Despite clinical relevance of immunological activation due to histone leakage into the serum following cardiac surgery, long-term data describing their longitudinal dynamic are lacking. Therefore, this study examines the serum levels of histone 3 (tH3) and its modifications (H3K4me3 and H3K27ac) alongside immune system activation during the acute and convalescence phases of cardiac surgery.pre-op) and 24 h (t24hr), seven days (t7d), and three months (t3m) post-procedure to examine serum levels of tH3, H3K4me3, and H3K27ac. Serum heat shock protein-60 (HSP-60) was a surrogate of the cellular damage marker. Serum C-reactive protein (CRP) and interleukin 6 (IL-6) assessed smoldering inflammation. TNFα and IL-6 production by whole blood in response to lipopolysaccharide (LPS) evaluated immunological activation. Electronic medical records provided demographic, peri-operative, and clinical information. Paired longitudinal analyses were employed with data expressed as mean and standard deviation (X ± SD) or median and interquartile range , post-operative serum tH3 significantly (p > 0.0001) increased after heart surgery , remained elevated at 7 days , and at 3 months . Serum H3K27ac was elevated at 24 h  and seven days  as compared to baseline hours (H3K27acPre-op = 0.55 ± 0.54). Serum H3K4me3 was significantly diminished at three months . tH3 correlated significantly with the duration of anesthesia (r2 = 0.38). In contrast, HSP-60 normalized seven days after surgery. Peri-operative intake of acetaminophen, but no acetylsalicylic acid (ASA), acid, ketorolac or steroids, resulted in the significant depression of serum H3K4me3 at 24 h . CRP, but not IL-6, remained elevated at 3 months compared to pre-surgical levels and correlated with tH324hrs (r2 = 0.43), tH37d , H3K4me37d (r2 = 0.53), and H3K27ac7d (r2 = 0.49). Production of TNFα by whole blood in response to LPS was associated with serum tH324hrs (r2 = 0.67). Diminished H3K4me324hrs, H3K27ac24hrs, and H3K27ac3m, accompanied the emergence of liver failure.Compared to pre-operative levels (tH3We demonstrated a prolonged elevation in serum histone 3 three months after cardiac surgery. Furthermore, histone 3 modifications had a discrete time evolution indicating differential immune activation. Anesthesia, tissue damage, reperfusion, hypoxemia, thermal shock, and exposure to large artificial surfaces trigger a complex inflammatory reaction during cardiac surgery –4. InducPrior research has demonstrated elevated serum histone levels up to three days post-operatively , 23. HowHowever, there are no data on baseline levels of histones or their composite landscape in cardiac surgery patients despite several factors in histone milieu , 25, 26.DAMP-mediated immune activation can be mitigated by direct or indirect defensin mechanisms limiting histones' detrimental effect , 35–38. The evolution of peri-operative inflammation impacts the long-term resolution of the inflammation and outcomes of cardiac surgery or any critical care illness , 15, 33.This study investigated knowledge gaps regarding the long-term release of histones after cardiac surgery. We hypothesized that histone 3 (H3) would be released predominantly during the acute period after cardiac surgery, similar to other DAMP (HSP-60) , 22. ConA total of 59 patients were enrolled in this observational study. The University of Pennsylvania Institutional Review Board approved the study . All patients scheduled for non-emergent cardiac surgery were approached for consent. Exclusion criteria were < 18 years of age, emergent surgery, lack of consent, and transplant or immunosuppressed patients.Demographics and clinical data were obtained from electronic health records (EHR), including surgical, anesthesia, and peri-operative records. The Charlson Comorbidity Index (CCI) was calculated to assess chronic disease burden . Acute PPre-op). Subsequent blood procurements took place 24 h (t24hr) and seven days (t7d) post-surgery, with a final follow-up at three months (t3m).After consent was secured, patient blood was collected before the onset of the surgery . Blood was collected in heparinized tubes and stored at 4 °C for up to 2 h. Plasma was obtained after 5 min at a 1200xCirculating levels of tH3, H3K27ac, and H3K4me3 were measured using enzyme-linked immunosorbent assay (ELISA) kits  and read on BioTek Synergy H1  at 450 nm with 570 nm correction. CRP was also measured with an ELISA kit  and read on BioTek Synergy H1  as specified above. TNFα, IL-6, and HSP-60 were measured using a multiplex assay  and analyzed on 3D FlaxAmp .A total of 0.5 mL of whole blood obtained at all time points was stimulated with lipopolysaccharide (LPS) [50 ng/mL]  or left unstimulated for 18 h at 37 °C. Plasma was obtained after 5 min at a 1200xg spin, and supernatants were collected. TNFα and IL-6 were measured with ELISA kits  and read on BioTek Synergy H1 .t-test for two variables, while ANOVA was used for multiple comparisons. For nonparametric variables, median and interquartile ranges  were utilized with the U-Mann–Whitney test employed to compare such variables. r2 and ρ Spearman correlation coefficients were used to assess relationships for parametric and nonparametric variables, respectively. k-means clustering was employed where appropriate. Both-sided p-values less than 0.05 were considered statistically significant for all tests unless a specific null hypothesis was formulated. Statistical analyses were performed with Statistica 11.0  or Statistical Package for the Social Sciences v26 .Shapiro–Wilk W and K-S tests were used to test the normality of the distribution variables and assess the distribution of these variables. Parametric variables are expressed as mean and standard deviation (X ± SD) and compared using A total of 59 patients were enrolled in the study. Their demographic, clinical, and peri-operative characteristics are presented in Table over60 = 1.7 ± 2.22 versus tH3under60 = 4.1 ± 5.72; t ), post-operative serum tH3 significantly  increased after heart surgery , remained elevated at 7 days , and 3 months   to be significantly diminished at 3 months   and 7 days post-surgery (p = 0.0078), and three months (p = 0.028)  and seven days  as compared to baseline hours (H3K27acPre-op = 0.55 ± 0.54)  and tH37d , but not H3K4me3 or H3K27ac at any time points.When examining correlations between patient demographics and clinical characteristics with serum levels of tH3 and its modifications, age correlated with tH324hr and tH37d correlated highly with the duration of anesthesia  did not affect serum tH3, H3K4me3, or H3K27ac in the post-operative period (data not shown). When examining correlations between anesthesia, surgery, and peri-operative parameters with serum levels of tH3 and its modifications, the serum levels of tH3r2 = 0.46; p < 0.05) and packed red blood cells (PRBC)  used for resuscitation during anesthesia. H3K27ac at seven days correlated with the volume of PRBC . The serum level of H3K4me3 failed to demonstrate any difference between surgical parameters and their serum level at any time;Serum H3K27ac at 24 h correlated with the volume of crystalloids , although the correlation between the dose and the serum level of H3K4me3 was below statistical significance. Neither acetylsalicylic acid, ketorolac, or steroid intake affected tH3, H3K4me3, or H3K27ac levels at any studied time point (data not shown).Peri-operative intake of acetaminophen significantly decreased serum levels of H3K4me3 at 24 h .HSP-60 was elevated 24 h after surgery, but at 7 days and 3 months post-surgery, the levels drop to below pre-surgical values  , and H3K27ac7d (r2 = 0.49 p < 0.05). Cluster analysis revealed three distinct groups of patients 24 h post-surgery  had the highest serum levels of CRP, IL-6, and tH3. Cluster #2 (n = 40) and Cluster #3 (n = 10) both had lower levels of tH3 than Cluster #1, but their serum CRP levels were different, with Cluster #3 having higher levels than Cluster #2  contained patients with higher levels of tH3 and CRP than Cluster #2 patients (n = 19).When examining correlations between general inflammatory markers and serum levels of tH3 and its modifications, no significant correlations were observed between levels of serum IL-6 and tH3, H3K4me3, or H3K27ac. There were significant correlations between CRP24hr , but not with tH324h or H3K27ac24h. At 24 h post-surgery, the production of TNFα in LPS-stimulated blood obtained at the same time significantly correlated with serum levels of tH324h   Fig. . The pro24hrs  and the length of stay in the ICU correlated with H3K4me324hrs . There were no significant correlations between APACHE scores at admission to the ICU or 24 h or 48 h and any of the histones (data not shown).The length of stay in hospital correlated with tH3F), respiratory failure (RF) or acute renal failure (AKIF) as defined by MODS definitions