In this work, a novel, porous Zn0.5Mg0.5FeMnO4 spinel oxide was successfully prepared via the sol–gel method and then studied as an anode material for Li-ion batteries (LIBs). Its crystal structure, morphology, and electrochemical properties were, respectively, analyzed through X-ray diffraction, high-resolution scanning electron microscopy, and cyclic voltammetry/galvanostatic discharge/charge measurements. From the X-ray diffraction, Zn0.5Mg0.5FeMnO4 spinel oxide was found to crystallize in the cubic structure with Fdm symmetry. However, the Zn0.5Mg0.5FeMnO4 spinel oxide exhibited a porous morphology formed by interconnected 3D nanoparticles. The porous Zn0.5Mg0.5FeMnO4 anode showed good cycling stability in its capacity during the initial 40 cycles with a retention capacity of 484.1 mAh g−1 after 40 cycles at a current density of 150 mA g−1, followed by a gradual decrease in the range of 40–80 cycles, which led to reaching a specific capacity close to 300.0 mAh g−1 after 80 cycles. The electrochemical reactions of the lithiation/delithiation processes and the lithium-ion storage mechanism are discussed and extracted from the cyclic voltammetry curves.Due to the low capacity, low working potential, and lithium coating at fast charging rates of graphite material as an anode for Li-ion batteries (LIBs), it is necessary to develop novel anode materials for LIBs with higher capacity, excellent electrochemical stability, and good safety. Among different transition-metal oxides, AB Recently, the extensive demand for consumer electronics and electric vehicles has led to a significant increase in the demand for rechargeable lithium-ion batteries (LIBs) owing to their high energy density, long cycle life, environmental friendliness, and no memory effect ,2,3. Sin−1), its low working potential (<0.2 V vs. Li+/Li), and its lithium coating at fast charging rates, graphite-based LIBs are limited and cannot meet the demand for fast-chargeable batteries with high energy density [In commercialized, traditional LIBs, graphite has been the most extensively used anode material to date due to its low cost and long cycle life ,8. Due t density ,10. Ther density ,12,13.xOy  have received significant attention as promising high-performance anode materials to substitute the graphite material in LIBs due to their distinctive properties such as higher specific capacity than that of the commercially used graphite, good safety, environmental friendliness, and low price [2 [2 [2 [2O3 [2 [2O4-type spinels as mixed transition-metal oxides with a greater capacity than graphite anodes (800–1000 mAh g−1) have received a lot of attention; they are considered interesting and promising electrochemically active materials for next-generation LIBs, which can combine two transition metals [2O4 spinel oxides as anode materials for LIBs use the conversion mechanism for Li+ ion storage [2O4-type spinel oxides with the desired morphology can be easily synthesized through several methods such as solid-state reaction [3O4 [2O4 [2O4 [2O4 [In recent decades, transition-metal oxides (TMOs) with the general formula Mow price ,15. Withow price , NiO [17ow price , TiO2 [1price [2 , MnO2 [2ce [2 [2 , SiO2 [2[2 [2 [2 , Fe2O3 [ [2 [2O3 , and SnO [2O3 [2 . Among tn metals ,25. Gene storage . In genereaction , sol–gelreaction , co-precreaction , hydrothreaction , electroreaction , low-temreaction , microwareaction , and Pecreaction  methods,ion [3O4 , copper 3O4 [2O4 , cobalt 2O4 [2O4 , nickel 2O4 [2O4 , etc. 2O4 nanoparticles prepared through the sol–gel method, as anode materials for LIBs, have shown a specific capacity near 474 mAh g−1 after 50 cycles under a current density of 90 mA g−1 [2O4 spinel [2O4 with mesoporous microsphere morphology has been reported by Z. Zhang et al. [−1 at 0.2 C and 0.8 C, respectively, after 50 cycles, suggesting good performance for fast charging. Another spinel ferrite, zinc ferrite ZnFe2O4, has gained much attention as an anode material for LIBs due to its great electrochemical performance with different morphologies, such as hollow spherical [Recently, nanostructured spinel ferrites have received tremendous interest for application in LIBs . Among n0 mA g−1 . In this4 spinel . The mang et al. . It has pherical , microsipherical , macropopherical , porous pherical , and fibpherical  morpholo2O4 spinel oxides have also attracted much interest as alternative electrode materials for LIBs owing to their excellent electrochemical performance [2O4 microsheets were synthesized by K. Cai et al. using a microwave-assisted hydrothermal route [−1 after 40 cycles at a rate of 0.1 C. Another spinel manganite, the MgMn2O4 spinel, was investigated by Z. Wang et al. [2O4 nanoparticles with particle sizes ranging from 20 to 30 nm prepared using the sol–gel method have shown a discharge capacity of 483.6 mAh g−1 after 100 cycles.Additionally, manganese-based ABformance ,48. Amonal route . From thg et al. . The MgM2O4−x with oxygen vacancies as an anode material for LIBs, which has shown a specific capacity of 650.5 mAh g−1 at 2 A g−1 after 1200 cycles, i.e., excellent reversible capacity and good cycling stability. K. Tian et al. [3O4  are high-performance anodes for LIBs. On the other hand, Nb-based oxides such as Nb2O5, Nb12O29, TiNb2O7, Ti2Nb10O29, etc., have attracted much attention as anode materials for high-rate lithium-ion energy storage [In recent years, Co-, Ni-, Zn-, Fe-, Cr-, Mn-, and Li-based multi-component spinel oxides have attracted great attention as high-performance anodes for LIBs and long-life lithium-ion batteries ,52. L. D storage . 0.5Mg0.5FeMnO4 spinel oxide using sol–gel method with a theoretical capacity of 976.3 mAh g−1 as an anode electrode for LIBs. Herein, Mg is used as an inactive stabilized pillar, which can produce good cycling stability. The synthesized active material is characterized by different techniques such as X-ray diffraction, thermogravimetry analysis, scanning electron microscopy, and the nitrogen adsorption–desorption isotherm to report, respectively, its crystallographic structure, thermal stability, morphology, and specific surface area. The electrochemical performance of the prepared porous Zn0.5Mg0.5FeMnO4 spinel oxide is tested by cyclic voltammetry and galvanostatic discharge/charge measurements.From the above, to simultaneously improve the stability, energy density, and high-rate capability of anode materials for LIBs, a novel, 3D interconnected structure belonging to transition-metal-oxide-type spinels is urgently needed. In this work, we report a facile route to synthesize 3D porous Zn0.5Mg0.5FeMnO4 sample. 0.5Mg0.5FeMnO4 sample acquired at room temperature. From this figure, the XRD data reveal the formation of the AB2O4-type spinel phase as all the experimental XRD peaks observed at different 2θ values are well matching with standard peaks of the MgFe2O4 spinel phase that crystallizes in the cubic Fdm structure with a lattice parameter of a = 8.3827 Å (JCPDS card: 88-1936) [0.5Mg0.5FeMnO4 sample exhibits a single spinel phase with the cubic Fdm system. However, no additional peaks from any impurities could be identified in the XRD pattern, revealing a high purity of the synthesized spinel phase and confirming that the organic species of CA have been completely removed. In addition, the synthesized spinel shows sharp XRD peaks with small full width at half maximum (FWHM), suggesting good crystallization of the synthesized Zn0.5Mg0.5FeMnO4 spinel oxide. Besides, the widening of the obtained XRD peaks provides proof of the existence of nanoscale-dimension spinel particles.The X-ray powder diffraction (XRD) technique was first employed to analyze the crystallographic structure, formation, and phase purity of the as-synthesized Zn88-1936) , which i0.5Mg0.5FeMnO4 spinel ferrite was refined using the Rietveld method [Fdm as a space group. The Rietveld refinement plot of the synthesized Zn0.5Mg0.5FeMnO4 spinel is displayed in obs) pattern is shown by open black circles, the simulated XRD  pattern by the Rietveld method is represented by red solid lines, and the difference curve  is indicated by pink solid lines. From this figure, it can be seen that the observed XRD pattern shows great agreement with the calculated XRD pattern, revealing the success of a realized structural refinement for the synthesized Zn0.5Mg0.5FeMnO4 spinel ferrite. Moreover, the quality of the structural refinement was evaluated by the consideration of the different R-factors such as structure factor (RF), Bragg factor (RB), profile factor (RP), expected factor (Rexp), and profile factor (Rwp) [0.5Mg0.5FeMnO4 spinel oxide in the Rietveld refinement such as structural parameters, profile parameters, and different reliability R-factors are summarized in 0.5Mg0.5FeMnO4 spinel oxide crystallizes in the cubic Fdm structure with a lattice parameter of a = 8.4265 (2) Å.For further deep analysis of structural parameters, the XRD pattern of the synthesized Znd method ,56. The or (Rwp) ,58,59,600.5Mg0.5FeMnO4 spinel oxide was drawn. 