Highly water-soluble and tumor-targeted photosensitizers for photodynamic therapy

Article abstract of DOI:10.1039/c5ob01035g

Biological uses of photosensitizers in photodynamic therapy (PDT) often suffer from a lack of tumor selectivity; a strategy based on molecule-targeted cancer therapies could provide a promising solution. To synthesize new water-soluble phthalocyanines (Pc

Full text of DOI:10.1039/c5ob01035g

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Highly water-soluble and tumor-targeted
photosensitizers for photodynamic therapy†
Cite this: Org. Biomol. Chem., 2015,
13, 7681
Yuxi Li,‡a,b Jin Wang,‡b Xiaoxiao Zhang,b Wenjun Guo,b Fu Li,a Min Yu,b Xiuqi Kong,b
Wenjie Wu*a and Zhangyong Hong*b
Biological uses of photosensitizers in photodynamic therapy (PDT) often suffer from a lack of tumor
selectivity; a strategy based on molecule-targeted cancer therapies could provide a promising solution.
To synthesize new water-soluble phthalocyanines (Pcs) for bio-conjugation with peptides or antibodies,
we developed a method to synthesize asymmetrically substituted Pcs with both high water solubility and
one monoamino group for conjugation with biological agents for tumor homing, using folic acid as the
ligand model to direct the modified Pcs into target cells. Here, we report studies on the syntheses and
characterization of these Pcs. In vitro and in vivo assays prove that the high solubility characteristic can
greatly increase the tumor targeting capability of Pcs by reducing non-specific uptake. This newly
designed photosensitizer accumulated almost completely in tumor regions, with a negligible signal found
in other tissues in the xenograft tumor model. These initial data provide strong evidence of the high
specificity tumor targeting of Pcs with folate and tri-glycerol substitutions. Theoretically, the synthesized
Pcs could be conveniently conjugated to many other ligands, endorsing the broad applicability of this
method for tumor-targeted PDT.
Received 22nd May 2015,
Accepted 4th June 2015
DOI: 10.1039/c5ob01035g
www.rsc.org/obc
would likely increase the success of therapy. The natural con-
nection of near-infrared (NIR) fluorescence imaging with
1. Introduction
Photodynamic therapy (PDT) is a therapeutic procedure utili- photodynamic therapy (PDT) forms a beneficial union for
zing a photosensitizer activated by light to create irreversible application as a noninvasive tool for cancer therapy.5
photo-damage to tissues, which has become a well-established
The synthesis of photosensitizers with desired properties is
therapeutic modality for the treatment of a variety of premalig- considered to be an important bottleneck in PDT therapy.6
nant and malignant diseases.1 As a non-invasive modality, Ideal photosensitizers are non-toxic to the host in the absence
PDT combines three individually harmless components, a of light, accumulate preferentially in tumor tissue, and most
photosensitizer, light, and molecular oxygen, to generate reac- importantly, have a high molar absorption coefficient and
tive oxygen species (ROS) leading to cellular and tissue high singlet oxygen generation efficiency in the biological
damage.2 PDT also has the advantages of repeated dose toler- wavelength window (650–900 nm) for deeper penetration into
ance and high specificity, which are achieved through the biological tissues.7 Traditionally, porphyrin-based photosensi-
precise application of light.3 Furthermore, PDT agents act as tizers have dominated the PDT field, and they usually have a
fluorescent probes to be used for fluorescence imaging relatively weak satellite absorption band (Q-band) in the region
technology, which could offer superior sensitivity and real- of 600–650 nm.8 Phthalocyanines (Pcs), as next generation
time imaging for in vivo cancer diagnoses and presentations.4 photosensitizers, are among the most promising candidates
Clearly identifying cancer cells before or during treatment for PDT and have received considerable attention.9 They offer
multiple desirable characteristics, such as strong light absorp-
tion at long wavelengths (650 nm to 850 nm), high efficiency
aCollege of Material Science and Chemical Engineering, Tianjin University of Science
of singlet oxygen generation, extraordinary stability and bio-
and Technology, Tianjin 300457, P. R. China. E-mail: wwjie@tust.edu.cn;
compatibility.10 Furthermore, the spectral and photophysical
Tel: +86 022 23502875
bState Key Laboratory of Medicinal Chemical Biology, College of Life Sciences,
properties of Pcs can be easily tuned by varying the substitu-
ents around the Pc aromatic core or the central metal.11
For most Pcs, extremely low solubility and aggregation
phenomena in water render them photodynamically inactive
in aqueous medium and thus significantly restrict their in vivo
biological and medical applications.12 Pcs have not been syste-
Nankai University, Tianjin 300071, P. R. China. E-mail: hongzy@nankai.edu.cn;
Tel: +86 022 23498707
†Electronic supplementary information (ESI) available: Synthetic procedure for
resin-bound phthalonitrile, HPLC chromatogram, ESI-HRMS and NMR data of
the phthalocyanines. See DOI: 10.1039/c5ob01035g
‡Yuxi Li and Jin Wang contributed equally to this work.
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matically studied in aqueous solutions. In the literature,
For the above reasons, new types of Pcs with high water
detailed attention has been given to the absorption and emis- solubility, as well as with one or more functional groups for
sion properties of Pcs in DMSO solutions, in which extremely conjugation with biological agents, have been anxiously
low solubility could be obtained.13 To become promising awaited. Here, we report our study on the synthesis and charac-
photosensitizers, high singlet oxygen quantum yields in terization of only such an asymmetrically substituted highly
aqueous medium are necessary, thus water-solubility rep- water-soluble Pc with two different types of peripheral substi-
resents an important characteristic of good photosensitizers.
tuents: one reactive group for conjugation and others for solu-
Many studies have focused on the optimization of Pcs to bility. The functionalized amine group can be conveniently
use Pcs in aqueous media.11,14 A historical approach was to conjugated with ligands, herein with folic acid as a model, for
incorporate insoluble Pcs primarily in liposomes, biodegrad- tumor targeting purposes.27 The high solubility characteristic
able polymeric nanoparticles, or emulsions with the use of a can greatly reduce non-specific uptake and thus reduce the
surfactant, e.g., Cremophor EL.15 Other efficient strategies background. However, the tumor homing ligand can selectively
include chemical modification of Pcs through the attachment bring the PDT agents inside tumor cells. The evaluation of
of appropriate hydrophilic substituents. Until recently, most of their photophysical and photochemical properties both in vitro
the chemical modifications have been performed based on the and in vivo proves that they have high potential as tumor-selec-
attachment of ionic substituents, such as sulfonates16 and car- tive PDT agents. These preliminary studies may offer a useful
boxylates17 to form anionic Pcs, or quaternized amino18 and strategy in the quest for more efficient tumor-selective Pcs for
aromatic groups19 to form cationic Pcs. Ionic substitutions can PDT.
strongly affect the characteristics of Pcs, leading to increased
water solubility and a reduced degree of aggregation as well as
a high rate of singlet oxygen generation. However, they also
2. Results and discussion
have serious drawbacks, including interaction with the con-
2.1. Molecular design of water-soluble Pcs
stituents of biological fluids (e.g., plasma proteins) and inter-
faces20 (e.g., cell membrane) and causing serious side effects.
