Structural characterization of copper and manganese oxide nanoparticles-doped calcium silicate (CaSiO 3 ) for synthesized via sol-gel method

Structural characterization of copper and manganese oxide nanoparticles-doped calcium silicate (CaSiO 3 ) for synthesized via sol-gel method | 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 Structural characterization of copper and manganese oxide nanoparticles-doped calcium silicate (CaSiO 3 ) for synthesized via sol-gel method Nanor Gragousian, Sahar Amiri, Hossein Ali khonakdar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8214924/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 Copper oxide (CuO)- and manganese oxide (MnO 2 ) doped calcium silicate (CaSiO 3 ) nanoparticles were synthesized by a sol-gel method. The synthesized doped nanoparticles were evaluated using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM) and energy dispersive Xrays (EDX). The bioactivity of the prepared nanoparticles was investigated after immersion in simulated body fluid (SBF) by means of inductively coupled plasma (ICP). The size and bioactivity of the prepared nanoparticles after 15 days of immersion in SBF was dependent on the ion concentrations. Furthermore, biocompatibility and antibacterial assays demonstrated that the synthesized microstructures exhibited favorable performance against both Gram-positive and Gram-negative bacterial strains. Based on the obtained results, these nanoparticles, with their promising biocompatibility and antibacterial activity, hold potential for application in cancer therapy. Finally, the cellular test was carried out, the results of which demonstrated non-cytotoxicity of the samples towards MDA-MB-231 Cells. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction In recent years, the development of bioactive materials with enhanced physicochemical and biological properties has attracted significant attention in the field of cancer treatment. Among these materials, calcium silicate-based compounds have emerged as promising candidates due to their excellent biocompatibility, bioactivity, and ability to promote cell adhesion and proliferation. Their structural versatility and ability to incorporate various dopants make them suitable platforms for designing multifunctional materials for biomedical applications [ 1 , 2 ]. Doping CaSiO 3 with transition metal oxides such as copper oxide (CuO) and manganese oxide (MnO₂) has been shown to significantly improve its physicochemical and biological properties. Copper ions play a crucial role in angiogenesis, antimicrobial activity, and reactive oxygen species (ROS) generation, which are beneficial in targeted cancer therapy [ 3 , 4 ]. The synergistic effect of Cu and Mn dopants may enhance structural stability, surface reactivity, and therapeutic efficacy of CaSiO 3 matrices [ 5 ]. Some researchers prepared CuO and MnO₂ doped CaSiO 3 through sol–gel route, solid-state reaction, co-precipitation approach, and the spray pyrolysis technique, but the most used and the simplest one is the well-known wet-chemistry protocol that involves the transition from a sol to a gel, subsequently completed by a thermal treatment [ 6 – 8 ]. Obtained nanoparticles from sol–gel showed good results in terms of mechanical strength, bioactivity, antibacterial activity, as well as cell adhesion, proliferation and differentiation, uniform mixing of the precursors, which leads to the obtaining of homogeneous products at low temperatures [ 8 – 10 ]. Our research focused on the addition of various CuO and MnO₂ concentrations to the CaSiO 3 during the preparation process using the wet precipitation method (economical and flexible method). CuO and MnO₂ doping was expected to affect the reaction and the final properties of the resulting materials. Under this perspective, doping of CaSiO 3 with copper would promote bone cancer treatment and induce the bone regeneration. Obtained nanoparticles were investigated by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) provide valuable insights into the crystalline structure, bonding characteristics, and nanoscale morphology of the materialsXRD, FTIR, SEM& EDX .The findings of this study are expected to provide fundamental insights into the design of multifunctional biomaterials for future cancer therapy applications. Materials and methods Calcium nitrate tetrahydrate (Ca(NO 3 ) 2 4H 2 O, 99%), tetraethyl orthosilicate (TEOS, 99%), Manganese (II) nitrate tetrahydrate (Mn (NO 3 ) 2 ·4H 2 O,99%), silica gel, nitric acid (HNO 3 ,99%) and calcium carbonate (CaCO 3 ,99%) were pueched from Merck and were used as received. Copper carbonate (CuCO 3 ,99%) and silica gel (SiO 2 ) pueched from Merck and were used as received. As regard to the phosphate buffer the following ingredients were utilized KH 2 PO 4 , NaCl and KH 2 PO 4 from Bio Basic Company (Markham, Canada). Synthesis of CuO-doped CaSiO 3 (CuCaSi) To synthesize CuCaSi, 0.3 g of CaCO 3 was first added to a 50 mL flask containing 2.5 mL of a water/ HNO 3 solution and stirred in a water bath for 24 hours. Separately, 0.02 g of CuCO 3 was added to another 50 mL flask containing 2.5 mL of the same water/ HNO 3 solution and also stirred in a water bath for 24 hours. After 24 hours, 0.51 g of silica gel was added to the CaCO 3 solution. Once the silica gel was fully dispersed in the CaCO 3 solution, the resulting mixture was added to the CuCO 3 solution and stirred at room temperature for 24 hours using a magnetic stirrer. Finally, the mixture was dried in an oven at 100°C for 24 hours, and the resulting powder was calcined in a furnace at 500°C for 3 hours [ 11 – 13 ]. Synthesis of MnO₂-doped CaSiO (MnCaSi) To synthesize MnCaSi, 7.81 g of tetraethyl orthosilicate (TEOS) was added to a 100 mL beaker containing 15 mL of a water/ HNO 3 solution (1:75 v/v) and stirred magnetically for 30 minutes to complete the hydrolysis of the silane ethoxy groups under acidic conditions. Subsequently, 1.88 g of Mn (NO 3 ) 2 ·4H 2 O was added to the solution. After complete dissolution of the manganese nitrate, 26.57 g of Mn (NO 3 ) 2 ·4H 2 O was added, and the reaction proceeded for 1 hour. The final solution was poured into a Petri dish and placed in an oven at 60°C. After 24 hours, the oven temperature was increased to 120°C and maintained for an additional 24 hours to complete the sol-gel process. Finally, the resulting powder was calcined in a furnace at 900°C for 3 hours[ 11 – 13 ]. Characterization Methods In order to study the effect of CuO and MnO₂ on the physicochemical properties of the prepared CuCaSi and MnCaSi nanoparticles, infrared spectra were obtained using Fourier transformer infrared spectrophotometer (FT-IR). Sample preparation involved mixing the synthesized powders with KBr and pressing the mixture into pellets (model FT/IR- 6100 type A, Germany). Crystallinity of the obtained samples were investigated with X-ray diffraction (XRD) using a wide-angle X-ray scattering (WAXS) system at room temperature over a 2θ range of 5°–60° (FK60-04 D5000, Siemens, Germany). Scanning Electron Microscopy (SEM) was used to examine the morphology and particle size of the synthesized nanostructures. The surface of the samples was coated with gold ((JEOL JXA-840A, Electron Probe Micro-Analyzer, Japan) at 20 kV. In