Evaluation of the performance of Fe 3 O 4 /MnO 2 hybrid nanozymes with doxorubicin on multicellular structure and their therapeutic management to limit the growth of human breast cancer cells

Evaluation of the performance of Fe 3 O 4 /MnO 2 hybrid nanozymes with doxorubicin on multicellular structure and their therapeutic management to limit the growth of human breast cancer cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of the performance of Fe 3 O 4 /MnO 2 hybrid nanozymes with doxorubicin on multicellular structure and their therapeutic management to limit the growth of human breast cancer cells Majid Sharifi, Mohammad Kamalabadi-Farahani, Amir-Abas Salmani, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4417286/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Sep, 2024 Read the published version in Cancer Nanotechnology → Version 1 posted 16 You are reading this latest preprint version Abstract Overwhelming evidence suggests that nanozymes show great promise in cancer therapy due to their stable catalytic properties and cost-effectiveness. However, the diverse responses of nanozymes in therapy have presented challenges. After designing pH-sensitive Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes with catalytic properties, we analyzed their characteristics using various techniques such as SEM, TEM, DLS, XRD, TGA, EDS, etc. We evaluated the nanozymes' toxicity on MCF-7 cells and their spheroids through MTT and flow cytometry assays, while also exploring their synergistic effects with photothermal therapy (PTT). The findings reveal that the 150–270 nm Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes demonstrate stable DOX release and catalytic activity in generating O 2 and ° OH, effectively inhibiting the growth of MCF-7 cells. It was found that the effective concentration for MCF-7 cells had to be raised from 2.13 to 4.64 µg/mL to inhibit spheroid growth. Because of the toxicity of this concentration on normal cells, using synergistic approaches is crucial to minimize side effects. Also, the results of cytotoxicity mechanism in spheroids highlight the significant impact of PTT with Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes in enhancing pro-inflammatory cytokines like TNF-α, CASP9, CASP7, and CASP3. Ultimately, optimizing the concentration of pH-sensitive hybrid nanozymes with PTT synergistic effects shows great potential for cancer treatment. Breast cancer Spheroids Hybrid nanozymes Doxorubicin Photothermal therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Breast cancer is currently one of the deadliest cancers in women, with around a third of affected women dying even after mastectomy. The main treatment involves drugs, which have been very effective in improving survival rates. However, about half of patients undergoing drug therapies suffer a relapse [ 1 ]. The main reason for treatment failure is resistance to anticancer drugs, caused by poor drug penetration due to significant hypoxia and blockages in tumor areas. Alongside traditional treatments like surgery, chemotherapy, and radiation therapy, nanozymes are being investigated as a less invasive option with catalytic properties. In addition to their ability to targeted release of drugs in tumor environments with a pH 6.5, nanozymes exhibit promise in drug-resistant tumors treatment by increasing O 2 levels to improve drug penetration, producing radicals (O 2 °¯ and ° OH), and synergizing with photothermal therapy (PTT) [ 2 , 3 ]. Among nanozymes mimicking peroxidase and catalase, the use of Fe 3 O 4 in tumor therapy is interesting due to high compatibility and Fenton reactions [ 4 ]. Fe 3 O 4 nanozymes are amazing for producing ° OH in acidic conditions through peroxidase-like activity on H 2 O 2 , as well as generating hydrogen peroxyradicals in neutral and alkaline conditions, similar to catalase-like activity for the production of O 2 and H 2 O [ 5 ]. To enhance the catalytic performance of Fe 3 O 4 nanozymes, the incorporation of MnO 2 nanozymes is proposed [ 6 ]. The addition of MnO 2 not only effective in Fe + 2 regeneration, but also reduces tumor hypoxia by converting H 2 O 2 to H 2 O and O 2 in acidic conditions [ 7 ]. Despite challenges like low selectivity and biocompatibility linked to MnO 2 [ 8 ], its capacity to convert superoxide into ° OH and H 2 O 2 during catalysis has increased its potential for use in cancer therapy [ 6 , 7 , 9 ]. Furthermore, Fe 3 O 4 and MnO 2 nanoparticles exhibit enhanced catalytic pathways under photothermal and photodynamic therapy activities, as evidenced in studies by Du et al. [ 10 ], Chen et al. [ 11 ], Cun et al. [ 12 ] and Zhang et al. [ 6 ]. The utilization of PTT technique to enhance thermal energy from laser irradiation in the tumor microenvironment is a non-invasive and precise method that can boost confidence in cancer treatment through synergistic interaction with nanozymes. It has been found that the heat generated by PTT enhances the glucose oxidase activity for starvation, peroxidase- and catalase-like activities of nanozymes to generate ° OH and O 2 , and also restricts cancer cell growth by activating the extrinsic apoptosis pathways [ 13 – 15 ]. As a result, it is anticipated that the treatment of drug-resistant tumors will be expedited through the inherent functionality of nanozymes and the cascading amplification of radicals, O 2 , and heat production induced by PTT. In addition to the significance of nanozymes and PTT in the therapeutic effects on breast cancers, the selection of the drug type is crucial. The standard treatment for advanced breast cancers involves doxorubicin (DOX), an anthracycline antibiotic [ 16 ]. DOX disrupts DNA and RNA centers, offering a reliable treatment route for solid tumors [ 17 ]. However, its usage is restricted due to heart tissue toxicity [ 18 ], and drug resistance to DOX remains inevitable [ 19 ]. Therefore, extensive research is required to reduce DOX toxicity in non-target tissues while maintaining high efficacy in tumor tissues. Numerous studies demonstrate that nanozymes with DOX effectively inhibit the growth of breast cancer cells [ 20 – 22 ]. The screening of anticancer drugs has traditionally been carried out using two-dimensional cell cultures due to their cost-effectiveness, speed, and multiple assays [ 23 ]. However, two-dimensional cultures do not accurately represent cell-cell and cell-extracellular matrix (ECM) interactions [ 24 ]. As a result, drug functions in this system can lead to false predictions of tumor response and clinical failures. Thus, it is noted that only around 5% of anticancer compounds advance to the clinical phase because of inadequate pharmacokinetics or uncertain efficacy [ 25 ]. Meanwhile, some argue that nanotechnology is on an uncertain path, relying on animal models without a full understanding of the intricate interactions of drug-resistant cancer cells events and the various biological barriers in human tissues [ 26 ]. To address this challenge, three-dimensional cell culture from human cancer cells, known as spheroid models, have been recommended to better mimic the uniform tissue of cancer cells [ 24 , 27 ]. Despite limitations such as lack of interaction with the extracellular environment, lower physical resistance, and insufficient cell diversity, studies have shown that therapeutic responses in this model closely resemble in vivo conditions [ 6 , 22 , 23 , 28 ]. In different scenarios, it has been revealed that the lack of complete penetration of therapeutic compounds due to high cell density and the presence of ECM, the change of oxygen gradient from the surface to the center, and the overexpression of anti-apoptotic proteins in the center of spheroids, anti-tumor therapeutic responses are significantly different compared to two-dimensional cultures [ 6 , 29 – 31 ]. In this research, we developed Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes with dual functionality for drug delivery and catalytic activity to inhibit MCF-7 cells. We then explored their antitumor effects in both two-dimensional culture and spheroid models. Alongside analyzing the Fe 3 O 4 /MnO 2 @DOX nanozymes' physicochemical characteristics and DOX loading/release capabilities, we investigated the enhanced efficacy against MCF-7 cells through synergistic actions with PTT. Our findings suggest that using Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes with catalytic properties and targeted drug release can create a new therapeutic opportunity for treating drug-resistant cells. However, focusing on cell organizational structures is crucial for advancing therapeutic objectives. 2. Material and methods 2.1. Materials All chemical and biological materials utilized in this research were procured from Merck, Germany. Human breast cancer MCF-7 cells were acquired from Shahroud University of Medical Sciences. 2.2. Synthesis of Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes The Fe 3 O 4 nanozymes were synthesized using the solvothermal approach. FeCl 3 .6H 2 O (3.25 g) and tri-sodium citrate (1.3 g) were dispersed in 100 mL of ethylene glycol and stirred continuously for 30 minutes. Subsequently, NaAc (6.0 g) was added to the mixture and sonicated for 1 hour. The resulting solution was then transferred to an autoclave set at 220°C and left for 10 hours. Following cooling, the product was washed three times with ethanol and distilled water before being dispersed in deionized water. Next, PAA (20 mg, 0.2 g/mL) and 150 µL of NH 3 .H 2 O (5 M) were added to the Fe 3 O 4 solution. The mixture was stirred for 20 minutes. Subsequently, 80 mL of Isopropanol was gradually introduced to the solution with vigorous shaking to produce Fe 3 O 4 @PAA. Following this, 100 µL of Mn (II) acetate tetrahydrate (50 mg/mL) was carefully dripped into the solution and stirred for 3 hours. The resulting Fe 3 O 4 /MnO 2 hybrid nanozymes were separated by centrifugation and washed with deionized water. The resulting precipitate was dried at 60°C, and the Fe 3 O 4 /MnO 2 hybrid nanozymes were then calcined at 450°C in an N 2 atmosphere for 4 hours. To load DOX (Doxorubicin), 10 mg of Fe 3 O 4 /MnO 2 hybrid nanozymes were added to 25 mL of dimethyl sulfoxide solution with 9 mg of DOX and kept for 24 hours with gentle agitation. The Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes were subsequently air-dried for 24 hours at room temperature and washed with PBS for further use. 2.3. Characterization of Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes To analyze the surface morphology and internal structures, field-emission scanning electron microscopy (FESEM; MIRA3, TESCAN) and high-resolution transmission electron microscopy (TEM; HRTEM, JEM-2010) were utilized with an acceleration voltage of 200 kV. The Zetasizer system (Malvern Instruments, UK) was employed to determine hydrodynamic size and zeta potential at 25°C. N 2 absorption isotherms of the samples at liquid nitrogen temperature (-196°C) were measured using Nova's Quantachrome automatic gas absorption system to assess the nanozyme cavities. The pore size distribution was considered from the desorption branch of the isotherm through the Barth-Joyner-Holland process. X-ray diffraction patterns of nanozymes were obtained through the XRD method. X-ray examination was conducted with a D/max- using Cu Kα radiation (Rigaku, Japan) in continuous scan mode ranging from 20°-70° with a step size of 0.02° and speed of 2°/min. Energy dispersive X-ray spectroscopy (EDS) and elemental mapping studies were carried out on a JEOL JEM2010 electron microscope at 100 kV. Also, the peroxidase-like activity of nanozymes were assessed (an UV-vis spectrometer, Shimadzu UV-2600) by studying the oxidation of TMB (3,3′,5,5′-tetramethylbenzidine) as a reaction substrate. To do this, 2 µL of 100 µg/mL Fe 3 O 4 nanozymes, Fe 3 O 4 /MnO 2 hybrid nanozymes, and Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes without and with PTT (Photothermal therapy: 808 nm laser light: 2 W/cm 2 for 3 minutes) were mixed with 500 µL of sodium acetate buffer at pH 6.5, along with 2 µL of 20 mg/mL TMB and 2 µL of 30% H 2 O 2 (hydrogen peroxide). Subsequently, the solutions were incubated in darkness for 10 minutes to carry out the peroxidase-like activity. Furthermore, the rise in ambient temperature due to the use of PTT (808 nm laser light: 2 W/cm 2 for 3 minutes) on Fe 3 O 4 /MnO 2 hybrid nanozymes (5 mg/mL in water distiller) were monitored with a thermometer while being exposed to 808 nm wavelength irradiation at various intervals: 0, 5, 10, 20, 40, 80, 160, 320, and 640 seconds. 2.4. Loading and validation tests, and drug release study To determine the loading capacity, 100 µg of Fe 3 O 4 /MnO 2 hybrid nanozymes were placed in drug solution with concentrations of 30, 60, 90, 120 and 150 µg for 24 hours at room temperature with gentle shaking (at 100 rpm). After separation of Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes by magnet and washing by PBS, the remaining solution was assessed and analyzed (based on Eq. 1) by fluorescence spectroscopy as the initial solution (Hitachi F 2500 spectrometer). Equation 1: Loading efficiency (%) = [(DIS − DRS)/DRS] × 100; DIS is the total amount of DOX in initial solution and DRS is the amount of DOX remaining in the solution. To explore drug loading, thermogravimetric analysis (TGA) was assessed using Perkin-Elmer TGA-7 under N 2 at a heating rate of 5°C/min in the range of 70–450°C. A superconducting quantum interference device (SQUID, MPMS-XL) was used to assess the magnetic properties of Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes, ranging from − 8,000 to + 8,000 G at 298 m. In the following, in vitro DOX release from Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes were conducted at 37°C in a shaker at 150 rpm in PBS solution with pH of 7.2 and 6.5, both with and without PTT (808 nm laser irradiation: 2 W/cm 2 for 3 minutes). At specified time intervals (0.37, 0.75, 1.5, 3, 6, 12 and 24 hours), 2 mL of the sample was withdrawn from each container and replaced with an equal volume of PBS to maintain a constant volume. The drug release extent was assessed by measuring the absorbance at 428 nm using a UV–vis spectrophotometer. The total DOX release was calculated using the following Eq. (2): Equation 2: Cumulative DOX release (%) = \(\frac{5 \times {\sum }_{\text{i}-1}^{\text{n}-1}{\text{C}}_{\text{i}}+50 \times {\text{C}}_{\text{n}}}{\text{w}\text{e}\text{i}\text{g}\text{h}\text{t} \text{o}\text{f} \text{D}\text{O}\text{X} \text{o}\text{n}\text{F}\text{e}3\text{O}4/\text{M}\text{n}\text{O}2@\text{D}\text{O}\text{X}}\times 100\) ; C i and C n refer to the of DOX concentration at time i and n, respectively. 2.5. O 2 and ° OH generation O 2 generation in aqueous solutions was assessed using a portable dissolved oxygen meter (Seven2GO pro S9 DO, Mettler Toledo). To conduct the experiment, 100 µg/mL of Fe 3 O 4 nanozymes, Fe 3 O 4 /MnO 2 hybrid nanozymes, Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes with and without PTT (808 nm laser irradiation: 2 W/cm 2 for 3 minutes) were introduced into 10 mL of sodium acetate buffer with 200 µL of 30% H 2 O 2 . The O 2 production (mg/mL) was measured at various intervals: 0, 18, 37, 75, 150, 225, 300, 450, and 600 seconds. Also, terephthalic acid was utilized to measure the ° OH level. Following the interaction of terephthalic acid with Fe 3 O 4 nanozymes, Fe 3 O 4 /MnO 2 hybrid nanozymes, Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes with and without PTT (808 nm laser irradiation: 2 W/cm 2 for 3 minutes), and its conversion to 2-hydroxy terephthalic acid, the ° OH level was assessed at a wavelength of 430–435 nm. 2.6. Cell culture The NIH3T3 and MCF-7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (GIBCO, New York) with 10% fetal bovine serum (FBS, AusGeneX) and 1% penicillin/streptomycin. Cell flasks were maintained in an incubator at 37°C with 5% CO2 and 95% humidity. For cell transfer to new culture medium, trypsinization (0.25% trypsin-EDTA) and re-suspension in DMEM medium were performed. 