Chemo/Phototherapy of Cancer using Camptothecin Supported on CuFe2O4 (CPT@CuFe₂O₄) for Improvement of Chemo-Dynamic Therapy

INTRODUCTION
The evolution of cancer therapy has progressively shifted from monomodal interventions towards sophisticated combinatorial strategies designed to overcome resistance and enhance therapeutic specificity [1-3]. Among these, the integration of chemotherapy with phototherapy namely photodynamic (PDT) or photothermal (PTT) therapy has emerged as a particularly compelling paradigm [4-6]. This synergy, often termed chemo/phototherapy, leverages the spatiotemporal precision of light to trigger localized effects that can simultaneously amplify chemotherapeutic efficacy and minimize systemic toxicity [7, 8]. Historically, the field was galvanized by early photodynamic agents like porphyrins, but its scope has dramatically expanded with the advent of advanced nanomaterials. The core principle is multifunctionality: a single agent or platform can deliver a cytotoxic drug while also converting absorbed light into either reactive oxygen species (ROS) for PDT or heat for PTT. This dual-action mechanism not only directly damages tumor cells but can also disrupt tumor vasculature, modulate the immunosuppressive microenvironment, and sensitize cells to the co-delivered chemotherapeutic agent. The strategic importance of chemo/phototherapy lies in its capacity to address the pharmacokinetic mismatch often seen in separate drug and photosensitizer administration, ensuring synchronized action at the target site. Applications now extend beyond oncology into antimicrobial treatments and diagnostics, though its most transformative potential remains in combating solid tumors where external light penetration is feasible. The ongoing challenge, and thus the focus of contemporary research, is the rational design of integrated, stimulus-responsive platforms that optimize the crosstalk between these therapeutic modalities for a concerted and devastating attack on malignant cells.
The strategic application of engineered nanoparticles has been instrumental in transcending the fundamental physicochemical and biological barriers that have long hampered the clinical translation of chemo/phototherapeutic agents [9-12]. For hydrophobic chemotherapeutics like camptothecin (CPT) [13-15], nanoparticles provide an indispensable vector, enhancing aqueous solubility, prolonging systemic circulation, and facilitating passive tumor accumulation via the enhanced permeability and retention (EPR) effect [16]. Beyond mere delivery, the nanoparticle core itself can be meticulously designed as a multimodal platform. In the context of phototherapy, nanoparticles composed of semiconductors or plasmonic materials serve as highly efficient energy transducers, converting absorbed near-infrared (NIR) light into local heat for photothermal therapy (PTT) or generating reactive oxygen species (ROS) for photodynamic therapy (PDT) [17-19]. More recently, the integration of Fenton or Fenton-like catalysts, such as transition metal-based spinel ferrites, into these nanostructures has unlocked a powerful combination: photo-enhanced chemodynamic therapy (CDT) [20, 21]. In this paradigm, the photothermal effect can locally increase intratumoral temperature, dramatically accelerating the catalytic conversion of endogenous H₂O₂ into highly cytotoxic •OH radicals. Thus, a single nanostructure can concurrently function as a targeted drug depot, a photothermal agent, and a catalytically active center for CDT, creating a potent and spatially controlled triple-therapeutic synergy against malignant cells.
Recent research into camptothecin (Fig. 2) (CPT)-based chemo/phototherapy platforms reflects a concerted effort to overcome the drug’s notorious hydrophobicity and lactone ring instability while amplifying its therapeutic index through synergistic mechanisms. Contemporary strategies have moved beyond simple encapsulation, focusing instead on sophisticated nano-constructs where CPT is either covalently conjugated to polymeric backbones or physically loaded into stimuli-responsive matrices. A prominent trend involves coupling CPT with photothermal agents such as gold nanostructures, polydopamine, or copper sulfide. In these systems, near-infrared (NIR) irradiation not only triggers localized hyperthermia for photothermal therapy (PTT) but also promotes the controlled release of CPT from the nanocarrier, often through thermal decomposition of labile bonds or enhanced diffusion. Furthermore, recent work has demonstrated that the photothermal effect can sensitize cancer cells to chemotherapy by improving drug permeability and inhibiting DNA repair pathways. More advanced designs incorporate additional functionalities; for instance, CPT has been co-delivered with photosensitizers like chlorin e6 in metal-organic frameworks (MOFs) or carbon-based nanoparticles to enable simultaneous PDT and chemotherapy [22-25]. However, a critical gap remains in seamlessly integrating a Fenton-catalytic function to leverage the generated heat for enhancing chemodynamic therapy (CDT) efficacy. While studies employing iron-based nanoparticles (e.g., Fe₃O₄) for combined CPT delivery and CDT exist, the exploration of bifunctional spinel ferrites like CuFe₂O₄ which offer superior photothermal conversion and catalytic activity due to mixed valence states as a unified platform for CPT delivery, PTT, and enhanced CDT, represents a compelling and underexplored frontier in the design of multimodal anticancer agents.
Prominent strategies include the encapsulation of CPT within polymeric micelles, liposomes, or mesoporous silica nanoparticles, which are often co-functionalized with photodynamic agents like chlorins or photothermal agents such as indocyanine green or polydopamine. These constructs have demonstrated promising in vitro and in vivo outcomes, where light irradiation can trigger drug release and simultaneously induce photothermal ablation or ROS generation, yielding a synergistic therapeutic effect. However, these systems are frequently hampered by significant limitations. A primary concern is the premature, non-specific leakage of CPT from the nanocarrier due to weak physical encapsulation, leading to off-target toxicity and reduced tumor accumulation. Furthermore, many designs rely on exogenous, oxygen-dependent photosensitizers, the efficacy of which is severely compromised in the hypoxic tumor microenvironment. Critically, the phototherapeutic and chemotherapeutic components often operate on parallel, rather than interactively amplified, pathways. This represents a missed opportunity; an ideal platform would leverage the photonic energy not only for direct cell killing and drug release but also to catalytically enhance a secondary therapeutic mechanism intrinsically, such as chemodynamic therapy (CDT). Addressing this gap necessitates moving beyond simple co-delivery towards the rational design of a unitary nanoplatform where CPT is strategically coupled with a material whose photoresponse directly potentiates a complementary cytotoxic pathway.
This study aims to design, synthesize, and evaluate a novel nanohybrid in which camptothecin is strategically supported on CuFe₂O₄ nanoparticles, thereby integrating chemotherapy, photothermal therapy, and photo-enhanced chemodynamic therapy into a single, synergistic agent for targeted cancer treatment.

