Quantitative Intracellular Delivery of Anticancer Nanodrugs Via an Immunoassay Employing Pt-SiO₂ Janus-Peroxidase Nanozyme

The accurate and efficient quantification of nanodrug dosage is crucial for early anticancer therapy. The enzyme-linked immunosorbent assay (ELISA) has emerged as a robust tool for detecting anticancer nanodrug dosage; however, the development of sensing elements to quantify anticancer nanodrugs still poses a challenge. To overcome this problem, we utilize polysuccinimide-loaded curcumin (CUR @PSI_OAm) as a model to employ an ELISA based on peroxidase nanozyme Pt-SiO₂ Janus nanoparticles (Pt-SiO₂ JNPs) for the indirect quantitative analysis of intracellular anticancer nanodrug dosage. This novel approach employs an immunoassay to indirectly quantify the dosage of anticancer nanodrugs while preserving its structural integrity. The silica components of Pt-SiO₂ JNPs adsorb intermediates, while the Pt NP components exhibit high catalytic activity. Pt-SiO₂ JNPs are functionalized with anti-PSI_OAm antibody (Pt-SiO₂ JNPs-Ab) to serve as an immunosensor capable of specific recognition of CUR @PSI_OAm. Additionally, we employed cytotoxicity assays and confocal imaging techniques to demonstrate the excellent biocompatibility of CUR @PSI_OAm, as well as its specific uptake by cancer cells. According to the experimental results, the limit of detection (LOD) for the immunoassay of Pt-SiO₂ JNPs as a marker for detecting CUR @PSI_OAm is approximately 4.5-fold lower than that of horseradish peroxidase. Therefore, by optimizing the conditions, we established a direct competitive ELISA using Pt-SiO₂ JNPs as colorimetric indicators for the quantitative detection of intracellular CUR @PSI_OAm. The LOD for this ELISA was determined to be 0.01 ng/mL, while the loaded CUR amount calculated from the drug loading capacity was found to be 0.22 pg/mL. Furthermore, the recoveries obtained from this established ELISA ranged between 94.0 and 108%, demonstrating excellent accuracy. Consequently, the peroxidase mimic Pt-SiO₂ JNPs-based ELISA exhibits significant potential for precise quantification of intracellular anticancer nanodrug dosages.