Relaxivities of Suspended Iron Oxides
Commonly extrapolated NMRD profiles are used to estimate the relaxivities of iron oxide contrast agents at high field strengths. Experimental data on iron oxides above 7 T is lacking. In this study, we found a plateau of r2 and a decrease of r1 between 7 T and 17.6 T. This is in line with the observations of others and the estimations obtained from nuclear magnetic relaxation dispersion (NMRD) profiles.
The molar relaxivities of iron oxide nanoparticles often depend on the size and composition of their iron oxide core (16). Both r1 and r2 increase with the size of the Fe3O4/Fe2O3 core; however, r1/r2 decreases due to the stronger increase in r2. This fact might explain the higher T2 relaxivities of AMNPs observed by us and others (9) when compared with that of VSOPs or SPIOs reported elsewhere (3, 10). As shown in Table 2, our results on r1 and r2 are consistent with the values reported by Billotey et al. (9) at lower field strengths. Furthermore, the size and composition of the coating material has been shown to significantly influence the T1 and T2 behavior of SPIOs (16). This is discussed below in detail.
Finally, the diffusion coefficients of the media used to dilute the contrast agent impacts their “apparent” relaxivities (5). This fact may be accountable for differences in the relaxivities obtained in our study when compared with measurements performed in pure water. To be able to provide constant results on free and internalized contrast agents, we chose to perform all our experiments in Ficoll solution.
Relaxivities of Intracellular Iron Oxides
Importantly, no clusters of cells or local differences in signal intensities due to inhomogeneous distribution of cells in Ficoll solution were observed within a 30-min MR-measurement time. Rather than using histological evaluation to exclude local differences in cell distribution, we preferred the use of susceptibility-sensitive gradient echo sequences with a long TE of 6 ms. Preparation of histological samples (smear slides) can potentially disperse cell clusters in Ficoll solution. Thus, clusters that can be detected with MR measurements might be undetectable in histology.
Previous studies at lower field strengths have covered the topic of changes in the relaxation behavior of SPIOs after cellular internalization (10, 17). These observed differences impact not only the choice of pulse sequences and imaging parameters in cell-tracking studies but also the quantification of contrast agent uptake via in vitro or in vivo relaxometry depends on knowledge of changes in relaxivities after cellular uptake (18). While Simon et al. (10) showed a decreased difference for r1 between 1.5 T (−79%) and 3 T (−48%), and a constant 2- to 3-fold reduction in r2, Brisset et al. (4) observed increasing differences between 4.7 T (r1: −62%; r2: −33%) and 7 T (r1: −69%; r2: −55%).
Measurements on intracellular relaxivities are more prone to changes in the experimental setup when compared with relaxometry on freely dispersed iron oxides. An extended number of parameters must be considered, such as the absolute number of cells, the amount of intracellular iron, and the volume fraction of the contrast agent in proportion to the surrounding medium. Our results suggest a pronounced r2 reduction after cellular internalization at field strengths above 7 T and a constant r1 reduction for Rh-AMNPs measured above 7 T. These results extend the observations made by Brisset et al. beyond 7 T.
Comparison of Relaxivities: Internalized Versus Suspended
In the experiments, a decrease of r1 was observed for suspended and internalized contrast agents by increasing field strength. This is in line with the behavior explained by the outer sphere relaxation theory (19, 20). As previously stated, the decrease of r1 with internalization has also been observed and previously described. This is explained by multicompartment exchange models (4, 9). Because of the internalization of the nanoparticles into endosomes, an intraendosomal compartment with large r1 but small volume fraction and another extraendosomal compartment with large volume fraction but small r1 are present. Although water protons are exchanged between these compartments, the effects of intraendosomal compartmentalization manifest in a reduced “apparent” r1 of the entire system. However, the overall relaxation time also decreases by increasing field strength because of the significant decrease of the relaxation time of the inner compartment containing the contrast agent at the fields strengths investigated in this work.
In our experiments, the behavior of r2 with increasing field strength was different for the internalized contrast agent when compared with noninternalized. While the r2 of the free contrast agent shows a plateau, the internalized contrast agent exhibits a significant decrease of r2 with increasing field strength. A possible explanation is that at the fields strengths investigated in this work, two different processes with different behaviors contribute to r2. The first is the quantum mechanical contribution described by outer sphere relaxation theory (6, 19–21). The second is the classical incoherent spin dephasing while diffusing in an inhomogeneous magnetic field (22–24).
The second effect has been thoroughly theoretically (22–26) and experimentally (9, 11) investigated. The incoherent spin dephasing depends on the size of the particle and the susceptibility difference between the nanoparticle and the surrounding water. For the field strengths investigated in this work (7 T up to 17.6 T), the magnetization of the superparamagnetic particles is saturated and the susceptibility difference does not alter significantly. Thus, one assumes that the part of r2 arising from incoherent spin dephasing should exhibit no dependence on the field strength of the external field. Incoherent spin dephasing, however, shows a significant dependence on the particle size.
The correlation time of the spin diffusion process inside such field inhomogeneities is given by τ = R2/D, where R is the typical size of the object creating the field inhomogeneity and D is the diffusion coefficient (27). To efficiently refocus the spins in this case and thus suppress the incoherent spin dephasing, the echo time of the spin echo must be shorter than the correlation time (22, 28). For suspended contrast agents, the correlation time ranges from nanoseconds to microseconds. Thus, with normal imaging sequences, the incoherent spin dephasing is unsuppressed and significantly contributes to r2.
