Biodistribution of therapeutics into the brain is severely hindered by the blood-brain barrier (BBB) and extracellular space (ECS) present in the brain parenchyma. BBB is formed by tightly regulated brain capillary endothelial cells, which impedes the exchange of most small and macro molecules between blood vessels and brain parenchyma. Any endogenous and exogenous substances that can transport across BBB and further distribute to the target site of the brain still need to overcome the steric hindrance, electrostatic interaction and hydrophobic adhesion generated from extracellular matrix (ECM) in the brain ECS. Based on the fact, enriched peptides identified from phage display CX7C heptamer library biopanned against in vitro BBB model hCMEC/D3 cells were selected to validate the function of the BBB shuttling and brain ECM diffusion in the in-vitro and in-vivo models.
1. Cloning of peptide-presenting M13 KE phages and transcytosis validation of M13 clones Lyophilized DNA oligonucleotides (synthesized by IDT) that encode for selected BBB shuttling peptides (Pep1- Pep11) and scrambled controls were inserted into backbone M13 DNA vector using standard molecular cloning techniques. Phage clones were incubated on hCMEC/D3, an in vitro BBB model, cultured on a transwell, and their transcytosis was quantified by standard double-layer agarose plaque assays.
2. Validation of BBB-shuttling and ECM diffusion for select peptides identified from phage display. The ability of FAM-peptides to shuttle across the BBB was validated on hCMEC/D3 cells. Peptides identified from phage display screening were synthesized and fluorescently-labeled with 5-FAM at N-terminus (Lifetein Inc.). 100 µM of each peptide was prepared in 100µL basal medium and added to the donor compartment of the transwell, with 600 µL medium was replenished to the receiver compartment. At 5, 10, 15, 30, 45, 60, 90 and 120 minutes after adding FAM-labeled peptides, transwell inserts were temporarily removed, and fluorescence in the receiver compartment was read by a plate reader at each time point. The output/input (ratio of fluorescence of receiver reservoir over donor reservoir) was calculated to compare the transcytosis efficiency of each peptide. To study diffusion through ECS-like space, µ-Slide 0.4 VI chamber (ibidi) was incubated with 30 µL Matrigel in each channel. After gelation, endothelial basal medium was added to hydrate the gel for 10 min. 60 µL 100 µM of each peptide was added to one reservoir of the chamber, 60 µL of basal medium was replenished to the other reservoir. Olympus IX83 fluorescence microscope was set-up to monitor FAM-peptide diffusion behavior by recording fluorescent images of the gel-filled chamber channel every 30 min, up to 12 hours, and diffusivity was quantified from image analysis.
3. In vivo biodistribution of Cy7 conjugated and FAM-labeled peptides. 100µL of 500µM of Cy7- and FAM-labeled peptides were injected intravenously of female 4-6 week old Balb/c and NU/NU nude mice, respectively, and allowed to circulate for 1 hr prior to sacrifice. Brain, liver, lung, and heart tissues were harvested, sectioned and stained with an anti-CD31 antibody and DAPI. Distribution of the peptides in the brain vasculature and the parenchyma were imaged by fluorescence microscopy.
Jasmim Leal– Graduate Research Assistant, University of Texas at Austin, Austin
Jae You Kim– University of Texas at Austin
Xinquan Liu– Graduate Research Assistant, University of Texas at Austin, Austin
Debadyuti Ghosh– Assistant Professor, University of Texas at Austin