Efficient Delivery of Streptavidin to Mammalian Cells:  Clathrin-Mediated Endocytosis Regulated by a Synthetic Ligand

Abstract

The efficient delivery of macromolecules to living cells presents a formidable challenge to the development of effective macromolecular therapeutics and cellular probes. We describe herein a novel synthetic ligand termed "Streptaphage" that enables efficient cellular uptake of the bacterial protein streptavidin by promoting noncovalent interactions with cholesterol and sphingolipid-rich lipid raft subdomains of cellular plasma membranes. The Streptaphage ligand comprises an N-alkyl derivative of 3 beta-cholesterylamine linked to the carboxylate of biotin through an 11-atom tether. Molecular recognition between streptavidin and this membrane-bound ligand promotes clathrin-mediated endocytosis, which renders streptavidin partially intracellular within 10 min and completely internalized within 4 h of protein addition. Analysis of protein uptake in Jurkat lymphocytes by epifluorescence microscopy and flow cytometry revealed intracellular fluorescence enhancements of over 300-fold (10 microM ligand) with >99% efficiency and low toxicity. Other mammalian cell lines including THP-1 macrophages, MCF-7 breast cancer cells, and CHO cells were similarly affected. Structurally related ligands bearing a shorter linker or substituting the protonated steroidal amine with an isosteric amide were ineffective molecular transporters. Confocal fluorescence microscopy revealed that Streptaphage-induced uptake of streptavidin functionally mimics the initial cellular penetration steps of Cholera toxin, which undergoes clathrin-mediated endocytosis upon binding to the lipid raft-associated natural product ganglioside GM1. The synthetic ligand described herein represents a designed cell surface receptor capable of targeting streptavidin conjugates into diverse mammalian cells by hijacking the molecular machinery used to organize cellular membranes. This technology has potential applications in DNA delivery, tumor therapy, and stimulation of immune responses.

Carbon nanotubes as cancer therapeutic carriers and mediators

“…For example, streptavidin, a protein purified from the bacterium Streptomyces avidinii, shows anticancer activity. 100 However, it cannot penetrate the cells because of its large molecular weight (~60 kDa). Shi Kam et al 101 conjugated streptavidin to an SWCNT-biotin transporter to internalize streptavidin into promyelocytic leukemia (HL60) cells and human T-cells (Jurkat) through the endocytosis pathway (Figure 4).…”

Carbon Nanotubes in Cancer Therapy and Drug Delivery

“…In another study, CNTs cationically functionalized with polyethylene imines have been shown capable of complexing with siRNA and generating a silencing activity of up to 30% and cytotoxicity of up to 60% [44]. Streptavidin is a protein that has anticancer activity [45]; however, due to its very large molecular weight (approximately 60,000 Da), it does not penetrate through cells. Kam and coworkers [12] have used a conjugate of streptavidin with SWNTs-biotin, which resulted in internalization of the protein into model cancer cells by adsorption-mediated endocytosis.…”

Biotinylation Facilitates the Uptake of Large Peptides by Escherichia coli and Other Gram-Negative Bacteria

“…Avidin drugs that bind to biotinylated vectors are being used to promote delivery across the blood-brain barrier (6,30,41), while antitumor toxins or imaging agents coupled to streptavidin are being delivered with biotinylated antibodies (17,37). Biotinylation has also been shown to promote the delivery of polyethylene glycol-camptothecin conjugates into human ovarian carcinoma cells (24) and increase the cellular uptake of polyethylene glycol-TAT nonapeptide conjugates into human Caco and CHO cells (38).…”