Structurally Engineered Ferrous Metal–Organic Framework as a Chemodynamic Therapy Nanoagent for Concurrent Hydroxyl Radical and Singlet Oxygen Generation

Abstract

Chemodynamic therapy (CDT) is an emerging cancer treatment that employs transition metal–based nanoagents to catalyze the conversion of elevated intracellular hydrogen peroxide in malignant cells into cytotoxic hydroxyl radicals (•OH) via Fenton-like reactions. Recent developments have also introduced CDT agents that generate singlet oxygen (¹O₂) through the Russell mechanism. However, current nanoplatforms efficiently produce either •OH or ¹O₂, but not both, and often exhibit suboptimal catalytic activity, thereby limiting the sufficient production of reactive oxygen species (ROS) required for cancer eradication. This report introduces a ferrous metal–organic framework, Fe(II)-TCPP (tetrakis(4-carboxyphenyl)porphyrin), as the first nanoagent capable of simultaneously and effectively generating •OH and ¹O₂ through dual catalytic pathways. Its nanoneedle-like morphology increases the surface area and promotes enhanced ROS production. Cell studies demonstrated selective intracellular generation of •OH and ¹O₂ in cancer cells, resulting in targeted cytotoxicity while sparing non-malignant cells. Systemic administration of Fe(II)-TCPP in a breast cancer mouse model resulted in preferential tumor accumulation, robust intratumoral ROS generation, cancer eradication, and prevention of recurrence without systemic toxicity. These findings mark a foundational advance in CDT nanoagents by integrating Fenton and Russell mechanisms into a single platform, enabling the design of multifunctional catalysts with enhanced ROS output and therapeutic efficacy.

Graphical Abstract

Fe(II)-TCPP nanoneedles enable dual ROS generation via Fenton reaction and Russell mechanisms, achieving highly efficient chemodynamic therapy against breast cancer and excellent biosafety under a simple intravenous monotherapy regimen.

Introduction

The advent of nanotechnology has significantly expanded the landscape of cancer therapeutics, enabling the development of novel treatment strategies such as photothermal therapy (PTT), photodynamic therapy (PDT), and sonodynamic therapy (SDT).^[1–2] These nanomedicine-based modalities offer distinctive advantages, including tumor-localized activation and reduced systemic toxicity.^[3] However, their clinical application remains constrained by several key limitations. Notably, PTT, PDT, and SDT require external energy sources, such as light or ultrasound, which can increase treatment complexity and introduce variability in therapeutic outcomes.^{[1–2, 4]} Moreover, both PTT and PDT suffer from poor tissue penetration of light, which restricts their effectiveness against deep-seated tumors and limits their translational potential.^[4]

Chemodynamic therapy (CDT), first introduced in 2016,^[5] has gained attention as a novel ROS-mediated therapy that circumvents the need for exogenous stimuli. Like PDT and SDT, CDT eliminates tumor cells by generating ROS. However, CDT uniquely exploits endogenous biochemical reactions, most notably the Fenton reaction, under the acidic and hydrogen peroxide (H₂O₂)-rich conditions of the tumor microenvironment (TME), allowing for in situ ROS generation without external activation.^[6] This intrinsic activation mechanism in cancer cells, combined with the efficient tumor accumulation of CDT nanoagents via passive targeting, offers great therapeutic precision, while avoiding the reliance on external triggers required by PDT and SDT.^[4]

Nanoagents used in CDT typically include nanostructures that incorporate transition metals such as iron (Fe), copper (Cu), or manganese (Mn), which catalyze Fenton or Fenton-like reactions to generate cytotoxic hydroxyl radicals (•OH) within the TME.^[7–10] In this context, metal–organic frameworks (MOFs) with transition metals have recently emerged as a versatile class of nanozymes and catalytic platforms for CDT applications, owing to their tunable metal centers, porous architectures, and high catalytic accessibility.^[11–12] Recent advances have also introduced CDT agents capable of producing singlet oxygen (¹O₂) through alternative pathways, including the Russell mechanism^[13–14] and ClO− + H₂O₂ coupling.^[15] However, current CDT nanoagents efficiently generate either •OH or ¹O₂, but not both.^[16] In addition, many commonly used CDT agents, particularly dense or nonporous nanoparticles, have low specific surface area, which limits the accessibility of catalytic sites and reduces ROS generation efficiency.^[17] These limitations often result in suboptimal catalytic activity and insufficient ROS output for effective cancer treatment.^[18] Therefore, there is a critical need for CDT nanoagents with enlarged surface area and dual catalytic functionality capable of simultaneously generating both •OH and ¹O₂ with high efficiency. Notably, previous studies have demonstrated that combining CDT, which generates •OH, with PDT, which produces ¹O₂, results in synergistic anticancer effects.^[19–21] However, most reported synergistic PDT/CDT systems rely on external light irradiation and multi-modal therapeutic components to achieve dual-ROS generation, which inevitably increases treatment complexity.^[22–23] Moreover, the dependence of PDT on light activation limits the clinical applicability of this combinatorial PDT/CDT approach for internal tumors where light penetration is inadequate. In contrast, a single CDT nanoagent capable of concurrently generating •OH and ¹O₂ through endogenous reactions alone would offer a more streamlined therapeutic paradigm. Such a modality-free strategy has the potential to retain the benefits of dual-ROS synergy while eliminating the dependence on photodynamic activation, thereby simplifying treatment protocols and improving robustness in vivo.

Herein, we report the development of a novel metal–organic framework nanoagent, Fe(II)-TCPP, whose molecular structure enables the simultaneous generation of •OH and ¹O₂ through Fenton and Russell mechanisms under tumor-relevant conditions. In addition, its nanoneedle-like morphology provides an enlarged specific surface area that facilitates greater catalytic accessibility and improves overall ROS production efficiency. In contrast to previously reported CDT agents such as Cu-TCPP^[14] and Fe(III)-TCPP,^[24] which predominantly generate either ¹O₂ or •OH, respectively, Fe(II)-TCPP simultaneously produces both ROS species with greater catalytic efficiency than either control. Screening across ten cancer cell lines and two non-malignant cell lines demonstrated that Fe(II)-TCPP induces potent cytotoxicity in the majority of cancer cells while exhibiting negligible toxicity toward healthy cells. In vivo evaluation of Fe(II)-TCPP using a breast cancer xenograft model revealed its efficient tumor accumulation following systemic administration, leading to robust intratumoral ROS generation, complete tumor regression, and long-term prevention of recurrence, all without observable systemic toxicity. This therapeutic performance not only surpassed that of the tested Cu-TCPP but also outperformed all previously reported CDT nanoagents.^{[13–15, 25–45]} Our findings establish the first dual-mechanism CDT platform that integrates Fenton and Russell chemistry within a nanoneedle framework, representing a significant advance in the design of therapeutic nanomaterials with high ROS-generating capacity and superior anticancer efficacy.

2. Results and Discussion

2.1. Synthesis and Characterization of Fe(II)-TCPP

The MOF-based CDT nanoagent, Fe(II)-TCPP, was rationally designed to enable the concurrent generation of •OH and ¹O₂, leveraging the distinct catalytic capabilities of its components. It consists of ferrous ions (Fe²+) coordinated with 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (TCPP) ligands (Figure 1A). Fe²+ was selected over other transition metal ions due to its superior redox properties and favorable kinetics in Fenton-type reactions,^[46–47] enabling efficient •OH generation under the acidic and hydrogen peroxide–rich conditions typical of the TME. In the synthesized Fe(II)-TCPP, TCPP functions as both a structural and functional ligand, offering multiple coordination sites for metal ions while self-assembling into extended two-dimensional architectures that provide increased surface area.^[48] This morphology enhances access to catalytic sites and promotes efficient ROS generation. Moreover, previous studies have demonstrated that TCPP can coordinate with metal ions such as Cu²+ to form MOF structures capable of catalyzing ¹O₂ production via the Russell mechanism.^{[14, 49]} By integrating a Fenton-active center (Fe²+) with a TCPP scaffold known to support Russell-type catalysis, Fe(II)-TCPP combines both ROS-generating pathways within a single nanoplatform.

Figure 1. Structural and physicochemical characterization of Cu-TCPP, Fe(III)-TCPP, and Fe(II)-TCPP.

(A) Schematic illustration of the Fe(II)-TCPP synthesis strategy and resulting molecular structure. (B–F) Comparative analysis of Cu-TCPP, Fe(III)-TCPP, and Fe(II)-TCPP, including: (B) TEM images, (C) DLS size distribution, (D) zeta potential measurements, (E) UV–VIS absorption spectra, and (F) FTIR spectra. (G) High-resolution O 1s XPS spectra of TCPP and Fe(II)-TCPP. The black dashed lines indicate the characteristic binding energies of the two major oxygen species: C–OH and C=O. Green and purple curves correspond to the fitted C–OH and C=O components, respectively; the blue line represents the fitted baseline; the red line denotes the total fitted envelope; and gray dots show the experimental data. (H) XPS Fe 2p spectra of Fe(III)-TCPP and Fe(II)-TCPP for comparison of oxidation states.

