In this work, we investigate the exfoliation feasibility down to the monolayer of VCl2(pyz)2 and CrCl2(pyz)2 by means of first-principles calculations. We explore their structural, electronic, magnetic and vibrational properties, as well as the effect of halide substitution. Then, we provide a full analysis of the spin-phonon coupling (SPC) in both 2D derivatives. Our calculations reveal a low SPC and thermal evolution of the magnetic exchange interactions and single-ion anisotropy mainly governed by low-frequency phonon modes. Finally, we provide chemical insights to improve the performance of these magnetic 2D MOFs based on the effective manipulation of the phonon modes that can present a major impact on their magnetic properties.Layered magnetic metal-organic frameworks (MOFs) are an emerging class of materials that can combine the advantages of both MOFs and 2D magnetic crystals. The recent discovery of large coercivity and long-range magnetic ordering up to 515 K in a layered MOF of general formula MCl This structural arrangement gives rise to highly versatile coordination compounds with a large variety of astonishing features such as structural, electronic and magnetic tunability, sizable porosity and the presence of very large surfaces ,2,3. Thaoperties . In addioperties ,11.d orbitals of metal centers and radicals. This situation leads to ultra-high operative temperatures, e.g., TC = 400 K (V(TCNE)x where x ≈ 2) [C = 515 K in a reduced chromium derivative of a family of coordination solids of general formula MCl2(pyz)2  [A particular class that is gaining increasing interest is that of the so-called MOF magnets ,13. Compe x ≈ 2)  or, moreyrazine) ,18,19. TMoreover, this family of MOF magnets is composed of electrically neutral magnetic layers that are exclusively stacked by van der Waals interactions. This enables the possibility of mechanical exfoliation to a few layers ,21,22,232(pyz)2 neutral 2D layered MOFs. First, we determine the feasibility of the exfoliation of these nanomaterials, as well as the effects on the electronic structure when reducing the dimensionality of the system. Then, we substitute the Cl ligands for heavier halides, exploring a promising way to tune their electronic, structural and magnetic properties by means of chemical design. Finally, we systematically investigate the lattice vibrations and corresponding spin-phonon coupling and thermal evolution of magnetic exchange and single-ion anisotropy parameters. This allows us to provide chemical insights for their further rational design to make them operative at higher temperatures.Here, we present a first-principles study based on density functional theory (DFT) of the Cr and V derivatives of the family of MCl−3 Ry and the energy difference between two consecutive relaxation steps was less than 10−4 Ry. To avoid unphysical interactions between images along the non-periodic directions, we added a vacuum of 18 Å in the z direction in all single-layer calculations. The Brillouin zone was sampled by a fine Γ-centered 10 × 10 × 1 (10 × 10 × 10) and 20 × 20 × 1 (20 × 20 × 20) k-point Monkhorst–Pack mesh [d electrons of V and Cr using the simplified version proposed by Dudarev et al. [We performed first-principles spin-polarized density functional theory (DFT) calculations as implemented in the QuantumEspresso package . The excack mesh  to simulack mesh . To propv et al. .In addition, we carried out broken-symmetry and vibrational DFT calculations using the Gaussian09 package in its revision D01 . For mag2(pyz)2 and CrCl2(pyz)2 are layered materials stacked by van der Waals interactions and crystallize in tetragonal I4/mmm and orthorhombic Immm space groups, respectively. Within each layer, VCl2(pyz)2 presents a square lattice of pyrazine-bridged metal centers with pyz rings disordered on two positions (cis- and trans-oriented) and chloride ligands aligned along the c crystallographic direction. Meanwhile, CrCl2(pyz)2 is almost isostructural with VCl2(pyz)2 but the Cr-Cr and Cr-N bond distances slightly deviate from a perfect 2D square network, as determined from synchrotron X-ray powder diffraction. For CrCl2(pyz)2, the magnetic intralayer interactions take place between the Cr(III) ions (S = 3/2) and pyrazine radical spins (S = 1/2), both coupled antiferromagnetically, which results in a net ferrimagnetic state (S = 1 per molecule) below ~55 K. In the case of VCl2(pyz)2, adjacent V(II) metallic centers are coupled antiferromagnetically—mediated by neutral pyrazine ligands—and preserve the global antiferromagnetic behavior up to ~120 K.VCl0 is the optimized separation between layers in the bulk. This allows us to obtain the cleavage energy of both nanomaterials 2 and CrCl2(pyz), respectively. This indicates that at higher interlayer distances both layers are not interacting anymore and thus the total energy of the system is the result of two independent monolayers. The obtained values are smaller than the calculated for prototypical 2D layered materials such as graphite (0.39 J/m2) or MoS2 (0.27 J/m2) [2) [3 (0.3 J/m2) [2(pyz)2 and CrCl2(pyz) are likely to be easily exfoliated in the laboratory.Recently, it has been proposed that these layered materials could be easily exfoliated down to the 2D limit due to the neutral nature of the layers . In ordeaterials c,d. The 27 J/m2)  whereas /m2) [2)  or CrI3 .3 J/m2) . Therefo2(pyz)2 and CrBr2(pyz)2, and with I, which leads to VI2(pyz)2 and CrI2(pyz)2 (see 2(pyz)2 and CrCl2(pyz)2 reveal torsion angles of ~25–26°, which implies that the pyrazine rings are less tilted than in their bulk counterparts (~33–35°). This is because van der Waals forces play a major role in the interlayer interaction of the pyrazine rings and stabilize a more planar configuration with respect to the c-axis. The analysis of the 2D systems, when moving from Cl to Br and I, evidence an increase in the torsion angles of the pyrazine and the metal-halogen distances due to stronger repulsion interactions between the atoms. This is a consequence of the larger ionic radius. The substitution of the halide plays also an important role in the bond distance between the metal and the nitrogen of the pyrazine ring. This results in longer distances when moving from Cl (2.33 Å) to Br (2.55 Å) and I (2.77 Å) in the case of the Cr-based family. This can be explained by the higher covalent nature of the bond when moving down the periodic table.Next, we perform a full optimization of their atomic coordinates and lattice vectors by means of DFT and investigate the effect of halogen substitution in the monolayer. This is done by replacing the Cl ions with Br, thus forming VBryz)2 see . Compare2(pyz)2 and CrCl2(pyz)2 bulk compounds, respectively 2 (2(pyz)2 (2(pyz)2 (2(pyz)2 (2(pyz)2 (2(pyz)2 bulk compound. Conversely CrI2(pyz)2 is metallic 2 a, VBr2(p(2(pyz)2 b and VI2(2(pyz)2 c, respec(2(pyz)2 d and CrB(2(pyz)2 e, the symetallic f and preorbitals .2(pyz)2 and CrCl2(pyz)2 (2(pyz)2 family , magnetic exchange is due to the interaction of the V centers in the system because in these compounds the pyrazine ligand is not reduced to a radical. From DFT, we calculate J = −40.57 cm−1 in the VCl2(pyz)2 system (within the −2J∑ijiSjS Hamiltonian definition). This is compatible with previously reported J values for the bulk [S = 3/2 − S’ = −3/2. This provides magnetic moment values of 2.84 μB and −2.84 μB for each V(II) center. As expected, one can observe that the neutral bridge pyrazine holds a negligible spin density, and thus there is superexchange coupling mediated by the pyrazine ring. This results in a moderate change of J values, as shown in In order to explore the magnetic properties of the targeted systems, we determine the magnetic exchange interactions (J) between the magnetic centers. The J values are calculated using the Gaussian09 package on a representative fragment formed by 75 and 43 atoms for VCll2(pyz)2 , respectthe bulk ,19. The 2(pyz)2 family , the magnetic intralayer interactions take place between each Cr(III) metal center (S = 3/2) and the pyrazine radicals (S = 1/2). According to our calculations, this scenario gives rise to a final ferrimagnetic state with an isotropic J value of −2034.13 cm−1, which is ~50 times higher than that of CrCl2(pyz)2. We obtain spin moments of 2.75 μB on Cr(III) and −0.72 μB on the pyrazine ligands, which are in good agreement with those expected for a broken-symmetry case S = 3/2 − S’ = −1/2. The total spin density on the pyrazine radicals is mostly due to the local magnetic moments of the N atoms (−0.18 for each pyrazine) and a small contribution of the magnetic moments of C, while the local magnetic moments of Cl are almost negligible. Upon halogen substitution, the magnetic exchange coupling substantially decreases by 27.2% for CrBr2(pyz)2 and by a drastic change of 72.9% for CrI2(pyz)2 (2(pyz)2 family case because the magnetic interaction mainly takes place between the Cr(III) and the radical. Concretely, the competition between a ferrimagnetic interaction Cr(III)-radical versus a ferromagnetic one between Cr3+ ions (t2g–eg) when decreasing the ligand field from Cl-Br-I is enhanced. This combined with the small increase in the distance between the magnetic centers (See 2(pyz)2.Conversely, in the case of the CrXI2(pyz)2 . This isters See  leads to2(pyz)2 series, the values of the single-ion anisotropy are 0.331 (Cl), −0.242 (Br) and −0.298 (I) cm−1 . These values follow the trend experimentally observed in similar octahedral Cr compounds, where there is a significant change in the behavior of D from Cl to Br/I, due to the higher ligand field of the former [2(pyz)2 family, the calculated D values are 0.140 (Cl), 0.190 (Br) and 0.228 (I) cm−1 . As in the previous case, the observed effect of the substitution with heavier halides is to increase the value of D, due to the enhancement in SOC in the ligands.Halogen substitution in the first coordination sphere has a direct effect on the ligand field around the metal. Thus, we determine the single-ion anisotropy (D) of the six compounds presented in this work. For that, we use NEVPT2 as implemented in the Orca package, which is a second-order perturbation theory using a CAS-like reference wavefunction (See Methods). In the case of the CrXe former . This haC, i.e., ~120 K and ~55 K for VCl2(pyz)2 and CrCl2(pyz)2, respectively (see 2(pyz)2) and 198 cm−1 (CrCl2(pyz)2). In the case of VCl2(pyz)2, one can observe that modes 23 and 24 cause the maximum variation for both negative and positive oscillation phases. This fact is easily explained because these lattice vibration modes directly affect V-N bond elongation and compression, both in-phase (2(pyz)2, the most drastic effects are observed for mode 10, which reaches almost 198 cm−1 of variation. This mode consists of the in-phase movement of the pyrazines towards the closer pyrazine ring. In addition, mode 18 also gives a noticeable variation in magnetic exchange due to the movement of the Cr(III) central ions towards the vicinity of two pyrazine rings in each extreme of the harmonic oscillation. This could be exploited by means of an external activation at low temperatures of specific lattice vibration modes, thus allowing an efficient tuning of the magnetic properties of the system. In addition, the opportunity of chemical substitution opens a window to change the resonant frequency of a selected lattice vibration mode to another region. We checked this possibility by calculating the vibrational lattice modes of VI2(pyz)2. In the latter, mode 24 is red-shifted from 675 cm−1 in VCl2(pyz)2 to 640 cm−1. This is due to the ratio k/m between both systems, k being the force constant and m being the reduced mass, which decreases from 0.267 to 0.24 cm−1.To investigate the effect of the lattice on the spin states, we explore spin-phonon coupling (SPC) in both families. First, we calculate the phonon normal modes in Gaussian09 using periodic boundary conditions. This allows us to generate a series of distorted coordinates following each mode until the differential energy of the distorted coordinates is equal to the next normal mode see . From eavely see . For eacvely see . We can in-phase  and out-in-phase . Hence, 2(pyz)2 as a case study. We recalculate both parameters at four finite displacements of the atomic coordinates following the eigenvectors while varying kQ, where kQ is a given real value of the distortion coordinate of the vibrational mode k. Then, we estimate the second derivative of the variation in these parameters with respect to kQ 2 compound because it resonates at even lower frequencies (98.9 cm−1) in CrI2(pyz)2) compared with 140.1 cm−1 in CrCl2(pyz)2, due to the heavier mass of I. As a synthetic insight to limit the effect of this phonon mode on the magnetic behavior, we suggest the introduction of steric repulsion on the pyrazine rings, e.g., by substituting H with methyl groups. This will eventually act over the effect of mode 6 by restricting the pyrazine movement, which will lead to quenching or diminishing of spin-phonon coupling through this mode.The effect of each phonon normal mode on the thermal evolution of J and D is represented in illation , causing2(pyz)2 appealing candidates in research fields such as spintronics or magnonics [x magnetic material, which is achieving a remarkable performance in the field of magnonics [x contains several magnon modes with high quality factors, such as long lifetime and low Gilbert damping arising from the small SOC of the system. In V(TCNE)x the V(II) centers are coupled with an unpaired electron lying in the C-C bond in an S = 3/2 − S’= 1/2 magnetic configuration that is quite similar to the one found in CrCl2(pyz)2. Although we expect that VCl2(pyz)2 compound will not display magnon transport due to the large distance between the V(II) magnetic centers, the magnetic exchange interaction between them could be modified by lattice-strain engineering [2(pyz)2 could be more interesting because there is antiferromagnetic coupling between Cr(III) and the delocalized radical between the pyrazine rings. So, these unpaired spins are located in a similar disposition to those of V(TCNE)x. Moreover, magnetic exchange interactions that take place in CrCl2(pyz)2 are in the same energy range as the calculated J in V(TCNE)x [The high synthetic versatility of MOFs represents a good opportunity to quench/enhance lattice vibrations that affect either positively or negatively the magnetic parameters. This permits the tailoring of the magnetic behavior by designing new MOF magnets with the aim of hardening/softening specific lattice vibrations. Furthermore, the described magnetic and electronic properties of the studied compounds and the prominent new applications of the novel 2D MOFs make these MXagnonics ,15. Chemagnonics . This isineering , which cineering . ConversV(TCNE)x ,58. This2(pyz)2 and VCl2(pyz)2, by means of first principles at the 2D limit. Firstly, we demonstrated the feasibility of isolating a single layer, as their simulated cleavage energies are similar to those reported for paradigmatic 2D materials. Regarding their magnetic properties, our results reveal robust intralayer magnetic exchange interactions, reaching −2034.13 and −40.57 cm−1 for the Cr and V derivatives, respectively. Then, we simulate the effect of halogen substitution by Br and I on the properties, which results in a decrease in J, due to the lengthening of the bonds in the systems, and an enhancement of single-ion anisotropy, due to the larger SOC of the heavier halides. Finally, we perform a full analysis of spin-phonon coupling in CrCl2(pyz)2 and VCl2(pyz)2 by applying a finite displacement approach. This allowed us to unveil the most coupled phonon modes and their thermal evolution. Overall, we observed modest spin-phonon coupling in this class of materials, which is consistent with their excellent magnetic performance. Our results pave the way to their improvement by effective manipulation of the phonon modes that have a major impact on the magnetic properties of this family, e.g., moving them towards other resonant frequencies where they may be easily activated or triggered by external stimuli.We investigated the structural, electronic and magnetic properties of two appealing magnetic MOFs, namely CrCl


The effects of molecular weight, the molar ratio of the raw materials, the catalyst dosage, and the different co-solvents on the properties of the prepared curing agent were systematically explored. The infrared absorption spectra of E20, EP1K, and the water-based epoxy curing agent were compared and analyzed. The coating properties of the waterborne epoxy varnish, which was based on water-based epoxy curing agents to emulsify and cure the resin E44, were systematically tested. The results demonstrated that with a molar ratio of 1:1:4 of PEG1000, E20, and MXDA, the boron trifluoride etherate (BF Water-based coatings employed water instead of organic solvent as the dispersion medium, which had a low volatile organic compound (VOC) content . CompareWater-based epoxy resin used water as the continuous phase, and the epoxy resin was dispersed in water by some method and mixed with water in the form of droplets or particles to form a stable and uniform emulsion . Water-bCuring agents also are known as hardeners, which are a class of substances that enhance or control curing reactions ,11. The Polyethylene glycol (PEG) is non-toxic and environmentally friendly; having an excellent water solubility, its introduction into the epoxy resin molecular chain could result in water-based epoxy resin ,22,23,243·Et2O: analytical pure, Shanghai Aladdin Biochemical Technology Co., Ltd. ; MXDA: analytical pure, Shanghai Maclin Biochemical Technology Co., Ltd. ; propylene glycol methyl ether (PM): analytical pure, Shanghai Maclin Biochemical Technology Co., Ltd.; absolute ethanol: analytical pure, Sinopharm Chemical Reagent Co., Ltd. ; mesityl oxide: analytical pure, Shanghai Maclin Biochemical Technology Co., Ltd. ; benzyl alcohol: analytical pure, Shanghai Maclin Biochemical Technology Co., Ltd. ; glycerol: analytical pure, Shanghai Macklin Biochemical Technology Co., Ltd. ; ethylene glycol butyl ether: analytical pure, Shanghai Maclin Biochemical Technology Co., Ltd. ; deionized water: homemade; perchloric acid (HClO4): analytical pure, Tianjin Damao Chemical Reagent Factory ; glacial acetic acid (HAc): analytical pure, Tianjin Jiangtian Chemical Co., Ltd. ; tetraethyl ammonium bromide (NEt4Br): analytical purity, Tianjin Jiangtian Chemical Co., Ltd. ; trichlormethane (CHCl3): analytical purity, Tianjin Jiangtian Chemical Co., Ltd. ; crystal violet: analytical pure, Shanghai Maclin Biochemical Technology Co., Ltd. PEG : analytical pure, Tianjin Chemio Chemical Reagent Co., Ltd. ; epoxy resin : industrial grade, Zhejiang Tiannu Group Paint Co., Ltd. ; BFConstant temperature heating agitator: DF-101S, Changzhou Putian Instrument Manufacturing Co., Ltd. ; constant temperature drying oven: DHG-9076A, Shanghai Jinghong Experimental Equipment Co., Ltd. ; Fourier transform infrared spectrometer: Nicolet 5700, Thermo Fisher Scientific, Waltham, MA, USA; adjustable film coated: KTQ-II., Dongguan Youtu Instrument Co., Ltd.