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SURFACE CHEMISTRY MANIPULATION OF GOLD 
NANORODS DISPLAYS HIGH CELLULAR UPTAKE 
IN VITRO WHILE PRESERVING OPTICAL PROPERTIES 
FOR BIO-IMAGING AND PHOTO-THERMAL APPLICATIONS 

ANTHONY B. POLITO III, Maj, USAF, BSC, PhD, MT(ASCP)SBB 

March 2016 

Final Report for March 2015 to DEC 2015 



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4. TITLE AND SUBTITLE 

SURFACE CHEMISTRY MANIPULATION OF GOLD 
NANORODS DISPLAYS HIGH CELLULAR UPTAKE IN 
VITRO WHILE PRESERVING OPTICAL PROPERTIES 
FOR BIO-IMAGING AND PHOTO-THERMAL 
APPLICATIONS. 


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7/2012-1/2016 


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6. AUTHOR(S) 

ANTHONY B. POLITO III. Maj, USAF, BSC, PhD, MT(ASCP)SBB 


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Air Force Institute of Technology (AF1T) Civilian Institution Programs (C1P) 
2950 Flobson Way 
WPAFB OH 45433-7765 


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Clinical Investigations 

Office of the Air Force Surgeon General SG5M, 

Research and Innovations 
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Falls Church, VA 22042-5164 


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14. ABSTRACT 

Due to their anisotropic shape, gold nanorods (GNRs) possess a number of advantages for biosystem use, including enhanced surface area and 
tunable optical properties within the near infrared region (NIR). However, a combination of cetyltrimethylammonium bromide (CTAB) related 
cytotoxicity, overall poor cellular uptake following surface chemistry modifications and loss of NIR optical properties due to material intracellular 
aggregation remain as obstacles for nano-based biomedical GNR applications. The current report demonstrated that in vitro exposure to tannic acid 
(TA) coated 11-mercaptoundecyl trimethylammonium bromide (MTAB) GNRs (MTAB-TA) in A549 human alveolar epithelial cells showed no 
significant decrease in cell viability or stress activation. In addition, MTAB-TA GNRs demonstrate a substantial level of cellular uptake while 
displaying a unique intracellular clustering pattern. This clustering pattern significantly reduces intracellular aggregation, preserving the GNRs NIR 
optical properties, vital for biomedical applications. MTAB-TA GNRs demonstrated significantly greater two photon luminescence microscopy 
image intensity and photo-thermal cellular ablation compared to bare MTAB GNRs. These results demonstrate how TA surface chemistry 
modification enhances biocompatibility and allows for a high rate of internalization while preserving the GNRs NIR optical properties. These 
findings identify MTAB-TA GNRs as prime candidates for use in nano-based bio-imaging and photo-thermal applications. 


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Standard Form 298 (Rev. 8-98) 

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TABLE OF CONTENTS 


1.0 Summary.1 

2.0 Introduction.2 

2.1 Objective.3 

3.0 Methods.4 

3.1 Synthesis of GNRs.4 

3.2 PEG functionalization of GNRs.4 

3.3 TA functionalization of GNRs.4 

3.4 Characterization of GNRs.4 

3.5 Cell culture conditions.4 

3.6 Cellular viability assessment.5 

3.7 ROS assay.5 

3.8 RT-PCR for surfactant protein A and inflammatory gene expression.5 

3.9 Human cytokine immunoassay.5 

3.10 Quantification of cellular uptake of GNRs by ICP-MS.6 

3.11 Darkfield microscopy.6 

3.12 Transmission electron microscopy.6 

3.13 Hyperspectral microscopy.7 

3.14 Two-photon luminescence microscopy.7 

3.15 Plasmonic photo-thennal cells ablation.7 

3.16 Statistical Analysis.7 

4.0 Results and Discussion.8 

4.1 GNR characterization.8 

4.2 MTAB-TA GNRs display enhanced biocompatibility.11 

4.3 Cellular association and in vitro intracellular hyperspectral signature.15 

4.4 High uptake with unique clustering pattern of MTAB-TA GNRs.18 

4.5 Low intracellular aggregation of MTAB-TA GNRs.21 

4.6 MTAB-TA GNRs exhibit superior two-photon luminescence image intensity.25 

4.7 MTAB-TA GNRs demonstrate higher efficiency for photo-thermal cellular ablation ....26 

5.0 Conclusions.31 

6.0 References.33 

List of Acronyms.38 


IV 

































LIST OF FIGURES 


Figure 1. GNR characterization.9 

Figure 2. MTAB-TA GNRs demonstrate enhanced biocompatibility.12 

Figure 3. MTAB-TA GNRs do not alter inflammatory gene expression.13 

Figure 4. MTAB-TA GNRs do not alter cytokine release.15 

Figure 5. Intracellular MTAB-TA GNRs retain NIR optical properties.17 

Figure 6. Uptake and retention of MTAB-TA GNRs.20 

Figure 7. Visualization of MTAB-TA GNR uptake.21 

Figure 8. Visualization of intracellular clustering pattern of MTAB-TA GNRs.22 

Figure 9. Visualization of MTAB-TA GNRs cellular retention.23 

Figure 10. Analysis of intracellular MTAB-TA GNR clusters.24 

Figure 11. MTAB-TA GNRs display greater two-photon luminescence intensity.26 

Figure 12. MTAB-TA GNR shows efficiency as agent for photo-thermal therapy.28 

Figure 13. Visual comparison of MTAB-TA GNRs efficiency for photo-thennal 

cellular ablation.30 

Figure 14. MTAB-TA GNRs demonstrate greatest efficiency for photo-thennal 

cellular ablation.31 

Figure 15. MTAB-TA GNRs have enhanced bio-imaging and photo- thennal 

properties.32 


v 


















LIST OF TABLES 


Table 1. Characterization of GNRs 


10 


VI 




PREFACE 


Funding for this project was provided through the Air Force Surgeon General Clinical 
Investigations Program. 


The authors would like to acknowledge the Biomedical Sciences PhD program at wright State 
University. The authors also wish to tha nk Dr. David Cool, Dr. Courtney Sulentic, Dr. Nancy 
Bigley and Dr. Shannila Mukhopadhyay. 



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1.0 SUMMARY 


Due to their anisotropic shape, gold nanorods (GNRs) possess a number of advantages 
for biosystem use, including enhanced surface area and tunable optical properties within 
the near infrared region (NIR). However, a combination of cetyltrimethylammonium 
bromide (CTAB) related cytotoxicity, overall poor cellular uptake following surface 
chemistry modifications and loss of NIR optical properties due to material intracellular 
aggregation remain as obstacles for nano-based biomedical GNR applications. The 
current report demonstrated that in vitro exposure to tannic acid (TA) coated 11- 
mercaptoundecyl trimethylammonium bromide (MTAB) GNRs (MTAB-TA) in A549 
human alveolar epithelial cells showed no significant decrease in cell viability or stress 
activation. In addition, MTAB-TA GNRs demonstrate a substantial level of cellular 
uptake while displaying a unique intracellular clustering pattern. This clustering pattern 
significantly reduces intracellular aggregation, preserving the GNRs NIR optical 
properties, vital for biomedical applications. MTAB-TA GNRs demonstrated 
significantly greater two photon luminescence microscopy image intensity and photo- 
thermal cellular ablation compared to bare MTAB GNRs. These results demonstrate how 
TA surface chemistry modification enhances biocompatibility and allows for a high rate 
of internalization while preserving the GNRs NIR optical properties. These findings 
identify MTAB-TA GNRs as prime candidates for use in nano-based bio-imaging and 
photo-thermal applications. 


