In Situ Complexation of sgRNA and Cas12a Improves the Performance of a One-Pot RPA-CRISPR-Cas12 Assay.

Due to their ability to selectively target pathogen-specific nucleic acids, CRISPR-Cas systems are increasingly being employed as diagnostic tools. "One-pot" assays that combine nucleic acid amplification and CRISPR-Cas systems (NAAT-CRISPR-Cas) in a single step have emerged as one of the most popular CRISPR-Cas biosensing formats. However, operational simplicity comes at a cost, with one-pot assays typically being less sensitive than corresponding two-step NAAT-CRISPR-Cas assays and often failing to detect targets at low concentrations. It is thought that these performance reductions result from the competition between the two enzymatic processes driving the assay, namely, Cas-mediated cis-cleavage and polymerase-mediated amplification of the target DNA. Herein, we describe a novel one-pot RPA-Cas12a assay that circumvents this issue by leveraging in situ complexation of the target-specific sgRNA and Cas12a to purposefully limit the concentration of active Cas12a during the early stages of the assay. Using a clinically relevant assay against a DNA target for HPV-16, we show how this in situ format reduces competition between target cleavage and amplification and engenders significant improvements in detection limit when compared to the traditional one-pot assay format, even in patient-derived samples. Finally, to gain further insight into the assay, we use experimental data to formulate a mechanistic model describing the competition between the Cas enzyme and nucleic acid amplification. These findings suggest that purposefully limiting cis-cleavage rates of Cas proteins is a viable strategy for improving the performance of one-pot NAAT-CRISPR-Cas assays.

Multiparametric Orthogonal Characterization of Extracellular Vesicles by Liquid Chromatography Combined with In-Line Light Scattering and Fluorescence Detection.

Extracellular vesicles (EVs) are membrane-enclosed biological nanoparticles with potential as diagnostic markers and carriers for therapeutics. Characterization of EVs poses severe challenges due to their complex structure and composition, requiring the combination of orthogonal analytical techniques. Here, we demonstrate how liquid chromatography combined with multi-angle light scattering (MALS) and fluorescence detection in one single apparatus can provide multiparametric characterization of EV samples, including concentration of particles, average diameter of the particles, protein amount to particle number ratio, presence of EV surface markers and lipids, EV shape, and sample purity. The method requires a small amount of sample of approximately 107 EVs, limited handling of the sample and data analysis time in the order of minutes; it is fully automatable and can be applied to both crude and purified samples.

High-Yield Production of Extracellular Vesicle Subpopulations with Constant Quality Using Batch-Refeed Cultures.

The conventional manufacturing of extracellular vesicles (EVs) is characterized by low yields and batch-to-batch variability, hampering fundamental research on EVs and their practical applications. Perfusion operations have huge potential to address these limitations and increase the productivity and quality of EVs. In this study, perfusion cultures are simulated with batch-refeed systems and their productivity is compared with that achieved using batch cultures. It is shown that a shift from batch to batch-refeed system can increase the space-time yields of a target EV subpopulation characterized by CD81 and CD63 biomarkers by threefold. Moreover, it is demonstrated that the method facilitates the consistent production of the target EVs from cells maintained under constant conditions for 13 days. These results indicate that the use of perfusion cultures is a promising strategy to increase the manufacturing yield of EVs and control the production of specific EV subpopulations with constant quality attributes, thereby improving reproducibility.

High-Yield Separation of Extracellular Vesicles Using Programmable Zwitterionic Coacervates.

Programmable coacervates based on zwitterionic polymers are designed as dynamic materials for ion exchange bioseparation. These coacervates are proposed as promising materials for the purification of soft nanoparticles such as liposomes and extracellular vesicles (EVs). It is shown that the stimulus-responsiveness of the coacervates and the recruitment of desired molecules can be independently programmed by polymer design. Moreover, the polymeric coacervates can recruit and release intact liposomes, human EVs, and nanoalgosomes in high yields and separate vesicles from different types of impurities, including proteins and nucleic acids. This approach combines the speed and simplicity of precipitation methods and the programmability of chromatography with the gentleness of aqueous two-phase separation, thereby guaranteeing product stability. This material represents a promising alternative for providing a low-shear, gentle, and selective purification method for EVs.

Programmable Zwitterionic Droplets as Biomolecular Sorters and Model of Membraneless Organelles.

Increasing evidence indicates that cells can regulate biochemical functions in time and space by generating membraneless compartments with well-defined mesoscopic properties. One important mechanism underlying this control is simple coacervation driven by associative disordered proteins that encode multivalent interactions. Inspired by these observations, programmable droplets based on simple coacervation of responsive synthetic polymers that mimic the "stickers-and-spacers" architecture of biological disordered proteins are developed. Zwitterionic polymers that undergo an enthalpy-driven liquid-liquid phase separation process and form liquid droplets that remarkably exclude most molecules are developed. Starting from this reference material, different functional groups in the zwitterionic polymer are progressively added to encode an increasing number of different intermolecular interactions. This strategy allowed the multiple emerging properties of the droplets to be controlled independently, such as stimulus-responsiveness, polarity, selective uptake of client molecules, fusion times, and miscibility. By exploiting this high programmability, a model of cellular compartmentalization is reproduced and droplets capable of confining different molecules in space without physical barriers are generated. Moreover, these biomolecular sorters are demonstrated to be able to localize, separate, and enable the detection of target molecules even within complex mixtures, opening attractive applications in bioseparation, and diagnostics.

