Water-soluble sulfonate porphyrin functionalized hyaluronic acid with comb-like structure: Potential photosensitizers for photodynamic therapy

Article abstract of DOI:10.1016/j.dyepig.2019.01.034

A water-soluble comb-like porphyrin polymer based on hyaluronic acid (HA) as the main chain and sulfonate substituted tetraphenylporphyrin (TASP) as side groups, named as HA-g-NH-TPP-(SO₃Na) [(HA-g-TASP)], was designed and synthesized, which co

Full text of DOI:10.1016/j.dyepig.2019.01.034

Accepted Manuscript
Water-soluble sulfonate porphyrin functionalized hyaluronic acid with comb-like
structure: Potential photosensitizers for photodynamic therapy
Xu Cui, Yanhui Li, Yanwei Li, Baoguo Qiu, Qian Duan
PII:
S0143-7208(18)30665-X
https://doi.org/10.1016/j.dyepig.2019.01.034
DYPI 7307
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 27 March 2018
Revised Date: 27 July 2018
Accepted Date: 22 January 2019
Please cite this article as: Cui X, Li Y, Li Y, Qiu B, Duan Q, Water-soluble sulfonate porphyrin
functionalized hyaluronic acid with comb-like structure: Potential photosensitizers for photodynamic
therapy, Dyes and Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.01.034.
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Graphical Abstract
A water-soluble comb-like porphyrin polymer based on hyaluronic acid (HA) as
the main chain and sulfonato substituted tetraphenylporphyrin (TASP) as side groups,
named as HA-g-TASP, was designed and synthesized, which could be widely used as
a potential photosensitizer in photodynamic therapy (PDT). HA-g-TASP is the
polymer with comb-like structure which can avoid π-π stacting between TASP units,
and the photosensitizer displayed high-water solubility, low dark cytotoxicity, high
photocytoxiticy and high singlet oxygen yield.

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Water-soluble sulfonate porphyrin functionalized hyaluronic acid
with comb-like structure: potential photosensitizers for photodynamic
therapy
Xu Cui a, Yanhui Li a, Yanwei Li a,b, Baoguo Qiu a and Qian Duan a,b*
a School of Materials Science and Engineering, Changchun University of Science and Technology,
Changchun, 130022, China
b Engineering Research Center of Optoelectronic Functional Materials, Ministry of Education,
Changchun, 130022, China
Tel: +86 431 85583015; Fax: +86 431 85583015
E-mail address: duanqian88@hotmail.com (Qian Duan)
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Abstract: A water-soluble comb-like porphyrin polymer based on hyaluronic acid (HA) as the
main chain and sulfonate substituted tetraphenylporphyrin (TASP) as side groups, named as
HA-g-NH-TPP-(SO3Na) [(HA-g-TASP)], was designed and synthesized, which could be used as a
potential photosensitizer in photodynamic therapy (PDT). This photosensitizer displayed high-water
solubility, low dark cytotoxicity, high photocytoxiticy and high singlet oxygen yield (Φ∆=0.86). The
HA-g-TASP was carefully characterized by UV-Vis, FTIR, and 1H NMR. UV-Vis spectra showed
the Q bands of the HA-g-TASP spectrum had a significant red shift about 50 nm to near-infrared
(NIR) region compared with TASP, the HA-g-TASP had smaller energy band gap. The NIR region
is known as the transparent window of the organisms, NIR irradiation shows larger penetration
distance in tissue. As a novel photosensitizer, HA-g-TASP exhibited low cytotoxicity in dark and
also low photocytotoxicity towards normal cells, but performed enhanced photodynamic activity
against cancer cells (HeLa cells). As a result, HA-g-TASP with comb-like structure may be a useful
photosensitizer for practical photodynamic therapy in the future.
Keywords: Photosensitizer, Photodynamic therapy, Porphyrin, Hyaluronic acid, Comb-like
structure.
