Nanoinjection: A Platform for Innovation in Ex Vivo Cell Engineering.

ConspectusIn human cells, intracellular access and therapeutic cargo transport, including gene-editing tools (e.g., CRISPR-Cas9 and transposons), nucleic acids (e.g., DNA, mRNA, and siRNA), peptides, and proteins (e.g., enzymes and antibodies), are tightly constrained to ensure healthy cell function and behavior. This principle is exemplified in the delivery mechanisms of chimeric antigen receptor (CAR)-T cells for ex-vivo immunotherapy. In particular, the clinical success of CAR-T cells has established a new standard of care by curing previously incurable blood cancers. The approach involves the delivery, typically via the use of electroporation (EP) and lentivirus, of therapeutic CAR genes into a patient's own T cells, which are then engineered to express CARs that target and combat their blood cancer. But the key difficulty lies in genetically manipulating these cells without causing irreversible damage or loss of function─all the while minimizing complexities of manufacturing, safety concerns, and costs, and ensuring the efficacy of the final CAR-T cell product.Nanoinjection─the process of intracellular delivery using nanoneedles (NNs)─is an emerging physical delivery route that efficiently negotiates the plasma membrane of many cell types, including primary human T cells. It occurs with minimal perturbation, invasiveness, and toxicity, with high efficiency and throughput at high spatial and temporal resolutions. Nanoinjection promises greatly improved delivery of a broad range of therapeutic cargos with little or no damage to those cargos. A nanoinjection platform allows these cargos to function in the intracellular space as desired. The adaptability of nanoinjection platforms is now bringing major advantages in immunomodulation, mechanotransduction, sampling of cell states (nanobiopsy), controlled intracellular interrogation, and the primary focus of this account─intracellular delivery and its applications in ex vivo cell engineering.Mechanical nanoinjection typically exerts direct mechanical force on the cell membrane, offering a straightforward route to improve membrane perturbation by the NNs and subsequent transport of genetic cargo into targeted cell type (adherent or suspension cells). By contrast, electroactive nanoinjection is controlled by coupling NNs with an electric field─a new route for activating electroporation (EP) at the nanoscale─allowing a dramatic reduction of the applied voltage to a cell and so minimizing post-EP damage to cells and cargo, and overcoming many of the limitations of conventional bulk EP. Nanoinjection transcends mere technique; it is an approach to cell engineering ex vivo, offering the potential to endow cells with new, powerful features such as generating chimeric antigen receptor (CAR)-T cells for future CAR-T cell technologies.We first discuss the manufacturing of NN devices (Section 2), then delve into nanoinjection-mediated cell engineering (Section 3), nanoinjection mechanisms and interfacing methodologies (Section 4), and emerging applications in using nanoinjection to create functional CAR-T cells (Section 5).

Electroactive nanoinjection platform for intracellular delivery and gene silencing.

BACKGROUND: Nanoinjection-the process of intracellular delivery using vertically configured nanostructures-is a physical route that efficiently negotiates the plasma membrane, with minimal perturbation and toxicity to the cells. Nanoinjection, as a physical membrane-disruption-mediated approach, overcomes challenges associated with conventional carrier-mediated approaches such as safety issues (with viral carriers), genotoxicity, limited packaging capacity, low levels of endosomal escape, and poor versatility for cell and cargo types. Yet, despite the implementation of nanoinjection tools and their assisted analogues in diverse cellular manipulations, there are still substantial challenges in harnessing these platforms to gain access into cell interiors with much greater precision without damaging the cell's intricate structure. Here, we propose a non-viral, low-voltage, and reusable electroactive nanoinjection (ENI) platform based on vertically configured conductive nanotubes (NTs) that allows for rapid influx of targeted biomolecular cargos into the intracellular environment, and for successful gene silencing. The localization of electric fields at the tight interface between conductive NTs and the cell membrane drastically lowers the voltage required for cargo delivery into the cells, from kilovolts (for bulk electroporation) to only ≤ 10 V; this enhances the fine control over membrane disruption and mitigates the problem of high cell mortality experienced by conventional electroporation. RESULTS: Through both theoretical simulations and experiments, we demonstrate the capability of the ENI platform to locally perforate GPE-86 mouse fibroblast cells and efficiently inject a diverse range of membrane-impermeable biomolecules with efficacy of 62.5% (antibody), 55.5% (mRNA), and 51.8% (plasmid DNA), with minimal impact on cells' viability post nanoscale-EP (> 90%). We also show gene silencing through the delivery of siRNA that targets TRIOBP, yielding gene knockdown efficiency of 41.3%. CONCLUSIONS: We anticipate that our non-viral and low-voltage ENI platform is set to offer a new safe path to intracellular delivery with broader selection of cargo and cell types, and will open opportunities for advanced ex vivo cell engineering and gene silencing.