vitro bioactivity test The evaluation of in vitro bioactivity was performed by immersing obtained CuCaSia and MnCaSi in a simulated body fluid (SBF) solution and placed in a shaker (37°C, 170 rpm). The concentrations of Ca, P, Si, and Cu elements after each soaking time in SBF were determined using an inductively coupled plasma spectrometer (ICP, Optima 7300 DV). MTT Cell Viability Assay To evaluate the cytotoxicity of the synthesized nanostructures, the MTT assay was performed. The test utilized high-purity DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics. MDA-MB-231 cells were counted and diluted before being seeded into 96-well plates at a density of 1×10⁴ cells per well. The incubator was set to 37°C with 95% air and 5% CO₂. After overnight incubation, the medium was replaced with formulations containing CaSi, IR-783 (a fluorescent dye), and Mn-CaSi/FA-IR-783 or Cu-CaSi/FA-IR-783 in concentrations ranging from 50 to 350 µg/mL. Following 24 hours of incubation, MTT solution was added to each well and the plates were incubated in the dark for 4 hours. A microplate reader (490 nm) was then used to record the optical density (OD) and determine the percentage of cell viability[ 13 ]. Cell viability was calculated using the following formula: $$\:cell\:viability=\frac{mean\:OD\:od\:samples}{mean\:OD\:of\:negative\:control}\times\:100$$ Antibacterial Assay This assay was conducted to determine the microbial inhibition rate, bacterial growth percentage, and the inhibition zone diameter. The antibacterial tests were performed at Absar Kavir Yazd Company in accordance with the AATCC Test Method 100–2004 standard. The experiment was carried out under microbiological laboratory conditions using sterile plastic Petri dishes specifically designed for solid culture media preparation. The synthesized samples were placed in contact with a bacterial suspension that was prepared based on the 0.5 McFarland standard. The samples were then incubated at 37°C for 24 hours. To quantitatively assess bacterial activity, 100 µL of the bacterial suspension was inoculated onto tryptic soy agar (TSA) plates using the spread plate method. The inoculated plates were subsequently incubated at 37°C for an additional 24 hours to allow potential bacterial growth and proliferation. After the incubation period, the bacterial growth percentage and inhibition zones were measured to evaluate the antibacterial performance of the samples[ 14 ]. Results and Discussion Synthesis and Characterization of CuCaSi Microstructures The incorporation of transition metals, including CuO ions, into CaCO 3 microstructures can enhance their thermal, biological, and mechanical properties. Among the most widely reported antibacterial agents in scientific literature, CuO and MnO 2 -doped nanostructures have demonstrated significant antimicrobial activity, which can be effectively imparted to the target microstructure. FTIR Spectrum Analysis of CuCaSi Microstructure The FTIR spectrum of the CuCaSi microstructure is presented in Fig. 1 CaSiO 3 and doped nanoparticles showed approximately the same bands with slight shifts in the wavelength bands 1147 cm − 1 and 1105 cm − 1 for 3% and 5% samples, respectively exhibited six infrared bands located at: 613, 673, 892, 933, 981, and 1147cm − 1 which related to the silicate network and respectively ascribed to the Si-O symmetric stretching of bridging oxygen atoms between tetrahedrons, Si-O stretching of nonbridging oxygen atoms, Si-O-Si symmetric stretching, and the longitudinal-optic mode of Si-O-Si asymmetric stretching, respectively. A weak transmission band at 1609 cm − 1 corresponds to an asymmetric stretching vibration of the C-O bond and the broad band at 3254 cm − 1 can be ascribed to the stretching mode in the hydroxide group. Additionally, the bands between the 2130–3419 and the 3468 cm − 1 may be due to Si-OH stretching in Si-OH groups or is due to stretching vibrations of O-H groups in H 2 O or hydroxyls with a wide range of hydrogen bond strengths [ 15 – 17 ]. The band at 1637 cm − 1 was due to H-O-H bending vibration of molecular H 2 O. This could improve the hydrophilicity of the produced composites, thus, in turn would increase the bioactivity and the cell adherence. Also, the bands at 1428–1442 cm − 1 were assigned to carbonate which relatively more intense for the samples doped. The spectra exhibit three vibrations occurring at wavenumbers 474, 563, and 593 cm − 1 for the sample 3% CuO which can be attributed to the vibrations of Cu-O; whereas samples doped with 5% CuO shows peaks at 476, 559, and 592 cm − 1 confirming the formation of highly pure CuO NPs. Both samples showed a moderate band at around 1025cm − 1 which may be attributed to the CuO phase. The FTIR analysis results confirmed that doping copper into the CaSiO 3 structure did not adversely affect the chemical integrity of the host matrix[ 15 – 17 ]. SEM Imaging of Synthesized CuCaSi Microstructures The SEM and EDX images of the synthesized CuO-based microstructures are shown in Fig. 2 . According to the results, most of the particles were observed to form agglomerates. High-resolution SEM scans clearly revealed the formation of nanoparticles in the nanometer range. Using ImageJ software for image analysis, the particle sizes were estimated to be in the range of 40–200 nanometers. One of the key parameters influencing the antibacterial properties of CuCaSi structures is the uniform dispersion of copper oxide within the matrix. To assess the elemental distribution and confirm the presence of Cu, Ca, and Si in the synthesized microstructures, energy-dispersive X-ray spectroscopy (EDX) analysis was performed, and the results are presented in Fig. 2 .b [ 18 , 19 ]. The EDX spectrum exhibited three peaks at approximately 1, 8, and 8.9 keV corresponding to the presence of copper. Additionally, two peaks located at around 0.5 and 3.6 keV confirmed the presence of calcium, while a peak at approximately 1.8 keV indicated the presence of silicon [93]. These findings confirm the successful incorporation and uniform distribution of the target elements within the CuCaSi structure [ 18 , 19 ]. XRD Characterization of Synthesized CuCaSi One of the key methods used to identify the synthesized microstructures is morphological analysis, which was conducted using X-ray diffraction (XRD). The XRD pattern of the CuCaSi microstructure is presented in Fig. 3 [ 18 , 20 , 21 ]. The diffraction pattern of CaSiO 3 displayed three characteristic peaks at 2θ values of 29.3°, 34.1°, and 49.7°, which correspond to the crystalline phases of tobermorite. A sharp peak observed at 2θ = 35° is the primary characteristic of crystalline CuO structures. Additional peaks at 2θ values of 57°, 58°, 60°, and 62° are also indicative of CuO phases. These results confirm the successful doping of CuO into the CaSiO 3 structure [ 18 , 20 , 21 ]. Quantitative Evaluation of Biocompatibility Using MTT Assay The biocompatibility of the synthesized microstructures was quantitatively assessed by the MTT assay, and the results are presented in Fig. 4 . According to the cytotoxicity test outcomes, both CaSiO 3 and CuCaSi microstructures exhibited favorable biocompatibility with MDA-MB-231 cells, indicating their suitability for biomedical applications. However, at