Biological uses of photosensitizers in PDT often suffer from a
lack of tumor selectivity. Taking advantage of highly specific
receptor–ligand interactions to direct the photosensitizers into
target cells leads to efficient strategies to solve this problem.
To synthesize new water-soluble Pcs for bio-conjugation with
peptides or antibodies, asymmetrically substituted Pcs with
two different types of peripheral substituents, one reactive
group for conjugation and others for solubility, were designed
(Fig. 1, PcZn1-lys and PcZn2-lys). For conjugation, an asym-
metric monoamino group was added using the strategy of
solid phase synthesis of un-symmetric AB3 type Pcs. Folic acid
was selected to covalently conjugate to these asymmetrically
substituted Pcs as model tumor-homing ligands for tumor tar-
geting purposes (Fig. 1, PcZn1-lys-FA and PcZn2-lys-FA). For
comparison, two types of hydrophilic substitutions, a triethy-
lene glycol monomethyl ether group and a glycerol group, were
adopted to increase solubility and reduce aggregation by
attachment to peripheral positions of the macrocycle. The sym-
metrical tetra-substituted PcZn1 and PcZn2 were also syn-
thesized for comparison. The water solubility and
photochemistry of these Pcs were tested and compared.
Theoretically, the synthesized Pcs could be conveniently conju-
gated to many other ligands for targeted PDT.
Non-ionic water-soluble Pcs represent another efficient way to
solve the solubility problems of Pcs, although studies of their
synthesis and application are rare. Recently several non-ionic
water-soluble Pcs have been synthesized through modification
with functional groups, such as carbohydrate substitution and
polyhydroxylate substitution.21
Conventional PDT for cancer therapy is based on the prefer-
ential accumulation of a photosensitizer in tumors with
minimal damage to normal tissues. However, because the
existing photosensitizers lack tumor selectivity, considerable
damage occurs in normal tissues, which leads to unwanted
toxicity. Thus, current methods of PDT would be improved if
more selective targeting of the photosensitizer was possible,
which would increase the uptake of PDT agents by the targeted
cancer cells. Most attempts at targeting have been performed
through encapsulation in liposomes and polymeric nano-
particles via an enhanced permeability and retention (EPR)
effect of tumor accumulation.22 Conjugation with various
tumor-specific vehicles, such as epidermal growth factor,
monoclonal antibodies23 and small molecule ligands (e.g.,
short peptides or peptidomimetics) would provide another
promising strategy to increase the selectivity of photosensiti-
zers with precise targeting properties. However, most of the
synthesized Pcs are not suitable for use in bio-conjugation, as
2.2. Synthesis of symmetric tetra-substituted Pcs
unique functional groups on the structure of Pcs that are Scheme 1 shows the synthesis route used to prepare the tetra-
needed for attachment are missing. The asymmetrically substi- substituted PcZn1 and PcZn2. 4-Nitrophthalonitrile reacts with
tuted A3B-type Pcs with one reactive group for conjugation are triethyleneglycol monomethylether 8 and S-(+)-2,2-dimethyl-
ideally suited for these applications,24 but difficulties in the 1,3-dioxolane-4-methanol 10, leading, respectively, to mono-
synthesis and isolation of these Pcs have limited their appli- substituted phthalonitriles 9 and 11 in a 74% yield and 71%
cation.25 Recently reported solid-phase synthesis of Pcs pre- yield. These monosubstituted phthalonitriles were cyclotetra-
sents an efficient method for the synthesis of pure AB3-type merized into PcZn1 and isopropylidene protected Pc 11 in the
mono-functionalized Pcs.26
presence of Zn(OAc)2·2H2O and 1,8-diazabicyclo[5.4.0]undec-7-
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Fig. 1 The structure of the designed Pcs.
Scheme 1 The syntheses of the symmetric Pcs with hydrophilic moiety substitutions.
ene (DBU) in n-hexyl alcohol. The isopropylidene group of 12 substitution has stronger effects; glycerol substituted PcZn2
could be removed readily upon treatment with a mixture of tri- has an even higher water solubility than the respective triethy-
fluoroacetic acid (TFA) and H2O (9 : 1 v/v), generating PcZn2 in leneglycol monomethylether substituted PcZn1. The structures
93% yield. The prepared PcZn1 and PcZn2 can be easily dis- of the new compounds were confirmed by NMR and high
1
solved in water. These oxygen-rich substituents added to the resolution mass spectrometry (HRMS). The H NMR spectra of
peripheral positions of the macrocycles can greatly enhance PcZn1 and PcZn2 show that the Pc protons appear at a lower
the solubility of PcZn1 and PcZn2 in water. However, glycerol field as a set of three multiplets, two multiplets between d 8.5
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and 9.1 ppm due to the resonances of the eight Pc-alpha 2.4. Conjugate of folic acid with the asymmetric tetra-
protons and a multiplet at d 7.6 and 7.8 ppm due to the four substituted Pcs
Pc-beta protons. The ESI-HRMS mass spectrum of PcZn1 and
The synthesized asymmetric PcZn1-lys and PcZn2-lys could be
conveniently conjugated to biomolecules, such as antibodies,
proteins or peptide ligands for selective tumor targeting pur-
poses in PDT. To test the application potential of these asym-
PcZn2 displayed ion peaks at m/z = 1247.4251 [M + Na]+ and
937.2102 [M + H]+, respectively, which confirmed the proposed
structures.
metric Pcs, we selected folic acid as the tumor homing ligand
to synthesize asymmetrically substituted PcZn1-lys-FA and
PcZn2-lys-FA. Two coupling strategies were tested for this
2.3. Synthesis of asymmetric tetra-substituted Pcs using
solid-phase synthesis
purpose. In the first (Scheme 3), the coupling reaction was
conducted on the solid support. The Mtt group was selectively
removed by treatment with a 2% trifluoroacetic acid solution
(TFA : DCM : TIS = 2 : 96 : 2) without cleaving the Pc from the
solid support. Then, folate was coupled in the presence of
HBTU and DIEA in a solution of DMF/DMSO (1 : 1). Finally,
polymer-bound AB3-type Pc was treated with 95% TFA
(TFA : H2O = 95 : 5) to yield the desired PcZn1-lys-FA or PcZn2-
lys-FA. In the second strategy (Scheme 4), the coupling of
folate was performed directly in the solution phase by the reac-
tion of PcZn1-lys or PcZn2-lys with NHS-activated folate in
DMSO solution, and the product was precipitated with diethyl
ether and purified through HPLC to produce PcZn1-lys-FA in
75% yield or PcZn2-lys-FA in 80% yield.