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1. Introduction
Nowadays, photodynamic therapy (PDT) has been widely used in many areas, such as cancer
therapy [1, 2], cancer detection [3, 4] and skin diseases [5, 6]. In the process of PDT, the activated
photosensitizers (PS) could react with the oxygen in the tissue under irradiation, causing the
formation of cytotoxic species such as singlet oxygen, resulting in toxicity to malignant cells [7]. PS
plays an important role in PDT treatment which determines the efficiency of PDT. Thus, it is still a
challenge to find new photosensitizers with high water solubility, low dark cytotoxicity, high
capacity to penetrate the plasma membrane and generate 1O2, and an ability to interact with specific
cellular targets [8-12]. Recent efforts to develop third-generation photosensitizers containing a
covalently attached tumor targeting to improve PDT efficacy, because the selectivity for tumors
remains insufficient in vivo for clinical applications [13]. So far, porphyrin as a common
photosensitizer is undisputed for its therapeutic abilities. Their excellent photophysical properties
and relative ease of both synthesis and functionalization make them widely used in the field of PDT
[14, 15]. While, as a widely used photosensitizer, porphyrin has poor selectivity for malignant
tissues, and low solubility inducing aggregation in aqueous media [16]. To overcome these
obstacles, many researchers investigated a variety of photosensitizer nanoparticles, nanoscale
organic-inorganic hybrid photosensitizer and polymeric photosensitizers, such as porphyrin-bridged
silsesquioxane theranostic nanoparticles [17], liposomes [18] and porphyrin-based metal-organic
framework [19] are efficient ways. But, these methods require significant chemical manipulation
(more synthetic steps) or complex formulation strategies (such as grafting liposomes to improve
biocompatibility). Li et al. [20] reported a dendrimer-based photosensitizer, dendrimer porphyrin
(DP), in which the focal porphyrin was surrounded by the third generation of poly (benzyl ether)
dendrons. In their work, they improved a part of water-ability of porphyrin, while, the larger
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molecular weight makes it hard to enter cells and its cell targeting is bad. Zheng et al. [21-24]
synthesized a liposome-like structure porphysomes using phospholipid-porphyrin conjugates as the
precursor, which has many advantages such as high loading capacity and excellent biocompatibility,
and these porphysomes exhibited potential applications in the photodiagnosis and phototherapy. Na
et al. [25-28] constructed a series of polymeric PSs using hydrophilic polymers such as hyaluronic
acid, chondroitin sulfate, pluronic F127, polyethyleneimine, and they found that it could effectively
improve the water solubility and enhance cellular internalization. Hyaluronic acid, as one of the
extracellular matrix polysaccharide, has been regarded as the excellent biomaterial for its
nonimmunogenic, biocompatible and biodegradable [29]. Intriguingly, it has been used as a drug
carrier or targeting moiety due to the specific interaction with CD44 (hyaluronic acid receptor) on
tumor cell [30].
In this study, we presented the synthesis and property of water-soluble hyaluronic
acid-porphyrin (HA-g-TASP). (SO3Na)2-TPP-(NH2)2 (TASP) is chosen as the precursor and
prepare HA-g-TASP with two-block structures: amino-sulfonic porphyrin as side groups for singlet
oxygen generation and hyaluronic acid as the main chain to target the cancer cells and reducing
1
cytotoxicity and maintain high O2 productivity. The synthesis of HA-g-TASP was described in
Scheme 1.
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Scheme 1. Synthesis of comb-like porphyrin polymer HA-g-TASP.
In contrast with conventional photosensitizers, HA-g-TASP had many advantages: first,
hydrophilic sulfonic group can improve the water solubility; second, HA was tumor targeting and
increase biocompatibility; third, HA as the main chain can ensure the efficacy of singlet oxygen
production even at an extremely high concentration because the comb-like structure of HA-g-TASP
can prevent porphyrin aggregation.
2. Experimental section
2.1 Materials
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Pyrrole was purchased from Aladdin, purified by distillation before use. The benzaldehyde,
sodium sulfide nonahydrate, boric acid, hyaluronic acid (HA) were purchased from Acros or
Aladdin Reagent Company and used without further purification. The dehydrated solvents were
obtained after treating tetrahydrofuran (THF) and dichloromethane (DCM) solvents with standard
procedures. All other analytical grade chemicals and reagents were purchased from Sinopharm
Chemical Reagent Co. and used as received.