higher doses, the biocompatibility of the copper oxide-doped samples decreased. These findings suggest that the biocompatibility of copper CuCaSi structures is dose-dependent, which is consistent with reports in previous studies on copper oxide nanostructures [ 13 , 14 , 22 ]. Synthesis and Characterization of Mn-dopped CaSiO 3 (MnCaSi) The FTIR spectrum of the Mn, CaSiO 3 and MnCaSi nanostructure is shown in Fig. 5 . According to the obtained results, the presence of a broad peak at 3424 cm⁻¹ corresponds to the stretching vibrations of hydroxyl groups (Si–OH) in the calcium silicate structure. Two peaks observed at 845 cm⁻¹ and 945 cm⁻¹ are attributed to the Si–Ca framework. Additionally, a broad peak at 945 cm⁻¹ is related to the stretching vibrations of Si–O–Si bonds. Furthermore, the peak observed at 1445 cm⁻¹ is characteristic of C–O stretching vibrations. The presence of Mn–O bonds was confirmed by a peak at 518 cm⁻¹ [ 20 , 22 , 23 ]. The distinctive absorption peaks were observed at 3260 cm − 1 , 2331 cm − 1 , 2109 cm − 1 , 1635 cm − 1 , 515 cm − 1 and 480 cm − 1 . The peak at 3260 cm − 1 may be due to O-H stretching and the peak at 2331 cm − 1 may be due to O = C = O stretching. The peak at 2109 cm − 1 is ascribed to the stretching vibration of N = C = S whereas the peak at 1635 cm − 1 is attributed to the stretching vibration of the C = C bond. The absorption bands at 480 cm − 1 and 515 cm − 1 correspond to the Mn-O bond, which confirms the formation of MnO nanoparticles. This analysis confirms that there are very low or no impurities present in the as-synthesized sample [ 20 , 22 , 23 ]. One of the characterization methods for the synthesized nanostructures was phase identification using X-ray diffraction (XRD) analysis. The XRD pattern of the MnCaSi nanostructure is shown in Fig. 6 . The peaks observed at 2θ angles of 37°, 42°, 47°, 54°, and 60° correspond to the crystalline phases of manganese. Additionally, the peaks appearing at 2θ angles of 32°, 39°, 44°, 51°, and 52° are attributed to the crystalline structures of CaSiO 3 , confirming the successful synthesis of MnCaSi[ 24 , 25 ]. Characterization of MnO 2 nanostructured film The SEM and EDX images of the prepared copper oxide-based nanostructures are shown in Fig. 7 . The SEM results indicated that most particles formed as agglomerates. In higher-resolution SEM scans, the formation of nanoparticles is clearly visible, and using ImageJ software, the particle sizes were estimated to range between 47 and 250 nanometers [ 20 , 26 ]. One of the important factors influencing the antibacterial properties and efficacy of Mn-containing structures is the uniform and proper distribution of manganese within the MnCaSi structure. Therefore, to investigate the distribution and confirm the presence of Mn, Ca, and Si elements in the synthesized nanostructures, EDX analysis was performed, and the results are presented in Fig. 7 . b. The EDX spectrum exhibited two peaks at 0.5 keV and 5.9 keV corresponding to manganese [ 20 , 21 , 26 ]. Additionally, two peaks at 0.5 keV and 3.6 keV were attributed to calcium, and the peak appearing at 1.8 keV confirmed the presence of silicon [93]. Based on the obtained results from this analysis, the presence of the targeted elements was confirmed, and their uniform distribution within the MnCaSi structure is clearly evident. For quantitative evaluation of the biocompatibility of the prepared microstructures, the MTT assay was used, and the results are shown in Fig. 8 . According to the cytotoxicity results, the CaSiO 3 microstructures and MnCaSi exhibited good biocompatibility with MDA-MB-231 cells and are suitable for medical applications. Moreover, at higher doses, the biocompatibility of the manganese-doped samples showed only a slight decrease. Based on the obtained results, the biocompatibility of the manganese-doped calcium silicate structures indicated that using higher doses, up to 300 µg/mL of these microstructures, does not lead to cytotoxicity or reduced biocompatibility performance [ 13 , 14 , 20 , 22 ]. Bioactivity in SBF ICP results. The variations in ion concentrations in the SBF after immersing the samples were measured using ICP (Table.1) which indicated that the calcium ion concentration in the SBF for all samples increased to about 200–350 ppm and showed a notable rise in calcium ion concentration (was detected in the SBF for samples containing higher copper levels. This indicates that higher Cu content enhances the release of Ca from the samples into the SBF. In contrast, the phosphorus ion concentration for all samples showed a significant decrease during the first 7 days of immersion and then stayed relatively constant until the end of the experiment, likely due to ion exchange between the samples and the SBF. This result suggested the positive effect of higher concentration of Cu on the Ca leakage from the samples into the SBF However, the phosphorus ions concentration for all the samples showed significant decrease during the 7 days immersion period. The dissolution of nanoparticles into the SBF was confirmed by the increased concentrations of Si and Cu ions in the solution, which depended on both the immersion duration and the Cu content in the nanoparticles. It is important to note that the interaction of silicate-based materials with physiological fluids follows general mechanisms reported previously. These mechanisms involve the leaching of ions from the silicate matrix, the breakdown of Si–O bonds in the material’s network, and the subsequent reprecipitation of Ca ions on the sample surfaces in a sequential manner. Table.1. ICP results of average values of ion release after immersing in SBF for different time intervals (in ppm) No Time (hrs) CaSiO 3 CuCaSi MnCaSi Ca P Si Ca P Si Cu Ca P Si Mn 0 0 33 0 0 33 0 0 0 33 0 0 1 3 230 35 22 250 36 19 27 205 35 23 28 2 6 330 10 25 330 13 22 42 315 7 27 45 3 9 400 8 31 390 10 28 48 420 10 31 49 4 12 440 15 37 460 9 34 50 450 9 39 52 5 15 580 10 42 540 8 45 53 570 9 46 57 Antibacterial Properties Antibacterial activity is one of the critical properties of nanoparticles used in medical applications. The antibacterial performance of the synthesized microstructures was evaluated against two bacterial strains, Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli , as well as the effect of manganese on this activity, using the zone of inhibition test (Fig. 9 ). The results demonstrated that the synthesized microstructures exhibited good antibacterial efficacy against both Gram-positive and Gram-negative bacteria. Furthermore, as expected and consistent with previously reported antibacterial properties of copper oxide [99], doping copper into the CaSiO 3 structures significantly enhanced the antibacterial performance of the microstructures. The synthesized microstructures showed better antibacterial activity against Gram-positive bacteria compared to Gram-negative bacteria, which can be attributed to the double-layered cell membrane of E. coli [ 27 , 28 ]. Conclusion In this study, CuO- and MnO₂-doped calcium silicate nanoparticles were successfully synthesized via the sol–gel method and thoroughly characterized using XRD, FTIR, SEM, and EDX analysis. The results confirmed the formation of well-defined doped nanostructures, whose size and bioactivity were strongly influenced by Cu content, particularly after immersion in SBF for 15 days. The doped nanoparticles exhibited excellent bioactivity, favorable biocompatibility, and notable antibacterial performance against both Gram-positive and Gram-negative bacteria. Moreover, cellular assays confirmed their non-cytotoxic nature toward MDA-MB-231 cells. Overall, the combination of bioactivity, antibacterial efficacy, and cytocompatibility suggests that these doped calcium silicate nanoparticles represent promising candidates for future biomedical applications. Declarations Funding “The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.” Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions “All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Nanor Gragousian, Sahar Amiri, Hossein Ali khonakdar. The first draft of the manuscript was written by Sahar Amiri and all authors commented on previous versions of the manuscript. 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1","display":"","copyAsset":false,"role":"figure","size":47901,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFTIR spectrum of (a) CaSiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e, (b) CuO and (c) CuCaSi\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8214924/v1/30d44e693e0afebd6daecdad.png"},{"id":99206516,"identity":"7984db4d-a509-4ca7-9c02-616cd3484744","added_by":"auto","created_at":"2025-12-30 06:46:02","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1426932,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM and EDX images of CuCaSi\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8214924/v1/f9d0cf09b2d8247d4f18a39a.jpeg"},{"id":99206515,"identity":"43181a2e-f54b-4b1a-ac24-077edb95096b","added_by":"auto","created_at":"2025-12-30 06:46:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":42119,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXRD spectrum of (a) CaSiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e, (b) CuO and (c) CuCaSi\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8214924/v1/34140552f7244a04eb170a75.png"},{"id":99319126,"identity":"3a88c64e-990a-4a9a-84bc-1e5c0e37e432","added_by":"auto","created_at":"2025-12-31 16:36:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":46368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytotoxicity (%) of control, CaSiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and CuCaSi against MDA-MB-231 cells\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8214924/v1/f51172e8c6ba7dbdea80d0d9.png"},{"id":99206519,"identity":"608666b2-cb74-47d3-912f-fc15de41b7a7","added_by":"auto","created_at":"2025-12-30 06:46:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":45267,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFTIR spectrum of (a) CaSiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e, (b) Mn and (c) MnCaSi\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8214924/v1/e66edf6690bc07d11fa505e3.png"},{"id":99206523,"identity":"57b67e6f-7396-4acc-a5e4-3d3a80354a78","added_by":"auto","created_at":"2025-12-30 06:46:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":39520,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXRD spectrum of (a) CaSiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e, (b) Mn and (c) \u003c/strong\u003eMnCaSi\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8214924/v1/53861f1b7270abd915293e24.png"},{"id":99319139,"identity":"9cea9b30-a065-4046-8976-81434640e054","added_by":"auto","created_at":"2025-12-31 16:36:23","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1705366,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM and EDX images of MnCaSi\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8214924/v1/c255de36a09dcbe918c3b47d.jpeg"},{"id":99206529,"identity":"96605925-a554-4c4e-bcda-127f2e6ebbbd","added_by":"auto","created_at":"2025-12-30 06:46:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":69421,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytotoxicity (%) of control, CaSiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and MnCaSi against MDA-MB-231 cells\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8214924/v1/4f24868252df1123ce5c8f08.png"},{"id":99206534,"identity":"440e0f3b-f80f-450d-9295-dddc6895f66d","added_by":"auto","created_at":"2025-12-30 06:46:02","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":985568,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition zone of CuCaSi and MnCaSi against (a) S. aureus and (b) Ecoli (1: CaSiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e, 2: CuCaSi, 3: MnCaSi)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8214924/v1/b962dc5c5d779ab7ab2363b8.jpeg"},{"id":104295472,"identity":"131970f8-117d-4755-859b-0b25ba3c16a2","added_by":"auto","created_at":"2026-03-10 07:43:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7684852,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8214924/v1/8dc4e03f-885f-4842-a750-e4f40205d635.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Structural characterization of copper and manganese oxide nanoparticles-doped calcium silicate (CaSiO 3 ) for synthesized via sol-gel method","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, the development of bioactive materials with enhanced physicochemical and biological properties has attracted significant attention in the field of cancer treatment. Among these materials, calcium silicate-based compounds have emerged as promising candidates due to their excellent biocompatibility, bioactivity, and ability to promote cell adhesion and proliferation. Their structural versatility and ability to incorporate various dopants make them suitable platforms for designing multifunctional materials for biomedical applications [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDoping CaSiO\u003csub\u003e3\u003c/sub\u003e with transition metal oxides such as copper oxide (CuO) and manganese oxide (MnO₂) has been shown to significantly improve its physicochemical and biological properties. Copper ions play a crucial role in angiogenesis, antimicrobial activity, and reactive oxygen species (ROS) generation, which are beneficial in targeted cancer therapy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The synergistic effect of Cu and Mn dopants may enhance structural stability, surface reactivity, and therapeutic efficacy of CaSiO\u003csub\u003e3\u003c/sub\u003e matrices [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSome researchers prepared CuO and MnO₂ doped CaSiO\u003csub\u003e3\u003c/sub\u003e through sol\u0026ndash;gel route, solid-state reaction, co-precipitation approach, and the spray pyrolysis technique, but the most used and the simplest one is the well-known wet-chemistry protocol that involves the transition from a sol to a gel, subsequently completed by a thermal treatment [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Obtained nanoparticles from sol\u0026ndash;gel showed good results in terms of mechanical strength, bioactivity, antibacterial activity, as well as cell adhesion, proliferation and differentiation, uniform mixing of the precursors, which leads to the obtaining of homogeneous products at low temperatures [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur research focused on the addition of various CuO and MnO₂ concentrations to the CaSiO\u003csub\u003e3\u003c/sub\u003e during the preparation process using the wet precipitation method (economical and flexible method). CuO and MnO₂ doping was expected to affect the reaction and the final properties of the resulting materials. Under this perspective, doping of CaSiO\u003csub\u003e3\u003c/sub\u003e with copper would promote bone cancer treatment and induce the bone regeneration. Obtained nanoparticles were investigated by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) provide valuable insights into the crystalline structure, bonding characteristics, and nanoscale morphology of the materialsXRD, FTIR, SEM\u0026amp; EDX .The findings of this study are expected to provide fundamental insights into the design of multifunctional biomaterials for future cancer therapy applications.