Vascular inflammation on a chip: A scalable platform for trans-endothelial electrical resistance and immune cell migration
Haley Ehlers, Arnaud Nicolas, Frederik Schavemaker, Jeroen P. M. Heijmans, Martin Bulst, Sebastiaan J. Trietsch and Lenie J. van den Broek
The vasculature system plays a critical role in inflammation processes in the body. Vascular inflammatory mechanisms are characterized by disruption of blood vessel wall permeability together with increased immune cell recruitment and migration. There is a critical need to develop models that fully recapitulate changes in vascular barrier permeability in response to inflammatory conditions. We developed a scalable platform for parallel measurements of trans epithelial electrical resistance (TEER) in 64 perfused microfluidic HUVEC tubules under inflammatory conditions. Over 250 tubules where exposed to Tumor necrosis factor alpha (TNFα) and interferon gamma (INF-γ) or human peripheral blood mononuclear cells. The inflammatory response was quantified based on changes TEER and expression of ICAM and VE-cadherin. We observed changes in barrier function in the presence of both inflammatory cytokines and human peripheral blood mononuclear cells, characterized by decreased TEER values, increase in ICAM expression as well changes in endothelial morphology. OrganoPlate 3-lane64 based HUVEC tubules provide a valuable tool for inflammatory studies in an automation compatible manner. Continuous TEER measurements enable long term, sensitive assays for barrier studies. We propose the use of our platform as a powerful tool for modelling endothelial inflammation in combination with immune cell interaction that can be used to screen targets and drugs to treat chronic vascular inflammation.
EdU sensing: The Raman way of following endothelial cell proliferation in vitro and ex vivo
Basseem Radwan, Stefano Rocchetti, Ewelina Matuszyk, Magdalena Sternak, Maciej Stodulski, Robert Pawlowski, Jacek Mlynarski, Krzysztof Brzozowski, Stefan Chlopicki, and Malgorzata Baranska
Biosensors and Bioelectronics 2022
Endothelial cells line the lumen of all vessels in the body and maintain vascular homeostasis. In particular, endothelial cell regeneration in response to insult sustain functional endothelial layer. EdU (5-ethynyl-2′-deoxyuridine) is an alkyne-tagged proliferation probe that incorporates into newly synthesized DNA and is used for fluorescence imaging of cell proliferation with the use of “click chemistry” reaction with a fluorescent azide. Here, we utilized EdU as a click-free Raman probe for tracking endothelial cell proliferation. Raman imaging of EdU was performed in live endothelial cells, showing an advantage over fluorescence imaging of EdU, as this technique did not require sample fixation and permeabilization. To validate Raman-based imaging of EdU to study endothelial cell proliferation, we showed that when endothelial cells were treated with cycloheximide or doxorubicin to impair the proliferation of endothelial cells, the Raman-based signal of EdU was diminished. Furthermore, endothelial cells proliferation detected using EdU-labelled Raman imaging was compared with fluorescence imaging. Finally, the method of Raman–based EdU imaging was used in the isolated murine aorta ex vivo. Altogether, our results show that Raman-based imaging of EdU provides a novel alternative for fluorescence-based assay to assess endothelial proliferation and regeneration.
Multimodal Investigation into the Interaction of Quinacrine with Microcavity-Supported Lipid Bilayers
Nirod Kumar Sarangi, Amrutha Prabhakaran, and Tia E. Keyes
Quinacrine is a versatile drug that is widely recognized for its antimalarial action through its inhibition of the phospholipase enzyme. It also has antianthelmintic and antiprotozoan activities and is a strong DNA binder that may be used to combat multidrug resistance in cancer. Despite extensive cell-based studies, a detailed understanding of quinacrine’s influence on the cell membrane, including permeability, binding, and rearrangement at the molecular level, is lacking. Herein, we apply microcavity-suspended lipid bilayers (MSLBs) as in vitro models of the cell membrane comprising DOPC, DOPC:Chol(3:1), and DOPC:SM:Chol(2:2:1) to investigate the influence of cholesterol and intrinsic phase heterogeneity induced by mixed-lipid composition on the membrane interactions of quinacrine. Using electrochemical impedance spectroscopy (EIS) and surface-enhanced Raman spectroscopy (SERS) as label-free surface-sensitive techniques, we have studied quinacrine interaction and permeability across the different MSLBs. Fluorescent lifetime correlation spectroscopy (FLCS) provides further biophysical insight, revealing that quinacrine binding increases the lipid diffusivity of DOPC and the ternary membrane while remarkably decreasing the lipid diffusivity of the DOPC:Chol membrane. Overall, because of its adaptability to multimodal approaches, the MSLB platform provides rich and detailed insights into drug–membrane interactions, making it a powerful tool for in vitro drug screening.