Solid-phase synthesis was utilized for the preparation of asym-
metric highly water-soluble AB3-type PcZn1-lys and PcZn2-lys
with monoamino substitution (as shown in Scheme 2) for the
convenient purification of the isotypes. The syntheses started
from an amine-functionalized, solid-supported phthalonitrile
13 (see the ESI† for the synthesis of 13). Briefly, a suspension
of polymer-bound phthalonitrile 13, triethyleneglycol mono-
methylether-substituted phthalonitrile 9 or glycerol-substi-
tuted phthalonitrile 11, Zn(OAc)2, and DBU in n-hexyl alcohol
were heated to 160 °C for 5 h to generate polymer-bound AB3-
type Pc. The resin was washed with methanol and CH2Cl2
until a colorless filtrate was collected, cleaved in 95% TFA with
triisopropylsilane (TIS) (TFA : H2O : TIS = 95 : 2.5 : 2.5), and was
then precipitated with diethyl ether and purified with HPLC to
afford AB3-type asymmetrical PcZn1-lys in approximately
20–30% yield. In addition to the resin, the 4-methyltrityl (Mtt)
group was also cleaved through TFA treatment. PcZn2-lys with
2.5. Ground state electron absorption and aggregation
behavior
glycerol-substituted chains was synthesized similarly, but the The electronic spectra of the synthesized Pcs consist of an
cutting reagent was changed to a solution of TFA/H2O (95 : 5) intense and sharp absorption band (Q band) at 682 nm and a
without TIS, as TIS could interfere with the isopropylidene broad Soret band (B band) at approximately 350 nm in DMSO
group and result in a very complex product. Synthesis of (Table 1 and Fig. 2), which are characteristic absorptions of
ZnPc1-lys and PcZn2-lys supports the applicability of this Pcs. Compared with the non-substituted ZnPc (∼670 nm, as
method to many other types of asymmetric Pcs.
shown in the ESI†), the Q band area is red-shifted during its
Scheme 2 Solid-phase syntheses of the asymmetric Pcs with monoamino group and hydrophilic moiety substitutions.
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Scheme 3 The conjugation of folate with the water soluble asymmetric Pcs on resin.
peripheral substitution, as can been explained by the attach- ranging from 1.0 × 10−5 to 2.0 × 10−6 M, as aggregation is
ment of the electron-donating alkoxy groups at the peripheral always concentration dependent (Fig. 2). The spectra had no
positions, reducing the energy gap of the Highest Occupied new bands appearing (normally blue-shifted) because the
Molecular Orbital (HOMO)–Lowest Unoccupied Molecular aggregated species and the normalized spectra at all concen-
Orbital (LUMO) of the Pc ring.
trations could be superimposed without a change in the shape
Pcs are notorious for their strong tendency to aggregate, of the Q-band. The intensity of the absorption of the Q band
which can significantly decrease their photosensitizing ability increased with the increase in concentration, obeying the
through self-quenching. Hydrophilic substitutions can reduce Beer–Lambert law, which suggested that aggregation of these
the aggregation tendency of the Pc core. The UV–Vis spectra of compounds in DMSO is negligible.
the Pcs were highly sensitive to the aggregation characteristics,
DMSO is known to be able to greatly reduce aggregation
which can be used to indicate the aggregation status. The elec- because it binds axially to zinc(II) Pcs as a coordinating
tronic spectra of all six of the synthesized Pcs in DMSO solvent. However, the electronic spectra of the formed Pcs in
showed completely monomeric behavior, as evidenced by a water showed differences (Fig. 3), although all of these Pcs are
single and narrow Q band (Fig. 2), typical for non-aggregated soluble in water. Their absorption spectra suggest cofacial
Pcs. However, in the spectra of the un-substituted ZnPc aggregation in water, as evidenced by the presence of a higher
(shown in the ESI, Fig. S5†), in addition to intense Q absorp- energy (blue-shifted) band at 620–640 nm in the Q band
tion bands at 670 nm, weaker absorptions at 621 nm were also region. This is evidence of the fact that both types of hydro-
present, which are generally observed in the presence of aggre- philic substitutions cannot completely solve the aggregation
gated species. The aggregation behavior of the Pcs in DMSO problems of Pcs in water, even though all of the synthesized
were also investigated through the analysis of the concen- Pcs have high water solubility. However, complete de-aggrega-
tration dependence of their UV–Vis spectra in concentrations tion of Pcs in pure water solution may not be necessary, as the
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Scheme 4 The conjugation of folate with the water soluble asymmetric Pcs in solution.
through adhesion to the cell membrane.23a Therefore, Triton
X-100 was added to the water solution of the six substituted
Pcs (concentration = 0.5 × 10−5 M) to mimic the lipid environ-
ment of cell membranes, and its ability to de-aggregate Pcs
was determined. The results (Fig. 3) showed that the aggrega-
tion of these molecules can be broken up in the presence of
Triton X-100 (10%). It is interesting to note the difference
between PcZn1-lys-FA and PcZn2-lys-FA compounds. The
addition of Triton X-100 can only partially break up the
aggregation of PcZn1-lys-FA (Fig. 3C), yet it completely broke
up PcZn2-lys-FA (Fig. 3F) aggregates, indicating that a glycol
chain substitution on Pcs has a stronger capability to decrease
the aggregation tendency of Pcs in aqueous media compared
to triethyleneglycol monomethylether substitution. Alterna-
tively, FA conjugation may reduce the water solubility and then
increase the aggregation of these conjugates.
Table 1 Absorption spectral data of PcZn1, PcZn1-lys, PcZn1-lys-FA,
PcZn2, PcZn2-lys and PcZn2-lys-FA in DMSO, H2O and a H2O + Triton
X-100 solution. For ZnPcs in DMSO, λmax = 672 nm, log ε = 5.14
Compound
PcZn1
Solvent
λmax/nm (log ε)
DMSO
H2O
H2O + Triton X-100
DMSO
H2O
H2O + Triton X-100
DMSO
H2O
H2O + Triton X-100
DMSO
H2O
682 (4.85)
629, 689
616, 684
682 (4.73)
631, 677
614, 682
682 (4.89)
641, 687
—, 685
PcZn1-lys
PcZn1-lys-FA
PcZn2
683 (4.69)
627, —
H2O + Triton X-100
DMSO
H2O
H2O + Triton X-100
DMSO
H2O
617,685
682 (4.65)
630, —
615, 684
682 (4.70)
630, —
PcZn2-lys
PcZn2-lys-FA
2.6. Fluorescence spectroscopy
H2O + Triton X-100
614, 683
The fluorescence excitation and emission spectra of all Pcs
were determined in DMSO, as shown in Fig. 4. All of the syn-
thesized Pcs display a fluorescent excitation band at approxi-
mately 684 nm and an emission band at approximately
694 nm in DMSO. They all showed similar fluorescence behav-
ior, and the shape of their excitation spectra was similar to
that of the absorption spectra.
