Supporting Information

Payload drug vs. Nanocarrier Biodegradation by Myeloperoxidase- and

Peroxynitrite-Mediated Oxidations: Pharmacokinetic Implications

Wanji Seo, a Alexandr A. Kapralov,

b Galina V. Shurin,

c Michael R. Shurin,

c,d Valerian E. Kagan

b,

Alexander Star*a

a. Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA

b. Department of Environmental and Occupational Health, University of Pittsburgh, 100 Technology Drive, Pittsburgh, PA 15219,

USA

c. Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA 15261, USA

d. Department of Immunology, University of Pittsburgh Medical Center, Pittsburgh, PA 15261, USA

Correspondence email: astar@pitt.edu

Table of contents Page

1. General information S1–S2

2. Synthesis of Materials S3

3. Characterization of phospholipid-poly(ethylene) glycol (PL-PEG) S4

4. Characterization of oxidized HiPco SWCNTs (ox-SWCNT) S5–S7

5. Characterization of DOX-SWCNT S8–S9

6. Myeloperoxidase-catalysed degradation experiments with hydrogen peroxide,

and chloride (MPO/H2O2/Clˉ) S10

7. TEM images of degradation under MPO/H2O2/Clˉ S11

8. Proposed degradation products of DOX S12–S13

9. Identification of a degradation product S14–S16

10. Degradation experiments with peroxynitrite S17

11. Characterization of peroxynitrite-mediated oxidation S18

12. Zeta potential titration with MPO S19

13. Methods of in vitro studies S20–S21

14. DOX release in MDSC cell medium S22

15. Ex vivo pH-dependent DOX release S23

16. MDSC abrogated cytotoxic/cytostatic effect of free DOX, but not DOX-SWCNT,

on 3LL cells in vitro S24

17. References S25

S1

1. General information

Materials

HiPco SWCNT was purchased from NanoIntegris®. Lyophilized human myeloperoxidase

(MPO) was received from Athens Research and Technology, INC. (Athens, GA, USA). N-

(aminopropylpolyethyleneglycol)carbamyl-disteaoyl phosphatidylethanolamine (DSPE-050PA) was

purchased from NOF Corporation. (Methyl-PEG12)3-PEG-NHS Ester (TMS(PEG)12) was obtained

from Thermo Scientific. N3-(2-Hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (Papa

NONOate) was purchased from Cayman Chemical Company (Ann Arbor, MI). All other chemicals

were purchased from Sigma Aldrich, and were used without further purification. All samples were

prepared by dispersing dry solid in either nanopure water or phosphate buffer (pH 7.4).

Instrumental

Nanopure water was collected from Thermo Scientific BarnsteadTM

NanopureTM

. Branson

5510 was used for ultrasonication. Thermo Scientific Savant SPD 1010 SpeedVac was employed to

dry aqueous samples (pressure: 5.6 Torr, temperature: 45 ˚C). The size distribution and the

morphology were analyzed with Transmission Electron Microscopy (FEI-Morgani, 80 keV). All

UV-Vis-NIR spectra were acquired using a Lambda 900 spectrophotometer (PerkinElmer).

Fluorescent spectra were taken using a Horiba Jovin Yobin Fluoromax 3. Renishaw inVia Raman

microscope was utilized to collect Raman spectra (laser λexcitation: 633 nm). Fourier Transform

spectroscopy (FTIR) was performed employing an IR-Prestige spectrophotometer (Shimadzu

Scientific) outfitted with an EasiDiff accessory (Pike Technologies). X-ray photoelectron

S2

spectroscopy (XPS) was obtained via a Thermo Scientific ESCALAB 250xi photoelectron

spectrometer using monochromated Al K Alpha X-rays as the source. A reverse-phase LC/MS

(LC/MS-2020 Shimadzu) equipped with a Phenomenex C18 column and a photodiode array (PDA)

detector was utilized. MALDI mass spectra were recorded on a MALDI, Voyager-DE PRO

Instrument. Zeta potential was measured using a Brookhaven ZetaPals at 25 ˚C under specified

conditions of pH. NMR spectra were acquired on a Bruker Avance III 400MHz NMR. Chemical

shifts were reported in ppm (δ) relative to residual solvent peaks (DMSO-d6 = 2.50 ppm for 1H).

