Multi-compartment encapsulation of communicating droplets and droplet networks in hydrogel as a model for artificial cells

www.nature.com/scientificreports OPEN received: 01 November 2016 accepted: 20 February 2017 Published: 03 April 2017 Multi-compartment encapsulation of communicating droplets and droplet networks in hydrogel as a model for artificial cells Mariam Bayoumi1, Hagan Bayley2, Giovanni Maglia1,3 & K. Tanuj Sapra4 Constructing a cell mimic is a major challenge posed by synthetic biologists. Efforts to this end have been primarily focused on lipid- and polymer-encapsulated containers, liposomes and polymersomes, respectively. Here, we introduce a multi-compartment, nested system comprising aqueous droplets stabilized in an oil/lipid mixture, all encapsulated in hydrogel. Functional capabilities (electrical and chemical communication) were imparted by protein nanopores spanning the lipid bilayer formed at the interface of the encapsulated aqueous droplets and the encasing hydrogel. Crucially, the compartmentalization enabled the formation of two adjoining lipid bilayers in a controlled manner, a requirement for the realization of a functional protocell or prototissue. Synthetic biology seeks to build cells and modify them to understand how life began, functions, and evolves1,2, as well as to engineer and exploit new life forms3,4. The top-down approach boasts of synthesizing a minimal genetic blueprint5–7, ‘creating’ a cell8, and taming metabolic pathways for biotechnological applications9–11. With equally grand ambitions, bottom-up synthetic biology is focused on the de novo design of a cell12,13 with the specific aims of building minimal structures14,15 and mimicking complex cellular functions16–19. Toward the realization of synthetic cellular systems, success has been achieved in the bottom-up design of a protocell20, and intriguing possibilities have been demonstrated for a functional prototissue21,22. The success of the biological cell depends on compartmentalization. A direct consequence of compartmentalization is chemical and electrical signaling, which are key factors in imparting emergent properties to biological cells and tissues. Consequently, a mandatory feature of a protocell, and the success of its translation into a prototissue, is compartmentalization and communication between its multiple compartments23. As in natural cells, enclosing DNA, RNA, and proteins within protocells ensures protection from degradation24 while providing the required concentrations for optimal function25,26. For protocellular systems, delimiting the active contents from their environment bestows the possibility of functional engineering27 by means of spatial28 and temporal control over the system22,29. In recent years, protocells have been introduced for applications in drug delivery and nanotechnology30 (e.g., nanometer-sized lipid vesicles31, giant unilamellar vesicles32, polymersomes33, capsosomes34, proteinosomes35, vesosomes36). Recently, aqueous droplets in oil have been proposed as protocell models37. The droplet protocells in an oil/lipid bath are connected through lipid bilayers at the contact interfaces38. Bilayer-linked aqueous droplets in a network are capable of electrical and chemical communication with each other39 and with surrounding aqueous medium40 via protein nanopores. Droplet networks have been shown to exhibit emergent properties of electrical41 and mechanical nature21, the first steps toward formation of a prototissue. The aqueous droplets can be replaced by millimeter-sized hydrogel pieces in oil with stable bilayers at their interfaces42,43. Here, we incorporated aqueous droplets, stabilized in an oil/lipid bath, inside a hydrogel, which might serve as the basic unit for the bottom-up construction of a protocell, and a collection of these might make prototissue (Fig. 1). The use of a firm hydrogel matrix was key to forming multiple compartments inside the same hydrogel unit. The stable encapsulation of aqueous droplets in different oil compartments held in the hydrogel enabled the 1 Department of Chemistry, KU Leuven, Celestijnenlaan 200G, 3001 Leuven, Belgium. 2Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA, United Kingdom. 3Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands. 4Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. Correspondence and requests for materials should be addressed to G.M. (email: ) or K.T.S. (email: ) Scientific Reports | 7:45167 | DOI: 10.1038/srep45167 1 www.nature.com/scientificreports/ Figure 1. Concept of bottom-up design of artificial cells and modular tissues. Akin to a biological tissue, we define an ensemble of protocells as a prototissue, and a network of prototissues is considered a proto-organ. A simple bottom-up hierarchical construction would be – proto-organelle →  protocell →  prototissue →  protoorgan. The proto-organelle is simply an aqueous droplet immersed in an oil/lipid bath to coat it with a lipid monolayer. To produce a system with hierarchical properties, the proto-organelle in oil is encased in a hydrogel. The aqueous droplet-hydrogel interface is stabilized by a lipid bilayer capable of functionalization with membrane proteins (e.g., chemical and electrical communication, sensing). Because protocells contain protoorganelles, multi-compartment encapsulation of single aqueous droplets (proto-organelles) in a hydrogel will form a protocell (i). An assembly of such hydrogel-protocells, ideally connected through lipid bilayers42, will form a prototissue (ii). Alternatively, multiple aqueous droplets (proto-organelles) in the same oil compartment (encased in hydrogel) will also constitute a protocell; multi-compartmentalization of the protocells in a hydrogel will constitute a prototissue (iii). A collection of such prototissues would be a proto-organ (iv). The crucial element of the proposed system is multiple levels of compartmentalization, modularity and spatial flexibility. The modular design has the advantage of structure-function interconversion; an agarose unit can be a protoorganelle, a protocell or a prototissue. Because the hydrogel pieces can be spatially manipulated42, a protocell can be introduced into or removed from a prototissue, e.g., by adding or removing a piece of hydrogel (i ↔  ii); injecting aqueous droplets can convert a protocell into a prototissue (i → iii); and prototissues can be assembled into or removed from a proto-organ (again by adding or removing a piece of hydrogel) (iii ↔  iv). formation of two bilayers close to each other – a first step toward engineering organelles and cell mimics for controlled electrical and chemical communication. A major advantage of the present strategy is the ease of hierarchical encapsulation, thereby offering a clear demarcation between a proto-organelle, a protocell and a prototissue. The concept We started with the premise that the scaffold of a protocell should also act as the basic unit of (...truncated)