Additive effect of heparin on the photoinactivation of Escherichia coli using tricationic P-porphyrins

Article abstract of DOI:10.1016/j.bmcl.2017.10.032

Polycationic porphyrins have received substantial attention in developing singlet oxygen-sensitizers for biological use such as in the photoinactivation of bacteria and photodynamic therapy (PDT) of tumor cells because they have strong binding affinities for DNA and proteins. However, these strong cellular interactions can retard elimination of the drug after PDT. Therefore, the studies on the interactions of porphyrins with other molecules present much interest, in order to modulate the sensitizers’ activity or even remove them from the human body after PDT. Here, we studied the additive effect of heparin on the photoinactivation by polycationic porphyrins using Escherichia coli as a model cell. Tricationic P-porphyrin sensitizers substituted with an N-alkylpyridinium group (alkyl = pentyl (1a), hexyl (1b), and heptyl (1c)) or N-hexylammonium (1d) as the axial ligand were used. Additionally, dicationic Sb-porphyrin substituted with an N-hexylpyridinium group (1e) was prepared. We studied the additive effect of heparin on the photoinactivation of E. coli by 1a–1e. The bactericidal activities were evaluated using the half-life (T_1/2 in min) of E. coli and the minimum effective concentrations ([P]) of the porphyrin sensitizers. In the absence of heparin, the [P] values were determined to be 0.4–0.5 μM for 1a?1c and 2.0 μM for 1d?1e. The bactericidal activity of 1a?1c was completely retarded by the addition of heparin (1.0 μM). However, the addition of heparin (1.0 μM) could not completely retard the bactericidal activity of 1d?1e whose [P] values were relatively large. It is suggested that tricationic 1a?1c adsorbed onto the anionic heparin through electrostatic interactions. The adsorption of 1 on heparin disturbs the uptake of 1 into E. coli cells. Thus, the addition of heparin was found to be a useful method for retarding photoinactivation.

Full text of DOI:10.1016/j.bmcl.2017.10.032

Accepted Manuscript
Additive effect of heparin on the photoinactivation of Escherichia coli using
tricationic P-porphyrins
Jin Matsumoto, Kou Suzuki, Hidekazu Uezono, Kaho Watanabe, Masahide
Yasuda
PII:
S0960-894X(17)31017-X
https://doi.org/10.1016/j.bmcl.2017.10.032
BMCL 25361
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 June 2017
12 October 2017
15 October 2017
Please cite this article as: Matsumoto, J., Suzuki, K., Uezono, H., Watanabe, K., Yasuda, M., Additive effect of
heparin on the photoinactivation of Escherichia coli using tricationic P-porphyrins, Bioorganic & Medicinal
Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.10.032
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“2nd Revision”
Additive effect of heparin on the photoinactivation of Escherichia coli using tricationic
P-porphyrins
Jin Matsumoto, Kou Suzuki, Hidekazu Uezono, Kaho Watanabe, Masahide Yasuda*
Department of Applied Chemistry, Faculty of Engineering, University of Miyazaki,
Gakuen-Kibanadai, Miyazaki 889-2192, Japan
Keywords: heparin; polycationic P-porphyrin; Escherichia coli; photoinactivation
Abstract
Polycationic porphyrins have received substantial attention in developing singlet
oxygen-sensitizers for biological use such as in the photoinactivation of bacteria and
photodynamic therapy (PDT) of tumor cells because they have strong binding affinities for
DNA and proteins. However, these strong cellular interactions can retard elimination of the
drug after PDT. Therefore, the studies on the interactions of porphyrins with other molecules
present much interest, in order to modulate the sensitizers` activity or even remove them
from the human body after PDT. Here, we studied the additive effect of heparin on the
photoinactivation by polycationic porphyrins using Escherichia coli as a model cell.
Tricationic P-porphyrin sensitizers substituted with an N-alkylpyridinium group (alkyl =
pentyl (1a), hexyl (1b), and heptyl (1c)) or N-hexylammonium (1d) as the axial ligand were
used. Additionally, dicationic Sb-porphyrin substituted with an N-hexylpyridinium group
(1e) was prepared. We studied the additive effect of heparin on the photoinactivation of E.
coli by 1a-1e. The bactericidal activities were evaluated using the half-life (T1/2 in min) of E.
coli and the minimum effective concentrations ([P]) of the porphyrin sensitizers. In the

