*3.2. Magnetic Characteristics of USPION-Treated Tissues*

Samples of the liver, left heart ventricle, kidneys, aorta, and whole arterial blood were prepared according to the procedures described above (Section 2). Magnetisation (*M*) measurements were realised similarly to the measurement of USPION dispersion. Hysteresis curves measured at 2 K for control and USPION-treated group, respectively (Figure 10a) showed almost no differences between these two groups, the saturation magnetisation (*M*s) and the magnetic coercivity (*H*c) were almost the same. However, the measurement of the temperature dependence of *M* (Figure 10b) showed considerable differences between control and USPION-treated group. ZFC and FC curves for the liver of USPION-treated rats were similar to that determined in the USPION dispersion per se at the higher temperature values and confirmed the presence of the USPIONs in the liver. There is also clearly visible maximum for the ZFC curve at 10 K (Inset of Figure 10b), which is characteristic for ferritin and confirms its presence in the liver. Liver of the control group showed only a contribution of naturally occurring ferritin and diamagnetism at the higher temperature (Figure 10b). As the temperature dependence measurement is time and liquid helium (used for cooling the MPMS) consuming, it was decided to measure the contribution of USPIONs at 300 K by measuring shortened *M*(*H*) dependence.

**Figure 10.** Magnetic properties of the liver of control and USPION-treated rats. (**a**) The mass magnetisation vs. applied magnetic field measurement at a temperature of 2 K, (**b**) the mass magnetisation vs. the temperature at the applied magnetic field of 4000 A/m. The inset shows the peak at the ZFC (zero field cooled) curve both for control and USPIONs group. Curves are the average of 5 measurements per group.

Parameters of sequence used for measurement are: RSO, scan length 4 cm, 2 × 5 scans per measurement, corresponding to every single point in the figures, frequency 1.5 Hz, measurement length of the partial *M*(*H*) dependences was 20 min, the applied field increased one-way only from 0 to 1 T. This setup, together with improved sample mounting, allows us to perform rapid and precise measurements of magnetic properties of the samples. Result of the measurements of liver samples is shown in Figure 11a. The presented data are averaged of control (*n* = 11) and USPION-treated (*n* = 9) group. In the control group, *M* decreases with the applied magnetic field linearly, which is caused by prevailing diamagnetism of the investigated samples. Data from USPION-treated rats showed initial curvature at low fields, which originated from the USPIONs. After this curvature the slope of the curve goes similar to the control group, indicating the still prevailing diamagnetism in the tissue samples over the contribution of the USPIONs, whose magnetisation saturates at the higher applied magnetic field.

Determination of USPIONs content in the treated tissue was done by subtracting of the averaged value of the mass magnetisation of control group values (*M*control) from each of the USPION samples values (Figure 11b):

$$M\_{\text{sample}}' = M\_{\text{sample}} - M\_{\text{control}} \tag{1}$$

and comparing *M*- sample with the mass magnetisation of the USPION (*M*USPIONs) dispersion measured at 300 K and 1 T field. The iron content in the USPIONs treated samples was determined using the following relation:

$$c\left[\frac{\mu\_{\rm Fe}}{\rm g}\right] = \frac{M\_{\rm sample}^{\prime} \times m\_{\rm Fe}}{M\_{\rm USPONs} \times m\_{\rm sample}} \times 10^6\tag{2}$$

where *c* is the USPIONs content in the sample (in μg of Fe per gram of the sample dry weight), *m*Fe is the mass of iron in USPION dispersion (in our case: *m*Fe = 10 μg) and *m*sample is the mass of the dried sample.

The same procedure of the measurement as for the liver was used for the samples from left heart ventricle (Figure 12) and kidney (Figure 13). In the case of kidney, the smaller cutting tool was used, with a diameter of 3.5 mm.

**Figure 11.** Magnetic properties of the liver of control and USPION-treated rats. (**a**) The partial *M*(*H*) dependences at 300 K. Data are presented as the averaged curve from the control group (*n* = 11) and USPION-treated group (*n* = 9). The mass of dry samples was in the range of 12–30 mg. (**b**) Corresponding *M*' data for individual rats of the USPION-treated group after subtraction of the control group average.

