Sequence Defined Disulfide-Linked
Shuttle for Strongly Enhanced Intracellular Protein Delivery
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Abstract
Intracellular protein transduction
technology is opening
the door for a promising alternative to gene therapy. Techniques have
to address all critical steps, like efficient cell uptake, endolysosomal
escape, low toxicity, while maintaining full functional activity of
the delivered protein. Here, we present the use of a chemically precise,
structure defined three-arm cationic oligomer carrier molecule for
protein delivery. This carrier of exact and low molecular weight combines
good cellular uptake with efficient endosomal escape and low toxicity.
The protein cargo is covalently attached by a bioreversible disulfide
linkage. Murine 3T3 fibroblasts could be transduced very efficiently
with cargo nlsEGFP, which was tagged with a nuclear localization signal.
We could show subcellular delivery of the nlsEGFP to the nucleus,
confirming cytosolic delivery and expected subsequent subcellular
trafficking. Transfection efficiency was concentration-dependent in
a directly linear mode and 20-fold higher in comparison with HIV-TAT-nlsEGFP
containing a functional TAT transduction domain. Furthermore, β-galactosidase
as a model enzyme cargo, modified with the carrier oligomer, was transduced
into neuroblastoma cells in enzymatically active form