SialoPen peptides are new cationic foldamers with remarkable cell permeability

Abstract

The ability to access intracellular targets is of vital importance as the number of identified druggable intracellular targets increases every year. However, intracellular delivery poses a formidable barrier, as many potential therapeutics are impermeable to cell membranes, which hinders their practical application in drug development. Herein we present de novo-designed unnatural cell penetrating peptide foldamers utilizing a 2,3-Didehydro-2-deoxyneuraminic acid (Neu2en) scaffold. Conveniently, this scaffold is amenable to standard Fmoc-based solid-phase peptide synthesis, with the advantages of tunable secondary structures and enhanced biostability. Flow cytometry and live-cell confocal microscopy studies showed that these Neu2en-based peptides, hereinafter termed SialoPen peptides, have significantly superior uptake in HeLa and primary neuronal hippocampal cells, outperforming the classical cell permeable peptides penetratin and HIV-TAT.

Keywords: Biological sciences; Cargo transport; Cell penetrating peptides; Drug delivery; Foldamer; Health sciences; Helical; Infectious disease; Neuron translocation; Polymer; Public health; SPPS; Sialic acid; Unnatural amino acid; Virology.

Figure 1

Sialic acid derivative Fmoc-Neu2en.N-(9-fluorenylmethoxycarbonyl)-2,3-dehydro-8,9-isopropylidene neuraminic acid 1 the α-δ unnatural amino acid used to build the SialoPen peptides for use in Fmoc based SPPS.

Figure 2

Chart showing the peptide design and fluorophore cargo attachment, and schematic showing structure of SialoPen peptides.

Figure 3

SialoPen peptides translocate HeLa cells efficiently as assessed by flow cytometry. Mean fluorescence intensity (MFI) of HeLa cells indicating that SialoPen peptides (6, 7, 8) show enhanced uptake vs peptides Pentratin P and TATp T especially at 5.0 μM. Error represented as ±SD, n = 3 biological replicates (triplicate wells per condition per replicate). Significance determined using unpaired t-tests reporting two-tailed p-values (95% confidence interval) additional values found in SI (Table S1). ∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001, ∗∗∗∗P ≤ 0.0001.

Figure 4

SialoPen peptides are taken up efficiently in primary and immortalized cell lines. Confocal microscopy images taken after 30 min incubation at 37 °C in imaging buffer showing uptake of SialoPen in primary rat hippocampal neurons and HeLa cells compared to TATp, all conjugated to the fluorophore Cy5. Representative images shown were taken at identical settings with no post processing. (a) Primary hippocampal neuronal cells incubated with TATp and SialoPen 6–8 taken at 63x magnification show no uptake of TATp by cells, (b) HeLa cells incubated with TATp and SialoPen 6–8 taken at 20x magnification. Three biological replicates (duplicate wells per replicate) per condition.

Figure 5

SialoPen peptides enter cells via an energy dependent pathway. Flow cytometry profile of HeLa cells incubated at 4 and 37 °C showing that peptides are likely taken up via an energy dependent endocytic pathway. Mean fluorescence intensity chart of parallel experiments showing normalized fluorescence intensity of HeLa cells treated with Cy5 labeled peptides at various concentrations. Error bars represented as ± SD, n = 3 biological replicates (triplicate wells per condition per biological replicate).

Figure 6

SialoPen peptides enter cells via non-classical endocytosis. Effects of endocytosis inhibitors on uptake of CPPs at 2.5 μM showing the SialoPen peptides are taken up via non-classical endocytic pathways compared to penetratin and TATp. Error represented as ±SD, n = 3 biological replicates (triplicate wells per condition per biological replicate). Uptake normalized to cells incubated in media only measured via flow cytometry. Further statistical analysis can be found in SI (Table S1).

Figure 7

SialoPen peptides show no apparent cytotoxic or lytic effects: MTT assay, and Membrane integrity: LDH assay.A.) Chart showing that SialoPen peptides are not toxic to HeLa cells up to 20 times (100 μM) the maximum tested concentration used in the flow cytometry assay. B.) Chart showing that SialoPen peptides have an insignificant effect on membrane integrity of HeLa cells up to 100 μM. Error represented as ± SD, n = 3 biological replicates (triplicate wells per biological repliate).

Figure 8

Circular dichroism studies show SialoPen peptides adopt helical conformations. Mean residue ellipticity (MRE) graph of SialoPen peptides showing SialoPen peptides are helical foldamers. Insert: Peak to trough analysis of SialoPen peptides showing that the helicity of SialoPen peptides increase with length.