The organizational complexity of biominerals has long fascinated scientists seeking to understand biological programming and implement new developments in biomimetic materials chemistry. Nonclassical crystallization pathways have been observed and analyzed in typical crystalline biominerals, involving the controlled attachment and reconfiguration of nanoparticles and clusters on organic templates. However, the understanding of templated amorphous silica mineralization remains limited, hindering the rational design of complex silica-based materials. Here, we present a systematic study on the stabilization of self-capping cationic silica cluster (CSC) and their assembly dynamics using DNA nanostructures as programmable attachment templates. By tuning the composition and structure of CSC, we demonstrate high-fidelity silicification at single-cluster resolution, revealing a process of adaptive templating involving cooperative adjustments of both the DNA framework and cluster morphology. Our results provide a unified model of silicification by cluster attachment and pave the way towards the molecular tuning of pre- and post-nucleation stages of sol-gel reactions. Overall, our findings provide new insights for the design of silica-based materials with controlled organization and functionality, bridging the gap between biomineralization principles and the rational design of biomimetic material.

Liquid crystalline droplets with molecularly crowded interiors capable of selective biomolecular sequestration and interfacial wetting have been recently developed for the construction of artificial cells (protocells) and chain-like protocell networks1-3. The controlled division of these synthetic protocells4-16, however, remains a challenge. Symmetric fission of vesicles and droplets has been shown17-26 using thermal gradients, dissipative self-assembly, wetting energies and chemical reactions27-31, but asymmetric division is rare. For example, osmotic pressure has been used to induce the asymmetric division of lipid vesicles containing a polyethylene glycol-dextran aqueous two-phase system32 as well as giant unilamellar vesicles prepared with lipid-phase-separated bilayers33. Here we show that structured liquid droplets exhibit asymmetric division in the absence of reconstituted protein machinery. In the presence of alkaline phosphatase or multivalent metal cations, individual multilamellar droplets split to produce two morphologically distinct progeny (droplet and vesicle). We show that heteromorphic division occurs by circumferential growth of a single surface caveola along a latent core-shell domain boundary because of induced changes in lipid headgroup-nucleotide counterion interactions and demonstrate that functional biomolecules are transferred between the different protocell generations. Taken together, our results provide a step towards the bottom-up assembly of proliferating artificial cells.

ABSTRACT In eukaryotic cells, membraneless organelles reorganize through regulated interactions with the plasma membrane and its underlying cortex, where cytoskeletal coupling and inner‐leaflet biochemistry tune condensate positioning, wetting, and function. Recreating such adaptive, cortex‐mediated control in synthetic systems remains a challenge, requiring a chassis that combines interfacial programmability with the mechanical resilience necessary to withstand the osmotic and electrostatic stresses of bottom‐up assembly. Here, we introduce polysaccharidosomes (P‐somes); semipermeable, mechanically robust protocells that function as membrane‐programmable chassis for directing coacervate–membrane coupling. By establishing a thin, cortex‐like protein layer on the inner membrane leaflet via template‐directed assembly, we demonstrate that in situ protein succinylation enables precise tuning of surface charge and coacervate–membrane wetting. Together with the systematic variation of membrane building blocks, this platform allows for fine control over coacervate wetting, morphology, and spatial organization. The uptake of external DNA adds a second tier of regulation: on nonpassivated membranes, DNA‐reconfigured coacervates generate interfacial protrusions that bridge neighboring P‐somes to promote tissue‐like clustering, whereas on passivated membranes, they coalesce into a single, nonwetting, nucleus‐like droplet centered within the lumen. This membrane–cortex‐inspired framework integrates mechanical resilience with chemical programmability, providing a scalable route to constructing membranized protocells with self‐organizing interiors and emergent collective behaviors.

Cytotoxic T lymphocytes play a crucial role in anti-tumour immunity, with granzyme B (GrB) being a decisive factor in this process. Developing a GrB-based delivery system to mimic T cell-based immunotherapy holds promise but remains challenging. Here, we present an artificial metalloenzyme (nanozyme) with GrB-like protease activity capable of inducing caspase-dependent cell apoptosis. The nanozyme is based on the site-specific binding of Pd(II) ions to recombinant human heavy chain apo-ferritin nanocages to generate a binuclear catalytic centre consisting of two Pd atoms with bridging cysteine ligands, monodentate methionine and histidine residues, and two water molecules. We show that encapsulation of the Pd-ferritin complex within membrane-fused lipid nanoparticles comprising surface-displayed single-chain antibodies affords GrB-mimicking nanozyme-based nanovesicles capable of receptor-mediated delivery of the nanozyme into the cytoplasm of tumour cells and induction of caspase-dependent apoptosis. This study provides valuable insights into the construction of nano-delivery systems with artificial GrB activity and presents a promising therapeutic option for solid tumours. Granzyme B is an important factor in cytotoxic T lymphocytes anti-tumour immunity. Here, the authors report on a Pd-FTn granzyme B-mimicking nanozyme with a binuclear catalytic centre, delivered by functionalised nanovesicles to selectively trigger caspase-dependent apoptosis for a T cell-inspired cancer therapy.

