Optimizing Isothermal Titration Calorimetry for the Novel Characterization of a Hydrogen Peroxide Sensitive Nanoparticle

Acute kidney injury (AKI) is the partial or complete loss of kidney ability to filter waste, and is linked to the overproduction of a reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), hydroxyl radical (HO•), singlet oxygen (1O2), and the superoxide anion (O2−). APP-103 is an intravenously administered nanoparticle consisting of a core of a vanillyl alcohol (VA)-containing copolymer, named PVAX, core and a polyvinyl alcohol (PVA) shell. APP-103 offers both site specific treatment through ROS consumption and prevention of future injury by releasing VA, an anti-inflammatory. While there is an existing quantitative assay to illustrate the consumption of ROS by APP-103, the assay detects H2O2 consumption in incremental measurements that fail to detail reaction kinetics. Isothermal titration calorimetry (ITC) is a promising method that can be applied to the APP-103 and ROS reaction, providing continual quantification of the reduction of H2O2. The aim of this research is to establish and evaluate the optimal parameters for ITC measurements. Furthermore, the identity of VA anti-inflammatory byproduct of the reaction was verified using liquid-chromatography mass spectrometry (LC-MS). A mass spectrum of the APP-103/ROS reaction product was generated and compared with the known mass spectrum of VA. Similar notable peaks suggest the successful release and elution of VA byproducts following the ROS/APP-103 reaction. This research provides a framework for the quantification of ROS consumption with a polymer-based nanoparticle.