Label-Free Analysis Reveals In Vivo Premature Drug Release Governed by Diffusion from Polymeric Micelles

Premature drug release in the bloodstream significantly limits the clinical use of polymeric micelles (PMs). However, whether this release results primarily from drug diffusion or micelle dissociation is still unclear because current techniques cannot track both the drug and carrier simultaneously and without labels. To address this, we developed an integrated label-free platform combining stable isotope-assisted size-exclusion chromatography, ultrafiltration, and mass spectrometry. This platform enables the absolute quantification of five critical circulatory species: micelle-encapsulated drug, free drug, protein-bound drug, intact micelles, and disassembled unimers. By applying this methodology to paclitaxel-loaded methoxypoly(ethylene glycol)-block-poly(d,l-lactide) micelles, we systematically deciphered the in vivo kinetics of drug release and micelle dissociation. Our results support diffusion-driven drug leakage as the primary factor reducing the circulatory efficacy of PMs under the tested conditions. This work provides valuable insights into the circulatory behavior of PMs and establishes a framework for their pharmacokinetic studies.