Glass-Like Characteristics of Intracellular Motion in Human Cells

The motion in the cytosol of microorganisms such as bacteria and yeast has been observed to undergo a dramatic slowing down upon cell energy depletion. These observations have been interpreted as the motion being “glassy,” but whether this notion is useful also for active, motor-protein-driven transport in eukaryotic cells is less clear. Here, we use fluorescence microscopy of beads in human (HeLa) cells to probe the motion of membrane-surrounded structures that are carried along the cytoskeleton by motor proteins. Evaluating several hallmarks of glassy dynamics, we show that at short length scales, the motion is heterogeneous, is nonergodic, is well described by a model for the displacement distribution in glassy systems, and exhibits a decoupling of the exchange and persistence times. Overall, these results suggest that the short length scale behavior of objects that can be transported actively by motor proteins in human cells shares features with the motion in glassy systems

Simultaneous Exposure of Different Nanoparticles Influences Cell Uptake

Drug delivery using nano-sized carriers holds tremendous potential for curing a range of diseases. The internalisation of nanoparticles by cells, however, remains poorly understood, restricting the possibility for optimising entrance into target cells, avoiding off-target cells and evading clearance. The majority of nanoparticle cell uptake studies have been performed in the presence of only the particle of interest; here, we instead report measurements of uptake when the cells are exposed to two different types of nanoparticles at the same time. We used carboxylated polystyrene nanoparticles of two different sizes as a model system and exposed them to HeLa cells in the presence of a biomolecular corona. Using flow cytometry, we quantify the uptake at both average and individual cell level. Consistent with previous literature, we show that uptake of the larger particles is impeded in the presence of competing smaller particles and, conversely, that uptake of the smaller particles is promoted by competing larger particles. While the mechanism(s) underlying these observations remain(s) undetermined, we are partly able to restrain the likely possibilities. In the future, these effects could conceivably be used to enhance uptake of nano-sized particles used for drug delivery, by administering two different types of particles at the same time

Extending the analogy between intracellular motion in mammalian cells and glassy dynamics

How molecules, organelles, and foreign objects move within living cells has been studied in organisms ranging from bacteria to human cells. In mammalian cells, in particular, cellular vesicles move across the cell using motor proteins that carry the vesicle down the cytoskeleton to their destination. We have recently noted several similarities between the motion of such vesicles and that in disordered, “glassy”, systems, but the generality of this observation remains unclear. Here we follow the motion of mitochondria, the organelles responsible for cell energy production, in mammalian cells over timescales from 50 ms to 70 s. Qualitative observations show that single mitochondria remain within a spatially limited region for extended periods of time, before moving longer distances relatively quickly. The displacement distribution is roughly Gaussian for shorter distances (≲ 0.05 µm) but exhibits exponentially decaying tails at longer distances (up to 0.40 µm). This behaviour is well-described by a model developed to describe the motion in glassy systems. These observations are extended to in total 3 different objects (mitochondria, lysosomes and nano-sized beads enclosed in vesicles), 3 different mammalian cell types (HEK 293, HeLa, and HT22), from 2 different organisms (human and mouse). Further evidence that supports glass-like characteristics of the motion is a difference between the time it takes to move a longer distance for the first time and subsequent times, as well as a weak ergodicity breaking of the motion. Overall, we demonstrate the ubiquity of glass-like motion in mammalian cells, providing a different perspective on intracellular motion

Sources of Variability in Nanoparticle Uptake by Cells

Understanding how nano-sized objects are taken up by cells is important for applications within medicine (nanomedicine), as well as to avoid unforeseen hazard due to nanotechnology (nanosafety). Even within the same cell population, one typically observes a large cell-to-cell variability in nanoparticle uptake, raising the question of the underlying cause(s). Here we investigate cell-to-cell variability in polystyrene nanoparticle uptake by HeLa cells, with generalisations of the results to silica nanoparticles and liposomes, as well as to A549 and primary human umbilical vein endothelial cells. We show that uptake of nanoparticles is correlated with cell size within a cell population, thereby reproducing and generalising previous reports highlighting the role of cell size in nanoparticle uptake. By repeatedly isolating (using fluorescence-activated cell sorting) the cells that take up the most and least nanoparticles, respectively, and performing RNA sequencing on these cells separately, we examine the underlying gene expression that contributes to high and low polystyrene nanoparticle accumulation in HeLa cells. We can thereby show that cell size is not the sole driver of cell-to-cell variability, but that other cellular characteristics also play a role. In contrast to cell size, these characteristics are more specific to the object (nanoparticle or protein) being taken up, but are nevertheless highly heterogeneous, complicating their detailed identification. Overall, our results highlight the complexity underlying the cellular features that determine nanoparticle uptake propensity

Drug Transport in Responding Lipid Membranes Can Be Regulated by an External Osmotic Gradient

In this paper, we demonstrate, for the first time, how an external osmotic gradient can be used to regulate diffusion of solutes across a lipid membrane. We present experimental and theoretical studies of the transport of different solutes across a monoolein membrane in the presence of an external osmotic gradient. The osmotic gradient introduces phase transformations in the membrane, and it causes nonlinear transport behavior. The external gradient can thus act as a kind of switch for diffusive transport in the skin and in controlled release drug formulations