0.5Mg0.5FeMnO4 spinel oxide with the cubic structure in Fdm symmetry. As clearly shown in the figure, the crystal structure of Zn0.5Mg0.5FeMnO4 spinel oxide is constructed by 3D interconnected tetrahedral and octahedral polyhedra, where Zn2+ and Mg2+ cations are located in the tetrahedral sites , Fe3+ and Mn3+ are located in the octahedral sites , and the red balls represent the O2− oxygen anions. Moreover, this structure shows three Wyckoff sites—the 8a, 16d, and 32e sites, which correspond to the tetrahedral, octahedral, and oxygen sites, respectively. Only 1/8 of the tetrahedral sites are occupied by Zn2+/Mg2+ cations, 1/2 of the octahedral sites are occupied by Fe3+/Mn3+ cations, and the 32e sites are totally occupied by oxygen anions. Therefore, the spinel structure exhibits 7/8 of the tetrahedral sites and 1/2 of the octahedral sites that are empty. During the first discharge of electrochemical cells, the lithium ions can be inserted into the unit cell to occupy the lacunar sites.From the obtained structural parameters by Rietveld refinement, the crystal structure of Zn0.5Mg0.5FeMnO4 spinel. In the case of the xerogel sample (red curve), two weight losses take place in the temperature range from room temperature to 350 °C. The first loss is observed before 140 °C, which is due to the evaporation of the water used as a solvent during the synthesis and the physically adsorbed water on the surface. The second weight loss observed in the temperature range of 140–350 °C is attributed to the decomposition of the organic species of the citric acid (CA) associated with the elimination of nitrogen dioxide (NO2↑). Further, no weight loss is present above 350 °C. Moreover, the thermal stability of the synthesized spinel was examined by thermogravimetric analysis (TGA). For the calcined sample at 700 °C (blue curve), its TG curve demonstrates no weight loss in the whole temperature range of 20–700 °C, indicating good thermal stability of the synthesized porous Zn0.5Mg0.5FeMnO4 spinel oxide and confirming that the organic molecules of the citric acid (CA) have been completely removed after calcination at 700 °C.In order to understand the mechanism of the calcination of the synthesized xerogel, the xerogel sample was characterized by thermogravimetric analysis (TGA) in a wide temperature range of 20–700 °C. 0.5Mg0.5FeMnO4 spinel oxide, which demonstrates its surface morphology. From the figure, it can be seen that the prepared spinel shows a microsized porous morphology with different pore sizes ranging from 2 to 5 μm. Moreover, the porous morphology can make the electrolyte used in the cells of LIBs penetrate easily, which leads to the acceleration of lithium ions diffusion, enhancing the electrochemical performance. For further deep analysis of the surface morphology, the synthesized spinel was characterized by high-resolution scanning electron microscopy (HR-SEM). The obtained HR-SEM image is presented in 0.5Mg0.5FeMnO4 spinel is composed of interconnected nanosized spherical particles with sizes ranging from 50 to 100 nm.The microstructural morphology of the synthesized spinel was revealed by scanning electron microscopy (SEM). 0.5Mg0.5FeMnO4 spinel oxide, an energy-dispersive X-ray (EDX) spectrometer was utilized. The results of this investigation are displayed in To carry out an investigation on the purity of the synthesized porous Zn0.5Mg0.5FeMnO4 spinel oxide collected at a liquid nitrogen temperature of 77 K. The analysis of the nitrogen adsorption–desorption isotherm was performed to obtain the specific surface area of the synthesized porous Zn0.5Mg0.5FeMnO4 spinel oxide using the Brunauer–Emmett–Teller (BET) method. From the nitrogen adsorption–desorption isotherm, the porous spinel shows a type IV nitrogen sorption isotherm with a small hysteresis loop at high partial pressures (P/P0), suggesting a characteristic of a mesoporous material. Using the Brunauer–Emmett–Teller (BET) method, the specific surface area of the synthesized porous Zn0.5Mg0.5FeMnO4 spinel oxide was found to be 13.54 m2 g−1. The obtained small specific surface area is probably due to the high aggregation of nanoparticles, as shown in the HR-SEM image  measurements were conducted over the 0.01–3.00 V voltage range vs. Li+/Li under ambient conditions. 0.5Mg0.5FeMnO4 spinel oxide collected at a sweep rate of 0.05 mV s−1. Specifically, in the initial cathodic sweep , two dominant peaks at 0.35 and 0.7 V are observed in the first cycle. In agreement with previous studies [2+, Fe3+, and Mn3+ ions together, which are incorporated in Zn0.5Mg0.5FeMnO4 spinel phase to their metallic states Zn, Fe, and Mn associated with the formation of MgO and amorphous Li2O, as demonstrated in Equation (1). After the reduction of Zn0.5Mg0.5FeMnO4, the formed Zn metal can react with Li+ ions, giving the reversible formation of LiZn alloys (Equation (2)) [2O3, and MnO to Zn, Fe, and Mn, respectively, as previously reported [In order to further investigate the beneficial effects of the Zn studies ,63, the ion (2)) . The othion (2)) ,65. For reported ,67. The reported ,69.First cycle:Subsequent cycles:2+ (ZnO), Fe3+ (Fe2O3), and Mn2+ (MnO), respectively, as represented in Equation (4) [In the charge process (delithiation), the distinct anodic peaks are between 1.6 V and 1.8 V for all cycles, which probably contribute to the various oxidations of metallic Zn, Fe, and Mn to their oxide states Zntion (4) ,70. Thertion (4) ,72:0.5 Z0.5Mg0.5FeMnO4 anode.On the other hand, the first cycle is easily distinct from the subsequent cycles. Besides, the difference between the redox peaks in the first and subsequent cycles is due to the capacity loss of the anode spinel during the first cycle. From the third cycle, the CV curves are similar and the redox peaks remain consistent, revealing good reversibility of the Zn0.5Mg0.5FeMnO4 spinel oxide acquired at a current density of 150 mA g−1 over the potential region of 0.01–3.00 V (vs. Li+/Li) for various cycles  are shown in 0.5Mg0.5FeMnO4 spinel oxide is similar to those of ZnFe2O4 and MgFe2O4 [0.5Mg0.5FeMnO4 spinel oxide has a theoretical capacity of 976.3 mAh g−1 as an anode electrode for LIBs. From −1, respectively. Note that the initial discharge capacity is higher than the theoretical capacity. This can be explained by the lithium-ion storage through a surface faradic redox reaction (pseudocapacitive). The irreversible capacity loss between discharge and charge of the first cycle is normally due to the formation of SEI film [2+, Fe3+, and Mn3+ transition metals to metallic states of Zn, Fe, and Mn, respectively [0.5Mg0.5FeMnO4 nanoparticles, leading to poor electrical contacts between Zn0.5Mg0.5FeMnO4 nanoparticles and that between the active material and current collector [0.5Mg0.5FeMnO4 anode at the current density of 150 mA g−1 during 80 cycles is displayed in 0.5Mg0.5FeMnO4 anode shows good cycling stability in its capacity during the initial 40 cycles, with a specific capacity of 484.1 mAh g−1 after 40 cycles followed by a gradual decrease in the range of 40–80 cycles. Moreover, the Zn0.5Mg0.5FeMnO4 anode exhibits a specific capacity close to 300.0 mAh g−1 after 80 cycles at an applied current density of 150 mA g−1. For the first cycle, the anode gives a Coulombic efficiency of 60.2%, which is owing to the abovementioned capacity loss in the first cycle. From the second cycle, the anode provides a stable value of Coulombic efficiency, which is more than 98% during 80 cycles. In order to compare the obtained performance with the literature, 0.5Mg0.5FeMnO4 spinel oxide with previously studied anodes.Subsequently, the results of the galvanostatic discharge/charge cycling of the synthesized porous Zn MgFe2O4 . It is wSEI film ,75. The ectively . During ollector . The cyc0.5Mg0.5FeMnO4 anode is examined by increasing the scan rates from 150 to 400 mA g−1 and then back to 150 mA g−1. For the first current density (150 mA g−1), the Zn0.5Mg0.5FeMnO4 anode reaches a specific capacity of 569.1 mAh g−1 after 10 cycles. When gradually increasing the current density, the specific capacity of the Zn0.5Mg0.5FeMnO4 anode gradually decreases and reaches a specific capacity of 533.2 mAh g−1 after 20 cycles at a current density of 200 mA g−1, and a specific capacity of 474.6 mAh g−1 after 30 cycles at a current density of 300 mA g−1, with good cycling stability. For the subsequent 10 cycles, the Zn0.5Mg0.5FeMnO4 anode exhibits a specific capacity of 365.3 mAh g−1 after 40 cycles at a current density of 400 mA g−1. When the current density is switched back to 150 mA g−1, the specific capacity of the studied anode increased rapidly to reach 481.8 mAh g−1. In the range of 40–80 cycles, the Zn0.5Mg0.5FeMnO4 anode was found to show a gradual decrease during discharge/charge cycling and reached a specific capacity close to 300.0 mAh g−1 after 80 cycles at an applied current density of 150 mA g−1.As presented in 0.5Mg0.5FeMnO4 anode were acquired at various scan rates ranging from 0.05 to 0.40 mV s−1, as shown in i) for the cathodic and anodic sweeps at various scan rates (v) was used to analyze the lithium-ion intercalation kinetics of the studied Zn0.5Mg0.5FeMnO4 anode. For the cathodic and anodic sweeps, the peak current  and the scan rates  can be related according to the Dunn relation, as expressed in Equation (6) [For further deep analysis of the kinetics of electrochemical lithiation/delithiation processes, the CV