Supercooled Smectic Nanoparticles: A Potential Novel Carrier System for Poorly Water Soluble Drugs

PURPOSE: The possibility of preparing nanoparticles in the supercooled thermotropic liquid crystalline state from cholesterol esters with saturated acyl chains as well as the incorporation of model drugs into the dispersions was investigated using cholesteryl myristate (CM) as a model cholesterol ester. METHODS: Nanoparticles were prepared by high-pressure melt homogenization or solvent evaporation using phospholipids, phospholipid/ bile salt, or polyvinyl alcohol as emulsifiers. The physicochemical state and phase behavior of the particles was characterized by particle size measurements (photon correlation spectroscopy, laser diffraction with polarization intensity differential scattering), differential scanning calorimetry, X-ray diffraction, and electron and polarizing light microscopy. The viscosity of the isotropic and liquid crystalline phases of CM in the bulk was investigated in dependence on temperature and shear rate by rotational viscometry. RESULTS: CM nanoparticies can be obtained in the smectic phase and retained in this state for at least 12 months when stored at 230C in optimized systems. The recrystallization tendency of CM in the dispersions strongly depends on the stabilizer system and the particle size. Stable drug-loaded smectic nanoparticles were obtained after incorporation of 10% (related to CM) ibuprofen, miconazole, etomidate, and 1% progesterone. CONCLUSIONS: Due to their liquid crystalline state, colloidal smectic nanoparticles offer interesting possibilities as carrier system for lipophilic drugs. CM nanoparticles are suitable model systems for studying the crystallization behavior and investigating the influence of various parameters for the development of smectic nanoparticles resistant against recrystallization upon storage.     

The physical state of lipid nanoparticles influences their effect on in vitro cell viability

Although lipid nanoparticles represent potent drug carriers, for many formulations toxicity data are rare. Thus, in this study, the effect of different lipid nanoparticles on the cell viability of L929 mouse fibroblasts was systematically investigated using the MTT assay. The formulations were composed of trimyristin, tristearin or cholesteryl myristate stabilized with poloxamer 188, polysorbate 80, polyvinyl alcohol or a blend of soybean phospholipid and sodium glycocholate. Depending on lipid and storage conditions, the nanoparticles were prepared in different physical states or crystal modifications leading to different particle shapes. The cell viability was influenced considerably by the physical state of the particle matrix with crystalline nanoparticles causing a stronger decrease in viability than the corresponding liquid or liquid crystalline particles. Effects on the cell viability were also related to the type of matrix lipid, stabilizer and the particle shape. However, the effects of differently shaped particles of different polymorphic modifications of crystalline tristearin were comparable. The low viability caused by poloxamer 188-stabilized particles could be correlated with a strong cell uptake which was investigated by confocal laser scanning microscopy.     

Drug release from differently structured monoolein/poloxamer nanodispersions studied with differential pulse polarography and ultrafiltration at low pressure

Aqueous colloidal monoolein/poloxamer dispersions are under investigation as drug delivery systems. Depending on the composition and preparation procedure these dispersions may either contain predominantly vesicular particles or nanoparticles of cubic inner structure. To study the influence of ultrastructure on drug release, corresponding dispersions loaded with the model drugs diazepam (two different concentrations) and chloramphenicol were prepared by high-pressure homogenization with or without subsequent heat treatment. The dispersions were characterized with regard to particle size and their ultrastructure was confirmed with small angle X-ray diffractometry. Two techniques with high time resolution, differential pulse polarography (DPP) and ultrafiltration at low pressure were compared for their suitability to monitor rapid release from the dispersions. Instantaneous release was found for both drugs independent on the type of particle structure with the amount of released drug being controlled by the partition coefficient. Both release methods were suitable to monitor the rapid appearance of the releasable drug in the release medium.     

