Synergistic nanotheranostic platform for fluorescence/magnetic resonance bimodal imaging-guided near infrared photoimmuno-chemodynamic thermal targeting therapy

Matsuoka, Kohei; Yamada, Mizuki; Okada, Ryu; Shimada, Mibuko; Yoshida, Mikina; Fukushima, Rika; Umeda, Kanna; Ohashi, Ayano; Oka, Chiemi; Kato, Ayako; Shimizu, Misae; Koike, Chiaki; Ito, Akira; Sato, Mitsuo; Sato, Kazuhide

doi:10.1186/s12951-026-05148-w

Synergistic nanotheranostic platform for fluorescence/magnetic resonance bimodal imaging-guided near infrared photoimmuno-chemodynamic thermal targeting therapy

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Published: 14 October 2026

Volume 24, article number 975 (2026)
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Synergistic nanotheranostic platform for fluorescence/magnetic resonance bimodal imaging-guided near infrared photoimmuno-chemodynamic thermal targeting therapy

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Kohei Matsuoka^1,2,3^na1,
Mizuki Yamada^1,2,3^na1,
Ryu Okada^2,3,4,
Mibuko Shimada⁵,
Mikina Yoshida^1,2,3,
Rika Fukushima^1,2,3,
Kanna Umeda^1,2,3,
Ayano Ohashi^1,2,3,
Chiemi Oka⁶,
Ayako Kato³,
Misae Shimizu³,
Chiaki Koike³,
Akira Ito⁶,
Mitsuo Sato^1,2 &
…
Kazuhide Sato ORCID: orcid.org/0000-0003-3025-088X^2,3,4,7,8

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Abstract

Near-infrared (NIR) photoimmunotherapy (PIT) is a promising cancer treatment strategy involving antibody–photoabsorber conjugates. However, the imaging depth and photobleaching susceptibility of the IR700 fluorescence used for treatment monitoring are limited, and antigen heterogeneity within tumors compromises the efficacy of strictly antigen-restricted therapies. We developed a synergistic nanotheranostic platform that integrates NIR-PIT, photothermal therapy, and chemodynamic therapy (CDT) into a single MRI-visible agent (NIR-PICT³). To achieve NIR-PICT³, conjugation of mAb–IRDye700DX (mAb-IR700) to dextran-coated superparamagnetic iron oxide nanoparticles (nanomag-D-spio) yielded nanomag-D-spio-mAb-IR700, enabling dual imaging (IR700 fluorescence/MRI) and targeted cytotoxicity upon 690-nm irradiation. The conjugate retained antigen selectivity, exhibited activatable NIR fluorescence after cellular uptake, generated negative T1- and T2-weighted MRI contrast, and demonstrated robust, cycle-enhanced photothermal performance. Iron-mediated redox catalysis promoted ·OH generation, supporting CDT. In vivo, systemic administration led to marked tumor accumulation, as confirmed by multimodal analyses. Repeated light dosing exploited the NIR-PIT–induced super-enhanced permeability and retention (SUPR) effect, enhancing nanoparticle retention, tumor heating, and therapeutic efficacy. In EGFR+ xenografts, NIR-PICT³ achieved superior tumor regression and survival while inducing photothermal bystander effects in antigen-heterogeneous models. These results establish a modular, image-guided strategy for platform therapy to overcome monitoring depth limitations and antigen heterogeneity in solid tumor NIR-PIT.

Graphical abstract

Photoimmunotherapy for Cancer Treatment

In vivo imaging of acute physiological responses after treatment of cancer with near-infrared photoimmunotherapy

Article 16 May 2023

Photoimmunotherapy for Cancer Treatment

Introducution

Superparamagnetic iron oxide nanoparticles (SPIONs) are versatile theranostic agents that can serve as magnetic resonance imaging (MRI) contrast enhancers while enabling magnetically induced hyperthermia [1]. Their magnetic properties permit noninvasive visualization of in vivo biodistribution and spatiotemporally controlled delivery of localized heating under alternating magnetic fields, highlighting the potential of SPIONs for both diagnosis and treatment delivery [2]. Growing evidence has linked intracellular iron handling and lipid peroxidation to ferroptosis, a regulated, iron-dependent, non-apoptotic cell death pathway of increasing interest in oncology. This has motivated a focused investigation into elucidating how the iron-mediated redox activity of SPIONs influences therapeutic efficacy, oxidative stress–driven cytotoxicity, and systemic safety profiles [3,4,5,6].

Photo-triggered cancer immunotherapeutic approaches have been developed to leverage photodynamic therapy and photothermal therapy (PTT) to potentiate antitumor immunity and induce tumor regression [7]. Near-infrared (NIR) photoimmunotherapy (PIT) is a promising treatment strategy based on the use of antibody–photoabsorber conjugates (APCs). These conjugates consist of a tumor cell surface antigen-specific monoclonal antibody covalently linked to the silicon phthalocyanine-based photoabsorber IRDye700DX (IR700) [8, 9]. Following the selective binding of the APC to its cognate membrane antigen, irradiation with NIR light at 690 nm triggers photochemical activation, which induces rapid and highly specific cytotoxicity [8,9,10,11,12,13,14,15]. Notably, NIR-PIT transiently enhances the permeability of tumor vasculature, leading to the induction of a super-enhanced permeability and retention (SUPR) effect. This effect augments the intratumoral accumulation of nanoscale agents up to approximately 24-fold relative to the baseline enhanced permeability and retention (EPR) effect, thereby creating a therapeutic window that enables the efficient delivery of concurrently administered or subsequent nanomedicines. This modality spares adjacent non-targeted cells, underscoring its therapeutic precision.

Advancements in NIR-PIT reflect an interdisciplinary endeavor that integrates expertise from molecular biology, photophysics, and chemistry. Based on its potent and selective cytotoxic effects, as well as its distinct mechanism of photochemosis [16], NIR-PIT was granted conditional approval by the Pharmaceuticals and Medical Devices Agency of Japan for the treatment of recurrent or previously treated head and neck squamous cell carcinoma [17, 18]. Furthermore, preclinical and early clinical studies of NIR-PIT have systematically examined a range of molecular targets across different tumor types, demonstrating that therapeutic efficacy depends on target expression, tumor accessibility, and intratumoral heterogeneity [19,20,21,22,23,24]. These findings highlight both the strengths and limitations of antigen-dependent NIR-PIT and provide the rationale for developing platform strategies, such as the present study, to address incomplete target coverage and enhance treatment outcomes.

Despite these advances, two major challenges persist. First, the imaging depth and photobleaching susceptibility of the IR700 fluorescence used for treatment monitoring are limited. Second, antigen heterogeneity within tumors compromises the efficacy of strictly antigen-restricted therapies. To address these limitations, NIR-PIT can be integrated with iron-based nanotheranostics, which would facilitate MRI-based tracking, ferroptosis induction, PTT, and chemodynamic therapy (CDT).

In this study, we developed a multifunctional antibody–photoabsorber–SPION conjugate that unifies tumor-targeted NIR-PIT, PTT, and CDT into a single therapeutic platform. This construct was designed to provide dual-modality imaging via IR700 fluorescence and MRI, exploit the SUPR effect to enhance nanoparticle retention and treatment efficacy, and extend the therapeutic benefits to antigen-negative tumor populations through photothermal bystander effects. This study demonstrated the feasibility of such a synergistic nanotheranostic system for precise, multimodal, and image-guided cancer therapies.

Results

Production and characterization of nanomag-D-spio-pan-IR700

A pan-IR700 conjugate was synthesized for EGFR-targeted NIR-PIT. The dye to antibody ratio (DAR) was arranged for approximately 2.5. Then, pan-IR700 was conjugated to 20 nm-sized nanomag-D-spio via cross-linking of carboxylic acids (Fig. 1A). The measured antibody-to-particle ratio was approximately 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed major protein bands at approximately 150–180 kDa for panitumumab, pan-IR700, nanomag-D-spio-pan, and nanomag-D-spio-pan-IR700, corresponding to the expected molecular weight of panitumumab. The band of the pan-IR700 complex showed a slightly higher molecular weight band than panitumumab and showed NIR fluorescence in the same SDS-PAGE gel, representing the additional molecular weight and signal of IR700 (Figure S1). NIR-PIT induces photochemosis via IR700 aggregation, as previously demonstrated [16, 25]. The 690 nm-irradiated pan-IR700 showed smearing and fading protein bands in SDS-PAGE due to the IR700 photochemical reaction. Under these conditions, nanomag-D-spio-pan-IR700 showed fading and smearing bands, suggesting that IR700-ligand silanol release also occurred (Figure S1).

