Synthesis, characterization, and bioactivity of carboxylic acid-functionalized titanium dioxide nanobelts

Abstract

Background: Surface modification strategies to reduce engineered nanomaterial (ENM) bioactivity have been used successfully in carbon nanotubes. This study examined the toxicity and inflammatory potential for two surface modifications (humic acid and carboxylation) on titanium nanobelts (TNB).

Methods: The in vitro exposure models include C57BL/6 alveolar macrophages (AM) and transformed human THP-1 cells exposed to TNB for 24 hrs in culture. Cell death and NLRP3 inflammasome activation (IL-1β release) were monitored. Short term (4 and 24 hr) in vivo studies in C57BL/6, BALB/c and IL-1R null mice evaluated inflammation and cytokine release, and cytokine release from ex vivo cultured AM.

Results: Both in vitro cell models suggest that the humic acid modification does not significantly affect TNB bioactivity, while carboxylation reduced both toxicity and NLRP3 inflammasome activation. In addition, short term in vivo exposures in both C57BL/6 and IL-1R null mouse strains demonstrated decreased markers of inflammation, supporting the in vitro finding that carboxylation is effective in reducing bioactivity. TNB instillations in IL-1R null mice demonstrated the critical role of IL-1β in initiation of TNB-induced lung inflammation. Neutrophils were completely absent in the lungs of IL-1R null mice instilled with TNB for 24 hrs. However, the cytokine content of the IL-1R null mice lung lavage samples indicated that other inflammatory agents, IL-6 and TNF-α were constitutively elevated indicating a potential compensatory inflammatory mechanism in the absence of IL-1 receptors.

Conclusions: Taken together, the data suggests that carboxylation, but not humic acid modification of TNB reduces, but does not totally eliminate bioactivity of TNB, which is consistent with previous studies of other long aspect ratio nanomaterials such as carbon nanotubes.

Figure 1

SEM image of TiO ₂ nanomaeterials; (A) the bare nanobelts, (B) the COOH-functionalized nanobelts, (C) the humic acid-coated nanobelts.

Figure 2

XRD pattern of the bare TiO ₂ nanobelts.

Figure 3

Ti 2p and O 1 s core levels of the XPS spectra obtained from the bare TiO ₂ nanobelts.

Figure 4

C 1 s, O 1 s, Si 2p, and Ti 2p core levels of the XPS spectra obtained from the COOH-TiO ₂ nanobelts.

Figure 5

Survey scan, C 1 s, O 1 s and Ti 2p core levels of the XPS spectra obtained from the humic acid-treated TiO ₂ nanobelts.

Figure 6

FT-IR spectra of the bare, the COOH-terminated and the humic acid (HA)-treated TiO ₂ nanobelts (NBs).

Figure 7

Viability and IL-1β release following 24-h exposure to TiO₂nanobelts in C57BL/6 alveolar macrophages co-cultured with 20 ng/mL LPS. A) Mean ± SEM percent viable cells relative to no particle control. B) Mean ± SEM IL-1β release. Asterisks indicate significance at *** P < 0.001 or * P < 0.05 compared to baseline condition. Daggers indicate significance at †† P < 0.01 or † P < 0.05 compared to the two other nanobelt variants at the same concentration.

Figure 8

TEM of TNP taken up by C57BL/6 alveolar macrophages 1.5 h in vitro post-exposure (25 μg/mL). A) No particle control AM. B) TNB-exposed AM. Arrow indicates abnormal phagolysosomal enlargement. C) High magnification of enlarged phagolysosome in TNB-exposed AM. D) Humic acid-modified TNB-exposed AM. E) Carboxylated TNB-exposed AM. F) High magnification of carboxylated TNB-exposed AM. Black spotted/speckled areas indicate areas of particle retention in the cytoplasm of the macrophage cells.

Figure 9

Summary data of lavage fluid contents 4 h following TiO₂nanobelt instillations in the lungs of C57BL/6 mice. A) Mean ± SEM IL-1β release. B) Mean ± SEM relative fluorescence units (RFU) corresponding to active cathepsin B. Asterisks indicate significance at **P < 0.01, or *P < 0.05 compared to dispersion media vehicle (DM) and control particle TiO₂ nanospheres (TNS).

Figure 10

Cell count and differentials 4 and 24 h following TiO₂nanobelt instillations in C57BL/6 wild-type mice and IL-1R null mice (24 hr only). A) Mean ± SEM neutrophil count at 4 and 24 h in WT mice only. B) Mean ± SEM total cell count at 24 h. C) Mean ± SEM alveolar macrophage count at 24 h. D) Mean ± SEM neutrophil count at 24 h. Asterisks indicate significance at ***P < 0.001, **P < 0.01 or *P < 0.05 compared to corresponding dispersion media vehicle (DM). Daggers indicate significance at ††† P < 0.001, †† P < 0.01, or † P < 0.05 compared to corresponding IL-1R particle exposure condition.

Figure 11

Summary data of lavage fluid cytokines 24 h following TiO₂nanobelt instillations in the lungs of C57BL/6 mice and IL-1R null mice. A) Mean ± SEM IL-18 release. B) Mean ± SEM IL-33 release. C) Mean ± SEM IL-6 release. D) Mean ± SEM TNF-α release. Asterisks indicate significance at *** P < 0.001, ** P > 0.01 or * P < 0.05 compared to corresponding dispersion media vehicle (DM). Daggers indicate significance at †† P < 0.01, or † P < 0.05 compared to the corresponding wild-type particle instillation condition. Symbols §§§ indicate significance at P < 0.001, or § P < 0.05 compared to the corresponding TiO₂ –COOH nanobelt (TNB COOH) instillation.

Figure 12

Viability and IL-1β release following 24-h exposure to TiO₂nanobelts in transformed human THP-1 cell line. A) Mean ± SEM percent LDH release relative to total cell lysis. B) Mean ± SEM percent viable cells relative to no particle control. C) Mean ± SEM IL-1β release. Asterisks indicate significance at ***P < 0.001, **P < 0.01, or *P < 0.05 compared to no particle 0 μg/ml condition. Daggers indicate significance at ††† P < 0.001, †† P < 0.01 or † P < 0.05 compared to the TiO₂ –COOH nanobelt (TNB COOH) at the same concentration. Symbols §§ indicate significance at P < 0.01 compared to the humic acid TiO₂ nanobelt (TNB HA) condition at the same concentration.

Figure 13

IL-1β release following 24-h exposure to TiO₂nanobelts in comparative murine AM cultures co-stimulated with 20 ng/mL LPS. Data indicate mean ± SEM IL-1β release in isolated culture media. Asterisks indicate significance at ***P < 0.001, or *P < 0.05 compared to no particle 0 μg/ml condition. Daggers indicate significance at ††† P < 0.001 compared to the -COOH TiO₂ nanobelt (-COOH TNB) exposure in the Balb/c AM. Symbols indicate significance at §§§ P < 0.001, or §§ P < 0.01 compared to the C57BL/6 AM TiO₂ nanobelt (TNB) exposure condition.