In vivo evaluation of nanodimensional drug delivery systems via positron emission tomography
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Abstract
In vivo tracking is crucial for the pre-clinical evaluation of drug delivery systems (DDSs), in order to understand structure-property relationships of the DDSs and their resulting pharmacokinetic profile. Among nuclear medical imaging tools, positron emission tomography (PET) is a noninvasive technique, allowing for quantification and providing an excellent spatial and temporal resolution. Because of these advantages, PET bears great potential in the evaluation of DDSs. In the herein presented work, different nanodimensional DDSs were evaluated via PET and/or biodistribution studies in mice and rats. By the utilization of the radionuclides 18F, 131I and 64Cu, various research questions could be answered.
First, random (ran) copolymers consisting of N-(2-hydroxypropyl)methacrylamide (HMPA) and lauryl methacrylate (LMA), which had shown favorable short-term in vivo characteristics in former studies, were radiolabeled with 131I. The longer-lived radioisotope 131I enabled tracking of their pharmacokinetics in tumor-bearing rats over three days. Despite a polymer clearance from the blood within this time span, there was an increase in tumor uptake observable over time, which strongly emphasizes the enhanced permeability and retention (EPR) effect.
Another part of this work focused on the in vivo evaluation of stealth liposomes. These spherical vesicles exhibit an excellent biocompatibility as well as a low toxicity. A polymer shell prevents them from being detected by the mononuclear phagocyte system (MPS). For this purpose, one needs to ensure a stable anchorage into the lipid bilayer, ideally paired with multifunctionality, which enables the linkage of different modalities (e.g. radiolabel and targeting vector). Thus, different multifunctional hyperbranched polyglycerols (hbPG) with a dialkyl anchor and their respective liposomal formulations were investigated in PET and biodistribution studies in mice, with respect to liposome stability. To compare hbPG and the gold standard PEG regarding their shielding properties, also a PEG lipid was examined. The polyether lipids were rapidly cleared via the renal system, whereas the corresponding liposomes circulated in the blood stream over the period of investigation. Overall, liposomes shielded by the hbPG lipids exhibited a favorable biodistribution, rendering them promising novel nanovesicles for drug transport and targeting.
Radiolabeling of the polyether lipids was carried out prior to the liposome formation via copper-catalyzed alkyne-azide cycloaddition (CuAAC) using 1-azido-2-(2-(2-[18F]fluoroethoxy)ethoxy)ethane ([18F]F-TEG-N3). This approach ensured the separation of cytotoxic copper, but entailed a time-consuming radiosynthesis on the other hand. Thus, a direct radiolabeling of preformed liposomes was tested. Herein, a main emphasis lay on the quantification of the retained amount of copper upon purification. For this purpose, 64Cu was utilized as catalyst for the CuAAC between liposomes and non-radioactive F-TEG-N3. In this way, the final copper content of the liposomes could straightforwardly be quantified by gamma spectrometry, revealing that only 0.018 % of the added radioactivity was still associated with the liposome after purification. The ration of retained copper is so low, that an in vivo application of the liposomes is absolutely reasonable.
In the last part of this work, hbPG-shielded liposomes, bearing multiple carbohydratic trimannose molecules on their surface, were investigated, regarding their potential to address dendritic cells (DCs) in vivo. DCs are professional antigen-presenting cells, which initialize all antigen-specific immune responses. Thus, they are a compelling target in cancer immunotherapy. The in vivo fate of the trimannosylated liposomes was evaluated via in vivo PET and ex vivo biodistribution studies in mice and the results were compared to similar liposomes bearing no trimannose on their surface. Here, the multifunctionality of hbPG was beneficial, as the 18F-label for tracking and the trimannose-label for DC-targeting could be implemented on the same molecule. It was found that the mannosylated liposomes highly accumulated in liver, spleen and bone marrow, compared to their non-mannosylated counterparts. These findings suggest, that the mannosylated liposomes were recognized by DCs, expressed in the mentioned organs. However, it cannot be excluded that other antigen-presenting cells, besides DCs, have also be targeted. Future cell studies on murine splenic immune cells have to elucidate which cell types are addressed in particular and to which extent. For this purpose, Oregon Green 488-labeled polyether lipids and respective liposomal formulations have already been synthesized.