Suppression of myeloid YAP antagonizes adverse cardiac remodeling during pressure overload stress.

Inflammation is an integral component of cardiovascular disease and is thought to contribute to cardiac dysfunction and heart failure. While ischemia-induced inflammation has been extensively studied in the heart, relatively less is known regarding cardiac inflammation during non-ischemic stress. Recent work has implicated a role for Yes-associated protein (YAP) in modulating inflammation in response to ischemic injury; however, whether YAP influences inflammation in the heart during non-ischemic stress is not described. We hypothesized that YAP mediates a pro-inflammatory response during pressure overload (PO)-induced non-ischemic injury, and that targeted YAP inhibition in the myeloid compartment is cardioprotective. In mice, PO elicited myeloid YAP activation, and myeloid-specific YAP knockout mice (YAPF/F;LysMCre) subjected to PO stress had better systolic function, and attenuated pathological remodeling compared to control mice. Inflammatory indicators were also significantly attenuated, while pro-resolving genes including Vegfa were enhanced, in the myocardium, and in isolated macrophages, of myeloid YAP KO mice after PO. Experiments using bone marrow-derived macrophages (BMDMs) from YAP KO and control mice demonstrated that YAP suppression shifted polarization toward a resolving phenotype. We also observed attenuated NLRP3 inflammasome priming and function in YAP deficient BMDMs, as well as in myeloid YAP KO hearts following PO, indicating disruption of inflammasome induction. Finally, we leveraged nanoparticle-mediated delivery of the YAP inhibitor verteporfin and observed attenuated PO-induced pathological remodeling compared to DMSO nanoparticle control treatment. These data implicate myeloid YAP as an important molecular nodal point that facilitates cardiac inflammation and fibrosis during PO stress and suggest that selective inhibition of YAP may prove a novel therapeutic target in non-ischemic heart disease.

Nanoparticle biointerfacing by platelet membrane cloaking.

Development of functional nanoparticles can be encumbered by unanticipated material properties and biological events, which can affect nanoparticle effectiveness in complex, physiologically relevant systems. Despite the advances in bottom-up nanoengineering and surface chemistry, reductionist functionalization approaches remain inadequate in replicating the complex interfaces present in nature and cannot avoid exposure of foreign materials. Here we report on the preparation of polymeric nanoparticles enclosed in the plasma membrane of human platelets, which are a unique population of cellular fragments that adhere to a variety of disease-relevant substrates. The resulting nanoparticles possess a right-side-out unilamellar membrane coating functionalized with immunomodulatory and adhesion antigens associated with platelets. Compared to uncoated particles, the platelet membrane-cloaked nanoparticles have reduced cellular uptake by macrophage-like cells and lack particle-induced complement activation in autologous human plasma. The cloaked nanoparticles also display platelet-mimicking properties such as selective adhesion to damaged human and rodent vasculatures as well as enhanced binding to platelet-adhering pathogens. In an experimental rat model of coronary restenosis and a mouse model of systemic bacterial infection, docetaxel and vancomycin, respectively, show enhanced therapeutic efficacy when delivered by the platelet-mimetic nanoparticles. The multifaceted biointerfacing enabled by the platelet membrane cloaking method provides a new approach in developing functional nanoparticles for disease-targeted delivery.

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.

Biomimetic Targeting of Nanoparticles to Immune Cell Subsets via Cognate Antigen Interactions.

Within the body, cellular recognition is mediated in large part by receptor-ligand interactions that result from the surface marker expression of the participant cells. In the case of immune cells, these interactions can be highly specific, enabling them to carry out their protective functions in fighting off infection and malignancy. In this work, we demonstrate the biomimetic targeting of antigen-specific immune cell populations by using nanoparticles functionalized with natural membrane derived from cells expressing the cognate antigen. Using red blood cell (RBC)-specific B cells as a model target, it is shown that RBC membrane-coated nanoparticles exhibit enhanced affinity compared with control nanoparticles. The concept is further demonstrated using murine models of alloimmunity and autoimmunity, where B cells elicited against RBCs can be positively labeled using the biomimetic nanoparticles. This strategy for antigen-specific immune cell targeting may have utility for the detection and treatment of various autoimmune conditions, and it may additionally have implications for the prevention of immune cell malignancies.

Multi-antigen avian influenza a (H7N9) virus-like particles: particulate characterizations and immunogenicity evaluation in murine and avian models.

