Mannosylated Liposomes for Intracellular Delivery of Protein to Macrophages

Abstract

Background: Intracellular protein delivery poses a major challenge due to the large molecular size and stability issues which lead to poor cellular uptake or lysosome digestion. Nanoparticles attract increasing attention for their potential in intracellular protein delivery, among which liposomes are widely investigated. Recently, pH sensitive liposomes (PSLs) were foundwith increased cytoplasmic cargo release utilising the low pH in endosomes (pH<6), where non-pH sensitive liposomes (nPSLs) may be entrapped in endosomes. PSLs consisting a fusogenic lipid were also found with increased cellular uptake. Macrophages play a critical role in inflammation and immune response, which can be adjusted via intracellular delivery of modulating proteins using PSLs. Surface modification of mannose, a ligand targeting to mannose receptors overexpressed on macrophages may facilitate specific cell delivery. Aim: This study aimed to investigate the potential of mannosylated PSLs (MAN-PSLs) for improved intracellular protein delivery to macrophages. It was hypothesised that mannosylation would promote specific internalisation of the liposomes by macrophages through the mannose receptors, and upon entering the cells, the pH-sensitivity of liposomes would promote a rapid release of the protein cargo to cytoplasm. PSLs and nPSLs served as reference formulations. Green fluorescent protein (GFP), a widely used fluorescent marker in biomedical research, served as a model protein also a fluorescent marker in this study. Methods: The influence of pH (5.0-8.0) on GFP fluorescence intensity, and GFP stability as a function of pH, temperatures with or without light exposure were investigated. PSLs were prepared with 1,2-dioleoyl-sn-glycerol-3-phosphatidylamine (DOPE, a fusogenic lipid) and cholesteryl hemisuccinate (CHEMS) at 3:2 molar ratio based on a previous study. MAN-PSLs were developed by addition of 0.5% 16:0 PA-PEG3-mannose to the formula of PSLs. The nPSLs composed 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol at 55:45 molecular ratio. GFP was passively loaded into the PSLs, MAN-PSLs and nPSLs using thin film hydration method. To enhance the entrapment efficiency (EE) and drug loading (DL) of GFP, loading conditions were optimised including lipid concentration, extrusion cycles and GFP concentration of loading solution. Cellular uptake of different liposomal formulations labelled with Nile red fluorescence by the RAW 264.7 macrophage cell line was evaluated over periods of times from 0.5-3 hours and 6- 24 hours using confocal microscopy. The fluorescence intensities of Nile red and GFP in the confocal images were analysed by the ImageJ™ software. Intracellular trafficking of GFP encapsulated in liposomes were observed as the sign of punctuates or homogenous distribution of its fluorescence in cytoplasm. Results: GFP fluorescence intensity was increased with the increase of pH from 5.0-8.0 with an Sshaped pH-depended fluorescence intensity curve. GFP had a half-life of 11 days at 4°C, pH 7.0-8.0 and 9.3 days at pH 5.0 when protected from light. However, the half-life was reduced to around 2 days when temperature increased to 20°C or without light protection. GFP loaded liposomes prepared in this study were mainly large unilamellar vesicles (LUVs) with comparable characters. Mannosylation did not significantly increase the size of PSLs (approximately 130 nm). However, GFP loading significantly increased the particle size from 130 to about 150 nm for all liposome formulations. The nPSLs exhibited lower zeta potential compared to PSLs and MAN-PSLs (-8 mV, -34 mV and -30 mV respectively), and was less affected by GFP loading. For all formulations, the optimised EE (20%) and DL (2.2‰) were achieved under optimised conditions: lipid concentration 20 mg/mL hydrated with 200 ug/mL GFP following 10 extrusion cycles. All formulations exhibited high stability in size and zeta potential during storage. However, only about 40% of GFP remained entrapped at 30 days. In vitro release study demonstrated the pH-responsive GFP release from PSLs and MAN-PSLs. PSLs and MAN-PSLs exhibited more rapid and higher efficiency of cellular uptake by RAW 264.7 cells (almost reach the maximal level at 1 hour) compared to nPSLs (peaked at 12 hours). Mannosylation further slightly prompted the cellular uptake of PSLs. The results of cellular uptake confirmed the fusogenicity of DOPE in PSLs. Besides, both PSLs and MAN-PSLs showed rapid cytoplasmic GFP release with GFP fluorescence peaked at 3 hours (endosome escape). Mannosylation showed less influence on the endosome escape ability of GFP entrapped in PSLs. In contrast, nPSLs exhibited delayed cytoplasmic GFP release with the highest GFP fluorescence only at 12 hours. Conclusion: GFP exhibited a pH-dependent fluorescence intensity, making it a suitable protein fluorescence marker for intracellular trafficking study. GFP was highly sensitive to light and temperature. In this study, both PSLs and MAN-PSLs exhibited improved cellular uptake by macrophages and cytoplasmic protein release for combined fusogenicity and pH sensitivity. Mannosylation further slightly enhanced the cellular uptake of PSLs without negative impact on the endosome escape ability. For the first time, PSLs (with or without ligand) were proved with high efficiency in intracellular protein delivery mediated by endosome escape.