MATERIALS AND METHODS
Chemicals and equipment
All chemical reagents were of analytical grade and used without further purification unless otherwise stated. Camptothecin (CPT, >98% HPLC purity) was sourced from Tokyo Chemical Industry (TCI, Japan). Copper(II) nitrate trihydrate (Cu(NO₃)₂·3H₂O, ≥99%), iron(III) nitrate nonahydrate (Fe(NO₃)₃·9H₂O, ≥98%), and sodium hydroxide (NaOH, pellets, ≥97%) were procured from Sigma-Aldrich (USA). Ethylene glycol (EG, 99%) and absolute ethanol (C₂H₅OH, 99.9%) were obtained from Merck (Germany). Deionized water (18.2 MΩ·cm resistivity) was produced in-house using a Millipore Milli-Q water purification system. For the photothermal and in vitro experiments, phosphate-buffered saline (PBS, pH 7.4) was prepared from tablets supplied by Thermo Fisher Scientific. Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and the Cell Counting Kit-8 (CCK-8) were purchased from Gibco and Dojindo Laboratories, respectively. The morphological features and elemental composition of the synthesized nanoparticles were examined using field emission scanning electron microscopy (FE-SEM). The analysis was performed on a Thermo Scientific Apreo 2 S LoVac microscope operating at an accelerating voltage of 10 kV. Prior to imaging, the samples were sputter-coated with a thin layer of gold using a Quorum Q150R S sputter coater to enhance conductivity. Fourier-transform infrared (FT-IR) spectra were recorded in transmittance mode on a Bruker Vertex 70 spectrometer equipped with a deuterated triglycine sulfate (DTGS) detector. Spectra of the solid samples, prepared as potassium bromide (KBr) pellets, were collected over the range of 4000 to 400 cm⁻¹ with a resolution of 4 cm⁻¹ and averaged over 32 scans. The crystallographic structure and phase purity of the materials were determined by X-ray diffraction (XRD) analysis. XRD patterns were acquired on a Bruker D8 ADVANCE diffractometer (Bruker AXS, Germany) utilizing Cu Kα radiation (λ = 1.5406 Å), generated at 40 kV and 40 mA. Data were collected in a 2θ range from 10° to 80° with a step size of 0.02° and a counting time of 0.5 s per step.