curves of the studied Zntion (6) ,80:i = aBoth sides of Equation (6) were taken in the logarithm function, which can be written asa and b are adjustable parameters. In general, three lithium-ion intercalation kinetics can be distinguished depending on the b-value. In particular, when the b-value approaches 0.5, the lithium-ion storage mechanism is controlled by the diffusion process. When the b-value is close to 1, the lithium-ion storage mechanism is through a surface faradic redox reaction (pseudocapacitive); whereas, when the b-value is between 0.5 and 1, the lithium-ion storage mechanism is a combination of a pseudocapacitive process and diffusion-controlled insertion [b-value was determined by plotting Log(i) against Log(v) for cathodic and anodic peaks, as shown in b-value for the cathodic and anodic sweeps corresponds to the slope of linear fitting of the peak currents versus scan rates. According to the estimated slopes 2·6H2O, iron nitrate nonahydrate Fe(NO3)3·9H2O, manganese nitrate tetrahydrate Mn(NO3)2·4H2O, and citric acid monohydrate C6H8O7·H2O of analytical grade were purchased from Sigma Aldrich. All purchased reagents were directly used as received without further purification. Zinc chloride ZnCl0.5Mg0.5FeMnO4 spinel oxide was synthesized using a facile sol–gel method. Initially, specific molar ratios of ZnCl2, Mg(NO3)2·6H2O, Fe(NO3)3·9H2O, and Mn(NO3)2·4H2O were dissolved together in deionized water under vigorous magnetic stirring for 30 min to obtain a homogeneous solution. To this homogeneous solution, citric acid (CA) dissociated in deionized water was added at a molar ratio of CA/cations = 2.5. Then, the solution was kept on the hot plate at 90 °C until a viscous gel formed followed by cooling slowly down to room temperature. Subsequently, the resulting gel was dried in an oven at 120 °C for 12 h to obtain the xerogel powder. Finally, the as-synthesized powder was annealed in the furnace at 700 °C for 12 h at a heating rate of 3 °C min−1 in air to obtain the porous Zn0.5Mg0.5FeMnO4 spinel oxide. During this synthesis, the Zn0.5Mg0.5FeMnO4 spinel oxide was formed from the reagents according to the following chemical reaction:The porous Zn0.5Mg0.5FeMnO4 spinel was firstly characterized by powder X-ray diffraction  with Cu-Kα (λ1 = 1.54056 Å and λ2 = 1.54439 Å) radiations. The XRD analysis was carried out at room temperature over a 2 theta range from 10° to 80° at a scan rate of 0.02°/s. The structural refinement of the prepared sample was carried out by the Rietveld method using Full-prof software (version 7.95) [−1 under a constant air purging flow. The surface morphology and surface elemental composition of the prepared powder spinel were evaluated by employing a scanning electron microscope interconnected with an energy-dispersive X-ray spectrometer . For further deep analysis of the surface morphology, a high-resolution scanning electron microscopy (HR-SEM) image was obtained on a JEOL JSM-IT500HR instrument. To obtain the Brunauer–Emmett–Teller (BET) specific surface area of the synthesized porous spinel, the nitrogen adsorption–desorption isotherm was collected at a liquid nitrogen temperature of 77 K using a Micromeritics Instrument.For the structural analysis and the phase identification, the synthesized porous Znon 7.95) . Thermal0.5Mg0.5FeMnO4 spinel, dry powders of the synthesized spinel , carbon black, and carboxymethyl cellulose (CMC) binder were mixed at a weight ratio of 80:10:10, respectively. To form a homogeneous slurry, the mixture was dispersed in ultrapure water and mixed for 2 h under vigorous magnetic stirring. Subsequently, the above slurry mixture was cast on a copper foil current collector with a thickness of 0.1 mm and a mass loading of 1~1.2 mg cm−2 using the doctor-blade technique. The loaded copper foil was dried at 60 °C under vacuum for 4 h to eliminate the water used as a solvent and then cut into disks of 11 mm in diameter. The produced electrodes were dried at 80 °C under vacuum for 12 h. All the electrochemical measurements of Zn0.5Mg0.5FeMnO4 spinel as anode material for LIBs were performed at room temperature using CR2032 coin cells in a half-cell configuration. Zn0.5Mg0.5FeMnO4//Li coin cells were assembled in an Ar (99.999%)-filled glove box  with high-purity metallic lithium disks as the counter and reference electrodes, Whatman as a separator, and a solution of LiPF6 (2 mol L−1) dissolved in EMC as the electrolyte. Their electrochemical performance was examined using a BioLogic Science multi-channel battery cycler for testing cyclic voltammetry (CV), galvanostatic discharge/charge, long-term cycling, and rate capability over the 0.01–3.00 V voltage range (vs. Li+/Li). The cyclic voltammetry (CV) data were measured at various scan rates: 0.05, 0.10, 0.20, 0.30, and 0.40 mV s−1. The galvanostatic discharge/charge and long-term cycling of the studied spinel were performed at a current density of 150 mA g−1. Moreover, the rate capability analysis was realized at different current densities of 150, 200, 300, and 400 mA g−1. The assembled coin cells were set still for 7 h before testing. In this work, the discharging and charging are considered as the lithiation and delithiation processes, respectively.To make the working electrodes of Zn0.5Mg0.5FeMnO4 spinel oxide elaborated by a facile sol–gel route as anode material for LIBs was investigated in detail. As demonstrated by the XRD, Rietveld method, SEM, and EDX results, the product has a high-purity phase with the desired chemical composition. From the structural analysis, the formation of a single AB2O4-type spinel phase of the synthesized sample was confirmed. Rietveld refinement evidenced the cubic spinel with Fdm symmetry. Under flowing air, the synthesized porous Zn0.5Mg0.5FeMnO4 spinel oxide showed a good thermal stability as the temperature increased to 700 °C. The SEM image confirmed the successful formation of porous morphology with pore sizes ranging from 2 to 5 μm. From the HR-SEM image, the 3D interconnection of the nanosized spherical particles led to a porous morphology. According to the BET method, the specific surface area of the synthesized porous Zn0.5Mg0.5FeMnO4 spinel oxide was found to be 13.54 m2 g−1. For the electrochemical performance, the Zn0.5Mg0.5FeMnO4 anode showed good cycling stability in its capacity during the initial 40 cycles, with a retention capacity of 484.1 mAh g−1 after 40 cycles at a current density of 150 mA g−1, followed by a gradual decrease in the range of 40–80 cycles, which led to reaching a specific capacity close to 300.0 mAh g−1 after 80 cycles at a current density of 150 mA g−1. Besides, the electrochemical reactions of the lithiation/delithiation processes were extracted from the CV curves. For the lithium-ion storage mechanism, a combination of diffusion-controlled intercalation and pseudocapacitance behavior was found. In general, this work is a way forward to develop inexpensive, highly efficient, and eco-friendly transition-metal oxide as a promising anode material for LIBs.In this work, the electrochemical performance of a porous Zn


Pantoea agglomerans uam8, was isolated from the ionic liquid (IL) Choline NTF2 and identified by molecular biology. A biodegradation study was performed at osmolarity conditions . These had an important influence on the growth of the strain, exopolysaccharide (EPS) production, and biodegradation (1303 mg/L max production and 80% biodegradation at 0.6 M). These conditions also had an important influence on the morphology of the strain and its EPSs, but not in the chemical composition. The EPS ) produced at 0.6 M was further characterized using different techniques. The obtained EPSs presented important differences in the behavior of the emulsifying activity for vegetable oils (olive (86%), sunflower (56%) and coconut (90%)) and hydrocarbons (diesel (62%), hexane (60%)), and were compared with commercial emulsifiers. The EPS produced at 0.6 M had the highest emulsifying activity overall. This EPS did not show cytotoxicity against the tested cell line (<20%) and presented great advantages as an antioxidant  (85%), hydroxyl radical (OH) (99%), superoxide anion (O2−) (94%), chelator (54%), and antimicrobial product (15 mm). The osmolarity conditions directly affected the capacity of the strain to biodegrade IL and the subsequently produced EPS. Furthermore, the EPS produced at 0.6 M has potential for environmental applications, such as the removal of hazardous materials by emulsification, whilst resulting in positive health effects such as antioxidant activity and non-toxicity.A specific microorganism,  The (2-Hydroxyethyl) trimethylammonium bis(trifluoromethylsulfonyl)imide Iolitec 99% Choline NTFEscherichia coli (E. coli CECT 516), Staphylococcus aureus (S. aureus CECT 8753), Enterococcus faecalis , Pseudomonas aeruginosa (P. aeruginosa CECT 108), and Salmonella typhimurium (S. thyphimurium CECT 409) were obtained from Colección Española de Cultivos (Spain). API 20E was obtained from BioMerieux España S.A. The HeLa cell line was purchased from CLS . The UltraClean microbial DNA isolation kit was obtained from MO BIO Labs., Inc. , and DNA purification JetQuick kit from Genomed .Trypticase soya agar (TSA), Luria–Bertani (LB), Dulbecco’s modified Eagle’s medium (DMEM) fetal bovine serum (FBS), L-glutamine, penicillin, streptomycin, catalase kit, and MTT  kit were obtained from Sigma-Aldrich . 