Positron Emission Tomography Reveals the Impact of Polymer Structure of PEG Alternatives on Accelerated Blood Clearance of Liposomes

Poly(ethylene glycol) (PEG) lipids are essential components for lipid-based nanocarriers for drug and gene delivery, providing colloidal stabilization and prolonged circulation upon first administration. However, repeated administrations of PEGylated nanocarriers can trigger accelerated blood clearance (ABC), mediated by anti-PEG antibodies (APAs). To address concerns regarding APA-mediated responses, randomized PEG (rPEG) has recently been introduced. rPEG is a polyether structurally similar to PEG and designed to evade established APAs binding mechanisms. Here, we investigate the in vivo pharmacokinetics of first and second administration of gallium-68-labeled polymer lipid (PL)-liposomes using quantitative, dynamic positron emission tomography (PET) imaging over 90 min, and ex vivo biodistribution. We evaluated a PL library, comprising PEG, rPEG, poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), poly(2-methyl-2-oxazine), poly(sarcosine) and poly(N,N-dimethylacrylamide). After the first administration, polymer structure strongly influenced liposome blood pool retention at 90 min. Upon second administration 7 days later, rPEG liposomes, in contrast to all other PL formulations, showed no significant change in pharmacokinetic behavior and retained the highest blood pool activity at 90 min. Consistently, only rPEG showed no increase in protein-associated activity in the plasma protein binding assay, indicating that formation of anti-rPEG antibodies under the conditions of this study is unlikely. Notably, rPEG pharmacokinetics also remained unchanged when mice were pretreated with PEG liposomes. This, together with no quantifiable interactions of APAs with rPEG in the plasma protein binding assay, supports low cross-reactivity between rPEG and APAs in vivo. Overall, our results indicate that the random copolymer design of rPEG offers a promising strategy for preserving pharmacokinetics under repeated-dosing conditions.