A Cascade‐Activatable Prodrug Nanomedicine for Photothermally Augmented in Situ Synergistic Chemo/Chemodynamic Therapy

ABSTRACT

Prodrug nanomedicine represents a promising strategy to enhance tumor‐selective drug activation while minimizing systemic toxicity in cancer treatment. Herein, we develop a cascade‐activatable prodrug nanoplatform, AuSt@MOS‐Cu₂O/DSF, constructed by encapsulating Au nanostars (AuSts) within a tetrasulfide bond‐rich mesoporous organosilica (MOS) shell co‐loaded with disulfiram (DSF) and Cu₂O nanoparticles (NPs) for photothermally augmented chemotherapy and chemodynamic therapy. This nanomedicine is designed for sequential activation within the tumor microenvironment (TME). Specifically, glutathione (GSH)‐triggered cleavage of the MOS shell releases Cu₂O NPs and DSF, followed by a Cu⁺‐mediated Fenton‐like reaction in the acidic TME to generate substantial ·OH for chemodynamic therapy. Concurrently, the resulting Cu²⁺ ions chelate with DSF to form cytotoxic bis(diethyldithiocarbamate)‐copper (CuET) in situ, enabling targeted chemotherapy. The system further amplifies therapeutic efficacy through intracellular GSH depletion via the tetrasulfide‐rich MOS and Cu²⁺ as well as near‐infrared photothermal effects, collectively enhancing drug release and catalytic activity. In a triple‐negative breast cancer murine model, AuSt@MOS‐Cu₂O/DSF achieves significant tumor growth inhibition with negligible systemic toxicity under near‐infrared irradiation. This work presents a synergistic strategy for repurposing clinical drugs into precision nanomedicines for the treatment of aggressive cancers.

A cascade‐activatable prodrug nanoplatform is developed by encapsulating Au nanostars within a tetrasulfide bond‐rich mesoporous organosilica shell co‐loaded with disulfiram and Cu₂O nanoparticles. This nanomedicine is sequentially activated within tumor microenvironment to generate substantial ∙OH for chemodynamic therapy and toxic CuET for targeted chemotherapy, achieving significant tumor growth inhibition and good biosafety.

1. Introduction

Despite tremendous advances in oncology, cancer remains a leading cause of global mortality [1]. Projections indicate a sharp rise in annual cancer deaths, expected to reach approximately 18.6 million by 2050 worldwide, largely driven by demographic shifts like aging and population growth [2]. Currently, chemotherapy serves as a first‐line treatment for a wide spectrum of malignant cancers. However, its efficacy is severely limited by several inherent challenges [3, 4], including (1) nonspecific targeting that results in systemic toxicity and dose‐limiting side effects, impairing patients’ quality of life and treatment continuity; (2) the multidrug resistance development; and (3) low tumor delivery efficiency, with typically less than 1% of the intravenously administered dose accumulating in solid tumors. This inefficient targeting not only compromises therapeutic outcomes but also causes significant “off‐target” damage to healthy tissues. Notably, nanomedicines have emerged as a promising strategy to improve drug delivery, employing organic or inorganic nanomaterials such as polymers, liposomes, metal‐organic frameworks, and mesoporous silica, designed in response to endogenous tumor stimuli (e.g., acidic pH, H₂O₂, elevated glutathione (GSH)) [5, 6, 7, 8, 9]. Nevertheless, many conventional nanomedicines are prone to premature drug release under physiological conditions and exhibit “always‐on” activity, causing unintended toxicity before reaching the tumor site. These limitations underscore the urgent need for smarter, more efficient drug delivery systems capable of truly tumor‐specific activation and controlled drug release to maximize therapeutic efficacy while minimizing adverse effects.

To address the limitations of “always‐on” conventional nanocarriers, prodrug strategies have garnered considerable attention for cancer treatment due to their capability for selective activation within the tumor microenvironment (TME) [10, 11]. These approaches involve administering pharmacologically inactive derivatives that can be precisely converted into active drugs upon exposure to tumor‐specific stimuli. Prodrug nanomedicines offer several key advantages, including high drug loading efficiency, minimized systemic toxicity, enhanced tumor‐targeting capability, in situ generation of active therapeutics, and controllable drug release profiles. Over the past decades, a variety of stimuli‐responsive drug delivery systems have been developed to improve targeted delivery and spatiotemporal activation of prodrugs in response to characteristic tumor biochemical cues, including overexpressed enzymes, mild acidity, elevated levels of H₂O₂ and GSH as well as hypoxia conditions [12, 13, 14, 15]. In particular, repurposing clinically approved drugs has emerged as a feasible strategy for constructing novel prodrug nanomedicines against cancers [16, 17]. Such approved drugs can cooperate with other components co‐loaded in the delivery systems to undergo a “nontoxic‐to‐toxic” transition specifically at the tumor site, thereby significantly improving therapeutic safety and efficacy.

Among these clinically approved drugs, disulfiram (DSF), a US Food and Drug Administration (FDA)‐approved agent for chronic alcoholism, has gained significant attention as a potent chemotherapeutic prodrug [16, 18, 19, 20, 21, 22]. A key advantage of DSF is its favorable safety profile; under physiological conditions, DSF itself exhibits low systemic toxicity. Its potent anticancer activity is, however, contingent upon activation via complexation with Cu²⁺ to form bis(diethyldithiocarbamate)‐copper (CuET) [19, 20, 21]. The primary pharmacologic mechanism involves CuET binding to nuclear protein localization protein 4 (NPL4), triggering its aggregation and consequently inhibiting the p97‐NPL4‐mediated protein degradation pathway, which ultimately induces apoptosis in cancer cells. Additionally, the DSF/Cu²⁺ combination disrupts cellular redox homeostasis through Cu⁺‐catalyzing Fenton‐like reaction, generates ·OH, and can promote immunogenic cell death, thereby further augmenting its anticancer efficacy [19, 20, 21]. Despite these promising effects, the clinical translation of DSF prodrug‐based chemotherapy faces several formidable challenges, including the poor metabolic stability of DSF in vivo, insufficient endogenous Cu²⁺ levels within tumors, potential systemic toxicity of preformed CuET, low aqueous solubility, and lack of tumor specificity. Consequently, the development of advanced DSF prodrug nanomedicines that enable coordinated delivery of DSF and Cu²⁺ to tumor sites for in situ generation of the active CuET species represents an urgent and attractive research direction.

Herein, we report a cascade‐activatable prodrug nanomedicine for photothermally augmented chemo‐ and chemodynamic synergistic therapy against triple‐negative breast cancer (TNBC). This system, termed AuSt@MOS‐Cu₂O/DSF, is constructed based on tetrasulfide bond‐rich mesoporous organosilica (MOS)‐encapsulated Au nanostar (AuSt) co‐loaded with DSF and Cu₂O nanoparticles (NPs) (Scheme 1a). As a prodrug nanoplatform, AuSt@MOS‐Cu₂O/DSF exhibits a cascade activation behavior within the TME for spatiotemporally controlled therapeutic delivery (Scheme 1b). Upon systemic administration, the prodrug nanomedicine preferentially accumulates at tumor sites via the enhanced permeability and retention (EPR) effect. Following cellular uptake, the tetrasulfide bond‐rich MOS framework undergoes rapid degradation triggered by intracellular GSH [23, 24, 25]. leading to the synchronous release of loaded DSF and Cu₂O NPs. In the acidic TME, the released Cu⁺ ions from Cu₂O NPs not only initiate a Fenton‐like reaction to generate ∙OH for chemodynamic therapy but also are oxidized to Cu²⁺, which subsequently coordinates with DSF to form the highly toxic CuET complex in situ for chemotherapy. Importantly, both the tetrasulfide bonds in MOS and the generated Cu²⁺ contribute to effective depletion of intracellular GSH, which in turn enhances ∙OH production by reducing antioxidant defense. Furthermore, the photothermal effect mediated by AuSt under near‐infrared irradiation significantly accelerates the reaction kinetics of the Fenton‐like process, promoting more efficient ∙OH generation. In vivo evaluations demonstrate that AuSt@MOS‐Cu₂O/DSF enables cascade activation specifically within the TME, resulting in potent suppression of TNBC tumor growth while exhibiting favorable biosafety profiles in a murine model.

SCHEME 1.

Cascade‐activatable prodrug nanomedicines for photothermally augmented in situ chemo‐/chemodynamic synergistic therapy. (a) Schematic illustration of the preparation process of the AuSt@MOS‐Cu₂O/DSF, involving the MOS coating onto AuSts and sequential loading of Cu₂O nanoparticles and DSF. (b) Schematic illustration of the therapeutic mechanism of the AuSt@MOS‐Cu₂O/DSF as a prodrug nanomedicine through cascade activation exclusively in tumors and photothermal augmentation under near‐infrared laser irradiation.

2. Results and Discussion

2.1. Synthesis and Characterization of Prodrug Nanomedicines

The synthesis process of the AuSt@MOS‐Cu₂O/DSF is schematically illustrated in Scheme 1a. Initially, near‐infrared plasmonic AuSts were prepared via a seed‐mediated growth method, as previously established in our laboratory [26, 27]. To prevent interference from residual polyvinylpyrrolidone (PVP) during the subsequent growth of the tetrasulfide‐rich MOS shell, a thin nonporous organosilica (OS) layer was first coated onto the AuSts, resulting in intermediate AuSt@OS. The MOS shell was then grown on the AuSt@OS surface via a one‐pot method with cetyltrimethylammonium bromide (CTAB) and sodium salicylate as the structure‐directing agent [28], yielding AuSt@MOS. Subsequently, Cu₂O NPs were grown within the mesopores of the MOS shell using CuCl₂ as the precursor and 1,1,3,3‐tetramethyldisiloxane (TMDS) as the reducing agent [29], followed by the loading of DSF, ultimately forming the prodrug nanomedicine AuSt@MOS‐Cu₂O/DSF. The drug loading capacity and encapsulation efficiency of DSF in the final nanomedicine were determined to be 19.0% and 47.5%, respectively.

