The behavior of glyceride-fatty acid mixtures in bile salt solution: Studies by gel filtration

Bile salt lipid emulsions were prepared which simulated the emulsified oil-micellar phase system of the small intestinal content during fat digestion. Application of such emulsions to gel columns prepared and eluted with 6 mM sodium taurodeoxycholate separated an emulsion phase and a micellar phase. The distribution of lipid solutes into the two phases under these conditions was measured. Micellar dimensions were larger as lipid concentrations were increased. Inclusion of multiple lipid classes resulted in larger micellar particles. Monoglyceride and fatty acids were eluted completely in the micellar phase under these conditions. Minimal measurable amounts of triolein were recovered in micellar solution. This was confirmed by extraction, chromatographic separation and quantitative analysis. As diolein concentration was increased, less was recovered in the micellar phase. When monoglyceride was added, more diolein entered the micellar phase. Addition of triglyceride enhanced the distribution of diolein into the emulsion phase.     

Phase behavior and hydrated solid structure in lysophospholipid/long-chain alcohol/water system and effect of cholesterol addition

Phase behavior in lysophospholipid/long-chain alcohol/water system at 80°C was investigated using hexanol and oleyl alcohol as the long-chain alcohol. Similarly to hydrophilic surfactant, a micellar phase in a lysophospholipid/water system transitioned to a lamellar liquid-crystalline phase by the addition of long-chain alcohol. In the oleyl alcohol system the lamellar liquid-crystalline phase was observed in wider region compared to the hexanol system. The effect of cholesterol addition on the phase behavior was also studied. The region of liquid-crystalline phase and (reverse micellar + liquid-crystalline + water) phase shifted towards higher lysophospholipid concentrations. The structure of hydrated solid as well as the transition between lamellar liquid-crystalline phase and hydrated solid was analyzed by X-ray scattering measurement and differential scanning calorimetry measurement. It was revealed that the hydrated solid was α-type crystals with lamellar structure. The hydrated solid (gel)-liquid crystal transition temperature gradually decreased with increasing oleyl alcohol concentration and the decrement was enhanced by the addition of cholesterol.     

Equilibrium of taurodeoxycholate between mixed micellar and aqueous phases: Effect of amphiphile

The equilibration of taurodeoxycholate between mixed micellar and aqueous phases has been studied by equilibrium dialysis. The presence of amphiphiles in the form of lecithin, long chain monoglyceride, and fatty acid in the bile salt solution will greatly decrease the bile salt concentration in the aqueous (intermicellar) phase. At high amphiphile concentration relative to bile salt, the concentration of bile salt in the aqueous phase will be below the critical micellar concentration (CMC) of the pure bile salt solution. Under these conditions, few simple micelles will be present and no binding of bile salts to protein takes place as indicated by experiments with colipase. The lowering of the concentration of bile salt in the aqueous phase by the presence of amphiphile may be a physiological mechanism to regulate bile salt absorption during the digestive phase of fat absorption.     

Effect of long and medium chain length lipids upon aqueous solubility of α-tocopherol

The efficiency by which α-tocopherol is solubilized in vitro into mixed bile salt micelles containing different lipids was studied. Alterations in solubility due to addition to the incubation media of triglyceride, free fatty acid, monoglyceride, and lecithin of either long or medium chain length were examined. Results are expressed as a partition ratio between a micellar and an oil phase. The triglyceride of both long and medium chain length fatty acids greatly decreased the solubility of α-tocopherol in bile salt solutions. When added singly, monoglyceride and lecithin of long chain length fatty acids increased the α-tocopherol solubilized four- to fivefold; fatty acids of either chain length and medium chain monoglyceride when added singly had no significant effect upon the tocopherol solubilized. An additive effect was observed when a combination of long chain monoglyceride and lecithin was added. Addition of fatty acid to this combination, however, significantly decreased the α-tocopherol solubility into the micellar phase. Although the solubility of α-tocopherol was increased by all combinations of medium chain length polar lipids, except the fatty acid-monoglyceride pair, the effect was three to seven times less than for the corresponding long chain mixture.     

