Novel Blood-Brain Barrier Shuttle Peptides Discovered through the Phage Display Method

Abstract

Delivery of therapeutic agents into the brain is a major challenge in central nervous system drug development. The blood-brain barrier (BBB) prevents access of biotherapeutics to their targets in the central nervous system and, therefore, prohibits the effective treatment of many neurological disorders. To find blood-brain barrier shuttle peptides that could target therapeutics to the brain, we applied a phage display technology on a primary endothelial rat cellular model. Two identified peptides from a 12 mer phage library, GLHTSATNLYLH and VAARTGEIYVPW, were selected and their permeability was validated using the in vitro BBB model. The permeability of peptides through the BBB was measured by ultra-performance liquid chromatography-tandem mass spectrometry coupled to a triple-quadrupole mass spectrometer (UHPLC-MS/MS). We showed higher permeability for both peptides compared to N-C reversed-sequence peptides through in vitro BBB: for peptide GLHTSATNLYLH 3.3 × 10^-7 cm/s and for peptide VAARTGEIYVPW 1.5 × 10^-6 cm/s. The results indicate that the peptides identified by the in vitro phage display technology could serve as transporters for the administration of biopharmaceuticals into the brain. Our results also demonstrated the importance of proper BBB model for the discovery of shuttle peptides through phage display libraries.

Keywords: blood–brain barrier; phage display; shuttle peptides.

Figure 1

Bio-panning experiments. Selection of phages specific for endothelial cells. Ph.D.-12^TM Phage Display Peptide Library was first incubated with neuroblastoma cells to exclude promiscuous binding peptides. The non-bound phages were then screened on primary rat brain endothelial cells. We performed four rounds of bio-panning.

Figure 2

The number of phages recovered from endothelial cells increased after each round of bio-panning.

Figure 3

Identification of peptides by high-resolution mass spectrometry analysis. Total ion chromatogram (A), fragmentation spectra of N–C reversed sequence peptide WPVYIEGTRAAV and VAARTGEIYVPW (B), fragmentation spectra of peptide GLHTSATNLYLH, and peptide N-C reversed sequence peptide HLYLNTASTHLG (C) are shown.

Figure 4

Selected reaction monitoring (SRM) chromatograms of peptides: (A) WPVYIEGTRAAV; (B) GLHTSATNLYLH; (C) VAARTGEIYVPW; and (D) HLYLNTASTHLG.

Figure 5

Confocal microscopy of endothelial cells after 1 h of co-incubation with VAARTGEIYVPW (A–C), GLHTSATNLYLH (D–F), WPVYIEGTRAAV (G–I) and HLYLNTASTHLG (J–L) peptides. Biotinylated peptides are shown in green and pan-laminin signal is shown in red. White arrows indicate examples of the internalization of peptides into endothelial cells. Scale bar = 50 µm.

Figure 6

The transport of peptides across the in vitro BBB model was affected by temperature. To test whether the transport of peptides was performed by active uptake, endothelial cells were incubated with peptides at 37 °C and also at 4 °C.