absence of heparin, the [P] values were determined to be 0.4 - 0.5M for 1a−1c and 2.0 M
for 1d−1e. The bactericidal activity of 1a−1c was completely retarded by the addition of
heparin (1.0 M). However, the addition of heparin (1.0 M) could not completely retard the
bactericidal activity of 1d−1e whose [P] values were relatively large. It is suggested that
tricationic 1a−1c adsorbed onto the anionic heparin through electrostatic interactions. The
adsorption of 1 on heparin disturbs the uptake of 1 into E. coli cells. Thus, the addition of
heparin was found to be a useful method for retarding photoinactivation.
---------------------------------------------------------------------------------------------------------------
Porphyrins have received substantial attention in developing singlet oxygen sensitizers
for the photoinactivation of bacteria1 and for the photodynamic therapy (PDT) of tumor cells.2
In particular, it is believed that polycationic porphyrins are promising 1O2-sensitizers, because
they are water-soluble and have strong binding affinities for DNA3-5 and proteins6.
Polycationic porphyrins can effectively photoinactivate even gram-negative bacteria (e.g.,
Escherichia coli) that have an impermeable barrier to antimicrobial agents.7 Therefore, the
studies on the interactions of porphyrins with other molecules present much interest, in order
to modulate the sensitizers' activity or even remove them from the human body after PDT.
Herein, we studied the additive effect of heparin on the photoinactivation by polycationic
porphyrins using Escherichia coli as a model cell.
Heparin (Scheme 1), a naturally occurring polysaccharide, is often incorporated into
cancer therapy in view of its unique biological and physicochemical properties such as anti-
inflammation, anti-angiogenesis, and anti-tumor cell proliferation.8 Huh et al. have
extensively prepared heparin-photosensitizer conjugates to control the activity of the
sensitizer and develop a new drug delivery system.9-11 The abundance of sulfate and
carboxylate groups endow heparin with high negative charge, mediating its electrostatic

interaction with cationic molecules. As a new functionality of heparin, we studied the
scavenging effect of anionic heparin on the photoinactivation of E. coli by polycationic
porphyrins.
Scheme 1. General structure of heparin
We selected tricationic P-porphyrins (1a-1c) bonded with alkylpyridinium groups
through axial hydroxo-ligands of P(tpp)(OH)2 complex (tpp = tetraphenylporphyrinato ligand).
The structures of bis[3-(1-alkyl-4-pyridinio)-1-propoxo]-5,10,15,20-tetraphenylporphyrinato-
phosphorus (V) dibromide, chloride (1a-1c) are shown in Scheme 2. These P-porphyrins
were water-soluble and their water-solubility (CW) values are shown in Table 1. These axially
alkylpyridinium-substituted P-porphyrins have already been successfully applied to the
inactivation of Saccharomyces cerevisiae12 and E. coli13 under visible-light irradiation. S.
cerevisiae was photoinactivated in concentrations that were lower than those in the cases of E.
coli: the effective minimum concentration of 1b was found to be 40 nM for S. cerevisiae and
500 nM for E. coli. Therefore, in the present study, E. coli was selected as a model cell in
order to examine the additive effect for porphyrin in higher concentration.
Scheme 2 Tricationic P-porphyrins (1a-1c) bound axially to the alkylpyridinium group

First, 1 was subjected to the photoinactivation of E. coli in the absence of heparin. E. coli
K-12 (IFO 3335) was cultured aerobically at 30 °C for 8 h in a basal medium (pH 6.5).13 After
the centrifugation of the cultured broth, the harvested cells were washed with physiological
saline (NaCl, 7 g L−1) and then the E. coli was suspended in physiological saline. The cell
concentration was determined by turbidity (absorbance) at 600 nm. A typical procedure for
the photoinactivation of E. coli using 1b in the absence of heparin is described as follows:13
An L-type glass tube containing E. coli cells (2 × 104 cell mL−1) and 1b (0.5 M) in
phosphate buffer solution (0.1 M, pH 7.6, 10 mL) was set on a reciprocal shaker and shaken
at 160 rpm at room temperature under dark conditions for 2 h to adsorb 1b onto the E. coli
cells. Irradiation was performed using a fluorescent lamp (= 400–723 nm, 10.5 W cm–2,
maximum intensity: 545 nm) for 2 h at room temperature under aerobic conditions with
shaking. Aliquots (0.1 mL) of the reaction mixture were taken from the L-type glass tube at
20-min intervals and plated on a solid agar medium. The amount of living cells (B) in colony
formation unit (CFU mL−1) was defined as the average number of E. coli colonies that
appeared after incubation for 24 h at 30 °C in three replicate plates.
Figure 1 shows the time−course plots of the survival ratio (100B/B0) where B0 is the
initial amount of E. coli. The survival ratio gradually decreased as irradiation time increased.
The bactericidal activity of 1b was evaluated by the half-life (T1/2 in min), which was the time
required to reduce B to 0.5 B0. The T1/2 was determined from the slope in the time−course
plots. Moreover, the minimum concentrations of the porphyrins ([P]) were adjusted so that
the T1/2 had values between 20 and 120 min. Thus, the [P] and T1/2 values for 1b were
determined to be 0.5 M and 27 min, respectively. Similarly, the [P] and values T1/2 were
determined for 1a and for 1c, as shown in Table 1. The survival plots in the absence of
heparin are shown in the Supporting Information. Details of the synthesis of 1a, which was

