Targeting the choroid plexus-CSF-brain nexus using peptides identified by phage display

Abstract

Drug delivery to the central nervous system requires the use of specific portals to enable drug entry into the brain and, as such, there is a growing need to identify processes that can enable drug transfer across both blood-brain and blood-cerebrospinal fluid barriers. Phage display is a powerful combinatorial technique that identifies specific peptides that can confer new activities to inactive particles. Identification of these peptides is directly dependent on the specific screening strategies used for their selection and retrieval. This chapter describes three selection strategies, which can be used to identify peptides that target the choroid plexus (CP) directly or for drug translocation across the CP and into cerebrospinal fluid.

Fig. 1

Targeting cultured CP epithelial cells and explants with EGF phage is shown. Using the methods described here, control (untargeted phage) and EGF-targeted phage were incubated with either cultured CP epithelial cells (a–f), explants of mouse CP (k), or injected i.v. (i) to demonstrate the feasibility of CP targeting with phage. Controls (a, c, e and i) used untargeted phage, whereas EGF-targeted phage was evaluated in b, d, f, h and j. When the particles are added to cells after adding exogenous EGF (1 μg/mL), then specificity can be demonstrated by eliminating the internalization (k). (l) In another approach, PCR can be used to assess recoveries from CSF in control untreated animals (CSF) and in three treatment groups (CSF-1, CSF-2, and CSF-3) as long as there is a positive control.

Fig. 2

Strategies for drug targeting to the CP are shown. There are three ways to target drugs to the brain via the CP. Firstly, drugs could be translocated directly into CSF from the apical (ventricular) side of the epithelium to find their targets via CSF bulk flow. In the second mechanism, the CP itself is the drug target, and the epithelium could be the therapeutic target of the drug and modulate its natural functions. The third mechanism targets the CP, for example with a gene, with the goal of exploiting the CPs natural ability to produce CSF and secretes biotherapeutic factors. Phage display could be used to identify each of the three categories of CP targeting agents depending on the different biopanning screens deployed. In the first, the CP is targeted “transchoroidally” from blood to CSF, in the second, it is targeted to the basolateral (blood-facing) epithelium, and in the third to the apical (CSF-facing) epithelium.

Fig. 3

(a, b) Dissection of brain for choroid plexus sampling: Three cuts with a flat edged razor blade enable dissection of the CP from the brain. While it is possible to accomplish this on the bench top, a dissecting microscope is highly recommended.

Fig. 4

Template for immunostaining. A 24 well plate is prepared prior to immunostaining using a template like the one shown here. Tissues are carefully transferred from one well to the next rather than washing in a single well and risking losing tissue.