Alexander M. Xu

Nanostraw–Electroporation System for Highly Efficient Intracellular Delivery and Transfection

Nondestructive introduction of genes, proteins, and small molecules into mammalian cells with high efficiency is a challenging, yet critical, process. Here we demonstrate a simple nanoelectroporation platform to achieve highly efficient molecular delivery and high transfection yields with excellent uniformity and cell viability. The system is built on alumina nanostraws extending from a track-etched membrane, forming an array of hollow nanowires connected to an underlying microfluidic channel. Cellular engulfment of the nanostraws provides an intimate contact, significantly reducing the necessary electroporation voltage and increasing homogeneity over a large area. Biomolecule delivery is achieved by diffusion through the nanostraws and enhanced by electrophoresis during pulsing. The system was demonstrated to offer excellent spatial, temporal, and dose control for delivery, as well as providing high-yield cotransfection and sequential transfection.

Shape Matters: Intravital Microscopy Reveals Surprising Geometrical Dependence for Nanoparticles in Tumor Models of Extravasation

Delivery is one of the most critical obstacles confronting nanoparticle use in cancer diagnosis and therapy. For most oncological applications, nanoparticles must extravasate in order to reach tumor cells and perform their designated task. However, little understanding exists regarding the effect of nanoparticle shape on extravasation. Herein we use real-time intravital microscopic imaging to meticulously examine how two different nanoparticles behave across three different murine tumor models. The study quantitatively demonstrates that high-aspect ratio single-walled carbon nanotubes (SWNTs) display extravasational behavior surprisingly different from, and counterintuitive to, spherical nanoparticles although the nanoparticles have similar surface coatings, area, and charge. This work quantitatively indicates that nanoscale extravasational competence is highly dependent on nanoparticle geometry and is heterogeneous.

Quantification of nanowire penetration into living cells

High-aspect ratio nanostructures such as nanowires and nanotubes are a powerful new tool for accessing the cell interior for delivery and sensing. Controlling and optimizing cellular access is a critical challenge for this new technology, yet even the most basic aspect of this process, whether these structures directly penetrate the cell membrane, is still unknown. Here we report the first quantification of hollow nanowires-nanostraws-that directly penetrate the membrane by observing dynamic ion delivery from each 100-nm diameter nanostraw. We discover that penetration is a rare event: 7.1±2.7% of the nanostraws penetrate the cell to provide cytosolic access for an extended period for an average of 10.7±5.8 penetrations per cell. Using time-resolved delivery, the kinetics of the first penetration event are shown to be adhesion dependent and coincident with recruitment of focal adhesion-associated proteins. These measurements provide a quantitative basis for understanding nanowire-cell interactions, and a means for rapidly assessing membrane penetration.

Plasma Membrane and Actin Cytoskeleton as Synergistic Barriers to Nanowire Cell Penetration

Nanowires are a rapidly emerging platform for manipulation of and material delivery directly into the cell cytosol. These high aspect ratio structures can breach the lipid membrane; however, the yield of penetrant structures is low, and the mechanism is largely unknown. In particular, some nanostructures appear to defeat the membrane transiently, while others can retain long-term access. Here, we examine if local dissolution of the lipid membrane, actin cytoskeleton, or both can enhance nanowire penetration. It is possible that, during cell contact, membrane rupture occurs; however, if the nanostructures do not penetrate the cytoskeleton, the membrane may reclose over a relatively short time frame. We show with quantitative analysis of the number of penetrating nanowires that the lipid bilayer and actin cytoskeleton are synergistic barriers to nanowire cell access, yet chemical poration through both is still insufficient to increase long-term access for adhered cells.

Fabrication of Sealed Nanostraw Microdevices for Oral Drug Delivery

The oral route is preferred for systemic drug administration and provides direct access to diseased tissue of the gastrointestinal (GI) tract. However, many drugs have poor absorption upon oral administration due to damaging enzymatic and pH conditions, mucus and cellular permeation barriers, and limited time for drug dissolution. To overcome these limitations and enhance oral drug absorption, micron-scale devices with planar, asymmetric geometries, termed microdevices, have been designed to adhere to the lining of the GI tract and release drug at high concentrations directly toward GI epithelium. Here we seal microdevices with nanostraw membranes-porous nanostructured biomolecule delivery substrates-to enhance the properties of these devices. We demonstrate that the nanostraws facilitate facile drug loading and tunable drug release, limit the influx of external molecules into the sealed drug reservoir, and increase the adhesion of devices to epithelial tissue. These findings highlight the potential of nanostraw microdevices to enhance the oral absorption of a wide range of therapeutics by binding to the lining of the GI tract, providing prolonged and proximal drug release, and reducing the exposure of their payload to drug-degrading biomolecules.

Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2

Summary Human in vitro generated monocyte‐derived dendritic cells (moDCs) and macrophages are used clinically, e.g., to induce immunity against cancer. However, their physiological counterparts, ontogeny, transcriptional regulation, and heterogeneity remains largely unknown, hampering their clinical use. High‐dimensional techniques were used to elucidate transcriptional, phenotypic, and functional differences between human in vivo and in vitro generated mononuclear phagocytes to facilitate their full potential in the clinic. We demonstrate that monocytes differentiated by macrophage colony‐stimulating factor (M‐CSF) or granulocyte macrophage colony‐stimulating factor (GM‐CSF) resembled in vivo inflammatory macrophages, while moDCs resembled in vivo inflammatory DCs. Moreover, differentiated monocytes presented with profound transcriptomic, phenotypic, and functional differences. Monocytes integrated GM‐CSF and IL‐4 stimulation combinatorically and temporally, resulting in a mode‐ and time‐dependent differentiation relying on NCOR2. Finally, moDCs are phenotypically heterogeneous and therefore necessitate the use of high‐dimensional phenotyping to open new possibilities for better clinical tailoring of these cellular therapies. HighlightsIn vitro monocyte cultures model in vivo inflammatory dendritic cells and macrophagesMonocyte‐derived dendritic cells integrate interleukin‐4 signaling time dependentlyNCOR2 controls differentiation of in vitro generated monocyte‐derived dendritic cellsIn vitro generated monocyte‐derived cells are phenotypically heterogeneous &NA; Monocyte‐derived cellular derivatives are used clinically and are a crucial tool in basic research. Sander and colleagues now show that they transcriptionally relate to in vivo inflammatory monocytes, that they integrate differentiation cues time dependently, and that in vitro differentiated monocytes are phenotypically heterogeneous.

Direct Intracellular Delivery of Cell Impermeable Probes of Protein Glycosylation Using Nanostraws

Bioorthogonal chemistry is an effective tool for elucidating metabolic pathways and measuring cellular activity, yet its use is currently limited by the difficulty of getting probes past the cell membrane and into the cytoplasm, especially if more complex probes are desired. Here we present a simple and minimally perturbative technique to deliver functional probes of glycosylation into cells by using a nanostructured “nanostraw” delivery system. Nanostraws provide direct intracellular access to cells through fluid conduits that remain small enough to minimize cell perturbation. First, we demonstrate that our platform can deliver an unmodified azidosugar, N‐azidoacetylmannosamine, into cells with similar effectiveness to a chemical modification strategy (peracetylation). We then show that the nanostraw platform enables direct delivery of an azidosugar modified with a charged uridine diphosphate group (UDP) that prevents intracellular penetration, thereby bypassing multiple enzymatic processing steps. By effectively removing the requirement for cell permeability from the probe, the nanostraws expand the toolbox of bioorthogonal probes that can be used to study biological processes on a single, easy‐to‐use platform.

Cellular reprogramming of human monocytes is regulated by time-dependent IL4 signalling and NCOR2

The clinical and therapeutic value of human in vitro generated monocyte-derived dendritic cell (moDC) and macrophages is well established. However, in line with recent findings regarding myeloid cell ontogeny and due to our limited understanding of their physiological counterparts, transcriptional regulation and heterogeneity, the full potential of these important cellular systems is still underestimated. In this study, we use cutting edge high-dimensional analysis methods to better understand the transcriptional organization, phenotypic heterogeneity and functional differences between human ex vivo isolated and in vitro generated mononuclear phagocytes with the aim to better realize their full potential in the clinic. We demonstrate that human monocytes activated by MCSF or GMCSF most closely resemble inflammatory macrophages identified in vivo, while IL4 signalling in the presence of GMCSF generates moDCs resembling inflammatory DCs in vivo, but not steady state cDC1 or cDC2. Moreover, these reprogramming regimes lead to activated monocytes that present with profoundly different transcriptomic, metabolic, phenotypic and functional profiles. Furthermore, we demonstrate that CD14+ monocytes are integrating multiple exogenous activation signals such as GMCSF and IL4 in a combinatorial and temporal fashion, resulting in a high-dimensional cellular continuum of reprogrammed monocytes dependent on the mode and timing of cytokine exposure. Utilizing nanostraw-based knockdown technology, we demonstrate that the IL4-dependent generation of moDCs relies on the induction, nuclear localization and function of the transcriptional regulator NCOR2. Finally, we unravel unappreciated heterogeneity within the clinically moDCs population and propose a novel high-dimensional phenotyping strategy to better tailor clinical quality control strategies for patient need and culture conditions to enhance therapeutic outcome.