2.2 Synthesis
2.2.1 Synthesis of 5, 10, 15, 20-tetraphenyl-21H, 23H-porphyrin (TPP)
The TPP precursor was synthesized by Adler’s method [31] (as shown in Scheme 1). A mixture
of benzaldehyde (5.0 mL, 0.050 mol), pyrrole (3.5 mL, 0.050 mol), and DMSO (6.0 mL) was added
to propionic acid (0.12 L), 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 water until the filtrate was neutral
(pH≈6.8) to obtain purple crystals. The crude product was dried under vacuum (80 °C) for 12 h. The
crude compound was purified by silica gel column chromatography with dichloromethane as the
1
eluent. Yield: 2.4 g, 31%. H NMR (400 MHz, CDCl3), δ ppm: 8.84 (s, 8H), 8.25-8.18 (m, 8H),
7.81-7.69 (m, 12H), -2.76 (s, 2H). (Figure S1)
2.2.2 Synthesis of di(4-aminophenyl)-diphenyl porphyrin [TPP-(NH2)2] (TAPP)
Synthesized from the corresponding TPP by a literature method [32] (as shown in Scheme 1).
The TPP (2.0 g, 3.3 mmol) was added to CH2Cl2 (60 mL) subsequently added nitric acid fuming (5.7
g, 90 mmol), the mixture solution was stirred at 0 °C for 3 h. Then added ammonia liquor and
deionized water until the filtrate was neutral (pH=7-8) to obtain purple crystals. The crude product
was dried under vacuum (80 °C) for 12 h. The crude compound was purified by silica gel column
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chromatography with dichloromethane-petroleum ether (1:4 v/v) as eluent to afford the desired dye as
purple solid (TNPP).
The as-obtained TNPP was reduced into TAPP. Typically, TNPP (0.90 g, 1.3 mmol) and
Na2S·9H2O (6.1 g, 25 mmol) were dissolved in 35 mL DMF and then reacted at 60 °C with a
magnetic stirring. TLC test results were collected, after 4 h the raw material point disappeared and
generated amino porphyrin mixture. Then the flask was removed from the oil bath and the mixture
was dissolved in the right amount of distilled water. The precipitate was collected and vacuum drying
for 24 h, resulting in a bright purple product. The crude compound was purified by silica gel column
chromatography using dichloromethane-ethyl acetate (200:1 v/v) as eluent to afford a mixture
product of cis- and trans- diaminotetraphenylporphyrin as purple solid. It is difficult to separate cis-
and trans- diaminotetraphenylporphyrin by column chromatography, due to the close polarity of the
1
products. Yield: 0.63 g, 31 %. H NMR (400 MHz, CDCl3) δ = 8.92 (d, J = 3.6 Hz, 4H), 8.82 (d, J =
3.2 Hz, 4H), 8.21 (d, J = 6.7 Hz, 4H), 7.99 (d, J = 7.8 Hz, 4H), 7.74 (q, J = 6.6 Hz, 6H), 7.05 (d, J = 7.8
Hz, 4H), 4.02 (s, 4H), -2.73 (s, 2H). (Figure S2)
2.2.3 Synthesis of di(4-aminophenyl)-di(4-sulfophenyl)-diphenyl-porphyrin
[(SO3)2-TPP-(NH2)2] (TASP)
TASP was synthesized as shown in Scheme 1. TAPP (0.050 g, 0.080 mmol) was added into
H2SO4 (1.2 mL, 0.020 mmol) and the mixture was stirred for 4 h at 120 °C. After naturally cooling
down to room temperature, the mixture was poured into 300 mL deionized water, then the pH of the
solution was regulated to 8-9 by using NaOH (3.0 mol L-1) and NaHCO3 (1.0 mol L-1).
Subsequently, the mixture solution was concentrated and filtered to remove the Na2SO4. Then the
methanol was added into the as-obtained crude filtrate, and the mixture was filtered to remove the
precipitated Na2SO4. Finally, the crude compound was recrystallized using methanol-acetone (1:10
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v/v) three times to get the purified TASP. The precipitate was collected and vacuum drying for 24 h,
1
resulting in bright purple product TASP. Yield: 0.020 g, 30 %. H NMR (400 MHz, DMSO-d6)
δ=9.03 (s, 2H), 8.94 (s, 2H), 8.82 (s, 4H), 8.18 (s, 4H), 8.06 (s, 4H), 7.87 (d, J = 7.6 Hz, 2H), 7.34 (s,
2H), 7.18 (s, 2H), 7.02 (d, J = 6.9 Hz, 2H), 4.39 (s, 4H), -2.75 - -2.83 (m, 2H). (Figure S3).
