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Article Cite This: ACS Omega 2019, 4, 242−256

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Identifying Trends in Gold Nanoparticle Toxicity and Uptake: Size, Shape, Capping Ligand, and Biological Corona Catherine Carnovale,†,‡ Gary Bryant,‡ Ravi Shukla,† and Vipul Bansal*,† †

Sir Ian Potter NanoBioSensing Facility, NanoBiotechnology Research Laboratory, School of Science, and ‡Centre for Molecular and Nanoscale Physics, School of Science, RMIT University, GPO Box 2476, Melbourne, Victoria 3001, Australia

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S Supporting Information *

ABSTRACT: The drive behind the growing interest in understanding gold nanoparticle (AuNP) cytotoxicity originates from the promise of AuNPs for diverse biological applications across the fields of drug delivery, biosensing, biological imaging, gene therapy, and photothermal therapy. Although we continue to investigate the novel biomedical applications of AuNPs, progress is currently stalled at the periphery of understanding the forces that govern critical nano−bio interactions. In this work, we systematically probe the size, shape, and surface capping effects of nanogold by designing a set of eight unique AuNPs. This allowed us to undertake a systematic study involving each of these parameters in the context of their influence on the cytotoxicity and cellular uptake by human prostate cancer cells (PC3) as a model biological system. While studying the influence of these parameters, our study also investigated the influence of serum proteins in forming different levels of biological corona on AuNPs, thereby further influencing the nano−bio interface. As such, increased cellular uptake (by nanoparticle number) was observed with decreasing the AuNP size and increased uptake levels were observed for gold nanospheres (of the same size) stabilized with amino acids compared to citrate or cetyltrimethylammonium bromide (CTAB). Spherical particles were found to be taken up in greater numbers compared to the shapes with broad flat faces. When measuring cytotoxicity, CTAB-stabilized rod- and cube-shaped particles were well tolerated by the cells, whereas toxicity was observed in the case of CTAB-stabilized spherical and prismatic particles. These effects, however, are underpinned by different mechanisms. Further, it is demonstrated that it is possible for different chemical stabilizers to elicit varied cytotoxic effects. Although we find the limited role of serum proteins in mediating toxicity, they do play a critical role in influencing the cellular uptake of AuNPs, with lower levels of uptake generally observed in the presence of serum. Our findings offer a useful step in the direction of predicting the biological interactions of AuNPs based on specific parameters of the AuNP design.

1. INTRODUCTION Widespread use of AuNPs in biomedical applications hinges on the ability of researchers to validate the safety of these particles for therapeutic use.1 Although many studies have shown negligible toxicity to cells on exposure to AuNPs,2−5 there are also studies that present conflicting results.6−9 Although gold particles of micron size or larger are generally thought of as noncatalytic, stable, inert, and biocompatible, the biological activity of AuNPs remains an issue of ongoing complexity and debate.10 Accounting for key variables such as the synthesis method, size, and shape of the nanoparticle as well as the cell type and endpoint examined,11 researchers have begun to form a matrix of results based on these findings.1 Whilst some studies compare results generated within a single research group,12 other reviews collate and compare the effects of particles produced by various research groups, using vastly different synthesis methods.1,10,13−18 The validity of these comparisons is clouded by the possibility that modifiable parameters such as size, shape, and characteristics of surface capping agents are vital in determining the final mode of © 2019 American Chemical Society

interaction of each AuNP with its biological surrounding and eventuated endpoint effect. As such, the importance of a systematic approach, sequentially modifying a single variable of the nanoparticle’s profile, is paramount in order to gain a clear understanding of the effect of each parameter on biological activity. One such approach undertaken in the current study is shown in Figure 1 with a note that although some of the other studies have included much larger library of particles for comparison,19 the work presented here is the first study that systematically varies one parameter (among size, shape, or surface ligand) to independently assess the influence of these parameters. Although the majority of research has been conducted on the cellular uptake of spherical AuNPs,4,20−22 a smaller body of work details the uptake of rod-shaped AuNPs with varying aspect ratio, surface charge, and surface functionalizaReceived: November 19, 2018 Accepted: December 21, 2018 Published: January 4, 2019 242

DOI: 10.1021/acsomega.8b03227 ACS Omega 2019, 4, 242−256

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ogy.33 Another study from our group on Ag nanoparticles of similar shape and size yet of different capping agents has revealed that the surface ligands also greatly influence the nanoparticle−serum interactions.34 Such independent observations, for instance, the influence of nanoparticle characteristics on their interactions with serum, and the effect of the presence of serum on nanoparticle cellular uptake and toxicity, emphasize on the importance of controllably varying single nanoparticle characteristic one-at-a-time to obtain a more detailed understanding of the nano−bio interactions. As such, an aspect that is clearly evident from previous studies is that comparison between outcomes obtained from different research groups is not a simple task with diverse cell lines used, various cell viability assays employed, and different chemical routes utilized to synthesize AuNPs of different shapes and sizes, impacting outcomes and leading to conflicting results. In the current study, we address this problem with a systematic study of AuNP variables (size, shape, and surface coating), while keeping the other variables consistent. A careful design then allowed us to investigate the combined influence of these physicochemical characteristics of nanoparticles along with the available serum proteins in producing a biological response. The approach illustrated in Figure 1 provides new insights into the effects of these variables on AuNP-cellular behavior.

Figure 1. Schematic representation of synthesized AuNPs designed to allow systematic comparison of size, shape, and surface-capping agents [denoted by color where cetyltrimethylammonium bromide (CTAB) is represented with green, citrate with orange, tyrosine with pink, and tryptophan with blue] on their biological action in context of interaction of these nanoparticles with different serum protein conditions mimicking different biological scenarios. Detailed physicochemical characterization of particles used in this study is provided in the Supporting Information through Figures S1−S4 and Table S1.

2. RATIONALE FOR DETERMINING THE EFFECT OF SERUM PROTEINS In typical cell culture conditions, cells are maintained in an environment of 10% serum [most commonly fetal bovine serum (FBS)] to promote growth and enhance the survival rate of cells.35 The serum is rich in proteins and growth factors which make it effective in mimicking the protein-rich environment of the blood. When nanoparticles are exposed to an in vivo biological environment, the serum proteins tend to form a protein corona on the nanoparticle surface, which is believed to give nanoparticles a biological identity.34,36 Therefore, it is important to understand the role of serum proteins in influencing the biological action of nanoparticles. In the current study, the importance of a protein corona in mediating cellular uptake and toxicity has been determined by performing toxicity and cellular uptake studies in three environments with different serum levels. As such, three potential scenarios were considered for cytotoxicity and nanoparticle uptake measurements (Table 1). Although the serum-supplemented medium is consistent with normal cell culture conditions mimicking a healthy in vivo environment, the serum-free condition and the serum pre-incubation followed by serum-deprivation condition mimic the scenarios

tion.6,23−25 The uptake of alternate shapes such as cubic and prismatic AuNPs has been studied to a lesser extent and remains largely unexplored. Gaining attention more recently is the importance of the cell line selected for cellular uptake studies.26,27 The specialized and diverse nature of cells means that not all cell types possess the means necessary to perform uptake through all possible endocytotic pathways.28 Such cells might be considered unsuitable for uptake studies as they would provide exceptionally poor points of comparison. Unsurprisingly, researchers have found that the rate and extent of uptake can vary dramatically between cell lines.29 Such evidence suggests that comparison of uptake levels between different cell lines must be made with caution. In the current study, we have chosen PC3 (prostate cancer) cells as a model system, as the interactions between gold nanoparticles and PC3 cells have been explored by a number of groups, particularly from the perspective of treating prostate tumors via thermal ablation due to the unique surface plasmon resonance (SPR) properties of gold nanoparticles.30 Through the course of such studies, it has been demonstrated that originally CTAB-coated gold nanorods are only minimally internalized by PC3 cells if their CTAB coating is replaced with polyethylene glycol, which may make them unsuitable for the intended applications.31,32 Studies of PC3 cells have also shown decreased internalization of particles in the presence of serum.6 When our group recently compared the interaction of gold nanoparticles of different morphologies (spheres, cubes, prisms, and rods) yet coated with the same surfactant (CTAB) with the human serum, we noted that the strength of serum−nanoparticle interactions is remarkably influenced by the particle morphol-

Table 1. Conditions of Varying Protein Levels Utilized for Cell Culture Experiments tested conditions mimicking different in vivo environments serum-free serum pre-incubated particles serum-supplemented media

243

media

gold nanoparticles

RPMI 1640 media without supplements RPMI 1640 media without supplements

pristine gold nanoparticles

RPMI 1640 media supplemented with 10% FBS

pristine gold nanoparticles incubated for 3 h in 5% FBS pristine gold nanoparticles


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Figure 2. TEM images representative of (a) AuNS(CTAB), (b) AuNC(CTAB), (c) AuNR(CTAB), (d) AuNPr(CTAB), (e) CTAB/citrate-AuNS, (f) citrate-AuNS, (g) tryptophan-AuNS, and (h) tyrosine-AuNS. Scale bars correspond to 50 nm.

