Polynucleotides in Cellular Mimics: Coacervates and Lipid Vesicles

Current Opinion in Colloid & Interface Science 26 (2016) 50–57

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Current Opinion in Colloid & Interface Science

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Polynucleotides in cellular mimics: Coacervates and lipid vesicles

Jeffrey R Vieregg a, T-Y Dora Tang b,⁎ a Institute for Molecular Engineering, University of Chicago, 5640 South Ellis Avenue, Chicago, IL 60637, USA b Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany article info abstract

Article history: In this review, we examine the interaction of nucleic acids with cell-like structures based on liquid–liquid phase Received 10 June 2016 separation of charged molecules (complex coacervation) and amphiphilic self-assembly (lipid vesicles). We Received in revised form 22 August 2016 discuss the mechanisms of their assembly and describe how they can be used as models for origin of life studies Accepted 2 September 2016 and for understanding two recently-described phenomena in modern cells: membrane-free organelles and Available online 28 September 2016 exosomes. Hybrid cells with increased structural complexity are highlighted and we then brieﬂy explore how strategies based on electrostatic and hydrophobic assembly can be used for designing and synthesizing delivery Keywords: Coacervate agents for therapeutic nucleic acids. While the physical mechanisms of self-assembly vary, both strategies Lipid vesicle provide viable routes for generating minimal compartmentalized systems, modeling cellular pathways, and for Self-assembly rational design of new synthetic cells for technological applications. Protocells © 2016 Elsevier Ltd. All rights reserved. Nucleic acids Drug delivery Therapeutics Synthetic cells

1. Introduction provide localization for heterogeneous reactions and are essential for the electrochemical gradients used for essential modern processes such Cells are the basic unit of self-replicating life on Earth. However, as ATP synthesis, but can require complex protein machinery to create, the complete mechanistic pathways that drive replication, homeostasis, maintain, and regulate flows of chemicals that cannot pass through them. information propagation and evolution, are still poorly understood. In Modern cellular biochemistry also provides clues with respect to addition, how biological life might have evolved from simple chemical information propagation in early life scenarios. Of the various biological processes remains an open, unanswered question. Therefore, synthesiz- molecules, only nucleic acids (RNA and DNA) have the capability to ing abiotic cellular analogs, or “protocells”, is an important route for catalyze chemical reactions and template their own genetic propaga- describing and understanding biological mechanisms and offers in- tion. The “RNA World” hypothesis imagines a scenario on prebiotic triguing models to describe evolutionary pathways in the pre-biotic earth where self-replicating RNA molecules were both enzymes and world [1,2]. In order to synthesize suitable protocells it is important genes; today, these functionalities are primarily fulfilled by proteins to connect molecular structure with self-assembly processes capable and DNA. As there is no material evidence for the “RNA World” hypoth- of supporting key features required for life, including concentration of esis it remains speculative [6], however the discovery that ribosomes functional molecules, creation of distinct chemical environments, and are, at their core, ribozymes [7•], strongly indicates that RNA preceded information propagation. proteins in the chemical evolution of early life, thus highlighting the Compartmentalization provides a mechanism for increasing local importance of considering polynucleotides in early life scenarios and concentrations of enzymes and substrates sufficiently to drive chemical in modern natural and synthetic self-assembled structures. reactions, and is a necessary feature for enriching functional molecules In this review, we discuss the interaction of nucleic acids with two and their precursors. One plausible scenario for prebiotic compartmental- types of self-assembled systems that display cell-like properties: complex ization describes the spontaneous assembly and concomitant chemical coacervates and hydrophobic assemblies such as micelles and vesicles. enrichment of charged molecules to form membrane free droplets called For each type, we briefly discuss their mechanisms of formation and coacervates [3••]. An alternative route to chemical compartmentalization how these processes may be applied to early life scenarios, compartmen- is via the formation of lipid membrane bound compartments, analogous talization phenomena in modern biology, and for therapeutics. All of to the membrane structure of modern cells [1,4,5]. Membranes also these topics are exciting areas of research in their own right, and space does not permit a full exploration of any of them here. We instead attempt to highlight recent research results and interesting connections, ⁎ Corresponding author. while referring the reader to reviews for more comprehensive explana- E-mail address: [email protected] (T.-Y.D. Tang). tions of the underlying phenomena.

