Title : Degradation-free intracellular delivery of nanomolar protein cargoes with a ganglioside GM1 specific recognition tag
There is a pressing need to develop ways to deliver therapeutic macromolecules to their intracellular targets. Lipid raft-mediated/caveolar endocytosis is exploited by viruses (simian virus 40, murine polyoma virus and echovirus 1), bacterial toxins (cholera and tetanus), and some endogenous proteins to deliver macromolecules in their functional form without degradation. We aimed to mimick this entrapment- and lysosomal degradation free endocytic pathway. These proteins of viral and bacterial origins trigger the desired intracellular delivery through binding to gangliosides at the caveolar entry points. We targeted the most abundant ganglioside GM1 in the caveolar pits to trigger the degradation-free endocytosis pathway. GM1 is overexpressed in cancer cells. Because therapeutic protein levels in the extracellular fluid do not exceed the nanomolar range (clinical protocols yield 100-500 nM), high-affinity target-carrier interaction is necessary to create a cell surface enrichment that facilitates sufficient material flux in potential clinical applications. We found a pentapeptide sequence WYKYW that specifically captures the glycan moiety of GM1 (KD = 24 nM). The WYKYW-tag facilitated the GM1-dependent selective caveolar endocytosis of proteins in which the cargo-loaded caveosomes did not fuse with lysosomes. An immunoglobulin G complex (580 kDa) was successfully delivered into live HeLa cells at extracellular concentrations ranging from 20 to 160 nM. The molecular recognition regions (Fv and Fc) of the antibodies remained functional after 24 hours, and escape of the functional cargo protein to the cytosol was observed. We found that a single pentapeptidic segment was sufficient to trigger the entrapment- and lysosomal degradation free lipid raft-mediated/caveolar endocytosis in live cells. Our endocytosis routing sequence was not toxic even in the high micromolar region. Our receptor-based approach is a useful alternative cell delivery method, because the very short, easily applicable and non-toxic tag facilitates the advantageous lipid raft-mediated/caveolar endocytosis in a carrier-triggered manner. It works at therapeutically relevant concentrations for many cell types expressing ganglioside GM1. Overexpressing ganglioside GM1 is a characteristic of tumor cells, and selectivity towards cancer cells are of great interest. The endocytosis routing tag can be attached with the already existing chemistry for the PEGylation protocols.