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

Contents lists available at ScienceDirect

Current Opinion in Colloid & Interface Science

j ourna l homepage: www.e lsev ie r .com/ locate /coc is

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, USAb Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany

⁎ Corresponding author.E-mail address: tang@mpi-cbg.de (T.-Y.D. Tang).

http://dx.doi.org/10.1016/j.cocis.2016.09.0041359-0294/© 2016 Elsevier Ltd. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 10 June 2016Received in revised form 22 August 2016Accepted 2 September 2016Available online 28 September 2016

In this review, we examine the interaction of nucleic acids with cell-like structures based on liquid–liquid phaseseparation of charged molecules (complex coacervation) and amphiphilic self-assembly (lipid vesicles). Wediscuss the mechanisms of their assembly and describe how they can be used as models for origin of life studiesand for understanding two recently-described phenomena in modern cells: membrane-free organelles andexosomes. Hybrid cells with increased structural complexity are highlighted and we then briefly explore howstrategies based on electrostatic and hydrophobic assembly can be used for designing and synthesizing deliveryagents for therapeutic nucleic acids. While the physical mechanisms of self-assembly vary, both strategiesprovide viable routes for generating minimal compartmentalized systems, modeling cellular pathways, and forrational design of new synthetic cells for technological applications.

© 2016 Elsevier Ltd. All rights reserved.

Keywords:CoacervateLipid vesicleSelf-assemblyProtocellsNucleic acidsDrug deliveryTherapeuticsSynthetic cells

1. Introduction

Cells are the basic unit of self-replicating life on Earth. However,the completemechanistic pathways that drive replication, homeostasis,information propagation and evolution, are still poorly understood. Inaddition, how biological life might have evolved from simple chemicalprocesses remains an open, unanswered question. Therefore, synthesiz-ing abiotic cellular analogs, or “protocells”, is an important route fordescribing and understanding biological mechanisms and offers in-triguing models to describe evolutionary pathways in the pre-bioticworld [1,2]. In order to synthesize suitable protocells it is importantto connect molecular structure with self-assembly processes capableof supporting key features required for life, including concentration offunctional molecules, creation of distinct chemical environments, andinformation propagation.

Compartmentalization provides a mechanism for increasing localconcentrations of enzymes and substrates sufficiently to drive chemicalreactions, and is a necessary feature for enriching functional moleculesand their precursors. Oneplausible scenario for prebiotic compartmental-ization describes the spontaneous assembly and concomitant chemicalenrichment of chargedmolecules to formmembrane free droplets calledcoacervates [3••]. An alternative route to chemical compartmentalizationis via the formation of lipid membrane bound compartments, analogousto the membrane structure of modern cells [1,4,5]. Membranes also

provide localization for heterogeneous reactions and are essential forthe electrochemical gradients used for essential modern processes suchas ATP synthesis, but can require complex protein machinery to create,maintain, and regulate flows of chemicals that cannot pass through them.

Modern cellular biochemistry also provides clues with respect toinformation propagation in early life scenarios. Of the various biologicalmolecules, only nucleic acids (RNA and DNA) have the capability tocatalyze chemical reactions and template their own genetic propaga-tion. The “RNA World” hypothesis imagines a scenario on prebioticearth where self-replicating RNA molecules were both enzymes andgenes; today, these functionalities are primarily fulfilled by proteinsand DNA. As there is nomaterial evidence for the “RNAWorld” hypoth-esis it remains speculative [6], however the discovery that ribosomesare, at their core, ribozymes [7•], strongly indicates that RNA precededproteins in the chemical evolution of early life, thus highlighting theimportance of considering polynucleotides in early life scenarios andin modern natural and synthetic self-assembled structures.

In this review, we discuss the interaction of nucleic acids with twotypes of self-assembled systems that display cell-like properties: complexcoacervates and hydrophobic assemblies such as micelles and vesicles.For each type, we briefly discuss their mechanisms of formation andhow these processesmay be applied to early life scenarios, compartmen-talization phenomena in modern biology, and for therapeutics. All ofthese topics are exciting areas of research in their own right, and spacedoes not permit a full exploration of any of them here. We insteadattempt to highlight recent research results and interesting connections,while referring the reader to reviews for more comprehensive explana-tions of the underlying phenomena.

51J.R. Vieregg, T.-Y.D. Tang / Current Opinion in Colloid & Interface Science 26 (2016) 50–57

2. Electrostatic assembly: complex coacervate protocells

Interactions between oppositely charged molecules can lead tophase separation, forming either liquid droplets (complex coacervation)or solid precipitates depending on the length, charge density and typeof macromolecules. Complex coacervation refers to spontaneous forma-tion of a polymer-rich liquid phase in dynamic equilibrium with apolymer-poor phase [8,9••] via electrostatic interactions between oppo-sitely charged molecules in aqueous solution. This process was firstdescribed by Bungenberg de Jong in the 1920s in mixtures of gelatin(polycation) and gum arabic (polyanion) [10].While a complete quanti-tative model is still lacking, many aspects of complex coacervation arequalitatively understood for long polyelectrolytes. The formation ofmacroion pairs between oppositely charged molecules is driven by anincrease of entropy from the release of low valence counterions andwater rearrangement as they associate. Themacroion pairs then assem-ble into larger clusters, which leads to macroscopic phase separation(Fig. 1a) [9••,11]. Significantly, the dense phase, while highly enrichedin polymer, remains highly hydrated with a large concentration ofcounterions. The relative importance of ion pairing, hydration effects,and long-range electrostatics remains uncertain [12–14••] as do thefactors determining whether a particular macroion pair will form aliquid or a solid condensed complex [15]. As polyelectrolytes can bechemically diverse the process of complex coacervation is molecularlynon-specific. Indeed, since the 1920s complex coacervation has beenobserved between hundreds of different natural and synthetic polymers[8,16,17••]. Their properties including ease of formation, high viscosity,strong adhesion and high encapsulation efficiencies have led to arange of industrial applications including food additives [18] and elec-tronic ink [19], as well as therapeutic assemblies (see Section 5).

