leonid vigderman et al- quantitative replacement of cetyl trimethylammonium bromide by cationic...

8/3/2019 Leonid Vigderman et al- Quantitative Replacement of Cetyl Trimethylammonium Bromide by Cationic Thiol Ligands

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Gold NanorodsDOI: 10.1002/anie.201107304

Quantitative Replacement of Cetyl Trimethylammonium Bromide byCationic Thiol Ligands on the Surface of Gold Nanorods and TheirExtremely Large Uptake by Cancer Cells**

Leonid Vigderman, Pramit Manna, and Eugene R. Zubarev*

Over the past decade, gold nanorods (Au NRs) have received

broad attention as possible therapeutic and diagnostic agents

because of their anisotropic physical properties.[18] However,

as-synthesized Au NRs based on the most common synthetic

approach, the seed-mediated synthesis,[9, 10] are not directly

useable for these applications. They are synthesized in a

concentrated cetyl trimethylammonium bromide (CTAB)

solution and are noncovalently coated with a CTAB

bilayer.[11] For CTAB-capped rods to remain soluble, the

concentration of free CTAB in solution must remain above acertain value and there is a constant dynamic exchange of

CTAB molecules between the solution and NR surfaces.[12]

This feature limits the usefulness of these rods for many

biological applications because free CTAB is known to be

highly cytotoxic.[13, 14] Various strategies have been developed

to functionalize gold nanorods with a variety of different

ligands to reduce their cytotoxicity.[12, 1520] Further reports

have dealt with the problems of stability and biocompatibility

of Au NRs by using various polymer shells,[21] polyelectro-

lytes,[22, 23] peptides,[24] surfactants,[25] and lipids[14,26] to modify

the Au NR surface. Partial replacement of CTAB was

qualitatively confirmed in many of these cases, but the exact

surface composition of nanorods and the amount of residualCTAB was either unknown or not possible to determine. For

that reason, there is always some ambiguity in interpretation

of in vitro and in vivo experiments involving such nanorods.[27]

Beyond just biocompatibility and low toxicity, there has

been great interest in understanding the cellular uptake of Au

NRs.[2832] For example, if CTAB is replaced by nonionic

polyethylene glycol, the cytotoxicity is substantially reduced,

but the cellular uptake drops as much as 94%, and virtually

no nanorods can enter the cells.[30] When polyelectrolytes are

randomly wrapped around the CTAB bilayer, the uptake

increases and the average number of rods that enter each cell

ranges from hundreds[31] to one hundred thousand.[29] How-

ever, even polyelectrolyte-coated Au NRs are taken by cellsin picogram quantities, which is only 0.1% of a typical cell

mass. This small weight fraction may explain a somewhat

limited success of photothermal oblation of tumor cells,[33]

which is strongly dependent on the actual number of nano-

rods present inside the cells. Therefore, an obvious goal is to

design stable and nontoxic (CTAB free) nanorods that could

enter cancer cells in much larger quantities.

Here we report a strategy for complete exchange of

CTAB for its thiolated analogue (16-mercaptohexadecyl)tri-

methylammonium bromide (MTAB) and we directly deter-

mine the chemical composition of the surface coating.

Through

1

H NMR analysis, we are able to prove that CTABis fully replaced by a covalent MTAB monolayer. By

combining thermogravimetric analysis (TGA) with TEM

size analysis of the nanorods, we are able to accurately

determine the packing density of the self-assembled thiol

monolayer on the surface of Au NRs. Cytotoxicity of these

novel nanorods was studied by MTT assay on breast cancer

cells (MCF-7), which were imaged by correlated optical and

scanning electron microscopy (SEM). Extremely large

number of nanorods (about 2 106 NRs per cell) were

found inside the viable cells as confirmed by transmission

electron microscopy (TEM) and quantified by inductively

coupled plasma-optical emission spectrometry (ICP-OES).

