Polyvinyl alcohol 1788 (PVA; Alcoholysis degree: 87.0–89.0%), iron (III) chloride anhydrous (FeCl₃; 99.9%) and pyrrole (99%) were purchased from Aladdin chemistry Co., Ltd. (Shanghai, China). Absolute ethyl alcohol (C₂H₅OH; AR) and ammonium hydroxide solution (NH₃·H₂O; 28.0–30.0%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Deionized water (H₂O) was prepared by a Milli-Q water purification system (Millipore, Bedford, MA, United States). All the chemical reagents were used without further purification.

We synthesized the PPy@Fe₃O₄ NPs by a very facile electrostatic adsorption method. First, PVA (0.75 g) was added in the deionized water (10 ml) and heated to 95°C until the solution was entirely dissolved. Later, FeCl₃ (0.373g, 2.30 mmol) was mixed homogeneously with the above mixture under strong magnetic stirring for 1 h. Next, pyrrole monomer (69.2μl, 0.9970 mmol) was slowly dropped, and the reaction was maintained for 4 h at 4°C under magnetic stirring. The final solution turned dark green, indicating that PPy NPs were successfully synthesized. Subsequently, the solution (2.5 ml) was taken out, mixed with deionized water (15 ml) and ethanol (2 ml) under stirring at 70°C. Afterwards, 1 ml 1.0 wt% aqueous ammonia liquid was dropped into the solution followed by stirring for 30 min. Then 1 ml of 1.0 wt% aqueous ammonia liquid was added dropwise again, and the mixture lastly maintained for 30 min at 70°C. Finally, after being washed three times by deionized water, the PPy@Fe₃O₄ NPs were collected by centrifugation (11000rpm) for 50 min.

The morphology and size of PPy@Fe₃O₄ NPs were determined using a JEM-200 transmission electron microscopy (TEM, JEOL, Tokyo, Japan) at 200kV acceleration voltage. The characteristic functional group and crystal structures of nanomaterials were measured using Fourier-Transformed Infrared (FTIR) spectrometer and X-ray powder diffraction (XRD) respectively.

Briefly, aqueous solutions of PPy@Fe₃O₄ NPs with different concentrations (100, 200 and 400 μg/ml) were separately irradiated by an 808 nm near-infrared (NIR) laser (1.0W/cm²). The temperature profiles were monitored and recorded using a thermal imaging camera (Fotric, Shanghai, China) over time. Then the PPy@Fe₃O₄ NPs (400 μg/ml) solutions were irradiated with the NIR laser for 10 min, and cooled down to room temperature. Last, we calculated the photothermal conversion efficiency (η) by the temperature curves according to published study. (Leng et al., 2018)

We evaluated the cytotoxicity of the PPy@Fe₃O₄ NPs by cell counting kit-8 (CCK-8) assays. In detail, normal human bronchial epithelial cells (BEAS-2B) and Human lung adenocarcinoma cells (A549) were respectively seeded into 96-well plates at a density of 10⁴ cells per well and cultured in pH7.4. The following day, we replaced the medium with fresh medium (pH7.4) containing the PPy@Fe₃O₄ NPs of different concentrations (25, 50, 100, 200, 400 μg/ml) for another 24 h. Then we changed the medium with 100 μl serum-free medium and 10 μl CCK-8 reagent. Following incubation for 2 h, we measured the absorbance value of wells at 450 nm using a microplate reader (Thermo, United States). The calculated formulas were listed in Supplementary Material.

The A549 cells were incubated in the culture plates and divided into four groups: 1) control; 2) PPy@Fe₃O₄ (400 μg/ml); 3) H₂O₂ (100 μM); 4) PPy@Fe₃O₄ (400 μg/ml) + H₂O₂ (100 μM). After 24 h of treatment, we used the probe solution 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime Biotechnology, Shanghai, China) to estimate the intracellular reactive oxygen species (ROS). After DCFH-DA treatment for 20 min, the cells were washed three times by phosphate buffered saline (PBS) buffer and blank medium was added. Finally, we acquired images using Confocal laser scanning Microscopy (CLSM, Leica Microsystems, Mannheim, Germany).

