A STUDY OF THE BEHAVIOR AND LOCALIZATION OF PT(II) AZIDE AND ALKYNE-MODIFIED DERIVATIVES IN CELLS USING BIOORTHOGONAL CHEMISTRY AND FLUORESCENCE MICROSCOPY
MetadataShow full item record
Despite their ubiquitous use, Pt(II) anti-cancer drugs still suffer from many issues such as off-drug target effects, renal and nephrotoxicity as well as acquired and intrinsic drug resistance. To obtain a better understanding of how to mitigate these deleterious effects can be mitigated we first must know all the targets of these drugs. Highlighted in this dissertation is previous work performed by groups exploring the localization of Pt in cells using fluorescence microscopy. While Pt drugs such as cisplatin contain no native fluorescence, a great deal of work has been done to covalently modify complexes with fluorescent tags. From studies using this technique, it been reported that Pt can target a number of compartments within the cell ranging from the nucleus to the cytoplasm. With each different derivative being observed in varied cell lines it becomes difficult to deconvolute a universal pattern to where Pt localizes, furthermore, the connected fluorophore could also bias Pt localization. To add general functionality and eliminate the bias of a pre-tethered fluorophore our lab has developed a number of different azide and alkyne-modified complexes that append a “reactive handle” to Pt compounds. This modification allows for use of the bioorthogonal azide-alkyne click reaction we are able to observe Pt localization after treatment. The focus of this work includes method development to conjugate a fluorophore to our Pt complexes in vitro and in cell cultures. We examined a number of different cell lines and observed frequent localization in the nucleolus of the cell. Also in this work is the development of methods to append multiple fluorophores to each Pt site to increase our ability to visualize these complexes in cells. Finally, we have also constructed a new Pt-azide that exhibits slower exchange kinetics due to a chelating exchangeable group. The use of this new complex will enable studies to determine whether changing the leaving group results in differential localization of Pt drugs in cells.