2HPO4 0.5, KH2PO4 0.04, NaCl 0.1, CaCl2 2H2O 0.002, (NH4) 2SO4 0.2, MgSO4 7H2O 0.02, and FeSO4 0.001, and supplemented with glucose and Choline NTF2 compound as a carbon source at the concentrations of 4 g/L and 50 mg/L, respectively. The media were exposed in the Universidad Autónoma de Madrid (Spain), with orientation 40°32′32.8″ N/3°41′31.0″ W for 72 h. Every 24 h, samples of 100 µL were taken and cultured in trypticase soya agar (TSA) and grown at 30 °C to isolate microorganisms. After the preliminary morphological and biochemical characterization, as used by Abrusci et al. [https://blast.ncbi.nlm.nih.gov/Blast.cgi). The selected sequences were aligned with CLUSTAL X [A medium was prepared as described in Abrusci et al.  (MGM), g2 produced in the biodegradation at time, t, and the theoretical amount of carbon dioxide. Formula (1):The aerobic biodegradation of the compound as a carbon source was prepared following the method described in 550 nm). Microbial cells were evaluated as a colony-forming unit (CFU) by different dilution plating incubated at 30 °C for 72 h with TSA agar medium. A Thermo Orion pH Meter  was used to determine the pH values during a period of 72 h [2) was determined by high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) using an Agilent Technologies 1100 series—6410B (TQ). An ACE Excel 3 C18-Amide column as a stationary phase was used with a mobile phase of 0.1% formic acid in water. The flow rate was 0.2 mL/min. The temperature for analysis was set at 40 °C. Three independent assays were performed.Bacterial growth was monitored with a spectrophotometer Biowave II+ (Biochrom Ltd.) to an optical density of 550 nm (OD of 72 h . The bio7 cells/mL concentration) was inoculated into flasks of 1000 mL capacity filled with 100 mL of this medium. The flasks were incubated at 30 °C and 110 rpm for 72 h, at which time the stationary phase was reached. Three independent assays were performed. The cultures obtained from the uam8 strain were centrifuged at 13,154× g for 30 min at 4 °C Duppont—RC5 [The strain was taken from the stock culture and inoculated in a trypticase soya agar medium (TSA). The plates were incubated at 30 °C for 24 h, after which the strain was transferred directly from the plate into flasks of 100 mL capacity filled with 20 mL of minimal growth medium prepared according to the method described in pont—RC5 . The EPS2 over the uam8 strain [TEM studies were undertaken to study the influence of Choline NTF8 strain . The spe2. The micrographs were obtained using a Philips XL 30 scanning electron microscope operating in a conventional high-vacuum mode at an accelerating voltage of 25 kV. Previously, EPS was coated with a 3 nm thick gold/palladium layer [Scanning electronic microscopy (SEM) studies were undertaken to investigate the exopolymers produced by the uam8 strain both with and without Choline NTFum layer .The molecular weight of EPS was obtained by matrix-assisted laser desorption/ionization/time-of-flight (MALDI TOF/TOF) analyzer equipped with a Nd:YAG 355-nm laser  as described previously . Mass spGas chromatography  coupled with a mass spectrometry detector (GC-MS) was used to determine monosaccharides following the procedure described in the literature . EPS wasThe IR spectra of EPS for the determination of the structural functional groups  were obtv/v. The tubes were stirred in a vortex at 2400 rpm for 2 min. After 24, 168 h, the emulsification index  was determined as follows by Formula (2):The emulsifying capabilities of the exopolymers was evaluated using the procedure described in . The ass2−•) were evaluated as indicators of the free radical scavenging activities of the EPS at different concentrations . The tests were carried out following the calculations and the procedure described in Sánchez-León et al. [Nonenzymatic antioxidants assays for 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH•), hydroxyl radical (•OH), and superoxide anion , and H2O2 . The O2 assay was effected with different concentrations of EPS (0.3 mL) which were mixed with 2.6 mL of phosphate buffer  and 90 μL of pyrogallol (3 mM), dissolved in HCl (10 mM). Absorbances were measured using a FLUOstar Omega spectrophotometer (DPPH/OD525 nm), (OH/OD510 nm) and (O2−/OD325 nm). Ascorbic acid (Vc) was used as a positive control.The OH assay was performed with the EPS diluted to various concentrations (40 μL) which were mixed with 40 μL of 9 mM ethanol-salicylic acid solution, FeSO1 represents the reaction mixture, A2 refers to the reaction mixture without DPPH, and A0 denotes the reaction mixture with DPPH and without EPS.Formula (3) was used to determine the percentage of radical-scavenging activity for DPPH, where A1 represents the reaction mixture, A2 refers to the reaction mixture without salicylic acid, and A0 denotes the reaction mixture with salicylic acid and without EPS.Formula (3) was used to determine the percentage of radical-scavenging activity for OH, where A2−•), where A0 and A10 represent the reaction mixture at 0 and 10 min, respectively; C0 and C10 represent the reaction mixture without pyrogallol at 0 and 10 min, respectively.Formula (4) was used to determine the percentage of superoxide scavenging activity (O5 cells/mL). The OD590 nm (FLUOstar Omega spectrophotometer) was recorded using a microplate reader.Toxicity assay was determined using the MTT  test (Sigma Aldrich) on HeLa cells. The toxicity of the EPS was tested at different concentrations (0–400 μg/mL) for 24 h, by the reduction of the MTT reagent to formazan as described by Perez-Blanco et al. . HeLa ce2O2 on HeLa cells was based on the procedure described by Huang-lin et al. [× 104 cells per well for 24 h. The medium was removed and replaced with 100 μL of H2O2 (0.25–2 mM) for 1 h at 37 °C, which was removed after this time. Cell viability was measured using the MTT method as described in The methodology used to measure the damage of the non-radical Hn et al. . For thi1 represents the absorbance of HeLa cells treated with H2O2 and the MTT solution, while A2 represents the absorbance of HeLa cells that were not subjected to any treatment with the MTT solution.HeLa cell viability was computed using Formula (5):2O2 was evaluated following the method described by Huang-lin et al. [× 104 cells per well for 24 h. DMEM solutions were removed and replaced by EPS diluted in DMEM at different concentrations (25–400 μg/mL) for 1 h. They were removed and 2 mM H2O2 was added and incubated for 1 h. The MTT method described in The ability of EPS to protect HeLa cells against the non-radical Hn et al. . For thi1 refers to cells that were treated with both H2O2 and EPS, and subsequently exposed to the MTT solution; and A2 refers to cells that did not receive any treatment and were exposed to the MTT solution.HeLa cell viability was calculated with Formula (6):2O2 was carried out. All images were captured with a 10x objective and analyzed with Olympus DP-23 software .Cell morphology was determined using an Olympus CKX53 fluorescence microscope with a DAPI filter. The same procedure as that performed with the measurement of the non-radical H× 105 HeLa cells were seeded in 24-well plates and incubated for 24 h. The medium was subsequently discarded, and the cells were washed with phosphate-buffered saline (PBS). Then, cells were treated with EPS at various concentrations  that were dissolved in PBS and incubated for 1 h at 37 °C. Once the treatment had elapsed, the supernatants were collected and used for the determination of CAT. This activity was determined using a test kit  according to the protocol.The protective capacity of the EPS against oxidative stress was investigated using HeLa cells . A total2 (2 mM), 0.2 mL of ferrozine (5 mM), and 2.75 mL of Milli-Q water and incubated at room temperature for 10 min. Ethylenediaminetetraacetic acid (EDTA) was used as a positive control. An OD562 nm (FLUOstar Omega spectrophotometer) was used.The chelating capacity of metals was carried out following the calculations and the operational procedure tested by Huang-lin et al.  against The chelating ability on ferrous ion was calculated according to Formula (7):0 = OD562nm of the deionized water.A1 = OD562nm of the reaction mixture.A2 = OD562nm of the reaction mixture but without FeCl2.AE. coli CECT 516, Staphylococcus aureus CECT 8753, Enterococcus faecalis CECT 184, Pseudomonas aeruginosa CECT 108, and Salmonella typhimurium CECT 409 strains were used as indicator microorganisms to evaluate the antibacterial activity of EPS. The plates prepared with the Luria–Bertani (LB) medium were inoculated with 100 μL of bacterial suspension (107 CFU/mL). Wells (4 mm in diameter) were made, where 60 mL of EPS was deposited at different concentrations (1–10 mg/mL). The LB agar plates were kept at 4 °C for 1 h and incubated at 30 °C for 24 h. The antimicrobial activity was determined by measuring the diameter of the inhibition zone around the wells.Agar well diffusion assay was used to evaluate the antibacterial activity of EPS following the method reported