+
prepared from P(tpp)Cl2 Cl− according to the preparation method described in a previous
report,14 are also described in the Supporting Information.
Table 1. Additive effect of heparin on the photoinactivation of E. coli by polycationic
P-porphyrins (1a-1e) a)
1
1a
3.8
0.5
29
>120
1b
5.8
1c
6.0
1d
6.1
2.0
43
1e
5.2
2.0
24 e)
73
CW/mM b)
[P] /Mc)
T1/2 /min d)
0.5
0.4
[Heparin]/M = 0.0
[Heparin]/M = 1.0
27 e)
>120
26 e)
>120
65
a) Photoinactivation was performed in buffer solutions (10 mL) containing E. coli
(1×104 cell mL–1), heparin (0 and 1.0 M), and 1 (0.4, 0.5, and 2.0 M) under
irradiation from a fluorescent lamp.
b) CW = Water solubility in mM. The CW values of 1b, 1c, and 1e were obtained from
Ref 14.
c) [P] = Minimum effective concentration of 1 in M
d) T1/2 = Half-life in min.
e) Data was obtained from Ref 13.
120
100
80
60
40
20
0
0
20 40 60 80 100 120
Irradiation time/min
Figure 1. Additive effect of heparin on the photoinactivation of E.coli using 1b (0.5M).
[Heparin] = 0.0 (○), 0.50 (△), and 1.0 M (▲).
To examine the additive effect of heparin, the photoinactivation of E. coli by 1 was
performed in the presence of various heparin concentrations. The concentration of 1 was
adjusted to [P]. A typical procedure for a run using 1b (0.5 M) and heparin (1.0 M) is

described as follows: An aqueous heparin solution (500 M) was prepared by dissolving 50
mg of sodium heparin (average MW 2.0 × 104, Wako Chemicals, Japan) in water (5 mL). The
aqueous heparin solution (500 M, 80 L) was mixed with an aqueous solution of 1b (50 M,
400 L) in measuring flask; then, the volume was adjusted to 2.0 mL using phosphate buffer
(0.1 M, pH 7.6). The solution was allowed to stand for 12 h. The resulting 1b/heparin solution
(0.5 mL) and an E. coli suspension (2 105 cells mL−1, 1.0 mL) were mixed in phosphate
buffer (8.5 mL) in an L-type glass tube. The L-type glass tube containing E. coli cells (2 × 104
cells mL−1), 1b (0.5 M), and heparin (1.0 M) in buffer (10 mL) was shaken at 160 rpm
under dark conditions for 2 h and then irradiated with a fluorescent lamp for 2 h. Time−course
plots of survival ratios for photoinactivation by 1b in the presence of heparin (1.0M) were
overlaid onto Fig. 1. The T1/2 values were drastically changed from 27 min to >120 min due to
the addition of 1.0 M of heparin. Thus, the addition of 1.0 M of heparin completely
retarded the bactericidal activity of 1b. When 0.50 M of heparin was added, the T1/2 value
was determined to be 101 min. The addition of 0.50 M of heparin increased the T1/2 value
but did not completely retard it. Therefore, the additive effects of heparin towards 1a and 1c
were examined using 1.0 M of heparin. Survival plots in the presence of heparin are shown
in the Supporting Information. It was found that the T1/2 values in the presence of heparin (1.0
M) were > 120 min and that they were larger than the cases without heparin. Thus, the
bactericidal activity of tricationic 1a−1c was markedly retarded by the addition of 1.0 M of
heparin.
Moreover, the additive effect of heparin was applied to other types of polycationic
P-porphyrins (Scheme 3): tricationic bis[3-(hexyldimethylammonio)-1-propoxo]-5,10,15,20-
tetraphenylporphyrinatophosphorus (V) trichloride (1d) and dicationic -(methoxo)-β- [3-(1-
hexyl-4-pyridinio)-1-propoxo]-5,10,15,20-tetraphenylporphyrinatoantimony (V) dibromide
(1e). The details of the preparation and the spectral data of 1d and 1e are shown in the