In 1924, Alexander Oparin put forward the idea that colloidalmicrodroplets formed via coacervation were the earliest metabolicunits in a reducing ‘prebiotic soup’ [20]. Over the next few decades,Oparin and co-workers demonstrated chemical enrichment within thedroplets, in-situ enzymatic reactions, and droplet growth and fissionreminiscent of cellular life [21••]. This ‘metabolism-first’ approach, how-ever, provided no clear connection to genetic evolution and informationpropagation via nucleic acids that would have been a key step at theonset of life. Their experiments also presumed the existence of largemacromolecules and polymers that are unlikely to have existed in aprebiotic environment.

In order for coacervates to be viable protocells, with the ability tosustain both chemical and genetic evolution, they must be able to

Fig. 1. Complex coacervation of chargedmolecules. (a) Oppositely-chargedmacromolecules for(b) Poly(lysine), (c) Adenosine triphosphate (ATP) and (d) deoxyribonucleic acid (DNA, sequewhile nucleotide polyphosphates have one negative charge per phosphate group.

form from small molecular weight molecules, particularly nucleotidesand their activated derivatives. This was first demonstrated in 2011 byKoga et al. [22••], who showed that coacervate microdroplets (Fig. 2a)could be formed from nucleoside triphosphates (ATP), diphosphates(ADP, FAD, NAD), and monophosphates (AMP) when mixed withshort (2–10 amino acid (aa)) lysine polypeptides (OLys) that mightplausibly be produced by prebiotic processes [23]. This study showedthat phase separation of small molecular weight ions has many similari-ties to complexation of larger polyelectrolytes. The dependence on elec-trostatic interactions, for example, is shown by the increase of the criticalconcentration required for coacervation (CCC) with decreasing negativecharge from ATP N ADP N AMP (Fig. 2b). In addition, they found thatincreasing the molecular weight of Poly(diallyldimethylammonium)(Poly(DADMAC)) from 150 to 275 kDa increased charge neutralization(with ATP) from 70 to 90%, These results suggest that increased hydro-phobicity, decreased solubility and increased orientational freedomfrom longer polymer chains all contribute to increasing charge neutrali-zation at the CCC.

Nucleotide coacervate droplets exhibit dynamic behaviors such ascoalescence and surface wetting (Fig. 2a) but remain stable over abroad pH and temperature range (between pH 2 and 10 and up to80 °C respectively). The coacervates also produce substantial chemicalenrichment of charged molecules, with OLys (2–10 aa)/ATP dropletsdisplaying a twenty-fold increase in ATP concentration relative to thedilute phase [22] and 15 kD poly(allylamine) droplets providing a300× increase of fluorescent solutes, as well as substantial enrichmentof Mg2+ and RNA oligonucleotides [24]. The dielectric constant of thedroplet interior is lower than that of the dilute phase, which providesa mechanism for sequestering low-charge molecules such as proteinenzymes. Sequestration provides a molecular route to accelerate bio-chemical reactions, primitive metabolism, and information processingby increasing the concentrations of substrates, as well as by crowding.This was demonstrated by Crosby et al. for the enzyme actinorhodinpolyketide synthase, which displayed an 18× rate increase whensequestered into ATP–PDDA coacervate droplets [25]. Significantly,mRNA transcription and sustained translation into protein have alsobeen demonstrated inside coacervates (Fig. 2c) [26•]. Taken together,these studies support the argument that coacervate microdropletsformed from nucleotide anions are viable platforms for protocell evolu-tion. Indeed, it has recently been shown that RNA within RNA–peptidecoacervates will elongate via temperature cycling. These results demon-strate a plausible scenario where compartmentalization is coupled withchemical evolution of polynucleotides [27].

m ion pairs that aggregate into membrane-free coacervate droplets. Molecular structure ofnce 5′-AGC-3′). At neutral pH, poly(lysine) and DNA each contain one charge per residue,

Fig. 2.Membrane free artificial cells. (a) Mixing polycations (PDDA) and anions (ATP) leads to phase separation, as shown by turbidity and droplet formation (4:1molar ratio PDDAwithATP, 50mM, pH8). Brightfieldmicroscopy image (scale bar 20 μm) showswetting behavior ofmicrodroplets on a glass slide. (b) Critical coacervation concentration (CCC) vs anion chargefor nucleotide polyphosphates: AMP (1−), ADP (2−), ATP (3−), modified from [22••]. (c) Fluorescent protein (mCherry) expression in coacervate phase vs time [26•] published from theRoyal 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•].

52 J.R. Vieregg, T.-Y.D. Tang / Current Opinion in Colloid & Interface Science 26 (2016) 50–57

The coacervates discussed thus far are formed from ribonucleotidemonomers, but longer nucleic acids are also capable of forming com-plexes with polycations, as has been demonstrated with polyribonucle-otides and tRNA [28••,29]. Experience with synthetic polyelectrolytes,as well as theoretical considerations, suggests that polynucleotideswill experience stronger confinement than individual monomers, andthat their affinity for the coacervate phase will increase with theirlength [8,14]. This may overcome one challenge for coacervates asfunctional protocells, namely that the lack of a membrane leads to lowconfinement times [30], while coupling the chemical evolution of poly-nucleotides to more efficient compartmentalization.

2.1. Membrane-free organelles: intracellular phase separation

The facilemechanisms of synthetic droplet formation via electrostaticself-assembly have also been observed in present day biological systems.For example, DNA is condensed to near crystalline density by basicproteins and small polyamines in the nucleoid of bacteria, spermatozoaheads, and viral capsids by the same electrostatic interactions thatdrive coacervate formation [31]. Over the last decade, it has been pro-posed that liquid–liquid phase separation is the mechanism drivingthe formation of RNA granules, processing (P) bodies, Cajal bodies, andthe nucleolus and other membrane-free compartments, in eukaryoticcells (Fig. 3) [32–37 (34••,35•)]. Like the synthetic coacervates thesemembrane-free organelles are dynamic but appear to have significantinvolvement in essential cellular processes such as replication, signaling,stress response, and disease.

