Cell Penetrating Peptides: A System for Delivery

One aspect of design of RNA molecules that serve specific functions is figuring out how to get those molecules inside the cell. In this post, Adrien delves into a system used for drug delivery, known as CPP, or cell-penetrating peptides. Analogues of such technology may be used for delivering RNA molecules developed in vitro into cells.

The ability to have a drug that can be taken up to a very specific location in the body is a long-standing problem. In 1906, the concept of the “magic bullet” was imagined by Paul Ehrlich (a Nobel Laureate for his work in immunology). This concept supposes that we could have what is known as  a magic bullet bring a drug, in a specific way, toward the active site of the drug.

Paul Ehrlich is considered the father of chemotherapy.

The field of drug delivery systems has improved during the last century and there are several types of nano-carriers which are already developed. One of these systems is the Cell Penetrating Peptides (or CPP). CPP are short peptides that are designed to carry molecules (nucleic acids, fluorophore, proteins…) inside the cells by going through the membrane.

Peptides are a short strand of amino acids. They can be defined as very small proteins. Peptides are composed of less than 50 amino acids. Small peptide sequences have many functions: they can act as hormones (ex. insulin); neuropeptids (ex. substance P, which is involved in pain mediation); or toxins (ex. amanitin in mushroom). Some of these toxins also have the capability to interact with the cell membrane and create pores in them.

Mechanisms of translocation used by CPP to be integrated into cells.

Ways to penetrate membranes have evolved in viruses, microorganisms and parasites alike. Infectious organisms, such as viruses and bacteria, possess proteins endowed with properties to penetrate living cells (Pugsley 1996). Pore-forming toxins such as the tetanus toxin (Boquet and Duflot 1982), the anthrax toxin (Ballard et al. 1996), cytolysins (Rossjohn et al. 1997), and the diphtheria toxin (Stenmark et al. 1991) facilitate the cellular entry for pathogens or help them to employ substrates from the host’s cytosol for their own metabolism. The wasp venom peptide toxin, mastoparan (see picture below), induces cell death by severely perforating the plasma membrane (Matsuzaki et al. 1996).


The 1989 paper by Ruben et al. showed that the TAT protein (a HIV protein that enhance the efficiency of viral transcription) has a short amino acid motif (Glycine-Arginine-Lysine-Lysine-Arginine-Glutamine-Arginine-Arginine-Arginine) that is highly enriched in basic amino acids. They observed that this motif promotes the export of the TAT protein from infected cells. And once excreted, the proteins are able to be internalized by a variety of different cell types. Since this breakthrough, this specific motif started to be study for its properties, and now is widely used as a CPP. Arginine is the most important amino acid for CPP as you can see it repeated several times on the TAT sequence. This is due to the positively charged guanidinium residue which is on the side chain of arginine. This residue can interact with the negatively charged phospholipid bilayer of the cell membrane. With this important breakthrough, scientists tried to attach different types of cargos (see the scheme below, from: Kelly M. Stewart, Kristin L. Horton and Shana O. Kelley, 2008) to this TAT basic domain (or other polyarginines peptides) and observed that this domain could mediate their efficient cellular internalization.


An example of protein delivery mediated by a CPP. The GFP (green fluorescent protein) was coupled to a R7 sequence (7 arginine residues). The first row is a negative control (without the R7) and the second row is GFP combined with CPP. The blue dye is DAPI used to stain the nucleus of the cells. Only the proteins combined with the CPP are internalised by the cells. (From Jo et al. 2014).

CPPs are wonderful tools that can be used for the delivery of several kinds of cargos, from nucleic acids to proteins to quantum dots. However, they do have some drawbacks. The most important is cytotoxicity. CPP can induce a membrane leakage or membrane perturbation depending on the nature of the cargo. Where oligonucleotides seem to not have high toxicity, proteins or fluorophores can be more lethal for the cell as a cargo (El-Andaloussi et al. 2007). Another problem is that the bigger is the cargo, the higher chance they have to be captured and trapped in the endosome. An alternative to avoid both of these problems is the use of CPDs (Cell Penetrating poly(Disulfide)s). They contain a polysulfide backbone, instead of a polyamide backbone (present in normal peptides, see figure below). This polysulfide backbone has shown interesting results in cellular uptake of proteins and in low cytotoxicity (Giulio Gasparini and Stefan Matile, 2015).

The backbone of CPD is represented where a disulfide monomer contains one side chain.
The regular polyamide chain of peptides is represented here.

 Finally, the last problem with CPPs that I will talk about is their low stability. Oligoarginines are extremely vulnerable to trypsin proteolysis. 5 min at 25 °C with a 1/800 ratio Trypsin/12-mer oligoarginine is enough to degrade all peptides. One approach that has been tried for increasing their stability is the use of “stitched” and “stapled” peptides. The idea is to connect, in a peptide, the n position to the n+4 position (which is equivalent to one turn in the alpha helix structure of a protein) or to the n+7 position (which is two turns of helix). This staple increases the alpha helix nucleation (see figures below), the stability of the peptide and the endocytosis (Hilinski et al. 2014).

Schematic representation of the “stitched” peptides. The hydrocarbon tether is used instead of the side chain of the amino acid.
Molecular dynamics representation of a stapled peptide (left) and a wild type peptide (right). The presence of the tether enhances the nucleation of the alpha helix. (Guo et al 2010).

Twenty years ago, the discovery of peptides that are able to cross cellular membranes launched a novel field in molecular delivery based on these non-invasive vectors, most commonly called cell-penetrating peptides (CPPs). These peptides were shown to efficiently transport various biologically active molecules inside living cells, and thus are considered promising devices for medical and biotechnological developments. CPPs can be also one alternative to carry RNA biosensors that we are developing with MetaRNA. We could maybe attach a RNA single strand molecule to a CPP via a covalent bond (disulfide bond or peptide bond for example). Next, the CPP could deliver the RNA biosensor inside the cell to detect some of our targets that we are interested in with MetaRNA.


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