Impact of nanoparticle surface functionalization on the protein corona and cellular adhesion, uptake and transport

Abstract

Background: Upon ingestion, nanoparticles can interact with the intestinal epithelial barrier potentially resulting in systemic uptake of nanoparticles. Nanoparticle properties have been described to influence the protein corona formation and subsequent cellular adhesion, uptake and transport. Here, we aimed to study the effects of nanoparticle size and surface chemistry on the protein corona formation and subsequent cellular adhesion, uptake and transport. Caco-2 intestinal cells, were exposed to negatively charged polystyrene nanoparticles (PSNPs) (50 and 200 nm), functionalized with sulfone or carboxyl groups, at nine nominal concentrations (15-250 μg/ml) for 10 up to 120 min. The protein coronas were analysed by LC-MS/MS.

Results: Subtle differences in the protein composition of the two PSNPs with different surface chemistry were noted. High-content imaging analysis demonstrated that sulfone PSNPs were associated with the cells to a significantly higher extent than the other PSNPs. The apparent cellular adhesion and uptake of 200 nm PSNPs was not significantly increased compared to 50 nm PSNPs with the same surface charge and chemistry. Surface chemistry outweighs the impact of size on the observed PSNP cellular associations. Also transport of the sulfone PSNPs through the monolayer of cells was significantly higher than that of carboxyl PSNPs.

Conclusions: The results suggest that the composition of the protein corona and the PSNP surface chemistry influences cellular adhesion, uptake and monolayer transport, which might be predictive of the intestinal transport potency of NPs.

Keywords: Cellular adhesion and uptake; High throughput screening; Label-free LC–MS/MS; Nanoparticles; Quantitative proteomics.

Fig. 1

Caco-2 cell viability after exposure to a concentration series of 50 nm- (-SM), (-CM), (-CP), and 200 nm (-CP) PSNPs for a 3 h and b 24 h using the WST-1 viability assay. Viability is given as a percentage of the control (% ± SD; n = 3)

Fig. 2

Confocal microscopy images of Caco-2 cells a w/o exposure to PSNPs—as control for the (-SM) and (–CM) PSNPs. After exposure for 24 h to a nominal concentration of 25 µg/ml b 50 nm (-SM) and c 50 nm (-CM). d Caco-2 cells—w/o exposure to PSNPs—as control for the (-CP) PSNPs. After exposure for 24 h to e 50 nm (-CP) and f 200 nm (-CP). Nuclei were stained in blue, lysosomes in red and PSNPs in green

Fig. 3

Cellular association of PSNPs with a different surface chemistry by Caco-2 cells. Adhesion and uptake was determined by single-cell HC image analysis of PSNP fluorescence. a Time dependent adhesion and uptake of four types of PSNPs in Caco-2 cells exposed to a nominal concentration of 50 µg PSNPs/ml for 10 up to 120 min. b Cellular association distribution profiles of 50 nm (-SM) PSNPs in the entire cell population at exposure to nominal concentration ranging from 15 to 250 µg/ml for 30 min. Note that the readings at the highest concentrations are hampered by saturation of the HC signal. c Concentration dependent cellular association of four types of PSNPs after 30 min of exposure (//; the fluorescence intensities of 50 nm (-SM) is plotted on the left y-axis while the rest of the PSNPs fluorescence intensities are plotted on the right y-axis). ^#Significant difference versus all lower concentrations (P < 0.05). *Significant difference between indicated concentrations (P < 0.05)

Fig. 4

Transport of PSNPs with a different surface chemistry by Caco-2 cells. Transport of 4 types of PSNPs in Caco-2 cells exposed to a nominal concentration of 250 µg PSNPs/ml for 24 h. *Significant difference from all and ^#significant difference from 50 nm (-CM) and 200 nm (-CP) PSNPs (P < 0.05)

Fig. 5

Composition of the protein corona on PSNPs with a different surface chemistry determined with LC–MS/MS. The proteins were classified into seven groups according to their biological function using proteomics databases. a Distribution of the protein groups in DMEM⁺ and in the protein coronas of the PSNPs, expressed as a percentage of the total protein mass. b Number of protein molecules per particle clustered per protein group. c Top 20 proteins with the highest protein adsorption on the respective PSNP. Proteins are ordered alphabetically. The colour code indicates the protein group and the size of the spot represents the mass fraction (%), which is also given in numbers

Fig. 6

Comparison of differently adsorbed proteins to the surface of 50 nm PSNPs with a different surface chemistry. Only proteins with 2 or more copy numbers on at least 1 of the 3 different PSNPs were considered. Asterisks indicate significance difference (P < 0.05)