Controlled Bacteria - Gold Nanorod Interactions for Enhancement of Optoacoustic Contrast

Anton Liopoa, Paul J. Derryb, Boris Ermolinskyc, Richard Sua, André Conjusteaua, Sergey Ermilova, Eugene R. Zubarevb and Alexander Oraevskya

aTomoWave Laboratories Inc., Houston, TX, b Department of Chemistry, Rice University, Houston, TX cUniversity of Texas at Brownsville, TX

1. ABSTRACT

Gold-based contrast agents, gold nanorod (GNR), were designed for the enhancement of optoacoustic signal. After synthesis, the GNR-CTAB complexes were modified by pegylation (PEG), or replacement of CTAB (cetyl trimethylammonium bromide) with MTAB (16-mercaptohexadecyl trimethylammonium bromide) for coverage of gold nanorods with heparin (GNR-HP). Modified GNR are purified through centrifugation and filtration. GNR-CTAB can be used as a model of positively charged gold surface for quantitative optoacoustic sensing in GNR-bacteria interactions, whereas GNR-PEG and GNR-HP can be used as negatively charged gold surface models. We studied controlled agglomeration of contrast agents with the bacteria E.Coli and Vibrio Cholerae. For bacterial sensing, the localized plasmon resonance peak shifts as a function of electrostatic binding, which was detected with two different wavelengths through 3D optoacoustic imaging.

Key words: CTAB, PEG, MTAB, gold nanorods, optoacoustic imaging and sensing, bacteria particle targeting

2. INTRODUCTION

The threat of contaminants such as bacterial, viral, and chemical toxins has long been recognized as a serious public health concern. Foodborne disease has been a persistent and serious threat to public health. During 2011, 47.8 million illnesses caused by foodborne diseases were reported to the CDC. Of those cases, 128,000 resulted in hospitalization and just over 3,000 people died. (CDC 2011*) The most popular detection methods are cell culture and colony counting methods, polymerase chain reaction (PCR), and immunology-based or two-photon Rayleigh scattering methods and biosensors [1-4]. However, these techniques are labor intensive and time consuming often requiring professional operation optically clean media. An alternative method, optoacoustic detection, can be used in optically turbid media with greater sensitivity than existing methodologies such as colorimetric reactions and fluorescence [5]. Furthermore, it is the only viable method of detection when studying heavily light-scattering samples such as milk or ground water. The principle of optoacoustic detection relies on the occurrence of thermal confinement which happens when the laser pulse duration is small compared to the transit time of sound through the penetration depth of the light. In this case, instantaneous heating of the medium can be assumed. The interaction of nanoparticles with biological systems ranging from biomolecules to biological cells is significant for a range of applications such as high-resolution biomedical imaging, gene sequencing for molecular diagnostics, and sensitive electronic devices. [3, 6-8]. Noble metal nanostructures generate significant interest because of their unique properties, including large optical field enhancements resulting in the strong scattering and absorption of light. The absorption spectra of GNRs exhibit two surface plasmon absorption bands, the transverse and the longitudinal surface plasmon resonance (LSPR). The latter is very sensitive to a large number of factors and is often utilized in sensing applications [9]. GNRs have also attracted great interest as a novel platform for nanobiotechnology and biomedicine because of convenient surface bioconjugation with molecular probes and remarkable optical properties related with the LSPR [10-13]. GNRs of various size and aspect ratio are promising for biomedical applications [9,10,14-19]. GNRs can absorb light about thousand times more strongly than an equivalent volume of an organic dye [20,21]. In addition, GNRs are ideal optoacoustic contrast agents because their

Photons Plus Ultrasound: Imaging and Sensing 2014, edited by Alexander A. Oraevsky, Lihong V. Wang, Proc. of SPIE Vol. 8943, 894368 · © 2014 SPIE · CCC code: 1605-7422/14/$18 · doi: 10.1117/12.2044628

