3 research outputs found
Acid-Labile Traceless Click Linker for Protein Transduction
Intracellular delivery of active proteins presents an
interesting
approach in research and therapy. We created a protein transduction
shuttle based on a new traceless click linker that combines the advantages
of click reactions with implementation of reversible pH-sensitive
bonds. The azidomethyl-methylmaleic anhydride (AzMMMan) linker was
found compatible with different click chemistries, demonstrated in
bioreversible protein modification with dyes, polyethylene glycol,
or a transduction carrier. Linkages were stable at physiological pH
but reversible at the mild acidic pH of endosomes or lysosomes. We
show that pH-reversible attachment of a defined endosome-destabilizing
three-arm oligo(ethane amino)amide carrier generates an effective
shuttle for protein delivery. The cargo protein nlsEGFP, when coupled
via the traceless AzMMMan linker, experiences efficient cellular uptake
and endosomal escape into the cytosol, followed by import into the
nucleus. In contrast, irreversible linkage to the same shuttle hampers
nuclear delivery of nlsEGFP which after uptake remains trapped in
the cytosol. Successful intracellular delivery of bioactive ß-galactosidase
as a model enzyme was also demonstrated using the pH-controlled shuttle
system
Sequence Defined Disulfide-Linked Shuttle for Strongly Enhanced Intracellular Protein Delivery
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
Toward Artificial Immunotoxins: Traceless Reversible Conjugation of RNase A with Receptor Targeting and Endosomal Escape Domains
The
specific transport of bioactive proteins into designated target
cells is an interesting and challenging perspective for the generation
of innovative biopharmaceuticals. Natural protein cytotoxins perform
this task with outstanding efficacy. They enter cells with receptor-targeted
specificity, respond to changing intracellular microenvironments,
and by various mechanisms translocate their cytotoxic protein subunit
into the cytosol. Here we imitate this toxin-based delivery strategy
in an artificial setting, by bioreversible conjugation of a cytotoxic
cargo protein (RNase A) with receptor-targeting PEG-folate and the
pH-specific endosomolytic peptide INF7 as synthetic delivery domains.
Covalent modification of the cargo protein was achieved using the
pH-labile AzMMMan linker and copper-free click chemistry with DBCO-modified
delivery modules. This linkage is supposed to enable traceless intracellular
release of the RNase A after exposure to the endosomal weakly acidic
environment. Delivery of RNase A via this polycation-free delivery
strategy resulted in high cytotoxicity against receptor-positive KB
tumor cells only when both PEG-folate and INF7 were attached