Anti-inflammatory Effects of PEGylated Phosphatidylserine-containing Liposomes in RAW 264.7 Macrophages

Objectives: Polyethylene glycol (PEG) have been widely utilized in the preparation of liposomes which can prolong the circulation time by interfering the macrophage recognition. However, the biological effects of PEGylated liposomes on macrophages have been rarely explored up to now. The aim of this study was to evaluate the effects of PEGylated phosphatidylserine-containing liposomes (PEG-PSLs) on the expression of inflammatory-related cytokines: TNF-α and TGF-β, in macrophages of the murine RAW 264.7 cell line.
Methods: PSLs (PS, L-a-phosphatidylcholine [PC], and cholesterol [CH] at a molar ratio of 2:1:1) were prepared using a mini-extruder with polycarbonate filters (50-nm, 100-nm, 200-nm, and 400-nm pore size). The PEG-PSLs were prepared identically, with the exception that DSPE-PEG-2000 was added with different concentrations (0.25%, 0.5%, 1%). RAW 264.7 cells were seeded at a density of 3 × 10⁵ cells/ml, then, to analyze TGF-β gene expression, the cells were directly treated with PSLs and PEG-PSLs. To analyze TNF-α gene expression, the liposomes were added to cells after pre-treatment with LPS for 1 or 3 h. The incorporation of TRITC-DHPE-labeled liposomes into cells was assessed using fluorescence-activated cell sorting (FACS) and fluorescence microscopy. Statistics were performed using analysis of variance (ANOVA).
Results: We found that 100-nm PSLs at a concentration of 12.5 μg/ml had the most significant effect. PEGylation differentially affected TNF-α and TGF-β levels. The PSL-mediated inhibitory effect on TNF-α secretion was enhanced by PEGylation, and PEG-PSLs decreased TGF-β levels compared with non-PEGylated PSLs. FACS analysis revealed that 1% PEGylation disturbed the incorporation of PSLs into macrophages. Fluorescent images also confirmed that the interaction between PSLs and RAW 264.7 cells was affected by PEGylation.
Conclusions: PEG-PSLs can prevent TNF-α secretion without increasing TGF-β levels in macrophages, and they support the potential clinical use of PEG-PSLs as anti-inflammatory agents in periodontitis and peri-implantitis with a relatively low potential to induce tissue fibrosis.