Chapter 8
Cardiac Targeting Peptide: From Identification to Validation
to Mechanism of Transduction
Kyle S. Feldman, Maria P. Pavlou, and Maliha Zahid
Abstract
Cell-penetrating peptides (CPPs), also known as protein transduction domains, were first identified
25 years ago. They are small, ~6–30 amino acid long, synthetic, or naturally occurring peptides, able to
carry a variety of cargoes across the cellular membranes in an intact, functional form. These cargoes can
range from other small peptides, full-length proteins, nucleic acids including RNA and DNA, nanoparticles,
and viral particles as well as radioisotopes and other fluorescent probes for imaging purposes. However, this
ability to enter all cell types indiscriminately, and even cross the blood–brain barrier, hinders their development into viable vectors. Hence, researchers have adopted various strategies ranging from pH activatable
cargoes to using phage display to identify tissue-specific CPPs. Use of this phage display strategy has led to
an ever-expanding number of tissue-specific CPPs. Using phage display, we identified a 12-amino acid,
non-naturally occurring peptide that targets the heart with peak uptake at 15 min after a peripheral
intravenous injection, that we termed Cardiac Targeting Peptide (CTP). In this chapter, we use CTP as
an example to describe techniques for validation of cell-specific transduction as well as provide details on a
technology to identify binding partner(s) for these ever-increasing plethora of tissue-specific peptides.
Given the myriad cargoes CTP can deliver, as well as rapid uptake after an intravenous injection, it can be
applied to deliver radioisotopes, miRNA, siRNA, peptides, and proteins of therapeutic potential for acute
cardiac conditions like myocardial infarction, where the window of opportunity for salvaging at-risk
myocardium is limited to 6 hrs.
Key words Cardiac targeting peptide, Protein transduction domains, Cell-penetrating peptides,
Phage display, TriCEPS
1 Introduction
The cell plasma membrane is a semi-permeable barrier that is
essential for cell integrity and survival but at the same time presents
a barrier to delivery of cargoes. Hence, the ability of trans-activator
of transcription (Tat) protein of the human immunodeficiency virus
to enter cultured cells and promote viral gene expression was met
with great enthusiasm [1, 2]. Shortly thereafter, Antennapedia
homeodomain, a homeobox transcription factor of Drosophila melanogaster, was shown to enter nerve cells and regulate neural
Kumaran Narayanan (ed.), Bio-Carrier Vectors: Methods and Protocols, Methods in Molecular Biology, vol. 2211,
https://doi.org/10.1007/978-1-0716-0943-9_8, © Springer Science+Business Media, LLC, part of Springer Nature 2021
97
Cardiac Targeting Peptide: From Identification to Validation
to Mechanism of Transduction
Kyle S. Feldman, Maria P. Pavlou, and Maliha Zahid
Abstract
Cell-penetrating peptides (CPPs), also known as protein transduction domains, were first identified
25 years ago. They are small, ~6–30 amino acid long, synthetic, or naturally occurring peptides, able to
carry a variety of cargoes across the cellular membranes in an intact, functional form. These cargoes can
range from other small peptides, full-length proteins, nucleic acids including RNA and DNA, nanoparticles,
and viral particles as well as radioisotopes and other fluorescent probes for imaging purposes. However, this
ability to enter all cell types indiscriminately, and even cross the blood–brain barrier, hinders their development into viable vectors. Hence, researchers have adopted various strategies ranging from pH activatable
cargoes to using phage display to identify tissue-specific CPPs. Use of this phage display strategy has led to
an ever-expanding number of tissue-specific CPPs. Using phage display, we identified a 12-amino acid,
non-naturally occurring peptide that targets the heart with peak uptake at 15 min after a peripheral
intravenous injection, that we termed Cardiac Targeting Peptide (CTP). In this chapter, we use CTP as
an example to describe techniques for validation of cell-specific transduction as well as provide details on a
technology to identify binding partner(s) for these ever-increasing plethora of tissue-specific peptides.
Given the myriad cargoes CTP can deliver, as well as rapid uptake after an intravenous injection, it can be
applied to deliver radioisotopes, miRNA, siRNA, peptides, and proteins of therapeutic potential for acute
cardiac conditions like myocardial infarction, where the window of opportunity for salvaging at-risk
myocardium is limited to 6 hrs.
Key words Cardiac targeting peptide, Protein transduction domains, Cell-penetrating peptides,
Phage display, TriCEPS
1 Introduction
The cell plasma membrane is a semi-permeable barrier that is
essential for cell integrity and survival but at the same time presents
a barrier to delivery of cargoes. Hence, the ability of trans-activator
of transcription (Tat) protein of the human immunodeficiency virus
to enter cultured cells and promote viral gene expression was met
with great enthusiasm [1, 2]. Shortly thereafter, Antennapedia
homeodomain, a homeobox transcription factor of Drosophila melanogaster, was shown to enter nerve cells and regulate neural
Kumaran Narayanan (ed.), Bio-Carrier Vectors: Methods and Protocols, Methods in Molecular Biology, vol. 2211,
https://doi.org/10.1007/978-1-0716-0943-9_8, © Springer Science+Business Media, LLC, part of Springer Nature 2021
97