Spontaneous confocal Raman microscopy--a tool to study the uptake of nanoparticles and carbon nanotubes into cells

Confocal Raman microscopy as a label-free technique was applied to study the uptake and internalization of poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) and carbon nanotubes (CNTs) into hepatocarcinoma human HepG2 cells. Spontaneous confocal Raman spectra was recorded from the cells exposed to oxidized CNTs and to PLGA NPs. The Raman spectra showed bands arising from the cellular environment: lipids, proteins, nucleic acids, as well as bands characteristic for either PLGA NPs or CNTs. The simultaneous generation of Raman bands from the cell and nanomaterials from the same spot proves internalization, and also indicates the cellular region, where the nanomaterial is located. For PLGA NPs, it was found that they preferentially co-localized with lipid bodies, while the oxidized CNTs are located in the cytoplasm

Nanoparticle uptake and their co-localization with cell compartments: a confocal Raman microscopy study at single cell level

Confocal Raman Microscopy, a non-invasive, non-destructive and label-free technique, was employed to study the uptake and localization of nanoparticles (NPs) in the Hepatocarcinoma human cell line HepG2 at the level of single cells. Cells were exposed to carbon nanotubes (CNTs) the surface of which was engineered with polyelectrolytes and lipid layers, aluminium oxide and cerium dioxide nanoparticles. Raman spectra deconvolution was applied to obtain the spatial distributions of NPs together with lipids/proteins in cells. The colocalization of the NPs with different intracellular environments, lipid bodies, protein and DNA, was inferred. Lipid coated CNTs associated preferentially with lipid rich regions, whereas polyelectrolyte coated CNTs were excluded from lipid rich regions. Al2O3 NPs were found in the cytoplasm. CeO2 NPs were readily taken up and have been observed all over the cell. Raman z-scans proved the intracellular distribution of the respective NPs

Solvent Effects on the Structure−Property Relationship of Redox-Active Self-Assembled Nanoparticle−Polyelectrolyte−Surfactant Composite Thin Films: Implications for the Generation of Bioelectrocatalytic Signals in Enzyme-Containing Assemblies

The search for strategies to improve the performance of bioelectrochemical platforms based on supramolecular materials has received increasing attention within the materials science community, where the main objective is to develop low-cost and flexible routes using self-assembly as a key enabling process. Important contributions to the performance of such bioelectrochemical devices have been made based on the integration and supramolecular organization of redox-active polyelectrolyte−surfactant complexes on electrode supports. Here, we examine the influence of the processing solvent on the interplay between the supramolecular mesoorganization and the bioelectrochemical properties of redox-active self-assembled nanoparticle−polyelectrolyte−surfactant nanocomposite thin films. Our studies reveal that the solvent used in processing the supramolecular films and the presence of metal nanoparticles not only have a substantial influence in determining the mesoscale organization and morphological characteristics of the film but also have a strong influence on the efficiency and performance of the bioelectrochemical system. In particular, a higher bioelectrochemical response is observed when nanocomposite supramolecular films were cast from aqueous solutions. These observations seem to be associated with the fact that the use of aqueous solvents increases the hydrophilicity of the film, thus favoring the access of glucose, particularly at low concentrations. We believe that these results improve our current understanding of supramolecular nanocomposite materials generated via polyelectrolyte−surfactant complexes, in order to use the processing conditions as a variable to improve the performance of bioelectrochemical devices.Facultad de Ciencias ExactasInstituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

Lipid layers on polyelectrolyte multilayer supports

The mechanism of formation of supported lipid layers from phosphatidylcholine and phosphatidylserine vesicles in solution on polyelectrolyte multilayers was studied by a variety of experimental techniques. The interaction of zwitterionic and acidic lipid vesicles, as well as their mixtures, with polyelectrolyte supports was followed in real time by micro-gravimetry. The fabricated lipid–polyelectrolyte composite structures on top of multilayer coated colloidal particles were characterized by flow cytometry and imaging techniques. Lipid diffusion over the macroscopic scale was quantified by fluorescence recovery after photobleaching, and the diffusion was related to layer connectivity. The phospholipid–polyelectrolyte binding mechanism was investigated by infrared spectroscopy. A strong interaction of polyelectrolyte primary amino groups with phosphate and carboxyl groups of the phospholipids, leading to dehydration, was observed. Long-range electrostatic attraction was proven to be essential for vesicle spreading and rupture. Fusion of lipid patches into a homogeneous bilayer required lateral mobility of the lipids on the polyelectrolyte support. The binding of amino groups to the phosphate group of the zwitterionic lipids was too weak to induce vesicle spreading, but sufficient for strong adsorption. Only the mixture of phosphatidylcholine and phosphatidylserine resulted in the spontaneous formation of bilayers on polyelectrolyte multilayers. The adsorption of phospholipids onto multilayers displaying quarternary ammonium polymers produced a novel 3D lipid polyelectrolyte structure on colloidal particles.<br/

Cytotoxicity effects of metal oxide nanoparticles in human tumor cell lines

Metallic and metal oxide nanoparticles (Nps) have a wide range of applications in various settings including household, cosmetics and chemical industries, as well as for coatings. Nevertheless, an in-depth study of the potential toxic effects of these Nps is still needed, in order to fulfill the mandatory requirement of ensuring the safety of workers, patients and the general public. In this study, Quick Cell colorimetric assays were used to evaluate the in vitro toxicity of different metal oxide Nps [Fe(II,III)Ox, TiOx, ZnO and CeO2] in several cell lines. The ZnO Nps were found to be highly toxic, with a lethal dose ≥100 μg/ml for all the cell lines studied. Western blot was also used to test the ability of the different Nps to activate the complement pathway. However, no activation of this cascade was observed when the Nps were added. In addition, the aggregation state and charge of the Nps in culture media was studied by dynamic light scattering (DLS) and measurement of zeta potential. Transmission Electron Microscopy was used to analyze Np uptake and localization at the cellular level

