Dissipative Self-Assembly of Dynamic Multicompartmentalized Microsystems with Light-Responsive Behaviors
Natural living cells are compartmentalized chemical systems that operate far from thermodynamic equilibrium and use energy and fuel from the environment to carry out their functions. Mimicking some aspects of natural living systems by including an artificial pathway for energy dissipation, we demonstrate the autonomous generation and self-assembly of active polymeric microsystems through polymerization-induced self-assembly, driven by chemical fuel. These selforganized synthetic systems exhibit a dynamic change in their multicompartmental structures when exposed to light.
INTRODUCTION<!>The Bigger Picture<!>RESULTS<!>DISCUSSION<!>EXPERIMENTAL PROCEDURES<!>Nonequilibrium Behaviors of Multicompartmental Microstructures Driven by the Light Influx<!>SUPPLEMENTAL INFORMATION
<p>As the basic building unit of life, the cell is a compartmentalized microstructure that can extract energy from the external environment and provide specialized cellular functions. The segregation provided by compartments is indispensable for living cells because it generates a free-energy gradient that enables them to operate far from thermodynamic equilibrium. 1 The self-assembly of their substructures is closely related to their dynamical properties resulting from nonequilibrium. [2][3][4][5] In the past decade, the design and construction of artificial compartmentalized systems that mimic cellular structure or perform cell-like functions has attracted substantial attention. [6][7][8][9][10][11] It not only sheds light onto the potential compartmentation mechanisms that may have operated in the origin of life [12][13][14][15] but also has extensive technological applications in many fields, such as materials for energy harvesting and transduction, environmental remediation, catalysis, and biomedicine. [16][17][18][19] In nature, extant living systems have compartments constructed with specialized phospholipid amphiphiles generated by a complex series of biochemical reactions. In the laboratory, natural amphiphiles and different types of materials could be used to investigate the origins of membranes and compartments in cellular life. ''A protocell is any experimental or theoretical model that involves a self-assembled compartment linked to chemical processes taking place around or within it, aimed at explaining how more complex biological cells or alternative forms of cellular organization may come about.'' 20 So far, various protocells assembled using lipids, [21][22][23] fatty acids, 24,25 and polymers [26][27][28][29][30][31][32] or nanoparticles [33][34][35] have been built. However, in these contexts,</p><!><p>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-ofconcept 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.</p><p>the formation of synthetic ''cells'' was performed under close to equilibrium conditions by self-assembly of pre-formed building blocks. Living systems always operate far from equilibrium. 36 Thus, mimicking the multicompartmental structures and dynamical aspects of living cells in a synthetic out-of-equilibrium system is an extremely attractive challenge.</p><p>Herein, we present an experimental strategy for the autonomous generation of primitive and active polymeric microstructures that can dissipate energy to control their multicompartmental structures. A natural living system uses a complex metabolic network to consume food and dissipate energy and to eventually perform complicated cellular functions. 37,38 Similarly, we have designed and implemented an artificial pathway (a sequence of chemical reactions) connecting the process of energy dissipation with the dynamic self-assembly of our synthetic protocell-like systems. To achieve this goal, we designed a Ru(bpy) 3 catalyst conjugated reversible addition-fragmentation chain-transfer polymer. It plays dual roles as both the catalyst and the building block in a dissipative polymerization-induced self-assembly (PISA) process. 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.</p><!><p>Polymers are excellent candidates for the self-assembly of synthetic compartments. Especially as an emerging chemical method, PISA provides a convenient route to generate artificial autonomous systems by the synthesis and self-assembly of polymeric amphiphiles in one step, driven by the energy of heat, light, or chemical fuel. [39][40][41][42] The BZ reaction is a prototypical nonequilibrium and dynamical chemical network, 43 and it is a demonstrated chemical fuel for driving polymerization and self-assembly. [44][45][46][47] We make use of PISA driven by the BZ reaction to construct systems endowed with dynamic structures and behaviors. To achieve this goal, we designed and introduced a mechanism for autonomous generation and dissipative self-assembly of our active microstructures. First, a multifunctional polymer (which is photosensitive, and we call PRAFT; Figures S1 and S2) tagged with both photosensitizers (Ru(bpy) 3 ) and reversible addition-fragmentation chain-transfer (RAFT) agents was synthesized. 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.</p><p>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 ( 1 H-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).</p><p>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) 48,49 among the Ru(bpy) 3 moieties in the blocks with PRAFT. The dark volume (or subcompartment) is the water phase, which is stabilized by the self-assembly of block copolymer amphiphiles and does not contain any fluorescent moieties, hence its appearance as a dark spot. To further demonstrate this point, we used a hydrophilic dye (sulfo-cyanine5.5 carboxylic acid) to monitor the distribution of the water phase within our systems. As shown in Figure S12, the sulfo-cyanine5.5 carboxylic acid was observed in the above dark spots, and the intensity was much higher than that in the polymer-enriched phase. This demonstrates the phase separation of the polymer-enriched and water phases. It mimics in a primitive way some of the biophysical properties of natural cells, where phase separation is thought to be the driving force mediating the formation of compartmental structures and sub-organelles. 