did not affect the serum level of measured histones at any point . S. S52]. SWe demonstrated for the first time the presence of circulating histone three months after elective heart surgery among adult individuals. Previous data were limited to three days of follow-up and focused on children undergoing surgery for congenital heart disease .The initial presence of tH3 signifies the necrosis and apoptosis secondary to iatrogenic surgical insult , 21, 22.We demonstrated that both H3 and CRP have prolonged presences in serum after cardiac surgery. However, the clinical importance of this finding is unclear. IL-6, CRP, and tH3 coalesce along the initial cluster to demonstrate unique patterns early after surgery and late in convalescence. Considering the complex interplay between these factors, the deployment of clustering techniques revealed higher order data that otherwise would not be apparent. The complex interplay between these factors stems from multiple roles of CRP, IL-6, and tH3, which are very context and level dependent , 58, 59.We explored whether histone modification may be linked with the emergence of the immunological status in patients undergoing heart surgery , 47. PriSeveral medications exhibit frequently underappreciated epigenetic activities , 30. We In contrast to prior research, we did not demonstrate a correlation between elevated serum levels of histones and several features of clinical demise , 57, 73.We found that the emergence of liver failure was related to a decrease in the level of total histone 3, while prior studies demonstrated that liver failure was related to increased serum histone, but these studies examine liver injury as the primary source of the insult . In our The interesting clinical finding of this study is the perseverance of circulating histones well into recovery after surgery, which may contribute to the emergence or acceleration of atherosclerosis , 44. In This study's longitudinal design accounting for pre-operative baseline measurements is a strength. This longitudinal analysis allowed for comparing the changes in histone levels and modifications between baseline and follow-up measurements for individual patients as the epigenome is highly variable inter-individually , 46, 76.Future studies must incorporate a significantly larger study sample to correlate circulating histone levels with organ failure. In addition, diet and tobacco exposure would need to be incorporated into analyses to control for epigenetic mediators. Finally, focusing on a subset of cardiac surgeries will reduce the heterogeneity of the insult, as we saw the difference related to age which is likely related to surgical intervention.We demonstrated a prolonged elevation of serum histone three levels after cardiac surgery in an adult population. Furthermore, histone three modifications had a discrete time evolution and correlated with the activation of the immune system at 24 h. The dominant factors in histone elevation were related to the duration of surgery and not etiology leading to the procedure. The relationship between changes in H3Kme4 and monocyte-altered responsiveness to bacterial pathogens necessitates further study to demonstrate whether this increases susceptibility to organ failure.


Up to 30% Gd and 20% Bi were simultaneously substituted in the cubic ZrO2 lattice  by employing a solution combustion method followed by multiple calcinations at 900 °C. Phase purity and composition of the material is confirmed by powder XRD and EDX measurements. The formation of an oxygen vacant Gd/Bi co-doped cubic zirconia lattice was also confirmed by Raman spectroscopy study. With the incorporation of Bi3+ and Gd3+ ions, the cubic Zrx−y1−BixGdyOδ2− phase showed relaxor type high κ dielectric behaviour (ε′ = 9725 at 600 °C at applied frequency 20 kHz for Zr0.6Bi0.2Gd0.2O1.8) with Tm approaching 600 °C. The high polarizability of the Bi3+ ion coupled with synergistic interaction of Bi and Gd in the host ZrO2 lattice seems to create the more labile oxide ion vacancies that enable superior oxide-ion transport resulting in high oxide ion conductivity  at relatively lower temperatures.Solid oxide fuel cells (SOFCs) offer several advantages over lower temperature polymeric membrane fuels cells (PMFCs) due to their multiple fuel flexibility and requirement of low purity hydrogen. In order to decrease the operating temperature of SOFCs and to overcome the high operating cost and materials degradation challenges, the Cubic phase of ZrO 3+ ion coupled with synergistic interaction of Bi and Gd in the host ZrO2 lattice seems to create the more labile oxide ion vacancies that enable high oxide ion conductivity at lower temperatures.The high polarizability of the Bi The fast oxygen ion diffusion of Na0.5Bi0.5TiO3 (NBT) is attributed to the high polarizability of Bi3+ and is mediated by oxygen vacancies1 that can be introduced either by changing the NBT compositions through Bi deficiency or by Mg doping.1,2 Dielectric leakage or relaxor-like characteristics of ferroelectrics or high κ dielectric materials reveal as a strong temperature and frequency dependence in the maximum of both real and imaginary parts of the dielectric permittivity. However, relaxors not only show particular and intriguing behaviours in the dielectric response, but also show promising activity in fast-ion conduction to be applied as oxide-ion conductors for application in solid oxide fuel cells (SOFCs), oxygen separation membranes, oxygen sensors and oxygen pumps.3–9Since after the discovery of superior oxide-ion conductivity in Mg doped perovskite structure Na10–15 However, significant increases in power losses factors especially ohmic and activation losses, due to relatively high temperature (T > 800 °C) operations reduce the cell performance.16–19 The ohmic and activation losses are primarily related to oxide-ion transport through the electrolyte and the sluggish reaction kinetics on the electrode surfaces. These losses can be reduced by using the electrolyte materials with high ionic conductivity at low temperatures, reducing electrolyte thickness, increasing reactant concentration, and a number of potential reaction sites, and decreasing the activation barrier. In the past few decades, significant research has been done in the development of perovskite and fluorite based oxide ionic conductors, e.g., LaGaO3 based (Sr and Mg doped) perovskites,20–23 rare earth doped ceria based materials,6,9,24–26 Na0.5Bi0.5Tix1−MgxOδ3−,1–3,27,28 δ-Bi2O3,4,29–32 KTa0.4Ti0.3Ge0.3O2.7.5Intermediate temperature solid oxide fuel cells (IT-SOFCs) have gained recent attention due to their potential long-term durability, shorter start-up times and economic competitiveness for a wide range of applications, such as small-scale portable devices, automotive auxiliary power units and large distributed power generation systems.4+ into Ce3+ at low oxygen partial pressures.33,34 This chemical instability of ceria restricts the application of the electrolyte resulting the issue of the stability of the cell. Further, high cost of gallium and formation of inactive secondary phases during the preparation of LaGaO3-based electrolyte is a serious concern that hamper the applicability of the material as an oxide-ion electrolyte in SOFCs. That is why more attention is given on the fabrication of thin electrolytes supported SOFCs relying on yttria-stabilized zirconia (YSZ) that has been widely used as an electrolyte material at high temperatures rather than the ceria-based electrolyte.35–40Ceria-based materials, especially rare earth-doped ceria (GDC and SDC), have been considered strong candidates for IT-SOFCs electrolytes due to their high ionic conductivity in intermediate temperature range. But their performance suffers/degrades due to electronic conduction resulting through partial reduction of Ce2-based materials, combination of high dielectric permittivity and thermal stability with low leakage current due to a reasonably high barrier height that limits electron tunnelling, counts it to further research as oxide ion conductor for SOFCs application.36–38 Also, Bi-based based oxide ion conductors demonstrate the remarkable ionic conductivity due to high-concentration of intrinsic oxygen vacancies and high polarizability of Bi3+ with 6s2 lone pair electrons.39 A zirconia-based electrolyte (YSZ) is considered the most effective candidate as a solid electrolyte for electrochemical cells working either in open-circuit mode (oxygen sensor) or in a power application (oxygen pump and solid-oxide fuel cell) due to its robustness.35–40 However; it seems to be an arduous task to achieve IT-SOFCs at a commercial scale using YSZ based electrolytes due to its relatively low oxide-ion conductivity at intermediate temperatures.In ZrO3+ ion seems to play a directive role in providing superior oxide-ion transport throughout