Coupling constants (J) were reported in Hz. C18 column chromatography was performed on a C18-

reversed phase silica gel purchased from Sigma-Aldrich.

S3

2. Synthesis of materials

Raw HiPco (NanoIntegris®) single-walled carbon nanotubes (25 mg) were oxidized in 50

mL of an acid mixture (H2SO4 /HNO3, 3/1, v/v) in an ultrasonic bath set at 25 ˚C over 3 h 20 min.

After thorough washing with distilled water several times, the oxidized nanotubes were dried under

vacuum over 24 h, which yielded 18 mg of OX-SWCNTs. A phospholipid functionalized PEG

(PL-PEG) was prepared by an amide coupling with DSPE-050PA (115 mg) and TMS(PEG)12 (71

mg) in anhydrous dichloromethane (3.0 mL). After 12 h, in the case when much of the reactants

still remained due to mostly the hydrolyzed –N-hydroxysuccinimide (NHS) group of TMS(PEG)12,

N,N-Dicyclohexylcarbodiimide (DMAP) (20 mg) and 4-dimethylaminopyridine (10 mg) were

added, and amide formation proceeded . After 24 h of stirring at room temperature, the solvent was

dried, and the reaction mixture was washed with nanopure water (61 mL) and was collected through

vacuum filtration. Excess DMAP was further removed through dialysis. This reaction gave an

average MW of ca. 7.4–8.2 kDa of PL-PEG (Fig. S1).

The ox-SWCNTs were sonicated with PL-PEG in phosphate buffer (pH 8.2) for 1 h and then

stirred at room temperature. The PEGylated ox-SWCNT was washed low molecular weight PEG

chains with ultracentrifugation (11,000 rpm, 30 min × 3) using a 100 kDa Amicon centrifugal filter.

Doxorubicin (4.8 mg) was dissolved in 10 mL of phosphate buffer (0.1 M, pH 8.2). The DOX

solution was sonicated for 30 min and was stirred overnight. After thorough washing with the same

buffer solution through centrifugation, the amount of DOX wash-off was calculated from a UV-Vis

calibration curve. The reaction gave about 2.3 mg of DOX bound to 3.0 mg of ox-SWCNT/PL-

PEG, which gave a 77% of drug loading. This estimation differs by 23% from the UV-Vis titration

(Fig. S6).

S4

3. Characterization of phospholipid-polyethylene glycol (PL-PEG)

MALDI mass spectrometry A matrix solution was prepared with α-Cyano-4-hydroxycinnamic

acid (10.0 mg) in a 2 mL mixture of equal volumes of 0.2 % of trifluoroacetic acid (TFA) in H2O

and CH3CN. A PL-PEG was dispersed in water (1 mg/mL) and then mixed with the prepared

CH3CN/TFA solution (1:1, v/v).

Fig. S1 MALDI mass spectrum of PL-PEG. The average molecular weight was estimated around 7.4– 8.2

KDa.

3000.0 4800.2 6600.4 8400.6 10200.8 12001.0

Mass (m/z)

0

153.2

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sity

Voyager Spec #1=>BC=>NF0.7[BP = 2344.2, 2295]

7414.2999

7235.0125 8263.12986697.6969

8027.88118652.9011

9618.6464

6367.02383039.0970 6964.3100 8645.33908035.35546414.6480 9881.07609296.69117427.0163

