Tiron-loaded silver nanoparticles: Synthesis, physico-chemical and biological characterization for pharmacologically apt properties

Tiron-loaded silver nanoparticles: Synthesis, physico-chemical and biological characterization for pharmacologically apt properties | 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 Tiron-loaded silver nanoparticles: Synthesis, physico-chemical and biological characterization for pharmacologically apt properties Pratima Dutta, Satendra Kumar Nirala, Pavitra Behra, Asim Amitabh Sahu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7479494/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Dec, 2025 Read the published version in BioNanoScience → Version 1 posted 9 You are reading this latest preprint version Abstract Nanotechnology offered synthesis of nanoparticles by unique combination of metals with natural and synthetic products for therapeutic uses with improved drug delivery. We synthesized tiron-loaded silver nanoparticles (TAgNPs), and performed their physico-chemical and biological characterization to understand pharmacologically apt properties. Synthesis of TAgNPs was confirmed by change in color to golden yellow with peak range of 430–440 nm under UV-visible spectroscopy. The particle size was found in the range of 14.9-12143.7 nm with a spherical shape under electron microscopy. Crystalline phase of tiron-loaded silver nanoparticles was observed by X-ray powder diffraction and thermal stability was confirmed by thermogravimetric analysis. Functional groups were validated under FTIR spectroscopy, which quantified functional groups on the surfaces of TAgNPs. The ability of TAgNPs to scavenge free radicals was evaluated with H 2 O 2, DPPH and ABTS assays, which suggested its excellent antioxidant potential. In vitro cytotoxicity through MTT assay and genotoxicity through comet assay on rat lymphocytes suggested outstanding safety profile of TAgNPs. Thus, TAgNPs have pharmacologically apt physico-chemical and biological properties and may offer as a promising agent for medicinal use against toxic manifestations in terms of oxidative stress associated disorders. Nanotechnology silver nanoparticles tiron cytotoxicity genotoxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Nanoscience defines the atomic structures and molecules ranging between 1 and 100 nm or more, whereas nanomedicine established the foundation of nanoscience by improving drug delivery, diagnosis and treating diseases [ 1 ]. Nanotechnology involves size, shape, surface, electrical, thermal, and magnetic properties, which on integration with nanobiotechnology, plays important roles in drug delivery, disease diagnosis and its treatments [ 2 ]. Researchers developed nano-medicines like liposomes, dendrimers, nanotubes etcetera for targeted drug delivery in afflicted sites in the body [ 3 ]. Drugs embedded in the nano-carriers established several advantages, including increased solubility and bioavailability of drugs, targeted site delivery, raised stability of drug due to their smaller size and wider surface area [ 4 ]. Antimicrobial [ 5 ], antibacterial [ 5 ], antiviral [ 5 ], anticoagulant [ 5 ], anticancer [ 6 ], wound-healing [ 7 ], antifungal [ 8 ], antidiabetic [ 9 ], and anti-inflammatory properties [ 9 ] of various materials have been transformed into various nanoparticles for better efficacy and drug delivery. Chelating agents effectively bind and eliminate toxic metals from the body [ 10 ]. Deferoxamine chelates free iron and aluminum, labile iron pools, hemosiderin and ferritin and excretes them in urine [ 10 ]. Deferiprone binds excess ferric ions (Fe III) and excretes in urine [ 11 ]. Ethylene diamine tetra-acetic acid (EDTA) is used to chelate cadmium, arsenic and mercury [ 12 ]. Penicillamine chelates copper, lead and mercury [ 13 ] by forming metal complex with divalent cations, which get easily solubilized in water and excreted through urination [ 14 ]. Tiron (4,5-Dihydroxy-1,3-benzenedisulphonic acid, disodium salt) is a compound with strong potential to from coordinate complexes with various metal ions. It consists of two hydroxyl groups and two sulfonic acid groups attached in a benzene ring. The attached functional group helps in donating electrons to neutralize free radicals. Tiron alone significantly decreases accumulation of aluminum in brain; however, combination of tiron and glutathione was found better against aluminum induced toxicity [ 15 ]. Individual and combined treatment of tiron with propolis [ 16 ] and α-tocopherol, propolis, and piperine [ 17 ] were reported to protect liver and kidney from beryllium induced intoxications. The few studies are available on fabricated nanoparticles of chelating compounds with their biological effects [ 18 ]. Tiron, with its immense antioxidant and chelating property due to its structural excellence to donate electron from hydroxyl groups, has never been synthesized as nanoparticles of silver or other metals. Therefore, this investigation aimed fabrication of tiron-loaded silver nanoparticles with physico-chemical and biological characterization for pharmacologically apt properties 2. Materials and methods 2.1 Chemicals : All the chemicals and reagents of analytical grade were used in the study. Silver nitrate, tiron, and aluminum nitrate, were procured from Hi-Media Laboratories, India. Beryllium nitrate was procured from Alpha-Chemika, India. 2.2 Synthesis of tiron-loaded silver nanoparticles : Silver nitrate solution (8 mM) was prepared by dissolving silver nitrate crystals in deionized water and stored in an amber colored bottle. Tiron (4 mM /5 mL) was dissolved in deionized water and maintained p H of 10.5 at room temperature; added freshly prepared silver nitrate solution and placed on a magnetic stirrer for 2 hours. Conversion of colorless solution into golden yellow color indicated completion of synthesis of tiron-loaded nanoparticles. Obtained suspension was further centrifuged for 20 min at 10,000 rpm and washed thrice with deionized water to collect nanoparticle pellets, which were dried in an oven at 37° C and kept at 4° C for physico-chemical and biological characterization [ 19 ]. 2.3 Physico-chemical characterization of tiron-loaded silver nanoparticles 2.3.1 UV-visible spectroscopy The UV-visible spectroscopy is based on the absorption of ultraviolet or visible light, which excites electrons from lower to higher energy level. The amount of light absorbed at specific wavelength was determined by Beer-Lambert law. During synthesis of tiron-loaded silver nanoparticles, silver ions are reduced from Ag + to Ag 0 , which was confirmed by scanning the solution of nanoparticles at wavelength 400–500 nm with distilled water as reference. The wavelength range of tiron-loaded silver nanoparticles was determined using a UV-visible spectrophotometer (Systronics, UV-VIS Spectrophotometer Model 117). 2.3.2 Scanning electron microscopy (SEM) with energy dispersive X-ray (EDX) The SEM works by focusing a beam of high energy electrons onto surface of samples and detecting signals produced by the interaction of beam with the sample, which are used to form high a resolution image showing surface topography and composition. Surface topography of nanoparticles was examined by SEM. The purity of tiron-loaded silver nanoparticles and its aggregation with tiron was inferred from SEM. The SEM (NOVA NANO SEM 450 FEI CO. OF USA (S.E.A.) PTE LTD., SINGAPORE) was operated with an accelerating voltage of 20.0 kV. The EDX analyzed the surface elemental composition in a sample. 2.3.3 Transmission electron microscopy (TEM) The principle of TEM is based on transmitting a beam of high energy electrons through a thin layered sample. The transmitted electrons from the sample are used to form a highly magnified image exhibiting internal structure and morphology. Internal morphology of silver nanoparticles was observed under TEM (G2F30 S-Twin at 300kV), which assured the size, morphology, and fabrication of silver nanoparticles. 2.3.4 Particle size analysis (PSA) Particle size analysis provides average size information of nanoparticles without damaging their property. It relies on the interaction of light energy with particles, or separating particles based on their size and density. The particle size and its distribution were determined using particle size analyzer (METROHM AU B B.V., NETHERLANDS). 2.3.5 Zeta potential Zeta potential determines the electrical charge on particles surface dispersed in a liquid. It provides potential stability of colloidal system and measures electrostatic repulsion/ attraction between the particles. The zeta potential analysis is used to predict long-term stability of nanoparticles in solution. Zeta potential of tiron-loaded silver nanoparticles was analyzed with the help of zeta potential analyzer, NanoPlus. 2.3.6 Thermogravimetric analysis Thermogravimetric analysis determined thermal stability and strength of catalysts in nanoparticles by measuring weight/mass change with respect to time and temperature. These mass changes reveal thermal stability, composition and decomposition properties of sample. Differential scanning calorimetry (DSC) measures temperature difference between a sample and a reference material as they are heated or cooled. The instrument used to analyze thermogravimetry of nanoparticles was NETZSCH STA 449F1 STA449F1A-01. 2.3.7 Fourier Transform Infrared Spectroscopy (FTIR) The FTIR works by passing infrared light through a sample and measuring the absorbed wavelengths. It converts the raw data into a spectrum, providing molecular structure and functional groups in samples. The FTIR spectrum provides absorption peaks in relation to frequencies of vibration within the atoms of nanoparticles. It characterizes nature of material and functional groups present in samples. Functional groups responsible for Ag + reduction, capping/stability of silver nanoparticles were determined with FTIR (SN 340, BRUKER OPTIK GmbH, GERMANY). 