Effect of structural variation on tumor targeting efficacy of cationically charged porphyrin derivatives: Comparative in-vitro and in-vivo evaluation for possible potential in PET and PDT

Article abstract of DOI:10.1016/j.ejmech.2021.113184

Tetracationic (TMPyP) and tricationic porphyrin (TriMPyCOOHP) derivatives were synthesized, characterized and investigated for binding with DNA by Isothermal Titration Calorimetry as well as by UV–Vis spectroscopy in order to study the effect of structural variation on tumor targeting efficacy of cationically charged porphyrin derivatives. Fluorescence cell imaging studies performed in cancer cell lines corroborated the findings of aforementioned studies. Photocytotoxicity experiments in A549 cell lines revealed relatively higher light dependent cytotoxic effects exerted by TMPyP compared to TriMPyCOOHP. In-vivo experiments in tumor bearing animal model revealed relatively longer retention of ⁶⁸Ga-TMPyP in tumorous lesion compared to that of ⁶⁸Ga-TriMPyCOOHP. The study reveals that removal of one of the positive charges of the tetracationic porphyrin derivatives significantly reduces their DNA binding ability and cytotoxicity as well as brings changes in the pharmacokinetic pattern and tumor retention in small animal model.

Full text of DOI:10.1016/j.ejmech.2021.113184

European Journal of Medicinal Chemistry 213 (2021) 113184
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Effect of structural variation on tumor targeting efficacy of cationically
charged porphyrin derivatives: Comparative in-vitro and in-vivo
evaluation for possible potential in PET and PDT
Mohini Guleria a, Shishu K. Suman a, Jyotsna B. Mitra a, Sandeep B. Shelar b,
,
, d, *
Jeyachitra Amirdhanayagam a, Haladhar D. Sarma c, Ashutosh Dash a d, Tapas Das a
a Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085, India
b Chemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085, India
c Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085, India
d Homi Bhabha National Institute, Anushaktinagar, Mumbai, 400094, India
a r t i c l e i n f o
a b s t r a c t
Article history:
T
etracationic (TMPyP) and tricationic porphyrin (TriMPyCOOHP) derivatives were synthesized, charac-
Received 28 May 2020
Received in revised form
7 January 2021
Accepted 7 January 2021
Available online 16 January 2021
terized and investigated for binding with DNA by Isothermal Titration Calorimetry as well as by UVeVis
spectroscopy in order to study the effect of structural variation on tumor targeting efficacy of cationically
charged porphyrin derivatives. Fluorescence cell imaging studies performed in cancer cell lines
corroborated the findings of aforementioned studies. Photocytotoxicity experiments in A549 cell lines
revealed relatively higher light dependent cytotoxic effects exerted by TMPyP compared to TriMPy-
COOHP. In-vivo experiments in tumor bearing animal model revealed relatively longer retention of 68Ga-
TMPyP in tumorous lesion compared to that of 68Ga-TriMPyCOOHP. The study reveals that removal of one
of the positive charges of the tetracationic porphyrin derivatives significantly reduces their DNA binding
ability and cytotoxicity as well as brings changes in the pharmacokinetic pattern and tumor retention in
small animal model.
Keywords:
Cationic porphyrin
Radiolabeled porphyrin
Photocytotoxicity
PET
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
PDT and palliative treatment of various types of cancers such as,
oesophageal and non-small cell lung cancer [4e12]. Despite this,
Porphyrin and its derivatives have drawn considerable attention
for quite a long time owing to their interesting physicochemical and
biological properties and these macrocyclic molecules have found
applications in several fields such as catalysis, photo-sensitization,
molecular electronics and sensors in addition to their use as anti-
oxidants and anti-microbials [1e3]. Apart from these, certain
porphyrin derivatives have been found to be suitable for targeted
therapy of tumorous lesions [4e12]. A combination of photo-
sensitizing potential along with tumor localizing property of
porphyrin derivatives makes them suitable for use in photody-
namic therapy (PDT). US FDA (Food and Drug Administration of
United States of America) has approved the use of two porphyrin
derivatives namely, Foscan® and Photofrin® (porfimer sodium) for
wide-spread applications of the porphyrin derivatives for the
routine clinical exploitation are yet to be achieved.
One of the primary hurdles, which have deterred the pervasive
usage of these agents in clinical domain, is the lipophilicity of the
porphyrin core which results undue and non-specific accumulation
of these agents in several non-target organs such as, lungs, liver and
gastrointestinal tracks (GIT) [9e12]. Therefore, attempts have been
directed to design the suitable porphyrin derivatives having
reduced lipophilicity and consequently with enhanced hydrophi-
licity. This can be accomplished either by introducing hydrophilic
moieties in the peripheral region of the porphyrin skeleton or by
the introduction of positive or negative charge in the porphyrin
moiety [13e16]. As lipid bi-layer of cell membrane is negatively
charged, it is expected that an oppositely charged molecule will be
more suitable to find its way inside the cells [13e16]. Based on
these facts, an attempt was made to synthesize two cationically
charged porphyrin derivatives, one tetracationic and the other tri-
cationic, by the incorporation of pyridiniumyl moieties at meso
* Corresponding author. Radiopharmaceuticals Division, Bhabha Atomic Research
Centre, Trombay, Mumbai, 400085, India.
E-mail address: tdas@barc.gov.in (T. Das).
https://doi.org/10.1016/j.ejmech.2021.113184
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

