A folate-conjugated platinum porphyrin complex as a new cancer-targeting photosensitizer for photodynamic therapy

Article abstract of DOI:10.1039/c9ob00698b

A new folate-conjugated platinum porphyrin complex (Por 4) was synthesized and characterized. The singlet oxygen production of the conjugates was evaluated through a 1,3-diphenylisobenzofuran method. The targeting ability and subcellular localization of P

Full text of DOI:10.1039/c9ob00698b

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
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DOI: 10.1039/C9OB00698B
Folate-Conjugated Platinum Porphyrin Complex as a New
Cancer-Targeting Photosenitizer for Photodynamic Therapy
a
a
a
b
a
*
Mengqian Yang , Jingran Deng , Ding Guo , Jie Zhang , Lixia Yang , Fengshou
a
*
Wu
a
Key Laboratory for Green Chemical Process of the Ministry of Education, School of Chemical
Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan, 430205, P. R. China. Email:
wfs42@126.com; fswu@wit.edu.cn.
b
Department of Chemistry, School of Pharmaceutical and Chemical Engineering, Taizhou
University, Taizhou, 318000, P. R. China. Email: zhang_jie@tzc.edu.cn.
Abstract
A new folate-conjugated platinum porphyrin complex (Por 4) was synthesized and characterized.
The singlet oxygen production of the conjugates was evaluated through
1,3-diphenylisobenzofuran method. The targeting ability and subcellular localization of Por 4
were confirmed by confocal laser scanning microscope in HeLa cells (overexpression of FR) as
well as A549 cells (low expression of FR). The results suggested that the modification of the
carboxyl group with porphyrin compound did not decrease the binding affinity of folic acid with
FR positive cancer cells. Moreover, the MTT assay using HeLa cells and A549 cells verified the
low cytotoxicity of Por 4 in the dark. Upon irradiation, Por 4 showed noticeable improvement in
toxicity against cancer cells with overexpression of FR. Upon the treatment of Por 4 at the
concentration of 20 μM, the cell viability was determined as 22% and 75% for HeLa and A549
cells, respectively, indicating that the folate-conjugated platinum porphyrin complex could be a
promising PDT agent for cancer with overexpresion of folate receptor.
Keywords: Porphyrin; targeting; photosensitizers; folic acid; PDT
1. Introduction
Cancer is one of the deadliest diseases threatening the human beings’ health around the world.
Among the emerging cancer therapy methods, photodynamic therapy (PDT) has gained
considerable attentions because it is noninvasive in nature, has fewer side effects, and causes

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negligible drug resistance.[1] The therapeutic mechanism of PDT is that the photosensitizer can
generate reactive oxygen species (ROS), such as singlet oxygen (1O2) under the irradiation of
specific wavelength light, which will effectively damage the structure and function of cancer
cells.[2-4] Porphyrin and its derivatives have been widely studied as the efficient photosensitizers
in photodynamic therapy (PDT). Some of them have been approved by the multinational
governments drug regulatory authorities and were widely applied in clinic due to their strong
photosensitizing effect, low toxicity and low side effects to the human body.[5,6] However, it is still
confronted with several challenges, such as low biocompatibility and singlet oxygen yield and
inadequate selectivity toward cancer tissues.[7] Accordingly, a PDT agent with good selectivity
and high 1O2 quantum yield is highly desirable.[8]
DOI: 10.1039/C9OB00698B
To enhance the therapeutic efficiency of photosensitizers and avoid the severe side effects on
healthy tissues, the targeting delivery of anticancer drugs to tumor sites in a passive or active
manner, is an effective method. Although passive targeting approaches provide a significant basis
for clinical therapeutic treatments, they still suffer from several drawbacks, such as prolonged
photosensitivity and photoalergic reactions.[9] In contrast, active targeting involves the attachment
of targeting ligands to photosensitizer that can specifically recognize receptors overexpressed on
the membrane of tumor cells. The targeting ligands, including monoclonal antibodies (anti-HER2,
anti-EGFR[9,10]), small biomolecules ( biotin[11]), peptides (TAT peptide[12] and RGD peptide[13]),
and oligonucleotides (aptamers[14,15]) were widely reported in previous literatures. Folic acid (FA)
is also a targeting agent which has been widely studied in the field of imaging or diagnostics [16-18]
and for the treatment of certain cancers. Numerous cancer cell lines, such as prostate, brain, lung,
[16,17]
nose, ovary, colon cancer
over-express FA receptors (FRs) because of their fast growth and
cell division.[19] On the contrary, the folate receptors (FRs) have very low expression on normal
cells.[20] Therefore, the anti-cancer drugs conjugated with FA can selectively accumulate in tumor
tissue.[21,22]
1
On the other hand, O2 is generated by energy transfer between the triplet excited state of
photosensitizers and the ground state of O2.[23-25] Thus, the introduction of heavy metal ions into
porphyrins could enhance the probability of intersystem crossing, which in turn improves their
capacity of singlet oxygen generation.[26,27] In our previous studies, we synthesized a series of
1
metal porphyrin complexes and investigated the metal effect of porphyrins on their O2 quantum

