Synthesis of Magnetic Copper Ferrite Nanosheet for Highly Efficient ROS generation and Photothermal Cancer Therapy

Synthesis of Magnetic Copper Ferrite Nanosheet for Highly Efficient ROS generation and Photothermal Cancer Therapy | 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 Synthesis of Magnetic Copper Ferrite Nanosheet for Highly Efficient ROS generation and Photothermal Cancer Therapy Srinivasan Rajasekar, Mohammad Iqbal Farheena, Chithira Appanan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7778334/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A primary challenge in preclinical cancer research is developing innovative, adaptable, and effective treatments that prioritise patient safety. This is particularly relevant for investigating nanoplatform-based therapies. This study demonstrates that Magnetic Copper Ferrite Nanosheets (CuFe 2 O 4 NS) possess the unique capability to function as both highly effective Reactive Oxygen Species (ROS) generators and agents for Photothermal Cancer Therapy. CuFe 2 O 4 NSs were successfully synthesised using a facile hydrothermal method. The morphological properties of the synthesised CuFe 2 O 4 NSs were characterised using scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HR-TEM), and energy-dispersive X-ray spectroscopy (EDAX). Notably, the CuFe 2 O 4 NSs exhibited enhanced ROS-generating activity at pH 5.0, as evidenced by the results of an in vitro investigation of ROS generation. Culturing HepG2 cancer cells with CuFe 2 O 4 NSs resulted in a marked increase in their mortality rate owing to the production of ROS and nuclear fragmentation during photothermal therapy. These results demonstrate the exceptional potential of CuFe 2 O 4 NSs as highly effective catalysts for both photothermal therapy (PTT) and ROS production. Furthermore, this study established that CuFe 2 O 4 NSs are promising novel platforms for cancer therapy. Photothermal therapy Nanosheet ROS generation and CuFe2O4 NS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction The World Health Organization has identified cancer as the leading cause of mortality worldwide, accounting for approximately 10 million deaths annually, representing nearly one-sixth of all global fatalities [ 1 ]. Despite the use of various medications in clinical settings to aid cancer patients, significant challenges remain in extending progression-free survival and improving post-treatment prognosis [ 2 ]. Standard therapeutic approaches for this condition include surgical tumour removal, radiation therapy, hormonal treatment, and combinations of chemotherapeutic drugs [ 3 ]. However, many of these strategies induce mild-to-severe adverse reactions in patients or are ineffective when the disease has advanced. Consequently, research has focused on the development of noninvasive cancer treatments. Photothermal therapy (PTT) is widely regarded as a highly promising form of hyperthermia owing to its high level of externally controlled specificity and exceptional safety [ 4 ]. PTT involves the use of photothermal conversion agents (PTAs) within tumour cells to convert light energy from near-infrared (NIR) irradiation into heat energy. This process generates heat within tumour cells, potentially leading to cell death via necrosis or apoptosis [ 5 ]. PTT offers several advantages for tumour treatment, including being minimally invasive, noninvasive, and highly effective. Despite these advantages, challenges such as the uneven distribution of injected PTT agents and NIR light hinder their ability to penetrate deep cancer tissues; thus, PTT alone has not achieved more effective tumour therapy. PTT is a potent method for effectively eradicating cancer when it is integrated with other cancer treatments [ 6 ]. The distinctive characteristics of two-dimensional (2D) nanomaterials, including their minimal thickness, extensive lateral dimensions, and ability to retain electrons without interlayer interactions, set them apart from zero-, one-, and three-dimensional structures, significantly enhancing the effectiveness of cancer therapy [ 7 , 8 ]. While a variety of 2D nanomaterials are currently utilised in diverse biomedical applications, 2D nanosheets (NSs) with suitable nanocargoes exhibit substantial potential for overcoming the challenges in cancer treatment. Generally, NSs are regarded as versatile systems with a broad array of surface-functionalized moieties owing to their large surface area and ease of modification. NSs fulfil the criteria for nanosystems owing to their biodegradability and biocompatibility [ 9 , 10 , 11 ]. A previous study demonstrated that biomimetic copper peroxide nanosheets (CPNS), formed through the self-assembly of nanozymes, were capable of delivering H 2 O 2 in acidic environments and initiating a series of intracellular biochemical reactions to generate ROS under both normoxic and hypoxic conditions without the need for external stimulation [ 11 ]. Another innovative approach involves the use of MoS 2 nanosheets combined with chitosan (CS). These functionalized MoS 2 nanosheets were engineered to act as nanocarriers for chemotherapeutic drugs, facilitating drug delivery in response to near-infrared (NIR) photothermal stimulation. This strategy integrates chemotherapy and photothermal therapy into a single system for cancer treatment. In vitro and in vivo studies of tumour ablation have demonstrated a more potent therapeutic effect when employing a combination of treatments, as opposed to chemotherapy or photothermal therapy alone [ 12 ]. Diverse arrays of nanostructures (NSs) have garnered considerable attention from researchers, particularly ferrite-based nanomaterials. [ 13 ]. These materials have been extensively investigated owing to their distinctive magnetic properties and relatively low toxicity. Notably, iron oxide-based nanomaterials have been approved by the US Food and Drug Administration for clinical applications, including use as iron supplements, magnetic resonance contrast agents, and drug carriers [ 14 ]. Ferrite nanomaterials primarily comprise ferric oxide as a core component, supplemented with one or more metal-based oxides, such as those derived from manganese, copper, nickel, cobalt, or zinc. In the acidic milieu of tumours, ferrite nanomaterials exhibit peroxidase-like activity, facilitating the catalysis of the Fenton reaction of H 2 O 2 to produce highly toxic •OH, thereby inducing tumour cell death [ 15 , 16 ]. Peroxidase activity is modulated by both the intrinsic properties of ferrite nanomaterials (including their chemical composition, crystal structure, and particle size) and extrinsic factors associated with the ROS-containing bio-microenvironment (such as physiological pH and buffers, biogenic reducing agents, and other organic compounds) [ 16 ]. This study investigated the application of magnetic copper ferrite nanosheets (CuFe 2 O 4 NSs) as nanomaterials for cancer therapy. These nanosheets can generate free radicals and facilitate photothermal ablation, both of which contribute to their remarkable antitumour efficacy. CuFe 2 O 4 NSs were synthesised via a hydrothermal method using precursors such as copper hexanitrite (CuH 2 N 2 O 7 ) and iron (III) nitrate nonahydrate (Fe (NO 3 ) 3 .9H 2 O). The physicochemical properties of the nanostructures were assessed using various characterisation techniques. In vitro studies of CuFe 2 O 4 NSs were conducted using HepG2 cancer cells, employing ROS generation detection methods. 