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.

Droplet Microfluidics for the Label-Free Extraction of Complete Phase Diagrams and Kinetics of Liquid-Liquid Phase Separation in Finite Volumes.

Liquid-liquid phase separation of polymer and protein solutions is central in many areas of biology and material sciences. Here, an experimental and theoretical framework is provided to investigate the thermodynamics and kinetics of liquid-liquid phase separation in volumes comparable to cells. The strategy leverages droplet microfluidics to accurately measure the volume of the dense phase generated by liquid-liquid phase separation of solutions confined in micro-sized compartments. It is shown that the measurement of the volume fraction of the dense phase at different temperatures allows the evaluation of the binodal lines that determine the coexistence region of the two phases in the temperature-concentration phase diagram. By applying a thermodynamic model of phase separation in finite volumes, it is further shown that the platform can predict and validate kinetic barriers associated with the formation of a dense droplet in a parent dilute phase, therefore connecting thermodynamics and kinetics of liquid-liquid phase separation.

A Graphene-Based Supramolecular Nanoreactor for the Fast Synthesis of Imines in Water.

The confinement of organic synthesis within waterborne nanoreactors is regarded with increasing attention to improve its yield and reduce the environmental impact. However, many catalysts, such as graphene, are barely dispersible in aqueous media and many chemical reactions cannot be performed in the presence of water due to thermodynamic limitations. Therefore, there is an urgent need to develop novel strategies to carry out these processes in more sustainable conditions. To pursue this goal, in this work, a waterborne supramolecular nanoreactor is developed. The system comprises a polymeric micelle obtained from the self-assembly of pyrrole-based amphiphilic block copolymers. The active catalytic component is represented by few graphene layers, functionalized with pyrrole to enhance their interaction with the micelle core and hence their nanoencapsulation. Using this nanoreactor, it is possible to synthesize imines starting from primary amines and aldehydes or ketones with high yield and in short time (Y = 90% after 5 min) at room temperature. Moreover, an efficient strategy to recycle the reactor is proposed, thus increasing the potential of this technology.

A Nanoparticle-Based Assay To Evaluate Surface-Induced Antibody Instability.

Protein stability against aggregation represents a major quality attribute for the successful development of biopharmaceuticals. Increasing evidence indicates that the formation of protein aggregates in aqueous solutions is often triggered by interactions between proteins and interfaces. Yet, in contrast to a large number of methods available to test protein bulk properties, high-throughput assays to investigate protein instability at interfaces remain much less developed. Major challenges include the control of the amount and type of surfaces, as well as the presence of synergistic effects between interfaces and hydrodynamic flows. Here, we describe and develop a highly controlled surface-mediated stress assay of protein instability based on polymeric nanoparticles. We show that hydrophobic nanoparticles are remarkably powerful in destabilizing large proteins such as antibodies. We further show that this approach can be implemented on a high-throughput microfluidic platform by compartmentalizing the protein solution in picoliter droplets surrounded by an oil phase. Our method allows the evaluation of protein instability at hydrophobic interfaces in a time scale of minutes and requires amounts of the sample in the order of a few hundred micrograms. We demonstrate that our assay represents a good mimic of air-water interfaces and finds application as a screening tool to optimize protein stability toward surface-induced aggregation. We provide a concrete example by identifying the optimal concentration range of Tween 80 that prevents antibody instability in the presence of interfaces. Overall, our hydrophobic nanoparticle surface-mediated stress assay (HNSSA) represents an attractive tool for accelerated tests of protein instability at interfaces under both stagnant and flow conditions, with implications for the optimization of buffer composition and the selection of stable biotherapeutic candidate molecules during early stage development.

Adaptive Chemoenzymatic Microreactors Composed of Inorganic Nanoparticles and Bioinspired Intrinsically Disordered Proteins.

