HP-β-CD (MW 1396), TQ (MW 164), ferroptosis inhibitor (Ferrostatin-1) and NF-κB activator (PMA) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Cell Counting Kit-8 (CCK-8) reagent (cat. no. AB0101), BeyoClick™ EdU-594 Kit (cat. no. C0071S), Hochest 33258 staining Kit (cat. no. C1011), DCFH-DA (cat. no. S0036S), dimethyl sulfoxide (DMSO) (cat. no. ST038), MDA Assay Kit (cat. no. S0131S), hematoxylin-eosin (H&E) staining Kit (cat. no. C02-04004), normal goat serum blocking solution (cat. no. P0260), HRP-labeled goat anti-rabbit secondary antibody (cat. no. A0181) and reduced glutathione (GSH) Assay Kit (cat. no. S0053) were obtained from Beyotime Institute of Biotechnology. C11-BODIPY 581/591 kit (cat. no. D3861) was obtained from Invitrogen; Thermo Fisher Scientific, Inc.. Anti-p-NF-κB (cat. no. ab207297) and anti-Ki-67 (cat. no. ab15580) were purchased from Abcam.

TQ/HP-β-CD was prepared by the freeze-drying method following the protocol previously reported (22,23). TQ/HP-β-CD inclusion complex was characterized by Fourier transform infrared (FT-IR) spectrophotometry (Avatar 370, Thermo-Nicolet; Thermo Fisher Scientific, Inc.), Powder X-ray diffraction (XRD) (Shimadzu Corporation), differential scanning calorimetry (DSC) (3500 Sirius, NETZSCH Group), and scanning electron microscopy (SEM) (Nova Nano SEM 230; Thermo Fisher Scientific, Inc.) as previously reported (22–24).

The phase solubility study was conducted according to the methods described in previous literature (22). Briefly, excess TQ or TQ/HP-β-CD inclusion complex is added to distilled water with a pH of 7.0 and stirred continuously at 500 × g for 24 h at 37°C. After filtering the suspension, the phase solubility is detected using an ultraviolet (UV)-visible spectrometer. The apparent stability constant (K) and complexation efficiency (CE) are calculated from the solubility diagram using the following equations: K=Slope/S₀. (1-Slope) and CE=Slope/(1-Slope). S₀ is the solubility of TQ in the absence of HP-β-CD, and Slope is the corresponding slope of the phase solubility diagram.

A total of 0.1 g of TQ and 0.94 g of TQ/HP-β-CD inclusion complex (containing 0.1 g of TQ) were added each to 50 ml of PBS (pH=7.4) and then stirred continuously at a speed of 100 × g for 24 h at 37°C. At the predetermined time, 1 ml of the supernatant solution was removed and replaced with an equal volume of fresh medium. The drug release rate was determined by a UV-visible spectrometer and calculated using the following equation: Drug release rate (%)=D₁/D₂ ×100%. D₁ is the amount of released drug within a certain period and D₂ is the total amount of drug.

Heparin sodium was added to the collected fresh blood samples to prevent coagulation. The venous blood was divided into three layers after centrifuging at 2,000 × g for 5 min. The red blood cells were separated from plasma and the remaining red blood cells were washed three times with normal saline to remove residual white blood cells. After that, the red blood cells were diluted with normal saline to prepare a 4% red blood cell solution.

A total of 300 µl of TQ/HP-β-CD solution with different concentrations (50, 100, 200, 400, 800, 1,500, 3,000 µmol/l), 1.2 ml of erythrocyte solution, and 1.2 ml of saline were added to 5-ml centrifuge tubes. A total of 1.5 ml of 4% red blood cell solution and 1.5 ml of deionized water were added to the test tube of the positive control group. In total, 1.5 ml of 4% red blood cell solution and 1.5 ml of normal saline were added to the test tube of the negative control group. The test tube was incubated in a 37°C constant-temperature incubator for 1 and 24 h, respectively. The tubes were centrifuged at a speed of 2,000 × g for 5 min to precipitate red blood cells. The absorbance value of the supernatant at 450 nm was detected with a microplate reader.

