Dimensions of Cellulose Nanocrystals from Cotton and Bacterial Cellulose: Comparison of Microscopy and Scattering Techniques.

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.

Sideways propelled bimetallic rods at the water/oil interface.

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.

Sulfobetaine-based ultrathin coatings as effective antifouling layers for implantable neuroprosthetic devices.

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.

Nanocomposite Hydrogels as Functional Extracellular Matrices.

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.

Stress-controlled shear flow alignment of collagen type I hydrogel systems.

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.

Label-Free Imaging of Membrane Potentials by Intramembrane Field Modulation, Assessed by Second Harmonic Generation Microscopy.

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 χeff(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.

Taylor Dispersion Analysis and Atomic Force Microscopy Provide a Quantitative Insight into the Aggregation Kinetics of Aß (1-40)/Aß (1-42) Amyloid Peptide Mixtures.

Aggregation of amyloid ß 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 Aß (1-40) and Aß (1-42). The current work aims at exploring in detail the aggregation of amyloid Aß (1-40):Aß (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.

Versatile and Robust Method for Antibody Conjugation to Nanoparticles with High Targeting Efficiency.

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.

Dual photonic bandgap hollow sphere colloidal photonic crystals for real-time fluorescence enhancement in living cells.

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.

Chlorite oxidized oxyamylose differentially influences the microstructure of fibrin and self assembling peptide hydrogels as well as dental pulp stem cell behavior.

Tailored hydrogels mimicking the native extracellular environment could help overcome the high variability in outcomes within regenerative endodontics. This study aimed to evaluate the effect of the chemokine-binding and antimicrobial polymer, chlorite-oxidized oxyamylose (COAM), on the microstructural properties of fibrin and self-assembling peptide (SAP) hydrogels. A further goal was to assess the influence of the microstructural differences between the hydrogels on the in vitro behavior of human dental pulp stem cells (hDPSCs). Structural and mechanical characterization of the hydrogels with and without COAM was performed by atomic force microscopy and scanning electron microscopy to characterize their microstructure (roughness and fiber length, diameter, straightness, and alignment) and by nanoindentation to measure their stiffness (elastic modulus). Then, hDPSCs were encapsulated in hydrogels with and without COAM. Cell viability and circularity were determined using confocal microscopy, and proliferation was determined using DNA quantification. Inclusion of COAM did not alter the microstructure of the fibrin hydrogels at the fiber level while affecting the SAP hydrogel microstructure (homogeneity), leading to fiber aggregation. The stiffness of the SAP hydrogels was sevenfold higher than the fibrin hydrogels. The viability and attachment of hDPSCs were significantly higher in fibrin hydrogels than in SAP hydrogels. The DNA content was significantly affected by the hydrogel type and the presence of COAM. The microstructural stability after COAM inclusion and the favorable hDPSCs' response observed in fibrin hydrogels suggest this system as a promising carrier for COAM and application in endodontic regeneration.

Label-Free Iron Oxide Nanoparticles as Multimodal Contrast Agents in Cells Using Multi-Photon and Magnetic Resonance Imaging.

INTRODUCTION: 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.

Glycine betaine grafted nanocellulose as an effective and bio-based cationic nanocellulose flocculant for wastewater treatment and microalgal harvesting.

Flocculation is a widely used technology in industry including for wastewater treatment and microalgae harvesting. To increase the sustainability of wastewater treatment, and to avoid contamination of the harvested microalgal biomass, there is a need for bio-based flocculants to replace synthetic polymer flocculants or metal salt coagulants. We developed the first cellulose nanocrystalline flocculant with a grafted cationic point charge, i.e. glycine betaine (i.e. N,N,N-trimethylglycine) grafted cellulose nanocrystals (CNCs) effective for the flocculation of kaolin (a model system for wastewater treatment), the freshwater microalgae Chlorella vulgaris, and the marine microalgae Nannochloropsis oculata. We successfully grafted glycine betaine onto CNCs using a one-pot reaction using a tosyl chloride activated esterification reaction with a degree of substitution ranging from 0.078 ± 0.003 to 0.152 ± 0.002. The degree of substitution is controlled by the reaction conditions. Flocculation of kaolin (0.5 g L-1) required a dose of 2 mg L-1, a comparable dose to commercial polyacrylamide-based flocculants. Flocculation was also successful for freshwater as well as marine microalgae (biomass concentration about 300 mg L-1 dry matter), although the flocculation efficiency of the latter remained below 80%. The dose to induce flocculation (DS = 0.152 ± 0.002) was 20 mg L-1 for the freshwater Chlorella vulgaris and 46 mg L-1 for the marine Nannochloropsis oculata, comparable to other bio-based flocculants such as chitosan or TanFloc.