1 



2.0 INTRODUCTION 


Nanomaterials are increasingly being developed for use in industrial, military and 
consumer products, including a vast array of biomedical applications (Adlakha-Hutcheon 
et al., 2009; Barreto et al., 2011). Recent advances in solution chemistries for synthesis of 
solid phase nanomaterial technology make it possible to manipulate gold nanomaterials 
into different sizes, shapes and surface structures (Sun & Xia, 2002). Gold nanorods 
(GNRs) are of particular interest due to their unique optical region absorbance, emission 
and electronic properties. GNR optical properties are tunable by preparing nano-featured 
structure based upon their dimensional aspect ratio (AR) or through surface modification 
chemistry (Bouhelier et al., 2005). The AR describes the two dimensional proportional 
relationship between the nanomaterial’s width and height. Depending on the GNR’s AR, 
a narrow range of light frequencies induces conduction band electron oscillation, termed 
surface plasmon resonance (SPR) (Liang et al., 2012). The spectral signature of the 
GNRs longitudinal plasmon resonance extends into the near-infrared (NIR) region and 
GNRs with a longitudinal SPR between 650 and 950 mn fall within the non-absorbing 
region of water and carbon based substances, termed the “water window” (Weissleder, 
2001). This feature allows for deep-tissue penetration and sensing with GNRs and makes 
them useful for nano-based biomedical applications. As such, GNRs have been used in a 
vast array of biomedical applications, including diagnostic imaging, photo-therapies, and 
drug/gene delivery (Agarwal et al., 2011; Nagesha et al., 2007; Pandey et al., 2013; 
Pissuwan et al., 2008). Further, GNRs also show great potential for “theragnostic” uses 
that combined diagnostic imaging and therapeutic applications (Choi et al., 2012; Jelveh 
& Chithrani, 2011; Wang et al., 2009; Yang et al., 2013). GNRs synthesized with AR 4 
will have longitudinal SPR around 800nm, allowing for the interaction with readily 
available 800nm lasers and therefore making GNRs with an approximate AR of 4 an 
ideal theragnostic platform. However, the combination elicited toxicity, poor cellular 
uptake and loss of NIR optical properties due to intracellular aggregation remain as 
obstacles for using GNRs in many nano-based biomedical applications (Alkilany & 
Murphy, 2010; Panyala et al., 2009). 

The toxicity of GNRs is largely a product of free and possibly surface associated cetyl 
trimethylammonium bromide (CTAB), which is a cationic surfactant used in the aqueous 
synthesis of GNRs (Takahashi et al., 2005; L. Wang et al., 2013a; Wang et al., 2011). 
During synthesis, CTAB provides a growth micelle environment around the gold 
seedings stabilizing them to form a rod (Jana et al., 2001; Nikoobakht & El-Sayed, 2003) 
and is present, in both the supernatant and as a bi-layer around the GNRs after synthesis. 
Two strategies have been reported to overcome this surfactant’s cytotoxicity: 1) 
replacement by post-synthesis ligand exchange or non-covalent overcoating by chemical 
cover layering via electrostatic attraction (Vigderman et al., 2012a). Unfortunately, some 
commonly used surface modifications (e.g. polyethylene glycol; PEG) can significantly 
lower cellular uptake of the GNRs into cells, thus reducing their utility in biomedical 
applications (Grabinski et al., 2011; Huff et al., 2007). In addition, other surface 
modifications (e.g. polymer coatings, peptide functionalization etc.) are prone to particle 
aggregation when they are taken up by cells (Untener et al., 2013; Zhang et al., 2013c) 
and result in alteration and/or loss of key optical properties (Kelly et al., 2003; Sosa et al., 
2003). Furthermore, over-coatings can break down in biological environments over time 


2 



(Ejima et al., 2013). This can result in surface leaching of the CTAB and therefore does 
not guarantee that the CTAB toxicity is completely mitigated by over-coating. Finally, 
over-coating and surface replacement procedures often require complicated multi-step 
functionalization processes (e.g. silica over-coating) that are difficult to scale up for 
biomedical applications (Gui & Cui, 2012). 

Previous studies found that TA coated GNRs have reduced toxicity, demonstrate a 
distinctive form of endosomal uptake and display a unique intracellular distribution 
pattern that reduces particle aggregation (Debrosse et al., 2013; Untener et al., 2013). 
Unfortunately, as the CTAB-TA GNR’s AR increases so does its toxicity, possibly due to 
any remaining CTAB. Therefore procedures for exhaustive removal or exchange of the 
CTAB from the GNRs may be essential to help lower toxicity. GNRs coated with MTAB 
(11-mercaptoundecyltrimethylammonium bromide), a thiol analogue of CTAB, represent 
a less toxic alternative though its biocompatibility and characterization within biological 
matrices has not yet been fully determined. 

Vigdennan et al. (2012b) recently used proton nuclear magnetic resonance spectroscopy 
to determine that complete replacement of CTAB with MTAB is possible due to its 
analogous chemical structure (Vigdennan et al., 2012b). MTAB replacement occurs as 
CTAB micelle bilayer around the GNR is exchanged with a monolayer of MTAB that 
strongly binds to the GNR. This study found that MTAB GNRs had no toxicity in the 
human breast adenocarcinoma cell line, MCF-7 even at high concentrations (Vigdennan 
et al., 2012b). In addition, 40% of MTAB GNR treatment was taken up by the cells, 
compared to less than 1% of their pegylated analogues, exceeding previously reported 
GNR uptake values (Vigdennan et al., 2012b). However, Vigdennan et al. (2012b) 
published TEM images that showed extensive aggregation of intracellular MTAB 
GNRs.This aggregation results in blue shift of plasmon resonance emissions, due to close 
proximity side by side assembly of GNRs, which moves the GNRs spectra MTAB GNRs 
out of the target NIR “water window” (Jain et al., 2006; Park, 2006). Therefore, new 
methods/techniques are needed to prevent the aggregation of MTAB GNRs in biological 
environment before they can be efficiently used in biomedical applications. 

While the low toxicity and increased cellular uptake of MTAB GNRs are improvements 
over other GNR preparations, the loss of NIR optical properties described above limit 
their utility for biomedical applications. 

2.1 Objective 

The aim of this study is to address this limitation by combining MTAB replacement with 
TA over-coating (i.e. MTAB-TA GNRs) and comparing their properties relative to 
MTAB and Silica GNRs, as well as CTAB GNRs with and without TA over-coating. TA 
coated GNRs taken up by cells display a unique intracellular distribution pattern that 
appears to reduce particle aggregation (Alkilany et al., 2009; Untener et al., 2013). Due to 
this, it is hypothesized that MTAB-TA GNRs will exhibit enhanced biocompatibility and 
cellular uptake, while preventing particle aggregation to preserve key NIR optical 
properties within the A549 adenocarcinomic human alveolar basal epithelial cell line. 

The A549 cell line retains significant alveolar phenotype, and has been thoroughly 
characterized and used in numerous nano biocompatabilty, bio-imaging and therapeutic 


3 



studies (Foster et al, 1998; Kuo et al, 2012; Mason & Williams, 1980; Uboldi et al, 
2009; Zhang et al., 2012b). 


3.0 METHODS 

3.1 Synthesis of gold nanorods 

GNRs with an approximate AR 4 (MTAB, MTAB-TA, and Silica) were purchased from 
Nanopartz (Loveland, CO, USA). GNRs of approximately AR 3 (CTAB, CTAB-TA, 
PEG) were synthesized according to a modified seed mediated procedure reported by 
Park and Vaia (2008). Briefly, a seed solution of CTAB (0.1 M) and chlorauric acid (0.1 
M) is combined at room temperature with a growth solution of CTAB (0.1 M), chlorauric 
acid (0.1 M) silver nitrate (0.1 M) ascorbic acid (0.1 M). The CTAB was purchased from 
GFS chemicals (Powell, OH, USA). The chloroauric acid, ascorbic acid, silver nitrate, 
sodium borohydride, sodium Chloride, MOPS buffer and tannic acid were obtained from 
Sigma Aldrich (St Louis, MO, USA). 