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Downloaded From: http://proceedings.spiedigitallibrary.org/ on 09/02/2014 Terms of Use: http://spiedl.org/terms optical absorption spectra can be tuned over a broad wavelength range in the near infrared (NIR) spectral region [22-25] and detecting the acoustic waves resulting from the optoacoustic effect [21]. It was previously demonstrated that positively charged GNRs stabilized by CTAB are effective for self-assembling into an electrically percolating monolayer of different nanoshapes on gram-positive bacterium, such as Bacillus cereus [26]. In this report we found that CTAB-capped GNRs can successfully be deposited on gram-negative bacteria, such as E. coli and V. cholerae and this effect has significant implications for optoacoustic sensing. * http://www.cdc.gov/foodborneburden/PDFs/FACTSHEET_A_FINDINGS_updated4-13.pdf

3. MATERIALS AND METHODS

Fabrication of Gold Nanorods (GNRs) We previously described a general strategy for the synthesis and stabilization of GNRs with thiol-terminal polyethylene glycol (mPEG-thiol, or PEG in this report), which displaces the original CTAB surfactant bilayer to provide biocompatibility of the resulting optoacoustic contrast agent [10,12,27-29]. To produce charged, biocompatible GNRs we used a thiolated analogue of CTAB, mercapto-cetyltrimethylammonium bromide (MTAB) as reported by Zubarev in 2012 [10]. These MTAB-GNRs have dramatically lower cytotoxicity; however, they exhibit poor stability in PBS and other high ionic strength buffers. To resolve this problem, we report the preparation of a new heparin-functionalized MTAB-GNRs with significantly improved saline stability. Presented below are the details of our GNR modification protocol adapted from previously reported methodologies [10,27,30,31].

CTAB-GNR Fabrication In a typical synthesis, gold seed particles were prepared by adding 0.250 mL of an aqueous 0.01 M solution of HAuCl4 • 3H2O to 7.5 mL of a 0.1 M CTAB solution in a 15 mL test tube. Afterwards, 0.600 mL of an ice-cold, aqueous 0.01 M NaBH4 solution was added all at once. This seed solution was used 2-4 h after its preparation. Next, a growth solution was prepared consisting of 4.75 mL 0.10 M CTAB, 0.200 mL of 0.01 M HAuCl4 • 3H2O, and 0.030 mL of 0.01 M AgNO3 solutions added in sequence and gently mixed by inversion. The solution at this stage had a bright orange color. Next, 0.032 mL of 0.10 M ascorbic acid was added to the Au(III)/CTAB solution and the combined solution became colorless upon mixing. After 10 min, a solution of gold seed nanoparticles was added to the growth solution and gently mixed for 10 s and left undisturbed for 1-3 h. Afterwards, the solution was left under thermostatic conditions for 24 hours at 30°C. Prior to surface functionalization with mPEG-SH or MTAB and heparin, the CTAB-GNRs were centrifuged at 1000 g for 10 min to separate aggregates. The pellet was removed and only the supernatant fraction was retained.

Surface Modification of CTAB-GNRs with mPEG-SH PEGylated-GNRs [27,32] were prepared using previously synthesized CTAB coated GNRs. The GNRs were centrifuged at 14000 g for 10 min and resuspended in 9 mL milli-Q (18.0 MΩ) H2O (optical density ~ 1.0). Next, 0.1 mL of 2 mM K2CO3 was added to 1 mL of aqueous GNR solution and 0.1 mL of 0.1 mM mPEG-SH-5000 (Laysan Bio Inc., Arab, AL). The resulting mixture was kept on a rocking platform at room temperature overnight. Excess mPEG-SH was removed from solution by two rounds of centrifugation (12000×g 10 min) and resuspended in a NaCl solution (pH 7.4) in final concentration 0.5 nM (OD near 2.0). UV-vis extinction spectra were measured by Thermo Scientific Evolution 201 spectrophotometer.

Surface Functionalization of CTAB-GNRs with MTAB In a typical synthesis, 40 mL of 0.25 mg/mL CTAB-GNRs, (Liz-Marzan 2011) were centrifuged at 12,000 g for 10 min and resuspended in 40 mL of milli-Q (18.0 MΩ) H2O. In a separate vial, 20 mg of previously prepared MTAB [10] was dissolved in 2 mL milli-Q H2O by gentle heating and then added at once to the CTAB-GNR solution. The solution of CTAB-GNRs was incubated overnight at 30°C and then centrifuged at 12,000 g for 10 min and redispersed in 40 mL milli-Q H2O six times.

