Balancing Stealth and Targetability: cCPP‐ and PEG‐Modified Liposomes for the Targeted Delivery of Anti‐HER2 Nanobodies

ABSTRACT Nanobodies (Nbs) are considered promising antibody fragments for overcoming limitations in precision oncology due to their high specificity and deep tissue penetration. However, their therapeutic potential remains limited by their rapid renal clearance. In this study, anti‐HER2 Nb‐loaded liposomes with dual functionalization combining polyethylene glycol 2000 (PEG) and cyclic cell‐penetrating peptides are developed to ameliorate their pharmacokinetic behavior while retaining binding specificity. Liposomal formulations with high encapsulation efficiencies are produced with dual centrifugation. Biophysical characterization reveals that PEGylation effectively mitigates cCPP‐induced membrane destabilization, ensuring structural integrity. In vitro assays confirm that, despite the steric shielding by PEG, the encapsulated Nbs retain their functionality and specific binding to HER2‐overexpressing cells. In vivo studies in zebrafish larvae demonstrate excellent biocompatibility and lack of immunogenicity. Crucially, liposomal encapsulation significantly modulates the pharmacokinetic profile of Nbs in rats, reducing renal accumulation compared to free Nbs. This study presents a robust liposomal platform that successfully balances the stealth properties of PEG with the functional benefits of cCPPs. Consequently, this platform offers an effective strategy to enhance the therapeutic window of low‐molecular‐weight biologics.