Figure 3. Cytotoxicity profiles for chemically distinct AuNS with different surface coatings: (a) citrate-AuNS, (b) tyrosine-AuNS, (c) tryptophanAuNS, and (d) CTAB/citrate-AuNS, as determined using MTT assay. Data are presented as mean ± standard deviation (SD). Asterisks indicate significant differences (P < 0.05) between different conditions at the same dose point.

3. RATIONALE FOR DETERMINING THE EFFECT OF NANOPARTICLE SHAPE To date, there has been no study that simultaneously undertakes the synthesis of spherical, rod-shaped, prismatic, and cubic AuNPs, importantly, using chemically similar routes for the purpose of subsequently comparing their uptake and toxicity profile in mammalian cells. To fully understand the direct influence of nanoparticle morphology, it is critical that all of the tested nanoparticles are synthesized using chemically similar routes to avoid the complexity of surface capping and corona effects. Such a study is carried out here, whereby spherical (AuNS), cubic (AuNC), rod-shaped (AuNR), and prismatic (AuNPr) particles (Figure 2a−d) were synthesized using similar chemical routes and a common chemical

of potentially subjecting the cells to a state of nutrient deprivation. The latter two scenarios may arise under diseased and unhealthy in vivo conditions. A time course study of PC3 cells was carried out to determine the maximal time point of unaffected viability in serum-free media and was determined to be 16 h using optical microscopy and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assays (Supporting Information, Figures S5 and S6). Results taken at this time point of 16 h ensure that any toxic effects observed are primarily due to the presence of nanoparticles and not the absence of serum. 244


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Table 2. Comparison of Physicochemical Parameters and Biological Effects for Each AuNPa lowest Au concentration at which cellular toxicity is observed [μM Au equivalent/% viability]

relative AuNP uptake [ % of dose taken-up per million cells]

shape

surface coating

hydrodynamic diameter from DLS

zeta potential charge [mV]

SFa

SPIa

SSa

SFa

SPaI

SSa

sphere sphere sphere sphere sphere rod prism cube

citrate tyrosine tryptophan CTAB/citrate CTAB CTAB CTAB CTAB

20 ± 5 21 ± 5 21 ± 9 27 ± 20 80 ± 28 n/a 90 ± 16 115 ± 77

−38.4 −22.6 −27.6 +21.9 +44.9 +38.1 +52.2 +30.4

100/77.3 100/52.2 100/85.2 0.5/70.1 100/0 100/84.8 100/33.4 n/a

100/73.8 100/79.9 100/89.45 0.5/57.1 10/72.0 n/a 100/31.4 n/a

n/a n/a n/a 10/0 10/72.1 n/a 100/24.7 n/a

12.50 36.94 23.93 5.73 33.30 4.37 2.57 16.81

11.24 23.60 24.15 5.58 23.19 2.90 2.17 15.25

2.95 10.79 3.61 2.63 12.92 3.17 1.42 8.66

a SF, SPI, and SS correspond to serum-free, serum-pre-incubated, and serum-supplemented medium conditions outlined in Table 1, respectively. Toxicity is defined as viability AuNC > AuNR inversely correlates with the number of particles internalized. This suggests that the cellular receptor sites may significantly diminish after repositioning to internalize particles with large contact areas, highlighting the effect of shape on cellular uptake of AuNPs. 6.4. Impact of AuNP surface ligand on toxicity and cellular uptake. The small spherical particles of similar size but distinct surface chemistries prepared in our study further allow direct examination of the effect of surface coating on cellular uptake and toxicity (Figure 5a,b). Most obviously, the effect of high levels of CTAB within the CTAB/citrate-AuNS generated the highest level of toxicity across all serum environments. As noted earlier, CTAB/citrate particles are significantly different from all other AuNS with the lowest biocompatibility of 0.1 μM (Figure 3). Comparison of citrate, tryptophan and tyrosine-capped AuNS (all produced using chemicals generally deemed biocompatible) in typical serumsupplemented cell culture conditions (Figure 3) show no significant difference, with all AuNPs well tolerated at 100 μM gold concentration (99.6, 101.9, and 106.2% cellular viability for citrate, tryptophan, and tyrosine, respectively). These results are in agreement with previous observations.2,9,22,68 By contrast, significant differences were seen in serum-free conditions, which have been less widely studied previously. Most remarkably, tyrosine particles, which are biocompatible in serum, cause significant loss of viability (52.2%) in serumfree conditions, whereas citrate and tryptophan exhibited smaller declines in viability. Figure 6 compares the cellular uptake profile of various nanoparticles. Previously, researchers have attempted to correlate the cellular uptake profile of nanoparticles with the charge on the nanoparticle surface.69,70 This is based on the rationale that because the mammalian cell membrane is negatively charged, the electrostatic interactions with positively charged particles should facilitate higher uptake. Although higher cellular uptake is generally reported with cationic particles,69,71 it is now increasingly recognized that anionic particles may also be taken up by the cells in high numbers.72 It has also been proposed that the magnitude of the charge may also be a critical factor, with higher uptake observed for particles with a surface charge of greater magnitude, regardless of whether they were positively or negatively charged.73 These observations indicate that although uptake may, in part, be influenced by surface charge, it is most likely dominated by other factors (e.g., nanoparticle size, shape, and surface ligands). The outcomes of our study also reaffirm this belief as we did not notice a charge-dependent common trend associated with the cellular uptake of nanoparticles, such that high and low uptakes of nanoparticles were observed both in the positively and negatively charged groups (Table 2). In fact,

Figure 7. TEM images of PC-3 cells after exposure to CTABstabilized AuNPs (a) AuNS, (b) AuNR, (c) AuNPr, and (d) AuNC (AuNPs indicated by red arrows and circle). 249