3.2 PEG functionalization of GNRs 

CTAB GNRs were functionalized with PEG as previously reported (Untener et al., 2013) 
with modifications. Briefly, the GNRs were functionalized overnight with 1 mM thiol 
PEG (Nanocs, Boston, MA) two times (MW 20000 followed by MW 5000) to displace 
the surface bound CTAB molecules. The GNR samples were centrifuged at 8,000g and 
the supernatant was removed and replaced with sterile water to remove residual free 
CTAB. 

3.3 TA functionalization of GNRs 

CTAB GNRs were functionalized with TA according to a modified procedure reported 
by Ejima et al. (2013) stepwise with TA (24mM), and MOPS buffer (100 mM, pH 7.4) 
with vortexing after each addition. The GNR samples were then centrifuged at 3,000g 
and the supernatant was removed and replaced with sterile water to remove residual TA. 
The MOPS buffer and tannic acid were obtained from Sigma Aldrich (St Louis, MO, 
USA). 

3.4 Characterization of GNRs 

The purity and spectral signature of the GNRs were analyzed before use with UV-Vis 
spectrometry on a Bio TEK Synergy HT (Winooski, VT, USA) instrument. For 
evaluation of rod size and morphology, nanoparticles in solution were placed onto a 
formvar carbon coated copper TEM grid (Electron Microscopy Sciences, Hatfield, PA, 
USA) and dried. They were imaged with transmission electron microscopy (TEM) using 
a Hitachi H-7600 with an accelerating voltage of 120 kV. To assess the surface charge of 
the GNRs, zeta potential measurements were taken using laser Doppler electrophoresis 
on a Malvern Zetasizer, Nano-ZS. Agglomerate sizes of the GNRs in media were 
detennined through dynamic light scattering (DLS), also on a Malvern Zetasizer 
(Malvern Instruments, MA, USA). 

3.5 Cell culture conditions 


4 



The A549 human lung cell line (American Type Culture Collection (ATCC), Manassas, 
VA, USA) was maintained in RPMI 1640 cell culture media (Life Technologies, Grand 
Island, NY, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, 
USA) and 1% penicillin streptomycin. Cells were maintained in a humidified incubator 
controlled at 37 °C and 5% C02. The same media composition was used for all GNR 
exposure procedures with the exception of the photo-thermal cellular ablation 
experiments where RPMI 1640 cell culture media without Phenol Red (Life 
Technologies, Grand Island, NY, USA) was used. 

3.6 Cellular viability assessment 

A549 human lung cell viability was evaluated using the CellTiter 96 Aqueous One 
Solution (MTS) (Promega, Madison, WI, USA) which monitors mitochondrial function 
and MultiTox-Glo Assay (LCDC) (Promega, Madison, WI, USA), which sequentially 
measures two protease activities; one is a marker of cell viability, and the other is a 
marker of cytotoxicity. Cells were seeded into a 96-well plate at a concentration of 2 x 
10 3 cells per well and the following day treated with the stated GNR conditions. After 
exposure period, the cells viability was determined in accordance with the manufacturer’s 
protocol. Result represents three independent trials with the average ± the standard error 
reported. 

3.7 ROS assay 

The intracellular generation of reactive oxygen species (ROS) after GNR exposure was 
evaluated using CM-H2DCFDA ( Life Technologies, Grand Island, NY, USA) 
Technologies, Grand Island, NY) which is based on intracellular esterases and oxidation 
that yields a fluorescent product that is trapped inside the cell. Cells were seeded into a 
96-well plate at a concentration of 2 x 10 3 cells per well and the following day treated 
with the stated GNR conditions. After 1 h, 6 h, and 24 h the intracellular ROS generation 
was determined in accordance with the manufacturer’s protocol. Results represent three 
independent trials with the average ± the standard error reported. 

3.8 RT-PCR for surfactant protein A and inflammatory gene expression 

The A549 human lung cells were seeded in 6 well plates at a cell density of 6 x 10 5 
cells/well cells/well. Following seeding and overnight incubation, the cells were treated 
with 20 pg/mL of GNRs, while untreated cells served as a negative control. Following 8 
and 24 h of exposure, RNA was isolated using the RNeasy Mini Kit from Qiagen 
according to the manufacturer’s protocols. The RNA quantity and purity was assessed 
using a NanoDrop 1000 and 1 pg of RNA was converted to cDNA using the High- 
Capacity cDNA Reverse Transcription Kit (Life Technologies, Grand Island, NY). The 
cDNA was then used in subsequent TaqMan® PCR assays to determine changes in gene 
expression for SPA1,11-6,11-6 and TNF -a and, while Hprtl was used as a housekeeping 
gene to normalize expression changes. The gene expression was presented as a fold 
change determined using the 2AAC(T) method (Livak & Schmittgen, 2001) and 
represents three independent trials with the average ± the standard error reported. 

3.9 Human cytokine immunoassay 


5 



Secreted inflammatory proteins following GNR exposure were evaluated using the Bio- 
Plex Pro Human Cytokine 8-Plex Immunoassay (BIO-RAD, Hercules, CA). The 
multiplex assay detects the following cytokines: GM-CSF, IFN-y, IL-2, IL-4, IL-6, IL-8, 
IL-10 and TNF-a. A549 cells were seeded in a 6-well plate at 6 x 10 5 cells/well for 24 h 
then exposed to GNRs (20 pg/mL) for 8 h. Next, the cells were washed three times with 
wann PBS, media was replaced and cells were incubated for 16 h. The media was then 
collected and cytokine concentration was determined in accordance with the 
manufacturer’s protocol. Results represent three independent trials with the average ± the 
standard error reported. 

3.10 Quantification of cellular uptake of GNRs by ICP-MS 

A total of 1 x 10 5 cells/well were seeded on 12mm diameter glass slides in a 24-well plate 
in triplicate then dosed with 5 pg/mL GNRs for 24 h. The cell samples were then washed 
three times with warm PBS and digested with an aqueous solution containing 0.05% 
Triton X-100, 3% HC1, and 1% HNO 3 . For GNR retention study the cell samples were 
then washed three times after 24 h post-exposure with wann PBS and media was replaced 
and repeated on day 4 and day 8. The intracellular gold concentration was determined 
through inductively coupled plasma mass spectrometry (ICP-MS) on a Perkin-Ehner 
ICP-MS 300D instrument (Santa Clara, CA). ICP-MS was conducted in standard mode 
with 20 sweeps per reading, at one reading per replicate, and three replicates per sample 
with a dwell time of 100 ms. A calibration curve was obtained using four gold standard 
solutions and the addition of an internal standard was done to ensure that no interferences 
were occurring. Results represent three independent trials with the average ± the standard 
error reported. 

3.11 Darkfield microscopy 

A549 human lung cells were seeded at 1.25 x 10 5 cells per chamber on a 2-well 
chambered slide and grown for 24 h. The following day the cells were dosed with 20 
pg/mL GNRs for 24 h. After 24 h, the cells were fixed with 4% parafonnaldehyde and 
incubated with Alexa Fluor 555-phalloidin for actin staining and DAPI for nuclear 
staining (Life Technologies, Grand Island, NY). The slides were then sealed and imaged 
using a CytoViva 150 ultra resolution attachment on an Olympus BX41 microscope 
(Aetos Technologies, Opelika, AL). All experiments were performed at a minimum of 
three times. Care was taken to ensure full evaluation of each slide for well represented 
images. 