Several reports have shown that even with systems thathave successfully functionalized gold nanorods, CTAB is still

observed to be present on the NRs.[28] We hypothesized that

we could use a molecule with a structure very similar to

CTAB to completely exchange the CTAB, but with the ability

to bind more strongly to the NR surface. Thus, we chose to

synthesize a thiolated CTAB analogue (MTAB) whose

synthesis is shown in Scheme 1. Commercially available

1,16-hexadecanediol was converted to a corresponding dibro-

mide under standard bromination conditions, which was

followed by the synthesis of its monothioester. Cleavage of

the acetyl group was carried out in anhydrous methanol using

Scheme 1. Synthesis of (16-mercaptohexadecyl)trimethylammoniumbromide (MTAB; NBS=N-bromosuccinimide, THF= tetrahydrofuran).

[*] L. Vigderman, Dr. P. Manna, Prof. E. R. ZubarevDepartment of Chemistry, Rice UniversityHouston, TX 77005 (USA)E-mail: [email protected]: http://www.owlnet.rice.edu/~zubarev/

[**] This work was supported by the NSF (grant numbers DMR-0547399and DMR-1105878).

Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/anie.201107304.

Angew. Chem. Int. Ed. 2011, 50, 1 7 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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in situ generated hydrogen chloride. The resulting 16-bromo-

1-hexadecanethiol was subjected to exhaustive methylation to

yield the final product (see the Supporting Information).

Importantly, the purified MTAB compound was found to be

water-soluble, which offers an opportunity to perform ligand

exchange in aqueous media. The MTAB ligand contains an

entire CTAB moiety, but installs a pendant thiol group to be

able to strongly anchor it to the gold surface. The cross-

section of this molecule is small enough to form a compact

monolayer on the surface of nanorods, thus providing their

solution stability.

Stabilization of gold nanorods is generally difficult

because of their small surface-to-volume ratio, and finding a

compound that can directly exchange with CTAB is further

complicated by the high-density positive charge and the

amphiphilic nature of the CTAB bilayer (Figure 1). In the

past, researchers have exchanged only the tips of goldnanorods because of relatively weak CTAB binding there

and have noted that it is much stronger on the sides.[3436]

Furthermore, as the CTAB on the sides of the rods begins to

exchange, it appears that the bilayer structure breaks down

and rods tend to aggregate prematurely. However, choosing a

cationic ligand with a chemical structure similar to native

CTAB allows for the direct exchange in water to proceed

smoothly as shown schematically in Figure 1. The noncova-

lent bilayer of CTAB is replaced with a compact thiolate

monolayer of MTAB which is covalently anchored to the

surface through goldsulfur bonds. UV/Vis absorbance spec-

troscopy is often used to assess the stability of gold nanorods

in solution as the intensity, the peak shape, and the peakposition of the longitudinal plasmon resonance (LSPR) are

sensitive to any aggregation that may occur. The UV/Vis

spectrum (see Figure S1A in the Supporting Information)

shows the typical absorbance spectrum for Au NRs and

demonstrates what happens when excess CTAB is removed

from solution through multiple rounds of centrifugation

followed by dispersion in pure water. As expected, after just

four rounds, the NRs have lost all solubility as evidenced by

the lack of any absorbance. In stark contrast, when MTAB-

modified NRs are purified by the same technique, there is no

appreciable change in the absorbance spectrum even after

five rounds of centrifugation and dispersion (see Figure S1B

in the Supporting Information), indicating their much

improved solution stability while also completely purifying

the product.

Additional experiments show that these rods can be

completely dried and kept in the solid state indefinitely

without losing their water solubility. Surprisingly, a standard

lyophilization technique can be applied to aqueous solution of

MTAB NRs to produce a fluffy dark brown powder. Fig-

ure 2a shows a photograph of 15 mg of lyophilized Au NRs

that occupy an area of several square centimeters. Most

importantly, when a small amount of this powder is placed on

the surface of pure water, rapid dissolution takes place as

manifested by the concentration swirls and a continuous

diffusion of the colored solute (Figure 2 b). The nanorods

dissolve in a matter of seconds without any heating or

sonication. It is sufficient to flip the vial only once to form a

homogeneous solution which does not contain any free

organic molecules. The solution remains intact for at least

several months as confirmed by UV/Vis analysis. This data

clearly shows that a dense monolayer of small cationic thiol

MTAB is capable of stabilizing large metallic particles in purewater and preventing their flocculation through entropically

driven depletion interactions.[37] This data also showes for the

first time that the presence of free surfactant in the solution of

gold nanorods is not necessary as long as their surface is

covalently functionalized by a dense organic shell.

