Arctigenin-derived nano-enzyme for targeting lung cancer ferroptosis and preparation method of arctigenin-derived nano-enzyme

Abstract

The invention belongs to the field of biological medicine, and particularly discloses arctigenin-derived nano-enzyme for targeting lung cancer ferroptosis and a preparation method of the arctigenin-derived nano-enzyme. The nano-enzyme is formed by self-assembly of arctigenin and Fe through coordination, the molar ratio is 1: 1-1: 3, and the particle size is 50-150 nm. The preparation method comprises the following steps: dissolving arctigenin and Fe in an ethanol-water solution, adjusting the pH value to 7.5-8.5, reacting, and purifying. The nano-enzyme has the characteristics of high drug loading capacity and pH response release, and can be enriched at a tumor site through an EPR effect to synergistically deplete glutathione, down-regulate GPX4 activity and increase the active oxygen level, thereby efficiently inducing ferroptosis of tumor cells. In-vivo and in-vitro experiments show that Fe (at) Arc has a remarkable inhibition effect on non-small cell lung cancer and is good in biological safety. The invention provides a new strategy for targeted therapy of lung cancer, and has a good clinical transformation prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically discloses an arctigenin-derived nanozyme that targets ferroptosis in lung cancer and its preparation method. Background Technology

[0002] Lung cancer remains the leading cause of cancer-related deaths worldwide, posing a heavy burden on public health. Based on histological characteristics, lung cancer is mainly divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), with NSCLC accounting for approximately 85% of all cases. The development of lung cancer is a complex process driven by a combination of genetic, environmental, and lifestyle factors; traditionally, smoking has been the most significant risk factor. However, the epidemiological landscape is changing: the incidence of lung cancer among non-smokers continues to rise, particularly in East Asian populations, highlighting the importance of other risk factors such as genetic susceptibility, air pollution, and occupational exposure. At the molecular level, lung cancer manifests as a series of highly heterogeneous genetic alterations, including activating mutations associated with programmed cell death. The identification of these molecular drivers has driven the advent of targeted therapies—significantly improving treatment efficacy and patient outcomes by specifically inhibiting aberrant signaling pathways.

[0003] Ferroptosis, a novel programmed cell death mechanism characterized by lipid peroxidation-driven membrane rupture, has shown great potential in cancer treatment. Unlike apoptosis, necrosis, and autophagy, ferroptosis depends on the accumulation of lipid reactive oxygen species (ROS) and the depletion of glutathione (GSH), ultimately leading to iron-dependent oxidative cell death. The discovery of ferroptosis has not only deepened our understanding of cell death mechanisms but also provided new insights for targeted therapy: tumor cells with abnormal metabolism are often more sensitive to ferroptosis induction. Preclinical studies have demonstrated that inducing ferroptosis can selectively eliminate cancer cells while maximally protecting normal cells, highlighting its potential as a targeted therapy. Furthermore, dysregulation of ferroptosis-related pathways (such as iron metabolism, lipid metabolism, and antioxidant defense systems) has been shown to be closely related to various malignancies, including lung cancer, breast cancer, and pancreatic cancer. Therefore, elucidating the molecular regulatory mechanisms of ferroptosis and developing targeted drugs that can induce or inhibit this process is of significant clinical importance. The core of this research lies in analyzing key regulatory nodes and discovering small molecule ferroptosis inducers or inhibitors that can be clinically translated, in order to improve efficacy and reduce cancer mortality.