6756.59053091.9522 9682.51147224.3874 8596.40016089.5578 7730.6214 10471.95576562.2478 11221.05029701.55068752.38858210.32557170.79663212.6215 6633.69295469.6942 10791.92638829.1984 9657.91347616.5667 8338.20894233.7504 7147.8929 10116.71886293.05085208.2513 9408.10198927.17293176.8301 7837.9905 8423.743810268.07626672.47336025.8038 9098.7391 9547.58997204.83734035.9017 10756.70648536.61727963.78255519.1138 10201.31713466.2160 4475.9673 6049.6676 9528.12356490.7109 8504.82957778.1333 9022.6351 10517.49463222.8462 9627.29145995.4234 8404.95587357.4191 11262.354810709.09707829.81224837.5296 9072.95703919.4699 10133.76605827.5514 8445.35976395.14743631.9501 11346.84139396.6212 10340.60404879.7171 5687.8982

10405.6904

S5

4. Characterization of oxidized HiPco SWCNTs (ox-SWCNT)

Raman spectroscopy Dried nanotubes were drop-cast on a microscope slide. Spectra were

collected with a 10 second exposure time and averaged across 5 scans per location. The collected

spectra were normalized to 1 with respect to the maximum intensity. After 3 h 20 min of an acid

treatment (H2SO4: HNO3, 3:1, v/v) with ultrasonication, the D band became wider and was shifted

to a higher wavenumber. ID/IG of the ox-SWCNT was substantially larger than that of the pristine

SWCNT, indicating the higher degree of functionalization.

Fig. S2 Raman spectroscopy of pristine HiPco SWCNT and ox-SWCNT.

Nanotubes D band (cm-1

) G band (cm-1

) ID/IG

intensity ratio

Pristine

SWCNT 1309 1591 0.08

ox-SWCNT 1327 1596 0.35

0

0.2

0.4

0.6

0.8

1

1200 1400 1600 1800 2000 2200 2400 2600 2800

No

rmali

zed

In

ten

sit

y

Wavenumber (cm-1)

ox-SWCNT

Pristine SWCNT

S6

Fourier transform spectroscopy (FTIR) ox-SWCNTs were homogeneously mixed with KBr.

Using KBr as the background and taking 32 scans per sample, a spectrum was obtained over the

range of 800 to 4000 cm-1

with a resolution of 4 cm-1

.

Fig. S3 Fourier transform infrared spectrum of ox-SWCNT.

X-ray photoelectron spectroscopy (XPS) The spot size of the sample was 400 µm (microns)

prepared on an aluminum plate. Charge compensation was provided by a low energy electron source

and Ar+ ions. Survey scans were collected using a pass energy of 150 eV, and high resolution scans

were collected using a pass energy of 50 eV. The average percentage indicates a mean obtained by

analyzing three different sample spots.

Peak (cm-1

) Functional group

3587 Ar–OH

2684–3433 (C=O)–OH

1728 (C=O)–OH

1567 –C=C–

1334 –S=O–

800 1300 1800 2300 2800 3300 3800

KM

Wavenumber (cm-1)

S7

0 200 400 600 800 1000 1200

Binding Energy (eV)

O KLL

Fig. S4 X-ray photoelectron spectroscopy (XPS) of ox-SWCNT. (a) Survey scan of the sample, (b) High

resolution spectra of C 1s and fitting curves corresponding to different functional groups, and (c) High

resolution spectra of O 1s and fitting curves.

Element Binding

Energy (eV)

Relative

Abundance (%)

C 1s 285.12 70.37

O 1s 532.47 25.22

S 2p 168.11 3.26

Surface functional

group

Binding

Energy (eV)

Relative

Abundance (%)

C–C (graphitic) 284.40 58.50

C=O 286.19 21.64

O=C–O 288.59 14.04

C–OH 285.14 5.85

Surface functional

group

Binding

Energy (eV)

Relative

Abundance (%)

O–C=O* 531.78 61.73

O=C–O* 533.23 35.80

C–O*H 532.64 2.47

O 1s

C 1s

S 2p

525 530 535 540

Binding Energy (eV)

O 1s

C=O*

O=C-O*

C-O*H

C KL1

280 285 290 295

Binding Energy (eV)

C 1s

C-C (graphitic)

C=O

O=C-O

C-OH

(a)

(b)

(c)

S8

5. Characterization of DOX-SWCNT

Fig. S5 Fluorescence emission spectra of DOX-SWCNT conjugate in comparison to free DOX of varying

concentrations.

