A pass to the cell: designing peptides for drug delivery.

Title: Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide

Authors: Misao Akishiba, Toshihide Takeuchi, Yoshimasa Kawaguchi, et al. Year: 2017

Year: 2017

Journal: Nature Chemistry



Antibodies and other cellular macromolecules can potentially be used for medical purposes. Moreover, antibodies have intrinsic targeting properties that could be exploited for an easy recognition of their targets.

In order to be effective medicines, they have to be transported to their designated destination cell.

However, the delivery of these molecules into the cellular environment can be quite challenging. In fact, macromolecules are usually unable of passing the cellular membrane on their own. They are, instead, captured by some membrane-bound compartments, called endosomes. These endosomes, originating from the cellular membrane, are part of the transportation mechanism of the cell. In theory, they can inglobate a macromolecule outside the cell and liberate it inside it.

It is not really that easy, though: once the antibodies have been trapped in the endosomes, they are not easily released into the cytosol (the liquid inside the cell), where they should perform their therapeutic activity.
In fact, they could be set free, by breaking (or lysing) the membrane of the endosomes where they are trapped. But this does not happen.  A common way to tackle this issue is the use of some pH-sensitive peptides (small macromolecules). These peptides change their structures with the pH variation that naturally occurs in the cell. In fact, inside the endosomal compartments, the pH is more acidic than it is in the extracellular environment. Once these peptides have entered the endosome, their structure changes and they are supposed to break the endosomal membrane and enter the cytosol.

In their active form, they usually have helical structures with amphiphilic character (one face of the helix is hydrophobic and the other hydrophilic,  i. e., one hates the water and the other likes it). The cellular membrane has hydrophobic character, and it strongly interacts with the hydrophobic face of the helix, this interaction weakens the chemical bonds that keep the membrane together, until the membrane breaks.
The cellular membrane also carries a global negative charge as most of the peptides currently studied do. This is not a chemical interaction as before:  opposite charges attract each other, whilst equal charges repel each other. In other words, if the peptide and the membrane have the same charge, they don’t efficiently come close enough to form the endosomes.
This paper describes the design of new endosomolytic (endosome-breaking) agents that can deliver the antibodies into the cytosol in a more efficient way. In this new approach, cationic peptides (positively charged) are chosen, their positive charge improves the interaction with the cellular membrane,  therefore increasing the intake of the cell.

Figure 1: Design concept and the proposed working scheme of the endosomolytic peptide. The chosen peptide, with strong lytic activity, was modified with a negatively charged residue (Glu) on the hydrophobic face, becoming non-lytic (top). In the endosomes, a lower pH neutralizes the charge, making the face of the helix hydrophobic again and causing a stronger interaction with the external membrane (bottom).

Figure 1 describes the proposed mechanism of the cellular intake and endosomal lysis of these peptides. Their structures, and therefore their strong interaction with the external membrane, would make them able to break it, causing cellular death. Therefore, the authors added a negatively charged amino acid (a peptide building block), the glutamine (Glu) on the hydrophobic side of the helix. According to the suggested mechanism, this residue is negatively charged in the extracellular environment, thus preventing the hydrophobic interaction with the membrane. Then, the glutamine becomes neutral in the more acidic endosomal environment and the peptide is able to perform its lytic activity.
For this study, a natural cationic amphiphilic peptide was chosen, the M-lycotoxin. Different mono and di-substituted analogues were synthesized by replacing one or two amino acids on the hydrophobic face of the helix with Glu. After evaluation of the toxicity and the endosomolytic activities of these peptides, the analogue with best results was chosen for further essays. This confirmed the feasibility of this approach and laid the groundwork for further development and improvement of biomacromolecules delivery.


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