The AuSt@MOS‐Cu₂O/DSF was characterized using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The nanomedicines exhibit good monodispersity and possess a spherical core/shell nanostructure comprising a AuSt core and a dendritic mesoporous shell, with an average diameter of approximately 122.1 (± 0.7) nm (Figure 1a, b; Figure S1). Elemental composition analysis was performed by high‐angle annular dark‐field scanning TEM (HAADF‐STEM) and energy‐dispersive X‐ray spectroscopy (EDS) elemental mapping analysis (Figure 1c). The EDS elemental maps show the co‐existence of Au, Si, S, Cu, and O within a single nanostructure, verifying the successful construction of AuSt@MOS‐Cu₂O/DSF. The line‐scan profiles of Au, Cu, and Si further reveal the core/shell architecture with the Au signal localized at the center, surrounded by Si, while Cu is distributed throughout the nanostructure (Figure 1d), indicating the successful loading of Cu₂O into the MOS shell.

FIGURE 1.

Synthesis and characterization of AuSt@MOS‐Cu₂O/DSF. (a) TEM image and (b) SEM image of AuSt@MOS‐Cu₂O/DSF. (c) HAADF‐STEM image and elemental mapping images of Au, Si, S, O and Cu in the AuSt@MOS‐Cu₂O/DSF and (d) the corresponding line‐scan concentration profiles of Au, Si and Cu along the indicated white arrow in the merged elemental mapping image (c). (e) Powder XRD patterns of MOS NPs, MOS‐Cu₂O NPs and AuSt@MOS‐Cu₂O/DSF. Note: the blue vertical lines represent the standard XRD pattern of cubic Au with JCPDS #04‐0784, and the yellow vertical lines represent the standard XRD pattern of Cu₂O with the JCPDS #05‐0667. (f) (i) Hydrodynamic size and (ii) ζ‐potential and (g) extinction spectra in water of AuSts, AuSt@OS, AuSt@MOS, AuSt@MOS‐Cu₂O, and AuSt@MOS‐Cu₂O/DSF. Data were presented as mean ± standard deviation (SD) (n = 3).

Powder X‐ray diffraction (XRD) analysis of AuSt@MOS‐Cu₂O/DSF clearly displays characteristic diffraction peaks corresponding to the AuSt core, while the MOS shell exhibits an amorphous structure (Figure 1e). To better visualize the formation of Cu₂O NPs within the mesopores, MOS‐Cu₂O NPs were prepared under identical conditions using pre‐synthesized MOS NPs without the AuSt core and a higher concentration of CuCl₂. The XRD pattern of these MOS‐Cu₂O NPs shows broaden diffraction peaks attributable to Cu₂O, consistent with their small crystallite size. In a control experiment where the tetrasulfide‐rich MOS was replaced with tetrasulfide‐free mesoporous silica, yellow solids adhered to the glass vessel wall (Figure S2). TEM imaging clearly identifies the presence of small Cu₂O NPs embedded in the mesopores of MOS‐Cu₂O and MOS‐Cu₂O/DSF NPs. In contrast, Cu₂O NPs were not observed within the mesopores of the mesoporous silica nanoparticle (MSN). Instead, the formed Cu₂O NPs appeared as aggregates that were distinctly separate from the MSN. These results collectively demonstrate that the tetrasulfide‐bonds within the MOS shell are essential for reducing Cu²⁺ to form Cu₂O NPs in the mesopore.

The hydrodynamic size and ζ‐potential of the nanoparticles were monitored throughout the synthesis of AuSt@MOS‐Cu₂O/DSF to track the successful stepwise construction of the nanomedicine. As illustrated in Figure 1f(i), the hydrodynamic size increased progressively from 49.7 (± 15.0) nm for the initial AuSts to 600.2 (± 116.9) nm following the sequential deposition of the nonporous OS layer, the MOS shell, and the loading of Cu₂O. Notably, the AuSt@MOS‐Cu₂O exhibited significant aggregation in aqueous media, resulting in a large hydrodynamic size. This value decreased to 201.7 (± 125.6) nm upon subsequent loading of DSF, yielding the final AuSt@MOS‐Cu₂O/DSF, which suggests improved dispersion stability in aqueous media. Concurrently, ζ‐potential measurements revealed a significant negative shift to −39.5 (± 5.1) mV after coating the AuSts with the OS layer (AuSt@OS), compared to −14.5 (± 2.4) mV of the as‐synthesized AuSts. The ζ‐potential became less negative for AuSt@MOS (−23 (± 2.9) mV), AuSt@MOS‐Cu₂O (−10.6 (± 2.4) mV), and AuSt@MOS‐Cu₂O/DSF (‐21.7 (± 2.7) mV), reflecting the changes in surface chemistry at each stage (Figure 1f(ii).

2.2. Photophysical Properties and Cascade Activation for Drug Releases

The as‐synthesized AuSts exhibit a strong near‐infrared plasmon band centered at 757 nm (Figure 1g). Upon sequential coating with the thin nonporous OS layer and the MOS shell, the plasmon band shifts to 805 and 818 nm, respectively, which is attributable to the increased dielectric constant of the surrounding medium [30]. Following the loading of DSF, the plasmon band of the final AuSt@MOS‐Cu₂O/DSF remains largely unchanged. Owing to its strong near‐infrared plasmonic characteristics, the photothermal performance of AuSt@MOS‐Cu₂O/DSF was evaluated under 808 nm‐laser irradiation. The infrared thermal images and corresponding temperature profiles of the aqueous suspensions were recorded at varying concentrations (0, 20, 40, 60, and 80 µg/mL) under 808 nm laser irradiation at a power density of 1.0 W/cm². As shown in Figure 2a(i and ii), AuSt@MOS‐Cu₂O/DSF exhibits a pronounced concentration‐dependent temperature increase upon 808 nm‐laser irradiation. Specifically, the temperature of the suspension at 80 µg/mL rapidly rises to over 63°C within 10 min of irradiation, whereas pure water under the same conditions remains at approximately 28°C. The photothermal conversion efficiency (η) of AuSt@MOS‐Cu₂O/DSF is calculated to be 61% based on the previously established method using the maximum steady‐state temperature and the system's time constant for heat transfer (Figure 2a (iii)) [31, 32]. Furthermore, no significant decrease in the steady‐state temperature was observed over four consecutive laser on/off cycles, indicating excellent photostability of the prodrug nanomedicine (Figure 2a(iv).

FIGURE 2.

Photothermal performance and responsive release behaviors of AuSt@MOS‐Cu₂O/DSF. Photothermal characterization: (a) (i) Infrared thermal images and (ii) temperature profiles of aqueous suspensions of the AuSt@MOS‐Cu₂O/DSF at various concentrations (0, 20, 40, 60, and 80 µg/mL) under 808 nm laser irradiation (1.0 W/cm²) for different times, (iii) the heating‐cooling curve and the plot of t versus ‐ln(θ) during the cooling after laser switch‐off of the AuSt@MOS‐Cu₂O/DSF aqueous suspension (80 µg/mL), and (iv) photostability of the AuSt@MOS‐Cu₂O/DSF aqueous suspension over four laser on/off cycles under 808 nm laser irradiation (1.0 W/cm²). Stimuli‐responsive release: (b) Schematic illustration of photothermally enhanced drug releases of Cu₂O NPs and DSF under near‐infrared laser irradiation, where the generated Cu²⁺ from released Cu₂O NPs rapidly reacts with DSF to form CuET with a characteristic absorption at 432 nm in the absorption spectra, used for spectrometric monitoring of DSF release. (c) Optical absorption spectra of CuCl₂, DSF, the equimolar mixture of CuCl₂ and DSF, AuSt@MOS‐Cu₂O/DSF, and AuSt@MOS‐Cu₂O/DSF at pH 5.5 in the presence of 10 mM GSH, all dissolved in water. (d) The release profiles of (i) Cu₂O NPs at pH 7.4 or 5.5 heated at 37 or 50°C and (ii) DSF at pH 7.4 or 5.5 with/without 808 nm laser irradiation (1.0 W/cm², 45 min) in the absence or presence of 10 mM GSH from the AuSt@MOS‐Cu₂O/DSF (50 µg/mL). (e) Kinetic constant of the DSF release from the AuSt@MOS‐Cu₂O/DSF under various conditions, fitted to a first‐order kinetic model. Data were presented as mean ± SD (n = 3).

