Elucidation of endocytosis , intracellular transport and release by microscopy

(Project SFB in Q2 1066 " Nanodimensional polymer therapeutics for tumor therapy " by the German Research Foundation )

Project: PD Dr. V. Mailänder , together with Dr. I. Lieberwirth and Dr. K. Koynov

This cross-sectional project is concerned with the stability, uptake and release of nanocarriers (NC) with embedded active ingredients such as siRNA, mRNA , plasmids, and low-molecular substances on microscopic, cellular level.

The processes at the microscopic level, which take place between NC application into the vein or the cell culture medium and the effect of the active ingredients, have so far hardly been studied. However, these are essential for the effectiveness of transport as well as for an advanced design rationale of NC. Here Q2 leads to a deeper understanding of the barriers to a successful drug transport. Thus, the loading efficiency of NC , decay or coagulation upon contact with blood components and interaction with the target cell membrane , the processes of cellular uptake and maturation of endosomal vesicles until to their arrival on the target molecules, for example mRNA or protein, are elucidated.

We study the kinetics of formation, decomposition and aggregation of the nano- carriers of the A projects in aqueous solution, plasma and blood. The success of payload transport through its decisive stations of uptake, distribution and release in the target cell is elucidated by high-resolution microscopic methods. The subcellular processes are described and quantified in detail in this project by the microscopic methods STED (stimulated emission depletion) , FCS and TEM (transmission electron microscopy) . E.g., various routes of uptake via clathrin- or caveolin-pathways and macropinocytosis were shown to allow the passing of the first barrier in the form of the cell membrane. These are also dependent on the type of NC and cells.

The investigation of early routing through various early and late endosomes and other intracellular retention (qualitative and quantitative) has as a goal the identification of NC properties, which can escape in high quantity from the endosomes. This is in turn done by high-resolution microscopy and fluorescent labeling of intracellular compartments. In addition, a 3D correlative microscopy method will be established through representation of the same cell in fluorescence microscopy and TEM or TEM imaging as well as the superposition of resulting frames, allowing statements with a resolution in the range of about 1 nm .

Partners: Dr. I. Lieberwirth and Dr. K. Koynov (MPI for Polymer Research, Mainz)