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Mechanism of Enhanced Cellular Uptake and Cytosolic Retention of MK2 Inhibitory Peptide Nano-polyplexes

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Abstract

Electrostatic complexation of a cationic MAPKAP kinase 2 inhibitory (MK2i) peptide with the anionic, pH-responsive polymer poly(propylacrylic acid) (PPAA) yields MK2i nano-polyplexes (MK2i-NPs) that significantly increase peptide uptake and intracellular retention. This study focused on elucidating the mechanism of MK2i-NP cellular uptake and intracellular trafficking in vascular smooth muscle cells. Small molecule inhibition of various endocytic pathways showed that MK2i-NP cellular uptake involves both macropinocytosis and clathrin mediated endocytosis, whereas the free peptide exclusively utilizes clathrin mediated endocytosis for cell entry. Scanning electron microscopy studies revealed that MK2i-NPs, but not free MK2i peptide, induce cellular membrane ruffling consistent with macropinocytosis. TEM confirmed that MK2i-NPs induce macropinosome formation and achieve MK2i endo-lysosomal escape and cytosolic delivery. Finally, a novel technique based on recruitment of Galectin-8-YFP was utilized to demonstrate that MK2i-NPs cause endosomal disruption within 30 min of uptake. These new insights on the relationship between NP physicochemical properties and cellular uptake and trafficking can potentially be applied to further optimize the MK2i-NP system and more broadly toward the rational engineering of nano-scale constructs for the intracellular delivery of biologic drugs.

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Acknowledgments

We thank Dr. Felix Randow and Dr. Bob Weinburg for kind gifts of plasmids via AddGene.com. We thank Dr. Janice Williams for imaging support and electron microscopy expertise. Confocal imaging, transmission electron microscopy, and scanning electron microscopy were performed in part through the use of the Vanderbilt University Medical Center Cell Imaging Shared Resource (supported by NIH Grants CA68485, DK20593, DK58404, DK59637 and EY08126). Dynamic light scattering was conducted at the Vanderbilt Institute of Nanoscale Sciences and Engineering. This work was supported by the American Heart Association (11SDG4890030), National Institutes of Health/National Heart, Lung, and Blood Institute (R21 HL110056 and R01 HL122347), and a National Science Foundation Graduate Research Fellowship to K.V.K. (0909667 and 1445197).

Conflict of Interest

KVK, BCE, CMB, and CLD report grant support from the National Institutes of Health and the American Heart Association; KVK additionally reports grant support from National Science Foundation Graduate Research Fellowship Program. During the conduct of the study, authors disclose non-financial support from Moerae Matrix, Inc., outside the submitted work. CMB is chief scientific officer and a shareholder of Moerae Matrix, Inc. BCE, CMB, and CLD are inventors listed on patent PCT/US2014/033873, licensed by Moerae Matrix, Inc. MK2i is known commercially known as MMI-0100 and is being developed by Moerae Matrix, Inc. for clinical use (ClinicalTrials.gov Identifier: NCT02515396).

Ethical Standards

No human or animal studies were carried out by the authors for the completion of this work.

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Authors and Affiliations

  1. Department of Biomedical Engineering, Vanderbilt University, 5919 Stevenson Center, VU Mailbox: PMB 351631, Nashville, TN, 37235-1631, USA

    Kameron V. Kilchrist & Craig L. Duvall

  2. Division of Vascular Surgery, Department of Surgery, Vanderbilt University Medical Center, Nashville, TN, 37232, USA

    Brian C. Evans & Colleen M. Brophy

  3. Veterans Affairs Medical Center, VA Tennessee Valley Healthcare System, Nashville, TN, 37212, USA

    Colleen M. Brophy

Authors
  1. Kameron V. Kilchrist
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  2. Brian C. Evans
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  4. Craig L. Duvall
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Correspondence to Craig L. Duvall.

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Associate Editor Tejal Desai oversaw the review of this article.

Craig L. Duvall is an Associate Professor of Biomedical Engineering (BME) at Vanderbilt University. He completed his undergraduate studies at the University of Kentucky in 2001 and immediately started his doctoral studies in BME at Georgia Tech and Emory University. Robert Guldberg, a Mechanical/Biomedical Engineer from Georgia Tech, and W. Robert Taylor, a cardiologist from Emory, jointly directed Dr. Duvall’s Ph.D. work. In 2007, Dr. Duvall joined the Bioengineering laboratories of Patrick Stayton and Allan Hoffman at the University of Washington developing polymeric drug delivery technologies as a postdoctoral researcher. Based on the foundations built from these combined experiences, the Duvall Advanced Therapeutics Laboratory (ATL) was launched at Vanderbilt in 2010. The ATL is funded by grants from NIH, DOD, NSF, AHA, and ADA and focuses on the development of novel drug delivery technologies for applications in regenerative medicine and breast cancer therapy.

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Kilchrist, K.V., Evans, B.C., Brophy, C.M. et al. Mechanism of Enhanced Cellular Uptake and Cytosolic Retention of MK2 Inhibitory Peptide Nano-polyplexes. Cel. Mol. Bioeng. 9, 368–381 (2016). https://doi.org/10.1007/s12195-016-0446-7

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