Christina Graf

Multivalency as a Chemical Organization and Action Principle

Multivalent interactions can be applied universally for a targeted strengthening of an interaction between different interfaces or molecules. The binding partners form cooperative, multiple receptor-ligand interactions that are based on individually weak, noncovalent bonds and are thus generally reversible. Hence, multi- and polyvalent interactions play a decisive role in biological systems for recognition, adhesion, and signal processes. The scientific and practical realization of this principle will be demonstrated by the development of simple artificial and theoretical models, from natural systems to functional, application-oriented systems. In a systematic review of scaffold architectures, the underlying effects and control options will be demonstrated, and suggestions will be given for designing effective multivalent binding systems, as well as for polyvalent therapeutics.

A General Method To Coat Colloidal Particles with Silica

We describe a general one-pot method for coating colloidal particles with amorphous titania. Various colloidal particles such as silica particles, large silver colloids, gibbsite platelets, and polystyrene spheres were successfully coated with a titania shell. Although there are several ways of coating different particles with titania in the literature, each of these methods is applicable to only one type of material. The present method is especially useful for giving the opportunity to cover many types of colloidal particles with titania and forgoes the use of a coupling agent or a precoating step. We can produce particles with a smooth titania layer of tunable thickness. The monodispersity, which improves during particle growth, and the high refractive index of titania make these particles potential candidates for photonic crystal applications. We also describe various ways of fabricating hollow titania shells, which have been intensively studied in the literature for their applications in electronics, catalysis, separations, and diagnostics. Note that our method initially produces amorphous shells on the particles, but these can be easily turned into crystalline titania by a calcination step. We also find that the growth of titania is a surface-reaction-limited process.

Skin Penetration and Cellular Uptake of Amorphous Silica Nanoparticles with Variable Size, Surface Functionalization, and Colloidal Stability

In this study, the skin penetration and cellular uptake of amorphous silica particles with positive and negative surface charge and sizes ranging from 291 ± 9 to 42 ± 3 nm were investigated. Dynamic light scattering measurements and statistical analyses of transmission electron microscopy images were used to estimate the degree of particle aggregation, which was a key aspect to understanding the results of the in vitro cellular uptake experiments. Despite partial particle aggregation occurring after transfer in physiological media, particles were taken up by skin cells in a size-dependent manner. Functionalization of the particle surface with positively charged groups enhanced the in vitro cellular uptake. However, this positive effect was contrasted by the tendency of particles to form aggregates, leading to lower internalization ratios especially by primary skin cells. After topical application of nanoparticles on human skin explants with partially disrupted stratum corneum, only the 42 ± 3 nm particles were found to be associated with epidermal cells and especially dendritic cells, independent of their surface functionalization. Considering the wide use of nanomaterials in industries and the increasing interest for applications in pharmaceutics and cosmetics versus the large number of individuals with local or spread impairment of the skin barrier, e.g., patients with atopic dermatitis and chronic eczema, a careful dissection of nanoparticle-skin surface interactions is of high relevance to assess possible risks and potentials of intended and unintended particle exposure.

Surface Functionalization of Silica Nanoparticles Supports Colloidal Stability in Physiological Media and Facilitates Internalization in Cells

The influence of the surface functionalization of silica particles on their colloidal stability in physiological media is studied and correlated with their uptake in cells. The surface of 55 ± 2 nm diameter silica particles is functionalized by amino acids or amino- or poly(ethylene glycol) (PEG)-terminated alkoxysilanes to adjust the zeta potential from highly negative to positive values in ethanol. A transfer of the particles into water, physiological buffers, and cell culture media reduces the absolute value of the zeta potential and changes the colloidal stability. Particles stabilized by L-arginine, L-lysine, and amino silanes with short alkyl chains are only moderately stable in water and partially in PBS or TRIS buffer, but aggregate in cell culture media. Nonfunctionalized, N-(6-aminohexyl)-3-aminopropyltrimethoxy silane (AHAPS), and PEG-functionalized particles are stable in all media under study. The high colloidal stability of positively charged AHAPS-functionalized particles scales with the ionic strength of the media, indicating a mainly electrostatical stabilization. PEG-functionalized particles show, independently from the ionic strength, no or only minor aggregation due to additional steric stabilization. AHAPS stabilized particles are readily taken up by HeLa cells, likely as the positive zeta potential enhances the association with the negatively charged cell membrane. Positively charged particles stabilized by short alkyl chain aminosilanes adsorb on the cell membrane, but are weakly taken up, since aggregation inhibits their transport. Nonfunctionalized particles are barely taken up and PEG-stabilized particles are not taken up at all into HeLa cells, despite their high colloidal stability. The results indicate that a high colloidal stability of nanoparticles combined with an initial charge-driven adsorption on the cell membrane is essential for efficient cellular uptake.