2.2.4 Synthesis of hyaluronic acid-aminophenyl-aminophenyl-di(4-sulfophenyl)-diphenyl
porphyrin [(SO3)2-TPP(NH2)-HA] (HA-g-TASP)
TASP (1.0 g, 1.2 mmol) and HA (0.45 g) were added to 20 mL of deionized water with stirring
at room temperature. After stirring for 10 minutes, boric acid (0.25 g, 4.0 mmol) was added in and
stirred at 25 °C for 24 h. The resulting polymer was dialyzed against water using a dialysis bag with a
molecular weight cut-off of 1000 (product of USA), unreacted TASP was removed thoroughly by
dialyzing against water for 7 days until there were hardly any TASP outside dialysis tube, and then
freeze-dried overnight to obtain the target HA-g-TASP polymer. Yield: 0.28 g, 21 %.
2.3 Characterization
Fourier transform infrared spectra (FT-IR) of the samples were performed by a
Shimadzu-8400S FT-IR spectrometer in KBr plates with a resolution of 4 cm-1 scanning
from 400-4000 cm-1. Ultraviolet-visible (UV-Vis) spectra were measured on a UV Mini
1240 (Shimadzu) spectrophotometer. 1H Nuclear Magnetic Resonance NMR) analysis was
(
recorded in CDCl3 or DMSO-d6 at 25 °C on an AV-400 NMR spectrometer (Bruker),
Tetramethylsilane (TMS) was used as internal standard. Fluorescence spectra were obtained
by an LS-55 fluorospectro photometer (Perkin Elmer).
2.4 Singlet oxygen generation
The singlet oxygen quantum yields (Φ∆) of TAPP and HA-g-TASP in DMSO were
determined by using 1,3-diphenylisobenzofuran (DPBF) as a scavenger and TPP as a standard
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[33-39]. The illumination source consisted of a 20 W xenon lamp and a color glass filter cut-on 610
nm. To avoid chain reactions induced by quenchers (DPBF) in the presence of singlet oxygen, the
concentration of DPBF was lowered than 5.0 × 10−5 mol L-1. The DPBF decay was monitored at
410 nm, and the experiments were carried out in DMSO. The singlet oxygen quantum was
calculated according to eq. (1):
ꢉꢊꢋ
ꢄꢅ
ꢀ∆ = ꢀ∆ꢁꢂꢃ
(1)
ꢆꢇꢈ
ꢉꢊꢋ
ꢄ
ꢅ
ꢆꢇꢈ
where ꢀ∆ꢁꢂꢃ is the singlet oxygen quantum yields for the standard TPP; ꢌꢁꢂꢃ and R are the DPBF
ꢁꢂꢃ
ꢎꢏꢐ
photobleaching rates in the presence of the standard and the analyte, respectively; ꢍ
the rates of light absorption by the standard and the analyte, respectively.
2.5 Dark cytotoxicity of HA-g-TASP
and ꢍꢎꢏꢐ are
L929 cells were chosen to evaluate the dark cytotoxicity of TASP. The cells were seeded in a
96-well plate. After culturing for 24 h, the L929 cells were respectively treated with HA-g-TASP,
TASP and TAPP at different doses in DMEM at 37 °C for 24 h in the dark, washed three times
with sterilized phosphate buffer saline (PBS), and the HA-g-TASP or TASP or TAPP solution was
replaced with fresh DMEM and 20 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) solution (5 mg mL-1) for 4 h. Then the MTT-containing medium was removed, and
0.20 mL DMSO was added to each well to extract the formazan products with agitation for 10 min.
