Water-soluble dendritic polyaspartic porphyrins: Potential photosensitizers for photodynamic therapy

Article abstract of DOI:10.1039/c1nj20733d

We prepared these photosensitizers with two-block structures: porphyrin cores for singlet oxygen generation and branched dendritic polyaspartic as hydrophilic shells for inducing water solubility, thus reducing cytotoxicity and giving high singlet oxygen

Full text of DOI:10.1039/c1nj20733d

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
NJC
Cite this: New J. Chem., 2012, 36, 28–31
www.rsc.org/njc
LETTER
Water-soluble dendritic polyaspartic porphyrins: potential
photosensitizers for photodynamic therapyw
Baoxiang Gao,*ac Yueling Liu,a Huijuan Yin,b Yingxin Li,*b Qianqian Baia and
Licui Zhanga
Received (in Montpellier, France) 22nd August 2011, Accepted 14th October 2011
DOI: 10.1039/c1nj20733d
We prepared these photosensitizers with two-block structures:
porphyrin cores for singlet oxygen generation and branched dendritic
polyaspartic as hydrophilic shells for inducing water solubility, thus
reducing cytotoxicity and giving high singlet oxygen yields.
at an extremely high concentration because the dendritic envelope
of DP can prevent aggregation of the central porphyrin.12
Among the requirements for a successful photosensitizer, in
addition to singlet oxygen generation capacity, good water
solubility and biocompatibility are of paramount importance.
Generally, water solubility has been imported by either introdu-
cing ionizable hydrophilic groups (carboxylic acid, phosphonic
acid, sulfonic acid, and ammonium groups) within the core
structure of a fluorescent dye or by grafting the hydrophobic
fluorophore to hydrophilic (bio)polymers (carbohydrates,
oligonucleotides and polyethylene glycol). Very recently, Akkaya
and coworkers reported a series of water-soluble photosensitizers
with amino acid modification, and these photosensitizers display
significant light-induced cytotoxic effects on the human
erythroleukemia cell line.13 Considering that the more biocom-
patible peptide-based dendrimers have demonstrated promising
characteristics as components of vaccines, as well as antiviral and
antibacterial agents, we have designed and synthesized dendritic
polyaspartic encapsulation of perylene bisimides, which showed
good water solubility and biocompatibility.14
Photodynamic therapy (PDT) is a new clinical treatment for
superficial tumors and age-related muscular degeneration.
This technique involves the systemic administration of a
photosensitive drug, by which singlet oxygen is generated after
light irradiation, to engender oxidative damage to cells.1,2 The
PDT strategy utilizes a photosensitizer, working as a light-sensitive
drug, to treat the target tissue locally upon the irradiation of light
with appropriate wavelengths. One of the key components in
effective and efficient PDT is the photosensitizer, also called
PDT drugs. The high singlet oxygen quantum yield of the
photosensitizers is significantly important for PDT. Most of
the conventional photosensitizers have large p-conjugation
domains to extend their absorption cross sections and basically
have hydrophobic characteristics. Therefore, photosensitizers
form aggregates easily, which produce the self-quenching of the
excited state in aqueous medium, because of their p–p interaction
and hydrophobic characteristics.3,4 A few approaches have
been proposed to enhance singlet oxygen quantum yield of the
photosensitizers. Quantum dots, gold and silica nanoparticles
modified with porphyrin photosensitizer, were developed to
undergo energy transfer and to generate singlet oxygen.5–9
Polymer micelles encapsulating porphyrin were also prepared
to induce singlet oxygen production by the FRET mechanism.10
Recently, Kazunori Kataoka and coworkers reported a
dendrimer-based photosensitizer, dendrimer porphyrin (DP), in
which the focal porphyrin is surrounded by the third generation of
poly(benzyl ether) dendrons.11 Unlike conventional photosensiti-
zers, the DP ensures the efficacy of singlet oxygen production even
In this study, we present the synthesis and property of
water-soluble dendritic polyaspartic porphyrins (DPAPs).
