Loose macroporous iron-based nano catalytic compound for inducing tumor para-apoptosis as well as preparation method and application of loose macroporous iron-based nano catalytic compound

Abstract

The invention discloses a loose macroporous iron-based nano catalytic compound for inducing tumor para-apoptosis and a preparation method and application thereof.The nano catalytic compound (TF-Fe at LC) is prepared by taking a porous Fe3O4 nano cluster composed of single crystals as a carrier, modifying the surface of the carrier with an acid-responsive tannic acid-iron (TA-Fe < 3 + >) network and co-loading lactate oxidase (LOD) and a mitochondrial uncoupling agent CCCP in the carrier. The TF-Fe-coated LC constructed by the invention does not need additional chemotherapeutic drugs, and tumor cells are induced to generate a large amount of active oxygen through catalytic cascade and mitochondrial oxidative stress, so that parapoptosis of the tumor cells is triggered; the parapoptotic cells can play a role of a tumor vaccine, induce other tumor cells to generate immunogenic death, and activate an organism immune system to recognize and kill tumors. In addition, the nano catalytic system can be combined with drugs and immunosuppressants to realize a synergistic anti-tumor effect.

Description

Technical Field

[0001] This invention belongs to the field of functional nanomaterial preparation and its biological application technology, and relates to a loose macroporous iron-based nanocatalytic complex that induces tumor paraapoptosis, its preparation method and application. Background Technology

[0002] Chemokinetic therapy (CDT), as an emerging cancer treatment strategy, relies on the core mechanism of metal-based nanocatalysts (such as Fe) to achieve its therapeutic effect. 3+ Cu 24 In the tumor microenvironment (TME), CDT triggers the Fenton reaction, catalyzing the conversion of endogenous hydrogen peroxide (H₂O₂) in tumor cells into highly reactive and toxic hydroxyl radicals (·OH), thereby achieving specific oxidative killing of tumor cells. However, CDT induces ROS-mediated tumor cell apoptosis via the Fenton reaction, which mainly depends on the activation of caspase, and tumor cells can easily evade apoptosis through various mechanisms. Therefore, developing cancer treatment strategies that do not rely on apoptosis pathways could help overcome tumor drug resistance.

[0003] Paraapoptosis, as a novel non-apoptotic programmed cell death mechanism, shows great potential for application. Characterized by endoplasmic reticulum and mitochondrial vacuolation, paraapoptosis possesses strong immunogenic potential and is expected to become a more effective pathway for tumor cell killing. Activation of paraapoptosis is closely related to protein homeostasis imbalance caused by abnormal ROS accumulation, with its core mechanisms involving endoplasmic reticulum stress and mitochondrial dysfunction. Current nanomedicines for inducing paraapoptosis mainly rely on Cu... 2+ mediated formation of toxic complexes, or Ca 2+ Mitochondrial dysfunction caused by overload. However, the above strategies are difficult to achieve safely and efficiently inducing paraapoptosis due to the uncontrollable toxicity of metal ions and toxic complexes or the lack of spatiotemporal synergy in multi-organelle damage. Therefore, developing novel nanoplatforms that can synergistically regulate the endoplasmic reticulum-mitochondrial dual pathway and circumvent heavy metal toxicity is a key direction to overcome current technological bottlenecks.

[0004] Iron(III) oxide (Fe3O4) has passed the technical review of the drug master file (DSP), gaining authoritative recognition for its biosafety as a medical material, and has not exhibited the cytotoxic characteristics of traditional heavy metals. As a typical Fenton reaction catalyst, Fe3O4 shows significant application potential in chemokinetic therapy (CDT). However, Fe3O4 iron-based nanomaterials still face many unresolved issues. On the one hand, the relatively limited specific surface area of ​​Fe3O4 prevents the full exposure of its surface active sites, directly leading to low catalytic efficiency. On the other hand, in complex physiological environments, iron-based nanomaterials are prone to aggregation, masking the original active sites and further deactivating them, thus further reducing catalytic efficiency. Summary of the Invention

[0005] To address the problems and deficiencies in the existing technologies, this invention provides a porous macroporous iron-based nanocatalytic complex for inducing tumor paraapoptosis, its preparation method, and its applications. This invention uses porous iron(III) oxide (P-Fe3O4) as a carrier, loads lactate oxidase (LOD) and mitochondrial uncoupling agent (CCCP), and modifies the surface with tannic acid-iron (Fe3O4). 3+ The TA, TF) metal polyphenol network, based on the idea of ​​CDT-driven paraapoptosis-immune activation, drives the occurrence of paraapoptosis through the "dual engine" of mitochondrial oxidative stress-catalytic cascade, and achieves the dual anti-tumor effect of "oxidative killing-immune activation" by combining immune checkpoint blockade.

[0006] In a first aspect, the present invention provides a loose macroporous iron-based nanocatalytic complex that induces tumor paraapoptosis, the nanocatalytic complex comprising a support and a catalytic sensitizing component loaded on the support;

[0007] The carrier is porous iron oxide modified with an iron ion-polyphenol complex;

[0008] The catalytic sensitizing components are lactate oxidase and carbonyl cyanide m-chlorophenylhydrazone.

[0009] Furthermore, in the loose macroporous iron-based nanocatalytic complex for inducing tumor paraapoptosis provided by the present invention, the particle size of the nanocatalytic complex is 100-300 nm.

[0010] The pore size of the unmodified porous magnetite with tannic acid-iron metal polyphenol network is 10-40 nm.

[0011] The lactate oxidase loading is 5-10%;

[0012] The loading of the carbonyl cyanide m-chlorophenylhydrazone is 5-15%.