2. Electrostatic assembly: complex coacervate protocells form from small molecular weight molecules, particularly nucleotides and their activated derivatives. This was first demonstrated in 2011 by Interactions between oppositely charged molecules can lead to Koga et al. [22••], who showed that coacervate microdroplets (Fig. 2a) phase separation, forming either liquid droplets (complex coacervation) could be formed from nucleoside triphosphates (ATP), diphosphates or solid precipitates depending on the length, charge density and type (ADP, FAD, NAD), and monophosphates (AMP) when mixed with of macromolecules. Complex coacervation refers to spontaneous forma- short (2–10 amino acid (aa)) lysine polypeptides (OLys) that might tion of a polymer-rich liquid phase in dynamic equilibrium with a plausibly be produced by prebiotic processes [23]. This study showed polymer-poor phase [8,9••] via electrostatic interactions between oppo- that phase separation of small molecular weight ions has many similari- sitely charged molecules in aqueous solution. This process was first ties to complexation of larger polyelectrolytes. The dependence on elec- described by Bungenberg de Jong in the 1920s in mixtures of gelatin trostatic interactions, for example, is shown by the increase of the critical (polycation) and gum arabic (polyanion) [10]. While a complete quanti- concentration required for coacervation (CCC) with decreasing negative tative model is still lacking, many aspects of complex coacervation are charge from ATP N ADP N AMP (Fig. 2b). In addition, they found that qualitatively understood for long polyelectrolytes. The formation of increasing the molecular weight of Poly(diallyldimethylammonium) macroion pairs between oppositely charged molecules is driven by an (Poly(DADMAC)) from 150 to 275 kDa increased charge neutralization increase of entropy from the release of low valence counterions and (with ATP) from 70 to 90%, These results suggest that increased hydro- water rearrangement as they associate. The macroion pairs then assem- phobicity, decreased solubility and increased orientational freedom ble into larger clusters, which leads to macroscopic phase separation from longer polymer chains all contribute to increasing charge neutrali- (Fig. 1a) [9••,11]. Significantly, the dense phase, while highly enriched zation at the CCC. in polymer, remains highly hydrated with a large concentration of Nucleotide coacervate droplets exhibit dynamic behaviors such as counterions. The relative importance of ion pairing, hydration effects, coalescence and surface wetting (Fig. 2a) but remain stable over a and long-range electrostatics remains uncertain [12–14••] as do the broad pH and temperature range (between pH 2 and 10 and up to factors determining whether a particular macroion pair will form a 80 °C respectively). The coacervates also produce substantial chemical liquid or a solid condensed complex [15]. As polyelectrolytes can be enrichment of charged molecules, with OLys (2–10 aa)/ATP droplets chemically diverse the process of complex coacervation is molecularly displaying a twenty-fold increase in ATP concentration relative to the non-specific. Indeed, since the 1920s complex coacervation has been dilute phase [22] and 15 kD poly(allylamine) droplets providing a observed between hundreds of different natural and synthetic polymers 300× increase of fluorescent solutes, as well as substantial enrichment [8,16,17••]. Their properties including ease of formation, high viscosity, of Mg2+ and RNA oligonucleotides [24]. The dielectric constant of the strong adhesion and high encapsulation efficiencies have led to a droplet interior is lower than that of the dilute phase, which provides range of industrial applications including food additives [18] and elec- a mechanism for sequestering low-charge molecules such as protein tronic ink [19], as well as therapeutic assemblies (see Section 5). enzymes. Sequestration provides a molecular route to accelerate bio- In 1924, Alexander Oparin put forward the idea that colloidal chemical reactions, primitive metabolism, and information processing microdroplets formed via coacervation were the earliest metabolic by increasing the concentrations of substrates, as well as by crowding. units in a reducing ‘prebiotic soup’ [20]. Over the next few decades, This was demonstrated by Crosby et al. for the enzyme actinorhodin Oparin and co-workers demonstrated chemical enrichment within the polyketide synthase, which displayed an 18× rate increase when droplets, in-situ enzymatic reactions, and droplet growth and fission sequestered into ATP–PDDA coacervate droplets [25]. Significantly, reminiscent of cellular life [21••].This‘metabolism-first’ approach, how- mRNA transcription and sustained translation into protein have also ever, provided no clear connection to genetic evolution and information been demonstrated inside coacervates (Fig. 2c) [26•]. Taken together, propagation via nucleic acids that would have been a key step at the these studies support the argument that coacervate microdroplets onset of life. Their experiments also presumed the existence of large formed from nucleotide anions are viable platforms for protocell evolu- macromolecules and polymers that are unlikely to have existed in a tion. Indeed, it has recently been shown that RNA within RNA–peptide prebiotic environment. coacervates will elongate via temperature cycling. These results demon- In order for coacervates to be viable protocells, with the ability to strate a plausible scenario where compartmentalization is coupled with sustain both chemical and genetic evolution, they must be able to chemical evolution of polynucleotides [27].

Fig. 1. Complex coacervation of charged molecules. (a) Oppositely-charged macromolecules form ion pairs that aggregate into membrane-free coacervate droplets. Molecular structure of (b) Poly(lysine), (c) Adenosine triphosphate (ATP) and (d) deoxyribonucleic acid (DNA, sequence 5′-AGC-3′). At neutral pH, poly(lysine) and DNA each contain one charge per residue, while nucleotide polyphosphates have one negative charge per phosphate group. 52 J.R. Vieregg, T.-Y.D. Tang / Current Opinion in Colloid & Interface Science 26 (2016) 50–57

Fig. 2. Membrane free artificial cells. (a) Mixing polycations (PDDA) and anions (ATP) leads to phase separation, as shown by turbidity and droplet formation (4:1 molar ratio PDDAwith ATP, 50 mM, pH 8). Brightfield microscopy image (scale bar 20 μm) shows wetting behavior of microdroplets on a glass slide. (b) Critical coacervation concentration (CCC) vs anion charge for nucleotide polyphosphates: AMP (1−), ADP (2−), ATP (3−), modified from [22••]. (c) Fluorescent protein (mCherry) expression in coacervate phase vs time [26•] published from the • Royal Society of Chemistry. (d) Fluorescence microscopy images of polylysine-RNA coacervate (scale bar 2 μm) with BODIPY FL C16 [69 ]. (e) Fatty acid coated coacervate microdroplets. Heterogeneous distribution of lipophilic dye indicates fatty acid coating on the outer surface of the coacervate droplets (scale bar 2 μm) [69•].

The coacervates discussed thus far are formed from ribonucleotide formation [32]. In vitro results support this hypothesis, as exemplified monomers, but longer nucleic acids are also capable of forming com- by the Ddx4 protein, which forms nuage bodies in humans and P gran- plexes with polycations, as has been demonstrated with polyribonucle- ules in worms [39]. Liquid droplets formed by the N-terminal domain of otides and tRNA [28••,29]. Experience with synthetic polyelectrolytes, Ddx4 are destabilized to a similar extent by increased salt concentration as well as theoretical considerations, suggests that polynucleotides and by neutralization (via methylation) of multiple positively charged will experience stronger confinement than individual monomers, and arginine residues, although cation–pi interactions also appear to play that their affinity for the coacervate phase will increase with their a role in phase separation. Interestingly, membrane-free Ddx4 liquid length [8,14]. This may overcome one challenge for coacervates as droplets also exhibited differential chemical environments as single- functional protocells, namely that the lack of a membrane leads to low stranded DNA was concentrated within the droplets while double- confinement times [30], while coupling the chemical evolution of poly- stranded DNA was largely excluded. Measurements with the RNA- nucleotides to more efficient compartmentalization. binding proteins LAF-1 [40••], hnRNPA1 [41], and Whi3 [42] also point to electrostatics as a key driving force for droplet formation, as well as 2.1. Membrane-free organelles: intracellular phase separation the crucial role of RNA. For example in-vitro experiments with liquid droplets formed from Whi3 showed a phase transition to gels and The facile mechanisms of synthetic droplet formation via electrostatic solid fibers under certain conditions. Similar phenomena were observed self-assembly have also been observed in present day biological systems. with a prion-like protein FUS [43–45•] (Fig. 3d), where ALS-associated For example, DNA is condensed to near crystalline density by basic mutations accelerated the solidification process. These results indicate proteins and small polyamines in the nucleoid of bacteria, spermatozoa that in some instances intracellular phase transitions may also be asso- heads, and viral capsids by the same electrostatic interactions that ciated with disease when proteins or RNA concentrations are mis- drive coacervate formation [31]. Over the last decade, it has been pro- regulated. posed that liquid–liquid phase separation is the mechanism driving the formation of RNA granules, processing (P) bodies, Cajal bodies, and 3. Hydrophobic assembly: vesicles the nucleolus and other membrane-free compartments, in eukaryotic cells (Fig. 3) [32–37 (34••,35•)]. Like the synthetic coacervates these Modern cells are physically defined by semi-permeable lipid bilayers membrane-free organelles are dynamic but appear to have significant composed primarily of phospholipid amphiphiles (two hydrocarbon involvement in essential cellular processes such as replication, signaling, chains linked by a phosphate head group, Fig. 4ai) and sterols (e.g. choles- stress response, and disease. terol, Fig. 4aii), along with numerous proteins. As this structural element The mechanisms of phase separation are not fully understood is ubiquitous across all kingdoms it is likely that a similar lipid membrane but it appears that intrinsically disordered proteins (IDPs) with low- was present in the last common ancestor. Self-assembly of lipid amphi- complexity sequence domains and charged residues are intimately philes into membrane-delineated compartments for protocell models involved in the formation of these membrane-free organelles [34,38]. and therapeutic assemblies has therefore attracted strong interest over In addition, many of these compartments either contain large fractions the last half a century. Above a critical concentration, amphiphilic lipid of RNA or require it for assembly, as in the case of stress granules monomers will self-assemble in water, driven by the hydrophobic effect, (SGs). The presence of these charged molecules suggests that electro- to form micelles (Fig. 4bi)orvesicles(Fig. 4bii) with bilayer membranes statics play an important role in liquid droplet formation. Moreover, and an aqueous interior. The latter strongly resembles the basic structure studies have shown that increasing the negative charge of liquid of modern cells and organelles, and a large number of studies have forming protein domains by serine phosphorylation inhibits SG examined vesicular behaviors such as budding and fission [46],aswell J.R. Vieregg, T.-Y.D. Tang / Current Opinion in Colloid & Interface Science 26 (2016) 50–57 53

