Biomolecular fingerprints of the effect of zoledronic acid on prostate cancer stem cells: Comparison of 2D and 3D cell culture models.

Revealing the potential of candidate drugs against different cancer types without disrupting normal cells depends on the drug mode of action. In the current study, the drug response of prostate cancer stem cells (PCSCs) to zoledronic acid (ZOL) grown in two-dimensional (2D) and three-dimensional (3D) culture systems was compared using Fourier transform-infrared (FT-IR) spectroscopy which is a vibrational spectroscopic technique, supporting by biochemical assays and imaging techniques. Based on our data, in 2D cell culture conditions, the ZOL treatment of PCSCs isolated according to both C133 and CD44 cell surface properties induced early/late apoptosis and suppressed migration ability. The CD133 gene expression and protein levels were altered, depending on culture systems. CD133 expression was significantly reduced in 2D cells upon ZOL treatment. FT-IR data revealed that the integrity, fluidity, and ordering/disordering states of the cell membrane and nucleic acid content were altered in both 2D and 3D cells after ZOL treatment. Regular protein structures decrease in 2D cells while glycogen and protein contents increase in 3D cells, indicating a more pronounced cytotoxic effect of ZOL for 2D cells. Untreated 3D PCSCs exhibited an even different spectral profile associated with IR signals of lipids, proteins, nucleic acids, and glycogen in comparison to untreated 2D cells. Our study revealed significant differences in the drug response and cellular constituents between 2D and 3D cells. Exploring molecular targets and/or drug-action mechanisms is significant in cancer treatment approaches; thus, FT-IR spectroscopy can be successfully applied as a novel drug-screening method in clinical research.

Abinitiocomputation of low-temperature miscibility gap of V(Se,Te)2.

Monolayers of quasi-binary transition metal dichalcogenides are a focus of attention as they are expected to exhibit many exciting physical properties, but not much is known about their thermodynamic stability. In this study, we use a combination of global energy landscape exploration, local minimization using density functional theory, and thermodynamic analysis, to compute the composition-temperature phase diagram of the quasi-binary V(Se,Te)2system, both for a 2H monolayer and for the analogous bulk material. We find that the phase diagram exhibits a miscibility gap, with a critical temperatureTc= 500 K andTc= 650 K for monolayer and bulk, respectively, indicating that the system prefers to form solid solution phases. In particular, at room temperature, the thermodynamically stable phase of the monolayer would correspond to a decomposition into two solid solution monolayers, with ca. 90% Se and Te content, respectively.

A three-dimensional spheroid-specific role for Wnt-ß-catenin and Eph-ephrin signaling in nasopharyngeal carcinoma cells.

One of the greatest unmet needs hindering the successful treatment of nasopharyngeal carcinomas (NPCs) is for representative physiological and cost-effective models. Although Epstein-Barr virus (EBV) infection is consistently present in NPCs, most studies have focused on EBV-negative NPCs. For the first time, we established and analyzed three-dimensional (3D) spheroid models of EBV-positive and EBV-negative NPC cells and compared these to classical two-dimensional (2D) cultures in various aspects of tumor phenotype and drug responses. Compared to 2D monolayers, the 3D spheroids showed significant increases in migration capacity, stemness characteristics, hypoxia and drug resistance. Co-culture with endothelial cells, which mimics essential interactions in the tumor microenvironment, effectively enhanced spheroid dissemination. Furthermore, RNA sequencing revealed significant changes at the transcriptional level in 3D spheroids compared to expression in 2D monolayers. In particular, we identified known (VEGF, AKT and mTOR) and novel (Wnt-ß-catenin and Eph-ephrin) cell signaling pathways that are activated in NPC spheroids. Targeting these pathways in 3D spheroids using FDA-approved drugs was effective in monoculture and co-culture. These findings provide the first demonstration of the establishment of EBV-positive and EBV-negative NPC 3D spheroids with features that resemble advanced and metastatic NPCs. Furthermore, we show that NPC spheroids have potential use in identifying new drug targets.

