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Journal ArticleDOI

A biocompatible redox MRI probe based on a Mn( ii )/Mn( iii ) porphyrin

05 Mar 2019-Dalton Transactions (The Royal Society of Chemistry)-Vol. 48, Iss: 10, pp 3249-3262

TL;DR: Water soluble fluorinated Mn-porphyrin derivative Mn-3 acts as an ascorbate specific turn-on MRI probe, which in turn can be re-oxidized by oxygen, and the relaxivity increase from the oxidized to the reduced form is considerably improved at medium frequencies.

AbstractFor the development of redox responsive MRI probes based on the MnIII/MnII couple, stable complexation of both reduced and oxidized forms of the metal ion and appropriate tuning of the redox potential in the biologically relevant range are key elements. The water soluble fluorinated Mn-porphyrin derivative Mn-3 satisfies both requirements. In aqueous solutions, it can reversibly switch between MnIII/MnII oxidation states. In the presence of ascorbic acid or β-mercaptoethanol, the MnIII form undergoes reduction, which is slowly but fully reversed in the presence of air oxygen. A UV-Vis kinetic study of MnIII/MnII reduction under oxygen-free conditions yielded second-order rate constants, k2, of 46.1 M−1 s−1 and 13.8 M−1 s−1 for the reaction with ascorbic acid and β-mercaptoethanol, respectively. This could correspond, in the absence of oxygen, to a half-life of a few minutes in blood plasma and a few seconds in circulating immune cells where ascorbic acid reaches 20–40 μM and a few mM concentrations, respectively. In contrast to expectations based on the redox potential, reduction with glutathione or cysteine does not occur. It is prevented by the coordination of the glutathione carboxylate group(s) to MnIII in the axial position, as was evidenced by NMR data. Therefore, MnIII-3 acts as an ascorbate specific turn-on MRI probe, which in turn can be re-oxidized by oxygen. The relaxivity increase from the oxidized to the reduced form is considerably improved at medium frequencies (up to 80 MHz) with respect to the previously studied Mn-TPPS4 analogues; at 20 MHz, it amounts to 150%. No in vitro cytotoxicity is detectable for Mn-3 in the typical MRI concentration range. Finally, 19F NMR resonances of MnIII-3 are relatively sharp which could open further opportunities to exploit such complexes as paramagnetic 19F NMR probes.

Topics: Ascorbic acid (59%), Redox (53%), Fluorine-19 NMR (52%), Carboxylate (50%)

Summary (2 min read)

Introduction

  • Submitted on 18 Nov 2020 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not.
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Journal Name

  • After several extractions of the crude with dichloromethane/water, the organic layer was dried with anhydrous sodium sulfate and concentrated under vacuum giving 3 in 82% yield (Scheme 1).
  • The absence of the sharp sextet characteristic of the free aqueous Mn2+ cation even at a very high amplification, led to the conclusion that the MnIII-3 sample was free of aqueous Mn2+ .
  • If some MnII-3 were present in the sample, a broad signal at giso ~2.0 would be observable at room temperature,62 which was not the case.

Results and Discussion

  • Synthesis of the biocompatible porphyrin-based probes Reagents and conditions: i) DMF, NaH, room temperature, 2h; ii) acetic acid/sodium acetate, Mn(II)acetate tetrahydrate, 80ºC, 2h.
  • In order to obtain free base porphyrin 2, nucleophilic aromatic substitution of 1 using mono methyl protected polyethylene glycol with Mw = ~500 g/mol (PEG500) was performed, in DMF as solvent and NaH as base, at room temperature for two hours.
  • The product was obtained in 65% yield, after purification, and fully characterized by 1H, 19F NMR spectroscopy and ESI-FIA-TOF mass spectrometry (see Figs. 1, S1, S2 and S3, ESI).

Proton and 19F NMR

  • The proton NMR spectrum of MnIII-3 in CDCl3 (Fig. S6a) shows a broad signal at -22.76 ppm corresponding to the -pyrrole protons, characteristic of MnIII high-spin meso-porphyrins.
  • 63, 64 The PEG chain CH2 protons at 3.66 ppm are unshifted and hardly broadened relative to the signal at 3.65 ppm for free PEG due to their long distance from the paramagnetic center.
  • This set of signals corresponds to the fluorine atoms at the meta positions of the meso-phenyl groups in the five possible combinations of the chain lengths of the four polydisperse PEG chains with n = 10- 11.
  • The broad signal around -126.2 ppm results from the overlap of the broader signals of the fluorine atoms at the ortho position, which are closer to the metal center.
  • 65, 66 Upon reduction of MnIII-3 to MnII-3, all the 19F resonances are completely broadened out, in accordance with the efficient paramagnetic relaxation of MnII (Fig. S6c).

Relaxivity studies

  • The relaxation properties have been investigated for both the oxidized MnIII and the reduced MnII form of the porphyrin complex 3. MnII-3 was obtained by adding 30 equivalents of ascorbic acid to the MnIII analogue.
  • Full conversion of MnIII-3 to MnII-3 was evidenced by recording UV-Vis spectra before the relaxivity measurements.
  • The NMRD profiles recorded at 25 °C for the reduced and for the oxidized form are presented in Fig.