Self-assembled multicompartment liquid crystalline lipid carriers for protein, peptide, and nucleic acid drug delivery

Lipids and lipopolymers self-assembled into biocompatible nano- and mesostructured functional materials offer many potential applications in medicine and diagnostics. In this Account, we demonstrate how high-resolution structural investigations of bicontinuous cubic templates made from lyotropic thermosensitive liquid-crystalline (LC) materials have initiated the development of innovative lipidopolymeric self-assembled nanocarriers. Such structures have tunable nanochannel sizes, morphologies, and hierarchical inner organizations and provide potential vehicles for the predictable loading and release of therapeutic proteins, peptides, or nucleic acids. This Account shows that structural studies of swelling of bicontinuous cubic lipid/water phases are essential for overcoming the nanoscale constraints for encapsulation of large therapeutic molecules in multicompartment lipid carriers. For the systems described here, we have employed time-resolved small-angle X-ray scattering (SAXS) and high-resolution freeze-fracture electronic microscopy (FF-EM) to study the morphology and the dynamic topological transitions of these nanostructured multicomponent amphiphilic assemblies. Quasi-elastic light scattering and circular dichroism spectroscopy can provide additional information at the nanoscale about the behavior of lipid/protein self-assemblies under conditions that approximate physiological hydration. We wanted to generalize these findings to control the stability and the hydration of the water nanochannels in liquid-crystalline lipid nanovehicles and confine therapeutic biomolecules within these structures. Therefore we analyzed the influence of amphiphilic and soluble additives (e.g. poly(ethylene glycol)monooleate (MO-PEG), octyl glucoside (OG), proteins) on the nanochannels' size in a diamond (D)-type bicontinuous cubic phase of the lipid glycerol monooleate (MO). At body temperature, we can stabilize long-living swollen states, corresponding to a diamond cubic phase with large water channels. Time-resolved X-ray diffraction (XRD) scans allowed us to detect metastable intermediate and coexisting structures and monitor the temperature-induced phase sequences of mixed systems containing glycerol monooleate, a soluble protein macromolecule, and an interfacial curvature modulating agent. These observed states correspond to the stages of the growth of the nanofluidic channel network. With the application of a thermal stimulus, the system becomes progressively more ordered into a double-diamond cubic lattice formed by a bicontinuous lipid membrane. High-resolution freeze-fracture electronic microscopy indicates that nanodomains are induced by the inclusion of proteins into nanopockets of the supramolecular cubosomic assemblies. These results contribute to the understanding of the structure and dynamics of functionalized self-assembled lipid nanosystems during stimuli-triggered LC phase transformations.     

Lipid Cubic Mesophases Combined with Superparamagnetic Iron Oxide Nanoparticles: A Hybrid Multifunctional Platform with Tunable Magnetic Properties for Nanomedical Applications

Hybrid materials composed of superparamagnetic iron oxide nanoparticles (SPIONs) and lipid self-assemblies possess considerable applicative potential in the biomedical field, specifically, for drug/nutrient delivery. Recently, we showed that SPIONs-doped lipid cubic liquid crystals undergo a cubic-to-hexagonal phase transition under the action of temperature or of an alternating magnetic field (AMF). This transition triggers the release of drugs embedded in the lipid scaffold or in the water channels. In this contribution, we address this phenomenon in depth, to fully elucidate the structural details and optimize the design of hybrid multifunctional carriers for drug delivery. Combining small-angle X-ray scattering (SAXS) with a magnetic characterization, we find that, in bulk lipid cubic phases, the cubic-to-hexagonal transition determines the magnetic response of SPIONs. We then extend the investigation from bulk liquid-crystalline phases to colloidal dispersions, i.e., to lipid/SPIONs nanoparticles with cubic internal structure (“magnetocubosomes”). Through Synchrotron SAXS, we monitor the structural response of magnetocubosomes while exposed to an AMF: the magnetic energy, converted into heat by SPIONs, activates the cubic-to-hexagonal transition, and can thus be used as a remote stimulus to spike drug release “on-demand”. In addition, we show that the AMF-induced phase transition in magnetocubosomes steers the realignment of SPIONs into linear string assemblies and connect this effect with the change in their magnetic properties, observed at the bulk level. Finally, we assess the internalization ability and cytotoxicity of magnetocubosomes in vitro on HT29 adenocarcinoma cancer cells, in order to test the applicability of these smart carriers in drug delivery applications.     

