DOI: 10.1002/cbic.200900017 Mitochondria-Penetrating Peptides: Sequence Effects and Model Cargo Transport Lema F. Yousif, [a] Kelly M. Stewart, [a] Kristin L. Horton, [a] and Shana O. Kelley* [a, b] Introduction Inefficient translocation of the cell membrane can limit the effi- cacy of drug molecules. [1] However, the cellular uptake of bio- active molecules can be increased by synthetically modifying physicochemical parameters or through the use of delivery vectors. One class of delivery vectors, cell-penetrating peptides (CPPs), has been used with significant success in preclinical studies and is effective with a wide range of impermeable cargo molecules, [2] including proteins, [3] nanocrystals, [4] nucleic acids, [5] and small-molecule drugs. [6] Given the utility of CPPs, significant effort has been directed towards optimizing their properties and understanding their mechanism of action. While CPPs are able to translocate the plasma membrane, most are not able to cross intracellular membranes; this limits organellar targeting of bioactive cargos. Mitochondria, for in- stance, are organelles implicated in many human diseases, such as diabetes, [7] Parkinson’s disease, [8] and cancer, [9] and it is therefore critical to develop strategies to deliver therapeutics to this intracellular compartment. Indeed, efforts have been made to use CPPs to deliver cargo to mitochondria, but avail- able sequences could not penetrate the organelle. Instead, other transporters, such as lipophilic cations, [10] sorbitol-based transporters, [11] and oligoguanidinium vectors [12] have been ex- plored as molecular mitochondrial transporters; however, of the targeting agents tested, limitations in cargos delivery were reported. Recently, we successfully engineered a set of CPPs that ex- hibit efficient cellular uptake as well as selective mitochondrial localization. These mitochondria-penetrating peptides (MPPs) [13] feature alternating basic and hydrophobic amino acids and were systematically engineered to elucidate the exact features needed for mitochondrial localization. These peptides appear to enter cells via a direct mode of uptake and bypass endocytic uptake, thereby avoiding endosomal/lysoso- mal sequestration that would prohibit their ability to accumu- late into mitochondria. The study of a family of sequences with varying charges and lipophilicities revealed that peptides must meet a minimal charge-dependent lipophilicity threshold for mitochondrial localization. Indeed, a critical balance of charge and lipophilicity was shown to be required for the uptake across mitochondrial membranes. Obtaining a broader understanding of the essential charac- teristics of MPPs is critical for their development as delivery vectors. Open questions remain about these peptides, includ- ing: 1) is an alternating hydrophobic-cationic motif necessary for mitochondrial localization and cargo transport; and 2) must cargos possess a particular level of polarity, charge, or hydro- phobicity for effective transport? The answers to these ques- tions would guide the application of these peptides in biology and medicine. While early studies provided a direction for MPP design, [13] a more detailed understanding of their physiochemi- cal properties is necessary to better optimize them as molecu- lar delivery agents for diverse chemical cargos. Herein, we describe a series of studies that characterize the MPP scaffold and how its exact structure correlates with mito- chondrial localization. We also tested the effect of various model cargo molecules on the intracellular accumulation of MPPs. By addressing the effects of sequence, charge distribu- tion, lipophilicity and cargo on the localization of MPPs, we gained information on the properties that permit access into the mitochondria and provided evidence that MPPs are suita- ble mitochondrial delivery vectors. A class of mitochondria-penetrating peptides (MPPs) was stud- ied in an effort to optimize their applications in the delivery of bioactive cargo to this therapeutically important organelle. The sequence requirements for mitochondrial entry were moni- tored, and it was discovered that while an alternating cationic/ hydrophobic residue motif is not required, the inclusion of a stretch of adjacent cationic amino acids can impede access to the organelle. In addition, a variety of N- and C-terminal cargo were tested to determine if there are limitations to the lipophi- licity, charge, or polarity of compounds that can be transported to mitochondria by MPPs. The results reported demonstrate that these peptide sequences are versatile transporters that will have a range of biological applications. [a] L. F. Yousif, K. M. Stewart, K. L. Horton, Prof. Dr. S. O. Kelley Department of Biochemistry, Faculty of Medicine University of Toronto, Toronto, ON M5S 1A8 (Canada) Fax: (+ 1) 416-978-2979 E-mail : shana.kelley@utoronto.ca [b] Prof. Dr. S. O. Kelley Department of Pharmaceutical Sciences, Faculty of Pharmacy University of Toronto, Toronto, ON, M5S 3M2 (Canada) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.200900017. ChemBioChem 2009, 10, 2081 – 2088  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 2081