(Dongguan China); portable pencil scratch hardness tester: QHQ-A Dongguan Jiuli Electronic Technology Co., Ltd. ; T-type bending machine tester: WZJ-II., Tianjin Shibo Weiye Chemical Instrument Co., Ltd. ; high-speed centrifuge: TG16-WS, Changsha Xiangzhi Instrument Co., Ltd. ; electrochemical workstation: CHI660E, Shanghai Chenhua Instrument Co., Ltd. ; viscosity tester: NDJ-9S, Bangxi Instrument Technology Co., Ltd. .The difference between E44 and E20, which are both epoxy resin of the bisphenol A type, and their difference lay in the different degrees of polymerization which resulted in various epoxy values. The basic parameters of E44 and E20 are displayed in In this experiment, E20 and PEG1000 were used to realize the hydration of epoxy resin, which was followed by a reaction with MXDA’s active hydrogen to create a water-based epoxy curing agent. Ultimately, the E44 was cured with the self-made curing agent in order to determine the performance of it. The reaction mechanism of the prepared nonionic waterborne epoxy curing agent was as follows: two carbon atoms and one oxygen atom in the epoxy group were in the same plane to form a ternary ring structure. Due to the differences in the atoms or groups connected by two carbon atoms in the ternary ring, there was a distinction in the electronegativity between the two carbon atoms and the oxygen atoms, which led to the electrostatic polarization. This action increased the density of the electron clouds around the oxygen atoms, which made the oxygen atom vulnerable to the attack of electrophilic reagents, the carbon atom vulnerable to the attack of nucleophilic reagents, and finally broke the C-O bond. Finally, epoxy-based rings were opened. In 3·Et2O was added at 70 °C. After 3 h of dripped, the 70 °C was kept for a while. Then, the temperature was raised to 95 °C and the reaction was maintained at that temperature for 9 h. Eventually, the temperature was decreased to 60 °C and the solvents were added and diluted to 50% of the solid content to obtain EP1K.In a nitrogen atmosphere, a four-mouth flask with a stirring paddle, a thermometer, a constant pressure drip funnel, and a spherical condenser tube was filled with PEG1000, E20, and a precise amount of solvent PM. The temperature was raised to 70 °C to dissolve it at 500 rpm and stir evenly. Afterwards, the BFA certain proportion of MXDA was added to a four-mouth flask, which was equipped with a stirring paddle, a thermometer, a constant pressure drip funnel, and a spherical condenser tube, and was heated to 75 °C. Then, a certain amount of EP1K was added dropwise at 75 °C and kept it warm for a period of time to obtain the target product.The tinplate sheets were used as coating substrates and sanded with 800-mesh sandpaper before being used, which was then washed with absolute ethanol and wiped clean. The self-made self-emulsifying non-ionic waterborne epoxy resin curing agent and E44 were combined at high speed according to the ratio of the epoxy group and active hydrogen equivalent of 0.9. Appropriate deionized water was added to prepare the water-based epoxy varnish. According to the standard “General preparation of paint film” (GB/T1727-1992), the coatings were prepared. The varnish was coated on the polished and cleaned tinplate sheet with a 50 μm coating rod and was placed in a constant temperature and humidity box at 25 °C for 7 days.4-HAc method. The CHI 660E electrochemical workstation was used to conduct the AC impedance (EIS) test. The sample’s viscosity was measured using an NDJ-9S viscosity tester. The value of porosity was estimated using the buoyancy method.The Nicolet 5700 Fourier-transformed infrared spectrometer was used to identify the IR distinctive absorption peaks of various compounds. A clear line-of-sight observation approach was used to assess the curing agents’ appearance status. The thermal stability, room-temperature stability, and low-temperature stability of the curing agent were tested by placing it in an environment of 60 °C, 25 °C, and −4 °C. To evaluate the centrifugal stability, a sample of curing agent diluted with water was placed in a centrifuge and spun at room temperature for 30 min at a speed of 3000 r/min. For the proper quantity of the water-based curing agent sample, it was mixed evenly with water to 20% of the solid content, and then placed for 24 h in order to test the dilution stability. The identical amount of curing agents and deionized water were both added into a beaker to ascertain the water solubility. The drying time was determined according to the standard “Methods of test for drying time of coatings, paints and putties” (GB/T1728-79). According to the standard “Paints and varnishes-Determination of resistance to liquids” (GB/T9274-88), acid, alkali, and water resistance were evaluated. Coating hardness was characterized according to the standard “Paints and varnishes determination of film hardness by pencil test” (GB/T6739-2006). Adhesion performance was tested according to the standard “Paints and varnishes-Cross cut test for films” (GB/T9286-1998). Impact resistance was tested according to the standard “Determination of impact resistance of the film” (GB/T1732-1993). The epoxy conversion rate was measured by the HClO−1 was weakened but did not disappear, indicating that, in the first stage, PEG and epoxy resin were partially ring-opened and the epoxy group was retained. A significant ether bond absorption peak at 1150 cm−1 shows that the alcohol hydroxyl group of PEG100 and the epoxy group successfully polymerized to produce more hydrophilic ether bonds. Because the initial reaction opened the ring of the epoxy group to generate hydroxyl, the intermolecular interaction strengthens with the increase in hydroxyl content, and a broad absorption peak of hydroxyl appeared at approximately 3400 cm−1. The epoxy characteristic peak of the self-made waterborne epoxy curing agent disappears, suggesting that the EP1K’s epoxy group underwent a ring-opening reaction in the reaction of the second step, which left no epoxy group residue. Additionally, despite the fact that the primary amine should have two peaks at 3400 cm−1 and 3500 cm−1, only one broad peak was formed here. The N-H stretching vibration absorption peak of the primary amine only showed up at 3520 cm−1 in the spectrogram of the water-based curing agent; this could be explained by the close proximity and the overlap of the absorption peaks. The N-H vibration absorption peak of secondary amines, which appeared at 3250 cm−1, indicates that MXDA and EP1K had successfully polymerized to form secondary amines. The study of the infrared spectrum revealed that the reaction process followed the anticipated reaction path.The Fourier infrared spectra of E20, EP1K, and the self-made waterborne epoxy curing agent are displayed in 3·Et2O as a catalyst under constant reaction temperature and time, the optimal molecular weight of PEG was determined. The effects of various PEG molecular weights on the effectiveness of waterborne epoxy curing agents and coating films are displayed in By controlling a single variable, the molar ratio of PEG, E20, and MXDA with different molecular weights was 1:1:4. By conducting experimental studies employing 0.3% BFThe comprehensive properties of the curing agents were related to the proportion of hydrophilic and lipophilic chain segments. 3·Et2O is toxic, but its catalytic effect is good, and it is the most commonly used and easily obtained Lewis acid. Therefore, BF3·Et2O was selected as the catalyst in this paper and we avoided the adverse effects by controlling its dosage. Under the condition that the molar ratio of E20 to PEG1000 was 1:1, experiments were conducted by adding 0.1%, 0.3%, 0.5%, and 1% BF3·Et2O to the reaction system, respectively. The effect of the amount of BF3·Et2O on the epoxy conversion and stability of the curing agent was tested. The outcomes are displayed in Although EP1K with a self-emulsifying property can be developed by employing epoxy groups and hydroxyl groups for reactions, this reaction was difficult to occur. BF3·Et2O was too little and the dissociated H+ was insufficient, it resulted in low reaction rates for alcohols and epoxy resins and the reactions could not be carried out adequately. With the increase in BF3·Et2O dosages, the stability of the curing agent increased. When the BF3·Et2O dosages were approximately 0.3% of the total mass of PEG1000 and E20, the curing agents were stable for more than 30 days. When the dosage of the catalyst reached 0.5%, the reaction viscosity increased, and the stability of the curing agent was very poor. In addition, BF3·Et2O would also participate in the cross-linking reaction of epoxy. A lot of the dissolved H+ would cause the epoxy resin to undergo a self-polymerization reaction when there was too much BF3·Et2O, which formed the colloidal substance. All the experiments showed that the BF3·Et2O content was 0.3%, and the curing agent had good performance in all aspects.There were many types of amine curing agents; most common were aromatic amines, fatty amines, and others. Aromatic amines’, due to the existence of the rigid benzene ring structure, cured film had better heat resistance, corrosion resistance, and mechanical strength than the cured film made with aliphatic amines ,26. SincThe molar ratio of E20 to MXDA primarily impacted the stability of the curing agents and the applicable period of the emulsion obtained by emulsifying E44. When the MXDA concentration was too low, the reaction between the primary amine and the epoxy group was insufficient, which left EP1K in the system, making it easier for the curing agent to layer. When the content of MXDA was too high, the application period of emulsion was too short due to the strong reaction activity of the system. During the preparation of the curing film, it was found that orange peel, fish eyes, shrinkage holes, shrinkage edges, and other phenomena would appear within the film’s surface. Several types of co-solvents were added to this experiment under the premise process optimization in order to lower viscosity, adjust surface tension, optimize coating surface state, and produce superior experimental results. After repeated tests, the coating leveling was the best in the coating surface state with the addition of 10% mesityl oxide. It could be uniform without bubbles, shrinkage, etc. The results of the experiment are displayed in When no co-solvent was added, there were some small resin particles in the emulsion, resulting in an uneven coating surface and poor adhesion. Although the adhesion greatly increased with the addition of 5% mesityl oxide, the additive quantity was insufficient, and bubbles were likely to form. It can be clearly observed in The basic parameters of the self-emulsifying non-ionic waterborne epoxy curing agent prepared in this experiment are shown in In order to further systematically and intuitively understand the relevant properties of the self-emulsifying non-ionic waterborne epoxy curing agent prepared in this experiment, the coating properties of the self-made self-emulsifying non-ionic waterborne epoxy curing agent (CA1), a commercially available nonionic waterborne epoxy curing agent with E20 modified triethylenetetramine (CA2), and a commercially available ionic waterborne epoxy curing agent (CA3) were compared according to the molar ratio of active hydrogen to epoxy group 0.9:1. The coating surface and performance test results are shown in Nyquist pictures of the coatings cured with two commercially available curing agents and the self-made curing agents in 3.5 wt% NaCl and 3.5 wt% KOH solutions were compared to further confirm the anti-corrosion effectiveness of the coatings. 