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Subphthalocyanines: addressing water-solubility, nano-encapsulation, and activation for optical imaging of B16 melanoma model. Yann Bernhard,a Pascale Winckler,b Rémi Chassagnon, c Philippe Richard,a Élodie Gigot,a Jean-Marie Perrier-Cornet, b and Richard A. Decréau*a 5
DOI: 10.1039/b000000x
a Institut de Chimie Moléculaire de l’Université de Bourgogne (ICMUB), UMR 6302 CNRS-Université de Bourgogne, BP 47870, F-21078, Dijon Cedex, France; E-mail: Richard.Decreau@u-bourgogne.fr b Université de Bourgogne, AgroSup Dijon, Dimacell Imaging Ressource Center, UMR A 02.102 PAM, F-21000 Dijon, France c Laboratoire Interdisciplinaire Carnot Bourgogne, UMR CNRS 6303- Université de Bourgogne, F-21078 Dijon, France10
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Outline
A. Chemistry and spectroscopic studies A1. Abbreviations .................................................................................................... 3
A2. Chemicals ............................................................................................................ 3
A3. Chromatography .............................................................................................. 4
A4. Characterizations methods ....................................................................... 4
A5. Synthesis ............................................................................................................. 6
A6. Purification ........................................................................................................ 11
A7. Analyses ............................................................................................................. 12
B. Liposomes B1. Liposome Preparation ................................................................................ 18
B2. Liposome First characterisation ........................................................... 18
B3. Liposome Purification by HiTRAP ....................................................... 19
B4. Liposome SIZE: DLS and TEM ............................................................... 19
B5. Liposome CONTENT: UV/Vis .................................................................. 20
C. Spectroscopic studies and Activation…………………………………………..21
D. Biological studies D1. Cells (culture, incubation, fixation) ..................................................... 25
D2. Confocal Microscopy .................................................................................. 26
D3. Biphoton Microscopy .................................................................................. 26
D4. Cell Viability Assay ....................................................................................... 27
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A-‐ CHEMISTRY AND SPECTROSCOPIC STUDIES
A1. Abbreviations
For clarity purposes simple labels are used all along the manuscript text:
• Initial Subphthalocyanine (SubPc) bearing chlorine atom are labelled SubPc-Cl (2)
• Second stage SubPc with a distal phenoxy picket are labelled according to the
substituent in para/meta position: SubPc-NO2 (1a), SubPc-NH2 (1b), SubPc-
(N((CH2)3SO3Na)2 (1c).
• Lipidic nanoparticle is labelled Np
A2. Chemicals
Chemicals used in this study are from various providers: Acros Organics [1,2-
dicyanobenzene (98 %, ref. 174012500), 4-aminophenol (97 %, ref. 104272500), 4-
nitrophenol (99 %, ref. 157052500), iodomethane (stabilized, 99 %, ref. 122371000),
bromoethane (98 %, ref. 154215000)], Sigma Aldrich [boron trichloride (1 M in p-xylene, ref.
345458), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (≥ 99 %, ref. P4329), 1,3-
propanesultone (98 %, ref. P50706)], Alfa Aesar [palladium 10 % on carbon (ref. A12012),
acetic anhydride (≥ 99 %, ref. 320102)], Fisher Scientific [trifluoroacetic anhydride (≥ 99 %,
ref. 147815000)], TCI [3-(dimethylamino)phenol (> 97 %, ref. D0657), N,N-diethyl-3-
aminophenol (> 97 %, ref. D0470)]. All chemicals and solvents were used as supplied without
further purification.
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A3. Chromatography
Compounds 1a-c, 2 and 3a-b were purified on column chromatography using silica gel
(60A, SDS) and a specific mixture of solvents as described in section 2. Compound 3a-b, 1c
were purified on column chromatography using alumina gel (60A, SDS) and a specific
mixture of solvent as described in section 2.
Compound 1c (sulfonated species) was purified and separated using on Dionex Ultimate
3000 semi-preparative column equipped with a C18 column. The method employed was the
following: eluent A: CH3CN/ 0.1 % TFA; eluent B: H2O/ 0.1 % TFA; flow: 2.8 mL/min; ramp
from A/B 10:90 to 50:50 in 40 min then A/B 50:50 during 5 min; return in 1 min to initial
conditions; detector: 200 nm, 300 nm, 565 nm.
