The luminescence performance of persistent luminescence nanoparticles is closely related to their defect structures, while the mechanisms by which different nanostructuring strategies regulate defect types and trap structures remain unclear. In this work, Zn₂SiO₄: Mn²⁺, Yb³⁺, Li⁺ persistent luminescence nanoparticles were prepared via top-down (TD) and bottom-up (BU) strategies to investigate the relationships among crystal structure, defect structure, trap behavior, and photodynamic therapy (PDT) performance. The TD strategy effectively reduced the particle size but introduced severe surface disorder and grain-boundary defects, resulting in the depletion of effective shallow traps and significant degradation of luminescence and afterglow performance. In contrast, the BU strategy produced uniformly dispersed nanoparticles (~150 nm) with abundant free-volume defects and open interfacial structures. Thermoluminescence analysis indicates that shallow traps at approximately 0.74 eV are the key energy-storage centers responsible for room-temperature persistent luminescence, while positron annihilation lifetime spectroscopy confirms that the BU samples contain more free-volume defects, facilitating carrier transport and interfacial energy transfer. As a result, the BU samples exhibit superior PDT performance with higher HeLa cell killing efficiency than the TD samples. These results demonstrate that the PDT performance of persistent luminescence nanoparticles is determined by the synergistic effects of defect types, trap structures, and interfacial energy transfer rather than luminescence intensity alone. This work provides new physical insights into the defect engineering of persistent luminescence nanomaterials for biomedical applications.