2. Materials and Methods 2.1 Materials CuH 2 N 2 O 7 , Fe (NO 3 ) 3 .9H 2 O, 2′ -7′ dichlorofluorescin diacetate (DCFH-DA), hydrogen peroxide (H 2 O 2 ), polyvinylpyrrolidone (PVP), phosphate-buffered saline (PBS), 4′,6- diamidino-2-phenylindole (DAPI), sodium borohydride, 3,3′,5,5′ -tetramethylbenzidine (TMB), sodium borohydride (≥ 98.0%), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich. 2.2 Preparation of Copper ferrite nanosheet (CuFe 2 O 4 NS) Copper ferrite nanosheets (CuFe 2 O 4 NS) were synthesised using a hydrothermal method. In this procedure, 30 mL of an aqueous solution containing 0.04 g of CuH 2 N 2 O 7 , 0.085 g of Fe (NO 3 )3.9H 2 O, and 0.2 g of PVP was dissolved in 20 mL of an aqueous solution of 0.45 g of sodium borohydride at 180°C for 12 h. The resultant product was obtained by centrifugation at 8000 rpm for 20 min and dried at 60°C overnight. The resulting brown product was calcined at 780°C for 5 h. Finally, the synthesised CuFe 2 O 4 NS were characterised. 2.3 Characterization of CuFe 2 O 4 NS The physical and chemical properties of NS, including its dimensions, chemical constituents, and elemental composition, were evaluated using spectroscopic and microscopic techniques. The morphology and size of the CuFe 2 O 4 NSs were examined using high-resolution transmission electron microscopy (HR-TEM) at an accelerating voltage of 200 kV. For HR-TEM analysis, a 5 µL (1 mg/mL) diluted suspension of CuFe 2 O 4 NSs was deposited on a carbon-coated copper grid, and the sample was dried in a hot air oven at 37°C for 5 h. For field-emission scanning electron microscopy (FE-SEM) analysis, a dilute suspension of CuFe 2 O 4 NSs (50 µL, 1 mg mL −) was prepared. This suspension was placed on a silicon wafer and dried in a hot air oven at 37°C for 5 h to ensure complete moisture removal. Subsequently, the samples were analysed using a field-emission scanning electron microscope (FE-SEM, ZEISS) operating at an accelerating voltage of 5 kV. The crystallinity of the synthesised NSs was assessed using X-ray diffraction (XRD) at 2θ values ranging from 10° to 90°, employing a D/max-2550 PC XRD device (Rigaku). X-ray photoelectron spectroscopy (XPS) was performed using a monochromatic Al Kα X-ray source to determine the purity and composition of the synthesised CuFe 2 O 4 NSs. 2.4 In vitro photothermal efficacy of CuFe 2 O 4 NSs An aqueous dispersion of CuFe 2 O 4 NSs was irradiated with a near-infrared (NIR) laser with a wavelength of 808 nm for 5 min. The cuvette contained varying concentrations of NSs (50, 100, 150, 200, and 300 µg mL-1), and the results were compared with those of the control experiments conducted with distilled water. An infrared thermal imager was used to record the temperature changes at 20-second intervals. The photothermal stability and conversion efficiency (η) of the CuFe 2 O 4 NSs were assessed using an on/off laser experiment, as previously reported [ 17 , 18 , 19 ]. 2.5 Extracellular reactive oxygen species (ROS) detection assay TMB was used to detect ROS in the presence of H 2 O 2 . In brief, approximately 5 µL of TMB, dissolved in DMSO at a concentration of 20 mg/mL, was combined with 1 mL of CuFe 2 O 4 NS suspension containing 100 µM H 2 O 2 at varying pH levels of PBS (pH = 5.0, 7.4). The blue oxidation product was measured at an absorbance of 652 nm to evaluate the ROS generation potential of TMB + H 2 O 2 (control), TMB + H 2 O 2 + CuFe 2 O 4 NS (pH 5.0), and TMB + H 2 O 2 + CuFe 2 O 4 NS (pH 7.4) [ 19 , 20 , 21 ]. 2.6 ROS detection assay using HepG2 cancer cells DAPI was used to stain cells in a 6-well plate, with each well containing 2 × 10 6 cells. The cells were subsequently treated with 10 mM DMSO as a control, 100 µg of bare CuFe 2 O 4 NS, or 100 µg of bare CuFe 2 O 4 combined with 50 and 100 µM H 2 O 2 for 24 h. The DAPI staining procedure followed the methodologies outlined in previous studies. The cells were exposed to 5 µg/mL DCFH-DA and incubated for 30 min. Subsequently, the cells were rinsed with PBS, and ROS production was evaluated using a fluorescence microscope (Nikon Eclipse, Inc., Japan) at excitation and emission wavelengths of 488 and 530 nm. The mean fluorescence intensity of DCF was measured to determine the ROS levels within the cells. 2.7 In vitro antitumor effect HepG2 cells were incubated at 37 ◦C for 12 h at a density of 2 × 10 5 cells per well in a 12-well plate. Subsequently, DMEM containing both control and CuFe 2 O 4 NS was added to the cell culture at a concentration of 75 µg/mL. The cells were cultured for an additional 4 h, after which they were exposed to an NIR laser (808 nm, 1.5 W cm − 2 ) for 5 min or left untreated. The cells were then washed thrice with PBS and stained with 50 µL of AO (10 mg/mL) and PI (10 mg/mL) for 20 min. Stained cells were examined using an inverted fluorescence microscope (LSM700, Zeiss, Germany) [ 19 , 21 ]. 2.8 Statistical analysis All experiments were performed in triplicate, and the results are presented as mean ± standard deviation (SD). 3. Results and discussion Two-dimensional (2D) copper ferrite nanosheets (CuFe 2 O 4 NSs) were synthesised via a hydrothermal process using a combination of chemical components, including CuH 2 N 2 O 7 , Fe (NO 3 ) 3 .9H 2 O, polyvinylpyrrolidone, and sodium borohydride. The formation of nanosheets was accomplished in an aqueous solution, and it was hypothesised that the interaction between the precursors and the reducing agent may be the mechanism responsible for the development of the prominent 2D CuFe 2 O 4 NSs. Figure 1 provides a detailed illustration of the general synthesis approach and practical applications of the 2D CuFe 2 O 4 NSs. The dimensions and morphology of the synthesised CuFe 2 O 4 NSs were examined using FE-SEM and HR-TEM, respectively. As illustrated in Fig. 2 A, the synthesised CuFe 2 O 4 NSs exhibited an irregular nanosheet morphology with rough surfaces and a tendency to adhere together. This agglomeration is attributed to the application of high-temperature heat treatment and the influence of the magnetic forces. The diameters of these nanosheets were broadly distributed, ranging from 130 nm to 850 nm. Furthermore, the TEM image corroborates the irregular nanosheet morphology, as shown in Fig. 2 B. The magnetic CuFe 2 O 4 NSs demonstrated remarkable resilience and stability, even after ultrasonication for over one hour prior to SEM and TEM measurements, indicating their promising potential for practical applications in large-scale production. Figure 2 C presents the SAED pattern of the magnetic CuFe 2 O 4 NSs with a spinel ferrite structure. The SAED pattern of the spinel crystal lattice in the CuFe 2 O 4 samples revealed diffraction rings at the (220), (311), (400), (422), and (511) planes, indicating that the samples were polycrystalline in nature with a similar crystal structure [ 21 – 23 ]. In addition, the presence of bright spots supports this conclusion. The chemical composition of the synthesised magnetic CuFe 2 O 4 NS was determined by qualitative EDX analysis, as shown in Fig. 3 A. EDX spectrum analysis revealed that the synthesised magnetic CuFe 2 O 4 NS comprised copper (38.56%), iron (33.22%), and oxygen (28.22%), thereby confirming its composition. Additional signals from silica were attributed to the use of a silica substrate during SEM