Although these Pcs have a quite low fluorescence intensity
in water, the addition of Triton X-100 to the water solutions of
these Pcs can greatly increase the intensity of these complexes
pure water solution cannot completely mimic physiological
conditions and PDT agents could be de-aggregated to recover
their functions once they form complexes with lipids, the
important constituents of the cell membrane or organelle
membranes. Recently Makoto Mitsunaga found that photo-
sensitizers can be effectively activated to kill tumor cells
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Fig. 2 Absorption spectra of PcZn1 (a), PcZn1-lys (b), PcZn1-lys-FA (c), PcZn2 (d), PcZn2-lys (e) and PcZn2-lys-FA (f) at various concentrations in
DMSO. The inset plots are Q-band absorbance versus the concentration of the corresponding compounds.
due to the decreased aggregation of these complexes. To test
the influence of the presence of Triton X-100 on the fluore-
scence properties of these Pcs, the fluorescence emission
spectra of PcZn1-lys-FA were determined in the aqueous
medium (concentration = 1.0 × 10−5 M) with the addition of
varied concentrations of Triton X-100. As shown in Fig. 5,
PcZn1-lys-FA exhibited very low emission in water (approxi-
mately 1/100 of the intensity in DMSO), and the addition of
Triton X-100 to the aqueous solution resulted in a significant
increase in the fluorescence intensity. Addition of Triton X-100
to 1.25% increased the fluorescence intensity 20-fold, while an
increase to 10% increased the fluorescence intensity 100-fold,
with an intensity close to half of that in DMSO.
2.7. Fluorescence quantum yields, lifetimes and singlet
oxygen quantum yields
Fluorescence quantum yield refers to the ratio of the number
of photons emitted to the number of photons absorbed, and
fluorescence lifetime refers to the average time that a molecule
remains in its excited state before returning to its ground
state. Upon excitation at 621 nm in DMSO, compounds 1–6
showed a fluorescence emission at 692–696 nm with a
quantum yield of 0.12–0.14 and a fluorescence lifetime of
approximately 3.0 ns. The quantum yields are low when com-
pared to non-substituted ZnPc (quantum yield of 0.28 in
DMSO). This is in accordance with the general observation
Fig. 3 Absorption spectra of PcZn1 (a), PcZn1-lys (b), PcZn1-lys-FA (c),
that the lower the energy of the Q band, the smaller the
quantum yield value.28
PcZn2 (d) PcZn2-lys (e) and PcZn2-lys-FA (f) in DMSO (black), water (red)
and water + Triton X-100 (blue) at 5 μM.
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Fig. 4 Absorption, excitation and emission spectra of PcZn1 (a), PcZn1-lys (b), PcZn1-lys-FA (c), PcZn2 (d) PcZn2-lys (e) and PcZn2-lys-FA (f) in
DMSO. Excitation wavelength: 615 nm.
nations, indicating that the Pcs were not damaged by the gene-
rated singlet oxygen.
2.8. In vitro cellular uptake assays with confocal microscopy
Folate can act as the targeted ligand to enhance the specificity
and cellular uptake of the cargo into folate receptor (FR)-over-
expressing cancer cells. Therefore, the cellular uptake behavior
of these folate-conjugated Pcs was investigated with human
cervical cancer (HeLa) cells and murine embryonic fibroblast
(NIH3T3) cells by analysis with fluorescence microscopy. After
the cells were exposed to 50, 100 and 200 μM concentrations of
PcZn1-lys-FA or PcZn2-lys-FA for 3 h, the Pc fluorescence could
be detected and highly distributed inside the cells, indicating
an efficient uptake of the Pcs by tumor cells (Fig. 6). Both com-
Fig. 5 Emission spectra of PcZn1-lys-FA in water with varied concen-
trations of Triton X-100. Excitation wavelength: 615 nm.
pounds showed similar intracellular fluorescent patterns, with
strong punctate fluorescence primarily distributed in the cyto-
plasm. These results are promising for the possible develop-
ment of such conjugates for PDT. It should be noted that even
these two Pcs, which are almost non-fluorescent in aqueous
medium due to aggregation, show very strong fluorescence
inside the cytoplasm. Cellular uptake can significantly increase
the fluorescence intensity of Pcs, presumably due to
monomerization of the aggregated dye species inside the cells.
These compounds showed some selectivity for the FA receptor
on the tumor cells. HeLa cells that have higher FA receptor
expression levels took up more PcZn1-lys-FA and PcZn2-lys-FA
than the NIH3T3 cells. However, the selectivity was not high
and further improvements are needed to increase selectivity.
A good photosensitizer must be very efficient in generating
singlet oxygen. This is quantified by the parameter of the
singlet oxygen quantum yield. The singlet oxygen quantum
yields for Pcs 1–6 were determined using a chemical method
by monitoring the disappearance of 1,3-diphenylisobenzo-
furan (DPBF) in DMSO with a UV–Vis spectrophotometer
(shown in the ESI†). Non-substituted ZnPc was used as the
reference. The results showed that all six of the synthesized
Pcs have roughly the same singlet oxygen quantum yield
values in the range 0.45–0.55, as shown in Table 2. These
values are in a similar range when compared to the non-substi-
tuted ZnPc. The substituent ions have a very moderate influ-
ence on the singlet oxygen quantum yield. It should be noted
that there was no decrease in the Q-band or formation of new
bands during the singlet oxygen quantum yield determi-
2.9. In vivo imaging of the distribution of Pcs in tumor-
bearing mice
To examine the tumor-targeting capability of PcZn1-lys-FA and
PcZn2-lys-FA in vivo, we prepared a xenograft tumor model by
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Table 2 Photophysical and photochemical parameters of PcZn1, PcZn1-lys, PcZn1-lys-FA, PcZn2, PcZn2-lys and PcZn2-lys-FA in DMSO. ΦF: fluore-
scence quantum yield, τF: fluorescence lifetimes, τ0: natural radiative lifetime, κF: rate constants for fluorescence, ΦΔ: singlet oxygen quantum yield.
For ZnPcs, ΦF = 0.18, ΦΔ = 0.67
Compound
λEx/nm
λEm/nm
ΔStokes (nm)
ΦF
τF/ns
τ0/ns
κF/s−l (×107)
ΦΔ
PcZn1
682
684
686
683
682
683
692
692
696
696
695
696
10
10
14
15
13
14
0.13
0.12
0.13
0.14
0.13
0.14
3.33
3.02
3.06
3.73
3.19
3.49
25.6
25.2
23.5
26.6
24.5
24.9
3.9
4.0
4.2
3.8
4.1
4.0
0.50
0.50
0.49
0.53
0.56
0.54
PcZn1-lys
PcZn1-lys-FA
PcZn2
PcZn2-lys
PcZn2-lys-FA
Fig. 6 Confocal images of cultured HeLa cells and NIH3T3 cells incubated with different concentrations of PcZn1-lys-FA and PcZn2-lys-FA (red).