Fe(II)-TCPP was synthesized using a nitrogen-assisted solvothermal method (Figure 1A), which, to our knowledge, has not been previously applied to MOF synthesis. This strategy has been successfully employed in the preparation of other Fe(II)-containing nanomaterials,^[50–51] as the nitrogen atmosphere plays a crucial role in preventing the oxidation of Fe²+ to Fe³+ during synthesis. In our approach, iron(II) chloride tetrahydrate (FeCl₂·4H₂O) and TCPP were dissolved in N,N-dimethylformamide (DMF), purged with nitrogen for 5 minutes, stirred for an additional 5 minutes, and then heated at 70°C for 2 hours. The resulting product was collected by centrifugation and washed with deionized water. The iron content of Fe(II)-TCPP was quantified using the ferrozine assay and determined to be 9.4 wt%.

To enable comparative evaluation of the structural and catalytic properties of Fe(II)-TCPP, two reference CDT nanoagents, Cu-TCPP and Fe(III)-TCPP, were synthesized using the same procedure but without the nitrogen purging step. Cu-TCPP, previously reported as an effective CDT agent, predominantly catalyzes ¹O₂ production via the Russell mechanism.^[14] Fe(III)-TCPP, which exhibits modest CDT activity through •OH generation,^[24] was included to directly assess how the oxidation state of iron influences the dual ROS-generating capability and catalytic performance of Fe(II)-TCPP.

Transmission electron microscopy (TEM) analysis revealed distinct morphologies among the synthesized CDT nanoagents (Figure 1B). Cu-TCPP formed extended two-dimensional sheetlike structures with an average length and hydrodynamic diameter of approximately 300 nm, as confirmed by TEM and dynamic light scattering (DLS) (Figure 1B and C). Fe(III)-TCPP assembled into uniform nanorods with dull ends, exhibiting an average length of approximately 400 nm and a DLS-measured size of about 600 nm. In contrast, Fe(II)-TCPP formed sharp, needlelike nanostructures with a significantly shorter length of approximately 100 nm and a hydrodynamic size of around 270 nm (Figure 1B and C). Although both Fe(II)-TCPP and Fe(III)-TCPP exhibited rod-like shapes, distinct morphological differences were evident. Fe(II)-TCPP showed pointed tips and a shorter length (Figure 1B and S1), whereas Fe(III)-TCPP formed longer, blunter rods lacking the sharp needlelike features (Figure 1B and S2).

All three CDT nanoagents displayed negatively charged surfaces, with zeta potentials of −11.2 mV for Fe(II)-TCPP, −18.8 mV for Fe(III)-TCPP, and −21.9 mV for Cu-TCPP, which can be attributed to the deprotonated carboxylic acid groups present in the TCPP structure (Figure 1D). The solution stability of Fe(II)-TCPP was evaluated by time-dependent TEM after incubation in water, PBS, cell culture medium, and serum for up to 15 days (Figure S3). Fe(II)-TCPP maintains its morphology in water, PBS, and culture medium without framework collapse or irreversible aggregation, while partial clustering in serum is attributed to protein adsorption. Overall, the nanostructures remain structurally intact under physiologically relevant conditions.

UV–visible (UV-VIS) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and O 1s X-ray photoelectron spectroscopy (XPS) were used to determine how metal ions are coordinated within the prepared TCPP-based CDT nanoagents. As shown in the UV–VIS spectra (Figure 1E), Cu-TCPP exhibited a characteristic single Q-band peak in the 500–700 nm range, consistent with the coordination of copper ions not only at the center of the porphyrin ring, but also acting as metal nodes to construct the MOF framework.^[52] In contrast, both Fe(II)-TCPP and Fe(III)-TCPP displayed four Q-band peaks, suggesting that both Fe²+ and Fe³+ serve primarily as metal nodes coordinated to the peripheral carboxyl groups of TCPP, rather than occupying the central porphyrin site.^[53] This coordination behavior was further supported by FTIR analysis (Figure 1F). The Cu–N stretching vibration in Cu-TCPP appeared at 999 cm−¹, while the N–H stretching vibration was observed at 966 cm−¹ for both Fe(III)-TCPP and Fe(II)-TCPP. These results indicate that copper ions are coordinated at the porphyrin core in addition to serving as structural nodes within the MOF network, whereas iron ions are coordinated externally as structural nodes.^[52] To further confirm that Fe²+ serves as a coordination node, high-resolution O 1s XPS spectra were collected for TCPP and Fe(II)-TCPP (Figure 1G). Peaks at 532.2 eV and 533.4 eV were assigned to the C=O and C–OH groups in TCPP, respectively. Upon formation of Fe(II)-TCPP, the intensity of the C–OH peak was significantly reduced, confirming coordination of Fe²+ to the carboxylic groups.^[54] The residual C–OH signal indicates that some carboxyl groups remain uncoordinated, consistent with the expected framework structure.

To confirm the oxidation states of iron in the synthesized CDT nanoagents, high-resolution XPS analysis of the Fe 2p region was conducted for both Fe(II)-TCPP and Fe(III)-TCPP (Figure 1H and S4). In Fe(III)-TCPP, the Fe 2p_3/2 and Fe 2p_1/2 peaks were observed at 711.8 eV and 725.4 eV, respectively, consistent with ferric iron (Fe³+).^[55] In comparison, Fe(II)-TCPP exhibited lower binding energies at 710.8 eV and 723.4 eV, consistent with the presence of ferrous iron (Fe²+).^[55] These spectral shifts indicate that Fe(II)-TCPP is rich in Fe²⁺, while Fe(III)-TCPP contains predominantly Fe³⁺. To further validate this result, the classical phenanthroline assay, specific for detecting Fe²+, was employed.^[56] As shown in Figure S5, Fe(II)-TCPP displayed a markedly stronger absorbance at 509 nm than Fe(III)-TCPP, providing additional evidence of its enriched Fe²+ content. Finally, to corroborate the Fe²⁺-enriched chemical environment in Fe(II)-TCPP, a ferrozine-based assay—highly selective for Fe^2+[57]—was additionally performed. As shown in Figure S6, Fe(II)-TCPP exhibited a pronounced ferrozine response in the visible region, whereas Fe(III)-TCPP showed a negligible signal under identical conditions. This result indicates substantially higher Fe²⁺ accessibility in Fe(II)-TCPP compared to Fe(III)-TCPP. Together with XPS and phenanthroline analysis, these results consistently demonstrate that Fe(II)-TCPP is enriched in ferrous iron species, a critical feature for maximizing Fenton reactivity and •OH generation.

Room-temperature ^57Fe Mössbauer spectroscopy was conducted to examine the iron coordination environment in Fe(II)-TCPP (Figure S7). The absence of sextet features or magnetic splitting confirms that iron exists predominantly as highly dispersed, coordination-bound species, effectively excluding magnetically ordered iron oxides or metallic iron impurities.^[58]

X-ray diffraction (XRD) analysis revealed notable structural differences between Fe(II)-TCPP and Fe(III)-TCPP (Figure S8). Fe(III)-TCPP displayed a broad diffraction peak centered at approximately 2θ ≈ 15.4°, consistent with a predominantly amorphous structure.^[59] In contrast, Fe(II)-TCPP exhibited a distinct peak near 2θ ≈ 87.6°, indicating partial crystallinity and enhanced structural ordering.^[60]

Taken together, the combined results from TEM, DLS, XPS, FTIR, XRD, and UV–VIS spectroscopy confirm the successful synthesis of a structurally and chemically distinct Fe(II)- based MOF. Unlike Fe(III)-TCPP, Fe(II)-TCPP features ferrous ions as the primary coordination nodes and exhibits enhanced structural order and well-defined nanoneedle morphology, establishing it as a novel CDT nanoagent platform with unique physicochemical characteristics.

2.2. Evaluation of ROS Generation Efficiency of Fe(II)-TCPP in Solution

Previous studies have shown that Cu-TCPP primarily generates ¹O₂ through the Russell mechanism, whereas Fe(III)-TCPP predominantly produces •OH through the Fenton reaction.^{[14, 61]} In contrast, Fe(II)-TCPP was specifically designed to support both ROS-generation pathways, and its needlelike morphology should provide a high surface area to enhance catalytic efficiency. Based on these features, we anticipated that Fe(II)-TCPP would generate both •OH and ¹O₂ under mildly acidic, hydrogen peroxide–rich conditions with higher efficiency than either Cu-TCPP or Fe(III)-TCPP.

The generation efficiency of ¹O₂ and •OH by Fe(II)-TCPP was compared with that of Cu-TCPP and Fe(III)-TCPP under identical conditions. All three nanoagents were evaluated in phosphate-buffered saline (PBS, pH 5.5) containing H₂O₂ to mimic the TME.^[62] Fluorescence-based assays were performed using hydroxyphenyl fluorescein (HPF) and singlet oxygen sensor green (SOSG) to detect •OH and ¹O₂, respectively.^[63] Additionally, electron spin resonance (ESR) spectroscopy with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidine (TEMP) spin-trapping agents was used to validate the generation of •OH and ¹O₂, respectively.

Fluorescence-based ROS assays confirmed distinct reactivity profiles for the three nanoagents. As expected, Fe(III)-TCPP produced higher levels of •OH compared to Cu-TCPP (Figure 2A), while Cu-TCPP generated markedly greater amounts of ¹O₂ than Fe(III)-TCPP (Figure 2B). These findings are consistent with previous reports, reinforcing that Fe(III)-TCPP primarily supports Fenton-type •OH production, whereas Cu-TCPP favors Russell-type ¹O₂ generation.^{[14, 24]} Remarkably, Fe(II)-TCPP outperformed Cu-TCPP and Fe(III)-TCPP by efficiently producing both ROS species at substantially higher levels (Figure 2A and B).

Figure 2. Detection of ROS generated by Cu-TCPP, Fe(III)-TCPP, and Fe(II)-TCPP in solution.

