A method for reproducible high-resolution imaging of 3D cancer cell spheroids.

Multicellular tumour cell spheroids embedded within three-dimensional (3D) hydrogels or extracellular matrices (ECM) are widely used as models to study cancer growth and invasion. Standard methods to embed spheroids in 3D matrices result in random placement in space which limits the use of inverted fluorescence microscopy techniques, and thus the resolution that can be achieved to image molecular detail within the intact spheroid. Here, we leverage UV photolithography to microfabricate PDMS (polydimethylsiloxane) stamps that allow for generation of high-content, reproducible well-like structures in multiple different imaging chambers. Addition of multicellular tumour spheroids into stamped collagen structures allows for precise positioning of spheroids in 3D space for reproducible high-/super-resolution imaging. Embedded spheroids can be imaged live or fixed and are amenable to immunostaining, allowing for greater flexibility of experimental approaches. We describe the use of these spheroid imaging chambers to analyse cell invasion, cell-ECM interaction, ECM alignment, force-dependent intracellular protein dynamics and extension of fine actin-based protrusions with a variety of commonly used inverted microscope platforms. This method enables reproducible, high-/super-resolution live imaging of multiple tumour spheroids, that can be potentially extended to visualise organoids and other more complex 3D in vitro systems.

A quantitative approach for determining the role of geometrical constraints when shaping mesenchymal condensations.

In embryogenesis, mesenchymal condensation is a critical event during the formation of many organ systems, including cartilage and bone. During organ formation, mesenchymal cells aggregate and undergo compaction while activating developmental programmes. The final three-dimensional form of the organ, as well as cell fates, can be influenced by the size and shape of the forming condensation. This process is hypothesized to result from multiscale cell interactions within mesenchymal microenvironments; however, these are complex to investigate in vivo. Three-dimensional in vitro models that recapitulate key phenotypes can contribute to our understanding of the microenvironment interactions regulating this fundamental developmental process. Here we devise such models by using image analysis to guide the design of polydimethylsiloxane 3D microstructures as cell culture substrates. These microstructures establish geometrically constrained micromass cultures of mouse embryonic skeletal progenitor cells which influence the development of condensations. We first identify key phenotypes differentiating face and limb bud micromass cultures by linear discriminant analysis of the shape descriptors for condensation morphology, which are used to guide the rational design of a micropatterned polydimethylsiloxane substrate. High-content imaging analysis highlights that the geometry of the microenvironment affects the establishment and growth of condensations. Further, cells commit to establish condensations within the first 5 h; condensations reach their full size within 17 h; following which they increase cell density while maintaining size for at least 7 days. These findings elucidate the value of our model in dissecting key aspects of mesenchymal condensation development.

Gold-silica quantum rattles for multimodal imaging and therapy.

Gold quantum dots exhibit distinctive optical and magnetic behaviors compared with larger gold nanoparticles. However, their unfavorable interaction with living systems and lack of stability in aqueous solvents has so far prevented their adoption in biology and medicine. Here, a simple synthetic pathway integrates gold quantum dots within a mesoporous silica shell, alongside larger gold nanoparticles within the shell's central cavity. This "quantum rattle" structure is stable in aqueous solutions, does not elicit cell toxicity, preserves the attractive near-infrared photonics and paramagnetism of gold quantum dots, and enhances the drug-carrier performance of the silica shell. In vivo, the quantum rattles reduced tumor burden in a single course of photothermal therapy while coupling three complementary imaging modalities: near-infrared fluorescence, photoacoustic, and magnetic resonance imaging. The incorporation of gold within the quantum rattles significantly enhanced the drug-carrier performance of the silica shell. This innovative material design based on the mutually beneficial interaction of gold and silica introduces the use of gold quantum dots for imaging and therapeutic applications.

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.

Surface presentation of functional peptides in solution determines cell internalization efficiency of TAT conjugated nanoparticles.

Functionalizing nanoparticles with cell-penetrating peptides is a popular choice for cellular delivery. We investigated the effects of TAT peptide concentration and arrangement in solution on functionalized nanoparticles' efficacy for membrane permeation. We found that cell internalization correlates with the positive charge distribution achieved prior to nanoparticle encountering interactions with membrane. We identified a combination of solution based properties required to maximize the internalization efficacy of TAT-functionalized nanoparticles.