50,51 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) 3 Cl 2 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.</p><p>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 G 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.</p><p>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) dp / p* transition. 52 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). 53,54 Since  Article the oxidized Ru(bpy) 3 is more hydrophilic, 55,56 the PRAFT block becomes more hydrophilic when bathed in this light. Thus, some of the Ru(bpy) 3 segments inserted in the polymer phase of the microstructure could be released into the water phase; this results in phase separation and, consequently, the reorganization of the multicompartmental structure until a new equilibrium state is reached (Figure 3F).</p><p>We confirm our interpretation that the multicompartmental structure evolution is the result of dissipative (or transient) self-assembly and reorganization of building blocks due to adjustment of the composition of the building blocks making up our ordered system. We assume that a higher content of PRAFT would result in a richer polymer phase in the resulting compartments. With this in mind, we tuned the ratios of the two different macro-RAFT materials (PRAFT or PEG-CTA) for the PISA generation of our multi-compartmentalized objects. As expected, a high ratio of PRAFT in the source materials led to a high content of polymer phase while also generating a small number and low volume of sub-compartments (Figures 4A-4C and S21). These results indicate that the slightly different wettability of the building blocks is responsible for variation in the multi-compartmentalized structure. Therefore, under the inflow of energy, the continuous change in the wettability of the Ru(bpy) 3 enriched polymer phase would actually lead to the active self-assembly and reorganization of the building blocks. It ultimately resulted in the evolution of the multicompartmental structure of our synthetic protocells and represents a form of dissipative nonequilibrium behavior.</p><p>To better understand how the illumination drives dissipative assembly in the synthetic multicompartments, we focused our attention on the structural dynamic change of the pure Ru(bpy) 3 conjugated polymer phase. Microstructures were synthesized and assembled by PRAFT initiated PISA. As expected, microstructures with Ru(bpy) 3 conjugated polymer phase were obtained (Figure S22). These microstructures have a built-in artificial pathway for energy dissipation. Therefore, we expected that the inflow of light would activate the microstructure, drive the dissipative self-assembly and reorganization, and finally lead to the generation of a multicompartmental structure (Figure 4D). Figures 4E, 4F, and S23 show optical microscopy images of a typical spherical microstructure before and after light exposure. By exposing them to light, the smooth surface of the microstructure became coarse, and fluorescence profiles reveal the generation of some cavities within the microstructures (Figure 4G). Clearly, the multicompartmental structure was formed upon illumination with light of the appropriate wavelength and intensity. This is because some of the chains of PRAFT with Ru(bpy) 3 moieties in the polymer phase become more hydrophilic under light exposure and lead to the reorganization of the building blocks. These results, again, demonstrate that the availability of external energy drives the dissipative self-assembly inside the compartment and induces our self-assembled system to dynamically change its structure.</p><!><p>Living systems are compartmentalized out-of-equilibrium systems, and their active behaviors and cellular functions are fueled by a continuous flow of energy and matter. 36,38 The construction of compartmentalized protocell systems has both basic science and technological values. Although some excellent examples of synthetic compartments with cell-like properties (growth and biomimetic behaviors) have been documented, the process to build a synthetic and self-assembled system that can dissipate energy for their autonomous generation and nonequilibrium behaviors is still in its infancy. The results presented here provide a synthetic approach ll 1166 Chem 6, 1160-1171, May 14, 2020 Article to construct dissipatively microscopic self-assembling systems in order to bridge this gap. In our system, we mimic some simple aspects of natural cells by the integration of a simplified artificial pathway into systems. We designed a PRAFT polymer with tagged Ru(bpy) 3 and RAFT agents that plays a dual role as both building blocks and catalysts in the ongoing dynamic (or transient) self-assembly. The multicompartmental structure of these ''protocells'' is closely linked to energy dissipation. By dissipation of chemical fuel (BZ reaction) or through light irradiation from the environment, macromolecular crowded compartments are generated autonomously in one pot, which show active multicompartmental structures. Such characteristics Article differentiate these systems from other reported artificial cell models operating under close to equilibrium conditions. The multicompartmental structure that results from the phase separation of the Ru(bpy) 3 moieties enriched polymer and the water phases demonstrates a possible mechanism to mimic the onset of compartmentalization with macromolecules in natural living systems. 