2.4 Free radical scavenging activity of tiron-loaded silver nanoparticles 2.4.1 Hydrogen peroxide scavenging activity: Hydrogen peroxide (H 2 O 2 ) scavenging activity was performed [ 20 ] on tiron-loaded silver nanoparticles at concentrations of 2, 4, 6, 8 and 10 µg that was added to 0.1 M phosphate buffer (pH 7.4) and mixed with 43 mM hydrogen peroxide. After 10 min, absorbance of reaction mixture was recorded at λ 230. Ascorbic acid was taken as standard. Where A b represents blank and A a represents absorption of sample 2.4.2 The DPPH free radical scavenging activity: The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay was performed [ 21 ]. Samples at different concentrations of 2, 4, 6, 8, 10 and 12 µg of nanoparticle samples of which 100 µl concentration solution sample was added to DPPH solution of 0.3 mM in methanol/ethanol. When sample was added to DPPH solution, the change of color was observed from dark purple to pale yellowish and brownish color, which confirmed that the compound pertains antioxidant property. After 30 min. of incubation in the dark, absorbance of reaction mixture was recorded by spectrophotometer at λ 517 nm. Ascorbic acid was taken as standard. Where A b represents blank and A a represents absorption of sample 2.4.3 The ABTS free radical scavenging activity: The 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay was performed [ 22 ]. The ABTS stock solution was prepared by adding 7 mM ABTS solution with 2.45 mM potassium persulfate solution reacted for 12 hours in the dark at room temperature. The 2 mL of ABTS stock solution was diluted with 50 mL ethanol. Sample at concentrations of 2, 4, 6, 8 and 10 µg was taken. Samples of 300 µl concentration was added to 180µl of ABTS stock solution. When sample was added with ABTS solution, the greenish-blue color of ABTS solution turned colorless in contact with sample molecules, which confirmed antioxidative capacity of the compound. The absorbance was recorded after 5 min. of incubation at λ 734 nm. Where A b represents blank and A a represents absorption of sample 2.5 Animal maintenance and lymphocyte culture Adult female Wistar rats (8–10 weeks old, 150 ± 10 g of body weight) were housed in disinfected polypropylene cages under controlled conditions (14 h light/10 h dark cycle, 25 ± 2°C, 55–70% humidity) with free access to standard pelleted diet (Akhoorath Ventures, Dehradun) and water ad libitum . Peripheral blood was taken from rats and lymphocytes were isolated and cultured in RPMI culture media for 24 h. 2.6 In-vitro cytoprotective and geno-protective potential of tiron-loaded silver nanoparticles Cytoprotective potential of was carried with MTT assay and geno-protective potential (DNA damage) with comet assays. Cultured lymphocytes were transferred to 96-well plates and their viability was assessed using the trypan blue exclusion method [ 23 ]. The entire well plate was assigned into seven groups. Group 1 served as control that did not receive any treatment, group 2 received co-exposure to combination of Al (1 µg/ml) and Be (6.5 µg/ml), group 3 received tiron-loaded silver nanoparticles 1 µg/ml along with combination of toxicants, group 4 received tiron-loaded silver nanoparticles 2 µg/ml along with combination of toxicants, group 5 received tiron-loaded silver nanoparticles 3 µg/ml along with combination of toxicants, group 6 received tiron-loaded silver nanoparticles 4 µg/ml along with combination of toxicants, group 7 received tiron-loaded silver nanoparticles 5µg/ml along with combination of both toxicants and incubated at 37° C for 24 h [ 24 ]. The MTT assay After 24 h of incubation, cells were treated with 5 mg/ml solution of diphenyltetrazolium bromide (MTT) for 4 h. Afterward, 100 µl of dimethyl sulfoxide (DMSO) was added to dissolve violet crystals [ 25 ]. The absorbance was determined by a microplate reader at λ 532 nm, and cell viability was determined with following formula, where C represents the absorbance of the control group and T is the absorbance of the treated group. Cell viability (%) = (C − T)/C × 100 Comet assay The comet assay was performed on lymphocytes in seven groups as mentioned in MTT assay [ 26 ]. The cell suspension was combined with an equal amount of 1% low-melting-point agarose and layered onto clean glass slides that had been pre-coated with 1% normal-melting-point agarose. Following electrophoresis, the slides were stained with ethidium bromide solution (1.5 mg/ml), and comet images were captured in bitmap format with fluorescence microscope (Leica DM-2500. Different parameters, including comet length, height, area, head diameter, % DNA in head, tail length, tail area, % DNA in tail, tail moment, and olive tail moment were analyzed. The mean values were calculated using TriTek comet analysis software. 2.7 Statistical analysis The data were expressed as mean ± standard error (SE). Statistical significance was analyzed with one-way ANOVA with a significance level at p ≤ 0.05 followed by Tukey’s HSD post hoc test at p ≤ 0.05 to compare differences between multiple groups. 3. Results 3.1 Characterization of tiron-loaded silver nanoparticles 3.1.1 Synthesis of tiron-loaded silver nanoparticles At the end point of synthesis, change in color from colorless to golden yellow confirmed the synthesis of tiron-loaded silver nanoparticles. Tiron molecules donate electrons to silver ions (Ag + ) in the solution to reduce them into Ag 0 that leads to nucleation and growth into nanoparticles (Fig. 1 a, b). 3.1.2 UV-visible spectroscopy UV- visible spectroscopy confirmed the formation of tiron-loaded silver nanoparticles showing absorbance peak at 430–440 nm. The peak refers to a specific optical property of synthesized tiron-loaded silver nanoparticles as a stabilizing or reducing agent and its presence can greatly influence the size, shape and optical properties of silver nanoparticles (Fig. 1 d) 3.1.3 Scanning electron microscope (SEM) with energy dispersive X-ray In SEM, it was remarkably observed that the nanoparticles were primarily spherical and more or less elongated in shape. Field emission SEM images of tiron-loaded silver nanoparticles at 4µm scale with HV 10.00 kV and working distance of 6.3 mm at 20000X magnification showing spherical shape nanoparticles (Fig. 2 a). The EDX analysis of tiron-loaded silver nanoparticles with dominated silver element (red dot) over the samples (Fig. 2 b). Figure 2 c demonstrates the EDX analysis of tiron-loaded silver nanoparticles with sharp peak at 2.60 keV energy level. 3.1.4 Transmission electron microscope (TEM) The TEM provided complete information of size, distribution and morphological background of nanoparticles. Figure 3 a showed selected area electron diffraction (SAED) pattern. The concentric rings with dots indicating crystalline nature of nanoparticles add plane. In this pattern, the nanoparticles exhibited polycrystalline nature and face centered cubic structure. The average size of silver nanoparticles in terms of area is 21.94093 nm. Figure 3 b illustrated spherical shaped silver nanoparticles with size area ranging from 5–50 nm. Figure 3 c & d exhibited high resolution polycrystalline lattice fringes at 10 nm scale with visible lattice planes. Polycrystalline denotes that the materials of nanoparticles are composed of multiple small crystalline grains. The lattice fringes exhibit regular and periodic patterns of contrast, which result from diffraction of electrons passing through the crystalline lattice of the nanoparticle material. 3.1.5 Particle size analysis The average size diameter in length of nanoparticles synthesized from tiron was 14.9, 109.4, 1305.5, and 12143.7 nm implies broad range of particle sizes. Different particle size shows nucleation and growth of nanoparticles. Particle size 14.9 nm could be the newly formed nuclei while 109.4, 1305.5, and 12143.7 nm could represent subsequent growth stages and aggregation within the nanoparticles. Figure 4 a showed size dispersion of tiron-loaded silver nanoparticles with intensity. The nanosize diameter indicated that tiron has strong capacity to reduce silver during nanoparticle synthesis. 3.1.6 Zeta potential The zeta potential of synthesized tiron-loaded silver nanoparticles was determined in water as diluent. The zeta potential was found to be -98.17mV. High negative charge confirmed the higher electrostatic repulsion within the particles preventing them aggregation and showed higher stability of nanoparticle formulation and extremely stable colloidal dispersion. The high negative zeta potential revealed that the surface of silver nanoparticles was strongly negatively charged as shown in Fig. 4 b. 3.1.7 Thermogravimetric analysis Thermogravimetric analysis/ differential scanning colorimetry (TG/ DSC) of tiron-loaded silver nanoparticles are shown within temperature range from 20° C to 560° C as displayed in Fig. 5 . Initial weight loss obtained at 100° C was 21.98% due to evaporation of water adsorbed by the silver nanoparticles. Second weight loss obtained at 180° C was 5.4% and ended weight loss was marked at 540° C, which showed that tiron compound present in nanoparticles get decomposed successfully. The total weight loss was around 27.38%. The results of DSC curve illustrated sharp exothermic peak at 142.7° C along with appearance of endothermic peak at 162° C. The exothermic peak indicated flow of less heat towards the sample while endothermic peak showed flow of more heat towards the sample. Exothermic peak at 142.7° C represented crystallization of silver nanoparticles and could be transitioning from amorphous particles to more ordered crystalline structured particles. 3.1.8 The FTIR analysis Presence of different functional groups in a compound determines the chemical reactions, reduction, capping/ stability of the compound. The FTIR spectrum of tiron-loaded silver nanoparticles was taken recorded. Various band range was found between 500 and 4000 absorption spectra as shown in Fig. 6 . Absorption spectra of 3776.68 cm − 1 showed the presence of hydroxyl group (-OH) stretching vibration with broad peak bound to tiron-loaded silver nanoparticles. Band at 3687.08, 3655.36 and 3627.75 cm − 1 assigned to (-OH) stretching alcohol group with medium sharp peak and 1589.21 cm − 1 revealed cyclic alkene (C = C) stretching with medium peak. 3.2 Free radical scavenging activity of tiron-loaded silver nanoparticles 3.2.1 Hydrogen peroxide scavenging activity Tiron-loaded silver nanoparticles showed concentration dependent H 2 O 2 free radical scavenging activity at different concentrations of 2, 4, 6, 8, and 10 µg. Increasing order of concentration of nanoparticles exhibited increasing order of H 2 O 2 free radical scavenging activity (Fig. 7 a). The IC 50 value of tiron-loaded silver nanoparticles was recorded as 6.183 µg. 3.2.2 The DPPH free radical scavenging activity Tiron-loaded silver nanoparticles showed concentration dependent DPPH free radical scavenging activity at different concentrations of 2, 4, 6, 8, 10 and 12 µg. The increasing order of concentration of tiron-loaded silver nanoparticles showed high DPPH free radical scavenging activity, which assured its antioxidative property at increasing concentrations (Fig. 7 b). The IC 50 value of tiron-loaded silver nanoparticles against DPPH free radical assay was noted as 4.317 µg. 3.2.3 The ABTS free radical scavenging activity Tiron-loaded silver nanoparticles also exhibited concentration dependent ABTS free radical scavenging activity at 2, 4, 6, 8, and 10 µg concentrations. The increasing order of concentration of tiron-loaded silver nanoparticles showed increasing order of ABTS free radical scavenging activity, which assured its increasing antioxidative potential at increasing concentrations (Fig. 7 c). The IC 50 value of tiron-loaded silver nanoparticles against DPPH free radical assay was noted as 1.021µg. 3.3 Cell viability assay In vitro cell viability study was performed with MTT assay. After 4 h of incubation period, significant difference was noted among control, toxicants and different treatment groups. Control group showed regular cytotoxicity, whereas combination of aluminum and beryllium significantly increased cytotoxicity of lymphocytes. The tiron-loaded silver nanoparticles at different concentrations 1,2,3,4 and 5 µg/ml with co-exposure to Al and Be significantly decreased cytotoxicity, thus increased viability of lymphocytes (Fig. 8 a) 3.4 Geno-protective potential of tiron-loaded silver nanoparticles To determine geno-protective potential of tiron-loaded silver nanoparticles, comet assay was performed. The comet assay signified