Cristina Bolzati

Folic Acid-Conjugated Europium Complexes as Luminescent Probes for Selective Targeting of Cancer Cells

We report the synthesis of three optical probes (Eu(3+)⊂1, Eu(3+)⊂2, and Eu(3+)⊂3) having a luminescent Eu complex (signaling unit) bonded in different positions to folic acid (FA), the folate receptor (FR) targeting unit. The structures of the two regioisomers Eu(3+)⊂1 and Eu(3+)⊂2 were assigned by mass spectrometric experiments. The optical properties and stability of these probes were assessed in phosphate-buffered saline, cell culture medium, rat serum, and cellular lysate, and results indicated that they are chemically and photophysically stable. Cytotoxicity was studied with ovarian cancer cells having high (SKOV-3), intermediate (OVCAR-3), low (IGROV-1), or null (A2780) expression of FRs. The internalized probe, evaluated in SKOV-3, IGROV-1, and A2780 cells, was in the order Eu(3+)⊂2 > Eu(3+)⊂1 > Eu(3+)⊂3. No internalization was observed for A2780 cells. Such results, together with those obtained in competition experiments of FA versus Eu(3+)⊂2 and FA or Eu(3+)⊂2 versus (3)H-FA, indicate that internalization is receptor-mediated and that Eu(3+)⊂2 shows high selectivity and specificity for FR.

Synthesis, characterization and biological evaluation of [188Re(N)(cys∼)(PNP)]+/0 mixed-ligand complexes as prototypes for the development of 188Re(N)-based target-specific radiopharmaceuticals

We report on an efficient procedure for the preparation of [(188)Re(N)(PNP)]-based complexes (where PNP is diphosphinoamine) useful in the development of target-specific radiopharmaceuticals. The radiochemical yield of the compounds was optimized considering such reaction parameters as nature of the nitrido nitrogen donor, reaction times and pH level. The chemical identity of the (188)Re agents was determined by high-performance liquid chromatography comparison with the corresponding well-characterized cold Re compounds. (188)Re(N) mixed compounds have been evaluated with regard to stability toward transchelation with GSH and degradation by serum enzymes. The clearance of selected radiocompounds from normal tissues and their in vivo stability were evaluated in rats by biodistribution and imaging studies. [(188)Re(N)(cys ∼)(PNP)](+/0) mixed-ligand compounds were efficiently prepared in aqueous solution from perrhenate using a multistep procedure based on the preliminary formation of the labile (188)Re(III)-EDTA species, which easily undergo oxidation/ligand exchange reaction to afford the [(188)Re(V) ≡ N](2+) core in the presence of dithiocarbazate. The final mixed-ligand compounds were obtained, at 100 °C, by adding the two bidentate ligands to the buffered [(188)Re(V) ≡ N](2+) solution (pH 3.2-3.6). However, a relatively high amount of cys ∼ ligand was required to obtain a quantitative radiochemical yield. The complexes were stable toward reoxidation to perrhenate and ligand exchange reactions. In vivo studies showed rapid distribution and elimination of the complexes from the body. No specific uptakes in sensitive tissues/organs were detected. A positive correlation of the distribution of the complexes estimated with biodistribution studies (%ID) and with micro-SPECT semiquantification imaging analysis (standard uptake values) was observed. These results support the possibility of applying [(188)Re(N)(PNP)] technology to the preparation of target-specific agents.

Transglutaminase-mediated conjugation and nitride-technetium-99m labelling of a bis(thiosemicarbazone) bifunctional chelator

An assessment study involving the use of the transglutaminase (TGase) conjugation method and the nitride-technetium-99m labelling on a bis(thiosemicarbazone) (BTS) bifunctional chelating agent is presented. The previously described chelator diacetyl-2-(N4-methyl-3-thiosemicarbazone)-3-(N4-amino-3-thiosemicarbazone), H2ATSM/A, has been functionalized with 6-aminohexanoic acid (ε-Ahx) to generate the bifunctional chelating agent diacetyl-2-(N4-methyl-3-thiosemicarbazone)-3-[N4-(amino)-(6-aminohexanoic acid)-3-thiosemicarbazone], H2ATSM/A-ε-Ahx (1), suitable for conjugation to glutamine (Gln) residues of bioactive molecules via TGase. The feasibility of the TGase reaction in the synthesis of a bioconjugate derivative was investigated using Substance P (SP) as model peptide. Compounds 1 and H2ATSM/A-ε-Ahx-SP (2) were labelled with nitride-technetium-99m, obtaining the complexes [99mTc][Tc(N)(ATSM/A-ε-Ahx)] (99mTc1) and [99mTc][Tc(N)(ATSM/A-ε-Ahx-SP)] (99mTc2). The chemical identity of 99mTc1 and 99mTc2 was confirmed by radio/UV-RP-HPLC combined with ESI-MS analysis on the respective carrier-added products 99g/99mTc1 and 99g/99mTc2. The stability of the radiolabelled complexes after incubation in various environments was investigated. All the results were compared with those obtained for the corresponding 64Cu-analogues, 64Cu1 and 64Cu2. The TGase reaction allows the conjugation of 1 with the peptide, but it is not highly efficient due to instability of the chelator in the required conditions. The SP-conjugated complexes are unstable in mouse and human sera. However, indeed the BTS system can be exploited as nitride-technetium-99m chelator for highly efficient technetium labelling, thus making compound 1 worthy of further investigations for new targeted technetium and copper radiopharmaceuticals encompassing Single Photon Emission Computed Tomography and Positron Emission Tomography imaging.