Towards understanding the design of dual-modal MR/fluorescent probes to sense zinc ions

A series of gadolinium complexes were synthesised in order to test the design of dual-modal probes that display a change in fluorescence or relaxivity response upon binding of zinc. A dansyl-DO3ATA gadolinium complex [GdL1] displayed an increase and a slight blue-shift in fluorescence in the presence of zinc; however, a decrease in relaxation rate was observed. Consequently, the ability of the well-known zinc chelator, BPEN, was assessed for relaxivity response when conjugated to the gadolinium chelate. The success of this probe [GdL2], lead to the inclusion of the same zinc-probing moiety alongside a longer wavelength emitting fluorophore, rhodamine [GdL3], to arrive at the final iteration of these first generation dual-modal zinc-sensing probes. The compounds give insight into the design protocols required for the successful imaging of zinc ions

Dual-modal imaging agents for zinc ion sensing

The area of MRI/optical imaging has received a lot of attention as their combination brings together the high spatial resolution of MRI with the high sensitivity of optical imaging. Changes in pancreatic β-cell mass contribute to the development of both type 1 and type 2 diabetes. Whilst the processes of β-cell loss are fairly well established for type 1, both the extent of the loss and the underlying mechanisms are relatively unknown for type 2. Zinc ions are highly concentrated in the insulin granules that are contained within β-cells. Few robust approaches currently exist to monitor changes in β-cell mass in vivo, and as such, this project aims to develop responsive lanthanide complexes to bind selectively and respond to zinc levels in this target area. The introductory chapter considers the fundamental aspects of molecular imaging, with a particular focus on magnetic resonance and optical imaging, as well as the intrinsic properties of the lanthanide elements, such as magnetism and luminescence. The subsequent results chapters contain more detailed introductions, relevant to the topics covered within the chapter. The synthesis of dual-modal MR/optical probes is described in chapter two. Three rhodamine-based [GdDO3A] complexes are described and their relaxivity and fluorescence properties are established. The Eu3+ and Tb3+ analogues are also studied. Two of the complexes, which show superior water solubilities, are further studied in in vitro and in vivo experiments. One probe displays a fluorescence pH sensitivity that allows for the differentiation of healthy cells from malignant cells due to their difference in pH whilst the other probe displays fluorescence at all pH’s. Both probes show accumulation in the mitochondria. Chapter three discusses the synthesis of an MR zinc sensor using a BPEN chelator as the zinc-binding moiety. Showing high selectivity for zinc, this probe is then further functionalised with the rhodamine fluorophore derivative previously described to give a dual- modal MR/fluorescent zinc sensor. This probe only shows an MR response in the presence of zinc. In vitro experiments show the localisation of the probe to differ from the results of the dual-modal probes discussed in chapter two, showing cytosol localisation. Finally, chapter four concerns the synthesis of a fluorescent zinc sensor and its conjugation to a [GdDO3A] scaffold to give a dual-modal MR/optical zinc sensor. This probe displays an improved response to zinc showing increases in both relaxivity and fluorescence. In vitro experiments with both INS1 and HEK cells show the probe to localise in the lysosome and mitochondria respectively.Open Acces

Lanthanide(III) complexes of rhodamine-DO3A conjugates as agents for dual-modal imaging

Two novel dual-modal MRI/optical probes based on a rhodamine-DO3A conjugate have been prepared. The bis aqua-Gd(III) complex Gd.L1 and mono aqua-Gd(III) complex Gd.L2 behave as dual-modal imaging probes (r1 = 8.5 and 3.8 mM-1s-1 for Gd.L1 and Gd.L2 respectively; λex = 560 nm and λem = 580 nm for both complexes). The rhodamine fragment is pH sensitive and upon lowering of pH an increase in fluorescence intensity is observed as the spirolactam ring opens to give the highly fluorescent form of the molecule. The ligands are bimodal when coordinated to Tb(III) ions, inducing fluorescence from both the lanthanide center and the rhodamine fluorophore, on two independent time-frames. Confocal imaging experiments were carried out to establish the localization of Gd.L2 in HEK cells. Co-localisation with MitoTracker® Green confirmed that Gd.L2 compartmentalizes in the mitochondria. Gd.L2 was also evaluated as an MRI probe for imaging tumors in BALB/c nude mice bearing M21 xenografts. A 36.5% decrease in T1 within the tumor was observed 30 minutes post injection showing that Gd.L2 is preferentially up taken in the tumor. Gd.L2 is the first small molecule MR/fluorescent dual-modal imaging agent to display an off-on pH switch upon its preferential uptake within the more acidic micro-environment of tumor cells