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eCalcium nitrate tetrahydrate (Ca(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e4H\u003csub\u003e2\u003c/sub\u003eO, 99%), tetraethyl orthosilicate (TEOS, 99%), Manganese (II) nitrate tetrahydrate (Mn (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;4H\u003csub\u003e2\u003c/sub\u003eO,99%), silica gel, nitric acid (HNO\u003csub\u003e3\u003c/sub\u003e,99%) and calcium carbonate (CaCO\u003csub\u003e3\u003c/sub\u003e,99%) were pueched from Merck and were used as received. Copper carbonate (CuCO\u003csub\u003e3\u003c/sub\u003e,99%) and silica gel (SiO\u003csub\u003e2\u003c/sub\u003e) pueched from Merck and were used as received. As regard to the phosphate buffer the following ingredients were utilized KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, NaCl and KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e from Bio Basic Company (Markham, Canada).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of CuO-doped CaSiO\u003csub\u003e3\u003c/sub\u003e (CuCaSi)\u003c/h2\u003e \u003cp\u003eTo synthesize CuCaSi, 0.3 g of CaCO\u003csub\u003e3\u003c/sub\u003e was first added to a 50 mL flask containing 2.5 mL of a water/ HNO\u003csub\u003e3\u003c/sub\u003e solution and stirred in a water bath for 24 hours. Separately, 0.02 g of CuCO\u003csub\u003e3\u003c/sub\u003e was added to another 50 mL flask containing 2.5 mL of the same water/ HNO\u003csub\u003e3\u003c/sub\u003e solution and also stirred in a water bath for 24 hours. After 24 hours, 0.51 g of silica gel was added to the CaCO\u003csub\u003e3\u003c/sub\u003e solution. Once the silica gel was fully dispersed in the CaCO\u003csub\u003e3\u003c/sub\u003e solution, the resulting mixture was added to the CuCO\u003csub\u003e3\u003c/sub\u003e solution and stirred at room temperature for 24 hours using a magnetic stirrer. Finally, the mixture was dried in an oven at 100\u0026deg;C for 24 hours, and the resulting powder was calcined in a furnace at 500\u0026deg;C for 3 hours [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSynthesis of MnO₂-doped CaSiO (MnCaSi)\u003c/h3\u003e\n\u003cp\u003eTo synthesize MnCaSi, 7.81 g of tetraethyl orthosilicate (TEOS) was added to a 100 mL beaker containing 15 mL of a water/ HNO\u003csub\u003e3\u003c/sub\u003e solution (1:75 v/v) and stirred magnetically for 30 minutes to complete the hydrolysis of the silane ethoxy groups under acidic conditions. Subsequently, 1.88 g of Mn (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;4H\u003csub\u003e2\u003c/sub\u003eO was added to the solution. After complete dissolution of the manganese nitrate, 26.57 g of Mn (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;4H\u003csub\u003e2\u003c/sub\u003eO was added, and the reaction proceeded for 1 hour. The final solution was poured into a Petri dish and placed in an oven at 60\u0026deg;C. After 24 hours, the oven temperature was increased to 120\u0026deg;C and maintained for an additional 24 hours to complete the sol-gel process. Finally, the resulting powder was calcined in a furnace at 900\u0026deg;C for 3 hours[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eCharacterization Methods\u003c/h3\u003e\n\u003cp\u003eIn order to study the effect of CuO and MnO₂ on the physicochemical properties of the prepared CuCaSi and MnCaSi nanoparticles, infrared spectra were obtained using Fourier transformer infrared spectrophotometer (FT-IR). Sample preparation involved mixing the synthesized powders with KBr and pressing the mixture into pellets (model FT/IR- 6100 type A, Germany). Crystallinity of the obtained samples were investigated with X-ray diffraction (XRD) using a wide-angle X-ray scattering (WAXS) system at room temperature over a 2θ range of 5\u0026deg;\u0026ndash;60\u0026deg; (FK60-04 D5000, Siemens, Germany). Scanning Electron Microscopy (SEM) was used to examine the morphology and particle size of the synthesized nanostructures. The surface of the samples was coated with gold ((JEOL JXA-840A, Electron Probe Micro-Analyzer, Japan) at 20 kV.\u003c/p\u003e\n\u003ch3\u003eIn vitro bioactivity test\u003c/h3\u003e\n\u003cp\u003eThe evaluation of in vitro bioactivity was performed by immersing obtained CuCaSia and MnCaSi\u003c/p\u003e \u003cp\u003ein a simulated body fluid (SBF) solution and placed in a shaker (37\u0026deg;C, 170 rpm). The concentrations of Ca, P, Si, and Cu elements after each soaking time in SBF were determined using an inductively coupled plasma spectrometer (ICP, Optima 7300 DV).\u003c/p\u003e\n\u003ch3\u003eMTT Cell Viability Assay\u003c/h3\u003e\n\u003cp\u003eTo evaluate the cytotoxicity of the synthesized nanostructures, the MTT assay was performed. The test utilized high-purity DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics. MDA-MB-231 cells were counted and diluted before being seeded into 96-well plates at a density of 1\u0026times;10⁴ cells per well. The incubator was set to 37\u0026deg;C with 95% air and 5% CO₂. After overnight incubation, the medium was replaced with formulations containing CaSi, IR-783 (a fluorescent dye), and Mn-CaSi/FA-IR-783 or Cu-CaSi/FA-IR-783 in concentrations ranging from 50 to 350 \u0026micro;g/mL. Following 24 hours of incubation, MTT solution was added to each well and the plates were incubated in the dark for 4 hours. A microplate reader (490 nm) was then used to record the optical density (OD) and determine the percentage of cell viability[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Cell viability was calculated using the following formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:cell\\:viability=\\frac{mean\\:OD\\:od\\:samples}{mean\\:OD\\:of\\:negative\\:control}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial Assay\u003c/h2\u003e \u003cp\u003eThis assay was conducted to determine the microbial inhibition rate, bacterial growth percentage, and the inhibition zone diameter. The antibacterial tests were performed at Absar Kavir Yazd Company in accordance with the AATCC Test Method 100\u0026ndash;2004 standard. The experiment was carried out under microbiological laboratory conditions using sterile plastic Petri dishes specifically designed for solid culture media preparation. The synthesized samples were placed in contact with a bacterial suspension that was prepared based on the 0.5 McFarland standard. The samples were then incubated at 37\u0026deg;C for 24 hours. To quantitatively assess