Dose dependent distribution and aggregation of gold nanoparticles within human lung adeno-carcinoma cells

Abstract

In this work, we discuss the distribution, aggregation and cytotoxicity of different treatment doses, 0.01, 0.05, 0.1, 0.2 and 0.5 nM, of poly(allylamine hydrochloride) (PAH) coated gold nanoparticles (Au NPs) with a human lung adeno-carcinoma cell line – A549 cells. By taking colorful scattering images and employing chromatic analysis, the evolution of Au NPs during cellular endocytosis and their distribution were revealed. With the lower treatment doses, 0.01 and 0.05 nM, Au NPs were mostly endocytosed and then clustered as larger aggregates inside cells. When the treatment dose was increased to 0.1 or 0.2 nM, a number of Au NPs were stuck on the membrane and formed two scattering color bands, yellow for larger aggregates inside cells and green for individual NPs on the membrane. By comparing with the cells treated with 0.1 nM Au NPs and dynasore, we find that the PAH coated Au NPs were up-taken into cells via dynamin dependent endocytosis. For the 0.5 nM dose, different from the 0.1 and 0.2 nM, there are large numbers of Au NPs stuck on the membrane, and furthermore, owing to the periodic lamellipodial contraction with rearward actin polymerization on the membrane, these stuck Au NPs were moved to and accumulated on the top of cells. From scanning electron microscopy (SEM) images, we find that the number and density of the Au NPs stuck on the membrane increases with the increasing treatment dose. Dual-beam focus ion beam (DBFIB) images showed the 2D covering and 3D stacking of the aggregated Au NPs on the membrane and inside endocytic vesicles respectively. Cytotoxicity testing indicates that the stuck Au NPs on the membrane would efficiently impact the cell viability. This work highlights the importance of an overall distribution of Au NPs in the NPs–cell interactive system.

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Introduction

Over the past decades, gold nanoparticles (Au NPs) have been widely used in biomedical applications as either contrast agents or drug/gene carriers.^1–5 All of these applications are based on the interactions of the Au NPs and cells, for example, adhering, endocytosis, and even penetrating. Previous studies suggest that the differences in uptake and distribution are dependent on the characteristics of Au NPs, such as size and surface modification.^6–8 Furthermore, these different interactions would further change cellular proliferation, regulation and so on.⁹ It also indicates that Au NPs can be designed for specialized uses. However, despite the rapid progress and early acceptance of Au NPs in biomedical trials,^10,11 concerns about their potential toxicity have also arisen. Compared to other nanoparticle systems, such as quantum dots (CdSe/ZnS) and carbon nanoparticles, Au NPs show a better biocompatibility than the others.^12–14 However, investigations have also brought forth notable data regarding the cytotoxicity of Au NPs dependent on the size, surface modification and, especially, the exposed dose.^15–17 Of the treatment of cells with Au NPs, too low dose could not achieve the expected result, while with over treating, side effects would be caused. For example, T. Mironava et al. found that with an increasing concentration of treatment Au NPs, cells show a notable increasing percentage in apoptosis.¹⁸ J. Davda et al. pointed out that over treating would reduce the efficiency of the NPs that enter cells.¹⁹

Aside from the treatment dose, recent literature also points out that the distribution of different types of Au NPs hugely impacts their cytotoxicity.^20–22 For instance, Au NPs that moved inside cells by the process of endocytosis have been demonstrated to finally accumulate in the lysosomes,²³ and toxicity is mainly caused by the release of the toxic ion Au^1+/3+ in this acidic environment. In comparison, no obvious toxicity was caused from Au NPs which are scattered in the cytosol. Furthermore, aggregations of NPs and their distribution in a biological system might result in a different consequence. For example, in the system of NP-based drug/gene delivery, if the aggregation of NPs occurred before being endocytosed, it might reduce the uptake of NPs, which would reduce the efficiency and increase their cytotoxicity.^24,25 On the other side, if the aggregation occurred after endocytosis, i.e. in the endosome, it might facilitate the release of the NPs from the endosome and increase the efficiency.²⁶ Therefore, a thorough understanding of the distribution and aggregation of Au NPs and their consequent cytotoxicity is a key issue for their biomedical applications.

