Iron (III) acetylacetonate, dibenzyl ether, oleylamine, anhydrous ethanol, and the WL-12 peptide were obtained from Shanghai Carboxphyll Bio-Tech Co., Ltd. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were supplied by Aladdin Bio-Tech Co., Ltd. All other chemicals were received in good amounts and were of reagent quality.

In the synthesis process, iron (III) acetylacetonate (2 g), dibenzyl ether (20 mL), and oleylamine (20 mL) were mixed in a dry three-neck round-bottom flask. While stirring the mixture, nitrogen gas was slowly introduced to create an inert atmosphere. The mixture was then placed on a heated magnetic stirrer and stirred to ensure homogeneity. The temperature was gradually increased to 100°C and maintained for 1 h to ensure complete dissolution of the components.

Next, the temperature was gradually increased to 295°C at a rate of 5°C every 10 min. Once the temperature reached 295°C, the reaction was allowed to proceed for 30 min to ensure completion. After the reaction was complete, the heat was turned off, and the mixture was allowed to cool naturally to room temperature.

The Fe₃O₄ nanoparticles were subsequently separated via an external magnetic field, and the product was subsequently washed several times with anhydrous ethanol until the supernatant became clear. The washed product was then placed in a vacuum drying oven and dried at 60°C for 12 h, yielding the final SPION.

First, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were dissolved in phosphate-buffered saline (PBS) and mixed at a 1:1 M ratio. The mixture was then subjected to continuous stirring at room temperature for 1 h to facilitate the reaction. Next, an equimolar amount of the Fe₃O₄ nanoparticle dispersion was added to the reaction mixture, and the resulting solution was incubated overnight at 4°C.

After the reaction, the mixture was centrifuged at high speed to remove the supernatant, and the precipitate was washed three times with distilled water, with centrifugation performed after each wash. The supernatant was then discarded, and the precipitate was resuspended in PBS. Subsequently, WL-12 was dissolved in PBS at a molar ratio of 2:1 (WL-12:Fe₃O₄ nanoparticles) and reacted with the Fe₃O₄ nanoparticle suspension at 4°C overnight.

Finally, the covalently bound product was subjected to centrifugation and filtration, followed by three additional washes with distilled water to remove any residual reactants. The resulting precipitate was freeze-dried to obtain WL-12@Fe₃O₄.

The morphology and distribution of the nanoparticles were examined by scanning electron microscopy (SEM). Fe₃O₄ and WL-12@Fe₃O₄ nanoparticles (1 mg each) were separately dissolved in 2 mL of deionized water, followed by ultrasonic treatment at 40% power for three cycles, each lasting 5 min. A 50 µL aliquot of the resulting suspension was then diluted with 2 mL of deionized water for further analysis. Dynamic light scattering (DLS) was employed to measure the particle size distribution and zeta potential of the nanoparticles.

The WL-12@Fe₃O₄ nanoparticles were diluted with saline and 100% FBS solutions to different concentrations (0, 20, 40, 60, 80, and 160 μg/mL) and placed into EP tubes with a total volume of 1 mL per tube. The Zeta potential of the WL-12@Fe₃O₄ nanoparticles was measured at 0, 12, and 24 h at room temperature to assess stability.

The cell lines used in this study included the human epithelial-type MPM cell line H226, the human biphasic MPM cell line MSTO-211H, and the human lung cancer cell lines A549 and H460, all of which were obtained from the Cell Bank of the Typical Culture Preservation Committee of the Chinese Academy of Sciences. All the cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 10,000 U/mL penicillin, and 10 mg/mL streptomycin under standard conditions (37°C and 5% CO₂).

Female BALB/c nude mice (four to six weeks old, SCXK (Dian) K2020-004) were purchased from the Laboratory Animal Department of Kunming Medical University. The mice were housed under specific pathogen-free (SPF) conditions with ad libitum access to sterile food and water and maintained on a 12-h light/dark cycle, with the temperature and humidity controlled at 24°C–26°C and 30%–50%, respectively. The animal experiments in this study were approved by the Animal Experiment Ethics Review Committee of Kunming Medical University (Approval No. KMMU20220931).

H226 and MSTO-211H cells in the logarithmic growth phase were harvested via trypsinization with 0.25% trypsin. After centrifugation, the cells were resuspended in saline, and the cell suspension concentration was adjusted to 12 × 10⁶ cells/50 µL. The cell suspension was mixed with Matrigel (50 µL) and subcutaneously injected into the right axilla of the nude mice. The body weights and tumor volumes of the tumor-bearing mice were recorded daily, and tumor growth was monitored for an additional 4 weeks once the tumors reached a maximum diameter of 1 cm.

