14. Somers, D. C., Nelson, S. B. & Sur, M. J. Neurosci. 15, 5448 (1995).

15. Ben-Yishai, R., Bar-Or, R. L. & Sompolinsky, H. Proc. Natl Acad. Sci. USA 92, 3844–3848 (1995).

16. Jain, V., Seung, H. S. & Turaga, S. C. Curr. Opin. Neurobiol. 20, 653–666 (2010).

17. Seung, H. S. Neuron 62, 17–29 (2009). 18. Fukushima, K. Biol. Cybernet. 36, 193–202

(1980). 19. Mesulam, M. Ann. Neurol. 57, 5–7 (2005).

Neurosci. 27, 419–451 (2004). 9. Sohya, K., Kameyama, K., Yanagawa, Y., Obata, K. &

Tsumoto, T. J. Neurosci. 27, 2145–2149 (2007). 10. Niell, C. M. & Stryker, M. P. J. Neurosci. 28,

7520–7536 (2008). 11. Kerlin, A. M., Andermann, M. L., Berezovskii, V. K. &

Reid, R. C. Neuron 67, 858–871 (2010). 12. Runyan, C. A. et al. Neuron 67, 847–857 (2010). 13. Douglas, R. J., Koch, C., Mahowald, M., Martin, K. A.

& Suarez, H. H. Science 269, 981–985 (1995).

M AT E R I A L S S C I E N C E

Bubble wrap of cell-like aggregatesUsing a microfluidic device, tiny polymeric capsules have been made in which different compounds can be isolated in separate, membrane-bound compartments — a prerequisite for the development of artificial cell aggregates.

T A K A M A S A H A R A D A & D E N N I S E . D I S C H E R

Adding milk to coffee or tea leads to a homogeneous (and tasty) mixture, but how could one keep such fluids apart

when they are combined? Aqueous fluids tend to flow and mix together even on the micro­metre scale of biological cells, and so keeping such fluids apart requires a robust partition. Cells in tissues achieve this by using flexible membranes not only to define and delimit each cell, but also to compartmentalize their nuclei and other organelles. Reporting in Angew andte Chemie, Shum et al.1 describe similar hier archical assemblies of synthetic cell­like structures, which use polymeric

surfactants as the building blocks of mem­branes instead of natural lipids and proteins. These robust, deformable microcapsules adhere tightly to each other while confining distinct aqueous fluids, in the same way that cells cluster together in tissues while enclosing separate portions of cytoplasm.

To achieve this feat, Shum et al. used a microfluidic device to make water­in­oil­in­water (W/O/W) double emulsions as tem­plates for the polymeric assemblies (Fig. 1). Such double emulsions consist of a water drop­let (or droplets) surrounded by an oil phase, which is itself suspended in a second aqueous solution. The authors precisely controlled the initial size of their emulsions by changing the

volume fraction of each phase being passed through the glass capillaries of the microfluidic device, and/or by changing the capillary diam­eter and flow rate. Importantly, the number of water droplets incorporated into the double emulsions could also be varied by controlling the flow rate of the phases.

The real key to their success, however, was in the choice of ingredients for the oil phase. The authors used two organic solvents mixed with a block copolymer (a polymer in which two or more chemically distinct polymer chains are connected together). The solubility of the copolymer was greater in one of the sol­vents (chloroform) than in the other (hexane). Because the copolymer was amphiphilic — consisting of a hydrophobic chain covalently linked to a hydrophilic chain — it became concentrated at the oil–water interfaces in the W/O/W double emulsions.

Once the double emulsions had formed, the volatile chloroform started to evaporate. Shum et al.1 observed that, as the chloroform left the system, the inner water droplets became coated with a dense monolayer (leaflet) of copolymer. Because the solubility of the copolymer in the remaining hexane was lower than in the origi­nal chloroform–hexane mixture, the water droplets started to stick together. The droplets also adhered to the interface of the oil phase with the surrounding water, where another leaflet of copolymer had formed.

The authors then eliminated the remaining hexane either by evaporation or by using shear in the microfluidic flow of their system. The end result was a highly cohesive assembly of polymer vesicles (polymersomes), wrapped together in a shared outer copolymer leaflet. The orientations of the inner droplets to each other and the contact angles of the interfaces between the droplets determined the eventual shapes of the polymersomes in the aggregates. This meant that the authors could make poly­mersomes of different shapes, but with the same number of compartments.

