Bifunctional Ligand Systems for the Radiolabeling of Nanoparticles and Biomolecules with the fac-[99mTc(CO)3]+-Core

Abstract

The application of nanoparticles (NPs) in the medical field is increasingly growing in importance. NPs such as mesoporous silica NPs, polymeric NPs, magnetic NPs, gold NPs (AuNPs), and quantum dots (QDs) are promising candidates for the design of novel imaging, drug delivery or theranostic agents. A major challenge after administration of NPs to living organisms is the exact localization. Among the various techniques to assess the biodistribution of functional nanostructures, radiolabeling and subsequent detection of the emitted γ-photons allows for noninvasive imaging by single photon emission computed tomography (SPECT) or positron emission tomography (PET). Moreover, radiolabeling provides an accurate quantification of nanostructure localization in different organs or tissues by performing an ex vivo biodistribution. For the 99mTc labeling of AuNPs and QDs a novel coating ligand was synthesized, containing a thiol group as an anchor for the NP surface, a spacer (e.g. polyethylene glycol PEG) and the 2,3-diaminopropionic acid (DAP) chelator for the [99mTc(CO)3]+ fragment. This ligand is multi-functional; it combines the metal chelate with conjugating functions to biological vectors in one single molecule. The concept allows coupling of any targeting function to the chelator. An example with a small molecule target for the prostate specific membrane antigen (PSMA) is given. Derivatized AuNPs and QDs can directly be labeled in one step with [99mTc(OH2)3(CO)3]+. The AuNPs in particular are highly stable in buffer and serum, a prerequisite for in vivo studies excluding misinterpretation of the biodistribution data. AuNPs with differing sizes (7 nm and 14 nm core diameter) were administered intravenously into nude NMRI mice bearing LNCaP xenografts. MicroSPECT images show for both probes rapid clearance from the blood pool via the hepatobiliary pathway. The 7 nm AuNPs revealed a significantly higher bone uptake than the 14 nm AuNPs. The high affinity towards bone mineral is further confirmed in vitro with hydroxyapatite. In case NPs have an intrinsic property for contrast, radiolabeling automatically leads to multi-modal imaging agents. Particularly the development of NP-based dual-modality probes for magnetic resonance imaging (MRI)/PET or MRI/SPECT is intensively investigated. One of the most commonly used radionuclide for clinical SPECT imaging is 99m Tc and the labeling of Fe3O4 NPs with 99mTc was shown to be a successful strategy to obtain dual-modality imaging agents. The focus in this thesis is on the 99mTc labeling of gold containing magnetic nanomaterials (Fe3O4-Au core-shell and heterostructured Fe3O4-Au Dumbbell-like NPs). The key elements for the labeling are novel coating ligands, consisting of mono- or dithiol anchors for the gold surface, a PEG linker and various chelators for the [99mTc(CO)3]+ fragment. In a variety of labeling experiments, favorable coatings were examined and their stability tested. The findings presented herein can form the basis for the development of potential, NP-based SPECT/MRI dual- modality imaging agents. The versatility of the established ligand system was further shown with an intermediate along the synthethic pathway. The terminal hydroxyl group of the triethylene glycol linker conjugated to DAP was combined with L-tyrosine via a Mitsunobu reaction to the para-hydroxy of L-tyrosine. The 99mTc-L-tyrosine derivative was evaluated in healthy and nude NMRI mice bearing a C6 tumor xenograft. Radiolabeled amino acid derivatives in general are highly interesting for tumor imaging due to enhanced amino acid metabolism and protein synthesis in tumors compared to normal peripheral tissue. However, uptake in the tumor and pancreas at 1 h post injection (p.i.) was low, indicating that the tracer was not recognized as an amino acid analogue. Although the 99mTc-L-tyrosine derivative was not suitable for tumor imaging, it may be useful to image and to assess the functionality of the gallbladder. At 1 h p.i., the uptake in the gallbladder was unexpectedly high and the microSPECT images showed a high uptake after already 15 min p.i. In addition, the bifunctional ligand system HS-PEG-DAP is not only well suitable for NP coating, but also for the labeling of biomolecules with a free thiol group. The basis for this novel labeling approach is the well known thiol-disufide exchange reaction. Therefore, the complex HS-PEG-DAP-99mTc(CO)3 was activated with 2,2’- dithiodipyridine to form the pyridyl disulfide derivative. This compound allows the radiolabeling of biomolecues bearing a thiol functionality, exemplified with thioglucose, cysteine and glutathione. The scope of this labeling strategy can be extended to small peptides or even proteins containing a free thiol group.