A4. Characterization methods
MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionisation - Time of Flight
Mass Spectroscopy):
Measurements were performed on Ultraflex II LRF 2000 (BRUKER), using dithranol or DHB
as a matrix. Solutions were prepared by dissolving 1 mg of compound into 1 mL of
appropriate solvent.
ESI-Q MS (ElectroSpray Ionisation-Quadripole Mass Spectroscopy): Measurements were performed on LTQ Orbitrap XL (THERMO) coupled to HPLC Ultimate
3000 (DIONEX). The solution was prepared from 1 mg compound dissolved in 1 mL of an
appropriate solvent, and subsequently diluted 100 times with methanol.
Fluorescence spectroscopy: Fluorescence measurements were performed on a Jasco FP-8500 spectrofluorometer equipped
with a Xe source. Fluorescence quantum yields were calculated using Rhodamine 6G in
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methanol as a reference (ΦF = 0.94). Excitation was performed at 488 nm for both sample and
reference. Emission spectra were recorded for an absorbance at 488 nm comprise between
0.03 and 0.07. Fluorescence quantum yields (ΦF) were determined by the comparison method,
using the following equation:
𝜙! = 𝜙! 𝑆𝑡𝑑 × !! !"#
!× !!!"!!"#
!!!"!!"# !"#× ! !"#
!
With:
Std correspond to standard (Rhodamine 6G)
ΦF and ΦF(Std): fluorescence quantum yields
η and η(Std): refractive index of the solvent (MeOH for standard; DCM, DMF or water for samples)
Abs and Abs(Std): absorbances at excitation wavelength (488 nm)
A and A(Std): areas under the fluorescence curves
HPLC (High Performance Liquid Chromatography):
Hydrophilic compound (1c) was analyzed on Dionex Ultimate 3000, equipped with a
Chromolith High Resolution RP-18 column (5-4.6 mm, Merck). The Method used was the
following: eluent A: CH3CN + 0.1 % TFA; eluent B: H2O + 0.1 % TFA; flow: 3 mL/min;
equilibrate for 1 min 45 min afterwards; ramp from 100 % B to 100 % A; duration: 5 min;
keep constant for 1 min; return in 1.5 min to initial conditions; detector: 214 nm, 230 nm, 254
nm, 565 nm.
NMR spectroscopy: Measurements were performed on a Bruker Dalton X, at 300 MHz (1H), 500 MHz (1H), 75
MHz (13C) or 96 MHz (11B) in various deuterated solvents (CDCl3, DMSO-d6, D2O, acetone
d6), with chemical shifts reported as δ in ppm relative to TMS (residual chloroform from
deuterated chloroform chemical shift was set at 7.26 ppm, deuterated dimethylsufoxyde,
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deuterated acetone at 2.05 and deuterated water at 4.79) and coupling constants expressed in
Hz. The following abbreviations were used to describe spin multiplicity: s = singlet, d =
doublet, t = triplet, m = multiplet.
UV-Visible spectroscopy:
SubPc spectra (in solution or liposome suspension) were performed on a Shimadzu UV-
2550 spectrophotometer. Free subphthalocyanine spectra were recorded in DCM, DMF (1ab)
or in water/buffer (1c or 1ab entrapped in liposome); in glass cuvettes 1x1x3 cm (1 cm path).
X-Ray Diffraction:
Experimental. Single crystals of H18C30N7OB (1b) were obtained by recrystallisation in
dichloromethane. A suitable crystal (0.22×0.15×0.07 mm3) was selected and mounted on a
mylar loop on a Bruker APEX-II CCD diffractometer. The crystal was kept at 115 K during
data collection. Using Olex2 [(Dolomanov et al., 2009)], the structure was solved with the
ShelXS [(Sheldrick, 2008)] structure solution program, using the Direct Methods solution
method. The model was refined with the ShelXL [(Sheldrick, 2008)] refinement package
using Least Squares minimisation.
A5. Synthesis
N-‐(4-‐hydroxyphenyl)acetamide (4):
To a solution of 4-aminophenol (2 g, 18.3 mmol) in 30 mL of absolute ethanol was added
acetic anhydride (1.74 mL, 18.3 mmol). The solution was stirred for 15 min at room
HN
OH
O
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temperature, then evaporated to dryness. The resulting solid was purified by silica gel column
chromatography (eluent: DCM/MeOH 95:5) to give N-(4-hydroxyphenyl)acetamide as a
white powder (2.65 g, 96 %). 1H NMR (300 MHz, acetone-d6, 300 K): δ (ppm)= 2.03 (s, 3H);
6.75 (d, 3J= 9.0 Hz, 2H); 7.43 (d, 3J= 9.0 Hz, 2H); 8.21 (s, 1H); 8.94 (s, 1H).
