detect hydroxyl radicals (·OH) and superoxide radicals (O 2 ·-) is generated.

[0153] like Figure 3 As shown in f, at pH 5.4, even without adding H 2 O 2 , the characteristic ·OH signal peak with an intensity ratio of about 1:2:2:1 can still be observed in the MNO2R@FEPDAC solution. 2 O 2 After 24h, the ·OH signal increased significantly, indicating that the material has intrinsic oxidase-like activity under acidic conditions and can catalyze H 2 O 2 Decomposition produces a large amount of OH.

[0154] Similarly, under the same conditions without adding H 2 O 2 Weak O can be detected 2 -signal, and when there is H 2 O 2 When 2 -Signal strength increases dramatically ( Figure 3 g).

[0155] These findings suggest that MNO2R@FEPDAC plays an important role in regulating the 2 ·- has significant catalytic potential in free radicals, indicating its broad application prospects in the field of tumor treatment.

[0156] Example 10 Synergistic effect test of near infrared irradiation and MNO2R@FEPDAC

[0157] This embodiment provides a test for the enhancement effect of near-infrared (NIR) irradiation on the rate of Fenton / Fenton-like reactions and the generation of reactive oxygen species (ROS).

[0158] Specific:

[0159] At pH 5.4, MNO2R@FEPDAC was introduced into H 2 O 2 After the solution was added, the appearance of the characteristic absorption peak at 652 nm confirmed the generation of ROS ( Figure 3 h).

[0160] The intensity of this characteristic peak increased significantly after NIR irradiation. The experimental results show that near-infrared radiation can accelerate the rate of Fenton / Fenton-like reactions and increase the ROS yield.

[0161] Therefore, the synergistic effect of near-infrared irradiation and MNO2R@FEPDAC provides a powerful strategy to amplify the ferroptosis effect of tumor cells.

[0162] Example 11 Testing the ability of MNO2R@FEPDAC's cRGD tumor targeting peptide to internalize into tumor cells

[0163] 5×10 4 HN6 cells were seeded into confocal culture dishes and cultured overnight.

[0164] After incubation with 20 μg / mL IR780-labeled MNO2R@FEPDAC for 6 h, the cells were washed with PBS and stained with Hoechst 33342 for 10 min.

[0165] Confocal images were collected using a Leica Stellaris 5 microscope.

[0166] This example evaluates the ferroptosis-mediated killing efficiency of MNO2R@FEPDAC on oral squamous cell carcinoma (OSCC) cells. Efficient cellular endocytosis of nanomaterials is a key step in achieving tumor cell clearance.

[0167] To visualize the cellular uptake process, IR-780-labeled MNO2R@FEPDAC was used to observe its internalization behavior in HN6 cells using confocal laser scanning microscopy (CLSM).

[0168] After 6 hours of incubation, dense red fluorescent signals were observed around the cell nuclei ( Figure 4 a), indicating that the cRGD tumor targeting peptide modified on the surface of the nanomaterial can effectively promote its efficient internalization by tumor cells.

[0169] This example also first evaluated the free HM-MnO 2 The potential cytotoxicity of HM-MnO 2 After 24 hours of treatment, the cell survival rate remained above 90% ( Figure 4 b), fully demonstrated its good biocompatibility.

[0170] Example 12 Near-infrared response cell killing rate test of MNO2R@FEPDAC

[0171] HN6 cells were plated at 1×10 4 The cells were seeded in 96-well plates at a density of 10 cells / mL and cultured overnight.

[0172] The next day, cells were exposed to different concentrations of MNO2R@FEPDAC (0, 10, 25, 50, 100 μg / mL) for 24 h to evaluate cytotoxicity.

[0173] In the anti-tumor experiment of this example, the cells were first incubated with a medium containing 40 μg / mL MNO2R@FEPDAC for 6 h, and then fresh medium was replaced with a power density of 0.5 W / cm 2 808 nm laser irradiation for 5 min.

[0174] After culturing for 24 hours, the cell activity was detected using the CCK-8 kit.