analysis. Furthermore, the elemental mapping images presented in Fig. 3 B illustrate the distribution of Cu, Fe, and O within the designed magnetic CuFe 2 O 4 NS, corroborating the uniform distribution of these elements. The chemical composition and surface characteristics of the CuFe 2 O 4 NS were comprehensively analysed using XPS, as shown in Fig. 4 A. The survey spectrum clearly revealed the presence of Cu, Fe, and O in the samples. The O 1s peak in the XPS spectra was located at approximately 529.74 eV, indicative of both O 2+ ions and -OH groups in the sample. The primary peak at 710.9 eV and the secondary peak at 724.6 eV correspond to Fe 2p 3/2 and Fe 2p 1/2 , respectively. The binding energies of Cu 2p 3/2 and Cu 2p 1/2 were observed at 933.5 eV and 954.2 eV, respectively, with a characteristic satellite peak of Cu 3+ at 943.3 eV [ 23 ]. These findings confirmed the successful synthesis of the CuFe 2 O 4 NS. The magnetisation value of the magnetic CuFe 2 O 4 NS annealed at 750°C is shown in Fig. 4 B. The sample exhibited ferromagnetic characteristics, with a saturation magnetisation of approximately 15.02 emu/g. This sample exhibited sufficient magnetic properties, enabling it to be easily attracted to a laboratory magnet, thereby rendering it an ideal candidate for the core of recyclable catalysts. These findings suggest that these catalysts can be reclaimed and reused after the catalytic process. These results highlight the significant impact of annealing temperature on the magnetic properties of the synthesised CuFe 2 O 4 NS. To assess the photothermal properties of the CuFe 2 O 4 NS, the temperature changes in 0.4 mL aqueous solutions with varying concentrations of CuFe 2 O 4 NS (50, 100, 150, 200, and 300 µg/mL) were monitored under NIR laser irradiation at a wavelength of 808 nm. As shown in Fig. 5 A, the increase in temperature exhibited a concentration-dependent trend. Notably, at a concentration of 300 µg/mL, the temperature reached 78.7°C upon NIR laser irradiation. In contrast, the temperature of the distilled water control group remained stable, showing no significant change (2.6°C), thereby confirming that NIR irradiation did not adversely affect the normal tissues. The CuFe 2 O 4 NS exhibited a significant photothermal effect, sufficient to induce thermal damage in cancerous tissues. Consequently, they are regarded as promising candidates for use as highly effective photothermal therapy agents in cancer treatment. A colorimetric detection method was used to demonstrate the ROS generation capacity of the CuFe 2 O 4 NS. Colourless TMB substrate was used as a probe. In the presence of H 2 O 2 , colourless TMB was oxidised to blue oxidised TMB (oxTMB), which was detected at an absorbance peak of 652 nm. The CuFe 2 O 4 NSs were evaluated for ROS generation across various pH levels, including 7.4 and 5.0. The CuFe 2 O 4 NSs exhibited enhanced peroxidase activity at pH 5.0, as indicated by the increased oxTMB absorption and resultant blue colouration (Fig. 5 B). Consequently, CuFe 2 O 4 NSs demonstrate exceptional sensitivity to a wide range of temperatures and pH values, as well as low concentrations of H 2 O 2 , thereby enabling efficient catalysis of peroxidase-like reactions [ 24 – 26 ]. The intracellular nuclear morphology and ROS generation of the control, bare CuFe 2 O 4 NS, CuFe 2 O 4 NS + 50 µM H 2 O 2 , and CuFe 2 O 4 NS + 100 µM H 2 O 2 samples were assessed using DAPI and DCFH-DA staining (Fig. 6 ). HepG2 cancer cells exhibited moderate cytotoxicity under control conditions, whereas exposure to CuFe 2 O 4 NSs resulted in pronounced nuclear fragmentation and elevated reactive oxygen species (ROS) production. These findings suggest that CuFe 2 O 4 NSs significantly contribute to the death of HepG2 cancer cells through ROS generation and nuclear fragmentation during photothermal therapy. The application of CuFe 2 O 4 NSs as a Fenton-like catalyst is advantageous when cancer cells produce high levels of lactic acid, as it facilitates the dismutation of H 2 O 2 into •OH. The production of ROS, such as O2- and H 2 O 2 , through the dismutation of molecular O 2 or CO 2 in cancer cells, induces oxidative stress, ultimately leading to cell death. Therefore, CuFe 2 O 4 NSs represent a unique therapeutic agent that can aid in the treatment of cancer. Figure 7 shows the data on the photothermal cytotoxicity of both the control and CuFe 2 O 4 NS at a concentration of 75 µg mL − 1 in HepG2 cells, assessed via the live/dead staining technique. This method employs acridine orange (AO) and propidium iodide (PI) dyes, which are essential tools in cell biology research, to distinguish between live and dead cells. AO is an intercalating dye capable of penetrating both living and dead cells, staining all nucleated cells, and emitting a green fluorescence. Conversely, propidium iodide can only permeate dead cells with compromised membrane integrity, staining all deceased nucleated cells. The findings indicated that HepG2 cells treated with the control exhibited viability, as evidenced by the green fluorescence. In contrast, treatment with CuFe 2 O 4 NSs resulted in the effective destruction of cancer cells when exposed to an NIR laser at 808 nm wavelength. Furthermore, HepG2 cells treated with CuFe 2 O 4 NSs demonstrated strong red fluorescence upon 5-minute NIR irradiation. These results substantiate the efficacy of CuFe 2 O 4 NSs for the treatment of HepG2 cancer cells. The ability of metallic ferrite to store oxygen and facilitate electron transfer is pivotal for the formation of the active electronic configuration of CuFe 2 O 4 NSs. This characteristic enables efficient heat transfer via surface electrons during irradiation, which is critical for the overall performance of these materials. Additionally, magnetic nanomaterials are considered superior candidates for use as PTT agents in cancer therapy. 4. Conclusion The designed nanosheets were intended to incorporate all the critical factors necessary for the efficient generation of ROS and photothermal cancer therapy while also mitigating adverse effects within a single multifunctional synthesised nanoplatform, termed CuFe 2 O 4 NSs. This study introduces a straightforward method for synthesising an effective photothermal agent, namely, CuFe 2 O 4 NSs, using a hydrothermal technique. Advanced microscopic techniques, including HR-TEM, SEM, and EDAX mapping, were employed to verify the morphological characteristics of the synthesised CuFe 2 O 4 NSs. The diameter of the CuFe 2 O 4 nanosheets typically ranged from 130 to 850 nm. Using a colorimetric measurement approach, we demonstrated that the synthesised CuFe 2 O 4 NSs exhibited enhanced peroxidase activity at pH 5.0. These results indicate that CuFe 2 O 4 NSs significantly contribute to the apoptosis of HepG2 cancer cells by generating ROS and inducing nuclear fragmentation during photothermal therapy. In conclusion, CuFe 2 O 4 NSs are potent agents for dual-action cancer treatment, encompassing both ROS production and PTT. Declarations Conflicts of interest The authors declare no conflicts of interest. Ethics declaration Not applicable Funding The authors declare that no funding was received from any organisation or agency to support this research. Author Contribution Srinivasan Rajasekar: conceptualization, design of methodology, development of the synthesis protocol, supervision, and the writing and editing of the manuscript. Mohammad Iqbal Farheena: Conducted the experimental execution, ROS generation assays, photothermal therapy studies, and data collection. Chithira Appanan: material characterization, data analysis, and figure preparation. Santhi Kuppusamy: contributed to the literature review, manuscript proofreading, and provided support for the experimental design. Acknowledgement Srinivasan Rajasekar: conceptualization, design of methodology, development of the synthesis protocol, supervision, and the writing and editing of the manuscript. Mohammad Iqbal Farheena: Conducted the experimental execution, ROS generation assays, photothermal therapy studies, and data collection. Chithira Appanan: material characterization, data analysis, and figure preparation. 