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

Abstract

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 °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.

Graphical abstract

Keywords:

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

Author contributions

ZW and AJD conceived the study. ZW, AJD, and HHC contributed to the study design and supervised the project; KY performed the experiment and prepared the manuscript. MT conducted image and co-localisation analysis and wrote the sections. All authors reviewed and revised the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by a Faculty Research Development Fund (FRDF 3716807) from The University of Auckland, New Zealand.