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DOI: 10.1039/C9OB00698B
yield. Among the various metal porphyrin complexes, the platinated porphyrin showed the highest
therapeutic activity compared to free base porphyrin and other metal porphyrin complexes,
1
probably due to its high efficiency of O2 generation.[28] In this context, here we designed and
synthesized a new platinum porphyrin-folate conjugate as an efficient photosensitizer for
tumor-targeting PDT. Specifically, nine methoxyl groups were introduced into the porphyrin
component to improve the biocompatibility of PDT agent.[29] And the platinum is coordinated with
porphyrin conjugate to enhance the photodynamic therapeutic effect of agent by the heavy atom
effect. Moreover, a targeting molecule (folic acid) is bonded to the platinum porphyrin complex to
enhance the tumor-targeting of the photosensitizer. As expected, the folate-conjugated platinum
porphyrin complex (Por 4) displayed the high singlet oxygen quantum yield and strong targeting
ability. The cytotoxicity and subcellular localization of Por 4 in cancer cells were further
evaluated through MTT method and confocal laser scanning microscope, respectively.
Results and Discussion
Synthesis
The synthetic routes of folic acid-platinum porphyrin conjugate (Por 4) were demonstrated in
Scheme 1. Firstly, Por 1 was synthesized by condensation of pyrrole with 3,4,5-tri
methoxybenzaldehyde and 4-formylbenzoic acid (3:1) in a refluxing of propionic acid. The Por 1
was then coordinated with potassium tetrachloroplatinate to obtain platinum porphyrin complex
(Por 2). The activation of the carboxylic acid group was performed with thionyl chloride in dry
pyridine, yielding the corresponding porphyrinic acyl chloride (Por 3). Due to the poor solubility
of FA, its direct addition to the porphyrinic acyl chloride solution did not afford the desired
product. Further addition of N-hydroxysuccinimide (NHS) at 50 °C afforded the activated ester
Por 3 (Scheme 1). Finally, in the presence of N,N-diisopropylethylamine (DIEA), the
carboxyl-activated folic acid was linked to the carboxyl-activated ester platinum porphyrin by
ethylenediamine to synthesize a folic acid-platinum porphyrin conjugate (Por 4).

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OCH3
OCH3
H3CO
OCH
DOI: 10.1039/C9OB00698B
H3CO
OCH3
3
OCH3
CHO
CHO
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
H
N
N
N
Pt
NH
N
N
O
H3CO
OCH3
O
(i)
(ii)
N
4
3
OH
OH
N
HN
COOH
H3CO
OCH3
H3CO
OCH3
OCH3
OCH3
Por 1
Por 2
OCH3
OCH3
OCH3
H3CO
H3CO
OCH3
H
N
O
H2
N
N
N
H3CO
H3CO
H3CO
O
O
H
(vi)
N
N
N
(iii)
OCH3
OCH3
OCH3
N
N
N
Pt
N
N
N
O
H
H
Pt
O
N
N
N
O
N
H
N
O
H
O
O
HO
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
Por 3
Por 4
H
H2N
N
N
N
H
N
H
H2
N
N
N
N
N
H
N
O
H2
N
N
N
H
H
N
(iv)
(v)
N
N
O
O
O
H
O
OH
N
N
H
O
O
H
O
N
N
O
H
N
O
NH2
HO
N
O
H
O
O
H
O
H
O
HO
FA
1
HO
FA
3
FA
2
(i) acetic anhydride, propionic acid, 130 °C, 1.5 h, yield 6%; (ii) K2PtCl4, benzonitrile, 110 °C, 24
h, yield 78%; (iii) SOCl2, NHS, pyridine, 50 °C, 3 h, yield 73%; (iv) DCC, NHS, DMSO, N2, 6 h
(v) Ethylenediamine, pyridine, rt, 12 h, yield 60%; (vi) FA 3, DIEA , DMF, N2, 24 h, yield 34%.
Scheme 1. Synthetic routes of Por 1, Por 2, Por 3, FA 2, FA 3, and Por 4.
Photophysical properties
1.5x105
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Por 1
Por 2
Por 4
a
b
Por 1
Por 2
Por 4
1.0x105
5.0x104
0.0
300
400
500
600
700
800
600
650
700
750
800
Wavelength (nm)
Wavelength (nm)
Fig. 1. (a) UV-vis absorption spectrum, and (b) emission spectrum of the intermediates and
targeted compound.
Since Por 3 was not very stable, its photophysical properties were not recorded and investigated.
The photophysical properties of Por 1, Por 2 and Por 4 were studied in DMF solution. The UV-vis
absorption spectrum of Por 1 (Fig. 1a) exhibited a sharp Soret band centered at 422 nm and weak

