Dual peptide conjugation strategy for improved cellular uptake and mitochondria targeting

Abstract

Mitochondria are critical regulators of cellular function and survival. Delivery of therapeutic and diagnostic agents into mitochondria is a challenging task in modern pharmacology because the molecule to be delivered needs to first overcome the cell membrane barrier and then be able to actively target the intracellular organelle. Current strategy of conjugating either a cell penetrating peptide (CPP) or a subcellular targeting sequence to the molecule of interest only has limited success. We report here a dual peptide conjugation strategy to achieve effective delivery of a non-membrane-penetrating dye 5-carboxyfluorescein (5-FAM) into mitochondria through the incorporation of both a mitochondrial targeting sequence (MTS) and a CPP into one conjugated molecule. Notably, circular dichroism studies reveal that the combined use of α-helix and PPII-like secondary structures has an unexpected, synergistic contribution to the internalization of the conjugate. Our results suggest that although the use of positively charged MTS peptide allows for improved targeting of mitochondria, with MTS alone it showed poor cellular uptake. With further covalent linkage of the MTS-5-FAM conjugate to a CPP sequence (R8), the dually conjugated molecule was found to show both improved cellular uptake and effective mitochondria targeting. We believe these results offer important insight into the rational design of peptide conjugates for intracellular delivery.

Scheme 1. Schematic Illustrations of (A) the Three Peptide Conjugates: MTS-5-FAM, 5-FAM-H₃R₈, and MTS-(5-FAM)-H₃R₈, and (B) Different Cellular Uptake of the Three Conjugates

MTS-5-FAM possessed α-helical secondary structure and showed no cell membrane-penetrating capability, but attaching to the cell membrane only. Cell penetrating peptide 5-FAM-H₃R₈ exhibited limited cellular uptake. Significant endocytosis was only observed for MTS-(5-FAM)-H₃R₈ containing both MTS and H₃R₈.

Figure 1

Representative flow cytometry results and circular dichroism (CD) spectra. (A) Quantitative comparison of the fluorescence intensity (in Geo mean) of three conjugates 5-FAM-H₃R₈ (red column), MTS-5-FAM (green column), and MTS-(5-FAM)-H₃R₈ (blue column) in MCF-7 breast cancer cell line after 4 h treatment with 1 μM peptide conjugates, and untreated cells were used as control. CD spectra of (B) 50 μM 5-FAM-H₃R₈, (C) 50 μM MTS-5-FAM (Insertion: schematic illustration of positive charge distribution in MTS, red: methionine, blue: arginine.), and (D) 50 μM MTS-(5-FAM)-H₃R₈. All the conjugates studied in CD analysis were dissolved in 20 mM sodium phosphate buffer (pH 7.4, with 20 mM SDS) at 37 °C.

Figure 2

Cellular uptake of 5-FAM-H₃R₈, MTS-5-FAM, and MTS-(5-FAM)-H₃R₈ in MCF-7 cells with lysosome (red, Lysotracker Red) and nucleus (blue, Hoechst 33342) after 4 h incubation with the respective conjugate (1 μM). An enlarged image is also included in the Supporting Information (Figure S7).

Figure 3

Investigation of secondary structure and its function preformed on cellular uptake study of designed peptide conjugate 5-FAM-(RLL)₂R₈. (A) CD spectrum of 5-FAM-(RLL)₂R₈. (B) Representative flow cytometry results of 5-FAM-H₃R₈ (red column) and 5-FAM-(RLL)₂R₈ (green column). (C) Cellular uptake of 5-FAM-(RLL)₂R₈ in MCF-7 cells with lysosome (red, Lysotracker Red) and nucleus (blue, Hoechst 33342) after 4 h incubation with the conjugate (1 μM).

Figure 4

Subcellular colocalization of 5-FAM-H₃R₈, MTS-5-FAM, and MTS-(5-FAM)-H₃R₈ in MCF-7 cells with mitochondria (red, Mitotracker) and nucleus (blue, Hoechst 33342) after 2 h incubation of the respective conjugates (5 μM). An enlarged image is also included in the Supporting Information (Figure S9).

Figure 5

Cell viability of MCF-7 breast cancer cells after 4 h treatment with 5-FAM-H₃R₈, MTS-5-FAM, MTS-(5-FAM)-H₃R₈, and 5-FAM-(RLL)₂R₈ at three different concentrations (1, 2, and 5 μM). The results exhibited near nontoxicity of four conjugates at all three concentrations.