The IR700 fluorescence appeared following incubation of the SDS sample buffer, suggesting that the conjugates were disrupted (Figure S1). The aggregation sizer confirmed that nanomag-D-spio-pan-IR700 with 16 (J/cm²) NIR-light irradiation increased particle size owing to photochemical reaction-induced aggregation (Figure S2). Transmission electron micrographs of nanomag-D-spio and nanomag-D-spio-pan-IR700 showed similar round particles of 20 nm size (Fig. 1B). The absorption spectra showed that nanomag-D-spio and nanomag-D-spio-pan-IR700 showed similar absorption spectra, suggesting that the absorption of pan-IR700 was quenched (Fig. 1C). Zeta potential analysis indicated that conjugation with pan-IR700 shifted nanomag-D-spio to a slightly positive potential (Fig. 1D). To confirm the conjugation of pan-IR700 to nanomag-D-spio, image analysis was performed using atomic force microscopy. Although nanomag-D-spio formed a spherical shape, nanomag-D-spio-pan-IR700 exhibited a dumpling-like shape, suggesting that nanomag-D-spio-pan-IR700 was successfully produced (Fig. 1E). The combination with pan-IR700 reduced the magnetization per unit mass. However, it still exhibited magnetic susceptibility (Fig. 1F). The brightness of T₁- and T₂-weighted MR images weakened with the concentration of nanomag-D-spio-pan-IR700, and the signal decreased in a concentration-dependent manner (T₁-weighted: R² = 0.92394 and T₂-weighted: R² = 0.84807), suggesting that nanomag-D-spio-pan-IR700 can be used as a negative MRI contrast agent and T₂-weighting is more sensitive than T₁-weighting (Fig. 1G).

We examined the photothermal performance under NIR-irradiation using a 690 nm laser. The solution temperature positively correlated with the concentration of nanomag-D-spio-pan-IR700 and laser energy (Figure S3A). The IR thermal image of nanomag-D-spio-pan-IR700 solution gradually brightened, and its temperature considerably increased from 25 to 83 °C (Figure S3B). The power conversion efficiency (PCE; η) of nanomag-D-spio-pan-IR700 was 29.21% (Figure S3C), suggesting that nanomag-D-spio-pan-IR700 can use the photothermal agent. The photothermal performance of nanomag-D-spio solution was also examined. The solution temperature positively correlated with the concentration of nanomag-D-spio and laser energy (Figure S3D).

In addition, nanomag-D-spio-pan-IR700 exhibited excellent photothermal stability for at least five cycles of heating by irradiation and subsequent natural cooling, indicating its potential for application in PTT (Fig. 1H).

To investigate the Fenton-like catalytic efficiency of nanomag-D-spio-pan-IR700, methylene blue (MB), which is degraded with ·OH and decreases absorbance, was selected as an indicator to detect the ·OH generation level following a previous report [26]. MB gradually faded under the condition of nanomag-D-spio (20 µg/mL) and H₂O₂ (20 µM) along with the incubation time, suggesting that the generation of ·OH was induced by the Fenton-like reaction (Fig. 1I). With the condition that the MB absorbance remained almost constant following incubation with nanomag-D-Spio, nanomag-D-spio + GSH, or nanomag-D-spio + H₂O₂ (Fig. 1J). Under these conditions, to verify the photothermal-intensified ·OH generation, we assumed 50 °C as the temperature that could be reached for PTT, and rapid MB degradation was observed at high temperatures. Correspondingly, MB degradation was strengthened by laser irradiation (Fig. 1J). These results suggest the potential application of nanomag-D-spio as an efficient photothermally enhanced CDT agent in cancer theranostics.

In vitro characterization of nanomag-D-spio-pan-IR700

To characterize nanomag-D-spio-pan-IR700 in vitro, its dequenching capacity was tested. In the absence of 1% SDS, IR700 fluorescence was not detected in the nanomag-D-spio-pan-IR700 solution. However, with the addition of 1% SDS, IR700 fluorescence appeared in this solution. The IR700 fluorescence increased by 2–3-fold compared to that in the control (n = 3, p < 0.001, Student’s t-test) (Fig. 2A). To test the stabilization of nanomag-D-spio-pan-IR700 conjugation in blood, nanomag-D-spio-pan-IR700 in mouse serum was imaged. IR700 fluorescence was not detected, suggesting that nanomag-D-spio-pan-IR700 was not degraded in the serum and was stable (n = 3, ns, Student’s t-test) (Fig. 2B). These results suggest that nanomag-D-spio-pan-IR700 is an activatable probe for IR700 fluorescence after internalization into target cells.

To test the fluorescence and targeting ability of nanomag-D-spio-pan-IR700 on EGFR-overexpressing cancer cells or no-EGFR-expressing cells, in vitro flow cytometric analysis was performed (Fig. 2C). Both pan-IR700 and nanomag-D-spio-pan-IR700 showed high IR700 fluorescence in EGFR-overexpressing A431 and MDA-MB-468 cells, which was inhibited by an excess of panitumumab. Neither pan-IR700 nor nanomag-D-spio-pan-IR700 showed IR700 fluorescence in the EGFR-negative H661 and 3T3 mouse cells (Fig. 2C). Nanomag-D-spio-pan-IR700 bound to the EGFR-overexpressing cells, whereas nanomag-D-spio-cont-IR700 (control IgG2) did not (Figure S4A). We also measured photobleaching susceptibility of IR700 in A431 cells incubated with pan-IR700 or nanomag-D-spio-pan-IR700. The photobleaching susceptibility of IR700-fluorescence was almost same with pan-IR700 as with nanomag-D-spio-pan-IR700 (Figure S4B). These results suggest that pan-IR700 and nanomag-D-spio-pan-IR700 are bound specifically to EGFR-expressing cells, and nanomag-D-spio-pan-IR700 was activated by IR700 fluorescence upon binding to these cells. After cell surface targeting, nanomag-D-spio-pan-IR700 colocalized with lysosomes, suggesting that nanomag-D-spio-pan-IR700 was internalized and catabolized by lysosomes (Figure S5).

To assess the magnetic effect of nanomag-D-spio-pan-IR700 binding after targeting the cells, MR images were taken of the targeting cell pellet (A431 cell pellets) in the tube after incubation with pan-IR700, nanomag-D-spio-pan-IR700, and nanomag-D-spio-pan (Fig. 2D). Both T₁- and T₂-weighted images showed negative contrast effects with nanomag-D-spio-pan-IR700 and nanomag-D-spio-pan, whereas pan-IR700 did not show any effect compared to the control (Fig. 2D). Quantitative analysis showed that nanomag-D-spio-pan-IR700 had a negative contrast effect, similar to that of nanomag-D-spio-pan (n = 3, p < 0.0001, analysis of variance ANOVA with Tukey’s test) (Fig. 2E). IR700 fluorescence was detected in pan-IR700 and nanomag-D-spio-pan-IR700 incubated with A431 cell pellets, and pan-IR700 showed higher fluorescence than nanomag-D-spio-pan-IR700 (n = 3, p < 0.0001, p = 0.0015 < 0.005, ANOVA with Tukey’s test) (Fig. 2F).

Oxidative stress was evaluated with CellROX Green. Pan-IR700 did not induce significant oxidative stress upon NIR light irradiation (Fig. 2G). nanomag-D-spio-pan-IR700 and nanomag-D-spio-pan enhanced oxidative stress, indicating that nanomag-D-spio induced the CDT effect (Fig. 2G). We named this combination NIR-PIT with CDT NIR-PICT³. The in vitro NIR-PICT³ cytotoxic effect was observed microscopically, and a cytotoxic effect was detected after staining dead cells with propidium iodide for EGFR-expressing A431, MDA-MB-468, and PC-9 cells (Figure S6).