3.12 Transmission electron microscopy 

A549 human lung cells were seeded in a 6-well plate at 6 x 10 5 cells/well for 24 h then 
exposed to the stated GNRs concentration (5 and 20 pg/mL) and washed three times with 
wann PBS. The cells then fixed overnight in 2% paraformaldehyde and 2% 
glutaraldehyde after indicated duration (24 h, 4 days or 8 days). The cells were then 
stained with 1% osmium tetroxide, washed, and subsequently dehydrated with ethanol 
dilutions ranging from 50 to 100%. The cells were then embedded in LR White resin and 
cured overnight at 60 °C under a vacuum, after which the samples were sectioned using a 
Leica EM UC7 Ultramicrotome. Cell sections of 70 mn in thickness were placed on a 
Formvar carbon coated copper TEM grid (Electron Microscopy Sciences, Hatfield, PA) 


6 



and were imaged. Transmission electron microscopy (TEM) was performed using a 
Hitachi H-7600 with an accelerating voltage of 120kV. All experiments were perfonned 
at minimum three times. Care was taken to ensure full evaluation of each sectioned 
sample for well represented images. 

3.13 Hyperspectral microscopy 

A549 human lung cells were seeded at 1.25 x 10 5 cells per chamber on a 2-well 
chambered slide and grown for 24 h and the following day was exposed to GNR 
(20ug/ml). After 24 h, the cells were fixed with 4% paraformaldehyde. The slides were 
then sealed and imaged using a CytoViva Hyperspectral Imaging System (Auburn, AL). 
Image capture times and setting remained constant for all samples. Finally, hyperspectral 
analysis was perfonned using CytoViva’s hyperspectral image analysis software. Results 
represent three independent trials with the average ± the standard error reported. Care 
was taken to ensure full evaluation of each slide for well represented images. 

3.14 Two-photon luminescence microscopy 

A549 cells were seeded at 1.25 x 10 5 cells per chamber on a 2-well chambered slide and 
grown for 24 h and the following day was exposed to GNR (20pg/ml). After 24 h, the 
cells were fixed with 4% paraformaldehyde. The slides were then sealed and imaged 
using an Olympus fvlOO multi-photon confocal microscopy Imaging System with a 25x 
(NA 1.05) water immersion objective (Center Valley, PA). Ti-sapphire laser (Mia-Tai 
laser, Spectra-Physics, Mountain View, CA) at 810 mn set at 0.5% transmissivity was 
used as excitation light source and a two-photon luminescence emission was detected by 
a photomultiplier tube with an ET660/40m-2p (640-680 mn) band pass filter (Chroma 
Technology, Bellows Falls, VT, USA). Cells were imaged at four times zoom and 0.15 
pm/slices though the cells were captured. Image capture setting remained constant for all 
samples. Finally, image processing and intensity analysis was perfonned using 
Olympus’s Fluoview (Center Valley, PA) image analysis software. Extracellular MTAB 
GNRs were used as an image intensity control. Results represent two independent trials 
(minimum of 8 replicates per trial) with the average ± the standard error reported. 

3.15 Plasmonic photo-thermal cells ablation 

A549 cells were seeded in a 6-well plate at 6 x 10 5 cells/well for 24 h then exposed to 
GNRs (20 pg/mL) and washed three times with warm PBS. Cells were then exposed to 
the cell-permeant calcein AM (2 pM) (Life Technologies, Grand Island, NY) and the 
non-cell-permeant ethidium homodimer-1 (4 pM) (Life Technologies, Grand Island, NY) 
and after 15 min cells were irradiated with an 810nm 3W Ti-sapphire laser (Mia-Tai 
laser, Spectra-Physics, Mountain View, CA) for 60 sweeps at indicated power level. 
Cellular ablation was measured at the 0, 1,5, 10 min time points. Results represent four 
independent trial with the average reported. Care was taken to ensure full evaluation of 
each slide for well represented images. 


3.16 Statistical analysis 

All experimental results represent a minimum of three independent trials unless otherwise 
stated. Data were expressed as the mean ± the standard error of the mean (SEM). 


7 



Statistical calculations were performed using SAS (Version 9.1) or GraphPad Prism 
(version 5.02, GraphPad Software Inc. La Jolla, CA, USA) to determine statistical 
significance at p values of <0.05 (*), <0.01 (**) , or <0.001 (***). 


4.0 RESULTS AND DISCUSSION 

4.1 GNR characterization 

GNR characterization was performed to determine their key physicochemical properties 
and to verify particle uniformity prior to experiments. TEM images demonstrated that 
GNR sets were unifonn in size and morphology (Figure 1 and Table 1). The AR 4 GNRs 
had a diameter 24.5 ±1.1 mn and a length of 104 ±1.2 mn on average. UV-Vis analysis 
confirmed predicted SPR peaks based on calculated AR (Figure ID & H) (Jun et al., 
2008). To determine GNR surface charge, zeta potential analysis was performed on each 
particle (Table 1). From this analysis, it was shown that MTAB GNRs were positively 
charged as expected due to MTABs quaternary ammonium cation. MTAB-TA GNRs 
displayed a negative surface charge, indicating that functionalization with TA was 
successful. When the GNRs were exposed to a protein rich environment (culture media) 
both MTAB and MTAB-TA GNRs displayed a negative surface charge, -15.5 and -18.1 
mV respectively. Hydrodynamic size of GNRs in culture media showed that TA coated 
GNRs were on average larger than MTAB GNRs. 






inoLOOioounotno 

■^■LfiincOCONNCOCOO) 

Wavelength (nm) 


9 


950 
















TTinintocoNNcocooiO) 

Wavelength (nm) 


Figure 1. GNR characterization. 

Representative TEM images of A. MTAB, B. MTAB-TA and C. Silica; D. UY-Vis 
absorption spectra of MTAB (gold), MTAB-TA (light blue) and Silica (pink) GNRs. 
Representative TEM images of E. CTAB F. CTAB-TA and G. PEG. H. UV-Vis 
absorption spectra of CTAB (red), CTAB-TA (dark blue) and PEG (green) GNRs. 


Table 1. Characterization of GNRs 


Name 

Primary 

Size (nm) 

Aspect 

Raito 

Surface 

Chemistry 

Surface 
Charge (mV) 

Hydrodynamic 
Diameter 
in Media (nm) 

MTAB 

25x102 ±4.0 

4.1 

MTAB 

36.0 

288.2 ±9.5 

MTAB-TA 

25x104 ±2.8 

4.1 

MTAB-TA 

-15.7 

509.6 ±66.9 

Silica 

25x106 ±3.0 

4.2 

Mesoporous 

Silica 

11.6 

455.8 ±11.3 


10 

























CTAB 

25x72 ±3.1 

2.9 

CTAB 

37.2 

318.7 ±4.3 

CTAB-TA 

22x60 ±2.1 

2.8 

CTAB-TA 

-19.4 

416.4 ±35.2 

PEG 

19x48 ±2.6 

2.5 

PEG 

2.1 

75.2 ±0.9 


4.2 MTAB-TA GNRs display enhanced biocompatibility 

Since GNRs are kn own to exhibit toxicity linked to their physiochemical properties, we 
compared the biocompatibility of CTAB and MTAB GNRs with and without TA coating 
by evaluating membrane integrity and mitochondrial function (Figure 2). Overcoating 
CTAB and MTAB GNRs with TA resulted in enhanced biocompatibility, with no 
significant decrease in viability with exposure to MTAB-TA GNRs concentrations as 
high as 320 pg/mL (Figure 2B). Following exposure to MTAB and MTAB-TA GNRs 
(20 ug/ml), A549 cells showed no significant effects on their cellular viability with 
viability remaining over 95% after 24 h and 48 h (Figure 2 A & C). The viability data 
demonstrated that the reported toxicity of CTAB-TA was not due to the TA over-coating, 
negative surface charge or the AR of these GNRs, suggesting that the residual CTAB 
bilayer was the cause of the toxicity (Alkilany et ah, 2009). 

