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.

Dendritic cells (DCs) are a compelling target in cancer immunotherapy as they initialize strong antigen‐specific immune responses. Drug delivery systems (DDSs) such as liposomes provide the opportunity to deliver antigens and immunostimulatory molecules to DCs, which in turn initiate an antigen‐specific immune response. To address predominantly DCs, DDSs need to be equipped with targeting moieties. This study evaluates liposomes, bearing the oligosaccharide trimannose on their surface, for their ability to address DCs in vitro and in vivo. Trimannose as a saccharidic structure is known to be recognized by receptors on the surface of DCs. To obtain trimannosylated liposomes, azide‐bearing trimannose is coupled to alkyne‐functionalized hyperbranched polyglycerol (hbPG) with a bis(hexadecyl)glycerol (BisHD) anchor in a Cu(I)‐catalyzed alkyne‐azide cycloaddition (CuAAC). To enable tracking of the liposomes in vivo, the trimannosylated BisHD‐hbPG lipids are radiolabeled with 18F in a CuAAC. Subsequently, liposomes are produced via the thin‐film hydration method followed by extrusion. The behavior of the trimannosylated liposomes is evaluated in in vitro cell binding assays and in vivo µPET and ex vivo biodistribution studies in healthy C57BL/6 mice and the results are compared to similar liposomes not bearing trimannose on their surface.

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The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is a powerful, highly reliable and selective reaction which allows for a rapid synthesis in high yields and under mild conditions (pH, temperature). However, the cytotoxicity of copper requires its complete removal prior to an application in vivo. This is an issue especially when it comes to CuAAC reactions on macromolecular structures or drug delivery systems, as copper might be retained by these systems. Thus, a quantification of the final copper content of these systems is inevitable, which we exemplified for a CuAAC reaction on liposomes using 64Cu-spiked Cu(I). In this respect, a Cu(II) nitrate solution was irradiated at the TRIGA Mark II research reactor Mainz to obtain c.a. [64Cu]Cu(II). The irradiated solution was directly used for a CuAAC on liposomes. After purification, their copper content was calculated utilizing γ-ray spectrometry. Only 0.018% of the added 64Cu-activity was still present in the liposome containing fractions after purification. This refers to a total amount of copper of 0.17 ng. The amount of retained copper is so low, that an in vivo application of the liposomes is absolutely reasonable. Besides this particular study, the experimental methodology may be applied to study many other CuAAC reactions, used for the synthesis of radiolabeled or non-radioactive species, which are intended for human applications.