Role of actin cytoskeleton in cargo delivery mediated by vertically aligned silicon nanotubes.

Nanofabrication technologies have been recently applied to the development of engineered nano-bio interfaces for manipulating complex cellular processes. In particular, vertically configurated nanostructures such as nanoneedles (NNs) have been adopted for a variety of biological applications such as mechanotransduction, biosensing, and intracellular delivery. Despite their success in delivering a diverse range of biomolecules into cells, the mechanisms for NN-mediated cargo transport remain to be elucidated. Recent studies have suggested that cytoskeletal elements are involved in generating a tight and functional cell-NN interface that can influence cargo delivery. In this study, by inhibiting actin dynamics using two drugs-cytochalasin D (Cyto D) and jasplakinolide (Jas), we demonstrate that the actin cytoskeleton plays an important role in mRNA delivery mediated by silicon nanotubes (SiNTs). Specifically, actin inhibition 12 h before SiNT-cellular interfacing (pre-interface treatment) significantly dampens mRNA delivery (with efficiencies dropping to 17.2% for Cyto D and 33.1% for Jas) into mouse fibroblast GPE86 cells, compared to that of untreated controls (86.9%). However, actin inhibition initiated 2 h after the establishment of GPE86 cell-SiNT interface (post-interface treatment), has negligible impact on mRNA transfection, maintaining > 80% efficiency for both Cyto D and Jas treatment groups. The results contribute to understanding potential mechanisms involved in NN-mediated intracellular delivery, providing insights into strategic design of cell-nano interfacing under temporal control for improved effectiveness.

Optically transparent vertical silicon nanowire arrays for live-cell imaging.

Programmable nano-bio interfaces driven by tuneable vertically configured nanostructures have recently emerged as a powerful tool for cellular manipulations and interrogations. Such interfaces have strong potential for ground-breaking advances, particularly in cellular nanobiotechnology and mechanobiology. However, the opaque nature of many nanostructured surfaces makes non-destructive, live-cell characterization of cellular behavior on vertically aligned nanostructures challenging to observe. Here, a new nanofabrication route is proposed that enables harvesting of vertically aligned silicon (Si) nanowires and their subsequent transfer onto an optically transparent substrate, with high efficiency and without artefacts. We demonstrate the potential of this route for efficient live-cell phase contrast imaging and subsequent characterization of cells growing on vertically aligned Si nanowires. This approach provides the first opportunity to understand dynamic cellular responses to a cell-nanowire interface, and thus has the potential to inform the design of future nanoscale cellular manipulation technologies.

Efficient Transmission Electron Microscopy Characterization of Cell-Nanostructure Interfacial Interactions.

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.

Cellular Deformations Induced by Conical Silicon Nanowire Arrays Facilitate Gene Delivery.

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.

Compression and deposition of microgel monolayers from fluid interfaces: particle size effects on interface microstructure and nanolithography.