UV-Vis titration determining the binding ratio of ox-SWCNT/PL-PEG with DOXS1

The

maximum binding ratio of DOX to ox-SWCNT/PL-PEG was determined from the fitting curve.

When the value of ΔA (ΔA = Abound DOX – AfreeDOX) reaches the maximum, the binding of DOX to

the nanotube is saturated, in which the wt equiv value (x) of the maximum is 0.964. Therefore, a

1:1 weight ratio of DOX to ox-SWCNT/PL-PEG was obtained from the fitting equation below.

(R2 = 0.9870)

Fig. S6 UV-Vis titration of ox-SWCNT/PL-PEG with DOX. The fitting curve was found using SigmaPlot

11.0.

Fitting

parameter Coefficient STD Error

a 0.0665 0.0049

b 0.3698 0.0382

xo 1.0960 0.0386

yo 0.0065 0.0026

2D Graph 3

Weight equivalent of [DOX]/ [SWNT-COOH/PL-PEG]

0.0 1.0 2.0 3.0 4.0 5.0

Absorb

ance

(= A

DO

X-S

WN

T -

Afr

ee

DO

X)

0.00

0.02

0.04

0.06

0.08

Col 2 vs Col 3

Col 2 vs Col 3: 1.4000

x column 3 vs y column 3

0.0

100,000.0

200,000.0

300,000.0

400,000.0

500,000.0

600,000.0

510 560 610 660

Em

issio

n I

nte

nsit

y (

A.U

)

Wavelength (nm)

DOX: 15.0 nM

DOX: 7.6 nM

DOX: 0.76 nM

DOX: 2.3 μM (DOX-SWCNT)

S9

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

Zeta

pote

ntial (m

V)

-40

-30

-20

-10

0

10

Col 2 vs Col 3

x column 16 vs y column 16

Weight equivalent of [DOX]/ [SWNT-COOH/PL-PEG]

Zeta potential titration of ox-SWCNT/PL-PEG with DOX A solution of ox-SWCNT/PL-PEG

in nanopure water (0.2 mg/mL) was prepared, and a 3 mL of the aliquot was transferred to a 20 mL

scintillation vial. Then the varying amount of DOX solution in water (1.3 mg/mL) was added to the

vial. After 30 min of sonication, the zeta potential of the solution mixture was measured with DLS.

Using a graphical linear fitting curve near the saturation point at y-axis (0 mV), the corresponding

wt equiv value was estimated. Approximately at 1.124 equiv, the zeta potential remains constant.

Here, a 1.1:1 binding ratio of DOX to ox-SWCNT/PL-PEG was found. The graphical linear fit was

obtained by OriginPro 8.5.

Fig. S7 Zeta potential titration of ox-SWCNT/PL-PEG with DOX.

6. Degradation experiments with myeloperoxidase, hydrogen peroxide, and chloride

(MPO/H2O2/Clˉ)

Each DOX-SWCNT sample was prepared by dispersing 0.03 mg of DOX-SWCNT in 720

µL of phosphate buffer (pH 7.4, 0.1 M). The concentration of free DOX was 0.02 mg/mL. Stock

solutions of DTPA and NaCl were added to the DOX-SWCNT solution, and their final

concentrations were adjusted to 0.38 mM and 0.14 M respectively. The concentration of DOX-

SWCNT was 0.04 mg/mL. For the experiment of +MPO/+H2O2/+Clˉ, 4.4 µg of MPO was added

every 24 h, and the H2O2 stock solution (18.75 mM) was added every 4 h (total volume of 30 µL

S10

per day). For −MPO/−H2O2/+Clˉ, the same amount of the pH 7.4 buffer solution was added. The

samples were stored in a standard cell culture incubator at 37 ˚C. For UV-Vis-NIR analysis,

samples were cooled at ambient temperature for about 10 min. Each spectrum was collected with

700 µL of a sample solution in a quartz sample holder (path length: 1 cm), and further absorbance

was recorded after each new addition of H2O2. A 0.02 mg/mL of ox-SWCNT/PL-PEG solution was

prepared in the same buffer condition. Then the same procedure was used to monitor the

degradation of the nanocarrier.