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morphologies studied. The other two shapes AuNR and AuNC showed higher biocompatibility than both AuNPr and AuNS. One distinguishing feature between these two groups of shapes is the richness of AuNPr and AuNS in [111] facets, whereas the AuNR and AuNC are rich in [100] and [110] facets. It is well known that a variety of chemical species, including CTAB, have different strengths of interactions with different crystal planes because of the difference in the surface energies of these facets, and it would, therefore, be reasonable to assume that cell surface ligands and biomolecules are also likely to have such preferred modes of interactions with nanomaterials. Thus, the way that cellular ligands and intracellular biomolecules interact with these crystal facets may play a role in contributing to the toxicity and uptake profile of different shapes of nanomaterials. Another possible explanation for the higher toxicity of morphologies rich in [111] facets may be that CTAB tends to preferentially bind to the [111] facets over other crystal planes.76 The higher cellular uptake of these CTAB-coated AuNS might lead to a build-up of toxic CTAB molecules within the cell, eventually leading to cell death. We make this suggestion based on our observation that although 20 nm AuNS prepared using different reducing agents/surface coatings were mostly biocompatible, those capped with CTAB in that group of materials were highly toxic, despite showing lower levels of uptake. However, this hypothesis, based on the cellular accumulation of CTAB, does not explain the high toxicity of AuNPr. This indicates a complex interplay of different mechanisms that induce cellular toxicity. An important aspect that is clear from our study is that despite conflicting reports, CTAB-coated rod and cube-shaped particles are very well tolerated by PC3 cells, especially if these particles are thoroughly washed to remove free CTAB molecules. This may have important implications in the biological application of gold-based nanomaterials, particularly considering the ability to fine-tune the position of the SPR bands of these materials in the near-infrared region. In regards to the uptake profile of different shapes of AuNPs, the geometry of nanoparticles seems to play a dominant role: our results suggest that the shapes with broad flat faces (AuNPr and AuNR) are less efficiently taken up by cells compared to those containing a higher degree of curvature (AuNS and AuNC). This is likely to be due to the difference in the accessibility of nanoparticles by cell surface receptors, as simulations have shown that the particle internalization energies follow the trend of spheres < cubes < rods < disks.63 Overall, it is noted that spherical particles are taken up preferentially by the cells, as their geometry requires making fewer contacts with cell surface receptors, while also requiring less free energy for particle wrapping and subsequent endocytosis. Regarding the influence of AuNP size on toxicity, while our study comparing only two sizes, did not offer sufficient enough data to draw broad conclusions, the trend for increased uptake of smaller particles (by number, not necessarily by total gold) was supported by this study. This may be explained by assuming that smaller particles (with larger curvature) require less energy to be internalized and need to interact with a lower number of cell surface receptors compared with larger particles. Conversely, larger particles require a greater number of cellular receptors such that over time, there may be insufficient coverage of receptors on the cell surface which may limit the uptake of additional particles.

appear to enter into the nucleus. Instead, AuNPs appeared to be localized within vesicles, with multiple AuNP-containing vesicles present inside a given cell. Closer inspection of the AuNPs showed un-agglomerated particles present within the vesicles (Figure 8). Although ICP−MS cannot discern

Figure 8. (a) AuNR and (b) AuNPr show preservation of AuNP morphology after internalization, with enlarged areas shown as insets.

between particles associated with the cellular membrane and those actually internalized, TEM images showed that AuNPs are predominantly located within the cell, whereas a small proportion co-exists on the cellular surface. These observations agree with previous studies where AuNPs were observed to internalize in the cellular matrix without necessarily internalizing inside the nucleus.5

7. CONCLUSIONS The current work, for the first time, provides a systematic study on the influence of AuNPs size, shape, and surface coating on cellular uptake and toxicity while attempting to keep the other variables consistent. Equally important, we systematically study the important role played by serum proteins in dictating the AuNP uptake and toxicity profile by PC3 cells. As a general observation, lower levels of uptake were observed in the presence of serum, whereas serum-deprived cells underwent higher levels of internalization of AuNPs. On the basis of these observations, it may be inferred that the presence of serum leads to the formation of a biological corona on the nanoparticle surface which reduces AuNP/membrane interactions. Alternatively, in a serum-rich environment, AuNPs may need to compete with free serum proteins for the cellular binding sites, compromising the interaction of AuNPs with the cells and reducing subsequent internalization. Interestingly, the commonly perceived idea that serum reduces the toxic effects of nanomaterials is not fully supported by our study, as similar biocompatibility trends were generally observed in both serum-rich and serum-deprived conditions. When considering the effect of the shape for AuNPs of approx. 70 nm size coated with CTAB, a universal correlation between the cellular uptake and the toxicity was not observed. These findings are counter to the common belief that higher uptake of nanomaterials leads to higher levels of toxicity. We instead noted that not all shapes of gold nanoparticles are equally biocompatible and certain shapes may elicit a higher toxic response than others. For instance, despite very low levels of uptake of AuNPr by PC3 cells (both in terms of the number of particles and the amount of gold per cell), these particles induced significant cellular toxicity. By contrast, AuNS exhibited an equivalently high toxicity (Figure 4) despite the amount of gold taken up was the highest of all of the 250


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bringing 100 mL of 0.25 mM HAuCl4 solution to boil while stirring. To the solution, 0.7 mL of 0.17 M sodium citrate solution was added before being left to stir on the heat until the solution turned red wine color, after which the heat was turned off and the solution was allowed to stir for a further 10 min. The flask was sealed with a plastic paraffin film and allowed to stabilize for a further 24 h before a 10 mL solution of 0.001 M CTAB solution was prepared and stirred, whereas the entire volume of the previously prepared citrate AuNP solution was added dropwise. The final solution was allowed to stabilize for a further 24 h. 8.2.4. Synthesis of CTAB-Stabilized Gold Nanospheres. Preparation of gold nanospheres was achieved by following the method described by Rayavarapu et al.80 Gold seeds were created by combining 9.75 mL of 0.1 M CTAB with 250 μL of 10 mM HAuCl4. To this solution, 600 μL of ice-cold freshly prepared 10 mM NaBH4 was injected while stirring vigorously. The solution was stirred for a further 2 min to allow excess NaBH4 to break down before being used within 5 min in the following reaction. To prepare the growth solution, 38 mL of 0.1 M CTAB solution was brought to a temperature of 25 °C before adding 2.0 mL of 10 mM HAuCl4, 210 μL of 0.1 M Lascorbic acid, and 48 μL of gold seed solution. The final solution was gently stirred before being left at 25 °C for 24 h. Following this period, the solutions were washed to remove excess CTAB and unreacted constituents. The solutions were first centrifuged at 2000 rpm for 6 min to settle excess CTAB, the supernatants were collected, and the process was repeated, whereas the pellet (made up of precipitated excess CTAB) was discarded. Following this, the supernatant was centrifuged at 6000 rpm, 12 min, 25 °C, repeated three times, each wash discarding the supernatants and redispersing the pellet in MilliQ water. 8.2.5. Synthesis of CTAB-Stabilized Rod-Shaped Gold Nanoparticles. A modified version of the method described by Liu and Guyot-Sionnest was employed to synthesize rodshaped gold nanoparticles.81 In a typical synthesis, a gold seed was created using a method devised by Nikoobakht and ElSayed82 whereby 10 mL of 0.1 M CTAB was brought to 30 °C, to which 250 μL of 10 mM HAuCl4 was added. To this solution, 600 μL of freshly prepared 10 mM NaBH4 was injected while stirring vigorously. The solution was stirred for further 5 min to allow excess NaBH4 to break down, before being used as a seed in the following reaction. CTAB solution (40 mL, 0.1 M) was brought to 30 °C to which 2.0 mL of 10 mM HAuCl4, 0.4 mL of 10 mM AgNO3, 800 μL of 0.1 M HCl, and 320 μL of 0.1 M L-ascorbic acid were added, stirring after each addition. Last, 96 μL of gold seed solution was injected into the growth solution and gently stirred before being left for 2 h at 30 °C. The solution was allowed to stabilize at room temperature for 24 h before being washed to remove excess CTAB and unreacted constituents. The solutions were first centrifuged at 2000 rpm for 6 min to settle excess CTAB, and the supernatants were collected, whereas the pellet (made up of precipitated excess CTAB) was discarded. Following this, the collected supernatant was centrifuged at 8000 rpm, 15 min, 12 °C, repeated three times, each wash discarding the supernatants and redispersing the pellet in Milli-Q water. 8.2.6. Synthesis of CTAB-Stabilized Prismatic Gold Nanoparticles. The method devised by Millstone et al. was employed to synthesize prismatic-shaped gold nanoparticles.83 In a typical synthesis, a 5 nm gold seed was prepared by adding 1 mL of 0.01 M sodium citrate dihydrate, 1 mL of 0.01 M

It was found that AuNP surface coating elicited remarkably different cellular uptakes and cytotoxic responses. Overall, increased uptake levels were observed for AuNS stabilized with tryptophan and tyrosine amino acids compared to those stabilized with citrate and CTAB/citrate. It has long been known that certain amino acids, particularly those rich in aromatic functional groups, may influence the binding and uptake of extracellular material into the cell. Our findings support the idea that the presence of aromatic groups within an amino acid may alter cellular uptake by affecting affinity and binding between the AuNP and the cell surface receptors. Further, opposed to the commonly perceived views that cationic nanoparticles typically show higher cellular uptake, our work did not find a direct correlation between the nanoparticle surface charge and their cellular uptake, further suggesting the involvement of a myriad of molecular forces beyond simple electrostatic interactions in determining their cellular uptake profile. The interaction of nanomaterials with the biological world is dictated by a complex interplay of a multitude of factors and molecular forces. Overall, the outcomes of the current study highlight the importance of a systematic approach to assess the biological profile of newly designed nanomaterials. It is hoped that this work will form the basis of ongoing systematic studies leading to the development of guiding principles governing cellular uptake and toxicity of nanomaterials, allowing for robust predictions of biological interaction with future nanomaterials.