Preparation and characterization of n-dodecyl-ferulate-loaded solid lipid nanoparticles (SLN)

Solid lipid nanoparticles (SLN) containing a novel potential sunscreen n-dodecyl-ferulate (ester of ferulic acid) were developed. The preparation and stability parameters of n-dodecyl-ferulate-loaded SLN have been investigated concerning particle size, surface electrical charge (zeta potential) and matrix crystallinity. The chemical stability of n-dodecyl-ferulate at high temperatures was also assessed by thermal gravimetry analysis. For the selection of the appropriated lipid matrix, chemically different lipids were melted with 4% (m/m) of active and lipid nanoparticles were prepared by the so-called high pressure homogenization technique. n-Dodecyl-ferulate-loaded SLN prepared with cetyl palmitate showed the lowest mean particle size and polydispersity index, as well as the highest physical stability during storage time of 21 days at 4, 20 and 40 degrees C. These colloidal dispersions containing the sunscreen also exhibited the common melting behaviour of aqueous SLN dispersions.     

Transformation of vesicular into cubic nanoparticles by autoclaving of aqueous monoolein/poloxamer dispersions.

The ultrastructure of aqueous colloidal dispersions of the cubic monoolein/poloxamer 407/water phase, in particular the particle size distribution and presence of an additional vesicular fraction, highly depends on composition and preparation parameters. Therefore, the effect of autoclaving on such dispersions was investigated. Before autoclaving at 121 degrees C, a dispersion of 4.6% monoolein/0.4% poloxamer predominantly consists of cubic particles beside a fraction of non-cubic particles. The small vesicular particles disappear almost completely upon autoclaving whereas larger particles with cubic structure remain in the sample. In contrast, a 4.4% monoolein/0.6% poloxamer dispersion contains predominantly small vesicular particles before heat treatment. After autoclaving, the majority of the particles is larger and of cubic structure and only a few small non-cubic particles remain. The effect can already be observed at short autoclaving times (e.g., 5 min) but a temperature of at least 90 degrees C is required to induce a major change in the ultrastructure. Results from temperature dependent small angle X-ray diffraction investigations indicate that temperatures corresponding to an isotropic phase are required for particle transformation. Heat treatment of monoolein/poloxamer dispersions can thus be used to transform vesicular dispersions into dispersions of cubic phase or to improve the cubic/non-cubic particle ratio in dispersions already containing particles with cubic internal structure.     

Liquid and semisolid SLN dispersions for topical application: rheological characterization.

Aqueous dispersions of solid lipid nanoparticles (SLN) are promising drug carrier systems for topical application. A drawback, however, is the need of incorporating the SLN dispersion in commonly used dermal carriers (creams, gels) to obtain the required semisolid consistency for dermal application. This study describes the production of SLN dispersions having the desired semisolid consistency by a one-step process. Physical characterization of these systems in terms of particle size and rheological properties revealed some interesting features. Despite the high lipid content it was possible to produce colloidal dispersions by high pressure homogenization. Continuous flow measurements revealed systems with yield point, plastic flow and thixotropy. Oscillation measurements proved the viscoelastic microstructure of the SLN dispersions. Higher concentrated SLN dispersions were found to have a prevailing elastic component in contrast to lower concentrated systems. Viscoelastic properties of a 40% SLN dispersion were found to be comparable to standard dermal preparations. Storage stability at room temperature in terms of particle size could be demonstrated over a 6-month period. The development of the gel structure of semisolid SLN dispersions is delayed comparable to commercial O/W creams with non-ionic emulsifiers. Parameters like concentration of the dispersed phase, particle size and particle shape were identified as significant factors influencing the microstructure of these complex semisolid systems.     

Preparation of semisolid drug carriers for topical application based on solid lipid nanoparticles

Aqueous dispersions of solid lipid nanoparticles (SLN) show some interesting features in topical drug delivery. However, to get a semisolid carrier having the appropriate consistency for topical application, the liquid SLN dispersions have to be incorporated in convenient topical dosage forms like hydrogels or creams. This is a time-consuming production process with several disadvantages. A new one-step production process delivering a semisolid topical formulation including SLN is presented avoiding these disadvantages. The semisolid SLN dispersions were produced by high-pressure homogenization using an APV Lab 40 homogenizer. The resulting dispersions were characterized concerning their particle size and rheological properties. Despite the high lipid content of the SLN dispersions, they retained their colloidal particle size. Viscoelastic measurements proved the existence of a gel-like structure with a prevailing elastic component.     