2,2,2-‐trifluoro-‐N-‐(4-‐hydroxyphenyl)acetamide (5):
A solution of 4-aminophenol (5 g, 45.8 mmol) in THF (70 mL) was cooled in an ice bath
for 15 min. Trifluoroacetic anhydride (19.6 mL, 0.141 mol) was added dropwise under
stirring for 30 min. The reaction mixture was stirred in an ice bath for an additional 1.5 h. The
solvent was removed under reduced pressure, the residue was then dissolved in ethyl acetate
(200 mL). The solution was washed with a saturated aqueous sodium bicarbonate solution
(3×200 mL) and water (3×100 mL), then dried with magnesium sulfate, filtered off, and
concentrated under vacuum. The product was purified by silica gel column chromatography
(eluent: DCM/MeOH 95:5) to give 5 (3.1 g, 62 %). 1H NMR (300 MHz, DMSO-d6, 300 K): δ
(ppm)= 6.77 (d, 3J= 9.0 Hz, 2H); 7.43 (d, 3J= 9.0 Hz, 2H); 9.49 (s, 1H); 10.97 (s, 1H).
B-‐chloro[subphtalocyaninato]boron(III) (2):
BCl3 (4 mL, 1 M solution in p-xylene) was added to dry phthalonitrile (0.5 g, 4 mmol),
under an argon atmosphere. The reaction mixture was poured into a preheated oil bath
(150°C), then stirred and left to reflux for 30 min. The solvent was removed under reduced
HN
OH
CF3
O
N
N N
N N
NB
Cl
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pressure and the resulting solid was subjected to silica gel column chromatography (eluent:
DCM) (50 mg, 9 %). 1H NMR (300 MHz, CDCl3, 300 K): δ (ppm)= 7.97 (m, 6H); 8.81 (m,
6H). 11B NMR (96 MHz, CDCl3, 300 K): δ (ppm)= 15.12 (s, 1B). UV-Vis (CHCl3), λmax
(nm) (logε)= 306 (4.709), 565 (5.005).
B-‐(4-‐nitrophenoxy)[subphtalocyaninato]boron(III) (1a):
A mixture of B-chloro[subphtalocyaninato]boron(III) (50 mg, 0.12 mmol) and the
corresponding phenol (1.2 mmol) in toluene (5 mL) was heated to reflux for 15 hours. After
evaporation of the solvent, the residue was subjected to alumina gel column chromatography
(eluent: DCM) and then recristallized into DCM/ heptane mixture (50:50 vol.) by slow
evaporation of DCM, to give 3a as a bronze cristalline solid (50 mg, 78 %). Analysis: same as
described below.
General procedure for the synthesis of phenoxy substitued subphtalocyanine starting from
phthalonitrile (1a, 3a-‐b):
BCl3 (4 mL, 1 M solution in p-xylene, 4 mmol) was added to dry phthalonitrile (0.5 g, 4
mmol), under an argon atmosphere. The reaction mixture was placed in a preheated oil bath
(150°C), then stirred and left to reflux for 30 min. The solvent was removed under reduced
pressure and the resulting solid was suspended in toluene (30 mL). Excess of the
corresponding phenol (12 mmol) was added and the mixture was heated under reflux during
15 hours. After evaporation to dryness, the residue was subjected to short alumina gel column
chromatography (eluent: DCM) to remove unreacted phenol.
N
N N
N N
NB
O NO2
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B-‐(4-‐nitrophenoxy)[subphtalocyaninato]boron(III) (1a):
The product was recrystallized into a DCM/heptane mixture (50:50 vol.) by slow
evaporation of DCM, to give (4-nitrophenoxy)[subphtalocyaninato]boron(III) (3a) as a bronze
crystalline solid (200 mg, 29 %). 1H NMR (300 MHz, CDCl3, 300 K): δ (ppm)= 5.39 (d, 3J=
9.1 Hz, 2H); 7,66 (d, 3J= 9.1 Hz, 2H); 7.91 (m, 6H); 8.86 (m, 6H). 13C NMR (75 MHz, CDCl3,
300 K): δ (ppm)= 118.3, 122.1, 125.0, 129.9, 130.7, 141.3, 151.2, 158.5. 11B NMR (96 MHz,
CDCl3, 300 K, SubBCl): δ (ppm)= -14.93 (s, 1B). UV-Vis (DCM), λmax (nm) (logε ; M-
1cm-1)= 305 (4.615), 564 (4.753).