M. Guleria, S.K. Suman, J.B. Mitra et al.
European Journal of Medicinal Chemistry 213 (2021) 113184
positions of the porphyrin structure.
generator [1.11 GBq, 30 mCi] purchased from ITG (Germany). C-18
reversed phase Sep-Pak® cartridges for purification of radiolabeled
porphyrin complexes were procured from Waters (India). The High
Performance Liquid Chromatography (HPLC) system (PU 1580)
used for the present study was obtained from JASCO (Japan). A C-18
reverse phase HiQSil (250 ꢀ 4 mm) column was used and the
elution profile was monitored by detecting the radioactivity signal
using a NaI(Tl) detector coupled with the HPLC system. All the
solvents used for HPLC analyses were of HPLC grade and filtered
prior to use. All radioactive countings, except the countings related
to the biodistribution studies, were performed using a well-type
NaI(Tl) scintillation counter, manufactured by Electronic Corpora-
tion of India Limited (India). The baseline of the detector was kept
at 450 keV and a window of 100 keV was used in order to utilize the
511 keV annihilation photo-peak of 68Ga.
All chemicals and reagents like MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide), sodium acetate, sodium
chloride, sodium bicarbonate, HEPES [(4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid)], DMEM (Dulbecco’s Modified Ea-
gle Medium) media, CT-DNA (Calf Thymus - Deoxyribo Nucleic
Acid) and trypan blue were procured from Sigma Chemical Inc.
(USA). DAPI (40,6-diamidino-2-phenylindole) was procured from
Merck (India). Fetal Bovine Serum (FBS) was purchased from GIBCO
Laboratories (USA). POLAR star Omega Plate Reader UVeVis Spec-
trophotometer, obtained from BMG LABTECH (Germany), was used
to measure the spectrometric readings. A549 cells were procured
from National Centre for Cell Sciences (India). For photocytotoxicity
experiments, cells were exposed to light generated by a set-up
which included LED (Light Emitting Diode) bulbs (9 W and 14 W,
Philips, India) as light source. The light doses received by the cells
were measured using an irradiance meter (Megger PVM210,
Taiwan) and are expressed as kJ/cm2. Cell imaging studies were
5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin
(TMPyP) is a well-studied tetracationic porphyrin derivative, with
four pyridiniumyl units, whose synthesis and characterization have
been previously reported by the authors in connection with their
study to develop a radiolabeled porphyrin derivative for PET
(positron emission tomography) imaging of tumorous lesions
[10,17]. During the course of present work, authors have synthe-
sized the same tetracationic porphyrin derivative along with a new
tricationic
porphyrin
derivative,
namely
5-
carboxymethyleneoxyphenyl-10,15,20-tris(1-methylpyridinium-4-
yl)porphyrin (TriMPyCOOHP) by replacing one of the pyridiniumyl
moieties by carboxymethyleneoxyphenyl group (HOOC-CH2-O-
C6H4-) in order to study the effect of structural modification as well
as variation of overall charge of the porphyrin derivatives on their
tumor affinity. Both the charge bearing porphyrin derivatives were
examined for their interaction with DNA (Deoxyribo Nucleic Acid),
if any, by ultra violet-visible (UVeVis) spectroscopy, isothermal
titration calorimetry (ITC) as well as by in-vitro fluorescence cell
imaging studies. Photocytotoxicity studies at two different light
doses were also conducted to evaluate the possible potential of
these derivatives in PDT. A comparison of in-vivo tumor targeting
efficacy of these two derivatives was carried out in Fibrosarcoma
tumor bearing Swiss mice model after radiolabeling the derivatives
with 68Ga. Gallium-68 [T1/2 ¼ 68 min, bþ
¼ 1.89 MeV (89%)] is a
(max)
generator produced radionuclide which is widely used for PET
imaging of various human ailments [18e21]. Availability from a
68Ge/68Ga radionuclide generator system as well as possibility of
multiple elutions of 68Ga in a day from this generator system are
the two prime factors responsible for the wide use of 68Ga for the
preparation of various radiotracers for PET imaging [18e21].
In the present study, our attempts were directed towards
comparing two structurally different cationically charged
porphyrin derivatives for their DNA targeting ability as well as tu-
mor affinity to understand the role played by the positive charge in
the structure of porphyrin moiety with an aim to shed light on the
notion that uptake of porphyrin derivatives is solely directed by
their lipophilic character.
carried out in
a Fluorescence Microscope (Olympus, Model:
B ꢀ 53F) at 10ꢀ magnification. ITC was performed with MicroCal
ITC200 system obtained from Malvern Instruments (UK).
Swiss mice (6e8 weeks age, 20e25 g weight) bearing fibrosar-
coma tumors were used as animal models for the biological studies.
The fibrosarcoma cell line used for raising the tumors in the ani-
mals was also purchased from National Centre for Cell Sciences
(India). All the animals used for the present study were bred and
reared in the laboratory animal house facility of our Institute
following the standard protocol. Radioactive countings associated
with the animal studies were performed using a flat-type NaI(Tl)
scintillation detector, procured from Electronic Corporation of India
Limited (India), employing the same counting set-up mentioned
earlier. The animal studies reported in the present article were
approved by the Institutional Animal Ethics Committee (IAEC) of
Bhabha Atomic Research Centre (BARC) and all the animal experi-
ments were carried out in strict compliance with the institutional
(IAEC) guidelines following the relevant national laws related to the
conduct of animal experimentation (Prevention of Cruelty to Ani-
mals Act, 1960).
2. Experimental
2.1. Materials and methods
4-Pyridinecarboxaldehyde,
tetrahydrofuran,
N,N0-dime-
thylformamide (DMF) and Di-phenylisobenzofuran (DPBF) were
procured from Sigma-Aldrich (USA). Ethyl bromoacetate, propionic
acid, nitrobenzene, iodomethane, dimethylsulphoxide (DMSO),
sodium hydroxide and anhydrous potassium carbonate were pro-
cured from S.D. Fine Chemicals (India). Thin Layer Chromatography
(TLC) was performed using silica gel 60 F254 coated aluminium
sheets, procured from Merck (India). Fourier Transform Infra-Red
(FT-IR) spectra were recorded in a Bruker Spectrophotometer
(Germany). UVeVis spectra were recorded using LABINDIA
Analytical (UV 3092) UVeVis Spectrophotometer, while fluores-
cence spectra were recorded using JASCO FP-8500 Spectrofluo-
rometer (Japan). Nuclear Magnetic Resonance (NMR) spectra were
acquired using VARIAN Mercury Plus 300 MHz NMR spectrometer
(USA) and Bruker 800 US, 800 MHz NMR spectrometer
(Switzerland). Mass spectra were recorded using Varian 410 Prostar
Binary LC Mass Spectrometer (USA) employing Electron Spray
Ionization (ESI) technique. MALDI-TOF (Matrix-Assisted Laser
Desorption/Ionization - Time Of Flight) Mass Spectrometry (MS)
was carried out using MALDI-TOF mass spectrometer (Ultra-
fleXtreme, Bruker, Germany).
All biological experiments were performed twice with each
sample in triplicates. All data are represented as mean standard
deviation. The differences between two groups were analyzed by
using the Student’s t-test. ANOVA was used for comparisons among
multiple groups to compare between different pair of treatments
for determining the significance value; p < 0.05 was considered to
be statistically significant.
2.2. Synthesis and characterization of 5,10,15,20-tetrakis(1-
methylpyridinium-4-yl)porphyrin (TMPyP) [2]
Tetracationic porphyrin derivative namely, 5,10,15,20-
tetrakis(1-methylpyridinium-4-yl)porphyrin (2) was synthesized
Gallium-68 was obtained from
a
68Ge/68Ga radionuclide
2