The mechanisms of phase separation are not fully understoodbut it appears that intrinsically disordered proteins (IDPs) with low-complexity sequence domains and charged residues are intimatelyinvolved in the formation of these membrane-free organelles [34,38].In addition, many of these compartments either contain large fractionsof RNA or require it for assembly, as in the case of stress granules(SGs). The presence of these charged molecules suggests that electro-statics play an important role in liquid droplet formation. Moreover,studies have shown that increasing the negative charge of liquidforming protein domains by serine phosphorylation inhibits SG

formation [32]. In vitro results support this hypothesis, as exemplifiedby the Ddx4 protein, which forms nuage bodies in humans and P gran-ules inworms [39]. Liquid droplets formed by the N-terminal domain ofDdx4 are destabilized to a similar extent by increased salt concentrationand by neutralization (via methylation) of multiple positively chargedarginine residues, although cation–pi interactions also appear to playa role in phase separation. Interestingly, membrane-free Ddx4 liquiddroplets also exhibited differential chemical environments as single-stranded DNA was concentrated within the droplets while double-stranded DNA was largely excluded. Measurements with the RNA-binding proteins LAF-1 [40••], hnRNPA1 [41], and Whi3 [42] also pointto electrostatics as a key driving force for droplet formation, as well asthe crucial role of RNA. For example in-vitro experiments with liquiddroplets formed from Whi3 showed a phase transition to gels andsolidfibers under certain conditions. Similar phenomenawere observedwith a prion-like protein FUS [43–45•] (Fig. 3d), where ALS-associatedmutations accelerated the solidification process. These results indicatethat in some instances intracellular phase transitions may also be asso-ciated with disease when proteins or RNA concentrations are mis-regulated.

3. Hydrophobic assembly: vesicles

Modern cells are physically defined by semi-permeable lipid bilayerscomposed primarily of phospholipid amphiphiles (two hydrocarbonchains linkedby a phosphate head group, Fig. 4ai) and sterols (e.g. choles-terol, Fig. 4aii), along with numerous proteins. As this structural elementis ubiquitous across all kingdoms it is likely that a similar lipidmembranewas present in the last common ancestor. Self-assembly of lipid amphi-philes into membrane-delineated compartments for protocell modelsand therapeutic assemblies has therefore attracted strong interest overthe last half a century. Above a critical concentration, amphiphilic lipidmonomers will self-assemble in water, driven by the hydrophobic effect,to form micelles (Fig. 4bi) or vesicles (Fig. 4bii) with bilayer membranesand an aqueous interior. The latter strongly resembles the basic structureof modern cells and organelles, and a large number of studies haveexamined vesicular behaviors such as budding and fission [46], as well

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 embryoover ~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 proteinin vitro [39]. Methylation of multiple arginine residues (red line) destabilizes droplets to a similar extent as doubling the NaCl concentration, consistent with a primarily electrostaticinteraction. (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].

53J.R. Vieregg, T.-Y.D. Tang / Current Opinion in Colloid & Interface Science 26 (2016) 50–57

as encapsulation of complex biochemical reactions such as proteinexpression [47–49••] as a proxy to model cellular processes. Moreover,studies that have coupled RNA encapsulation and RNA interactionswith cell membranes give new insights into origins of life, as well as pro-viding tools for therapeutic delivery of nucleic acids.

3.1. Vesicle protocells in early life

While essential for modern life, phospholipid membranes haveseveral characteristics thatmake themproblematic for early-life scenarios[1,4]. The bilayer formed by phospholipids is highly impermeable tocharged compounds, which enables high electrochemical potentials

Fig. 4.Hydrophobic protocells. (a) Examples of lipidmembrane building blocks (i) Phospholipidacid), (iv) cationic lipid N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methy(i) micelles or (ii) vesicles with bilayer membranes depending on their structure and concentrswells vesicles, causing them to grow at the expense of empty vesicles. In the presence of excto mechanical stress or changing environment. The vesicles containing self-replicating RNA gro

for energy harvesting and signaling in modern cells but also preventsimportation of activated nucleosides and essential ions. Phospholipidbilayers are also very stable, making growth and division difficultwithoutthe aid of modern enzymes such as flippases and permeases. By contrast,single-chain fatty acids (Fig. 4aiii) form dynamic bilayers with leafletexchange times on the order of seconds and residence times on theorder of minutes, enabling growth/decay dynamics and selective perme-ability via transient defect formation [50]. Fatty acids are also relativelyeasy to synthesize abiotically, being detected inmeteorites at abundances20 times that of amino acids [4]. At certain concentrations fatty acidswill form micelles or vesicles depending on the pKa of the acid and thepH of the solution. The chemistry and dynamics of fatty acid assemblies

, palmitoyl-oleoyl-sn-phosphatidylcholine (POPC), (ii) cholesterol, (iii) fatty acid (gadoleicl-sulfate (DOTAP). (b) In aqueous solution, amphiphiles can self-assemble into eitheration. (c) A plausible scenario for coupled protocell growth and division. RNA elongationess amphiphile and micelles, vesicles grow and become unstable, leading to splitting duew, 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-tions related to their interactions with polynucleotides or origin oflife studies.

The first of these relates to activated ribonucleosides and chainelongation. Protein nucleic acid polymerases use basic residues to stabi-lize the high negative charge transition state containing a nucleosidetriphosphate (NTP) and nucleic acid in close proximity. Lacking thisability, ribozyme polymerases rely on divalent cations such as Mg2+

at concentrations up to hundreds of mM [51], which causes precip-itation of fatty acids, membrane destruction [5] and polynucleotidehydrolysis. Non-enzymatic template elongation reactions also requirehigh Mg2+ concentrations, making it difficult to reconcile NTP poly-merization with the physical requirements of fatty acid self-assembly.One way around this problem is to use nucleotides with alternateleaving groups such as imidazole, 2-methylimidazole (2MeIm), and1-hydroxy-7-azabenzotriazole, which carry a lower charge (1− vs 3−

for NTPs) and react more readily than NTPs. If citrate is includedas a chelating agent, 2MeIm-activated nucleotides polymerize non-enzymatically on a single-stranded template at a rate of 0.67 h−1 insolution and copy short templates overnight in oleic acid vesicles[52••]. This does not solve the problem for ribozymes, however, and nei-ther citrate nor the alternate activated nucleosides have been producedby plausible abiotic reactions. It may be that more efficient ribozymepolymerases can be found; ribozyme ligases have been identified thatfunction with Mg2+ concentrations as low as 2 mM in the presenceof fatty acid vesicles and low-concentration EDTA as a chelator [53].Another attractive possibility is charge reduction through complexationof either the NTP or polynucleotides by basic peptides, analogous to(or even composed of) the coacervates discussed above [27].