The use of liposomes for the preparation of protein-free lipid emulsions models of chylomicron remnants.

Artificial chylomicron remnants were investigated as a new drug carrier system for the targeting of hepatic parenchymal cells. The emulsions presented here are similar in particle size and composition to natural lipoproteins. The preparations contained triolein, phospholipid, cholesterol and cholesteryl oleate. Egg yolk lecithin was either used to form multilamellar or unilamellar liposomes or it was incorporated into a lipid film prior to emulsification. Typically the lipid film contained triolein, cholesterol and cholesteryl oleate. When multilamellar liposomes were used however, cholesterol and cholesteryl oleate were incorporated into the vesicles. The emulsions were prepared by ultrasonication or by means of a microemulsifier. The unilamellar liposomes used with the microemulsifier yielded the best particle distribution, i.e. in the range of 40-60 as determined by quasi-elastic light scattering. The advantage of the method results from the complete emulsification of the components. The particle size remained unchanged during storage, although flocculation was observed. The results show that the synthesis of artificial chylomicron remnants in a microemulsifier is possible and reproducible.     

Effect of drug loading on the transformation of vesicular into cubic nanoparticles during heat treatment of aqueous monoolein/poloxamer dispersions.

Colloidal dispersions of the pre-equilibrated cubic phase in the monoolein/poloxamer 407/water system, which are under investigation as potential drug carriers, often contain a considerable fraction of undesired non-cubic particles, particularly when prepared with high concentrations of poloxamer. Recent investigations revealed that the non-cubic particles can be transformed into particles of cubic internal structure by heat treatment. The present study investigates the effect of drug loading on the non-cubic to cubic transformation process during autoclaving of the dispersions. The results indicate that the process can also proceed in dispersions loaded with different concentrations of ubidecarenone, tocopheryl acetate, betamethasone-17-valerate, chloramphenicol or miconazole. At low concentration, none of the drugs had pronounced influence on the autoclaved dispersions whereas with increasing drug concentration different effects were observed. Depending on the type of drug no effects (betamethasone-17-valerate), increasing particle size of the dispersions (chloramphenicol, miconazole) or phase separation upon autoclaving (high load of miconazole) was observed. Except for loading with high amounts of chloramphenicol, which led to the formation of cubic phase particles already without additional heat treatment, the properties of the thermally untreated dispersions were virtually unaffected by drug incorporation.     

Flocculated Amorphous Nanoparticles for Highly Supersaturated Solutions

Purpose  To recover polymer-stabilized amorphous nanoparticles from aqueous dispersions efficiently by salt flocculation and to show that the particles redisperse and dissolve rapidly to produce highly supersaturated solutions. Methods  Nanoparticle dispersions of itraconazole stabilized by nonionic polymers were formed by antisolvent precipitation and immediately flocculated with sodium sulfate, filtered and dried. The size after redispersion in water, crystallinity, and morphology were compared with those for particles produced by spray drying and rapid freezing. Results  Particle drug loading increased to ∼90% after salt flocculation and removal of excess polymer with the filtrate. The formation of the flocs at constant particle volume fraction led to low fractal dimensions (open flocs), which facilitated redispersion in water to the original primary particle size of ∼300 nm. Amorphous particles, which were preserved throughout the flocculation–filtration–drying process, dissolved to supersaturation levels of up to 14 in pH 6.8 media. In contrast, both spray dried and rapidly frozen nanoparticle dispersions crystallized and did not produce submicron particle dispersions upon addition to water, nor high supersaturation values. Conclusions  Salt flocculation produces large yields of high surface area amorphous nanoparticle powders that de-aggregate and dissolve rapidly upon redispersion in pH 6.8 media, for supersaturation levels up to 14.     

