Living material assembly of bacteriogenic protocells.

Advancing the spontaneous bottom-up construction of artificial cells with high organizational complexity and diverse functionality remains an unresolved issue at the interface between living and non-living matter1-4. Here, to address this challenge, we developed a living material assembly process based on the capture and on-site processing of spatially segregated bacterial colonies within individual coacervate microdroplets for the endogenous construction of membrane-bounded, molecularly crowded, and compositionally, structurally and morphologically complex synthetic cells. The bacteriogenic protocells inherit diverse biological components, exhibit multifunctional cytomimetic properties and can be endogenously remodelled to include a spatially partitioned DNA-histone nucleus-like condensate, membranized water vacuoles and a three-dimensional network of F-actin proto-cytoskeletal filaments. The ensemble is biochemically energized by ATP production derived from implanted live Escherichia coli cells to produce a cellular bionic system with amoeba-like external morphology and integrated life-like properties. Our results demonstrate a bacteriogenic strategy for the bottom-up construction of functional protoliving microdevices and provide opportunities for the fabrication of new synthetic cell modules and augmented living/synthetic cell constructs with potential applications in engineered synthetic biology and biotechnology.

Reprogramming macrophages with R848-loaded artificial protocells to modulate skin and skeletal wound healing.

After tissue injury, inflammatory cells are rapidly recruited to the wound where they clear microbes and other debris, and coordinate the behaviour of other cell lineages at the repair site in both positive and negative ways. In this study, we take advantage of the translucency and genetic tractability of zebrafish to evaluate the feasibility of reprogramming innate immune cells in vivo with cargo-loaded protocells and investigate how this alters the inflammatory response in the context of skin and skeletal repair. Using live imaging we show that protocells loaded with R848 cargo (which targets TLR7/8 signalling), are engulfed by macrophages resulting in their switching to a pro-inflammatory phenotype and altering their regulation of angiogenesis, collagen deposition and re-epithelialization during skin wound healing, as well as dampening osteoblast and osteoclast recruitment and bone mineralization during fracture repair. For infected skin wounds, R848-reprogrammed macrophages exhibited enhanced bactericidal activities leading to improved healing. We replicated our zebrafish studies in cultured human macrophages, and showed that R848-loaded protocells similarly reprogramme human cells, indicating how this strategy might be used to modulate wound inflammation in the clinic.

Membranized Coacervate Microdroplets: from Versatile Protocell Models to Cytomimetic Materials.

Although complex coacervate microdroplets derived from associative phase separation of counter-charged electrolytes have emerged as a broad platform for the bottom-up construction of membraneless, molecularly crowded protocells, the absence of an enclosing membrane limits the construction of more sophisticated artificial cells and their use as functional cytomimetic materials. To address this problem, we and others have recently developed chemical-based strategies for the membranization of preformed coacervate microdroplets. In this Account, we review our recent work on diverse coacervate systems using a range of membrane building blocks and assembly processes. First, we briefly introduce the unusual nature of the coacervate/water interface, emphasizing the ultralow interfacial tension and broad interfacial width as physiochemical properties that require special attention in the judicious design of membranized coacervate microdroplets. Second, we classify membrane assembly into two different approaches: (i) interfacial self-assembly by using diverse surface-active building blocks such as molecular amphiphiles (fatty acids, phospholipids, block copolymers, protein-polymer conjugates) or nano- and microscale objects (liposomes, nanoparticle surfactants, cell fragments, living cells) with appropriate wettability; and (ii) coacervate droplet-to-vesicle reconfiguration by employing auxiliary surface reconstruction agents or triggering endogenous transitions (self-membranization) under nonstoichiometric (charge mismatched) conditions. We then discuss the key cytomimetic behaviors of membranized coacervate-based model protocells. Customizable permeability is achieved by synergistic effects operating between the molecularly crowded coacervate interior and surrounding membrane. In contrast, metabolic-like endogenous reactivity, diffusive chemical signaling, and collective chemical operations occur specifically in protocell networks comprising diverse populations of membranized coacervate microdroplets. In each case, these cytomimetic behaviors can give rise to functional microscale materials capable of promising cell-like applications. For example, immobilizing spatially segregated enzyme-loaded phospholipid-coated coacervate protocells in concentrically tubular hydrogels delivers prototissue-like bulk materials that generate nitric oxide in vitro, enabling platelet deactivation and inhibition of blood clot formation. Alternatively, therapeutic protocells with in vivo vasoactivity, high hemocompatibility, and increased blood circulation times are constructed by spontaneous assembly of hemoglobin-containing cell-membrane fragments on the surface of enzyme-loaded coacervate microdroplets. Higher-order properties such as artificial endocytosis are achieved by using nanoparticle-caged coacervate protocell hosts that selectively and actively capture guest nano- and microscale objects by responses to exogenous stimuli or via endogenous enzyme-mediated reactions. Finally, we discuss the current limitations in the design and programming of membranized coacervate microdroplets, which may help to guide future directions in this emerging research area. Taken together, we hope that this Account will inspire new advances in membranized coacervate microdroplets and promote their application in the development of integrated protocell models and functional cytomimetic materials.

