Comparison of two strategies for conferring water solubility to a zinc phthalocyanine substituted with 1,2-diethylamino

Article abstract of DOI:10.1016/j.dyepig.2013.05.020

Polyamines are naturally occurring compounds that play important roles in multiple cell processes. In most cases, phthalocyanine-polyamine (Pc-polyamine) conjugates have good anticancer activity. However, low solubility in water limits their application i

Full text of DOI:10.1016/j.dyepig.2013.05.020

Dyes and Pigments 99 (2013) 348e356
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Comparison of two strategies for conferring water solubility to a zinc
phthalocyanine substituted with 1,2-diethylamino
*
Ao Wang, Yanjie Ma, Lin Zhou, Shan Lu, Yun Lin, Jiahong Zhou, Shaohua Wei
College of Chemistry and Materials Science, Jiangsu Key Laboratory of Biofunctional Materials, Key Laboratory of Applied Photochemistry, Nanjing Normal
University, Nanjing 210046, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Polyamines are naturally occurring compounds that play important roles in multiple cell processes. In
most cases, phthalocyanine-polyamine (Pc-polyamine) conjugates have good anticancer activity. How-
ever, low solubility in water limits their application in photodynamic therapy (PDT). The common
method to solve this problem is quaternizing the nitrogen atoms to obtain cationic derivatives. An
alternative strategy by preparing hydrochloride is easily neglected. In this paper, a Pc-polyamine con-
jugate substituted with 1,2-diethylamino (ZnPc1) was conferred water solubility by two different stra-
tegies, which are preparing hydrochloride (ZnPc2) and quaternizing the nitrogen atoms (ZnPc3). The
synthetic methods and properties of the two derivatives were compared and discussed. Results indicated
that both ZnPc2 and ZnPc3 possessed good solubility in water. However, strategy of preparing hydro-
chloride is much easier than quaternization process and the singlet oxygen generation ability, the
photostability and anticancer activity of ZnPc2 in vitro were also superior to the ZnPc3.
Received 9 April 2013
Received in revised form
16 May 2013
Accepted 21 May 2013
Available online 31 May 2013
Keywords:
Phthalocyanine
Polyamine
Photodynamic therapy
Water solubility
Hydrochloride
Quaternizing
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
always observed in water, which has strong influences on their
in vivo distribution, the photobiological and photochemical activ-
Photodynamic therapy (PDT) is a novel and promising nonin-
vasive modality for treatment of a variety of malignant diseases [1].
It uses the combined action of photosensitizer, light and molecular
oxygen to cause irreversible photodamage to tumor cells and tis-
sues [2,3]. Among the three individually components, behaviors of
the photosensitizer determine greatly the final therapeutic
outcome [4]. During the past decades, a great number of potential
photosensitizers for PDT have been studied. Phthalocyanines (Pcs),
ities, and finally limit their potential applications in PDT [12]. The
common means for preparing water soluble Pcs with high effi-
ciency is to attach hydrophilic groups like polyethylene glycol,
amino acid, carbohydrates [13e15], ionic groups including cationic
and anionic substituent [16e18] at the peripheral and axial posi-
tions of Pc ring.
Polyamines are important compounds that play multifunctional
roles in cell proliferation and differentiation processes [19]. Poly-
amine conjugate with photosensitizers such as porphyrins and Pcs
have been reported [20,21]. In most cases, their photochemical
properties, selectivity for tumor cells and anticancer activity are
enhanced. However, Pc-polyamine conjugates also have low solu-
bility in water due to the hydrophilic nature of amino groups is
limited, can’t suppress the intrinsic hydrophobic property of Pc
macrocycle. To confer water solubility to Pcs substituted with
amino groups, the common method is quaternizing the nitrogen
atoms to obtain cationic derivatives. The quaternized Pcs not only
have good solubility in water, but also have low aggregation and
high anticancer activity [22,23]. While, the Pc-polyamine conju-
gates can be easily conferred water solubility by preparing hydro-
chloride because of the existence of amino groups in their
structure. This alternative strategy takes advantages of the easily
the two-dimensional 18
p-electron aromatic synthetic analogs of
porphyrins, have attracted a great deal of interest due to their
unique physical and chemical properties [5,6]. Especially, Pcs are
considered as the promising photosensitizers for PDT owing to
their intense absorption in the phototherapeutic window (600e
900 nm), high efficiency to generate reactive oxygen species (ROS),
high phototoxicity and low darktoxicity [7e10].
