Protein corona: implications for nanoparticle interactions with pulmonary cells

Abstract

Background: We 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.

Methods: CeO₂, 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.

Results: We 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.

Conclusions: We 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.

Keywords: Biokinetics; Engineered nanoparticles; Lung macrophage; Nanotoxicity; Protein corona.

Fig. 1

Transmission electron micrographs (TEM) of nanoparticle suspensions in DI water. a CeO₂. b Si-CeO₂. Nanothin coatings of amorphous silica are shown (arrows). A higher magnification of additional Si-CeO₂ NPs are shown in the inset. The silica coating appears as less electron dense coating (arrowheads) surrounding the core CeO₂ NPs. c BaSO₄. d ZnO nanorods

Fig. 2

Analysis of NP-bound proteins by 1D gel electrophoresis and mass spectrometry. a The molecular weights (kDa) of reference proteins are shown in lane MW. A representative gel from one experiment is shown. NP-corona components identified by LC-MS are indicated on right. b LC-MS profiles of the most abundant BAL proteins and influence of NP type on corona profile. The nine most abundant proteins are shown. The total proteins per mg NPs were in the order of CeO₂ = Si-CeO₂ = BaSO₄ < ZnO. (* P < 0.05, significant variations in each protein among 4 NP types). c When expressed as % of total bound protein, transferrin, albumin and α-1-antitrypsin were greater in the corona of ZnO and BaSO₄ than of both CeO₂ NPs. (* P < 0.05, significant variations in each protein among 4 NP types). d When expressed as bound proteins per surface area of NPs, transferrin and α-1-antitrypsin were greater in the corona of ZnO than of CeO₂ NPs. Bound albumin was greater in the coronas of ZnO and BaSO₄ than of both CeO₂ NPs. Data are mean ± SD, n = 3 rats per NP)

Fig. 3

Transmission electron micrographs of lavaged cells at 24 h from rats instilled with NPs at a dose of 1 mg/kg body weight. Macrophage uptake was scored as +, ++, or +++ when 1–2, 3–4 or ≥5 particle-containing phagosomes were observed in macrophages, respectively. a CeO₂, b Si-CeO₂, c BaSO₄ and d. ZnO. No identifiable ZnO NPs were observed in lavaged cells from ZnO-instilled rats. e Morphometric analysis of macrophage uptake of NPs (n = 248, CeO₂, n = 273, Si-CeO₂, n = 295, BaSO₄, n = 250, ZnO). A significantly smaller fraction of lavaged macrophages showed uptake of BaSO₄ NPs compared with CeO₂ and Si-CeO₂ NPs. Data are mean ± SE of % of macrophages (n = 3 rats per NP). * P < 0.05, BaSO₄ vs. CeO₂ and Si-CeO₂ NPs

Fig. 4

a Transmission electron micrograph of citrate-coated Au NPs. b UV-vis spectra of citrate-coated Au NPs in water (black curve) and albumin-coated Au NPs in PBS (red curve). The Au NPs show a red-shift in the peak absorbance wavelength after albumin coating

Fig. 5

Analysis of Au NPs after incubation with BAL fluid. a NP-bound rat BAL proteins were analyzed by 1D gel electrophoresis and mass spectrometry. The molecular weights (kDa) of reference proteins are shown in lane MW. A representative gel from one experiment is shown. Three proteins identified by LC-MS are indicated on right. b Quantification of albumin, Tf and Tubb2A adsorbed on Au NPs. Data are mean ± SE (n = 4 rats per NP)

Fig. 6

Tissue distribution of Au at 6 and 24 h after IT instillation of citrate- and albumin-coated Au NPs in rats. The bulk of measured Au was found in the lungs. Measured Au levels in the lungs were not different between the two NPs at both time points. Low percentages of instilled Au were measured in the liver, spleen and tracheobronchial lymph nodes. The translocation of Au to the spleen was significantly lower in rats instilled with albumin-coated Au NPs than in rats instilled with citrate-coated Au NPs at 24 h. Data are mean ± SE (n = 12 rats per NP). Note: Y-axis in log scale

Fig. 7

Transmission electron micrographs of lavaged cells at 24 h from rats instilled with Au NP suspensions at a dose of 1 mg/kg body weight. a Macrophage uptake of citrate-coated Au NPs and b albumin-coated Au NPs. Micrographs in 6A and 6B insets are higher magnification of the areas shown. c Morphometric analysis of NP uptake. Macrophage uptake was scored similarly as described in Fig. 3. No significant difference in macrophage uptake of NPs (n = 185, citrate-coated Au NPs, n = 191, albumin-coated Au NPs) was found. Data are mean ± SE of % of macrophages (n = 3 rats per NP)