ABSTRACT
Antifibrinolytic site-specific pharmaco-laser therapy (SSPLT) is an experimental treatment modality for refractory port wine stains (PWS). Conceptually, antifibrinolytic drugs encapsulated in thermosensitive liposomes are delivered to thrombi that form in semi-photocoagulated PWS blood vessels after conventional laser treatment. Local release of antifibrinolytics is induced by mild hyperthermia, resulting in hyperthrombosis and complete occlusion of the target blood vessel (clinical endpoint). In this study, 20 thermosensitive liposomal formulations containing tranexamic acid (TA) were assayed for physicochemical properties, TA:lipid ratio, encapsulation efficiency, and endovesicular TA concentration. Two candidate formulations (DPPC:DSPE-PEG, DPPC:MPPC:DSPE-PEG) were selected based on optimal properties and analyzed for heat-induced TA release at body temperature (T), phase transition temperature (Tm), and at T > Tm. The effect of plasma on liposomal stability at 37 °C was determined, and the association of liposomes with platelets was examined by flow cytometry. The accumulation of PEGylated phosphocholine liposomes in laser-induced thrombi was investigated in a hamster dorsal skinfold model and intravital fluorescence microscopy. Both formulations did not release TA at 37 °C. Near-complete TA release was achieved at Tm within 2.0-2.5 min of heating, which was accelerated at T > Tm. Plasma exerted a stabilizing effect on both formulations. Liposomes showed mild association with platelets. Despite positive in vitro results, fluorescently labeled liposomes did not sufficiently accumulate in laser-induced thrombi in hamsters to warrant their use in antifibrinolytic SSPLT, which can be solved by coupling thrombus-targeting ligands to the liposomes.
ABSTRACT
The aim of this work was to create human liver microtissue spheroids metabolically active by using a hollow fiber membrane bioreactor whose design and structural features ensure a uniform microenvironment and adequate oxygenation. Human hepatocyte spheroids with uniform size and shape were formed through self-assembling and cultured into the bioreactor. Adjacent spheroids fused, giving rise to larger microstructures around the fibers forming liver-like tissue, which retained functional features in terms of urea synthesis, albumin production, and diazepam biotransformation up to 25days. The overall data strongly corroborates that within the bioreactor a proper oxygenation and supply of nutrients were provided to the cells ensuring a physiological amount even in the spheroids core. The oxygen uptake rate and the mathematical modelling of the mass transfer directly elucidated that liver microtissue spheroids are not exposed to any oxygen mass transfer limitation. The minimum oxygen concentration reached at the center of multiple spheroids with diameter of 200µm is significantly higher than the one of the perivenous zone in vivo, while for larger microtissues (400µm diameter) the oxygen concentration drops to values that are equal to the maximum concentration found in the liver periportal zone. Both experimental and modelling investigations led to the achievement of significant results in terms of liver cell performance. Indeed, the creation of a permissive microenvironment inside the bioreactor supported the formation and long-term maintenance of functional human liver microtissues.