S11

0

0.01

0.02

0.03

0.04

0.05

0.06

900 950 1000 1050 1100

Ab

so

rban

ce (

A.U

)

Wavelength (nm)

0 h

48 h

96 h

7. Degradation under MPO/H2O2/Clˉ

Each of original sample was diluted 1:20 or 1:50 times with ethanol, and 3 μL of the diluted

solution was placed on a lacey carbon copper grid and then permitted to dry in ambient conditions

over 24 h.

Fig. S8 (a) NIR spectral change in the S11 band of ox-SWCNT/PL-PEG and TEM images of degradation in

phosphate buffer (0.1M, pH 7.4) of the same sample over (b) 0 h, (c) and (d) 24 h, (e) 48 h, (f) 72 h, and (g)

96 h.

(a) (b)

)

(a)

(c)

)

(a)

(d)

)

(a)

(f)

)

(a)

(e)

)

(a)

(d)

)

(a)

(b)

)

(a)

(c)

)

(a)

(a)

)

(a)

S11

) (a)

(g)

)

(a)

S12

8. Proposed degradation products of DOX formed under the conditions of +MPO/+H2O2/+Cl‾

and −MPO/−H2O2/+Cl‾ .

Scheme S1. (a) Degradation products of DOX possibly formed in the MPO-catalyzed oxidative and the

control (non-oxidative) conditions (b) ox-SWCNT and its functional group transformations upon reaction

with HOCl.

HOCl produced from MPO/H2O2/Clˉ can induce both oxidation and chlorination of DOX.

The –OH groups undergo a transformation into the carbonyl groups of 6,S2

and the primary amine

group are chlorinated selectively in 7.S3

Simultaneously, compound 11 can be formed from

phenolic (–OH) groups of ox-SWCNTs. If the nanotubes contain dangling bonds terminated with –

C=C– although this constituent is not abundant,S4

compound 12 may provide another competing

reaction with DOX.

(b)

(a)

S13

Compounds 8 and 9 were previously identified in MPO/H2O2/NO2ˉ, in which nitrite, a

strong oxidant (or cofactor), promotes reduction of the hydroquinone moiety of the B-ring (Scheme

1).S5

However, this pathway seems to less likely occur in our MPO/H2O2/Clˉ system.

Compound 3 is a known metabolite resulting from cleavage of the daunosamine by

hydrolysisS6

although its mechanism has not been well understood. However, 3 was not found in

the control condition. In the case of in vivo experiments, we may see more different degradation

pathways. Compounds 3 and 5 can be formed under reductive environments in cells. Cellular

enzymes such as NADPH-cytochrome P450 reductase and flavoenzymes initiate transformation of

the quinone of DOX to semiquinone (1eˉ reduction) or hydroquinone (2eˉ reduction).S7

Compound

5 is formed by conversion of a ketone to an alcohol in ring A. Compound 3 is associated with

various pathways, including electron transfer and quinone methide formation.S8

No matter how the

reduction process proceeds, DOX will eventually cleave the daunosamine, and a new bond is

formed with nucleophiles, electrophiles, or radicals besides the hydroxyl group at C7 depending on

the degradation pathway.

S14

9. Identification of a degradation product (compound 4)

1H NMR analysis In order to collect a large amount of sample for analysis, the weight and volume

of each Doxorubicin solution was scaled up by 48 times while maintaining the same concentration

used in the UV-Vis-NIR experiments. After incubation, the water in the samples was removed

using SpeedVac over 6.5 h. The collected solid contained Doxorubicin degradation products and

phosphate salt. Using methanol, N,N-dimethylformamide (DMF) and toluene, the collected

samples were washed thoroughly and dried with a rotary evaporator and then a high vacuum pump

over 24 h.

