Dissipative Self-Assembly of Dynamic Multicompartmentalized Microsystems with Light-Responsive Behaviors

Abstract

•Autonomous self-assembly of polymeric multicompartmental microstructures•Out-of-equilibrium structural evolution powered by energy dissipation•Spatially controllable structures and functionalities The design and self-assembly of biomimetic compartments not only have the potential to help uncover the mechanisms that may have occurred during the origin of life but also offer new inspiration for technology and applications in many fields. An intriguing aspect of the compartmental structure in extant living systems is, for example, the ubiquity of dynamic features driven by energy flow. Thus, mimicking the multicompartmental structures and some dynamical aspects of living systems is extremely attractive. In this paper, we develop a strategy using chemical fuel for the autonomous generation of active polymeric microsystems that can dissipate energy to control their multicompartmental structures. Overall, the proposed proof-of-concept principles illustrate a strategy to design autonomous microsystems with complex and dynamic behaviors and introduce a way to engineer active and functional materials for broad applications in biosensors, microreactors, or molecular delivery. Living cells are compartmentalized systems operating far from thermodynamic equilibrium and consume energy from the environment to carry out their functions. The construction of biomimetic synthetic compartments has both scientific and technological value. Despite many advances, the assembly of synthetic compartments that present nonequilibrium behaviors based on dissipative (or transient) self-assembly remains a grand challenge. Here, we describe the design and synthesis of multicompartmental systems with an active microstructure and complex behaviors. By including an artificial pathway for energy dissipation, we demonstrate the autonomous generation of active polymeric systems using a strategy based on polymerization-induced self-assembly (PISA), driven by chemical fuel. Remarkably, these self-organized synthetic systems exhibit a dynamic change in their multicompartmental structures when exposed to light. This work introduces a strategy toward the design and construction of synthetic compartments with a dynamic structure and also presents pathways to engineer materials with spatially controllable structures and functionalities. Living cells are compartmentalized systems operating far from thermodynamic equilibrium and consume energy from the environment to carry out their functions. The construction of biomimetic synthetic compartments has both scientific and technological value. Despite many advances, the assembly of synthetic compartments that present nonequilibrium behaviors based on dissipative (or transient) self-assembly remains a grand challenge. Here, we describe the design and synthesis of multicompartmental systems with an active microstructure and complex behaviors. By including an artificial pathway for energy dissipation, we demonstrate the autonomous generation of active polymeric systems using a strategy based on polymerization-induced self-assembly (PISA), driven by chemical fuel. Remarkably, these self-organized synthetic systems exhibit a dynamic change in their multicompartmental structures when exposed to light. This work introduces a strategy toward the design and construction of synthetic compartments with a dynamic structure and also presents pathways to engineer materials with spatially controllable structures and functionalities. 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The unique building blocks in the synthetic system provide a pathway for the absorption and dissipation of either chemical fuel or light energy. More specifically, we demonstrate the autonomous generation of our systems through dissipative (or transient) polymerization and self-assembly driven by chemical fuel (Belousov-Zhabotinsky [BZ] reaction), which also show complex structural changes under exposure to light. Overall, mimicking some aspects of nature, the proposed proof-of-concept principles not only illustrate an experimental strategy to design autonomous self-assembled and active ordered polymeric and artificial systems with complex and dynamic behaviors but also present the way to engineer active materials with spatially separated and controllable modules and functionalities. Polymers are excellent candidates for the self-assembly of synthetic compartments. 