Supporting Information. The survival plots of 1d and 1e are shown in Fig. 2A and 2B,
respectively. In the absence of heparin, the [P] values of 1d and 1e were determined to be 2.0
M. The [P] and T1/2 values are summarized in Table 1. In the cases of 1d and 1e in the
presence of heparin (1.0 M), the T1/2 values were 65 and 73 min, respectively. The addition
of heparin could not prolong the T1/2 of 1d and 1e to >120 min, despite the addition of 1.0 M
of heparin completely retarding the bactericidal activity with 1a-1c.
Scheme 3.Tricationic P-porphyrin bound axially to ammonium (1d) and dicationic
Sb-porphyrin (1e).
(A) 1d (2.0 M)
(B) 1e (2.0 M)
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0 20 40 60 80 100120
Irradiation time/min
0 20 40 60 80 100120
Irradiation time/min
Figure 2. Additive effect of heparin on the photoinactivation of E.
coli using 1d (A) and 1e (B). [Heparin] = 0.0 (○) and 1.0 M (▲),
[1] = 2.0M.
The interaction of porphyrin with heparin was examined via monitoring the porphyrin
absorption spectral changes after the addition of heparin. The peak width (HW) at half height
of the Soret band was measured at 415 nm after the addition of aqueous heparin solution (0 −

20 nM) to an aqueous solution of tricationic 1b (0.5 M) and dicationic 1e (0.5 M). Figure
3 shows the plots of HW against the concentration of heparin. The addition of heparin caused
6 nm of HW broadening for 1b (from 19 nm to 25 nm). The HW broadening was due to the
interaction of heparin with 1b.15 Therefore, it is suggested that tricationic 1b was adsorbed
onto anionic heparin and this intervened between the aggregation of heparins to each other
through electrostatic interactions. The adsorption onto heparin induced aggregation of 1b,
resulting in a broadening of the HW. In the case of 1e, broadening of the HW from 13 to 19
nm occurred. However, the spectral change of 1e was more gentle compared with tricationic
1b, suggesting that the electrostatic force of dicationic 1e was weak for heparin aggregation.
It is suggested that the adsorption of 1 onto heparin disrupts the uptake of 1 into E. coli cells.
26
24
22
20
18
16
14
12
10
0 2 4 6 8 10 12 14 16 18 20
[heparin]/nM
Figure 3. Dependence of peak width at half height (HW) of the Soret band on heparin
concentration (0-20 nM) in the absorption spectra of 1b (0.5 M, ○) and 1e (0.5 M, ▲).
The incorporation of porphyrins inside E. coli was confirmed from fluorescence
microscopy images as follows: A phosphate buffer solution containing 1b (1 M), E. coli (ca.
2  105 cell mL−1), and heparin (0 and 1 M) was left to stand for 2 h at 25 °C. The mixture (1
mL) was centrifuged for 15 min at 12,000 rpm and the supernatant solution (0.9 mL) was
removed to obtain a sample solution. The sample solution (2 L) was placed on an agar

pad13,16 set on a cover glass (24 mm 50 mm) and covered with another cover glass (18 mm
18 mm). Fluorescence microscopy images were obtained with an Olympus FV-300 confocal
laser scanning microscope (CLSM) equipped with a 60 water immersion objective lens
(numerical aperture = 1.2) under laser excitation at 543 nm. As shown in Fig. 4A, a
fluorescence image depicting the emission of 1b was observed inside E. coli in the absence of
heparin. It was found that 1b was incorporated inside the cell in the absence of heparin. On
the contrary, in the presence of heparin (1 M), fluorescence from within cells was not
observed, as shown in Fig. 4D. Therefore, in the presence of heparin, 1b was not incorporated
within the cells. These observations demonstrate that the aggregation of 1b with heparin
disturbs the uptake of 1b into E. coli. Thus, photoinactivation was retarded via the addition of
heparin.
(A)
(B)
(C)
(D)
(E)
(F)
Figure 4. Fluorescence images of E. coli with the addition of 1b. These images were obtained
with a CLSM under laser-excitation at 543 nm. Fluorescence from within the cells was
observed in the absence of heparin (A) but not observed in the presence of heparin (D).
Transmission images of E. coli containing 1b (E) and 1b and heparin (B). The image in C was