bacterial activity, 100 \u0026micro;L of the bacterial suspension was inoculated onto tryptic soy agar (TSA) plates using the spread plate method. The inoculated plates were subsequently incubated at 37\u0026deg;C for an additional 24 hours to allow potential bacterial growth and proliferation. After the incubation period, the bacterial growth percentage and inhibition zones were measured to evaluate the antibacterial performance of the samples[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis and Characterization of CuCaSi Microstructures\u003c/h2\u003e \u003cp\u003eThe incorporation of transition metals, including CuO ions, into CaCO\u003csub\u003e3\u003c/sub\u003e microstructures can enhance their thermal, biological, and mechanical properties. Among the most widely reported antibacterial agents in scientific literature, CuO and MnO\u003csub\u003e2\u003c/sub\u003e-doped nanostructures have demonstrated significant antimicrobial activity, which can be effectively imparted to the target microstructure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFTIR Spectrum Analysis of CuCaSi Microstructure\u003c/h2\u003e \u003cp\u003eThe FTIR spectrum of the CuCaSi microstructure is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e CaSiO\u003csub\u003e3\u003c/sub\u003e and doped nanoparticles showed approximately the same bands with slight shifts in the wavelength bands 1147 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1105 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 3% and 5% samples, respectively exhibited six infrared bands located at: 613, 673, 892, 933, 981, and 1147cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e which related to the silicate network and respectively ascribed to the Si-O symmetric stretching of bridging oxygen atoms between tetrahedrons, Si-O stretching of nonbridging oxygen atoms, Si-O-Si symmetric stretching, and the longitudinal-optic mode of Si-O-Si asymmetric stretching, respectively. A weak transmission band at 1609 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to an asymmetric stretching vibration of the C-O bond and the broad band at 3254 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e can be ascribed to the stretching mode in the hydroxide group. Additionally, the bands between the 2130\u0026ndash;3419 and the 3468 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e may be due to Si-OH stretching in Si-OH groups or is due to stretching vibrations of O-H groups in H\u003csub\u003e2\u003c/sub\u003eO or hydroxyls with a wide range of hydrogen bond strengths [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The band at 1637 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was due to H-O-H bending vibration of molecular H\u003csub\u003e2\u003c/sub\u003eO. This could improve the hydrophilicity of the produced composites, thus, in turn would increase the bioactivity and the cell adherence. Also, the bands at 1428\u0026ndash;1442 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were assigned to carbonate which relatively more intense for the samples doped. The spectra exhibit three vibrations occurring at wavenumbers 474, 563, and 593 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the sample 3% CuO which can be attributed to the vibrations of Cu-O; whereas samples doped with 5% CuO shows peaks at 476, 559, and 592 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e confirming the formation of highly pure CuO NPs. Both samples showed a moderate band at around 1025cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e which may be attributed to the CuO phase. The FTIR analysis results confirmed that doping copper into the CaSiO\u003csub\u003e3\u003c/sub\u003e structure did not adversely affect the chemical integrity of the host matrix[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSEM Imaging of Synthesized CuCaSi Microstructures\u003c/h2\u003e \u003cp\u003eThe SEM and EDX images of the synthesized CuO-based microstructures are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. According to the results, most of the particles were observed to form agglomerates. High-resolution SEM scans clearly revealed the formation of nanoparticles in the nanometer range. Using ImageJ software for image analysis, the particle sizes were estimated to be in the range of 40\u0026ndash;200 nanometers. One of the key parameters influencing the antibacterial properties of CuCaSi structures is the uniform dispersion of copper oxide within the matrix. To assess the elemental distribution and confirm the presence of Cu, Ca, and Si in the synthesized microstructures, energy-dispersive X-ray spectroscopy (EDX) analysis was performed, and the results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.b [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe EDX spectrum exhibited three peaks at approximately 1, 8, and 8.9 keV corresponding to the presence of copper. Additionally, two peaks located at around 0.5 and 3.6 keV confirmed the presence of calcium, while a peak at approximately 1.8 keV indicated the presence of silicon [93]. These findings confirm the successful incorporation and uniform distribution of the target elements within the CuCaSi structure [\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=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eXRD Characterization of Synthesized CuCaSi\u003c/h2\u003e \u003cp\u003eOne of the key methods used to identify the synthesized microstructures is morphological analysis, which was conducted using X-ray diffraction (XRD). The XRD pattern of the CuCaSi microstructure is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe diffraction pattern of CaSiO\u003csub\u003e3\u003c/sub\u003e displayed three characteristic peaks at 2θ values of 29.3\u0026deg;, 34.1\u0026deg;, and 49.7\u0026deg;, which correspond to the crystalline phases of tobermorite. A sharp peak observed at 2θ\u0026thinsp;=\u0026thinsp;35\u0026deg; is the primary characteristic of crystalline CuO structures. Additional peaks at 2θ values of 57\u0026deg;, 58\u0026deg;, 60\u0026deg;, and 62\u0026deg; are also indicative of CuO phases. These results confirm the successful doping of CuO into the CaSiO\u003csub\u003e3\u003c/sub\u003e structure [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\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=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Evaluation of Biocompatibility Using MTT Assay\u003c/h2\u003e \u003cp\u003eThe biocompatibility of the synthesized microstructures was quantitatively assessed by the MTT assay, and the results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. According to the cytotoxicity test outcomes, both CaSiO\u003csub\u003e3\u003c/sub\u003e and CuCaSi microstructures exhibited favorable biocompatibility with MDA-MB-231 cells, indicating their suitability for biomedical applications. However, at higher doses, the biocompatibility of the copper oxide-doped samples decreased. These findings suggest that the biocompatibility of copper CuCaSi structures is dose-dependent, which is consistent with reports in previous studies on copper oxide nanostructures [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis and Characterization of Mn-dopped CaSiO\u003csub\u003e3\u003c/sub\u003e (MnCaSi)\u003c/h2\u003e \u003cp\u003eThe FTIR spectrum of the Mn, CaSiO\u003csub\u003e3\u003c/sub\u003e and MnCaSi nanostructure is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. According to the obtained results, the presence of a broad peak at 3424 cm⁻\u0026sup1; corresponds to the stretching vibrations of hydroxyl groups (Si\u0026ndash;OH) in the calcium silicate structure. Two peaks observed at 845 cm⁻\u0026sup1; and 945 cm⁻\u0026sup1; are attributed to the Si\u0026ndash;Ca framework. Additionally, a broad peak at 945 cm⁻\u0026sup1; is related to the stretching vibrations of Si\u0026ndash;O\u0026ndash;Si bonds. Furthermore, the peak observed at 1445 cm⁻\u0026sup1; is characteristic of C\u0026ndash;O stretching vibrations. The presence of Mn\u0026ndash;O bonds was confirmed by a peak at 518 cm⁻\u0026sup1; [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe distinctive absorption peaks were observed at 3260 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2331 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2109 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1635 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 515 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 480 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The peak at 3260 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e may be due to O-H stretching and the peak at 2331 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e may be due to O\u0026thinsp;=\u0026thinsp;C\u0026thinsp;=\u0026thinsp;O stretching. The peak at 2109 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is ascribed to the stretching vibration of N\u0026thinsp;=\u0026thinsp;C\u0026thinsp;=\u0026thinsp;S whereas the peak at 1635 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is attributed to the stretching vibration of the C\u0026thinsp;=\u0026thinsp;C bond. The absorption bands at 480 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 515 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e correspond to the Mn-O bond, which confirms the formation of MnO nanoparticles. This analysis confirms that there are very low or no impurities present in the as-synthesized sample [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOne of the characterization methods for the synthesized nanostructures was phase identification using X-ray diffraction (XRD) analysis. The XRD pattern of the MnCaSi nanostructure is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The peaks observed at 2θ angles of 37\u0026deg;, 42\u0026deg;, 47\u0026deg;, 54\u0026deg;, and 60\u0026deg; correspond to the crystalline phases of manganese. Additionally, the peaks appearing at 2θ angles of 32\u0026deg;, 39\u0026deg;, 44\u0026deg;, 51\u0026deg;, and 52\u0026deg; are attributed to the crystalline structures of CaSiO\u003csub\u003e3\u003c/sub\u003e, confirming the successful synthesis of MnCaSi[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of MnO\u003csub\u003e2\u003c/sub\u003e nanostructured film\u003c/h2\u003e \u003cp\u003eThe SEM and EDX images of the prepared copper oxide-based nanostructures are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. The SEM results indicated that most particles formed as agglomerates. In higher-resolution SEM scans, the formation of nanoparticles is clearly visible, and using ImageJ software, the particle sizes were estimated to range between 47 and 250 nanometers [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOne of the important factors influencing the antibacterial properties and efficacy of Mn-containing structures is the uniform and proper distribution of manganese within the MnCaSi structure. Therefore, to investigate the distribution and confirm the presence of Mn, Ca, and Si elements in the synthesized nanostructures, EDX analysis was performed, and the results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. b. The EDX spectrum exhibited two peaks at 0.5 keV and 5.9 keV corresponding to manganese [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdditionally, two peaks at 0.5 keV and 3.6 keV were attributed to calcium, and the peak appearing at 1.8 keV confirmed the presence of silicon [93]. Based on the obtained results from this analysis, the presence of the targeted elements was confirmed, and their uniform distribution within the MnCaSi structure is clearly evident.\u003c/p\u003e \u003cp\u003eFor quantitative evaluation of the biocompatibility of the prepared microstructures, the MTT assay was used, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. According to the cytotoxicity results, the CaSiO\u003csub\u003e3\u003c/sub\u003e microstructures and MnCaSi exhibited good biocompatibility with MDA-MB-231 cells and are suitable for medical applications. Moreover, at higher doses, the biocompatibility of the manganese-doped samples showed only a slight decrease. Based on the obtained results, the biocompatibility of the manganese-doped calcium silicate structures indicated that using higher doses, up to 300 \u0026micro;g/mL of these microstructures, does not lead to cytotoxicity or reduced biocompatibility performance [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBioactivity in SBF ICP results.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe variations in ion concentrations in the SBF after immersing the samples were measured using ICP (Table.1) which indicated that the calcium ion concentration in the SBF for all samples increased to about 200\u0026ndash;350 ppm and showed a notable rise in calcium ion concentration (was detected in the SBF for samples containing higher copper levels. This indicates that higher Cu content enhances the release of Ca from the samples into the SBF. In contrast, the phosphorus ion concentration for all samples showed a significant decrease during the first 7 days of immersion and then stayed relatively constant until the end of the experiment, likely due to ion exchange between the samples and the SBF. This result suggested the positive effect of higher concentration of Cu on the Ca leakage from the samples into the SBF However, the phosphorus ions concentration for all the samples showed significant decrease during the 7 days immersion period.\u003c/p\u003e \u003cp\u003eThe dissolution of nanoparticles into the SBF was confirmed by the increased concentrations of Si and Cu ions in the solution, which depended on both the immersion duration and the Cu content in the nanoparticles. It is important to note that the interaction of silicate-based materials with physiological fluids follows general mechanisms reported previously. These mechanisms involve the leaching of ions from the silicate matrix, the breakdown of Si\u0026ndash;O bonds in the material\u0026rsquo;s network, and the subsequent reprecipitation of Ca ions on the sample surfaces in a sequential manner.