Targeting cell cytotoxicity was tested in an in vitro mixed culture of non-targeting 3T3-RFP cells and targeting EGFR-overexpressing A431 cells (Fig. 2H). EGFR-targeting cytotoxicity was detected using pan-IR700 and nanomag-D-spio-pan-IR700 1 h after NIR light irradiation. IR700 fluorescence decreased after NIR light irradiation, suggesting the occurrence of IR700-induced photochemosis. Targeting cytotoxicity was also evaluated in EGFR-expressing MDA-MB-468 and PC-9 cells, and only specific targeted cell death was detected (Figure S7).

In vitro cytotoxicity was evaluated quantitatively using a luciferase activity 1 h after NIR light irradiation, and nanomag-D-spio-pan-IR700 showed higher cytotoxicity than pan-IR700 at 0.5 J/cm². nanomag-D-spio-pan did not show significant cytotoxicity in this short time (n = 3, p < 0.05, Student’s t-test) (Fig. 2I). Similar cytotoxicity was confirmed in EGFR-overexpressing MDA-MB-468-luc-green fluorescence protein (GFP) cells and PC-9-luc cells (Figure S8). The PC-9 cell line expresses a moderate amount of EGFR, and the NIR-PICT³–induced cytotoxicity was significantly more pronounced in PC-9 cells than that in A431 or MDA-MB-468 cells, suggesting enhanced contributions from chemodynamic and photothermal effects.

To investigate whether ferroptosis contributes to the cell death induced by nanomag-D-spio-pan, intracellular ferrous iron generation, a key biochemical hallmark of ferroptosis, was examined using FerroOrange staining. The combination of nanomag-D-spio-pan and NIR-light irradiation resulted in a clear increase in intracellular ferrous iron levels, which was consistently observed in both confocal imaging and flow cytometric analyses (Figure S9). This suggests that NIR exposure facilitates spio-derived iron release, thereby potentially enhancing ferroptotic signaling.

Further experiment for evaluation of ferroptosis involvement in nanomag-D-spio-pan treated cell, immunoblotting analysis of GPX4 expression was performed. Nanomag-D-spio-pan incubation also showed slight GPX4 decrease compared to control or NIR light (4 J/ cm²). With the treatment of Nanomag-D-spio-pan + NIR light irradiation, GPX4 expression was gradually decreased from 1 to 48 h after NIR-light irradiation (Figure S10 and S11). These data suggested that Nanomag-D-spio-pan incubation induces ferroptotic signaling, and adding NIR-light irradiation enhanced the signaling.

To address whether other cell death pathway involved in this NIR-PICT³ therapy, we analyzed the apoptosis/ pyroptosis/ necrosis pathway signal protein expression. Control, NIR-light and nanomag-D-spio-pan did not show any change of Caspase-3, Cleaved Caspase-3 (19 kDa), Cleaved Caspase-3 (17 kDa). On the other hand, Caspase-3 gradually increased from 1 h to 48 h after nanomag-D-spio-pan + NIR-light irradiation. Cleaved Caspase-3 (17k Da) slightly increased at 24 h after nanomag-D-spio-pan + NIR-light irradiation (Figure S12 and S13). GSDMD, SPTAN1, Cleaved SPTAN1 did not show any change compared to control or NIR-light with nanomag-D-spio-pan + NIR-light irradiation. These data suggested that nanomag-D-spio-pan + NIR-light could induce not necrosis or pyroptosis but apoptosis.

By changing the targeting antibodies to human epidermal growth factor receptor 2 (HER2) or podoplanin, similar targeting NIR-PICT³ effects were confirmed (Figure S14-19). NIR-PICT³ exhibits a photothermal cytotoxic effect and functions as a versatile therapeutic platform adaptable to different monoclonal antibodies.

In vivo biodistribution of nanomag-D-spio-pan-IR700

To assess the biodistribution of nanomag-D-spio-pan-IR700 in A431-xenografted mouse models, MRI and IR700 fluorescence imaging were performed. The A431 tumors were imaged before the intravenous injection of nanomag-D-spio-pan-IR700 via the tail vein at 1, 3, 6, 9, and 12 h and 1, 2, 3, 4, 5, 6, and 7 d after injection (Fig. 3A). IR700 fluorescence was found to have accumulated gradually after 1 h, and the highest IR700 fluorescence was detected at 12 h, which was maintained for 2 d. After 2 d, IR700 fluorescence gradually decreased from 3 d after injection (Fig. 3B, upper lanes; more detailed time points are shown in Figure S20). MRI showed that nanomag-D-spio-pan-IR700 accumulated 1 h after injection as a negative MRI contrast agent, showing a white tumor turning black on both T₁- and T₂-weighted MRI. The black contrast-enhanced area gradually expanded and reached a plateau 6 h after the nanomag-D-spio-pan-IR700 injection. The contrast-enhanced area persisted for 3 d and gradually cleared away from the tumor area 4 d after injection.

Quantitative analysis revealed that the contrast-enhanced effect on MRI was the highest at 6 h and gradually cleared after 7 d. In T₂-weighted images, the black contrast-enhanced area was more sensitive than that in T₁-weighted images. IR700 fluorescence peaked at 12 h to 2 d and cleared gradually up to 7 d after injection (Fig. 3C). MRI of ex vivo tumor was confirmed at 1 d after nanomag-D-spio-pan-IR700 injection (Figure S21). The hypointense regions were predominantly distributed along intratumoral vessels. ICP-MS was used to quantify iron and confirmed that nanomag-D-spio-pan-IR700 accumulated in the tumors and liver (n = 3, p = 0.0395 < 0.05, p = 0.0002 < 0.0005, Student’s t-test) (Fig. 3D). Iron staining and Berlin Blue staining were performed on fixed tumor specimens, and Berlin Blue was stained in the area along the tumor vessels (Fig. 3E). Iron staining of the tumor was also confirmed using LA-ICP-MS ⁵⁶Fe imaging (Figure S22). To analyze nanomag-D-spio-pan-IR700 accumulation in organs, hematoxylin and eosin (HE) staining and Berlin Blue staining were performed on the liver, lung, spleen, kidney, and intestine (Figure S23). nanomag-D-spio-pan-IR700 was detected in the spleen and liver, indicating that nanomag-D-spio-pan-IR700 was catabolized by the reticuloendothelial macrophages in these organs.

Collectively, the results suggest that nanomag-D-spio-pan-IR700 successfully accumulated in the targeted tumors at 6 h to 2 d, followed by clearance to 7 d. Accumulation was detected and quantified via fluorescence imaging and MRI. Based on these results, we decided to perform NIR light irradiation repeatedly until 5 d after injection.

In vivo evaluation of antitumor effect with NIR-PICT³

To investigate the antitumor effect of NIR-PICT³ with nanomag-D-spio-pan-IR700, bioluminescence imaging (BLI) was performed, and tumor temperature was monitored in EGFR-overexpressing A431-luc-GFP xenografted mouse tumor models. The 690 nm NIR-light was irradiated five times as biodistribution revealed that nanomag-D-spio-pan-IR700 was detected until 5 d after intravenous injection (Fig. 4A). Representative bioluminescence images indicated that the tumors treated with phosphate-buffered saline (PBS) and PBS + NIR-light gradually increased with increasing luciferase activity. Under these conditions, the tumors treated with pan-IR700, nanomag-D-spio-pan, and nanomag-D-spio-pan-IR700 without NIR light also showed no apparent differences compared to the controls. Tumors treated with pan-IR700, nanomag-D-spio-pan, and nanomag-D-spio-pan-IR700 with repeated NIR-light irradiation showed significant antitumor effects (Fig. 4B). The temperature of the tumors increased daily after NIR-light irradiation in NIR-PICT³ tumors (nanomag-D-spio-pan-IR700 with repeated NIR-light irradiation), whereas that for nanomag-D-spio-pan did not, suggesting that NIR-PICT³ should be utilized as a repeated therapy given the enhanced photothermal effect (Fig. 4C).

