Gold Nanocluster Extracellular Vesicle Supraparticles: Self-Assembled Nanostructures for Three-Dimensional Uptake Visualization.

Extracellular vesicles (EVs) are secreted by the vast majority of cells and are being intensively studied due to their emerging involvement in a variety of cellular communication processes. However, the study of their cellular uptake and fate has been hampered by difficulty in imaging EVs against the cellular background. Here, we show that EVs combined with hydrophobic gold nanoclusters (AuNCs) can self-assemble into supraparticles, offering an excellent labeling strategy for high-resolution electron microscopic imaging in vitro. We have tracked and visualized the reuptake of breast cancer cell-derived EV AuNC supraparticles into their parent cells, from early endocytosis to lysosomal degradation, using focused ion beam-scanning electron microscopy (FIB-SEM). The presence of gold within the EVs and lysosomes was confirmed via DF-STEM EDX analysis of lift-out sections. The demonstrated formation of AuNC EV supraparticles will facilitate future applications in EV imaging as well as the EV-assisted cellular delivery of AuNCs.

Angular Approach Scanning Ion Conductance Microscopy.

Scanning ion conductance microscopy (SICM) is a super-resolution live imaging technique that uses a glass nanopipette as an imaging probe to produce three-dimensional (3D) images of cell surface. SICM can be used to analyze cell morphology at nanoscale, follow membrane dynamics, precisely position an imaging nanopipette close to a structure of interest, and use it to obtain ion channel recordings or locally apply stimuli or drugs. Practical implementations of these SICM advantages, however, are often complicated due to the limitations of currently available SICM systems that inherited their design from other scanning probe microscopes in which the scan assembly is placed right above the specimen. Such arrangement makes the setting of optimal illumination necessary for phase contrast or the use of high magnification upright optics difficult. Here, we describe the designs that allow mounting SICM scan head on a standard patch-clamp micromanipulator and imaging the sample at an adjustable approach angle. This angle could be as shallow as the approach angle of a patch-clamp pipette between a water immersion objective and the specimen. Using this angular approach SICM, we obtained topographical images of cells grown on nontransparent nanoneedle arrays, of islets of Langerhans, and of hippocampal neurons under upright optical microscope. We also imaged previously inaccessible areas of cells such as the side surfaces of the hair cell stereocilia and the intercalated disks of isolated cardiac myocytes, and performed targeted patch-clamp recordings from the latter. Thus, our new, to our knowledge, angular approach SICM allows imaging of living cells on nontransparent substrates and a seamless integration with most patch-clamp setups on either inverted or upright microscopes, which would facilitate research in cell biophysics and physiology.

Biophysical Regulations of Epigenetic State and Notch Signaling in Neural Development Using Microgroove Substrates.

A number of studies have recently shown how surface topography can alter the behavior and differentiation patterns of different types of stem cells. Although the exact mechanisms and molecular pathways involved remain unclear, a consistent portion of the literature points to epigenetic changes induced by nuclear remodeling. In this study, we investigate the behavior of clinically relevant neural populations derived from human pluripotent stem cells when cultured on polydimethylsiloxane microgrooves (3 and 10 µm depth grooves) to investigate what mechanisms are responsible for their differentiation capacity and functional behavior. Our results show that microgrooves enhance cell alignment, modify nuclear geometry, and significantly increase cellular stiffness, which we were able to measure at high resolution with a combination of light and electron microscopy, scanning ion conductance microscopy (SICM), and atomic force microscopy (AFM) coupled with quantitative image analysis. The microgrooves promoted significant changes in the epigenetic landscape, as revealed by the expression of key histone modification markers. The main behavioral change of neural stem cells on microgrooves was an increase of neuronal differentiation under basal conditions on the microgrooves. Through measurements of cleaved Notch1 levels, we found that microgrooves downregulate Notch signaling. We in fact propose that microgroove topography affects the differentiation potential of neural stem cells by indirectly altering Notch signaling through geometric segregation and that this mechanism in parallel with topography-dependent epigenetic modulations acts in concert to enhance stem cell neuronal differentiation.

Substrate Stiffness-Driven Membrane Tension Modulates Vesicular Trafficking via Caveolin-1.

