X. Chen et al. / Dyes and Pigments 114 (2015) 93e104
95
purified by passing through a sillica gel column with dichloro-
methane/methyl alcohol (1:5). Yield: 1.576 g (80.0%). M.P. >200 ꢀC.
IR (KBr, cmꢁ1): 3490, 3425, 3130, 1620, 1400, 1230, 1040, 710, 486.
processes [15,16]. 1O2 was generated by photoexcitation of the
ZnPcs molecules in their soret band with laser pulses at
k ¼ 355 nm followed by the energy transfer between the triplet
state of ZnPcs and the ground state of the solvated O2 molecules.
1O2 generation was determined by the ADPA bleaching method.
ZnPcs (5 ꢂ 10ꢁ6 M) and ADPA (5.5 ꢂ 10ꢁ6 M) were mixed and
irradiated. The reaction was monitored spectrophotometrically by
measuring the decrease in optical density every 1 min at an
absorbance maximum of 378 nm of ADPA. All samples were air
equilibrated.
1H NMR (400 MHz, DMSO-d6):
d (ppm) 7.61e7.59 (br, 16H, Pc-H),
7.56 (s, 4H, Pc-H), 7.50 (s, 4H, Pc-H), 7.39e7.28 (m, 60H, Pc-H),
7.08 (s, 4H, Pc-H), 3.36 (s, 8H, CH2). 13C NMR (100 MHz, DMSO-
d6):
d (ppm) 146.1, 136.4, 129.6, 128.7, 128.0, 127.8, 127.6, 126.4,
120.3, 119.4, 119.2, 117.0, 102.7, 98.9, 77.4, 51.6, 49.5, 47.5, 45.8, 43.1,
31.9, 29.7, 9.8, 6.8. Anal. Calcd. For C136H100N12O4: C, 80.40; H, 4.96;
N, 8.27. Found: C, 80.20; H, 4.78, N, 8.28.
2.2.4. 2(3), 9(10), 16(17), 23(24)-tetra-((amino)methyl)phenoxy)
phthalocyaninato -zinc(ZnPc)
2.5. Cellular uptake of ZnPc1, ZnPc2 and ZnPc3 in HeLa cells
Under the condition of ice-water bath, compound 5 (500 mg,
0.25 mol) and excess trifluoroacetic acid (TFA) (0.5 mL) were dis-
solved in CH2Cl2 (10 mL) and stirred for 1 h. Then, the reaction
mixture was heated to room temperature and left to stir for another
2 h. The crude product was collected by filtration and washed
successively with CH2Cl2. Thereafter, the green solid was dissolved
in H2O and precipitated by adjusting PH to 9e10. The residue
product collected by filtration was washed successively with H2O
and CH3OH. The product was vacuum-dried at 50 ꢀC for 12 h to
afford the final. M.P. >200 ꢀC. IR (KBr, cmꢁ1): 3410, 3240, 2840
(CH2), 2230 (CN), 1630, 1420, 1120, 1005, 620. 1H NMR (400 MHz,
HeLa cells were incubated under the same experimental con-
ditions with ZnPc1, ZnPc2 and ZnPc3 (the concentration: 1.5 mM)
for respectively 2, 4, 6 and 24 h in the dark. After respectively 2, 4, 6
and 24 h incubation, the drug concentration remaining in the
medium was detected and calculated. All cellular uptake amounts
were calculated according to the standard curves.
2.6. Intracellular ROSs detection by DCFH-DA
In order to evaluate the ability of the ZnPcs to generate ROS
in vitro, the probe DCFH-DA was used as a fluorescent probe [17,18].
The non-fluorescent DCFH-DA can be oxidized to the fluorescent
DCF in the presence of ROSs. At first, cells (with about 60%
confluence) in a 6-well plate suspension in the medium were
combined with ZnPc1, ZnPc2 and ZnPc3, and incubated in incu-
bator (37 ꢀC, 5% CO2) for 24 h. Then, the plates were washed with
sterile PBS, and the DCFH-DA was added into the wells. After in-
cubation for 0.5 h, cells were washed with PBS and illuminated for
about 15 min.
