Manganese-52: applications in cell radiolabelling and liposomal nanomedicine PET imaging using oxine (8-hydroxyquinoline) as an ionophore

Article abstract of DOI:10.1039/c8dt00100f

The ionophore 8-hydroxyquinoline (oxine) has been used to radiolabel cells and liposomal medicines with ¹¹¹In and, more recently, ⁸⁹Zr, for medical nuclear imaging applications. Oxine has also shown promising ionophore activity for the positron-emitting radionuclide ⁵²Mn that should allow imaging of labelled cells and nanomedicines for long periods of time (>14 days). However, to date, the radiometal complex formed and its full labelling capabilities have not been fully characterised. Here, we provide supporting evidence of the formation of [⁵²Mn]Mn(oxinate)₂ as the metastable complex responsible for its ionophore activity. The cell labelling properties of [⁵²Mn]Mn(oxinate)₂ were investigated with various cell lines. The liposomal nanomedicine, DOXIL (Caelyx) was also labelled with [⁵²Mn]Mn(oxinate)₂ and imaged in vivo using PET imaging. [⁵²Mn]Mn(oxinate)₂ was able to label various cell lines with moderate efficiency (15-53%), however low cellular retention of ⁵²Mn (21-25% after 24 h) was observed which was shown not to be due to cell death. PET imaging of [⁵²Mn]Mn-DOXIL at 1 h and 24 h post-injection showed the expected pharmacokinetics and biodistribution of this stealth liposome, but at 72 h post-injection showed a profile matching that of free ⁵²Mn, consistent with drug release. We conclude that oxine is an effective ionophore for ⁵²Mn, but high cellular efflux of the isotope limits its use for prolonged cell tracking. [⁵²Mn]Mn(oxinate)₂ is effective for labelling and tracking DOXIL in vivo. The release of free radionuclide after liposome extravasation could provide a non-invasive method to monitor drug release in vivo.