Polycyclic hydrocarbon and polychlorinated biphenyl solubilization in aqueous solutions of mixed micelles

To determine the physiochemical behavior of xenobiotic hydrocarbons in simulated intestinal content, we examined the partition of 7,12-dimethylbenzanthracene (DMBA), 3-methyl cholanthrene (MC), benzo(a)pyrene, and a polychlorinated biphenyl compound (PCB, Aroclor 1242) between an emulsified oil phase and a mixed micellar solution. In a mixed lipid-bile salt system, negligible amounts of hydrocarbon were present in aqueous solution below the critical micellar concentration (CMC) of sodium taurocholate. Once the CMC was obtained, the 4 hydrocarbons exhibited nearly identical partitions from the lipid into the micellar system which was enhanced by increased concentrations of bile salt, reduction of triglyceride concentration and the formation of mixed rather than pure bile salt micelles. The partition of DMBA and MC into micelles was optimized by long-chain monounsaturated oleic acid and monooleoylglycerol as compared to their octanoic or linoleic counterparts. Linoleic acid and monolinoleoylglycerol maximized the partition of PCB from the oil into the micellar phase. In a mixed micellar system excluding an oil phase and an excess of DMBA, a molar saturation ratio (mol hydrocarbon:mol bile salt) was calculated by regression analysis to be 0.162. This indicates that more than one molecule of hydrocarbon is solubilized per mixed micelle and that the aqueous solubilization of hydrocarbon may be attributed to true micellar solubilization.     

Enzymatische Herstellung von festen Fettsäuremonoglyceriden

Enzymatic Preparation of Solid Fatty Acid Monoglycerides Lipases can be used to synthesize monoglycerides from solid fatty acids and glycerol. We have examined the conditions of such reaction systems with a view to developing a simple technical process. The selectivity of the Penicillium cyclopium and Rhizopus sp. lipases were studied at high rates of fatty acid turnover. By the correct choice of lipase, temperature and water content, the reaction may be steered in the direction of either monoglycerides of diglycerides. Using the Penicillium lipase under narrowly defined reaction conditions a highly selective monoglyceride synthesis is possible. Di glycerides are almost the sole products when using the Rhizopus lipase at 40°C. At 20°C, but under otherwise identical conditions, the main products are monoglycerides. The Penicillium lipase catalyzes the synthesis of monoglycerides at both 20°C and 40°C, provided that the water content of the glycerine is less than 10%. At a glycerol concentration of 80% the selectivity changes such that more diglycerides are formed. The enzymatic synthesis of glycerides can be so regulated that more than 95% of the available fatty acid is incorporated into monoglyceride. After melting the reaction mixture and allowing it to stand for less than an hour, the phases separate and excess glycerol can be separated very simply. A product conforming to industry specifications can then be produced by distilling off the trace amounts of remaining glycerol from the lipid phase.     