As illustrated in Figure 2b, the AuSt@MOS‐Cu₂O/DSF functions as a prodrug nanomedicine whose tetrasulfide‐rich MOS shell can be cleaved by GSH [23, 24, 25], leading to shell degradation and the subsequent release of encapsulated Cu₂O and DSF. In the acidic TME, Cu⁺ ions from Cu₂O NPs can directly catalyze a Fenton‐like reaction, generating ·OH for chemodynamic therapy while simultaneously producing Cu²⁺. The resulting Cu²⁺ then reacts with the liberated DSF to form cytotoxic CuET, which exhibits a characteristic absorption peak at 432 nm in the optical absorption spectrum (Figure 2c). This distinct absorbance enables real‐time monitoring of DSF release. Notably, the characteristic absorption at 432 nm of CuET is absent in the AuSt@MOS‐Cu₂O/DSF, but emerges clearly after incubation at pH 5.5 in the presence of GSH, confirming GSH‐responsive degradation of the nanomedicine, a process further promoted by tumor acidity. We further investigated the payload release kinetics from AuSt@MOS‐Cu₂O/DSF under different pH values (pH 7.4 and 5.5) and temperatures (37°C or 50°C). The release of Cu₂O NPs was quantified by inductively coupled plasma‐optical emission spectrometer (ICP‐OES) analysis of the supernatant collected from treated AuSt@MOS‐Cu₂O/DSF suspensions. The release of DSF was assessed by monitoring the characteristic absorption at 432 nm corresponding to the formed CuET complex. As shown in Figure 2d(i) and Figure S3, significantly more Cu and DSF are released at pH 5.5 compared to pH 7.4, and heating at 50°C further accelerates their releases. Moreover, both laser irradiation and the presence of GSH substantially enhance the release kinetics of Cu₂O and DSF (Figure 2d(ii)). The release kinetic constant and maximum release amount of DSF under various conditions, fitted to a first‐order kinetic model, are summarized in Figure 2e and Table S1. The results unequivocally verify that both heating and laser irradiation promote payload release, with the most efficient release occurring at pH 5.5 under laser irradiation in the presence of GSH.

2.3. In Vitro Therapeutic Mechanism and Anticancer Effects

As illustrated in Figure 3a(i), the tetrasulfide bonds within the MOS shell enable AuSt@MOS‐Cu₂O/DSF to undergo rapid degradation in response to GSH, thereby releasing Cu⁺ ions that catalyze a Fenton‐like reaction to generate substantial amounts of ·OH [33]. Furthermore, both the tetrasulfide bonds themselves and Cu²⁺ produced via the Fenton‐like reaction contribute to GSH depletion, which in turn enhances the efficacy of ·OH‐mediated chemodynamic therapy. To evaluate the ·OH generation in the presence of H₂O₂, 3,3′,5,5′‐tetramethylbenzidine (TMB) was employed as a chromogenic probe. ·OH oxidizes colorless TMB to blue oxidized TMB (oxTMB), characterized by an absorption peak at 652 nm [34]. We first assessed ·OH production by AuSt@MOS‐Cu₂O/DSF in the presence of H₂O₂ at different pH values (pH 7.4, 6.5, and 5.5). As shown in Figure 3a(ii and iii), the most efficient ·OH generation occurs at pH 5.5, whereas it is negligible at physiological pH (pH 7.4). No significant ·OH signal is detected in the absence of AuSt@MOS‐Cu₂O/DSF (control), confirming that the Fenton‐like reaction is effectively promoted under acidic conditions. Additionally, electron spin resonance (ESR) spectroscopy using 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) as a spin‐trapping agent was conducted [35]. As displayed in Figure 3b, AuSt@MOS‐Cu₂O/DSF exhibits the characteristic 1:2:2:1 quartet signal of the DMPO/·OH adduct, which is significantly stronger than that of AuSt@MOS, further verifying efficient ·OH generation due to the loading of Cu₂O. To reveal the change in the Cu valence state within AuSt@MOS‐Cu₂O/DSF, X‐ray photoelectron spectroscopy (XPS) analysis was performed (Figure S4). The results indicate that the Cu in the as‐prepared AuSt@MOS‐Cu₂O/DSF exists predominantly as Cu⁺ (83.3%), with a minor contribution of Cu²⁺ (16.7%), confirming that Cu is primarily in a reduced state. After treatment with GSH and H₂O₂, however, the proportion of Cu⁺ decreases to 45.1%, while that of Cu²⁺ increases to 54.9%. Thus, we demonstrate the oxidation of Cu⁺ into Cu²⁺ via the Fenton‐like catalytic reaction.

FIGURE 3.

In situ ·OH generation, GSH depletion, cytotoxicity, and in vitro anticancer effects of AuSt@MOS‐Cu₂O/DSF. (a) (i) Schematic illustration of the responsive release behaviors, in situ ·OH generation, and GSH depletion by AuSt@MOS‐Cu₂O/DSF, along with their detection methods. (ii) Measurement of ·OH: absorption spectra of 0.25 mM TMB aqueous solution treated with 18 µg/mL AuSt@MOS‐Cu₂O/DSF in the presence of 2.5 mM H₂O₂ at different pH values (pH 7.4, 6.5, and 5.5) for 30 min, and (iii) the corresponding change of the absorbance at 652 nm of oxTMB with the reaction time at different pH values. Note: control (ctr) represents the absence of the AuSt@MOS‐Cu₂O/DSF at pH 5.5, and the inset in panel (iii) is the photograph of 0.25 mM TMB aqueous solution after reaction for 30 min under corresponding conditions. (b) ESR spectra of DMPO/·OH adducts in the aqueous suspensions of AuSt@MOS or AuSt@MOS‐Cu₂O/DSF both at 40 µg/mL in the presence of 10 mM H₂O₂ with DMPO as the ·OH spin‐trapping agent. Measurement of GSH: (c) (i) Absorption spectra of 120 µM DTNB mixed with the reaction solution of AuSt@MOS‐Cu₂O/DSF and GSH for different time intervals (0, 6, 18, 24, and 48 h), and (ii) the corresponding GSH level change as a function of the treatment time after treatment with 1.0 mg/mL AuSt@MOS or AuSt@MOS‐Cu₂O/DSF. (d) TEM images of the AuSt@MOS‐Cu₂O/DSF after incubation at pH 7.4 or pH 5.5 in the presence of 10 mM GSH (pH 5.5 + GSH) for 24 h. (e) (i) Cell viability of 4T1 cells treated with various concentrations of AuSt@MOS, AuSt@MOS‐Cu₂O and AuSt@MOS‐Cu₂O/DSF with or without 808 nm‐laser irradiation (1.0 W/cm², 5 min), and (ii) the corresponding fluorescence images of 4T1 cells after various treatments at an identical concentration of 30 µg/mL, followed by staining with Calcein‐AM (green, live cells)/PI (red, dead cells). Data are presented as mean ± SD (n = 3), and the statistical significance of differences among multiple groups was carried out using one‐way analysis of variance (***p < 0.001).

The GSH depletion capability was evaluated using Ellman's assay, where 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (DTNB) reacts with GSH to yield yellow 2‐nitro‐5‐thiobenzoate (NTB) with a characteristic absorption at 412 nm (Figure 3a(i)) [35, 36]. As depicted in Figure 3c(i), the absorbance at 412 nm progressively decreased with increasing reaction time between AuSt@MOS‐Cu₂O/DSF and GSH, accompanying by a rise in absorbance at 320 nm, indicating effective GSH consumption. GSH depletion was also observed in AuSt@MOS lacking Cu₂O, confirming the role of tetrasulfide bonds in this process (Figure 3c(ii); Figure S5). Notably, the incorporation of Cu₂O significantly enhanced the GSH depletion ability of AuSt@MOS‐Cu₂O/DSF. After 24 h of incubation, AuSt@MOS‐Cu₂O/DSF maintains its structural integrity under physiological conditions (pH 7.4) but shows significant degradation at pH 5.5 in the presence of GSH, as shown in TEM images (Figure 3d). Collectively, these results demonstrate the effective GSH depletion by AuSt@MOS‐Cu₂O/DSF, attributable to the combined action of tetrasulfide bonds in in MOS shell and Cu²⁺ generated from Cu⁺‐catalyzing Fenton‐like reaction.

The in vitro anticancer efficacy of the prodrug nanomedicine was evaluated against 4T1 breast cancer cells using the MTT cell viability assay. As presented in Figure 3e(i), both AuSt@MOS and AuSt@MOS‐Cu₂O display negligible cytotoxicity with the cell viability exceeding 87% after 24 h of incubation at a concentration of 30 µg/mL. In contrast, AuSt@MOS‐Cu₂O/DSF exhibits pronounced concentration‐dependent cytotoxicity, reducing the viability of 4T1 cells to 61% at 10 µg/mL, significantly lower than that observed for AuSt@MOS (99%) and AuSt@MOS‐Cu₂O (97%) at the same concentration. This enhanced cytotoxicity is attributed to the in situ generation of highly cytotoxic CuET from the released Cu²⁺ and DSF within the cellular environment, as confirmed in Figure S6. The IC₅₀ value of CuET formed from the complexation of DSF and Cu²⁺ is one to two orders‐of‐magnitude lower than those of DSF or Cu²⁺ alone, demonstrating its significantly enhanced cytotoxic potency. Notably, under 808 nm‐laser irradiation (denoted as (+)), all three drug formulations show a marked decreases in cell viability with increasing concentrations. Specifically, at 30 µg/mL under laser exposure (+), the viability of 4T1 cells decreases to 64% for AuSt@MOS, 56% for AuSt@MOS‐Cu₂O, and 21% for AuSt@MOS‐Cu₂O/DSF, all of which were significantly lower than the corresponding values without laser irradiation (denoted as (−)). We further evaluated the synergistic effect among the individual therapeutic components based on the cytotoxicity data. As shown in Figure S7, the IC₅₀ values were determined as follows: 43.02 µg/mL for AuSt@MOS (+), 98.19 µg/mL AuSt@MOS‐Cu₂O (−), 31.09 µg/mL for AuSt@MOS‐Cu₂O (+), 19.52 µg/mL for AuSt@MOS‐Cu₂O/DSF (−), and 10.87 µg/mL for AuSt@MOS‐Cu₂O/DSF (+), respectively. Using the median‐effect equation [37], the calculated combination index (CI) of the photothermal effect, CDT and chemotherapy in AuSt@MOS‐Cu₂O/DSF was 0.81, which indicates a synergistic interaction among the photothermal therapy, chemodynamic therapy, and chemotherapy. The superior therapeutic performance of AuSt@MOS‐Cu₂O/DSF was further corroborated by live/dead cell co‐staining assay using Calcein‐AM and propidium iodide (PI) (Figure 3e(ii). In summary, the excellent in vitro anticancer efficacy of AuSt@MOS‐Cu₂O/DSF stems from the synergistic combination of ·OH generation‐mediated chemodynamic therapy and CuET‐induced chemotherapy, collectively potentiated by effective GSH depletion and near‐infrared photothermal effects.