Fig. 3. Liquid-phase separations in vivo. (a) Freely-diffusing RNA and protein form phase-separated liquid droplets (ii) that can mature into ordered solids (iii). Modified from [39]. (b) RNA–protein liquid droplets (P granules) in a dividing Caenorhabditis elegans germ cell [37]. Droplets are actively transported to the posterior (P) end of the single-cell embryo over ~10 min prior to division into a germ and a somatic cell. Green: GFP-tagged PGL-1 protein; Red: DIC image. (c) Temperature-concentration phase diagram of Ddx4 protein in vitro [39]. Methylation of multiple arginine residues (red line) destabilizes droplets to a similar extent as doubling the NaCl concentration, consistent with a primarily electrostatic interaction. (d) In vitro, GFP-tagged FUS protein forms small liquid droplets (left, t = 0) that coalesce (middle, t = 4 h) and mature into fibrous solids (right, t = 8 h) [45]. as encapsulation of complex biochemical reactions such as protein for energy harvesting and signaling in modern cells but also prevents expression [47–49••] as a proxy to model cellular processes. Moreover, importation of activated nucleosides and essential ions. Phospholipid studies that have coupled RNA encapsulation and RNA interactions bilayers are also very stable, making growth and division difficult without with cell membranes give new insights into origins of life, as well as pro- the aid of modern enzymes such as flippases and permeases. By contrast, viding tools for therapeutic delivery of nucleic acids. single-chain fatty acids (Fig. 4aiii) form dynamic bilayers with leaflet exchange times on the order of seconds and residence times on the 3.1. Vesicle protocells in early life order of minutes, enabling growth/decay dynamics and selective permeability via transient defect formation [50]. Fatty acids are also relatively While essential for modern life, phospholipid membranes have easy to synthesize abiotically, being detected in meteorites at abundances several characteristics that make them problematic for early-life scenarios 20 times that of amino acids [4]. At certain concentrations fatty acids [1,4]. The bilayer formed by phospholipids is highly impermeable to will form micelles or vesicles depending on the pKa of the acid and the charged compounds, which enables high electrochemical potentials pH of the solution. The chemistry and dynamics of fatty acid assemblies

