Reverse Size Dependences of the Cellular Uptake of Triangular and

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Reverse Size Dependences of Triangular and Spherical Gold Nanoparticles on the Cellular Uptake Katsuyuki Nambara, Kenichi Niikura, Hideyuki Mitomo, Takafumi Ninomiya, Chie Takeuchi, Jinjian Wei, Yasutaka Matsuo, and Kuniharu Ijiro Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02064 • Publication Date (Web): 21 Sep 2016 Downloaded from http://pubs.acs.org on September 22, 2016

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Article for Langmuir

Reverse Size Dependences of the Cellular Uptake of Triangular and Spherical Gold Nanoparticles

Katsuyuki Nambara,† Kenichi Niikura,*,‡ Hideyuki Mitomo,‡ Takafumi Ninomiya,§ Chie Takeuchi,‡ Jinjian Wei,† Yasutaka Matsuo,‡ and Kuniharu Ijiro*,‡

†

Graduate School of Chemical Sciences and Engineering, Hokkaido University, Kita 13, Nishi 8, Kita-Ku, Sapporo 060-8628, Japan ‡

Research Institute for Electronic Science (RIES), Hokkaido University, Kita 21, Nishi 10, Kita-Ku, Sapporo 001-0021, Japan §

Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan

Corresponding Authors *E-mail: [email protected], [email protected]

Notes The authors declare no competing financial interest.

1 ACS Paragon Plus Environment

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Abstract Gold nanoparticles (GNPs) show promise as both drug and imaging carriers with application to both diagnosis and therapy. For the safe and effective use of such gold nanomaterials in the biomedical field, it is crucial to understand how the size and shape of the nanomaterials affect their biological features, such as in vitro cellular uptake speed and accumulation as well as cytotoxicity. Herein, we focus on triangular gold nanoparticles (TNPs) of four different sizes (side length, 46, 55, 72, and 94 nm; thickness, 30 nm) and compare the cellular internalization efficiency with those of spherical nanoparticles (SNPs) of various diameters (22, 39, and 66 nm). Both surfaces were coated with anionic thiol ligands. Inductively coupled plasma emission spectrometer (ICP-ES) data demonstrated that TNPs with longer sides showed higher levels of uptake into RAW264.7 and HeLa cells. On the other hand, in the case of SNPs, those with smaller diameters showed higher levels of uptake in both cells. Our results support the notion of a reverse size dependence of TNPs and SNPs in terms of cellular uptake. For HeLa cells, in particular, 20-fold more efficient internalization was observed for TNPs with longer sides (72 nm in side length) compared to SNPs (66 nm) with a similar surface area. These results highlight the importance of the shape of nanomaterials on their interactions with cells and provide a useful guideline for the use of TNPs.


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Introduction Cells are the basic unit of life, and are enclosed by a cell membrane made up of a lipid bilayer. This lipid bilayer acts as a barrier to the delivery of functional nanoparticles into cells. An understanding of the interactions between cell membranes, including artificial membranes,1 and nanomaterials is important for the rational design of drug carriers. Gold nanomaterials show promise as carriers not only for drug delivery,2 but also for imaging and therapy3 based on Raman or light scattering4-6 and photothermal conversion,7-9 respectively.

Recently, the

synthesis of gold nanoparticles (GNPs) of various shapes and sizes has been explored.10,11 These nanomaterials of different shapes and sizes are expected to interact differently with the biomembrane,12 affecting cellular uptake efficiency13 and biological responses, such as cytotoxicity,14,15 in vivo biodistribution,16,17 and subsequent immune responses.18,19

Therefore,

it is important to understand how the size and shape of GNPs affect their interactions with cells.20

The effects of the size and shape of nanoparticles on cellular uptake have been widely

investigated based on experimental19,21 and simulation studies.22

For example, both

experimental and simulation studies have shown the effect of the diameter of spherical gold nanoparticles on cellular uptake level in various cells.23-25

Herein, we focus on triangular gold

nanoparticles (referred to as TNPs) and demonstrate the effect of side length on their cellular internalization in comparison with spherical nanoparticles (Scheme 1). The benefits of TNPs over spherical gold nanoparticles (SNPs) for biomedical applications are the higher drug payload due to the higher surface ratio to volume, and the plasmon peaks of TNPs around the near infrared (NIR) region with large scattering properties26 enable efficient in vivo imaging through biological windows and photothermal therapy. Åberg et al. reported that nanoparticle uptake is a two-step process; first, nanoparticles adhere to the cell surface and, next, they are internalized via energy-dependent endocytosis.27 Therefore, the trade-off between “adhesion


