Liposome-encapsulated superoxide dismutase mimetic: theranostic potential of an MR detectable and neuroprotective agent

Abstract

Endogenous manganese based superoxide dismutase (Mn-SOD) provides the primary defense against excess production of potentially toxic superoxide anion (O2 (-) ). M40401 is a synthetic enzyme mimetic that has a catalytic activity rate exceeding that of the native SOD enzymes. The presence of a paramagnetic Mn(II) cation in M40401 suggests that the delivery and spatial distribution of this enzyme mimetic in vivo may be directly detectible using magnetic resonance imaging (MRI); however, the cardiotoxicity of Mn(II) severely limits the use of free M40401 in living systems. To deliver M40401 in vivo in amounts sufficient for MRI detection and to limit potential cardiotoxicity, we encapsulated M40401 into 170 nm liposomes composed of phosphatidylcholine and PEGylated phosphatidylethanolamine to achieve extended circulation in the bloodstream. The obtained liposomes efficiently catalyzed superoxide dismutation in vitro. Using 3 T MRI we investigated the biokinetics of liposome-encapsulated M40401 in mice and found that, in addition to catalyzing superoxide dismutation in vitro, M40401 caused differential and region-specific enhancement of mouse brain after systemic administration. Thus, liposome encapsulated M40401 is an ideal candidate for development as a theranostic compound useful for simultaneous MRI-mediated tracking of delivery as well as for neuroprotective treatment of ischemic brain.

Keywords: M40401; MRI; Mn-SOD; liposomes; manganese complex; mouse brain.

Fig. 1

Synthesis of manganese-based superoxide dismutase mimetic, M40401 Manganese(II) dichloro [2S,21S-dimethyl-3,10,13,20,26-pentaazatetracyclo[20.3.1.0.^4,90^14,19] hexacosa-1(26),22(23),24-triene] (compound 5), ref. (47).

Fig. 2

(A) Transmission electron micrograph of DSPC/DSPE-PEG liposomes loaded with M40401 (magnification ×25,000). (B) Schematic of DSPC/DSPE-PEG liposomes with M40401 in the aqueous layer. (C) Chemical formula of M40401 Mn complex.

Fig. 3

(A) Relaxivity of M40401 in DPBS at 0.47 T, r1 = 4.41 ± 0.05 [mM.s]⁻¹. (B) Longitudinal T₁ relaxation time of intact and lysed M40401 liposomes at 0.47 T. (C) 3 T saturation recovery images of four phantoms (1, M40401 liposomes plus detergent; 2, DPBS solution; 3, free M40401; 4, M40401 liposomes in DPBS solution ) using spin-echo sequence at different repetition times (TR) and TE/NEX = 8.2 ms 1 1. The T₁ values of each phantom are: 1, 695 ± 33 ms; 2, 2833 ± 263 ms; 3, 176 ± 12; 4 , 2981± 169. (D) T₁-weighted image of the four phantoms shown in (C) at 3 T using gradient-echo sequence (TR/TE/FA/NEX = 192 ms/5.5ms/75°/4).

Fig. 4

The V_max of superoxide dismutation measured as a function of M40401 and SOD concentrations. The reaction kinetics were measured as an increase of resorufin absorbance generated from Amplex Red in the presence of peroxidase and superoxide generating system (xanthine/xanthine oxidase). Data is represented (n=2) as mean+SD (▲), mean–SD (□), and mean±SD (×).

Fig. 5

ROI definitions for different brain regions and the pituitary gland. Normalized T₁-WT signal intensities were measured in different regions of the mouse brain (n=5) after the I.V. injection of M40401 liposomes (12 μmol M40401/kg). Representative axial slices from T₁-weighted image of a mouse brain prior to liposome injection are shown (C) corresponding to the shaded region in the coronal slices (A) which correlate with schematic brain slices (B) from the mouse brain atlas (63). The schematic brain slices approximately correspond to the central region of the 1-mm-thick MR image slices. The different ROI regions are: CER – cerebellum; COR – cortex; SC – subcortex; CN – caudate nucleus; HP – hippocampus; PIT – pituitary gland; OB – olfactory bulb. Panels (D) and (E) show change in signal intensities normalized as percent change relative to the pre-contrast image in the different ROI regions. The graphs show the change in MR signal intensity up to approximately 40 minutes. Data are represented on both axes as mean±SEM. ANOVA test for mixed models showed a significant effect of M40401 liposome injection on the seven different brain regions (p<0.0001) and time points after liposome injection (p<0.01).

Fig. 6

MR signal enhancement in the mouse brain with SNR as a function of time after IV injection of M40401 liposomes. The inset shows T₁-WT mouse brain images corresponding to the signal intensity curve (time in minutes). Data is shown from a representative animal across several slices in the cortex and subcortex regions as mean–SD (◆) and mean+SD (×), respectively.