by Rajoka et al. . The E. ® Inc., Chicago, IL, USA). p < 0.05 was considered statistically significant.Analysis of variance test (ANOVA) was performed to undertake the statistical comparisons by using the Statistical Package for the Social Sciences version 21   (2/2.91 ± 0.04) (2/4.168 ± 0.06) (2/2.554 ± 0.04) (2/9.43 ± 0.2), (2/9.47 ± 0.3), (2/7.98 ± 0.17), (2 (pH 7 to just 6.4))  (2/900 mg L−1) (2/1100 mg L−1)  [Kocuria rosea ZJUQH (2008 mg L−1) [Lactobacillus confuses  [2 (2 (2 and 1.0 M Choline NTF2) (2), its biodegradation was independent of the choline cation. Under the conditions of 0.6 M Choline NTF2, 42% of the anion was residual, whereas for 1.0 M Choline NTF2, only 25% of the anion was residual. These results confirmed a larger utilization by P. agglomerans uam8 of the anion (NTF2) at a higher osmolarity. This could be because the high concentration of sodium chloride favored the solubility of the anion and its bioavailability. Gram-negative bacteria  have been effective in biodegrading toxic compounds such as phospohonium-based ILs [The experiments  were con2) a in diff ± 0.06) b and the ± 0.04) c in the  ± 0.06) e with re ± 0.06) d. With a ± 0.04) f. In the ± 0.2), c; (0.6 M ± 0.3), e; and (1± 0.17), f. For 0.st 6.4)) e, no acucidified f. pH wou mg L−1) e, compar mg L−1) c and (1. mg L−1) f. In thentoea sp , high le mg L−1) , Kocuria mg L−1) , and Lac06) 2/2.5 ± 0.04 (.06) 2/2.4 ± 0.04  L−1) [2 e and 1.01) [2 (2 f. On thene NTF2) g, but inuses 25,9 mg L−1 [ased ILs  and chloased ILs .2, in the first 24 h. With 0.6 M Control , which made for more interesting environmental conditions.The EPS produced from the biodegradation of 0.6 M Choline NTF2 presented an isolated peak, 2, and 1.0 M Choline NTF2) also presented this characteristic. This highlighted that the obtained polymers were pure. This is an important feature of bacterial exopolysaccharides [2 showed a molecular weight of 5221.5 Da.  [The EPS produced by s strain  showed a. (BM39) ,70 produPantoea sp. have also been described in the EPSs of the genus Pantoea YU16-S3 [Less common monomers such as the heteropolysaccharide of  YU16-S3  with glu2 is shown in 2, 0.6 M Control and 1.0 M Choline NTF2), shown in −1 and 2928 cm−1 indicated O-H stretching vibration and C-H stretching vibration, respectively [−1 and 1401 cm−1 was attributed to the ionic carboxyl group (COO-) and C-H bond vibration, respectively [−1 could be attributed to the pyranose ring [−1 region indicated C–O–C stretching vibration and the peak at 1018 cm−1 was due to C–O–H stretching vibration [Pantoea agglomerans [Pantoea alhagi NX-11 [FT-IR spectroscopy is an analytical technique that allows the identification of functional groups from absorption peaks . The FT-ectively ,74. The ectively . Accordiose ring . Absorptibration . FT-IR slomerans , and in gi NX-11 .2 medium) experienced an initial weight loss of 11.04% due to moisture content at approximately 180 °C. This is due to the carboxyl groups of the polysaccharide, which have an affinity for water molecules. Subsequently, a weight loss of around 61% occurred at 365 °C, which was attributed to the degradation of the sample itself. Furthermore, the DSC analysis (2 was lower than the purified EPS from Pantoea sp. BCCS 001 GH (weight loss (69%) at 318 °C) [Pantoea sp. YU16-S3 (weight loss (20%) at 200 °C) [One of the important factors to take into account for an efficient environmental application of bacterial exopolysaccharides is the study of their thermal stability. The TGA analysis showed b that thanalysis c of this 318 °C) ,79 and P 200 °C) , which c2 medium) emulsification study was carried out at different times (24 h (E24) and 168 h (E168)), concentrations , and at pH 7.0, and with both vegetable oils  and hydrocarbons .The emulsifying activity of the exopolysaccharides is crucial for its environmental application. This activity is determined by measuring the retention of the emulsion of vegetable oils or hydrocarbons in water after a certain time period. The EPS (0.6 M Choline NTFp < 0.05), high emulsification values were given for olive oil ((E24) 86.4%), sunflower oil ((E24) 56%), and coconut oil ((E24) 90%). However, for 168 h, the activity was only maintained for coconut oil ((E168) 90%). Pantoea sp. [The results indicated that EPS was very efficient at 2 mg/mL compared with the results obtained by toea sp. , which rtoea sp. .In order to see the environmental advantages of using natural polymers over commercial compounds, the EPS was also tested with three commercial emulsifiers , p < 0.05) activity for diesel ((E24) 60%) compared with commercial emulsifiers (Triton X-100 ((E24) 50%), Tween 20 ((E24) 50%) and SDS ((E24) 37.5%)). However, for a concentration of 2 mg, the emulsifying activity of the EPS was significantly (p < 0.05) improved. This increased for olive oil ((E24) 86.4%), sunflower ((E24) 56.5%), coconut ((E24) 90%), diesel ((E24) 62%), and hexane (E24) 60%), compared with commercial emulsifiers. For the latter, only Tween 20 obtained a very slight improvement for olive oil ((E24) 90%), only 4% more when compared with EPS.The results indicated that at a concentration of 1 mg, the EPS only presented a better emulsifying , at a concentration of 1 mg. This is shown in 2 are compared. The results indicated that for the vegetable oils and hydrocarbons, the EPSs of 0.2 M, and 1.0 M Choline NTF2 did not have a notable emulsifying activity, with values that did not reach 20% in most cases. However, the EPS obtained from 0.6 M Choline NTF2, presented 47% higher activity than the other EPSs for vegetable oils, and even higher in hydrocarbons, with a 60% increase in emulsifying activity for 0.6 M Choline NTF2. This indicated that the morphology of the EPS had a decisive role in this activity [The emulsifying activity study was also carried out with the other EPSs produced from biodegrading different concentrations of Choline NTFactivity .2−)) have been used as instruments to explore the antioxidant activity of exopolysaccharides [Nonenzymatic antioxidant assays as shown in charides .2 was tested in the range of 0.1 to 10 mg/mL, for all assays. For radical scavenging activity DPPH, 2-), shown in Tetragenococcus halophilus SNTH-8 with DPPH and hydroxyl radicals activity of 63.53% and 50.19%, respectively, at 12 mg/mL [Rhodococcus qingshengii QDR4-2 resulted in DPPH, hydroxyl and superoxide radicals of 49.09 ± 1.54%, 30.24 ± 2.82%, and 39.18 ± 2.70%, respectively, at 3 mg/mL [The antioxidant capacity of the EPS obtained from 0.6 M Choline NTF12 mg/mL . Rhodoco 3 mg/mL . These r2 medium), with all the tested concentrations, maintained cell viability, above 90% and very close to control p < 0.05). EPS did not cause damage to the tested cell line. Studies carried out in this same genus revealed that for the EPS of Pantoea sp. YU16-S3 [2, in a wide range of concentrations, gives EPS a great advantage for environmental and public health applications.The cytotoxicity results showed that EPS  had occurred [Cell viability and morphological study was carried out to evaluate the possible effect of a non- radical Hoccurred .2O2-induced damage with a viability of more than 70%. This could be directly related to the morphology of the EPS (p < 0.05). The increase in the CAT activity by the EPS allowed the prevention of the formation of hydroxyl radicals and stimulated the enzymatic defence of the cells [Lactobacillus kimchi SR8 (8.78 ± 0.31 U/mL at 50 mg/Kg) [Lactococcus lactis (8.19 ± 1.36 U/mL at 40 mg/Kg) [On the other hand, cells previously incubated with EPS exhibited a morphology typical of control cells, with good development of filipodia,  the EPS , where ihe cells . The abi0 mg/Kg)  and in o0 mg/Kg) . This re2 presented an iron chelation  [EPS obtained from the biodegradation of 0.6 M Choline NTFhelation a,b capacto 30.5% . The che0.5% [−) . FurtherStaphylococcus aureus CECT 8753 (15.0 ± 0.4 mm), On the other hand, the results of the antimicrobial activity of EPS showed a strong zone of inhibition with a halo over 15 mm where the bacterial growth was inhibited for one of the tested strains. EPS was able to prevent the growth of Pantoea. agglomerans uam8 isolated from ionic liquid Choline NTF2 is presented as a good candidate for its biodegradation under saline stress conditions. This study provides valuable information on the behavior of the exopolysaccharides produced from the biodegradation of an ionic liquid. Varying the osmolarity conditions of the medium had an effect on the morphology of the produced exopolysaccharides whilst the chemical composition remained unchanged. The exopolysaccharide produced in 0.6 M conditions had the highest emulsifying activity for natural oils and hydrocarbons compared with those produced in different osmolarities. The 0.6 M Choline NTF2 exopolysaccharide did not present cytotoxicity and had antioxidant capabilities. Furthermore, this EPS had other important biotechnological advantages, such as chelating and antimicrobial properties. The role of these polymers produced by bacteria in biodegradation processes opens up an important avenue of research, to glimpse how they can influence the environment where they are produced, and their direct relationship with the environment.