A second problem involves coupling vesicle growth to replication ofthe interior contents, a key requirement for effective chemical evolu-tion. In this case, polynucleotides can provide a solution, via osmoticpressure. Due to their higher charge density, polynucleotides complexmore counterions compared to monomers, particularly if they arefolded. It has been proposed that RNA elongation generates a decreasein osmotic pressure that drives vesicle growth at the expense of lessactive vesicles [54] successfully coupling polymerase evolution to com-petition at the protocellular scale (Fig. 4c). Larger vesicles are alsomorelikely to be split by external shear stress and, if they are multilamellar,are subject to “pearling” instabilities, both of which provide plausiblepathways for division [1].

A final problem relates to the eventual transition from simple fattyacid vesicles to more stable membranes required for evolution ofmore sophisticated reactions. As mentioned above, phospholipid bilay-ers are impermeable to charged molecules. Modern cells solve thisproblem by using protein pores and channels to selectively regulateflows into and out of the cell, sustaining concentration differencesrequired for metabolism. Several results indicate that RNA is also capa-ble of forming pores in phospholipid bilayers. Vlassov et al. showed thatmultimeric RNA sequences are capable of permeabilizing liposomesand membranes via interaction with the phosphate head groups [55],and subsequent work demonstrated selective transport of tryptophanamino acids, thus demonstrating that the RNAworld can support mem-brane transport [50].

3.2. Exosomes: protocells in the modern world?

Exosomes are small (30–120 nm diameter) lipid vesicles thatlack internal organelles and circulate independently in the extracellularenvironment; as such, they sharemany of the physico-chemical charac-teristics of lipid membrane based protocells despite very differentbiogenesis and functions. Originally thought to be cellular waste prod-ucts, they are now known to function in intercellular communicationand have become the subject of intense interest due to their abilityto alter the phenotype of distant cells via transport of proteins andmicroRNAs (miRNAs) [56,57].

Exosomes are found in diverse body fluids, and are producedby many types of cells. In contrast to microvesicles [58], exosomesare derived from late endosomes, also known as multi-vesicularbodies, and released into the environment via SNARE-mediatedfusion within the plasma membrane. This process is initiated byvarying signaling pathways in different cell types, and is regulatedby RAB and ESCRT proteins [57]. Compared to the plasma mem-brane, exosome membranes are enriched in raft-type lipids suchas cholesterol and sphingomyelin [58] and possess generic andcell-type specific membrane protein and glycosylation signatures[59]. The latter can provide a “delivery address” for tissue-specifictargeting. A key discovery showed that exosomes contained mRNAand miRNA that were both active in target cells [60–63••]. Further-more, exosomal RNA populations differ from those of the parentcell, a characteristic of functional signaling [64]. Much remains tobe discovered about the content of exosomal messages, but theyhave been implicated in pathological processes such as tumor metas-tasis, viral infection, drug resistance, and immune response, as wellas embryonic development [57,64]. A deeper understanding of therole and functions of exosomes in biology should help unlock theirpotential for therapeutics and non-invasive diagnostics. As an exam-ple, researchers have attempted to deliver therapeutic RNAs withexosomes, either by loading purified exosomes and injecting theminto the body or by transfecting host cells with therapeutic RNAsto be packaged and exported [65•]. Currently, exosome-mediated de-livery lags behind engineered vesicle systems (Section 5) [66,67],but the intense interest in the field suggests that progress may berapid in this area.

4. Hybrid systems: electrostatic and hydrophobic assembly together

Combining electrostatic and hydrophobic assembly allows forcreation of more complex systems with enhanced capabilities. Lipidswith cationic head groups are commonly used to co-assemble lipidvesicles with nucleic acid polyanions (Section 5), and electrostatically-assembled nanoparticles can be further stabilized by functionalizing theanionic RNA component with cholesterol [68•]. Hybrid electrostatic–hydrophobic assembly is also interesting for early-life protocellstudies, as coacervates and lipid vesicles are in many ways comple-mentary in terms of capabilities required for self-replication. Coac-ervation readily concentrates charged molecules such as nucleicacids and activated nucleotides, but does not allow for internalstructure or heterogeneous catalysis and has limited capability tosustain electrochemical gradients at the boundary. Lipid vesiclesprovide powerful models for compartmentalization, but lack amechanism for enriching their interiors with ions and hydrophilicbiomolecules. Co-assembly of coacervates and vesicles is a promis-ing route to more complete cellularity, as is encapsulation of pre-formed coacervates. The latter was recently demonstrated by addingsodium oleate to positively-charged coacervates formed from RNAor ATP and polylysine or poly(DADMAC) [69•]. In the absence ofoleic acid, a lipophilic dye (BODIPY FL C16) partitioned homoge-neously within the coacervate droplets due to the lower dielectricconstant of the interior compared to the dilute exterior phase. Afterthe addition of the fatty acid, fluorescence was observed at the drop-let boundary only, indicating the formation of a lipid membrane.Small angle X-ray scattering measurements showed Bragg peaksconsistent with the formation of a multilamellar membrane, anddye uptake measurements showed differential permeability basedon charge and molecular weight. Increasing the exterior ionicstrength resulted in droplet disassembly and growth, reminiscentof the encapsulated PEG/dextran systems discussed earlier. Whilemuch work remains, this study shows that very simple chemical andphysical processes are capable of transforming various membrane-freecoacervate droplets, including OLys/RNA, into membrane-encapsulatedprotocells.

55J.R. Vieregg, T.-Y.D. Tang / Current Opinion in Colloid & Interface Science 26 (2016) 50–57

5. Electrostatic assemblies and engineering lipid vesicles fortherapeutics

Therapeutic nucleic acids come in several forms, including DNA(gene therapy), RNA duplexes (siRNA), and oligonucleotides formicroRNA and antisense pathways, but they face common challengesas drugs. To be effective, within the target tissue, the nucleic acidsmust evade clearance by the liver and kidney, cross the hydrophobiccell membrane, and avoid degradation by nuclease enzymes bothoutside and inside the cell [70]. The same electrostatic and hydrophobicinteractions that give rise to protocells can also be harnessed to produceself-assembling delivery systems that offer solutions to several of theseproblems. A detailed description of these systems is beyond the scopeof this review, instead, we mention the basic chemical and physicalstrategies and provide references for the interested reader.