AOT逆胶束体系脂肪酶催化合成油酸乙酯

在二-(2-乙基己基)琥珀酸酯磺酸钠和异辛烷构建的逆胶束体系中,以Lipex脂肪酶为催化剂,合成了油酸乙酯,考察了各影响因素对其产率的影响,并进行了脂肪酶紫外荧光检测和体系粒度分析. 结果表明,Lipex脂肪酶具有良好的催化活性,反应条件优化选用异辛烷为溶剂,在反应温度25℃、缓冲液pH 6.5、水/表面活性剂(摩尔比)10、乙醇/油酸(摩尔比)20及Lipex脂肪酶浓度0.035 g/L、油酸浓度0.005 mol/L、摇床转速150 r/min、反应36 h的条件下,油酸乙酯产率达到71.25%. 逆胶束粒度和酶构象直接影响酶活性,最适逆胶束粒度约为80 nm.     

Influence of temperature-induced phase transitions on fat emulsions

The polymorphic behavior of a commercial monoglyceride emulsifier together with a liquid vegetable oil and water has been investigated by means of microscopic examination and differential scanning calorimetry. The results show that the stability regions of the liquid crystalline phases formed are highly dependent on the temperature and related both to the solubility of the monoglyceride emulsifier in the oil phase and to its polymorphic behavior. The formation and stability of emulsions are discussed in connection with the results obtained. Presented at the ISF-AOCS World Congress, Chicago, September 1970.     

Neutral lipid composition ofCulex quinquefasciatus andCulex tritaeniorhynchus cells at two phases of growth

The mosquito cellsCulex quinquefasciatus andCulex tritaeniorhynchus were grown in spinner cultures. The fatty acid profiles of the neutral lipid classes were analyzed, and comparisons were made between the two species at logarithmic and stationary phases of growth and between the species and the medium. The results from the data suggest an increase in the average chain length of the fatty acids in the free fatty acid and sterol ester fractions of theCulex quinquefasciatus cells with aging. From the logarithmic to stationary phase, both Culex cells showed some increase in desaturation of acids in the di- and triglyceride and free fatty acid fractions. TheCulex tritaeniorhynchus cells also showed some increase in desaturation of acids in the monoglyceride fraction. Differences between the twoCulex species at the logarithmic phase of growth were observed in the fatty acid profiles of all the neutral lipid fractions examined and at the stationary phase of growth in the free fatty acid, triglyceride, and sterol ester fractions.     

Interaction of a model skin surface lipid with a modified triglyceride

A study of the interaction between a model skin surface lipid (SSL) and an oil base skin softener (G2) was made by comparing the phase behavior of these substances in combination with the water/triethanolamine: oleic acid (1:1 wt ratio) system. A principle feature of the water/G2/triethanolamine:oleic acid (TEA:OL) system was the formation of a lamellar liquid crystal region that incorporated an average of 40% G2 over the 10–45% water range. The phase behavior of the water/SSL/TEA:OL system demonstrated only over a 4–9% water range. At greater than 5% SSL, there is the complete absence of single phase regions above 10’% water. From comparison of the phase regions and the small angle X-ray diffraction results, it is found that for G2/SSL mixtures the phase behavior of the lamellar liquid crystal is dominated by the SSL and the interlayer spacing of the lamellar liquid crystal is dominated by G2.     

油酸辅助水热合成纳米钛酸锶的性能调控与表征

采用油酸辅助一步水热法,以廉价易得的锐钛矿型TiO2为钛源、Sr(OH)2?8H2O为锶源合成SrTiO3纳米颗粒,通过XRD, TEM和SEM研究了SrTiO3纳米颗粒的形貌,考察了反应时间、Sr/Ti摩尔比和油酸浓度对SrTiO3纳米颗粒性能的影响,以所制SrTiO3为催化剂降解亚甲基蓝考察其光催化性能。结果表明,钙钛矿型立方相SrTiO3纳米颗粒具有均匀的尺寸和规则的形貌。实验范围内,随反应时间和Sr/Ti摩尔比增加,SrTiO3纳米粒子的晶粒尺寸呈增加趋势;随油酸浓度增加,SrTiO3颗粒粒度呈下降趋势。添加油酸可调控制备出具有规则形貌的钙钛矿型立方相结构的SrTiO3纳米颗粒;Sr/Ti摩尔比为0.75时,产品纯度高、结晶度好。油酸、反应时间和Sr/Ti摩尔比对SrTiO3纳米粒子微观结构的影响可分别用颗粒间作用力、奥斯特瓦尔德熟化和晶体成核理论解释。     

