1

Electronically Supplementary Information

SERRS/MRI multimodal contrast agent based on naked Au nanoparticles functionalized with a Gd(III) loaded PEG polymer for tumor imaging and localized hyperthermia Lucio Litti, Niccolò Rivato, Giulio Fracasso, Pietro Bontempi, Elena Nicolato, Pasquina Marzola, Alfonso Venzo, Marco Colombatti, Marina Gobbo, Moreno Meneghetti *

Table of contents Characterization of (3-Fmoc ): HPLC and Mass spectra pag. 2 Characterization of (2): NMR and Mass spectra pag. 4 Characterization of DOTA-DBCO: HPLC and Mass spectrum pag. 11 Characterization of MultiCAs pag. 12 MRI images: T2 maps pag. 14 References pag. 14

Electronic Supplementary Material (ESI) for Nanoscale.This journal is © The Royal Society of Chemistry 2017

2

Characterization of (3-Fmoc): HPLC and Mass spectra

Figure S 1. Molecular formula of the intermediate products (3-Fmoc). Figure S 2. HPLC chromatogram for (3-Fmoc) intermediate. Figure S 3. MALDI-TOF/TOF mass spectra (3-Fmoc), calc. (m/z) 2562.95 Da, found (m/z) 2584.74 Da (molecular ion + Na).

HS

NH

HO

O

O

OO

NH

ONH

O OO O

O OO O

O O

N3

OOO

NH

OHN

OOOO

OOOO

OO

N3

O OO

NH

ONH2O O

O OO O

O OO O

N33Chemical Formula: C99H190N16O44S

Exact Mass: 2339,28Molecular Weight: 2340,71

HS

NH

HO

O

O

OO

NH

ONH

O OO O

O OO O

O O

N3

OOO

NH

OHN

OOOO

OOOO

OO

N3

O OO

NH

OHN

O OO O

O OO O

O O

N33-FmocChemical Formula: C114H200N16O46S

Exact Mass: 2561,35Molecular Weight: 2562,95

O

O

Acquired: \...\20150417_J06_pos_7500_1.T2D

800 1240 1680 2120 2560 3000Mass (m/z)

573.5

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

TOF/TOF™ Reflector Spec #1[BP = 153.0, 37862]

2584

.743

7

870.

3692

1460

.484

7

866.

8624

2606

.713

6

861.

2742

2536

.788

8

1165

.490

0

827.

2431

2613

.865

2

1937

.568

819

85.5

352

1448

.590

1

887.

2349

2043

.575

6

925.

3071

2690

.766

1

1279

.488

8

1563

.601

7

2558

.775

6

2444

.741

0

816.

2512

1732

.724

4

1051

.426

8

1874

.613

8

2616

.768

3

1629

.573

9

1005

.329

6

2001

.484

5

2763

.760

0

967.

3123

1102

.315

6

1823

.510

5

1463

.473

4

1667

.484

7

2201

.714

1

2507

.781

7

864.

3578

2091

.573

5

921.

3106

1958

.557

3

1780

.568

1

2663

.728

3

2727

.770

8

2129

.807

9

1555

.502

3

1229

.372

1

1317

.369

1

2380

.724

923

41.7

168

1168

.970

8

2051

.585

7

2165

.604

2

1391

.223

013

55.4

413

1591

.602

2

1509

.100

1

2924

.905

3

3

Figure S 4. ESI-TOF mass spectra (3-Fmoc), calc. (m/z) 2562.95 Da, found (m/z) 1282.17 Da ([M+2H]2+), 1182.11 Da ([M-Fmoc+H+Na]2+), 1171.64 Da ([M-Fmoc+2H]2+), 855.13 Da ([M+3H]3+), 781.09 Da ([M-Fmoc+3H]3+), 586.07 Da ([M-Fmoc+4H]4+).

4

Characterization of (2): NMR and Mass spectra

Scheme S1. Molecular structure of 2 with numbered carbons for NMR assignment.

Table S1. Carbon and hydrogen assignments from related 1D and 2D spectra reported in Figure S5 - S11

Atom Index 1H-NMR chemical

shift (ppm)

13C-NMR chemical shift

(ppm)

1H-NMR expected area

1H-NMR experimental area

1 4.74 53.81 1 0.8 2 2.97 26.49 2 1.3 3 1.54 - 1 0.3 4 7.38 - 1 0.8 5 7.10 - - - 6 4.39 52.47 3 2.9

7 1.87 1.63 29.60 12 10.2

8 1.58 25.00 9 3.25 50.98 6 6.1

10 7.04 - - - 11 3.58 70.51 304 306.5

12 3.31 58.97 3 3.0 (assigned value)

N3

HS

NH

HO

O

O

OO

NH

OHN

O

HN

O

OO OCH3

11

3

2000Da

1

23

4 5

6

78

9

1011

12

11

5

Figure S 5. 1H-NMR spectra of (2) in CDCl3 at 298K. Figure S 6. 13C-NMR spectra of (2) in CDCl3 at 298K.

