Correlations between functional porphyrin positions and accumulation in cancer cells

Article abstract of DOI:10.1016/j.bmcl.2020.127437

Porphyrin is accumulated in tumours due to its interaction with protein. Cancer therapy with porphyrin as a carrier molecule is attracting attention. Porphyrin displays two functional sites termed β- and meso-positions. A correlation between the functiona

Full text of DOI:10.1016/j.bmcl.2020.127437

Bioorganic&MedicinalChemistryLetters30(2020)127437
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Correlations between functional porphyrin positions and accumulation in
cancer cells
⁎
Toshifumi Tojoa, Koshi Nishidaa, Takeshi Kondoa,b, Makoto Yuasaa,b,
a Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
b Research Institute for Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Porphyrin is accumulated in tumours due to its interaction with protein. Cancer therapy with porphyrin as a
carrier molecule is attracting attention. Porphyrin displays two functional sites termed β- and meso-positions. A
correlation between the functional position on the porphyrin molecule and the ability to accumulate in cancer
cells is observed in the present study. The accumulation of porphyrin derivatives was determined by measuring
fluorescence intensity after incubation for 2 and 24 h. The accumulation of cancer cells depended on the position
and length of functional groups. Estimated binding constants between porphyrin and bovine serum albumin
suggest that the position of functional groups leads to changes in binding affinity and influences the accumu-
lation of porphyrin derivatives in cancer cells.
Porphyrin
Tumour accumulation
Fluorescence quenching
π-π stacking interaction
Currently, biocompatibility is an important issue for the identifi-
cation of chemicals with useful pharmacological properties.1 Bio-
compatibility can best be addressed by using compounds taken from
relevant organisms like mammals. An example is porphyrin, which is a
basic structure constituent of heme. Porphyrin is composed of four
pyrrole subunits and has 18π-electrons that form a planer. Porphyrin
can form conjugates with metals (Fe, Ni, and Co)2–5 that define elec-
trochemical and oxidation–reduction characteristics. These character-
istics are measured to determine the amounts of active oxygen in an
organism.6,7 Porphyrin is also a focus for drug development because it
shows selective accumulation in tumours.8 This accumulation is due to
π–π stacking between the π-conjugated molecule structure of por-
phyrin, aromatic amino acid residues of blood proteins and by activated
endocytosis of proteins (LDL,9–11 albumin12,13 and transferrin14,15) fa-
cilitated by receptors activated in cancer cells. Anti-cancer drug therapy
is regarded as an effective method for cancer treatment. Still, side ef-
fects caused by toxicity in normal cells can be problematic, and drug
delivery systems (DDS) that transport drug selectively to cancer cells
are of particular importance. The accumulation of porphyrin in tumour
cells might be used in a DDS for anti-cancer agents.8,16 Porphyrin
photosensitivity can generate anti-cancer activity.17–19 Porphyrin is
excited by light irradiation, and active oxygen species are formed by
energy transfer to oxygen when porphyrin returns to ground. Active
oxygen species are toxic and can show anti-cancer activity.20 In addi-
tion, anti-cancer drugs and active oxygen species can act synergistically
to enhance anti-cancer effects.16,21 Thus, using porphyrin to carry anti-
cancer drugs may be an effective strategy for drug delivery. Several
studies have reported the accumulation of porphyrin in tumour cells
focusing on functional groups and conjugated metal.16,22,23 These re-
ports emphasised the importance of electrostatic interactions between
cell membranes and electric charge on functional groups. Porphyrin has
two functional sites, termed the β- and meso-positions (Fig. 1). Little is
known about the influence of these functional positions on porphyrin
accumulation in tumours. In this work, β- and meso-positions were used
to elucidate correlations between functional porphyrin positions and
accumulation in cancer cells. Understanding the influence of different
positions for tumour accumulation can help define steric effects and
lead to the discovery of new molecules with high tumour accumulation.
Four porphyrin derivatives were used in the current study (Fig. 1).
These compounds have a basic tetraphenylporphyrin structure. Tetra-
phenylporphyrin can be inexpensively obtained, and the introduction of
functional groups is relatively easy.16 The steric effect was explored
with different alkyl chains as functional groups (carbon number: 1 or
15). Each alkyl chain is introduced to tetraphenylporphyrin by an
amide bond.
The synthesis of test compounds β-P1 (1), β-P15 (2), meso-P1 (3)
and meso-P15 (4) are shown in Scheme 1. First, H2TPP (5) was con-
verted to compound 6 using copper nitrate trihydrate and sulphuric
acid.24 Compound 7 was then synthesised from compound 6 using re-
duction with SnCl2·2H2O and hydrochloric acid.25 Finally, compound 7
⁎ Corresponding author.
E-mail address: yuasa@rs.tus.ac.jp (M. Yuasa).
https://doi.org/10.1016/j.bmcl.2020.127437
Received 23 March 2020; Received in revised form 17 July 2020; Accepted 21 July 2020
Availableonline25July2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

