Fusogenic Liposome-Assisted Drug Delivery Across Physiological Barriers

Abstract

Fusogenic liposomes (FL) are biocompatible, cationic nano-carriers, consisting of neutral and positively charged lipids, as well as an aromatic compound. They were previously used for the delivery of nucleic acids, proteins and functionalized lipids in vitro, highlighting their potential for drug delivery. Drugs and their delivery vehicles encounter a variety of interaction partners after administration. This includes components of the bloodstream and the vascular wall, as well as extracellular matrix (ECM) during tissue accumulation. Thus, an understanding of those interactions is highly relevant for FL-mediated drug delivery. In the present work, these interactions were investigated partially in vitro and in vivo, using epithelial cell lines and primary endothelial cells, as well as a mouse model of vascular aging.<br /> FL enable the cell delivery of cargo by membrane fusion compared to commonly used liposomes using endocytosis. FL are thus permitting a direct release of cargo to the cytosol. The benefits of membrane fusion were investigated based on cargo localization inside the FL formulation and cargo basicity using confocal laser-scanning microscopy. Here, membrane fusion enabled passive uptake of membrane-impermeable cargo, although the latter was not entrapped in FL, presumably by induction of transient membrane pores. Additionally, anti-proliferative effects of anti-cancer therapeutics could be improved by direct release into the cytosol, if cargo was sensitive for lysosomal degradation due to higher basicity. This highlights the benefit of FL delivery for drugs with intracellular targets.<br /> Basement membrane (BM) lesions and ECM remodeling are crucial markers for breast cancer progression. Nano-carriers that can adapt to the dynamically changing tumor matrix could therefore improve delivery of anti-cancer therapeutics. Interactions of FL with the ECM were thus examined in gels of ECM-relevant proteins. Furthermore, FL delivery was investigated in the MCF 10A acini model, mimicking breast acini with an adjustable BM, and spheroids of the highly invasive breast cancer cell line MDA-MB-231. Investigation of tracer dye uptake in the MCF 10A acini model using confocal laser-scanning microscopy and flow cytometry indicated a size-restrictive delivery of tracer dye. Furthermore, FL-mediated delivery of anti-cancer drug doxorubicin could improve anti-proliferative effects in comparison to commonly used liposomes if BM was lesioned or less matured. However, the passage of FL through ECM was vastly affected by opposed charges of FL and ECM, and protein secretion of the MDA-MB-231 spheroid model further reduced FL uptake. While this work demonstrated increasing drug performance by FL delivery if BM is less matured, the formulation is still in need of further optimization to effectively traverse tumor matrix.<br /> The ability to leave systemic circulation is highly relevant for tissue accumulation. It is critically affected by protein interactions and vascular permeability, which is vastly characterized by the endothelial cell barrier. In the present work, a model of the vascular endothelial barrier consisted of a primary human umbilical vein endothelial cell layer with two compartments to measure the passage of FL using fluorescence spectroscopy. Here, FL passage was increased if endothelial hyperpermeability was generated. Additionally, FL mediated the tracer dye delivery to endothelial cell layers, though membrane fusion was retarded by protein-liposome interactions. Furthermore, the use of FL for endothelial delivery was evaluated. Pharmacologically relevant effects of the antioxidant resveratrol were investigated in vitro using fluorescence spectroscopy and plate reader measurements, as well as in vivo using laser speckle contrast imaging. Resveratrol delivery reduced an endothelial hyperpermeability in vitro, most likely due to the observed reduction of reactive oxygen species activity. In vivo, the adaptability of the vascular tone to neuronal stimulation could be rescued by FL-mediated resveratrol delivery in a mouse model of vascular aging. In addition, biodistribution of FL was evaluated. The vascular delivery of liposomal tracer dye and cerebral circulation of FL were assessed by intravital imaging in a mouse model. Tracer dye was localized in the cerebral vasculature 1 h after injection of FL, with a decay after 24 h. Plate reader measurements of tracer dye fluorescence in dissected organs also demonstrated an accumulation of FL in the liver. Accordingly, FL clearance from the circulation within 24 h was postulated, presumably by hepatic elimination.<br /> The present work provides a clearer understanding of FL interaction with the physiological barriers ECM and vascular wall. Steric and electrostatic interactions presumably modified FL passage through ECM in vitro and also modulated FL delivery and performance of drug cargo doxorubicin. Hyperpermeability increased FL passage through the endothelial barrier in vitro, indicating an enhanced permeability at target sites with leaky vasculatures. FL also enabled endothelial delivery of antioxidants in vitro and in vivo and are thus not only proposed as drug delivery vehicles for tissue accumulation in the periphery but for drug delivery to the vasculature, e.g., in cerebral endothelial dysfunction-related pathophysiologies.