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Supporting Information for:

Gold Nanoparticle Coated Carbon Nanotube Ring

with Enhanced Raman Scattering and Photothermal

Conversion Property for Theranostic Applications

Jibin Song,1 Feng Wang,

2 Xiangyu Yang,

1 Bo Ning,

3 Mary G. Harp,

4 Stephen H. Culp,

4 Song

Hu,3 Peng Huang,

1 Liming Nie,

1 Jingyi Chen,

2 Xiaoyuan Chen

1*

1 Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of

Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda,

Maryland 20892, United States.

2 Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, United States.

3 Department of Biomedical Engineering, University Of Virginia, Charlottesville, VA 22903.

4 Department of Urology, University Of Virginia, Charlottesville, VA 22903.

Materials and Characterization

Potassium gold(III) chloride (KAuCl4) was purchased from Alfa Aesar. Copper(I) bromide

(CuBr), 4-acetoxystyrene, N,N,N’,N’,N’’-pentamethyl diethylenetriamine (PMDETA),

polyvinylpyrolidone (PVP), phospholipid-polyethylene glycol (MW=5000), methoxy-

poly(ethylene glycol)-thiol (MPEG-SH) with a molecular weight of 5 kDa, polyvinyl alcohol

(PVA, MW 9,000~10,000), and hydrazine hydrate (50-60%) were purchased from Sigma-

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Aldrich. 4-Acetoxystyrene was dried with CaH2 overnight and distilled under vacuum.

Radiometal [64

Cu] was produced by the PET Department, NIH Clinical Center. All solvents

unless specified were obtained from Sigma-Aldrich and used as received.

Scanning electron microscopy images were obtained on a Hitachi SU-70 Schottky field

emission gun Scanning Electron Microscope (FEG-SEM). Transmission Electron Microscopy

(TEM) was conducted on a Jeol JEM 2010 electron microscope at an acceleration voltage of 300

kV. UV-vis absorption spectra were recorded by using a Shimadzu UV-2501 spectrophotometer.

Gel permeation chromatography (GPC) was measured on a Shimadzu HPLC system using

chloroform as the eluent, and the molecular weight was calibrated with polystyrene standards.

Thermogravimetric analysis (TGA) was performed on a Perkin-Elmer Diamond TG/DTA.

Samples were placed in platinum sample pans and heated under a nitrogen atmosphere at a rate

of 10 °C/min to 100 °C and held for 40 min to completely remove residual solvent. Samples

were then heated to 700 °C at a rate of 10 °C/min. 1H NMR spectra were obtained on a Bruker

AV300 scanner using CDCl3 as the solvent. Dark-field images of live cells were carried out on

an Olympus71 inverted microscope with an oil-immersion dark field condenser at 100×

magnification, and fluorescence images were collected using a Photometrics CoolSNAP-cf

cooled CCD camera.

Preparation of ATRP Initiator, (2-(2-Bromoisobutyryloxy)ethyl) Pyrenemethylamine)

(BiBEP)

2-Bromoisobutyryl bromide (13.4 mL, 0.1 mol) and triethylamine (15.5 mL, 0.1 mol) were

added to 120 mL of methylene chloride; then, 21.3 g (0.08 mol) of 1-pyrenemethylamine

hydrochloride was slowly added to the reaction mixture at 0 ºC. The reaction was held at room

temperature overnight; then, the white precipitate was filtered off, and the clear filtrate was

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extracted with saturated sodium bicarbonate in water (2 × 100 mL), followed by 100 mL water.

The solution was then dried over anhydrous MgSO4 overnight. The final product was obtained

after filtration and evaporation of the solvent (yield: 85%).

NH2

+ Br

O

Br

NHO

Br

N(CH2CH3)3

Scheme S1. Synthesis of 2-(2-bromoisobutyryloxy)ethyl) pyrenemethylamine.

Preparation of BiBEP attached carbon nanotube ring (CNTR@BiBEP)

10 mg BiBEP was added into 10 mL solution of CNTR in DMF (0.2 mg/mL), followed by

sonication for 30 min at room temperature. The resulting suspension was then centrifuged at

18,000 rpm for 20 min for three times to remove the free BiBEP.

TGA analysis of the CNTR@PvPH

Thermogravimetry analysis (TGA) was conducted on a Perkin-Elmer Diamond TG/DTA.