Triplet–Triplet Annihilation Upconversion by Polymeric Sensitizers
Keshav Kumar Jha, Amrutha Prabhakaran, Christopher S. Burke, Marcus Schulze, Ulrich S. Schubert, Tia E. Keyes, Michael Jäger, and Benjamin Dietzek-Ivanšić
Journal of Physical Chemistry C 2022
This contribution studies the effect of integrating Ru(dqp)2-inspired molecular sensitizers into the side chains of a PMMA polymer, which─as a macromolecular photosensitizer─is codissolved with 9,10-diphenylanthracene as an annihilator. We study the effect of confining the sensitizers into a comparably small volume on the triplet–triplet annihilation upconversion (TTAUC) process and compare the results to those of an upconversion system using various concentrations of a monomeric annihilator. We show that our approach of using a macromolecular photosensitizer allows for upconversion at extremely low excitation power densities. Furthermore, the onset of a strong annihilation regime, that is, a regime in which the intensity of the upconverted light scales linearly with the increase of the excitation power, is significantly reduced using the polymeric sensitizer; however, the upconversion intensity sits below those of the monomeric counterparts.
Shorter Alkyl Chains Enhance Molecular Diffusion and Electron Transfer Kinetics between Photosensitisers and Catalysts in CO2-Reducing Photocatalytic Liposomes
David M. Klein, Dr. Santiago Rodríguez-Jiménez, Marlene E. Hoefnagel, Andrea Pannwitz, Amrutha Prabhakaran, Maxime A. Siegler, Tia E. Keyes, Erwin Reisner,Albert M. Brouwer, Sylvestre Bonnet
Chemistry – A European Journal 2021
A series of Ru photosensitisers (RuCn) and Re catalysts (ReCn) covalently functionalised with alkyl chains of different lengths (n=9, 12, 15, 17, and 19) were immobilised on liposomes to perform photocatalytic CO2 reduction using ascorbate (HAsc−) as electron donor. A higher turnover number for CO production (TONCO) was found for systems with shorter alkyl tails, which was due to the higher mobility of the molecules on the lipid bilayer, leading to faster electron transfer between RuCn and ReCn.
Radiative lifetime of a BODIPY dye as calculated by TDDFT and EOM-CCSD methods: solvent and vibronic effects
Rengel Cane E. Sia, Ruben Arturo Arellano-Reyes, Tia E. Keyes and Julien Guthmuller
Physical Chemistry Chemical Physics 2021
The radiative emission lifetime and associated S1 excited state properties of a BODIPY dye are investigated with TDDFT and EOM-CCSD calculations. The effects of a solvent are described with the polarizable continuum model using the linear response (LR) approach as well as state-specific methods. The Franck–Condon (FC), Herzberg–Teller (HT) and Duschinsky vibronic effects are evaluated for the absorption and emission spectra, and for the radiative lifetime. The transition energies, spectra shapes and radiative lifetime are assessed with respect to experimental results. It is found that the TDDFT transition energies are overestimated by about 0.4–0.5 eV, whereas EOM-CCSD improves the vertical emission energy by about 0.1 eV in comparison to TDDFT. The solvatochromic and Stokes shifts are better reproduced by the state-specific solvation methods, which show that these methods are more suited than the LR model to describe the solvent effects on the BODIPY dye. The vibronic effects lead to an increase of the radiative lifetime of about 0.4 to 1.0 ns depending on the theoretical approach, which highlights the importance of such effects. Moreover, the HT effects are negligible on both the spectra and lifetime, which demonstrates that the FC approximation is accurate for the BODIPY dye. Finally, the comparison with experimental data shows that the radiative lifetimes predicted by EOM-CCSD and TDDFT have comparable accuracy.
Langmuir–Blodgett Films of Diketopyrrolopyrroles with Tunable Amphiphilicity
Maximilian L. Hupfer, Beata Koszarna, Soumik Ghosh, Daniel T. Gryko, and Martin Presselt
In this work, we present the formation of H- and J-aggregates of amphiphilic centrosymmetric diketopyrrolopyrroles containing aliphatic or aromatic amino groups. The inherent amphiphilicity of these dyes predestines their assembly at interfaces to form ordered supramolecular structures. We employed the Langmuir–Blodgett (LB) technique to generate, manipulate, and deposit such supramolecular structures. The aforementioned amines provide an additional means to control the formation of the supramolecular assemblies. In the resulting LB films, both H- and J-aggregates of the dyes can be realized, leading to very broad absorption spectra. In contrast to many reports on H- and J-aggregates, the interactions between the symmetric diketopyrrolopyrroles are controlled via interface assembly and π-stacking and not by dipolar interactions.