The absorbance at 492 nm was measured by an enzyme-linked immuno sorbent assay (ELISA)
microplate reader (Bio-Rad). The cell viability (%) was calculated by the following Eq. (2):
Cell viability (%) = (Xsample /Xcontrol) × 100%
Where Xsample is the absorbance of the cells incubated in DMEM, Xcontrol is the absorbance
without treatment.
(2)
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2.6 Phototoxicity measurement
The photocytotoxicity against L929 and HeLa cells carried out by the same way of the dark
cytotoxicity as described above. Briefly, after the addition of HA-g-TASP into the plate, all the
procedures operated under the light. The cells plate was exposed to red light (610 nm) for 10 min.
Then irradiated cells were back to the incubator for 24 h and assayed cell viability. The subsequent
procedures were the same as described in the cytotoxicity assay.
3 Results and discussion
3.1 Synthesis and characterization
The HA-g-TASP was synthesized according to the following steps as schematically illustrated
in Scheme 1. First, the TPP was prepared via Adler’s method with benzaldehyde and pyrrole.
Second, TPP was nitrated by nitric acid fuming to get TNPP, and then TNPP was reduced by
Na2S·H2O to get TAPP. Third, TAPP was sulfonated with concentrated sulfuric acid to obtain
TASP. Last, TASP grafted on HA to get HA-g-TASP via an amidation reaction catalyzed by boric
acid. HA-g-TASP was water-soluble with comb-like structure, which could act as potential
photosensitizers for the photodynamic therapy.
To identify the construct of the as-prepared materials, the synthesized products were
characterized by FTIR spectra. As shown in Figure 1, for (a) TPP showed the absorption
bands at 3317 cm-1 which was attributed to N-H stretching vibration of the pyrrole ring,
three absorption bands at 1594, 1473 and 1434 cm-1 which were related to the skeletal
vibration of the benzene rings in the porphyrin, and the absorption bands at 799 cm-1 and
702 cm-1 which were assigned to C-H bending vibration of the benzene rings. For (b) TAPP
,
the peak at 3374 cm-1 was assigned to the amino groups of the benzene rings. After the
sulfonation reaction, the strong absorption bands at 1040 cm-1 and 636 cm-1 were observed,
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which were assigned to the sulfoacid stretching vibration [Figure 1(c)]. After the amidation
reaction, there was a strong absorption band at 1634 cm-1 in TASP, which was assigned to
1
the C=O stretching vibration of acylamino groups. [Figure 1(d)]. H NMR spectra further
1
supported the successful preparation of all the products. H NMR spectra of TPP
,
TAPP,
1
and TASP were shown in the experimental section and Figures S1-S3. The H NMR
spectrum of HA-g-TASP and the corresponding signal assignments were shown in Figure 2,
S(a) is the area of the signals at -2.92 ppm (a) assigned to pyrrole ring protons; S(c) is the
area of the signals appearing at 8.35 ppm (c) assigned to the protons of the acylamino groups.
The integral area ratio [S(a):S(c)] was calculated to 1:0.55, indicating the success grafting of
1
porphyrin monomer on a hyaluronic acid chain. The results of IR spectra and H NMR
spectra both further demonstrated that HA-g-TASP was successfully prepared.
Figure 1. FTIR spectra of (a) TPP, (b) TAPP, (c) TASP and (d) HA-g-TASP.
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Figure 2. 1H NMR spectrum of HA-g-TASP in DMSO-d6.
3.2 Photophysical properties of porphyrin derivatives and HA-g-TASP
Figure 3. (a) UV-vis absorption spectra of TPP and TAPP in CH2Cl2 solution. (b) UV-vis absorption spectra of
TASP and HA-g-TASP in H2O solution. (c) Emission spectra of TPP and TAPP with excitation at 380 nm
(1.3×10-5 g mL-1 in CH2Cl2 solution ). (d) Emission spectra of TASP and HA-g-TASP with excitation at 250 nm
(1.3×10-5 g mL-1 in H2O solution).