Controlling the microstructure of monolayers of microgels confined at a water/oil interface is the key to their successful application as nanolithography masks after deposition on a solid substrate. Previous work demonstrated that compression of the monolayer can be used to tune the microgel arrangement and to explore the full two-dimensional area-pressure phase diagram of the particles trapped at the interface. Here, we explore a new size range, using microgels with 210 nm and 1.45 µm bulk diameters, respectively. We start by investigating the properties of isolated particles in situ at the interface by freeze-fracture cryo-SEM, and after deposition using an atomic force microscope. We then study their collective behavior in a compressed monolayer and highlight significant differences in terms of the accessible structural phases and their transitions. More specifically, the larger microgels behave similar to colloids with a hard core and a soft polymeric shell, exhibiting capillarity driven clustering at a large specific area and a solid-solid phase transition between two hexagonal lattices at higher compressions. The smaller particles instead show no aggregation and a smooth transition from a hexagonal lattice to a dense disordered monolayer. Finally, we demonstrate that the larger microgels can be effectively turned into masks for the fabrication of vertically aligned silicon nanowires by means of metal-assisted chemical etching. These findings highlight the subtle interplay between particle architecture, adsorption and interactions at the interface, the understanding and harnessing of which are at the basis of their successful use as nanopatterning tools.

Fully Tunable Silicon Nanowire Arrays Fabricated by Soft Nanoparticle Templating.

We demonstrate a fabrication breakthrough to produce large-area arrays of vertically aligned silicon nanowires (VA-SiNWs) with full tunability of the geometry of the single nanowires and of the whole array, paving the way toward advanced programmable designs of nanowire platforms. At the core of our fabrication route, termed "Soft Nanoparticle Templating", is the conversion of gradually compressed self-assembled monolayers of soft nanoparticles (microgels) at a water-oil interface into customized lithographical masks to create VA-SiNW arrays by means of metal-assisted chemical etching (MACE). This combination of bottom-up and top-down techniques affords excellent control of nanowire etching site locations, enabling independent control of nanowire spacing, diameter and height in a single fabrication route. We demonstrate the fabrication of centimeter-scale two-dimensional gradient photonic crystals exhibiting continuously varying structural colors across the entire visible spectrum on a single silicon substrate, and the formation of tunable optical cavities supported by the VA-SiNWs, as unambiguously demonstrated through numerical simulations. Finally, Soft Nanoparticle Templating is combined with optical lithography to create hierarchical and programmable VA-SiNW patterns.

Surface-assisted laser desorption/ionization mass spectrometry using ordered silicon nanopillar arrays.

Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) is ideally suited for the high-throughput analysis of small molecules in bodily fluids (e.g. saliva, urine, and blood plasma). A key application for this technique is the testing of drug consumption in the context of workplace, roadside, athlete sports and anti-addictive drug compliance. Here, we show that vertically-aligned ordered silicon nanopillar (SiNP) arrays fabricated using nanosphere lithography followed by metal-assisted chemical etching (MACE) are suitable substrates for the SALDI-MS detection of methadone and small peptides. Porosity, length and diameter are fabrication parameters that we have explored here in order to optimize analytical performance. We demonstrate the quantitative analysis of methadone in MilliQ water down to 32 ng mL(-1). Finally, the capability of SiNP arrays to facilitate the detection of methadone in clinical samples is also demonstrated.

The Roles of Micro- and Nanoscale Materials in Cell-Engineering Systems.

Customizable manufacturing of ex vivo cell engineering is driven by the need for innovations in the biomedical field and holds substantial potential for addressing current therapeutic challenges; but it is still only in its infancy. Micro- and nanoscale-engineered materials are increasingly used to control core cell-level functions in cellular engineering. By reprogramming or redirecting targeted cells for extremely precise functions, these advanced materials offer new possibilities. This influences the modularity of cell reprogramming and reengineering, making these materials part of versatile and emerging technologies. Here, the roles of micro- and nanoscale materials in cell engineering are highlighted, demonstrating how they can be adaptively controlled to regulate cellular reprogramming and core cell-level functions, including differentiation, proliferation, adhesion, user-defined gene expression, and epigenetic changes. The current reprogramming routes used to achieve pluripotency from somatic cells and the significant potential of induced pluripotent stem cell technology for translational biomedical research are covered. Recent advances in nonviral intracellular delivery modalities for cell reprogramming and their constraints are evaluated. This paper focuses on emerging physical and combinatorial approaches of intracellular delivery for cell engineering, revealing the capabilities and limitations of these routes. It is showcased how these programmable materials are continually being explored as customizable tools for inducing biophysical stimulation. Harnessing the power of micro- and nanoscale-engineered materials will be a step change in the design of cell engineering, producing a suite of powerful tools for addressing potential future challenges in therapeutic cell engineering.