[0013] Secondly, this invention provides a method for preparing a loose macroporous iron-based nanocatalytic complex that induces tumor paraapoptosis, comprising: preparing iron(III) oxide by hydrothermal method using urea, ferric chloride hexahydrate, sodium polyacrylate, and sodium citrate dihydrate as raw materials; etching iron(III) oxide with an ethylene glycol solution containing citric acid to obtain porous iron(III) oxide; dispersing the porous iron(III) oxide in PBS solution, sequentially adding lactate oxidase solution and carbonyl cyanide m-chlorophenylhydrazone solution, stirring in the dark to obtain a mixture; and sequentially adding Fe to the mixture. 3+ The solution and tannic acid solution were reacted in the dark, followed by washing and magnetic separation to obtain the nano-catalytic complex.

[0014] Furthermore, in the preparation method of the loose macroporous iron-based nanocatalytic complex that induces tumor paraapoptosis provided by the present invention, the molar ratio of urea, ferric chloride hexahydrate, sodium polyacrylate, and sodium citrate dihydrate is 6:1:(1-5):4.

[0015] The hydrothermal reaction temperature is 180–210℃, and the reaction time is 4–12 h.

[0016] Furthermore, in the preparation method of the loose macroporous iron-based nanocatalytic complex for inducing tumor paraapoptosis provided by the present invention, the concentration of the PBS solution containing porous iron oxide is 0.25–1.0 mg / mL.

[0017] The concentration of the lactate oxidase solution is 20–200 U;

[0018] The concentration of the carbonyl cyanide m-chlorophenylhydrazone solution is 100–200 μg / mL;

[0019] Fe 3+ The molar ratio of tannic acid to tannins is 1:1;

[0020] The stirring process, which is carried out in the dark, is conducted at a temperature of 4–25°C for 6–12 hours.

[0021] The light-protected reaction is carried out at a temperature of 4–25°C for 6–12 hours.

[0022] Thirdly, this invention provides the application of loose macroporous iron-based nanocatalytic complexes that induce tumor paraapoptosis in the preparation of antitumor drugs.

[0023] Furthermore, in the application of the loose macroporous iron-based nanocatalytic complex that induces tumor paraapoptosis provided by the present invention in the preparation of antitumor drugs, the antitumor drugs are tumor immunotherapy drugs.

[0024] Furthermore, in the application of the loose macroporous iron-based nanocatalytic complex that induces tumor paraapoptosis provided by the present invention in the preparation of antitumor drugs, the tumor immunotherapy drugs include the nanocatalytic complex and immune checkpoint inhibitors.

[0025] The immune checkpoint inhibitor is a CTL4, PD-1, or PD-L1 inhibitor.

[0026] Fourthly, this invention provides the application of loose macroporous iron-based nanocatalytic complexes that induce tumor paraapoptosis in the preparation of antibacterial drugs.

[0027] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0028] (1) In the nanocatalytic composite (TF-Fe@LC) provided by this invention, Fe is modified on the surface of porous Fe3O4 nanoclusters. 3 + -TA networks can utilize pH-sensitive metal-phenol coordination bonds (Fe 3+ The -O bond dissociates in the microacidic environment of the tumor, enabling targeted and specific responsive release of the drug / CDT catalyst; simultaneously, the pyrogallol structure of TA acts as an electron donor, accelerating the release of Fe. 3+ / Fe 2 + Redox cycles enhance the catalytic efficiency of the Fenton reaction. Porous Fe3O4 nanoclusters, composed of ultra-small Fe3O4 nanocrystals, function as both drug carriers and Fenton catalysts. They can dissociate into ultra-small Fe3O4 nanocrystals in response to the slightly acidic environment of tumors, simultaneously releasing Fe... 2+ / Fe 3+ Catalyzing the Fenton reaction induces oxidative stress in tumor cells, and the conversion from large-particle nanoclusters to small-particle nanocrystals can also balance long-term circulation and infiltration within the nanoparticles. LOD catalyzes the generation of H2O2 from excess lactate in the tumor microenvironment (TME), overcoming the limitations of CDT efficacy and achieving self-supply of catalytic raw materials. In TF-Fe@LC, CCCP uncoupling releases superoxide anions (O2) from the mitochondrial electron transport chain. ·- The ·OH generated by Fe-based nanomaterials forms a spatially complementary oxidative damage network, causing mitochondrial oxidative stress and dysfunction, leading to vacuolization of mitochondria and the endoplasmic reticulum (ER), ultimately driving non-caspase-dependent paraapoptosis. This invention effectively triggers immunogenic cell death (ICD) through CDT-induced paraapoptosis, enhancing its ability to recognize and precisely kill tumor cells. Furthermore, the constructed nanocatalytic system exhibits good compatibility and can be used in combination with antitumor drugs and immunosuppressants to achieve significant synergistic antitumor effects.

[0029] (2) This invention utilizes citric acid to etch the originally dense Fe3O4, transforming the Fe3O4, which was originally composed of interconnected ultra-small nanocrystals, into loose and porous P-Fe3O4 nanoclusters. This loose and porous structure can be completely decomposed in the physiological environment and excreted from the body through normal metabolic pathways, greatly reducing the risks of toxic reactions and immune reactions that may be caused by long-term material residues in the body, and further enhancing its biosafety in biomedical applications. At the same time, this loose and porous structure allows H2O2 to accumulate in its pores and near its surface, increasing the contact area between H2O2 and P-Fe3O4 nanoclusters, making the H2O2 decomposition reaction more efficient and significantly improving the decomposition efficiency, thereby greatly enhancing the tumor killing effect.

