Home | Legals | Sitemap | KIT
Anne Ulrich
Project Leader
Prof. Dr. Anne Ulrich


Karlsruher Institut für Technologie (KIT)
Institut für Organische Chemie
Fritz-Haber-Weg 6

76131 Karlsruhe, Germany

---------

Karlsruher Institut für Technologie (KIT)
Institut für Biologische Grenzflächen (IBG-2)
Hermann-von-Helmholtz-Platz 1

76344 Eggenstein-Leopoldshafen Germany

---------

Phone:
+49 (0)721 608 43912

anne ulrich∂kit edu

Homepage

E1: Transport of Nanoparticles through Membranes

Our vision in project E1 is to study and manipulate cellular functions by using nanomaterials, e.g., by optical tracking of quantum dots, by stimulation of metallic/magnetic particles via external electric or magnetic fields, or by destabilizing bacteria with silver nanoparticles. As a prerequisite, these non-natural cargoes have to be delivered into the cells. Their action is often limited by their failure to cross the hydrophobic cellular membrane and any cell wall present (as in plants and bacteria). There exist, however, a number of natural cell-penetrating peptides that can overcome these barriers via various routes, and they are often able to carry cargo along. Such transporters and their synthetic analogues are therefore highly useful as delivery agents in pharmacological applications. However, synthetic analogues are usually selected empirically, as no guidelines exist to optimize their composition, structure, and interactions with the cargo. Neither can they be easily targeted against a specific cell type nor against specific intracellular compartments. In addition, ordinary polypeptides are rapidly degraded in a biological environment by proteolysis. Therefore, our ongoing studies are focused on: (i) elucidating the molecular transport mechanisms to provide rules for rational design of more effective carrier-constructs, (ii) chemically modifying the transporters for improved performance, (iii) delivering cargo to specific intracellular targets, and (iv) implementing functionalized devices, e.g. to trigger reactions or release the cargo.

Mechanisms of Translocation

It is generally accepted that the physical interactions of cationic amphiphilic transporters with the cell membrane lead to a re-organization and local permeabilization of the lipid bilayer. To elucidate these steps, it is necessary to describe the 3D structures of the membrane-bound peptides in a lipid bilayer at quasi-atomic level. A highly sensitive solid-state 19F-NMR method has been developed to determine the conformation of a peptide, its membrane alignment and its dynamic behavior (see E1.2: Structure-Function Analysis of Membrane-Active Peptides). Molecular dynamics simulations, utilizing these NMR parameters, yield a comprehensive picture of these structures, both in coarse-grained approaches and at the atomic level (see E1.6: Simulation of Membrane Penetrating Peptides and Nanoparticles). Different types of mechanisms have thus been demonstrated, such as an assembly into transient pores, the formation of inverted micelles in the lipid bilayer, or a polar flip-flop between monolayers. For the first time, solid-state NMR was able to detect and resolve these 19F-labeled molecules not only in synthetic lipid model membranes, but also in native biomembranes from human erythrocytes and bacterial protoplasts.

Chemical Modifications for Improved Performance

Attempts to improve the serum stability of cell-penetrating transporters generally rely on biomimetic strategies, using for example D-amino acids, b-peptides, and other types of linear or branched molecular frameworks. Peptoids and polyamine transporters are explored here, based on combinatorial synthesis and click reactions for bioconjugation to cargoes (see E1.1: Novel Carriers for Nano-Structured Material through Membranes). The novel peptoidic structures were screened for organ or organelle specificity in mice, human cervix carcinoma cells, and plant cells. By coupling bleaching-resistant fluorescent dyes as cargo to such transporters, high-performance 4D spinning disk and STED microscopy of peptoids has become possible for the first time. Incorporation of two different dyes within the same structure enabled FRET experiments, which allowed for observing the cleavage within the transporter.

Translocation into Plant Cells

The different kinds of transporters are tested to deliver cargo into tobacco cells and to target specific intracellular structures. This includes synthetic peptoids with or without guanidinium side chains (see E1.1: Novel Carriers for Nano-Structured Material through Membranes), as well as various peptides from natural origin or medium-throughput screens (see E1.2: Structure-Function Analysis of Membrane-Active Peptides). In first tests with tobacco cells, it was demonstrated that certain transporters can enter plant cells without damaging the acidic vacuole (see E1.5: Use of Nanoparticles to Study and Manipulate the Polarity of Plant Cells). Regarding target-specific delivery, fluorescently labeled peptide carriers were fused to an actin-binding peptide. This step allowed, for the first time in plant cells, the visualization of the actin cytoskeleton in vivo without the need for genetic engineering. The cellular uptake mechanisms of these chemically engineered carriers have been fully characterized.

Design of Functionalized Nano-Bio Conjugates

With various transporters at hand and a range of nanoparticles with different properties from other CFN projects, the ultimate step is to combine them into nano-bio conjugates with novel biological properties. In this context, biofunctionalized TiO2 nanoparticles have been prepared to generate reactive oxygen species and induce apoptosis (see JRG: Light Activable Nanoparticles and Biomolecules). Their effects on the cellular surface, their cell uptake and cytotoxicity are being examined in plant cells (see E1.5: Use of Nanoparticles to Study and Manipulate the Polarity of Plant Cells). Another construction principle has been explored for designing stable bio-nano conjugates, by making use of DNA to form combinatorial nanoarrays of proteins on planar gold substrates. These arrays enable the parallel characterization of proteins in complex biological samples, such as blood serum.