⁹⁹ᵐTc SPECT imaging agent based on cFLFLFK for the detection of FPR1 in inflammation

Non-invasive imaging of the inflammatory process can provide a great deal of insight into a wide variety of diseases states, aiding diagnosis, evaluation and effective targeted treatment. During inflammation, blood borne leukocytes are recruited, through a series of activation and adhesion steps, to the site of injury or infection where they migrate across the blood vessel wall into the tissue. Thus, tracking leukocyte recruitment and accumulation provides a dynamic and localised read out of inflammatory events. Current leukocyte imaging techniques require ex vivo labelling of patient blood, involving laborious processing and potential risks to both patient and laboratory staff. Utilising high affinity ligands for leukocyte specific receptors may allow for injectable tracers that label leukocytes in situ, omitting potentially hazardous ex vivo handling. Formyl peptide receptors (FPRs) are a group of G-protein coupled receptors involved in the chemotaxis and inflammatory functioning of leukocytes. Highly expressed on leukocytes, and up regulated during inflammation, these receptors provide a potential target for imaging inflammatory events. Herein we present the synthesis and initial in vitro testing of a potential Single Photon Emission Computed Tomography (SPECT) leukocyte tracer. The FPR1 antagonist cFLFLFK-NH₂, which displays high affinity with little physiological effect, has been linked via a PEG motif to a ⁹⁹ᵐTc chelate. This tracer shows in vitro binding to human embryonic kidney cells expressing the FPR1 receptor, and functional in vitro tests reveal cFLFLFK-NH₂ compounds to have no effect on inflammatory cell functioning. Overall, these data show that ⁹⁹ᵐTc.cFLFLFK-NH₂ may be a useful tool for non-invasive imaging of leukocyte accumulation in inflammatory disease states

Probing Unexpected Reactivity in Radiometal Chemistry: Indium-111-Mediated Hydrolysis of Hybrid Cyclen-Hydroxypyridinone Ligands

Chelators based on hydroxypyridinones have utility in incorporating radioactive metal ions into diagnostic and therapeutic agents used in nuclear medicine. Over the course of our hydroxypyridinone studies, we have prepared two novel chelators, consisting of a cyclen (1,4,7,10-tetraazacyclododecane) ring bearing two pendant hydroxypyridinone groups, appended via methylene acetamide motifs at either the 1,4-positions (L1) or 1,7-positions (L2) of the cyclen ring. In radiolabeling reactions of L1 or L2 with the γ-emitting radioisotope, [111In]In3+, we have observed radiometal-mediated hydrolysis of a single amide group of either L1 or L2. The reaction of either [111In]In3+ or [natIn]In3+ with either L1 or L2, in aqueous alkaline solutions at 80 °C, initially results in formation of [In(L1)]+ or [In(L2)]+, respectively. Over time, each of these species undergoes In3+-mediated hydrolysis of a single amide group to yield species in which In3+ remains coordinated to the resultant chelator, which consists of a cyclen ring bearing a single hydroxypyridinone group and a single carboxylate group. The reactivity toward hydrolysis is higher for the L1 complex compared to that for the L2 complex. Density functional theory calculations corroborate these experimental findings and importantly indicate that the activation energy required for the hydrolysis of L1 is significantly lower than that required for L2. This is the first reported example of a chelator undergoing radiometal-mediated hydrolysis to form a radiometalated complex. It is possible that metal-mediated amide bond cleavage is a source of instability in other radiotracers, particularly those in which radiometal complexation occurs in aqueous, basic solutions at high temperatures. This study highlights the importance of appropriate characterization of radiolabeled products