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energy” and “membrane-bending energy during endocytosis” is a key factor in determining the level of cellular uptake. Nanomaterials with a large flat face, such as TNPs and nanodisks, can generate a stronger adhesion force to the membrane than SNPs due to the wide contact area.28 Furthermore, the results of experimental and simulation studies support the idea that membrane wrapping, beginning from a geometrical region with high local curvature, accelerates the cellular internalization of nanomaterials.29-33

There are two regions of high curvature on TNPs

(the sharp vertices and edges), so it is expected that the geometrical features of TNPs could contribute to enhanced cellular internalization. These curvatures on the TNPs are independent of their side length, while the curvature of SNPs depends on their diameter. These differences might lead to differences in size dependence between TNPs and SNPs.

We synthesized TNPs

with various side lengths and explored the size dependence of their cellular internalization in comparison to that of SNPs with a similar surface area or volume. Recently, Fuente and coworkers demonstrated that the light irradiation on TNP-internalized cells induced cellular apoptosis, but not necrosis, indicating the unique merits of TNPs in comparison to other nanomaterials available for biomedical applications.34

Despite the rapid progress in the

development of synthetic methods for the production of TNPs,35,36 no quantitative or systematic studies on the cellular internalization and biological applications of TNPs of various sizes have been reported. In this paper, we present the effect of the side length of TNPs on their cellular uptake into two types of cells (RAW264.7 and HeLa cells). RAW.264.7 cells are a cell line established from mouse monocyte macrophage, thus these cells intrinsically uptake foreign particles via phagocytosis. In contrast, HeLa cells, which are derived from cervical cancer cells, were used as nonphagocytic cells. It was reported that HeLa cells internalize particles of various sizes and shapes through different endocytotic pathways.37-39

The difference in cellular uptake


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mechanism between RAW264.7 and HeLa cells might be involved. the surface charge also affects cellular uptake.40,41

Further, it is known that

It is known that nanoparticles which are

negatively charged are more slowly taken into cells through endocytosis or pinocytosis than positively charged nanoparticles.42,43 cytotoxicity.

However, positively charged nanoparticles show greater

Therefore, we choose nitrilotriacetic acid (NTA)-terminated alkanethiol

(NTA-SH) (see structure in Scheme 1) as an anionic surface ligand as GNPs with anionic ligands generally show an absence of cytoxicity and the NTA group enables specific modification with proteins through metal ions for further protein delivery.

We found a reverse

size dependence on cellular uptake in which are increase in the side length of TNPs increased their cellular uptake, while increases in the diameter of SNPs resulted in lower cellular uptake.


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Experimental Section Materials HAuCl4·3H2O,

L-Ascorbic

acid,

NaI,

NaBH4,

and

N-[Nα,Nα-Bis(carboxymethyl)-L-lysine]-12-mercaptododecanamide (NTA-SH) were purchased from

Sigma-Aldrich

(USA).

Hexadecyltrimethylammonium

chloride

(CTAC),

hexadecyltrimethylammonium bromide (CTAB), 1.0 mol/L NaOH, ethanol, trisodium citrate dehydrate, potassium ferrocyanide, gold standard solution (Au 1000) (1 mg/mL = 1,000 ppm), hydrochloric acid, and nitric acid were purchased from Wako Pure Chemical Industries, Ltd. (Japan). Ultrapure water (18.2 MΩcm-1, Milli-Q, Millipore, USA) was used for all solution preparations

and

experiments.

