Microwave-assisted green synthesis of superparamagnetic nanoparticles using fruit peel extracts: surface engineering, T 2 relaxometry, and photodynamic treatment potential

Abstract

Superparamagnetic iron oxide nanoparticles (SPIONs) have the potential to be used as multimodal imaging and cancer therapy agents due to their excellent magnetism and ability to generate reactive oxygen species when exposed to light. We report the synthesis of highly biocompatible SPIONs through a facile green approach using fruit peel extracts as the biogenic reductant. This green synthesis protocol involves the stabilization of SPIONs through coordination of different phytochemicals. The SPIONs were functionalized with polyethylene glycol (PEG)-6000 and succinic acid and were extensively characterized by X-ray diffraction analysis, field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, atomic force microscopy, Rutherford backscattering spectrometry, diffused reflectance spectroscopy, fluorescence emission, Fourier-transform infrared spectroscopy, ultraviolet-visible spectroscopy, and magnetization analysis. The developed SPIONs were found to be stable, almost spherical with a size range of 17-25 nm. They exhibited excellent water dispersibility, colloidal stability, and relatively high R 2 relaxivity (225 mM(-1) s(-1)). Cell viability assay data revealed that PEGylation or carboxylation appears to significantly shield the surface of the particles but does not lead to improved cytocompatibility. A highly significant increase of reactive oxygen species in light-exposed samples was found to play an important role in the photokilling of human cervical epithelial malignant carcinoma (HeLa) cells. The bio-SPIONs developed are highly favorable for various biomedical applications without risking interference from potentially toxic reagents.

Keywords: MRI contrast agent; bio synthesis; biocompatible; green approach; microwave incubation; multifunctional; photosensitive SPIONs; surface modification.

Figure 1

Schematic diagram of the work. Abbreviations: MRI, magnetic resonance imaging; PDT, photodynamic therapy; SPIONs, superparamagnetic iron oxide nanoparticles.

Figure 2

Structural and optical characterization of SPIONs. Notes: (A) XRD patterns of the samples, (B) Rutherford backscattering spectra, and (C, D) diffuse reflectance spectra showing the composition and bandgap energies of the synthesized bio-SPIONs. Abbreviations: AP-SPIONs, apple peel-mediated SPIONs; LP-SPIONs, lemon peel-mediated SPIONs; OP-SPIONs, orange peel-mediated SPIONs; PP-SPIONs, pomegranate peel-mediated SPIONs; SPIONs, superparamagnetic iron oxide nanoparticles; XRD, X-ray diffraction.

Figure 3

Fluorescence spectra, magnetization response and FTIR spectra. Notes: (A) Fluorescence emission spectra where λ_ex =270 nm and (B) magnetization curve of bio-SPIONs. FTIR spectra of (C) PEG-SPIONs and (D) SA-SPIONs functionalized through MW incubation. Abbreviations: AP-SPIONs, apple peel-mediated SPIONs; FTIR, fourier transform infrared; LP-SPIONs, lemon peel-mediated SPIONs; MW, microwave; OP-SPIONs, orange peel-mediated SPIONs; PEG, polyethylene glycol; PP-SPIONs, pomegranate peel-mediated SPIONs; SA, succinic anhydride; SPIONs, superparamagnetic iron oxide nanoparticles.

Figure 4

FESEM images of (A) PEG-PP-SPIONs, (B) PEG-LP-SPIONs, (C) PEG-LP-SPIONs, and (D) PEG-LP-IONPs functionalized through MW incubation. Note: Transparent thin shell surrounding the NPs indicates successful PEGylation. Abbreviations: FESEM, field emission scanning electron microscopy; IONP, iron oxide nanoparticles; LP-SPIONs, lemon peel-mediated SPIONs; MW, microwave; NPs, nanoparticles; PEG, polyethylene glycol; PP-SPIONs, pomegranate peel-mediated SPIONs; SPIONs, superparamagnetic iron oxide nanoparticles.

Figure 5

AFM images of (A) SA-PP-SPIONs, (B) SA-OP-SPIONs, (C) SA-AP-SPIONs, and (D) SA-LP-SPIONs functionalized through MW incubation. Abbreviations: AFM, atomic force microscopy; AP-SPIONs, apple peel-mediated SPIONs; LP-SPIONs, lemon peel-mediated SPIONs; MW, microwave; OP-SPIONs, orange peel-mediated SPIONs; PP-SPIONs, pomegranate peel-mediated SPIONs; SA, succinic anhydride; SPIONs, superparamagnetic iron oxide nanoparticles.

Figure 6

MR studies of SPIONs. Notes: (A) T₂-weighted images of cell suspensions with different metal ion (Fe in millimoles) concentrations at 1.5 T. (B) Linear fitting of 1/T₂ as a function of Fe concentration. (C) MR transverse relaxivity (R₂) values of PEG-SPIONs functionalized through MW incubation. Abbreviations: AP-SPIONs, apple peel-mediated SPIONs; LP-SPIONs, lemon peel-mediated SPIONs; MR, magnetic resonance; MW, microwave; OP-SPIONs, orange peel-mediated SPIONs; PEG, polyethylene glycol; PP-SPIONs, pomegranate peel-mediated SPIONs; SPIONs, superparamagnetic iron oxide nanoparticles.

Figure 7

Biocompatibility of SPIONs. Notes: (A) Hemolysis results of bio-SPIONs. Relative cell viability curves of (B) bio-SPIONs, (C) PEG-SPIONs, and (D) SA-SPIONs functionalized through MW incubation. Percentage viabilities (mean ± SD) were calculated (P<0.05; two-tailed t-test). Abbreviations: AP-SPIONs, apple peel-mediated SPIONs; LP-SPIONs, lemon peel-mediated SPIONs; OP-SPIONs, orange peel-mediated SPIONs; NTC, nontreated cell; PP-SPIONs, pomegranate peel-mediated SPIONs; PEG, polyethylene glycol; SA, succinic anhydride; SPIONs, superparamagnetic iron oxide nanoparticles; SD, standard deviation; PC, positive control.

Figure 8

Photo-toxicity studies. Notes: (A) PDT effects of bio-SPIONs on HeLa cell viability. (B) Cell viability ratios in light and dark reactions (CV+/CV−) vs concentration. (C) Changes in the surviving fractions of the treated HeLa cells preincubated with and without the scavengers (NaN₃ and mannitol). (D) TBA assay results for SPION exposure. Data are expressed as percentage (mean ± SD) relative to the NTC sample (P<0.05; two-tailed t-test). Abbreviations: AP-SPIONs, apple peel-mediated SPIONs; CV, cell viability; HeLa, human cervical epithelial malignant carcinoma; LP-SPIONs, lemon peel-mediated SPIONs; NTC, nontreated cell; OP-SPIONs, orange peel-mediated SPIONs; PDT, photodynamic therapy; PP-SPIONs, pomegranate peel-mediated SPIONs; SPION, superparamagnetic iron oxide nanoparticle; TBA, thiobarbituric acid; TBARS, TBA-reactive substances; SD, standard deviation.