Vladimir P. Torchilin, Robert A. Schatz, Akio Ohta
Date of Award
Master of Science
Department or Academic Unit
Bouvé College of Health Sciences. Department of Pharmaceutical Sciences.
Sub-cellular targeting, Pharmaceutical sciences
Pharmacy and Pharmaceutical Sciences
The last century has witnessed dramatic advances in targeted delivery of therapeutic molecules. Current drug delivery approaches have successfully achieved tissue-specific and even cell-specific delivery of therapeutic molecules. However, in many cases such advances have resulted in less than the expected dramatic improvement in drug action. It is likely that in such cases, in spite of cell specific delivery, the drug molecules were not successfully delivered to their sub-cellular target inside the cell. It would appear reasonable that for the desired improvement in biological action of such a drug it is necessary to control the sub-cellular distribution of the drug molecule. To achieve such sub-cellular targeting, we have been exploring the use of subcellularly targeted lipid-based nanocarriers. We have already shown that liposome's surface, modified with mitochondriotropic stearyl triphenylphosphonium (STPP) cations, target mitochondria. We also demonstrated the potential of such a targeted delivery system in enhancing the therapeutic outcome of a drug molecule known to act on mitochondria when incorporated into mitochondria-targeted nanocarriers. We studied the effect of sub-cellular targeting on the proapoptotic and cytotoxic action of sclareol. Sclareol (labd-14-ene-8, 13-diol) is a ditertiary alcohol, a member of the labdane type diterpenes with demonstrated antitumor activity by virtue of its proapoptotic action on tumor cell lines. The apoptotic action of sclareol is mediated in part by activation of the mitochondrial apoptosis pathway. As sclareol acts on mitochondria, we hypothesized that sclareol incorporated in mitochondria targeted liposomes will improve its proapoptotic activity and cytotoxic action on cancer cells. We successfully incorporated sclareol into non-targeted liposomes and mitochondria targeted liposomes and assessed the in vitro cytotoxic activity and proapoptotic activity of sclareol incorporated in mitochondria-targeted liposomes compared to sclareol incorporated in non-targeted liposomes. Results from liposomal characterization showed preparations of stable liposomal formulations. A cell proliferation assay showed a significant increase in cytotoxicity of sclareol when incorporated into mitochondria-targeted liposomes compared to non-targeted liposomes. Using nuclei condensation by staining DNA of treated cells using Hoechst dye, we found a significant number of cells having apoptotic nuclei at 48 hrs when treated with sclareol incorporated into non-targeted liposomes. Nuclei condensation is a late apoptotic event which provided a time line to detect early apoptotic events such as caspase activation and detection of apoptotic cells by flow cytometry. As sclareol induces apoptosis partly via mitochondrial pathway, we measured the activation of mitochondrial pathway by measuring caspase-9 activation at 24 hrs. There was a significant increase in activation of caspase-9 in cells treated with sclareol incorporated into targeted liposomes compared to cells treated with sclareol incorporated into non-targeted liposomes. This increase in activation of caspase-9 reflected the molecular targeting of sclareol to its subcellular site of action mitochondria. Also, the cells undergoing apoptosis were counted by Vybrant ® Apoptosis assay kit #3 from invitrogen using flow cytometry and showed an increased number of cells undergoing apoptosis when treated with sclareol incorporated into targeted liposomes compared to sclareol incorporated into non-targeted liposomes. In our earlier studies, we have already shown potential of mitochondria-targeted liposomes in delivering ceramide, drug known to act on mitochondria, to its subcellular site of action. In the present study, we found that sclareol incorporated into mitochondria-targeted liposomes exhibits increased cytotoxic and proapoptotic activity compared to sclareol incorporated into non-targeted liposomes. By doing so, we demonstrated broad applicability of mitochondria targeted liposomes in delivering therapeutic molecules with diverse physicochemical properties to its subcellular target site of action.
Niravkumar R. Patel
Patel, Niravkumar R., "Subcellular targeting for improved drug action : incorporation of sclareol into mitochondria targeted liposomes" (2008). Pharmaceutical Science Master's Theses. Paper 10. http://hdl.handle.net/2047/d10018906
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