The assembly of protein and inorganic nanoparticles represents an attractive approach to generate composite materials with multiple functions. Herein, we functionalize inorganic nanoparticles with intrinsically disordered protein domains associated with the formation of membraneless compartments in cells. These protein sequences, defined as low complexity domains (LCDs), encode intermolecular interactions that drive highly controlled, dynamic self-assembly in response to environmental changes. We show that the properties of the LCDs can be transferred to inorganic nanoparticles, inducing controlled phase separation that is dynamic and responsive to ionic strength and pH. Specifically, we hybridize magnetic nanoparticles with multi-domain proteins consisting of LCD domains and a globular enzyme, generating dynamic protein-composite compartments that locally confine hybrid chemoenzymatic reactions and respond to external magnetic fields and changes in solution conditions.

Dynamics of Synthetic Membraneless Organelles in Microfluidic Droplets.

Cells can form membraneless organelles by liquid-liquid phase separation. As these organelles are highly dynamic, it is crucial to understand the kinetics of these phase transitions. Here, we use droplet-based microfluidics to mix reagents by chaotic advection and observe nucleation, growth, and coarsening in volumes comparable to cells (pL) and on timescales of seconds. We apply this platform to analyze the dynamics of synthetic organelles formed by the DEAD-box ATPase Dhh1 and RNA, which are associated with the formation of processing bodies in yeast. We show that the timescale of phase separation decreases linearly as the volume of the compartment increases. Moreover, the synthetic organelles coarsen into one single droplet via gravity-induced coalescence, which can be arrested by introducing a hydrogel matrix that mimics the cytoskeleton. This approach is an attractive platform to investigate the dynamics of compartmentalization in artificial cells.

Zwitterionic Polyester-Based Nanoparticles with Tunable Size, Polymer Molecular Weight, and Degradation Time.

Biodegradable polymer nanoparticles are an important class of materials used in several applications for their unique characteristics. In particular, the ones stabilized by zwitterionic materials are gaining increased interest in medicine as alternative to the more common ones based on poly(ethylene glycol) thanks to their superior stability and ability to avoid both the accelerated blood clearance and allergic reactions. In this work, a novel class of zwitterionic based NPs has been produced, and a method to independently control the nanoparticle size, degradation time, and polymer molecular weight has been developed and demonstrated. This has been possible by the synthesis and the fine-tuning of zwitterionic amphiphilic block copolymers obtained via the combination of ring-opening polymerization and reversible addition-fragmentation chain transfer polymerization. The final results showed that when two block copolymers contain the same number of caprolactone units, the one with longer oligoester lateral chains degrades faster. This phenomenon is in sharp contrast with the one seen so far for the common linear polyester systems where longer chains result in longer degradation times, and it can be used to better tailor the degradation behavior of the nanoparticles.

Synthesis and characterization of pH-sensitive drinkable nanoparticles for oral delivery of ibuprofen.

Ibuprofen (IBU) is a widespread drug used to treat both acute and chronic disorders. It is generally taken orally but the free drug can induce the irritation of the gastric mucosa due to its acid nature. In literature, different approaches have been adopted to prevent the release in the stomach, such as physical entrapment with film-coated tablets and drug-conjugates. Nevertheless, these solutions have many disadvantages, including the fast release of the drug and the difficulty to swallow the tablet, especially for children who may vomit or refuse the tablet. For this reason, in this work, novel formulations are proposed that do not require the encapsulation of the drug into a solid form and, in turn, their assumption as a pill. IBU has been linked to different types of methacrylates via ester bond in order to produce pH-responsive macromolecular monomers. The novelty is related to the use of these drug-conjugates macromonomer for the production of nanoparticles (NPs) via emulsion polymerization (EP), using water as solvent. The final emulsion is able to load up to 30 mg ml-1 of IBU, so less than 10 ml is required to be assumed to reach the minimum therapeutic dose of the drug (200 mg). Finally, the release of IBU from these novel drinkable formulations has been investigated in the gastric and intestinal simulated fluids to show the preferential release of IBU from the NPs in basic conditions. A comparison with an existing oral suspension has been performed to highlight the slower release in acid environment of these new formulations. Afterwards, the IBU loaded NPs were tested in vitro showing lower toxicity compared to the free drug.

Use of RAFT macro-surfmers for the synthesis of transparent aqueous colloids with tunable interactions.

