Artificial Receptor in Synthetic Cells Performs Transmembrane Activation of Proteolysis.

The design of artificial, synthetic cells is a fundamentally important and fast-developing field of science. Of the diverse attributes of cellular life, artificial transmembrane signaling across the biomolecular barriers remains a high challenge with only a few documented successes. Herein, the study achieves signaling across lipid bilayers and connects an exofacial enzymatic receptor activation to an intracellular biochemical catalytic response using an artificial receptor. The mechanism of signal transduction for the artificial receptor relies on the triggered decomposition of a self-immolative linker. Receptor activation ensues its head-to-tail decomposition and the release of a secondary messenger molecule into the internal volume of the synthetic cell. Transmembrane signaling is demonstrated in synthetic cells based on liposomes and mammalian cell-sized giant unilamellar vesicles and illustrates receptor performance in cell mimics with a diverse size and composition of the lipid bilayer. In giant unilamellar vesicles, transmembrane signaling connects exofacial receptor activation with intracellular activation of proteolysis. Taken together, the results of this study take a step toward engineering receptor-mediated, responsive behavior in synthetic cells.

Mechanisms of Degradation for Polydisulfides: Main Chain Scission, Self-Immolation, Or Chain Transfer Depolymerization.

Organic polydisulfides hold immense potential for the design of recyclable materials. Of these, polymers based on lipoic acid are attractive, as they are based on a natural, renewable resource. Herein, we demonstrate that reductive degradation of lipoic acid polydisulfides is a rapid process whereby the quantity of added initiator relative to the polymer content defines the mechanism of polymer degradation, through the main chain scission, self-immolation, or "chain transfer" depolymerization. The latter mechanism is defined as the one during which a thiol group released through the decomposition of one polydisulfide chain initiates depolymerization of the neighbor macromolecule. The chain transfer mechanism afforded the highest yields of recovery of the monomer in its pristine form, and just one molecule of the reducing agent to initiate polymer degradation afforded recovery of over 50% of the monomer. These data are important to facilitate the development of polymer recycling and monomer reuse schemes.

Transmembrane signaling by a synthetic receptor in artificial cells.

Signal transduction across biological membranes is among the most important evolutionary achievements. Herein, for the design of artificial cells, we engineer fully synthetic receptors with the capacity of transmembrane signaling, using tools of chemistry. Our receptors exhibit similarity with their natural counterparts in having an exofacial ligand for signal capture, being membrane anchored, and featuring a releasable messenger molecule that performs enzyme activation as a downstream signaling event. The main difference from natural receptors is the mechanism of signal transduction, which is achieved using a self-immolative linker. The receptor scaffold is modular and can readily be re-designed to respond to diverse activation signals including biological or chemical stimuli. We demonstrate an artificial signaling cascade that achieves transmembrane enzyme activation, a hallmark of natural signaling receptors. Results of this work are relevant for engineering responsive artificial cells and interfacing them and/or biological counterparts in co-cultures.

Chemical zymogens for the protein cysteinome.

We present three classes of chemical zymogens established around the protein cysteinome. In each case, the cysteine thiol group was converted into a mixed disulfide: with a small molecule, a non-degradable polymer, or with a fast-depolymerizing fuse polymer (ZLA). The latter was a polydisulfide based on naturally occurring molecule, lipoic acid. Zymogen designs were applied to cysteine proteases and a kinase. In each case, enzymatic activity was successfully masked in full and reactivated by small molecule reducing agents. However, only ZLA could be reactivated by protein activators, demonstrating that the macromolecular fuse escapes the steric bulk created by the protein globule, collects activation signal in solution, and relays it to the active site of the enzyme. This afforded first-in-class chemical zymogens that are activated via protein-protein interactions. We also document zymogen exchange reactions whereby the polydisulfide is transferred between the interacting proteins via the "chain transfer" bioconjugation mechanism.

Macromolecular Viral Entry Inhibitors as Broad-Spectrum First-Line Antivirals with Activity against SARS-CoV-2.

