Studies towards elucidating the potential of 5,10,15,20-tetrakis(: P -carboxy-methyleneoxyphenyl)porphyrin as a theranostic agent for applications in PET and PDT

Article abstract of DOI:10.1039/c7md00433h

Porphyrins, owing to their inherent tendency to accumulate in tumorous lesions, are considered suitable for developing agents for theranostic applications involving tumor diagnosis and targeted tumor therapy. The aim of the present work is to study the po

Full text of DOI:10.1039/c7md00433h

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DOI: 10.1039/C7MD00433H.
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DOI: 10.1039/C7MD00433H
Studies towards elucidating the potential of 5,10,15,20-tetrakis(p-carboxy-
methyleneoxyphenyl)porphyrin as a theranostic agent for applications
in PET and PDT
a
a
* a,b
a
Mohini Guleria, Chandan Kumar, Tapas Das,
Jeyachitra Amirdhanayagam,
a
c
a,b
Rohit Sharma, Haladhar D. Sarma and Ashutosh Dash
a
Radiopharmaceuticals Division, Bhabha Atomic Research Centre,
Trombay, Mumbai ꢀ 400085, India
b
Homi Bhabha National Institute, Anushaktinagar, Mumbai ꢀ 400094, India
c
Radiation Biology and Health Sciences Division,
Bhabha Atomic Research Centre, Trombay, Mumbai ꢀ 400085, India
*
Author for Correspondence
Tapas Das
Radiopharmaceuticals Division
Bhabha Atomic Research Centre
Trombay, Mumbai ꢀ 400085, INDIA
91ꢀ22ꢀ2559 0613
Telephone
Fax
91ꢀ22ꢀ2550 5151
eꢀmail
tdas@barc.gov.in
1

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Abstract
Porphyrins, owing to their inherent tendency to accumulate in tumorous lesions, are
considered suitable for developing agents for theranostic applications involving tumor
diagnosis and targeted tumor therapy. The aim of the present work is to study the potential
of
a
porphyrin
derivative
namely,
5,10,15,20ꢀtetrakis(pꢀ
carboxymethyleneoxyphenyl)porphyrin (SPTA) as a theranostic agent for applications in
positron emission tomography (PET) and photodynamic therapy (PDT). SPTA was
synthesized inꢀhouse following a threeꢀstep reaction process and characterized by using
1
13
spectroscopic techniques viz. UVꢀVis, FTꢀIR, HꢀNMR, CꢀNMR spectroscopy as well as
6
8
by mass spectrometry. SPTA was labeled with Ga, a generator produced PET radioisotope
6
8
and the radiolabeled product was characterized by HPLC. The GaꢀSPTA complex was
prepared with a radiochemical purity >95 % under the optimized conditions. Diagnostic
68
potential of GaꢀSPTA was evaluated by cell uptake studies in two different tumor cell lines
6
8
(
HT1080 and AT549) which revealed affinity of GaꢀSPTA towards the cancer cells. The
biodistribution studies carried out in Swiss mice bearing fibrosarcoma tumors exhibited
accumulation of the radiotracer in tumor. The therapeutic potential of SPTA was evaluated
by determining its photoꢀcytotoxicity employing MTT assay in HT1080 and AT549 cell
lines using three different light doses, which indicated significant cytotoxicity of SPTA in
presence of light. The present study indicates the possible potential of SPTA in radionuclidic
imaging as well as in photodynamic therapy (PDT) thus confirming the promising
theranostic nature of the porphyrin derivative.
68
Keywords: Ga, PDT, PET, Photoꢀcytotoxicity, Porphyrin, Theranostic
2

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Introduction
Porphyrins offer excellent potential for developing tumorꢀavid agents as they are
biocompatible, their metal complexes are thermodynamically stable and kinetically inert,
and they exhibit preferential affinity for tumors, both with or without the presence of a metal
1
ꢀ3
ion in its central core. This property of the porphyrin derivatives has been wellꢀexploited
in designing and exploring several tetrapyrrolic macrocyclic derivatives for their possible
3
ꢀ11
usage in diagnosis and therapy of tumorous lesions.
However, porphyrin moiety being
sufficiently lipophilic in nature, porphyrinꢀbased agents usually exhibit high nonꢀspecific
uptake, which eventually limits the scope of their wideꢀspread clinical applications. This
problem can be circumvented, at least partially, by the introduction of hydrophilic
substituents on the peripheral positions of porphyrin core, as the presence of such
substituents not only increases the hydrophilicity of the porphyrin derivatives, which in turn
helps in improving their pharmacokinetic behavior; but also makes them water soluble, a
9
,12ꢀ15
preferred prerequisite for clinical applications.
The suitability of porphyrin core to accommodate various metal ions viz. Cu, Ga, Pd,
etc. makes it a useful targeting agent for developing radiometallated porphyrin derivative by
6
4/67
incorporating the radioactive counterparts of the metal ions in the porphyrin moiety (
Cu,
6
7/68
109
9,11,16ꢀ19
Ga, Pd, etc.).
Upon radiolabeling these metal ions become an integral part of
the porphyrin structure and this rules out the requirement of any structural modification or
attachment of a biꢀfunctional chelating agent (BFCA), which may affect the tumorꢀaffinity
exhibited by the porphyrin derivative. In addition to the tumor avidity, porphyrin derivatives
also act as excellent photosensitizers and this property has long been utilized in the field of
2
,14ꢀ16,20,21
photodynamic therapy (PDT).
PDT employs the effects of lightꢀmediated inꢀsitu
generation of the singlet oxygen, through an efficient energy transfer mechanism from a
2
,17,21
photosensitizer to the triplet oxygen, for exerting the therapeutic action.
These dual
3