During the PDT administration, the drug (Pc) is injected into the
patient’s blood stream [4], and since the blood itself is a hydrophilic
system, water solubility becomes crucial for drugs in PDT [11].
However, for most Pcs, low solubility and high aggregation are
* Corresponding author. Tel./fax: þ86 025 85891761.
E-mail address: shwei@njnu.edu.cn (S. Wei).
0143-7208/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dyepig.2013.05.020

A. Wang et al. / Dyes and Pigments 99 (2013) 348e356
349
soluble of hydrochloride in water, has been reported rarely.
Furthermore, the differences between these two strategies and the
two hydrophilic derivatives have been rarely compared.
ZnPc3 was obtained by methylation of ZnPc1 in CH3OH. IR (KBr,
cmꢀ1): 3438, 2990, 1595, 1468, 1230, 1080, 860, 740. 1H NMR
(400 MHz, DMSO-d6):
d (ppm) 9.42 (d, 4H, 2.88 Hz, Pc-H), 9.03 (d,
So, here, to improve water-solubility, we synthesized the qua-
ternized form (ZnPc3) and hydrochloride form (ZnPc2) of a zinc Pc
substituted with 1,2-diethylamino (ZnPc1), which was prepared in
our previous studies (unpublished results). Their photochemical
and photo-induced anticancer activity were also compared and
discussed. Results indicate that both the two strategies, quater-
nizing and preparing hydrochloride, can greatly improve the
water-solubility of ZnPc1. However, strategy of preparing hydro-
chloride is much easier than quaternization process and the
singlet oxygen generation ability and in vitro anticancer activity of
hydrochloride derivative are also superior to the quaternized form.
It is expected that the comparison of the two different strategies
for preparing water soluble and high efficient Pc-polyamine con-
jugates can promote the studies on Pc-based photosensitizers for
PDT.
4H, 14.69 Hz, Pc-H), 7.94e8.03 (m, 4H, Pc-H), 7.70e7.84 (m, 8H, Ar),
7.49e7.58 (m, 8H, Ar), 4.80 (s, 8H, CH2), 4.08(s,16H, CH2), 3.38e3.48
(br, 16H, CH2), 3.12e3.18 (br, 36H, CH3), 1.31e1.34 (m, 24H, CH3). 13
C
NMR (100 MHz, DMSO-d6):
d (ppm) 8.1, 8.4, 9.3, 47.8, 49.9, 52.7,
55.4, 56.2, 56.5, 57.4, 67.1,112.9,113.4,119.1,119.3,121.8,122.7,122.8,
124.6,130.4, 134.2, 135.9, 140.0, 151.9,157.4, 159.7, 159.9. Anal. Calcd.
For C96H126I8N16O4Zn: C, 43.53; H, 4.79; N, 8.46. Found: C, 42.90; H,
4.88; N, 8.40.
2.2. Hydrochloride derivative of 2(3),9(10),16(17),23(24)-tetra-
(((2-(diethylamino) ethylamino) methyl) phenoxy)
phthalocyaninato-zinc (Ⅱ) (ZnPc2)
ZnPc1 (0.20 g, 0.14 mmol) was suspended in 5 mL redistilled
water in a reaction bulb and warmed to reflux. Excess 5% HCl
aqueous was added into the solution drop-wise until ZnPc1 was
totally dissolved. The solution was concentrated and poured in to
20 mL acetone. The solid blue product was collected by filtration,
then thoroughly washed by dichloromethane and dried in vacuum.
The title product ZnPc2 was obtained as a dark blue solid. Yield:
0.23 g (93.8%). To verify the relative content of elemental chlorine of
ZnPc2, the EDS was performed. Quantitative analysis shows that
the mean atomic ratio of Zn/Cl of ZnPc2 is 0.120. Compared with the
standard value 0.125, the results confirm the prediction made by us
that one ZnPc2 molecule contains eight HCl in its structure.
