Abstract

Clinical translation of nanomedicines is greatly hindered by insufficient understanding of their in vivo process, yet a key challenge lies in quantifying the encapsulated versus free drug forms in tissues and cells. Herein, we present a facile, versatile anti-PEG single-chain variable-fragment antibody (PEG-scFv)-based method enabling quantitative measurement of both forms in various biofluids ( e . g ., interstitial fluid, cytoplasm). By this method, we map the in vivo process of PEGylated liposomal doxorubicin (sLip/Dox) at unprecedented resolution. In the bloodstream, doxorubicin remains largely encapsulated in liposomes (>99%). In liver as the main organ for drug elimination, less drug was distributed in the interstitium (>80% encapsulated) but more in liver cells (mainly in Kupffer cells) released in a time-dependent manner, accompanying doxorubicin transferred to hepatocytes most in free form by 12 h postinjection. After extravasation into tumors, there was a limited access of sLip/Dox to tumor cells, confining most of the drug in the interstitium mainly being encapsulated (more than 75%), and the internalized fraction underwent a gradual release process in both tumor-associated macrophages and tumor cells. These findings revealed that for sLip/Dox, which primarily underwent drug release intracellularly, cellular internalization rates could be the key factor in determining its in vivo performance. Given widespread PEGylation on developing nanomedicines and the cost-effectiveness of scFv production, PEG-scFv offers a broadly applicable tool for dissecting in vivo processes of nanomedicines to establish dose–effect relationships like small-molecule drugs, further to guide rational nanotherapeutic design.