the lattice at temperature close to dielectric relaxation temperatures.1,4,47 ZrO6 octahedra was a feudal point in developing superior high κ dielectric/ferroelectric materials especially in PZT based perovskite structures. Relaxor type of high dielectric materials were also reported for doped cubic zirconia phases.41–44 We have envisaged that high polarizability of Bi3+ ion couple with high κ dielectric relaxation (high dielectric leakage) can generate superior oxide-ion conduction near Tm (the temperature of the maximum dielectric permittivity). To realize the concept, we attempted the suitable dopping of Bi3+ and Gd3+ ions into ZrO2 lattice to stabilize cubic phase of zirconia and found that the synergistic interaction by introducing a secondary substituent (Gd3+ ions) enhances the oxide-ion vacancy transport within the percolation limit of ion transport inside the host structure at lower temperatures. In cubic ZrO2, the theoretical ratio of the ionic radius of the cation to anion (O2−) for fully packed FCC lattice is 0.73 at room temperature, but the ratio is 0.59 for tetragonal phase of ZrO2 stabilized at room temperature.45 Hence, doping of other elements with larger ionic radius than Zr at Zr site is an efficient way to stabilize the high-temperature cubic phase at room temperature by the formation of solid solutions. Our study show that the co-doping of Bi3+ and Gd3+ ions 46 in ZrO2 lattice resulted the formation of Zr–Bi–Gd–O solid solution in cubic fluorite structure at room temperature and also it resulted the superior oxide-ion transport (oxide ion conductivity ∼ 10−2 S cm−1 above 500 °C) at lower temperatures. Material also showed relaxor type dielectric nature of solid solution coupled with synergistic interaction of Gd and Bi in solid solution Zrx−y1−BixGdyOδ2−. Here, we present the synthesis, characterization, permittivity and oxide-ion conductivity studies of Bi3+ and Gd3+ substituted cubic zirconia in this manuscript.Recent studies demonstrate that the relaxor nature of high κ dielectricity and higher polarizability of Bix−y1−BixGdyOδ2− samples were synthesized by employing solution combustion method by dissolving stoichiometric amount of ZrO(NO3)·xH2O, Bi2O3 and Gd2O3 in 100 ml of 40% nitric acid solution with continuous stirring at 90 °C for 4–5 hours. Further for auto-combustion, glycine was used as the fuel and was added in a molar ratio of 1.5 : 1 to total moles of metal ions present in the solution. The temperature of the hot plate-magnetic stirrer was increased to 250 °C for combustion to start. Reaction ends up with vigorous combustion after the evaporation of water at gelation point due to constant heating. The material left behind after combustion was collected, and multiple calcinations were carried out at 900 °C for 12 hours to get single-phase materials. For conductivity measurement, the powder was made into pallets of 10 mm diameter and ∼0.2 cm thickness by pressing it to ∼8 ton weight on a hydraulic press. These pallets were fired at 1000 °C for 10 hours for densification. Density of the pellet was measured by using Archimedes method and it was found to be ∼97% of the apparent density obtained from geometrical analysis.Zrλ = 1.54 Å) in the range 2θ ∼ 10–90° with a step size of 0.02°. The structures were refined by the Rietveld refinement method using the FULLPROF suite software package and cubic fluorite ZrO2 (space group: Fm3̄m) as model structure. The microstructures of the sintered samples were investigated by using scanning electron microscopy (EVO – scanning electron microscope MA15/18). The average grain size was calculated using the linear intercept method. The composition of the compounds was examined by energy dispersive X-ray (EDX) spectroscopy with a probe attached to the SEM instrument. Raman spectroscopy of powdered sample was carried out by using STR-300 micro-Raman spectrometer with a laser excitation wavelength of 532 nm and step size of 1.9 cm−1.The phase formation study was carried out through Rigaku Miniflex desktop X-ray diffractometer (XRD) with Cu Kα radiation  in different combinations and several solid solutions were synthesized in cubic fluorite structure. The synthesized Bi3+ and Gd3+ substituted ZrO2 powder was in off-white in colour. The crystal structure and phase purity of the material was analyzed by powder XRD study. Powder XRD pattern of Zr0.6Bi0.2Gd0.2Oδ2− (B20G20), Zr0.6Bi0.15Gd0.25Oδ2− (B15G25), Zr0.65Bi0.15Gd0.20Oδ2− (B15G20), Zr0.7Bi0.15Gd0.15Oδ2− (B15G15), and Zr0.6Bi0.10Gd0.30Oδ2− (B10G30), are shown 2O3, Bi2O3 or any other phases of pure ZrO2. Thus single phase materials were synthesized using solution combustion route with multiple calcinations at 900 °C for 12 hours. Crystal structures of Bi and Gd substituted ZrO2 were refined using the Rietveld method. 0.6Bi0.2Gd0.2O1.8 and (b) Zr0.6Bi0.1Gd0.3O1.8. Fitted profile matched well with the observed XRD pattern. The structural parameters obtained from Rietveld refinement of powder XRD pattern is given in 3+ and Gd3+ cations on Zr site in ZrO2 lattice, there was increase in the lattice parameter of the materials with increase in concentration of dopants.As Gd0.6Bi0.2Gd0.2O1.8  are shown in 0.6Gd0.2Bi0.2Oδ2− sample . The Raman spectrum for cubic ZrO2 is characterized by a narrow band at 145 cm−1 and broad bands centered around 250, 305, 440, and ∼601 cm−1. The stabilized ZrO2 sample in this study clearly showed the broad peak between 500 to 650 cm−1 that is related to the disordered oxygen sub-lattice along with mass-related disorder indicating of a large disorder in the cationic cage upon Gd and Bi ion substitution in cubic ZrO2 lattice whereas monoclinic ZrO2 exhibits several well defined sharp bands because of the symmetry reduction.48 Since the cations are much heavier than the oxygen atoms, they are the major contributors to the vibrations associated with the acoustic branches indicating a periodic arrangement of the vacancies in stabilized Gd/Bi co-doped cubic-zirconia the lattice.49–51 A careful examination of the Raman spectra also shows weak bands around 620, 660, and 815 cm−1 that could be associated with the rearrangement of the anionic sub-lattice, i.e. oxygen ions and vacancies containing Bi cage in stabilized Bi/Gd codoped cubic zirconia.52 Further observed bands around 535 nm and 790 nm can be assigned to Raman vibrations of Gd containing sub lattice of stabilized Bi/Gd codoped cubic zirconia.53 Thus the Raman spectroscopy study clearly reveal the formation of oxygen vacant Gd/Bi co-doped cubic zirconia lattice.2 structure, impedance spectroscopy was utilized to study the oxide-ion vacancy conduction process and dielectric constant of the materials at various temperatures in different environments. 2i.e. (i) Zr0.6Bi0.2Gd0.2O1.8 (B20G20), (ii) Zr0.6Bi0.15Gd0.25O1.8 (B15G25), (iii) Zr0.65Bi0.15Gd0.2O1.825 (B15G20), (iv) Zr0.7Bi0.15Gd0.15O1.85 (B15G15), and (v) Zr0.6Bi0.10Gd0.30O1.8 (B10G30). The total electrical conductivity was found continuously increasing with increasing temperature for all the compositions. The best electrical conductivity of this series was observed for the composition Zr0.6Bi0.2Gd0.2O1.8. At 550 °C, the measured conductivity was ∼10−2 S cm−1 for Zr0.6Bi0.2Gd0.2O1.8, which is better than that of Zr0.92Y0.08O2 (YSZ) at 670 °C at and of La0.8Sr0.2Ga0.83Mg0.17O3 (LSGM) at 600 °C. Here, a careful study was made on development to superior ZrO2 based electrolyte. As Gd can stabilized the ZrO2 in cubic fluorite structure, role or promoting effect of Bi3+ ion were utilized to develop superior oxide-ion conductors. Systematically, we carried out ionic conductivity study of Gd stabilized ZrO2 for 15%, 20%, 25%, 30% Gd3+ ions doping in ZrO2 and ionic conductivity data of these materials are provided in 2 showed almost similar conductivities. Further to improve the conductivity of Gd stabilized cubic ZrO2, Gd3+ and Bi3+ion co-substituted cubic zirconia was synthesized. We have found that at max, total 40% ions can be substituted at Zr site to make single phase cubic fluorite material. In first attempt additional 15% Bi3+ co-doping was attempted along with Gd3+ ions. However, in case of 30% Gd stabilized zirconia, only 10% additional Bi3+ can be doped in single phase. Among them, we Zr0.65Gd0.20Bi0.15Oδ2− showed highest conductivity. Further we extended Bi3+ ion substitution in Gd stabilized cubic zirconia and found that highest conductivity can be achieved with Zr0.6Gd0.2Bi0.2Oδ2− sample. Thus this study can confirm that maximum 40% substitution in ZrO2 lattice can be achieved using Gd3+ and Bi3+ ions together, and the highest conductivity was achieved for cubic fluorite Zr0.6Gd0.2Bi0.2Oδ2− sample. The data for oxide-ion conductivity of different samples of Zrx−y1−BixGdyOx+y)/22− was observed for Zr0.6Gd0.2Bi0.2Oδ2−. As evident from the study, the conductivity of the materials was increased with increasing Gd content in the cubic fluorite ZrO2 lattice. At initiation of co-doping, the promoting effect of Bi is clearly visible on co-doping of Bi along with Gd in ZrO2 lattice and we found that the maximum conductivity was observed for Zr0.6Gd0.2Bi0.2Oδ2− sample as maximum co-doping or simultaneous substitution of Gd and Bi in cubic fluorite ZrO2 lattice is limited to 40%.To see the effect of Gd and Bi substitution in the cubic fluorite ZrO0.6Bi0.2Gd0.2O1.8 was also carried out at different temperatures in dry hydrogen (UHP H2) and dry nitrogen (UHP N2) environment  is very much comparable to Sr and Ga doped LaGaO3; LSGM and KTa0.4Ti0.3Ge0.3O2.7 (KTTGO). The activation energy for oxide-ion conductivity was found as low as 0.42 eV. In the case of all samples, a sudden increase in oxide-ion conductivity was found at or around 450 °C.Impedance study of Zrironment  also to e sample . The linnia only . Furtherented in . Oxide-i2 atmosphere. 