Soft Synthetic Cells with Mobile Membrane Ligands for Ex Vivo Expansion of Therapy-Relevant T Cell Phenotypes.

The expansion of T cells ex vivo is crucial for effective immunotherapy but currently limited by a lack of expansion approaches that closely mimic in vivo T cell activation. Taking inspiration from bottom-up synthetic biology, a new synthetic cell technology is introduced based on dispersed liquid-liquid phase-separated droplet-supported lipid bilayers (dsLBs) with tunable biochemical and biophysical characteristics, as artificial antigen presenting cells (aAPCs) for ex vivo T cell expansion. These findings obtained with the dsLB technology reveal three key insights: first, introducing laterally mobile stimulatory ligands on soft aAPCs promotes expansion of IL-4/IL-10 secreting regulatory CD8+ T cells, with a PD-1 negative phenotype, less prone to immune suppression. Second, it is demonstrated that lateral ligand mobility can mask differential T cell activation observed on substrates of varying stiffness. Third, dsLBs are applied to reveal a mechanosensitive component in bispecific Her2/CD3 T cell engager-mediated T cell activation. Based on these three insights, lateral ligand mobility, alongside receptor- and mechanosignaling, is proposed to be considered as a third crucial dimension for the design of ex vivo T cell expansion technologies.

Cell-Like Capsules with "Smart" Compartments.

Eukaryotic cells have inner compartments (organelles), each with distinct properties and functions. One mimic of this architecture, based on biopolymers, is the multicompartment capsule (MCC). Here, MCCs in which the inner compartments are chemically unique and "smart," i.e., responsive to distinct stimuli in an orthogonal manner are created. Specifically, one compartment alone is induced to degrade when the MCC is contacted with an enzyme while other compartments remain unaffected. Similarly, just one compartment gets degraded upon contact with reactive oxygen species generated from hydrogen peroxide (H2 O2 ). And thirdly, one compartment alone is degraded by an external, physical stimulus, namely, by irradiating the MCC with ultraviolet (UV) light. All these specific responses are achieved without resorting to complicated chemistry to create the compartments: the multivalent cation used to crosslink the biopolymer alginate (Alg) is simply altered. Compartments of Alg crosslinked by Ca2+ are shown to be sensitive to enzymes (alginate lyases) but not to H2 O2 or UV, whereas the reverse is the case with Alg/Fe3+ compartments. These results imply the ability to selectively burst open a compartment in an MCC "on-demand" (i.e., as and when needed) and using biologically relevant stimuli. The results are then extended to a sequential degradation, where compartments in an MCC are degraded one after another, leaving behind an empty MCC lumen. Collectively, this work advances the MCC as a platform that not only emulates key features of cellular architecture, but can also begin to capture rudimentary cell-like behaviors.

Synthetic Cells: From Simple Bio-Inspired Modules to Sophisticated Integrated Systems.

Bottom-up synthetic biology is the science of building systems that mimic the structure and function of living cells from scratch. To do this, researchers combine tools from chemistry, materials science, and biochemistry to develop functional and structural building blocks to construct synthetic cell-like systems. The many strategies and materials that have been developed in recent decades have enabled scientists to engineer synthetic cells and organelles that mimic the essential functions and behaviors of natural cells. Examples include synthetic cells that can synthesize their own ATP using light, maintain metabolic reactions through enzymatic networks, perform gene replication, and even grow and divide. In this Review, we discuss recent developments in the design and construction of synthetic cells and organelles using the bottom-up approach. Our goal is to present representative synthetic cells of increasing complexity as well as strategies for solving distinct challenges in bottom-up synthetic biology.

Multicompartment Polymeric Nanocarriers for Biomedical Applications.

Multicompartment polymeric nanocarriers which mimic the compartmentalized architecture of living cells have received considerable research attention in the biomedical field. The advancement of synthetic polymeric chemistry has allowed multicompartment polymeric nanocarriers to be tailored for biomedical applications such as drug delivery, encapsulated catalysis, and artificial cellular mimics. In this review, polymer-based multicompartment nanocarriers (multicompartment micelles, multicompartment polymersomes, and capsosomes) have been discussed. This review focuses on multicompartment systems applied to biomedical applications over the last ten years. The synthetic procedures and structural properties that impact the specific application are also highlighted.

Control of Nanoparticle Release by Membrane Composition for Dual-Responsive Nanocapsules.