Dulbecco's

modified

Eagle’s

medium

(DMEM),

phosphate-buffered saline (PBS, pH 7.4), and Trypsin-EDTA solution 1X (0.05% trypsine/0.02% EDTA) were purchased from Sigma-Aldrich (USA). Fetal bovine serum (FBS) and penicillin-streptomycin were purchased from Thermo Fisher Scientific (USA). Solution glutaraldehyde (25%), 2% w/v solution osmium tetroxide, Epon 812 resin, dodecenyl succinic anhydride (DDSA), methyl nadic anhydride (MNA), and 2,4,6-tri(dimethylaminomethyl) phenol (DMP30) were purchased from TAAB Laboratories Equipment, Ltd. (England). BioCoat poly-D-lysine 8-well CultureSlides were purchased from CORNING (USA). Scanning transmission electron microscopic (STEM) images were obtained using a STEM HD-2000 system (Hitachi High-Tech Manufacturing & Service Co., Ltd., Japan) with 200 kV acceleration voltage. UV-vis spectra were measured with a UV-vis spectrophotometer (UV-2600; Shimadzu Corporation, Japan). Dynamic light scattering (DLS) analysis was performed with DelsaNano HC (Beckman Coulter, Inc, USA) and ELSZ-2000 (Otsuka Electronics Co., Ltd., Japan) particle analyzers. Measurements of ζ potential were performed with DelsaNano HC, and the GNP solutions were dissolved in water (final concentration: ca. 0.1 nM).


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The analysis of Au concentration taken up into cells was performed using an inductively coupled plasma emission spectrometer (ICP-ES) (ICPE-9000; Shimadzu Corporation, Japan). Sample preparation for ICP-ES was performed with a microwave sample preparation system (ETHOS One, Milestone, Italy). Transmission electron microscopic (TEM) images were obtained using a JEM-1400 transmission electron microscope (JEOL, Japan) with 80 kV acceleration voltage.

Synthesis of triangular gold nanoparticles coated with CTA Triangular gold nanoparticles were synthesized following a protocol based on a seeding growth method reported by Liz-Marzán et al.35 (1) Preparation of the seed solution HAuCl4·3H2O (25 µL, 0.05 M) was added to a CTAC aqueous solution (4.7 mL, 0.1 M) in a 50-mL plastic tube. The solution was manually stirred until the solution appeared yellow. Freshly prepared NaBH4 solution (300 µL, 0.01 M) was then injected under vigorous stirring by voltex mixer. The solution was then left for 2 h at room temperature prior to use. The solution (100 µL) was diluted 10 times with CTAC solution (900 µL, 0.1 M) to produce the seed solution. (2) Growth reaction We prepared the following two growth solutions: (1) an aqueous solution containing 1.2 mM HAuCl4·3H2O, 15.2 µM NaI, and 16.2 mM CTAC; and (2) an aqueous solution containing 61.3 mM HAuCl4·3H2O, 73.5 µM NaI, and 4.9 mM CTAC. Subsequently, ascorbic acid solutions (80 µL and 1.6 mL, 0.1 M) were added to solution (1) and (2), respectively. The solutions became colorless after the addition and mixing of the ascorbic acid. Finally, the seed solution (100, 200, 400, or 450 µL) was quickly added to the colorless solution (1), and this solution (12.8 mL) was immediately added to the colorless solution (2) and manually stirred for a few seconds by gentle inversion. The solution was left undisturbed at room temperature for 2 h.


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(3) Purification The as-prepared triangular gold nanoparticle solutions were centrifugated (2,900g, 60 min, 25 °C), and the supernatant was removed. The residue was transferred to a 15-mL tube, then Milli-Q water (5 mL) and 25 wt% CTAC solution (1.0, 1.2, 2.0, or 2.5 mL) were added. Flocculation of the triangular gold nanoparticles was completed by standing overnight at room temperature. The supernatant was then removed, and the precipitates were redispersed in Milli-Q water (10 mL) and CTAC solution (30 mL, 0.1 M). Synthesis of spherical gold nanoparticles coated with CTA (22 and 39 nm in dia.) Spherical gold nanoparticles were prepared according to the method44 reported by Wang et al. with some modifications. (1) Preparation of seed solution HAuCl4·3H2O (250 µL, 0.01 M) was added to a CTAC solution (7.5 mL, 0.1 M) in a 50-mL plastic tube. The solution was manually stirred. The solution appeared bright orange. Then, a freshly prepared ice-cold NaBH4 solution (600 µL, 0.01 M) was injected under vigorous stirring by voltex mixer. The tube was then left for 2 h at room temperature until future use. The solution (100 µL) was diluted 10 times with CTAC solution (900 µL, 0.1 M) to produce the seed solution. (2) Growth reaction The growth solution was prepared in a 50-mL tube by the sequential addition of CTAB solution (3.0 mL, 0.1 M), CTAC solution (3.0 mL, 0.1 M), Milli-Q water (30 mL), followed by HAuCl4·3H2O (740 µL, 0.01 M). Subsequently, an ascorbic acid solution (3.6 mL, 0.1 M) was added to the growth solution. The solution became colorless after mixing with the ascorbic acid. Finally, the seed solution (200 µL for 22-nm diameter, or 18.6 µL for 39-nm diameter) was quickly added to the growth solution and manually stirred for a few seconds by gentle inversion. The spherical gold nanoparticle solution was left undisturbed overnight at 30 °C.


