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.

Mechanism Matters: A Taxonomy of Cell Penetrating Peptides

“…Much work has been done, mostly by trial and error, to modify sequences to improve internalization of CPPs, with some examples of success [1–6]. Here we discuss the many overlapping mechanisms by which actively and passively internalized peptides, and attached cargo, interact with lipid bilayer membranes and bypass or disrupt membranes to gain access to the cell cytosol.…”

Self-assembling peptide-based building blocks in medical applications

“…The conjugation of the non-peptidic group can be performed during solid phase peptide synthesis on resin. The orthogonal groups of the amino acids provide specific regions for conjugation within a peptide sequence, or the option for conjugation onto the N-terminus [60]. …”

Molecular design and synthesis of self-assembling camptothecin drug amphiphiles

“…The use of peptides in therapeutic applications, either as drugs themselves [1][2][3] or as a delivery vector for another bioactive agent [4][5][6][7][8][9][10][11][12] , is a widely explored area within the drug delivery community, especially now that their synthesis has become more facile and cost effective [13,14] . The versatility in peptide design that the twenty naturally occurring amino acids offer, coupled with the potential for high affinity associations toward target receptors, make their usage highly attractive, especially in the creation of bioactive supramolecular nanomaterials [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] .…”