Cell-Penetrating Molecules for Drug Delivery

Many biologically active compounds cannot be used as therapeutics or chemical probes, because they cannot penetrate the cell membrane. Cell-penetrating peptides (CPPs) provide a potential solution to this problem. Conventional CPPs such as Tat (GRKKRRQRRRPQ), penetratin (RQIKIWFQNRRMKWKK), and nonaarginine (RRRRRRRRR) are short, cationic and/or amphipathic peptides. They have been used to transport a wide variety of cargo molecules across the eukaryotic cell membrane. However, linear CPPs are susceptible to proteolytic degradation in vivo, have very poor cytosolic delivery efficiencies, and have poor bioavailability/biodistribution. We discovered a family of small amphipathic cyclic peptides as exceptionally active CPPs, e.g., cyclo(phe-Nal-Arg-arg-Arg-arg-Gln) (CPP9) and cyclo(Phe-phe-Nal-Arg-arg-Arg-arg-Gln) (CPP12) (Figure 5a,b) [4-6]. These cyclic CPPs exhibit excellent cytosolic delivery efficiencies (up to 120%), proteolytic stability, bioavailability, and broad biodistribution. Mechanistic studies revealed that cyclic CPPs bind directly to the plasma membrane phospholipids, enter cells by endocytosis, and efficiently escape from early endosomes into the cytosol by a novel vesicle budding and collapse mechanism (Figure 6) [6]. We have shown that cyclic CPPs can be used to effectively deliver a wide variety of cargos including small molecules, peptides, proteins, nucleic acids, and protein/nucleic acid complexes into the cytosol of mammalian cells. We have also discovered a family of non-peptidic cell-penetrating motifs (CPMs), which are capable of specifically and efficiently delivering attached cargos into the mitochondrial matrix (Figure 5c) [7].

Current and future studies in this area include:

  • Further investigation into the molecular mechanism by which cyclic CPPs and CPMs escape from the endosome;

  • Elucidation of the mechanism by which CPMs enter the mitochondrial matrix;

  • Discovery of additional cyclic CPPs or CPMs with improved properties (e.g., selectivity for cancer cells);

  • Application of CPPs and CPMs to deliver other cargos as therapeutic agents and chemical probes;

  • Mechanism of endosomal escape by noneveloped viruses, bacterial toxins, and other nonviral delivery systems, i.e., whether they exit the endosome by vesicle budding and collapse [8].


Key Publications:

Efficient Delivery of Cyclic Peptides into Mammalian Cells with Short Sequence Motifs

Ziqing Qian, Tao Liu, Yu-Yu Liu, Roger Briesewitz, Amy M. Barrios, Sissy M. Jhiang, and Dehua Pei. ACS Chem. Biol.20138 (2), 423–431


Cyclic peptides hold great potential as therapeutic agents and research tools, but their broad application has been limited by poor membrane permeability. Here, we report a potentially general approach for intracellular delivery of cyclic peptides. Short peptide motifs rich in arginine and hydrophobic residues (e.g., FΦRRRR, where Φ is l-2-naphthylalanine), when embedded into small- to medium-sized cyclic peptides (7–13 amino acids), bound to the plasma membrane of mammalian cultured cells and were subsequently internalized by the cells. Confocal microscopy and a newly developed peptide internalization assay demonstrated that cyclic peptides containing these transporter motifs were translocated into the cytoplasm and nucleus at efficiencies 2–5-fold higher than that of nonaarginine (R9). Furthermore, incorporation of the FΦRRRR motif into a cyclic peptide containing a phosphocoumaryl aminopropionic acid (pCAP) residue generated a cell permeable, fluorogenic probe for detecting intracellular protein tyrosine phosphatase activities.


 Early Endosomal Escape of a Cyclic Cell-Penetrating Peptide Allows Effective Cytosolic Cargo Delivery

Ziqing Qian, Jonathan R. LaRochelle, Bisheng Jiang, Wenlong Lian, Ryan L. Hard, Nicholas G. Selner, Rinrada Luechapanichkul, Amy M. Barrios, and Dehua Pei. Biochemistry201453 (24), 4034–4046