Fig. S9 1H NMR spectrum of free Doxorubicin (dimethyl sulfoxide-d6, 400 MHz) at 0 h.

0 h

1

S15

Fig. S10 (a) 1H NMR spectrum of free DOX (−MPO/−H2O2) without purification after 32 h and (b) the

aromatic proton shift region (δ7.0–8.3) of the same spectrum. The protons of compound 4 were assigned

based on a precedent analysisS9

and a predicted NMR data using Advanced Chemistry Development, INC.

(ACS/Labs) Software V11.01 (©1994–2013 ACD/Labs).

H6

δ8.06 (d, J = 3.6)

H4 (d, J = 8.4) H5 (dd, J = 7.6 )

4 4

After 32 h

(a)

(b)

S16

3.500e5

3.000e5

2.500e5

2.000e5

1.500e5

1.000e5

5.000e4

0m/ z1500.01000.0500.0

Sample: was27_20130812_01_002

Data File Name: C:\ LabSolutions\ Data\ Project1\ was27\ was27_20130812_01_002.lcd

Date and Time: Monday, August 12, 2013 3:27:34 PM

Int

389.20

390.20

533.55335.00

155.05534.40

1.200e5

1.000e5

8.000e4

6.000e4

4.000e4

2.000e4

0m/ z1500.01000.0500.0

Sample: was27_20130619_02_001

Data File Name: C:\ LabSolutions\ Data\ Project1\ was27\ was27_20130619_02_001.lcd

Date and Time: Thursday, June 20, 2013 12:12:19 PM

Int 100.65

354.15

141.85

359.15

360.95

355.05 652.30

LC/MS analysis Electro spray ionization (ESI-MS) was used to measure the mass of the

degradation products. The samples were scanned in both positive and negative modes. H2O and

CH3CN were used as eluents.

MW=336.30 g/mol

Fig. S11 LC/MS chromatograms and mass spectra of the control sample after 32 h. To remove the

phosphate salt, these samples were purified with C18 mini-column chromatography. (a) The peak 359.15

(m/z) is an adduct of [MW + Na]. (b) The peak of 335 (m/z) is indicative of a negative adduct [MW−H].

Negative mode

(a)

(b)

4

Positive mode

S17

10. Degradation experiments with peroxynitrite

Each DOX-SWCNT sample had a concentration of 0.03 mg of DOX-SWCNT in 808 µL of

phosphate buffer (pH 7.4, 0.1 M), and 0.02 mg/mL of free DOX was prepared in the same buffer.

Stock solutions of all other reagents were prepared every day. Then 7.5 μL of a xanthine oxidase

(XO) solution (×50 diluted from the original enzyme) containing 0.15–0.3 mU of XO, was added

per day, followed by additions of 7.5 μL of xanthine solution (7.0 mM) and 7.5 μL of a PAPA

NONOate solution (3.5 mM) every 2 h (total 6 times per day). Due to the dilution effect, the

concentrations of both xanthine and PAPA NONOate solutions were raised after 6 additions,

maintaining the same amount of each reagent relative to the total volume of the DOX-SWCNT

solution. After incubation over a given time period, all the samples were filtered using Amicon

centrifuge filters (size: 1,000 Da). The filtrate and the concentrate were separately collected by

ultracentrifuge (14,000 × g, 10 min), and the concentrate was further diluted in phosphate buffer

(0.1M, pH 7.4) for proper analysis. Then fluorescence emission spectroscopy (λexcitation: 488 nm,

λemission: 592 nm) was utilized to measure the concentration of DOX-SWCNT within a linear

calibration range at room temperature. Due to some loss of DOX-SWCNT after the ultracentrifugal

filtration, a separate test determining the average recovery rate of DOX-SWCNT was performed to

estimate the concentration accurately. The average recovery rate of five replicated samples was 62

± 4 (%) from the original amount.