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(3) Purification The as-prepared spherical gold nanoparticle solutions were centrifugated (10,000g for 22-nm, 4,000g for 39-nm, 20 min, 25 °C), and the supernatants were removed. The residue was redispersed in Milli-Q water (10 mL). Synthesis of spherical gold nanoparticles coated with citrate (66 nm in dia.) Spherical gold nanoparticles of 66 nm in diameter coated with citrate were synthesized following a protocol based on a seeding growth method reported by Puntes et al.45 A sodium citrate solution (150 mL, 2.2 mM) was heated in a 250 mL three-necked round-bottomed flask for 15 min at 100 °C under vigorous stirring. HAuCl4·3H2O (1 mL, 25 mM) was then injected, and the solution was left for 10 min. After the reaction solution was cooled to 90 °C, HAuCl4·3H2O solution (1 mL, 25 mM) was again injected. After 30 min, HAuCl4·3H2O solution (1 mL, 25 mM) was injected once more, and the reaction was continued for 30 min at 90 °C. The sample was diluted by extracting 55 mL of this sample and adding Milli-Q water (53 mL) and 60 mM sodium citrate (2 mL). This solution was then used as a new seed solution for repeating the growth reaction, and this process were repeated four times. NTA modification of triangular and spherical gold nanoparticles NTA-SH (22.1 mg, 4.6 × 10-5 mol) was dissolved in NaOH aqueous solution (1.2 × 10-4 mol, 10.12 mL). The aqueous dispersions of the various GNPs synthesized by the above protocols (1.0 nM, 500 µL) were washed by centrifugation (1,500g for triangles, 2,000g for spheres). After the supernatant was removed, the residue was dispersed in Milli-Q water (500 µL). After the second centrifugation, the precipitate was dispersed in Milli-Q water (500 µL), and the NTA solution (100 µL, 21 µmol) was added to the solution. The solution was stirred at room temperature for 24 h. After the reaction, the solution was purified by centrifugation to remove excess NTA-SH, and the precipitate was finally redispersed in 1.0 mM phosphate buffer (pH 7.4, 1.0 mL).


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Cell Culture RAW264.7 and HeLa cells were maintained in monolayer cultures in 100-mm tissue culture dishes. The cells were grown under humidified 5% CO2 / 95% air at 37 ºC in DMEM supplemented with 10% (v/v) FBS, penicillin (500 units/mL), and streptomycin (500 µg/mL). The cells were harvested from the dishes for transfer by treatment with 0.05% trypsine/0.02% EDTA. RAW264.7 and HeLa cells were maintained by seeding 5.0 × 105 cells per 100-mm tissue culture dish and the cells were subcultured every 2-3 days. TEM Observation of the sliced RAW264.7 and HeLa cells RAW264.7 and HeLa cells were seeded at 2.0 × 104 cells in BioCoat poly-D-lysine 8-well CultureSlides and cultured for 1 day under the abovementioned conditions. The cells were incubated in the presence of the various GNPs (final particle concentration: 5.0 pM; 3.0 x 109 particles/mL) for 24 h in DMEM. After washing three times with phosphate buffer, the cells were fixed in 2.5% glutaraldehyde/0.1M phosphate buffer (pH 7.4) overnight at 4 ºC, postfixed in a mixed aqueous solution of 1% osmium tetroxide and 1.5% potassium ferrocyanide for 1 h at room temperature, dehydrated in a graded ethanol series, and embedded in Epon 812. Ultrathin sections were then cut on a RMC Ultramicrotome MTX. The sections were stained with uranyl acetate followed by lead citrate and examined by TEM. Quantitative analysis of the cellular uptake of the various GNPs RAW264.7 and HeLa cells were seeded at 5.0 × 104 cells in 35-mm tissue culture dishes and cultured for 1 day under the abovementioned conditions. The cells were washed twice with PBS (1.0 mL), and cells were incubated in the 10% FBS-containing DMEM (2.0 mL) and the solution of various gold nanostructures (10 µL, 1.0 nM of TNPs or SNPs) were added. The final particle concentration of TNPs and SNPs in the culture media was 5.0 pM. After incubation at 37 ºC for


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1, 3, or 24 h, the cells were washed twice with PBS (1.0 mL), removed from the dishes by treatment with 0.05% trypsine/0.02% EDTA at 37 ºC for 5 min, and the solution collected in a 15-mL tube. The cell suspension was added to aqua regia (HCl: 3.0 mL, HNO3: 1.0 mL) to dissolve the GNPs and diluted with Milli-Q water up to 10 mL. A calibration curve was prepared using gold standard solutions (0, 10, 20, 50, 100, 500, and 1000 ppb). All ICP-ES measurements were repeated independently three times.