The chemical structures of DPAPs are shown in Scheme 2.
We prepared these DPAPs with two-block structures:
porphyrin cores for singlet oxygen generation, and branched
dendritic polyaspartic as hydrophilic shells for inducing water
solubility and reducing cytotoxicity. These DPAPs are
expected to have biocompatibility, water solubility, and high
singlet oxygen yields.
Most porphyrin-cored dendrimers have been synthesized
through a divergent or a convergent approach. The divergent
synthetic approach may often suffer from the disadvantage
that the final products contain structural defects. This problem
may be avoided by convergent approach. Thus, we chose the
convergent approach to dendritic polyaspartic porphyrins.
The polyaspartic-dendrons were synthesized according to
the procedures reported in the literature.15 The peptide-
conjugated benzaldehyde 5, 6 and 7 was obtained by amida-
tion of the carboxybenzaldehyde using equivalent of peptide
and the coupling reagents DCC-BtOH in dichloromethane
(Scheme 1).
a College of Chemistry and Environmental Science, Hebei University,
Baoding, 071002, P. R. China. E-mail: bxgao@hbu.edu.cn;
Fax: +86 3125079317
b Institute of Biomedical Engineering, Chinese Academy of Medical
Science & Peking Union Medical College, Tianjin, 300192,
P. R. China. E-mail: yingxinli@bme.org.cn; Fax: +86 22 87891131
c Key Laboratory of Medicinal Chemistry and Molecular Diagnosis,
Ministry of Education, Hebei University, Baoding, 071002, P. R. China
w Electronic supplementary information (ESI) available: See DOI:
10.1039/c1nj20733d
c
28 New J. Chem., 2012, 36, 28–31
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012

View Article Online
porphyrins, the bands in aqueous solutions are broadened. It
appears that the attached dendritic cages seem to have almost
no influence on the encapsulated porphyrin absorption bands.
The photoluminescence spectra of DPAPs were recorded in
5 ꢁ 10ꢂ6 mol Lꢂ1 aqueous solution (Fig. 2). The porphyrin
P1-COOH and P2-COOH have similar fluorescence emission
spectra with two distinct peaks at 646 nm and 704 nm.
However, the porphyrin P1-COOH shows an additional small
peak at 606 nm. Most likely this emission originates exclusively
from the mono-protonated porphyrin core.16 Furthermore, we
determined the fluorescence quantum yields of the DPAPs in
the aqueous solution (5 ꢁ 10ꢂ6 M). P1-COOH exhibited a low
fluorescence quantum yield (8%), which could be attributed to
fluorescence quenching by strong aggregation in water.15 With
increasing dendron generation, there was a continuous increase
in fluorescence quantum yields from 8% for P1-COOH to 11%
for P2-COOH and up to 14% for P3-COOH. The bulky
dendritic polyaspartic shells may prevent the quenchers from
interacting with the porphyrin core.
Scheme 1 Synthesis of the dendritic polyaspartic substituted
benzaldehyde.
Singlet oxygen generation capacities of these DPAPs were
studied in water. Relative rates of singlet oxygen generation
were evaluated by monitoring the rates of decay of the 1,3-
diphenyl-isobenzofuran (the singlet oxygen quencher). Three
porphyrin compounds can induce photodegradation of the
1,3-diphenyl-isobenzofuran. Using tetraphenyporphine in
benzene as the reference (FD = 0.63),17 the singlet oxygen
yields of compound P1-COOH, P2-COOH and P3-COOH
are 0.48, 0.61 and 0.89, respectively. The higher singlet oxygen
yield of compound P3-COOH may be ascribed to the
branched dendritic polyaspartic groups that somewhat hinder
the excited state quench.