M. Guleria, S.K. Suman, J.B. Mitra et al.
European Journal of Medicinal Chemistry 213 (2021) 113184
by N-methylation of 5,10,15,20-tetra(4-pyridyl)porphyrin (1)
following the procedure reported earlier [10]. In brief, compound
(1) was synthesized by refluxing equimolar ratio of pyridine-4-
carboxyaldehyde (4.28 g, 40 mmol) and pyrrole (2.67 g, 40 mmol)
in presence of nitrobenzene (10 mL) using propionic acid (50 mL) as
solvent for a period of 2 h. The reaction mixture was allowed to
attain room temperature and residual solvents were removed by
vacuum distillation. The solid residue, thus obtained, was washed
with petroleum ether and subsequently purified by silica gel
chromatography using 0.5% methanol in chloroform as the eluting
solvent.
CH2-), 113.19, 116.94, 117.40, 119.03 (>C ¼ , methine bridges), 120.29
(m-Ar), 121.33 (o-Ar), 122.21 (b-pyrrole), 129.44 (p-Ar), 134.05,
134.87, 135.66 (b-pyrrole), 143.76, 148.21 (p-pyridine), 150.15,
150.62 (
a
-pyrrole), 158.06 (m-pyridine), 168.99 (o-pyridine), 184.21
(>C¼O). ESI-MS: C45H33N7O3 (calcd.) 719.79; MS (ESIþ): observed
[MþH] 720.9. MALDI-TOF: observed 720.285 [MþH].
2.3.2. Synthesis and characterization of 5-
carboethoxymethyleneoxyphenyl-10,15,20-tris(1-
methylpyridinium-4-yl)porphyrin [4]
Compound (4) was synthesized by room temperature stirring of
compound (3) (0.1 g, 0.13 mmol) with excess of CH3I using
dichloromethane as solvent. Post-reaction, the solvent was
removed by using a rotary evaporator and the residue left behind
was re-dissolved in dry DMF. The solution was stirred at room
temperature after the addition of excess CH3I for a period of 24 h.
Progress of the reaction was monitored by TLC using 15% MeOH in
CHCl3 (Rf ¼ 0.1). Finally, the solvent was removed by rotary evap-
orator following repetitive washings with ether. The purified
compound (4) was characterized by FT-IR, UVeVis, NMR spec-
troscopy and mass spectrometry. Compound (4): (yield ¼ 75 mg,
80%). UVeVis (MeOH, lmax nm, log e): 427 (4.05), 518 (3.89), 560
(3.84), 593 (3.49), 648 (3.25). FT-IR (MeOH, ῡ cmꢁ1): 3411.94
(MeOH), 3040.11, 1736.42, 1636.71, 1562.12, 1507.06, 1448.13,
Compound (1): (yield ¼ 450 mg, 2.4%). UVeVis (CHCl3, lmax
nm): 415, 512, 544, 587, 642. 1H NMR (CDCl3,
d
ppm): ꢁ2.92 (2H,
s, -NH), 8.18 (8H, d, J ¼ 4.5 Hz, m-pyridyl-H), 8.87 (8H, s, pyrrole-
H), 9.06 (8H, d, J ¼ 5.7 Hz, o-pyridyl-H). ESI-MS: C40H26N8
(calcd.) 618.69, MS (ESIþ): observed 619.8 [MþH]þ.
Compound (2): (yield ¼ 263 mg, 80%). UVeVis (H2O, lmax nm,
log e): 422 (4.39), 516 (3.59), 551 (3.41), 584 (3.40), 656 (3.29).
1H NMR (DMSO,
d
ppm): ꢁ3.10 (2H, s, -NH), 4.72 (12H, s, þN-
CH3), 9.00 (8H, d, J ¼ 6.6 Hz, m-pyridyl-H), 9.20 (8H, s, pyrrole-
H), 9.47 (8H, d, J ¼ 6.6 Hz, o-pyridyl-H). ESI-MS: C44H38N8
(calcd.) 678.83, MS (ESIþ): observed [M]4þ 169.1, [M-CH3]3þ
221.2, [M-2CH3]2þ 324.2, [M-3CH3]þ 633.2. MALDI-TOF:
observed 633.339 [M-3CH3]þ.
1321.40, 1216.69, 1184.58. 1H NMR (DMSO,
d
ppm): ꢁ3.01 (2H, s,
-NH),1.30 (3H, t, J ¼ 7.2 Hz), 4.27e4.30 (2H, q, J ¼ 7.2 Hz), 4.70 (9H, s,
N-CHþ3 ), 5.08 (2H, s), 7.02 (2H, s, pyrrole-H), 7.41e7.42 (2H, d,
J ¼ 8.0 Hz, m-Ar-H), 8.14e8.15 (2H, d, J ¼ 8.0 Hz, o-Ar-H), 8.39e8.40
(3H, d, J ¼ 6.4 Hz, pyridyl-H), 8.96e8.98 (6H, m, pyrrole-H),
9.17e9.18 (3H, d, J ¼ 6.4 Hz, pyridyl-H), 9.42e9.43 (6H, d,
2.3. Synthesis and characterization of 5-
carboxymethyleneoxyphenyl-10,15,20-tris(1-methylpyridinium-4-
yl)porphyrin (TriMPyCOOHP) [5]
J ¼ 6.4 Hz, pyridyl-H). 13C NMR (DMSO,
d ppm): 14.62 (-CH3), 48.48
Synthesis of 5-carboxymethyleneoxyphenyl-10,15,20-tris(1-
methylpyridinium-4-yl)porphyrin [5] was accomplished by
following a three-step reaction procedure mentioned below:
(CH3-Nþ), 61.39 (Ph-O-CH2), 65.50 (O-CH2-), 113.90, 114.83, 115.70
(>C ¼, methine bridges), 121.32 (m-Ar), 123.18 (o-Ar), 126.93 (
pyrrole), 132.60 (p-Ar), 133.65, 134.94, 136.02 ( -pyrrole), 144.65,
147.09 (p-pyridine), 150.71, 156.98, 158.45 ( -pyrrole), 162.91 (m-
b-
b
a
2.3.1. Synthesis and characterization of 5-
carboethoxymethyleneoxyphenyl-10,15,20-tris(4-pyridyl)porphyrin
[3]
pyridine), 169.36 (o-pyridine), 181.92(>C¼O). ESI-MS: C48H42N7O3
(calcd.) 764.89; MS (ESIþ): observed 254.9 [M]3þ, 374.7 [M-CH3]2þ
MALDI-TOF: observed 734.299 [M-2CH3]þ.
.
Compound (3) was synthesized by refluxing a mixture of pyrrole
(5.2 mL, 0.08 mol), 4-pyridinecarboxaldehyde (5.4 mL, 0.06 mol)
and p-carboethoxymethyleneoxybenzaldehyde (4 g, 0.02 mol) in a
molar ratio of 4:3:1, in presence of nitrobenzene (10 mL), using
propionic acid (40 mL) as solvent. p-carboethoxymethyleneox-
ybenzaldehyde (1a), used as one of the ingredients for the above-
mentioned reaction, was synthesized by following a procedure
reported in the literature [9]. The refluxing was continued for 2 h
and the reaction mixture was allowed to attain room temperature,
subsequent to which it was stored at 4 ꢂC overnight. The residual
solvent of the reaction mixture was removed by distillation. The
residue, thus obtained, was extracted in chloroform followed by
removal of the solvent using rotary evaporator. The desired product
was purified by carrying out repeated column chromatography
procedures using a maximum of 4% methanol in chloroform as the
eluting solvent (Rf ¼ 0.4). This resulted into the formation of
~250 mg of compound (3), which was characterized by FT-IR,
UVeVis, NMR spectroscopy and Mass spectrometry. Compound
(3): (yield ¼ 250 mg, 2%). UVeVis (CHCl3, lmax nm, log e): 414 (4.13),
511 (2.80), 545 (2.35), 588 (2.26), 643 (1.93). FT-IR (CHCl3, ῡ cmꢁ1):
3316.97, 2922.77, 1756.95, 1653.10, 1593.07, 1473.87, 1289.72,
2.3.3. Synthesis and characterization of 5-
carboxymethyleneoxyphenyl-10,15,20-tris(1-methylpyridinium-4-
yl)porphyrin [5]
Compound (5) was synthesized by alkaline hydrolysis of com-
pound (4), which was carried out by room temperature stirring of
compound (4) (0.05 g, 0.065 mmol) in a bi-phasic solvent medium
consisting of DMF and 2 N NaOH in 1:1 (v/v) ratio. The reaction was
magnetically stirred for 48 h at room temperature. Post-reaction,
the solvent was evaporated using a rotary evaporator and the res-
idue left behind was treated with 2 N hydrochloric acid (HCl) fol-
lowed by drying using rotary evaporator to remove the solvent. The
residue thus obtained was extracted using dry methanol repeatedly
so as to extract the compound (5) leaving behind the sodium salt.
The compound (5), thus obtained, was characterized by FT-IR,
UVeVis spectroscopy and mass spectrometry. Compound (5):
(yield ¼ 43 mg, 90%). UVeVis (H2O, lmax nm, log e): 417 (3.86), 518
(3.07), 558 (3.01), 584 (2.99), 645 (2.90). FT-IR (MeOH, ῡ cmꢁ1):
3559.77, 1637.79, 1589.05, 1378.00, 994.26. ESI-MS: C46H38N7O3
(calcd.) 736.84 MS (ESIꢁ): observed 736.6 [M]. MALDI-TOF:
observed 782.610 [Mþ4Na-H-3CH3].
1200.94.1H NMR (CDCl3,
d
ppm): ꢁ2.88 (2H, s, -NH), 1.41e1.43 (3H,
t, J ¼ 7.1 Hz), 4.41e4.43 (2H, q, J ¼ 6.7 Hz), 4.93 (2H, s), 6.91 (1H, s,
pyrrole-H), 7.31e7.32 (2H, d, J ¼ 8.4 Hz, m-Ar-H), 7.72 (3H, s (poorly
resolved), pyrrole-H), 8.12e8.13 (2H, d, J ¼ 8.4 Hz, o-Ar-H), 8.17 (6H,
s, pyridyl-H), 8.85e8.87 (4H, m, pyrrole-H), 9.06 (6H, s, pyridyl-H).
2.4. Isothermal titration calorimetry (ITC)
For ITC, porphyrin derivatives [TMPyP (0.75 mM), TriMPy-
COOHP (0.75 mM)] were prepared in 1 ꢀ TE buffer (10 mM tris,
13C NMR (CDCl3,
d
ppm): 14.31 (-CH3), 61.66 (Ph-O-CH2), 65.73 (O-
1 mM EDTA, pH ¼ 8). Calorimeter cell (200
mL) was loaded with CT-
3

