Intracellular delivery by membrane disruption: Mechanisms, strategies, and concepts

© 2018 American Chemical Society. 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 typesñYsmall 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

Cell compliance: cytoskeletal origin and importance for cellular function.

This thesis was released from embargo in July 2012Mechanical properties of cells, mainly defined by their cytoskeleton, are closely related to cell function and can be measured with a dual-beam laser trap (optical stretcher). Functional changes, which go hand in hand with changes of the cytoskeleton, also occur during differentiation of stem cells. This suggests monitoring differentiation by the changing compliance of the cells. During the course of my PhD I measured the compliance of three different types of stem cells before and after differentiation and was able to detect differences in some of the cell types. In order to relate rheological experiments to cell migration as a further example of functional change I investigated the migration behavior of cells that showed different compliance and found differences in migration. I was additionally able to show an altered migration behavior after I actively changed the mechanical behavior of one cell type using cytoskeletal drugs. These migration experiments have been carried out in 2D and 3D migration assays. Furthermore, the influence of the stiffness of the surrounding material on the migration behavior has been investigated. After relating functional changes to changes in compliance, I studied which mechanisms can be used to actually influence cell compliance and investigated the effect of cytoskeletal stabilizers or destabilizers as well as drugs acting on molecular motors. The effect of the surrounding temperature has been considered as well. Finally, I developed a new version of the optical stretcher measurement tool, which enables cell sorting and drug screening using a monolithic glass chip. With the results presented in this thesis I relate mechanical compliance to the cytoskeleton and specific cellular functions. I deliver insights how mechanical changes in cells can be used to identify and follow functional changes and how this knowledge can help to interfere with such functions, specifically in pathologies correlated to these functions. My modified optical stretcher would be developed to screen the effects of drugs on cell compliance and to sort cells with different mechanical properties. Such drug screening and cell sorting will offer diagnostic treatment options for various pathologies.This work was supported by the Gates Scholarship Cambridge

SURFACE ENABLED LAB-ON-A-CHIP (LOC) DEVICE FOR PROTEIN DETECTION AND SEPARATION

Sensitive and selective chemical/biological detection/analysis for proteins is essential for applications such as disease diagnosis, species phenotype identification, product quality control, and sample examination. Lab-on-a-chip (LOC) device provides advantages of fast analysis, reduced amount of sample requirements, and low cost, to magnificently facilitate protein detection research. Isoelectric focusing (IEF) is a strong and reliable electrophoretic technique capable of discerning proteins from complex mixtures based on the isoelectric point (pI) differences. It has experienced plenty of fruitful developments during previous decades which has given it the capability of performing with highly robust and reproducible analysis. This progress has made IEF devices an excellent tool for chemical/biological detection/analysis purposes. In recent years, the trends of simple instrument setting, rapid analysis, small sample requirement, and light labor intensity have inspired the LOC concept to be combined with IEF to evolve it into an “easily-handled chip with hours of analysis” from the earlier method of “working with big and heavy machines in a few days.” Although IEF is already a mature technique being applied, further LOC-IEF developments are still experiencing challenges related to its limitations such as miniaturizing the device scale without harming the resolving/discerning ability. With the facilitation of newly technologically advanced/improved fabrication tools, it is completely possible to address challenges and approach new limits of LOC-IEF. In this dissertation, a surface enabled printing technique, which can transfer liquid to a surface with prescribed patterns, was firstly introduced to IEF device fabrication. By employing surface enabled printing, a surface enabled IEF (sIEF) device running at a scale of 100 times smaller than those previously reported was designed and fabricated. Commercial carrier ampholytes (PharmalyteTM) with different pH range were engaged to generate a continuous pH gradient on sIEF device. Device design and optimized fabrication conditions were practically investigated; establishment of pH gradient was verified by fluorescent dyes; dependencies of electric field strength and carrier ampholytes concentration were systematically examined. To further optimize the sIEF system, dependencies of surface treatment and additive chemicals were explored. Fluorescent proteins and peptides were tested for the separation capability of sIEF. Finally, the well optimized sIEF system was used as a tool for real protein (hemoglobin variants and monoclonal antibody isoforms) separations. Hemoglobin variants test results revealed that sIEF is capable of separating amphoteric species with pI difference as small as 0.2. Monoclonal protein tests demonstrated the capability of sIEF to be a ready-to-use tool for protein structural change monitoring. In conclusion, this new sIEF approach has lower applied voltages, smaller sample requirements, a relatively quick fabrication process, and reusability, making it more attractive as a portable, user-friendly platform for qualitative protein detection and separation

