How Can Phage Display Peptides Be Used in Nano-Drug Carriers

Nanoparticles have received extensive attention as drug carriers in the diagnosis of specific diseases, the detection and monitoring of therapeutic agents, and targeted therapies. The primary strategy for targeting drug carriers is to achieve targeting by utilizing specific recognition capabilities such as antibodies and antigens, ligands and receptors. This type of targeting is often referred to as “active targeting” by functionalizing the surface of the nanoparticle to achieve targeted behavior.

Currently, researchers have developed new nanoparticle ligand drug carriers using phage display technology. The phage display technology is to clone a polypeptide or a protein coding gene or target gene fragment into a proper position of a phage coat protein structural gene, and to make the exogenous polypeptide or protein is fused to the coat protein, and the fusion protein is displayed on the surface of the phage with the reassembly of the daughter phage. The displayed polypeptide or protein can maintain a relatively independent spatial structure and biological activity to facilitate recognition and binding of the target molecule. Expression of a foreign peptide or protein variant library can be fused to a phage coat protein using phage display technology, such that each variant is displayed on the surface of the virion. The strongly bound phage particles are then biopanning and recovered. Finally, each peptide ligand is identified by DNA sequencing and can then be applied to the nanoparticle drug carrier by chemical synthesis.

However, it has been found during the actual testing that phage-derived peptides conjugated to drug carriers are targeted and do not always have good targeting capabilities, for example, a 15 amino acid peptide (hereinafter referred to as a GYR peptide) is cloned on the fd coat protein (p3) of a filamentous phage, and the clone has high in human brain capillary endothelium hCMEC / D3 cells and mouse brain endothelium in vivo. However, when the researchers impart this property to liposomes, they have weak binding to the target regardless of the density of the GYR peptide on the surface of the liposome.  The reason was analyzed and various factors were found to influence this process, such as particle shape, possible changes in the functionalization process, structure and orientation of the peptide, and masking of the ligand by plasma/serum proteins. In addition, peptide alignments that differ in the phage microenvironment may also be one of the causes of these differences. Thus simply simply attaching the synthetic phage peptide to a drug carrier does not necessarily mimic those structural displays that confer target recognition and specificity on the surface of the phage.

In order to solve this problem, the researchers made further research and exploration. Further studies of the p3 protein on the surface of filamentous phage fd revealed a copy of the five p3 proteins present on the surface of the phage, and the distance between them was very close. The interdomain interactions and disulfide bonds of the p3 protein can affect the structure of p3, thereby promoting intermolecular binding between adjacent p3 proteins, forming a multivalent domain with high target binding affinity. By using similar principles, the researchers proposed the hypothesis that the phage peptide can be multimerized to increase its targeting affinity. This hypothesis was verified by GYR peptides mentioned above, guided by GYR self-assembly to core-shell nanoparticles and multiple crossed β-sheet nanofibrils (NLCs). When NLC targets more than two receptors on brain endothelial cells, the drug carrier can cross the blood-brain barrier (BBB) and deliver functional nucleic acids to the brain without adverse reactions and toxicity. Therefore, it provides a new solution for the low targeting affinity of phage display peptides.