57,58 Therefore, our approach not only solves the problem of autonomous generation of synthetic multicompartments but also provides an example of endogenous protocell-like processes operating under a continuous flux of energy from the environment.</p><p>Compartments internal to natural cells (i.e., nucleus, endosomes, or other organelles) and internal molecular reorganizations are used to perform complex cellular functions. The basis for these is the phase separation of intracellular macromolecules due to the interactions among them. 50,51,59 They are controlled by their metabolic reaction networks driven by energy dissipation. Given the complex regulatory networks in living cells and the role played by evolution and natural selection in their current configurations, it is not possible at this time to construct such a level of complexity in synthetic systems. Our multicompartmental microstructures with their simplified artificial pathway mimic, albeit in a primitive way, incorporate some of the basic dynamic features of the compartmental structure. They can use external light energy and give rise to dynamic self-assembly and phase separation as a simplified manner of structural changes, including the generation of new subcompartments and the fusion of subcompartments. Our results also suggest that the building blocks for PRAFT play a central role in the formation of multicompartmental structures and the dynamical evolution of their structure. Though the building blocks used in this study involve specialized macroRAFT polymer and monomer, the principles underlying their behavior, however, are general and should also apply to other molecules and synthetic systems. In summary, using dissipative (or transient) PISA driven by chemical fuel, we have presented a class of biomimetic microsystem with a dynamic multicompartmental structure. Specifically, a multifunctional macromolecular precursor molecule was designed to be both catalyst and building block to initiate the autonomous synthesis, dissipative self-assembly, and then the structural evolution of these microstructures. By integration of an artificial pathway in the dynamic system for the absorption and dissipation of energy provided by chemical fuel or light, synthetic multicompartments were autonomously generated through a self-controlled BZ-PISA. We have seen that the dynamic formation and complex behaviors of the resulting multicompartmental structures can be attributed to the phase segregation and consequent structural reorganization of the building blocks under the inflow of energy. Our results not only constitute a strategy toward the design and construction of synthetic multicompartments and simple, non-biochemistry based biomimetic protocells of a primitive form but also shed some light on potential compartmentalization mechanisms in the origin and evolution of natural life by providing a mechanism to describe the nonequilibrium induced phase separation. Finally, considering the modular components of the above compartments, our work also provides useful insights to design advanced materials for many applications in chemistry-based technologies (e.g., biosensors, microreactors, or molecular delivery).</p><!><p>Synthesis of the PRAFT 500 mg of 4arm-PEG-NH 2 was dissolved in 1 mL of dichloromethane and 18.8 mg of CTA-NHS was dissolved in 250 mL of dichloromethane, respectively. Under vigorous ll agitation, the CTA-NHS solution was added dropwise to the 4arm-PEG-NH 2 solution, and the mixture was further stirred overnight. The product (CTA-4arm-PEG) was precipitated into excess diethyl ether and purified several times. 36 mg of the purified CTA-4arm-PEG was dissolved in 4 mL of DMF. Protected from light, 15 mg of Ru(bpy) 3 -NHS was added in the above solution and followed by adding 10 mL of triethylamine. The mixture was stirred at room temperature for 24 h. Afterward, the mixture was dialyzed in water to remove the solvent and unreacted chemicals. The product solution was lyophilized to obtain the final PRAFT agents.</p><p>Dissipative Self-Assembly of Multicompartmental Microstructures through BZ-PISA 100 mL of PRAFT solution (1 mg mL À1 ) and the PEG-CTA (1 mg mL À1 ) at the designed ratio were added in water with a total volume of 2 mL. Under magnetic stirring, 125 mL of malonic acid (MA) solution (0.6 M), 150 mL of H 2 SO 4 solution (5M), and 12.5 mL of BA were added to the mixture. Then, 250 mL of sodium bromate solution (1 M) was added. The mixture was protected from light and stirred at 200 rpm at room temperature. The redox potential of the system was monitored by a Micro-Redox electrode (Microelectrodes, Inc.), and the data were collected by a LabQuest 2 (Vernier). After reaction for 2 h, the products were purified by dialysis, and the sample suspension was stored in the refrigerator (4 C) and protected from light. The procedure for the construction of microstructures using PEG-CTA as the macro-RAFT agent alone is similar. Instead of using the mixed macro-RAFT agents, 200 mL PEG-CTA (1 mg mL À1 ) and 30 mL of Ru(bpy) 3 Cl 2 (8.5 mM) were added, and other procedures are the same. After preparation, the samples are used within one week.</p><!><p>The irradiation light was separated by filters (UV, 377 G 50 nm; blue, 472 G 20 nm; green, 562 G 40 nm; and red, 628 G 40 nm) from a light source (X-Cite 120, Excelitas) for the desired wavelength, and the light intensity was calibrated by a power meter (Nova II, Ophir). 60 mL of sample suspension (2.5 mg mL À1 in aqueous solution, pH 6.0-7.0) was transferred to a glass slide with a Frame-Seal chamber (SLF0601, Bio-Rad), and it was further sealed by a coverslip. The slide was loaded on a microscope stage and exposed to spectrally separated light for the determined time. Afterward, the microscopy images were recorded to monitor the changes and visualize the compartmental evolution.</p><!><p>Supplemental Information can be found online at https://doi.org/10.1016/j.chempr. 2020.02.009.</p>