570 Biowissenschaften, Biologie
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Interference of silica nanoparticles with the traditional Limulus amebocyte lysate gel clot assay
(2014)
Endotoxin contaminations of engineered nanomaterials can be responsible for observed biological responses, especially for misleading results in in vitro test systems, as well as in vivo studies. Therefore, endotoxin testing of nanomaterials is necessary to benchmark their influence on cells. Here, we tested the traditional Limulus amebocyte lysate gel clot assay for the detection of endotoxins in nanoparticle suspensions with a focus on possible interference of the particles with the test system. We systematically investigated the effects of nanomaterials made of, or covered by, the same material. Different types of bare or PEGylated silica nanoparticles, as well as iron oxide-silica core shell nanoparticles, were tested. Detailed inhibition/enhancement controls revealed enhanced activity in the Limulus coagulation cascade for all particles with bare silica surface. In comparison, PEGylation led to a lower degree of enhancement. These results indicate that the protein-particle interactions are the basis for the observed inhibition and enhancement effects. The enhancement activity of a particle type was positively related to the calculated particle surface area. For most silica particles tested, a dilution of the sample within the maximum valid dilution was sufficient to overcome non-valid enhancement, enabling semi-quantification of the endotoxin contamination.
In engineered living materials (ELMs) non-living matrices encapsulate microorganisms to acquire capabilities like sensing or biosynthesis. The confinement of the organisms to the matrix and the prevention of overgrowth and escape during the lifetime of the material is necessary for the application of ELMs into real devices. In this study, a bilayer thin film hydrogel of Pluronic F127 and Pluronic F127 acrylate polymers supported on a solid substrate is introduced. The inner hydrogel layer contains genetically engineered bacteria and supports their growth, while the outer layer acts as an envelope and does not allow leakage of the living organisms outside of the film for at least 15 days. Due to the flat and transparent nature of the construct, the thin layer is suited for microscopy and spectroscopy-based analyses. The composition and properties of the inner and outer layer are adjusted independently to fulfil viability and confinement requirements. We demonstrate that bacterial growth and light-induced protein production are possible in the inner layer and their extent is influenced by the crosslinking degree of the used hydrogel. Bacteria inside the hydrogel are viable long term, they can act as lactate-sensors and remain active after storage in phosphate buffer at room temperature for at least 3 weeks. The versatility of bilayer bacteria thin-films is attractive for fundamental studies and for the development of application-oriented ELMs.
The fascinating properties of natural biominerals, such as stiffness combined with toughness, are difficult to mimick in synthetic pathways. Therefore, this work investigated the biosynthesis of biomineralization proteins in different organisms for producing synthetic minerals under the control of a protein phase. The soluble nacre protein perlucin from Haliotis was produced in bacteria but remained insoluble after native extraction. It was partially solubilized when fused to a GFP, which was additionally identified to be a strong inhibitor of calcium carbonate formation. Various forms of synthetic biominerals were obtained in simple calcium carbonate precipitation assays depending on the first ionic interaction partner, assay volume and protein concentrations. In comparison to bacteria as less complex organisms, the biomineralization proteins perlucin, N16N and ovocleidin-17 were also expressed in the plant cell wall of Arabidopsis and Nicotiana, which has not been described in the literature so far. This work demonstrated that N16N-GFP and ovocleidin-17-GFP can be expressed in epidermal leaf cells of Nicotiana. In perlucin-GFP transformed Arabidopsis, the plant morphology changed although neither the protein nor RNA were detected. The expression of foreign biomineralization proteins in biotechnologically relevant plants was demonstrated for the first time, opening routes toward new and improved materials.
The inhalation of nanoparticles can cause interactions with pulmonary structures. Human alveolar epithelial cells type II organize the alveolar epithelium and thus can be regarded as barrier against pulmonary nanoparticle uptake. Within the present work, interactions of differently sized gold nanoparticles with A549 cells, a model for type II human alveolar epithelial cells, were studied. The intracellular location of the fluorescently labeled gold particles was analyzed by STED (stimulated emission depletion) and electron microscopy. Gold nanoparticles were detected inside the cell nucleus and the Golgi complex. A nanoparticle accumulation was observed at the perinuclear region. The association of gold nanoparticles and cytoskeletal filaments indicated an active intracellular nanoparticle transport. A vesicle-based transport of gold nanoparticles was revealed by live cell imaging. Besides the intracellular particle location, an impact of gold nanoparticles on cell viability and cell proliferation was studied. Cell-based assays revealed a different cytotoxic potential of the gold nanoparticle sizes used. The last part of the work describes the development of an approach of correlative light and electron microscopy (CLEM) for studying nanoparticle-cell interactions. The used method allowed for a correlative imaging of the nanoparticle fluorescence in relation to the nanoparticle core.