The condensation of peptide-conjugated benzaldehyde 5 or
6 with distilled pyrrole using BF3ꢀEt2O as catalyst and
dichloromethane as solvent in an aerobic dark environment,
following by DDQ oxidation and Et3N neutralization,
afforded porphyrins P1-OCH3 and P2-OCH3 with 11% yield
(Scheme 2). However, the same reaction conditions did not
produce the target compound P3-OCH3 because of the
extremely low solubility of the condensation intermediate. By
using high boiling point 1,1,2,2-tetrachloroethane, which is
usually a good solvent of p-conjugated molecules, the target
compound P3-OCH3 was obtained in yields of 13%. Hydrolysis
of porphyrins P-OCH3 gave the water-soluble porphyrin
compound P-COOH in a nearly quantitative yield. Porphyrin
compound P3-COOH could not be precipitated from the
aqueous solution even upon strong acidification, and therefore,
the solution had to be purified from the excess salt by extensive
dialysis. Subsequent lyophilization produced the green powder.
Porphyrin compound P3-COOH has in total 28 aspartic units
and 32 peripheral carboxylate groups.
The biocompatibility of DPAPs was evaluated for Hela cells
using MTT cell-viability assay. Fig. 3 summarizes the viability
of Hela cells after being cultured with PEPPs solutions at a
concentration of 5.0 ꢁ 10ꢂ5 for 24 and 72 h. Three porphyrin
compounds had very low cytotoxicity (over 90% viability)
within 72 h incubation time. It indicates that water-soluble
dendritic polyaspartic porphyrins have high biocompatibility.
We ascribed the exceptionally low cytotoxicity to dendritic
polyaspartic, which protects the dyes from interacting
non-specifically with the extracellular proteins and triggering
immunogenicity and antigenicity inside the cells.18
Optical absorption spectra of porphyrins contain a Soret
band (416 nm) and four Q-bands (517, 554, 581, 635 nm) in the
UV-vis region (Fig. 1). The spectra of the porphyrin dendrimers
resemble those of the cores, although, typical of water-soluble
In order to study photodynamic activities of DPAPs, Hela
cells were incubated at different dose and then submitted to
Scheme 2 Synthesis of DPAPs.
Fig. 1 UV-vis absorption spectra of DPAPs in aqueous solutions.
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012
New J. Chem., 2012, 36, 28–31 29

View Article Online
Fig. 4 In vitro viability of Hela cells with illumination (532 nm,
15 mW cmꢂ2, 30 J cmꢂ2).
Fig. 2 Photoluminescence spectra of DPAPs in aqueous solutions.
Experimental
The starting material, 4-carboxybenzaldehyde was purchased
from Acros, and used without any further purification.
Solvents were purchased from Sinopharm Chemical Reagent
Co. Ltd, and used without any further purification. L-Aspartic
acid and N-carbobenzyloxy-L-aspartic acids were purchased
from Yangzhou Baosheng Bio-Chemical Co. Ltd. N-(3-Di-
methylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDCI),
4-dimethylaminopyridine and 1-hydroxybenzotrizole were
purchased from Shanghai Medpep Co. Ltd. Compounds 2, 3
and 4 were synthesized according to the reported methods.15
The 1H NMR spectra were recorded at 20 1C on 400 MHz
NMR spectrometer (Bruker). Chemical shifts are reported in
ppm at room temperature using CDCl3, or DMSO as solvent
and tetramethylsilane as internal standard unless indicated
otherwise. Abbreviations used for splitting patterns are
s = singlet, br = broad, d = dublet, m = multiplet. Mass
spectra were carried out using MALDI-TOF/TOF matrix
assisted laser desorption ionization mass spectrometry with
autoflexIII smartbeam (Bruker Daltonics Inc).
Fig. 3 In vitro viability of Hela cells treated with P1-COOH and
P2-COOH and P3-COOH solution at 5.0 ꢁ 10ꢂ5mol Lꢂ1 for 24 and
72 h, respectively.
irradiation (532 nm; 15 mW cmꢂ2) for 20 min, whereas cells
not treated by DPAPs and only exposed to the laser were used
as controls for cytotoxicity. MTT assay was performed after
irradiation, to establish the cytotoxicity of DPAPs. Fig. 4
showed the dose-dependent cells viability for these compounds.
It can be seen that the photocytotoxicity depends greatly on
the dose. As the concentration increases from 0 mM to 50 mM,
the DPAPs showed enhanced photocytotoxicity (cell viability
P1-COOH: from 98% to 75%; P2-COOH: from 96% to 25%;
P3-COOH: from 94% to 1%). The photocytotoxicity of
P3-COOH was much higher than that of P1-COOH and
P2-COOH under the same experimental conditions. Further-
more, the photocytotoxicity of DPAPs was in good agreement
with the trend observed for the singlet oxygen yields of these
compounds.
Synthesis and characterization of DPAPs
General procedure for synthesis of precursor: dendritic
polyaspartic substituted benzaldehyde
0.289 g 4-carboxybenzaldehyde (2 mmol) was dissolved in
60 mL of dichloromethane, 0.575 g 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (3 mmol) was added , and the
mixture was stirred at 0 1C for 30 min. 3 mmol polyaspartic-
dendrons (3 mmol) and 0.456 g HOBt (3 mmol) were added and
the mixture stirred at room temperature for 24 h. Solvent was
removed by rotary evaporation. The crude product was purified
by column chromatography on silica gel with methylene chloride:
ethanol as an eluent to afford dendritic polyaspartic substituted
benzaldehyde as a white solid.
In conclusion, we reported the synthesis and properties of
water-soluble dendritic polyaspartic porphyrins. With increasing
dendron generation, the singlet oxygen yields of these DPAPs
were improved from 48% to 89%. The hydrophilic peptides
induced DPAPs with water solubility and biocompatibility.
The photodynamic activities of these compounds were further
evaluated. These results provide motivation to further explore
the anticancer potential of water-soluble dendritic polyaspartic
porphyrins.
Compound 5: 66% yield. 1H NMR (CDCl3, 400 MHz):
d 10.09 (s,1 H, CHO), 7.97 (s, 4 H), 7.36 (d, 1 H, CONH),
5.09–5.04 (m, 1 H, CH), 3.81 (s, 3 H, CH3), 3.72 (s, 3 H,
OCH3), 3.21–2.96 (m,
2 H, CH2). Anal. Calcd for
C14H15NO6: C, 57.34; H, 5.16; N, 4.78. found: C, 57.08;
H, 5.27; N, 4.97. m/z [MALDIꢂTOF]: 294.1 (M+H+).
Compound 6: 51% yield. 1H NMR (CDCl3, 400 MHz):
d 10.09 (s, 1 H, CHO), 8.27–6.89 (m, 3 H, CONH), 7.99–7.76
c
30 New J. Chem., 2012, 36, 28–31
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012

View Article Online
(m, 4 H, CH), 5.08–4.85 (m, 3 H, CH), 3.76–3.64 (m, 12 H,
OCH3), 3.02–2.66 (m, H, CH2). Anal. Calcd for
temperature for 4 h, and the solvent was evaporated under
vacuum at room temperature. Water was added to the remain-
ing solid, and the mixture was stirred at room temperature for
additional 2 h. The solution was neutralized by HCl, filtered,
dialyzed against distilled water for 4 days, and lyophilized.
Compound P3-COOH: 13% yield. 1H NMR (DMSO,
400 MHz): d 8.88 (s, 8 H, CH), 8.21 (br, 16 H, CH),
9.05–7.97 (m, 28 H, CONH), 4.93–4.45 (m, 28 H, CH),
2.78–2.55 (m, 56 H, CH2), ꢂ2.97 (s, 2 H, NH). Anal. Calcd
for C160H170N32O92: C, 47.88; H, 4.27; N, 11.17. found: C,
47.69; H, 4.58; N, 11.36. m/z [MALDIꢂTOF]: 4036.7
(M+Na+).