that combined exposure to Al and Be significantly increased genotoxicity as indicated by increase in tail length as compared to control group. Tiron-loaded silver nanoparticles at concentrations of 1, 2, 3, 4 and 5 µg/ml along with combined exposure to Al and Be offered protection to DNA as indicated by decrease in tail length (Fig. 8 b). Different parameters of comet assays, including comet length, comet height, comet area, head diameter, % DNA in head, tail length, tail area, % DNA in tail, tail movement, and olive moment have been given in Table 1 . Figure 9 and Table 1 jointly represented that tiron-loaded silver nanoparticles have excellent potential to protect DNA from damage; thus, reduced genotoxicity. Table 1 Geno-protective potential of TAgNPs using comet assay Parameter Control Al + Be TAgNPs 1mg TAgNPs 2mg TAgNPs 3mg TAgNPs 4mg TAgNPs 5mg ANOVA (F-value) Comet length 136 ± 7.51 677 ± 37.4 a 297 ± 16.4 ab 250 ± 13.8 ab 221 ± 12.2 abc 185 ± 10.2 b c 168 ± 9.28 bcd 127 @ Comet height 103 ± 5.69 605 ± 33.4 a 309 ± 17.0 ab 260 ± 14.3 ab 249 ± 13.7 ab 198 ± 10.9 abc 180 ± 9.95 abcd 106 @ Comet area 47 ± 2.59 484 ± 26.7 a 311 ± 17.1 ab 252 ± 13.9 abcd 71 ± 3.92 bcd 58 ± 3.20 bcd 71 ± 3.92 bcd 191 @ Head diameter 51 ± 2.81 96 ± 5.30 a 166 ± 9.17 ab 176 ± 9.72 ab 59 ± 3.26 bcd 93 ± 5.14 acde 103 ± 5.69 acde 68.9 @ % DNA in head 100 ± 5.52 20.3 ± 1.12 a 91.5 ± 5.05 b 92.3 ± 5.10 b 93.1 ± 5.14 b 93.4 ± 5.16 b 99.1 ± 5.48 b 40.6 @ Tail length 02 ± 0.11 818 ± 45.2 a 19 ± 1.05 b 15 ± 0.82 b 10 ± 0.55 b 05 ± 0.27 b 03 ± 0.16 b 384 @ Tail area 02 ± 0.11 51 ± 2.81 a 13 ± 0.71 ab 11 ± 0.60 ab 07 ± 0.38 abc 02 ± 0.11 bcde 01 ± 0.05 bcde 290 @ % DNA in tail 11.9 ± 0.66 97.4 ± 5.38 a 11 ± 0.60 b 10 ± 0.55 b 0.01 ± 0.00 bcd 9.62 ± 0.53 be 4.44 ± 0.24 b 321 @ Tail movement 0 939 ± 51.9 a 5 ± 0.27 b 6 ± 0.33 b 3 ± 0.16 b 2 ± 0.11 b 2 ± 0.11 b 390 @ Olive movement 0 109 ± 6.02 a 4 ± 0.22 b 7 ± 0.38 b 5 ± 0.27 b 4 ± 0.22 b 1 ± 0.05 b 366 @ Data are presented at mean ± SE, *control vs Al + Be at P ≤ 0.05, TAgNPs + (Al + Be) for tukey’s HSD post hoc test at P ≤ 0.05; Significant at 5% level for ANOVA. Abbreviation: Al + Be (aluminium + beryllium) at 1 µg + 6.5 µg, TAgNPs = Tiron loaded silver nanoparticles at 1–5 mg; a Control vs Al + Be, TAgNPs 1–5 mg/kg, b Al+Be vs TAgNPs 1–5 mg/kg, c TAgNPs 1 mg/kg vs TAgNPs 2–5 mg/kg, d TAgNPs 2 mg/kg vs TAgNPs 3–5 mg/kg, e TAgNPs 3 mg/kg vs TAgNPs 4–5 mg/kg, f TAgNPs 4 mg/kg vs TAgNPs 5 mg/kg 4. Discussion Tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt) has been reported as an excellent chelator and therapeutic agent against aluminum [ 15 , 27 ] and beryllium [ 16 , 17 , 28 ] induced toxic manifestations. Silver nanoparticles of various compounds exhibit antimicrobial, bactericidal, anticancer, and wound-healing properties [ 29 ]. Synthesis of tiron-loaded silver nanoparticles may offer an innovative approach against combined exposure to Al and Be induced toxic manifestations in liver and kidney. Tiron, acted as reducing and capping agent during synthesis of silver nanoparticle, contributing antioxidant properties to the nanoparticles by chelating metals and scavenging reactive oxygen species (ROS). Tiron stabilizes nanoparticles and prevents aggregation and form smaller uniform particles. Presence of hydroxyl group, stretched alcohol group and cyclic alkenes primarily contribute to reduction of Ag⁺ ion forming tiron-loaded silver nanoparticles. Chelating and antioxidant properties of tiron protect liver [ 28 ], kidney [ 28 ] and brain [ 15 ] from metal toxicity. In view of this, the present work aimed to synthesize and characterize tiron-loaded silver nanoparticles and evaluate their in vitro antioxidant and protective activity against toxicity induced by combined exposure to Al and Be. The change in color of tiron-loaded AgNPs was relevant to the change in color noticed in econazole nitrate synthesized silver nanoparticles [ 30 ]. This change was attributed to the surface plasmon resonance of silver nanoparticles, and the color intensity corresponds to the number of electrons released during the reduction of Ag⁺ to Ag⁰ [ 31 , 32 ]. Tiron loaded silver nanoparticles revealed surface plasmon resonance shifting, which confirmed interaction of tiron on the surface of silver nanoparticle. The observed peak at wavelength 430–440 nm was a characteristic feature of silver nanoparticles as reported in previous findings [ 33 ]. Spherical shape of silver nanoparticles indicated uniform nucleation and growth, which exhibited homogenous size distribution [ 34 ]. Spherical shape offers enhanced cellular uptake of silver nanoparticles, may improve circulation and biodistribution with more predictable and controlled drug release [ 35 ]. Its interaction with tiron could enhance pharmacological performance. The EDX analysis confirmed the presence of silver metal ions, which dominated surface of the samples that indicated the capping of silver nanoparticles with tiron. The EDX spectrum indicated prominent signal for silver metals. This analysis was relevant to earlier work [ 36 ]. In the present study, tiron-loaded silver nanoparticles showed promising potential in drug delivery and size uniformity as analyzed and confirmed by TEM. The average size area of silver nanoparticles often shows increased surface area with effective biological interactions. The spherical shape ensures uniform distribution and good biocompatibility. The average size of tiron-loaded silver nanoparticles in terms of area was found 21.94093 nm while spherical shaped with size area was in a range from 5–50 nm. Presence of lattice fringes showed polycrystalline nature, which provides stability, reactivity and electron sharing ability. Polycrystalline structure of tiron-loaded silver nanoparticles was due to rapid nucleation, growth and stabilization of growing nanoparticles. Tiron inhibited aggregation and growth rate by acting as a capping agent resulting in polycrystallinity, which enhanced surface activity and reactivity of silver nanoparticles leading their potential use in pharmacology [ 37 , 38 ]. Particle size analysis of tiron-loaded silver nanoparticles manifested broad size distribution 109.4, 1305.5, and 12143.7 nm can occur due to agglomeration over specific area or variations in parameters such as pH, temperature and reaction time [ 39 , 40 , 41 ]. This reflected the hydrodynamic diameter of capping agent and solvation layer around the particles which makes them suitable for drug delivery. Zeta potential of nanoparticles provides an important information about their surface charge and stability in suspension and electrostatic repulsion or attraction among the nanoparticles. In this investigation, zeta potential was observed − 98.17mV, which indicated high stability with high repulsion between the nanoparticles. Tiron having two hydroxyl and sulfonate groups, strongly ionize in solvent media, resulting in negatively charged surface showing colloidal stability due to impact arrangement of tiron molecules or strong chelation. This could be due to the highly negative charged stabilizers or functional groups adsorbed on the surface of nanoparticles [ 42 , 43 ]. The TGA helps in monitoring of sample in terms of weight loss with respect to temperature. In tiron-loaded silver nanoparticles, initial weight loss indicated adsorbed water and loosely bound surface molecules (volatile molecules). Secondary weight loss reflected partial decomposition of weakly organic molecules and final decomposition results in complete degradation of tiron at 540℃ [ 44 , 45 ]. The sharp exothermic peak of DSC curve revealed oxidative decomposition while endothermic peak at 162 ℃ signified thermal disruptions among the molecules. This study confirmed that tiron-loaded silver nanoparticles revealed complete decomposition and crystallization of the nanoparticles. This thermostability supports its suitability for pharmaceutical application. Tiron-loaded silver nanoparticles showed specified functional groups, including hydroxyl groups and cyclic alkene [ 46 , 47 , 48 ]. This suggested multiple functional groups participated in reducing, capping and stabilization of tiron-loaded silver nanoparticles and also raised solubility, antioxidant activity and bio-interaction of nanoparticles and enhanced its applications for pharmaceutical uses [ 49 ]. Tiron showed its significant applications in both pharmacy and pharmaceutical industries due to their specific complex formation with toxic metals [ 15 ] inhibiting ROS generation and minimizing oxidative stress and cellular injury [ 50 ]. This features primarily showed its antioxidant and metal chelating property [ 51 ]. The H 2 O 2 , DPPH and ABTS induced free radical scavenging activity of tiron-loaded silver nanoparticles corroborated its potential as an excellent antioxidant for medicinal uses. The in vitro safety profile assessment provided an alternative method of animal testing for evaluating toxicological and/ or therapeutic effects of chemical entities. Combined exposure to Al and Be to lymphocytes has been implicated in producing damage to mitochondria and its dysfunction [ 52 , 53 ]. Combination of Al and Be disrupted cellular redox system by prompting oxidative stress, mitochondrial dysfunctions and alteration in DNA entity. It is further associated with the initiation of lipid peroxidation process and cytoplasmic enzymes leakage like lactate dehydrogenase and transaminases showing disturbance in membrane integrity [ 54 ]. The hydroxyl and sulfonate groups in tiron play crucial role in metal binding. Tiron-loaded silver nanoparticles exhibited remarkable antioxidant probably through its metal-chelating behavior. Cellular macromolecules such as lipids, proteins and DNA were negatively affected when exposed to Al and Be [ 55 ]. Tiron-loaded silver nanoparticles possess unique physicochemical characteristics that could help in preventing oxidative stress and DNA damage caused by free radicals and contribute to the stabilization of both cell membranes and DNA itself. 5. Conclusion This study concluded that tiron-loaded silver nanoparticles may offer excellent therapeutic potential at relatively lower doses in comparison to previously used doses of tiron alone. Tiron-loaded silver nanoparticles have pharmacologically apt physico-chemical and biological properties; thus, may offer as a promising agent for medicinal use against combination of Al and Be induced toxic manifestations in terms of oxidative stress associated disorders. Declarations The study was approved by institutional animal ethics committee (CPCSEA/994/GO/Re/S/06). Conflict of interest: The authors declare no competing interests. Funding: Partial financial support from Guru Ghasidas University by providing non-NET fellowship to PD, PB, AM and AAS and for procuring selected chemicals. Author Contribution PD, PB AM and AAS collectively done bench work of the experiment, PD prepared draft of the manuscript, SKN and MB conceptualized and prepared experimental design, arranged essential facilities, materials and supervision to complete the experimentation. Data Availability No datasets were generated or analyzed during the current study. References Durgam, L. K., & Oroszi, T. L. