Length and aspect ratio represent important toxicity determinants of fibrous nanomaterials. We have previously shown that anatase TiO₂ nanofibers (TiO₂ NF) cause a dose-dependent decrease of cell viability as well as the loss of epithelial barrier integrity in polarized airway cell monolayers. Herein we have investigated the impact of fiber shortening, obtained by ball-milling, on the biological effects of TiO₂ NF of industrial origin. Long TiO₂ NF (L-TiO₂ NF) were more cytotoxic than their shortened counterparts (S-TiO₂ NF) toward alveolar A549 cells and bronchial 16HBE cells. Moreover, L-TiO₂ NF increased the permeability of 16HBE monolayers and perturbed the distribution of tight-junction proteins, an effect also mitigated by fiber shortening. Raw264.7 macrophages efficiently internalized shortened but not long NF, which caused cell stretching and deformation. Compared with L-TiO₂ NF, S-TiO₂ NF triggered a more evident macrophage activation, an effect suppressed by the phagocytosis inhibitor cytochalasin B. Conversely, a significant increase of inflammatory markers was detected in either the lungs or the peritoneal cavity of mice exposed to L-TiO₂ NF but not to S-TiO₂ NF, suggesting that short-term macrophage activation in vitro may not be always a reliable indicator of persistent inflammation in vivo. It is concluded that fiber shortening mitigates NF detrimental effects on cell viability and epithelial barrier competence in vitro as well as inflammation development in vivo. These data suggest that fiber shortening may represent an effective safe-by-design strategy for mitigating TiO₂ NF toxic effects.

长度和纵横比代表了纤维纳米材料的重要毒性决定因素。先前我们已经表明，锐钛矿型TiO ₂纳米纤维（TiO ₂ NF）引起剂量依赖性的细胞生存力下降，以及极化气道细胞单层中上皮屏障完整性的丧失。本文中，我们研究了通过球磨获得的纤维缩短对工业来源的TiO ₂ NF的生物学效应的影响。长的TiO ₂ NF（L-TiO ₂ NF）对肺泡A549细胞和支气管16HBE细胞的毒性比短的TiO ₂ NF （S-TiO ₂ NF）高。此外，L-TiO ₂NF增加了16HBE单层的渗透性并扰乱了紧密连接蛋白的分布，纤维缩短也减轻了这种影响。Raw264.7巨噬细胞有效地内化了缩短但不长的NF，从而导致细胞拉伸和变形。与L-TiO ₂ NF相比，S-TiO ₂ NF触发了更明显的巨噬细胞激活，这一作用被吞噬抑制剂细胞松弛素B抑制。相反，在小鼠的肺或腹膜腔中检测到炎性标记物显着增加暴露于L-TiO ₂ NF而不暴露于S-TiO ₂ NF，这表明体外短期巨噬细胞活化可能并不总是持续炎症的可靠指标体内。结论是纤维缩短减轻了NF对体外细胞活力和上皮屏障能力的有害影响，以及体内炎症的发展。这些数据表明，缩短纤维可能代表减轻TiO ₂ NF毒性效应的有效的安全设计策略。