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The utility of messenger RNA (mRNA) as a therapy is gaining a broad interest due to its potential for addressing a wide range of diseases, while effective delivery of mRNA molecules to various tissues still poses a challenge. This study reports on the design and characterization of new ionizable amino-polyesters (APEs), synthesized via ring opening polymerization (ROP) of lactones with tertiary amino-alcohols that enable tissue and cell type selective delivery of mRNA. With a diverse library of APEs formulated into lipid nanoparticles (LNP), structure-activity parameters crucial for efficient transfection are established and APE-LNPs are identified that can preferentially home to and elicit effective mRNA expression with low in vivo toxicity in lung endothelium, liver hepatocytes, and splenic antigen presenting cells, including APE-LNP demonstrating nearly tenfold more potent systemic mRNA delivery to the lungs than vivo-jetPEI. Adopting tertiary amino-alcohols to initiate ROP of lactones allows to control polymer molecular weight and obtain amino-polyesters with narrow molecular weight distribution, exhibiting batch-to-batch consistency. All of which highlight the potential for clinical translation of APEs for systemic mRNA delivery and demonstrate the importance of employing controlled polymerization in the design of new polymeric nanomaterials to improve in vivo nucleic acid delivery.

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.

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.

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.

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Biophysical and biochemical characterisation of microalgae-derived extracellular vesicles.

The production of new proteins with messenger RNA (mRNA) has gained a broad interest due to its potential for addressing a wide range of diseases. Here, the design and characterization of novel ionizable poly(β-amino ester)-co-poly(caprolactone) terpolymers, synthesized via the combination of the ring opening polymerization and the Michael step-growth polymerization, are reported. The versatility of this method is demonstrated by varying the number of caprolactone units attached to each poly(β-amino ester) (PBAE) terpolymer. The ability of the novel poly-caprolactone (PCL)-based PBAE materials to deliver mRNA is shown to depend on the physiochemical characteristics of the material, such as lipophilicity, as well as the formulation method used to complex the polymer with the oligonucleotide. This latter variable represents a previously unstudied aspect of PBAE library screens that can play an important role in identifying true top candidates for nucleic acid delivery. The most stable terpolymer is injected intravenously (IV) in mice and shows a transfection efficacy several times higher than the polyethylenimine (PEI) which is focused in the spleen, opening the possibility to use these biodegradable carriers in the intravenous delivery of antigen-encoding mRNA for cancer immunotherapy and vaccination.