Secondary Modification of MTAB-GNRs by Heparin Sulfate 20 mg porcine intestine-derived heparin sulfate (HS) (Sigma-Aldrich, St. Louis, MO) was dissolved in 2 mL milli-Q H2O and then added to 10 mL of MTAB-GNR stock. The combined solution was mixed briefly and incubated at 30°C for 1 h. The HS modified MTAB-GNR solution was then centrifuged four times at 12,000 g for 10 min. ζ = -50.9 mV.

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Downloaded From: http://proceedings.spiedigitallibrary.org/ on 09/02/2014 Terms of Use: http://spiedl.org/terms Bacterial Cell Culturing Vibrio cholerae classical strain 0395 and E. coli K802 were grown in a Luria-Bertani (LB) broth at 37°C with shaking (200 rpm) overnight. Each bacterial strain was diluted to 1:100 in 3 mL of fresh LB and grown until they reached the mid-logarithmic phase of growth (optical density at 600 nm [OD600] about 0.6). Bacterial cultures were centrifuged at 1500 × g for 5 min to separate cells from supernatant. Incubation both bacteria with modified GNR in final concentration 0.25 nN (OD around 1) were in 0.9% Saline (Sodium Chloride), pH 7.3 from 5 min till 72 hours at room temperature.

Optoacoustic (OA) imaging system and measurements bacteria GNR interaction In these studies we used a commercial prototype of a three-dimensional optoacoustic tomography system developed for preclinical research at TomoWave Laboratories (Houston, TX), and introduced in our earlier publications [29,33-37]. The OA imaging system consists of four main components: fiber-optic light delivery, mouse holder with translation and rotation, detector array of 64 transducers, and data acquisition and imaging electronics [36,37]. A commercial dual wavelength SpectraWave laser (collaboration between TomoWave, Houston, Texas, and Quanta Systems, Solbiate, Italy) with a tunable Ti:Saph (12ns pulse) and Nd:YAG (12ns pulse) was used for these experiments. The laser was tuned from ~750 to ~825 nm with a repetition rate of 10 Hz and an output energy of 70 mJ/pulse. The light delivery from the laser was delivered into a bifurcated fiber bundle where the rectangular outputs were placed outside of the water tank orthogonal to the probe. The arc shaped probe consisted of 64 channels spread across a 152° arc angle with a arc radius of 65 mm. The water tank was held at a constant 27°C during scans where each acquisition was averaged 32 times with a sampling rate of 25 MHz over 1536 samples. Amplification was 80 dB. The phantom had three vertical garolite rods between two acrylic discs with four polytetrafluoroethylene tubes each with an internal diameter of 0.635 mm and a wall thickness of 0.051mm. Optoacoustic (OA) tomography was applied for the visualization of signals generated from bacteria-CTAB-GNR solutions. The optoacoustic assay calibration was developed from the experimental optical properties prior to and after the incubation of CTAB-GNRs with V. cholerae. OA images collected from plasmon excitation at 748 and 824 nm of samples of V. cholerae incubated with CTAB-GNRs. The optoacoustic response of the CTAB-GNR probes was compared to the response after incubation of the probes with bacteria, both at 748 nm (short wavelength) and 824 nm (long wavelength). The actual calibration curve is established, for both wavelengths, from the ratio of optoacoustic responses of the incubated probes with free CTAB-GNR probes. Signal processing was handled in a similar manner to our prior publication [36, 38]. Reconstruction was handled by the described CUDA software [36,37] and visualization was done in Volview 2.0 (Kitware, Clifton Park, NY). The image processing parameters were held constant between all tubes and scans such that one can directly compare the image brightness across all tubes.

Scanning Electron Microscope Imaging Scanning electron micrographs were collected on a FEI Quanta 4 ESEM FEG at 20 kV acceleration under high vacuum conditions. Samples were prepared by drop casting fixed E. coli-GNR solutions onto acetone-washed, diced silicon wafers (University Wafer, Cambridge, MA.)

Zeta-potential The zeta-potentials (ζ) of the GNRs before and after functionalization were measured with a high performance particle sizer (Malvern Instruments Ltd., Southborough, MA, USA) at 25oC, and ten 20-second runs were performed for each sample.