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7479494","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512737150,"identity":"d1fa34ac-38c2-4d0b-8d73-068ac8757c8b","order_by":0,"name":"Pratima Dutta","email":"","orcid":"","institution":"Guru Ghasidas University","correspondingAuthor":false,"prefix":"","firstName":"Pratima","middleName":"","lastName":"Dutta","suffix":""},{"id":512737151,"identity":"581f8767-5214-48df-9b28-dc66c4218d44","order_by":1,"name":"Satendra Kumar 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tiron-loaded silver nanoparticles\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7479494/v1/738773b34ba862f8ca433a22.png"},{"id":91465820,"identity":"1a3ae322-3c77-424a-9ff2-dc040c18e1ef","added_by":"auto","created_at":"2025-09-16 18:40:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":514493,"visible":true,"origin":"","legend":"\u003cp\u003eField emission scanning electron microscopy (SEM) with energy dispersive X-ray (EDX): \u003cstrong\u003ea\u003c/strong\u003e SEM images of tiron loaded silver nanoparticles showing spherical shapes at 4µm scale. \u003cstrong\u003eb\u003c/strong\u003e Silver (represented in red dots) distribution over the sample. \u003cstrong\u003ec\u003c/strong\u003e EDX analysis of tiron loaded silver nanoparticles shows sharp peak at 2.60 keV (Energy).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7479494/v1/e04181a8bce509fbf0d3463c.png"},{"id":91467263,"identity":"c2d3652c-2cc3-47e9-8479-e710e2f23553","added_by":"auto","created_at":"2025-09-16 18:56:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":380919,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission electron microscopy of tiron loaded silver nanoparticles: \u003cstrong\u003ea\u003c/strong\u003e HR-TEM image of selected area electron diffraction (SAED) pattern of tiron loaded silver nanoparticles confirmed its polycrystalline and face centered cubic structure. \u003cstrong\u003eb\u003c/strong\u003e Biosynthesized silver nanoparticles. \u003cstrong\u003ec-d\u003c/strong\u003e High resolution polycrystalline lattice image of tiron-loaded silver nanoparticles\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7479494/v1/c86063cc85cbcac7d28c6d43.png"},{"id":91465260,"identity":"1eae7223-8af1-44be-91e2-1f46b510bd82","added_by":"auto","created_at":"2025-09-16 18:32:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":197318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eParticle size distribution (DLS) of tiron loaded silver nanoparticles. \u003cstrong\u003eb\u003c/strong\u003e Zeta potential analysis of tiron loaded silver nanoparticles\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7479494/v1/a226083abd1b80e7672ec058.png"},{"id":91467525,"identity":"498429d9-73cb-4922-99f8-f3e7898259e3","added_by":"auto","created_at":"2025-09-16 19:04:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":33173,"visible":true,"origin":"","legend":"\u003cp\u003eThermogravimetric analysis of tiron-loaded silver nanoparticles.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7479494/v1/44b5eff1faeedf9c776322e0.png"},{"id":91466606,"identity":"22128282-0292-46eb-88c2-b57edbfed6bc","added_by":"auto","created_at":"2025-09-16 18:48:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":26309,"visible":true,"origin":"","legend":"\u003cp\u003eThe FTIR of tiron-loaded silver nanoparticles\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7479494/v1/343351952486d7332d350b9d.png"},{"id":91465269,"identity":"91b803b5-b578-4113-986d-e592ddc3de5d","added_by":"auto","created_at":"2025-09-16 18:32:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":114431,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging activity of tiron-loaded silver nanoparticles. Data are presented at mean ±SE. \u003cstrong\u003eb \u003c/strong\u003eDPPH free radical scavenging activity of tiron-loaded silver nanoparticles. Data are presented at mean ±SE. \u003cstrong\u003ec \u003c/strong\u003eABTS activity of tiron-loaded silver nanoparticles. Data are presented at mean ±SE\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7479494/v1/edd6121aa8f499a14f7138cb.png"},{"id":91465826,"identity":"f1907173-5bc7-4a1a-b9c8-29303d958574","added_by":"auto","created_at":"2025-09-16 18:40:50","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":96656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003e\u003cem\u003eIn-vitro\u003c/em\u003e MTT assay showing cell viability. \u003cstrong\u003eb \u003c/strong\u003eDNA damage percentage of lymphocytes\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7479494/v1/fa5e1b095cf37fb37ef4e206.png"},{"id":91465829,"identity":"020a9312-469b-4d32-906d-0d7169534362","added_by":"auto","created_at":"2025-09-16 18:40:51","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":60406,"visible":true,"origin":"","legend":"\u003cp\u003eDNA damage detected by comet assay and therapeutic potential of tiron-loaded silver nanoparticles. \u003cstrong\u003ea\u003c/strong\u003e Control, \u003cstrong\u003eb\u003c/strong\u003e Toxicant, \u003cstrong\u003ec\u003c/strong\u003etoxicant+TAgNPs 1 mg, \u003cstrong\u003ed\u003c/strong\u003etoxicant+TAgNPs 2 mg, \u003cstrong\u003ee\u003c/strong\u003etoxicant+TAgNPs 3 mg, \u003cstrong\u003ef\u003c/strong\u003etoxicant+TAgNPs 4 mg, \u003cstrong\u003eg \u003c/strong\u003etoxicant+TAgNPs 5 mg\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7479494/v1/3c4b2d75f19f60d3a422b3f1.png"},{"id":98814237,"identity":"0e6c09a9-aeb5-4a2b-b385-d794f8167c8e","added_by":"auto","created_at":"2025-12-22 16:12:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3104740,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7479494/v1/14868476-83c9-484d-a5f9-5f77b61c8b9b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tiron-loaded silver nanoparticles: Synthesis, physico-chemical and biological characterization for pharmacologically apt properties","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNanoscience defines the atomic structures and molecules ranging between 1 and 100 nm or more, whereas nanomedicine established the foundation of nanoscience by improving drug delivery, diagnosis and treating diseases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Nanotechnology involves size, shape, surface, electrical, thermal, and magnetic properties, which on integration with nanobiotechnology, plays important roles in drug delivery, disease diagnosis and its treatments [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Researchers developed nano-medicines like liposomes, dendrimers, nanotubes etcetera for targeted drug delivery in afflicted sites in the body [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Drugs embedded in the nano-carriers established several advantages, including increased solubility and bioavailability of drugs, targeted site delivery, raised stability of drug due to their smaller size and wider surface area [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Antimicrobial [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], antibacterial [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], antiviral [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], anticoagulant [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], anticancer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], wound-healing [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], antifungal [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], antidiabetic [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and anti-inflammatory properties [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] of various materials have been transformed into various nanoparticles for better efficacy and drug delivery.\u003c/p\u003e\u003cp\u003eChelating agents effectively bind and eliminate toxic metals from the body [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Deferoxamine chelates free iron and aluminum, labile iron pools, hemosiderin and ferritin and excretes them in urine [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Deferiprone binds excess ferric ions (Fe III) and excretes in urine [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Ethylene diamine tetra-acetic acid (EDTA) is used to chelate cadmium, arsenic and mercury [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Penicillamine chelates copper, lead and mercury [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] by forming metal complex with divalent cations, which get easily solubilized in water and excreted through urination [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Tiron (4,5-Dihydroxy-1,3-benzenedisulphonic acid, disodium salt) is a compound with strong potential to from coordinate complexes with various metal ions. It consists of two hydroxyl groups and two sulfonic acid groups attached in a benzene ring. The attached functional group helps in donating electrons to neutralize free radicals. Tiron alone significantly decreases accumulation of aluminum in brain; however, combination of tiron and glutathione was found better against aluminum induced toxicity [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Individual and combined treatment of tiron with propolis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and α-tocopherol, propolis, and piperine [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] were reported to protect liver and kidney from beryllium induced intoxications.\u003c/p\u003e\u003cp\u003eThe few studies are available on fabricated nanoparticles of chelating compounds with their biological effects [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Tiron, with its immense antioxidant and chelating property due to its structural excellence to donate electron from hydroxyl groups, has never been synthesized as nanoparticles of silver or other metals. Therefore, this investigation aimed fabrication of tiron-loaded silver nanoparticles with physico-chemical and biological characterization for pharmacologically apt properties\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Chemicals\u003c/strong\u003e: All the chemicals and reagents of analytical grade were used in the study. Silver nitrate, tiron, and aluminum nitrate, were procured from Hi-Media Laboratories, India. Beryllium nitrate was procured from Alpha-Chemika, India.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Synthesis of tiron-loaded silver nanoparticles\u003c/strong\u003e: Silver nitrate solution (8 mM) was prepared by dissolving silver nitrate crystals in deionized water and stored in an amber colored bottle. Tiron (4 mM /5 mL) was dissolved in deionized water and maintained \u003cem\u003ep\u003c/em\u003eH of 10.5 at room temperature; added freshly prepared silver nitrate solution and placed on a magnetic stirrer for 2 hours. Conversion of colorless solution into golden yellow color indicated completion of synthesis of tiron-loaded nanoparticles. Obtained suspension was further centrifuged for 20 min at 10,000 rpm and washed thrice with deionized water to collect nanoparticle pellets, which were dried in an oven at 37\u0026deg; C and kept at 4\u0026deg; C for physico-chemical and biological characterization [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Physico-chemical characterization of tiron-loaded silver nanoparticles\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section3\"\u003e\n \u003cdiv class=\"Heading\"\u003e2.3.1 UV-visible spectroscopy\u003c/div\u003e\n \u003cp\u003eThe UV-visible spectroscopy is based on the absorption of ultraviolet or visible light, which excites electrons from lower to higher energy level. The amount of light absorbed at specific wavelength was determined by Beer-Lambert law. During synthesis of tiron-loaded silver nanoparticles, silver ions are reduced from Ag\u003csup\u003e+\u003c/sup\u003e to Ag\u003csup\u003e0\u003c/sup\u003e, which was confirmed by scanning the solution of nanoparticles at wavelength 400\u0026ndash;500 nm with distilled water as reference. The wavelength range of tiron-loaded silver nanoparticles was determined using a UV-visible spectrophotometer (Systronics, UV-VIS Spectrophotometer Model 117).