Engineered Microparticles Suggest Proteolysis as a Critical Prerequisite for Chromatin Clearance in Macrophage Phagosomes

Defective clearance of phagocytosed DNA contributes to inflammation, yet the molecular factors governing DNA degradation within phagosomes remain unclear. Here, we present a materials-based platform using engineered microparticles to dissect how DNA is processed inside macrophage phagosomes. Using microcontact printing, we fabricated two classes of DNA-containing microparticles: thermoresponsive poly( N -isopropylacrylamide) (PNIPAM) microspheres encapsulating intercalator-labeled DNA and chromatin-mimetic particles composed of multilayered histone–DNA assemblies with tunable cross-linking. These structures provide precise control over DNA accessibility, protein association, and degradability. Upon phagocytosis by macrophages, DNA embedded within hydrated PNIPAM networks remained intact, indicating restricted diffusion of phagosomal enzymes. In contrast, DNA electrostatically complexed with histone was efficiently degraded but only after proteolytic removal of the histone barrier. When histone was chemically cross-linked, DNA degradation was inhibited. These results demonstrate that proteolysis of DNA-bound proteins is a critical prerequisite for DNase II-mediated cleavage in macrophage phagosomes. This modular microparticle platform offers a reductionist approach for probing the biochemical and physical determinants of DNA degradation within phagocytes and enables a systematic investigation of how protein–DNA interactions, cross-linking, or pathological stabilization of chromatin-like structures influences intracellular DNA persistence and inflammatory signaling.