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.

Enhancing the Sensitivity of DNA and Aptamer Probes in the Dextran/PEG Aqueous Two-Phase System.

Increasing the local concentration of DNA-based probes is a convenient way to improve the sensitivity of biosensors. Instead of using organic solvents or ionic liquids that phase-separate with water based on hydrophobic interactions, we herein studied a classic aqueous two-phase system (ATPS) comprising polyethylene glycol (PEG) and dextran. Polymers of higher molecular weights and higher concentrations favored phase separation. DNA oligonucleotides are selectively enriched in the dextran-rich phase unless the pH was increased to 12. A higher volume ratio of PEG-to-dextran and a higher concentration of PEG also enrich more DNA probes in the dextran-rich phase. The partition efficiency of the T15 DNA was enriched around seven times in the dextran phase when the volume ratio of dextran and PEG reached 1:10. The detection of limit improved by 3.6-fold in a molecular beacon-based DNA detection system with the ATPS. The ATPS also increased the sensitivity for the detection of Hg2+ and adenosine triphosphate, although these target molecules alone distributed equally in the two phases. This work demonstrates a simple method using water soluble polymers to improve biosensors.

Invasion and Defense Interactions between Enzyme-Active Liquid Coacervate Protocells and Living Cells.

The design and construction of mutual interaction models between artificial microsystems and living cells have the potential to open a wide range of novel applications in biomedical and biomimetic technologies. In this study, an artificial form of invasion-defense mutual interactions is established in a community of glucose oxidase (GOx)-containing liquid coacervate microdroplets and living cells, which interact via enzyme-mediated reactive oxygen species (ROS) damage. The enzyme-containing coacervate microdroplets, formed via liquid-liquid phase separation, act as invader protocells to electrostatically bind with the host HepG2 cell, resulting in assimilation. Subsequently, the glucose oxidation in the liquid coacervates initiates the generation of H2 O2 , which serves as an ROS resource to block cell proliferation. As a defense strategy, introduction of catalase (CAT) into the host cells is exploited to resist the ROS damage. CAT-mediated decomposition of H2 O2 leads to the ROS scavenging and results in the recovery of cell viability. The results obtained in the current study highlight the remarkable opportunities for the development of mutual interacting communities on the interface of artificial protocells/living cells. They also provide a new approach for engineering cellular behaviors through exploiting artificial nonliving microsystems.

Coacervate Microdroplets as Synthetic Protocells for Cell Mimicking and Signaling Communications.

Synthetic protocells are minimal systems that mimic certain properties of natural cells and are used to research the emergence of life from a nonliving chemical network. Currently, coacervate microdroplets, which are formed via liquid-liquid phase separation, are receiving wide attention in the context of cell biology and protocell research; these microdroplets are notable because they can provide liquid-like compartment structures for biochemical reactions by creating highly macromolecular crowded local environments. In this review, an overview of recent research on the formation of coacervate microdroplets through phase separation; the design of coacervate-based stimuli-responsive protocells, multichamber protocells, and membranized protocells; and their cell mimic behaviors, is provided. The simplified protocell models with precisely defined and tunable compositions advance the understanding of the requirements for cellular structure and function. Efforts are then discussed to establish signal communication systems in protocell and protocell consortia, as communication is a fundamental feature of life that coordinates matter exchanges and energy fluxes dynamically in space and time. Finally, some perspectives on the challenges and future developments of synthetic protocell research in biomimetic science and biomedical applications are provided.

Enzyme-active liquid coacervate microdroplets as artificial membraneless organelles for intracellular ROS scavenging.

Artificial organelles are microcompartments capable of performing catalytic reactions in living cells to replace absent or lost cellular functions. Coacervate microdroplets, formed via liquid-liquid phase separation, have been developed as membraneless organelles that mimic the dynamical organization of liquid organelles. However, the further studies focusing on cellular implanting of coacervate microdroplets in living cells to supplement the dysfunction of natural cells are still rare. Here catalase (CAT)-containing coacervate microdroplets, developed as artificial membraneless organelles with unique liquid compartments, were integrated into living cells to scavenge intracellular massive reactive oxygen species (ROS) and recover cell viability. The enzyme-containing coacervate microdroplets were constructed by sequestering CAT in poly(dimethyldiallylammonium chloride) (PDDA)/polyacrylic acid (PAA) coacervate microdroplets; their liquid-like fluidity was revealed by fluorescence recovery after bleaching, and coalescence experiment in vitro and in living cells. After cellular internalization, the coacervate microdroplets remained in the polymer-rich dense phase and retained enzymatic activities. CAT-mediated H2O2 removal and ROS scavenging in living cells decreased the cytotoxicity of H2O2, improving cell viability. The cell internalization of coacervate microdroplets in vitro provides a novel approach for designing artificial membraneless organelles in living cells. The strategy of using artificial organelle-mediated enzymatic reactions to supplement cellular dysfunctions can be exploited for their further biomedical applications.