Liver fibrosis, a condition characterized by extensive deposition and cross-linking of extracellular matrix (ECM) proteins, is idiosyncratic in cases of chronic liver injury. The dysregulation of ECM remodeling by hepatic stellate cells (HSCs), the main mediators of fibrosis, results in an elevated ECM stiffness that drives the development of chronic liver disease such as cirrhosis and hepatocellular carcinoma. Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) is a key element in the regulation of ECM remodeling, which modulates the degradation and turnover of ECM components. We have previously reported that a rigid, fibrotic-like substrate can impact TIMP-1 expression at the protein level in HSCs without altering its mRNA expression. While HSCs are known to be highly susceptible to mechanical stimuli, the mechanisms through which mechanical cues regulate TIMP-1 at the post-translational level remain unclear. Here, we show a mechanism of regulation of plasma membrane tension by matrix stiffness. We found that this effect is orchestrated by the ß1 integrin/RhoA axis and results in elevated exocytosis and secretion of TIMP-1 in a caveolin-1- and dynamin-2-dependent manner. We then show that TIMP-1 and caveolin-1 expression increases in cirrhosis and hepatocellular carcinoma. These conditions are associated with fibrosis, and this effect can be recapitulated in 3D fibrosis models consisting of hepatic stellate cells encapsulated in a self-assembling polypeptide hydrogel. This work positions stiffness-dependent membrane tension as a key regulator of enzyme secretion and function and a potential target for therapeutic strategies that aim at modulating ECM remodeling in chronic liver disease.

IL-1ß mediated nanoscale surface clustering of integrin α5ß1 regulates the adhesion of mesenchymal stem cells.

Clinical use of human mesenchymal stem cells (hMSCs) is limited due to their rapid clearance, reducing their therapeutic efficacy. The inflammatory cytokine IL-1ß activates hMSCs and is known to enhance their engraftment. Consequently, understanding the molecular mechanism of this inflammation-triggered adhesion is of great clinical interest to improving hMSC retention at sites of tissue damage. Integrins are cell-matrix adhesion receptors, and clustering of integrins at the nanoscale underlies cell adhesion. Here, we found that IL-1ß enhances adhesion of hMSCs via increased focal adhesion contacts in an α5ß1 integrin-specific manner. Further, through quantitative super-resolution imaging we elucidated that IL-1ß specifically increases nanoscale integrin α5ß1 availability and clustering at the plasma membrane, whilst conserving cluster area. Taken together, these results demonstrate that hMSC adhesion via IL-1ß stimulation is partly regulated through integrin α5ß1 spatial organization at the cell surface. These results provide new insight into integrin clustering in inflammation and provide a rational basis for design of therapies directed at improving hMSC engraftment.

Multiplexing physical stimulation on single human induced pluripotent stem cell-derived cardiomyocytes for phenotype modulation.

Traditional in vitro bioengineering approaches whereby only individual biophysical cues are manipulated at any one time are highly inefficient, falling short when recapitulating the complexity of the cardiac environment. Multiple biophysical cues are present in the native myocardial niche and are essential during development, as well as in maintenance of adult cardiomyocyte (CM) phenotype in both health and disease. This study establishes a novel biofabrication workflow to study and manipulate hiPSC-CMs and to understand how these cells respond to a multiplexed biophysical environment, namely 3D shape and substrate stiffness, at a single cell level. Silicon masters were fabricated and developed to generate inverse patterns of the desired 3D shapes in bas relief, which then were used to mold the designed microwell arrays into a hydrogel. Polyacrylamide (PAAm) was modified with the incorporation of acrylic acid to provide a carboxylic group conjugation site for adhesion motifs, without compromising capacity to modulate stiffness. In this manner, two individual parameters can be finely tuned independently within the hydrogel: the shape of the 3D microwell and its stiffness. The design allows the platform to isolate single hiPSC-CMs to study solely biophysical cues in the absence of cell-cell physical interaction. Under physiologic-like physical conditions (3D shape resembling that of adult CM and 9.83 kPa substrate stiffness that mimics muscle stiffness), isolated single hiPSC-CMs exhibit increased Cx-43 density, cell membrane stiffness and calcium transient amplitude; co-expression of the subpopulation-related MYL2-MYL7 proteins; and higher anisotropism than cells in pathologic-like conditions (flat surface and 112 kPa substrate stiffness). This demonstrates that supplying a physiologic or pathologic microenvironment to an isolated single hiPSC-CM in the absence of any physical cell-to-cell communication in this biofabricated platform leads to a significantly different set of cellular features, thus presenting a differential phenotype. Importantly, this demonstrates the high plasticity of hiPSC-CMs even in isolation. The ability of multiple biophysical cues to significantly influence isolated single hiPSC-CM phenotype and functionality highlights the importance of fine-tuning such cues for specific applications. This has the potential to produce more fit-for-purpose hiPSC-CMs. Further understanding of human cardiac development is enabled by the robust, versatile and reproducible biofabrication techniques applied here. We envision that this system could be easily applied to other tissues and cell types where the influence of cellular shape and stiffness of the surrounding environment is hypothesized to play an important role in physiology.