Cyclic heptapeptide cyclo(FΦRRRRQ) (cFΦR4, where Φ is l-2-naphthylalanine) was recently found to be efficiently internalized by mammalian cells. In this study, its mechanism of internalization was investigated by perturbing various endocytic events through the introduction of pharmacologic agents and genetic mutations. The results show that cFΦR4 binds directly to membrane phospholipids, is internalized into human cancer cells through endocytosis, and escapes from early endosomes into the cytoplasm. Its cargo capacity was examined with a wide variety of molecules, including small-molecule dyes, linear and cyclic peptides of various charged states, and proteins. Depending on the nature of the cargos, they may be delivered by endocyclic (insertion of cargo into the cFΦR4 ring), exocyclic (attachment of cargo to the Gln side chain), or bicyclic approaches (fusion of cFΦR4 and cyclic cargo rings). The overall delivery efficiency (i.e., delivery of cargo into the cytoplasm and nucleus) of cFΦR4 was 4–12-fold higher than those of nonaarginine, HIV Tat-derived peptide, or penetratin. The higher delivery efficiency, coupled with superior serum stability, minimal toxicity, and synthetic accessibility, renders cFΦR4 a useful transporter for intracellular cargo delivery and a suitable system for investigating the mechanism of endosomal escape.


Discovery and Mechanism of Highly Efficient Cyclic Cell-Penetrating Peptides

Ziqing Qian, Agnieszka Martyna, Ryan L. Hard, Jiang Wang, George Appiah-Kubi, Christopher Coss, Mitch A. Phelps, Jeremy S. Rossman, and Dehua Pei Biochemistry 2016 DOI: 10.1021/acs.biochem.6b00226.


Previous cell-penetrating peptides (CPPs) generally have low cytosolic delivery efficiencies, because of inefficient endosomal escape. In this study, a family of small, amphipathic cyclic peptides was found to be highly efficient CPPs, with cytosolic delivery efficiencies of up to 120% (compared to 2.0% for Tat). These cyclic CPPs bind directly to the plasma membrane phospholipids and enter mammalian cells via endocytosis, followed by efficient release from the endosome. Their total cellular uptake efficiency correlates positively with the binding affinity for the plasma membrane, whereas their endosomal escape efficiency increases with the endosomal membrane-binding affinity. The cyclic CPPs induce membrane curvature on giant unilamellar vesicles and budding of small vesicles, which subsequently collapse into amorphous lipid/peptide aggregates. These data suggest that cyclic CPPs exit the endosome by binding to the endosomal membrane and inducing CPP-enriched lipid domains to bud off as small vesicles. Together with their high proteolytic stability, low cytotoxicity, and oral bioavailability, these cyclic CPPs should provide a powerful system for intracellular delivery of therapeutic agents and chemical probes.


Non‐Peptidic Cell‐Penetrating Motifs for Mitochondrion‐Specific Cargo Delivery

George Appiah Kubi, Dr. Ziqing Qian, Dr. Souad Amiar, Ashweta Sahni, Prof. Dr. Robert V. Stahelin, Prof. Dr. Dehua Pei; Angewandte Chemie. 10.1002/ange.201811940.

Mitochondrial dysfunction is linked to a variety of human illnesses, but selective delivery of therapeutics into the mitochondrion is challenging. Now a family of amphipathic cell‐penetrating motifs (CPMs) is presented, consisting of four guanidinium groups and one or two aromatic hydrophobic groups (naphthalene) assembled through a central scaffold (a benzene ring). The CPMs and CPM‐cargo conjugates efficiently enter the interior of cultured mammalian cells and are specifically localized into the mitochondrial matrix, as revealed by high‐resolution confocal microscopy. With a membrane‐impermeable peptide as cargo, the CPMs exhibited ≥170‐fold higher delivery efficiency than previous mitochondrial delivery vehicles. Conjugation of a small‐molecule inhibitor of heat shock protein 90 to a CPM resulted in accumulation of the inhibitor inside the mitochondrial matrix with greatly enhanced anticancer activity. The CPMs showed minimal effect on the viability or the mitochondrial membrane potential of mammalian cells.


 Overcoming Endosomal Entrapment in Drug Delivery

Dehua Pei and Marina Buyanova; Bioconjugate Chemistry; Just Accepted Manuscript; DOI: 10.1021/acs.bioconjchem.8b00778

Intracellular delivery of biological agents such as peptides, proteins, and nucleic acids generally rely on the endocytic pathway as the major uptake mechanism, resulting in their entrapment inside
the endosome and lysosome. The recent discovery of cell-penetrating molecules of exceptionally high endosomal escape and cytosolic delivery efficiencies and elucidation of their mechanism of
action represent major breakthroughs in this field. In this Topical Review, we provide an overview of the recent progress in understanding and enhancing the endosomal escape process and the new opportunities opened up by these recent findings.