DMSO-d6):
(s, 4H, Pc-H), 8.37 (s, 4H, Pc-H), 7.78e7.66 (br, 8H, Pc-H), 7.54e7.45
(br, 8H, Pc-H), 3.36 (s, 8H, CH2). 13C NMR (100 MHz, CDCl3):
(ppm)
d (ppm) 12.7 (s, 8H, NH2), 8.98e8.96 (br, 4H, Pc-H), 8.55
d
159.4, 159.07, 158.7, 157.9, 157.7, 135.0, 131.7, 130.3, 129.2, 127.1,
124.6, 121.5, 119.8, 119.4, 117.5, 114.6, 115.0, 108.2, 103.4, 100.1, 45.9.
Anal. Calcd. For C60H44N12O4: C, 67.83; H, 4.17; N, 15.82. Found: C,
67.44; H, 4.22; N, 15.70.
2.2.5. Hydrochloride, hydrobromide and hydriodate derivative of
2(3), 9(10), 16(17), 23(24)-tetra-((amino)methyl)phenoxy)
phthalocyaninato-zinc (ZnPc1, ZnPc2 and ZnPc3)
2.7. Cell morphology
ZnPc1 (ZnPc2, ZnPc3) was suspended in 5 mL redistilled water
in a reaction bulb and warmed to reflux. Excess 5% HCl (HBr, HI)
aqueous was added into the solution drop-wise until ZnPc1
(ZnPc2, ZnPc3) was totally dissolved under 40 ꢀC. After adding
solution into the acetone, the crude precipitation was collected by
filtration, then thoroughly washed by dichloromethane and dried
in vacuum. The title product ZnPc1 (ZnPc2, ZnPc3) was obtained as
dark blue solid. Yield: 0.23 g (93.8%). The relative content of
elemental chlorine (bromine and iodine) in ZnPc1 (ZnPc2, ZnPc3)
was analyzed through EDS. Quantitative analysis shows that the
mean atomic ratio of Cl/Zn (Br/Zn and I/Zn) of ZnPc1 (ZnPc2,
ZnPc3) respectively was 0.188 (0.186 and 0.186). Compared with
the standard value 0.125, the results confirmed our desired out-
comes that one ZnPc1 (ZnPc2, ZnPc3) molecule contains eight HCl
(HBr and HI) in its structure. Since the elemental analysis, FT-IR, 1H
NMR spectroscopy, electronic spectroscopy and mass spectra of
three ZnPcs are analogous to those of compound ZnPc, they are not
described in detail here.
For routine maintenance, HeLa cells were cultured in suspension
in DMEM medium supplemented with 10% FCS and incubated at
37 ꢀC in humidified air with 5% CO2. Cell growth inhibition was
evaluated using a standard colorimetric MTT assay [19]. Before
treatment with ZnPcs, the cells were seeded in 96-well plates and
incubated overnight. The density of cells in the medium was about
10,000 cells per well. After treatment with ZnPcs overnight and
being irradiated by light, cells were washed with PBS three times
and the cell morphology changes were observed under a fluores-
cence microscope.
2.8. Hoechst 33342 staining
Chromatin condensation was detected by nuclear staining with
Hoechst 33342 [20]. After treatment with ZnPcs overnight and
being irradiated by light, cells were washed with PBS three times
and treated with 25 m
g mLꢁ1 Hoechst 33342 at 37 ꢀC with 5% CO2 in
the dark for 30 min. Nuclear morphology change was observed
under a fluorescence microscope.
2.3. Photodegradation studies
Photostability studies of ZnPcs used in this work were carried
out in H2O by monitoring the decrease in the Q-band absorption
before and after irradiation with 665 nm LED using UVeVis
spectrophotometer.
2.9. Darktoxicity
In the cytotoxicity test, the same concentrations of ZnPcs were
suspended in DMEM and were ultrasonicated for 30 s to prevent
agglomeration. To determine the darktoxicity, HeLa cells were
seeded into 96 well plates at a density of 5 ꢂ 105 cells/cm2 and
incubated for 24 h in growth medium to allow for attachment. Cell
cytotoxicity was assessed by using the classical MTT colorimetric
assay [21,22].
2.4. 1O2 generation detection
1O2 is one of the most active traits among the reactive oxygen
and holds a prominent role in various biological and chemical