Co-assembling de novo designed peptide with high-payload drug protein for noninvasive treatment of corneal neovascularization

The specificity and low toxicity of protein drugs are significant for disease treatment but are strongly limited by their weak tissue penetrative capacity. Although formulating proteins with nanoparticle is an alternative strategy, the low encapsulation efficiency (EE) and loading capacity (LC) of protein drugs and their potential for protein inactivation remain significant challenges. Herein, the de novo designed peptide (Arg-His-Cys-Arg-His-Cys-Arg-His-Cys) (RHC)₃, zinc ions (Zn²⁺), and the anti-neovascular protein drug Bevacizumab (Beva) were co-assembled to form PZA@Beva (peptide and Zn²⁺ assemblies encaspsulated Beva) nanomedicine, aiming to overcome the challenges associated with corneal neovascularization (CNV) model. The optimized size of PZA@Beva is approximately 162.5 nm, with EE% and LC% of Beva 92.7 % and 55.8 %, respectively. The bioactivity of encapsulated Beva was preserved, protecting it from proteolytic degradation, and the release of Beva from PZA@Beva exhibited pH-dependent kinetics. In vitro, PZA@Beva demonstrated effective penetration across the ocular barrier via both the paracellular pathway (by opening corneal tight junctions) and the transcellular pathway (through rapid cellular endocytosis). Additionally, PZA@Beva exhibited no cytotoxicity in vitro or in vivo, coupled with prolonged ocular retention, collectively yielding promising results for the treatment of CNV. This study contributes to non-invasive protein delivery across ocular bio-barriers for the treatment of diseases in the anterior segment.