Muthukrishnan, Nandhini (2014-05). Intracellular Delivery Using Fluorophore-CPP Conjugates and Light: Mechanisms and Implications. Doctoral Dissertation. | Thesis individual record
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.

etd chair
publication date
2014