Synthesis and characterization of aminoporphyrin-end-functionalized poly(N-isopropylacrylamide) with photodynamic and thermoresponsive effects

Article abstract of DOI:10.1002/asia.201301513

The novel aminoporphyrin-end-functionalized poly(N-isopropylacrylamide) (PNIPAM) polymer H₂N-TPP-PNIPAM (TPP=5,10,15,20-tetraphenyl-21H,23H- porphyrin) behaves as a multifunctional platform that displays a photodynamic effect, thermosensitivity

Full text of DOI:10.1002/asia.201301513

FULL PAPER
DOI: 10.1002/asia.201301513
Synthesis and Characterization of Aminoporphyrin-End-Functionalized
Poly(N-isopropylacrylamide) with Photodynamic and Thermoresponsive
Effects
Yang Yang,[a, b] Yanhui Li,[a] Nannan Qiu,[a] Guihua Cui,[a] Toshifumi Satoh,[c] and
Qian Duan*[a]
Abstract: The novel aminoporphyrin-
end-functionalized poly(N-isopropyla-
crylamide) (PNIPAM) polymer H2N-
TPP-PNIPAM (TPP=5,10,15,20-tetra-
phenyl-21H,23H-porphyrin) behaves as
a multifunctional platform that displays
a photodynamic effect, thermosensitivi-
ty, and fluorescence properties. The
polymer was designed by using an
asymmetrical aminoporphyrin (i.e.,
H2N-TPP-Cl) as the initiator for the
atom-transfer radical polymerization of
N-isopropylacrylamide (NIPAM). The
polydispersity index (PDI) obtained by
gel-permeation chromatography indi-
cated that the molecular-weight distri-
bution was narrow (1.09<PDI<1.27).
The lower critical solution tempera-
tures of H2N-TPP-PNIPAM showed
a decreasing trend as the molecular
weight was increased as a result of the
incorporation of the porphyrin group
at the end of the chain. The fluores-
cence spectra revealed the luminescent
properties of the materials. The results
of confocal laser scanning microscopy
showed that the polymer could enter
the cytoplasm through endocytosis. In
addition, the multifunctional platform
exhibited low toxicity against normal
cells (L929) and cancer cells (Hela)
and enhanced photodynamic activity
towards HeLa cells, without significant
necrocytosis towards L929 cells; as
a result this material may be useful in
the future for practical photodynamic
therapy.
Keywords: amphiphiles · biocom-
patibility · photodynamic effect ·
radical reactions · thermosensitivity
1. Introduction
plasms.[2] Porphyrins have been investigated extensively and
used in this field owing to their superior photoelectric prop-
erties and photodynamic therapy.[1] Although porphyrin de-
rivatives have interesting photodynamic properties, their
low solubility in aqueous media and their systemic biotoxici-
ty are pendent problems that preclude their direct applica-
tion in PDT treatments. Introducing a hydrophilic group
that has good biocompatibility to the porphyrin is an effec-
tive strategy to increase the biocompatibility and water solu-
bility of porphyrin derivatives.[3] It is well known that
poly(N-isopropylacrylamide) (PNIPAM) has attracted much
attention as a thermoresponsive polymer, the state of which
could change from a clear solution to a turbid suspension at
a lower critical solution temperature (LCST) of 328C.[4] Es-
pecially, PNIPAM shows well-established water solubility
and biological low toxicity. These outstanding properties
have encouraged the exploitation of PNIPAM in many ap-
plications, such as adsorption/desorption sheets for cell cul-
tures, drug delivery, tunable optical devices, and chromato-
graphic separation.[5] Thus, one very promising strategy is to
construct porphyrin-functionalized PNIPAM toward inte-
grating several different functionalities.
Photodynamic therapy (PDT), a medical procedure based
on the combined action of a photosensitizer (dye) and light,
has been known as a promising method for the treatment of
solid tumors, and it is usually composed of a photosensitizer
to generate cytotoxic singlet oxygen (1O2) during light irra-
diation in the presence of triplet oxygen (3O2).[1] Over the
past 20 years, it has been increasingly employed in the treat-
ment of cancer and other skin diseases, such as lung cancer,
esophageal cancer, gastric cancer, bladder carcinoma, chol-
angiocarcinoma, malignant mesothelioma, and brain neo-
[a] Y. Yang, Y. Li, N. Qiu, G. Cui, Prof. Q. Duan
School of Materials Science and Engineering
Changchun University of Science and Technology
Changchun 130022 (China)
Fax : (+86)431-85583015
E-mail: duanqian88@hotmail.com
[b] Y. Yang
School of Chemistry and Environmental Engineering
Changchun University of Science and Technology
Changchun 130022 (China)
Recently, controlled/“living” radical polymerization
(CRP) has been widely developed, and this has provided an
efficient way of synthesizing polymers with a functionalized
structure and narrow molecular-weight distribution. Atom-
transfer radical polymerization (ATRP), one of the most in-
vestigated CRP methods, can provide polymers with de-
[c] Prof. T. Satoh
Division of Biotechnology and Macromolecular Chemistry
Graduate School of Engineering, Hokkaido University
Sapporo 060-8628 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201301513.