We next examined the cellular uptake and intracellular drug release behaviors of the AuSt@MOS‐Cu₂O/DSF in 4T1 cells. For fluorescent visualization, AuSt@MOS‐Cu₂O/DOX was prepared following the same synthetic protocol, but with doxorubicin (DOX) loaded as a red‐fluorescent probe in place of DSF. DOX can target the cell nucleus after being released from the MOS shell. Therefore, co‐localization analysis of DOX with the nucleus can be used to assess both the cellular uptake of AuSt@MOS‐Cu₂O/DOX and the release status of its DOX cargo. As shown in Figure 4a, after 1 h of incubation, red fluorescence is predominantly localized in the cytoplasm, indicating successful internalization of AuSt@MOS‐Cu₂O/DOX by 4T1 cells. Notably, the red fluorescence of DOX is well separated from the blue nuclear staining (Hoechst 33342) with a Pearson's correlation coefficient (𝜌) of 0.35, which differs significantly from the pattern observed in free DOX/Hoechst 33342 co‐stained cells (Figure S8). This suggests that DOX remains largely encapsulated within the nanocarrier at the 1 h time‐point. Even after 4 h of incubation, only a slight increase in nuclear red fluorescence is observed (ρ = 0.40). In contrast, upon 808 nm‐laser irradiation (1.0 W/cm², 5 min), a substantial enhancement of red fluorescence in the nuclei is detected (ρ = 0.84), indicating laser‐enhanced release of DOX from the nanomedicine. The endocytic uptake was further investigated using AuSt@MOS^Cy3‐Cu₂O/DSF, which was labeled with red fluorescent dye Cy3, followed by lysosomal staining with Lyso‐Tracker Green. As displayed in Figure S9, significant colocalization of the red fluorescence from the nanomedicine with the green fluorescence of Lyso‐Tracker Green was observed, with ρ = 0.58 at 1 h post‐incubation. This confirms the internalization of the nanomedicine and its accumulation in cellular lysosomes. After 4 h of incubation, however, the ρ value decreases to 0.21, indicating the diffusion of Cy3 into the cytosol, which is attributed to the acidity‐triggered degradation of the MOS shell.

FIGURE 4.

Cellular uptake, drug release, and in vitro anticancer mechanism. (a) Cellular uptake and drug release of the AuSt@MOS‐Cu₂O/DOX (30 µg/mL) by 4T1 cells after incubation for 1 or 4 h under no laser or 808 nm‐laser irradiation (1.0 W/cm², 5 min), co‐stained with Hoechst 33342 (nucleus, blue), and the corresponding colocalization scatter plot. (b) Fluorescence images of DCFH‐DA‐stained 4T1 cells for intracellular ·OH generation after various treatments: PBS (−), PBS (+), AuSt@MOS (30 µg/mL) (−), AuSt@MOS (30 µg/mL) (+), AuSt@MOS‐Cu₂O (30 µg/mL) (−), AuSt@MOS‐Cu₂O (30 µg/mL) (+), AuSt@MOS‐Cu₂O/DSF (30 µg/mL) (−), and AuSt@MOS‐Cu₂O/DSF (30 µg/mL) (+). Note: (−) indicates no laser irradiation; (+) indicates 808 nm laser irradiation (1.0 W/cm², 5 min). (c) Fluorescence images of JC‐1 dye‐stained 4T1 cells after treatment with PBS, AuSt@MOS (30 µg/mL), AuSt@MOS‐Cu₂O (30 µg/mL) or AuSt@MOS‐Cu₂O/DSF (30 µg/mL). (d) Fluorescence quantitative analysis of (i) DCF and (ii) JC‐1. (e) Fluorescence images of acridine orange (AO)‐stained 4T1 cells for lysosome integrity analysis after treatment with PBS (control), AuSt@MOS (30 µg/mL), AuSt@MOS‐Cu₂O (30 µg/mL) or AuSt@MOS‐Cu₂O/DSF (30 µg/mL). (f) Relative intracellular GSH levels in 4T1 cells after treatment with various concentrations (0, 25, 50, and 100 µg/mL) of AuSt@MOS or AuSt@MOS‐Cu₂O/DSF for 6 h. Data are presented as mean ± SD (n = 3), and the statistical significance of differences among multiple groups was carried out using one‐way analysis of variance (**p < 0.01, ***p < 0.001).

As established previously, the cascade‐activation of AuSt@MOS‐Cu₂O/DSF in the TME enables ·OH generation via a Cu⁺‐catalyzed Fenton‐like reaction, which is further promoted by GSH depletion and photothermal effects. Subsequently, intracellular ·OH production was assessed using 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA) as a fluorescent probe, which is oxidized by ·OH to form 2,7‐dichlorofluorescein (DCF) emitting green fluorescence [38]. As shown in Figure 4b,d(i), cells treated with AuSt@MOS show minimal green fluorescence with or without laser irradiation, comparable to the PBS (−) and PBS (+) control groups. In contrast, cells treated with AuSt@MOS‐Cu₂O exhibit a clearly elevated ·OH level, as indicated by brighter fluorescence, which is further enhanced under the laser irradiation. Notably, the most intense green fluorescence is observed in the AuSt@MOS‐Cu₂O/DSF (+) group, underscoring the synergistic effect of Cu⁺‐mediated Fenton‐like reaction, GSH depletion, and photothermal enhancement. As is well‐known, the generated ∙OH typically triggers a cascade of intracellular damage. For instance, ∙OH can induce intracellular lipid peroxidation (LPO). Therefore, C11‐BODIPY was employed as a ratiometric fluorescent probe to assess LPO in 4T1 cells following various treatments. C11‐BODIPY readily penetrates into the lipid layer of cells, and its red fluorescence shifts to green as a result of LPO [39]. As displayed in Figure S10, treatment with either AuSt@MOS‐Cu₂O or AuSt@MOS‐Cu₂O/DSF leads to enhanced green fluorescence and diminished red fluorescence in 4T1 cells, indicating effective LPO induced by ∙OH generation. Furthermore, laser irradiation intensifies LPO due to the elevated ∙OH level. These observations align with the changes in intracellular ∙OH levels presented in Figure 4b and d (i). The peroxidation of polyunsaturated fatty acids is a hallmark of ferroptotic cell death. Given that our nanomedicine simultaneously depletes GSH and enhances ∙OH generation via the Fenton‐like reaction, we conclude that a ferroptosis‐like mechanism significantly contributes to the enhanced therapeutic efficacy. To assess mitochondrial dysfunction, the mitochondrial membrane potential (MMP) was measured using a JC‐1 staining assay, where JC‐1 dye forms red fluorescent J‐aggregates in healthy mitochondria but reverts to green fluorescent monomers upon depolarization [40]. As shown in Figure 4c,d(ii), cells treated with AuSt@MOS‐Cu₂O also exhibit obvious green fluorescence, while the strongest green fluorescence and weakest red fluorescence can be seen in cells treated with AuSt@MOS‐Cu₂O/DSF, indicating severe mitochondrial membrane collapse after the AuSt@MOS‐Cu₂O/DSF treatment. Lysosomal integrity was evaluated using acridine orange (AO), which emits red fluorescence in intact acidic lysosomes but shifts to green upon leakage into the less acidic cytosol [41]. As shown in Figure 4e, cells treated with PBS, AuSt@MOS, or AuSt@MOS‐Cu₂O display strong red fluorescence, indicating preserved lysosomal membranes. In contrast, cells incubated with AuSt@MOS‐Cu₂O/DSF display negligible red fluorescence and intense green fluorescence, suggesting severe lysosomal membrane disruption induced by in situ formed CuET. Furthermore, intracellular GSH depletion is found to be concentration‐dependent and more pronounced in cells treated with AuSt@MOS‐Cu₂O/DSF compared to AuSt@MOS alone (Figure 4f), consistent with the dual GSH‐consuming capacity of the tetrasulfide bonds and Cu²⁺ ions.

Subsequently, flow cytometry was performed to analyze the mechanism of cell death following various treatments [14]. As shown in Figure S11, the total apoptosis rate (Q2 + Q3) for 4T1 cells treated with AuSt@MOS‐Cu₂O and AuSt@MOS‐Cu₂O/DSF reaches 20.66% and 24.50%, respectively, which is significantly higher than that in the PBS (2.41%) and AuSt@MOS (3.11%) control groups. Notably, the AuSt@MOS‐Cu₂O/DSF group exhibits a substantially higher late apoptosis rate (20.90%) compared to the AuSt@MOS‐Cu₂O group (7.66%), underscoring the enhanced pro‐apoptotic effect conferred by the DSF‐derived CuET complex. Furthermore, a scratch assay was conducted to evaluate the potential anti‐migratory effect of AuSt@MOS‐Cu₂O/DSF on 4T1 cells. As presented in Figure S12, the wound area in the control group (G1) decreases significantly after 24 h of incubation relative to the initial baseline (0 h), a trend similarly observed in the AuSt@MOS‐treated group (G2), indicating minimal impact on cell motility. In contrast, wound healing was significantly inhibited in both the AuSt@MOS‐Cu₂O (G3) and AuSt@MOS‐Cu₂O/DSF (G4) treatment groups. Remarkably, in the AuSt@MOS‐Cu₂O/DSF group, the wound area shows negligible reduction over the 24 h period, demonstrating a strong inhibitory effect on the migration ability of 4T1 cells.