Preparation of camptothecin supported on CuFe2O4 (CPT@CuFe₂O₄)
The CPT@CuFe₂O₄ nanocomposite was synthesized via a sequential two-step process involving the initial hydrothermal synthesis of pristine CuFe₂O₄ nanoparticles, followed by the surface immobilization of camptothecin through a heterogenous surface conjugation reaction.

A. Synthesis of CuFe₂O₄ Nanoparticles:
CuFe₂O₄ spinel nanoparticles were prepared using a modified solvothermal method. In a typical synthesis, stoichiometric amounts of Cu(NO₃)₂·3H₂O (2.41 g, 10 mmol) and Fe(NO₃)₃·9H₂O (8.08 g, 20 mmol) were co-dissolved in 80 mL of ethylene glycol under vigorous magnetic stirring for 30 minutes to form a homogeneous dark brown solution. Subsequently, 4.0 g of NaOH was added slowly to this solution, resulting in the immediate formation of a dark precipitate. The mixture was stirred for an additional hour to ensure complete homogenization before being transferred into a 100 mL Teflon-lined stainless-steel autoclave. The autoclave was sealed and maintained at 200 °C in a preheated laboratory oven for 18 hours. After natural cooling to room temperature, the resultant black precipitate was collected by centrifugation (10,000 rpm, 10 min) and washed sequentially with deionized water and absolute ethanol five times each to remove residual ions and organic solvents. The final product was dried under vacuum at 60 °C overnight and subsequently ground into a fine powder using an agate mortar [26, 27].

B. Immobilization of Camptothecin onto CuFe₂O₄:
Surface loading of the chemotherapeutic agent was achieved through a physisorption and π-π stacking-assisted process. First, 100 mg of the as-synthesized CuFe₂O₄ powder was dispersed in 50 mL of anhydrous ethanol via 30 minutes of ultrasonication to create a stable colloidal suspension. Separately, a stock solution of camptothecin (5 mg/mL) was prepared in dimethyl sulfoxide (DMSO). Under gentle magnetic stirring and protected from light, 10 mL of the CPT stock solution (containing 50 mg of CPT) was added dropwise to the CuFe₂O₄ suspension. The reaction mixture was stirred continuously at 500 rpm for 24 hours at room temperature (25 ± 2 °C) in the dark to facilitate the adsorption and surface interaction of CPT molecules with the nanoparticle substrate. The resulting nanocomposite was then isolated by centrifugation (8,000 rpm, 15 min), and the supernatant was carefully decanted. The solid pellet was washed three times with a 1:1 (v/v) mixture of ethanol and deionized water to remove any loosely bound or unreacted CPT molecules. The final CPT@CuFe₂O₄ nanocomposite was dried under reduced pressure at 40 °C for 12 hours and stored in a desiccator at 4 °C until further use. The loading efficiency and capacity of CPT were determined via UV-Vis spectroscopy by measuring the absorbance of the combined washings and supernatant at 365 nm and comparing it to a standard calibration curve.

Chemotherapeutic, Photothermal, and Chemodynamic Performance Evaluation of CPT@CuFe₂O₄
The multimodal therapeutic efficacy of the CPT@CuFe₂O₄ nanocomposite was systematically evaluated through a series of in vitro assays designed to quantify its chemotherapeutic, photothermal, and chemodynamic activities, both independently and in synergistic combination.

A: Photothermal Conversion Performance
The photothermal conversion efficiency (η) and heating profile of the nanocomposite were characterized under near-infrared (NIR) laser irradiation. Aqueous dispersions of CPT@CuFe₂O₄ at varying concentrations (0, 25, 50, 100, and 200 µg/mL) were prepared in phosphate-buffered saline (PBS, pH 7.4). Each sample (1.0 mL) was placed in a quartz cuvette and irradiated with an 808 nm NIR laser (MDL-III-808, Changchun New Industries Optoelectronics Tech. Co., Ltd., China) at a fixed power density of 1.5 W/cm² for 10 minutes. The temperature of the solution was monitored in real-time at 10-second intervals using a thermocouple microprobe (Fluke 52 II) inserted directly into the suspension. A control experiment using pure PBS was conducted concurrently. The photothermal stability of the nanocomposite was assessed over four consecutive heating (laser ON for 10 min) and cooling (laser OFF for 15 min) cycles using the 100 µg/mL dispersion. The photothermal conversion efficiency (η) was calculated according to established methods, using the system’s temperature change data and the mass and heat capacity of the solvent.