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Qian Duan et al.
signed structures by using suitable initiators.[6] For example,
Baek et al. designed a thermoresponsive amphiphilic star
block copolymer photosensitizer that was used as a smart re-
mover of benzene, toluene, ethyl benzene, and xylenes.[7]
Ren et al. synthesized star-shaped amphiphilic and thermor-
esponsive copolymers with a porphyrin core and performed
further investigations in drug release.[8] To the best of our
knowledge, there are few reports on linear-porphyrin-func-
tionalized PNIPAM polymers that possess a reactive site for
further reactions.
In the present study, we designed and prepared a novel
aminoporphyrin-end-functionalized poly(N-isopropylacryla-
mide), H2N-TPP-PNIPAM (TPP=5,10,15,20-tetraphenyl-
21H,23H-porphyrin), that behaves as a multifunctional plat-
form that displays a photodynamic effect, thermosensitivity,
and fluorescence properties. The multifunctionalized H2N-
TPP-PNIPAM polymer was synthesized by using an asym-
metrical aminoporphyrin (i.e., H2N-TPP-Cl) as the initiator
for the ATRP of N-isopropylacrylamide (NIPAM). The
target polymer exhibited a photodynamic effect, thermosen-
sitivity, and fluorescence properties. At the same time, the
biocompatibility and water solubility of the photosensitizer
were remarkably increased after the porphyrin was grafted
to PNIPAM. Besides, H2N-TPP-PNIPAM demonstrated
a high potentiality of binding acidic cancer cells, in particu-
lar because of the alkalinity of the aminoporphyrin. The si-
mulated process of killing cancer cells is shown in Figure 1.
We anticipated the use of the product in PDT; thus, the cell
cytotoxicity and phototoxicity of the polymer were also in-
vestigated.
Figure 1. Simulated process of killing cancer cells by polymers for photo-
dynamic therapy.
trans-TPP-(NH2)2 as the precursor is because of its higher
yield in the reduction reaction of the previous step. First,
the H2N-TPP-Cl initiator was synthesized by the reaction of
2-chloropropionyl chloride with cis-TPP-(NH2)2 in the pres-
ence of triethylamine. Asymmetrically substituted H2N-
TPP-Cl was more difficult to obtain because of a low yield.
The dosage of 2-chloropropionyl chloride was inadequate,
because the two amino groups on cis-TPP-(NH2)2 have
equal opportunity to react, and thus a TPP-Cl2 byproduct
1
was inevitably generated (the H NMR spectrum of TPP-Cl2
is shown in Figure S3, Supporting Information). To improve
the yield of H2N-TPP-Cl, the dropping speed of the reagent
was as low as possible, and the mixture was sufficiently
stirred during the process. It was necessary to monitor the
progress of the reaction by TLC continually. After the sepa-
ration of the byproducts by column chromatography, the
monoamino-substituted H2N-TPP-Cl initiator was obtained,
and the yield reached up to 17.8%. The FTIR spectra of cis-
TPP-(NH2)2 and H2N-TPP-Cl are shown in Figure 2A,B. In
Figure 2A, the absorption bands at 1627 and 1482 cmꢀ1
were attributed to the skeletal vibration of the benzene
rings in the porphyrin, and the absorption bands at 975 and
789 cmꢀ1 were assigned to the pyrrole ring. After the amida-
tion reaction of cis-TPP-(NH2)2 with 2-chloropropionyl chlo-
ride, there was a strong absorption band at 1668 cmꢀ1, which
was assigned to the carbonyl stretching vibration, and the
strong characteristic absorbance of the methyl group at
2. Results and Discussion
2.1. Synthesis and Characterization
The synthesis strategy to H2N-TPP-PNIPAM is described in
Scheme 1. The reason we chose cis-TPP-(NH2)2 rather than
Abstract in Chinese:
Figure 2. FTIR spectra of A) cis-TPP-(NH2)2, B) H2N-TPP-Cl, and
C) H2N-TPP-PNIPAM (Mn,GPC =5820) in KBr plates.
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Scheme 1. Synthetic route to the well-defined H2N-TPP-Cl initiator and H2N-TPP-PNIPAM.
1
2950 cmꢀ1 clearly appeared (Figure 2B). The H NMR spec-
trum of H2N-TPP-Cl and the corresponding signal assign-
ments are shown in Figure 3. The signals appearing at ꢀ2.85
and 10.78 ppm were assigned to the NH of the pyrrole ring
and the acylamino group of the porphyrin, respectively. All
signals characteristic of both the porphyrin skeleton and the
peripheral substations are clearly observed.
Next, the target H2N-TPP-PNIPAM polymer was pre-
pared through ATRP of H2N-TPP-Cl and NIPAM
(Scheme 1). To the best of our knowledge, there are no re-
ports on the ATRP of NIPAM by using an initiator with a re-
active aniline on the periphery of the porphyrin. Data for
the polymerization reactions are summarized in Table 1.