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characteristics of the porphyrin derivatives i.e. core radiometallation as well as
photosensitizing action, combined with their natural tumor targeting ability, makes them an
excellent choice for developing agents for theranostic applications, wherein in unlabeled
form, they can be used as photosensitizers in PDT for targeted tumor therapy while
diagnosis of tumorous lesions can be undertaken by employing radionuclidic imaging
technique using the same porphyrin derivative labeled with a suitable diagnostic
6
4
68
67
4
radionuclide ( Cu, Ga, Ga).
In the present study, an attempt was made to synthesize a symmetrically substituted
porphyrin derivative, namely 5,10,15,20ꢀtetrakis(pꢀcarboxymethyleneoxyphenyl)porphyrin
(SPTA) and to evaluate its potential towards theranostic application. The rationale behind
choosing this porphyrin derivative is the presence of four carboxyl substituents in the
periphery of the porphyrin ring which is expected to reduce the lipophilicity and thus
improve the pharmacokinetic behavior exhibited by the porphyrin. For evaluating the
6
8
diagnostic potential of the porphyrin derivative, it was radiolabeled with Ga. Galliumꢀ68
T½ = 68 min) is a positron emitting radioisotope and is used for radiolabeling a variety of
molecules for the preparation of various PET (positron emission tomography) imaging
(
2
2ꢀ26
68
agents for clinical applications
such as, GaꢀDOTAꢀTATE (DOTA: 1,4,7,10ꢀ
3
68
tetraazacyclododecaneꢀ1,4,7,10ꢀtetraacetic acid, TATE: Tyr ꢀOctreotate), GaꢀDOTAꢀTOC
1
3
68
3
(
TOC: DꢀPhe ꢀTyr ꢀoctreotide) and GaꢀDOTAꢀNOC (NOC: Nal ꢀOctreotide) for imaging
6
8
of neuroendocrine tumors;
GaꢀCitrate for quantification of pulmonary vascular
((4ꢀ{{bisꢀ(posphonomethyl))carbamoyl]ꢀmethyl}ꢀ7,10ꢀ
6
8
permeability;
GaꢀBPAMD
bis(carboxymethyl)ꢀ1,4,7,10ꢀtetraazacyclododecꢀ1ꢀyl)acetic acid) for imaging of bone
6
8
metastases and GaꢀPSMAꢀ11 (prostate specific membrane antigen) for imaging of prostate
2
2ꢀ26
cancer.
In vitro cell uptake studies were carried out in two different cell lines viz.
HT1080 (human fibrosarcoma) and A549 (human lung carcinoma) to evaluate the tumor
4

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6
8
affinity of the Gaꢀlabeled SPTA. The radiolabeled agent was also administered in
fibrosarcoma tumor bearing Swiss mice to investigate its potential as a PET imaging agent.
An attempt was also made to determine the photoꢀcytotoxicity of the porphyrin derivative
which was measured in the same tumor cell lines in absence as well as in presence of light
using different doses to evaluate its suitability for application in PDT.
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Experimental
Materials and methods
Pyrrole, 4ꢀhydroxybenzaldehyde and ethylbromoacetate used for the synthesis of the
porphyrin derivative were purchased from Aldrich Chemical Company (USA). Propionic
acid and nitrobenzene were obtained from S.D. Fine Chemicals (India). Silica gel (60ꢀ120
mesh size), used for the column chromatography and silica gel plates (Silica Gel 60 F254),
required for analytical thinꢀlayer chromatography (TLC) were procured from Merck (India).
All other chemicals used in the present study were purchased from reputed local
manufacturers and were of analytical grade.
UltravioletꢀVisible (UVꢀVis) spectra were recorded using a Jasco Vꢀ530 UV/Vis
spectrophotometer (Japan). Photoluminescence measurements were carried out using a
Hitachi Fꢀ4500 spectrofluorimeter at room temperature. Fourier transform infra red (FTꢀIR)
spectra were recorded in a Jasco FT/IRꢀ420 spectrophotometer (Japan). Proton nuclear
1
magnetic resonance ( HꢀNMR) spectra were acquired using a 300 MHz Varian VXR 300s
spectrometer (USA). Mass spectra were recorded using 410 Prostar Binary LC mass
spectrometer (Varian, USA) using electron spray ionization (ESI). Galliumꢀ68 was eluted
6
8
68
from a 1.11 GBq (30 mCi) Ge/ Ga radionuclide generator obtained from Eckert & Ziegler
(Germany) and purified, prior to its use, by using Strata™ XꢀC cation exchange column,
purchased from Phenomenex (India). All radioactive counting, associated with the
radiochemical studies, was carried out by using a wellꢀtype NaI(Tl) scintillation counter,
obtained from Electronic Corporation of India Limited (ECIL, India). The high performance
liquid chromatography (HPLC) system (PU 1580) was obtained from Jasco (Japan). Elution
profiles were monitored by detecting the radioactivity signal using a wellꢀtype NaI(Tl)
detector (Jasco, Japan) coupled with the HPLC system. All the solvents used for HPLC
studies were degassed and filtered prior to use and were of HPLC grade. Final purification
6