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable.1. ICP results of average values of ion release after immersing in SBF for different time intervals (in ppm)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003cp\u003e(hrs)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eCaSiO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e \u003cp\u003eCuCaSi\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c13\" namest=\"c10\"\u003e \u003cp\u003eMnCaSi\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eSi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eMn\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e230\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e330\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e330\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e315\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e390\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e420\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e440\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e460\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e450\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e540\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e570\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial Properties\u003c/h2\u003e \u003cp\u003eAntibacterial activity is one of the critical properties of nanoparticles used in medical applications. The antibacterial performance of the synthesized microstructures was evaluated against two bacterial strains, Gram-positive \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and Gram-negative \u003cem\u003eEscherichia coli\u003c/em\u003e, as well as the effect of manganese on this activity, using the zone of inhibition test (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The results demonstrated that the synthesized microstructures exhibited good antibacterial efficacy against both Gram-positive and Gram-negative bacteria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, as expected and consistent with previously reported antibacterial properties of copper oxide [99], doping copper into the CaSiO\u003csub\u003e3\u003c/sub\u003e structures significantly enhanced the antibacterial performance of the microstructures. The synthesized microstructures showed better antibacterial activity against Gram-positive bacteria compared to Gram-negative bacteria, which can be attributed to the double-layered cell membrane of E. coli [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, CuO- and MnO₂-doped calcium silicate nanoparticles were successfully synthesized via the sol\u0026ndash;gel method and thoroughly characterized using XRD, FTIR, SEM, and EDX analysis. The results confirmed the formation of well-defined doped nanostructures, whose size and bioactivity were strongly influenced by Cu content, particularly after immersion in SBF for 15 days. The doped nanoparticles exhibited excellent bioactivity, favorable biocompatibility, and notable antibacterial performance against both Gram-positive and Gram-negative bacteria. Moreover, cellular assays confirmed their non-cytotoxic nature toward MDA-MB-231 cells. Overall, the combination of bioactivity, antibacterial efficacy, and cytocompatibility suggests that these doped calcium silicate nanoparticles represent promising candidates for future biomedical applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e“The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.”\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e“All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Nanor Gragousian, Sahar Amiri, Hossein Ali khonakdar.\u003c/p\u003e\n\u003cp\u003eThe first draft of the manuscript was written by Sahar Amiri and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.”\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAndrada-Elena A, Costea CC, Surdu VA, Voicu G, Jinga SI, Busuioc C (2022) Gels. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/gels8090574\u003c/span\u003e\u003cspan address=\"10.3390/gels8090574\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNo Y, Li J, Zreiqat H (2017) Materials 10(2):153\u0026ndash;190\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi K, Cao H, Huang H, Tang S, Wang H, Yang Q, Hu Y, Weng J, Chen X (2025) Regen Biomater. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/rb/rbaf014\u003c/span\u003e\u003cspan address=\"10.1093/rb/rbaf014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePouroutzidou GK, Theodorou GS, Kontonasaki E, Tsamesidis I, Pantaleo A, Patsiaoura D, Papadopoulou L, Rhoades J, Likotrafiti E, Lioutas CB, Chrissafis K, Paraskevopoulos KM (2019) J Mater Sci Mater Med. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10856-019-6297-8\u003c/span\u003e\u003cspan address=\"10.1007/s10856-019-6297-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang F, Zhou M, Gu W, Shen Z, Ma X, Lu F, Yang X, Zheng Y, Gou Z (2020) J Mater Chem B 8:1060\u0026ndash;1070\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu Z, Zhuang H, Ma B, Xiao Y, Koc B, Zhu Y, Wu C (2021) Manganese-Doped Calcium Silicate Nanowire Composite Hydrogels for Melanoma Treatment and Wound Healing. 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M, Etemadi S, Hatam R, Khavid A, Atena S, Mohsen B (2025) Nanofabrication. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.37819/nanofab.010.2038\u003c/span\u003e\u003cspan address=\"10.37819/nanofab.010.2038\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe Q, Yuan H, Bu Y, Hu J, Olatunde OZ, Gong L, Wang P, Hu T, Li Y, Lu C, Molecules (2024) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules29132960\u003c/span\u003e\u003cspan address=\"10.3390/molecules29132960\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":"","lastPublishedDoi":"10.21203/rs.3.rs-8214924/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8214924/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCopper oxide (CuO)- and manganese oxide (MnO\u003csub\u003e2\u003c/sub\u003e) doped calcium silicate (CaSiO\u003csub\u003e3\u003c/sub\u003e) nanoparticles were synthesized by a sol-gel method. The synthesized doped nanoparticles were evaluated using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM) and energy dispersive Xrays (EDX). The bioactivity of the prepared nanoparticles was investigated after immersion in simulated body fluid (SBF) by means of inductively coupled plasma (ICP). The size and bioactivity of the prepared nanoparticles after 15 days of immersion in SBF was dependent on the ion concentrations. Furthermore, biocompatibility and antibacterial assays demonstrated that the synthesized microstructures exhibited favorable performance against both Gram-positive and Gram-negative bacterial strains. Based on the obtained results, these nanoparticles, with their promising biocompatibility and antibacterial activity, hold potential for application in cancer therapy. Finally, the cellular test was carried out, the results of which demonstrated non-cytotoxicity of the samples towards MDA-MB-231 Cells.\u003c/p\u003e","manuscriptTitle":"Structural characterization of copper and manganese oxide nanoparticles-doped calcium silicate (CaSiO 3 ) for synthesized via sol-gel method","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-30 06:45:53","doi":"10.21203/rs.3.rs-8214924/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":"bbd9e5ca-804c-49a9-89ab-8018930b7d06","owner":[],"postedDate":"December 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-10T07:42:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-30 06:45:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8214924","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8214924","identity":"rs-8214924","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}