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Results and Discussion Preparation of GNPs of various shapes and sizes Triangular and spherical GNPs of various sizes (TNPX and SNPX; X represents the average side length for TNPs or diameter for SNPs) were synthesized according to previous protocols based on a seeding growth method35,44,45 (see Experimental section). These protocols gave TNPs coated with cetyltrimethylammonium chloride (referred to as TNPs-CTA).

SNPs were

synthesized by two methods depending on diameter, producing SNPs coated with (1) cetyltrimethylammonium chloride/bromide (SNPs-CTA) and (2) citrate (SNPs-Citrate). The size and shape of the as-prepared GNPs were confirmed by transmission electron microscopy (TEM) using a scanning transmission electron microscope (STEM) (Figure S1). TEM images showed that the side length of the TNPs-CTA was 46, 55, 72, and 94 nm, respectively. The thickness of the TNP72-CTA was 29 ± 4 nm, based on Figure S2E, and this thickness was in good agreement with that produced using the same protocol in a previous report.35 not identify the thickness of the other TNPs-CTA from the TEM images.

We could

However, the good

agreement between the UV-vis spectra and side length of the TNPs-CTA and those in the previous report34 suggests that the thickness of the other TNPs-CTA was also around 30 nm (Figure 2). Hereafter, we regard all the TNPs-CTA as 30 nm in thickness.

We estimated the

purity of the TNPs from TEM images. Although there were some irregularly shaped particles, which we regarded as impurities, the purity of the TNPs-CTA was sufficiently high; ≥ 90% (TNP46-CTA), 83% (TNP55-CTA), 77% (TNP72-CTA), and 70% (TNP94-CTA), based on at least 500 counts of particles on the TEM images (Figure S2). The mean diameters of the SNPs-CTA were 22 and 39 nm, and SNPs-Citrate was 66 nm (Figure S1). The detailed physicochemical properties of the prepared GNPs are shown in Table 1. TNP46 and SNP39 possess similar surface areas and volumes. TNP72 and SNP66 have similar surface area, and


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TNP94 and SNP66 possess similar volumes. Surface modification of GNPs with NTA-SH. We functionalized the surface of the gold nanostructures with NTA-SH. TNPs-CTA, SNPs-CTA, and SNPs-Citrate were added to the NTA solution and the mixtures were stirred for 24 h at room temperature. The resultant solutions were washed twice via centrifugation, and the nanoparticles were resuspended in Milli-Q water. We confirmed the size and shape of the NTA-functionalized GNPs by STEM in TEM mode (Figure 1). TEM images clearly showed that the size and shape of the TNPs and SNPs were not changed after the ligand exchange reaction.

The ζ potentials of all GNPs were also measured (Table 1).

As-prepared GNPs,

except for SNP66-Citrate, were positively charged, and the surface charge was changed to negative after ligand exchange, indicating successful ligand exchange.

For TNPs-CTA, the

localized surface plasmon resonance (LSPR) wavelength was between 631-672 nm, depending on the side length. For SNPs-CTA, the LSPR wavelength was between 522-542 nm, depending on the diameter (Figure 2).

After the ligand exchange reaction with NTA-SH, the LSPR

wavelength peaks (wavelength of maximum absorbance) of all the GNPs were largely unchanged. UV-vis spectra of TNP94- and SNP66-NTA showed a little difference after ligand exchange. However, as we confirmed almost no change in TEM images of TNP94- and SNP66-NTA, we used these NTA-functionalized GNPs in the following experiment. Dynamic light scattering (DLS) analyses before and after the ligand exchange support the fact that there was almost no change in the hydrodynamic diameters (Table 1). These results support the idea that no aggregations occurred during the ligand exchange process. We investigated the ligand densities on TNPs and SNPs by ICP-ES (Table S2). We found that ligand densities showed unique tendencies depending on size and shape.