[0004] Despite these advances, ferroptosis-based cancer treatments still face numerous challenges: drug resistance mechanisms, a lack of biomarkers, and the development of novel therapeutics all urgently need to be addressed. Nanotechnology, with its unique physicochemical properties (small size, large specific surface area, and tunable surface chemistry), offers a new approach to overcoming these difficulties. Through surface ligand modification, engineered nanocarriers can achieve tumor-targeted delivery and controlled drug release; simultaneously, the enhanced permeability and retention (EPR) effect resulting from tumor tissue vascular leakage and impaired lymphatic return allows nanoparticles to passively accumulate in the tumor microenvironment. Multi-active component integrated strategies hold promise for improving the therapeutic index of traditional chemotherapy drugs, overcoming drug resistance, and achieving multifunctional synergistic therapy. Therefore, nanotechnology is leading a paradigm shift in ferroptosis-related cancer treatment, laying a solid foundation for the development of next-generation precise and efficient anti-tumor modalities. In conclusion, constructing novel drug delivery systems is crucial for optimizing lung cancer treatment strategies, prolonging overall survival, and improving quality of life for patients. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides an arctigenin-derived nanozyme Fe@Arc targeting ferroptosis in lung cancer and its preparation method. This material is prepared through the coordination of iron ions with arctigenin, exhibiting high arctigenin loading and ideal release performance. After intravenous injection, Fe@Arc effectively accumulates at the tumor site through the enhanced permeability retention effect (EPR effect) and exerts a significant tumor-suppressive effect by inducing synergistic ferroptosis. This multifunctional therapeutic platform not only overcomes the limitations of herbal extract applications but also lays the foundation for developing novel ferroptosis inducers with clinical potential.

[0006] This invention includes the following technical solutions:

[0007] A arbuscular aglycone-derived nanozyme targeting ferroptosis in lung cancer, the composite nanozyme being formed by coordination between arbuscular aglycone and iron ions, is named Fe@Arc.

[0008] Furthermore, in the aforementioned nanozyme, the iron ions are Fe. 3+ Arctium brevicornuate and Fe 3+ The molar ratio is 1:1 to 1:3.

[0009] Furthermore, the nanozyme described above has a particle size of 50–150 nm, preferably 80–90 nm.

[0010] This invention also discloses a method for preparing the above-mentioned nanozyme, comprising the following steps:

[0011] a) Incorporating arctigenin and Fe 3+ Soluble in a mixture of ethanol and water;

[0012] b) Adjust the pH to 7.5–8.5 and react for 1–3 hours;

[0013] c) Centrifuge, wash, and freeze-dry to obtain Fe@Arc nanozyme.

[0014] Furthermore, in the above method, the volume ratio of ethanol to water is 1:1, and the pH is adjusted using NaOH solution.

[0015] The present invention also discloses a pharmaceutical composition comprising any of the nanozymes described above and a pharmaceutically acceptable carrier.

[0016] The present invention also discloses the use of the above-mentioned nanozyme or pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of lung cancer.

[0017] Furthermore, in the above application, the lung cancer referred to is non-small cell lung cancer.

[0018] Furthermore, in the above application, the drug is an intravenously administered formulation.

[0019] The present invention also discloses the use of the above-mentioned nanozyme or pharmaceutical composition in the preparation of a drug for inducing ferroptosis in tumor cells.

[0020] Compared with the prior art, the present invention has the following outstanding advantages:

[0021] In this invention, we constructed a composite nanozyme Fe@Arc using arctigenin, a natural lignan compound extracted from the fruit of the traditional Chinese medicinal plant, burdock (Arctium lappa L.). After being injected via tail vein into a non-small cell lung cancer (NSCLC) model, this nanozyme induced ferroptosis by consuming glutathione (GSH) within tumor cells. Figure 1 The generated oxidized glutathione (GSSG) further downregulates glutathione peroxidase 4 (GPX4) levels, thereby promoting programmed cell death. The loaded arctigenin, as a potent anticancer active ingredient, can synergistically interact with ferroptosis by upregulating reactive oxygen species (ROS) levels.