Integrating Porous Silicon Nanoneedles within Medical Devices for Nucleic Acid Nanoinjection.

Porous silicon nanoneedles can interface with cells and tissues with minimal perturbation for high-throughput intracellular delivery and biosensing. Typically, nanoneedle devices are rigid, flat, and opaque, which limits their use for topical applications in the clinic. We have developed a robust, rapid, and precise substrate transfer approach to incorporate nanoneedles within diverse substrates of arbitrary composition, flexibility, curvature, transparency, and biodegradability. With this approach, we integrated nanoneedles on medically relevant elastomers, hydrogels, plastics, medical bandages, catheter tubes, and contact lenses. The integration retains the mechanical properties and transfection efficiency of the nanoneedles. Transparent devices enable the live monitoring of cell-nanoneedle interactions. Flexible devices interface with tissues for efficient, uniform, and sustained topical delivery of nucleic acids ex vivo and in vivo. The versatility of this approach highlights the opportunity to integrate nanoneedles within existing medical devices to develop advanced platforms for topical delivery and biosensing.

Opto-Lipidomics of Tissues.

Lipid metabolism and signaling play pivotal functions in biology and disease development. Despite this, currently available optical techniques are limited in their ability to directly visualize the lipidome in tissues. In this study, opto-lipidomics, a new approach to optical molecular tissue imaging is introduced. The capability of vibrational Raman spectroscopy is expanded to identify individual lipids in complex tissue matrices through correlation with desorption electrospray ionization (DESI) - mass spectrometry (MS) imaging in an integrated instrument. A computational pipeline of inter-modality analysis is established to infer lipidomic information from optical vibrational spectra. Opto-lipidomic imaging of transient cerebral ischemia-reperfusion injury in a murine model of ischemic stroke demonstrates the visualization and identification of lipids in disease with high molecular specificity using Raman scattered light. Furthermore, opto-lipidomics in a handheld fiber-optic Raman probe is deployed and demonstrates real-time classification of bulk brain tissues based on specific lipid abundances. Opto-lipidomics opens a host of new opportunities to study lipid biomarkers for diagnostics, prognostics, and novel therapeutic targets.

Lithiated porous silicon nanowires stimulate periodontal regeneration.

Periodontal disease is a significant burden for oral health, causing progressive and irreversible damage to the support structure of the tooth. This complex structure, the periodontium, is composed of interconnected soft and mineralised tissues, posing a challenge for regenerative approaches. Materials combining silicon and lithium are widely studied in periodontal regeneration, as they stimulate bone repair via silicic acid release while providing regenerative stimuli through lithium activation of the Wnt/ß-catenin pathway. Yet, existing materials for combined lithium and silicon release have limited control over ion release amounts and kinetics. Porous silicon can provide controlled silicic acid release, inducing osteogenesis to support bone regeneration. Prelithiation, a strategy developed for battery technology, can introduce large, controllable amounts of lithium within porous silicon, but yields a highly reactive material, unsuitable for biomedicine. This work debuts a strategy to lithiate porous silicon nanowires (LipSiNs) which generates a biocompatible and bioresorbable material. LipSiNs incorporate lithium to between 1% and 40% of silicon content, releasing lithium and silicic acid in a tailorable fashion from days to weeks. LipSiNs combine osteogenic, cementogenic and Wnt/ß-catenin stimuli to regenerate bone, cementum and periodontal ligament fibres in a murine periodontal defect.

CRISPR/Cas-Assisted Nanoneedle Sensor for Adenosine Triphosphate Detection in Living Cells.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) (CRISPR/Cas) systems have recently emerged as powerful molecular biosensing tools based on their collateral cleavage activity due to their simplicity, sensitivity, specificity, and broad applicability. However, the direct application of the collateral cleavage activity for in situ intracellular detection is still challenging. Here, we debut a CRISPR/Cas-assisted nanoneedle sensor (nanoCRISPR) for intracellular adenosine triphosphate (ATP), which avoids the challenges associated with intracellular collateral cleavage by introducing a two-step process of intracellular target recognition, followed by extracellular transduction and detection. ATP recognition occurs by first presenting in the cell cytosol an aptamer-locked Cas12a activator conjugated to nanoneedles; the recognition event unlocks the activator immobilized on the nanoneedles. The nanoneedles are then removed from the cells and exposed to the Cas12a/crRNA complex, where the activator triggers the cleavage of an ssDNA fluorophore-quencher pair, generating a detectable fluorescence signal. NanoCRISPR has an ATP detection limit of 246 nM and a dynamic range from 1.56 to 50 µM. Importantly, nanoCRISPR can detect intracellular ATP in 30 min in live cells without impacting cell viability. We anticipate that the nanoCRISPR approach will contribute to broadening the biomedical applications of CRISPR/Cas sensors for the detection of diverse intracellular molecules in living systems.