We propose a new method to produce fluorinated nanoparticles (NPs) based on ab initio reversible addition-fragmentation chain transfer (RAFT) emulsion polymerization without the use of toxic surfactants. NP size, surface charge, and chemistry can be controlled via the adoption of different macromolecular transfer agents produced via RAFT polymerization of amphiphilic monomers. Thanks to this versatility, interparticle interactions can be easily tuned by changing solvent composition and temperature. In addition, the refractive index and density of the solvent can simultaneously match those of the NPs by adding sodium polytungstate, an organic salt widely used for density gradient centrifugation. These colloids may be used as model systems for the study of self-assembly and aggregation in aqueous media when optical methods are required.

RNA modulates hnRNPA1A amyloid formation mediated by biomolecular condensates.

Several RNA binding proteins involved in membraneless organelles can form pathological amyloids associated with neurodegenerative diseases, but the mechanisms of how this aggregation is modulated remain elusive. Here we investigate how heterotypic protein-RNA interactions modulate the condensation and the liquid to amyloid transition of hnRNPA1A, a protein involved in amyothropic lateral sclerosis. In the absence of RNA, formation of condensates promotes hnRNPA1A aggregation and fibrils are localized at the interface of the condensates. Addition of RNA modulates the soluble to amyloid transition of hnRNPA1A according to different pathways depending on RNA/protein stoichiometry. At low RNA concentrations, RNA promotes both condensation and amyloid formation, and the catalytic effect of RNA adds to the role of the interface between the dense and dilute phases. At higher RNA concentrations, condensation is suppressed according to re-entrant phase behaviour but formation of hnRNPA1A amyloids is observed over longer incubation times. Our findings show how heterotypic nucleic acid-protein interactions affect the kinetics and molecular pathways of amyloid formation.

Comparison of the Protective Effect of Polysorbates, Poloxamer and Brij on Antibody Stability Against Different Interfaces.

Therapeutic proteins and antibodies are exposed to a variety of interfaces during their lifecycle, which can compromise their stability. Formulations, including surfactants, must be carefully optimized to improve interfacial stability against all types of surfaces. Here we apply a nanoparticle-based approach to evaluate the instability of four antibody drugs against different solid-liquid interfaces characterized by different degrees of hydrophobicity. We considered a model hydrophobic material as well as cycloolefin-copolymer (COC) and cellulose, which represent some of the common solid-liquid interfaces encountered during drug production, storage, and delivery. We assess the protective effect of polysorbate 20, polysorbate 80, Poloxamer 188 and Brij 35 in our assay and in a traditional agitation study. While all nonionic surfactants stabilize antibodies against the air-water interface, none of them can protect against hydrophilic charged cellulose. Polysorbates and Brij increase antibody stability in the presence of COC and the model hydrophobic interface, although to a lesser extent compared to the air-water interface, while Poloxamer 188 has a negligible stabilizing effect against these interfaces. These results highlight the challenge of fully protecting antibodies against all types of solid-liquid interfaces with traditional surfactants. In this context, our high-throughput nanoparticle-based approach can complement traditional shaking assays and assist in formulation design to ensure protein stability not only at air-water interfaces, but also at relevant solid-liquid interfaces encountered during the product lifecycle.

The interface of condensates of the hnRNPA1 low-complexity domain promotes formation of amyloid fibrils.

The maturation of liquid-like protein condensates into amyloid fibrils has been associated with several neurodegenerative diseases. However, the molecular mechanisms underlying this liquid-to-solid transition have remained largely unclear. Here we analyse the amyloid formation mediated by condensation of the low-complexity domain of hnRNPA1, a protein involved in amyotrophic lateral sclerosis. We show that phase separation and fibrillization are connected but distinct processes that are modulated by different regions of the protein sequence. By monitoring the spatial and temporal evolution of amyloid formation we demonstrate that the formation of fibrils does not occur homogeneously inside the droplets but is promoted at the interface of the condensates. We further show that coating the interface of the droplets with surfactant molecules inhibits fibril formation. Our results reveal that the interface of biomolecular condensates of hnRNPA1 promotes fibril formation, therefore suggesting interfaces as a potential novel therapeutic target against the formation of aberrant amyloids mediated by condensation.

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.

Sequestration within biomolecular condensates inhibits Aß-42 amyloid formation.