2.4. In Vivo Biosafety and Anticancer Efficacy

We further evaluated the in vivo anticancer performance and biosafety of AuSt@MOS‐Cu₂O/DSF using a TNBC murine model. The experimental timeline for establishing the 4T1 tumor model and subsequent treatments is outlined in Figure 5a. Prior to in vivo studies, a hemolysis assay was conducted to assess the hemocompatibility of AuSt@MOS‐Cu₂O/DSF. As presented in Figure S13, the hemolysis rate remains <5.0% even at a high concentration of 100 µg/mL, indicating excellent hemocompatibility and minimal impact on erythrocyte integrity. Subsequently, the biodistribution and blood pharmacokinetics of AuSt@MOS‐Cu₂O/DSF were investigated following intravenous administration into 4T1 tumor‐bearing mice. The blood circulation profile followed a two‐compartment model, with distribution and elimination half‐lives of t_1/2α = 0.31 h and t_1/2β = 6.3 h, respectively (inset, Figure 5b), indicating a sufficiently long circulation time for effective tumor accumulation. Biodistribution analysis in major organs (heart, liver, spleen, lung, and kidney) and tumors at 12, 24, and 48 h post‐intravenous administration reveals predominant accumulation in the liver, spleen, and kidney, consistent with clearance by the reticuloendothelial system (Figure 5b). Importantly, significant nanomedicine accumulation is observed at the tumor sites, reaching its peak at 24 h after administration.

FIGURE 5.

In vivo anticancer efficacy assessment. (a) The experimental timeline for 4T1 tumor murine model establishment and therapeutic treatments. (b) Biodistribution of Au in 4T1 tumor‐bearing mice at 12, 24 and 48 h post‐intravenous administration of 15 mg/kg AuSt@MOS‐Cu₂O/DSF (n = 3). Inset: the corresponding blood circulation curve fitted to a two‐compartment model for circulation half‐life analysis. (c) (i) Infrared thermal images at various time‐points (0–5 min) of 4T1 tumor‐bearing mice and (ii) the tumor temperature profiles after intravenous administration of PBS or 15 mg/kg AuSt@MOS‐Cu₂O/DSF under 808 nm laser irradiation (1.0 W/cm², 5 min). (d) Representative photographs of 4T1 tumor‐bearing mice after various treatments, and (e) (i) the tumor volume growth curves and (ii) body weight change curves of the mice after various treatments (n = 5). (f) (i) Photographs of excised tumors on day 14 post‐treatment and (ii) tumor weights harvested on day 14 from the mice after various treatments (n = 5). (g) The corresponding H&E‐staining images and (h) TUNEL staining images of tumor slices harvested on day 14 after various treatments. Data are presented as mean ± SD, and the statistical significance of differences among multiple groups was carried out using one‐way analysis of variance (***p < 0.001).

For the in vivo anticancer efficacy study, 4T1 tumor‐bearing mice with tumor volumes of approximately 100 mm³ were randomly divided into six groups (n = 5) and treated as follows: (1) PBS (200 µL), (2) CuET (3.0 mg/kg), (3) AuSt@MOS (15 mg/kg), (4) AuSt@MOS (15 mg/kg) + laser, (5) AuSt@MOS‐Cu₂O/DSF (15 mg/kg), and (6) AuSt@MOS‐Cu₂O/DSF (15 mg/kg) + laser. For groups receiving laser irradiation (+), tumors were exposed to an 808 nm‐laser at a power density of 1.0 W/cm² for 5 min after 24 h of intravenous administration. The tumor temperature in the AuSt@MOS‐Cu₂O/DSF (+) group rapidly increases and reaches a steady‐state temperature of 48°C, significantly higher than that in the PBS group (38°C), confirming effective photothermal conversion (Figure 5c). Tumor volume and body weight were monitored every other day. As shown in Figure 5d–f, the CuET (−), AuSt@MOS (+) and AuSt@MOS‐Cu₂O/DSF (−) groups all exhibits superior tumor growth inhibition compared to the AuSt@MOS (−) and PBS groups. The most potent suppression is observed in the AuSt@MOS‐Cu₂O/DSF (+) group, which achieved the highest tumor growth inhibition rate of 71%. No significant body weight fluctuations are observed in all these groups during the treatment period, indicating minimal systemic toxicity (Figure 5e(ii)). Notably, two mice in the CuET (−) group succumbed to the high systemic toxicity of the preformed drug, underscoring the safety advantage of the in situ activated prodrug system. These results collectively demonstrate that the combination of AuSt@MOS‐Cu₂O/DSF and laser irradiation achieves synergistic anticancer efficacy with markedly improved biosafety, attributable to the cascade‐activation of combined chemotherapy and chemodynamic therapy, augmented by GSH depletion and photothermal effects.

To further corroborate the therapeutic outcome, tumor sections were analyzed via hematoxylin & eosin (H&E) staining and terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling (TUNEL) assay. As displayed in Figure 5g, h, the most severe cellular damage and the highest abundance of apoptotic cells are observed in the AuSt@MOS‐Cu₂O/DSF (+) group, whereas other treatments induce moderate damage. Furthermore, H&E staining of major organs (heart, liver, spleen, lung, and kidney) collected after treatment reveals no significant pathological abnormalities (Figure S14). Consistent with this, blood biochemical analysis and liver/kidney function tests showed no notable deviation from normal ranges after various treatments (Figure S15), confirming the excellent in vivo biosafety profile of the nanomedicine. Taken altogether, the AuSt@MOS‐Cu₂O/DSF prodrug nanomedicine demonstrates satisfactory biosafety and superior therapeutic efficacy for TNBC treatment, leveraging a synergistic combination of TME‐responsive drug cascade activation, chemodynamic therapy, chemotherapy, and photothermal augmentation.

3. Conclusions

In summary, we have successfully developed a cascade‐activatable prodrug nanomedicine, AuSt@MOS‐Cu₂O/DSF, for tumor‐selective and synergistic treatment of aggressive TNBC via photothermally augmented chemotherapy and chemodynamic therapy. This nanoplatform was rationally engineered to undergo TME‐specific degradation, initiated by GSH‐induced cleavage of the tetrasulfide‐rich MOS shell, leading to the controlled release of encapsulated Cu₂O NPs and DSF. In the acidic TME, Cu⁺ ions liberated from Cu₂O NPs effectively catalyzed a Fenton‐like reaction, generating substantial amounts of ·OH for chemodynamic therapy while simultaneously producing Cu²⁺ ions. These Cu²⁺ ions subsequently chelated with the released DSF to form highly cytotoxic CuET in situ, enabling targeted chemotherapy against cancer cells without significant harm to surrounding normal tissues. Importantly, this system achieved effective depletion of intracellular GSH through the combined action of the tetrasulfide bonds in the MOS shell and the generated Cu²⁺, which in turn amplified ·OH production for enhanced chemodynamic therapy. The anticancer performance was further augmented by the intrinsic photothermal effect under near‐infrared irradiation, which also promoted the drug release and Fenton‐like catalytic activity. In vivo studies demonstrated remarkable tumor growth inhibition in a TNBC murine model, achieving a high inhibition rate of 71% under near‐infrared laser irradiation, along with excellent biosafety. This study provides a promising strategy for repurposing clinically approved drugs into safe and effective prodrug nanomedicines for targeted treatment of aggressive cancers.

4. Experimental Section

4.1. Chemicals and Reagents

All chemicals and solvents were used directly without any further purification, unless noted otherwise. Ultrapure water (resistivity: 18.2 MΩ·cm) used throughout all experiments was produced with a Millipore Direct‐Q3 UV system. Tetraethyl orthosilicate (TEOS, 98%), bis[3‐(triethoxysilyl) propyl] tetrasulfide (BTES, 90%), 3‐mercaptopropyltriethoxysilane (MPTES, 96%), ammonia hydroxide (NH₃∙H₂O, 25%–28% (v/v)), triethanolamine (TEA, 99%), cetyltrimethylammonium bromide (CTAB, 99%), disulfiram (DSF, 97%), doxorubicin hydrochloride (DOX∙HCl, 98%), 3,3′,5,5′‐tetramethylbenzidine (TMB, 98%), 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO, 97%), and 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA, 97%) all were purchased from Aladdin Co., Ltd. 1,1,3,3‐Tetramethyldisiloxane (TMDS, 98%) was ordered from Shanghai Adamas Reagent Co., Ltd. Polyvinyl pyrrolidone (PVP, average molecular weight = 10000 g/mol) were provided from TCI (Shanghai) Development Co., Ltd. Sodium salicylate (99.5%) and sodium chloride (NaCl, 99.5%) were purchased from Macklin, Inc. Trisodium citrate dihydrate (C₆H₅Na₃O₇∙2H₂O, 99.0%), N,N‐dimethylformamide (DMF, 99.8%), hydrogen peroxide solution (H₂O₂, 30% (w/w)), hydrochloric acid (HCl, 36.5‐38.0%), nitric acid (HNO₃, 65.0‐68.0%), cupric chloride dihydrate (CuCl₂∙2H₂O, 99%), and sodium borohydride (NaBH₄, 99%) were obtained from Sinopharm Chemical Reagent Co., Ltd. Chloroauric acid tetrahydrate (HAuCl₄·4H₂O, 99.0% trace metals basis) was ordered from Shanghai Civi Chemical Technology Co. Ltd. Roswell Park Memorial Institute 1640 medium (RPMI‐1640) was acquired from Shanghai VivaCell Biosciences Ltd. Fetal bovine serum (FBS) was ordered from Shanghai Excell Biological Technology Co., Ltd. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) cell viability assay kit, Calcein‐AM/Propidium Iodide (PI) cell viability/cytotoxicity assay kit, Annexin V‐FITC/Propidium Iodide (PI) apoptosis detection kit, Lipid Peroxidation Assay Kit with BODIPY C11, Hoechst 33342, JC‐1 mitochondrial membrane potential assay kit, and acridine orange were from Beyotime Biotechnology Co., Ltd.