2.6.1. Toxicity evaluation Cytotoxicity tests were conducted using the MTT method on NIH3T3 and MCF-7 cells. Initially, a density of 8×10 3 NIH3T3 and MCF-7 cells were plated in a 96-well plate and incubated for 12 hours at 37°C in 5% CO 2 . Subsequently, NIH3T3 cells were exposed to specific concentrations of 0.27, 0.54, 1.08, 2.16, 4.32, and 8.65 µg/mL of Fe 3 O 4 /MnO 2 nanozymes. Also, MCF-7 cells were exposed to various concentrations of DOX (0.31, 0.62, 1.25, 2.5, 5, and 10 µM), Fe 3 O 4 /MnO 2 hybrid nanozymes (0.27, 0.54, 1.08, 2.16, 4.32, and 8.65 µg/ml), and Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes (0.27, 0.54, 1.08, 2.16, 4.32, and 8.65 µg/ml). Additionally, a set of MCF-7 cells received treatment with an 808 nm laser at 2 W/cm 2 for 3 minutes following an 8-hour exposure to Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes (0.27, 0.54, 1.08, 2.16, 4.32, and 8.65 µg/ml). Then, the cells were then incubated for 48 hours. Afterward, they were rinsed and 100 µL of fresh medium containing 20 µL of MTT solution (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide: 0.5 mg/mL) was introduced into each well. The culture medium was once again incubated in darkness at 37°C for 4 hours. The resultant purple formazan crystals were dissolved in 100 µL of DMSO (dimethyl sulfoxide), and the absorbance was assessed at 570 nm with a multi-well plate reader. The survival rates of both treated and control cells were calculated using Eq. 3. Equation 3: cell viability (%)= [[Optical density of dosing cells-Optical density of blank]÷[ Optical density of control-Optical density of blank]]×100 2.6.2. Apoptosis and ROS assays To further study the inhibition of MCF-7 cell growth by Fe 3 O 4 /MnO 2 nanozymes and DOX, the apoptosis level was assessed using the Annexin-V/PI Apoptosis Analysis Kit (Yeasen, Inc., China) and flow cytometry. MCF-7 cells were seeded in a 6-well plate at a density of 5×10 5 as described in section 2.6 . and then incubated for 12 hours. Afterward, the old culture medium was replaced with a new one containing DOX (2.5 µM), Fe 3 O 4 /MnO 2 hybrid nanozymes (2.16 µg/mL), and Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes (2.16 µg/mL) with and without PTT (808 nm laser irradiation, 2 W/cm 2 for 3 minutes) onto the plates. The MCF-7 cells were then cultured at 37°C with 5% CO2 for 48 hours. After incubation, MCF-7 cells were harvested by centrifugation at 1000g (5 min) and washed thrice in cold PBS. The cells were trypsinized, resuspended in 200 µL of binding buffer, and then stained with Annexin V-FITC/Alexa Fluor 488 (5 µL) and propidium iodide (PI: 10 µL) following the manufacturer's instructions. Subsequently, the stained cells were analyzed using flow cytometry in the absence of light. To assess intracellular ROS levels, 5×10 5 cells per well were seeded in a 6-well plate. After 12 hours of incubation at 37°C with 5% CO2, the cells were treated with DOX (2.5 µM), Fe 3 O 4 /MnO 2 nanozymes (2.16 µg/mL), Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes (2.16 µg/mL) without or with PTT (808 nm laser irradiation, 2 W/cm 2 for 3 minutes). The MCF-7 cells were re-incubated for 48 hours. Subsequently, the cells were rinsed with PBS and treated with 10 µM 2,7-dichlorodihydrofluorescein. After a 30-minute incubation period, the cells underwent two PBS washes. The level of ROS was then assessed using FACscan (BD Bioscience, USA) by measuring the fluorescence intensity of 2,7-dichlorofluorescein produced through the oxidation of 2,7-dichlorodihydrofluorescein. 2.6.3. MCF-7 spheroids formation, cytotoxicity and morphometry To generate MCF-7 3D spheroids, a method involving reducing FBS levels on non-adherent surfaces was employed. Briefly, MCF-7 cells were cultured at a density of 10 3 cells in ultra-low-attachment 24-well plates with DMEM medium supplemented with high glucose, 0.5% FBS, and 2% penicillin-streptomycin (all sourced from Gibco, USA) at 37°C in a 5% CO 2 . The MCF-7 spheroids were then incubated for six days. Afterwards, the MCF-7 spheroids were exposed to varying concentrations of DOX (0.31, 0.62, 1.25, 2.5, 5, and 10 µM), Fe 3 O 4 /MnO 2 hybrid nanozymes (0.27, 0.54, 1.08, 2.16, 4.32, and 8.65 µg/ml), and Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes (0.27, 0.54, 1.08, 2.16, 4.32, and 8.65 µg/ml) for 48 hours. Moreover, a set of MCF-7 spheroids received treatment with an 808 nm laser irradiation at 2 W/cm 2 for 3 minutes following an 8-hour exposure to Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes (0.27, 0.54, 1.08, 2.16, 4.32, and 8.65 µg/ml). Proliferation and viability of MCF-7 spheroids were evaluated 48 hours post-treatment with therapeutic compounds utilizing Alamar Blue cell viability reagent. As per the manufacturer's instructions, 10 µg/mL (one-tenth of the total culture medium volume) of Alamar Blue was added to each MCF-7 spheroid and kept to incubator for 24 hours. Subsequent to the incubation period, fluorescence intensity was measured on plates at 535 nm excitation and 595 nm emission using a DTX 880 microplate reader from Beckman Coulter in Brea, CA. Furthermore, to study the growth inhibition of MCF-7 spheroids by DOX (5 µM), Fe 3 O 4 /MnO 2 hybrid nanozymes (4.32 µg/mL), Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes (4.32 µg/mL) without or with PTT (an 808 nm laser irradiation at 2 W/cm 2 for 3 minutes), the dimensions of spheroids were evaluated using images with ImageJ software on the 6th day (equivalent to the first day of treatment) and the 11th day (5 days after treatment). 2.6.4. Mechanisms of cytotoxicity Total RNA extraction was performed using the Trizol reagent (Sinaclon Bio Science, Iran) on spheroids 5-day post-treatment. The concentration and purity of RNA samples were assessed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA). DNase I was utilized to eliminate genomic contamination from the isolated RNA. Complementary DNA (cDNA) was generated using the BONmiR™ qRT-PCR miRNA Detection Kit (Stem Cell Technology Research Center, Tehran, Iran) following the manufacturer's instructions. Quantitative real-time PCR was conducted using SYBR Premix Ex Taq™ II (Takara Bio, Japan) and monitored with the Applied Biosystems® StepOneTM instrument and ABI7500 thermocycler following this protocol: initial activation at 95°C for 5 min, 40 cycles of denaturation at 95°C for 5 seconds, and annealing/extension at 60°C for 30 seconds. PCR primer pair 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´; Caspase-3: F-5´-GGAAGCGAATCAATGGACTCTGG-3´, R-5´- GCATCGACATCTGTACCAGACC-3´; β-actin served as the internal reference gene for normalizing gene expression. mRNA expression level was determined using the 2 −(ΔΔCT) method. All reactions were conducted in triplicate. 2.8. Statistical analysis The data were analyzed using one-way analysis of variance. Statistical significances were assessed with the Statistical Package for Social Science (version 20) and Tukey’s multiple comparison tests. P-values below 5% were deemed statistically significant. 3. Results 3.1. Morphology and chemical properties The morphology and size features of the engineered hybrid nanozymes, as revealed by SEM (Fig. 1 a) and TEM (Fig. 1 c) analyses, indicate that the Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes, ranging from 150 to 270 nm, consist of two fused nanospheres with surface cavities. While the DLS results indicate that the size of Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes ranges vary from 90 to 370 nm, the highest concentration can be seen in the range of 140 to 270 nm. Meanwhile, the porous structure of the Fe 3 O 4 /MnO 2 hybrid nanozymes was verified through nitrogen absorption and desorption output, as depicted in Fig. 1 E. The N 2 adsorption-desorption isotherm outcomes exhibit type IV behavior with noticeable residual rings within the 0.30–0.44, 0.45–0.78 and 0.79–0.97 P/P0 range. The surface area of the Fe 3 O 4 /MnO 2 hybrid nanozymes was calculated at 65.266 m 2 /g, featuring cavities ranging from 1.2 to 34.7 nm with an average size of 7.34 nm (Fig. 1 F). Moreover, the zeta potential measurements reveal that Fe 3 O 4 nanozymes and Fe 3 O 4 /MnO 2 hybrid nanozymes exhibit − 3.14 mV and − 25.2 mV values, respectively, under neutral conditions, demonstrating the effective incorporation of MnO 2 into the Fe 3 O 4 nanozymes. Also, Fig. 1 H illustrates the XRD patterns of Fe3O4 nanozymes and Fe 3 O 4 /MnO 2 hybrid nanozymes. The Fe 3 O 4 nanozymes (black curve) shows diffraction peaks at (2θ = 30.1°: 220), (2θ = 35.0°: 311), (2θ = 44.6°: 400), (2θ = 54.6°: 422), (2θ = 56.6°: 511), and (2θ = 63.5°: 440) corresponding to Fe 3 O 4 (PDF#19–629). In contrast, the hybrid nanozymes (red curve) exhibits these Fe 3 O 4 peaks along with two additional peaks at 2θ = 27.3° and 2θ = 36.9°, indicating the presence of MnO 2 nanozymes (PDF#30–0820). This suggests that Mn(Ⅱ) acetate was calcined to produce Fe 3 O 4 /MnO 2 . In the following, elemental mapping from EDS analysis reveals the atomic distribution of metal ions on Fe 3 O 4 /MnO 2 hybrid nanozymes (Fig. 2 A and 2 B) with a Mn: Fe: O weight ratio of 37.3%: 22.6%: 40.1%. The mapping indicates that Fe is concentrated in the smaller nanospheres while Mn is predominant in the larger one (Fig. 2 C). The Fig. 2 D data indicates that exposing the nanozymes to 808 nm laser irradiation at 2 W/cm 2 for 3 minutes’ results in a temperature rise across various pH levels. Transitioning from a neutral (pH 7.2) to an acidic (pH 6.5) environment, akin to the acidity found in cancerous tumors, effectively triggers heat generation from Fe 3 O 4 /MnO 2 hybrid nanozymes. Also, the investigating the peroxidase-like activity revealed that Fe 3 O 4 /MnO 2 hybrid nanozymes exhibit favorable peroxidase characteristics in the presence of H 2 O 2 and at a pH of 6.5. In Fig. 2 E, TMB is converted by nanozymes into the blue oxTMB product. As anticipated, the inclusion of MnO 2 on the Fe 3 O 4 nanozymes enhances the catalytic performance. Furthermore, the synergism of PTT with Fe 3 O 4 /MnO 2 hybrid nanozymes significantly enhances the catalytic efficiency. 3.2. Loading and validation tests, and drug release DOX loading content in Fig. 3 A revealed that as the concentration of DOX increased, the rate of drug loading in Fe 3 O 4 /MnO 2 hybrid nanozymes also significantly increased with nanozymes constant concentration. However, as anticipated, the drug loading efficiency percentage decreased with higher DOX concentration. In summary, the findings indicate that the highest drug loading efficiency percentage (over 51.1%) is achieved with 60 µg of drug in 100 µg of Fe 3 O 4 /MnO 2 hybrid nanozymes. In this regard, TGA confirmed DOX presence in Fe 3 O 4 /MnO 2 hybrid nanozymes. As shown in Fig. 3 B, Fe 3 O 4 /MnO 2 hybrid nanozymes exhibit high stability at temperatures up to 450°C. The slight mass decrease of Fe 3 O 4 /MnO 2 hybrid nanozymes between 100–300°C in the thermogram, accounting for ~ 4.9% by weight, is attributed to water loss within the pores and minor structural damage to Fe 3 O 4 /MnO 2 hybrid nanozymes. However, the stability of Fe 3 O 4 /MnO 2 hybrid nanozymes in terms of weight percentage between 300°C and 450°C demonstrates their robustness. Concurrently, the TGA curve reveals a weight loss of up to 42.48% for Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes, confirming the presence of DOX on Fe 3 O 4 /MnO 2 hybrid nanozymes. The weight percentage decrease in Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes exhibits a two-step transition: a gradual decline from 95°C to 190°C (14.9% weight reduction) followed by a steeper decrease from 195°C to 260°C (27.4% weight reduction). This behavior is likely attributed to the structural segments and functional groups of the drug molecule undergoing degradation. Furthermore, to support the aforementioned results, it was observed that the magnetic saturation of Fe 3 O 4 nanozymes consistently reduces from 64.9 emu/g to 45.0 emu/g and 42.6 emu/g in the presence of MnO 2 nanozymes and DOX. Hence, the assertion regarding drug loading on Fe 3 O 4 /MnO 2 hybrid nanozymes is substantiated. The Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes' capability to release DOX was studied at 37°C under varying acidity levels. Figure 2 D illustrates that DOX release from the nanozymes is time-dependent. Generally, DOX release is higher in acidic conditions (51.9%) compared to neutral conditions (25.8%). Moreover, the synergism of PTT with acidity boosts DOX release up to 81.19% by enhancing the peroxidase-like activities of the nanozymes. Furthermore, a higher burst release of DOX from Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes is observed at pH 6.5 (17.93%), exceeding the release at pH 7.2 (12.98%). Despite the somewhat negative impact of PTT in an acidic environment on the burst release of DOX, with an increase of up to 38.12%, controlled release improvement can help maintain a stable therapeutic dosage over 24 hours. Furthermore, by enhancing Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes performance in acidic conditions, particularly through synergistic effects, it becomes feasible to boost DOX delivery in tumor areas with pH levels ranging from 6 to 6.5. This can also elevate drug efficacy by concentrating it in tumor tissues. Evidence for this was seen in the significant reduction of drug release at pH 7.2. 3.3. O 2 generation Evaluation of catalytic activity in Fig. 3 E indicates that the presence of MnO 2 nanozymes on the Fe 3 O 4 nanozymes enhances the nanozyme's catalytic activity for O 2 generation by nearly twofold, increasing from 8.4 to 16.2 mg/L. Despite the negative impact of DOX loading on the catalytic activity of Fe 3 O 4 /MnO 2 hybrid nanozymes in O 2 generation (14.1 mg/L), this study indicates that the combined effect of PTT with Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes notably enhances the catalytic activity for O 2 generation (19.8 mg/L) (Fig. 3 E). Overall, this discovery indicates that boosting O 2 generation through Fe 3 O 4 /MnO 2 hybrid nanozymes post DOX release, or utilizing Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes alongside PTT (808 nm laser irradiation: 2 W/cm 2 for 3 minutes), can mitigate hypoxia and enhance drug permeation. In the following, the results depicted in Fig. 3 F indicate that the Fe 3 O 4 /MnO 2 hybrid nanozymes effectively enhance the ° OH level compared to non-hybrid nanozymes. This enhancement is evidenced by the increased fluorescence intensity of 2-hydroxyterephthalic acid. While the presence of DOX on the Fe 3 O 4 /MnO 2 hybrid nanozymes decreases the ° OH level, it seems that the release of the drug at pH 6.5 leads to an increase in ° OH levels. Moreover, the study revealed that the Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes, in synergism with PTT, significantly elevate the ° OH level compared to other experimental groups. 3.4. Cytotoxicity of Fe 3 O 4 /MnO 2 @DOX To investigate the toxicity effect of nanozymes with and without DOX loading, MTT and flow cytometry techniques were employed on NIH-3T3 cells and MCF-7 cells. Figure 4 A shows that NIH-3T3 cells exhibited no significant cytotoxicity when treated with Fe 3 O 4 /MnO 2 hybrid nanozymes at concentrations of 0.27, 0.54, 1.08, and 2.16 µg/mL. However, toxicity increased when the concentration was raised to 4.32 µg/mL or higher. This study demonstrates that Fe 3 O 4 /MnO 2 hybrid nanozymes exhibit favorable biocompatibility as both a catalytic agent and a carrier for delivering anticancer drugs, particularly within a specific range. Similar to the toxicity output in NIH-3T3 cells, it was found in Fig. 4 B that the toxicity of DOX, Fe 3 O 4 /MnO 2 hybrid nanozymes with and without DOX, combined with PTT (808 nm: 2 W/cm 2 for 3 minutes) on MCF-7 cells, is dose-dependent. The study indicates that the most significant suppression of MCF-7 cells by Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes occurs at doses of 2.16, 4.32, and 8.64 µg/mL compared to the control group (60.3%, 70.8% and 72.8% growth inhibition). However, based on the toxicity dose of Fe 3 O 4 /MnO 2 hybrid nanozymes in Fig. 4 A, it is recommended to use a dose of 2.16 µg/mL (equivalent to 2.5 µM DOX). In addition, it was discovered that the synergistic effect of Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes with PTT (808 nm: 2 W/cm 2 for 3 minutes) enhances the toxicity level of nanozymes on cancer cells from 2.16 µg/ml (65.9% growth inhibition) to 1.08 µg/ml (57.3% growth inhibition). It was also revealed that nanozymes, as nanocarriers, significantly increase the toxicity of DOX in all doses compared to DOX. Ultimately, the dose-dependent cytotoxicity of DOX in MCF-7 cells can be enhanced by two methods: loading on nanozymes and synergy with PTT, without inducing toxicity in non-cancerous cells. After determining the toxicity dose from Fig. 4 B, to analyze the percentage of apoptotic and necrotic cells during therapeutic activities, MCF-7 cells were exposed to 2.16 µg/ml concentrations of Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes with and without PTT, along with 2.5 µmol DOX. Flow cytometry results in Fig. 4 C indicate that while DOX led to an increase in late and early apoptotic cells compared to the control (Q2: 33.4% vs 4.11%; Q3: 13.7% vs 5.53%), employing Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes as a drug carrier effectively raised late and early apoptotic cell levels by 34.9% and 13.3%, respectively. Also, it was found that the combination of PTT (808 nm: 2 W/cm 2 for 3 minutes) with Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes induced the highest levels of early (Q2: 36.0%) and late (Q3: 15.3%) apoptotic cells in MCF-7 cells. In support of the crucial role of apoptosis, Fig. 4 D demonstrates that the Fe 3 O 4 /MnO 2 hybrid nanozymes and DOX significantly raised the intracellular ROS level compared to the control. Furthermore, loading DOX on Fe 3 O 4 /MnO 2 hybrid nanozymes resulted in a higher intracellular ROS level. Figure 4 D illustrates that the greatest intracellular ROS is achieved through the synergy of Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes with PTT, confirming the heightened apoptotic level in this particular group. Thus, it was discovered that DOX, Fe 3 O 4 /MnO 2 hybrid nanozymes, Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes with and without PTT, induce the demise of MCF-7 cells by elevating intracellular ROS levels, leading to apoptosis. Supporting this conclusion, the morphological alterations (like changes in volume and shape) and the quantity of cancer cells exposed to DOX, Fe 3 O 4 /MnO 2 hybrid nanozymes, Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes with and without PTT, as depicted in Fig. 4 E, indicate an anticipated cell demise. 