Another indirect method that has often been used to

characterize Au NR complexes is the zeta potential measure-

ment, which can qualitatively describe the charge surrounding

a nanoparticle. The zeta potential of the MTAB NRs was

found to be +55 mV, demonstrating the cationic structures as

expected from the high concentration of quaternary ammo-

nium groups positioned around the NR surface. However, the

Figure 1. Exchange of the CTAB bilayer for the MTAB thiol monolayer. Figure 2. a) Photograph of lyophilized powder of MTAB-functionalizedAu NRs and b) its spontaneous dissolution in pure water. The imageswere taken consecutively within intervals of five second.

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zeta potential is unable to distinguish between CTAB and

MTAB on the surface of the rods given that they both have

the same cationic headgroup exposed to the solution and so is

useful only as a secondary confirmation of structure. We

chose 1H NMR spectroscopy to analyze the organic compo-

nent of the MTAB NRs which should be able to differentiate

between the CTAB and MTAB molecules. We used KCN to

dissolve the gold NR core and release any surface-bound

organic material into solution[39] after having rigorously

purified the functionalized NRs. Because of the low surface-

to-volume ratio as well as high density of the gold core, we

performed an oxidative dissolution of 105 mg of Au NRs in

one milliliter of D2O to get enough organic material for a

sufficiently strong 1H NMR signal. The dissolution proceeded

very slowly, taking approximately two weeks to dissolve all of

the gold, suggesting a high packing density for the MTAB

surface functionality.[40] On the contrary, CTAB-coated NRs

dissolved in about one hour under similar conditions. The

dissolution procedure caused the organic component to

precipitate from the D2O solution, apparently because of

the formation of a Au/CN/MTAB complex. The NMRspectrum of the D2O supernatant showed no signals, con-

firming that the entire organic component had precipitated

from the solution. The NMR spectrum of this precipitate

dissolved in deuterated methanol, as well as that of pure

CTAB and MTAB thiol, is shown in Figure 3. Comparing the

spectra of CTAB and MTAB, it is clear that the isolated signal

from the terminal methyl group of CTAB at 0.91 ppm is not

present in the MTAB spectrum, making it a convenient

marker for the presence of any CTAB in solution. Analysis of

the NMR spectrum of the dissolved NRs (spectrum C in

Figure 3) clearly shows that there is no signal corresponding

to this methyl group, proving that no CTAB remained on the

surface of the NRs after the exchange and the following

purification. Further analysis of the NMR spectrum of the

dissolved NRs shows the formation of the expected disulfide

of MTAB as evidenced by the triplet at 2.75 ppm correspond-

ing to the a-disulfide protons (instead of 2.5 ppm for a-thiol

protons). Importantly, the NMR technique is capable of

detecting small organic molecules at concentration close to

105m, whereas the concentration of our solution was 102m.

This implies that even if there were some residual CTAB

undetectable by NMR spectroscopy, it would not exceed

0.1%. Therefore, for all practical purposes one can conclude

that the method described hear offers an opportunity to

quantitatively replace CTAB by its thiolated analogue.

Because the exact chemical composition of the NR

surface agents was determined, it was possible to use TGA

to further characterize the MTAB NRs. TGA analysis is a

highly useful analytical technique for hybrid inorganic

organic nanostructures because it allows us to accurately

quantify the weight percentage of a nanostructure that is

organic compared to inorganic core.[39,41, 42] For our study,

more than 100 mg of MTAB NRs were synthesized and

functionalized to be used for TGA and NMR measurements,

which is quite a large amount when it comes to Au NR

synthesis, requiring a synthesis on the four liter scale.