Advances in high-resolution microscopy for the study of intracellular interactions with biomaterials.

The study of sophisticated biomaterials and their cellular targets requires visualization methods with exquisite spatial and temporal resolution to discern cell organelles and molecular events. Monitoring cell-material interactions at high resolution is key for the continued development and optimization of biomaterials, for monitoring cell uptake of cargo, and for understanding the cell response to extracellular cues. This review evaluates the advantages and disadvantages of different forms of electron microscopy and super-resolution microscopy in elucidating how biomaterial surface chemistry and topography can affect intracellular events at the nanoscale.

Porous Silicon Nanoneedles Modulate Endocytosis to Deliver Biological Payloads.

Owing to their ability to efficiently deliver biological cargo and sense the intracellular milieu, vertical arrays of high aspect ratio nanostructures, known as nanoneedles, are being developed as minimally invasive tools for cell manipulation. However, little is known of the mechanisms of cargo transfer across the cell membrane-nanoneedle interface. In particular, the contributions of membrane piercing, modulation of membrane permeability and endocytosis to cargo transfer remain largely unexplored. Here, combining state-of-the-art electron and scanning ion conductance microscopy with molecular biology techniques, it is shown that porous silicon nanoneedle arrays concurrently stimulate independent endocytic pathways which contribute to enhanced biomolecule delivery into human mesenchymal stem cells. Electron microscopy of the cell membrane at nanoneedle sites shows an intact lipid bilayer, accompanied by an accumulation of clathrin-coated pits and caveolae. Nanoneedles enhance the internalization of biomolecular markers of endocytosis, highlighting the concurrent activation of caveolae- and clathrin-mediated endocytosis, alongside macropinocytosis. These events contribute to the nanoneedle-mediated delivery (nanoinjection) of nucleic acids into human stem cells, which distribute across the cytosol and the endolysosomal system. This data extends the understanding of how nanoneedles modulate biological processes to mediate interaction with the intracellular space, providing indications for the rational design of improved cell-manipulation technologies.

Rolling Circle Transcription-Amplified Hierarchically Structured Organic-Inorganic Hybrid RNA Flowers for Enzyme Immobilization.

Programmable nucleic acids have emerged as powerful building blocks for the bottom-up fabrication of two- or three-dimensional nano- and microsized constructs. Here we describe the construction of organic-inorganic hybrid RNA flowers (hRNFs) via rolling circle transcription (RCT), an enzyme-catalyzed nucleic acid amplification reaction. These hRNFs are highly adaptive structures with controlled sizes, specific nucleic acid sequences, and a highly porous nature. We demonstrated that hRNFs are applicable as potential biological platforms, where the hRNF scaffold can be engineered for versatile surface functionalization and the inorganic component (magnesium ions) can serve as an enzyme cofactor. For surface functionalization, we proposed robust and straightforward approaches including in situ synthesis of functional hRNFs and postfunctionalization of hRNFs that enable facile conjugation with various biomolecules and nanomaterials (i.e., proteins, enzymes, organic dyes, inorganic nanoparticles) using selective chemistries (i.e., avidin-biotin interaction, copper-free click reaction). In particular, we showed that hRNFs can serve as soft scaffolds for ß-galactosidase immobilization and greatly enhance enzymatic activity and stability. Therefore, the proposed concepts and methodologies are not only fundamentally interesting when designing RNA scaffolds or RNA bionanomaterials assembled with enzymes but also have significant implications on their future utilization in biomedical applications ranging from enzyme cascades to biosensing and drug delivery.