The quantitative relative light units (RLU) in BLI showed a significant decrease in tumors treated with NIR-PICT³ (p = 0.0215 < 0.05 [day 4], 0.0125 < 0.05 [day 7], Kruskal–Wallis and Dunn’s multiple comparison tests, n = 10 in each group) (Fig. 4D). Body weight did not change significantly with treatment (Fig. 4E). Tumor volume significantly decreased with NIR-PICT³ (p = 0.0002 < 0.0005 [day 14], Kruskal–Wallis and Dunn’s multiple comparison tests, n = 10 in each group) (Fig. 4F). Ex vivo treated tumors were imaged (Figure S24). Survival was significantly prolonged in NIR-PICT³ compared to the other groups (p = 0.0001, log-rank test, n = 10). EGFR-expressing MDA-MB-469-luc-GFP and PC-9-luc xenografted tumors were also evaluated, and significant antitumor effects were observed (Figure S25 and S26).

These results suggest that EGFR-targeted NIR-PICT³ showed significant antitumor effects and that the tumor temperature increased with repeated NIR light irradiation.

In vivo evaluation of blood and tumor histology with NIR-PICT³

Blood tests were performed to evaluate side effects in NIR-PICT³-treated mice. Serum ferritin levels increased with the injection of nanomag-D-spio-pan or nanomag-D-spio-pan-IR700 on days 1, 7, and 14, although the increase was not significant. Serum iron levels were not elevated in the treated mice. Liver blood chemistry [lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (TB)] was also measured, and no significant differences were detected among the groups (Fig. 5A). Other blood chemistry parameters [total protein (TP), cholinesterase (ChE), triglyceride (TG), high-density lipoprotein (HDL), direct bilirubin (D-bil), blood urea nitrogen (BUN), alkaline phosphatase (ALP), leucine aminopeptidase (LAP), total cholesterol (T-cholesterol), and total bile acid (TBA)] were also evaluated and showed no significant differences between groups (Figure S27). Therefore, no significant changes were observed in the blood.

To evaluate long-term toxicity of NIR-PICT³-treated mice, Liver blood chemistry was also done at 40 days after injection of nanomag-D-spio-pan. TP, ChE, AST, ALT, T-Bil、LAP and D-Bil did not significantly change compared to control. ALP and HDL-chol significantly decreased in the nanomag-D-spio-pan intravenously injected mice, however, we thought this decrease did not due to liver damage (Figure S28). Overall, the long-term toxicity could be permissible, and manageable.

To confirm the antitumor effect of NIR-PIT, nanomag-D-spio-pan with NIR-light (photothermal effect and CDT), and NIR-PICT³, treated tumors were stained with HE at 1 d after the first NIR-light irradiation (Fig. 5B). The tumors appeared intact in the control, NIR light, pan-IR700, nanomag-D-spio-pan, and nanomag-D-spio-pan-IR700 groups without NIR light. In NIR-PIT-treated tumors (pan-IR700 with NIR light), approximately half of the tumor population died. nanomag-D-spio-pan with NIR light (photothermal effect and CDT) caused little hemorrhage, and 20% of the population died. NIR-PICT³-treated (nanomag-D-spio-pan-IR700 with NIR light) mice showed little hemorrhage, and 90% of the population died (Fig. 5B). These histological results are compatible with the quantification of the antitumor effect (Fig. 4). The hemorrhage was caused by the photothermal effect. Histological confirmation of EGFR-targeted NIR-PICT³-treated tumor specimens showed that the tumor cells were completely destroyed on day 7 (Figure S29).

Mechanism of in vivo enhanced PTT in NIR-PICT³

When nanoparticles are administered intravenously, nanoscale drugs circulate throughout the body via blood vessels. Therefore, nanoscale drugs preferentially leak into the tumor tissue from the abundant, highly permeable tumor vasculature and remain within the tumor owing to limited efflux via the underdeveloped lymphatics. This is known as the EPR effect [27] which has been applied to delivery systems designed to maintain anticancer drug concentrations in tumors. Antibody–drug conjugates, micellar drugs, and liposomal drugs are clinically used for this purpose. In tumors treated with NIR-PIT, the EPR effect of nanoparticles was enhanced approximately 24-fold owing to the increased permeability caused by rapid cell death in the perivascular tumor tissue [27]. This enhanced EPR effect induced by NIR-PIT is known as the SUPR effect. Recently, both nanosized and microsized particles have been found to accumulate with NIR-PIT [28].

To demonstrate the accumulation of nanoparticles after NIR-PICT³, QD800, a quantum-dot-based fluorescent nanoparticle with a mean diameter of 20 nm (approximately the same size as nanomag-D-spio-pan-IR700), was used to evaluate the SUPR effect. After performing NIR-PICT³ in an A431-luc-GFP-transplanted bilateral tumor mouse model, QD800 was administered intravenously. One tumor (right side) was irradiated, whereas the contralateral tumor (left side) was shielded. SUPR was evaluated by externally detecting the QD800 fluorescence signal (800 nm fluorescence). QD800 was administered intravenously 1 h after treatment based on prior research [28]. Following the intravenous infusion of QD800, changes in the fluorescence signal of QD800 within the tumor were tracked for 60 min (Fig. 6A). In QD800 fluorescence imaging performed 1 h after NIR light irradiation, the QD800 fluorescence signal was distributed uniformly throughout the body immediately after administration. However, in tumors treated with NIR-PIT or NIR-PICT³, the signal intensity was maintained to a greater extent than that in non-irradiated tumors (Fig. 6B). This suggests that the NIR-PIT-induced photochemosis enhances the retention and permeability of the injected QD800 nanoparticles within the tumors, which reflects the SUPR effect. QD800 fluorescence signals were higher in NIR-PICT³-treated tumors than in NIR-PIT-treated tumors, suggesting that the additional photothermal and CDT effects enhanced the SUPR effect. Quantitative analysis indicated that the highest QD800 accumulation was detected in NIR-PICT³-treated tumor. Around 5-fold higher accumulation in the NIR-PICT³-treated tumor was measured as nanomag-D-spio-pan and NIR-light PTT (n = 3 in each group, mean ± standard error of the mean, p < 0.005, nanomag-D-spio-pan-IR700 vs. PBS, nanomag-D-spio-pan, respectively; ANOVA with Tukey’s test) (Fig. 6C).

To confirm nanomag-D-spio accumulation in the treated tumors, ICP-MS (iron) of ex vivo treated tumors was performed (n = 3, p = 0.007 < 0.01, Student’s t-test) (Fig. 6D). Quantitative analysis indicated a 5-fold increase in iron in NIR-PICT³-treated tumors compared with that in the others, which is similar to the results for QD800 accumulation.

Overall, these results suggest that combining NIR-PIT with iron oxide nanoparticles (IONPs) enhances iron nanoparticle accumulation through the synergistic effects of NIR-PIT, PTT, and CDT, thereby achieving an ideal effect. Therefore, NIR-PICT³ can be detected using MRI and a combination of NIR-PIT, enhanced PTT, and CDT, achieving theranostic-integrated photoenhanced targeting therapy.

Evaluation of in vivo photothermal bystander effect in NIR-PICT³

Antibody-based targeted therapy is useful because it can treat tumors that highly express the target antigen. However, human tumors contain areas of both high and low antigen expression [29]. The higher the antibody specificity, the more selectively the high-expression areas are damaged, leading to a tendency for tumors with low antigen expression to recur. This type of heterogeneity has recently been identified as a cause of tumor recurrence and is implicated in the resistance to all anticancer therapies [30, 31]. Therefore, the balance between targeting ability and bystander effect on non-targeted cancer cells is crucial for antibody–drug conjugates conjugated with anticancer drugs. Owing to the high specificity of photochemosis, NIR-PIT may leave non-EGFR-targeted cancer cells intact for conditionally approved EGFR drugs, raising concerns as a potential cause of recurrence [12, 31]. To overcome this limitation, a combination therapy with other anticancer therapies such as cancer immunotherapies is anticipated, and clinical trials are currently being planned.