3·Et2O as the catalyst, a waterborne epoxy curing agent was produced. Adding 10% mesityl oxide in the system could improve the leveling property of the coating. The curing agent had a solid content of 50 ± 5%, viscosity of 4600 mpa·s, active hydrogen equivalent of 159 g/mol, and storage stability of more than 180 days. The varnish was prepared by emulsifying and curing E44 with self-made water-based epoxy curing agent according to the ratio of active hydrogen/epoxy base 0.9:1. The hardness of the coating was 5 H, the impact strength was 50 kg·cm, the flexibility was 0 mm, the adhesion was grade 0, and the acid, alkali, and water resistance of the coating were qualified.In this paper, ring-opening polymerization reaction was carried out using E20 and PEG1000 as the raw ingredients. The self-emulsifying system EP1K was created by introducing a PEG1000 hydrophilic flexible chain segment. EP1K was then added to MXDA to create a non-toxic, room-temperature curable, non-ionic waterborne epoxy curing agent with self-emulsifying qualities. The experimental conditions for the preparation of EP and final products were also discussed. The IR spectra of E20, EP1K, and self-made waterborne epoxy curing agents were analyzed to verify the chemical reaction process. The factors affecting the stability of the curing agents, the mechanical properties, and corrosion resistance of the coating were studied through comparative experiments. Through the test and analysis of the comprehensive performance of the self-made waterborne epoxy curing agent and the emulsion curing E44 coating, the experimental formula and experimental conditions of the best performance were finally selected: the ratio of n (PEG1000):n (E20):n (MXDA) was 1:1:4, and the ring-opening polymerization was completed in two steps. Using PM as the solvent and 0.3 wt% BF


Adrenocortical carcinoma (ACC) is a rare, often deadly cancer arising from the adrenal gland. Mortality associated with ACC has remained unchanged over the last several decades. The rarity of ACC, an incomplete understanding of its molecular basis, and limited availability of pre-clinical models have hampered the development of novel therapeutic approaches. The present work aims to address these gaps with a focus on the Wnt/β-catenin cell signaling pathway, which is aberrantly activated in ~40% of ACC tumors. We discovered a novel ECM program activated in ACC that is associated with Wnt/β-catenin activation and poor survival. Wnt/β-catenin inhibition disrupted the expression of ECM genes and induced the loss of cancer cell viability. To extend these findings, we developed an orthotopic mouse model of rapidly progressive ACC and demonstrated that disruption of the Wnt/β-catenin axis with the novel small molecule inhibitor Tegavivint is a potential effective therapeutic strategy to reduce ACC tumor burden in vivo.COL11A1 expression originates specifically from cancer cells and is strongly correlated with both Wnt/β-catenin activation and poor patient survival. Inhibition of constitutively active Wnt/β-catenin signaling in the human ACC cell line, NCI-H295R, significantly reduced the expression of COL11A1 and other ECM components and decreased cancer cell viability. To investigate the preclinical potential of Wnt/β-catenin inhibition in the adrenal microenvironment, we developed a minimally invasive orthotopic xenograft model of ACC and demonstrated that treatment with the newly developed Wnt/β-catenin:TBL1 inhibitor Tegavivint significantly reduced tumor growth. Together, our data support that the inhibition of aberrantly active Wnt/β-catenin disrupts transcriptional reprogramming of the microenvironment and reduces ACC growth and survival. Furthermore, this β-catenin-dependent oncogenic program can be therapeutically targeted with a newly developed Wnt/β-catenin inhibitor. These results show promise for the further clinical development of Wnt/β-catenin inhibitors in ACC and unveil a novel Wnt/β-catenin-regulated transcriptome.Adrenocortical carcinoma (ACC) is a rare but highly aggressive cancer with limited treatment options and poor survival for patients with advanced disease. An improved understanding of the transcriptional programs engaged in ACC will help direct rational, targeted therapies. Whereas activating mutations in Wnt/β-catenin signaling are frequently observed, the β-catenin-dependent transcriptional targets that promote tumor progression are poorly understood. To address this question, we analyzed ACC transcriptome data and identified a novel Wnt/β-catenin-associated signature in ACC enriched for the extracellular matrix (ECM) and predictive of poor survival. This suggested an oncogenic role for Wnt/β-catenin in regulating the ACC microenvironment. We further investigated the minor fibrillar collagen, collagen XI alpha 1 (COL11A1), and found that  Adrenocortical carcinoma (ACC) is a rare cancer arising from the adrenal cortex. The overall 5-year survival rate for ACC is 35% , and 5-yAXIN2 [LEF1 [APCDD1 [APC and ZNRF3 and gain-of-function (GOF) mutations in the gene encoding β-catenin, CTNNB1.One molecular pathway that has been long associated with ACC is the Wnt/β-catenin pathway. This pathway is essential for development and homeostasis of many tissues, including the adrenal cortex ,5. Wnt lAXIN2 , LEF1 [7N2 [LEF1 , and APC [APCDD1 . In the APC. Enhanced Wnt/β-catenin activation was further implicated in ACC by later studies that found CTNNB1 GOF mutations and nucleo-cytoplasmic β-catenin accumulation in a relatively high proportion of human ACC cases [CTNNB1 (~16%), LOF alterations in APC (2–3%), and LOF alterations in ZNRF3 (~20%). Moreover, increased Wnt/β-catenin activity, determined by the presence of a genetic alteration or nucleo-cytoplasmic β-catenin staining, is associated with poor prognosis in ACC patients [A connection between aberrant Wnt/β-catenin activation and ACC pathogenesis was first suggested by the increased incidence of adrenocortical tumors in patients with Familial Adenomatous Polyposis (FAP) . FAP is CC cases . More reCC cases ,13. Thespatients ,15.CTNNB1 are frequent in ACC, precluding the use of such inhibitors. Strategies that directly target downstream β-catenin activity are essential to effectively block pathway activation in these tumors. Whereas several previously developed small molecules have been shown to inhibit nuclear β-catenin binding to TCF/LEF [CTNNB1 GOF mutations at the phosphorylation site [Given the high prevalence of genetic alterations that enhance Wnt/β-catenin signaling in ACC, there has been considerable interest in therapeutically targeting this pathway. Whereas upstream inhibitors that block Wnt ligand activation of cell surface receptors are attractive, activating mutations in  TCF/LEF , the cliion site ,18,19,20ion site ,18,21,226 cells) while failing to mimic the endogenous tumor microenvironment. Whereas genetic mouse mutants that spontaneously develop metastatic ACC have recently been described, the stochastic nature of tumor development limits the utility of such models in timely preclinical studies designed to test new therapeutic agents [Successfully advancing new therapeutic agents in the clinic requires in vivo models that faithfully recapitulate human disease. In ACC, the development of accurate preclinical xenograft mouse models has proven challenging. Most approaches rely on subcutaneous, renal subcapsular, or splenic injection of tumor cells ,24,25,26c agents ,28. Orthc agents ,30,31.ECM proteins are key components of the tumor microenvironment that have been shown to play driving roles in both carcinogenesis and tumor progression ,33. CellCTNNB1 GOF mutation. To translate and extend our findings in vivo, we implemented and characterized a novel ultrasound-guided orthotopic xenograft model and found that Tegavivint treatment decreased β-catenin levels and significantly inhibited tumor growth in mice. Taken together, these studies support further clinical development of Wnt/β-catenin inhibitors that target β-catenin-dependent transcriptional activity in ACC.In the present work, we identified a novel transcriptional signature of aberrant Wnt/β-catenin activation that is enriched in ECM components and associated with poor survival in ACC. We tested two independent inhibitors of Wnt/β-catenin, PKF115-584 and Tegavivint, and demonstrated that both effectively repressed Wnt/β-catenin activity, decreased the expression of a distinct subset of ECM genes, and coordinately inhibited the in vitro growth of ACC cells harboring a http://www.R-project.org/; accessed on 6 April 2021, version 4.1) using software packages from the Bioconductor portal . We downloaded harmonized RNA-seq counts data from the Genomic Data Commons (GDC) portal using TCGABiolinks (version 2.22.4) [EdgeR; version 3.36.0 [MineICA  to perform Independent Component Analysis (ICA). We used the Mann–Whitney test to interrogate the association between each gene signature (component) identified by ICA and the presence of CTNNB1 mutations by comparing the loading values of a “witness” gene  in CTNNB1-mutated and CTNNB1-negative samples. We