IR (KCl, cmꢀ1): 3413, 2923, 2644, 1605, 1519, 1477, 1232, 1096,
2. Experiments
2.1. Materials and characteristics
All necessary reagents and solvents were of analytical grade
quality obtained from commercial suppliers. All reagents were
purified according to reported procedures before use [24]. Diso-
dium salt of 9,10-anthracenedipropionic acid (ADPA) and [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT)
were purchased from SigmaeAldrich. Dulbecco’s modified Eagle’s
medium (DMEM) was from Gibco.
1045, 834. 1H NMR (400 MHz, DMSO-d6):
d (ppm) 9.99e10.29 (br,
For all the experiments recorded in water, the water soluble Pcs
(ZnPc2 and ZnPc3) were dissolved in water and diluted to the final
concentrations; in the case of Pc insoluble (ZnPc1) in water, the
stock solution was prepared in methanol and diluted with water to
the final concentrations. IR spectra were recorded on IR-
Spectrometer Nicolet Nexus 670. 1H NMR and 13C NMR spectra
were recorded on a Bruker Advance 400 MHz NMR spectrometer.
Elemental analyses were taken with Vario MICRO, Elementar. The
relative content of zinc and chlorine of the ZnPc2 were obtained
using energy dispersive spectrometer (EDS, Noran Vantage, Themo
Noran). UVevis spectra were recorded on spectrophotometer Cary
5000, Varian. Fluorescence spectra were recorded on Perkin Elmer
LS 50B fluorescence spectrophotometer. Zeta potential was recor-
ded on Zetasizer Nano 90 light scattering, Malvern. The pH values
were taken with FE20/EL20 pH meter, Mettler Toledo. Cell
morphology changes were observed under a Zeiss Observer fluo-
rescence microscope. A 665 nm LED lamp was used as light source.
ZnPc1 was prepared by a four-step procedure. First, N0,N0-
diethylethane-1,2-diamine reacted with 4-hydroxybenzaldehyde
used K2CO3 as the basic catalyst to obtain an imine intermediate.
Then, the imine intermediate was deoxidated with NaBH4 in
protic solvents. The precursor of ZnPc1 was prepared by a base
catalyzed nucleophilic ipso-nitro substitution reaction of 4-
nitrophthalonitrile with the product of previous step. Finally,
ZnPc1 was obtained by the reaction of precursor compound with
zinc acetate in the presence of 1,8-diazabicyclo [5,4,0]-undec-7-ene
(DBU). IR (KBr, cmꢀ1): 3440, 2970,1660,1580,1460, 1223,1080, 740.
4H, NH$HCl), 9.02 (s, 4H, Pc-H), 8.53 (s, 4H, Pc-H), 7.78e7.90 (m, 8H,
Ar), 7.73 (s, Pc-H, 4H), 7.44e7.60 (m, 8H, Ar), 4.32 (s, 8H, CH2), 3.59
(s, 16H, CH2), 3.24 (s, 16H, CH2), 1.31 (d, 24H, 6.40 Hz, CH3). 13C NMR
(100 MHz, DMSO-d6):
d (ppm) 145.2,130.1,129.5,124.6,123.5,121.0,
119.5, 54.0, 53.1, 51.9, 50.0, 47.5, 44.9, 44.5, 31.2, 29.5, 11.9. Anal.
Calcd. For C84H104Cl8N16O4Zn: C, 57.62; H, 5.99; N, 12.80. Found: C,
57.10; H, 6.08; N, 12.66.
2.3. Photodegradation studies
Photodegradation studies were carried out in water by moni-
toring the decrease in the Q-band absorption of the Pcs using UVe
vis spectrophotometer.
2.4. Singlet oxygen generation detection
The singlet oxygen generation abilities of the Pcs were deter-
mined using the experimental described in literature [25,26]. The
absorption intensity of ADPA continuously decreased as the irra-
diation time increasing. Pcs and ADPA were mixed and irradiated.