Confinement-guided shaping of semiconductor nanowires and nanoribbons: "writing with nanowires".

To fully exploit their full potential, new semiconductor nanowire building blocks with ab initio controlled shapes are desired. However, and despite the great synthetic advances achieved, the ability to control nanowire's geometry has been significantly limited. Here, we demonstrate a simple confinement-guided nanowire growth method that enables to predesign not only the chemical and physical attributes of the synthesized nanowires but also allows a perfect and unlimited control over their geometry. Our method allows the synthesis of semiconductor nanowires in a wide variety of two-dimensional shapes such as any kinked (different turning angles), sinusoidal, linear, and spiral shapes, so that practically any desired geometry can be defined. The shape-controlled nanowires can be grown on almost any substrate such as silicon wafer, quartz and glass slides, and even on plastic substrates (e.g., Kapton HN).

Biorecognition layer engineering: overcoming screening limitations of nanowire-based FET devices.

Detection of biological species is of great importance to numerous areas of medical and life sciences from the diagnosis of diseases to the discovery of new drugs. Essential to the detection mechanism is the transduction of a signal associated with the specific recognition of biomolecules of interest. Nanowire-based electrical devices have been demonstrated as a powerful sensing platform for the highly sensitive detection of a wide-range of biological and chemical species. Yet, detecting biomolecules in complex biosamples of high ionic strength (>100 mM) is severely hampered by ionic screening effects. As a consequence, most of existing nanowire sensors operate under low ionic strength conditions, requiring ex situ biosample manipulation steps, that is, desalting processes. Here, we demonstrate an effective approach for the direct detection of biomolecules in untreated serum, based on the fragmentation of antibody-capturing units. Size-reduced antibody fragments permit the biorecognition event to occur in closer proximity to the nanowire surface, falling within the charge-sensitive Debye screening length. Furthermore, we explored the effect of antibody surface coverage on the resulting detection sensitivity limit under the high ionic strength conditions tested and found that lower antibody surface densities, in contrary to high antibody surface coverage, leads to devices of greater sensitivities. Thus, the direct and sensitive detection of proteins in untreated serum and blood samples was effectively performed down to the sub-pM concentration range without the requirement of biosamples manipulation.

Si nanowires forest-based on-chip biomolecular filtering, separation and preconcentration devices: nanowires do it all.

The development of efficient biomolecular separation and purification techniques is of critical importance in modern genomics, proteomics, and biosensing areas, primarily due to the fact that most biosamples are mixtures of high diversity and complexity. Most of existent techniques lack the capability to rapidly and selectively separate and concentrate specific target proteins from a complex biosample, and are difficult to integrate with lab-on-a-chip sensing devices. Here, we demonstrate the development of an on-chip all-SiNW filtering, selective separation, desalting, and preconcentration platform for the direct analysis of whole blood and other complex biosamples. The separation of required protein analytes from raw biosamples is first performed using a antibody-modified roughness-controlled SiNWs (silicon nanowires) forest of ultralarge binding surface area, followed by the release of target proteins in a controlled liquid media, and their subsequent detection by supersensitive SiNW-based FETs arrays fabricated on the same chip platform. Importantly, this is the first demonstration of an all-NWs device for the whole direct analysis of blood samples on a single chip, able to selectively collect and separate specific low abundant proteins, while easily removing unwanted blood components (proteins, cells) and achieving desalting effects, without the requirement of time-consuming centrifugation steps, the use of desalting or affinity columns. Futhermore, we have demonstrated the use of our nanowire forest-based separation device, integrated in a single platform with downstream SiNW-based sensors arrays, for the real-time ultrasensitive detection of protein biomarkers directly from blood samples. The whole ultrasensitive protein label-free analysis process can be practically performed in less than 10 min.