(A–B) Fluorescence detection of •OH (A) and ¹O₂ (B) using HPF and SOSG probes, respectively, in PBS (pH 5.5) containing 1 mM H₂O₂ and the indicated CDT nanoagent. (C–D) ESR spectra of •OH and ¹O₂ detected using DMPO and TEMP as spin-trapping agents, respectively, in PBS (pH 5.5) containing 10 mM H₂O₂ and the indicated CDT nanoagent. All nanoagents were tested at a concentration of 20 μg/mL. Controls included H₂O₂ alone, as well as Cu-TCPP, Fe(III)-TCPP, and Fe(II)-TCPP in PBS (pH 5.5) without H₂O₂. (E) Schematic representation of Fe(II)-TCPP–mediated Fenton and Russell reactions generating •OH and ¹O₂.

ESR spectroscopy further corroborated the dual ROS-generating capability of Fe(II)-TCPP. Compared to Cu-TCPP and Fe(III)-TCPP, Fe(II)-TCPP exhibited markedly stronger and well-resolved ESR signals, including a distinct 1:2:2:1 quartet peak from DMPO–OH adducts^[64] (Figure 2C) and a characteristic 1:1:1 triplet peak from TEMP-derived nitroxide^[65] (Figure 2D), confirming efficient generation of both •OH and ¹O₂. Collectively, these results demonstrate that Fe(II)-TCPP enables robust, simultaneous generation of multiple reactive oxygen species, achieving higher production levels than both Fe(III)-TCPP and Cu–TCPP.

Additional experiments demonstrated that the ROS generation efficiency of Fe(II)-TCPP is influenced by both pH and reaction time (Figure S9 and S10). Generation of both •OH and ¹O₂ was markedly enhanced at pH 5.5 and progressively declined at pH 6.5 and 7.4. In addition, prolonged incubation times resulted in progressively increased ROS levels, indicating that Fe(II)-TCPP maintains robust catalytic function over time.

Based on prior reports^{[14, 24]} and our experimental findings (Figure 2 and S9), we propose that the developed Fe(II)-TCPP generates •OH and ¹O₂ via Fenton- and Russell-type pathways, respectively (Figure 2E).

The Fenton reaction is a well-established iron-catalyzed process in which ferrous ions (Fe²+) react directly with H₂O₂ to produce highly reactive •OH.^[66] This reaction proceeds most efficiently under acidic conditions, where the reaction rate and radical yield are significantly higher than at neutral pH.^[67]

$$\mathbf{\text{Fenton}}\mathbf{\text{reaction}}:{\text{Fe}}^{2+}+{\text{H}}_2{\text{O}}_2+{\text{H}}^{+}\to {\text{Fe}}^{\text{3+}}+{\text{H}}_2\text{O}+•\text{OH}$$

Consistent with this, our data provides strong evidence that Fe(II)-TCPP mediates •OH generation through a Fenton-like mechanism. In PBS at pH 5.5, the addition of H₂O₂ to Fe(II)-TCPP led to a pronounced increase in fluorescence of the •OH-specific probe HPF, whereas minimal and comparable signals were observed with Fe(II)-TCPP or H₂O₂ alone (Figure 2A). These results were further validated by ESR spectroscopy using the •OH-specific spin-trapping agent DMPO (Figure 2C). Importantly, we observed strong pH dependence of this process: robust •OH generation at pH 5.5 with a progressive decline at pH 6.5 and 7.4 (Figure S9A), consistent with the established pH profile of classical Fenton chemistry.^[68] Although the ferrous ions in Fe(II)-TCPP are framework-bound and thus protected from precipitation typical of free iron salts, acidic conditions still favor the reaction by maintaining H₂O₂ in its neutral, more reactive form, preserving a favorable Fe²+/Fe³+ redox potential, and stabilizing the protonation state of the porphyrin–carboxylate environment, thereby enhancing substrate accessibility to the catalytic sites.^[69] Collectively, these findings support that Fe(II)-TCPP functions as a Fenton-like catalyst, cycling between Fe²+ and Fe³+ states to decompose H₂O₂ and generate •OH. Many ferrous-based nanoagents have been reported to generate hydroxyl radicals via the Fenton reaction under comparable conditions,^[70] which is in full agreement with our findings and further corroborates that Fe(II)-TCPP produces •OH through a Fenton-like pathway.

In the classical Russell mechanism, organic substrates are first oxidized to hydroperoxides (ROOH), which then undergo redox reactions to generate peroxyl radicals (ROO•). Two such radicals recombine to form an unstable tetroxide (ROOOOR) intermediate that decomposes, releasing ¹O₂.^[71–72]

$$\mathbf{\text{Russell}}\mathbf{\text{mechanism}}:\text{2ROH}\stackrel{{\text{H}}_2{\text{O}}_2+{\text{H}}^{+}}{\to }\text{2ROOH}\stackrel{\text{Fe}\left(\text{II}\right){\text{-TCPP+H}}^{\text{+}}}{\to }\text{2ROO•}\to \text{ROOOOR}\to 2^1{\text{O}}_2$$

We attribute the ¹O₂ production observed for Fe(II)-TCPP in the presence of H₂O₂ at an acidic pH of 5.5 to this Russell-type pathway, consistent with the mechanism previously reported for Cu-TCPP MOFs, which are built with the same TCPP ligand.^[14] Wang et al. experimentally demonstrated that at pH 5.5, the benzoic acid groups of TCPP ligands in Cu-TCPP can be peroxidized by H₂O₂ into perbenzoic acid species, which was strongly pH-dependent and most pronounced under acidic conditions.^[14]

In XPS experiments (Figure 1G), we demonstrated that not all TCPP ligands in Fe(II)-TCPP bind to Fe²⁺ via their carboxyl groups; a fraction of TCPP ligands contain free carboxyl groups, which form the basis for TCPP peroxidation (Figure 2E). Furthermore, common lipids (such as phospholipids) in cells may also be oxidized via the same pathway to form lipid hydroperoxides in an H₂O₂-abundant and acidic environment,^{[13, 72]} which may participate in subsequent reactions. The peroxidized benzoic acid groups of TCPP in the Fe(II)-TCPP or lipid hydroperoxide could be converted into ROO• species by the trace amount of catalytic Fe²⁺, which is also from Fe(II)-TCPP.^[72] Subsequently, toxic ¹O₂ can be produced in the spontaneous recombination reaction of two ROO• species through the Russell mechanism (Figure 2E).^[72] When we replaced Fe(II)-TCPP with free TCPP and Fe²+ ions in a solution under identical H₂O₂ and pH 5.5 PBS conditions, robust ¹O₂ generation was observed (Figure S11), whereas no ¹O₂ occurred in the absence of Fe²+. These results suggest that the Fe²+ centers in Fe(II)-TCPP are the primary catalytic sites and function equivalently to free Fe²+ ions.

Importantly, our fluorescence (SOSG probe) and ESR (TEMP spin trap) studies were performed under the same conditions (pH 5.5 and H₂O₂ concentration) as those reported by Wang et al., and similarly demonstrated that Fe(II)-TCPP produces substantial ¹O₂ only when combined with H₂O₂, whereas Fe(II)-TCPP or H₂O₂ alone showed negligible signal (Figure 2B and D). Moreover, we observed a clear pH dependence of this process, with ¹O₂ production highest at pH 5.5 and progressively decreasing at pH 6.5 and 7.4 (Figure S9B). Together, these findings support that Fe(II)-TCPP undergoes ligand peroxidation under acidic H₂O₂ conditions, forming peroxyl radicals that recombine via the Russell mechanism to release ¹O₂.

To quantitatively assess the catalytic kinetics of •OH and ¹O₂ generation by Cu-TCPP, Fe(III)-TCPP, and Fe(II)-TCPP, a 3,3′,5,5′-tetramethylbenzidine (TMB) chromogenic assay was employed in the presence of specific ROS scavengers: sodium azide (NaN₃) for ¹O₂ and isopropanol for •OH.^[14] The reaction kinetics were analyzed using the Michaelis–Menten model to determine the catalytic efficiencies (k_cat·mass/K_m, the core indicator of overall catalytic efficiency) of the CDT nanoagents.^[73] As shown in the Michaelis–Menten plots (Figure 3A) and summarized in the kinetic parameters table (Figure 3B), Fe(II)-TCPP demonstrated the highest catalytic efficiency for •OH generation, with a (k_cat·mass/K_m) value of 3.871 L·mg−¹·min−¹, exceeding Cu-TCPP (0.208 L·mg−¹·min−¹) and Fe(III)-TCPP (0.475 L·mg−¹·min−¹) by 18.6- and 8.2-fold, respectively. Fe(II)-TCPP also demonstrated superior catalytic performance for ¹O₂ generation, with a catalytic efficiency (k_cat·mass/K_m) of 0.575 L·mg−¹·min−¹. This was 1.3-fold higher than Cu-TCPP (0.435 L·mg−¹·min−¹) and 4.6-fold higher than Fe(III)-TCPP (0.126 L·mg−¹·min) (Figure 3C and D). These findings not only support the fluorescence assays and ESR results (Figure 2) demonstrating that Fe(II)-TCPP is a dual-pathway CDT nanoagent with exceptional efficiency in generating both •OH and ¹O₂, but also reinforce the distinct mechanistic roles of the control nanoagents. Specifically, Fe(III)-TCPP exhibited significantly higher catalytic efficiency for •OH generation (0.475 L·mg−¹·min−¹) than Cu-TCPP (0.208 L·mg−¹·min−¹), consistent with its Fenton-dominated activity. In contrast, Cu-TCPP showed superior efficiency in ¹O₂ generation (0.435 L·mg−¹·min−¹) compared to Fe(III)-TCPP (0.126 L·mg−¹·min−¹), supporting its Russell-type catalytic behavior.