The anti-cancer effect of PPy@Fe₃O₄ NPs was evaluated by the Calcine-AM/propidium iodide (PI) test (Beyotime Biotechnology, Shanghai, China) and the Annexin V-FITC/PI apoptosis kit (Multi Sciences, Hangzhou, China). To investigate the PTT effect of the PPy@Fe₃O₄ NPs, A549 cells were incubated in the culture plates at pH6.5 and divided into 6 groups; group 1 with cells only; group 2 cultured with NIR only; group 3 cultured with H₂O₂ (100 μM) only; group 4 cultured with PPy@Fe₃O₄ NPs (400 μg/ml) + NIR; group 5 cultured with PPy@Fe₃O₄ NPs (400 μg/ml) + H₂O₂ (100 μM); group6 cultured with PPy@Fe₃O₄ NPs (400 μg/ml) + H₂O₂ (100 μM) + NIR. After incubation with PPy@Fe₃O₄ NPs (400 μg/ml) for 12 h, the cells of group 2,4 and 6 were exposed to an 808 nm NIR laser (1.0 W/cm²) for 10 min. Afterwards, we stained the A549 cells with Calcein-AM and PI for about 15 min, and images were acquired by CLSM.

And we detected cell apoptosis by Annexin V-FITC Apoptosis kit. The A549 cells in different groups were harvested and washed with PBS and Binding Buffer. Then the cells were resuspended in 500 μl Bind Buffer with 5 μl Annexin V-FITC and 10 μl PI solution to mix evenly for 5 min at room temperature in darkness. And we measured cell apoptosis immediately by flow cytometry (cytoflex LX, Beckman Coulter).

In vitro migration assay, 24-well Transwell chambers with a polycarbonate filter membrane of 8 μm pore size (Corning, United States) were used to assess the effect of the PPy@Fe₃O₄ NPs on A549 cell migration. Cells were divided into control group and PPy@Fe₃O₄ group. After starvation of A549 cells for 24 h, we resuspended cells in serum-free RPMI 1640 medium and serumfree RPMI 1640 medium containing the PPy@Fe₃O₄ NPs at a concentration of 400 μg/ml respectively. Next, the A549 cells were inoculated into the upper chamber and the RPMI 1640 medium was placed in the lower chamber. After culturing for 24 h, the A549 cells that migrated to the bottom of the membranes were immobilized and stained with 1% crystal violet (Beyotime Biotechnology, Shanghai, China). Finally, the migrated cells of each groups were counted under a microscope (Leica, Germany).

A549 cells (5*10⁵) were seeded into 6-well plate and divided into the control group and the PPy@Fe₃O₄ group (400 μg/ml). After incubation for 24 h, the cells were washed by PBS and lysed in RIPA buffer. Then we removed the cell debris by centrifugation, and the supernatants were harvested and stored at −80°C. Equivalent amounts of protein (25 µg) were separated on 10% SDS-PAGE and transferred onto 0.22 um polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and subsequently incubated with primary antibodies including anti-MMP2/MMP9/MMP13 (Abclonal, China, 1:1,000 dilution) and anti-β-actin (Proteintech, China, 1:10,000 dilution). The following day, we washed the membranes three times for 10 min each time by Tris-buffered saline with Tween20 (TBST), and then incubated them in the corresponding secondary antibody (goat anti-rabbit IgG horseradish peroxidase (HRP), goat anti-mouse IgG-HRP, 1:1,000, Affinity Biosciences, China) at room temperature for 2 h. Following washing three times for 10 min with TBST buffer, the membranes were treated with enhanced chemiluminescence (ECL, EpiZyme, Shanghai, China) reagent for exposure.

