pH-responsive Fe/Mn-doped mesoporous silica nanozymes for synergistic chemophotothermal therapy in breast cancer via hypoxia alleviation and controlled drug release

2.1. Materials

All chemical compounds used were obtained from Sigma-Aldrich (St Louis, MO, USA). Human breast cancer cell (MDA-MB-231) and human umbilical vein endothelial cell (HUVEC) were obtained from the Pasteur Institute of Tehran.

2.2. Synthesis of mSiO₂-Fe/Mn@DOX hybrid nanozymes

The mSiO₂-Fe/Mn@DOX hybrid nanozyme was synthesized via a three-phase process. First, a solution of cetyltrimethylammonium chloride (CTAC, 24 mL, 25 wt%) and triethanolamine (0.18 g) in water (36 mL) was stirred at 60°C for 1 h. Subsequently, tetraethyl orthosilicate (TEOS, 20 mL of a 20 v/v% solution in 1-octadecene) was carefully layered onto the mixture and magnetically stirred at 60°C for 12 h. The upper 1-octadecene phase was then replaced with a 20% (v/v) TEOS solution in decahydronaphthalene, and the reaction was continued for a further 12 h. This step was repeated by replacing the oil layer with a TEOS solution in cyclohexane under the same conditions for an additional 12 h. The resulting mesoporous silica nanoparticles (mSiO₂) were collected by centrifugation, washed repeatedly with ethanol, and the CTAC template was removed by refluxing in a 0.6 wt% ethanolic ammonium nitrate solution at 60°C for 6 h. For metal doping, the purified mSiO₂ was dispersed in 25 mL of water, followed by the addition of MnCl₂·4H₂O (0.25 mmol), FeSO₄·7H₂O (0.5 mmol), and NH₄Cl (15 mmol). After the addition of ammonia solution (1 mL) and brief sonication at room temperature, the mixture was transferred to a Teflon-lined stainless-steel autoclave and heated at 140°C for 16 hrs. The resulting mSiO₂-Fe/Mn product was washed three times with distilled water and ethanol, then dried at 60°C. Finally, for drug loading, 10 mg of the mSiO₂-Fe/Mn nanozyme was added to 25 mL of a dimethyl sulfoxide (DMSO) solution containing 9 mg of DOX and gently stirred for 24 h. The final product, mSiO₂-Fe/Mn@DOX, was collected by centrifugation, washed with phosphate-buffered saline (PBS) to remove unencapsulated drug, and dried at room temperature for 24 h.

2.3. Characterization of mSiO₂-Fe/Mn and mSiO₂-Fe/Mn@DOX nanozymes

The morphology and dimensions of the mSiO₂-Fe/Mn hybrid nanozymes were characterized using scanning electron microscopy (FE-SEM; TESCAN MIRA3 FRENCH) at an accelerating voltage of 20 kV and transmission electron microscopy (TEM; HRTEM, JEM-2010) at 100 kV. Hydrodynamic size and zeta potential measurements for both mSiO₂-Fe/Mn and mSiO₂-Fe/Mn@DOX were conducted using a Zetasizer (Malvern Instruments, UK). The specific surface area and pore characteristics were evaluated from N₂ adsorption-desorption isotherms measured at liquid nitrogen temperature (77 K) using a Quantachrome Nova automated gas adsorption system. The pore size distribution was derived from the adsorption branch of the isotherm using the Barrett-Joyner-Halenda (BJH) method, complemented by density functional theory (DFT) calculations. Crystalline structure was analyzed by X-ray diffraction (XRD) on a Rigaku D/max diffractometer with Cu Kα radiation. Data were collected in a continuous scanning mode over a 2θ range of 20° to 80° with a step size of 0.05° and a scanning speed of 2° min^-1. Finally, elemental composition and distribution were assessed by energy-dispersive X-ray (EDX) spectroscopy and elemental mapping, performed on the powdered samples (TESCAN MIRA3 FRENCH).

2.4. Drug loading and release

The drug loading capacity of the mSiO₂-Fe/Mn@DOX hybrid nanozymes was evaluated by incubating 100 μg of the nanozymes with varying concentrations of DOX (20, 40, 60, 80, and 120 μg mL^-1) under gentle shaking (100 rpm, 23°C) for 24 h. After magnetic separation and two washes with PBS, the concentration of unbound DOX in the supernatant was quantified by fluorescence spectroscopy (Hitachi F-2500 spectrometer), and the loading capacity was calculated using Eq. (1).

A is the total amount of DOX in the initial solution, and B is the amount of DOX remaining in the solution.

The drug loading was further confirmed by thermogravimetric analysis (TGA) using a Perkin-Elmer TGA-7 instrument. Samples were heated from 50 to 450°C under a nitrogen atmosphere at a rate of 5°C min^-1.

