Sulfobetaine-Phosphonate Block Copolymer Coated Iron Oxide Nanoparticles for Genomic Locus Targeting and Magnetic Micromanipulation in the Nucleus of Living Cells.

Exerting forces on biomolecules inside living cells would allow us to probe their dynamic interactions in their native environment. Magnetic iron oxide nanoparticles represent a unique tool capable of pulling on biomolecules with the application of an external magnetic field gradient; however, their use has been restricted to biomolecules accessible from the extracellular medium. Targeting intracellular biomolecules represents an additional challenge due to potential nonspecific interactions with cytoplasmic or nuclear components. We present the synthesis of sulfobetaine-phosphonate block copolymer ligands, which provide magnetic nanoparticles that are stealthy and targetable in living cells. We demonstrate, for the first time, their efficient targeting in the nucleus and their use for magnetic micromanipulation of a specific genomic locus in living cells. We believe that these stable and sensitive magnetic nanoprobes represent a promising tool to manipulate specific biomolecules in living cells and probe the mechanical properties of living matter at the molecular scale.

Multiomic Droplet-Based Assay for Ultralow Input Samples.

The extraction and separation of cellular compounds are crucial steps in numerous biological protocols, particularly in multiomics studies, where several cellular modalities are examined simultaneously. While magnetic particle extraction is commonly used, it may not be applicable for ultralow input samples. Microfluidics has made possible the analysis of rare or low-materiality samples such as circulating tumor cells or single cells through miniaturization of numerous protocols. In this study, a microfluidics workflow for separating different cellular modalities from ultralow input samples is presented. This approach is based on magnetic tweezers technology, allowing the extraction and resuspension of magnetic particles between consecutive nanoliter droplets to perform multistep assays on small volumes. The ability to separate and recover mRNA and gDNA in samples containing less than 10 cells is demonstrated, achieving separation efficiency comparable to the one obtained with conventional pipetting but with a significantly lower amount of starting material, typically 1-2 orders of magnitude less.

A continuum model for magnetic particle flows in microfluidics applicable from dilute to packed suspensions.

The manipulation of magnetic microparticles has always been pivotal in the development of microfluidic devices, as it encompasses a broad range of applications, such as drug delivery, bioanalysis, on-chip diagnostics, and more recently organ-on-chip development. However, predicting the behavior and trajectory of these particles remains a recurring and partly unresolved question. Magnetic particle-laden flows can display intricate collective behaviors, such as packed plugs, column-shaped aggregates, or fluidization, which are difficult to predict. In this study, we introduce a finite-element model to simulate highly dense flows of magnetic microparticles. Our method relies on an interpenetrating continuum approach, where both the liquid and particle phases are described by the Navier-Stokes equations, in which the magnetic force, interphase friction, and interparticle forces were included. We demonstrate its applicability across the entire range of particle packing densities and compare the results with experimental data from real microfluidic application cases. The model successfully replicates complex behaviors, such as particle aggregation, plug formation and fluidization. This approach has potential to accelerate microfluidic device development by reducing the need for costly and time-consuming experimental optimization.

Magnetic Microtweezers for High-Throughput Bioseparation in Sub-Nanoliter Droplets.

Multiomics studies at single-cell level require small volume manipulation, high throughput analysis, and multiplexed detection, characteristics that droplet microfluidics can tackle. However, the initial step of molecule bioseparation remains challenging. Here, we describe a unique magnetic device to trap and extract magnetic particles in sub-nanoliter droplets, for compartmentalisation of detection steps. Relying on electrodeposition of NiFe structures and microfluidic manipulation, the extraction of 1 µm diameter magnetic particles was achieved at high throughput (20 droplets per second) with an efficiency close to 100% in 450 pL droplets. The first demonstration of its adaptability to single-cell analysis is demonstrated with the extraction of mRNA. Using a purified nucleic acid solution, this unique magnetic configuration was able to reach a RNA extraction rate of 72%. This is the first demonstration of a physical separation in droplets at high throughput at single-cell scale.

Modular microfluidic system for on-chip extraction, preconcentration and detection of the cytokine biomarker IL-6 in biofluid.

The cytokine interleukin 6 (IL-6) is involved in the pathogenesis of different inflammatory diseases, including cancer, and its monitoring could help diagnosis, prognosis of relapse-free survival and recurrence. Here, we report an innovative microfluidic approach that uses the fluidization of magnetic beads to specifically extract, preconcentrate and fluorescently detect IL-6 directly on-chip. We assess how the physical properties of the beads can be tuned to improve assay performance by enhancing mass transport, reduce non-specific binding and multiply the detection signal threefold by transitioning between packed and fluidization states. With the integration of a full ELISA protocol in a single microfluidic chamber, we show a twofold reduction in LOD compared to conventional methods along with a large dynamic range (10 pg/mL to 2 ng/mL). We additionally demonstrate its application to IL-6 detection in undiluted serum samples.