Shum and colleagues’ ensembles resemble aggregates of soap bubbles and also adherent cells such as those seen in the eye2. The com­parison with adherent cells is telling, because the formation of structured subsets of cells in tissues is a complex process that is also controlled to some extent by the physics of adhesion. Networks of biochemical reactions direct cell crawling and other key processes, but developmental biologists have also noted that simple mixtures of cells sort themselves in a manner reminiscent of the behaviour of immiscible liquids in emulsions. It has been thought for decades that cell sorting in vivo might occur because cells preferentially adopt configurations that minimize the surface and bulk mechanical energies of cell clus­ters. Evidence of this has come most recently from in vitro studies3 in which the adhesion strengths of cells were altered in systems con­taining small numbers of different cell types.

Multi-compartmentpolymersome

OilWater

Water

a b c

d

Copolymer

Figure 1 | The synthesis of polymeric cell-like aggregates. Shum et al.1 have used a microfluidic device to prepare multi­compartment polymer vesicles. a, The authors began by making water­in­oil­in­water (W/O/W) double emulsions — water droplets suspended in an oil phase, which is itself suspended in water. The oil phase consisted of two solvents and a copolymer, which concentrates at the interfaces of the emulsions as monolayers. b, As the more volatile solvent in the oil phase evaporates, the monolayers become adhesive and stick to each other. c, The authors then removed the remaining solvent from the oil phase, generating multi­compartment vesicles called polymersomes, whose membranes consist of bilayers of copolymer molecules. (Graphics in a– c adapted from Scheme 1 of ref. 1.) d, This picture overlays optical and fluorescent images of polymersomes. One compartment contains a fluorescent solute, with the other containing a non­fluorescent solute. No cross­contamination occurs. Scale bar, 200 µm. (Image from ref. 1.)

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The results of these studies indicated that cells sort themselves according to the surface tension of their aggregates.

Inspired by cells, materials scientists have spent more than a decade making micro­capsules in which self­assembled polymer bilayers surround a single compartment. Such polymersomes are now made from a wide range of amphiphilic polymers, using methods4 that range from the hydration of dried films of a copolymer to solvent extrac­tion from a polymer­solubilizing mixture of water and solvent. Polymersomes are gener­ally more robust than lipid vesicles, and exhibit a wide range of properties that are governed by both the chemical properties and molecu­lar masses of the polymer from which they are made. What’s more, polymersomes have been constructed5 in which calcium or copper metal ions induce the formation of domains similar to the rafts hypothesized to exist in cell membranes.

One reason for the broad interest in poly­mersomes is that they might eventually be used to deliver drugs to target tissues in humans. Shum and colleagues1 also have this goal in mind, because they used a bio­compatible copolymer (PEG­b­PLA) that has been approved by the US Food and Drug Administration for drug­delivery purposes. Indeed, PEG­b­PLA has previously been used to make polymersomes that have delivered therapeutic nucleic acids, such as interfering RNA and antisense oligonucleotides, to cells in animals6.

Many other applications of polymersomes are also being developed, including syn­thetic cells, but Shum and colleagues’ vesicle aggregates1 offer intriguing new possibili­ties: unlike conventional vesicles, they could encapsulate several different cargoes without cross­contamination. The authors hypoth­esize that their vesicle aggregates could serve as micrometre­scale reactors, for example, in which different reactants are loaded into separate compartments, the number of which sets the stoichiometry of the reaction. The loaded polymersomes could be delivered to a target site, whereupon controlled degrada­tion of the polymer triggers a reaction as the encapsulated chemicals are released.

To prove that they could indeed load differ­ent compounds into different compartments of the same polymersome, Shum et al. con­structed a microfluidic device that uses sepa­rate microchannels to inject water droplets containing different solutes — one of which was fluorescent — into the oil phase of dou­ble emulsions. The authors made polymer­somes from the emulsions as before, and then used optical and fluorescent microscopy to prove that the fluorescent cargo was trapped exclusively in one compartment.