The drug release profile was investigated under different physiological conditions. mSiO₂-Fe/Mn@DOX was dispersed in PBS at two pH values (7.2 and 6.5), with and without the application of PTT (808 nm laser, 2 W cm^-2, 3 min), while shaking at 100 rpm. At predetermined time intervals (22.5, 45, 90, 180, 360, 720, and 1440 min), 5 mL aliquots were withdrawn and replaced with an equal volume of fresh pre-warmed buffer. The amount of DOX released was determined by measuring the absorbance at 480 nm, and the cumulative release percentage was calculated using Eq. (2).

C_i and C_n refer to the DOX concentration at time i and n, respectively.

2.5. Peroxidase and catalase activity

The peroxidase-like activity of the nanozymes was evaluated by UV-Vis spectroscopy (Shimadzu UV-2600). The reaction mixture, with a total volume of 504 μL, contained 2 μL of either mSiO₂-Fe/Mn or mSiO₂-Fe/Mn@DOX (both at 100 μg/mL), 500 μL of sodium acetate buffer (pH 4.5), 2 μL of 3,3′,5,5′-tetramethylbenzidine (TMB; 20 mg mL^-1), and 2 μL of H₂O₂ (30%). For conditions involving PTT, the nanozymes were subjected to laser irradiation (808 nm, 2 W/cm²) prior to addition. The solutions were incubated in the dark for 10 min, after which the absorbance was measured to quantify the peroxidase-mediated oxidation of TMB.

Furthermore, oxygen generation was monitored in aqueous solutions at pH 6.5 using a portable dissolved oxygen meter (Seven2GO pro S9 DO, Mettler Toledo). A solution of 10 mL sodium acetate buffer containing 200 μL of H₂O₂ (30%) was prepared, and the initial O₂ concentration was recorded. Upon the addition of the nanozymes (mSiO₂-Fe/Mn or mSiO₂-Fe/Mn@DOX, 100 μg mL^-1), with or without a prior PTT treatment (808 nm laser, 2 W cm^-2, 3 min), the O₂ production (mg L^-1) was measured at time points of 0, 18, 37, 75, 150, 225, 300, 450, and 600 s.

2.6. Fe and Mn ions release

The fabricated mSiO₂-Fe/Mn’s biodegradation was assessed at pH 7.4 (physiological) and 6.5 (tumor) to evaluate its pH-responsive behavior. The mSiO₂-Fe/Mn@DOX was incubated in PBS at pH 6.5 (with and without PTT) and 7.4 at 37°C with shaking. Inductively coupled plasma mass spectrometry (ICP-MS) was used to measure Fe and Mn ion concentrations in the supernatant at 0, 3, 6, 12, 24, and 48 h.

2.7. In vitro trials

Human umbilical vein endothelial cells (HUVEC) and MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco) and maintained in an incubator at 37°C with 5% CO₂. For passage, cells were trypsinized (0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) and resuspended in DMEM.

2.8. Spheroid formation and cytotoxicity

MDA-MB-231 spheroids were generated by seeding 10³ cells in low-adhesion 24-DMEM penicillin-streptomycin, followed by incubation at 37°C under 5% CO₂ for 7 days. Spheroids exceeding 100 μm in diameter reliably model the hypoxia and interstitial permeability barriers of solid tumors, thereby replicating the associated physical and microenvironmental drug resistance [17]. After the 7-day formation period, spheroids were treated for 48 h with a range of concentrations of free DOX (0.31–10 μM), mSiO₂-Fe/Mn (0.27–8.65 μg mL^-1), and mSiO₂-Fe/Mn@DOX (0.27–8.65 μg mL^-1), with or without PTT. PTT was applied 8 h post-treatment initiation using an 808 nm laser at 2 W/cm² for 3 min. Spheroid viability was assessed 48 h post-treatment using an Alamar Blue assay. Spheroids were incubated with Alamar Blue (10% v/v of the culture volume) for 24 h, and fluorescence intensity was measured (excitation: 535 nm, emission: 595 nm) using a Beckman Coulter DTX 880 microplate reader.

Based on the established optimal nanozyme concentration that minimizes toxicity to healthy cells while controlling proliferation, we further investigated the growth inhibition of MDA-MB-231 spheroids. Spheroids were treated with selected concentrations of DOX (2.5 and 5 μM), mSiO₂-Fe/Mn (2.16 and 4.32 μg mL^-1), and mSiO₂-Fe/Mn@DOX (2.16 and 4.32 μg mL^-1), with and without PTT. Spheroid size was monitored by imaging with ImageJ software on day 7 (designated as treatment day 1) and day 13 (6-day post-treatment).