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Q bands at 516, 552, 591 and 648 nm, respectively. The two peaks in the Q bands of Por 2 and Por
4 disappeared, probably due to the metallization of porphyrin. Meanwhile, the Soret band of
porphyrin showed a certain hypsochromic shift after coordination with metal. As shown in Fig. 1a,
the Soret band of Por 1 was located at 422 nm, while Por 2 and Por 4 shifted to 406 nm, ascribed
to the strong dπ-eg(π*) π-dative interaction between Pt ion and porphyrin macrocycle.[30] The
fluorescence emission of Por 1, Por 2 and Por 4 was tested in DMF solution. As shown in Fig. 1b,
both of platinum porphyrin complexes (Por 2 and Por 4) exhibited red emission (λem= 650, 740 nm)
with fluorescence intensity lower than that of free base porphyrin (Por 1), probably due to the
heavy metal effect. The fluorescence quantum yields of Por 2 and Por 4 were determined to be
0.34 and 0.23, respectively. These results suggested that the conjugation of folic acid did not
affect the wavelength of emission peaks.
Singlet oxygen detection
1.00
0.96
0.92
0.88
0.84
DPBF
DPBF+TPP
DPBF+Por 1
0.80
DPBF+Por 2
DPBF+Por 4
0.76
0.72
0
10
20
30
40
50
60
Illumination Time (sec)
Fig. 2 The changes of absorbance at the characteristic peak of DPBF (418 nm) as a function of
irradiation time.
The singlet oxygen (1O2) generation ability of Por 1, Por 2 and Por 4 was studied with the
singlet-oxygen trap molecule 1,3-diphenylisobenzofuran (DPBF). DPBF is a good singlet
1
oxygen scavenger, which was widely used in the measurement of O2. The relative
consumption of the capture agent, that is, the degree of attenuation of the absorbance, reflects
the generation efficiency of singlet oxygen. As shown in Fig. 2, the absorbance of DPBF
displayed a time-dependent decrease in reaction with singlet oxygen, generated from

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porphyrins when irradiated with a LED lamp. Using tetraphenylporphyrin (TPP) as a
reference, the singlet oxygen quantum yield of Por 1, Por 2, and Por 4 was calculated to be
0.72, 0.85 and 0.88, respectively. Under the same condition, the absorbance intensity of
DPBF that mixed with Por 1 decreased more compared with that of tetraphenylporphyrin after
irradiation for 60 s, suggesting the methoxyporphyrin had the higher singlet oxygen quantum
yield, probably due to the better biocompatibility. In addition, since the capacity of
photosensitizer to generate 1O2 is dependent on the efficiency of the intersystem crossing (ISC)
from 1PS* to 3PS*, the platinated porphyrins (Por 2 and Por 4) showed higher singlet oxygen
quantum yield related to that of Por 1, due to the heavy-atom effect of metal platinum.
FR-mediated targeting for HeLa Cells
Fig. 3 Laser scanning confocal microscopy images (excited at 488 nm laser) of HeLa and A549
cells incubated with Por 4 at a concentration of 2.0 µM in the cell culture medium at 37 °C for 4 h.
(The scale bar is 20 μm)
Next, we evaluated the targeting ability of Por 4, where the sample was incubated with
FR-positive cell lines (HeLa cells) and FR-negative cell lines (A549 cells) at 2.0 µM, respectively,
and then analyzed by confocal fluorescence imaging. As shown in Fig. 3, the HeLa cells (the first
row of Fig. 3) displayed strong red fluorescent signals after 4 h of incubation with Por 4. In
contrast, very weak red fluorescent signals were observed in A549 cells (the second row of Fig. 3)
under the same condition, indicating that Por 4 could target specifically the cancer cells with
overexpression of FR via FR-mediated endocytosis.
Subcellular localization