The influence of dysfunctional actin on polystyrene-nanotube-mediated mRNA nanoinjection into mammalian cells.

The advancement of nanofabrication technologies has transformed the landscape of engineered nano-bio interfaces, especially with vertically aligned nanoneedles (NNs). This enables scientists to venture into new territories, widening NN applications into increasingly more complex cellular manipulation and interrogation. Specifically, for intracellular delivery application, NNs have been shown to mediate the delivery of various bioactive cargos into a wide range of cells-a physical method termed "nanoinjection". Silicon (Si) nanostructures demonstrated great potential in nanoinjection, whereas the use of polymeric NNs for nanoinjection has rarely been explored. Furthermore, the underlying mechanism of interaction at the cell-NN interface is subtle and multifaceted, and not fully understood-underpinned by the design versatility of the NN biointerface. Recent studies have suggested that actin dynamic plays a pivotal role influencing the delivery efficacy. In this study, we fabricated a new class of NNs-a programmable polymeric nanotubes (NTs)-from polystyrene (PS) cell cultureware, designed to facilitate mRNA delivery into mouse embryonic fibroblast GPE86 cells. The PSNT delivery platform was able to mediate mRNA delivery with high delivery efficiency (∼83%). We also investigated the role of actin cytoskeleton in PSNTs mediated intracellular delivery by introducing two actin inhibitors-cytochalasin D (Cyto D) and jasplakinolide (Jas)-to cause dysfunctional cytoskeleton, via inhibiting actin polymerization and depolymerization, respectively (before and after the establishment of cell-PSNT interface). By inhibiting actin dynamics 12 h before cell-PSNT interfacing (pre-interface treatment), the mRNA delivery efficiencies were significantly reduced to ∼3% for Cyto D-treated samples and ∼1% for Jas-treated sample, as compared to their post-interface (2 h after cell-PSNT interfacing) counterpart (∼46% and ∼68%, respectively). The added flexibility of PSNTs have shown to help withstand mechanical breakage stemming from cytoskeletal forces in contrast to the SiNTs. Such findings will step-change our capacity to use programmable polymeric NTs in fundamental cellular processes related to intracellular delivery.

Engineering Efficient CAR-T Cells via Electroactive Nanoinjection.

Chimeric antigen receptor (CAR)-T cell therapy has emerged as a promising cell-based immunotherapy approach for treating blood disorders and cancers, but genetically engineering CAR-T cells is challenging due to primary T cells' sensitivity to conventional gene delivery approaches. The current viral-based method can typically involve significant operating costs and biosafety hurdles, while bulk electroporation (BEP) can lead to poor cell viability and functionality. Here, a non-viral electroactive nanoinjection (ENI) platform is developed to efficiently negotiate the plasma membrane of primary human T cells via vertically configured electroactive nanotubes, enabling efficient delivery (68.7%) and expression (43.3%) of CAR genes in the T cells, with minimal cellular perturbation (>90% cell viability). Compared to conventional BEP, the ENI platform achieves an almost threefold higher CAR transfection efficiency, indicated by the significantly higher reporter GFP expression (43.3% compared to 16.3%). By co-culturing with target lymphoma Raji cells, the ENI-transfected CAR-T cells' ability to effectively suppress lymphoma cell growth (86.9% cytotoxicity) is proved. Taken together, the results demonstrate the platform's remarkable capacity to generate functional and effective anti-lymphoma CAR-T cells. Given the growing potential of cell-based immunotherapies, such a platform holds great promise for ex vivo cell engineering, especially in CAR-T cell therapy.

Non-covalent monolayer-piercing anchoring of lipophilic nucleic acids: preparation, characterization, and sensing applications.