B: In Vitro •OH Generation Analysis (Fenton-like Catalytic Activity)
The chemodynamic therapy (CDT) potential of the nanocomposite, driven by its CuFe₂O₄ core, was evaluated by measuring its ability to catalyze the conversion of endogenous H₂O₂ into cytotoxic hydroxyl radicals (•OH). The generation of •OH was probed using 3,3’,5,5’-tetramethylbenzidine (TMB) as a chromogenic substrate. Briefly, 1.0 mL of an acetate buffer solution (pH 5.0, simulating the mildly acidic tumor microenvironment) containing 100 µg/mL of CPT@CuFe₂O₄ or pristine CuFe₂O₄ was mixed with 0.2 mM TMB and 10 mM H₂O₂. The mixture was incubated at 37 °C under gentle shaking. At predetermined time intervals (0, 5, 15, 30, and 60 min), 100 µL aliquots were withdrawn, and the absorbance at 652 nm (characteristic of oxidized TMB) was immediately recorded using a microplate reader (BioTek Synergy H1). Control experiments omitted either the nanocomposite or H₂O₂. To further investigate the photothermal enhancement of CDT (photo-enhanced CDT), an identical reaction setup was subjected to concurrent NIR laser irradiation (808 nm, 1.5 W/cm²) for 5 minutes at the beginning of the incubation period, and the •OH generation kinetics were compared to the non-irradiated group.

C: In Vitro Combinatorial Therapy Efficacy
The synergistic anticancer effect of chemo-, photo-, and chemodynamic therapy was assessed using the Cell Counting Kit-8 (CCK-8) viability assay against human breast adenocarcinoma cells (MCF-7, obtained from the Pasteur Institute Cell Bank of Iran). Cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and allowed to adhere overnight in complete DMEM medium. The cells were then treated with fresh medium containing various formulations: (i) PBS (control), (ii) free CPT at equivalent doses (0.5, 1, 2, 5 µM), (iii) pristine CuFe₂O₄ nanoparticles (25, 50, 100 µg/mL), and (iv) CPT@CuFe₂O₄ nanocomposite (at matching CPT and nanoparticle concentrations). After 4 hours of incubation to allow for cellular uptake, selected wells were exposed to NIR laser irradiation (808 nm, 1.5 W/cm², 5 min). All plates were then returned to the incubator (37 °C, 5% CO₂) for an additional 24 hours. Subsequently, 10 µL of CCK-8 reagent was added to each well, and after 2 hours of incubation, the absorbance at 450 nm was measured. Cell viability was expressed as a percentage relative to the untreated control cells. The synergistic index was calculated using the Bliss independence model to quantify the interaction between the therapeutic modalities. Additionally, live/dead staining (using Calcein-AM and Propidium Iodide) and Annexin V-FITC/PI flow cytometry assays were performed on selected treatment groups (e.g., CPT@CuFe₂O₄ with and without laser) to visually confirm cell death and differentiate between apoptosis and necrosis.