Next, cellular stress was examined by measuring changes in reactive oxygen species 
(ROS) after cellular exposure to the GNRs. The 6 h time point was chosen based on 
maximum ROS response before cell death. A549 cells showed no significant increase in 
ROS levels after exposure to MTAB and MTAB-TA GNRs, even at four times the 
treatment concentration of the CTAB GNRs that significantly increased ROS levels 
(Figure 2D). 

Finally, GNR surface chemistry has been shown to alter cellular response, and gene and 
protein expression analysis has been used to detect subtle changes after exposure to 
GNRs and other NMs (Grabinski et ah, 2011; Hauck, et ah, 2008b). Therefore, we 
evaluated the inflammatory response of exposure to MTAB and MTAB-TA GNRs in 
A549 human lung cells by measuring changes in surfactant protein A (SPA1) and 
inflammatory cytokine (SPA1, IL-6, IL-8 and TNF-a) mRNA expression and GM-CSF, 
IFN-y, IL-2, IL-4, IL-6, IL-8, IL-10 and TNF-a cytokine release. 

A549 cells showed no significant change in the measured mRNA (SPA1, IL-6, IL-8 and 
TNF -a) levels after 8 h and 24 h (Figure 3) exposure to MTAB and MTAB-TA GNRs 
(20ug/mL). However, a lower concentration of CTAB GNRs (2.5pg/mL) resulted in a 
significant increase of IL-6, IL-8 and Tnf-a mRNA after 8 h and SPA1, IL-8 and Tnf-a 
mRNA after 24 h. 

In addition there was no significant change in GM-CSF, IFN-y, IL-2, IL-4, IL-6, 
IL-8, IL-10 and TNF-a cytokine release after exposure to MTAB and MTAB-TA GNRs 
(20ug/mL). Overall cytokine release was low with only GM-CSF and IL-8 cytokines 
reaching concentrations greater than lpg/mL (Figure 4). Together these results 
demonstrated the high in vitro biocompatibility of both MTAB and MTAB-TA GNRs. 

In view of their biocompatibility based on these initial findings and their longitudinal 
SPR peaks in the NIR “water window”, we chose to further explore the cellular 
association and in vitro hyperspectral signature of MTAB GNRs with and without a TA 
coating. 


11 







"~5 10 20 40 80 160 320 

GNR Concentration (ug/mL) 




12 




















































































Figure 2. MTAB-TA GNRs demonstrate enhanced biocompatibility. 

A549 human lung cells were exposed to MTAB and MTAB-TA GNRs for 24 h or 48 h. 
Cell viability was assessed using LCDC or MTS assays and was represented relative to 
the control cells. A. LCDC assay showed no significant decrease in cell viability 
following exposure (20 pg/mL) for 24 h.. However, exposure to CTAB and CTAB-TA 
GNRs (20 pg/mL) resulted in a significant decrease in cell viability. B. 24 h MTS assay 
results indicated that the TA coating enhanced the biocompatibility of both CTAB and 
MTAB GNRs. MTAB -TA showed no significant decrease in viability at concentrations 
as high as 320 pg/mL C. 48 h MTS assay showed that the cytotoxicity of CTAB-TA 
GNRs (20 pg/mL) significantly increases over time whereas there was no significant 
decrease in viability after exposure to MTAB and MTAB-TA GNRs. D. ROS assay 
demonstrated no significant increase in ROS after exposure to CTAB-TA, MTAB and 
MTAB-TA GNRs. Taken together, these results demonstrate the high biocompatibility of 
both MTAB and MTAB-TA GNRs. Statistical significance was determined using a one 
way ANOVA with Dunnett’s post-hoc tests. 



13 










































E 


IL-8 


F 


IL-8 



Figure 3. MTAB-TA GNRs do not alter inflammatory gene expression. 

A549 human lung cells were exposed to GNRs for 8 h and inflammatory gene expression 
was assessed at 8 h and 24 h using RT-PCR. MTAB and MTAB-TA GNRs (20ug/mL) 
resulted in no significant change in A-B. SPA1, C-D. IL-6, E-F. IL-8 and G-H. TNF -a 
mRNA levels after 8 h or 24 h. In contrast, CTAB GNRs (2.5ug/mL) resulted in a 
significant increase of IL-6, IL-8 and TNF -a mRNA after 8 h and SPA1, IL-8 and TNF - 
a mRNA after 24 h in A549 human lung cells. Statistical significance was determined 
using a one-way ANOVA with a tukey post-hoc tests. 


14 

































Figure 4. MTAB-TA GNRs do not alter cytokine release. 

A549 human lung cells were exposed to GNRs for 8 h and inflammatory gene expression 
was assessed at 24 h using a multiplex bead-based flow cytometric immunoassay. MTAB 
and MTAB-TA GNR exposures (20ug/mL) resulted in no significant change in released 
A) 1L-8 and B) GM-CSF protein levels. Exposure to CTAB GNRs (2.5pg/mL) resulted in 
a significant increase of released 1L-8 and GM-CSF protein in A549 human lung cells. 
Statistical significance was determined using a one-way ANOVA with a tukey post-hoc 
tests. 


4.3 Cellular association and in vitro intracellular hyperspectral signature of MTAB-TA 
GNRs 

Visualization of cellular association of MTAB and MTAB-TA GNRs was examined 
using darkfield microscopy (Figure 5A-C). Both GNRs interacted with A549 cells and 
had a high level of cellular association. MTAB and MTAB-TA GNRs appeared to be 
densely packed, suggesting the associated and/or internalized GNRs are in clusters. In 
addition the morphology of A549 cells were retained, further confirming the 
biocompatibility of the GNRs. 

Next, we performed in vitro hyperspectral imaging (HSI) microscopy to investigate the 
GNR optical properties after cellular association. HSI combines the use of darkfield 
microscopy and spectroscopy for the measurement of the reflectance spectrum at 
individual pixels in a micrograph. HSI analysis has successfully been used for 
characterizing gold nanoparticle aggregation, protein adsorption and cell uptake in 
biological/cellular environments (Grabinski et ah, 2013). This in vitro analysis of MTAB 
and MTAB-TA GNRs is critical, as many studies have shown that biological/cellular 
environments can alter the optical properties of GNRs and other nanomaterials. HSI 
analysis demonstrated that both GNRs had a strong association to A549 cells and 
appeared to indicate clustering of both MTAB and MTAB-TA GNRs (Figure 5D-E). 
However, the MTAB GNRs displayed a vast array of colors when compared to the 
primarily white appearance of the MTAB GNRs, indicating a shift out of the NIR to the 
visible spectra. Further, MTAB-TA GNRs have a more uniform clustering that allows for 
easier identification and delineation of the GNR clusters. Next, the reflectance spectrum 
of individual GNR clusters were measured and compiled to create a hyperspectral profile 


15 


























for both MTAB and MTAB-TA GNRs (Figure 5G). The hyperspectral analysis revealed 
that the spectral maximas were decreased in both hyperspectral profiles, as compared to 
their spectral profiles as synthesized (Figure 13). This blue shift in spectral maximas 
could have resulted from GNR intracellular aggregation with side-by-side assembly (Jain 
et ah, 2006; Stacy et ah, 2013). This would effectively lower the GNR AR resulting in 
the changes seen in hyperspectral profile. The hyperspectral profile of MTAB-TA GNRs 
(n=385) revealed a sharper peak still within the NIR target “water window” (—732 nm). 
In contrast, the hyperspectral profiles of MTAB GNRs (n=194) and control (n=335) 
displayed broad peaks that were located primarily outside the target NIR window (-630- 
679 nm and -550-634 nm, respectively). In addition, MTAB-TA GNRs demonstrated a 
greater than 2.5 fold increase in scattering intensity after cellular association over the 
MTAB GNRs. Together, these results suggest that the MTAB-TA GNR form unifonn 
GNR clusters that are able to preserve their NIR optical properties after cellular uptake. 
This finding is significant for nano-based bio-imaging and therapeutic applications as a 
strong spectral profile in the NIR “water window” after exposure to biological/cellular 
environments is required for optimum efficacy in biomedical applications. 

