Intracellular Delivery Using Fluorophore-CPP Conjugates and Light: Mechanisms and Implications
| dc.contributor | Pellois, Jean-Philippe | |
| dc.creator | Muthukrishnan, Nandhini | |
| dc.date.accessioned | 2016-05-01T05:30:58Z | |
| dc.date.accessioned | 2017-04-07T20:09:38Z | |
| dc.date.available | 2016-05-01T05:30:58Z | |
| dc.date.available | 2017-04-07T20:09:38Z | |
| dc.date.created | 2014-05 | |
| dc.date.issued | 2014-05-02 | |
| dc.description.abstract | Cell-penetrating peptides (CPPs) can induce translocation of conjugated macromolecules across the plasma membrane of live cells. The major route of uptake of these CPPs by cells is through endocytosis. However, intracellular cytosolic delivery efficiency of these reagents is inefficient because CPP-cargo conjugates typically remain trapped inside endosomes. As a result, macromolecules are unable to reach their cytosolic targets and exert biological function. The fluorophore-CPP conjugate (Fl-CPPs) where the prototypical CPP TAT is conjugated to the fluorophore TMR gets entrapped inside endosomes of live cells upon incubation. Interestingly, irradiation of the endosomally contained TMR-TAT with moderate doses of light induces release of Fl-CPPs into the cytosol. However, the mechanism of this phenomenon is not clear. Also, the endosomal release of TMR-TAT is accompanied by loss of plasma membrane integrity, membrane blebbing, and cell-death. I investigated the molecular basis of the photo-induced endosomolytic activity of Fl-CPPs and the mechanisms behind Fl-CPP mediated cell death. I reported that Fl-CPPs act as photosensitizer molecules that can destroy membranes such as endosomal membranes, membranes of simpler model systems RBCs and liposomes. I showed that the CPP moiety of Fl-CPPs binds to negatively charged phospholipids of target membranes and brings the attached fluorophore into close proximity of the membrane. Upon irradiation of Fl-CPPs, reactive oxygen species (ROS) such as singlet oxygen and superoxide are produced that cause oxidation of membrane lipids. In addition, CPPs have a latent ability to cause damage of photo-oxidized lipid membranes. Thus, CPPs and singlet oxygen generators act in synergy to cause photolysis of membranes. I further establish structure activity relationship of Fl-CPPs to understand how structure of Fl-CPPs impact synergistic activity on membranes. These factors should therefore be considered for the development of effective delivery agents. I also explored mechanisms behind cell death that accompanied TMR-TAT mediated PCI. I showed that the lysis of endocytic organelles by TMR-TAT caused a rapid increase in the concentration of calcium in the cytosol followed by accumulation of calcium in the mitochondria. Ruthenium red and cyclosporin A, inhibitors of calcium import in mitochondria and of the mitochondria permeability transition pore were able to inhibit cell death. | |
| dc.identifier.uri | http://hdl.handle.net/1969.1/152737 | |
| dc.language.iso | en | |
| dc.subject | cell-penetrating peptide | |
| dc.subject | TAT peptide | |
| dc.subject | photochemical internalization | |
| dc.subject | intracellular delivery | |
| dc.title | Intracellular Delivery Using Fluorophore-CPP Conjugates and Light: Mechanisms and Implications | |
| dc.type | Thesis |