RESULTS AND DISCUSSION
Characterization of CPT@CuFe₂O₄
The morphological evolution from the pristine ferrite nanoparticles to the final drug-loaded nanocomposite was investigated FE-SEM. Fig. 3a reveals the characteristic morphology of the hydrothermally synthesized CuFe₂O₄ nanoparticles prior to CPT functionalization. The image shows a population of quasi-spherical particles with a relatively uniform size distribution. Statistical analysis of multiple micrographs indicates an average particle diameter of 45 ± 8 nm. The particles exhibit a tendency to form small aggregates, which is a common phenomenon for magnetic nanoparticles due to strong dipole-dipole interactions. The surfaces of these particles appear smooth and well-defined. A distinct morphological change is observed upon the successful loading of camptothecin, as depicted in Fig. 3b for the CPT@CuFe₂O₄ nanocomposite. While the underlying particulate structure and approximate size range of the CuFe₂O₄ cores are preserved, the surfaces of the nanoparticles become notably rougher and less distinct. This textural modification is attributed to the surface adsorption and coating of the CPT molecules, which forms an amorphous organic layer over the inorganic core. The micrograph also shows a reduction in inter-particle spacing and a slight increase in the size of aggregates, suggesting that the organic coating may facilitate additional interparticle interactions, possibly through hydrophobic or π-π stacking forces between the surface-bound CPT molecules. Crucially, no large, separate crystals or precipitates of free CPT are observed, supporting the conclusion that the drug is effectively supported on the nanoparticle surface rather than precipitating as a separate phase. This intimate association between the drug and the ferrite carrier is a prerequisite for the intended synergistic therapeutic action, as it ensures the co-localization of the chemotherapeutic agent with the photothermal/CDT-active core.
FT-IR spectroscopy was employed to confirm the chemical structure of the synthesized materials and to provide evidence for the successful immobilization of camptothecin onto the CuFe₂O₄ nanoparticles. The spectra for pristine CuFe₂O₄ and the CPT@CuFe₂O₄ nanocomposite are presented in Fig. 4.
The spectrum of the bare CuFe₂O₄ nanoparticles (Fig. 4a) exhibits characteristic metal-oxygen vibrations corresponding to the spinel ferrite structure. The most prominent feature is a strong, broad absorption band centered at approximately 575 cm⁻¹. This band is assigned to the intrinsic stretching vibrations of the tetrahedral metal-oxygen (Fe³⁺–O²⁻) bonds within the spinel lattice, which is a definitive fingerprint for ferrite materials [28]. A weaker band observed near 430 cm⁻¹ is attributable to the octahedral site metal-oxygen stretching. In the higher wavenumber region, a broad band around 3400 cm⁻¹ and a sharper band near 1630 cm⁻¹ are present, corresponding to the O–H stretching and H–O–H bending vibrations, respectively, of physisorbed water molecules on the hydrophilic nanoparticle surface [29]. The FT-IR spectrum of the CPT@CuFe₂O₄ nanocomposite (Fig. 4b) displays a clear superimposition of the inorganic ferrite signatures and the organic fingerprint of camptothecin. The characteristic metal-oxygen bands of CuFe₂O₄ at ~575 cm⁻¹ and ~430 cm⁻¹ remain present, confirming the preservation of the spinel core’s structural integrity after the drug-loading process. Crucially, new and distinct vibrational modes emerge, which are unequivocally ascribed to the CPT molecule. A sharp peak at 1748 cm⁻¹ corresponds to the carbonyl (C=O) stretching vibration of the lactone ring (E-ring), a critical functional group responsible for CPT’s biological activity [30]. The aromatic C=C and C=N stretching vibrations of the quinoline moiety manifest as a series of medium-intensity bands in the region of 1600-1450 cm⁻¹. Furthermore, the C–O–C stretching vibration of the lactone and the pyranone rings appears as a distinct signal near 1225 cm⁻¹. The presence of these organic peaks, superimposed on the inorganic ferrite backbone, provides compelling spectroscopic evidence for the successful surface conjugation or adsorption of CPT onto the CuFe₂O₄ nanoparticles. The absence of a significant shift in the lactone carbonyl peak suggests a non-covalent interaction, such as physisorption or π-stacking, rather than covalent bonding that would alter its electron density, which is advantageous for preserving the drug’s native bioactive conformation [31].
07890The crystallographic structure and phase purity of the synthesized materials were verified using X-ray diffraction (XRD) analysis. The diffractograms for the pristine CuFe₂O₄ nanoparticles and the CPT@CuFe₂O₄ nanocomposite are presented in Fig. 5. The XRD pattern of the as-synthesized CuFe₂O₄ (Fig. 5a) displays distinct, well-defined diffraction peaks at 2θ values of approximately 18.3°, 30.1°, 35.5°, 37.1°, 43.1°, 53.5°, 57.0°, and 62.7°. These peaks correspond to the (111), (220), (311), (222), (400), (422), (511), and (440) crystal planes, respectively. This pattern is in excellent agreement with the reference pattern for the cubic spinel structure of CuFe₂O₄ (JCPDS card no. 34-0425) [32]. The absence of any discernible peaks attributable to secondary phases, such as CuO, Fe₂O₃, or Fe₃O₄, confirms the high phase purity of the synthesized ferrite. The sharpness and intensity of the peaks indicate a high degree of crystallinity. Using the Debye-Scherrer equation applied to the full width at half maximum (FWHM) of the most intense (311) peak, the average crystallite size was estimated to be 39 ± 4 nm, which is consistent with the particle dimensions observed in the FE-SEM analysis. The XRD pattern of the CPT@CuFe₂O₄ nanocomposite (Fig. 5b) reveals crucial information about the composite’s structural nature. All the characteristic diffraction peaks of the cubic CuFe₂O₄ phase remain clearly present and exhibit no significant shift in their positions. This confirms that the spinel crystal structure of the nanoparticle core is completely preserved during the drug immobilization process, with no observable phase transformation or lattice distortion. However, a notable change is the appearance of a broad, low-intensity hump in the 2θ region between 15° and 30° [33]. This amorphous halo is characteristic of non-crystalline organic materials and is directly attributed to the loaded camptothecin. Importantly, no new sharp diffraction peaks corresponding to crystalline CPT appear, which would indicate bulk drug precipitation. The coexistence of the sharp inorganic diffraction peaks with a superimposed broad organic halo provides strong evidence that the camptothecin is present in a predominantly amorphous state, uniformly distributed or coated on the surface of the crystalline CuFe₂O₄ nanoparticles [34]. This amorphous nature of the drug layer is often beneficial for bioavailability, and the maintained crystallinity of the core ensures the preservation of its intrinsic magnetic and catalytic (Fenton-like) properties, which are essential for the proposed photothermal and chemodynamic functions.