Understanding pharmacokinetics (PK) and pharmacodynamics (PD) of the sustained-release liothyronine (SR-T3) is of paramount importance to design therapeutic regimens that are able to simulate normal thyroid hormone secretion while avoiding excursions in the T3 serum concentration. Here, we designed a parallel randomized clinical trial to characterize the PK and PD of the combined preparations of LT4 + SR-T3 in hypothyroid patients.0 − 24 were calculated at the last visit.Radioiodine-treated hypothyroid patients over 20 years of age, who attained euthyroidism with LT4 monotherapy were recruited from the Endocrine Clinic in Tehran. The patients were allocated to two intervention groups of group A: 9 µg SR-T3 plus 68.5 μg LT4 (ratio 1:7.5) and group B: 12 µg SR-T3 plus 60 µg LT4 (ratio 1:5), and a control group with LT4 monotherapy. For PD study, thyroid hormone profile was evaluated at 8 and 12 weeks intervals after intervention. To assess PK properties of SR-T3, T3-Cmax, T3-Tmax and AUC0 − 24 in the combined sustained-release preparation were 4.38 ± 1.1 h., 101.0 ± 5.7 ng/dl and 2257 ± 110 ng.h/L, respectively which were significantly different from the control group.Serum T4 and FT4 concentrations decreased in the intervention groups after 3 months. No significant difference was observed in serum T3 and FT3 concentrations before and after intervention. Serum T3/T4 ratio increased significantly in the intervention groups after intervention, with the highest increase in group B from 8.6 ± 2.03 at baseline to 12.2 ± 1.6. Comparison of trial groups at follow-up showed no differences in serum TSH, T4, T3 and T3/T4 concentrations among different groups. During 24 h, minimal variation in serum T3 concentration was observed in group B with mean ∆T3 of 15.4 ± 10.5 ng/dl. T3-Tmax, T3-Cmax and AUCCombined treatment with a single dose of SR-T3 plus LT4 is associated with increased serum T3/T4 ratio and minimal excursions in serum T3 concentration during 24 h; however, it was not significantly different from the control group. To incorporate sustained-release T3 in the management of hypothyroidism, a higher ratio of SR-T3 to LT4 than that of the previously recommended by the international organizations is suggested.IRCT20100922004794N13. https://www.irct.ir/search/result?query=IRCT20100922004794N13. Registration date: 08/12/2021.The online version contains supplementary material available at 10.1186/s12902-023-01434-y. Levothyroxine monotherapy is considered the standard of care for hypothyroidism by professional organizations, whereby the patients achieve the state of euthyroidism through the normalization of TSH, a reliable proxy of euthyroidism . In euthClinically, a sizable minority of patients adequately treated with LT4 complain of residual symptoms –10. HighThe above-mentioned evidence led to the growing interest in combination therapy with LT4 plus liothyronine(LT3); however, multiple randomized controlled clinical trials addressing objective and subjective clinical outcomes did not provide conclusive results , 13. TheTo design clinical trials with a greater likelihood of indicating efficacy, the American, European, and British thyroid associations have recently published a consensus statement to promote the development of future clinical trials . The worThe study was conducted at the Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran, from January 1, 2022 to 31 June, 2022. Radioiodine-treated hypothyroid patients over 20 years of age, who attained euthyroid status with LT4 monotherapy and serum TSH concentration of 0.5-5 mU/L were recruited from the Endocrine Clinic and referred to the Research Institute for Endocrine Sciences (RIES). The patients with pregnancy, chronic kidney or liver disease, congestive heart failure, cancer, and those taking methimazole, propylthiouracil (PTU), tamoxifen, estrogen, progesterone, and corticosteroids were excluded. Dorsa Pharmaceutical Company and Tavan Institute formulated sustained-release tablets with 7.5, 9 and 12 µg potency.A parallel randomized controlled clinical trial was applied. Initially, the patients were allocated to the three intervention and one control groups. Intervention groups received three different combinations of LT4 + LT3 yielding ratios of 1:10 (75 µg LT4 + 7.5 µg SR-T3), 1:7.5(9 µg SR-T3 + 68.5 µg LT4) and 1:5 (12 µg SR-T3 + 60 µg LT4). As during follow-up, serum TSH concentration significantly increased in the patients who received 75 µg LT4 + 7.5 µg SR-T3, this group were excluded from the study. Therefore, trial groups in this survey consisted of group A with a daily intake of 9 µg SR-T3 plus 68.5 µg LT4 (ratio 1:7.5), group B with a daily intake of 12 µg SR-T3 plus 60 µg LT4 (ratio 1:5) and a control group with LT4 monotherapy (group C).Figure At the baseline visit, after taking informed written consent, blood pressure, weight, and height were measured. The trained staff filled the questionnaire regarding demographic and thyroid symptoms. Patients were allocated to three intervention groups and one control group using stratified randomization. Two stratifications were made based on gender. Under each sex subgroup, patients were randomly assigned to three treatment groups using the random table allocation.After implementation of randomization and specific coding, the subjects were assigned to two intervention and one control groups using allocation concealment. The standard method of ensuring allocation concealment was sequentially numbered. The staff member not involved in the trial was instructed to keep the list private and only revealed a treatment allocation after receiving information showing that the patient is eligible and has consented to the trial. The patients and the investigators were kept from knowing who was assigned to which treatment. All groups received identical tablets in physical appearance, taste, and smell to fulfill blinding.th. and 12th. weeks. The patients were visited after 6 weeks to measure serum TSH and assess therapy adherence. To ensure compliance with drug therapy, the responsible person checked the drug package and counted the number of pill intakes by direct questioning in two weeks intervals by phone call and pill counting at the last visit. Drug dosage was adjusted to maintain serum TSH concentration within 0.5-5 mU/L. At baseline and two follow-ups 8 and 12 weeks later, body weight and blood pressure were measured and venous blood samples were collected after a 12-hour fasting. The serum samples maintained in microcentrifuge tubes at -20ºC to measure serum TSH, total T3, total T4, free T4, and free T3. After fasting blood sampling at the last visit, the patients received the allocated treatment at 8 am, and the blood sampling was taken 6 times during a 24 h period at 9 am, 10 am and 12 am, 14–16 pm, and the next day at 8 am, to measure serum TSH, T4, free T4, T3, and free T3 concentrations in all samples to calculate T3/T4 ratio, time for T3 to reach maximum concentration (T3-Tmax), maximum serum concentration of T3 (T3-Cmax) and the area under the plasma concentration time curve over 24 h (AUC0 − 24).The participants were evaluated at baseline and two consequent follow-ups on the 8https://www.irct.ir/search/result?query=IRCT20100922004794N13”. This study was conducted in concordance with the Helsinki Declaration ethical principles, and all procedures on the study participants were approved by the National Research Council of the Islamic Republic of Iran (IR.SBMU.ENDOCRINE.REC.1400.031), the Human Research Review Committee of the Endocrine Research Center, Shahid Beheshti University, Tehran, Iran. Trial participants signed informed consent forms at baseline, and their personal information would remain strictly confidential.Primary outcome was set to find the best LT4 + SRT3 combination to maintain normal serum concentration of TSH, T4, T3, free T4, free T3 and T3/T4 ratio. Secondary outcome was to find the best LT4 + SRT3 combination which provide most favorable T3- Cmax, T3-Tmax, and AUC and patient preferences. The study design was registered by the Iranian Registry of Clinical Trials, IRCT20100922004794N13 (dated: 08/12/2021) available at: “The formulation of liothyronine sodium sustained-release tablets was developed by Noor Research & Educational Institute (TAVAN). Using Avicel PH-102 and HPMC in formulation of tablets sustained the drug release of T3 for over 10 h. The in vitro dissolution study was carried out in 500 ml, pH 6.8 phosphate buffer using United States Pharmacopeia Apparatus I, basket method, at a speed of 100 rpm and temperature of 37 C. Levothyroxine sodium (LT4) plus SR-T3 was administered based on the previous levothyroxine dosage which was at a weight-based dose of ~ 1.6 mcg/kg. Each combination was equal to 100 µg Levothyroxine.The 2012 ETA, suggested L-T4 + L-T3 combination treatment with a dose ratio between 13:1 and 20:1 by weight , howeverAll laboratory measurements were determined on -20ºC stored serum samples. The thyroid function tests including TSH, T3, T4, and free hormones were measured by