The relative molecular weight (Mn) and polydispersity index
(PDI) were obtained through gel-permeation chromatogra-
phy (GPC). Elution times of the samples are shown in Fig-
ure S4. From Table 1, it can be concluded that the conver-
sion and the molecular weight increased as the polymeri-
zation time was increased. The PDI remained narrow with
values between 1.09 and 1.27, which revealed that the poly-
merization of NIPAM was a controlled process.[9] The value
of Mn,MS (the MALDI-TOF mass spectrum of the P1 sample
is shown in Figure S5) was much lower than the value of
Mn,GPC, whereas it was close to the value of Mn,theor. The re-
sults further illustrated that the molecular weight deter-
mined by GPC was just a relative value. Normally, the mo-
lecular weight of PNIPAM determined by GPC by using
polystyrene (PS) as a calibrating agent deviates from the
actual value. Overall, we found that the value of Mn,MS was
somewhat higher than that of Mn,theor, which could be attrib-
Figure 3. 1H NMR spectrum of H2N-TPP-Cl in DMSO.
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Table 1. Data for the polymerization reactions[a] and the LCST of H2N-TPP-PNIPAM.
the part of porphyrin (d=7.20–
8.15 ppm) with those of the me-
thine protons of NIPAM (d=
[c]
[d]
[e]
Sample
t [h]
Conv.[b] [%]
Mn,GPC
Mw/Mn
AHCTUNGTERG(NNUN GPC)
Mn,theor
Mn,MS
LCST[f] [8C]
P1
P2
P3
P4
P5
2
6
12
18
24
17
28
36
45
51
1830
5820
9230
13100
16500
1.23
1.09
1.19
1.24
1.27
2660
3900
4800
5800
6500
3280
4100
5300
6120
6750
36.5
34.9
33.2
32.8
31.8
4.02 ppm).
The
molecular
weight of H2N-TPP-PNIPAM
(Mn,GPC =5820) estimated by
1H NMR spectroscopy was
4580, which was in good agree-
ment with Mn,MS. Thus, it was
suggested that well-defined
H2N-TPP-PNIPAM with an
aminoporphyrin end group was
successfully prepared by ATRP.
[a] Reaction temperature=608C, solvent=DMF, [H2N-TPP-Cl] /
[b] Conv.=conversion, as determined by gravimetric measurement. [c] Determined by GPC by using THF as
the eluent and relative to polystyrene standards. [d] Mn,theor =MNIPAMA[NIPAM]0conv./100[H2N-TPP-Cl]0 +
. [e] Determined by MALDA-TOF MS. [f] Determined by UV/Vis spectroscopy through turbidime-
0 ACHTUNGTRENNU[NG Me6TREN]0/ACHUTNRTEG[NNGUN CuCl]0/ACHTUNGTERN[NUGN NIPAM]0 =1:5:5:100.
CTHUNGTRENNUNG
M
H2NꢀTPPꢀCl
try.
uted to the existence of a trace amount of the deactivated
initiator.
The structure of H2N-TPP-PNIPAM was also character-
ized by FTIR spectroscopy (Figure 2c). The characteristic
absorption bands of PNIPAM are clearly observed, as evi-
denced by the presence of a carbonyl stretching vibration
2.2. Photophysical Properties of Porphyrin Derivatives and
Polymers
It is known that the photophysical properties of porphyrin
derivatives have a direct impact on their applications in
PDT.[11] It is clearly seen from Figure 5A that all of the por-
phyrin derivatives exhibit the characteristic absorption
bands (one B band located at ꢁ410 nm and 2–4 Q bands lo-
(the secondary amide I band, nC O) at 1657 cmꢀ1 and the N
H bending vibration (the secondary amide II band, nNꢀH) at
ꢀ
=
1534 cmꢀ1. The 1H NMR spectrum of H2N-TPP-PNIPAM
and the corresponding signal assignments are shown in
Figure 4. All characteristic signals of the NIPAM units are
Figure 4. 1H NMR spectrum of H2N-TPP-PNIPAM (Mn,GPC =5820) in
CDCl3.
clearly observed. Moreover, the signals appearing at 9.8 and
5.5 ppm were assigned to the NH of the acylamino group
and the aniline adjacent to benzene ring of the porphyrin,
which were discernible after enlarging the photograph. With
the spectrum of H2N-TPP-Cl as a reference, the signal at
ꢀ2.85 ppm, which could be assigned to the pyrrole NH
group of the porphyrin, disappeared. This was attributed to
the partial metallization of the porphyrin core by CuII
during the ATRP process.[10] To further confirm the above
conclusion, we measured the concentration of copper in the
polymer by elemental analysis by using the P2 sample as an
example, and the value was 1992 mggꢀ1. The molecular
weight was estimated by comparing the resonating signals in
Figure 5. A) UV/Vis spectra of cis-TPP-(NH2)2, H2N-TPP-Cl, and H2N-
TPP-PNIPAM (Mn,GPC =5820) in CH2Cl2 solution (0.075 mgmLꢀ1).