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\n \u003cdiv class=\"Heading\"\u003e2.3.2 Scanning electron microscopy (SEM) with energy dispersive X-ray (EDX)\u003c/div\u003e\n \u003cp\u003eThe SEM works by focusing a beam of high energy electrons onto surface of samples and detecting signals produced by the interaction of beam with the sample, which are used to form high a resolution image showing surface topography and composition. Surface topography of nanoparticles was examined by SEM. The purity of tiron-loaded silver nanoparticles and its aggregation with tiron was inferred from SEM. The SEM (NOVA NANO SEM 450 FEI CO. OF USA (S.E.A.) PTE LTD., SINGAPORE) was operated with an accelerating voltage of 20.0 kV. The EDX analyzed the surface elemental composition in a sample.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\n \u003cdiv class=\"Heading\"\u003e2.3.3 Transmission electron microscopy (TEM)\u003c/div\u003e\n \u003cp\u003eThe principle of TEM is based on transmitting a beam of high energy electrons through a thin layered sample. The transmitted electrons from the sample are used to form a highly magnified image exhibiting internal structure and morphology. Internal morphology of silver nanoparticles was observed under TEM (G2F30 S-Twin at 300kV), which assured the size, morphology, and fabrication of silver nanoparticles.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n \u003cdiv class=\"Heading\"\u003e2.3.4 Particle size analysis (PSA)\u003c/div\u003e\n \u003cp\u003eParticle size analysis provides average size information of nanoparticles without damaging their property. It relies on the interaction of light energy with particles, or separating particles based on their size and density. The particle size and its distribution were determined using particle size analyzer (METROHM AU B B.V., NETHERLANDS).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n \u003cdiv class=\"Heading\"\u003e2.3.5 Zeta potential\u003c/div\u003e\n \u003cp\u003eZeta potential determines the electrical charge on particles surface dispersed in a liquid. It provides potential stability of colloidal system and measures electrostatic repulsion/ attraction between the particles. The zeta potential analysis is used to predict long-term stability of nanoparticles in solution. Zeta potential of tiron-loaded silver nanoparticles was analyzed with the help of zeta potential analyzer, NanoPlus.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n \u003cdiv class=\"Heading\"\u003e2.3.6 Thermogravimetric analysis\u003c/div\u003e\n \u003cp\u003eThermogravimetric analysis determined thermal stability and strength of catalysts in nanoparticles by measuring weight/mass change with respect to time and temperature. These mass changes reveal thermal stability, composition and decomposition properties of sample. Differential scanning calorimetry (DSC) measures temperature difference between a sample and a reference material as they are heated or cooled. The instrument used to analyze thermogravimetry of nanoparticles was NETZSCH STA 449F1 STA449F1A-01.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n \u003cdiv class=\"Heading\"\u003e2.3.7 Fourier Transform Infrared Spectroscopy (FTIR)\u003c/div\u003e\n \u003cp\u003eThe FTIR works by passing infrared light through a sample and measuring the absorbed wavelengths. It converts the raw data into a spectrum, providing molecular structure and functional groups in samples. The FTIR spectrum provides absorption peaks in relation to frequencies of vibration within the atoms of nanoparticles. It characterizes nature of material and functional groups present in samples. Functional groups responsible for Ag\u003csup\u003e+\u003c/sup\u003e reduction, capping/stability of silver nanoparticles were determined with FTIR (SN 340, BRUKER OPTIK GmbH, GERMANY).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch3\u003e2.4 Free radical scavenging activity of tiron-loaded silver nanoparticles\u003c/h3\u003e\n \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\n \u003ch3\u003e2.4.1 Hydrogen peroxide scavenging activity:\u003c/h3\u003e\n \u003cp\u003eHydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) scavenging activity was performed [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e] on tiron-loaded silver nanoparticles at concentrations of 2, 4, 6, 8 and 10 \u0026micro;g that was added to 0.1 M phosphate buffer (pH 7.4) and mixed with 43 mM hydrogen peroxide. After 10 min, absorbance of reaction mixture was recorded at \u0026lambda; 230. Ascorbic acid was taken as standard.\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img1758047021.png\" style=\"width: 382px;\"\u003e\u003c/p\u003e\n \u003cp\u003eWhere A\u003csub\u003eb\u003c/sub\u003e represents blank and A\u003csub\u003ea\u003c/sub\u003e represents absorption of sample\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n \u003ch3\u003e2.4.2 The DPPH free radical scavenging activity:\u003c/h3\u003e\n \u003cp\u003eThe 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay was performed [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. Samples at different concentrations of 2, 4, 6, 8, 10 and 12 \u0026micro;g of nanoparticle samples of which 100 \u0026micro;l concentration solution sample was added to DPPH solution of 0.3 mM in methanol/ethanol. When sample was added to DPPH solution, the change of color was observed from dark purple to pale yellowish and brownish color, which confirmed that the compound pertains antioxidant property. After 30 min. of incubation in the dark, absorbance of reaction mixture was recorded by spectrophotometer at \u0026lambda; 517 nm. Ascorbic acid was taken as standard.\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img1758047416.png\" style=\"width: 358px;\"\u003e\u003c/p\u003e\n \u003cp\u003eWhere A\u003csub\u003eb\u003c/sub\u003e represents blank and A\u003csub\u003ea\u003c/sub\u003e represents absorption of sample\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n \u003ch3\u003e2.4.3 The ABTS free radical scavenging activity:\u003c/h3\u003e\n \u003cp\u003eThe 2,2\u0026apos;-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay was performed [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. The ABTS stock solution was prepared by adding 7 mM ABTS solution with 2.45 mM potassium persulfate solution reacted for 12 hours in the dark at room temperature. The 2 mL of ABTS stock solution was diluted with 50 mL ethanol. Sample at concentrations of 2, 4, 6, 8 and 10 \u0026micro;g was taken. Samples of 300 \u0026micro;l concentration was added to 180\u0026micro;l of ABTS stock solution. When sample was added with ABTS solution, the greenish-blue color of ABTS solution turned colorless in contact with sample molecules, which confirmed antioxidative capacity of the compound. The absorbance was recorded after 5 min. of incubation at \u0026lambda; 734 nm.\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img1758047434.png\" style=\"width: 361px;\"\u003e\u003c/p\u003e\n \u003cp\u003eWhere A\u003csub\u003eb\u003c/sub\u003e represents blank and A\u003csub\u003ea\u003c/sub\u003e represents absorption of sample\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch3\u003e2.5 Animal maintenance and lymphocyte culture\u003c/h3\u003e\n \u003cp\u003eAdult female Wistar rats (8\u0026ndash;10 weeks old, 150\u0026thinsp;\u0026plusmn;\u0026thinsp;10 g of body weight) were housed in disinfected polypropylene cages under controlled conditions (14 h light/10 h dark cycle, 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 55\u0026ndash;70% humidity) with free access to standard pelleted diet (Akhoorath Ventures, Dehradun) and water \u003cem\u003ead libitum\u003c/em\u003e. Peripheral blood was taken from rats and lymphocytes were isolated and cultured in RPMI culture media for 24 h.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch3\u003e2.6 \u003cem\u003eIn-vitro\u003c/em\u003e cytoprotective and geno-protective potential of tiron-loaded silver nanoparticles\u003c/h3\u003e\n \u003cp\u003eCytoprotective potential of was carried with MTT assay and geno-protective potential (DNA damage) with comet assays. Cultured lymphocytes were transferred to 96-well plates and their viability was assessed using the trypan blue exclusion method [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. The entire well plate was assigned into seven groups. Group 1 served as control that did not receive any treatment, group 2 received co-exposure to combination of Al (1 \u0026micro;g/ml) and Be (6.5 \u0026micro;g/ml), group 3 received tiron-loaded silver nanoparticles 1 \u0026micro;g/ml along with combination of toxicants, group 4 received tiron-loaded silver nanoparticles 2 \u0026micro;g/ml along with combination of toxicants, group 5 received tiron-loaded silver nanoparticles 3 \u0026micro;g/ml along with combination of toxicants, group 6 received tiron-loaded silver nanoparticles 4 \u0026micro;g/ml along with combination of toxicants, group 7 received tiron-loaded silver nanoparticles 5\u0026micro;g/ml along with combination of both toxicants and incubated at 37\u0026deg; C for 24 h [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eThe MTT assay\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAfter 24 h of incubation, cells were treated with 5 mg/ml solution of diphenyltetrazolium bromide (MTT) for 4 h. Afterward, 100 \u0026micro;l of dimethyl sulfoxide (DMSO) was added to dissolve violet crystals [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. The absorbance was determined by a microplate reader at \u0026lambda; 532 nm, and cell viability was determined with following formula, where C represents the absorbance of the control group and T is the absorbance of the treated group.\u003c/p\u003e\n \u003cp\u003eCell viability (%) = (C\u0026thinsp;\u0026minus;\u0026thinsp;T)/C \u0026times; 100\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eComet assay\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe comet assay was performed on lymphocytes in seven groups as mentioned in MTT assay [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. The cell suspension was combined with an equal amount of 1% low-melting-point agarose and layered onto clean glass slides that had been pre-coated with 1% normal-melting-point agarose. Following electrophoresis, the slides were stained with ethidium bromide solution (1.5 mg/ml), and comet images were captured in bitmap format with fluorescence microscope (Leica DM-2500. Different parameters, including comet length, height, area, head diameter, % DNA in head, tail length, tail area, % DNA in tail, tail moment, and olive tail moment were analyzed. The mean values were calculated using TriTek comet analysis software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch3\u003e2.7 Statistical analysis\u003c/h3\u003e\n \u003cp\u003eThe data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE). Statistical significance was analyzed with one-way ANOVA with a significance level at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 followed by Tukey\u0026rsquo;s HSD post hoc test at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 to compare differences between multiple groups.