Intracellular delivery to mammalian cells has become critical to both fundamental research applications and cell-based therapies. In this Review, Klavs Jensen and colleagues discuss recent work on developing in vitro and ex vivo strategies for cytosolic delivery of macromolecules, concentrating on membrane-disruption-based methods and the use of nanotechnology, microfluidics and laboratory-on-a-chip technology.

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In vitro and ex vivo strategies for intracellular delivery.

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 a...

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Intracellular Delivery by Membrane Disruption: Mechanisms, Strategies, and Concepts.

Untethered mobile microrobots have the potential to leverage minimally invasive theranostic functions precisely and efficiently in hard-to-reach, confined, and delicate inner body sites. However, such a complex task requires an integrated design and engineering, where powering, control, environmental sensing, medical functionality, and biodegradability need to be considered altogether. The present study reports a hydrogel-based, magnetically powered and controlled, enzymatically degradable microswimmer, which is responsive to the pathological markers in its microenvironment for theranostic cargo delivery and release tasks. We design a double-helical architecture enabling volumetric cargo loading and swimming capabilities under rotational magnetic fields and a 3D-printed optimized 3D microswimmer (length = 20 μm and diameter = 6 μm) using two-photon polymerization from a magnetic precursor suspension composed from gelatin methacryloyl and biofunctionalized superparamagnetic iron oxide nanoparticles. At nor...

3D-Printed Biodegradable Microswimmer for Theranostic Cargo Delivery and Release.

Mapping brain activity has received growing worldwide interest because it is expected to improve disease treatment and allow for the development of important neuromorphic computational methods. MEMS and microsystems are expected to continue to offer new and exciting solutions to meet the need for high-density, high-fidelity neural interfaces. Herein, the state-of-the-art in recording and stimulation tools for brain research is reviewed, and some of the most significant technology trends shaping the field of neurotechnology are discussed. Understanding even the most basic brain functions will require considerable advances in the MEMS-based tools that are used in brain research. Sensors that are capable of monitoring single neurons or mapping the complex neural networks responsible for faculties such as memory or learning will be crucial for furthering our knowledge. As the human brain contains around 85 million neurons and 100 trillion synapses, the challenge is enormous. John Seymour and Euisik Yoon and colleagues at the University of Michigan, United States, review the state of the art in microsystem devices that are used to record and stimulate the brain. They highlight innovations in multimodal sensor arrays and illustrate the need for further innovation in packaging and microsystems to match the scale of the neuronal circuits under study. Ultimately the teamwork between neurotechnologists and neuroscientists will lead to critical breakthroughs in brain research over the next decade.

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State-of-the-art MEMS and microsystem tools for brain research

In nature, various types of animals will form self-organised large-scale structures. Through designing wireless actuation methods, microrobots can emulate natural swarm behaviours, which have drawn extensive attention due to their great potential in biomedical applications. However, as the prerequisite for their in-vivo applications, whether microrobotic swarms can take effect in bio-fluids with complex components has yet to be fully investigated. In this work, we first categorise magnetic active swarms into three types, and individually investigate the generation and navigation behaviours of two types of the swarms in bio-fluids. The influences of viscosities, ionic strengths and mesh-like structures are studied. A strategy is then proposed to select the optimised swarms in different fluidic environments based on their physical properties, and the results are further validated in various bio-fluids. Moreover, we also realise the swarm generation and navigation in bovine eyeballs, which also validates the proposed prediction in the ex-vivo environment. Although microrobotic swarms are attractive for biomedical applications, controlling their collective movement in specific bio-fluids is difficult. Here, the authors report navigated locomotion and collective behaviors of magnetic field- and medium-induced micro-robotic swarms in various bio-fluids.

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Active generation and magnetic actuation of microrobotic swarms in bio-fluids.

A long-standing question in natural reproduction is how mammalian sperm navigate inside female reproductive tract and finally reach the egg cell, or oocyte. Recently, fluid flow was proposed as a long-range guidance cue for sperm navigation. Coitus induces fluid flow from oviduct to uterus, and sperm align themselves against the flow direction and swim upstream, a phenomenon termed rheotaxis. Whether sperm rheotaxis is a passive process dominated by fluid mechanics, or sperm actively sense and adapt to fluid flow remains controversial. Here we report the first quantitative study of sperm flagellar motion during human sperm rheotaxis and provide direct evidence indicating that sperm rheotaxis is a passive process. Experimental results show that there is no significant difference in flagellar beating amplitude and asymmetry between rheotaxis-turning sperm and those sperm swimming freely in the absence of fluid flow. Additionally, fluorescence image tracking shows no Ca(2+) influx during sperm rheotaxis turning, further suggesting there is no active signal transduction during human sperm rheotaxis.

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Human sperm rheotaxis: a passive physical process.

