Dy2O@C82 isomers show SMM behavior with broad magnetic hysteresis, but the temperature and magnetization relaxation depend strongly on the fullerene cage. The short Dy−O distances and the large negative charge of the oxide ion in Dy2O@C82 result in the very strong magnetic anisotropy of Dy ions. Their magnetic moments are aligned along the Dy−O bonds and are antiferromagnetically (AFM) coupled. At low temperatures, relaxation of magnetization in Dy2O@C82 proceeds via the ferromagnetically (FM)‐coupled excited state, giving Arrhenius behavior with the effective barriers equal to the AFM‐FM energy difference. The AFM‐FM energy differences of 5.4–12.9 cm−1 in Dy2O@C82 are considerably larger than in SMMs with {Dy2O2} bridges, and the Dy∙∙∙Dy exchange coupling in Dy2O@C82 is the strongest among all dinuclear Dy SMMs with diamagnetic bridges. Dy‐oxide clusterfullerenes provide a playground for the further tuning of molecular magnetism via variation of the size and shape of the fullerene cage.A new class of single‐molecule magnets (SMMs) based on Dy‐oxide clusterfullerenes is synthesized. Three isomers of Dy The smallest fragment of dysprosium oxide is stabilized inside a fullerene. Three isomers of oxide clusterfullerenes Dy2O@C82 are synthesized and characterized by single‐crystal X‐ray diffraction. The compact size of the Dy2O cluster leads to unusually short Dy—O bonds, strong magnetic anisotropy, and enhanced antiferromagnetic interactions between Dy ions. Dy2O@C82 isomers behave as single molecule magnets with broad magnetic hysteresis. The same group soon isolated another type of oxide clusterfullerene, Sc2O@Cs(6)‐C82, in which the Sc—O—Sc cluster has a bent shape with µ2‐coordinated oxide ion.2 to the atmosphere in the arc‐discharge reactor, Feng and Chen et al. obtained and structurally characterized a series of Sc2O@C2n molecules with 2n ranging from 70 to 82.3+)2O2−@C2n4−, and therefore the preferable fullerene cages for encapsulation of Sc2O are those, which form stable tetraanions. Compared to the extensively studied Sc2O@C2n, the information on the oxide clusterfullerenes of metals other than Sc is very scarce. Mixed‐metal ScGdO@C82 was synthesized and characterized spectroscopically.2O@C2n species for M = Y, Lu was proved by mass‐spectrometry,[qv: 12b] but none of them has been isolated so far. Very recently, Ho2O@C74 was obtained and its molecular structure with an almost linear Ho2O cluster was elucidated by single‐crystal X‐ray diffraction.Oxide clusterfullerenes were first discovered in 2008 by Stevenson et al., who reported the isolation of Sc9 electrons, Dy3+ is a Kramers ion, which ensures that its ground magnetic state is a doublet with orthogonal wave functions. This combination of a bistable magnetic ground state and a strong single‐ion magnetic anisotropy are essential for the single molecule magnetism (SMM).2N@C80 in 2012,In this work, we report on the synthesis and characterization of the first Dy‐oxide clusterfullerenes. The recent interest in Dy‐EMFs is caused by their robust magnetic properties.2ScN@C80‐hI has been the strongest SMM among the clusterfullerenes so far. It has the blocking of magnetization at TB = 8 K, and above 60 K its spin relaxation follows the Orbach mechanism with the thermal barrier of 1206 ± 15 cm−1 corresponding to the 5th Kramers doublet of Dy ions.[qv: 19c] The computational studies revealed that Dy‐oxide clusterfullerenes may have superior SMM properties with thermal barriers up to 1400 cm−1.[qv: 19e] Here we provide the first experimental study of the magnetic properties of three isomers of Dy2O@C82. We demonstrate that Dy2O‐clusterullerenes are excellent single molecule magnets exhibiting broad magnetic hysteresis and the strongest superexchange coupling between Dy ions ever reported for nonradical bridged compounds.The nitride clusterfullerene Dy22.12O@C82 (Cs(6), C3v(8), C2v(9)) were synthesized by a modified Krätschmer‐Huffman DC arc‐discharge method under a He/CO2 atmosphere (200 Torr of helium with 20 Torr of CO2 added).2) under an argon atmosphere for 12 h. The crude extract was treated with TiCl4 and most of the empty fullerenes were removed .2O@C82 isomers with Cs(6), C3v(8), and C2v(9) fullerene cages were isolated and purified by multistage high‐performance liquid chromatography (HPLC) . In Figure2O@C82, which confirms their molecular formulas.Dy2.22 solution of the Dy2O@C82 isomers. X‐ray diffraction of Dy2O@Cs(6)‐C82∙Ni(OEP)∙2(C6H6) was measured at 100 K using the wavelength of 0.82653 Å with a CCD detector at the beamline BL17B of the Shanghai Synchrotron Radiation Facility (SSRF); Dy2O@C3v(8)‐C82∙Ni(OEP)∙1.5(C6H6)∙CS2 and Dy2O@C2v(9)‐C82∙Ni(OEP)∙C6H6 were measured with Bruker APEX II at room temperature and 173 K, respectively. The structures were solved by direct methods and refined using all data (based on F2) by SHELX 2016.FigureCs, C3v, and C2v isomers, respectively, which is similar to the previous reports.Crystals suitable for X‐ray diffraction analysis were grown by layering the benzene solution of nickel (II) octaethylporphyrin (Ni(OEP)) onto the CS2O@Cs(6)‐C82∙Ni(OEP)∙2(C6H6) contains two fullerene sites A and B, as well as two Ni(OEP) sites. This phenomenon was already reported for two similar crystals Sc2S@Cs(6)‐C82∙Ni(OEP)∙2(C6H6)2C2@Cs(6)‐C82∙Ni(OEP)∙2(C6H6),2O cluster with the major site occupancies of Dy of 0.76 and two minor sites with occupancy of 0.15 and 0.09. In the site B the Dy2O cluster is less ordered with the site occupancies of 0.52, 0.29, and 0.19 . The cage and oxygen sites also show some sign of disorder, although the structure still can be modeled satisfactorily using a single cage orientation. Structural parameters of the encapsulated Dy2O cluster can be determined reliably from the major site in the well‐ordered structure A. The Dy—O bond lengths are 2.009(9) and 2.002(9) Å, and the Dy—O—Dy bond angle is 138.8(5)°.The asymmetric unit of Dy2O@C3v(8)‐C82∙Ni(OEP)∙1.5(C6H6)∙CS2, the asymmetric unit contains one intact Ni(OEP) and one fullerene molecule with ordered carbon cage. The encapsulated Dy2O is strongly disordered, with 8 sites refined for Dy with occupancies ranging from 0.518(3) to 0.137(3) as shown in Figure 2O@C2v(9)‐C82∙Ni(OEP)∙C6H6 contains a half of the fullerene molecule and a half of the Ni(OEP) molecule. The crystallographic mirror plane bisects both the Ni(OEP) and the fullerene cage through the mirror plane of the molecules. Oxygen atom is refined as a single site located on the crystallographic mirror plane near the center of the carbon cage. Dy atoms are severely disordered with 14 refined sites ‐C82 and Dy2O@C2v(9)‐C82 precludes precise determination of the cluster structural parameters for these isomers. Average Dy—O distances (weighted with Dy site occupancies) are 1.978(9) and 1.944(8) Å in C3v(8) and C2v(9)‐C82 isomers. The average Dy—O distances in Dy2O@Cs(6)‐C82 isomer are 1.985(11) Å for the site A and 1.939(12) Å for the less ordered site B, which shows that the disorder in the structure reduces the apparent Dy—O distances. Thus, based on the most reliable parameters determined for the site A of Dy2O@Cs(6)‐C82, we conclude that the Dy—O bond lengths in Dy2O@C82 are close to 2.0 Å. This value is much shorter than 2.30 Å, which might be the expected based on the covalent radii of Dy (1.67 Å) and O (0.63 Å).2O@C82 are indeed among the shortest ever reported for molecular Dy compounds 13], featuring Dy5 pyramids with the shortest Dy—O bond of 1.951(18) Å.A strong disorder of the Dy2O@C82 are rather common and occur in different types of EMFs, including oxide,Cs(6)‐C82, C3v(8)‐C82, and C2v(9)‐C82 cages characterized by single‐crystal X‐ray diffraction in the form of co‐crystals with M(OEP)  is given in Table S3 and Figures S7–S46 (Supporting Information). Analysis of these crystal structures shows that the orientation of the same cage isomer toward the porphyrin moiety is not fixed but can vary from one EMF to another. For 17 EMF structures with Cs(6)‐C82 cage, we found four different ways how the cage is oriented with respect to Ni(OEP). The EMF∙Ni(OEP) orientation found in this work for Dy2O@Cs(6)‐C82 is similar to several other EMFs, including carbides, sulfides, and dimetallofullerenes. Eight EMFs with the C3v(8) cage reported so far show three different orientation types. The cage‐Ni(OEP) orientation in Dy2O@C3v(8)‐C82∙Ni(OEP) appears to be unique and does not fit any of these three. The EMFs with C2v(9)‐C82 cage demonstrate the largest structural diversity with 6 orientations in 14 reported crystal structures. The Dy2O@C2v(9)‐C82 system adds to this diversity yet another unique cage‐Ni(OEP) orientation.All three fullerene cage isomers found in this work for Dy2.32O clusters inside the fullerenes. As the endohedral clusters may have multiple energy minima inside the carbon cages, it is important to have a comprehensive sampling including all possible structural configurations. We have recently shown that rotation of the endohedral cluster along the Fibonacci sphere nodes provides such efficient and homogenous sampling for structural studies of EMFs.2O@C82 at the PBE‐D level with PAW potentials using the VASP 5.0 code.Cs(6) , five for C3v(8) , and eight for C2v(9) . Note that the results of the calculations for Y2O@C82 and re‐optimization results for Dy2O@C82 are quite close to each other , which confirms once again that Y is a good model for Dy in computational studies of EMFs.Whereas the fullerene cage structures are unambiguously elucidated by single‐crystal X‐ray diffraction, the strong disorder in positions of Dy atoms does not allow precise determination of the endohedral cluster positions. Therefore, we performed DFT calculations to find the preferable geometries and orientations of the Dy2O@C82 analogs with atomic masses of Dy assigned to Y. Single point energies and forces were calculated at the PBE/TZ2P level using Priroda code. These forces were used to propagate the system in the canonical ensemble (NVT) using Nose‐Hoover algorithm as implemented in the Python Atomic Simulation Environment libraries (ASE 3.0).−1 for each isomer  were propagated with time step 1 fs for 18–20 ps using the initial conformers geometries as starting points and initial velocities assigned randomly from Maxwell‐Boltzmann distribution at 300 K. For the sole conformer of Cs isomer, the propagation was continued for 80 ps. These trajectories were multiplied then by the cage symmetries and combined to evaluate the spatial distribution of O and Dy atoms at a given unit volume inside the fullerene with discretization of 0.042 × 0.042 × 0.042 Å3. The probability isosurfaces obtained by this approach for three isomers of Dy2O@C82 are plotted in FigureThe static DFT optimizations allow finding of the energy minima, but not provide sufficient information on the internal dynamics in the molecule. The presence of several energetically close minima may indicate a flat potential energy surface, which in due turn may result in complex internal dynamics of the endohedral cluster and its freezing in multiple conformers when the compound is cooled down. DFT‐based Born‐Oppenheimer molecular dynamics (BOMD) simulations were performed to shed more light onto this question. All BOMD calculations were performed for Y2O@Cs(6)‐C82 isomer, DFT calculations favor one particular conformer, which is more stable than all other six structures by 20–40 kJ mol−1. This conformer coincides with the major Dy1A–O1–Dy2A site resolved by X‐ray diffraction in Dy2O@Cs(6)‐C82∙Ni(OEP)∙2(C6H6) ‐C82,[qv: 4c] Sc2S@Cs(6)‐C82,2C2@Cs(6)‐C82,2@Cs(6)‐C82,[qv: 36c] Tm2@Cs(6)‐C82,[qv: 33b] Tm2C2@Cs(6)‐C82,[qv: 33b] and Er2@Cs(6)‐C82.