Figure 3. Catalytic kinetics of ROS generation by Cu-TCPP, Fe(III)-TCPP, and Fe(II)-TCPP.

(A) Michaelis–Menten curves for •OH generation. (B) Comparison of kinetic parameters for •OH generation. (C) Michaelis–Menten curves for ¹O₂ generation. (D) Comparison of kinetic parameters for ¹O₂ generation. Reactions were conducted in PBS (pH 5.5) containing 0.8 M H₂O₂ and varying concentrations of TMB (0–2 mM). Error bars represent the standard error of the mean from three independent experiments. v is the initial reaction velocity. [E] is the concentration of the CDT nanoagents. K_m is the Michaelis constant, ν_max is the maximal reaction velocity, and k_cat·mass is the catalytic constant, where k_cat·mass = ν_max/[E], and the k_cat·mass /K_m value indicates the catalytic efficiency of the nanoagents.

The time-dependent absorbance curves, zoomed-in linear segments, Michaelis–Menten plots, and Lineweaver–Burk plots collectively support that all three CDT agents follow classic enzyme-like kinetics, as evidenced by their well-fitted hyperbolic and linear profiles (Figures S12 and S13).^[74] The superior performance of Fe(II)-TCPP may be attributed to its stronger redox reactivity and efficient Fenton-like behavior.^[46–47] The relatively low Michaelis constant (K_m) and high maximal reaction velocity (v_max) values are critical indicators for CDT because they reflect the nanocatalyst’s efficiency to generate ROS in the TME. A low K_m reflects a strong substrate affinity, allowing Fe(II)-TCPP to efficiently catalyze ROS production even at the low endogenous H₂O₂ concentrations typically found in cancer tissues (~50–100 μM), compared to ~20 nM in healthy cells.^[75] Meanwhile, a high v_max indicates rapid catalytic velocity, supporting sustained and robust generation of cytotoxic •OH and ¹O₂ within biologically relevant timeframes.^[73] Fe(II)-TCPP has the highest v_max value among the three nanoagents. Collectively, these findings suggest that Fe(II)-TCPP possesses the optimal catalytic profile for effective CDT, capable of achieving high ROS yields under physiological conditions relevant to disease environments.

2.3. In Vitro Evaluation of ROS Generation and CDT Efficacy of Fe(II)-TCPP

To assess the therapeutic potential of the synthesized nanoagents, intracellular ROS generation and CDT anticancer efficacy were investigated for Fe(II)-TCPP, Cu(II)-TCPP, and Fe(III)-TCPP across a panel of ten cancer cell lines and two non-malignant control cell lines. The cell line panel and corresponding details are summarized in Table S1.

Intracellular ROS detection was performed using 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) as a fluorescence probe for total ROS measurement, while specific detection of •OH and ¹O₂ was achieved using HPF and SOSG probes, respectively.

The results revealed that Fe(II)-TCPP induced significantly higher levels of intracellular total ROS than either Cu-TCPP or Fe(III)-TCPP in seven of the ten tested cancer cell lines (Figure S14). This enhanced ROS generation was particularly pronounced in the triple-negative breast cancer cell line MDA-MB-231, where Fe(II)-TCPP treatment led to a 5.7-fold increase in total ROS levels at 20 μg/mL (Figure 4B) compared with the control group (0 μg/mL). In comparison, the same concentration of Cu-TCPP and Fe(III)-TCPP resulted in only 2.5-fold and 1.4-fold increases, respectively (Figure 4B). Notably, this trend persisted across the tested concentration range (2.5–100 μg/mL), with Fe(II)-TCPP consistently inducing substantially higher intracellular ROS levels than either of the control agents (Figure 4B). As the concentration increased, the disparity in ROS generation widened, further emphasizing the superior catalytic activity of Fe(II)-TCPP in promoting oxidative stress within cancer cells and its greater ROS-generating capacity among the two control nanoagents. Live-cell fluorescence imaging further corroborated the above-discussed results, visually confirming the enhanced intracellular ROS production induced by Fe(II)-TCPP when compared to Cu-TCPP and Fe(III)-TCPP (Figure S15).

Figure 4. In vitro evaluation of intracellular ROS generation and therapeutic efficacy of CDT nanoagents in non-malignant and cancer cell lines.

(A–B) Quantification of intracellular total ROS levels in MCF-10A non-malignant cells (A) and MDA-MB-231 cancer cells (B) using the DCFH-DA fluorescent probe. (C–D) Quantification of intracellular •OH levels using the HPF fluorescent probe (C) and ¹O₂ levels using the SOSG fluorescent probe (D) in MDA-MB-231 cells. For ROS detection assays, cells were treated with Cu-TCPP, Fe(III)-TCPP, or Fe(II)-TCPP at concentrations ranging from 0 to 100 μg/mL for 24 h. RFI: relative fluorescence intensity. (E–F) Dose-response curves showing viability of MCF-10A (E) and MDA-MB-231 (F) cells assessed 24 h after treatment with Cu-TCPP, Fe(III)-TCPP, and Fe(II)-TCPP at concentrations from 0 to 100 μg/mL. Curves represent nonlinear regression analysis used to determine IC₅₀ values for each treatment condition. (G) Summary of IC₅₀ values for Cu-TCPP, Fe(III)-TCPP, and Fe(II)-TCPP in two non-malignant cell lines (HEK293 and MCF-10A) and ten cancer cell lines after 24 h treatment. Data are presented as mean ± SD (n = 6). Statistical significance was determined by two-way ANOVA followed by Tukey’s post-hoc test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

To further dissect the contributions of individual ROS, intracellular levels of •OH and ¹O₂ were assessed in MDA-MB-231 cells using HPF and SOSG probes, respectively. In agreement with the total ROS measurements, Fe(II)-TCPP significantly elevated intracellular levels of both •OH and ¹O₂ across the tested concentration range (0–100 μg/mL), outperforming both Cu-TCPP and Fe(III)-TCPP (Figure 4C and D). For example, at a concentration of 20 μg/mL, Fe(II)-TCPP increased intracellular •OH and ¹O₂ by 3.7- and 3.9-fold, respectively, relative to untreated cells, indicating its ability to efficiently promote both Fenton and Russell-type ROS generation pathways. In contrast, Cu-TCPP increased •OH and ¹O₂ by only 1.1- and 1.8-fold, while Fe(III)-TCPP raised these levels by 1.3- and 1.2-fold, respectively. The disproportionate increases in ¹O₂ and •OH production by Cu-TCPP and Fe(III)-TCPP, respectively, further support their preferential engagement in Russell-type (Cu-TCPP) and Fenton-type (Fe(III)-TCPP) pathways. The distinct ROS generation patterns observed for Fe(II)-TCPP, Cu-TCPP, and Fe(III)-TCPP remained consistent across all tested concentrations (Figure 4C and D). Overall, the observed intracellular ROS-generating trends align with the solution-based findings (Figure 2 and 3), demonstrating that Fe(II)-TCPP comparably produces both •OH and ¹O₂ at significantly higher levels than the control nanoagents.

Live-cell fluorescence imaging further confirmed that Fe(II)-TCPP primarily generates •OH and ¹O₂ in MDA-MB-231 cancer cells. Fluorescence microscopy with the total ROS probe DCFH-DA showed a pronounced and comparable decrease in cellular fluorescence following co-treatment with either NaN₃ (¹O₂ scavenger) or isopropanol (•OH scavenger). Notably, simultaneous application of both scavengers nearly abolished fluorescence, demonstrating that both ¹O₂ and •OH are major contributors to ROS generation by Fe(II)-TCPP (Figure S16). In contrast, Cu-TCPP mainly produced ¹O₂, while Fe(III)-TCPP produced relatively more •OH (Figure S16).

Notably, total ROS measurements revealed that Fe(II)-TCPP did not increase intracellular ROS levels across the tested concentration range (0–100 μg/mL) in non-malignant human cell lines, including the mammary epithelial MCF-10A and embryonic kidney HEK293 cells (Figure 4A and S14). This selective ROS activation underscores the cancer-specific responsiveness of Fe(II)-TCPP, in stark contrast to the control CDT nanoagents. Both Cu-TCPP and Fe(III)-TCPP induced concentration-dependent increases in ROS in non-malignant cells, with Fe(III)-TCPP exhibiting a notably higher effect (Figure 4A and S14). For example, in MCF-10A cells at 20 μg/mL, Fe(III)-TCPP increased ROS levels by 2.1-fold, reaching a maximum 4.2-fold increase at 80 μg/mL (Figure 4B). Cu–TCPP led to a lower ROS elevation in the same cells, with a 1.2-fold increase at 20 μg/mL and 3.4-fold at 80 μg/mL. These results highlight the superior selectivity of Fe(II)-TCPP, which efficiently generates ROS in cancer cells while sparing healthy cells, a desirable feature for minimizing off-target oxidative damage in therapeutic applications.

