Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Oct 3;103(40):14707-12.
doi: 10.1073/pnas.0606749103. Epub 2006 Sep 26.

Calcium-sensitive MRI Contrast Agents Based on Superparamagnetic Iron Oxide Nanoparticles and Calmodulin

Affiliations
Free PMC article

Calcium-sensitive MRI Contrast Agents Based on Superparamagnetic Iron Oxide Nanoparticles and Calmodulin

Tatjana Atanasijevic et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

We describe a family of calcium indicators for magnetic resonance imaging (MRI), formed by combining a powerful iron oxide nanoparticle-based contrast mechanism with the versatile calcium-sensing protein calmodulin and its targets. Calcium-dependent protein-protein interactions drive particle clustering and produce up to 5-fold changes in T2 relaxivity, an indication of the sensors' potency. A variant based on conjugates of wild-type calmodulin and the peptide M13 reports concentration changes near 1 microM Ca(2+), suitable for detection of elevated intracellular calcium levels. The midpoint and cooperativity of the response can be tuned by mutating the protein domains that actuate the sensor. Robust MRI signal changes are achieved even at nanomolar particle concentrations (<1 microM in calmodulin) that are unlikely to buffer calcium levels. When combined with technologies for cellular delivery of nanoparticulate agents, these sensors and their derivatives may be useful for functional molecular imaging of biological signaling networks in live, opaque specimens.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Calcium sensor mechanism. (A) SA-MACS-CaM (red) and SA-MACS-M13 (green) nanoparticles form binary aggregates in the presence, but not the absence, of calcium. SPIO aggregation is predicted to lead to T2 changes. (B) The calcium-dependent interaction of CaM (red) and M13 (green) polypeptides underlies the aggregation effect, which requires that each nanoparticle be polydentate in one of these proteins. Protein surfaces are biotinylated (arrows) and attached to SPIOs via tight biotin/streptavidin binding.
Fig. 2.
Fig. 2.
Light-scattering and AFM analysis of calcium-dependent behavior of the SPIOs conjugates. (A) Mean particle radii are graphed for SA-MACS-CaM, SA-MACS-M13, SA-MACS-RS20, and binary mixtures. CaM conjugates mixed in 1:1 ratio with M13 and RS20 conjugates are denoted 1C:1M and 1C:1R, respectively; 3:1 ratios are denoted 3C:1M and 3C:1R. Radii are expressed relative to the radius of SA-MACS-CaM for each mixture in 500 μM CaCl2 (gray bars) and 500 μM EDTA (white bars). Error bars denote SEM (n = 3). Strong calcium-dependent effects are observed for all mixed SPIO samples. Ratios of 1:1 produce the largest aggregates but also show a tendency toward calcium-independent clustering. 3:1 binary sensors show little aggregation in the EDTA condition but still display a robust calcium-induced response. (B) Size distributions of the 3C:1M sensor in 500 μM EDTA (dotted line) and 500 μM CaCl2 (solid line). Distribution histograms were calculated from DLS data, assuming polydisperse but monomodal size populations, and represent the percentage mass estimated in each size interval. (C) Atomic force micrographs of 3C:1M samples obtained after preincubation in 50 μM EDTA (Left) or 50 μM CaCl2 (Right) and indicating a change in clustering. (Scale bar: 500 nm.)
Fig. 3.
Fig. 3.
T2 relaxation rate changes measured by MRI. (A) SPIO conjugates were arrayed into microtiter plates and imaged by MRI. A spin echo pulse sequence was used with Carr-Purcell-Meiboom-Gill (CPMG) acquisition to collect images at multiple echo times for each excitation (see Materials and Methods for details). The four images shown indicate signal dependence on TE for PBS buffer, SA-MACS-CaM, SA-MACS-M13, and a calcium sensor formed by mixing these two species in a 3:1 ratio, each in the presence of either 500 μM Ca2+ (+) or 500 μM EDTA (−). Unmixed CaM conjugates have the highest relaxivity and produce the most diminished signal at high echo times. The calcium sensor shows high relaxivity in the absence