Biomolecular condensates are emerging as an efficient strategy developed by cells to control biochemical reactions in space and time by locally modifying composition and environment. Yet, local increase in protein concentration within these compartments could promote aberrant aggregation events, including the nucleation and growth of amyloid fibrils. Understanding protein stability within the crowded and heterogeneous environment of biological condensates is therefore crucial, not only when the aggregation-prone protein is the scaffold element of the condensates but also when proteins are recruited as client molecules within the compartments. Here, we investigate the partitioning and aggregation kinetics of the amyloidogenic peptide Abeta42 (Aß-42), the peptide strongly associated with Alzheimer's disease, recruited into condensates based on low complexity domains (LCDs) derived from the DEAD-box proteins Laf1, Dbp1 and Ddx4, which are associated with biological membraneless organelles. We show that interactions between Aß-42 and the scaffold proteins promote sequestration and local increase of the peptide concentration within the condensates. Yet, heterotypic interactions within the condensates inhibit the formation of amyloid fibrils. These results demonstrate that biomolecular condensates could sequester aggregation-prone proteins and prevent aberrant aggregation events, despite the local increase in their concentration. Biomolecular condensates could therefore work not only as hot-spots of protein aggregation but also as protective reservoirs, since the heterogenous composition of the condensates could prevent the formation of ordered fibrillar aggregates.

Synthesis and Characterization of a "Clickable" PBR28 TSPO-Selective Ligand Derivative Suitable for the Functionalization of Biodegradable Polymer Nanoparticles.

Reactive microgliosis is a pathological hallmark that accompanies neuronal demise in many neurodegenerative diseases, ranging from acute brain/spinal cord injuries to chronic diseases, such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD) and age-related dementia. One strategy to assess and monitor microgliosis is to use positron emission tomography (PET) by exploiting radioligands selective for the 18 kDa translocator protein (TSPO) which is highly upregulated in the brain in pathological conditions. Several TSPO ligands have been developed and validated, so far. Among these, PBR28 has been widely adopted for PET imaging at both preclinical and clinical levels, thanks to its high brain penetration and high selectivity. For this reason, PBR28 represents a good candidate for functionalization strategies, where this ligand could be exploited to drive selective targeting of TSPO-expressing cells. Since the PBR28 structure lacks functional moieties that could be exploited for derivatization, in this work we explored a synthetic pathway for the synthesis of a PBR28 derivative carrying an alkyne group (PBR-alkyne), enabling the fast conjugation of the ligand through azide-alkyne cycloaddition, also known as click-chemistry. As a proof of concept, we demonstrated in silico that the derivatized PBR28 ligand maintains the capability to fit into the TSPO binding pocked, and we successfully exploited PBR-alkyne to decorate zwitterionic biodegradable polymer nanoparticles (NPs) resulting in efficient internalization in cultured microglia-like cell lines.

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.

Surfactant-free and rinsing-resistant biodegradable nanoparticles with high adsorption on natural fibers for the long-lasting release of fragrances.

Synthetic polymers are attracting growing attention as additives for laundry and personal care products. In particular, the high volatility of many common fragrances requires the development of polymeric particles for their encapsulation and controlled release. Unfortunately, the vast majority of these carriers is made from polymers that are not biodegradable. This poses severe concerns about the accumulation of nano- and microplastics. Hence, such particles are expected to be banned from the market in the coming years. Therefore, biodegradable particles enabling a long-lasting release of the fragrances are urgently needed. In this work, we produced biodegradable nanoparticles (NPs) that are structurally composed of lactones, i.e. well known perfumes that occur naturally and that are already considered safe by regulatory agencies. We polymerized these lactones via ring opening polymerization (ROP) using an ionizable tertiary amine as initiator to produce in a single step amphiphilic oligoesters able to directly self-assemble into NPs once nanoprecipitated in water. In this way, we can produce biodegradable NPs with a perfume loading up to 85 % w/w without the need for additional surfactants. Subsequently we show that the ionizable group is able to confer a positive charge to our nanoparticles and, in turn, a high adsorption capacity on natural fibers (i.e. hairs and cotton fabric). Finally, we demonstrate the nanoparticle resistance to rinsing and their ability to confer a long-lasting fragrance perception to treated hair swatches for at least 3 weeks.