Peptide synthesis
Peptides were synthesized via automated peptide synthesis using a syro I peptide synthesis robot (Manufacturer). Peptides were assembled on a RINK-amide resin in parallel 96-well tip synthesis format using 10 mg of resin per tip. The resin was primed for synthesis by three cycles of resin incubation with 200 µL dimethylformamide (DMF) for 30 minutes. FMOC deprotection was performed at each stage of synthesis by treating the resin three times with 80 µL of a 40% piperidine/60% DMF mixture then washing the resin 8 times with 100 µL of DMF. Coupling was performed by incubating the resin with 6 molar equivalents of a preactivated mixture of each FMOC protected amino acid for 30 minutes. Coupling steps were performed twice, first using a 2/2/1 mixture of 450 mM FMOC amino acid in DMF/ 450 mM N,N'-Diisopropylcarbodiimide in DMF/ 900 mM Oxyma in DMF, and secondly using a 2/2/1 mixture of 450 mM FMOC amino acid in DMF/430 mM (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate in DMF/ 1.8 M N,N-Diisopropylethylamine in N-Methyl-2-pyrrolidone. Each coupling step was performed using 100 µL of each preactivation mixture, and was followed with washing 4 times with 110 µL of DMF and a deprotection step as described previously. After the final amino acid coupling, two additional rounds of coupling and deprotection were performed to create an N terminal Fluorescein label connected to the peptide chain via a Beta-alanine linker. The beta alanine linker coupling was performed as specified above for the individual amino acid building block steps, and the fluorophore coupling step was performed by incubating the resin overnight in 100 µL of 3 molar equivalents of Fluorescein isothiocyanate isomer I dissolved in a mixture of DMF and 6 molar equivalents of DIPEA followed by washing the resin 8 times with 110 µL DMF. Cleavage of the peptide from the resin backbone was performed by washing the resin with Dichloromethane, then treating the resin for 2 hours with 200 µL of a mixture composed of 92.5% TFA, 2.5% TIS, 2.5% DODT, 2.5% Water. The labeled peptides were collected in a deep well 96 well plate, precipitated using cold ethyl ether and then centrifuged at 1000 RPM for 10 minutes to pellet the precipitate. Pelleted peptides were then resuspended in cold ethyl ether and centrifuged again in a similar fashion for two additional cycles to wash the peptides of residual cleavage cocktail. The peptides were then resuspended in 10 mM HCl, frozen overnight at -80°C, and lyophilized to remove water and trifluoroacetic acid. The resulting peptide powders were sealed and stored at -80°C.
Adjustment of peptide concentrations
One milliliter of 20% ethanol was added to each well of the 96 well plates containing the lyophilized peptides and the plates were then re-sealed. Sealed plates were placed until halfway submerged into a bath sonicator for 30 minutes to aid resuspension. The plates were centrifuged at 1000 RPM for 10 minutes to pellet any undissolved material. A 100 µL volume of each peptide were transferred into a clear bottom 96 well plate containing an internal standard of FITC in 80% water/20% ethanol, 10 mM HCl. Each peptide was quantified using FITC absorbance as a proxy for peptide concentration. Each peptide was normalized to 50 µM in a new deep well 96 well plate by adding calculated amounts of 20% ethanol and labelled peptide, then aliquoted to opaque black 96 well daughter plates that were sealed with foil and stored at -80°C.
Cell culture
Human B3 corneal epithelial cells and ARPE19 cells were obtained from ATCC. Cells were grown to confluency using DMEM at 37°C, 5% CO2. ARPE-19 and B3 cells for uptake experiments were seeded into a dark walled, clear bottom 96 well plate at 200 cells per well. All solutions were warmed to 37°C, and CPP incubations were performed at 37°C, 5% CO2.
CPP uptake
CPP working solutions were prepared by diluting the samples from the 50 µM stock plate to 10 µM in cell growth media. Cells were incubated with 100 µL of the 10 µM CPP solution for 2 hours, after which the CPP solution was aspirated from the wells, which were then washed three times with phosphate buffered saline. Cells were then incubated with 100 uL of PBS for analysis. Raw Cell fluorescence values were monitored using a spectramax I3 plate reader using an excitation wavelength of 495 nm and an emission wavelength of 530 nm. An Incucyte live cell analysis microscopy system was used to extract brightfield and fluorescence images of the cells as well, using a 20X objective microscope for brightfield images and a green fluorescent channel with an excitation wavelength of 460 nm (passband 440,480 nm) and an emission wavelength of 524 nm (passband 504,544 nm). Incucyte software was used to determine green calibrated units and phase area.
Confocal Microscopy
Three representative sequences: TAT (YGRKKRRQRRR), Penetratin (RQIKIWFQNRRMKWKK), Nicastrin (RLPRCVRSTARLARALSPAF) were selected for comparative confocal microscopy analysis to the same CPP bearing lysosomal targeting sequences (LYSTAT- CSEWAYGRKKRRQRRR; LYSPenetratin- SLLKGRQGIYRQIKIWFQNRRMKWKK; LYSNicastrin- SLLKGRQGIYRLPRCVRSTARLARALSPAF) to assess whether Localization to the lysosome was increased by the inclusion of lysosomal targeting sequences. A control CPP consisting of FITC- GGGGGGGGGG was used to establish background signal intensity for the Green channel. Confluent B3 and ARPE19 Cells seeded on coverslips were treated with 1 µM of each peptide in 1 mL of media in a 6 well dish for 2 hours at 37°C. The cells were then washed 3 times in PBS before being incubated with LysoTracker™ Red DND-99 according to the manufacture’s instructions. The cells were fixed with 4% Paraformaldehyde in PBS for 10 minutes at room temperature. The nuclear fraction was stained blue using a 1:5000 dilution of Hoescht stain in PBS. The microscope slides were imaged on Leica SP8 confocal microscope utilizing 63X oil immersion objective with UV laser 405nm, VIS laser 488nm, and VIS laser 638.