Abstract
Phagocytosis performed by a macrophage involves complex membrane trafficking and reorganization among various membranous cellular structures including phagosomes and vesicles derived from the phagosomes known as phagosome-derived vesicles. The present work reports on development of a technique that allows to specifically label the phagosome-derived vesicles in macrophages with a membrane dye. The technique is based on the use of microfabricated microparticles that are made of a thermosensitive nonbiodegradable polymer poly(N-isopropylacrylamide) (PNIPAM) or its derivative and contain a membrane dye 1,1'-dialkyl-3,3,3',3'-tetramethylindodicarbocyanine (DiI). The microparticles can be phagocytosed by RAW264.7 macrophages into their phagosomes, resulting in formation of intracellular DiI-positive vesicles derived from the phagosomes. The DiI-positive vesicles are motile and acidic; can be stained by fluorescently labelled dextran added in the culture medium; and can accumulate around new phagosomes, indicating that they possess properties of lysosomes. This technique is also applicable to another membrane dye 3,3'-dioctadecyloxacarbocyanine (DiO) and holds great potential to be useful for advancing our understanding of phagocytosis. STATEMENT OF SIGNIFICANCE: Phagocytosis performed by macrophages is a cellular process of great importance to various applications of biomaterials such as drug delivery and medical implantation. This work reports on a technique for characterizing phagocytosis based on the use of poly(N-isopropylacrylamide), which is a major biomaterial with numerous applications. This technique is the first of its kind and has generated an original finding about phagocytosis. In addition to drug delivery and medical implantation, phagocytosis plays critical roles in diseases, injuries and vaccination. This work could thus attract immediate and widespread interests in the field of biomaterials science and engineering.