Dopamine hydrochloride (PDA) and Manganese (II) sulfate monohydrate (MnSO₄·H₂O) were sourced from Aladdin Reagent (Shanghai, China). Tris-(hydroxymethyl)-aminomethane (Tris-HCL) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (Missouri, United States). The CCK-8 reagent was sourced from Dojindo Laboratories (Japan), and Calcein AM/PI Cell Staining Kit was supplied by Soleibio (Shanghai, China). All reagents were analytically pure and used without further modification. Distilled water was used throughout the experiments.

Dopamine hydrochloride (90 mg) and manganese (II) sulfate monohydrate (3 mg) were suspended in deionized water and stirred continuously for 24 h. Subsequently, this pre-formed chelate solution (0.6 mL) was introduced into a water/ethanol mixture (6 mL each) under ultrasonication. The pH of the mixture was adjusted to 8.5–9 using Tris buffer and mixed continuously for 6 h. The resulting Mn-PDA NPs were recovered by centrifugation and washed sequentially with ethanol and water twice.

Size distribution and zeta potential were determined by Malvern laser diffraction analysis. Transmission electron microscopy (TEM, JEM-2100) revealed morphology. Chemical bond changes were analyzed using Fourier-transform infrared spectroscopy (FT-IR, Thermo Fisher Scientific). Manganese oxidation states were determined using X-ray photoelectron spectroscopy (XPS) and electron paramagnetic resonance (EPR). The manganese concentration was measured using inductively coupled plasma atomic emission spectrometry (ICP-AES, Agilent 5100). To monitor the release of manganese ions from Mn-PDA NPs, they were placed in pH 7.4 and pH 6.0 PBS buffer solutions with GSH at 10 mM respectively, and stirred in a 37°C water bath. Samples were taken at fixed time points (0, 2, 4, 6, 8, 10, 12, 24, 48, 72 h) and the manganese ion content was analyzed using ICP-AES.

To evaluate photothermal performance, the suspensions of deionized water (H₂O), DOPA NPs, and Mn-PDA NPs were exposed to an 808 nm laser (1 W/cm², 5 min) while monitoring temperature at 30-s intervals. Separately, we assessed the dose-dependent response by irradiating Mn-PDA NPs at concentrations of 0, 50, 100, and 200 μg/mL under identical laser conditions for 10 min. Thermal stability was evaluated by subjecting Mn-PDA NPs to four consecutive laser irradiation cycles (808 nm, 1 W/cm²), recording temperature throughout.

For phantom studies, solutions of Mn-PDA NPs were prepared at varying Mn(II) concentrations (0, 0.01, 0.03, 0.06, 0.13, 0.25 mM). Imaging was performed on a 3.0T MRI scanner (GE Signa Architect) using T1-weighted (T1WI), T2-weighted (T2WI), and MAGiC sequences, quantitative T1map and T2map were derived from the MAGiC data (Zhao et al., 2025). Specific scanning parameters are listed in Table 1. Regions of interest (ROIs) were consistently positioned across samples at the same level to measure longitudinal and transverse relaxation times (T1 and T2). Then longitudinal and transverse relaxivity (r₁ and r₂) were determined from linear regressions correlating nanoparticle concentration with reciprocal relaxation times.

Statistical analyses were conducted using Origin software (version 2021) and GraphPad Prism (version 10.1.1). Independent two-tailed t-tests were used to analyze the differences between two groups, while one-way analysis of variance (ANOVA) was employed to evaluate the differences among multiple groups. Data were collected in triplicate and presented as mean ± standard deviation (mean ± SD). Differences were established as statistically significant at the following thresholds of significance: p < 0.05 (∗), p < 0.01 (∗∗), p < 0.001 (∗∗∗), and p < 0.0001 (∗∗∗∗).