The hemolysis rate of the positive control group was set as 100%, and the hemolysis rate of the TQ/HP-β-CD group was calculated according to the following equation: Hemolysis rate (%)=(A₁/A₀) ×100%. A₁ is the absorbance value of the TQ/HP-β-CD group, and A₀ is the absorbance value of the positive control group.

Two immortalized NSCLC cell lines (A549 and HCC827) were obtained from the American Type Culture Collection and were incubated as previously described (11).

A549 cells were plated at a density of 5×10⁴ cells per well onto transparent microporous membrane inserts (Thincerts™; 1 µm pore size, 1.13 cm² surface area) within 12-well culture plates. Growth medium was introduced into the basolateral (1.0 ml) and apical (0.5 ml) compartments and refreshed at two-day intervals throughout a 21-day differentiation phase. Cultures were incubated at 37°C with 90% humidity. Monolayers achieved experimental readiness when transepithelial electrical resistance stabilized within the 200–300 Ω·cm² range.

Both TQ solution and the TQ/HP-β-CD inclusion complex were diluted in Hanks' solution to yield equivalent final TQ concentrations of 25 µmol/l. These test formulations were apically administered to the differentiated monolayers. Permeability assessments commenced by adding 0.5 ml of test solution to the apical chamber and 1.0 ml of Hanks' solution to the basolateral chamber. Aliquots were periodically collected from the basolateral compartment for quantitative analysis via high performance liquid chromatography.

The apparent permeability coefficient (Papp, cm/s) was derived using the equation: Papp=dQ/(dt × A × C₀). dQ/dt is the steady-state permeation rate (mass/time), C₀ is the initial apical drug concentration, and A is the monolayer surface area (cm²).

The increasing concentrations of TQ, HP-β-CD, and TQ/HP-β-CD in the treatment group were 12.5, 25, 50, 100, 200, 300, and 400 µmol/l. TQ was dissolved in DMSO to prepare a 50 mmol/l mother solution.

Different concentrations of TQ samples were obtained by diluting the mother liquor with a DMEM medium and labeling it as the TQ/DMSO group. TQ was dissolved in a DMEM medium to obtain TQ samples of different concentrations labeled as TQ/H₂O group. HP-β-CD was dissolved in a DMEM medium to obtain different concentrations of HP-β-CD samples, which were labeled as the HP-β-CD group. TQ/HP-β-CD inclusion complex was dissolved in DMEM medium to obtain TQ/HP-β-CD samples of different concentrations, labeled as TQ/HP-β-CD group.

A total of 1×10⁴ A549 or HCC827 cells were cultivated in 96-well plates with different concentrations of TQ, HP-β-CD, and TQ/HP-β-CD as aforementioned for 24 h. A total of 10 µl of CCK-8 reagent was added to each well of a 96-well plate, followed by incubation at 37°C for 1 h, and the absorbance value was detected at 570 nm with a microplate reader. The value of IC₅₀ was analyzed using GraphPad software 8.0 software (Dotmatics). The growth inhibitory rate was calculated according to the following equation: Growth inhibitory rate (%)=(1-A₁/A₀) ×100%. A₁ is the absorbance value of the treatment group, and A₀ is the absorbance value of the negative control group.

A549 or HCC827 cells at logarithmic growth phase were inoculated into a 96-well plate and treated with different drugs for 24 h. EdU working solution was added and cells remained for 2 h in a 37°C incubator. Pre-cooled 4% paraformaldehyde was added, and cells were incubated at room temperature for 30 min. A total of 0.5% Triton X-100 was used to permeate the membrane for 20 min and react with Hoechst 33342 reaction solution in the dark for 30 min. Finally, the image was analyzed using ImageJ software (version 1.51j8; National Institutes of Health).

The proliferation and migration activities were measured in A549 or HCC827 cells by the collagen sprout outgrowth assay as previously described (25).

A549 or HCC827 cells at logarithmic growth phase were inoculated into a 6-well plate, treated with different drugs for 24 h at 37°C, and then washed three times with PBS. Then, ROS was measured using fluorescence microscopy and flow cytometry, respectively. DCFH-DA was used to detect the ROS levels according to the manufacturer's protocol. In brief, A549 or HCC827 cells at logarithmic growth phase were seeded in 6-well plates and cultured with different drugs for 24 h at 37°C. After treatment, cells were harvested and washed three times with PBS and labeled with 20 µmol/l DCFH-DA under 37°C for 30 min in the dark. The cells were then washed with PBS, followed by fluorescence microscopy imaging (Zeiss LSM800; Zeiss GmbH). ROS levels were detected using BD FACS-Calibur flow cytometer (BD Biosciences), and the fluorescence intensity of ROS was analyzed with FlowJo software (FlowJo LLC).