The reaction was monitored spectrophotometrically by measuring
the decrease in optical density at absorbance maximum of 378 nm
of ADPA. The rate of singlet oxygen generation is calculated by the
following Eq. [25]:
À
Á
ln ½ADPAꢁt=½ADPAꢁ0 ¼ ꢀkt
1H NMR (400 MHz, DMSO-d6):
d (ppm) 9.37 (t, 4H, 23.82 Hz, Pc-H),
where [ADPA]t and [ADPA]0 are the concentrations of ADPA after
and prior irradiation, respectively. Values of k are the rate of singlet
oxygen generation and t is the time of irradiation.
8.91 (t, 4H, 13.44 Hz, Pc-H), 7.81e7.89 (m, 4H, Pc-H), 7.72 (t, 8H,
11.10 Hz, Ar), 7.45e7.53 (m, 8H, Ar), 4.34 (d, 8H, 11.00 Hz, CH2), 3.46
(s, 16H, CH2), 3.25 (t, 16H, 6.36 Hz, CH2), 1.25 (t, 24H, 5.66 Hz, CH3).
13C NMR (100 MHz, CDCl3):
d
(ppm) 11.8, 29.7, 44.2, 45.3, 47.0, 51.5,
2.5. Zeta potential measurement (ZP)
52.5, 53.4, 120.1, 123.7 (br), 129.8 (br). Anal. Calcd. For
C84H96N16O4Zn: C, 69.14; H, 6.63; N, 15.36. Found: C, 68.95; H, 6.86;
N, 15.58.
The ZP of the HeLa cells (cervical carcinoma cell line) and ZnPcs
were measured by a Malvern Zetasizer Nano 90 light scattering. For

350
A. Wang et al. / Dyes and Pigments 99 (2013) 348e356
the preparation of samples, the HeLa cells and ZnPcs were dilute
with DMEM (Dulbecco’s modified eagle’s medium). The experi-
ments were performed three times and an average of the results
was used.
methylate DNA [15]. While, the hydrochloride derivative ZnPc2 was
obtained by protonating the ZnPc1 in water. The protonating agent
is dilute hydrochloric acid which is commonly low toxicity. To
obtain pure product, the quaternization derivative ZnPc3 was fil-
trated while hot, and then thoroughly washed with ethanol and
methanol. ZnPc2 was poured into acetone, then, filtrated. The
quaternization derivatives ZnPc3 is dark green in color, while hy-
drochloride derivative ZnPc2 is dark blue.
2.6. Photodynamic activity in vitro
2.6.1. Darktoxicity
The darktoxicity of the Pcs was determined by means of the
colorimetric MTT assay [27,28]. HeLa cells were seeded into 96 well
plates at a density of 5 ꢂ 105 cells cmꢀ2 and incubated for 24 h in
growth medium to allow for attachment. After 24 h, cellular sur-
vival was measured.
The solubility of the three compounds in some common organic
and water was measured at room temperature to give some in-
dications about their amphipathic property. The amphipathic is an
important characteristic of an ideal PDT drug [29]. ZnPc1 is soluble
in the common organic solvents, such as methanol, chloroform and
dimethyl formamide (DMF) et al. However, it is insoluble in water
due to the hydrophilic nature of amino groups is limited, can’t
suppress the intrinsic hydrophobic property of Pc macrocycle. By
contrast, both ZnPc2 and ZnPc3 have good water solubility. Inter-
estingly, the two derivatives also have solubility in some polarity
organic solvents like DMF and dimethyl sulfoxide (DMSO). ZnPc2
also can be soluble in methanol and ethanol. To a certain degree,
ZnPc2 and ZnPc3 show an amphipathic property because of their
solubility both in water and organic solvents.
2.6.2. Phototoxicity
For photo-induced anticancer experiments, HeLa cells were
incubated as described above. After 24 h, the old medium was
replaced by fresh medium (without calf serum) with the three Pcs,
separately. Cells were then incubated at 37 ꢃC under 5% CO2 over-
night. The cells were then rinsed three times with PBS (phosphate
buffered saline) and refilled with by fresh medium (without calf
serum). The cells were immediately exposed to 665 nm LED light
for 5 min and after 24 h incubation cellular survival was measured
as described above.
