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Development of Novel Peptides for Mitochondrial Drug Delivery: Amino Acids Featuring Delocalized Lipophilic Cations

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ABSTRACT

Purpose

To create a new class of mitochondria-penetrating peptides (MPPs) that would facilitate drug delivery into the organelle through the inclusion of delocalized lipophilic cations (DLCs) in the peptide sequence.

Methods

We synthesized two novel amino acids featuring DLCs and incorporated them into peptides. Systematic studies were conducted to compare peptides containing these residues to those with natural cationic amino acids. Diastereomers were compared to determine the most advantageous arrangement for these peptides. Peptide lipophilicity, cellular uptake and mitochondrial specificity were compared for a variety of peptides.

Results

Synthetic DLC residues were found to increase mitochondrial localization of MPPs due to higher overall hydrophobicity. MPP stereochemistry was important for cellular uptake rather than subcellular localization. This study reaffirmed the importance of uniform overall charge distribution for mitochondrial specificity.

Conclusions

DLCs can be incorporated into synthetic peptides and facilitate mitochondrial drug delivery. Lipophilicity and charge distribution must be carefully balanced to ensure localization within mitochondria.

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Abbreviations

CPP:

cell-penetrating peptide

DLC:

delocalized lipophilic cation

MPP:

mitochondria-penetrating peptide

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Author information

Authors and Affiliations

  1. Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, M5S 3M2, Canada

    Shana O. Kelley & Kelly M. Stewart

  2. Department of Pharmaceutical Sciences Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, M5S 3M2, Canada

    Shana O. Kelley & Rida Mourtada

Authors
  1. Shana O. Kelley
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  2. Kelly M. Stewart
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  3. Rida Mourtada
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Corresponding author

Correspondence to Shana O. Kelley.

ELECTRONIC SUPPLEMENTARY MATERIALS

Below is the link to the electronic supplementary material.

Fig. S1

Synthesis of carboxy-derivatized thiazole orange dye. See Materials and Methods for synthesis protocols. (PDF 9 kb)

Fig. S2

Structures of (a) Zincke and (b) peptide alkylation reaction intermediates as detected by ESI Mass Spectrometry. aDue to peptide reconstruction algorithms not recognizing the pyridinium formal charges, m/z detected was always equal to the calculcated m/z minus the total number of formal positive charges. (PDF 178 kb)

Fig. S3

Subcellular localization of the cation panel in unfixed HeLa cells. Representative confocal images of the peptide panel taken with a 63x objective (a) and 20x objective (b) for clarity. (PDF 1312 kb)

Fig. S4

Uniformity of peptide uptake in HeLa cells. The uniformity of peptide uptake across all cells is displayed by comparing the relative width and shape of flow cytometry histograms of HeLa cells treated with 4 μM peptide for 60 min. The DLC containing peptides exhibited sharper Gaussian distribution, which corresponds to a more consistent cellular uptake and confirms the even cellular internalization profiles seen with confocal microscopy. Note: Peptide 1c exhibits histograms with shapes similar to 1 d and is omitted from graph for clarity. See Materials and Methods for the flow cytometry protocol. (PDF 103 kb)

Fig. S5

Structures of MPP-conjugates used in optimization studies. See Materials and Methods for synthesis protocols. (PDF 191 kb)

Fig. S6

Structures of the polymeric DLC MPPs. See Materials and Methods for synthesis and purification protocols. (PDF 26 kb)

Table S1

ESI mass spectrometry and HPLC characterization for peptide conjugates. (PDF 57.1 kb)

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Kelley, S.O., Stewart, K.M. & Mourtada, R. Development of Novel Peptides for Mitochondrial Drug Delivery: Amino Acids Featuring Delocalized Lipophilic Cations. Pharm Res 28, 2808–2819 (2011). https://doi.org/10.1007/s11095-011-0530-6

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  • DOI: https://doi.org/10.1007/s11095-011-0530-6

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