performed gene set enrichment analysis using the online GSEA tool  to calculate enrichment scores using the Canonical Pathways collection from MSigDB, which is comprised of several curated gene lists from different sources, including Biocarta, KEGG, and Reactome, representing a broad collection of pathways and biologic processes. We used ComplexHeatmap (version 2.16.0) [GSVA  default algorithm [COL11A1 expression, we plotted the log2-CPM values of the gene as a function of the natural log-transformed hazard-ratio using smoothHR . Using an additive Cox model, we defined the optimal cutoff as the point where the lower limit of the confidence interval of the natural log-transformed hazard ratio crossed the baseline. To perform the comparative analysis between the ACC-TCGA 340-gene Wnt signature identified by ICA and other models of constitutive Wnt activation, including the dataset by Leng et al. [limma  to perform class comparison analysis between β-Catenin-GOF and WT mice, and between nuclear CTNNB1+ and CTNNB1- human ACC. To compare the lists of genes upregulated by Wnt signaling in both human ACC and the murine model, we built a Venn diagram using the ggVennDiagram function . To generate the chromatin accessibility track from ACC-TCGA samples, we downloaded 18 bigwig files from 9 ACC samples that were generated by Corces et al. [https://github.com/ucscGenomeBrowser/kent; accessed on 13 May 2020, version 357). To generate the track with the accessibility peaks, we combined the coordinates of the corresponding peaks reported by Corces et al. into a single .bed file using bedtools . To annotate these peaks, we downloaded .bed files of ChIP-seq experiments for TCF/LEF transcription factors in several different human cell lines from the UniBind database . To demonstrate regions of open and active chromatin, as well as binding sites of CTNNB1 in the NCI-H295R cell line, we downloaded the bigwig files corresponding to ATAC-seq, H3K27ac, and CTNNB1 ChIP-seq from Mohan et al. [https://github.com/JetBrains-Research/jbr; accessed on 13 May 2020, version 1.0.5641) to generate the figures overlaying these tracks in the genomic regions of interest   and perfn 3.36.0  after co 2.16.0)  to generlgorithm  to calculgorithm  and the s et al.  and combn et al. , as wellinterest .2 at 37 °C. NCI-H295R cells were grown in DMEM/Ham’s F-12 (1:1)  supplemented with 10% NuSerum I , 1% Insulin-Transferrin-Selenium-Ethanolamine (ThermoFisher Scientific), and 1% penicillin/streptomycin (ThermoFisher Scientific). The human NCI-H2935R cell line has been authenticated using short tandem repeat profiling within the last three years. Y1 cells were cultured in High-Glucose DMEM (ThermoFisher Scientific) supplemented with 2.5% fetal bovine serum , 7.5% horse serum (ThermoFisher Scientific), and 1% penicillin/streptomycin. All experiments were performed with mycoplasma-free cells. Tegavivint was a gift from Iterion Therapeutics, INC , and PKF115-584 was obtained from Tocris . All compounds were solubilized in DMSO.NCI-H295R (RRID:CVCL_0458) and Y1 cell lines were obtained from American Type Culture Collection  and cultured in a humidified incubator containing 5% COCells were plated at a density of 300,000 cells per well in a clear 24-well plate or 30,000 cells per well in a 96-well plate one day prior to treatment. At the endpoint, cells were incubated with the colorimetric reagent alamarBlue (ThermoFisher Scientific) in accordance with the manufacturer’s instructions. Absorbance at 570 nm and 600 nm was measured after 3 h.HPRT1 as a housekeeping gene. The primers used are listed in RNA was extracted using a RNeasy Mini Kit . Reverse transcription was performed using a High-Capacity cDNA Reverse Transcription Kit  according to the manufacturer’s instructions. The cDNA obtained was then used as the template for qPCR analysis using a Power SYBR Green PCR Master Mix (Applied Biosystems) and an Applied Biosystems 7300 Real-Time PCR System. Expression levels were normalized using the ΔΔCt method using g for 20 min at 4 °C. Protein concentrations were measured using a Pierce BCA protein assay kit (ThermoFisher Scientific), and equal amounts of total protein were separated by NuPAGE 4–12% Bis-TRIS gel in MES SDS running buffer (ThermoFisher Scientific). Protein was transferred from the gel to a nitrocellulose membrane and blocked with Odyssey Blocking Buffer  for 1 h. Membranes were probed with antibodies against β-catenin , Active β-Catenin , or β-actin  at 4 °C overnight. The next day, the membrane was washed three times with TBS-T buffer, incubated with the secondary antibodies for 1 h at room temperature, and washed three times with TBS-T and one time with PBS. Images were acquired using the LiCor Odyssey imaging system. Images were cropped to reorder samples run together on the same membrane.Cells were harvested in RIPA buffer (ThermoFisher Scientific) containing cOmplete Protease Inhibitor Cocktail , PhosSTOP phosphatase inhibitors (Roche), and 50 uM ETDA, on ice for 30 min, and centrifuged at 16,000× Stable NCI-H295R and Y1 cell lines expressing pLVX-EF1α-LUC2-IRES-mCherry , a gift NSG mice were housed and maintained in specific pathogen-free conditions and facilities accredited by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the United States Department of Agriculture, United States Department of Health and Human Services. Animal care was overseen by the Unit for Laboratory Animal Medicine at the University of Michigan.3, 1011 photons/s/cm2/sr, 120 days post-injection, ≥15% weight loss, or all other mice in the cohort had reached endpoint. For studies testing Tegavivint treatment, 200,000 NCI-H295R cells were injected. Treatment was initiated when the tumor volume reached approximately 40–100 mm3. Tumor volumes were extrapolated from last ultrasound using the NCI-H295R tumor doubling time of 6 days. Tegavivint was suspended in 5% dextrose, and an intraperitoneal (i.p.) injection of 50 mg/kg Tegavivint or vehicle was administered 5 days a week for two weeks. At necropsy, the tumor volume was calculated as an ellipsoid volume = 4/3 × π × (0.5 × D1) × (0.5 × D2) × (0.5 × D3), where D1, D2, D3 are the longest measurements in the X, Y, and Z axis, respectively.Intra-adrenal injections were performed on 6–13-week-old male immunodeficient NSG mice  as described previously . UltrasoTissues were fixed in 10% neutral-buffered formalin for 24 h at room temperature, processed, paraffin embedded, and cut into 5 µm sections. Tissue characterization following routine hematoxylin and eosin staining was performed by a board-certified veterinary pathologist (MJH). For immunohistochemical staining, slides were labeled with antibodies against proCOL11A1 , β-catenin , SF-1 (Proteintech Group (PTGlabs) custom-made AB_2716716, 1:1000, 10mM NaCitrate + 0.05% Tween-20 pH6), Ki67 , or Human Nucleoli . Images were acquired on an Nikon Optiphot-2 microscope with an Olympus DP-70 camera. Scoring methodology: proCOL11A1 and β-catenin specimens were independently assessed in a blinded fashion by two observers following these criteria: proCOL11A1 immunostaining was evaluated according to the cytoplasmatic signal, scored on a 0–3 scale, with a panel of normal and non-malignant tissues used as negative controls, and pancreatic ductal adenocarcinoma used as a positive control. In total, 97 tumors, each with triplicate cores, were evaluated. For statistical analysis, tumors stained 0–2 were grouped. β-catenin immunostaining was evaluated as present or absent in the membranous, cytoplasmic, and nuclear compartments for each sample. For samples treated with Tegavivint, β-catenin nuclear signal was additionally evaluated in 4 HPF per sample and scored on a 0–3 scale. KI67 immunolabeling was quantified using the Aperio whole-slide imaging and digital analysis system. To quantify labeling, the nuclear algorithm provided was used following visual optimization and tuning to the labeling on KI67-stained slides.For immunofluorescent staining, slides were blocked for 1 h at room temperature followed by primary antibody incubation overnight at 4 °C, and primary antibodies were detected with HRP-polymer solution  and Alexa fluor tyramide reagent . Nuclei were counterstained with DAPI. Nonspecific staining was blocked using 2.5% horse serum for non-mouse antibodies. The M.O.M. kit (Vector Laboratories) was used for all primary mouse antibodies according to the manufacturer’s instructions. IF slides were mounted using ProLong Gold  and imaged on a Nikon Optiphot-2 microscope with a CoolSNAP Myo camera.p-values ≤ 0.05 were assigned significance, and data are expressed as mean ± SD. For comparison of Kaplan–Meier survival, a log-rank test was performed. For comparison between ACC cells or tumors treated with different experimental conditions, a two-tailed Student’s t-test or two-tailed Welch’s t-test  was performed.Statistical analyses were performed using R (version 4.1), Graphpad Prism 7, or Microsoft Excel software (versions 16.71). CTNNB1 .To identify transcriptional programs that are engaged in human ACC tumors, we analyzed RNA-seq data from 78 ACC samples included in The Cancer Genome Atlas Project (ACC-TCGA) . Using uand LGR5 A. To invGSVA to calculate a score that quantified the expression of these 340 genes in each sample. We divided the ACC-TCGA cohort into two groups according to the median of the gene score. We observed a significant difference in both overall survival and disease-free survival between the two groups   C. These CTNNB1 mutation, we hypothesized that the signature, in which ECM biology is among the most significantly represented processes, was Wnt/β-catenin-driven. To further investigate this hypothesis, we performed a correlation analysis of select ECM genes and known Wnt/β-catenin target genes. We analyzed the ECM genes that provided the highest component projections in the signature identified by ICA in the classes of collagen, laminin, and integrin: COL11A1, COL26A1, LAMC3, and ITGA2  (p = 0.0003) and DFS (p = 0.0075) (High