Genome-wide forward genetic screening to identify receptors and proteins mediating nanoparticle uptake and intracellular processing.

Understanding how cells process nanoparticles is crucial to optimize nanomedicine efficacy. However, characterizing cellular pathways is challenging, especially if non-canonical mechanisms are involved. In this Article a genome-wide forward genetic screening based on insertional mutagenesis is applied to discover receptors and proteins involved in the intracellular accumulation (uptake and intracellular processing) of silica nanoparticles. The nanoparticles are covered by a human serum corona known to target the low-density lipoprotein receptor (LDLR). By sorting cells with reduced nanoparticle accumulation and deep sequencing after each sorting, 80 enriched genes are identified. We find that, as well as LDLR, the scavenger receptor SCARB1 also mediates nanoparticle accumulation. Additionally, heparan sulfate acts as a specific nanoparticle receptor, and its role varies depending on cell and nanoparticle type. Furthermore, some of the identified targets affect nanoparticle trafficking to the lysosomes. These results show the potential of genetic screening to characterize nanoparticle pathways. Additionally, they indicate that corona-coated nanoparticles are internalized via multiple receptors.

Antiretroviral adherence and treatment outcomes among patients living with HIV at an Indonesian HIV clinic: a cross-sectional study / Cumplimiento de los antirretrovirales y resultados del tratamiento entre pacientes que viven con el VIH en una clínica de VIH de Indonesia: un estudio transversal

Objective: This study assessed antiretroviral adherence and treatment outcomes among outpatients with human immunodeficiency virus (HIV). Methods: A cross-sectional study was performed on patients with HIV over 18 years old receiving antiretroviral therapy for at least six months at an Indonesian clinic, from January to March 2021. The previously validated self-reported adherence questionnaire was used to recall antiretroviral use. Viral load and CD4 were indicators of treatment outcomes. Binary logistic regression was used to explore factors associated with nonadherence and poor treatment outcomes. Results: Ninety-five patients were included in the study (male 70.5%, median [interquartile range, IQR] age 35 [29–42] years, and median [IQR] treatment duration 29 [15–49] months). Adherence greater than 95% was observed in 89.5%, 88.4%, 95.8% of the patients in the past week, month, and three months, respectively. Patients with secondary education or lower were associated with low adherence (adjusted odds ratio, aOR: 7.73, 95%CI: 1.12– 53.19). Viral suppression and improved CD4 were observed in 83.2% and 68.4% of the patients, respectively. Taking non-nucleoside reverse transcriptase inhibitors (NNRTIs)-based regimen was associated with viral suppression (aOR: 0.01, 95%CI: 0.00–0.14) as well as high CD4 count (aOR: 0.16, 95%CI: 0.03– 0.83). Being diagnosed with stage 4 of HIV (aOR: 72.38, 95%CI: 3.11–1687.28) and having adherence of 95% or lower (aOR: 68.84, 95%CI: 4.86–974.89) were associated with non-suppressed viral load, and having HIV stage 3 (aOR: 7.81, 95%CI: 1.26–48.40) or 4 (aOR: 26.15, 95%CI: 3.42–200.10) at diagnosis was associated with low CD4. Conclusion: Rates of self-reported adherence and treatment outcomes were high. Secondary education or lower was a predictor of low adherence. Using NNRTIs-based therapy was associated with good treatment outcomes; meanwhile, stage 3 or 4 of HIV at diagnosis and low adherence were predictors of poor outcomes. Therefore, strategies to improve adherence and treatment outcomes are warranted.(AU)

Rapid Internalization of Nanoparticles by Human Cells at the Single Particle Level.

Nanoparticle uptake by cells has been studied for applications both in nanomedicine and in nanosafety. While the majority of studies have focused on the biological mechanisms underlying particle internalization, less attention has been given to questions of a more quantitative nature, such as how many nanoparticles enter cells and how rapidly they do so. To address this, we exposed human embryonic kidney cells to 40-200 nm carboxylated polystyrene nanoparticles and the particles were observed by live-cell confocal and super-resolution stimulated emission depletion fluorescence microscopy. How long a particle remained at the cell membrane after adsorbing onto it was monitored, distinguishing whether the particle ultimately desorbed again or was internalized by the cell. We found that the majority of particles desorb, but interestingly, most of the particles that are internalized do so within seconds, independently of particle size. As this is faster than typical endocytic mechanisms, we interpret this observation as the particles entering via an endocytic event that is already taking place (as opposed to directly triggering their own uptake) or possibly via an as yet uncharacterized endocytic route. Aside from the rapidly internalizing particles, a minority of particles remain at the membrane for tens of seconds to minutes before desorbing or being internalized. We also followed particles after cell internalization, observing particles that appeared to exit the cell, sometimes as rapidly as within tens of seconds. Overall, our results provide quantitative information about nanoparticle cell internalization times and early trafficking.

