Comparison of Linear and Hyperbranched Polyether Lipids for Liposome Shielding by ¹⁸F-Radiolabeling and Positron Emission Tomography

Abstract

Multifunctional and highly biocompatible polyether structures play a key role in shielding liposomes from degradation in the bloodstream, providing also multiple functional groups for further attachment of targeting moieties. In this work hyperbranched polyglycerol ( hbPG) bearing lipids with long alkyl chain anchor are evaluated with respect to steric stabilization of liposomes. The branched polyether lipids possess a hydrophobic bis(hexadecyl)glycerol membrane anchor for the liposomal membrane. hbPG was chosen as a multifunctional alternative to PEG, enabling the eventual linkage of multiple targeting vectors. Different hbPG lipids ( M = 2900 and 5200 g mol) were examined. A linear bis(hexadecyl)glycerol-PEG lipid ( M = 3000 g mol) was investigated as well, comparing hbPG and PEG with respect to shielding properties. Radiolabeling of the polymers was carried out using 1-azido-2-(2-(2-[F]fluoroethoxy)ethoxy)ethane ([F]F-TEG-N) via copper-catalyzed alkyne-azide cycloaddition with excellent radiochemical yields exceeding 95%. Liposomes were prepared by the thin-film hydration method followed by repeated extrusion. Use of a custom automatic extrusion device gave access to reproducible sizes of the liposomes (hydrodynamic radius of 60-94 nm). The in vivo fate of the bis(hexadecyl)glycerol polyethers and their corresponding assembled liposome structures were evaluated via noninvasive small animal positron emission tomography (PET) imaging and biodistribution studies (1 h after injection and 4 h after injection) in mice. Whereas the main uptake of the nonliposomal polyether lipids was observed in the kidneys and in the bladder after 1 h due to rapid renal clearance, in contrast, the corresponding liposomes showed uptake in the blood pool as well as in organs with good blood supply, that is, heart and lung over the whole observation period of 4 h. The in vivo behavior of all three liposomal formulations was comparable, albeit with remarkable differences in splenic uptake. Overall, liposomes shielded by the branched polyglycerol lipids show a favorable biodistribution with greatly prolonged blood circulation times, rendering them promising novel nanovesicles for drug transport and targeting.

Targeting of Immune Cells with Trimannosylated Liposomes

“…[ 19 ] As a main result of this work, it emerged that the liposomes stay intact and circulate in the blood over the investigated period of 4 h. Thus, it was evident to apply a targeting vector, namely the aforementioned trimannose, in order to address DCs. For this purpose, the already evaluated alkyne 4 ‐BisHD‐ hb PG 63 [ 19 ] appeared to be an ideal choice, since it exhibited on average four alkyne functionalities, allowing for simultaneous linkage of both trimannose and of a radiolabel to track the system in vivo. To this end, we synthesized [ 18 F]FTT‐trimannose‐alkyne‐BisHD‐ hb PG 63 following the synthesis routes illustrated in Scheme .…”

“…Reaction conditions were adopted from the radiolabeling of alkyne 4 ‐BisHD‐ hb PG 63 as specified in a previous report. [ 19 ] Radiolabeling and fluorescence labeling of alkyne 4 ‐BisHD‐ hb PG 63 was accomplished in the same manner as for its trimannosylated counterpart (Figures S2 and S3, Supporting Information).…”

“…Liposomes were prepared by the thin film hydration method followed by repeated extrusion through polycarbonate membranes with pore diameters of 400 nm, 100 nm, and then 50 nm. Extrusion was performed using an automatic extrusion device described in a previous report, [ 19 ] which allows for a reproducible pressure during the extrusion process and is regulated by a separate control module. Via this automatization, it was possible to obtain liposomes with reproducible sizes (see below).…”

“…The radiolabeling procedure for alkyne 4 ‐BisHD‐ hb PG 63 is described in a previous report. [ 19 ] Trimannose‐alkyne‐BisHD‐ hb PG 63 was radiolabeled in the same fashion.…”

“…This multifunctional polyether lipid has recently been investigated by our group and featured a stable anchorage in the lipid bilayer, while corresponding liposomes exhibited a favorable biodistribution with a prolonged circulation in the bloodstream. [ 19 ] To obtain trimannosylated liposomes, (1‐(2‐(2‐(2‐(2‐azidoethoxy)ethoxy)ethoxy)ethyl)‐1 H ‐1,2,3‐triazole‐4‐yl)methoxy)‐3,6‐di‐ O ‐α‐ d ‐mannopyranosyl‐α‐ d ‐mannopyranoside (trimannose azide) was reacted with alkyne 4 ‐BisHD‐ hb PG 63 in a copper‐catalyzed alkyne‐azide cycloaddition reaction (CuAAC). To track the liposomes in vitro, the trimannosylated polyether lipids were labeled with the fluorescent dye Oregon Green (OG)488, whereas for in vivo experiments, trimannose‐alkyne‐BisHD‐ hb PG 63 was radiolabeled with the prosthetic group 1‐azido‐2‐(2‐(2‐[ 18 F]fluoroethoxy)ethoxy)ethane ([ 18 F]F‐TEG‐N 3 ) via CuAAC, resulting in an [ 18 F]F‐TEG‐triazole ([ 18 F]FTT) group.…”