Steam sterilisation of vesicular phospholipid gels

Vesicular phospholipid gels (VPGs), highly concentrated phospholipid dispersions of semisolid consistency and vesicular morphology are under investigation as potential implantable depots for sustained release of drugs and as intermediates for subsequent dilution into 'conventional' liposome dispersions. It was investigated here if VPGs can be steam sterilised. VPGs prepared from 400 mg/g egg-phosphatidylcholine by high-pressure homogenisation retained their vesicular structure but showed a slight increase in vesicle size (freeze-fracture electron microscopy). However, autoclaving slowed down both, the in vitro release of the hydrophilic marker carboxyfluorescein and vesicles from VPGs. This was assumed to be due to bigger vesicle sizes and corresponding increase in packing density of the vesicular matrix. Upon dilution into a liposome dispersion both negative staining electron microscopy and dynamic laser light scattering analysis confirmed a distinct increase in liposome size, mainly due to fusion of small (20 nm) vesicles with unfavourable curvature. This was consistent with the observed increase in encapsulation efficiency of carboxyfluorescein. Phospholipid hydrolysis during autoclaving was negligible with lysophosphatidylcholine formation of less than 2% (thin layer chromatography). Despite significant change of their morphological and functional properties during autoclaving VPGs retained their main characteristics, such as vesicular structure, sustained release and dilutability to liposome dispersions, and are, therefore, considered as autoclavable.     

Impaired clearance, elimination, and metabolism of plasma cholesterol esters associated with hypercholesteremia in mice fed cyclopropenoid fatty acids

Swiss-Webster mice were fed corn oil control diet or 0.7% cyclopropenoid fatty acid (CPFA) for 8 weeks and dosed iv with an equimolar suspension of [3H]cholesteryl palmitate and [14C]cholesteryl palmitoleate. Blood decline of labeled sterol was biphasic. There were no differences in vivo plasma cholesterol ester metabolism, elimination kinetics, or fecal elimination rate for labeled sterol from [3H]cholesteryl palmitate or [14C]cholesteryl palmitoleate within CPFA or control groups. However, compared to controls, CPFA animals diverted significantly more labeled sterol into saturated and diunsaturated cholesterol esters, less into mono- and tetraunsaturated esters, and showed decreased blood clearance and fecal elimination of labeled sterol. Biliary elimination was probably not impaired by depressed hepatic cholesterol esterase activity in CPFA-fed mice. The fundamental effect of CPFA on serum cholesterol concentration appears to reside in a severely imbalanced cholesterol ester profile. Results indicate that CPFA alter normal fatty acid profile of serum cholesterol esters by proportionally altering the C-2 fatty acyl composition of serum phospholipid, which is the substrate for lecithin:cholesterol acyltransferase, the major source of plasma cholesterol esters.     

莪术油固体脂质纳米粒的制备

目的研究影响莪术油固体脂质纳米粒制备的主要因素。方法采用高压均质法制备莪术油固体脂质纳米粒混悬液,以单因素考察和正交设计法筛选出比较理想的处方和工艺,并考察其形态、粒径、载药量及包封率。结果所制得的固体脂质纳米粒为圆整的类球形实体粒子,表面光滑,平均粒径为80.3nm,载药量为11.82%,包封率为81.75%。结论高压均质法可用于莪术油类液体药物固体脂质纳米粒的制备。     

含胆盐脂质体体外稳定性影响因素的研究

以钙黄绿素为小分子水溶性荧光探针,采用薄膜分散法制备了平均粒径为150～200 nm的胆盐脂质体(含甘氨胆酸钠,SGC-Lip)和普通脂质体(含胆固醇,CH-Lip)。通过比较两种脂质体在生理范围内的pH、渗透压、胆盐溶液环境中药物的泄漏率,粒径及多分散系数(PDI)的变化来考察胆盐脂质体的稳定性。结果表明,pH和胆盐浓度是影响脂质体稳定性的主要因素,渗透压对脂质体的稳定性影响较小。SGC-Lip在pH 1.2、2.0及低浓度牛黄胆酸钠(<5 mmol/L)溶液中比CH-Lip更稳定。SGC-Lip在不同溶液中粒径和PDI无显著改变,提示胆盐对脂质膜的作用可能是通过对膜的溶蚀形成孔道,而不是使脂质体完全破裂。     

