Maximizing Synergistic Activity When Combining RNAi and Platinum-Based Anticancer Agents

摘要

RNAi approaches have been widely combined with platinum-based anticancer agents to elucidate cellular responses and to target gene products that mediate acquired resistance. Recent work has demonstrated that platination of siRNA prior to transfection may negatively influence RNAi efficiency based on the position and sequence of its guanosine nucleosides. Here, we used detailed spectroscopic characterization to demonstrate rapid formation of Pt-guanosine adducts within 30 min after coincubation of oxaliplatin [OxaPt(Ⅱ)] or cisplatin [CisPt(II)] with either guanosine monophosphate or B-cell lymphoma 2 (BCL-2) siRNA. After 3 h of exposure to these platinum(II) agents, >50% of BCL-2 siRNA transcripts were platinated and unable to effectively suppress mRNA levels. Platinum-(IV) analogues [OxaPt(lV) or CisPt(IV)] did not form Pt-siRNA adducts but did display decreased in vitro uptake and reduced potency. To overcome these challenges, we utilized biodegradable methoxyl-poly(ethylene glycol)-block-poly(ε-caprolactone)-block-poly(L-lysine) (mPEG-b-PCL-b-PLL) to generate self-assembled micelles that covalendy conjugated OxaPt(IV) and/or electrostatically complexed siRNA. We then compared multiple strategies by which to combine BCL-2 siRNA with either OxaPt(Ⅱ) or OxaPt(IV). Overall, we determined that the concentrations of siRNA (nM) and platinum(Ⅱ)-based anticancer agents (μM) that are typically used for in vitro experiments led to rapid Pt-siRNA adduct formation and ineffective RNAi. Coincorporation of BCL-2 siRNA and platinum(IV) analogues in a single micelle enabled maximal suppression of BCL-2 mRNA levels (to <10% of baseline), augmented the intracellular levels of platinum (by ~4x) and the numbers of resultant Pt-DNA adducts (by >5x), increased the cellular fractions that underwent apoptosis (by ~4x), and enhanced the in vitro antiproliferative activity of the corresponding platinum(II) agent (by 10-100X, depending on the cancer cell line). When combining RNAi and platinum-based anticancer agents, this generalizable strategy may be adopted to maximize synergy during screening or for therapeutic delivery.