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.

The effect of biomolecular corona on adsorption onto and desorption from a model lipid membrane.

The current lack of insight into nanoparticle-cell membrane interactions hampers smart design strategies and thereby the development of effective nanodrugs. Quantitative and methodical approaches utilizing cell membrane models offer an opportunity to unravel particle-membrane interactions in a detailed manner under well controlled conditions. Here we use total internal reflection microscopy for real-time studies of the non-specific interactions between nanoparticles and a model cell membrane at 50 ms temporal resolution over a time course of several minutes. Maintaining a simple lipid bilayer system across conditions, adsorption and desorption were quantified as a function of biomolecular corona, particle size and fluid flow. The presence of a biomolecular corona reduced both the particle adsorption rate onto the membrane and the duration of adhesion, compared to pristine particle conditions. Particle size, on the other hand, was only observed to affect the adsorption rate. The introduction of flow reduced the number of adsorption events, but increased the residence time. Lastly, altering the composition of the membrane itself resulted in a decreased number of adsorption events onto negatively charged bilayers compared to neutral bilayers. Overall, a model membrane system offers a facile platform for real-time imaging of individual adsorption-desorption processes, revealing complex adsorption kinetics, governed by particle surface energy, size dependent interaction forces, flow and membrane composition.

Electronic Smart Blister Packages to Monitor and Support Medication Adherence: A Usability Study.

Purpose: An electronic version of the Dosepak® (EDP) which records date and time of dosing events has been developed to monitor adherence to medication packaged in blisters. This study aimed to evaluate its usability and acceptance and to monitor dose-taking adherence for optimal implementation in future clinical trials and practice. Methods: Healthy volunteers aged over 18 years were asked to dispense placebo tablets twice daily from EDPs equipped with a re-usable electronic module for a total duration of four weeks. Afterwards, subjects were asked to complete an online questionnaire and partake in a short one-on-one interview. The usability of the EDP was assessed using the System Usability Scale (SUS), while dose-taking adherence was monitored by EDP records, pill counting, and self-report. The short interview explored user experiences in more detail. Results: Twenty subjects with median [IQR] age 41.5 [32-49.8] years, 55% female, 45% healthcare professionals, and 20% chronic medication users completed the study and found the EDP easy to use, with a mean [SD] SUS score of 78.0 [11.2]. Median [IQR] dose-taking adherence was 89% [82-95%] based on EDP records, 96.5% [89-100%] based on pill counting, 92% [91-96%] based on self-report, and the levels differed significantly (p < 0.05). Four themes emerged from the interviews: user preference, experience, patient burden, and ideas for improvement. Most participants preferred smaller sized blisters. They found the EDP simple to use and did not see any patient burden for its use in trials or clinical practice. Some reported forgetfulness and suggested reminders built into the blister or sent to their mobile phones. Adequate information or instruction should also be provided for older people and polypharmacy patients. Conclusion: EDP had good perceived usability, was well accepted, and differed significantly from other adherence measurement methods. This study provides input to further guide scale-up of the blister packages.

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.

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.

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.

Single-molecule localisation microscopy: accounting for chance co-localisation between foci in bacterial cells.

Using single-molecule fluorescence microscopes, individual biomolecules can be observed within live bacterial cells. Using differently coloured probes, physical associations between two different molecular species can be assessed through co-localisation measurements. However, bacterial cells are finite and small (~ 1 µm) relative to the resolution limit of optical microscopes (~ 0.25 µm). Furthermore, the images produced by optical microscopes are typically two-dimensional projections of three-dimensional objects. These limitations mean that a certain proportion of object pairs (molecules) will inevitably be assigned as being co-localised, even when they are distant at molecular distance scales (nm). What is this proportion? Here, we attack this problem, theoretically and computationally, by creating a model of the co-localisation expected purely due to chance. We thus consider a bacterial cell wherein objects are distributed at random and evaluate the co-localisation in a fashion that emulates an experimental analysis. We consider simplified geometries where we can most transparently investigate the effect of a finite size of the cell and the effect of probing a three-dimensional cell in only two dimensions. Coupling theory to simulations, we also study the co-localisation expected due to chance using parameters relevant to bacterial cells. Overall, we show that the co-localisation expected purely due to chance can be quite substantial and describe the parameters that it depends upon.

Imaging of nanoparticle uptake and kinetics of intracellular trafficking in individual cells.