Samples were placed in platinum sample pans and heated under a nitrogen atmosphere at a rate

of 10 °C/min to 100 °C and held for 40 min to completely remove residual solvent.

Samples were then heated to 700 °C at a rate of 10 °C/min.

To gain the sample for TGA analysis, 20 mL dispersion of CNTR@PvPH was filtered

through a PTFE micro-porous membrane (100 nm). CNTR@PvPH left on the membrane were

then washed by DMF solution and dried for 12 h at 60 ºC under vacuum.

NIR Laser Irradiation of CNTR@AuNP and CNTR@AuNS

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A total of 500 µL CNTR@AuNP, CNTR@AuNS based on the same concentration of CNT

in 1 mL Eppendorf vial was irradiated with a 808 nm diode laser (spot size: 1 cm) at a power

density of 0.25 or 0.5 W/cm2

for 6 min, respectively. Real-time thermographic images and

temperature elevation of the vesicle aqueous solution were taken by an infrared thermographic

camera as a function of irradiation time.

Preparation of Radioactive 64

Cu-Labeled CNTR@AuNP and CNTR@AuNS

To prepare radiometal 64

Cu-doped PEGylated CNTR@AuNP, 4 µL 64

CuCl2 was pre-mixed

with 1.5 mg of Na-ascorbate (in 0.1 M borate buffer pH 8.6). Then 200 µL of sample (1 mg/mL)

was added.1-2

The mixture was shaken at 37 ºC for 1 h. The resulting 64

Cu-labeled

CNTR@AuNP were purified by centrifugation (10,000 g, 10 min) for three times to remove the

unreacted 64

Cu and excess amount of reagents. The purified 64

Cu-labeled samples were finally

dispersed in PBS. The labeling efficiency was determined by using the instant thin-layer

chromatography (ITLC) plates and 0.1 M citric acid pH 5 as an eluent.3

64Cu labeling of

CNTR@AuNS followed the same approach.

Raman Test

A RENISHAW Raman microscope with WIRE 2.0 software and 808 nm (maximum energy:

50 mW) emission line of an air-cooled He-Ne laser was used for SERS measurements. The laser

beam with a laser spot size of 2−5 µm was focused by a 20× objective. A single scan with an

integration time of 15 s was performed. The encoder feedback controlled the grating stage with a

holographic grating of 1800 lines/mm on an interchangeable magnetic mount. The Raman signal

of the CNT, CNTR, CNTR@AuNS, CNTR@AuNP and CNT@AuNP based on the same

concentration of CNT in water were tested.

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Calculation of SERS Enhancement Factor (EF)

The EF of individual CNTR@AuNP was determined by computing the ratio of SERS to

normal Raman scattering of CNT using the following equation, EF = (ISERS × CNormal)/(INormal ×

CSERS), where ISERS and INormal are the Raman intensities at 1588 cm-1

for CNTR@AuNP and

CNT, CSERS and CNormal are the concentrations of CNTR@AuNP and CNT in pure solution.

ICP-MS Sample Preparation and Measurement

The PEGylated CNTR@AuNP (0.1 nM) were incubated with U87MG cancer cells in 24 well

plates (106

cells/well) for 2, 4, 8, 12, and 16 h. After incubation, cells were washed three times

with PBS buffer and then lysis buffer (300 µL) was added to the cells. The resultant cell lysate

was digested using 3 mL of HNO3 and 1 mL of H2O2 for 24 h. 3 mL of aqua regia was then

added and the sample was allowed to react for another 4 h. This solution was diluted in 100 mL

de-ionized water with 5% aqua regia, and centrifuged at 3000 rpm for 5 min to remove any

cellular debris. All experiments were repeated 3 times, and each replicate was measured 3 times

by ICP-MS. Afterwards, the concentration of Au was converted into the number of

CNTR@AuNP using equation S1, where MCNTR@AuNP refers to the molar mass of the

CNTR@AuNP and M refers to the measured number of Au ions (atoms) from ICP-MS analysis.

