Correlative light, electron, and ion microscopy provides a powerful multiscale framework for analyzing single cells, but integrating these methods remains challenging due to divergent specimen preparation requirements, especially for rare cell populations where low yield and sample loss limit success. Herein, we introduce a femtosecond (fs) laser ablation strategy that enables rapid material removal and targeted fabrication of 3D sub-100-µm microstructures on resin-embedded biological substrates. The workflow is demonstrated using flow-sorted mouse CD4+ T-cell subsets: regulatory (Treg) and effector (Teff) cells. Following resin embedding, fs-laser ablation was used to fabricate a half-grid geometry featuring an array of micropillars (∼30 µm tip diameter), each containing single or multiple target cells while limiting laser-induced damage to ∼3 µm. We validated the approach by preparing electron-transparent membranes using a hybrid fs‑laser/focused ion beam scanning electron microscopy (FIB-SEM) workflow and performing 3D FIB-SEM tomography on single cells positioned on the fabricated micropillar tips. These analyses revealed well-preserved subcellular architecture and enabled direct ultrastructural comparison between the T-cell subsets. The grid geometry is also compatible with additional downstream analytical techniques, expanding opportunities for nanoscale chemical analysis. Overall, this work establishes a practical pathway that integrates precision micro/nanoengineering with high‑resolution correlative imaging for robust analysis of rare and limited cell populations.

The development of point-of-care wearable devices capable of measuring insulin concentration has the potential to significantly improve diabetes management and life quality of diabetic patients. However, the lack of a suitable point-of-care device for personal use makes regular insulin level measurements challenging, in stark contrast to glucose monitoring. Herein, we report an electrochemical transdermal biosensor that utilizes a high-density polymeric microneedle array (MNA) to detect insulin in interstitial fluid (ISF). The biosensor consists of gold-coated polymeric MNA modified with an insulin-selective aptamer, which was used for extraction and electrochemical quantification of the insulin in ISF. In vitro testing of biosensor, performed in artificial ISF (aISF), showed high selectivity for insulin with a linear response between 0.01 nM and 4 nM (sensitivity of similar to 65 Omega nM(-1)), a range that covers both the physiological and the pathological concentration range. Furthermore, ex vivo extraction and quantification of insulin from mouse skin showed no impact on the biosensor's linear response. As a proof of concept, an MNA-based biosensing platform was utilized for the extraction and quantification of insulin on live mouse skin. In vivo application showed the ability of MNs to reach ISF, extract insulin from ISF, and perform electrochemical measurements sufficient for determining insulin levels in blood and ISF. We believe that our MNA-based biosensing platform based on extraction and quantification of the biomarkers will help move insulin assays from traditional laboratory approaches to personalized point-of-care settings.

Pluripotent stem cells (PSCs), comprised of embryonic stem cells and induced PSCs, hold tremendous therapeutic potential. This has been driven by transformative advances in cell engineering and manufacturing, from fundamental research to clinical therapies. These innovations have come from a deeper understanding of developmental cell biology, the ability to recapitulate complex biochemical, mechanical, and topographical cues necessary for precise cell differentiation and functional maturation, and the deployment of advanced biotechnological approaches. For example, recent advances in micro‐ and nanotopographical engineering have introduced novel biomimetic approaches to enhance PSC adhesion, self‐renewal, lineage specification, and spatial organization, while continued development of PSC manufacturing—including 3D bioreactor systems, microfluidic confinement devices, and scalable automation technologies—is driving a considerable shift beyond 2D culture and biochemical signaling methods. This mini‐review examines the impact of recent developments in the application of micro‐ and nanotopographical cues in controlling core PSC fate and functions, including proliferation, adhesion, pluripotency, and differentiation. A gene expression profile can be altered by these topographical cues, and evaluate current strategies to integrate topographical control in PSC technology is highlighted.

Central nervous system (CNS) diseases pose a significant global health challenge, currently affecting one in six individuals worldwide. Despite extensive research into their molecular and cellular mechanisms, effective treatment remains elusive due to the inherent complexity of CNS disorders and the protective constraints of the blood‐brain barrier (BBB).Conventional monotherapies often fail to produce satisfactory outcomes. In recent years, nanomaterials have emerged as promising therapeutic platforms—offering enhanced pharmacokinetics, improved biodistribution, and reduced systemic toxicity. More importantly, multifunctional nanomaterials can be engineered to actively target the brain parenchyma and lesion sites, co‐deliver multiple therapeutics, and modulate neuroimmune responses synergistically. Their tunability and adaptability to diverse pathological contexts make them well‐suited to address the multifactorial nature of CNS diseases. This review discusses the mechanisms contributing to therapeutic resistance in common brain disorders and highlights the potential of engineered nanomaterials that can address the multifaceted disease microenvironment. We also identify current challenges and prospects for the rational design of multifunctional nanoplatforms tailored to CNS disease treatment.

