Biocompatibility of tungsten disulfide inorganic nanotubes and fullerene-like nanoparticles with salivary gland cells

Abstract

Impaired salivary gland (SG) function leading to oral diseases is relatively common with no adequate solution. Previously, tissue engineering of SG had been proposed to overcome this morbidity, however, not yet clinically available. Multiwall inorganic (tungsten disulfide [WS2]) nanotubes (INT-WS2) and fullerene-like nanoparticles (IF-WS2) have many potential medical applications. A yet unexplored venue application is their interaction with SG, and therefore, our aim was to test the biocompatibility of INT/IF-WS2 with the A5 and rat submandibular cells (RSC) SG cells. The cells were cultured and subjected after 1 day to different concentrations of INT-WS2 and were compared to control groups. Growth curves, trypan blue viability test, and carboxyfluorescein succinimidyl ester (CFSE) proliferation assay were obtained. Furthermore, cells morphology and interaction with the nanoparticles were observed by light microscopy, scanning electron microscopy and transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy. The results showed no significant differences in growth curves, proliferation kinetics, and viability between the groups compared. Moreover, no alterations were observed in the cell morphology. Interestingly, TEM images indicated that the nanoparticles are uptaken by the cells and accumulate in cytoplasmic vesicles. These results suggest promising future medical applications for these nanoparticles.

FIG. 1.

Typical TEM images of (A) IF-WS₂ and (B) INT-WS₂. IF, inorganic fullerene; INT, inorganic nanotubes; TEM, transmission electron microscopy; WS₂, tungsten disulfide.

FIG. 2.

Growth curves of cells with and without IF/INT-WS₂. The density variation by day of A5 (A) and RSC (B) cells cultured with different concentrations of INT-WS₂ (A) and IF-WS₂ (B) until reaching confluence. Data are expressed as the average cell density±standard error of the mean (n≥4). RSC, rat submandibular cells. Color images available online at www.liebertpub.com/tea

FIG. 3.

CFSE proliferation assay on A5 cells with and without INT-WS₂. CFSE dilution on A5 cells cultured with different concentrations of INT-WS₂ until reaching confluence on day 3. As the cells divide, there is a reduction in the amount of fluorescence. Unstained cells have a small amount of autofluorescence as demonstrated and served as a control. Data demonstrate one representative result from three duplicates of each group with negligible standard deviations. CFSE, carboxyfluorescein succinimidyl ester. Color images available online at www.liebertpub.com/tea

FIG. 4.

A5 cells morphology is unaffected by the nanoparticles. Light microscopy (A, B) and SEM (C, D) images of A5 cells with INT-WS₂. (A) Second day from culture. (B) Fourth day from culture. Arrow in (A) indicates a single nanotube, whereas arrow in (B) most likely points to a platelet of WS₂. Scale bars: (A, B) 100 μm, (C) 20 μm, (D) 5 μm. SEM, scanning electron microscopy.

FIG. 5.

RSC cells morphology is unaffected by the nanoparticles. Light microscopy (A, B) and SEM (C, D) images of RSC cells with IF-WS₂. (A) Second day from culture. (B) Fourth day from culture. The arrows probably point to platelets of WS₂. Scale bars: (A, B) 100 μm, (C) 20 μm, (D) 5 μm.

FIG. 6.

The cellular area of A5 and RSC cells with/without the nanoparticles. Light microscopy images of A5 cells (A) and RSC cells (B) upon reaching confluence (day 5 for A5 cells and day 4 for RSC cells) were analyzed. Cells were chosen in a random manner from each image and measured using NIH ImageJ software. Data are expressed as the average cell area±standard error of the mean (number of cells=35).

FIG. 7.

Contact between A5 cells and INT-WS₂. (A) Environmental scanning electron microscopy (ESEM) images (using backscattered (BSE) and secondary (SE) detectors as indicated in the black box) of an A5 cell grown in a medium with 35.2 μg/mL INT-WS₂. The EDS of areas 1 (white rectangle, control) and 2 (black rectangle, nanotube aggregate) are shown in panel (B). The compounds, tungsten (W, M, and L shells) and sulfur (S and K shells), show peaks in the EDS of (B2)—area of cell in contact with nanotube but do not appear in (B1)—control area of cell. EDS, energy dispersive X-ray spectroscopy.

FIG. 8.

Contact between a RSC cell and IF-WS₂. (A) ESEM images (using BSE and SE detectors as indicated in the black box) of a RSC cell grown in a medium with 100 μg/mL IF-WS₂. The EDS of areas 1 (control) and 2 (IF-WS₂) are shown in panel (B). The elements tungsten (W, M, and L shells) and sulfur (S and K shells) show peaks in the EDS of (B2)—area of cell in contact with the nanoparticle but do not appear in (B1)—control area of cell.

FIG. 9.

INT-WS₂ are uptaken by A5 cells. TEM images of A5 cells without (I) or with (II–VI) 35.2 μg/mL INT-WS₂. The dashed circles delimit an area that is enlarged in another panel (as indicated). The white arrows are pointing at the nanoparticles, whereas the black arrows show the intracellular membrane surrounding them. The dashed line in panel (IV) is located on a tight junction between two cells.

FIG. 10.

IF-WS₂ are uptaken by RSC cells. TEM images of RSC cells without (I) or with (II–VI) 100 μg/mL IF-WS₂. The dashed circles delimit an area that is enlarged in another panel (as indicated). The white arrows pointing to nanoparticle aggregates.