[0175] In addition, under the same treatment conditions, the cells were stained with 1 μg / mL calcein-AM / PI double staining reagent at 37°C for 15 min in the dark, and finally the live / dead cell fluorescence images were captured using an Olympus IX71 fluorescence microscope from Japan.

[0176] Compared with the control group, the group receiving only near-infrared (NIR) irradiation (0.5 W / cm 2 The cell survival rate was still close to 100% ( Figure 4 c), indicating that the phototoxicity of near-infrared radiation under this parameter is extremely low.

[0177] When treated with MNO2R@FEPDAC at a concentration of 40 μg / mL, the cell survival rate decreased by 31.8%, showing a moderate ferroptosis effect.

[0178] After near-infrared irradiation, the cell killing rate reached 95.2%, which is speculated to be due to the outbreak of photothermal-activated ferroptosis effect.

[0179] To visually verify the therapeutic effect, live / dead cell staining experiments were performed using calcein-AM (green fluorescence to label live cells) and propidium iodide (red fluorescence to label dead cells).

[0180] The results showed that the cells in the MNO2R@FEPDAC combined with near-infrared irradiation group completely showed red fluorescence ( Figure 4 d), which is highly consistent with the cell viability data.

[0181] This example shows that the synergistic combination of photothermal effect and ferroptosis mechanism provides prospects for the application of anti-tumor drugs, especially drugs for the treatment of oral squamous cell carcinoma (OSCC).

[0182] Example 13 Test of multi-enzyme catalytic activity and photothermal response of RSL3 release characteristics of MNO2R@FEPDAC under tumor microenvironment (TME) conditions

[0183] This embodiment includes:

[0184] 1. Detection of intracellular reactive oxygen species (ROS) and lipid peroxides (LPO):

[0185] HN6 cells were cultured at 1×10 4 The cells were inoculated at a density of 100 μM H 2 O 2 and fresh culture medium (pH = 6.5) containing 40 μg / mL MNO2R@FEPDAC solution.

[0186] The cells in the near-infrared treatment group were exposed to a power density of 0.5 W / cm 2 After irradiation with 808 nm laser for 5 minutes, the cells were cultured overnight. Then, the cells were incubated with DCFH-DA (ROS probe) and Liperfluo (LPO probe) for 30 minutes, washed with PBS three times, and observed under a fluorescence microscope.

[0187] 2. Detection of cell malondialdehyde (MDA), glutathione (GSH) and reduced coenzyme I (NADH) levels:

[0188] Commercial kits were used to quantitatively detect the contents of MDA, GSH and NADH.

[0189] Specifically, HN6 cells were seeded in a 96-well plate and precultured for 24 h, and then co-cultured with different treatment groups (PBS group, NIR group, MNO2R@FEPDAC group, and MNO2R@FEPDAC+NIR combined group) for 24 h.

[0190] After the culture, the cells were collected by digestion and centrifugation, and the corresponding kits were used to perform quantitative analysis of each index.

[0191] Specifically, the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe detection revealed that a large amount of ROS ( Figure 4 e).

[0192] The production of lipid peroxide (LPO), a key trigger of ferroptosis, was visualized by fluorescence imaging, with the MNO2R@FEPDAC+NIR group showing the most significant intracellular LPO level ( Figure 4 f).

[0193] In addition, the LPO production was verified by detecting the LPO decomposition product malondialdehyde (MDA).

[0194] like Figure 4 As shown in (g), near-infrared irradiation alone had no effect on MDA levels, while MNO2R@FEPDAC treatment significantly increased MDA levels, and this indicator was further increased after combined near-infrared treatment.

[0195] Glutathione (GSH) depletion is also critical for enhancing ferroptosis.

[0196] Specifically, the relative GSH level in HN6 cells treated with MNO2R@FEPDAC+NIR decreased by 75%, which was significantly lower than that in other experimental groups ( Figure 4 h), which indicates that the nanomaterial has excellent glutathione peroxidase (GPx) mimetic activity and can effectively consume intracellular GSH.

[0197] 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.

[0198] Therefore, we evaluated the NADH oxidase (NOx) biomimetic activity 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 the production of intracellular GSH by consuming NADH.

