Thesis (M.Sc. in Pharm)--Chulalongkorn University, 2008

Macrophages play a predominant role in eliminating invading microorganisms by phagocytosis and secretion of cytotoxic substances such as nitric oxide (NO) and other free radicals. Excessive production of these radicals can cause oxidative damages to surrounding host tissues, which lead to severe pathological conditions. Antioxidants may help to alleviate these problems. Due to several disadvantages in the physicochemical properties of antioxidants, a feasible delivery system for intracellular delivery of these molecules to macrophages is desirable. The aim of this study was to investigate whether liposomes would be a good candidate for intracellular delivery of antioxidants to macrophage cells. α-Tocopherol and Nacetylcysteine (NAC) were used as models for lipophilic and hydrophilic antioxidants in the study, respectively. The effects of incorporation of antioxidants into liposomes on NO production in lipopolysaccharide (LPS)-stimulated J774A.1 cells were investigated. Effects of liposome composition and inclusion of negatively charged lipids [dicetylphosphate (DCP) and phosphatidylglycerol] at 10 mol% into liposome bilayers were also examined. J774A.1 macrophage cells were incubated in the presence of antioxidants, blank liposomes, or antioxidant-loaded liposomes, as well as in the co-existence of free antioxidants and liposome preparations for 24 hr., followed by cell stimulation using 0.125 μg/ml LPS for another 24 hr. NO production was detected by Griess reaction. NO production from LPS-stimulated J774A.1 cells was used as the baseline control. The inhibitory effect of the treatment on NO production was compared among the treatments. The results suggested that the presence of antioxidants, either encapsulated in or coincubated with liposomes, did not have any synergistic nor additive effect with liposomes. On the contrary, the inhibitory effect on NO production seen with blank liposomes was reduced by both antioxidants. Both neutral and negatively charged liposomes gave comparable results. In addition, NAC-loaded negatively charged liposomes in the presence of free NAC, but not co-incubation of NAC solution with blank liposomes or NACloaded negatively charged liposomes without free NAC, exerted severe cytotoxic effect on J774A.1 cells. Further investigation with liposomes containing DCP and calcein solution indicated that NAC-encapsulated negatively charged liposomes might cause cytotoxicity by increasing free NAC uptake into the cells. However, a more refined experiment would be necessary to establish the exact mechanism. Due to the intrinsic inhibitory effect of liposomes on NO production, the overall results of this study indicated that an antagonistic effect might occur if liposomes were used concomitantly with antioxidants. Thus, liposomes might not be a good candidate for antioxidant delivery into the macrophage cells.