Periodic Reporting for period 4 - ActiDrops (Synthetic Active Droplets Inspired by Life)
Periodo di rendicontazione: 2024-08-01 al 2025-01-31
Active droplets are synthetic compartments formed by converting inactive precursors into droplet-forming molecules using chemical fuel. These droplets are inherently unstable and disappear once the fuel is depleted, making them dynamic, energy-dependent systems. Inspired by their crucial role in cell biology, the project aims to recreate and study such life-like behaviors—including spontaneous formation and disassembly, optimal size convergence, collective dynamics, and even self-division—through synthetic analogs. By translating these behaviors into synthetic materials, we aim to obtain a better understanding of the mechanism underlying active self-assembly and its role in biology and the origin of life.
To achieve this, four main objectives are addressed:
1. Develop a library of active modules to control droplet lifetime.
2. Synthesize fuel-dependent active droplets.
3. Elucidate the mechanism behind their accelerated ripening and spatial regulation.
4. Explore how reaction kinetics influence droplet size and enable spontaneous self-division.
To achieve this, four main objectives are addressed:
1. Develop a library of active modules to control droplet lifetime.
2. Synthesize fuel-dependent active droplets.
3. Elucidate the mechanism behind their accelerated ripening and spatial regulation.
4. Explore how reaction kinetics influence droplet size and enable spontaneous self-division.
The successful development of ActiDrops has enabled the achievement of most of the research goals, ensuring the delivery of key milestones and generating significant scientific impact.
A diverse library of chemical reaction cycles (CRCs) has been established, leading to the formation of active modules with a wide range of residence times (Chen et al., JACS, 2023). These reactions have been carefully optimized to minimize the formation of undesired by-products (Chen et al., Chem Sci, 2023). CRCs typically involve the activation of a carboxylate using fuel, producing a transient anhydride that subsequently hydrolyzes. The reduction in negative charge upon activation promotes self-assembly.
These CRCs have been successfully integrated into various self-assembling systems, resulting in the formation of active oil droplets (Schwarz et al., Chem Sci, 2021) and complex coacervates (Donau et al., Nat Commun, 2020), both of which have been studied extensively.
Active oil droplets were developed using long aliphatic precursors that phase-separate from water. Since these droplets are water-excluded, both activation and deactivation occur in the surrounding aqueous environment. Remarkably, these droplets exhibit accelerated ripening, growing significantly faster than classical models would predict. The growth rate can be tuned by adjusting the residence time of the active module (Tena-Solsona et al., ChemSystemsChem, 2021). Additionally, unexpected behaviors such as parasitic and self-emulative dynamics were observed (Schwarz et al., Chem Sci, 2021).
In contrast, active complex coacervates were formed by combining a cationic peptide—generated upon fuel addition—with an anionic polymer, such as RNA. These coacervates, which can consist of up to 95% water, display distinct spatial organization: activation occurs in the external medium, while deactivation takes place within the droplets via hydrolysis. They assemble spontaneously and disassemble in the absence of fuel. Notably, they exhibit life-like behaviors such as fusion, vacuole formation, and transient enrichment of functional RNA (Donau et al., Nat Commun, 2020). These systems have laid a strong foundation for the rational design of fuel-driven, self-assembled compartments (Donau et al., Angew Chem, 2022; Späth et al., Angew Chem, 2022).
The fuel-dependent nature of these compartments has revealed behaviors that challenge classical thermodynamic paradigms. For instance, accelerated ripening in active oil droplets occurs at rates several orders of magnitude higher than predicted by Ostwald ripening (Tena-Solsona et al., ChemSystemsChem, 2020). In addition, liquid spherical shells have been shown to emerge as non-equilibrium steady states (Bergmann et al., Nat Commun, 2023), emphasizing the unique potential of energy-driven assembly processes.
Among the most recent discoveries is the spontaneous emergence of spatio-temporal oscillations in the number, size, and position of droplets within synthetic cells. In these systems, coacervate-based droplets sediment and fuse, followed by controlled shrinkage via a size-regulation mechanism that leads to the expulsion of internal material. This behavior marks a significant step toward the development of life-like, dynamic materials (Sastre et al., Nat Commun, 2025).
Building upon these findings, we expanded our approach to include the formation of active vesicles, which serve as valuable models for synthetic cells. A key characteristic of these vesicles is that their formation and disassembly are regulated by the presence or absence of fuel (Zambrano et al., JACS, 2024; Zozulia et al., Angew Chem, 2024). This dynamic control has generated considerable interest in the synthetic biology field.
We have also achieved significant progress in the area of vesicle self-division. Fatty acid-based vesicles respond to chemical fuel by undergoing membrane budding and division, without requiring complex molecular machinery. (Zambrano et al., JACS, 2024). A complementary manuscript on cell division has recently been accepted in Chem.
A diverse library of chemical reaction cycles (CRCs) has been established, leading to the formation of active modules with a wide range of residence times (Chen et al., JACS, 2023). These reactions have been carefully optimized to minimize the formation of undesired by-products (Chen et al., Chem Sci, 2023). CRCs typically involve the activation of a carboxylate using fuel, producing a transient anhydride that subsequently hydrolyzes. The reduction in negative charge upon activation promotes self-assembly.
These CRCs have been successfully integrated into various self-assembling systems, resulting in the formation of active oil droplets (Schwarz et al., Chem Sci, 2021) and complex coacervates (Donau et al., Nat Commun, 2020), both of which have been studied extensively.