3.2. Ground state electronic absorption
2.7. Cell morphology
The aim of UVevis experiments was to provide the information
about the electron density of the Pcs, their aggregation and
maximum absorbance [30]. Pcs exhibit typical electronic spectra
with B band at 300e400 nm and Q band at 600e700 nm, both
The HeLa cells were maintained in DMEM containing glucose
supplemented with 110 mg Lꢀ1 sodium pyruvate, 0.1 mg mLꢀ1
penicillin, 0.1 mg mLꢀ1 streptomycin,
L
-glutamine, 10% (v/v) FBS
correlate to
pe
p* transitions [31]. The UVevis spectra of metallic
(fetal bovine serum) and pyridoxine hydrochloride in a humidified
atmosphere at 37 ꢃC and 5.0% CO2. The cells were treated with Pcs
overnight. The cells were irradiated by 665 nm LED immediately
and then washed with PBS three times. Changes of cell morphology
were observed under the fluorescence microscope.
mono-Pcs in solution display a narrow Q band, but the splitting or
two bands observed in these complexes indicates the presence of
both monomeric and aggregate forms of the complexes in solution
[32]. For ZnPcs, they usually showed monomer behavior evidenced
by a single (narrow) Q band at w680 nm while aggregate behavior
by an additional Q band at 630e640 nm [26,33]. The aggregation
behavior of Pcs is dependent on the nature of solvent and substit-
uent et al. [34].
2.8. Statistical analysis
Statistical analysis was done and the mean, standard deviation,
standard error, and the significant changes were expressed as the
mean ꢄ S. All biochemical experiments were performed three
times and an average of the results were used. P < 0.05 was
considered necessary as statistical significance.
The ground state electronic absorption spectra of ZnPc1, ZnPc2
and ZnPc3 were performed in water and shown in Fig. 2. The
spectra superposed in Fig. 2 indicating that all of the three ZnPcs
are typical aggregate Pcs because of the appearance of the addi-
tional Q band at 640 nm. Such spectral features are typical for Pc
dimmers or aggregates [33]. Comparison studies indicated that
only the quaternization derivative ZnPc3 has an obvious monomer
band at 680 nm, which may be result from the effects of steric
hindrance and electrical charges. It seems evident that the charged
substituents induce mutual repulsion reducing the aggregation of
ZnPc3. However, hydrochloride derivative ZnPc2 shows serious
aggregation, with intense absorbance at 640 nm and negligible
band at 680 nm. It is obvious that good water solubility and pro-
tonation did not reduce the aggregation of ZnPc2 in water.
3. Results and discussion
The pH values would have an important impact on ZnPc2 since
protonation is reversible. So, the pH values were measured by a pH
meter. The data were performed three times and the average of the
results was used. At the concentration of 3 ꢂ 10ꢀ6 M (experimental
concentration), the pH values of the aqueous solutions of ZnPc1,
ZnPc2, ZnPc3 and redistilled water were 6.89, 6.75, 6.82 and 6.81
respectively. The results showed that there were only tiny changes
after the drugs added into redistilled water, and these tiny changes
would have little influence on properties and existence forms of the
drugs.
3.3. Fluorescence spectra and properties
Since fluorescence can be used to obtain information about
photosensitizer localization and distribution as well as release from
tissues [35], the fluorescence properties of Pcs intended for use in
PDT is especially important. In a particular solvent, the fluorescence
properties of Pcs can depend on many factors, including aggrega-
tion, substituent and pH et al.