4. RESULTS AND DISCUSSION

GNRs were prepared in an aqueous solution of CTAB using a seed-mediated method and purified by centrifugation and washing as previously described [10,29,38]. The resulting gold nanorods have CTAB, a cationic surfactant bound to their surfaces in the form of a bilayer. The CTAB-GNR zeta potential (ζ) was approximately ζ+50 mV. After surface modification with mPEG-SH or MTAB and heparin sulfate the ζ reversed the polarity. mPEG-SH-GNRs typically presented ζ ≈ -17 mV and HS-MTAB-GNRs presented around ζ ≈ -51 mV. CTAB-GNRs rapidly deposited onto the bacterial substrate. Fig. 1 shows UV-vis extinction spectra of E. coli incubated with CTAB-GNRs from 0.1 to 72 hours. The visible-NIR spectra measured for CTAB-GNRs before and during their incubation with bacteria show that the optical properties of the CTAB-GNRs changed dramatically over a short period of time (Fig. 1). The as-prepared CTAB-GNRs have a strong, narrow longitudinal plasmon band that decreased in intensity and red shifted after 5-10 min of incubation. Overnight incubation resulted in a stabilization of the LSPR.

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Downloaded From: http://proceedings.spiedigitallibrary.org/ on 09/02/2014 Terms of Use: http://spiedl.org/terms 1.200 -GNR-NaCI 0.1h 1.000 - GNR-NaCI 72 h - -GNR-E.coli0.1h GNR-E.coli 1.0h 0.800 oo-GNR-E.coli 15h -- -GNR-E.coli 72h 0.600 -E.coli along

0.400

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600 800 1000 Wavelength (nm)

Figure 1. UV-vis absorbance spectra of incubation of E. coli with CTAB-GNR from 0.1 to 72 hours.

In order to verify the deposition of the CTAB-GNRs onto the E. coli, SEM micrographs were collected (Fig. 2) before (2a) and after (2b) incubation. As shown in Fig. 2b, the CTAB-GNRs were deposited onto the E. coli and very few were found in the supernatant. This finding demonstrated the strong binding affinity of CTAB-GNRs towards the negatively- charged E. coli cell wall. E. coli and V. cholerae are both gram-negative bacterial species with highly negative surface charges. The negative surface charge is caused by the lipopolysaccharides that make up a majority of the peptidoglycan layer in the cell walls. Figure 2b shows the coverage monolayer of CTAB-GNRs well below the percolation threshold of 30-40 % areal coverage for random orientation. Interestingly, the deposited CTAB-GNRs trend towards a longitudinal alignment. The cause of this trend is likely due to stronger surface adhesion along the longitudinal axis of the bacterial cell wall with the longitudinal axis of the CTAB-GNRs. The red shift and reduction in intensity of the LSPR bands are consistent with the SEM imaging showing end-to-end alignment. This effect is also consistent with other reports of longitudinal GNR alignment in solution [39,40]. The reduction in the LSPR intensity is consistent with the large number of CTAB-GNRs electrostatically adhered to the bacteria instead of being suspended in the supernatant. As a result, the red shift and the decrease in LSPR intensity can be controlled.

Figure 2. a) SEM microphotograph of E. coli prior to incubation with CTAB-GNRs. b) After incubation with CTAB-GNRs (deposition of gold nanorods on bacterial wall shown in yellow), Scale bar is 1 µm.

In a subsequent experiment, the interaction between GNRs and V. cholerae was studied. As with E. coli, V. cholerae is a gram-negative bacteria with a negatively charged cell wall. Two GNR controls were used with a neutral (PEG-SH) and