0.6Bi0.2Gd0.2O1.8 sample preheated at 120 °C. The lack of physically adsorbed water on the sample was demonstrated as marginal weight loss was observed up to 150 °C followed by very little weight loss (∼0.5%) up to 900 °C. The TGA analysis confirms the relatively low hygroscopicity or dry nature of the material. The DSC curve shown in 2 samples. 0.6Bi0.2Gd0.2O1.8 sample preheated at 120 °C for about 1 hour. Absence of peaks between 3300 and 4000 cm−1 clearly suggests the absence or insignificant presence of hydroxide ions or physio-adsorbed water on the surface of the material. This confirms that the conductivity observed for cubic fluorite Zrx−y1−BixGdyOx+y)/22− analysis at a constant heating rate of 10 °C per minute in the temperature range of 30–900 °C in N0.6Bi0.2Gd0.2O1.8 sample show a significant  (maximum permittivity) equivalent to ∼9725 at 600 °C at applied frequency 20 kHz, much higher than those for the pure ZrO2. With increasing applied frequency, the Tm had varied and observed to decrease. This gradual decrease with increasing applied frequency confirms the relaxor behaviour of this high κ dielectric material. Also, these compositions show a rather high dielectric loss (tan δ > 100) above ∼400 °C that increases exponentially with temperature above 600 °C  seems to be associated with conduction or migration of oxide vacancy.The sudden change or activation of oxygen vacancy migration above 450 °C confirms the oxide-ion transport within the percolation limit of a conductive phase transition coupled with thermal activation. Further to understand the effect of dielectric polarizability on oxide-ion conductivity, the dielectric constant in the frequency range of 20 kHz to 100 kHz at different temperatures is plotted in e 600 °C  suggesti4+ and Zr4+ cation from its mean position in octahedral coordination was shown to have the high dielectric constant and relaxor type behaviour and associated high oxide-ion conductivity in KTa0.4Ti0.3Ge0.3O2.7.5 Here, Bi3+ and Gd3+ ions were doped into ZrO2 lattice to stabilize the materials in cubic phase and the lone pair of Bi3+ ions can also play a vital role in enhancing the polarizability of the solid solution. Further, the synergistic interaction by introducing a secondary substituent (Gd3+ ions) seem to play important role to enhance the oxide-ion vacancy transport within the percolation limit ion transport inside the host structure at lower temperatures. High polarizability of Bi3+ with 6s2 lone pair electrons has been viewed as a key factor for high ionic conductivity in Bi-based oxide conductors, e.g., δ-Bi2O3,30 Bi4Ti3O12,5 γ-Bi4V2O11.47 Similarly, in the present material, the 6s2 lone pairs of Bi3+ ions can be oppositely pointed toward a vacant central of the Zr plane of ZrO8 polyhedra in the parent-phase as shown in 0.5Bi0.5TiO3,1–3,27,28 KTax−y1−TixGeyOδ3−,5 20% Sm doped CeO2(Ce0.8Sm0.2Oδ2−)6 and La2Mo2O9.7 However further studies are necessary to validate the relationship of dielectric relaxation and associated phase transitions that provide shorter conduction pathways for material's superior oxide-ion conductivity.It is well known that higher concentration of oxide-ion vacancies at lattice sites and their high mobility are two key factors for achieving high ionic conductivity in typical oxide ion conductors. For cubic zirconium oxides, oxide ionic conduction is primarily associated with the conducting passageway through a cubic block and the movement of oxygen vacancies dominates. Similar to PZT-based ferroelectric ceramics, the vibration of a smaller-sized Ti0.6Bi0.2Gd0.2O1.8 was synthesized by solution combustion synthesis route and characterized as high κ relaxor dielectric as well as an oxide-ion conductor for IT-SOFCs applications using various characterization techniques such as powder XRD, SEM-EDX and impedance spectroscopy. Only up to 40% ions can be substituted at Zr site to make single phase cubic fluorite material using Gd3+ and Bi3+ as simultaneous substituent. Up to 20% of Bi and 30% Gd was co-substituted in ZrO2 lattice  in different combinations and several solid solutions were synthesized in cubic fluorite structure. Zr0.6Bi0.2Gd0.2O1.8 sample showed superior oxide-ion conductivity with the lower activation energy in the temperature range of 300–650 °C. The oxide-ion conductivity of Zr0.6Bi0.2Gd0.2O1.8 was found 10−2 S cm−1 at or above 500 °C. Considering the robustness of ZrO2 based systems, the materials can act as an possible candidates as an oxide-ion electrolyte for intermediate temperature solid oxide fuel cells (IT-SOFCs), as the material requires low processing cost and delivers high conductivity at relatively lower temperatures. Nonetheless, more studies are required to determine the applicability of the materials as an oxide-ion electrolyte for the production of IT-SOFCs. The cubic Zrx−y1−BixGdyOδ2− phase also showed relaxor type high κ dielectric behaviour (ε′ = 9725 at 600 °C at applied frequency 20 kHz for Zr0.6Bi0.2Gd0.2O1.8) with Tm approaching to 600 °C. The polarizability of Bi3+ ion coupled with high κ dielectric relaxation (high dielectric leakage) can utilize as new tool to develop superior oxide-ion conduction near Tm (the temperature of the maximum dielectric permittivity) and effort can be made to bring down the Tm to achieve higher ionic conductivity at lower temperatures.Cubic ZrThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Homozygous loss of SAMD1 was embryonic lethal, with no living animals seen after embryonic day 18.5. At embryonic day 14.5, organs were degrading and/or incompletely developed, and no functional blood vessels were observed, suggesting failed blood vessel maturation. Sparse red blood cells were scattered and pooled, primarily near the embryo surface. Some embryos had malformed heads and brains at embryonic day 15.5. In vitro, SAMD1 absence impaired neuronal differentiation processes. Heterozygous SAMD1 knockout mice underwent normal embryogenesis and were born alive. Postnatal genotyping showed a reduced ability of these mice to thrive, possibly due to altered steroidogenesis. In summary, the characterization of SAMD1 knockout mice suggests a critical role of SAMD1 during developmental processes in multiple organs and tissues.SAM domain-containing protein 1 (SAMD1) has been implicated in atherosclerosis, as well as in chromatin and transcriptional regulation, suggesting a versatile and complex biological function. However, its role at an organismal level is currently unknown. Here, we generated SAMD1 SAMD1 may participate in LDL retention and uptake, and its knockdown suppresses vascular smooth muscle cell (VSMC) differentiation and proliferation, which affects foam cell development3. However, SAMD1 is widely expressed in most organs and tissues, both in mice and humans . The extensiveness of expression suggests roles in adult homeostasis, but the molecular function of SAMD1 has been minimally studied so far. SAMD1 was found to directly associate with CpG islands at the chromatin, acting as a transcriptional regulator, and be required for proper embryonic stem (ES) cell differentiation4. Earlier reports further support epigenetic functions for SAMD1. SAMD1 was enriched at nucleosomes and H3K4me3-possessing chromatin regions, as revealed by mass spectrometry6. It was also among the highest 5% of proteins that associate with both H3K4me3-modified and bivalently H3K4me3/H3K27me3-modified chromatin7.Human SAMD1 (SAM domain-containing protein 1) is a 538 amino acid protein that has orthologs in most vertebrates, including zebrafish. Originally, SAMD1 was identified as a protein involved in atherosclerotic low-density lipoprotein (LDL) binding in humans4. The N-terminal domain belongs to a group of related winged helix domains that bind to unmethylated CpG motifs8, explaining the enrichment of SAMD1 at CpG islands on the genome9. SAMD1 associates with repressive chromatin regulatory complexes