Fig. 6.
The role of stereochemistry and proteolysis in transcellular cargo delivery across MDCKII cells. A. The permeability of cargo as a function of concentration for the CL-5FAM peptide and the CL peptide D-isomer (DCL-5FAM). The dashed line represents Papp of free 5FAM added at 10 μM apical concentration. <D.L. = below the detection limit. The sample size n represents the number of transwell replicates: 2.5 μM CL-5FAM (n=6); all other concentrations (n=3). Data represent mean ± SD. B. The permeability of peptide-cargo conjugates (CL-5FAM, CL-6TAMRA, DCL-5FAM, and DCL-6TAMRA) and for free cargo (5FAM and 5(6)TAMRA). The apical concentrations were 2.5 μM for the peptide-cargo conjugates, 10 μM for 5FAM, and 20 μM for 5(6)TAMRA. The sample size n represents the number of transwell replicates: CL-6TAMRA and DCL-6TAMRA (n=4), all other treatments (n=3). Data represent mean ± SD. C. The permeability of peptide-cargo conjugates (CL-6TAMRA and DCL-6TAMRA) and Lucifer yellow in tissue-engineered microvessels. The concentrations were 2.5 μM for the peptide-cargo conjugates and 100 μM for Lucifer yellow. The sample size n represents the number of microvessels: CL-6TAMRA (n=3), DCL-6TAMRA (n=3), LY (n=6). Data represent mean ± SD. D. Confocal microscopy images of the uptake of 2.5 μM CL-6TAMRA and DCL-6TAMRA by MDCKII cells after 1-hour incubation at 37 ˚C (see Fig. S13 for nuclear stain and additional images). Both peptide-cargo conjugates showed diffuse and punctate fluorescence inside the cells. DCL-6TAMRA had an additional localization inside the nucleus, which was not observed for CL-6TAMRA. The D-isomer of the CL peptide can deliver cargo into cells but has low Papp across the biological barrier. E. HPLC spectra of the basolateral solution 1 hour after introducing CL-5FAM or CL-6TAMRA into the apical chamber. The input solution is the peptide-conjugate solution prior to addition into the apical chamber. The CL-6TAMRA spectrum is expanded 11-fold for comparison to CL-5FAM. F. The proposed mechanism for transcellular transport of CL-cargo conjugates. The CL peptide delivers the conjugated cargo into cells, where the peptide undergoes proteolysis. One of the proteolysis products is the cargo, conjugated to the N-terminal amino acid of the peptide, which exits the cells on the basolateral side, as well as the apical side.