Based on the results of the in vivo enhanced PTT and increased drug accumulation, we hypothesized that the targeting disadvantage of NIR-PIT could be overcome by NIR-PICT³. Therefore, we created a mixed tumor model mimicking human tumor antigen heterogeneity and evaluated it preclinically. We established a mixed tumor model comprising EGFR+ A431 and EGFR− H661-luc cells, and the antitumor cytotoxicity-enhanced photothermal-bystander effect (non-targeted tumor cells) was monitored by measuring the luciferase activity of H661-luc cells. First, we evaluated mixed tumors using HE staining and EGFR immunostaining (Fig. 7A). EGFR immunostaining revealed that the mixed tumors were well mixed, with diffuse EGFR + and EGFR− lesions, indicating successful establishment of the antigen-heterogeneous mixed tumor model.

After characterization and confirmation of the antigen-heterogeneous mixed tumor model, the antitumor cytotoxic enhanced photothermal-bystander effect (non-targeted H661-luc tumor cells) was evaluated using the same regimen as shown in Figs. 4A and 7B. To precisely compare the in vivo cytotoxic bystander effects induced by NIR-PICT³, we developed a bilateral mixed-tumor mouse model, in which only the right side of the mixed tumor was irradiated with NIR light. A representative BLI is shown in Fig. 7C. The irradiated right side of the mixed tumor, the left side in the control, and the pan-IR700 administered group did not show any antitumor effects against the non-targeted tumor populations, which exhibited a gradual increase in luciferase activity (Fig. 7C). The BLI intensity on the right side of the mixed tumor in nanomag-D-spio-pan-injected mice slightly decreased with NIR light irradiation on day 2, indicating limited in vivo cytotoxic photothermal bystander effects (Fig. 7C). Under these conditions, the BLI intensity of the right side of the mixed tumor in nanomag-D-spio-pan-IR700-injected mice and NIR-PICT³-treated mixed tumor decreased with NIR-light irradiation, indicating considerable in vivo cytotoxic photothermal bystander effects (Fig. 7C).

For quantitative analyses, the luciferase activity (RLU ratio) was used (non-EGFR-targeted H661-luc population in the mixed tumor). The results revealed that NIR-PICT³ exerted antitumor effects on the non-targeting H661-luc population in the mixed tumors, whereas the other treatments showed no significant effects (p = 0.0298 < 0.05, Kruskal–Wallis test with Dunn’s post-hoc test) (Fig. 7D). The same quantitative analyses for comparing the NIR-light irradiated with pan-IR700, nanomag-D-spio-pan, and nanomag-D-spio-pan-IR700 groups showed a significant decrease in RLU in NIR-PICT³-treated tumors (p = 0.0483 < 0.05 vs. nanomag-D-spio + NIR-light; p = 0.0111 < 0.05 vs. pan-IR700 + NIR-light; Kruskal–Wallis test with Dunn’s post-hoc test) (Fig. 7E).

Tumor volume was quantified to compare the antitumor effects on whole mixed tumors. Both NIR-PIT (pan-IR700 + NIR-light) and nanomag-D-spio-pan + NIR-light led to whole tumor reduction due to the targeted photochemosis (NIR-PIT effect) or photothermal effect, respectively (Fig. 7F). NIR-PICT³ exerted a greater antitumor effect on whole mixed tumors than NIR-PIT or nanomag-D-spio-pan + NIR light (p < 0.05, Kruskal–Wallis test with Dunn’s post-hoc test) (Fig. 7F). When comparing the NIR-light irradiated with pan-IR700 (NIR-PIT), nanomag-D-spio-pan, and nanomag-D-spio-pan-IR700 (NIR-PICT³) groups, a significant decrease in tumor volume was detected in NIR-PICT³-treated tumors (p = 0.0062 < 0.01 vs. nanomag-D-spio + NIR-light, p = 0.0241 < 0.05 vs. pan-IR700 + NIR-light, Kruskal–Wallis test with Dunn’s post-hoc test) (Fig. 7G). This indicated that NIR-PICT³ exerted the strongest antitumor effect on whole mixed tumors, suggesting that it exhibits antitumor effects on both the EGFR-targeting A431 cells and non-EGFR-targeting H661-luc cells and confirming the in vivo cytotoxic photothermal bystander effect. Furthermore, survival was significantly prolonged in the NIR-PICT³ group (p = 0.0001, log-rank test) (Fig. 7H), with complete response in half of the mice.

Collectively, these results demonstrate that NIR-PICT³ exerts potent antitumor effects in vivo through both target-specific cytotoxicity and photothermal bystander effects in antigen-heterogeneous tumors.

Discussion

We developed a magnetically traceable EGFR-targeted NIR-PIT platform by integrating panitumumab–IR700 with dextran-coated superparamagnetic iron oxide (nanomag-D-spio), yielding a single theranostic agent (nanomag-D-spio-pan-IR700) that was physically characterized, MRI-visible, and cytotoxic upon NIR irradiation. In vitro, the conjugate preserved EGFR selectivity and induced stronger cell death than pan-IR700 alone, suggesting that retaining spio synergistically augments the photochemosis-based cell death mechanism of NIR-PIT, combined with additional PTT, CDT, and ferroptosis effects. These multiple cell death mechanisms lead to enhanced antitumor effects.

Systemic administration achieved tumor accumulation confirmed by dual imaging (MRI and IR700 fluorescence), ICP-MS, and immunohistology, establishing true enhanced intratumoral delivery rather than mere vascular retention. In vivo therapy with fractionated 200 J cm⁻² light produced significant tumor regression versus comparators, accompanied by a measurable surface temperature increase at the treatment site, which can be considered an enhancement of photothermal effect. Together with the dose-sparing in vitro results, these data indicate that the spio co-formulation can potentiate NIR-PIT and confer an advantage over conventional NIR-PIT that lacks an intrinsic imaging handle (Fig. 8A).

Notably, therapeutic efficacy was preserved in heterogeneous xenograft models comprising both EGFR-positive and EGFR-negative cells with the photothermal bystander effect. Although pan-IR700 combined with light irradiation proved to be insufficient, treatment with nanomag-D-spio-pan-IR700 combined with NIR light resulted in significant tumor regression. This outcome is consistent with the enhanced localized damage within the irradiation field, which may help overcome the challenge of antigen heterogeneity, a well-recognized limitation of antigen-restricted PIT monotherapy (Fig. 8B).

Therapeutic monitoring capitalizes on the SUPR phenomenon elicited by NIR-PIT. When QD800 was employed as a nanotracer, we detected substantial post-treatment retention within the ablated tumor bed, accompanied by superior therapeutic outcomes relative to that observed in the pan-IR700 controls. These observations corroborate those of previous reports indicating that NIR-PIT transiently augments intratumoral nanoagent delivery and further suggest that SUPR-based imaging readouts may function as quantitative metrics for evaluating therapeutic responses [28].

Safety evaluations demonstrated elevated ferritin levels without overt abnormalities in hepatic or renal enzyme profiles, consistent with the established reticuloendothelial system processing of iron oxide probes, which are widely utilized in both clinical and preclinical MRI applications. Although these findings are promising, comprehensive investigations are necessary to delineate the long-term fate of iron deposits, potential immunogenic consequences, and the relative contributions of photochemical and photothermal mechanisms to the observed therapeutic effects.

Despite these promising results, the limitations of this study should be considered. First, the therapeutic depth of NIR-PIT remains constrained by the limited tissue penetration of 690 nm light, which typically reaches several centimeters into soft tissue. This inherent optical attenuation may restrict treatment efficacy in deeply seated or large tumors, necessitating the use of interstitial or endoscopic light-delivery systems to extend the clinical applicability. Recently, new light-based devices have been developed that may overcome these limitations [32,33,34,35]. Second, although transient elevations in serum ferritin levels were observed without acute toxicity, long-term monitoring is essential to assess potential iron overload, oxidative stress, or macrophage dysfunction associated with repeated spio exposure. Our results demonstrate that iron accumulates and is deposited in the target tumor after a single administration, and sufficient efficacy can be achieved with repeated light irradiation alone, potentially avoiding iron overload. Finally, although the xenograft models provided valuable proof-of-concept data, they did not fully represent the complexity of human tumor immunology or the pharmacokinetic interplay between antibody–spio conjugates and the host immune system. Future studies should aim to develop immunocompetent or orthotopic tumor models to clarify these aspects and optimize dosing schedules and safety margins before clinical translation.