2.9. Mechanisms of cytotoxicity

To investigate the toxicity mechanism, RNA was extracted from drug-treated spheroids using Trizol reagent (Sinaclon Bio Science, Iran). RNA concentration and purity were then assessed via Nanodrop spectrophotometry (Thermo Fisher Scientific, USA), followed by DNase I treatment to remove genomic DNA contamination. Subsequently, cDNA was synthesized using the BONmiR™ miRNA qRT-PCR Detection Kit (Stem Cell Technology Research Center, Tehran, Iran) following the manufacturer’s protocol. Real-time quantitative PCR was performed on an ABI PRISM 7500 Fast Sequence Detection System (USA) using Power SYBR®Green PCR Master Mix (Applied Biosystems, USA) with the following cycling conditions: 95°C for 5 min, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. β-Actin was used as an internal reference gene. mRNA expression levels were determined using the 2^−(ΔΔCT) method, with all reactions performed in triplicate. Primer sequences are:

β-actin: F- 5´-CTTCTACAATGAGCTGCGTG-3´, R-5´-TCATGAGGTAGTCAGTCAGG-3´;

TNF-α: F-5´-CTCTTCTGCCTGCTGCACTTTG-3´, R-5´-ATGGGCTACAGGCTTGTCACTC-3´;

CASP7: F-5´-CGGAACAGACAAAGATGCCGAG-3´, R- 5´-AGGCGGCATTTGTATGGTCCTC-3´;

Bcl-2: F-5´-ATCGCCCTGTGGATGACTGAGT-3´, R-5´-GCCAGGAGAAATCAAACAGAGGC-3´;

CASP9: F-5´-GTTTGAGGACCTTCGACCAGCT-3´, R-5´-CAACGTACCAGGAGCCACTCTT-3´;

CASP3: F-5´-GGAAGCGAATCAATGGACTCTGG-3´, R-5´-GCATCGACATCTGTACCAGACC-3´.

2.10. Statistical analysis

Statistical analysis was performed using GraphPad Prism 6.01 (GraphPad Software, San Diego, CA, USA). Data are presented as mean ± SD. Multiple group comparisons were analyzed by analysis of variance (ANOVA), and individual group comparisons by Student’s t-test. Significance was defined as p < 0.05.

3.1. Morphology and mSiO₂-Fe/Mn@DOX properties

Scanning electron microscope (SEM) (Figure 1a) and transmission electron microscope (TEM) (Figure 1b) images show that the mSiO₂-Fe/Mn hybrid nanozymes are spherical with rough surfaces and a relatively uniform size of 30–60 nm. dynamic light scattering (DLS) analysis (Figure 1c) confirmed a narrow hydrodynamic size distribution with an average diameter of 40.9 nm, predominantly ranging from 30 to 50 nm. The nanozymes exhibited a zeta potential of -25.1 mV at neutral pH (Figure 1d), indicating high colloidal stability favorable for in vivo applications. This negative surface charge within the -5 to -30 mV range at pH 7.4 promotes nanozyme dispersion, efficient drug loading, and delivery in aqueous environments [10]. Nitrogen adsorption-desorption analysis revealed a type IV isotherm with distinct hysteresis loops in the P/P₀ ranges of 0.36–0.52 and 0.52–0.97 (Figure 1e), confirming the mesoporous structure of the mSiO₂-Fe/Mn hybrid nanozymes. The material possessed a specific surface area of 52.26 m² g^-1, with pore sizes ranging from 1.1 to 18.2 nm and an average pore width of 5.26 nm (Figure 1e(i)). XRD patterns of mSiO₂ nanospheres and mSiO₂-Fe/Mn hybrid nanozymes are shown in Figure 1(f). The mSiO₂ nanospheres (black curve) displayed a broad bulge between 22° and 32°, characteristic of their amorphous silica structure, consistent with known patterns superimposing on quartz Bragg reflections [18]. For the mSiO₂-Fe/Mn nanozymes, the presence of distinct peaks at 2θ = 33.7° (220), 36.2° (311), 45.3° (400), 54.3° (422), 57.4° (511), and 63.5° (440) confirms the successful deposition of iron oxide. Additional peaks at 2θ = 27.9° and 36.2° indicate the incorporation of manganese oxide, suggesting that calcination resulted in the formation of the mSiO₂-Fe/Mn composite. The XRD pattern of the drug-loaded system (mSiO₂-Fe/Mn@DOX) showed no new diffraction peaks compared to the unloaded nanozymes (Figure 1f). This indicates that the DOX is in an amorphous state, confined within the mesoporous channels rather than forming a crystalline phase on the surface. The absence of drug-derived crystalline features confirms that the loading process preserves the structural integrity of the nanozyme, affirming the stability of the hybrid architecture for drug delivery. energy-dispersive x-ray (EDX) spectroscopy and elemental mapping (Figures 2a and b) corroborate the XRD results. The EDX spectrum revealed a Si:O:Fe:Mn weight ratio of 33.2%:34.9%:21.8%:10.1%, confirming the co-deposition of Fe and Mn. Furthermore, the elemental maps (Figure 2b) indicate a more extensive surface coverage of iron compared to manganese on the mSiO₂ nanospheres.

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Figure 1.