M. Guleria, S.K. Suman, J.B. Mitra et al.
European Journal of Medicinal Chemistry 213 (2021) 113184
DNA (0.03 mM) and the solutions of porphyrin derivatives were
injected into CT-DNA until the signal saturation was achieved. In-
2.7. Cytotoxicity assays
jections were performed in the sequential aliquots of 19 ꢀ 2
m
L. In
For cytotoxicity assays, A549 cells were cultured in DMEM
medium containing 10% FBS and grown up to 60e70% confluence.
After harvesting, 1 ꢀ 105 cells were seeded in 12-well tissue culture
plates and allowed to attach to the plate overnight. Thereafter,
A549 cells were treated with different concentrations of porphyrin
order to minimize the impact of equilibration artifacts, results ob-
tained corresponding to first injection of 0.4 mL was not considered
during analysis. All the titrations were performed at 25 ꢂC. Analysis
of all ITC experiments was carried out using the single site model
with ITC 200 software after subtracting the buffer titrations. These
isotherms were fitted with single site binding model using ITC 200
software. The model assumes the presence of independent binding
derivatives (TMPyP and TriMPyCOOHP) viz. 0.05, 1, 10, 50, 100 mM
and were incubated for various time intervals for different assays.
Control set of cells were left untreated. All treatments were carried
out in triplicates.
sites for ligand to give stoichiometry (n),
stant (K) of their respective interactions.
DH and association con-
2.8. MTT assay
MTT assay is a colorimetry based technique used to measure
cellular metabolic activity and thus provides information regarding
the cell viability. Viable cells that contain NAD(P)H-dependent
oxidoreductase enzymes reduce the MTT reagent to formazan
and generate a deep purple coloration, while non-viable cells fail to
2.5. Determination of interaction of TMPyP and TriMPyCOOHP with
CT-DNA by UVeVis spectroscopy
UVeVis spectra of TMPyP and TriMPyCOOHP as well as their
mixtures with CT-DNA were recorded in tris buffer (10 mM tris,
1 mM EDTA, pH ¼ 8) and shift in absorption maxima of Soret band
was monitored, if any, by following the procedure reported else-
where [22]. In the cuvette containing ~2 ꢀ 10ꢁ6 M solution of the
produce any color change. For carrying out MTT assay, 50
mL of
A549 cells (~0.5 ꢀ 105 cells) were harvested in serum free media,
treated with different concentrations of porphyrin derivatives and
incubated at 37 ꢂC for 3 h after mixing with 50
mL of MTT solution
porphyrin derivatives in tris buffer (2.0 mL), 50
replaced gradually with small aliquots (50
and the corresponding UVeVis spectra were recorded after 10 min
of each addition. A total of 150 L of CT-DNA was added in each
mL of buffer was
(5 mg/mL in PBS). After incubation, 150
mL of MTT solvent (4 mM
mL) of CT-DNA (0.03 mM)
HCl, 0.1% NP40 in isopropanol) was added and incubated for 15 min
under shaking condition for dissolution of formazan. Reaction was
stopped by adding 1/10 volume of 1 N HCl and the absorbance of
the reaction mixtures was monitored at 590 nm. Percent cell
viability was calculated from these data [ratio of ODs (optical
density) of treated to control sample, multiplied by 100] to find out
the magnitude of cell proliferation. This data was used to plot
percentage cell proliferation at different concentrations for both
the porphyrin derivatives.
m
cuvette. The experiment was performed similarly for both the
porphyrin derivatives. Attempts were also made to analyze the
interaction of TMPyP with CT-DNA by gradually increasing the CT-
DNA to TMPyP ratio viz. 0.18, 0.37, 0.56, 0.75, 0.93 and 1.12 and
monitoring the corresponding changes in absorption and emission
spectra. Additionally, attempts were made to observe change in the
absorption spectrum of TMPyP-CT-DNA solution mixture at higher
buffer strength (100 mM instead of 1 mM). Absorption spectra of
TMPyP-CT-DNA mixture were also analyzed after addition of
different concentrations of NaCl salt (10 mM, 100 mM and 200 mM
in 2 mL tris buffer) to investigate any shift in the absorption max-
ima (436 nm) of porphyrin-DNA complex.
2.9. Trypan Blue Exclusion Viability assay
Trypan blue exclusion assay was carried out to determine the
cell viability wherein the intact cells appear transparent while the
dead cells take up the trypan blue dye and appear blue when
viewed under the microscope. For this assay, 10 mL of 0.4% trypan
blue solution was mixed with 10
and treated with different concentrations of the porphyrin de-
m
L of A549 cells (~1 ꢀ 104 cells)
2.6. Determination of singlet oxygen formation by porphyrin
derivatives
rivatives. The samples were counted for viability using
hemocytometer.
a
Both the porphyrin derivatives (TMPyP and TriMPyCOOHP)
were analyzed for their ability to produce singlet oxygen by car-
rying out irradiation at one of the Q-bands (516 and 518 nm,
respectively) in presence of DPBF for different time points (between
30 and 540 s for TMPyP and 30e1980 s for TriMPyCOOHP). For
present experiment, stock solutions of TMPyP (1 mM) and DPBF
(10 mM) were prepared in DMF whereas owing to low solubility of
TriMPyCOOHP in DMF, UVeVis absorption and emission spectra for
TriMPyCOOHP were recorded by preparing its stock solution
(1 mM) and that of DPBF (10 mM) in DMSO. For irradiation
2.10. Determination of photo-cytotoxicity of TMPyP and
TriMPyCOOHP by MTT assay in A549 cell lines
Photo-cytotoxicity of TMPyP and TriMPyCOOHP was deter-
mined in A549 cancer cell lines by following the procedure
mentioned below. Three different cell experiments were set up. In
the first set, cells were incubated with the porphyrin derivative
(1
mM) in absence of light and designated as ‘vehicle control-1
(Vcontrol-1)’, while in second set, cells were incubated in pres-
ence of light, but without the porphyrin derivative and was
designated as ‘vehicle control-2 (Vcontrol-2)’. On the other hand, in
third set of experiment, cells were exposed to light in presence of
experiment, 4
L of DPBF was used in 2 mL of DMF/DMSO resulting into final
concentration of porphyrin derivative and DPBF in cuvette as 2
and 25 M respectively. Relative changes (decrease) in the ab-
mL of each porphyrin derivative was utilized whereas
5
m
mM
m
1 mM of either TMPyP or TriMPyCOOHP and were designated as
sorption of DPBF at 325 nm was monitored at different irradiation
time points to mark the transformation of DPBF to an endoperoxide
which decomposes to 1,2-dibenzoylbenzene upon reaction with
singlet oxygen. Results of present experiment were plotted by
taking ln(ODt/OD0) as Y-axis and time of irradiation (in seconds) as
X-axis, where ODt ¼ optical density after ‘t’ time of irradiation and
OD0 ¼ optical density without irradiation.
‘treated’. Cells were exposed to light corresponding to two different
light doses viz. 0.01 and 0.04 kJ/cm2. The irradiation was continued
for 90 min and light dose received by the cells was calculated by
multiplying the light intensity (in W/m2) with time of exposure.
Vcontrol-1 and treated set were incubated with TMPyP and TriM-
PyCOOHP for 6 h and subsequently treated set was exposed to light.
The treated cell lines were incubated for another 18 h post-
4