electrochemiluminescence immunoassay method using Roche Diagnostics kits on the Cobas e- 411 automated analyzer . The sensitivity of TSH, T3, T4, FT3, and FT4 assays were 0.005 µIU/mL, 0.195 ng/mL, 0.420 µg/dL, 0.6 pmol/L, and 0.5 pmol/L, respectively. Lyophilized quality control materials  in three different concentrations were used to monitor accuracy of measurements. The intra- and inter-assay coefficients of variation (CVs) were respectively 2.8% and 3.1%.for TSH, 2.3% and 3.0% for T3, 1.7% and 2.6% for T4, 2.1% and 3.9% for FT3, and 1.0% and 2.9% for FT4. Mean values and the reference ranges of thyroid hormones obtained from the Tehran Thyroid Study (TTS) were TSH  and FT4  . Normal All analyzes were performed using STATA software version 14. Quantitative variables were summarized using mean, standard deviation and median. Qualitative variables were reported in frequency and percentage. Baseline characteristics between the three groups were tested using ANOVA. The comparison within groups was performed by two-tailed paired Student’s t-test or with the Wilcoxon rank t test. Chi-square person test were used to compare the frequencies between classification variables. The difference between the three groups was tested using the ANOVA test and Benferoni post hoc test. P value less than 0.05 was considered statistically significant. The maximum observed concentration (C-max) and the time at which this occurred (T-max) were calculated. The area under the plasma concentration–time curves from zero to the last sampling time AUC0-24 was calculated according to the trapezoidal rule using the actual times of measurements. Analysis of covariance (ANCOVA) was performed on AUC, Tmax and Cmax using STATA software version 14. ANOVA results revealed the effect of the sources of variation like sequence, subjects in a sequence, period and formulation on the bioequivalence data. The results of ANCOVA were calculated at 5% level of significance.2. There was no difference in age, sex, BMI, and serum concentrations of thyroid hormones among trial groups at baseline.Nineteen hypothyroid patients adhered to the allocated therapy until the end of the study. The baseline characteristics of the intervention and control groups were presented in Table Two combined preparations of LT4 + SR-T3 did not have enough potency to attain normal TSH values; however, after increasing the dosage, serum TSH concentrations reached values within the normal ranges. Table 0 − 24, T-max, and C-max for T3, T4 and T3/T4 ratio in the intervention and control groups are presented in Table The results of the serum concentrations of TSH, total T4, total T3, and T3/T4 ratio in the intervention and control groups in serial measurements for the PK study at last follow-up are presented in Fig. At the end of the study, among 9 patients who underwent combined therapy, three preferred LT4 monotherapy, one preferred combination therapy and five patients reported no preference.Decreased serum T4 concentrations and increased serum T3 concentration in treatment with 12 µg SR-T3 plus 60 µg LT4 led to a significant increase in serum T3/T4 ratio after intervention; however, serum T3/T4 ratio was lower than the normal serum T3/T4 ratio in euthyroid subjects (~ 1:14–15) and no difference was observed in serum T3, T4 and T3/T4 ratio after intervention between CBT (combination therapy) and LT4 monotherapy groups. Single dose administration of LT4 + SR-T3 was associated with more improved T3-derived Tmax(~ 4–5 h) and Cmax (~ 105 ng/dl) while serum T3 concentration remained within normal ranges with no fluctuation for 24 h after achieving maximum level.The only study by Hennemann et al. with randomized crossover design on fifteen patients (mean age of 50 y.) with primary hypothyroidism showed that T4 plus sustained-release T3 (1:20 ratio) improved serum TSH, T4, and T3 concentrations and the T4/T3 ratio compared to treatment with LT4 alone, while non-physiologic T3 peaks were avoided . In the Although serum TSH values are comparable and within normal ranges in LT4-treated patients, LT4 as an exogenous pro-drug cannot simulate serum thyroid hormone profile and circadian rhythm in the native euthyroid state.Serum TSH values in the previous clinical trials varied in patients under LT4 + LT3 combination therapy . In this0− 48, was proportional to the given dosage and the authors suggested that adequate administration of oral T3-S might consider a more suitable treatment for hypothyroidism [The studies on the PK properties of sustained-release liothyronine are more coordinated; however, apart from the current study, there is only one other study which assessed PK of SR-T3 in combined preparation with LT4 . In the yroidism . Howeveryroidism .The current study is strengthened by including hypothyroid patients under LT4 monotherapy due to radioactive iodine intake for treating Graves’ Disease with no or negligible residual secretion of endogenous T3 and T4 and using the modern technique for measurement of thyroid hormones. Evaluation of the two combinations of the LT4 + SR-T3 enabled us to choose a more appropriate combination based on the participant’s body weight and not a fixed dosage such as the previous studies. Also, to achieve equilibrium in thyroid hormones, the PK study was performed after three months of treatment and not by a single-dose drug intake. However, the study is limited by evaluating PK study only within 24 h and the low potency of the LT4 component in the combined therapy which made us to increase the total dosage to restore euthyroidism.In conclusion, combined treatment with levothyroxine plus sustained-release liothyronine in hypothyroid patients with ratios of 1:5 might restore euthyroidism in hypothyroid patients yielding more improved T3/T4 serum ratio and T3-derived pharmakikinetics,while keeping serum T3 concentrations relatively stable within 24 h with minimal excursions. Single dose administration of LT4 + SR-T3 maintains serum T3 concentration approximate to normal ranges up to 24 h with minimal variation in serum T3 not exceeding 25 ng/dl indicates longer duration of the SR-T3 release in the intestine compared to other preparations. Although maximum serum T3 concentration (Tmax) achieves during 3–4 h, there is minimal difference in serum T3 concentrations at serial measurements during 24 h. Significant increase in serum T3/T4 ratio in combined therapy which is still lower than mean values in euthyroid individuals, could be translated into the assumption that administration of higher ratios of SR-T3 in combination therapy should be provided to achieve the physiological ratio of T3/T4 in treated hypothyroid patients. After attainment of the best appropriate preparation, designing and conducting appropriate randomized controlled clinical trials to compare the efficacy outcomes of combination therapy with sustained- release liothyronine are highly warranted to ensure sustainable delivery of T4 and T3 to thyroid hormone target issues.Below is the link to the electronic supplementary material.Supplementary Material 1


Complement component 3a and its receptor (C3a/C3aR) and the nucleotide-binding oligomerization domain-like receptor protein-3 (NLRP3) inflammasome contribute to epithelial-mesenchymal transition (EMT). However, the relationship between C3a/C3aR and the NLRP3 inflammasome in EMT remains unclear. This study aimed to elucidate the roles of C3a/C3aR and the NLRP3 inflammasome involved in TGF-β-induced EMT.Mouse renal tubular epithelial cells (TCMK-1) were exposed to C3a and TGF-β for 48 h. C3aR antagonist, MCC950, an inhibitor of the NLRP3 inflammasome and PD98059, an inhibitor of ERK signaling, were respectively applied to pretreat the cells at 30 min before C3a and TGF-β administration.The cells were collected for western blot, immunofluorescence staining and ELISA. Unilateral ureteral obstruction (UUO) models were established using male C57BL/6 wild-type (WT) mice and age-matched C3aR-deficient mice. MCC950 was intraperitoneally injected in UUO mice. Kidney samples were collected for immunohistochemistry staining.In vitro, C3a synergized with TGF-β to promote EMT and the activation of the NLRP3 inflammasome. Inhibition of C3aR attenuated EMT and the activation of the NLRP3 inflammasome. Inhibition of the NLRP3 inflammasome alleviated EMT but didn’t affect the expression of C3aR. Inhibition of ERK signaling inhibited the activation of the NLRP3 inflammasome. In vivo, the expression of IL-1β was significantly higher in UUO mice compared to the sham-operated mice. C3aR deficiency and inhibition of the NLRP3 Inflammasome contributed to decreased IL-1β in UUO mice.Our data revealed that C3a/C3aR synergies with TGF-β to activate the NLRP3 inflammasome to promote epithelial-mesenchymal transition of renal tubular epithelial cells through ERK signaling, and the way in which C3aR activates the inflammasome is to promote the assembly of the NLRP3 inflammasome. Chronic kidney disease (CKD), on account of its high morbidity and mortality, has become a global health burden. Globally, the prevalence of CKD was 9.1% and 1.2 million people died from CKD in 2017 . EpithelComplement C3 is a crucial component of the complement system which contributes to a variety of tissue injuries. The smaller fragment of C3, known as C3a, has been proved to play a vital role in the pathogenesis of several kidney diseases by binding to its receptor (C3aR) . Our preThe nucleotide-binding oligomerization domain-like receptor protein-3 inflammasome (NLRP3 inflammasome) is a protein oligomeric complex composed of NLRP3, apoptosis associated speck-like protein (ASC), and pro-Caspase-1. Once the NLRP3 inflammasome is activated, pro-Caspase-1 undergoes cleavage to form active Caspase-1.This active Caspase-1 then regulates the maturation of IL-1β and IL-18, leading to inflammation and fibrosis . AdditioAlthough both C3a/C3aR and the NLRP3 inflammasome contribute to EMT, the relationship between C3a/C3aR and the NLRP3 inflammasome in EMT remains unclear. Recently, we proposed that C3aR aggravated renal interstitial fibrosis by regulating the activation of the NLRP3 inflammasome (particularly regulating the assembly of the inflammasome) in the unilateral ureteral obstruction (UUO) model . We alsoMouse renal tubular epithelial cells  were purchased from American type culture collection  and were cultivated in MEM/EBSS medium  with 10% fetal bovine serum (FBS). 