3.5. Toxicity in MCF-7 spheroids To investigate the impact of nanozymes toxicity on spheroids from MCF-7 cells, the MTT technique was employed. Figure 5 A illustrates that both DOX and Fe 3 O 4 /MnO 2 hybrid nanozymes exhibit dose-dependent toxicity, with no significant variance observed across different concentrations. The study revealed that the highest toxicity towards MCF-7 spheroids occurs at concentrations of 5 µM for DOX (49.1% growth inhibition) and 4.32 µg/mL for Fe 3 O 4 /MnO 2 hybrid nanozymes (49.8% growth inhibition) and above (58.8% and 60.1% growth inhibition in 10 µM for DOX and 8.64 µg/mL for Fe 3 O 4 /MnO 2 hybrid nanozymes). While, loading DOX onto Fe 3 O 4 /MnO 2 hybrid nanozymes enhances toxicity levels to 2.16 µg/mL (equivalent to 2.5 µM of DOX) (53.3% growth inhibition). Also, Fig. 5 A clearly indicates that utilizing PTT (808 nm: 2 W/cm 2 for 3 minutes) in synergism with Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes exhibits superior anticancer effects across various concentrations. Despite the heightened toxicity of the synergized group at 4.32 (72.8% growth inhibition of spheroids) and 8.65 (75.5% growth inhibition of spheroids) µg/mL, toxicity levels rise until reaching 1.08 µg/mL (55.1% growth inhibition of spheroids). After establishing the toxic dose of DOX and nanozymes in spheroids (Fig. 5 B), MCF-7 spheroids were treated with 5 µM of DOX and 4.32 µg/mL Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes, both with and without PTT. Subsequently, their growth inhibition was assessed. In Fig. 5 B and 5 C, rapid growth is observed in the spheroids of the control group, which are generally dense with a size of 168.3 ± 6.5 µm visible on the 5th day (103 ± 8.5 µm on the first day). Although there is no significant difference between the DOX and Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes groups in inhibiting spheroid growth, both groups effectively suppressed spheroid growth compared to the control group (113.3 ± 8 and 116.6 ± 8.1 µm compared to 163 ± 6.5 µm, respectively). However, the most significant inhibition to MCF-7 spheroid growth was achieved with the Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes (50.69% growth inhibition) and its combined effect with PTT (65.14% growth inhibition). 3.6. Cytotoxicity mechanisms The toxicity mechanism depicted in Fig. 5 D illustrates that Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes, with or without PTT, are highly effective in triggering apoptosis through both intrinsic and extrinsic pathways. However, in terms of synergistic activity, the upregulation of TNF-α (17.64 vs. 10.38) and CASP7 (5.20 vs. 2.88) associated with the extrinsic pathway, along with the downregulation of Bcl-2 (0.21 vs. 0.38) and the elevation of CASP9 (8.10 vs. 5.12) compared to the Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes, offer greater promise in the treatment process. Despite the lack of significant differences between the DOX and Fe 3 O 4 /MnO 2 hybrid nanozymes groups in apoptosis pathways, DOX not only enhances apoptosis induction by increasing CASP3 (4.35 vs. 2.77), but also influences the extrinsic pathway by boosting TNF-α (5.55 vs. 3.20). In conclusion, these findings affirm that synergistic therapeutic approaches yield more dependable outcomes in cancer treatment. 4. Discussion Nowadays, the use of nanozymes with varied catalytic capabilities controlled by pH and constituent elements, along with their integration with therapeutic techniques like PTT, has garnered significant interest. Nonetheless, beyond environmental obstacles and sustained catalytic performance [ 32 ], their efficacy within the therapeutic window dictated by the biological conditions of tumors or tumor cells poses a challenge. By enhancing controllable catalytic activity through element combinations and addressing environmental hurdles [ 33 ], it is hoped that tumor treatment procedures can be enhanced by enhancing drug penetration via reducing hypoxia levels, and modifying the tumor microenvironment. With this premise, following the study of Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes and their structural and physicochemical analysis (Figs. 2 and 3 ), it was discovered that these nanozymes, with improved catalytic activity, lead to a greater O 2 and ° OH levels in hypoxic environments compared to non-hybrid nanozymes [ 7 , 34 ]. According to the findings of Xu et al. [ 35 ], it appears that increasing O 2 levels by enhancing drug permeability and increasing ° OH radical through inducing apoptosis can speed up the drug resistance cancer treatment process. This effect is particularly remarkable when combined synergistically with PTT and radiotherapy, as noted by Lv et al. [ 14 ] and Li et al. [ 36 ]. Also, besides enhancing O 2 and ° OH levels, pH-responsive Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes offer a hopeful strategy for targeted drug delivery in drug-resistant cancers, ensuring efficient drug release at pH 6.5, particularly in synergistic actions, similar to the discoveries of Cheng et al. [ 22 ], Meng et al. [ 37 ], and Chen et al. [ 7 ]. In line with findings of Wang et al. [ 34 ] and Zhu et al. [ 38 ], it was discovered that Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes, particularly in synergistic activity, demonstrates substantial toxicity against MCF-7 cells by promoting apoptosis through enhanced ROS. Nevertheless, in agreement with the findings of Nie et al. [ 39 ] and Zeng et al. [ 40 ], it was found that the Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes, as opposed to DOX administration, could improve the treatment process by targeting of drug release, triggering intracellular ROS, and raising O 2 levels. This increase in the aforementioned events without synergistic effects by PTT could raise hopes for treating tumors inaccessible to laser irradiation. Despite the successes in limiting MCF-7 cell growth or removing them, the low efficacy of drugs or nanocarriers in clinical trials and human treatments has made researchers uncertain about their use. Although descriptive and analytical studies highlight the ineffectiveness of therapeutic approaches due to low EPR (enhanced permeability and retention), drug pump issues, high hypoxia from vessel blockage or excessive O 2 consumption, and physiological differences between animals and humans, and the cells derived from them [ 26 ], the analysis of therapeutic dosage based on the state of organization of cells in the early stages has received less attention. During this study, it was discovered that the optimal treatment dosage for MCF-7 spheroid structures under 200 µm differs significantly from that of two-dimensional cultures. The choice to select spheroids under 200 µm is based on the limitation of oxygen diffusion in tissues larger than 200 µm, as outlined in the empirical and mathematical modeling [ 41 ]. In the following, it was demonstrated that the optimal dosage of DOX and Fe 3 O 4 /MnO 2 hybrid nanozymes needs to be doubled when transitioning from two-dimensional culture to spheroids, going from 2.5 to 5 µM and from 2.16 to 4.32 µg/mL, respectively (Fig. 5 A). Similarly, it was disclosed that the efficient dose of Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes, in altering cellular structure from a two-dimensional setup to a spheroid, also doubled (Fig. 5 A). While, the results in Fig. 4 A indicate that raising the Fe 3 O 4 /MnO 2 hybrid nanozymes dosage to 4.32 µg/mL can lead to adverse effects on biological functions. Therefore, in line with the findings of Al-Kattan et al. [ 28 ] and the results of Fig. 5 A, which confirm the use of synergistic activities with PTT to create the desired toxicity for spheroids with the optimal concentration of nanozymes, it is recommended to utilize synergistic activities. PTT synergy with Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes appears to effectively restrict spheroid growth through various actions, such as elevating O2 and °OH radical levels, enhancing drug release, and raising ambient temperature to 45°C (Fig. 3 F) to increase both intrinsic and extrinsic apoptosis pathways (Fig. 5 D). Furthermore, in line with the aforementioned discovery, Cheng et al. [ 22 ], Zhang et al. [ 6 ] and Emami et al. [ 31 ] demonstrated that altering the organizational structure of MCF-7 and MCF-7/ADR, 4T1 and BT-20 cells from two-dimensional to spheroidal culture can impose less restrictions on the growth of drug-resistant cancer cells at the same dosage, due to decreased drug permeability. Confirming this discovery, it was found that as the dimensions of MCF-7 spheroids increase, the permeability of both the DOX and nanocarriers decreases [ 42 ]. In this regard, Reynolds et al. [ 29 ] demonstrated that while the growth of spheroids' periphery decreased, the core of spheroids significantly increased with the presence of cancer drugs. The limited drug access to the spheroids' core, attributed to higher cell and collagen accumulation, appears to be the primary cause for reduced drug effectiveness. Based on finding of Brancato et al. [ 43 ], it appears that the formation of ECM, caused by changing the culture medium from two-dimensional culture to spheroids, creates more complex barriers to drug penetration. Thus, it is anticipated that the dosage of therapeutic compounds will rise in spheroids as a result of altered drug/nanocarriers penetration patterns, potentially impacting their function through the presence of ECM. Cellular organizations like spheroids versus two-dimensional cultures or larger spheroids versus small spheroids are more resistant to therapeutic activities due to reduced drugs/nanozymes penetration caused by a more compact ECM, changes in O 2 slope, and increased hypoxia in the center of the structure, as well as excessive expression of anti-apoptosis proteins (Bcl-2, BAX, etc.) in the center of cellular clusters [ 29 , 41 , 44 ]. Therefore, it is recommended to use patterns closer to resistant tumors such as spheroids or organoids in academic/pharmaceutical centers to reduce the costs of the research-production process and explain the effects of drugs/Nano-compounds. 5. Conclusions In this study, following the creation and synthesis of Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes, we explored their combined performance with PTT. Physicochemical assessments verified the existence of iron and magnesium, showcasing their enzymatic functions in O 2 and ° OH production. The findings indicated that 150–270 nm-sized Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes could effectively partake in therapeutic tasks. These nanozymes exhibited thermal and biological stability at pH 7.2, featuring 7.34 nm pores, suggesting their potential as drug carriers in biological systems. Moreover, their capability to loading and release DOX reliably in an acidic environment (pH 6.5) similar to tumor tissues, particularly in the pH-responsive of Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes, enabled targeted drug delivery. In the following, the MTT and flow cytometry assessment indicates the high efficacy of Fe 3 O 4 /MnO 2 @DOX hybrid nanozymes, particularly in synergy with PTT to inhibit cancer cell growth. However, toxicity evaluations of these nanozymes in MCF-7-derived spheroids demonstrate reduced toxicity levels at similar concentrations. While the synergistic effect is promising in limiting spheroid growth, escalating the Fe 3 O 4 /MnO 2 @DOX dose with associated side effects is deemed unacceptable. These findings underscore the importance of considering cellular organizational structures alongside the notable therapeutic responses of nanozymes. Declarations Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgements The statements made herein are the sole responsibility of the authors. Author information Authors and Affiliations Student Research Committee, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran Majid Sharifi Department of Tissue Engineering, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran Majid Sharifi, Mohammad Kamalabadi-Farahani, Amir Abas Salmani & Mahmoud Malaki Contributions MS, AAS, MM: conceptualization, methodology, revision; MS, MKF: analysis, validation, supervision; writing. All authors read and approved the final manuscript. Corresponding authors Correspondence to Majid Sharifi Ethics declarations Ethics approval and consent to participate Consent for publication All authors read and approve the final manuscript. Competing interests All of the authors declare that they have no conflict of interest. Funding The present study was supported by Shahroud University of Medical Sciences, Shahroud, Iran as a Ph.D. thesis (Grant number: 200159). This research was carried out with the ethical code of IR.SHMU.AEC.1402.008. References Wang X, Teng Z, Wang H, Wang C, Liu Y, Tang Y, Wu J, Sun J, Wang H, Wang J (2014) Increasing the cytotoxicity of doxorubicin in breast cancer MCF-7 cells with multidrug resistance using a mesoporous silica nanoparticle drug delivery system. Int J Clin Exp Pathol 7(4):1337 Falahati M, Sharifi M, Hagen TLT (2022) Explaining chemical clues of metal organic framework-nanozyme nano-/micro-motors in targeted treatment of cancers: benchmarks and challenges. J Nanobiotechnol 20(1):153 Cui M, Xu B, Wang L (2024) Recent advances in multi-metallic‐based nanozymes for enhanced catalytic cancer therapy. BMEMat 2(1):e12043 Chen M, Deng G, He Y, Li X, Liu W, Wang W, Zhou Z, Yang H, Yang S (2019) Ultrasound-enhanced generation of reactive oxygen species for MRI-guided tumor therapy by the Fe@ Fe3O4-based peroxidase-mimicking nanozyme. ACS Appl Bio Mater 3(1):639–647 Sun Y, Xu L, Liu X, Shen Y, Zhang Y, Gu N, Xiong F (2022) Coronal relay reactor Fe3O4@CeO2 for accelerating ROS axial conversion through enhanced Enzyme-like effect and relay effect. Chem Eng J 429:132303 Zhang Y, Yang Y, Shi J, Wang L (2021) A multimodal strategy of Fe 3 O 4@ ZIF-8/GOx@ MnO 2 hybrid nanozyme via TME modulation for tumor therapy. Nanoscale 13(39):16571–16588 Chen X, Tao H, Guo Y, Wang Z, Li R, Zhao Y, Liu C, Zhao X, Wang X, Duan S (2023) Anti-CD44 antibodies grafted immunoaffinity Fe3O4@ MnO2 nanozymes with highly oxidase-like catalytic activity for specific detection of triple-negative breast cancer MDA-MB-231 cells. Anal Chim Acta 1249:340947 Sisakhtnezhad S, Rahimi M, Mohammadi S (2023) Biomedical applications of MnO2 nanomaterials as nanozyme-based theranostics. Biomed Pharmacother 163:114833 Li J, Cai X, Zhang Y, Li K, Guan L, Li Y, Wang T, Sun T, ChemistrySelect (2022) 7(23) pp e202201127 Du W, Chen W, Wang J, Zhang H, Song L, Hu Y, Ma X (2023) A dual-nanozyme-loaded black phosphorus multifunctional therapeutic platform for combined photothermal/photodynamic/starvation cancer therapy. J Mater Chem B 11(23):5185–5194 Chen K, Yan R, Yang H, Xia Y, Shang Y, Song J, Peng Z, Yang G (2023) Multifunctional Nanozymes by Amplifying Intracellular Oxidative Stress for Enhanced Photothermal–Photodynamic Therapy. ACS Appl Nano Mater 6(22):20855–20865 Cun J-E, Pan Y, Zhang Z, Lu Y, Li J, Pan Q, Gao W, Luo K, He B, Pu Y (2022) Photo-enhanced upcycling H2O2 into hydroxyl radicals by IR780-embedded Fe3O4@ MIL-100 for intense nanocatalytic tumor therapy. Biomaterials 287:121687 de Melo-Diogo D, Pais‐Silva C, Dias DR, Moreira AF, Correia IJ (2017) Strategies to improve cancer photothermal therapy mediated by nanomaterials. Adv Healthc Mater 6(10):1700073 Lv W, Cao M, Liu J, Hei Y, Bai J (2021) Tumor microenvironment-responsive nanozymes achieve photothermal-enhanced multiple catalysis against tumor hypoxia. Acta Biomater 135:617–627 Wu C-Y, Hsu Y-H, Chen Y, Yang L-C, Tseng S-C, Chen W-R, Huang C-C, Wan D (2021) Robust O2 Supplementation from a Trimetallic Nanozyme-Based Self-Sufficient Complementary System Synergistically Enhances the Starvation/Photothermal Therapy against Hypoxic Tumors. ACS Appl Mater Interfaces 13(32):38090–38104 Sohail M, Sun Z, Li Y, Gu X, Xu H (2021) Research progress in strategies to improve the efficacy and safety of doxorubicin for cancer chemotherapy. Expert Rev Anticancer Ther 21(12):1385–1398 Sritharan S, Sivalingam N (2021) A comprehensive review on time-tested anticancer drug doxorubicin. Life Sci 278:119527 Sharifi M, Jafari S, Hasan A, Paray BA, Gong G, Zheng Y, Falahati M (2020) Antimetastatic Activity of Lactoferrin-Coated Mesoporous Maghemite Nanoparticles in Breast Cancer Enabled by Combination Therapy. ACS Biomaterials Sci Eng 6(6):3574–3584 Ye S, Chen S, Yang X, Lei X (2024) Drug resistance in breast cancer is based on the mechanism of exocrine non-coding RNA. Discover Oncol 15(1):138 Ning S, Zheng Y, Qiao K, Li G, Bai Q, Xu S (2021) Laser-triggered combination therapy by iron sulfide-doxorubicin@ functionalized nanozymes for breast cancer therapy. J Nanobiotechnol 19:1–14 Li J-x, Zhang L-m, Liu C-c, Wu Q-n, Li S-p, Lei X-p (2021) Y.-g. Huang, G.-n. Feng, X.-y. Yu, X.