M. Guleria, S.K. Suman, J.B. Mitra et al.
European Journal of Medicinal Chemistry 213 (2021) 113184
irradiation. Cells were harvested for determining the cytotoxicity in
presence of light by MTT and Trypan Blue Exclusion Viability assay
as mentioned earlier and data for % viability was plotted for both
the porphyrin derivatives at two light doses.
injected subcutaneously on the dorsum of each animal. The tumors
were allowed to grow until the size of the tumors reached about
10 mm in diameter, subsequent to which the animals were used for
the experiment. Each animal, weighing 20e25 g, was intravenously
injected with 100 mL of the radiolabeled preparation (~50 mCi, 1.85
2.11. Cellular imaging by fluorescence
MBq) through one of the lateral tail veins. Biological distribution of
both the radiolabeled complexes was studied at two different post-
administration time points viz. 30 and 60 min. Three animals were
used for each time point. The animals were sacrificed at the
designated post-administration time points through CO2 asphyxia.
Blood samples of the animals were collected by cardiac puncture
immediately after sacrificing the animals. Subsequently, the or-
gans/tissues were excised, washed with normal saline, dried,
weighed in a weighing balance and radioactivity associated with
each organ/tissue was measured using a flat-type NaI(Tl) counter.
The percentage of injected activity (%IA) accumulated in various
organs/tissue was calculated from the above data and expressed as
%IA per g (%IA/g) of organ/tissue. The total activity accumulated in
blood, skeleton and muscles were calculated by considering 7, 10
and 40% of the animal body weight are constituted by these organs/
tissue, respectively [23,24]. Percentage of activity excreted through
urine was calculated by subtracting the activity accounted for all
the organs from the total injected activity.
A549 cells (~0.4 ꢀ 106 cells) were cultured in DMEM containing
10% fetal bovine serum and 1% penicillin and streptomycin in a
humidified CO2 (5%) incubator at 37 ꢂC. For cell uptake studies,
A549 cells were plated in 6-well plate format and cultured to
70e80% confluence. Cells were treated with 10 mM concentration of
either TMPyP or TriMPyCOOHP for 4e5 h. Subsequently, cells were
washed gently with PBS and fixed with 4% paraformaldehyde in
PBS for 15e20 min at room temperature. Cells were again washed
with PBS and permeabilized with 0.1% Triton ꢀ 100 for 10 min and
then stained with DAPI (5 mg/mL) for 5e10 min. Fluorescence im-
ages of A549 cells incubated with porphyrin and those stained with
DAPI were captured on fluorescence microscope.
2.12. Preparation of 68Ga-TMPyP and 68Ga-TriMPyCOOHP
Porphyrin derivatives, namely TMPyP and TriMPyCOOHP were
radiolabeled with 68Ga by following a generalized procedure.
Freshly eluted 68GaCl3 (74 MBq, 2 mCi, 200
mL), obtained by eluting
3. Results
a
68Ge/68Ga generator using 0.05 N HCl, was added to 0.5 mg of
TMPyP (0.808
mmol) or TriMPyCOOHP (0.679
mmol) dissolved in
3.1. Syntheses and characterization of porphyrin derivatives
milliQ water (0.5 mL). The pH of the reaction mixture was adjusted
to 5.5 using 2 N aqueous sodium acetate solution prior to its in-
cubation in a boiling water bath for 30 min. Post-incubation, an
Both the porphyrin derivatives were synthesized by following
Alder-Longo method, a commonly adopted procedure used for the
synthesis of porphyrin derivatives [25] (Schemes 1 and 2).
aliquot of reaction mixture (5e10 mL) was injected in a RP-HPLC to
determine the percentage radiochemical yield (% RCY). For further
purification, 68Ga-labeled porphyrin derivatives (68Ga-TMPyP and
68Ga-TriMPyCOOHP) were separately loaded on a C-18 reversed
phase Sep-Pak® cartridge. The cartridge was pre-conditioned by
passing 4.0 mL of ethanol followed by 2.0 mL of double distilled
water prior to the loading of the radiolabeled preparations. Free
GaCl3 was eluted using 6.0 mL of double distilled water and sub-
sequently radiolabeled porphyrin complex was eluted out from the
column using 1.0 mL of ethanol. Ethanol present in the purified
preparation was removed by gentle warming and the preparation
was reconstituted with normal saline. The radiochemical purity of
the purified 68Ga-labeled porphyrins was determined by HPLC
studies employing gradient elution technique following the quality
control method reported elsewhere [10].
UVeVis spectra consisting of one intense Soret band at
418e422 nm followed by four less intense Q-bands in between
500 nm and 700 nm along with a signal corresponding to two
protons in the negative region of 1H NMR spectra provided
conclusive evidence in favour of the formation of porphyrin moiety
in various porphyrin derivatives synthesized in the present study.
Presence of an ester group in the porphyrin structure (3) was
confirmed by the presence of the peak at 1756.95 cmꢁ1 in FT-IR
spectrum. Its formation was further confirmed by the presence of
a mass peak at 720.285 (obtained using MALDI-TOF instrument)
corresponding to molecular ion [MþH]. Mass spectra of compounds
(2) and (4) recorded in positive mode (ESI-MS), exhibited m/z peaks
at 169.1 [M]4þ and 254.9 [M]3þ respectively, whereas 633.339 [M-
3CH3]þ and 734.299 [M-2CH3]þ were the most abundant peaks
corresponding to compound (2) and (4) in the mass spectra ac-
quired using MALDI-TOF mass spectrometer, indicating the for-
mation of tetracationic and tricationic molecular ions respectively
providing an evidence in favour of the formation of four and three
quaternary nitrogen centres, respectively. FT-IR spectrum of com-
pound (5), obtained by basic hydrolysis of compound (4), exhibited
peaks characteristic to -OH and >C¼O of carboxylic group at
3559.77 (broad) and 1637.79 cmꢁ1, respectively. Observation of a
sharp singlet at 4.72 ppm corresponding to 12 protons arising from
2.13. Determination of partition coefficient (log Po/w
)
Partition coefficients of 68Ga-TMPyP and 68Ga-TriMPyCOOHP
complexes were determined in octanol-water solvent system in
order to ascertain their lipophilicity/hydrophilicity. For this, the
radiolabeled preparation (100
(900 L) and octanol (1 mL) and the resulting solution was vortexed
thoroughly. This mixture was subsequently centrifuged at
4000 rpm for 5 min. Aliquots of 100 L were withdrawn from both
mL) was added to a mixture of water
m
m
the methyl groups attached with quaternary nitrogen centre in 1
H
water and octanol layers and counted separately using the well-
type NaI(Tl) counter. Partition coefficient of the 68Ga-labeled
porphyrin derivatives were calculated from this data.
NMR spectrum of compound (2) confirmed the methylation of all
the four pyridyl nitrogens present in the porphyrin structure.
Symmetrical structure of compound (2) resulted into a singlet for
all the eight hydrogens of pyrrole rings. Similarly, eight hydrogens
each at ortho- and meta-positions of pyridine rings exhibited two
doublets in the 1H NMR spectrum. In 1H NMR spectrum of com-
pound (3), observation of a triplet, quartet and singlet at chemical
shift positions of 1.42 ppm, 4.42 ppm and 4.93 ppm, respectively
provided evidence in favour of presence of an aliphatic chain
(-OeCH2eCOOeCH2eCH3) in its structure. Additionally, presence
2.14. Bio-distribution studies
Pharmacokinetic evaluation of 68Ga-labeled porphyrin de-
rivatives was carried out by bio-distribution studies in fibrosar-
coma bearing Swiss mice. For creating the tumors on the animals,
about 106 cells of HSDM1C1 murine fibrosarcoma (100
mL) were
5

