Self-reproduction of fatty acid vesicles: a combined experimental and simulation study.

Dilution of a fatty acid micellar solution at basic pH toward neutrality results in spontaneous formation of vesicles with a broad size distribution. However, when vesicles of a defined size are present before dilution, the size distribution of the newly formed vesicles is strongly biased toward that of the seed vesicles. This so-called matrix effect is believed to be a key feature of early life. Here we reproduced this effect for oleate micelles and seed vesicles of either oleate or dioleoylphosphatidylcholine. Fluorescence measurements showed that the vesicle contents do not leak out during the replication process. We hypothesized that the matrix effect results from vesicle fission induced by an imbalance of material across both leaflets of the vesicle upon initial insertion of fatty acids into the outer leaflet of the seed vesicle. This was supported by experiments that showed a significant increase in vesicle size when the equilibration of oleate over both leaflets was enhanced by either slowing down the rate of fatty acid addition or increasing the rate of fatty acid transbilayer movement. Coarse-grained molecular-dynamics simulations showed excellent agreement with the experimental results and provided further mechanistic details of the replication process.

High cytotoxicity of cisplatin nanocapsules in ovarian carcinoma cells depends on uptake by caveolae-mediated endocytosis.

PURPOSE: Cisplatin nanocapsules, nanoprecipitates of cisplatin encapsulated in phospholipid bilayers, exhibit increased in vitro toxicity compared with the free drug toward a panel of human ovarian carcinoma cell lines. To elucidate the mechanism of cell killing by nanocapsules and to understand the cell line dependence of nanocapsule efficacy, the route of uptake and the intracellular fate of the nanocapsules were investigated. EXPERIMENTAL DESIGN: Intracellular platinum accumulation and cisplatin-DNA-adduct formation were measured in cell lines that differ in sensitivity to cisplatin nanocapsules. Confocal fluorescence microscopy in combination with down-regulation with small interfering RNA was used to map the route of cellular uptake of nanocapsules containing fluorescein-labeled cisplatin. RESULTS: In sensitive cell lines, cisplatin from nanocapsules is taken up much more efficiently than the free compound. In IGROV-1 cells, the increased platinum accumulation results in augmented cisplatin-DNA-adduct formation. Confocal fluorescence microscopy revealed that the uptake of nanocapsules is energy dependent. Colocalization with markers of early and late endosomes indicated uptake via endocytosis. Down-regulation of caveolin-1 with small interfering RNA inhibited the uptake and cytotoxic effect of nanocapsules in IGROV-1 cells. Ovarian carcinoma cells, in which the nanocapsules are less effective than in IGROV-1 cells, do not internalize the nanocapsules (OVCAR-3) or accumulate them in an endocytic compartment after clathrin-mediated endocytosis (A2780). CONCLUSIONS: The high cytotoxicity of cisplatin nanocapsules requires caveolin-1-dependent endocytosis that is followed by release of the drug from a late endosomal/lysosomal compartment and cisplatin-DNA-adduct formation. The findings may be applied in predicting the efficacy of nanoparticulate anticancer drug delivery systems in treating different tumor types.

Antitumor activity and biodistribution of cisplatin nanocapsules in nude mice bearing human ovarian carcinoma xenografts.

Cisplatin nanocapsules represent a novel lipid formulation of the anticancer drug cis-diamminedichloridoplatinum(II) (cisplatin), characterized by an unprecedented cisplatin-to-lipid molar ratio, and exhibiting strongly increased in-vitro cytotoxicity compared with the free drug. In this study, antitumor efficacy and biodistribution of PEGylated cisplatin nanocapsules were compared with those of the free drug in a mouse tumor model. Nude mice bearing human ovarian carcinoma OVCAR-3 xenografts were treated twice with a 1-week interval by intravenous administration of cisplatin nanocapsules or cisplatin in solution, and the growth inhibitory effects were determined by measurement of tumor volumes. At a dose of 3 mg cisplatin/kg, corresponding to the maximum tolerated dose of cisplatin nanocapsules, cisplatin nanocapsules and cisplatin in solution exhibited similar therapeutic effectiveness, reducing tumor growth by 90% at day 20 after first injection. The platinum biodistribution was assayed by analyzing plasma and tissues for total platinum content by nonflame atomic absorption spectroscopy. Plasma and tumor concentrations of platinum were similar for both formulations. During the first hour after injection of cisplatin nanocapsules, the platinum content of the kidney was 40% less than that after administering the free drug. Platinum from nanocapsules showed rapid and 4.5-fold higher accumulation in the liver compared with free cisplatin, and, at a slower rate, accumulation to a high concentration in the spleen. We conclude that the formulation of cisplatin nanocapsules inhibits the growth of OVCAR-3 xenografts in nude mice, albeit to a similar extent as free cisplatin. The results suggest that the antitumor efficacy of the nanocapsules could be improved by preventing rapid clearance from circulation.

Is there a preferential interaction between cholesterol and tryptophan residues in membrane proteins?