8. MATERIALS AND METHODS 8.1. Materials. All chemicals used for AuNP synthesis were purchased from Sigma-Aldrich and used without further modification. Reactions were carried out using Milli-Q water in a glassware washed with aqua regia and rinsed thoroughly with deionized water. The resulting nanoparticle samples were dialyzed or centrifuged to ensure removal of unreacted species prior to characterization. 8.2. Nanoparticle Synthesis. 8.2.1. Synthesis of CitrateStabilized Spherical Gold Nanoparticles. A method adapted from Li et al.77 was employed whereby 2.0 mL of 25 mM HAuCl4 was mixed with 7.7 mL of 20 mM NaOH before bringing the total volume up to 20 mL with Milli-Q water. The tube was heated to 85 °C before 600 μL of 0.0283 M trisodium citrate was injected while vigorously stirring. The solution was allowed to stabilize for further 24 h at room temperature. 8.2.2. Synthesis of Tryptophan- and Tyrosine-Stabilized Spherical Gold Nanoparticles. A modified version of the method described by Daima et al. was used to create both tryptophan- and tyrosine-reduced AuNPs.78,79 In a typical synthesis, 92 mL of Milli-Q water, 1 mL of KOH, and 5 mL of 0.01 M tryptophan or tyrosine were added in a flask which was heated and rapidly stirred for approximately 4 min, before adding 2 mL of 0.01 M HAuCl4. The solution was left to stir on the heat until it turned a red wine color, after which the heat was turned off and the solution was allowed to stir for a further 10 min. The flask was sealed with a plastic paraffin film and allowed to stabilize for a further 24 h. The particles thus obtained are negatively charged because of the zwitterionic nature of amino acids with the corresponding isoelectric points of tyrosine and tryptophan as 5.66 and 5.89, respectively, leading to a net negative charge at the physiological pH. 8.2.3. Synthesis of CTAB/Citrate-Stabilized Spherical Gold Nanoparticles. Citrate-stabilized AuNPs were synthesized by 251


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were used for all cell culture studies described in this chapter. PC-3 cells have been well characterized since their isolation in 1979 and have demonstrated suitability in viability assays for various compounds since this time.6,85,86 Generally speaking, macrophage cells, cancer cells, and/or epithelial cells are employed for uptake studies87 making them suitable for both purposes. 8.5. Cytotoxicity Testing. MTT is a yellow tetrazole which is capable of being reduced by living organisms into a purple formazan product.88,89 The MTT assay is used to assess viability by spectrophotometrically measuring the amount of insoluble formazan crystals which are produced when a viable cell cleaves the MTT tetrazolium ring using mitochondrial dehydrogenase. AuNP toxicity was determined by seeding 100 μL into a 96well cell culture plate at a seeding density of 100 000 cells/mL. The plate was incubated (37 °C, 5% CO2, 85% RH, 24 h) before the media were removed and replaced with 90 μL fresh warmed media in fulfillment of the conditions outlined in Table 1. Following the replenishment of media, 10 μL of nanoparticle solution or vehicle control (Milli-Q water) was added to the wells in triplicate. The plate was reincubated for 16 hours before the addition of 10 μL of 5 mg/mL MTT solution, diluted in Dulbecco’s phosphate-buffered saline (DPBS) solution. The plate was then wrapped in an aluminum foil to protect the light-sensitive dye and reincubated for a further 4 h. Following this period, the contents of the plate were carefully removed without disturbing the formazan crystals which form within the cells. To each well, 100 μL of dimethyl sulfoxide was added and the contents resuspended to ensure dissolution of the formazan crystals before being read using a PerkinElmer Multimode Spectrophotometer at 595 nm. The percentage of viable cells was determined as the relative absorbance of AuNP-treated cells compared with the absorbance of untreated control cells averaged over the triplicate wells. Each assay was performed three times independently. 8.6. Determination of Gold Nanoparticle Uptake by ICP−MS. The extent of nanoparticle uptake was determined using ICP−MS following treatment of PC-3 cells with AuNPs in conditions of varying protein. To perform uptake experiments, PC-3 cells were seeded into multiple 75 cm2 flasks at a seeding density of 100 000 cells/mL. The flasks were incubated (37 °C, 5% CO2, 85% RH, 24 h) before the media were removed and replaced with fresh media to fulfill the conditions described in Table 1. Following the replenishment of media, AuNPs were added to the flask. The highest common tolerated dose among each group of AuNPs was used (i.e., a final concentration of 1 μM for chemically different AuNS and 10 μM for different shaped AuNPs). The flask was reincubated (37 °C, 5% CO2, 85% RH, 6 h), which allowed for sufficient time for AuNP uptake to occur.12,90 Following this period, the cells were washed three times with DPBS to ensure removal of free nanoparticles before treatment with TrypLE to dislodge the cells. Cells were resuspended in cell culture media and centrifuged at 3500 rpm for 10 min. Following centrifugation, the supernatant was discarded and the cell containing pellet redispersed in the cell culture medium before a cell count was performed. The sample was repeatedly centrifuged, resuspending in solutions of increasing ethanol concentration before being oven dried, dissolved in aqua-regia, and filtered prior to assessment. Gold content was determined using ICP−MS, with bismuth of 10 ng/mL as an internal standard in addition

