4.4 Article

Mannosylation of pH-sensitive liposomes promoted cytoplasmic delivery of protein to macrophages: green fluorescent protein (GFP) performed as an endosomal escape tracer

Journal

PHARMACEUTICAL DEVELOPMENT AND TECHNOLOGY
Volume 26, Issue 9, Pages 1000-1009

Publisher

TAYLOR & FRANCIS LTD
DOI: 10.1080/10837450.2021.1969406

Keywords

Green fluorescent protein; pH-dependent fluorescence; mannosylated pH-sensitive liposome; macrophage-targeting; endosome escape; entrapment efficiency

Funding

  1. University of Auckland, New Zealand [FRDF 3716807]

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Comparative study between non-pH-sensitive liposomes and mannosylated pH-sensitive liposomes for protein delivery to macrophages revealed that mannosylation enhanced cellular uptake and endosomal escape without affecting pH-responsiveness. GFP fluorescence acted as a probe for endosomal escape of liposomal cargo, showing efficient performance of mannosylated liposomes in facilitating intracellular delivery.
Conventional non-pH-sensitive liposomes for cytoplasmic delivery of protein suffer from poor efficiency. Here we investigated mannosylated pH-sensitive liposomes (MAN-PSL) for cytoplasmic delivery of protein to macrophages RAW 264.7 using PSL and non-pH-sensitive liposomes for comparison. We characterised the pH-dependent fluorescence of green fluorescent protein (GFP) and encapsulated it in liposomes as an intracellular trafficking tracer. GFP showed a reversed 'S'-shaped pH-fluorescence curve with a dramatic signal loss at acidic pH. GFP stored at 4 degrees C with light protection showed a half-life of 10 days (pH 5-8). The entrapment efficiency of GFP was dominated by the volume ratio of intraliposomal core to external medium for thin-film hydration. Mannosylation did not affect the pH-responsiveness of PSL. Confocal microscopy elucidated that mannosylation promoted the cellular uptake of PSL. For both these liposomes, the strongest, homogeneously distributed GFP fluorescence in the cytoplasm was found at 3 h, confirming efficient endosomal escape of GFP. Conversely, internalisation of non-pH-sensitive liposomes was slow (peaked at 12 h) and both Nile Red and GFP signals remained weak and punctuated in the cytosol. In conclusion, GFP performed as a probe for endosome escape of liposomal cargo. Mannosylation facilitated the internalisation of PSL without compromising their endosomal escape ability.

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