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- ItemQuantifying ligand-cell interactions and determination of the surface concentrations of ligands on hydrogel films: The measurement challenge(Melville, NY : AIP Publishing, 2015) Beer, Meike V.; Hahn, Kathrin; Diederichs, Sylvia; Fabry, Marlies; Singh, Smriti; Spencer, Steve J.; Salber, Jochen; Möller, Martin; Shard, Alexander G.; Groll, JürgenHydrogels are extensively studied for biomaterials application as they provide water swollen noninteracting matrices in which specific binding motifs and enzyme-sensitive degradation sites can be incorporated to tailor cell adhesion, proliferation, and migration. Hydrogels also serve as excellent basis for surface modification of biomaterials where interfacial characteristics are decisive for implant success or failure. However, the three-dimensional nature of hydrogels makes it hard to distinguish between the bioactive ligand density at the hydrogel-cell interface that is able to interact with cells and the ligands that are immobilized inside the hydrogel and not accessible for cells. Here, the authors compare x-ray photoelectron spectrometry (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), enzyme linked immunosorbent assay (ELISA), and the correlation with quantitative cell adhesion using primary human dermal fibroblasts (HDF) to gain insight into ligand distribution. The authors show that although XPS provides the most useful quantitative analysis, it lacks the sensitivity to measure biologically meaningful concentrations of ligands. However, ToF-SIMS is able to access this range provided that there are clearly distinguishable secondary ions and a calibration method is found. Detection by ELISA appears to be sensitive to the ligand density on the surface that is necessary to mediate cell adhesion, but the upper limit of detection coincides closely with the minimal ligand spacing required to support cell proliferation. Radioactive measurements and ELISAs were performed on amine reactive well plates as true 2D surfaces to estimate the ligand density necessary to allow cell adhesion onto hydrogel films. Optimal ligand spacing for HDF adhesion and proliferation on ultrathin hydrogel films was determined as 6.5 ± 1.5 nm.
- ItemReconstruction of Ultra-thin Alveolar-capillary Basement Membrane Mimics(Weinheim : Wiley-VCH, 2021) Jain, Puja; Nishiguchi, Akihiro; Linz, Georg; Wessling, Matthias; Ludwig, Andreas; Rossaint, Rolf; Möller, Martin; Singh, SmritiAlveolar-capillary basement membrane (BM) is ultra-thin (<2 µm) extracellular matrix that maintains integral epithelial-endothelial cell layers. In vitro reconstructions of alveolar-capillary barrier supported on synthetic scaffolds closely resembling the fibrous and ultra-thin natural BM are essential in mimicking the lung pathophysiology. Although BM topology and dimensions are well known to significantly influence cellular behavior, conventionally used BM mimics fail to recreate this natural niche. To overcome this, electrospun ultra-thin 2 µm poly(caprolactone) (PCL) nanofibrous mesh is used to establish an alveolar-capillary barrier model of lung endothelial/epithelial cells. Transepithelial electrical resistance (TEER) and permeability studies reveal integral tight junctions and improved mass transport through the highly porous PCL meshes compared to conventional dense membranes with etched pores. The chemotaxis of neutrophils is shown across the barrier in presence of inflammatory response that is naturally impeded in confined regions. Conventional requirement of 3 µm or larger pore size can lead to barrier disruption due to epithelial/endothelial cell invasion. Despite high porosity, the interconnected BM mimic prevents barrier disruption and allows neutrophil transmigration, thereby demonstrating the physiological relevance of the thin nanofibrous meshes. It is envisioned that these bipolar cultured barriers would contribute to an organ-level in vitro model for pathological disease, environmental pollutants, and nanotoxicology. © 2021 The Authors. Advanced Biology published by Wiley-VCH GmbH
- Item3D-Printing of Structure-Controlled Antigen Nanoparticles for Vaccine Delivery(Columbus, Ohio : American Chemical Soc., 2020) Nishiguchi, Akihiro; Shima, Fumiaki; Singh, Smriti; Akashi, Mitsuru; Moeller, MartinTargeted delivery of antigens to immune cells using micro/nanocarriers may serve as a therapeutic application for vaccination. However, synthetic carriers have potential drawbacks including cytotoxicity, low encapsulation efficiency of antigen, and lack of a morphological design, which limit the translation of the delivery system to clinical use. Here, we report a carrier-free and three-dimensional (3D)-shape-designed antigen nanoparticle by multiphoton lithography-based 3D-printing. This simple, versatile 3D-printing approach provides freedom for the precise design of particle shapes with a nanoscale resolution. Importantly, shape-designed antigen nanoparticles with distinct aspect ratios show shape-dependent immune responses. The 3D-printing approach for the rational design of nanomaterials with increasing safety, complexity, and efficacy offers an emerging platform to develop vaccine delivery systems and mechanistic understanding.
- ItemBioactive Nanogels Mimicking the Antithrombogenic Nitric Oxide-Release Function of the Endothelium(Weinheim : Wiley-VCH, 2023) Hosseinnejad, Aisa; Ludwig, Nadine; Mersmann, Sina; Winnerbach, Patrick; Bleilevens, Christian; Rossaint, Rolf; Rossaint, Jan; Singh, SmritiNitric oxide (NO) plays a significant role in controlling the physiology and pathophysiology of the body, including the endothelial antiplatelet function and therefore, antithrombogenic property of the blood vessels. This property of NO can be exploited to prevent thrombus formation on artificial surfaces like extracorporeal membrane oxygenators, which when come into contact with blood lead to protein adsorption and thereby platelet activation causing thrombus formation. However, NO is extremely reactive and has a very short biological half-life in blood, so only endogenous generation of NO from the blood contacting material can result into a stable and kinetically controllable local delivery of NO. In this regards, highly hydrophilic bioactive nanogels are presented which can endogenously generate NO in blood plasma from endogenous NO-donors thereby maintaining a physiological NO flux. It is shown that NO releasing nanogels could initiate cGMP-dependent protein kinase signaling followed by phosphorylation of vasodilator-stimulated phosphoprotein in platelets. This prevents platelet activation and aggregation even in presence of highly potent platelet activators like thrombin, adenosine 5′-diphosphate, and U46619 (thromboxane A2 mimetic).