Rapid,Room Temperature Nanoparticle Drying and Low-Energy Reconstitution via Electrospinning

Nanoparticle formulations offer advantages over free drugs; however, stability of the nanoparticle dispersions is a significant obstacle, and drying is often required for long-term size stability. The main limitation of current drying methods is particle aggregation upon reconstitution which can be overcome with sonication (impractical in a clinical setting) or large amounts of cryoprotectants (result in hypertonic dispersions). Therefore, new approaches to nanoparticle drying are necessary. We demonstrate conversion of nanoparticle dispersions to a dry, thermostable form via electrospinning. As a proof-of-concept, polyethylene glycol stabilized nanoparticles and polyvinyl alcohol were blended and electrospun into ～300 nm fibers. Following electrospinning, nanoparticles were stored for at least 7 months and redispersed with low osmolarity to their original size without sonication. The nanoparticles redisperse to their original size when the fiber diameter and nanoparticle diameter are comparable (nanoparticle:nanofiber ratio ～1). Nanoparticles with liquid cores and larger particles better maintained their size when compared to nanoparticles with solid cores and smaller particles, respectively. Storing the nanoparticles within nanofibers appears to prevent Ostwald ripening improving thermostability. Overall, this novel approach enables rapid, continuous drying of nanoparticles at room temperature to facilitate long-term nanoparticle storage. Improved nanoparticle drying techniques will enhance clinical translation of nanomedicines.     

The influence of the incorporation of cholesterol and water on the particle size, bilayer thickness, melting behavior, and relative sucrose ester composition of reversed vesicles

The influence of the incorporation of cholesterol and water on the particle size, bilayer thickness, melting behavior, and relative sucrose ester composition of reversed vesicles was studied. Reversed vesicles (RVs) were prepared of sucrose ester in silicon oil by sonication. The RVs were characterized by polarized light microscopy, laser diffraction, high-performance liquid chromatography, small angle X-ray scattering (SAXS), and differential scanning calorimetry. The particle size distributions of the studied dispersions were bimodal with peaks at 5 and 0.4 microm. There was no significant difference in the sucrose ester composition of these two size categories of RVs. The incorporation of cholesterol and water had no effect on the size distribution of the RVs. The SAXS results showed that the RVs prepared without cholesterol and water consisted of bilayers with fully interdigitated alkyl chains. The incorporation of high concentrations of cholesterol caused a phase separation within the bilayers. The incorporation of water also resulted in a phase separation within the bilayers but at a lower cholesterol concentration. The presence of two different size classes of RVs in one RVs dispersion and the phase separation within the bilayers of certain compositions can have consequences for the application of RVs.     

Investigations on 5-fluorouracil solid lipid nanoparticles (SLN) prepared by hot homogenization

The purpose of this study was to investigate the effect of different formulation factors on the properties of solid lipid nanoparticles (SLN) prepared by a hot homogenization method. Using the particle size, physical stability constant (K(e)) and zeta-potential as standards, the stability of SLNs was investigated as a function of phospholipid and poloxamer contents. It was demonstrated that the content of phospholipid had a significant influence on the zeta-potential, which increased considerably with increasing phospholipid content. However, the particle size increased remarkably when the phospholipid content was as high as 1.5% due to the increased viscosity. Poloxamer 188 exhibited no remarkable influence on particle size when the concentration was as low as 1.0%. The influence of the phospholipid and poloxamer content on the embedding ratios of drug substances was further studied using 5-fluorouracil (5-Fu) as a model drug. It was shown that the embedding ratio increased considerably with phospholipid content and independent of poloxamer content, implying that 5-Fu was incorporated into the phospholipid bilayer membrane.     

