Surface Functionalized Polyhydroxyalkanoate Nanoparticles via SpyTag–SpyCatcher System for Targeted Breast Cancer Treatment

<b>Background/Objectives:</b> Biodegradable polymers have emerged as promising platforms for drug delivery. Produced by microbiomes, polyhydroxyalkanoates (PHAs) offer excellent biocompatibility, biodegradability, and environmental sustainability. In this study, we report the surface functionalization of PHA-based nanoparticles (NPs) using the SpyTag-SpyCatcher system to enhance cellular uptake. <b>Methods:</b> Initial conjugation with mEGFP-SpyTag enabled visualization, followed by decoration with HER2-specific Affibody-SpyCatcher and/or TAT-SpyCatcher peptides. The prepared NPs retained a diameter of <200 nm and a negatively charged surface. <b>Results:</b> Affibody-functionalized NPs significantly enhanced internalization and cytotoxicity in HER2-overexpressing SK-BR-3 cells, whereas TAT-functionalized NPs promoted uptake across various cell types, independently of HER2 expression. Dual-functionalized NPs exhibited synergistic or attenuated effects based on the HER2 expression levels, highlighting the critical role of ligand composition in targeted delivery. <b>Conclusions:</b> The results of this study demonstrate that the SpyTag-SpyCatcher-mediated surface engineering of PHA NPs offers a modular and robust strategy for active targeting in nanomedicine.