B) Emission spectra (lEx =420 nm) of cis-TPP-(NH2)2, H2N-TPP-Cl, and
H2N-TPP-PNIPAM (Mn,GPC =5820) in CH2Cl2 solution (0.03 mgmLꢀ1).
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cated at ꢁ500–700 nm) in the near-UV and visible region. It
is interesting that the Q bands did not occur simultaneously.
Compared with the initiator, the Q band of H2N-TPP-
PNIPAM is reduced to two bands in the visible region,
which could be attributed to the metallization of the por-
phyrin core by CuII during the ATRP process. As illustrated
in the UV/Vis spectrum of H2N-TPP-PNIPAM (Figure 5A),
the lowest-energy Q band at approximately 600 nm, with an
absorptivity in the tens of thousands, is the transition of in-
terest for PDT, as it is beneficial to the transmission of
energy in PDT. In addition, porphyrins have been associated
with high affinity for tumor sites and with the efficient for-
mation of reactive oxygen species.[12] The fluorescence emis-
sion spectra of the porphyrin derivatives and the polymers
in CH2Cl2 solution are shown in Figure 5B, and they reveal
the luminescent properties of the polymer. It is clearly seen
from Figure 5B that all of the porphyrin derivatives exhibit
two emission bands at 450 and 650 nm in the near-UV and
visible region. The 650 nm emission band is followed by
a second smaller one at 718 nm. Relative to the emission
band of the H2N-TPP-Cl initiator, the emission band of
H2N-TPP-PNIPAM at 650 nm is somewhat blueshifted. This
was attributed to metallization of the porphyrin core by CuII
during the ATRP process. Fluorescence quenching of the
final polymer was also observed. The fluorescence proper-
ties of the porphyrin polymer are suited for detection imag-
ing in PDT.
of PNIPAM revealed that the polymers experienced dehy-
dration and collapsed from a hydrated, extended coil into
a hydrophobic globule. Notably, increasing the temperature
through the CP ultimately resulted in intermolecular aggre-
gation.[4] Table 1 shows that the LCST of H2N-TPP-
PNIPAM decreased as the molecular weight increased. The-
oretical research demonstrated that hydrophobic end groups
decrease the thermal phase-transition temperature of
PNIPAM.[9] The results deviate from the above theory, but
they are consistent with our previous reports,[13] and this can
be explained from three different aspects. First, H2N-TPP-
PNIPAM could form a hydrophobic core consisting of the
porphyrin with a diffuse corona of PNIPAM chains to facili-
tate the formation of a micelle, and this would effectively
isolate the hydrophobic groups from water and hence dra-
matically suppress the hydrophobic effects of the material.[14]
Second, steric hindrance also plays an important role. The
tendency of H2N-TPP-PNIPAM to form packed globules
arising from an increase in the temperature is hindered by
the huge planar structure and the substitutions on the pe-
ripheral benzene rings of the porphyrin. Third, the porphy-
rin core of H2N-TPP-PNIPAM can be easily protonated,
which would result in the formation of hydrogen bonds in
aqueous solution. These effects jointly result in an increase
in the LCST on the whole. However, the LCST values are
relatively low and approach 328C, which is commonly re-
ported for PNIPAM, as the molecular weight increases. This
was ascribed to the end-group effect on the performance of
the polymer, which is less remarkable for higher molecular-
weight samples. If the molecular weight is increased, the
proportion of porphyrin end groups in the polymer chain
decreases and the end effect of the porphyrin group on
H2N-TPP-PNIPAM becomes less significant.
2.3. Thermoresponsive Properties of H2N-TPP-PNIPAM
In the current study, the LCST was arbitrarily defined as the
temperature corresponding to a 10% decrease in transmit-
tance. The temperature dependence of the cloud point (CP)
for H2N-TPP-PNIPAM in aqueous solution is shown in
Figure 6. Clearly, there is a sharp transition in the CP curve,
which is indicative of the thermoresponsive properties of
H2N-TPP-PNIPAM. Investigations into the phase transition
2.4. In Vitro Cytotoxicity Studies
The cytotoxicity of the free PNIPAM and the polymers was
tested against normal cells (i.e., L929 cell lines) and cancer
cells (i.e., Hela cell lines) by the methyl thiazolyl tetrazoli-
um (MTT) assay. Figure 7 shows that the cytotoxicity of the
H2N-TPP-PNIPAM polymers increased slightly as the con-
centration of the cell lines was increased, whereas no necro-
cytosis was observed for various concentrations of free
PNIPAM. Figure 7A indicates that the cytotoxicity of the
P2 sample (Mn,GPC =5820) to Hela cells was higher than that
of the P3 sample (Mn,GPC =9230). The same result was ob-
tained with the L929 cells, as shown in Figure 7B. Changes
in the cytotoxicity caused by changes in the molecular
app:ds:weight were attributed to the peripheral PNIPAM
polymer chains. The cytotoxicity of H2N-TPP-PNIPAM
mainly arises from the porphyrin core, as free PNIPAM has
hardly any cytotoxicity. The proportion of porphyrin in the
polymer chain decreases as the molecular weight increases,
which leads to the higher cytotoxicity of the P2 sample rela-
tive to that of the P3 sample. On the whole, the cell cytotox-
icity results indicate that the polymer exhibits low toxicity
Figure 6. Temperature dependence of optical transmittance at a wave-
length of 500 nm obtained for aqueous solutions of H2N-TPP-PNIPAM
(1.5 mgmLꢀ1). Mn,GPC =1827, LCST=36.58C (&); Mn,GPC =5820, LCST=
34.98C (*); Mn,GPC =9230, LCST=33.28C (~); Mn,GPC =13050, LCST=
32.88C (!); Mn,GPC =16498, LCST=31.88C (3).