-q. Sun, Doxorubicin-loaded hydrogen peroxide self-providing copper nanodots for combination of chemotherapy and acid-induced chemodynamic therapy against breast cancer. J Colloid Interface Sci 593:323–334 Cheng X, Li D, Sun M, He L, Zheng Y, Wang X, Tang R (2019) Co-delivery of DOX and PDTC by pH-sensitive nanoparticles to overcome multidrug resistance in breast cancer. Colloids Surf B 181:185–197 Mó I, Sabino IJ, Melo-Diogo Dd, Lima-Sousa R, Alves CG, Correia IJ (2020) The importance of spheroids in analyzing nanomedicine efficacy. Nanomedicine 15(15):1513–1525 Carvalho MR, Lima D, Reis RL, Correlo VM, Oliveira JM (2015) Evaluating biomaterial-and microfluidic-based 3D tumor models. Trends Biotechnol 33(11):667–678 Wilhelm S, Tavares AJ, Dai Q, Ohta S, Audet J, Dvorak HF, Chan WC (2016) Analysis of nanoparticle delivery to tumours. Nat reviews Mater 1(5):1–12 Sharifi M, Cho WC, Ansariesfahani A, Tarharoudi R, Malekisarvar H, Sari S, Bloukh SH, Edis Z, Amin M, Gleghorn JP (2022) An updated review on EPR-based solid tumor targeting nanocarriers for cancer treatment. Cancers 14(12):2868 Chen G, Liu W, Yan B (2022) Breast cancer MCF-7 cell spheroid culture for drug discovery and development. J cancer therapy 13(3):117 Al-Kattan A, Ali LMA, Daurat M, Mattana E, Gary-Bobo M (2020) Biological assessment of laser-synthesized silicon nanoparticles effect in two-photon photodynamic therapy on breast cancer MCF-7 cells. Nanomaterials 10(8):1462 Reynolds DS, Tevis KM, Blessing WA, Colson YL, Zaman MH, Grinstaff MW (2017) Breast cancer spheroids reveal a differential cancer stem cell response to chemotherapeutic treatment. Sci Rep 7(1):10382 Law S, Leung AW, Xu C (2020) Folic acid-modified celastrol nanoparticles: synthesis, characterization, anticancer activity in 2D and 3D breast cancer models. Artif Cells Nanomed Biotechnol 48(1):542–559 Emami F, Pathak S, Nguyen TT, Shrestha P, Maharjan S, Kim JO, Jeong J-H, Yook S (2021) Photoimmunotherapy with cetuximab-conjugated gold nanorods reduces drug resistance in triple negative breast cancer spheroids with enhanced infiltration of tumor-associated macrophages. J Controlled Release 329:645–664 Khan S, Sharifi M, Bloukh SH, Edis Z, Siddique R, Falahati M (2021) In vivo guiding inorganic nanozymes for biosensing and therapeutic potential in cancer, inflammation and microbial infections. Talanta 224:121805 Sharifi M, Kheradmandi R, Alizadeh M (2023) Two birds with one stone: triple negative breast cancer therapy by PtCo bimetallic nanozyme coated with gemcitabine-hyaluronic acid-polyethylene glycol. Cancer Nanotechnol 14(1):41 Wang X, Liu C, Chen X, Zhao X, Wu J, Chen H, Wan M, Zhao S, Li X, Li N, Duan S (2024) A Smart Nitric Oxide (NO) Generating Immuno-Trimetallic Nanocatalyst Triggering Chemodynamic Therapy in Breast Cancer Treatment, Advanced Functional Materials, n/a(n/a) pp 2316186 Xu Q, Zhang Y, Yang Z, Jiang G, Lv M, Wang H, Liu C, Xie J, Wang C, Guo K (2022) Tumor microenvironment-activated single-atom platinum nanozyme with H2O2 self-supplement and O2-evolving for tumor-specific cascade catalysis chemodynamic and chemoradiotherapy. Theranostics 12(11):5155 Li Y, Yun K-H, Lee H, Goh S-H, Suh Y-G, Choi Y (2019) Porous platinum nanoparticles as a high-Z and oxygen generating nanozyme for enhanced radiotherapy in vivo. Biomaterials 197:12–19 Meng L, Cheng Y, Tong X, Gan S, Ding Y, Zhang Y, Wang C, Xu L, Zhu Y, Wu J, Hu Y, Yuan A (2018) Tumor Oxygenation and Hypoxia Inducible Factor-1 Functional Inhibition via a Reactive Oxygen Species Responsive Nanoplatform for Enhancing Radiation Therapy and Abscopal Effects. ACS Nano 12(8):8308–8322 Zhu X, Liu Y, Yuan G, Guo X, Cen J, Gong Y, Liu J, Gang Y (2020) In situ fabrication of MS@ MnO 2 hybrid as nanozymes for enhancing ROS-mediated breast cancer therapy. Nanoscale 12(43):22317–22329 Nie Z, Vahdani Y, Cho WC, Bloukh SH, Edis Z, Haghighat S, Falahati M, Kheradmandi R, Jaragh-Alhadad LA, Sharifi M (2022) 5-Fluorouracil-containing inorganic iron oxide/platinum nanozymes with dual drug delivery and enzyme-like activity for the treatment of breast cancer. Arab J Chem 15(8):103966 Zeng Z, Wang Z, Chen S, Xiao C, Liu M, Zhang J, Fan J, Zhao Y, Liu B (2022) Bio-nanocomplexes with autonomous O2 generation efficiently inhibit triple negative breast cancer through enhanced chemo-PDT. J Nanobiotechnol 20(1):500 Riffle S, Hegde RS (2017) Modeling tumor cell adaptations to hypoxia in multicellular tumor spheroids. J Experimental Clin Cancer Res 36:1–10 Holub AR, Huo A, Patel K, Thakore V, Chhibber P, Erogbogbo F (2020) Assessing advantages and drawbacks of rapidly generated ultra-large 3D breast cancer spheroids: studies with chemotherapeutics and nanoparticles. Int J Mol Sci 21(12):4413 Brancato V, Gioiella F, Imparato G, Guarnieri D, Urciuolo F, Netti PA (2018) 3D breast cancer microtissue reveals the role of tumor microenvironment on the transport and efficacy of free-doxorubicin in vitro. Acta Biomater 75:200–212 Nunes AS, Barros AS, Costa EC, Moreira AF, Correia IJ (2019) 3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs. Biotechnol Bioeng 116(1):206–226 Additional Declarations No competing interests reported. Supplementary Files floatimage1.jpeg Cite Share Download PDF Status: Published Journal Publication published 28 Sep, 2024 Read the published version in Cancer Nanotechnology → Version 1 posted Editorial decision: Revision requested 21 Jul, 2024 Reviews received at journal 20 Jul, 2024 Reviews received at journal 16 Jul, 2024 Reviews received at journal 15 Jul, 2024 Reviewers agreed at journal 10 Jul, 2024 Reviews received at journal 04 Jul, 2024 Reviews received at journal 30 Jun, 2024 Reviewers agreed at journal 24 Jun, 2024 Reviewers agreed at journal 23 Jun, 2024 Reviewers agreed at journal 23 Jun, 2024 Reviewers agreed at journal 23 Jun, 2024 Reviewers agreed at journal 30 May, 2024 Reviewers invited by journal 30 May, 2024 Editor assigned by journal 21 May, 2024 Submission checks completed at journal 21 May, 2024 First submitted to journal 14 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4417286","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":308752923,"identity":"83dc1e07-f691-4a72-b530-0fad1affad6d","order_by":0,"name":"Majid Sharifi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBAC+RlAIsFGQo5BAirCRkiLwQ2QljQJYxK0gFWmMSQ2SBBSCtci3fzsw4MEi/T5s7vTJBhq7Bj4pA/g1yI/55jxjIQEidwNd85uk2A4lszAxpdAwJobCcYMiT+AWiRygVrYDjCw8RBy2Y30zwxAW9LlZ4C0/CNKS44xSEsCww2gFsY2IrQY3MgpBmkx3HAjd7NFYl8yD0Et8jPSNzP+SKiTBzps440P3+zk5HsIOQwFJDAwEPTJKBgFo2AUjAIiAACeXjrDtwkf/QAAAABJRU5ErkJggg==","orcid":"","institution":"Shahroud University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Majid","middleName":"","lastName":"Sharifi","suffix":""},{"id":308752924,"identity":"654c412b-6f66-49e8-8a57-d5851c078af7","order_by":1,"name":"Mohammad Kamalabadi-Farahani","email":"","orcid":"","institution":"Shahroud University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"","lastName":"Kamalabadi-Farahani","suffix":""},{"id":308752925,"identity":"3448c09e-0343-4ee6-ad59-0f6526d235fc","order_by":2,"name":"Amir-Abas Salmani","email":"","orcid":"","institution":"Shahroud University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Amir-Abas","middleName":"","lastName":"Salmani","suffix":""},{"id":308752926,"identity":"3fdcf1b2-2e83-4b44-a943-9cba38ae7d24","order_by":3,"name":"Mahmoud Malaki","email":"","orcid":"","institution":"Shahroud University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mahmoud","middleName":"","lastName":"Malaki","suffix":""}],"badges":[],"createdAt":"2024-05-14 07:36:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4417286/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4417286/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12645-024-00293-z","type":"published","date":"2024-09-28T15:57:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57644366,"identity":"043947f8-0d42-488e-bd63-7cdc1bdcd2cb","added_by":"auto","created_at":"2024-06-03 18:47:11","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":540930,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterizations of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes. (A) SEM images of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2 \u003c/sub\u003ehybrid nanozymes and (B) Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes. (C) TEM images of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes. (D) the size distribution and (E) N\u003csub\u003e2\u003c/sub\u003e adsorption-desorption isotherms [(F): The size of pores on Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2 \u003c/sub\u003ehybrid nanozymes is indicated by the inset] of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2 \u003c/sub\u003ehybrid nanozymes. (G) Zeta potential values of Fe3O4 nanozymes and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2 \u003c/sub\u003ehybrid nanozymes. (H) XRD patterns of the synthesized Fe3O4 nanozymes and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2 \u003c/sub\u003ehybrid nanozymes.\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4417286/v1/a70d765f8ed9eb437967fc88.jpeg"},{"id":57644368,"identity":"928aa9cb-6b53-4625-93e1-b689171f9cba","added_by":"auto","created_at":"2024-06-03 18:47:11","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":474062,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Representative TEM images of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2 \u003c/sub\u003ehybrid nanozymes, (B) EDS and (C) element mapping of nanozyme. (D) Temperature versus irradiation time of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2 \u003c/sub\u003ehybrid nanozymes suspensions with concentrations of 5 mg/mL in distilled water. (E) Peroxidase-like activities of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanozymes, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2 \u003c/sub\u003ehybrid nanozymes with and without photothermal therapy (PTT).\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4417286/v1/e0bdab8f113fd670a5138769.jpeg"},{"id":57644370,"identity":"d48010e8-2263-4f45-b98a-679e87c1a3c5","added_by":"auto","created_at":"2024-06-03 18:47:11","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":395358,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Drug loading and its efficiency. (B) Thermogravimetric analysis of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2 \u003c/sub\u003ehybrid nanozymes and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes, and their weight loss between 70-450 °C heating. (C) Magnetization curves at room temperature for\u0026nbsp;Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4 \u003c/sub\u003enanozymes, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2 \u003c/sub\u003ehybrid nanozymes and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes. (D) Quantitative analyses of DOX release at 37 °C at different pH with and without photothermal therapy (PTT). (E) O\u003csub\u003e2\u003c/sub\u003e generation in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e nanozymes, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes with and without PTT. (F) Fluorescence spectra of terephthalic acid incubated with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e nanozymes, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes with and without PTT in the hypoxic condition to show the presence of \u003csup\u003e°\u003c/sup\u003eOH.\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4417286/v1/10c487b846e58a3c25edf5cf.jpeg"},{"id":57644369,"identity":"ae635fb8-d460-4ccd-ab02-926177b70ef9","added_by":"auto","created_at":"2024-06-03 18:47:11","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":632292,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Cytotoxicity assay of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes on NIH-3T3 cells by MTT assay.\u0026nbsp;\u003csup\u003e*\u003c/sup\u003eP\u0026lt; 0.05,\u0026nbsp;and \u003csup\u003e**\u003c/sup\u003eP\u0026lt; 0.01. (B) Viability of MCF-7 cells after incubation with different concentrations of DOX, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes with and without photothermal therapy (PTT) for 48 h. (C) Flow cytometric analysis of live and dead MCF-7 cells in different treatment groups: control, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes (2.16 μg/mL), DOX (2.16 μM), Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes (2.16 μg/mL) with and without PTT. Cell necrosis and apoptosis measured using propidium iodide (PI) and Annexin V-FITC staining. (D) Representative DCFH staining of MCF-7 cells in different treatments for ROS evaluation. (E) Optical microscopy images of MCF-7 cells treated with different treatments.\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4417286/v1/dfd51e4deac2153bf489810f.jpeg"},{"id":57644371,"identity":"98dd7669-da45-40e3-ace7-50cbd5837f15","added_by":"auto","created_at":"2024-06-03 18:47:12","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":500938,"visible":true,"origin":"","legend":"\u003cp\u003e(A)\u003cstrong\u003e \u003c/strong\u003eViability of MCF-7 spheroids after incubation with different concentrations of DOX, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes with and without photothermal therapy (PTT) for 48 h. (B) Average of diameter of MCF-7 spheroids in different treatment groups. (C) Images of MCF-7 spheroids incubated in different treatment groups: control, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes (4.32 μg/mL), DOX (5 μM), Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes (4.32 μg/mL) with and without PTT. (D) The effect of DOX, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes with and without PTT on the extrinsic and intrinsic mechanisms of apoptosis by examining the expression of TNF-α, CASP7, Bcl-2, CASP9, and CASP3 in MCF-7 spheroids. \u003csup\u003e*\u003c/sup\u003eP\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt; 0.01 and \u003csup\u003e***\u003c/sup\u003eP\u0026lt; 0.001 indicate significant differences.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4417286/v1/98498465b24b805c40221210.jpeg"},{"id":65627102,"identity":"3fb2acc7-de73-4612-a338-d700a6802cc4","added_by":"auto","created_at":"2024-09-30 16:11:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3443247,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4417286/v1/16549caf-c9bc-4145-b48b-e693802766c0.pdf"},{"id":57644367,"identity":"b78b86f1-866e-4fda-97a9-97a264d0f98f","added_by":"auto","created_at":"2024-06-03 18:47:11","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":288509,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4417286/v1/3212a4ec2d52105a021e0815.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of the performance of Fe 3 O 4 /MnO 2 hybrid nanozymes with doxorubicin on multicellular structure and their therapeutic management to limit the growth of human breast cancer cells","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBreast cancer is currently one of the deadliest cancers in women, with around a third of affected women dying even after mastectomy. The main treatment involves drugs, which have been very effective in improving survival rates. However, about half of patients undergoing drug therapies suffer a relapse [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The main reason for treatment failure is resistance to anticancer drugs, caused by poor drug penetration due to significant hypoxia and blockages in tumor areas. Alongside traditional treatments like surgery, chemotherapy, and radiation therapy, nanozymes are being investigated as a less invasive option with catalytic properties. In addition to their ability to targeted release of drugs in tumor environments with a pH 6.5, nanozymes exhibit promise in drug-resistant tumors treatment by increasing O\u003csub\u003e2\u003c/sub\u003e levels to improve drug penetration, producing radicals (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026deg;\u0026macr;\u003c/sup\u003e and \u003csup\u003e\u0026deg;\u003c/sup\u003eOH), and synergizing with photothermal therapy (PTT) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among nanozymes mimicking peroxidase and catalase, the use of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e in tumor therapy is interesting due to high compatibility and Fenton reactions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanozymes are amazing for producing \u003csup\u003e\u0026deg;\u003c/sup\u003eOH in acidic conditions through peroxidase-like activity on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, as well as generating hydrogen peroxyradicals in neutral and alkaline conditions, similar to catalase-like activity for the production of O\u003csub\u003e2\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. To enhance the catalytic performance of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanozymes, the incorporation of MnO\u003csub\u003e2\u003c/sub\u003e nanozymes is proposed [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The addition of MnO\u003csub\u003e2\u003c/sub\u003e not only effective in Fe\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e regeneration, but also reduces tumor hypoxia by converting H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to H\u003csub\u003e2\u003c/sub\u003eO and O\u003csub\u003e2\u003c/sub\u003e in acidic conditions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Despite challenges like low selectivity and biocompatibility linked to MnO\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], its capacity to convert superoxide into \u003csup\u003e\u0026deg;\u003c/sup\u003eOH and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e during catalysis has increased its potential for use in cancer therapy [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Furthermore, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e and MnO\u003csub\u003e2\u003c/sub\u003e nanoparticles exhibit enhanced catalytic pathways under photothermal and photodynamic therapy activities, as evidenced in studies by Du et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], Chen et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], Cun et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and Zhang et al. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The utilization of PTT technique to enhance thermal energy from laser irradiation in the tumor microenvironment is a non-invasive and precise method that can boost confidence in cancer treatment through synergistic interaction with nanozymes. It has been found that the heat generated by PTT enhances the glucose oxidase activity for starvation, peroxidase- and catalase-like activities of nanozymes to generate \u003csup\u003e\u0026deg;\u003c/sup\u003eOH and O\u003csub\u003e2\u003c/sub\u003e, and also restricts cancer cell growth by activating the extrinsic apoptosis pathways [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. As a result, it is anticipated that the treatment of drug-resistant tumors will be expedited through the inherent functionality of nanozymes and the cascading amplification of radicals, O\u003csub\u003e2\u003c/sub\u003e, and heat production induced by PTT.