As shown in Fig. 3, the fluorescence emission spectra of the
three ZnPcs were performed in water with the excitation wave-
length of 600 nm. The fluorescence emission of ZnPc1 was
completely quenched in water. The result can be attributed to the
3.1. Synthesis and comparison
Fig. 1 showed a brief schematic for the synthesis of quaterni-
zation and hydrochloride derivatives of ZnPc1. Cationic derivative
ZnPc3 was obtained by the quaternization of aliphatic nitrogen
atom of ZnPc1 in methanol. The quaternizing agent methyl iodide
was commonly used. The quaternizing agent methyl iodide is
potentially dangerous and must be used with care, as it is likely to

A. Wang et al. / Dyes and Pigments 99 (2013) 348e356
351
R3
R3
N
N
N
N
N
N
N
Zn
N
R1
R1
H
O
H
N
C
N
N
N
N
R3
R3
N
N
Zn
N
N
I-
N
I-
O
ZnPc3
R3=
R1
R1
R2
R2
N
N
HN
R1=
O
ZnPc1
N
N
N
N
N
N
Zn
N
R2
R2
N
•
2HCl
HN
O
ZnPc2 R2=
Fig. 1. Brief schematics for the synthesis of quaternization and hydrochloride phthalocyanine derivatives.
quenching of the excited singlet states by the diethylamino sub-
stituent as a result of effective intramolecular photoinduced elec-
tron transfer (PET) and aggregation [36,37]. Aggregated Pcs are not
known to fluorescence since aggregation lowers the fluorescence
intensity of molecules through dissipation of energy [5]. A common
feature of the quaternization derivative ZnPc3 and hydrochloride
derivative ZnPc2 is the strong fluorescence emission. In the case of
ZnPc3, the quaternization efficiently reduces the aggregation as
well as inhibits the intramolecular PET process, resulting in a
stronger fluorescence emission. Contrary to most Pc aggregate [6],
the hydrochloride derivative ZnPc2 show moderate fluorescence
emission intensity though it is aggregate in water. The observation
may be result from the competitive effect between intramolecular
PET and aggregation. The formation of hydrochloride can greatly
prevent the PET process by protonation, resulting in the increase of
fluorescence intensity. While, it is believed that the protonation of
amine groups did not reduce the aggregation evidently, resulting in
the quenching of fluorescence. The final fluorescence emission
spectra of ZnPc2 indicate that fluorescence quenching by aggre-
gation is lesser compared to the fluorescence enhancement by the
forbidden of PET.
0.5
500
ZnPc1
ZnPc1
ZnPc2
ZnPc2
0.4
ZnPc3
400
ZnPc3
0.3
0.2
0.1
0.0
300
200
100
0
300
400
500
600
700
800
640 660 680 700 720 740 760 780 800
Wavelength(nm)
Wavelength(nm)
Fig. 2. Ground state electronic absorption spectra of ZnPc1, ZnPc2 and ZnPc3 in water
(Concentration ¼ 3 ꢂ 10ꢀ6 M).
Fig. 3. Fluorescence emission spectra of ZnPc1, ZnPc2 and ZnPc3 in water
(Concentration ¼ 3 ꢂ 10ꢀ6 M; Excitation wavelengths: 600 nm).

352
A. Wang et al. / Dyes and Pigments 99 (2013) 348e356
3.4. Photodegradation studies and comparison
665 nm LED light. All the ZnPcs studied showed a decrease in the Q
band. However, the photostability of them are different. The result
can be attributed to a number of factors, including the substituent,
exist form of Pcs, solvent and light intensity et al. [39]. Different
substituents exhibit different effects on the photostability of ZnPcs.
While some substituents stabilize the ring against photo-
degradation, some make it more vulnerable to oxidative attack
[33,38].
As can be observed in Fig. 5, the hydrochloride derivative ZnPc2
showed better photostability than the quaternization derivative
ZnPc3. Generally, the result may be attributed to the following two
factors: 1) the existence form of ZnPc2 is mainly aggregate while
ZnPc3 shows apparent monomer; commonly, aggregate is more
stable than monomer in the same condition. 2) It is obviously that
the quaternization of amine groups resulted in the decrease in the
stability of the zinc Pc complexes, while the hydrochloride de-
rivative increased the stability [5]. From the Fig. 5, we can also
observe that the aggregate form of ZnPc3 is more stable than its
monomer form, which agrees with the same ZnPcs reported in
literature [26].