B-‐(4-‐acetamidophenoxy)[subphtalocyaninato]boron(III) (3a):
(90 mg, 16 %). 1H NMR (300 MHz, CDCl3, 300 K): δ (ppm)= 1.97 (s, 3H); 5.32 (d, 3J= 8.8
Hz, 2H); 6.84 (d, 3J= 9.2 Hz, 2H); 7.84 (m, 6H); 8.79 (m, 6H).
B-‐(4-‐(2,2,2-‐trifluoro)acetamidophenoxy)[subphtalocyaninato]boron(III) (3b):
N
N N
N N
NB
O NO2
N
N N
N N
NB
O NH
O
N
N N
N N
NB
O NH
OCF3
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(150 mg, 19 %). 1H NMR (300 MHz, CDCl3, 300 K): δ (ppm)= 1.97 (s, 3H); 5.32 (d, 3J=
8.8 Hz, 2H); 6.84 (d, 3J= 9.2 Hz, 2H); 7.84 (m, 6H); 8.79 (m, 6H).
B-‐(4-‐aminophenoxy)[subphthalocyaninato]boron(III) (1b):
A mixture of B-(4-nitrophenoxy)[subphtalocyaninato]boron(III) 3a (240 mg, 0,45 mmol)
and activated palladium 10% on carbon (20 mg, 0.17 mmol) in a DCM/ MeOH mixture (1:1
vol., 20 mL) was stirred under hydrogen atmosphere during 72 hours. The mixture was
filtered off on celite to remove palladium and charcol, then the mixture was evaporated to
dryness. The resulting solid was subjected to silica gel column chromatography (eluent:
DCM/MeOH 99:1) to obtain 1a as a bronze solid (200 mg, 88 %). 1H NMR (300 MHz,
CDCl3, 300 K): δ (ppm)= 3.62 (s, 2H); 5.25 (d, 3J= 8.8 Hz, 2H); 6.16 (d, 3J= 8.8 Hz, 2H); 7.87
(m, 6H); 8.83 (m, 6H,). 13C NMR (75 MHz, CDCl3, 300 K): δ (ppm)= 116.7, 120.2, 122.4,
129.9, 131.1, 139.2, 145.6, 151.4. HR-MS ESI: m/z= 504.1740 [M+H]+ (calcd for
C30H19BN7O+: 504.1744). UV-Vis (DCM), λmax (nm) (log ε ; M-1.cm-1): 305 (4.596), 562
(4.898). UV-Vis (DMF), λmax (nm) (log ε ; M-1.cm-1): 303 (4.656), 563 (4.931).
B-‐(4-‐(N,N-‐bis(sulfopropyl))aminophenoxy)[subphthalocyaninato]boron(III) (1c):
To a solution of 1b (50 mg, 0.1 mmol) in DMF (5 mL) was added 1,3-propanesultone (61
mg, 0.5 mmol). The reaction mixture was stirred at 50°C during 72 h, then evaporated under
N
N N
N N
NB
O NH2
N
N N
N N
NB
O N
SO3H
SO3H
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reduced pressure. The residue was subjected to silica gel column chromatography (eluent:
DCM/MeOH from 98:2 vol. to 70: 30 vol.) and then by semi-preparative reverse phase
column chromatography (see section 1) to obtain 1c (10 mg, 12 %). 1H NMR (600 MHz,
DMSO-d6, 300 K): δ (ppm)= 1.58 (m, 3J= 7.4 Hz, 4H); 2.32 (t, 3J= 7.4 Hz, 4H); 3.05 (t, 3J=
7.4 Hz, 4H); 6.16 (d, 3J= 8.8 Hz, 2H); 7.87 (m, 6H); 8.83 (m, 6H,). MS MALDI-TOF: m/z=
748.036 [M+H]+ (calcd for C36H31BN7O7S2+: 748.18), 770.03 [M+Na]+ (calcd for
C36H30BN7O7S2Na+: 770.16), 786.01 [M+K]+ (calcd for C36H30BN7O7S2K+: 786.14). HR-MS
ESI: m/z= 372.57899 [M-2H]2- (calcd for C36H28BN7O7S2+: 372.57900). HPLC: Rt (min)=
2.267 (98.9 % at 254 nm; 99.4 % at 565 nm). UV-Vis (H2O), λmax (nm) (log ε ; M-1.cm-1): 315
(4.149), 566 (4.696).