One possible explanation of the differences in hyperspectral signature may be due to 
differences in GNR uptake. Another possible explanation could be due to differences in 
aggregation states after cellular association and/or internalization (Aaron et ah, 2009). To 
test these possibilities, we set out to examine MTAB and MTAB-TA GNR uptake by 
Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and intracellular aggregation 
states by TEM. 


16 




17 



Wavelength (nm) 

Figure 5. Intracellular MTAB-TA GNRs retain NIR optical properties. 

Representative darkfield and hyperspectral images of A549 cells with analysis of 
intracellular GNR optical properties. Fluorescent images following 24 h exposure to 
GNR (20 pg/mL) A. Control, B. MTAB and C. MTAB-TA. Fluorescent images 
illustrate clustering of GNRs with the morphology of A549 cells retained. A549 cells 
underwent actin (red) and nuclear (blue) staining with GNRs (reflecting white). 
Hyperspectral images following 24 h exposure to GNR (20 ug/mL) D. Control, E. 

MTAB and F. MTAB-TA. Results revealed that MTAB-TA presented a more uniform 
clustering and spectral profile based on their appearance in the hyperspectral images. G. 
Analysis of in vivo optical properties. MTAB hyperspectral profile (red) (n=194) 
displayed low intensity and loss of NIR optical properties of GNRs. In contrast, the 
MTAB-TA hyperspectral profile (green) (n=385) showed preserved NIR properties with 
intensity greater than 2.5 times that of the MTAB hyperspectral profile. Control (blue) 
(n=335). 

4.4 High uptake with unique clustering pattern of MTAB-TA GNRs 
Quantification of cellular uptake in the MTAB and MTAB-TA GNRs (5 pg/ml) was 
determined by ICP-MS. Both MTAB and MTAB-TA GNRs showed a high level (40% 
and 39% of treatment respectively) of cellular uptake compared to 1.5% with PEG GNRs 
or 15% with silica coated GNRs (Figure 6A). These results are similar to the 40% uptake 
of MTAB GNRs in MCF-7 cells reported by Vigderman et al (Vigderman et ah, 2012b). 
In addition, this finding suggests that the TA over-coating negative surface charge has 
minimal impact on cellular uptake of MTAB-TA GNRs. These findings appear to differ 
from other studies that report that GNRs with a positive surface charge have a greater 
cellular association and uptake than GNRs with a negative surface charge (Hauck et al., 
2008a; Qiu et al., 2010). It was originally proposed that the positive surface charge on 
the GNRs was attracted to the negatively charged membrane of the cell resulting in 
higher GNR cellular association (Hauck et al., 2008a). However, when GNRs of 


18 




















differing charge are placed in a biological environment (or simulated biological 
environment such as culture media) the GNRs will take on the charge of the biological 
environment (Qiu et ah, 2010). This is in agreement with our finding that both MTAB 
and MTAB-TA GNRs displayed a negative surface charge, -15.5 and -18.1 mV, 
respectively. It has been more recently proposed that the greater uptake levels seen with 
GNRs with a positive surface charge is because of their greater affinity for protein and 
formation of a protein corona that strongly influences the GNRs cellular uptake (Nel et 
al., 2009; Qiu et al., 2010; Walkey & Chan, 2012; Walkey et al., 2014). TA has a strong 
attraction for protein and cellular membranes that has been well documented (Van Buren 
& Robinson, 1969; Wagner, 1976). In addition, it has been reported that TA coated gold 
NMs have strong cellular association and unique form of cellular uptake (Mukhopadhyay 
et al., 2012; Untener et al., 2013). Therefore the MTAB-TA GNRs may fonn a 
distinctive protein corona that may account for their uptake properties; thus, further 
research on the impact of the protein corona of TA GNRs and other NM is needed. The 
finding that there is no significant difference in uptake of the two GNRs further suggests 
that the difference seen in hyperspectral profiles is not significantly impacted by 
differences in cellular uptake of the two GNRs. 

The concentration of GNR remained at that level for up to 8 days post exposure with 93% 
and 90% retention of the MTAB and MTAB-TA GNRs, respectively (Figure 6B). This 
suggests that there is minimal exocytosis of the GNRs and/or high reuptake of 
exocytosed GNRs as supported by the previous finding on GNR trafficking (Zhang, W. et 
al., 2013a). Therefore, we investigated the intracellular state of the GNRs, as previous 
studies have shown that the aggregation state of GNRs can affect their spectral profiles 
(Kelly et al., 2003; Sosa et al., 2003). 

The distribution of MTAB and MTAB-TA GNRs (5 pg/ml) within the cell was 
observed using TEM (Figure 7). MTAB-TA GNRs demonstrated low aggregation of 
GNRs with a unique distribution pattern/clusters within the A549 cells. In contrast, 
MTAB GNRs appeared aggregated in dense clusters and/or tightly packed crest shape 
groupings. This suggests aggregation of the MTAB GNRs in the cell may account for the 
differences seen in the two GNRs spectral profiles after cellular association. 


19 




MTAB MT AB-T A 

Figure 6. Uptake and retention of MTAB-TA GNRs 

A549 human lung cells were exposed to GNRs (5 jag/m L) for 24 h, GNR uptake was 
quantified using ICP-MS. A. Results show high level of uptake of 40% for MTAB and 
39% for MTAB-TA GNRs compared to PEG (1.5%) and Silica (15%) GNRs. B. After 8 
days post exposure 93% and 90% of the MTAB and MTAB-TA GNRs were retained, 
respectively. Statistical significance was determined using a one-way ANOVA with a 
Tukey post-hoc tests. 


20 

























































Figure 7. Visualization of MTAB-TA GNR uptake. 

Representative TEM Images of A549 cells after 24 h GNR exposure (5pg/ml). A. MTAB 
B. MTAB-TA C. Control. Results demonstrated intracellular aggregation of MTAB 
GNRs and low aggregation of MTAB-TA GNRs. 