[0030] (3) The TF-Fe@LC provided by the present invention has little effect on blood cells, reducing the risk of blood-related adverse reactions when applied in vivo; TF-Fe@LC does not significantly interfere with the overall physiological function of mice and will not disrupt the normal metabolism and physiological balance of the body; H&E staining results of major organs show no obvious tissue damage or pathological changes, and TF-Fe@LC will not cause substantial damage to important organs in vivo, and has good organ compatibility.

[0031] (4) The TF-Fe@LC provided by this invention can specifically target tumor tissue, which is conducive to its accumulation at the tumor site and exert anti-tumor activity, improving the therapeutic effect while reducing damage to normal tissues; TF-Fe@LC can directly act on tumor cells and can also exert anti-tumor effects by regulating the immune system; it is proposed that paraapoptosis-induced ICD can enhance the efficacy of immune checkpoint inhibitors, providing a potential possibility for enhancing ICD activation of anti-tumor immunotherapy through paraapoptosis, and expanding the strategies and methods of tumor immunotherapy.

[0032] (5) In the unilateral anti-tumor experiment, TF-Fe@LC can effectively inhibit the growth of tumors in mice, which once again proves its good biocompatibility and does not produce serious side effects while effectively treating tumors. In the bilateral tumor inhibition experiment, the in situ tumor growth was slowed when TF-Fe@LC was used in combination with αPD-L1, and the distant tumor was inhibited. This indicates that the combination of TF-Fe@LC and αPD-L1 antibody can relieve the inhibitory signal and achieve the synergistic effect of "oxidative killing-activated immunity" in anti-tumor treatment. Attached Figure Description

[0033] Figure 1The morphology and particle size characterization of TF-Fe@LC are shown below. (A) TEM image of P-Fe3O4; (B) TEM image of TF-Fe@LC; (C) Elemental energy distribution diagram of TF-Fe@LC; (D) Particle size distribution diagram of P-Fe3O4, Fe@LC and TF-Fe@LC; (E) Particle size variation of P-Fe3O4, Fe@LC and TF-Fe@LC in PBS solution over time; P-Fe is P-Fe3O4, n=3.

[0034] Figure 2 The in vivo antitumor effect of TF-Fe@LC is shown. (A) Changes in body weight of mice in each group during treatment; (B) Changes in tumor volume of mice in each group during treatment; (C) Tumor mass of mice in each group after treatment; (D) Tumor photographs; (E) Photographs of tumor-bearing mice in each group after treatment; (F) H&E staining images, immunohistochemical and immunofluorescence staining results of major organs and tumor tissues of mice, scale bar is 100 μm; n = 5.

[0035] Figure 3 The results of the in vivo immunostimulation assay for TF-Fe@LC are shown. (A) Drainage lymph node DCsCD86 after TF-Fe@LC stimulation. + Flow cytometry; (B) TF-Fe@LC stimulation of draining lymph node DCs CD80 + Flow cytometry; (C) TF-Fe@LC stimulated drainage of lymph node DCs CD80 + / CD86 + Statistical chart of expression; (D) PBS stimulation of draining lymph node DCs CD86 + Flow cytometry; (E) PBS stimulation of draining lymph node DCs CD80 + Flow cytometry; (F) PBS stimulation of draining lymph node DCs CD80 + / CD86 + Expression statistics; (G)PBS and TF-Fe@LC stimulation of spleen CD3 + CD8 + T cell flow cytometry; (H) PBS and TF-Fe@LC stimulation of spleen CD3 + CD8 + T cell analysis diagram; (I) Splenic CD3 stimulation with PBS and TF-Fe@LC. + CD4 + T cell flow cytometry; (J) PBS and TF-Fe@LC stimulation of spleen CD3 + CD4 + T cell analysis diagram; (K) tumor volume in tumor prevention experimental mice; (L) weight change in tumor prevention experimental mice; n=5.

[0036] Figure 4 The bilateral antitumor effect of TF-Fe@LC is shown. (A) Changes in proximal tumor volume during treatment; (B) Changes in distal tumor volume during treatment; (C) Photographs of proximal (upper) and distal (lower) tumors after treatment; (D) Spleen weight in each group after treatment; (E) Spleen photographs in each group after treatment; G1 is PBS, G2 is CCCP, G3 is TF-Fe@L, G4 is TF-Fe@LC, and G5 is TF-Fe@LC+αPD-L1; n = 3.

[0037] Figure 5 The change in body weight in mice during bilateral antitumor treatment with TF-Fe@LC was 3.

[0038] Figure 6 Flow cytometry was used to detect the expression levels of dendritic cells (DCs) in lymph nodes during TF-Fe@LC bilateral antitumor therapy. (A) CD80 expression levels in different groups. + (A) Representative flow cytometry plots; (B) Different groups of CD80 + Quantitative analysis of expression; (C) shows CD86 in different groups. + Representative flow cytometry plots; (D) shows different groups of CD86 + Quantitative analysis of expression; n=3, G1 is PBS, G2 is CCCP, G3 is TF-Fe@L, G4 is TF-Fe@LC, G5 is TF-Fe@LC+αPD-L1.

[0039] Figure 7 The expression level of lymphocytes in the spleen during TF-Fe@LC bilateral antitumor therapy. (A) CD3 expression levels in different groups. + CD8 + Representative flow cytometry images of T cells; (B) shows different groups of CD3. + CD8 + Statistical analysis of T cells; (C) shows different groups of CD3 + CD4 + Representative flow cytometry images of T cells; (D) shows different groups of CD3. + CD4 + Statistical analysis of T cells; n=3, G1 is PBS, G2 is CCCP, G3 is TF-Fe@L, G4 is TF-Fe@LC, G5 is TF-Fe@LC+αPD-L1.

