Spatially resolved analysis of microenvironmental gradient impact on cancer cell phenotypes.

Despite the physiological and pathophysiological significance of microenvironmental gradients, e.g., for diseases such as cancer, tools for generating such gradients and analyzing their impact are lacking. Here, we present an integrated microfluidic-based workflow that mimics extracellular pH gradients characteristic of solid tumors while enabling high-resolution live imaging of, e.g., cell motility and chemotaxis, and preserving the capacity to capture the spatial transcriptome. Our microfluidic device generates a pH gradient that can be rapidly controlled to mimic spatiotemporal microenvironmental changes over cancer cells embedded in a 3D matrix. The device can be reopened allowing immunofluorescence analysis of selected phenotypes, as well as the transfer of cells and matrix to a Visium slide for spatially resolved analysis of transcriptional changes across the pH gradient. This workflow is easily adaptable to other gradients and multiple cell types and can therefore prove invaluable for integrated analysis of roles of microenvironmental gradients in biology.

Interlaboratory Comparison on Absolute Photoluminescence Quantum Yield Measurements of Solid Light Converting Phosphors with Three Commercial Integrating Sphere Setups.

Scattering luminescent materials dispersed in liquid and solid matrices and luminescent powders are increasingly relevant for fundamental research and industry. Examples are luminescent nano- and microparticles and phosphors of different compositions in various matrices or incorporated into ceramics with applications in energy conversion, solid-state lighting, medical diagnostics, and security barcoding. The key parameter to characterize the performance of these materials is the photoluminescence/fluorescence quantum yield (Φf), i.e., the number of emitted photons per number of absorbed photons. To identify and quantify the sources of uncertainty of absolute measurements of Φf of scattering samples, the first interlaboratory comparison (ILC) of three laboratories from academia and industry was performed by following identical measurement protocols. Thereby, two types of commercial stand-alone integrating sphere setups with different illumination and detection geometries were utilized for measuring the Φf of transparent and scattering dye solutions and solid phosphors, namely, YAG:Ce optoceramics of varying surface roughness, used as converter materials for blue light emitting diodes. Special emphasis was dedicated to the influence of the measurement geometry, the optical properties of the blank utilized to determine the number of photons of the incident excitation light absorbed by the sample, and the sample-specific surface roughness. While the Φf values of the liquid samples matched between instruments, Φf measurements of the optoceramics with different blanks revealed substantial differences. The ILC results underline the importance of the measurement geometry, sample position, and blank for reliable Φf data of scattering the YAG:Ce optoceramics, with the blank's optical properties accounting for uncertainties exceeding 20%.

Influence of nanoparticle encapsulation and encoding on the surface chemistry of polymer carrier beads.

Surface-functionalized polymer beads encoded with molecular luminophores and nanocrystalline emitters such as semiconductor nanocrystals, often referred to as quantum dots (QDs), or magnetic nanoparticles are broadly used in the life sciences as reporters and carrier beads. Many of these applications require a profound knowledge of the chemical nature and total number of their surface functional groups (FGs), that control bead charge, colloidal stability, hydrophobicity, and the interaction with the environment and biological systems. For bioanalytical applications, also the number of groups accessible for the subsequent functionalization with, e.g., biomolecules or targeting ligands is relevant. In this study, we explore the influence of QD encoding on the amount of carboxylic acid (COOH) surface FGs of 2 µm polystyrene microparticles (PSMPs). This is done for frequently employed oleic acid and oleylamine stabilized, luminescent core/shell CdSe QDs and two commonly used encoding procedures. This included QD addition during bead formation by a thermally induced polymerization reaction and a post synthetic swelling procedure. The accessible number of COOH groups on the surface of QD-encoded and pristine beads was quantified by two colorimetric assays, utilizing differently sized reporters and electrostatic and covalent interactions. The results were compared to the total number of FGs obtained by a conductometric titration and Fourier transform infrared spectroscopy (FTIR). In addition, a comparison of the impact of QD and dye encoding on the bead surface chemistry was performed. Our results demonstrate the influence of QD encoding and the QD-encoding strategy on the number of surface FG that is ascribed to an interaction of the QDs with the carboxylic acid groups on the bead surface. These findings are of considerable relevance for applications of nanoparticle-encoded beads and safe-by-design concepts for nanomaterials.

Dual color pH probes made from silica and polystyrene nanoparticles and their performance in cell studies.

