Engineering the Spatial Distribution of Amphiphilic Molecule within Complex Coacervate Microdroplet via Modulating Charge Strength of Polyelectrolytes.

The investigation of the interplay between complex coacervate microdroplets and amphiphilic molecules offers valuable insights into the processes of prebiotic compartmentalization on the early Earth and presents a promising avenue for future advancements in biotechnology. Herein, the interaction between complex coacervate microdroplets and amphiphilic molecule (decanoic acid) is systematically investigated by varying charge strengths of negatively charged polyelectrolytes (DNA and PAA) and positively charged polyelectrolytes (PDDA and DEAE-Dextran). It is found that the interaction between amphiphilic molecule and complex coacervate microdroplets depended on the delicate balance between the interaction between decanoic acid and polyelectrolyte and the interaction between two polyelectrolytes. The different spatial distribution of amphiphilic molecule can result in differences in the internal microenvironment, which can further alter the uptake or exclusion of small molecules and biomolecules with different charges and polarities and functional biological process.

Interface Binding Mechanism of Nanoclay Hybridized Coacervate Microdroplets for the Controllable Construction of Protocells.

Coacervate microdroplets, a protocell model in exploring the origin of life, have gained significant attention. Clay minerals, catalysts during the origin of life, are crucial in the chemical evolution of small molecules into biopolymers. However, our understanding of the relationship between clay minerals and the formation and evolution of protocells on early Earth remains limited. In this work, the nanoclay montmorillonite nanosheet (MMT-Na) was employed to investigate its interaction with coacervate microdroplets formed by oligolysine (K10) and adenine nucleoside triphosphate (ATP). As an anionic component, MMT-Na was noted to promote the formation of coacervate microdroplets. Furthermore, the efficiency of ssDNA enrichment and the degree of ssDNA hybridization within these microdroplets were significantly improved. By combining inorganic nanoclay with organic biopolymers, our work provides an efficient way to enrich genetic biomolecules in the primitive Earth environment and builds a nanoclay-based coacervate microdroplets, shedding new light on life's origin and protocell evolution.

Study on the detection method of biological characteristics of hepatoma cells based on terahertz time-domain spectroscopy.

Liver cancer usually has a high degree of malignancy and its early symptoms are hidden, therefore, it is of significant research value to develop early-stage detection methods of liver cancer for pathological screening. In this paper, a biometric detection method for living human hepatocytes based on terahertz time-domain spectroscopy was proposed. The difference in terahertz response between normal and cancer cells was analyzed, including five characteristic parameters in the response, namely refractive index, absorption coefficient, dielectric constant, dielectric loss and dielectric loss tangent. Based on class separability and variable correlation, absorption coefficient and dielectric loss were selected to better characterize cellular properties. Maximum information coefficient and principal component analysis were employed for feature extraction, and a cell classification model of support vector machine was constructed. The results showed that the algorithm based on parameter feature fusion can achieve an accuracy of 91.6% for human hepatoma cell lines and one normal cell line. This work provides a promising solution for the qualitative evaluation of living cells in liquid environment.

Regulation of species metabolism in synthetic community systems by environmental pH oscillations.

Constructing a synthetic community system helps scientist understand the complex interactions among species in a community and its environment. Herein, a two-species community is constructed with species A (artificial cells encapsulating pH-responsive molecules and sucrose) and species B (Saccharomyces cerevisiae), which causes the environment to exhibit pH oscillation behaviour due to the generation and dissipation of CO2. In addition, a three-species community is constructed with species A' (artificial cells containing sucrose and G6P), species B, and species C (artificial cells containing NAD+ and G6PDH). The solution pH oscillation regulates the periodical release of G6P from species A'; G6P then enters species C to promote the metabolic reaction that converts NAD+ to NADH. The location of species A' and B determines the metabolism behaviour in species C in the spatially coded three-species communities with CA'B, CBA', and A'CB patterns. The proposed synthetic community system provides a foundation to construct a more complicated microecosystem.

Reversible modulation of protocell volume via collective response of functional protein in its membrane.