4.2. Characterization

Transmission electron microscopy (TEM) images were acquired with an FEI Tecnai G² F20 S‐TWIN TMP transmission electron microscope at 200 kV. High angle annular dark field‐scanning transmission electron microscopy (HAADF‐STEM) and elemental mapping analysis were performed on a Thermo Fisher Scientific Talos^TM F200X scanning/transmission electron microscope at 200 kV. Scanning electron microscopy (SEM) images were taken with a Hitachi Regulus 8230 field‐emission scanning electron microscope at 15 kV. Powder X‐ray diffraction (XRD) was used to analyze the crystal phase on a Rigaku D/Max 2500VB 18 kW X‐ray diffractometer at 40 kV and 250 mA with Cu Kα radiation (λ = 1.5406 Å). Optical extinction or absorption spectra were recorded on an Agilent Cary 5000 UV‐vis‐NIR spectrophotometer. Dynamic light scattering (DLS) was performed to determine the hydrodynamic size of the samples, electrophoretic light scattering (ELS) was used to measure ζ‐potential, both on a Malvern Zetasizer Nano ZSE ZEN3700 instrument. Photothermal performance was assessed using a FLIR A35 infrared thermal imaging camera under a CW‐808 nm‐laser. The quantitative analysis of Au and Cu was performed with either an Agilent 5100 inductively coupled plasma‐optical emission spectrometer (ICP‐OES) or a PerkinElmer NEXION 2000 inductively coupled plasma‐mass spectrometer (ICP‐MS). The fluorescence imaging was conducted on a Zeiss Axio Vert. A1 inverted fluorescence microscope. Cell viability and hemolysis assays were performed on a Tecan Spark multimode microplate reader. Electron‐spin‐resonance (ESR) spectra were measured on a Bruker Magnettech ESR5000 spectrometer.

4.3. Preparation of AuSt@MOS‐Cu₂O/DSF

First, AuSts were synthesized using a seed‐mediated growth method well‐established in our laboratory [26, 27]. The molar concentration of as‐synthesized AuSts were determined based on the Au seed concentration with an extinction coefficient of 3.07 × 10⁷ M⁻¹∙cm⁻¹ at 519 nm [42]. Considering the interference of pre‐existing PVP in as‐synthesized AuSts on the growth of ordered MON layer, the as‐synthesized AuSts were initially coated with a thin layer of organosilica (OS). Specifically, 0.32 mL of MPTES was mixed with 3.0 mL NH₃∙H₂O, which was added dropwise to 320 mL AuSt aqueous suspension (71 pM) under vigorous stirring. After 3 min reaction, the mixture was kept still at 30°C for 8 h. The reaction mixture was purified through centrifugation at 8000 rpm for 15 min and washing twice with H₂O, yielding AuSt@OS redispersed in 1 mL H₂O at 4300 µg/mL. Subsequently, the MOS layer was further grown on the AuSt@OS surface via a one‐pot method with cationic surfactant CTAB and sodium salicylate as the structure directing agent [28]. In brief, 15 mg of TEA, 64 mg of CTAB, 20 mg of sodium salicylate, and 4.3 mg of AuSt@OS were added to 20 mL H₂O under magnetic stirring, which was fully homogenized by ultrasonication. Then, a mixture of 70 µL TEOS and 56 µL BTES was added dropwise, and after the reaction proceeded at 40°C for 16 h, the reaction mixture was centrifuged at 800 rpm and washed three times with 1.0 wt% NaCl methanolic solution, yielding AuSt@MOS dispersed in 1 mL ethanol at 2900 µg/mL.

Afterward, 5.0 mg of CuCl₂∙2H₂O was added to 10 mL ethanol containing 290 µg/mL AuSt@MOS, along with the introduction of 0.2 mL TMDS as a reducing agent [29]. The reaction proceeded at room temperature for 6 h under magnetic stirring, and then the reaction mixture was centrifuged at 800 rpm and washed twice ethanol. The resultant AuSt@MOS‐Cu₂O was redispersed in 9.0 mL ethanol, which was added with 1.0 mL DSF ethanolic solution (8.0 mg/mL). The reaction proceeded for 2 h under magnetic stirring, and the mixture was centrifuged and washed successively with ethanol and H₂O, yielding the final product – AuSt@MOS‐Cu₂O/DSF. The loading capacity and encapsulation efficiency were determined by the following equations [43]:

$$\mathrm{Loading}\mathrm{capacity}\left(\%\right)=\frac{WeightofDSF}{Weightofthenanomedicine}\times 100$$ (1)

$$\mathrm{Encapsulation}\mathrm{efficiency}\left(\%\right)=\frac{WeightofDSF}{WeightofaddedDSF}\times 100$$ (2)

4.4. In Vitro Analysis of the Photothermal Performance

To evaluate the photothermal performance of the AuSt@MOS‐Cu₂O/DSF, 0.2 mL of aqueous suspensions with varied concentrations (0, 20, 40, 60, and 80 µg/mL) was added to microcentrifuge tubes, which was continuously irradiated with an 808 nm‐laser at a laser power density of 1.0 W/cm² for 10 min. The temperature profile and infrared thermal images were longitudinally recorded with an FLIR A35 infrared thermal imaging camera. To test the photothermal stability, the aqueous suspension of 80 µg/mL AuSt@MOS‐Cu₂O/DSF was irradiated under 808 nm‐laser for four consecutive laser on/off cycles with the temperature profile recorded in real‐time. The photothermal conversion efficiency was calculated using the following equations [31, 32]:

$$\eta =\frac{hS\left(T_{\max }-T_{\mathrm{amb}}\right)-Q_0}{I\left(1-{10}^{-A_{\lambda }}\right)}$$ (3)

$$t=-\frac{m_{\mathrm{s}}{C}_{\mathrm{s}}}{hS}\ln \theta$$ (4)

$$\theta =\frac{T-T_{\mathrm{amb}}}{T_{\max }-T_{\mathrm{amb}}}$$ (5)

where T _max and T _amb represent the steady‐state temperature and ambient temperature, respectively; h and S represent the heat transfer coefficient and heat transfer surface area, respectively; m _s and C _s were the mass and heat capacity (4.2 J/g) of the water solvent, respectively; Q ₀ was the heat generated by the solvent (water) absorbing the incident laser at λ = 808 nm, which can be calculated by Q ₀ = 5.4×10⁻⁴×I (J/s); I was the laser power; and A_λ represents the absorbance at the laser wavelength λ = 808 nm.

4.5. In Vitro Analysis of Responsive Releases

The responsive releases of Cu²⁺ and DSF from the AuSt@MOS‐Cu₂O/DSF were analyzed with varied pH values in the absence or presence of GSH. First, the AuSt@MOS‐Cu₂O/DSF (50 µg/mL) was dispersed in 0.8% SDS‐containing PBS at varied pH values (pH 7.4, 6.5, and 5.5) in the absence or presence of 10 mM GSH to make the aqueous suspensions for release analysis of Cu₂O and DSF. For in vitro analysis of Cu₂O release, 20 mL of the suspensions were kept at 37°C under gentle magnetic stirring (200 rpm), 1.0 mL of which was taken at certain time intervals and centrifuged for collection of the supernatants. The Cu amount in the collected supernatants was determined by ICP‐OES. To investigate the temperature effect on the releases of Cu₂O and DSF, 2.5 mL of the aqueous suspensions were placed in a standard quartz cuvette that was kept at 37 or 50°C in a water‐bath under gentle magnetic stirring (200 rpm). The released Cu₂O and DSF eventually formed CuET with a characteristic absorption at 432 nm in the absorption spectra, which was used for quantitative analysis of Cu₂O and DSF.

To investigate the effect of the laser irradiation on the releases of Cu₂O and DSF, 220 µL of the aqueous suspensions was added to each well of 96‐well plates, which was kept in the dark (denoted as (−)) or irradiated with an 808 nm‐laser at a power density of 1.0 W/cm² for 45 min (denoted as (+)). The absorbance at 432 nm of each well was tested at different time‐points, and the time‐dependent release profiles were fitted through the first‐order kinetics equation:

$$\mathrm{\Delta }{A}_t=\mathrm{\Delta }{A}_{\infty }\left(1-e^{-kt}\right)$$ (6)

$$\mathrm{C}{\mathrm{u}}_t=\mathrm{C}{\mathrm{u}}_{\infty }\left(1-e^{-kt}\right)$$ (7)

where t and k represent the release time and the release kinetic constant, respectively; ∆A _t was the absorbance change at 432 nm at time t relative to that before laser irradiation, and ∆A _ꝏ was the steady‐status absorbance change relative to that before laser irradiation, which represent the concentration of the formed CuET; Cu_t was the amount of released Cu₂O at time t, and Cu_ꝏ was the maximum amount of released Cu₂O.