A6. Purification
Fig. S1. HPLC chromatograms of the followings (from bottom to top): Chromatogram-1
(line 1, in black): Mixture before purification; Chromatogram-2 (line 2, in blue): SubPc-
(propsulf)2 (1c), Chromatogram 3 (line 3, in pink): SubPc-propsulf (detection at 565 nm)
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A7. Analyses
Fig. S2. 11B NMR spectrum of Sub-‐NO2 (1a) in the presence of a low quantity of Sub-‐Cl (2)
(CDCl3, 96 MHz, 300K)
Fig. S3. 1H NMR spectrum of Sub-‐NO2 (1a) (CDCl3, 300 MHz, 300K)
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Fig. S4. 13C NMR spectrum of Sub-‐NO2 (1a) (CDCl3, 75 MHz, 300K)
Fig. S5. 1H NMR spectrum of Sub-‐NH2 (1b) (CDCl3, 300 MHz, 300K)
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Fig. S6. 13C NMR spectrum of Sub-‐NH2 (1b) (CDCl3, 75 MHz, 300K)
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Fig. S7. HR-‐MS ESI spectrum of Sub-‐NH2 (1b)
Experimental,
Calculated,Experimental,
Calculated,
[M#2Na]2#(
[M#2Na+H]#(
[M#Na]#(
N
N N
N N
NB
O N
SO3Na
SO3Na
Chemical Formula: C36H28BN7Na2O7S2Exact Mass: 791,14
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Fig. S8. HR-‐MS ESI spectrum of Sub-‐NH2 (1c)
Experimental,
Calculated,
[M32Na]23,
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Compound 1b
CCDC 1014064 Formula H18C30N7OB D calc./g cm-‐3 1.451 ⎧/mm-‐1 0.092 Formula Weight 503.32 Colour orange Size/mm3 0.22×0.15×0.07 T/K 115 Crystal System triclinic Space Group P-‐1 a/Å 10.1660(4) b/Å 10.7702(3) c/Å 11.7522(5) 〈/° 86.653(2)
/° 78.280(2)
/° 66.200(2) V/Å3 1152.38(8) Z 2 Theta min/° 3.172 Theta max/° 27.413 Measured Refl. 9428 Independent Refl. 5191 Reflections Used 4180 R(int) 0.0269 Parameters 358 Restraints 1 Largest Peak 0.285 Deepest Hole -‐0.265 GooF 1.085 wR2(all data) 0.1039 wR2 0.0934 R1(all data) 0.0674 R1 0.0489
Table S1. X-‐ray details. Fig. S9. Crystallographic structure of Sub-‐NH2 (1b)
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B-‐ LIPOSOMES
B1. Liposome Preparation Liposomes were prepared by the injection method using a solution of 1,2-dipalmitoyl-sn-
glycero-3-phosphocholine (14.6 mg) in ethanol (1 mL)(solution A) and a solution of
Subphthalocyanine (5 μmol) in chloroform (10 mL)(solution B). A mixture of 100 μL of
SubPc solution (B) and 100 μL of DPPC solution (A) was quickly injected using an Hamilton
syringe in 10 mL of buffer solution (PBS or NaCl) under vigourous agition at 60°C. The
mixture was left under agitation during 2 min at the same temperature, then cooled down to
room temperature and further used for desired application.
B2. Liposome First characterisation
Fig. S10. Absorbtion (left) and fluorescence emission (right) of Sub-‐NO2 (1a) in different media
THF: 100 μL of SubPc solution B + 100 μL of ethanol in 10 mL of THF H2O: 100 μL of SubPc solution B + 100 μL of ethanol in 10 mL of water
PBS: 100 μL of SubPc solution B + 100 μL of DPPC solution A in 10 mL of PBS
0"
0,05"
0,1"
0,15"
0,2"
0,25"
0,3"
0,35"
0,4"
300" 400" 500" 600" 700" 800"
""""""
DPPC"/"PBS"
H20"THF"
0"
50"
100"
150"
200"
250"
300"
350"
500" 550" 600" 650" 700"Wavelenghts"(nm)"
Fluorescence"
Wavelenghts"(nm)"
Absorbance"
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B3. Liposome Purification by HiTRAP The purification of the liposomes was achieved on a Äkta pure (GE). The liposome
suspension (500 µL) was injected on a 1 mL loop, and elution was performed on a Hi-‐
trap column (GE) using water as the eluent with a flow rate set at 3 mL/min. The
detection wavelengths were set at 204 nm, 300 nm, and 580 nm. Upon collection of
fractions, 4 mL of suspension were collected, and subsequently lyophilized.