Volume change plays an important role in biological cells to regulate their internal microenvironment. To adapt to the rapid variation of the surface area during the volume change, the lipid membrane is dynamically modulated via membrane folding invagination, or spontaneous uptake or release of lipid molecules under osmotic pressure. Here, we demonstrate an alternative approach to design a functional protocellular system capable of dynamically adjusting its volume and intracellular microenvironment in response to the alteration of pH. By assembling and subsequently cross-linking pH-responsive caseinate at the water-oil interface, the caseinate-based protocell with more than ten thousand caseinate units in its membrane was established and showed a reversible volume and pore size change to pH variation due to the collective response of the caseinate in the membrane, which could be used to control the spatial distribution of proto-organelle by regulating of the viscosity inside the protocell.

Hierarchical Structuration in Protocellular System.

Spatial control is one of the ubiquitous features in biological systems and the key to the functional complexity of living cells. The strategies to achieve such precise spatial control in protocellular systems are crucial to constructing complex artificial living systems with functional collective behavior. Herein, the authors review recent advances in the spatial control within a single protocell or between different protocells and discuss how such hierarchical structured protocellular system can be used to understand complex living systems or to advance the development of functional microreactors with the programmable release of various biomacromolecular payloads, or smart protocell-biological cell hybrid system.

Power-controlled acoustofluidic manipulation of microparticles.

Recently, surface acoustic wave (SAW) based acoustofluidic separation of microparticles and cells has attracted increasing interest due to accuracy and biocompatibility. Precise control of the input power of acoustofluidic devices is essential for generating optimum acoustic radiation force to manipulate microparticles given their various parameters including size, density, compressibility, and moving velocity. In this work, an acoustophoretic system is developed by employing SAW based interdigital electrode devices. Power meters are applied to closely monitor the incident and reflected powers of the SAW device, which are associated with the separation efficiency. There exists a range of input powers to migrate the microparticles to the pressure node due to their random locations when entering the SAW field. Theoretical analysis is performed to predict a proper input power to separate mixtures of polystyrene microspheres, and the end lateral position of microspheres being acoustically separated. The separation efficiency of four sizes of microspheres, including 20 µm, 15 µm, 10 µm, and 5 µm, is calculated and compared with experimental results, which suggest the input power for separating the mixture of these microspheres. The study provides a practical guidance on operating SAW devices for bioparticle separation using the incident power as a control parameter.

Open source board based acoustofluidic transwells for reversible disruption of the blood-brain barrier for therapeutic delivery.

BACKGROUND: Blood-brain barrier (BBB) is a crucial but dynamic structure that functions as a gatekeeper for the central nervous system (CNS). Managing sufficient substances across the BBB is a major challenge, especially in the development of therapeutics for CNS disorders. METHODS: To achieve an efficient, fast and safe strategy for BBB opening, an acoustofluidic transwell (AFT) was developed for reversible disruption of the BBB. The proposed AFT was consisted of a transwell insert where the BBB model was established, and a surface acoustic wave (SAW) transducer realized using open-source electronics based on printed circuit board techniques. RESULTS: In the AFT device, the SAW produced acousto-mechanical stimulations to the BBB model resulting in decreased transendothelial electrical resistance in a dose dependent manner, indicating the disruption of the BBB. Moreover, SAW stimulation enhanced transendothelial permeability to sodium fluorescein and FITC-dextran with various molecular weight in the AFT device. Further study indicated BBB opening was mainly attributed to the apparent stretching of intercellular spaces. An in vivo study using a zebrafish model demonstrated SAW exposure promoted penetration of sodium fluorescein to the CNS. CONCLUSIONS: In summary, AFT effectively disrupts the BBB under the SAW stimulation, which is promising as a new drug delivery methodology for neurodegenerative diseases.

Engineering the Coacervate Microdroplet Interface via Polyelectrolyte and Surfactant Complexation.

Complex coacervate microdroplets, which are formed via spontaneous liquid-liquid phase separation by mixing two oppositely charged polyelectrolytes in water, have emerged as a new paradigm in the fields of origin of life, membraneless subcellular compartmentalization, bioreactors, and drug delivery. However, how to further improve its stability and enhance its selectivity in one particular coacervate system remains a challenge. By generating a membrane-like layer at the surface of coacervate microdroplets via electrostatic interactions between oppositely charged surfactants and polyelectrolytes, we here achieve tunable permeability and enhanced stability of the coacervates at the same time. Depending on the surfactants used, membrane-like layer-coated coacervate microdroplets exhibit different selectivity over solute sequestration and can promote or inhibit DNA hybridization. Our approach provides a practical tool to engineer functional bioinspired microcompartments with potential applications in the fields of controlled drug release and microreactor technology.