\u003c/p\u003e \u003cp\u003eIn addition to the significance of nanozymes and PTT in the therapeutic effects on breast cancers, the selection of the drug type is crucial. The standard treatment for advanced breast cancers involves doxorubicin (DOX), an anthracycline antibiotic [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. DOX disrupts DNA and RNA centers, offering a reliable treatment route for solid tumors [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, its usage is restricted due to heart tissue toxicity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and drug resistance to DOX remains inevitable [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Therefore, extensive research is required to reduce DOX toxicity in non-target tissues while maintaining high efficacy in tumor tissues. Numerous studies demonstrate that nanozymes with DOX effectively inhibit the growth of breast cancer cells [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe screening of anticancer drugs has traditionally been carried out using two-dimensional cell cultures due to their cost-effectiveness, speed, and multiple assays [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, two-dimensional cultures do not accurately represent cell-cell and cell-extracellular matrix (ECM) interactions [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. As a result, drug functions in this system can lead to false predictions of tumor response and clinical failures. Thus, it is noted that only around 5% of anticancer compounds advance to the clinical phase because of inadequate pharmacokinetics or uncertain efficacy [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Meanwhile, some argue that nanotechnology is on an uncertain path, relying on animal models without a full understanding of the intricate interactions of drug-resistant cancer cells events and the various biological barriers in human tissues [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. To address this challenge, three-dimensional cell culture from human cancer cells, known as spheroid models, have been recommended to better mimic the uniform tissue of cancer cells [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Despite limitations such as lack of interaction with the extracellular environment, lower physical resistance, and insufficient cell diversity, studies have shown that therapeutic responses in this model closely resemble in vivo conditions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In different scenarios, it has been revealed that the lack of complete penetration of therapeutic compounds due to high cell density and the presence of ECM, the change of oxygen gradient from the surface to the center, and the overexpression of anti-apoptotic proteins in the center of spheroids, anti-tumor therapeutic responses are significantly different compared to two-dimensional cultures [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this research, we developed Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes with dual functionality for drug delivery and catalytic activity to inhibit MCF-7 cells. We then explored their antitumor effects in both two-dimensional culture and spheroid models. Alongside analyzing the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX nanozymes' physicochemical characteristics and DOX loading/release capabilities, we investigated the enhanced efficacy against MCF-7 cells through synergistic actions with PTT. Our findings suggest that using Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes with catalytic properties and targeted drug release can create a new therapeutic opportunity for treating drug-resistant cells. However, focusing on cell organizational structures is crucial for advancing therapeutic objectives.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eAll chemical and biological materials utilized in this research were procured from Merck, Germany. Human breast cancer MCF-7 cells were acquired from Shahroud University of Medical Sciences.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Synthesis of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes\u003c/h2\u003e \u003cp\u003eThe Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanozymes were synthesized using the solvothermal approach. FeCl\u003csub\u003e3\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO (3.25 g) and tri-sodium citrate (1.3 g) were dispersed in 100 mL of ethylene glycol and stirred continuously for 30 minutes. Subsequently, NaAc (6.0 g) was added to the mixture and sonicated for 1 hour. The resulting solution was then transferred to an autoclave set at 220\u0026deg;C and left for 10 hours. Following cooling, the product was washed three times with ethanol and distilled water before being dispersed in deionized water. Next, PAA (20 mg, 0.2 g/mL) and 150 \u0026micro;L of NH\u003csub\u003e3\u003c/sub\u003e.H\u003csub\u003e2\u003c/sub\u003eO (5 M) were added to the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e solution. The mixture was stirred for 20 minutes. Subsequently, 80 mL of Isopropanol was gradually introduced to the solution with vigorous shaking to produce Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@PAA. Following this, 100 \u0026micro;L of Mn (II) acetate tetrahydrate (50 mg/mL) was carefully dripped into the solution and stirred for 3 hours. The resulting Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes were separated by centrifugation and washed with deionized water. The resulting precipitate was dried at 60\u0026deg;C, and the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes were then calcined at 450\u0026deg;C in an N\u003csub\u003e2\u003c/sub\u003e atmosphere for 4 hours. To load DOX (Doxorubicin), 10 mg of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes were added to 25 mL of dimethyl sulfoxide solution with 9 mg of DOX and kept for 24 hours with gentle agitation. The Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes were subsequently air-dried for 24 hours at room temperature and washed with PBS for further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Characterization of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes\u003c/h2\u003e \u003cp\u003eTo analyze the surface morphology and internal structures, field-emission scanning electron microscopy (FESEM; MIRA3, TESCAN) and high-resolution transmission electron microscopy (TEM; HRTEM, JEM-2010) were utilized with an acceleration voltage of 200 kV. The Zetasizer system (Malvern Instruments, UK) was employed to determine hydrodynamic size and zeta potential at 25\u0026deg;C. N\u003csub\u003e2\u003c/sub\u003e absorption isotherms of the samples at liquid nitrogen temperature (-196\u0026deg;C) were measured using Nova's Quantachrome automatic gas absorption system to assess the nanozyme cavities. The pore size distribution was considered from the desorption branch of the isotherm through the Barth-Joyner-Holland process. X-ray diffraction patterns of nanozymes were obtained through the XRD method. X-ray examination was conducted with a D/max- using Cu Kα radiation (Rigaku, Japan) in continuous scan mode ranging from 20\u0026deg;-70\u0026deg; with a step size of 0.02\u0026deg; and speed of 2\u0026deg;/min. Energy dispersive X-ray spectroscopy (EDS) and elemental mapping studies were carried out on a JEOL JEM2010 electron microscope at 100 kV. Also, the peroxidase-like activity of nanozymes were assessed (an UV-vis spectrometer, Shimadzu UV-2600) by studying the oxidation of TMB (3,3\u0026prime;,5,5\u0026prime;-tetramethylbenzidine) as a reaction substrate. To do this, 2 \u0026micro;L of 100 \u0026micro;g/mL Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanozymes, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes, and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes without and with PTT (Photothermal therapy: 808 nm laser light: 2 W/cm\u003csup\u003e2\u003c/sup\u003e for 3 minutes) were mixed with 500 \u0026micro;L of sodium acetate buffer at pH 6.5, along with 2 \u0026micro;L of 20 mg/mL TMB and 2 \u0026micro;L of 30% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (hydrogen peroxide). Subsequently, the solutions were incubated in darkness for 10 minutes to carry out the peroxidase-like activity. Furthermore, the rise in ambient temperature due to the use of PTT (808 nm laser light: 2 W/cm\u003csup\u003e2\u003c/sup\u003e for 3 minutes) on Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes (5 mg/mL in water distiller) were monitored with a thermometer while being exposed to 808 nm wavelength irradiation at various intervals: 0, 5, 10, 20, 40, 80, 160, 320, and 640 seconds.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Loading and validation tests, and drug release study\u003c/h2\u003e \u003cp\u003eTo determine the loading capacity, 100 \u0026micro;g of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes were placed in drug solution with concentrations of 30, 60, 90, 120 and 150 \u0026micro;g for 24 hours at room temperature with gentle shaking (at 100 rpm). After separation of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes by magnet and washing by PBS, the remaining solution was assessed and analyzed (based on Eq.\u0026nbsp;1) by fluorescence spectroscopy as the initial solution (Hitachi F 2500 spectrometer).\u003c/p\u003e \u003cp\u003eEquation 1: Loading efficiency (%) = [(DIS\u0026thinsp;\u0026minus;\u0026thinsp;DRS)/DRS] \u0026times; 100; DIS is the total amount of DOX in initial solution and DRS is the amount of DOX remaining in the solution.\u003c/p\u003e \u003cp\u003eTo explore drug loading, thermogravimetric analysis (TGA) was assessed using Perkin-Elmer TGA-7 under N\u003csub\u003e2\u003c/sub\u003e at a heating rate of 5\u0026deg;C/min in the range of 70\u0026ndash;450\u0026deg;C. A superconducting quantum interference device (SQUID, MPMS-XL) was used to assess the magnetic properties of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes, ranging from \u0026minus;\u0026thinsp;8,000 to +\u0026thinsp;8,000 G at 298 m. In the following, in vitro DOX release from Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes were conducted at 37\u0026deg;C in a shaker at 150 rpm in PBS solution with pH of 7.2 and 6.5, both with and without PTT (808 nm laser irradiation: 2 W/cm\u003csup\u003e2\u003c/sup\u003e for 3 minutes). At specified time intervals (0.37, 0.75, 1.5, 3, 6, 12 and 24 hours), 2 mL of the sample was withdrawn from each container and replaced with an equal volume of PBS to maintain a constant volume. The drug release extent was assessed by measuring the absorbance at 428 nm using a UV\u0026ndash;vis spectrophotometer. The total DOX release was calculated using the following Eq.\u0026nbsp;(2):\u003c/p\u003e \u003cp\u003eEquation 2: Cumulative DOX release (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{5 \\times {\\sum }_{\\text{i}-1}^{\\text{n}-1}{\\text{C}}_{\\text{i}}+50 \\times {\\text{C}}_{\\text{n}}}{\\text{w}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t} \\text{o}\\text{f} \\text{D}\\text{O}\\text{X} \\text{o}\\text{n}\\text{F}\\text{e}3\\text{O}4/\\text{M}\\text{n}\\text{O}2@\\text{D}\\text{O}\\text{X}}\\times 100\\)\u003c/span\u003e\u003c/span\u003e; C\u003csub\u003ei\u003c/sub\u003e and C\u003csub\u003en\u003c/sub\u003e refer to the of DOX concentration at time i and n, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. O\u003csub\u003e2\u003c/sub\u003e and \u003csup\u003e\u0026deg;\u003c/sup\u003eOH generation\u003c/h2\u003e \u003cp\u003eO\u003csub\u003e2\u003c/sub\u003e generation in aqueous solutions was assessed using a portable dissolved oxygen meter (Seven2GO pro S9 DO, Mettler Toledo). To conduct the experiment, 100 \u0026micro;g/mL of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanozymes, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes with and without PTT (808 nm laser irradiation: 2 W/cm\u003csup\u003e2\u003c/sup\u003e for 3 minutes) were introduced into 10 mL of sodium acetate buffer with 200 \u0026micro;L of 30% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The O\u003csub\u003e2\u003c/sub\u003e production (mg/mL) was measured at various intervals: 0, 18, 37, 75, 150, 225, 300, 450, and 600 seconds. Also, terephthalic acid was utilized to measure the \u003csup\u003e\u0026deg;\u003c/sup\u003eOH level. Following the interaction of terephthalic acid with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanozymes, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes with and without PTT (808 nm laser irradiation: 2 W/cm\u003csup\u003e2\u003c/sup\u003e for 3 minutes), and its conversion to 2-hydroxy terephthalic acid, the \u003csup\u003e\u0026deg;\u003c/sup\u003eOH level was assessed at a wavelength of 430\u0026ndash;435 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Cell culture\u003c/h2\u003e \u003cp\u003eThe NIH3T3 and MCF-7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (GIBCO, New York) with 10% fetal bovine serum (FBS, AusGeneX) and 1% penicillin/streptomycin. Cell flasks were maintained in an incubator at 37\u0026deg;C with 5% CO2 and 95% humidity. For cell transfer to new culture medium, trypsinization (0.25% trypsin-EDTA) and re-suspension in DMEM medium were performed.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1. Toxicity evaluation\u003c/h2\u003e \u003cp\u003eCytotoxicity tests were conducted using the MTT method on NIH3T3 and MCF-7 cells. Initially, a density of 8\u0026times;10\u003csup\u003e3\u003c/sup\u003e NIH3T3 and MCF-7 cells were plated in a 96-well plate and incubated for 12 hours at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. Subsequently, NIH3T3 cells were exposed to specific concentrations of 0.27, 0.54, 1.08, 2.16, 4.32, and 8.65 \u0026micro;g/mL of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e nanozymes. Also, MCF-7 cells were exposed to various concentrations of DOX (0.31, 0.62, 1.25, 2.5, 5, and 10 \u0026micro;M), Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes (0.27, 0.54, 1.08, 2.16, 4.32, and 8.65 \u0026micro;g/ml), and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes (0.27, 0.54, 1.08, 2.16, 4.32, and 8.65 \u0026micro;g/ml). Additionally, a set of MCF-7 cells received treatment with an 808 nm laser at 2 W/cm\u003csup\u003e2\u003c/sup\u003e for 3 minutes following an 8-hour exposure to Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes (0.27, 0.54, 1.08, 2.16, 4.32, and 8.65 \u0026micro;g/ml). Then, the cells were then incubated for 48 hours. Afterward, they were rinsed and 100 \u0026micro;L of fresh medium containing 20 \u0026micro;L of MTT solution (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide: 0.5 mg/mL) was introduced into each well. The culture medium was once again incubated in darkness at 37\u0026deg;C for 4 hours. The resultant purple formazan crystals were dissolved in 100 \u0026micro;L of DMSO (dimethyl sulfoxide), and the absorbance was assessed at 570 nm with a multi-well plate reader. The survival rates of both treated and control cells were calculated using Eq.\u0026nbsp;3.\u003c/p\u003e \u003cp\u003eEquation 3: cell viability (%)= [[Optical density of dosing cells-Optical density of blank]\u0026divide;[ Optical density of control-Optical density of blank]]\u0026times;100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2. Apoptosis and ROS assays\u003c/h2\u003e \u003cp\u003eTo further study the inhibition of MCF-7 cell growth by Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e nanozymes and DOX, the apoptosis level was assessed using the Annexin-V/PI Apoptosis Analysis Kit (Yeasen, Inc., China) and flow cytometry. MCF-7 cells were seeded in a 6-well plate at a density of 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e as described in section \u003cspan refid=\"Sec8\" class=\"InternalRef\"\u003e2.6\u003c/span\u003e. and then incubated for 12 hours. Afterward, the old culture medium was replaced with a new one containing DOX (2.5 \u0026micro;M), Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes (2.16 \u0026micro;g/mL), and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes (2.16 \u0026micro;g/mL) with and without PTT (808 nm laser irradiation, 2 W/cm\u003csup\u003e2\u003c/sup\u003e for 3 minutes) onto the plates. The MCF-7 cells were then cultured at 37\u0026deg;C with 5% CO2 for 48 hours. After incubation, MCF-7 cells were harvested by centrifugation at 1000g (5 min) and washed thrice in cold PBS. The cells were trypsinized, resuspended in 200 \u0026micro;L of binding buffer, and then stained with Annexin V-FITC/Alexa Fluor 488 (5 \u0026micro;L) and propidium iodide (PI: 10 \u0026micro;L) following the manufacturer's instructions. Subsequently, the stained cells were analyzed using flow cytometry in the absence of light.