Although carrier-mediated delivery systems offer promise for nucleic acid transfection in vivo 1,2 , membrane-disruption-based modalities are attractive candidates for universal delivery systems in vitro and ex vivo. In this review, we begin with motivations driving next-generation intracellular delivery strategies and suggest relevant requirements for future systems. Next, a broad overview of current delivery concepts covering salient strengths, challenges and opportunities is presented. Following that, our focus shifts to prevalent mechanisms of membrane disruption and recovery in the context of intracellular delivery. Finally,

During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round. Mitotic cell rounding is thought to facilitate organization within the mitotic cell and be necessary for the geometric requirements of division. However, the forces that drive this shape change remain poorly understood in the presence of external impediments, such as a tissue environment. Here we use cantilevers to track cell rounding force and volume. We show that cells have an outward rounding force, which increases as cells enter mitosis. We find that this mitotic rounding force depends both on the actomyosin cytoskeleton and the cells' ability to regulate osmolarity. The rounding force itself is generated by an osmotic pressure. However, the actomyosin cortex is required to maintain this rounding force against external impediments. Instantaneous disruption of the actomyosin cortex leads to volume increase, and stimulation of actomyosin contraction leads to volume decrease. These results show that in cells, osmotic pressure is balanced by inwardly directed actomyosin cortex contraction. Thus, by locally modulating actomyosin-cortex-dependent surface tension and globally regulating osmotic pressure, cells can control their volume, shape and mechanical properties.

Intracellular delivery is a key step in biological research and has enabled decades of biomedical discoveries. It is also becoming increasingly important in industrial and medical applications ranging from biomanufacture to cell-based therapies. Here, we review techniques for membrane disruption-based intracellular delivery from 1911 until the present. These methods achieve rapid, direct, and universal delivery of almost any cargo molecule or material that can be dispersed in solution. We start by covering the motivations for intracellular delivery and the challenges associated with the different cargo types-small molecules, proteins/peptides, nucleic acids, synthetic nanomaterials, and large cargo. The review then presents a broad comparison of delivery strategies followed by an analysis of membrane disruption mechanisms and the biology of the cell response. We cover mechanical, electrical, thermal, optical, and chemical strategies of membrane disruption with a particular emphasis on their applications and challenges to implementation. Throughout, we highlight specific mechanisms of membrane disruption and suggest areas in need of further experimentation. We hope the concepts discussed in our review inspire scientists and engineers with further ideas to improve intracellular delivery.

Actomyosin-dependent mitotic rounding occurs in both cell culture and tissue, where it is involved in cell positioning and epithelial organization. How actomyosin is regulated to mediate mitotic rounding is not well understood. Here we characterize the mechanics of single mitotic cells while imaging actomyosin recruitment to the cell cortex. At mitotic onset, the assembly of a uniform DIAPH1-dependent F-actin cortex coincides with initial rounding. Thereafter, cortical enrichment of F-actin remains stable while myosin II progressively accumulates at the cortex, and the amount of myosin at the cortex correlates with intracellular pressure. Whereas F-actin provides only short-term (<10 s) resistance to mechanical deformation, myosin sustains intracellular pressure for a longer duration (>60 s). Our data suggest that progressive accumulation of myosin II to the mitotic cell cortex probably requires the Cdk1 activation of both p21-activated kinases, which inhibit myosin recruitment, and of Rho kinase, which stimulates myosin recruitment to the cortex.

Nuclear transfection of DNA into mammalian cells is challenging yet critical for many biological and medical studies. Here, by combining cell squeezing and electric-field-driven transport in a device that integrates microfluidic channels with constrictions and microelectrodes, we demonstrate nuclear delivery of plasmid DNA within 1 hour after treatment, the most rapid DNA expression in a high-throughput setting (up to millions of cells per minute per device). Passing cells at high speed through microfluidic constrictions smaller than the cell diameter mechanically disrupts the cell membrane, allowing a subsequent electric field to further disrupt the nuclear envelope and drive DNA molecules into the cytoplasm and nucleus. By tracking the localization of the ESCRT-III (endosomal sorting complexes required for transport) protein CHMP4B, we show that the integrity of the nuclear envelope is recovered within 15 minutes of treatment. We also provide insight into subcellular delivery by comparing the performance of the disruption-and-field-enhanced method with those of conventional chemical, electroporation, and manual-injection systems.

The deployment of molecular to microscale carriers for intracellular delivery has tremendous potential for biology and medicine, especially for in vivo therapies. The field remains limited, however, by a poor understanding of how carriers gain access to the cell interior. In this review, we provide an overview of the different types of carriers, their speculated modes of entry, putative pathways of vesicular transport, and sites of endosomal escape. We compare this alongside pertinent examples from the cell biology of how viruses, bacteria, and their effectors enter cells and escape endosomal confinement. We anticipate insights into the mechanisms of cellular entry and endosomal escape will benefit future research efforts on effective carrier-mediated intracellular delivery. WIREs Nanomed Nanobiotechnol 2016, 8:465-478. doi: 10.1002/wnan.1377 For further resources related to this article, please visit the WIREs website.

Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells.

Despite the importance of mitotic cell rounding in tissue development and cell proliferation, there remains a paucity of approaches to investigate the mechanical robustness of cell rounding. Here we introduce ion beam-sculpted microcantilevers that enable precise force-feedback-controlled confinement of single cells while characterizing their progression through mitosis. We identify three force regimes according to the cell response: small forces (∼5 nN) that accelerate mitotic progression, intermediate forces where cells resist confinement (50-100 nN), and yield forces (>100 nN) where a significant decline in cell height impinges on microtubule spindle function, thereby inhibiting mitotic progression. Yield forces are coincident with a nonlinear drop in cell height potentiated by persistent blebbing and loss of cortical F-actin homogeneity. Our results suggest that a buildup of actomyosin-dependent cortical tension and intracellular pressure precedes mechanical failure, or herniation, of the cell cortex at the yield force. Thus, we reveal how the mechanical properties of mitotic cells and their response to external forces are linked to mitotic progression under conditions of mechanical confinement.