[0040] Figure 8 (A) Lymphocyte expression levels in distal tumor tissues after bilateral antitumor therapy with TF-Fe@LC. (B) CD3 expression levels in each group of distal tumors. + CD8 + Representative flow cytometry images of T cells; (B) CD3 groups in distal tumors.+ CD8 + Statistical analysis of T cells; (C) shows the CD3 groups in the distal tumor. + CD4 + Representative flow cytometry images of T cells; (D) shows the CD3 groups in the distal tumor. + CD4 + Statistical analysis of T cells; n=3, G1 is PBS, G2 is CCCP, G3 is TF-Fe@L, G4 is TF-Fe@LC, G5 is TF-Fe@LC+αPD-L.

[0041] Figure 9 After the completion of bilateral antitumor therapy with TF-Fe@LC, CD4 in tumor tissue + (Green) / CD8 + Immunofluorescence staining of (red) T lymphocytes and tumor cell apoptosis (TUNEL); scale bar is 100 μm.

[0042] In the above figure, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001. Detailed Implementation

[0043] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially.

[0044] The Balb / C mice (female, 6 weeks old) used in the following examples were purchased from the Experimental Animal Center of Air Force Medical University. All animal experiments were conducted in accordance with the guidelines of the Experimental Animal Ethics Committee of Air Force Medical University (certification number: 251260).

[0045] Example 1

[0046] This embodiment provides a method for preparing nanocatalytic composites.

[0047] 1. Preparation of porous iron oxide nanoclusters

[0048] Urea, ferric chloride hexahydrate, sodium polyacrylate, and sodium citrate dihydrate were dissolved in deionized water at a molar ratio of 6:1:1:4 with vigorous magnetic stirring. After the solution was completely mixed, it was transferred to a reaction vessel and reacted at 180°C for 12 hours. After the reaction was completed and the solution was cooled, it was washed with deionized water, centrifuged, magnetically separated, and freeze-dried to obtain black Fe3O4 powder for later use. Fe3O4 and citric acid were dispersed sequentially in 300 mL of ethylene glycol, with the Fe3O4 content being 120 mg and the citric acid concentration being 0.3 M. After uniform dispersion, the mixture was heated to 180°C in an oil bath and mechanically stirred at 200 r / min for 50 min, with N2 protection throughout the stirring process. After the reaction, aeration was continued, and after cooling, the mixture was removed, washed with deionized water, centrifuged, and magnetically separated to obtain monodisperse porous Fe3O4 nanoclusters with a pore size of 36.7 nm, named P-Fe3O4 (P-Fe).

[0049] 2. Preparation of nanocatalytic composites

[0050] 10 mg of P-Fe3O4 nanoclusters were weighed and added to 20 mL of PBS solution, and dispersed by sonication. 200 U of LOD (lactate oxidase) and 200 μg / mL of CCCP (carbonyl cyanide m-chlorophenylhydrazone, mitochondrial uncoupling agent) solution were added dropwise to the dispersion, and the mixture was stirred mechanically at 25 °C in the dark for 6 h to obtain a mixed solution. 30 mM FeCl3·6H2O (Fe...) was then prepared separately. 3+ ) solution and tannic acid (TA) solution (Fe 3+ The molar ratio of Fe to tannic acid is 1:1. Fe is then processed stepwise. 3+ The solution and TA solution were added to the mixture, and the mixture was mechanically stirred at 25°C in the dark for 6 hours. After washing with ultrapure water and magnetic separation, the Fe nanocatalytic complex was obtained. 3+ -TA-P-Fe3O4@LOD / CCCP(TF-Fe@LC), freeze-dried in the dark for later use.

[0051] Example 2

[0052] This embodiment provides a method for preparing a nanocatalytic composite and its anti-tumor therapeutic effect.

[0053] 1. Preparation of porous iron oxide nanoclusters

[0054] 1.44 g urea, 1.08 g ferric chloride hexahydrate, 1.2 g sodium polyacrylate, and 4.8 g sodium citrate dihydrate (coordinated, with a molar ratio of 6:1:3:4 for urea, ferric chloride hexahydrate, sodium polyacrylate, and sodium citrate dihydrate) were dissolved in deionized water under vigorous magnetic stirring. After the solution was completely mixed, it was transferred to a reaction vessel and reacted at 200°C for 4 hours. After the reaction was completed and the solution was cooled, it was washed with deionized water, centrifuged, magnetically separated, and freeze-dried to obtain black Fe3O4 powder for later use. Fe3O4 and citric acid were then dispersed sequentially in 250 mL of ethylene glycol, with Fe3O4 content of 100 mg and citric acid concentration of 0.5 M. After uniform dispersion, the mixture was heated in an oil bath to 160°C and mechanically stirred at 400 r / min for 60 min, with N2 protection throughout the stirring process. After the reaction, continuous aeration was maintained, and the material was removed after cooling. It was then washed with deionized water, centrifuged, and magnetically separated to collect the nanoclusters, ultimately yielding monodisperse porous Fe3O4 nanoclusters, named P-Fe3O4 (P-Fe). This process utilizes citric acid to etch Fe3O4, transforming the original Fe3O4 composed of interconnected ultra-small nanocrystals (3-6 nm) into loose, porous P-Fe3O4 nanoclusters. The pore size of P-Fe3O4 reaches 16.9 nm, enabling effective loading of biomacromolecules at the level of demand (LOD).