HAuCl4, and 1 mL of freshly prepared 0.1 M NaBH4 to 36 mL of Milli-Q water while stirring vigorously. The gold seeds were allowed to mix for a further 1 min then left undisturbed for 2 hours before use. An iodide-doped CTAB mixture was prepared by adding 2.733 g of CTAB and 75 μL of 0.1 M KI to 150 mL of Milli-Q water. The solution was sealed, heated, and sonicated to dissolve CTAB before being set for 2 h. Following this period, three growth solutions were prepared, such that growth solutions 1 and 2 were each made up of 9 mL of prepared iodide-doped CTAB solution, 250 μL of 0.01 M HAuCl4, 50 μL of 0.1 M NaOH, and 50 μL of 0.1 M L-ascorbic acid, whereas solution 3 contained 90 mL of prepared iodidedoped CTAB solution, 2.5 mL of 0.01 M HAuCl4, 500 μL of 0.1 M NaOH, and 500 μL of 0.1 M L-ascorbic acid. To commence the reaction, 1 mL of seed solution was added to growth solution 1 which was gently stirred before transferring 1 mL into growth solution 2, which was stirred before adding its total contents into growth solution 3. The reaction was allowed to proceed undisturbed for 30 min. The solution was left to stabilize for 24 h before being washed to remove excess CTAB and unreacted constituents. The solutions were first centrifuged at 2000 rpm for 6 min to settle excess CTAB, and the supernatant was removed and kept, whereas the pellet (made up of precipitated excess CTAB) was discarded. Following this, the supernatant was centrifuged at 8000 rpm for 3 min at room temperature. This process was repeated three times, discarding the supernatants and redispersing the pellet in Milli-Q water after each wash. 8.2.7. Synthesis of CTAB-Stabilized Cubic Gold Nanoparticles. A modified version of the method detailed by Kim et al. was employed whereby 40 mL of 10 mM CTAB was combined with 500 μL of 10 mM HauCl4. The solution was mixed before 200 μL of 100 mM L-ascorbic acid was added, followed by repeated inversion to mix.84 The solution was allowed to stand for 30 min after which 60 μL of 100 mM NaOH was slowly added to the bottom of each reaction vessel and allowed to stand undisturbed at 25 °C for 6 h before the tubes were inverted. The solution was allowed to stabilize for 24 h before being washed to remove excess CTAB and unreacted constituents. The solutions were first centrifuged at 2000 rpm, 6 min, room temperature to settle excess CTAB, and the supernatants were kept, whereas the pellet (made up of precipitated excess CTAB) was discarded. Following this, the solutions were centrifuged at 9000 rpm, 3 min, repeated three times, discarding the supernatants and redispersing the pellet in Milli-Q water after each wash. 8.3. Nanoparticle Preparation for Biological Testing. To prepare AuNPs for biological testing, all nanoparticles were thoroughly washed (via dialysis or centrifugation) and concentrated as previously outlined. These purified AuNPs are referred to as pristine AuNPs hereafter. Microwave plasmaatomic emission spectroscopy was employed to determine the concentration of each AuNP sample prior to testing. AuNP dilutions were carried out approximately 3 h prior to toxicity experiments using deionized Milli-Q water as the diluent. For nanoparticles requiring pre-incubation in 5% FBS v/vthe dilutions were modified such that the first dilution (1000 μM) contained 5% FBS v/v and all subsequent dilutions were performed with Milli-Q water supplemented with 5% FBS. The AuNP solutions were left to incubate at 37 °C until required. 8.4. Cell Line. PC-3 cells (human prostate cancer cells) derived from bone metastasis of a grade IV prostatic adenocarcinoma isolated from a 62-year-old male Caucasian 252


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one-way ANOVA assuming significance at P = 0.05 (indicated by an asterisk).

to a range of standard gold solutions. Using the recorded cell count and correcting for dilution, the amount of gold per sample was determined in each case. Further calculations were performed to relate this value to the number of gold nanoparticles taken up. Each sample was collected independently, in duplicate. 8.7. Cellular Uptake Calculations. To compare the degree of AuNP uptake, data are first presented as the amount of gold (pg) taken up per cellby dividing the total amount of gold detected (determined using ICP−MS) by the total number of cells collected (determined by a cell count performed after washing the cells thoroughly post-treatment). Second, the number of AuNPs taken up per cell (Pcell) is presented by calculating the average weight of a single nanoparticle from each sample based on TEM measurements using the following equations. For spherical AuNPs, the total number of atoms per particle (Nparticle) is given by Nparticle = V

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b03227. Cellular toxicity time-point optimization studies; cytotoxicity studies of capping ligands; particle size calculations; and extensive characterization of AuNPs including UV−visible absorbance spectroscopy, dynamic light scattering, TEM particle size distribution, X-ray diffraction, and zeta potential measurements (PDF)

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*E-mail: [email protected]. Phone: +61 3 9925 2121 (V.B.).

ρ ·NA M

ORCID

Catherine Carnovale: 0000-0003-1250-3302 Vipul Bansal: 0000-0002-3354-4317 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

π ρ ·NA 3 = D = 30.89·D3 6 M

where D is the average diameter of the spherical particle (determined via TEM measurements). For cubic, rod-shaped, and prismatic particles, the total number of atoms per particle (Nparticle) was determined similarly after calculating the volume (V) of each particle using the relevant volume equation for each shape. To determine the weight of each particle, the weight of a singular gold atom was calculated; matom =

AUTHOR INFORMATION

Corresponding Author

where V is the volume of a sphere, defined by the density (Au ρ = 1.93 × 107 g/m3), molar mass (Au M = 196.97 g/mol), and Avogadro’s constant (NA) which can be simplified to Nparticle

ASSOCIATED CONTENT

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS V.B. acknowledges the Australian Research Council (ARC) for a Future Fellowship (FT140101285V.B.) and funding support through a Linkage grant (LP130100437V.B. and R.R.). V.B. thanks the valuable support of Ian Potter Foundation in establishing Sir Ian Potter NanoBioSensing Facility at RMIT University. The authors acknowledge the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility at the RMIT Microscopy & Microanalysis Facility, at RMIT University.

M 196.97 = = 3.27 × 10−22 g NA 6.022 × 1023

Thus, particle weight mAuNP was determined using the following equation (results provided in Table S1, Supporting Information);

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mAuNP = matom ·Nparticle

ABBREVIATIONS AuNC, gold nanocubes; AuNP, gold nanoparticle; AuNPr, gold nanoprisms; AuNR, gold nanorods; AuNS, gold nanospheres; CTAB, cetyltrimethylammonium bromide; FBS, fetal bovine serum; ICP−MS, inductively coupled plasma-mass spectrometry; MP-AES, microwave plasma-atomic emission spectroscopy; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TEM, transmission electron microscopy

8.8. TEM of Cells. Cells were exposed to AuNPs for 6 h, before thorough washing with DPBS to remove free AuNPs. Cells were then fixed in a solution of 1% osmium tetroxide + 1.5% potassium ferrocyanide for 60 min before being dehydrated in solutions of increasing ethanol concentration before finishing in absolute acetone. After dehydration, the cells were transferred to a resin−acetone mixture, moved to 100% resin, and placed under vacuum before being embedded at 70 °C overnight for polymerization. To image cells, a microtome was used to cut thin sections which were placed on TEM grids. In order to have a reasonable number of AuNPs to allow visualization of the uptake process, cells were subjected to 0.1 μM equivalent gold concentration. 8.9. Statistical Processing of Data. The cellular viability data were gathered from three independent experiments conducted in triplicate wells. Uptake data were determined from two independent samples. Data from both experiments are presented as mean ± SD. Statistical analysis was performed in Excel, with differences determined using two-tailed t-tests or

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REFERENCES

(1) Carnovale, C.; Bryant, G.; Shukla, R.; Bansal, V. Size, shape and surface chemistry of nano-gold dictate its cellular interactions, uptake and toxicity. Prog. Mater. Sci. 2016, 83, 152−190. (2) Connor, E. E.; Mwamuka, J.; Gole, A.; Murphy, C. J.; Wyatt, M. D. Gold Nanoparticles Are Taken Up by Human Cells but Do Not Cause Acute Cytotoxicity. Small 2005, 1, 325−327. (3) Qu, Y.; Lü, X. Aqueous synthesis of gold nanoparticles and their cytotoxicity in human dermal fibroblasts−fetal. Biomed. Mater. 2009, 4, 025007. (4) Brandenberger, C.; Rothen-Rutishauser, B.; Mühlfeld, C.; Schmid, O.; Ferron, G. A.; Maier, K. L.; Gehr, P.; Lenz, A.-G. Effects