In this work, we study the distribution, aggregation and cytotoxicity of Au NPs with different treatment concentrations (0, 0.01, 0.05, 0.1, 0.2 and 0.5 nM). Au NPs with a diameter of 50 nm and human lung adeno-carcinoma cells A549 were used here. Thanks to the strong scattering property of Au NPs, the clusters of Au NPs can be clearly observed under dark field illumination. Furthermore, owing to the local surface plasmon resonance (LSPR) coupling effect of Au NPs,²⁷ the number of Au NPs and their clusters in cells could be estimated by using chromatic analysis as reported in our previous effort.²⁸ Sectional dark field microscopy,¹ scanning electron microscopy (SEM, FEI Nova 200) and dual-beam focus ion beam (DBFIB, FEI Helios 600) were used to verify the distribution of Au NPs within cells. To understand the differences in the cellular uptake processes of Au NPs with different treatment concentrations, the trails of the adhesion and following evolution were also observed under dark field illumination. Cell viability with different treatment concentrations of Au NPs was also determined using a MTT assay. The relationship between the distribution, aggregation and cytotoxicity of Au NPs with different treatment concentrations was then compared. This work highlights the significant consequences of different dose treatments of Au NPs. As either a drug/gene carrier or contrast agent, the dose-optimization of the efficacy and safety of NPs are stressed in nanomedicine and nanotoxicology. By inspecting the distribution and aggregation of the NPs and their cytotoxicity, it helps us to think about the use of NPs, as well as improve or redesign the functions of the NPs used in nanomedicine.

Experimental section

Materials

Gold nanospheres with a diameter of 50 nm were obtained from Nanopartz with original particle concentrations of approximately 5.38 × 10¹⁰ particles per milliliter. Poly(allylamine hydrochloride) (PAH, M_w ∼ 15 000), dynasore, glutaraldehyde and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich. F-12K cell culture medium, fetal bovine serum (FBS), and antibiotic penicillin streptomycin amphotericin (PSA) were from Gibco, Invitrogen. 99.99% ethanol was purchased from Merck, Taiwan.

Cell culture

A549 adeno-carcinoma cells were cultured in the F-12K containing 10% FBS and 1% PSA. Cells were incubated in a 37 °C and 5% CO₂ incubator. In the experiment, the cells (10⁶ cells per mL) were implanted into the microchip with a cell-culture cavity (1 cm × 3 cm × 120 μm) for overnight.

Setup

The setup was based on the Olympus upright microscope BX41. 100× (N.A. = 0.6–1.3) oil type objective lenses were used in the sectional dark field microscopy. A 40× air type (N.A. = 0.6) was used in the dynamic trail experiment. Different to the 40× air type objective, the 100× oil objective lens was mounted on a PZT stage controlled by a function generator. A zig-zag voltage ramp was generated to scan along the Z axis for the axial section. Once the CCD was activated, the shutter would be opened and then a 20 W metal halide light was introduced obliquely into the sample in the Cyto Viva setup, and the frame grabber began the synchronic image sequence recording. Cells were implanted inside a homemade microfluidic chip which was configured by acrylic junctions, glass and double-sided tape as shown as shown in Fig. S1.† During the experiment, fresh medium was kept flowing into the chamber at a rate of 1 μL min⁻¹ using a syringe pump to keep a fluidic environment. At the beginning of the experiment, Au NPs (100 μL with different concentration, 0.01, 0.05, 0.1, 0.2 and 0.5 nM) were injected into the chip. The microscope, the chip containing cells and the syringe pump with medium were placed inside an incubator at 37 °C and with a 5% CO₂ atmosphere, as shown in Fig. S1.†