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.

Acoustically accelerated neural differentiation of human embryonic stem cells.

Human embryonic stem cells (hESCs) and their derived products offer great promise for targeted therapies and drug screening, however, the hESC differentiation process of mature neurons is a lengthy process. To accelerate the neuron production, an acoustic stimulator producing surface acoustic waves (SAWs) is proposed and realized by clamping a flexible printed circuit board (PCB) directly onto a piezoelectric substrate. Neural differentiation of the hESCs is greatly accelerated after application of the acoustic stimulations. Acceleration mechanisms for neural differentiation have been explored by bulk RNA sequencing, quantitative polymerase chain reaction (qPCR) and immunostaining. The RNA sequencing results show changes of extracellular matrix-related and physiological activity-related gene expression in the low or medium SAW dose group and the high SAW dose group, respectively. The neural progenitor cell markers, including Pax6, Sox1, Sox2, Sox10 and Nkx2-1, are less expressed in the SAW dose groups compared with the control group by the qPCR. Other genes including Alk, Cenpf, Pcdh17, and Actn3 are also found to be regulated by the acoustic stimulation. Moreover, the immunostaining confirmed that more mature neuron marker Tuj1-positive cells, while less stem cell marker Sox2-positive cells, are presented in the SAW dose groups. These results indicate that the SAW stimulation accelerated neural differentiation process. The acoustic stimulator fabricated by using the PCB is a promising tool in regulation of stem cell differentiation process applied in cell therapy. STATEMENT OF SIGNIFICANCE: Human embryonic stem cells (hESC) are used for investigating the complex mechanisms involved in the development of specialized biological cells and organs. Different types of hESCs derived cell products can be used for cell therapy procedures aiming to regenerate functional tissues in patients who suffer from various degenerative diseases. Accelerating the hESCs' differentiation process can considerably benefit the clinical utilization of these cells. This study develops a highly effective acoustic stimulator working at ∼20 MHz to investigate what roles do acousto-mechanical stimuli play in the differentiation of hESCs. Our results show that acoustic dose alters the extracellular matrix and physiological activity-related gene expression, which indicates that the acoustic stimulation is an important tool for regulating the stem cells' differentiation processes in cell therapy.

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.

Construction of protocell-based artificial signal transduction pathways.

The maintenance of an orderly and controllable multicellular society depends on the communication and signal regulation between various types of biological cells. How to replicate complicated signal transduction pathways in synthetic protocellular communities remains a key challenge in bottom-up synthetic biology. Herein, we review recent advances in the design and construction of interactive protocell communities, or protocell communities and biological communities, and explore the ways of designing and constructing artificial paracrine-like signaling pathways and juxtacrine-like signaling pathways. Key molecules involved in the signaling pathways that can be used to connect two or more spatially separated communities, and diverse signal outputs generated by the communication are summarized. We also propose the limitations, challenges and opportunities in this field.

Chemical-mediated translocation in protocell-based microactuators.

Artificial cell-like communities participate in diverse modes of chemical interaction but exhibit minimal interfacing with their local environment. Here we develop an interactive microsystem based on the immobilization of a population of enzyme-active semipermeable proteinosomes within a helical hydrogel filament to implement signal-induced movement. We attach large single-polynucleotide/peptide microcapsules at one or both ends of the helical protocell filament to produce free-standing soft microactuators that sense and process chemical signals to perform mechanical work. Different modes of translocation are achieved by synergistic or antagonistic enzyme reactions located within the helical connector or inside the attached microcapsule loads. Mounting the microactuators on a ratchet-like surface produces a directional push-pull movement. Our methodology opens up a route to protocell-based chemical systems capable of utilizing mechanical work and provides a step towards the engineering of soft microscale objects with increased levels of operational autonomy.

Chemical Information Exchange in Organized Protocells and Natural Cell Assemblies with Controllable Spatial Positions.