Ratiometric green-red fluorescent nanosensors for fluorometrically monitoring pH in the acidic range were designed from 80 nm-sized polystyrene (PS) and silica (SiO2) nanoparticles (NPs), red emissive reference dyes, and a green emissive naphthalimide pH probe, analytically and spectroscopically characterized, and compared regarding their sensing performance in aqueous dispersion and in cellular uptake studies. Preparation of these optical probes, which are excitable by 405 nm laser or LED light sources, involved the encapsulation of the pH-inert red-fluorescent dye Nile Red (NR) in the core of self-made carboxylated PSNPs by a simple swelling procedure and the fabrication of rhodamine B (RhB)-stained SiO2-NPs from a silane derivative of pH-insensitive RhB. Subsequently, the custom-made naphthalimide pH probe, that utilizes a protonation-controlled photoinduced electron transfer process, was covalently attached to the carboxylic acid groups at the surface of both types of NPs. Fluorescence microscopy studies with the molecular and nanoscale optical probes and A549 lung cancer cells confirmed the cellular uptake of all probes and their penetration into acidic cell compartments, i.e., the lysosomes, indicated by the switching ON of the green naphthalimide fluorescence. This underlines their suitability for intracellular pH sensing, with the SiO2-based nanosensor revealing the best performance regarding uptake speed and stability.

Multicolor Polystyrene Nanosensors for the Monitoring of Acidic, Neutral, and Basic pH Values and Cellular Uptake Studies.

A first tricolor fluorescent pH nanosensor is presented, which was rationally designed from biocompatible carboxylated polystyrene nanoparticles and two analyte-responsive molecular fluorophores. Its fabrication involved particle staining with a blue-red-emissive dyad, consisting of a rhodamine moiety responsive to acidic pH values and a pH-inert quinoline fluorophore, followed by the covalent attachment of a fluorescein dye to the particle surface that signals neutral and basic pH values with a green fluorescence. These sensor particles change their fluorescence from blue to red and green, depending on the pH and excitation wavelength, and enable ratiometric pH measurements in the pH range of 3.0-9.0. The localization of the different sensor dyes in the particle core and at the particle surface was confirmed with fluorescence microscopy utilizing analogously prepared polystyrene microparticles. To show the application potential of these polystyrene-based multicolor sensor particles, fluorescence microscopy studies with a human A549 cell line were performed, which revealed the cellular uptake of the pH nanosensor and the differently colored emissions in different cell organelles, that is, compartments of the endosomal-lysosomal pathway. Our results demonstrate the underexplored potential of biocompatible polystyrene particles for multicolor and multianalyte sensing and bioimaging utilizing hydrophobic and/or hydrophilic stimuli-responsive luminophores.

Analyzing the surface of functional nanomaterials-how to quantify the total and derivatizable number of functional groups and ligands.

Functional nanomaterials (NM) of different size, shape, chemical composition, and surface chemistry are of increasing relevance for many key technologies of the twenty-first century. This includes polymer and silica or silica-coated nanoparticles (NP) with covalently bound surface groups, semiconductor quantum dots (QD), metal and metal oxide NP, and lanthanide-based NP with coordinatively or electrostatically bound ligands, as well as surface-coated nanostructures like micellar encapsulated NP. The surface chemistry can significantly affect the physicochemical properties of NM, their charge, their processability and performance, as well as their impact on human health and the environment. Thus, analytical methods for the characterization of NM surface chemistry regarding chemical identification, quantification, and accessibility of functional groups (FG) and surface ligands bearing such FG are of increasing importance for quality control of NM synthesis up to nanosafety. Here, we provide an overview of analytical methods for FG analysis and quantification with special emphasis on bioanalytically relevant FG broadly utilized for the covalent attachment of biomolecules like proteins, peptides, and oligonucleotides and address method- and material-related challenges and limitations. Analytical techniques reviewed include electrochemical titration methods, optical assays, nuclear magnetic resonance and vibrational spectroscopy, as well as X-ray based and thermal analysis methods, covering the last 5-10 years. Criteria for method classification and evaluation include the need for a signal-generating label, provision of either the total or derivatizable number of FG, need for expensive instrumentation, and suitability for process and production control during NM synthesis and functionalization.

Safe-by-Design part II: A strategy for balancing safety and functionality in the different stages of the innovation process.

