Development of Engineered Microparticles for Investigating Enzymatic Degradation of Proteins and Peptides within Phagosomes

Phagocytosis involves the engulfment and enzymatic degradation of particulate objects by phagocytes in a compartment called a phagosome. The degradation mechanisms for natural phagocytic objects such as proteins and peptides are not well understood. To explore this, we developed a novel method using microparticles made of proteins and/or peptides and poly(<i>N</i>-isopropylacrylamide) (PNIPAM) via microfabrication. These microparticles were fed to phagocytes, and the presence of fluorescently labeled fragments in phagosome-derived vesicles (PDVs) was characterized. Using ovalbumin (OVA) and poly-l-lysine as test proteins and peptides, we found that RAW264.7 macrophages engulfed these microparticles, leading to fluorescent PDVs, indicating enzymatic degradation of OVA. We extended this approach to other proteins such as histone and immunoglobulin G and to different phagocytes such as BV2 microglial cells and mouse bone marrow-derived macrophages. Our method also allows for induction of phagosomal rupture and membrane labeling of PDVs.