With an

increase in the side length of TNPs, the ligand densities decreased from 3.1 molecules nm-2


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(TNP46-NTA) to 1.1 molecules nm-2 (TNP94-NTA). With an increase in the diameter of SNPs, the ligand densities decreased from 5.4 molecules nm-2 (SNP22-NTA) to 2.6 molecules nm-2 (SNP66-NTA). These results indicate that smaller particles were covered with a higher density of NTA-SH. Residual CTAB/CTAC might affect cellular uptake and cytotoxicity, thus the residual surfactant level was estimated by 1H NMR (Figure S3). The results showed that the ratio of residual CTAB on the GNP surface was less than 1.4%. Therefore, we concluded that the residual CTAB/CTAC was negligible. The internalization of TNPs and SNPs in RAW264.7 and HeLa cells RAW264.7 and HeLa cells were incubated with TNP46- and SNP39-NTA (the combination of similar surface areas and volumes) for 24 h. The particle concentration of TNPs and SNPs in the cell culture media was 5.0 pM. After washing with phosphate buffer three times, phase-contrast images of the cells were obtained in the culture media (DMEM containing 10% FBS) (Figure 3A-F).

Phase-contrast images clearly showed that the

morphology of the cells was not changed. Additionally, cell counting kit-8 (CCK) assay did not show any differences between with and without nanoparticles in either type of cells for all GNPs, supporting no significant cytotoxicity of nanoparticles (Figure S4). We then confirmed the cellular uptake and intercellular distribution of TNPs and SNPs by TEM. TNP46- and SNP39-NTA, the surface areas and volumes of which are similar (Table 1), were applied to cells and incubated for 24 h at 37 °C. The monodispersity of TNP46- and SNP39-NTA in a culture media was confirmed by DLS (Table S1). For TEM observation, after washing with phosphate buffer three times, cells were fixed with glutaraldehyde and embedded in epoxy resin.

Ultrathin slices of these samples (thickness; ca. 70 nm) were

prepared by ultra-microtome and TEM images of the sliced samples were obtained (Figure 3 and Figure S5).

The TEM images indicated that the triangular shape of the TNPs was maintained


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even after cellular uptake in both cells (Figure 3G and I). In particular, Figure 3G-J clearly show that TNP46- and SNP39-NTA were distributed intercellular, especially in the endosomes, suggesting that both cells were internalized via endocytotic pathway. Quantitative estimation of the cellular uptake of TNPs- and SNPs-NTA We investigated the relationship between the side length of TNP-NTA and the level of cellular uptake by ICP-ES (Figure 4).

ICP-ES analyses afford a quantitative internalized

number of nanoparticles based on the concentration of Au atoms in a cell. The average number of nanoparticles in single RAW264.7 cell was 120, 130, 210, and 240 × 103 particles for TNP46-, TNP55-, TNP72-, and TNP94-NTA, respectively, based on the calculation according to a previous report45 (Figure 4A). TNP94-NTA, the largest particle used in this experiment, was the most efficiently internalized. On the other hand, the cellular uptake level of SNP-NTA was 290, 120, and 74 × 103 particles/cell for SNP22-, SNP39-, and SNP66-NTA, respectively. SNP22-NTA, the smallest particle, was most efficiently internalized among our spherical GNPs. These results demonstrate the shape dependence of cellular uptake in RAW264.7 cells.

For

HeLa cells, the internalized number was 4.0, 11, 79, and 190 × 103 for TNP46-, TNP55-, TNP72-, and TNP94-NTA, respectively, and 32, 4.1, and 4.0 × 103 particles for SNP22-, SNP39- and SNP66-NTA, respectively (Figure 4B).

The size dependence of TNPs on cellular

uptake in HeLa cells was more significant than that in RAW264.7 cells; TNP94-NTA was internalized at about 48-fold that of TNP46-NTA.

A comparison of TNP72-NTA and

SNP66-NTA, the surface areas of which are similar, showed that HeLa cells preferentially internalized the triangular GNPs at about 20-fold that of spherical GNPs.