Cytotoxic CD8+ T cells, essential in combating viral infections and cancer, become dysfunctional from prolonged antigen exposure. Precursors of exhausted T (TPEX) cells are pivotal in sustaining immune responses in chronic diseases and mediating immunotherapy efficacy. They also control viral infection within B-cell follicles, facilitated by CXCR5 expression. How cytokines regulate TPEX cell fate and follicular entry is not well understood. We reveal that IL-2 treatment enhances CD8+ T cell effector functions in chronic LCMV infection but hinders CXCR5+ TPEX cell formation and infection control within B-cell follicles. Mechanistically, IL-2 suppresses TPEX cell differentiation in a STAT5 and BLIMP1-dependent manner. Using an IL-2 fusion protein targeting CD122, we shifted the differentiation towards CX3CR1+ T cells with increased effector function. Clinical observations with low-dose IL-2 in autoimmune disease confirmed IL-2’s inhibitory effect on CXCR5+ TPEX cells, underscoring IL-2’s crucial regulatory role and therapeutic potential in modulating TPEX and effector T cell generation.### Competing Interest StatementThe authors have declared no competing interest.

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.

In 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).

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.

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.

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. Graphical abstract

Surface charge of biomaterials is one of the most influential parameters on regulating the complex processes of cell responses in tissue engineering. This study explores the contributions of xTa (x ​= ​5; 10; 30 in wt%) interface with the Ti as matrix on the in-vitro cell growth when such composites were produced through cold spray additive manufacturing. Preliminary results reveal that formation of intimate contact between deposited Ti and Ta splats provides meaningful differences in work function, results in an estimated surface potential variation around 50 ​mV, that ultimately influence cell growth. Increasing mass fraction of Ta in the chosen cold sprayed Ti-xTa composites was beneficial to initial cell attachment and proliferation upon the surface. Electrochemical response of cold sprayed coatings indirectly proves that Ta may act as anode and Ti performs as cathode in the electrochemical cells with possible surface charge gradient that allow to design and adapt biomaterials surfaces to a specific application. Understanding the mechanism of cell growth upon the surface of cold sprayed Ti-xTa composites will contribute to design of biomaterial surface for promising osseointegrity in biomedical applications.

Cell-based immunotherapy such as chimeric antigen receptor (CAR)-T therapy holds great promise in treating cancer and other diseases; but the current viral-based method represents a significant cost and safety hurdle. Here, we show for the first time successful CAR transfection into primary T cells via vertically aligned silicon nanotube (SiNT) arrays. SiNT-mediated transfection achieves comparable or even higher delivery efficiency (20–37%) and expression efficiency (18–24%) to that achieved by electroporation. Scanning electron microscopy imaging after focused ion beam milling demonstrated the tight T cell–SiNT interface. The induced membrane invaginations and the proximity between individual SiNTs and the nucleus might enhance endocytic pathways, and enable direct delivery of CAR construct into the nucleus, thus resulting in higher CAR expression efficiency. SiNT-interfacing also results in faster proliferation of T cells compared to cells transfected by electroporation; non-activated T (N_SiNT) cells undergo higher numbers of cell division than pre-activated ones (A_SiNT). By co-culturing with target lymphoma Raji cells, we prove that SiNT-transfected CAR-T cells can suppress Raji cell growth, indicated by significant increase in effector:target (E:T) ratio (by up to 30.7-fold). While SiNTs induce an overall upregulation of cytokine production in T cells, N_SiNT T cells exhibited high increase in secretion of IFNγ and IL-6, and relatively high in TNFα, which could contribute to their enhanced killing ability (∼96% cytotoxicity), demonstrated by their stronger inhibition on target Raji cells through luciferase assay. The results demonstrate the capacity of SiNT-mediated transfection of generating effective anti-lymphoma CAR-T cells. Considering the growing potential of cell-based therapies, we expect that a non-viral nanoinjection platform such as ours will facilitate the full realization of their therapeutic promise.