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than that of P3 (Figure 8C,F). This is because the photody-
namic effect of H2N-TPP-PNIPAM mainly arises from the
porphyrin core. Therefore, the polymers with lower molecu-
lar app:ds:weights always exhibit efficient phototoxicity.
However, this could be attributed to the peripheral
PNIPAM polymer chain. 1O2 was partly quenched during
the process of bulky PNIPAM chain crossing, which would
lead to decreased phototoxicity for the porphyrin. The
PNIPAM and porphyrin exhibited no apparent cytotoxicity
without irradiation. The results show that the cancer cells
were killed by the reactive oxygen species produced from
the porphyrin core under light irradiation.
2.6. Confocal Laser Scanning Microscopy (CLSM) of the
Polymer
To assess the uptake of the porphyrin polymers in the two
cell lines, CLSM was used to investigate the endocytosis of
normal cells (i.e., mouse fibroblasts, L929) and cancer cells
(i.e., Hela). The labeled polymer (green) was incubated for
4 h with the two cell lines, and then the cell nuclei were
stained with 4’,6-diamidino-2-phenylindole (DAPI, blue)
and the membrane was stained with Alexa Fluor 555 phal-
loidin (red). The fluorescent image is shown in Figure 9.
From the fluorescent imaging, we can find that only a few
green fluorescent signals in the cytoplasm are observed for
the L929 cell after 4 h incubation, but signals in the cyto-
plasm for the Hela cells are clearly observed (Figure 9A),
especially in comparison to the L929 group (Figure 9C).
The results show that the polymer can enter into the cyto-
plasm through endocytosis. Moreover, only a small amount
of polymer accumulated in the normal cytoplasm, and most
Figure 7. Cell viability after incubation in free PNIPAM and polymer
aqueous solutions: A) HeLa cells (PNIPAM, Mn,GPC =8800 (&); H2N-
TPP-PNIPAM,
M
n,GPC =5820 (*); H2N-TPP-PNIPAM,
Mn,GPC =9230
(~) and B) L929 cells (PNIPAM,
M
n,GPC =8800 (&); H2N-TPP-
PNIPAM, Mn,GPC =5820 (*); H2N-TPP-PNIPAM, Mn,GPC =9230 (~).
against both normal cells (i.e., L929) and cancer cells (i.e.,
Hela).
2.5. Phototoxicity of the
Polymers
Porphyrin medications used in
PDT are required to possess
high lethality to tumor cells.
Moreover, possible injury to
normal tissues must also be
avoided. Photodynamic therapy
was studied by investigating the
phototoxicity of the polymers
(P2, Mn,GPC =5820; P3, Mn,GPC
=
9230) against HeLa cells and
L929 cells by the MTT assay
under 400–700 nm irradiation.
No significant necrocytosis was
observed against the normal
cells (i.e., L929), as shown in
Figure 8B,E. In contrast, vast
necrocytosis was observed in
the Hela cells, as shown in Fig-
ure 8A,D. The phototoxicity of
Figure 8. A) Light microscopic images of Hela cells after incubation with P3 (0.2 mgmLꢀ1); B) light microscop-
ic images of L929 cells after incubation with P3 (0.2 mgmLꢀ1); C) cell viability after incubation with P3;
D) light microscopic images of Hela cells after incubation with P2 (0.2 mgmLꢀ1); E) light microscopic images
of L929 cells after incubation with P2 (0.2 mgmLꢀ1); F) cell viability after incubation with P2. Error bars rep-
resent standard deviation of three replicates for the test. Phototoxicity represented as the percent of cell death
P2 to cancer cells was higher after 24 h treatment with the polymers and illumination at 400–700 nm (1.66 mWcmꢀ2, 15 min).
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Experimental Section
Materials and Instruments
NIPAM (Aldrich, 99%) was recrystallized twice from benzene/hexane
(3:2 v/v) prior to use; tris[2-(dimethylamino)ethyl]amine (Me6TREN)
was synthesized from trisACTHNUGRTNEUNG(2-amino)ethylamine (TREN, Aldrich, 99%) ac-
cording to the literature.[15] CuCl (Aldrich, 99%) was successively
washed with acetic acid and ether and then dried and stored under a ni-
trogen atmosphere. Pyrrole (Acros, 99%) was purified by distillation.