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15:47:51","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":82738,"visible":true,"origin":"","legend":"","description":"","filename":"f72f6d996d6e423e906ece6b9d06d8451structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7778334/v1/fcd324ccb0e09aa4ea6544b5.xml"},{"id":94473924,"identity":"980bfe0a-4f2f-4d40-add3-433452f8acc4","added_by":"auto","created_at":"2025-10-27 15:46:18","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":87557,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7778334/v1/936196f90274fadcfea9ba2d.html"},{"id":94473983,"identity":"a8e61156-fbf7-4041-a03e-120970812363","added_by":"auto","created_at":"2025-10-27 15:46:32","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":80540,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the formation mechanism of the novel CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs assembly and its potential applications.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7778334/v1/1e025c0714238f79b93fe451.jpeg"},{"id":94473717,"identity":"d707f53c-6ba5-4858-a83e-0fbad0d9cc0e","added_by":"auto","created_at":"2025-10-27 15:45:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1677624,"visible":true,"origin":"","legend":"\u003cp\u003eElectron microscopy images illustrate the morphology and microstructural characteristics of the magnetic CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS. (A) Representative FE-SEM images of CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS reveal the presence of irregularly sized, sheet-like materials that adhere together. (B) TEM image and (C) SAED pattern of the CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7778334/v1/b91703ebac63b5ff7b3f617d.png"},{"id":94474081,"identity":"a83083bc-eb47-4825-bb9c-84d9de0d6f3f","added_by":"auto","created_at":"2025-10-27 15:47:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3490639,"visible":true,"origin":"","legend":"\u003cp\u003eThe EDX spectra and elemental mapping images illustrate the elemental distribution within the magnetic nanosheets. (A) EDX analysis identifies the elements present in the magnetic CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS, and (B (i)) presents a TEM image of CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS. (ii-v) Elemental mapping revealed the homogeneous distribution of elements such as copper (Cu), iron (Fe), and oxygen (O) within the magnetic CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7778334/v1/99fba29bb329a5777c60dfe3.png"},{"id":94473825,"identity":"aa7d93f9-dbb0-4766-ba5f-85b749778c8c","added_by":"auto","created_at":"2025-10-27 15:45:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":650342,"visible":true,"origin":"","legend":"\u003cp\u003e(A) XPS and (B) magnetic hysteresis loop of CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7778334/v1/60ff29a950ab448310f5548e.png"},{"id":94474279,"identity":"c8c5d390-7070-48c0-bc58-f6104b58488f","added_by":"auto","created_at":"2025-10-27 15:48:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":491473,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Temperature elevation curves for distilled water (control) and CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS at varying concentrations (50, 100, 150, 200, and 300 μg mL\u003csup\u003e−1\u003c/sup\u003e) and (B) in vitro ROS generation. The catalytic oxidation of TMB by CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs at different pH values was measured using Vis–NIR absorbance.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7778334/v1/83d2c6698a0adfd05f8b7654.png"},{"id":94474327,"identity":"9a4b175f-74e3-4d3c-a8d0-4bc67a38a068","added_by":"auto","created_at":"2025-10-27 15:48:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4503315,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence images depicting ROS generation, nuclear fragmentation, and ROS production were observed in HepG2 cancer cells stained with DAPI and DCFH-DA following treatment with control, bare CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs, CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS combined with 50 μM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS combined with 100 μM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at a magnification of 20x.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7778334/v1/da3c8233b37797c9fe85eec6.png"},{"id":94474009,"identity":"f8fcee5d-9890-495c-92a7-9270418ed640","added_by":"auto","created_at":"2025-10-27 15:46:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3121042,"visible":true,"origin":"","legend":"\u003cp\u003eThe photothermal cytotoxicity of both the control and CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4 \u003c/sub\u003eNS in the presence or absence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at a concentration (50 and 100 µM) towards the HepG2 cell, was evaluated using the acridine orange (AO) and propidium iodide (PI) dyes for live/dead staining technique.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7778334/v1/61e38d4573f58bf209606e50.png"},{"id":94505717,"identity":"93a2af3c-5ecd-4ef5-b938-7988c69dc8d3","added_by":"auto","created_at":"2025-10-28 16:22:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12804653,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7778334/v1/a14063fc-186b-4feb-a174-8fcb30b36a37.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis of Magnetic Copper Ferrite Nanosheet for Highly Efficient ROS generation and Photothermal Cancer Therapy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe World Health Organization has identified cancer as the leading cause of mortality worldwide, accounting for approximately 10\u0026nbsp;million deaths annually, representing nearly one-sixth of all global fatalities [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite the use of various medications in clinical settings to aid cancer patients, significant challenges remain in extending progression-free survival and improving post-treatment prognosis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Standard therapeutic approaches for this condition include surgical tumour removal, radiation therapy, hormonal treatment, and combinations of chemotherapeutic drugs [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, many of these strategies induce mild-to-severe adverse reactions in patients or are ineffective when the disease has advanced. Consequently, research has focused on the development of noninvasive cancer treatments. Photothermal therapy (PTT) is widely regarded as a highly promising form of hyperthermia