Architecture-engineered nano-in-micro biomaterials enabling sequential therapeutic activation through spatially compartmentalized design.

Spatial organization within biomaterial systems plays critical role in regulating the timing and interaction of multiple therapeutic agents; however, achieving controllable sequential exposure within a single carrier architecture remains challenging. Here, a multilayer nano-in-micro (NIM) biomaterial system was engineered via layer-by-layer assembly to investigate how architectural compartmentalization governs mass transport and functional activation. Polydopamine nanoparticles were employed as a core template, while gold nanoparticles and nanostructured lipid carriers (NLC) were sequentially deposited to establish spatially separated functional domains. Quantitative release studies demonstrated architecture-mediated transport differentiation, where outer-layer components exhibited rapid release under physiological condition (31,2% within 8 min), while inner-confined agents remained diffusion restricted (6.6%), enabling controlled sequential exposure. Acidic environments selective enhanced inner-layer transport confirming stimulus-responsive modulation of interlayer diffusion resistance. The engineered capsule further exhibited reproducible photothermal heating under near infrared irradiation and efficient photodynamic reactive oxygen species generation, serving as a functional validation of staged activation enabled multilayer organization. Photothermal heating further induces localized softening of lipid-based layers promoting architecture-dependent permeability changes and spatially enhanced release, highlighting the functional role of NLC carriers beyond structural assembly and thermally configurable lipid interfaces. In vitro cellular studies demonstrated enhanced therapeutic response arising from temporally separated agent exposure compared with nonstructural systems. Collectively, the results establishing layered NIM assemblies as programmable biomaterial architectures in which structure-defined spatial organization governs transport behavior and multimodal activation. This architectural strategy provides a generalizable framework for designing biomaterials requiring controlled spatiotemporal delivery and activation.