Patterning of Protein-Sequestered Liquid-Crystal Droplets Using Acoustic Wave Trapping.

Development of spatially organized structures and understanding their role in controlling kinetics of multistep chemical reactions are essential for the successful design of efficient systems and devices. While studies that showcase different types of methodologies for the spatial organization of various colloidal systems are known, design and development of well-defined hierarchical assemblies of liquid-crystal (LC) droplets and subsequent demonstration of biological reactions using such assemblies still remain elusive. Here, we show reversible and reconfigurable one-dimensional (1D) assemblies of protein-bioconjugate-sequestered monodisperse LC droplets by combining microfluidics with noninvasive acoustic wave trapping technology. Tunable spatial geometries and lattice dimensions can be achieved in an aqueous medium comprising ≈19 or 62 µm LC droplets. Different assemblies of a mixed population of larger and smaller droplets sequestered with glucose oxidase (GOx) and horseradish peroxidase (HRP), respectively, exhibit spatially localized enzyme kinetics with higher initial rates of reaction compared with GOx/HRP cascades implemented in the absence of an acoustic field. This can be attributed to the direct substrate transfer/channeling between the two complementary enzymes in close proximity. Therefore, our study provides an initial step toward the fabrication of LC-based devices for biosensing applications.

Autonomic Integration in Nested Protocell Communities.

The self-driven organization of model protocells into higher-order nested cytomimetic systems with coordinated structural and functional relationships offers a step toward the autonomic implementation of artificial multicellularity. Here, we describe an endosymbiotic-like pathway in which proteinosomes are captured within membranized alginate/silk fibroin coacervate vesicles by guest-mediated reconfiguration of the host protocells. We demonstrate that interchange of coacervate vesicle and droplet morphologies through proteinosome-mediated urease/glucose oxidase activity produces discrete nested communities capable of integrated catalytic activity and selective disintegration. The self-driving capacity is modulated by an internalized fuel-driven process using starch hydrolases sequestered within the host coacervate phase, and structural stabilization of the integrated protocell populations can be achieved by on-site enzyme-mediated matrix reinforcement involving dipeptide supramolecular assembly or tyramine-alginate covalent cross-linking. Our work highlights a semi-autonomous mechanism for constructing symbiotic cell-like nested communities and provides opportunities for the development of reconfigurable cytomimetic materials with structural, functional, and organizational complexity.

Osmotic-Induced Reconfiguration and Activation in Membranized Coacervate-Based Protocells.

The design and construction of synthetic protocells capable of stimuli response and homeostatic regulation is an important challenge for synthetic protobiology. Here, we develop a step toward the construction of model protocells capable of a hypotonic stress-induced volume response that facilitates an increase in membrane permeability and the triggering of endogenous enzyme reactions. We describe a facile self-transformation process for constructing single- or multichambered molecularly crowded protocells based on the osmotic reconfiguration of lipid-coated coacervate droplets into multicompartmentalized coacervate vesicles. Hypotonic swelling broadens membrane permeability and increases transmembrane transport such that protease-based hydrolysis and enzyme cascades can be triggered and enhanced within the protocells by osmotically induced expansion. Specifically, we demonstrate how the enhanced production of nitric oxide (NO) within the swollen coacervate vesicles can be used to induce in vitro blood vessel vasodilation in thoracic artery rings. Our approach provides opportunities for designing reconfigurable model protocells capable of homeostatic volume regulation, dynamic structural reorganization, and adaptive functionality in response to changes in environment osmolarity, and could find applications in biomedicine, cellular diagnostics, and bioengineering.