BACKGROUND: Human infection with avian influenza A virus (H7N9) was first reported in China in March 2013. Since then, hundreds of cases have been confirmed showing severe symptoms with a high mortality rate. The virus was transmitted from avian species to humans and has spread to many neighboring areas, raising serious concerns over its pandemic potential. Towards containing the disease, the goal of this study is to prepare a virus-like particle (VLP) that consists of hemagglutinin (HA), neuraminidase (NA) and matrix protein 1 (M1) derived from the human isolate A/Taiwan/S02076/2013(H7N9) for potential vaccine development. RESULTS: Full length HA, NA, and M1 protein genes were cloned and expressed using a baculoviral expression system, and the VLPs were generated by co-infecting insect cells with three respective recombinant baculoviruses. Nanoparticle tracking analysis and transmission electron microscopy were applied to verify the VLPs' structure and antigenicity, and the multiplicity of infection of the recombinant baculoviruses was adjusted to achieve the highest hemagglutination activity. In animal experiments, BALB/c mice and specific-pathogen-free chickens receiving the VLP immunization showed elevated hemagglutination inhibition serum titer and antibodies against NA and M1 proteins. In addition, examination of cellular immunity showed the VLP-immunized mice and chickens exhibited an increased splenic antigen-specific cytokines production. CONCLUSIONS: The H7N9 VLPs possess desirable immunogenicity in vivo and may serve as a candidate for vaccine development against avian influenza A (H7N9) infection.

Clearance of pathological antibodies using biomimetic nanoparticles.

Pathological antibodies have been demonstrated to play a key role in type II immune hypersensitivity reactions, resulting in the destruction of healthy tissues and leading to considerable morbidity for the patient. Unfortunately, current treatments present significant iatrogenic risk while still falling short for many patients in achieving clinical remission. In the present work, we explored the capability of target cell membrane-coated nanoparticles to abrogate the effect of pathological antibodies in an effort to minimize disease burden, without the need for drug-based immune suppression. Inspired by antibody-driven pathology, we used intact RBC membranes stabilized by biodegradable polymeric nanoparticle cores to serve as an alternative target for pathological antibodies in an antibody-induced anemia disease model. Through both in vitro and in vivo studies, we demonstrated efficacy of RBC membrane-cloaked nanoparticles to bind and neutralize anti-RBC polyclonal IgG effectively, and thus preserve circulating RBCs.

Nanoparticle-Based Antivirulence Vaccine for the Management of Methicillin-Resistant Staphylococcus aureus Skin Infection.

With the rising threat of antibiotic-resistant bacteria, vaccination is becoming an increasingly important strategy to prevent and manage bacterial infections. Made from deactivated bacterial toxins, toxoid vaccines are widely used in the clinic as they help to combat the virulence mechanisms employed by different pathogens. Herein, the efficacy of a biomimetic nanoparticle-based anti-virulence vaccine is examined in a mouse model of methicillin-resistant Staphylococcus aureus (MRSA) skin infection. Vaccination with nanoparticle-detained staphylococcal α-hemolysin (Hla) effectively triggers the formation of germinal centers and induces high anti-Hla titers. Compared to mice vaccinated with control samples, those vaccinated with the nanoparticle toxoid show superior protective immunity against MRSA skin infection. The vaccination not only inhibits lesion formation at the site of bacterial challenge, but also reduces the invasiveness of MRSA, preventing dissemination into other organs. Overall, this biomimetic nanoparticle-based toxin detainment strategy is a promising method for the design of potent anti-virulence vaccines for managing bacterial infections.

Modulating antibacterial immunity via bacterial membrane-coated nanoparticles.

Synthetic nanoparticles coated with cellular membranes have been increasingly explored to harness natural cell functions toward the development of novel therapeutic strategies. Herein, we report on a unique bacterial membrane-coated nanoparticle system as a new and exciting antibacterial vaccine. Using Escherichia coli as a model pathogen, we collect bacterial outer membrane vesicles (OMVs) and successfully coat them onto small gold nanoparticles (AuNPs) with a diameter of 30 nm. The resulting bacterial membrane-coated AuNPs (BM-AuNPs) show markedly enhanced stability in biological buffer solutions. When injected subcutaneously, the BM-AuNPs induce rapid activation and maturation of dendritic cells in the lymph nodes of the vaccinated mice. In addition, vaccination with BM-AuNPs generates antibody responses that are durable and of higher avidity than those elicited by OMVs only. The BM-AuNPs also induce an elevated production of interferon gamma (INFγ) and interleukin-17 (IL-17), but not interleukin-4 (IL-4), indicating its capability of generating strong Th1 and Th17 biased cell responses against the source bacteria. These observed results demonstrate that using natural bacterial membranes to coat synthetic nanoparticles holds great promise for designing effective antibacterial vaccines.

