Cholesterol Content Of Liposomes Research Articles

Cholesterol-Containing Liposomes Decorated With Au Nanoparticles as Minimal Tunable Fusion Machinery.

Membrane fusion is essential for the basal functionality of eukaryotic cells. In physiological conditions, fusion events are regulated by a wide range of specialized proteins, operating with finely tuned local lipid composition and ionic environment. Fusogenic proteins, assisted by membrane cholesterol and calcium ions, provide the mechanical energy necessary to achieve vesicle fusion in neuromediator release. Similar cooperative effects must be explored when considering synthetic approaches for controlled membrane fusion.Weshow that liposomes decorated with amphiphilic Au nanoparticles (AuLips) can act as minimal tunable fusion machinery. AuLips fusion is triggered by divalent ions, while the number of fusion events dramatically changes with, and can be finely tuned by, the liposome cholesterol content.Wecombine quartz-crystal-microbalance with dissipation monitoring (QCM-D), fluorescence assays, and small-angle X-ray scattering (SAXS) with molecular dynamics (MD) at coarse-grained (CG) resolution, revealing new mechanistic details on the fusogenic activity of amphiphilic Au nanoparticles (AuNPs) and demonstrating the ability of these synthetic nanomaterials to induce fusion regardless of the divalent ion used (Ca2+ or Mg2+ ). The results provide a novel contribution to developing new artificial fusogenic agents for next-generation biomedical applications that require tight control of the rate of fusion events (e.g., targeted drug delivery).

Cholesterol Microdomain Enhances the Biofilm Eradication of Antibiotic Liposomes.

Resistance and tolerance of biofilms to antibiotics is the greatest challenge in the treatment of bacterial infections. Therefore, developing an effective strategy against biofilms is a top priority. Liposomes are widely used as antibiotic drug carriers; however, common liposomes lack affinity for biofilms. Herein, biofilm-targeted antibiotic liposomes are created by simply adjusting their cholesterol content. The tailored liposomes exhibit significantly enhanced bacterial inhibition and biofilm eradication effects that are positively correlated with the cholesterol content of liposomes. The experiments further demonstrate that this enhanced effect can be ascribed to the effective drug release through the pores, which are formed by the combination of cholesterol microdomains in liposomal lipid bilayers with membrane-damaged toxins in biofilms. Consequently, liposome encapsulation with a high cholesterol concentration improves noticeably the pharmacodynamics and biocompatibility of antibiotics after pulmonary administration. This work may provide a new direction for the development of antibiofilm formulations that can be widely used for the treatment of infections caused by bacterial biofilms.

Experimental design for in vitro skin penetration study of liposomal superoxide dismutase

A computer-based technique using a 2 (5−2) fractional factorial design was applied for screening the factors affecting the penetration effectiveness of liposomal recombinant human-Cu/Zn-superoxide dismutase. Unilamellar liposomes, containing recombinant rh-Cu/Zn-SOD in the aqueous core, are aimed at enhancing the penetration of the drug applied topically. Factors that mainly influence the chemical and physical characteristics of liposomes such as charge, molar content of cholesterol, size, surfactant and lipid were evaluated at two levels. In vitro skin penetration studies with pigskin were carried out in Franz-type diffusion cells over a period of 4 and 8 h. The response variables, namely the amounts of rh-Cu/Zn-SOD penetrated into the different skin layers, were analyzed by ELISA (enzyme linked immunosorbent assay). Analysis of variance showed that the size and the cholesterol content of liposomes as well as the duration of the penetration studies have a statistically significant influence on the amount of protein found in deeper skin layers.

リポソーム膜中のコレステロール含量が及ぼす補体第３成分（Ｃ３）の結合と体内動態への影響

In this study we analyzed the binding ability of complement component 3 (C3) associated with liposomes after incubation in rat serum in order to determine the effect of cholesterol content of liposomes on the complement system liposomes interactions and the fate of liposomes in vivo. Liposomes having different cholesterol content showed no difference in clearances from blood circulation, while in both hepatic and renal clearances the liposomes showed marked difference. The hepatic clearance increased with increasing cholesterol content and the renal clearance decreased with increasing cholesterol content. Further, it appeared that those liposomes had different C3 binding potentials : cholesterol-rich (44 mol%) liposomes had higher affinity for C3 than cholesterol-poor (22 mol%) liposomes. Interestingly, there was a good correlation between the C3 binding ability and hepatic clearance of liposomes, while there was an inverse correlation between the C3 binding ability and renal clearance of liposomes. These results suggested that the extent of interaction of liposomes with complement system directly related to the distribution of liposomes in liver, leading to a possibility that the liposomes having high affinity for C3 are phagocytosed by Kuppfer cells via complement receptors. Release of encapsulated carboxy fluorescein from liposomes was inhibited in heat-treated rat serum, less done in EDTA-treated one. This finding indicated that degradation of liposomes in in vivo or in vitro was attributed to a heat-labile serum component, but not complement system. We clarified in this study that the complement-liposome interactions play a critical role on the hepatic uptake of liposomes, but not on the degradation of liposomes in blood circulation.