Acoustic Trapping: An Emerging Tool for Microfabrication Technology.

The high throughput deposition of microscale objects with precise spatial arrangement represents a key step in microfabrication technology. This can be done by creating physical boundaries to guide the deposition process or using printing technologies; in both approaches, these microscale objects cannot be further modified after they are formed. The utilization of dynamic acoustic fields offers a novel approach to facilitate real-time reconfigurable miniaturized systems in a contactless manner, which can potentially be used in physics, chemistry, biology, as well as materials science. Here, the physical interactions of microscale objects in an acoustic pressure field are discussed and how to fabricate different acoustic trapping devices and how to tune the spatial arrangement of the microscale objects are explained. Moreover, different approaches that can dynamically modulate microscale objects in acoustic fields are presented, and the potential applications of the microarrays in biomedical engineering, chemical/biochemical sensing, and materials science are highlighted alongside a discussion of future research challenges.

Orthogonal Light-Dependent Membrane Adhesion Induces Social Self-Sorting and Member-Specific DNA Communication in Synthetic Cell Communities.

Developing orthogonal chemical communication pathways in diverse synthetic cell communities is a considerable challenge due to the increased crosstalk and interference associated with large numbers of different types of sender-receiver pairs. Herein, the authors control which sender-receiver pairs communicate in a three-membered community of synthetic cells through red and blue light illumination. Semipermeable protein-polymer-based synthetic cells (proteinosomes) with complementary membrane-attached protein adhesion communicate through single-stranded DNA oligomers and synergistically process biochemical information within a community consisting of one sender and two different receiver populations. Different pairs of red and blue light-responsive protein-protein interactions act as membrane adhesion mediators between the sender and receivers such that they self-assemble and socially self-sort into different multicellular structures under red and blue light. Consequently, distinct sender-receiver pairs come into the signaling range depending on the light illumination and are able to communicate specifically without activation of the other receiver population. Overall, this work shows how photoswitchable membrane adhesion gives rise to different self-sorting protocell patterns that mediate member-specific DNA-based communication in ternary populations of synthetic cells and provides a step towards the design of orthogonal chemical communication networks in diverse communities of synthetic cells.

A Photo-degradable Crosslinker for the Development of Light-responsive Protocell Membranes.

The achievement of light-responsive behaviours is an important target for protocell engineering to allow control of fundamental protocellular processes such as communication via diffusible chemical signals, shape changes or even motility at the flick of a switch. As a step towards this ambitious goal, here we describe the synthesis of a novel poly(ethylene glycol)-based crosslinker, reactive towards nucleophiles, that effectively degrades with UV light (405 nm). We demonstrate its utility for the fabrication of the first protocell membranes capable of light-induced disassembly, for the photo-generation of patterns of protocells, and for the modulation of protocell membrane permeability. Overall, our results not only open up new avenues towards the engineering of spatially organised, communicating networks of protocells, and of micro-compartmentalised systems for information storage and release, but also have important implications for other research fields such as drug delivery and soft materials chemistry.

Chemical Communication and Protocell-Matrix Dynamics in Segregated Colloidosome Micro-Colonies.

Despite an emerging catalogue of collective behaviours in communities of homogeneously distributed cell-like objects, microscale protocell colonies with spatially segregated populations have received minimal attention. Here, we use microfluidics to fabricate Janus-like calcium alginate hydrogel microspheres with spatially partitioned populations of enzyme-containing inorganic colloidosomes and investigate their potential as integrated platforms for domain-mediated chemical communication and programmable protocell-matrix dynamics. Diffusive chemical signalling within the segregated communities gives rise to increased initial enzyme kinetics compared with a homogeneous distribution of protocells. We employ competing enzyme-mediated hydrogel crosslinking and decrosslinking reactions in different domains of the partitioned colonies to undertake selective expulsion of a specific protocell population from the community. Our results offer new possibilities for the design and construction of spatially organized cytomimetic consortia capable of endogenous chemical processing and protocell-environment interactivity.