obtained by overlapping images A and B. The image in F was obtained by overlapping
images D and E.
In conclusion, we succeeded to control the bactericidal reactivity of the polycationic
porphyrins through the addition of heparin. The additive effect of heparin to photoinactivation
using various porphyrins is currently underway in our laboratories. In a future, the studies on
the use of heparin for the controlled release and activity modulation will continue. In addition,
we will study the possible use of heparin for elimination of the sensitizer from a human body
after PDT. These studies will be reported in due course.
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Research (C) (16K05847) from
the Japan Society for the Promotion of Science (JSPS).
References
1. Hamblin MR, Hasan T. Photodynamic therapy: A new antimicrobial approach to
infectious disease?, Photochem. Photobiol. Sci., 3 (2004) 436–450.
2. Alves E, Faustino MAF, Neves MGPMS, Cunha T, Nadais H, Almeida A. Potential
applications of porphyrins in photodynamic inactivation beyond the medical scope, J.
Photochem. Photobiol., C, 22 (2014) 34–57.
3. Trommel JS, Marzilli LG. Synthesis and DNA binding of novel water-soluble cationic
methylcobalt porphyrins, Inorg. Chem., 40 (2001) 4374–4383.
4. Haeubl M, Reith LM, Gruber B, Karner U, Müller N, Knör G, Schoefberger W. DNA
interactions and photocatalytic strand cleavage by artificial nucleases based on
water-soluble gold(III) porphyrins, J. Biol. Inorg. Chem., 14 (2009) 1037–1052.

5. Tada-Oikawa S, Oikawa S, Hirayama J, Hirakawa K, Kawanishi S. DNA damage and
apoptosis induced by photosensitization of 5,10,15,20-tetrakis
(N-methyl-4-pyridyl)-21H,23H-porphyrin via singlet oxygen generation, Photochem.
Photobiol., 85 (2009) 1391–1399.
6. Gyulkhandanyan A, Gyulkhandanyan L, Ghazaryan R, Fleury F, Angelini M,
Gyulkhandanyan G, Sakanyan V. Assessment of new cationic porphyrin binding to
plasma proteins by planar microarray and spectroscopic methods, J. Biomol. Struct. Dyn.,
31 (2013) 363–375.
7. Banfi S, Caruso E, Buccafurni L, Battini V, Zazzaron S, Barbieri P, Orlandi V.
Antibacterial activity of tetraaryl-porphyrin photosensitizers: An in vitro study on Gram
negative and Gram positive bacteria, J. Photochem. Photobiol., B, 85 (2006) 28–38.
8. Yang X, Du H, Liu J, Zhai G. Advanced nanocarriers based on heparin and its derivatives
for cancer management, Biomacromolecules,16, 423-436 (2015)
9. Li L, Cho H, Kim S, Kang HC, Huh KM, Polyelectrolyte nanocomplex formation of
heparin-photosensitizer conjugate with polymeric scavenger for photodynamic therapy,
Carbohydrate Polymers, 121, 122-131 (2015)
10. Li L, Bae B-C, Tran TH, Yoon KH, Na K, Huh KM, Self-quenchable biofunctional
nanoparticles of heparin-folate- photosensitizer conjugates for photodynamic
therapy,Carbohydrate Polymers, 86, 708-715 (2011)
11. Tran TH, Bae B-C, Lee Y-K, Na K, Huh KM. Heparin-folate-retinoic acid bioconjugates
for targeted delivery of hydrophobic photosensitizers, Carbohydrate Polymers, 92,
1615-1624 (2013).
12. Matsumoto J, Kai Y, Yokoi H, Okazaki S, Yasuda M. Assistance of human serum
albumin to photo-sensitized inactivation of Saccharomyces cerevisiae with axially
pyridinio–bonded P-porphyrins, J. Photochem. Photobiol. B: Biol. 161, 279-283 (2016)

13. Matsumoto J, Suemoto Y, Kanemaru H, Takemori K, Shigehara M, Miyamoto A, Yokoi
H, Yasuda M. Alkyl substituent effect on photosensitized inactivation of Escherichia coli
by pyridinium-bonded P-porphyrin, J. Photochem. Photobiol. B: Biol. 168, 124-131
(2017).
14. Matsumoto J, Kubo T, Shinbara T, Matsuda N, Shiragami T, Fujitsuka M, Majima T,
Yasuda M. Spectroscopic analysis of the interaction of human serum albumin with
tricationic phosphorus porphyrins bearing axial pyridinio groups, Bull. Chem. Soc. Jpn.,
86 (2013) 1240–1247.
15. Matsumoto J, Tanimura S, Shiragami T, Yasuda M. Water-solubilization of
alkyloxo(methoxo)porphyrinatoantimony bromides, Physical Chemistry Chemical
Physics, 11, 9766-9771 (2009).

Highlights
* Heparin could strongly interact with polycationic P-porphyrins through Coulombic
interactions.
* Heparin (1.0 M) could retard the bactericidal activity of tricationic P-porphyrins.
* This is an interesting finding on the scavenging effect of heparin on the photoinactivation of
E. coli.