Road Map for the Use of Electron Spin Resonance Spectroscopy in the Study of Functionalized Magnetic Nanoparticles

Abstract

Electron paramagnetic resonance (EPR) spectroscopy is gaining increasing recognition in research on various nanostructures. In the case of iron oxide nanoparticles, EPR measurements offer the possibility of determining the magnetic phase and the exact type (Fe₃O₄, γ-Fe₂O₃, α-Fe₂O₃, or a combination) of the core material. Furthermore, the EPR technique enables the study of relaxation processes, estimation of the effective and surface anisotropy constants, and assessment of the influence of sample aging on the magnetic properties of nanoparticles. The scope of the information obtained can be further expanded by utilizing spin labeling of polymer-coated nanoparticles. By analyzing the signals from the attached nitroxide, one can determine certain properties of the coating and its interactions with the environment (e.g., body fluids, cells, tissues) and also perform imaging of nanoparticles in various media. In some cases, EPR can help monitor the encapsulation of active substances and their release processes. Unfortunately, despite the enormous potential, not all of the possibilities offered by EPR are routinely used in nanoscience. Therefore, the present article aims not only to present the current applications and existing trends but also to indicate directions for future EPR research, constituting a road map.

Keywords: EPR imaging; EPR spectroscopy; drug delivery; functionalization; iron oxide; magnetic nanoparticles.

Figure 1

EPR spectra of PEG-coated and doxorubicin-functionalized magnetite nanoparticles (core diameter of 8–12 nm) in water. Measurements performed using Bruker EMX-10 spectrometer (Billerica, MA, USA) (sweep range 650 mT, modulation amplitude 0.5 mT, time constant 20.48 ms) after field cooling procedure (5 min in 500 mT) at 230 K for the orientations of 0° and 90° with respect to the direction of the external magnetic field (authors, unpublished).

Figure 2

Result of the numerical processing of the X-band EPR signal of PEG-coated and doxorubicin-functionalized magnetite nanoparticles (core diameter of 8–12 nm) in water. CREM (computer resolution enhancement method) analysis allowed the extraction of a narrow line (g = 1.99) from the broad core signal (authors’ unpublished results).

Figure 3

X-band EPR spectra of TEMPO-labeled and silane-coated Fe₃O₄ nanoparticles in: (a) water; (c) human serum; (e) whole human blood. The narrow-sweep-range signals from nitroxides attached to nanoparticles in: (b) water, (d) serum, and (f) whole blood. All spectra recorded at 293 K (authors’ unpublished results).

Figure 4

EPR spectra of the 4-amino-TEMPO spin label attached to silane-coated magnetite nanoparticles in water: (a) at 283 K; (b) at 230 K; (c) at 230 K after CREM analysis; (d) EasySpin spectrum simulation at 230 K (experimental data adapted from [34]).

Figure 5

Results of EPR imaging of PEG-coated and 4-amino-TEMPO-functionalized Fe₃O₄ nanoparticles (core diameter 14 nm) in hydrogel (sodium alginate and calcium chloride). Measurements were carried out using an L-band (1 GHz) continuous-wave Bruker ELEXSYS E540L spectrometer (field gradient 1 mT/cm) in 30 min time intervals. Pseudocolor surface plots show the changes in the signal intensity distribution of 10 mL of the nanoparticle dispersion (5 mmol/mL) in the sample over time: (a) 0 min, (b) 30 min, (c) 60 min and (d) 90 min (authors’ unpublished results).

Figure 6

EPR spectroscopy supported by the spin-labeling technique can facilitate the study of the interaction of nanoparticles with biological surroundings. The signal from nitroxide (TEMPO or its derivatives) clearly varies depending on the environment. The powder spectrum has the form of a single line. In solution, we observe a characteristic triplet. When TEMPO is attached to the nanoparticle, the high field line is distorted due to hindered rotation of the nitroxide and the anisotropic components of the ĝ and  tensors not being fully averaged. EPR is an efficient tool for monitoring nanoparticle endocytosis and redox processes occurring inside cells. For example, incubation of yeast in the presence of TEMPO-functionalized Fe₃O₄ particles performed at 310 K results in the recombination of spin labels (figure based on authors’ own research).