B4. Liposome SIZE: DLS and TEM
DLS (Dynamic Light Scattering):
Hydrodynamic diameter measurements were performed on a Zeta-Nanosizer (Malvern) into
10-2 M NaCl solutions.
TEM (Transmission Electron Microscopy):
Transmission Electron Microscopy images were obtained from a JEOL JEM 2100 LaB6
operating at 200 kV (point resolution 2.5 Å). The copper grids were dipped in dilute
suspension of samples in ethanol and naturally dried.
Morphology and structure were observed (/Observations were performed) by Transmission
Electron Microscopy (TEM) using a JEOL JEM 2100 LaB6 microscope operating at 200 kV
and with a Scherzer resolution of 0.25 nm. The images were recorded with an on-line charged
coupled device camera, and the analyses of the results were performed using the Digital
Micrograph software. The samples for TEM observation have been prepared by negative
staining as described/outlined below/hereafter: A drop (5-10µl) of the suspension (/of
liposomes) was placed onto a carbon-coated copper grid. When the suspension has partly
dried, the grid is flipped over; a droplet of the staining solution placed in a Petri dish and held
in contact for 30 seconds.
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The staining solution is a 1-2% ammonium molybdate solution in distilled water with the
pH adjusted with ammonium or sodium hydroxide to pH 7.0.
2% (w/v) Ammonium molybdate, pH 7.0 (in a 1.5 ml Eppendorf tube: 0.02 g in 0.8 mL
distilled water, and a few drops of 10 N NaOH followed by adjustment of the pH to 7.0 (using
pH paper), the final volume was brought to 1.0 mL; and the resulting solution was filtered
through a 0.2 micron filter.
B5. Liposome CONTENT: UV/Vis
Fig. S11. Absorption spectrum of Sub-‐NO2 (1a) containing liposome after purification by
HITRAP and lyophilisation (spectrum in DCM).
Calculation of SubPc incorporation ratio in liposome: The incorporation ratio is calculated as follow:
𝑅 =𝑛 𝑖𝑛𝑐𝑜𝑟𝑝𝑜𝑟𝑒𝑑𝑛(𝑖𝑛𝑗𝑒𝑐𝑡𝑒𝑑) ×100
Where
𝑛(𝑖𝑛𝑐𝑜𝑟𝑝𝑜𝑟𝑒𝑑) =𝑉×𝐴𝜀×𝑙
𝑛 𝑖𝑛𝑗𝑒𝑐𝑡𝑒𝑑 = 𝑉 𝑖𝑛𝑗𝑒𝑐𝑡𝑑 ×𝑐(𝑙𝑖𝑝𝑜𝑠𝑜𝑚𝑖𝑎𝑙 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛)
And where: R: incorporation ratio (%) n(incorporated): quantities of Sub-‐NO2 imbebbed in liposome (moles)
0"
0,01"
0,02"
0,03"
0,04"
0,05"
0,06"
0,07"
0,08"
0,09"
300" 350" 400" 450" 500" 550" 600" 650" 700" 750" 800"Wavelenghts"(nm)"
Absorbance"