[qv: 36b] The BOMD trajectory started from the optimized structure of this conformer shows oscillation of the cluster near the starting position for at least 80 ps without rearrangement of the cluster. This dynamical behavior resembles that in Y2S@C82 with Cs cage symmetry reported by some of us recently.[qv: 4c] Thus, both experiment and theory agree in that the Cs(6)‐C82 cage has two well‐defined positions for endohedral metal atoms common for different types of clusterfullerenes and dimetallofullerenes and that metal atoms tend to reside in these positions without extensive motion of the cluster. From two minor sites of the Dy2O cluster in Dy2O@Cs(6)‐C82∙Ni(OEP)∙2(C6H6), the one with the occupancy of 0.15 is close to the third most stable DFT‐optimized conformer with the relative energy of 24 kJ mol−1 . At this time we could not locate energy minimum close to the structure of the third minor site.For Dy2O@C3v(8)‐C82 our calculations located five unique conformers with three of them lying within 1 kJ mol−1, and two others being at ≈10 kJ mol−1 higher . C3v point group further multiplies the Dy2O sites by symmetry operations, resulting in the dense filling of the space inside the fullerene with multiple quasienergetic positions of the cluster. The BOMD simulations reveal that the cluster easily rearranges itself between different minima, forming the egg‐shaped probability isosurface closely following the shape of the fullerene cage ‐C82 cage.13C NMR spectroscopy also suggests free rotation of the endohedral cluster in M2C2@C3v(8)‐C82 and M2@C3v(8)‐C82 C3v symmetry of the fullerene.For Dye Figure . The onl2O@C3v(8)‐C82∙Ni(OEP)∙1.5(C6H6)∙CS2 crystal are disordered. In fact, the relative location of Dy sites and the shapes of thermal ellipsoids indicate that the Dy2O cluster is probably moving continuously, so that Dy atoms are “delocalized” in some regions inside the fullerene. The group of Dy sites  with combined occupancy of 1.0 corresponds to one such region, whereas the sites  demark moving areal of another Dy atom. Comparison with DFT‐optimized conformers shows that there is indeed a clustering of Dy positions of the two most stable conformers and one conformer with the energy of 9 kJ mol−1 in each of these regions . The DFT‐optimized Dy—O bonds length and Dy—O—Dy angles in the two most stable conformers of Dy2O@C3v(8)‐C82 are 2.017 Å/2.044 Å/139° and 2.014 Å/2.035 Å/145°.Thus, it comes as no surprise that the metal positions in the Dy2O@C2v(9)‐C82 located eight unique structures , two of which are almost isoenergetic within 2 kJ mol−1, five structures have close relative energies in the range of 11–16 kJ mol−1, and one is rather unstable with ΔE at 63 kJ mol−1. The cluster dynamics in the C2v(9)‐C82 isomer presents an intermediate situation between localized motions as in Cs(6)‐C82 and almost completely delocalized trajectory as in C3v(8)‐C82: BOMD simulations reveal that the regions of high probability density are more extended than in the Cs isomer but are not as delocalized as in C3v. Comparison to the diffraction data allows assignment of the real energy minima to sites Dy1–O–Dy2 and Dy1–O–Dy3, as well as their symmetry equivalents Dy1'–O–Dy2 and Dy1'–O–Dy3. The combined occupancy of these sites is 0.72. There is no theoretical minimum close the experimental Dy1–O–Dy2 cluster configuration. Remarkably, the relative energies of the conformers corresponding to the sites Dy1–O–Dy2' and Dy1–O–Dy3 are 14 and 16 kJ mol−1, respectively, and their Dy—O bonds length and Dy—O—Dy angles are 2.004 Å/2.040 Å/155° and 2.010 Å/2.047 Å/149°. Thus, unlike in Cs(6) and C3v(8) isomers, the major experimental sites of Dy atoms in Dy2O@C2v(9)‐C82∙Ni(OEP)∙C6H6 do not correspond to the lowest energy conformers of the free Dy2O@C2v(9)‐C82 molecule.The conformer search for Dy2O cluster positions inside the fullerene cages. For the C3v isomer, this leads to the presence of a limited number of cluster positions from the multitude that might be expected based on the close energies of several conformers and a high symmetry of the cage. For the C2v isomer, the observed conformations are not the most stable ones predicted theoretically. Besides, the application of the symmetry operations of the fullerene cage to the cluster positions should also give much more equivalent cluster sites than found experimentally. Our recent computational study of the interaction of M3N@C80 clusterfullerenes with Ni(OEP) in molecular complexes and crystals demonstrates that the intermolecular interactions result in the spread of the relative energies of the formally equivalent conformers in the range up to 20 kJ mol−1.To summarize, although there is an agreement between the DFT‐predicted conformers and experimentally determined positions of metal atoms inside the fullerene cages, it is also clear that intermolecular interactions in the crystals noticeably affects distribution of the DyC2v(9)‐C82 cages.Thus, the packing of the fullerene cages and the realization of certain cluster conformers in the crystals is a result of a subtle balance between the conformer stability and the cage‐Ni(OEP) interactions. When fullerene cage provides different conformations for the position of the endohedral cluster, the cluster can adjust its orientation during crystallization to minimize the energy for a given EMFs‐Ni(OEP) orientation. Furthermore, the minimization of the total crystal energy can be achieved for higher‐energy conformers of free EMF molecules, when they provide a more favorable spatial distribution of the electrostatic potential and optimal intermolecular interactions. Besides, if the EMF molecule has equally stable conformers with different location of metal atoms, a large variety of possible fullerene‐Ni(OEP) orientations may be also realized in different crystals as found for the EMFs with C2v(9) isomer is obviously the most polymorphic one. On the other side is the Cs(6) isomer with the ratio of 17:4. In fact, the vast majority of the crystal structures of EMFs with the C82‐Cs(6) cage belong to only two orientation types: one is typical for EMFs with two endohedral metal atoms, whereas another type is found for EMFs with one metal (see the Supporting Information for more details). Thus, we conclude that the enhanced stability of one particular conformer in the isolated EMF molecule also results in the reduced variety of the crystal structures realized for this EMF, whereas the conformational flexibility also leads to a structural diversity.The ratio of the total number of reported crystal structures to the number of orientation types can be used as a criterion of the structural diversity. With the ratio of 15:7, the 2.42O@C82 isomers were studied by means of vis–NIR absorption spectroscopy and electrochemistry (cyclic voltammetry). Full details of these studies are presented in Supporting Information . In brief, absorption features of the studied compounds are similar to those of other metallofullerenes with C82 cages in a formal 4– charge state. The overall redox behavior of the three isomers of Dy2O@C82 varies from cage to cage and show noticeable deviation in redox potential from Sc2O@C82 analogs. DFT calculations show that frontier orbitals of all three isomers are localized on the fullerene with negligible contribution from the Dy2O cluster .Electronic properties of Dy2.52.5.12O@C82 were studied by DC SQUID magnetometry using a Quantum Design VSM MPMS3 magnetometer. All three isomers showed magnetic hysteresis at low temperatures  and is closing near 8 K. The C2v isomer shows the broadest hysteresis at 2 K , which is observed up to 8 K. Hysteresis loops of all three compounds have inflections at 1–1.5 T, which are most pronounced for the C2v isomer. The nature of these features is discussed further in the text.Magnetic properties of DyTB) were determined by comparing magnetic susceptibility  of the sample measured during cooling in the field of 0.2 T (FC) to the curve measured during in‐field warming of the sample preliminary cooled down in zero field (ZFC). χZFC curves measured with a temperature sweep rate of 5 K min−1 have well defined maxima at 4.4 K (Cs), 7.4 K (C3v), and 5.8 K (C2v). At the same time, χZFC and χFC curves diverge up to somewhat higher temperatures, 10 K (Cs), 9 (C3v), and 8 K (C2v). Again, the shape of the carbon cage has a strong influence on the SMM properties of the encapsulated Dy2O cluster  were measured by DC magnetometry. The magnetization of the samples was first saturated in the field of 7 T, then the field was scanned to zero field (or to a certain finite field) with the highest possible sweep rate of 70 mT s−1 allowed by the magnetometer, and then the decay of magnetization was recorded while the sample was slowly restoring its equilibrium magnetization. The decay curves were then fitted with a stretched exponential function to determine an average relaxation time .To get the information on the relaxation of magnetization in DyFigureT−1). A significant variation of the properties in dependence on the fullerene cage isomer can be well seen again. Cs and C2v isomers show exponential decay of relaxation time with temperature (straight line in log(τ)‐vs‐T−1 coordinates) in the whole accessible temperature range. Such a temperature dependence of relaxation time is usually associated with an Orbach relaxation mechanismUeff is the energy barrier corresponding to the excited spin state involved in the spin reversal, and τ0 is the attempt time. Fit of the experimental data with Equation Ueff = 10.8 ± 0.1 K and τ0 = 2.1 ± 0.1 s for the Cs isomer and Ueff = 18.6 ± 0.2 K and τ0 = 0.63 ± 0.06 s for the C2v isomer. More complex temperature dependence is found for the C3v isomer. Its relaxation times can be best fitted by a combination of two linear processes with Ueff/τ0 parameters of 7.7 ± 0.3 K/165 ± 27 s (prevails at the lowest temperatures) and 22.6 ± 0.8 K/2.63 ± 0.35 s. An onset of another relaxation mechanism can be also seen near 6 K, but shorter relaxation times cannot be measured reliably with DC magnetometry. Alternatively, a temperature dependence of the relaxation times of the C3v isomer can be also described by a power function of temperature, τ−1(T) = CTn, with parameters C = (7.1 ± 0.2) × 10−6 s−1 K−n and n = 4.12 ± 0.03.Relaxation times measured in zero field are plotted in 2O@C82 isomers is hundreds of K (see discussion below). Hence, the Orbach relaxation via single‐ion LF‐excited states of Dy can be excluded as an explanation of the Arrhenius behavior at low temperatures. Instead, one has to consider the spin states formed by the intramolecular interaction of two Dy ions as had been already found in Dy2ScN and Dy2S clusterfullerenes.The attempt times of the linear processes are much longer than normally observed for the Orbach mechanism involving a ligand‐field excited state of a lanthanide ion. Besides, the ligand field splitting for Dy ions in Dy2.5.22O@C82. For each Dy2O@C82 structure, one of the Dy ions was replaced by Y, and then the multiplet structure for Dy (6H15/2) in DyYO@C82 was calculated at the CASSCF/SO‐RASSI level using SINGLEANISO routine to extract ab initio ligand‐field parameters.2O@C82 isomers gave similar results. Figure2O@C82‐Cs (major conformer). Analogous data for different conformers of other Dy2O@C82 isomers are summarized in the Supporting Information . The oxide ion at the distance of ≈2 Å from Dy3+ imposes a very strong uniaxial anisotropy in the latter. The quantization axes for Dy ions are almost coinciding with corresponding Dy—O bonds, and the overall LF splitting is in the range of 1360–1490 cm−1. The energy of the first excited Kramers doublet (KD) varies from 386 to 515 cm−1. In terms of the | J, Jm〉 basis, the first KDs have nearly pure composition , and substantial mixing of Jm functions starts from the fifth or sixth KD. Accordingly, transition probabilities within one KD attain significant values only in the fifth‐sixth KDs, which shows that the Orbach relaxation via the ligand‐field excited states should have a barrier of at least 1200–1300 cm−1 (≈1700–1900 K). Similar findings were reported by Singh and Rajaraman [qv: 19e] in a computational study of DyLuO@C2n and DyScO@C2n  molecules. Unfortunately, the isolable amount of Dy2O@C82 is not sufficient for its study by AC magnetometry, which is required for the measurement of relaxation times above TB. At the temperatures accessible for DC magnetometry, the relaxation of magnetization in Dy2O@C82 is not governed by excited ligand‐field states.Ab initio calculations were performed to get information on the single‐ion magnetic anisotropy of Dy ions in Dy2O@C82 structures we studied are similar, a variation of the LF energies by 100–200 cm−1 should be pointed out. There seems to be no difference between cage isomers in this regard, i.e., the changes between different conformers are comparable for each cage isomer. The LF parameters may be influenced by the variation of geometrical parameters of the Dy2O cluster. However, we could not find any correlation between Dy—O distances and the energy of the first excited KD or the total LF splitting . Most likely, the differences are caused by the fullerene cage, and in particular by the arrangement of carbon atoms at the Dy coordination sites.Although the overall anisotropy patterns for Dy ion in all Dy6 coordination. More often, Dy is displaced from the position above the center of a hexagon toward one of the pentagon/hexagon edges. In this situation, coordination motif changes to η4 or even η2. In the third coordination type, Dy is located close to the pentagon/hexagon/hexagon (acenaphthylene) moiety and exhibits η2 coordination to the hexagon/hexagon edge . In general, there is a quasicontinuum of Dy‐cage coordination geometries as the outlined motifs are interconvertible via small displacements of Dy ions. At this moment, we could not find any obvious correlation between Dy‐fullerene coordination site and variation of the LF splitting.Analysis of more than twenty Dy‐cage coordination sites in DFT‐optimized structures  revealed three main motifs. In some conformers, Dy is located above the center of a cage hexagon and exhibits ηC2v isomer . Here Dy has a unique η4 coordination to a pentagon, in which metal atom is not exactly above the center of the latter, but is close to one of the 5/6 edges. This type of metal‐cage coordination imposes a stronger influence on the spin properties of Dy ion as its quantization axis is displaced noticeably from the Dy−O bond. Interestingly, the η5 coordination of Dy to cyclopentadienyl ring is known to produce the largest LF splitting in Dy‐SMMs, including Dy‐metallocenes with the highest blocking temperatures of magnetization among SMMs.The highest LF splitting is realized for a high‐energy conformer of the 2.5.32O@C82 clusterfullerene is a Kramers doublet with essentially pure Jm = ±15/2 composition and magnetic moment aligned along the Dy–O bond . The total magnetic moment is then aligned along the z axis. In the other doublet, the moments have opposite orientation along the z axis, but inevitably have the same orientation in the perpendicular direction (defined as y‐axis hereafter). The total magnetic moment is then aligned along the y‐axis. In the following, the states with the moment along the z‐axis will be defined as ferromagnetic (FM), whereas the states with the moment along the y‐axis are defined as antiferromagnetic (AFM) since magnetic moment of the FM state is larger than that of the AFM state as long as the ∠Dy—O—Dy angle exceeds π/2.According to ab initio calculations, magnetic ground state for each Dy ion in Dynd Figure8a. For2O@C82 can be reasonably well described by the following spin HamiltonianH^LFi are single‐ion ligand‐field Hamiltonians , j12 is the coupling constant between the lanthanide moments, Ji^, and H^ZEE describes Zeeman splitting. The energy difference between FM and AFM states can be then computed asJm = ±15/2 ground state of Dy3+. The shape of the χT curves  parameters – they should be on the iso‐energy lines plotted in Figure 2O@C82. Using Hamiltonian Eq. j12/α values: −0.052 cm−1/50° for the Cs isomer, −0.048 cm−1/60° for the C3v isomer, and −0.093 cm−1/52° for the C2v isomer . Note that if the sign of j12 is reversed (favoring then the FM state), the χT curves develop a characteristic sharp peak at low temperatures. Such peaks are not observed experimentally, proving that the ground magnetic state of all three Dy2O@C82 isomers is indeed AFM. The optimal angles α should be understood as an average value since there is most probably a distribution of Dy—O—Dy angles in the real structures  µB = 18.1 µB between 1 and 2 T as the FM state becomes lower in energy. If the field is applied along the y‐axis, the FM state remains intact, whereas the AFM state undergoes Zeeman splitting. The magnetization increases fast from zero to the saturation at 10∙2sin(α/2) µB = 8.5 µB.The inflections in the low‐temperature magnetization curves of DyT = 1.8 K reproduces a similar inflection in the real experimental curve . For instance, the QTM is usually the fastest relaxation process for single‐ion magnets in zero field. Dy∙∙∙Dy interactions in dinuclear systems quench the zero‐field QTM. But the QTM may be operational in the finite fields, at which the level crossing takes place. To analyze this possibility, we studied the field dependence of relaxation times. The dependencies were measured at 2.5 K to have reasonably but not extremely long relaxation times (accuracy of the measured times decrease as they become shorter than ≈100 s). For the 2O@C82 isomers exhibit a gradual decrease of the relaxation time with the increase of the field. This behavior can be attributed to the increasing contribution of the direct relaxation mechanism, in which the spin flip is caused by the phonon matching the Zeeman splitting. With further increase of the magnetic field, all isomers show an abrupt acceleration of the relaxation. For the Cs isomer the change of the relaxation mechanism starts already at 0.4 T, for the C3v and C2v isomers the changes become apparent at 0.7–0.8 T. At further increase of the field, relaxation times increase again and then turn to a gradual decay. The dips (negative peaks) in the τ‐H dependencies are ≈1 T broad and correspond to the inflections in the magnetization curves. We propose that they are caused by the QTM at the AFM‐FM level crossing. The large width of these “resonances” is caused by the distribution of the molecular orientations in the powder sample as well as by the distribution of dipolar fields.Figure C3v and C2v isomers, the QTM is dominant up to 3 K, whereas thermally activated processes start to contribute above that temperature. For C3v, we could fit the whole dependence by a combination of QTM with τQTM of 394 ± 6 s and an Orbach process with Ueff of 19.3 ± 0.7 K and τ0 of 2.3 ± 0.4 s. Note that parameters of this Orbach process are similar to those observed for the second Orbach process in zero field. For the C2v isomer, the τQTM is found to be 140 ± 5 s.To prove the QTM mechanism, we studied the temperature dependence of relaxation times at the fields close to the centers of the dips. The characteristic feature of the QTM is the temperature independence of the relaxation times  = CTn, with C = (8.4 ± 0.4) × 10−3 s−1 K−n and n = 0.75 ± 0.06. Such a small exponent in the power function may correspond to the direct process . Alternatively, this temperature dependence can be also described by an Orbach process with Ueff = 1.7 ± 0.1 K and τ0 = 29.8 ± 1.5 s. But since the relaxation times of the Cs isomer are already rather short and therefore are not very precise, we prefer to restrain ourselves from a further discussion of the reason for the temperature dependence of the in‐field relaxation times in the Cs isomer. It is suffice to note that in‐field relaxation times of the Cs isomer are much faster than zero‐field values and that the temperature variation is substantially less pronounced than in zero field.For the 2O@C82 have AFM ground state and exhibit sufficiently slow relaxation of magnetization to develop a magnetic hysteresis. In zero field, relaxation of magnetization follows the Orbach mechanism involving the FM excited state, which allows precise determination of the AFM‐FM energy splitting. The latter, in due turn, simplifies determination of the coupling constant describing Dy∙∙∙Dy interaction and the average angle between magnetic moments of Dy ions. As the FM state shows stronger field dependence, this state becomes prevalent at high fields. At the fields corresponding to the AFM‐FM level crossing, all isomers exhibit QTM, which is apparent from the “dips” in the field dependence of the relaxation times as well as from the temperature‐independent in‐field relaxation times.To summarize this section, we showed that all three isomers of Dy2.5.52O@C82 to other SMMs based on Dy‐clusterfullerenes. Structurally, oxide clusterfullerenes are similar to sulfide clusterfullerenes. Both Dy2O and Dy2S species transfer four electrons to the fullerene cage and hence prefer the same cage isomers, and both clusters have an angular shape . Earlier we isolated Dy2S@C82 with Cs(6) and C3v(8) cage isomers and studied their SMM properties.