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Fig. 4 Laser scanning confocal microscopy images (excited at 488 nm laser) of HeLa cells
incubated with Por 4 for 4 h. Lysosomes (green) were stained with LysoTracker Green (50 μM).
The nucleus (blue) was stained with Hoechst 33342 (1 μg/mL). (The scale bar is 20 μm).
To further figure out the subcellular localization of Por 4 in HeLa cells, LysoTracker Green and
Hoechst staining was applied to visualize cell lysosomes (green) and nucleus (blue), respectively.
As shown in Fig. 4, the red emission from Por 4 is almost overlapped with that of green
fluorescence of LysoTracker Green, demonstrated the conjugate mainly entered the lysosomes of
cancer cells, probably ascribed to the endocytosis mediated by folate receptor.
In vitro dark cytotoxicity and photocytotoxicity
120
100
80
60
40
20
0
120
100
80
60
40
20
0
Dark
Irradiration
Dark
Irradiation
a
b
1
2
1
2
5
10
5
10
0
20
0
20
Concentration (M)
Concentration (M)
Fig. 5 HeLa (a), and A549 (b) cells viability at different concentrations (0, 1, 2, 5, 10, and 20 μM)
of Por 4 for 4 h at 37 °C without or with irradiation for 30 min.
HeLa and A549 cells were utilized to evaluate the in vitro cytotoxicity of Por 4 via MTT assays.
As shown in Fig. 5, Por 4 exhibited minor cytotoxicity toward HeLa and A549 cells in the dark
with the increase of Por 4 from 0 to 20 μM. The surviving fractions of two cancer cells was both
over 90% with the incubation of 20 μM Por 4, demonstrating that Por 4 had favorable
biocompatibility with cells in all testing concentrations. Meanwhile, the photocytotoxicity of the
synthesized porphyrin was also evaluated by a similar protocol. Obviously, Por 4 showed the
significantly therapeutic efficacy against HeLa cells in a dose-dependent manner (Fig. 5b), with

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the half maximal inhibitory concentration (IC50) about 5.78 μM upon irradiation. When the
concentration of sample was evaluated to 20 μM, the surviving fractions of Hela cells were only
22.25 ±4.75%, while the cell survival rate of A549 cells was as high as 75.25 ±4.75%, further
indicating the selectively accumulation of Por 4 in cancer cells high expression of folate
receptors.[31] These results clearly confirmed that the synthesized Por 4 with active targeting group
could be used as an efficient photosensitizer for cancer therapy.
Conclusions
In summary, we have synthesized and characterized a new photosensitizer (Por 4), where the
methoxyporphyrin (Por 2) was conjugated with folic acid using ethylenediamine as a linker. Por 4
exhibited high efficiency of singlet oxygen generation because of the heavy atom effect. Moreover,
due to the conjugation of folic acid, Por 4 could target specifically the cancer cells with
overexpression of FR (HeLa cells) via FR-mediated endocytosis. Accordingly, Por 4 can
selectively accumulate in HeLa cells, leading to a remarkable photodynamic therapeutic effect
under irradiation. Thus, the synthesized Por 4 with good biocompatibility, remarkable
photodynamic therapeutic effect and tumor-targeting properties, could be used as a potential
photosensitizer for cancer with overexpresssion of folate receptor.
Experimental sections
Materials and Instruments
All chemicals were of reagent-grade, purchased from Sigma-Aldrich and used without further
purification. All analytical-grade solvents were dried by standard procedures, distilled and
deaerated before use. 1H NMR spectra were recorded on a Varian Mercury-VX 300 spectrometer.
The UV-vis spectra were carried out on a UV-vis Spectrophotometer (Shimadzu). The
fluorescence spectra were performed on a PE LS55 fluorescence spectrometer. The chemical
shifts were referenced to tetramethylsilane, TMS (d = 0.00). Mass spectra, reported as m/z, were
obtained either on a Bruker Autoflex MALDI-TOF mass spectrometer or a Finnigan TSQ 710
(FAB-MS) mass spectrometer.
Synthesis of carboxyl acid porphyrin (Por 1)