\u003c/p\u003e \u003cp\u003eTo assess intracellular ROS levels, 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well were seeded in a 6-well plate. After 12 hours of incubation at 37\u0026deg;C with 5% CO2, the cells were treated with DOX (2.5 \u0026micro;M), Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e nanozymes (2.16 \u0026micro;g/mL), Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes (2.16 \u0026micro;g/mL) without or with PTT (808 nm laser irradiation, 2 W/cm\u003csup\u003e2\u003c/sup\u003e for 3 minutes). The MCF-7 cells were re-incubated for 48 hours. Subsequently, the cells were rinsed with PBS and treated with 10 \u0026micro;M 2,7-dichlorodihydrofluorescein. After a 30-minute incubation period, the cells underwent two PBS washes. The level of ROS was then assessed using FACscan (BD Bioscience, USA) by measuring the fluorescence intensity of 2,7-dichlorofluorescein produced through the oxidation of 2,7-dichlorodihydrofluorescein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.6.3. MCF-7 spheroids formation, cytotoxicity and morphometry\u003c/h2\u003e \u003cp\u003eTo generate MCF-7 3D spheroids, a method involving reducing FBS levels on non-adherent surfaces was employed. Briefly, MCF-7 cells were cultured at a density of 10\u003csup\u003e3\u003c/sup\u003e cells in ultra-low-attachment 24-well plates with DMEM medium supplemented with high glucose, 0.5% FBS, and 2% penicillin-streptomycin (all sourced from Gibco, USA) at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e. The MCF-7 spheroids were then incubated for six days. Afterwards, the MCF-7 spheroids were exposed to varying concentrations of DOX (0.31, 0.62, 1.25, 2.5, 5, and 10 \u0026micro;M), Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes (0.27, 0.54, 1.08, 2.16, 4.32, and 8.65 \u0026micro;g/ml), and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes (0.27, 0.54, 1.08, 2.16, 4.32, and 8.65 \u0026micro;g/ml) for 48 hours. Moreover, a set of MCF-7 spheroids received treatment with an 808 nm laser irradiation at 2 W/cm\u003csup\u003e2\u003c/sup\u003e for 3 minutes following an 8-hour exposure to Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes (0.27, 0.54, 1.08, 2.16, 4.32, and 8.65 \u0026micro;g/ml). Proliferation and viability of MCF-7 spheroids were evaluated 48 hours post-treatment with therapeutic compounds utilizing Alamar Blue cell viability reagent. As per the manufacturer's instructions, 10 \u0026micro;g/mL (one-tenth of the total culture medium volume) of Alamar Blue was added to each MCF-7 spheroid and kept to incubator for 24 hours. Subsequent to the incubation period, fluorescence intensity was measured on plates at 535 nm excitation and 595 nm emission using a DTX 880 microplate reader from Beckman Coulter in Brea, CA. Furthermore, to study the growth inhibition of MCF-7 spheroids by DOX (5 \u0026micro;M), Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes (4.32 \u0026micro;g/mL), Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes (4.32 \u0026micro;g/mL) without or with PTT (an 808 nm laser irradiation at 2 W/cm\u003csup\u003e2\u003c/sup\u003e for 3 minutes), the dimensions of spheroids were evaluated using images with ImageJ software on the 6th day (equivalent to the first day of treatment) and the 11th day (5 days after treatment).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.6.4. Mechanisms of cytotoxicity\u003c/h2\u003e \u003cp\u003eTotal RNA extraction was performed using the Trizol reagent (Sinaclon Bio Science, Iran) on spheroids 5-day post-treatment. The concentration and purity of RNA samples were assessed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA). DNase I was utilized to eliminate genomic contamination from the isolated RNA. Complementary DNA (cDNA) was generated using the BONmiR\u0026trade; qRT-PCR miRNA Detection Kit (Stem Cell Technology Research Center, Tehran, Iran) following the manufacturer's instructions. Quantitative real-time PCR was conducted using SYBR Premix Ex Taq\u0026trade; II (Takara Bio, Japan) and monitored with the Applied Biosystems\u0026reg; StepOneTM instrument and ABI7500 thermocycler following this protocol: initial activation at 95\u0026deg;C for 5 min, 40 cycles of denaturation at 95\u0026deg;C for 5 seconds, and annealing/extension at 60\u0026deg;C for 30 seconds. PCR primer pair sequences are: β- actin: F-5\u0026acute;-CTTCTACAATGAGCTGCGTG-3\u0026acute;, R-5\u0026acute;-TCATGAGGTAGTCAGTCAGG-3\u0026acute;; TNF-α: F-5\u0026acute;-CTCTTCTGCCTGCTGCACTTTG-3\u0026acute;, R-5\u0026acute;-ATGGGCTACAGGCTTGTCACTC-3\u0026acute;; CASP7: F-5\u0026acute;-CGGAACAGACAAAGATGCCGAG-3\u0026acute;, R-5\u0026acute;-AGGCGGCATTTGTATGGTCCTC-3\u0026acute;; Bcl-2: F-5\u0026acute;-ATCGCCCTGTGGATGACTGAGT-3\u0026acute;, R-5\u0026acute;-GCCAGGAGAAATCAAACAGAGGC-3\u0026acute;; CASP9: F-5\u0026acute;-GTTTGAGGACCTTCGACCAGCT-3\u0026acute;, R-5\u0026acute;-CAACGTACCAGGAGCCACTCTT-3\u0026acute;; Caspase-3: F-5\u0026acute;-GGAAGCGAATCAATGGACTCTGG-3\u0026acute;, R-5\u0026acute;- GCATCGACATCTGTACCAGACC-3\u0026acute;;\u003c/p\u003e \u003cp\u003eβ-actin served as the internal reference gene for normalizing gene expression. mRNA expression level was determined using the 2\u003csup\u003e\u0026minus;(ΔΔCT)\u003c/sup\u003e method. All reactions were conducted in triplicate.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe data were analyzed using one-way analysis of variance. Statistical significances were assessed with the Statistical Package for Social Science (version 20) and Tukey\u0026rsquo;s multiple comparison tests. P-values below 5% were deemed statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Morphology and chemical properties\u003c/h2\u003e \u003cp\u003eThe morphology and size features of the engineered hybrid nanozymes, as revealed by SEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) and TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) analyses, indicate that the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes, ranging from 150 to 270 nm, consist of two fused nanospheres with surface cavities. While the DLS results indicate that the size of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes ranges vary from 90 to 370 nm, the highest concentration can be seen in the range of 140 to 270 nm. Meanwhile, the porous structure of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes was verified through nitrogen absorption and desorption output, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE. The N\u003csub\u003e2\u003c/sub\u003e adsorption-desorption isotherm outcomes exhibit type IV behavior with noticeable residual rings within the 0.30\u0026ndash;0.44, 0.45\u0026ndash;0.78 and 0.79\u0026ndash;0.97 P/P0 range. The surface area of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes was calculated at 65.266 m\u003csup\u003e2\u003c/sup\u003e/g, featuring cavities ranging from 1.2 to 34.7 nm with an average size of 7.34 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Moreover, the zeta potential measurements reveal that Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanozymes and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes exhibit \u0026minus;\u0026thinsp;3.14 mV and \u0026minus;\u0026thinsp;25.2 mV values, respectively, under neutral conditions, demonstrating the effective incorporation of MnO\u003csub\u003e2\u003c/sub\u003e into the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanozymes. Also, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH illustrates the XRD patterns of Fe3O4 nanozymes and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes. The Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanozymes (black curve) shows diffraction peaks at (2θ\u0026thinsp;=\u0026thinsp;30.1\u0026deg;: 220), (2θ\u0026thinsp;=\u0026thinsp;35.0\u0026deg;: 311), (2θ\u0026thinsp;=\u0026thinsp;44.6\u0026deg;: 400), (2θ\u0026thinsp;=\u0026thinsp;54.6\u0026deg;: 422), (2θ\u0026thinsp;=\u0026thinsp;56.6\u0026deg;: 511), and (2θ\u0026thinsp;=\u0026thinsp;63.5\u0026deg;: 440) corresponding to Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e (PDF#19\u0026ndash;629). In contrast, the hybrid nanozymes (red curve) exhibits these Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e peaks along with two additional peaks at 2θ\u0026thinsp;=\u0026thinsp;27.3\u0026deg; and 2θ\u0026thinsp;=\u0026thinsp;36.9\u0026deg;, indicating the presence of MnO\u003csub\u003e2\u003c/sub\u003e nanozymes (PDF#30\u0026ndash;0820). This suggests that Mn(Ⅱ) acetate was calcined to produce Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e. In the following, elemental mapping from EDS analysis reveals the atomic distribution of metal ions on Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) with a Mn: Fe: O weight ratio of 37.3%: 22.6%: 40.1%. The mapping indicates that Fe is concentrated in the smaller nanospheres while Mn is predominant in the larger one (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD data indicates that exposing the nanozymes to 808 nm laser irradiation at 2 W/cm\u003csup\u003e2\u003c/sup\u003e for 3 minutes\u0026rsquo; results in a temperature rise across various pH levels. Transitioning from a neutral (pH 7.2) to an acidic (pH 6.5) environment, akin to the acidity found in cancerous tumors, effectively triggers heat generation from Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes. Also, the investigating the peroxidase-like activity revealed that Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes exhibit favorable peroxidase characteristics in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and at a pH of 6.5. In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, TMB is converted by nanozymes into the blue oxTMB product. As anticipated, the inclusion of MnO\u003csub\u003e2\u003c/sub\u003e on the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanozymes enhances the catalytic performance. Furthermore, the synergism of PTT with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes significantly enhances the catalytic efficiency.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Loading and validation tests, and drug release\u003c/h2\u003e \u003cp\u003eDOX loading content in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA revealed that as the concentration of DOX increased, the rate of drug loading in Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes also significantly increased with nanozymes constant concentration. However, as anticipated, the drug loading efficiency percentage decreased with higher DOX concentration. In summary, the findings indicate that the highest drug loading efficiency percentage (over 51.1%) is achieved with 60 \u0026micro;g of drug in 100 \u0026micro;g of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes. In this regard, TGA confirmed DOX presence in Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes exhibit high stability at temperatures up to 450\u0026deg;C. The slight mass decrease of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes between 100\u0026ndash;300\u0026deg;C in the thermogram, accounting for ~\u0026thinsp;4.9% by weight, is attributed to water loss within the pores and minor structural damage to Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes. However, the stability of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes in terms of weight percentage between 300\u0026deg;C and 450\u0026deg;C demonstrates their robustness. Concurrently, the TGA curve reveals a weight loss of up to 42.48% for Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes, confirming the presence of DOX on Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes. The weight percentage decrease in Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes exhibits a two-step transition: a gradual decline from 95\u0026deg;C to 190\u0026deg;C (14.9% weight reduction) followed by a steeper decrease from 195\u0026deg;C to 260\u0026deg;C (27.4% weight reduction). This behavior is likely attributed to the structural segments and functional groups of the drug molecule undergoing degradation. Furthermore, to support the aforementioned results, it was observed that the magnetic saturation of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanozymes consistently reduces from 64.9 emu/g to 45.0 emu/g and 42.6 emu/g in the presence of MnO\u003csub\u003e2\u003c/sub\u003e nanozymes and DOX. Hence, the assertion regarding drug loading on Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes is substantiated.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes' capability to release DOX was studied at 37\u0026deg;C under varying acidity levels. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD illustrates that DOX release from the nanozymes is time-dependent. Generally, DOX release is higher in acidic conditions (51.9%) compared to neutral conditions (25.8%). Moreover, the synergism of PTT with acidity boosts DOX release up to 81.19% by enhancing the peroxidase-like activities of the nanozymes. Furthermore, a higher burst release of DOX from Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes is observed at pH 6.5 (17.93%), exceeding the release at pH 7.2 (12.98%). Despite the somewhat negative impact of PTT in an acidic environment on the burst release of DOX, with an increase of up to 38.12%, controlled release improvement can help maintain a stable therapeutic dosage over 24 hours. Furthermore, by enhancing Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes performance in acidic conditions, particularly through synergistic effects, it becomes feasible to boost DOX delivery in tumor areas with pH levels ranging from 6 to 6.5. This can also elevate drug efficacy by concentrating it in tumor tissues. Evidence for this was seen in the significant reduction of drug release at pH 7.2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. O\u003csub\u003e2\u003c/sub\u003e generation\u003c/h2\u003e \u003cp\u003eEvaluation of catalytic activity in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE indicates that the presence of MnO\u003csub\u003e2\u003c/sub\u003e nanozymes on the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanozymes enhances the nanozyme's catalytic activity for O\u003csub\u003e2\u003c/sub\u003e generation by nearly twofold, increasing from 8.4 to 16.2 mg/L. Despite the negative impact of DOX loading on the catalytic activity of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes in O\u003csub\u003e2\u003c/sub\u003e generation (14.1 mg/L), this study indicates that the combined effect of PTT with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes notably enhances the catalytic activity for O\u003csub\u003e2\u003c/sub\u003e generation (19.8 mg/L) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Overall, this discovery indicates that boosting O\u003csub\u003e2\u003c/sub\u003e generation through Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes post DOX release, or utilizing Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes alongside PTT (808 nm laser irradiation: 2 W/cm\u003csup\u003e2\u003c/sup\u003e for 3 minutes), can mitigate hypoxia and enhance drug permeation. In the following, the results depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF indicate that the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes effectively enhance the \u003csup\u003e\u0026deg;\u003c/sup\u003eOH level compared to non-hybrid nanozymes. This enhancement is evidenced by the increased fluorescence intensity of 2-hydroxyterephthalic acid. While the presence of DOX on the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes decreases the \u003csup\u003e\u0026deg;\u003c/sup\u003eOH level, it seems that the release of the drug at pH 6.5 leads to an increase in \u003csup\u003e\u0026deg;\u003c/sup\u003eOH levels. Moreover, the study revealed that the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes, in synergism with PTT, significantly elevate the \u003csup\u003e\u0026deg;\u003c/sup\u003eOH level compared to other experimental groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Cytotoxicity of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX\u003c/h2\u003e \u003cp\u003eTo investigate the toxicity effect of nanozymes with and without DOX loading, MTT and flow cytometry techniques were employed on NIH-3T3 cells and MCF-7 cells. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA shows that NIH-3T3 cells exhibited no significant cytotoxicity when treated with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes at concentrations of 0.27, 0.54, 1.08, and 2.16 \u0026micro;g/mL. However, toxicity increased when the concentration was raised to 4.32 \u0026micro;g/mL or higher. This study demonstrates that Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes exhibit favorable biocompatibility as both a catalytic agent and a carrier for delivering anticancer drugs, particularly within a specific range. Similar to the toxicity output in NIH-3T3 cells, it was found in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB that the toxicity of DOX, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes with and without DOX, combined with PTT (808 nm: 2 W/cm\u003csup\u003e2\u003c/sup\u003e for 3 minutes) on MCF-7 cells, is dose-dependent. The study indicates that the most significant suppression of MCF-7 cells by Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes occurs at doses of 2.16, 4.32, and 8.64 \u0026micro;g/mL compared to the control group (60.3%, 70.8% and 72.8% growth inhibition). However, based on the toxicity dose of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, it is recommended to use a dose of 2.16 \u0026micro;g/mL (equivalent to 2.5 \u0026micro;M DOX). In addition, it was discovered that the synergistic effect of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes with PTT (808 nm: 2 W/cm\u003csup\u003e2\u003c/sup\u003e for 3 minutes) enhances the toxicity level of nanozymes on cancer cells from 2.16 \u0026micro;g/ml (65.9% growth inhibition) to 1.08 \u0026micro;g/ml (57.3% growth inhibition). It was also revealed that nanozymes, as nanocarriers, significantly increase the toxicity of DOX in all doses compared to DOX. Ultimately, the dose-dependent cytotoxicity of DOX in MCF-7 cells can be enhanced by two methods: loading on nanozymes and synergy with PTT, without inducing toxicity in non-cancerous cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter determining the toxicity dose from Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, to analyze the percentage of apoptotic and necrotic cells during therapeutic activities, MCF-7 cells were exposed to 2.16 \u0026micro;g/ml concentrations of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes with and without PTT, along with 2.5 \u0026micro;mol DOX. Flow cytometry results in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC indicate that while DOX led to an increase in late and early apoptotic cells compared to the control (Q2: 33.4% vs 4.11%; Q3: 13.7% vs 5.53%), employing Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes as a drug carrier effectively raised late and early apoptotic cell levels by 34.9% and 13.3%, respectively. Also, it was found that the combination of PTT (808 nm: 2 W/cm\u003csup\u003e2\u003c/sup\u003e for 3 minutes) with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes induced the highest levels of early (Q2: 36.0%) and late (Q3: 15.3%) apoptotic cells in MCF-7 cells. In support of the crucial role of apoptosis, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD demonstrates that the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes and DOX significantly raised the intracellular ROS level compared to the control. Furthermore, loading DOX on Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes resulted in a higher intracellular ROS level. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD illustrates that the greatest intracellular ROS is achieved through the synergy of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes with PTT, confirming the heightened apoptotic level in this particular group. Thus, it was discovered that DOX, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes with and without PTT, induce the demise of MCF-7 cells by elevating intracellular ROS levels, leading to apoptosis. Supporting this conclusion, the morphological alterations (like changes in volume and shape) and the quantity of cancer cells exposed to DOX, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes with and without PTT, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, indicate an anticipated cell demise.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Toxicity in MCF-7 spheroids\u003c/h2\u003e \u003cp\u003eTo investigate the impact of nanozymes toxicity on spheroids from MCF-7 cells, the MTT technique was employed. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA illustrates that both DOX and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes exhibit dose-dependent toxicity, with no significant variance observed across different concentrations. The study revealed that the highest toxicity towards MCF-7 spheroids occurs at concentrations of 5 \u0026micro;M for DOX (49.1% growth inhibition) and 4.32 \u0026micro;g/mL for Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes (49.8% growth inhibition) and above (58.8% and 60.1% growth inhibition in 10 \u0026micro;M for DOX and 8.64 \u0026micro;g/mL for Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes). While, loading DOX onto Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes enhances toxicity levels to 2.16 \u0026micro;g/mL (equivalent to 2.5 \u0026micro;M of DOX) (53.3% growth inhibition). Also, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA clearly indicates that utilizing PTT (808 nm: 2 W/cm\u003csup\u003e2\u003c/sup\u003e for 3 minutes) in synergism with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes exhibits superior anticancer effects across various concentrations. Despite the heightened toxicity of the synergized group at 4.32 (72.8% growth inhibition of spheroids) and 8.65 (75.5% growth inhibition of spheroids) \u0026micro;g/mL, toxicity levels rise until reaching 1.08 \u0026micro;g/mL (55.1% growth inhibition of spheroids).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter establishing the toxic dose of DOX and nanozymes in spheroids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), MCF-7 spheroids were treated with 5 \u0026micro;M of DOX and 4.32 \u0026micro;g/mL Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes, both with and without PTT. Subsequently, their growth inhibition was assessed. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, rapid growth is observed in the spheroids of the control group, which are generally dense with a size of 168.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5 \u0026micro;m visible on the 5th day (103\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5 \u0026micro;m on the first day). Although there is no significant difference between the DOX and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes groups in inhibiting spheroid growth, both groups effectively suppressed spheroid growth compared to the control group (113.3\u0026thinsp;\u0026plusmn;\u0026thinsp;8 and 116.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1 \u0026micro;m compared to 163\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5 \u0026micro;m, respectively). However, the most significant inhibition to MCF-7 spheroid growth was achieved with the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes (50.69% growth inhibition) and its combined effect with PTT (65.14% growth inhibition).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Cytotoxicity mechanisms\u003c/h2\u003e \u003cp\u003eThe toxicity mechanism depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD illustrates that Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes, with or without PTT, are highly effective in triggering apoptosis through both intrinsic and extrinsic pathways. However, in terms of synergistic activity, the upregulation of TNF-α (17.64 vs. 10.38) and CASP7 (5.20 vs. 2.88) associated with the extrinsic pathway, along with the downregulation of Bcl-2 (0.21 vs. 0.38) and the elevation of CASP9 (8.10 vs. 5.12) compared to the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes, offer greater promise in the treatment process. Despite the lack of significant differences between the DOX and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes groups in apoptosis pathways, DOX not only enhances apoptosis induction by increasing CASP3 (4.35 vs. 2.77), but also influences the extrinsic pathway by boosting TNF-α (5.55 vs. 3.20). In conclusion, these findings affirm that synergistic therapeutic approaches yield more dependable outcomes in cancer treatment.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eNowadays, the use of nanozymes with varied catalytic capabilities controlled by pH and constituent elements, along with their integration with therapeutic techniques like PTT, has garnered significant interest. Nonetheless, beyond environmental obstacles and sustained catalytic performance [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], their efficacy within the therapeutic window dictated by the biological conditions of tumors or tumor cells poses a challenge. By enhancing controllable catalytic activity through element combinations and addressing environmental hurdles [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], it is hoped that tumor treatment procedures can be enhanced by enhancing drug penetration via reducing hypoxia levels, and modifying the tumor microenvironment. With this premise, following the study of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes and their structural and physicochemical analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), it was discovered that these nanozymes, with improved catalytic activity, lead to a greater O\u003csub\u003e2\u003c/sub\u003e and \u003csup\u003e\u0026deg;\u003c/sup\u003eOH levels in hypoxic environments compared to non-hybrid nanozymes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. According to the findings of Xu et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], it appears that increasing O\u003csub\u003e2\u003c/sub\u003e levels by enhancing drug permeability and increasing \u003csup\u003e\u0026deg;\u003c/sup\u003eOH radical through inducing apoptosis can speed up the drug resistance cancer treatment process. This effect is particularly remarkable when combined synergistically with PTT and radiotherapy, as noted by Lv et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and Li et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Also, besides enhancing O\u003csub\u003e2\u003c/sub\u003e and \u003csup\u003e\u0026deg;\u003c/sup\u003eOH levels, pH-responsive Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes offer a hopeful strategy for targeted drug delivery in drug-resistant cancers, ensuring efficient drug release at pH 6.5, particularly in synergistic actions, similar to the discoveries of Cheng et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], Meng et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], and Chen et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In line with findings of Wang et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and Zhu et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], it was discovered that Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes, particularly in synergistic activity, demonstrates substantial toxicity against MCF-7 cells by promoting apoptosis through enhanced ROS. Nevertheless, in agreement with the findings of Nie et al. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] and Zeng et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], it was found that the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes, as opposed to DOX administration, could improve the treatment process by targeting of drug release, triggering intracellular ROS, and raising O\u003csub\u003e2\u003c/sub\u003e levels. This increase in the aforementioned events without synergistic effects by PTT could raise hopes for treating tumors inaccessible to laser irradiation. Despite the successes in limiting MCF-7 cell growth or removing them, the low efficacy of drugs or nanocarriers in clinical trials and human treatments has made researchers uncertain about their use. Although descriptive and analytical studies highlight the ineffectiveness of therapeutic approaches due to low EPR (enhanced permeability and retention), drug pump issues, high hypoxia from vessel blockage or excessive O\u003csub\u003e2\u003c/sub\u003e consumption, and physiological differences between animals and humans, and the cells derived from them [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], the analysis of therapeutic dosage based on the state of organization of cells in the early stages has received less attention. During this study, it was discovered that the optimal treatment dosage for MCF-7 spheroid structures under 200 \u0026micro;m differs significantly from that of two-dimensional cultures. The choice to select spheroids under 200 \u0026micro;m is based on the limitation of oxygen diffusion in tissues larger than 200 \u0026micro;m, as outlined in the empirical and mathematical modeling [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In the following, it was demonstrated that the optimal dosage of DOX and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes needs to be doubled when transitioning from two-dimensional culture to spheroids, going from 2.5 to 5 \u0026micro;M and from 2.16 to 4.32 \u0026micro;g/mL, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Similarly, it was disclosed that the efficient dose of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes, in altering cellular structure from a two-dimensional setup to a spheroid, also doubled (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). While, the results in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA indicate that raising the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e hybrid nanozymes dosage to 4.32 \u0026micro;g/mL can lead to adverse effects on biological functions. Therefore, in line with the findings of Al-Kattan et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and the results of Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, which confirm the use of synergistic activities with PTT to create the desired toxicity for spheroids with the optimal concentration of nanozymes, it is recommended to utilize synergistic activities. PTT synergy with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes appears to effectively restrict spheroid growth through various actions, such as elevating O2 and \u0026deg;OH radical levels, enhancing drug release, and raising ambient temperature to 45\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) to increase both intrinsic and extrinsic apoptosis pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Furthermore, in line with the aforementioned discovery, Cheng et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], Zhang et al. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and Emami et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] demonstrated that altering the organizational structure of MCF-7 and MCF-7/ADR, 4T1 and BT-20 cells from two-dimensional to spheroidal culture can impose less restrictions on the growth of drug-resistant cancer cells at the same dosage, due to decreased drug permeability. Confirming this discovery, it was found that as the dimensions of MCF-7 spheroids increase, the permeability of both the DOX and nanocarriers decreases [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In this regard, Reynolds et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] demonstrated that while the growth of spheroids' periphery decreased, the core of spheroids significantly increased with the presence of cancer drugs. The limited drug access to the spheroids' core, attributed to higher cell and collagen accumulation, appears to be the primary cause for reduced drug effectiveness. Based on finding of Brancato et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], it appears that the formation of ECM, caused by changing the culture medium from two-dimensional culture to spheroids, creates more complex barriers to drug penetration. Thus, it is anticipated that the dosage of therapeutic compounds will rise in spheroids as a result of altered drug/nanocarriers penetration patterns, potentially impacting their function through the presence of ECM.\u003c/p\u003e \u003cp\u003eCellular organizations like spheroids versus two-dimensional cultures or larger spheroids versus small spheroids are more resistant to therapeutic activities due to reduced drugs/nanozymes penetration caused by a more compact ECM, changes in O\u003csub\u003e2\u003c/sub\u003e slope, and increased hypoxia in the center of the structure, as well as excessive expression of anti-apoptosis proteins (Bcl-2, BAX, etc.) in the center of cellular clusters [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Therefore, it is recommended to use patterns closer to resistant tumors such as spheroids or organoids in academic/pharmaceutical centers to reduce the costs of the research-production process and explain the effects of drugs/Nano-compounds.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn this study, following the creation and synthesis of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes, we explored their combined performance with PTT. Physicochemical assessments verified the existence of iron and magnesium, showcasing their enzymatic functions in O\u003csub\u003e2\u003c/sub\u003e and \u003csup\u003e\u0026deg;\u003c/sup\u003eOH production. The findings indicated that 150\u0026ndash;270 nm-sized Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes could effectively partake in therapeutic tasks. These nanozymes exhibited thermal and biological stability at pH 7.2, featuring 7.34 nm pores, suggesting their potential as drug carriers in biological systems. Moreover, their capability to loading and release DOX reliably in an acidic environment (pH 6.5) similar to tumor tissues, particularly in the pH-responsive of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes, enabled targeted drug delivery. In the following, the MTT and flow cytometry assessment indicates the high efficacy of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes, particularly in synergy with PTT to inhibit cancer cell growth. However, toxicity evaluations of these nanozymes in MCF-7-derived spheroids demonstrate reduced toxicity levels at similar concentrations. While the synergistic effect is promising in limiting spheroid growth, escalating the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX dose with associated side effects is deemed unacceptable. These findings underscore the importance of considering cellular organizational structures alongside the notable therapeutic responses of nanozymes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe statements made herein are the sole responsibility of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003eStudent Research Committee, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran\u003c/p\u003e\n\u003cp\u003eMajid Sharifi\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDepartment of Tissue Engineering, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran\u003c/p\u003e\n\u003cp\u003eMajid Sharifi,\u0026nbsp;Mohammad Kamalabadi-Farahani, Amir Abas Salmani\u0026nbsp;\u0026amp;\u0026nbsp;Mahmoud Malaki\u003c/p\u003e\n\u003cp\u003eContributions\u003c/p\u003e\n\u003cp\u003eMS, AAS, MM: conceptualization, methodology, revision; MS, MKF: analysis, validation, supervision; writing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eCorresponding authors\u003c/p\u003e\n\u003cp\u003eCorrespondence to Majid Sharifi\u003c/p\u003e\n\u003cp\u003eEthics declarations\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eAll authors read and approve the final manuscript.