Encapsulation by coacervate and solid polyelectrolyte complexesformed from nucleic acids and cationic polymers (polyplexes) hasbeen extensively studied as non-viral transfection vectors both in vitroand in vivo [65•,71–73]. One strategy to prevent aggregation, reduceunwanted interactions with serum proteins, and promote longer circu-lation time is based on functionalizing the polycations with neutralhydrophilic polymers such as polyethylene glycol (PEG). In the case ofpolyplexes this leads to the formation of nanoparticleswith a coacervateor solid complex core surrounded by a PEG corona and typical diametersbetween 20 and 200 nm [74]. Layer-by-layer electrostatic assemblycan also be used to produce nanoparticles and functionalize surfacesof implanted devices [75,76].

Hydrophobically-assembled micelles, or liposomes, have alsobeen extensively engineered to be effective delivery platforms fornucleic acids [65•]. Cationic and ionizable lipids enable efficient load-ing of anionic polynucleotides and release upon endocytosis, thoughin vivo potency and toxicity remain challenging [77,78]. As withpolyplexes, PEG-functionalized liposomes (stable nucleic acid lipidparticles, or SNALPS) display improved circulation time, reducedtoxicity and immunogenicity and have been used in multiple clinicaltrials [70,79•]. Other strategies for modifying and tuning the deliveryvehicles include incorporation of targeting moieties and/or labilelinkages for intracellular disassembly [80]. Nucleic acids can also bemade amphiphilic by conjugating them with hydrophobic groups,enabling interesting new types of self-assembled nanostructures[81••,82].

6. Conclusion

In this review, we have discussed how cellular mimics can beproduced by self-assembly of charged biomolecules (complexcoacervation) and lipid amphiphiles (vesicles) and discussed theirutility for prebiotic pathways, biological organization and (briefly)therapeutic applications. Both mechanisms incorporate essentialfeatures of modern cells, and their spontaneous assembly propertiessuggest that they might have been viable routes for cellular evolutionon early earth and these possibilities are still under investigation. Addi-tionally, the incorporation of synthetic biology and high-throughputapproaches [83–85 (84••)] provide a valuable complement to the morephysico-chemical approaches described here, raising hopes for under-standing the basic principles governing how cells evolved, and howwe can use cellular mimics in the modern world.

• Of special interest.•• Of outstanding interest.

Acknowledgments

T-Y T's work was supported by the MaxSynBio, MPG(M.IF.A.MOZGDT80). We would like to acknowledge F. Friedrichat MTO (Media Technologies and Outreach) group at MPI-CBGfor assistance with the graphical abstract.

References and recommended reading•,••

[1] Schrum JP, Zhu TF, Szostak JW. The origins of cellular life. Cold Spring Harb PerspectBiol 2010;2:a002212. http://dx.doi.org/10.1101/cshperspect.a002212.

[2] Mann S. Systems of creation: the emergence of life from nonliving matter. Acc ChemRes 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-lipidmicro-compartments and protocell models. Curr Opin Chem Biol 2014;22:1–11. http://dx.doi.org/10.1016/j.cbpa.2014.05.018. A review summarizingthe state of the art of synthetic cells specifically focusing on colloidosomes,proteinosomes and coacervates.

[4] Chen IA, Walde P. From self-assembled vesicles to protocells. Cold Spring HarbPerspect Biol 2010;2, a002170–0. http://dx.doi.org/10.1101/cshperspect.a002170.

[5] Monnard P-A,Walde P. Current ideas about prebiological compartmentalization. Life2015;5:1239–63. http://dx.doi.org/10.3390/life5021239.

[6] Robertson MP, Joyce GF. The origins of the RNA world. Cold Spring Harb PerspectBiol 2012;4:a003608. http://dx.doi.org/10.1101/cshperspect.a003608.

•[7] Steitz TA, Moore PB. RNA, the first macromolecular catalyst: the ribosome is aribozyme. Trends Biochem Sci 2003;28:411–8. http://dx.doi.org/10.1016/S0968-0004(03)00169-5. Review paper which summarises the structural evidenceobtained over the last 60 years that confirms that RNA holds genetic informationand is an enzyme.

[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.http://dx.doi.org/10.1016/j.jcis.2011.05.080.

••[9] Priftis D, Laugel N, Tirrell M. Thermodynamic characterization of polypeptidecomplex coacervation. Langmuir 2012;28:15947–57. http://dx.doi.org/10.1021/la302729r. Experimental paper which probes the thermodynamics ofcomplex coacervation using isothermal calorimetry and shows that the process isprimarily endothermic and is driven by entropic contributions.

[10] de Jong HB, Kruyt HR. Coacervation (partial miscibility in colloid systems). Proc KNed Akad Wet 1929;32:849–56.

[11] Burgess DJ. Practical analysis of complex coacervate systems. J Colloid Interface Sci1990;140:227–38. http://dx.doi.org/10.1016/0021-9797(90)90338-O.

[12] Kudlay A, Ermoshkin AV, Olvera de la Cruz M. Complexation of oppositely chargedpolyelectrolytes: effect of ion pair formation. Macromolecules 2004;37:9231–41.http://dx.doi.org/10.1021/ma048519t.

[13] Spruijt E, Westphal AH, Borst JW, Cohen Stuart MA, van der Gucht J. Binodalcompositions of polyelectrolyte complexes. Macromolecules 2010;43:6476–84.http://dx.doi.org/10.1021/ma101031t.

••[14] Fu J, Schlenoff JB. Driving forces for oppositely charged polyion association in aqueoussolutions: enthalpic, entropic, but not electrostatic. J Am Chem Soc 2016;138:980–90.http://dx.doi.org/10.1021/jacs.5b11878. This paper suggests that electrostatic fieldtheory gives a qualitative description of complex-coacervation and breaks downwhen considering close range interactions between polyelectrolytes.

[15] Wang Q, Schlenoff JB. The polyelectrolyte complex/coacervate continuum. Macro-molecules 2014;47:3108–16. http://dx.doi.org/10.1021/ma500500q.

[16] de Kruif CG, Weinbreck F, de Vries R. Complex coacervation of proteins and anionicpolysaccharides. Curr Opin Colloid Interface Sci 2004;9:340–9. http://dx.doi.org/10.1016/j.cocis.2004.09.006.

••[17] Miller DR, Das S, Huang K-Y, Han S, Israelachvili JN,Waite JH.Mussel coating protein-derived complex coacervates mitigate frictional surface damage. ACS BiomaterSci Eng 2015;1:1121–8. http://dx.doi.org/10.1021/acsbiomaterials.5b00252. Thiswork explores the effect of coacervation on the coefficient of friction between puri-fied proteins frommussels and hyaluronic acid as adhesive models for mussels.