Two-Photon Light Trigger siRNA Transfection of Cancer Cells Using Non-Toxic Porous Silicon Nanoparticles.

The concept of using two-photon excitation in the NIR for the spatiotemporal control of biological processes holds great promise. However, its use for the delivery of nucleic acids has been very scarcely described and the reported procedures are not optimal as they often involve potentially toxic materials and irradiation conditions. This work prepares a simple system made of biocompatible porous silicon nanoparticles (pSiNP) for the safe siRNA photocontrolled delivery and gene silencing in cells upon two-photon excitation. PSiNP are linked to an azobenzene moiety, which possesses a lysine group (pSiNP@ICPES-azo@Lys) to efficiently complex siRNA. Non-linear excitation of the two-photon absorber system (pSiNP) followed by intermolecular energy transfer (FRET) to trans azobenzene moiety, result in the photoisomerization of the azobenzene from trans to cis and in the destabilization of the azobenzene-siRNA complex, thus inducing the delivery of the cargo siRNA to the cytoplasm of cells. Efficient silencing in MCF-7 expressing stable firefly luciferase with siRNAluc against luciferase is observed. Furthermore, siRNA against inhibitory apoptotic protein (IAP) leads to over 70% of MCF-7 cancer cell death. The developed technique using two-photon light allows a unique high spatiotemporally controlled and safe siRNA delivery in cells in few seconds of irradiation.

Multi-stage delivery nano-particle systems for therapeutic applications.

BACKGROUND: The daunting task for drug molecules to reach pathological lesions has fueled rapid advances in Nanomedicine. The progressive evolution of nanovectors has led to the development of multi-stage delivery systems aimed at overcoming the numerous obstacles encountered by nanovectors on their journey to the target site. SCOPE OF REVIEW: This review summarizes major findings with respect to silicon-based drug delivery vectors for cancer therapeutics and imaging. Based on rational design, well-established silicon technologies have been adapted for the fabrication of nanovectors with specific shapes, sizes, and porosities. These vectors are part of a multi-stage delivery system that contains multiple nano-components, each designed to achieve a specific task with the common goal of site-directed delivery of therapeutics. MAJOR CONCLUSIONS: Quasi-hemispherical and discoidal silicon microparticles are superior to spherical particles with respect to margination in the blood, with particles of different shapes and sizes having unique distributions in vivo. Cellular adhesion and internalization of silicon microparticles is influenced by microparticle shape and surface charge, with the latter dictating binding of serum opsonins. Based on in vitro cell studies, the internalization of porous silicon microparticles by endothelial cells and macrophages is compatible with cellular morphology, intracellular trafficking, mitosis, cell cycle progression, cytokine release, and cell viability. In vivo studies support superior therapeutic efficacy of liposomal encapsulated siRNA when delivered in multi-stage systems compared to free nanoparticles. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.

Discoidal Porous Silicon Particles: Fabrication and Biodistribution in Breast Cancer Bearing Mice.

Porous silicon (pSi) is emerging as a promising material in the development of nanovectors for the systemic delivery of therapeutic and imaging agents. The integration of photolithographic patterning, typical of the semiconductor industry, with electrochemical silicon etching provides a highly flexible strategy to fabricate monodisperse and precisely tailored nanovectors. Here, a microfabrication strategy for direct lithographic patterning of discoidal pSi particles is presented that enables precise and independent control over particle size, shape, and porous structure. Discoidal pSi nanovectors with diameters ranging from 500 to 2600 nm, heights from 200 to 700 nm, pore sizes from 5 to 150 nm, and porosities from 40 to 90% are demonstrated. The degradation in serum, interaction with immune and endothelial cells in vitro, and biodistribution in mice bearing breast tumors are assessed for two discoidal nanovectors with sizes of 600 nm × 400 nm and 1000 nm × 400 nm. It is shown that both particle types are degraded after 24 h of continuous gentle agitation in serum, do not stimulate cytokine release from macrophages or affect endothelial cell viability, and accumulate up to about 10% of the injected dose per gram tissue in orthotopic murine models of breast cancer. The accumulation of the discoidal pSi nanovectors into the breast tumor mass is found to be up to five times higher than for spherical silica beads with similar diameters.