that contain the histone demethylase KDM1A and several other SAM domain proteins, including the Polycomb group-related proteins L3MBTL3 and SFMBT112. Consequently, the absence of SAMD1 typically leads to the derepression of its target genes9. SAMD1 is often upregulated in cancer tissues and correlates with worse prognosis in some cancer types, such as adenoid cystic carcinoma (ACC) and liver cancer13. A CRISPR screen in K562 cells suggested that SAMD1 is required for the efficient proliferation of these cells14. Further work demonstrated that SAMD1 deletion in HepG2 hepatocellular carcinoma cells leads to a global readjustment of the active H3K4me2 chromatin mark and a more favorable gene signature, supporting an oncogenic role in this context9. SAMD1 has also been described to play a role in muscle adaptation15. Taken together, these findings suggest that SAMD1 likely has a function in multiple biological processes and diseases, but the biological role of SAMD1 at the organismal level has yet to be investigated.The SAMD1 protein has two globular domains. At the C-terminus, it possesses a SAM domain that can interact with other SAM domain-containing proteins, such as L3MBTL3, and allows multimerization−/−) embryos were observed at embryonic day (E) 14.5, and none were observed after E18.5. Development appeared grossly normal at E12.5, but at E14.5 the investigated embryos exhibited numerous defects, including degrading endothelial cell (EC) tubes, degrading inner organs and an almost complete absence of red blood cells (RBCs). The heterozygous (SAMD1+/−) mice had a less severe phenotype and approximately 70% of the mice survived past week 3 postnatally but they exhibited reduced weight gain and hormonal changes.Here, we report the first knockout of SAMD1 in mice. Fewer homozygous (SAMD1+/+), heterozygous , and knockout  mice were close to the expected Mendelian ratios through E12.5. All embryos removed at E12.5 had heartbeats. The first dead SAMD1 KO embryos and abnormal phenotypes were observed at E14.5  also showed no KO (p = 5.1 × 10–13)  (Supplementary Table +/+ mice were crossed with SAMD1 ± mice (p = 0.003)  embryos were not noticeably different from that of the WT embryos, and HET mice were born alive  tubes, as well as complete regional network regression, leaving behind "sleeves” of collagen and apoptotic ECs21. During maturation, progenitor/pericyte/smooth muscle cells connect to EC tubes, providing stability needed to control regression20. The presence of RBCs in the KO skin implies that a functional circulatory system existed prior to an apparent abnormal regression of the EC tube vascular plexus in the KO embryo.When the yolk sac was removed, the KO embryo within the amnion appeared developmentally delayed and visually smaller than the WT embryo Fig. e. BloodySimilar to the embryo, the KO placenta was pale compared to the WT Fig. e, probab22. The E15.5 embryo was less pale than the E14.5 embryo, had longer and larger red lines, and had a few apparently intact blood vessels. Some areas of the embryo surface in and beneath the skin, and above the brain, had small hemorrhages and lines of RBCs that may have once been contained in vessels, suggesting a failing circulatory system. Since the E14.5 KO had a skull vault and was paler, the investigated E15.5 KO embryo is likely an example of a different SAMD1 KO phenotype, but the surface RBC patterns appear similar to the E14.5 KO. Failed skull vault development may be caused by failure of neural tube closure. Clear fluid leakage due to failing lymphatic and blood vessels likely produced edema  staining on sagittal slices of an E14.5 embryo Fig. . Hematoxans Fig. . AlthougThe KO lung also appeared to be fragmenting, and RBCs were not apparent, in contrast to the WT lung where RBCs filled large vessels Fig. b. BronchThe KO liver had lobes and was large enough to have developed at least until E12.5 , which line all blood and lymph vessels, from primitive endothelial tubes to fully mature vessels. We used H&E, CD31 or VEGFR2 staining (with hematoxylin counterstain) to mark ECs and provide morphological identification of other cells. Cluster of differentiation 31  is widely used as a marker for ECs, staining the cell surface. Vascular endothelial growth factor 2  stains the cytoplasm of ECs. Using these different markers, we investigated the organs and tissues of the SAMD1 KO embryos.Looking first at the embryo surface, H&E staining of the WT embryo showed groups of RBCs in the skin and RBCs in a larger blood vessel beneath the subcutaneous tissue. Here, CD31 stained ECs of capillaries and larger vessels  cells have nuclei, and begin to mix with definitive RBCs from approximately E12.5, and enucleating EryPs at E12.531. A mix of nucleated and definitive RBCs remains in circulation until gestation. Obviously, nucleated and enucleated RBCs could not have arrived at these locations unless a functional circulatory system existed later than 12.5. This observation suggests circulation failure trapped EryPs and definitive RBCs in a nonfunctioning vessel around E13.5, when approximately 50% of RBCs would not have nuclei31. Some KO RBCs appeared faded  in a blood vessel above a WT rib do not have nuclei, and few if any lymphocytes are seen Fig. d. RBCs aded Fig. d, implyiTaken together, our work so far suggests that the absence of SAMD1 leads to improper development of all investigated organs and tissues in the embryo. We have summarized our findings in Table 4. Although SAMD1 functions mainly as a transcriptional repressor, we found both up and downregulated pathways upon SAMD1 deletion, suggesting direct and indirect consequences of SAMD1 deletion. Thus, it appears that SAMD1 plays a rather pleiotropic role during differentiation processes, consistent with the multifaceted mouse phenotype.To better understand the molecular source of the many defects upon SAMD1 deletion, we investigated our previously published RNA-Seq data upon undirected ES cell differentiation in the absence of SAMD14 and several genes related to brain development, such as Cbln1, Ntrk2, and Plxna4, Eph4, become derepressed upon SAMD1 deletion during differentiation , Pecam1 (CD31), Thy1 and Tie1, which are critical factors for angiogenesis34, were strongly downregulated in SAMD1 KO cells, both in differentiated and undifferentiated cells  and Mef2c35  and Nestin (Nes), were dysregulated upon SAMD1 KO  embryo was not noticeably different from that in the WT embryo  and LDL (low-density lipoprotein) were higher in the HETs, while HDL (high-density lipoprotein) was lower in the HETs Fig. h. ProperCollectively, these data suggest that although the heterozygous deletion of SAMD1 has no substantial consequences on embryogenesis, it still has an impact on the ability of the mice to develop normally after birth.9. Upon mouse ES cell differentiation, the absence of SAMD1 enhances the expression of genes involved in neuronal pathways, while factors important for metabolism and angiogenesis are downregulated  FS-23]. The Pfizer Institutional Animal Care and Use Committee (IACUC) approved all of the animal procedures and protocols according to the criteria stated by the National Academy of Sciences National Research Council (NRC) publication 86-23, 1985. All methods were performed in accordance with the relevant guidelines and regulations, as well as in accordance with the ARRIVE guidelines.51 was used to replace 2396 bp of the mouse SAMD1 (Accession ID D3YXK1) gene encompassing the 3′ 615 bp of exon 1, exons 2–4 and all of exon 5 except for the 3′ 83 bp with a neomycin phosphotransferase cassette in a 9433 bp genomic subclone obtained from a C57BL/6J BAC . The vector was introduced into Bruce4 (mixed C57Bl/6N:C57BL/6J) mouse embryonic stem cells52 using a standard homologous recombination technique. Southern blot analysis was used to identify correctly targeted mouse embryonic stem cell clones. Chimeric mice were generated by injection of the targeted ES cells into Balb/c blastocysts. Chimeric mice were bred with C57Bl/6J mice to produce F1 heterozygotes. Germline transmission was confirmed by PCR analysis . F1 heterozygous males and females were mated to produce F2 mice. The colony was maintained by intercrossing under specific pathogen-free conditions with unrestricted access to food and water.SAMD1 knockout mice were genetically engineered by Pfizer . To generate the SAMD1 targeting vector, recombineeringRNA and cDNA measurements were performed at the Brigham & Women’s Hospital . Synthesis of cDNA (ThermoScript RT-PCR System (Invitrogen Cat 11146-016)). RT-PCR was performed in a MyiQ single-color real-time PCR system . Total ribonucleic acid (RNA) from 250,000 cells was reverse transcribed by Superscript II (Invitrogen) according to the manufacturer's instructions. Quantitative PCR was performed with SYBR green PCR mix (primers for SAMD1 