expression of minor fibrillar collagen  cancers . We ther and DFS . To foll= 0.006) B, indica 0.0075) C.COL11A1 expression were correlated with poor outcomes in patients, we next wanted to evaluate the effects of Wnt/β-catenin inhibition on the ECM gene signature and investigate the utility of inhibition as a therapeutic strategy in ACC. We performed experiments on the NCI-H295R human ACC cell line, which harbors an activating S45P CTNNB1 mutation [Having established that Wnt/β-catenin activity and mutation ,21, or Pmutation  previousmutation . Both inmutation ,18, we iAXIN2, LEF1, and APCDD1   D. We als= 0.877) E. Taken At necropsy, tumors were further characterized to identify the utility of the model for future studies. Histologically, tumors formed from NCI-H295R and Y1 xenografts modeled characteristics of high-grade ACC, designated by 20 or greater mitotic figures per 50 high-power fields (≥20/50 HPF) A,D. NCI-3, after which they were treated with 50 mg/kg Tegavivint (n = 8), or vehicle (n = 7) (p = 0.936), and Tegavivint was well tolerated by mice. After two weeks of Tegavivint treatment, a striking 65% reduction in tumor weight and 58% reduction in tumor volume was observed over vehicle-treated controls  A. There CTNNB1 mutations that is highly correlated with both Wnt/β-catenin activity and uniquely associated with ECM expression. The strong transcriptional signature associated with the activation of this pathway and its major contribution to decreased patient survival suggest that pharmacologically targeting Wnt/β-catenin is an attractive therapeutic approach that can fulfill the urgent need to improve the standard of care for advanced ACC [Over the last two decades, a large body of evidence has implicated sustained Wnt/β-catenin signaling as a key driver event in the molecular pathogenesis of ACC . Indeed,nced ACC ,3. IndeeCOL11A1, COL26A1, LAMC3, and ITGA2 was correlated with Wnt/β-catenin pathway activation (Our analysis sought to better understand the role of Wnt/β-catenin signaling in ACC. Interestingly, we observed a spectrum of Wnt/β-catenin signaling across ACC, with patient tumors bearing ligand-independent GOF mutations in β-catenin exhibiting the highest level of downstream Wnt signaling, whereas tumors with ligand-dependent LOF mutations in ZNRF3 exhibited more moderate Wnt/β-catenin activation, as previously observed . ACCs witivation D. ThoughCTNNB1 and ZNRF3 mutations. This is likely to be the case as the observation that a significant proportion of ACC bear nuclear β-catenin staining not explained by CTNNB1 mutation status preceded the discovery of ZNRF3 mutations in ACC [Though mutation data are not available for the TMA tumors we characterized, we assume that nuclear β-catenin staining captures tumors with a spectrum of Wnt/β-catenin activity, including those with either s in ACC . Our chaITGA2, where we observed a variable response to Wnt/β-catenin inhibitors. Indeed, the regulation of ITGA2 expression has been previously linked to not only β-catenin but also the transcription factor FOXL2, as well as AP1, AP2, and GATA [We speculate that the correlation between Wnt/β-catenin and ECM is more broadly applicable. Indeed, differential ECM secretion has been linked to Wnt/β-catenin signaling in Ewing Sarcoma cells . Whereasand GATA .COL11A1 expression is present in both NCI-H295R cells and in the neoplastic cells present in human ACC samples. Moreover, TCGA data confirm that more aggressive subtypes of ACC are both enriched for Wnt/β-catenin and exhibit the lowest level of stromal cell infiltration, consistent with a cell autonomous role of Wnt/β-catenin activity in ECM deposition [In other studies, COL11A1 expression has been almost exclusively localized to cancer-associated stromal cells ,56. In cposition ,50. ThouCOL11A1 transcript and protein expression is associated with shortened OS and DFS is consistent with findings in ovarian cancer [COL11A1 expression is associated with chemoresistance in human tumors [Our finding that n cancer  and compn tumors . These rGiven our desire to ultimately target therapy against the Wnt/β-catenin pathway, we developed a model to translate ACC in vitro findings in vivo. We implemented and characterized a novel model of ACC that captures features of high-grade ACC in patients. The high mitotic rate and Ki67 labeling index of both NCI-H295R and Y1 tumors capture histologic features of patient tumors with high risk for recurrence and decreased overall survival . We alsoCTNNB1 mutation. Our results are consistent with and complimentary to prior studies in which β-catenin knockdown reduced Wnt/β-catenin transcriptional activity in ACC cells and impaired the function of β-catenin in cell-cell adhesion [We used our preclinical tumor model to extend our in vitro findings and confirm that the therapeutic inhibition of Wnt/β-catenin significantly inhibits the growth of tumors harboring a GOF adhesion ,39. Takeadhesion .CTNNB1 mutation. Importantly, we developed a novel orthotopic xenograft model to investigate the preclinical implications of these findings and found that the in vivo inhibition of Wnt/β-catenin:TBL1 signaling with Tegavivint significantly reduced tumor growth. Collectively, our studies demonstrate the preclinical efficacy of a Wnt/β-catenin inhibitor in ACC and underscore the rationale for therapeutically translating Wnt/β-catenin inhibition in ACC patients.In conclusion, we identified that ACC with high Wnt/β-catenin activity exhibits a unique expression pattern of ECM components associated with poor survival. Moreover, Wnt/β-catenin inhibition disrupts ECM expression and reduces growth in ACC with an activating


Microneedles (MNs) have drawn rising attention owing to their merits of convenience, noninvasiveness, flexible applicability, painless microchannels with boosted metabolism, and precisely tailored multifunction control. MNs can be modified to serve as novel transdermal drug delivery, which conventionally confront with the penetration barrier caused by skin stratum corneum. The micrometer-sized needles create channels through stratum corneum, enabling efficient drug delivery to the dermis for gratifying efficacy. Then, incorporating photosensitizer or photothermal agents into MNs can conduct photodynamic or photothermal therapy, respectively. Besides, health monitoring and medical detection by MN sensors can extract information from skin interstitial fluid and other biochemical/electronic signals. Here, this review discloses a novel monitoring, diagnostic, and therapeutic pattern by MNs, with elaborate discussion about the classified formation of MNs together with various applications and inherent mechanism. Hereby, multifunction development and outlook from biomedical/nanotechnology/photoelectric/devices/informatics to multidisciplinary applications are provided. Programmable intelligent MNs enable logic encoding of diverse monitoring and treatment pathways to extract signals, optimize the therapy efficacy, real-time monitoring, remote control, and drug screening, and take instant treatment. Lethal contagions and massive other diseases are threatening human health and quality of living. Pharmaceuticals have to pass through the stratum corneum (SC) and viable epidermis before entering the capillaries and circulatory system . Three rMicroneedles (MNs) generally consist of arrays of micrometer-sized needles with a height that ranged from 25 to 1,500 μm ,7. The mMicroneedle therapy (MNT) refers to a diagnostic and therapeutic strategy based on material science and biomedical and flexible electronics/optoelectronics using MN platform for diagnosis and treatment of targeted diseases  via the multifunctionalization of MNs, involving medical diagnosis, health monitoring, medical beauty, transcutaneous immunization (TCI), and other multidisciplinary functions Fig. . This reThe MN fabrication methods include lithography, cast micromolding, injection molding, hot embossing, droplet-born air blowing (DAB), drawing lithography, centrifugal lithography (CL), 3-dimensional (3D) printing methods, and so on. MNs are generally classified into solid MNs, coated MNs, hollow MNs, dissolving MNs, and hydrogel-forming MNs (HFMNs) Fig. . AbundanSolid MNs, the earliest type of MNs for TDD, are usually made up of silicon, metals, and polymers . PracticCoated MNs are prepared by coating drug formulation onto the surface of MNs . After iHollow MNs, which resemble microsyringes, deliver specific drugs into skin by injecting fluid formulations into deep tissue through multiple needles at the same time . ContinuDissolving MNs are made by micromolding water-soluble biodegradable, biocompatible polymer, or polysaccharides to enclose drugs in MNs . After iHFMNs were first reported in 2012 and typically consist of swellable polymers (crosslinked hydrogels) . They ar2 is deposited on the Si wafer, the photosensitizer is coated on the SiO2 surface, ultraviolet (UV) light is irradiated on the photosensitizer through a mask to form a microstructure, and then the fine microstructure preparation of the Si surface is realized by etching and removing the photosensitizer  technology is a common method to fabricate silicon MNs . First, zer Fig. A. Wang eCast micromolding is the MN forming process by filling a solution into a pre-prepared MN mold before solidification Fig. B 48]. E. E48]. EInjection molding is a molding method to form MN structures by injecting polymer solutions into molds Fig. C 52]. M. M52]. MHot embossing forms MNs by melting the polymer into a liquid with heat and then applying pressure to fill in the mold Fig. D 58]. P. P58]. PThe drawing lithography method is to coat a negative photoresist on a glass substrate, the photoresist is stretched into a needle-like structure as a solid MN mold, a metal coating is formed on the surface of MN mold by electroplating, and then the photoresist was removed to obtain hollow metal MNs Fig. E 62]. B. B62]. BDAB and CL both obtain MNs by forming hourglass-shaped droplets