Cancer cell membrane-coated nanoparticles for anticancer vaccination and drug delivery.

Cell-derived nanoparticles have been garnering increased attention due to their ability to mimic many of the natural properties displayed by their source cells. This top-down engineering approach can be applied toward the development of novel therapeutic strategies owing to the unique interactions enabled through the retention of complex antigenic information. Herein, we report on the biological functionalization of polymeric nanoparticles with a layer of membrane coating derived from cancer cells. The resulting core-shell nanostructures, which carry the full array of cancer cell membrane antigens, offer a robust platform with applicability toward multiple modes of anticancer therapy. We demonstrate that by coupling the particles with an immunological adjuvant, the resulting formulation can be used to promote a tumor-specific immune response for use in vaccine applications. Moreover, we show that by taking advantage of the inherent homotypic binding phenomenon frequently observed among tumor cells the membrane functionalization allows for a unique cancer targeting strategy that can be utilized for drug delivery applications.

Erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform.

Efforts to extend nanoparticle residence time in vivo have inspired many strategies in particle surface modifications to bypass macrophage uptake and systemic clearance. Here we report a top-down biomimetic approach in particle functionalization by coating biodegradable polymeric nanoparticles with natural erythrocyte membranes, including both membrane lipids and associated membrane proteins for long-circulating cargo delivery. The structure, size and surface zeta potential, and protein contents of the erythrocyte membrane-coated nanoparticles were verified using transmission electron microscopy, dynamic light scattering, and gel electrophoresis, respectively. Mice injections with fluorophore-loaded nanoparticles revealed superior circulation half-life by the erythrocyte-mimicking nanoparticles as compared to control particles coated with the state-of-the-art synthetic stealth materials. Biodistribution study revealed significant particle retention in the blood 72 h following the particle injection. The translocation of natural cellular membranes, their associated proteins, and the corresponding functionalities to the surface of synthetic particles represents a unique approach in nanoparticle functionalization.

Cell-penetrating peptides for transmucosal delivery of proteins.

Enabling non-invasive delivery of proteins across the mucosal barriers promises improved patient compliance and therapeutic efficacies. Cell-penetrating peptides (CPPs) are emerging as a promising and versatile tool to enhance protein and peptide permeation across various mucosal barriers. This review examines the structural and physicochemical attributes of the nasal, buccal, sublingual, and oral mucosa that hamper macromolecular delivery. Recent development of CPPs for overcoming those mucosal barriers for protein delivery is summarized and analyzed. Perspectives regarding current challenges and future research directions towards improving non-invasive transmucosal delivery of macromolecules for ultimate clinical translation are discussed.

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.

Overcoming the nutritional immunity by engineering iron-scavenging bacteria for cancer therapy.

Certain bacteria demonstrate the ability to target and colonize the tumor microenvironment, a characteristic that positions them as innovative carriers for delivering various therapeutic agents in cancer therapy. Nevertheless, our understanding of how bacteria adapt their physiological condition to the tumor microenvironment remains elusive. In this work, we employed liquid chromatography-tandem mass spectrometry to examine the proteome of E. coli colonized in murine tumors. Compared to E. coli cultivated in the rich medium, we found that E. coli colonized in tumors notably upregulated the processes related to ferric ions, including the enterobactin biosynthesis and iron homeostasis. This finding indicated that the tumor is an iron-deficient environment to E. coli. We also found that the colonization of E. coli in the tumor led to an increased expression of lipocalin 2 (LCN2), a host protein that can sequester the enterobactin. We therefore engineered E. coli in order to evade the nutritional immunity provided by LCN2. By introducing the IroA cluster, the E. coli synthesizes the glycosylated enterobactin, which creates steric hindrance to avoid the LCN2 sequestration. The IroA-E. coli showed enhanced resistance to LCN2 and significantly improved the anti-tumor activity in mice. Moreover, the mice cured by the IroA-E. coli treatment became resistant to the tumor re-challenge, indicating the establishment of immunological memory. Overall, our study underscores the crucial role of bacteria's ability to acquire ferric ions within the tumor microenvironment for effective cancer therapy.

Bacteria colonization in tumor microenvironment creates a favorable niche for immunogenic chemotherapy.