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UV-vis absorption spectra and fluorescence spectra were used to characterize the optical
properties of the as-prepared materials. It can be clearly seen from Figure 3(a) and 3(b) that
different porphyrin derivatives and HA-g-TASP in different solvents exhibited the entirely different
characteristic absorption bands (one B band located at ≈410 nm and 1-4 Q bands located at
≈500-700 nm) in the near-UV and visible region. It can be seen in Figure 3(a) that the TAPP
presented a red-shift of the Soret band and Q bands in comparison with the TPP, ascribed to the
effect of an electron donor, in other word, amino groups as auxochromic group inducing smaller
energy band gap and redshift of the optical absorption. Figure 3(b) showed the UV-vis absorption
spectra of TASP and HA-g-TASP in H2O solution. The Q band of HA-g-TASP was reduced to
two bands and presented a large red shift (about 50 nm) to the NIR region in comparison with the
TASP, which could be attributed to the comb-like structure cause energy transmission between
TASP and HA, or HA contains large hydroxy and carboxy groups as auxochromic group inducing
longer wavelength of absorbed photon, smaller energy band gap, and redshift of the optical
absorption. The least-energy Q band was the transition of interest for PDT, as the NIR region with
the minimal light absorption and scattering is known as the transparent window of the organisms,
NIR irradiation showed lower photodamage effect, larger penetration distance in tissue, and higher
signal-to-noise ratio [40].
The fluorescence emission spectra of the porphyrin derivatives and HA-g-TASP in CH2Cl2
and H2O solution were shown in Figure 3(c) and 3(d), respectively. And they reveal the luminescent
properties of the porphyrin derivatives and HA-g-TASP. It was clearly seen that the TPP, TAPP
with excitation at 380 nm in CH2Cl2 solution exhibited emission bands at 650 nm, 660 nm in the
visible region, and the TASP, HA-g-TASP with excitation at 250 nm in H2O solution exhibited
emission bands at 340 nm, 370 nm in the near-UV. Relative to the emission band of the TPP in
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CH2Cl2 at 650 nm, the emission band of TAPP was somewhat redshifted. Compared to the
emission band of the TASP in H2O at 340 nm, the emission band of HA-g-TASP was redshifted
nearly 25 nm. It could be attributed to the changing solvent and the comb-like structure cause
energy transmission between TASP and HA. The fluorescence properties of the porphyrin polymer
might be suited for detection imaging in PDT.
3.3 The singlet oxygen quantum yields
The generation of singlet oxygen was an imperative prerequisite for photodynamic [31-33].
The singlet oxygen could be produced when the energy transfers between the triplet state and
ground state of photosensitizers. Measurements of singlet oxygen quantum yields can assess the
effectiveness of newly synthesized photosensitizers. The Φ∆ values were determined using DPBF as
a scavenger and TPP as a standard. The DPBF decay at 410 nm was monitored every 10 min. The
peak intensity of the mixture of quenchers (DPBF) with TAPP [Figure 4(a)] and HA-g-TASP
[Figure 4(b)] in DMSO was tested using a UV-Vis spectrophotometer. Using tetraphenyl porphine
in benzene as the reference (Φ∆=0.63) [41], the singlet oxygen quantum was calculated according to
eq. (1), the singlet oxygen yields of TAPP and HA-g-TASP were 0.52 and 0.86, respectively. It was
obvious that the singlet oxygen quantum yields of HA-g-TASP had significantly improved and
were higher than the TAPP. In contrast with most photosensitizers, HA-g-TASP had high singlet
oxygen yield (Φ∆=0.86) [42-45]. Moreover, we have investigated the photostability of TAPP and
HA-g-TASP in DMSO. As shown in figure S4, all the UV spectra of the samples tested at different
time maintain the same shape, inducing spectra overlap into one curve as seen in figure S4(a) and
(c). And B bands of TAPP and HA-g-TASP remain unchanged with the increase of illumination
time as seen in figure S4(b) and (d), indicating excellent photostability. The high singlet oxygen
yield could be ascribed to the comb-like structure which prevented the quenchers from interacting
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with the porphyrin core, and then somewhat hindered the excited state quench, on the other hand,
the comb-like structure, not only can avoid the aggregation of TASP, but also can achieve the
"highly condensed" owing to the polymer effect and avoid π-π stacking between TASP units. As a
result, HA-g-TASP was a promising photosensitizer for PDT.