The Versatile Electronic, Magnetic and Photo-Electro Catalytic Activity of a New 2D MA2 Z4 Family*.

The recent successful growth of MoSi2 N4 and WSi2 N4 monolayers led to the discovery of a new class of the two-dimensional (2D) MA2 Z4 materials with no known 3D layered allotropes, which renders great possibilities to integrate diverse properties by proper design of sandwiched "MZ2 " building blocks and "A-Z" passivation layers. In this work, the dynamic stability, electronic properties, and surface reactivity of the new MA2 Z4 family, in which M is Ti, Zr, Hf, V, Nb, Ta, Cr, Mo, W, A refers to Si or Ge, and Z is N, P or As, is theoretically probed. Among the proposed 54 possible combinations, about 42 compositions are dynamically stable, which vary from non-magnetic, anti-ferromagnetic, to ferromagnetic semiconductors, metals and half-metals. In particular, the VB (V, Nb, Ta) MA2 Z4 possesses robust intrinsic ferromagnetism that is essential for spintronics applications. In regard to surface activity, most MA2 Z4 , particularly N- or P-terminated IVB and VB MA2 Z4 , have high catalytic potential for hydrogen evolution, and the ▵GH of non-magnetic MA2 Z4 is highly correlated to the highest occupied p electronic states of the surface Z atoms. The photocatalytic activity is also evaluated. MoSi2 N4 and WSi2 N4 within 4 % tensile strain are capable of photocatalytic overall water splitting. The findings indicate the new 2D MA2 Z4 family has fascinating properties and possesses strong potential for applications but not limited to electronics, spintronics and catalysts, which will stimulate the interests of experimental synthesis.

Generation of equine enteroids and enteroid-derived 2D monolayers that are responsive to microbial mimics.

Enteroid cultures are three-dimensional in vitro models that reflect the cellular composition and architecture of the small intestine. One limitation with the enteroid conformation is the enclosed lumen, making it difficult to expose the apical surface of the epithelium to experimental treatments. The present study was therefore conducted to generate cultures of equine enteroids and to develop methods for culture of enteroid-derived cells on a two-dimensional plane, enabling easy access to the apical surface of the epithelium. Equine enteroids were established from small intestinal crypts within 7-9 days of culture. Transcriptional analysis of cell type markers confirmed the presence of enterocytes, stem-, Paneth-, proliferative-, enteroendocrine-, goblet- and tuft cells. This cellular composition was maintained over multiple passages, showing that the enteroids can be kept for prolonged periods. The transfer from 3D enteroids to 2D monolayers slightly modified the relative expression levels of the cell type markers, indicating a decrease of goblet- and Paneth cells in the monolayers. Stimulation with the TLR2, 3 and 4 agonists Pam3CSK4, Poly I:C and LPS, respectively, induced the pro-inflammatory cytokines TNF-α and IL-8, while the TLR5 agonist FliC only induced TNF-α. In addition, an up-regulation of TGF-ß, IL-33 and IFN-ß was recorded after exposure to lipofected Poly I:C that also affected the monolayer integrity. Thus, the equine enteroid-derived 2D monolayers described in the present study show both genetic and functional similarities with the equine intestine making it an interesting in vitro model for studies demanding access to the apical surface, e.g. in studies of host-microbe interactions.

Sn3C2monolayer with transition metal adatom for gas sensing: a density functional theory studies.