from QIAGEN N.V.) and analysis performed with StepOne Software . Levels of mRNA were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. cDNAs were synthesized using a High-Capacity RNA-to-cDNA™ Kit . Total 2 µg RNA/100 µl cDNA synthesis reaction and 1 µl cDNA per TaqMan reaction. GAPDH was used as an endogenous control .53. In short, embryos from WT, SAMD1+/−, and SAMD1−/− mice were isolated between E12.5 and E18.5. Internal organs, heads, tails, and limbs were removed from embryos prior to treatment with trypsin to dissociate the cells. Cells were seeded into T-75 cell culture dishes in 15 ml of MEF media: KO DMEM , 15% FBS (Life Technologies), nonessential amino acids (Life Technologies 111140-050), 2 mM l-glutamine , 0.1 mM β-mercaptoethanol (Sigma M7522), and 0.25 mg/ml gentamicin .Mouse embryonic fibroblasts (MEFs) were isolated as described+/+ to SAMD1+/− were performed by Caliper Life Sciences  based on the Phenotype Pfinder platform54.All behavioral, physical, hormonal, and response phenotype comparisons of SAMD118. In short, tissues were collected immediately after E14.5 embryo collection, fixed in 10% buffered formalin and embedded in paraffin. Four micrometer serial sections were cut and mounted on glass slides. One section from each lesion was stained with hematoxylin and eosin (H&E). Other slices were stained for CD31 (ab28364) or VEGFR2 (ab2349) with hematoxylin counterstaining following Abcam’s protocols.Antibodies against CD31 and VEGFR2 were purchased from Abcam . Immunohistochemical staining procedures were performed by Pfizer  and at the Brigham & Women’s Hospital . E14.5 has been established as the optimal time point for mouse developmental disorder phenotype analyses4. The WT and SAMD1 KO mESCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and GlutaMAX , 15% fetal calf serum (FCS) , 1 × nonessential amino acids , 1 × sodium pyruvate , 1 × penicillin/streptomycin , 10 µM β-mercaptoethanol , and LIF  on 0.2% gelatin-coated plates.E14 mESCs (E14TG2a) were provided from the lab of Jacqueline Mermoud . SAMD1 KO E14 mESCs were established previously6 on 0.1% gelatin-coated and dried 100 mm cell culture dishes for 3 days in mESC medium, and the medium was changed every 24 h. To start neurodifferentiation, 1 × 106 dissociated ES cells were incubated on nonadhesive bacterial dishes (100 mm) for 6–8 days in mESC medium without LIF, and the medium was changed every 2 d. Embroid bodies (EBs) were collected and redistributed to 4 wells of a 6-well plate containing 2 ml differentiation medium containing DMEM , 20% FCS, 2 mM l-glutamine , 1 × penicillin/streptomycin, 1 × nonessential amino acids, 50 µM β-mercaptoethanol, and 0.5 µM trans-retinoic acid  and incubated for 2 days. EBs from two wells each were collected and redistributed to a 100 mm cell culture dish, coated with 20 μg/ml poly-l-ornithine hydrobromide in PBS  and incubated for 7 days in neurobasal medium  with 1 × L-glutamine, 1 × penicillin/streptomycin, and 1 × B-27 supplement 55.mESCs were plated at a density of 1 × 10l-glutamine, 1 × penicillin/streptomycin, 50 µg/ml BSA , 20 µg/ml insulin , 10 ng/ml EGF , and 10 ng/ml FGF  on 0.1% gelatin-coated and subsequently 5 μg/ml laminin-coated  six-well tissue culture plates and passaged with Accutase  (adapted from56).The established neural precursor cells (NPCs) were cultured in Euromed-N medium  supplemented with 1 × N-2 , 1 × 5) into gelatinized (0.1% in H2O) and poly-l-ornithine hydrobromide-coated (20 μg/ml in PBS) six-well tissue culture plates for differentiation with N2B27 medium, consisting of DMEM/F12  and neurobasal medium (1:1), with 1 × N-2, 1 × B-27, 100 µM β-mercaptoethanol and the addition of 10 ng/ml FGF  (adapted from57). Analysis of the cells was performed with immunofluorescence staining and RT-qPCR at chosen time points.To promote the neurogenic capacity of NPCs and allow the formation of mature neurons, WT and SAMD1 KO NPCs were seeded at high density  according to the manufacturer’s manual, including an on-column DNA digest . The PrimeScript RT Reagent Kit  was used to transcribe mRNA into cDNA according to the manufacturer’s manual. Samples were incubated for 30 min at 37 °C followed by 5 min at 85 °C to inactivate PrimeScript RT enzymes. Subsequently, cDNA was diluted 1:20 to be used in RT-qPCR. For analysis by real-time quantitative PCR, MyTaq Mix  was used. For gene expression analysis, values were normalized to mActb and mGapdh expression. The qPCR primers used are presented in Supplementary Table 4.The following commercial antibodies were used: H3K4me2 , TUJ1/TUBB3 , and GFAP . The antibody for SAMD1 is custom-made. It is directed against the SAM domain of human SAMD1 (amino acids 452 to 538) and was produced as described previously5 cells per well. Cell viability was determined at 1, 3, and 6 days after seeding using the MTT assay by adding 90 µl of 5 mg/ml thiazolyl blue ≥ 98%  to each well. The medium was aspirated after 1 h, and stained cells were dissolved in 400 µl of lysis buffer . Absorption was measured at 595 nm using a plate reader. All values were normalized to day 1 to compensate for variations in seeding density. The mean value of three biological replicates was determined.To determine proliferation rates, cells were seeded on 6-well plates at a density of 1 × 10l-ornithine hydrobromide-coated coverslips Cells were fixed with 4% formaldehyde (w/v), methanol-free , and subsequently permeabilized with wash buffer (0.5% Triton X-100 in PBS). Blocking was performed with 10% FCS in wash buffer. Primary antibodies were diluted 1:500 in blocking solution and incubated in a wet chamber overnight at 4 °C. Three washing steps of the cells were performed before incubation with secondary antibodies, using Alexa Fluor 488 goat anti-rabbit IgG (H + L) and Alexa Fluor 546 goat anti-mouse IgG (H + L) , at a 1:2000 dilution each. Following three washing steps, the coverslips were mounted onto microscopy slides using VECTASHIELD® Antifade Mounting Medium with DAPI  and sealed. Microscopy was performed using a Leica DM5500 microscope, and data were analyzed using ImageJ (Fiji).WT or SAMD1 KO mESCs were seeded on 0.2% gelatin-coated coverslips. WT or SAMD1 KO differentiated neural precursor and neural cells were seeded on 0.1% gelatin-coated and 20 μg/ml poly-To determine the cellular fluorescence in microscopy images using ImageJ, cells of interest were outlined and measured for their area, integrated intensity and mean gray value. Adjacent background regions were measured for the same parameters to calculate the corrected total cell fluorescence (CTCF) according to the following formula: CTCF = integrated density – . Average CTCF values and standard deviations for 5 cells each in 3 different fluorescence microscopy images per sample are presented in a graph.https://www.encodeproject.org/, under “Mouse Development”)24 and from GSE4571923. GSEA was performed with standard settings58 using previously published data (GSE144396)4.Gene expression data from mouse embryo tissues were obtained from ENCODE . Deviation from the mendelian ratio was evaluated using Chi Square tests. The significance of differences between two independently measured groups were evaluated using a two-tailed unpaired t-test. Two-way ANOVA followed by Tukey’s postdoc test was applied for multiple group comparisons. Significance of the Lipoprint analysis was evaluated using a two-tailed Mann–Whitney Supplementary Information.


Rapa@Ti3C2 was prepared via electrostatic self-assembly of Ti3C2 and Rapa, with a loading capacity of Rapa at 92%. Ti3C2 was used as a drug delivery reservoir of Rapa. Rapa@Ti3C2-IOL was designed to have the synergistic photothermal and near infrared (NIR)-controllable drug release property. As a result, Rapa@Ti3C2-IOL exhibited the advantages of simple preparation, high light transmittance, excellent photothermal conversion capacity, and NIR-controllable drug release behavior. The Rapa@Ti3C2 coating effectively eliminated the LECs around Rapa@Ti3C2-IOL under a mild 808-nm NIR laser irradiation (1.0 W/cm−2). Moreover, NIR-controllable Rapa release inhibited the migration of LECs and suppressed the inflammatory response after photothermal therapy in vitro. Then, Rapa@Ti3C2-IOL was implanted into chinchilla rabbit eyes, and the effectiveness and biocompatibility to prevent PCO were evaluated for 4 weeks. The Rapa@Ti3C2-IOL implant exhibited excellent PCO prevention ability with the assistance of NIR irradiation and no obvious pathological damage was observed in surrounding healthy tissues. In summary, the present study offers a promising strategy for preventing PCO via ultrathin Ti3C2 MXene nanosheet-based IOLs with synergistic photothermal and NIR-controllable Rapa release properties.Posterior capsule opacification (PCO) is one of the most frequent late-onset complications after cataract surgery. Several