The construction of protocell networks with self-regulated spatial dynamics and functions is an important challenge in the emerging field of colloidal systems chemistry. Existing strategies predominantly produce protocell networks with fixed or randomly distributed spatial organization, relying on direct surface interactions or externally imposed conditions, while largely overlooking dynamic interactions with the surrounding environment, thereby limiting the emergence of reconfigurable network behaviours. Here we demonstrate chemical strategies for implementing the spontaneous segregation and selective translocation of binary/ternary populations of enzyme-containing proteinosomes in dextran droplet/polyethylene glycol aqueous phase-separated media. The segregated proteinosomes exhibit tunable membrane wettability, engage in cross-community chemical signalling and undergo signal-induced reversals in phase compatibility to produce reconfigurable networks capable of protocell-mediated recruitment and dispatchment. We exploit the protocell phase dynamics to spatiotemporally modulate DNase I activity in a subpopulation of translocating proteinosomes. Our methodology provides a platform for developing protocell communities with self-regulated spatiotemporal order and offers opportunities in cytomimetic modelling and colloidal systems chemistry.

Protein coacervates formed by liquid-liquid phase separation are emerging as active force generators, independent of ATP-driven motors. Nevertheless, the coordination and force scaling of protein coacervates remain largely unexplored. Here, we engineer a temperature-responsive elastin-based protocell model displaying temperature-modulated contractility and attendant force harnessing. By leveraging the phase separation properties, we modulate the protocell dynamics associated with volume contraction and membrane budding. Crosslinking of the elastin-based membrane influences the contraction dynamics such that the accumulation of mechanical forces in the protocells results in the spontaneous expulsion of internally trapped protein liquid-liquid phase separation (LLPS) complexes. We use a simple mathematically model to show how protein coacervation can amplify small piconewton-scale forces to perform large-scale mechanical work, highlighting the mechanical potential of protein coacervation dynamics. Taken together, our results provide a model framework for harnessing protein coacervates-driven forces and offer a step to future applications in synthetic biology, biomaterials and next-generation soft robotics.

Dynamic sub-compartmentalization and internal organization are important assets of living cells to control functional complexity. Mimicking these features in artificial cells provides a platform to effectively respond to external cues by changing internal structure, thereby emulating life-like behavior. Here, we present a strategy to construct sub-compartmentalized artificial cells by converting multiphase coacervate droplets (MCDs) into nested coacervate vesicles (NCVs), in which the outer host domain is electrostatically reconfigured into a continuous semipermeable shell, while the internal guest droplets are preserved. The generated artificial cells exhibit spatial segregation of coacervate constituents and encapsulated fluorescent dyes, enzymes, and gold nanoparticles, and remain morphologically stable under different conditions. The membranized artificial cells display artificial metabolic features by means of poly(N-isopropylacrylamide) (PNIPAAm) synthesis and subsequent temperature-dependent aggregation, leading to emergent behavior including self-regulated photothermal transitions, feedback-mediated photocatalysis, and spatiotemporal organization of internal cargoes. Overall, our approach establishes a robust artificial cell platform that combines sub-compartmentalization with self-regulating properties, integrating functionality with structural complexity.

Liquid-liquid phase separation plays an important role in many natural and technological processes. Herein, we implement lateral microphase separation at the surface of oil micro-droplets suspended in water to prepare a range of discrete floating protein/polymer continuous two-dimensional (2D) heterostructures with variable interfacial domain structures and dynamics. We show that gel-like domains of bovine serum albumin (BSA) co-exist with fluid-like polyvinyl alcohol (PVA) regions at the oil droplet surface to produce floating heterostructures comprising a 2D phase-separated protein mesh or an array of discrete mobile protein rafts depending on the conditions employed. Enzymes are embedded in the discontinuous BSA domains to produce droplet-supported microphase-separated 2D reaction scaffolds that can be tuned for interfacial catalysis. Taken together, our work has general implications for the structural and functional augmentation of oil droplet interfaces and contributes to the surface engineering and functionality of droplet-based micro-reactors.

