Direct Cytosolic Delivery of Polar Cargo to Cells by Spontaneous Membrane-translocating Peptides

• Published in: The Journal of biological chemistry 2013-10, Vol.288 (41), p.29974-29986
• Main Authors: He, Jing, Kauffman, W. Berkeley, Fuselier, Taylor, Naveen, Somanna K., Voss, Thomas G., Hristova, Kalina, Wimley, William C.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Biological Transport; Cell Membrane - metabolism; Cell Survival - drug effects; Cell-penetrating Peptides; CHO Cells; Cricetinae; Cricetulus; Cytosol - metabolism; Drug Delivery; Drug Delivery Systems - methods; Endocytosis; Female; Fluorescent Dyes - administration & dosage; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; Membrane Biology; Membrane Biophysics; Membrane Transport; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Peptides - administration & dosage; Peptides - chemistry; Peptides - metabolism; Quinolinium Compounds - administration & dosage; Quinolinium Compounds - chemistry; Quinolinium Compounds - metabolism; Reproducibility of Results; Rhodamines - administration & dosage; Rhodamines - chemistry; Rhodamines - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.488312

Direct cellular entry of potentially useful polar compounds into cells is prevented by the hydrophobic barrier of the membrane. Toward circumventing this barrier, we used high throughput screening to identify a family of peptides that carry membrane-impermeant cargos across synthetic membranes. Here we characterize the plasma membrane translocation of these peptides with polar cargos under a variety of conditions. The spontaneous membrane-translocating peptides (SMTPs) delivered the zwitterionic, membrane-impermeant dye tetramethylrhodamine (TAMRA) into cells even when the conditions were not permissive for endocytosis. They also delivered the larger, anionic membrane-impermeant dye Alexa Fluor 546 but did not deliver a quantum dot nanoparticle. Under all conditions, the SMTP-cargo filled the cytoplasm with a diffuse, non-punctate fluorescence that was partially excluded from the nucleus. d-Amino acid peptides behaved identically in vitro, ruling out proteolysis as an important factor in the diffuse cellular distribution. Thus, cytosolic delivery of SMTP-cargo conjugates is dominated by direct membrane translocation. This is in sharp contrast to Arg9-TAMRA, a representative highly cationic, cell-penetrating peptide, which entered cells only when endocytosis was permitted. Arg9-TAMRA triggered large scale endocytosis and did not appreciably escape the endosomal compartments in the 1-h timescales we studied. When injected into mice, SMTP-TAMRA conjugates were found in many tissues even after 2 h. Unconjugated TAMRA was rapidly cleared and did not become systemically distributed. SMTPs are a platform that could improve delivery of many polar compounds to cells, in the laboratory or in the clinic, including those that would otherwise be rejected as drugs because they are membrane-impermeant. Background: Spontaneous membrane-translocating peptides were discovered by screening in synthetic lipid vesicles. Results: The translocating peptides carry membrane-impermeant cargos directly across cell membranes and drive systemic biodistribution in small animals. Conclusion: These peptides constitute a new class of delivery vehicle for membrane-impermeant cargos. Significance: Spontaneous membrane-translocating peptides could expand the universe of useful drugs.