However, even this amount of material is difficult to handle,

forming an almost unusable thin film when dried normally. Tosolve this problem, the MTAB NRs were lyophilized from an

aqueous solution, forming a low density powder which could

be easily handled, as mentioned previously (Figure 2). TGA

analysis of the lyophilized material (see Figure S2 in the

Supporting Information) shows a weight loss of 5.3% in the

range between 200 and 4508C corresponding to the percent-

age of organic material in the structure. Since the NMR

analysis proved that no residual CTAB was left, this weight

must be entirely because of MTAB ligands covalently

attached to the surface of rods.

We then performed a careful TEM analysis by measuring

over 300 nanorods, which determined that the average length

and width of MTAB NRs were 41.88 and 9.87 nm, respec-tively. With this information, we were able to calculate a

grafting density of about 3.7 moleculesnm2 for an MTAB

monolayer and 5013 MTAB molecules residing on each Au

NR (see the Supporting Information for detailed calcula-

tions). This is close to the grafting density of 4.5 mole-

culesnm2 for neutral alkanethiols on flat gold substrates[43]

and may be slightly lower because of repulsive forces between

the positively charged headgroups. We believe this is the first

proof of a dense self-assembled thiol monolayer on gold

nanorods with a calculated grafting density. Estimation of the

extent of thiol functionalization has been carried out on

DNA-functionalized Au NRs by measuring fluorescence

intensity, but such structures contained only about 40 DNAmolecules per nanorod[17] as compared to over 5000 MTAB

molecules.

With the characterization complete, we performed cyto-

toxicity and cell uptake experiments to see if the MTAB NRs

could be useful for biological applications. We first measured

their in vitro cytotoxicity compared to regular CTAB-capped

NRs using the standard MTT assay on MCF-7 breast cancer

cells (see Figure S3 in the Supporting Information). These

studies reaffirmed the cytotoxicity of regular CTAB-capped

gold nanorods and showed the decreased cytotoxicity of the

thiolated rods, which are not cytotoxic even up to concen-

trations of 0.1 gL1. This finding supports previous assertions

Figure 3. 1H NMR spectra in deuterated methanol of CTAB (A), pureMTAB (B), and the organic product released upon oxidative dissolu-tion of MTAB NRs (C).

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that surface charge may not be a key factor for the