The mechanisms underlying the probing and response of cells to direct cell-presented mechanical signals generated in the local microenvironment are important controllers of diverse cell behaviours. Here we construct a model artificial pathogen cell with the similar compartmentalization architecture and same range of tunable rigidity as found in natural cells. By incubating the artificial cells with macrophages, we investigate the mechanisms of mechano-crosstalk between living cells and model protocells. We show that macrophages are equipped with distinct pseudopodia that facilitate the probing of cell-presented mechanical signals. Increasing the rigidity of the artificial pathogen cells enhances the proinflammatory polarization of the macrophages by promoting the docking of the mechanosensitive molecular clutch, actin assembly, and pseudopodia extension. The relationship between cell morphology and functional plasticity involves a mechano-transduction axis including artificial cell rigidity, pseudopodia, and macrophage inflammatory response. Taken together, our model protocells provide a new platform to decouple cell-presented mechanical signals and highlight their role in governing protocell-living cell mechano-crosstalk.

Artificial cells (ACs) offer a powerful platform to reprogram metabolic signaling in complex tissue environments by replicating key biological functions without the full complexity of living cells. However, achieving autonomous metabolite exchange and stable integration with living tissues remains a major challenge. Here, we report the development of proteinosome-based ACs equipped with a minimal metabolism to mediate bidirectional communication with glycolytic tumor cells. These tumors accumulate lactate, a metabolic byproduct that promotes immunosuppression and metastasis. Although lactate oxidase (LOx) can degrade lactate, its oxidation product, pyruvate, may inadvertently fuel tumor growth. To overcome this limitation, we engineered dual-processor ACs coencapsulating LOx and pyruvate decarboxylase (PDC), enabling selective conversion of lactate into cytotoxic acetaldehyde while suppressing pyruvate and hydrogen peroxide accumulation. These ACs demonstrate sustained catalytic activity, maintain reactive oxygen species homeostasis, and remain functional when integrated in 3D tumor spheroids. Crucially, they engage in autonomous, bidirectional metabolite exchange, preferentially with cancer cells over normal cells, dynamically rewiring important metabolites of the tumor microenvironment and suppressing cell viability. This work establishes synthetic metabolic biointerfaces as programmable actuators capable of reshaping pathological signaling in cancer tissues.

Liquid-liquid phase separation (LLPS) has been achieved in various cytomimetic (protocell) models, but controlling molecular condensation using noninert crowders to systematically alter protocell function remains challenging. Intracellular ATP levels influence protein-protein interactions, and dysregulation of ATP can alter cellular crowding dynamics, thereby disrupting the normal formation or dissolution of condensates. Here, we develop a membranized protocell model capable of endogenous LLPS and liquid-gel-like phase separation through precise manipulation of intermolecular interactions within semipermeable polysaccharide-based microcapsules (polysaccharidosomes, P-somes), prepared using microtemplate-guided assembly. We demonstrate that intraprotocellular diffusion-mediated LLPS can be extended into the liquid-gel-like domain by the uptake of the biologically active crowder ATP, resulting in a range of modalities dependent on the fine-tuning of molecular condensation. Endogenous enzyme activity in these crowded polysaccharidosomes is enhanced compared to free enzymes in solution, though this enhancement diminishes at higher levels of intraprotocellular condensation. Additionally, increased molecular crowding inhibits intraprotocell DNA strand displacement reactions. Our findings introduce an expedient and optimized approach to the batch construction of membranized protocell models with controllable molecular crowding and functional diversity. Our mix-incubate-wash protocol for inducing endogenous LLPS in membranized protocells offers potential applications in microreactor technology, environmental sensing, and the delivery and sustained release of therapeutics.

Coupling molecular-level chemical networks to macroscopic functions provides a step toward the implementation of rudimentary forms of agency in life-like objects and materials. Here, a negative feedback circuit within a networked binary protocell community is linked to higher-level spatial dynamic behaviors by exposing the consortium to a unidirectional reaction-diffusion gradient of an activator. The activator initiates artificial lysis and release of protocell-encapsulated enzymes, which spatiotemporally restrict membrane disassembly to generate distinct protocell survival boundaries within homogeneously distributed or segregated arrangements of the binary populations. We track the collective defense responses under nonequilibrium conditions and show that spatial symmetry breaking is related to the local generation of a hydrogen peroxide gradient and is strongly influenced by confinement and edge effects that spatially modulate the protocell survival networks formed within the hydrogels. In each case, the results are validated by computer simulations based on reaction-diffusion modeling. Overall, our work presents a general approach to implementing and decoding the spatial dynamic behaviors and collective responses of chemically networked protocell communities operating under nonequilibrium conditions and provides a pathway to self-protective cytomimetic systems that exhibit quasi-intelligent spatiotemporal behaviors in response to gradient-mediated activation.