The tumor microenvironment (TME) presents differential selective pressure (DSP) that favors the growth of cancer cells, and monovalent therapy is often inadequate in reversing the cancer cell dominance in the TME. In this work, we introduce bacteria as a foreign species to the TME and explore combinatorial treatment strategies to alter DSP for tumor eradication. We show that cancer-selective chemotherapeutic agents and fasting can provide a strong selection pressure against tumor growth in the presence of bacteria. Moreover, we show that an immunogenic drug (oxaliplatin), but not a non-immunogenic one (5-FU), synergizes with the bacteria to activate both the innate and adaptive immunity in the TME, resulting in complete tumor remission and a sustained anti-tumor immunological memory in mice. The combination of oxaliplatin and bacteria greatly enhances the co-stimulatory and antigen-presenting molecules on antigen-presenting cells, which in turn bridge the cytotoxic T cells for cancer-cell killing. Our findings indicate that rational combination of bacterial therapy and immunogenic chemotherapy can promote anticancer immunity against the immunosuppressive TME.

High-Affinity Superantigen-Based Trifunctional Immune Cell Engager Synergizes NK and T Cell Activation for Tumor Suppression.

The development of immune cell engagers (ICEs) can be limited by logistical and functional restrictions associated with fusion protein designs, thus limiting immune cell recruitment to solid tumors. Herein, a high affinity superantigen-based multivalent ICE is developed for simultaneous activation and recruitment of NK and T cells for tumor treatment. Yeast library-based directed evolution is adopted to identify superantigen variants possessing enhanced binding affinity to immunoreceptors expressed on human T cells and NK cells. High-affinity superantigens exhibiting improved immune-stimulatory activities are then incorporated into a superantigen-based tri-functional yeast-display-enhanced multivalent immune cell engager (STYMIE), which is functionalized with a nanobody, a Neo-2/15 cytokine, and an Fc domain for tumor targeting, immune stimulation, and prolonged circulation, respectively. Intravenous administration of STYMIE enhances NK and T cell recruitment into solid tumors, leading to enhanced inhibition in multiple tumor models. The study offers design principles for multifunctional ICEs.

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.

Engineering Cyborg Bacteria Through Intracellular Hydrogelation.

Natural and artificial cells are two common chassis in synthetic biology. Natural cells can perform complex tasks through synthetic genetic constructs, but their autonomous replication often causes safety concerns for biomedical applications. In contrast, artificial cells based on nonreplicating materials, albeit possessing reduced biochemical complexity, provide more defined and controllable functions. Here, for the first time, the authors create hybrid material-cell entities termed Cyborg Cells. To create Cyborg Cells, a synthetic polymer network is assembled inside each bacterium, rendering them incapable of dividing. Cyborg Cells preserve essential functions, including cellular metabolism, motility, protein synthesis, and compatibility with genetic circuits. Cyborg Cells also acquire new abilities to resist stressors that otherwise kill natural cells. Finally, the authors demonstrate the therapeutic potential by showing invasion into cancer cells. This work establishes a new paradigm in cellular bioengineering by exploiting a combination of intracellular man-made polymers and their interaction with the protein networks of living cells.

Lymph Node Follicle-Targeting STING Agonist Nanoshells Enable Single-Shot M2e Vaccination for Broad and Durable Influenza Protection.

The highly conserved matrix protein 2 ectodomain (M2e) of influenza viruses presents a compelling vaccine antigen candidate for stemming the pandemic threat of the mutation-prone pathogen, yet the low immunogenicity of the diminutive M2e peptide renders vaccine development challenging. A highly potent M2e nanoshell vaccine that confers broad and durable influenza protectivity under a single vaccination is shown. Prepared via asymmetric ionic stabilization for nanoscopic curvature formation, polymeric nanoshells co-encapsulating high densities of M2e peptides and stimulator of interferon genes (STING) agonists are prepared. Robust and long-lasting protectivity against heterotypic influenza viruses is achieved with a single administration of the M2e nanoshells in mice. Mechanistically, molecular adjuvancy by the STING agonist and nanoshell-mediated prolongation of M2e antigen exposure in the lymph node follicles synergistically contribute to the heightened anti-M2e humoral responses. STING agonist-triggered T cell helper functions and extended residence of M2e peptides in the follicular dendritic cell network provide a favorable microenvironment that induces Th1-biased antibody production against the diminutive antigen. These findings highlight a versatile nanoparticulate design that leverages innate immune pathways for enhancing the immunogenicity of weak immunogens. The single-shot nanovaccine further provides a translationally viable platform for pandemic preparedness.