cytotoxicity of nanoparticle systems.[31] Although previous

reports have shown that it is mostly free CTAB that

contributes to the cytotoxicity of CTAB NRs in the short

term,[31] it is unknown what may happen to surface-bound

CTAB in the longer term in in vivo experiments. Our system

has the advantage of having quantitative CTAB replacement

such that this is no longer an issue. Low cytotoxicity is

particularly useful if the NRs can be taken up by cells

efficiently. To study the uptake, MCF-7 cells were treated with

a 20 mg mL1 solution of MTAB NRs in Eagles minimum

essential medium for 24 h. The cells were subsequently rinsed

multiple times with PBS solution to remove the excess of NRs

that did not enter the cells. After that, the cells were

trypsinized, redispersed into medium, and carefully plated

onto a new culture slide. This approach allowed us to create a

clear background without any free nanorods, which is

critically important for differentiating between the internal-

ized particles and those which are not associated with the

cells. The dark field optical image (Figure 4A) clearly shows a

large uptake of MTAB NRs and demonstrates their high

scattering efficiency. It also appears that NRs are clustered

together inside the cells rather than being evenly distributed

throughout their interior, suggesting that the MTAB NRs

enter the cells through an endosomal pathway. To perform a

correlated SEM imaging of the exact same group of cells, we

fixed the sample with glutaraldehyde and further cross-linked

the cells with osmium tetroxide. This enabled us to prevent

the collapse of cells under high vacuum conditions required

for the SEM. The SEM imaging correlated with the optical

experiments (Figure 4B) shows areas of increased brightness

inside the cells because of clusters of internalized MTAB

NRs. The locations of the NR clusters match up well with the

bright spots seen in the optical micrograph, although there are

some morphological changes that occurred during the fixation

and dehydration processes. Most importantly, SEM imaging

reveals that there are no MTAB NRs on the surface of the

cells, which would be impossible to confirm by the optical

microscopy alone. In contrast, the visualization of nanorods

residing in thicker cell areas is more efficient by optical

microscopy, displaying their strong potential as imaging

contrast agents.To further characterize the cellular uptake, we performed

transmission electron microscopy (TEM) imaging of cells

treated with the MTAB NRs as shown in Figure 5. Unlike

SEM and optical microscopy, TEM images of the microtomed

cross-sections reveal precise location and spatial distribution

of individual nanorods. We prepared 75 nm thick sections that

were cut through the center of cells. The TEM image of the

entire cell cross-section (Figure 5A) shows an extremely large

amount of NRs, which appear as dark particles. The volume of

this cross-sectional sample is approximately 0.5% of the total

cell volume (20 mm in diameter), which means that the total

number of nanorods inside the cell is approximately 200 times

Figure 4. A) Dark field optical micrograph and B) SEM image of cellstreated with MTAB NRs.

Figure 5. TEM images of microtomed MCF-7 cancer cells treated withthe MTAB NRs (75 nm thick). A) View of an entire cell cross-section.B) Magnified image of the area outlined by the black box in panel A;C) Magnified image of the area outlined by the dashed box in panel B.

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greater than that present in this image (about 10000 NRs).

Thus, even on the basis of TEM one can roughly estimate that

there are at least two million nanorods per cell. If confirmed

by quantitative methods, it would be significantly larger than

the numbers reported in the literature for nanorods of similar

size, which range from several hundred[31] to one-hundred-

fifty thousand NRs per cell.[29] Even at low magnification one

can see that NRs do not enter the nucleus and are clustered in

endosomes. Higher magnification of regions outlined by the

boxes (Figure 5B and C, respectively) clearly shows individ-

ual nanorods that do not appear to be free in the cyto-

plasm.[29,4446] The amount of uptake was compared to another

popular NR system, pegylated nanorods (PEG NRs), which

are known to resist cellular uptake and thus form a good

negative control for our system. To quantify the average

number of NRs taken by cells, we performed ICP-OES

analysis on dissolved cells which had been treated with both

MTAB NRs and their pegylated analogues. Based on these

results, while almost no PEG NRs were taken up (less than

1%), about 40% of MTAB NRs in solution were taken up by

the cells. This corresponds to a level of about 2.17 millionMTAB NRs per cell that have been treated with a solution of

nanorods (see the Supporting Information for detailed

calculations). Such a great amount of metallic gold increases

the mass of cancer cell by 0.13 ng, which is approximately

13% of a typical cell mass (1 ng). It is particularly remarkable

that the cells retain their viability and continue to proliferate

with such a significant amount of gold nanostructures inside

them. These results show that the MTAB NRs have a very low

cytotoxicity and suggest that they may be promising for

further biomedical applications such as drug/gene delivery

and photothermal therapy.

In conclusion, we have synthesized highly stable, func-

tionalized gold nanorods as an alternative to CTAB-cappedNRs through a direct quantitative exchange with a thiolated

CTAB analogue. The MTAB NRs showed a highly increased

stability to multiple rounds of purification compared to

CTAB NRs. We were able to quantitatively prove the

complete removal of CTAB through the use of 1H NMR

spectroscopy, and thus determined the exact composition of

the organic component of the hybrid nanostructures. We also

successfully performed a TGA analysis to measure the exact

organic versus inorganic composition of hybrid nanostruc-

tures and determined a grafting density for the MTAB thiol,

proving the formation of a compact self-assembled monolayer

on gold nanorods. The MTAB NRs were not only found to be

nontoxic, but were also efficiently taken up by cancer cellsin vitro in very large amounts as shown by a comprehensive

combination of optical microscopy, SEM, TEM, and ICP-

OES data.