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Downloaded From: http://proceedings.spiedigitallibrary.org/ on 09/02/2014 Terms of Use: http://spiedl.org/terms negatively charged (heparin sulfate, HP) surface modifications. Figure 3 shows the UV-vis extinction spectra of those GNRs during 6 hour incubation with V. cholerae. Both the mPEG-SH-GNRs and HP-MTAB-GNRs initially have intense LSPRs (PEG-GNR and HP-GNR respectively, on fig. 3) and after 6 hours of incubation with V. cholera and E. coli, exhibit no significant change in the intensity, wavelength, or FWHM of either control sample. When CTAB-GNRs were incubated with V. cholerae there was a significant reduction in intensity and red shift of the LSPR (Fig. 4) indicating electrostatic deposition as seen with E. coli. The decrease in intensity and the red shift in the LSPR appear at higher concentrations (108 cfu mL-1) of bacteria, but the optimization of CTAB-GNR:bacteria ratio could decrease the sensitivity of optical and optoacoustic detection of bacteria. Bacteria are much larger in size as compared with GNRs and several hundred CTAB coated GNRs are attached to the E. coli, and V. cholerae surface.

1.200 - PEG-GNR-Oh - - HP- GNR -Oh 1.000 PEG- GNR -0.1 h --HP- GNR -0.1h 0.800 - PEG-GNR-1 h --HP- GNR -1h

0.600 - PEG-GNR-6h - - HP-GNR-6h

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Figure 3. UV-vis absorbance spectra of PEGylated (PEG-GNR) and heparinized (HP-MTAB-GNR) gold nanorods during 6 hours incubation with V. cholerae 1.000 GNR-CTAB GNR-V.Cholerae 0.800 V. Cholerae alone

U g 0.600

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Figure 4. UV-vis absorbance spectra of 5 min incubation of V. cholerae with CTAB-GNRs.

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Optoacoustic (OA) tomography was applied for the visualization of signals generated from bacteria-CTAB-GNR solutions. We have demonstrated the interaction between bacteria and CTAB-GNR contrast agents for solution-based optoacoustic assays. The optoacoustic assay calibration was developed from the experimental optical properties prior to and after the incubation of CTAB-GNRs with V. cholerae (Figure 5).

GNR- GNR- CTAB CTAB Bacteria

i A

824nm 824nm

f B

Figure 5. Optoacoustic images at 748 and 824 nm of samples of V. cholerae incubated with CTAB-GNRs showing switch in brightness.

OA images collected from plasmon excitation at 748 and 824 nm of samples of V. cholerae incubated with CTAB-GNRs show a switch in signal return intensity as illustrated in Figure 5a. The optoacoustic response of the CTAB-GNR probes is also compared to the response after incubation of the probes with bacteria, both at 748 nm (short wavelength) and 824 nm (long wavelength) (See Fig. 5b). The actual calibration curve is established, for both wavelengths, from the ratio of optoacoustic responses of the incubated probes with free CTAB-GNR probes. The brightness of representative small volumes ζ was observed in the short wavelength case, for GNR-CTAB (0.786), and GNR-CTAB-Bacteria (0.541). In the long wavelength case, the values are 0.714 and 0.845, respectively. These results address the nonspecific binding of modified GNRs and bacteria. However, instances of acute infection, bioterrorism, contaminated bio-liquids and food such as beverages, water and milk. More importantly, we investigated the use of CTAB-coated GNR bioprobes and their interaction with different gram-negative bacteria without any modification of the contrast agent, sample manipulation such as centrifugation and washing, or the inclusion of additional reagents. Described effects have mechanism of binding with gram-negative bacteria through their membrane outside peptidoglycan layer. The outer leaflet of the outer membrane bilayer is composed of an unusual lipid, lipopolysaccharides (LPS). LPS is composed of three parts: a proximal hydrophobic lipid A region, a core oligosaccharide region connecting a distal O-antigen polysaccharide region to lipid A. This distal region protrudes in the medium. All the fatty acid chains present in LPS are saturated which significantly reduces the fluidity. Also, the LPS molecule contains six or seven covalently linked fatty acid chains, in contrast to the glycerophospholipid that contains only two fatty acid residues [41]. In conclusion, we have demonstrated for the first time a fast and highly promising assay for gram negative bacteria detection using positively-charged gold nanorods and 3D optoacoustic technology. This bioassay is rapid and takes less than 10-15 min from the introduction of bacteria to detection solution. The strong electrostatic interaction between bacteria and CTAB coating on nanorods results in changes of optoacoustic signal by a shift in the LSPR wavelength which could be used for sensing bacterial pathogens.

