Interaction of liposomes with bile salts investigated by asymmetric flow field-flow fractionation (AF4): A novel approach for stability assessment of oral drug carriers.

For oral drug delivery the stability of liposomes against intestinal bile salts is of key importance. Here, asymmetric flow field-flow fractionation (AF4) coupled to multi-angle laser light scattering (MALLS) and a differential refractive index (dRI) detector was employed to monitor structural re-arrangement of liposomes upon exposure to the model bile salt taurocholate. For comparison, a conventional stability assay was employed using a hydrophilic marker and size exclusion chromatography (SEC) to separate released from liposome-entrapped dye. Calcein-containing liposomes with and without cholesterol were compared in terms of their in vitro stability upon exposure to bile salts by separating liposomes from co-existing colloidal species emerging after stress test using AF4/MALLS/dRI. Dynamic light scattering (DLS) was utilized in parallel. Our AF4/MALLS/dRI results suggested that exposure of egg-phospholipid liposomes to bile salts at physiological concentrations led to the formation of two new species of colloidal associates, likely (mixed) micelles. Subjecting cholesterol-containing liposomes to the same bile media did not lead to any new colloidal structures, indicating increased stability of these liposomes. Our SEC-based release assay largely confirmed these findings, indicating that AF4/MALLS/dRI is a suitable technique for prediction of in vitro oral stability of liposomal formulations. Moreover, the powerful AF4/MALLS/dRI technique appears promising to improve the understanding of the underlying mechanisms during bile salt-induced liposomal breakdown.

(Sub)micron particles forming in aqueous dispersions of amorphous solid dispersions of the poorly soluble drug ABT-199: A combined particle optical counting and field-flow fractionation study.

The dispersive behavior of three different amorphous solid dispersion (ASD) formulations of the poorly soluble ABT-199 (Venetoclax) were studied in aqueous and biomimetic media and spontaneously forming supramolecular associates and particles analysed. To this end, the aqueous dispersions were fractionated into a submicron (colloidal) and micrometer-sized particle-fraction by bench-top centrifugation. The submicron fraction was characterized by Asymmetric Flow Field-Flow Fractionation in conjunction with Multi-angle Laser Light Scattering (AF4-MALLS), Dynamic Light Scattering (DLS) and zeta potential analysis. The micron particle fraction was characterized by Single Particle Optical Sensing (SPOS) and light microscopy. Furthermore, drug contents were monitored in terms of total dispersed drug and apparently dissolved drug in the submicron fraction. Despite the fact, that all three formulations showed decent dispersive behavior with almost the complete drug content rapidly dispersed, substantial differences were identified between two of the formulations and the third one: ABT-199/12 and ABT-199/20 showed pronounced precipitation of the drug in form of micrometer particles, a phenomenon described as glass liquid phase separation (GLPS) and only a marginal fraction of the drug was found in the submicron-fraction, i.e. associated with 3 to 4 different supramolecular assemblies (micelles), irrespective whether buffer or fasted state simulated intestinal fluid (FaSSIF) were used as dispersion media. In contrast, ABT-199/40 showed pronounced formation of a wide variety of supramolecular assemblies (micelles) along with substantial association of the drug with all of these, but reduced glass liquid phase separation.

Co-existing colloidal phases of human duodenal aspirates: Intraindividual fluctuations and interindividual variability in relation to molecular composition.

We investigated the ultrastructural pattern of colloidal phases in human duodenal fluids. Aspirates were collected from three volunteers in both fasted and fed nutritional states. Analysis methods comprised the combination of asymmetric flow field-flow fractionation (AF4) and multi-angle laser light scattering (MALLS). Furthermore, dynamic light scattering (DLS) and diffusion-ordered NMR spectroscopy (DOSY-NMR) were employed as alternative analytical approaches for comparison. By AF4/MALLS, up to four, and in some cases up to five distinct co-existing fractions could be differentiated in the sub-micron size-range, which, in accordance with a previous study (Elvang et al., 2018), may be assigned to three main types, namely small bile salt micelles, intermediate size mixed bile salt/phospholipid micelles and large phospholipid aggregates / vesicles. Although more or less the same colloidal phases were found to co-exist in all aspirates, their prevalence was found to vary, both over time and between the three individual human volunteers. Any uniform changes of patterns of colloidal phases over time, however, could not be identified. On the other hand, prevalence of specific colloidal phases was identified for aspirates of individual volunteers, which correlated reasonably well with the prevalence of certain lipid species in their molecular composition. It remains to be investigated whether such prevalence of specific colloidal phases influences drug solubilizing capacity as well as drug absorption. If so, this may help to better understand the substantial inter-individual variability seen in many drug absorption profiles.