While, for

RAW264.7 cells, the cellular uptake level of TNP72-NTA was about 3-fold that of SNP66-NTA. This means that HeLa cells are more sensitive to the shape of GNPs. It is of great interest that the cellular uptake level of TNPs with longer sides was


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higher than that of TNPs with shorter sides, while the cellular uptake level of SNPs with a larger diameter was lower than that of SNPs with a smaller diameter. There are several possible factors determining cellular uptake level, such as surface contact area,30,47 membrane deformation energy,47,48 sedimentation,47,49 and biological pathway.37,38

A triangular shape

could provide a large contact area with a flat surface due to the high surface ratio to volume compared to a spherical shape. Remembering that the cellular uptake of nanoparticles is a two-step process in which the nanoparticles initially adhere to the cell membrane and are subsequently internalized by the cells via energy-dependent pathways,27 the stronger adhesion force of the larger TNPs might contribute toward the observed trend in which " a larger size shows a larger uptake".

Nanodisks also have flat surfaces, and Roy et al. reported that the

uptake level of hydrogel nanodisks (ca. 100-300 nm in diameter, around 100 nm in height) was higher than that of nanorods (400-800 nm in length) in several mammalian cells due to lower membrane deformation energy, and the uptake level was increased when the diameter of the disk was increased, probably due to the larger adhesion area.47

Recently, a simulation study

has provided evidence in support of the notion that the larger volume of oblate ellipsoids is effective in inducing efficient endocytosis due to the lower level of rotation.25

These factors

might also contribute to the favorable uptake of TNPs with longer sides observed in our study. In addition to the larger contact area, based on the previous data on nanomaterial cellular uptake,29 the edges and vertices of TNPs with high local curvature could accelerate their cellular internalization. The sedimentation could be a factor to enhance the cellular uptakes. GNP solutions in cuvettes were monitored using UV-vis spectrometry for 0-24 h at room temperature (Figure S6). Larger GNPs (TNP94-NTA and SNP66-NTA) showed some sedimentation at 12 h. A time-course study on the cellular uptake of TNPs- and SNPs-NTA showed that the cellular


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uptake-dependences on TNP and SNP size were similar after incubation for 3 h and for 24 h (Figure 5), suggesting that internalization of GNPs occurred prior to sedimentation. Although, in general, the sedimentation effect of nanoparticles is important, these results indicated that the sedimentation in our system is not a major factor in the cellular uptake of GNPs.

From a

biological view point, endocytotic pathway could be an important to determine the cellular uptake level of GNPs. In fact, negatively charged nanoparticles were internalized into HeLa cells via different pathways that depend on nanoparticle sizes.37-39

Based on TEM images in

Figure 3, it seems that both TNPs and SNPs were localized in the endosomes. However, at the moment, we cannot conclude the difference in the detailed mechanism (clathrin or caveolin mediation) of endocytosis between the TNPs and SNPs uptakes.

The relationship between GNP surface area / volume and cellular uptake level The effects of size or shape on cellular uptake level were discussed for nanomaterials with similar surface areas or volumes.19

We summarized uptake levels of GNPs in Figure 4 as

functions of surface area or volume (Figure 6). It is noted that TNPs with a larger surface area are more effective in taken up while SNPs are less efficiently taken up in both cell types. This reverse dependence was also observed when the particle volume was used as a function. For TNPs and SNPs, smaller particles showed higher ligand densities, thus reverse dependence cannot be explained by ligand densities. This indicates that the shape of GNPs is a major factor in the high level of cellular uptake of TNPs. With regard to biomedical applications, as TNPs simultaneously enable a higher drug payload due to a large surface area and more efficient uptake. TNPs could provide a good platform for nanocarriers in drug transportation systems.


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Conclusion We synthesized triangular and spherical GNPs of various sizes that were subsequently functionalized with anionic NTA ligands.

When the surface area was more than 0.5 × 104 nm2,

the TNPs showed 3-fold more effective cellular uptake than did SNPs with a similar surface area in RAW 264.7 cells.

For HeLa cells, in particular, the TNPs showed 20-fold more

efficient internalization than did SNPs. TNPs with longer sides showed higher levels of uptake into RAW264.7 and HeLa cells. On the other hand, in the case of SNPs, those with smaller diameters showed higher levels of uptake in both cells. Our results provide support to the notion of reverse size dependences between TNPs and SNPs in terms of cellular uptake number. The edges and vertices of TNPs with high local curvature are important factors in accelerating their cellular internalization. The cellular uptake properties of TNPs shown here support the fact that nanomaterials with a triangular shape may afford a good platform for efficient drug delivery into cells for which the uptake of spherical nanoparticles is limited.