Oleic acid modulates the partitioning of cholesterol from micellar bile salt solution

The effects of monounsaturated fatty acid (oleic acid) on cholesterol monomer activity and on the rate of cholesterol influx were studied in vitro. A polyethylene disc method was employed to determine cholesterol monomer activity in constant sodium taurocholate-cholesterol micellar solution containing different oleic acid concentration levels at pH 5.5, 6.5 and 7.2. In addition, the effect of oleic acid on the rates of cholesterol influx was determined using an everted rat jejunal sac technique. At pH 5.5, increased oleic acid concentration from 5 to 10 mM resulted in significant decreased apparent cholesterol monomer activity (3.8±0.21 nmol/disc vs 1.0±0.08, P<0.001). At pH 6.5, apparent cholesterol monomer activity was 2.3±0.19 nmol/disc at 5 mM and 0.5±0.09 at 16 mM oleic acid level (P<0.001). Apparent monomer activity of cholesterol in micellar solutions at pH 7.2 used for the influx study at 5 and 15 mM oleic acid concentration level was 1.8±0.14 and 0.7±0.08 nmol/disc, respectively (P<0.001). Thus there was a significant decrease in cholesterol monomer activity by the addition of oleic acid at each pH. The rate of cholesterol influx across the brush border membrane of the rat jejunum at 5 and 15 mM oleic acid concentration level was 3.2±0.31 and 1.5±0.21 nmol/100 mg dry weight tissues/min, respectively (P<0.001). It is concluded that the addition of oleic acid decreases both monomer activity and the rate of influx of cholesterol from micellar solution. This effect is primarily attributable to the inhibition of the release of cholesterol monomers from the mixed micelle.     

CTAB逆胶束体系中Lipex脂肪酶催化合成油酸乙酯

研究了CTAB逆胶束体系的适宜构建条件以及Lipex脂肪酶催化合成油酸乙酯的适宜反应条件。探讨了有机溶剂、底物配比、pH值、水含量W₀、酸浓度、反应时间等因素对反应的影响。研究表明,在以CTAB为表面活性剂时,以异辛烷为溶剂,正己醇作助溶剂条件下,能得到较为稳定的逆胶束体系,更有利于Lipex脂肪酶的活性发挥;在该反应体系下反应温度25 ℃,缓冲液pH7. 0,W₀10,酸醇比1∶4（摩尔比）,油酸浓度0. 01 mol·L^-1,摇床转速150 r·min^-1,反应36 h,油酸乙酯产率达到79%。并建立了逆胶束体系中双底物作用的动力学模型,经验证模拟计算结果与实验结果吻合良好。     

Synthesis of triacylglycerol by lipase in phosphatidylcholine reverse micellar system

Triacylglycerols were synthesized from 1,2-diacylglycerol and fatty acids by lipase entrapped in phosphatidylcholine reverse micelles in n-hexane. In the reaction system without reverse micelles, however, 1,2-diacylglycerol was hydrolyzed into 2-monoacylglycerol and fatty acid, and triacylglycerol was not synthesized. The maximum activity of synthetic reaction was obtained at Wo=10 (Wo=mol water/mol surfactant), which was the water content of this reverse micellar system. Though the optimal pH of theR. delemar lipase reaction is about pH 5.6 in a bulk water system, the enzyme was active for triacylglycerol synthesis at pH’s from 5 to 9 in the reverse micellar system. For the synthesis of triacylglycerols, lauric, myristic, palmitic, stearic, oleic and arachidic acids were effectively used as the fatty acid substrate. 2-Monoacylglycerol was also effective as a substrate of triacylglycerol synthesis. Furthermore, 1,2-diacylglycerol could be replaced by several kinds of aliphatic alcohols as fatty acid acceptors in the reverse micellar system. In this case, those alcohols with chain length more than 4 carbons were effectively used for ester formation.     