Influence of Cholesterol on Physical Properties of Egg Phosphatidylcholine Liposomes

Physical properties of unilamellar vesicles (ULVs) and multilamellar vesicles (MLVs) prepared from egg phosphatidylcholine have been investigated as a function of cholesterol content. Molecular dimensions of ULVs and MLVs with or without cholesterol have been measured by dynamic laser light scattering method. Inclusion of cholesterol in the liposomal bilayer structure significantly affected the mean diameter of vesicles suspended in buffer. The mean molecular dimension of MLVs was found to increase by 28% whereas that of ULVs decreased by 52% at the highest concentration of cholesterol (50 mole%)). Studies on stability of unilamellar vesicles containing cholesterol were carrried out by monitoring the release of encapsulated carboxyfluorescein (CF) after suspension in phosphate buffered saline (PBS) and human blood plasma at 37°C. Vesicles with cholesterol displayed greater stability than controls in buffer as well as in human plasma. Moreover, the mean size of these vesicles significantly decreased after suspension in human plasma compared with phosphate buffered saline (PBS) which was dependent on the concentration of choleserol. However, vesicles without cholesterol showed larger decrease of size in plasma than in buffer. Furthermore, cholesterol-free vesicles were found to be more stable in buffer than in plasma. These results suggest that the cholesterol content of liposomes affects their molecular size and stability in biological fluid environment which may govern the fate of vesicles in body circulation.

Study of liposomal drug delivery systems 3. Dynamic in-vitro release of steroids from multilamellar liposomes

The effects of (a) drug input concentration, (b) liposome composition and (c) hydrophobicity of the encapsulated drug on dynamic in-vitro drug release were studied. Two corticosteroids with different hydrophobicities, triamcinolone (TRM) and hydrocortisone (HC), were encapsulated into multilamellar liposomes (MLVs) by a modified hydration method. Both the input drug concentration and the liposomal composition (i.e. phospholipid (PL) and cholesterol (Chol) molar ratios) were varied independently. The liposomes were prepared with, and the dynamic in-vitro release studies conducted in, distilled water (without any additives), using a USP dissolution apparatus under simulated, finite sink conditions at 37°C. The concentration of the drug released (in aliquot samples taken for 24 h) was determined by UV-spectrophotometry. The in-vitro release of steroids from MLVs indicated a biphasic, apparent first-order release which, in the initial phase, seemed to be diffusion-controlled. The release of drug was dependent upon the drug input concentration and the liposomal composition (i.e. cholesterol content), and was influenced by the hydrophobicity of the encapsulated drug. The results showed that a faster drug release was observed for the less hydrophobic HC-containing preparations than for the hydrophobic TRM-loaded vesicles. It was, however, noted that a higher percentage of HC than TRM remained within the liposomes in all preparations after 24 h. The increasing cholesterol content in liposomes at constant drug input provided a minimum release rate for preparations with a PL/Chol = 1 0.5 (M) ratio. However, a further increase of the cholesterol content promoted the release of hydrophobic steroids from the liposome membrane, indicating a displacement of the steroids by cholesterol within the bilayers of the lipid membrane.

Specific drug binding by purified lipid-reconstituted P-glycoprotein: Dependence on the lipid composition

We fused P-glycoprotein with β-galactosidase at the C-terminus aiming to study the mechanism of drug binding of P-glycoprotein in reconstitution experiments. Expression of the fusion protein in NIH 3T3 cells conferred a multidrug-resistant phenotype, suggesting that β-galactosidase fusion at the C-terminus does not affect the functions of P-glycoprotein. The fusion protein was partially purified by simple immunoprecipitation with anti-β-galactosidase polyclonal antibody, and its [ 3H]azidopine binding property was investigated in the presence of various compositions of liposomes. The purified P-glycoprotein, after reconstitution into liposomes, was capable of binding [ 3H]azidopine. When the cholesterol content of liposomes was increased to a weight ratio of 20%, the specific binding activity of the partially purified fusion protein was stimulated, and when the cholesterol content was increased higher, the binding activity decreased. The binding was specifically decreased by competition with vinblastine. Stigmasterol was less effective, and ergosterol was the least effective in stimulating the specific binding.