Active oil droplets were developed using long aliphatic precursors that phase-separate from water. Since these droplets are water-excluded, both activation and deactivation occur in the surrounding aqueous environment. Remarkably, these droplets exhibit accelerated ripening, growing significantly faster than classical models would predict. The growth rate can be tuned by adjusting the residence time of the active module (Tena-Solsona et al., ChemSystemsChem, 2021). Additionally, unexpected behaviors such as parasitic and self-emulative dynamics were observed (Schwarz et al., Chem Sci, 2021).
In contrast, active complex coacervates were formed by combining a cationic peptide—generated upon fuel addition—with an anionic polymer, such as RNA. These coacervates, which can consist of up to 95% water, display distinct spatial organization: activation occurs in the external medium, while deactivation takes place within the droplets via hydrolysis. They assemble spontaneously and disassemble in the absence of fuel. Notably, they exhibit life-like behaviors such as fusion, vacuole formation, and transient enrichment of functional RNA (Donau et al., Nat Commun, 2020). These systems have laid a strong foundation for the rational design of fuel-driven, self-assembled compartments (Donau et al., Angew Chem, 2022; Späth et al., Angew Chem, 2022).
The fuel-dependent nature of these compartments has revealed behaviors that challenge classical thermodynamic paradigms. For instance, accelerated ripening in active oil droplets occurs at rates several orders of magnitude higher than predicted by Ostwald ripening (Tena-Solsona et al., ChemSystemsChem, 2020). In addition, liquid spherical shells have been shown to emerge as non-equilibrium steady states (Bergmann et al., Nat Commun, 2023), emphasizing the unique potential of energy-driven assembly processes.
Among the most recent discoveries is the spontaneous emergence of spatio-temporal oscillations in the number, size, and position of droplets within synthetic cells. In these systems, coacervate-based droplets sediment and fuse, followed by controlled shrinkage via a size-regulation mechanism that leads to the expulsion of internal material. This behavior marks a significant step toward the development of life-like, dynamic materials (Sastre et al., Nat Commun, 2025).
Building upon these findings, we expanded our approach to include the formation of active vesicles, which serve as valuable models for synthetic cells. A key characteristic of these vesicles is that their formation and disassembly are regulated by the presence or absence of fuel (Zambrano et al., JACS, 2024; Zozulia et al., Angew Chem, 2024). This dynamic control has generated considerable interest in the synthetic biology field.
We have also achieved significant progress in the area of vesicle self-division. Fatty acid-based vesicles respond to chemical fuel by undergoing membrane budding and division, without requiring complex molecular machinery. (Zambrano et al., JACS, 2024). A complementary manuscript on cell division has recently been accepted in Chem.
ActiDrops has marked a turning point in the development of synthetic active compartments, enabling breakthroughs in fuel-driven self-assembly that were previously unattainable. By integrating a diverse library of chemical reaction cycles (CRCs)—based on carboxylate activation and anhydride hydrolysis—ActiDrops established a versatile platform for generating active modules with tunable lifetimes, successfully applied to oil droplets, complex coacervates, and vesicles.
Among its key contributions is the first-ever synthesis of active oil droplets and active complex coacervate droplets, whose contrasting water content leads to distinct behaviors. Oil droplets show accelerated ripening and dynamic instabilities that challenge classical thermodynamic models, while coacervates exhibit life-like properties such as fusion and RNA compartmentalization, driven by fuel-dependent assembly and disassembly.
A particularly striking discovery was the emergence of spatio-temporal oscillations in coacervate droplets, highlighting the essential role of chemical fuel in sustaining non-equilibrium dynamics. These insights were further expanded using a microfluidic setup, enabling the detailed tracking of individual droplets in confined volumes (Bergmann et al., Angew Chem, 2022).
ActiDrops also advanced the field with the development of fuel-driven vesicles capable of self-division through membrane budding, without requiring complex molecular machinery. These structures represent a significant step toward mimicking protocell behavior and advancing synthetic cell models.
By bridging chemical reactivity and dynamic self-assembly, ActiDrops has redefined the boundaries of synthetic biology, providing a robust platform for the bottom-up construction of life-like systems. Its influence is already evident, inspiring new theoretical models and experimental approaches across disciplines.
Among its key contributions is the first-ever synthesis of active oil droplets and active complex coacervate droplets, whose contrasting water content leads to distinct behaviors. Oil droplets show accelerated ripening and dynamic instabilities that challenge classical thermodynamic models, while coacervates exhibit life-like properties such as fusion and RNA compartmentalization, driven by fuel-dependent assembly and disassembly.
A particularly striking discovery was the emergence of spatio-temporal oscillations in coacervate droplets, highlighting the essential role of chemical fuel in sustaining non-equilibrium dynamics. These insights were further expanded using a microfluidic setup, enabling the detailed tracking of individual droplets in confined volumes (Bergmann et al., Angew Chem, 2022).
ActiDrops also advanced the field with the development of fuel-driven vesicles capable of self-division through membrane budding, without requiring complex molecular machinery. These structures represent a significant step toward mimicking protocell behavior and advancing synthetic cell models.
By bridging chemical reactivity and dynamic self-assembly, ActiDrops has redefined the boundaries of synthetic biology, providing a robust platform for the bottom-up construction of life-like systems. Its influence is already evident, inspiring new theoretical models and experimental approaches across disciplines.