Cell-penetrating peptides (CPPs) are short sequences — typically 5–30 residues — that cross the plasma membrane and can carry covalent or non-covalent cargo into the cytoplasm. They have become a standard tool in cell biology where transfection reagents are unsuitable, and they are an active area of therapeutic research.
The main classes
| Class | Example | Length | Net charge | Origin |
|---|---|---|---|---|
| Cationic | TAT (47–57) | 9 | +8 | HIV-1 trans-activator |
| Cationic | Penetratin (Antp 43–58) | 16 | +7 | Drosophila Antennapedia |
| Cationic, oligoArg | R8, R9 | 8–9 | +8/+9 | Synthetic |
| Amphipathic | MPG, Pep-1 | 21–27 | varies | Chimeric |
| Hydrophobic | C105Y | 10 | 0 | α1-antitrypsin |
Uptake mechanisms
CPP uptake is concentration-, sequence-, cargo- and cell-type-dependent. At low concentration (≤1 µM), uptake is dominantly endocytic (macropinocytosis, caveolae- or clathrin-mediated, depending on cell type). At higher concentration (>5 µM), direct translocation through transient inverted micelles becomes appreciable.
Practical consequence: a CPP that works at 10 µM via direct translocation may not work at 1 µM via endocytosis if the cargo is endosomally degraded. Always titrate.
Cargo strategies
Covalent conjugation
- N-terminal extension of the cargo peptide with the CPP sequence (ideal for short peptide cargos).
- Disulfide linkage (cleaved in the reducing cytosol — releases free cargo).
- Maleimide–thiol conjugation to a Cys-containing cargo.
- Click chemistry (azide–alkyne) for orthogonal labelling.
Non-covalent complexation
- MPG and Pep-1 form non-covalent complexes with siRNA, plasmids and proteins by electrostatics.
- Rapid to set up, but stoichiometry is harder to control.
Research applications
- Intracellular peptide inhibitors. Bring a 10–20 mer inhibitor of a protein–protein interaction into the cytoplasm without transfection (e.g. the AIP-CaMKII inhibitor used in synaptic plasticity work; see AIP-CaMKII inhibitor).
- Imaging probes. CPP-fluorophore conjugates label live cells without electroporation.
- siRNA delivery. CPP–siRNA complexes for primary cells refractory to lipoplex transfection.
- Mitochondrial targeting. Adding a triphenylphosphonium tail to a CPP can direct cargo to the inner mitochondrial membrane.
Pitfalls
- Trypan-blue artefact. Many cationic CPPs permeabilise damaged cells faster than intact ones. Confirm uptake by orthogonal microscopy and a viability stain other than trypan blue.
- Cytotoxicity at high concentration. All cationic CPPs perturb membranes above ~10–20 µM; titrate downwards rather than up.
- Endosomal trapping. A fluorescent puncta-only pattern indicates endosomal trapping; pair with a small-molecule endosomal escape enhancer or switch CPP class.
- Serum binding. Some CPPs bind serum albumin and lose activity in 10% FBS; pre-test in serum-free or low-serum conditions and add serum stepwise.
Sequence selection in practice
Start with TAT (47–57) or R8 for proof-of-principle. If endosomal trapping dominates, switch to penetratin or an amphipathic class such as MPG. If the cargo is sensitive to disulfide reduction, use a stable thioether or click linkage rather than a disulfide.