Poly(ethylenimine) (PEI) is frequently used as transfection agent for delivery of nucleic acids to the cytosol. After endocytosis of complexes of PEI and nucleic acids, a fraction of them can escape endosomes/lysosomes and reach the cytosol. One proposed mechanism is the so-called proton sponge effect, which involves buffering of the lysosomal pH by PEI. There are however also reports that report the absence of such buffering. In this work, the buffering capacity of PEI of the lysosomal pH was investigated in situ by combining PEI and pH-sensing ratiometric fluorophores in a single carrier particle. As carrier particles, hereby capsules were used, which were composed of polyelectrolyte walls based on layer-by-layer assembly, with the pH sensors located inside the capsule cavities. In this way, the local pH around individual particles could be monitored during the whole process of endocytosis. Results demonstrate the pH-buffering capability of PEI, which prevents the strong acidification of lysosomes containing PEI. This effect was related to the presence of PEI and was not related to the overall charge of the carrier particles. In case PEI was added in molecular form, no buffering of pH could be observed by endocytosed encapsulated pH-sensing ratiometric fluorophores. Co-localization experiments demonstrated that this was due to the fact that internalized free PEI and the encapsulated pH-sensing ratiometric fluorophores were not located in the same lysosomes. Missing co-localization might explain why also in other studies no pH buffering was found; in the case of co-delivery of PEI, the pH sensors could be clearly observed.
Keywords: chloroquine; intracellular pH detection; layer-by-layer particles; particle-based delivery; poly(ethylenimine) delivery; polymer capsules; proton sponge effect.