Inhibitors of viral cell entry based on poly(styrene sulfonate) and its core-shell nanoformulations based on gold nanoparticles are investigated against a panel of viruses, including clinical isolates of SARS-CoV-2. Macromolecular inhibitors are shown to exhibit the highly sought-after broad-spectrum antiviral activity, which covers most analyzed enveloped viruses and all of the variants of concern for SARS-CoV-2 tested. The inhibitory activity is quantified in vitro in appropriate cell culture models and for respiratory viral pathogens (respiratory syncytial virus and SARS-CoV-2) in mice. Results of this study comprise a significant step along the translational path of macromolecular inhibitors of virus cell entry, specifically against enveloped respiratory viruses.

Electrostatics Trigger Interfacial Self-Assembly of Bacterial Ice Nucleators.

Ice active bacteria can catalyze water freezing at high subzero temperatures using ice nucleating proteins (INPs) located at their outer cell walls. INPs are the most effective ice nucleators known and are of significant interest for agriculture, climate research, and freeze/antifreeze technologies. The aggregation of INPs into large ice nucleation sites is a key step for effective ice nucleation. It has been proposed that ice active bacteria can drive the aggregation of INPs and thereby trigger ice nucleation. However, the mechanism of INP aggregate assembly and the molecular processes behind the activation are still unclear. Both biochemical pathways and activation through electrostatics have been proposed based on experiments with lysed ice active bacteria. For a more direct view on the assembly of INPs, we follow the structure and water interactions of a synthetic model INP of the well-studied ice bacterium Pseudomonas syringae at the air-water interface as a function of the subphase pH. By combining sum frequency generation spectroscopy with two-dimensional infrared spectra, we conclude that self-assembly and electrostatic interactions drive the formation of ordered INP structures capable of aligning interfacial water.

Synthetic Artificial Apoptosis-Inducing Receptor for On-Demand Deactivation of Engineered Cells.

The design of a fully synthetic, chemical "apoptosis-inducing receptor" (AIR) molecule is reported that is anchored into the lipid bilayer of cells, is activated by the incoming biological input, and responds with the release of a secondary messenger-a highly potent toxin for cell killing. The AIR molecule has four elements, namely, an exofacial trigger group, a bilayer anchor, a toxin as a secondary messenger, and a self-immolative scaffold as a mechanism for signal transduction. Receptor installation into cells is established via a robust protocol with minimal cell handling. The synthetic receptor remains dormant in the engineered cells, but is effectively triggered externally by the addition of an activating biomolecule (enzyme) or in a mixed cell population through interaction with the surrounding cells. In 3D cell culture (spheroids), receptor activation is accessible for at least 5 days, which compares favorably with other state of the art receptor designs.

Chemical Artificial Internalizing Receptors for Primary T Cells.

The newest generation of cell-based technologies relies heavily on methods to communicate to the engineered cells using artificial receptors, specifically to deactivate the cells administered to a patient in the event of adverse effects. Herein, artificial synthetic internalizing receptors are engineered that function in mammalian cells in 2D and in 3D and afford targeted, specific intracellular drug delivery with nanomolar potency in the most challenging cell type, namely primary, donor-derived T cells. Receptor design comprises a lipid bilayer anchor for receptor integration into cell membrane and a small xenobiotic molecule as a recognition ligand. Artificial receptors are successfully targeted by the corresponding antibody-drug conjugate (ADC) and exhibit efficient cargo cell entry with ensuing intracellular effects. Receptor integration into cells is fast and robust and affords targeted cell entry in under 2 h. Through a combination of the receptor design and the use of ADC, combined benefits previously made available by chimeric artificial receptors (performance in T cells) and the chemical counterpart (robustness and simplicity) in a single functional platform is achieved. Artificial synthetic receptors are poised to facilitate the maturation of engineered cells as tools of biotechnology and biomedicine.

S-nitrosothiol-terminated poly(vinyl alcohol): Nitric oxide release and skin blood flow response.