253


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and uptake of gold nanoparticles deposited at the air-liquid interface of a human epithelial airway model. Toxicol. Appl. Pharmacol. 2010, 242, 56−65. (5) Shukla, R.; Bansal, V.; Chaudhary, M.; Basu, A.; Bhonde, R. R.; Sastry, M. Biocompatibility of Gold Nanoparticles and Their Endocytotic Fate Inside the Cellular Compartment: A Microscopic Overview. Langmuir 2005, 21, 10644−10654. (6) Arnida; Malugin, A.; Ghandehari, H. Cellular uptake and toxicity of gold nanoparticles in prostate cancer cells: a comparative study of rods and spheres. J. Appl. Toxicol. 2010, 30, 212−7. (7) Goodman, C. M.; McCusker, C. D.; Yilmaz, T.; Rotello, V. M. Toxicity of Gold Nanoparticles Functionalized with Cationic and Anionic Side Chains. Bioconjugate Chem. 2004, 15, 897−900. (8) Pan, Y.; Leifert, A.; Ruau, D.; Neuss, S.; Bornemann, J.; Schmid, G.; Brandau, W.; Simon, U.; Jahnen-Dechent, W. Gold nanoparticles of diameter 1.4 nm trigger necrosis by oxidative stress and mitochondrial damage. Small 2009, 5, 2067−2076. (9) Daima, H. K.; Selvakannan, P. R.; Shukla, R.; Bhargava, S. K.; Bansal, V. Fine-Tuning the Antimicrobial Profile of Biocompatible Gold Nanoparticles by Sequential Surface Functionalization Using Polyoxometalates and Lysine. PLoS One 2013, 8, No. e79676. (10) Khlebtsov, N.; Dykman, L. Biodistribution and toxicity of engineered gold nanoparticles: a review of in vitro and in vivo studies. Chem. Soc. Rev. 2011, 40, 1647−1671. (11) Beddoes, C. M.; Case, C. P.; Briscoe, W. H. Understanding nanoparticle cellular entry: A physicochemical perspective. Adv. Colloid Interface Sci. 2015, 218, 48−68. (12) Chithrani, B. D.; Ghazani, A. A.; Chan, W. C. W. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett. 2006, 6, 662−668. (13) Ding, L.; Yao, C.; Yin, X.; Li, C.; Huang, Y.; Wu, M.; Wang, B.; Guo, X.; Wang, Y.; Wu, M. Size, Shape, and Protein Corona Determine Cellular Uptake and Removal Mechanisms of Gold Nanoparticles. Small 2018, 14, 1801451. (14) Sharifi, S.; Behzadi, S.; Laurent, S.; Laird Forrest, M.; Stroeve, P.; Mahmoudi, M. Toxicity of nanomaterials. Chem. Soc. Rev. 2012, 41, 2323−2343. (15) Alkilany, A. M.; Murphy, C. J. Toxicity and cellular uptake of gold nanoparticles: what we have learned so far? J. Nanopart. Res. 2010, 12, 2313−2333. (16) Mahmoudi, M.; Lynch, I.; Ejtehadi, M. R.; Monopoli, M. P.; Bombelli, F. B.; Laurent, S. Protein−Nanoparticle Interactions: Opportunities and Challenges. Chem. Rev. 2011, 111, 5610−5637. (17) Carnovale, C.; Bryant, G.; Shukla, R.; Bansal, V. Gold Nanoparticle Biodistribution and Toxicity: Role of Biological Corona in Relation with Nanoparticle Characteristics. Metal Nanoparticles in Pharma; Springer, 2017; pp 419−436. (18) Sangabathuni, S.; Vasudeva Murthy, R.; Chaudhary, P. M.; Surve, M.; Banerjee, A.; Kikkeri, R. Glyco-gold nanoparticle shapes enhance carbohydrate-protein interactions in mammalian cells. Nanoscale 2016, 8, 12729−12735. (19) Walkey, C. D.; Olsen, J. B.; Song, F.; Liu, R.; Guo, H.; Olsen, D. W. H.; Cohen, Y.; Emili, A.; Chan, W. C. W. Protein Corona Fingerprinting Predicts the Cellular Interaction of Gold and Silver Nanoparticles. ACS Nano 2014, 8, 2439−2455. (20) Cai, H.; Yao, P. Gold nanoparticles with different amino acid surfaces: serum albumin adsorption, intracellular uptake and cytotoxicity. Colloids Surf., B 2014, 123, 900−906. (21) Vetten, M. A.; Tlotleng, N.; Tanner Rascher, D.; Skepu, A.; Keter, F. K.; Boodhia, K.; Koekemoer, L.-A.; Andraos, C.; Tshikhudo, R.; Gulumian, M. Label-free in vitro toxicity and uptake assessment of citrate stabilised gold nanoparticles in three cell lines. Part. Fibre Toxicol. 2013, 10, 50. (22) Khan, J. A.; Pillai, B.; Das, T. K.; Singh, Y.; Maiti, S. Molecular Effects of Uptake of Gold Nanoparticles in HeLa Cells. ChemBioChem 2007, 8, 1237−1240. (23) Alkilany, A. M.; Nagaria, P. K.; Hexel, C. R.; Shaw, T. J.; Murphy, C. J.; Wyatt, M. D. Cellular Uptake and Cytotoxicity of Gold

Nanorods: Molecular Origin of Cytotoxicity and Surface Effects. Small 2009, 5, 701−708. (24) Alkilany, A. M.; Nagaria, P. K.; Wyatt, M. D.; Murphy, C. J. Cation Exchange on the Surface of Gold Nanorods with a Polymerizable Surfactant: Polymerization, Stability, and Toxicity Evaluation. Langmuir 2010, 26, 9328−9333. (25) Chithrani, B. D.; Chan, W. C. W. Elucidating the mechanism of cellular uptake and removal of protein-coated gold nanoparticles of different sizes and shapes. Nano Lett. 2007, 7, 1542−1550. (26) Schrand, A. M.; Dai, L.; Schlager, J. J.; Hussain, S. M.; Osawa, E. Differential biocompatibility of carbon nanotubes and nanodiamonds. Diamond Relat. Mater. 2007, 16, 2118−2123. (27) Sohaebuddin, S. K.; Thevenot, P. T.; Baker, D.; Eaton, J. W.; Tang, L. Nanomaterial cytotoxicity is composition, size, and cell type dependent. Part. Fibre Toxicol. 2010, 7, 22. (28) Kuhn, D. A.; Vanhecke, D.; Michen, B.; Blank, F.; Gehr, P.; Petri-Fink, A.; Rothen-Rutishauser, B. Different endocytotic uptake mechanisms for nanoparticles in epithelial cells and macrophages. Beilstein J. Nanotechnol. 2014, 5, 1625−1636. (29) Cartiera, M. S.; Johnson, K. M.; Rajendran, V.; Caplan, M. J.; Saltzman, W. M. The uptake and intracellular fate of PLGA nanoparticles in epithelial cells. Biomaterials 2009, 30, 2790−2798. (30) Stern, J. M.; Stanfield, J.; Kabbani, W.; Hsieh, J.-T.; Cadeddu, J. A. Selective Prostate Cancer Thermal Ablation With Laser Activated Gold Nanoshells. J. Urol. 2008, 179, 748−753. (31) Leung, J.; Wu, S.; Chou, K.; Signorell, R. Investigation of Sub100 nm Gold Nanoparticles for Laser-Induced Thermotherapy of Cancer. Nanomaterials 2013, 3, 86−106. (32) Wolfe, T.; Chatterjee, D.; Lee, J.; Grant, J. D.; Bhattarai, S.; Tailor, R.; Goodrich, G.; Nicolucci, P.; Krishnan, S. Targeted gold nanoparticles enhance sensitization of prostate tumors to megavoltage radiation therapy in vivo. Nanomedicine 2015, 11, 1277−1283. (33) Carnovale, C.; Bryant, G.; Shukla, R.; Bansal, V. Impact of nanogold morphology on interactions with human serum. Phys. Chem. Chem. Phys. 2018, 20, 29558−29565. (34) Abraham, A. N.; Sharma, T. K.; Bansal, V.; Shukla, R. Phytochemicals as Dynamic Surface Ligands To Control Nanoparticle-Protein Interactions. ACS Omega 2018, 3, 2220−2229. (35) Murakami, H.; Masui, H.; Sato, G. H.; Sueoka, N.; Chow, T. P.; Kano-Sueoka, T. Growth of hybridoma cells in serum-free medium: ethanolamine is an essential component. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 1158−1162. (36) Carnovale, C.; Bryant, G.; Shukla, R.; Bansal, V. Impact of nanogold morphology on interactions with human serum. Phys. Chem. Chem. Phys. 2018, 20, 29558. (37) De Paoli Lacerda, S. H.; Park, J. J.; Meuse, C.; Pristinski, D.; Becker, M. L.; Karim, A.; Douglas, J. F. Interaction of gold nanoparticles with common human blood proteins. ACS Nano 2009, 4, 365−379. (38) Brewer, S. H.; Glomm, W. R.; Johnson, M. C.; Knag, M. K.; Franzen, S. Probing BSA Binding to Citrate-Coated Gold Nanoparticles and Surfaces. Langmuir 2005, 21, 9303−9307. (39) Si, S.; Mandal, T. K. Tryptophan-Based Peptides to Synthesize Gold and Silver Nanoparticles: A Mechanistic and Kinetic Study. Chem.Eur. J. 2007, 13, 3160−3168. (40) Dubey, K.; Anand, B. G.; Badhwar, R.; Bagler, G.; Navya, P. N.; Daima, H. K.; Kar, K. Tyrosine- and tryptophan-coated gold nanoparticles inhibit amyloid aggregation of insulin. Amino Acids 2015, 47, 2551−2560. (41) García, I.; Sánchez-Iglesias, A.; Henriksen-Lacey, M.; Grzelczak, M.; Penadés, S.; Liz-Marzán, L. M. Glycans as Biofunctional Ligands for Gold Nanorods: Stability and Targeting in Protein-Rich Media. J. Am. Chem. Soc. 2015, 137, 3686−3692. (42) Tarantola, M.; Pietuch, A.; Schneider, D.; Rother, J.; Sunnick, E.; Rosman, C.; Pierrat, S.; Sönnichsen, C.; Wegener, J.; Janshoff, A. Toxicity of gold-nanoparticles: Synergistic effects of shape and surface functionalization on micromotility of epithelial cells. Nanotoxicology 2011, 5, 254−268. 254