Conclusions

  • A new water-soluble fluorinated porphyrin derivative conjugated with PEG chains was synthesized.
  • Interestingly, 19F NMR resonances of MnIII-3 are relatively sharp which could open further possibilities to exploit such complexes as paramagnetic 19F NMR probes.93.
  • The present MnII/MnIII-porphyrin couple satisfies many of the criteria required for a redox imaging probe: good water solubility, biocompatibility, non-toxicity, thermodynamic and kinetic stability in both metal oxidation states, a redox half-cell potential accessible to biologically relevant reducing agents with fast kinetics (adapted to the imaging time scale), and a strong “turn on” relaxivity response upon reduction.
  • 80, 81 Therefore, the role of ascorbic acid should not be ignored either in maintaining the extracellular redox state.
  • Furthermore, MRI quantification of ascorbate could be also achieved by separating the effect of the in vivo concentration of the probe from the redox effect on the observed contrast.

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A biocompatible redox MRI probe based on a Mn( ii
)/Mn( iii ) porphyrin
Sara Pinto, Mário Calvete, Mariana Ghica, Sérgio Soler, Iluminada Gallardo,
Agnès Pallier, Mariana Laranjo, Ana Cardoso, M. Margarida C. A. Castro,
Christopher Brett, et al.
To cite this version:
Sara Pinto, Mário Calvete, Mariana Ghica, Sérgio Soler, Iluminada Gallardo, et al.. A biocompatible
redox MRI probe based on a Mn( ii )/Mn( iii ) porphyrin. Dalton Transactions, Royal Society of
Chemistry, 2019, 48 (10), pp.3249-3262. �10.1039/c8dt04775h�. �hal-02071232�

Journal Name
ARTICLE
This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins
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Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A Biocompatible Redox MRI Probe Based on a Mn(II)/Mn(III)
Porphyrin
Sara M. A. Pinto
a,b,
*, Mário J. F. Calvete
a,b
, Mariana E. Ghica,
a
Sérgio Soler
c
, Iluminada Gallardo
c
,
Agnès Pallier
d
, Mariana B. Laranjo
b,e
, Ana M.S. Cardoso
f
, M. Margarida C. A. Castro
b,e
, Christopher
M.A. Brett,
a
Mariette M. Pereira,
a,b
*Éva Tóth
d,
* and Carlos F. G. C. Geraldes
b,e,
*
For the development of redox responsive MRI probes based on the Mn
III
/Mn
II
couple, stable complexation of both reduced
and oxidized forms of the metal ion and appropriate tuning of the redox potential in the biologically relevant range are key
elements. The water soluble fluorinated Mn-porphyrin derivative Mn-3 satisfies both requirements. In aqueous solution, it
can reversibly switch between Mn
III
/Mn
II
oxidation states. In the presence of ascorbic acid or β-mercaptoethanol, the Mn
III
form undergoes reduction, which is fully reversed in the presence of oxygen. A UV-Vis kinetic study of Mn
III
/Mn
II
reduction
yielded second-order rate constants, k
2
, of 46.1 M
-1
s
-1
and 13.8 M
-1
s
-1
for the reaction with ascorbic acid and β-
mercaptoethanol, respectively. This could correspond to a half-life of a few minutes in blood plasma and a few seconds in
circulating immune cells where ascorbic acid reaches 20-40 M and a few mM concentrations, respectively. Contrary to
expectations based on the redox potential, reduction with glutathione or cysteine does not occur. It is prevented by the
coordination of the glutathione carboxylate group(s) to Mn
III
in axial position, as it was evidenced by NMR data. Therefore,
Mn
III
-3 acts as an ascorbate specific turn-on MRI probe, which in turn can be re-oxydized by oxygen. The relaxivity increase
from the oxidized to the reduced form is considerably improved at medium frequencies (up to 80 MHz) with respect to the
previously studied Mn-TPPS
4
analogues; at 20 MHz, it amounts to 150%. No in vitro cytotoxicity is detectable for Mn-3 in the
typical MRI imaging concentration range. Finally,
19
F NMR resonances of Mn
III
-3 are relatively sharp which could open further
opportunities to exploit such complexes as paramagnetic
19
F NMR probes.
Introduction
In any living body, both extracellular and intracellular redox
environments are tightly regulated
1-3
and their dysregulation may be
associated with a wide range of pathophysiological conditions,
including chronic inflammation,
4
neoplastic growth
5
and ischemia,
6
since several triggered biochemical cascade events can damage
cellular or tissue components, promoting disease progression.
7-9
The
redox environment in a
biological fluid, organelle, cell, or tissue is
maintained by a number of redox couples present which are
linked to each other. Among these, glutathione (GSH) is the
major thiol-disulfide redox buffer of the cell with an average
concentration of 111 mM in the cytosol, much higher than
most other redox active compounds.
10
Therefore, the redox
state of the glutathione disulfide-glutathione couple
(GSSG/2GSH) can serve as an indicator of the intracellular redox
environment. Changes of the half-cell reduction potential (E
hc
)
of the GSSG/2GSH couple appear to correlate with the biological
status of the cell: proliferation E
hc
~ -240 mV; differentiation E
hc
~ - 200 mV; or apoptosis E
hc
~ -170 mV.
10
The extracellular space
is typically more oxidized than the cytosol. In cell extracts, the
total concentration of reduced (GSH) and oxidized (GSSG)
glutathione present can be assessed by HPLC or fluorescent
assays.
11, 12
The development of imaging techniques capable of following in
vivo tissue redox environment/activity is of utmost interest in
clinical research and medical practice, particularly in diagnosis,
a.
Department of Chemistry, Faculty of Science and Technology, University of
Coimbra, Rua Larga, 3004-535, Coimbra, Portugal
b.
Coimbra Chemistry Centre, CQC, University of Coimbra, Rua Larga, 3004-535,
Coimbra, Portugal
c.
Departament de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra,
Barcelona, Spain
d.
Centre de Biophysique Moléculaire, CNRS, UPR 4301, Université d’Orléans,
Orléans, France
e.
Department of Life Sciences, Faculty of Science and Technology, University of
Coimbra, Calçada Martim de Freitas, 3000-393, Coimbra, Portugal
f.
Center of Neurosciences and Cell Biology, CNC, University of Coimbra, Rua Larga,
Faculty of Medicine, 3004-504, Coimbra, Portugal
*Corresponding authors: C.F.G.C. Geraldes: geraldes@ci.uc.pt; E. Tóth: Eva
.JAKABTOTH@cnrs.fr; S.M.A.Pinto: smpinto@qui.uc.pt
g.
Electronic supplementary information (ESI) available. Figs. S1-S12: 1H, 19F NMR
and ESI-FIA-TOF mass spectra of Porphyrin 2; ESI-FIA-TOF mass spectra of MnIII-
3; EPR spectra at 25ºC of MnCl2 and MnIII3 in PBS (pH 7.4); 1H and 19F NMR
spectra of MnIII-3 and MnII-3 in CDCl3; Cyclic voltammograms of ZnII-3 and
MnIII-3; UV-Vis spectra of the re-oxidation of MnII-3 with hydrogen peroxide; UV-
Vis spectrum of 0.041mM MnIII-3 and of MnII-3 24 h after addition of 25.3
equivalents of ascorbic acid; Kinetic results for the reduction of MnIII-3 to MnII-3
with ascorbic acid and with mercaptoethanol in PBS (25 ºC, pH = 7.4); 13C NMR
of glutathione without and with 0.1 equiv of MnIII-3 in D2O; Table S1: Reduction
potentials of ZnII-3 and MnIII-3 and comparison with literature values for some
perfluorinated tetraphenylporphyrins. Equations used to analyze the 1H NMRD
profiles. This material is available free of charge via the Internet at
http://pubs.acs.org
.

