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Hendrik Reynders,1 Indra Van Zundert,1 Rui Silva,1– 3 Bram Carlier,3 Olivier Deschaume,4 Carmen Bartic,4 Susana Rocha,1 Sergey Basov,5 Margriet J Van Bael,5 Uwe Himmelreich,3 Thierry Verbiest,1 Ana Zamora1,3 1Molecular Imaging and Photonics, KU Leuven, Leuven, Belgium; 2Engineering Department, Oporto University, Porto, Portugal; 3Biomedical MRI, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium; 4Laboratory for Soft Matter and Biophysics, KU Leuven, Leuven, Belgium; 5Quantum Solid State Physics, KU Leuven, Leuven, BelgiumCorrespondence: Ana Zamora Email anamaria.zamoramartinez@kuleuven.beIntroduction: The inherent fluorescence properties of iron oxide nanoparticles (IONPs) were characterized, and their applicability for multiphoton imaging in cells was tested in combination with their magnetic resonance imaging (MRI) capabilities.Methods: Superparamagnetic iron oxide nanoparticles were synthesized and subsequently coated with polyethylene glycol to make them water-dispersible. Further characterization of the particles was performed using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), dynamic light scattering (DLS), superconducting quantum interference device (SQUID) and magnetic resonance relaxivity measurements. MRI and fluorescence properties of bare IONPs were first studied in solution and subsequently in A549-labeled cells.Results: The particles, with a core size of 11.3 ± 4.5 nm, showed a good negative MRI contrast in tissue-mimicking phantoms. In vitro studies in mammalian A549 cells demonstrate that these IONPs are biocompatible and can also produce significant T2/T2* contrast enhancement in IONPs-labeled cells. Furthermore, excitation-wavelength dependent photoluminescence was observed under one- and two-photon excitation.Discussion: The obtained results indicated that IONPs could be used for fluorescence label-free bioimaging at multiple wavelengths, which was proven by multiphoton imaging of IONPs internalization in A549 cancer cells.Keywords: iron oxide nanoparticles, photoluminescence, biomedical imaging, MRI, cancer cells, multimodal imaging

In this work, we report the design and fabrication of a light-addressable, paper-based nanocomposite scaffold for optical pacing and read-out of in vitro grown cardiac tissue. The scaffold consists of paper cellulose microfibers functionalized with gold nanorods (GNRs) and semiconductor quantum dots (QDs), embedded in a cell-permissive collagen matrix. The GNRs enable cardiomyocyte activity modulation through local temperature gradients induced by modulated near-infrared (NIR) laser illumination, with the local temperature changes reported by temperature-dependent QD photoluminescence (PL). The micrometer-sized paper fibers promote the tubular organization of HL-1 cardiac muscle cells, while the NIR plasmonic stimulation modulates reversibly their activity. Given the nanoscale spatial resolution and facile fabrication, paper-based nanocomposite scaffolds with NIR modulation offer excellent alternatives to electrode-based or optogenetic methods for cell activity modulation, at the single cell level, and are compatible with 3D tissue constructs. Such paper-based optical platforms can provide new possibilities for the development of in vitro drug screening assays and heart disease modeling.

Alkali and quaternary ammonium cations interact with negatively charged cellulose nanocrystals (CNCs) bearing sulfated or carboxylated functional groups. As these are some of the most commonly occurring cations CNC encounter in applications, the thermodynamic parameters of these CNC-counterion interactions were evaluated with isothermal titration calorimetry (ITC). Whereas the adsorption of monovalent counterions onto CNCs was thermodynamically favourable at all evaluated conditions as indicated by a negative Gibbs free energy, the enthalpic and entropic contributions to the CNC-ion interactions were found to be strongly dependent on the hydration characteristics of the counterion and could be correlated with the potential barrier to water exchange of the respective ions. The adsorption of chaotropic cations onto the surface was exothermic, while the interactions with kosmotropic cations were endothermic and completely entropy-driven. The interactions of CNCs with more bulky quaternary ammonium counterions were more complex, and the mechanism of interaction shifted from electrostatic interactions with surface charged groups of CNCs towards adsorption of alkyl chains onto the CNC hydrophobic planes when the alkyl chain length increased., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Different microscopy and scattering methods used in the literature to determine the dimensions of cellulose nanocrystals derived from cotton and bacterial cellulose were compared to investigate potential bias and discrepancies. Atomic force microscopy (AFM), small-angle X-ray scattering (SAXS), depolarized dynamic light scattering (DDLS), and static light scattering (SLS) were compared. The lengths, widths, and heights of the particles and their respective distributions were determined by AFM. In agreement with previous work, the CNCs were found to have a ribbon-like shape, regardless of the source of cellulose or the surface functional groups. Tip broadening and agglomeration of the particles during deposition cause AFM-derived lateral dimensions to be systematically larger those obtained from SAXS measurements. The radius of gyration determined by SLS showed a good correlation with the dimensions obtained by AFM. The hydrodynamic lateral dimensions determined by DDLS were found to have the same magnitude as either the width or height obtained from the other techniques; however, the precision of DDLS was limited due to the mismatch between the cylindrical model and the actual shape of the CNCs, and to constraints in the fitting procedure. Therefore, the combination of AFM and SAXS, or microscopy and small-angle scattering, is recommended for the most accurate determination of CNC dimensions.