For in vivo MRI measurements, we intratumorally injected the A549 tumor-bearing mice with 200 μl of 3 mg/ml PPy@Fe₃O₄ NPs, when tumor size reached visible size (7–9 mm in diameter). Then we scanned the mice before and 3 h after injection. Thus, we successfully acquired the high-resolution T2-weighted MRI scan images of mice by a 3.0T MRI system (Ingenia 3.0T CX, Philips Healthcare). The T2-weighted MRI parameters were as follows: pulse waiting time (TR) = 2,800 ms, echo time (TE) = 60 ms, slice width (SW) = 5.0 mm.

When the tumors reached 7–9 mm in diameter, we divided the mice into four groups (n = 6 per group): 1) control; 2) PBS + NIR; 3) PPy@Fe₃O₄ NPs (3 mg/ml); 4) PPy@Fe₃O₄ NPs (3 mg/ml) + NIR. The mice of group3 and 4 were injected intravenously with 200ul PPy@Fe₃O₄ NPs (3 mg/ml) and the mice of group2 was injected intravenously with 200ul PBS. Then 8 h after injection, the mice of group2 and 4 were irradiated with 808 nm laser (1W/cm²) for 10 min. Meanwhile, we measured the temperature changes of the tumor by a FLIR A300 thermal imaging camera. Simultaneously, tumor volume and body weight were monitored every 2 days. On day 14, the mice were sacrificed. Then the major organs were isolated for photograph or histological analysis (hematoxylin and eosin (H and E) staining), and the tumor was stained subsequently by immunohistochemistry (Ki67) and immunofluorescence (TUNEL).

The tumor tissues specimens of mice were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Then the tissue blocks were cut into sections of 3-5 um thickness. For Ki67 staining, the sections were immunostained overnight at 4°C with an anti-Ki67 antibody (Abcam). After washing in PBS, the sections were subsequently incubated with the second antibody for 1 h. Then the slides were stained with 3, 3-diaminobenzidine (DAB) and hematoxylin separately, dehydrated, and mounted. Images were captured using a microscope (Leica, Leica DMi8). For TUNEL staining, the proportion of apoptotic cells were performed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining kit (Servicebio, Wuhan, China). We obtained all images using a CLSM.

All results and measurements were shown as the mean ± SD (standard deviation). Comparison between the mean values of different groups were analyzed by one way analysis of variance (ANOVA) or Student’s t-test: (*) p < 0.05 was considered statistically significant; (**) p < 0.01 and (***) p < 0.001 was considered highly statistically significant; (****) p < 0.0001 was considered extremely statistically significant.

We used TEM to determine the sizes and morphologies of PPy@Fe₃O₄ NPs. The PPy@Fe₃O₄ NPs are shown in Figure 1A, Figure 1B and Supplementary Figure S1. As we can see, the mean diameter of PPy@Fe₃O₄ NPs was measured to be 71.0 nm, and many tiny Fe₃O₄ nano-crystals were surface adsorbed on PPy NPs. The FTIR spectrum showed clearly the characteristic absorption peaks of PPy, indicating the successful formation of PPy (Figure 1C). And XRD patterns showed that the PPy@Fe₃O₄ NPs had characteristic peaks (Figure 1D), which could be indexed to the Fe₃O₄ nanocrystals (JCPDS No. 65–3,107). (Wang et al., 2020) The above results demonstrated that the PPy@Fe₃O₄ NPs had been successfully prepared.

