Mechanism of Action and Gold Nanoparticle Delivery of Pt(IV) Prodrugs of Cisplatin

The antitumor effects of platinum(IV) complexes, considered prodrugs for cisplatin, are believed to be due to biological reduction of Pt(IV) to Pt(II), with the reduction products binding to DNA and other cellular targets. In this work we used pBR322 DNA to capture the products of reduction of oxoplatin, c,t,c-[PtCl2(OH)2(NH3)2], 2, and a carboxylate-modified analog, c,t,c-[PtCl2(OH)(O2CCH2CH2CO2H)(NH3)2], 3, by ascorbic acid (AsA) or glutathione (GSH). Since carbonate plays a significant role in the speciation of platinum complexes in solution, we also investigated the effects of carbonate on the reduction/DNA-binding process. In pH 7.4 buffer in the absence of carbonate, both 2 and 3 are reduced by AsA to cisplatin (confirmed using 195Pt NMR), which binds to and unwinds closed circular DNA in a manner consistent with the formation of the well-known 1, 2 intrastrand DNA crosslink. However, when GSH is used as the reducing agent for 2 and 3, 195Pt NMR shows that cisplatin is not produced in the reaction medium. Although the Pt(II) products bind to closed circular DNA, their effect on the mobility of Form I DNA is different from that produced by cisplatin. When physiological carbonate is present in the reduction medium, 13C NMR shows that Pt(II) carbonato complexes form which block or impede platinum binding to DNA. Another Pt(IV) complex, 1-adamantanemethylamine-Pt(IV), 4, which is also a potential prodrug of cisplatin was synthesized by tethering 1-adamantanemethylamine to oxoplatin using a dicarboxylate linker. To achieve higher anticancer activity and better targeting, gold nanoparticle (AuNP) coated with the host ligand per-6-thio-β- cyclodextrin, was synthesized as a drug delivery vehicle for complex 4. The results of cytotoxicity studies show that the loaded delivery vehicle inhibits the growth of human neuroblastoma cells and optical microscopy indicates that the nanoparticles localize in the nucleus of the cell. The binding of complex 4 and β-cyclodextrin is also investigated using 1H NMR, and a model with a 1:1 stoichiometry with formation of adamantane dimers is proposed. To achieve better understanding of the effect of recovery time on cell cytotoxicity, the toxic effects of three metal complexes containing the 2,2\u27-bypyridine ligand, Cu(bpy)(NCS)2, 8, [Cu(bpy)2(H2O)](PF6)2, 9, and Zn(bpy)2(NCS)3, 10, were investigated toward human neuroblastoma cells (SK-N-SH) and ovarian cancer cells (OVCAR-3) using two different cytotoxicity assays. The cells were exposed to various concentrations of the compounds for 1 h and the percent inhibition of cell growth, I, measured for various times after exposure, i.e., as a function of the recovery time t. After developing the theory showing the relationship between I and t, the cytotoxicity data were analyzed to reveal that the two copper complexes, 8 and 9, cause the cells to divide at a slower rate than the controls during the recovery period, but the zinc complex, 10, had little or no effect on cell division during the recovery period. The usual metric for reporting cytotoxicity is IC50, which is the concentration of agent required to inhibit cell growth to 50% of the control population. However, since IC50 can depend on the recovery time, t, as is the case for 8 and 9, reporting IC50 for a single recovery time can hide important information about the long-time effects of a cytotoxic agent on the health of the cell population