It remains challenging to precisely decipher the structural and functional characteristics of protein coronas. To overcome the drawbacks frequently occurring in the traditional separation methods, an anti-PEG single-chain variable fragment (PEG-scFv) based affinity chromatography (AfC) was developed to achieve precise and efficient separation of protein coronas on PEGylated liposomes (sLip). His-tagged PEG-scFv could readily capture sLip without affecting protein corona compositions, and separate sLip/protein complex from plasma protein aggregates and endogenous vesicles through the Ni-NTA column. AfC demonstrated 43-fold higher protein corona collecting efficiency than centrifugation, which was extremely crucial for separation of in vivo protein coronas due to the limitation of sample size. AfC evaded contamination by endogenous vesicles and protein aggregates occurring in centrifugation, and reserved the loosely bound proteins, providing an unprecedented approach to deeply decipher protein coronas. The scFv-based AfC also paves new avenues for the separation of protein coronas formed on other nanomedicines.

PEGylated nanocarriers have gained increasing attention due to reduced toxicity and enhanced circulation compared with free drugs. According to guidances of drug regulatory departments worldwide, it is crucial to determine free and liposomal drug concentrations; however, the conventional used separation methods including dialysis, ultrafiltration, and solid-phase extraction (SPE) have drawbacks of time-consuming, drug leakage, environmental pollution or error bias of trace level drug. Here we developed a facile PEG-scFv-based separation method combined with HPLC to quantify free doxorubicin (DOX) and liposomal DOX in plasma. Anti-PEG single chain variable fragment antibody (PEG-scFv) was adopted to sediment PEGylated liposomes by simple incubation and low speed centrifugation. Compared to SPE, it demonstrated sufficient accuracy and sensitivity to evaluate free and liposomal DOX with intact liposomes. Therefore, it can serve as an alternative approach of SPE, which is suitable for quality assessment and pharmacokinetics evaluation of PEGylated liposomal drugs and possible other PEGylated nanocarriers.

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