Tetrahydrofuran (THF, Duksan, 99.9%) was submicron filtered before
use. 2-Chloropropionyl chloride (99%) and other chemicals were pur-
chased from Sigma–Aldrich, France.
1H NMR spectra were measured with a Bruker Avance 400 MHz spec-
trometer at ambient temperature by using CDCl3 or [D6]DMSO as the
solvent and tetramethylsilane for 1H calibration. Elemental analysis was
obtained with a Carlo Erba-MOD1106 instrument. Molecular weights
(Mn,GPC) and polydispersity (Mw/Mn) were measured with a gel-permea-
tion chromatograph with a Waters e2695 separations module and two
Waters styragel columns (HR4 and HT3, 300ꢁ7.8 mm, 5 mm particles;
exclusion limits: ꢁ5000–30000 and ꢁ500–30000 gmolꢀ1, respectively)
and a Waters 2414 refractive index detector maintained at 358C. The
polymer molar masses were determined by using linear polystyrene as
a calibration standard. THF was used as a mobile phase at a flow rate of
1.0 mLminꢀ1. The UV/Vis absorption spectra of the polymers were deter-
mined with a Shimadzu-1240 spectrophotometer. Infrared spectra were
recorded by measuring samples in KBr disks with a Shimadzu IR-8400S
spectrometer. A Shimadzu RF-5301PC fluorescence spectrophotometer
was used to measure the fluorescence spectra (excitation/emission slit: 5/
5 nm). Matrix-assisted laser desorption ionization time-of-flight mass
spectrometry (MALDI-TOF MS) measurements were performed by
using an AutoflexIII Smartbeam mass spectrometer, and a 20 kV acceler-
ation voltage was used. The positive ions were detected in the reflector
mode (20 kV). A Smartbeam laser (355 nm, 200 ns pulse width) operat-
ing at 3 Hz was used to produce laser desorption, and 500 shots were
summed. The spectra were externally calibrated by using 2,5-dihydroxy-
benzoic acid as a linear calibration. Samples for MALDI-TOF MS were
prepared by mixing the polymer and a cationizing agent (sodium trifluor-
oacetate, 0.1%) in acetonitrile/H2O (1:1 v/v) solution.
Figure 9. Fluorescent imaging of labeled polymer (green, P3,
0.25 mgmLꢀ1) against HeLa cells and L929 cells. The cell nuclei were
stained with DAPI (blue), and the membrane was stained with Alexa
Fluor 555 phalloidin (red).
of the polymer accumulated in the cancer cytoplasm. This is
beneficial to the PDT process.
3. Conclusions
Syntheses
A porphyrin-end-functionalized PNIPAM (i.e., H2N-TPP-
PNIPAM) was designed by using an asymmetrical amino-
porphyrin (i.e., H2N-TPP-Cl) as the initiator for the ATRP
of NIPAM. H2N-TPP-PNIPAM with a reactive aniline
group on the periphery of the porphyrin can be used in fur-
ther reactions as a potential reaction site. The lower LCST
of H2N-TPP-PNIPAM showed a decreasing trend accompa-
nying an increase in the molecular weight as a result of the
incorporation of the porphyrin group at the end of the
chain. After combination of the aminoporphyrin with
PNIPAM, the polymer exhibited well-defined fluorescence,
high biocompatibility, and preferential photodynamic effects
to tumor cells. Furthermore, no significant necrocytosis was
observed after the normal cells were exposed to the polymer
through light-toxicity processing, and thus, possible injury to
normal tissue was effectively avoided. The multi-functional-
ized porphyrin polymer showed excellent potential for fluo-
rescence detection and photodynamic therapy.
5,10,15,20-Tetraphenyl-21H,23H-porphyrin (TPP, 1)
The TPP precursor was synthesized by Adlerꢂs method (as shown in
Scheme 1).[16]
A mixture of benzaldehyde (5 mL, 0.05 mol), pyrrole
(3.5 mL, 0.05 mol), and DMSO (6 mL) was added to propionic acid
(120 mL), and the resulting mixture was heated at reflux for 30 min and
then cooled to room temperature. The reaction mixture was filtered
under vacuum, washed thoroughly with methanol until the filtrate
became clear, and then washed with warm water until the filtrate was
neutral (pHꢁ6.8) to obtain a purple crystals. The crude product was
dried under vacuum (808C) for 12 h. The crude compound was purified
by silica gel column chromatography (dichloromethane) to afford desired
dye 1 as a purple solid. Yield: 2.39 g, 31.12%. 1H NMR (500 MHz,
DMSO): d=ꢀ2.76 (2H, d; pyrrole-NH), 7.78–8.22 (15H, d; phenyl),
8.88 ppm (8H, s; pyrrole-H). FTIR (KBr): n˜ =3020 (nArꢀH), 1606, 1591,
1510 (nC CꢀC), 969, 725 cmꢀ1 (dNꢀH). UV/Vis (CH2Cl2): lmax =415, 514,
=
547, 588, 643 nm. Elemental analysis calcd (%) for C44H30N4: C 85.97, H
4.92, N 9.11; found: C 85.93, H 4.58, N 9.49.