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68
®
of Gaꢀlabeled porphyrin was carried out using Cꢀ18 reversed phase SepꢀPak cartridges
procured from Waters (India).
Chemicals for biological assays were purchased from Sigma Chemical Inc. (USA)
unless stated otherwise. HT1080 (human fibrosarcoma) and A549 (human lung carcinoma)
cells were obtained from the National Center for Cell Sciences (NCCS, India). These cells
were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10 %
fetal calf serum (Invitrogen Carlsbad, USA) and antibiotic solution (10 mL/L of 100×
2
solution) and incubated at 37 °C in humidified 5 % CO atmosphere of an incubator. For the
measurement of photoꢀcytotoxicity, cells were exposed to light generated by a set up which
included LED bulbs (9 W and 14 W, Philips, India) as light source. The irradiation was
continued for a period of 4 h and light doses received by the cells were measured using an
irradiance meter (PVM210, Taiwan). The fibrosarcoma cell line used for raising the tumors
in the animals was also purchased from NCCS (India). Radioactive counting, associated
with the animal studies, was carried out using a flatꢀtype NaI(Tl) scintillation counter (ECIL,
India). Animal studies being reported in the present article were approved by the
Institutional Animal Ethics Committee (IAEC) and all the animal experiments were carried
out in strict compliance with the institutional (IAEC) guidelines and following the relevant
national laws relating to the conduct of animal experimentation (Prevention of Cruelty to
Animals Act, 1960).
Synthesis of 5,10,15,20ꢀtetrakis(pꢀcarboxymethyleneoxyphenyl)porphyrin (SPTA)
5,10,15,20ꢀtetrakis(pꢀcarboxymethyleneoxyphenyl)porphyrin
(SPTA)
was
synthesized by following a three step procedure briefly described below:
Synthesis of 4ꢀcarboethoxymethyleneoxybenzaldehyde (i)
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4ꢀcarboethoxymethyleneoxybenzaldehyde was synthesized by following the procedure
8
reported in the literature. The crude product was purified by silica gel column
chromatography
using
CHCl
3
as
eluting
solvent
whereby
pure
4ꢀ
carboethoxymethyleneoxybenzaldehyde (i) was obtained as a colorless viscous liquid (5.7 g,
1
yield 70%): R = 0.5 (CHCl ); HꢀNMR (300 MHz, CDCl ): δ =1.24 (t, 3H, J = 7.8 Hz, ꢀ
f
3
3
COOCH
2
CH
3
), 4.20 (q, 2H, ꢀCOOCH
2
CH
3
2
), 4.75 (s, 2H, ꢀOCH ), 6.97 (d, 2H, J = 7.2 Hz,
1
3
ArH), 7.85 (d, 2H, J = 7.1 Hz, ArH), 9.90 ppm (s, 1H, >CHO); CꢀNMR (75 MHz,
]DMSO): δ=14.02 (CH ꢀCH ), 60.84 (OꢀCH ꢀCH ), 64.75 (OꢀCH ꢀC=O), 115.06 (mꢀ
phenyl), 130.16 (pꢀphenyl), 131.72 (oꢀphenyl), 162.48 (pꢀOꢀphenyl), 168.21 (O=CꢀO),
[
D
6
2
3
2
3
2
ꢀ1
1
2
91.36 ppm (O=CHꢀ); FTꢀIR (neat, ν cm ) = 1754; ESIꢀMS (m/z): calcd for C H O :
11 12 4
08.21, found: [M+1] 209.15.
Synthesis of 5,10,15,20ꢀtetrakis(pꢀcarboethoxymethyleneoxyphenyl)porphyrin (ii)
,10,15,20ꢀtetrakis(pꢀcarboethoxymethyleneoxyphenyl)porphyrin was synthesized by the
dropꢀwise addition of pyrrole (1.30 g, 19.2 mmol) to the refluxing mixture of (i) (3 g, 14.4
5
27
mmol), propionic acid (25 mL) and nitrobenzene (8 mL). The refluxing was continued for
an additional hour and the resultant reaction mixture was stored overnight at 4 °C.
Subsequently nitrobenzene and propionic acid were removed from the reaction mixture by
distillation to obtain the crude product which was purified by silica gel column
chromatography using 1% MeOH in CHCl as eluting solvent and thus 294 mg of pure (ii)
3
1
was obtained (yield 2%). R = 0.8 (CHCl /MeOH 9.7:0.3); HꢀNMR (300 MHz, CDCl ): δ =
f
3
3
1
8
8
.41 (t, 12H, J = 7.5 Hz, ꢀCOOCH
2
CH
3
), 4.42 (q, 8H, J = 4.5 Hz, ꢀCOOCH
2
CH
3
), 4.91 (s,
2
H, ꢀOCH ), 7.51ꢀ7.50 (d, 8H, J = 7.5 Hz, mꢀArH), 8.13ꢀ8.12 (d, 8H, J = 8.0 Hz, oꢀArH),
13
.84 ppm (s, 8H, pyrrole). CꢀNMR (75 MHz, [D]CHCl
3
2 3
): δ = 9.63 (CH ꢀCH ), 61.57 (Oꢀ
CH ꢀCH ), 65.71 (OꢀCH ꢀC=O), 112.94 (oꢀphenyl), 119.8 (βꢀpyrrole), 124.10 (=C<),
2
3
2
128.98 (pꢀphenyl), 137.94 (αꢀpyrrole), 157.70 (=CꢀO), 172.12 ppm (O=C<). FTꢀIR (neat, ν
8