H226 and MSTO-211H cells were seeded into 24-well plates at a density of 7 × 10⁴ cells per well and incubated at 37°C with 5% CO₂ for 24 h. After the cells were washed three times with PBS, 30 µL of WL-12@Fe₃O₄ nanoparticles or Fe₃O₄ nanoparticles (at a concentration of 1 mg/mL) were added to each well, followed by incubation for an additional hour. The deposition of iron particles in both cell types was then observed via Prussian blue staining.

H460 cells (high PD-L1 expression) and A549 cells (low PD-L1 expression) were also seeded into 24-well plates at a density of 7 × 10⁴ cells per well and incubated at 37°C with 5% CO₂ for 24 h. After three PBS washes, 100 µL of WL-12@Fe₃O₄ nanoparticles or Fe₃O₄ nanoparticles (at a concentration of 1 mg/mL) were added to each well, with the control group receiving regular culture medium. The cells were incubated for an additional 6 h. The concentration of iron in the dialysate was measured via inductively coupled plasma‒mass spectrometry (ICP-MS).

For the in vivo targeting study, tumor-bearing mice were injected via the tail vein with 100 µL of WL-12@Fe₃O₄ nanoparticles or Fe₃O₄ nanoparticles (1 mg/mL, dissolved in saline). After 1 h, the mice were sacrificed by cervical dislocation, and major organs, including the heart, kidneys, liver, lungs, spleen, and tumor tissues, were harvested. The iron content in the tissue lysates was measured via ICP‒MS.

After anaesthesia, the tumor-bearing mice were positioned on a 3.0T GE MRI scanner for T1-weighted imaging (T1WI), T2-weighted imaging (T2WI), and T2 mapping. The specific scanning parameters are provided in Table 1. Following the initial scans, 100 µL of WL-12@Fe₃O₄ nanoparticles or Fe₃O₄ nanoparticles (1 mg/mL concentration) were injected intravenously via the tail vein. The injection concentration was determined on the basis of previous studies (Huang et al., 2024) and related literature review (Shen and Yu, 2023). T2 mapping scans were repeated at 0.5, 1, 1.5, and 2 h postinjection to evaluate the MR imaging effects of nontargeted (Fe₃O₄) and targeted nanoparticles (WL-12@Fe₃O₄) in H226 and MSTO-211H tumor-bearing mice. The mice were divided into nontargeted and targeted nanoprobe groups, with 5 mice per group and a total of 20 mice. For four consecutive weeks, the ability of targeted MRI to detect PD-L1 expression levels in the two types of tumors was assessed, with 20 mice per group and a total of 40 mice.

The raw data from the tumor-bearing mice were processed and analysed via a GE postprocessing workstation (Advantage Windows 4.6, GE Medical Systems, United States). T1WI and T2WI images were used to observe the location, morphology, and size of the tumors. Regions of interest (ROIs) were delineated on the largest tumor slice and two adjacent slices, covering more than 90% of the total tumor area while avoiding necrotic regions. The T2 relaxation time (T2 value) of the tumor tissues was measured via T2 maps. A double-blind method was employed for data measurement on both plain and contrast-enhanced images, with two attending physicians independently conducting measurements. Each physician measured the images three times, and the average value was taken. The T2 difference (ΔT2 value) between the plain and enhanced scans was calculated as follows: ΔT2 = T2 value (unenhanced) - T2 value (enhanced).

Following MRI scanning, the tumor-bearing mice were euthanized via cervical dislocation, and the tumor tissues were harvested. The tissues were then fixed in 10% neutral formalin solution. Immunohistochemical staining was performed to assess PD-L1 expression levels in the tumor tissues. An Aipathwell AI-based digital pathology image analysis system (provided by Servicebio, China) was used to quantify the target areas under high magnification. The system automatically calculates the proportion of positive cells (percentage of positive cells = number of positive cells/total number of cells × 100%). The average PD-L1 expression levels from the largest tumor section and its two adjacent sections were selected as the study’s metric.

Statistical analysis and graphical plotting were performed via SPSS software version 26.00 and R language (version v4.2.1). The data are presented as the means ± standard deviations (SDs). For normally distributed data, an independent sample t-test was used to compare two groups, whereas one-way analysis of variance (ANOVA) was applied for comparisons among multiple groups. For nonnormally distributed data, the Mann‒Whitney U test was used. A P-value of <0.05 was considered to indicate statistical significance.