T. Tojo, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127437
Fig. 1. Structures of β-P1 (1), β-P15 (2), meso-P1 (3) and meso-P15 (4).
provided β-P1 (1) and β-P15 (2) by using condensation with corre-
sponding carboxylic acid chlorides. Target compounds were purified by
chromatography on silica gel (CHCl3).26 The preparation of meso-P1 (3)
and meso-P15 (4) is also shown in Scheme 1. Nitration of compound 5
afforded compound 8 using sodium nitrate,27 and compound 8 was
treated with SnCl2·2H2O and hydrochloric acid to obtain compound
9.25 Then, meso-P1 (3) and meso-P15 (4) were synthesised using the
same conditions for obtaining β-P1 (1) and β-P15 (2), respectively.26 β-
P1 (1), β-P15 (2), meso-P1 (3) and meso-P15 (4) were purified by
chromatography on silica gel (CHCl3), and identities of the four por-
phyrin derivatives were confirmed with 1H-NMR, 13C NMR and ESI-MS.
The accumulation of β-P1 (1), β-P15 (2), meso-P1 (3) and meso-P15
(4) in cancer cells was determined using human breast cancer cells
(MCF-7). Cells were cultured in Dulbecco’s Modified Eagle Medium
containing 10% heat-inactivated fetal bovine serum, penicillin and
streptomycin without phenol red. MCF-7 cells (5.0 × 105 cells/2 mL/
dish) were dispensed into 6-well plates and cultured for 24 h at 37℃ in
a humidified atmosphere of 5% CO2 in air. Cells were treated with
porphyrins in DMSO solutions at a final concentration of 10 μM for a
short duration (2 h) and long duration (24 h) to evaluate time-depen-
dent changes. After incubation, cell lysates were adjusted with SDS
buffer, and the fluorescence intensity was measured to determine por-
phyrin accumulation. Fig. 2 shows the results for each porphyrin {β-P1:
3.3 × 10−2 μM (2 h), 4.3 × 10−2 μM (24 h); meso-P1: 4.6 × 10−2 μM
Fig. 2. Accumulation of test compounds against MCF-7 cells (β-P1, meso-P1, β-
P15 and meso-P15) against MCF-7 (A) incubation time: 2 h. (B) incubation time:
24 h.
(2 h),
5.8 × 10−2 μM
(24 h)
β-P15:
1.7 × 10−2 μM
(2 h),
1.7 × 10−2 μM (24 h); meso-P15: 3.0 × 10−2 μM (2 h), 4.4 × 10−2 μM
(24 h)}. β-P15 showed the lowest accumulation, and meso-P1 showed
the highest regardless of incubation time. The accumulation of meso-
P15 and β-P1 were similar (meso-P1 > meso-P15, β-P1 > β-P15).
Based on these results, the accumulation of porphyrin derivatives in
MCF-7 cells is likely to depend on the position of functional groups in
Scheme 1. Synthesis of β-P1 (1), β-P15 (2), meso-P1 (3) and meso-P15 (4).α (αReagents and conditions: (a) CH3COOH, (CH3CO)2O, Cu(NO3)2·3H2O, CHCl3, r.t., 16 h,
then concH2SO4, CH2Cl2, r.t., 20 min; (b) SnCl2·2H2O, HCl, 65 °C, 5 h, 46% over 3 steps; (c) corresponding carboxylic acid chloride, Et3N, CH2Cl2, r.t., 3 h, 34%-
quant; (d) CH3COOH, CCl3COOH, NaNO2, r.t., 2 h; (e) SnCl2·2H2O, HCl, 65 °C, 5 h, 50% over 2 steps; (f) corresponding carboxylic acid chloride, Et3N, CH2Cl2, r.t.,
3 h, 49%-quant.)
2