kinds of drug-eluting intraocular lenses (IOL) were designed for sustainable drug release to suppress ocular inflammation, the proliferation of lens epithelial cells (LECs) and the development of PCO after cataract surgery. Despite previous advances in this field, the drug-loaded IOLs were limited in ocular toxicity, insufficient drug-loading capacity, and short release time. To prevent PCO and to address these drawbacks, a novel drug-loaded IOL (Rapa@Ti Posterior capsule opacification (PCO) is a common complication and the primary cause of non-refractive visual loss after lens extraction combined with intraocular lens (IOL) implantation . It is cSeveral strategies, including surgical techniques, IOL materials, IOL with square edge design and drug-eluting IOL, were performed for the prophylaxis of PCO . The int2 nanorods, was fabricated for eliminating residual LECs after cataract surgery (2), the PCO occurrence is reduced to 30–40% in a rabbit model. However, as nanorods were coated on the rim of IOLs, the temperature in the center of IOL gradually decreased to 38°C, which would not reach the temperature of killing the proliferating LECs in the optics of IOLs. Therefore, it is essential to find a material with high light transmittance to evenly coat the whole optic surface of IOL for the photothermal ablative effect on the optic axis. Moreover, photothermal therapy (PTT) gives rise to the inflammatory response  irradiation, such as graphene oxide for infected wounds , gold na surgery . After Nresponse , which iresponse . This maresponse . The NIR3C2, are featured with high photothermal-conversion capacity, which is developed as an effective photothermal nanoplatform upon NIR irradiation. Moreover, the ultrathin planar nanostructure of MXene provides a large anchoring site for numerous drug molecules. Therefore, in the field of biomedicine, MXenes make themselves outstanding in biosensors, antibacterial activity, drug delivery, photothermal therapy and hemodialysis , namely Rapa@Ti3C2-IOL, for PCO prevention. The ultrathin Ti3C2 nanosheets were prepared by wet etching the Ti3AlC2 MAX phase. Then, Rapa-loaded Ti3C2 nanosheets (Rapa@Ti3C2) were synthesized via electrostatic self-assembly and were coated onto the optic surface of the commercial hydrophobic IOL by an easy two-step spin coating method. This Rapa@Ti3C2-IOL showed the performance of simple preparation, high light transmittance, excellent photothermal ablative efficacy, and NIR-controllable Rapa release behavior, which effectively prevents PCO both in vitro and in vivo. This study presented the therapeutic effect of Rapa@Ti3C2-IOL upon NIR irradiation and provided a promising application for PCO prevention.In the present study, we designed and fabricated novel ultrathin Ti3AlC2  was bought from Beijing Forsman Scientific (China). Ethanol absolute (≥99.7%) and HF  were bought from Sinopharm Chemical Reagent Co., Ltd. . Tetrapropylammonium hydroxide (TPAOH) (25 wt.% in water) and Rapa were bought from Sinopharm Chemical Reagent Co., Ltd. All chemicals were used as received without further treatment unless otherwise stated.Ti3AlC2 powder was etched at 10 ml 40% HF at room temperature to remove the Al layer. The precipitate was then centrifuged three times in turn with water and ethanol for 3 min at 3500 rpm. The monolayer Ti3C2 nanosheets were intercalated with 20 ml TPAOH(25 wt. % in water)and were vacuum-dried and stored at 4°C.According to the methods reported previously . Briefly3C2 was dispersed in phosphate buffered saline (PBS = 7.2) to prepare suspensions of different concentrations. A certain amount of Rapa was added to the Ti3C2 dispersion and stirred for 2 h. Then, the Rapa-loaded Ti3C2 nanosheets (Rapa@Ti3C2) were collected by centrifugation for 3 min at 3500 rpm and stored at 4°C. The absorption peak intensity of Rapa was measured by UV-vis–NIR spectrometer, and the absorption peak was observed at 278 nm. The loading capacity of Rapa can be calculated according to:Monolayer TiThe Rapa loading capacity was 92%.3C2 solution and Rapa@Ti3C2 solution (the concentration ratio of Rapa/Ti3C2 = 0.92:1) were coated onto the optic surface of hydrophobic acrylic IOLs supplied by Eyebright Medical Technology (Beijing) Co., Ltd. via a two-step spin coating method. In brief, the loops of IOLs were shielded with scotch tape and the optic surface of hydrophobic IOLs was cleaned and activated with oxygen plasma by an OKSUN plasma etcher . 40 μL of Ti3C2 or Rapa@Ti3C2 solution was evenly deposited on the optic surface by a spin coater  at 5,000 rpm for 2 min, followed by drying the coating at 6000 rpm for 1 min. There are three types of IOLs in this study: C-IOLs = commercial IOLs as the control, Ti3C2-IOLs = IOLs were coated with Ti3C2; Rapa@Ti3C2-IOLs = IOLs were coated with Rapa@Ti3C2.As described previously , the Ti33C2 MXenes and Rapa@Ti3C2 were characterized by Scanning Electron Microscopy , Transmission Electron Microscope  and Atomic Force Microscope . The formation of Ti3C2 was also confirmed by X-ray diffraction . Size and zeta potential were acquired by Malvern Zetasizer Nano ZS90 (Britain). UV–vis absorption spectra were captured by a SHIMADZU UV3600 spectrometer.Ti3C2 solution was irradiated by a NIR laser (808 nm). The absorbance of the solution was determined at λ = 278 nm at given time intervals to calculate the Rapa releasing percentage.When evaluating NIR-controlled drug release, the Rapa@Ti3C2 solution at different concentrations was directly irradiated by NIR light at different powers density for 180 s. The temperatures of Rapa@Ti3C2 solution (25 μg/ml) with different laser power densities ranging from 0.1 to 3.0 W/cm2 were recorded by an infrared thermal imager . The temperatures of Rapa@Ti3C2-IOLs with various concentrations of Ti3C2  were detected under irradiation with different power densities. Finally, the photothermal stability of Rapa@Ti3C2-IOL was evaluated by three cycles of the laser on/off. The changes in temperature were recorded every 10 s by an infrared thermal imager.Rapa@TiThe anterior capsule of the C57 mouse  was cut into a square of 1 mm × 1 mm and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 2% fetal bovine serum. Once the cells grew out of the capsule, they were digested and harvested for subculture. NIH/3T3 (CRL-1658) was obtained from American Type Culture Collection (ATCC), and cultured in the same condition.3C2. Mouse LECs and NIH/3T3 cells were seeded in the 96-well plate at 8 × 103 per well in 2% FBS-supplemented and 1% penicillin/streptomycin-supplemented DMEM. After being cultured for 24 h, a fresh medium containing free Rapa, free Ti3C2, or Rapa@Ti3C2 at different concentrations  was added with or without the 808 nm laser irradiation. Then, the CCK-8 solution was diluted 1:10 with DMEM for evaluating the cytotoxicity.The CCK-8 assay kit (Dojindo) was used for testing the cytotoxicity of Rapa@Ti2) for 10 min. After that, calcein-AM/PI double-staining of live/dead assay was used to label cells for 10 min and washed three times with PBS. Finally, cells were observed and captured by a fluorescence microscope.To evaluate the photothermal ablative effect of nano-IOLs, three types of IOLs were carefully put on top of the cells seeded in 24 well plates and irradiated by NIR laser (1.0 W/cm6 mouse LECs were seeded in 24-well plates and cultured with IOL for 24 h. They were divided into 5 groups as follows: 1) C-IOL; 2) Ti3C2-IOL; 3) Ti3C2-IOL + NIR; 4) Rapa@Ti3C2-IOL; 5) Rapa@Ti3C2-IOL + NIR. After the adhesion of cells, a sterile pipette tip was used to scratch the adhesion surface to evaluate cell migration, and the images were captured by microscopy at 0 h, 24 h and 48 h after scratching. ImageJ software was used to measure the wound area: more than three random fields were selected for evaluation, and the wound area at 0 h time point was taken as the reference.About 1 × 10via Transwell migration chambers (8 μm pore size) from BD Biosciences. Cells were grown to 70% confluence and serum was starved for 24 h. Following detachment with trypsin and washing with PBS, cells were re-suspended in serum-free medium and added to the upper chamber. Serum-free medium containing various solutions or IOLs were added to the bottom chamber. After 24 and 48 h, the cells that had not migrated were removed from the upper face of the filters with cotton swabs, and the migrated cells were fixed and stained with crystal violet/methanol solution.The Transwell assay was performed 6 mouse LECs were seeded in 24-well plates and cultured with C-IOL, Ti3C2-IOL and Rapa@Ti3C2-IOL upon NIR irradiation (1.0 W/cm2) for 10 min. After 24 h, the supernatant was collected for analysis. 