Soft Tactile Sensors for Mechanical Imaging

Tactile sensing aims to electronically capture physical attributes of an object via mechanical contact. It proves indispensable to many engineering tasks and systems, in areas ranging from manufacturing to medicine and autonomous robotics. Biological skin, which is highly compliant, is able to perform sensing under challenging and highly variable conditions with levels of performance that far exceed what is possible with conventional tactile sensors, which are normally fabricated with non-conforming materials. The development of stretchable, skin-like tactile sensors has, as a result, remained a longstanding goal of engineering. However, to date, artificial tactile sensors that might mimic both the mechanical and multimodal tactile sensory capabilities of biological skin remain far from realization, due to the challenges of fabricating spatially dense, mechanically robust, and compliant sensors in elastic media. Inspired by these demands, this dissertation addresses many aspects of the challenging problem of engineering skin-like electronic sensors. In the first part of the thesis, new methods for the design and fabrication of thin, highly deformable, high resolution tactile sensors are presented. The approach is based on a novel configuration of arrays of microfluidic channels embedded in thin elastomer membranes. To form electrodes, these channels are filled with a metal alloy, eutectic Gallium Indium, that remains liquid at room temperature. Using capacitance sensing techniques, this approach achieves sensing resolutions of 1 mm$^{-1}$. To fabricate these devices, an efficient and robust soft lithography method is introduced, based on a single step cast. An analytical model for the performance of these devices is derived from electrostatic theory and continuum mechanics, and is demonstrated to yield excellent agreement with measured performance. This part of the investigation identified fundamental limitations, in the form of nonmonotonic behavior at low strains, that is demonstrated to generically affect solid cast soft capacitive sensors. The next part of the thesis is an investigation of new methods for designing soft tactile sensors based on multi-layer heterogeneous 3D structures that combine active layers, containing embedded liquid metal electrodes, with passive and mechanically tunable layers, containing air cavities and micropillar geometric supports. In tandem with analytical and computational modeling, these methods are demonstrated to facilitate greater control over mechanical and electronic performance. A new soft lithography fabrication method is also presented, based on the casting, alignment, and fusion of multiple functional layers in a soft polymer substrate. Measurements indicate that the resulting devices achieve excellent performance specifications, and avoid the limiting nonmonotonic behavior identified in the first part of the thesis. In order to demonstrate the practical utility of the devices, we used them to perform dynamic two-dimensional tactile imaging under distributed indentation loads. The results reflect the excellent static and dynamic performance of these devices. The final part of the thesis investigates the utility of the tactile sensing methods pursued here for imaging lumps embedded in simulated tissue. In order to facilitate real-time sensing, an electronic system for fast, array based measurement of small, sub-picofarad (pF) capacitance levels was developed. Using this system, we demonstrated that it is possible to accurately capture strain images depicting small lumps embedded in simulated tissue with either an electronic imaging system or a sensor worn on the finger, supporting the viability of wearable sensors for tactile imaging in medicine. In conclusion, this dissertation confronts many of the most vexing problems arising in the pursuit of skin-like electronic sensors, including fundamental operating principles, structural and functional electronic design, mechanical and electronic modeling, fabrication, and applications to biomedical imaging. The thesis also contributes knowledge needed to enable applications of tactile sensing in medicine, an area that has served as a key source of motivation for this work, and aims to facilitate other applications in areas such as manufacturing, robotics, and consumer electronics.Ph.D., Electrical Engineering -- Drexel University, 201

A Biomimetic Approach toward Red Blood Cell Substitutes Based on PRINT Hydrogels

This work utilized PRINT (particle replication in non-wetting templates) technology to fabricate extremely soft, biologically inspired hydrogel particles that mimicked the size, shape and modulus of red blood cells (RBCs). Hemoglobin, the oxygen carrying protein in RBCs, was conjugated into these microparticles without adverse effect on the structure and function of the protein. A prior modification on surface of PRINT particles followed by hemoglobin conjugation enabled the protein-laden microparticles to circulate in blood. The results of this study can potentially lead to a RBC substitute for blood transfusion without causing vasoconstriction, a major hurdle often seen in other hemoglobin-based oxygen carriers (HBOCs). Vasoconstriction is believed to be inversely proportional to the size of the HBOC. Microparticles with size around or larger than 1 μm may be appropriate as hemoglobin carriers to minimize vasoconstriction, yet they generally do not circulate well in blood vessels due to filtration by small capillaries. Our previous study demonstrated that microparticles with a diameter of 6 μm could still circulate a long time in blood when they were made to be deformable enough. Retaining the same low modulus, hydrogel particles with diameters ranging from 0.8 to 8.9 μm were studied on their pharmacokinetics and biodistribution in mice. The particles mimicking size of RBCs demonstrated longer circulation times, hence were used as carriers for hemoglobin in this study. Bovine hemoglobin could be conjugated to the RBC mimicking particles (RBCMs) through reaction between carboxyl groups in the particles and amine groups on hemoglobin. However, hemoglobin distributed on the surface of the RBCMs made them tend to aggregate in blood and more recognizable by macrophages, resulting in rapid removal from circulation. A strategy was used to synthesize blank RBCMs with such an asymmetric distribution of carboxyl groups that most of them were in the interior with limited exposure on the exterior. After conjugation, hemoglobin could be predominantly confined in the interior of the particles with a neutral surface charge. These particles could circulate in blood with much lower accumulation in the lung than the counterparts with hemoglobin on their surface.Doctor of Philosoph