Figure 4. UV-Vis absorption spectra of DPBF with TAPP (a) and HA-g-TASP (b) after irradiation for every 10
min, down from 10 to 80 min (inset: plot of absorbance versus concentration).
3.4 Cytotoxicity Assays
Figure 5. Dark toxicity against L929 cells determined by the MTT assay with TAPP, TASP, and HA-g-TASP.
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To investigate the dark toxicity and biocompatibility of TAPP, TASP, and HA-g-TASP in vitro,
the cytotoxicity against of L929 cells was determined by the MTT assay without light. As shown in
Figure 5, the dark toxicity of TAPP, TASP, and HA-g-TASP at varying concentrations (0 to 0.256 mg
mL-1) of porphyrins derivate was tested. In dark, the viability of L929 cells in the TAPP, TASP, and
HA-g-TASP was almost up to 90% in the range of 0.78-
1000 µg mL-1. By contrast, the HA-g-TASP
displayed significantly low dark toxicity, and the cells viability was about 94%. Therefore, the result
proved that HA-g-TASP had good biocompatibility to be a promise polymeric material.
Figure 6. Light microscopic images of (a) HeLa cells, (b) L929 cells after incubation with HA-g-TASP.
Normally, photosensitizers used in PDT possess high phototoxicity to tumor cells, while can’t
damage normal tissues and cells [46]. To investigate the phototoxicity of HA-g-TASP in vitro, the
phototoxicity against L929 and HeLa cells were determined by the MTT assay under light irradiated.
Phototoxicity of HA-g-TASP against HeLa cells and L929 cells were at the same concentrations
and irradiated for 30 min. Light microscopic images of HeLa cells and L929 cells indicated that
HA-g-TASP could effectively generate the cytotoxic substance to kill the HeLa cells [Figure 6(a)],
while showed very low phototoxicity against the L929 cells [Figure 6(b)].
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Figure 7. In vitro viability of HeLa cells treated TAPP (a), TASP (b), and HA-g-TASP (c) at 7.8-1000 µg mL-1
for 24 h, and illumination at 400-700 nm for 30 min respectively. (Black is dark toxicity, red is phototoxicity.)
In addition, the in vitro cytotoxicity on HeLa cells with or without light treatment was
appraised by MTT assay. The dark cytotoxicity and phototoxicity of TAPP [Figure 7(a)], TASP
[Figure 7(b)], HA-g-TASP [Figure 7(c)] were investigated in HeLa cells at varying concentrations
(7.8 to 1000 μg mL-1). As shown in Figure 7, with the cell viability decreased with the increase of
porphyrins derivate. However, TAPP, TASP, and HA-g-TASP had no significant toxicity to HeLa
cells even at a concentration of 1000 μg mL-1 in the dark. The MTT assay results with light
conditions revealed that the cell viability decreased with the increasing concentration of porphyrins
derivates. At a concentration of 1000 μg mL-1, the HeLa cells survival in HA-g-TASP was 20%,
indicated that HA-g-TASP had significant phototoxicity to HeLa cells. These results are in good
agreement with the singlet oxygen quantum yields of the porphyrin polymer. (The microscopic
images see the Supporting Information, Figure S5.) HA-g-TASP is the polymer with a comb-like
structure which can avoid π-π stacking between TASP units, and increase effective density of TASP
units to ensure high singlet oxygen yield under light irradiated. For HA had good water-soluble and
biocompatibility, HA-g-TASP with HA as a main chain make itself dissolving in water and insure
cells survival rate as shown in the discussion. As shown in Figure 7, because the molecular weight
of HA is very high, resulting in the HA-g-TASP with even higher molecular weight，therefore the
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HA-g-TASP required to test came up to 1000 µg mL-1. Consequently, the HA-g-TASP may
contribute to the development of a new generation of photosensitizer for PDT.