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−1
cm ): 3291, 3258, 3198, 2976, 1758, 1666. ESIꢀMS (m/z): calcd for C60
54 12 4
H O N : 1022.37,
found: [M] 1022.3.
Synthesis of 5,10,15,20ꢀtetrakis(pꢀcarboxymethyleneoxyphenyl)porphyrin (SPTA) (iii)
,10,15,20ꢀtetrakis(pꢀcarboxymethyleneoxyphenyl)porphyrin was prepared by room
temperature stirring of (ii) (0.1 g, 0.09 mmol) in 10 mL of 2 N NaOH for 2 days. Compound
5
(iii) in purified form was obtained by removing unreacted (ii) via repetitive extractions with
13
3 2 2
CHCl (62 mg, yield 70%). CꢀNMR (75 MHz, D O): δ=77.87 (OꢀCH ꢀCOOH), 114.21 (oꢀ
phenyl), 122.28 (βꢀpyrrole), 125.67 (pꢀphenyl), 129.28 (αꢀpyrrole), 181.61 ppm (ꢀCOOH);
ꢀ1
FTꢀIR ( neat, ν cm ): 3448, 1666, 1599, 1483, 1178, 1064. UV/Vis (H O): (λmax, nm): 416,
2
5
16, 553, 591, 649; ESIꢀMS (m/z): calcd for C H N O : 909.8, found: [M+Na] 932.6
52 38 4 12
68
Elution and purification of Ga
6
8
68
Gaꢀ68, used for the present study, was eluted from a 1.11 GBq (30 mCi) Ge/ Ga
68
radionuclide generator. The generator was eluted with 5 mL of 0.1 N HCl. GaCl , thus
3
™
obtained, was purified by using a strata XꢀC cation exchange column following the
procedure mentioned below. The column was preꢀconditioned by passing 0.5 mL CH
3
OH
6
8
followed by 0.5 mL of 0.1 N HCl prior to the loading of GaCl
3
on the column. The column
6
8
was washed with 20% 0.1 N HCl in acetone and finally Ga was eluted using 0.2% 0.05 N
HCl in acetone.
68
Preparation, purification and characterization of Gaꢀlabeled SPTA
Gaꢀ68ꢀlabeled SPTA was prepared by incubating SPTA (0.5 mg, 0.5 ꢁmol),
68
3
dissolved in ammonium acetate buffer (300 ꢁL, pH=5.0), with GaCl (200 µL, 0.4 mCi) in
6
8
a boiling water bath for a period of 45 min. The GaꢀSPTA complex, thus obtained, was
®
further purified using Sepꢀpak cartridge following the protocol mentioned below. The
9

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cartridge was preꢀconditioned by passing 4 mL of ethyl alcohol followed by 2 mL of double
68
distilled water before the loading of GaꢀSPTA complex on the column. Uncomplexed
6
8
Ga, if any, was eluted using 5 mL of double distilled water, subsequent to which the pure
GaꢀSPTA complex was eluted by washing the column with 1 mL of ethyl alcohol. The
6
8
alcohol was slowly evaporated by gentle warming under the inert gas flow and the purified
6
8
GaꢀlabeledꢀSPTA complex was reconstituted in normal saline for the subsequent studies.
The radiolabeled porphyrin complex was characterized by reversed phase HPLC
using water (A) and acetonitrile (B), both mixed with 0.1 % trifluoroacetic acid, as the
mobile phase employing gradient elution technique (0ꢀ4 min 5 % B, 4ꢀ15 min 5 % B to 95
%
B, 15ꢀ25 min 95 % B, 25ꢀ30 min 95 % B to 5 % B). About 20 µL of the radiolabeled
preparation was injected into the column and the elution was monitored by observing the
radioactivity profile using a wellꢀtype NaI(Tl) scintillation detector attached with the HPLC
system. Flow rate was maintained at 1 mL/min during the experiment.
nat
Preparation of Galliumꢀlabeled SPTA (iv)
Inactive gallium complex of the porphyrin derivative (SPTA) was also synthesized in
6
8
68
order to have an idea about the position occupied by Ga in the GaꢀSPTA complex. The
procedure involved the overnight refluxing of equimolar amount of SPTA (10 mg, 0.01
mmol) and gallium nitrate (28 mg, 0.1 mmol) in ammonium acetate buffer of pH=5.0 for a
period of 12 h. The product, thus obtained, was further purified by removing unreacted
®
gallium, if any, using Sepꢀpak cartridge following the same protocol used for the
purification of the corresponding radiolabeled complex. The formation of GaꢀSPTA
complex was characterized by UVꢀVis spectroscopy as well as by mass spectrometry.
UV/Vis (H O): (λ , nm): 421, 559, 603; ESIꢀMS (m/z): calcd for C H GaN O : 977.16,
2
max
52 36
4
12
+
found: (M ) 977.3
1
0

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Determination of partition coefficient (Log Po/w
)
68
Partition coefficient of GaꢀSPTA complex was determined in octanolꢀwater solvent
system in order to ascertain the lipophilicity/hydrophilicity of the complex. For this, the
radiolabeled preparation (200 µL) was added to a mixture of water (800 µL) and octanol (1
mL) and the resulting solution was vortexed thoroughly. This mixture was subsequently
centrifuged at 3000 rpm for 5 min. Aliquots of 100 µL were withdrawn from both water and
octanol layers and counted separately using the wellꢀtype NaI(Tl) counter.
In vitro stability studies
6
8
In vitro stability of the radiolabeled preparation ( GaꢀSPTA complex) was
determined by following a generalized procedure. In brief, an aliquot (200 µL) was
withdrawn from the radiolabeled complex and incubated with human serum (300 µL) at 37
°C up to 2 h. Acetonitrile (500 µL) was added to the reaction mixture postꢀincubation, for
precipitating serum proteins. The resultant mixture was centrifuged at 10000 rpm for 4 min
for the separation of pellet from the supernatant. The supernatant was analyzed by HPLC
using the protocol mentioned above.
nat
Determination of photo luminous quantum (PL) yield of SPTA (iii) and Galliumꢀlabeled
SPTA (iv)
The photo luminous quantum (PL) yield (QY) of the samples was determined based
on the comparative method by using equation (1). An ethanolic solution of coumarin 153 (Φ
=
0.544) was used as the standard reference for the present study was.
(1)
1
1