The level of lipid peroxidation in 1.0×10⁵ A549 or HCC827 cells was measured using the C11-BODIPY 581/591 kit according to the manufacturer's protocol.

After 2.0×10⁵ A549 or HCC827 cells were treated with different drugs for 24 h, the content of MDA and GSH/GSSG ratio were determined according to the manufacturer's protocol.

Nuclear extracts were extracted from A549 cells and incubated with IRDye-700-labeled NF-κb oligonucleotides for electrophoretic mobility shift assay, as previously described (26).

A total of 15 female BALB/c nude mice (6 weeks old, ~22 g) were purchased from Beijing Weitong Lihua Company. Animals were housed under specific pathogen-free conditions at a controlled temperature of 22±2°C, 50±10% relative humidity, and a 12/12-h light-dark cycle with ad libitum access to autoclaved water and standard rodent chow. Environmental enrichment (for example, nesting material and shelters) was provided. All animal experiments followed the ARRIVE guidelines and were approved (approval no. 20240215001) by the Animal Care and Ethics Committee of Ma'anshan People's Hospital (Ma'anshan, China) conducted in strict accordance with the UK Animals (Scientific Procedures) Act 1986 and relevant institutional guidelines.

Each mouse was subcutaneously injected with 5×10⁶ A549 cells under brief isoflurane anesthesia (4% induction, 2% maintenance in oxygen). Tumor size was measured daily using digital calipers with volume calculated as (Length × Width²)/2. Animal health and behavior (posture, mobility, grooming, spontaneous activity, and signs of pain/distress) were assessed twice daily throughout the study. Pre-defined humane endpoints requiring immediate euthanasia included: Tumor volume >1,500 mm³, tumor ulceration or necrosis >15% surface area, body weight loss >20% from baseline, inability to access food/water, or severe lethargy/distress.

When tumors reached ~100 mm³, mice were randomly assigned to treatment groups (n=5/group): untreated control, TQ group, or TQ/HP-β-CD group. The experimental duration post-grouping was 28 days. The TQ group received intraperitoneal injections of 100 µl solution containing 3 mg TQ every other day. The TQ/HP-β-CD group received intraperitoneal injections of 100 µl solution containing 28.2 mg TQ/HP-β-CD (equivalent to 3 mg TQ) every other day. The control group received no treatment. All mice survived to the scheduled endpoint. On day 28, mice were euthanized by cervical dislocation under deep isoflurane anesthesia (5% induction in oxygen). Death was confirmed by absence of detectable heartbeat (cardiac palpation), absence of respiratory movements for >5 min, and fixed dilated pupils. Liver, kidney and tumor tissues were immediately collected post-mortem for analysis. Welfare considerations included: i) Anesthesia for tumor implantation and euthanasia (isoflurane: 4% induction/2% maintenance for implantation; 5% for euthanasia) ii) strict humane endpoints iii) twice-daily health monitoring iv) environmental enrichment and v) minimized injection volumes. No analgesics were administered during treatment as injections caused only transient discomfort without observable distress. No animals required early euthanasia or were found dead during the study.

The liver, kidney, and tumor tissues were placed in 4% paraformaldehyde for 48 h. Then, the tissues were embedded in paraffin and cut into thin slices (4 µm). Routine H&E and Perl staining were then performed on the slices. IHC for Ki-67 and NF-κb as also performed on paraffin-embedded tissue sections. Paraffin-embedded tissue sections were baked at 65°C, dewaxed, hydrated, and subjected to antigen retrieval using citrate sodium buffer. Endogenous peroxidase activity was blocked with 3% H₂O₂. Sections were blocked with normal goat serum blocking solution at room temperature for 30 min. Primary antibodies were then applied: Ki-67 (1:200) or NF-κB p65 (1:100), and incubated at 4°C overnight. After washing with PBS, HRP-labeled goat anti-rabbit secondary antibody (1:200) was added and incubated at room temperature for 1 h. DAB was used for color development, followed by counterstaining with hematoxylin. Finally, the sections were dehydrated, cleared, and mounted. Slices were observed and images were captured using an Olympus BX41 optical microscope (Olympus Corporation).