ARTICLE Journal Name
2 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx
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prognosis or monitoring therapeutic response.
13, 14
The
fundamental goal of developing molecular probes capable of
imaging redox activity has seen some clinical success. In
particular, positron emission tomography (PET) probes capable
of targeting hypoxic tissue, such as the radiotracers
64
Cu
II
-
diacetyl-bis(N4-methylthiosemi-carbazone) (
64
Cu
II
-ATSM) and
18
F-fluoro-misonidazole (
18
F-MISO), showed capacity to predict
treatment outcome in patients undergoing radiotherapy.
15-17
The same hypoxia targeting mechanism of probe uptake
(irreversible reaction and retention in oxygen-deprived tissue)
has been extended to MRI contrast agents and fluorescent
probes for optical imaging.
18, 19
Redox-sensitive nitroxide
derivative probes were also used in electron paramagnetic
resonance (EPR) imaging and spectroscopy to detect reductive
environment (thiols and other reducing species).
20, 21
Magnetic resonance imaging (MRI) is an attractive modality to
monitor redox dynamics thanks to its exceptional spatial
resolution (<100
m using modern high-field equipment), lack
of invasiveness and use of ionizing radiation, and its ability to
simultaneously report physiological and anatomical
information. Not surprisingly, the interest in the development
of redox sensitive MRI probes has grown over the last years.
14,
22-26
These can be based on redox active ligands, undergoing a
change in the ligand structure or the molecular rotation upon
reduction/oxidation, while the oxidation state of the
paramagnetic metal remains the same (e.g. merocyanine unit
linked to a GdDO3A which isomerizes to the spirooxazine
derivative by the redox stimulus of NADH).
27
Alternatively,
complexes of redox active paramagnetic metals, like europium
and manganese, can be also explored, in which the different
oxidation states have different magnetic properties.
11, 22
Utterly, both types of probes must fulfill several requirements:
i) biocompatibility, ii) a redox half-cell potential which is
compatible with the redox potential of biological reducing
agents; iii) strong signal change, if possible signal increase upon
activation (“turn-on” probe); and iv) rapid redox kinetics as
compared to the imaging time scale. Importantly, the redox
active metal ion needs to be complexed by a ligand that is
capable of efficiently chelating both oxidation states such that
reduction or oxidation does not result in the decomposition of
the complex.
Among redox active metal ions, manganese is particularly
attractive for the development of redox responsive MRI probes,
whose efficiency is measured by their proton relaxivity r
i
(i = 1,2)
(paramagnetic enhancement of the water proton relaxation
rates, R
i
= T
i
-1
, where T
1
and T
2
are, respectively, the longitudinal
and transverse relaxation times, referred to 1 mM
concentration of the paramagnetic ion). Manganese is a
biogenic element and in its +2 oxidation state, Mn
2+
, is a very
efficient paramagnetic relaxation agent. Mn
2+
(d
5
configuration,
high spin S = 5/2), with long (in the 0.1 1 ns range) longitudinal
electronic relaxation times and labile water exchange, is an
attractive alternative to Gd
3+
in MRI probes.
28, 29
Indeed, in the
last years, there has been intensive research to identify suitable
ligand structures for Mn
2+
chelation.
30, 31
Mn
3+
(d
4
configuration)
is also paramagnetic, usually giving rise to high spin S = 2
compounds. While water exchange is also fast,
14
the
longitudinal electronic relaxation times are shorter (≤ 10 ps),
making them less efficient relaxation agents.
32
The nuclear
relaxation mechanism of Mn
3+
is different from that of Mn
2+
and
therefore their relaxation efficiency shows a very distinct
dependence on the external magnetic field B
0
. While for the S =
5/2 Mn
2+
complexes, the classical Solomon-Bloembergen-
Morgan (SBM) formalism describes well nuclear relaxation,
leading to a decrease of r
1
at high field, for the S = 2 Mn
3+
complexes the analysis of the water proton relaxation rates is
much less straightforward. Water soluble and air stable Mn
3+
-
porphyrins have been investigated as tumor targeted MRI
agents.
33
For these compounds, the electron relaxation times
are relatively long due to their higher symmetry.
32
For instance,
the Mn
III
-TPPS
4
(meso-tetra(4-sulfonatophenyl)porphine
chloride) chelate
34
displays an anomalous r
1
relaxivity, with a
peak above approximately 2 MHz. It was shown that the NMR
relaxation mechanism has several unique aspects, including the
unusual role of large rhombic zero-field splitting (ZFS)
interactions, which are of the order of the nuclear Zeeman
energy. The effect of these interactions on the electron spin
dynamics leads to those relaxivity anomalies at high field.
35,36
As
a consequence of the different relaxation mechanisms, the
proton relaxivity, and thus the MRI efficiency can be either
higher or lower for the reduced Mn
2+
relative to the oxidized
Mn
3+
form, depending on the external magnetic field and on the
chelating ligand.
Porphyrins, which are macrocyclic ligands per excellence and
widely used in a multitude of applications, such as catalysis,
37-39
materials science
40-43
and biomedicine, including MRI,
30, 44-48
offer the possibility to obtain very stable chelates to host many
metal ions, including paramagnetic ones. Such
metalloporphyrins having a much reduced risk of
transmetallation, may avoid potential in vivo metal release
during the MRI examination which would cause toxicity, such as
for instance gadolinium induced fibrosis disorders,
49
or
manganese induced neurotoxicity.
50 50 50 50 50 50 50 50 50 50 50
Many
studies have been reported of Mn
III
-porphyrin dimers,
supramolecular and macromolecular conjugates as MRI
contrast agents,
33, 51-58
including enzyme
55, 56
and Zn
2+
-
responsive
57, 58
agents. So far, the only studies on Mn-
porphyrins as redox MRI probes were reported by Aime and
collaborators,
59
who explored a Mn
II
/Mn
III
-based water-soluble
porphyrin (TPPS
4
) as an oxygen sensor. However, at clinically
relevant fields (
1
H frequencies of 60-120 MHz), and at 25 ºC, the
r
1
relaxivity
values revealed to be very close for the Mn
II
and
Mn
III
analogues. On the other hand, the formation of
macromolecular conjugates, through the encapsulation of the
Mn
II
-TPPS
4
and Mn
III
-TPPS
4
complexes in commercially
available, water-soluble poly-
-cyclodextrin (CD), led to a
marked difference in relaxivity at 20 MHz between the reduced
and oxidized state.