Chromatic analysis of Au NPs clusters

The analysis of the aggregated Au NPs is based on our previous study in ref. 28. Briefly, the chromatogram of the different aggregated number of Au NPs was first established by cross-referencing their scattering image and SEM images. As a result of the coupling of the LSPR in high order mode,²⁷ the increase in the aggregated number of Au NPs would induce their scattering color to redden, i.e. from green to yellow-orange, and red. Once the relationship between the scattering color and their aggregated number was established, we can estimate the number of Au NPs inside each bright spot (Au NP clusters) under the dark field illumination even within the cell environment. The scattering images of Au NPs within cells were first transferred from RGB to HSB (hue, saturation and brightness) color space using a Matlab program. The ratio of the brightness to hue of each Au NP cluster was then calculated. By applying the chromatogram-aggregated number relation established before, the number of every cluster was calculated. This chromatic analysis method was also validated by inductively coupled plasma mass spectrometry (ICP-MS) as described in ref. 28.

Functionalization of Au NPs

The commercial Au NPs were initially stabilized by citrate anions and have negative charges on the surface. Compared to the citrate coating, the PAH coating can increase the attachment to the cells, because of the negative charge of the cellular membrane,²⁹ and its stabilization in the culture medium. Fig. S2(a) and (b)† shows the cells treated with citrate coated Au NPs. We find that there are only few Au NPs attached on the cells, while these Au NPs present as large aggregates in the culture medium. Thus, in the following experiments, we modified the surface of the Au NPs with PAH to increase their stability and cellular adsorption. PAH coated Au NPs were made by adding 1 wt% PAH into citrate stabilized gold nanosphere solution (900 μL, ∼4.5 × 10¹⁰ NPs mL⁻¹) for two hours (the final PAH concentration was 0.1 wt%). The unbound polymer was then removed and the Au NPs (1 mL in microtube) were subsequently washed twice by adding deionized water (DIW) and concentrated, with the final concentration being 2 nM, ∼1.2 × 10¹¹ NPs mL⁻¹. Au NPs were then added into the medium at different concentrations (0.01, 0.05, 0.1, 0.2 and 0.5 nM) and subsequently injected into the microchip for the following experiments. The UV-Vis spectra of these Au NPs with different surface modifications and concentrations were shown in Fig. S3.†

Inductively coupled plasma mass spectrometry (ICP-MS) analysis

For the ICP-MS experiments, the cells were cultured in a 48 well culture dish at an initial density of ∼10⁴ cells per millilitre. After 24 hours, different doses of PAH-coated Au NPs were added to the medium. After 5 minutes, cells were washed with culture medium thrice to remove the unbound NPs. After 8 hours, cells were trypsinized, collected and then sonicated for 10 minutes to disrupt the cell membranes. Au NPs were dissolved by adding 0.3 mL aqua regia. The concentration of gold was determined by ICP-MS (ICP-MS Xseries II, Thermo) and converted to the number of Au NPs per cell.

Specimen preparation for scanning electron microscopy (SEM)

For the inspection for the SEM, the cells were first treated with different concentrations of Au NPs and cultured for 8 hours in the micro-fluidic environment as described in the setup section. The cells were then fixed using 2.5% glutaraldehyde for 2 hours, and dehydrated using 99.99% ethanol. Samples were dried using a critical point dryer (Leica EM CPD300) and subsequently coated with 20 nm gold film by sputtering for either EM or DBFIB inspection.

Cytotoxicity evaluation

To evaluate the cytotoxicity of Au NPs with different concentrations, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used. The A549 cells were incubated in 96 well plates with a density of 10⁴ cells per well overnight at 37 °C and in a 5% CO₂ atmosphere. Different concentrations of Au NPs were then added into each well. After 5 minutes, the suspended Au NPs were washed away twice using F-12K cell culture medium rinsing. For the endocytosis-inhibited experiment, the cells were pre-treated with 160 μM dynasore for 1 hour. The culture medium used in the endocytosis-inhibited experiments before the MTT test always contained 160 μM dynasore. Cell viability was then evaluated using MTT after an extra 8 hours’ interaction with Au NPs. The medium in each well was first removed and replaced with 100 μL fresh culture medium. 10 μL of 12 mM MTT dissolved in PBS was added into each well. The culture plates were incubated at 37 °C and in a 5% CO₂ atmosphere for 4 hours. After removal of all but 50 μL of the medium, 100 μL of DMSO was added to each well and the sample was incubated at 37 °C for 10 minutes to dissolve the dye. The absorbance at 540 nm was measured using a microplate reader.