The gas sensing properties of pristine Sn3C2monolayer and different transition metal adatom (TM-Sn3C2, where TM = Fe, Co, Ni, Cu, Ru, Rh, Pd and Ag) was investigated using van der Waals corrected density functional theory. The understanding and potential of use of Sn3C2monolayers as sensors or adsorbent for CO, CO2, NO, NO2and SO2gaseous molecules is evaluated by calculating the adsorption and desorption energetics. From the calculated adsorption energies, we found that the pristine Sn3C2monolayer and 3dTM has desirable properties for removal of the considered molecules based on their high adsorption energy, however the 4dTM is applicable as recoverable sensors. We applied an Arrhenius-type equation to evaluate the recovery time for the desorption of the molecules on the pristine and TM adatom on Sn3C2monolayer. We found that the negative adsorption energies from -1 to -2 eV of the molecules resulted in easier recovery of the adsorbed gases at reasonable temperatures compared to adsorption energies in between 0 and -1 eV (weakly physiosorbed) and below -2 eV (strongly chemisorbed). Hence, we obtained that the Rh-Sn3C2, Ru-Sn3C2, Pd-Sn3C2, Pd-Sn3C2, and Rh-Sn3C2monolayers are good recoverable scavengers for the CO, CO2, NO, NO2, and SO2molecules. The current theoretical calculations provide new insight on the effect of TM adatoms on the structural, electronic, and magnetic properties of the Sn3C2monolayer and different transition metal adatom as well as shed light on their application as gas sensors/scavengers.

Nonlinear Dark-Field Imaging of One-Dimensional Defects in Monolayer Dichalcogenides.

One-dimensional defects in two-dimensional (2D) materials can be particularly damaging because they directly impede the transport of charge, spin, or heat and can introduce a metallic character into otherwise semiconducting systems. Current characterization techniques suffer from low throughput and a destructive nature or limitations in their unambiguous sensitivity at the nanoscale. Here we demonstrate that dark-field second harmonic generation (SHG) microscopy can rapidly, efficiently, and nondestructively probe grain boundaries and edges in monolayer dichalcogenides (i.e., MoSe2, MoS2, and WS2). Dark-field SHG efficiently separates the spatial components of the emitted light and exploits interference effects from crystal domains of different orientations to localize grain boundaries and edges as very bright 1D patterns through a Cerenkov-type SHG emission. The frequency dependence of this emission in MoSe2 monolayers is explained in terms of plasmon-enhanced SHG related to the defect's metallic character. This new technique for nanometer-scale imaging of the grain structure, domain orientation and localized 1D plasmons in 2D different semiconductors, thus enables more rapid progress toward both applications and fundamental materials discoveries.

Oxygen Is an Ambivalent Factor for the Differentiation of Human Pluripotent Stem Cells in Cardiac 2D Monolayer and 3D Cardiac Spheroids.

Numerous protocols of cardiac differentiation have been established by essentially focusing on specific growth factors on human pluripotent stem cell (hPSC) differentiation efficiency. However, the optimal environmental factors to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Here, we hypothesized that low oxygen exposure enhances the molecular and functional maturity of the cardiomyocytes. We aimed at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hPSCs in 2D- and 3D-based protocols. hPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed. The mitochondrial localization and metabolic properties were evaluated. The intracellular Ca2+ handling and contractile properties were also monitored. The 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac spheroids containing hPSC-CMs in LOE further exhibited cardiac markers, hypertrophy, steadier SR Ca2+ release properties revealing a better SR Ca2+ handling, and enhanced contractile force. Preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac spheroids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.

Gas Adsorption Investigation on SiGe Monolayer: A First-Principle Calculation.

The gas adsorption behaviors of CO, CO2, SO2, NO2, NO, NH3, H2, H2O, and O2 on SiGe monolayer are studied using the first-principles calculation method. Three special adsorption sites and different gas molecule orientations are considered. Based on adsorption energy, band gap, charge transfer, and the electron localization function, the appropriate physical adsorptions of SO2, NO, NH3, and O2 are confirmed. These gases possess excellent adsorption properties that demonstrate the obvious sensitiveness of SiGe monolayer to these gases. Moreover, SiGe may be used as a sensing material for some of them. NO2 adsorption in different adsorption sites can be identified as chemical adsorption. Besides, the external electric field can effectively modify the adsorption strength. The range of 0 ~ - 2 V/nm can create a desorption effect when NH3 adsorbs at the Ge site. The NH3 adsorption models on Ge site are chosen to investigate the properties of the I-V curve. Our theoretical results indicate that SiGe monolayer is a promising candidate for gas sensing applications.