6
C24H29N3O12: C, 52.27; H, 5.30; N, 7.62. found: C, 52.09;
H, 5.41; N, 7.87. m/z [MALDIꢂTOF]: 552.6 (M+H+).
Compound 7: 51% yield. 1H NMR (DMSO, 400 MHz):
d 10.08 (s, 1 H, CHO), 8.72 (d, J = 7.6 Hz, 1 H, CONH),
8.32–8.99 (m, 2 H, CONH), 8.22–8.16 (m, 4 H, CONH),
8.03–7.97 (m, 4 H, CH), 4.74–4.54 (m, 7 H, CH), 3.60–3.58
(m, 24 H, OCH3), 2.77–2.40 (m, 14 H, CH2). Anal. Calcd for
C44H57N7O24: C, 48.49; H, 5.38; N, 9.18. found: C, 48.20; H,
5.50; N, 9.35. m/z [MALDIꢂTOF]: 1068.8 (M+H+).
General procedure for synthesis of P1-COOH and P2-COOH
To a stirred mixture of dendritic polyaspartic substituted
benzaldehyde compound 5 or compound 6 (0.5 mmol) and
pyrrole (0.5 mmol) in dichloromethane (200 mL) at room
temperature was added boron trifluoride etherate (0.2 mmol).
After the mixture was stirred for an additional 2 h, DDQ
(1.2 mmol) was added and the reaction mixture was allowed to
stir for 30 min at room temperature. The solvent was removed
under reduced pressure, and the residue was chromatographed
over silica gel using dichloromethane as eluent. The obtained
product was redissolved in dichloromethane and precipitated
with methanol. Excess of NaOH was added to a solution of
compound P1-OCH3 and P2-OCH3 (100 mg) in 10 mL of
THF. The mixture was stirred at room temperature for 4 h,
and the solvent was evaporated under vacuum at room
temperature. Water was added to the remaining solid, and
the mixture was stirred at room temperature for additional
2 h. The solution was neutralized by HCl, filtered, dialyzed
against distilled water for 4 days, and lyophilized.
Compound P1-COOH: 12% yield.1H NMR (DMSO,
400 MHz): d 12.8 (br, 8 H, COOH), 9.15 (s, 4 H, CONH),
8.88 (s, 8 H, CH), 8.35 (s, 16 H, CH), 4.94–4.92 (m, 4 H, CH),
2.96–2.87 (m, 8 H, CH2), ꢂ2.93 (s, 2 H, NH). Anal. Calcd for
C64H50N8O20: C, 61.44; H, 4.03; N, 8.96. found: C, 61.23; H,
4.21; N, 9.12. m/z [MALDIꢂTOF]: 1251.4 (M+H+).
Compound P2-COOH: 11% yield. 1H NMR (DMSO,
400 MHz): d 12.66 (br, 16 H, COOH), 8.88 (br, 8 H, CH),
8.32 (br, 16 H, CH), 8.88–7.95 (m, 12 H, CONH), 5.00–4.62
(m, 12 H, CH), 2.89–2.73 (m, 24 H, CH2), ꢂ2.91 (s, 2 H, NH).
Anal. Calcd for C93H90N16O38: C, 54.76; H, 4.45; N, 10.99.
found: C, 54.56; H, 4.53; N, 11.10. m/z [MALDIꢂTOF]:
2040.9 (M+H+).
This work was supported by the Key Project of Chinese
Ministry of Education (No. 210015), National Natural Science
Foundation of China (No. 20804012), Natural Science
Foundation of Hebei Province (No. B2009000169), the
Science Research Project of Department of Education of
Hebei Province (No. 2007413), and the Natural Science
Foundation of Hebei University (No. 2007105).
Notes and references
1 M. Hoebeke and X. Damoiseau, Photochem. Photobiol. Sci., 2002,
1, 283.
2 X. Damoiseau, F. Tfibel, M. Hoebeke and M. P. Fontaine-Aupart,
Photochem. Photobiol., 2002, 76, 480.