4.5 Low intracellular aggregation of MTAB-TA GNRs. 

Since concentration can influence nanoparticle aggregation, we studied this cellular 
patterning at 20 pg/ml. At this new concentration, MTAB GNRs displayed small and 
large tightly packed clusters (Figure 8 A1,A4), crest (Figure 8 A3,5,6) and doughnut 
(Figure 8 A2) shaped groupings with aggregation of GNRs. MTAB-TA GNRs again 
displayed distinctive GNR cluster patterns with low aggregation of GNRs (Figure 8 B). 
Most GNRs are taken up through receptor-mediated endocytosis and further trafficked 
via an endo-lysosomal pathway (Chithrani et ah, 2009). Further, it appears that the 
MTAB-TA GNR clusters are less compressed/density packed than the MTAB GNR 
groupings. After 8 day post exposure the number of GNR clusters/groupings per cell in 
both the MTAB and MTAB-TA exposed cells decreased possibly due to consolidation of 
the GNR groupings and by cell division (Figure 9). However, there is only a slight 
decrease in the total amount of GNRs present in the sample (Figure 6B). This supports 
the finding of Zhang et al (2013 a) that demonstrated the long tenn retention of gold 
nanoparticles in NDA-MB-231 breast cancer cells with the concentration of GNRs in 
cells correlating with their rate of cellular division rather than exocytosis. 


21 








Figure 8. Visualization of intracellular clustering pattern of MTAB-TA GNRs. 

Representative TEM Images of A549 cells after 24 h GNR exposure (20ug/ml). A. 
MTAB and B. MTAB-TA GNRs. Results demonstrate intracellular aggregation of 
MTAB GNRs and low aggregation and unique clustering of MTAB GNRs. 


22 






Figure 9. Visualization of MTAB-TA GNRs cellular retention. 

Representative TEM Images of A549 cells 8 day post exposure after 24 h GNR exposure 
(20ug/ml). Representative images of A. MTAB and B. MTAB-TA GNRs 8 days post 
exposure. Results show a decrease in the number of GNR clusters/groupings per cell in 
both the MTAB and MTAB-TA exposed cells. 

Next, we analyzed the GNRs clusters/groupings with ImageJ software (Schneider 
et ah, 2012) (Figure 10). The MTAB GNR groupings (n=28) were more densely packed 
than the MTAB-TA GNR groupings (n=24) as reflected in the area fraction of GNRs 
over total area of clusters/groupings values of 64% vs 50%, respectively (p<0.001). In 
addition, the MTAB GNR groupings had a smaller average diameter (calculated as the 
mean of smallest and largest diameter measurement for each grouping) than the average 
MTAB-TA GNR cluster at 343 nm vs 534 nm, respectively (p<0.001). The total area of 
the MTAB-TA GNR clusters, as measured by ImageJ, appears to trend larger; however, 
the difference was determined not to be statistically significant (p=0.099). Taken 
together, these results indicate that the differences in the intracellular pattern may account 
for the preserved NIR spectral profiles of the MTAB-TA GNR clusters. Further, we 
examined the average diameter of extracellular GNR clusters in the TEM image with 
ImageJ. Results indicated that the diameter of extracellular MTAB-TA GNR clusters was 
larger than MTAB GNR clusters, 310 nm vs 165 nm, respectively (Figure 10 F). Based 
on these finding, we tested if these differences in the intracellular MTAB and MTAB-TA 
GNR clusters spectral profiles had any beneficial effect with nano-based biomedical 
applications, specifically two-photon luminescence microscopy and photo-thennal 
cellular ablation. 


23 




★ ★★ 


A. _ C. 



Intracellular GNR Clusters 



24 


Diameter (nm) GNR Cluster Area (urn 2 ) 


D. 



Intracellulaer GNR clusters 



Extracellular GNR Clusters 




Figure 10. Analysis of intracellular MTAB-TA GNR clusters. 

Representative images of intracellular clusters in A549 cells after 24 h GNR exposure 
(20pg/ml) and clusters analysis using ImageJ software. A. MTAB B. MTAB-TA C. 
Average intracellular cluster density D. Average intracellular cluster area E. Average 
intracellular cluster diameter F. Average extracellular clusters diameter. Results 
demonstrated that MTAB-TA GNR clusters (n=24) are on average less densely packed 
(50% vs 64%) and have a large average diameter (534 mn vs 343 nm) compared to 
MTAB GNR groupings (n=28) in A549 cells. However, there was no statistically 
significant difference in the average intracellular cluster area, 70 ±16 pm 2 (MTAB) vs 
111±19 pm 2 (MTAB-TA). The average diameter of extracellular MTAB-TA GNR 
clusters were larger than the average diameter of extracellular MTAB GNR clusters, 310 
nm vs 165 nm, respectively. Statistical significance was determined using t-tests. 

4.6 MTAB-TA GNRs exhibit superior two-photon luminescence image intensity 
The use of GNRs has been described in a variety of bio-imaging modalities (Boca & 
Astilean, 2010; Eghtedari et ah, 2007; von Maltzahn et ah, 2009; Wang et ah, 2005; T. 
Wang et ah, 2013b). Of all these, nano-based bio-imaging two-photon luminescence 
imaging has been shown to provide the highest contrast and spatial resolution (Tong et 
al., 2009). Therefore, two-photon luminescence imaging was the focus of our bio¬ 
imaging studies. A strong two-photon luminescence intensity essential for bio-imaging as 
it allows for deeper imaging in tissues with less power. 

Two-photon luminescence microscopy was used to capture 3-D images of A549 human 
lung cells that were exposed to GNRs (20 pg/mL) for 24 h. Next, we analyzed the 
intracellular MTAB and MTAB-TA GNR clusters with Fluoview software (Olympus, 
Pittsburgh, PA). The 3-D images were converted into 2-D images (Figure 11A-B) and the 
average intensity of MTAB and MTAB-TA GNR clusters was determined. Results 
demonstrated that intracellular MTAB-TA GNRs produced stronger two-photon 
luminescence intensity (798 ± 40.9 AU (161% of control)) when compared to 
intracellular MTAB (546 ± 25.1 AU (111% of control)). This indicated that the TA 
coating was able to reduce intracellular aggregation, protecting the GNRs key optical 
properties and NIR spectral signature, and in turn enhancing the GNRs bio-imaging 
capabilities. 


25 



A 

4 

• • • w 

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* 

V 

0 




10 [im 

B ■* 


•:*i£ 

1L 

# 

* Jjjw 

9 ST 

> 

9 

* • 

■ 1000 

1 f 

10 nm 

_ Si 

c 


0 




*** 



Figure 11. MTAB-TA GNRs display greater two-photon luminescence intensity. 

Representative images of A549 cells after GNR exposure (20pg/ml) for 24 h A. MTAB 
B. MTAB-TA GNRs. C. Average image intensity D. Average image intensity (% of 
control). Results demonstrated, on average, that the image intensity for cells with MTAB- 
TA GNR (798 ± 40.9 AU and 161% of control) is greater than cells with MTAB (546 ± 
25.1 AU and 111% of control). Statistical significance was detennined using t-tests. 


