Anja Kroeger

Incorporation of Nanoparticles into Polymersomes: Size and Concentration Effects

Because of the rapidly growing field of nanoparticles in therapeutic applications, understanding and controlling the interaction between nanoparticles and membranes is of great importance. While a membrane is exposed to nanoparticles its behavior is mediated by both their biological and physical properties. Constant interplay of these biological and physicochemical factors makes selective studies of nanoparticles uptake demanding. Artificial model membranes can serve as a platform to investigate physical parameters of the process in the absence of any biofunctional molecules and/or supplementary energy. Here we report on photon- and fluorescence-correlation spectroscopic studies of the uptake of nanosized SiO(2) nanoparticles by poly(dimethylsiloxane)-block-poly(2-methyloxazoline) vesicles allowing species selectivity. Analogous to the cell membrane, polymeric membrane incorporates particles using membrane fission and particles wrapping as suggested by cryo-TEM imaging. It is revealed that the incorporation process can be controlled to a significant extent by changing nanoparticles size and concentration. Conditions for nanoparticle uptake and controlled filling of polymersomes are presented.

Mechanical properties of poly(dimethylsiloxane)-block-poly(2-methyloxazoline) polymersomes probed by atomic force microscopy.

Poly(dimethylsiloxane)-block-poly(2-methyloxazoline) (PDMS-b-PMOXA) vesicles were characterized by a combination of dynamic light scattering (DLS), cryogenic transmission electron microscopy (cryo-TEM), and atomic force microscopy imaging and force spectroscopy (AFM). From DLS data, a hydrodynamic radius of ~150 nm was determined, and cryo-TEM micrographs revealed a bilayer thickness of ~16 nm. In AFM experiments on a silicon wafer substrate, adsorption led to a stable spherical caplike conformation of the polymersomes, whereas on mica, adsorption resulted also in vesicle fusion and formation of bilayer patches or multilayer stacks. This indicates a delicate balance between the mechanical stability of PDMS-b-PMOXA polymersomes on one hand and the driving forces for spreading on the other. A Youngs modulus of 17 ± 11 MPa and a bending modulus of 7 ± 5 × 10(-18) J were derived from AFM force spectroscopy measurements. Therefore, the elastic response of the PDMS-b-PMOXA polymersomes to external stimuli is much closer to that of lipid vesicles compared to other types of polymersomes, such as polystyrene-block-poly(acrylic acid) (PS-b-PAA).

Probing bioinspired transport of nanoparticles into polymersomes.

Transmembrane transport: A combination of photon and fluorescence correlation spectroscopies is used to study the internalization of nanoparticles into a minimal model system based on poly(dimethylsiloxane)‐block‐poly(2‐methyloxazoline) vesicles (see picture). These techniques provide information about the kinetics and dynamics of this transport process in real time.

Block copolymers of PS-b-PEO co-assembled with azobenzene-containing homopolymers and their photoresponsive properties

A new concept of the efficient fabrication and design of three layer photoresponsive core–shell–corona (CSC) nanostructures by the co-assembly of diblock copolymers with azobenzene-containing homopolymers is presented. Depending on the azobenzene-containing homopolymer content, the formation of CSC vesicles and CSC micelle-like nanostructures are observed, which are characterized by UV–vis absorption spectroscopy, transmission electron microscopy (TEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). The observed nanostructures confirm the recent computer simulations of Xu et al. (J. Phys. Chem. B, 2010, 114, 1257). All co-assemblies are photoresponsive and show reversible cis–transphotoisomerization. Irradiation of the spherical CSC micelle-like nanostructures by polarized light induces their deformation to ellipsoidal-core-within-ellipsoidal-shell and ellipsoidal-core-within-spherical-shell nanostructures, respectively. The type of photoinduced deformation depends on the initial size of the spherical CSC micelle-like nanostructures. These CSC nanostructures could be used as photo-controlled templates for complex nanostructures.

Detergent Properties Influence the Stability of the Glycophorin A Transmembrane Helix Dimer in Lysophosphatidylcholine Micelles

Detergents might affect membrane protein structures by promoting intramolecular interactions that are different from those found in native membrane bilayers, and fine-tuning detergent properties can be crucial for obtaining structural information of intact and functional transmembrane proteins. To systematically investigate the influence of the detergent concentration and acyl-chain length on the stability of a transmembrane protein structure, the stability of the human glycophorin A transmembrane helix dimer has been analyzed in lyso-phosphatidylcholine micelles of different acyl-chain length. While our results indicate that the transmembrane protein is destabilized in detergents with increasing chain-length, the diameter of the hydrophobic micelle core was found to be less crucial. Thus, hydrophobic mismatch appears to be less important in detergent micelles than in lipid bilayers and individual detergent molecules appear to be able to stretch within a micelle to match the hydrophobic thickness of the peptide. However, the stability of the GpA TM helix dimer linearly depends on the aggregation number of the lyso-PC detergents, indicating that not only is the chemistry of the detergent headgroup and acyl-chain region central for classifying a detergent as harsh or mild, but the detergent aggregation number might also be important.