Resistive Memory Devices at the Thinnest Limit: Progress and Challenges.

The Si-based integrated circuits industry has been developing for more than half a century, by focusing on the scaling-down of transistor. However, the miniaturization of transistors will soon reach its physical limits, thereby requiring novel material and device technologies. Resistive memory is a promising candidate for in-memory computing and energy-efficient synaptic devices that can satisfy the computational demands of the future applications. However, poor cycle-to-cycle and device-to-device uniformities hinder its mass production. 2D materials, as a new type of semiconductor, is successfully employed in various micro/nanoelectronic devices and have the potential to drive future innovation in resistive memory technology. This review evaluates the potential of using the thinnest advanced materials, that is, monolayer 2D materials, for memristor or memtransistor applications, including resistive switching behavior and atomic mechanism, high-frequency device performances, and in-memory computing/neuromorphic computing applications. The scaling-down advantages of promising monolayer 2D materials including graphene, transition metal dichalcogenides, and hexagonal boron nitride are presented. Finally, the technical challenges of these atomic devices for practical applications are elaborately discussed. The study of monolayer-2D-material-based resistive memory is expected to play a positive role in the exploration of beyond-Si electronic technologies.

Experimental and Computational Analysis of High-Intensity Focused Ultrasound Thermal Ablation in Breast Cancer Cells: Monolayers vs. Spheroids.

High-intensity focused ultrasound (HIFU) is a non-invasive therapeutic modality that uses precise acoustic energy to ablate cancerous tissues through coagulative necrosis. In this context, we investigate the efficacy of HIFU ablation in two distinct cellular configurations, namely 2D monolayers and 3D spheroids of epithelial breast cancer cell lines (MDA-MB 231 and MCF7). The primary objective is to compare the response of these two in vitro models to HIFU while measuring their ablation percentages and temperature elevation levels. HIFU was systematically applied to the cell cultures, varying ultrasound intensity and duty cycle during different sonication sessions. The results indicate that the degree of ablation is highly influenced by the duty cycle, with higher duty cycles resulting in greater ablation percentages, while sonication duration has a minimal impact. Numerical simulations validate experimental observations, highlighting a significant disparity in the response of 2D monolayers and 3D spheroids to HIFU treatment. Specifically, tumor spheroids require lower temperature elevations for effective ablation, and their ablation percentage significantly increases with elevated duty cycles. This study contributes to a comprehensive understanding of acoustic energy conversion within the biological system during HIFU treatment for 2D versus 3D ablation targets, holding potential implications for refining and personalizing breast cancer therapeutic strategies.

Quaternary 2D monolayer Cu2Cl2Se2Hg2: anisotropic carrier mobility and tunable bandgap for transistor and photocatalytic applications.

Despite the advantages of quaternary two-dimensional (2D) materials, fewer studies have been done on them than binary 2D materials. Calculations of quaternary 2D monolayer Cu2Cl2Se2Hg2based on density functional theory and Green's function surface analysis provide insights into its structural, dynamic, and thermal stability. This material has a direct band gap of 0.91/2.0 eV (Perdew-Burke-Ernzerhof/Heyd-Scuseria-Ernzerhof) and demonstrates anisotropic carrier mobility. The electron mobility in theadirection is 1.2 × 103cm2V-1s-1, which is significantly higher than the hole mobility of 0.48 × 103cm2V-1s-1. In thebdirection, the electron mobility is 1.01 × 103cm2V-1s-1and is 8.9 times larger than the hole mobility of 0.11 × 103cm2V-1s-1. The light absorption coefficients of Cu2Cl2Se2Hg2are 1.0 × 105cm-1and 2.5 × 105cm-1in the visible and ultraviolet ranges, respectively. Uniaxial strain leads to an anisotropic alteration in the band gap and band edge position. By manipulating the strain direction and level in Cu2Cl2Se2Hg2, it is possible to increase the current ON/OFF ratio for field-effect transistors (FETs) and to facilitate photocatalytic water splitting through a redox reaction. The research reveals that Cu2Cl2Se2Hg2, a 2D monolayer in the quaternary form, has promising capabilities as an alternative for creating crystal-oriented FETs and photocatalytic water splitting systems.