ACS Omega

Article

(43) Lundqvist, M.; Stigler, J.; Elia, G.; Lynch, I.; Cedervall, T.; Dawson, K. A. Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 14265−14270. (44) Boulos, S. P.; Davis, T. A.; Yang, J. A.; Lohse, S. E.; Alkilany, A. M.; Holland, L. A.; Murphy, C. J. Nanoparticle-Protein Interactions: A Thermodynamic and Kinetic Study of the Adsorption of Bovine Serum Albumin to Gold Nanoparticle Surfaces. Langmuir 2013, 29, 14984−14996. (45) Giljohann, D. A.; Seferos, D. S.; Patel, P. C.; Millstone, J. E.; Rosi, N. L.; Mirkin, C. A. Oligonucleotide loading determines cellular uptake of DNA-modified gold nanoparticles. Nano Lett. 2007, 7, 3818−3821. (46) Dutta, D.; Sundaram, S. K.; Teeguarden, J. G.; Riley, B. J.; Fifield, L. S.; Jacobs, J. M.; Addleman, S. R.; Kaysen, G. A.; Moudgil, B. M.; Weber, T. J. Adsorbed proteins influence the biological activity and molecular targeting of nanomaterials. Toxicol. Sci. 2007, 100, 303−315. (47) Bajaj, A.; Samanta, B.; Yan, H.; Jerry, D. J.; Rotello, V. M. Stability, toxicity and differential cellular uptake of protein passivatedFe3O4 nanoparticles. J. Mater. Chem. 2009, 19, 6328−6331. (48) Arnida; Janát-Amsbury, M. M.; Ray, A.; Peterson, C. M.; Ghandehari, H. Geometry and surface characteristics of gold nanoparticles influence their biodistribution and uptake by macrophages. Eur. J. Pharm. Biopharm. 2011, 77, 417−423. (49) Patel, P. C.; Giljohann, D. A.; Daniel, W. L.; Zheng, D.; Prigodich, A. E.; Mirkin, C. A. Scavenger Receptors Mediate Cellular Uptake of Polyvalent Oligonucleotide-Functionalized Gold Nanoparticles. Bioconjugate Chem. 2010, 21, 2250−2256. (50) Verma, A.; Stellacci, F. Effect of Surface Properties on Nanoparticleâ€″Cell Interactions. Small 2010, 6, 12−21. (51) Conner, S. D.; Schmid, S. L. Regulated portals of entry into the cell. Nature 2003, 422, 37−44. (52) Rappoport, J.; Smith; Giroud; Wiggins; Gower, F.; Thorley, J. A.; Stolpe, B.; Mazzolini; Dyson. Cellular entry of nanoparticles via serum sensitive clathrin-mediated endocytosis, and plasma membrane permeabilization. Int. J. Nanomed. 2012, 7, 2045−2055. (53) Cedervall, T.; Lynch, I.; Foy, M.; BerggÅrd, T.; Donnelly, S. C.; Cagney, G.; Linse, S.; Dawson, K. A. Detailed identification of plasma proteins adsorbed on copolymer nanoparticles. Angew. Chem., Int. Ed. 2007, 46, 5754−5756. (54) Muthusamy, B.; Hanumanthu, G.; Suresh, S.; Rekha, B.; Srinivas, D.; Karthick, L.; Vrushabendra, B. M.; Sharma, S.; Mishra, G.; Chatterjee, P.; Mangala, K. S.; Shivashankar, H. N.; Chandrika, K. N.; Deshpande, N.; Suresh, M.; Kannabiran, N.; Niranjan, V.; Nalli, A.; Prasad, T. S. K.; Arun, K. S.; Reddy, R.; Chandran, S.; Jadhav, T.; Julie, D.; Mahesh, M.; John, S. L.; Palvankar, K.; Sudhir, D.; Bala, P.; Rashmi, N. S.; Vishnupriya, G.; Dhar, K.; Reshma, S.; Chaerkady, R.; Gandhi, T. K. B.; Harsha, H. C.; Mohan, S. S.; Deshpande, K. S.; Sarker, M.; Pandey, A. Plasma Proteome Database as a resource for proteomics research. Proteomics 2005, 5, 3531−3536. (55) Lynch, I.; Salvati, A.; Dawson, K. A. What does the cell see? Nat. Nanotechnol. 2009, 4, 546−547. (56) Wang, S.; Lu, W.; Tovmachenko, O.; Rai, U. S.; Yu, H.; Ray, P. C. Challenge in understanding size and shape dependent toxicity of gold nanomaterials in human skin keratinocytes. Chem. Phys. Lett. 2008, 463, 145−149. (57) Rayavarapu, R. G.; Petersen, W.; Hartsuiker, L.; Chin, P.; Janssen, H.; van Leeuwen, F. W. B.; Otto, C.; Manohar, S.; Manohar, S.; van Leeuwen, T. G. In vitrotoxicity studies of polymer-coated gold nanorods. Nanotechnology 2010, 21, 145101. (58) Hauck, T. S.; Ghazani, A. A.; Chan, W. C. W. Assessing the effect of surface chemistry on gold nanorod uptake, toxicity, and gene expression in mammalian cells. Small 2008, 4, 153−159. (59) Singh, A.; Shukla, R.; Hassan, S.; Bhonde, R. R.; Sastry, M. Cytotoxicity and Cellular Internalization Studies of Biogenic Gold Nanotriangles in Animal Cell Lines. Int. J. Green Nanotechnol. 2011, 3, 251−263.