Recently, several indications have been found that suggest a preferential interaction between cholesterol and tryptophan residues located near the membrane-water interface. The aim of this study was to investigate by direct methods how tryptophan and cholesterol interact with each other and what the possible consequences are for membrane organization. For this purpose, we used cholesterol-containing model membranes of dimyristoylphosphatidylcholine (DMPC) in which a transmembrane model peptide with flanking tryptophans [acetyl-GWW(LA)8LWWA-amide], called WALP23, was incorporated to mimic interfacial tryptophans of membrane proteins. These model systems were studied with two complementary methods. (1) Steady-state and time-resolved Förster resonance energy transfer (FRET) experiments employing the fluorescent cholesterol analogue dehydroergosterol (DHE) in combination with a competition experiment with cholesterol were used to obtain information about the distribution of cholesterol in the bilayer in the presence of WALP23. The results were consistent with a random distribution of cholesterol which indicates that cholesterol and interfacial tryptophans are not preferentially located next to each other in these bilayer systems. (2) Solid-state 2H NMR experiments employing either deuterated cholesterol or indole ring-deuterated WALP23 peptides were performed to study the orientation and dynamics of both molecules. The results showed that the quadrupolar splittings of labeled cholesterol were not affected by an interaction with tryptophan-flanked peptides and, vice versa, that the quadrupolar splittings of labeled indole rings in WALP23 are not significantly influenced by addition of cholesterol to the bilayer. Therefore, both NMR and fluorescence spectroscopy results independently show that, at least in the model systems studied here, there is no evidence for a preferential interaction between cholesterol and tryptophans located at the bilayer interface.

Carboplatin nanocapsules: a highly cytotoxic, phospholipid-based formulation of carboplatin.

Platinum-based drugs are widely used in cancer chemotherapy. However, their clinical use is limited by systemic toxicity, rapid blood clearance, and the occurrence of resistance. Our research is aimed at increasing the therapeutic index of these drugs by encapsulation in a lipid formulation. Previously, we developed a method for efficient encapsulation of cisplatin in a lipid formulation, yielding cisplatin nanocapsules. Here, we show that carboplatin, a cisplatin-derived anticancer drug with different chemical properties, can be efficiently encapsulated in a lipid formulation by a similar method. The carboplatin nanocapsules exhibit a very high cytotoxicity in vitro: the IC(50) value of carboplatin nanocapsules is up to a 1,000-fold lower than that of conventional carboplatin when tested on a panel of carcinoma cell lines. Cellular platinum content analysis and confocal fluorescent imaging of the interaction of the carboplatin nanocapsules with IGROV-1 cells indicate that the improved cytotoxicity is due to increased platinum accumulation in the cells, resulting from uptake of the formulation by endocytosis.

Cisplatin nanocapsules.

Cisplatin nanocapsules represent a novel lipid formulation of the anticancer drug cis-diamminedichloroplatinum(II), in which nanoprecipitates of cisplatin are covered by a phospholipid bilayer coat consisting of an equimolar mixture of phosphatidylcholine and phosphatidylserine. Cisplatin nanocapsules are characterized by an unprecedented cisplatin-to-lipid molar ratio and exhibit strongly improved cytotoxicity against tumor cells in vitro compared with the free drug. Here, methods for preparing and characterizing cisplatin nanocapsules are reported.

Preparation and stability of lipid-coated nanocapsules of cisplatin: anionic phospholipid specificity.

Cisplatin nanocapsules represent a novel lipid formulation of the anti-cancer drug cis-diamminedichloroplatinum(II) (cisplatin), in which nanoprecipitates of cisplatin are coated by a phospholipid bilayer consisting of a 1:1 mixture of zwitterionic phosphatidylcholine (PC) and negatively charged phosphatidylserine (PS). Cisplatin nanocapsules are characterized by an unprecedented cisplatin-to-lipid ratio and exhibit increased in vitro cytotoxicity compared to the free drug [Nat. Med. 8, (2002) 81]. In the present study, the stability of the cisplatin nanocapsules was optimized by varying the lipid composition of the bilayer coat and monitoring in vitro cytotoxicity and the release of contents during incubations in water and in mouse serum. The release of cisplatin from the PC/PS (1:1) nanocapsules in water increased with increasing temperature with a t(1/2) of 6.5 h at 37 degrees C. At 4 degrees C, cisplatin was retained in the nanocapsules for well over 8 days. Replacement of PS by either phosphatidylglycerol or phosphatidic acid revealed that nanocapsules prepared of PS were more stable, which was found to be due to the ability of PS to form a stable cisplatin-PS coordination complex. Mouse serum had a strong destabilizing effect on the cisplatin nanocapsules. The PC/PS formulation lost over 80% of cisplatin within minutes after resuspension in serum. Incorporation of poly(ethylene glycol 2000) (PEG)-derivatized phosphatidylethanolamine and cholesterol in the bilayer coat extended the lifetime of the cisplatin nanocapsules in mouse serum to almost an hour. The results demonstrate that specificity in the interaction of cisplatin with anionic phospholipids is an important criterium for the formation and stability of cisplatin nanocapsules.

Nanocapsules: lipid-coated aggregates of cisplatin with high cytotoxicity.

Cisplatin is one of the most widely used agents in the treatment of solid tumors, but its clinical utility is limited by toxicity. The development of less toxic, liposomal formulations of cisplatin has been hampered by the low water solubility and low lipophilicity of cisplatin, resulting in very low encapsulation efficiencies. We describe a novel method allowing the efficient encapsulation of cisplatin in a lipid formulation; it is based on repeated freezing and thawing of a concentrated solution of cisplatin in the presence of negatively charged phospholipids. The method is unique in that it generates nanocapsules, which are small aggregates of cisplatin covered by a single lipid bilayer. The nanocapsules have an unprecedented drug-to-lipid ratio and an in vitro cytotoxicity up to 1000-fold higher than the free drug. Analysis of the mechanism of nanocapsule formation suggests that the method may be generalized to other drugs showing low water solubility and lipophilicity.