Mn-PDA NPs were synthesized based on previously reported methods (Zhang et al., 2023), and the average diameter and zeta potential values of the NPs were obtained by the Dynamic Light Scattering (DLS). As shown in Figure 1A, TEM demonstrates that Mn²⁺ ions initially chelate with PDA to form particulate complexes, exhibiting a uniform size distribution centered at ∼2 nm. Subsequently, under alkaline conditions, these complexes undergo spontaneous oxidative polymerization to form homogeneous nanospheres with an average diameter of 210.3 ± 18.4 nm (PDI = 0.18; Figures 1B,C). The zeta potential of DOPA was −33.9 ± 1.1 mV. After chelating manganese ions, the zeta potential of Mn-PDA NPs increased to −18.7 ± 1.2 mV (Figure 1D),We speculate in a DOPA-rich system, carboxyl groups dominate the surface charge. Despite Mn ²⁺ coordination and protonated amines, the high density of -COO- ensures an overall negative potential. The FT-IR spectra (Figure 1E) provide critical evidence for Mn²⁺ chelation: (i) Broadening and reduction in intensity of the N-H (∼3,350 cm⁻¹) and C=C peaks (blue-shifted to 1,630 cm⁻¹); (ii) Disappearance of SO₄ ²⁻ signatures (1,110 cm⁻¹); (iii) Emergence of new Mn-O bonds (560 cm⁻¹), confirming successful coordination between Mn²⁺ and catechol groups. Constituent elements of Mn-PDA NPs was examined by XPS revealed peaks at 285, 400, 531, and 637 eV, corresponding to C1s, N1s, O1s, and Mn2p3s, respectively (Figure 1F). As shown in Supplementary Figure S1C,D, the EPR spectrum of Mn-PDA NPs exhibits a characteristic six-line hyperfine splitting pattern (centered at g = 2.0090), indicative of isolated Mn²⁺ (3d⁵, S = 5/2) in a high-symmetry ligand field. In contrast, the EPR spectrum of crystalline MnSO₄·H₂O (Supplementary Figure S1A,B) displays only broad peaks due to pronounced ZFS anisotropy induced by low-symmetry distortion, which obscures the hyperfine splitting. Furthermore, the molar concentration of Mn(II) in Mn PDA NPs was determined via ICP-AES as 5.8%, confirming the successful preparation of Mn-PDA NPs.As shown in Supplementary Figure S2, manganese ion (Mn²⁺) release was minimal in the pH 7.4 environment, remaining below approximately 0.5 μg/mL. In contrast, under conditions of pH 6.0 and 10 mM GSH, the Mn²⁺ release increased significantly by 6 h and stabilized at levels between 2 and 4 μg/mL, even after 72 h, the cumulative release accounted for only about 5% of the total potential. The results demonstrated that the release of Mn²⁺ from Mn-PDA NPs was minimal under the tested conditions, indicating the excellent stability of the nanoparticles in vitro.

PDA can chelate various metal ions, such as Mn²⁺ and Fe³⁺, and absorb and convert near-infrared (NIR) light into heat energy. Previous studies have demonstrated that Mn²⁺ coordination in PDA significantly enhances photothermal conversion efficiency (η > 45%), enabling effective tumor ablation at reduced laser doses (Wu et al., 2021). As shown in Figure 1G, a series of concentrations of the nanoparticles in an aqueous solution were irradiated with an 808 nm laser (1 W/cm², 5 min), with ultrapure water used as a control. Minimal temperature change was observed in water under laser irradiation. In contrast, the temperature change in Mn-PDA NPs aqueous solutions occurred in a concentration-dependent manner, with the maximum temperature reaching 48.5°C (8.0°C higher than that in DOPA NPs at 100 μg/mL). Furthermore, as the Mn-PDA NPs concentration increased, the temperature rose gradually (Figure 1H).