[qv: 4c] Cs isomer of Dy2S@C82 was found to be a softer SMM than the C3v isomer showing a similar effect of the fullerene cage isomer as found in this work for Dy2O@C82.It is instructive to compare the magnetic properties of Dy2O@C82 are substantially different from those of Dy2S@C82. As can be seen from comparison of magnetic hysteresis curves of Dy2O@C82 and Dy2S@C82 with Cs and 3Cv cage isomers shown in FigureTB/TB100 temperatures of the C3v isomer of Dy2S@C82 are 4.0/2.0 K, whereas for Dy2O@C82 they are increased to 7.4/5.9 K. Thus, replacement of S by O in otherwise similar structures results in a dramatic improvement of the SMM performance. The strength of Dy∙∙∙Dy exchange interactions in Dy2S cluster is similar to those in Dy2O but has an opposite sign . Furthermore, the Dy—S bonds of 2.4–2.5 Å are much longer than Dy−O bonds, and the LF in sulfide clusterfullerenes is almost twice smaller. Ab initio calculations predict that the overall LF splitting in Dy2S@C82 is 880–970 cm−1, whereas the energy of the first excited KD is 220–300 cm−1.But in other aspects, magnetic properties of Dy2O@C82 is similar or even higher than in Dy2ScN@C80‐hI. So far, the latter has been the strongest SMM among the clusterfullerenes.[qv: 18a] Note that Dy2@C80(CH2Ph), which is the strongest SMM among all Dy‐EMFs, not considered as a clusterfullerene.[qv: 18b]TB of Dy2O@C82‐C3v studied in this work is close to that of Dy2ScN@C80, and the TB100 value of the oxide is even higher than for the nitride clusterfullerene. Apparently, similarity of the LF also results in comparable SMM properties, at least at low temperatures. Both types of EMF‐SMMs also exhibit similar spin‐relaxation mechanism at low temperature featuring the exchange‐excited state of the dinuclear cluster. But in the Dy2ScN cluster, as in the aforementioned Dy2S, the ground state is FM,[qv: 22b] whereas Dy2O favors AFM coupling.The size of the LF splitting in DyhI for Dy2ScN@C80 or C3v for Dy2S@C82) are better SMMs than the analogous EMFs with less symmetric cages. The results of these work on three isomers of Dy2O@C82 perfectly follow this trend. The C3v isomer with the most pronounced motions of the Dy2O cluster  relaxation behavior.qv: 4c,22a] As insignificant variations of the LF are not relevant at these low temperatures, we hypothesized that the fullerene cage contributes to the relaxation of magnetization via the spin‐phonon coupling and the energy transfer from the spin system to the lattice. Free motion of the endohedral cluster indicates that the potential energy surface is flat, and that the cluster vibrations are weakly coupled to the fullerene modes. The spin‐lattice energy transfer is then not very efficient, i.e., the fullerene cage shields the endohedral cluster not only from chemically active environment but also from the lattice phonons. On the contrary, restricted motion of the endohedral cluster points to the stronger coupling to the cage vibrations, which facilitates the spin‐lattice relaxation. As a result, the EMFs with more “isotropic” symmetric cages  by affecting magnetic anisotropy of oxygen‐bonded Dy ions and (ii) by mediating Dy∙∙∙Dy exchange interactions via µLF Splitting: When a Dy—O bond is short and oxygen bears a considerable negative charge, a strong LF splitting evolves for the Dy ion. In the computational study, Ungur and Chibotaru predicted+ would have a very short Dy−O bond length of 1.74 Å and a large spin relaxation barrier exceeding 2100 cm−1. Whereas [DyO]+ cannot be obtained as an isolated entity, the closest experimental realization of such unit was reported by Kazin et al. via partial substitution of Ca or Sr in corresponding apatite lattices,+ fragments with the bond length of 2.05–2.14 Å. The solids then show Dy‐based magnetic hysteresis with blocking temperatures up to 11 K and thermal barriers of relaxation of 780–1040 cm−1 assigned to the 4th or 5th Kramers doublets.TabletBu)2(py)5][BPh4] salt with TB of 14 K and the relaxation barrier of 1261 cm−1, corresponding to the 5th‐8th Kramers doublets. The average axial Dy−O bond length in this molecule is 2.111(2) Å.Among the molecular compounds  in the temperature range of 60–76 K.[qv: 19c] Importantly, large LF splitting in Dy2O@C82 ensures that Dy ions remain in their ground magnetic state and that there are no efficient relaxation channels via LF excited state.As already discussed above, Dy—O bonds of ≈2.0 Å in DyExchange Interactions: Bridging oxo‐ligand is a ubiquitous element in the structure of polynuclear SMMs. Dozens of di‐nuclear Dy SMMs and a number of SMMs with higher nuclearity containing {Dy2O2} bridges have been reported,2O@C82 with one Dy—(µ2‐O)—Dy exchange channel to the exchange parameters in other SMMs with {Dy2O2} bridges, which have two such channels, is of high interest . It is not difficult to see that the FM‐AFM energy difference with this Hamiltonian is ΔEAFM − FM = 0.5Jtotcos(α) and that Jtot in Equation (4) and j12 in Equation Jtot = 450 j12 . Jtot, which is usually determined from the fit to experimental magnetization data, is often further divided into dipolar and exchange contributions, Jdip and Jexch. Dipolar term can be computed from the energy of the dipole‐dipole interaction:nr→ is the normal of the radius vector connecting two magnetic moments μ1→ and μ2→, and R12 is the distance between them. E12dip can be computed exactly using experimental or DFT‐computed atomic coordinates and ab initio derived orientations of the magnetization axes and g‐tensors of individual Dy centers. The exchange term can be then determined as a difference of Jtot and Jdip. Comprehensive EPR and magnetometry study showed that taking Jtot in Equation Jtot was treated as a tensor.Jtot. In the vast majority of magnetic studies of {Dy2O2} complexes the coupling was treated as isotropic, and we follow this approach in this work .The most common approach to the exchange interactions in {Dy2O@C82 isomers to a list of selected dinuclear SMMs with {Dy2O2} bridge described in the literature. The list includes all {Dy2O2}‐SMMs with ΔEAFM−FM exceeding 2 cm−1, as well as few other illustrative examples. The largest ΔEAFM−FM difference in complexes with {Dy2O2} bridges was reported recently by Gao et al. for the chain compound, in which dinuclear moieties are further linked via chloride bridges.EAFM−FM value for the {Dy2O2} fragment in this compound is 6 cm−1, Jtot amounts to 11.4 cm−1 (positive sign means FM interactions are favored), and Jdip/Jexch constants are both positive and of similar size. Four more {Dy2O2} compounds have |ΔEAFM−FM| values exceeding 3 cm−1, of them three feature antiferromagnetic coupling in the ground‐state. Again, Jdip/Jexch constants in the compounds with the strongest overall coupling have the same sign. For the vast majority of {Dy2O2} compounds, the |ΔEAFM−FM| is less than 3 cm−1. In some cases , Jdip and Jexch have relatively large values but opposite signs, leading to small overall Jtot. In most cases, dipolar interactions favor FM coupling, while exchange coupling tends to be of AFM nature. To our knowledge, there is no uniform model relating structural parameters and coupling constants in {Dy2O2} bridges so far. Analysis of the structural data in Table 3 does not reveal any obvious correlation between angles, bond lengths, Dy∙∙∙Dy distances, and J‐constants.Table 2O2} complexes to Dy2O@C82, one can find that the strongest coupling in the former is of the same size as in the C3v isomer of Dy2O@C82, which has the weakest Dy∙∙∙Dy coupling among the three Dy2O@C82 isomers. The coupling in Cs and especially C2v isomer is much stronger than in any {Dy2O2} complex reported to date. Further, dipolar interactions in Dy2O@C82 isomers all favor FM coupling, and because of the relatively short Dy∙∙∙Dy distances, Jdip values are comparably large and range from 8.6 to 10.2 cm−1. But these strong FM dipolar interactions are outweighed by even stronger AFM exchange coupling, so that the overall Dy∙∙∙Dy coupling in Dy2O@C82 is antiferromagnetic. To compensate positive Jdip constants, Jexch constants take the values from −31.8 cm−1 in C3v isomer and −32.6 cm−1 in Cs isomer to −50.5 cm−1 in C2v. These are the largest exchange coupling constants in polynuclear Dy compounds aside from the complexes with radical bridges.Comparing then {Dy32O@C82, and in depth studies of their magnetic properties. In accordance with the fourfold electron transfer expected for the Dy2O cluster, the three cage isomers isolated for Dy2O@C82 are Cs(6), C3v(8), and C2v(9). Single crystal X‐ray diffraction studies revealed that endohedral Dy2O cluster in Dy2O@C82 has bent shape and features unusually short Dy—O bonds of ≈2.0 Å.In this work, we succeeded in the synthesis of the first Dy‐oxide clusterfullerenes, the structural characterization of the three isomers of Dy2O clusters in Dy2O@C82 leads to both strong axial ligand field and unprecedentedly strong antiferromagnetic exchange coupling between Dy ions. All three isomers of Dy2O@C82 exhibit slow relaxation of magnetization and develop a magnetic hysteresis at low temperature. In zero field, the relaxation of magnetization follows the Orbach mechanism involving the ferromagnetic excited state. Thus, the effective barriers determined from Arrhenius plots of the temperature dependence of the relaxation times are equal to the energy difference between AFM‐ and FM‐coupled states. These differences in Dy2O@C82 are considerably higher than in any SMM with Dy—O—Dy bridges reported to date. At the fields corresponding to the AFM‐FM level crossing, all isomers of Dy2O@C82 exhibit quantum tunneling of magnetization, which is apparent from the negative peaks in the field dependence of the relaxation times as well as from the temperature‐independent in‐field relaxation times.The compact geometry of Dy2O clusters, though not fully understood, is quite considerable. First, the size of exchange coupling constant in the Dy2O cluster varies considerably with the cage isomer. Second, the fullerene cage isomerism affects internal dynamics of the Dy2O cluster, which appears to correlate strongly with the relaxation of magnetization. The C3v isomer, in which the cluster is moving almost freely, is found to be the strongest SMM among all three studied structures, whereas the Cs isomer with the fixed position of the endohedral cluster has the shortest magnetization relaxation times. In addition to dynamical properties, the size and the shape of the fullerene cage can also affect the structural parameters of the endohedral cluster, such as Dy—O bond lengths and Dy—O—Dy angle. Thus, further exploration of the Dy2O@C2n clusterfullerene family may reveal even better SMMs.The role of the fullerene cage in the magnetic properties of encapsulated DyThe authors declare no conflict of interest.SupplementaryClick here for additional data file.