Modular Metal-Quinone Networks with Tunable Architecture and Functionality.

Nanostructured materials with tunable structures and functionality are of interest in diverse areas. Herein, metal ions are coordinated with quinones through metal-acetylacetone coordination bonds to generate a class of structurally tunable, universally adhesive, hydrophilic, and pH-degradable materials. A library of metal-quinone networks (MQNs) is produced from five model quinone ligands paired with nine metal ions, leading to the assembly of particles, tubes, capsules, and films. Importantly, MQNs show bidirectional pH-responsive disassembly in acidic and alkaline solutions, where the quinone ligands mediate the disassembly kinetics, enabling temporal and spatial control over the release of multiple components using multilayered MQNs. Leveraging this tunable release and the inherent medicinal properties of quinones, MQN prodrugs with a high drug loading (>89 wt %) are engineered using doxorubicin for anti-cancer therapy and shikonin for the inhibition of the main protease in the SARS-CoV-2 virus.

Triggerable Protocell Capture in Nanoparticle-Caged Coacervate Microdroplets.

Controlling the dynamics of mixed communities of cell-like entities (protocells) provides a step toward the development of higher-order cytomimetic behaviors in artificial cell consortia. In this paper, we develop a caged protocell model with a molecularly crowded coacervate interior surrounded by a non-cross-linked gold (Au)/poly(ethylene glycol) (PEG) nanoparticle-jammed stimuli-responsive membrane. The jammed membrane is unlocked by either exogenous light-mediated Au/PEG dissociation at the Au surface or endogenous enzyme-mediated cleavage of a ketal linkage on the PEG backbone. The membrane assembly/disassembly process is used for the controlled and selective uptake of guest protocells into the caged coacervate microdroplets as a path toward an all-water model of triggerable transmembrane uptake in synthetic protocell communities. Active capture of the guest protocells stems from the high sequestration potential of the coacervate interior such that tailoring the surface properties of the guest protocells provides a rudimentary system of protocell sorting. Our results highlight the potential for programming surface-contact interactions between artificial membrane-bounded compartments and could have implications for the development of protocell networks, storage and delivery microsystems, and microreactor technologies.

The Surgical Skills and Technology Elective Program and Medical Student Career Choice.

INTRODUCTION: The Surgical Skills and Technology Elective Program (SSTEP) is a bootcamp for preclinical medical students that uses simulation learning methodology to provide exposure to procedural specialties and enhance surgical skills. Despite the widespread adoption of similar bootcamps, evidence is lacking regarding their impact on students' decision to pursue surgical/procedural careers. METHODS: This exploratory analysis consisted of a retrospective, cross-sectional assessment of SSTEP involving a survey examining engagement during clerkship rotations and career decision-making was sent to all SSTEP participants since the program's inception (n = 184). In addition, publicly available data through the Canadian Resident Matching Service (CaRMS) were used to compare match data between all SSTEP participants who have participated in the CaRMS match (n = 144) and students in corresponding years who did not participate in SSTEP (n = 351). RESULTS: Seventy-four SSTEP participants (40.2%) responded to the survey. Of the respondents, the majority agreed or strongly agreed that SSTEP influenced participants to engage in more procedural opportunities during clerkship (73%) and that they felt more confident performing procedural tasks during clerkship because of SSTEP (92%). Fifty percent of participants agreed that their anxiety decreased regarding clinical specialty decision. Thirty percent of participants indicated that SSTEP influenced them to pursue a procedural career. Examination of CaRMS data showed that 42% of SSTEP participants matched into direct-entry procedural specialties compared with 32% of non-SSTEP graduating medical students at our institution (P = 0.048). CONCLUSIONS: Our analysis supports the utility of preclerkship surgical bootcamps. By providing early exposure to procedural skills, SSTEP promoted engagement with procedural skills during clerkship. Participation in SSTEP influenced student career choice, which may have contributed to the increased match rate into procedural specialties for SSTEP participants.