of calcium but not when calcium is present, where the signal remaining at 80 ms TE for the sensor is comparable to that observed for PBS alone. All SPIO solutions contained 7.9 mg/liter Fe. (B) Normalized T2-weighted MRI signal observed for PBS; for unmixed SPIO conjugates with CaM, M13, and RS20; and for the sensor variants described in Results and Discussion: 3C:1M, 1C:1M, 3C:1R, and 1C:1R. For each species, the top row shows signal observed in the presence of 500 μM CaCl2, and the bottom row presents signal in 500 μM EDTA. This image was obtained by dividing MRI intensities observed at TE = 30 ms by intensities at TE = 10 ms. (C) T2 values obtained by fitting monoexponential decay curves to MRI signal as a function of TE (odd echoes only: TE = 10, 30, 50, and 70 ms), using the data from A and B. For each solution, the T2 observed in the presence (gray bars) and absence (white bars) of calcium is reported. Calcium-dependent T2 changes are greatest for the 3:1 conjugate ratio calcium sensors 3C:1M and 3C:1R.
Fig. 4.
Fig. 4.
Time courses and reversibility of calcium-dependent changes. (A) Representative time courses of particle association and dissociation for the 3C:1M aggregation-based calcium sensor. To measure association (filled circles), 3C:1M mixed particles were preincubated in 100 μM EDTA for 1 h before dilution into a final concentration of 500 μM CaCl2. To measure dissociation (open circles), particles were premixed in 100 μM CaCl2 and diluted into 500 μM EDTA. The horizontal gray line approximates the starting point for disaggregation, measured by diluting particles preincubated in 100 μM CaCl2 into the same buffer before DLS. (B) Time courses of changes in T2 relaxation time after calcium-induced aggregation and EDTA-induced disaggregation. Preincubations and dilutions were performed as for the DLS experiments in A, but larger volumes were used. Filled circles correspond to dilution from EDTA into calcium, and open circles correspond to dilution from CaCl2 to EDTA. Measurements were taken continuously over 1 h using the methods of Fig. 3, with each image requiring 8 min to acquire and a mixing and setup dead time of 4 min. The horizontal gray line approximates the initial T2 of disaggregating SPIOs, again measured by control dilution of particles in 100 μM CaCl2.
Fig. 5.
Fig. 5.
Calcium sensitivity range and tuning of calcium sensor variants. (A) Calcium titrations were performed to determine the EC50 of the SPIO calcium sensor variants. EGTA-buffered solutions were used for 0–1.35 μM and 39 μM Ca2+ concentrations, and unbuffered calcium solutions were used for the remainder of concentrations in the 1.5–500 μM range. The EC50 determined for 3C:1M from three independent titrations (one shown here) was 1.4 ± 0.1 μM Ca2+. (B) A normalized MRI image analogous to Fig. 3B, showing relative signal obtained from 7.9 mg/liter 3C:1M sensor in the presence of 0–5.0 μM calcium ions. The transition in signal amplitude occurs near 1 μM, as indicated by the DLS data. (C) T2 values computed from the MRI data of B. (D) Calcium titration of sensors constructed from mutant XCaM proteins 3X1:1M (filled circles) and 3X2:1M (open circles). The mutant CaM used in the 3X2:1M sensor has a lower affinity for the M13 sequence; coupling between calcium sensitivity and binding affinity is likely to reduce this variant's calcium-binding affinity (EC50 of 10 ± 2 μM, n = 2). (E) MRI signal intensity at calcium concentrations from 0 to 15 μM (TE = 40 ms, normalized as in B but with color scale from 0 to 0.8). The 3X1:1M variant shows similar behavior to 3C:1M but with more gradual transition in intensity with increasing [Ca2+], as in the DLS titration curve. Variant 3X2:1M shows transition onset at a higher calcium concentration, and even at 15 μM Ca2+ has apparently not reached a saturated response. (F) T2 values computed from the image data of E, for sensor variants 3X1:1M (filled circles) and 3X2:1M (open circles).

Similar articles

See all similar articles

Cited by 58 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback