Nanoparticle Wettability Influences Nanoparticle-Phospholipid Interactions

Abstract

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 CeO_2, 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.

Figure 1.

Transmission electron micrographs of sonicated NP suspensions in DI water. (a) CeO₂. (b) Si-CeO₂. Nanothin coatings of amorphous silica are shown (arrows). (c) BaSO₄. (d) ZnO nanorods.

Figure 2.

Surface hydrophobicity of CeO₂, Si-CeO₂, BaSO4, and ZnO NPs determined by Rose Bengal partitioning. The relationships between NP surface areas and PQ are plotted. The data were then analyzed by performing linear regression. Following model fitting, the slopes of PQ for each NP were compared using SAS statistical software (SAS Institute, Inc. Cary, NC). The slopes of the linear regression lines are proportional to the relative hydrophobicity of NPs. The relative surface hydrophobicity was on the order of CeO₂ > ZnO = BaSO₄ > Si-CeO₂ NPs.

Figure 3.

Tandem quadruple mass spectroscopy analyses of lipids adsorbed on NPs after 30 min incubation in the cell-free BAL fluid. (a) Total adsorbed PLs were calculated per surface area as 1.76 ± 0.06 (CeO₂), 1.19 ± 0.06 (Si—CeO₂), 0.90 ± 0.10 (BaSO₄), and 1.14 ± 0.02 (ZnO) mg/m². (b) Amounts of different PL classes are shown. PC was the most abundant class bound to all NPs (*CeO₂ higher than the other three NPs, ^#BaSO4 lower than the other three NPs, MANOVA, *^#P < 0.05). The amounts of NP-bound individual PL class also significantly varied among the four NP types (MANOVA, ^@P < 0.05). Data are mean ± standard deviation, n = 3 replicates (PC: phosphatidylcholine; LPC: lysophosphatidylcholine; PA: phosphatidic acid; LPA: lysophosphatidic acid; PI: phosphatidylinositol; PG: phosphatidylglycerol; PE: phosphatidyethanolamine; PS: phosphatidylserine).

Figure 4.

Cryogenic TEM micrographs of CeO₂ and BaSO₄ NPs postincubation in rat BALf showing interaction of intact liposomes with agglomerates of CeO₂ (a) and BaSO₄ (b) NPs.

Figure 5.

Cryo-TEM micrographs of CeO₂ (a,b) and BaSO₄ (c,d) NPs postincubation in a 2:1 mixture of DPPC and DPPA. Multilamellar and unilamellar vesicles are seen adsorbed to clusters of CeO₂ NPs (a,b). Agglomerates of BaSO₄ NPs interacting with PL vesicles with polyhedral shapes (c) and lipid bilayer (d) are shown.

Figure 6.

Atomic force microscopy analyses of CeO₂ (a-c) and BaSO₄ (d-f) NP interaction with a DPPC/DPPA mixture. (a,d) Three-dimensional rendering of AFM height images. (b,e) Top view of the same images. (c,f) Height profiles across the dashed line in panels b and e.