Synthetic Immunogenic Cell Death Mediated by Intracellular Delivery of STING Agonist Nanoshells Enhances Anticancer Chemo-immunotherapy.

Many favorable anticancer treatments owe their success to the induction immunogenic cell death (ICD) in cancer cells, which results in the release of endogenous danger signals along with tumor antigens for effective priming of anticancer immunity. We describe a strategy to artificially induce ICD by delivering the agonist of stimulator of interferon genes (STING) into tumor cells using hollow polymeric nanoshells. Following intracellular delivery of exogenous adjuvant, subsequent cytotoxic treatment creates immunogenic cellular debris that spatiotemporally coordinate tumor antigens and STING agonist in a process herein termed synthetic immunogenic cell death (sICD). sICD is indiscriminate to the type of chemotherapeutics and enables colocalization of exogenously administered immunologic adjuvants and tumor antigens for enhanced antigen presentation and anticancer adaptive response. In three mouse tumor models, sICD enhances therapeutic efficacy and restrains tumor progression. The study highlights the benefit of delivering STING agonists to cancer cells, paving ways to new chemo-immunotherapeutic designs.

Viromimetic STING Agonist-Loaded Hollow Polymeric Nanoparticles for Safe and Effective Vaccination against Middle East Respiratory Syndrome Coronavirus.

The continued threat of emerging, highly lethal infectious pathogens such as Middle East respiratory syndrome coronavirus (MERS-CoV) calls for the development of novel vaccine technology that offers safe and effective prophylactic measures. Here, a novel nanoparticle vaccine is developed to deliver subunit viral antigens and STING agonists in a virus-like fashion. STING agonists are first encapsulated into capsid-like hollow polymeric nanoparticles, which show multiple favorable attributes, including a pH-responsive release profile, prominent local immune activation, and reduced systemic reactogenicity. Upon subsequent antigen conjugation, the nanoparticles carry morphological semblance to native virions and facilitate codelivery of antigens and STING agonists to draining lymph nodes and immune cells for immune potentiation. Nanoparticle vaccine effectiveness is supported by the elicitation of potent neutralization antibody and antigen-specific T cell responses in mice immunized with a MERS-CoV nanoparticle vaccine candidate. Using a MERS-CoV-permissive transgenic mouse model, it is shown that mice immunized with this nanoparticle-based MERS-CoV vaccine are protected against a lethal challenge of MERS-CoV without triggering undesirable eosinophilic immunopathology. Together, the biocompatible hollow nanoparticle described herein provides an excellent strategy for delivering both subunit vaccine candidates and novel adjuvants, enabling accelerated development of effective and safe vaccines against emerging viral pathogens.

Dengue viral protease interaction with NF-κB inhibitor α/ß results in endothelial cell apoptosis and hemorrhage development.

Hemorrhagic manifestations occur frequently accompanying a wide range of dengue disease syndromes. Much work has focused on the contribution of immune factors to the pathogenesis of hemorrhage, but how dengue virus (DENV) participates in the pathogenic process has never been explored. Although there is no consensus that apoptosis is the basis of vascular permeability in human dengue infections, we showed in dengue hemorrhage mouse model that endothelial cell apoptosis is important to hemorrhage development in mice. To explore the molecular basis of the contribution of DENV to endothelial cell death, we show in this study that DENV protease interacts with cellular IκBα and IκBß and cleaves them. By inducing IκBα and IκBß cleavage and IκB kinase activation, DENV protease activates NF-κB, which results in endothelial cell death. Intradermal inoculation of DENV protease packaged in adenovirus-associated virus-9 induces endothelial cell death and dermal hemorrhage in mice. Although the H51 activity site is not involved in the interaction between DENV protease and IκB-α/ß, the enzymatic activity is critical to the ability of DENV protease to induce IκBα and IκBß cleavage and trigger hemorrhage development. Moreover, overexpression of IκBα or IκBß protects endothelial cells from DENV-induced apoptosis. In this study, we show that DENV protease participates in the pathogenesis of dengue hemorrhage and discover IκBα and IκBß to be the new cellular targets that are cleaved by DENV protease.

Synthetic control of living cells by intracellular polymerization.

An emerging cellular engineering method creates synthetic polymer matrices inside cells. By contrast with classical genetic, enzymatic, or radioactive techniques, this materials-based approach introduces non-natural polymers inside cells, thus modifying cellular states and functionalities. Here, we cover various materials and chemistries that have been exploited to create intracellular polymer matrices. In addition, we discuss emergent cellular properties due to the intracellular polymerization, including nonreplicating but active metabolism, maintenance of membrane integrity, and resistance to environmental stressors. We also discuss past work and future opportunities for developing and applying synthetic cells that contain intracellular polymers. The materials-based approach will usher in new applications of synthetic cells for broad biotechnological applications.