Herein, the construction of a nanocatalytic medicine, CaO2@DOX@ZIF‐67, via a bottom‐up approach is described. CaO2@DOX@ZIF‐67 simultaneously supplies O2 and H2O2 to achieve improved chemo/chemodynamic therapy. In the weakly acidic environment within tumors, CaO2@DOX@ZIF‐67 is broken down to rapidly release the Fenton‐like catalyst Co2+ and the chemotherapy drug doxorubicin (DOX). The unprotected CaO2 reacts with H2O to generate both O2 and H2O2. The generated O2 relieves the hypoxia in the tumor and further improve the efficacy of DOX. Meanwhile, the generated H2O2 reacts with Co2+ ions to produce highly toxic •OH through a Fenton‐like reaction, resulting in improved chemodynamic therapy.Combined chemo/chemodynamic therapy is a promising strategy to achieve an improved anticancer effect. However, the hypoxic microenvironment and limited amount of H 2O2 in most solid tumors severely restrict the efficacy of chemotherapy and chemodynamic therapy, respectively. Herein, a nanocatalytic medicine, CaO2@DOX@ZIF‐67, is synthesized via a bottom‐up approach. By virtue of simultaneously producing O2 and H2O2 under acidic conditions, the fabricated nanocatalytic medicine can eliminate hypoxic tumors via enhanced chemo/chemodynamic therapy.The hypoxic microenvironment and limited amount of H The zeolitic imidazolate framework ZIF‐67 is stable in physiologically neutral conditions, and can therefore prevent CaO2 from reacting prematurely. However, ZIF‐67 should decompose in the acidic environment within tumor and rapidly release Co2+, which can then catalyze the decomposition of H2O2 to produce •OH through a Fenton‐like reactionScheme2 nanoparticles were first prepared with PEG‐200 as solvent and template. Then, DOX was coated onto the surface of the CaO2 via coordination between DOX and Ca to form CaO2@DOX. To further isolate and protect the CaO2@DOX from external water, ZIF‐67 was finally constructed on the surface of CaO2@DOX by an in situ synthesis method. This yielded the pH‐responsive nanocatalytic medicine CaO2@DOX@ZIF‐67. The outermost ZIF‐67 layer will be disassembled in the acidic environment of tumor due to its pH sensitivity, resulting in rapid release of Co2+ and DOX. At the same time, the unprotected CaO2 will react with water to produce O2 and H2O2. The generated H2O2 will then be further catalyzed by Co2+ ions to produce highly toxic •OH through a Fenton‐like reaction, while the generated O2 will improve the efficacy of DOX by alleviating the hypoxic conditions in the tumor. Therefore, this nanocatalytic medicine provides a novel strategy for improving the effectiveness of combined chemo/chemodynamic therapy.Herein, we designed a nanocatalytic medicine for coencapsulation of DOX and CaO2@DOX@ZIF‐67 was synthesized by a bottom‐up approach. First, CaO2 was prepared via a hydrolysis–precipitation procedure reported in the literature.2 to form CaO2@DOX by coordination reaction.2@DOX exhibited a significant red‐shift and two bands were observed at around 550 and 590 nm, which are characteristic of the Ca‐DOX complex .2@DOX by an in situ synthesis method. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of CaO2@DOX@ZIF‐67 revealed monodisperse nanospheres with a mean diameter of 200 nm  analysis of CaO2 and CaO2@DOX@ZIF‐67 is presented in Figure 2 exhibited distinct peaks (2θ) at 30.1°, 35.6°, and 47.3°, which were identical with the values reported in the literature for CaO2 (card number 03‐0865).2 are not obvious, which might be ascribed to the low crystallinity of CaO2. In contrast, ZIF‐67 is highly crystalline, and we can observe its dominant peaks in CaO2@DOX@ZIF‐67.2@DOX@ZIF‐67 was calculated to be 508.8 m2 g−1, which is much lower than that of ZIF‐67 (1492.5 m2 g−1). X‐ray photoelectron spectroscopy (XPS) was further used to characterize the chemical composition and chemical state of CaO2@DOX@ZIF‐67. The XPS survey spectrum showed that CaO2@DOX@ZIF‐67 is composed of Ca, O, Co, N, and C , the loading capacity of DOX was quantitatively determined to be 5.2 wt%. Even in CaO2@DOX, the loading efficiency of DOX was only 7.2%. This low loading capacity might be because the coordination sites on surface of the CaO2 were completely occupied. The release of DOX from CaO2@DOX@ZIF‐67 was measured in buffers of different pH. As shown in Figure2+ within 2 min at pH 5.0. Then, the stability of CaO2@DOX@ZIF‐67 in serum and phosphate buffered saline (pH 7.4) was also investigated using DLS. Figure S4 (Supporting Information) showed that no detectable changes in particle size was observed within 24 h.UV–vis spectrophotometry was used to determine the loading of DOX in CaO2 is predicted to react with water to produce O2 and H2O2. The generation of H2O2 arising from the reaction of CaO2 and water was evaluated using an H2O2 assay kit. As shown in Figure 22+) was obtained in the presence of CaO2@DOX@ZIF‐67. The next prediction is that the generated H2O2 will further react with Co2+ ions released from ZIF‐67 through a Fenton‐like reaction to produce highly toxic •OH. To prove this, we used 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO), a •OH trapping agent. The production of •OH was measured by electron spin‐resonance (ESR) spectrometry, in which the DMPO/•OH adduct presents a characteristic 1:2:2:1 four‐line signal.2@DOX@ZIF‐67 is stable in physiologically neutral conditions, while under acidic conditions, •OH radicals are effectively generated by Co2+‐catalyzed decomposition of the produced H2O2 through a Fenton‐like reaction. As control, the production of •OH was also measured in the presence of CaO2@DOX and ZIF‐67 + H2O2, respectively. The results further demonstrated that Co2+ ions acted as the necessary Fenton‐like reaction catalyst driving generation •OH radicals . In addition, the generation of •OH was further evaluated at pH 5.0 using 3,3′,5,5′‐tetramethylbenzidine (TMB), which is oxidized by •OH to form ox‐TMB.2@DOX@ZIF‐67. In order to quantitatively determine the amount of H2O2 generated from CaO2@DOX@ZIF‐67, the absorbance values of the ox‐TMB were measured at 370 nm and a standard curve was plotted . According to the standard curve, 58.4 µmol of H2O2 is produced by 1 mg of CaO2@DOX@ZIF‐67. This would greatly improve the efficacy of CDT.After the decomposition of ZIF‐67, the unprotected CaO2@DOX@ZIF‐67 was also determined. Twenty milligrams of CaO2@DOX@ZIF‐67 were added to 20 mL of buffer solution (pH 5.0) and the concentration of dissolved oxygen was monitored by a portable oxygen meter in real time. As shown in Figure 2@DOX@ZIF‐67 had a higher oxygen concentration (78 µmol g−1), which would improve the efficacy of DOX treatment. In order to visualize the formation of oxygen bubbles, a video was taken of the reaction of CaO2@DOX@ZIF‐67 with H2O at pH 5.0 (Video S1 is available in the Supporting Information). In addition, the exclusive formation of O2 was confirmed using gas chromatography (GC). Figure 2@DOX@ZIF‐67 had the same retention time as pure O2. These results indicate the excellent and specific O2‐generating capacity of CaO2@DOX@ZIF‐67. In summary, our data demonstrate that CaO2@DOX@ZIF‐67 has many properties which hold promise for enhanced chemo/chemodynamic therapy.The amount of oxygen generated by CaO2, we further investigated the in vitro anticancer effect of CaO2@DOX@ZIF‐67. The viability of MCF‐7 cells treated with CaO2@DOX@ZIF‐67 was evaluated by standard methyl thiazolyl tetrazolium (MTT) assay. As shown in Figure2@DOX@ZIF‐67 and the pH value. At the same concentration of CaO2@DOX@ZIF‐67, the cytotoxic effect was greater under slightly acidic conditions (pH 6.5) than under neutral conditions (pH 7.4). These results indicate that the cytotoxic effect of CaO2@DOX@ZIF‐67 is mainly triggered by acidic conditions. The colocalization assay demonstrated the acidic condition was mainly provided by the lysosome of MCF‐7 cells .Encouraged by the efficient production of •OH and O2@DOX@ZIF‐67 on MCF‐7 cells is independent of the concentration of oxygen owing to the capacity of CaO2@DOX@ZIF‐67 to relieve hypoxia. Figure 2@DOX@ZIF‐67 exhibited similarly high levels of cytotoxicity toward MCF‐7 cells under both normoxic (21% O2) and hypoxic (1% O2) conditions. The cytotoxic effects of CaO2@DOX and CaO2@ZIF‐67 on MCF‐7 cells were also independent of the oxygen concentration. In contrast, the viability of cells treated with free DOX or DOX@ZIF‐67 is clearly higher under hypoxic conditions than under normoxic conditions. All the above results confirm that the oxygen generated by CaO2 is beneficial for improving the efficacy of DOX. The excellent cytotoxicity of CaO2@DOX@ZIF‐67 compared to the other groups can be ascribed to the combined chemo/chemodynamic treatment effect.As expected, the cytotoxic effect of CaO2O2 by Co2+ to generate •OH. However, strong green fluorescence was observed in MCF‐7 cells treated with CaO2@DOX@ZIF‐67. These results further indicate that CaO2@DOX@ZIF‐67 can significantly increase the intracellular level of H2O2 owing to the presence of CaO2, and the H2O2 then disintegrates to yield •OH through a Fenton‐like reaction catalyzed by Co2+ ions.To verify the effective intracellular production of •OH, we used the ROS fluorescence probe 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA) Figure c. Compar2@DOX@ZIF‐67 was further evaluated in MCF‐7 derived multicellular tumor spheroids (MCTSs) using alive‐dead cell staining assay with calcein acetoxymethyl ester  and propidium iodide  . In comparison, only partial apoptosis was observed in the MCTSs incubated with free‐DOX, DOX@ZIF‐67, CaO2@DOX, and CaO2@ZIF‐67. These results are in accordance with the MTT assay, and further demonstrate the combined chemo/chemodynamic treatment effect of CaO2@DOX@ZIF‐67.In addition, the combined treatment effect of CaO) Figure d. Compar2@DOX@ZIF‐67 against MCF‐7 breast tumor xenografts in nude mice. MCF‐7 tumor‐bearing female Nu/Nu nude mice were randomly separated into groups when the tumor volume reached 100 mm3, and used to evaluate different treatments after intratumoral injection. To validate the generation of •OH in vivo, we first used the near‐infrared (NIR) fluorescence dye Cy7, which is degraded by •OH and therefore acts as a sensor. Tumor‐bearing mice received an intratumoral injection of CaO2@DOX@ZIF‐67 + Cy7 or Cy7 alone. In the group treated with Cy7 only, no obvious change in fluorescence was observed. In contrast, the fluorescence of the group treated with CaO2@DOX@ZIF‐67 + Cy7 decreased quickly  at 850 nm with a photoacoustic (PA) imaging system. Tumor‐bearing mice received an intratumoral injection of saline (control), DOX@ZIF‐67, and CaO2@DOX@ZIF‐67, respectively. As shown in Figure 2 in the group treated with CaO2@DOX@ZIF‐67 is obviously higher. Compared with the saline (control) and DOX@ZIF‐67‐treated groups, the amount of intratumoral oxygen in the CaO2@DOX@ZIF‐67‐treated tumors increased approximately eightfolds and fourfolds, respectively . These results were further verified by immunofluorescence staining assay using an antibody against hypoxia inducible factor‐1α (HIF‐1α)  Figure d. The gr2@DOX, CaO2@ZIF‐67, and CaO2@DOX@ZIF‐67. In the groups treated with free DOX and DOX@ZIF‐67, there was modest inhibition of tumor growth compared to the control group , free DOX, DOX@ZIF‐67, CaOp Figurea. These t Figure c. Hematos Figure d. Corress Figure d. Furthes Figure b or majo2@DOX@ZIF‐67, was successfully constructed via a bottom‐up approach for combined chemo/chemodynamic therapy of tumors. Under weakly acidic conditions, CaO2@DOX@ZIF‐67 can be broken down to rapidly release the Fenton‐like catalyst Co2+ and DOX. Subsequently, the unprotected CaO2 reacts with H2O to simultaneously generate O2 and H2O2, which will improve the efficacy of DOX and CDT, respectively. Both the in vitro and in vivo results demonstrated that CaO2@DOX@ZIF‐67 has an excellent antitumor performance and low systemic toxicity. Therefore, this nanocatalytic medicine could be a promising candidate for combined pH‐responsive chemo/chemodynamic therapy.In summary, a pH‐triggered nanocatalytic medicine, CaOThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supplemental Video 1Click here for additional data file.