Fig. 4. Hydrophobic protocells. (a) Examples of lipid membrane building blocks (i) Phospholipid, palmitoyl-oleoyl-sn-phosphatidylcholine (POPC), (ii) cholesterol, (iii) fatty acid (gadoleic acid), (iv) cationic lipid N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP). (b) In aqueous solution, amphiphiles can self-assemble into either (i) micelles or (ii) vesicles with bilayer membranes depending on their structure and concentration. (c) A plausible scenario for coupled protocell growth and division. RNA elongation swells vesicles, causing them to grow at the expense of empty vesicles. In the presence of excess amphiphile and micelles, vesicles grow and become unstable, leading to splitting due to mechanical stress or changing environment. The vesicles containing self-replicating RNA grow, thus spiraling toward increased functionality. 54 J.R. Vieregg, T.-Y.D. Tang / Current Opinion in Colloid & Interface Science 26 (2016) 50–57 are amply reviewed elsewhere; here we focus on a few key ques- Exosomes are found in diverse body fluids, and are produced tions related to their interactions with polynucleotides or origin of by many types of cells. In contrast to microvesicles [58],exosomes life studies. are derived from late endosomes, also known as multi-vesicular The first of these relates to activated ribonucleosides and chain bodies, and released into the environment via SNARE-mediated elongation. Protein nucleic acid polymerases use basic residues to stabi- fusion within the plasma membrane. This process is initiated by lize the high negative charge transition state containing a nucleoside varying signaling pathways in different cell types, and is regulated triphosphate (NTP) and nucleic acid in close proximity. Lacking this by RAB and ESCRT proteins [57].Comparedtotheplasmamem- ability, ribozyme polymerases rely on divalent cations such as Mg2+ brane, exosome membranes are enriched in raft-type lipids such at concentrations up to hundreds of mM [51], which causes precip- as cholesterol and sphingomyelin [58] and possess generic and itation of fatty acids, membrane destruction [5] and polynucleotide cell-type specific membrane protein and glycosylation signatures hydrolysis. Non-enzymatic template elongation reactions also require [59]. The latter can provide a “delivery address” for tissue-specific high Mg2+ concentrations, making it difficult to reconcile NTP poly- targeting. A key discovery showed that exosomes contained mRNA merization with the physical requirements of fatty acid self-assembly. and miRNA that were both active in target cells [60–63••].Further- One way around this problem is to use nucleotides with alternate more, exosomal RNA populations differ from those of the parent leaving groups such as imidazole, 2-methylimidazole (2MeIm), and cell, a characteristic of functional signaling [64]. Much remains to 1-hydroxy-7-azabenzotriazole, which carry a lower charge (1− vs 3− bediscoveredaboutthecontentofexosomalmessages,butthey for NTPs) and react more readily than NTPs. If citrate is included have been implicated in pathological processes such as tumor metas- as a chelating agent, 2MeIm-activated nucleotides polymerize non- tasis, viral infection, drug resistance, and immune response, as well enzymatically on a single-stranded template at a rate of 0.67 h−1 in as embryonic development [57,64]. A deeper understanding of the solution and copy short templates overnight in oleic acid vesicles role and functions of exosomes in biology should help unlock their [52••]. This does not solve the problem for ribozymes, however, and nei- potential for therapeutics and non-invasive diagnostics. As an exam- ther citrate nor the alternate activated nucleosides have been produced ple, researchers have attempted to deliver therapeutic RNAs with by plausible abiotic reactions. It may be that more efficient ribozyme exosomes, either by loading purified exosomes and injecting them polymerases can be found; ribozyme ligases have been identified that into the body or by transfecting host cells with therapeutic RNAs function with Mg2+ concentrations as low as 2 mM in the presence to be packaged and exported [65•]. Currently, exosome-mediated de- of fatty acid vesicles and low-concentration EDTA as a chelator [53]. livery lags behind engineered vesicle systems (Section 5) [66,67], Another attractive possibility is charge reduction through complexation but the intense interest in the field suggests that progress may be of either the NTP or polynucleotides by basic peptides, analogous to rapid in this area. (or even composed of) the coacervates discussed above [27]. A second problem involves coupling vesicle growth to replication of 4. Hybrid systems: electrostatic and hydrophobic assembly together the interior contents, a key requirement for effective chemical evolution. In this case, polynucleotides can provide a solution, via osmotic Combining electrostatic and hydrophobic assembly allows for pressure. Due to their higher charge density, polynucleotides complex creation of more complex systems with enhanced capabilities. Lipids more counterions compared to monomers, particularly if they are with cationic head groups are commonly used to co-assemble lipid folded. It has been proposed that RNA elongation generates a decrease vesicles with nucleic acid polyanions (Section 5), and electrostatically- in osmotic pressure that drives vesicle growth at the expense of less assembled nanoparticles can be further stabilized by functionalizing the active vesicles [54] successfully coupling polymerase evolution to com- anionic RNA component with cholesterol [68•]. Hybrid electrostatic– petition at the protocellular scale (Fig. 4c). Larger vesicles are also more hydrophobic assembly is also interesting for early-life protocell likely to be split by external shear stress and, if they are multilamellar, studies, as coacervates and lipid vesicles are in many ways comple- are subject to “pearling” instabilities, both of which provide plausible mentary in terms of capabilities required for self-replication. Coac- pathways for division [1]. ervation readily concentrates charged molecules such as nucleic A final problem relates to the eventual transition from simple fatty acids and activated nucleotides, but does not allow for internal acid vesicles to more stable membranes required for evolution of structure or heterogeneous catalysis and has limited capability to more sophisticated reactions. As mentioned above, phospholipid bilay- sustain electrochemical gradients at the boundary. Lipid vesicles ers are impermeable to charged molecules. Modern cells solve this provide powerful models for compartmentalization, but lack a problem by using protein pores and channels to selectively regulate mechanism for enriching their interiors with ions and hydrophilic flows into and out of the cell, sustaining concentration differences biomolecules. Co-assembly of coacervates and vesicles is a promis- required for metabolism. Several results indicate that RNA is also capa- ing route to more complete cellularity, as is encapsulation of pre- ble of forming pores in phospholipid bilayers. Vlassov et al. showed that formed coacervates. The latter was recently demonstrated by adding multimeric RNA sequences are capable of permeabilizing liposomes sodium oleate to positively-charged coacervates formed from RNA and membranes via interaction with the phosphate head groups [55], or ATP and polylysine or poly(DADMAC) [69•].Intheabsenceof and subsequent work demonstrated selective transport of tryptophan oleic acid, a lipophilic dye (BODIPY FL C16) partitioned homoge- amino acids, thus demonstrating that the RNA world can support mem- neously within the coacervate droplets due to the lower dielectric brane transport [50]. constant of the interior compared to the dilute exterior phase. After the addition of the fatty acid, fluorescence was observed at the drop- 3.2. Exosomes: protocells in the modern world? let boundary only, indicating the formation of a lipid membrane. Small angle X-ray scattering measurements showed Bragg peaks Exosomes are small (30–120 nm diameter) lipid vesicles that consistent with the formation of a multilamellar membrane, and lack internal organelles and circulate independently in the extracellular dye uptake measurements showed differential permeability based environment; as such, they share many of the physico-chemical charac- on charge and molecular weight. Increasing the exterior ionic teristics of lipid membrane based protocells despite very different strength resulted in droplet disassembly and growth, reminiscent biogenesis and functions. Originally thought to be cellular waste prod- of the encapsulated PEG/dextran systems discussed earlier. While ucts, they are now known to function in intercellular communication much work remains, this study shows that very simple chemical and and have become the subject of intense interest due to their ability physical processes are capable of transforming various membrane-free to alter the phenotype of distant cells via transport of proteins and coacervate droplets, including OLys/RNA, into membrane-encapsulated microRNAs (miRNAs) [56,57]. protocells. J.R. Vieregg, T.-Y.D. Tang / Current Opinion in Colloid & Interface Science 26 (2016) 50–57 55

