Characterization and antitumor effect of doxorubicin-loaded Fe3O4-Au nanocomposite synthesized by electron beam evaporation for magnetic nanotheranostics

Abstract

Magnetic nanocomposites (MNC) are promising theranostic platforms with tunable physicochemical properties allowing for remote drug delivery and multimodal imaging. Here, we developed doxorubicin-loaded Fe₃O₄-Au MNC (DOX-MNC) using electron beam physical vapor deposition (EB-PVD) in combination with magneto-mechanochemical synthesis to assess their antitumor effect on Walker-256 carcinosarcoma under the influence of a constant magnetic (CMF) and electromagnetic field (EMF) by comparing tumor growth kinetics, magnetic resonance imaging (MRI) scans and electron spin resonance (ESR) spectra. Transmission (TEM) and scanning electron microscopy (SEM) confirmed the formation of spherical magnetite nanoparticles with a discontinuous gold coating that did not significantly affect the ferromagnetic properties of MNC, as measured by vibrating-sample magnetometry (VSM). Tumor-bearing animals were divided into the control (no treatment), conventional doxorubicin (DOX), DOX-MNC and DOX-MNC + CMF + EMF groups. DOX-MNC + CMF + EMF resulted in 14% and 16% inhibition of tumor growth kinetics as compared with DOX and DOX-MNC, respectively. MRI visualization showed more substantial tumor necrotic changes after the combined treatment. Quantitative analysis of T₂-weighted (T₂W) images revealed the lowest value of skewness and a significant increase in tumor intensity in response to DOX-MNC + CMF + EMF as compared with the control (1.4 times), DOX (1.6 times) and DOX-MNC (1.8 times) groups. In addition, the lowest level of nitric oxide determined by ESR was found in DOX-MNC + CMF + EMF tumors, which was close to that of the muscle tissue in the contralateral limb. We propose that the reason for the relationship between the observed changes in MRI and ESR is the hyperfine interaction of nuclear and electron spins in mitochondria, as a source of free radical production. Therefore, these results point to the use of EB-PVD and magneto-mechanochemically synthesized Fe₃O₄-Au MNC loaded with DOX as a potential candidate for cancer magnetic nanotheranostic applications.

Fig. 1. Schematic representation of Fe₃O₄–Au MNC synthesis via EB-PVD: (A) iron and sodium chloride are vaporized by an electron beam, then the vapors condense on a cooled substrate, forming iron nanoparticles embedded in NaCl matrix; (B) following oxidation the nanoparticles are coated with gold evaporated from a reactor.

Fig. 2. TEM image (A) and electron pattern (B) of Fe₃O₄ nanoparticles synthesized using EB-PVD.

Fig. 3. SEM images with increasing magnification from (A) and (B) of Fe₃O₄–Au MNC: EB-PVD formed island structures of gold (bright spots) on the surface of magnetite nanoparticles (dark spots).

Fig. 4. TEM, FFT images and size distributions of Fe₃O₄–Au MNC before (A, A′, C and E) and after DOX loading (B, B′, D and F): TEM images with increasing magnification from A to A′ and B to B′; FFT images (C and D): yellow marks correspond to the contributions of gold and red marks correspond to the contributions of Fe₃O₄; size (nm) distributions (E and F) of Au and Fe₃O₄ nanoparticles comprising MNC.

Fig. 5. Hydrodynamic size (D) of Fe₃O₄–Au MNC in aqueous solution.

Fig. 6. Magnetic hysteresis loop measured for Fe₃O₄–Au MNC at room temperature.

Fig. 7. Growth kinetics of Walker-256 carcinosarcoma: (1) – control (no treatment); (2) – DOX; (3) – DOX-MNC; (4) – DOX-MNC + CMF + EMF.

Fig. 8. Changes in body weight of Walker-256 carcinosarcoma-bearing animals relative to their weight before implantation. *Significant difference from control, p < 0.05; ^&significant difference from DOX, p < 0.05; ^§significant difference from DOX-MNC, p < 0.05.

Fig. 9. Representative T₂W coronal whole-body MRI scans of Walker-256 carcinosarcoma-bearing rats acquired at 18 days after tumor implantation: control (A), DOX (B), DOX-MNC (C), DOX-MNC + CMF + EMF (D). Red circles (1) indicate the tumor ROI; green circles (2) denote the muscle ROI in the contralateral limb; yellow circles outline the background ROI (3).

Fig. 10. Image histograms of tumor ROIs on T₂W MRI scans: control (A); DOX (B); DOX-MNC (C); DOX-MNC + CMF + EMF (D).

Fig. 11. Electron spin resonance spectra of Walker-256 carcinosarcoma 18 days after implantation: (1) – control (no treatment); (2) – DOX; (3) – DOX-MNC; (4) – DOX-MNC + CMF + EMF; T = 77 K.

Fig. 12. Redox state in Walker-256 carcinosarcoma 18 days after implantation: *significant difference from control, p < 0.05; ^&significant difference from DOX, p < 0.05; ^§significant difference from DOX-MNC, p < 0.05; ^#significant difference from DOX-MNC + CMF + EMF, p < 0.05.