However, if the contrast agent is internalized, the iron oxide nanoparticles concentrate in the cells, forming a large object the size of an endosome or even an entire cell. These objects are in the micrometer range with correspondingly large field inhomogeneities. Thus, the correlation time of the diffusion around these objects (cell or endosome) ranges from microseconds to milliseconds. For such correlation times, incoherent spin dephasing is suppressed using multispin echo sequences (22, 28–30). Thus, at all field strengths, a significant drop in r2 occurs if the nanoparticles are internalized in cells.
The quantum mechanical contribution described by outer sphere theory shows a similar behavior due to internalization of the contrast agent in cells. This effect is analogous to r1 (4, 9) and should lead to a drop in r2 if the contrast agent is internalized. The dependence on the field strength, however, is different when compared with that of incoherent spin dephasing. Analogous to r1, a decrease in r2 with increasing field strength is theoretically predicted (6, 19–21). In contrast to r1, however, r2 decreases to a nonvanishing value in the high field limit.
Taking both contributions for the relaxation rate r2 into account might explain the different behavior for internalized and free iron oxide nanoparticles. For free iron oxide nanoparticles, the experiments show a plateau in r2 and no significant dependence on the field strength. This implies that, in this case, incoherent spin dephasing dominates the r2 relaxivity.
For internalized iron oxide nanoparticles, the experiments show a significant drop of r2 at all field strengths when compared with the suspended contrast agent. This is in agreement with both mechanisms, the incoherent spin dephasing and the outer sphere relaxation theory when it is combined with multicompartment exchange models. The observable decrease of r2 with increasing field strength, however, may indicate that incoherent spin dephasing (9, 11, 23, 24) is suppressed to an extent where the effects leading to a field strength dependence of the r2 relaxation (6, 19–21) could be observed.
It should be noted that the decrease in r2 with increasing field strength observed in our study is stronger than that expected from theory. A quantitative description, however, of this effect is difficult as, to the best of the authors' knowledge, a single theoretical model that includes both contributions for contrast agents internalized in cells is unavailable. Thus, further investigation must be performed to fully understand this behavior.
It would be of interest to investigate the dependence of the r1/r2 ratio on different field strengths for suspended and internalized contrast agents. While in the suspended case the ratio decreases with increasing field strength, it remains relatively constant when the contrast agent is internalized. This is surprising as this effect is neither predicted by outer sphere theory nor by the incoherent spin dephasing. However, as the r1/r2 ratio is very low at these field strengths (cf. Table 1), the r1 effect of the investigated internalized contrast agent might be of no practical interest. Nevertheless, this behavior is interesting from a theoretical point of view and thus should be further investigated.
The determination of r2* was beyond the scope of this study. This is mainly due to technical difficulties when trying to quantify T2* on high-field systems. The homogeneity of the main magnetic field becomes critically necessary at field strengths up to 17.6 T. So far, we cannot guarantee sufficient field homogeneity for all three field strengths used in this study.
Comparison of Rh-AMNPs with AMI-25 and VSOP-C200
As previously shown, size (31, 32) and surface charge are major determinants of the cellular uptake of iron oxides (33). Although we used a standardized label protocol for all contrast agents, the iron content (picogram per cell) showed variations between the different types of contrast agents. Remarkably, smaller iron oxides (AMNP, VSOP) showed higher uptake, which is most likely due to their anionic surface coating (32–34).
The variation of intracellular iron concentrations might hamper comparability between the three contrast agents. Importantly, regarding Rh-AMNPs, we investigated the impact of constant iron content (picogram per cell) and varying amounts of cells. The impact of the opposite situation (constant numbers of cells, varying pg Fe per cell) on r1 and r2 relaxivities was not within the scope of this study.
We believe that our experiments more accurately represent the in vivo situation or cell transplantation studies as a defined mean iron concentration per cell would likely be achieved in these scenarios. Kuhlpeter et al. (18) stated that only the total internalized iron amount per volume influences r2/r2*. Bowen et al., however, demonstrated a difference in the R2 relaxation rates for a constant total internalized iron amount. The difference is dependent on whether the cell number is varied or the iron load of the cells (11). This issue has not been covered in our work and should be investigated in further studies.
The different coating materials, hydrodynamic diameters, and iron cores must also be accounted for as they predominately impact the relaxation properties of SPIOs. Differences in iron core diameters might further explain the less pronounced decrease in r2 of commercially available AMI-25 and VSOPs when compared with Rh-AMNPs. The 2- to 30-fold reduction of the molar relaxivities of three iron oxide contrast agents at 17.6 T, however, allows us to conclude that internalization of iron oxides leads to a distinct decrease in r1 and r2 at very high field strengths.
Although the volume fractions of cells seem low in our experimental setup (0.17% to 1.32% by volume), the absolute numbers and volumes used in this study seem appropriate with respect to possible in vivo conditions (e.g., in vivo labeled macrophages) or cell transplantation experiments (10). Nevertheless, results may significantly vary when performed with different volume fractions of cells.
Finally, we recognize that the iron loading levels of 0.16–1.47 pg Fe per cell are lower than those typically found with similar nanoparticles and similar phagocytic cell lines (35). Higher levels may be achieved by varying the iron oxide concentration (36) or incubation times. The r1 and r2 findings in this article must be understood in this context. Furthermore, the principal finding of strongly decreased r1 and r2 on cellular compartmentalization at very high field may vary with higher cellular iron oxide loading levels.