Highly monodisperse water-dispersable iron oxide nanoparticles for biomedical applications

We demonstrate a unique approach for preparing high quality iron oxide (Fe3O4) nanoparticles functionalized by newly developed multifunctional dendron ligands for biomedical applications. These particles are suitable for magnetic resonance imaging (MRI), highly stable in aqueous solutions as well as physiological media and not cytotoxic. In particular, oleic acid capped Fe3O4 particles (d = 12 ± 0.8 nm) were modified in a ligand exchange process by investigating several dendron ligands of variable size and an adjustable number of polar end groups. The dendron based ligands lead only to a slight increase in hydrodynamic diameter of the nanoparticles after the ligand exchange process (∼6 nm). They also allow an adjustment of the particle polarity as well as a gradually variable surface functionalization. Light scattering, transmission electron microscopy, and visible spectroscopy studies show consistently that the dendron-capped iron oxide nanoparticles exhibit excellent stability in various physiological media as well as aqueous solutions in a broad pH range. It is also demonstrated by magnetic resonance studies that the magnetic relaxivity is almost not affected by the ligand exchange. Therefore, such small particles might be of specific interest for cardiovascular MRI and MRI of extravascular targets. In addition, the present approach opens new possibilities for the specific linking of biomolecules to the particle surface, which can be beneficial for various biological sensing and therapeutic applications. The cytotoxicity of the Fe3O4 nanoparticles was evaluated using the WST-8 assay. In the examined concentration range up to 100 μg Fe/mL no significant decrease in cell viability was detected.

Photoactivation of CdSe/ZnS Quantum Dots Embedded in Silica Colloids

A study of the influence of the local environment on the light-induced luminescence enhancement of CdSe/ZnS quantum dots (QD) embedded in silica colloids that are dispersed in various solvents is presented. The photoluminescence of the embedded QD is enhanced up to a factor of ten upon photoactivation by ultraviolet or visible light. This enhancement is strongly dependent on the local environment. The thickness-dependent permeability of the silica shell covering the QD controls the influence of the solvent on the QD. If foreign ions are present the activation state is stabilized after termination of the activation, whereas in their absence the process is partially reversible. A new qualitative model for the photoactivation of QD in various environments is developed. It comprises light-induced passivation and subsequent oxidation processes. The embedded QD also retain their fluorescence quantum yield inside living cells. Moreover, they can be activated for many hours in living cells by laser radiation in the visible regime.

PVP-coated, negatively charged silver nanoparticles: A multi-center study of their physicochemical characteristics, cell culture and in vivo experiments

Summary PVP-capped silver nanoparticles with a diameter of the metallic core of 70 nm, a hydrodynamic diameter of 120 nm and a zeta potential of −20 mV were prepared and investigated with regard to their biological activity. This review summarizes the physicochemical properties (dissolution, protein adsorption, dispersability) of these nanoparticles and the cellular consequences of the exposure of a broad range of biological test systems to this defined type of silver nanoparticles. Silver nanoparticles dissolve in water in the presence of oxygen. In addition, in biological media (i.e., in the presence of proteins) the surface of silver nanoparticles is rapidly coated by a protein corona that influences their physicochemical and biological properties including cellular uptake. Silver nanoparticles are taken up by cell-type specific endocytosis pathways as demonstrated for hMSC, primary T-cells, primary monocytes, and astrocytes. A visualization of particles inside cells is possible by X-ray microscopy, fluorescence microscopy, and combined FIB/SEM analysis. By staining organelles, their localization inside the cell can be additionally determined. While primary brain astrocytes are shown to be fairly tolerant toward silver nanoparticles, silver nanoparticles induce the formation of DNA double-strand-breaks (DSB) and lead to chromosomal aberrations and sister-chromatid exchanges in Chinese hamster fibroblast cell lines (CHO9, K1, V79B). An exposure of rats to silver nanoparticles in vivo induced a moderate pulmonary toxicity, however, only at rather high concentrations. The same was found in precision-cut lung slices of rats in which silver nanoparticles remained mainly at the tissue surface. In a human 3D triple-cell culture model consisting of three cell types (alveolar epithelial cells, macrophages, and dendritic cells), adverse effects were also only found at high silver concentrations. The silver ions that are released from silver nanoparticles may be harmful to skin with disrupted barrier (e.g., wounds) and induce oxidative stress in skin cells (HaCaT). In conclusion, the data obtained on the effects of this well-defined type of silver nanoparticles on various biological systems clearly demonstrate that cell-type specific properties as well as experimental conditions determine the biocompatibility of and the cellular responses to an exposure with silver nanoparticles.