(a) SEM and (b) TEM images of mSiO₂-Fe/Mn hybrid nanozymes, (c) the size distribution of mSiO₂-Fe/Mn@DOX hybrid nanozymes, (d) Zeta potential values of mSiO₂-Fe/Mn and mSiO₂-Fe/Mn hybrid nanozymes, (e) N₂ adsorption–desorption isotherms (i) The size of pores on mSiO₂-Fe/Mn is indicated by the inset) of mSiO₂-Fe/Mn hybrid nanozymes, and (f) XRD patterns of the synthesized mSiO₂, mSiO₂-Fe/Mn hybrid nanozymes and mSiO₂-Fe/Mn@DOX hybrid nanozymes.

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Figure 2.

(a) EDX spectra and (b) element mapping of mSiO₂-Fe/Mn hybrid nanozymes, (c) Standard absorption curve of DOX in different concentrations, (d) DOX loading and its efficiency, (e) TGA curves of mSiO -Fe/Mn and mSiO₂-Fe/Mn@DOX hybrid nanozymes, and (f) Quantitative analyses of DOX release at 37°C at different pH with or without PTT.

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3.2. Drug loading and release

Figures 2(c,d) demonstrate that while the absolute DOX loading in the mSiO₂-Fe/Mn hybrid nanozymes increases with higher DOX concentrations, the corresponding loading efficiency decreases. The highest loading efficiency (exceeding 50%) was achieved at initial DOX amounts of 20 and 40 μg per 100 μg of nanozyme. To maintain a safety margin for nanozyme administration in normal cells, a loading ratio of 60 μg DOX per 100 μg mSiO₂-Fe/Mn was selected for subsequent experiments, yielding a loading efficiency of 40.9%. This observed plateau in loading is attributed to the saturation of the available nanopore volume and surface area. A loading capacity of approximately 40% indicates that the physical and chemical adsorption sites within the porous architecture were fully occupied, precluding further DOX incorporation. This finding is consistent with the measured specific surface area, which is influenced by the successful incorporation of Fe/Mn species within the silica matrix. TGA data (Figure 2e) corroborate the loading results, indicating a total DOX content of 39.9 wt% in the mSiO₂-Fe/Mn@DOX sample based on the observed weight loss. The degradation of the loaded drug occurred in two distinct phases: a rapid weight loss of 31.52% between 90°C and 180°C, followed by a slower decline of 9.37% from 180°C to 310°C. This two-stage mass loss is likely due to the sequential thermal decomposition of different structural components and functional groups within the DOX molecules. In contrast, the TGA profile of the unloaded mSiO₂-Fe/Mn nanozymes indicates high thermal stability between 300°C and 450°C, with negligible weight change. The minor weight loss of 7.1 wt% observed between 100°C and 300°C is attributed to the evaporation of residual pore water and minimal structural decomposition.

Figure 2(f) demonstrates that DOX release from the nanozyme at 37°C is both pH-responsive and time-dependent, with a significantly higher cumulative release observed under acidic conditions compared to neutral pH (55.5% vs. 37.2%). The combination of an acidic environment and PTT further enhanced the release to 83.9%. This synergistic effect is likely attributable to PTT-induced amplification of the nanozyme’s peroxidase-like activity (Figure 2f). Although the initial DOX release from mSiO₂-Fe/Mn@DOX at pH 6.5 (15.98%) was only marginally higher than at pH 7.2 (11.37%), the application of PTT under acidic conditions substantially increased this initial release by 28.43%. This pH-triggered enhancement promotes the targeted and synergistic release of DOX within the acidic tumor microenvironment, potentially increasing therapeutic efficacy by concentrating the drug in cancerous tissue. The data suggest that the accelerated drug release is primarily driven by the degradation of the nanoparticle framework, rather than merely enhanced DOX diffusion. This is supported by the strong correlation between the DOX release profile and the leaching kinetics of Fe and Mn ions under acidic conditions (Figures 2f). The synergistic effect of PTT, which boosts peroxidase-like activity (Figure 3a), concurrently accelerates the dissolution of the inorganic matrix and the rapid release of the therapeutic payload. This correlation indicates that nanoparticle disintegration is the principal mechanism governing drug release.

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Figure 3.

(a) Peroxidase-like activities of mSiO₂-Fe/Mn and mSiO₂-Fe/Mn@DOX hybrid nanozymes with or without PTT, (b) Catalase-like activities of mSiO₂-Fe/Mn and mSiO₂-Fe/Mn@DOX hybrid nanozymes with or without PTT, (c) Fe and (d) Mn ions release profiles from mSiO₂-Fe/Mn@DOX hybrid nanozymes with or without PTT at different pH.