5,10-Di(4-aminophenyl)-15,20-diphenylporphyrin [cis-TPP-(NH2)2, 3]
Synthesized from the corresponding TPP by a literature method (as
shown in Scheme 1).[17] Yield: 629 mg, 31.45%. 1H NMR (500 MHz,
CDCl3): d=ꢀ2.73 (2H, s; pyrrole-NH), 3.97 (4H, s; aminophenyl-NH),
7.03–8.22 (18H, m; phenyl), 8.81 (4H, s; pyrrole-H), 8.92 ppm (4H, s;
pyrrole-H). FTIR (KBr): n˜ =3470 (nNꢀH), 3030 (nArꢀH), 1627, 1482, (nC
=
CꢀC), 975, 789 cmꢀ1 (dNꢀH). UV/Vis (CH2Cl2): lmax =424, 518, 557, 593,
Chem. Asian J. 2014, 9, 1379 – 1387
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Qian Duan et al.
650 nm. Elemental analysis calcd (%) for C44H32N6: C 81.96, H 5.00,
N13.03; found: C 82.13, H 5.14, N12.73.
plate was then returned to the incubator. After 4 h, the MTT solution
was carefully removed from each well and DMSO (200 mL) was added to
dissolve the MTT formazan crystals that had formed. The above opera-
tion was completed in the dark. The plate was incubated for an addition-
al 10 min before the absorbance was read at 492 nm and recorded by an
enzyme-linked immuno sorbent assay (ELISA) microplate reader (Bio-
Rad). The cell viability was calculated according to Equation (1):
5-(4-Aminophenyl)-10-[4-(2-chloropropionylamino)phenyl]-15,20-
diphenylporphyrin (H2N-TPP-Cl, 4)
cis-TPP-(NH2)2 (200 mg, 3.12ꢁ10ꢀ4 mol), triethylamine (43.2 mL, 3.16ꢁ
10ꢀ4 mol), and dichloromethane (100 mL) were mixed under a nitrogen
atmosphere in an ice bath. 2-Chloropropionyl chloride (31.6 mL, 3.12ꢁ
10ꢀ4 mol) in dichloromethane (10 mL) was added dropwise over 30 min.
TLC analysis of the reaction mixture showed that the spots correspond-
ing to the starting materials disappeared and that two new spots corre-
sponding to the required dyes arose. After removal of the solvent, the
crude compound was purified by silica gel column chromatography (di-
chloromethane/ethyl acetate=100:1) to afford desired dye 4 as a purple
solid. Yield: 40.8 mg, 17.8%. 1H NMR (500 MHz, DMSO): d=ꢀ2.85
Asample
Acontrol
ð1Þ
Cell viability ½%ꢂ ¼
ꢃ 100 %
in which Asample is the absorbance of the cells incubated in DMEM and
the polymer mixture and Acontrol is the absorbance of the cells incubated
in DMEM.
Phototoxicity Measurements
(2H, s, pyrrole-NH), 1.76 [3H, d; CH
ACHTUNGTREN(NUGN CH3)Cl], 4.87 [4H, q; CH-
The phototoxicity was performed with HeLa and L929 cell lines by MTT
assay. For the phototoxicity assay, the polymers were added and then the
plate with cells was placed on ice and exposed to light from an 18 W
(1.66 mWcmꢀ2) lamp that filtered through a 400–700 nm long-pass filter
for 15 min. The cells were returned to the incubator for 24 h and assayed
for viability, and the other conditions were the same as those for the cy-
totoxicity measurements.
ACHTUNGTRENNUNG
8.80–8.96 (8H, m; pyrrole-H), 10.78 ppm (1H, s; acid amide-H). FTIR
(KBr): n˜ =3480 (nNꢀH), 3030 (nArꢀH), 2950 (nCꢀH), 1668 (nC O), 1532
=
(dCꢀNꢀH), 1478, (nC CꢀC), 975, 789 cmꢀ1 (dNꢀH). UV/Vis (CH2Cl2): lmax
=
=
422, 517, 556, 593, 649 nm. Elemental analysis calcd (%) for
C47H35ClN6O: C 76.77, H 4.8, N 11.43, O 2.18, Cl 4.82; found: C 76.52, H
4.91, N 11.22, O 2.37, Cl 4.97.