Flexo-Ferroelectricity and a Work Cycle of a Two-Dimensional-Monolayer Actuator.

Well recognized mechanical flexibility of two-dimensional (2D) materials is shown to bring about unexpected behaviors to the recently discovered monolayer ferroelectrics, especially those displaying normal, off-plane polarization. A "ferro-flexo" coupling term is introduced into the energy expression, to account for the connection of ferroelectricity and bending (strain gradient) of the layer, to predict and quantify its spontaneous curvature and how it affects the phase transitions. With InP as a chemically specific representative example, the first-principles calculations indeed reveal strong coupling ∼P·Ï° between the ferroelectric polarization (P) and the curvature of the layer (Ï° ≡ 1/r), having profound consequences for both mechanics and ferroelectricity of the material. Due to flexural relaxation, the spontaneous polarization and the transition barrier rise significantly, leading to large changes in the Curie temperature, coercive field, and domain wall width and energy, based on Monte Carlo simulations. On the other hand, the polarization switching, characteristic to ferroelectrics, does induce an overall layer bending, enabling a conversion of electrical signal to movement as an actuator; its possible work-cycles and maximum work-efficiency are briefly discussed.

Molecular Triplet Sensitization of Monolayer Semiconductors in 2D Organic/Inorganic Hybrid Heterostructures.

Hybrid heterostructures (HSs) comprising organic and two-dimensional (2D) monolayer semiconductors hold great promise for optoelectronic applications. So far, research efforts on organic/2D HSs have exclusively focused on coupling directly photoexcited singlets to monolayer semiconductors. It remains unexplored whether and how the optically dark triplets in organic semiconductors with intriguing properties (e.g., long lifetime) can be implemented for modulating light-matter interactions of hybrid HSs. Herein, we investigate the triplet sensitization of monolayer semiconductors by time-resolved spectroscopic studies on Pd-octaethylporphyrin (PdOEP)/WSe2 and PdOEP/WS2 HSs with type I and type II band alignment, respectively. We show that PdOEP triplets formed in ∼5 ps from intersystem crossing can transfer energy or charge to WSe2 or WS2 monolayers, respectively, leading to a significant photoluminescence enhancement (180%) in WSe2 or long-lived charge separation (>2 ns) in WS2. The triplet transfer occurs in ∼100 ns, which is more than 3 orders of magnitude slower than singlet and can be attributed to its tightly localized nature. Further study of thickness dependence reveals the dictating role of triplet diffusion for triplet sensitization in organic/2D HSs. This study shows the great promise of much less explored molecular triplets on sensitizing 2D monolayer semiconductors and provides the guidance to achieve long-range light harvesting and energy migration in organic/2D HSs for enhanced optoelectronic applications.

Efficient transgenesis and homology-directed gene targeting in monolayers of primary human small intestinal and colonic epithelial stem cells.