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Tris(bipyridine)ruthenium (Ru(bpy)3) moieties can catalyze the BZ reaction to generate radicals which are required for PISA. Under light exposure, the photophysical and chemical properties of the Ru(bpy)3 moieties can lead to reorganization in the set of building blocks as a route for energy dissipation. We show in the following that the integration of the PRAFT into the supramolecular structure opens an artificial pathway to dissipate external energy that is capable of driving the self-assembly of synthetic multi-compartmentalized micron-sized systems and also change their structures dynamically. As both raw material and catalyst, PRAFT can initiate the chemical-fuel-driven PISA for synthesis and autonomous self-assembly of the macromolecular components (Figure 1). More specifically, the PRAFT can initiate the BZ reaction. The BZ reaction subsequently generates the radicals (Figure S3) needed for polymerization and enables the polymerization of monomer (butyl acrylate, BA) with the PRAFT and Poly(ethylene glycol) 4-cyano-4-(phenylcarbonothioylthio)pentanoate (PEG-CTA) to produce the amphiphiles, which eventually lead to the autonomous self-assembly of synthetic multicompartments. Proton nuclear magnetic resonance (1H-NMR) shows the formation of polymeric amphiphiles (Figure S4). Notably, dissipative chemical energy from the BZ-reaction is critical for the self-organized formation of our microsystems. As shown in Figure S5, when the BZ reaction was not activated, no microstructure formed regardless of the presence of a similar ionic environment. Furthermore, the dynamics of the BZ reaction is also affected and controlled by the formation of the systems themselves. As seen from the BZ oscillation profile (Figures 2A and S6), the BZ reaction coupled to the PISA synthetic system resulted in a delayed onset of chemical oscillations, indicating the consumption of radicals for polymerization. After that, the oscillation amplitude gradually decreased because the loss of free Ru(bpy)3 moieties as catalysts during self-assembly of synthetic systems leads to the termination of chemical reactions as well as energy dissipation. In other words, energy dissipation is also affected by the formation of synthetic self-assembled systems. The continuous growth of the hydrophobic block leads to the generation and growth of microcompartments (Figures 2B and S7).Figure 2Chemical-Fuel-Driving PISA for the Dissipative Self-Assembly of Multicompartmental Endogenous and Self-Assembled Active Synthetic MicrostructuresShow full caption(A) Oscillation profiles of the pure BZ reaction (blue) and BZ-PISA (red). BZ reaction as the chemical energy source drives the dynamic PISA.(B) Mean size variation with time of the generated microstructures by BZ-PISA. The microstructure continuously grows during BZ-PISA.(C–H) Morphology characterization of the multicompartmental systems produced by this mode of BZ-PISA: (C) bright-field microscopy image, (D) fluorescence microscopy image, (E) 3D reconstruction confocal microscopy image, (F) confocal images with orthogonal views, and (G and H) cryo-SEM images. Note: (H) presents the enlarged regions of interest in (G). Scale bars: 5 μm (C–F) and 1 μm (G and H).(I) Schematic illustration of the multicompartmental structures pointing out the water phase and polymer-enriched phase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Oscillation profiles of the pure BZ reaction (blue) and BZ-PISA (red). BZ reaction as the chemical energy source drives the dynamic PISA. (B) Mean size variation with time of the generated microstructures by BZ-PISA. The microstructure continuously grows during BZ-PISA. (C–H) Morphology characterization of the multicompartmental systems produced by this mode of BZ-PISA: (C) bright-field microscopy image, (D) fluorescence microscopy image, (E) 3D reconstruction confocal microscopy image, (F) confocal images with orthogonal views, and (G and H) cryo-SEM images. Note: (H) presents the enlarged regions of interest in (G). Scale bars: 5 μm (C–F) and 1 μm (G and H). (I) Schematic illustration of the multicompartmental structures pointing out the water phase and polymer-enriched phase. We then characterized the morphology and structure of the formed synthetic objects. Figures 2C and S8 show bright-field images of the multicompartmental microstructures thus generated. We noticed that the resulting synthetic polymeric microstructures have a rough surface. Because of the fluorescence properties of the Ru(bpy)3 moieties (Figure S9), we were also able to take advantage of both fluorescence microscopy and confocal microscopy (Figures 2D–2F and S10). The multicompartmental character of the structures was clearly observed and confirmed by cryogenic scanning electron microscopy (cryo-SEM) (Figures 2G and 2H) and cryogenic transmission electron microscopy (cryo-TEM) imaging (Figure S11). Clearly, our systems are composed of two well-defined phases (Figure 2I). The fluorescent part is the polymer enriched phase, which is formed through the interactions (e.g., coordination and hydrophobicity)48Li H. Yang P. Pageni P. Tang C. Recent advances in metal-containing polymer hydrogels.Macromol. Rapid Commun. 2017; 38: 1700109Crossref Scopus (66) Google Scholar,49Tamate R. Ueki T. Shibayama M. Yoshida R. Self-oscillating vesicles: spontaneous cyclic structural changes of synthetic diblock copolymers.Angew. 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Darzacq X. Karpen G.H. Phase separation drives heterochromatin domain formation.Nature. 2017; 547: 241-245Crossref PubMed Scopus (946) Google Scholar,51Larson A.G. Elnatan D. Keenen M.M. Trnka M.J. Johnston J.B. Burlingame A.L. Agard D.A. Redding S. Narlikar G.J. Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin.Nature. 2017; 547: 236-240Crossref PubMed Scopus (884) Google Scholar Notably, the resulting multicompartmental microstructures were relatively stable, and no significant morphology and size changes were observed (Figure S13) even after they had been stored and protected from light for several weeks. It is important to note that the integrated artificial pathway in these protocell systems is critical for the formation of a multicompartmental structure. As shown in Figures S14A–S14C, in absence of the PRAFT polymer, no microstructure was formed because the chemical energy could not be utilized by the system for dissipative self-assembly. Although the BZ reaction activated by free Ru(bpy)3Cl2 can also initiate the polymerization, in the absence of PRAFT polymers, no multicompartmental microstructure was observed (Figures S14D–S14F) because energy consumption is not controlled by the self-assembly. Our synthetic multicompartments differ from most of the current synthetic protocell models built by self-assembly of preformed blocks because an artificial dissipation pathway is implemented in our systems, which can also dissipate light energy due to the unique photosensitivity of the integrated Ru(bpy)3 moieties. Therefore, we conclude that access to externally available energy drives the dynamic self-assembly and reorganization inside these systems. As a result, these synthetic systems exhibit structural changes and show dissipative stimulus responsiveness. Without light irradiation, no apparent change was observed in our systems (Figure S15). However, under illumination with light (wavelength 472 ± 20 nm), the dark spots changed actively (Figures 3A and 3B ; Video S1). Some new dark spots emerged, and certain small dark spots coalesced into larger dark spots. Interestingly, the volume of the active microstructures changed under the inflow of light (Figure S16), which can be ascribed to the generation of new subcompartments and ejection of the water phase to the bulk solution. In all cases, our self-assembled systems were highly active, continuously changing their structures and configurations. We also studied the effects of changes of local temperature upon irradiation. As shown in Figure S17, in a few minutes, the temperature fluctuation is less than 1°C. Furthermore, under the defined conditions (25°C–45°C), the temperature has no apparent influence on the multicompartmental structure of the synthetic system. We interpret this as evidence that these systems cannot dissipate the thermal energy for functionality. Since our designed Ru(bpy)3 moieties have different absorption for light of different wavelengths (Figure S18), we expected a variety of structural changes at different levels of photo exposure (Figure 3C). Moving across the different absorption gradients of light shows a clearer picture of the structural transformation taking place. The Ru(bpy)3 moieties have a strong absorbance in the UV, which is relatively high-energy light, and thus we observed significant changes of the multicompartmental structure, decrease of fluorescence intensity, and increased volume of the subcompartments (dark spots; Figures 3D and S19). Similarly, we also observed a slight change of multicompartmental structure under green light irradiation. However, no apparent structural change was observed under red light exposure. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJlMGY3OWNmNDI0ZjA0NTA1NTQ3YTlhZjY2MGRhZDJhZCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc5MzEyMTU1fQ.oLfPwzFYtvpG6d8j3cvQVCMq2Hdij1oJ3B4QFOAGsGEnr03APugAQDz5zqfi3LlL91TVwZAIl2ALRCudJovJxrvM6Hl3DsmA_PAUIA7Z4qeTMONyhnTMxpB_0LTjM9BJNA8FX94O_xrSR3LZpNSr8_daFSyglELY-ugyGhPSVK94omM7DAUpdfJqmmEFzBeMaaSgj6PQy8Mml21-am2fIlSLc0kPf1l2WknEkae7cA5axZ_a4CrF3eXS6CYcbba8PJPG3g5YQaYTBYbA1ChORFrsEQSTFdqFB8r296CpydY6WtPHP9vgn9OjFD3hQIf3pnPTdoBSmxNLWqQGHRN0Lw Download .mp4 (3.78 MB) Help with .mp4 files Video S1. Nonequilibrium Structure Evolution of Our Synthetic Microstructures under Light Illumination (2× Speed) The above results demonstrate that our dissipative self-organized systems exhibit active behaviors. This can be ascribed to the integration of Ru(byp)3 enriched building blocks into the system which provides an appropriate pathway for the dissipation of light energy (Figure 3E). Notably, [Ru(bpy)3]2+ has strong absorbance centered at ∼455 nm (Figure S18) due to the metal-to-ligand charge-transfer (MLCT) dπ → π∗ transition.52Bhasikuttan A.C. Suzuki M. Nakashima S. Okada T. Ultrafast fluorescence detection in Tris(2,2‘-bipyridine)ruthenium(II) complex in solution: relaxation dynamics involving higher excited states.J. Am. Chem. Soc. 2002; 124: 8398-8405Crossref PubMed Scopus (287) Google Scholar Under irradiation with light, it can produce a redox-active photoexcited state ([Ru∗(bpy)3]2+), which then reacts with an electron acceptor (e.g., oxygen) to generate an oxidized ground state ([Ru(bpy)3]3+) (Figures 3E and S20).53Yoon T.P. Ischay M.A. Du J. Visible light photocatalysis as a greener approach to photochemical synthesis.Nat. 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