Angiopoietin-1 Mimetic Nanoparticles for Restoring the Function of Endothelial Cells as Potential Therapeutic for Glaucoma

A root cause for the development and progression of primary open-angle glaucoma might be the loss of the Schlemm’s canal (SC) cell function due to an impaired Angiopoietin-1 (Angpt-1)/Tie2 signaling. Current therapeutic options fail to restore the SC cell function. We propose Angpt-1 mimetic nanoparticles (NPs) that are intended to bind in a multivalent manner to the Tie2 receptor for successful receptor activation. To this end, an Angpt-1 mimetic peptide was coupled to a poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) block co-polymer. The modified polymer allowed for the fabrication of Angpt-1 mimetic NPs with a narrow size distribution (polydispersity index < 0.2) and the size of the NPs ranging from about 120 nm (100% ligand density) to about 100 nm (5% ligand density). NP interaction with endothelial cells (HUVECs, EA.hy926) as surrogate for SC cells and fibroblasts as control was investigated by flow cytometry and confocal microscopy. The NP–cell interaction strongly depended on the ligand density and size of NPs. The cellular response to the NPs was investigated by a Ca2+ mobilization assay as well as by a real-time RT-PCR and Western blot analysis of endothelial nitric oxide synthase (eNOS). NPs with a ligand density of 25% opposed VEGF-induced Ca2+ influx in HUVECs significantly which could possibly increase cell relaxation and thus aqueous humor drainage, whereas the expression and synthesis of eNOS was not significantly altered. Therefore, we suggest Angpt-1 mimetic NPs as a first step towards a causative therapy to recover the loss of SC cell function during glaucoma.

Role of Ligand Distribution in the Cytoskeleton-Associated Endocytosis of Ellipsoidal Nanoparticles

Nanoparticle (NP)–cell interaction mediated by receptor–ligand bonds is a crucial phenomenon in pathology, cellular immunity, and drug delivery systems, and relies strongly on the shape of NPs and the stiffness of the cell. Given this significance, a fundamental question is raised on how the ligand distribution may affect the membrane wrapping of non-spherical NPs under the influence of cytoskeleton deformation. To address this issue, in this work we use a coupled elasticity–diffusion model to systematically investigate the role of ligand distribution in the cytoskeleton-associated endocytosis of ellipsoidal NPs for different NP shapes, sizes, cytoskeleton stiffness, and the initial receptor densities. In this model, we have taken into account the effects of receptor diffusion, receptor–ligand binding, cytoskeleton and membrane deformations, and changes in the configuration entropy of receptors. By solving this model, we find that the uptake process can be significantly influenced by the ligand distribution. Additionally, there exists an optimal state of such a distribution, which corresponds to the fastest uptake efficiency and depends on the NP aspect ratio and cytoskeleton stiffness. We also find that the optimal distribution usually needs local ligand density to be sufficiently high at the large curvature region. Furthermore, the optimal state of NP entry into cells can tolerate slight changes to the corresponding optimal distribution of the ligands. The tolerance to such a change is enhanced as the average receptor density and NP size increase. These results may provide guidelines to control NP–cell interactions and improve the efficiency of target drug delivery systems.

Polymeric Nanoparticles Properties and Brain Delivery

Safe and reliable entry to the brain is essential for successful diagnosis and treatment of diseases, but it still poses major challenges. As a result, many therapeutic approaches to treating disorders associated with the central nervous system (CNS) still only show limited success. Nano-sized systems are being explored as drug carriers and show great improvements in the delivery of many therapeutics. The systemic delivery of nanoparticles (NPs) or nanocarriers (NCs) to the brain involves reaching the neurovascular unit (NVU), being transported across the blood–brain barrier, (BBB) and accumulating in the brain. Each of these steps can benefit from specifically controlled properties of NPs. Here, we discuss how brain delivery by NPs can benefit from careful design of the NP properties. Properties such as size, charge, shape, and ligand functionalization are commonly addressed in the literature; however, properties such as ligand density, linker length, avidity, protein corona, and stiffness are insufficiently discussed. This is unfortunate since they present great value against multiple barriers encountered by the NPs before reaching the brain, particularly the BBB. We further highlight important examples utilizing targeting ligands and how functionalization parameters, e.g., ligand density and ligand properties, can affect the success of the nano-based delivery system.