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n(injected): quantities of Sub-‐NO2 injected for HITRAP purification (moles) V: volume of solubilisation for UV measurement (400 μL DCM) A: absorption at maximum of solution in DCM after purification (0.045) ε: molar extinction coefficient of Sub-‐NO2 (56700 L.mol-‐1.cm-‐1) l: cuvettes length (1 cm) V(injected): injected volume for HITRAP purification (500 μL) c(liposomial solution): Sub-‐NO2 concentration in intial liposomial solution (5 μM)
𝑅 =𝑉×𝐴
𝜀×𝑙×𝑉(𝑖𝑛𝑗𝑒𝑐𝑡𝑒𝑑)×𝑐(𝑙𝑖𝑝𝑜𝑠𝑜𝑚𝑖𝑎𝑙 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛) =400. 10!!×0.045
56700×1×500. 10!!×5. 10!!×100
𝑹 = 𝟏𝟐.𝟕 %
C-‐ SPECTROSCOPIC STUDIES and ACTIVATION
Compounds Solvent
Absorption maximum
wavelength (λmax, nm)
Emission maximum
wavelength (λem, nm)
Molar extinction coefficient of Q
band (10-3.Mol.L-1.cm-1)
Fluorescence quantum yield
(ΦF)
Factor of increase
SubBCl (2) CHCl3 565 571 101.2 0.25 /
SubPc-NO2 (1a) CHCl3 563 572 56.7 0.139 /
SubPc-NH2 (1b) CHCl3 562 571 79.1 0.0074 20.7
CHCl3 + TFA 564 572 81.0 0.153
SubPc-NH2 (1b) DMF 563 572 85.4 0.0065 18.6
DMF + H2SO4 564 573 88.2 0.121
SubPc-(propsult)2 (1c) DMF 563 572 46.8 0.0016 /
SubPc-(propsult)2 (1c) pH 8 566 575 49.7 0.0115 /
SubPc-(propsult)2 (1c) pH 4 566 575 49.7 0.0358 3
Table. S2. Optical Properties of SubPcs 1a-‐c and 2 in various solvents
Compounds in liposome Solvent
Absorption maximum
wavelength (λmax, nm)
Emission maximum
wavelength (λem, nm)
Fluorescence quantum yield
(ΦF)
Factor of increase
SubPc-NO2 (1a) PBS 569 571 0.052 /
SubPc-NH2 (1b) pH 3.6 568 572 0.0007 52
pH 6.6 568 572 0.0365
Table. S3. Optical properties of SubPcs 1a-‐b entrapped in liposomes
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Fig. S11. UV-Vis spectrum of SubPc-NH2 (1b) in DCM (298 K), and in the presence of
increasing amounts of pure trifluoroacetic acid (from 10 μL to 2/3)
Fig. S12. Fluorescence intensity of SubPc-NH2 (1b) in CHCl3 (298 K), as a function of
quantities of trifluoroacetic acid solution added (0-70 μL of a 0,1 % TFA solution -volume- in
CHCl3) in a constant total volume of 3 mL.
0"
0,02"
0,04"
0,06"
0,08"
0,1"
0,12"
0,14"
0,16"
0,18"
0" 10" 20" 30" 40" 50" 60" 70"
Fluo
rescen
ce*intensity
*
Volume*of*TFA*1%*added*(μL)*
0"
0,2"
0,4"
0,6"
0,8"
1"
1,2"
1,4"
1,6"
300" 350" 400" 450" 500" 550" 600" 650" 700" 750" 800"
SubNH21DCM"
+10"TFA"
+20"TFA"
+30"TFA"
+40"TFA"
+50"TFA"
+100"TFA"
+150"TFA"
+200"TFA"
+300"TFA"
+500"TFA"
1/3"TFA"
2/3"TFA"
Absorbance
Wavelenght (nm)
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Fig. S13. UV-Vis spectrum of SubPc-NH2 (1b) in DMF (298 K), and in the presence of
sulfuric acid (100 μL of a 0,01 % solution -volume- in DMF)
Fig. S14. Relative fluorescence intensity of liposomal suspension of Sub-‐NH2 (1b) as a function of pH (phosphate-‐citrate buffer).