Three independent experiments were conducted, with data presented as the mean ± SD of absolute values or control percentages. Statistical analysis was conducted using GraphPad Prism 8.0 software (Dotmatics). The differences between two or more groups were confirmed through one-way ANOVA followed by Tukey's post hoc test. P-<0.05 (on both sides) was considered to indicate a statistically significant difference.

Based on previous studies (22,23), the freeze-drying method was employed in the present study to prepare TQ/HP-β-CD inclusion complexes in a 1:1 molar ratio and characterize and biologically validate them.

In previous studies, FTIR was used to investigate the interaction between guest molecules and CDs in inclusion complexes (22–24). The FTIR spectra of TQ, HP-β-CD and TQ/HP-β-CD are demonstrated in Fig. 2A. HP-β-CD spectrum has unique absorption signals at ~3,318 cm⁻¹ and 1,020 cm⁻¹, representing OH group and C-O-C glucose unit bending, respectively. Pure TQ exhibits an intense sharp band at 1,647 cm⁻¹, corresponding to the stretching of carbonyl groups. CH₂ produces stretching vibration displayed at 2,970 cm⁻¹. The spectral band of 1,450-1,020 cm⁻¹ represents C=C bending vibration (aliphatic) and C-O-C stretching vibration. After the formation of the TQ/HP-β-CD inclusion complex, it can be observed that the carbonyl group of TQ located at 1,520 cm⁻¹ disappears from the spectrum of the inclusion complex due to stretching. Meanwhile, the characteristic broad band of the OH groups in HP-β-CD shifted from 3,318 cm⁻¹ to 3,401 cm⁻¹. The signal changes related to the polar functional groups of TQ and HP-β-CD can indicate the partial embedding of TQ into the HP-β-CD cavity, thereby confirming the successful formation of the inclusion complex (Fig. 1).

As revealed in Fig. 2B, XRD was used further to investigate the inclusion complex of TQ and HP-β-CD. No diffraction peaks in the X-ray spectrum of HP-β-CD indicate that it is amorphous. The XRD pattern of TQ showed a series of strong and sharp diffraction peaks, indicating that the compound is essentially crystalline. However, the inclusion complex of TQ with HP-β-CD exhibited a well-differentiated diffusion X-ray pattern similar to HP-β-CD, indicating that TQ no longer exists in crystalline form and its inclusion complex with HP-β-CD exists in an amorphous state. The transformation into an amorphous form is important evidence for TQ to be embedded in the HP-β-CD cavity in a molecular state (20,21).

The DSC curves of TQ, HP-β-CD and TQ/HP-β-CD inclusion complex are shown in Fig. 2C. The DSC spectrum of HP-β-CD showed two peaks at 72 and 158°C, corresponding to the glass transition (Tg) and melting point (Tm) temperatures, respectively. TQ has a sharp endothermic peak at 48.44°C, which is the Tm of the crystal. The DSC spectrum of the TQ/HP-β-CD inclusion complex is highly similar to the HP-β-CD, but the difference is that the TQ/HP-β-CD inclusion complex has lower Tg and Tm. In addition, the endothermic peak of TQ disappeared in the DSC curve of the inclusion complex, which is attributed to the transition of crystalline TQ to an amorphous state. The aforementioned results indicated that the TQ/HP-β-CD inclusion complex exhibits reduced thermal stability and crystallization trend, consistent with the XRD results.

Under a scanning electron microscope (Fig. 2D), HP-β-CD exhibits an amorphous spherical structure with cavities. TQ presents an irregularly sized rectangular crystal structure, and inclusion complexes exhibited irregularly shaped aggregates or clumps. The change in the morphology of the inclusion complex is mainly attributed to the embedding of partial TQ molecules into the cavity of HP-β-CD, which confirms the formation of the inclusion complex morphologically, similar to previous literature (21).