T. Tojo, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127437
Fig. 3. Fluorescence quenching of BSA treated with (A) β-P1 (B) meso-P1 (C) β-P15 (D) meso-P15.
Table 1
Binding parameters between BSA and test compounds.
Ksv (L mol−1
a
)
kq (L mol−1
Kα (L mol−1
c
)
Accumulation in
MCF-7d (μM)
b
s−1
)
β-P1
4.33 × 104
4.96 × 104
3.03 × 104
4.33 × 1012
4.96 × 1012
3.03 × 1012
4.09 × 1012
2.86 × 104
3.81 × 104
1.98 × 104
3.12 × 104
4.3 × 10−2
5.8 × 10−2
1.7 × 10−2
4.4 × 10−2
meso-P1
β-P15
meso-P15 4.09 × 104
a
Ksv: Stern–Volmer fluorescence quenching constant, b Kq: BSA biomolecular
quenching rate constant, c Ka: binding constant against BSA, d Incubation time:
24 h.
Meso-porphyrin derivatives showed high accumulation, which de-
creased with longer carbon chains. Porphyrin is π-electron rich mole-
cule, and it has been reported that porphyrin shows higher affinity for
albumin,12,13 and endocytosis of albumin is the primary pathway for
accumulation of porphyrin.28,29 The accumulation was caused by π–π
stacking between the π-conjugated molecule structure of porphyrin,
aromatic amino acid residues of blood proteins and by activated en-
docytosis of albumin facilitated by receptors activated in cancer cells.
Based on these results, we expected that binding affinity between al-
bumin and porphyrin affects the accumulation in cancer cells and tried
to calculate binding constants between bovine serum albumin (BSA)
and porphyrin.
Calculation binding constants between test compounds (meso-P1,
meso-P15, β-P1 or β-P15) and BSA use Stern–Volmer plots.30 Porphyr-
in–BSA complexes are taken up by cancer cells using endocytosis.8 The
use of Stern–Volmer plots for measuring interactions is called ‘fluores-
cence quenching’. BSA fluorescence is excited at 280 nm, while por-
phyrin does not. When a fluorescent compound forms a ground-state
complex with a non-fluorescent compound, the fluorescence intensity
of the complex is decreased and can be used to calculate the binding
Fig. 4. (A) Stem-Volumer plots and (B) Leher plots for the quenching of BSA
treated with test compounds (β-P1, meso-P1, β-P15 and meso-P15).
porphyrin and the lengths of alkyl chains. Focusing on the functional
position, meso-porphyrin derivatives (meso-P1 and meso-P15) showed
higher accumulation than β-porphyrin derivatives (β-P1 and β-P15).
Especially, the accumulation of meso-P15 was three times higher than
β-P15. Thus, longer alkyl chains exaggerate differences in the accu-
mulation of porphyrin derivatives.
constant of the complex. The addition of
a
porphyrin solution
decrease in
(0.80 mM) to BSA solution (9.27 μM) causes
a
a
3

T. Tojo, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127437
fluorescence due to the aromatic amino acid residue of BSA (Fig. 3).
Using these results, Stern–Volmer plots were generated using the
Stern–Volmer Eq. (1):
Acknowledgments
The authors would like to thank Prof. Shin Aoki for the technical
support.
F0
F
= Ksv [Por] + 1
This research did not receive any specific grant from funding
agencies in public, commercial or not-for-profit sectors.
(1)
where F0 and F are BSA fluorescence intensities in the absence and
presence of porphyrin; Ksv is the Stern–Volmer fluorescence quenching
constant; [Por] is the concentration of porphyrin. The Stern–Volmer Eq.
(1) was incorporated into the Lehrer Eq. (2):
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127437.
F0
1
1
=
+
F0
F
Ka [Por]
(2)
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Declaration of Competing Interest
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The authors declare that they have no known competing financial
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