M. Guleria, S.K. Suman, J.B. Mitra et al.
European Journal of Medicinal Chemistry 213 (2021) 113184
Scheme 1. Reaction scheme for the synthesis of compound TMPyP (2): (a) nitrobenzene, propionic acid, reflux, 2 h; (b) CH3I, RT, Stirring for 48 h.
Scheme 2. Reaction scheme for the synthesis of compound TriMPyCOOHP (5): (a) nitrobenzene, propionic acid, reflux, 2 h; (b) CH3I, RT, Stirring for 48 h; (c) DMF:2 N NaOH (1:1 v/
v), RT, Stirring for 48 h.
of carbonyl ester carbon [single such carbon in compound (3)] was
confirmed by 13C NMR spectroscopy where a peak at chemical shift
of 184.21 ppm was observed. Methylation of nitrogen in all the
three pyridyl rings, present in the structure of compound (4),
leading to formation of three cationic centres, was confirmed by
presence of a sharp singlet corresponding to nine hydrogens at
6

M. Guleria, S.K. Suman, J.B. Mitra et al.
European Journal of Medicinal Chemistry 213 (2021) 113184
4.44 ppm in 1H NMR spectrum. Ortho and meta-hydrogens of a
single benzene ring present in the structure of compound (4)
exhibited two doublets at 7.42 and 8.14 ppm corresponding to
integration for two hydrogens each. Due to insertion of cationic
centres in the structure of compound (4), chemical shifts corre-
sponding to hydrogens attached to pyridyl carbons including others
in the aromatic region were observed to be shifted downfield
relative to those present in the structure of compound (3). Similar
observation was also made in 1H NMR spectra obtained for com-
pound (1) and (2).
into CT-DNA resulted in insignificant amount of heat signatures as
compared to TMPyP-CT-DNA interaction, which indicated absence
of interaction between TriMPyCOOHP and CT-DNA.
3.3. Determination of interaction of TMPyP and TriMPyCOOHP with
CT-DNA by UVeVis spectroscopy
UVeVis spectra of TMPyP and TriMPyCOOHP as well as their
reaction mixtures with CT-DNA recorded in tris buffer revealed a
bathocromic shift of ~14 nm for Soret band (436 from 422 nm)
corresponding to the reaction mixture of TMPyP with CT-DNA
(Fig. 2(a)). However, no change in absorption maxima of Soret
band in UVeVis spectrum was observed when TriMPyCOOHP was
allowed to react with CT-DNA. This observation also indicated the
absence of any interaction between TriMPyCOOHP and CT-DNA.
Further investigation of effect on absorption and emission
behavior of TMPyP-CT-DNA complex at different molar ratio (CT-
DNA to TMPyP) revealed no further shift in the absorption maxima
except for decrease in the absorption (hypochromicity), whereas in
the corresponding emission spectra an increase in the emission
intensity (hyperchromicity) was observed both at 654 nm as well as
at shoulder emission of 714 nm (Fig. 2(b) and (c)). Attempts to carry
out the similar studies using higher buffer strength (100 mM)
revealed no significant difference with the observations made
earlier when tris buffer of relatively lower strength (10 mM) was
3.2. Isothermal titration calorimetry (ITC)
ITC measurements were carried out to understand thermody-
namics of porphyrin and CT-DNA interaction for both the porphyrin
derivatives. The interaction between TMPyP and CT-DNA was found
to be exothermic, which is evident from negative value of the
enthalpy observed when TMPyP was allowed to interact with CT-
DNA (Fig. 1). Saturation of binding sites on DNA was achieved af-
ter eleven injections. The isotherm obtained by plotting heat
change vs. molar ratio (porphyrin and CT-DNA) was used to
calculate stoichiometry (n), change of enthalpy (
DH), change of
entropy ( S) and association constant (K) of TMPyP-DNA binding
D
(Fig. 1). These values were found to be similar to those reported in
the literature [22]. On the other hand, injections of TriMPyCOOHP
Fig. 1. ITC analysis of porphyrin-CT-DNA interaction. Upper panels depict the raw calorimetric data of the titration of TMPyP (0.75 mM) and TriMPyCOOHP (0.75 mM) into CT-DNA
(0.03 mM) at 25 ꢂC in the 1 ꢀ TE buffer (10 mM tris, 1 mM EDTA, pH ¼ 8), whereas lower panels show the corresponding buffer corrected integrated injection heats.
7

M. Guleria, S.K. Suman, J.B. Mitra et al.
European Journal of Medicinal Chemistry 213 (2021) 113184
Fig. 2. Graphs: (a) Overlaid UVeVis spectra of TMPyP (2
mM) and TMPyP-CT-DNA reaction mixture depicting bathochromic shift; (b) Overlaid absorption pattern obtained cor-
responding to TMPyP-CT-DNA mixture examined at different CT-DNA to TMPyP molar ratios viz. 0.18, 0.37, 0.56, 0.75, 0.93 and 1.12; (c) Overlaid emission spectra of TMPyP-CT-DNA
reaction mixtures depicting rise in emission intensity with increasing CT-DNA to TMPyP molar ratios.
utilized for such studies. Additionally, attempts to study the effect
of presence of salt on interaction behavior of TMPyP and CT-DNA
revealed no effect on the position of the absorption maxima, con-
firming the well-observed mode of interaction between the two as
intercalative rather than external groove binding [26,27].
form singlet oxygen indirectly by monitoring the decomposition of
DPBF leading to decrease in the absorption of the latter upon
irradiation at a wavelength specific to porphyrin derivative (Figs. 3
and 4). Graphs plotted between ln(ODt/OD0) and time of irradiation
(in seconds) for TMPyP and TriMPyCOOHP revealed the require-
ment of longer periods of irradiation to observe appreciable decline
in absorbance for latter than the former, which clearly indicates the
relatively higher potential of TMPyP towards generation of singlet
oxygen compared to TriMPyCOOHP. This observation also corrob-
orates the results obtained during photo-cytotoxicity experiments
3.4. Determination of singlet oxygen formation by porphyrin
derivatives
Both the porphyrin derivatives were analyzed for their ability to
Fig. 3. Graphs (a) and (b) depicting variation in absorption at a wavelength specific to DPBF (325 nm) in presence of TMPyP (2 mM) upon irradiation at different time points ranging
from 30 to 540 s.
8

