Mechanical regulation of extracellular vesicle activity during tumour progression.

Extracellular vesicles (EVs) are naturally occurring membrane-bound vesicles secreted by cells. Functionalized with surface-targeting molecules and carrying signalling proteins and nucleic acids as cargo, EVs can rewire pathways and alter biological processes in recipient cells. Tumour-derived EVs have key roles in cancer progression, particularly in metastasis, by promoting tumour cell invasion and the establishment of pre-metastatic niches.

Extracellular Vesicles for Clinical Diagnostics: From Bulk Measurements to Single-Vesicle Analysis.

Extracellular vesicles (EVs) play a crucial role in intercellular communication, signaling pathways, and disease pathogenesis by transporting biomolecules such as DNA, RNA, proteins, and lipids derived from their cells of origin, and they have demonstrated substantial potential in clinical applications. Their clinical significance underscores the need for sensitive methods to fully harness their diagnostic potential. In this comprehensive review, we explore EV heterogeneity related to biogenesis, structure, content, origin, sample type, and function roles; the use of EVs as disease biomarkers; and the evolving landscape of EV measurement for clinical diagnostics, highlighting the progression from bulk measurement to single vesicle analysis.

Brain biomarker profiles vary with semi-synthetic and grain-based diets in healthy and mTBI mice.

Across a range of neurological disorders, there is a growing appreciation for how the gut influences brain health, but few ways of monitoring its effects. Although nutrition influences traumatic brain injury (TBI) recovery, its influence on biomarkers-whether as an intervention or confounder-is poorly understood. Beyond specialized diets, standard rodent diets may also affect brain function.

Brain-derived extracellular vesicle microRNAs in Lewy body and Alzheimer's disease.

Introduction: Robust plasma-based biomarkers to distinguish Lewy body disease (LBD) and Alzheimer's disease (AD) are currently lacking. We applied track-etch magnetic nanopore (TENPO) sorting for enrichment of brain-derived extracellular vesicle (EV) signatures as potential biomarkers to address this gap.

Methods: We analyzed plasma from 137 autopsy-confirmed patients [30 LBD, 31 AD, 30 AD/LBD, 19 AD with amygdala Lewy bodies (AD/ALB), and 27 controls], sequencing miRNAs from TENPO-isolated GluR2-positive (neuron-enriched) and GLAST-positive (astrocyte-enriched) EVs, and measuring plasma proteins (Aβ40, Aβ42, tau, p-Tau181, p-Tau231) via SIMOA.

Automated and parallelized microfluidic generation of large and precisely-defined lipid nanoparticle libraries.

Building on the success of lipid nanoparticles (LNPs) in vaccines, LNPs are being developed for a broad set of therapeutic applications by changing both the structures of the lipids used to formulate each LNP and their relative proportions. Because lipid synthesis and screening have been parallelized using combinatorial chemistry and LNP barcoding respectively, the manual and sequential microfluidic formulation of LNPs has become the rate-limiting step in the discovery process. In this work, we present a high-throughput, automated microfluidic platform capable of generating large, precisely-defined LNP libraries in parallel at a rate of one distinct formulation every three seconds.

miRNA panel from HER2+ and CD24+ plasma extracellular vesicle subpopulations as biomarkers of early-stage breast cancer.

Background: Mammography screening has improved early breast cancer detection, leading to reduced mortality and lower rates of advanced breast cancer. However, mammography has a high false positive rate that results in over a million invasive breast biopsies of benign lesions in the US each year. Therefore, there is a need for noninvasive, blood-based diagnostics that can accurately assess risk of malignancy for women with indeterminate lesions identified by mammography, such as BI-RADS category 4 breast lesions.

Combining time domain modulation optofluidics and high dynamic range imaging for multiplexed, high throughput digital droplet assays.

Digital enzyme-linked immunoassays (dELISA) have been successfully applied to the ultrasensitive quantification of analytes, including nucleic acids, proteins, cells, and extracellular vesicles, achieving robust detection limits in complex clinical specimens such as blood, and demonstrating utility across a broad range of clinical applications. The ultrasensitivity of dELISA comes from partitioning single analytes, captured onto a microbead, into millions of compartments so that they can be counted individually. There is particular interest in using dELISA for multiplexed measurements, but generating and detecting the billions of compartments necessary to perform multiplexed ultrasensitive dELISA remains a challenge.

Agarose Microgel-Based In Situ Cleavable Immuno-Rolling Circle Amplification for Multiplexed Single-Molecule Quantitation on Single Extracellular Vesicles.