owing to its high level of externally controlled specificity and exceptional safety [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. PTT involves the use of photothermal conversion agents (PTAs) within tumour cells to convert light energy from near-infrared (NIR) irradiation into heat energy. This process generates heat within tumour cells, potentially leading to cell death via necrosis or apoptosis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. PTT offers several advantages for tumour treatment, including being minimally invasive, noninvasive, and highly effective. Despite these advantages, challenges such as the uneven distribution of injected PTT agents and NIR light hinder their ability to penetrate deep cancer tissues; thus, PTT alone has not achieved more effective tumour therapy. PTT is a potent method for effectively eradicating cancer when it is integrated with other cancer treatments [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe distinctive characteristics of two-dimensional (2D) nanomaterials, including their minimal thickness, extensive lateral dimensions, and ability to retain electrons without interlayer interactions, set them apart from zero-, one-, and three-dimensional structures, significantly enhancing the effectiveness of cancer therapy [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. While a variety of 2D nanomaterials are currently utilised in diverse biomedical applications, 2D nanosheets (NSs) with suitable nanocargoes exhibit substantial potential for overcoming the challenges in cancer treatment. Generally, NSs are regarded as versatile systems with a broad array of surface-functionalized moieties owing to their large surface area and ease of modification. NSs fulfil the criteria for nanosystems owing to their biodegradability and biocompatibility [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. A previous study demonstrated that biomimetic copper peroxide nanosheets (CPNS), formed through the self-assembly of nanozymes, were capable of delivering H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in acidic environments and initiating a series of intracellular biochemical reactions to generate ROS under both normoxic and hypoxic conditions without the need for external stimulation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Another innovative approach involves the use of MoS\u003csub\u003e2\u003c/sub\u003e nanosheets combined with chitosan (CS). These functionalized MoS\u003csub\u003e2\u003c/sub\u003e nanosheets were engineered to act as nanocarriers for chemotherapeutic drugs, facilitating drug delivery in response to near-infrared (NIR) photothermal stimulation. This strategy integrates chemotherapy and photothermal therapy into a single system for cancer treatment. In vitro and in vivo studies of tumour ablation have demonstrated a more potent therapeutic effect when employing a combination of treatments, as opposed to chemotherapy or photothermal therapy alone [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDiverse arrays of nanostructures (NSs) have garnered considerable attention from researchers, particularly ferrite-based nanomaterials. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These materials have been extensively investigated owing to their distinctive magnetic properties and relatively low toxicity. Notably, iron oxide-based nanomaterials have been approved by the US Food and Drug Administration for clinical applications, including use as iron supplements, magnetic resonance contrast agents, and drug carriers [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Ferrite nanomaterials primarily comprise ferric oxide as a core component, supplemented with one or more metal-based oxides, such as those derived from manganese, copper, nickel, cobalt, or zinc. In the acidic milieu of tumours, ferrite nanomaterials exhibit peroxidase-like activity, facilitating the catalysis of the Fenton reaction of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to produce highly toxic \u0026bull;OH, thereby inducing tumour cell death [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Peroxidase activity is modulated by both the intrinsic properties of ferrite nanomaterials (including their chemical composition, crystal structure, and particle size) and extrinsic factors associated with the ROS-containing bio-microenvironment (such as physiological pH and buffers, biogenic reducing agents, and other organic compounds) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study investigated the application of magnetic copper ferrite nanosheets (CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs) as nanomaterials for cancer therapy. These nanosheets can generate free radicals and facilitate photothermal ablation, both of which contribute to their remarkable antitumour efficacy. CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs were synthesised via a hydrothermal method using precursors such as copper hexanitrite (CuH\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e) and iron (III) nitrate nonahydrate (Fe (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e.9H\u003csub\u003e2\u003c/sub\u003eO). The physicochemical properties of the nanostructures were assessed using various characterisation techniques. In vitro studies of CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs were conducted using HepG2 cancer cells, employing ROS generation detection methods.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials\u003c/h2\u003e\u003cp\u003eCuH\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e, Fe (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e.9H\u003csub\u003e2\u003c/sub\u003eO, 2\u0026prime; -7\u0026prime; dichlorofluorescin diacetate (DCFH-DA), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), polyvinylpyrrolidone (PVP), phosphate-buffered saline (PBS), 4\u0026prime;,6- diamidino-2-phenylindole (DAPI), sodium borohydride, 3,3\u0026prime;,5,5\u0026prime; -tetramethylbenzidine (TMB), sodium borohydride (\u0026ge;\u0026thinsp;98.0%), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Preparation of Copper ferrite nanosheet (CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS)\u003c/h2\u003e\u003cp\u003eCopper ferrite nanosheets (CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS) were synthesised using a hydrothermal method. In this procedure, 30 mL of an aqueous solution containing 0.04 g of CuH\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e, 0.085 g of Fe (NO\u003csub\u003e3\u003c/sub\u003e)3.9H\u003csub\u003e2\u003c/sub\u003eO, and 0.2 g of PVP was dissolved in 20 mL of an aqueous solution of 0.45 g of sodium borohydride at 180\u0026deg;C for 12 h. The resultant product was obtained by centrifugation at 8000 rpm for 20 min and dried at 60\u0026deg;C overnight. The resulting brown product was calcined at 780\u0026deg;C for 5 h. Finally, the synthesised CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS were characterised.