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Por 1 was synthesized according to the method reported in the literature with minor
modification. [32] To a 250 mL three-neck flask, 3, 4, 5-trimethoxybenzaldehyde (2.94 g ，15 mmol),
4-formylbenzoic acid (0.76 g, 5 mmol) and 180 mL propionic acid were added. The resulting
solution was then heated to reflux, followed by the addition of freshly distilled pyrrole (1.92 mL,
20 mmol) within 15 min. After refluxing for another 1 h, the solvent was evaporated in vacuo. The
residue was dissolved in CH2Cl2 and extracted with saturated aqueous NaHCO3 solution until the
organic layer was neutral. The organic layer was then concentrated and the residue was purified by
silica gel column chromatography (MeOH in CH2Cl2, 0-1% v/v) to get the desired product, yield
10%. 1HNMR (400 MHz, CDCl3): δ (ppm) = 8.99 (d, 8H), 8.40 (d, 4H), 7.49 (s, 6H), 3.98 (t, 27H),
-2.79 (br s, 2H); MALDI-TOF MS: calcd for: 929.98, found: 929.28; UV-vis (DMF): λ= 422, 516,
552, 591, 648 nm.
Synthesis of platinum porphyrin complex (Por 2)
Por 1 (100 mg, 0.108 mmol) and potassium tetrachloroplatinate (89.7 mg, 0.215 mmol) were
dissolved in 15 mL benzonitrile. The mixture was then heated to reflux for 48 h. The completion
of metal insertion was verified by UV-vis spectroscopy and TLC. The solvent was evaporated in
vacuo and the residue was subjected to silica column chromatography (MeOH in CH2Cl2: 0-2%
v/v). The pure fraction was collected and concentrated to give the product 82 mg, yield 78%.
1HNMR (400 MHz, CDCl3): δ (ppm) = 8.83 (d, J = 7.8 Hz, 8H), 8.33 (d, J = 10.6 Hz, 4H), 7.40 (s,
6H), 3.82 (s, 27H); MALDI-TOF MS: calcd for: 1121.28, found: 1121.28; UV-vis (DMF): λ= 406,
511, 540 nm.
Synthesis of carboxyl-activated ester porphyrin (Por 3)
In the dark and under an inert atmosphere, Por 2 (30 mg, 0.027 mmol) was dissolved in a
mixture of 8 mL pyridine and 0.2 mL dichlorosulfoxide, After stirring at 50 °C in the dark for 30
min, N-hydroxysuccinimide (180 mg, 0.06 mmol) was added and then stirred for another 3 h. The
purification of the crude compound was performed using a silica gel column with CH2Cl2 as an
eluent. After recrystallization in CH2Cl2/hexane solutions, the desired product was obtained in
1
yield 73%. HNMR (400 MHz, CDCl3): δ (ppm) = 8.90 (d, J = 7.8 Hz, 6H), 8.68 (d, J = 4.9 Hz,
2H), 8.54 (d, J = 7.8 Hz, 2H), 8.32 (d, J = 8.0 Hz, 2H), 7.43 (s, 6H), 4.06 (s, 27H), 2.99 (s, 4H);
MS (ESI): m/z =1219 [M]+, 1026.9 [M-Pt+2H]+. Anal. Calcd for C58H49N5O13Pt:ꢀ C, 57.14; H,
4.05; N, 5.74. Found:ꢀ C, 57.19; H, 4.02; N, 5.78.