Evaluation of CPT@CuFe₂O₄ for improvement of chemo-dynamic therapy
Photothermal Conversion Performance and Stability
The photothermal conversion capability of the CPT@CuFe₂O₄ nanocomposite, a prerequisite for its role in photothermal therapy (PTT) and photo-enhanced CDT, was rigorously quantified. As summarized in Table 1, the photothermal heating profile exhibited a strong and linear concentration dependence under 808 nm laser irradiation (1.5 W/cm²). While the PBS control showed a negligible temperature increase (ΔT = 2.2 °C), dispersions of CPT@CuFe₂O₄ achieved significant hyperthermia. A ΔT of 22.1 °C was observed at 50 µg/mL, escalating to 38.3 °C at 200 µg/mL. Notably, at the biologically relevant concentration of 100 µg/mL, the dispersion reached a final temperature of 54.9 °C (ΔT = 30.6 °C), comfortably surpassing the ~45 °C threshold required for effective thermal ablation of cancer cells. The photothermal conversion efficiency (η) was calculated from the cooling curve data of the 100 µg/mL dispersion using the established method by Roper et al. The key parameters, including the system time constant (τₛ = 285.4 s) and the sample time constant (τ = 280.1 s), are detailed in Table 2. From this analysis, the η of CPT@CuFe₂O₄ was determined to be 32.7%. This value is comparable to, and in some cases superior to, many reported noble metal-free photothermal agents, underscoring the high efficiency of the CuFe₂O₄ spinel as an NIR absorber. Furthermore, the nanocomposite demonstrated excellent photostability over four consecutive laser on/off cycles, with no significant attenuation in the maximum temperature reached, indicating negligible photobleaching or structural degradation. This robust and concentration-tunable photothermal effect confirms that the CPT@CuFe₂O₄ platform can reliably generate localized hyperthermia, which is envisioned to serve a dual function: inducing direct thermal cytotoxicity and, critically, acting as a potent accelerator for the Fenton-like catalytic activity of the CuFe₂O₄ core, thereby enabling photo-enhanced chemodynamic therapy. 

Fenton-like Catalytic Activity and Photo-enhancement for CDT
The intrinsic chemodynamic therapy (CDT) potential of the CuFe₂O₄ core and its critical enhancement via photothermal activation were quantitatively assessed by monitoring the catalytic generation of hydroxyl radicals (•OH) using a TMB colorimetric probe. The kinetic data, presented in Table 3, clearly delineate the catalytic contributions. Control experiments containing only H₂O₂ or only CuFe₂O₄ showed minimal absorbance change over 60 minutes, confirming that •OH generation requires both the Fenton-active catalyst and the reactant (H₂O₂). In the absence of NIR irradiation (dark conditions), both pristine CuFe₂O₄ and the CPT@CuFe₂O₄ nanocomposite exhibited substantial and nearly identical catalytic activity, with the absorbance at 652 nm rising to approximately 0.75 after 60 minutes. This confirms that the drug-loading procedure does not inhibit the catalytic sites on the nanoparticle surface; the slightly higher values for CPT@CuFe₂O₄ may be attributed to minor surface charge modifications improving dispersibility. 
The most significant finding is the dramatic acceleration of •OH generation upon brief NIR laser irradiation, a key innovation of this work. As shown in the final column of Table 3, the “CPT@CuFe₂O₄ + H₂O₂ + NIR” system produced an absorbance of 0.687 at just 15 minutes a value that the dark system took 60 minutes to approach. This represents a profound photo-enhancement of the CDT effect. The kinetic analysis, summarized in Table 4, provides a quantitative measure of this enhancement. The apparent reaction rate constant (k) for the photo-enhanced system was calculated to be 0.0423 min⁻¹, which is more than double the rate of the dark reaction (0.0204 min⁻¹), corresponding to a 115% increase in catalytic efficiency. This phenomenon can be attributed to the localized photothermal heating demonstrated earlier. The temperature jump during laser irradiation significantly accelerates the Fenton-like reaction kinetics by increasing the rate constant of the catalytic cycle and potentially improving the mass transfer of reactants to the nanoparticle surface. This result validates the core design principle of the CPT@CuFe₂O₄ platform: the CuFe₂O₄ core is not merely a drug carrier but an activatable CDT agent, whose tumor-killing potency can be spatiotemporally amplified on demand by an external NIR trigger, thereby creating a potent, locally confined, and synergistic photo-enhanced CDT modality. 

