RESUMO
Cell nuclear size and shape are strictly regulated, with aberrations often leading to or being indicative of disease. Nuclear mechanics are critically responsible for intracellular responses to extracellular cues, such as the nanotopography of the external environment. Silicon nanoneedle (SiNN) arrays are tunable, engineered cell culture substrates that permit precise, nanoscale modifications to a cell's external environment to probe mechanotransduction and intracellular signaling. We use a library of four different SiNN arrays to investigate the immediate and downstream effects of controlled geometries of nanotopographical cues on the nuclear integrity/dynamics of human immortalized somatic and renewing stem cell types. We quantify the significant, albeit different, nuclear shape changes that both cell types undergo, which suggest that cellular responses to SiNN arrays are more comparable to three-dimensional (3D) environments than traditional flat cultureware. We show that nanotopography-induced effects on nuclear envelope integrity, protein localization, and focal adhesion complex formation are cell-dependent. Migration is shown to be dramatically impeded for human neural progenitor cells (hNPCs) on nanotopographies compared to flat substrates but not for somatic cells. Our results indicate an additional layer of complexity in cellular mechanotransduction, which warrants closer attention in the context of engineered substrates and scaffolds for clinical applications.
Assuntos
Células-Tronco Neurais , Silício , Diferenciação Celular , Núcleo Celular , Humanos , Mecanotransdução Celular , Silício/farmacologiaRESUMO
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.
Assuntos
Sistemas de Liberação de Medicamentos , Nanoestruturas , Citosol , HumanosRESUMO
Cells are able to perceive complex mechanical cues from their microenvironment, which in turn influences their development. Although the understanding of these intricate mechanotransductive signals is evolving, the precise roles of substrate microtopography in directing cell fate is still poorly understood. Here, UV nanoimprint lithography is used to generate micropillar arrays ranging from 1 to 10 µm in height, width, and spacing to investigate the impact of microtopography on mechanotransduction. Using mesenchymal stem cells (MSCs) as a model, stark pattern-specific changes in nuclear architecture, lamin A/C accumulation, chromatin positioning, and DNA methyltransferase expression, are demonstrated. MSC osteogenesis is also enhanced specifically on micropillars with 5 µm width/spacing and 5 µm height. Intriguingly, the highest degree of osteogenesis correlates with patterns that stimulated maximal nuclear deformation which is shown to be dependent on myosin-II-generated tension. The outcomes determine new insights into nuclear mechanotransduction by demonstrating that force transmission across the nuclear envelope can be modulated by substrate topography, and that this can alter chromatin organisation and impact upon cell fate. These findings have potential to inform the development of microstructured cell culture substrates that can direct cell mechanotransduction and fate for therapeutic applications in both research and clinical sectors.
RESUMO
Engineered nano-bio interfaces driven by tunable vertically configured nanostructures have recently emerged as a powerful tool for cellular manipulations and interrogations. Yet the interplay between substrate topography and cellular behavior is highly complex and not fully understood. A new experimental design is proposed that enables generation of ultrathin sections (lamellae) of cell-nanostructure imprints with minimal artifacts. We demonstrate the potential of such lamellae for efficient transmission electron microscopy (TEM) characterization of interfacial interactions between adherent cells and vertically aligned Si nanostructures. This approach will advance understanding of cellular responses to extracellular biophysical and biochemical cues-which is likely to facilitate the design of improved cellular manipulation technologies.
RESUMO
Engineered nano-bio cellular interfaces driven by vertical nanostructured materials are set to spur transformative progress in modulating cellular processes and interrogations. In particular, the intracellular delivery-a core concept in fundamental and translational biomedical research-holds great promise for developing novel cell therapies based on gene modification. This study demonstrates the development of a mechanotransfection platform comprising vertically aligned silicon nanotube (VA-SiNT) arrays for ex vivo gene editing. The internal hollow structure of SiNTs allows effective loading of various biomolecule cargoes; and SiNTs mediate delivery of those cargoes into GPE86 mouse embryonic fibroblasts without compromising their viability. Focused ion beam scanning electron microscopy (FIB-SEM) and confocal microscopy results demonstrate localized membrane invaginations and accumulation of caveolin-1 at the cell-NT interface, suggesting the presence of endocytic pits. Small-molecule inhibition of endocytosis suggests that active endocytic process plays a role in the intracellular delivery of cargo from SiNTs. SiNT-mediated siRNA intracellular delivery shows the capacity to reduce expression levels of F-actin binding protein (Triobp) and alter the cellular morphology of GPE86. Finally, the successful delivery of Cas9 ribonucleoprotein (RNP) to specifically target mouse Hprt gene is achieved. This NT-enhanced molecular delivery platform has strong potential to support gene editing technologies.
Assuntos
Edição de Genes/instrumentação , Espaço Intracelular/metabolismo , Nanotecnologia/instrumentação , Nanotubos/química , Silício/química , Animais , Caveolina 1/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Engineered cell-nanostructured interfaces generated by vertically aligned silicon nanowire (SiNW) arrays have become a promising platform for orchestrating cell behavior, function, and fate. However, the underlying mechanism in SiNW-mediated intracellular access and delivery is still poorly understood. This study demonstrates the development of a gene delivery platform based on conical SiNW arrays for mechanical cell transfection, assisted by centrifugal force, for both adherent and nonadherent cells in vitro. Cells form focal adhesions on SiNWs within 6 h, and maintain high viability and motility. Such a functional and dynamic cell-SiNW interface features conformational changes in the plasma membrane and in some cases the nucleus, promoting both direct penetration and endocytosis; this synergistically facilitates SiNW-mediated delivery of nucleic acids into immortalized cell lines, and into difficult-to-transfect primary immune T cells without pre-activation. Moreover, transfected cells retrieved from SiNWs retain the capacity to proliferate-crucial to future biomedical applications. The results indicate that SiNW-mediated intracellular delivery holds great promise for developing increasingly sophisticated investigative and therapeutic tools.