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Title page for ETD etd-03282016-154853

Type of Document Master's Thesis
Author Kilchrist, Kameron V.
Author's Email Address kameron.v.kilchrist@vanderbilt.edu
URN etd-03282016-154853
Title Mechanism of Enhanced Cellular Uptake and Cytosolic Retention of MK2 Inhibitory Peptide Nano-polyplexes
Degree Master of Science
Department Biomedical Engineering
Advisory Committee
Advisor Name Title
Craig Duvall Committee Chair
Hak-Joon Sung Committee Member
  • macropinocytosis
  • pH-responsive
  • endosome escape
  • nanoparticle
  • Drug delivery
  • vascular therapeutic
Date of Defense 2016-03-28
Availability unrestricted
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 utilizes clathrin mediated endocytosis alone 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 developed and utilized to demonstrate that MK2i-NPs cause endosomal disruption within 30 minutes 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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