Results and discussion

Scattering images and chromatic analysis

Fig. 1 shows a series of scattering images of cells treated with different concentrations of Au NPs at t = 8 hours. Compared to the cells, the Au NPs clusters show a fantastic color (green and yellow spots) under the dark field illumination. According to previous studies, the different colors of the Au NPs clusters indicates a different aggregated number.^28,30 To quantify the relation between the treatment concentration of Au NPs and their cellular uptake, chromatic analysis was employed.²⁵ Chromatic analysis can be used to calculate the total number of Au NPs per cell by summing the number of Au NPs in every cluster. Fig. 2(a) shows the calculated total number of Au NPs per cell using chromatic analysis and ICP-MS. We find that these two analysis methods show a similar result, thus, as the treatment concentration was increasing, the total number of Au NPs per cell was also increasing. This indicates the reliability of the chromatic analysis, and this result is in good agreement with the scattering image as shown in Fig. 1. In this work, as Au NPs were only applied as a treatment at the beginning, it can be considered as a “source-limited” case. Compared to other previous works which were “source-sufficient”, i.e. cells were always immersed in the NP containing medium during the experiment.^24,31,32 However, both of these two cases show similar results. Generally, with the same interaction time, a higher concentration of NPs has a higher chance of interacting with cells and therefore increases the number taken up.

Fig. 1 Scattering images of A549 cells after treatment with different concentrations of Au NPs for 8 hours. Scale bar = 20 μm.

Fig. 2 (a) Total number per cell, (b) spot (cluster) number, and (c) spot (cluster) percentage of Au NPs with different treatment concentrations per cell. Data represented as standard error of the mean in (b), sample number = 45.

In our previous study,³³ we found that the PAH coated Au NPs (0.1 nM) were up-taken into cells via dynamin dependent endocytosis, and the color change was due to the progressive sorting by the fusion of the endocytosis in cells. As shown in Fig. S2(c and d) and Movie 1,† Au NPs were firstly scattered on the cell as green dots, and then the green dots start decreasing and yellow spots start appearing, indicating the aggregation of Au NPs. In comparison, in the case of cells treated with dynasore, an inhibitor of dynamin dependent endocytosis,^34,35 there is no obvious aggregation happening. Again, this result clarifies that the Au NPs were taken up into cells via dynamin dependent endocytosis, and different color spots indicate the different sizes of Au NPs clusters, or in general, the cargos in the cellular vesicle at different endocytic states. Vesicle formation provides a means of cellular entry for extracellular substances. Although ICP-MS is excellent in quantification, it is difficult to distinguish the aggregation of Au NPs. In comparison, chromatic analysis can provide additional information on the aggregation of Au NPs within cells. In the following experiment, to simplify the vesicle formation, here, we categorized the number of Au NPs in the cluster into four groups. They are the aggregate number n = 1–3, 4–6, 7–12 and n > 12 respectively. Every group is roughly twice the size of the prior one. The categorization is based on the assumption of the homotypic endocytosis process, that is, that the endocytic vesicles containing cargos (Au NPs in this work) would undergo either homotypic fusion to form a new, larger endosome which carries the combined cargos of the original ones, or homotypic fusion to form two smaller new endosomes with each containing half of the cargo of the original one.³⁶ Fig. 2(b) shows the spot number of each aggregated group with different treatment doses. We observe that with the increasing concentration, the numbers of color spots (Au NP clusters) of each group were also increasing. However, the rate of increase of each group seems not to be homogeneous. There are abrupt increases within the range from 0.05 to 0.1 nM, while a plateau was reached in the range from 0.2 to 0.5 nM of n = 1–3. For n = 7–12, the spot number rises slowly at concentrations less than 0.2 nM, but abruptly increases at 0.5 nM. In comparison, the others show a much more stable expansion with the increase of the treatment dose.

