2.4 |. Quantification of peptide cell permeability

The quantification of peptide uptake was performed using a protocol previously developed by Qian and co-workers^[16] to evaluate peptide permeability efficiency in a time-, concentration-, and temperature-dependent manner. Prior to experimentation, all peptides were reconstituted in sodium phosphate buffer (2.26 mM NaH₂PO₄ H₂O and 8.43 mM Na₂H PO₄ 7H₂O). Stock peptide concentration was determined using a Nano-Drop (Thermo Scientific) at the wavelength of 492 nm using the UV-vis function. Three days prior to the experiment, HeLa cells were seeded at a density of 1 × 10⁴ cells/mL in 12-well plates (Corning). On the day of experiment, peptides were diluted to the desired final concentrations in extracellular buffer (ECB; 5.036 mM HEPES pH 7.4, 136.89 mM NaCl, 2.68 mM KCl, 2.066 mM MgCl₂ 6H₂O, 1.8 mM CaCl₂ 2H₂O, and 5.55 mM glucose). The cells were washed 2× with ECB (1 mL/well) followed by the addition of the peptide solutions (500 μL/well) for indicated time points at the indicated temperatures. All plates were wrapped in aluminum foil to avoid deactivating the FAM tag on the peptide. Following the incubation time, the peptide solution was removed, and the cells were washed 2× with ECB. The cells were trypsinized (200 μL/well) for 10 minutes at room temperature to detach the cells from the wells and remove any remaining peptide adhered to the extracellular surface. The cells were then resuspended in ECB (1 mL/well), thoroughly mixed, and transferred to microcentrifuge tubes. The samples were centrifuged (1800 rcf, 2.5 minutes, at room temperature) to isolate the cells. Following centrifugation, the supernatant was discarded, and the pellet was lysed with 0.1 M NaOH (250 μL/tube). While the fluorescent intensity of FAM has been demonstrated to be dependent on pH, it was confirmed that the 0.1 M NaOH had negligible effects on the intensity profile of RWRWR, OWRWR, and S-OWRWR peptides at both 10 and 30 μM concentrations (Supporting Information Figure S3).

Each sample was then transferred to a 96-well plate and quantified by fluorometry (Perkin Elmer (Waltham, MA) Wallac 1420 VICTOR2 multilabel HTS counter). The FAM tag was quantified using an excitation filter of 490 nm and an emission filter of 535 nm. During every experiment, a no peptide control was performed (eg, cells incubated with ECB only). To measure the background signal of each peptide, the peptide solutions used in each experiment was analyzed by fluorometry to normalize the observed fluorescent signal for each sample. All experimentation conditions were performed in triplicate with every experiment performed twice to ensure reproducibility. The fluorescent signal measured by the plate reader was normalized using Equation (1):

where F denotes the fluorescent signal of cells incubated with peptide, C denotes the fluorescent signal of cells incubated with no peptide, P denotes the average fluorescent signal of the peptide in suspension, and B denotes the average fluorescent signal of the ECB. This analysis was performed to allow for a comparison between the different peptides used in this study. Data reported for each peptide are the average of triplicate samples. Standard deviations were calculated based on the triplicate samples to obtain the error bars and perform analysis of variance. Each experiment was repeated twice to confirm the results.

Statistical analysis of uptake was performed using ANOVA F-statistics with SAS software. The III-67B peptide, a sequence determined to be impermeable to intact cells, was used as negative control. To determine the statistical significance of the permeability efficiency of each peptide, the normalized signals obtained from that peptide were assessed by statistical t-tests compared the negative control. For the time-dependent studies, 10 μM peptide was incubated with HeLa cells for at 10-minute intervals between 10 and 100 minutes at 37°C. For the peptides that demonstrated insignificant time-dependence (rapid internalization; RWRWR, OWRWR, RWOWR, TAT, and ARG) comparison against the negative control was carried out based on the 20-minute time point. For the peptides demonstrating time-dependence (slow internalization), namely OWOWO and WKWK, this analysis was performed based on the 100-minute time point. For the temperature-dependent studies, 10 μM peptide solution was incubated with HeLa cells for 60 minutes at 3 different temperature (37, 25, and 4°C). For the concentration-dependent studies, 4 different concentrations of peptide (5, 10, 20, and 30 μM) solution were incubated with HeLa cells at 37°C for 60 minutes. ANOVA F-statistics were performed to assess the statistical significance of the effect of incubation time, incubation temperature, and peptide concentration on the normalized fluorometry signals.