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3.3. Peroxidase- and catalase-mimic activity

Nanozymes exhibiting peroxidase-like activity catalyze the decomposition of H₂O₂, generating reactive radicals that oxidize the substrate TMB to blue-colored oxTMB (Figure 3a). The lack of significant absorption in a control mixture of TMB and H₂O₂ confirms that this oxidation is nanozyme-dependent. The mSiO₂-Fe/Mn hybrid nanozyme successfully catalyzed this reaction, as evidenced by a distinct color change and the characteristic absorption peak at 645 nm (Figure 3a), confirming its intrinsic peroxidase-like activity. While drug loading partially reduced the catalytic activity of the mSiO₂-Fe/Mn@DOX nanozyme, its synergy with PTT (808 nm laser, 2 W/cm², 3 min) significantly enhanced the peroxidase-like activity, overcoming the initial suppression caused by DOX. Furthermore, the nanozymes demonstrated catalase-like activity by generating O₂, a key function for mitigating tumor hypoxia. As illustrated in Figure 3(b), the O₂ production of the drug-loaded nanozyme (mSiO₂-Fe/Mn@DOX) was measured at 9.3 mg L^-1. However, when combined with PTT, the O₂ production was significantly boosted to 14.2 mg L^-1. This represents a 1.27-fold increase compared to the unloaded mSiO₂-Fe/Mn nanozymes, indicating a potent strategy for ameliorating tumor hypoxia. The results indicate that the released Fe²⁺/Fe³⁺ and Mn²⁺ ions act as catalytic centers, mimicking catalase activity to decompose endogenous H₂O₂ into O₂. This reaction serves a dual purpose: it directly alleviates hypoxic conditions by generating O₂, and it simultaneously depletes the antioxidant H₂O₂, thereby disrupting cellular redox homeostasis. This dual action collectively fosters a pro-oxidant environment that initiates and perpetuates a therapeutic cascade of ROS.

3.4. Fe and Mn ions release

ICP-MS analysis (Figures 3c, d) revealed a distinct time-dependent release profile for Fe and Mn ions from the nanozymes, which was significantly influenced by environmental stimuli. The release kinetics were biphasic, characterized by an initial burst within the first 8 h, followed by a more sustained and gradual release over the remaining 48 h period. The acidic environment (pH 6.5), which mimics the tumor microenvironment, markedly accelerated the degradation of the mSiO₂-Fe/Mn nanozymes, leading to a significantly higher release of both Mn and Fe ions compared to neutral conditions (Fe: 8.8% vs. 3.5%; Mn: 24.1% vs. 12.9%). The highest degree of ion release was observed for the mSiO₂-Fe/Mn@DOX formulation in synergy with PTT, resulting in cumulative releases of 31.3% for Fe and 39.9% for Mn. Notably, the DOX release profile under acidic conditions (Figure 2f) closely mirrored the leaching kinetics of the metal ions. This strong correlation suggests a reciprocal relationship where the degradation of the inorganic framework, indicated by ion release, is the principal driver for the concomitant release of the encapsulated drug.

3.5. Cell viability

The cytotoxicity of mSiO₂-Fe/Mn hybrid nanozymes, both with and without DOX loading, was evaluated in HUVEC (normal) and MDA-MB-231 (cancer) cells using a CCK-8 assay. In HUVEC cells, the mSiO₂-Fe/Mn nanozymes demonstrated no significant cytotoxicity at concentrations up to 2.16 μg mL^-1 (Figure 4a). Notable toxicity to these normal cells was only observed at higher concentrations (≥ 4.32 μg mL^-1). This indicates a favorable biocompatibility profile within a specific concentration window, supporting its potential as a catalytic agent and drug carrier. In MDA-MB-231 breast cancer cells, the toxicity of free DOX, mSiO₂-Fe/Mn, and mSiO₂-Fe/Mn@DOX, both with and without PTT (808 nm, 2 W cm^-2, 3 min)—exhibited a clear dose-dependent response (Figure 4b). The mSiO₂-Fe/Mn@DOX nanozymes were the most effective, achieving 63.5%, 73.2%, and 76.5% growth inhibition at concentrations of 2.16, 4.32, and 8.64 μg mL^-1, respectively. Based on the biocompatible threshold established in HUVEC cells (Figure 4a), a concentration of 2.16 μg mL^-1 of nanozymes (equivalent to 2.5 μM DOX) was identified as optimal for further study. At this biocompatible concentration, the combination of mSiO₂-Fe/Mn@DOX and PTT resulted in superior cancer cell death (69.2% growth inhibition) compared to either treatment alone. Notably, significant cell inhibition (59.2%) was still achieved even at a lower, and likely safer, nanozyme concentration of 1.08 μg/mL when combined with PTT. In summary, the cytotoxic effect of DOX on MDA-MB-231 cells was significantly enhanced in a dose-dependent manner by nanozyme-based delivery and synergistic PTT, while maintaining lower toxicity in non-cancerous cells.

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Figure 4.

(a) Cell viability of human umbilical vein endothelial cell (HUVEC) incubated with mSiO₂-Fe/Mn hybrid nanozymes, (b) MDA-MB-231 cells viability after incubation with different concentrations of DOX, mSiO₂-Fe/Mn, mSiO₂-Fe/Mn@DOX hybrid nanozymes with and without PTT for 48 h. *p<0.05, and **p<0.01.

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3.6. Apoptosis and ROS

Optimized doses of the mSiO₂-Fe/Mn nanozyme (2.16 μg mL^-1) and free DOX (2.5 μM), selected based on minimal HUVEC toxicity and effective MDA-MB-231 cell death, were used to analyze apoptosis and necrosis via flow cytometry (Figure 5a). DOX treatment increased the population of both early (Q3: 9.15% vs. control 5.0%) and late (Q2: 18.5% vs. control 4.57%) apoptotic cells compared to the control. The mSiO₂-Fe/Mn nanozyme alone induced less apoptosis (early: 6.43%; late: 13.8%) than free DOX. However, loading DOX onto the nanozyme (mSiO₂-Fe/Mn@DOX) enhanced its efficacy, resulting in higher levels of both early (Q3: 12.3%) and late (Q2: 22.3%) apoptosis compared to either agent alone. As anticipated, the combination of mSiO₂-Fe/Mn@DOX with PTT (808 nm, 2 W cm^-2, 3 min) produced the greatest pro-apoptotic effect, with early and late apoptotic populations rising to 13.2% and 26.7%, respectively, indicating promising therapeutic potential. This enhanced apoptosis is mechanistically linked to intracellular ROS generation. The mSiO₂-Fe/Mn@DOX nanozyme significantly increased ROS levels by 1.27-fold and 1.22-fold compared to the mSiO₂-Fe/Mn nanozyme and free DOX, respectively (Figure 5b). The highest ROS level (90.6) was achieved through the synergy of mSiO₂-Fe/Mn@DOX with PTT, directly corroborating the observed apoptosis data. Overall, the combination therapy shifted the cell death pattern, enhancing the transition from early to late apoptosis (evident as a shift from Q1/Q4 to Q2 quadrants). Consistent with these findings, morphological assessment of MDA-MB-231 cells (Figure 5c) revealed characteristic signs of cell death, such as shrinkage and reduced cell number, following treatment with DOX, mSiO₂-Fe/Mn, or mSiO₂-Fe/Mn@DOX, with the most pronounced effects observed in the combination group with PTT.

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Figure 5.

(a) Flow cytometric analysis of live and dead MDA-MB-231 cells in different treatment groups: control, DOX (2.5 μM), mSiO₂-Fe/Mn (2.16 μg mL^-1), mSiO₂-Fe/Mn@DOX (2.16 μg mL^-1) hybrid nanozyme with and without PTT. Cell necrosis and apoptosis were measured using propidium iodide (PI) and Annexin V-FITC staining. (b) Representative DCFH staining of MDA-MB-231 cells in different treatments for ROS evaluation. (c) Optical microscopy images of MDA-MB-231 cells treated with different treatments (scale bar: 100 μm).

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3.7. Spheroids formation and cytotoxicity

Alamar Blue viability assays on MDA-MB-231 spheroids revealed a dose-dependent toxicity for both free DOX and the mSiO₂-Fe/Mn nanozyme. Maximum growth inhibition was observed at 10 μM DOX (61.8%) and 8.64 μg mL^-1 mSiO₂-Fe/Mn (62.0%) (Figure 6a). However, initial toxic effects were noted at lower concentrations of 5 μM DOX (51.9% inhibition) and 4.32 μg mL^-1 mSiO₂-Fe/Mn (52.3% inhibition), which were previously established as toxic to normal HUVEC cells and are therefore unsuitable for therapeutic use. Loading DOX onto the nanozyme (mSiO₂-Fe/Mn@DOX) improved the therapeutic index, achieving significant toxicity at the biocompatible concentration of 2.16 μg mL^-1 (equivalent to 2.5 μM DOX). Furthermore, combining mSiO₂-Fe/Mn@DOX with PTT (808 nm, 2 W cm^-2, 3 min) synergistically enhanced the anticancer effect, resulting in 65.4% growth inhibition at this safe dosage. This synergy also enabled effective therapy at an even lower, safer nanozyme dose of ≤1.08 μg mL^-1.

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Figure 6.

(a) MDA-MB-231 spheroids viability after incubation with different concentrations of control, DOX (0.31-10 μM), mSiO₂-Fe/Mn (0.27-8.65 μg mL^-1), mSiO₂-Fe/Mn@DOX (0.27-8.65 μg mL^-1) hybrid nanozymes with and without PTT for 48 hrs. (b) Optical microscopy images of MDA-MB-231 spheroids incubated in different treatment groups: DOX (2.5 μM), mSiO₂-Fe/Mn (2.16 μg mL^-1), mSiO₂-Fe/Mn@DOX (2.16 μg mL^-1) hybrid nanozymes with and without PTT (Scale bar: 50 μm), and (c) Average of diameter of MDA-MB-231 spheroids in different treatment groups: DOX (2.5 μM), mSiO₂-Fe/Mn (2.16 μg mL^-1), mSiO₂-Fe/Mn@DOX (2.16 μg mL^-1) hybrid nanozymes with and without PTT.