DNA nanotubes in coacervate microdroplets as biomimetic cytoskeletons modulate the liquid fluidic properties of protocells.

Coacervate microdroplets, formed via liquid-liquid phase separation, have been proposed as a compartment model for the construction of artificial cells or organelles. However, these microsystems are very fragile and demonstrate liquid-like fluidity. Here, an artificial cytoskeleton based on DNA nanotubes was constructed in coacervate microdroplets to modulate the liquid fluidic properties of the microdroplets. The coacervate microdroplets were obtained from the association of oppositely charged polyelectrolytes through liquid-liquid phase separation, and DNA nanotubes were constructed by molecular tile self-assembly from six clip sequences. The DNA nanotubes were efficiently sequestered in the liquid coacervate microdroplets, and the rigid structure of the DNA nanotubes was capable of modulating the liquid fluidic properties of the coacervate protocell models, as indicated by coalescence imaging and atomic force microscopy analysis. Therefore, artificial cytoskeletons made from DNA nanotubes worked in modulating the liquid fluidic properties of coacervate microdroplets, in a manner akin to the cytoskeleton in the cell. DNA cytoskeletons have the potential to become an ideal platform with which how the liquid fluidic properties of cells are modulated by their cytoskeletons can be investigated, and the cell-sized coacervate microdroplets containing artificial cytoskeletons might be critical in developing a stable liquid-phase protocell model.

Signal processing and generation of bioactive nitric oxide in a model prototissue.

The design and construction of synthetic prototissues from integrated assemblies of artificial protocells is an important challenge for synthetic biology and bioengineering. Here we spatially segregate chemically communicating populations of enzyme-decorated phospholipid-enveloped polymer/DNA coacervate protocells in hydrogel modules to construct a tubular prototissue-like vessel capable of modulating the output of bioactive nitric oxide (NO). By decorating the protocells with glucose oxidase, horseradish peroxidase or catalase and arranging different modules concentrically, a glucose/hydroxyurea dual input leads to logic-gate signal processing under reaction-diffusion conditions, which results in a distinct NO output in the internal lumen of the model prototissue. The NO output is exploited to inhibit platelet activation and blood clot formation in samples of plasma and whole blood located in the internal channel of the device, thereby demonstrating proof-of-concept use of the prototissue-like vessel for anticoagulation applications. Our results highlight opportunities for the development of spatially organized synthetic prototissue modules from assemblages of artificial protocells and provide a step towards the organization of biochemical processes in integrated micro-compartmentalized media, micro-reactor technology and soft functional materials.

Coacervate microdroplet protocell-mediated gene transfection for nitric oxide production and induction of cell apoptosis.

Liquid coacervate microdroplets have been widely explored as membrane-free compartment protocells for cargo delivery in therapeutic applications. In this study, coacervate protocells were developed as gene carriers for transfection of nitric oxide synthase (NOS) and overproduction of nitric oxide (NO) for killing of cancer cells. The coacervate microdroplet protocells were formed via the liquid-liquid phase separation of oppositely charged diethylaminoethyl-dextran/polyacrylic acids. The coacervate microdroplet protocells were found to facilitate gene transfection, which was demonstrated by cell imaging of the internalized coacervate microdroplets containing plasmids of enhanced green fluorescent protein. Due to their high transfection capability, the coacervate protocells were subsequently utilized for the delivery of NOS plasmids (pNOS). The cellular internalization of pNOS-containing coacervate carriers was found to result in high NOS expression coupled with NO overproduction, which then induced cell apoptosis and decreased cell viability. The cell apoptosis is associated with NO-mediated mitochondrial damage. The enhanced gene transfection was attributed to coacervate microdroplets' unique high sequestration capability and liquid-like fluidity. Overall, the incorporation of genes in coacervate microdroplets was demonstrated as a viable and novel strategy for the development of cargo biocarriers for biomedical applications.

Photothermally Activated Coacervate Model Protocells as Signal Transducers Endow Mammalian Cells with Light Sensitivity.