5. Electrostatic assemblies and engineering lipid vesicles for References and recommended reading•,•• therapeutics [1] Schrum JP, Zhu TF, Szostak JW. The origins of cellular life. Cold Spring Harb Perspect Biol 2010;2:a002212. http://dx.doi.org/10.1101/cshperspect.a002212. Therapeutic nucleic acids come in several forms, including DNA [2] Mann S. Systems of creation: the emergence of life from nonliving matter. Acc Chem (gene therapy), RNA duplexes (siRNA), and oligonucleotides for Res 2012;45:2131–41. http://dx.doi.org/10.1021/ar200281t. [3]•• Li M, Huang X, Tang TYD, Mann S. Synthetic cellularity based on non-lipid microRNA and antisense pathways, but they face common challenges micro-compartments and protocell models. Curr Opin Chem Biol 2014;22: as drugs. To be effective, within the target tissue, the nucleic acids 1–11. http://dx.doi.org/10.1016/j.cbpa.2014.05.018. A review summarizing must evade clearance by the liver and kidney, cross the hydrophobic the state of the art of synthetic cells specifically focusing on colloidosomes, cell membrane, and avoid degradation by nuclease enzymes both proteinosomes and coacervates. [4] Chen IA, Walde P. From self-assembled vesicles to protocells. Cold Spring Harb outside and inside the cell [70]. The same electrostatic and hydrophobic Perspect Biol 2010;2, a002170–0. http://dx.doi.org/10.1101/cshperspect.a002170. interactions that give rise to protocells can also be harnessed to produce [5] Monnard P-A, Walde P. Current ideas about prebiological compartmentalization. Life – self-assembling delivery systems that offer solutions to several of these 2015;5:1239 63. http://dx.doi.org/10.3390/life5021239. [6] Robertson MP, Joyce GF. The origins of the RNA world. Cold Spring Harb Perspect problems. A detailed description of these systems is beyond the scope Biol 2012;4:a003608. http://dx.doi.org/10.1101/cshperspect.a003608. fi of this review, instead, we mention the basic chemical and physical [7]• Steitz TA, Moore PB. RNA, the rst macromolecular catalyst: the ribosome is a strategies and provide references for the interested reader. ribozyme. Trends Biochem Sci 2003;28:411–8. http://dx.doi.org/10.1016/S0968- 0004(03)00169-5. Review paper which summarises the structural evidence Encapsulation by coacervate and solid polyelectrolyte complexes obtained over the last 60 years that confirms that RNA holds genetic information formed from nucleic acids and cationic polymers (polyplexes) has and is an enzyme. been extensively studied as non-viral transfection vectors both in vitro [8] van der Gucht J, Spruijt E, Lemmers M, Stuart MAC. Polyelectrolyte complexes: – • – bulk phases and colloidal systems. J Colloid Interface Sci 2011;361:407 22. and in vivo [65 ,71 73]. One strategy to prevent aggregation, reduce http://dx.doi.org/10.1016/j.jcis.2011.05.080. unwanted interactions with serum proteins, and promote longer circu- [9]•• Priftis D, Laugel N, Tirrell M. Thermodynamic characterization of polypeptide lation time is based on functionalizing the polycations with neutral complex coacervation. Langmuir 2012;28:15947–57. http://dx.doi.org/10. 1021/la302729r. Experimental paper which probes the thermodynamics of hydrophilic polymers such as polyethylene glycol (PEG). In the case of complex coacervation using isothermal calorimetry and shows that the process is polyplexes this leads to the formation of nanoparticles with a coacervate primarily endothermic and is driven by entropic contributions. or solid complex core surrounded by a PEG corona and typical diameters [10] de Jong HB, Kruyt HR. Coacervation (partial miscibility in colloid systems). Proc K – between 20 and 200 nm [74]. Layer-by-layer electrostatic assembly Ned Akad Wet 1929;32:849 56. [11] Burgess DJ. Practical analysis of complex coacervate systems. J Colloid Interface Sci can also be used to produce nanoparticles and functionalize surfaces 1990;140:227–38. http://dx.doi.org/10.1016/0021-9797(90)90338-O. of implanted devices [75,76]. [12] Kudlay A, Ermoshkin AV, Olvera de la Cruz M. Complexation of oppositely charged – Hydrophobically-assembled micelles, or liposomes, have also polyelectrolytes: effect of ion pair formation. Macromolecules 2004;37:9231 41. http://dx.doi.org/10.1021/ma048519t. been extensively engineered to be effective delivery platforms for [13] Spruijt E, Westphal AH, Borst JW, Cohen Stuart MA, van der Gucht J. Binodal • nucleic acids [65 ]. Cationic and ionizable lipids enable efficient load- compositions of polyelectrolyte complexes. Macromolecules 2010;43:6476–84. ing of anionic polynucleotides and release upon endocytosis, though http://dx.doi.org/10.1021/ma101031t. [14]•• Fu J, Schlenoff JB. Driving forces for oppositely charged polyion association in aqueous in vivo potency and toxicity remain challenging [77,78].Aswith solutions: enthalpic, entropic, but not electrostatic. J Am Chem Soc 2016;138:980–90. polyplexes, PEG-functionalized liposomes (stable nucleic acid lipid http://dx.doi.org/10.1021/jacs.5b11878. This paper suggests that electrostatic field particles, or SNALPS) display improved circulation time, reduced theory gives a qualitative description of complex-coacervation and breaks down when considering close range interactions between polyelectrolytes. toxicity and immunogenicity and have been used in multiple clinical • [15] Wang Q, Schlenoff JB. The polyelectrolyte complex/coacervate continuum. Macro- trials [70,79 ]. Other strategies for modifying and tuning the delivery molecules 2014;47:3108–16. http://dx.doi.org/10.1021/ma500500q. vehicles include incorporation of targeting moieties and/or labile [16] de Kruif CG, Weinbreck F, de Vries R. Complex coacervation of proteins and anionic polysaccharides. Curr Opin Colloid Interface Sci 2004;9:340–9. http://dx.doi.org/10. linkages for intracellular disassembly [80]. Nucleic acids can also be 1016/j.cocis.2004.09.006. made amphiphilic by conjugating them with hydrophobic groups, [17]•• Miller DR, Das S, Huang K-Y, Han S, Israelachvili JN, Waite JH. Mussel coating protein- enabling interesting new types of self-assembled nanostructures derived complex coacervates mitigate frictional surface damage. ACS Biomater – [81••,82]. Sci Eng 2015;1:1121 8. http://dx.doi.org/10.1021/acsbiomaterials.5b00252. This work explores the effect of coacervation on the coefficient of friction between purified proteins from mussels and hyaluronic acid as adhesive models for mussels. [18] Gouin S. Microencapsulation. Trends Food Sci Technol 2004;15:330–47. 6. Conclusion http://dx.doi.org/10.1016/j.tifs.2003.10.005. [19] Dai R, Wu G, Li W, Zhou Q, Li X, Chen H. Gelatin/carboxymethylcellulose/dioctyl sulfosuccinate sodium microcapsule by complex coacervation and its application In this review, we have discussed how cellular mimics can be for electrophoretic display. Colloids Surf A Physicochem Eng Asp 2010;362:84–9. produced by self-assembly of charged biomolecules (complex http://dx.doi.org/10.1016/j.colsurfa.2010.03.041. [20] Oparin AI. Origin of life. New York: Macmillan; 1938. coacervation) and lipid amphiphiles (vesicles) and discussed their [21]•• Oparin AI, Gladilin KL. Evolution of self-assembly of probionts. Biosystems 1980;12: utility for prebiotic pathways, biological organization and (briefly) 133–45. Summary of the compartmentalisation properties required for the onset therapeutic applications. Both mechanisms incorporate essential of life on earth with experimental summary of these properties as demonstrated within coacervates. features of modern cells, and their spontaneous assembly properties – [22]•• Koga S, Williams DS, Perriman AW, Mann S. Peptide nucleotide microdroplets as suggest that they might have been viable routes for cellular evolution a step towards a membrane-free protocell model. Nat Chem 2011;3:720–4. http:// on early earth and these possibilities are still under investigation. Addi- dx.doi.org/10.1038/nchem.1110. First description of coacervate formation from low molecular weight, prebiotic molecules including nucleotides such as ATP and tionally, the incorporation of synthetic biology and high-throughput •• FAD. approaches [83–85 (84 )] provide a valuable complement to the more [23] Fox SW. The evolutionary significance of phase-separated microsystems. Orig Life physico-chemical approaches described here, raising hopes for under- 1976;7:49–68. standing the basic principles governing how cells evolved, and how [24] Frankel EA, Bevilacqua PC, Keating CD. Polyamine/nucleotide coacervates provide strong compartmentalization of Mg2+, nucleotides, and RNA. Langmuir 2016;32: we can use cellular mimics in the modern world. 2041–9. http://dx.doi.org/10.1021/acs.langmuir.5b04462. [25] Crosby J, Treadwell T, Hammerton M, Vasilakis K, Crump MP, Williams DS, et al. Stabilization and enhanced reactivity of actinorhodin polyketide synthase minimal complex in polymer–nucleotide coacervate droplets. Chem Commun 2012;48: Acknowledgments 11832–4. http://dx.doi.org/10.1039/c2cc36533b. [26]• Tang TYD, van Swaay D, deMello A, Anderson JLR, Mann S. In vitro gene expression T-Y T's work was supported by the MaxSynBio, MPG within membrane-free coacervate protocells. Chem Commun 2015;51:11429–32. (M.IF.A.MOZGDT80). We would like to acknowledge F. Friedrich at MTO (Media Technologies and Outreach) group at MPI-CBG • Of special interest. for assistance with the graphical abstract. •• Of outstanding interest. 56 J.R. Vieregg, T.-Y.D. Tang / Current Opinion in Colloid & Interface Science 26 (2016) 50–57