[0055] 2. Preparation of nanocatalytic composites

[0056] 5 mg of P-Fe3O4 nanoclusters were weighed and added to 10 mL of PBS solution, and dispersed by sonication. 100 U of LOD (lactic acid oxidase) and 100 μg / mL of CCCP (carbonyl cyanide m-chlorophenylhydrazone, mitochondrial uncoupling agent) solution were added dropwise to the dispersion, and the mixture was stirred mechanically at 4 °C in the dark for 12 h to obtain a mixture. 24 mM FeCl3·6H2O (Fe...) was then prepared... 3+ ) solution and tannic acid (TA) solution (Fe 3+ The molar ratio of Fe to tannic acid is 1:1. Fe is then processed stepwise. 3+ The solution and TA solution were added to the mixture, and the mixture was mechanically stirred at 4°C in the dark for 12 hours. After washing with ultrapure water and magnetic separation, the Fe nanocatalytic complex was obtained. 3+ -TA-P-Fe3O4@LOD / CCCP(TF-Fe@LC) was freeze-dried in the dark for later use. In addition, Fe3O4@LOD / CCCP(Fe@LC) and Fe were prepared according to the above steps. 3+ -TA-P-Fe3O4(TF-Fe), Fe 3+ -TA-P-Fe3O4@LOD(TF-Fe@L) and Fe 3+ -TA-P-Fe3O4@CCCP(TF-Fe@C).

[0057] 3. Pore size and loading analysis of the nanocatalytic composite

[0058] like Figure 1 As shown in (AC), after loading CCCP and LOD and encapsulating with TF, the morphology and structure of the nanoclusters did not change significantly, but the average particle size was larger (around 280 nm), and the edges of the nanoclusters were significantly smoother. Elemental mapping images show that TF-Fe@LC contains Fe, C, N, P, Cl, and O elements, indicating the successful preparation of the nanocatalytic composite. The particle sizes of the P-Fe3O4, Fe@LC, and TF-Fe@LC nanocascade systems are shown in Figure 1. Figure 1 As shown in (D, E) of the diagram, the particle size distribution shows that the hydrated particle size of Fe@LC increased significantly after drug loading, while the hydrated particle size of TF-Fe@LC increased slightly after TF coating, reaching approximately 300 nm. P-Fe3O4, Fe@LC, and TF-Fe@LC nanoclusters were continuously monitored in PBS for 7 days, and no significant increase or aggregation was observed, all exhibiting uniform particle size distribution and good colloidal stability. In this example, the LOD loading in 500 μg / mL TF-Fe@LC was approximately 38.1 U (7.62%), and the CCCP loading was approximately 51.7 μg / mL (10.34%).

[0059] 4. The therapeutic effect of nanocatalytic complex on tumors

[0060] Experimental method: 5×10 6 4T1 cell suspension was subcutaneously inoculated into the right groin of Balb / C mice, establishing a subcutaneous 4T1 tumor model after 7 days. When the tumor volume reached 80 mm²... 3 Thirty mice were randomly divided into six groups: PBS group, CCCP group (2.5 mg / kg), TF-Fe group, TF-Fe@L group, TF-Fe@C group, and TF-Fe@LC group (all administered at a ferric oxide concentration of 12.5 mg / kg). The mice were administered the drug every two days, and their body weight and tumor volume were recorded. After 14 days of treatment, the mice were euthanized and dissected. Major organs (heart, liver, spleen, lung, and kidney) and tumor tissues from different groups were collected, fixed in 4% paraformaldehyde, and stained with H&E. In addition, subcutaneous tumors were rapidly dissected, and the tumor tissues were photographed and weighed. Afterward, they were fixed in 4% paraformaldehyde, and subsequent histomorphological analysis (H&E staining) and cell death and proliferation detection (TUNEL fluorescent labeling and Ki-67 immunohistochemistry) were performed on these tumor tissue samples.

[0061] Experimental results: During the in vivo antitumor treatment with the nanocatalytic complex, the body weight of the mice in the six groups fluctuated slightly, but maintained an overall upward trend without significant changes, indicating good drug safety. Figure 2(A)). Changes in tumor volume in each group of mice during treatment are shown in Figure 1. Figure 2 As shown in (B), compared with the PBS group, the CCCP group showed no significant difference, while the other four groups all exhibited significant tumor-inhibiting effects. Among them, the TF-Fe@LC group showed the most significant inhibitory effect on tumor growth, and the average tumor weight, corresponding tumor images, and tumor-bearing mouse photographs also verified the good anti-tumor effect of TF-Fe@LC. Figure 2 (C, D, E)). After treatment, pathological analysis was performed on the major organs (heart, liver, spleen, lung, kidney) and tumor tissues of the mice. H&E staining results showed that the drug treatment group had no effect on normal tissues, while the tumor tissues showed obvious cell damage. Figure 2 (E in the text). The above data indicate that the nanocatalytic complex provided in this embodiment has a certain anti-tumor effect. The above data indicate that TF-Fe@LC can effectively inhibit tumor growth, has good anti-tumor efficacy, and can target tumor cells for therapy.