The photobleaching property is a measure of the photostability
of a photosensitizer and this is important for those potential for
use in PDT. It is indicated by a reduction in the absorbance in-
tensity without the emergence of new peaks on photo-irradiation
[38].
The decompositions of the three Pcs were analyzed following
the decrease in absorption of their Q bands. Fig. 4 shows the UVevis
spectra changes of the three Pcs in water upon irradiating with
0.4
A
0.3
0s
0.2
225s
3.5. Singlet oxygen generation ability
0.1
The production of singlet oxygen is result from the energy
transfer between the triplet state of photosensitizers and ground
state molecular oxygen. There is a necessity of high efficiency
singlet oxygen generation since the singlet oxygen generation
ability is an important indicator for the potential of photosensi-
tizers in PDT [40].
0.0
550
600
650
700
750
Wavelength(nm)
Singlet oxygen can be detected using a chemical method by the
photo-oxidation of ADPA to its endoperoxide derivative. The ability
of the three Pcs in generating singlet oxygen, as reflected by the rate
of decay of the singlet oxygen quencher ADPA, was recorded and
compared. As shown in Fig. 6, all of the Pcs can induce photo-
oxidation of ADPA (Fig. 6AeC) and the efficiency followed the or-
der kZnPc2 (0.0036) > kZnPc3 (0.0028) > kZnPc1 (0.0022) (Fig. 6D). It is
believed that the PET process of amino groups quench singlet ox-
ygen excited state of ZnPc1, resulting in the reduced singlet oxygen
production. In contrast, singlet oxygen generation increased when
the amino groups were quaternized or protonated, as the PET
pathway was switched off [41].
0.4
B
0.3
0.2
0.1
0.0
0s
225s
Comparative studies in the singlet oxygen production ability of
ZnPc2 and ZnPc3 show that both of them can efficiently increase
the production of singlet oxygen, while ZnPc2 is better than ZnPc3.
550
600
650
700
750
Wavelength(nm)
0.4
0.3
0.2
0.1
0.0
ZnPc2-a
50
ZnPc1-a
C
ZnPc1-m
40
ZnPc3-a
ZnPc3-m
0s
30
20
10
0
225s
0
50
100
150
200
250
550
600
650
700
750
Wavelength(nm)
Time(s)
Fig. 4. Absorption spectra changes of A: ZnPc1; B: ZnPc2; C: ZnPc3 irradiated with
665 nm LED in water (Concentration ¼ 3 ꢂ 10ꢀ6 M).
Fig. 5. Photobleaching properties of the three Pcs in water (a: aggregate form; m:
monomer form; Concentration ¼ 3 ꢂ 10ꢀ6 M).

A. Wang et al. / Dyes and Pigments 99 (2013) 348e356
353
1.5
1.0
0.5
0.0
1.5
A
C
0s
0s
1.0
150s
150s
0.5
0.0
300
350
400
450
300
350
400
450
Wavelength(nm)
Wavelength(nm)
1.5
1.0
0.5
0.0
0.0
ZnPc1
ZnPc2
ZnPc3
B
-0.1
-0.2
-0.3
-0.4
-0.5
-0.6
D
0s
150s
0
30
60
90
Time(s)
120
150
300
350
400
450
Wavelength(nm)
Fig. 6. UVevis spectrum for the determination of singlet oxygen generation ability of (A) ZnPc1; (B) ZnPc2; (C) ZnPc3 use ADPA as the quencher at the concentration of 3 ꢂ 10ꢀ6
M
(D) Plots of ADPA absorbance vs. time.
Many factors may be responsible for this result including: ability of
substituent, triplet excited state energy and the existence form of
the two Pcs. However, the predominant factor in explaining the
observed trends is difficult to identify.
since it is an important index in PDT. The Hoechst 33342 which is
part of blue fluorescent dyes is usually used to assess changes in
nuclear morphology, since it can bind to DNA. Fig. 8 showed the
nuclear morphology changes of HeLa cells after phototreatment. As
shown in Fig. 8A, when the cells were treated without drugs, there
is no significant change in their nuclear morphology. In contrast,
the cells showed obvious nuclear morphology changes, such as
nuclear shrinkage, chromatin condensation and fragmentation
when they were phototreated with ZnPc1 (Fig. 8B), ZnPc2 (Fig. 8C)
and ZnPc3 (Fig. 8D).
