Quantitative\nIntracellular Delivery of Anticancer\nNanodrugs Via an Immunoassay Employing Pt-SiO<sub>2</sub> Janus-Peroxidase\nNanozyme

The accurate and efficient quantification of nanodrug\ndosage is\ncrucial for early anticancer therapy. The enzyme-linked immunosorbent\nassay (ELISA) has emerged as a robust tool for detecting anticancer\nnanodrug dosage; however, the development of sensing elements to quantify\nanticancer nanodrugs still poses a challenge. To overcome this problem,\nwe utilize polysuccinimide-loaded curcumin (CUR @PSI<sub>OAm</sub>) as a model to employ an ELISA based on peroxidase nanozyme Pt-SiO<sub>2</sub> Janus nanoparticles (Pt-SiO<sub>2</sub> JNPs) for the indirect\nquantitative analysis of intracellular anticancer nanodrug dosage.\nThis novel approach employs an immunoassay to indirectly quantify\nthe dosage of anticancer nanodrugs while preserving its structural\nintegrity. The silica components of Pt-SiO<sub>2</sub> JNPs adsorb\nintermediates, while the Pt NP components exhibit high catalytic activity.\nPt-SiO<sub>2</sub> JNPs are functionalized with anti-PSI<sub>OAm</sub> antibody (Pt-SiO<sub>2</sub> JNPs-Ab) to serve as an immunosensor\ncapable of specific recognition of CUR @PSI<sub>OAm</sub>. Additionally,\nwe employed cytotoxicity assays and confocal imaging techniques to\ndemonstrate the excellent biocompatibility of CUR @PSI<sub>OAm</sub>, as well as its specific uptake by cancer cells. According to the\nexperimental results, the limit of detection (LOD) for the immunoassay\nof Pt-SiO<sub>2</sub> JNPs as a marker for detecting CUR @PSI<sub>OAm</sub> is approximately 4.5-fold lower than that of horseradish\nperoxidase. Therefore, by optimizing the conditions, we established\na direct competitive ELISA using Pt-SiO<sub>2</sub> JNPs as colorimetric\nindicators for the quantitative detection of intracellular CUR @PSI<sub>OAm</sub>. The LOD for this ELISA was determined to be 0.01 ng/mL,\nwhile the loaded CUR amount calculated from the drug loading capacity\nwas found to be 0.22 pg/mL. Furthermore, the recoveries obtained from\nthis established ELISA ranged between 94.0 and 108%, demonstrating\nexcellent accuracy. Consequently, the peroxidase mimic Pt-SiO<sub>2</sub> JNPs-based ELISA exhibits significant potential for precise\nquantification of intracellular anticancer nanodrug dosages.