24 h prior to treatment, TCMK-1 cells were starved in MEM/EBSS medium containing 0.5% FBS, followed by exposure to 1 μg/ml C3a  and 10 ng/ml TGF-β  for 48 h. 20 μM C3aRa , 10 μM MCC950  and 20 μM PD98059  were respectively applied to pretreat TCMK-1 cells at 30 min before C3a and TGF-β administration.Proteins from cultured TCMK-1 cells were extracted using RIPA buffer containing protease. Protein concentrations were quantified using a BCA protein assay kit. The protein samples were separated by 6–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane, followed by blocking with 5% skim milk for 1 h. Then, the membranes were incubated overnight at 4 °C with the following antibodies: C3aR , NLRP3 , Caspase 1–20 , p-ERK1/2 , ERK1/2 , and β-tubμlin  as an internal control. Next, the membranes were incubated with appropriate secondary antibodies for 1 h at room temperature. Specific bands were detected by enhanced chemiluminescence.The medium was removed and the cells were fixed for 40 min with 4% paraformaldehyde. Then the cells were stained according to standard immunofluorescence procedures. After incubation with anti-α-SMA  and anti-ZO-1 antibodies at 4 °C overnight, appropriate Alexa Fluor 488 secondary antibodies  were used for fluorescent labeling. The nuclei were stained with DAPI, and images were obtained using a fluorescence microscope . Image analysis was performed in ImageJ software.Protein level of IL-1β in supernatants of cell cultures were detected by ELISA Kits  according to the manufacturer’s instructions. The OD was measured by a multimode ELISA plate reader .The total number of mice used in the study was 60 and the number used in each group was 6. All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Fujian Medical University . The health status of the mice was monitored everyday. In the event of inter-mouse aggression, it was necessary to house them in separate cages. Euthanasia was conducted if the mice exhibited loss of tactile response, immobility of the eyes, severe weight loss (20% of body weight), dehydration (fur and eyes sinking), and other more severe abnormal behaviors. C57BL/6 mice with C3aR KO  and without C3aR KO [C57BL/6 wild-type (WT) mice] were purchased from GemPharmatech Co., Ltd. (China). CRISPR/Cas9 technology was used to knockout C3aR gene and generate chimera F0 mice. Positive F0 mice confirmed by PCR genotyping and sequence analysis were mated with wild-type C57BL/6 mice to generate F1 heterozygous mice. Positive F1 mice confirmed by PCR genotyping and sequence analysis were mated with each other to generate C3aR-KO homozygotes (C3aR KO mice). Animals were housed in a pathogen-free, temperature-controlled environment with a 12 h/12 h light/dark photocycle and had ad libitum access to food and tap water to avoid dehydration-induced hypovolemia. Male C3aR KO mice and age-matched C57BL/6 WT mice (8–12 weeks old) were used for experiments. Mice were anesthetized with ketamine (50 mg/kg body weight) and xylazine (100 mg/kg body weight), and then, a lateral incision on the back was created. UUO was achieved by double ligation of the renal pelvis and the upper 1/3 of the left ureter; this was followed by making a cut in the middle of the double ligation. Sham-operated mice, which underwent an identical procedure but without ureteric ligation, were used as sham control. Mice were euthanized at day 14 after the operation, and obstructed kidneys were collected.The NLRP3 inflammasome was inhibited in vivo by intraperitoneally injecting 20 mg/kg MCC950  in 200 µL of normal saline (NS) or vehicle (200 µL of NS) once daily from the day of operation to the day of euthanasia (UUO model).2O2. Sections were then incubated overnight at 4 °C with rabbit multiclonal antibodies against IL-1β , followed by incubation for 1 h at room temperature with appropriate secondary antibodies that were conjugated with horseradish peroxidase. Isotype-matched IgGs were used as a negative control. Immunohistological staining results were quantified using ImageJ software.The kidneys were fixed with 4% paraformaldehyde and embedded in paraffin. Then, 4-μm sections were prepared for immunohistochemistry staining. The paraffin-embedded sections were blocked with 3% bovine serum albumin  in phosphate-buffered saline (PBS). For staining involving horseradish peroxidase-conjugated secondary antibodies, endogenous peroxidase was blocked with Hpost-hoc assessments. Two-way ANOVA was used for the experiments invloving two factors. A value of P < 0.05 was considered statistically significant. All statistical analyses were calculated with SPSS 25.0.Data are expressed as means ± SD. Normality of distribution was tested using the Shapiro–Wilk test before analysis. Comparison between groups was performed by one-way ANOVA using the least significant difference test  or Dunnett’s T3 test  for Western blot showed that the expression of C3aR increased after TCMK-1 cells were stimulated by C3a and C3a combined with TGF-β, compared with the cells exposed to TGF-β alone  restrained the expression of C3aR induced by C3a combined with TGF-β in TCMK-1 cells . TGF-β, known as a canonical inducer of EMT, regulates transcription through both SMAD and non-SMAD signaling , 21. SMAThe complement system is a complex system consisting of over 30 types of soluble proteins, membrane-binding proteins, and complement receptors. It is present in serum and tissue fluid of human and animals. Upon activation, it exerts various biological effects including regulation of phagocytosis, cell lysis, inflammation mediation, immune regulation, and clearance of immune complexes. The complement system can be activated through three pathways: classical pathway, lectin pathway, and bypass pathway . During 2S blocks the activation of the NLRP3 inflammasome by down-regulating TGF-β1 signaling, thus improving vascular fibrosis [NLRP3 is a member of the nucleotide-binding oligomerized domain-like receptor family. The expression of NLRP3 increases when cells are exposed to pathogen-associated molecular patterns (PAMP) or damage-associated molecular pattern (DAMP) (priming stage) . Then apfibrosis . Neverthfibrosis . Our stufibrosis , 34. Whefibrosis . Our preIt has been reported that C3a/C3aR mediates the assembly of the NLRP3 inflammasome in human monocytes via ERK signaling . We hypoThere are some limitations to this study. Firstly, the role of C3a/C3aR in facilitating the NLRP3 inflammasome assembly through ERK signaling remains largely unexplored. Secondly, the mechanism by which NLRP3 induces EMT was not discussed. We hypothesize that IL-1β produced by the activation of the NLRP3 inflammasome may promote EMT. The activation of the NLRP3 inflammasome may also further enhance EMT promoted by TGF-β. We will explore the relationship between the NLRP3 inflammasome and TGF-β in the future study. Thridly, the relationship between C3aR and the NLRP3 inflammasome was not discussed in CKD specimens. We will collect renal biopsy samples of CKD patients to examine the expressions of C3aR and the NLRP3 inflammasome. Together with other nephropathy indicators such as proteinuria, blood pressure, blood C3 levels and renal pathology, we will further investigate the potential of C3aR and the NLRP3 inflammasome as diagnostic and prognostic markers of CKD.Our study demonstrated that C3a/C3aR synergies with TGF-β to activate the NLRP3 inflammasome to promote epithelial-mesenchymal transition of renal tubular epithelial cells through ERK signaling, and the way in which C3aR activates the inflammasome is to promote the assembly of the NLRP3 inflammasome. This will provide some new insights for the new biomarkers and therapeutic targets of epithelial-mesenchymal transition.