As shown in Figure 2A, the aqueous dispersion of PPy@Fe₃O₄ NPs had a broad absorption throughout the visible to the NIR region, which demonstrated that PPy@Fe₃O₄ NPs were good potential PTT agents. We further evaluate the photothermal properties of PPy@Fe₃O₄ NPs with NIR light irradiation. With the prolonging of the irradiation time and increased concentration of PPy@Fe₃O₄ NPs, the temperature rose rapidly (Figure 2B). Finally, when the concentration of PPy@Fe₃O₄ NPs reached 400 μg/ml, the temperature reached about 70°C after 5 min of irradiation, which was high enough for irreversible tumor ablation (Figure 2C). Next, we tested the photothermal conversion efficiency (ŋ) of PPy@Fe₃O₄ NPs, which can show the capability of converting energy of light into heat. As shown in Figure 2D, the ŋ value of PPy@Fe₃O₄ NPs was figured out to be ∼51.8%, which is much higher than traditional PTT agents, such as Cu_2-xSe nanocrystals, Cu₉S₅ nanocrystals and Au nanorods. (Hessel et al., 2011; Tian et al., 2011)

To assess the cytotoxic effects of PPy@Fe₃O₄ NPs, the CCK8 assay was performed on BEAS-2B and A549 cells. The cells were incubated with PPy@Fe₃O₄ NPs (0, 25, 50, 100, 200, 400 μg/ml) for 24 h to test the cell viability. The results indicated that PPy@Fe₃O₄ NPs exhibited lower cytotoxicity towards normal and cancer cells as high as 400 μg/ml (Figure 3), demonstrating that they have good biocompatibility.

Chemodynamic therapy is an effective therapeutic treatment that causes damage of tumor cells by producing ROS. To examine the effects of PPy@Fe₃O₄ NPs on ROS production, we detected ROS by fluorescent probe 2′, 7′-dichlorofluorescein-diacetate (DCFH-DA), which was oxidized to 2, 7′-dichlorofluorescein (DCF) in the presence of ROS. As displayed in Figure 4A, compared with control group, the intensity of DCF fluorescence in H₂O₂ group and PPy@Fe₃O₄ NPs group increased weakly. When treated with both H₂O₂ and PPy@Fe₃O₄ NPs, the cells displayed strong and extensive green fluorescence, indicating that PPy@Fe₃O₄ NPs could increase Fenton-induced ROS generation in the presence of H₂O₂.

Given the desirable photothermal conversion performance and significant synergistic effects, we further determined the in vitro therapeutic efficacy of PPy@Fe₃O₄ NPs through Calcine-AM/PI test. Living cells were stained with green fluorescent calcein AM, while dead cells were stained with red fluorescent PI (Figure 4B). Compared with the control group, A549 cells exhibited strong green fluorescence and no red fluorescence under exposure of NIR laser radiation, which indicated that the viability of the cells was not compromised. In contrast, H₂O₂ slightly increased the percentage of dead cells. And A549 cells showed a remarkable cell death after treatment by PPy@Fe₃O₄ NPs with NIR irradiation or PPy@Fe₃O₄ NPs with H₂O₂, suggesting that the photothermal therapy and chemodynamic therapy of the PPy@Fe₃O₄ NPs could effectively killed the A549 cells. When A549 cells were treated by the combination of PTT and CDT, very few living cells were observed. To sum up, PPy@Fe₃O₄NPs have satisfactory synergistic therapeutic effects.

We measured the influence of PPy@Fe₃O₄ NPs on cell apoptosis by using Annexin V/PI staining and flow cytometry (Figure 4C, Supplementary Figure S2). The early apoptosis cells were defined as Annexin V (+)/PI (−), and cells in the late stage of were considered as Annexin V (+)/PI (+). The flow cytometry results showed that NIR group had no evident difference compared to the control group. Nevertheless, after treatment with H₂O₂, the percentage of apoptosis cells increased to 8.9%. After treatment by PPy@Fe₃O₄ NPs with NIR irradiation and PPy@Fe₃O₄ NPs with H₂O₂, the percentage of apoptosis cells significantly increased to 42.75% and 27.35%. And most surprisingly, the apoptosis rate in the PPy@Fe₃O₄ NPs + H₂O₂ + NIR group increased highly to 76.99%, which further demonstrated that the PPy@Fe₃O₄ NPs + H₂O₂ + NIR group had the highest killing effect due to synergistic PTT and CDT.