[0199] These data suggest that the powerful multienzyme activity of MNO2R@FEPDAC can significantly enhance the ferroptosis of tumor cells.

[0200] Example 14: Ferroptosis effect in MNO2R@FEPDAC cells

[0201] It is known that ferroptosis induction can lead to oxidative damage to the inner mitochondrial membrane and induce significant morphological changes.

[0202] Transmission electron microscopy (TEM) images showed that HN6 cells treated with MNO2R@FEPDAC (whether or not combined with near-infrared irradiation) showed mitochondrial dysfunction characteristics associated with ferroptosis, including reduced mitochondrial volume, increased membrane density, reduced or disappeared cristae structure, and balloon-like morphology ( Figure 4 i).

[0203] To further explore the ferroptosis effect, we detected the expression levels of intracellular ferroptosis-related proteins by western blot.

[0204] Specifically, this embodiment uses Western blot experiment, and the steps are as follows:

[0205] 1. Lyse cells on ice using radioimmunoprecipitation buffer to obtain protein extracts.

[0206] 2. Protein concentration was quantified by BCA method (bicinchoninic acid method).

[0207] 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 μm.

[0208] 4. The membrane was blocked with bovine serum albumin (BSA) and then incubated with the primary antibody at 4°C overnight.

[0209] 5. After the washing steps, the membrane was incubated with horseradish peroxidase-labeled secondary antibody at room temperature for 1 hour.

[0210] 6. Protein bands were developed using a chemiluminescence imaging system (Bio-Rad, Singapore), and the band intensities were normalized to the β-actin level.

[0211] 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 phenomenon of decreased intracellular GSH levels.

[0212] Ferroptosis suppressor protein 1 (FSP1) converts coenzyme Q10 (CoQ10(H)) into CoQ10H 2 FSP1 plays an important role in scavenging LPO free radicals, but the oxidation process of NAD(P)H to NAD(P)+ will destroy the biological function of FSP1. The NADH oxidase (NOx) mimetic activity of MNO2R@FEPDAC effectively inhibits the FSP1 protein with anti-ferroptosis function.

[0213] In addition, MNO2R@FEPDAC treatment also suppressed the expressions of other ferroptosis-related proteins such as ferritin heavy chain 1 (FTH1) and ferredoxin 1 (FDX1).

[0214] In summary, MNO2R@FEPDAC triggers a strong outbreak of intracellular ferroptosis effects through multiple mechanisms such as efficient intracellular uptake, multi-enzyme mimetic activity, photothermal responsive ROS storm, lipid metabolism destruction and GPX4 / FSP1 pathway regulation, thereby significantly enhancing tumor cell ferroptosis.

[0215] Example 15 Evaluation of tumor targeting and anti-tumor ability of MNO2R@FEPDAC in vivo

[0216] The in vivo biodistribution and photothermal effect experiments of this embodiment specifically include:

[0217] 1. Tumor-bearing mice were given 10 mg / kg of IR780-labeled MNO2R@FEPDAC by intravenous injection.

[0218] 2. In vivo fluorescence images were collected at set time points (3, 6, 12, 24, and 48 hours) using the Caliper IVIS Spectrum Imaging System (PerkinElmer, USA).

[0219] 3. Detect ex vivo fluorescence imaging of the heart, liver, spleen, lung, kidney and tumor 48 hours after injection.

[0220] 4. The mice were anesthetized 48 hours after injection, and the tumor site was illuminated with 808nm laser at 0.5W / cm 2 Irradiate for 5 minutes.

[0221] 5. Use infrared thermal imaging to monitor temperature changes in the tumor area.

[0222] Specifically, based on the above experimental methods, the tumor targeting ability of MNO2R@FEPDAC was studied using an HN6 tumor-bearing mouse model, and after intravenous injection of IR-780-labeled MNO2R@FEPDAC, an in vivo fluorescence imaging system (IVIS) was used to monitor its biodistribution characteristics over time.