4.6. In Vitro Evaluation of ∙OH Generation

The in vitro ∙OH generation ability was assessed at varied pH values (pH 7.4, 6.5, and 5.5) in the presence of H₂O₂ using the TMB colorimetric assay [34]. Typically, AuSt@MOS‐Cu₂O/DSF was added at 18 µg/mL to PBS solutions containing 0.25 mM TMB and 2.5 mM H₂O₂. The absorption spectra were recorded as a function of reaction time, and the absorbance at 652 nm characteristic of oxidized TMB (oxTMB) was used for quantitative analysis of ∙OH.

4.7. Electron Spin Resonance (ESR) Spectroscopy Analysis

ESR spectroscopy was employed to detect ∙OH using DMPO as the spin‐trapping agent [35]. First, AuSt@MOS or AuSt@MOS‐Cu₂O/DSF (both at 40 µg/mL) were added to 100 µL H₂O containing 10 mM H₂O₂, followed by the addition of DMPO (1.0 µL). After thoroughly mixed, the suspensions were filled into quartz capillaries for collection of ESR spectra.

4.8. In Vitro Evaluation of the GSH Depletion Ability

The tetrasulfide bond in the MOS of AuSt@MOS‐Cu₂O/DSF could be cleaved by GSH, which triggered the degradation of MOS and thereby concomitant releases of Cu₂O and DSF. Thus, MOS and the formed Cu²⁺ from Cu₂O‐based Fenton‐like reaction enable the GSH depletion. The GSH depletion ability of both AuSt@MOS and AuSt@MOS‐Cu₂O/DSF was evaluated using Ellman's assay, in which 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (DTNB) as the molecular probe could react with GSH to yield yellow 2‐nitro‐5‐thiobenzoate (NTB) with a characteristic absorption at 412 nm [35, 36]. In brief, AuSt@MOS or AuSt@MOS‐Cu₂O/DSF (1.0 mg/mL) was added to 1.0 mL PBS of 10 mM GSH. After incubation at 37°C for 48 h, 0.1 mL of the reaction solution was taken at different time intervals and centrifuged at 8000 rpm to collect the supernatant for GSH detection. Specifically, 6.0 µL of 40 mM DTNB agent and 48 µL of the supernatant were mixed in 2.0 mL Tris‐HCl buffer, and incubated for 30 min before absorption measurements.

4.9. Cell Culture and Animal Tumor Model

4T1 murine breast cancer cell line was provided by the American Type Culture Collection (ATCC). 4T1 cells were cultured in the RPMI‐1640 medium supplemented with 10% (v/v) FBS and 1% (v/v) penicillin‐streptomycin in a humidified incubator containing 5% CO₂ at 37°C. All animal experiments were conducted in accordance with the approved protocols of the Institutional Animal Care and Use Committee at Central South University (Approval No.: CSU‐2022‐0472). Female BALB/c mice (3‐5 weeks old, 13–16 g) were purchased from Hunan Slack Laboratory Animal Co., Ltd. The 4T1 breast tumor xenograft murine model was established by subcutaneously injecting 100 µL PBS containing 1.0 × 10⁶ 4T1 cells into the right thigh of each mouse. When the tumor volume reached approximately 100 mm³ after growth for 6 days, the tumor model was established, and the 4T1 tumor‐bearing mice were used for in vivo studies.

4.10. In Vitro Assessment of Cytotoxicity and Anticancer Effects

For in vitro cytotoxicity assay, 4T1 cells were seeded in 96‐well plates at a cell density of 1.0×10⁴ cells/well, and cultured for 24 h. Subsequently, the cell culture medium was replaced with fresh RPMI‐1640 medium containing various formulations of AuSt@MOS, AuSt@MOS‐Cu₂O, and AuSt@MOS‐Cu₂O/DSF, each at different concentrations (0 (PBS, control), 10, 20, and 30 µg/mL, Au basis). For the laser irradiation (+) groups, the cells were irradiated with an 808 nm‐laser at a power density of 1.0 W/cm² for 5 min after 24 h incubation. The cell viability was determined by the MTT assay. Specifically, the cell culture medium was removed, and 100 µL of fresh RPMI‐1640 medium containing 0.5 mg/mL MTT agent was added to each well. After treatment for 4 h, the culture medium was replaced with 100 µL DMSO for each well, and the absorbance at 570 nm was detected for quantitative analysis of cell viability. The synergistic effect was evaluated from the cytotoxicity data using the Chou‐Talalay method [37]. The combination index (CI) was calculated according to the following equation [37]:

$$\mathrm{C}I=\sum _{j=1}^n\left(\frac{{\left(D\right)}_j}{{(D_x)}_j}\right)$$ (8)

where D represent the drug doses used in combination to achieve median‐effect, and D_x represent drug doses that would be required individually to achieve that same effect. Additionally, the live/dead cell co‐staining assay was performed on 4T1 cells after various treatments using the Calcein‐AM/PI cell live/dead assay kit. Specifically, 4T1 cells were seeded in 96‐well plates at a cell density of 1.0 × 10⁴ cells/well, and cultured for 24 h. Afterward, the cell culture medium was replaced with fresh RPMI‐1640 medium containing PBS (2.0 µL, control), AuSt@MOS, AuSt@MOS‐Cu₂O, and AuSt@MOS‐Cu₂O/DSF (30 µg/mL, Au basis). After incubation for 24 h, the cells were kept in the dark or irradiated with an 808 nm‐laser at a power density of 1.0 W/cm² for 5 min. Then, the culture medium was removed, followed by the addition of 2.0 µM Calcein‐AM and 8.0 µM PI for cell dying. Finally, the treated cells were observed using an inverted fluorescence microscope with the green channel (Excitation: 495 nm; Emission: 520 nm) and red channel (Excitation: 530 nm; Emission: 620 nm), respectively.

4.11. Analysis of Cellular Uptake Behaviors and Intracellular Releases

To study the cellular uptake and responsive releases of payloads, fluorescent DOX was loaded into AuSt@MOS‐Cu₂O to make AuSt@MOS‐Cu₂O/DOX. In brief, the AuSt@MOS‐Cu₂O was added at a final concentration of 0.2 mg/mL to an aqueous solution of 0.5 mg/mL DOX∙HCl. After magnetic stirring overnight, the reaction mixture was centrifuged and washed with water until a colorless supernatant was obtained, yielding AuSt@MOS‐Cu₂O/DOX. Then, 4T1 cells were seeded in 96‐well plates at a cell density of 1.0 × 10⁴ cells/well and cultured for 24 h. The cell culture medium was replaced with fresh RPMI‐1640 medium containing 30 µg/mL AuSt@MOS‐Cu₂O/DOX, and incubated for 1 or 4 h. To examine the effect of near‐infrared irradiation on the release of drug (DOX), the treated cells were irradiated with an 808 nm‐laser at a power density of 1.0 W/cm² for 5 min after incubation for 4 h. Subsequently, the cell culture medium was removed, and the cells were washed three times with PBS, followed by staining with Hoechst 33342 (nucleus, blue) for 10 min. Finally, the treated cells were subjected to fluorescence imaging.

For the further investigation of the endocytosis pathway, 4T1 cells were first cultured for 24 h for cell adhesion and then washed with PBS. Subsequently, 5 µg/mL AuSt@MOS^Cy3‐Cu₂O/DSF were added. The cells were incubated for 1 or 4 h and then co‐stained with Lyso‐Tracker Green (lysosomes, green), which were subsequently observed by a fluorescence microscope. The Pearson's colocalization coefficient of AuSt@MOS^Cy3‐Cu₂O/DSF (red) and lysosomes (green) was analyzed using ImageJ software.

4.12. Detection of Intracellular∙OH

The intracellular ∙OH generation was tested using DCFH‐DA as the molecular probe, which was oxidized into 2',7'‐dichlorofluorescein (DCF) emitting green fluorescence by ∙OH [38]. In brief, 4T1 cells were seeded in 96‐well plates at a cell density of 1.0 × 10⁴ cells/well, and cultured for 24 h. Then, the cells were treated with PBS (control), AuSt@MOS, AuSt@MOS‐Cu₂O, and AuSt@MOS‐Cu₂O/DSF (30 µg/mL, Au basis) for 6 h. For laser irradiation groups, the cells were subsequently irradiated with an 808 nm‐laser at a power density of 1.0 W/cm² for 5 min. Afterward, the cell culture medium was removed, and the cells were stained with 10 µM DCFH‐DA in fresh RPMI‐1640 medium for 30 min, followed by removal of the culture medium and rinsing three times with PBS. Finally, the treated cells were subjected to fluorescence imaging.

4.13. Detection of Lipid Peroxidation

Intracelluar LPO was assessed using C11‐BODIPY as a molecular probe with an excitation wavelength of 581 nm and a maximum emission wavelength of 591 nm. This lipophilic, ratiometric fluorescent dye incorporates into cell membranes. In its reduced state, the probe has excitation and emission maxima at 581 and 591 nm, respectively, exhibiting red fluorescence. Upon oxidation by lipid peroxides, its conjugates diene structure was modified, causing a shift in its fluorescence emission peak, with the excitation and emission maxima changing to approximately 488 nm and 510 nm, resulting primarily in green fluorescence [39]. Consequently, the degree of LPO was indicated by a decrease in the red‐to‐green fluorescence intensity ratio. Briefly, 4T1 cells were seeded in 96‐well plates at a density of 1.0 × 104 cells/well and cultured for 24 h. The cells were then treated for 6 h with PBS (control), AuSt@MOS, AuSt@MOS‐Cu₂O or AuSt@MOS‐Cu₂O/DSF (30 µg/mL, Au basis). For the laser irradiation groups, cells were subsequently irradiated with an 808 nm‐laser at a power density of 1.0 W/cm² for 5 min. Afterward, the cell culture medium was removed, and the cells were stained with 2 µM C11‐BODIPY in PBS for 30 min. Following incubation, the staining solution was discarded, and the cells were rinsed three times with PBS. Finally, the treated cells were observed under a fluorescence microscope.