Ionic strength controls long-term cell-surface interactions - A QCM-D study of S. cerevisiae adhesion, retention and detachment.

Understanding microbial adhesion and retention is crucial for controlling many processes, including biofilm formation, antimicrobial therapy as well as cell sorting and cell detection platforms. Cell detachment is inextricably linked to cell adhesion and retention and plays an important part in the mechanisms involved in these processes. Physico-chemical and biological forces play a crucial role in microbial adhesion interactions and altering the medium ionic strength offers a potential means for modulating these interactions. Real-time studies on the effect of ionic strength on microbial adhesion are often limited to short-term bacterial adhesion. Therefore, there is a need, not only for long-term bacterial adhesion studies, but also for similar studies focusing on eukaryotic microbes, such as yeast. Hereby, we monitored, in real-time, S. cerevisiae adhesion on gold and silica as examples of surfaces with different surface charge properties to disclose long-term adhesion, retention and detachment as a function of ionic strength using quartz crystal microbalance with dissipation monitoring. Our results show that short- and long-term cell adhesion levels in terms of mass-loading increase with increasing ionic strength, while cells dispersed in a medium of higher ionic strength experience longer retention and detachment times. The positive correlation between the cell zeta potential and ionic strength suggests that zeta potential plays a role on cell retention and detachment. These trends are similar for measurements on silica and gold, with shorter retention and detachment times for silica due to strong short-range repulsions originating from a high electron-donicity. Furthermore, the results are comparable with measurements in standard yeast culture medium, implying that the overall effect of ionic strength applies for cells in nutrient-rich and nutrient-deficient media.

The Importance of Excellent π-π Interactions in Poly(thiophene)s To Reach a High Third-Order Nonlinear Optical Response.

Poly(thiophene)s have an inherently large third-order nonlinear optical (TONO) response, but applications are not straightforward due to unoptimized materials. Therefore, several structure-property relationships (molar mass, branching, regioregularity) are investigated to unravel which structural modifications give the highest TONO response. Poly(3-hexylthiophene) with different molar masses, poly[3-(2-ethylhexyl)thiophene] with different molar masses, and random copolymers with different degrees of regioregularity are synthesized and measured by UV-vis spectroscopy and the third harmonic scattering technique. Every structural modification that leads to an increase in π-π interactions in poly(thiophene)s leads to an increase in the TONO response of the material. Therefore, a material with a high molar mass, an unbranched alkyl side chain, and a high regioregularity degree is preferably tested as a promising TONO material.

SANS study of mixed cholesteric cellulose nanocrystal - gold nanorod suspensions.

Self-assembly of cellulose nanocrystals (CNCs) doped with anisotropic gold nanorods (AuNRs) was studied by small-angle neutron scattering. Correlation distances and structured domains were analysed to determine the influence of CNC and AuNR concentration on structuring. The transfer of the nematic structure of CNCs to AuNRs is explained in terms of an entropy-driven evolution from an isotropic to a cholesteric phase, with small nematic domains already present in the "isotropic" phase in equilibrium with the chiral nematic phase.

Towards Mimicking the Fetal Liver Niche: The Influence of Elasticity and Oxygen Tension on Hematopoietic Stem/Progenitor Cells Cultured in 3D Fibrin Hydrogels.

Hematopoietic stem/progenitor cells (HSPCs) are responsible for the generation of blood cells throughout life. It is believed that, in addition to soluble cytokines and niche cells, biophysical cues like elasticity and oxygen tension are responsible for the orchestration of stem cell fate. Although several studies have examined the effects of bone marrow (BM) niche elasticity on HSPC behavior, no study has yet investigated the effects of the elasticity of other niche sites like the fetal liver (FL), where HSPCs expand more extensively. In this study, we evaluated the effect of matrix stiffness values similar to those of the FL on BM-derived HSPC expansion. We first characterized the elastic modulus of murine FL tissue at embryonic day E14.5. Fibrin hydrogels with similar stiffness values as the FL (soft hydrogels) were compared with stiffer fibrin hydrogels (hard hydrogels) and with suspension culture. We evaluated the expansion of total nucleated cells (TNCs), Lin-/cKit+ cells, HSPCs (Lin-/Sca+/cKit+ (LSK) cells), and hematopoietic stem cells (HSCs: LSK- Signaling Lymphocyte Activated Molecule (LSK-SLAM) cells) when cultured in 5% O2 (hypoxia) or in normoxia. After 10 days, there was a significant expansion of TNCs and LSK cells in all culture conditions at both levels of oxygen tension. LSK cells expanded more in suspension culture than in both fibrin hydrogels, whereas TNCs expanded more in suspension culture and in soft hydrogels than in hard hydrogels, particularly in normoxia. The number of LSK-SLAM cells was maintained in suspension culture and in the soft hydrogels but not in the hard hydrogels. Our results indicate that both suspension culture and fibrin hydrogels allow for the expansion of HSPCs and more differentiated progeny whereas stiff environments may compromise LSK-SLAM cell expansion. This suggests that further research using softer hydrogels with stiffness values closer to the FL niche is warranted.