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. 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 low-voltage, simple, scalable, 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 editing. 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. 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 ~63% (antibody), ~56% (mRNA), and ~52% (plasmid DNA), with minimal impact on cells’ viability post nanoscaleEP (>90%). We also show gene editing through the delivery of siRNA that targets TRIOBP, yielding gene knockdown efficiency of ~41%. We anticipate that our ENI platform is set to offer a new path to intracellular delivery with broader selection of cargo and cell types, and will open opportunities for advanced ex vivo cell engineering and gene editing.

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.

Tunable vertically aligned nanostructures, usually fabricated using inorganic materials, are powerful nanoscale tools for advanced cellular manipulation. However, nanoscale precision typically requires advanced nanofabrication machinery and involves high manufacturing costs. By contrast, polymeric nanoneedles (NNs) of precise geometry can be produced by replica molding or nanoimprint lithography—rapid, simple, and cost-effective. Here, cytocompatible polymeric arrays of NNs are engineered with identical topographies but differing stiffness, using polystyrene (PS), SU8, and polydimethylsiloxane (PDMS). By interfacing the polymeric NN arrays with adherent and suspension mammalian cells, and comparing the cellular responses of each of the three polymeric substrates, the influence of substrate stiffness from topography on cell behavior is decoupled. Notably, the ability of PS, SU8, and PDMS NNs is demonstrated to facilitate mRNA delivery to GPE86 cells with 26.8% ± 3.5%, 33.2% ± 7.4%, and 30.1% ± 4.1% average transfection efficiencies, respectively. Electron microscopy reveals the intricacy of the cell–NN interactions; and immunofluorescence imaging demonstrates that enhanced endocytosis is one of the mechanisms of PS NN-mediated intracellular delivery, involving the endocytic proteins caveolin-1 and clathrin heavy chain. The results provide insights into the interfacial interactions between cells and polymeric NNs, and their related intracellular delivery mechanisms.

Tunable vertically aligned nanostructures, usually fabricated using inorganic materials, are powerful nanoscale tools for advanced cellular manipulation. However, nanoscale precision typically requires advanced nanofabrication machinery and involves high manufacturing costs. By contrast, polymeric nanoneedles (NNs) of precise geometry can be produced by replica molding or nanoimprint lithography—rapid, simple, and cost‐effective. Here, cytocompatible polymeric arrays of NNs are engineered with identical topographies but differing stiffness, using polystyrene (PS), SU8, and polydimethylsiloxane (PDMS). By interfacing the polymeric NN arrays with adherent and suspension mammalian cells, and comparing the cellular responses of each of the three polymeric substrates, the influence of substrate stiffness from topography on cell behavior is decoupled. Notably, the ability of PS, SU8, and PDMS NNs is demonstrated to facilitate mRNA delivery to GPE86 cells with 26.8% ± 3.5%, 33.2% ± 7.4%, and 30.1% ± 4.1% average transfection efficiencies, respectively. Electron microscopy reveals the intricacy of the cell–NN interactions; and immunofluorescence imaging demonstrates that enhanced endocytosis is one of the mechanisms of PS NN‐mediated intracellular delivery, involving the endocytic proteins caveolin‐1 and clathrin heavy chain. The results provide insights into the interfacial interactions between cells and polymeric NNs, and their related intracellular delivery mechanisms.

Abstract 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.

Engineered nano–bio interfaces–driven by vertical micro/nanoneedles, nanoparticles, organ‐on‐chip devices, and a diversity of nanosubstrates for mass spectroscopy imaging–are spurring scientific and technological progress, from fundamental to transnational biomedical research. Each class has its own characteristic features, which is critical for their translational uptake, but they broadly share the same range of functionality and applicability at the forefront of modern research and medicine. The review provides insights into unique attributes of microneedle technology and its ability for efficient transdermal transport of therapeutic compounds. The uses of nanoneedle technology in precise manipulation of increasingly complex cellular processes at the cell–material interface and their potential for major improvements for many fundamental research applications and ex vivo cell‐based therapies are highlighted. A snapshot in the use of food and drug administration (FDA)‐approved nanoparticle therapeutics and their applications in nanomedicine is provided. The achievements in organ‐on‐chip technology, particularly at the preclinical stage, and its potential to efficiently screen diverse types of therapeutics are covered. The final section is dedicated to the use of nanomaterial‐enhanced mass spectrometry in drug discovery and imaging. Overall, this review aims to highlight those main rules in the design of bio–nano interfaces that have successfully achieved translation into the market.

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.