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ACKNOWLEDGEMENTS The authors acknowledge of Dr. D. Provenzano, UT at Brownsville, TX and Dr. T. Erova from UTMB at Galveston, TX, which provided V.Cholerae and E.Coli, respectively. This work was supported by the National Institutes of Health 1R43ES021629, R44CA110137, R44CA110137-S1 (AL, RS, AC, SE and AAO) and the National Science Foundation DMR-1105878 (PJD and ERZ).

5. REFERENCES

[1] Wang, Y., Z. Ye, C. Si, and Y. Ying. "Subtractive Inhibition Assay for the Detection of E. Coli O157:H7 Using Surface Plasmon Resonance," Sensors 11, 2728-39 (2011). [2] Wang, C., and J. Irudayaraj. "Gold Nanorod Probes for the Detection of Multiple Pathogens.," Small 4 (12), 2204–08 (2008). [3] Singh, A.K., D. Senapati, S. Wang, J. Griffin, A. Neely, P. Candice, K.M.Naylor, B. Varisli, J.R. Kalluri, and P.C. Ray. "Gold Nanorod Based Selective Identification of Escherichia Coli Bacteria Using Two-Photon Rayleigh Scattering Spectroscopy.," ACS Nano. 3 (7), 1906-12 (2009). [4] Alvarez-Puebla, R. A., A. Agarwal, P. Manna, B. P. Khanal, P. Aldeanueva-Potel, E. Carbo-Argibay, N. Pazos-Perez, L. Vigderman, E. R. Zubarev, N. A. Kotov, and L. M. Liz-Marzan. "Gold Nanorods 3d-Supercrystals as Surface Enhanced Raman Scattering Spectroscopy Substrates for the Rapid Detection of Scrambled Prions," Proc. Natl. Acad. Sci. U.S.A. 108, 8157-61 (2011). [5] Conjusteau, A., A.V. Liopo, D. Tsyboulski, S. A. Ermilov, W.R. Elliott, N. Barsalou, S.M. Maswadi, R.D. Glickman, and A.A. Oraevsky. "Optoacoustic Sensor for Nanoparticle Linked Immunosorbent Assay (Nanolisa)," Proceedings SPIE 7899, 789910 (2011). [6] Mirkin, C.A., C.S. Thaxton, and N.L. Rosi. "Nanostructures in Biodefense and Molecular Diagnostics," Expert Rev Mol Diag. 4 (6), 749– 51 (2004). [7] Kim, S., Y.T. Lim, E.G. Soltesz, A.M. De Grand, J. Lee, A. Nakayama, J.A. Parker, T. Mihaljevic, R.G. Laurence, D.M. Dor, L.H. Cohn, M.G. Bawendi, and J.V. Frangioni. "Near-Infrared Fluorescent Type Ii Quantum Dots for Sentinel Lymph Node Mapping," Nat. Biotechnol. 22 (1), 93-97 (2004). [8] Berry, V., S. Rangaswamy, and R.F. Saraf. "Highly Selective, Electrically Conductive Monolayer of Nanoparticles on Live Bacteria," Nano Lett. 4 (5), 939-42 (2004). [9] Vigderman, L., B.P. Khanal, and E.R. Zubarev. "Functional Gold Nanorods: Synthesis, Self-Assembly, and Sensing Applications," Adv. Mater. 