Investigation on the structure of the stabilizer layer of triglyceride nanocrystals and their mesoscopic structures in aqueous suspensions by small-angle X-ray and neutron scattering

Mit Hilfe von SAXS- und SANS-Messungen und einer verbesserten, um Neutronenstreuung erweiterten Variante der XPPSA-Methode wurde die Struktur der Lecithin-Stabilisatorschicht bei wäßrigen Triglycerid- und Tetracosan-Nanosuspensionen untersucht. Bislang konnten Nanosuspensionen erfolgreich untersucht werden, bei denen die Hauptphasenübergangstemperatur Tm des Lecithins hinreichend weit unterhalb der (durch homogene Nukleation bedingten) Kristallisationstemperatur Tc der jeweiligen Nanoemulsionen liegen. Im Fall der Tripalmitin-Nanodispersionen waren dies Lecithine mit hinreichend kurzen di-gesättigten (DLPC, di-C12:0) und einfach (DOPC, di-C18:1) oder mehrfach (gereinigtes Sojalecithin Lipoid S100) ungesättigten Azylketten. Bei diesen Lecithinen findet wahrscheinlich eine durch eine homogene Nukleation ausgelöste Kristallisation der Nanoemulsionen statt. Die Nanokristalle liegen dann überwiegend als Plättchen in der stabilen und bekannten Beta-Modifikation vor. Für Nanosuspensionen mit diesen Lecithinen stellte sich heraus, daß ein Modell, bei dem sich die Azylketten und Kopfgruppen der Lecithin-Moleküle sehr flach in einer Monolage an der Oberfläche der Nanokristalle anordnen, die SAXS- und SANS-Daten gut simultan reproduzieren kann. Ähnliche Ergebnisse konnten auch für DMPC-stabilisierte Tetracosan-Nanosuspensionen erzielt werden. Im Vergleich zur Nanosuspension nimmt die Dicke der DMPC-Stabilisatorschicht der entsprechenden Tetracosan-Nanoemulsion zu. Deren Struktur ähnelt in Übereinstimmung mit früheren molekulardynamischen Simulationsstudien der eines halben DMPC-Bilayers. Die Azylketten wechselwirken dabei nur geringfügig mit den oberflächennahen Tetracosan-Molekülen. Mangels bislang nicht erhältlicher deuterierter Varianten von DOPC und S100 (im Gegensatz zu DMPC bei den Tetracosannanodispersionen) konnte bei den Triglyceridnanosuspensionen bislang nur wenig Gebrauch von unterschiedlichen Neutronenstreukontrasten gemacht werden. Sobald diese verfügbar sind (DOPC und DLPC), sollten diese in künftigen Untersuchungen genutzt werden. Aus den Untersuchungen zeigte es sich, daß das Vorhandensein der hochaufgelösten Kristallstrukturen aller Modifikationen des Matrixlipids in den Nanokristallen unabdingbar ist. Diese Strukturen sind jedoch nicht immer bekannt (Alpha und unbekannte Triglyceridmodifikation, ungewöhnliche Kristallstruktur der Tetracosannanokristalle). Für die Triglyceridnanosuspensionen mit Lecithinen mit längeren di-gesättigten Azylketten (DMPC, DPPC und DSPC, typ. Tm>>Tc) sind der genaue Ablauf der Kristallisation, sowie die innere Struktur der Nanokristalle und deren Stabilisatorschicht noch unklar. Im Fall der genauer untersuchten Tripalmitin-Nanosuspensionen finden sich signifikante Mengen der unbekannten (bei DMPC) sowie Alpha- (DPPC, DSPC) Triglyceridmodifikation. In Übereinstimmung mit früheren Studien finden sich Hinweise darauf, daß die Kristallisation durch eine grenzflächengesteuerte heterogene Nukleation ausgelöst wird. Vermutlich bauen sich die Lecithin-Azylketten während deren Kokristallisation mit den oberflächennahen Triglyceridmolekülen tiefer in die Triglyceridmatrix ein. Für eine genauere Analyse von deren SAS-Kurven wären entsprechend neue Strukturmodelle für die Stabilisatorschicht nötig. Aber auch eine Bestimmung von hochaufgelösten Kristallstrukturen der Alpha- und unbekannten Triglyceridmodifikationen wäre für zukünftige Untersuchungen notwendig. Der Einfluß des Kostabilisators NaGC auf die Kristallisation und die Struktur der Lecithin-Stabilisatorschicht ist bislang für alle untersuchten Nanodispersionen noch nicht detailliert verstanden. Mit zunehmender Partikelkonzentrationen wird nicht nur die bereits bekannte Stapelbildung der Triglyceridplättchen beobachtet, sondern auch ein isotrop-nematischer Phasenübergang. Dessen kritische Konzentration läßt sich über die Ausbildung von anisotropen 2D-SAS-Streubildern auf mikroskopischer, und durch die Doppelbrechung der Proben unter gekreuzten Polarisatoren (bei etwas höheren Konzentrationen), auf makroskopischer Skala bestimmen. Die kritische Konzentration ist dabei abhängig vom Aspektverhältnis der Plättchen und damit u.a. vom verwendeten Triglycerid und Lecithin, sowie der Ionenstärke im Dispersionsmedium (beeinflußt das effektive Aspektverhältnis). Die nematische Phase besteht dabei aus kleinen Domänen von typ. einigen hundert Nanometern bis wenigen Mikrometern (max. 30 µm). Die kleineren Plättchen-Stapel unterhalb der kritischen Konzentration für den I-N Übergang können als die Vorläufer der Domänen der flüssigkristallinen Phase angesehen werden. Zumindest auf kurzen Zeitskalen (Tagen) ergibt sich keine I-N Phasenseparation, vermutlich aufgrund zu geringer Dichteunterschiede zwischen Wasser und den Lipidnanopartikeln sowie der erhöhten Viskosität der Proben. Triglyceridplättchen aus mit Hilfe des kationischen Kostabilisators DODAB hergestellten Triglyceridnanosuspensionen können stapelförmige Strukturen bilden, bei denen sich anionische DNA-Moleküle partiell zwischen den Plättchen einlagern. Die Wahl des Lecithin-Stabilisators, eines weiteren Kostabilisators (Tween ® 80 oder Poloxamer 188), sowie die Konzentration an DODAB erlauben dabei eine Variation der Plättchendicken und damit der inneren Struktur der Nanokomposite. Eine genauere Analyse der Stabilisatorschicht der Nanokristalle in den nativen Nanosuspensionen sowie in den DNA-Komplexen und eine genaue Lokalisierung der DNA in diesen mit Hilfe der SAS-Daten und der XNPPSA-Methode war aufgrund der komplizierten Kombination von drei Stabilisatoren und einem hohen Anteil der unbekannten Triglyceridmodifikation in den Nanokristallen