Membrane potential in liposomes measured by the transmembrane distribution of 86Rb +, tetraphenylphosphonium or triphenylmethylphosphonium: Effect of cholesterol in the lipid bilayer

Valinomycin-induced potassium diffusion potential (Δψ, inside negative) in the liposomes made of phosphatidylcholine and various amounts of cholesterol was measured by uptake of 86Rb +, tetraphenylphosphonium (TPP +) or triphenylmethylphosphonium (TPMP +). In any liposome, the values of membrane potential obtained by 86Rb + uptake (Δψ Rb) agreed well with those calculated from the imposed potassium concentration gradient using the Nernst equation, and were not affected by the presence of cholesterol. However, both Δψ TPP and Δψ TPMP showed smaller values than Δψ Rb when the cholesterol content in liposomes increased. Δψ TPMP at a stationary state was much smaler than Δψ TPP. The orientational order parameter of the lipids' bilayer with various cholesterol content was estimated from fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. The results indicated that the permeation of TPP + or TPMP + into liposomes containing a large amount of cholesterol is strongly restricted by the high ordering of phosphatidylcholine acyl chains.

Inhibitory effect of cholesterol on the uptake of liposomes by liver and spleen

The effect of cholesterol content of small unilamellar (SUV) and reverse phase (REV) liposomes on blood clearance and tissue distribution has been studied. [ 14C]Inulin has been used as an aqueous marker of liposomes to represent the uptake of intact liposomes in tissues. The blood clearance of the intravenously-injected SUV and REV liposomes depends on the cholesterol content of liposomes. The cholesterol-free (0 mol%) liposomes are cleared more readily from the circulation than the cholesterol-poor liposomes (20 mol%) and the cholesterol-poor are cleared more rapidly than the cholesterol-rich (46.6 mol%) liposomes. This clearance pattern of liposomes from the circulation is not attributed to the change of size of liposomes due to the increase in cholesterol content of liposomes. However, poor stability of cholesterol-free or cholesterol-poor liposomes in the circulation is partly responsible, but the predominant factor responsible for the observed blood clearance pattern is the inhibitory effect of cholesterol on the uptake of liposomes by reticuloendothelial-rich tissues liver and spleen. Uptake of liposomes by these organs is decreased with increasing cholesterol content of vesicles. It is suggested that to produce liposome preparations with a long circulating half life in vivo it is necessary to inhibit their uptake by liver and spleen.

Interaction of hapten-sensitized liposomes with cells bearing hapten-specific receptors.

The binding of liposomes sensitized with 2,4-dinitrophenyl-6-N-aminocaproylphosphatidylethanolamine (DNP-Cap-PE) to MOPC-315 cells, which secrete and bear on their surfaces anti-DNP immunoglobulins, was studied. The binding was affected by cholesterol content, phospholipid composition and hapten density of liposomes: The binding of distearoylphosphatidylcholine liposomes sensitized with 5 mol% hapten to the cells increased with increasing cholesterol content in liposomes. The amount of liposomes composed of phospholipid with a higher transition temperature (such as distearoylphosphatidylcholine), which bound to MOPC-315 cells, was much higher than that of liposomes composed of phospholipid with a lower transition temperature (such as egg yolk phosphatidylcholine). The amount of distearoylphosphatidylcholine liposomes containing equimolar cholesterol, which bound to the cells at 0 degrees C, increased with increasing amount of the hapten in liposomes up to 2.5 mol%. The binding became maximum at 2.5 mol% and decreased with higher hapten density in liposomes. The immunogenicity of hapten-sensitized liposomes is known to be affected by the liposomal composition such as cholesterol content, phospholipid composition and hapten density. This model study suggests that the binding of liposomes to cells is important for expressing the immunogenicity of hapten-sensitized liposomes.

Stability of liposomes in vivo and in vitro is promoted by their cholesterol content and the presence of blood cells.

Abstract The stability of liposomes containing 6-carboxyfluorescein or β-fructofuranosidase was measured in the blood of injected animals and also in vitro in the presence of whole blood or serum. Stability, monitored by measuring changes in the permeability of liposomes to 6-carboxyfluorescein and sucrose, was found to depend on the cholesterol content of liposomes and it was promoted by the presence of blood cells.