The capability to directly interrogate intracellular structures inside a single cell for measurement and manipulation is important for understanding subcellular and suborganelle activities, diagnosing diseases, and developing new therapeutic approaches. Compared with measurements of single cells, physical measurement and manipulation of subcellular structures and organelles remain underexplored. To improve intracellular physical measurement and manipulation, we have developed a multipole magnetic tweezers system for micromanipulation involving submicrometer position control and piconewton force control of a submicrometer magnetic bead inside a single cell for measurement in different locations (spatial) and different time points (temporal). The bead was three-dimensionally positioned in the cell using a generalized predictive controller that addresses the control challenge caused by the low bandwidth of visual feedback from high-resolution confocal imaging. The average positioning error was quantified to be 0.4 μm, slightly larger than the Brownian motion-imposed constraint (0.31 μm). The system is also capable of applying a force up to 60 pN with a resolution of 4 pN for a period of time longer than 30 min. The measurement results revealed that significantly higher stiffness exists in the nucleus' major axis than in the minor axis. This stiffness polarity is likely attributed to the aligned actin filament. We also showed that the nucleus stiffens upon the application of an intracellularly applied force, which can be attributed to the response of structural protein lamin A/C and the intracellular stress fiber actin filaments.

Intracellular manipulation and measurement with multipole magnetic tweezers

Robotic micromanipulation is a relatively young field. However, after three decades of development and evolution, the fundamental physics; techniques for sensing, actuation, and control; tool sets ...

Robotic Micromanipulation: Fundamentals and Applications

Properties of organelles and intracellular structures play important roles in regulating cellular functions, such as gene expression, cell motility and metabolism. The ability to directly interrogate intracellular structures inside a single cell for measurement and manipulation has significant implications in the understanding of subcellular and suborganelle activities, diagnosing diseases, and potentially developing new therapeutic approaches. In the past few decades, a number of technologies have been developed to study single-cell properties. However, methods of measuring intracellular properties and manipulating subcellular structures have been largely underexplored. Due to the even smaller size of intracellular targets and lower signal-to-noise ratio than that in whole-cell studies, the development of tools for intracellular measurement and manipulation is challenging. This paper reviews emerging microsystems and nanoengineered technologies for sensing and quantitative measurement of intracellular properties and for manipulating structures inside a single cell. Recent progress and limitations of these new technologies as well as new discoveries and prospects are discussed. Measuring and manipulating biological processes in the tiny compartments inside a cell is crucial for understanding their function. Much can go wrong if the localization of intracellular events is disturbed: for example, cardiac arrhythmia can arise when electrical currents spread incorrectly among cells in the heart. Yu Sun and colleagues at the University of Toronto, Canada, review emerging methods for conducting precise measurements and manipulation inside cells. They describe tools that comprise a sensor attached to a device outside the cell, such as nanowires for recording electrical activity. They also discuss unattached probes that are delivered into the cell, such as nanoparticles for temperature and pH sensing. The researchers conclude that improvements in the spatial and temporal resolution of these measurements will provide fresh insight into how the intracellular environment affects the health of cells.

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Voyage inside the cell: Microsystems and nanoengineering for intracellular measurement and manipulation

From single-pole magnetic tweezers to robotic magnetic-field generation systems, the development of magnetic micromanipulation systems, using electromagnets or permanent magnets, has enabled a multitude of applications for cellular and intracellular measurement and stimulation. Controlled by different configurations of magnetic-field generation systems, magnetic particles have been actuated by an external magnetic field to exert forces/torques and perform mechanical measurements on the cell membrane, cytoplasm, cytoskeleton, nucleus, intracellular motors, etc. The particles have also been controlled to generate aggregations to trigger cell signaling pathways and produce heat to cause cancer cell apoptosis for hyperthermia treatment. Magnetic micromanipulation has become an important tool in the repertoire of toolsets for cell measurement and stimulation and will continue to be used widely for further explorations of cellular/intracellular structures and their functions. Existing review papers in the literature focus on fabrication and position control of magnetic particles/structures (often termed micronanorobots) and the synthesis and functionalization of magnetic particles. Differently, this paper reviews the principles and systems of magnetic micromanipulation specifically for cellular and intracellular measurement and stimulation. Discoveries enabled by magnetic measurement and stimulation of cellular and intracellular structures are also summarized. This paper ends with discussions on future opportunities and challenges of magnetic micromanipulation in the exploration of cellular biophysics, mechanotransduction, and disease therapeutics.

Zhuoran Zhang

Papers

Human sperm rheotaxis: a passive physical process.

Intracellular manipulation and measurement with multipole magnetic tweezers

Robotic Micromanipulation: Fundamentals and Applications

Voyage inside the cell: Microsystems and nanoengineering for intracellular measurement and manipulation

Magnetic Measurement and Stimulation of Cellular and Intracellular Structures.