An unusual cell penetrating peptide identified using a plasmid display-based functional selection platform

Abstract

Cell penetrating peptides (CPPs) have tremendous potential for use in gene and drug delivery applications. The selection of new CPPs with desired capabilities from randomized peptide libraries is challenging, since the CPP phenotype is a complex selection target. Here we report the discovery of an unusual new CPP from a randomized peptide library using a functional selection system based on plasmid display (PD). After four rounds of screening of a 14-mer peptide library over PC12 cells, several peptides were identified and tested for their ability to deliver the green fluorescent protein (GFP). One peptide (SG3) exhibited a cell penetrating phenotype; however, unlike other well-known CPPs such as TAT or Penetratin, the newly identified peptide was not highly cationic. The PD protocol necessitated the addition of a cationic lipid (Lipofectamine2000), and in the presence of this compound, the SG3 peptide significantly outperformed the well-known TAT CPP in the delivery of GFP to PC12 cells and primary astrocytes. When the SG3 peptide was fused to the pro-apoptotic BH3 peptide from the Bak protein, significant cell death was induced in cultured primary astrocytes, indicating relevant, intracellular delivery of a functional cargo. The PD platform is a useful method for identifying functional new CPPs from randomized libraries with unique delivery capabilities.

Figure 1

Schematic of the selection protocol developed for identifying new CPPs from a randomized library using the PD platform.

Figure 2

Delivery of GFP to PC12 cells using peptides identified from the library selection. A. Fusion proteins (2.8 nmol) were added to PC12 cells in a 24-well plate and analyzed by flow cytometry. B. Dose-dependent uptake of fluorescent fusions by PC12 cells: GFP (■), GFP-TAT (▲) and GFP-SG3 (○). In both panels, each experiment was performed in triplicate (n=3) and error bars represent standard errors. ‘*’ indicates the value is significantly different from the GFP treated control cells. C–E. Confocal microscopy analysis of fusion protein uptake by PC12 cells (Green: GFP signal; Red: CellTracker Red CMTPX; Yellow arrows: a typical spot on the image of Z plane, orthogonal X and Y plane). C: incubation for 4 hours with GFP-TAT, resulting in an overlap coefficient of 0.983; D: incubation for 4 hours with GFP-SG3, resulting in an overlap coefficient of 0.994; E: incubation for 4 hours with GFP, resulting in an overlap coefficient of 0.978.

Figure 3

Effect of heparin treatment on CPP-mediated delivery of the fluorescent fusion proteins to PC12 cells. Black bars: untreated cells incubated with 2.8 nmol of protein for 4 hours at 37°C; White bars: cells pre-treated with 100ug/mL of heparin before incubation with each protein. Each experiment was performed in triplicate (n=3) and error bars represent standard error. ‘*’ indicates the value is significantly different from the heparin treated cells.

Figure 4

Effect of the cationic lipid, Lipofectamine2000 on CPP-mediated fusion protein delivery. A. Dose-dependent delivery of proteins to PC12 cells in the presence of 10μl Lipofectamine2000. After 4 hours of incubation with the protein/Lipofectamine2000 complex, cells were analyzed by flow cytometry. GFP (■), GFP-TAT (○) and GFP-SG3 (▲). B. Sample histograms from flow cytometry analysis of GFP and GFP-TAT with and without Lipofectamine2000. Protein (2.8nmol) with or without Lipofectamine was incubated with primary astrocytes under the same transduction conditions as panel A. C. Comparison of replicate flow cytometry results as described in panel B. For panels A and C, experiments were performed in triplicate (n=3) and error bars represent standard error.

Figure 5

CPP-mediated delivery of the BH3 death peptide into primary astrocytes. The number of dead cells was compared to the number of dead cells obtained upon treatment with solvent or solvent plus Lipofectamine2000 alone. Each experiment was performed in triplicate (n=3) and error bars represent standard error. ‘*’ indicates the value is significantly different from the cells transduced by fusion peptides without the presence of cationic lipid.