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SUPPORTING INFORMATION

Corroles and Corrole/Transferrin Nanoconjugates as Candidates for Sonodynamic Therapy

Vinay Kumar Sharma,a Atif Mahammed,a Matan Soll, a Boris Tumanskiia and Zeev

Grossa,*

aSchulich Faculty of Chemistry, Technion-Israel Institute of Technology, Haifa 32000, Israel.

E-mail: chr10zg@technion.ac.il

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2019

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Table of contents Page No.

[A] Instrumentation and Materials………………………………………………… 3

[B] General formulation of transferrin/corrole nanoconjugates ………………….. 4

[C] Characterization of NPs (HPLC traces)………………………………………. 4-6

[D] Spin detection assay…………………………………………………………... 6-7

[E] Singlet oxygen scavenging assay……………………………………………… 7-8

[F] Spin quantification assay………………………………………………………. 8-9

[G] UV-vis spectra………………………………………………………………… 10-11

[H] Cell proliferation assay and sonodynamic treatments…………………………. 11-13

[I] References ……………………………………………………………………… 13

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Instrumentation and Materials:

Commercially available chemicals were purchased from Sigma-Aldrich, Merck and Chem

Intel and used as received unless otherwise stated. Pyrrole was subjected to filtration on column

packed with neutral aluminium oxide before use, while the rest of the reagents used without

further purification. Unless mentioned separately, synthesis was performed at ambient

conditions. The Silica gel (230-400 mesh) used for column chromatography was obtained from

E. Merck Ltd. Either flash and preparative thin layer chromatography were performed to purify

the compounds. Nanoparticles were prepared in Dulbecco’s Phosphate Buffered Saline

purchased from Sigma Aldrich, dialysis was conducted using a Slide-A-Lyzer® Dialysis

cassette. The source of the DU-144 cells was ATCC, medium (DMEM from ATCC) and serum

from Biological Industry-Mishmar Haemek.

UV-vis Spectroscopy:

The UV-vis spectra of synthesized corroles were recorded on Agilent Technologies Cary 8454

UV-vis spectrophotometer. Quartz cuvettes of 1.0 cm thickness were used to measure the

samples.

EPR Spectroscopy:

The EPR spectra were recorded on a Bruker EMX-10/12 X-band (ν = 9.3 GHz) digital EPR

spectrometer. The spectra were recorded at a microwave power of 1 mW, 100 kHz magnetic

field modulation of 1 G amplitude. Digital field resolution was 2048 points per spectrum.

Spectra processing and simulation were performed with the Bruker WIN-EPR and SimFonia

Software.

DLS and Nanosight NS300 systems:

The size of the nanoparticles was measured using the Nanosight NS300 Analyser in accordance

with manufacturer’s instructions.

HPLC:

HPLC analysis was performed using a MERCK HITACHI HPLC system with a diode array

detector supported with HPLC Chromaster Driver for Waters® Empower™3 Software. 10 µL

samples were injected using the auto sampler. Size exclusion chromatography was done with

either a SuperoseTM 6 10/300 GL gel column or a sephadexTM 200 10/300 GL column (as

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noted), with 0.5 mL/min eluting rate and sterilized PBS (Sigma, sterile-filtered, isotonic, pH

7.2) as eluent.

Preparation of corroles sonosensitizers:

1-H3, 1-Al, 1-Ga, 1-Fe, 1-Mn, 2-H3, 2-Al, and 2-Ga were synthesised as previously described.1-

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General formulation of transferrin/corrole nanoconjugates:

1.8 ml of PBS containing 200 μL of 200 μM transferrin in PBS was taken in vial, stirred at 400

rpm in a 5 oC water bath. Subsequently, a solution of 200 μL of 1 mM corrole (either 2-H3, 2-

Al or 2-Ga) dissolved in DMSO was added dropwise into it. After 1 minute stirring, the solution

was transferred to the dialysis cassette for 24 h dialysis in a 1 litre PBS solution. After dialysis,

the transferrin/corrole nanoconjugates were characterized by size exclusion HPLC and DLS.

Sample preparation for in vitro US:

600 μL of either a 0.4 mM aqueous corrole (1-H3, 1-Al, 1-Ga, 1-Fe, or 1-Mn) solution or 400uL

of pre-prepared nanoconjugates of corrole (2-H3, 2-Al, or 2-Ga) were diluted in either 1.9 mL

or 10 μL (the NPs) of a phosphate buffered saline solution (PBS) containing TMPone (2,2,6,6-

tetramethyl-4-piperidone, Sigma-Aldrich, 50 mM and 425 mM). The pre-prepared solutions of

1-H3, 1-Al, and 1-Ga were also checked with 20 μM transferrin protein. Singlet oxygen

generation was monitored using electron spin resonance (EPR) spectroscopy and the spin

trapping technique, utilizing TMPone as the spin-probing agents for singlet oxygen.