[0223] like Figure 5 As shown in a, the fluorescence signal at the tumor site gradually increased over time, reaching a peak at 48 hours after injection and starting to decay after 72 hours. 48 hours of in vitro fluorescence imaging confirmed the effective accumulation of MNO2R@FEPDAC in tumor tissues, and this targeted aggregation may be attributed to both the size-dependent enhanced permeability and retention (EPR) effect and the active targeting ability mediated by cRGD.

[0224] By monitoring 0.5W / cm 2 The in vivo photothermal performance of MNO2R@FEPDAC was evaluated by the temperature change of the tumor site under near-infrared irradiation. Thermal imaging showed that the temperature of the tumor area gradually increased to a stable state of 48.2°C within 5 minutes. Figure 5 b, c), confirming that the nanomaterial has excellent photothermal effect in vivo.

[0225] Therefore, MNO2R@FEPDAC shows significant application potential in amplifying reactive oxygen species generation and promoting ferroptosis.

[0226] Example 16 Anti-tumor effect of MNO2R@FEPDAC in vivo

[0227] To confirm the in vivo therapeutic effect of MNO2R@FEPDAC, we evaluated its anti-tumor ability in the HN6 tumor-bearing mouse model.

[0228] Specifically, NOD-SCID mice were subcutaneously injected with 1×10 6 When the tumor volume reaches about 200mm 3 The mice were randomly divided into four groups (n=5 in each group): control group, near-infrared light group, MNO2R@FEPDAC group and MNO2R@FEPDAC+near-infrared light group.

[0229] On day 0, mice received an intravenous injection of PBS (100 μl) or MNO2R@FEPDAC (100 μl) containing 10 mg / kg.

[0230] After 48 hours, the tumor sites of the designated groups were irradiated with 808 nm laser (0.5 W / cm 2 Body weight was recorded every other day during the experiment, and tumor volume was measured every 2 days using a vernier caliper.

[0231] On day 14, mice were sacrificed, and tumors and major organs (heart, liver, spleen, lung, and kidney) were collected and fixed in 4% paraformaldehyde for histopathological and immunohistochemical analysis.

[0232] The formula for calculating tumor volume is V = long diameter × short diameter 2 / 2.

[0233] Based on the above experiments, the tumor inhibitory effect of different treatment regimens was evaluated by continuously measuring the tumor volume after treatment.

[0234] In the near-infrared (NIR) treatment group, 808 nm laser (0.5 W / cm 2 ) The tumor was irradiated for 5 minutes.

[0235] like Figure 5 As shown in (d), MNO2R@FEPDAC induced ferroptosis through its multienzyme mimetic activity in the tumor microenvironment (TME), thereby effectively inhibiting tumor growth.

[0236] In sharp contrast, the MNO2R@FEPDAC combined with NIR group showed complete inhibition and even ablation of tumor growth ( Figure 5 e).

[0237] At the end of the study, the tumor weight in the combined treatment group was significantly lower than that in the other groups ( Figure 5f). The excellent anti-tumor effect of MNO2R@FEPDAC combined with NIR was further confirmed by pathological analysis such as hematoxylin-eosin (H&E) staining, TUNEL staining, and Ki67 staining of tumor tissues ( Figure 5 g).

[0238] There was no significant difference in the weight of mice in each group during the whole experiment, which proved that the nanomaterial has good biocompatibility (see Fig. 22 ).

[0239] Example 17 In vivo biodegradation and biosafety of MNO2R@FEPDAC.

[0240] The in vivo clearance rate and biosafety evaluation experiments of this embodiment include:

[0241] Major organs were collected on days 0, 1, 3, 7, and 14 after intravenous injection of MNO2R@FEPDAC (10 mg / kg).

[0242] After nitric acid digestion, the iron and manganese contents of the organs were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) to evaluate their biodegradability.

[0243] On the 14th day, 0.6 mL of blood sample was collected through the orbital venous plexus, placed in a heparinized tube, and centrifuged at 3000 rpm for 15 min at 4°C to obtain serum.

[0244] Blood biochemical tests related to liver and kidney function were performed by Wuhan Xavier Biotechnology Co., Ltd. (China).

[0245] Major organs such as heart, liver, lung, spleen and kidney were collected at different time points after intravenous injection.