Confocal Laser Scanning Microscopy (CLSM)
H2N-TPP-PNIPAM
CLSM was used to investigate the uptake of the porphyrin polymers
against normal cells (mouse fibroblasts, L929) and cancer cells (Henrietta
Lacks, Hela). In brief, a solution of H2N-TPP-PNIPAM (2 mgmLꢀ1 in
H2O, 2 mL) was dropped into a solution of fluorescein isothiocyanate
(FITC, 2 mgmLꢀ1 in DMSO, 5 mL), and then the mixture was stirred for
2 h. The reaction mixture was processed by dialysis by using a cellophane
tube (MWCO=1000) in H2O. The residue was freeze dried to give the
FITC-labeled polymer. For microscopic observation, HeLa cells and
L929 cells were seeded into six-well plates at 1.5ꢁ105 per well in the
growth medium (90% DMEM, 10% FBS, 2 mL) and incubated for 24 h.
The medium was replaced with fresh DMEM (2 mL) before the FITC-la-
beled polymer was added. The cells were incubated at 378C for 4 h,
washed three times with PBS, and fixed with 4% paraformaldehyde for
10 min at room temperature. Cells were then rewashed three times with
PBS. The cell nucleus was stained with DAPI (1 mgmLꢀ1, 1 mL) for
10 min, the PBS washing step was repeated, and the membrane was
stained with Alexa Fluor 555 phalloidin for 20 min at 378C. Finally, the
cover slips were fully cleaned and enclosed with glycerol. The distribu-
tion of the polymer was visualized by using a confocal laser scanning mi-
croscope (ZEISS LSM 780).
The ATRP of NIPAM was performed at 608C in DMF. The molar ratio
of initiator, Me6TREN/CuCl, and monomer was 1:5:100. A mixture of
CuCl (4.95 mg, 0.05 mmol) and Me6TREN (12 mL, 0.05 mmol) in DMF
(1 mL) was placed in one side of an H-shaped glass ampoule and stirred
under a nitrogen atmosphere. The H2N-TPP-Cl (7.35 mg, 0.01 mmol) ini-
tiator and the NIPAM (113 mg, 1 mmol) monomer in DMF (1.0 mL)
were placed in the other side of the ampoule. Nitrogen was bubbled
through both mixtures for 5 min to remove oxygen. Three freeze–pump–
thaw cycles were then performed to degas the solutions. Both mixtures
were mixed and left to stand at 608C for several hours. The polymeri-
zation reaction was terminated by exposure to air. The reaction mixture
was passed through a neutral Al2O3 column by using THF as the eluent
to remove the copper complex. The resulting polymer was purified by di-
alysis by using a cellophane tube [molecular-weight cutoff (MWCO)=
2000] in DMF. After removing the solvents, the residue was dried under
vacuum (408C) for 24 h to give the target H2N-TPP-PNIPAM polymer.
1H NMR (500 MHz, DMSO): d=1.17 (CH3), 1.71 (CH2), 2.48 (CH), 3.98
(CH) 5.56 (phenyl-NH2), 6.03–7.10 (br., Me2CHNH), 7.20–8.15 (porphy-
rin), 10.78 ppm (phenyl-NH-C=O). FTIR (KBr): n˜ =3465, 3338 (nNꢀH),
2960, 2837 (nCꢀH), 1657 (nC O), 1534 (dCꢀNꢀH), 1288, (nC CꢀC), 1079,
=
=
735 cmꢀ1 (dNꢀH). UV/Vis (CH2Cl2): lmax =416, 541, 612 nm.
Acknowledgements
Cloud Points Measurements
Cloud points (CPs) of the H2N-TPP-PNIPAM aqueous solutions were
measured by UV/Vis spectroscopy through turbidimetry. The sample dis-
solved in water (1.5 mgmLꢀ1) was poured into a thermally controlled cell
(1ꢁ4 cm). Transmittance of the aqueous solution at 500 nm was moni-
We are grateful to the National Natural Science Foundation of China
(50903009), Jilin Science & Technology Department, Science and Tech-
nology Development Project (20070556, 20100115, and 201201120), and
Science and Technology Bureau of Changchun City Project (2008280 and
2013060) Foundation for Strategical Research for financial support. The
authors are also grateful to Junwei Huang for help with FTIR analyses
and Xiuwen Guan for measuring in vitro cytotoxicity. The authors would
like to thank all reviewers of this article for their comments and sugges-
tions.
tored with a heating rate of 18Cminꢀ1
.
Cell Viability Measurements
Cell viability was used to investigate the relative cytotoxicity of the poly-
mers through the MTT assay against normal cells (mouse fibroblasts,
L929) and cancer cells (Henrietta Lacks, Hela). In brief, cells were cul-
tured in 96-well plates with Dulbeccoꢂs modified Eagleꢂs medium
(DMEM), and the polymers were mixed with high-content glucose, sup-
plemented with 10% (v) heat-inactivated fetal bovine serum (FBS), peni-
cillin (100 unitsmLꢀ1), and streptomycin (100 mgmLꢀ1) in a 5% CO2 in-
cubator at 378C under 95% humidity. The concentrations of the poly-
mers solutions ranged from 0.05 to 0.2 mgmLꢀ1. In total, three solution
concentrations were obtained. After 24 h incubation, an aliquot (20 mL)
of the MTT solution (5 mgmLꢀ1 in PBS) was added to each well. The
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