Manufactured nanomaterials have the potential to impact an exceedingly wide number of industries and markets ranging from energy storage, electronic and optical devices, light-weight construction to innovative medical approaches for diagnostics and therapy. In order to foster the development of safer nanomaterial-containing products, two main aspects are of major interest: their functional performance as well as their safety towards human health and the environment. In this paper a first proposal for a strategy is presented to link the functionality of nanomaterials with safety aspects. This strategy first combines information on the functionality and safety early during the innovation process and onwards, and then identifies Safe-by-Design (SbD) actions that allow for optimisation of both aspects throughout the innovation process. The strategy encompasses suggestions for the type of information needed to balance functionality and safety to support decision making in the innovation process. The applicability of the strategy is illustrated using a literature-based case study on carbon nanotube-based transparent conductive films. This is a first attempt to identify information that can be used for balancing functionality and safety in a structured way during innovation processes.

High-dose intranasal application of titanium dioxide nanoparticles induces the systemic uptakes and allergic airway inflammation in asthmatic mice.

BACKGROUND: Titanium dioxide nanoparticles (TiO2 NPs) have a wide range of applications in several industrial and biomedical domains. Based on the evidence, the workers exposed to inhaled nanosized TiO2 powder are more susceptible to the risks of developing respiratory diseases. Accordingly, this issue has increasingly attracted the researchers' interest in understanding the consequences of TiO2 NPs exposure. Regarding this, the present study was conducted to analyze the local effects of TiO2 NPs on allergic airway inflammation and their uptake in a mouse model of ovalbumin (OVA)-induced allergic airway inflammation. METHODS: For the purpose of the study, female BALB/c mice with or without asthma were intranasally administered with TiO2 NPs. The mice were subjected to histological assessment, lung function testing, scanning electron microscopy (SEM), inductively coupled plasma mass spectrometry (ICP-MS), and NP uptake measurement. In addition, T helper (Th) 1/Th2 cytokines were evaluated in the lung homogenate using the enzyme-linked immunosorbent assay. RESULTS: According to the results, the mice receiving OVA alone or OVA plus TiO2 NPs showed eosinophilic infiltrates and mucus overproduction in the lung tissues, compared to the controls. Furthermore, a significant elevation was observed in the circulating Th2 cytokines, including interleukin (IL)-4, IL-5, and IL-13 after NP exposure. The TiO2 NPs were taken up by alveolar macrophages at different time points. As the results of the SEM and ICP-MS indicated, TiO2 NPs were present in most of the organs in both asthmatic and non-asthmatic mice. CONCLUSION: Based on the findings of the current study, intranasally or inhalation exposure to high-dose nanosized TiO2 particles appears to exacerbate the allergic airway inflammation and lead to systemic uptake in extrapulmonary organs. These results indicate the very important need to investigate the upper limit of intranasally or inhalation exposure to nanosized TiO2 particles in occupational and environmental health policy.

Mechanical strain mimicking breathing amplifies alterations in gene expression induced by SiO2 NPs in lung epithelial cells.

The effects of engineered nanomaterials on human health are still intensively studied in order to facilitate their safe application. However, relatively little is known how mechanical strain as induced in alveolar epithelial cells by breathing movements modifies biological responses to nanoparticles (NPs). In this study, A549 cells as a model for alveolar epithelial cells were exposed to 25 nm amorphous colloidal silica NPs under dynamic and static culture conditions. Gene array data, qPCR, and ELISA revealed an amplified effect of NPs when cells were mechanically stretched in order to model the physiological mechanical deformation during breathing. In contrast, treatment of cells with either strain or NPs alone only led to minor changes in gene expression or interleukin-8 (IL-8) secretion. Confocal microscopy revealed that stretching does not lead to an increased internalization of NPs, indicating that elevated intracellular NP accumulation is not responsible for the observed effect. Gene expression alterations induced by combined exposure to NPs and mechanical strain showed a high similarity to those known to be induced by TNF-α. This study suggests that the inclusion of mechanical strain into in vitro models of the human lung may have a strong influence on the test results.

Distribution of SiO2 nanoparticles in 3D liver microtissues.

