Multifunctional liposomes: Microscale formulation, modification and in vitro interaction
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Abstract
Liposomes have been in clinical application for over two decades and are the arguably most important class of nanoparticulate therapeutics. However, the investigation of functionalized liposome systems is yet limited by synthetic access as well as by material and time consumption. Dual centrifugation (DC) has previously been established as a valuable method for the rapid formulation of liposomes in small batch sizes. However, standard laboratory-scale syntheses of novel polymeric amphiphiles typically only allow for a relatively limited number of preparations. Here, DC formulation was scaled down to the single-digit milligram scale of total lipids, while beneficial properties such as high encapsulation efficiencies (EE) were preserved. This enabled the detailed investigation of the formulation parameter space of liposomes that contained rare prototype compounds.
Efficient surface derivatization routes are key requirements for the use of valuable targeting ligands. After having established CuAAC chemistry for surface modification of liposomes recently, this tool was used to investigate the in vitro interaction of a new class of polyglycerol amphiphiles. Live cell imaging revealed a rapid desorption of cholesterol-based amphiphiles from the liposome surface. While the sterical stabilization of these vesicles was intended to prevent unspecific interaction in biological systems, the observed instability rendered the use of such amphiphiles questionable. The permutation of the amphiphile structures, membrane anchors and reactive groups, and the application of orthogonal derivatization via CuAAC and IEDDA revealed that solely the cholesterol anchorage caused the mentioned desorption effects. Consistently, less effective active targeting of folic acid-containing cholesterol-amphiphiles was found when compared to dialkyl-substituted anchors. However, these more stable membrane anchors were also found to desorb after binding to cellular folate receptors, which suggested fundamental implications for the rational design of liposome membrane anchors.
While sterical stabilization provides “stealth” properties to liposomes to overcome rapid clearance from the blood stream in vivo, it typically also decreases interaction with and delivery to cells in the target tissue. As a potential solution, a novel class of acid-cleavable amphiphiles that contained acetal or ketal groups was investigated for stabilization of liposomes. Half lifes at lysosomal-like pH of 5.4 were found to be 20 h and 15 min, respectively, and therefore illustrated the suitability of these systems for the controlled shedding of liposomes.
Enabled by the substantial decrease of material consumption, polymersomes and polymeric micelles have been prepared via micro scale DC formulation in a proof-of-concept study, while characteristics of DC formulations like narrow size distributions and high EE were maintained. In contrast to conventional formulation methods, such as solvent switching, substantial benefits regarding time consumption and preparation yields rendered micro scale DC formulation a highly valuable tool for such systems.