Fig. 2(c) shows the spot percentage of each aggregated group. From the data, we can further observe the abrupt change in n = 1–3 and n = 7–12 at the treatment doses of 0.1 and 0.5 nM. In order to have a more deep comprehension of the formation of these different clusters of Au NPs, sectional scattering images and the trail of the Au NPs in cellular uptake were inspected. In the receptor-mediated endocytosis evolution of the NPs, mono-dispersed NPs are usually found on or near the membrane. As the endocytosis progresses, the NPs are expected to be sorted and clustered in endo-lysosomes inside the cell.^30,32,37 However, in the case of 0.1, 0.2 and 0.5 nM concentrations, the Au NPs show some different endocytic behaviours. For the 0.1 and 0.2 nM, there are two kinds of color bands, one is yellow and scattered near the nucleus, and the other one is on the membrane which is green (indicating individual nanoparticles) as indicated in Fig. 3(a) and (b). For the 0.5 nM, all the spots show as yellow, while distributed mainly in two different areas, beside the nucleus and attached to the membrane as indicated in Fig. 3(c).

Fig. 3 Sectional scattering images at different focal planes z = 0.96 and 4.8 μm with treatment doses (a) 0.1, (b) 0.2 and (c) 0.5 nM. Scale bar = 20 μm.

Trails of the cellular uptake and following evolution with different treatment doses (0.1, 0.2 and 0.5 nM) of Au NPs are shown in Movie 1–3,† respectively. In the cases of 0.1 nM and 0.2 nM treatment, green spots, the mono-dispersed Au NPs, are scattered all over the cells in the beginning. As time goes on, Au NPs start to move toward the center of the cell, and yellow spots, the clustering NPs, begin to form simultaneously. After a while, the number of green and yellow spots seems to come to a stable state, i.e. no more new clustering of Au NPs was achieved. Finally, two different aggregated areas were formed. By inspecting the scattering images at different focal planes (z = 0.96 and z = 4.8 μm) as shown in Fig. 3(a) and (b), the locations of the aggregated areas were further revealed. We can observe that there are two different areas in particular, yellow mostly beside the nucleus in the bottom of the cells, and green on the membrane (indicated by the arrowheads in the bottom rows of Fig. 3(a) and (b)). In comparison, in the case of 0.5 nM treatment as Movie 3† shows, although the green spots moved to the center and the yellow spots were revealed simultaneously in the beginning, the same as in 0.1 and 0.2 nM, no green spots remained. As a result, there are still two areas in particular where the spots are, but both of them contain yellow spots instead. Fig. 3(c) shows the different focal planes (z = 0.96 and z = 4.8 μm) of the scattering images in the case of 0.5 nM, and these two aggregated areas were found in different locations; one is beside the nucleus in the bottom of the cell, the other is on the membrane as indicated by the arrowhead in the bottom rows of Fig. 3(c). We attributed this difference to the cooperation of the actin polymerization and endocytic processes at the plasmon membrane.^38,39 As described before, Au NPs were expected to be clustered inside cells and result in the change of the scatter color from green to yellow. However, when the number of the treatment Au NPs was too large, cells would find it very difficult to deal with all of them through endocytosis from the membrane into cells. Meanwhile, the periodic lamellipodial contraction with actin polymerization is still at work on the membrane, and so the un-endocytosed Au NPs would be thus concentrated to the center, at the top of the cell as seen in Fig. 4(a) and (b). As the treatment dose was still increased to 0.5 nM, the coupling of the LSPR would also be induced by the highly concentrated un-endocytosed Au NPs on the membrane, meaning the scatter color would also be changed from green to yellow.