The nuclei of the cells were stained with DAPI (blue). Upper panels, incubated with PcZn1-lys-FA; lower panels, incubated with PcZn2-lys-FA.
subcutaneously inoculating mice with human epidermoid mice showed strong fluorescence, visualized throughout the
carcinoma (KB) cells (1.0 × 106 cells per mouse) in the whole body of the mice in 5 min, but an intense fluorescence
dorsum. KB cells have high expression levels of the folate signal was primarily located in the liver, kidney and lung.
receptor. When tumors grew to approximately 50 mm3 in After 3 h post-injection, the fluorescence signals began to
volume, PcZn1-lys-FA or PcZn2-lys-FA (250 µg per mouse) decrease. However, the tumor-to-background ratios increased
was intra-venously injected into the mice via the tail vein. and accumulation in the tumor could be observed. Yet at 7 h
The fluorescence signal and intensity distribution of the Pcs post-injection, only very weak fluorescence signals could be
were monitored continuously with an in vivo fluorescence detected and were primarily located in the tumor and lung.
imaging system (IVIS Lumina II, Xenogen, Alameda, CA, USA). In contrast, the tumor distribution of PcZn2-lys-FA was signifi-
As shown in Fig. 7A and C, PcZn1-lys-FA injected into the cantly different (as shown in Fig. 7B and D). In the tumor
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Fig. 7 Distribution of PcZn1-lys-FA and PcZn2-lys-FA (red) in vivo and in different organs. The BALB/c nude mice bearing KB human tumour xeno-
grafts at their spike were intravenously injected with PcZn1-lys-FA and PcZn2-lys-FA via the tail vein. (A) In vivo images of the mice treated with
PcZn1-lys-FA; (B) In vivo images of the mice treated with PcZn2-lys-FA; (C) PcZn1-lys-FA and (D) PcZn2-lys-FA distribution in different organs: liver,
spleen, heart, kidney, lung and tumor (from left to right).
region, the fluorescence intensity of PcZn2-lys-FA was in vivo studies of these Pcs. PcZn2-lys-FA can completely dis-
much stronger than that of PcZn1-lys-FA. At 3 hours post criminate between healthy and tumor tissues in a sub-
injection, accumulation of the PcZn2-lys-FA fluorescence cutaneous xenograft tumor model, making this approach a
signals in the tumor regions was already very strong. After promising therapeutic and diagnostic agent for the treatment
3 hours post injection, the fluorescence intensity in the of cancer. The ability to covalently conjugate many different
tumors gradually increased, while the signal in other regions ligands to Pcs means that this may be a highly flexible plat-
(including liver, spleen, heart, kidney and lung) were rapidly form to synthesize Pcs for targeted PDT. The present investi-
reduced, resulting in significantly increased tumor-to-back- gations are preliminary studies in the search for more efficient
ground ratios. After 7 hours post injection, the signals were photodynamic therapy agents based on Pcs.
almost completely located in the tumor area; only negligible
signals could be found in other tissues. These initial data
provided strong evidence of high-specificity tumor targeting
4. Experimental section
of PcZn2-lys-FA with folate and tri-glycerol substitutions.
4.1. Chemicals and materials
The high hydrophilicity of glycerol moieties may have the
4-Nitrophthalonitrile, triethyleneglycol monomethylether, S-
(+)-2,2-dimethyl-1,3-dioxolane-4-methanol and 4-hydroxy-
ability to greatly reduce the non-specific affinity to normal
tissues.
benzoic acid were purchased from Alfa Aesar (Tianjin, China).
Triton X-100 and non-substituted ZnPc were purchased from
Aldrich. 1,3-Diphenylisobenzofuran (DPBF) was purchased
from J&K (Beijing, China). RPMI-1640 without folic acid was
3. Conclusion
In conclusion, we developed a target-specific Pc based on a purchased from Sigma-Aldrich (St. Louis, MO, USA). Other
highly water-soluble folate-Pc conjugate. Here, we report the chemical agents were purchased from Alfa Aesar (Tianjin,
synthesis, basic photophysical properties, and in vitro and China).
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4.2. Cells and animals
4.18 (dt, J = 9.9, 4.9 Hz, 1H), 4.09 (qd, J = 9.7, 5.3 Hz, 2H), 3.89
(dt, J = 14.4, 7.2 Hz, 1H), 1.44 (s, 3H, CH3), 1.40 (s, 3H, CH3).
Synthesis of PcZn1 (1). A solution of phthalonitrile 9
(145 mg, 0.5 mmol) and Zn(OAc)2·2H2O (60.4 mg, 0.25 mmol)
in n-hexyl alcohol (2 mL) was heated to 120 °C to completely
dissolve the salt before DBU (83.8 μL) was added. The mixture
was stirred at 160 °C for 5 h. The product was solidified by
pouring into 20 mL of petroleum ether and draining the solu-
tion. The green product was purified by silica gel chromato-
graphy using a dichloromethane : methanol mixture (20 : 1) as
an eluent (94 mg, 62%). HRMS (ESI): calcd for
C60H72N8NaO16Zn [M + Na]+ 1247.4250, found 1247.4251. 1H
NMR (400 MHz, DMSO-d6) δ 8.81 (m, 4H, Ar-H), 8.34 (m, 4H,
Ar-H), 7.58 (m, 4H, Ar-H), 4.63 (m, 8H, CH2), 4.13 (m, 20H,
CH2 and CH3), 3.91–3.81 (m, 8H, CH2), 3.75 (m, J = 3.0 Hz, 8H,
CH2), 3.67 (m, J = 2.1 Hz, 8H, CH2), 3.54 (m, 8H, CH2). 13C
NMR (DMSO-d6) overlapping signals at 159.85, 151.36, 139.69,
130.97, 122.99, 117.32, 105.25, 71.34, 69.71, 67.78, 58.00.
HeLa, NIH3T3 and KB cells were obtained from Saierbio
(Tianjin, China). The cells were continuously cultured in folic
acid-free RPMI 1640 medium supplemented with 10% fetal
bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C
under a humidified atmosphere containing 5% CO2. The final
folic acid concentration (with FBS as the only source of folic
acid) falls in the range of the physiological concentration of
human serum. BALB/c nude mice (4–6 weeks of age) were pur-
chased from Vital River Laboratory Animal Technology Co. Ltd
(Beijing, China) and maintained in a germ-free environment
with free access to food and water. All animal procedures were
conducted under a protocol approved by the Institutional
Animal Care and Use Committee of Nankai University
(Tianjin, China).
4.3. Instruments
1H and 13C NMR spectra were recorded in DMSO-d6 solutions
on a Bruker AV400 400 MHz spectrometer. The mass spectra
were recorded on Varian 7.0 T FTMS. Optical spectra in the
UV-visible region were recorded with USA Cary 5000 using a
1 cm path length cuvette at room temperature. Fluorescence
measurements were performed on a PTI QM/TM/NIR system
(Photon Technology International, Birmingham, NJ, USA)
equipped with a quartz cell (1 cm × 1 cm).