0"
0,1"
0,2"
0,3"
0,4"
0,5"
0,6"
0,7"
0,8"
0,9"
1"
300" 350" 400" 450" 500" 550" 600" 650" 700" 750" 800"
SubPc2NH2"(DMF)"
SubPc2NH2"(DMF"+"H2SO4)"
Absorbance*(a.u.)*
Wavelenghts*(nm)*
SubPcNH2)(DMF))
SubPcNH2)(DMF)+)H2SO4))
0"
0,2"
0,4"
0,6"
0,8"
1"
1,2"
2,6" 3,1" 3,6" 4,1" 4,6" 5,1" 5,6" 6,1" 6,6"pH"
F/Fmax"
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Fig. S15. UV-Vis spectrum of SubPc-(propsulf)2 (1c) in DMF and water (298 K)
Fig. S16. Normalized absorption , emission and excitation spectrum of SubPc-‐(propsulf)2 (1c) in water (298 K)
Absorbance*(a.u.)*
Wavelenghts*(nm)*
0"
0,1"
0,2"
0,3"
0,4"
0,5"
0,6"
0,7"
0,8"
0,9"
1"
300" 350" 400" 450" 500" 550" 600" 650" 700" 750" 800"
SubPc2(propsulf)3"(DMF)"
SubPc2(propsulf)3"(H2O)"
SubPc(propsulf)2/(DMF)/
SubPc(propsulf)2/(H2O)/
0"
0,2"
0,4"
0,6"
0,8"
1"
1,2"
310" 360" 410" 460" 510" 560" 610" 660" 710" 760"
Absorb1on"
Emission"
Excita1on"
Fluo
rescen
ce*intensity
*or*a
bsorbance*(a.u.)*
Wavelenghts*(nm)*
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Fig. S17. Relative fluorescence intensity of Sub-‐(propsulf)2 (1c) as a function of pH (phosphate-‐citrate buffer)
D-‐ BIOLOGICAL STUDIES
D1. Cells (culture, incubation, fixation) B16-‐F10 Melanoma Cells were grown in RPMI supplemented with Foetal Calf Serum and
1% streptavidine. Cells were platted in 8 chambers polystyrene vessel (with tissue
culture treated glass slides; BD Falcon Culture slides Ref 3541 18) two days before the
experiment. Then the medium was removed and replaced with non-‐supplemented RPMI
medium mixed with the solution of subphthalocyanine. The resulting concentration in
SubPc was 10 µM. The time of incubation was set at 1h, then the cells were rinsed with
PBS. The overall process of cell fixation was achieved as follows: after removing PBS,
cold (-‐30°C) methanol (100 µL per well) was subsequently added, the plate was stored
in the fridge (4°C) for 5 min, then the methanol was removed. Cold PBS (4°C) was added
and the plate was stored in an ice-‐container until microscopic measurement. PBS was
0"
0,2"
0,4"
0,6"
0,8"
1"
1,2"
2" 3" 4" 5" 6" 7" 8" 9"pH"
F/Fmax"
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removed by tilting the plate, the top part (plastic walls) of the plate was removed, and a
small layer of PBS was added to cover the cells, then a µm-‐thin glass plate (for
microscopy use) was placed on top of the PBS layered cells.
D2. Confocal Microscopy Confocal images were collected using a Nikon C1Si Eclipse TE 2000 confocal microscope
(Nikon, Japan). Imaging was carried out with a ×100 PlanApo objective (NA: 1.4, oil,
Nikon, Japan). Two laser diodes were used as light sources, which delivered 488 and
561 nm wavelength light. Fluorescence emission was collected by a spectral detector,
using collection bands of [489-‐648]nm with 488nm excitation and [566-‐721]nm for
561nm excitation.
Fig. S18. Left to right : Transmission, superposition and fluorescence (confocal) images of B16F10 cells incubated with a solution of 1c in PBS / RPMI (excitation at 561 nm).
D3. Biphoton Microscopy Biphotonic images were collected on a Nikon A1-‐MP scanning microscope (Nikon,
Japan). Imaging was carried out with a ×25 Apo LWD objective (NA: 1.1, Water
Immersion, Nikon, Japan) at a scanning speed of 0.5 frame per second. An IR laser
(Chameleon, Coherent) was used to provide a 780nm excitation. Fluorescence emission
was collected on four detection channels (FF01-‐492/SP, FF03-‐525/50, FF01-‐
575/25,FF01-‐629/56, Semrock).
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Fig. S19. Fluorescence of B16 cells upon incubation with a solution of 1a (biphoton microscope). Left to right: images acquired on 492nm, 525nm and 629nm
detection channels, respectively. D4. Cell Viability Assay Cells were platted on 96-‐wells culture plates two days before the experiment. The
percentage of cell viability was assessed by the tetrazolium colorimetric assay (MTT) as
described by T. Mossman (J. Immunol. Methods, 1983, 65, 55-‐63). Immediately after
incubation of the cells with 1a, 1c , cells were rinsed with PBS, then culture medium was
added to the wells, and cells were left at 37°C for 6h. The MTT solution was then added
to the culture plate, and subsequent incubation was allowed for 2h, allowing the
formation of blue formazan crystals. Then, the experiment was stopped : the medium
was removed, and DMSO was added to dissolve the crystals. A purple solution was
obtained, the absorbance of which was examined at 570 nm (using a Multiscan GO
spectrometer). No toxicity was found at 10 µM and below.
Recommended