The motion of self-propelling microswimmers is significantly affected by confinement, which can enhance or reduce their mobility and also steer the direction of their propulsion. While their interactions with solid boundaries have already received considerable attention, many aspects of the influence of liquid-liquid interfaces (LLI) on active particle propulsion still remain unexplored. In this work, we studied the adsorption and motion of bimetallic Janus sideways propelled rods dispersed at the interface between an aqueous solution of hydrogen peroxide and oil. The wetting properties of the bimetallic rods result in a wide distribution of their velocities at the LLI. While a fraction of rods remain immotile, we note a significant enhancement of motility for the rest of the particles with velocities of up to 8 times higher in comparison to those observed near a solid wall. Liquid-liquid interfaces, therefore, can provide a new way to regulate the propulsion of bimetallic particles.

Foreign body response (FBR), inflammation, and fibrotic encapsulation of neural implants remain major problems affecting the impedance of the electrode-tissue interface and altering the device performance. Adhesion of proteins and cells (e.g., pro-inflammatory macrophages, and fibroblasts) triggers the FBR cascade and can be diminished by applying antifouling coatings onto the implanted devices. In this paper, we report the deposition and characterization of a thin (±6 nm) sulfobetaine-based coating onto microfabricated platinum electrodes and cochlear implant (CI) electrode arrays. We found that this coating has stable cell and protein-repellent properties, for at least 31 days in vitro, not affected by electrical stimulation protocols. Additionally, its effect on the electrochemical properties relevant to stimulation (i.e., impedance, charge injection capacity) was negligible. When applied to clinical CI electrode arrays, the film was successful at inhibiting fibroblast adhesion on both the silicone packaging and the platinum/iridium electrodes. In vitro, in fibroblast cultures, coated CI electrode arrays maintained impedance values up to five times lower compared to non-coated devices. Our studies demonstrate that such thin sulfobetaine containing layers are stable and prevent protein and cell adhesion in vitro and are compatible for use on CI electrode arrays. Future in vivo studies should be conducted to investigate its ability to mitigate biofouling, fibrosis, and the resulting impedance changes upon long-term implantation in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Over recent years, nano-engineered materials have become an important component of artificial extracellular matrices. On one hand, these materials enable static enhancement of the bulk properties of cell scaffolds, for instance, they can alter mechanical properties or electrical conductivity, in order to better mimic the in vivo cell environment. Yet, many nanomaterials also exhibit dynamic, remotely tunable optical, electrical, magnetic, or acoustic properties, and therefore, can be used to non-invasively deliver localized, dynamic stimuli to cells cultured in artificial ECMs in three dimensions. Vice versa, the same, functional nanomaterials, can also report changing environmental conditions-whether or not, as a result of a dynamically applied stimulus-and as such provide means for wireless, long-term monitoring of the cell status inside the culture. In this review article, we present an overview of the technological advances regarding the incorporation of functional nanomaterials in artificial extracellular matrices, highlighting both passive and dynamically tunable nano-engineered components.