(60) Dharmatti, R.; Phadke, C.; Mewada, A.; Thakur, M.; Pandey, S.; Sharon, M. Biogenic gold nano-triangles: Cargos for anticancer drug delivery. Mater. Sci. Eng., Proc. Conf. 2014, 44, 92−98. (61) Van Dong, P.; Ha, C. H.; Binh, L.-T.; Kasbohm, J. Chemical synthesis and antibacterial activity of novel-shaped silver nanoparticles. Int. Nano Lett. 2012, 2, 1−9. (62) Saha, S.; Malik, M. M.; Qureshi, M. S. Comparative Study of Synergistic Effects of Antibiotics with Triangular Shaped Silver Nanoparticles, Synthesized Using UV-Light Irradiation, on Staphylococcus aureus and Pseudomonas aeruginosa. J. Biomater. Nanobiotechnol. 2014, 05, 186−193. (63) Li, Y.; Kröger, M.; Liu, W. K. Shape effect in cellular uptake of pegylated nanoparticles: comparison between sphere, rod, cube and disk. Nanoscale 2015, 7, 16631−16646. (64) Bansal, V.; Li, V.; O’Mullane, A. P.; Bhargava, S. K. Shape dependent electrocatalytic behaviour of silver nanoparticles. CrystEngComm 2010, 12, 4280−4286. (65) Lu, F.; Wu, S.-H.; Hung, Y.; Mou, C.-Y. Size Effect on Cell Uptake in Well-Suspended, Uniform Mesoporous Silica Nanoparticles. Small 2009, 5, 1408−1413. (66) Gao, H.; Shi, W.; Freund, L. B. From The Cover: Mechanics of receptor-mediated endocytosis. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9469−9474. (67) Albanese, A.; Tang, P. S.; Chan, W. C. W. The effect of nanoparticle size, shape, and surface chemistry on biological systems. Annu. Rev. Biomed. Eng. 2012, 14, 1−16. (68) Villiers, C. L.; Freitas, H.; Couderc, R.; Villiers, M.-B.; Marche, P. N. Analysis of the toxicity of gold nano particles on the immune system: effect on dendritic cell functions. J. Nanopart. Res. 2009, 12, 55−60. (69) Fröhlich, E. The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. Int. J. Nanomed. 2012, 7, 5577. (70) Stojiljković, A.; Kuehni-Boghenbor, K.; Gaschen, V.; Schüpbach, G.; Mevissen, M.; Kinnear, C.; Möller, A. M.; Stoffel, M. H. High-content analysis of factors affecting gold nanoparticle uptake by neuronal and microglial cells in culture. Nanoscale 2016, 8, 16650−16661. (71) Fromen, C. A.; Rahhal, T. B.; Robbins, G. R.; Kai, M. P.; Shen, T. W.; Luft, J. C.; DeSimone, J. M. Nanoparticle surface charge impacts distribution, uptake and lymph node trafficking by pulmonary antigen-presenting cells. Nanomedicine 2016, 12, 677−687. (72) Schleh, C.; Semmler-Behnke, M.; Lipka, J.; Wenk, A.; Hirn, S.; Schäffler, M.; Schmid, G.; Simon, U.; Kreyling, W. G. Size and surface charge of gold nanoparticles determine absorption across intestinal barriers and accumulation in secondary target organs after oral administration. Nanotoxicology 2011, 6, 36−46. (73) Xiao, K.; Li, Y.; Luo, J.; Lee, J. S.; Xiao, W.; Gonik, A. M.; Agarwal, R. G.; Lam, K. S. The effect of surface charge on in vivo biodistribution of PEG-oligocholic acid based micellar nanoparticles. Biomaterials 2011, 32, 3435−3446. (74) Davis, C. G.; Van Driel, I.; Russell, D.; Brown, M. S.; Goldstein, J. L. The low density lipoprotein receptor. Identification of amino acids in cytoplasmic domain required for rapid endocytosis. J. Biol. Chem. 1987, 262, 4075−4082. (75) Yang, H.; Fung, S.-Y.; Liu, M. Programming the Cellular Uptake of Physiologically Stable Peptide-Gold Nanoparticle Hybrids with Single Amino Acids. Angew. Chem., Int. Ed. 2011, 50, 9643− 9646. (76) Grzelczak, M.; Pérez-Juste, J.; Mulvaney, P.; Liz-Marzán, L. M. Shape control in gold nanoparticle synthesis. Chem. Soc. Rev. 2008, 37, 1783−1791. (77) Li, C.; Li, D.; Wan, G.; Xu, J.; Hou, W. Facile synthesis of concentrated gold nanoparticles with low size-distribution in water: temperature and pH controls. Nanoscale Res. Lett. 2011, 6, 440. (78) Daima, H. K.; Selvakannan, P.; Homan, Z.; Bhargava, S. K.; Bansal, V. , 2011 International Conference on Tyrosine mediated gold, silver and their alloy nanoparticles synthesis: Antibacterial activity toward gram positive and gram negative bacterial strains, Nanoscience, Technology and Societal Implications (NSTSI); IEEE, 2011; pp 1−6. 255


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Article

(79) Selvakannan, P. R.; Ramanathan, R.; Plowman, B. J.; Sabri, Y. M.; Daima, H. K.; O’Mullane, A. P.; Bansal, V.; Bhargava, S. K. Probing the effect of charge transfer enhancement in off resonance mode SERS via conjugation of the probe dye between silver nanoparticles and metal substrates. Phys. Chem. Chem. Phys. 2013, 15, 12920−12929. (80) Rayavarapu, R. G.; Ungureanu, C.; Krystek, P.; van Leeuwen, T. G.; Manohar, S. Iodide Impurities in Hexadecyltrimethylammonium Bromide (CTAB) Products: Lot−Lot Variations and Influence on Gold Nanorod Synthesis. Langmuir 2010, 26, 5050−5055. (81) Liu, M.; Guyot-Sionnest, P. Mechanism of silver(I)-assisted growth of gold nanorods and bipyramids. J. Phys. Chem. B 2005, 109, 22192−22200. (82) Nikoobakht, B.; El-Sayed, M. A. Preparation and growth mechanism of gold nanorods (NRs) using seed-mediated growth method. Chem. Mater. 2003, 15, 1957−1962. (83) Millstone, J. E.; Wei, W.; Jones, M. R.; Yoo, H.; Mirkin, C. A. Iodide Ions Control Seed-Mediated Growth of Anisotropic Gold Nanoparticles. Nano Lett. 2008, 8, 2526−2529. (84) Kim, M.; Park, H. J.; Han, S. W.; Park, J.; Yun, W. S. Shape Transformation of Gold Nanoparticles from Octahedron to Cube Depending on in situ Seed-Growth Time. Bull. Korean Chem. Soc. 2013, 34, 2243−2244. (85) Schaeublin, N. M.; Braydich-Stolle, L. K.; Maurer, E. I.; Park, K.; MacCuspie, R. I.; Afrooz, A. R. M. N.; Vaia, R. A.; Saleh, N. B.; Hussain, S. M. Does Shape Matter? Bioeffects of Gold Nanomaterials in a Human Skin Cell Model. Langmuir 2012, 28, 3248−3258. (86) Hattori, Y.; Maitani, Y. Folate-Linked Lipid-Based Nanoparticle for Targeted Gene Delivery. Curr. Drug Delivery 2005, 2, 243−252. (87) Lai, Y.; Chiang, P.-C.; Blom, J. D.; Li, N.; Shevlin, K.; Brayman, T. G.; Hu, Y.; Selbo, J. G.; Hu, L. G. Comparison of In vitro Nanoparticles Uptake in Various Cell Lines and In vivo Pulmonary Cellular Transport in Intratracheally Dosed Rat Model. Nanoscale Res. Lett. 2008, 3, 321−329. (88) Kroll, A.; Pillukat, M. H.; Hahn, D.; Schnekenburger, J. Current in vitro methods in nanoparticle risk assessment: Limitations and challenges. Eur. J. Pharm. Biopharm. 2009, 72, 370−377. (89) Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55−63. (90) Coulter, J. A.; Jain, S.; Butterworth, K. T.; Taggart, L. E.; Dickson, G. R.; McMahon, S. J.; Hyland, W. B.; Muir, M. F.; Trainor, C.; Hounsell, A. R.; O’Sullivan, J. M.; Schettino, G.; Currell, F. J.; Hirst, D. G.; Prise, K. M. Cell type-dependent uptake, localization, and cytotoxicity of 1.9 nm gold nanoparticles. Int. J. Nanomed. 2012, 7, 2673−2685.

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