Unterkühlt-Smektische Lipid-Nanopartikel: Entwicklung und Charakterisierung eines neuartigen Trägersystems für schwer wasserlösliche Arzneistoffe

Kolloidale Lipid-Emulsionen und Dispersionen fester Lipid-Nanopartikel, die als Trägersysteme für schwer wasserlösliche Arzneistoffe untersucht werden, erscheinen insbesondere im Hinblick auf ihre physiologische Verträglichkeit gegenüber anderen kolloidalen Trägersystemen wie beispielsweise Polymer-Nanopartikeln von Vorteil. Kolloidale Fettemulsionen werden seit langem für die parenterale Ernährung eingesetzt und sind auch geeignet für die Solubilisierung lipophiler Arzneistoffe [Prankerd und Stella 1990, Floyd und Jain 1996, Klang und Benita 1998]. Während der flüssige Zustand der Lipidmatrix einerseits eine hohe Aufnahmekapazität für Arzneistoffe aufweist, ist er jedoch mit einigen Nachteilen wie beispielsweise der hohen Mobilität inkorporierter Arzneistoffmoleküle und einem möglichen Partikelgrößenwachstum aufgrund von Koaleszenz der Emulsionspartikel verbunden. Um eine langsamere Freisetzung von inkorporierten Arzneistoffen sowie auch eine höhere Stabilität der Nanopartikel zu erzielen, dabei aber auch die Vorteile der kolloidalen Fettemulsionen insbesondere im Hinblick auf die physiologische Verträglichkeit zu bewahren, wurden Anfang der 90er Jahre Lipid-Nanopartikel mit einer festen Matrix eingeführt [Westesen 2000, Müller et al. 2000, Mehnert und Mäder 2001]. Aufgrund der meist hochgeordneten, kristallinen Matrix ist die Aufnahmekapazität für Arzneistoffe in diese Nanopartikel jedoch begrenzt. Um die Aufnahmekapazität von Arzneistoffen in feste Lipid-Nanopartikel zu erhöhen, wurden Lipid-Nanopartikel aus Gemischen von festen und flüssigen Lipiden entwickelt, wobei angenommen wird, dass die flüssigen Lipide als "Nano-Kompartimente" in der festen Lipidmatrix eingebettet vorliegen [Müller et al. 2002]. Untersuchungen zur Ultrastruktur von Compritol-Nanopartikeln, denen unterschiedliche Mengen mittelkettiger Trigylceride (MCT zugemischt wurden, haben allerdings gezeigt, dass es zumindest bei höheren Konzentrationen von MCT zu einem Austritt der flüssigen Lipide aus der kristallinen Matrix kommt [Jores et al. 2003, 2004]. Eine weitere, viel versprechende Alternative bietet der flüssigkristalline Zustand der Lipidmatrix. Der Ordnungsgrad der flüssigkristallinen Struktur bedingt eine vergleichsweise hohe Viskosität und damit eine eingeschränkte Beweglichkeit eingeschlossener Arzneistoffmoleküle. Andererseits sollte durch die Fluidität auf molekularer Ebene die Aufnahmekapazität für Fremdmoleküle im Vergleich zum hoch geordneten kristallinen Zustand höher sein. Ziel der vorliegenden Arbeit war die Entwicklung und Charakterisierung eines wässrigen kolloidalen Arzneistoffträgersystems, dessen Lipidpartikel in einem thermotrop flüssigkristallinen (smektischen) Zustand vorliegen und das insbesondere auch für eine parenterale Applikation geeignet sein sollte. Für die Herstellung der Nanopartikel wurden Cholesterolester verwendet

Molecular Networks and Macromolecular Molar Mass Distributions for Preliminary Characterization of Danish Craft Beers

Beer is one of the most widely consumed beverages containing up to 200,000 unique small molecules and a largely uncharacterized macromolecular and particulate space. The chemical profiling of beer is difficult due to its complex nature. To address this issue, we have used various state-of-the-art methods to determine the physicochemical characteristics of beer. Specifically, we have successfully generated an LC-MS-based molecular network with minimal sample preparation to profile indoles in beer and confirmed their presence using 1H-NMR. In addition, we have identified different macromolecular signatures in beer of different colors by utilizing AF4-MALS. These preliminary findings lay the foundation for further research on the physicochemical nature of beer

Feasibility of the preparation of cochleate suspensions from naturally derived phosphatidylserines

IntroductionCochleates are cylindrical particles composed of dehydrated phospholipid bilayers. They are typically prepared by addition of calcium ions to vesicles composed of negatively charged phospholipids such as phosphatidylserines (PS). Due to their high physical and chemical stability, they provide an interesting alternative over other lipid-based drug formulations for example to improve oral bioavailability or to obtain a parenteral sustained-release formulation.MethodsIn the present study, the feasibility to prepare cochleate suspensions from soy lecithin-derived phosphatidylserines (SPS) was investigated and compared to the “gold standard” dioleoyl-phosphatidylserine (DOPS) cochleates. The SPS lipids covered a large range of purities between 53 and >96% and computer-controlled mixing was evaluated for the preparation of the cochleate suspensions. Electron microscopic investigations were combined with small-angle x-ray diffraction (SAXD) and Laurdan generalized polarization (GP) analysis to characterize particle structure and lipid organization.ResultsDespite some differences in particle morphology, cochleate suspensions with similar internal lipid structure as DOPS cochleates could be prepared from SPS with high headgroup purity (≥96%). Suspensions prepared from SPS with lower purity still revealed a remarkably high degree of lipid dehydration and well-organized lamellar structure. However, the particle shape was less defined, and the typical cochleate cylinders could only be detected in suspensions prepared with higher amount of calcium ions. Finally, the study proves the feasibility to prepare suspensions of cochleates or cochleate-like particles directly from a calcium salt of soy-PS by dialysis