Rapid plasma membrane isolation via intracellular polymerization-mediated biomolecular confinement.

Plasma membrane isolation is a foundational process in membrane proteomic research, cellular vesicle studies, and biomimetic nanocarrier development, yet separation processes for this outermost layer are cumbersome and susceptible to impurities and low yield. Herein, we demonstrate that cellular cytosol can be chemically polymerized for decoupling and isolation of plasma membrane within minutes. A rapid, non-disruptive in situ polymerization technique is developed with cell membrane-permeable polyethyleneglycol-diacrylate (PEG-DA) and a blue-light-sensitive photoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). The photopolymerization chemistry allows for precise control of intracellular polymerization and tunable confinement of cytosolic molecules. Upon cytosol solidification, plasma membrane proteins and vesicles are rapidly derived and purified as nucleic acids and intracellular proteins as small as 15 kDa are stably entrapped for removal. The polymerization chemistry and membrane derivation technique are broadly applicable to primary and fragile cell types, enabling facile membrane vesicle extraction from shorted-lived neutrophils and human primary CD8 T cells. The study demonstrates tunable intracellular polymerization via optimized live cell chemistry, offers a robust membrane isolation methodology with broad biomedical utility, and reveals insights on molecular crowding and confinement in polymerized cells. STATEMENT OF SIGNIFICANCE: Isolating the minute fraction of plasma membrane proteins and vesicles requires extended density gradient ultracentrifugation processes, which are susceptible to low yield and impurities. The present work demonstrates that the membrane isolation process can be vastly accelerated via a rapid, non-disruptive intracellular polymerization approach that decouples cellular cytosols from the plasma membrane. Following intracellular polymerization, high-yield plasma membrane proteins and vesicles can be derived from lysis buffer and sonication treatment, respectively. And the intracellular content entrapped within the polymerized hydrogel is readily removed within minutes. The technique has broad utility in membrane proteomic research, cellular vesicle studies, and biomimetic materials development, and the work offers insights on intracellular hydrogel-mediated molecular confinement.

In Situ Hydrogelation of Cellular Monolayers Enables Conformal Biomembrane Functionalization for Xeno-Free Feeder Substrate Engineering.

The intricate functionalities of cellular membranes have inspired strategies for deriving and anchoring cell-surface components onto solid substrates for biological studies, biosensor applications, and tissue engineering. However, introducing conformal and right-side-out cell membrane coverage onto planar substrates requires cumbersome protocols susceptible to significant device-to-device variability. Here, a facile approach for biomembrane functionalization of planar substrates is demonstrated by subjecting confluent cellular monolayer to intracellular hydrogel polymerization. The resulting cell-gel hybrid, herein termed GELL (gelated cell), exhibits extraordinary stability and retains the structural integrity, membrane fluidity, membrane protein mobility, and topology of living cells. In assessing the utility of GELL layers as a tissue engineering feeder substrate for stem cell maintenance, GELL feeder prepared from primary mouse embryonic fibroblasts not only preserves the stemness of murine stem cells but also exhibits advantages over live feeder cells owing to the GELL's inanimate, non-metabolizing nature. The preparation of a xeno-free feeder substrate devoid of non-human components is further shown with HeLa cells, and the resulting  HeLa GELL feeder effectively sustains the growth and stemness of both murine and human induced pluripotent stem cells. The study highlights a novel bio-functionalization strategy that introduces new opportunities for tissue engineering and other biomedical applications.

Nanotechnology advances in pathogen- and host-targeted antiviral delivery: multipronged therapeutic intervention for pandemic control.

The COVID-19 pandemic's high mortality rate and severe socioeconomic impact serve as a reminder of the urgent need for effective countermeasures against viral pandemic threats. In particular, effective antiviral therapeutics capable of stopping infections in its tracks is critical to reducing infection fatality rate and healthcare burden. With the field of drug delivery witnessing tremendous advancement in the last two decades owing to a panoply of nanotechnology advances, the present review summarizes and expounds on the research and development of therapeutic nanoformulations against various infectious viral pathogens, including HIV, influenza, and coronaviruses. Specifically, nanotechnology advances towards improving pathogen- and host-targeted antiviral drug delivery are reviewed, and the prospect of achieving effective viral eradication, broad-spectrum antiviral effect, and resisting viral mutations are discussed. As several COVID-19 antiviral clinical trials are met with lackluster treatment efficacy, nanocarrier strategies aimed at improving drug pharmacokinetics, biodistributions, and synergism are expected to not only contribute to the current disease treatment efforts but also expand the antiviral arsenal against other emerging viral diseases.