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Characterization of CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS\u003c/h2\u003e\u003cp\u003eThe physical and chemical properties of NS, including its dimensions, chemical constituents, and elemental composition, were evaluated using spectroscopic and microscopic techniques. The morphology and size of the CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs were examined using high-resolution transmission electron microscopy (HR-TEM) at an accelerating voltage of 200 kV. For HR-TEM analysis, a 5 \u0026micro;L (1 mg/mL) diluted suspension of CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs was deposited on a carbon-coated copper grid, and the sample was dried in a hot air oven at 37\u0026deg;C for 5 h. For field-emission scanning electron microscopy (FE-SEM) analysis, a dilute suspension of CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs (50 \u0026micro;L, 1 mg mL \u0026minus;) was prepared. This suspension was placed on a silicon wafer and dried in a hot air oven at 37\u0026deg;C for 5 h to ensure complete moisture removal. Subsequently, the samples were analysed using a field-emission scanning electron microscope (FE-SEM, ZEISS) operating at an accelerating voltage of 5 kV. The crystallinity of the synthesised NSs was assessed using X-ray diffraction (XRD) at 2θ values ranging from 10\u0026deg; to 90\u0026deg;, employing a D/max-2550 PC XRD device (Rigaku). X-ray photoelectron spectroscopy (XPS) was performed using a monochromatic Al Kα X-ray source to determine the purity and composition of the synthesised CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003e2.4 In vitro photothermal efficacy of\u003c/em\u003e CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs\u003c/h2\u003e\u003cp\u003eAn aqueous dispersion of CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs was irradiated with a near-infrared (NIR) laser with a wavelength of 808 nm for 5 min. The cuvette contained varying concentrations of NSs (50, 100, 150, 200, and 300 \u0026micro;g mL-1), and the results were compared with those of the control experiments conducted with distilled water. An infrared thermal imager was used to record the temperature changes at 20-second intervals. The photothermal stability and conversion efficiency (η) of the CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs were assessed using an on/off laser experiment, as previously reported [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Extracellular reactive oxygen species (ROS) detection assay\u003c/h2\u003e\u003cp\u003eTMB was used to detect ROS in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. In brief, approximately 5 \u0026micro;L of TMB, dissolved in DMSO at a concentration of 20 mg/mL, was combined with 1 mL of CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS suspension containing 100 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at varying pH levels of PBS (pH\u0026thinsp;=\u0026thinsp;5.0, 7.4). The blue oxidation product was measured at an absorbance of 652 nm to evaluate the ROS generation potential of TMB\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (control), TMB\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS (pH 5.0), and TMB\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS (pH 7.4) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 ROS detection assay using HepG2 cancer cells\u003c/h2\u003e\u003cp\u003eDAPI was used to stain cells in a 6-well plate, with each well containing 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells. The cells were subsequently treated with 10 mM DMSO as a control, 100 \u0026micro;g of bare CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS, or 100 \u0026micro;g of bare CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e combined with 50 and 100 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 24 h. The DAPI staining procedure followed the methodologies outlined in previous studies. The cells were exposed to 5 \u0026micro;g/mL DCFH-DA and incubated for 30 min. Subsequently, the cells were rinsed with PBS, and ROS production was evaluated using a fluorescence microscope (Nikon Eclipse, Inc., Japan) at excitation and emission wavelengths of 488 and 530 nm. The mean fluorescence intensity of DCF was measured to determine the ROS levels within the cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 In vitro antitumor effect\u003c/h2\u003e\u003cp\u003eHepG2 cells were incubated at 37 ◦C for 12 h at a density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well in a 12-well plate. Subsequently, DMEM containing both control and CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS was added to the cell culture at a concentration of 75 \u0026micro;g/mL. The cells were cultured for an additional 4 h, after which they were exposed to an NIR laser (808 nm, 1.5 W cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) for 5 min or left untreated. The cells were then washed thrice with PBS and stained with 50 \u0026micro;L of AO (10 mg/mL) and PI (10 mg/mL) for 20 min. Stained cells were examined using an inverted fluorescence microscope (LSM700, Zeiss, Germany) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e\u003cp\u003eAll experiments were performed in triplicate, and the results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003eTwo-dimensional (2D) copper ferrite nanosheets (CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs) were synthesised via a hydrothermal process using a combination of chemical components, including CuH\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e, Fe (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e.9H\u003csub\u003e2\u003c/sub\u003eO, polyvinylpyrrolidone, and sodium borohydride. The formation of nanosheets was accomplished in an aqueous solution, and it was hypothesised that the interaction between the precursors and the reducing agent may be the mechanism responsible for the development of the prominent 2D CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e provides a detailed illustration of the general synthesis approach and practical applications of the 2D CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe dimensions and morphology of the synthesised CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs were examined using FE-SEM and HR-TEM, respectively. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the synthesised CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs exhibited an irregular nanosheet morphology with rough surfaces and a tendency to adhere together. This agglomeration is attributed to the application of high-temperature heat treatment and the influence of the magnetic forces. The diameters of these nanosheets were broadly distributed, ranging from 130 nm to 850 nm. Furthermore, the TEM image corroborates the irregular nanosheet morphology, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. The magnetic CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs demonstrated remarkable resilience and stability, even after ultrasonication for over one hour prior to SEM and TEM measurements, indicating their promising potential for practical applications in large-scale production. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC presents the SAED pattern of the magnetic CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs with a spinel ferrite structure. The SAED pattern of the spinel crystal lattice in the CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e samples revealed diffraction rings at the (220), (311), (400), (422), and (511) planes, indicating that the samples were polycrystalline in nature with a similar crystal structure [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In addition, the presence of bright spots supports this conclusion.