24 (36), 4811-41 (2012). [10] Vigderman, L., P. Manna, and 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," Angew. Chem. Int. Ed. 51, 636- 41 (2012). [11] Pastoriza-Santos, I., J. Perez Juste, and L.M. Liz-Marzan. "Silica-Coating and Hydrophobation of Ctab-Stabilized Gold Nanorods," Chem Mater. 18 (10), 2465-7 (2006). [12] Eghtedari, M., Liopo, A. V., Copland, J. A., Oraevsky, A. A., Motamedi, M. "Engineering of Hetero-Functional Gold Nanorods for the in Vivo Molecular Targeting of Breast Cancer Cells," Nano Lett 9 (1), 287-91 (2009). [13] Xu, J., J. Reumers, J. Couceiro, F. De Smet, R. Gallardo, S. Rudyak, A. Cornelis, J. Rozenski, A. Zwolinska, J-C. Marine, D. Labrechts, Y-A. Suh, and F.J.S. Rousseau. "Gain of Function of Mutant P53 by Coaggregation with Multiple Tumor Suppressors," Nature Chem Biol 7, 285-95 (2011). [14] Liao, H., and J. H. Hafner. "Gold Nanorod Bioconjugates," Chemistry of Materials 17 (18), 4636-41 (2005). [15] Fernandez-Lopez, C. , C. Mateo-Mateo, R.A. Alvarez-Puebla, J. Perez-Juste, I. Pastoriza-Santos, and L.M. Liz-Marzan. "Highly Controlled Silica Coating of Peg-Capped Metal Nanoparticles and Preparation of Sers-Encoded Particles," Langmuir 25 (24), 13894-9 (2009). [16] Lasagna-Reeves, C., D. Gonzalez-Romero, M. A. Barria, I. Olmedo, A. Clos, V. M. Sadagopa Ramanujam, A. Urayama, L. Vergara, M. J. Kogan, and C. Soto. "Bioaccumulation and Toxicity of Gold Nanoparticles after Repeated Administration in Mice," Biochem Biophys Res Commun 393 (4), 649-55 (2010). [17] Alkilany AM, Murphy CJ. . "Toxicity and Cellular Uptake of Gold Nanoparticles: What We Have Learned So Far? ," Journal of nanoparticle research 12 (7), 2313-33 (2010). [18] Huang, X., I. H. El-Sayed, W. Qian, and M. A. El-Sayed. "Cancer Cell Imaging and Photothermal Therapy in the near-Infrared Region by Using Gold Nanorods," J Am Chem Soc 128 (6), 2115-20 (2006). [19] Tiwary, R., W. Yu, L.A. De Graffenried, B.G. Sanders, and K. Kline. "Targeting Cholesterol-Rich Microdomains to Circumvent Tamoxifen-Resistant Breast Cancer," Breast cancer research 13 (6), R120 (2011). [20] Urbanska, K., B. Romanowska-Dixon, and Z. Matuszak. "Indocyanine Green as a Prospective Sensitizer for Photodynamic Therapy of Melanomas," Acta. Biochim. Pol. 49, 387-91 (2002). [21]Oraevsky, A. "Gold and Silver Nanoparticles as Contrast Agents for Optoacoustic Imaging." In Photoacoustic Imaging and Spectroscopy, edited by L. Wang. New York: Taylor and Francis Group, (2009).