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Where, ‘Φ’ is the quantum yield, ‘A’ is the integrated PL intensity, ‘OD’ is the optical
density, and ‘n’ is the refractive index. The subscript ‘S’ and ‘R’ refers to the sample and
reference, respectively. The wavelength, where the OD (or absorbance) of both the sample
and reference matched with each other, was chosen as the excitation wavelength for
performing the study. Excitation was carried out at 418 nm for SPTA whereas 405 nm was
nat
selected as the excitation wavelength for GaꢀlabeledꢀSPTA. The emission was recorded to
be mainly at 680 and 730 nm for the former whereas the latter was found to exhibit emission
at 500 and 680 nm.
In vitro cell uptake and viability studies
In vitro cell uptake studies were carried out in HT1080, A549 and PBL (peripheral
blood lymphocytes) cells. For determination of cell uptake in cancer cell lines viz. HT1080
6
and A549, about 1×10 cells were plated in 6 wellꢀplates for overnight and approximately
6
8
1
0000 and 20000 cpm of radioactivity ( GaꢀSPTA) were added separately to both the cell
68
types and incubated for 1 h at 4 °C. On the other hand, for determining the uptake of Gaꢀ
SPTA in healthy cell lines, PBL were isolated from healthy human blood using a solution
containing polysucrose and sodium diatrizoate equivalent to FicollꢀPaque™ Plus and
Histopaque® 1077. Mononuclear cells (Lymphocytes and Monocytes) were washed twice
with phosphate buffered saline (PBS) and finally reꢀsuspended in RPMI media. Cells were
6
counted and around 1×10 cells were added in tubes containing 10000 and 20000 cpm of
radioactivity. After incubation, cells were washed thrice with iceꢀcold PBS and radioactivity
associated with the cell pellets was measured in NaI(Tl) detector. Cell uptake of radiolabeled
porphyrin was calculated as the percentage of total radioactivity associated with the cell
pellet. Attempt was also made to determine the viability of healthy PBL cells postꢀ
6
8
incubation with GaꢀSPTA. For this study, PBL cells were isolated by following the
1
2