Flow cytometry analysis using the DCFH-DA probe was further performed to validate intracellular ROS generation at the single-cell level in non-malignant (MCF-10A) and cancer (MDA-MB-231) cells (Figure S17). In line with the above-discussed results (Figure 4A, B, and S15), treatment with Fe(II)-TCPP (25 μg/mL) resulted in a 6.8-fold increase in mean fluorescence intensity (MFI) in MDA-MB-231 cancer cells compared to untreated controls (Figure S17H). By contrast, Cu-TCPP and Fe(III)-TCPP induced considerably weaker responses in cancer cells, with MFI increases of only 2.0-fold and 1.6-fold, respectively. Crucially, Fe(II)-TCPP did not elevate ROS levels in non-malignant MCF-10A cells, with MFI remaining at baseline levels (Figure S17D), demonstrating its cancer-selective activity. Similarly, Cu-TCPP showed minimal effect on healthy cells (1.1-fold increase in MFI). In contrast, Fe(III)-TCPP induced a marked 2.7-fold increase in MFI in MCF-10A cells, confirming its lack of selectivity as observed in the fluorescence-based ROS quantification assays. Together, these data provide quantitative single-cell evidence that Fe(II)-TCPP combines potent ROS-generating efficacy in cancer cells with a favorable selectivity profile that spares non-malignant cells.

To further evaluate the selective anticancer activity of Fe(II)-TCPP, half-maximal inhibitory concentrations (IC₅₀) were determined 24 hours post-treatment across both cancer and non-malignant cell lines and compared with Cu-TCPP and Fe(III)-TCPP (Figure 4E–G and S18). Among the three CDT nanoagents tested, Fe(II)-TCPP demonstrated superior cytotoxicity, showing significantly enhanced anticancer activity in 8 out of 10 cancer cell lines compared to the other formulations (Figure 4G and S18). Fe(II)-TCPP exhibited the most potent anticancer efficacy against human triple-negative breast cancer (MDA-MB-231), hepatocellular carcinoma (HepG2), and prostate carcinoma (DU-145) cell lines, with IC₅₀ values of 8.96, 8.62, and 6.62 μg/mL, respectively. Cu-TCPP exhibited moderate therapeutic efficacy, achieving its lowest IC₅₀ value of 16.8 μg/mL in MDA-MB-231 cells, which was 1.9-fold higher than that of Fe(II)-TCPP (8.96 μg/mL) (Figure 4F and G). In contrast, Fe(III)-TCPP demonstrated limited anticancer activity, with IC₅₀ values exceeding the maximum tested concentration of 100 μg/mL in 6 out of 10 cancer cell lines, including MDA-MB-231 (Figure 4F and G). These results establish Fe(II)-TCPP as the most potent CDT agent for multiple cancers among the tested formulations.

Importantly, Fe(II)–TCPP exhibited minimal cytotoxicity toward non-malignant cells, including MCF-10A and HEK293, with no significant decrease in viability observed at concentrations up to 60 μg/mL, and even at the highest tested dose, viability was reduced by only ~24.6% in MCF-10A cells and 20.9% in HEK293 cells (Figure 4E, G, and S18). In contrast, Cu–TCPP exhibited moderate cytotoxicity, with IC₅₀ values of 72.5 μg/mL in MCF-10A and 84.3 μg/mL in HEK293 (Figure 4E, G, and S18). Notably, Fe(III)–TCPP was the most toxic to normal cells, with IC₅₀ values of 10.8 μg/mL and 16.0 μg/mL in MCF-10A and HEK293, respectively. These findings closely align with the intracellular ROS data, reinforcing that Fe(II)–TCPP efficiently and selectively induces robust ROS production in cancer cells while sparing normal cells. Conversely, Fe(III)–TCPP was associated with elevated ROS levels and cytotoxicity in non-malignant cells but showed limited ROS generation and therapeutic efficacy in most cancer lines. Finally, the hemocompatibility of Fe(II)–TCPP and Cu–TCPP was evaluated to further assess their safety profiles. Consistent with their minimal cytotoxicity in non-malignant cells, both nanoagents demonstrated acceptable blood compatibility across the entire tested concentration range (10–100 μg/mL, Figure S19). Notably, Fe(II)–TCPP exhibited markedly superior hemocompatibility, with a hemolysis rate of only 4% at 100 μg/mL, below the commonly accepted 5% threshold for blood compatibility (according to the American Society for Testing and Materials, less than 5% hemolysis is considered null).^[76] In contrast, Cu–TCPP induced a significantly higher hemolysis rate of 10.2% at the same concentration (Figure S19). These findings reinforce the excellent biocompatibility of Fe(II)–TCPP and further support its promise as a selective and safe CDT nanoagent.

2.4. In Vivo Evaluation of ROS Generation and CDT Efficacy of Fe(II)-TCPP

Given the superior in vitro ROS generation and cytotoxicity observed for Fe(II)-TCPP and Cu-TCPP in cancer cells, and following the 3R principles (Replacement, Reduction, and Refinement) for ethical animal research,^[77] we restricted in vivo testing to these two nanoagents, excluding Fe(III)-TCPP due to its comparatively lower efficacy. Among the ten cancer cell lines evaluated, Cu-TCPP demonstrated the highest anticancer potency, with the lowest IC₅₀ value of 16.8 μg/mL observed in MDA-MB-231 triple-negative breast cancer (TNBC) cells (Figure 4G). As TNBC represents a highly aggressive and therapeutically resistant subtype, and Cu-TCPP demonstrated its strongest in vitro activity in this model, evaluating Fe(II)-TCPP alongside Cu-TCPP in the MDA-MB-231 animal model provides a rigorous and meaningful benchmark for assessing its therapeutic potential. Therefore, the antitumor efficacy of both Fe(II)-TCPP and Cu-TCPP was evaluated in mice bearing MDA-MB-231 xenografts in the mammary fat pad.

A previous study has demonstrated that Cu-TCPP efficiently accumulates in mouse cancer xenografts 24 hours after intravenous (IV) injection.^[14] Accordingly, we first assessed the biodistribution of Fe(II)-TCPP in mice bearing MDA-MB-231 xenografts 24 hours post-IV administration to ensure its tumor accumulation before evaluating therapeutic efficacy. Prussian blue staining of excised tissues revealed prominent Fe(II)-TCPP accumulation in both tumor and spleen tissues (Figure 5A). These findings were further confirmed by quantitative iron measurements in tissues using the ferrozine assay, which revealed the high iron accumulation in the spleen (115.5 ± 12.3 μg/g) and tumor (57.6 ± 4.5 μg/g) (Figure 5B), significantly exceeding the levels observed in the control group (30.1 ± 4.3 μg/g in spleen and 13.5 ± 3.1 μg/g in tumor). In contrast, iron concentrations in the liver, kidneys, lungs, and heart were comparable to those of untreated controls, indicating no off-target accumulation in these major organs. The efficient tumor accumulation of Fe(II)-TCPP is likely mediated by the enhanced permeability and retention (EPR) effect, while the elevated splenic uptake is attributed to the negative surface charge of Fe(II)-TCPP, promoting recognition and clearance by the reticuloendothelial system.^[78]

Figure 5. In vivo evaluation of biodistribution and intratumoral ROS detection of Fe(II)-TCPP.

(A) Prussian blue-stained histological sections of tumors and organs resected from mice bearing MDA-MB-231 xenografts 24 h post-IV injection of PBS (control) and Fe(II)-TCPP at a dose of 4 mg/kg (n=3 per group). Dark blue staining indicates iron deposits. (B) Iron content (μg) per gram of tissue from samples described in (A), quantified by ferrozine assay (Fig. S16). Data are presented as mean ± SD (n = 3). Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc test (*p < 0.05, ****p < 0.0001). (C) Fluorescence imaging of live mice with MDA-MB-231 xenografts 24 h post-IV injection of PBS (control), Cu(II)-TCPP, and Fe(II)-TCPP (4 mg/kg for each nanoagent). Whole-body fluorescence images were recorded before (“-DCFH-DA”) and 15 min after (“+DCFH-DA”) intratumoral injection of DCFH-DA fluorescence probe for total ROS detection. Tumors are outlined with red circles. (D) Quantification of fluorescence intensity generated by DCFH-DA fluorescence probe in tumors from (C). Data are presented as mean ± SD (n = 3). Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc test (****p < 0.0001).

To investigate the in vivo ROS-generating capability of the nanoagents, we evaluated intratumoral ROS levels in MDA-MB-231 xenografts following systemic administration of Fe(II)-TCPP or Cu-TCPP. Twenty-four hours after IV injection of the nanoagents, the fluorescence probe DCFH-DA for the total ROS detection was directly injected into the tumors. As shown in Figure 5C, both Fe(II)-TCPP and Cu-TCPP induced detectable fluorescence within the tumor tissue, indicating successful intratumoral ROS production. Notably, tumors treated with Fe(II)-TCPP exhibited markedly stronger fluorescence compared to those treated with Cu-TCPP. Quantitative analysis revealed that Fe(II)-TCPP induced a 6.6-fold increase in fluorescence relative to untreated controls, whereas Cu-TCPP induced only a 2.2-fold increase (Figure 5D), indicating that Fe(II)-TCPP generated approximately three times more intratumoral ROS than Cu-TCPP. These results are consistent with our in vitro findings in MDA-MB-231 cells (Figure 4B) and further support the superior ROS-generating efficiency of Fe(II)-TCPP within the TME. To further evaluate the tissue selectivity of in vivo ROS generation by Fe(II)-TCPP, we examined ROS levels in the spleen, a major reticuloendothelial organ in which Fe(II)-TCPP accumulates following systemic administration. As shown in Figure S21, no significant increase in ROS-associated fluorescence was detected in splenic tissue from Fe(II)-TCPP–treated mice compared with control animals. Quantitative analysis confirmed comparable fluorescence intensities between the two groups.

