Phenotypic study of gingival fibroblasts labeled with superparamagnetic anionic nanoparticles

Methods: We evaluated after labeling transcription and secretion of connective tissue remodeling molecules, i.e. Matrix Metalloproteinases (MMPs), Tissue Inhibitors of Metalloproteinases (TIMPs) and cytokines controlling their activation/inhibition, i.e. Transforming Growth Factor β (TGF-β1), Tumor Necrosis Factor α (TNF-α), Interleukins 1-β and 4 (IL-1β and IL-4). Proliferation kinetic realized, cellular uptake was studied by Perls coloration and magnetophoresis on labelled culture. Then Dot Blotting, Western Blotting and Zymography were used to detect MMP-1, -2, -3, TIMP-1 and -2 secretions in culture supernatants, and RT-PCR was performed to detect the mRNA expression of these molecules. Elisa tests were used to determine TGF-β1, TNF-α, IL-1β and IL-4 levels.

Results: Our data indicated high (15.3±5.8 pg/cell) but heterogeneous distribution of nanoparticles in HGF. Twenty four hours after labeling, MMP-1, -2, -3 and TIMP-2 secretion increased (p<0.001) with RT-PCR confirmation at h12; while TIMP-1 did not. IL-1β increased at day 1 (D1) (p<0.001) and IL-4 at day 3 (D3) (p<0.01), but not TGF-β1 and TNF-α.

Conclusions: After labeling with these maghemite nanoparticles, HGF increased secretion of IL-1β at D1, probably inducing MMP-1, -2, -3 and TIMP-2 increase. IL-4 secretion increase began with MMPs and TIMPs synthesis decrease at D3. Despite this transitory inflammatory reaction the three days following internalization, maghemite nanoparticles do not affect HGF phenotype, authorizing their use as labellers.