RCC worldwide has increased by about 2% over the past two decades. The homeobox protein Six2 has been shown to promote the stemness of breast cancer cells and play a role in kidney development, but its involvement in RCC progression has not previously been investigated. Here, we found that six2 expression was significantly increased in RCC tissues and negatively correlated with the overall survival of patients with RCC. In addition, six2 expression exhibited a remarkably higher level relative to that in normal renal cells. Functional experiments showed that six2 knockdown attenuated the stemness of RCC cells, which was evident by decreased spheroid formation ability and stemness marker (sox2 and nanog) expression. Mechanistic studies indicated that Six2 directly bound to the enhancer of sox2, promoting sox2 expression and downstream effector expression of nanog. Furthermore, overexpression of sox2 rescued the inhibitory effects of six2 on the stemness of RCC cells. Notably, six2 expression is positively correlated with sox2 and nanog expression in RCC tissues. Collectively, our results point toward a six2/sox2 axis responsible for RCC cell stemness.Renal cell carcinoma (RCC) accounts for 2–3% of adult malignant tumors, and the incidence of  CSCcancer stem cellkdknockdownoeoverexpressionOSoverall survivalqPCRquantitative real‐time PCRRCCrenal cell carcinomasix2‐kdsix2kd lentivirus vector for IL‐2 and IFN‐α immunotherapy, but the effective rate for patients is only 5–27%, and the adverse effects are great; in recent years, a series of targeted drugs has been developed and rapidly applied in clinic Renal cell carcinoma (RCC) accounts for 2–3% of adult malignant tumors, and the incidence of RCC worldwide has increased by about 2% over the past two decades et al. SIRT2 is highly expressed in RCC cells with stemness, and knockdown (kd) of SIRT2 reduced the stemness and chemoresistance of RCC cells; Huang et al. CXCR2 expression; and Xiao et al. SDF‐1/CXCR4 axis. However, there are still no drugs that could effectively kill stem‐like RCC cells, which means other biomarkers or signaling is critical for RCC stemness. Mutations of the transcription factor six2 have been associated with renal hypodysplasia, renal cysts or vesicoureteric reflux and provoked to be tested in patients with isolated congenital abnormalities of kidneys six2 promotes the progenitor maintenance of nephron progenitors six2 is activated in renal neoplasms and influences cellular proliferation and migration six2 has been shown to promote the stemness of breast cancer cells six2 in RCC tumor progression are still unclear.Cancer stem cells (CSCs) or cells with stemness have been proved to contribute to cell heterogeneity, tumor recurrence, metastasis and drug resistance six2 expression was significantly increased in RCC tissues and negatively correlated with the overall survival (OS) of patients with RCC. Further functional experiments indicated that kd of six2 attenuated the stemness of RCC cells. Mechanistic studies revealed that Six2 directly bound to the enhancer of sox2, a critical regulator for CSC progression, which is responsible for six2‐induced stemness of RCC cells. Our results suggest that six2 may be a potential target for RCC treatments or prognosis.In this study, we showed that −1 penicillin and 0.08 mg·mL−1 streptomycin under humidified atmosphere with 5% CO2 at 37 °C.Twenty‐six pairs of RCC and normal adjacent paraffin‐embedded tissue samples were obtained from the Qianfoshan Hospital of Shandong Province between March 2016 and September 2018. The experiments were undertaken with the understanding and written consent of each subject. Written informed consent from all patients and approval of the hospital ethics review committees were obtained. The study methodologies conformed to the standards set by the Declaration of Helsinki. RCC cell lines SLR23 and SLR20 and human renal epithelial cell line HREpiC were purchased from the Chinese Academy of Sciences Cell Bank. All of the cell lines were cultured in 1640 medium  containing 10% FBS (Thermo Fisher Scientific), 80 U·mLr2: Genomics Analysis and Visualization Platform (http://r2.amc.nl) was used for analyzing the correlation between six2 expression and the OS of patients with RCC. In this platform, the only two kidney tumor datasets, Tumor Kidney Renal Clear Cell Carcinoma  and Tumor Renal Papillary Cell Carcinoma , were used for analysis, and the minimum group size was six (Gene ID: 10736).The six2 and overexpression (oe) lentivirus vector for sox2 were designated six2‐kd and sox2‐oe, respectively. In addition, control empty lentivirus vector served as a control group in this work. The stable cell lines with six2‐kd were screened by culturing with puromycin.The lentivirus package was constructed by Shanghai Genechem Co., Ltd. . The kd lentivirus vector for −△△ct method.Total RNAs from tissues and cells were extracted with TRIeasyTM Total RNA Extraction Reagent TRIeasyTM  following the manufacturer's recommendation. Then, cDNA was reversely synthesized using HiScript® Q Select RT SuperMix for quantitative real‐time PCR  according to the standard procedure. Quantitative RT‐PCR was performed on the StepOne Plus PCR system with AceQ® Universal SYBR® qPCR Master Mix (Vazyme). Glyceraldehyde‐3 phosphate dehydrogenase served as an internal reference. The relative expression levels of transcripts were calculated using the 2six2 , sox2 , nanog  and β‐actin .The detailed procedure was described previously −1 basic fibroblast growth factor (Sigma), 20 ng·mL−1 epidermal growth factor (Sigma), 4 μg·mL−1 heparin (Sigma) and antibiotics at 37 °C under a 5% humidified CO2 atmosphere. After 10 days, the number and size of spheroid were evaluated and quantified under a microscope.The detailed procedure was mentioned in the previous work Sox2 DNA contains two evolutionally conserved enhancers, SRR1 and SRR2. The SRR2 domain was inserted into the pGL3‐Enhancer vector, named pGL3‐sox2. The empty vector served as a control vector. In addition, we constructed a single point mutation (T27G) within the putative six2 binding region of the SRR2 enhancer based on a six2 binding site that was previously identified via Mut Express MultiS Fast Mutagenesis Kit (Vazyme) sox2‐T27G. The pGL3‐sox2, pGL3‐sox2‐T27G or empty vector and Renilla luciferase construct were cotransfected into RCC cells with six2 stable oe using Lipofectamine 3000 (Thermo Fisher Scientific) following the manufacturer's recommendation. Seventy‐two hours later, cell lysates were collected, and the luciferase activity was measured using the Dual‐Luciferase Reporter Assay System . The activity of Renilla luciferase was used for normalization.sox2 by qPCR assay. The ChIP antibody for six2 and relative negative antibody were purchased from Proteintech (Cat No. 11562‐1‐AP).ChIP‐qPCR analysis was performed using the ChIP kit  according to the standard procedure. DNA produced using this kit was used for detecting the enrichment of SRR2 of A600 value of the control group, which was regarded as 1.Cell viability was assessed by the Cell Counting Kit‐8  assay. Cells with different treatments were plated in a 96‐well plate at the density of 3000 cells per well and incubated for 24, 48 and 72 h. The number of viable cells was evaluated by the Cell Counting Kit‐8, and 600 nm absorbance was measured using a Microplate Reader . All values were normalized to the graphpad prism 6 . Standard error mean calculations were used for generating error bars. An unpaired one‐tailed Student's t‐test was performed to assay the significance between two groups. One‐way ANOVA followed by a Bonferroni comparison was used for experiments with three or more conditions. Asterisks represent the significance of difference from the control group: *P < 0.05, **P < 0.01.Data analysis was carried out using six2 expression in RCC and normal adjacent tissues, and found that six2 was significantly increased in RCC tissues compared with that in normal adjacent tissues (Fig. six2 displayed a higher level in RCC cells relative to the normal renal epithelial cells (Fig. r2 platform showed that six2 expression was negatively correlated with the OS of patients with RCC (Fig. six2 may hold promoting roles in RCC progression.First, we detected ues Fig. A. In agrlls Fig. B,C. NotaRCC Fig. D,E. Thessix2 facilitates the stemness of RCC cells. First, we detected the six2 level in nonadherent RCC spheroid cells and found that six2 exhibited a remarkably higher level relative to the adherent cells (Fig. six2 expression was higher in RCC cells and tissues, we chose to knock down six2 expression in RCC cells by lentivirus infection, and stable cell line construction, qPCR and western blot confirmed the kd efficiency of six2‐kd (Fig. six2‐kd had no effects on RCC cell viability (Fig. six2‐kd reduced the expression of stemness regulators (sox2 and nanog; Fig. six2‐kd, characterized as the decrease of spheroid size and number (Fig. six2 positively regulates the stemness of RCC cells.Because CSCs contribute to tumor progression, recurrence and metastasis, we wondered whether six2 regulates the stemness of RCC cells. Two recent studies showed that six2 promotes the stemness of breast cancer cells via different mechanisms; one indicated that six2 activates the competing endogenous RNA network between CYP4Z1 and pseudogene CYP4Z2P via directly binding to their promoters sox2 SRR2 enhancer region sox2 SRR2 enhancer region. As expected, the basal pGL3‐Sox2 activity was higher in RCC cells than that in normal HREpiC cells, and six2‐kd reduced the luciferase activity of pGL3‐sox2 in RCC cells, whereas the activity of pGL3‐sox2‐T27G was unaffected (Fig. six2‐kd reduced the SRR2 enrichment (Fig. sox2 in RCC cells.Then, we explored the mechanism by which six2 regulates the stemness of RCC cells through sox2. Sox2 was overexpressed in RCC cells with six2 stable kd via lentivirus infection, and the infection efficiency was confirmed by western blot and quantitative RT‐PCR (Fig. nanog was rescued by sox2‐oe (Fig. six2‐kd‐mediated decrease of spheroid formation ability was partially reversed by sox2‐oe (Fig. six2 positively regulates the stemness of RCC cells in a sox2‐dependent manner.We further investigated whether PCR Fig. A,B. As e‐oe Fig. C,D. In a‐oe Fig. E,F. Collsix2, sox2 and nanog expression in clinical samples and online datasets from the r2 platform. We found that the expression of six2 and sox2 or nanog exhibited a positive correlation in clinical RCC and normal tissues (Fig. r2 platform (Fig. Finally, to further confirm our conclusion, we detected the correlation between ues Fig. A–D. Notaorm Fig. E–H.sox2. Although the promoting roles of six2 have been established in other tumors, to our knowledge, this is the first work to reveal the roles of six2 in RCC cell stemness.This work indicated that transcription factor Six2 positively regulated the stemness of RCC cells via directly binding to the SRR2 enhancer of stemness master regulator et al. six2 positively regulates the stemness of RCC cells. Evaluating the previous works that reveal that six2 promotes the stemness of breast cancer cells six2 on cancer cell stemness are common in other tumors. Future work should be performed to resolve this issue, which could facilitate six2 as the drug target.CSCs have been regarded as the root of tumor progression, recurrence, metastasis and drug resistance. A previous study showed that salinomycin exhibits a specific killing capacity on CSCs by sequestering iron in lysosomes nanog acts as the downstream effector of sox2 in breast cancer six2 in nephrogenesis, coupled with six2 expression in RCC, indicate that six2 may be a critical regulator of both renal cell stemness and RCC stemness, and it will be of interest to determine whether six2 similarly regulates sox2 and the downstream effector nanog in the context of normal renal development. Importantly, we must admit that six2, like sox2, is a transcriptional factor and thus not an ideal drug target. Collectively, this work identifies that six2 plays a critical role in promoting RCC cell stemness through six2/sox2 signaling. Notably, the relationship between six2 and sox2 or nanog expression is positively relevant, and six2 expression is negatively correlated with the OS of patients with RCC. Therefore, it will be important to identify more targetable downstream effectors of six2 that are critical in mediating its ability to promote RCC cell stemness.Notably, the previous study has shown that The authors declare no conflict of interest.NC, HL, YH and SS conceived and designed the project. NC, HL and YH acquired the data. NC and HL analyzed and interpreted the data. NC and HL wrote the paper