[18] Gouin S. Microencapsulation. Trends Food Sci Technol 2004;15:330–47.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/dioctylsulfosuccinate sodium microcapsule by complex coacervation and its applicationfor electrophoretic display. Colloids Surf A Physicochem Eng Asp 2010;362:84–9.http://dx.doi.org/10.1016/j.colsurfa.2010.03.041.

[20] Oparin AI. Origin of life. New York: Macmillan; 1938.

••[21] Oparin AI, Gladilin KL. Evolution of self-assembly of probionts. Biosystems 1980;12:133–45. Summary of the compartmentalisation properties required for the onsetof life on earth with experimental summary of these properties as demonstratedwithin coacervates.

••[22] Koga S, Williams DS, Perriman AW, Mann S. Peptide–nucleotide microdroplets asa step towards a membrane-free protocell model. Nat Chem 2011;3:720–4. http://dx.doi.org/10.1038/nchem.1110. First description of coacervate formation fromlow molecular weight, prebiotic molecules including nucleotides such as ATP andFAD.

[23] Fox SW. The evolutionary significance of phase-separated microsystems. Orig Life1976;7:49–68.

[24] Frankel EA, Bevilacqua PC, Keating CD. Polyamine/nucleotide coacervates providestrong compartmentalization of Mg2+, nucleotides, and RNA. Langmuir 2016;32:2041–9. 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 minimalcomplex in polymer–nucleotide coacervate droplets. Chem Commun 2012;48:11832–4. http://dx.doi.org/10.1039/c2cc36533b.

•[26] Tang TYD, van Swaay D, deMello A, Anderson JLR, Mann S. In vitro gene expressionwithin membrane-free coacervate protocells. Chem Commun 2015;51:11429–32.

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-step reactions such as protein expression within membrane-free coacervates.

[27] Jia TZ, Fahrenbach AC, Kamat NP, Adamala KP, Szostak JW. Oligoarginine peptidesslow strand annealing and assist non-enzymatic RNA replication. Nat Chem 2016.http://dx.doi.org/10.1038/nchem.2551.

••[28] Aumiller WM, Keating CD. Phosphorylation-mediated RNA/peptide complexcoacervation as a model for intracellular liquid organelles. Nat Chem 2016;8:129–37. http://dx.doi.org/10.1038/nchem.2414. First extensive study of RNA/peptide based coacervates and how they can be repeatedly dissolved and formedvia phosphorylation and dephosphorylation

[29] Porschke D. The binding of Arg-and Lys-peptides to single stranded polyribonucleo-tides and its effect on the polymer conformation. Biophys Chem 1979;10:1–16.http://dx.doi.org/10.1016/0301-4622(79)80001-0.

[30] Jia TZ, Hentrich C, Szostak JW. Rapid RNA exchange in aqueous two-phase systemand coacervate droplets. Orig Life Evol Biosph 2014;44:1–12. http://dx.doi.org/10.1007/s11084-014-9355-8.

[31] Teif VB, Bohinc K. Condensed DNA: condensing the concepts. Prog Biophys Mol Biol2011;105:208–22. http://dx.doi.org/10.1016/j.pbiomolbio.2010.07.002.

[32] Kedersha N, Ivanov P, Anderson P. Stress granules and cell signaling: more than justa passing phase? Trends Biochem Sci 2013;38:494–506. http://dx.doi.org/10.1016/j.tibs.2013.07.004.

[33] Brangwynne CP. Phase transitions and size scaling of membrane-less organelles.J Cell Biol 2013;203:875–81. http://dx.doi.org/10.1083/jcb.201308087.

••[34] Toretsky JA, Wright PE. Assemblages: functional units formed by cellular phaseseparation. J Cell Biol 2014;206:579–88. http://dx.doi.org/10.1083/jcb.201404124.Review describing the role of intrinsically disordered proteins and their structuralfunction in cytoplasmic organisation and role in cellular disease.

•[35] Zhu L, Brangwynne CP. Nuclear bodies: the emerging biophysics of nucleoplas-mic phases. Curr Opin Cell Biol 2015;34:23–30. http://dx.doi.org/10.1016/j.ceb.2015.04.003. Review summarising the role of RNA-protein bodies for regulationwithin the cell.

[36] Wallace EWJ, Kear-Scott JL, Pilipenko EV, Schwartz MH, Laskowski PR, Rojek AE,et al. Reversible, specific, active aggregates of endogenous proteins assemble uponheat stress. Cell 2015;162:1286–98. http://dx.doi.org/10.1016/j.cell.2015.08.041.

[37] Brangwynne CP, Eckmann CR, Courson DS, Rybarska A, Hoege C, Gharakhani J, et al.Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science 2009;324:1729–32. http://dx.doi.org/10.1126/science.1172046.

[38] Uversky VN, Kuznetsova IM, Turoverov KK, Zaslavsky B. Intrinsically disordered pro-teins as crucial constituents of cellular aqueous two phase systems and coacervates.FEBS Lett 2015;589:15–22. http://dx.doi.org/10.1016/j.febslet.2014.11.028.

[39] Nott TJ, Petsalaki E, Farber P, Jervis D, Fussner E, Plochowietz A, et al. Phase transitionof a disordered nuage protein generates environmentally responsive membranelessorganelles. Mol Cell 2015;57:936–47. http://dx.doi.org/10.1016/j.molcel.2015.01.013.

••[40] Elbaum-Garfinkle S, Kim Y, Szczepaniak K, Chen CC-H, Eckmann CR, Myong S, et al.The disordered P granule protein LAF-1 drives phase separation into droplets withtunable viscosity and dynamics. Proc Natl Acad Sci U S A 2015;112:7189–94.http://dx.doi.org/10.1073/pnas.1504822112. This work describes the role of RNAwithin LAF-1 droplets for altering thematerial and internal properties of the droplets

[41] Lin Y, Protter DSW, Rosen MK, Parker R. Formation and maturation of phase-separated liquid droplets by RNA-binding proteins. Mol Cell 2015;60:208–19.http://dx.doi.org/10.1016/j.molcel.2015.08.018.

[42] Zhang H, Elbaum-Garfinkle S, Langdon EM, Taylor N, Occhipinti P, Bridges AA,et al. RNA controls polyQ protein phase transitions. Mol Cell 2015;60:220–30.http://dx.doi.org/10.1016/j.molcel.2015.09.017.