NCNTR@AuNP=M/MCNTR@AuNP (Eq. S1)

Calculation of the Photothermal Conversion Efficiency of CNTR@AuNP

The photothermal conversion efficiency (η) of straight CNT@AuNP and CNTR@AuNP

were calculated according to the energy balance of the system as follows:

η = (hS△Tmax - Qs) / I(1-10-A808

) (Eq. S2)

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τs = mDCD / hS (Eq. S3)

In which h is the heat-transfer coefficient, S is the surface area of the container, △DTmax is the

temperature change of the vesicle solution at the maximum steady-state temperature, I is the laser

power, A808 is the absorbance of the CNT@AuNPs or CNTR@AuNPs at 808 nm, and Qs is the

heat associated with light absorption by the solvent. The variable τs is the sample-system time

constant, and mD and CD are the mass (0.15 g) and heat capacity (4.2 J/g) of the deionized water

used as the solvent. According to equations S2 and S3 and Fig. S21, the η values of the

CNTR@AuNP and CNT@AuNP were determined to be 76% and 61%, respectively. Based on

the same method, the photothermal conversion efficiencies of CNTR@AuNS (52 %) and CNTR

(14%) were also calculated.

In Vitro Photothermal Therapy by PEGylated CNTR@AuNP

A standard Cell Counting Kit-8 (CCK-8) was utilized to analyze the cytotoxicity of

PEGylated CNTR@AuNP following a general protocol. Briefly, the U87MG cells were seeded

in a 96-well plate (1×104 cells/well). After incubation at 37 °C for 24 h, PEGylated

CNTR@AuNP or CNTR@AuNS were added and incubated for 4 h, the cells were then washed

with PBS and 100 µL fresh medium was added. The cells were exposed to an 808 nm laser at

0.125, 0.25 and 0.5 W/cm2 for 200 s, respectively. The standard CCK-8 assay was applied to test

the viability of U87 cancer cells at 24 h after treatment. All experimental results were averaged

based on triplicated experiments.

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Figure S1. Schematic illustration of the self-assembly of carbon nanotubes (CNTs) into CNT

ring (CNTR) and further growth of redox-active polymer brushes poly(4-vinylphenol) (PvPH) by

surface-atom-transfer radical-polymerization (ATRP) method to reduce Au3+

to Au0 and growth

of gold nanoparticles onto CNTR (CNTR@AuNP). The CNTR@AuNP was further labeled with

positron emitting radionuclide 64

Cu.

Figure S2. TEM image of CNT with the length of 0.5-2 µm.

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Figure S3. CNT ring with the average diameter of ~90 (a), ~180 (b) and ~290 nm (c).

Figure S4. CNT ring with the average diameter of ~230 nm (a) and ~720 nm (b) were prepared

by using CNT with the length of 0.5-5 µm and 5-9 µm, respectively (scale bar: 100 nm).

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Figure S5. GPC trace of the PvPH detached from CNTR@PvPH (Mn = 14.5 kDa, PDI = 1.21).

Figure S6. TGA traces of CNTR and CNTR@PvPH.

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Figure S7. Solubility of CNT, CNTR and CNTR@PvPH.

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Figue S8 Representative TEM images of CNTR (a), CNTR@PvPH (b), CNTR@AuNP at

different reaction times: (c) 10 min, (d) 20 min, (e) 30 min, (f) 40 min. Scale bar: 20 nm.

Figure S9. Representative HR-TEM images of a single CNTR@AuNP. The AuNP interparticle

distance is 0.85 ± 0.45 nm.

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Figure S10. TEM images (a-e) and corresponding UV-vis spectra (f) of 140 nm CNTR@AuNP

made from 5 different batches showed high reproducibility of the synthetic procedure (scale bar:

100 nm).

Figure S11 Representative HR-SEM images of CNTR (a), CNTR@AuNP (b), CNTR@AuNS (c)

and CNT@AuNP (d) (scale bar: 50 nm).

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Figure S12. Representative AFM images of CNTR (a), CNTR@AuNP (b), CNTR@AuNS, (c)

and CNT@AuNP (d).

Figure S13. Hydrodynamic diameter distribution of different sized CNTR@AuNP (black line:

Dh= 125 nm, red line: Dh= 240 nm and green line: Dh= 320 nm).

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Figure S14. UV-vis-NIR spectra of (a) CNT@AuNP, CNT@AuNP and CNTR@AuNS, and

CNTR (b) based on the same mass concentration. (c) UV-vis-NIR spectra of different sized

CNTR@AuNP: ~125 nm (red line), 240 nm (green line) and ~320 nm (blue line).

a b

Figure S15. TEM images of CNTR@AuNP with the size of 240 nm (a) and 320 nm (b).

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Figure S16. TEM images of straight CNT@AuNP.

Figure S17. Dark-field images of CNTR (a) and 125 nm CNTR@AuNP.