Conclusions

In conclusion, these findings establish nanomag-D-spio-pan-IR700 as a prototype of a single-agent, image-guided NIR-PIT platform that (i) facilitates precise tumor localization under MRI guidance, (ii) elicits photochemosis as a distinct mode of NIR-PIT cell death, (iii) induces mixed CDT and ferroptosis cytotoxic effects, (iv) exerts enhancement photothermal effect with repeated NIR-light irradiation, (v) maintains therapeutic efficacy in the context of antigenic heterogeneity, and (vi) enables real-time monitoring of treatment dynamics through SUPR-responsive nano-tracers. Considering the clinical advancement of EGFR-targeted NIR-PIT and the well-documented utility of spios as MRI contrast agents, this modular approach represents a feasible and translational strategy for precision-oriented, image-monitoring NIR-PIT for solid tumors.

Materials and methods

Experimental Design

We developed a synergistic nanotheranostic platform that integrates NIR-PIT, photothermal therapy, and CDT into a single MRI-visible agent (NIR-PICT³). With this platform, the in vitro and in vivo effects were evaluated and a new concept for enhanced PTT and the photo-thermal bystander effect was established.

Ethics statement

All animal experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animal Resources of the Nagoya University Animal Care and Use Committee (Nagoya, Japan; approval numbers 2021, M210770-001; 2022, M220370-003; 2023, M230269-003; 2024, M240295-002; and 2025, M250149-003). The animals were maintained in accordance with the Act on Welfare and Management of Animals, Standards Relating to the Care and Keeping and Reducing Pain of Laboratory Animals, and Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions. This study complies with the ARRIVE 2.0 guidelines for animal research.

Reagents

The IRDye 700DX-NHS ester was purchased from LI-COR Biosciences, Lincoln, NE, USA. Panitumumab, a fully humanized IgG2 mAb directed against EGFR, was purchased from Amgen [36], Thousand Oaks, CA, USA. Nanomag^®-D-spio was purchased from micromod Partikeltechnologie GmbH, Rostock, Germany. Qdot 800 ITK Amino (PEG) Quantum Dots were purchased from Thermo Fisher Scientific, Waltham, MA, USA. D-Luciferin was obtained from Goryo Chemical Sapporo, Japan. For luciferase transfection, RediFect-Red-FLuc-puromycin was purchased from Perkin Elmer, Waltham, MA, USA. A 10% SDS solution was purchased from NIPPON GENE, Tokyo, Japan. FerroOrange was obtained from Dojindo Laboratories, Kumamoto, Japan, and CellROX Green Reagent was purchased from Thermo Fisher Scientific.

Cell culture

All cell lines were obtained from the American Type Culture Collection, Manassas, VA, USA. A431-luc-GFP cells (human epidermoid skin cancer cells) with genes encoding firefly luciferase and GFP [13, 15], MDA-MB-468-luc-GFP cells (human breast cancer cells) with genes encoding firefly luciferase and GFP (12, 36), and 3T3-luc (murine fibroblast, NIH-3T3), PC9-luc (human lung cancer cells), and H661-luc (human lung cancer cells) cells with genes encoding firefly luciferase were cultured in RPMI-1640 (Thermo Fisher Scientific) [23] supplemented with 10% fetal bovine serum and penicillin (100 IU/mL)–streptomycin (100 µg/mL; Thermo Fisher Scientific).

Conjugation of IR700 to panitumumab

Conjugation was performed as previously reported. Panitumumab (147 kDa)(6.8 nmol) was incubated with IR700 NHS-ester (1.9 kDa)(30.8 nmol, LI-COR Biosciences) in 0.1 mol/L Na₂HPO₄ (pH 8.6) at 25 °C for 1 h [37, 38] The mixture was separated and purified with a Sephadex G50 column (PD-10; GE Healthcare, Piscataway, NJ, USA) [23]. Protein concentration was determined using a Coomassie Plus Protein Assay Kit (Thermo Fisher Scientific) by measuring the absorption at 595 nm using a spectrophotometer (UV-1900; Shimadzu, Kyoto, Japan) [37, 39]. IR700 concentration was measured via absorption at 689 nm using a spectrophotometer (UV-1900) to determine the number of fluorophore molecules conjugated to the mAb (dye-mAb ratio) [37, 40, 41]. Protein concentration was further analyzed and quantified using SDS-PAGE [42]. Panitumumab-IR700 was referred to as pan-IR700. The bioactivity of the conjugated products was determined by testing their binding to A431, MDA-MB-468, and PC9 cells, which overexpressed EGFR, using flow cytometry. The cells (1 × 10⁵) were incubated with pan-IR700 (10 µg/mL) in the RPMI-1640 complete medium for 6 h at 37 °C. To confirm the binding specificity of the new conjugates, a competition assay was performed by adding excess panitumumab (1 µg). The cells were analyzed using a flow cytometer (DxFLEX; Beckman Coulter, Inc., Fullerton, CA, USA), and the results were processed using FlowJo v10 (Beckman Coulter).

Conjugation and characterization of pan-IR700 to nanomag-D-spio

nanomag-D-spio-streptavidin (20 nm size) was conjugated to pan-IR700 via the cross-linking of carboxylic acids through carbodiimide compounds. Transmission electron micrographs of nanomag-D-spio-pan-IR700 were captured using a JEM-F200 microscope (Nihon Denshi, Tokyo, Japan). The zeta potential was measured using a Zetasizer Lab (Malvern Panalytical, Great Malvern, UK). Magnetic properties were detected using a vibrating sample magnetometer (Riken Denshi, Tokyo, Japan). MR images were acquired using an MR imager (MR VivoLVA, Japan REDOX, Fukuoka, Japan). Photobleaching susceptibility of IR700-fluorescence was measured with a PEARL imager. The quantity of IR700-fluorescence of after NIR-irradiation was divided that of before NIR-light irradiation. After, A431 cells were incubated pan-IR700 or nanomag-D-spio-pan-IR700, NIR-light irradiation was performed. The ratio of IR700-fluorescence A.U after NIR-light irradiation to before was evaluated.

Fluorescence microscopy

To detect antigen-specific localization of the IR700 conjugate nanomag-D-spio, fluorescence microscopy was performed (A1Rsi; Nikon Instech, Tokyo, Japan). A431, MDA-MB-468, or PC9 cells (2 × 10⁴) were seeded in glass-bottom dishes and incubated for 24 h. Then, 10 µg/mL nanomag-D-spio-pan-IR700 was added onto the culture medium, and the cells were incubated at 37 °C for 6 h. The cells were then washed twice with PBS. Propidium iodide (final concentration 2 µg/mL; Thermo Fisher Scientific), SYTOX blue (final concentration: 2 µg/mL; Thermo Fisher Scientific), FerroOrange (final concentration: 1 µM; Dojindo Laboratories), or Hoechst (final concentration: 500 nM) was added 20 min before microscopic observation [43]. The cells were exposed to NIR light (16 J/cm²), and serial microscopic images were obtained.

Measurement of ·OH generation

nanomag-D-spio or nanomag-D-spio-pan-IR700 were mixed with 1 mM GSH solution for 30 min. The GSH mixture was tested with 10 µg/mL MB and 20 µM H₂O₂ at different pH values (5.4, 6.4, or 7.4) and different temperatures. To investigate the enhanced ·OH generation under NIR-light irradiation, 10 µg/mL MB and 20 µM H₂O₂ at pH 5.4 were added followed by laser irradiation (200 mW/cm²). Each sample of the same concentration was mixed with 10 µg/mL MB as a control group. The ·OH generation was evaluated according to MB degradation, which was measured via the absorbance decrease using spectroscopy (UV-1900; Shimadzu, Kyoto, Japan).

ROS generation analysis

PC9-luc, A431, and 3T3 Her2 cells (1 × 10⁵) were seeded onto 24-well plates and allowed to grow for 24 h before incubation in a medium containing 10 µg/mL nanomag-D-spio-pan-IR700 for 12 h at 37 °C. Untreated cells were used as the control group (blank). The solution was removed, and the cells were rinsed carefully with PBS twice. CellROX Green (5 µM)-containing medium was used for another 30 min of incubation. The cells were gently collected by scraping with a cell scraper [44]. CellROX fluorescence was evaluated in 10,000 cells using a flow cytometer (DxFLEX; Beckman Coulter).