[43] Schwartz JC, Wang X, Podell ER, Cech TR. RNA seeds higher-order assembly of FUSprotein. Cell Rep 2013;5:918–25. http://dx.doi.org/10.1016/j.celrep.2013.11.017.

[44] Murakami T, Qamar S, Lin JQ, Schierle GSK, Rees E, Miyashita A, et al. ALS/FTDmutation-induced phase transition of FUS liquid droplets and reversible hydrogelsinto irreversible hydrogels impairs RNP granule function. Neuron 2015;88:678–90.http://dx.doi.org/10.1016/j.neuron.2015.10.030.

•[45] Patel A, Lee HO, Jawerth L, Maharana S, Jahnel M, Hein MY, et al. A liquid-to-solidphase transition of the ALS protein FUS accelerated by disease mutation. Cell 2015;162:1066–77. http://dx.doi.org/10.1016/j.cell.2015.07.047. In vitro experimentsprobing the effects of patient modifications to FUS protein on the liquid to solidphase transitions associated to disease.

[46] Döbereiner HG, Käs J, Noppl D, Sprenger I, Sackmann E. Budding andfission of vesicles.Biophys J 1993;65:1396–403. http://dx.doi.org/10.1016/S0006-3495(93)81203-7.

[47] Nomura S-IM, Tsumoto K, Hamada T, Akiyoshi K, Nakatani Y, Yoshikawa K. Geneexpression within cell-sized lipid vesicles. Chembiochem 2003;4:1172–5.http://dx.doi.org/10.1002/cbic.200300630.

[48] Noireaux V, Bar-Ziv R, Godefroy J, Salman H, Libchaber A. Toward an artificialcell based on gene expression in vesicles. Phys Biol 2005;2:P1–8. http://dx.doi.org/10.1088/1478-3975/2/3/P01.

••[49] Murtas G, Kuruma Y, Bianchini P, Diaspro A, Luisi PL. Protein synthesis in liposomeswith a minimal set of enzymes. Biochem Biophys Res Commun 2007;363:12–7.http://dx.doi.org/10.1016/j.bbrc.2007.07.201. Steps towards a minimal syntheticcell containing only 36 enzymes and ribosomes for the expression of GFP andacyltransferases.

[50] Mansy SS. Membrane transport in primitive cells. Cold Spring Harb Perspect Biol2010;2:a002188. http://dx.doi.org/10.1101/cshperspect.a002188.

[51] Glasner ME, Yen CC, Ekland EH, Bartel DP. Recognition of nucleoside triphosphatesduring RNA-catalyzed primer extension. Biochemistry 2000;39:15556–62. http://dx.doi.org/10.1021/bi002174z.

••[52] Adamala K, Szostak JW. Nonenzymatic template-directed RNA synthesis insidemodel protocells. Science 2013;342:1098–100. http://dx.doi.org/10.1126/science.

1241888. Work which describes the use of citrate to reconcile the disruptive effectsof high concentration Mg2+ on fatty acid vesicles to successfully allow RNA copyingwithin fatty acid vesicles.

[53] Anella F, Danelon C. Reconciling ligase ribozyme activity with fatty acid vesiclestability. Life 2014;4:929–43. http://dx.doi.org/10.3390/life4040929.

[54] Chen IA, Roberts RW, Szostak JW. The emergence of competition between modelprotocells. Science 2004;305:1474–6. http://dx.doi.org/10.1126/science.1100757.

[55] Vlassov A, Khvorova A, Yarus M. Binding and disruption of phospholipid bilayers bysupramolecular RNA complexes. Proc Natl Acad Sci U S A 2001;98:7706–11. http://dx.doi.org/10.2307/3056063?ref=search-gateway:098e00639238a272052788e5c598ac28.

[56] Vlassov AV, Magdaleno S, Setterquist R, Conrad R. Exosomes: current knowledge oftheir composition, biological functions, and diagnostic and therapeutic potentials.Biochim Biophys Acta Gen Subj 1820;2012:940–8. http://dx.doi.org/10.1016/j.bbagen.2012.03.017.

[57] Azmi AS, Bao B, Sarkar FH. Exosomes in cancer development, metastasis, and drugresistance: a comprehensive review. Cancer Metastasis Rev 2013;32:623–42.http://dx.doi.org/10.1007/s10555-013-9441-9.

[58] Raposo G, StoorvogelW. Extracellular vesicles: exosomes, microvesicles, and friends.J Cell Biol 2013;200:373–83. http://dx.doi.org/10.1083/jcb.201211138.

[59] Batista BS, Eng WS, Pilobello KT, Hendricks-Muñoz KD, Mahal LK. Identification of aconserved glycan signature for microvesicles. J Proteome Res 2011;10:4624–33.http://dx.doi.org/10.1021/pr200434y.

[60] Ratajczak J, Wysoczynski M, Hayek F, Janowska-Wieczorek A, Ratajczak MZ.Membrane-derived microvesicles: important and underappreciated mediatorsof cell-to-cell communication. Leukemia 2006;20:1487–95. http://dx.doi.org/10.1038/sj.leu.2404296.

[61] Mittelbrunn M, Gutiérrez-Vázquez C, Villarroya-Beltri C, González S, Sánchez-CaboF, González MÁ, et al. Unidirectional transfer of microRNA-loaded exosomes fromT cells to antigen-presenting cells. Nat Commun 2011;2:282. http://dx.doi.org/10.1038/ncomms1285.

[62] Montecalvo A, Larregina AT, Shufesky WJ, Stolz DB, Sullivan MLG, Karlsson JM,et al. Mechanism of transfer of functional microRNAs between mouse dendriticcells via exosomes. Blood 2012;119:756–66. http://dx.doi.org/10.1182/blood-2011-02-338004.

••[63] Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediatedtransfer of mRNAs and microRNAs is a novel mechanism of genetic exchange be-tween cells. Nat Cell Biol 2007;9:654–9. http://dx.doi.org/10.1038/ncb1596.Experimental evidence for the transportation of active RNA between cell types with-in exosomes.

[64] Ramachandran S, Palanisamy V. Horizontal transfer of RNAs: exosomes asmediatorsof intercellular communication. Wiley Interdiscip Rev RNA 2011;3:286–93.http://dx.doi.org/10.1002/wrna.115.