http://dx.doi.org/10.1039/C5CC04220H. Successful demonstration of complex multi- 1241888. Work which describes the use of citrate to reconcile the disruptive effects step reactions such as protein expression within membrane-free coacervates. of high concentration Mg2+ on fatty acid vesicles to successfully allow RNA copying [27] Jia TZ, Fahrenbach AC, Kamat NP, Adamala KP, Szostak JW. Oligoarginine peptides within fatty acid vesicles. slow strand annealing and assist non-enzymatic RNA replication. Nat Chem 2016. [53] Anella F, Danelon C. Reconciling ligase ribozyme activity with fatty acid vesicle http://dx.doi.org/10.1038/nchem.2551. stability. Life 2014;4:929–43. http://dx.doi.org/10.3390/life4040929. [28]•• Aumiller WM, Keating CD. Phosphorylation-mediated RNA/peptide complex [54] Chen IA, Roberts RW, Szostak JW. The emergence of competition between model coacervation as a model for intracellular liquid organelles. Nat Chem 2016;8: protocells. Science 2004;305:1474–6. http://dx.doi.org/10.1126/science.1100757. 129–37. http://dx.doi.org/10.1038/nchem.2414. First extensive study of RNA/ [55] Vlassov A, Khvorova A, Yarus M. Binding and disruption of phospholipid bilayers by peptide based coacervates and how they can be repeatedly dissolved and formed supramolecular RNA complexes. Proc Natl Acad Sci U S A 2001;98:7706–11. http:// via phosphorylation and dephosphorylation dx.doi.org/10.2307/3056063?ref=search-gateway: [29] Porschke D. The binding of Arg-and Lys-peptides to single stranded polyribonucleo- 098e00639238a272052788e5c598ac28. tides and its effect on the polymer conformation. Biophys Chem 1979;10:1–16. [56] Vlassov AV, Magdaleno S, Setterquist R, Conrad R. Exosomes: current knowledge of http://dx.doi.org/10.1016/0301-4622(79)80001-0. their composition, biological functions, and diagnostic and therapeutic potentials. [30] Jia TZ, Hentrich C, Szostak JW. Rapid RNA exchange in aqueous two-phase system Biochim Biophys Acta Gen Subj 1820;2012:940–8. http://dx.doi.org/10.1016/j. and coacervate droplets. Orig Life Evol Biosph 2014;44:1–12. http://dx.doi.org/10. bbagen.2012.03.017. 1007/s11084-014-9355-8. [57] Azmi AS, Bao B, Sarkar FH. Exosomes in cancer development, metastasis, and drug [31] Teif VB, Bohinc K. Condensed DNA: condensing the concepts. Prog Biophys Mol Biol resistance: a comprehensive review. Cancer Metastasis Rev 2013;32:623–42. 2011;105:208–22. http://dx.doi.org/10.1016/j.pbiomolbio.2010.07.002. http://dx.doi.org/10.1007/s10555-013-9441-9. [32] Kedersha N, Ivanov P, Anderson P. Stress granules and cell signaling: more than just [58] Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. a passing phase? Trends Biochem Sci 2013;38:494–506. http://dx.doi.org/10.1016/j. J Cell Biol 2013;200:373–83. http://dx.doi.org/10.1083/jcb.201211138. tibs.2013.07.004. [59] Batista BS, Eng WS, Pilobello KT, Hendricks-Muñoz KD, Mahal LK. Identification of a [33] Brangwynne CP. Phase transitions and size scaling of membrane-less organelles. conserved glycan signature for microvesicles. J Proteome Res 2011;10:4624–33. J Cell Biol 2013;203:875–81. http://dx.doi.org/10.1083/jcb.201308087. http://dx.doi.org/10.1021/pr200434y. [34]•• Toretsky JA, Wright PE. Assemblages: functional units formed by cellular phase [60] Ratajczak J, Wysoczynski M, Hayek F, Janowska-Wieczorek A, Ratajczak MZ. separation. J Cell Biol 2014;206:579–88. http://dx.doi.org/10.1083/jcb.201404124. Membrane-derived microvesicles: important and underappreciated mediators Review describing the role of intrinsically disordered proteins and their structural of cell-to-cell communication. Leukemia 2006;20:1487–95. http://dx.doi.org/10. function in cytoplasmic organisation and role in cellular disease. 1038/sj.leu.2404296. [35]• Zhu L, Brangwynne CP. Nuclear bodies: the emerging biophysics of nucleoplas- [61] Mittelbrunn M, Gutiérrez-Vázquez C, Villarroya-Beltri C, González S, Sánchez-Cabo mic phases. Curr Opin Cell Biol 2015;34:23–30. http://dx.doi.org/10.1016/j.ceb. F, González MÁ, et al. Unidirectional transfer of microRNA-loaded exosomes from 2015.04.003. Review summarising the role of RNA-protein bodies for regulation T cells to antigen-presenting cells. Nat Commun 2011;2:282. http://dx.doi.org/ within the cell. 10.1038/ncomms1285. [36] Wallace EWJ, Kear-Scott JL, Pilipenko EV, Schwartz MH, Laskowski PR, Rojek AE, [62] Montecalvo A, Larregina AT, Shufesky WJ, Stolz DB, Sullivan MLG, Karlsson JM, et al. Reversible, specific, active aggregates of endogenous proteins assemble upon et al. Mechanism of transfer of functional microRNAs between mouse dendritic heat stress. Cell 2015;162:1286–98. http://dx.doi.org/10.1016/j.cell.2015.08.041. cells via exosomes. Blood 2012;119:756–66. http://dx.doi.org/10.1182/blood- [37] Brangwynne CP, Eckmann CR, Courson DS, Rybarska A, Hoege C, Gharakhani J, et al. 2011-02-338004. Germline P granules are liquid droplets that localize by controlled dissolution/ [63]•• Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated condensation. Science 2009;324:1729–32. http://dx.doi.org/10.1126/science. transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange be- 1172046. tween cells. Nat Cell Biol 2007;9:654–9. http://dx.doi.org/10.1038/ncb1596. [38] Uversky VN, Kuznetsova IM, Turoverov KK, Zaslavsky B. Intrinsically disordered pro- Experimental evidence for the transportation of active RNA between cell types with- teins as crucial constituents of cellular aqueous two phase systems and coacervates. in exosomes. FEBS Lett 2015;589:15–22. http://dx.doi.org/10.1016/j.febslet.2014.11.028. [64] Ramachandran S, Palanisamy V. Horizontal transfer of RNAs: exosomes as mediators [39] Nott TJ, Petsalaki E, Farber P, Jervis D, Fussner E, Plochowietz A, et al. Phase transition of intercellular communication. Wiley Interdiscip Rev RNA 2011;3:286–93. of a disordered nuage protein generates environmentally responsive membraneless http://dx.doi.org/10.1002/wrna.115. – organelles. Mol Cell 2015;57:936 47. http://dx.doi.org/10.1016/j.molcel.2015.01.013. [65]• Juliano RL. The delivery of therapeutic oligonucleotides. Nucleic Acids