Role of SR-BI in regulating lipoprotein structure and metabolism

The scavenger receptor class B type I (SR-BI) is a cell surface lipoprotein receptor that mediates physiologically relevant selective cholesteryl ester uptake from high-density lipoprotein (HDL). SR-BI appears to be required for the maintenance of reverse cholesterol transport, normal HDL levels and HDL structure by means of hepatic-selective HDL-cholesterol and phospholipid uptake in mice. SR-BI can also promote the selective uptake of cholesteryl ester from apoB-containing lipoproteins; however, unlike the effect of SR-BI on HDL-cholesterol, the effect on low-density lipoprotein is limited in vivo. Furthermore, SR-BI plays a protective role against atherosclerotic development. A pharmacological agent that increased reverse cholesterol transport by targeting SR-BI could become a powerful tool for prevention of or therapeutic intervention in atherosclerotic cardiovascular diseases.     

Semisolid SLN dispersions for topical application: influence of formulation and production parameters on viscoelastic properties.

Aqueous solid lipid nanoparticle (SLN) dispersions with a high lipid content up to 35% and viscous to semisolid consistency were produced by a high pressure homogenization process. Despite their high lipid content and viscosity these dispersions preserved their colloidal size range. The SLN dispersions were compared to nanoemulsions and microparticle dispersions with regard to particle size, viscoelastic properties and formation of a semisolid gel structure. Viscoelastic measurements including oscillation stress sweep tests and oscillation frequency sweep tests demonstrated that the existence of a solid particle matrix with a particle size in the nanometer range is a prerequisite to form a semisolid dispersion having the appropriate consistency for topical application. Striking differences were observed between solid lipid micro- and nanodispersions of the same composition. Particle size reduction resulted in an 80-fold increase of the elastic modulus. Particle size distribution, the physical state of the dispersed lipid phase and the emulsifier concentration have been identified as further key factors for the viscoelastic properties and gel structure of the lipid nanodispersions. By conducting oscillation measurements it was possible to relate the stability of lipid dispersions to specific rheological parameters therefore providing a sensitive tool in stability assessment. Changing the production process from a 40 ml batch to a 2 l batch turned out to have an influence on the colloidal structures of semisolid SLN dispersions. Consistency increased but particle size and ratio of elastic to viscous properties stayed in the same range.     

Ezetimibe selectively inhibits intestinal cholesterol absorption in rodents in the presence and absence of exocrine pancreatic function

1. Ezetimibe potently inhibits the transport of cholesterol across the intestinal wall, thereby reducing plasma cholesterol in preclinical animal models of hypercholesterolemia. The effect of ezetimibe on known absorptive processes was determined in the present studies. 2. Experiments were conducted in the hamster and/or rat to determine whether ezetimibe would affect the absorption of molecules other than free cholesterol, namely cholesteryl ester, triglyceride, ethinylestradiol, progesterone, vitamins A and D, and taurocholic acid. In addition, to determine whether exocrine pancreatic function is involved in the mechanism of action of ezetimibe, a biliary anastomosis model, which eliminates exocrine pancreatic function from the intestine while maintaining bile flow, was established in the rat. 3. Ezetimibe reduced plasma cholesterol and hepatic cholesterol accumulation in cholesterol-fed hamsters with an ED(50) of 0.04 mg kg(-1). Utilizing cholesteryl esters labelled on either the cholesterol or the fatty acid moiety, we demonstrated that ezetimibe did not affect cholesteryl ester hydrolysis and the absorption of fatty acid thus generated in both hamsters and rats. The free cholesterol from this hydrolysis, however, was not absorbed (92 - 96% inhibition) in the presence of ezetimibe. Eliminating pancreatic function in rats abolished hydrolysis of cholesteryl esters, but did not affect the ability of ezetimibe to block absorption of free cholesterol (-94%). Ezetimibe did not affect the absorption of triglyceride, ethinylestradiol, progesterone, vitamins A and D, and taurocholic acid in rats. 4. Ezetimibe is a potent inhibitor of intestinal free cholesterol absorption that does not require exocrine pancreatic function for activity. Ezetimibe does not affect the absorption of triglyceride as a pancreatic lipase inhibitor (Orlistat) would, nor does it affect the absorption of vitamin A, D or taurocholate, as a bile acid sequestrant (cholestyramine) would.