M. Guleria, S.K. Suman, J.B. Mitra et al.
European Journal of Medicinal Chemistry 213 (2021) 113184
Fig. 4. Graphs (a) and (b) depicting variation in absorption at a wavelength specific to DPBF (325 nm) in presence of TriMPyCOOHP (2 mM) upon irradiation at different time points
ranging from 30 to 1980 s.
(described below) where TMPyP induced greater cell death when
exposed to similar radiation doses compared to that of
TriMPyCOOHP.
proliferation at both the light doses. Here again TMPyP exhibited
relatively higher photo-cytotoxicity than TriMPyCOOHP when
exposed to two different light doses (47.71 0.71 and 43.98 0.42
versus 77.67 0.98 and 61.30 4.59% cell proliferation at 0.01 and
0.04 kJ/cm2 light doses, respectively) (Fig. 6) indicating the rela-
tively higher potency of the former derivative.
3.5. Cytotoxicity and photo-cytotoxicity experiments
Cytotoxicity experiments carried out in A549 cancer cell line
revealed the non-toxic nature of both the porphyrin derivatives
3.6. Cellular imaging by fluorescence
towards A549 cells at the lowest tested concentration i.e. 0.05 mM
Attempts towards fluorescence imaging of TMPyP and TriMPy-
COOHP in A549 cancer cell lines revealed the preferential cell up-
take exhibited by TMPyP over that of TriMPyCOOHP (Fig. 7). Also,
ability of the former to accumulate in cell nucleus was confirmed by
merging the cell images corresponding to TMPyP and that of DAPI.
(Fig. 5). However, cytotoxicity caused by both the porphyrin de-
rivatives in absence of light was found to be increasing propor-
tionately with the increasing concentration of the porphyrin
derivatives and at highest tested concentration (100
mM), cell
proliferation was observed to be reduced down to less than 50%.
Moreover, TMPyP was observed to exhibit comparatively higher
cytotoxicity than TriMPyCOOHP at lower concentrations (p < 0.05).
Nonetheless, at higher concentrations, percentage cell proliferation
was observed to be similar for both the porphyrin derivatives
(p > 0.05) (Fig. 5).
Photo-cytotoxic experiments revealed the light dependent
cytotoxic effects of both the porphyrin derivatives on A549 cells
(Fig. 6). In comparison to both Vcontrol-1 as well as Vcontrol-2,
treated set of cell lines exhibited lesser percentage of cell
3.7. Preparation of 68Ga-TMPyP and 68Ga-TriMPyCOOHP
Both the porphyrin derivatives were radiolabeled with 68Ga,
eluted from a 68Ge/68Ga radionuclide generator and the maximum
radiolabeling yield achieved under the optimized reaction condi-
tions were 85.54 0.52% and 70.30 1.50% for 68Ga-TMPyP and
68Ga-TriMPyCOOHP, respectively. Therefore, attempts were made
to purity the radiolabeled porphyrin derivatives using pre-
conditioned C18-Sep-pak® cartridge columns. Post-purification,
68Ga-TMPyP could be obtained with >90% radiochemical purity,
while the same for 68Ga-TriMPyCOOHP improved to >95%. Per-
centage radiochemical purity of the radiolabeled preparations were
determined using HPLC radio-chromatograms, where free 68GaCl3
eluted with a retention time (Rt) of 3.5 0.24 min, whereas both
the 68Ga-labeled porphyrin complexes exhibited almost similar
retention time of ~8.0 min (Fig. 8).
3.8. Determination of partition coefficient (log Po/w
)
Partition coefficients of 68Ga-TMPyP and 68Ga-TriMPyCOOHP
complexes were observed to be ꢁ4.30 and ꢁ1.55, respectively
indicating hydrophilic nature of both the complexes. However, it is
evident from the results that the former radiolabeled complex is
relatively more hydrophilic than the latter one.
3.9. Bio-distribution studies
Bio-distribution studies carried out in fibrosarcoma bearing
animal model revealed to some extent differential pharmacokinetic
behavior for the two 68Ga-labeled porphyrin derivatives (Fig. 9).
Out of the two time points chosen for animal studies viz. 30 and
60 min, it was at the higher time point where differential uptake in
Fig. 5. Graphical representation of dark toxicity in terms of percentage cell prolifer-
ation (with respect to control) at varying concentration of TMPyP and TriMPyCOOHP
obtained by MTT assay in A549 cell lines.
9

M. Guleria, S.K. Suman, J.B. Mitra et al.
European Journal of Medicinal Chemistry 213 (2021) 113184
Fig. 6. Graphical representation of cell toxicity (with respect to control) exhibited by 1
m
M of TMPyP and TriMPyCOOHP in A549 cell lines at two different light doses viz. 0.01 and
0.04 kJ/cm2 (‘Control’ as cells without porphyrin and without light exposure, ‘Vcontrol1’ as cells with porphyrin and without light exposure, ‘Vcontrol2’ as cells without porphyrin
and exposed to light, while ‘treated’ as cells with porphyrin and exposed to light) obtained by MTT assay.
Fig. 7. Bright field and fluorescence images of A549 cells (~0.4 ꢀ 106 cells) incubated with DAPI (5
mg/mL), porphyrin derivatives (10 mM) and co-incubated with DAPI and porphyrin
derivatives.
various organs was observed between the two radiolabeled com-
plexes. Uptake of 68Ga-TriMPyCOOHP in liver (6.39
1.06 and
for both the complexes at early post-administration time point
(6.47 0.87 versus 5.34 0.73% IA/g). However, at higher post-
administration time point, 68Ga-TMPyP exhibited significantly
9.41 1.71% IA/g at 30 and 60 min post-administration, respec-
tively) at both the time points was relatively higher that was
observed for 68Ga-TMPyP (4.80 0.18 and 3.47 0.51% IA/g at 30
and 60 min post-administration, respectively) (Fig. 9). This obser-
vation can be supported by the partition coefficient values
observed for both the complexes where the former complex was
found to be relatively less hydrophilic than that of latter. Initially
accumulated activity from various organs underwent clearance
both via hepatobiliary as well as renal pathway, though clearance
via latter route was observed to be predominant in case of 68Ga-
TMPyP (15.31 0.56% IA/g at 60 min post-administration) than that
observed in case of 68Ga-TriMPyCOOHP (8.10 0.31% IA/g at 60 min
post-administration). Similar uptake in tumor mass was observed
higher (p < 0.05) retention (6.07
0.03 %IA/g at 60 min post-
administration) compared to that of 68Ga-TriMPyCOOHP
(2.09 1.20 %IA/g at 60 min post-administration).
This has been reflected in the resulting tumor to blood and tu-
mor to muscle ratios as these values for 68Ga-TMPyP (1.80 0.14
and 9.34
significantly higher than those for 68Ga-TriMPyCOOHP complex
(0.49 0.23 and 4.43 0.97 respectively at 60 min post-
1.10, respectively at 60 min post-administration) are
administration) (Table 1). However, high tumor to muscle ratio
observed for both the complexes at all the time points indicated
about their preferential ability to accumulate in the tumorous
lesion (Table 1).
10