Hybrid confocal Raman endomicroscopy for morpho-chemical tissue characterization.

Confocal laser endomicroscopy (CLE) offers imaging of tissue microarchitecture and has emerged as a promising tool for in vivo clinical diagnosis of cancer across many organs. CLE, however, can show high inter-observer dependency and does not provide information about tissue molecular composition. In contrast, Raman spectroscopy is a label-free optical technique that provides detailed biomolecular compositional information but offers limited or no morphological information. Here we present a novel hybrid fiber-optic confocal Raman endomicroscopy system for morpho-chemical tissue imaging and analysis. The developed confocal endomicroscopy system is based on a novel detection scheme for rejecting Raman silica fiber interference permitting simultaneous CLE imaging and Raman spectral acquisition of tissues through a coherent fiber bundle. We show that this technique enables real-time microscopic visualization of tissue architecture as well as simultaneous pointwise label-free biomolecular characterization and fingerprinting of tissue paving the way for multimodal diagnostics at endoscopy.

Axonal Length Determines Distinct Homeostatic Phenotypes in Human iPSC Derived Motor Neurons on a Bioengineered Platform.

Stem cell-based experimental platforms for neuroscience can effectively model key mechanistic aspects of human development and disease. However, conventional culture systems often overlook the engineering constraints that cells face in vivo. This is particularly relevant for neurons covering long range connections such as spinal motor neurons (MNs). Their axons extend up to 1m in length and require a complex interplay of mechanisms to maintain cellular homeostasis. However, shorter axons in conventional cultures may not faithfully capture important aspects of their longer counterparts. Here this issue is directly addressed by establishing a bioengineered platform to assemble arrays of human axons ranging from micrometers to centimeters, which allows systematic investigation of the effects of length on human axonas for the first time. This approach reveales a link between length and metabolism in human MNs in vitro, where axons above a "threshold" size induce specific molecular adaptations in cytoskeleton composition, functional properties, local translation, and mitochondrial homeostasis. The findings specifically demonstrate the existence of a length-dependent mechanism that switches homeostatic processes within human MNs. The findings have critical implications for in vitro modeling of several neurodegenerative disorders and reinforce the importance of modeling cell shape and biophysical constraints with fidelity and precision in vitro.

Nanoneedles Induce Targeted siRNA Silencing of p16 in the Human Corneal Endothelium.

Nanoneedles can target nucleic acid transfection to primary cells at tissue interfaces with high efficiency and minimal perturbation. The corneal endothelium is an ideal target for nanoneedle-mediated RNA interference therapy aimed at enhancing its proliferative capacity, necessary for tissue regeneration. This work develops a strategy for siRNA nanoninjection to the human corneal endothelium. Nanoneedles can deliver p16-targeting siRNA to primary human corneal endothelial cells in vitro without toxicity. The nanoinjection of siRNA induces p16 silencing and increases cell proliferation, as monitored by ki67 expression. Furthermore, siRNA nanoinjection targeting the human corneal endothelium is nontoxic ex vivo, and silences p16 in transfected cells. These data indicate that nanoinjection can support targeted RNA interference therapy for the treatment of endothelial corneal dysfunction.

Porcine Organotypic Epicardial Slice Protocol: A Tool for the Study of Epicardium in Cardiovascular Research.

