Osmotically Rupturing Phagosomes in Macrophages Using PNIPAM Microparticles

Abstract

The rupture of macrophage phagosomes has been implicated in various human diseases and plays a critical role in immunity. However, the mechanisms underlying this process are complex and not yet fully understood. This study describes the development of a robust engineering method for rupturing phagosomes based on a welldefined mechanism. The method utilizes microfabricated microparticles composed of uncrosslinked linear poly(N-isopropylacrylamide) (PNIPAM) as phagocytic objects. These microparticles are internalized into phagosomes at 37 °C. By exposing the cells to a cold shock at 0 °C, the vast majority of the microparticle-containing phagosomes rupture. The percentage of phagosomal rupture decreases with the increase of the cold-shock temperature. The osmotic pressure in the phagosomes and the tension in the phagosomal membrane are calculated using the Flory−Huggins theory and the Young−Laplace equation. The modeling results indicate that the osmotic pressure generated by dissolved microparticles is probably responsible for phagosomal rupture, are consistent with the experimentally observed dependence of phagosomal rupture on the cold-shock temperature, and suggest the existence of a cellular mechanism for resisting phagosomal rupture. Moreover, the effects of various factors including hypotonic shock, chloroquine, tetrandrine, colchicine, and L-leucyl-L-leucine O-methyl ester (LLOMe) on phagosomal rupture have been studied with this method. The results further support that the osmotic pressure generated by the dissolved microparticles causes phagosomal rupture and demonstrated usefulness of this method for studying phagosomal rupture. This method can be further developed, ultimately leading to a deeper understanding of phagosomal rupture.