Ferrous iron detection

FerroOrange (Dojindo Laboratories) was used to detect intracellular ferrous iron according to the manufacturer’s protocol. Briefly, the harvested cells were washed three times with Hanks’ balanced salt solution (HBSS; Thermo Fisher Scientific) after removing the culture medium under treatment conditions and then co-incubated with 1 µM FerroOrange working solution at 37 °C for 30 min. Fluorescence was evaluated using a flow cytometer (DxFLEX, Beckman Coulter). Fluorescent images were captured using a fluorescence microscope (A1Rsi; Nikon Instech). A fluorospectrophotometer was used at excitation and emission wavelengths of 543 and 580 nm, respectively. The ferrous iron analysis was performed by measuring the fluorescence intensity under each treatment condition.

In vitro NIR-PICT³

For EGFR-targeted-NIR-PICT³, A431, MDA-MB-468, and PC9 (1 × 10⁵) were seeded onto 12-well plates and incubated in a medium containing 10 µg/mL nanomag-D-spio-pan-IR700 for 12 h at 37 °C. After washing twice with PBS, the cells were irradiated with a 690 nm NIR light-laser (MLL-III-690; Changchun New Industries Optoelectronics Tech, Co., Ltd., Changchun, China) [14] The actual power densities (mW/cm²) were measured using an optical power meter (PM100; Thorlabs, Newton, NJ, USA) [34].

Photothermal cytotoxic effects were assessed by measuring the luciferase activity. To monitor luciferase activity, 150 µg/mL D-luciferin-containing medium (Goryo Chemical) was administered to PBS-washed cells at 1 h after NIR- PICT³. The cells were analyzed using a plate reader to detect their bioluminescence (Cytation 5; BioTek, Winooski, VT, USA) [45].

Ferroptotic signaling evaluation

A431 cells (1 × 10⁵) were seeded onto 12-well plates and incubated in a medium containing 0.225 mg/mL nanomag-D-spio-panfor 12 h at 37 °C. After washing twice with PBS, the cells were irradiated with a 690 nm NIR light-laser (4 J/cm2). After incubation for indicated duration, cells were harvested with 1 x SDS sample buffer and subjected to immunoblotting. Anti-GPX4 (ab125066, abcam), and anti-Histon H3 (proteintech MABI 0301) were used.

Apoptosis, pyroptosis, necrosis signaling evaluation

A431 cells (1 × 10⁵) were seeded onto 12-well plates and incubated in a medium containing 10 µg/mL nanomag-D-spio-pan-IR700 or 0.225 mg/mL nanomag-D-spio-pan for 12 h at 37 °C. After washing twice with PBS, the cells were irradiated with a 690 nm NIR light-laser (4 J/cm2). After incubation for indicated duration, cells were harvested with 1 x SDS sample buffer and subjected to immunoblotting. Anti-SPTAN1, Cleaved SPTAN1 (proteintech, 31676-1-AP), anti-GSDMD (proteintech, 20770-1-AP), anti-Caspase3, Cleaved Caspase3 (proteintech, 19677-1-AP), anti- Cleaved Caspase3 (proteintech, 68773-1-lg) and anti-Histon H3 (proteintech MABI 0301) were used.

Animals and tumor models

All in vivo procedures were conducted in compliance with the institutional guidelines of the Animal Care and Use Committee of Nagoya University. All mice were purchased from SLC Japan, Inc. (Tokyo, Japan). During all experimental procedures, mice were anesthetized using isoflurane (Wako, Osaka, Japan). Approximately 8–12-week-old Balb/C-Slc-nu/nu mice were inoculated with A431-luc-GFP (2 × 10⁶), MDA-MB-468-luc-GFP (1 × 10⁷), or PC9-luc cells (5 × 10⁶) on the right, left, or both dorsa. Animals were randomly assigned to each experimental group to achieve the highest possible consistency in tumor luciferase activity among all groups. The number of mice used is indicated in the figure captions.

In vivo NIR-PICT³

In vivo NIR-PICT³ assay was performed 10 d after tumor cell inoculation. Mice were intravenously injected with 100 µg nanomag-D-spio-pan-IR700. On the day following injection, mice were irradiated with NIR-light (690 nm) at 200 J/cm² using a 690 nm NIR light-laser (MLL-III-690; Changchun New Industries Optoelectronics Tech, CO., Ltd) [power was 1.0 W at 690 nm], unless otherwise specified, applied on the right tumor [46,47,48,49]. After 4 d (total 5 d), the treated tumors were irradiated repeatedly at 200 J/cm², as indicated in the figure regimen.

In vivo evaluation of nano drug delivery after NIR-PICT³

Following NIR-light irradiation, 32 pmol of pegylated non-targeted quantum dots (QD800; Qdot 800 ITK™ Amino (PEG) Quantum Dots; Thermo Fisher Scientific) were injected intravenously, and in vivo imaging was performed for each.

In vivo IR700 QD800 imaging

IR700 and QD800-fluorescence were detected before and after therapy using a fluorescence imager (Pearl Imager; LI-COR Biosciences) [50,51,52].

In vivo BLI

For BLI, 2.5 mg of d-luciferin (Goryo Chemical) was injected intraperitoneally, and 10 min after injection, the mice were imaged on a bioluminescence imager (IVIS, Perkin Elmer) [53, 54].

MRI

MR images were acquired before injection and every day post-injection using a 1.5 T MRI system with the following parameters: T₁-weighted sequence, spin echo; repetition time/echo time, 500/9.0 ms; image size, 128 × 256; slice thickness, 1 mm; slice number, 18; interval, 0.5 mm; and field of view, 60 mm × 30 mm. The acquisition time for each scan was 01 min 05 s. MR images were analyzed using ImageJ software for quantitative analysis [55]. Percentage signal enhancement (% SE) was calculated as the percentage difference ratio between the tumor signal intensity (SI) at each time point, t, and the SI before injection (t₀): % SE(t) = [(SI(t) − SI(t₀)]/SI(t₀) × 100%.

Serum mice blood chemistry

Mice were anesthetized, and blood was drawn via puncture of the superior maxillary vein. After the blood cells and serum have been separated, the serum is frozen. The samples were then analyzed via Screening set of ORIENTAL YEAST CO., LTD (Tokyo, Japan).

Tumor tissue staining

Mice were euthanized by carbon dioxide inhalation using a gradual-fill method in accordance with the institutional review board’s guidelines. Death was confirmed prior to tissue collection. The tumors or organs of the euthanized mice were harvested and fixed in 10% formalin. The samples were embedded in paraffin and sliced for staining with HE or Prussian Blue. The slides were imaged, and Prussian Blue staining was assessed using a fluorescence digital microscope (BZ-X810; Keyence Corporation, Osaka, Japan).

Statistical analysis

Data are expressed as means ± standard error of the mean as determined by a minimum of three experiments, unless otherwise indicated. Statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA) and Python (https://www.python.org/). An unpaired t-test was used for two-group comparisons. Correlations were determined using Pearson product-moment correlation coefficients. For linear regression, p < 0.05 was considered to indicate a significant difference.

Data availability

All data are available in the main text or the supplementary materials.