2-Monoglyceride as an aid to the absorption of cholesterol into the thoracic lymph

Absorption of radioactive cholesterol given in triolein was nearly three-fold that of a similar dose given in ethyl oleate or oleic acid. The difference appears to reflect a need for monoglyceride, since addition of 2-monoolein to ethyl oleate improved cholesterol absorption.     

Temperature shifts in regulation of lipids accumulated byLipomyces starkeyi

The effect of temperature on the accumulating triglycerides ofLipomyces starkeyi was studied in 10-L fermentation experiments. The temperature during the growth, lipid accumulation and postaccumulation phases was altered. The cellular lipid content, the glucose conversion efficiency and the specific lipid production rate were highest when the growth phase temperature was 28°C, instead of 16–18°C. The temperature of the accumulation phase had an influence on the lipid quality at the end of the accumulation. Oleic acid content increased from 52 to over 60% when the accumulation phase temperature was decreased from 28 to 15°C and, concomitantly, palmitic acid decreased from 33 to 26%. The degree of fatty acid unsaturation was the highest (0.75 Δmol⁻¹) when the accumulation phase temperature was the lowest (15°C) andvice versa. The temperature shift after the lipid accumulation phase affected neither the composition nor the amount of the accumulated lipids. In conclusion, the temperature profile for the highest lipid yield with the most desired composition should be: (i) a growth phase temperature that gives the maximum growth rate and (ii) an accumulation phase temperature that produces the desired ratio of palmitic to oleic acid.     

Über den Einfluß des kolloichemischen Aufbaus von Trägergrundlagen auf die cutane Aufnahme eines Pharmakons. I. Entwicklung von potentiellen Transportsystemen auf der Basis von Kohlenwasserstoff als Ölkomponente

Influence of the Colloidal Structure of Vehicles on the Percutaneous Drug Absorption. I. Development of Potential Transport Systems with Hydrocarbon as Oil Component In this part the development and colloidal differentiation of some model systems is presented containing dodecane as oil, water and polyoxyethylene(7)laurylether as nonionic surfactant. In specific concentration ranges mixtures of water and surfactant give stable solutions being micellar or inverse-micellar, and two mesophases, a gel with hexagonal structures (phase M) and a region with a onedimensional lamellar lattice (phase N). Dodecane being the third component will be integrated within the unpolar regions of the structures, enlarging the diameter of the rods in the case of phase M and the thickness of lipid bilayer in the case of phase N. The formation of emulsions, creams and gels is highly depending on the presence of the hydrocarbon. Their different structures will be described, proposing a specific three-phase structure of the creams and a cubic lattice in the case of the isotropic, visco-elastic gel.     

Intestinal cholesterol and oleic acid uptake from solutions supersaturated with lipids

To test the role of nonmicellar phases in lipid absorption, intestinal uptake of fatty acids and cholesterol has been studied in vitro from supersaturated and micellar solutions. The micellar solubility limit at equilibrium was established for cholesterol and oleate/monoolein (2∶1) at pH 6.7 with 10 mM taurocholate. Uptake by rat intestinal everted sacs was measured during incubation of 5 min. Cholesterol uptake increased linearly with the cholesterol content of micellar or supersaturated solutions up to a supersaturation of 150%. Oleate uptake, by contrast, remained essentially the same from either saturated or supersaturated (130–280%) mixtures. The difference between cholesterol and oleate uptake rates is explained by their distinct effects on micellar size, which is unchanged by cholesterol supersaturation but is increased by oleate. Solutions largely supersaturated (280%) with oleatemonoolein are polydisperse and contain viscous isotropic and paracrystalline phases similar to those observed during lipid absorption. These results suggest that, in the presence of such solutions, uptake occurs from both the micellar saturated and nonmicellar supersaturated phases.