Received: October 17, 2011

Published online:&& &&, &&&&

.Keywords: cancer cells cellular uptake gold nanorods surface chemistry

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Communications


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Communications

Gold Nanorods

L. Vigderman, P. Manna,

E. R. Zubarev* &&&&&&&&

Quantitative Replacement of Cetyl

Trimethylammonium Bromide by

Cationic Thiol Ligands on the Surface of

Gold Nanorods and Their Extremely

Large Uptake by Cancer Cells

Stable and biocompatible : The chemical

composition of synthesized cationic thi-

olate-monolayer-protected gold nanorods

was precisely determined. In vitro cell

culture experiments showed no cytotox-

icity of these nanorods, and the number

of nanorods that were taken up by each

cancer cell exceeded two million particles

(see picture).


http://www.angewandte.org/http://www.angewandte.org/


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Supporting Information

Wiley-VCH 2011

69451 Weinheim, Germany

Quantitative Replacement of Cetyl Trimethylammonium Bromide byCationic Thiol Ligands on the Surface of Gold Nanorods and TheirExtremely Large Uptake by Cancer Cells**

Leonid Vigderman, Pramit Manna, and Eugene R. Zubarev*

anie_201107304_sm_miscellaneous_information.pdf


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1

Supporting Information

Synthesis of MTAB. Synthesis of 1,16-dibromohexadecane, 1. A 50 mL solution of

triphenylphosphine (3.93 g, 15 mmol) in anhydrous THF was added to a stirred solution ofN-

bromosuccinamide (2.67 g, 15 mmol) in 50 mL THF at 0C. Upon vigorous stirring, a solution of

hexadecane-1,16-diol (1 g, 3.9 mmol) in 25 mL THF was slowly added to the mixture of NBS and

Ph3P. The resulting solution was warmed to room temperature and then heated at 60C for 3.5 hours.

THF was removed by rotary evaporation and the residue was re-crystallized form ethanol to obtain 1.1

g of2 as white powder (70 % isolated yield).1H NMR (CDCl3, 400 MHz): 1.26-1.46 (m, 24 H), 1.85

(q, 4H), 3.41 (t, 4H).

Synthesis of 16-bromo-1-hexadecanethioacetate, 2. One gram (2.60 mmol) of 1 was dissolved in 40

mL methanol in a three-neck flask and the solution was degassed for one hour. Separately, 124 mg of

sodium methoxide was dissolved in 12 mL dry, ice-cold methanol and mixed with 204 mg (2.6 mmol)

of thioacetic acid. This mixture was transferred into a funnel. The solution in the three-neck flask was

refluxed under argon atmosphere and the content in the funnel was slowly added to the solution over

the course of 4 hours. After the reaction was complete, the content of the flask was cooled to room

temperature and methanol was removed under reduced pressure. The yellow oil was further purified by

column chromatography (20 % ethyl acetate in hexane) to obtain 480 mg of3 (50 % yield).

Synthesis of 16-bromo-1-hexadecanethiol 3. To a stirred solution of2 (400 mg, 1.05 mmol) in 10 mL

of methanol, 4 mL of acetyl chloride was added drop-wise and the reaction mixture was kept at 50

C

for 4 hours. After the reaction was complete, 200 mL of CH2Cl2 was added to the reaction mixture and

excess acetyl chloride and HCl were removed by multiple extractions with DI water. The mixture was


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2

dried over sodium sulfate. Methylene chloride was evaporated under reduced pressure to obtain 284

mg of3 as colorless oil (80 % isolated yield).1H NMR (CDCl3, 400 MHz): 1.26-1.46 (m, 25H), 1.60

(m, 2H), 1.85 (q, 2H), 2.52 (q, 2H), 3.41 (t, 2H).

Synthesis of 16-mercaptohexadecyltrimethylammonium bromide, MTAB. To a solution of3 (284 mg,

0.85 mmol) in 5 mL of ethyl acetate, 3 mL of 4.2 M ethanolic solution of trimethylamine was added.