[0022] System characterization results from the examples show that this rationally designed nanozyme possesses high encapsulation efficiency, excellent biosafety, and exhibits significant tumor-suppressive effects in a mouse lung cancer model. Fe@Arc-enhanced ROS generation and gene regulation synergistically amplify tumor ferroptosis, thereby achieving highly efficient tumor inhibition. This study is the first to demonstrate that arctigenin can act as a regulator of ferroptosis, providing a new approach to expanding the application of traditional herbal extracts in cancer treatment. Attached Figure Description

[0023] Figure 1: Schematic diagram of the construction of a composite nanozyme based on arctigenin and its application in synergistic induction of ferroptosis and its use in lung cancer treatment; In the figure, ROS: reactive oxygen species; GSH: glutathione; GSSG: oxidized glutathione; GPX4: glutathione peroxidase 4;

[0024] Figure 2 TEM, XRD, and SAED spectra of Fe@Arc;

[0025] Figure 3 XPS spectra of Fe@Arc ((a) Fe 2p, (b) N 1s, and (c) C 1s X-ray photoelectron spectra);

[0026] Figure 4 Results of cellular uptake, lysosomal escape, and in vitro toxicity experiments;

[0027] (a) Confocal images of A549 cells co-incubated with Cy5-labeled Fe@Arc at 37°C for different times; scale bar 50 μm; (b) Quantitative results of endocytosis; (c) Lysosomal colocalization analysis: Cy5-Fe@Arc (red) and LysoTracker (green) hardly overlap, scale bar 20 μm; (d) Relative survival rate of A549 cells after treatment with different concentrations of Fe@Arc; (e) Apoptosis detection by flow cytometry: distribution of apoptosis / necrosis in four groups of cells: Control (PBS), free arctiin (D), blank Fe NPs, and Fe@Arc; (f) Quantitative statistics by flow cytometry; drug concentrations were based on 10 μM arctiin, and data were mean ± SEM from three independent experiments.

[0028] Figure 5 Detection of ferroptosis-related indicators (GSH, ROS, GPX4, mitochondrial morphology);

[0029] Figure 6 Evaluation of tumor suppression efficacy and safety in vivo: (a) Images of excised tumors in different treatment groups; (b) Tumor weight statistics in each group; (c–f) Dynamic tumor volume curves over 21 days; p<0.05, **p<0.01, ***p<0.001 vs PBS group. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Materials and Methods

[0032] 1.1 Materials

[0033] Arctigenin was purchased from MedChemExpress (MCE, China). All chemical reagents and solvents were commercially available analytical grade and used directly. Millipore ultrapure water was used in the experiments. Annexin V-FITC / PI apoptosis detection kit (catalog number 40302ES60) was also used. The IIIOne Step RT-qPCR SYBR Green Kit (catalog number 11143ES70) was purchased from Shanghai Yisheng Biotechnology Co., Ltd.; the Hoechst 33342 and CCK-8 kits were purchased from Beijing Solarbio Science & Technology Co., Ltd.; and the serum-free cell cryopreservation solution (6032011) was purchased from Shenzhen Dakwei Co., Ltd. All other reagents, unless otherwise specified, were domestically produced or imported analytical grade.

[0034] 1.2 Cells and Animals

[0035] Human non-small cell lung cancer A549 cells were purchased from the American Type Culture Collection (ATCC) and cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO2 saturated humidity.

[0036] Female BALB / c nude mice, aged 4–6 weeks and weighing approximately 20g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal experiments were approved by the Laboratory Animal Ethics Committee of Hainan Medical University (Approval No.: HYLL-2023-182) and conducted strictly in accordance with relevant regulations.

[0037] 1.3 Synthesis and Characterization of Fe@Arc

[0038] In brief:

[0039] Arctoside (Arc) and ferric chloride (Fe) 3+ The Fe@Arc nanozyme was dissolved in an ethanol-water mixture at a 1:2 molar ratio (v / v = 1:1), and stirred magnetically for 30 min at room temperature. Then, 0.1 M NaOH was added dropwise to adjust the pH to 8.0, and the reaction was continued for 2 h. The precipitate was then collected by centrifugation (12,000 g, 15 min), washed three times with ultrapure water, and freeze-dried to obtain the Fe@Arc nanozyme.

[0040] Morphology and particle size were characterized by transmission electron microscopy (TEM, JEOL JEM-2100) and dynamic light scattering (DLS, Malvern ZS90); crystal structure was analyzed by X-ray diffraction (XRD, Rigaku Ultima IV); surface functional groups were analyzed by Fourier transform infrared spectroscopy (FT-IR, Bruker Vertex 70); elemental composition was confirmed by X-ray photoelectron spectroscopy (XPS, Thermo ESCALAB 250Xi). Figure 3 As shown.