S18

0

0.02

0.04

0.06

0.08

0.1

350 450 550

Ab

so

rban

ce U

nit

Wavelength (nm)

0 h

48 h

11. Characterization of peroxynitrite-mediated oxidation

Fig. S12 (a) Degradation of free DOX after 48 h. Absorption increased at 480 nm probably due to the

formation of a degradation product with the same absorption properties. (b) NIR absorption spectra of ox-

SWCNT/PL-PEG and (c) UV-Vis-NIR absorption spectra of DOX-SWCNT (The spectra were normalized at

862 nm).

Fig. S13 TEM images of peroxynitrite-mediated degradation in phosphate buffer (0.1M, pH 7.4) of ox-

SWCNT/PL-PEG over (a) 0 h, (b) 24 h, (c) 48 h, (d) 72 h, and (e) 96 h. TEM samples were prepared using

the same method described in page S11.

0

0.005

0.01

0.015

0.02

0.025

0.03

900 950 1000 1050 1100

Ab

so

rban

ce U

nit

Wavelength (nm)

0 h

48 h

96 h

(a) (b) (c) (d)

(e)

0

0.04

0.08

0.12

0.16

0.2

350 550 750 950

Ab

so

rban

ce U

nit

Wavelength (nm)

0 h

48 h

(a) (c) (b)

S19

Table 1. Zeta potential changes upon sequential addition of each

component of DOX-SWNT at pH 7.4.

12. Zeta potential titration with MPO

The laser of the zeta potential analyzer was set at 532 nm. The zeta potential of unbound

pure MPO was measured separately, which gave a negative potential (−9.0 ± 1.7 mV). A rapid

color change to a very bright yellow upon the laser irradiation suggests the photosensitive heme of

MPO.S10

After each titration, a sample containing DOX-SWCNT and MPO solutions was placed

under ambient temperature and pressure over 1 h until their binding reached equilibrium, and then

zeta potential was recorded. For the titration sample, solutions of DOX-SWCNT (0.3 mg/mL) and

MPO were prepared in a mixture of nanopure water and 0.05 M, pH 7.4 phosphate buffer (17:1,

v/v).

Sample Name Zeta Potential

(mV)

SWNT-COOH -48.3 ± 1.6

SWNT-COOH/PL-PEG -34.3 ± 1.1

SWNT-COOH/PL-PEG/DOX -14.2 ± 2.0

Free DOX +9.2 ± 1.2

S20

13. In vitro cell studies

Mice Pathogen-free C57BL/6 mice (7–8 wk old) mice from Jackson Labs (Bar Harbor, ME, USA)

were individually housed and acclimated for 2 weeks. Animals were supplied with water and food

ad libitum and housed under controlled light, temperature, and humidity conditions. All animal

studies were conducted under a protocol approved by the Institutional Animal Care and Use

Committee.

Cell B16 melanoma cells were obtained from American Type Culture Collection (ATCC, Manassas,

VA, USA) and maintained in RPMI 1640 medium that was supplemented with 2 mM L-glutamine,

100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 10% heat-inactivated FBS, 0.1 mM

nonessential amino acids, and 1 mM sodium pyruvate (Invitrogen Life Technologies, Inc., Grand

Island, NY, USA). Tumor conditioned medium was collected from sub-confluent cultures,

centrifuged (300 g, 15 min) and cell-free supernatant was collected, aliquoted and used to treat

MDSC.

For MDSC generation, bone marrow cells from tibia were isolated, filtered through a 70 m

cell strainer, and red blood cells were lysed with lysing buffer (155 mM NH4Cl in 10 mM Tris-HCl

buffer pH 7.5, 25°C) for 3 min. After RBC lysis, cells were washed and used for MDSC sorting.

CD11b+ Gr-1

+ MDSC were isolated from the bone marrow cell suspensions by magnetic cell

sorting using a mouse MDSC Isolation Kit (MACS, Miltenyi Biotec, Auburn, CA, USA) according

to the manufacturer’s instructions. Isolated MDSC were cultured in supplemented RPMI 1640

medium with 25% (vol/vol) B16 conditioned medium for 48 h to generate tumor-activated MDSC

expressing high levels of MPO.