4 Conclusions
In summary, a novel water-soluble comb-like porphyrin polymer HA-g-TASP was
1
synthesized successfully. The results of FT-IR, UV-Vis and H NMR have shown the
structure of HA-
the HA- -TASP had a significant red shift to the near-infrared (NIR), which is known as the
transparent window of the organisms. And from the fluorescence spectra of HA- -TASP
the porphyrin polymer might be suited for detection imaging in PDT. HA- -TASP had high
singlet oxygen yield (Φ∆=0.86) than most photosensitizers. According to the MTT assay,
HA- -TASP had low dark toxicity and intensive phototoxicity and also low
g-TASP. In comparison with the UV-Vis spectrum of TASP, the Q bands of
g
g
,
g
g
photocytotoxicity towards normal cells, but performed enhanced photodynamic activity
against cancer cells (HeLa cells). Thus, possible injury to normal tissue was effectively
avoided. The successful introduction of sulfonyl and HA dramatically improved the water
solubility and tumor targeting of the porphyrin as a potential reaction site. Besides, it also
ensured the efficacy of singlet oxygen production even at an extremely high concentration
because the water-soluble can prevent aggregation of the central porphyrin. All of the
advantages mentioned above promoted the delivery of porphyrin drugs into cancer cells for
PDT.
Acknowledgments
The authors are thankful to the National Natural Science Foundation of China
(21153003), Jilin Science & Technology Department (20140204017GX, 20150520025JH),
China Post-doctoral Science Foundation (no. 2014M561268), Science & Technology Bureau
18

ACCEPTED MANUSCRIPT
of Changchun City project (2013060), the Education Department of Jilin Province science
project (JJKH20170606KJ), Changchun University of Science and Technology Innovation
Foundation (XJJLG-2016-05, XJJLG-2016-06) for nancial support to this work.
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Captions：
Scheme 1. Synthesis of comb-like porphyrin polymer HA-g-TASP.
Captions to Figures
Figure 1. FTIR spectra of (a) TPP, (b) TAPP, (c) TASP and (d) HA-g-TASP.
Figure 2. 1H NMR spectrum of HA-g-TASP in DMSO-d6.
Figure 3. (a) UV-vis absorption spectra of TPP and TAPP in CH2Cl2 solution. (b) UV-vis
absorption spectra of TASP and HA-g-TASP in H2O solution. (c) Emission spectra of TPP and
TAPP with excitation at 380 nm (0.01275 mg mL-1 in CH2Cl2 solution ). (d)Emission spectra of
TASP and HA-g-TASP with excitation at 250 nm (0.01275 mg mL-1 in H2O solution).
Figure 4. UV-Vis absorption spectra of DPBF with TAPP (a) and HA-g-TASP (b) after irradiation
for every 10 min, down from 10 to 80 min (inset: plot of absorbance versus concentration).
Figure 5. Dark toxicity of L929 cells determined by the MTT assay after treatment of TAPP,
TASP, and HA-g-TASP.
Figure 6. Light microscopic images of (a) HeLa cells, (b) L929 cells after incubation with
HA-g-TASP.
Figure 7. In vitro viability of HeLa cells treated TAPP (a), TASP (b), and HA-g-TASP (c) at
7.8-1000 µg mL-1 for 24 h, and illumination at 400-700 nm for 30 min respectively. (Black is dark
toxicity, red is phototoxicity.)
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Scheme 1. Synthesis of comb-like porphyrin polymer HA-g-TASP.
Figure 1. FTIR spectra of (a) TPP, (b) TAPP, (c) TASP and (d) HA-g-TASP.
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Figure 2. 1H NMR spectrum of HA-g-TASP in DMSO-d6.
Figure 3. (a) UV-vis absorption spectra of TPP and TAPP in CH2Cl2 solution. (b) UV-vis absorption spectra of
TASP and HA-g-TASP in H2O solution. (c) Emission spectra of TPP and TAPP with excitation at 380 nm
(0.01275 mg mL-1 in CH2Cl2 solution ). (d) Emission spectra of TASP and HA-g-TASP with excitation at 250 nm
(0.01275 mg mL-1 in H2O solution).
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Figure 4. UV-Vis absorption spectra of DPBF with TAPP (a) and HA-g-TASP (b) after irradiation for every 10
min, down from 10 to 80 min (inset: plot of absorbance versus concentration).
Figure 5. Dark toxicity of L929 cells determined by the MTT assay after treatment of TAPP, TASP, and
HA-g-TASP.
Figure 6. Light microscopic images of (a) HeLa cells, (b) L929 cells after incubation with HA-g-TASP.
28