Qualitative detection of single submicron and nanoparticles in human skin by scanning transmission x-ray microscopy

First results on single particle detection in human skin samples by x-ray microscopy are reported. 94+/-6 and 161+/-13 nm gold core particles with silica shells and 298+/-11 nm silica particles coated with a gold shell on ultramicrotome sections of human skin were determined. The particles were applied on fresh intact skin samples, which were sectioned prior to imaging. After screening the sections by conventional microscopy techniques, defined areas of interest were qualitatively investigated by scanning transmission x-ray microscopy at the Swiss Light Source. In studies on the percutaneous penetration of 161+/-13 nm particles on human skin samples, x-ray microscopy yielded high-resolution images of single particles spreading on the superficial layer of the stratum corneum and on the epithelium in superficial parts of hair follicles. No deeper penetration was observed. The present work using x-ray microscopy provides the unique opportunity to study qualitative penetration processes and membrane-particle interactions on the level of single particles. This goes beyond present approaches using optical microscopy. Further improvement of this approach will allow one to study particles with different physicochemical properties and surface modifications, including responses of the exposed tissue.

Skin barrier disruptions in tape stripped and allergic dermatitis models have no effect on dermal penetration and systemic distribution of AHAPS-functionalized silica nanoparticles

The skin is a potential site of entry for nanoparticles (NP) but the role of disease-associated barrier disturbances on the path and extent of skin penetration of NP remains to be characterized. Silica nanoparticles (SiO2-NP) possess promising potential for various medical applications. Here, effects of different skin barrier disruptions on the penetration of N-(6-aminohexyl)-aminopropyltrimethoxysilane (AHAPS) functionalized SiO2-NP were studied. AHAPS-SiO2-NP (55±6 nm diameter) were topically applied on intact, tape stripped or on inflamed skin of SKH1 mice with induced allergic contact dermatitis for one or five consecutive days, respectively. Penetration of AHAPS-SiO2-NP through the skin was not observed regardless of the kind of barrier disruption. However, only after subcutaneous injection, AHAPS-SiO2-NP were incorporated by macrophages and transported to the regional lymph node only. Adverse effects on cells or tissues were not observed. In conclusion, AHAPS-SiO2-NP seem to not cross the normal or perturbed mouse skin. From the clinical editor: Skin is a potential site of entry for nanoparticles; however, it is poorly understood how skin diseases may alter this process. In tape-stripped skin and allergic contact dermatitis models the delivery properties of AHAPS-SiO2 nanoparticles remained unchanged, and in neither case were these NP-s able to penetrate the skin. No adverse effects were noted on the skin in these models and control mice.

Overview about the localization of nanoparticles in tissue and cellular context by different imaging techniques

Summary The increasing interest and recent developments in nanotechnology pose previously unparalleled challenges in understanding the effects of nanoparticles on living tissues. Despite significant progress in in vitro cell and tissue culture technologies, observations on particle distribution and tissue responses in whole organisms are still indispensable. In addition to a thorough understanding of complex tissue responses which is the domain of expert pathologists, the localization of particles at their sites of interaction with living structures is essential to complete the picture. In this review we will describe and compare different imaging techniques for localizing inorganic as well as organic nanoparticles in tissues, cells and subcellular compartments. The visualization techniques include well-established methods, such as standard light, fluorescence, transmission electron and scanning electron microscopy as well as more recent developments, such as light and electron microscopic autoradiography, fluorescence lifetime imaging, spectral imaging and linear unmixing, superresolution structured illumination, Raman microspectroscopy and X-ray microscopy. Importantly, all methodologies described allow for the simultaneous visualization of nanoparticles and evaluation of cell and tissue changes that are of prime interest for toxicopathologic studies. However, the different approaches vary in terms of applicability for specific particles, sensitivity, optical resolution, technical requirements and thus availability, and effects of labeling on particle properties. Specific bottle necks of each technology are discussed in detail. Interpretation of particle localization data from any of these techniques should therefore respect their specific merits and limitations as no single approach combines all desired properties.