between 2 parallel substrates before solidification. The DAB method uses the movement of parallel substrates to connect viscous droplets between the upper and lower substrates, and directly blows the polymer solution through air to solidify it to form MNs Fig. F 66]. S. S66]. SCL uses centrifugal method to elongate the polymer droplets on the surface of the lower substrate under centrifugal force Fig. G. After 3D printing, also known as additive manufacturing, can rapidly, accurately, and stably prepare materials with fine structure, and as an emerging technology, it can facilitate the development of material and biomedical science  was tested by in vitro cell migration experiments, and MOF-GO-Ag-MN had the best effect of promoting cell migration. The wound healing in diabetic mice was evaluated, and it was found that MOF-GO-Ag-MN treatment had the smallest wound area, the largest tissue regeneration, and the fastest new angiogenesis rate, demonstrating the best wound healing effect. Zn-MOFs can eliminate bacteria by releasing zinc ions and have good biocompatibility and antibacterial properties [Active ingredients can be effectively delivered to the lesion site through MNs, achieving efficient delivery and better therapeutic effects. Chronic nonhealing wounds are colonized by bacteria that often develop into biofilms and act as a physicochemical barrier to internal bacteria, inducing chronic inflammation and tissue hypoxia . Removinoperties . The Zn-operties . The Zn-Chronic diabetic wounds can induce skin ulcers and impaired angiogenesis, ROS overexpression, and persistent inflammation, which make wounds difficult to heal . TherefoPremna microphylla and Centella asiatica, and loaded asiatic acid (AA) as a pharmaceutical active ingredient into MNs  staining discovered that the thickness of regenerated granulation tissue in CHMN group was the largest. Immunohistochemical analysis of the expression of the pro-inflammatory factor interleukin-6 (IL-6) in the wound area discovered that the CHMN group had the lowest IL-6 expression. Masson's trichrome staining showed that the CHMN group had the most collagen deposition, and immunofluorescence staining showed that the wound site of the CHMN group formed more new blood vessels. These results substantiate that CHMN has excellent ability to promote wound healing and has valuable application potential. Chitosan is a derivative of chitin, which not only has good biocompatibility but also has antibacterial effect. Using chitosan as the matrix of MNs can enable the antibacterial function [S. aureus and E. coli cocultured with the CSMNA patch indicated its excellent antibacterial performance. In vivo animal experiments in rats showed that CSMNA patch could significantly promote wound healing by favoring the granulation tissue formation and collagen deposition, inhibiting the expression of pro-inflammatory factors, and accelerating angiogenesis, with high potential for practical applications.In addition to the synthetic active ingredients, traditional Chinese medicine is a time-honored, ancient, and unique drug system, which has achieved considerable therapeutic effects from China to all over the world . CombiniMNs Fig. C. In vitfunction . Wound hfunction . ControlPTT is a treatment method in which photothermal agents can convert light energy into heat under NIR laser irradiation to kill bacteria or pathogens . Adjusti1O2, superoxide anion O2−, and hydroxyl radical OH•) under light so as to eliminate target cells, and since no drug is used, no drug resistance occurs [S. aureus and E. coli with high antibacterial activity [2+ doped Zn2GeO4 (ZGC) and combined it with HA MN to prepare ZGC@MN patches that promote wound healing  by biomimetic mineralization and prepared CINP@SiO2 NPs with good bioactivity, which then combined with MeHA MN patches to achieve the dual efficacy of tumor PTT and wound healing  method. The migration experiments of HUVECs showed that the CINP@SiO2-HA MN group had the highest mobility and could promote the migration of HUVECs. The expressions of hypoxia-inducible factor 1α (HIF-1α), VEGF, kinase insertion domain receptor (KDR), and endothelial NO (eNOs) were also measured by enzyme-linked immunosorbent assay (ELISA), and it was found that the CINP@SiO2-HA MN group had the highest expression effect, indicating that CINP@SiO2-HA MNs had the ability to promote angiogenesis. In vitro antitumor experiments showed that the CINP@SiO2-HA MN group had the best photothermal treatment effect. In vivo animal experiments confirmed that CINP@SiO2-HA MNs could simultaneously achieve antitumor and skin tissue regeneration effects through PTT, ROS scavenging, and up-regulation of angiogenic gene expression. Accordingly, the CINP@SiO2-HA MN patch has valuable application potential.It is usually difficult to achieve the best therapeutic effect by a single antitumor therapy, and a more effective treatment can often be achieved by adopting a synergistic therapy. Surgical removal of superficial tumors and surrounding skin tissue is the mainstay of treatment for malignant skin tumors, but the risk of postoperative residual infiltrating tumor cells and wound infection still remains . Lei et ing Fig. A. 3- are one of the constituents of melanoma, which can lead to melanoma recurrence and ineffective treatment . The aPDInsulin injections are the most common treatments for diabetes, but the pain and discomfort caused by intravenous injections induce the physical and mental inconvenience for diabetic patients . MNT canOral administration is the most convenient and common way of administration, but the difficulty in overcoming the complex gastrointestinal barrier and in achieving efficiently targeted absorption of macromolecular drugs still remains a challenge . Using aObesity is a serious problem facing mankind all over the world, which trigger a variety of metabolic disorders and numerous diseases such as hyperglycemia and blood pressure increase, insulin resistance, type 2 diabetes, cancer, and cardiovascular and cerebrovascular diseases . ExtensiVascular insufficiency and/or oxidative stress in peri-follicular microenvironment can cause androgenetic alopecia (AGA) . DeliverIn addition to traditional drugs, some natural active ingredients combined with MN can also effectively treat AGA. β-Sitosterol is a natural phytosterol that exists in multiple parts of plants and has various pharmacological activities such as angiogenesis, antioxidant, and immunomodulation . EncapsuPropionibacterium acnes is one of the causes of acne [P. acnes by PDT to achieve excellent anti-acne effect , and other difficult diseases will have high possibility of immunization, diagnosis, and therapy. However, the industrialization of pharmaceutical products requires a long process. Several improvements may speed up this process.Approved drugs and materials have been clinically and lawfully permitted for in vivo use, which are more secure in terms of biosafety, and thus, MNs prepared using these substances are more likely to be approved for applications in clinical trials, contributing to the early realization of MNT.To shorten the time of complex pharmacological research, PDT and PTT based on photosensitized and photothermal materials/NPs are rising fields adopting biosafe component/nanostructures that catalytically engender ROS and energy, respectively, to kill viruses, microbials, pathogens, and malignant cells.Personalized and customized MNs are designed to accommodate that MNs can be prepared at home, beauty salon, and health consulting and clinic centers. The prepolymer solutions containing different active ingredients can be purchased directly, and people only need to add the ingredient solution to a small MN preparation machine to prepare MN patches loaded with active ingredients for the treatment of corresponding diseases, healthcare, and beauty and skin problems.The design of flexible MNs that can support electronic/optoelectronic devices will become a future trend. The existing MN patches are generally small in size and can be directly pressed into the skin for real-time monitoring, diagnosis, information transmission, human–machine interaction, and controllable treatment. Designing an MN patch with a larger size can not only effectively increase the content of active ingredients but also facilitate the functional integration of the MN patch with larger area. It is also necessary to develop the insertion device optimizing the MN patch to achieve optimal attachment. For example, the flexible MN batteries using biocompatible electrolyte  and electrodes  could be developed to power the implantable and wearable devices such as heart pacemakers, body chips, artificial electronic cochlear implant, wearable sensors, and electronic tablets, avoiding the hazard risk of nonbiocompatible corrosive and poisonous electrolyte from Li-ion batteries otherwise. The unique semi-implantable property enables the MN sensors to monitor both surface and internal bioinformation simultaneously.Intelligent MN patches with highly integrated functions are awaiting development. The combination of MN patch and intelligent electronics can not only realize real-time health monitoring, remote control, and big-data healthcare but also take corresponding disease treatment or countermeasures against abnormal conditions. For example, the MN patch can monitor the blood glucose value in the body in real time and automatically deliver an appropriate amount of insulin to the body when the blood glucose value exceeds a certain threshold to reduce blood glucose. Through real-time monitoring of the electrocardiogram of patients with angina pectoris, if abnormal heart rate occurs, the MN patch can deliver the loaded angina pectoris emergency medicine  to the body intelligently, which can greatly reduce the mortality of patients. It is believed that the intelligent MN patches will greatly benefit the healthcare and quality of human life.Flourishing development of brain–computer interfaces, artificial