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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"

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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.