Two-dimensional (2D) cultures of intestinal and colonic epithelium can be generated using human intestinal stem cells (hISCs) derived from primary tissue sources. These 2D cultures are emerging as attractive and versatile alternatives to three-dimensional organoid cultures; however, transgenesis and gene-editing approaches have not been developed for hISCs grown as 2D monolayers. Using 2D cultured hISCs we show that electroporation achieves up to 80% transfection in hISCs from six anatomical regions with around 64% survival and produces 0.15% transgenesis by PiggyBac transposase and 35% gene edited indels by electroporation of Cas9-ribonucleoprotein complexes at the OLFM4 locus. We create OLFM4-emGFP knock-in hISCs, validate the reporter on engineered 2D crypt devices, and develop complete workflows for high-throughput cloning and expansion of transgenic lines in 3-4 weeks. New findings demonstrate small hISCs expressing the highest OLFM4 levels exhibit the most organoid forming potential and show utility of the 2D crypt device to evaluate hISC function.

Engineering the Optoelectronic Properties of 2D Hexagonal Boron Nitride Monolayer Films by Sulfur Substitutional Doping.

Tuning the optical and electrical properties of two-dimensional (2D) hexagonal boron nitride (hBN) is critical for its successful application in optoelectronics. Herein, we report a new methodology to significantly enhance the optoelectronic properties of hBN monolayers by substitutionally doping with sulfur (S) on a molten Au substrate using chemical vapor deposition. The S atoms are more geometrically and energetically favorable to be doped in the N sites than in the B sites of hBN, and the S 3p orbitals hybridize with the B 2p orbitals, forming a new conduction band edge that narrows its band gap. The band edge positions change with the doping concentration of S atoms. The conductivity increases up to 1.5 times and enhances the optoelectronic properties, compared to pristine hBN. A photodetector made of a 2D S-doped hBN film shows an extended wavelength response from 260 to 280 nm and a 50 times increase in its photocurrent and responsivity with light illumination at 280 nm. These enhancements are mainly due to the improved light absorption and increased electrical conductivity through doping with sulfur. This S-doped hBN monolayer film can be used in the next-generation electronics, optoelectronics, and spintronics.

High-efficient serum-free differentiation of endothelial cells from human iPS cells.

INTRODUCTION: Endothelial cells (ECs) form the inner lining of all blood vessels of the body play important roles in vascular tone regulation, hormone secretion, anticoagulation, regulation of blood cell adhesion and immune cell extravasation. Limitless ECs sources are required to further in vitro investigations of ECs' physiology and pathophysiology as well as for tissue engineering approaches. Ideally, the differentiation protocol avoids animal-derived components such as fetal serum and yields ECs at efficiencies that make further sorting obsolete for most applications. METHOD: Human induced pluripotent stem cells (hiPSCs) are cultured under serum-free conditions and induced into mesodermal progenitor cells via stimulation of Wnt signaling for 24 h. Mesodermal progenitor cells are further differentiated into ECs by utilizing a combination of human vascular endothelial growth factor A165 (VEGF), basic fibroblast growth factor (bFGF), 8-Bromoadenosine 3',5'-cyclic monophosphate sodium salt monohydrate (8Bro) and melatonin (Mel) for 48 h. RESULT: This combination generates hiPSC derived ECs (hiPSC-ECs) at a fraction of 90.9 ± 1.5% and is easily transferable from the two-dimensional (2D) monolayer into three-dimensional (3D) scalable bioreactor suspension cultures. hiPSC-ECs are positive for CD31, VE-Cadherin, von Willebrand factor and CD34. Furthermore, the majority of hiPSC-ECs express the vascular endothelial marker CD184 (CXCR4). CONCLUSION: The differentiation method presented here generates hiPSC-ECs in only 6 days, without addition of animal sera and at high efficiency, hence providing a scalable source of hiPSC-ECs.

2D Sb2C3 monolayer: A promising material for the recyclable gas sensor for environmentally toxic nitrogen-containing gases (NCGs).