Bone Morphogenetic Protein 2 (BMP-2) Aggregates Can be Solubilized by Albumin-Investigation of BMP-2 Aggregation by Light Scattering and Electrophoresis

Bone morphogenetic protein 2 (BMP-2) has a high tendency to aggregate at physiological pH and physiological ionic strength, which can complicate the development of growth factor delivery systems. The aggregation behavior in differently concentrated BMP-2 solutions was investigated using dynamic and static light scattering. It was found that at higher concentrations larger aggregates are formed, whose size decreases again with increasing dilution. A solubilizing effect and therefore less aggregation was observed upon the addition of albumin. Imaged capillary isoelectric focusing and the simulation of the surface charges of BMP-2 were used to find a possible explanation for the unusually low solubility of BMP-2 at physiological pH. In addition to hydrophobic interactions, attractive electrostatic interactions might be decisive in the aggregation of BMP-2 due to the particular distribution of surface charges. These results help to better understand the solubility behavior of BMP-2 and thus support future pharmaceutical research and the development of new strategies for the augmentation of bone healing

Interaction of dispersed cubic phases with blood components

The interaction of aqueous nanoparticle dispersions, e.g. based on monoolein/poloxamer 407, with blood components is an important topic concerning especially the parenteral way of administration. Therefore, the influence of human and porcine plasma on dispersed cubic phases was investigated. Particle size measurements of mixtures with plasma indicated a decrease in particle size. In cryo-transmission electron micrographs, different structures could be found, which arose from the dispersed cubic phases under plasma contact. Non-cubic structures on the particle surface were decomposed first. Several phase transitions with the formation of smaller and sometimes larger particle fractions were observed beside remaining cubic structures. A very low but detectable hemolytic activity was found for the dispersed cubic phases based on monoolein and poloxamer 407, when compared to the hemolytic activity of cubic phases based on monoolein and poloxamer 188, on soy phosphatidylcholine, glycerol dioleate and polysorbate 80 or the parenteral fat emulsion Lipofundin MCT 20%

Comparative SAXS and DSC study on stratum corneum structural organization in an epidermal cell culture model (ROC):Impact of cultivation time

Cell cultured skin equivalents present an alternative for dermatological in vitro evaluations of drugs and excipients as they provide the advantage of availability, lower variability and higher assay robustness compared to native skin. For penetration/permeation studies, an adequate stratum corneum barrier similar to that of human stratum corneum is, however, a prerequisite. In this study, the stratum corneum lipid organization in an epidermal cell culture model based on rat epidermal keratinocytes (REK organotypic culture, ROC) was investigated by small-angle X-ray scattering (SAXS) in dependence on ROC cultivation time and in comparison to native human and rat stratum cornea. In addition, the thermal phase behavior was studied by differential scanning calorimetry (DSC) and barrier properties were checked by measurements of the permeability of tritiated water. The development of the barrier of ROC SC obtained at different cultivation times (7, 14 and 21 days at the air–liquid interface) was connected with an increase in structural order of the SC lipids in SAXS measurements: Already cultivation for 14 days at the air–liquid interface resulted overall in a competent SC permeability barrier and SC lipid organization. Cultivation for 21 days resulted in further minor changes in the structural organization of ROC SC. The SAXS patterns of ROC SC had overall large similarities with that of human SC and point to the presence of a long periodicity phase with a repeat distance of about 122 Å, e.g. slightly smaller than that determined for human SC in the present study (127 Å). Moreover, SAXS results also indicate the presence of covalently bound ceramides, which are crucial for a proper SC barrier, although the corresponding thermal transitions were not clearly detectable by DSC.Due to the competent SC barrier properties and high structural and organizational similarity to that of native human SC, ROC presents a promising alternative for in vitro studies, particularly as it can be obtained under overall rather straightforward cell culture conditions and thus low assay costs

Phosphatidylinositol Stabilizes Fluid-Phase Liposomes Loaded with a Melphalan Lipophilic Prodrug

Previously, a liposomal formulation of a chemotherapeutic agent melphalan (Mlph) incorporated in a fluid lipid bilayer of natural phospholipids in the form of dioleoylglyceride ester (MlphDG) was developed and the antitumor effect was confirmed in mouse models. The formulation composed of egg phosphatidylcholine (ePC), soybean phosphatidylinositol (PI), and MlphDG (8:1:1, by mol) showed stability in human serum for at least 4–5 h. On the contrary, replacing PI with pegylation of the liposomes, promoted fast dissociation of the components from the bilayer. In this work, interactions of MlphDG-liposomes with the most abundant plasma protein—albumin—in function of the presence of PI in the formulation were explored using Fourier transform infrared spectroscopy. The release of MlphDG from the liposomes was studied by asymmetrical flow field-flow fractionation (AF4) using micelles formed by a polyethylene glycol conjugate with phosphatidylethanolamine to mimic the physiological lipid sink like lipoproteins. Our results show that PI actually protects the membrane of MlphDG-liposomes from the protein penetration, presumably due to pairing between the positively charged MlphDG and negatively charged PI, which compensates for the heterogeneity of the lipid bilayer. The AF4 technique also evidences high stability of the formulation as a drug carrier