neural network, implantable/wearable devices, artificial intelligence, and software robotics requires the intimate alliance, interaction, and integration between technology and living organisms. Because the basic building blocks of living organisms are different from those used in electronic devices, the ability to connect artificial devices to biological systems is critical to the success in these fields. MN chips could be expected to have the integrated structure of perception, storage and computing, large-scale parallel processing, and event-driven operation, enabling the accurate perception, response, and treatment to real-time events in a robust and energy-efficient manner. MNs as the integrated platform for efficient and low-power processing of big data could combine neuromorphic system to build a biomimetic cognitive system, matching the biological neural perception and processing capabilities. MN chips combining the biological/artificial neural networks could perform tasks such as pattern classification and feature extraction to analyze the photosignals/electrosignals/chemical signals of bioinformation with real-time solving problems of disease, accidents, uncertain conditions, and abnormal states.Programmable photoelectronic MNs combine the polarized light, infrared treatment, and stereo-dynamic interference electrical therapy, which are highly integrated, and can be carried out independently or at the same time, almost targeted at all pain lesions. They show advantages including no pain, no injury, no risk of infection, short treatment time, wide range of adaptation, no side effects, and complications. Polarized light emitted by MNs can be used in elderly patients who have allergic reactions to drugs and hemorrhagic symptom and other patients who are not suitable for nerve block therapy. Infrared polarized light emitted by MNs can be used as an alternative therapy for nerve block to treat exercise pain (a variety of subacute chronic muscle and joint pain), pain caused by various bone and joint degenerative diseases , and various arthritis. Additionally, infrared MNs can reduce inflammation and improve the immune ability and disease resistance. Combination of various wavelength lasers can perform better than single-wavelength laser for therapy. Besides, stereo-dynamic interference electro-therapeutic MNs can relieve pain and quickly eliminate depression pain or exercise fatigue. Hence, the programmable photoelectronic MNs combining polarized light, infrared, and stereo-dynamic interference electrical therapy can process the logic encoding of diverse photoelectric therapeutic pathways to adapt to different conditions and optimize the therapy efficacy.MN chips refer to integrated platform that is implantable/wearable to body and capable of collecting photo-/electro-/chemical signals, analyzing, computing, and processing treatment in real time. According to diverse functions, the MN chips could be classified into DNA chips, RNA chips, protein chips, tissue chips, organ chips, neural chips, multifunctional chips, etc. The MN chips could load high density of precisely fixed biomolecules, integrated circuits, and devices, which show advantages of high throughput, miniaturization, and fast processing speed, and accordingly could be widely used in the fields of cell analysis, disease diagnosis, drug screening, individualized therapy, and gene sequencing. For example, the MN RNA chips could perform RNA sequencing and detection for the real-time virus  detection, analysis, diagnosis, treatment, drug screening, and delivery to ensure the security of personal health safety. The MN cell chips using breast cancer-specific peptide as probe molecules could perform breast cancer cell detection in real time. MN organ chips could realize the dynamic monitoring of normal/abnormal signals of certain organs and the drug metabolism/efficacy pathways on target organs. The development of MN chips depends largely on the multidisciplinary integration of multiple technologies. The pursuit of higher detection sensitivity, lower detection cost, and more refined detection/treatment performance is the unlimited driving force for the upgrading of MN chip technology. The wearable/implantable MN chips could be promising for real-time monitoring/diagnosis/treatment of either body surface or internal organs. The advantages of implantable MN chips include the remote sensing and control of biological information, which can be implanted into human body and perform a series of tasks. The future of MN chips requires the combination of novel techniques such as biological computers and artificial intelligence to achieve biological information extraction, data storage, computing, processing, intelligent decision-making, and instruction execution.Optogenetic MNs could possess advantages of noninvasive, reversible, and spatial–temporal precision, offering novel approach and concepts for disease treatment. As a good inducer of gene expression, light can manipulate gene expression and cell behavior with unprecedented spatial and temporal precision. With further exploration of optogenetic MN techniques, the personalized precision therapy and clinical transformation based on optogenetics could become possible. Programmable light-emitting MNs as the optogenetic tools can correspond to different light wavelengths for applications in precision therapy for the neurological diseases, tumors, cardiovascular diseases, diabetes, and intestinal diseases, as well as applications for controlling gene transcription expression, gene editing, gene recombination, and organelle movement. At the same time, multifunctional combination of optogenetic MNs and intelligent electronic equipment will be used in implantable/portable intelligent bioelectronic medicine, artificial intelligence diagnosis, and treatment, which are promising to be realized. The remote controllability, reversibility, and nontoxicity of the optical control system provide a solid foundation for the application of optogenetics in biomedicine. The success of these approaches will have a long-lasting influence on precision medicine for practice in future.MN electrodes applied with certain electric potentials could induce electrochemical catalytic reactions to reduce inflammatory factors, remove vascular thrombosis, and eliminate gastric alcohol or other target substances in organs, in vitro and in vivo. Subsequently, MN bioelectrodes could treat a variety of diseases through electrical stimulation therapy and bioelectrocatalytic reactions, such as electrocatalytic oxygen evolution reaction for blood oxygen supply and hydrogen evolution reaction to reduce peroxide inflammatory cytokines avoiding the tissue damage or aging. Besides, MN biofuel cells or biobatteries with biocompatible electrolytes are demanded for wearable and implantable devices , avoiding the poisonous and corrosive electrolytes from Li-ion batteries, and especially avoiding the tedious surgery every few years to replace battery of heart pacemakers. Bioenergy from inner or surface system could be directly converted into electric power by MN biofuel cells. Safe and secure electrodes could be guaranteed to eliminate hazard risks. Various other applications such as the microplastic beauty, microdisplay light-emitting devices, and programmable matrix array devices are awaiting for future exploration to benefit scientific research and industrial development.Owing to the rapid development of micromachining technology, growing studies of MNs are creating approaches for multifunctional therapy, diagnosis, monitoring, immunization, medical beauty, electronic/optoelectronic devices, intelligent integrated circuits, and so on. Combining the advantages of multidisciplinary techniques including biomedical, nanomedical, photonic, electronic, photoelectronic, catalytic, and nanomaterial technologies, optogenetics, electronic information, computing, and artificial intelligence, MNs become promising to improve human health and quality of life, expanding to various intelligent devices and facilities. Accordingly, numerous diseases such as cancer, diabetes, cardiac–cerebral vascular disease, wound infection, contagions, and arthritis could be healed by the multifunctional MN diagnosis and therapy. Ascribed to the various merits including painless, minimally invasive, flexible, portable, simple operation, precise control, accurate detection, and strong adaptability, MNs will become a significant future trend among hotspots involving nanotechnology, flexible devices, photo-/electro-chemical catalytic therapy, optogenetics, remote control, healthcare, big data, human–machine interaction, brain–computer interfaces, artificial neural networks, Internet plus artificial intelligence, and so on.Programmable, intelligent, and photoelectronic MNs allying PDT, PTT, polarized infrared/photoelectronic devices, integrated circuits, and nanotechnology could process the logic encoding of diverse monitoring and treatment pathways to adapt to different conditions, optimize the therapy efficacy, realize the real-time health monitoring, remote control, and big-data healthcare, and also take instant disease treatment. MN chips show advantages of high throughput, miniaturization, and fast processing speed and could be used in cell analysis, disease diagnosis, drug screening, individualized therapy, nucleic acid detection, and RNA/DNA sequencing. MN chips combining the biological/artificial neural networks and neuromorphic system for efficient and low-power processing of big data could build a biomimetic cognitive system, matching the biological neural perception and processing capabilities. Programmable photoelectronic MNs as the intelligent optogenetic tools can control different light wavelengths for precision therapy of neurological diseases, tumors, cardiovascular disease, diabetes, and intestinal diseases, as well as applications for the control of gene transcription expression, gene editing, gene recombination, and organelle movement.