The mixture was vigorously stirred under argon for 4 days. The resulting white precipitate was filtered

and the washed several times with ethyl acetate to remove excess trimethylamine. The residue was

vacuum dried to obtain 270 mg of MTAB (80 % isolated yield).1H NMR (CDCl3, 400 MHz): 1.26-

1.46 (m, 25H), 1.60 (m, 2H), 1.85 (m, 2H), 2.52 (q, 2H), 3.5 (s, 9H), 3.55-3.7 (m, 2H).

Gold nanorods synthesis. Gold nanorods were synthesized according to procedure described

elsewhere.[41]

Briefly, a seed solution was created by adding 60 L of a 0.1 M NaBH4 solution to 10

ml of 5.0x10-4

M HAuCl4 in 0.1 M CTAB solution upon rigorous stirring. Separately, the growth

solution was prepared by adding 2 mL of 0.1 M AgNO3 to 2 liters of 5.0x10-4

M HAuCl4 in 0.1 M

CTAB solution. To this solution was added 11.5 mL of 0.1 M ascorbic acid solution and hand-stirred

until the solution became clear, followed immediately by the addition of 3.2 mL of seed solution. A

dark brown color indicating the presence of gold nanorods was visible after about 30 min and the

solution was allowed to sit overnight before functionalization.

Gold nanorods functionalization with MTAB thiol. Four liters of Au NRs solution was

concentrated to about 9 mL using centrifugation at 13,000 rpm. The concentrated NRs solution was

purified by two cycles of centrifuging at 13,000 rpm, removing the supernatant and redispersing the

precipitate in pure water. To this solution was added 60 mg of MTAB and the solution was stirred for


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2 days. The MTAB-NRs were purified by five cycles of centrifugation as described earlier and finally

dispersed in 4 mL of water to a concentration of 15 mg/mL as determined by ICP-OES measurements.

Figure S1.UV-vis absorbance spectrum of CTAB-NRs (A) and MTAB-NRs (B) after purification steps by

high-speed centrifugation and redispersion into pure water.

Analysis of MTAB-NRs. For TGA analysis (TA Q-600 TGA/DSC), MTAB-NRs were first

lyophilized to obtain a useable powder. 1 mL of 15 mg/mL NR solution was placed into an Eppendorf

tube, flash frozen with liquid nitrogen, and lyophilized. In the TGA measurement, the temperature was

held at 120 C for 1 hour to remove any residual water and then ramped at 10 C/min to 850 C. For

1H-NMR analysis (Bruker 400 MHz), 1.5 mL of 15 mg/mL NR solution was transferred into D2O by

two rounds of centrifugation at 13,000 rpm and redispersion into D2O. To the D2O solution was added

50 mg KCN and the solution stirred for two days until the solution was colorless and a white

precipitate had formed. The precipitate was isolated by centrifugation and dissolved in deuterated

methanol. UV-Vis analysis was carried out on a Varian Cary 500 UV-Vis-NIR Spectrophotometer.

Zeta Potential measurements were carried out on a Malvern Instruments Zetasizer Nano-ZS.


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Figure S2.TGA curve of MTAB-NRs. Residual water is lost at 100 C. Main weight loss of about 5.3 %

from organic component occurs from 200 to 450 C.

Calculation of MTAB binding parameters from TGA data. From TEM size analysis of ~200 NRs,

average length and width are 41.88 nm and 9.87 nm, respectively. Assume NR = cylindrical middle

with radius (r) and height (h) plus 2 spherical caps with base radius (r) and height (a). Based on TEM,

the spherical caps extend for about 2 nm on each side so h = 37.88 nm, r = is 4.935 nm, and a = 2 nm.