US exposure:

All the solutions (containing 1-H3, 1-Al, 1-Ga, 1-Fe, 1-Mn, 2-H3/TF-NPs, 2-Al/TF-NPs, 2-Ga-

/TF-NPs, 1-H3 + Transferrin, 1-Al + Transferrin and 1-Ga + Transferrin) were exposed to US

in a polystyrene tube for 5 min using a plane wave transducer, operating at 50% of 100 Hz

wave mode at f = 1 MHz under US power of 3.0 W/cm2. After the irradiation, EPR spectra

were immediately recorded, using 50 μL of the solution.

HPLC graph:

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Fig S1: HPLC chromatogram of 2-H3/TF-NPs. The red and blue curves represent corrole

(measured at 421 nm) and protein (measured at 280 nm) respectively. The 2-H3/TF-NPs

fraction eluted at 16.22 min and the corrole free transferrin peak was obtained latter at 27.42

min

Fig S2: HPLC chromatogram of 2-Al/TF-NPs. The red and blue curves represent corrole

(measured at 421 nm) and protein (measured at 280 nm), respectively. The 2-Al/TF-NPs

fraction eluted at 16.11 min and the corrole free transferrin peak was obtained latter at 27.42

min

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Fig S3: HPLC chromatogram of 2-Ga/TF-NPs. The red and blue curves represent corrole

(measured at 421 nm) and protein (measured at 280 nm) respectively. The 2-Ga/TF-NPs

fraction eluted at 16.14 min and the corrole free transferrin peak was obtained latter at 27.42

min

Spin detection:

The short-lived singlet oxygen produced by the sonsensitizers was identified by its reaction

with the effective spin trap TMPone, which leads to the paramagnetic TMPone/1O2 adduct

known as TMPone nitroxide Scheme S1 (long lived-form), which can be observed at room

temperature using conventional EPR instrument. The characteristic three peak EPR signal (aN

15.7 G and g-factor of 2.0062) was obtained upon the irradiation of 1-H3, 1-Al, 1-Ga, 2-H3/TF-

NPs, 2-Al/TF-NPs, 2-Ga-/TF-NPs, 1-H3 + Transferrin, 1-Al + Transferrin and 1-Ga +

Transferrin, in the presence of TMPone (Fig. 3 in the main text) while no signals were obtained

in the cases of 1-Fe, 1-Mn, and control (TMPone + buffer and buffer + protein) with and

without US exposure.

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Scheme S1: Generation of TMPone/1O2 adduct by reaction with spin trap TMPone and the

singlet oxygen produced by the sonosensitizers upon US exposure.

Singlet oxygen scavenging assay:

1-H3, 1-Al, and 1-Ga, 2-H3/TF-NPs, 2-Al/TF-NPs, 2-Ga-/TF-NPs were also examined in the

presence of the singlet-oxygen scavenger sodium azide (1M NaN3) during the exposure of US,

in order to deduce the role that singlet oxygen plays in EPR signal production.

Fig S4: Effect of quencher (1 M NaN3) on EPR signal intensity of sonosensitizers (100 µM) in

the presence of TMPone (38 Mm) in PBS solution following irradiation by US power at 3

W/cm2 for 5 min at 1 MHz.

1-Al

1-Ga

1-H3

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2-Al/TF-NPs

2-Ga/TF-NPs

2-H3/TF-NPs

Fig S5: Effect

of quencher (1

M NaN3) on

singlet oxygen

produced by US

power at 3

W/cm2 for 5

min at 1 MHz.

Spin

quantification:

Spin

quantification

was done by

comparing the

intensity of

unknown

Concentration of unknown = * concentration of

𝑝𝑒𝑎𝑘 𝑡𝑜 𝑝𝑒𝑎𝑘 𝑎𝑚𝑝𝑙𝑖𝑡𝑢𝑑𝑒 𝑜𝑓 𝑢𝑛𝑘𝑛𝑜𝑤𝑛𝑝𝑒𝑎𝑘 𝑡𝑜 𝑝𝑒𝑎𝑘 𝑎𝑚𝑝𝑙𝑖𝑡𝑢𝑑𝑒 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑

standard

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samples with a known sample as standard. TMPone nitroxide was used as the standard sample,

in 10-5 M and 10-6 M concentration in our work (Fig. S6) for the technique called relative

measurement. So, the concentration of singlet oxygen produced by the sonosensitizers were

calculated by using the formula given below. The peak to peak amplitude were calculated as

shown in the Fig S7. One important note to bare in mind during this comparison that the EPR

signals must have the same linewidth. According to the formula, the concentration of singlet

oxygen produced by sonosensitizers are shown in the main text Fig 4. 1-H3 + Transferrin shows

highest intense signal which correspond to the amount of singlet oxygen formed in the system.

Fig S6: EPR signals of TMPone nitroxide as standard with concentration of (a) 10-6 M and (b)

10-5 M in PBS solution. Ultrasonic parameters were: US power, 3 W/cm2; time, 5 min; wave

frequency, 1 MHz.