[0062] Fe 3+ -TA network is a pH-sensitive metal-phenol coordination bond (Fe 3+ -O bonds can target the tumor microacidic environment and, under weakly acidic conditions, gradually disintegrate the metal polyphenol network, releasing Fe. 2+ It also exposes P-Fe3O4, catalyzing the limited H2O2 within tumor cells to generate ·OH. TF-Fe@LC releases LOD, catalyzing the generation of H2O2 from LA in the microacidic environment of the tumor, thereby reacting with Fe. 2+ A cascade reaction occurs (lactic acid metabolism reprogramming - H2O2 self-supply); simultaneously, TF-Fe@LC releases trace amounts of CCCP to induce O2 through the uncoupling of the electron transport chain. - The leakage forms a positive feedback loop with the generated ·OH, causing a significant increase in the level of ROS in tumor cells, leading to mitochondrial damage in tumor cells (mitochondrial oxidative stress). The two work together to exert an anti-tumor effect through oxidative stress.

[0063] At the cellular level, ROS can precisely regulate the activity of the autophagy pathway. Autophagy, as an important intracellular degradation mechanism, plays a role in clearing damaged organelles (such as mitochondria) and misfolded proteins. TF-Fe@LC can induce tumor cells to produce large amounts of ROS, and excessive ROS can block the normal functioning of the autophagy pathway. After the autophagy pathway is blocked, damaged mitochondria and misfolded proteins that rely on autophagy for clearance cannot be degraded in time. Damaged mitochondria not only lose their normal energy metabolism function, but also continue to produce more ROS, forming a vicious cycle of "ROS production - autophagy blockage - accumulation of damaged mitochondria - further increase in ROS". At the same time, the accumulation of misfolded proteins will disrupt the homeostasis of intracellular proteins and affect normal cellular physiological processes. As the intracellular ROS level continues to rise, excessive ROS will act on the endoplasmic reticulum, interfering with the correct folding and processing of proteins in the endoplasmic reticulum, thereby activating the endoplasmic reticulum stress response. When endoplasmic reticulum stress persists and cannot be effectively relieved, it leads to paraapoptosis in tumor cells.

[0064] Example 3

[0065] This embodiment demonstrates the efficacy of the nanocatalytic complex in tumor immunotherapy.

[0066] 1. Evaluation of unilateral antitumor activity and immune response in mice

[0067] Experimental methods: Mouse 4T1 cells were seeded into T25 culture flasks. After the cells reached the logarithmic growth phase, they were treated with TF-Fe@LC for 24 hours. Dead cells were collected, washed three times with PBS, and the cell concentration was adjusted to 1×10⁻⁶. 7Single-cell suspensions were prepared at 100 μL / mL. Twenty 6-week-old female Balb / C mice were randomly divided into two groups (n=10 per group): In the experimental group, each mouse received a subcutaneous injection of 100 μL of 4T1 dead cells treated with TF-Fe@LC in the inner thigh (near the groin area); in the control group, each mouse received a subcutaneous injection of 100 μL of PBS in the inner thigh. Inoculations were performed weekly for a total of three weeks (days 0, 7, and 14). Seven days after the last inoculation (day 21), five mice from each group were euthanized, and inguinal lymph nodes and spleens were collected. The cells were placed in sterile PBS, gently ground using a 5 mL syringe plunger, and filtered through a 70 μm cell sieve to prepare single-cell suspensions. The lymph node single-cell suspensions were then incubated with anti-CD11c, anti-CD80, and anti-CD86 antibodies at 4°C in the dark for 30 min for flow cytometry analysis of dendritic cell maturation. Splenic single-cell suspensions were collected, cleaved, and then anti-CD3, anti-CD4, and anti-CD8 antibodies were added. The mixture was incubated at 4°C in the dark for 30 minutes and then used for flow cytometry to detect T-cell activation. The remaining 10 mice were implanted with tumors subcutaneously in the opposite thigh using standard 4T1 cells. Body weight and tumor growth were monitored in both groups of mice.

[0068] Experimental Results: In this embodiment, TF-Fe@LC-treated 4T1 dead cells were used as a tumor vaccine. Through in vivo tumor vaccination and re-challenge experiments, the activation effect on the immune system of Balb / C mice was preliminarily evaluated. Figure 3 Mature dendritic cells (DCs) play an important role in activating T cells, promoting T cell infiltration, and enhancing anti-tumor immune responses. Therefore, flow cytometry was used to analyze the maturation level of DCs in mouse drainage lymph nodes collected after three immunizations. Figure 3 The (AF) results showed that, compared with the PBS group, mice immunized with lethal 4T1 cells treated with TF-Fe@LC exhibited significantly enhanced expression of the co-stimulatory molecules CD80 and CD86 on the surface of dendritic cells (DCs). Mature DCs can effectively promote the activation and proliferation of CD8+ T cells, thereby enhancing the anti-tumor immune response. Based on this, this example used flow cytometry to quantitatively detect the infiltration of CD4+ T cells and CD8+ T cells in the spleen. Figure 3 As shown in (GJ), the treatment group significantly increased CD3 compared to the PBS group. + CD4 + T cells and CD3 + CD8 +T cell infiltration reached 28.2±1.4% and 14.0±0.6%, respectively, demonstrating that TF-Fe@LC-treated 4T1 dead cells could promote the infiltration of cytotoxic T cells in mice. After immunization, normal 4T1 cells were used to implant tumors subcutaneously in mice. Mouse body weight and tumor volume were recorded daily for 5 days, and mice were sacrificed after 21 days. The results showed that mice immunized with TF-Fe@LC-treated "4T1-Vaccine" experienced slow tumor growth after tumor implantation, and mouse body weight was not significantly affected. Figure 3 (K, L) in the middle.