Manganese-based nanoparticles, leveraging their inherent paramagnetism, have emerged as promising T1-MRI contrast agents, offering an alternative to Gd-DTPA. Recent studies highlight the synergistic effects between Mn²⁺ and the PDA matrix in enhancing proton spin relaxation (Ma et al., 2024). To evaluate Mn-PDA NPs for MRI-guided PTT in tumor theranostics, their MR performance was assessed using a 3.0 T MR scanner. As shown in Figure 2A, Mn-PDA NPs exhibited a concentration-dependent brightening effect on T1-MR images and a darker effect on T2-MR images. T1map revealed a longitudinal relaxation rate (r1) of 9.05 mM⁻¹s⁻¹ (Figure 2B), nearly double that of Gd-DTPA (r1 = 3.5–5.6 mM⁻¹s⁻¹ at 3.0 T) (Wahsner et al., 2019), and a transverse relaxation rate (r2) of 94.62 mM⁻¹s⁻¹ (Figure 2C), slightly lower than SPIO NPs (r2 = 118.2 mM⁻¹s⁻¹) (Choi et al., 2012), yet still meeting clinical standards.

Biocompatibility is crucial in therapeutic applications, so the cytotoxicity of Mn-PDA NPs was evaluated in 2 cell lines (MeT-5A and MSTO-211H) using the CCK-8 assay. Even at a concentration as high as 400 μg/mL, the viability of both cell lines exceeded 80.0%, demonstrating favorable biocompatibility (Figure 2E). Furthermore, the in vitro therapeutic effects of DOPA NPs and Mn-PDA NPs were evaluated by labeling MSTO-211H cells with Calcein-AM/PI and CCK-8 assay. In Figure 2D, the majority of MSTO-211H cells exhibited high viability in the absence of NIR irradiation, as indicated by intense green fluorescence. However, under 808-nm NIR irradiation (1 W/cm², 5 min), MSTO-211H cells treated with Mn-PDA NPs showed significant damage, with cell viability dropping to 19.7% ± 1.1% (from 95.5% ± 2.4% in non-irradiated controls) and a marked increase in red fluorescence. In contrast, cells treated with DOPA NPs exhibited only minimal signs of apoptosis, with viability moderately reduced to 52.5% ± 1.2% (from 97.2% ± 3.7% of controls) (Figure 2F).In conclusion,the Mn-PDA-mediated phototherapy could result in significant combined antitumor efficacy of Mn-PDA NPs against MPM.

Given the advantageous characteristics of Mn-PDA NPs, we conducting in vivo MRI studies in MPM tumor-bearing BALB/c nude mice. Following intravenous injection, 3.0 T MRI scans revealed time-dependent signal changes at the tumor site over 24 h. T2-weighted signal slightly decreased, while T1-weighted signal progressively intensified, peaking at 8 h post-injection (Figure 3A). The corresponding longitudinal relaxation time (T1) decreased significantly from 1,293 ± 62.6 ms (pre-injection) to 737 ± 82.6 ms at 8 h post-injection. This peak enhancement, likely influenced by Mn-PDA NP biodegradation, represented a 42.9% ± 4.1% reduction in T1, corresponding to an increase in T1 -weighted signal intensity. Although the signal intensity declined thereafter, the T 1 value remained elevated at 24 h (1,058 ± 59.6 ms), which was 18.2% ± 1.3% lower than the pre-injection T1 values. This confirmed efficient tumor accumulation and sustained MRI contrast enhancement by the Mn-PDA NPs (Figure 3B).

The Mn²⁺ concentration in the tumor was quantified by ICP-AES analysis at 8 h post-injection. The control group administered with saline had a concentration of 0.31 ± 0.03 mg/kg, whereas the Mn-PDA NPs group showed a significantly higher level of 1.12 ± 0.07 mg/kg (Figure 3C). Mn-PDA NPs are shown to be an effective and reliable tumor MR imaging agent with enhanced imaging capabilities. Compared to the control group, Mn²⁺ accumulation was observed in other organs, including the heart (1.07 ± 0.09 mg/kg), liver (1.27 ± 0.11 mg/kg), spleen (0.64 ± 0.05 mg/kg), lung (1.08 ± 0.1 mg/kg), and kidney (0.59 ± 0.03 mg/kg). This distribution is consistent with the expected biological pattern for non-targeted nanoparticles eliminated via phagocytosis.