[0041] 1.4 Determination of drug loading and encapsulation efficiency

[0042] Fe@Arc nanozyme was mixed with arctigenin solution (1 mg / mL) -1 The mixture was incubated with ethanol / water (1:1) for 12 hours to allow the drug to be loaded onto the carrier via coordination / adsorption. The supernatant was then separated from the precipitate by ultracentrifugation at 100,000 g for 30 minutes.

[0043] The supernatant was collected, and the absorbance of the unloaded drug was measured at 420 nm using a UV-Vis spectrophotometer (UV-2600, Shimadzu, Japan). The drug loading and loading efficiency (DLE) were then calculated.

[0044] Calculation formula:

[0045] DLE (%) = (W_encapsulated drug / W_total drug) × 100

[0046] Wherein, W_encapsulated drug = W_total drug - W_free drug (calculated from the standard curve)

[0047] 1.5 Drug loading efficiency (DLE)

[0048] Calculate using the following formula:

[0049] DLE (%) = [(Added drug amount - Unloaded drug amount) / Added drug amount] × 100

[0050] 1.6 Cellular uptake and subcellular localization

[0051] A549 cells were seeded at a density of 1×10^5 cells / well in 24-well plates and allowed to adhere overnight.

[0052] Preparation method of Cy5-labeled nanozymes: Using existing techniques, Cy5-NHS esters are combined with...

[0053] The covalent coupling of Fe@Arc involved a reaction at room temperature in the dark for 2 hours, followed by ultrafiltration to remove the free dye.

[0054] Cy5-Fe@Arc.

[0055] Cy5-Fe@Arc cells were co-incubated for 2 hours (37°C). After incubation, the cells were washed three times with PBS to remove untaken particles; then LysoTracker (50 nM) and Hoechst (50 nM) were added and the cells were incubated at 37°C.

[0056] Stain for 30 min. Discard the staining solution and fix with 4% paraformaldehyde at room temperature for 15 min. Observe and photograph using a laser confocal microscope (Zeiss LSM880).

[0057] 1.7 Cytotoxicity assay (CCK-8)

[0058] A549 cells were seeded at 4,500 cells / well in 96-well plates and allowed to adhere overnight. The culture medium was then replaced with fresh medium containing different concentrations of the drug. After 48 hours of culture, the supernatant was discarded, and 100 μL of fresh medium containing 10% CCK-8 was added to each well. The plates were incubated at 37°C for 1 hour. The absorbance was measured at 450 nm using a microplate reader (reference wavelength 630 nm).

[0059] 1.8 Analysis of in vivo therapeutic effects

[0060] All animal experiments were approved by the Experimental Animal Ethics Committee of Hainan Medical University, document number [number missing].

[0061] HYLL-2023-182 approved.

[0062] A549 cells (1×10^6 cells / 100μL PBS) were subcutaneously inoculated into the right flank of nude mice. Tumors were monitored daily, and the volume was calculated using the following formula:

[0063] Volume = (major axis × minor axis 2) / 2.

[0064] When the average tumor volume is approximately 100 mm 3 Mice were randomly divided into 4 groups (n=5). Drugs were administered via tail vein every 3 days for a total of 21 days. Tumor volume and body weight were measured every 3 days. At the experimental endpoint, mice were sacrificed, tumors were harvested, weighed, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for H&E staining and immunohistochemical analysis.

[0065] Stain for 30 min. Discard the staining solution and fix with 4% paraformaldehyde at room temperature for 15 min. Observe and photograph using a laser confocal microscope (Zeiss LSM880).

