An in vitro study of liposomal curcumin: stability, toxicity and biological activity in human lymphocytes and Epstein-Barr virus-transformed human B-cells.

Curcumin is a multi-functional and pharmacologically safe natural agent. Used as a food additive for centuries, it also has anti-inflammatory, anti-virus and anti-tumor properties. We previously found that it is a potent inhibitor of cyclosporin A (CsA)-resistant T-cell co-stimulation pathway. It inhibits mitogen-stimulated lymphocyte proliferation, NFkappaB activation and IL-2 signaling. In spite of its safety and efficacy, the in vivo bioavailability of curcumin is poor, and this may be a major obstacle to its utility as a therapeutic agent. Liposomes are known to be excellent carriers for drug delivery. In this in vitro study, we report the effects of different liposome formulations on curcumin stability in phosphate buffered saline (PBS), human blood, plasma and culture medium RPMI-1640+10% FBS (pH 7.4, 37 degrees C). Liposomal curcumin had higher stability than free curcumin in PBS. Liposomal and free curcumin had similar stability in human blood, plasma and RPMI-1640+10% FBS. We looked at the toxicity of non-drug-containing liposomes on (3)H-thymidine incorporation by concanavalin A (Con A)-stimulated human lymphocytes, splenocytes and Epstein-Barr virus (EBV)-transformed human B-cell lymphoblastoid cell line (LCL). We found that dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) were toxic to the tested cells. However, addition of cholesterol to the lipids at DMPC:DMPG:cholesterol=7:1:8 (molar ratio) almost completely eliminated the lipid toxicity to these cells. Liposomal curcumin had similar or even stronger inhibitory effects on Con A-stimulated human lymphocyte, splenocyte and LCL proliferation. We conclude that liposomal curcumin may be useful for intravenous administration to improve the bioavailability and efficacy, facilitating in vivo studies that could ultimately lead to clinical application of curcumin.

Multivariate toxicity screening of liposomal formulations on a human buccal cell line.

The influence of various formulation factors on the in vitro cellular toxicity of liposomes on human buccal cells (TR146), were studied by using the concept of statistical experimental design and multivariate evaluation. The factors investigated were the type of main phospholipid (egg-PC, DMPC, DPPC), lipid concentration, the type of charge, liposome size, and amount and nature of the charged component (diacyl-PA, diacyl-PG, diacyl-PS, stearylamine (SA), diacyl-TAP) in the liposomes. Both full factorial design and D-optimal designs were created. Several significant main factors and interactions were revealed. Positively charged liposomes were shown to be toxic. The toxicity of negatively charged liposomes was relatively low. Diacyl-TAP was less toxic than SA, and DPPC was less toxic than DMPC. Low level of positively charged component was favourable and essential when using egg-PC as the main lipid. The amount of negatively charged component, the liposome size, and the total lipid concentration did not affect the toxicity within the experimental room. DPPC appeared to be a good candidate when formulating both positively and negatively charged liposomes with low cellular toxicity. The concept of statistical experimental design and multivariate evaluation was shown to be a useful approach in cell toxicity screening studies.

[Specific inhibitory effect of hybrid liposomes on the growth of hybridoma cells in vitro].

Remarkably high inhibitory effects of the hybrid liposomes composed of L-alpha-dimyristoyl-phosphatidylcholine (DMPC) and polyoxyethylenealkyl ether (C14(EO)n, n = 6-8 and C12(EO)n, n = 8-12)) on the growth of human lymphoma-human B-lymphocyte hybridoma (HF) cells in vitro were obtained. The hybrid liposomes composed of 90 mol% DMPC/10 mol% C14(EO)n (n = 6-8) or C12(EO)n (n = 8-12) were more fluid as compared with 90 mol% DMPC/10 mol% C14(EO)4 or C12(EO)n (n = 4, 23) hybrid liposomes on the basis of fluorescence polarization measurements. These results suggest that the inhibitory effects of the hybrid liposomes on the growth of HF cells should be related to the membrane fluidity. No toxicity to normal rats in vivo was observed in the experiment using 90 mol% DMPC/10 mol% C14(EO)7 or 90 mol% DMPC/10 mol% C12(EO)12 hybrid liposomes.

Influence of phospholipid/amphotericin B ratio and phospholipid type on in vitro renal cell toxicities and fungicidal activities of lipid-associated amphotericin B formulations.

We studied the influence of the lipid/amphotericin B (AMB) ratio and the phospholipid type on the in vitro renal cell toxicity and antifungal efficacy of lipid-associated AMB (L-AMB). L-AMB was prepared at one of two different lipid/AMB ratios (1 and 40) by incubating AMB with empty small unilamellar vesicles, made from one of three different phospholipids: dipalmitoyl-, dimirystoyl-, and distearoylphosphatidylcholine (DPPC, DMPC, and DSPC, respectively). Renal cell toxicity, investigated through an assessment of the Na-dependent uptake of phosphate by proximal tubular cells, and fungicidal effect against Candida albicans were studied after 1 h of treatment at 37 degrees C. The amount of unbound AMB present in each L-AMB formulation was studied by use of circular dichroism. At a lipid/AMB ratio of 40, the three lipidic formulations were not toxic for renal cells but were less effective against C. albicans than AMB; however, DSPC-AMB, which contained 50% unbound AMB, was more effective against C. albicans than DPCC-AMB or DMPC-AMB, containing 0 and 13% unbound AMB, respectively. At a lipid/AMB ratio of 1, the antifungal effects of L-AMB and AMB were similar, whatever the phospholipid used, but only DMPC-AMB remained highly protective against AMB renal cell toxicity, despite the presence of the same amount of unbound AMB (50%) in DMPC-AMB and DPPC-AMB. We conclude that the in vitro activities and renal cell toxicities of different L-AMB formulations are influenced by the phospholipid type and the lipid/AMB ratio. The optimal ratio depends on the phospholipid itself. At a lipid/AMB ratio of 40, the antifungal activity depends mainly on the amount of unbound AMB in the formulation. At a lipid/AMB ratio of 1, the renal cell toxicity also depends on the fluidity of the phospholipid.

Remarkably enhanced inhibitory effects of three-component hybrid liposomes including sugar surfactants on the growth of lung carcinoma cells.

Sugar parts play important roles in recognizing molecules on the cell membranes. We successfully produced sugar-type micellar surfactants, lactonoalkylamide (LacCn), for the first time. Spherical vesicles, three-component hybrid liposomes, were obtained after the sonication of the mixture of L-alpha-dimyristoyl-phosphatidylcholine (DMPC), Tween 20 and LacCn (DMPC:Tween 20:LacCn=65:7:28). It is noteworthy that high inhibitory effects of the three-component hybrid liposomes composed of DMPC, Tween 20, and LacCn (DMPC:Tween 20:LacCn=65:7:28) on the growth of glioma (U251) and lung adenocarcinoma (RERF-LC-OK) cells were attained in vitro without any drug, although no significant inhibitory effects of any individual component (DMPC, Tween 20, LacCn) or the two-component hybrid liposomes of DMPC and Tween 20 on the growth of tumor cells examined were obtained.