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Fig S7: Peak to peak amplitude and linewidth of an EPR signal.

UV-vis spectra:

Fig S8: Change in the UV-vis spectra of 1-H3 upon addition of 20 % apo-transferrin protein.

The spectra were recorded in PBS buffer solution.

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0

0.05

0.1

0.15

0.2

0.25

350 450 550 650

Abs

orba

nce

Wavelength (nm)

1-Al1-Al+apo-Transferrin

Fig S9: Changes in the UV-vis spectra of 1-Al upon addition of 20 % apo-transferrin protein.

The spectra were recorded in PBS buffer solution.

0

0.1

0.2

350 400 450 500 550 600 650

Abs

orba

nce

Wavelength (nm)

1-Ga1-Ga+apo-Transferrin

Fig S10: Changes in the UV-vis spectra of 1-Ga upon addition of 20 % apo-transferrin protein.

The spectra were recorded in PBS buffer solution.

Cell proliferation assay and sonodynamic treatments:

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Tumor cells DU-145 were seeded in 96-well microtiter plates (8×103 cells per well; 100 μL per

well) 24 h before the addition of the 2-Ga NPs. At the time of drug treatment, stock solutions

of compounds were diluted 10-fold to the desired final concentrations with EMEM medium.

Aliquots of 10 μL of these diluted solutions were added to the appropriate microtiter wells

containing 90 μL of growth medium, resulting in the required final drug concentrations (5

concentrations per compound, ranging from 0.02 to 27 μM). All cells were incubated in the

dark throughout the 24-72 h treatment period and did not receive prolonged exposure to light.

Following 24 h incubation at 37 °C, cells were exposed to ultrasound waves using a Saniflex

transducer (Chattanooga, Guildford Surrey, UK) at 1 MHz, 0.3 w/cm2 50% for 2 min. 24 h post

exposure to ultrasound cells viability was determined using the MTT assay (Sigma Aldrich)

according to the manufacturer’s instructions. Absorbance was measured using a microplate

reader (Synergy 4; Biotek Instruments) at 570 nm/630 nm. Experiments were performed in

triplicates and each dose response represents the mean of three or more independent

experiments.

0102030405060708090

0.02 5 9 17 27 37

% o

f cel

l sur

viva

l

2-H3 NPs (µM)

* * **

Fig S11: Effects of sonodynamic treatment on DU-145 Tumor cells growth. DU-145 cells were

incubated for 24 h with the 2-H3 NPs (at various concentration ranging from 0.02 to 37 μM)

and then exposed to very low intensity US (0.3 W/cm2 for 2 min at 1 MHz. Cell proliferation

was evaluated after 24, using the MTT assay. Data points are reported as mean ± SEM *P<0.01

vs. reciprocal treatment without US. Representative of three independent results.

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0

20

40

60

80

100

120

2 9 15 27 57 107

% o

f sur

viva

l

1-Ga (µM)

* * *

Fig S12: Effects of sonodynamic treatment on DU-145 Tumor cells growth. DU-145 cells were

incubated for 24 h with the 1-Ga (at various concentration ranging from 2 to 107 μM) and then

exposed to very low intensity US (0.3 W/cm2 for 2 min at 1 MHz. Cell proliferation was

evaluated after 24, using the MTT assay. Data points are reported as mean ± SEM *P<0.05 vs.

reciprocal treatment without US. Representative of three independent results.

0102030405060708090

100

9 15 27 57 107

% o

f cel

l sur

viva

l

1-Al (µM)

Fig S13: Effects of sonodynamic treatment on DU-145 Tumor cells growth. DU-145 cells were

incubated for 24 h with the 1-Al (at various concentration ranging from 9 to 107 μM) and then

exposed to very low intensity US (0.3 W/cm2 for 2 min at 1 MHz. Cell proliferation was

evaluated after 24, using the MTT assay.

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References:

1. A. Mahammed, I. Goldberg and Z. Gross, Org. Lett., 2001, 3, 3443-3446.2. A. Kanamori, M.-M. Catrinescu, A. Mahammed, Z. Gross and L. A. Levin, J. Neurochem., 2010,

114, 488-498.3. M. Soll, O. Bar Am, A. Mahammed, I. Saltsman, S. Mandel, M. B. H. Youdim and Z. Gross, ACS

Chem. Neurosci., 2016, 7, 1374-1382.4. Z. Gross, N. Galili and I. Saltsman, Angew. Chem. Int. Ed., 1999, 38, 1427-1429.5. J. Bendix, I. J. Dmochowski, H. B. Gray, A. Mahammed, L. Simkhovich and Z. Gross, Angew.

Chem. Int. Ed., 2000, 39, 4048-4051.6. A. Mahammed and Z. Gross, J. Inorg. Biochem., 2002, 88, 305-309.