Purification and activity standardisation of a (166m)Ho solution.

As part of a project to use the long-lived (T(1/2)=1200a) (166m)Ho as reference source in its reference ionisation chamber, IRA standardised a commercially acquired solution of this nuclide using the 4pibeta-gamma coincidence and 4pigamma (NaI) methods. The (166m)Ho solution supplied by Isotope Product Laboratories was measured to have about 5% Europium impurities (3% (154)Eu, 0.94% (152)Eu and 0.9% (155)Eu). Holmium had therefore to be separated from europium, and this was carried out by means of ion-exchange chromatography. The holmium fractions were collected without europium contamination: 162h long HPGe gamma measurements indicated no europium impurity (detection limits of 0.01% for (152)Eu and (154)Eu, and 0.03% for (155)Eu). The primary measurement of the purified (166m)Ho solution with the 4pi (PC) beta-gamma coincidence technique was carried out at three gamma energy settings: a window around the 184.4keV peak and gamma thresholds at 121.8 and 637.3keV. The results show very good self-consistency, and the activity concentration of the solution was evaluated to be 45.640+/-0.098kBq/g (0.21% with k=1). The activity concentration of this solution was also measured by integral counting with a well-type 5''x5'' NaI(Tl) detector and efficiencies computed by Monte Carlo simulations using the GEANT code. These measurements were mutually consistent, while the resulting weighted average of the 4pi NaI(Tl) method was found to agree within 0.15% with the result of the 4pibeta-gamma coincidence technique. An ampoule of this solution and the measured value of the concentration were submitted to the BIPM as a contribution to the Système International de Référence.

Bioconjugated lanthanide luminescent helicates as multilabels for lab-on-a-chip detection of cancer biomarkers.

The lanthanide binuclear helicate [Eu(2)(L(C2(CO(2)H)))(3)] is coupled to avidin to yield a luminescent bioconjugate EuB1 (Q = 9.3%, tau((5)D(0)) = 2.17 ms). MALDI/TOF mass spectrometry confirms the covalent binding of the Eu chelate and UV-visible spectroscopy allows one to determine a luminophore/protein ratio equal to 3.2. Bio-affinity assays involving the recognition of a mucin-like protein expressed on human breast cancer MCF-7 cells by a biotinylated monoclonal antibody 5D10 to which EuB1 is attached via avidin-biotin coupling demonstrate that (i) avidin activity is little affected by the coupling reaction and (ii) detection limits obtained by time-resolved (TR) luminescence with EuB1 and a commercial Eu-avidin conjugate are one order of magnitude lower than those of an organic conjugate (FITC-streptavidin). In the second part of the paper, conditions for growing MCF-7 cells in 100-200 microm wide microchannels engraved in PDMS are established; we demonstrate that EuB1 can be applied as effectively on this lab-on-a-chip device for the detection of tumour-associated antigens as on MCF-7 cells grown in normal culture vials. In order to exploit the versatility of the ligand used for self-assembling [Ln(2)(L(C2(CO(2)H)))(3)] helicates, which sensitizes the luminescence of both Eu(III) and Tb(III) ions, a dual on-chip assay is proposed in which estrogen receptors (ERs) and human epidermal growth factor receptors (Her2/neu) can be simultaneously detected on human breast cancer tissue sections. The Ln helicates are coupled to two secondary antibodies: ERs are visualized by red-emitting EuB4 using goat anti-mouse IgG and Her2/neu receptors by green-emitting TbB5 using goat anti-rabbit IgG. The fact that the assay is more than 6 times faster and requires 5 times less reactants than conventional immunohistochemical assays provides essential advantages over conventional immunohistochemistry for future clinical biomarker detection.

Synthesis of F-18-labeled cyclic-[RGDfK] peptides for PET imaging of angiogenesis

Objectives: αvβ3 integrin is of great interest for tumor targeting because of its high concentration in tumor tissue. It recognizes ligands containing an arginine-glycine-aspartate motif (RGD), and a number of RGD-containing peptides have been developed as PET imaging probes of angiogenesis. We synthesized a series of 18F-labeled cyclic-[RGDfK] peptides for in vivo imaging of αvβ3 expression. Our F-18 labeled prosthetic groups were attached to the αvβ3 ligand via click chemistry, and the reaction conditions (time, temperature, solvent and pH) were optimized by using single modified amino acids.Methods: Seven amino acids were selected considering their different biochemical properties (polarity, total charge, presence of aromatic ring and heteroatom). All the amino acids were modified by the introduction of azido moiety to allow the interaction with alkyne prosthetic groups. Once the conditions of the click chemistry were optimized, the prosthetic groups were also coupled with the cyclic-[RGDfK] exhibiting an azido function. 4- Trimethylammonium-nitrobenzene triflate was used as precursor for the radiosynthesis of the prosthetic groups. The fluorination was carried out with K2CO3/K2.2.2 in CH3CN at 95 oC, and the nitro group was reduced with NaBH4 and Pd/C in MeOH. The resulting 18F-aniline was subsequently coupled to alkynoic acids to yield the final F-18 labeled prosthetic groups. Finally, the prosthetic groups were attached to the peptides via Huisgen's cycloaddition. Figure 1. F-18 labeled αvβ3 ligand.Results: Our new prosthetic groups were successfully clicked to the modified amino acids and to the cyclic- [RGDfK], and the reactions were almost quantitative within 1 to 3.5 h. The pH of the reaction did not influence the reaction kinetic and yield. The four steps of the F-18 labeling were completely automated providing the final products in quantities and yields practical for PET imaging. IC50 values of our ligands for αvβ3 and α5β1 demonstrated a high selectivity of our compounds towards αvβ3, as well as the negligible effect of the prosthetic groups on the affinity of the ligand to its receptor, as confirmed by the prediction of the molecular modeling.Conclusions: We have successfully synthesized novel F-18 labeled prosthetic groups, as well as novel PET imaging probes of αvβ3 expression. The reaction conditions of the Huisgen's cycloaddition were optimized with selected modified amino acids, and subsequently transposed to the cyclic-[RGDfK] peptide. IC50 data demonstrate that our 18F-labeled ligands were selective for αvβ3. In vivo microPET/CT studies in tumor bearing mice are underway.