The in vivo anti-tumor efficacy of Mn PDA NPs combined with PTT in the MSTO-211H mice modelwas evaluated. According to the MR imaging results, laser irradiation was performed 8 h after tail vein injection. As shown in Figure 4C, after 5 min of 808 nm laser exposure, the saline group showed only a slightly temperature increase (26.4 °C–32.4°C; ΔT ≈6.0 °C), the DOPA NPs group reached 42.5 °C which potentially insufficient for complete ablation due to tumor cell thermotolerance mediated by heat shock proteins (Hsps), while the Mn-PDA NPs group experienced a significant rise and rapidly reached 48.9°C (ΔT ≈22.4°C), confirming potent photothermal conversion and self-targeting capability. In addition, MR imaging provided critical information for treatment optimization by identifying tumor location, nanoparticle distribution, and monitoring peritumoral reactions such as exudation and edema (Jiang et al., 2022). As shown in Figures 4A–C, the MnPDA NPs + L group demonstrated the best therapeutic effect. Specifically, Figure 4B revealed significant tumor growth inhibition, with tumors appearing almost completely resolved while preserving the surrounding normal tissues. However, MRI images (Figure 4A) still revealed trace amounts of residual tumor tissue within the deep subcutaneous layers and the chest wall musculature. This finding underscores the distinct advantage of magnetic resonance imaging in detecting and monitoring residual disease to accurately assess the effectiveness of tumor treatment. Histological analysis (H&E and Ki-67 staining) of excised tumors further confirmed the therapeutic effects. Tumor cells in the saline and saline + L groups maintained close packing and normal morphology, whereas clear apoptotic features, including pyknotic nuclei and tissue vacuolation, were observed in both the DOPA NPs + L and Mn-PDA NPs + L groups. Moreover, Ki-67 staining corroborated these findings, showing markedly reduced expression levels and proliferation in Mn-PDA NPs + L groups compared to controls (Figure 4D).

Therapeutic efficacy of Mn-PDA NPs was evaluated in the MPM tumor model. Mice treated with Mn-PDA NPs + L, DOPA NPs + L, or saline (controls)+ L showed contrasting outcomes: treatment groups exhibited steady weight gain alongside tumor regression, whereas controls experienced weight loss concomitant with uncontrolled tumor growth. Critically, Mn-PDA NPs + L demonstrated the most potent tumor inhibition, recapitulating in vitro findings and confirming superior efficacy of photothermal therapy (Figures 4E,F).

Given the therapeutic efficacy of Mn-PDA NPs + L, its potential organ toxicity was assessed. H&E staining of major organs (heart, liver, spleen, lung, kidney) revealed no pathological abnormalities in any group (Figure 5A). As shown in Figure 5B, the hematological indices, including liver-associated enzymes (AST, ALT), renal function indicators (CRE, BUN), and cardiac biomarkers (CK-MB, LDH), all stayed within normal reference ranges across all experimental groups, suggesting minimal toxicity effects in the tumor-bearing mice. Collectively, Mn-PDA NPs demonstrated good biocompatibility and potent tumor suppression. However, their clinical translation is delayed due to potential toxicity risks, especially the accumulation of free Mn²+ in dopaminergic neurons, which may lead to neurotoxicity. Although some clinical trials (Zhang et al., 2023) and approved agents, like Mn-DPDP (Hamm, et al., 1992), suggest short-term safety ofmanganese-based nanocomposites, there is a lack of long-term toxicological data and animal models that closely mimic humans. Further research is needed to confirm their safety and effectiveness for clinical use.