These findings indicate that although Fe(II)-TCPP accumulates in the spleen, it does not induce appreciable ROS generation in this tissue. This observation is consistent with the absence of the acidic and H₂O₂-rich microenvironment required to activate Fenton- or Russell-type chemodynamic reactions outside the tumor milieu. Collectively, the pronounced intratumoral ROS generation together with negligible ROS activation in the spleen demonstrates tumor-selective CDT activation and a favorable in vivo biosafety profile of Fe(II)-TCPP, supporting its therapeutic potential for chemodynamic therapy.

To evaluate the therapeutic efficacy of both CDT nanoagents, orthotopic breast cancer models were established by injecting 2.5 million MDA-MB-231 cells into the fourth mammary fat pad of female mice. Fourteen days post-inoculation, mice received IV administrations of either PBS (control), Fe(II)-TCPP, or Cu-TCPP at an equivalent dose of 4 mg/kg every third day for a total of four injections (days 1, 4, 7, and 10) (Figure 6A). Remarkably, all tumors in the Fe(II)-TCPP-treated group completely regressed by day 10 following treatment initiation (Figure 6C). In comparison, maximized tumor regression was delayed in the Cu-TCPP group, occurring by day 13 in only 3 of 5 mice (Figure 6C). To assess long-term therapeutic durability and potential relapse, animals were monitored for another 30 days after the last injection of the CDT agents. By day 28, no tumor regrowth was observed in either treatment group; however, recurrence was noted by day 40 in one mouse per group (Figure 6C and S22). Ultimately, four of five mice in the Fe(II)-TCPP group remained tumor-free, compared to two of five in the Cu-TCPP group (Figure 6B). Final tumor volumes at day 40 averaged 555.9 mm³ in the control group, 54.5 mm³ in the Cu-TCPP group, and only 7.6 mm³ in the Fe(II)-TCPP group (Figure 6C), corresponding to tumor inhibition rates of 90.2% and 98.6% for Cu-TCPP and Fe(II)-TCPP, respectively. Post-mortem tumor excision (Figure 6D) and weighing confirmed these findings: the average tumor mass was 395.6 mg in controls, 31.8 mg in Cu-TCPP-treated mice, and only 2.0 mg in Fe(II)-TCPP-treated mice (Figures 6E), reflecting inhibition rates of 92.0% and 99.5% for Cu-TCPP and Fe(II)-TCPP, respectively. Hematoxylin and eosin (H&E) staining of collected tumor tissues showed intact morphology and dense cellularity in the control group (Figure S23). Cu-TCPP treatment induced mild cellular disruption and cytoplasmic vacuolization, indicating limited tumoricidal effect. In contrast, Fe(II)-TCPP treatment led to extensive structural disintegration, nuclear condensation and fragmentation, and widespread necrosis, demonstrating potent antitumor efficacy in vivo (Figure S23). Together, these data demonstrate the superior and durable therapeutic efficacy of Fe(II)-TCPP over Cu-TCPP, establishing its potential as a highly effective CDT agent.

Figure 6. In vivo evaluation of CDT efficacy mediated by Fe(II)-TCPP and Cu(II)-TCPP.

(A) Experimental design: Mice were inoculated with MDA-MB-231 cells 14 days prior to treatment initiation (t = −14 days), followed by four IV doses of PBS, Fe(II)-TCPP (4 mg/kg), or Cu(II)-TCPP (4 mg/kg) on day 1, 4, 7, and 10. Tissue collection occurred on day 40. (B) Photographs of mice on day 40. Tumors are outlined with red circles. (C) Changes in tumor volume of mice treated with PBS (control), Cu(II)-TCPP, and Fe(II)-TCPP as described in the experimental design (A). (D, E) Photographs (D) and weights (E) of tumors collected on day 40. (F) Changes in body weight of mice during the treatments. Results in (C), (E), and (F) are expressed as mean ± SD, tumor volumes (C) or weights (E) at day 40 were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. (n = 5). **p < 0.01, ***p < 0.001.

Throughout the 40-day treatment period, no significant changes in body weight were observed across all groups, indicating the absence of overt systemic toxicity (Figure 6F). This was further supported by comprehensive hematological and biochemical analyses conducted post-euthanasia. Key blood parameters, including red and white blood cell counts, protein and electrolyte levels, as well as markers of kidney (BUN, creatinine), liver (ALT, ALP), and muscle (creatine kinase) function, remained within normal ranges and showed no statistically significant differences compared to untreated controls (Figures S24 and S25). In addition, histopathological examination of major organs (heart, liver, spleen, lungs, and kidneys) via H&E staining revealed no noticeable tissue damage or abnormalities in either treatment group (Figure S23), further confirming the favorable biocompatibility and safety profile of both nanoagents.

To benchmark the therapeutic potential of our developed Fe(II)-TCPP against existing CDT agents, we conducted a comprehensive literature survey of all single CDT nanoplatforms published to date (Table S2). Among the 24 relevant studies identified (Table S2),^{[13–15, 25–45]} Twenty-one publications reported CDT nanoagents that catalyze Fenton-type reactions to generate •OH.^[25–45] Three reports described alternative approaches: Cu-TCPP and IO-LAHP generating ¹O₂ through the Russell mechanism,^[13–14] and ClO− loaded in a hybrid core–shell nanocarrier (zeolitic imidazolate framework with amphiphilic poloxamer 188) producing ¹O₂ via ClO− + H₂O₂ coupling.^[15] This comprehensive analysis reveals that Fe(II)-TCPP represents a unique CDT agent with the distinctive capability to efficiently and abundantly generate both •OH and ¹O₂ simultaneously without requiring external stimuli.

Regarding the in vivo evaluation of anticancer efficacy among the identified CDT agents, breast cancer animal models were most frequently employed, with 14 out of 24 studies (Table S2). In addition, 9 out of the 24 studies utilized intratumoral injection (Table S2), a route that typically yields enhanced therapeutic outcomes compared to IV administration. Our systemic treatment regimen consisted of four IV injections at a relatively low dose of 4 mg/kg, with only 5 of the 24 reported studies using a lower dose (Table S2). Notably, our treatment duration (10 days) was among the shortest documented. Despite these conservative conditions, Fe(II)-TCPP demonstrated superior therapeutic performance compared to all 24 studies surveyed. Remarkably, Fe(II)-TCPP represents the first CDT nanoagent to achieve complete tumor eradication in all treated mice (n = 5) during the 10-day treatment period and prevent cancer recurrence in 4 out of 5 animals within the next 30-day observation period. Importantly, the prevention of cancer recurrence by CDT nanoagents has not been previously evaluated, making this finding particularly significant. In terms of tumor mass inhibition, including the post-treatment recurrence period, Fe(II)-TCPP achieved 99.5% efficacy, ranking first among all published single-agent CDT studies to date (Table S2). These findings conclusively establish Fe(II)-TCPP as a leading CDT nanomedicine platform reported to date, delivering exceptional therapeutic efficacy under relatively conservative dosing conditions without requiring additional therapeutic modalities or external stimuli.

3. CONCLUSION

We designed the first nanoagent that overcomes two fundamental limitations of conventional CDT: suboptimal catalytic efficiency under physiological conditions and the inability to efficiently generate multiple reactive oxygen species concurrently. Our findings reveal that Fe(II)-TCPP achieves pH-responsive, dual-pathway ROS production, exploiting both Fenton and Russell mechanisms to yield robust levels of •OH and ¹O₂. In vitro, Fe(II)-TCPP demonstrated efficient and comparable generation of both species in cancer cells, negligible ROS production in non-malignant cells, and selectively induced cancer cell death with markedly higher efficacy than Cu–TCPP and Fe(III)-TCPP controls. This dual functionality translated into potent and selective anticancer effects in vivo, achieving complete tumor regression and long-term suppression of recurrence after systemic administration, without obvious toxicity. Beyond establishing a new benchmark for CDT efficacy, this work provides a blueprint for designing next-generation nanoagent-based therapeutics that couple multiple catalytic pathways for amplified oxidative stress within tumors.

4. Experimental Section

4.1. Chemicals

Iron(II) chloride tetrahydrate (FeCl₂·4H₂O), Copper(II) chloride dihydrate (CuCl₂·2H₂O), Iron(III) chloride hexahydrate (FeCl₃·6H₂O), 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (TCPP), dimethylformamide (DMF), 1,10-phenanthroline, hydrogen peroxide (H₂O₂, 30%), 3,3′,5,5′-tetramethylbenzidine (TMB), sodium azide (NaN₃), isopropanol, hydroxyphenyl fluorescein (HPF), 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMP), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), calcein acetoxymethyl ester (calcein-AM), propidium iodide (PI), Perl’s reagent, nuclear fast red (NFR), and eosin were purchased from Sigma-Aldrich Co., LLC (USA). TCPP, Singlet Oxygen Sensor Green (SOSG), and Megm Bullet Kit Growth Media were purchased from Thermo Fisher Scientific (USA). Dulbecco’s Modified Eagle Medium (DMEM), 0.25% Trypsin-EDTA, and phosphate-buffered saline (PBS, pH 7.4) were obtained from Gibco, a part of Thermo Fisher Scientific (USA). PBS solutions at pH 6.5 and 5.5 were prepared by adjusting pH 7.4 PBS with hydrochloric acid. Perl’s reagent and NFR were purchased from Polysciences, Inc. (USA), and hematoxylin was obtained from Biosciences International (USA). Deionized water was prepared using a Milli-Q purification system.