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe chemical composition of the synthesised magnetic CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS was determined by qualitative EDX analysis, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. EDX spectrum analysis revealed that the synthesised magnetic CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS comprised copper (38.56%), iron (33.22%), and oxygen (28.22%), thereby confirming its composition. Additional signals from silica were attributed to the use of a silica substrate during SEM analysis. Furthermore, the elemental mapping images presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB illustrate the distribution of Cu, Fe, and O within the designed magnetic CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS, corroborating the uniform distribution of these elements.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe chemical composition and surface characteristics of the CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS were comprehensively analysed using XPS, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. The survey spectrum clearly revealed the presence of Cu, Fe, and O in the samples. The O 1s peak in the XPS spectra was located at approximately 529.74 eV, indicative of both O\u003csup\u003e2+\u003c/sup\u003e ions and -OH groups in the sample. The primary peak at 710.9 eV and the secondary peak at 724.6 eV correspond to Fe 2p\u003csub\u003e3/2\u003c/sub\u003e and Fe 2p\u003csub\u003e1/2\u003c/sub\u003e, respectively. The binding energies of Cu 2p\u003csub\u003e3/2\u003c/sub\u003e and Cu 2p\u003csub\u003e1/2\u003c/sub\u003e were observed at 933.5 eV and 954.2 eV, respectively, with a characteristic satellite peak of Cu\u003csup\u003e3+\u003c/sup\u003e at 943.3 eV [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These findings confirmed the successful synthesis of the CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS. The magnetisation value of the magnetic CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS annealed at 750\u0026deg;C is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. The sample exhibited ferromagnetic characteristics, with a saturation magnetisation of approximately 15.02 emu/g. This sample exhibited sufficient magnetic properties, enabling it to be easily attracted to a laboratory magnet, thereby rendering it an ideal candidate for the core of recyclable catalysts. These findings suggest that these catalysts can be reclaimed and reused after the catalytic process. These results highlight the significant impact of annealing temperature on the magnetic properties of the synthesised CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess the photothermal properties of the CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS, the temperature changes in 0.4 mL aqueous solutions with varying concentrations of CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS (50, 100, 150, 200, and 300 \u0026micro;g/mL) were monitored under NIR laser irradiation at a wavelength of 808 nm. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, the increase in temperature exhibited a concentration-dependent trend. Notably, at a concentration of 300 \u0026micro;g/mL, the temperature reached 78.7\u0026deg;C upon NIR laser irradiation. In contrast, the temperature of the distilled water control group remained stable, showing no significant change (2.6\u0026deg;C), thereby confirming that NIR irradiation did not adversely affect the normal tissues. The CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS exhibited a significant photothermal effect, sufficient to induce thermal damage in cancerous tissues. Consequently, they are regarded as promising candidates for use as highly effective photothermal therapy agents in cancer treatment. A colorimetric detection method was used to demonstrate the ROS generation capacity of the CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS. Colourless TMB substrate was used as a probe. In the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, colourless TMB was oxidised to blue oxidised TMB (oxTMB), which was detected at an absorbance peak of 652 nm. The CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs were evaluated for ROS generation across various pH levels, including 7.4 and 5.0. The CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs exhibited enhanced peroxidase activity at pH 5.0, as indicated by the increased oxTMB absorption and resultant blue colouration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Consequently, CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs demonstrate exceptional sensitivity to a wide range of temperatures and pH values, as well as low concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, thereby enabling efficient catalysis of peroxidase-like reactions [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe intracellular nuclear morphology and ROS generation of the control, bare CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS, CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS\u0026thinsp;+\u0026thinsp;50 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS\u0026thinsp;+\u0026thinsp;100 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e samples were assessed using DAPI and DCFH-DA staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). HepG2 cancer cells exhibited moderate cytotoxicity under control conditions, whereas exposure to CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs resulted in pronounced nuclear fragmentation and elevated reactive oxygen species (ROS) production. These findings suggest that CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs significantly contribute to the death of HepG2 cancer cells through ROS generation and nuclear fragmentation during photothermal therapy. The application of CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs as a Fenton-like catalyst is advantageous when cancer cells produce high levels of lactic acid, as it facilitates the dismutation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into \u0026bull;OH. The production of ROS, such as O2- and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, through the dismutation of molecular O\u003csub\u003e2\u003c/sub\u003e or CO\u003csub\u003e2\u003c/sub\u003e in cancer cells, induces oxidative stress, ultimately leading to cell death. Therefore, CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs represent a unique therapeutic agent that can aid in the treatment of cancer.