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.

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.

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.

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.

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.

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.

Precision Surface Microtopography Regulates Cell Fate via Changes to Actomyosin Contractility and Nuclear Architecture.

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.

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.

Emerging Roles of 1D Vertical Nanostructures in Orchestrating Immune Cell Functions.

Engineered nano-bio cellular interfaces driven by 1D vertical nanostructures (1D-VNS) are set to prompt radical progress in modulating cellular processes at the nanoscale. Here, tuneable cell-VNS interfacial interactions are probed and assessed, highlighting the use of 1D-VNS in immunomodulation, and intracellular delivery into immune cells-both crucial in fundamental and translational biomedical research. With programmable topography and adaptable surface functionalization, 1D-VNS provide unique biophysical and biochemical cues to orchestrate innate and adaptive immunity, both ex vivo and in vivo. The intimate nanoscale cell-VNS interface leads to membrane penetration and cellular deformation, facilitating efficient intracellular delivery of diverse bioactive cargoes into hard-to-transfect immune cells. The unsettled interfacial mechanisms reported to be involved in VNS-mediated intracellular delivery are discussed. By identifying up-to-date progress and fundamental challenges of current 1D-VNS technology in immune-cell manipulation, it is hoped that this report gives timely insights for further advances in developing 1D-VNS as a safe, universal, and highly scalable platform for cell engineering and enrichment in advanced cancer immunotherapy such as chimeric antigen receptor-T therapy.

Silicon-Nanotube-Mediated Intracellular Delivery Enables Ex Vivo Gene Editing.

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.

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.

Tunable 2D binary colloidal alloys for soft nanotemplating.

The realization of non-close-packed nanoscale patterns with multiple feature sizes and length scales via colloidal self-assembly is a highly challenging task. We demonstrate here the creation of a variety of tunable particle arrays by harnessing the sequential self-assembly and deposition of two differently sized microgel particles at the fluid-fluid interface. The two-step process is essential to achieve a library of 2D binary colloidal alloys, which are kinetically inaccessible by direct co-assembly. These versatile binary patterns can be exploited for a range of end-uses. Here we show that they can for instance be transferred to silicon substrates, where they act as masks for the metal-assisted chemical etching of binary arrays of vertically aligned silicon nanowires (VA-SiNWs) with fine geometrical control. In particular, continuous binary gradients in both NW spacing and height can be achieved. Notably, these binary VA-SiNW platforms exhibit interesting anti-reflective properties in the visible range, in agreement with simulations. The proposed strategy can also be used for the precise placement of metallic nanoparticles in non-close-packed arrays. Sequential depositions of soft particles enable therefore the exploration of complex binary patterns, e.g. for the future development of substrates for biointerfaces, catalysis and controlled wetting.

Realization of Molecular-Based Transistors.

Molecular-based devices are widely considered as significant candidates to play a role in the next generation of "post-complementary metal-oxide-semiconductor" devices. In this context, molecular-based transistors: molecular junctions that can be electrically gated-are of particular interest as they allow new modes of operation. The properties of molecular transistors composed of a single- or multimolecule assemblies, focusing on their practicality as real-world devices, concerning industry demands and its roadmap are compared. Also, the capability of the gate electrode to modulate the molecular transistor characteristics efficiently is addressed, showing that electrical gating can be easily facilitated in single molecular transistors and that gating of transistor composed of molecular assemblies is possible if the device is formed vertically. It is concluded that while the single-molecular transistor exhibits better performance on the lab-scale, its realization faces signifacant challenges when compared to those faced by transistors composed of a multimolecule assembly.

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.

Ordered Silicon Pillar Arrays Prepared by Electrochemical Micromachining: Substrates for High-Efficiency Cell Transfection.

Ordered arrays of silicon nano- to microscale pillars are used to enable biomolecular trafficking into primary human cells, consistently demonstrating high transfection efficiency can be achieved with broader and taller pillars than reported to date. Cell morphology on the pillar arrays is often strikingly elongated. Investigation of the cellular interaction with the pillar reveals that cells are suspended on pillar tips and do not interact with the substrate between the pillars. Although cells remain suspended on pillar tips, acute local deformation of the cell membrane was noted, allowing pillar tips to penetrate the cell interior, while retaining cell viability.