2.3. Nanoparticle Characterization

The iron content of the particle dispersions was colorimetrically determined using the phenanthroline method [36]. The hydrodynamic diameter distribution, weighted by volume (d_V), the intensity-weighted mean hydrodynamic size (Z-Average), the polydispersity index (PDI), and the ζ-potential of the MNP were determined by dynamic light scattering (DLS) on a Zetasizer Nano ZS particle analyzer (Malvern Instruments, Worcestershire, UK). For DLS, PDI and Z-Average measurement, MNP dispersions were diluted with Milli-Q water to a final concentration of 1 mmol Fe/L. For ζ-potential measurement, MNP dispersions were diluted with 10 mM NaCl to a final concentration of 1 mmol Fe/L and adjusted to a pH of about 7.20 with NaOH. Short- and long-term stability of the MNP dispersions were investigated by visual inspection and by DLS. Nanoparticle size, morphology and phase were analyzed by transmission electron microscopy (TEM) using a TECNAI G2 20 S-Twin and a TITAN 80–300 (FEI Company, Hillsboro, OR, USA). ¹H-NMR T1- and T2-relaxation rates were measured with a Minispec MQ 40 Time-Domain Nuclear magnetic resonance (TD-NMR) spectrometer at 40 °C and 0.94 T (Bruker, Karlsruhe, Germany). Relaxivities (relaxation coefficients) r1 and r2 were determined by linear fitting of T1- and T2-relaxation rates as functions of iron concentrations. MNP were also analyzed by magnetic particle spectroscopy (MPS) to obtain information on their response to alternating magnetic fields. MPS measurements were performed on undiluted samples using a magnetic particle spectrometer (MPS-3, Bruker BioSpin, Germany) at 12 mT, 25 kHz and 37 °C for 10 s. For measurement the samples were filled in Life Technologies polymerase chain reaction (PCR) tubes with sample volumes of 30 μL. The amplitude of the magnetic moment was normalized to the iron content of each sample, resulting in the spectrum of the magnetization, M_k, which is given in Am²/mol(Fe).

To obtain an adequate reference for the immobilized state of MCP-PEG10K2 in organs, MNP were immobilized in polyacrylamide gel (PAA). For this, 94 µL acrylamide solution (30% in water), 94 µL N-N′-methylenebisacrylamide solution (2% in water), 9.5 µL ammonium persulfate (1% in water), 24 µL water, 60 µL MCP-PEG10K2 dispersion (10.6 mmol (Fe)/L), and 18.5 µL N,N,N′,N′-tetramethyletylenediamine (1:30 diluted with water (v/v)) were mixed and subsequently vortexed. Finally, 50 µL of the resulting dispersion was filled in a measuring cuvette and polymerized at 60 °C in a water bath for approx. 3 min. For M(H) measurement, 75 µL sample volumes were filled in a polycarbonate capsule. The magnetic moment of each sample was measured using an MPMS (Magnetic Property Measurement System, Quantum Design, San Diego, CA, USA) consecutively increasing the applied magnetic field from 0 to 5 T. The background signal caused by empty capsules, diamagnetic susceptibility of the dispersion medium, and demineralized water, was subtracted from the signal obtained for the samples. The resulting data represent MNP magnetization and were normalized to the iron content of the sample, which allows quantitative evaluation of the data. Since the M(H) did not saturate even at highest measurement fields, the value of M, at H = 448 kA/m was taken as a substitute of the saturation magnetization (M_S). This field value was found to be a good compromise between a small change of M with H and a low uncertainty. Note that the uncertainty increases with the field value because of potential errors during background subtraction. Because of possible small deviation of the sample from the center position in the MPMS, we give an uncertainty of the absolute values of 5%. FTIR measurements were performed using a Bruker ALPHA spectrometer (model A250/D, Bruker Optik GmbH, Leipzig, Germany) equipped with a diamond ATR sampling module (model A220/D-01, Bruker Optik GmbH, Leipzig, Germany). Measured data were processed using the OPUS 6.5 software package (Bruker Optik GmbH, Leipzig, Germany). Background and baseline correction as well as atmospheric compensation were applied to all spectra. Spectra were acquired in absorbance mode and converted to transmission mode from 23 coadded scans between 4000 and 375 cm⁻¹.