Nagarjun V. Konduru

Biokinetics and effects of barium sulfate nanoparticles

BackgroundNanoparticulate barium sulfate has potential novel applications and wide use in the polymer and paint industries. A short-term inhalation study on barium sulfate nanoparticles (BaSO4 NPs) was previously published [Part Fibre Toxicol 11:16, 2014]. We performed comprehensive biokinetic studies of 131BaSO4 NPs administered via different routes and of acute and subchronic pulmonary responses to instilled or inhaled BaSO4 in rats.MethodsWe compared the tissue distribution of 131Ba over 28 days after intratracheal (IT) instillation, and over 7 days after gavage and intravenous (IV) injection of 131BaSO4. Rats were exposed to 50 mg/m3 BaSO4 aerosol for 4 or 13 weeks (6 h/day, 5 consecutive days/week), and then gross and histopathologic, blood and bronchoalveolar lavage (BAL) fluid analyses were performed. BAL fluid from instilled rats was also analyzed.ResultsInhaled BaSO4 NPs showed no toxicity after 4-week exposure, but a slight neutrophil increase in BAL after 13-week exposure was observed. Lung burden of inhaled BaSO4 NPs after 4-week exposure (0.84 ± 0.18 mg/lung) decreased by 95% over 34 days. Instilled BaSO4 NPs caused dose-dependent inflammatory responses in the lungs. Instilled BaSO4 NPs (0.28 mg/lung) was cleared with a half-life of ≈ 9.6 days. Translocated 131Ba from the lungs was predominantly found in the bone (29%). Only 0.15% of gavaged dose was detected in all organs at 7 days. IV-injected 131BaSO4 NPs were predominantly localized in the liver, spleen, lungs and bone at 2 hours, but redistributed from the liver to bone over time. Fecal excretion was the dominant elimination pathway for all three routes of exposure.ConclusionsPulmonary exposure to instilled BaSO4 NPs caused dose-dependent lung injury and inflammation. Four-week and 13-week inhalation exposures to a high concentration (50 mg/m3) of BaSO4 NPs elicited minimal pulmonary response and no systemic effects. Instilled and inhaled BaSO4 NPs were cleared quickly yet resulted in higher tissue retention than when ingested. Particle dissolution is a likely mechanism. Injected BaSO4 NPs localized in the reticuloendothelial organs and redistributed to the bone over time. BaSO4 NP exhibited lower toxicity and biopersistence in the lungs compared to other poorly soluble NPs such as CeO2 and TiO2.

Bioavailability, distribution and clearance of tracheally instilled, gavaged or injected cerium dioxide nanoparticles and ionic cerium

Cerium oxide nanoparticles (NPs) have wide commercial applications. Understanding their fate in the body is fundamental to toxicological evaluations. We compared bioavailability, tissue distribution, clearance and excretion of radioactive 141Ce after intratracheal instillation (IT), gavage, or intravenous (IV) injection of neutron-activated 141CeO2 NPs and 141CeCl3 (ionic 141Ce) in Wistar Han rats. First, we evaluated pulmonary responses to IT-instilled CeO2 NPs and CeCl3 and observed dose-dependent inflammatory effects. Then, groups of rats were IT-instilled with 1 mg kg−1 of 141CeO2 NPs or 0.1 mg kg−1 141CeCl3. Sequential analyses of lungs over 28 days showed slow lung clearance of 141CeO2 NPs (half-life = ~140 days) and of ionic 141Ce (half-life = ~55 days). However, less than 1% and 6% of instilled 141Ce was measured in selected extrapulmonary organs in 141CeO2 NPs and ionic 141Ce groups, respectively. Following gavage (5 mg kg−1), nearly 100% of both test materials was excreted in the feces. Since detected 141Ce activity in tissues could be in nanoparticulate or dissolved form, we also compared the 141Ce tissue distribution post-IV injection with the IT and gavage data. Both IV-injected ionic 141CeCl3 and 141CeO2 NPs were predominantly retained in the liver, bone and spleen, all organs that typically remove circulating particles. We conclude that nanoceria is slowly cleared from the lungs but has minimal extrapulmonary accumulation. Potential risks from prolonged pulmonary retention need further investigation. Risk from ingested nanoceria is likely far lower due to very low absorption and rapid elimination of ceria not absorbed from the gastrointestinal tract.