[0246] The concentrations of manganese (Mn) and iron (Fe) were quantitatively analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES) to characterize the biodegradability of MNO2R@FEPDAC.

[0247] like Figure 6 As shown in a, the Mn and Fe contents increased significantly on the first day after injection, but gradually decreased from day 7. By day 14, the element levels returned to the baseline state before injection, indicating that the nanomaterial can achieve effective clearance during the treatment cycle.

[0248] Furthermore, blood biochemical analysis was performed on the 14th day after treatment to evaluate liver function (including ALT, AST, ALP, ALB and T-BIL, representing alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, albumin and total bilirubin, respectively) and renal function (UA, BUN and CR, corresponding to uric acid, blood urea nitrogen and creatinine, respectively). The levels of the above biomarkers in all groups were within the normal range, indicating that no abnormal liver and kidney function was found in any group ( Figure 6 b).

[0249] In addition, H&E staining results of major organs showed that no significant tissue damage was observed in the MNO2R@FEPDAC injection group compared with the control group (see Fig.23 ).

[0250] These results confirm the excellent biocompatibility and degradability of MNO2R@FEPDAC.

[0251] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A near-infrared induced explosive ferroptosis nanocomposite, characterized in that: The invention comprises nanoparticles loaded with an ferroptosis inducer and iron-doped dopamine and a targeting ligand modified on the surface of the nanoparticles. The nanoparticles use manganese dioxide with 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.

2. The nanocomposite according to claim 1, characterized in that The nanoparticles are hollow spherical structures with an average diameter of about 50-200 nm and a hydrodynamic diameter of 181±10.2 nm.

3. The nanocomposite according to claim 2, characterized in that The loading amount of ferroptosis inducer in the construct was 50-70%.

4. The nanocomposite according to claim 1 or 3, characterized in that: The specific surface area of ​​the manganese dioxide carrier is 20-60m2 / g, and the average pore diameter is 2-10nm.

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

6. The method for preparing the near-infrared induced burst ferroptosis nanocomposite according to any one of claims 1 to 5, characterized in that: The steps include: S1. Preparation of hollow mesoporous manganese dioxide nanoparticles; S2, dispersing the hollow mesoporous manganese dioxide nanoparticles and the ferroptosis inducer in methanol, stirring for 8-16 hours after ultrasonic treatment, and centrifuging to collect the first intermediate product; S3, dispersing the first intermediate product in a weak alkaline buffer, adding dopamine and iron salt, and stirring for 6-10 hours to obtain a second intermediate product; S4, adding a disulfide bond-containing cross-linking agent to the second intermediate product, and continuing stirring for 20-28 hours; S5, reacting the obtained product with a targeting ligand solution activated by an activator, and continuing stirring for 12-24 hours; S6. Collect the final product by centrifugation, wash it, and freeze-dry it to obtain the nanocomposite.

7. The method according to claim 6, characterized in that: The hollow mesoporous manganese dioxide nanoparticles described in step S1 are prepared by a template method; and / or The mass ratio of the hollow mesoporous manganese dioxide nanoparticles to the ferroptosis inducing agent in step S2 is (3.5-4.5):1, and the ultrasonic treatment time is 25-35 minutes; and / or The buffer in step S3 is tris-hydrochloric acid buffer with a pH of 8.3-8.7, and the iron salt is Fe(NO3)3·9H2O, and the amount added is 0.05-0.15 mL per 10 mg of the first intermediate product; and / or The disulfide bond-containing cross-linking agent in step S4 is NH2-SS-NH2, and the amount added is 8-12 mg per 10 mg of the second intermediate product; and / or In step S5, the targeting ligand is synthesized by a functionalized polyethylene glycol derivative, and the activator is a mixed solution of EDC and NHS; and / or The washing in step S6 uses a mixed solution of ethanol and water in a volume ratio of (1:1)-(3:1), and the number of washing times is 2-4 times.

8. Use of the near-infrared responsive nanocomposite according to any one of claims 1 to 5 in the preparation of anti-tumor drugs.

9. The use according to claim 8, wherein the tumor is head and neck squamous cell carcinoma.

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