We have developed a platform for the multiplexed and ultrasensitive profiling of individual extracellular vesicles (EVs) directly in plasma, which we call GDEVA─Agarose microel-based igital single-molecule-single ssay. GDEVA achieves single-molecule sensitivity and moderate multiplexing (demonstrated 3-plex), and can achieve a throughput of ∼10 EVs per minute necessary to resolve EVs directly in human plasma when read out using flow cytometry. Our platform integrates a rolling circle amplification (RCA) immunoassay of EV surface proteins, which are cleaved from single EVs, and amplified within agarose microgels, followed by flow cytometry-based readout or imaging after fluorescence-activated cell sorting (FACS).

Solution biophysics identifies lipid nanoparticle non-sphericity, polydispersity, and dependence on internal ordering for efficacious mRNA delivery.

Lipid nanoparticles (LNPs) are the most advanced delivery system currently available for RNA therapeutics. Their development has accelerated since the success of Patisiran, the first siRNA-LNP therapeutic, and the mRNA vaccines that emerged during the COVID-19 pandemic. Designing LNPs with specific targeting, high potency, and minimal side effects is crucial for their successful clinical use.

Enhanced Accumulation and Penetration of Magnetic Nanoclusters in Tumors Using an 8-Magnet Halbach Array Leads to Improved Cancer Treatment.

Nanoparticles have gained attention as drug delivery vehicles for cancer treatment, but often struggle with poor tumor accumulation and penetration. Single external magnets can enhance magnetic nanoparticle delivery but are limited to superficial tumors due to the rapid decline in the magnetic field strength with distance. We previously showed that a 2-magnet device could extend targeting to greater tissue depths.

Robust, Scalable Microfluidic Manufacturing of RNA-Lipid Nanoparticles Using Immobilized Antifouling Lubricant Coating.

Despite the numerous advantages demonstrated by microfluidic mixing for RNA-loaded lipid nanoparticle (RNA-LNP) production over bulk methods, such as precise size control, homogeneous distributions, higher encapsulation efficiencies, and improved reproducibility, their translation from research to commercial manufacturing remains elusive. A persistent challenge hindering the adoption of microfluidics for LNP production is the fouling of device surfaces during prolonged operation, which significantly diminishes performance and reliability. The complexity of LNP constituents, including lipids, cholesterol, RNA, and solvent mixtures, makes it difficult to find a single coating that can prevent fouling.

Multi-stage-mixing to control the supramolecular structure of lipid nanoparticles, thereby creating a core-then-shell arrangement that improves performance by orders of magnitude.

As they became the dominant gene therapy platform, lipid nanoparticles (LNPs) experienced nearly all their innovation in varying the structure of individual molecules in LNPs. This ignored control of the spatial arrangement of molecules, which is suboptimal because supramolecular structure determines function in biology. To control LNPs' supramolecular structure, we introduce multi-stage-mixing (MSM) to successively add different molecules to LNPs.

High-throughput, multiplexed quantification, and sorting of single EVs at single-molecule level.

We have developed a platform for the high-throughput, multiplexed, and ultra-sensitive profiling of individual extracellular vesicles (EVs) directly in plasma, which we call BDEVS - Agarose ead-based igital Single Molecule-Single orting. Unlike conventional approaches, BDEVS achieves single molecule sensitivity and moderate multiplexing (demonstrated 3-plex) without sacrificing the throughput (processing ten thousand of EVs per minute) necessary to resolve EVs directly in human plasma. Our platform integrates rolling circle amplification (RCA) of EV surface proteins, which are cleaved from single EVs, and amplified within agarose droplets, followed by flow cytometry-based readout and sorting, overcoming steric hindrance, non-specific binding, and the lack of quantitation of multiple proteins on EVs that have plagued earlier approaches.

Towards the clinical translation of a silver sulfide nanoparticle contrast agent: large scale production with a highly parallelized microfluidic chip.

Purpose: Ultrasmall silver sulfide nanoparticles (AgS-NP) have been identified as promising contrast agents for a number of modalities and in particular for dual-energy mammography. These AgS-NP have demonstrated marked advantages over clinically available agents with the ability to generate higher contrast with high biocompatibility. However, current synthesis methods for inorganic nanoparticles are low-throughput and highly time-intensive, limiting the possibility of large animal studies or eventual clinical use of this potential imaging agent.

Mitigation of Device Heterogeneity in Graphene Hall Sensor Arrays Using Per-Element Backgate Tuning.