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Characterization of tiron-loaded silver nanoparticles\u003c/h2\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e3.1.1 Synthesis of tiron-loaded silver nanoparticles\u003c/h2\u003e\u003cp\u003eAt the end point of synthesis, change in color from colorless to golden yellow confirmed the synthesis of tiron-loaded silver nanoparticles. Tiron molecules donate electrons to silver ions (Ag\u003csup\u003e+\u003c/sup\u003e) in the solution to reduce them into Ag\u003csup\u003e0\u003c/sup\u003e that leads to nucleation and growth into nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e3.1.2 UV-visible spectroscopy\u003c/h2\u003e\u003cp\u003eUV- visible spectroscopy confirmed the formation of tiron-loaded silver nanoparticles showing absorbance peak at 430\u0026ndash;440 nm. The peak refers to a specific optical property of synthesized tiron-loaded silver nanoparticles as a stabilizing or reducing agent and its presence can greatly influence the size, shape and optical properties of silver nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e3.1.3 Scanning electron microscope (SEM) with energy dispersive X-ray\u003c/h2\u003e\u003cp\u003eIn SEM, it was remarkably observed that the nanoparticles were primarily spherical and more or less elongated in shape. Field emission SEM images of tiron-loaded silver nanoparticles at 4\u0026micro;m scale with HV 10.00 kV and working distance of 6.3 mm at 20000X magnification showing spherical shape nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The EDX analysis of tiron-loaded silver nanoparticles with dominated silver element (red dot) over the samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec demonstrates the EDX analysis of tiron-loaded silver nanoparticles with sharp peak at 2.60 keV energy level.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e3.1.4 Transmission electron microscope (TEM)\u003c/h2\u003e\u003cp\u003eThe TEM provided complete information of size, distribution and morphological background of nanoparticles. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea showed selected area electron diffraction (SAED) pattern. The concentric rings with dots indicating crystalline nature of nanoparticles add plane. In this pattern, the nanoparticles exhibited polycrystalline nature and face centered cubic structure. The average size of silver nanoparticles in terms of area is 21.94093 nm. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb illustrated spherical shaped silver nanoparticles with size area ranging from 5\u0026ndash;50 nm. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec \u0026amp; d exhibited high resolution polycrystalline lattice fringes at 10 nm scale with visible lattice planes. Polycrystalline denotes that the materials of nanoparticles are composed of multiple small crystalline grains. The lattice fringes exhibit regular and periodic patterns of contrast, which result from diffraction of electrons passing through the crystalline lattice of the nanoparticle material.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e3.1.5 Particle size analysis\u003c/h2\u003e\u003cp\u003eThe average size diameter in length of nanoparticles synthesized from tiron was 14.9, 109.4, 1305.5, and 12143.7 nm implies broad range of particle sizes. Different particle size shows nucleation and growth of nanoparticles. Particle size 14.9 nm could be the newly formed nuclei while 109.4, 1305.5, and 12143.7 nm could represent subsequent growth stages and aggregation within the nanoparticles. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea showed size dispersion of tiron-loaded silver nanoparticles with intensity. The nanosize diameter indicated that tiron has strong capacity to reduce silver during nanoparticle synthesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\u003ch2\u003e3.1.6 Zeta potential\u003c/h2\u003e\u003cp\u003eThe zeta potential of synthesized tiron-loaded silver nanoparticles was determined in water as diluent. The zeta potential was found to be -98.17mV. High negative charge confirmed the higher electrostatic repulsion within the particles preventing them aggregation and showed higher stability of nanoparticle formulation and extremely stable colloidal dispersion. The high negative zeta potential revealed that the surface of silver nanoparticles was strongly negatively charged as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e3.1.7 Thermogravimetric analysis\u003c/h2\u003e\u003cp\u003eThermogravimetric analysis/ differential scanning colorimetry (TG/ DSC) of tiron-loaded silver nanoparticles are shown within temperature range from 20\u0026deg; C to 560\u0026deg; C as displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Initial weight loss obtained at 100\u0026deg; C was 21.98% due to evaporation of water adsorbed by the silver nanoparticles. Second weight loss obtained at 180\u0026deg; C was 5.4% and ended weight loss was marked at 540\u0026deg; C, which showed that tiron compound present in nanoparticles get decomposed successfully. The total weight loss was around 27.38%. The results of DSC curve illustrated sharp exothermic peak at 142.7\u0026deg; C along with appearance of endothermic peak at 162\u0026deg; C. The exothermic peak indicated flow of less heat towards the sample while endothermic peak showed flow of more heat towards the sample. Exothermic peak at 142.7\u0026deg; C represented crystallization of silver nanoparticles and could be transitioning from amorphous particles to more ordered crystalline structured particles.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003e3.1.8 The FTIR analysis\u003c/h2\u003e\u003cp\u003ePresence of different functional groups in a compound determines the chemical reactions, reduction, capping/ stability of the compound. The FTIR spectrum of tiron-loaded silver nanoparticles was taken recorded. Various band range was found between 500 and 4000 absorption spectra as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Absorption spectra of 3776.68 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e showed the presence of hydroxyl group (-OH) stretching vibration with broad peak bound to tiron-loaded silver nanoparticles. Band at 3687.08, 3655.36 and 3627.75 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e assigned to (-OH) stretching alcohol group with medium sharp peak and 1589.21 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e revealed cyclic alkene (C\u0026thinsp;=\u0026thinsp;C) stretching with medium peak.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Free radical scavenging activity of tiron-loaded silver nanoparticles\u003c/h2\u003e\u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1 Hydrogen peroxide scavenging activity\u003c/h2\u003e\u003cp\u003eTiron-loaded silver nanoparticles showed concentration dependent H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e free radical scavenging activity at different concentrations of 2, 4, 6, 8, and 10 \u0026micro;g. Increasing order of concentration of nanoparticles exhibited increasing order of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e free radical scavenging activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). The IC\u003csub\u003e50\u003c/sub\u003e value of tiron-loaded silver nanoparticles was recorded as 6.183 \u0026micro;g.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2 The DPPH free radical scavenging activity\u003c/h2\u003e\u003cp\u003eTiron-loaded silver nanoparticles showed concentration dependent DPPH free radical scavenging activity at different concentrations of 2, 4, 6, 8, 10 and 12 \u0026micro;g. The increasing order of concentration of tiron-loaded silver nanoparticles showed high DPPH free radical scavenging activity, which assured its antioxidative property at increasing concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). The IC\u003csub\u003e50\u003c/sub\u003e value of tiron-loaded silver nanoparticles against DPPH free radical assay was noted as 4.317 \u0026micro;g.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3 The ABTS free radical scavenging activity\u003c/h2\u003e\u003cp\u003eTiron-loaded silver nanoparticles also exhibited concentration dependent ABTS free radical scavenging activity at 2, 4, 6, 8, and 10 \u0026micro;g concentrations. The increasing order of concentration of tiron-loaded silver nanoparticles showed increasing order of ABTS free radical scavenging activity, which assured its increasing antioxidative potential at increasing concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). The IC\u003csub\u003e50\u003c/sub\u003e value of tiron-loaded silver nanoparticles against DPPH free radical assay was noted as 1.021\u0026micro;g.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Cell viability assay\u003c/h2\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e cell viability study was performed with MTT assay. After 4 h of incubation period, significant difference was noted among control, toxicants and different treatment groups. Control group showed regular cytotoxicity, whereas combination of aluminum and beryllium significantly increased cytotoxicity of lymphocytes. The tiron-loaded silver nanoparticles at different concentrations 1,2,3,4 and 5 \u0026micro;g/ml with co-exposure to Al and Be significantly decreased cytotoxicity, thus increased viability of lymphocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Geno-protective potential of tiron-loaded silver nanoparticles\u003c/h2\u003e\u003cp\u003eTo determine geno-protective potential of tiron-loaded silver nanoparticles, comet assay was performed. The comet assay signified that combined exposure to Al and Be significantly increased genotoxicity as indicated by increase in tail length as compared to control group. Tiron-loaded silver nanoparticles at concentrations of 1, 2, 3, 4 and 5 \u0026micro;g/ml along with combined exposure to Al and Be offered protection to DNA as indicated by decrease in tail length (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). Different parameters of comet assays, including comet length, comet height, comet area, head diameter, % DNA in head, tail length, tail area, % DNA in tail, tail movement, and olive moment have been given in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e and Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e jointly represented that tiron-loaded silver nanoparticles have excellent potential to protect DNA from damage; thus, reduced genotoxicity.