[0069] After TF-Fe@LC acts on 4T1 cells, it disrupts the endoplasmic reticulum and mitochondria through paraapoptosis, triggering an endoplasmic reticulum stress response. Endoplasmic reticulum stress (ICD) promotes the release of damage-associated molecular patterns (DAMPs) from cells. During ICD, tumor-associated antigens within 4T1 cells are released along with DAMPs. The released DAMPs induce dendritic cell (DC) maturation and antigen presentation, thereby activating T cell-mediated anti-tumor immune responses.

[0070] 2. Bilateral antitumor evaluation and immune response test in mice

[0071] Experimental methods: A bilateral 4T1 subcutaneous tumor model was established in female Balb / C mice. 1.0 × 10⁻⁶ tumor cells were implanted into the tumor cells. 6 4T1 cells in logarithmic growth phase were subcutaneously seeded into the right hind leg of Balb / C mice to investigate the antitumor therapeutic effect of TF-Fe@LC on proximal tumors. Three days later, 5.0 × 10⁶ cells were... 5 Four T1 cells were subcutaneously injected into the left hind leg of Balb / C mice to serve as a distal tumor for evaluating antitumor immune response. When the proximal tumor volume reached 50-80 mm², the tumor was considered to have a distal tumor. 3 Thirty mice were randomly divided into five groups: PBS group, CCCP group (2.5 mg / kg), TF-Fe@L group, TF-Fe@LC group, and TF-Fe@LC+αPD-L1 group (all administered at a ferric oxide concentration of 12.5 mg / kg, αPD-L1: 100 μg). Intratumoral injection of αPD-L1 was performed on day 0, followed by intraperitoneal injection on day 1. Administration was repeated every 3 days for a total of 5 administrations. Mouse weight and tumor volume were recorded every 2 days during the treatment period, for a total of 14 days. After treatment, three mice from each group were randomly euthanized, and tumors and spleens were rapidly collected and photographed. Tumors in the bilateral model were sectioned, fixed, and stained to observe the TUNEL and CD4 / CD8 ratios and infiltration. Three additional mice from each group were dissected in a clean bench, and lymph nodes, spleens, and tumors were collected for subsequent immunological testing of lymphocytes.

[0072] Lymph nodes and spleen were treated according to the method described in Example 1, "Evaluation of Unilateral Antitumor Activity and Immune Response Test in Mice," and corresponding single-cell suspensions were prepared. Flow cytometry was used to analyze the maturation of dendritic cells (DCs) and the activation of T lymphocytes in the spleen. For the detection of T lymphocytes in tumor tissue: tumor tissue lysis buffer was prepared in advance, and proximal and distal tumor fragments were removed and surgically minced to 0.1 mm. 3 Around 37°C, lysis buffer was added, and the cells were digested for 30-45 minutes. After digestion, the cell suspension was collected and filtered through a sieve to obtain a tumor cell suspension. This suspension was then centrifuged at 375g for 5 minutes to obtain a tumor single-cell suspension. Subsequent processing was the same as for the spleen: cells were blocked after red blood cell lysis, and stained with APC-conjugated CD3 antibody, FITC-conjugated CD4 antibody, and PE-conjugated CD8 antibody. The cells were incubated at 4°C in the dark for 30 minutes, centrifuged, washed, and collected. Finally, T cells in the tumor tissue were quantitatively detected by flow cytometry.

[0073] This embodiment uses a 4T1 bilateral tumor model to explore the synergistic antitumor effect of combined treatment with immune checkpoint blockade (αPD-L1). The right tumor was designated as the primary tumor, and the left tumor as a distant tumor for evaluating the antitumor immune response. After 5 treatment cycles, the results are as follows: Figure 4 As shown in (A, B), compared with the PBS group, CCCP group, and TF-Fe@L group, the TF-Fe@LC group showed significant inhibition of both proximal and distal tumors in mice. Further combination with αPD-L1 resulted in the highest inhibition rates for both proximal and distal tumors, at 90.17% and 84.5%, respectively. Figure 4 As shown in (C), the inhibitory trends of distal and proximal tumors in each treatment group were basically the same, indicating that the combined TF-Fe@LC and immune checkpoint blockade therapy had a very ideal effect.

[0074] The spleen is an important immune organ in the human body. Tumor stimulation leads to spleen enlargement, and spleen size reflects the body's immune function to some extent. As the largest peripheral immune organ, the spleen plays a crucial role in the immune response. Therefore, spleen tissue was collected from mice to examine whether it activated related immune responses. Due to tumor antigen stimulation, the spleen tissue of mice in both the PBS and CCCP groups significantly enlarged. This splenomegaly may trigger negative immune regulation, thereby accelerating tumor development. Figure 4 As shown in (D, E), the spleens of the TF-Fe@L and TF-Fe@LC groups were relatively smaller, while the TF-Fe@LC+αPD-L1 group successfully avoided splenomegaly. This phenomenon may be attributed to the combined use of paraapoptosis and immunosuppressants effectively activating anti-tumor immunity, thereby inhibiting abnormal splenic proliferation. Furthermore, during treatment, the mice showed no significant change in body weight. Figure 5This indicates that the drug-treated group had minimal adverse effects on the mice.

[0075] like Figure 6 As shown, the maturation level of dendritic cells (DCs) significantly increased after combined treatment with TF-Fe@LC and αPD-L1. DC activation effectively stimulated CD8+. + T cells, thereby enhancing adaptive immunity against solid tumors.