Facile Transformation of Murine and Human Primary Dendritic Cells into Robust and Modular Artificial Antigen-Presenting Systems by Intracellular Hydrogelation.

The growing enthusiasm for cancer immunotherapies and adoptive cell therapies has prompted increasing interest in biomaterials development mimicking natural antigen-presenting cells (APCs) for T-cell expansion. In contrast to conventional bottom-up approaches aimed at layering synthetic substrates with T-cell activation cues, transformation of live dendritic cells (DCs) into artificial APCs (aAPCs) is demonstrated herein using a facile and minimally disruptive hydrogelation technique. Through direct intracellular permeation of poly(ethylene glycol) diacrylate (PEG-DA) hydrogel monomer and UV-activated radical polymerization, intracellular hydrogelation is rapidly accomplished on DCs with minimal influence on cellular morphology and surface antigen display, yielding highly robust and modular cell-gel hybrid constructs amenable to peptide antigen exchange, storable by freezing and lyophilization, and functionalizable with cytokine-releasing carriers for T-cell modulation. The DC-derived aAPCs are shown to induce prolonged T-cell expansion and improve anticancer efficacy of adoptive T-cell therapy in mice compared to nonexpanded control T cells, and the gelation technique is further demonstrated to stabilize primary DCs derived from human donors. The work presents a versatile approach for generating a new class of cell-mimicking biomaterials and opens new venues for immunological interrogation and immunoengineering.

Inhibitory effects of safrole on phagocytosis, intracellular reactive oxygen species, and the activity of myeloperoxidase released by human polymorphonuclear leukocytes.

BACKGROUND: Safrole, a component of Piper betle inflorescence, inhibits bactericidal activity and the release of superoxide anion (O(2)(-)) by polymorphonuclear leukocytes (PMNs). This in vitro study further investigated the effects of safrole on phagocytic activity, the intracellular production of reactive oxygen species (ROS), and the activity of the lysosomal enzyme myeloperoxidase (MPO), which is released by human PMNs. METHODS: The possible effects of safrole on the phagocytic activity of PMNs against Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) were determined using flow cytometry. PMNs were treated with various concentrations of safrole, which was followed by treatment with Hanks balanced salt solution with or without cytochalasin B and fMet-Leu-Phe (CB/fMLP). Intracellular ROS was determined using 2',7'-dichlorofluorescein diacetate and a fluorometer, whereas MPO activity was determined using a substrate assay. RESULTS: Safrole significantly inhibited the phagocytic activity of PMNs in a dose-dependent manner. Approximately 50% of the phagocytic activity of PMNs was affected when 10 mM safrole was used. Exposure of the PMNs to safrole (up to 5 mM) did not directly affect the intracellular levels of ROS and the extracellular activity of MPO. However, the ability of CB/fMLP to trigger production of intracellular ROS and the activity of MPO released by human PMNs was significantly suppressed by safrole. CONCLUSIONS: Safrole reduced the uptake of A. actinomycetemcomitans by human PMNs. Safrole also impaired the normal activation activity of PMNs. Alterations in the defensive properties of PMNs by safrole might promote bacterial colonization, and this could result in periodontal infection.

Intracellular hydrogelation preserves fluid and functional cell membrane interfaces for biological interactions.

Cell membranes are an intricate yet fragile interface that requires substrate support for stabilization. Upon cell death, disassembly of the cytoskeletal network deprives plasma membranes of mechanical support and leads to membrane rupture and disintegration. By assembling a network of synthetic hydrogel polymers inside the intracellular compartment using photo-activated crosslinking chemistry, we show that the fluid cell membrane can be preserved, resulting in intracellularly gelated cells with robust stability. Upon assessing several types of adherent and suspension cells over a range of hydrogel crosslinking densities, we validate retention of surface properties, membrane lipid fluidity, lipid order, and protein mobility on the gelated cells. Preservation of cell surface functions is further demonstrated with gelated antigen presenting cells, which engage with antigen-specific T lymphocytes and effectively promote cell expansion ex vivo and in vivo. The intracellular hydrogelation technique presents a versatile cell fixation approach adaptable for biomembrane studies and biomedical device construction.