M. Guleria, S.K. Suman, J.B. Mitra et al.
European Journal of Medicinal Chemistry 213 (2021) 113184
Fig. 8. HPLC profiles of (a) unlabeled/free 68GaCl3, (b) TMPyP, (c) 68Ga-TMPyP, (d) TriMPyCOOHP, and (e) 68Ga-TriMPyCOOHP.
FT-IR, 1H NMR, 13C NMR spectroscopy as well as by mass spec-
trometry. Out of the two porphyrin derivatives, one is a symmet-
rically substituted tetracationic porphyrin (TMPyP), while the other
one is the functionally modified version of first one containing
three cationic centres and one aliphatic carboxylic moiety at the
peripheral position (TriMPyCOOHP). The very first step of synthesis
involving formation of tetrapyrrolic cavity of porphyrin is a limiting
reaction with respect to yield of the desired reaction product. In
case of compound (2) (TMPyP), it was 2.4%, whereas for compound
(3) it was observed to be only 2%. However, lower yield of porphyrin
core formation reactions is a quite common observation, especially
for polar porphyrin derivatives, as they exhibit relatively lesser
tendencies towards crystallization [25,28]. Despite the careful se-
lection of stochiometric ratios of all the ingredients, formation of
various other porphyrin derivatives of all possible combinations
with meso-substituents was observed along with the formation of
tar like polypyrrole products during the course of syntheses of the
porphyrin derivatives reported in the present study [25,28]. How-
ever, once porphyrin core is formed, rest of the reactions involving
participation of meso-substituents resulted into relatively higher
yields.
It is well-documented in the literature that porphyrins have an
inherent tendency to accumulate in the tumorous lesions [4e12]
and interact with duplex DNA as well as G-quadruplexes, which are
well-known structural elements of telomeres as well as promoters
of certain pro-oncogenes [22]. Therefore, interaction of porphyrin
with G-quadruplex can potentially abrogate DNA-telomerase
interaction and mitigate the expression of important oncogenes
[22]. In this line, the present work involves the attempts made to
carry out the comparative evaluation of two in-house synthesized
porphyrin derivatives (TMPyP and TriMPyCOOHP) towards accu-
mulation in tumorous lesions and more precisely, studying the
effect of removing one charged centre and replacing the same with
an aliphatic carboxylic acid moiety on DNA-porphyrin interaction.
The rationale behind carrying out the aforementioned modification
was due to the fact that porphyrin-based ligands are used for tar-
geting mostly solid tumors and such tumors usually have slightly
acidic microenvironment owing to the enhanced formation of lactic
acid [29]. Aliphatic carboxylic acids have a pKa of ~5.0 and at
physiological pH (i.e. in blood stream), it is expected to remain in
carboxylate ion form. However, after penetration through the cell
Fig. 9. Graphical representation of results of bio-distribution studies of 68Ga-TMPyP
and 68Ga-TriMPyCOOHP carried out in fibrosarcoma bearing Swiss mice at two
different post-administration time points viz. 30 and 60 min.
Table 1
Tumor to blood and tumor to muscle ratio values for 68Ga-TMPyP and 68Ga-TriM-
PyCOOHP at two different post-injection time points viz. 30 and 60 min.
Tumor/Organ ratio
68Ga-TMPyP
30 min
68Ga-TriMPyCOOHP
60 min
30 min
60 min
Tumor/Blood
Tumor/Muscle
0.89 0.04
4.41 0.62
1.80 0.14
9.34 1.10
0.86 0.29
6.43 0.97
0.49 0.23
4.43 0.97
4. Discussion
Two cationically charged porphyrin derivatives were synthe-
sized following multi-step reaction procedures and characterized
by employing standard spectroscopic techniques such as, UVeVis,
11

M. Guleria, S.K. Suman, J.B. Mitra et al.
European Journal of Medicinal Chemistry 213 (2021) 113184
membrane, it is expected that the porphyrin derivative will un-
dergo protonation and will be retained within the cell in the pro-
tonated form [29]. Therefore, to enhance the probability of cellular
penetration and retention, carboxylic acid derivative of phenyl
residue was chosen while designing the structure of TriMPyCOOH.
Both the porphyrin derivatives were screened for possible
interaction with CT-DNA by performing various experiments. As
revealed in ITC experiments, TMPyP exhibited binding with CT-
DNA which was reflected in the values of thermodynamic param-
eters such as, association constant (K), stoichiometry (n), enthalpy
5. Conclusion
Results of various experiments carried out during present study
involving two structurally different porphyrin derivatives viz.
TMPyP and TriMPyCOOHP indicate the important role played by
the structure as well as charge of the porphyrin derivatives in
controlling their pharmacokinetics, cytotoxicity and tumor affinity.
These results indicate the importance of preserving all the four
cationic centres in the structure of porphyrin derivative as removal
of one of them and its replacement with uncharged aliphatic
moiety interfered with its DNA binding ability. These observations
could be helpful in developing suitable porphyrin derivatives which
may have significant role in the management of cancers in the
coming days.
(
(K
D
H) and entropy
(DS) associated with the reaction
¼
16.60
3.56
ꢀ
104 Mꢁ1
,
N
¼
0.77
0.05,
D
H ¼ ꢁ5.96 0.54 kcal molꢁ1
,
D
S ¼ 3.88 cal molꢁ1 degꢁ1). The
thermodynamic data obtained in the present case is found to be
strikingly similar to that of interaction of TMPyP with G2-
quadruplex reported elsewhere [22]. However, it is interesting to
note that TriMPyCOOHP failed to exhibit any such interaction.
These observations were also corroborated by UVeVis spectros-
copy where interaction of CT-DNA with TMPyP resulted a bath-
ochromic shift of the Soret band by ~14 nm (436 from 422 nm), but
no such shift of the Soret band was observed when CT-DNA was
allowed to interact with TriMPyCOOHP. This observation also in-
dicates minimal or no interaction between the tricationic
porphyrin derivative with DNA. This observation infers that such
structural modification of the porphyrin moiety has a detrimental
effect with respect to its DNA-interacting capability. Further in-
vestigations of interaction between TMPyP and CT-DNA at higher
DNA/TMPyP molar ratios revealed serial increase in the fluores-
cence intensity, which is indicative of the phenomenon where
electrostatic interaction of TMPyP with DNA promotes quenching
of S1 state (first singlet excited state) of the photosensitizer and
promotes fluorescence (‘on state’ of photosensitizer) [30].
In agreement with the previous observations, during fluores-
cence imaging studies, TMPyP was observed to exhibit accumula-
tion in the nucleus of A549 cells, which was further confirmed by
co-incubation with DAPI; whereas TriMPyCOOHP failed to exhibit
similar behavior. Cell cytotoxicity studies in dark as well as in
presence of light carried out by two different assays revealed the
relatively higher cytotoxicity associated with TMPyP than that with
TriMPyCOOHP. This behavior could be attributed to the ability of
TMPyP to interact with DNA and thereby its ability to exert greater
cell damage. Though both TMPyP as well as TriMPyCOOHP
exhibited greater cytotoxicity in presence of light as compared to
dark, photo-cytotoxic effects were more pronounced in case of
former than those observed in case of latter. This observation again
could be an indicative of localization of oxidative radicals produced
as a result of porphyrin mediated energy transfer, closer to cell DNA
owing to TMPyP-DNA interaction. In order to evaluate the
comparative in-vivo efficacy of both the porphyrin derivatives, they
were radiolabeled with 68Ga, a well-known PET radionuclide and
the radiolabeled preparations were evaluated in tumor bearing
small animal model. The results of bio-distribution studies revealed
almost similar uptake of the radiotracers in per gram of tumor mass
at the early time point (30 min post-administration) for both 68Ga-
TMPyP and 68Ga-TriMPyCOOHP. However, the former exhibited
significantly higher retention in the tumor at longer time point
(60 min post-administration) compared to the latter. Moreover, the
relatively rapid clearance of activity from blood and liver, observed
in case of the 68Ga-TMPyP compared to that of 68Ga-TriMPyCOOHP
had resulted better tumor to background ratios for the former
radiolabeled complex. These observations indicate the important
role played by the structure as well as charge of the porphyrin
derivatives in controlling their cytotoxicity and tumor affinity.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgement
Bhabha Atomic Research Centre (BARC) is a constituent unit of
Department of Atomic Energy, Government of India and all the
research activities carried out at the Institute is fully funded by the
Government of India.
The authors gratefully acknowledge Dr. P. K. Pujari, Director,
Radiochemistry and Isotope Group, BARC for his constant support
and encouragement. The authors are thankful to Dr. Ankona Dutta,
Ms. Geetanjali Dhotre and the other staff members of the MALDI-
TOF facility of Tata Institute of Fundamental Research (TIFR),
Mumbai for allowing and helping us in recording the mass spectra
of the compounds using MALDI-TOF MS. The authors also express
their gratitude to Ms. Mamata Joshi of National NMR Facility, TIFR,
Mumbai for providing the facility of recording the NMR spectra
reported in the article. The authors also acknowledge Dr. P. A.
Hassan, Head Nanotherapeutics and Biosensors Section, Chemistry
Division, BARC for providing us the facility of fluorescence imaging.
The authors also thank Mr. Umesh Kumar of Radiopharmaceuticals
Division, BARC and all the staff members of the Animal House fa-
cility, Radiation Biology and Health Sciences Division, BARC for the
help provided during the course of the present study.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113184.
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