Effect of Artemisinin-Loaded Mesoporous Cerium-Doped Calcium Silicate Nanopowder on Cell Proliferation of Human Periodontal Ligament Fibroblasts

Abstract

Ion doping has rendered mesoporous structures important materials in the field of tissue engineering, as apart from drug carriers, they can additionally serve as regenerative materials. The purpose of the present study was the synthesis, characterization and evaluation of the effect of artemisinin (ART)-loaded cerium-doped mesoporous calcium silicate nanopowders (NPs) on the hemocompatibility and cell proliferation of human periodontal ligament fibroblasts (hPDLFs). Mesoporous NPs were synthesized in a basic environment via a surfactant assisted cooperative self-assembly process and were characterized using Scanning Electron Microscopy (SEM), X-ray Fluorescence Spectroscopy (XRF), Fourier Transform Infrared Spectroscopy (FT-IR), X-ray Diffraction Analysis (XRD) and N2 Porosimetry. The loading capacity of NPs was evaluated using Ultrahigh Performance Liquid Chromatography/High resolution Mass Spectrometry (UHPLC/HRMS). Their biocompatibility was evaluated with the MTT assay, and the analysis of reactive oxygen species was performed using the cell-permeable ROS-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). The synthesized NPs presented a mesoporous structure with a surface area ranging from 1312 m²/g for undoped silica to 495 m²/g for the Ce-doped NPs, excellent bioactivity after a 1-day immersion in c-SBF, hemocompatibility and a high loading capacity (around 80%). They presented ROS scavenging properties, and both the unloaded and ART-loaded NPs significantly promoted cell proliferation even at high concentrations of NPs (125 μg/mL). The ART-loaded Ce-doped NPs with the highest amount of cerium slightly restricted cell proliferation after 7 days of culture, but the difference was not significant compared with the control untreated cells.

Keywords: artemisinin loading; cerium doping; hemocompatibility; human periodontal ligament fibroblasts; mesoporous nanopowders.

Figure 1

Representative SEM micrographs of the synthesized mesoporous nanopowders. (a) Si-NP, (b) SiCa-NP, (c) SiCaCe1-NP, (d) SiCaCe2.5-NP, (e) SiCaCe5-NP.

Figure 2

FT-IR spectra of NPs.

Figure 3

Wide- (A) and small-angle (B) XRD of NPs.

Figure 4

N₂ adsorption-desorption isotherms of MSNs.

Figure 5

FTIR spectra of all NPs before (0 days) and after (1 day) immersion in simulated body fluid (c-SBF).

Figure 6

(A) Loading Capacity of Artemisinin in different groups of mesoporous NPs, (B) Artemisinin release capacity (%) of mesoporous NPs in a time range of up to 96 h.

Figure 7

Hemocompatibility assay of NPs and ART-loaded NPs after 60 min of incubation at 37 °C). ** indicates statistically significant difference (p < 0.001) between treated cells and untreated (controls), while different letters suggest statistically significant differences (p < 0.001) among concentrations.

Figure 8

MTT results of cell viability at different concentrations of nanopowders (NPs) (μg/mL). The lines with * above bars indicate statistically significant differences (p < 0.05) of cell viability among the treated cells with the different NPs and concentrations and the untreated cells (controls), while different letters above bars suggest statistically significant differences (p < 0.05) of cell viability among the cells treated with different concentrations of NPs. Same letters above the bars suggest that cell viability did not differ significantly among the specific NPs and associated concentrations.

Figure 9

Extracellular ROS (%) levels after 1, 3, 5 and 7 days of different concentrations (12.5, 60, 125 μg/mL)) of NPs in contact with hPDLFs. The lines with * above bars indicate statistically significant differences (p < 0.05) of ROS % among the treated cells with the different NPs and concentrations and the untreated cells (controls), while different letters above bars suggest statistically significant differences (p < 0.05) of ROS % among the cells treated with different concentrations of NPs. Same letters above the bars suggest that ROS production did not differ significantly among the specific NPs and associated concentrations.