Methylsulfone derivatized poly(ethylene) glycol (PEG) macromers can be biofunctionalized with thiolated ligands and cross-linked with thiol-based cross-linkers to obtain bioactive PEG hydrogels for in situ cell encapsulation. Methylsulfonyl-thiol (MS-SH) reactions present several advantages for this purpose when compared to other thiol-based cross-linking systems. They proceed with adequate and tunable kinetics for encapsulation, they reach a high conversion degree with good selectivity, and they generate stable reaction products. Our previous work demonstrated the cytocompatibility of cross-linked PEG-MS/thiol hydrogels in contact with fibroblasts. However, the cytocompatibility of the in situ MS-SH cross-linking reaction itself, which generates methylsulfinic acid as byproduct at the cross-linked site, remains to be evaluated. These studies are necessary to evaluate the potential of these systems for in vivo applications. Here we perform an extensive cytocompatibility study of PEG hydrogels during in situ cross-linking by the methylsulfonyl-thiol reaction. We compare these results with maleimide–thiol cross-linked PEGs which are well established for cell culture and in vivo experiments and do not involve the release of a byproduct. We show that fibroblasts and endothelial cells remain viable after in situ polymerization of methylsulfonyl-thiol gels on the top of the cell layers. Cell viability seems better than after in situ cross-linking hydrogels with maleimide–thiol chemistry. The endothelial cell proinflammatory phenotype is low and similar to the one obtained by the maleimide–thiol reaction. Finally, no activation of monocytes is observed. All in all, these results demonstrate that the methylsulfonyl-thiol chemistry is cytocompatible and does not trigger high pro-inflammatory responses in endothelial cells and monocytes. These results make methylsulfonyl-thiol chemistries eligible for in vivo testing and eventually clinical application in the future.
Each cell in a multicellular organism permanently adjusts the concentration of its cell surface proteins. In particular, epithelial cells tightly control the number of carriers, transporters and cell adhesion proteins at their plasma membrane. However, sensitively measuring the cell surface concentration of a particular protein of interest in live cells and in real time represents a considerable challenge. Here, we introduce a novel approach based on split luciferases, which uses one luciferase fragment as a tag on the protein of interest and the second fragment as a supplement to the extra-cellular medium. Once the protein of interest arrives at the cell surface, the luciferase fragments complement and generate luminescence. We compared the performance of split Gaussia luciferase and split Nanoluciferase by using a system to synchronize biosynthetic trafficking with conditional aggregation domains. The best results were achieved with split Nanoluciferase, for which luminescence increased more than 6000-fold upon recombination. Furthermore, we showed that our approach can separately detect and quantify the arrival of membrane proteins at the apical and basolateral plasma membrane in single polarized epithelial cells by detecting theluminescence signals with a microscope, thus opening novel avenues for characterizing the variations in trafficking in individual epithelial cells.
Unlike genomic alterations, gene expression profiles have not been widely used to refine cancer therapies. We analyzed transcriptional changes in acute myeloid leukemia (AML) cell lines in response to standard first-line AML drugs cytarabine and daunorubicin by means of RNA sequencing. Those changes were highly cell- and treatment-specific. By comparing the changes unique to treatment-sensitive and treatment-resistant AML cells, we enriched for treatment-relevant genes. Those genes were associated with drug response-specific pathways, including calcium ion-dependent exocytosis and chromatin remodeling. Pharmacological mimicking of those changes using EGFR and MEK inhibitors enhanced the response to daunorubicin with minimum standalone cytotoxicity. The synergistic response was observed even in the cell lines beyond those used for the discovery, including a primary AML sample. Additionally, publicly available cytotoxicity data confirmed the synergistic effect of EGFR inhibitors in combination with daunorubicin in all 60 investigated cancer cell lines. In conclusion, we demonstrate the utility of treatment-evoked gene expression changes to formulate rational drug combinations. This approach could improve the standard AML therapy, especially in older patients.
Moderne Nanomaterialien nehmen eine immer größere Bedeutung in industriell gefertigten Produkten ein. Der Mensch kommt deshalb über verschiedene Expositionswege, die z.T. auch den medizinischen Einsatz von Nanopartikeln in diagnostischen oder therapeutischen Anwendungen beinhalten, in Kontakt. Die Interaktion von Nanomaterialien mit biologischen Systemen des menschlichen Körpers wird von den physikalisch-chemischen Eigenschaften der Partikel bestimmt. Die Art der Interaktion mit einzelnen Körperzellen bzw. deren Bestandteilen entscheidet, ob positive oder negative Effekte auf die Gesundheit des Menschen entstehen.
