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 ( R 2 > 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.