Abbreviations

NIR:: Near-infrared
PIT:: Photoimmunotherapy
PTT:: Photothermal therapy
CDT:: Chemodynamic therapy
MRI:: Magnetic resonance imaging
NIR-PICT³ :: Near-infrared photoimmuno-chemodynamic thermal targeting therapy
IR700:: IRDye700DX
EGFR:: Epidermal growth factor receptor
SPIONs:: Superparamagnetic iron oxide nanoparticles
APCs:: Antibody–photoabsorber conjugates
SUPR:: Super-enhanced permeability and retention
EPR:: Enhanced permeability and retention
Pan:: Panitumumab
SDS-PAGE:: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
IR:: Infrared
PCE:: Power conversion efficiency
MB:: Methylene blue
GSH:: Glutathione
SDS:: Sodium dodecyl Sulfate
IgG:: Immunoglobulin
RFP:: Red fluorescence protein
GFP:: Green fluorescence protein
ICP-MS:: Inductively Coupled Plasma Mass Spectrometry
LA-ICP-MS:: Laser Ablation Inductively Coupled Plasma Mass Spectrometry
HE:: Hematoxylin and eosin
BLI:: Bioluminescence imaging
Luc:: Luciferase
Luc-GFP:: Luciferase-green fluorescence protein
PBS:: Phosphate-buffered saline
RLU:: Relative light units
BLI:: Bioluminescence imaging
GPX4:: Selenoprotein glutathione peroxidase 4
GSDMD:: Gasdermin D
SPTAN1:: Spectrin alpha, non-erythrocytic 1
LDH:: Lactate dehydrogenase
AST:: Aspartate aminotransferase
ALT:: Alanine aminotransferase
TB:: Total bilirubin
TP:: Total protein
ChE:: Cholinesterase
TG:: Triglyceride
HDL:: High-density lipoprotein
D-bil:: Direct bilirubin
BUN:: Blood urea nitrogen
ALP:: Alkaline phosphatase
LAP:: Leucine aminopeptidase
T-chol:: Total cholesterol
TBA:: Total bile acid
QD:: Quantum dot
IONPs:: Iron oxide nanoparticles
UV:: Ultraviolet
ROS:: Reactive oxygen species
HBSS:: Hanks’ balanced salt solution
PEG:: Polyethylene glycol

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Acknowledgements

This study was conducted as a joint research project with the JSR Corporation. We thank Dr. Nohara Satoshi for discussions pertaining to materials.

Funding

This study was supported by: The Advanced Research Infrastructure for Materials and Nanotechnology in Japan (ARIM JAPAN of MEXT): KS; KAKEN [21K07217, 25K03451, and 25K22916 (JSPS)]: KS; FOREST-Souhatsu [JPMJFR2017 (JST)]: KS; AMED SeedsA (26ym0126807j0005, A-226, AMED): KS; JST TONGALI: KS; Toyota Physical and Chemical Research Institute research scholarship: KS; Sumitomo Electric Group CSR Foundation research scholarship: KS; Takeda Science Foundation research scholarship: KS; SGH Foundation research scholarship: KS; Japanese Society for Thermal Medicine research scholarship: KS; Research Grant from The Chemo-Sero-Therapeutic Research Institute: KS; Suzuki-kenzo scholarship: KS; TERUMO Science Foundation research scholarship: KS, Foundation of Public Interest of Tatematsu, Tatematsu Foundation: KS; Iketani ScienceFoundation research scholarship: KS. Funders provided only financial support and had no role in the study design, data collection, data analysis, interpretation, or writing of the reports.

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Kohei Matsuoka and Mizuki Yamada contributed equally to this work.

Authors and Affiliations

Division of Host Defense Sciences, Dept. of Integrated Health Sciences, Graduate School of Medicine, Nagoya University, Nagoya, Japan
Kohei Matsuoka, Mizuki Yamada, Mikina Yoshida, Rika Fukushima, Kanna Umeda, Ayano Ohashi & Mitsuo Sato
Graduate School of Medicine, Nagoya University, Nagoya, Japan
Kohei Matsuoka, Mizuki Yamada, Ryu Okada, Mikina Yoshida, Rika Fukushima, Kanna Umeda, Ayano Ohashi, Mitsuo Sato & Kazuhide Sato
Institute for Advanced Research, Advanced Analytical and Diagnostic Imaging Center (AADIC) / Medical Engineering Unit (MEU), Nagoya University, B3 Unit Frontier, Nagoya, Japan
Kohei Matsuoka, Mizuki Yamada, Ryu Okada, Mikina Yoshida, Rika Fukushima, Kanna Umeda, Ayano Ohashi, Ayako Kato, Misae Shimizu, Chiaki Koike & Kazuhide Sato
Institute for Advanced Research, Nagoya University, Nagoya, Japan
Ryu Okada & Kazuhide Sato
JSR corporation, Tokyo, Japan
Mibuko Shimada
Graduate School of Engineering, Nagoya University, Nagoya, 464-8603, Japan
Chiemi Oka & Akira Ito
FOREST-Souhatsu, JST, Tokyo, Japan
Kazuhide Sato
Institute for Advanced Research, Graduate School of Medicine, Nagoya University, Nagoya, 466-8550, Aichi, Japan
Kazuhide Sato

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Kohei Matsuoka
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Mizuki Yamada
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Ryu Okada
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Mibuko Shimada
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Mikina Yoshida
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Rika Fukushima
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Kanna Umeda
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Ayano Ohashi
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Chiemi Oka
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Ayako Kato
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Misae Shimizu
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Chiaki Koike
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Akira Ito
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Mitsuo Sato
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Kazuhide Sato
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Contributions

KM and YM contributed equally to this work. KM and YM mainly conducted the experiments. RO and KS interpreted the experimental results. MS produced the IONP conjugates. KM, RO, MY, RF, MY, KU, AO, CO, AK, MS, CK, and KS analyzed the data. CO and AI analyzed the material data. AI and MS aided with the experiments and use of devices. MS, CK, and CK helped with the animal studies. KM, RO, and KS wrote the manuscript. KS designed and supervised the whole study. All authors have approved the final version of the manuscript. The manuscript was written through contributions of all authors.

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Correspondence to Kazuhide Sato.

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All animal experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animal Resources of the Nagoya University Animal Care and Use Committee (Nagoya, Japan; approval numbers 2021, M210770-001; 2022, M220370-003; 2023, M230269-003; 2024, M240295-002; and 2025, M250149-003). The animals were maintained in accordance with the Act on Welfare and Management of Animals, Standards Relating to the Care and Keeping and Reducing Pain of Laboratory Animals, and Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions. This study complies with the ARRIVE 2.0 guidelines for animal research.

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Matsuoka, K., Yamada, M., Okada, R. et al. Synergistic nanotheranostic platform for fluorescence/magnetic resonance bimodal imaging-guided near infrared photoimmuno-chemodynamic thermal targeting therapy. J Nanobiotechnol 24, 975 (2026). https://doi.org/10.1186/s12951-026-05148-w

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Received: 05 June 2026
Accepted: 28 September 2026
Published: 14 October 2026
Version of record: 21 October 2026
DOI: https://doi.org/10.1186/s12951-026-05148-w

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Synergistic nanotheranostic platform for fluorescence/magnetic resonance bimodal imaging-guided near infrared photoimmuno-chemodynamic thermal targeting therapy

Abstract

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Introducution

Results

Production and characterization of nanomag-D-spio-pan-IR700

In vitro characterization of nanomag-D-spio-pan-IR700

In vivo biodistribution of nanomag-D-spio-pan-IR700

In vivo evaluation of antitumor effect with NIR-PICT3

In vivo evaluation of blood and tumor histology with NIR-PICT3

Mechanism of in vivo enhanced PTT in NIR-PICT3

Evaluation of in vivo photothermal bystander effect in NIR-PICT3

Discussion

Conclusions

Materials and methods

Experimental Design

Ethics statement

Reagents

Cell culture

Conjugation of IR700 to panitumumab

Conjugation and characterization of pan-IR700 to nanomag-D-spio

Fluorescence microscopy

Measurement of ·OH generation

ROS generation analysis

Ferrous iron detection

In vitro NIR-PICT3

Ferroptotic signaling evaluation

Apoptosis, pyroptosis, necrosis signaling evaluation

Animals and tumor models

In vivo NIR-PICT3

In vivo evaluation of nano drug delivery after NIR-PICT3

In vivo IR700 QD800 imaging

In vivo BLI

MRI

Serum mice blood chemistry

Tumor tissue staining

Statistical analysis

Data availability

Abbreviations

References

Acknowledgements

Funding

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Ethics approval

Consent for publication

Competing interests

Additional information

Publisher’s note

Supplementary Information

Supplementary Material 1 (download PDF )

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About this article

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Keywords

Profiles

In vivo evaluation of antitumor effect with NIR-PICT³

In vivo evaluation of blood and tumor histology with NIR-PICT³

Mechanism of in vivo enhanced PTT in NIR-PICT³

Evaluation of in vivo photothermal bystander effect in NIR-PICT³

In vitro NIR-PICT³

In vivo NIR-PICT³

In vivo evaluation of nano drug delivery after NIR-PICT³