Disease research and drug screening platforms require in vitro model systems with cellular cues resembling those of natural tissues. Fibrillar alignment, occurring naturally in extracellular matrices, is one of the crucial attributes in tissue development. Obtaining fiber alignment in 3D, in vitro remains an important challenge due to non-linear material characteristics. Here, we report a cell-compatible, shear stress-based method allowing to obtain 3D homogeneously aligned fibrillar collagen hydrogels. Controlling the shear-stress during gelation results in low strain rates, with negligible effects on the viability of embedded SH-SY5Y cells. Our approach offers reproducibility and tunability through a paradigm shift: The shear-stress initiation moment, being the critical optimization parameter in the process, is related to the modulus of the developing gel, whereas state of the art methods often rely on a predefined time to initiate the alignment procedure. After curing, the induced 3D alignment is maintained after the release of stress, with a linear relation between the total acquired strain and the fiber alignment. This method is generally applicable to 3D fibrillar materials and stress/pressure-controlled setups, making it a valuable addition to the fast-growing field of tissue engineering. STATEMENT OF SIGNIFICANCE: Controlling fiber alignment in vitro 3D hydrogels is crucial for developing physiologically relevant model systems. However, it remains challenging due to the non-linear material characteristics of fibrillar hydrogels, limiting the scalability and repeatability. Our approach tackles these challenges by utilizing a stress-controlled rheometer allowing us to monitor structural changes in situ and determine the optimal moment for applying a shear-stress inducing alignment. By careful parameter control, we infer the relationship between time, induced strain, alignment and biocompatibility. This tunable and reproducible method is both scalable and generally applicable to any fibrillar hydrogel, therefore, we believe it is useful for research investigating the link between matrix anisotropy and cell behavior in 3D systems, organ-on-chip technologies and drug research., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Despite the importance of cell characterization and identification for diagnostic and therapeutic applications, developing fast and label-free methods without (bio)-chemical markers or surface-engineered receptors remains challenging. Here, we exploit the natural cellular response to mild thermal stimuli and propose a label- and receptor-free method for fast and facile cell characterization. Cell suspensions in a dedicated sensor are exposed to a temperature gradient, which stimulates synchronized and spontaneous cell-detachment with sharply defined time-patterns, a phenomenon unknown from literature. These patterns depend on metabolic activity (controlled through temperature, nutrients, and drugs) and provide a library of cell-type-specific indicators, allowing to distinguish several yeast strains as well as cancer cells. Under specific conditions, synchronized glycolytic-type oscillations are observed during detachment of mammalian and yeast-cell ensembles, providing additional cell-specific signatures. These findings suggest potential applications for cell viability analysis and for assessing the collective response of cancer cells to drugs., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Optical interrogation of cellular electrical activity has proven itself essential for understanding cellular function and communication in complex networks. Voltage-sensitive dyes are important tools for assessing excitability but these highly lipophilic sensors may affect cellular function. Label-free techniques offer a major advantage as they eliminate the need for these external probes. In this work, it is shown that endogenous second-harmonic generation (SHG) from live cells is highly sensitive to changes in transmembrane potential (TMP). Simultaneous electrophysiological control of a living human embryonic kidney (HEK293T) cell, through a whole-cell voltage-clamp reveals a linear relation between the SHG intensity and membrane voltage. The results suggest that due to the high ionic strengths and fast optical response of biofluids, membrane hydration is not the main contributor to the observed field sensitivity. A conceptual framework is further provided that indicates that the SHG voltage sensitivity reflects the electric field within the biological asymmetric lipid bilayer owing to a nonzero χ eff ( 2 ) tensor. Changing the TMP without surface modifications such as electrolyte screening offers high optical sensitivity to membrane voltage (≈40% per 100 mV), indicating the power of SHG for label-free read-out. These results hold promise for the design of a non-invasive label-free read-out tool for electrogenic cells., (© 2022 Wiley-VCH GmbH.)

Aggregation of amyloid β peptides is known to be one of the main processes responsible for Alzheimer's disease. The resulting dementia is believed to be due in part to the formation of potentially toxic oligomers. However, the study of such intermediates and the understanding of how they form are very challenging because they are heterogeneous and transient in nature. Unfortunately, few techniques can quantify, in real time, the proportion and the size of the different soluble species during the aggregation process. In a previous work (Deleanu et al . Anal. Chem. 2021, 93, 6523-6533), we showed the potential of Taylor dispersion analysis (TDA) in amyloid speciation during the aggregation process of Aβ (1-40) and Aβ (1-42). The current work aims at exploring in detail the aggregation of amyloid Aβ (1-40):Aβ (1-42) peptide mixtures with different proportions of each peptide (1:0, 3:1, 1:1, 1:3, and 0:1) using TDA and atomic force microscopy (AFM). TDA allowed for monitoring the kinetics of the amyloid assembly and quantifying the transient intermediates. Complementarily, AFM allowed the formation of insoluble fibrils to be visualized. Together, the two techniques enabled us to study the influence of the peptide ratios on the kinetics and the formation of potentially toxic oligomeric species.

To overcome the problems of refractive index matching and increased disorder when working with traditional heterostructure colloidal photonic crystals (CPCs) with dual or multiple photonic bandgaps (PBGs) for fluorescence enhancement in water, we propose the use of a chemical heterostructure in hollow sphere CPCs (HSCPCs). A partial chemical modification of the HSCPC creates a large contrast in wettability to induce the heterostructure, while the hollow spheres increase the refractive index difference when used in aqueous environment. With the platform, fluorescence enhancement reaches around 160 times in solution, and 72 times (signal-to-background ratio ~7 times) in cells during proof-of-concept live cardiomyocyte contractility experiments. Such photonic platform can be further exploited for chemical sensing, bioassays, and environmental monitoring. Moreover, the introduction of chemical heterostructures provides new design principles for functionalized photonic devices., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The application of antibodies in nanomedicine is now standard practice in research since it represents an innovative approach to deliver chemotherapy agents selectively to tumors. The variety of targets or markers that are overexpressed in different types of cancers results in a high demand for antibody conjugated-nanoparticles, which are versatile and easily customizable. Considering up-scaling, the synthesis of antibody-conjugated nanoparticles should be simple and highly reproducible. Here, we developed a facile coating strategy to produce antibody-conjugated nanoparticles using 'click chemistry' and further evaluated their selectivity towards cancer cells expressing different markers. Our approach was consistently repeated for the conjugation of antibodies against CD44 and EGFR, which are prominent cancer cell markers. The functionalized particles presented excellent cell specificity towards CD44 and EGFR overexpressing cells, respectively. Our results indicated that the developed coating method is reproducible, versatile, and non-toxic, and can be used for particle functionalization with different antibodies. This grafting strategy can be applied to a wide range of nanoparticles and will contribute to the development of future targeted drug delivery systems.