Functional interfaces of biomolecules and inorganic substrates like semiconductor materials are of utmost importance for the development of highly sensitive biosensors and microarray technology. However, there is still a lot of room for improving the techniques for immobilization of biomolecules, in particular nucleic acids and proteins. Conventional anchoring strategies rely on attaching biomacromolecules via complementary functional groups, appropriate bifunctional linker molecules, or non-covalent immobilization via electrostatic interactions. In this work, we demonstrate a facile, new, and general method for the reversible non-covalent attachment of amphiphilic DNA probes containing hydrophobic units attached to the nucleobases (lipid-DNA) onto SAM-modified gold electrodes, silicon semiconductor surfaces, and glass substrates. We show the anchoring of well-defined amounts of lipid-DNA onto the surface by insertion of their lipid tails into the hydrophobic monolayer structure. The surface coverage of DNA molecules can be conveniently controlled by modulating the initial concentration and incubation time. Further control over the DNA layer is afforded by the additional external stimulus of temperature. Heating the DNA-modified surfaces at temperatures >80 °C leads to the release of the lipid-DNA structures from the surface without harming the integrity of the hydrophobic SAMs. These supramolecular DNA layers can be further tuned by anchoring onto a mixed SAM containing hydrophobic molecules of different lengths, rather than a homogeneous SAM. Immobilization of lipid-DNA on such SAMs has revealed that the surface density of DNA probes is highly dependent on the composition of the surface layer and the structure of the lipid-DNA. The formation of the lipid-DNA sensing layers was monitored and characterized by numerous techniques including X-ray photoelectron spectroscopy, quartz crystal microbalance, ellipsometry, contact angle measurements, atomic force microscopy, and confocal fluorescence imaging. Finally, this new DNA modification strategy was applied for the sensing of target DNAs using silicon-nanowire field-effect transistor device arrays, showing a high degree of specificity toward the complementary DNA target, as well as single-base mismatch selectivity.

Silicon Nanoneedle-Induced Nuclear Deformation: Implications for Human Somatic and Stem Cell Nuclear Mechanics.

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.

Changing Fate: Reprogramming Cells via Engineered Nanoscale Delivery Materials.

The incorporation of nanotechnology in regenerative medicine is at the nexus of fundamental innovations and early-stage breakthroughs, enabling exciting biomedical advances. One of the most exciting recent developments is the use of nanoscale constructs to influence the fate of cells, which are the basic building blocks of healthy function. Appropriate cell types can be effectively manipulated by direct cell reprogramming; a robust technique to manipulate cellular function and fate, underpinning burgeoning advances in drug delivery systems, regenerative medicine, and disease remodeling. Individual transcription factors, or combinations thereof, can be introduced into cells using both viral and nonviral delivery systems. Existing approaches have inherent limitations. Viral-based tools include issues of viral integration into the genome of the cells, the propensity for uncontrollable silencing, reduced copy potential and cell specificity, and neutralization via the immune response. Current nonviral cell reprogramming tools generally suffer from inferior expression efficiency. Nanomaterials are increasingly being explored to address these challenges and improve the efficacy of both viral and nonviral delivery because of their unique properties such as small size and high surface area. This review presents the state-of-the-art research in cell reprogramming, focused on recent breakthroughs in the deployment of nanomaterials as cell reprogramming delivery tools.

Knocking down highly-ordered large-scale nanowire arrays.

The large-scale assembly of nanowire elements with controlled and uniform orientation and density at spatially well-defined locations on solid substrates presents one of the most significant challenges facing their integration in real-world electronic applications. Here, we present the universal "knocking-down" approach, based on the controlled in-place planarization of nanowire elements, for the formation of large-scale ordered nanowire arrays. The controlled planarization of the nanowires is achieved by the use of an appropriate elastomer-covered rigid-roller device. After being knocked down, each nanowire in the array can be easily addressed electrically, by a simple single photolithographic step, to yield a large number of nanoelectrical devices with an unprecedented high-fidelity rate. The approach allows controlling, in only two simple steps, all possible array parameters, that is, nanowire dimensions, chemical composition, orientation, and density. The resulting knocked-down arrays can be further used for the creation of massive nanoelectronic-device arrays. More than million devices were already fabricated with yields over 98% on substrate areas of up, but not limited to, to 10 cm(2).