bislang nicht möglich. Die bislang ungelöste starke Agglomeration der Nanokristalle bzw. der Stapel in den Komplexen (besonders bei hohen DODAB- und DNA-Konzentrationen) ist für mögliche praktische Anwendungen sehr problematisch.By means of SAXS and SANS and an improved version of the XPPSA-method (extended for neutron scattering), the structure of the lecithin stabilizer layer of triglyceride and tetracosan nanocrystals in aqueous suspensions was studied. The investigations were successful only for those nanosuspensions, which are stabilized by lecithins, whose main phase transition temperature Tm is sufficiently below the crystallization (caused by homogeneous nucleation) temperature Tc of the particular nanoemulsion. In case of tripalmitin nanodispersions these are lecithins with sufficiently short di-saturated (DLPC, di-C12:0) or mono- (DOPC, di-C18:1) and poly-unsaturated (purified soy lecithin Lipoid S100) acyl chains. In case of these lecithins, the crystallization is most probably triggered by a homogeneous nucleation in the nanoemulsions. After crystallization, nearly all nanocrystals adopt the platelet-like shape of the stable Beta-modification. The SAXS- and SANS-patterns of nanosuspensions with these lecithins are well reproduced with a model, where the acyl chains and the head groups of the lecithin molecules lie rather flatly in a monolayer at the surface of the nanocrystals. Similar results were obtained for DMPC-stabilized tetracosan nanosuspensions. By contrast with the nanosuspension, the thickness of the DMPC stabilizer layer rises for the corresponding tetracosan nanoemulsion. Their structure resembles, in accordance with previous molecular dynamic simulation studies, the structure of a half DMPC bilayer. Only minor interactions of the acyl chains with the near-surface tetracosan molecules were found. Since there are no deuterated versions of DOPC and S100 (in contrast to DMPC used for the tetracosan nanodispersions), the full potential of neutron scattering could not yet be utilized for the triglyceride nanosuspensions. Once they (DOPC and DLPC) are available, they should be used in future studies. As the investigations revealed, the availability of high-resolution crystal structures of all modifications of the matrix lipid is crucial. However, these structures are not always known (Alpha and unknown triglyceride modification, uncommon crystal structure for the tetracosan nanocrystals). For triglyceride nanosuspensions, stabilized by lecithins with longer di-saturated acyl chains (DMPC, DPPC and DSPC, typ. Tm>>Tc), the exact crystallization process and the inner structure of the nanocrystals and their stabilizer layer remain unclear. In case of the more thoroughly investigated tripalmitin nanosuspensions, significant amounts of the unknown (DMPC) and Alpha- (DPPC, DSPC) triglyceride modification were found. In full agreement with previous studies, evidence for a crystallization, triggered by surface-heterogeneous nucleation, is found. Presumably, there is a deeper integration of the lecithin acyl chains into the triglyceride matrix, upon their co-crystallization with the surface-near triglyceride molecules. For a more detailed analysis of their SAS-patterns, new structural models for the stabilizer layer must be developed. Furthermore, high-resolution crystal structures of the Alpha- and unknown triglyceride modifications must be determined. Finally, for all studied nanodispersions the influence of the co-stabilizer NaGC on the crystallization and the structure of the lecithin stabilizer layer remains to be determined, too. With rising platelet concentration the known stack formation of the triglyceride platelets is followed by an isotropic-nematic phase transition. The corresponding critical concentration is determined on the microscopic level by the onset of an anisotropic intensity distribution in the 2D-SAS-scattering patterns, and, on a macroscopic level when the samples turn birefringent between crossed polarizers (at slightly elevated concentrations). The critical concentration depends on the aspect ratio of the platelets, which is governed by, i.a., the choice of the matrix triglyceride and lecithin, and the ionic strength of the dispersion medium (affects the apparent aspect ratio). The nematic phase consists of rather small domains, ranging typically between several hundred nanometers to a few microns (max. 30 µm) in size. The smaller platelet stacks, found below the critical concentration for the I-N phase transition, can be regarded as the precursors of the domains of the liquid-crystalline phase. At least on short time scales (days) no I-N phase separation was found, most probably because of the very small difference between the gravimetric densities of water and the lipid nanoparticles and the elevated viscosity of the suspensions. Using the cationic co-stabilizer DODAB in the preparation of the triglyceride nanosuspensions, the triglyceride platelets can form stack-like structures, where anionic DNA molecules are partially sandwiched between the platelets. The choice of the lecithin stabilizer and another costabilizer (Tween 80 or Poloxamer 188), and the concentration of DODAB affect the thickness of the platelets and, thus, the inner structure of the nanocomposites. A more detailed analysis of the stabilizer layer of the nanocrystals in the native nanosuspensions and the DNA-complexes, and a precise localization of the DNA within the complexes with the SAS-patterns and the XNPPSA-method was not yet possible, due to the rather complicated combination of three stabilizers and a high fraction of the unknown triglyceride modification in the nanocrystals. The strong agglomeration of the nanocrystals and stacks in the complexes (particularly for high DODAB and DNA concentrations) limits their potential application so far

