Unraveling the Oligomer-specific Uptake Dynamics of PEG600 With 9-18 Subunits in HepG2 Cells by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry Coupled With Ammonium Adduct Strategy to Enhance Sensitivity.

Polyethylene glycol (PEG) has emerged as a predominant synthetic polymer in contemporary drug delivery systems due to its unique physicochemical properties and versatile functionalization capabilities. Deciphering chain-length-dependent biosafety and cellular uptake profiles of PEG polymers is vital for optimizing nanomedicine formulations. We established an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) methodology employing ammonium adduction for multiplex quantification of PEG600 oligomers (n = 9-18) in human hepatocellular carcinoma (HepG2) hepatocytes. The method demonstrated linear response (R2 > 0.99) across 10-1000 ng/mL for PEG600 oligomers (n = 9-18). PEG600 oligomers (n = 9-18) internalization in HepG2 cells was quantified by the validated UHPLC-MS/MS method. PEG600 polymers with 9-18 subunits could be absorbed into HepG2 cells with a small quantity (less than 0.04%). The cytotoxicity study suggested that the half-maximal inhibitory concentration of PEG600 on HepG2 cells is 31.42 mg/mL. PEG600 oligomers demonstrated negligible anti-proliferative effects in HepG2 hepatocytes. This study could provide analytical techniques and critical reference data for cytotoxicity and whole cellular uptake profiles of PEG600 polymers with 9-18 subunits in HepG2 cells. This work establishes an analytical framework for assessing the cytotoxicity and cellular uptake kinetics of PEG600 oligomers (n = 9-18) in HepG2 cells.