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGeno-protective potential of TAgNPs using comet assay\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" 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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAl\u0026thinsp;+\u0026thinsp;Be\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTAgNPs\u003c/p\u003e\u003cp\u003e1mg\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTAgNPs\u003c/p\u003e\u003cp\u003e2mg\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTAgNPs\u003c/p\u003e\u003cp\u003e3mg\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTAgNPs\u003c/p\u003e\u003cp\u003e4mg\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eTAgNPs\u003c/p\u003e\u003cp\u003e5mg\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eANOVA\u003c/p\u003e\u003cp\u003e(F-value)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eComet length\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e136\u0026thinsp;\u0026plusmn;\u0026thinsp;7.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e677\u0026thinsp;\u0026plusmn;\u0026thinsp;37.4\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e297\u0026thinsp;\u0026plusmn;\u0026thinsp;16.4\u003csup\u003e\u003cb\u003eab\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e250\u0026thinsp;\u0026plusmn;\u0026thinsp;13.8\u003csup\u003e\u003cb\u003eab\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e221\u0026thinsp;\u0026plusmn;\u0026thinsp;12.2\u003csup\u003e\u003cb\u003eabc\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e185\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2\u003csup\u003e\u003cb\u003eb\u003c/b\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e168\u0026thinsp;\u0026plusmn;\u0026thinsp;9.28\u003csup\u003e\u003cb\u003ebcd\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e127\u003csup\u003e@\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eComet height\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e103\u0026thinsp;\u0026plusmn;\u0026thinsp;5.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e605\u0026thinsp;\u0026plusmn;\u0026thinsp;33.4\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e309\u0026thinsp;\u0026plusmn;\u0026thinsp;17.0\u003csup\u003e\u003cb\u003eab\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e260\u0026thinsp;\u0026plusmn;\u0026thinsp;14.3\u003csup\u003e\u003cb\u003eab\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e249\u0026thinsp;\u0026plusmn;\u0026thinsp;13.7\u003csup\u003e\u003cb\u003eab\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e198\u0026thinsp;\u0026plusmn;\u0026thinsp;10.9\u003csup\u003e\u003cb\u003eabc\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e180\u0026thinsp;\u0026plusmn;\u0026thinsp;9.95\u003csup\u003e\u003cb\u003eabcd\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e106\u003csup\u003e@\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eComet area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e484\u0026thinsp;\u0026plusmn;\u0026thinsp;26.7\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e311\u0026thinsp;\u0026plusmn;\u0026thinsp;17.1\u003csup\u003e\u003cb\u003eab\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e252\u0026thinsp;\u0026plusmn;\u0026thinsp;13.9\u003csup\u003e\u003cb\u003eabcd\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e71\u0026thinsp;\u0026plusmn;\u0026thinsp;3.92\u003csup\u003e\u003cb\u003ebcd\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e58\u0026thinsp;\u0026plusmn;\u0026thinsp;3.20\u003csup\u003e\u003cb\u003ebcd\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e71\u0026thinsp;\u0026plusmn;\u0026thinsp;3.92\u003csup\u003e\u003cb\u003ebcd\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e191\u003csup\u003e@\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHead diameter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e51\u0026thinsp;\u0026plusmn;\u0026thinsp;2.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e96\u0026thinsp;\u0026plusmn;\u0026thinsp;5.30\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e166\u0026thinsp;\u0026plusmn;\u0026thinsp;9.17\u003csup\u003e\u003cb\u003eab\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e176\u0026thinsp;\u0026plusmn;\u0026thinsp;9.72\u003csup\u003e\u003cb\u003eab\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e59\u0026thinsp;\u0026plusmn;\u0026thinsp;3.26\u003csup\u003e\u003cb\u003ebcd\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e93\u0026thinsp;\u0026plusmn;\u0026thinsp;5.14\u003csup\u003e\u003cb\u003eacde\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e103\u0026thinsp;\u0026plusmn;\u0026thinsp;5.69\u003csup\u003e\u003cb\u003eacde\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e68.9\u003csup\u003e@\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e% DNA in head\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;5.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e91.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.05\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e92.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.10\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e93.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.14\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e93.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.16\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e99.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.48\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e40.6\u003csup\u003e@\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTail length\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e818\u0026thinsp;\u0026plusmn;\u0026thinsp;45.2\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e384\u003csup\u003e@\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTail area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e51\u0026thinsp;\u0026plusmn;\u0026thinsp;2.81\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003csup\u003e\u003cb\u003eab\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003csup\u003e\u003cb\u003eab\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003e\u003cb\u003eabc\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003e\u003cb\u003ebcde\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003e\u003cb\u003ebcde\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e290\u003csup\u003e@\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e% DNA in tail\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e97.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.38\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003e\u003cb\u003ebcd\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e9.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003e\u003cb\u003ebe\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e321\u003csup\u003e@\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTail movement\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e939\u0026thinsp;\u0026plusmn;\u0026thinsp;51.9\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e390\u003csup\u003e@\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOlive movement\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e109\u0026thinsp;\u0026plusmn;\u0026thinsp;6.02\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e366\u003csup\u003e@\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003eData are presented at mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE, *control vs Al\u0026thinsp;+\u0026thinsp;Be at P\u0026thinsp;\u0026le;\u0026thinsp;0.05, TAgNPs + (Al\u0026thinsp;+\u0026thinsp;Be) for tukey\u0026rsquo;s HSD post hoc test at P\u0026thinsp;\u0026le;\u0026thinsp;0.05; Significant at 5% level for ANOVA. Abbreviation: Al\u0026thinsp;+\u0026thinsp;Be (aluminium\u0026thinsp;+\u0026thinsp;beryllium) at 1 \u0026micro;g\u0026thinsp;+\u0026thinsp;6.5 \u0026micro;g, TAgNPs\u0026thinsp;=\u0026thinsp;Tiron loaded silver nanoparticles at 1\u0026ndash;5 mg; \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003eControl vs Al\u0026thinsp;+\u0026thinsp;Be, TAgNPs 1\u0026ndash;5 mg/kg, \u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003eAl+Be vs TAgNPs 1\u0026ndash;5 mg/kg, \u003csup\u003e\u003cb\u003ec\u003c/b\u003e\u003c/sup\u003eTAgNPs 1 mg/kg vs TAgNPs 2\u0026ndash;5 mg/kg, \u003csup\u003e\u003cb\u003ed\u003c/b\u003e\u003c/sup\u003eTAgNPs 2 mg/kg vs TAgNPs 3\u0026ndash;5 mg/kg, \u003csup\u003e\u003cb\u003ee\u003c/b\u003e\u003c/sup\u003eTAgNPs 3 mg/kg vs TAgNPs 4\u0026ndash;5 mg/kg, \u003csup\u003e\u003cb\u003ef\u003c/b\u003e\u003c/sup\u003eTAgNPs 4 mg/kg vs TAgNPs 5 mg/kg\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eTiron (4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt) has been reported as an excellent chelator and therapeutic agent against aluminum [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and beryllium [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] induced toxic manifestations. Silver nanoparticles of various compounds exhibit antimicrobial, bactericidal, anticancer, and wound-healing properties [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Synthesis of tiron-loaded silver nanoparticles may offer an innovative approach against combined exposure to Al and Be induced toxic manifestations in liver and kidney. Tiron, acted as reducing and capping agent during synthesis of silver nanoparticle, contributing antioxidant properties to the nanoparticles by chelating metals and scavenging reactive oxygen species (ROS). Tiron stabilizes nanoparticles and prevents aggregation and form smaller uniform particles. Presence of hydroxyl group, stretched alcohol group and cyclic alkenes primarily contribute to reduction of Ag⁺ ion forming tiron-loaded silver nanoparticles. Chelating and antioxidant properties of tiron protect liver [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], kidney [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and brain [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] from metal toxicity. In view of this, the present work aimed to synthesize and characterize tiron-loaded silver nanoparticles and evaluate their \u003cem\u003ein vitro\u003c/em\u003e antioxidant and protective activity against toxicity induced by combined exposure to Al and Be.