4.14. Analysis of the Intracellular GSH Depletion

For intracellular GSH detection, 4T1 cells were seeded in 6‐well plates at a cell density of 5.0 × 10⁵ cells/well and cultured for 24 h. Then, the cell culture medium was replaced with fresh RPMI‐1640 medium containing various concentrations (0, 25, 50, and 100 µg/mL) of AuSt@MOS or AuSt@MOS‐Cu₂O/DSF. After incubation for 6 h, the culture medium was removed, and the cells were washed three times with PBS, followed by the addition of 100 µL Triton‐X‐100 lysis buffer (0.4%) for cell lysis. Afterward, the lysates were centrifuged at 9000 rpm for 5 min, and 50 µL of the supernatant was mixed with 200 µL 0.5 mM DNTB, which was subjected to absorption measurements with the absorbance at 412 nm used for quantitative analysis of GSH levels.

4.15. Analysis of Mitochondrial Integrity

The mitochondrial integrity of 4T1 cells treated with the AuSt@MOS‐Cu₂O/DSF was examined through measuring the mitochondrial membrane potential (MMP) using JC‐1 mitochondrial membrane potential assay kit [40]. Specifically, 4T1 cells were seeded in 96‐well plates at a cell density of 1.0 × 10⁴ cells/well and cultured for 24 h. Then, the cell culture medium was replaced with fresh RPMI‐1640 medium containing PBS (control) or 30 µg/mL AuSt@MOS‐Cu₂O/DSF for 6 h. Afterward, the culture medium was removed, and the cells were treated with JC‐1 staining solution for 20 min according to the manufacturer's protocol. Finally, the cells were washed three times with PBS and subjected to fluorescence imaging using an inverted fluorescence microscope.

4.16. Analysis of Lysosomal Integrity

The lysosomal integrity of 4T1 cells treated with the AuSt@MOS‐Cu₂O/DSF was tested by acridine orange staining [41]. The green fluorescence of acridine orange could shift to red fluorescence due to its protonation and concentration in the acidic lysosomes. In brief, 4T1 cells were seeded in 96‐well plates at a cell density of 1.0 × 10⁴ cells/well and cultured for 24 h. Subsequently, the cell culture medium was replaced with RPMI‐1640 containing 30 µg/mL AuSt@MOS‐Cu₂O/DSF, and the cells were incubated for another 6 h. After removal of the culture medium and washing with PBS, 100 µL of acridine orange staining solution was added for staining at 37°C for 2–10 min. Then, the cells were washed twice with PBS, followed by the addition of 100 µL fresh culture medium and subsequently fluorescence imaging.

4.17. Cell Apoptosis Assay

For in vitro cell apoptosis analysis, 4T1 cells were seeded in 6‐well plates at a cell density of 5.0 × 10⁵ cells/well and cultured for 24 h. Then, the cells were treated with PBS (control), AuSt@MOS, AuSt@MOS‐Cu₂O, and AuSt@MOS‐Cu₂O/DSF (30 µg/mL, Au basis) for 12 h. Afterwards, the culture medium was removed, the cells were collected, and washed with PBS for cell apoptosis analysis using the Annexin V‐FITC/PI apoptosis detection kit [14]. Specifically, the cells were stained with annexin V‐FITC (5 µg mL⁻¹) and PI (100 µg mL⁻¹), and analyzed by flow cytometry.

4.18. Scratch Experiment

Scratch wound assay was used to study the migration and proliferation of 4T1 cells treated with different nanomedicine. Specifically, 4T1 cells were seeded in 6‐well plates at a cell density of 5.0 × 10⁵ cells/well and cultured for 24 h. Then, the cell monolayers were scratched with a sterile 10 µL pipet to create scratch wounds, and the cell culture medium was replaced with fresh RPMI‐1640 medium containing 15 µg/mL of AuSt@MOS, AuSt@MOS‐Cu₂O, and AuSt@MOS‐Cu₂O/DSF, respectively. After incubated for 24 h, the photographic images were recorded at 0 and 24 h using a Zeiss Axio Vert. A1 inverted fluorescence microscope. The scratch area was calculated using ImageJ software.

4.19. In Vivo Pharmacokinetic and Biodistribution Studies

The AuSt@MOS‐Cu₂O/DSF was intravenously administered into 4T1 tumor‐bearing mice at a dose of 15 mg/kg in 200 µL PBS (n = 3). For blood pharmacokinetic analysis, 25 µL of the blood was drawn from the tail vein of each mouse at different time‐points (10 min, 30 min, 1 h, 3 h, 9 h, 18 h, and 30 h). The blood was subsequently digested with aqua regia, and the Au content was determined by ICP‐MS. For in vivo biodistribution study, the mice were sacrificed at different time‐points (12 h, 24 h, and 48 h) post‐administration, and the major organs (heart, liver, spleen, lung, and kidney) and tumors were harvested, which were digested with aqua regia for quantitative analysis of the Au content by ICP‐MS. The Au percentage of injection dose per gram (Au %ID/g) was determined by the following equation:

$$\mathrm{Au}\%\mathrm{ID}/\mathrm{g}=\frac{\mathrm{Tested}\mathrm{amount}}{\mathrm{Injected}\mathrm{amount}}/\left(\mathrm{Sample}\mathrm{weight}\right)\times 100$$ (9)

where Tested amount was the amount of Au in the harvested organs or tumors determined by ICP‐MS, Injected amount was the amount of Au in the administered formulations, and Sample weight was the total weight of harvested organs or tumors.

4.20. In Vivo Anticancer Efficacy Evaluation

When the tumor volumes reached approximately 100 mm³, the mice were randomly divided into six groups (n = 5), which were intravenously administered with (1) PBS (200 µL), (2) CuET (3.0 mg/kg), (3) AuSt@MOS (15 mg/kg), (4) AuSt@MOS (15 mg/kg) + laser, (5) AuSt@MOS‐Cu₂O/DSF (15 mg/kg), and (6) AuSt@MOS‐Cu₂O/DSF (15 mg/kg) + laser. For the laser irradiation (+) groups, the tumors were irradiated with an 808 nm‐laser at a power density of 1.0 W/cm² for 5 min after 24 h of intravenous administration. During the laser irradiation, the tumor temperature was recorded in real‐time using an FLIR A35 infrared thermal imaging camera. The tumor volume and body weight were monitored every other day over a treatment course of 14 days. The tumor volume (V) was calculated based on V = (tumor length × (tumor width)²)/2, and the relative tumor volume was calculated as V/V₀ for each mouse, where V₀ was the initial tumor volume at the beginning of the treatment. On day 14 post‐administration, the mice were sacrificed, and the tumors were harvested. The tumors were weighted, photographed, and analyzed by hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling (TUNEL) staining analysis.

4.21. Hemolytic Study

The hemolysis assay was performed to evaluate the hemocompatibility of the AuSt@MOS‐Cu₂O/DSF. Briefly, whole blood was collected from healthy mice and centrifuged at 3500 rpm for 10 min to collect red blood cells (RBCs). The supernatant was removed and the pellet was washed with PBS several times until the supernatant was clear and colorless. Subsequently, various concentrations of the AuSt@MOS‐Cu₂O/DSF (10, 20, 40, 60, 80, and 100 µg/mL) were mixed with RBCs in PBS. RBCs dispersed in ultrapure water and PBS were set as the positive and negative controls, respectively. After incubation at 37°C for 12 h, all the samples were centrifuged at 3500 rpm for 10 min, and the absorbance (A) of the supernatant was detected at 416 nm for calculation of the hemolysis rate by the following equation:

$$\mathrm{Hemolysis}\mathrm{rate}\left(\%\right)=\frac{A_{\mathrm{sample}}-A_{\mathrm{negative}}}{A_{\mathrm{positive}}-A_{\mathrm{negative}}}\times 100$$ (10)

where A _sample, A _positive, and A _negative represent the absorbance at 416 nm of the supernatant in test sample, positive group, and negative group, respectively.

4.22. In Vivo Biosafety Assessment

The 4T1 tumor‐bearing mice were intravenously administered with PBS (200 µL, control), (2) CuET (3.0 mg/kg), (3) AuSt@MOS (15 mg/kg), and (4) AuSt@MOS‐Cu₂O/DSF (15 mg/kg). On day 14 post‐administration, the mice were sacrificed, and the major organs were excised for H&E staining analysis. Blood was collected on day 14 post‐administration for blood biochemical analysis and liver/kidney function examination, including alanine transaminase (ALT), aspartate transferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), and lactate dehydrogenase (LDH).

4.23. Statistical Analysis

Experimental data were presented as mean ± standard deviation (SD), and all experiments were carried out with at least three repeats independently unless noted otherwise. Data analysis was conducted with Origin 2019b and GraphPad Prism 8.3.0 software. The statistical significance of differences among multiple groups was calculated using one‐way analysis of variance (ANOVA) with *p < 0.05 considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: smll73875‐sup‐0001‐SuppMat.docx.

Li J., Liu B., Su H., and Li M., “A Cascade‐Activatable Prodrug Nanomedicine for Photothermally Augmented in Situ Synergistic Chemo/Chemodynamic Therapy.” Small 22, no. 39 (2026): e73875. 10.1002/smll.73875

Data Availability Statement

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