INTRODUCTION: Nanoparticles (NPs) are used in numerous products in technical fields and biomedicine; their potential adverse effects have to be considered in order to achieve safe applications. Besides their distribution in tissues, organs, and cellular localization, their impact and penetration during the process of tissue formation occurring in vivo during liver regeneration are critical steps for establishment of safe nanomaterials. MATERIALS AND METHODS: In this study, 3D cell culture of human hepatocarcinoma cells (HepG2) was used to generate cellular spheroids, serving as in vitro liver microtissues. In order to determine their differential distribution and penetration depth in HepG2 spheroids, SiO2 NPs were applied either during or after spheroid formation. The NP penetration was comprehensively studied using confocal laser scanning microscopy and scanning electron microscopy. RESULTS: Spheroids were exposed to 100 µg mL-1 SiO2 NPs either at the beginning of spheroid formation, or during or after formation of spheroids. Microscopy analyses revealed that NP penetration into the spheroid is limited. During and after spheroid formation, SiO2 NPs penetrated about 20 µm into the spheroids, corresponding to about three cell layers. In contrast, because of the addition of SiO2 NPs simultaneously to cell seeding, NP agglomerates were located also in the spheroid center. Application of SiO2 NPs during the process of spheroid formation had no impact on final spheroid size. CONCLUSION: Understanding the distribution of NPs in tissues is essential for biomedical applications. The obtained results indicate that NPs show only limited penetration into already formed tissue, which is probably caused by the alteration of the tissue structure and cell packing density during the process of spheroid formation.

Targeted T1 Magnetic Resonance Imaging Contrast Enhancement with Extraordinarily Small CoFe2O4 Nanoparticles.

Extraordinarily small (2.4 nm) cobalt ferrite nanoparticles (ESCIoNs) were synthesized by a one-pot thermal decomposition approach to study their potential as magnetic resonance imaging (MRI) contrast agents. Fine size control was achieved using oleylamine alone, and annular dark-field scanning transmission electron microscopy revealed highly crystalline cubic spinel particles with atomic resolution. Ligand exchange with dimercaptosuccinic acid rendered the particles stable in physiological conditions with a hydrodynamic diameter of 12 nm. The particles displayed superparamagnetic properties and a low r2/ r1 ratio suitable for a T1 contrast agent. The particles were functionalized with bile acid, which improved biocompatibility by significant reduction of reactive oxygen species generation and is a first step toward liver-targeted T1 MRI. Our study demonstrates the potential of ESCIoNs as T1 MRI contrast agents.

Kinetic and spectroscopic responses of pH-sensitive nanoparticles: influence of the silica matrix.

Intracellular pH sensing with fluorescent nanoparticles is an emerging topic as pH plays several roles in physiology and pathologic processes. Here, nanoparticle-sized pH sensors (diameter far below 50 nm) for fluorescence imaging have been described. Consequently, a fluorescent derivative of pH-sensitive hydroxypyrene with pK a = 6.1 was synthesized and subsequently embedded in core and core-shell silica nanoparticles via a modified Stöber process. The detailed fluorescence spectroscopic characterization of the produced nanoparticles was carried out for retrieving information about the environment within the nanoparticle core. Several steady-state and time-resolved fluorescence spectroscopic methods hint to the screening of the probe molecule from the solvent, but it sustained interactions with hydrogen bonds similar to that of water. The incorporation of the indicator dye in the water-rich silica matrix neither changes the acidity constant nor dramatically slows down the protonation kinetics. However, cladding by another SiO2 shell leads to the partial substitution of water and decelerating the response of the probe molecule toward pH. The sensor is capable of monitoring pH changes in a physiological range by using ratiometric fluorescence excitation with λ ex = 405 nm and λ ex = 488 nm, as confirmed by the confocal fluorescence imaging of intracellular nanoparticle uptake.

Silica Nanoparticles for Intracellular Protein Delivery: a Novel Synthesis Approach Using Green Fluorescent Protein.

In this study, a novel approach for preparation of green fluorescent protein (GFP)-doped silica nanoparticles with a narrow size distribution is presented. GFP was chosen as a model protein due to its autofluorescence. Protein-doped nanoparticles have a high application potential in the field of intracellular protein delivery. In addition, fluorescently labelled particles can be used for bioimaging. The size of these protein-doped nanoparticles was adjusted from 15 to 35 nm using a multistep synthesis process, comprising the particle core synthesis followed by shell regrowth steps. GFP was selectively incorporated into the silica matrix of either the core or the shell or both by a one-pot reaction. The obtained nanoparticles were characterised by determination of particle size, hydrodynamic diameter, ζ-potential, fluorescence and quantum yield. The measurements showed that the fluorescence of GFP was maintained during particle synthesis. Cellular uptake experiments demonstrated that the GFP-doped nanoparticles can be used as stable and effective fluorescent probes. The study reveals the potential of the chosen approach for incorporation of functional biological macromolecules into silica nanoparticles, which opens novel application fields like intracellular protein delivery.