Dendrimer Influenced Supramolecular Structure Formation of Block Copolymers? II. Dendrimer Concentration Dependence

The dendrimer concentration dependence of the supramolecular structure formation of polystyrene-block-poly(acrylic acid) in dioxane/THF was investigated as a function of water content. The distribution as well as the localization of the dendrimer units inside the formed aggregates were determined by comparative studies of turbidity measurements and transmission electron microscopy. The strong and specific interactions present between the amine groups of the dendrimer (PAMAM) and the carboxylic acid residues of PAA in the copolymer have a strong influence on the structure formation. The PAMAM concentration as well as the character of the terminal groups of the dendrimer influence the strength of these interactions and consequently affect the structure formation process. As shown by fluorescence quenching experiments, on all supramolecular hierarchical structure levels, and specifically in vesicles, the dendrimer is coated by the PAA chains of the block copolymer due to the strong interactions; since the PAA blocks are connected to the PS blocks, which form the corona, the dendrimer is surrounded by PS chains and is thus encapsulated into the hydrophobic regions of the block copolymer aggregates. A high-resolution transmission electron microscopy image of a micelle is shown, in which the individual dendrimer cores are seen to be localized in the center of these aggregates, and thus, the structure proposed in the previous publication (Kroeger, A.; Li, X.; Eisenberg, A. Langmuir 2007, 23, 10732) is confirmed. Furthermore, the sizes of the resulting aggregates depend on the relative concentration of dendrimer, expressed as RAm/Ac (the ratio of amine to acid groups). With increasing RAm/Ac values, not only the sizes of the micelles but also the vesicle dimensions, especially vesicle wall thicknesses, increase, and this effect suggests the encapsulation of the dendrimer into the vesicle walls. Thus, the constitution of the vesicle structure is determined precisely. This feature allows the potential incorporation of a wide range of species into the vesicle walls or the center of the micelle cores.

On the pathway of cellular uptake: new insight into the interaction between the cell membrane and very small nanoparticles

Summary For any living cell the exchange with its environment is vital. Therefore, many different kinds of cargo are able to enter cells via energy-dependent or -independent routes. Nanoparticles are no exemption. It is known that small silica nanoparticles with a diameter below 50 nm are taken up by cells and that their uptake exerts pronounced toxic effects beyond a certain concentration threshold. However, neither the exact uptake mechanism of these particles nor the actual reason for their toxicity has yet been elucidated. In this study we examined the uptake of silica nanoparticles with a diameter of 7, 12 and 22 nm by means of transmission electron microscopy, accompanied by toxicological assays. We show that for every particle diameter tested a different membrane morphology during uptake can be observed and that the amount of particles entering in one event is different for the three sizes. Silica particles with a diameter of 22 nm show single-particle internalization with a membrane wrapped around the particles in the cytosol, whereas 12 nm particles display row-like multi-particle uptake into elongated membrane structures and those with a diameter of 7 nm or less end up in tubular endocytic structures containing many particles. These membrane morphologies proved to be highly reproducible as we found them in five different cell lines. Additionally, we performed ATP and LDH assays to determine particle toxicity. Exceeding a certain concentration threshold the nanoparticles showed a high toxic potential both in the biochemical assay measurements and from morphological findings. We could not find any hint at the induction of apoptosis, neither morphologically nor biochemically. In this regard we discuss membrane damage and consumption as one possible mechanism of toxicity, linking morphological observations to toxicological findings to bridge the gap in understanding the mechanism of toxicity of small nanoparticles.

The Viscosity Law of Dendronized Linear Polymers

The intrinsic viscosity [η] of dendronized linear macromolecules dissolved in thermodynamically good solvents depends both on chain length that is contour length L(c) of the backbone of the polymer and on the persistence length l(p). Investigating a set of samples of identical L(c) but differing in the generation number for which data on lp are available, it becomes possible to establish a generalized viscosity law in the form [η] = K(M/M(p))(α) in which M(p) denotes the molar mass of the objects per l(p), M is the total molar mass, K is a constant, and α is the Mark-Howink exponent, which is found to be 0.55.