The effect of sedimentation and diffusion on cellular uptake of gold nanoparticles

Abstract

In vitro experiments typically measure the uptake of nanoparticles by exposing cells at the bottom of a culture plate to a suspension of nanoparticles, and it is generally assumed that this suspension is well-dispersed. However, nanoparticles can sediment, which means that the concentration of nanoparticles on the cell surface may be higher than the initial bulk concentration, and this could lead to increased uptake by cells. Here, we use upright and inverted cell culture configurations to show that cellular uptake of gold nanoparticles depends on the sedimentation and diffusion velocities of the nanoparticles and is independent of size, shape, density, surface coating and initial concentration of the nanoparticles. Generally, more nanoparticles are taken up in the upright configuration than in the inverted one, and nanoparticles with faster sedimentation rates showed greater differences in uptake between the two configurations. Our results suggest that sedimentation needs to be considered when performing in vitro studies for large and/or heavy nanoparticles.

Figure 1. Experimental setups and the gold nanoparticles used in this study

a, Schematic of the upright (left) and inverted (right) configurations for measuring cellular uptake of nanoparticles. Cells are not drawn to scale and each well of a 6-well culture plate contained only one glass coverslip on which cells are immobilized. b-g, TEM images showing the six types of gold nanoparticles used to examine the effects of size, shape and density on the disparity in cellular uptake between the two configurations: 15-nm (in diameter) nanospheres (b), 54-nm nanospheres (c), 100-nm nanospheres (d), 62-nm (in outer edge length) nanocages (e), 118-nm nanocages (f), nanorods (g, 16 nm×40 nm in diameter by length). The 50-nm scale bar applies to all images.

Figure 2. Uptake values of various types of gold nanoparticles for cells positioned in the upright and inverted configurations

The number (N) of particles taken up per cell was measured for both the as-prepared and PEGylated samples using the UV-vis method. The nanoparticles and their concentrations (based on particle number) used to incubate with the cells were: a, 15-nm nanospheres (120 pM); b, 54-nm nanospheres (20 pM); c, 100-nm nanospheres (2.8 pM); d, 62-nm nanocages (20 pM); e, 118-nm nanocages (2.6 pM); and f, nanorods (20 pM). Error bars shown in the plots are standard errors with n=4.

Figure 3. Comparison of the disparity in cellular uptake between the upright and inverted configurations for different types of gold nanoparticles

We express the disparity in uptakes between the two configurations as 1-(N_in/N_up), where N_up and N_in are the number of nanoparticles taken up per cell in the upright and inverted configurations, respectively. The disparity increased with the particle size for both the nanospheres and nanocages. In addition, the disparity was insensitive to the surface coating for all types of nanoparticles. Error bars shown in the plots are standard errors with n=4.

Figure 4. Different zones that are involved in cellular uptake of gold nanoparticles and the two factors affecting the uptake process

a, Schematic showing the transport, interaction and uptake zones when the cell is positioned in an upright (left) or inverted (right) configuration. The concentration of nanoparticles in the interaction zone for both configurations may differ depending on the sedimentation (S) and diffusion (D) characteristics of the nanoparticles. b, Diffusion (V_d) and sedimentation (V_s) velocities of the as-prepared nanoparticles. c, The ratios of sedimentation to diffusion velocities (V_s/V_d) for various types of nanoparticles. The ratios determine which factor is dominant in transporting the nanoparticles to the cell surface, thereby affecting the uptake process.

Figure 5. Disparity in uptake between the upright and inverted configurations as a function of the ratio of sedimentation to diffusion velocities

The disparity in uptake is expressed as 1-(N_in/N_up) and the plot includes all the 1-(N_in/N_up) values measured using both the UV-vis (Fig. 3 and Supplementary Figs. S6 and S9) and ICP-MS methods with the cells (Supplementary Table S1). The disparity increased with increasing V_s/V_d ratio regardless of particle size, shape, surface coating and initial concentration. The data can be grouped into two linear regions; the two dashed lines cross at V_s/V_d=3, corresponding to a value of 0.3 for disparity. In general, the effect of sedimentation must be taken into consideration for nanoparticles with V_s/V_d greater than 3.