Surface modification of zinc oxide nanoparticles with amorphous silica alters their fate in the circulation

Abstract Nanoparticle (NP) pharmacokinetics and biological effects are influenced by many factors, especially surface physicochemical properties. We assessed the effects of an amorphous silica coating on the fate of zinc after intravenous (IV) injection of neutron activated uncoated 65ZnO or silica-coated 65ZnO NPs in male Wistar Han rats. Groups of IV-injected rats were sequentially euthanized, and 18 tissues were collected and analyzed for 65Zn radioactivity. The protein coronas on each ZnO NP after incubation in rat plasma were analyzed by SDS-PAGE gel electrophoresis and mass spectrometry of selected gel bands. Plasma clearance for both NPs was biphasic with rapid initial and slower terminal clearance rates. Half-lives of plasma clearance of silica-coated 65ZnO were shorter (initial – <1 min; terminal – 2.5 min) than uncoated 65ZnO (initial – 1.9 min; terminal – 38 min). Interestingly, the silica-coated 65ZnO group had higher 65Zn associated with red blood cells and higher initial uptake in the liver. The 65Zn concentrations in all the other tissues were significantly lower in the silica-coated than uncoated groups. We also found that the protein corona formed on silica-coated ZnO NPs had higher amounts of plasma proteins, particularly albumin, transferrin, A1 inhibitor 3, α-2-hs-glycoprotein, apoprotein E and α-1 antitrypsin. Surface modification with amorphous silica alters the protein corona, agglomerate size, and zeta potential of ZnO NPs, which in turn influences ZnO biokinetic behavior in the circulation. This emphasizes the critical role of the protein corona in the biokinetics, toxicology and nanomedical applications of NPs.

The role of natural processes and surface energy of inhaled engineered nanoparticles on aggregation and corona formation

The surface chemistry of engineered nanoparticles (ENPs) becomes more important as their size decreases and enters the nanometer-range. This review explains the fundamental properties of the surface chemistry of nanoparticles, and argues that their agglomeration and the formation of corona around them are natural processes that reduce surface energy. ENP agglomeration and surface corona formation are further discussed in the context of inhaled ENPs, as the lung is a major port of ENP entry to the body. The pulmonary surfactant layer, which the inhaled ENPs first encounter as they land on the lung surface, represents a unique environment with a variety of well-defined biomolecules. Many factors, such as hydrophobicity, surface charge of ENPs, protein/phospholipid concentrations of the alveolar lining fluid, etc. influence the complex processes of ENP agglomeration and corona formation in the alveolar lining fluid, and these events occur even before the ENPs reach the cells. We suggest that molecular dynamic simulations can represent a promising future direction for research of the behavior of inhaled ENPs, complementing the experimental approaches. Moreover, we want to remind biologists working on ENPs of the importance relationship between ENP surface energy and size.

Pulmonary distribution of nanoceria: comparison of intratracheal, microspray instillation and dry powder insufflation

Abstract Particles can be delivered to the respiratory tract of animals using various techniques. Inhalation mimics environmental exposure but requires large amounts of aerosolized NPs over a prolonged dosing time, varies in deposited dose among individual animals, and results in nasopharyngeal and fur particle deposition. Although less physiological, intratracheal (IT) instillation allows quick and precise dosing. Insufflation delivers particles in their dry form as an aerosol. We compared the distribution of neutron-activated 141CeO2 nanoparticles (5 mg/kg) in rats after (1) IT instillation, (2) left intrabronchial instillation, (3) microspraying of nanoceria suspension and (4) insufflation of nanoceria dry powder. Blood, tracheobronchial lymph nodes, liver, gastrointestinal tract, feces and urine were collected at 5 min and 24 h post-dosing. Excised lungs from each rat were dried at room temperature while inflated at a constant 30 cm water pressure. Dried lungs were then sliced into 50 pieces. The radioactivity of each lung piece and other organs was measured. The evenness index (EI) of each lung piece was calculated [EI = (μCi/mgpiece)/(μCi/mglung)]. The degree of EI value departure from 1.0 is a measure of deposition heterogeneity. We showed that the pulmonary distribution of nanoceria differs among modes of administration. Dosing by IT or microspraying resulted in similar spatial distribution. Insufflation resulted in significant deposition in the trachea and in more heterogeneous lung distribution. Our left intrabronchial instillation technique yielded a concentrated deposition into the left lung. We conclude that animal dosing techniques and devices result in varying patterns of particle deposition that will impact biokinetic and toxicity studies.