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Based on the optimal dose determined from spheroid toxicity (Figure 6a) and normal cell biocompatibility (Figure 4a), we assessed the structural integrity of MDA-MB-231 spheroids treated with 2.5 μM DOX and 2.16 μg mL^-1 mSiO₂-Fe/Mn@DOX (Figure 6b). Control spheroids grew rapidly from 93.7±7.6 μm on day 1 to 153.3±8.5 μm on day 6 post-treatment. Both free DOX and mSiO₂-Fe/Mn nanozymes suppressed growth (109.6±6.0 μm and 110.3±7.5 μm, respectively), with no significant difference between them (Figure 6c). Although these treatments inhibited growth compared to the control, the fact that spheroids remained larger and denser on day 6 than on day 1 indicates limited efficacy. In contrast, mSiO₂-Fe/Mn@DOX treatment not only inhibited growth but significantly reduced spheroid size to 80.1±9.0 μm, demonstrating a more potent effect. The most profound outcome was achieved by combining mSiO₂-Fe/Mn@DOX with PTT, which reduced spheroid size to 58.6±4.5 μm, indicating a highly effective and disruptive therapeutic action.

3.8. Cytotoxicity mechanisms

Quantitative real-time polymerase chain reaction (qRT- PCR) analysis of MDA-MB-231 spheroids (Figure 7) confirmed that the nanozymes induce apoptosis through both the intrinsic and extrinsic pathways. While free DOX and the mSiO₂-Fe/Mn nanozymes activated the extrinsic pathway to a similar extent, DOX had a more pronounced effect on the intrinsic pathway. Loading DOX onto the nanozyme platform (mSiO₂-Fe/Mn@DOX) enhanced cancer cell death more effectively than either agent alone by concurrently activating both apoptotic pathways. This was evidenced by upregulation of extrinsic pathway markers (TNF-α and CASP7) and intrinsic pathway markers (increased CASP9 and decreased B-cell lymphoma 2 (BCL-2)). The combination of mSiO₂-Fe/Mn@DOX with PTT produced the most significant pro-apoptotic effect. Compared to mSiO₂-Fe/Mn@DOX alone, the PTT-synergized treatment led to a substantial upregulation of key apoptotic genes: TNF-α (19.14 vs. 11.01) and CASP7 (6.45 vs. 3.66) in the extrinsic pathway, and CASP9 (9.84 vs. 5.90) in the intrinsic pathway, alongside a marked downregulation of the anti-apoptotic protein Bcl-2 (0.14 vs. 0.26). This coordinated gene expression profile, culminating in increased caspase-3 activity, demonstrates a powerful synergistic mechanism for inducing tumor cell death.

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Figure 7.

The effect of DOX (2.5 μM), mSiO₂-Fe/Mn (2.16 μg/mL), mSiO₂-Fe/Mn@DOX (2.16 μg mL^-1) hybrid nanozymes with and without PTT on the extrinsic (expression of TNF-α, CASP7, and CASP3) and intrinsic (expression of BCL2 and CASP9) mechanisms of apoptosis in MDA-MB-231 spheroids. *p<0.05, **p<0.01 and ***p<0.001.

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Nanozymes that respond to tumor microenvironment (TME) cues such as acidic pH and elevated H₂O₂ hold significant promise for cancer therapy, particularly when combined with adjuvant treatments like PTT [19]. However, clinical translation faces challenges, including dosage control due to off-target interactions, limited tumor-specific accumulation, and the environmental instability of nanozymes. To address the issue of accumulation, the mSiO₂-Fe/Mn@DOX hybrid nanozymes were designed with a size under 80 nm (Figure 1c), aligning with findings that nanocarriers between 30-80 nm optimize tumor penetration through the enhanced permeability and retention (EPR) effect [20,21]. As emphasized in the field, controlling nanozyme size to overcome biological barriers and manipulating the tumor microenvironment (TME) for controlled catalysis are critical steps forward [3]. The functional efficacy of our platform is consistent with recent advances. Similar to a previous study [22], the mSiO₂-Fe/Mn@DOX nanozymes exhibited enhanced peroxidase-like (Figure 3a) and catalase-like (Figure 3b) activities under hypoxic conditions, leading to increased oxygen and free radical generation (Figure 3). This catalytic activity directly contributes to treating resistant cancers by improving drug permeability through oxygen generation and inducing apoptosis via •OH radical production [23]. Furthermore, in agreement with previous studies [24, 25], we found that the nanozymes, especially when synergized with PTT, significantly increased cancer cell apoptosis (Figure 5a), suggesting a potent strategy to overcome drug resistance. The mechanism underlying this efficacy was confirmed by the observed intracellular ROS production (Figure 5b), which signifies successful nanozyme penetration and directly contributes to apoptotic cell death. This ROS-mediated apoptosis confirms the effectiveness of the nanozymes in suppressing both 2D cultures and 3D spheroids [25]. Our results in Figure 5 also corroborate the findings of Fang et al. [26], demonstrating that the synergistic application of nanozymes and PTT co-activates apoptotic pathways and intracellular ROS to potently suppress cancer growth. Finally, the targeted drug delivery capability of our system is a key feature. The mSiO₂-Fe/Mn@DOX nanozymes are engineered for pH-responsive drug release, a strategy supported by Xu et al. [27] for treating resistant cancers. The DOX release profile (Figure 2f), characterized by an initial burst and sustained release, was highly dependent on environmental acidity. This pH-sensitivity facilitates targeted delivery by enhancing DOX solubility and accelerating the nanozyme’s degradation, thereby promoting the release of Fe and Mn ions (Figures 3c and d) that further drive catalytic therapy. This synergistic mechanism of ion release and drug delivery is consistent with the previous observations [19, 27].