[0066] Example 1

[0067] Construction and characterization of Fe@Arc

[0068] We constructed Fe@Arc nanozymes using arctigenin as a raw material via a one-step coordination-coprecipitation method. TEM analysis showed that the nanozymes exhibited uniform morphology and a particle size of approximately 82.3 ± 5.8 nm. Figure 2 a) No obvious aggregation. The XRD pattern shows characteristic Fe3O4 peaks at 2θ = 35° and 62°, confirming the crystal structure. Figure 2 b). FT-IR at 1630cm -1 A Fe-O stretching vibration peak appears at 1510 cm⁻¹, while the benzene ring skeletal vibration of arctigenin is preserved. -1 This indicates that the drug has been successfully coordinated. Figure 2 c).

[0069] Example 2

[0070] Drug encapsulation and release

[0071] Using a dual coordination-adsorption strategy, the encapsulation efficiency of arctigenin reached 87.4 ± 2.6%. In vitro release experiments (PBS, pH 7.4) showed that Fe@Arc was cumulatively released by about 65% within 48 hours, exhibiting pH-responsive accelerated release (85% release in 48 hours at pH 5.5), which is beneficial for triggering drug release in the tumor microenvironment.

[0072] Example 3

[0073] In vitro antitumor effects

[0074] To verify the cellular uptake capacity of Fe@Arc, we labeled the nanozyme with the fluorescent probe Cy5 and performed confocal imaging. Figure 4 As shown in ab, a significant red fluorescence signal is visible within A549 cells, indicating...

[0075] Fe@Arc can be efficiently taken up. Further investigation into its lysosomal escape efficiency revealed almost no colocalization between the red fluorescence of Fe@Arc and the green fluorescence of lysosomes (LysoTracker labeling). Figure 4 c) This suggests that the nanozyme successfully escaped from the lysosome, which is beneficial for efficient intracellular delivery.

[0076] Subsequently, the toxicity of different concentrations of Fe@Arc to A549 cells was evaluated using a CCK-8 assay. The results showed that cell viability decreased significantly with increasing concentration. Figure 4 d), and the half-maximal inhibitory concentration (IC50) of Fe@Arc. 50 The levels of Annexin V were significantly lower than those of free arctiin, indicating that the nanozyme significantly enhanced the drug's efficacy. Furthermore, Annexin V-FITC / PI live / dead staining further visually confirmed that, compared to the same dose of free drug, the Fe@Arc treatment group showed a large amount of Annexin V. + / PI + Double positive cells ( Figure 4The results suggest that Fe@Arc has a stronger ability to induce apoptosis / ferroptosis. These results collectively indicate that Fe@Arc achieves potent killing of lung cancer cells through efficient lysosomal escape and synergistic effects with the ROS-GSH-GPX4 axis.

[0077] Example 4

[0078] Inducing ferroptosis

[0079] Ferroprelation is a programmed cell death process characterized by the accumulation of iron-dependent lipid peroxides, ultimately leading to membrane rupture. It differs from apoptosis, necrosis, and autophagy, and is regulated by multiple metabolic and oxidative stress pathways, making it highly promising in lung cancer treatment. For example... Figure 5 As shown, the Fe@Arc group exhibited significantly decreased intracellular GSH levels, significantly increased lipid ROS (C11-BODIPY 581 / 591 probe), downregulated GPX4 protein expression, and marked morphological changes typical of ferroptosis, such as mitochondrial shrinkage and cristae reduction; while ferroptosis inhibitors...

[0080] Ferrostatin-1 can reverse the above changes, confirming that Fe@Arc exerts its anti-tumor effect through the ferroptosis pathway.

[0081] Example 5

[0082] In vivo anticancer therapy and microenvironment regulation

[0083] Encouraged by the excellent in vitro therapeutic effects of Fe@Arc, we further evaluated its in vivo anticancer potential in an A549 tumor-bearing nude mouse model. Mice were randomly divided into four groups and injected via tail vein: PBS, free arctigenin, blank Fe NP, and Fe@Arc (all at 10 mg / kg arctigenin). -1 (Dosage), administration time points are on days 0, 2, and 4, for a total of 3 times.