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Synthesis of ethyl folate (FA 3)
Folic acid (44.1 mg, 1.0 mmol) was activated with N,N'-dicyclohexylcarbodiimide (DCC) (24.8
mg, 1.2 mmol) and N-hydroxysuccinimide (NHS) (23 mg, 2.0 mmol) in 30 mL of dimethyl
sulfoxide (DMSO) at 50 °C for 6 h, which was then reacted with ethylenediamine (60 mg, 10
mmol) with pyridine (15 μL) as a catalyst at room temperature. After 24 h, the precipitate formed
was removed by filtration. 20 mL diethyl ether was added to the filtrate to obtain the yellow solid,
which was then washed with 50 mL of acetone/diethyl ether solution (30:70% v/v ratio) and
diethyl ether (3×50 mL) to remove traces of unreacted reagents and diisopropylethylammonium
1
(NHS) salt. The product was dried in vacuum overnight and the yield was 60%. HNMR (400
MHz, CDCl3): δ (ppm) = 8.68 (s, 1H ), 8.15-7.99 (m, 1H), 7.7-7.52 (m, 2H), 7.3-6.8 (m, 3H),
6.63-6.5 (m, 2H), 4.45 (br s, 2H), 4.26 (m, 1H), 3.32-3.19 (m, 4H), 3.1 (m, 2H), 2.15-1.79 (m, 4H).
MS (ESI): m/z =483.73 [M+H]+.
Synthesis of folic acid-platinum porphyrin conjugates (Por 4)
To a 50 mL two-neck flask, FA 3 (42.85 mg, 0.089 mmol), Por 3 (15 mg, 0.029 mmol),
N,N-diisopropylethylamine (47 mg, 0.36 mmol) and DMF (0.7 mL) were added in the dark under
a nitrogen atmosphere. After stirring at ambient temperature for 24 h, the mixture was gradually
poured into a vigorously stirred solution of anhydrous Et2O (20 mL).The dark brown precipitate
obtained was collected by filtration, washed with Et2O and CH2Cl2 and dried under vacuum to get
8.5 mg product, yield 34%. 1H NMR (400 MHz, CDCl3) δ 8.86 (s, 6H), 8.73-8.63 (m, 3H), 8.22 (s,
6H), 8.01 (s, 1H), 7.53 (d, J = 9.6 Hz, 2H), 7.37 (d, J = 14.2 Hz, 4H), 7.00 (s, 3H), 6.59 (s, 2H),
4.69 (s, 2H), 4.67-4.63 (m, 1H), 4.09 (dd, J = 8.7 Hz, 27H), 2.96 (s, 2H), 2.88 (s, 2H), 2.23 (d, J =
8.4 Hz, 4H). MS (ESI): m/z =1122.39 {M-[H2CCH2-NH-FA]}+. Anal. Calcd for C75H69N13O15Pt:ꢀ
C, 56.74; H, 4.38; N, 11.47. Found:ꢀ C, 56.29; H, 4.31; N, 11.17. UV-Vis (DMF): λ= 406, 511,
540 nm.
UV-vis absorption and fluorescence spectra
The UV-vis absorption spectrum and fluorescence spectrum of four porphyrin conjugates were
measured in DMF solution. Fluorescence quantum yield (φf) of an unknown sample is calculated
by using Eq. (1)[33]
.

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I f
I Rf nR
n


2

 f  Rf
(1)


Where, Rf is the known fluorescence quantum yield of the reference sample (referred by the
superscript ‘R’); I f and I Rf are the integrated fluorescence intensities of the unknown sample
and reference sample, respectively; A and AR are the absorbance of the unknown sample and
reference sample, respectively; n and nR are the refractive indices of the solvents for the unknown
sample and reference sample, respectively. The fluorescence quantum yield of the samples are
measured at room temperature relative to that of TPP in THF solution (Rf = 0.052)[33]
.
Singlet oxygen detection
The production of singlet oxygen was established qualitatively using 1,3-
diphenylisobenzofuran (DPBF) as a singlet oxygen fluorescent probe. The absorbance change of
DPBF at 418 nm could be assigned to the signal of consumption of singlet oxygen. During the
experiment, the absorbance of the 1O2 scavenger (DPBF) was adjusted to around 1.0 in
air-saturated DMSO. The experiments were initially performed with about 45 μM DPBF. The
photosensitizer was added and its concentration was adjusted to around 1 μM. After measurements
were taken in the dark, the cuvette was exposed to LED lamp at the peak absorption wavelength
for 10 seconds. The absorbance was measured several times after each irradiation. Each of the
photosensitizers was irradiated 6 times. The singlet oxygen quantum yields of the synthesized
porphyrins was calculated using Eq. (2) with 5,10,15,20-tetraphenylporphyrin (TPP) as the
reference (R = 0.64).
kS F R
kR F S
S  R
(2)
R is the singlet oxygen quantum yield of the reference. Superscript S and R indicate the
sample and reference compound respectively, k is the plot slop of the change in absorbance of
DPBF (at 410 nm) with the irradiation time and F is the absorption correction factor, which is
given by F=1-10-OD. OD is the corresponding absorbance at irradiation wavelength.[34]
Cell viability assays