DNA origami patterning of synthetic T cell receptors reveals spatial control of the sensitivity and kinetics of signal activation

Receptor clustering plays a key role in triggering cellular activation, but the relationship between the spatial configuration of clusters and the elicitation of downstream intracellular signals remains poorly understood. We developed a DNA-origami–based system that is easily adaptable to other cellular systems and enables rich interrogation of responses to a variety of spatially defined inputs. Using a chimeric antigen receptor (CAR) T cell model system with relevance to cancer therapy, we studied signaling dynamics at single-cell resolution. We found that the spatial arrangement of receptors determines the ligand density threshold for triggering and encodes the temporal kinetics of signaling activities. We also showed that signaling sensitivity of a small cluster of high-affinity ligands is enhanced when surrounded by nonstimulating low-affinity ligands. Our results suggest that cells measure spatial arrangements of ligands, translate that information into distinct signaling dynamics, and provide insights into engineering immunotherapies.

Control Cell Migration by Engineering Integrin Ligand Assembly

Advances in mechanistic understanding of integrin-mediated adhesion highlight the importance of precise control of ligand presentation in directing cell migration. Top-down nanopatterning limited the spatial presentation to sub-micron. To enhance it to molecular level, we propose a bottom-up nanofabrication strategy. Via self-assembly and co-assembly, precise control of ligand presentation is succeeded by varying the proportions of assembling ligand and nonfunctional peptide. Assembled nanofilaments fulfill multi-functions exerting enhancement to suppression effect on cell migration with tunable amplitudes. Self-assembled nanofilaments possessing super high ligand density selectively suppress cancer cell migration by preventing integrin/actin disassembly at cell rear, which provides new insights to ligand-density-dependent-modulation, revealing valuable inputs to therapeutic innovations in tumor metastasis.One-Sentence SummaryEngineering integrin ligand assembly from bottom-up offers a generalized tool to selectively control cell migration with tunable amplitudes.

LFA-1 and Kindlin-3 enable the collaborative transport of SLP-76 microclusters by myosin and dynein motors

Integrin engagement within the immune synapse enhances T cell activation, but our understanding of this process is incomplete. In response to T cell receptor (TCR) ligation, SLP-76 (LCP2), ADAP (FYB), and SKAP-55 (SKAP1) are recruited into microclusters and activate integrins via the effectors Talin-1 and Kindlin-3. We postulated that integrins influence the centripetal transport and signaling of SLP-76 microclusters via these linkages. We show that contractile myosin filaments surround and are co-transported with SLP-76 microclusters, and that TCR ligand density governs the centripetal movement of both structures. Centripetal transport requires formin activity, actomyosin contraction, microtubule integrity, and dynein motor function. Although immobilized VLA-4 (a4b1) and LFA-1 (aLb2) ligands arrest the centripetal movement of SLP-76 microclusters and myosin filaments, VLA-4 acts distally, while LFA-1 acts in the lamellum. Integrin b2, Kindlin-3, and Zyxin are required for complete centripetal transport, while integrin b1 and Talin-1 are not. CD69 upregulation is similarly dependent on integrin b2, Kindlin-3, and Zyxin, but not Talin-1. These findings highlight the integration of cytoskeletal systems within the immune synapse and reveal extracellular ligand-independent roles for LFA-1 and Kindlin-3.

Dietary Fiber-Tethered Gold Nanoparticles: An Innovative Analytical Tool for Probing Interactions

Epidemiological studies have recognized that daily consumption of dietary fiber-containing foods reduces the incidence of developing many chronic diseases, for example, by interacting with nutritionally relevant compounds. The low affinity nature that some of these interactions can have make the development of an analytical detection system for their study particularly difficult. Therefore, the mechanism of action of binding compounds, by which a dietary fiber exerts its potential health benefits, remains largely unknown. Here, a novel method based on glyco-nanotechnology is proposed for studying the interaction between galactomannan and target molecules. Starting from a bottom-up approach, gold nanoparticles and thiolated galactomannans of two different sizes were synthesized separately, and then mixed for auto-assembly of the two glyconanoparticle materials. In addition, a preliminary interaction study between the prepared glyconanoparticles and Concanavalin A was carried out using transmission electron microscopy (TEM) from which it could be deduced that the molecular weight and ligand density on the gold core play an important role in the interaction. Therefore, dietary fiber-tethered gold nanoparticles are a valuable tool to elucidate key parameters underlying dietary fiber interactions.