Journal Name ARTICLE
This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3
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Caravan and collaborators
30
explored
hydroxybenzylethylenediamine acetic acid derivatives for
Mn
II/III
-chelation to create redox active contrast agents. The
stability of the complexes was recently improved by using a
“Janus” ligand that readily isomerizes between binding modes
that favor either the Mn
3+
or Mn
2+
ion.
30
Rapid and reversible
interconversion between the two oxidation modes was
achieved by peroxidase activity (oxidation) and L-cysteine
(reduction), which was translated into an unprecedented 9-fold
relaxivity increase between the Mn
3+
and Mn
2+
analogue,
respectively. One concern, nevertheless, about these open-
chain complexes is the lack of a sufficiently high kinetic
inertness which is desirable for in vivo applications.
Based on previous experience in porphyrin chemistry, our
objective is to develop water soluble, biocompatible Mn-
porphyrin complexes with a redox potential that is adapted to
biological requirements. The approach to the development of
such specifically designed Mn
II/III
complexes consists of
modulating the redox potential by using appropriate electron
withdrawing substituents on the porphyrin structure. The fine-
tuning of the redox potential would be highly important since
most of the redox responsive MRI probes reported so far do not
undergo redox chemistry in a biologically relevant range.
22
The
capacity of porphyrin complexes to selectively localize in tumor
cells is well known and will be a great advantage. Indeed, it can
simplify probe design as no additional targeting and cell
internalizing moieties are required, although they might be later
added to the chemical structure to further improve these
properties.
Herein we report the synthesis and physical-chemical
characterization of a new fluorinated Mn
II/III
porphyrin
derivative which was conjugated with PEG chains for better
solubility and biocompatibility. Its redox properties were
studied by cyclic voltammetry. The Mn
III
reduction kinetics was
followed in the presence of ascorbic acid and
-
mercaptoethanol (BME) by UV-Vis. The relaxivity differences
between the reduced and oxidized forms were investigated
using
1
H Nuclear Magnetic Relaxation Dispersion (NMRD).
Cytotoxicity evaluation of the system on epithelial HeLa cells is
also reported.
Results and Discussion
S
ynthesis of the biocompatible porphyrin-based probes
The strategy adopted for the synthesis of the Mn
III
biocompatible probe
3
is depicted in
Scheme 1
.
Scheme 1.
Synthetic route for the preparation of the biocompatible porphyrin
3
. Reagents and conditions: i) DMF, NaH, room temperature, 2h; ii) acetic acid/sodium
acetate, Mn(II)acetate tetrahydrate, 80ºC, 2h.
Porphyrin
1
was synthesized by the NaY method, developed by
some of us,
60
where NaY acts as a reusable catalyst. After work-
up 12% of the isolated porphyrin was obtained. In order to
obtain free base porphyrin
2
, nucleophilic aromatic substitution
of
1
using mono methyl protected polyethylene glycol with Mw
= ~500 g/mol (PEG500) was performed, in DMF as solvent and
NaH as base, at room temperature for two hours. The product
was obtained in 65% yield, after purification, and fully
characterized by
1
H,
19
F NMR spectroscopy and ESI-FIA-TOF
mass spectrometry (see
Figs. 1, S1, S2 and S3
,
ESI
). Its main
characterization detailing feature was ascertained by observing
the loss of the characteristic triplet (p-F)
19
F NMR signal at
around -150 ppm (
Fig. 1
) for porphyrin
2
, when compared with
the
19
F NMR spectrum of
1
, which presents its three typical (m-
F), (p-F) and (o-F) signals at -160.2, -150.1 and -135.4 ppm,
respectively (
Fig. 1
), confirming the tetrapegylation pattern
obtained.