**Figure 12.** Magnetic properties of the left heart ventricle of control and USPION-treated rats. (**a**) The partial *M*(*H*) dependences at 300 K and (**b**) corresponding *M*' data for individual rats of the USPION-treated group after subtraction of the control group average. Data (a) are presented as the averaged curve from the control group (*n* = 10) and USPION-treated group (*n* = 7). The mass of dry samples was in the range of 14–25 mg.

**Figure 13.** Magnetic properties of the kidney of control and USPION-treated rats. (**a**) The partial *M*(*H*) dependences at 300 K, (**b**) corresponding *M*' data for individual rats of the USPION-treated group after subtraction of the control group average. Data (**a**) are presented as the averaged curve from the control group (*n* = 6) and USPION-treated group (*n* = 7). The mass of dry samples was in the range of 3–6 mg. The smaller cutting tool was used (diameter of 3.5 mm).

Samples of the aorta were cut to a length of 5 mm and prepared similarly to tissue samples. The measured mass magnetisation is presented in Figure 14.

**Figure 14.** Magnetic properties of the aorta of control and USPION-treated rats. (**a**) The partial *M*(*H*) dependences at 300 K, (**b**) corresponding *M*' data for individual rats of the USPION treated group after subtraction of the control group average. Data (a) are presented as the averaged curve from the control group (*n* = 4) and USPION-treated group (*n* = 4). The mass of dry samples was in the range of 1.8–2.5 mg.

Samples of blood were prepared according to Section 2.7, before preparation the blood was defrosted and homogenised using an ultrasonic bath for 60 s (50 kHz, 30 W). Measured mass magnetisation is presented in Figure 15.

**Figure 15.** Magnetic properties of the blood of control and USPION-treated rats. (**a**) The partial *M*(*H*) dependences at 300 K and (**b**) corresponding *M*' data for individual rats of the USPION treated group after subtraction of the control group average (b). Data (a) are presented as the averaged curve from the control group (*n* = 4) and USPION-treated group (*n* = 6). The mass of dry samples was in the range of 1.3–2.3 mg.

From measurements of *M* of the control groups, we derived the sensitivity of our method of determination of USPION-originated iron content as a variance of measurement changes with the magnetic field (Figure 16). Therefore, a line to the data was fitted by the weighted regression. In particular, we supposed *var*(*i*) = σ2*x*2, i.e., the weights *wi* = 1/*x*<sup>2</sup> *<sup>i</sup>* . Then, a confidence band around the fitted line *y*ˆ is determined as *y*ˆ ± 2σˆ*xi*, where the parameters of *y*ˆ and σˆ were estimated by the weighted least squares procedure. Then the upper border of the band around should be considered as a minimum value of *M* for a sample with USPION content. Value of this minimal iron content was determined using the same procedure as for the USPION treated samples. Determined sensitivity is presented in the Table 1 for each type of samples. The samples with the lower USPION content than the determined sensitivity were omitted and final mean USPION content (for dry and wet sample) is

presented in the Table 1 together with number of averaged samples. Presented are also dry and wet weights of the samples of each measured tissue and blood. For better clarity, the data for the USPION content in tissues presented also in the Figure 17.

**Figure 16.** Confidence band around (green lines) of *M* data of the liver control group (black lines for individual samples) and their mean value (red line).

**Table 1.** Summary of USPION-originated iron content in the tissues and whole blood of rats treated with USPIONs (1 mg/kg i.v., 100 min post-infusion). Mean sample mass is presented as dry and wet weight. Sensitivity of the measurement method is expressed in μg of USPION iron per g of dry sample weight. *n* is the number of samples where USPION content was determined. Results are presented as mean ± SEM.


**Figure 17.** Mean USPION-originated iron content determined in the tissues of liver, left heart ventricle (LHV), kidney, aorta and whole arterial blood. Results are presented as mean ± SEM.