50 μL of the sample was incubated with the primary antibodies against IL-1β, IL-6, TNF-α, and MMP-9 at 4°C. The standard curve ranges from 3.2 to 10,000 pg/ml.In our previous study, the inflammatory cytokines  were determined by using the Multiplex Luminex Assay  with Multiplex kits (Millipore). In brief, 1 × 10n = 12 rabbits/group): 1) C-IOL; 2) Ti3C2-IOL; 3) Ti3C2-IOL + NIR; 4) Rapa@Ti3C2-IOL; 5) Rapa@Ti3C2-IOL + NIR. All animals were anesthetized by intravenous injection of 3% pentobarbital sodium at 1 ml/kg body weight, and preoperative pupil dilation was performed with 1 drop of 0.5% tropicamide  3 times, 15 min apart. Following that, they all underwent phacoemulsification and IOL implantation in the left eyes by the same surgeon . A cornea incision was established by keratome. Phacoemulsification was performed with the Stellaris system , and different groups of IOL were implanted respectively.All animal experiments were performed following the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research  and approved by the Laboratory Animal Ethics Committee of the Third Medical Center of PLA General Hospital. Sixty female chinchilla rabbits up to 2.0–2.5 kg weight, 12 weeks old were used during the experiment, which was randomly divided into five groups  and dexamethasone-tobramycin  four times daily for 2 weeks. After surgery, a slit lamp photography system  was used to observe and photograph the experimental eyes at designated time points. The degrees of PCO was assessed using photographs by retro illumination according to the criteria listed in 2) for 10 min. The changes in temperature of implanted IOL were recorded every 10 s by a thermal imager.One week after surgery, the eyes of rabbits were irradiated by NIR laser . Furthermore, a histological examination of different ocular tissues was carried out, and the posterior capsules, corneas, ciliary bodies and retinas were evaluated using hematoxylin and eosin (H&E) staining.To detect the release of Rapa, 0.1 ml aqueous humor was collected in a centrifuge tube at day 1, 3, 8, 15, 22, and 29 after surgery. The concentration of Rapa in the aqueous humor was determined by high performance liquid chromatography  at a wavelength of 278 nm.® tonometer  was used to measure intraocular pressure (IOP). Anterior segment optical coherence tomography was used to measure the central corneal thickness (CCT). Corneal specular microscopy was used to count the number of corneal endothelial cells (CEC) . All experiments were repeated three times and the average value was recorded.A TonoVet Plusp < 0.05 was considered statistically significant.The data were analyzed using Graphpad Prism software version 9.2 and expressed as the mean ± standard error of mean. One-way ANOVA with post-hoc testing was used for comparison of multiple groups with Gaussian distribution. Kruskal–Wallis test was used for comparison of multiple groups without normal distribution. 3C2-IOL and photothermal conversion temperature. Ultrathin Ti3C2 MXene nanosheets were synthesized using HF etching and a TPAOH intercalator, which could produce nano-sized MXene nanosheets with high dispersion and sufficient planar size to meet the severe requirements for current and future biomedical applications  disappears and is replaced by the characteristic periodic peak originating from stacked Ti3C2 layers. Namely, Ti3C2 and Rapa@ Ti3C2 only have peak values at 2θ ≈ 6°  of Ti3C2 and absorbance at 808 nm , the extinction coefficient of Rapa@Ti3C2 was calculated to be 4.2256 L g−1cm−1 , indicating its probability to be used in further PTT. UV–vis spectrum showed that Ti3C2 nanosheets loaded with Rapa exhibited characteristic absorption peaks of Rapa molecules (λ = 278 nm)  and power density of irradiation   , indicatter 48 h . Especiateristic . The pho0 W/cm2) , indicatIn vitro cytotoxicity of Ti3C2 and Rapa@Ti3C2 was further evaluated by CCK-8 assay  for 10 min (3C2 or Rapa@Ti3C2 (the concentration of Ti3C2 was 25 μg/ml) upon NIR irradiation at different power densities for 10 min, nearly no cell alive at the power density ≥1.0 W/cm2 (-8 assay . After i studies . Howeverr 10 min . When LE.0 W/cm2 .in vitro (3C2 (the ratio of Rapa/Ti3C2 = 0.92:1) at different concentrations of Rapa for 72 h, The number of cells decreased significantly when the free Rapa concentration ≥1.0 μg/ml, while the anti-proliferative effect of Rapa@Ti3C2 is inferior to that of free Rapa in the same concentration  upon NIR irradiation. The temperature range of each heating process kept the same, indicating that Rapa@Ti3C2-IOL has high thermal stability  upon NIR irradiation , the cells were tested for dual live/dead staining assay. Fluorescence microscope images showed that the cells covered by Ti3C2-IOL or Rapa@Ti3C2-IOL were dead and stained with red signals, while cells away from the IOL were alive and stained with green signals  C-IOL; 2) Ti3C2-IOL; 3) Ti3C2-IOL + NIR; 4) Rapa@Ti3C2-IOL; 5) Rapa@Ti3C2-IOL + NIR. At the time point of 0 h, 24 h and 48 h after scratch, the changes in cell morphology and scratch wound areas were observed. The wound-healing assay displayed that the area of the remaining scratch wounds was much wider in the Rapa@Ti3C2-IOL + NIR group than that of the non-Rapa group, indicating that Rapa significantly suppressed cell migration in mouse LECs  was irradiated once a week post operation, and the temperature of IOL was recorded by a thermal imager in the first week after surgery. The data showed that the temperature of Ti3C2-IOL and Rapa@Ti3C2-IOL rapidly rose to 50°C in the 1st min after NIR irradiation , remarkably better than that of the C-IOL group (3.50 ± 0.19), Ti3C2-IOL group (3.42 ± 0.19), Ti3C2-IOL + NIR group (1.75 ± 0.17) and Rapa@Ti3C2-IOL group (0.83 ± 0.21). On day 28 post-operation, the Miyake-Apple view of the rabbits’ eyes showed no obvious proliferation materials on the posterior capsules of the Rapa@Ti3C2-IOL + NIR group  µg/ml on day 1 after surgery and decreased gradually. From the 15th day, Rapa was not detectable in aqueous samples. However, 1 day after NIR irradiation, the Rapa concentration in aqueous humor increased to about 3 μg/ml on day 8, day 15 and day 22. As NIR irradiation triggered Rapa release from Ti3C2 nanosheets for 3 cycles, the Rapa concentration in aqueous humor was nearly 1 μg/ml on day 29 (3C2-IOL + NIR group was (0.08 ± 0.29), remarkably better than that of the C-IOL group (0.75 ± 0.62), Ti3C2-IOL group (0.67 ± 0.49), Ti3C2-IOL + NIR group (1.67 ± 0.65) and Rapa@Ti3C2-IOL group (0.42 ± 0.52) (The Rapa concentrations in aqueous humor after Rapa@Tin day 29 . A high  ± 0.52) . All the2), compared with that of the control group. However, the subretinal fluid  was observed in groups exposed to the laser power at 2 and 3 W/cm2 (2 was safe for ocular treatment, and this NIR power density was much lower than that in several previous studies (3C2-IOL + NIR group was not significantly different from those of the control group, C-IOL group, Ti3C2-IOL group, Ti3C2-IOL + NIR group and Rapa@Ti3C2-IOL group at day 28 after surgery (3C2-IOL group and Rapa@Ti3C2-IOL + NIR group than those of the control group on day 3 and dropped to the baseline from day 7. However, the IOP levels in the C-IOL group, Ti3C2-IOL group and Ti3C2-IOL + NIR group were significantly higher compared to those of the control group on day 28 after surgery (3C2-IOL + NIR group were the same as those of the control group on day 28 after surgery (3C2-IOL, which may be an attractive strategy for future clinical applications for preventing PCO.We further assessed the histological changes of the retina under NIR irradiation with different power densities. The H&E staining of the retina in each group showed no significant changes with mild NIR power density (≤1.0 W/cm 3 W/cm2 , indicat studies . Moreove surgery . In addi surgery . In cont surgery . The res3C2 onto the surface of the optical part of commercial IOL, namely Rapa@Ti3C2-IOL, for combinational PTT and NIR-controllable drug releasing therapy for PCO prevention. This Rapa@Ti3C2-IOL system showed the characteristics of high light transmittance, moderate refractive index and comparable strain stress and elongation performances to commercial hydrophobic IOL. Notably, the Rapa@Ti3C2-IOL underwent controllable Rapa release behavior under 808 nm NIR irradiation, due to the high photothermal conversion efficiency of Ti3C2. The NIR-triggered Rapa release was beneficial for inhibiting the migration of LECs and suppressing the inflammatory response after photothermal therapy. The synergistic therapy studies of Rapa@Ti3C2-IOL show that BP- DOX@IOL possesses an excellent LECs killing ability and PCO prophylaxis both in vitro and in vivo. In addition, Rapa@Ti3C2-IOL had negligible toxicity for other intraocular tissues, which makes it promising for clinical translation. In summary, the present study provides evidence that this Rapa@Ti3C2-IOL nanosystem with excellent therapy efficiency and high biocompatibility may be applied as a promising strategy for preventing PCO.In this study, we coated Rapa@Ti