Rapid Characterization and Quantification of Extracellular Vesicles by Fluorescence-Based Microfluidic Diffusion Sizing.

Extracellular vesicles (EVs) are emerging as promising diagnostic and therapeutic tools for a variety of diseases. The characterization of EVs requires a series of orthogonal techniques that are overall time- and material-consuming. Here, a microfluidic device is presented that exploits the combination of diffusion sizing and multiwavelength fluorescence detection to simultaneously provide information on EV size, concentration, and composition. The latter is achieved with the nonspecific staining of lipids and proteins combined with the specific staining of EV markers such as EV-associated tetraspanins via antibodies. The device can be operated as a single-step immunoassay thanks to the integrated separation and quantification of free and EV-bound fluorophores. This microfluidic technique is capable of detecting and quantifying components associated to EV subtypes and impurities and thus to measure EV purity in a time scale of minutes, requiring less than 5 µL of sample and minimal sample handling before the analysis. Moreover, the analysis is performed directly in solution without immobilization steps. Therefore, this method can accelerate screening of EV samples and aid the evaluation of sample reproducibility, representing an important complementary tool to the current array of biophysical methods for EV characterization, particularly valuable for instance for bioprocess development.

Nanoalgosomes: Introducing extracellular vesicles produced by microalgae.

Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production.

Isolation of extracellular vesicles from microalgae: towards the production of sustainable and natural nanocarriers of bioactive compounds.

Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 µg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and ß-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles.

A Molecular Logic Gate Enables Single-Molecule Imaging and Tracking of Lipids in Intracellular Domains.

Photoactivatable dyes enable single-molecule imaging and tracking in biology. Despite progress in the development of new fluorophores and labeling strategies, many intracellular compartments remain difficult to image beyond the limit of diffraction in living cells. For example, lipid domains, e.g., membranes and droplets, remain difficult to image with nanometric resolution. To visualize these challenging subcellular targets, it is necessary to develop new fluorescent molecular devices beyond simple on/off switches. Here, we report a fluorogenic molecular logic gate that can be used to image single molecules associated with lipid domains, most notably droplets, with excellent specificity. This probe requires the subsequent action of light, a lipophilic environment, and a competent nucleophile to produce a fluorescent product. The combination of these inputs results in a probe that can be used to image the boundary of lipid droplets in three dimensions with resolution beyond the limit of diffraction. Moreover, this probe enables single-molecule tracking of lipid trafficking between droplets and the endoplasmic reticulum.

Macroporous Polymer⁻Protein Hybrid Materials for Antibody Purification by Combination of Reactive Gelation and Click-Chemistry.

Clickable core-shell nanoparticles based on poly(styrene-co-divinylbenzene-co-vinylbenzylazide) have been synthesized via emulsion polymerization. The 38 nm sized particles have been swollen by divinyl benzene (DVB) and 2,2'-azobis(2-methylpropionitrile) (AIBN) and subsequently processed under high shear rates in a Z-shaped microchannel giving macroporous microclusters (100 µm), through the reactive gelation process. The obtained clusters were post-functionalized by "click-chemistry" with propargyl-PEG-NHS-ester and propargylglicidyl ether, yielding epoxide or NHS-ester activated polymer supports for bioconjugation. Macroporous affinity materials for antibody capturing were produced by immobilizing recombinant Staphylococcus aureus protein A on the polymeric support. Coupling chemistry exploiting thiol-epoxide ring-opening reactions with cysteine-containing protein A revealed up to three times higher binding capacities compared to the protein without cysteine. Despite the lower binding capacities compared to commercial affinity phases, the produced polymer-protein hybrids can serve as stationary phases for immunoglobulin affinity chromatography as the materials revealed superior intra-particle mass transports.

Scalable Production and Isolation of Extracellular Vesicles: Available Sources and Lessons from Current Industrial Bioprocesses.

Potential applications of extracellular vesicles (EVs) are attracting increasing interest in the fields of medicine, cosmetics, and nutrition. However, the manufacturing of EVs is currently characterized by low yields. This limitation severely hampers progress in research at the laboratory and clinical scales, as well as the realization of successful and cost-effective EV-based products. Moreover, the high level of heterogeneity of EVs further complicates reproducible manufacturing on a large scale. In this review, possible directions toward the scalable production of EVs are discussed. In particular, two strategies are considered: i) the optimization of upstream unit operations and ii) the exploitation of well-established and mature technologies already in use in other industrial bioprocesses.