Journal Name
ARTICLE
This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 4
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Figure 1.
19
F NMR of porphyrin
1
(black spectrum) and porphyrin
2
(blue spectrum).
The preparation of Mn
III
-
3
proceeded by reacting
2
with excess
manganese diacetate tetrahydrate in sodium acetate/acetic
acid buffer solution at 80 ºC for two hours. After several
extractions of the crude with dichloromethane/water, the
organic layer was dried with anhydrous sodium sulfate and
concentrated under vacuum giving
3
in 82% yield (
Scheme 1
).
The Mn
III
-
3
complex was characterized by ESI-FIA-TOF mass
spectrometry (see
Fig. S4, ESI
), presenting the characteristic
poly-dispersed m/z peaks from PEGylated 1121.3612,
1319.9802, 1386.0195, 1493.6057 [M]
2+
(polydisperse
distribution). Additionally, Electron Paramagnetic Resonance
(EPR) experiments were also performed, in order to confirm the
absence of the Mn
II
salt used in the complexation reaction. The
absence of the sharp sextet characteristic of the free aqueous
Mn
2+
cation even at a very high amplification, led to the
conclusion that the Mn
III
-
3
sample was free of aqueous Mn
2+
(
Figure S5, ESI
).
47
The EPR spectrum of the Mn
III
-
3
was also silent
at room temperature, as expected from the fast electron
relaxation characteristic of the integer-spin (non-Kramers)
porphyrin compound.
61
Characteristic EPR resonances of Mn
II
-
porphyrins have been observed at liquid nitrogen temperature
(e.g. a strong sextet at g
~
5.9-5.2, a signal at g
~ 2.02.1, and
sometimes three other weak signals at 1.23, 0.77 and 0.54).
61,62
If some Mn
II
-
3
were present in the sample, a broad signal at g
iso
~2.0 would be observable at room temperature,
62
which was
not the case.
Proton and
19
F NMR
The proton NMR spectrum of Mn
III
-
3
in CDCl
3
(
Fig. S6a
) shows a
broad signal at -22.76 ppm corresponding to the
-pyrrole
protons, characteristic of Mn
III
high-spin meso-porphyrins. The
large upfield shift and large broadening observed arise from the
dominant contact mechanism, due to the small magnetic
anisotropy of the coordinated Mn
III
ion and consequent dipolar
shift induced.
63, 64
The PEG chain CH
2
protons at 3.66 ppm are
unshifted and hardly broadened relative to the signal at 3.65
ppm for free PEG due to their long distance from the
paramagnetic center.
The
19
F NMR spectrum of Mn
III
-
3
(
Fig. S6b
) consists of a broad
signal centered at -126.2 ppm and a set of five upfield shifted
(in the range of -149.7 to -157.7 ppm) sharper (linewidths
around 190 Hz) signals of different intensities. This set of signals
corresponds to the fluorine atoms at the meta positions of the
meso-phenyl groups in the five possible combinations of the
chain lengths of the four polydisperse PEG chains with n = 10-
11. The broad signal around -126.2 ppm results from the
overlap of the broader signals of the fluorine atoms at the ortho
position, which are closer to the metal center.
65, 66
Upon
reduction of Mn
III
-
3
to Mn
II
-
3
,
all
the
19
F resonances are
completely broadened out, in accordance with the efficient
paramagnetic relaxation of Mn
II
(
Fig. S6c
).

