Intracellular protein delivery is offering numerous possibilities in research and in
therapy. Aside gene therapy, protein delivery into living cells is one of the most
promising tools for the treatment of various so far immedicable diseases including
cancer. To develop a practicable protein delivery platform, a test system which allows
easy control of successful intracellular delivery is needed. Therefore a test system
based on two model proteins was established. A nuclear localization signal tagged
EGFP molecule is enabling fast control of cellular uptake and endosomal release.
The second model protein ß-galactosidase is evidencing that protein conformation is
not irreversible disturbed by modification with the carrier molecules. Protein
transduction technology is opening the door for a promising alternative to gene
therapy, as it is lacking of the potential malignant side effects of gene therapy. The
most limiting step in the development of a therapeutic drug remains the delivery
process. In the last decade, many techniques to deliver proteins into living cells were
developed. Although great efforts were made, so far no all-purpose technique is
available that addresses all critical steps, like efficient uptake, endo-lysosomal
escape, low toxicity, while maintaining enzymatic activity. Each method has got its
limitation, for example cell type dependence. Among the so far used carriers, the
most effective ones are cationic polymers like polyethylenimine. These carriers are
lacking of precise structure and often show high toxicity, dependent on the molecular
weight of the used polymer. In this thesis the properties of the three arm cationic
oligomer 386, which was previously designed for siRNA delivery was investigated in
regard of being applicable as a transduction carrier for protein delivery. This carrier
molecule, in contrast to other cationic polymers used for protein delivery, is of precise
structure, of low molecular weight and potentially degradable by proteases. The
transduction oligomer was covalently bound to the protein by a bioreversible bond.
Our results reveal that covalent coupling of the structure defined cationic oligomer
386 to a protein leads to a high efficient, serum insensitive and low toxic alternative to
established protein transduction technologies. For a general all-purpose delivery
system covalent coupling of the carrier to the cargo protein is indispensable. Protein
delivery requires special properties to the linker molecule. Therefore in this work a
new pH sensitive linker was developed which combines the advantages of click
reactions with the implementation of a traceless cleavable bond between two conjugated molecules. Three different click chemistries were performed which all are
compatible with the acid labile properties. A traceless cleavage may be a particularly
important feature in protein transduction strategies, to maintain full bioactivity of
enzymes and other proteins. The current example of 386 carrier-mediated cytosolic
delivery and subsequent nuclear import of released nls-EGFP demonstrates the
advantage of the traceless linker. To demonstrate that the modification does not
irreversibly affect structure and biological activity of proteins, 386-AzMMMan-ßgalactosidase
was delivered as a model enzyme. It exhibited cytosolic activity in the
transduced cells far higher than without shuttle. Aside from these encouraging
options for protein delivery and modification, the linker might have broader use in the
design of novel programmed, acid labile and biodegradable drug delivery systems.
Targeted therapeutics could, after delivery into acidic tumor areas or upon cellular
uptake into endosomes, be dismantled from their outer shell including targeting
ligands. Besides drug delivery, the linker may also be of interest for other
applications, such as reversible labeling of various biological and also chemical
molecules. The developed linking strategy and the presented concepts for
transduction shuttles may help to get a step closer in the design of an all-purpose
protein delivery platform, applicable on bench as on bedside