Second-order optimized regularized structured illumination microscopy (sorSIM) for high-quality and rapid super resolution image reconstruction with low signal level.

Structured illumination microscopy (SIM) is a widely used super resolution imaging technique that can down-modulate a sample's high-frequency information into objective recordable frequencies to enhance the resolution below the diffraction limit. However, classical SIM image reconstruction methods often generate poor results under low illumination conditions, which are required for reducing photobleaching and phototoxicity in cell imaging experiments. Although denoising methods or auxiliary items improved SIM image reconstruction in low signal level situations, they still suffer from decreased reconstruction quality and significant background artifacts, inevitably limiting their practical applications. In order to improve the reconstruction quality, second-order optimized regularized SIM (sorSIM) is designed specifically for image reconstruction in low signal level situations. In sorSIM, a second-order regularization term is introduced to suppress noise effect, and the penalty factor in this term is selected to optimize the resolution enhancement and noise resistance. Compared to classical SIM image reconstruction algorithms as well as to those previously used in low illumination cases, the proposed sorSIM provides images with enhanced resolution and fewer background artifacts. Therefore, sorSIM can be a potential tool for high-quality and rapid super resolution imaging, especially for low signal images.

QCM-D Study of Time-Resolved Cell Adhesion and Detachment: Effect of Surface Free Energy on Eukaryotes and Prokaryotes.

Cell-material interactions are crucial for many biomedical applications, including medical implants, tissue engineering, and biosensors. For implants, while the adhesion of eukaryotic host cells is desirable, bacterial adhesion often leads to infections. Surface free energy (SFE) is an important parameter that controls short- and long-term eukaryotic and prokaryotic cell adhesion. Understanding its effect at a fundamental level is essential for designing materials that minimize bacterial adhesion. Most cell adhesion studies for implants have focused on correlating surface wettability with mammalian cell adhesion and are restricted to short-term time scales. In this work, we used quartz crystal microbalance with dissipation monitoring (QCM-D) and electrical impedance analysis to characterize the adhesion and detachment of S. cerevisiae and E. coli, serving as model eukaryotic and prokaryotic cells within extended time scales. Measurements were performed on surfaces displaying different surface energies (Au, SiO2, and silanized SiO2). Our results demonstrate that tuning the surface free energy of materials is a useful strategy for selectively promoting eukaryotic cell adhesion and preventing bacterial adhesion. Specifically, we show that under flow and steady-state conditions and within time scales up to ∼10 h, a high SFE, especially its polar component, enhances S. cerevisiae adhesion and hinders E. coli adhesion. In the long term, however, both cells tend to detach, but less detachment occurs on surfaces with a high dispersive SFE contribution. The conclusions on S. cerevisiae are also valid for a second eukaryotic cell type, being the human embryonic kidney (HEK) cells on which we performed the same analysis for comparison. Furthermore, each cell adhesion phase is associated with unique cytoskeletal viscoelastic states, which are cell-type-specific and surface free energy-dependent and provide insights into the underlying adhesion mechanisms.

Visualization and characterization of metallo-aggregates using multi-photon microscopy.

A simple and cost-effective method based on multi-photon microscopy is presented for the preliminary screening of the general morphology, size range and heterogeneity of Ir(iii) nano-aggregate formulations.

Sensitive and specific detection of E. coli using biomimetic receptors in combination with a modified heat-transfer method.

We report on a novel biomimetic sensor that allows sensitive and specific detection of Escherichia coli (E. coli) bacteria in a broad concentration range from 102 up to 106 CFU/mL in both buffer fluids and relevant food samples (i.e. apple juice). The receptors are surface-imprinted polyurethane layers deposited on stainless-steel chips. Regarding the transducer principle, the sensor measures the increase in thermal resistance between the chip and the liquid due to the presence of bacteria captured on the receptor surface. The low noise level that enables the low detection limit originates from a planar meander element that serves as both a heater and a temperature sensor. Furthermore, the experiments show that the presence of bacteria in a liquid enhances the thermal conductivity of the liquid itself. Reference tests with a set of other representative species of Enterobacteriaceae, closely related to E. coli, indicate a very low cross-sensitivity with a sensor response at or below the noise level.