In dieser Arbeit wurden kolloidale Silikananopartikel, die aus Tetraethylorthosilicat nach der Synthese von Hartlen (2008) hergestellt wurden, auf ihre Fähigkeit untersucht, mit den Signaltransduktionswegen epithelialer Zellen zu interagieren. Im Versuchssystem mit Rattenlungenepithelzellen (RLE 6TN) wurden drei verschiedene Größen kolloidaler Silikananopartikel (Si15 – 15 nm; Si25 – 25 nm; Si80 – 80 nm) untersucht und mit Kontrollnanopartikeln verglichen. Zusätzlich zur Zytotoxizität der Partikel wurden vor allem Effekte auf die Zellproliferation und Signaltransduktion betrachtet. Neben den molekularen Mechanismen der Partikeleffekte wurden insbesondere die Aufnahmemechanismen der Silikananopartikel und die subzelluläre Lokalisation der Partikel in den Zellen untersucht.
Die kleinsten verwendeten Silikapartikel (Si15) zeigten einen bislang nicht beschriebenen inhibitorischen Effekt auf die durch den epidermalen Wachstumsfaktor- (EGF-)vermittelte Zellproliferation. Dieser Effekt war nicht durch die Induktion von Zelltod auf der Ebene von Apoptose oder Nekrose begründet. Vielmehr zeigte sich eine spezifische Interaktion von Silikananopartikeln mit der EGFR-abhängigen proliferativen Signaltransduktion. Si15 Nanopartikel führten zu einer Reduktion der Phosphorylierung des EGFRs am Tyrosinrest 1173. Die mit der Aktivierung des Rezeptors einhergehende Internalisierung und die Aktivierung von intrazellulären Signalkinasen, insbesondere Proteinkinase B (Akt), wurden durch die Nanopartikel signifikant verringert. Die molekularen Mechanismen der antiproliferativen Wirkung der Nanopartikel wurden auf der Ebene der Interaktion mit dem natürlichen Liganden EGF identifiziert. So konnte in zellfreien Untersuchungen mit fluoreszenzmarkiertem EGF gezeigt werden, dass dieser an Silikananopartikel bindet. Konfokalmikroskopische Untersuchungen zeigten im zellulären System eine Kolokalisation der Partikel und EGF in vesikulären Strukturen. Weitere Analysen mit den Endozytose-Inhibitoren Chlorpromazin und Filipin III haben gezeigt, dass die Silikananopartikel (SiNP) in Abwesenheit von EGF Clathrin-abhängig von den Zellen internalisiert werden. Durch Färbungen mit vesikulären Markern wurden die SiNP in frühen und späten Endosomen lokalisiert, sodass von einem Transport der Partikel zu den Lysosomen ausgegangen werden kann.
Nanoinjection—the process of intracellular delivery using vertically configured nanostructures—is a physical route that efficiently negotiates the plasma membrane, with minimal perturbation and toxicity to the cells. Nanoinjection, as a physical membrane-disruption-mediated approach, overcomes challenges associated with conventional carrier-mediated approaches such as safety issues (with viral carriers), genotoxicity, limited packaging capacity, low levels of endosomal escape, and poor versatility for cell and cargo types. Yet, despite the implementation of nanoinjection tools and their assisted analogues in diverse cellular manipulations, there are still substantial challenges in harnessing these platforms to gain access into cell interiors with much greater precision without damaging the cell’s intricate structure. Here, we propose a non-viral, low-voltage, and reusable electroactive nanoinjection (ENI) platform based on vertically configured conductive nanotubes (NTs) that allows for rapid influx of targeted biomolecular cargos into the intracellular environment, and for successful gene silencing. The localization of electric fields at the tight interface between conductive NTs and the cell membrane drastically lowers the voltage required for cargo delivery into the cells, from kilovolts (for bulk electroporation) to only ≤ 10 V; this enhances the fine control over membrane disruption and mitigates the problem of high cell mortality experienced by conventional electroporation.
Hydrogels with adjustable mechanical properties have been engineered as matrices for mammalian cells and allow the dynamic, mechano-responsive manipulation of cell fate and function. Recent research yields hydrogels, where biological photoreceptors translated optical signals into a reversible and adjustable change in hydrogel mechanics. While their initial application provides important insights into mechanobiology, broader implementation is limited by a small dynamic range of addressable stiffness. Herein, this limitation is overcome by developing a photoreceptor-based hydrogel with reversibly adjustable stiffness from ≈800 Pa to the sol state. The hydrogel is based on star-shaped polyethylene glycol, functionalized with the red/far-red light photoreceptor phytochrome B (PhyB), or phytochrome-interacting factor 6 (PIF6). Upon illumination with red light, PhyB heterodimerizes with PIF6, thus crosslinking the polymers and resulting in gelation. However, upon illumination with far-red light, the proteins dissociate and trigger a complete gel-to-sol transition. The hydrogel's light-responsive mechanical properties are comprehensively characterized and it is applied as a reversible extracellular matrix for the spatiotemporally controlled deposition of mammalian cells within a microfluidic chip. It is anticipated that this technology will open new avenues for the site- and time-specific positioning of cells and will contribute to overcome spatial restrictions.
