Extracellular Vesicle Refractive Index Derivation Utilizing Orthogonal Characterization.

The analysis of small particles, including extracellular vesicles and viruses, is contingent on their ability to scatter sufficient light to be detected. These detection methods include flow cytometry, nanoparticle tracking analysis, and single particle reflective image sensing. To standardize measurements and enable orthogonal comparisons between platforms, a quantifiable limit of detection is required. The main parameters that dictate the amount of light scattered by particles include size, morphology, and refractive index. To date, there has been a lack of accessible techniques for measuring the refractive index of nanoparticles at a single-particle level. Here, we demonstrate two methods of deriving a small particle refractive index using orthogonal measurements with commercially available platforms. These methods can be applied at either a single-particle or population level, enabling the integration of diameter and scattering cross section values to derive the refractive index using Mie theory.

Size Measurement of Extracellular Vesicles and Synthetic Liposomes: The Impact of the Hydration Shell and the Protein Corona.

Size characterization of extracellular vesicles (EVs) and drug delivery liposomes is of great importance in their applications in diagnosis and therapy of diseases. There are many different size characterization techniques used in the field, which often report different size values. Besides technological biases, these differences originate from the fact that various methods measure different physical quantities to determine particle size. In this study, the size of synthetic liposomes with nominal diameters of 50nm and 100nm, and red blood cell-derived EVs (REVs) were measured with established optical methods, such as dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA), and with emerging non-optical methods such as microfluidic resistive pulse sensing (MRPS) and very small-angle neutron scattering (VSANS). The comparison of the hydrodynamic sizes obtained by DLS and NTA with the sizes corresponding to the excluded volume of the particles by MRPS enabled the estimation of the thickness of the hydration shell of the particles. The comparison of diameter values corresponding to the boundary of the phospholipid bilayer obtained from VSANS measurements with MRPS size values revealed the thickness of the polyethylene glycol-layer in case of synthetic liposomes, and the thickness of the protein corona in case of REVs.

Detection and phenotyping of extracellular vesicles by size exclusion chromatography coupled with on-line fluorescence detection.

New methods for quantifying extracellular vesicles (EVs) in complex biofluids are critically needed. We report the development of a new technology combining size exclusion chromatography (SEC), a commonly used EV purification technique, with fluorescence detection of specifically labelled EVs. The resulting platform, Flu-SEC, demonstrates a linear response to concentration of specific EVs and could form the basis of a system with phenotyping capability. Flu-SEC was validated using red blood cell derived EVs (REVs), which provide an ideal EV model with monodisperse size distribution and high EV concentration. Microfluidic Resistive Pulse Sensing (MRPS) was used to accurately determine the size distribution and concentration of REVs. Anti-CD235a antibody, specific to glycophorin A, and the more general wheat germ agglutinin (WGA), were selected to label REVs. The results show the quantitative power of Flu-SEC: a highly linear fluorescence response over a wide range of concentrations. Moreover, the Flu-SEC technique reports the ratio of EV-bound and free-antibody molecules, an important metric for determining optimal labelling conditions for other applications. Flu-SEC represents an orthogonal tool to single-particle fluorescent methods such as flow cytometry and fluorescent NTA, for the quantification and phenotyping of EVs.

The p53 circuit board.

The p53 tumor suppressor is embedded in a large gene network controlling diverse cellular and organismal phenotypes. Multiple signaling pathways converge onto p53 activation, mostly by relieving the inhibitory effects of its repressors, MDM2 and MDM4. In turn, signals originating from increased p53 activity diverge into distinct effector pathways to deliver a specific cellular response to the activating stimuli. Much attention has been devoted to dissecting how the various input pathways trigger p53 activation and how the activity of the p53 protein itself can be modulated by a plethora of co-factors and post-translational modifications. In this review we will focus instead on the multiple configurations of the effector pathways. We will discuss how p53-generated signals are transmitted, amplified, resisted and eventually integrated by downstream gene circuits operating at the transcriptional, post-transcriptional and post-translational levels. We will also discuss how context-dependent variations in these gene circuits define the cellular response to p53 activation and how they may impact the clinical efficacy of p53-based targeted therapies.

A high-throughput label-free nanoparticle analyser.

Synthetic nanoparticles and genetically modified viruses are used in a range of applications, but high-throughput analytical tools for the physical characterization of these objects are needed. Here we present a microfluidic analyser that detects individual nanoparticles and characterizes complex, unlabelled nanoparticle suspensions. We demonstrate the detection, concentration analysis and sizing of individual synthetic nanoparticles in a multicomponent mixture with sufficient throughput to analyse 500,000 particles per second. We also report the rapid size and titre analysis of unlabelled bacteriophage T7 in both salt solution and mouse blood plasma, using just ~1 × 10⁻6 l of analyte. Unexpectedly, in the native blood plasma we discover a large background of naturally occurring nanoparticles with a power-law size distribution. The high-throughput detection capability, scalable fabrication and simple electronics of this instrument make it well suited for diverse applications.