Protein corona: implications for nanoparticle interactions with pulmonary cells

BackgroundWe previously showed that cerium oxide (CeO2), barium sulfate (BaSO4) and zinc oxide (ZnO) nanoparticles (NPs) exhibited different lung toxicity and pulmonary clearance in rats. We hypothesize that these NPs acquire coronas with different protein compositions that may influence their clearance from the lungs.MethodsCeO2, silica-coated CeO2, BaSO4, and ZnO NPs were incubated in rat lung lining fluid in vitro. Then, gel electrophoresis followed by quantitative mass spectrometry was used to characterize the adsorbed proteins stripped from these NPs. We also measured uptake of instilled NPs by alveolar macrophages (AMs) in rat lungs using electron microscopy. Finally, we tested whether coating of gold NPs with albumin would alter their lung clearance in rats.ResultsWe found that the amounts of nine proteins in the coronas formed on the four NPs varied significantly. The amounts of albumin, transferrin and α-1 antitrypsin were greater in the coronas of BaSO4 and ZnO than that of the two CeO2 NPs. The uptake of BaSO4 in AMs was less than CeO2 and silica-coated CeO2 NPs. No identifiable ZnO NPs were observed in AMs. Gold NPs coated with albumin or citrate instilled into the lungs of rats acquired the similar protein coronas and were cleared from the lungs to the same extent.ConclusionsWe show that different NPs variably adsorb proteins from the lung lining fluid. The amount of albumin in the NP corona varies as does NP uptake by AMs. However, albumin coating does not affect the translocation of gold NPs across the air-blood barrier. A more extensive database of corona composition of a diverse NP library will develop a platform to help predict the effects and biokinetics of inhaled NPs.

In vivo formation of Ce-phosphate Nanoparticles following Intratracheal Instillation of CeCl3: Subcellular sites, Nanostructures, Precipitation Mechanisms and Nanoparticle 3D-Alignment

We demonstrate the in vivo formation of nano-particulate Ce-containing structures in lungs instilled with 5 mg/kg CeCl3 using high resolution electron microscopy (HRTEM), energy loss spectroscopy (EELS), and elemental mapping (EDS). The observed high lung retention of Ce after instillation of CeCl3 (75-92% retained at 28 days) is unexpected since metal ions are usually readily transported across the air-blood barrier. The binding of cerium ions to lung constituents has been suggested [1], and the formation of cerium phosphate has been shown previously, but without discussions on the mechanisms involved in nanoparticle nucleation and growth [2]. Determining the form of Ce after lung exposure to CeCl3 has been challenging due to the difficulties in distinguishing ions from particulate forms when using radioactivity or ICP-MS. We have identified cerium nanoparticles at the sub-cellular level in lung macrophages after CeCl3 instillation. This observation provides insights on the cell structures and components that will help distinguish which cellular areas are the sites of in vivo nanoparticle formation.

Calcium co-Localization with in vivo Cerium Phosphate Nanoparticle Formation after Intratracheal Instillation Dosing with CeCl3 or CeO2 NPs

The bioprocessing of CeO2 nanoparticles after uptake in lung tissues is compared with the formation of nanoparticles after inhalation of CeCl3 aerosols. In both cases, high-resolution TEM/STEM analyses indicate that cerium phosphate nanoparticles (NPs) had precipitated in phagolysosomal regions within macrophages. Importantly, the primary particle size, morphology, and agglomeration tendencies of the NPs were strikingly similar. Formation of cerium phosphate NPs after bioprocessing of inhaled CeO2 crystals proceeds via dissolution, ion transport, followed by nucleation and growth [1]. Application of 2D and 3D elemental maps of the NP bioprocessing stages and corresponding tissue interactions provide insights on the breakdown mechanisms, formation of new precipitates, size, and morphology changes of the original delivered NPs, and ion transport phenomena that result in secondary particle formation. Particle size and morphology are very similar for all of the cerium phosphate NPs, suggesting a common underlying precipitation mechanism independent of whether the metal ions are derived from dissolution of nanoparticles (CeO2) or from instilled metal ions (CeCl3).

Nanoparticle Wettability Influences Nanoparticle-Phospholipid Interactions

We explored the influence of nanoparticle (NP) surface charge and hydrophobicity on NP-biomolecule interactions by measuring the composition of adsorbed phospholipids on four NPs, namely, positively charged CeO2 and ZnO and negatively charged BaSO4 and silica-coated CeO2, after exposure to bronchoalveolar lavage fluid (BALf) obtained from rats, and to a mixture of neutral dipalmitoyl phosphatidylcholine (DPPC) and negatively charged dipalmitoyl phosphatidic acid (DPPA). The resulting NP-lipid interactions were examined by cryogenic transmission electron microscopy (cryo-TEM) and atomic force microscopy (AFM). Our data show that the amount of adsorbed lipids on NPs after incubation in BALf and the DPPC/DPPA mixture was higher in CeO2 than in the other NPs, qualitatively consistent with their relative hydrophobicity. The relative concentrations of specific adsorbed phospholipids on NP surfaces were different from their relative concentrations in the BALf. Sphingomyelin was not detected in the extracted lipids from the NPs despite its >20% concentration in the BALf. AFM showed that the more hydrophobic CeO2 NPs tended to be located inside lipid vesicles, whereas less hydrophobic BaSO4 NPs appeared to be outside. In addition, cryo-TEM analysis showed that CeO2 NPs were associated with the formation of multilamellar lipid bilayers, whereas BaSO4 NPs with unilamellar lipid bilayers. These data suggest that the NP surface hydrophobicity predominantly controls the amounts and types of lipids adsorbed, as well as the nature of their interaction with phospholipids.