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Journal ArticleDOI
TL;DR: The unique theranostic role of porphyrin based conjugated systems and their respective applications in disease diagnosis and therapy is reviewed.
Abstract: The synthesis and application of porphyrins has seen a huge shift towards research in porphyrin bio-molecular based systems in the past decade. The preferential localization of porphyrins in tumors, as well as their ability to generate reactive singlet oxygen and low dark toxicities has resulted in their use in therapeutic applications such as photodynamic therapy. However, their inherent lack of bio-distribution due to water insolubility has shifted research into porphyrin-nanomaterial conjugated systems to address this challenge. This has broadened their bio-applications, viz. bio-sensors, fluorescence tracking, in vivo magnetic resonance imaging (MRI), and positron emission tomography (PET)/CT imaging to photo-immuno-therapy just to highlight a few. This paper reviews the unique theranostic role of porphyrins in disease diagnosis and therapy. The review highlights porphyrin conjugated systems and their applications. The review ends by bringing current challenges and future perspectives of porphyrin based conjugated systems and their respective applications into light.

45 citations


Cites background from "A biocompatible redox MRI probe bas..."

  • ...An ideal contrast agent is one that is able to affect radiofrequency pulses and enhance image contrast on relaxation time weighted MR image (properties that are present in co-ordinated metal ions) [11,34]....

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Journal ArticleDOI
TL;DR: The results in both cell line and animal models of PaC suggest that these NPs represent an ideal agent for mediating effective MRI-guided chemotherapy-PDT, giving them great promise for the clinical treatment ofPaC.
Abstract: As nanomedicine-based clinical strategies have continued to develop, the possibility of combining chemotherapy and singlet oxygen-dependent photodynamic therapy (PDT) to treat pancreatic cancer (PaC) has emerged as a viable therapeutic modality. The efficacy of such an approach, however, is likely to be constrained by the mechanisms of drug release and tumor oxygen levels. In the present study, we developed an Fe(III)-complexed porous coordination network (PCN) which we then used to encapsulate PTX (PCN-Fe(III)-PTX) nanoparticles (NPs) in order to treat PaC via a combination of chemotherapy and PDT. The resultant NPs were able to release drug in response to both laser irradiation and pH changes to promote drug accumulation within tumors. Furthermore, through a Fe(III)-based Fenton-like reaction these NPs were able to convert H2O2 in the tumor site to O2, thereby regulating local hypoxic conditions and enhancing the efficacy of PDT approaches. Also these NPs were suitable for use as a T1-MRI weighted contrast agent, making them viable for monitoring therapeutic efficacy upon treatment. Our results in both cell line and animal models of PaC suggest that these NPs represent an ideal agent for mediating effective MRI-guided chemotherapy-PDT, giving them great promise for the clinical treatment of PaC.