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procedure mentioned above and PHA (Phytohemagglutinin) was added to the plated cells.
68
2
0000 cpm of GaꢀlabeledꢀSPTA activity was added to the cells after an incubation period
of 24 h and the cell viability was determined using MTT assay.
Biodistribution studies
68
Tumor targeting potential as well as biological distribution of GaꢀSPTA complex
was studied by carrying out biodistribution studies in Swiss mice bearing fibrosarcoma
6
tumors. About 10 cells of HSDM1C1 murine fibrosarcoma (100 µL) were injected
subcutaneously on the dorsum of each animal. The tumors were allowed to grow until they
were approximately 10 mm in diameter subsequent to which the animals were used for the
experiment. Each animal, weighing 20ꢀ25 g, was intravenously injected with ~100 µL of the
radiolabeled preparation (~50 µCi, 1.85 MBq) through one of the lateral tail veins. The
animals were maintained at the normal laboratory atmosphere and sacrificed through CO
2
asphyxia after the designated postꢀadministration timeꢀpoints, e.g. 30 min, 1 h, and 2 h. All
the vital organs/tissue of the animals were excised, washed with normal saline, dried,
weighed in a weighing balance and radioactivity associated with each organ/tissue was
measured using a flatꢀtype NaI(Tl) counter. Activity accumulated in various organs/tissue
was calculated from these data and expressed as percentage injected activity per gram of
organ/tissue (% IA/g). For each time point, 3 animals were used. Activity excreted through
urine was indirectly calculated by subtracting the activity accounted in all the organs/tissue
from the total injected activity. For this purpose, the activity accumulated in skeleton, blood
and muscles were calculated by considering 10 %, 7 %, and 40 % of the animal body weight
2
8,29
are constituted by these organs/tissue, respectively.
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Study of cytotoxicity of SPTA by MTT assay in HT1080, A549 and PBL cell lines
Cell toxicity was determined by MTT [3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide] assay which was carried out following the protocol mentioned
3
below. Approximately, 2×10 cells were plated separately in 96 well plates and incubated at
3
7 °C in humidified 5 % CO
concentrations (2ꢀ1000 nM) of SPTA were added in the cells and incubated for 48 h at 37 °C
in humidified 5 % CO atmosphere. After incubation supernatant was removed and 10 µL of
2
atmosphere of an incubator for overnight. Different
2
MTT solution (5 mg/mL) was added in each well and incubated again for 4 h. Supernatant
solution was removed postꢀincubation and 100 µL of solubilizing buffer (20 % SDS in 50 %
DMF) was added to dissolve formazan formed after reduction of MTT dye in living cells.
The color was quantified at 570 nm wavelength with reference to 630 nm using BioTek
Universal Microplate Reader (BioTek USA, Winooski, VT). Percent cell viability was
calculated {ratio of ODs (optical density) of treated to control sample, multiplied by 100} to
find the magnitude of cell proliferation. MTT assay was also used to determine the
cytotoxicity of SPTA in healthy cell lines (PBL). For this study, PBL cells were isolated
from human blood and incubated for 24 h after the addition of PHA. Different
concentrations of SPTA ranging from 2 to 1000 nM were added in the cells followed by
incubation for 48 h at 37 °C in humidified 5 % CO atmosphere. Percent cell viability was
2
determined by MTT assay following the same protocol described above.
Determination of photoꢀcytotoxicity of SPTA by MTT assay in HT1080 and A549 cell lines
Photoꢀcytotoxicity of SPTA was determined in HT1080 and A549 cancer cell lines
5
by following the procedure mentioned below. Approximately, 2×10 cells were plated
separately in culture dish and incubated at 37 °C in humidified 5 % CO atmosphere in an
2
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incubator for overnight. Three set of cells were prepared for the assay. First set of cells were
incubated without porphyrin and without exposure of light and considered as ‘control’,
while second set of cells were incubated in the presence of light but without porphyrin and
was considered as ‘vehicle control’. On the other hand, third set of cells exposed to light in
presence of 1000 nM SPTA was considered as ‘treated’. Cells were exposed to light
2
corresponding to three different light doses viz. 0.02, 0.03 and 0.06 kJ/cm . The irradiation
was continued for 4 h and light dose received by the cells was calculated by multiplying the
2
light intensity (in W/m ) with time of exposure. Before exposing to light, the third set of
cells was incubated with SPTA for 24 h and subsequently exposed to light for a period of 4
h. The treated cell lines were incubated for another 20 h postꢀirradiation. Cells were
3
harvested and about 2×10 cells were plated separately in 96 wellꢀplates and 10 µL of MTT
solution (5 mg/mL) was added in each well followed by incubation for 4 h. MTT assay was
carried out as described in the previous section.
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Results
Synthesis and characterization of SPTA
Symmetrically substituted porphyrin derivative namely, 5,10,15,20ꢀtetrakis(pꢀ
carboxymethyleneoxyphenyl)porphyrin (SPTA) was synthesized following a threeꢀstep
procedure (Fig. 1). All the intermediates [(i) and (ii)] as well as the final product [(iii),
SPTA] were characterized employing the usual spectroscopic techniques viz. UVꢀVis, FTꢀ
1
13
IR, HꢀNMR and CꢀNMR spectroscopy. The observance of proton and carbon signals in
1
13
the expected positions of HꢀNMR and CꢀNMR spectra of all the intermediates as well as
final compound provided the evidence in favour of the formation of desired compounds.
This was further confirmed by observing the molecular ion peaks of the compounds at the
expected values in their mass spectra.
68
Preparation and characterization of GaꢀlabeledꢀSPTA
6
8
68
SPTA was labeled with Ga using an ‘integrated approach’ where Ga becomes a
part of the porphyrin structure without the need of any structural modification which is
5
possible due to the suitability of ionic radius of Ga(III) for porphyrin core. The radiolabeled
6
8
complex, GaꢀSPTA was obtained with radiochemical purity ~80 % under the optimized
68
reaction conditions. The radiochemical purity of the GaꢀSPTA complex was improved
further to > 95 % through purification using Sepꢀpak cartridge. The radiolabeled complex
was characterized by HPLC where it exhibited a retention time of ~15 min, while the
68
uncomplexed Ga eluted from the column within 4 min under identical conditions. A
68
typical HPLC chromatogram of GaꢀSPTA complex is shown in Fig. 2.
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nat
Preparation and characterization of Gaꢀlabeled SPTA
Preparation of cold gallium complex of SPTA was attempted in order to ascertain the
position occupied by gallium metal ion in the porphyrin moiety. Cold GaꢀSPTA complex