Immunogold FIB-SEM: Combining Volumetric Ultrastructure Visualization with 3D Biomolecular Analysis to Dissect Cell-Environment Interactions.

Volumetric imaging techniques capable of correlating structural and functional information with nanoscale resolution are necessary to broaden the insight into cellular processes within complex biological systems. The recent emergence of focused ion beam scanning electron microscopy (FIB-SEM) has provided unparalleled insight through the volumetric investigation of ultrastructure; however, it does not provide biomolecular information at equivalent resolution. Here, immunogold FIB-SEM, which combines antigen labeling with in situ FIB-SEM imaging, is developed in order to spatially map ultrastructural and biomolecular information simultaneously. This method is applied to investigate two different cell-material systems: the localization of histone epigenetic modifications in neural stem cells cultured on microstructured substrates and the distribution of nuclear pore complexes in myoblasts differentiated on a soft hydrogel surface. Immunogold FIB-SEM offers the potential for broad applicability to correlate structure and function with nanoscale resolution when addressing questions across cell biology, biomaterials, and regenerative medicine.

Nanoneedle-Mediated Stimulation of Cell Mechanotransduction Machinery.

Biomaterial substrates can be engineered to present topographical signals to cells which, through interactions between the material and active components of the cell membrane, regulate key cellular processes and guide cell fate decisions. However, targeting mechanoresponsive elements that reside within the intracellular domain is a concept that has only recently emerged. Here, we show that mesoporous silicon nanoneedle arrays interact simultaneously with the cell membrane, cytoskeleton, and nucleus of primary human cells, generating distinct responses at each of these cellular compartments. Specifically, nanoneedles inhibit focal adhesion maturation at the membrane, reduce tension in the cytoskeleton, and lead to remodeling of the nuclear envelope at sites of impingement. The combined changes in actin cytoskeleton assembly, expression and segregation of the nuclear lamina, and localization of Yes-associated protein (YAP) correlate differently from what is canonically observed upon stimulation at the cell membrane, revealing that biophysical cues directed to the intracellular space can generate heretofore unobserved mechanosensory responses. These findings highlight the ability of nanoneedles to study and direct the phenotype of large cell populations simultaneously, through biophysical interactions with multiple mechanoresponsive components.

High-resolution label-free 3D mapping of extracellular pH of single living cells.

Dynamic mapping of extracellular pH (pHe) at the single-cell level is critical for understanding the role of H+ in cellular and subcellular processes, with particular importance in cancer. While several pHe sensing techniques have been developed, accessing this information at the single-cell level requires improvement in sensitivity, spatial and temporal resolution. We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensitivity > 0.01 units, 2 ms response time, and 50 nm spatial resolution. The platform was integrated into a double-barrel nanoprobe combining pH sensing with feedback-controlled distance dependance via Scanning Ion Conductance Microscopy. This allows for the simultaneous 3D topographical imaging and pHe monitoring of living cancer cells. These classes of nanoprobes were used for real-time high spatiotemporal resolution pHe mapping at the subcellular level and revealed tumour heterogeneity of the peri-cellular environments of melanoma and breast cancer cells.

Spearhead Nanometric Field-Effect Transistor Sensors for Single-Cell Analysis.

Nanometric field-effect-transistor (FET) sensors are made on the tip of spear-shaped dual carbon nanoelectrodes derived from carbon deposition inside double-barrel nanopipettes. The easy fabrication route allows deposition of semiconductors or conducting polymers to comprise the transistor channel. A channel from electrodeposited poly pyrrole (PPy) exhibits high sensitivity toward pH changes. This property is exploited by immobilizing hexokinase on PPy nano-FETs to give rise to a selective ATP biosensor. Extracellular pH and ATP gradients are key biochemical constituents in the microenvironment of living cells; we monitor their real-time changes in relation to cancer cells and cardiomyocytes. The highly localized detection is possible because of the high aspect ratio and the spear-like design of the nano-FET probes. The accurately positioned nano-FET sensors can detect concentration gradients in three-dimensional space, identify biochemical properties of a single living cell, and after cell membrane penetration perform intracellular measurements.