[0076] To investigate the enhancing effect of immune checkpoint inhibitors on distal effects, flow cytometry was used to analyze CD4+ in mouse spleen and tumor tissue. + and CD8 + The infiltration of T lymphocytes. For example... Figure 7 As shown, the spleen contains CD3 + High T cell count, CD4 + / CD8 + The proportion of T lymphocytes in tumor tissue is relatively low. Among them, the TF-Fe@LC+αPD-L1 group has CD4... + / CD8 + The percentages of T lymphocytes were 27.13% and 11.55%, respectively, and CD8... + The number of T cells was 2.35, 1.88, 1.56, and 1.31 times that of the PBS group, CCCP group, TF-Fe@L group, and TF-Fe@LC group, respectively.

[0077] like Figure 8 As shown, in distal tumors, the TF-Fe@LC+αPD-L1 group showed tumor infiltration of CD4. + and CD8 + The highest percentages of T cells were 42.34% and 20.56%, respectively, consistent with the in situ tumor. Furthermore, Figure 9 The figure shows the CD4 levels in tumor tissues treated with different drug groups. + and CD8 + Immunofluorescence staining images of T lymphocyte infiltration clearly show CD4 in the PBS group and CCCP group. + (Green fluorescence) and CD8 + (Red fluorescence) T cells were less distributed, while lymphocyte infiltration was significantly increased in the TF-Fe@LC group and the TF-Fe@LC+αPD-L1 group, especially the TF-Fe@LC+αPD-L1 group, which showed a large amount of red fluorescence. The above results demonstrate that the combined administration of TF-Fe@LC and αPD-L1 can effectively promote the infiltration of cytotoxic T cells.

[0078] This embodiment also used TUNEL staining to examine the apoptosis or necrosis of cells within the primary and distant tumors in each treatment group. The results are as follows: Figure 9As shown, green fluorescence signals in TUNEL staining represent apoptotic or necrotic cells. In the PBS and CCCP groups, the green fluorescence signals were weak, while the green fluorescence signals in the TF-Fe@LC+αPD-L1 combined administration group were significantly enhanced, indicating that large-scale tumor cell death occurred and tumor growth was effectively inhibited.

[0079] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. A loosely porous macroporous iron-based nanocatalytic complex that induces tumor paraapoptosis, characterized in that, The nanocatalytic composite includes a support and a catalytic sensitizing component supported on the support; The carrier is porous iron oxide modified with an iron ion-polyphenol complex; The catalytic sensitizing components are lactate oxidase and carbonyl cyanide m-chlorophenylhydrazone.

2. The loosely porous macroporous iron-based nanocatalytic complex for inducing tumor paraapoptosis according to claim 1, characterized in that, The particle size of the nanocatalytic composite is 100–300 nm; The pore size of the unmodified porous iron oxide with tannic acid-iron polyphenol network is 10–40 nm.

3. The loosely porous macroporous iron-based nanocatalytic complex for inducing tumor paraapoptosis according to claim 1, characterized in that, The lactate oxidase loading is 5-10%; The loading of the carbonyl cyanide m-chlorophenylhydrazone is 5-15%.

4. A method for preparing a loosely porous macroporous iron-based nanocatalytic complex that induces tumor paraapoptosis, characterized in that, include: Iron oxide was prepared by hydrothermal method using urea, ferric chloride hexahydrate, sodium polyacrylate and sodium citrate dihydrate as raw materials. Porous magnetite was obtained by etching magnetite with an ethylene glycol solution containing citric acid. The porous magnetite was then dispersed in PBS solution, and lactate oxidase solution and m-chlorophenyl hydrazone carbonyl cyanide solution were added sequentially. The mixture was stirred in the dark to obtain a solution. Fe was then added sequentially to the solution. 3+ The solution and tannic acid solution were reacted in the dark, followed by washing and magnetic separation to obtain the nano-catalytic complex.

5. The method for preparing the loose macroporous iron-based nanocatalytic complex for inducing tumor paraapoptosis according to claim 4, characterized in that, The molar ratio of urea, ferric chloride hexahydrate, sodium polyacrylate, and sodium citrate dihydrate is 6:1:(1~5):4; The hydrothermal reaction temperature is 180–210℃, and the reaction time is 4–12 h.

6. The method for preparing the loose macroporous iron-based nanocatalytic complex for inducing tumor paraapoptosis according to claim 4, characterized in that, The concentration of the PBS solution containing porous iron oxide is 0.25–1.0 mg / mL; The concentration of the lactate oxidase solution is 20–200 U; The concentration of the carbonyl cyanide m-chlorophenylhydrazone solution is 100–200 μg / mL; Fe 3+ The molar ratio of tannic acid to tannins is 1:1; The stirring process, which is carried out in the dark, is conducted at a temperature of 4–25°C for 6–12 hours. The light-protected reaction is carried out at a temperature of 4–25°C for 6–12 hours.

7. The use of the loose macroporous iron-based nanocatalytic complex for inducing tumor paraapoptosis as described in any one of claims 1 to 3 in the preparation of antitumor drugs.

8. The application of the loose macroporous iron-based nanocatalytic complex for inducing tumor paraapoptosis according to claim 7 in the preparation of antitumor drugs, characterized in that, The anti-tumor drug is a tumor immunotherapy drug.

9. The application of the loose macroporous iron-based nanocatalytic complex for inducing tumor paraapoptosis according to claim 8 in the preparation of antitumor drugs, characterized in that, The tumor immunotherapy drug includes nanocatalytic complexes and immune checkpoint inhibitors; The immune checkpoint inhibitor is a CTL4, PD-1, or PD-L1 inhibitor.

10. The use of the loose macroporous iron-based nanocatalytic complex for inducing tumor paraapoptosis according to any one of claims 1 to 3 in the preparation of antibacterial drugs.

Images