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Downloaded From: http://proceedings.spiedigitallibrary.org/ on 09/02/2014 Terms of Use: http://spiedl.org/terms [22] Plech, A., V. Kotaidis, S. Gresillon, C. Dahmen, and G.von Plessen. "Laser-Induced Heating and Melting of Gold Nanoparticles Studied by Time-Resolved X-Ray Scattering," Phys Rev B. 70 (19), 195423 (2004). [23] Wang, Y., S. Teitel, and C. Dellago. "Surface-Driven Bulk Reorganization of Gold Nanorods," Nano letters 5 (11), 2174-8 (2005). [24] Petrova, H., J. Perez Juste, I. Pastoriza-Santos, G. V. Hartland, L. M. Liz-Marzan, and P. Mulvaney. "On the Temperature Stability of Gold Nanorods: Comparison between Thermal and Ultrafast Laser-Induced Heating," Phys Chem Chem Phys 8 (7), 814- 21 (2006). [25] Bayer, C. L., Y. S. Chen, S. Kim, S. Mallidi, K. Sokolov, and S. Emelianov. "Multiplex Photoacoustic Molecular Imaging Using Targeted Silica-Coated Gold Nanorods," Biomed Opt Express 2 (7), 1828-35 (2011). [26] Berry, V., A. Gole, S. Kundu, C.J. Murphy, and R.F. Saraf. "Deposition of Ctab-Terminated Nanorods on Bacteria to Form Highly Conducting Hybrid Systems," JACS 127, 17600-176001 (2005). [27]Liopo, A., A. Conjusteau, and A. Oraevsky. “Peg-Coated Gold Nanorod Monoclonal Antobody Conjugates in Preclinical Research with Optoacoustic Tomography, Photothermal Therapy, and Sensing,” Proceedings of SPIE 8223, 822344, (2012). [28] Su, R., A. V. Liopo, H. P. Brecht, S. A. Ermilov, and A. A. Oraevsky. "Gold Nanorod Distribution in Mouse Tissues after Intravenous Injection Monitored with Optoacoustic Tomography," Proceedings SPIE 7899, 78994B (2011). [29] Liopo, A., A. Conjusteau, O. Chumakova, S. Ermilov, R. Su, and A. Oraevsky. "Highly Purified Biocompatible Gold Nanorods for Contrasted Optoacoustic Imaging of Small Animal Models," Nanoscience and Nanotechnology Letters 4, 1-6 (2012). [30] Sau, T. K., and C. J. Murphy. "Seeded High Yield Synthesis of Short Au Nanorods in Aqueous Solution," Langmuir 20 (15), 6414-20 (2004). [31] Liopo, A. V., A. Conjusteau, M. Konopleva, M. Andreeff, and A. A. Oraevsky. "Photothermal Therapy of Acute Leukemia Cells in the near-Infrared Region Using Gold Nanorods Cd-33 Conjugates," Proceedings SPIE 7897, 789710 (2011). [32] Niidome, T., M. Yamagata, Y. Okamoto, Y. Akiyama, H. Takahashi, T. Kawano, Y. Katayama, and Y. Niidome. "Peg-Modified Gold Nanorods with a Stealth Character for in Vivo Applications," J Control Release 114 (3), 343-47 (2006). [33] Brecht, H-P., Su, R., Fronheiser, M., Ermilov, S.A., Conjusteau, A., Oraevsky, A.A. "Optoacoustic 3d Whole Body Tomography: Experiments in Nude Mice," Proceedings SPIE 7177, 0E1 (2009). [34] Ermilov, S. A., T. Khamapirad, A. Conjusteau, M. H. Leonard, R. Lacewell, K. Mehta, T. Miller, and A. A. Oraevsky. "Laser Optoacoustic Imaging System for Detection of Breast Cancer," J Biomed Opt 14 (2), 024007 (2009). [35] Su, R., A. V. Liopo, H. P. Brecht, S. A. Ermilov, K. Larin, and A. A. Oraevsky. "Optoacoustic Tomography in Preclinical Research: In Vivo Distribution of Highly Purified Peg-Coated Gold Nanorods," Proceedings SPIE 8089, 808902 (2011). [36] Tsyboulski, D. A., A. V. Liopo, R. Su, S. A. Ermilov, S. M. Bachilo, R. B. Weisman, and A. A. Oraevsky. "Enabling in Vivo Measurements of Nanoparticle Concentrations with Three-Dimensional Optoacoustic Tomography," J Biophotonics, (2013). [37] Su, R., S.A. Ermilov, A.V. Liopo, and A.A. Oraevsky. "Laser Optoacoustic Tomography: Towards New Technology for Biomedical Diagnostics," Nucl Instrum Methods Phys Res A 720, 58-61 (2013). [38] Su, R., S. A. Ermilov, A. V. Liopo, and A. A. Oraevsky. "Three-Dimensional Optoacoustic Imaging as a New Noninvasive Technique to Study Long-Term Biodistribution of Optical Contrast Agents in Small Animal Models," J Biomed Opt 17 (10), 101506 (2012). [39] Thomas, K.G., S. Barazzouk, B.I. Ipe, S.T.S. Joseph, and P.V. Kamat. "Unidirectional Plasmon Coupling through Longitudinal Self-Assembly of Gold Nanorods," J. Phys. Chem. B 108, 13066-68 (2004). [40] Perez-Juste, J., I. Pastoriza-Santos, L. M. Liz-Marzan, and P. Mulvaney. "Gold Nanorods: Synthesis, Characterization and Applications," Coordination Chemistry Reviews 249, 1870–901 (2005). [41]Cohen, G.N. "The Outer Membrane of Gram-Negative Bacteria and the Cytoplasm Membrane, Chapter 2." In Microbial Biochemistry, 500: Springer Science Business Media, (2011).

Proc. of SPIE Vol. 8943 894368-8

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