Spontaneous Membranization in a Silk-Based Coacervate Protocell Model.

Molecularly crowded coacervate micro-droplets are useful protocell constructs but the absence of a physical membrane limits their application as cytomimetic models. Auxiliary surface-active agents have been harnessed to stabilize the coacervate droplets by irreversible shell formation but endogenous processes of reversible membranization have received minimal attention. Herein, we describe a dynamic alginate/silk coacervate-based protocell model in which membrane-less droplets are reversibly reconfigured and inflated into semipermeable coacervate vesicles by spontaneous self-organization of amphiphilic silk polymers at the droplet surface under non-neutral charge conditions in the absence of auxiliary agents. We show that membranization can be reversibly controlled endogenously by programming the pH within the protocells using an antagonistic enzyme system such that structural reconfigurations in the protocell microstructure are coupled to the trafficking of water-soluble solutes. Our results open new perspectives in the design of hybrid protocell models with dynamical structural properties.

Protocellular CRISPR/Cas-Based Diffusive Communication Using Transcriptional RNA Signaling.

Protocells containing enzyme-driven biomolecular circuits that can process and exchange information offer a promising approach for mimicking cellular features and developing molecular information platforms. Here, we employ synthetic transcriptional circuits together with CRISPR/Cas-based DNA processing inside semipermeable protein-polymer microcompartments. We first establish a transcriptional protocell that can be activated by external DNA strands and produce functional RNA aptamers. Subsequently, we engineer a transcriptional module to generate RNA strands functioning as diffusive signals that can be sensed by neighboring protocells and trigger the activation of internalized DNA probes or localization of Cas nucleases. Our results highlight the opportunities to combine CRISPR/Cas machinery and DNA nanotechnology for protocellular communication and provide a step towards the development of protocells capable of distributed molecular information processing.

G-Quadruplex-Induced Liquid-Liquid Phase Separation in Biomimetic Protocells.

Biomolecular condensates comprised of specific proteins and nucleic acids are now recognized as one of the key organizing mechanisms in eukaryotic cells. However, the specific roles played by the nucleic acid secondary structure and sequence in biomolecular phase separation are still not clear. Here, utilizing giant membrane vesicles (GMVs) as a protocell model, we found that single-stranded DNA (ssDNA) with a parallel G-quadruplex structure could functionally cooperate with a G-quadruplex-binding protein to form speckle-like puncta inside the GMVs. The clustering behavior is dependent on the structural diversity of G-quadruplexes, and the reversible clustering behavior implicated a new pathway in dynamically regulating the formation of biomolecular condensates. This finding represents a potential link between G-quadruplex-binding proteins and the resulting G-quadruplex-mediated biomolecular phase separation, which would gain insight into a wide range of biological processes associated with nucleic acid-modulated phase separation inside living cells.

Giant Coacervate Vesicles As an Integrated Approach to Cytomimetic Modeling.

Although giant unilamellar vesicles (GUVs) have been extensively studied as synthetic cell-like microcompartments, their applicability as cytomimetic models is severely compromised by low levels of membrane permeability, low encapsulation efficiencies, and high physicochemical instability. Here, we develop an integrated cytomimetic model comprising a macromolecularly crowded interior with high sequestration efficiency and enclosed within a phospholipid membrane that is permeable to molecules below a molecular weight cutoff of ca. 4 kDa. The protocells are readily prepared by spontaneous assembly of a phospholipid membrane on the surface of preformed polynucleotide/polysaccharide coacervate microdroplets and are designated as giant coacervate vesicles (GCVs). Partial anchoring of the GCV membrane to the underlying coacervate phase results in increased robustness, lower membrane fluidity, and increased permeability compared with GUV counterparts. As a consequence, enzyme and ribozyme catalysis can be triggered in the molecularly crowded interior of the GCV but not inside the GUVs when small molecule substrates or inducers are present in the external environment. By integrating processes of membrane-mediated compartmentalization and liquid-liquid microphase separation, GCVs could offer substantial advantages as cytomimetic models, synthetic protocells, and artificial biomolecular microreactors.