Fig. 4 SEM images of A549 cells with different treatment doses of Au NPs. (a–c) Cells treated with 0, 0.01 and 0.05 nM concentrations of Au NPs, and (d–f) is the zoom-in of the red frame with the area ∼10 × 9 μm² in (a–c) respectively. (g–h) Cells treated with 0.1, 0.2 and 0.5 nM concentrations of Au NPs, and (j–l) shows the zoom-in of the red frame with the area ∼10 × 9 μm² as shown in (g and h) respectively. Note, the fibers shown in (d) and (e) are the cilia of A549. Scale bar = 10 μm.

Demonstration of the distribution of Au NPs by SEM and DBFIB

SEM was also used to demonstrate the distribution of the Au NPs on the membrane. As shown in Fig. 4, we can easily observe that the Au NPs aggregate on the top of the cells treated with the “over treatment” (0.2–0.5 nM). It is noted that even for the “under treatment” (0.01 and 0.05 nM), there still remained some individual NPs on the membrane. It indicates that, in the NP–cell interactions, especially in the studies of the endocytosis, that maybe not all of the NPs would be endocytosed and some amount of NPs would be left on the membrane somehow. With the increasing of the treatment dose, the number and density of the Au NPs on the membrane was also increased. Fig. 5 shows the density of the Au NPs in the aggregated bands on the top of cells. The data further verify the different colors present of the aggregated bands in the scattering images of Fig. 3.

Fig. 5 Density of Au NPs on the aggregated bands of cells treated with different doses of Au NPs. ‡p-value < 0.01, ⁂p-value < 0.005. Sample number = 15.

DBFIB was then used to determine the difference between the endocytosed and un-endocytosed Au NPs in and on cells, respectively. The cases of 0.1 and 0.5 nM were studied. Fig. 6 shows the scattering images and their corresponding SEM images. We can find the un-endocytosed Au NPs were scattered on the membrane more individually after the 0.1 nM treatment. As the treatment dose was increased to 0.5 nM, they formed a “2-D quilt” covering on the cell, similar to that as shown in Fig. 4(l). Using DBFIB cutting, Au NPs aggregated in the endocytic vesicle were also revealed. Different from the “2-D quilt” on the cells, Au NPs are observed as 3-D stacks inside both in 0.1 and 0.5 nM (stacks of SEM sectional images are shown in Fig. S4†). It indicates that Au NPs can form a larger aggregation inside cells than on the cell membrane. In other words, the concentrated density on the membrane is limited by the 2-D space. The special resolution of this optical system is about 0.25–0.3 μm, and after calculation, there are around 7–12 NPs under this resolution. It also explained why the biggest increase in spots number was the aggregated number n = 7–12 rather than n > 12 in Fig. 3(b) and (c), although both of them were increasing at 0.5 nM.

Fig. 6 Scattering images of cells treated with (a) 0.1 and (b) 0.2 nM Au NPs at (1) z = 0.96 and (2) z = 4.8 μm. As well as (3) their corresponding SEM images, (4) 52° tilted and (5) sectional SEM images cut by DBFIB. Note that because there is a 15 degree rotation in the scattering images in (a), to have a better comparison with the SEM images, the scattering images from the dark field microscope were rotated and cropped.

Dose dependent cytotoxicity test

The cytotoxicity at different treatment doses was examined using an MTT assay. Fig. 7(a) shows the result. With the increase in treatment dose, the cellular viability decreased. We attribute the cytotoxicity partly to the increase of the Au NP clusters inside the cells, especially in the lysosomes. In previous studies, evidence indicates that the cytotoxicity of metal NPs is mostly from the release of toxic ions, such as Ag⁺, Au^1+/3+, and Cd²⁺, in the acidic environment of lysosomes.^22,40–42 From the scattering images in Fig. 2, we can observe that with the increasing of the treatment dose, the number of Au NP clusters inside the cell also increases, and thus, the result is also consistent with previous efforts.⁴³

Fig. 7 Cell viability with different treatment doses of Au NPs (a) without and (b) with 160 μM dynasore treatment. *p-value < 0.05, ‡p-value < 0.01, ⁂p-value < 0.005. Data comes from 5 independent experiments.