An ultrasound-based platform is established to prepare homogenous arrays of giant unilamellar vesicles (GUVs) or red blood cell (RBCs), or hybrid assemblies of GUV/RBCs. Due to different responses to the modulation of the acoustic standing wave pressure field between the GUVs and RBCs, various types of protocell/natural cell hybrid assemblies are prepared with the ability to undergo reversible dynamic reconfigurations from vertical to horizontal alignments, or from 1D to 2D arrangements. A two-step enzymatic cascade reaction between transmitter glucose oxidase-containing GUVs and peroxidase-active receiver RBCs is used to implement chemical signal transduction in the different hybrid micro-arrays. Taken together, the obtained results suggest that the ultrasound-based micro-array technology can be used as an alternative platform to explore chemical communication pathways between protocells and natural cells, providing new opportunities for bottom-up synthetic biology.

Hydrogel-Immobilized Coacervate Droplets as Modular Microreactor Assemblies.

Immobilization of compartmentalized microscale objects in 3D hydrogels provides a step towards the modular assembly of soft functional materials with tunable architectures and distributed functionalities. Herein, we report the use of a combination of micro-compartmentalization, immobilization, and modularization to fabricate and assemble hydrogel-based microreactor assemblies comprising millions of functionalized polysaccharide-polynucleotide coacervate droplets. The heterogeneous hydrogels can be structurally fused by interfacial crosslinking and coupled as input and output modules to implement a UV-induced photocatalytic/peroxidation nanoparticle/DNAzyme reaction cascade that generates a spatiotemporal fluorescence read-out depending on the droplet number density, intensity of photoenergization, and chemical flux. Our approach offers a route to heterogeneous hydrogels with endogenous reactivity and reconfigurable architecture, and provides a step towards the development of soft modular materials with programmable functionality.

Catalytic processing in ruthenium-based polyoxometalate coacervate protocells.

The development of programmable microscale materials with cell-like functions, dynamics and collective behaviour is an important milestone in systems chemistry, soft matter bioengineering and synthetic protobiology. Here, polymer/nucleotide coacervate micro-droplets are reconfigured into membrane-bounded polyoxometalate coacervate vesicles (PCVs) in the presence of a bio-inspired Ru-based polyoxometalate catalyst to produce synzyme protocells (Ru4PCVs) with catalase-like activity. We exploit the synthetic protocells for the implementation of multi-compartmentalized cell-like models capable of collective synzyme-mediated buoyancy, parallel catalytic processing in individual horseradish peroxidase-containing Ru4PCVs, and chemical signalling in distributed or encapsulated multi-catalytic protocell communities. Our results highlight a new type of catalytic micro-compartment with multi-functional activity and provide a step towards the development of protocell reaction networks.

Photoswitchable Phase Separation and Oligonucleotide Trafficking in DNA Coacervate Microdroplets.

Coacervate microdroplets produced by liquid-liquid phase separation have been used as synthetic protocells that mimic the dynamical organization of membrane-free organelles in living systems. Achieving spatiotemporal control over droplet condensation and disassembly remains challenging. Herein, we describe the formation and photoswitchable behavior of light-responsive coacervate droplets prepared from mixtures of double-stranded DNA and an azobenzene cation. The droplets disassemble and reassemble under UV and blue light, respectively, due to azobenzene trans/cis photoisomerisation. Sequestration and release of captured oligonucleotides follow the dynamics of phase separation such that light-activated transfer, mixing, hybridization, and trafficking of the oligonucleotides can be controlled in binary populations of the droplets. Our results open perspectives for the spatiotemporal control of DNA coacervates and provide a step towards the dynamic regulation of synthetic protocells.

Acoustic deformation for the extraction of mechanical properties of lipid vesicle populations.

We use an ultrasonic standing wave to simultaneously trap and deform thousands of soft lipid vesicles immersed in a liquid solution. In our device, acoustic radiation stresses comparable in magnitude to those generated in optical stretching devices are achieved over a spatial extent of more than ten acoustic wavelengths. We solve the acoustic scattering problem in the long-wavelength limit to obtain the radiation stress. The result is then combined with thin-shell elasticity theory to form expressions that relate the deformed geometry to the applied acoustic field intensity. Using observation of the deformed geometry and this model, we rapidly extract mechanical properties, such as the membrane Young's modulus, from populations of lipid vesicles.