25 citations


Journal ArticleDOI
TL;DR: Overall, this work aims to provide the reader with a comprehensive view of how intracellular or extracellular redox buffer systems can be assessed by using MRI contrast agents based on lanthanide or transition metal ions using T1- weighted, T2-weighted, paraCEST 1H or 19F MRI.
Abstract: Given their potential in a better characterization and diagnosis of major pathologies like cancer or chronic inflammation, redox-activated Magnetic Resonance Imaging (MRI) probes have recently attracted much interest from chemists. Such redox responsive probes are capable of reporting on specific biomarkers that are related to tissue redox potential disruption or hypoxia. Lately, this research area has experienced remarkable development, including redox-responsive metal complexes and nanoparticles. Here we critically review the progress with a specific focus on metal-based probes and some nanoparticle examples. We demonstrate, via representative cases, the different molecular mechanisms that can generate a redox-modulated MRI response. They can be based on the redox activity of either the ligand or the metal center, provided the different oxidation states of the metal ion are endowed with different magnetic properties. A particular emphasis is given to recent advances and to the imaging probes that have attained in vivo validation. In overall, we aim to provide the reader with a comprehensive view of how intracellular or extracellular redox buffer systems can be assessed by using MRI contrast agents based on lanthanide or transition metal ions using T1-weighted, T2-weighted, paraCEST 1H or 19F MRI.

21 citations


Journal ArticleDOI
Abstract: The preparation of covalently linked manganese(III) meso-tetrakis(2,6-dichlorophenyl)porphyrin acetate to aminopropyl functionalized silica gel and its full characterization by standard spectroscopic methods including Fourier transform infrared spectroscopy (FTIR), thermogravimetry (TG) and scanning electronic microscopy (SEM) is described. The supported metalloporphyrin was evaluated as catalyst in the degradation of trimethoprim (TMP), using hydrogen peroxide as green oxidant. Catalyst was reused for five consecutive cycles without significant loss of activity or catalyst degradation/leaching. Degradation products were identified by UHPLC-MS, allowing to propose a plausible degradation pathway. Furthermore, ecotoxicity (EC50 and EC20) of TMP-based solutions (before and after degradation) were evaluated towards the bacterium V. fischeri, the microalgae R. subcapitata and the invertebrate B. calyciflorus.

7 citations


Journal ArticleDOI
Abstract: Four high-spin Fe(III) macrocyclic complexes, including three dinuclear and one mononuclear complex, were prepared toward the development of more effective iron-based magnetic resonance imaging (MRI) contrast agents. All four complexes contain a 1,4,7-triazacyclononane macrocyclic backbone with two hydroxypropyl pendant groups, an ancillary aryl or biphenyl group, and a coordination site for a water ligand. The pH potentiometric titrations support one or two deprotonations of the complexes, most likely deprotonation of hydroxypropyl groups at near-neutral pH. Variable-temperature 17O NMR studies suggest that the inner-sphere water ligand is slow to exchange with bulk water on the NMR time scale. Water proton T1 relaxation times measured for solutions of the Fe(III) complexes at pH 7.2 showed that the dinuclear complexes have a 2- to 3-fold increase in r1 relaxivity in comparison to the mononuclear complex per molecule at field strengths ranging from 1.4 T to 9.4 T. The most effective agent, a dinuclear complex with macrocycles linked through para-substitution of an aryl group (Fe2(PARA)), has an r1 of 6.7 mM-1 s-1 at 37 °C and 4.7 T or 3.3 mM-1 s-1 per iron center in the presence of serum albumin and shows enhanced blood pool and kidney contrast in mice MRI studies.

4 citations


References
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TL;DR: Estimates can be used to more fully understand the redox biochemistry that results from oxidative stress, which hopefully will provide a rationale and understanding of the cellular mechanisms associated with cell growth and development, signaling, and reductive or oxidative stress.
Abstract: Redox state is a term used widely in the research field of free radicals and oxidative stress. Unfortunately, it is used as a general term referring to relative changes that are not well defined or quantitated. In this review we provide a definition for the redox environment of biological fluids, cell organelles, cells, or tissue. We illustrate how the reduction potential of various redox couples can be estimated with the Nernst equation and show how pH and the concentrations of the species comprising different redox couples influence the reduction potential. We discuss how the redox state of the glutathione disulfide-glutathione couple (GSSG/2GSH) can serve as an important indicator of redox environment. There are many redox couples in a cell that work together to maintain the redox environment; the GSSG/2GSH couple is the most abundant redox couple in a cell. Changes of the half-cell reduction potential (E(hc)) of the GSSG/2GSH couple appear to correlate with the biological status of the cell: proliferation E(hc) approximately -240 mV; differentiation E(hc) approximately -200 mV; or apoptosis E(hc) approximately -170 mV. These estimates can be used to more fully understand the redox biochemistry that results from oxidative stress. These are the first steps toward a new quantitative biology, which hopefully will provide a rationale and understanding of the cellular mechanisms associated with cell growth and development, signaling, and reductive or oxidative stress.

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TL;DR: The paradoxical roles of the tumor microenvironment during specific stages of cancer progression and metastasis are discussed, as well as recent therapeutic attempts to re-educate stromal cells within the TME to have anti-tumorigenic effects.
Abstract: Cancers develop in complex tissue environments, which they depend on for sustained growth, invasion and metastasis. Unlike tumor cells, stromal cell types within the tumor microenvironment (TME) are genetically stable and thus represent an attractive therapeutic target with reduced risk of resistance and tumor recurrence. However, specifically disrupting the pro-tumorigenic TME is a challenging undertaking, as the TME has diverse capacities to induce both beneficial and adverse consequences for tumorigenesis. Furthermore, many studies have shown that the microenvironment is capable of normalizing tumor cells, suggesting that re-education of stromal cells, rather than targeted ablation per se, may be an effective strategy for treating cancer. Here we discuss the paradoxical roles of the TME during specific stages of cancer progression and metastasis, as well as recent therapeutic attempts to re-educate stromal cells within the TME to have anti-tumorigenic effects.

