SERS based pH nanosensors forin-vitropH measurement in multicellular 3D tumour spheroids

Tumor acidosis is a consequence of altered metabolism that primarily takes place due to lactate secretion from anaerobic glycolysis. As a result, many regions within the tumors are chronically hypoxic and acidic. To measure the intratumor pH dynamically, we have fabricated a biocompatible pH nanoparticle sensor using surface-enhanced Raman spectroscopy (SERS-pNPS) and monitored continuous pH levels in three-dimensional multicellular spheroids. The 3D multicellular spheroids were cultured using a micro-well array chip made of polydimethylsiloxane (PDMS). The SERS-pNPS were synthesized by linking 4-Mercaptobenzoic acid (4-MBA) to silver nanoparticles (AgNPs) of size 50 nm. The calibration curve demonstrates a linear correlation between the ratio of Raman peak intensities (1378 cm^-1/1620 cm^-1) with the pH level. The sensor exhibits a detection limit of pH 4.4 and demonstrates linearity within the physiological pH range (pH 4.4-pH 8.23). The SERS-pNPS was applied for pH measurement in different 3D co-cultured spheroid models such as lung cancer (A549-NIH3T3), breast cancer (MCF-NIH3T3), colon cancer (HCT8-NIH3T3) and mono-cultured spheroids using fibroblast (NIH3T3) cells. The detailed analysis indicated that the 3D co-cultured cancerous tumor models have 16% more acidic microenvironment as compared to 3D mono-cultured spheroid model. Also, a presence of a decreasing pH gradient from peripheral to the core region is observed in both the cases indicating acidosis in the core region. The SERS-pNPS platform facilitates a non-invasive and dynamic pH tracking, and thus offers an improved insight into the acidic microenvironment in various tumor models.