4.7 MTAB-TA GNRs demonstrated higher efficiency for photo-thermal cellular ablation. 
The MTAB, MTAB-TA and silica GNRs all have spectral profiles that demonstrate 
longitudinal absorbance at approximately 810 mn. When these GNRs are exposed to 
corresponding 810 mn laser irradiation, the GNRs absorbs photons and this energy is 
converted to heat. This photothennal effect results in the GNRs becoming an extremely 
localized heat source that can be used to kill cancer cells via photo-thermal therapy 
(Cobley et al., 2011; Shanmugam et ah, 2014; Zhang et ah, 2012). The preserved spectral 
profile found in the intracellular MTAB-TA GNR suggests that they would be an 
excellent agent for photo-thermal therapy. To determine if the TA coating enhances the 
GNR photo-thermal properties in vitro, we used A549 adenocarcinomic human lung cells 
to evaluate the efficacy of MTAB and MTAB-TA GNR for photo-thermal cellular 
ablation. A549 human lung cells were exposed to the GNRs (20 ug/mL) for 24 h and 


26 























washed three times. Next, calcein AM (2 pM) and ethidium homodimer-1 (4 pM) was 
added to RPMI 1640 cell culture media without Phenol Red and incubated for 15 min. 
The cells were then irradiated with 60 sweeps of an 810nm 3W Ti-sapphire laser (30- 
75mW). Cellular ablation was measured at the 0, 1,5, 10 min post irradiation time points. 
Results show that MTAB-TA is an effective photo-thennal therapeutic agent with cell 
viability decreasing as the laser power was increased (Figure 12). MTAB-TA GNR 
demonstrated the highest level of cell death and therefore the greatest efficacy for photo- 
thennal cellular ablation compared to MTAB and silica GNRs (Figure 13 & 26). Photo- 
thennal cellular ablation with silica GNRs resulted in non-unifonn cell death in the 
irradiated field. This may be due to lower cellular uptake of silica GNRs (15%) compared 
to MTAB (40%) and MTAB-TA (39%) GNRs. Based on the cellular uptake results, the 
effective dose in the cell was approximately 3 pg/mL for the silica GNRs compared to 
approximately 8 pg/mL for MTAB and MTAB-TA GNRs. These photo-thermal cellular 
ablation results further confirms that the TA coating of GNRs preserves their optical 
properties and enhances their efficacy for photo-thennal applications. 


27 



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28 


Figure 12. MTAB-TA GNR shows efficiency as agent for photo-thermal therapy. 

Representative images of A549 cells after exposure to MTAB-TA GNRs (20ug/ml) for 
24 h. Results show A549 cells before and after exposure to NIR laser irradiation. Results 
show viability of MTAB-TA GNR treated cell decreasing as the laser power was 
increased. Results demonstrate that MTAB-TA GNRs are an effective photo-thermal 
therapeutic agent. 


29 



Functionalized GNRs 



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• ft • * 

• # • 


y 4P r j 

Silica 

•4 r | > j t 

ft 

«4 J # - • • 

•4 f t 

GNRs 

^ * ***+> 4 , * <g' % . 

• * 

' * ■ * ' 

/, * ^ I 

/. > f •* * *i 



ft • 

"uUftlftftT * f 

f 

.* 

Control 

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J "ft ft ^ 

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; ^5/flLfrV^ 


30 




Figure 13. Visual comparison of MTAB-TA GNRs efficiency for photo-thermal cellular 
ablation. 

Representative images of A549 cells after exposure to GNRs (20 ug/ml) for 24 h. Results show 
A549 cells with MTAB, MTAB-TA, or silica GNRs before and after exposure to NIR laser 
irradiation. Results show that MTAB-TA has the greatest efficiency for photo-thermal cellular 
ablation as illustrated by the decrease in cell viability. Silica GNRs resulted in non-unifonn 
killing of cells and GNR-free control showed no decrease in cell viability. 

A. |_jve Cell B. Dead Cell 




Time (Min) Time (Min) 

Figure 14. MTAB-TA GNRs demonstrate greatest efficiency for photo-thermal cellular 
ablation. 

A549 human lung cells were exposed to GNRs (20 ug/ml) for 24 h and irradiated with 60 sweeps 
of an 810nm Ti-sapphire laser (75mW), cell viability was assessed using A. Mean Calcein AM 
fluorescence B. Mean ethidium homodimer-lflouescence. Results shows that photo-thermal 
cellular ablation with MTAB-TA GNRs results in both a significant decrease in mean Calcein 
AM fluorescence and a significant increase in mean ethidium homodimer-1 fluorescence. This 
indicates a significant decrease in cell viability and significant increase in cell death and 
demonstrates MTAB-TA GNRS superior photo-thermal cellular ablation properties. Statistical 
significance was determined using t-tests at 10 min post exposure. 


5.0 CONCLUSIONS 

In this study, the GNR surface chemistry was modified by replacing CTAB with MTAB and 
over-coating with TA. This created a novel GNR (MTAB-TA) that formed unique clusters, 
showed no decrease in cellular viability, no indication of cellular stress and no alteration of cell 
morphology, confirming its enhanced biocompatibility. Further, in the A549 human lung cancer 
cell line, MTAB-TA GNRs demonstrated a cellular uptake rate 26 times greater than the 
commonly used PEG GNRs and 2.5 times greater than silica coated GNRs (Figure 5). This high 
uptake rate would enable a lower effective diagnostic and therapeutic working concentration. 

The MTAB-TA GNRs displayed unique intracellular distribution patterns that not only preserved 
their NIR optical properties within the water but also enhanced their spectral intensity greater 
than 2.5 times that of uncoated GNRs. This finding is critical for bio-applications because it 


31 









allows for the use of minimally invasive NIR lasers, higher resolution imaging and more 
effective therapies. Finally, we demonstrated that the MTAB-TA GNRs had the greatest efficacy 
for photo-thermal cellular ablation compared to MTAB and silica GNRs (Figure 13 & 14). 

In conclusion, this study has identified the complete replacement of CTAB with MTAB and the 
use of TA to overcoat and create at soft shell around the GNR, reducing GNR aggregation, 
protecting and preserving the GNRs NIR optical properties intracellularly (Figure 15). Based on 
their biocompatible nature, high rate of in vitro cell internalization and low intracellular 
aggregation, MTAB-TA GNRs are prime candidates for use in vivo experimentation, nano-based 
bio-imaging and photo-thermal applications. 


MTAB-TA GNRs 


r 

v* 



Low 
intracellular 
aggregation 



MTAB GNRs 


High 

intracellular 

aggregation 





Preserved 

optical 

properties 



Enhanced Bio-Imaging 
and Photo Thermal 
Properties 


««00 

,Z A 

{: /\ 

£ f \ 

O MO > 

: / V 

^' 

two 

1090 

£> '•*’ 

| (990 
c <wo 

9 MO 

f/X 

Wtwtongth (iwn) 


Ml M0U04I90MM1 

wy. . .... i-» 

\r■ ii| 


Degraded 

optical 

properties 


J 




Figure 15. MTAB-TA GNRs have enhanced bio-imaging and photo-thermal properties. 

MTAB and MTAB-TA GNRs displayed high in vitro biocompatibility and cellular uptake. 
However, after internalization by A549 human lung cancer cells, the MTAB GNRs displayed 
high intracellular aggregation. This resulted in a degradation of the GNRs optical properties. On 
the contrary, TA coated MTAB GNRs (MTAB-TA) displayed low intracellular aggregation and 
preserved NIR optical properties. This results in greater two-photon image intensity and photo- 
thermal cellular ablation making them ideal nano-based bio-imaging and photo-thermal 
applications. 


32 



















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37 



LIST OF ACRONYMS 


ATCC 

AR 

CTAB 

DLS 

FBS 

GNMs 

GNRs 

HIS 

ICP-MS 

LCDC 

MTAB 

MTS 

NIR 

NM 

PBS 

PEG 

ROS 

SPR 

TA 

TEM 

UV-Vis 


American Type Culture Collection 
Aspect Ratio 

Cetyl trimethylammonium Bromide 

Dynamic Light Scattering 

Fetal Bovine Serum 

Gold Nanomaterials 

Gold Nanorods 

Hyperspectral Imaging 

Inductively Coupled Plasma Mass Spectrometry 
Live Cell Dead Cell 

11-mercaptohexadecyl trimethylammonium Bromide 
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- 

(4-sulfophenyl)-2H-tetrazolium) 

Near-Infrared 
Nanomaterial 
Phosphate Buffered Saline 
Polyethylene glycol 
Oxygen Species 
Surface Plasmon Resonance 
Tannic Acid 

Transmission Electron Microscopy 
Ultra-Violet Visible Spectroscopy 


38