Synthesis of phthalocyanine 12. A mixture of phthalonitrile
11 (145.2 mg, 0.56 mmol), Zn(OAc)2·2H2O (41.2 mg,
0.188 mmol) in n-hexyl alcohol (2 mL) was heated to 120 °C
for 10 min before DBU (76.5 μL) was added. The mixture was
stirred at 160 °C for 5 h. The product was solidified by pouring
into 20 mL of petroleum ether and draining the solution. The
green product was purified by silica gel chromatography using
1
PE : EA (2 : 3) as an eluent (107 mg, 70%). H NMR (400 MHz,
DMSO-d6) δ 9.06 (s, 2H, Ar-H), 8.69 (s, 2H, Ar-H), 7.75 (d, J =
8.6 Hz, 4H, Ar-H), 7.46–7.25 (m, 4H, Ar-H), 4.77 (s, 2H), 4.61 (s,
4H), 4.42 (d, J = 26.4 Hz, 4H), 4.24 (s, 2H), 4.14 (s, 6H), 3.79 (s,
4.4. Synthesis experiments
Synthesis of phthalonitrile 9. To a solution of 4-nitrophtha- 2H), 1.30 (dd, J = 34.7, 15.0 Hz, 24H, CH3).
lonitrile (1.0 g, 5.78 mmol) in dry DMF (8 mL) were added tri-
Synthesis of PcZn2 (2). Compound 12 (100 mg, 0.09 mmol)
ethyleneglycol monomethylether (1.42 g, 8.67 mmol) and was stirred in a solution of TFA : H2O = 95 : 5 for 10 min. The
finely ground K2CO3 (3.6 g, 26 mmol). The color of the solu- reaction mixture was then evaporated to dryness under
tion turned from yellow to dark brown. The mixture was reduced pressure, and the dark blue powder was washed suc-
stirred at room temperature for 24 h before being poured into cessively with ethyl acetate, hexane, dichloromethane and
350 mL of ice-water and left overnight. The resulting yellow chloroform to yield 2 as a dark blue powder (78 mg, 93%).
precipitate was isolated by filtration and purified by column HRMS (ESI): calcd for C44H41N8O12Zn [M + H]+ 937.2130,
1
chromatography on silica gel with ethyl acetate–petroleum found 937.2102. H NMR (400 MHz, DMSO-d6) δ 9.30 (m, 4H,
ether (1 : 1) as an eluting solvent to give a yellow solid (1.2 g) in Ar-H), 8.89 (m, 4H, Ar-H), 7.81 (m, 4H, Ar-H), 5.26 (m, 4H,
74% yield. 1H NMR (400 MHz, CDCl3) δ 7.70 (d, J = 8.8 Hz, 1H, CH2), 4.94 (m, 4H, CH2), 4.63 (m, 4H, CH), 4.51 (m, 4H, CH2),
Ar-H), 7.31 (d, J = 2.5 Hz, 1H, Ar-H), 7.22 (dd, J = 8.8, 2.6 Hz, 4.15 (m, 4H, CH2), 3.73 (s, 8H, O-H). 13C NMR (DMSO-d6) over-
1H, Ar-H), 4.22 (dd, J = 5.2, 3.9 Hz, 2H, CH2), 3.89 (dd, J = 5.2, lapping signals at 160.45, 152.07, 139.65, 130.81, 123.15,
3.9 Hz, 2H, CH2), 3.75–3.69 (m, 2H, CH2), 3.69–3.61 (m, 4H, 117.63, 105.56, 70.35, 63.00.
CH2), 3.57–3.52 (m, 2H, CH2), 3.38 (s, 3H, CH2).
Synthesis of PcZn1-lys (3). Phthalonitrile 9 (1.8 mmol) and
Synthesis of phthalonitrile 11. The solution of 4-nitrophtha- Zn(OAc)2 (0.6 mmol) were added to resin 13 (0.3 g,
lonitrile (1.0 g, 5.78 mmol), S-(+)-2,2-dimethyl-1,3-dioxolane-4- 0.0225 mmol) that was pre-swelled in anhydrous n-hexanol
methanol (764 μL, 5.78 mmol) and finely ground K2CO3 (3.6 g, (3 mL) for 30 min. The mixture was heated to 120 °C for
26 mmol) in dry DMF (8 mL) was stirred at 50 °C, checking for 10 min, and DBU (1.2 mmol, 163 μL) was added to the
the completion (approximately 6 h) of the reaction by TLC mixture. The reaction was carried out at 160 °C for 5 h. The
(silica gel, hexane/ethyl acetate = 1/1). The reaction mixture mixture was solidified by pouring it into 30 mL of petroleum
was poured into ice water and left overnight. The resulting ether and draining the solution. The resin was washed with
solid was filtered and purified by column chromatography dichloromethane and methanol until a colorless filtrate was
(silica gel, hexane/ethyl acetate = 3/1) to yield 1.05 g of 11 obtained. The resin was suspended into a solution of TFA/
1
(71%) as a white solid. H NMR (400 MHz, CDCl3) δ 7.73 (dd, H2O/triisopropylsilane (TIS) (95 : 2.5 : 2.5) and shaken for 1 h
J = 8.8, 4.2 Hz, 1H, Ar-H), 7.31 (d, J = 2.5 Hz, 1H, Ar-H), 7.23 at room temperature. The filtrate was evaporated to dryness,
(dd, J = 8.8, 2.6 Hz, 1H, Ar-H), 4.50 (dq, J = 11.0, 5.4 Hz, 1H), and the crude mixture was purified by filtration through high
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performance liquid chromatography (HPLC, LC-20AT, Shi- 2Pc-Ar-H), 7.09 (m, 2H, Ar-H), 6.64 (m, 2H, FA-Ar-H), 4.67–3.37
madzu, Kyoto, Japan) to yield the product PcZn1-lys as a blue (m, 36H, CH2), 3.10 (m, 9H, CH3), 1.79–1.34 (m, 6H, CH2).
solid (7.5 mg, 25% yield). HRMS (ESI): calcd for
Synthesis of PcZn2-lys-FA (6). The Mtt protecting group of
C66H75N11NaO15Zn [M + Na]+ 1348.4628, found 1348.4632. H resin 15 was deprotected in a solution of TFA/CH2Cl2/TIPS
NMR (400 MHz, DMSO-d6) δ 9.20 (m, 1H, NH), 9.04 (m, 3H, (1 : 98 : 1). Folic acid (0.11 mmol), HBTU (0.1 mmol) and DIEA
Ar-H), 8.71–8.51 (m, 3H, Ar-H), 8.18 (m, 2H, Ar-H), 7.69 (m, (0.22 mmol) were dissolved in 5 mL of DMF/DMSO (1 : 1) and
4H, Ar-H), 7.58–7.47 (m, 2H, Ar-H), 7.11 (m, 2H, Ar-H), 4.67 added to the resin. The mixture was agitated for 3 h in the
(m, 6H, CH2), 4.46 (m, 1H, CH), 4.10 (m, 6H, CH2), 3.82 (m, dark and then washed with DMF/DMSO (1 : 1) (5 × 8.0 mL) to
6H, CH2), 3.71 (m, 6H, CH2), 3.63 (m, J = 4.1 Hz, 6H, CH2), yield resin 17. The resin 17 was suspended in a cleavage cock-
3.50 (m, J = 4.1 Hz, 6H, CH2), 3.29–3.25 (m, 9H, CH3), 2.82 (m, tail of TFA/H2O = 95 : 5 and purified using HPLC to yield the
2H, CH2), 1.81 (m, 2H, NH2), 1.59 (m, 2H, CH2), 1.45 (m, 2H, product PcZn2-lys-FA as a blue solid (7.4 mg, 24% yield). In the
1
CH2), 1.24 (m, J = 11.2 Hz, 2H, CH2).