\u003c/p\u003e\u003cp\u003eThe change in color of tiron-loaded AgNPs was relevant to the change in color noticed in econazole nitrate synthesized silver nanoparticles [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This change was attributed to the surface plasmon resonance of silver nanoparticles, and the color intensity corresponds to the number of electrons released during the reduction of Ag⁺ to Ag⁰ [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTiron loaded silver nanoparticles revealed surface plasmon resonance shifting, which confirmed interaction of tiron on the surface of silver nanoparticle. The observed peak at wavelength 430\u0026ndash;440 nm was a characteristic feature of silver nanoparticles as reported in previous findings [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Spherical shape of silver nanoparticles indicated uniform nucleation and growth, which exhibited homogenous size distribution [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Spherical shape offers enhanced cellular uptake of silver nanoparticles, may improve circulation and biodistribution with more predictable and controlled drug release [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Its interaction with tiron could enhance pharmacological performance. The EDX analysis confirmed the presence of silver metal ions, which dominated surface of the samples that indicated the capping of silver nanoparticles with tiron. The EDX spectrum indicated prominent signal for silver metals. This analysis was relevant to earlier work [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In the present study, tiron-loaded silver nanoparticles showed promising potential in drug delivery and size uniformity as analyzed and confirmed by TEM. The average size area of silver nanoparticles often shows increased surface area with effective biological interactions. The spherical shape ensures uniform distribution and good biocompatibility. The average size of tiron-loaded silver nanoparticles in terms of area was found 21.94093 nm while spherical shaped with size area was in a range from 5\u0026ndash;50 nm. Presence of lattice fringes showed polycrystalline nature, which provides stability, reactivity and electron sharing ability. Polycrystalline structure of tiron-loaded silver nanoparticles was due to rapid nucleation, growth and stabilization of growing nanoparticles. Tiron inhibited aggregation and growth rate by acting as a capping agent resulting in polycrystallinity, which enhanced surface activity and reactivity of silver nanoparticles leading their potential use in pharmacology [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Particle size analysis of tiron-loaded silver nanoparticles manifested broad size distribution 109.4, 1305.5, and 12143.7 nm can occur due to agglomeration over specific area or variations in parameters such as pH, temperature and reaction time [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This reflected the hydrodynamic diameter of capping agent and solvation layer around the particles which makes them suitable for drug delivery.\u003c/p\u003e\u003cp\u003eZeta potential of nanoparticles provides an important information about their surface charge and stability in suspension and electrostatic repulsion or attraction among the nanoparticles. In this investigation, zeta potential was observed \u0026minus;\u0026thinsp;98.17mV, which indicated high stability with high repulsion between the nanoparticles. Tiron having two hydroxyl and sulfonate groups, strongly ionize in solvent media, resulting in negatively charged surface showing colloidal stability due to impact arrangement of tiron molecules or strong chelation. This could be due to the highly negative charged stabilizers or functional groups adsorbed on the surface of nanoparticles [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe TGA helps in monitoring of sample in terms of weight loss with respect to temperature. In tiron-loaded silver nanoparticles, initial weight loss indicated adsorbed water and loosely bound surface molecules (volatile molecules). Secondary weight loss reflected partial decomposition of weakly organic molecules and final decomposition results in complete degradation of tiron at 540℃ [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The sharp exothermic peak of DSC curve revealed oxidative decomposition while endothermic peak at 162 ℃ signified thermal disruptions among the molecules. This study confirmed that tiron-loaded silver nanoparticles revealed complete decomposition and crystallization of the nanoparticles. This thermostability supports its suitability for pharmaceutical application.\u003c/p\u003e\u003cp\u003eTiron-loaded silver nanoparticles showed specified functional groups, including hydroxyl groups and cyclic alkene [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. This suggested multiple functional groups participated in reducing, capping and stabilization of tiron-loaded silver nanoparticles and also raised solubility, antioxidant activity and bio-interaction of nanoparticles and enhanced its applications for pharmaceutical uses [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Tiron showed its significant applications in both pharmacy and pharmaceutical industries due to their specific complex formation with toxic metals [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] inhibiting ROS generation and minimizing oxidative stress and cellular injury [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. This features primarily showed its antioxidant and metal chelating property [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, DPPH and ABTS induced free radical scavenging activity of tiron-loaded silver nanoparticles corroborated its potential as an excellent antioxidant for medicinal uses.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e safety profile assessment provided an alternative method of animal testing for evaluating toxicological and/ or therapeutic effects of chemical entities. Combined exposure to Al and Be to lymphocytes has been implicated in producing damage to mitochondria and its dysfunction [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Combination of Al and Be disrupted cellular redox system by prompting oxidative stress, mitochondrial dysfunctions and alteration in DNA entity. It is further associated with the initiation of lipid peroxidation process and cytoplasmic enzymes leakage like lactate dehydrogenase and transaminases showing disturbance in membrane integrity [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The hydroxyl and sulfonate groups in tiron play crucial role in metal binding. Tiron-loaded silver nanoparticles exhibited remarkable antioxidant probably through its metal-chelating behavior. Cellular macromolecules such as lipids, proteins and DNA were negatively affected when exposed to Al and Be [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Tiron-loaded silver nanoparticles possess unique physicochemical characteristics that could help in preventing oxidative stress and DNA damage caused by free radicals and contribute to the stabilization of both cell membranes and DNA itself.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study concluded that tiron-loaded silver nanoparticles may offer excellent therapeutic potential at relatively lower doses in comparison to previously used doses of tiron alone. Tiron-loaded silver nanoparticles have pharmacologically apt physico-chemical and biological properties; thus, may offer as a promising agent for medicinal use against combination of Al and Be induced toxic manifestations in terms of oxidative stress associated disorders.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e The study was approved by institutional animal ethics committee (CPCSEA/994/GO/Re/S/06).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003ePartial financial support from Guru Ghasidas University by providing non-NET fellowship to PD, PB, AM and AAS and for procuring selected chemicals.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003ePD, PB AM and AAS collectively done bench work of the experiment, PD prepared draft of the manuscript, SKN and MB conceptualized and prepared experimental design, arranged essential facilities, materials and supervision to complete the experimentation.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eNo datasets were generated or analyzed during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDurgam, L. 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The interplay between inflammation, oxidative stress, DNA damage, DNA repair and mitochondrial dysfunction in depression. \u003cem\u003eProgress in Neuro-Psychopharmacology and Biological Psychiatry\u003c/em\u003e, \u003cem\u003e80\u003c/em\u003e, 309\u0026ndash;321. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.pnpbp.2017.06.036\u003c/span\u003e\u003cspan address=\"10.1016/j.pnpbp.2017.06.036\" 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":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Nanotechnology, silver nanoparticles, tiron, cytotoxicity, genotoxicity","lastPublishedDoi":"10.21203/rs.3.rs-7479494/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7479494/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNanotechnology offered synthesis of nanoparticles by unique combination of metals with natural and synthetic products for therapeutic uses with improved drug delivery. We synthesized tiron-loaded silver nanoparticles (TAgNPs), and performed their physico-chemical and biological characterization to understand pharmacologically apt properties. Synthesis of TAgNPs was confirmed by change in color to golden yellow with peak range of 430\u0026ndash;440 nm under UV-visible spectroscopy. The particle size was found in the range of 14.9-12143.7 nm with a spherical shape under electron microscopy. Crystalline phase of tiron-loaded silver nanoparticles was observed by X-ray powder diffraction and thermal stability was confirmed by thermogravimetric analysis. Functional groups were validated under FTIR spectroscopy, which quantified functional groups on the surfaces of TAgNPs. The ability of TAgNPs to scavenge free radicals was evaluated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e DPPH and ABTS assays, which suggested its excellent antioxidant potential. \u003cem\u003eIn vitro\u003c/em\u003e cytotoxicity through MTT assay and genotoxicity through comet assay on rat lymphocytes suggested outstanding safety profile of TAgNPs. Thus, TAgNPs have pharmacologically apt physico-chemical and biological properties and may offer as a promising agent for medicinal use against toxic manifestations in terms of oxidative stress associated disorders.\u003c/p\u003e","manuscriptTitle":"Tiron-loaded silver nanoparticles: Synthesis, physico-chemical and biological characterization for pharmacologically apt properties","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-16 18:32:46","doi":"10.21203/rs.3.rs-7479494/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-30T09:08:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-30T08:34:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"328875741067156329072067477235917689546","date":"2025-09-22T14:49:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-09T21:52:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"191978786119708112456166350397754371536","date":"2025-09-09T21:48:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-09T07:56:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-09T07:51:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-09T07:12:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioNanoScience","date":"2025-08-28T10:48:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fe222621-3476-47a8-8270-452351123994","owner":[],"postedDate":"September 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T16:06:51+00:00","versionOfRecord":{"articleIdentity":"rs-7479494","link":"https://doi.org/10.1007/s12668-025-02253-2","journal":{"identity":"bionanoscience","isVorOnly":false,"title":"BioNanoScience"},"publishedOn":"2025-12-17 15:58:33","publishedOnDateReadable":"December 17th, 2025"},"versionCreatedAt":"2025-09-16 18:32:46","video":"","vorDoi":"10.1007/s12668-025-02253-2","vorDoiUrl":"https://doi.org/10.1007/s12668-025-02253-2","workflowStages":[]},"version":"v1","identity":"rs-7479494","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7479494","identity":"rs-7479494","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}