•[65] Juliano RL. The delivery of therapeutic oligonucleotides. Nucleic Acids Res 2016,gkw236–31. http://dx.doi.org/10.1093/nar/gkw236. An extensive review whichdescribes the challenges for nucleotide therapies and some possible solutionsbased on oligo-nucleotide conjugates, lipid/polymer coated nanoparticles assolutions.

[66] Vader P, Kooijmans SA, Stremersch S, Raemdonck K. New considerations in thepreparation of nucleic acid-loaded extracellular vesicles. Ther Deliv 2014;5:105–7.http://dx.doi.org/10.4155/tde.13.142.

[67] Stremersch S, Vandenbroucke RE, Van Wonterghem E, Hendrix A, De Smedt SC,Raemdonck K. Comparing exosome-like vesicles with liposomes for the functionalcellular delivery of small RNAs. J Control Release 2016;232:51–61. http://dx.doi.org/10.1016/j.jconrel.2016.04.005.

•[68] Oe Y, Christie RJ, Naito M, Low SA, Fukushima S, Toh K, et al. Actively-targetedpolyion complex micelles stabilized by cholesterol and disulfide cross-linkingfor systemic delivery of siRNA to solid tumors. Biomaterials 2014;35:7887–95.http://dx.doi.org/10.1016/j.biomaterials.2014.05.041. This paper describes a strate-gy for improving complex micelle uptake into tumours to increase siRNA deliverybased on alteration of micelle composition and chemical cross-linking.

•[69] Tang TYD, Rohaida Che Hak C, Thompson AJ, Kuimova MK, Williams DS, PerrimanAW, et al. Fatty acid membrane assembly on coacervate microdroplets as a steptowards a hybrid protocell model. Nat Chem 2014;6:527–33. http://dx.doi.org/10.1038/nchem.1921. Example of the synthesis of hybrid protocells based oncomplex coacervation and amphiphile assembly.

[70] Lorenzer C, Dirin M, Winkler A-M, Baumann V, Winkler J. Going beyond the liver:progress and challenges of targeted delivery of siRNA therapeutics. J Control Release2015;203:1–15. http://dx.doi.org/10.1016/j.jconrel.2015.02.003.

[71] Miyata K, Nishiyama N, Kataoka K. Rational design of smart supramolecular assem-blies for gene delivery: chemical challenges in the creation of artificial viruses. ChemSoc Rev 2012;41:2562. http://dx.doi.org/10.1039/c1cs15258k.

[72] Merkel OM, Kissel T. Quo vadis polyplex? J Control Release 2014;190:415–23.http://dx.doi.org/10.1016/j.jconrel.2014.06.009.

[73] Lächelt U, Wagner E. Nucleic acid therapeutics using polyplexes: a journey of50 years (and beyond). Chem Rev 2015;115:11043–78. http://dx.doi.org/10.1021/cr5006793.

[74] Voets IK, de Keizer A, Cohen Stuart MA. Complex coacervate core micelles. AdvColloid Interface Sci 2009;147–148:300–18. http://dx.doi.org/10.1016/j.cis.2008.09.012.

[75] Hammond PT. Polyelectrolyte multilayered nanoparticles: using nanolayers forcontrolled and targeted systemic release. Nanomedicine 2012;7:619–22.http://dx.doi.org/10.2217/nnm.12.47.

[76] Roh YH, Lee JB, Shopsowitz KE, Dreaden EC, Morton SW, Poon Z, et al. Layer-by-layerassembled antisense DNA microsponge particles for efficient delivery of cancertherapeutics. ACS Nano 2014;8:9767–80. http://dx.doi.org/10.1021/nn502596b.

57J.R. Vieregg, T.-Y.D. Tang / Current Opinion in Colloid & Interface Science 26 (2016) 50–57

[77] Allen TM, Cullis PR. Liposomal drug delivery systems: from concept to clinicalapplications. Adv Drug Deliv Rev 2013;65:36–48. http://dx.doi.org/10.1016/j.addr.2012.09.037.

[78] Kanasty R, Dorkin JR, Vegas A, AndersonD. Deliverymaterials for siRNA therapeutics.Nat Mater 2013;12:967–77. http://dx.doi.org/10.1038/NMAT3765.

•[79] Ozcan G, Ozpolat B, Coleman RL, Sood AK, Lopez-Berestein G. Preclinical and clinicaldevelopment of siRNA-based therapeutics. Adv Drug Deliver Rev 2015;87:108–19.http://dx.doi.org/10.1016/j.addr.2015.01.007. Review summarising the challengesor SiRNA as therapeutics and some strategies to overcome these

[80] Wagner E. Polymers for siRNA delivery: inspired by viruses to be targeted, dynamic,and precise. Acc Chem Res 2012;45:1005–13. http://dx.doi.org/10.1021/ar2002232.

••[81] KwakM, Herrmann A. Nucleic acid amphiphiles: synthesis and self-assembled nano-structures. Chem Soc Rev 2011;40:5745–55. http://dx.doi.org/10.1039/c1cs15138j.Review which summaries the basic principles for the design and synthesis of DNAamphiphiles, their higher order structures when self assembled and their potentialapplications.

[82] Patwa A, Gissot A, Bestel I, Barthélémy P. Hybrid lipid oligonucleotide conjugates:synthesis, self-assemblies and biomedical applications. Chem Soc Rev 2011;40:5844–54. http://dx.doi.org/10.1039/c1cs15038c.

[83] Kim AK, DeRose R, Ueno T, Lin B, Komatsu T, Nakamura H, et al. Toward total synthe-sis of cell function: reconstituting cell dynamics with synthetic biology. Sci Signal2016;9, re1–re1. http://dx.doi.org/10.1126/scisignal.aac4779.

••[84] Miller D, Gulbis J. Engineering protocells: prospects for self-assembly and nanoscaleproduction-lines. Life 2015;5:1019–53. http://dx.doi.org/10.3390/life5021019.Review highlighting the prospects for designing and synthesising artificial cellsfrom biological components for real world applications.

[85] Fallah-Araghi A, Baret J-C, Ryckelynck M, Griffiths AD. A completely in vitroultrahigh-throughput droplet-based microfluidic screening system for proteinengineering and directed evolution. Lab Chip 2012;12:882–91. http://dx.doi.org/10.1039/c2lc21035e.