Cellular Binding of Anionic Nanoparticles is Inhibited by Serum Proteins Independent of Nanoparticle Composition

Abstract

Nanoparticles used in biological applications encounter a complex mixture of extracellular proteins. Adsorption of these proteins on the nanoparticle surface results in the formation of a "protein corona," which can dominate the interaction of the nanoparticle with the cellular environment. The goal of this research was to determine how nanoparticle composition and surface modification affect the cellular binding of protein-nanoparticle complexes. We examined the cellular binding of a collection of commonly used anionic nanoparticles: quantum dots, colloidal gold nanoparticles, and low-density lipoprotein particles, in the presence and absence of extracellular proteins. These experiments have the advantage of comparing different nanoparticles under identical conditions. Using a combination of fluorescence and dark field microscopy, flow cytometry, and spectroscopy, we find that cellular binding of these anionic nanoparticles is inhibited by serum proteins independent of nanoparticle composition or surface modification. We expect these results will aid in the design of nanoparticles for in vivo applications.

Keywords: colloidal gold; nanoparticles; opsonization; protein corona; quantum dots; serum proteins.

Figure 1

Formation of a protein corona on carboxylate-modified QDs was confirmed using gel electrophoresis (1% w/v agarose). To form the corona, QDs (green) were incubated with MEM supplemented with 10% FBS (QD + FBS). As a control, QDs were incubated in water (QD + H₂O) and MEM (QD + MEM) in the absence of protein.

Figure 2

Fluorescence microscopy images of carboxylate-modified QDs (green) bound to BS-C-1 cells. Cell nuclei are stained with DAPI (blue). QDs were incubated with cells for 10 minutes at 4 °C in (A) MEM, (B) MEM supplemented with FBS, and (C) MEM supplemented with BSA.

Figure 3

Flow cytometry was used to measure cellular binding of QDs in MEM, MEM supplemented with FBS (FBS), MEM supplemented with BSA (BSA), and cells in the absence of quantum dots (Cells only). Results were normalized against QD binding in MEM.

Figure 4

Formation of a protein corona on the surface of citrate-modified Au NPs after incubation in MEM supplemented with 10% FBS. Au NPs were washed via centrifugation five times and supernatants (S) were analyzed with SDS-PAGE. S1 was diluted to 1% and S2 was diluted to 10% v/v due to the high protein concentration. Protein is no longer visible in the supernatant after 4 wash steps. The protein corona is removed from the surface of the NP with SDS (NP + SDS). In the absence of SDS (NP + H₂O), no protein is evident on the gel. FBS is shown for comparison. Molecular weight (MW) marker values are 225, 150, 100, 75, 50, 35, 25, 15, 10, and 5 kDa.

Figure 5

Dark field microscopy images of citrate-modified Au NPs (yellow) bound to BS-C-1 cells after incubation for 30 minutes at 4 °C in (A) MEM and (B) MEM supplemented with FBS. Images of cells without Au NPs are presented in Figure S1, showing scatter due to cells alone.

Figure 6

Cellular binding of protein-Au NP complexes was measured using the absorption spectra of Au NPs before and after incubation with the cells. (A) Representative difference spectra show the relative cellular binding of Au NPs following incubation with cells in the presence (FBS) or absence (MEM) of serum proteins. The difference spectra of cells in the absence of Au NPs is shown for comparison (Cells only). (B) Binding of protein-Au NP complexes in MEM, MEM supplemented with FBS, and MEM supplemented with BSA. Absorbance of medium incubated with cells, but without NPs, was negligible. Binding was normalized to 100% for Au NPs incubated with cells in MEM (*p < 0.05; **p < 0.01; there was no statistically significant difference between FBS and BSA).

Figure 7

Fluorescence microscopy images of LDL fluorescently labeled with DiD (red) bound to BS-C-1 cells. Cell nuclei are stained with DAPI (blue). LDL-DiD was incubated with cells for 20 minutes at 4 °C in (A) MEM, (B) MEM supplemented with FBS, and (C) MEM supplemented with BSA.

Figure 8

Flow cytometry was used to measure the cellular binding of LDL particles in MEM, MEM supplemented with FBS (FBS), MEM supplemented with BSA (BSA), and cells in the absence of LDL (Cells only). Binding was normalized against LDL binding in MEM (*p < 0.05; ***p < 0.001).