Stimulus-responsive polymeric nanocapsules usable as cell mimics can be engineered to precisely control cargo release. This work reports the release behavior of post-loaded nanoparticles through permeable membranes of stable pH and temperature dual-responsive polymeric nanocapsules (CP1, CP2, and CP3) with the same membrane thickness but different membrane composition, prepared by layer-by-layer assembly and surface-initiated single electron transfer living radical polymerization, respectively. These nanocapsules differ in their tunable membrane permeability for post-loaded nanoparticles as protein mimics, tailored by pH and temperature stimuli. Release mechanisms are dominated by membrane composition, such as polyelectrolyte multilayer membrane for CP1, pure cationic membrane for CP2, and valve-like functions for CP3. Thus, one can postulate the main locations of post-loaded protein mimics in the different nanocapsules. Understanding the post-loading and diffusion mechanism of nanoparticles through permeable membranes in cell mimics paves the way for the construction of new "smart" synthetic protocells with control over the exchange of bioactive nanoparticles between different compartments.

Host Cell Mimic Polymersomes for Rapid Detection of Highly Pathogenic Influenza Virus via a Viral Fusion and Cell Entry Mechanism.

Highly pathogenic avian influenza virus (HPAIV) infections have occurred continuously and crossed the species barrier to humans, leading to fatalities. A polymerase chain reaction based molecular test is currently the most sensitive diagnostic tool for HPAIV; however, the results must be analyzed in centralized diagnosis systems by a trained individual. This requirement leads to delays in quarantine and isolation. To control the spread of HPAIV, rapid and accurate diagnostics suitable for field testing are needed, and the tests must facilitate a differential diagnosis between HPAIV and low pathogenic avian influenza virus (LPAIV), which undergo cleavage specifically by trypsin- or furin-like proteases, respectively. In this study, a differential avian influenza virus rapid test kit is developed and evaluated in vitro and using clinical specimens from HPAIV H5N1-infected animals. It is demonstrated that this rapid test kit provides highly sensitive and specific detection of HPAIV and LPAIV and is thus a useful field diagnostic tool for H5N1 HPAIV outbreaks and for rapid quarantine control of the disease.

Functional Cellular Mimics for the Spatiotemporal Control of Multiple Enzymatic Cascade Reactions.

Next-generation therapeutic approaches are expected to rely on the engineering of biomimetic cellular systems that can mimic specific cellular functions. Herein, we demonstrate a highly effective route for constructing structural and functional eukaryotic cell mimics by loading pH-sensitive polymersomes as membrane-associated and free-floating organelle mimics inside the multifunctional cell membrane. Metabolism mimicry has been validated by performing successive enzymatic cascade reactions spatially separated at specific sites of cell mimics in the presence and absence of extracellular organelle mimics. These enzymatic reactions take place in a highly controllable, reproducible, efficient, and successive manner. Our biomimetic approach to material design for establishing functional principles brings considerable enrichment to the fields of biomedicine, biocatalysis, biotechnology, and systems biology.

Mimic of the Cellular Antioxidant Defense System for a Sustainable Regeneration of Nicotinamide Adenine Dinucleotide (NAD).

The prolonged use of enzymes under oxidative stress is a major challenge in enabling effective enzymatic reaction pathways. Herein, we report a biomimetic antioxidant defensive strategy capable of providing adequate protection of enzymes against superoxide-mediated oxidation. Superoxide dismutase (SOD) and catalase (CAT) were chosen as scavengers and covalently encapsulated into silica nanoreactors, together with glucose dehydrogenase (GDH), which simultaneously should produce the coenzyme nicotinamide adenine dinucleotide (NADH, reduced form). By the enzymatic reactions of SOD and CAT, the interior of silica nanoreactors becomes a "ROS safe zone" to protect the glucose-dependent NADH production of coencapsulated GDH. We further combined this protected NADH-producing module with photocatalytic nanoparticles that enable the light-triggered oxidation of NADH back to NAD+ (oxidized form). In combination, these two modules allow interconversion between NAD+ and NADH by the addition of glucose or by light irradiation (LED lamp or sunlight). This protection and regeneration strategy is a versatile tool for enzyme applications for biological reactors, catalysis, or prototypes of artificial organelles or building blocks that contains fragile biomolecules and rely on the coenzyme NAD+/NADH.

Weak Multivalent Binding of Influenza Hemagglutinin Nanoparticles at a Sialoglycan-Functionalized Supported Lipid Bilayer.

Quantification of the multivalent interactions of influenza viruses binding at interfaces may provide ways to tackle key biological questions regarding influenza virulence and zoonoses. Yet, the deconvolution of the contributions of molecular and interfacial parameters, such as valency, interaction area, and receptor density, to the binding of whole viruses is hindered by difficulties in the direct determination of these parameters. We report here a chemical platform technology to study the binding of multivalent recombinant hemagglutinin (rHA) nanoparticles at artificial sialoglycan cell receptor-presenting interfaces in which all these parameters can be derived, thus allowing the desired full and quantitative binding analysis. SiO2 substrates were functionalized with supported lipid bilayers containing a targeted and tunable fraction of a biotinylated lipid, followed by the adsorption of streptavidin and biotinylated polyvalent 2,3- or 2,6-sialyl lactosamine (SLN). rHA nanoparticles were used as a virus mimic to provide a good prediction of the number of interactions involved in binding. Low nanomolar affinities and selectivities for binding at the 2,6-SLN platforms were observed for rHA particles from three different virus variants. When fitting the data to a multivalency model, the nanomolar overall affinity appears to be achieved by 6-9 HA-sugar molecular interaction pairs, which individually present a rapid association/dissociation behavior. This dynamic behavior may be an essential biological attribute in the functioning of the influenza virus.