Protocell research offers diverse opportunities to understand cellular processes and the foundations of life and holds attractive potential applications across various fields. However, it is still a formidable task to construct a true-to-life synthetic cell with high organizational and functional complexity. Here we present a protocol for constructing bacteriogenic protocells by employing prokaryotes as on-site repositories of compositional, functional and structural building blocks to address this challenge. This approach is based on the capture and processing of two spatially segregated bacterial colonies within individual coacervate microdroplets to produce membrane-bounded, molecularly crowded, compositionally, structurally and functionally complex synthetic cells. The bacteriogenic protocells inherit sufficient biological components from their bacterial building units to exhibit highly integrated life-like properties, including biocatalysis, glycolysis and gene expression. The protocells can be endogenously remodeled to acquire diverse proto-organelles including a spatially partitioned nucleus-like DNA/histone-based condensate to store genetic material, membrane-bounded water vacuoles to adjust cellular osmotic pressure, a three-dimensional network of F-actin proto-cytoskeleton to support structural stability and proto-mitochondria to generate endogenous ATP as source of energy. The protocells ultimately develop a nonspherical morphology due to the continuous biogeneration of metabolic products by implanted living bacteria cells. This protocol provides a novel living material assembly strategy for the construction of functional protoliving microdevices and offers opportunities for potential applications in engineered synthetic biology and biomedicine. The protocol takes ~27 d to complete and requires expertise in microbiology, phase separation, biochemistry and molecular biology related techniques. This protocol describes how to prepare membrane-bound, complex protocells with life-like capabilities by using building blocks from two distinct bacterial colonies spatially segregated within individual coacervate microdroplets.

The fabrication of cytomimetic materials capable of orchestrated and adaptive functions remains a significant challenge in bottom-up synthetic biology. Inspired by the cell/matrix integration of living bone, here we covalently tether distributed single populations of alkaline phosphatase-containing inorganic protocells (colloidosomes) onto a crosslinked organic network to establish viscoelastic tissue-like micro-composites. The prototissues are endogenously calcified with site-specific mineralization modalities involving selective intra-protocellular calcification, matrix-specific extra-protocellular calcification or gradient calcification. To mirror the interplay between osteoblasts and osteoclasts, we prepare integrated prototissues comprising a binary population of enzymatically active colloidosomes capable of endogenous calcification and decalcification and utilize chemical inputs to induce structural remodelling. Overall, our methodology opens a route to the chemically self-regulated calcification of homogeneous and gradient tissue-like mineral-matrix composites, advances the development of bottom-up synthetic biology in chemical materials research, and could provide potential opportunities in bioinspired tissue engineering, hydrogel technologies and bone biomimetics.

The design and implementation of collective actions in model protocell communities is an on-going challenge in synthetic protobiology. Herein, we covalently graft alginate or chitosan onto the outer surface of semipermeable enzyme-containing silica colloidosomes to produce hairy catalytic protocells with pH-switchable membrane surface charge. Binary populations of the enzymatically active protocells exhibit self-initiated stimulus-responsive changes in spatial organization such that the mixed community undergoes alternative modes of electrostatically induced self-sorting and reversible co-clustering. We demonstrate that co-clustering, but not self-sorting, mitigates signal attenuation in a binary community of enzyme-containing sender and receiver protocells due to increased proximity effects. The level of signal attenuation is correlated with a time-dependent pH-mediated switch in the spatial organization of the sender and receiver populations. Our results pave the way towards the development of programmable networks of adaptive life-like objects and could have implications for the development of interactive cytomimetic materials and agent-based robotics.

The construction of biomimetic agents capable of generating precise outcomes in response to specific molecular inputs is a central challenge for the development of programmable synthetic cells with integrated biomimetic functions. Here, we harness acoustic standing waves to generate periodic microarrays of enzyme-encoded coacervate microdroplets for the implementation of embodied enzyme logic circuits (EELCs). We describe a range of biocatalytic communication channels capable of performing localized and distributed Boolean logic functions in single or segregated populations of model protocells by using a range of molecular inputs, fluorescence or hydrogelation outputs, and programmable response dynamics. To implement long-range collective signal processing, we integrate EELC modules across spatially segregated protocell populations to generate distributed time-regulated logic operations involving negative feedback, pulse generation, and redirected output-input connectivity. Our results provide a step toward the non-DNA programming of model protocell communication and computational networks for miniaturized autonomous sensing devices capable of chemical-based information processing.

