Cell-penetrating peptides, also known as CPP, have the ability to cross plasma membranes. Ever since the discovery of this ability, it has been exploited for a variety of applications, including delivering bioactive molecules to stop the actions of diseases producing cellular mechanisms.

Through the selective delivery of drugs into the target cells, it is possible to achieve improved drug distribution, as well as a significant reduction of dosing and toxicity. In this review, we will look at some of the challenges being encountered in this specific field of application, as well as some of the factors that may influence the efficacy of the delivery.

About Cell-penetrating peptides

Cell-penetrating peptides feature short peptide sequences – usually made up to 30 amino acids in length. They have the unique feature of being able to go through the cellular membranes from where they can be equipped to deliver a variety of cargo. The very first discovery regarding cell-penetrating peptides, was in the antennapedia homeodomain protein and the HIV-1 TAT proteins.

This was due to the fact that these two protein types have the unique ability to enter inside of the cells. Following the discovery, the specific domains responsible for the cellular uptake were isolated, giving rise to a new group of transfection molecules, known as cell-penetrating peptides, which are also known as protein transduction domains, PTDs.

The very first cell-penetrating peptides were obtained from existing proteins, but with time, scientists and researchers have made attempts to modify the peptide sequences for greater efficacy. The main reason for doing so, was to remove the minimal functional domains, and to bring in expression vectors with very minimal interferences to the cloning capabilities of the vector.

The next step was to create a chimeric peptide, which featured cell-penetrating regions from wasp venom, also known as galanin protein. The last class of the cell-penetrating peptides featured the construction of a synthetic peptide that had better or improved cell penetration capabilities. Examples of these peptides include polyarginine, as well as some improved versions of the natural cell-penetrating peptides.

Cell-penetrating peptides have proven to be very effective in delivering a variety of cargo into the cells. These include, but are not limited to small therapeutic molecules, and large cargoes such as plasmids and proteins. They are known to have an extraordinary ability that makes them ideal candidates for both research and therapeutic applications.

Subtle evidence to this fact, is the increase in the use of the cell-penetrating peptides in lots of therapeutic applications, in fields such as antineoplastic therapies, and antimicrobial applications. Additionally, there are lots of CPP-based treatments and therapies that have already gone through clinical trials, and are being seen as very powerful, and potent delivery methods for a wide range of therapeutic agents.

The use of cell-penetrating peptides is due to the numerous advantages they have over other delivery methods. Currently, gene delivery vectors are mainly classified into either vector or non-vector deliveries. Both of these categories are widely used in a variety of applications, though viral vectors have always shown very high rates of gene expression and replication. However, they are seen not to come with certain biological threats.

There is also a great variation in non-viral delivery methods – they range from electroporation and microinjection to chemical approaches, such as calcium phosphate and cationic liposomes.

Of all these methods, it can be claimed that cell-penetrating peptides have given some of the most outstanding results when it comes to transinfections, with very high transinfection rates, as well as the versatility to be used in a wide variety of cell types.

Also, they have been demonstrated to have reduced cellular toxicity, which has made it possible for transinfection to be carried out via very simple methodologies.

The chemistry of cell-penetrating peptides

The very first cell-penetrating peptides to be used in active research were obtained from the sequences of membrane-interacting proteins, such as antimicrobial peptides, fusion proteins, transmembrane domains, and signal peptides. From these proteins, it was possible to identify short sequences that had the unique capability to efficiently cross over most of the cellular membranes.

The process of translocation happened in a carrier or a receptor-dependent method, which made it possible for the delivery of biomolecules and other cargo right inside the intracellular compartments.

One of the proteins – TAT from the HIV-1 virus, was realized to have the properties to behave like a carrier mediating the transportation of various cargo molecules through the plasma membranes of both in vivo and in vitro cells.

This unique ability to penetrate the plasmas membranes was later on associated with a very specific domain of the HIV-1 virus, and it was duly designated as PTD. Peptides with similar capabilities have also been designated as PTDs.

There are various other peptides that are known to have the ability to move across plasma membranes, and a good number of them have been found to have several arginine residues. It is critical to note that most of the polyarginine peptides have demonstrated very good rates of cellular uptakes. This unique ability of transduction, has been closely associated with the effects of the guanidinium groups usually found in the arginine residues.

It has also been noted that arginine oligomers have the ability to form bidentate bonds. These bonds are usually typical of the guanidinium groups. Studies show that the bonds may be the very initial steps in the transduction process. You should remember that the cellular association of guanidinium groups is not necessarily the only mechanism that is usually used to initiate the cellular uptake.

There are other well-documented initiation mechanisms that have been proven by the cell-penetrating peptides, and these mechanisms don’t contain arginine residues. However, transportation can still be done into the cells, just with the same efficiency as with the polyarginine.

However, from only the basic nature of peptides, there is not enough information to formulate a detailed explanation of the efficiency of translocation. One of the challenges with this, is that lysime-rich peptides have been known to possess highly reduced translocation efficiency in comparison to arginine-rich peptides. Conversely, some cargo poly-lysine peptides have been shown to have very high transduction efficiency.