The epicardium has recently gained interest in the cardiovascular field due to its capacity to support heart regeneration after ischemic injury. Models to study the epicardium of large animals in vitro are limited and mainly based on epicardial cell isolation/differentiation from stem cells, followed by 2D cells culture. In this method paper, we describe the procedure to obtain and culture 3D organotypic heart slices presenting an intact epicardium, as a novel model to study the epicardial physiology and activation. Epicardial slices are obtained from porcine hearts using a high-precision vibratome and retain a healthy epicardial layer embedded in its native extracellular environment and connected with other cardiac cells (cardiomyocytes, fibroblasts, vascular cells etc.). Epicardial slices can be cultured for 72 h, providing an ideal model for studying the epicardium physiology or perform pharmacological interventions/gene therapy approaches. We also report on methods to assesses the viability and composition of the epicardial slices, and evaluate their architecture in 3D through tissue decoloration. Finally, we present a potential application for a nanomaterial-based gene transfer method for tracking of epicardial cells within the slice. Crucially, given the similarity in morphology and physiology of porcine heart with its human counterpart, our system provides a platform for translational research while providing a clinically relevant and ethical alternative to the use of small animals in this type of research.

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.

Near-infrared imaging method for the in vivo assessment of the biodistribution of nanoporous silicon particles.

In the development of new nanoparticle-based technologies for therapeutic and diagnostic purposes, understanding the fate of nanoparticles in the body is crucial. We recently developed a multistage vector delivery system comprising biodegradable and biocompatible nanoporous silicon particles (first-stage microparticles [S1MPs]) able to host, protect, and deliver second-stage therapeutic and diagnostic nanoparticles (S2NPs) on intravenous injection. This delivery system aims at sequentially overcoming the biologic barriers en route to the target delivery site by separating and assigning tasks to the coordinated logic-embedded vectors constituting it. In this work, by conjugating a near-infrared dye on the surface of the S1MP without compromising the porous structure and potential loading of S2NPs, we were able to monitor the in vivo distribution of S1MPs in healthy mice using an optical imaging system. It was observed that particles predominantly accumulated in the liver and spleen at the end of 24 hours. Further quantification of S1MPs in the major organs of the animals by elemental analysis of silicon using inductively coupled plasma-atomic electron spectroscopy verified the accuracy of in vivo near-infrared imaging as a tool for evaluation of nanovector biodistribution.

Agarose surface coating influences intracellular accumulation and enhances payload stability of a nano-delivery system.

PURPOSE: Protein therapeutics often require repeated administrations of drug over a long period of time. Protein instability is a major obstacle to the development of systems for their controlled and sustained release. We describe a surface modification of nanoporous silicon particles (NSP) with an agarose hydrogel matrix that enhances their ability to load and release proteins, influencing intracellular delivery and preserving molecular stability. METHODS: We developed and characterized an agarose surface modification of NSP. Stability of the released protein after enzymatic treatment of loaded particles was evaluated with SDS-page and HPLC analysis. FITC-conjugated BSA was chosen as probe protein and intracellular delivery evaluated by fluorescence microscopy. RESULTS: We showed that agarose coating does not affect NSP protein release rate, while fewer digestion products were found in the released solution after all the enzymatic treatments. Confocal images show that the hydrogel coating improves intracellular delivery, specifically within the nucleus, without affecting the internalization process. CONCLUSIONS: This modification of porous silicon adds to its tunability, biocompatibility, and biodegradability the ability to preserve protein integrity during delivery without affecting release rates and internalization dynamics. Moreover, it may allow the silicon particles to function as protein carriers that enable control of cell function.

Tutorial: using nanoneedles for intracellular delivery.

Intracellular delivery of advanced therapeutics, including biologicals and supramolecular agents, is complex because of the natural biological barriers that have evolved to protect the cell. Efficient delivery of therapeutic nucleic acids, proteins, peptides and nanoparticles is crucial for clinical adoption of emerging technologies that can benefit disease treatment through gene and cell therapy. Nanoneedles are arrays of vertical high-aspect-ratio nanostructures that can precisely manipulate complex processes at the cell interface, enabling effective intracellular delivery. This emerging technology has already enabled the development of efficient and non-destructive routes for direct access to intracellular environments and delivery of cell-impermeant payloads. However, successful implementation of this technology requires knowledge of several scientific fields, making it complex to access and adopt by researchers who are not directly involved in developing nanoneedle platforms. This presents an obstacle to the widespread adoption of nanoneedle technologies for drug delivery. This tutorial aims to equip researchers with the knowledge required to develop a nanoinjection workflow. It discusses the selection of nanoneedle devices, approaches for cargo loading and strategies for interfacing to biological systems and summarises an array of bioassays that can be used to evaluate the efficacy of intracellular delivery.