Dissociation kinetics of macrocyclic trivalent lanthanide complexes of 1-oxa-4,7,10-triazacyclododecane-4,10-diacetic acid (H₂ODO2A)

<div><p>The dissociation kinetics of selected trivalent lanthanide (Ln³⁺, Ln=La, Pr, Eu, Er, Lu) complexes of the macrocyclic ligand H₂ODO2A (1-oxa-4,7,10-triazacyclododecane-4,10-diacetic acid), LnODO2A⁺, were studied in the [H⁺] range (0.1–2.4) × 10⁻⁴ M in the temperature range 15–45 °C. Excess Cu²⁺ ions were used as the scavenger for the ligand in acetate–acetic acid buffer medium. The dissociation reactions are independent of [Cu²⁺] and follow the rate law <i>k</i>_obs = <i>k</i>_d + <i>k</i>_AC[Acetate] + K′<i>k</i>_lim[H⁺]/(1 + K′[H⁺]), where <i>k</i>_d, <i>k</i>_AC, and <i>k</i>_lim are the respective dissociation rate constants for the [H⁺]-independent, acetate-assisted, and the [H⁺]-dependent limiting pathways; K′ is the equilibrium constant for the protonation reaction LnODO2A⁺ + H⁺ LnODO2AH²⁺. The dissociation rates of LnODO2A⁺ complexes are all faster than those of the corresponding LnDO2A⁺ complexes (DO2A²⁻ is the fully deprotonated dianion of the ligand H₂DO2A, 1,4,7,10-tetrazacyclo-dodecane-1,7-diacetic acid), consistent with the notion that LnODO2A⁺ complexes are kinetically more labile and thermodynamically less stable than the corresponding LnDO2A⁺ complexes, and H₂ODO2A is not pre-organized for Ln³⁺ ion complexation but H₂DO2A is.</p></div

MR scans of CSPIO-labeled islet isografts in non-diabetic mice.

<p>A: Grafts of CSPIO-labeled islets were visualized on MR scans as distinct hypointense areas homogeneously located at the upper pole of the left kidney at day 1 and week 1, 2, 3, 4, 5, 6, and 8 after syngeneic transplantation in non-diabetic mice. B: Compared with the same area on the contralateral kidneys, the MR signal intensity in CSPIO-labeled (solid triangle and square) was significantly lower than that of control (open triangle and square) islet grafts (81.9±14.0% vs. 103.8±15.4%, P = 3.68297E-05).</p

MR scans of CSPIO-labeled islet allgrafts in a non-diabetic (A) and a diabetic (B) mouse.

<p>(<b>A</b>) At day 3, 10, 17, 24, and 31 post-allotransplantation in a non-diabetic recipient, MR scans showed hypointense areas at the upper pole of the left kidney. (<b>B</b>) In a streptozotocin-induced diabetic recipient, there was no significant change of MR hypo-intensity at the upper pole of left kidney for 2–5 wks although its blood glucose levels decreased in the first 2 weeks and then went up. Our MR images could not differentiate the signal loss in grafted kidneys between the diabetic and non-diabetic mouse.</p

TEM micrographs and iron mapping of CSPIO-labeled islet isografts at 8 weeks after transplantation in non-diabetic mice.

<p>A: TEM showed several electron dense clumps distributed in the cytoplasm of a β-cell with intact ultrastructure. B: Electron energy-loss spectroscopy mapping of iron demonstrated significant signals of Fe recorded as bright areas in the islet graft.</p

Insulin secretion of islets incubated with and without CSPIO.

<p>Studies of static incubation at 2.8 and 16.7 mmol/L glucose (A) and perifusion with 2.8 and 16.7 mmol/L glucose (B) showed the islets incubated overnight with [black column (n = 3) and solid circles (n = 3)] and without [white column (n = 3) and open circles (n = 3)] CSPIO (10 µg/mL) had comparable insulin responses to high glucose challenges. Stimulation indices were calculated by dividing the amount of insulin released with 16.7 mmol/L glucose by that released with 2.8 mmol/L glucose.</p

Histology of CSPIO-labeled islet isografts at 8 weeks after transplantation in non-diabetic mice.

<p>Colocalization of insulin (A; brown) and iron (B; blue) staining in CSPIO-labeled islet grafts. Scale bar: 100 µm.</p

Death rates of NIT-1, βTC, and αTC1 cells as well as islets incubated with and without CSPIO.

<p>NIT-1 (A and D), βTC (B and E) and αTC1 (C and F) cells incubated with CSPIO did not increase death rates with increasing CSPIO concentrations up to 80 µg (A–C) or incubation time up to 72 h (D–F). In addition, overnight incubation of islets with CSPIO (10 µg/ml) did not affect cell viability (G, H). A–H were determined by propidium iodide (PI) and H by 7-aminoactinomycin D (7-AAD).</p

TEM micrographs of isolated islets incubated with CSPIO.

<p>At 1 h (A; ×10,000) and 4 h (B; ×10,000) after incubation, TEM showed CSPIO particles (indicated by the arrow) were within islets but between cells. However, CSPIO particles were observed in endocytotic vesicles of an alpha-cell at 8 h (C; ×10,000).</p

Histology of islet allografts at day 10, 17, 24, and 31 after transplantation in non-diabetic mice.

<p>Upper row: insulin staining of CSPIO-labeled islet grafts; middle row: Prussian blue staining of CSPIO-labeled islet grafts; lower row: insulin staining of unlabeled islet grafts. There was profound immune cell infiltration at day 10 and 17, and a decrease in graft size at day 24 and 31 in the CSPIO-labeled islet graft. However, colocalization of insulin staining (shown in brown) and iron staining (shown in blue) presented in the CSPIO-labeled islet graft all the time. In contrast, the unlabeled islet graft at day 24 and 31 contained many immune cells but no insulin-positive cells. Magnification: 100×; Scale bar: 100 µm.</p