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eAll of the authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study was supported by Shahroud University of Medical Sciences, Shahroud, Iran as a Ph.D. thesis (Grant number: 200159). This research was carried out with the ethical code of IR.SHMU.AEC.1402.008.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang X, Teng Z, Wang H, Wang C, Liu Y, Tang Y, Wu J, Sun J, Wang H, Wang J (2014) Increasing the cytotoxicity of doxorubicin in breast cancer MCF-7 cells with multidrug resistance using a mesoporous silica nanoparticle drug delivery system. Int J Clin Exp Pathol 7(4):1337\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFalahati M, Sharifi M, Hagen TLT (2022) Explaining chemical clues of metal organic framework-nanozyme nano-/micro-motors in targeted treatment of cancers: benchmarks and challenges. J Nanobiotechnol 20(1):153\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui M, Xu B, Wang L (2024) Recent advances in multi-metallic‐based nanozymes for enhanced catalytic cancer therapy. BMEMat 2(1):e12043\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen M, Deng G, He Y, Li X, Liu W, Wang W, Zhou Z, Yang H, Yang S (2019) Ultrasound-enhanced generation of reactive oxygen species for MRI-guided tumor therapy by the Fe@ Fe3O4-based peroxidase-mimicking nanozyme. ACS Appl Bio Mater 3(1):639\u0026ndash;647\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Y, Xu L, Liu X, Shen Y, Zhang Y, Gu N, Xiong F (2022) Coronal relay reactor Fe3O4@CeO2 for accelerating ROS axial conversion through enhanced Enzyme-like effect and relay effect. Chem Eng J 429:132303\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Yang Y, Shi J, Wang L (2021) A multimodal strategy of Fe 3 O 4@ ZIF-8/GOx@ MnO 2 hybrid nanozyme via TME modulation for tumor therapy. Nanoscale 13(39):16571\u0026ndash;16588\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen X, Tao H, Guo Y, Wang Z, Li R, Zhao Y, Liu C, Zhao X, Wang X, Duan S (2023) Anti-CD44 antibodies grafted immunoaffinity Fe3O4@ MnO2 nanozymes with highly oxidase-like catalytic activity for specific detection of triple-negative breast cancer MDA-MB-231 cells. Anal Chim Acta 1249:340947\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSisakhtnezhad S, Rahimi M, Mohammadi S (2023) Biomedical applications of MnO2 nanomaterials as nanozyme-based theranostics. Biomed Pharmacother 163:114833\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Cai X, Zhang Y, Li K, Guan L, Li Y, Wang T, Sun T, ChemistrySelect (2022) 7(23) pp e202201127\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDu W, Chen W, Wang J, Zhang H, Song L, Hu Y, Ma X (2023) A dual-nanozyme-loaded black phosphorus multifunctional therapeutic platform for combined photothermal/photodynamic/starvation cancer therapy. J Mater Chem B 11(23):5185\u0026ndash;5194\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen K, Yan R, Yang H, Xia Y, Shang Y, Song J, Peng Z, Yang G (2023) Multifunctional Nanozymes by Amplifying Intracellular Oxidative Stress for Enhanced Photothermal\u0026ndash;Photodynamic Therapy. ACS Appl Nano Mater 6(22):20855\u0026ndash;20865\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCun J-E, Pan Y, Zhang Z, Lu Y, Li J, Pan Q, Gao W, Luo K, He B, Pu Y (2022) Photo-enhanced upcycling H2O2 into hydroxyl radicals by IR780-embedded Fe3O4@ MIL-100 for intense nanocatalytic tumor therapy. Biomaterials 287:121687\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Melo-Diogo D, Pais‐Silva C, Dias DR, Moreira AF, Correia IJ (2017) Strategies to improve cancer photothermal therapy mediated by nanomaterials. Adv Healthc Mater 6(10):1700073\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLv W, Cao M, Liu J, Hei Y, Bai J (2021) Tumor microenvironment-responsive nanozymes achieve photothermal-enhanced multiple catalysis against tumor hypoxia. Acta Biomater 135:617\u0026ndash;627\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu C-Y, Hsu Y-H, Chen Y, Yang L-C, Tseng S-C, Chen W-R, Huang C-C, Wan D (2021) Robust O2 Supplementation from a Trimetallic Nanozyme-Based Self-Sufficient Complementary System Synergistically Enhances the Starvation/Photothermal Therapy against Hypoxic Tumors. ACS Appl Mater Interfaces 13(32):38090\u0026ndash;38104\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSohail M, Sun Z, Li Y, Gu X, Xu H (2021) Research progress in strategies to improve the efficacy and safety of doxorubicin for cancer chemotherapy. Expert Rev Anticancer Ther 21(12):1385\u0026ndash;1398\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSritharan S, Sivalingam N (2021) A comprehensive review on time-tested anticancer drug doxorubicin. Life Sci 278:119527\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharifi M, Jafari S, Hasan A, Paray BA, Gong G, Zheng Y, Falahati M (2020) Antimetastatic Activity of Lactoferrin-Coated Mesoporous Maghemite Nanoparticles in Breast Cancer Enabled by Combination Therapy. ACS Biomaterials Sci Eng 6(6):3574\u0026ndash;3584\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYe S, Chen S, Yang X, Lei X (2024) Drug resistance in breast cancer is based on the mechanism of exocrine non-coding RNA. Discover Oncol 15(1):138\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNing S, Zheng Y, Qiao K, Li G, Bai Q, Xu S (2021) Laser-triggered combination therapy by iron sulfide-doxorubicin@ functionalized nanozymes for breast cancer therapy. J Nanobiotechnol 19:1\u0026ndash;14\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J-x, Zhang L-m, Liu C-c, Wu Q-n, Li S-p, Lei X-p (2021) Y.-g. Huang, G.-n. Feng, X.-y. Yu, X.-q. Sun, Doxorubicin-loaded hydrogen peroxide self-providing copper nanodots for combination of chemotherapy and acid-induced chemodynamic therapy against breast cancer. J Colloid Interface Sci 593:323\u0026ndash;334\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng X, Li D, Sun M, He L, Zheng Y, Wang X, Tang R (2019) Co-delivery of DOX and PDTC by pH-sensitive nanoparticles to overcome multidrug resistance in breast cancer. Colloids Surf B 181:185\u0026ndash;197\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM\u0026oacute; I, Sabino IJ, Melo-Diogo Dd, Lima-Sousa R, Alves CG, Correia IJ (2020) The importance of spheroids in analyzing nanomedicine efficacy. Nanomedicine 15(15):1513\u0026ndash;1525\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarvalho MR, Lima D, Reis RL, Correlo VM, Oliveira JM (2015) Evaluating biomaterial-and microfluidic-based 3D tumor models. Trends Biotechnol 33(11):667\u0026ndash;678\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilhelm S, Tavares AJ, Dai Q, Ohta S, Audet J, Dvorak HF, Chan WC (2016) Analysis of nanoparticle delivery to tumours. Nat reviews Mater 1(5):1\u0026ndash;12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharifi M, Cho WC, Ansariesfahani A, Tarharoudi R, Malekisarvar H, Sari S, Bloukh SH, Edis Z, Amin M, Gleghorn JP (2022) An updated review on EPR-based solid tumor targeting nanocarriers for cancer treatment. Cancers 14(12):2868\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen G, Liu W, Yan B (2022) Breast cancer MCF-7 cell spheroid culture for drug discovery and development. J cancer therapy 13(3):117\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Kattan A, Ali LMA, Daurat M, Mattana E, Gary-Bobo M (2020) Biological assessment of laser-synthesized silicon nanoparticles effect in two-photon photodynamic therapy on breast cancer MCF-7 cells. Nanomaterials 10(8):1462\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReynolds DS, Tevis KM, Blessing WA, Colson YL, Zaman MH, Grinstaff MW (2017) Breast cancer spheroids reveal a differential cancer stem cell response to chemotherapeutic treatment. Sci Rep 7(1):10382\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaw S, Leung AW, Xu C (2020) Folic acid-modified celastrol nanoparticles: synthesis, characterization, anticancer activity in 2D and 3D breast cancer models. Artif Cells Nanomed Biotechnol 48(1):542\u0026ndash;559\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEmami F, Pathak S, Nguyen TT, Shrestha P, Maharjan S, Kim JO, Jeong J-H, Yook S (2021) Photoimmunotherapy with cetuximab-conjugated gold nanorods reduces drug resistance in triple negative breast cancer spheroids with enhanced infiltration of tumor-associated macrophages. J Controlled Release 329:645\u0026ndash;664\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan S, Sharifi M, Bloukh SH, Edis Z, Siddique R, Falahati M (2021) In vivo guiding inorganic nanozymes for biosensing and therapeutic potential in cancer, inflammation and microbial infections. Talanta 224:121805\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharifi M, Kheradmandi R, Alizadeh M (2023) Two birds with one stone: triple negative breast cancer therapy by PtCo bimetallic nanozyme coated with gemcitabine-hyaluronic acid-polyethylene glycol. Cancer Nanotechnol 14(1):41\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Liu C, Chen X, Zhao X, Wu J, Chen H, Wan M, Zhao S, Li X, Li N, Duan S (2024) A Smart Nitric Oxide (NO) Generating Immuno-Trimetallic Nanocatalyst Triggering Chemodynamic Therapy in Breast Cancer Treatment, Advanced Functional Materials, n/a(n/a) pp 2316186\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu Q, Zhang Y, Yang Z, Jiang G, Lv M, Wang H, Liu C, Xie J, Wang C, Guo K (2022) Tumor microenvironment-activated single-atom platinum nanozyme with H2O2 self-supplement and O2-evolving for tumor-specific cascade catalysis chemodynamic and chemoradiotherapy. Theranostics 12(11):5155\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Yun K-H, Lee H, Goh S-H, Suh Y-G, Choi Y (2019) Porous platinum nanoparticles as a high-Z and oxygen generating nanozyme for enhanced radiotherapy in vivo. Biomaterials 197:12\u0026ndash;19\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng L, Cheng Y, Tong X, Gan S, Ding Y, Zhang Y, Wang C, Xu L, Zhu Y, Wu J, Hu Y, Yuan A (2018) Tumor Oxygenation and Hypoxia Inducible Factor-1 Functional Inhibition via a Reactive Oxygen Species Responsive Nanoplatform for Enhancing Radiation Therapy and Abscopal Effects. ACS Nano 12(8):8308\u0026ndash;8322\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu X, Liu Y, Yuan G, Guo X, Cen J, Gong Y, Liu J, Gang Y (2020) In situ fabrication of MS@ MnO 2 hybrid as nanozymes for enhancing ROS-mediated breast cancer therapy. Nanoscale 12(43):22317\u0026ndash;22329\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNie Z, Vahdani Y, Cho WC, Bloukh SH, Edis Z, Haghighat S, Falahati M, Kheradmandi R, Jaragh-Alhadad LA, Sharifi M (2022) 5-Fluorouracil-containing inorganic iron oxide/platinum nanozymes with dual drug delivery and enzyme-like activity for the treatment of breast cancer. Arab J Chem 15(8):103966\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng Z, Wang Z, Chen S, Xiao C, Liu M, Zhang J, Fan J, Zhao Y, Liu B (2022) Bio-nanocomplexes with autonomous O2 generation efficiently inhibit triple negative breast cancer through enhanced chemo-PDT. J Nanobiotechnol 20(1):500\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiffle S, Hegde RS (2017) Modeling tumor cell adaptations to hypoxia in multicellular tumor spheroids. J Experimental Clin Cancer Res 36:1\u0026ndash;10\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolub AR, Huo A, Patel K, Thakore V, Chhibber P, Erogbogbo F (2020) Assessing advantages and drawbacks of rapidly generated ultra-large 3D breast cancer spheroids: studies with chemotherapeutics and nanoparticles. Int J Mol Sci 21(12):4413\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrancato V, Gioiella F, Imparato G, Guarnieri D, Urciuolo F, Netti PA (2018) 3D breast cancer microtissue reveals the role of tumor microenvironment on the transport and efficacy of free-doxorubicin in vitro. Acta Biomater 75:200\u0026ndash;212\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNunes AS, Barros AS, Costa EC, Moreira AF, Correia IJ (2019) 3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs. Biotechnol Bioeng 116(1):206\u0026ndash;226\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-nanotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cano","sideBox":"Learn more about [Cancer Nanotechnology](https://cancer-nano.biomedcentral.com/)","snPcode":"12645","submissionUrl":"https://submission.nature.com/new-submission/12645/3","title":"Cancer Nanotechnology","twitterHandle":"@CancerNanotech","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Breast cancer, Spheroids, Hybrid nanozymes, Doxorubicin, Photothermal therapy","lastPublishedDoi":"10.21203/rs.3.rs-4417286/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4417286/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOverwhelming evidence suggests that nanozymes show great promise in cancer therapy due to their stable catalytic properties and cost-effectiveness. However, the diverse responses of nanozymes in therapy have presented challenges. After designing pH-sensitive Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes with catalytic properties, we analyzed their characteristics using various techniques such as SEM, TEM, DLS, XRD, TGA, EDS, etc. We evaluated the nanozymes' toxicity on MCF-7 cells and their spheroids through MTT and flow cytometry assays, while also exploring their synergistic effects with photothermal therapy (PTT). The findings reveal that the 150\u0026ndash;270 nm Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes demonstrate stable DOX release and catalytic activity in generating O\u003csub\u003e2\u003c/sub\u003e and \u003csup\u003e\u0026deg;\u003c/sup\u003eOH, effectively inhibiting the growth of MCF-7 cells. It was found that the effective concentration for MCF-7 cells had to be raised from 2.13 to 4.64 \u0026micro;g/mL to inhibit spheroid growth. Because of the toxicity of this concentration on normal cells, using synergistic approaches is crucial to minimize side effects. Also, the results of cytotoxicity mechanism in spheroids highlight the significant impact of PTT with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MnO\u003csub\u003e2\u003c/sub\u003e@DOX hybrid nanozymes in enhancing pro-inflammatory cytokines like TNF-α, CASP9, CASP7, and CASP3. Ultimately, optimizing the concentration of pH-sensitive hybrid nanozymes with PTT synergistic effects shows great potential for cancer treatment.\u003c/p\u003e","manuscriptTitle":"Evaluation of the performance of Fe 3 O 4 /MnO 2 hybrid nanozymes with doxorubicin on multicellular structure and their therapeutic management to limit the growth of human breast cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-03 18:47:06","doi":"10.21203/rs.3.rs-4417286/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-21T07:19:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-20T14:13:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-16T19:38:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-15T15:12:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"155287099479921988981919637096668857397","date":"2024-07-10T13:53:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-04T06:44:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-01T02:18:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"107539193317936963551977479695540416804","date":"2024-06-24T07:37:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54505094033998245963788927221521066967","date":"2024-06-24T01:56:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"333728909395788175094380450526752061010","date":"2024-06-23T17:57:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172073012322774546461849874634148691232","date":"2024-06-23T14:11:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"177463108058389877932996473998031463315","date":"2024-05-30T16:00:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-30T11:37:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-21T12:14:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-21T12:14:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Nanotechnology","date":"2024-05-14T07:35:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-nanotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cano","sideBox":"Learn more about [Cancer Nanotechnology](https://cancer-nano.biomedcentral.com/)","snPcode":"12645","submissionUrl":"https://submission.nature.com/new-submission/12645/3","title":"Cancer Nanotechnology","twitterHandle":"@CancerNanotech","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"96bb631d-12ec-4c51-a201-bd201e18e124","owner":[],"postedDate":"June 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-30T15:59:33+00:00","versionOfRecord":{"articleIdentity":"rs-4417286","link":"https://doi.org/10.1186/s12645-024-00293-z","journal":{"identity":"cancer-nanotechnology","isVorOnly":false,"title":"Cancer Nanotechnology"},"publishedOn":"2024-09-28 15:57:00","publishedOnDateReadable":"September 28th, 2024"},"versionCreatedAt":"2024-06-03 18:47:06","video":"","vorDoi":"10.1186/s12645-024-00293-z","vorDoiUrl":"https://doi.org/10.1186/s12645-024-00293-z","workflowStages":[]},"version":"v1","identity":"rs-4417286","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4417286","identity":"rs-4417286","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}