We further demonstrated that each endophenotype is conserved among affected family members. This study expands our understanding of the mechanism underlying the Gi-coupled receptor signaling dysfunction occurring in AIS and provides the first evidence for its hereditary nature. Collectively, our findings offers a new perspective on Gαi hypofunctionality in a human disease by revealing specific serine phosphorylation signatures of Gαi isoforms that may facilitate the identification of AIS patients at risk of spinal deformity progression.Adolescent idiopathic scoliosis is the most prevalent spine deformity and the molecular mechanisms underlying its pathophysiology remain poorly understood. We have previously found a differential impairment of melatonin receptor signaling in AIS osteoblasts allowing the classification of patients into three biological endophenotypes or functional groups . Here, we provide evidence that the defect characterizing each endophenotype lies at the level of Gαi proteins leading to a systemic and generalized differential impairment of Gi-coupled receptor signaling. The three Gαi isoforms exhibited a selective serine phosphorylation patterns for each AIS endophenotype resulting in a differential reduction in Gαi protein activity as determined by cellular dielectric spectroscopy and small interfering RNA methods. We found that one endophenotype (FG2) with phosphorylated Gαi It is unclear why some curves progress with growth while others do not. Severe curves can present with substantial physical deformities with possible secondary cardiopulmonary problems that become life threatening when the curve exceeds a Cobb angle of 100° (the standard measure for curve magnitude)4. A dearth of information on the AIS pathophysiology has resulted in the use of the same treatment protocols that have existed for the last 30 years, i.e., observation only (for Cobb angles between 10° to 25°), external bracing (25° to 45°), and spinal surgery as a last resort (>45°)7. Early curve detection and an improved stratification of AIS patients are critical for better and effective treatment options.Adolescent idiopathic scoliosis (AIS) is a prevalent three-dimensional spinal deformity of unknown cause affecting an average of 2% to 4% of the pediatric population worldwide, with females affected more often in number and severity9. We have shown that AIS is also heterogeneous at the molecular level10. We demonstrated that osteoblasts of AIS patients exhibit different degrees of melatonin signaling that reduce the ability to inhibit cAMP production in response to melatonin receptor stimulation. Melatonin binds to melatonin receptors that are coupled to Gαi proteins and blocks the synthesis of cAMP produced by adenylate cyclases. We hypothesized that Gαi protein impairment could explain the melatonin-signaling pathway dysfunction10 and other neuroendocrine abnormalities observed in AIS13. We tested Gi-coupled receptor signaling using peripheral blood mononuclear cells (PBMCs) of AIS patients using cellular dielectric spectroscopy (CDS)14. The conformational changes in the G-protein-coupled receptors (GPCRs) that are activated by a signaling molecule  are measured by CDS as impedance (Ω). Based on the CDS measurements of the differential patterns of melatonin signaling, the AIS patients were classified into three distinct biological endophenotypes or functional groups 17. The CDS measurements for FG1, FG2, FG3, were 10–40 Ω, 40–80 Ω, and 80–120 Ω respectively. Melatonin signaling was normal when the CDS measurements exceeded 120 Ω20. The CDS measurements confirmed that the response of the cells of AIS patients to melatonin stimulation was mainly Gαi protein-dependent. From these observations, we concluded that we could reliably stratify the Gi signaling defects among AIS patients into three endophenotypes using melatonin receptor agonists14.AIS clinical heterogeneity is characterized by varied curve morphologies and magnitudes even among patients within the same family1, Gαi2, and Gαi3 isoforms at serine residues. The phosphorylation of Gαi proteins limits their signaling ability and suppresses the hormonal inhibition of cAMP production in various cell types23. Our clinical data indicate that the AIS patients classify in FG2 endophenotype were more prone to develop a severe scoliosis, contrasting significantly with AIS patients classified in FG3 or FG1 endophenotype. The present study provides the first evidence that differential Gi signaling impairment associated with each AIS endophenotype may have a hereditary component and prognostic usefulness to predict the risk of spinal deformity progression.The objectives of the current study were to analyze the differential patterns of Gi protein coupled receptor signaling and explore their contribution in AIS pathogenesis. We investigated the clinical relevance of Gαi proteins by studying 1196 patients with AIS from two independent longitudinal cohorts. We stratified AIS patients into one of the three endophenotypes . Osteoblasts, myoblasts, and PBMCs of a subset of the 1196 AIS patients and healthy control subjects were analyzed and we demonstrate that each AIS endophenotype exhibit a differential phosphorylation of GαiTable 14. The results obtained with the patients of the French-Canadian cohort are reported in Table P = 0.545), FG3 endophenotype was overrepresented among the moderate cases (P = 0.0001), and FG2 endophenotype was overrepresented among the severe cases (P = 0.0005). Indeed, among the surgical cases, FG2 was the most prevalent entity and FG3 represented the intermediate entity, while FG1 was less frequent, with proportions of distribution of 10% for FG1, 60% for FG2 and 30% for FG3.The prognostic value of our functional classification was evaluated with subjects of the French-Canadian cohort, and validated using the independent Italian cohort. To derive prognostic information from our functional classification, we examined the distribution of AIS groups among different stages of disease. For this purpose, we classified the AIS patients into one of the three AIS endophenotypes based on their impedance response measured by CDS using melatonin as agonist, as previously describedThe results obtained with the patients of the Italian cohort show that 6 patients were classified in the first endophenotype (FG1), 88 in the second one (FG2), and 84 in the third one FG3). Like the French-Canadian cohort, AIS patients from the Italian cohort presented also similar proportions of distribution in the mild-moderate stage, while FG2 was the most prevalent group in the severe stage with a proportion of 61% compared to 36% and 3% for FG3 and FG1, respectively  previously defined. Results illustrated in Fig. 18. However, the impedance profiles generated by the AIS osteoblasts briefly decreased below zero before quickly reverting to a positive slope. The transient negative phase associated with AIS was greater in the osteoblasts of the FG1 AIS patients compared with that of the FG2 or FG3 AIS patients. These results indicate that in AIS, the response induced by melatonin receptors through their interactions with Gαi proteins is differentially modified among the three patient endophenotypes. There was also a differential decrease in the magnitude of the overall positive component of the impedance response among the three AIS endophenotypes relative to the controls  was observed at 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 µM, 10 µM melatonin concentrations in the control and the AIS endophenotype groups  of melatonin between the groups were similar to those observed in Fig. 25. The impedance responses (Ω) observed in the control group treated with phenylmelatonin, iodomelatonin, and melatonin, were 145 ± 9.43, 119 ± 5.02, and 95 ± 2.03, respectively. The percent reduction in the impedance responses of the AIS osteoblasts compared with the controls, were 90% in FG1, 57% in FG2, and 37% in FG3 for phenylmelatonin; 80% in FG1, 49% in FG2, and 28% in FG3 for iodomelatonin; and 72% in FG1, 43% in FG2, and 21% in FG3 for melatonin. These observations exclude the possibility that the defective signaling observed in AIS cells results, from either a reduced activity of the melatonin receptors or a defective coupling of the Gαi proteins.Next, we examined the possibility of a change in the activity of the melatonin receptors. We compared the integrated response to melatonin in the control and the AIS osteoblasts with the response to the melatonin analogues phenylmelatonin (10 µM) and iodomelatonin (10 µM). The results illustrated in Fig. 26. If the number of the functional Gαi proteins is reduced in AIS, we would expect to observe changes in the inhibitory effect of this peptide in response to melatonin in the AIS osteoblasts. Results illustrated in Fig. 50 values differed between the groups . We found that the treatment with PTX not only altered the initial impedance response to melatonin in the AIS groups but also dramatically reduced the positive component in the FG1 subgroup , were tested, and the representative concentration-response curves for each of these compounds in the control and the AIS osteoblasts are illustrated in Fig. We examined whether this signaling defect is confined only to osteoblasts by extending our analysis to myoblasts and PBMCs. We found that both the cell types exhibited a response pattern similar to that obtained with the osteoblasts  to knock down the Gαs or Gαq expression before stimulating the cells. Quantitative real-time PCR (qRT-PCR) and Western blot analyses confirmed the efficiency of the siRNA approach , µ-opioid receptor (MOR), and LPA type 1 receptor (LPA1R)] were immunoprecipitated in the osteoblasts from the control subjects or the patients of each endophenotype group and blotted with an antibody against the Gαs or Gαq protein. As expected, we found that the Gαs protein was present in the precipitates of each of these receptors in the FG1 osteoblasts but not in the FG2 or FG3 cells, while Gαq was precipitated with the three receptors in all the AIS endophenotypes. The precipitates of each of these receptors contain neither Gαs nor Gαq proteins in the control group Fig. . Western34. We previously reported an increased phosphorylation of the serine residues of the Gαi1, Gαi2, and Gαi3 isoforms of the Gαi proteins in the osteoblasts from AIS patients10. Given that all the Gαi isoforms share the same properties in various cell types and that some isoforms are more efficient than others are, we hypothesized that the divergence in the response characterizing the three AIS endophenotypes is due to a structural modification of the three indicated Gαi protein isoforms. To address this hypothesis, we first examined the gene and protein expression levels of these isoforms as well as the expression of the Gαs and Gαq proteins in the osteoblasts from the control and the AIS patients. The qRT-PCR analysis revealed no significant change in the mRNA expression between the control and the AIS osteoblasts for the three isoforms of the Gαi proteins  as well as the Gαs or Gαq proteins  couple simultaneously to the Gαs and Gαi proteins.Since many receptors couple to only certain Gαi isoforms, we chose to stimulate cells with melatonin or LPA to activate their cognate receptors that were shown to couple to Gαi11. On average, AIS affects 2–4% of the global pediatric population11. It represents a serious and chronic health condition affecting individuals throughout their lives. More than seven million patients in the US are diagnosed with scoliosis . Most are diagnosed with scoliosis between the ages of 10 and 15, and one out of every six children will have a progressive curve that requires active treatment. Efforts to identify genomic variants that may predict the risk of spinal deformity progression have generated many variants with minimal effects (reviewed in35). Using multiple cell types  from AIS patients and healthy controls, we identified, for the first time, a common disease mechanism among all the AIS patients based on a differential impairment of Gi-coupled receptor signaling activities, which appears to be systemic and generalized. Such Gi-signaling dysfunction led to the classification of AIS patients into three