was synthesized following a simple procedure and characterized by UVꢀVis spectroscopy as
well as by mass spectrometry. Fig. 3 shows the overꢀlaid UVꢀVis spectra of SPTA and cold
GaꢀSPTA complex. The presence of only two Qꢀbands (instead of four bands seen in case of
SPTA), observed in the longer waveꢀlength region of the UVꢀVis spectrum of cold Gaꢀ
SPTA complex clearly indicates the presence of Ga metal ion in the core of the porphyrin
30
moiety. The structure of GaꢀSPTA complex was further confirmed by obtaining the
molecular ion peak of the complex in the desired position of the mass spectrum.
Partition coefficient (Log Po/w
)
6
8
Partition coefficient (Log Po/w) for GaꢀSPTA complex was determined to be ꢀ0.25
which indicates the hydrophilic nature of the complex.
In vitro stability studies
Galliumꢀ68ꢀlabeledꢀSPTA complex was found to maintain its radiochemical purity
(> 90 %) till 2 h postꢀincubation period, up to which the study was continued.
nat
Determination of photo luminous quantum (PL) yield of SPTA (iii) and Galliumꢀlabeled
SPTA (iv)
Photo luminous quantum yields were found out to be 0.105 for SPTA (iii) and 0.109
nat
for Gaꢀlabeled SPTA (iv). Similar quantum yields observed for both unmetallated SPTA
and GaꢀlabeledꢀSPTA indicated the absence of quenching of PL quantum yield in presence
of metal atom. The present observation is in the similar line with that documented by Azad
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nat
et al. where unmetallated and
Gaꢀmetallated protoporphyrinIXꢀpeptide conjugates
31
exhibited similar PL quantum yields.
In vitro cell uptake and viability studies
6
8
In vitro cell uptake studies for GaꢀSPTA were carried out in HT1080, A549 and
PBL cells and the cell bound activity was found to be ~7 % in both the cancer cell lines
(HT1080, A549) whereas only 0.42 % bound activity was observed in PBL cells. The results
68
of cell uptake studies are depicted in Fig. 4 (a) and (b). Higher uptake of GaꢀSPTA in both
the cancer cell lines relative to that observed in healthy cell lines (PBL) is indicative of
preferential affinity of the radiotracer towards cancer cells. No impact of inhibition of cell
uptake was observed in the presence of excess SPTA which indicates the probable nonꢀ
specific passive diffusion of radiolabeled porphyrin complex across the cell membranes of
13
all the three cell lines. Also, cell viability studies carried out in healthy cell lines revealed
the absence of any significant difference in the cell viability between the control cells (PBL
68
68
incubated without GaꢀlabeledꢀSPTA) and the PBL cells incubated with GaꢀlabeledꢀSPTA
(p>0.056).
Biodistribution Studies
68
Tumor targeting potential as well as the pharmacokinetic behaviour of GaꢀSPTA
was evaluated by carrying out bioꢀdistribution studies in Swiss mice bearing fibrosarcoma
tumors. The study revealed accumulation (1.82±0.65 %IA/g) of radiolabeled porphyrin in
the tumor within 30 min postꢀinjection (p.i.) which increased to 2.49±0.16% IA/g at 1 h p.i.
and retained therein till 2 h p.i. (2.50±0.66 % IA/g), up to which the study was continued.
The radiolabeled agent also exhibited significant accumulation in various organs/tissue such
as blood, lungs, heart, liver and spleen within 30 min p.i. However, the initially accumulated
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activities in these organs/tissue exhibited gradual clearance with time as the uptake in these
organs/tissue were found to decrease at 1 h and 2 h p.i. thus resulting gradual improvement
in the tumor to nonꢀtarget uptake ratios (2.71±0.37 at 30 min p.i. to 7.49±1.03 at 2 h p.i. for
tumor to muscle and 0.13±0.05 at 30 min p.i. to 0.28±0.12 at 2 h p.i for tumor to blood). The
results of the biodistribution studies are tabulated in Table 1. The radiolabeled porphyrin
exhibited significant urinary clearance as 41.53±1.58 of the % injected activity was cleared
through renal pathway at 2 h postꢀadministration.
Study of cytotoxicity of SPTA by MTT assay in HT1080, A549 and PBL cell lines
The MTT assay performed using SPTA showed that the porphyrin derivative is not
toxic to the cells at lower concentrations (below 1 ꢁM). However, about 13ꢀ24 % toxicity
was noted when the concentration of SPTA was increased to 1 ꢁM range in both HT1080
and A549 cell lines. The results of MTT assay performed with different concentrations of
SPTA in HT1080 and A549 cell lines are shown in Fig. 5. MTT assay performed to
determine the toxicity of SPTA towards healthy cell lines revealed that even at 1 ꢁM
concentration of SPTA, decrease in % cell proliferation was only 4ꢀ5% (Fig. 6). This
observation indicates that porphyrin derivative under study (SPTA) is largely nonꢀtoxic to
the healthy cells.
Determination of photoꢀcytotoxicity of SPTA by MTT assay in HT1080 and A549 cell lines
To find out the impact of light induced cytotoxicity of SPTA in HT1080 and A549
cell lines, these cells were treated with 1 ꢁM of SPTA and subsequently exposed to light
corresponding to different light doses. The light induced cytotoxicity, as determined by MTT
assay, was found out to be increasing with increase in the light dose received by the cells.
Also, extent of light induced cytotoxicity was found to be slightly more for HT1080 cell
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lines in comparison to that observed in A549 cell lines. For the highest light dose received
2
by both the cell lines (of the order of 0.06 kJ/cm ), cell toxicity was observed to be ~87 % in
HT1080 and ~83 % in A549 cell lines. The results of photoꢀinduced cell toxicity exhibited
by SPTA in comparison with control (absence of both SPTA and light) as well as vehicle
control (vehicle control/Vcontrol1 = absence of light and presence of SPTA and vehicle
control/Vcontrol2 = presence of light and absence of SPTA) are shown in Fig. 7. This clearly
indicates the potential of SPTA in inducing cytotoxicity when exposed to the light. The
results obtained in the experiment were found to be statistically significant when analyzed
by one way ANOVA (p< 0.05).
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Discussion
The term ‘theranostics’, coined by John Funkhouser in the year 1998, refers to the
application of same agent for the two interdependent applications, namely therapy and
32ꢀ34
diagnosis.
Porphyrins, with their inherent ability to preferentially accumulate in the
cancerous lesions, can be viewed as a crucial substance for developing the suitable agents
for theranostic applications. Porphyrin and porphyrin based assemblies have been
35
demonstrated to be used as multimodel agents exhibiting theranostic properties. However,
in radiopharmaceutical applications, there exists two distinct possibilities through which
porphyrins can be used for theranostic purpose. One obvious way is the use of suitable
porphyrin derivative, radiolabeled separately with a pair of radionuclides, one diagnostic and
the other therapeutic in nature. On the other hand, theranosis with porphyrin can also be
achieved by using a radiolabeled porphyrin for diagnostic purpose, while affecting the