The phase solubility diagram of TQ encapsulated into HP-β-CD was depicted in Fig. 3A, indicating that the water solubility of TQ increases proportionally with the increase of HP-β-CD molar concentration. The correlation coefficient (R²) of the linear regression equation is 0.98, showing that the system follows an A_L-type solubility plot. The apparent stability constant (K_1:1) of the inclusion complex is related to its stability. The K_1:1 value increases proportionally to the stability of the inclusion complex (24). The K_1:1 value of the TQ/HP-β-CD inclusion complex is 562.34 l/mol, demonstrating the preparation of a stable inclusion complex between HP-β-CD and TQ. The detection of solubility at room temperature (26°C) demonstrated that the solubility of TQ and TQ/HP-β-CD inclusion complex in aqueous solution were 0.024 and 37.42 mmol/l, respectively. The solubility of TQ/HP-β-CD was 1,559-fold that of TQ. The CE displays the ratio of dissolved complex concentration to dissolved free CD concentration and was employed to evaluate the solubility of hydrophobic molecules in CDs (27). According to the phase solubility diagram, the CE value of the inclusion complex is 1.942, indicating HP-β-CD is suitable for preparing TQ.

To investigate whether the surface modifier HP-β-CD can enhance the hydrophilicity of TQ as well as improve the release characteristics of TQ. Then, the in vitro release experiment of TQ was performed. The in vitro drug release curves of free TQ and TQ/HP-β-CD inclusion complex in PBS (pH=7.4) at 37°C for ~24 h were displayed in Fig. 3B. Specifically, the cumulative amount of TQ released from the TQ/HP-β-CD inclusion complex increased rapidly in the first h of the experiment, gradually increasing and reaching a plateau. A total of >52.5% of TQ was released from the inclusion complex within the first h, while free TQ was only released at 5.5%. After 24 h, the drug release rate in inclusion complex and free TQ were 84.3 and 9.3%, respectively. The aforementioned data confirmed the ability of the new formula (TQ/HP-β-CD) to improve the hydrophilicity and release characteristics of TQ.

According to the national evaluation standard (GB/T16886.4-2003), the hemolysis rate of medical biomaterials used in clinical applications should be <5% to meet the safety requirements of biomaterials (28). As demonstrated in Fig. 4A, the upper liquid of the test tube in the negative control group is clear and transparent, and erythrocytes precipitate at the bottom, indicating that hemolysis has not occurred. After 1 h of reaction, the upper liquid of the test tube in the positive control group was uniformly red, and there was no red blood cell precipitation at the bottom, indicating that all red blood cells had undergone hemolysis. After treatment with different concentrations of TQ/HP-β-CD inclusion complex (50–3,000 µmol/l) for 1 h, the test tube solution remained relatively clear. When the processing time of the inclusion complex was extended to 24 h, the test tube liquid of the low concentration TQ/HP-β-CD inclusion complex (50–400 µmol/l) group remained clear, while the upper liquid of the test tube of the high concentration TQ/HP-β-CD inclusion complex (800–3,000 µmol/l) group appeared red, indicating that the low concentration TQ/HP-β-CD inclusion complex (50–400 µmol/l) used in the present study had favorable blood compatibility.

In addition, the hemolysis degree of each group was quantitatively measured using spectrophotometry (Fig. 4B). The results showed that after 1 h of treatment, the hemolysis rate of the TQ/HP-β-CD groups with different concentrations was <1%. When the action time of the TQ/HP-β-CD is extended to 24 h, the red blood cell hemolysis rate of the low concentration TQ/HP-β-CD groups (50–400 µmol/l) is <5%. When the concentration of the TQ/HP-β-CD exceeds 400 µmol/l, the red blood cell hemolysis rate exceeds the threshold of 5%, indicating that the low-concentration inclusion complex inflicts damage to red blood cells and meets the safety requirements of national standards. The concentration of TQ/HP-β-CD inclusion complex used in subsequent experiments is within the safe range.