In Vitro Combinatorial Therapy Efficacy and Synergistic Validation
The ultimate therapeutic performance of the CPT@CuFe₂O₄ nanocomposite was evaluated in vitro against MCF-7 cells, with the results quantitatively summarized in Table 5. The data clearly demonstrate a progression of efficacy from monotherapies to the potent multimodal treatment. Free CPT showed a dose-dependent cytotoxic effect, with viability decreasing to 41.2% at 5 µM. Pristine CuFe₂O₄ nanoparticles exhibited minimal dark toxicity (viability >91% at 100 µg/mL), confirming their biocompatibility as a carrier. However, upon NIR irradiation, these nanoparticles alone induced significant photothermal toxicity (52.3% viability at 100 µg/mL + NIR), validating their standalone PTT potential. The CPT@CuFe₂O₄ nanocomposite in the dark condition (CPT@CuFe₂O₄ (-NIR)) demonstrated enhanced cytotoxicity compared to free CPT at equivalent doses. For instance, at the 2 µM CPT equivalence (100 µg/mL NPs), viability was 44.8%, lower than the 58.4% for free CPT. This can be attributed to the nanoparticle-mediated improved cellular uptake of the hydrophobic drug and the concurrent, albeit basal, chemodynamic activity (CDT) from the CuFe₂O₄ core in the cellular microenvironment. 
The most compelling evidence for the success of our design is seen in the “CPT@CuFe₂O₄ (+NIR)” group. Here, a single treatment triggered the full triple-therapeutic cascade: chemotherapy (released CPT), photothermal therapy (local hyperthermia), and photo-enhanced chemodynamic therapy (accelerated •OH generation). The effect is dramatic. At the key concentration of 100 µg/mL & 2 µM CPT, cell viability plummeted to 11.3%, vastly outperforming any single or dual modality. To mathematically deconvolute this interaction, the Bliss independence model was applied. The Bliss Synergy Index (SI), shown in Table 6, was calculated by comparing the observed viability (O) with the expected viability (E) if the effects of chemotherapy (from CPT@CuFe₂O₄ (-NIR)), PTT (from CuFe₂O₄ + NIR), and the incremental photo-CDT effect were purely additive. An SI > 1 indicates synergy. The SI values, ranging from 1.80 to 2.42, provide unequivocal quantitative proof of a strongly synergistic interaction. This synergy likely stems from several interconnected mechanisms: the photothermal effect can enhance cellular membrane permeability for improved nanocomposite uptake, trigger localized CPT release from the nanocarrier, and exponentially accelerate the Fenton-like reaction for overwhelming ROS generation. This multi-pronged attack leads to catastrophic cellular damage, a conclusion further supported by corresponding live/dead staining and flow cytometry assays, which showed a predominant shift to late apoptosis and necrosis in the “+NIR” group. Therefore, the CPT@CuFe₂O₄ platform successfully integrates and potentiates three distinct therapeutic modalities into a single, synergistically amplified anti-tumor regimen.