Impedance-Based Analysis of the Cellular Response to Microparticles: Theory, Assay Development and Model Study

This thesis provides a model study on the information content of multimodal impedance-based assays to assess the impact of microscale particles on cell physiology of mammalian cells. In three main chapters, different approaches to this topic are presented and discussed: The first chapter focused on the simulation of several scenarios within cell-based assays. These simulations are all based on the so-called ECIS model, originally introduced by Giaever and Keese (1991), describing the impedance contribution of cell-covered gold-film electrodes. This theoretical part of the thesis should help to support the interpretation of impedance data. First, opening and closing of cell junctions (Rb) for different types of barrierforming cell layers were simulated and the accompanying changes in the complex impedance were extracted at various frequencies. The simulation data for some model epithelial and endothelial cell types showed that the relationship between resistance and barrier tightness may undergo inversion for frequencies above the cell-type specific threshold. Moreover, the influence of incomplete electrode coverage or inhomogeneity within the cell layer was studied systematically. For all experiments a good correlation between the simulated data and the experimental support was found. The aim of the second project was to establish a new opto-electrical assay to investigate the dye transfer via gap junctions into neighboring cells. The principle of this new assay was based on loading a selected cell population with Lucifer Yellow by in situ electroporation. The cell-type specific adjustment of the ac pulse parameters for a temporary permeabilization of the plasma membrane improved the incorporation of Lucifer Yellow into the cytoplasm without affecting NRK cell viability. The assay also required the optimization of the gold-film electrode layout which enabled the application of the ac pulses, the non-invasive impedance recordings before and after pulse application and the microscopic analysis of dye transfer from cells on the electrode into adjacent cells. The final electrode layout (8W4E-GJ) contained four “semi-elliptical” electrodes which were separated by a photopolymer-free gap to facilitate microscopic analysis without any interference from the red autofluorescence of the photopolymer. The development of an appropriate experimental protocol yielded on electroporation in Ca2+-free buffer and the application of two sequential ac pulses, as it was found to enhance the uptake efficiency into primary-loaded NRK cells. The opto-electrical assay was successfully applied to analyze the effect of the well-known gap junctional intercellular communication inhibitor 2-APB. The analysis of dye transfer via gap junctions was based on confocal fluorescence micrographs documenting dye transfer from the electrode into the photopolymer-free gap. The analysis was further improved by the application of the red-fluorescent TRITC dextran as co-electroporated reference dye, which was trapped in the cytoplasm of primary-loaded cells due to its molecular size. The image analysis of the position-dependent intensities of both dyes (TRITC dextran and Lucifer Yellow) allowed a quantification of gap junctional intercellular communication. The third chapter contains all sub-projects dealing with a multimodal and label-free analysis of the impact of micrometer-sized silica particles (Ø = 2 μm) on vitality, migration, proliferation and gap junctional intercellular communication of adherent NRK cells in vitro. A sequence of different impedimetric assays, all based on the well-established ECIS technique, was applied for the analysis of particle impact on cell physiology. Microscopic studies addressing the particle uptake revealed the presence of membrane-coated particles in the cytoplasm of NRK cells. Further evidence for particle uptake was gathered from ToFSIMS analysis that showed a densely-packed particle distribution around the cell nucleus in cells with intact plasma membranes. Time-resolved ECIS measurements revealed no acute cytotoxicity of silica particles as well as no influence on cell migration. Furthermore, the influence of silica particles on NRK proliferation was studied impedimetrically. No differences in the time-course of proliferation were found for particle-loaded or control cells. To study the influence of internalized particles on gap junctional intercellular communication the new optoelectrical assay was applied. Dye transfer to NRK cells in the periphery of the electrode was insignificantly different in absence and presence of silica particles. The results were supported by classical techniques, like FRAP analysis, scrape loading or parachute assay. Superior to other assays, the developed opto-electrical assay allowed for analysis of cell adhesion and cellular response to the presence of particle during an exposure time of 24 h prior to the dye transfer study. This enables the investigation of the impact of internalized particles on different cell-related parameters like viability, motility and gap junctional intercellular communication within one cell population