second strategy, phthalocyanine 4 (4.2 mg, 0.0037 mmol) was
Synthesis of PcZn2-lys (4). Phthalonitrile 11 (1.8 mmol) and mixed with folic acid-NHS (3.0 mg, 0.0056 mmol) in DMSO
Zn(OAc)2 (0.6 mmol) were added to resin 13 (0.3 g, and then triethylamine (2.0 μL) was added. The mixture was
0.0225 mmol), which was pre-swelled in anhydrous n-hexanol stirred in the dark for 8 h and purified using HPLC to yield the
(3 mL) for 30 min. The mixture was heated to 120 °C, and DBU product PcZn2-lys-FA as a blue solid (4.5 mg, 80%). HRMS
(1.2 mmol, 163 μL) was added to the mixture. The reaction was (ESI): calcd for C73H69N18O17Zn+ [M + H]+ 1533.4374, found
1
conducted at 160 °C for 5 h. The mixture was solidified by 1533.4370. H NMR (400 MHz, DMSO-d6) δ 9.11 (m, 2H, Pc-Ar-
pouring it into 30 mL of petroleum ether and draining the H), 8.66–8.48 (m, 4H, 3Pc-Ar-H, 1FA-Ar-H), 8.19 (m, 3H, Pc-Ar-
solution. The resin was washed with dichloromethane and H), 7.88 (m, 4H, 2Pc-Ar-H, 2Ar-H), 7.66 (m, 4H, 2Pc-Ar-H,
methanol until a colorless filtrate was obtained. The resin was 2FA-Ar-H), 7.50 (m, 2H, Pc-Ar-H), 7.09 (m, 2H, Ar-H), 6.64 (m,
suspended in a solution of TFA/H2O = 95/5 and shaken for 1 h 2H, FA-Ar-H), 4.54–3.10 (m, 27H, 16CH2, 5CH, 6OH), 2.34–1.19
at room temperature. The filtrate was evaporated to dryness, (m, 10H, CH2).
and the crude mixture was purified by filtration through high
performance liquid chromatography (HPLC, LC-20AT, Shi-
4.5. Photophysical and photochemical properties
madzu, Kyoto, Japan) to yield PcZn2-lys as a blue solid (6.9 mg,
27% yield). HRMS (ESI): calcd for C54H51N11NaO12Zn+ [M +
UV-Vis spectra. Optical spectra in the UV-Vis region were
recorded in the wavelength range of 300–850 nm with a USA
1
Na]+ 1132.2902, found 1132.2908. H NMR (400 MHz, DMSO-
Cary 5000 using a 1 cm path length cuvette at room
temperature.
d6) δ 9.24 (m, 2H, Pc-Ar-H), 9.02–8.67 (m, 2H, Pc-Ar-H), 8.45
(m, 1H, Pc-Ar-H), 8.16 (m, 2H, Pc-Ar-H), 7.96 (m, 1H, Pc-Ar-H),
7.72 (m, 4H, 2Pc-Ar-H, 2Ar-H), 7.50 (m, 2H, Pc-Ar-H), 7.08 (m,
2H, Ar-H), 4.54 (m, 6H, CH2), 4.14 (m, 3H, CH), 3.73 (m, 6H,
CH2), 2.81 (m, 2H, CH2), 1.78 (m, 2H, CH2), 1.59 (m, 2H, CH2),
1.40 (s, 6H, O-H), 1.21 (m, J = 23.1 Hz, 2H, CH2).
Synthesis of PcZn1-lys-FA (5). The Mtt protecting group of
resin 14 was deprotected in a solution of TFA/CH2Cl2/TIPS
(1 : 98 : 1). Folic acid (0.11 mmol), HBTU (0.1 mmol) and DIEA
(0.22 mmol) were dissolved in 5 mL of DMF/DMSO (1 : 1) and
added to the resin. The mixture was agitated for 3 h in the
dark and then washed with DMF/DMSO (1 : 1) (5 × 8.0 mL) to
yield resin 16. The resin 16 was suspended in a solution of
TFA/H2O/TIPS (95 : 2.5 : 2.5; 2 mL) and agitated for 1 h. The
resin was removed by filtration. After removing the TFA of the
filtrate in a vacuum, the residue was dissolved in DMSO and
purified using high performance liquid chromatography
Excitation and emission spectra. Fluorescence excitation
and emission spectra were recorded in the wavelength range of
500–850 nm using 1 cm path length cuvettes at room tempera-
ture. The photomultiplier tube (PMT) voltage was set at 1074
V. The decayed curves of Pc emission at 692 nm were excited
by the high resolution laser (N2 laser at 337 nm tuned by the
laser dye PLD665) at 665 nm.
Quantum yield. The quantum yield (ΦF) of Pcs in DMSO
was determined on a PTI QM/TM/NIR system spectrometer
under excitation of 665 nm, using a comparative method with
the non-substituted ZnPc (ΦF = 0.18 in DMSO)29 as the refer-
ence,
FAstdn2
FStdAn2std
ΦF ¼ ΦFðStdÞ
(HPLC, LC-20AT, Shimadzu, Kyoto, Japan) to yield the product where F and FStd are the areas under the fluorescence emission
PcZn1-lys-FA as a blue solid (3.1 mg, 7.9% yield). In the second curves of the samples and the standard, respectively. A and
strategy, phthalocyanine 3 (5.0 mg, 0.0037 mmol) was mixed AStd are the respective absorbance of the samples and standard
with folic acid-NHS (3.0 mg, 0.0056 mmol) in DMSO and then at the excitation wavelengths. n2 and nstd are the refractive
2
triethylamine (2.0 μL) was added. The mixture was stirred in indices of solvents used for the sample and standard, respect-
the dark for 8 h and purified with HPLC to yield the product ively. The absorbance of the solutions at the excitation wave-
PcZn1-lys-FA as a blue solid (4.9 mg, 75%). HRMS (ESI): calcd length ranged between 0.03 and 0.05.
for C85H92N18NaO20Zn+ [M + Na]+ 1771.5919, found 1771.5925.
Natural radiative lifetimes. Natural radiative lifetimes (τ0)
1H NMR (400 MHz, DMSO-d6) δ 9.20 (m, 1H, NH), 9.01 (m, 3H, were determined using the PhotochemCAD program which
Pc-Ar-H), 8.62 (m, 4H, 3Pc-Ar-H, 1FA-Ar-H), 8.22 (m, 2H, Ar-H), uses the Strickler–Berg equation. The fluorescence lifetimes
τF
7.87 (m, 4H, 2Pc-Ar-H), 7.67 (m, 2H, 2FA-Ar-H), 7.51 (m, 2H, (τF) were evaluated using the equation: ΦF ¼
.