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DOI: 10.1039/C9OB00698B
The potentiality of Por 4 as photosensitizer was tested using human cervical cancer cell line
(HeLa cells) and human non-small cell lung cancer cell lines (A549 cells), where HeLa cells
highly expressed folate receptors while lowly expression in A549 cells . The cytotoxic activity of
the conjugate was evaluated using MTT assay. After treating the selected cell lines with Por 4 at
different concentrations (0, 1, 2, 5, 10, and 20 μM in DMEM) for 24 h at 37 °C, MTT assay was
performed to detect the surviving fraction of cells. HeLa cells and A549 cells were cultured in
DMEM (Dulbecco Modified Eagle Medium) supplemented with 5% FCS (fetal calf serum), 100
o
U/mL penicillin, 100 μg/mL streptomycin at 37 C and 6% CO2. HeLa cells were first seeded in
96-well plates for 24 h and then treated with sample overnight in the dark. Cytotoxicity was
determined by MTT reduction assay. The cell monolayer was washed twice with phosphate
buffered saline (PBS) and then incubated with 50 μL of MTT solution (0.5 mg/mL) at 37 oC for 3
h. After removing the medium, 100 μL of DMSO was added. The solution was shaken for 30 min
to dissolve the formed crystal in living cells. Absorbance was measured at a dual wavelength of
540 nm and 690 nm on a Labsystem Multiskan plate reader (Merck Eurolab, Switzerland). Each
dosing concentration was performed in triplicate wells and repeated twice for MTT assay.
The photocytotoxicity of samples in HeLa cells and A549 cells was assessed by a similar
protocol. Cells were first seeded in 96-well plates at 3× 103 cells per well for 24 h. The cells were
then treated with sample in the dark overnight. Afterwards, the cell was exposure to yellow light
(4 J/cm2) produced from a 400 W tungsten lamp fitted with a heat-isolation filter and a 500 nm
long-pass filter. The fluence rate was 6 mW/cm2. Cell viability was determined by the MTT
reduction assay.
FR-mediated targeting for HeLa cells
Laser scanning confocal microscope was applied to detect the FR-mediated targeting of the
sample for cancer cells. For comparison, A549 cells with low expression of FR and HeLa cells
with high expression of FR were selected for the biological studies, respectively.[35] Cancer cells
were seeded on 3.5 cm well with cover slips and then cultured in the DMEM medium (10%) for
two days. Afterward, cells were incubated with Por 4 at a concentration of 2.0 μM for 4 h at 37 °C
and 5% CO2. The cells were then washed three times with PBS solution before imaging on a
confocal microscope.
Subcellular localization

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HeLa cells were cultured in RPMI 1640 medium (GibcoTm) containing 10% fetal calf serum and
1% antibiotic penicillin and streptomycin (P/S), and cultured at 37 °C in a humidified 5% CO2
atmosphere. Cells (6 × 103 cells per well) were seeded in 6-well plates (with sterile coverslips in
each well) and incubated for two days at 37 °C in a humidified 5% CO2 atmosphere. After
renewal with the new medium, the cells were incubated with a photosensitizer at a concentration
of 1.0 μM for 4 h at 37 °C. After that, the supernatant was carefully removed and the cells were
washed three times with PBS. Subsequently, the slides were mounted and observed by a Zeiss
Laser Scanning Confocal Microscope (LSM7 DUO), and then analyzed using ZEN 2009 software
(Carl Zeiss).
DOI: 10.1039/C9OB00698B
Co-staining of porphyrin, LysoTracker Green and Hoechst 33342: HeLa cells were first
incubated with porphyrin at 37 °C for 4 h and then further incubated with LysoTracker Green (50
nM) and Hoechst 33342 (50 nM) for 10 min at 37 °C. The cells were washed three times with
PBS solution before imaging on a confocal microscope.
Acknowledgments
We are thankful for the support from National Natural Science Foundation of China (21601142),
Natural Science Foundation of Hubei Province (2017CFB689), Zhejiang Provincial Natural
Science Foundation of China (LY18B010002), and the 10th Graduate Innovative Fund of Wuhan
Institute of Technology (CX2018023).
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