Elasticity and adhesion of resting and lipopolysaccharide-stimulated macrophages

AbstractColloidal Force Microscopy was employed to study the viscoelastic and adhesive properties of macrophages upon stimulation with lipopolysaccharide (LPS). Force vs. distance measurements were performed. The adhesion of LPS-stimulated cells (separation force=37±3nN) was almost twice as high as that of resting macrophages (16±1nN). Upon retraction pulling of membrane tethers was observed. Tether lengths and forces at which rupture take place did not depend on stimulation. The reduced Young’s modulus K, a measure of cytoskeleton elasticity, was three times lower than that of the control. The data show that LPS has profound effects on cytomechanical and adhesion properties of macrophages

Donnan equilibrium and osmotic pressure in hollow polyelectrolyte microcapsules

A simple theoretical model of the Donnan equilibrium is applied to studies of distributions of ions between the external and internal volumes of hollow polyelectrolyte capsules as well as of concomitant osmotic pressure load on a capsule wall resulting from these distributions and the presence of polyanion. The model system consist of dispersed polyelectrolyte capsules in an electrolyte solution whereby two cases are considered with respect to the presence of the polyanion, either in the inner or external solution. It is assumed that the capsule wall is impermeable to polyanion, but water and all ions can freely penetrate. The model predictions are summarized by presenting the difference between the external pH, the pH of the inner solution and the osmotic pressure difference across the capsule wall, both in the dependence of sodium chloride concentration.Teoretičen model Donnanovega ravnovesja smo uporabili v študiji porazdelitve malih ionov med zunanjim in notranjim volumnom votlih polielektrolitskih kapsul, zaradi česar se spreminja tudi osmozna obremenitev stene kapsul ob prisotnosti poliiona. Model opisuje disperzijo polielektrolitskih kapsul v elektrolitski raztopini, kjer je poliion prisoten v zunanji ali notranji raztopini kapsule. Predpostavili smo, da je stena kapsule neprepustna za poliion in prepustna za vodo in majhne ione. Modelne napovedi so podane kot vrednosti pH notranje raztopine v odvisnosti od natrijevega klorida in razlike osmoznih tlakov na steno kapsule v odvisnosti od natrijevega klorida

Flow cytometry of HEK 293T cells interacting with polyelectrolyte multilayer capsules containing fluorescein-labeled poly(acrylic acid) as a pH sensor

Polyelectrolyte multilayer sensor capsules, 5 m in diameter, which contained fluorescein-labeled poly(acrylic acid) (PAAAF) as pH-sensitive reporter molecules, were fabricated and employed to explore their endocytotic uptake into HEK 293T cells by flow cytometry. The percentage of capsules residing in the endolysosomal compartment was estimated from the fluorescence intensity decrease caused by acidification. Capsules attached to the extracellular surface of the plasma membrane were identified by trypan blue quenching. The number of capsules in the cytoplasm was rather small, being below the detection limit of the method. The advantages of polyelectrolyte multilayer capsules are that the fluorophore is protected from interaction with cellular compartments and that the multilayer can be equipped with additional functions

Dynamic Light Scattering from Oriented, Rotating Particles: A Theoretical Study and Comparison to Electrorotation Data

In recent years, electrorotation has developed in the field of biology as a technique for characterization of single cell dielectric properties. Applications to colloidal particles are scarce, although the method provides information on the electrical structure of the particle's interior. The method explores the frequency-dependent polarizability difference of the particles and the suspension medium by using rotating field in the frequency range of 1 kHz to 200 MHz to induce individual particle rotation. To allow interpretation of the electrorotation spectra measured with light scattering techniques, the theoretical autocorrelation function of light scattered from rotating particles of cylindrical symmetry has been calculated. All particles were assumed to possess a single scattering site and the rotation axes were assumed to be parallel. An appropriate expression for the rotational diffusion around the longitudinal angle was also derived. Diffusion around the azimuthal angle could be neglected. The theoretical result consists of a well structured autocorrelation function. The theoretical limits of particle size and rotation speed that allow detection of electrorotation were explored. Experimental autocorrelation functions from electrorotation of human red blood cells could be explained theoretically when reasonable parameters for the cell's properties were assumed.Ces précédentes années, l'électrorotation a évolué comme une technique de caractérisation des attributs diélectriques d'une cellule unique. Il n'y a que peu d'applications pour les particules colloidales, bien que la méthode fournisse de l'information sur la structure électrique de l'intérieur de la particule. Cette méthode explore la différence entre la polarisabilité des particules et du moyen de suspension en utilisant des champs électriques tournants avec une fréquence de 1 kHz à 200 MHz pour induire une rotation individuelle de la particule. Afin de permettre l'interprétation des spectres de l'électrorotation mesuré à l'aide des techniques de lumière diffusée, la fonction d'autocorrelation théorique de la lumière diffusée par des particules tournant d'une symétrie cylindrique a été calculée. Nous supposons que toutes les particules ne possèdent qu'une côte diffusante et que les axes de rotation sont parallèles. Il en dérive une expression appropriée pour la diffusion rotationnelle autour de l'angle longitudinal. La diffusion des particules autour de l'angle azimutal est negligée. Le résultat théorique consiste en une fonction d'autocorrelation bien structurée. Les limites théoriques de la grandeur de particule ainsi que la vitesse de rotation, qui permettent la détection de l'électrorotation, ont été explorées. On peut expliquer les fonctions d'autocorrelation expérimentales de l'électrorotation des cellules rouges du sang humain quand on présuppose des paramètres raisonnables pour les attributs cellulaires