Graphene Hall-effect magnetic field sensors (GHSs) exhibit high performance comparable to state-of-the-art commercial Hall sensors made from III-V semiconductors. Graphene is also amenable to CMOS-compatible fabrication processes, making GHSs attractive candidates for implementing magnetic sensor arrays for imaging fields in biosensing and scanning probe applications. However, their practical appeal is limited by response heterogeneity and drift, arising from the high sensitivity of two-dimensional (2D) materials to local device imperfections.

Single-Mitochondrion Sequencing Uncovers Distinct Mutational Patterns and Heteroplasmy Landscape in Mouse Astrocytes and Neurons.

Background: Mitochondrial (mt) heteroplasmy can cause adverse biological consequences when deleterious mtDNA mutations accumulate disrupting 'normal' mt-driven processes and cellular functions. To investigate the heteroplasmy of such mtDNA changes we developed a moderate throughput mt isolation procedure to quantify the mt single-nucleotide variant (SNV) landscape in individual mouse neurons and astrocytes In this study we amplified mt-genomes from 1,645 single mitochondria (mts) isolated from mouse single astrocytes and neurons to 1. determine the distribution and proportion of mt-SNVs as well as mutation pattern in specific target regions across the mt-genome, 2.

Ultrasensitive quantification of PD-L1+ extracellular vesicles in melanoma patient plasma using a parallelized high throughput droplet digital assay.

The expression of programmed death-ligand 1 (PD-L1) on extracellular vesicles (EVs) is an emerging biomarker for cancer, and has gained particular interest for its role mediating immunotherapy. However, precise quantification of PD-L1+ EVs in clinical samples remains challenging due to their sparse concentration and the enormity of the number of background EVs in human plasma, limiting applicability of conventional approaches. In this study, we develop a high-throughput droplet-based extracellular vesicle analysis (DEVA) assay for ultrasensitive quantification of EVs in plasma that are dual positive for both PD-L1 and tetraspanin (CD81) known to be expressed on EVs.

High-Throughput Single-Cell, Single-Mitochondrial DNA Assay Using Hydrogel Droplet Microfluidics.

There is growing interest in understanding the biological implications of single cell heterogeneity and heteroplasmy of mitochondrial DNA (mtDNA), but current methodologies for single-cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single-cell genomic, RNA, and protein analysis, their application to sub-cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose-based droplet microfluidic approach for single-cell, single-mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells.

High-throughput single-cell, single-mitochondrial DNA assay using hydrogel droplet microfluidics.

There is growing interest in understanding the biological implications of single cell heterogeneity and intracellular heteroplasmy of mtDNA, but current methodologies for single-cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single-cell genomic, RNA, and protein analysis, their application to sub-cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose-based droplet microfluidic approach for single-cell, single-mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells.

Towards the Clinical Translation of a Silver Sulfide Nanoparticle Contrast Agent: Large Scale Production with a Highly Parallelized Microfluidic Chip.

Ultrasmall silver sulfide nanoparticles (Ag S-NP) have been identified as promising contrast agents for a number of modalities and in particular for dual-energy mammography. These Ag S-NP have demonstrated marked advantages over clinically available agents with the ability to generate higher contrast with high biocompatibility. However, current synthesis methods are low-throughput and highly time-intensive, limiting the possibility of large animal studies or eventual clinical use of this potential imaging agent.

Brain-derived extracellular vesicles as serologic markers of brain injury following cardiac arrest: A pilot feasibility study.

Aim: Assessment of neurologic injury within the immediate hours following out-of-hospital cardiac arrest (OHCA) resuscitation remains a major clinical challenge. Extracellular vesicles (EVs), small bodies derived from cytosolic contents during injury, may provide the opportunity for "liquid biopsy" within hours following resuscitation, as they contain proteins and RNA linked to cell type of origin. We evaluated whether micro-RNA (miRNA) from serologic EVs were associated with post-arrest neurologic outcome.

Throughput-scalable manufacturing of SARS-CoV-2 mRNA lipid nanoparticle vaccines.

Lipid nanoparticles (LNPs) are a potent delivery technology that have made it possible for the recent clinical breakthroughs in mRNA therapeutics and vaccines. A key challenge to the broader implementation of mRNA therapeutics and vaccines is the development of technology to produce precisely defined LNP formulations, with throughput that can scale from discovery to commercial manufacturing and meet the stringent manufacturing standards of the pharmaceutical industry. To address these challenges, we have developed a microfluidic chip that incorporates 1×, 10×, or 256× LNP-generating units that achieve scalable production rates of up to 17 L/h of precisely defined LNPs.