Thus, the surface area of the rod = (r2

+ a2) + 2rh = 1352.6 nm

2and the volume =( /3) a (3r

2+ a

2) +

r2h = 3059.6 nm

3. Mass of Au per NR = 3059.6 nm

3 59 (atoms/nm

3) 197 = 3.5610

7Da. Mass

MTAB = 0.053 (mass Au per NR + mass MTAB). Mass MTAB = 1.99106

Da. Number of MTAB

per NR = 1.99106

Da/ 397 (Da/molecule) = 5,013 MTAB molecules per NR. Molecular footprint =

1352.6 nm2

/ 5,013 molecules = 0.270 nm2

per molecule of MTAB or 3.7 MTAB molecules per nm2.


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Figure S3. Cytotoxicity plot based on MTT analysis, which shows the percent survival of MCF-7 cells when

treated with different concentrations of CTAB-NRs (blue) and MTAB-NRs (red).

Calculation of amount of NR uptaken per cell. 500,000 cells were treated with 2 mL of medium

containing 80 g/mL MTAB-NR in medium. From ICP-OES, the total amount of up-taken gold was

determined to be 40% of 160 g or 64 g Au / 500,000 cells = 1.2810-4

(g Au/cell). Given the

density of gold and the previously calculated volume per NR of 3060 nm3

1.9310-14

(g Au/nm3) =

5.9110-11 (g Au/NR). So, 1.2810-4 (g Au/cell) / 5.9110-11 (g Au/NR) = 2.17106 NR per cell.

TEM imaging of MTAB-NR uptake into cells. MCF-7 cells were plated into a 6-well plate at

500,000 cells per well and treated with 2 mL of a 20 g/mL solution of NRs per well in medium for 24


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hours. Cells were washed several times with PBS and fixed with a solution containing 3%

glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3, for 1 hour. After

fixation, the samples were washed and treated with 0.1% Millipore-filtered cacodylate buffered tannic

acid, postfixed with 1% buffered osmium tetroxide for 30 min, and stained en bloc with 1% Millipore-

filtered uranyl acetate. The samples were dehydrated in increasing concentrations of ethanol,

infiltrated, and embedded in LX-112 medium. The samples were polymerized in a 70oC oven for 2

days. Ultrathin sections were cut in a Leica Ultracut, stained with uranyl acetate and lead citrate in a

Leica EM Stainer, and examined in a JEM 1010 transmission electron microscope (JEOL) at an

accelerating voltage of 80 kV. Digital images were obtained using AMT Imaging System (Advanced

Microscopy Techniques Corp).

Cytotoxicity and cell uptake. MCF-7 cells were cultured in Eagles Minimum Essential Medium with

non-essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, and fetal bovine serum to a

final concentration of 10 %. MCF-7 cells were plated into a 96-well plate at 15,000 cells per well and

6 wells per each concentration. Cells were treated with an appropriate amount MTAB-NRs or CTAB-

NRs in medium for 24 hours, followed by the addition of 25 L of a 5 mg/mL MTT solution in PBS.

After incubating for 4 hours, the medium was removed, the cells were lysed with DMSO, and the

absorbance measured on a plate reader at 570 nm. For cell uptake imaging experiments, MCF-7 cells

were plated into a 6-well plate at 500,000 cells per well and treated with 2 ml of a 20 g/mL solution

of NRs in medium for 24 hours, trypsinized, and then plated onto a culture slide. For quantification of

NR uptake by ICP-OES, cells were treated with 2 mL of an 80 g/mL solution of NRs in medium for

24 hours, washed several times with PBS, trypsinized, pelleted, dissolved with aqua regia, and the gold

concentration measured by ICP. For SEM imaging cells were first fixed by using 2.5 % glutaraldehyde

in PBS for an hour followed by 0.4 % osmium tetroxide solution. Next, cells were dehydrated by


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consecutive treatment with 25 %, 50 %, 70 %, 95 %, and 100 % ethanol followed by 50 % and 100%

hexamethyldisilazane and allowed to dry overnight.

Figure S4. TEM image of MTAB-functionalized gold nanorods.

leonid vigderman et al- quantitative replacement of cetyl trimethylammonium bromide by cationic...

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