Monitoring excited-state relaxation in a molecular marker in live cells–a case study on astaxanthin
Tingxiang Yang, Avinash Chettri, Basseem Radwan, Ewelina Matuszyk, Malgorzata Baranska and Benjamin Dietzek
Chemical Communications 2021
Small molecules are frequently used as dyes, labels and markers to visualize and probe biophysical processes within cells. However, very little is generally known about the light-driven excited-state reactivity of such systems when placed in cells. Here an experimental approach to study ps time-resolved excited state dynamics of a benchmark molecular marker, astaxanthin (AXT), in live human cells is introduced. The photoinduced dynamics in AXT recorded in cellulo appears to be unchanged compared to the dynamics recorded in solution. Aside from indicating the absence of AXT aggregation in the cells, this points to the fact that structural distortion of the AXT molecules, which has been suggested based on shifts of the resonance Raman active C=C vibration, do not alter the transient absorption data recorded during the in cellulo experiments. Finally, in a methodological context we show that our approach to in cellulo transient absorption spectroscopy offers a valuable path to study the impact of local environments on the photoinduced dynamics in stains, markers and beyond this in light-activated drugs, e.g. for photodynamic therapy, and interactions between phototherapy agents and live cells
Toward Raman Subcellular Imaging of Endothelial Dysfunction
Adriana Adamczyk, Ewelina Matuszyk, Basseem Radwan, Stefano Rocchetti, Stefan Chlopicki, and Malgorzata Baranska
Journal of Medicinal Chemistry 2021
Multiple diseases are at some point associated with altered endothelial function, and endothelial dysfunction (ED) contributes to their pathophysiology. Biochemical changes of the dysfunctional endothelium are linked to various cellular organelles, including the mitochondria, endoplasmic reticulum, and nucleus, so organelle-specific insight is needed for better understanding of endothelial pathobiology. Raman imaging, which combines chemical specificity with microscopic resolution, has proved to be useful in detecting biochemical changes in ED at the cellular level. However, the detection of spectroscopic markers associated with specific cell organelles, while desirable, cannot easily be achieved by Raman imaging without labeling. This critical review summarizes the current advances in Raman-based analysis of ED, with a focus on a new approach involving molecular Raman reporters that could facilitate the study of biochemical changes in cellular organelles. Finally, imaging techniques based on both conventional spontaneous Raman scattering and the emerging technique of stimulated Raman scattering are discussed.
Multiplex Raman imaging of organelles in endothelial cells
Ewelina Matuszy, Adriana Adamczyk, Basseem Radwan, Anna Pieczara, Piotr Szcześniak, Jacek Mlynarski, Katarzyna Kamińska, Malgorzata Baranska
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 2021
The aim of this work is to visualize the main cell organelles in endothelial cells (HMEC-1) using established reporters (EdU and MitoBADY), but also to test a new Raman reporter, namely falcarinol, which exhibits lipophilic properties. Moreover, we tested the possibility to use Raman reporters as a probe to detect changes in distribution of certain organelles after induced endothelial dysfunction (ED) in in vitro models. In both cases, induced ED is characterized by the formation of lipid droplets in the cells, which is why a good tool for the detection of lipid-rich organelles is so important in these studies.
Astaxanthin as a new Raman probe for biosensing of specific subcellular lipidic structures: can we detect lipids in cells under resonance conditions?
Krzysztof Czamara, Adriana Adamczyk, Marta Stojak, Basseem Radwan, Malgorzata Baranska
Cellular and Molecular Life Sciences 2020
Here we report a new Raman probe for cellular studies on lipids detection and distribution. It is (3S, 3’S)-astaxanthin (AXT), a natural xanthophyll of hydrophobic properties and high solubility in lipids. We showed that AXT accumulates in lipidic structures of endothelial cells in time-dependent manner that provides possibility to visualize e.g. endoplasmic reticulum, as well as nuclear envelope. As a non-toxic reporter, it has a potential in the future studies on e.g. nucleus membranes damage in live cells in a very short measuring time.
Labeled vs. Label-Free Raman Imaging of Lipids in Endothelial Cells of Various Origins
Basseem Radwan, Adriana Adamczyk, Szymon Tott, Krzysztof Czamara, Katarzyna Kaminska, Ewelina Matuszyk, Malgorzata Baranska
Endothelial dysfunction has been recognized as a primary or secondary cause of many diseases and it manifests itself, among others, by increased lipid content or a change in the lipid composition in the EC. Therefore, the analysis of cellular lipids is crucial to understand the mechanisms of disease development. Tumor necrosis factor alpha (TNF-α)-induced inflammation of EC alters the lipid content of cells, which can be detected by Raman spectroscopy. We consider (3S,3′S)-astaxanthin (AXT), a natural dye with a characteristic resonance spectrum, as a new Raman probe for the detection of lipids in the EC of various vascular beds, i.e., the aorta, brain and heart. AXT colocalizes with lipids in cells, enabling imaging of lipid-rich cellular components in a time-dependent manner using laser power 10 times lower than that commonly used to measure biological samples.
Interaction of Miltefosine with Microcavity Supported Lipid Membrane: Biophysical Insights from Electrochemical Impedance Spectroscopy
Nirod Kumar Sarangi, Amrutha Prabhakaran, Tia E. Keyes
Miltefosine an alkylphosphocholine analogue, is the only drug taken orally for the treatment of leishmaniasis‐a parasitic disease caused by sandflies. Although it is believed that Miltefosine exerts its activity by acting at the lipid membrane, detailed understanding of the interaction of this drug with eukaryotic membranes is still lacking. Herein, we exploit microcavity pore suspended lipid bilayers (MSLBs) as a biomimetic platform in combination with a highly sensitive label‐free electrochemical impedance spectroscopy (EIS) technique to gain biophysical insight into the interaction of Miltefosine with host cell membrane as a function of lipid membranes composition.