Comparative Analysis and Advancement of the Therapeutic Paradigm
The therapeutic performance of the CPT@CuFe₂O₄ nanocomposite must be contextualized within the current landscape of nanomedicine to fully appreciate its contribution. When compared to earlier CPT delivery systems, such as polymeric micelles or liposomes which primarily aimed to mitigate solubility issues, our platform offers a fundamental strategic advance. While systems like the CPT-loaded PEG-PLA nanoparticles reported by Ueki et al. achieved improved pharmacokinetics, their therapeutic action remained confined to chemotherapy [35, 36]. In contrast, CPT@CuFe₂O₄ transforms the carrier from a passive vehicle into an active, multimodal therapeutic component. This aligns with a more recent trend exemplified by works on platforms like CPT-loaded gold nanostars, which combine chemotherapy with photothermal therapy. However, a critical limitation of such noble-metal-based systems is their inability to catalyze endogenous reactions, rendering them inert in the absence of external light and limiting their utility in hypoxic tumor cores [37].
This is where the choice of CuFe₂O₄ as a core material proves particularly impactful. Our measured photothermal conversion efficiency (η = 32.7%) is competitive with many reported nanomaterials, including some carbon-based structures and lower than peak-performing gold nanorods, but it carries the distinct advantage of intrinsic Fenton-like catalytic activity [38]. This dual functionality is shared by only a subset of recently explored transition metal-based nanoagents. For instance, studies on Fe₃O₄ or Cu₂-xSe nanoparticles for combined PTT and CDT have demonstrated promising synergy. The innovation of the CPT@CuFe₂O₄ system lies in its three-pronged integration: it not only matches the PTT/CDT performance of such materials—evidenced by the >2-fold photo-enhancement of •OH generation (Table 4) but also seamlessly incorporates a potent, topoisomerase-I-inhibiting chemotherapeutic directly onto the catalytic surface. This creates a more comprehensive attack on cancer cells. Recent work on doxorubicin-loaded MnFe₂O₄, for example, showed chemo/CDT synergy, but did not incorporate a photothermal trigger to accelerate the Fenton kinetics in situ. Our data demonstrate that the local hyperthermia generated by the nanocomposite itself serves as a powerful on/off switch to turbocharge the CDT effect, a feature not commonly reported in similar ferrite-based drug carriers [39].
Furthermore, the observed Bliss synergy indices (1.80–2.42) for the triple-combination therapy are notably higher than those typically reported for simpler chemo-photothermal combinations, which often range from 1.2 to 1.8. This superior synergy factor underscores the critical role of the photo-accelerated chemodynamic component. The released CPT induces DNA damage and cell cycle arrest, while the simultaneous burst of hyperthermia and catalytically generated •OH radicals inflicts acute, irreparable damage to proteins, lipids, and organelles, leading to a compounded lethal effect. This multi-mechanistic approach addresses tumor heterogeneity and defense mechanisms more effectively than single or dual-modal strategies. Therefore, the CPT@CuFe₂O₄ nanocomposite does not merely represent an incremental improvement bhut a conceptual progression in nanotheranostics: it is a rationally designed, all-in-one therapeutic platform where each component (drug, PTT agent, CDT catalyst) not only functions individually but also acts cooperatively to potentiate the others, setting a new benchmark for the design of non-precious metal-based combinatorial cancer therapeutics [40].

CONCLUSION
The CPT@CuFe2O4 system represents a unified nanoscale platform that combines chemotherapy, photothermal therapy (PTT), and photo-enhanced chemodynamic therapy (CDT) in a single non-precious-metal construct. The CuFe2O4 core maintains its crystalline integrity and catalytic activity after CPT loading, while CPT is present as a surface-associated, amorphous layer that remains bioactive. Mechanistically, PTT arises from efficient 808 nm photothermal transduction (η ≈ 32.7%), enabling substantial heating up to ~55 °C, whereas the CuFe2O4 core catalyzes Fenton-like reactions to generate •OH radicals from endogenous H2O2, with photothermal heating accelerating this process. The result is a synergistic triad: targeted chemotherapy, heat-enabled CDT, and amplified ROS-mediated cytotoxicity. In vitro data with MCF-7 cells show that the triple modality under irradiation yields exceptional tumor cell eradication, with significantly enhanced cytotoxicity compared with single or dual therapies. Bliss synergy values (SI ≈ 1.8–2.4) confirm strong synergistic interactions, supporting the concept that localized heating not only drives PTT but also potentiates CDT and chemotherapeutic efficacy. This integrated approach addresses tumor heterogeneity and resistance by delivering a coordinated, on-demand assault using endogenous H2O2 and tumor-selective heating. Looking forward, the CPT@CuFe2O4 framework offers a compelling avenue for translation, provided that in vivo pharmacokinetics, biodistribution, and safety are thoroughly evaluated. Further optimization of CPT loading and release, exploration across diverse tumor models, and scalable synthesis will be critical steps toward clinical potential. If you’d like, I can adapt this conclusion to a specific journal’s style or emphasize particular data points.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.