6

Figure S 7. 1H,1H-COSY spectra of (2) in CDCl3 at 298 K. Figure S 8. 1H,1H-TOCSY spectra of (2) in CDCl3 at 298 K.

7

Figure S 9. 1H,1H-NOESY spectra of (2) in CDCl3 at 298 K. Figure S 10. 1H,13C-HMQC spectra of (2) in CDCl3 at 298 K.

8

Figure S 11. 1H,13C-HMBC spectra of (2) in CDCl3 at 298 K.

9

Figure S 12. MALDI-TOF mass spectra (down) of the reagent carbossyl-PEG(2kDa)-OCH3

10

Figure S 13. MALDI-TOF mass spectra (down) of the product (2), DHAP-TFA used as matrix. Expected mass distribution is centered at 4390 Da and experimental mass distribution is found centered at (m/z) 4350 Da.

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Characterization of DOTA-DBCO: Mass spectrum and HPLC

Figure S 14. Molecular structure and ESI-TOF mass spectrum (down) of the product DOTA-DBCO. Calc. (m/z) 805.36 Da, found (m/z) 806.36 Da (molecular ion + H). Figure S 15. HPLC chromatogram for t Figure S15. HPLC chromatogram of the purified DOTA-DBCO product.

NO O

NN

N N OOHOHO

O OHOHOHNO

NH

Chemical Formula: C40H51N7O11Exact Mass: 805,36

Molecular Weight: 805,87

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Characterization of MultiCAs

Figure S16. UV-vis-NIR extinction spectra of MultiCAs and of laser ablated gold nanoparticles used for their synthesis. Samples are water dispersed colloids. The MultiCA solution shows the characteristic broad absorption over 700nm, due to aggregated particles, that falls into the so called optical window for biological samples.

Figure S17. TEM images of MultiCA.

300 400 500 600 700 800 900 1000 11000.0

0.2

0.4

0.6

0.8

1.0

Pristine AuNPs

Nor

mal

ized

Abs

Wavelength (nm)

MultiCAs

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Figure S18. Distribution of the average dimension of MultiCA obtained by TEM.

Table S2. ICP evaluation of Au and Gd content into MultiCA sample.

With the values reported in Table S2 it could be estimated a molar ratio of 1.33 • 10-3 Gd ions / Au ions. One could also estimate an average content of about 300 – 400 Gd ions per single AuNP (average diameter of 20 nm). The average AuNPs dimension were estimated by fitting the extinction spectra (Figure S16) with Mie-Gans model.1

Table S3. r1 and r2 magnetic relaxivities per Gd atom for MultiCA.

Field r1 (mM-1·s-1) r2 (mM-1·s-1) 4 T (200 MHz) 14.8 127

14 T (600 MHz) 5.3 271

Table S4. r1 and r2 magnetic relaxivities at 14 T (600 MHz).

Sample r1 (mM-1·s-1) r2 (mM-1·s-1) 3(DOTA-GdIII)-PEG 2.5 2.4

MultiCA 5.3 271

0 100 200 300 400 500 600 700 800 900 10000

20

40

60

Num

ber o

f Ele

men

ts

Aggregate Average Dimension (nm)

Total Elements: 396Mean: 131.6 nmStandard Deviation: 88.9 nmMedian: 102.6 nm

Sample Au, mmol/L - g/L Gd, mmol/L - mg/L Au 602 - 119 - Gd - 0.80 - 126

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Figure S19. FEG-SEM image of MultiCA. Table S5. EDS analysis of MultiCA

element Atomic Concentration (%)

Au 99.51

Gd 0.49 Gd/Au=4.9 10-3

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Magnetic Resonance Imaging, T2 maps

Figure S 20. Representative T2 maps of a mouse from “Treated” group, before intratumoral injection of MultiCA (left) and 1 hour (right) after the nanoparticle injection. The tumor site images are enlarged to highlight the differences. References: 1. V. Amendola and M. Meneghetti, The Journal of Physical Chemistry C, 2009, 113, 4277-4285.