[0084] like Figure 6 As shown in c–f, the tumor in the PBS control group grew rapidly, exceeding 1200 mm in volume within 21 days. 3 The free arctiin group showed slight inhibition, but the tumors continued to grow; the blank FeNP group showed limited inhibitory effect. In contrast, tumor growth in the Fe@Arc group was almost completely inhibited, with a mean volume of only 18.7% of that in the PBS group on day 21 (p<0.001). Post-tumor weighing results (…). Figure 6 (b) Consistent with the volume trend: the Fe@Arc group had the lowest average tumor weight (0.18±0.04g), significantly lower than the PBS group (1.34±0.21g, p<0.001). Furthermore, no significant decrease in body weight was observed in any group during treatment, and no significant pathological damage was found in the major organs via H&E staining, suggesting that Fe@Arc has good biocompatibility.

[0085] In summary, Fe@Arc nanozymes exhibit remarkable tumor-suppressing effects in vivo, providing strong evidence for the treatment of ferroptosis in lung cancer.

[0086] In summary, this invention constructs a composite nanozyme, Fe@Arc, with arctigenin, a traditional Chinese medicine active ingredient, as its core, for targeted ferroptosis therapy in lung cancer. This nanoplatform precisely induces ferroptosis in tumor cells through a triple mechanism of "glutathione depletion-GPX4 downregulation-lipid ROS burst." In vitro and in vivo experiments demonstrate that Fe@Arc possesses high loading efficiency, excellent biocompatibility, and significant tumor-suppressing effects, providing a feasible paradigm for the clinical translation of ferroptosis-targeted nanomedicines. This work not only expands the application boundaries of traditional Chinese medicine extracts in precision cancer treatment but also lays a theoretical and experimental foundation for future cancer nanomedicine design targeting "ferroptosis-metabolic reprogramming."

[0087] It is worth noting that the above description of the embodiments focuses on illustrating the technical solution of the present invention, rather than precisely defining its scope of protection. Those skilled in the art should understand that appropriate adjustments and optimizations can be made based on the technical details disclosed in the embodiments of the present invention, or equivalent substitutions can be implemented for individual or even all technical elements. Such adjustments and substitutions will not deviate from the core essence of the technical solution of the present invention and should be included within the technical protection scope of the embodiments of the present invention. In short, the protection of the present invention should not be limited to the concrete presentation of the above embodiments, but broadly covers all equivalent changes and improvements that do not depart from its basic concept. In summary, the protection definition of the present invention should be based on the statement of the claims, and the above embodiments are only used as a reference guide for understanding the present invention.

Claims

1. A burdock-derived nanozyme targeting ferroptosis in lung cancer, characterized in that, The composite nanozyme is formed by the coordination of arctigenin and iron ions, and is named Fe@Arc.

2. The nanozyme according to claim 1, characterized in that, The iron ions are Fe. 3+ Arctium brevicornuate and Fe 3+ The molar ratio is 1:1 to 1:

3.

3. The nanozyme according to claim 1, characterized in that, Its particle size is 50–150 nm, preferably 80–90 nm.

4. The method for preparing nanozymes according to any one of claims 1-3, characterized in that, Includes the following steps: a) Arctium brevicornuate and Fe 3+ Soluble in a mixture of ethanol and water; b) Adjust the pH to 7.5–8.5 and react for 1–3 hours; c) Centrifuge, wash, and freeze-dry to obtain Fe@Arc nanozyme.

5. The method according to claim 4, characterized in that, The volume ratio of ethanol to water is 1:1, and the pH is adjusted using NaOH solution.

6. A pharmaceutical composition, characterized in that, It comprises the nanozyme as described in any one of claims 1–3 and a pharmaceutically acceptable carrier.

7. The use of the nanozyme according to any one of claims 1–3 or the pharmaceutical composition according to claim 6 in the preparation of a medicament for the prevention and / or treatment of lung cancer.

8. The application according to claim 7, characterized in that, The lung cancer in question is non-small cell lung cancer.

9. The application according to claim 7, characterized in that, The drug is an intravenously administered preparation.

10. The use of the nanozyme according to any one of claims 1–3 or the pharmaceutical composition according to claim 6 in the preparation of a medicament for inducing ferroptosis in tumor cells.

Images