Res 2016, fi – [40]•• Elbaum-Gar nkle S, Kim Y, Szczepaniak K, Chen CC-H, Eckmann CR, Myong S, et al. gkw236 31. http://dx.doi.org/10.1093/nar/gkw236. An extensive review which The disordered P granule protein LAF-1 drives phase separation into droplets with describes the challenges for nucleotide therapies and some possible solutions tunable viscosity and dynamics. Proc Natl Acad Sci U S A 2015;112:7189–94. based on oligo-nucleotide conjugates, lipid/polymer coated nanoparticles as http://dx.doi.org/10.1073/pnas.1504822112. This work describes the role of RNA solutions. within LAF-1 droplets for altering the material and internal properties of the droplets [66] Vader P, Kooijmans SA, Stremersch S, Raemdonck K. New considerations in the [41] Lin Y, Protter DSW, Rosen MK, Parker R. Formation and maturation of phase- preparation of nucleic acid-loaded extracellular vesicles. Ther Deliv 2014;5:105–7. separated liquid droplets by RNA-binding proteins. Mol Cell 2015;60:208–19. http://dx.doi.org/10.4155/tde.13.142. http://dx.doi.org/10.1016/j.molcel.2015.08.018. [67] Stremersch S, Vandenbroucke RE, Van Wonterghem E, Hendrix A, De Smedt SC, [42] Zhang H, Elbaum-Garfinkle S, Langdon EM, Taylor N, Occhipinti P, Bridges AA, Raemdonck K. Comparing exosome-like vesicles with liposomes for the functional et al. RNA controls polyQ protein phase transitions. Mol Cell 2015;60:220–30. cellular delivery of small RNAs. J Control Release 2016;232:51–61. http://dx.doi.org/ http://dx.doi.org/10.1016/j.molcel.2015.09.017. 10.1016/j.jconrel.2016.04.005. [43] Schwartz JC, Wang X, Podell ER, Cech TR. RNA seeds higher-order assembly of FUS [68]• Oe Y, Christie RJ, Naito M, Low SA, Fukushima S, Toh K, et al. Actively-targeted protein. Cell Rep 2013;5:918–25. http://dx.doi.org/10.1016/j.celrep.2013.11.017. polyion complex micelles stabilized by cholesterol and disulfide cross-linking [44] Murakami T, Qamar S, Lin JQ, Schierle GSK, Rees E, Miyashita A, et al. ALS/FTD for systemic delivery of siRNA to solid tumors. Biomaterials 2014;35:7887–95. mutation-induced phase transition of FUS liquid droplets and reversible hydrogels http://dx.doi.org/10.1016/j.biomaterials.2014.05.041. This paper describes a strate- into irreversible hydrogels impairs RNP granule function. Neuron 2015;88:678–90. gy for improving complex micelle uptake into tumours to increase siRNA delivery http://dx.doi.org/10.1016/j.neuron.2015.10.030. based on alteration of micelle composition and chemical cross-linking. [45]• Patel A, Lee HO, Jawerth L, Maharana S, Jahnel M, Hein MY, et al. A liquid-to-solid [69]• Tang TYD, Rohaida Che Hak C, Thompson AJ, Kuimova MK, Williams DS, Perriman phase transition of the ALS protein FUS accelerated by disease mutation. Cell 2015; AW, et al. Fatty acid membrane assembly on coacervate microdroplets as a step 162:1066–77. http://dx.doi.org/10.1016/j.cell.2015.07.047. In vitro experiments towards a hybrid protocell model. Nat Chem 2014;6:527–33. http://dx.doi.org/10. probing the effects of patient modifications to FUS protein on the liquid to solid 1038/nchem.1921. Example of the synthesis of hybrid protocells based on phase transitions associated to disease. complex coacervation and amphiphile assembly. [46] Döbereiner HG, Käs J, Noppl D, Sprenger I, Sackmann E. Budding and fission of vesicles. [70] Lorenzer C, Dirin M, Winkler A-M, Baumann V, Winkler J. Going beyond the liver: Biophys J 1993;65:1396–403. http://dx.doi.org/10.1016/S0006-3495(93)81203-7. progress and challenges of targeted delivery of siRNA therapeutics. J Control Release [47] Nomura S-IM, Tsumoto K, Hamada T, Akiyoshi K, Nakatani Y, Yoshikawa K. Gene 2015;203:1–15. http://dx.doi.org/10.1016/j.jconrel.2015.02.003. expression within cell-sized lipid vesicles. Chembiochem 2003;4:1172–5. [71] Miyata K, Nishiyama N, Kataoka K. Rational design of smart supramolecular assem- http://dx.doi.org/10.1002/cbic.200300630. blies for gene delivery: chemical challenges in the creation of artificial viruses. Chem [48] Noireaux V, Bar-Ziv R, Godefroy J, Salman H, Libchaber A. Toward an artificial Soc Rev 2012;41:2562. http://dx.doi.org/10.1039/c1cs15258k. cell based on gene expression in vesicles. Phys Biol 2005;2:P1–8. http://dx.doi.org/ [72] Merkel OM, Kissel T. Quo vadis polyplex? J Control Release 2014;190:415–23. 10.1088/1478-3975/2/3/P01. http://dx.doi.org/10.1016/j.jconrel.2014.06.009. [49]•• Murtas G, Kuruma Y, Bianchini P, Diaspro A, Luisi PL. Protein synthesis in liposomes [73] Lächelt U, Wagner E. Nucleic acid therapeutics using polyplexes: a journey of with a minimal set of enzymes. Biochem Biophys Res Commun 2007;363:12–7. 50 years (and beyond). Chem Rev 2015;115:11043–78. http://dx.doi.org/10. http://dx.doi.org/10.1016/j.bbrc.2007.07.201. Steps towards a minimal synthetic 1021/cr5006793. cell containing only 36 enzymes and ribosomes for the expression of GFP and [74] Voets IK, de Keizer A, Cohen Stuart MA. Complex coacervate core micelles. Adv acyltransferases. Colloid Interface Sci 2009;147–148:300–18. http://dx.doi.org/10.1016/j.cis. [50] Mansy SS. Membrane transport in primitive cells. Cold Spring Harb Perspect Biol 2008.09.012. 2010;2:a002188. http://dx.doi.org/10.1101/cshperspect.a002188. [75] Hammond PT. Polyelectrolyte multilayered nanoparticles: using nanolayers for [51] Glasner ME, Yen CC, Ekland EH, Bartel DP. Recognition of nucleoside triphosphates controlled and targeted systemic release. Nanomedicine 2012;7:619–22. during RNA-catalyzed primer extension. Biochemistry 2000;39:15556–62. http:// http://dx.doi.org/10.2217/nnm.12.47. dx.doi.org/10.1021/bi002174z. [76] Roh YH, Lee JB, Shopsowitz KE, Dreaden EC, Morton SW, Poon Z, et al. Layer-by-layer fi [52]•• Adamala K, Szostak JW. Nonenzymatic template-directed RNA synthesis inside assembled antisense DNA microsponge particles for ef cient delivery of cancer model protocells. Science 2013;342:1098–100. http://dx.doi.org/10.1126/science. therapeutics. ACS Nano 2014;8:9767–80. http://dx.doi.org/10.1021/nn502596b. J.R. Vieregg, T.-Y.D. Tang / Current Opinion in Colloid & Interface Science 26 (2016) 50–57 57