To assess cell mobility in response to the PPy@Fe₃O₄ NPs, we used transwell cell cultures in vitro, which certified the effect of PPy@Fe₃O₄ NPs on suppressing the migration of lung cancer cells (Figures 5A,B).

MMP2/9/13 had been found to play vital functions in the invasion and metastasis of malignant tumors. Therefore, we examined the impact of PPy@Fe₃O₄ NPs on these protein expressions in A549 cells using western blotting (Figure 5C). The outcomes showed that the expressions of MMP2, MMP9 and MMP13 were obviously declined in the PPy@Fe₃O₄ NPs group compared to the control group. In summary, these experiments suggested that PPy@Fe₃O₄ NPs could suppress the growth and metastasis of A549 cells.

To identify the therapeutic efficacy of PPy@Fe₃O₄ NPs on tumor growth in vivo, we conducted further comparative studies. 1 × 10⁶ A549 cells were subcutaneously injected into BALB/c nude mice. When the volumes of tumors reached to approximately 100 mm³, we divided the mice into four groups randomly: 1) control; 2) PBS + NIR; 3) PPy@Fe₃O₄ NPs; 4) PPy@Fe₃O₄ NPs + NIR. Mice of the group 2 were injected with 200ul PBS via the tail vein, and mice of group 3, 4 were injected with PPy@Fe₃O₄ NPs (3 mg/ml) via the tail vein. After 8 h, the mice of group 2, 4 were irradiated by an 808 nm NIR laser (1.0W/cm²) for 10 min. Meanwhile, by using an infrared thermal camera, we found that the tumor temperature of mice injected with PPy@Fe₃O₄ NPs rapidly increased to ∼53°C during irradiation, which was sufficient for ablating tumors. However, the tumor temperature of the group 2 had negligible rise (Figures 6A,B). After 14 days, the mice were sacrificed and the tumor volumes and body weights were recorded (Figures 6C–E). The growth of tumors in group 2 was similar to the control group, demonstrating that NIR alone could not inhibit tumor growth. Whereas, tumor growth in the group 3 was partially inhibited due to CDT of PPy@Fe₃O₄ NPs. Noticeably, the tumor growth in group 4 was substantially inhibited because of the synergistic effects of PTT and CDT. Representative pictures of the tumors in different groups further determined the therapeutic efficacy. There was no obvious difference in body weight among the groups, suggesting that the adverse effect of PPy@Fe₃O₄ NPs was negligible. Additionally, the main tissues and organs of different groups were collected for H&E staining to estimate the safety of various treatments (Figure 6F). We could not find evident damage in the group 2, 3, 4, compared to the control group, illustrating that the PPy@Fe₃O₄ NPs were biologically safe in vivo.

Next, we evaluated the proliferation and apoptosis of tumor cells by immunocytochemistry and immunofluorescence (Figures 6G,H). We noticed that PPy@Fe₃O₄ NPs could slightly reduce the expression of the proliferation marker Ki67 and increase apoptosis, compared with the control group. Moreover, treatment of PPy@Fe₃O₄ NPs with NIR further significantly decreased the level of Ki67 and caused cell apoptosis more effectively. These results clearly showed that PPy@Fe₃O₄ NPs with NIR could suppress effectively tumor growth in vivo.

MRI offers excellent spatial resolution and tissue penetration, which is a noninvasive method for the identification and early cancer diagnosis. We used PPy@Fe₃O₄ NPs as contrast agents to evaluate the contrast-enhancing effect in vivo (Figure 7A). We treated the tumor-bearing mice with the PPy@Fe₃O₄ NPs by intratumor injection, and utilized T2 weighted MRI to observe the tumor before PPy@Fe₃O₄ NPs injection and 2 h post-injection (Figures 7B,C). Compared with pre-injection, we revealed that the signal intensity of tumor sites changed obviously 2 h after injection, which indicated that the PPy@Fe₃O₄ NPs might be ideal contrast agents for MRI in future applications.