Spatial and structural metrics for living cells inspired by statistical mechanics

Funding from the Irish Research Council for Science, Engineering and Technology (C.Å.); Science Foundation Ireland, 09/RFP/MTR2425 (J.A.V.; C.Å.) and 12/IA/1422 (K.A.D.); the European Union Seventh Framework Programme project NanoTransKinetics, grant agreement no. 266737 (C.Å.) and FutureNanoNeeds grant agreement no. 604602 (K.A.D.); and the Irish Research Council (L.W.F.) is gratefully acknowledged.Experimental observations in cell biology have advanced to a stage where theory could play a larger role, much as it has done in the physical sciences. Possibly the lack of a common framework within which experimentalists, computational scientists and theorists could equally contribute has hindered this development, for the worse of both disciplines. Here we demonstrate the usage of tools and concepts from statistical mechanics to describe processes inside living cells based on experimental data, suggesting that future theoretical/computational models may be based on such concepts. To illustrate the ideas, we describe the organisation of subcellular structures within the cell in terms of (density) pair correlation functions, and subsequently use the same concepts to follow nano-sized objects being transported inside the cell. Finally, we quantify an interesting subcellular re-organisation, not previously discerned by molecular biology methods.Publisher PDFPeer reviewe

Clinical Value of Emerging Bioanalytical Methods for Drug Measurements:A Scoping Review of Their Applicability for Medication Adherence and Therapeutic Drug Monitoring

INTRODUCTION: Direct quantification of drug concentrations allows for medication adherence monitoring (MAM) and therapeutic drug monitoring (TDM). Multiple less invasive methods have been developed in recent years: dried blood spots (DBS), saliva, and hair analyses. AIM: To provide an overview of emerging drug quantification methods for MAM and TDM, focusing on the clinical validation of methods in patients prescribed chronic drug therapies. METHODS: A scoping review was performed using a systematic search in three electronic databases covering the period 2000-2020. Screening and inclusion were performed by two independent reviewers in Rayyan. Data from the articles were aggregated in a REDCap database. The main outcome was clinical validity of methods based on study sample size, means of cross-validation, and method description. Outcomes were reported by matrix, therapeutic area and application (MAM and/or TDM). RESULTS: A total of 4590 studies were identified and 175 articles were finally included; 57 on DBS, 66 on saliva and 55 on hair analyses. Most reports were in the fields of neurological diseases (37%), infectious diseases (31%), and transplantation (14%). An overview of clinical validation was generated of all measured drugs. A total of 62 drugs assays were applied for MAM and 131 for TDM. CONCLUSION: MAM and TDM are increasingly possible without traditional invasive blood sampling: the strengths and limitations of DBS, saliva, and hair differ, but all have potential for valid and more convenient drug monitoring. To strengthen the quality and comparability of future evidence, standardisation of the clinical validation of the methods is recommended

Design and Properties of Genetically Encoded Probes for Sensing Macromolecular Crowding

Cells are highly crowded with proteins and polynucleotides. Any reaction that depends on the available volume can be affected by macromolecular crowding, but the effects of crowding in cells are complex and difficult to track. Here, we present a set of Forster resonance energy transfer (FRET)-based crowding-sensitive probes and investigate the role of the linker design. We investigate the sensors in vitro and in vivo and by molecular dynamics simulations. We find that in vitro all the probes can be compressed by crowding, with a magnitude that increases with the probe size, the crowder concentration, and the crowder size. We capture the role of the linker in a heuristic scaling model, and we find that compression is a function of size of the probe and volume fraction of the crowder. The FRET changes observed in Escherichia collare more complicated, where FRET increases and scaling behavior are observed solely with probes that contain the helices in the linker. The probe with the highest sensitivity to crowding in vivo yields the same macromolecularvolume fractions as previously obtained from cell dry weight. The collection of new probes provides more detailed readouts on the macromolecular crowding than a single sensor

Low uptake of silica nanoparticles in Caco-2 intestinal epithelial barriers

Cellular barriers, such as the skin, the lung epithelium or the intestinal epithelium, constitute one of the first obstacles facing nanomedicines or other nanoparticles entering organisms. It is thus important to assess the capacity of nanoparticles to enter and transport across such barriers. In this work, Caco-2 intestinal epithelial cells were used as a well-established model for the intestinal barrier, and the uptake, trafficking and translocation of model silica nanoparticles of different sizes were investigated using a combination of imaging, flow cytometry and transport studies. Compared to typical observations in standard cell lines commonly used for in vitro studies, silica nanoparticle uptake into well-developed Caco-2 cellular barriers was found to be very low. Instead, nanoparticle association to the apical outer membrane was substantial and these particles could easily be misinterpreted as internalised in the absence of imaging. Passage of nanoparticles through the barrier was very limited, suggesting that the low amount of internalised nanoparticles was due to reduced uptake into cells, rather than a considerable transport through them