Novel Fe3O4 Nanoparticles with Bioactive Glass-Naproxen Coating: Synthesis, Characterization, and In Vitro Evaluation of Bioactivity

Abstract

Immune response to biomaterials, which is intimately related to their surface properties, can produce chronic inflammation and fibrosis, leading to implant failure. This study investigated the development of magnetic nanoparticles coated with silica and incorporating the anti-inflammatory drug naproxen, aimed at multifunctional biomedical applications. The synthesized nanoparticles were characterized using various techniques that confirmed the presence of magnetite and the formation of a silica-rich bioactive glass (BG) layer. In vitro studies demonstrated that the nanoparticles exhibited bioactive properties, forming an apatite surface layer when immersed in simulated body fluid, and biocompatibility with bone cells, with good viability and alkaline phosphatase activity. Naproxen, either free or encapsulated, reduced nitric oxide production, an inflammatory marker, while the BG coating alone did not show anti-inflammatory effects in this study. Overall, the magnetic nanoparticles coated with BG and naproxen showed promise for biomedical applications, especially anti-inflammatory activity in macrophages and in the bone field, due to their biocompatibility, bioactivity, and osteogenic potential.

Keywords: biocompatibility; magnetic nanoparticles; naproxen; silica.

Figure 1

Schematics of Fe₃O₄ nanoparticles with bioactive glass-naproxen coating and the parameters considered in experimental design.

Figure 2

For iron oxide nanoparticles (mag0): (a) HRTEM images for mag0; and (b) SAD with inverted contrast. (c): Radial profiles of the SAD patterns and simulated electron diffraction profiles for standard magnetite and maghemite (both with a centric setting space group of *Fd-3 m) as a function of the reciprocal d *—spacings (dhkl* = dhkl ⁻¹). The intensities for all patterns were normalized in relation to that of (113) peak reflection.

Figure 3

TEM micrographs of the samples: (a) mag0; (b) mag1; (c) MBG.

Figure 4

SEM micrographs of heat-treated (700 °C) MBG samples: (a) before (MBG0) and after being soaked in SBF for different days: (b) 7 days (MBG7) and (c) 14 days (MBG14). The insets show the respective EDS spectra obtained for the samples’ surface.

Figure 5

ATR-FTIR spectra of (a) mag0; (b) TMAOH; (c) mag1; and of MBG samples soaked in SBF for different time periods: (d) 0 days (MBG0); (e) 7 days (MBG7); and (f) 14 days (MBG14).

Figure 6

Powder X-ray diffraction patterns of: (a) mag0; (b) mag1; and of coated MBG samples before (MBG0) (c) and after soaking immersion in SBF for 7 days, (MBG7) (d) and for 14 days (MBG14) (e).

Figure 7

ATR-FTIR spectra of samples: (a) naproxen; (b) MBG; (c) MBG–naproxen.

Figure 8

ATR-FTIR spectra of samples: (a) naproxen; (b) MBG; (c) MBG–naproxen.

Figure 9

Cell viability evaluated by MTT in RAW 264.7 (a,b), MC3T3-E1 (c,d), and Saos-2 (e,f) cells exposed to MBG, naproxen, and MBG–naproxen for 24 h and 48 h at the subsequent concentrations of 25, 50, and 100 μg.mL⁻¹. Results represent the mean ± SD of triplicates of the experiments, and the mean cell viability was normalized by the mean viability of the control group. The viability control used was PBS, and the cytotoxicity control used was TritonTM X-100 or DMSO 50%. (*) denotes a significant difference compared with the viability control (p ≤ 0.05), and (#) denotes a significant difference in relation to the cytotoxicity control (p ≤ 0.05) as determined by a two-way ANOVA followed by a Tukey post-test. A dashed line was used to identify the 70% viability limit according to ISO10993-5.

Figure 10

Nitrite levels evaluated in RAW 264.7 cells after 24 h (a) and 48 h (b) of MBG, naproxen and MBG–naproxen treatment at 25, 50, and 100 μg.mL⁻¹. (*) Statistical difference in relation to untreated, not stimulated with LPS + IFN-γ control (p ≤ 0.05) and (#) statistical difference in relation to untreated, stimulated with LPS + IFN-γ control (p ≤ 0.05), as determined by a one-way ANOVA followed by a Dunnett’s post-test. Each bar shows the mean ± SD of triplicates of experiments.

Figure 11

Prussian blue staining in RAW 264.7 cells for intracellular iron detection after 24 and 48 h of exposition with MBG, naproxen, and MBG–naproxen treatment at 25, 50, and 100 μg.mL⁻¹. Scale bar 10 μm.

Figure 12

Alkaline phosphatase activity evaluated by BCIP-NBT in MC3T3-E1 cells exposed with MBG, naproxen and MBG–naproxen after 3 (a) and 7 days (b) at 25 and 50 μg.mL⁻¹. Results represent the mean ± SD of duplicates of the experiments, and they were normalized by the control group (PBS). Stars denotes a significant difference compared with the control group (p ≤ 0.05) as determined by a one-way ANOVA followed by a Tukey post-test.