4.2. Synthesis of Fe(II)-TCPP, Cu-TCPP, and Fe(III)-TCPP

Fe(II)-TCPP nanoneedles were synthesized via a modified solvothermal method. Briefly, FeCl₂·4H₂O (30 μmol, 6.0 mg) and TCPP (10 μmol, 7.9 mg) were dissolved in 15 mL of DMF. The solution was purged with nitrogen gas for 5 minutes, stirred for an additional 5 minutes, and then heated at 70°C for 2 hours. The resulting product was collected by centrifugation and washed thoroughly with deionized water several times.

Cu-TCPP nanosheets were synthesized using the same procedure, except without nitrogen bubbling, and CuCl₂·2H₂O (30 μmol, 5.1 mg) was used instead of FeCl₂·4H₂O.

Fe(III)-TCPP nanorods were synthesized similarly, with FeCl₃·6H₂O (30 μmol, 8.1 mg) replacing FeCl₂·4H₂O and no nitrogen bubbling step.

4.3. Characterization

TEM images were acquired using a Tecnai T12 iCorr system (FEI Company, Hillsboro, OR, USA) with an accelerating voltage of 80 kV. The hydrodynamic size and zeta potential of the nanoparticles were measured by DLS using a Zetasizer ZEN3600 (Malvern Panalytical Ltd., Malvern, Worcestershire, United Kingdom). UV/visible absorption spectra were recorded using a UV-1800 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). FTIR spectra were recorded using a Nicolet iS5 spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). XPS spectra were obtained using a Thermo Scientific ESCALAB 250Xi system (Thermo Fisher Scientific Inc., Waltham, MA, USA). ^57Fe Mössbauer spectra were collected at room temperature (296 K) using a SeeCo Mössbauer spectrometer (SeeCo, Inc., Edina, MN, USA) with a velocity range of ±4 mm s−¹. XRD patterns were collected using a D8 Advance diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) in the 2θ range of 10–90°.

4.4. Detection of Ferrous Iron

The Fe²+ content in Fe(II)-TCPP was determined by colorimetric analysis using 1,10-phenanthroline.^[79] A saturated solution of 1,10-phenanthroline (1 mL) was added to 10 μL of Fe(II)-TCPP or Fe(III)-TCPP (1 mg/mL). After 1 hour of reaction, the absorbance at 509 nm was measured using a UV/vis spectrophotometer.

The ferrozine assay was also employed to evaluate the Fe²+ specificity and comparative Fe²+ accessibility in Fe–TCPP systems. Briefly, aqueous solutions of Fe²+ and Fe³+ ionic standards (0.4 mM FeCl₂·4H₂O or FeCl₃·6H₂O), as well as Fe(II)-TCPP and Fe(III)-TCPP dispersions (10 μg/mL) were prepared. Ferrozine reagent (final concentration is 0.5%) was then added to each sample under identical conditions, followed by gentle mixing and incubation at room temperature for 2 h to allow complex formation. Photographs were taken to record the colorimetric response. UV–vis absorption spectra were subsequently collected in the range of 450–700 nm.

4.5. Detection of ¹O₂ and •OH in Solution by Fluorescence Spectroscopy

Singlet oxygen (¹O₂) was detected using SOSG. In a typical procedure, 20 μL of Cu-TCPP, Fe(III)-TCPP, or Fe(II)-TCPP (1 mg/mL), 10 μL of H₂O₂ (100 mM), and 10 μL of SOSG (1 mM in DMSO) were mixed with 0.96 mL of PBS (pH 5.5). The PBS containing SOSG and further including H₂O₂, Cu-TCPP, Fe(III)-TCPP, or Fe(II)-TCPP, was used as a control. The mixture was incubated for 4 hours, and fluorescence was measured at 500 nm excitation and 527 nm emission.

For hydroxyl radical (•OH) detection, SOSG was replaced with HPF (final concentration: 5 μM), while all other conditions remained unchanged.

ROS generation of Fe(II)-TCPP was also assessed under varying pH conditions (5.5, 6.5, and 7.4) and at different time points (0, 1, and 4 hours) using the same reagent concentrations.

To detect the catalysis of Fe2+ in the production of ¹O₂, 10 μL of FeCl₂ aqueous solution (0.5 mM) was added to 1 mL of PBS containing 10 μM SOSG, 0.1 mM TCPP, and 1 mM H₂O₂, with the mixture without Fe2+ as a control, and the mixture was monitored at 0 and 8 hours.

4.6. Detection of ¹O₂ and •OH by Electron Spin Resonance

Electron spin resonance (ESR) was used to confirm ROS production. Measurements were performed on a Bruker E500 X-band electron paramagnetic resonance spectrometer (Bruker BioSpin GmbH, Rheinstetten, Baden-Württemberg, Germany), equipped with a super-X microwave bridge, a super-high-Q cavity, and a liquid nitrogen cryostat. For ¹O₂ detection, TEMP (6.2 mM) served as a spin trap. Aqueous Cu-TCPP, Fe(III)-TCPP, or Fe(II)-TCPP (20 μL, 1 mg/mL) and H₂O₂ (10 μL, 1 M) were added to 970 μL of PBS (pH 5.5) containing TEMP. After 2 hours, ESR signals were recorded.

For •OH detection, TEMP was replaced with DMPO (final concentration: 10 μL/mL). All samples were tested in parallel with single-component controls.

4.7. Hydroxyl Radicals and Singlet Oxygen Generation Kinetics

To evaluate the catalytic activity of Cu-TCPP, Fe(III)-TCPP, and Fe(II)-TCPP nanoparticles in generating ROS, including •OH and ¹O₂, we employed a TMB-based colorimetric assay and calculated the initial reaction rates (v₀) according to the Beer–Lambert law. The detailed procedure is as follows:

All catalytic reactions were conducted in PBS buffer (pH 5.5). H₂O₂ (0.8 M) was used as the ROS precursor, and TMB was used as the chromogenic substrate, with concentrations ranging from 0 to 2 mM. The reaction mixtures were incubated with Cu-TCPP, Fe(III)-TCPP, or Fe(II)-TCPP. The absorbance at 652 nm (indicative of oxidized TMB) was recorded every 10 seconds over a total of 200 s. During the process, NaN₃ (1 mM) or isopropanol (1 mM) was added as ¹O₂ or •OH quencher to ensure that TMB oxidation reflects only the activity of the targeted ROS.

The linear portion of the reaction-time curve (typically 0–30 s) was extracted and fitted to obtain the slope (ΔA/Δt). This slope was converted to the initial reaction rate (v₀) using the Beer–Lambert law:

$${\nu }_0=\text{C}/\text{t}=\Delta A/(\epsilon \times l\times \Delta t)=\mathit{\text{slope}}/(\epsilon \times l)$$

where ε = 39,000 M−¹·cm−¹ (molar absorption coefficient of oxTMB at 652 nm) and l = 0.6 cm (pathlength for microplate). The calculated v₀ values were plotted against the corresponding TMB concentrations and fitted with the Michaelis–Menten equation or the Lineweaver–Burk double-reciprocal method to obtain K_m and V_max. Catalytic constants were calculated as:

$$k_{\mathit{\text{cat}}·\mathit{\text{mass}}}={\nu }_{\mathit{\text{max}}}/\left[E\right],\text{and}\text{the}{k}_{\mathit{\text{cat}}·\mathit{\text{mass}}}/K_m$$

to evaluate catalytic efficiency.

4.8. Intracellular ROS Quantification

To quantify intracellular ROS, the two non-malignant cell lines (Hek293 and MCF-10A) and ten cancer cell lines (A549, A2780, ES2, MLM3, HeLa, KPC, MDA-MB-231, MCF-7, HepG2, and DU-145) were seeded in 96-well plates at 1 × 10⁴ cells/well and incubated for 24 hours. Cells were treated with Cu-TCPP, Fe(III)-TCPP, or Fe(II)-TCPP (2.5–100 μg/mL) for 24 hours. After treatment, the medium was replaced with 100 μL of PBS containing 10 μM DCFH-DA and incubated for 20 minutes. Intracellular fluorescence was excited at 485 nm and measured at 528 nm using a Synergy HT microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). For MDA-MB-231 cells, additional staining was performed to specifically detect different ROS types: 10 μM HPF was used to measure •OH, and 10 μM SOSG was used to quantify ¹O₂, following the same incubation protocol as DCFH-DA. ROS levels were normalized to the control group and presented as relative fluorescence intensity (RFI).

4.9. Detection of Intracellular ROS by Imaging

MCF-10A and MDA-MB-231 cells (2 × 10⁵ cells/dish) were incubated in 35 mm dishes for 24 hours, followed by treatment with 3 mL DMEM containing Cu-TCPP, Fe(III)-TCPP, or Fe(II)-TCPP (25 μg/mL). NaN₃ (1 mM) or isopropanol (1 mM) was added as a ¹O₂ or •OH quencher, separately or together. After 8 hours, cells were washed and incubated with 3 mL of PBS containing 1 μM DCFH-DA for 20 minutes at 37°C. Fluorescence imaging was performed using a Zeiss/Yokogawa CSU-X1 spinning disk confocal microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) with excitation at 488 nm and detection at 500–550 nm.

4.10. Flow cytometry analysis of intracellular ROS

Intracellular reactive oxygen species (ROS) levels were quantitatively analyzed by flow cytometry using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) as a general ROS probe. MCF-10A and MDA-MB-231 cells were seeded in 6-well plates and allowed to adhere overnight. Cells were then treated with Cu-TCPP, Fe(III)-TCPP, or Fe(II)-TCPP at an equivalent concentration (25 μg mL−¹) for 8 h under standard culture conditions. Untreated cells served as controls. After treatment, cells were washed twice with phosphate-buffered saline (PBS) and incubated with DCFH-DA (1 μM) in serum-free medium at 37 °C for 20 min in the dark. Excess probe was removed by washing with PBS, and cells were harvested by trypsinization, resuspended in PBS, and immediately subjected to flow cytometric analysis.

Flow cytometry was performed using a standard flow cytometer equipped with a 488 nm excitation laser, and DCF fluorescence was collected in the FITC channel. Cell populations were initially gated based on forward scatter (FSC-A) versus side scatter (SSC-A) to exclude debris, followed by FSC-A versus FSC-H gating to discriminate singlet cells. A minimum of 10,000 singlet events were collected per sample. Data were analyzed using FlowJo software, and intracellular ROS levels were quantified as the mean fluorescence intensity (MFI) of DCFH-DA. All experiments were performed in triplicate, and data are presented as mean ± SD.

4.11. Cell Viability Assay

The cytotoxicity of Cu-TCPP, Fe(III)-TCPP, and Fe(II)-TCPP was evaluated using a calcein-AM assay. Two non-malignant cell lines (HEK293 and MCF-10A) and ten cancer cell lines (A549, A2780, ES2, MLM3, HeLa, KPC, MDA-MB-231, MCF-7, HepG2, and DU-145) were seeded into 96-well plates at a density of 1 × 10⁴ cells per well and incubated for 24 hours in DMEM. Cells were then treated with Cu-TCPP, Fe(III)-TCPP, or Fe(II)-TCPP at various concentrations (0–100 μg/mL) for another 24 hours. After treatment, the culture medium was replaced with 100 μL of PBS containing 10 μM calcein-AM and incubated for 20 minutes at 37°C. Intracellular fluorescence was excited at 485 nm and measured at 528 nm using a Synergy HT microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). Cell viability was expressed as a percentage of the control group.

4.12. Hemolysis Assay

To assess the hemocompatibility of Cu-TCPP and Fe(II)-TCPP, 1 mL of diluted red blood cell (RBC) suspension was mixed with the nanoparticles at final concentrations of 10, 20, 30, 40, 50, and 100 μg/mL. PBS and deionized water served as negative and positive controls, respectively. After incubation at 37°C for 2 hours, samples were centrifuged at 3000 rpm for 5 minutes. Supernatant absorbance at 540 nm was measured to calculate hemolysis using the formula:

$$\text{Hemolysis}\left(\%\right)=\left[\left({\text{A}}_{\text{sample}}-{\text{A}}_{\text{negative}\text{control}}\right)/\left(A_{\text{positive}\text{control}}-A_{\text{negative}\text{control}}\right)\right]\times 100\%$$

A is absorbance. Each condition was tested in triplicate.

4.13. In Vivo Studies and Tumor Model Construction

Female Nu/Nu nude mice (6–8 weeks old, 20–25 g, purchased from Charles River Laboratories) were used for all animal studies. The Institutional Animal Care and Use Committee of Oregon Health and Science University approved all animal studies (IACUC protocol #: IP00000033).

MDA-MB-231 breast cancer cells were used to construct the tumor model. Specifically, after collecting MDA-MB-231 cells, 2.5 × 10⁶ cells were orthotopically injected into the fourth breast pad on the left side of the mice to simulate a more realistic breast cancer environment. After 14 days, the tumors in the mice had grown to approximately 50 mm³, which was deemed suitable for subsequent imaging or treatment experiments.

4.14. Biodistribution Imaging

To estimate the biodistribution of Fe(II)-TCPP, two groups of mice (n=3) were IV treated with PBS (100 μL) or Fe(II)-TCPP (100 μL, 4 mg/kg). Twenty-four hours post-injection, mice were euthanized via cardiac perfusion, and the tissues (tumor, liver, heart, spleen, lung, and kidney) were harvested. The tumor and organ sections were subjected to Prussian blue staining. Briefly, after the tissues were sectioned at 8 μm thickness, they were fixed in 4% paraformaldehyde. The sections were rehydrated through a graded ethanol series and incubated with a freshly prepared Perl’s Prussian blue solution (prepared by mixing equal volumes of 2% potassium ferrocyanide and 2% hydrochloric acid) for 30 minutes at room temperature. After rinsing with distilled water, the slides were counterstained with nuclear fast red for 5 min, dehydrated, cleared, and mounted. Iron deposits appeared as blue granules within the tissues. The images of various tissues were obtained using a BZ-X710 microscope (Keyence Corporation of America, Itasca, IL, USA).

4.15. Tissue Iron Quantification

Iron content was measured using a Ferrozine-based spectrophotometric assay. To quantitatively evaluate iron in cancer tumors and various tissues, major organs and tissues (tumor, liver, heart, spleen, lung, and kidney) were collected and digested in freshly prepared aqua regia. Digested tissue and iron calibration standards, ammonium iron (II) sulfate hexahydrate, were adjusted to contain between 0.1 and 1.0 mM Fe. Afterwards, 50 μL of 60% perchloric acid and 50 μL of 30% hydrogen peroxide were added to 50 μL of the tissue sample or standard, and the tubes were boiled in a water bath for 30 min to denature the protein. Then, 150 μL of hydroxylamine was slowly added to each tube, and the tubes were incubated for an additional 30 min in a 37°C water bath. Following this reaction, 500 μL Ferrozine and 500 μL pyridine were added and mixed. The absorbance was measured at 562 nm using a UV-1800 spectrophotometer (Shimadzu Corporation, Kyoto, Japan), with readings taken after a 2-hour incubation at room temperature.

4.16. In Vivo and Ex Vivo ROS Detection

The DCFH-DA probe was used to detect ROS in vivo. Once the tumor reached a volume of approximately 50 mm³, PBS, Cu-TCPP, or Fe(II)-TCPP (4 mg/kg) was injected intravenously. After 24 hours, DCFH-DA (50 μL, 10 μM in PBS) was intratumorally injected, and the mice were allowed to incubate for 15 minutes. Fluorescence images of the mice were captured before and after injecting DCFH-DA for each group by using an in vivo imaging system (IVIS) Lumina XRMS (PerkinElmer, Woodbridge, ON, Canada) with an excitation wavelength of 480 nm and an emission wavelength of 520 nm. Fluorescence intensity was quantified using ImageJ software.

To evaluate whether Fe(II)-TCPP induces off-target ROS generation in the spleen, an ex vivo splenic ROS fluorescence assay was performed using DCFH-DA as a general ROS probe. Mice were systemically treated with Fe(II)-TCPP or saline (control) following the same dosing regimen used in the biodistribution study. After 24 hours, DCFH-DA (10 μM in PBS, 50 μL) was intraparenchymally injected directly into the spleen. After 20 min of incubation, mice were euthanized and spleens were immediately excised, gently rinsed with PBS, and subjected to ex vivo fluorescence imaging using an IVIS imaging system under fixed acquisition settings. Data are presented as mean ± SD (n = 4 mice per group).

4.17. In Vivo Treatment

Once the tumors reached approximately 50 mm³ in size, the mice were randomly divided into three groups (n = 5 per group) and treated with PBS, Cu-TCPP, or Fe(II)-TCPP. Each group received a 100 μL intravenous injection of the respective solution every 3 days for a total of 4 injections, with Cu-TCPP and Fe(II)-TCPP doses of 4 mg/kg. Tumor sizes and body weight were measured every 3 days. Tumor volume was calculated using the equation:

$$\text{Tumor}\text{Volume}=\left(L\times W^2\right)/2$$

where L and W are the tumor length and width, respectively. On day 40, the mice were euthanized, and the tumors and other organs were dissected.

4.18. Hematoxylin and Eosin (H&E) Staining for Tissue

After the mice were euthanized, tumors and major organs were extracted and fixed in 10% formalin. The tumors and organs were then sliced to a thickness of 8 μm for H&E staining. The slices were imaged using a BZ-X710 microscope (Keyence Corporation of America, Itasca, IL, USA).

4.19. Blood Routine and Blood Biochemistry Test

1 mL of blood was collected from each mouse after treatment, and the samples were analyzed by IDEXX Laboratories.

4.20. Statistical Analysis

Data were presented as mean ± standard deviation (SD) based on at least three independent experiments. The sample size (n) for each study is specified in the figure legends. Statistical analysis was performed using two-way or one-way analysis of variance (ANOVA) followed by multiple comparisons within each row. Analyses were conducted using GraphPad Prism v10.3.0 (GraphPad Software, LLC, San Diego, CA, USA). A p-value of less than 0.05 was considered statistically significant.

4.21. Database of Literature

Relevant data were retrieved from the Web of Science database using the search terms: “chemodynamic therapy” AND (“tumor” OR “cancer”) AND (“in vivo” OR “mice” OR “mouse”) NOT “synergistic therapy” NOT “combination therapy,” covering the period from January 1950 to March 2025. The resulting dataset comprises studies published between 2017 and 2025. After manual screening and preprocessing of 92 eligible records, key features were extracted and standardized. Ultimately, 24 studies that fully matched our inclusion criteria were selected for further analysis.

Supplementary Material

Supporting Information is available from the Wiley Online Library or from the author.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.