Cultured MDA-MB-231 cells confirmed the favorable drug release and catalytic activity of the mSiO₂-Fe/Mn@DOX hybrid nanozyme in suppressing breast cancer cells, which aligns with the previous finding [28]. However, a key therapeutic challenge was identified: while higher doses of mSiO₂-Fe/Mn nanozymes increased cancer cell suppression (Figure 4b), concentrations exceeding 2.16 μg/mL induced significant toxicity in normal cells (Figure 4a). Furthermore, the doses deemed safe for normal cells (2.5 μM DOX and 2.16 μg mL^-1 mSiO₂-Fe/Mn) were effective in 2D monolayer cultures but proved inadequate against the more resistant MDA-MB-231 spheroids. Conversely, increasing the concentration to effective spheroid-suppressing levels (5 μM DOX, 4.32 μg mL^-1 nanozyme) created an unacceptably toxic environment for normal cells. This underscores the critical importance of the synergistic effect achieved by loading DOX onto the nanozyme. The mSiO₂-Fe/Mn@DOX platform at the safe dose of 2.16 μg mL^-1 minimized toxicity to normal cells while significantly enhancing growth inhibition in both MDA-MB-231 cells (62.56%) and their spheroids (54.33%) (Figures 4b, 6a). The therapeutic profile was further amplified by combining mSiO₂-Fe/Mn@DOX with PTT, which boosted spheroid suppression to 65.46% (Figure 6a). Notably, this synergy enabled effective treatment at an even lower, safer dose of 1.08 μg mL^-1, which achieved 56.29% spheroid growth inhibition while maintaining 72.89% viability in normal cells. This approach of combining nanozymes with PTT to enhance efficacy at biocompatible doses is strongly supported by the previous work [29]. It is well-established that 3D spheroid cultures reduce drug penetration and efficacy compared to 2D monolayers [30,31]. Our results demonstrate that the synergy between mSiO₂-Fe/Mn@DOX and PTT effectively overcomes this penetration barrier [32,33]. The mechanism involves the PTT-enhanced release of metal ions and the subsequent generation of •OH radicals, which improves drug permeability and initiates a powerful apoptotic cascade through both intrinsic and extrinsic pathways (Figure 7) [34,35]. Through this multifaceted action, the mSiO₂-Fe/Mn@DOX hybrid nanozyme successfully arrests and suppresses the growth of resistant MDA-MB-231 spheroids. While this study demonstrates the promising therapeutic potential of the mSiO₂^-Fe/Mn@DOX nanozyme platform, several limitations warrant acknowledgment to guide future research:

• Preclinical models: The findings are primarily based on 2D cell cultures and spheroids. Although spheroids offer greater complexity than monolayers, they cannot fully recapitulate the in vivo TME, which includes immune components, vascularization, and interstitial fluid pressure. Future work should prioritize in vivo studies to evaluate the nanozyme’s efficacy, biodistribution, and reliance on the EPR effect, providing a more clinically relevant assessment of its targeting and therapeutic performance.

• Long-term toxicity: The observed toxicity of higher nanozyme doses (>2.16 μg mL^-1) to normal cells, though reduced compared to free DOX, highlights the need for a comprehensive biosafety assessment. A critical next step is a detailed in vivo toxicological study to evaluate potential long-term accumulation of Fe/Mn ions in vital organs (e.g., liver, spleen, kidneys) and any associated chronic effects.

• Mechanistic insight: While we documented the upregulation of key apoptotic markers (TNFα, CASP3, CASP7, CASP9), the precise molecular cascades and signaling pathways initiated by the combined action of released DOX, metal ions, and ROS require further elucidation. Deeper mechanistic investigations, such as transcriptomic and proteomic analyses, are recommended to fully delineate the cell death pathways involved.

• Optimization of combination therapy: The synergy with PTT was crucial for enhancing efficacy and reducing the required drug dose. However, the parameters for this combination, such as the timing of PTT application relative to nanozyme accumulation and the optimal laser dosage and wavelength, were not exhaustively optimized. Future work should systematically explore these variables to establish a maximally effective and safe treatment protocol.