τ0
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Singlet oxygen quantum yields. Singlet oxygen quantum (no. 20110031110019) and the National Natural Science Foun-
yield (ΦΔ) values were determined by the comparative method dation of China (no. 31270926).
using 1,3-diphenylisobenzofuran (DPBF) as a singlet oxygen
chemical quencher in DMSO with ZnPcs as references:
Std
Notes and references
RI
ΦΔ ¼ ΦSΔtd
abs
RStdIabs
1 (a) H. Ali and J. E. van Lier, in Handbook of Porphyrin
where ΦSΔtd is the singlet oxygen quantum yield for the stan-
dard ZnPc (ΦSΔtd = 0.67 in DMSO). R and RStd are the DPBF
photobleaching rates in the presence of respective samples
and standards, respectively, and Iabs and I
light absorption by synthetic phthalocyanines and reference
substances, respectively. The degradation of the solutions was
monitored at 417 nm, and DPBF concentrations were lowered
to ∼2.5 × 10−5 mol L−1. The light intensity of 6.0 × 1015
photons per s per cm2 was used for ΦΔ determinations.
Science, ed. K. M. Kadish, K. M. Smith and R. Guilard,
World Scientific, 2010, vol. 4, pp. 1–120; (b) A. P. Castano,
P. Mroz and M. R. Hamblin, Nat. Rev. Cancer, 2006, 6, 535;
(c) D. E. Dolmans, D. Fukumura and R. K. Jain, Nat. Rev.
Cancer, 2003, 3, 380–387; (d) H. Ali and J. E. van Lier,
Chem. Rev., 1999, 99, 2379.
2 (a) A. Juarranz, P. Jaen, F. Sanz-Rodriguez, J. Cuevas and
S. Gonzalez, Clin. Transl. Oncol., 2008, 10, 148–154;
(b) B. W. Henderson and T. J. Dougherty, Photochem. Photo-
biol., 1992, 55, 145–157.
Std
abs
are the rates of
4.6. In vitro cellular uptake assays
3 S. B. Brown, E. A. Brown and I. Walker, Lancet Oncol., 2004,
5, 497.
HeLa and NIH3T3 cells that were continuously cultured in
folic acid-free RPMI 1640 medium supplemented with 10%
FBS and 1% penicillin/streptomycin at 37 °C and 5% CO2 were
seeded into 24-well chambered coverglass (Lab-Tek, Nunc,
USA) at a density of 5 × 104 cells per well (0.2 mL) 24 h before
initiating the experiments. Two hours before the experiments,
the medium was removed and replaced with 1.0 mL of fresh
folic acid-deficient RPMI-1640. After incubation with a series
of concentrations of PcZn1-lys-FA or PcZn2-lys-FA at 37 °C for
30 min, the cells were washed three times with phosphate
buffer saline (PBS). The cells were then fixed with 4% formal-
dehyde for 10 min at room temperature and washed three
times with PBS. Then, the cells were stained with 1 µg mL−1
DAPI for 3–5 min and washed three times with PBS. Confocal
images were acquired using a Confocal Laser Scanning Micro-
scope (TCS SP8, Leica, Wetzlar, Germany).
4 (a) G. M. van Dam, G. Themelis, L. M. Crane, N. J. Harlaar,
R. G. Pleijhuis, W. Kelder, A. Sarantopoulos, J. S. de Jong,
H. J. Arts, A. G. van der Zee, J. Bart, P. S. Low and
V. Ntziachristos, Nat. Med., 2011, 17, 10; (b) Q. T. Nguyen,
E. S. Olson, T. A. Aguilera, T. Jiang, M. Scadeng and
L. G. Ellies, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 4317–
4322; (c) H. Kobayashi, M. Ogawa, R. Alford, P. L. Choyke
and Y. Urano, Chem. Rev., 2010, 110, 2620–2640.
5 (a) L. B. Josefsen and R. W. Boyle, Theranostics, 2012, 2,
916–966; (b) J. P. Celli, B. Q. Spring, I. Rizvi, C. L. Evans,
K. S. Samkoe, S. Verma, B. W. Pogue and T. Hasan, Chem.
Rev., 2010, 110, 2795–2838.
6 P. Agostinis, K. Berg, K. A. Cengel, T. H. Foster,
A. W. Girotti, S. O. Gollnick, S. M. Hahn, M. R. Hamblin,
A. Juzeniene, D. Kessel, M. Korbelik, J. Moan, P. Mroz,
D. Nowis, J. Piette, B. C. Wilson and J. Golab, CA-Cancer
J. Clin., 2011, 61, 250–281.
4.7. In vivo imaging
7 (a) S. L. Gibbs, Quant. Imaging Med. Surg., 2012, 2, 177–187;
(b) N. M. Idris, M. K. Gnanasammandhan, J. Zhang,
P. C. Ho, R. Mahendran and Y. Zhang, Nat. Med., 2012, 18,
1580–1585; (c) M. Ethirajan, Y. Chen, P. Joshi and
R. K. Pandey, Chem. Soc. Rev., 2011, 40, 340–362; (d) S. Luo,
E. Zhang, Y. Su, T. Cheng and C. Shi, Biomaterials, 2011,
32, 7127–7138; (e) H. Kobayashi, M. Ogawa, R. Alford,
P. L. Choyke and Y. Urano, Chem. Rev., 2010, 110, 2620–
2640.
To set up the tumor model, Balb/c nude mice (8 weeks of age)
were implanted with KB cells (2 × 106 cells per mouse in a
volume of 100 μL) subcutaneously in the flank. When the
tumor volumes reached approximately 50 mm3 (approximately
10 days post-tumor inoculation), PcZn1-lys-FA or PcZn2-lys-FA
was injected into the mice via the tail vein at a dose of 250 µg
per mouse. Images were captured at 0, 5 min, 1 h, 4 h and 7 h
after injection using the Xenogen in vivo imaging system with
a Cy 5.5 filter (λex = 615–665 nm, λem = 695–770 nm). The
Xenogen images were obtained with the same settings (small
binning value 2, exposure time 5 s, F/stop 1). The nude mice
were sacrificed 7 or 9 h after injection. Then, the organs
including liver, spleen, heart, lung, kidney and tumor were col-
lected and analyzed by the Xenogen in vivo imaging system.
8 M. Ethirajan, Y. Chen, P. Joshi and R. K. Pandey, Chem.
Soc. Rev., 2011, 40, 340–362.
9 (a) J.-P. Taquet, C. Frochot, V. Manneville and M. Barberi-
Heyob, Curr. Med. Chem., 2007, 14, 1673–1687;
(b) T. Nyokong, Coord. Chem. Rev., 2007, 251, 1707;
(c) R. Hudson and R. W. J. Boyle, J. Porphyrins Phthalo-
cyanines, 2004, 8, 954–975.
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Acknowledgements
This work was supported by the Research Fund for the
Doctoral Program of Higher Education of China
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