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows the data on the photothermal cytotoxicity of both the control and CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS at a concentration of 75 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in HepG2 cells, assessed via the live/dead staining technique. This method employs acridine orange (AO) and propidium iodide (PI) dyes, which are essential tools in cell biology research, to distinguish between live and dead cells. AO is an intercalating dye capable of penetrating both living and dead cells, staining all nucleated cells, and emitting a green fluorescence. Conversely, propidium iodide can only permeate dead cells with compromised membrane integrity, staining all deceased nucleated cells. The findings indicated that HepG2 cells treated with the control exhibited viability, as evidenced by the green fluorescence. In contrast, treatment with CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs resulted in the effective destruction of cancer cells when exposed to an NIR laser at 808 nm wavelength. Furthermore, HepG2 cells treated with CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs demonstrated strong red fluorescence upon 5-minute NIR irradiation. These results substantiate the efficacy of CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs for the treatment of HepG2 cancer cells. The ability of metallic ferrite to store oxygen and facilitate electron transfer is pivotal for the formation of the active electronic configuration of CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs. This characteristic enables efficient heat transfer via surface electrons during irradiation, which is critical for the overall performance of these materials. Additionally, magnetic nanomaterials are considered superior candidates for use as PTT agents in cancer therapy.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe designed nanosheets were intended to incorporate all the critical factors necessary for the efficient generation of ROS and photothermal cancer therapy while also mitigating adverse effects within a single multifunctional synthesised nanoplatform, termed CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs. This study introduces a straightforward method for synthesising an effective photothermal agent, namely, CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs, using a hydrothermal technique. Advanced microscopic techniques, including HR-TEM, SEM, and EDAX mapping, were employed to verify the morphological characteristics of the synthesised CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs. The diameter of the CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanosheets typically ranged from 130 to 850 nm. Using a colorimetric measurement approach, we demonstrated that the synthesised CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs exhibited enhanced peroxidase activity at pH 5.0. These results indicate that CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs significantly contribute to the apoptosis of HepG2 cancer cells by generating ROS and inducing nuclear fragmentation during photothermal therapy. In conclusion, CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs are potent agents for dual-action cancer treatment, encompassing both ROS production and PTT.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflicts of interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe authors declare that no funding was received from any organisation or agency to support this research.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eSrinivasan Rajasekar: conceptualization, design of methodology, development of the synthesis protocol, supervision, and the writing and editing of the manuscript. Mohammad Iqbal Farheena: Conducted the experimental execution, ROS generation assays, photothermal therapy studies, and data collection. Chithira Appanan: material characterization, data analysis, and figure preparation. Santhi Kuppusamy: contributed to the literature review, manuscript proofreading, and provided support for the experimental design.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eSrinivasan Rajasekar: conceptualization, design of methodology, development of the synthesis protocol, supervision, and the writing and editing of the manuscript. Mohammad Iqbal Farheena: Conducted the experimental execution, ROS generation assays, photothermal therapy studies, and data collection. Chithira Appanan: material characterization, data analysis, and figure preparation. Santhi Kuppusamy: contributed to the literature review, manuscript proofreading, and provided support for the experimental design.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eQi, K., Sun, B., Liu, S., \u0026amp; Zhang, M. (2023). Research progress on carbon materials in tumor photothermal therapy. \u003cem\u003eBiomedicine \u0026amp; Pharmacotherapy\u003c/em\u003e, \u003cem\u003e165\u003c/em\u003e, 115070.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHu, Q., Sun, W., Wang, C., \u0026amp; Gu, Z. (2016). Recent advances of cocktail chemotherapy by combination drug delivery systems. \u003cem\u003eAdvanced drug delivery reviews\u003c/em\u003e, \u003cem\u003e98\u003c/em\u003e, 19\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eP\u0026eacute;rez-Herrero, E., \u0026amp; Fern\u0026aacute;ndez-Medarde, A. (2015). 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Tumor-targeted molybdenum disulfide@ barium titanate core\u0026ndash;shell nanomedicine for dual photothermal and chemotherapy of triple-negative breast cancer cells. \u003cem\u003eJournal of Materials Chemistry B\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(5), 1044\u0026ndash;1056.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Photothermal therapy, Nanosheet, ROS generation and CuFe2O4 NS","lastPublishedDoi":"10.21203/rs.3.rs-7778334/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7778334/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA primary challenge in preclinical cancer research is developing innovative, adaptable, and effective treatments that prioritise patient safety. This is particularly relevant for investigating nanoplatform-based therapies. This study demonstrates that Magnetic Copper Ferrite Nanosheets (CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NS) possess the unique capability to function as both highly effective Reactive Oxygen Species (ROS) generators and agents for Photothermal Cancer Therapy. CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs were successfully synthesised using a facile hydrothermal method. The morphological properties of the synthesised CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs were characterised using scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HR-TEM), and energy-dispersive X-ray spectroscopy (EDAX). Notably, the CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs exhibited enhanced ROS-generating activity at pH 5.0, as evidenced by the results of an in vitro investigation of ROS generation. Culturing HepG2 cancer cells with CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs resulted in a marked increase in their mortality rate owing to the production of ROS and nuclear fragmentation during photothermal therapy. These results demonstrate the exceptional potential of CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs as highly effective catalysts for both photothermal therapy (PTT) and ROS production. Furthermore, this study established that CuFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NSs are promising novel platforms for cancer therapy.\u003c/p\u003e","manuscriptTitle":"Synthesis of Magnetic Copper Ferrite Nanosheet for Highly Efficient ROS generation and Photothermal Cancer Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-27 14:30:54","doi":"10.21203/rs.3.rs-7778334/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"74915719-5e3b-4311-8af3-8a91668fef06","owner":[],"postedDate":"October 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-27T15:15:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-27 14:30:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7778334","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7778334","identity":"rs-7778334","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}