Construction and Functioning of Coacervate Protocells

Protocells (or artificial cells) are synthetic structures that can mimic features of eukaryotic cells, such as structure (form, organization, compartmentalization) and function (metabolism, growth, reproduction).1 An interesting strategy to construct protocells with an intracellular cytosolic-like environment is the use of coacervates.2 Coacervates have a crowded and highly concentrated environment capable of sequestering, incorporating, and freely exchanging different molecular and colloidal species with the external environment.2 The present study aimed to incorporate artificial organelles (AOs) and enzymes into a coacervate scaffold to construct membraneless and multicompartmentalized protocells capable of performing a pH-dependent metabolism-like cascade reaction.3 Four key components were integrated to obtain the protocell: i) Signal transduction component - Urease, which hydrolyzes exogenous urea into ammonia, raising the internal pH and acting as a chemical signal; ii) AO1 - A substrate reservoir consisting of fluorescein diphosphate-loaded phosphorylated poly(glycerol sebacate) nanoparticles, capable of protecting the substrate at pH = 5.5, and releasing it at pH = 7.4; iii) AO2 - Polymersomes loaded with alkaline phosphatase, which present low enzyme activity at pH = 5.5, but become enzymatically active at physiological pH to hydrolyze fluorescein diphosphate into fluorescein and; iv) Cytosol-like scaffold - Coacervate composed by poly(diallyldimethylammonium chloride) and carboxymethyl dextran, responsible for the incorporation of the AOs and urease. The results show that higher loading of AOs and urease into the coacervates were obtained when the coacervation process occurs in the presence of these components at pH = 5.5, rather than adding the components after coacervation. The resulting protocell has a diameter of around 5 - 8 μm and presents stability over time under continuous stirring. The metabolism-like cascade reaction was triggered by adding urea. Urease converted it into ammonia, increasing the internal pH. This signal induced the release of the substrate from AO1, while simultaneously activating AO2 to catalyze the production of fluorescein. Fluorescence monitoring confirmed sustained substrate conversion, indicating effective communication between the organelles. In summary, these protocells demonstrate stable operation under dynamic conditions and mimic cellular metabolic processes through coordinated signal transduction and organelle communication. Therefore, this platform offers a versatile strategy for designing synthetic cells with life-like functional behaviors.