Formation of liquid crystalline phases in aqueous suspensions of platelet-like tripalmitin nanoparticles

Suspensions of platelet-like shaped tripalmitin nanocrystals stabilized by the pure lecithin DLPC and the lecithin blend S100, respectively, have been studied by small-angle x-ray scattering (SAXS) and optical observation of their birefringence at different tripalmitin (PPP) concentrations φ PPP . It could be demonstrated that the platelets of these potential drug delivery systems start to form a liquid crystalline phase already at pharmaceutically relevant concentrations φ PPP of less than 10 wt. %. The details of this liquid crystalline phase are described here for the first time. As in a previous study [A. Illing et al. , Pharm. Res.21, 592 (2004)] some platelets are found to self-assemble into lamellar stacks above a critical tripalmitin concentration φstPPP of 4 wt. %. In this study another critical concentration φlcPPP≈7 wt. % for DLPC and φlcPPP≈9 wt. % for S100 stabilized dispersions, respectively, has been observed. φlcPPP describes the transition from a phase of randomly oriented stacked lamellae and remaining non-assembled individual platelets to a phase in which the stacks and non-assembled platelets exhibit an overall preferred orientation. A careful analysis of the experimental data indicates that for concentrations above φlcPPP the stacked lamellae start to coalesce to rather small liquid crystalline domains of nematically ordered stacks. These liquid crystalline domains can be individually very differently oriented but possess an overall preferred orientation over macroscopic length scales which becomes successively more expressed when further increasing φ PPP . The lower critical concentration for the formation of liquid crystalline domains of the DLPC-stabilized suspension compared to φlcPPP of the S100-stabilized suspension can be explained by a larger aspect ratio of the corresponding tripalmitin platelets. A geometrical model based on the excluded volumes of individual platelets and stacked lamellae has been developed and successfully applied to reproduce the critical volume fractions for both, the onset of stack formation and the appearance of the liquid crystalline phase

Designing optical elements from isotropic materials by using transformation optics