3.6. Zeta potential measurement
It was suggested that the zeta potential was important for
photosensitizers used in PDT since the charge of drugs was closely
related to cell uptake and finally affected the anticancer activity
[42]. The ZP of HeLa cells and the three Pcs were measured in
DMEM. The results showed that HeLa cells were electronegative
(ZP ¼ ꢀ7.34 mV), ZnPc1 was almost neutral (ZP ¼ 0.53 mV), while
ZnPc2 (ZP ¼ 12.30 mV) and ZnPc3 (ZP ¼ 8.76 mV) were both
electropositive. This result implied the high anticancer activity of
ZnPc2 and ZnPc3.
3.9. In vitro anticancer activity
The toxicity of Pcs without illumination was called darktoxicity.
It was an important biocompatibility indicator of Pcs. In order to
study the in vitro anticancer activity of ZnPc1 as well as its hydro-
chloride derivative ZnPc2 and quaternization derivative ZnPc3, the
darktoxicity and phototoxicity of them were evaluated toward HeLa
cells. To study darktoxicity of the ZnPcs, HeLa cells were treated
3.7. Cell morphology
The morphology of HeLa cells was observed by microscopy
before and after phototreatment. Drastic changes in the
morphology of HeLa cells, which was treated overnight with ZnPcs,
have been observed after irradiation. The representative results are
shown in Fig. 7. These results provided evidence about membrane
deformation and an increase in the volume of cells, indicating a
higher fragility after the photodynamic action.
with 10
mM drugs without illumination. The phototoxicity was
obtained by treating HeLa cells with 2
mination by 665 nm LED lamp.
mM drugs and 3 min illu-
As shown in Fig. 9, all of the Pcs are essentially noncytotoxic in
the absence of light, but exhibit different degrees of phototoxicity.
The low darktoxicity of the three ZnPcs indicate their good
biocompatibility. The phototoxicity of them follows the order
ZnPc2 > ZnPc3 > ZnPc1, which is in line with the trend of singlet
oxygen generation ability and ZP results mentioned above. The
hydrochloride derivative ZnPc2 and quaternization derivative
ZnPc3 are both particularly potent with the cell survival percentage
23.4% and 49.4%, separately.
3.8. Hoechst 33342 staining
PDT process will damage tumor cells as well as induce DNA
destruction in nuclear. There is a necessity of DNA damage research

354
A. Wang et al. / Dyes and Pigments 99 (2013) 348e356
Fig. 7. Photograph showing morphology of normal HeLa cells treated with drugs and irradiated for 5 min with 665 nm LED (A) control, without drugs; (B) treated with ZnPc1;
(C) treated with ZnPc2; (D) treated with ZnPc3. 40 microscope objective. Bar ¼ 100 m.
m
Fig. 8. Fluorescence micrographs of HeLa cells stained with Hoechst 33342 after being phototreated with ZnPcs (A) normal cells; (B) treated with ZnPc1; (C) treated with ZnPc2;
(D) treated with ZnPc3. Bar ¼ 100 m.
m

A. Wang et al. / Dyes and Pigments 99 (2013) 348e356
355
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∗∗
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0
ZnPc1
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Drugs
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efficiently improve the water solubility of the Pc-polyamine con-
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trogen atoms, the strategy of preparing hydrochloride is not only
much easier in the preparing process but also superior in the singlet
oxygen generation ability, photostability and in vitro anticancer
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particular interest because of its simple prepare process and
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Acknowledgment
This work was supported by the National Natural Science
Foundation of China (20973093, 21201102), The Natural Science
Foundation of Jiangsu Higher Education Institutions of China
(12KJB150015), The Scientific Research Foundation of Nanjing
Normal University (No. 2011103XG0249), The Priority Academic
Program Development of Jiangsu Higher Education Institutions
(PAPD).
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chemical modification of hypocrellins toward direct drug delivery and effec-
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