A Computational Investigation of In Vivo Cytosolic Protein Delivery for Cancer Therapy.

The ability to specifically block or degrade cytosolic targets using therapeutic proteins would bring tremendous therapeutic opportunities in cancer therapy. Over the last few years, significant progress has been made with respect to tissue targeting, cytosolic delivery, and catalytic inactivation of targets, placing this aim within reach. Here, we developed a mathematical model specifically built for the evaluation of approaches towards cytosolic protein delivery, involving all steps from systemic administration to translocation into the cytosol and target engagement. Focusing on solid cancer tissues, we utilized the model to investigate the effects of microvascular permeability, receptor affinity, the cellular density of targeted receptors, as well as the mode of activity (blocking/degradation) on therapeutic potential. Our analyses provide guidance for the rational optimization of protein design for enhanced activity and highlight the importance of tuning the receptor affinity as a function of receptor density as well as the receptor internalization rate. Furthermore, we provide quantitative insights into how enzymatic cargoes can enhance the distribution, extent, and duration of therapeutic activity, already at very low catalytic rates. Our results illustrate that with current protein engineering approaches, the goal of delivery of cytosolic delivery of proteins for therapeutic effects is well within reach.

A Hybrid In Silico and Tumor-on-a-Chip Approach to Model Targeted Protein Behavior in 3D Microenvironments.

To rationally improve targeted drug delivery to tumor cells, new methods combining in silico and physiologically relevant in vitro models are needed. This study combines mathematical modeling with 3D in vitro co-culture models to study the delivery of engineered proteins, called designed ankyrin repeat proteins (DARPins), in biomimetic tumor microenvironments containing fibroblasts and tumor cells overexpressing epithelial cell adhesion molecule (EpCAM) or human epithelial growth factor receptor (HER2). In multicellular tumor spheroids, we observed strong binding-site barriers in combination with low apparent diffusion coefficients of 1 µm2·s-1 and 2 µm2 ·s-1 for EpCAM- and HER2-binding DARPin, respectively. Contrasting this, in a tumor-on-a-chip model for investigating delivery in real-time, transport was characterized by hindered diffusion as a consequence of the lower local tumor cell density. Finally, simulations of the diffusion of an EpCAM-targeting DARPin fused to a fragment of Pseudomonas aeruginosa exotoxin A, which specifically kills tumor cells while leaving fibroblasts untouched, correctly predicted the need for concentrations of 10 nM or higher for extensive tumor cell killing on-chip, whereas in 2D models picomolar concentrations were sufficient. These results illustrate the power of combining in vitro models with mathematical modeling to study and predict the protein activity in complex 3D models.

VEGF (Vascular Endothelial Growth Factor) Functionalized Magnetic Beads in a Microfluidic Device to Improve the Angiogenic Balance in Preeclampsia.

Preeclampsia is a hypertensive pregnancy disease associated with a massive increase in sFlt-1 (soluble form of the vascular endothelial growth factor 1) in the maternal circulation, responsible for angiogenic imbalance and endothelial dysfunction. Pilot studies suggest that extracorporeal apheresis may reduce circulating sFlt-1 and prolong pregnancy. Nonspecific apheresis systems have potential adverse effects because of the capture of many other molecules. Our concept is based on a specific and competitive apheresis approach using VEGF (vascular endothelial growth factor) functionalized magnetic beads to capture sFlt-1 while releasing endogenous PlGF (placental growth factor) to restore a physiological angiogenic balance. Magnetic beads were functionalized with VEGF to capture sFlt-1. Experiments were performed using PBS, conditioned media from human trophoblastic cells, and human plasma. The proof of concept was validated in dynamic conditions in a microfluidic device as an approach mimicking real apheresis. Magnetic beads were functionalized with VEGF and characterized to evaluate their surface ligand density and recognition capabilities. VEGF-coated magnetic beads proved to be an efficient support in capturing sFlt-1 and releasing PlGF. In static conditions, sFlt-1 concentration decreased by 33±13%, whereas PlGF concentration increased by 27±10%. In dynamic conditions, the performances were improved, with 40% reduction of sFlt-1 and up to 2-fold increase of free PlGF. The sFlt-1/PlGF ratio was reduced by 63% in the plasma of preeclamptic patients. Apheresis was also associated with VEGF release. A ligand-based approach using VEGF-coated beads is an effective approach to the capture of sFlt-1 and the release of endogenous PlGF. It offers new perspectives for the treatment of preeclampsia.