Clusters of Nanoscale Liposomes Modulate the Release of Encapsulated Species and Mimic the Compartmentalization Intrinsic in Cell Structures.

We report the ability to place a high concentration of liposomes in a confined volume as a multicompartment cluster that mimics biological cells and allows for the modulation of release of encapsulated species. The formation of these coated multicompartmental structures is achieved by first binding liposomes into clusters before encapsulating them within a two-dimensional metal-organic framework composed of tannic acid coordinated with a metal ion. The essential feature is a molecularly thin skin over a ssystem of clustered liposomes in a pouch. The structural features of these pouches are revealed by small-angle scattering and electron microscopy. Through cryogenic electron microscopy, clusters with intact liposomes are observed that appear to be encapsulated within a pouch. Small-angle X-ray scattering shows the emergence of a relatively weak Bragg peak at q = 0.125 Å-1, possibly indicating the attachment of the bilayers of adjacent liposomes. The metal-phenolic network (MPN) forms a nanosized conformal coating around liposome clusters, resulting in the reduced release rate of the encapsulated rhodamine B dye. We further show the possibility of communication between the adjacent nanocompartments in the cluster by demonstrating enhanced energy transfer using fluorescence resonance energy transfer (FRET) experiments where the lipophilic donor dye 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) incorporated within one liposomal compartment transfers energy upon excitation to the lipophilic acceptor dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) in a neighboring liposomal compartment due to their close proximity within the multicompartmental cluster. These observations have significance in adapting these multicompartmental structures that mimic biological cells for cascade reactions and as new depot drug delivery systems.

Shape-Preserved Transformation of Biological Cells into Synthetic Hydrogel Microparticles.

The synthesis of materials that can mimic the mechanical, and ultimately functional, properties of biological cells can broadly impact the development of biomimetic materials, as well as engineered tissues and therapeutics. Yet, it is challenging to synthesize, for example, microparticles that share both the anisotropic shapes and the elastic properties of living cells. Here, a cell-directed route to replicate cellular structures into synthetic hydrogels such as polyethylene glycol (PEG) is described. First, the internal and external surfaces of chemically fixed cells are replicated in a conformal layer of silica using a sol-gel process. The template is subsequently removed to render shape-preserved, mesoporous silica replicas. Infiltration and cross-linking of PEG precursors and dissolution of the silica result in a soft hydrogel replica of the cellular template as demonstrated using erythrocytes, HeLa, and neuronal cultured cells. The elastic modulus can be tuned over an order of magnitude (≈10-100 kPa) though with a high degree of variability. Furthermore, synthesis without removing the biotemplate results in stimuli-responsive particles that swell/deswell in response to environmental cues. Overall, this work provides a foundation to develop soft particles with nearly limitless architectural complexity derived from dynamic biological templates.

Mussel-Inspired pH-Switched Assembly of Capsules with an Ultrathin and Robust Nanoshell.

Enclosed films, also called capsules, bearing an ultrathin and robust nanoshell have sparked much interest for use in many applications, for which facile preparation methods are urgently pursued. Inspired by the pH-programmed adhesion/cohesion of mussel-secreted foot proteins, polyphenol/polyamine capsules with an ultrathin and robust nanoshell are fabricated through a pH-switched assembly on sacrificial calcium carbonate (CaCO3) templates. Polyphenols adhere to the templates at pH 6.0 and rapidly cohere with polyamines at pH 8.0. The pH-switched assembly process is accomplished in only a few minutes where multiple instances of electrostatic interactions and chemical conjugation between polyphenols and polyamines occur. As a result, the capsules exhibit a nanoshell thickness of ∼10 nm and a superior mechanical strength of ∼1.575 GPa (elasticity modulus). Cell mimics are prepared through encasing enzymes in the lumen and present an activity recovery of ∼70% along with little activity decline during reuse. Amine or phenolic groups on the nanoshell of capsules are then applied to induce the generation of titania or silver nanoparticles, which may expand the applications of the capsules to the photo- and biorelated realms. Our study not only deepens the understanding of the adhering process of mussels but also offers a generic method toward functional materials for diverse applications.