A grander challenge for vesicle assembly is to prepare functional mimics of cell aggre­gates in tissues, in which each vesicle performs

a specialized function. Such differentiation of cells in close proximity is what makes tissues so complex. Shum and colleagues’ microfluidic method for assembling polymersome aggre­gates under mild reaction conditions is likely to stimulate much research in this area, and to be used for many other applications, for years to come. ■

Takamasa Harada and Dennis E. Discher are in the Department of Chemical and Biomolecular Engineering, University of

Pennsylvania, Philadelphia, Pennsylvania 19104, USA. e-mail: discher@seas.upenn.edu

1. Shum, H. C., Zhao, Y., Kim, S.-H. & Weitz, D. A. Angew. Chem. Int. Edn 50, 1648–1651 (2011).

2. Hayashi, T. & Carthew, R. W. Nature 431, 647–652 (2004).

3. Manning, M. L. et al. Proc. Natl Acad. Sci. USA 107, 12517–12522 (2010).

4. Discher, D. E. & Eisenberg, A. Science 297, 967–973 (2002).

5. Christian, D. A. et al. Nature Mater. 8, 843–849 (2009).6. Kim, Y. et al. J. Controlled Release 134, 132–140

(2009).

H O S T– PAT H O G E N I N T E R A C T I O N

Culprit within a culpritThe parasitic infection mucocutaneous leishmaniasis can vary in severity. It emerges that the levels of an RNA virus within the parasite affect both the host’s immune response and the parasite’s persistence.

M A R T I N O L I V I E R

Protozoan parasites of the genus Leish-mania are responsible for the infectious disease leishmaniasis. The disease mainly

affects those living in tropical and sub­tropi­cal parts of the world, and a unique form of it called mucocutaneous leishmaniasis causes a disfiguring disease in the Americas. Despite the century­old knowledge that Leishmania parasites of the Viannia subgenus cause leish­maniasis, how the disease progresses remains enigmatic. How does a primary cutaneous lesion lead to a secondary, metastasizing form reappearing months later in the nasopharyn­geal tissues? Writing in Science, Ives et al.1 shed light on this process, showing that the parasite recruits help from within to counteract the host’s immune responses, thus enabling it to spread farther afield.

Mucocutaneous leishmaniasis is associ­ated with a persistent inflammatory response characterized by increased expression of pro­inflammatory mediators (TNF­α and some chemokines)2,3 that play a pivotal part in recruiting immune­system cells such as macro phages to the site of infection. Using pre­vious approaches4,5, Ives et al. isolated clones of Leishmania guyanensis — a parasite of the Viannia subgenus — from infected hamsters.

The clones were either non­metastatic (L.g.M–) or metastatic (L.g.M+), and Ives et al. found that the L.g.M+ clones harboured a virus called Leishmania RNA virus 1 (LRV1). When the authors infected mouse macro phages with these clones, the cells rapidly expressed sev­eral cytokines and chemokines (including TNF­α and CXCL10) that are relevant to the pathology of mucocutaneous leishmaniasis. The strongest response was to L.g.M+ clones and to another clone isolated from patients

with mucocutaneous leishmaniasis. By con­trast, L.g.M– clones and their corresponding human isolates from cutaneous lesions led to only a modest, albeit significant, response compared with uninfected cells; this is consist­ent with earlier findings comparing different Leishmania species6.

Ives et al.1 show that the ability of the L.g.M+ clones to induce increased expression of pro­inflammatory molecules depends on their internalization into macrophages and their sequestration within phagolysosomes. A phagolysosome is an organelle in which engulfed pathogens can be destroyed; Leishma-nia, however, has evolved to survive its harsh environment.

An inflammatory immune response is often triggered by the recognition of a pathogen by specific receptors of the innate immune sys­tem. One such receptor family comprises the Toll­like receptors (TLRs), which are found on the cell surface or on vesicle membranes of macrophages and other cells of innate immu­nity. To determine how the inflammatory response to L. guyanensis comes about, Ives and colleagues studied macrophages that were functionally deficient in the various phago­somal TLRs (TLR3, 7 and 9) or in intracellular adaptors related to TLR signalling (MyD88 and TRIF).

They found that TLR3 and TRIF are essen­tial for maximal production not just of pro­inflammatory mediators induced by L.g.M+, but also of IFN­β — a cytokine produced in response to TLR3 activation and which poten­tially can cause organ damage. Intriguingly, previous work on virus­free Leishmania spe­cies suggested that IFN­β7 and TLR38 could be involved, respectively, in the progression of leishmaniasis and in the parasite’s recognition by IFN­γ­primed macrophages. Those studies

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