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.
Oleanolic acid derivative DKS26 has hypolipidemic, islet, and hepatoprotective effects. However, high lipophilicity and low water solubility led to DKS26 extremely low oral bioavailability. Herein, lipid-based nanocarriers, including lipid nanodiscs (sND/DKS26) and liposomes (sLip/DKS26), are prepared to improve DKS26 oral absorption. In comparison to free DKS26 (5.81%), the absolute oral bioavailabilities are significantly increased to 29.47% (sND/DKS26) and 37.25% (sLip/DKS26) without detectable toxicity or immunogenicity even after repeated administrations. Both sND/DKS26 and sLip/DKS26 significantly reduce the feeding glucose level and the AUC of OGTT in db/db diabetic mice. Aiding by the newly developed scFv-based nanocarrier separation methods, no intact nanocarriers are detected in blood circulation after oral administration, suggesting that both formulations are unable to penetrate the intestinal epithelium. They enhance DKS26 absorption mainly by improving intestinal cell uptake and rapid intracellular release of the payload. Since pre-existing anti-PEG is widely detected in humans, the present oral absorption pathway of both nanocarriers successfully avoids unfavorable immunological responses after interaction with anti-PEG antibodies. The application of lipid-based nanocarriers paves an efficient and safe avenue for the clinical translation and application of poorly soluble therapeutics derived from traditional Chinese medicine.
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