S21

Apoptosis assay CellTracker™ Orange CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino)

tetramethylrhodamine) (Molecular Probes) was utilized to label B16 melanoma cell prior to co-

culture experiments. The use of this fluorescent dye allowed distinguishing tumor cells and MDSC

and assessing apoptosis in tumor cells. B16 cells and cells in mixed cultures were stained with

Annexin V (Pharmingen) and the percentage of apoptotic Annexin V+ CMTMR+ cells was

determined by flow cytometry (FacsCalibur, BD Biosciences, San Jose, CA, USA) and analyzed by

FlowJo software.

Statistics Results were analyzed using one-way ANOVA and Student unpaired t-test with Welch's

correction for unequal variances. All experiments were done in triplicates or repeated at least twice,

and the results were presented as the means ± SEM (standard error of the mean). P values of < 0.05

were considered to be statistically significant.

Cell proliferation assay 3LL cells were labeled with CellTracker™ Orange CMTMR (5-(and-6)-

(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine) (Molecular Probes) and co-incubated

with free DOX and DOX-SWCNT in the presence of tumor-activated MDSC. Co-incubation of

3LL cells with SWCNT, MDSC or both served as controls. After 24 h of incubation, the number of

labeled 3LL cells was determined by flow cytometry (FacsCalibur) events calculated for 1 min.

Increased number of cells (versus control medium group) suggests an increase in cell proliferation,

while lower cell number reflects cytotoxic (cell death) and/or cytostatic (inhibition of cell

proliferation) effects on tumor cells.

S22

14. Concentrations of released DOX in cell medium

Fig. S14 MDSC were incubated in the medium containing 5 μM solutions of free DOX and DOX-SWCNT

(with 100% drug loading). After 25 h, each supernatant of the drug-incubated cell medium was collected by

centrifugation (10,000 g, 15 min), and the concentration of free DOX was estimated by measuring

fluorescence emission intensity at 590 nm (λexcitation=488 nm) using standard calibration fit.

0 5 20 25

0

1

2

3

4

5 Free DOX

DOX-SWCNT

DO

X c

on

ce

ntr

atio

n (

µM

)

Time (h)

S23

15. Ex vivo pH-dependent DOX release from SWCNT

Fig. S15 The same concentration of DOX-SWCNT (with 100% drug loading) used in the degradation

experiment was prepared in phosphate buffer (0.1 M, pH 7.4) and acetate buffer (0.1 M, pH 5.5) over 24 h,

respectively. The free DOX released from the nanotube carrier was collected by filtration through a 10kD

Amicon centrifugal filter (11,000 rpm, 10 min), and the concentration of free DOX was measured using UV-

Vis at 480 nm. The error bars indicate the means ± SD of three replicate measurements.

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25

No

rmalized

ab

so

rban

ce (

%)

Time (h)

pH 7.4

pH 5.5

S24

16. MDSC abrogated cytotoxic/cytostatic effect of free DOX, but not DOX-SWCNT, on 3LL

cells in vitro.

Fig. S16 3LL lung carcinoma cells and bone marrow-derived tumor-activated MDSC were

generated and cultured as described in M&M. Cells were co-cultured for 24 h in the presence of

soluble free DOX or DOX-SWCNT alone or together. ox-SWCNT/PL-PEG and MDSC+ ox-

SWCNT/PL-PEG served as controls. The number of tumor cells was determined by assessing flow

cytometry events for 60 sec as described in M&M. All cell cultures were set in triplicates and

results are shown as the mean SEM (N=2). *, p<0.01 versus control (medium) group (One way

ANOVA).

med

ium

DOX-f

ree

DOX-C

NT

CNT

MDSC

MDSC

+DOX-f

ree

MDSC

+DOX-C

NT

MDSC

+CNT

3L

L/F

AC

S/6

0s

ec

0

10000

20000

30000

40000

50000

60000

**

*

S25

17. References

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