Chemical modifications of cell-penetrating peptides

There are various alterations that can be done to cell-penetrating peptides with the alterations not being limited to the modifications of the amino acid’s functional groups in the peptide chain. It is very possible to substitute the amino acids to come up with a customized hydrophobicity, new cationic content, as well as other properties that will suit certain specific applications. In one in vitro study where a library of 15 amino-acid long viral peptides was used, it was observed that there was enhanced translocation efficiency with low concentrations.

This was mainly attributed to the presence of cationic amino acids. A common modification to the cell-penetrating peptides, may include replacing an existing residue with another residue that has a stronger affinity for the cell surface.

A typical example of such a modification could be substituting lysine for arginine. Another interesting modification to cell-penetrating peptides, is the use of histidine. This is because histidine is known to provide an acidic endosomal escape through what is commonly known as proton sponge effect.

There are other approaches that can greatly enhance the availability of the cell-penetrating peptides to deliver cargo, and this includes modifying the functional groups in the peptide. This is usually done through the formation of linkages or masking groups to highly active sites.

In most cases, the newly created bond should be easily broken under normal physiological conditions to make it possible to obtain the original CPP.

Through this approach, it was possible to modify the model amphipathic peptide using citraconic anhydride, which was used to block the e-amino group of the lysine residues. This was achieved through the formation of an acid labile amide group.

With the masking of the cationic charge of model amphipathic peptide, a notable decrease was realized in the non-specific binding and uptake. Additionally, it was observed that a series of peptides based on the TP10 peptide could be modified chemically using stearic acid. Such a modification led to an increase in the uptake and it also improved the endossomolytic properties.

Mechanisms of cell penetrations

There are various mechanisms through which cell-penetrating peptides are able to make the journey across the plasma membranes. They include, but are not limited to the following:

Endosomal and non-endosomal pathways
There have been debates on how the cell-penetrating peptides enter the cells for more than two decades now. Initially, metabolic energy-independent pathways were proposed to be the am in the mechanism, but this notion was soon dropped when studies showed that the cell-penetrating peptides could enter the cells through an energy-independent endocytosis mechanism.

Endocytosis has always been suggested as being one of the main internalization routes for many of the complex cell-penetrating peptides. As a result, there are so many endocytotic pathways that have been advanced to facilitate the uptake of complex peptides.

Interestingly, most of the cell-penetrating peptide complexes don’t just rely on one route to gain entry into the cells since they have the ability to use a variety of endocytic pathways at the same time. This has made the study of its entry mechanism a very detailed and complex process than it was initially considered.

The latest developments in this field tend to suggest that there is another mechanism that the cell-penetrating peptides use to go through the cell membranes. Apart from the normal physical translocation of the membrane, cell-penetrating peptides also have the ability to enter the cells via induction of endocytotic-like membrane invaginations – a process also known as physical endocytosis.

Different cell-penetrating peptides may have the ability to induce tubular structures or negative curvatures on the membranes, for non-artificial membranes. This type of entry is relatively new since it was just recently discovered.

According to this theory, it seems as though the cell-penetrating peptides start by binding to phospholipids before starting to form a negative curvature in the cellular membrane without using any of the cellular metabolic energy. Consequently, invagination starts to grow inwards until it is cleaved with dynamin, in a process that is very similar to cellular endocytosis.

Endocytotic release of conjugates

Endosomal escape is usually the very next step after the process of internalization of cell-penetrating peptides through endocytosis. This process must always take place to stop the cell-penetrating hormones as well as the cargoes from being degraded in lysosomes. There have been numerous proposals for modification strategies aimed at facilitating the escape of the cell-penetrating hormones from the endosomes.
Some of these proposals include adding endosome-disruptive sequences, acidifying the endosmotic membranes, and including molecules such as chloroquine to the sequence of the cell-penetrating peptides.

Sadly, the endocytic escape rate can be quite difficult to examine due to the cargo biological effects that may happen much later than the actual time of the cargo release from the endosome. In essence, it is possible for the cargo markers to show activity when they are still right within the endosomes.

To overcome this problem, scientists have created a fluorescent marker – CPP conjugate. This conjugate is bound by a disulfide bridge designed to impede fluorescence. This disulfide bridge is usually cleaved by plasmatic enzymes after the endosomal escape, giving room for the free marker to start emitting fluorescence, which is then possible to measure.

Through this method, it was observed that the results of cargo delivery by the cell-penetrating peptides can be as fast as ten minutes, and may reach a peak after thirty minutes. After this, the rate at which the molecule penetrates the cell membranes will be lower than the number of molecules being degraded.

Once the cytoplasm is reached, it is a must for the cargo complexes to be released. This can happen through cleavage or dissociation of the complexes. The mode chosen will always depend on the type of bond used in linking the cell-penetrating peptides to the cargo.