Scanning electron microscopy (SEM) images revealed that the WL-12@Fe₃O₄ nanoparticles exhibited a uniform spherical shape and homogeneous distribution (Figure 1A). Compared with that of Fe₃O₄, the electron-withdrawing ability of the WL-12@Fe₃O₄ nanoparticles decreased, which led to a reduction in the surface potential (−9.34 ± 0.54 mV vs. −20.20 ± 0.88 mV, P < 0.05) (Figure 1B). Both WL-12@Fe₃O₄ and Fe₃O₄ nanoparticles displayed unimodal size distributions. The average particle size of the WL-12@Fe₃O₄ nanoparticles (43.82 nm) was larger than that of the Fe₃O₄ nanoparticles (32.67 nm) (Figure 1C).

After the WL-12@Fe₃O₄ nanoparticles were diluted in physiological saline and 100% FBS, no significant aggregation or precipitation was observed at 0 h, 12 h, or 24 h at room temperature in either medium (Figure 1D). And, the Zeta potential of WL-12@Fe₃O₄ nanoparticles in different solutions did not change significantly (P > 0.05) (Figure 1E).

The XPS spectrum reveals the presence of Fe 2p, O 1s, C 1s, and N 1s peaks within the nanoparticles. Notably, the Fe 2p peak predominantly appears around 711.5 eV. When combined with XANES data, this indicates that iron primarily exists in the +3 oxidation state. The FT-IR spectrum demonstrates the occurrence of NH stretching vibration peaks at 3,453.13 cm⁻¹ and C=O stretching vibrations at 1,566.86 cm⁻¹ (Figure 2).

The cytotoxic effects of WL-12@Fe₃O₄ nanoparticles on H226 and MSTO-211H cells were evaluated via a CCK-8 assay. Both cell lines were cocultured with various concentrations of nanoparticles (0, 25, 50, 100, or 200 μg/mL) for 24 h, and the cell viability in both cases remained above 90% (Figure 3).

Hematoxylin‒eosin (HE) staining revealed no signs of necrosis, fibrosis, or other pathological changes in the major organs of the nude mice, including the brain, heart, lungs, liver, spleen, and kidneys (Figure 4).

When the Fe₃O₄ nanoparticles were incubated with the tumor cells, no visible blue particle deposition was observed on the tumor cells. In contrast, after incubation with WL-12@Fe₃O₄ nanoparticles, blue particle deposition was observed on the tumor cells, particularly in MSTO-211H cells (Figure 5A).

The concentration of iron ions in PD-L1-high H460 cells (930.22 ± 11.75 ng/mL) was significantly greater than that in PD-L1-low A549 cells (254.89 ± 17.33 ng/mL), and the difference was statistically significant (P < 0.05) (Figure 5B).

The iron concentration in tumor tissues injected with WL-12@Fe₃O₄ nanoparticles (141.02 ± 3.82 μg/g) was significantly greater than that in those injected with Fe₃O₄ nanoparticles (36.43 ± 3.56 μg/g, P < 0.05). However, no statistically significant differences in iron ion concentrations were detected between the two groups in other organs (P > 0.05) (Figure 5C).

After injection of the nanoparticles, the T2 values of H226 and MSTO-211H tumor tissues gradually decreased, reaching their lowest point at 1.5 h and 1 h, respectively, after which the T2 values began to increase. The T2 value changes in H226 tumors followed a “slow decrease-slow increase” pattern, whereas MSTO-211H tumors exhibited a “rapid decrease-slow increase” pattern. The ΔT2 values in the targeted group were significantly greater than those in the nontargeted group for both H226 and MSTO-211H tumors (12.38 ± 1.05 m, 16.26 ± 2.02 m vs. 8.04 ± 0.93 m, 11.38 ± 1.16 m; P< 0.05). Additionally, the ΔT2 values in MSTO-211H tumors were significantly greater than those in H226 tumors (P < 0.05) (Figure 6).

The targeted nanoparticles were injected via the tail vein, and MR scans (including T2WI and T2 mapping) were performed on H226 and MSTO-211H tumor-bearing mice prior to injection and at 1.5 h and 1 h post-injection, respectively. The results revealed that, as tumor growth progressed over time (1, 2, 3, and 4 weeks), both the ΔT2 value and PD-L1 expression of the tumor tissue increased gradually. Moreover, a strong positive correlation between ΔT2 value and PD-L1 expression was observed (R = 0.97, P < 0.05) (Figures 7, 8).