To investigate the potential of HP-β-CD to enhance the permeability and subsequent cellular uptake of TQ in A549 cells, permeability studies were conducted comparing free TQ to the TQ/HP-β-CD inclusion complex. The Papp values determined were 1.04×10⁻⁷ cm/s for free TQ and 5.2×10⁻⁷ cm/s for the TQ/HP-β-CD complex. This 5-fold increase in Papp demonstrates that complexation with HP-β-CD significantly enhances the transepithelial transport and cellular uptake of TQ in the A549 monolayer model. These findings indicate that HP-β-CD serves as an effective carrier system, facilitating the delivery of a greater amount of TQ into tumor cells.

Previous studies have confirmed that TQ significantly inhibits the proliferation, migration and invasion of various human cancer cells, including lung cancer (11–13). Meanwhile, compared with free TQ, the nano-emulsion loaded with TQ has higher cellular uptake and more potent anticancer activity (27). The present study compared the inhibitory effects of TQ/H₂O, TQ/DMSO, HP-β-CD and TQ/HP-β-CD inclusion complex on the proliferation of NSCLC cell lines, specifically A549 and HCC827 cells. As shown in Fig. 5A, TQ/DMSO exerted a strong inhibitory effect on the proliferation of A549 and HCC827 cells, with IC₅₀ values of 26.1 and 28.2 µmol/l, respectively, which are similar to previous findings (13). TQ/H₂O revealed a weaker inhibitory effect on the proliferation of A549 and HCC827 cells, with IC₅₀ values of 290.6 and 252.2 µmol/l, respectively. The inhibition rate of HP-β-CD on the proliferation of NSCLC cells was <3%, indicating that HP-β-CD was almost non-toxic. Combining TQ with HP-β-CD to form an inclusion complex had a stronger inhibitory effect on the proliferation of NSCLC cells than TQ/H₂O, with IC₅₀ values reduced to 48.2 and 61.7 µmol/l, respectively. This enhanced inhibitory activity aligns with the significantly improved permeability and cellular uptake of TQ/HP-β-CD observed in the A549 monolayer model, where its apparent permeability coefficient (Papp) was 5-fold higher than that of free TQ.

The EdU assay results showed that TQ/H₂O could effectively inhibit the proliferation of A549 and HCC827 cells (Fig. 5B), and the inhibitory effect of TQ/HP-β-CD at the same concentration on NSCLC cell proliferation was further enhanced. In addition, the collagen sprout outgrowth assay is an effective method for evaluating cell proliferation and migration, with longer buds indicating more vigorous cell proliferation and migration abilities (29). As shown in Fig. 5C, TQ/H₂O significantly inhibited the sprout outgrowth of A549 and HCC827 cells after 24 h of treatment. Compared with TQ/H₂O, the sprout length of A549 and HCC827 cells treated with TQ/HP-β-CD was further shortened.

The aforementioned results indicate that HP-β-CD can enhance the inhibitory effect of TQ on the proliferation and migration of NSCLC cells. Considering the excellent biocompatibility of TQ/HP-β-CD in the experimental concentration range, TQ/HP-β-CD can potentially become an anti-tumor agent for treating NSCLC.

To confirm whether TQ/HP-β-CD can induce ferroptosis in NSCLC cells, DHCA staining was first used to detect the generation of ROS. The results showed that TQ/HP-β-CD treatment increased ROS generation in A549 and HCC827 cells (Fig. 6A). Flow cytometry further showed that TQ/HP-β-CD increases the expression of ROS in NSCLC cells (Fig. 6B).

In addition, the present study further detected the hallmark of ferroptosis - lipid peroxidation level-in NSCLC cells. The dye C11-BODIPY 581/591 was used, which is sensitive to lipid peroxidation, for laser confocal scanning microscopy observation. The intensity of green fluorescence (oxidized C11-BODIPY) under the microscope was used to evaluate the level of lipid peroxidation. The results revealed that TQ/HP-β-CD treatment increased lipid peroxidation levels in A549 and HCC827 cells (Fig. 6C). Meanwhile, the level of lipid peroxidation product MDA in A549 and HCC827 cells was increased after TQ/HP-β-CD treatment. The present study found that TQ/HP-β-CD can significantly downregulate the level of GSH and the ratio of GSH/GSSG in NSCLC cells (Fig. 6D). These data indicated that TQ/HP-β-CD can induce ferroptosis in A549 and HCC827 cells.