Based on density functional theory investigation, we exposed the potential application of hexagonal Sb2C3 nanosheet as highly sensitive material for nitrogen-containing gases (NCGs) NH3, NO2 and NO molecules. Our rigorous simulations show that NH3, NO2 and NO molecules shows physisorption on the Sb2C3 nanosheet via vdW DFT-D3 interactions. The calculations were carried out by considering that the monolayer Sb2C3 as the sensor material modulated with its electrical conductivity when its surface adsorbs the gas molecules for their various orientations and positions. It is also found that the magnetic properties are induced in non-magnetic Sb2C3 nanosheet by adsorption of NO molecule. The interaction of the Sb2C3 nanosheet with the gas molecules is further analysed by the charge density difference (CDD), electrostatic potential (ESP) and Bader charge analysis. Our analysis indicates a strong possibility for the detection of NO2 and NO gas molecules by the Sb2C3 based sensor, due to the associated significant changes in the conductivity and reasonable adsorption energy. Also, in the visible region at T = 300 K, very low recovery times have been found as 431 µs, 785.01 s and 53.8 µs for NH3, NO2 and NO, respectively, which strongly suggest the Sb2C3 nanosheets as a better reversible multi-time gas sensor material towards the NCGs adsorption. We also explored the humidity effect on the NCGs based 2D Sb2C3 sensor material. The current-voltage (I-V) characteristics also confirmed the suitability of 2D Sb2C3 in real-time applications. Overall, present work reveals that the 2D Sb2C3 nanosheets as a promising material for semiconductor-based nano sensors for environmentally hazard pollutants like NCG molecules.

Traceable Nanocluster-Prodrug Conjugate for Chemo-photodynamic Combinatorial Therapy of Non-small Cell Lung Cancer.

In cancer treatment, image-guided combinatorial therapy is usually a more promising approach than conventional therapy because it may overcome the drawbacks of conventional cancer treatment, such as tumor recurrence and multidrug resistance. To achieve a high therapeutic effect in image-guided combinatorial therapy, the therapeutic material should be traceable, biocompatible, and yet highly effective in eradicating tumors. For this purpose, we developed a traceable nanocarrier consisting of atomically precise gold nanoclusters (Au NCs, Au22(SG)18, abbreviated as Au22 NCs, where SG stands for glutathione) and a biopolymer (i.e., chitosan). This traceable nanocarrier (Chito-Au22) was then combined with dual prodrugs (i.e., chemotherapeutic platinum (Pt(IV)) prodrug and photodynamic aminolevulinic acid (ALA) prodrug) through a bioconjugation method. It was found that the final nanocomposite (abbreviated as Pt(IV)-ALA-Chito-Au22) has a pH-responsive drug release behavior, and the cumulative drug release can exceed 50% within 12 h at an acidic pH of 5.0. After 15 min of white light irradiation, the nanocomposite showed a synergistic killing effect on the A549 non-small cell lung carcinoma cell line. The Pt(IV)-ALA-Chito-Au22 nanocomposite also showed a high cellular uptake capacity and reactive oxygen species (ROS) generation capability, resulting in a significant killing effect on three-dimensional (3D) multicellular A549 spheroids. In the presence of light, the volume of the multicellular spheroids treated by our nanocomposites was reduced more than two times compared with those treated by a single prodrug/component. The nanocomposite also showed good cell viability on normal lung cell lines. The multifunctional nanocomposites developed in this study have broad prospects in both therapeutic and diagnostic applications.

Directed Differentiation of Human Pluripotent Stem Cells for the Generation of High-Order Kidney Organoids.

Our understanding in the inherent properties of human pluripotent stem cells (hPSCs) have made possible the development of differentiation procedures to generate three-dimensional tissue-like cultures, so-called organoids. Here we detail a stepwise methodology to generate kidney organoids from hPSCs. This is achieved through direct differentiation of hPSCs in two-dimensional monolayer culture toward the posterior primitive streak fate, followed by induction of intermediate mesoderm-committed cells, which are further aggregated and cultured in three-dimensions to generate kidney organoids containing segmented nephron-like structures in a process that lasts 20 days. We also provide a concise description on how to assess renal commitment during the time course of kidney organoid generation. This includes the use of flow cytometry and immunocytochemistry analyses for the detection of specific renal differentiation markers.