Polymer-Surfactant Driven Interactions and the Resultant Microstructure in Protein-Containing Liquid Crystal Droplets.

Integration of molecular liquid crystals (LCs) with functional proteins can provide new class of materials for potential applications in optical biosensing. However, hydrophobic nematic LCs (length ∼ 1-2 nm) and hydrophilic proteins, size ∼ O (nm), do not intermix without chemical modification of at least one of them. Bioconjugation of proteins with a polyethylene glycol-based polymeric surfactant (PS) can provide a core-shell system that is sequestered within nonaqueous LC (4-cyano-4'-pentylbiphenyl) microdroplets. However, the nature of interactions between the components and detailed understanding of the resultant hybrid microstructure remains unclear. Here, using a combination of isothermal titration calorimetry (ITC), fluorescence microscopy, and infrared-imaging spectroscopy, we show that strong hydrophobic interactions between the LC and PS drives the sequestration of a myoglobin-PS (Mb-PS; dispersed in the aqueous phase) into the LC spherical microdroplets or even into a bulk LC phase. The average values of both, the binding constant and the standard molar enthalpy change, are increased by approximately a factor of 2.5 times when the unmodified Mb is conjugated to the PS. Small-angle X-ray scattering studies reveal that LC molecules act as a solvent for the Mb-PS conjugate; furthermore, the LC long-range order is disturbed due to mixing, as exemplified by the change in its coherence length from 8.9 to 5.7 nm. Detailed all-atomistic molecular dynamic simulations for a three-component PS-water-LC system show a change in interaction energy of -144 kJ mol-1 PS-1 upon the contact of PS chains (initially dispersed in water) with LC and agree with the ITC experiments.

The Next Surgical Skills and Technology Elective Program: The "Surgical Skills and Technology Elective Program" Decreases Cognitive Load During Suturing Tasks in Second Year Medical Students.

OBJECTIVE: The Surgical Skills and Technology Elective Program (SSTEP) is a one-week, simulation-based procedural skills bootcamp for preclinical medical students. Using cognitive load (CL) as a useful framework for understanding simulation in medical education, our aims were to (1) examine the ability of SSTEP to decrease medical students' CL during procedural skills training and (2) determine the impact of SSTEP on secondary learning. METHODS: In this prospective cohort study, twenty SSTEP participants and twenty controls were recruited. CL was assessed during a simple suturing task and a clinical vignette multitasking activity, where participants were required to suture and concurrently listen to a clinical vignette. CL was measured using the validated Subjective Rating of Mental Effort (SRME) and its impact on working memory was assessed using a knowledge test about the clinical vignette. RESULTS: Participants reported lower SRME scores while suturing following SSTEP, which persisted at 3 months (p = 0.002) and were significantly lower than controls (p = 0.031). Participants also reported lower SRME scores during the clinical vignette multitasking activity (p = 0.011), despite no improvement among controls (p = 0.63). Participants significantly outperformed controls on the clinical vignette knowledge test (p = 0.02). CONCLUSIONS: Surgical skills training through SSTEP was associated with lower reports of mental effort and increased performance on secondary learning tasks. Procedural skills bootcamps may better prepare students for the complex learning environments encountered during clinical clerkship.

A Novel Acid-Degradable PEG Crosslinker for the Fabrication of pH-Responsive Soft Materials.

The design and synthesis of a novel acid-degradable polyethylene glycol-based N-hydroxysuccinimide (NHS) ester-activated crosslinker is reported. The crosslinker is reactive towards nucleophiles and features a central ketal functional group that is stable at pH > 7.5 and rapidly hydrolyses at pH > 6.0. The crosslinker is used to (i) fabricate acid-degradable polysaccharide hydrogels that exhibit controlled degradation upon exposure to an acidic environment or via endogenous enzyme activity; and (ii) construct hydrogel-filled protein-polymer microcompartments (termed proteinosomes) capable of pH-dependent membrane disassembly. Taken together the results provide new opportunities for the fabrication of pH-responsive soft materials with potential applications in drug delivery, tissue engineering, and soft-matter bioengineering.