Acknowledgments This work was supported by The Canon Foundation and JSPS KAKENHI 16H03822. A part of this work was supported by "Nanotechnology Platform" Program of MEXT. We thank to Dr. G. Wang at RIKEN for helpful discussions of the synthesis of TNPs. ICP emission spectrometer, ζ potential, DLS, and STEM measurements were carried out at the OPEN FACILITY, Hokkaido University Sousei Hall.

Associated Content Supporting information is available free of charge via the Internet at http://pubs.acs.org//


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Figures

Scheme 1. Schematic illustration of this study and the chemical structure of NTA-SH.

Figure 1. TEM images of (A) TNP46-NTA, (B) TNP55-NTA, (C) TNP72-NTA, (D) TNP94-NTA, (E) SNP22-NTA, (F) SNP39-NTA, and (G) SNP66-NTA. Scale bars represent 100 nm.


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Figure 2. Normalized UV-vis spectra of various GNPs in Milli-Q water. LSPR wavelength peaks were normalized to 1.0.


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Figure 3. Phase-contrast images of (A)-(C) RAW264.7 and (D)-(F) HeLa cells after incubation with TNP46-NTA and SNP39-NTA for 24 h. TEM images of sliced (G), (H) RAW264.7 and (I), (J) HeLa cells after incubation with TNP46-NTA and SNP39-NTA for 24 h.


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Figure 4. Size- and shape-dependent cellular uptake of TNPs and SNPs into (A) RAW264.7 and (B) HeLa cells after incubation for 24 h. Uptake level was determined by ICP-ES. Bars represent mean values of three independent experiments and error bars show SD.

Figure 5. The cellular uptake of TNPs and SNPs into (A) RAW264.7 and (B) HeLa cells after incubation for 1, 3 and 24 h. Data are represented as mean values ± SD of three independent experiments.


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Figure 6. Cellular uptake level (24-h incubation) of TNPs and SNPs as a function of surface area or volume in (A), (B) RAW264.7 and (C), (D) HeLa cells.


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Table 1. The physicochemical characteristics of the various GNPs.

Name

Size (nm)

Wavelength of maximum absorbance (nm)

Hydrodynamic diameter (nm)

ζ potential (mV)

Surface area / particle (nm )

TNP46

l = 46 ± 4

CTA: 631 NTA: 631

CTA: 41 ± 8 NTA: 47 ± 7

CTA: 54 ± 5 NTA: -39 ± 7

6.0 × 10

TNP55

l = 55 ± 4

CTA: 635 NTA: 637

CTA: 50 ± 8 NTA: 53 ± 8

CTA: 30 ± 5 NTA: -66 ± 7

7.6 × 10

TNP72

l = 72 ± 5

CTA: 650 NTA: 655

CTA: 71 ± 10 NTA: 65 ± 9

CTA: 50 ± 3 NTA: -70 ± 6

1.1 × 10

TNP94

l = 94 ± 7

CTA: 672 NTA: 674

CTA: 90 ± 6 NTA: 78 ± 10

CTA: 40 ± 3 NTA: -66 ± 3

1.6 × 10

SNP22

d = 22 ± 4

CTA: 522 NTA: 523

CTA: 22 ± 4 NTA: 21 ± 5

CTA: 19 ± 3 NTA: -37 ± 5

1.5 × 10

SNP39

d = 39 ± 7

CTA: 527 NTA: 527

CTA: 43 ± 4 NTA: 47 ± 4

CTA: 19 ± 1 NTA: -46 ± 4

4.8 × 10

SNP66

d = 66 ± 7

Citrate: 542 NTA: 545

Citrate: 63 ± 12 NTA: 64 ± 14

Citrate: -47 ± 1 NTA: -29 ± 4

1.4 × 10

a

b

2 c

3

3

4

4

3

3

3

3 c

Volume / particle (nm ) 4

2.7 × 10

4

3.9 × 10

4

6.7 × 10

5

1.1 × 10

3

5.6 × 10

4

3.1 × 10

a: The size (side length; l , and diameter; d ) was calculated as the average of over 200 particles based on TEM images. b: ζ potential measurement was carried out by DelsaNano HC (Beckman Coulter, Inc, USA), and all gold nanoparticles measured were disolved in water at a final concentration of about 0.1 nM. c: Surface area and volume for TNPs were calculated based on the hypothesis that the thickness was 30 nm.


5

1.5 × 10

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Reverse Size Dependences of the Cellular Uptake of Triangular and

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