To further verify that ferroptosis is the main form of TQ/HP-β-CD induced NSCLC cell death, rescue experiments were conducted by Ferrostatin-1, a widely used ferroptosis inhibitor. It was found that Ferrostatin-1 can effectively alleviate the inhibitory effect of TQ/HP-β-CD on the proliferation of A549 and HCC827 cells (Fig. 7A). Meanwhile, pretreatment with Ferrostatin-1 can also reverse the accumulation of ROS mediated by TQ/HP-β-CD in the cytoplasm (Fig. 7B) and the increase in lipid peroxidation level (Fig. 7C). In addition, ELISA experiments identified that Ferrostatin-1 can effectively restore the elevated MDA level and decreased GSH level induced by TQ/HP-β-CD in A549 and HCC827 cells (Fig. 7D).

To elucidate the underlying mechanism by which TQ/HP-β-CD induces ferroptosis, NF-κB DNA binding activity was measured using EMSA (Fig. 8A). The results showed that treatment with TQ/HP-β-CD alone for 24 h effectively suppressed NF-κB DNA binding activity. To further investigate the dependence of TQ/HP-β-CD-induced ferroptosis on NF-κB, A549 cells were pretreated with the NF-κB activator PMA followed by TQ/HP-β-CD treatment. Notably, PMA pretreatment rescued the inhibitory effect of TQ/HP-β-CD on NF-κB activity.

Subsequently, the CCK-8 assay results indicated that PMA can partially reverse the inhibitory effect of TQ on A549 cell proliferation (Fig. 8C). Meanwhile, after TQ/HP-β-CD treatment, lipid peroxidation level and ROS generation increased in A549 cells. At the same time, PMA successfully weakened the effect of TQ/HP-β-CD on lipid peroxidation and ROS generation (Fig. 8B and D). In addition, PMA also alleviated the regulatory effect of TQ/HP-β-CD on the expression of MDA and GSH in A549 cells (Fig. 8E). The aforementioned results indicated that TQ/HP-β-CD may first inhibit the basal level of NF-κB DNA binding activity previously present in NSCLC cells, leading to the inactivation of downstream genes and ultimately inducing ferroptosis in NSCLC cells. However, the exact mechanism by which TQ/HP-β-CD regulates the NF-κB pathway in NSCLC cells remains unclear, and further research is needed to determine whether other molecular mechanisms are involved.

In the A549 ×enograft mouse model, both TQ and TQ/HP-β-CD significantly inhibited tumor growth compared with the control group. The tumor growth inhibition rates were 32.1% for TQ and 67.9% for TQ/HP-β-CD (Fig. 9A and B). H&E staining of tumor tissues revealed more extensive necrotic features (nuclear fragmentation, contraction and dissolution) in the TQ/HP-β-CD group compared with TQ and control groups (Fig. 9E). IIHC showed a significant reduction in Ki-67 positive cells (indicating proliferation) in TQ/HP-β-CD treated tumors compared with both control and TQ groups (Fig. 9F).

Furthermore, mice treated with TQ or TQ/HP-β-CD showed no significant adverse symptoms (vomiting, convulsions or bloating) and maintained stable body weights (Fig. 9C). H&E staining of liver and kidney tissues from all groups revealed normal architecture without significant pathological changes (Fig. 9E).

Analysis of subcutaneous tumors showed that both TQ and TQ/HP-β-CD treatment significantly decreased GSH levels and increased MDA levels compared with the control group, with TQ/HP-β-CD showing a stronger effect (Fig. 9D). Prussian blue staining revealed significantly increased free iron deposition in tumor tissues from both TQ and TQ/HP-β-CD treated mice compared with controls, again more pronounced in the complex group (Fig. 9F). IHC analysis showed significantly reduced positive expression of NF-κB in tumor tissues from TQ and TQ/HP-β-CD groups compared with control, with TQ/HP-β-CD exhibiting a stronger suppressive effect (Fig. 9F).

In summary, in vivo research results indicated that TQ/HP-β-CD has favorable biocompatibility and a stronger inhibitory effect on NSCLC growth than TQ, and NF-κB-mediated ferroptosis induction may be its primary mechanism of action, which is consistent with in vitro experimental results.