International audienceBy taking advantage of a conformal mapping technique, we propose designs for various optical elements such as directional antennas, flat lenses, or bends. In contrast to most of the existing design approaches, the elements can be implemented with isotropic materials, thus strongly facilitating their fabrication. We furthermore generalize the concept and show that under certain conditions previously suggested devices consisting of anisotropic materials may be replaced by isotropic ones using an appropriate transformation. The designs are double-checked by full-wave simulations. A comparison with their anisotropic counterparts reveals a similar performance

Mesoscopic Structures of Triglyceride Nanosuspensions Studied by Small-Angle X-ray and Neutron Scattering and Computer Simulations

Aqueous suspensions of platelet-like shaped tripalmitin nanocrystals are studied here at high tripalmitin concentrations (10 wt % tripalmitin) for the first time by a combination of small-angle X-ray and neutron scattering (SAXS and SANS). The suspensions are stabilized by different lecithins, namely, DLPC, DOPC, and the lecithin blend S100. At such high concentrations the platelets start to self-assemble in stacks, which causes interference maxima at low Q-values in the SAXS and SANS patterns, respectively. It is found that the stack-related interference maxima are more pronounced for the suspension stabilized with DOPC and in particular DLPC, compared to suspensions stabilized by S100. By use of the X-ray and neutron powder pattern simulation analysis (XNPPSA), the SAXS and SANS patterns of the native tripalmitin suspensions could only be reproduced simultaneously when assuming the presence of both isolated nanocrystals and stacks of nanocrystals of different size in the simulation model of the dispersions. By a fit of the simulated SAXS and SANS patterns to the experimental data, a distribution of the stack sizes and their volume fractions is determined. The volume fraction of stacklike platelet assemblies is found to rise from 70% for S100-stabilized suspensions to almost 100% for the DLPC-stabilized suspensions. The distribution of the platelet thicknesses could be determined with molecular resolution from a combined analysis of the SAXS and SANS patterns of the corresponding diluted tripalmitin (3 wt %) suspensions. In accordance with microcalorimetric data, it could be concluded that the platelets in the suspensions stabilized with DOPC, and in particular DLPC, are significantly thinner than those stabilized with S100. The DLPC-stabilized suspensions exhibit a significantly narrower platelet thickness distribution compared to DOPC- and S100-stabilized suspensions. The smaller thicknesses for the DLPC- and DOPC-stabilized platelets explain their higher tendency to self-assemble in stacks. The finding that the nanoparticles of the suspension stabilized by the saturated lecithin DLPC crystallize in the stable β-tripalmitin modification with its characteristic platelet-like shape is surprising and can be explained by the fact that the main phase transformation temperature for DLPC is, as for unsaturated lecithins like DOPC and S100, well below the crystallization temperature of the supercooled tripalmitin emulsion droplets

Liquid Crystalline Phase Formation in Suspensions of Solid Trimyristin Nanoparticles

The presence of liquid crystalline phases in suspensions of solid lipid nanoparticles can increase the risk of their gelling upon administration through fine needles. Here we study the formation of liquid crystalline phases in aqueous suspensions of platelet-like shaped solid lipid nanoparticles. A native lecithin-stabilized trimyristin (20 wt %) suspension was investigated at different dilution levels by small-angle X-ray scattering (SAXS) and visual inspection of their birefringence between two crossed polarizers. For trimyristin concentrations φ_MMM < 6 wt %, the dispersed platelets are well separated from each other whereas they start to self-assemble into stacked lamellae for 6 wt % ≤ φ_MMM < 12 wt %. For φ_MMM ≥ 12 wt %, the SAXS patterns become increasingly anisotropic, which is a signature of an evolving formation of a preferred orientation of the platelets on a microscopic scale. Simultaneously, the suspensions become birefringent, which proves the existence of an anisotropic liquid crystalline phase formed in the still low viscous liquid suspensions. Spatially resolved SAXS scans and polarization microscopy indicate rather small domains in the (sub)­micrometer size range in the nematic liquid crystalline phase and the presence of birefringent droplets (tactoids). The observed critical concentrations for the formation of stacks and the liquid crystalline phase are significantly higher as for equivalent suspensions prepared from triglycerides with longer chains. This can be explained with the lower aspect ratio of trimyristin platelets. Special emphasis is put on the isotropic–liquid crystalline phase transition as a function of the ionic strength of the dispersion medium and φ_MMM. Higher salt concentrations allow shifting of the phase transition to higher trimyristin concentrations. This can be attributed to a partial screening of the repulsive forces between the platelets, which allows higher packing densities within the platelet stacks and of remaining isolated platelets