The bottom-up construction of leaf-like materials capable of using visible light and water to produce oxygen and chemical energy is a major goal of artificial photosynthesis. In this work, we show that robust artificial cells (protocells) prepared by the membranization of polymer/nucleotide coacervate droplets with a bio-inspired Ru-based polyoxometalate (POM) catalytic oxygen-evolving center are capable of continuous light-assisted water oxidation at room temperature. We present a method to electrostatically assemble millions of the photocatalytic protocells into tissue-like protocellular sheets and spheroids and demonstrate that the protocellular assemblies exhibit enhanced rates of photocatalytic water oxidation compared with individual protocells. Our results highlight opportunities for synergistically integrating key aspects of photocatalysis, cytomimetics, and bottom-up engineering toward the development of modular photosynthetic active matter and applications in artificial photosynthesis and energy capture.

Self-assembly, a fundamental property of living matter, drives the interconnected cellular organization of tissues. Synthetic cell models have been developed as bionic materials to mimic inherent cellular features such as self-assembly. Here, we leverage co-assembly of synthetic and natural cells to create hybrid living 3D cancer cultures. We screened synthetic cell models, including giant unilamellar vesicles, coacervates, microdroplet emulsions, proteinosomes, and colloidosomes, for their ability to form hybrid tumoroids. Our results identify the balance of inter- and extracellular adhesion and synthetic cell surface tension as key material properties driving successful co-assembly of hybrids. We further demonstrate that these synthetic cells can establish artificial tumor immune microenvironments (ART-TIMEs), mimicking immunogenic signals within tumoroids. Using the ART-TIME approach, we identify co-signaling mechanisms between PD-1 and CD2 as a driver in immune evasion of pancreatic ductal adenocarcinoma. Our findings demonstrate the 3D bottom-up self-assembly of hybrid cancer microenvironments to replace immune components with defined bionic materials, pushing the boundaries to functionally integrating living and non-living matter. ### Competing Interest Statement The authors have declared no competing interest.

The design and construction of continuous flow biochemical reactors comprising immobilized biocatalysts have generated great interest in the efficient synthesis of value-added chemicals. Living cells use compartmentalization and reaction-diffusion processes for spatiotemporal regulation of biocatalytic reactions, and implementing these strategies into continuous flow reactors can offer new opportunities in reactor design and application. Herein, the fabrication of protocell-based continuous flow reactors for enzyme and whole-cell mediated biocatalysis is demonstrated. Semipermeable membranized coacervate vesicles are employed as model protocells that spontaneously sequester enzymes or accumulate living bacteria to produce embodied microreactors capable of single- or multiple-step catalytic reactions. By packing millions of the enzyme/bacteria-containing coacervate vesicles in a glass column, a facile, cost-effective, and modular methodology capable of performing oxidoreductase, peroxidase and lipolytic reactions, enzyme-mediated L-DOPA synthesis, and whole-cell glycolysis under continuous flow conditions, is demonstrated. It is shown that the protocell-nested enzymes and bacterial cells exhibit enhanced activities and stability under deleterious operating conditions compared with their non-encapsulated counterparts. These results provide a step toward the engineering of continuous flow reactors based on cell-like microscale agents and offer opportunities in the development of green and sustainable industrial bioprocessing.

An experimental pathway to the spontaneous generation of compositionally diverse synthetic protocells is presented. The pathway is initiated by flat giant unilamellar vesicles (FGUVs) that originate from compositionally different multilamellar lipid reservoirs and undergo spontaneous spreading across solid surfaces. On contact, the spreading FGUVs merge to produce a concentration gradient in membrane lipids across the fusion interface. Subsequent reconstruction through a series of shape transformations produces a network of nanotube-connected lipid vesicles that inherit different ratios of the membrane constituents derived from the bilayers of the parent FGUVs. The fusion process leads to the engulfment of small FGUVs by larger FGUVs, mimicking predator-prey behavior in which the observable characteristics of the prey are lost but the constituents are carried by the predator FGUV to the next generation of lipid vesicles. We speculate that our results could provide a feasible pathway to autonomous protocell diversification in origin of life theories and highlight the possible role of solid surfaces in the development of diversity and rudimentary speciation of natural protocells on the early Earth. We present a feasible pathway leading to a rudimentary type of protocell diversity and speciation based on the fusion of flat giant unilamellar lipid vesicles on solid surfaces. image