Deciphering the in vivo fate and biomacromolecular interactions of DSPE-PEG2000

DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-2000), a key excipient of stealth nano-formulations like Doxil®, prolongs nano-carrier blood residence by forming steric hydrophilic barriers. However, its assumed biological inertness faces challenges from emerging PEG-related risks. Limited by analytical methods, its in vivo fate and biomacromolecular interactions remain uncharacterized. Here, Liquid Chromatography-Quadrupole/Time-of-Flight Mass Spectrometry (LC-Q-TOF MS) and anti-PEG single-chain variable fragment (PEG-scFv) strategy were innovatively employed to decode DSPE-PEG2000's biological properties post-intravenous administration. Results reveal a short plasma half-life, tissue-specific distribution in lung, liver, and spleen, complement activation without adaptive antibody response, cleavage to DSPE/PEG2000 with renal excretion/degradation of PEG2000, and a plasma protein binding rate (PPB) of 63.2 ± 7.11 % that competitively displaces 50 % of doxorubicin binding. These findings establish its non-inert profile of tissue-specific distribution, innate immune activation, complex metabolism, and plasma protein-based excipient-drug interactions, highlighting DSPE-PEG2000's potential safety risks. The innovative methodology developed herein provides a paradigm for polymer tracking and offers critical insights to guide excipient safety assessments and optimization.