3 N. Tomioka, D. Takasu, T. Takahashi and T. Aida, Angew.
Chem., Int. Ed., 1998, 37, 1531.
4 N. Nishiyama, H. R. Stapert, G.-D. Zhang, D. Takasu,
D.-L. Jiang, T. Nagano, T. Aida and K. Kataoka, Bioconjugate
Chem., 2003, 14, 58.
5 G. F. Paciotti, L. Myer, D. Weinreich, D. Goia, N. Pavel,
R. E. McLaughlin and L. Tamarkin, Drug Delivery, 2004, 11, 169.
6 Y. Cheng, A. C. Samia, J. D. Meyers, I. Panagopoulos, B. Fei and
C. Burda, J. Am. Chem. Soc., 2007, 129, 4526.
7 S. H. Cheng, C. H. Lee, C. S. Yang, F. G. Tseng, C. Y. Moud and
L. W. Lo, J. Mater. Chem., 2009, 19, 1252.
8 S. Shen, A. E. Garcia-Bennett, Z. Liu, Q. Lu, Y. Shi, Y. Yan,
C. Yu, W. Liu, Y. Cai, O. Terasaki and D. Zhao, J. Am. Chem.
Soc., 2005, 127, 6780.
9 S. H. Wu, Y. S. Lin, Y. Hung, Y. H. Chou, Y. H. Hsu, C. Chang
and C. Y. Mou, ChemBioChem, 2008, 9, 53.
10 W.-D. Jang, N. Nishiyama, G.-D. Zhang, A. Harada, D.-L. Jiang,
S. Kawauchi, Y. Morimoto, M. Kikuchi, H. Koyama, T. Aida and
K. Kataoka, Angew. Chem., Int. Ed., 2005, 44, 419.
11 Y. Li, W. D. Jang, N. Nishiyama, A. Kishimura, S. Kawauchi,
Y. Morimoto, S. Miake, T. Yamashita, M. Kikuchi, T. Aida and
K. Kataoka, Chem. Mater., 2007, 19, 5557.
12 R. Ideta, F. Tasaka, W.-D. Jang, N. Nishiyama, G.-D. Zhang,
A. Harada, Y. Yanagi, Y. Tamaki, T. Aida and K. Kataoka, Nano
Lett., 2005, 5, 2426.
13 F. Yukruk, A. L. Dogan, H. Canpinar, D. Guc and E. U. Akkaya,
Org. Lett., 2005, 7, 2885.
14 B. X. Gao, H. X. Li, H. M. Liu, L. C. Zhang, Q. Q. Bai and
X. W. Ba, Chem. Commun., 2011, 47, 3894.
General procedure for synthesis of P3-COOH
To a stirred mixture of dendritic polyaspartic substituted
benzaldehyde compound 7 (0.5 mmol) and pyrrole (0.5 mmol)
in 1,1,2,2-tetrachloroethane (200 mL) at room temperature
was added boron trifluoride etherate (0.2 mmol). After the
mixture was stirred for an additional 2 h, DDQ (1.2 mmol)
was added and the reaction mixture was allowed to stir for
30 min at room temperature. The reaction mixture was
precipitated with methanol, and washed with chloroform.
Excess of NaOH was added to a solution of P3-OCH3
(100 mg) in 10 mL of THF. The mixture was stirred at room
15 B. X. Gao, H. X. Li, D. F. Xia, S. F. Sun and X. W. Ba Beilstein,
J. Org. Chem., 2011, 7, 198.
16 S. A. Vinogradov and D. F. Wilson, Chem.–Eur. J., 2000, 6, 2456.
17 M. C. DeRosa and R. J. Crutchley, Coord. Chem. Rev., 2002,
233–234, 351.
18 J. M. Harris and R. B. Chess, Nat. Rev. Drug Discovery, 2003,
2, 214.
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012
New J. Chem., 2012, 36, 28–31 31