[77] Allen TM, Cullis PR. Liposomal drug delivery systems: from concept to clinical [82] Patwa A, Gissot A, Bestel I, Barthélémy P. Hybrid lipid oligonucleotide conjugates: applications. Adv Drug Deliv Rev 2013;65:36–48. http://dx.doi.org/10.1016/j.addr. synthesis, self-assemblies and biomedical applications. Chem Soc Rev 2011;40: 2012.09.037. 5844–54. http://dx.doi.org/10.1039/c1cs15038c. [78] Kanasty R, Dorkin JR, Vegas A, Anderson D. Delivery materials for siRNA therapeutics. [83] Kim AK, DeRose R, Ueno T, Lin B, Komatsu T, Nakamura H, et al. Toward total synthe- Nat Mater 2013;12:967–77. http://dx.doi.org/10.1038/NMAT3765. sis of cell function: reconstituting cell dynamics with synthetic biology. Sci Signal – [79]• Ozcan G, Ozpolat B, Coleman RL, Sood AK, Lopez-Berestein G. Preclinical and clinical 2016;9, re1 re1. http://dx.doi.org/10.1126/scisignal.aac4779. – development of siRNA-based therapeutics. Adv Drug Deliver Rev 2015;87:108 19. [84]•• Miller D, Gulbis J. Engineering protocells: prospects for self-assembly and nanoscale http://dx.doi.org/10.1016/j.addr.2015.01.007. Review summarising the challenges production-lines. Life 2015;5:1019–53. http://dx.doi.org/10.3390/life5021019. or SiRNA as therapeutics and some strategies to overcome these Review highlighting the prospects for designing and synthesising artificial cells [80] Wagner E. Polymers for siRNA delivery: inspired by viruses to be targeted, dynamic, from biological components for real world applications. and precise. Acc Chem Res 2012;45:1005–13. http://dx.doi.org/10.1021/ar2002232. [85] Fallah-Araghi A, Baret J-C, Ryckelynck M, Griffiths AD. A completely in vitro fl [81]•• Kwak M, Herrmann A. Nucleic acid amphiphiles: synthesis and self-assembled nano- ultrahigh-throughput droplet-based micro uidic screening system for protein structures. Chem Soc Rev 2011;40:5745–55. http://dx.doi.org/10.1039/c1cs15138j. engineering and directed evolution. Lab Chip 2012;12:882–91. http://dx.doi.org/ Review which summaries the basic principles for the design and synthesis of DNA 10.1039/c2lc21035e. amphiphiles, their higher order structures when self assembled and their potential applications.