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TL;DR: The spectrophotometric/microplate reader assay method for glutathione (GSH) can assay GSH in whole blood, plasma, serum, lung lavage fluid, cerebrospinal fluid, urine, tissues and cell extracts and can be extended for drug discovery/pharmacology and toxicology protocols to study the effects of drugs and toxic compounds on glutATHione metabolism.
Abstract: The spectrophotometric/microplate reader assay method for glutathione (GSH) involves oxidation of GSH by the sulfhydryl reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) to form the yellow derivative 5'-thio-2-nitrobenzoic acid (TNB), measurable at 412 nm. The glutathione disulfide (GSSG) formed can be recycled to GSH by glutathione reductase in the presence of NADPH. The assay is composed of two parts: the preparation of cell cytosolic/tissue extracts and the detection of total glutathione (GSH and GSSG). The method is simple, convenient, sensitive and accurate. The lowest detection for GSH and GSSG is 0.103 nM in a 96-well plate. This method is rapid and the whole procedure takes no longer than 15 min including reagent preparation. The method can assay GSH in whole blood, plasma, serum, lung lavage fluid, cerebrospinal fluid, urine, tissues and cell extracts and can be extended for drug discovery/pharmacology and toxicology protocols to study the effects of drugs and toxic compounds on glutathione metabolism.

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TL;DR: The current understanding of how disturbance in redox homeostasis may affect cell death and contribute to the development of diseases such as cancer and degenerative disorders is reviewed and the basic knowledge on redox regulation of cell survival can be used to develop strategies for the treatment or prevention of those diseases.
Abstract: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important roles in regulation of cell survival. In general, moderate levels of ROS/RNS may function as signals to promote cell proliferation and survival, whereas severe increase of ROS/RNS can induce cell death. Under physiologic conditions, the balance between generation and elimination of ROS/RNS maintains the proper function of redox-sensitive signaling proteins. Normally, the redox homeostasis ensures that the cells respond properly to endogenous and exogenous stimuli. However, when the redox homeostasis is disturbed, oxidative stress may lead to aberrant cell death and contribute to disease development. This review focuses on the roles of key transcription factors, signal-transduction pathways, and cell-death regulators in affecting cell survival, and how the redox systems regulate the functions of these molecules. The current understanding of how disturbance in redox homeostasis may affect cell death and contribute to the development of diseases such as cancer and degenerative disorders is reviewed. We also discuss how the basic knowledge on redox regulation of cell survival can be used to develop strategies for the treatment or prevention of those diseases. Antioxid. Redox Signal. 10, 1343–1374.

1,378 citations


Journal ArticleDOI
TL;DR: A number of new therapeutic strategies currently under investigation for preventing myocardial reperfusion injury have the potential to improve clinical outcomes in patients with acute MI treated with PPCI.
Abstract: Acute myocardial infarction (MI) is a major cause of death and disability worldwide. In patients with MI, the treatment of choice for reducing acute myocardial ischemic injury and limiting MI size is timely and effective myocardial reperfusion using either thombolytic therapy or primary percutaneous coronary intervention (PPCI). However, the process of reperfusion can itself induce cardiomyocyte death, known as myocardial reperfusion injury, for which there is still no effective therapy. A number of new therapeutic strategies currently under investigation for preventing myocardial reperfusion injury have the potential to improve clinical outcomes in patients with acute MI treated with PPCI.

1,327 citations


Frequently Asked Questions (2)
Q1. What have the authors contributed in "A biocompatible redox mri probe based on a mn( ii )/mn( iii ) porphyrin" ?

Pinto et al. this paper proposed a new water-soluble fluorinated porphyrin derivative conjugated with PEG chains, which is capable of stabilizing both MnIII and MnII oxidation states in a biologically relevant range of redox potentials. 

Interestingly, 19F NMR resonances of MnIII-3 are relatively sharp which could open further possibilities to exploit such complexes as paramagnetic 19F NMR probes. In this respect, Mn-porphyrin redox responsive probes can be advantageous. The present MnII/MnIII-porphyrin couple satisfies many of the criteria required for a redox imaging probe: good water solubility, biocompatibility, non-toxicity, thermodynamic and kinetic stability in both metal oxidation states, a redox half-cell potential accessible to biologically relevant reducing agents with fast kinetics ( adapted to the imaging time scale ), and a strong “ turn on ” relaxivity response upon reduction. Their redox sensitive MRI probe might have potential to report, with fast kinetics ( few minutes half-life ), on extracellular ascorbate without the interference of the cysteine-cystine buffer.