The development of a novel photothermally activated coacervate model protocell is reported as a signal transducer to endow mammalian cells with light sensitivity. In this system, near-infrared light irradiation triggers H2 S release in coacervate model protocells, leading to modulation of the behavior of living cells. The functional coacervate model protocells are prepared by loading metal-alloyed plasmonic nanoparticles and an H2 S donor into the liquid coacervate microdroplets. Upon light irradiation, the H2 S signal messenger is released through the photothermal effect of plasmonic nanoparticles and photothermal mediated pyrolysis of the H2 S donor. The H2 S signal is delivered to the mammalian cell community to trigger depletion of reactive oxygen species, reduce the activity of lactate dehydrogenase and improve cell viability. This study provides a new approach to the implementation of chemical signaling in artificial cell colonies and protocell/living cell consortia. The photothermal protocell system offers a powerful platform for light modulation of the behavior of mammalian cells and shows great promise for biomedical applications.

Development and antibacterial activities of bacterial cellulose/graphene oxide-CuO nanocomposite films.

The absence of antibacterial activity of bacterial cellulose (BC) restricts its applications in the biomedical field. To introduce antimicrobial properties into BC, we studied the synthesis, structure, and antimicrobial properties of a novel nanocomposite film comprising BC, graphene oxide (GO), and copper-oxide (CuO) nanosheets. The nanocomposite film was synthesized by incorporating GO-CuO nanohybrids into BC matrix through homogenized blending. The CuO nanosheets, with a length range of 50 nm-200 nm and width range of 20 nm-50 nm, which were uniformly grown on the GO along with even distribution of GO-CuO nanohybrids on the surface of the cellulose fibers. The nanocomposites displayed better antibacterial activity against gram-positive than gram-negative bacteria. BC/GO-CuO nanocomposites showed higher antibacterial activity than BC/CuO. We also elucidated the mechanism of antibacterial activity of the nanocomposites. Further, the nanocomposites exhibited biocompatibility towards mice fibroblast cells. The nanocomposites might serve as an excellent source for development of antibacterial materials.

Enzyme-mediated nitric oxide production in vasoactive erythrocyte membrane-enclosed coacervate protocells.

The design and construction of synthetic therapeutic protocells capable of establishing cognate chemical communication channels with living cells is an important challenge for synthetic biology and bio-engineering. Here we develop a step towards protocell-mediated nitric-oxide-induced vasodilation by constructing a new synthetic cell model based on bio-derived coacervate vesicles with high haemocompatibility and increased blood circulation times. The hybrid protocells are prepared by the spontaneous self-assembly of haemoglobin-containing erythrocyte membrane fragments on the surface of preformed polysaccharide-polynucleotide coacervate micro-droplets containing glucose oxidase. We use the sequestered enzymes to program a spatially coupled glucose oxidase/haemoglobin reaction cascade, which in the presence of glucose and hydroxyurea generates a protocell-mediated flux of nitric oxide that we exploit for in vitro and in vivo blood vessel vasodilation. Taken together, our results provide new opportunities for the development of endogenously organized cell-like entities (biocompatible micro-bots) geared specifically towards active interfacing with individual living cells and cell communities.

Uricase-containing coacervate microdroplets as enzyme active membrane-free protocells for detoxification of uric acid in serum.

Based on the unique property of preferential sequestration of guest molecules, coacervate microdroplets are proposed as enzyme active membrane-free protocells, in which uricase is loaded for efficient detoxification of uric acid in serum.

Synthesis of boronate-decorated polyethyleneimine-grafted porous layer open tubular capillaries for enrichment of polyphenols in fruit juices.

A combination between modification with porous layer and grafting of polyethyleneimine (PEI) on the inner face of capillary was for the first time developed for boronate affinity in-tube solid-phase microextraction (SPME) material to enhance the extraction capacity for cis-diol-containing polyphenols. The successful synthesis of boronate-decorated polyethyleneimine-grafted porous layer open tubular (BPPLOT) capillary was confirmed by scanning electron micrograph, Fourier transform-infrared spectra and absorption experiments. The porous layer, PEI and boronate affinity provided high specific surface area, more binding sites for boronate groups and specific selectivity of BPPLOT capillary, respectively. The maximum binding quantity of BPPLOT capillary greatly improved, and ranged from 143 to 170 µg m-1 for cis-diol-containing polyphenols (catechin, chlorogenic acid, caffeic acid and epicatechin). A green method based on boronate affinity in-tube SPME was developed for separation and enrichment polyphenols, and some parameters of in-tube SPME were optimized. After in-tube SPME, HPLC with UV detection was used for quantitative determination of polyphenols. Recoveries of standard spiked cis-diol-containing polyphenols from fruit juice were between 80.9% and 102%, with intra-day and inter-day coefficient of variation ranging from 4.8% to 7.3% and 5.0% to 8.6%, respectively. Conversely, recovery of non-cis-diol-containing ferulic acid was no greater than 3.0%. These results suggested that the BPPLOT capillary could effectively separate and enrich cis-diol-containing polyphenols from real samples.