Live cell imaging is a powerful tool to understand how nano-sized objects, such as the drug carriers used for nanomedicine applications, are taken up and trafficked by cells. Here we visualized human HeLa cells as they took up and trafficked nanoparticles of different sizes and quantified nanoparticle colocalization with different fluorescently-labelled intracellular compartments over time. This allowed us to obtain kinetic profiles of nanoparticle transport towards the lysosomes in individual cells. With a simple theoretical model, we determined the typical departure time of nanoparticles from the cell membrane and typical lysosome arrival time. We compared these kinetics parameters for nanoparticles of different sizes and determined how they vary in individual cells. We also performed a similar analysis for early endocytic compartments through which nanoparticles transit and discuss challenges in quantifying the colocalization in this case. The results show a high variability in intracellular trafficking kinetics between individual cells. Additionally, they help us to understand how nanoparticle properties affect their cellular uptake and intracellular distribution kinetics.

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.

Kinetics of nanoparticle uptake into and distribution in human cells.

Whether one wishes to optimise drug delivery using nano-sized carriers or avoid hazard posed by engineered nanomaterials, the kinetics of nanoparticle uptake into human cells and their subsequent intracellular distribution is key. Unique properties of the nanoscale implies that such nanoparticles are taken up and trafficked in a different fashion compared to molecular species. In this review, we discuss in detail how to describe the kinetics of nanoparticle uptake and intracellular distribution, using previous studies for illustration. We also cover the extracellular kinetics, particle degradation, endosomal escape and cell division, ending with an outlook on the future of kinetic studies.

Asymmetry of nanoparticle inheritance upon cell division: Effect on the coefficient of variation.

Several previous studies have shown that when a cell that has taken up nanoparticles divides, the nanoparticles are inherited by the two daughter cells in an asymmetrical fashion, with one daughter cell receiving more nanoparticles than the other. This interesting observation is typically demonstrated either indirectly using mathematical modelling of high-throughput experimental data or more directly by imaging individual cells as they divide. Here we suggest that measurements of the coefficient of variation (standard deviation over mean) of the number of nanoparticles per cell over the cell population is another means of assessing the degree of asymmetry. Using simulations of an evolving cell population, we show that the coefficient of variation is sensitive to the degree of asymmetry and note its characteristic evolution in time. As the coefficient of variation is readily measurable using high-throughput techniques, this should allow a more rapid experimental assessment of the degree of asymmetry.

Time-Resolved Quantification of Nanoparticle Uptake, Distribution, and Impact in Precision-Cut Liver Slices.

Much effort within the nanosafety field is currently focused on the use of advanced in vitro models to reduce the gap between in vitro and in vivo studies. Within this context, precision-cut tissue slices are a unique ex vivo model to investigate nanoparticle impact using live tissue from laboratory animals and even humans. However, several aspects of the basic mechanisms of nanoparticle interactions with tissue have not yet been elucidated. To this end, liver slices are exposed to carboxylated and amino-modified polystyrene known to have a different impact on cells. As observed in standard cell cultures, amino-modified polystyrene nanoparticles induce apoptosis, and their impact is affected by the corona forming on their surface in biological fluids. Subsequently, a detailed time-resolved study of nanoparticle uptake and distribution in the tissue is performed, combining fluorescence imaging and flow cytometry on cells recovered after tissue digestion. As observed in vivo, the Kupffer cells accumulate high nanoparticle amounts and, interestingly, they move within the tissue towards the slice borders. Similar observations are reproduced in liver slices from human tissue. Thus, tissue slices can be used to reproduce ex vivo important features of nanoparticle outcomes in the liver and study nanoparticle impact on real tissue.

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.

Reciprocal upregulation of scavenger receptors complicates interpretation of nanoparticle uptake in non-phagocytic cells.

Nanoparticles have great potential as drug delivery vehicles or as imaging agents for treatment and diagnosis of various diseases. It is therefore crucial to understand how nanoparticles are taken up by cells, both phagocytic and non-phagocytic. Small interference RNA has previously been used to isolate the effect of particular receptors in nanoparticle uptake by silencing their expression. Here we show that, when it comes to receptors with overlapping function, interpretation of such data has to be done with caution. We followed the uptake of silica nanoparticles by scavenger receptors in A549 lung epithelial cells. While we successfully knocked-down gene expression of several different receptors within the scavenger receptor family (SR-A1, MARCO, SR-BI, LOX-1 and LDLR) this caused reciprocal up and down regulation of the other scavenger receptors. Subsequent nanoparticle uptake experiments in silenced cells exhibit a complex behaviour, which could easily be misinterpreted if reciprocal regulation is not considered. Preliminary identification of the actual scavenger receptors involved can be found by disentangling the effects mathematically. Finally, we show that the effects are still present under more realistic biological conditions, namely at higher serum concentrations.

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 Förster 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 coli are 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 macromolecular volume 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.

Spatial and Structural Metrics for Living Cells Inspired by Statistical Mechanics.

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.