Polymeric biomaterials capable of delivering nitric oxide (NO) topically can be used to enhance skin blood flow (SkBF) and accelerate wound healing. Herein, we used reversible addition-fragmentation chain transfer radical (RAFT) polymerization to synthesize the first poly(vinyl alcohol) (PVA) functionalized with terminal NO-releasing S-nitrosothiol (RSNO) groups for topical NO delivery. This strategy was based on the synthesis of a precursor amino-terminated PVA (PVA-NH2), which was next functionalized with iminothiolane yielding 4-imino-4-amino-PVA-butane-1-thiol (PVA-SH), and finally S-nitrosated yielding S-nitroso 4-imino-4-amino-PVA-butane-1-thiol (PVA-SNO). Real-time chemiluminescence NO detection showed that blended films of pure PVA with PVA-SNO with mass ratios 30:70, 50:50 and 70:30 release NO with initial rates ranging from 1 to 12 nmol g-1 min-1, and lead to a 2 to 10-fold dose-response increase in the SkBF, after topical application on the ventral forearm of volunteers. These results show that PVA-SNO is a potential platform for topical NO delivery in biomedical applications.

Macromolecular prodrugs of ribavirin: Polymer backbone defines blood safety, drug release, and efficacy of anti-inflammatory effects.

Macromolecular (pro)drugs hold much promise as broad-spectrum antiviral agents as either microbicides or carriers for intracellular delivery of antiviral drugs. Intriguing opportunity exists in combining the two modes of antiviral activity in the same polymer structure such that the same polymer acts as a microbicide and also serves to deliver the conjugated drug (ribavirin) into the cells. We explore this opportunity in detail and focus on the polymer backbone as a decisive constituent of such formulations. Fourteen polyanions (polycarboxylates, polyphosphates and polyphosphonates, and polysulfonates) were analyzed for blood pro/anti coagulation effects, albumin binding and albumin aggregation, inhibitory activity on polymerases, cytotoxicity, and anti-inflammatory activity in stimulated macrophages. Ribavirin containing monomers were designed to accommodate the synthesis of macromolecular prodrugs with disulfide-exchange triggered drug release. Kinetics of drug release was fast in all cases however enhanced hydrophobicity of the polymer significantly slowed release of ribavirin. Results of this study present a comprehensive view on polyanions as backbone for macromolecular prodrugs of ribavirin as broad-spectrum antiviral agents.

Macromolecular Antiviral Agents against Zika, Ebola, SARS, and Other Pathogenic Viruses.

Viral pathogens continue to constitute a heavy burden on healthcare and socioeconomic systems. Efforts to create antiviral drugs repeatedly lag behind the advent of pathogens and growing understanding is that broad-spectrum antiviral agents will make strongest impact in future antiviral efforts. This work performs selection of synthetic polymers as novel broadly active agents and demonstrates activity of these polymers against Zika, Ebola, Lassa, Lyssa, Rabies, Marburg, Ebola, influenza, herpes simplex, and human immunodeficiency viruses. Results presented herein offer structure-activity relationships for these pathogens in terms of their susceptibility to inhibition by polymers, and for polymers in terms of their anionic charge and hydrophobicity that make up broad-spectrum antiviral agents. The identified leads cannot be predicted based on prior data on polymer-based antivirals and represent promising candidates for further development as preventive microbicides.

Synthetic Polymer with a Structure-Driven Hepatic Deposition and Curative Pharmacological Activity in Hepatic Cells.

Synthetic polymers make strong contributions as tools for delivery of biological drugs and chemotherapeutics. The most praised characteristic of polymers in these applications is complete lack of pharmacological function such as to minimize the side effects within the human body. In contrast, synthetic polymers with curative pharmacological activity are truly rare. Moreover, such activity is typically nonspecific rather than structure-defined. In this work, we present the discovery of poly(ethylacrylic acid) (PEAA) as a polymer with a suit of structure-defined, unexpected, pharmacological, and pharmacokinetic properties not observed in close structural analogues. Specifically, PEAA reveals capacity to bind to albumin with ensuing natural hepatic deposition in vivo and exhibits concurrent inhibitory activity against the hepatitis C virus and inflammation in hepatic cells. Our findings provide a view on synthetic polymers as curative, functional agents and present PEAA as a unique biomedical tool with applications related to health of the human liver.