Bright fluorescent silica-nanoparticle probes for high-resolution STED and confocal microscopy.

In recent years, fluorescent nanomaterials have gained high relevance in biological applications as probes for various fluorescence-based spectroscopy and imaging techniques. Among these materials, dye-doped silica nanoparticles have demonstrated a high potential to overcome the limitations presented by conventional organic dyes such as high photobleaching, low stability and limited fluorescence intensity. In the present work we describe an effective approach for the preparation of fluorescent silica nanoparticles in the size range between 15 and 80 nm based on L-arginine-controlled hydrolysis of tetraethoxysilane in a biphasic cyclohexane-water system. Commercially available far-red fluorescent dyes (Atto647N, Abberior STAR 635, Dy-647, Dy-648 and Dy-649) were embedded covalently into the particle matrix, which was achieved by aminosilane coupling. The physical particle attributes (particle size, dispersion, degree of agglomeration and stability) and the fluorescence properties of the obtained particles were compared to particles from commonly known synthesis methods. As a result, the spectroscopic characteristics of the presented monodisperse dye-doped silica nanoparticles were similar to those of the free uncoupled dyes, but indicate a much higher photostability and brightness. As revealed by dynamic light scattering and ζ-potential measurements, all particle suspensions were stable in water and cell culture medium. In addition, uptake studies on A549 cells were performed, using confocal and stimulated emission depletion (STED) microscopy. Our approach allows for a step-by-step formation of dye-doped silica nanoparticles in the form of dye-incorporated spheres, which can be used as versatile fluorescent probes in confocal and STED imaging.

Metal-enhanced luminescence in colloidal solutions of CdSe and metal nanoparticles: investigation of density dependence and optical band overlap.

The photoluminescence (PL) of semiconductor nanoparticles (SNP) is strongly modified when the semiconductor is in the proximity of a metal surface or a metal nanoparticle (MNP). The effect may be due to two different phenomena which are (a) (Förster) resonant energy transfer ((F)RET) between the semiconductor and the metal and (b) the enhanced electric field around metallic structures that arises from surface plasmon oscillations. Here we present experimental evidence for enhancement and quenching of the PL of dilute SNP colloidal solutions depending on the amount of admixed MNP and the position of the MNP plasmon band with respect to the excitation wavelength and the optical bands in the SNP. The average distance between an MNP and its next neighbor MNP is varied between ∼0.1 and 2 µm by varying the MNP concentration, whereas that between MNP and SNP as well as between SNP and SNP is kept at about 0.1 µm. A model function based on the rate equations of the system is developed that yields a satisfactory description of the measured data by considering solely FRET between the particle species. The derived function is an extension of the Stern-Volmer equation, as it not only accounts for the energy transfer from the fluorescent SNPs to the MNPs, but also for the transfer of excitation from MNPs to SNPs and between MNPs. This theory provides a deeper insight into the mechanisms of metal-enhanced fluorescence and fluorescence quenching phenomena.

Synthesis of tumor-associated MUC1-glycopeptides and their multivalent presentation by functionalized gold colloids.

The mucin MUC1 is a glycoprotein involved in fundamental biological processes, which can be found over-expressed and with a distinctly altered glycan pattern on epithelial tumor cells; thus it is a promising target structure in the quest for effective carbohydrate-based cancer vaccines and immunotherapeutics. Natural glycopeptide antigens indicate only a low immunogenicity and a T-cell independent immune response; however, this major drawback can be overcome by coupling of glycopeptide antigens multivalently to immunostimulating carrier platforms. In particular, gold nanoparticles are well suited as templates for the multivalent presentation of glycopeptide antigens, due to their remarkably high surface-to-volume ratio in combination with their high biostability. In this work the synthesis of novel MUC1-glycopeptide antigens and their coupling to gold nanoparticles of different sizes are presented. In addition, the development of a new dot-blot immunoassay to test the potential antigen-antibody binding is introduced.