biological endophenotypes  based on the selective serine phosphorylation of one or more Gαi isoforms . As previously defined, these endophenotypes are representative of the genes and molecular pathways underlying AIS pathogenesis37. By studying two independent longitudinal cohorts in distinct Caucasian populations, we showed that AIS patients classified in FG2 endophenotype develop more often a severe scoliosis (Cobb angle > 45°) requiring a surgical treatment when compared to FG1 and FG3 endophenotype.AIS is a common, complex multifactorial disease and one of the most prevalent childhood deformities worldwide1 and Gαi2 were phosphorylated in FG2 osteoblasts, and Gαi2 and Gαi3 were phosphorylated in FG3 osteoblasts. The selective depletion of the Gαi3 and Gαi1 isoforms in the FG2 and FG3 osteoblasts respectively almost abolished their cellular responses toward three distinct Gi-coupled receptors in a similar manner, further confirming that Gαi3 and Gαi1 achieve the residual Gi signaling in FG2 and FG3 osteoblasts respectively. This is consistent with the concept that each Gαi isoform can partially rescue Gi-coupled receptor signaling. Nevertheless, FG2 osteoblasts exhibited a weaker residual Gi signaling when compared with FG3 osteoblasts, which further support our previous data obtained with cAMP assays showing that FG2 osteoblasts were almost unable to inhibit cAMP production induced by forskolin stimulation15. This is in agreement with several reports indicating that Gαi3 is less efficient to inhibit cAMP production when compared with the other Gαi isoforms40 and the fact that Gαi3 isoform is less abundant in human osteoblasts, as demonstrated in the present study. Of note, depletion of all three isoforms by siRNA interference almost abrogated the signaling impedance in FG2 and FG3 osteoblasts, contrasting with FG1 osteoblasts where 50% of Gαi signaling activity was maintained despite the fact that all three isoforms were phosphorylated. These results suggest a possible heterogeneity in the number and position of serine residues phosphorylated among Gαi isoforms in each AIS endophenotype.Indeed, we observed that all the Gαi isoforms were phosphorylated in the FG1 osteoblasts, whereas only Gαi41, we observed that the reduced level of the functional Gαi proteins enhances the ability of their cognate receptor to couple to Gαs or Gαq proteins in AIS. However, the magnitude of the response was not affected when Gαq proteins are depleted; eliminating the possibility, that Gαq signaling is acting as a compensatory signaling pathway when Gαi proteins are impaired in AIS. In contrast, depletion of the Gαs protein results in a significant reduction in the magnitude of the Gi signaling response in AIS osteoblasts. Indeed, several reports indicate that the inhibition of the Gαi proteins enhances Gs-coupled receptor signaling45. Our results reveal a similar relationship in AIS in particular with the FG1 endophenotype where the phosphorylation of the three Gαi isoforms results in the permissive coupling of Gαs with three different Gi-coupled receptors  as demonstrated by our co-immunoprecipitation assays. On a clinical point of view, our data are potentially interesting for the postsurgical pain management of scoliotic patients classified in FG1 endophenotype because scoliosis surgery is associated with considerable postoperative pain requiring more analgesia when compared for other major surgical procedures. Indeed, the uncoupling of Gαi proteins from μ-opioid receptors is one of the known mechanisms underlying morphine tolerance47. Normally, the activation of μ receptor by morphine and subsequent Gαi protein coupling results in the inhibition of adenylyl cyclase activity and the interruption of nociceptive signal49. Repeated administration of morphine causes a switch from Gαi protein to Gαs protein coupled to μ receptors causing their activation and leading to morphine tolerance and reduced analgesia51. The pre-existing mechanism underlying the switch from Gαi protein to Gαs protein in AIS cells in absence of morphine is not exclusive to μ receptors since MT2R and LPA1R are similarly affected. Therefore, it is conceivable that differential morphine tolerance and morphine-induced hyperalgesia  could potentially complicate the postoperative management of AIS patients classified in FG1 endophenotype.Given that GPCRs promiscuously couple to and activate signaling through multiple G proteins45, migraine with aura (MA), or migraine without aura (MO)52. Interestingly, X-ray examination revealed that 80.6% of the patients with FM exhibit scoliosis, contrasting with only 8.9% of the age-matched control population53. Furthermore, Brox et al., reported that migraine was the second most common comorbidity in middle-aged patients with idiopathic scoliosis54. More interestingly, the Gi protein hypofunctionality in FM patients and those with MO exhibit a cAMP-inhibitory curve response in the presence of Gpp(NH)p similar to the one obtained with FG2 AIS patients52. Of note, patients suffering from MA exhibit, in the same conditions, a cAMP-inhibitory curve response similar to the one obtained with AIS patients classified in the FG1 endophenotype52.The hypofunctionality of Gi proteins was previously reported in patients suffering from fibromyalgia (FM)in vitro methods with either recombinant proteins or transfected cells overexpressing wild-type or mutated Gαi isoforms. Clearly, this is not a trivial task and much more work is required to determine the molecular mechanisms underlying the selective phosphorylation of endogenous Gαi isoforms in AIS. Secondly, there is a need for validation of these endophenotypes in other replication cohorts and in different ethnic groups affected by AIS as well as in other diseases, like FM, where scoliosis is a frequent comorbidity.In the present study, we acknowledge some limitations. First, this study was not designed to determine the nature of the phosphorylated serine residues among Gαi protein isoforms tested. Our attempts by distinct phospho-proteomic approaches failed to revealed specific phospho-peptides among the endogenous Gαi isoforms, which could explain why most  previous studies used indirect In summary, Gαi protein hypofunctionality may represent a novel and unrecognized molecular mechanism underlying AIS pathogenesis and may explain several endocrine disturbances occurring in AIS as well as the higher prevalence of spinal deformity progression in patients classified in the FG2 endophenotype.A total of 1591 subjects were enrolled in two independent studies in Montreal (Canada) and Milan . For the first cohort , 1018 AIS patients were recruited consecutively between 2008 and 2013 from The Sainte-Justine University Hospital, The Montreal Children’s Hospital, and The Shriners Hospital for Children, in Canada  was approved by the Institutional Review Board (IRB) of Sainte-Justine University Hospital, The Montreal Children’s Hospital, The Shriners Hospital for Children and McGill University as well as by The Affluent School Board and The English School Board of Montreal. The study in Milan  was approved by the IRB ASL Città di Milano and Istituto Ortopedico Galeazzi. All aspects of this research were performed in accordance with the relevant guidelines and regulations.10, from the vertebral bone specimens of 36 AIS patients undergoing spine surgery and from the tibia or femur of 12 non-scoliotic patients subjected to surgery to treat a traumatic condition. The myoblasts were isolated from the skeletal muscle specimens obtained intraoperatively from 36 AIS patients and 12 non-scoliotic patients that were subjected to surgery. Each skeletal muscle specimen was cleared of fatty and connective tissue prior to being cut into small pieces. The tissues were then transferred to a phosphate-buffered saline (PBS) solution containing 0.01% collagenase, and digested for 45 min at 37 °C. After diluting in alpha-MEM (1:1), the solution was filtered through a 45 µm nylon filter and centrifuged at 280 x g for 5 min at room temperature. The pellet containing myoblasts was suspended in culture media  supplemented with 20% fetal bovine serum  and 1% penicillin/streptomycin . After two weeks, the culture media was replaced by fresh culture media supplemented with 10% FBS and 1% penicillin/streptomycin, and the myoblasts were allowed to grow to confluence. All the subjects were asked to provide a blood sample. Peripheral blood mononuclear cells (PBMC) were isolated from the whole blood of 36 AIS patients and 12 non-scoliotic patients by density gradient centrifugation , following the manufacturer’s standard procedure.The osteoblasts were prepared, as previously described11. The GPCR agonists melatonin, iodomelatonin, phenylmelatonin, lysophosphatidic acid (LPA), the selective μ-opioid agonist D-Ala2, NMe-Phe4, Glyol5-enkephalin (DAMGO), adenosine A1 receptor (NECA), type 2 cannabinoid receptor (CB65), a2 adrenergic receptor (UK14304), Somatostatin, MMK1, Apelin-17, BP554, and Quinpirole, used in the study at a concentration of 10 μM each, were from Tocris Bioscience Bio-Techne . Melatonin was also used at a submaximal concentration of 1 μM. The patients were classified, as previously described, and based on the decreased impedance response to melatonin receptor stimulation compared with the control subjects11. Similarly the impedance responses to the activator - GpAnt-2 , the competitor – mastoparan , and the inhibitor – PTX  were analyzed.The functionality of the Gαi, Gαs, and Gαq proteins was evaluated with the CDS assay, as previously described, in osteoblasts, myoblasts, and PBMCs of the AIS patients and the normal controls1, Gαi2, Gαi3, Gαs, Gαq, and scrambled siRNA (10 μM each) were obtained from Ambion . The sequences used for gene silencing are shown in Supplementary Table 2. Functional experiments were performed 48 h post transfection. The gene knockdown was evaluated by qRT-PCR.All Gαi1, Gαi2, Gαi3, Gαs, and Gαq genes, are shown in Supplementary Table RNA was isolated from the osteoblasts using TRIzol reagent  based on the manufacturer’s protocol. The total RNA (1 µg) was reverse-transcribed into cDNA using the Tetro cDNA synthesis Kit . Following cDNA synthesis, qRT-PCR was performed using a PCR master mix containing the QuantiTect SYBR Green PCR Master Mix 2 ×  . The amplification consisted of 45 cycles of the following program: denaturation at 95 °C for 195 s; annealing at 60 °C for 15 s, extension at 72 °C for 15 s and dissociation at 95 °C for 15 s, 60 °C for 15 s and 90 °C for 15 s. The transcript expression was assessed with Stratagene Mx3000P , and the calculations were performed according to the ΔΔCT method using β-actin as an internal control. The sequences of the forward and reverse primers used to identify the mRNA of the Gαi4 and the protease inhibitor cocktail . The immunoprecipitations (IP) were carried out with 1 mg of the total protein extract. The lysates were first pre-cleared with protein G beads for 1 h. The supernatant was then incubated with antibodies directed specifically against the melatonin receptor ; goat polyclonal; sc28453; Santa Cruz Biotechnology, Inc., TX, USA), the mu-opioid receptor , the LPA receptor , or phosphoserine/threonine α-tubulin , followed by a 1 h incubation with protein G beads with gentle rocking. The beads were washed 3 times with RIPA lysis buffer and the bound proteins were eluted with a Western blot loading buffer 2X and boiled at 100 °C for 5 minutes, before being separated on a 10% SDS-PAGE gel and blotted onto nitrocellulose. The blots were then examined using antibodies directed specifically against Gi1, Gi2, Gi3, Gs, or Gq . All the antibodies were used at a 1:1000 dilution and were purchased at Santa Cruz Biotechnology. The protein lysates were also subjected directly to Western blot analyses with specific antibodies to the melatonin receptor, mu-opioid receptor, LPA receptor, or α-tubulin . The bands were visualized using the SuperSignal chemiluminescent substrate .Osteoblasts from the AIS patients and the trauma control cases were lysed in RIPA buffer  containing 5 mM NaVOData reported represent mean values ± SEM. The statistical analysis was performed by multiple comparisons of means with the GraphPad Prism 5.0 software (GraphPad Software) using one-way ANOVA followed by a post-hoc Dunnett’s test. Only P values < 0.05 were considered significant. The clinical distributions were compared using a chi-square test.Supplemental Information