| [Background]Nanoparticles have multiple applications in various areas due to its unique physical, chemical and biological characterizations.. Their applications may bring us enormous economic benefits, but at the same time their potential biological and environmental safety problems also has aroused wide public concern around the world. Previous studies have shown that nanoparticles affected metabolism, apoptosis, cell cycle arrest, stress response cellular transport and inflammatory response. Intratracheal admin-istrated nanoparticles can be transferred to various extrapulmonary organs, including the vital organs such as brain and heart. The transportation may occur through simple physical displacement and chemical processes such as dissolution and protein-binding. And some research showed that20%intratracheal administrated nano-TiO2particles could be detected in macrophages in the lung lavage fluid, which meant the most of nano-TiO2particles were in the epithelial cells transferred to the mesenchyme or circulation system. However, there is no direct evidence to exhibit the transport processes at present.[Objective]In the present study, our aim is to show the morphologic evidence of the penetration of nano-TiO2particles through air-blood barrier in rats. We hope to provide some useful information in the biological safety evaluation, risk management, and the application of nano-TiO2particles materials in biomedical areas, and further promote the healthy development of nano technology and nano industry.[Methods]Rats were administrated by nano-TiO2particles through intratracheal instillation method. HE staining, the transmission electron microscopy (TEM), and the inductively coupled plasma mass spectrometry (ICP-MS) were employed in this study.(1) Lung tissue pathology:HE staining was used to observe the lung tissue structure change and general distribution of the nano-TiO2particles in lung tissue.(2) TEM observation:TEM was applied to observe the distribution in alveoli and the impacts of nano-TiO2particles on the ultrastructure of alveolar, especially the translocation of nanoparticles through lung air-blood barrier, and X-ray energy spectrometer was performed to indentify the Ti element in our interest regions.(3) ICP-MS measurement:ICP-MS was employed to detect the concentrations of nano-Ti02particles in venous blood at different time points (0,2,6,12,24, and48h).[Results]1. Material characterization results (TEM and X-ray diffraction (XRD))The ratio of anatase phase and rutile phase sample is80%：20%, nano-TiO2particles showed round crystal structures, the average particle size is about25nm.2. Optical microscope resultsFor control group, no significant inflammatory changes were found, rat alveolar showed normal structure, such as thin alveolar septa, clean alveolar space. For treatment group, there were no obvious pathological changes in lung tissue at2h after nano-TiO2perfusion, the alveolar showed a little shrinkage, a small amount of nanoparticles deposited in the alveoli surface and alveolar septa with aggregation state. Six hours after nano-TiO2perfusion, there were a large number of nano-TiO2agglomerates distributed in respiratory bronchioles, alveolar ducts, alveolar sacs, alveoli and pulmonary interstitium in rat lungs. Obvious pathological changes of lung tissue, hyperemia and hemorrhage, were found. And alveolar protein exudation and inflammatory cell infiltration greatly increased. In12h nano-TiO2perfusion group, the distribution of nano-TiO2agglomerates in the lung tissue showed decreased numbers than6h group, but more serious pathological changes existed, such as the destruction of parts of the alveolar integrity, the arranged disorder of alveolar structure or pulmonary consolidation, even the entire disappearing of alveolar structure. In24h nano-TiO2perfusion group, rare nano-TiO2particles deposition in alveolar surface and interstitial were found, and the situation of alveolar hyperemia and edema improved.3. Transmission-energy spectrum, electron microscopy resultsIn6h perfusion group, nano-TiO2particles in alveolar spaces formed big agglomerates or small chain agglomerates. The morphologies of free macrophages in alveolar spaces were normal, but there were a large number of nano-TiO2clusters without endocytic membrane in the cytoplasm. At the air-blood barrier, some nano-TiO2particle clusters adhered on the surface of alveolar epithelial cells. Parts of them were found in alveolar epithelial cells by crossing the air-blood barrier directly. nano-TiO2agglomerates with chain structures were across the alveolar epithelial cells and the basement membrane into the capillary endothelium. After12h of perfusion, the distribution of nano-TiO2in the lung tissue was similar to6h, but nano-TiO2particles may across the blood barrier into the capillary cavity in different ways. Agglomerates with chain structures were directly across the air-blood barrier, and massive agglomerates went through the air-blood barrier by damaging the cell membrane. Twenty-four hours after reperfusion, a large number of nano-TiO2particles went into the pulmonary interstitial, and alveolar septum was significantly thicker. The cells which contacted nano-TiO2particles showed cell damage, apoptosis and necrosis. A large number of vacuoles could be seen in the cell, the size of nucleus were reduced, and mitochondrial showed swelling and scattered ridge. In the type II alveolar epithelial cells containing nano-TiO2particles, there were a large number of lamellar bodies, and cell surface microvilli were disappeared.4. ICP-MS results.Comparing to the control group, whole blood Ti concentrations at0,2,6,12,24 and48h in nano-TiO2perfusion group were significantly increased. No obvious changes were found for the comparisons between time points.[Conclusion]Our TEM images showed nano-TiO2particles can penetrate air-blood barrier in rats directly. |
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