therapy through comparatively more established route of PDT using the same porphyrin
derivative in its unmetallated form. In the present work, we have attempted to explore the
second route in order to study the theranostic potential of porphyrin.
The prime reasons for selecting SPTA as the porphyrin derivative of choice for the
present work include its hydrophilic nature (due to the presence of four carboxylic acid
groups in the mesoꢀpositions of the porphyrin moiety), its similarity with hematoporphyrin
(native porphyrin derivative present in body as part of heme protein) and literature reports
claiming entrapment of acid derivatized porphyrins in solid tumors having intracellular
36
acidic environment. Galliumꢀ68, a generator produced PET radionuclide, was incorporated
1
8
in SPTA in order to make the porphyrin suitable for radiodiagnostic application. Unlike F,
6
8
the widely used PET radioisotope, which requires an onꢀsite cyclotron facility, Ga is
68
68
readily available from a radionuclide generator ( Ge/ Ga) system which makes it possible
to perform the PET imaging at centers which are far from the proximity of a cyclotron
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37,38
68
68
facility.
Moreover, the long shelfꢀlife of Ge/ Ga radionuclide generator along with
6
8
possibility of multiple elutions of Ga in a single day makes it an attractive choice for
3
7ꢀ39
68
developing PET agents for radiodiagnostic applications.
The radiolabeled product, Gaꢀ
SPTA, was characterized by HPLC employing gradient elution technique and it showed a
partition coefficient (Log P ) value of ꢀ0.25 in octanolꢀwater system indicating the
o/w
hydrophilic nature of the complex. The structure of radiolabeled SPTA complex was further
confirmed by synthesizing the corresponding inactive gallium complex of SPTA, whose
UVꢀVis spectrum indicated the presence of gallium metal ion within the porphyrin core.
68
Biodistribution studies carried out with GaꢀSPTA in Swiss mice bearing fibrosarcoma
tumors showed encouraging tumor accumulation and retention therein till 2 h postꢀ
administration up to which the studies were continued. The increase in tumor uptake and
clearance of activity from the nonꢀtarget organs with time gradually improved the tumor to
background ratio, which is important for a good imaging agent.
The potential of using SPTA for therapeutic application through PDT was also
evaluated by determining the photoꢀcytotoxic effect exerted by the unlabeled derivative on
human fibrosarcoma (HT1080) and human lung carcinoma (A549) cell lines. In the present
study, the light induced cytotoxicity imparted by SPTA, on both the cancer cell lines was
found to be quite significant indicating the potential of the porphyrin derivative for
therapeutic applications. Although the present study is preliminary in nature, it demonstrated
the possibility of using suitable porphyrin derivatives for theranostic applications and thus
realizing the goal of ‘Personalized Medicine’.
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Conclusions
The present study documents the preparation of a Gaꢀlabeled porphyrin derivative
6
8
68
(
SPTA) and its evaluation as a theranostic agent. GaꢀSPTA was found to accumulate in
tumorous lesion when administered in tumor bearing Swiss mice. In vitro cell studies to
investigate the photoꢀcytotoxic nature of unmetallated porphyrin derivative revealed the
light dependent cytotoxicity in human fibrosarcoma (HT1080) and human lung carcinoma
(A549) cell lines which indicated the potential of the SPTA towards its application in PDT.
These studies revealed the possible potential of using porphyrin derivatives as theranostic
agents. However, relatively slower in vivo clearance of nonꢀspecific uptake exhibited by the
porphyrin derivative, investigated in the present study, presses the need to explore other
porphyrin derivatives with superior characteristics for their theranostic applications
involving radionuclidic imaging and photodynamic therapy.
Acknowledgements
The authors thankfully acknowledge Dr. Apurav Guleria, Radiation and
Photochemistry Division, Bhabha Atomic Research Centre (BARC) for carrying out studies
related to the determination of photoluminous quantum yields. The help rendered by the
staff members of the Animal House Facility, Radiation Biology and Health Sciences
Division, BARC during animal experimentations is also acknowledged.
Conflict of interest
There are no conflicts of interest to declare.
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Figure legends
Fig. 1:
Scheme for synthesis of SPTA (iii): (a) K
2 3
CO , dry acetone, reflux, 8 h, (b)
propionic acid, nitrobenzene, reflux, (c) 2 N NaOH, stirring, R.T., 48 h
6
8
68
Fig. 2:
Fig. 3:
Fig. 4:
HPLC profile of (a) free Ga and, (b) Gaꢀlabeled SPTA
nat
Overlaid UVꢀVis profile of SPTA (iii) and Gaꢀlabeled SPTA (iv)
6
8
Graphical representation of percentage cell uptake exhibited by GaꢀSPTA:
(a) in two different cancer cell lines viz. HT1080 and A549 corresponding to
two different amounts of radioactivity (A) 10000 and (B) 20000 cpm, and (b)
in peripheral blood lymphocytes (PBL) corresponding to (C) 10000 and (D)
20000 cpm of added radioactivity.
Fig. 5:
Fig. 6:
Fig. 7:
Graphical representation of cell toxicity assay (MTT) for determination of
toxicity exhibited by SPTA at different concentrations in two different cancer
cell lines viz. HT1080 and A549.
Graphical representation of cell toxicity assay (MTT) for determination of
toxicity exhibited by SPTA at different concentrations in healthy cell line
(PBL).
Graphical representation of cell toxicity exhibited by SPTA in two different
cancer cell lines viz. (a) HT1080 and (b) A549 with increasing light dose,
considering ‘control’ as cells without SPTA and without light, ‘vehicle
control/Vcontrol1’ as cells with SPTA and without light, ‘vehicle
control/Vcontrol2’ as cells without SPTA and in presence of light, while
‘treated’ as cells with SPTA in presence of light.
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6
8
Table 1: Results of biodistribution studies of Ga-labeled SPTA in fibrosarcoma
bearing Swiss mice (n=3) at three different time points viz. 30 min, 1 h, and 2 h
Injected activity per gram (%IA/g ) of
organ/tissue
Organ
3
0 min
1 h
2 h
Blood
Lung
13.44±0.16
7.21±0.54
4.48±0.15
1.13±0.21
1.74±0.20
18.08±0.26
3.27±0.02
3.88±0.17
0.67±0.04
3.56±0.20
1.82±0.65
2.71±0.37
11.29±0.67
5.70±1.27
3.91±0.52
1.46±0.09
3.15±0.16
17.28±0.69
2.65±0.01
3.41±0.08
0.61±0.09
1.07±0.39
2.49±0.16
4.67±1.20
9.19±1.86
4.64±1.20
3.62±0.68
3.83±0.57
5.10±2.23
13.07±3.43
2.30±0.04
2.38±0.93
0.33±0.04
2.65±0.85
2.50±0.66
7.49±1.03
Heart
Stomach
GIT
Liver
Spleen
Kidney
Muscle
Bone
Tumor
Tumor/Muscle ratio
Tumor/Blood ratio
Excretion
0.13±0.05
0.22±0.00
34.5±2.54
0.28±0.12
21.05±1.52
41.53±1.58
2
8

Page 29 of 29
MedChemComm
View Article Online
DOI: 10.1039/C7MD00433H
Graphical abstract
Demonstration of theranostic potential of a porphyrin derivative: as a PET agent and a
photosensitizer in PDT