Furthermore, in most efforts, the toxicity is mainly regarded as the “Trojan horse effect”.^42,44,45 However, in this work, we find Au NPs would not only be stuck inside cells, but also stacked on the membrane. To evaluate whether the stacked Au NPs on the membrane would additionally impact the cell viability or not, a controlled experiment with cells treated with different doses of Au NPs while pre-treated with dynasore, was performed. Fig. 7(b) shows the result. Under the inhibition of receptor-mediated endocytosis caused by dynasore, all Au NPs are assumed to be stuck on the membrane. From the result, although the inhibition of endocytosis would affect the cell viability, a series of Au NP treatments on the membrane additionally impacts the cell viability. Unlike the acidic environment in lysosomes, the membrane should be a relatively neutral environment. It means that there should be no release of the toxic ions, Au^1+/3+. Therefore, we think it might be due to the physical influence, the pressure from the Au NPs on the membrane. With the increase of the loading on the membrane, Au NPs would hugely influence the cell metabolism.

Some studies have pointed out that both chemical and physical reasons would induce different kinds of cellular responses, such as apoptosis, proliferation, and migration.^46–48 However, there is still no exact explanation for the influence. In this work, we verify that the interaction between NPs and cells is not only inside cells, but also that un-endocytosed NPs on the membrane would affect the cellular behaviour. Both of these should be considered as important factors in NP–cell studies.

Conclusions

In summary, we show different evolutions of Au NP uptake with different treatment doses using dark field microscopy and SEM. In the lower treatment doses, 0.01 and 0.05 nM, most NPs can be endocytosed smoothly and clustered inside endocytic vesicles with a larger aggregated number. While with the higher treatment doses, 0.1 and 0.2 nM, a traffic jam of endocytosis occurred. Although the clusters inside cells increased, lots of Au NPs began to be stuck on the membrane. Accompanied by the periodic lamellipodial contraction with rearward actin polymerization on the membrane, the stuck Au NPs were then moved to the top of cells. This results in two distinct scatter color bands, yellow in the cells and green on the membrane. Furthermore, when the number of stuck Au NPs was too large, the highest treatment dose of 0.5 nM in this work, the stuck Au NPs further started to aggregate on the top of cells. Two bands with same scatter color were then shown. SEM images further validated the different aggregations of Au NPs on the membrane. Higher treatment doses resulted in the higher density of Au NPs. DBFIB and tilted SEM images indicated the different aggregated situations of Au NPs inside the cells and on the membrane, 3D stacking and 2D covering respectively. Fig. 8 illustrates the different aggregated situations with different treatment doses of Au NPs. MTT analysis also revealed the influence of Au NPs with the different treatment doses. Generally, higher treatment doses induces higher cytotoxicity. However, the dynasore pre-treated cytotoxicity test further proves that the higher cytotoxicity of the higher treatment dose did not only come from the Au NPs inside cells, but also from the stuck NPs on the membrane. High concentration of the Au NPs could increase the total amount of cellular uptake, but might lower the yield of Au NP entry due to a traffic jam of endocytosis and even increase the side-effects of the use of Au NPs as contrast agents or drug/gene carriers. In previous studies, major works focus on the endocytosed or penetrated Au NPs inside cells. The influence from the Au NPs stuck on the membrane is often limited. This work highlights the importance of an overall distribution of Au NPs in the NPs–cell interactive system.

Fig. 8 Illustration for different evolutions of cellular uptake with different treatment doses of Au NPs.

Acknowledgements

The authors thank Dr Chau-Hwang Lee and Miss Huei-Jyuan Pan for assistance with cell culture. The tilted and sectional SEM image was supported by the DBFIB core facility at the Research Center for Applied Sciences, Academia Sinica. This work was supported by the Ministry of Science and Technology of Taiwan, under Project MOST 103-2221-E-001-013-MY3. Technical support from the core facilities for nanoscience and nanotechnology, Academia Sinica in Taiwan, is acknowledged.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra18801f

‡ These authors contributed equally to this work.

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