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Figure S18. Single particle dark-field scattering spectra of CNTR@AuNP: a 125 nm sized

particle before (black line) and after incubation in water for 5 days (blue line), a 240 nm (green

line) and a 320 nm (red line) sized CNTR@AuNP.

Figure S19. SERS spectra of CNTR@AuNP and straight CNT@AuNP at the same

concentration illuminated with 808 nm laser.

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Figure S20. Temperature increase of CNTR@AuNP solution (based on the CNT concentration

of 1 µg/mL) with different power density laser irradiation.

Figure S21. (a) Temperature variation of the aqueous solution of CNTR@AuNP (OD@808 nm

= 0.5) irradiated with 808 nm laser (0.5 W/cm2) for 300 s, and then the laser was turned off. (b)

Time constant (Ts) for heat transfer from the system is determined to be 92.64 s by applying the

linear time data from the cooling period (after 300 s) versus negative natural logarithm of driving

force temperature obtained from the cooling stage of panel a.

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Figure S22. Near-field (a) and optical spectra (b) of the bare CNTR. A linear polarized light is

propagated along the direction normal to the ring (z-direction). The cross-section of the near field

plot is made slightly off-center in the XY-plane. In the optical spectra, the extinction curve

overlaps with the absorption curve since the scattering is very close to zero.

Figure S23 UV-vis spectra of PEGylated CNTR@AuNP in water, PBS, cell culture media, and

fetal bovine serum (FBS) for 5 days without obvious change.

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Figure S24 Time-dependent cell uptake of CNTR@AuNP measured by ICP-MS.

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Figure S25. SERS detection of U87MG cells treated with PEGylated CNTR@AuNP: (a) SERS

spectra. (b) Raman intensity at 1588 cm-1

.

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Figure S26. (a) Fluorescence images of U87MG cancer cells after incubation with PEGylated

CNTR@AuNP and irradiated by 808 nm laser of different power densities for 200 s. Cells were

stained with Calcein AM (live, green) and PI (dead, red) after 24 h after irradiation. (b) Viability

of U87MG cells after incubation with PEGylated CNTR@AuNP and exposure to 808 nm laser

of different power densities for 200 s.

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Figure S27. (a) Photograph of the UVA-C2 renal cell carcinoma (RCC) xenograft tumor model

on the mouse ear. (b) PAM images of the UVA-C2 tumor before and after treatment with

PEGylated CNTR@AuNP illuminated with 532 and 808 nm laser, respectively.

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Figure S28 (a) Whole-body PET images of U87MG tumor mice injected with 64

Cu-labeled

PEGylated CNTR@AuNP at 1, 10 and 24 h postinjection. (b) Region of interest (ROI)

quantification of CNTR@AuNP in the tumor, muscle and liver at 1, 10 and 24 h postinjection.

Figure S29 Blood clearance of 64

Cu-labeled PEGylated CNTR@AuNP in U87MG tumor-

bearing mice. The circulation half-life of the nanoring was estimated to be about 19 h.

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Figure S30. Biodistribution of PEGylated CNTR@AuNP in tumor mice at 24 h postinjection

measured by inductively coupled plasma mass spectrometric (ICP-MS) analysis of Au in

different organs and tissues.

Figure S31. Photographs of U87MG tumor mice at different days after injection with 200 µL

500 µg/mL PEGylated CNTR@AuNP after irradiation with 808 nm laser at 0.25 W/cm2.

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Figure S32. Kaplan-Meier plot of the five groups of U87MG tumor-bearing mice for the 50 days

after laser irradiation.

Figure S33. Hematoxylin and eosin stained tumor tissues of different groups of mice treated with

PBS and CNTR@AuNP irradiated with/without laser.

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Figure S34. The body weight changes of U87MG tumor-bearing mice as a function of time after

different treatments.

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Figure S35. H&E stained images of major organs collected from different groups of U87MG

tumor-bearing mice after different treatments.

References:

(1) Guo, W.; Sun, X.; Jacobson, O.; Yan, X.; Min, K.; Srivatsan, A.; Niu, G.; Kiesewetter, D.

O.; Chang, J.; Chen, X. ACS Nano 2014, 9, 488.

(2) Song, J.; Yang, X.; Jacobson, O.; Lin, L.; Huang, P.; Niu, G.; Ma, Q.; Chen, X. ACS

Nano 2015, 9, 9199.