Versatile Diphosphine Chelators for Radiolabeling Peptides with ^99mTc and ⁶⁴Cu

We have developed a diphosphine (DP) platform for radiolabeling peptides with 99mTc and 64Cu for molecular SPECT and PET imaging, respectively. Two diphosphines, 2,3-bis(diphenylphosphino)maleic anhydride (DP Ph) and 2,3-bis(di- p-tolylphosphino)maleic anhydride (DP Tol), were each reacted with a Prostate Specific Membrane Antigen-targeted dipeptide (PSMAt) to yield the bioconjugates DP Ph-PSMAt and DP Tol-PSMAt, as well as an integrin-targeted cyclic peptide, RGD, to yield the bioconjugates DP Ph-RGD and DP Tol-RGD. Each of these DP-PSMAt conjugates formed geometric cis/ trans-[MO 2(DP X-PSMAt) 2] + (M = 99mTc, 99gTc, natRe; X = Ph, Tol) complexes when reacted with [MO 2] + motifs. Furthermore, both DP Ph-PSMAt and DP Tol-PSMAt could be formulated into kits containing reducing agent and buffer components, enabling preparation of the new radiotracers cis/ trans-[ 99mTcO 2(DP Ph-PSMAt) 2] + and cis/ trans-[ 99mTcO 2(DP Tol-PSMAt) 2] + from aqueous 99mTcO 4 - in 81% and 88% radiochemical yield (RCY), respectively, in 5 min at 100 °C. The consistently higher RCYs observed for cis/ trans-[ 99mTcO 2(DP Tol-PSMAt) 2] + are attributed to the increased reactivity of DP Tol-PSMAt over DP Ph-PSMAt. Both cis/ trans-[ 99mTcO 2(DP Ph-PSMAt) 2] + and cis/ trans-[ 99mTcO 2(DP Tol-PSMAt) 2] + exhibited high metabolic stability, and in vivo SPECT imaging in healthy mice revealed that both new radiotracers cleared rapidly from circulation, via a renal pathway. These new diphosphine bioconjugates also furnished [ 64Cu(DP X-PSMAt) 2] + (X = Ph, Tol) complexes rapidly, in a high RCY (&gt;95%), under mild conditions. In summary, the new DP platform is versatile: it enables straightforward functionalization of targeting peptides with a diphosphine chelator, and the resulting bioconjugates can be simply radiolabeled with both the SPECT and PET radionuclides, 99mTc and 64Cu, in high RCYs. Furthermore, the DP platform is amenable to derivatization to either increase the chelator reactivity with metallic radioisotopes or, alternatively, modify the radiotracer hydrophilicity. Functionalized diphosphine chelators thus have the potential to provide access to new molecular radiotracers for receptor-targeted imaging. </p

Long-term evolution of the coupled boundary layers (STRATUS) mooring recovery and deployment cruise report NOAA Research Vessel R H Brown • cruise RB-01-08 9 October - 25 October 2001

This report documents the work done on cruise RB-01-08 of the NOAA R/V Ron Brown. This was Leg 2 of R/V Ron Brown’s participation in Eastern Pacific Investigation of Climate (EPIC) 2001, a study of air-sea interaction, the atmosphere, and the upper ocean in the eastern tropical Pacific. The science party included groups from the Woods Hole Oceanographic Institution (WHOI), NOAA Environmental Technology Laboratory (ETL), the University of Washington (UW), the University of California, Santa Barbara (UCSB), and the University Nacional Autonoma de Mexico (UNAM). The work done by these groups is summarized in this report. In addition, the routine underway data collected while aboard R/V Ron Brown is also summarized here.Funding was provided by the National Oceanic and Atmospheric Administration under Grant Numbers NA96GPO429 and NA17RJ1223

Global study of social odor awareness

Olfaction plays an important role in human social communication, including multiple domains in which people often rely on their sense of smell in the social context. The importance of the sense of smell and its role can however vary inter-individually and culturally. Despite the growing body of literature on differences in olfactory performance or hedonic preferences across the globe, the aspects of a given culture as well as culturally universal individual differences affecting odor awareness in human social life remain unknown. Here, we conducted a large-scale analysis of data collected from 10,794 participants from 52 study sites from 44 countries all over the world. The aim of our research was to explore the potential individual and country-level correlates of odor awareness in the social context. The results show that the individual characteristics were more strongly related than country-level factors to self-reported odor awareness in different social contexts. A model including individual-level predictors (gender, age, material situation, education and preferred social distance) provided a relatively good fit to the data, but adding country-level predictors (Human Development Index, population density and average temperature) did not improve model parameters. Although there were some cross-cultural differences in social odor awareness, the main differentiating role was played by the individual differences. This suggests that people living in different cultures and different climate conditions may still share some similar patterns of odor awareness if they share other individual-level characteristics