Full text of DOI:10.1039/c8dt00100f

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Manganese-52: applications in cell radiolabelling
and liposomal nanomedicine PET imaging using
oxine (8-hydroxyquinoline) as an ionophore†
Cite this: DOI: 10.1039/c8dt00100f
a
a
b
a
a
Peter Gawne,
Francis Man,
Jesper Fonslet, Riya Radia, Jayanta Bordoloi,
c
c
d
a
Matthew Cleveland, Pilar Jimenez-Royo, Alberto Gabizon, Philip J. Blower,
e
a
Nicholas Long
and Rafael T. M. de Rosales
*
The ionophore 8-hydroxyquinoline (oxine) has been used to radiolabel cells and liposomal medicines
1
11
89
with In and, more recently, Zr, for medical nuclear imaging applications. Oxine has also shown prom-
5
2
ising ionophore activity for the positron-emitting radionuclide Mn that should allow imaging of labelled
cells and nanomedicines for long periods of time (>14 days). However, to date, the radiometal complex
formed and its full labelling capabilities have not been fully characterised. Here, we provide supporting
5
2
evidence of the formation of [ Mn]Mn(oxinate)
2
as the metastable complex responsible for its ionophore
activity. The cell labelling properties of [ Mn]Mn(oxinate) were investigated with various cell lines. The
liposomal nanomedicine, DOXIL® (Caelyx) was also labelled with [ Mn]Mn(oxinate)
5
2
2
5
2
2
and imaged in vivo
5
2
using PET imaging. [ Mn]Mn(oxinate)
2
was able to label various cell lines with moderate efficiency
5
2
(
15–53%), however low cellular retention of Mn (21–25% after 24 h) was observed which was shown not
5
2
to be due to cell death. PET imaging of [ Mn]Mn-DOXIL at 1 h and 24 h post-injection showed the
expected pharmacokinetics and biodistribution of this stealth liposome, but at 72 h post-injection showed
5
2
Received 9th January 2018,
Accepted 20th March 2018
a profile matching that of free Mn, consistent with drug release. We conclude that oxine is an effective
52
52
ionophore for Mn, but high cellular efflux of the isotope limits its use for prolonged cell tracking. [ Mn]
Mn(oxinate) is effective for labelling and tracking DOXIL in vivo. The release of free radionuclide after
DOI: 10.1039/c8dt00100f
2
rsc.li/dalton
liposome extravasation could provide a non-invasive method to monitor drug release in vivo.
ligands are usually lipophilic and have low denticity. Binding
of the radiometal with the ionophore results in a complex that
Introduction
Ionophores are organic ligands that facilitate transport of is both lipophilic and uncharged, and able to passively cross
metal ions passively across lipid bilayers. In particular, iono- lipid bilayers (Scheme 1). The radiometal-ionophore complexes
phores have been widely used since the 1970s to directly label are commonly meta-stable and dissociate inside the cell/lipo-
and image both cells and liposomal medicines in vivo with
radiometals using clinical nuclear imaging techniques such as
single-photon emission tomography (SPECT) and more
recently positron emission tomography (PET). Ionophore
a
School of Biomedical Engineering & Imaging Sciences, King’s College London,
St Thomas’ Hospital, London, SE1 7EH, UK. E-mail: rafael.torres@kcl.ac.uk
b
The Hevesy Lab, Technical University of Denmark, 4000 Roskilde, Denmark
c
GSK Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire,
SG1 2NY, UK
d
Oncology Institute, Shaare Zedek Medical Center and Hebrew University-School of
Medicine, Jerusalem 9103102, Israel
e
Department of Chemistry, Imperial College London, South Kensington Campus,
London SW7 2AZ, UK
Scheme 1 Diagram showing the proposed mechanism of labelling cells
and liposomes using radio-ionophore complexes. (A) The neutral lipo-
philic radio-ionophore complex crosses lipid bilayer. (B) The meta-stable
complex dissociates and (C) the radio-metal binds to intracellular pro-
teins/macromolecules or drugs with chelating groups within liposomal
medicines.
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8dt00100f. The data supporting this research are openly available from the
King’s College London research data archive at http://dx.doi.org/10.18742/
RDM01-283.
This journal is © The Royal Society of Chemistry 2018
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5
2
some, at which point trapping occurs via the binding of the have shown that oxine has promising Mn ionophore activity
1
2
radiometal to intracellular proteins or intraliposomal drug in vitro using liposomes as a model. The radiolabelling yields
molecules – provided they have chelating groups – or other and in vitro serum stability properties where comparable to
2
89
4
metal-chelating ligands (Scheme 1). As such, effective radio- those obtained with [ Zr]Zr(oxinate) . However, the identity of
5
2
ionophore agents should facilitate fast uptake and slow radio- the [ Mn]Mn-oxine complex was not known and its cell label-
nuclide efflux, whilst not affecting the viability or function of ling and in vivo liposome tracking ability was unexplored. Here,
cells/liposomes.
we describe the synthesis and characterisation of the radiometal
5
2
The longitudinal imaging/tracking of living cells and lipo- complex [ Mn]Mn(oxinate) (Fig. 1C) and evaluated its cell-
2
somal nanomedicines within a living organism has appli- labelling properties. Additionally, the in vivo stability and bio-
5
2
cations in locating inflammation (labelled leukocytes) and distribution of Mn-labelled liposomes, radiolabelled with this
determining the biodistribution of therapeutic cells and radiotracer, were investigated in mice with PET imaging using
nanomedicines. To allow this, the choice of radionuclide is the clinically approved nanomedicine DOXIL® (Caelyx).
important. One of the most widely used radio-ionophore
complexes to date is the tris(oxinate) complex of the gamma-
1
11
emitting radionuclide
In (t1/2 = 2.8 days) (Fig. 1A). Known
Results and discussion
111
as “[ In]In-oxine” this compound has been used clinically since
5
2
Radiosynthesis of [ Mn]Mn(oxinate)
2
the 1980s to radiolabel autologous leukocytes for imaging of
infection and inflammation in vivo, and was recently with- [ Mn]Mn(oxinate) can be synthesised rapidly and reliably by
3
,4
52
2
5
2
drawn from the EU market due to the perceived low cost-effec- the addition of oxine (from a DMSO stock solution) to [ Mn]
5
tiveness by the supplier. More recently, it has regained atten- MnCl2 in dilute HCl, followed by neutralisation with 0.1 M
tion for its ability to directly label and track a variety of ammonium acetate solution (pH 7) and a brief heating step at
6
–11
immune cells.
With the growing availability of PET, focus 50 °C (Fig. 2A). Instant thin layer radiochromatography (iTLC)
has shifted towards the development of PET tracers for cell lab- analysis using a mobile phase of 25% methanol in chloroform
5
2
elling. PET imaging offers higher sensitivity than SPECT shows that whereas [ Mn]MnCl stays at the baseline (R = 0)
2
f
imaging as well as improved spatial resolution and quantifi- radioactivity of the product solution migrates with the solvent,
1
2
89
cation in the clinical setting. In particular, Zr complexes indicating the formation of the expected lipophilic compound.
+
(t1/2 = 3.27 days; β = 22.3%) have been investigated as PET R values for the oxine compound were inconsistent (R =
f f
1
11
alternatives to [ In]In-oxine. Zirconium and indium have 0.3–0.8) most likely due to varying amounts of DMSO present
similar preferred ligand types despite the different in the reaction mixture. The radiochemical yield (RCY) was
preferred oxidation states (In(III) and Zr(IV)) – as well as having 69 ± 20% (n = 3) based on iTLC analysis, which we also used as
–
1
89
4
similar half-lives, and the PET tracer [ Zr]Zr(oxinate) (also an estimate of the radiochemical purity. The lipophilicity of
8
9
52
known as [ Zr]Zr-oxine, Fig. 1B) has shown excellent cell [ Mn]Mn(oxinate) was confirmed with log P measurements
labelling and tracking properties.
Zr, combined with improved cellular retention of the radio- whereas the log P of MnCl showed the expected high hydro-
2
nuclide compared to
cell tracking with PET (7–14 days) with various cell types.
4 4
Zr]Zr(oxinate) has also been used to directly label and track over [ Zr]Zr(oxinate) : it does not require the solvent extrac-
2
1
,13
The longer half-life of using octanol/water solvent extraction (log P
= 1.5 ± 0.1),
water
8
9
52
1
11
13
In, allowed more prolonged in vivo philicity of a hydrated manganese ion (log P = −1.2 ± 0.3)
1
3–15
52
(Fig. 2B). The synthesis of [ Mn]Mn(oxinate) has benefits
2
8
9
89
[
liposomal medicines in vivo for up to 7 days, without the need tion step required to remove oxalate/oxalic acid from the final
8
9
for modification of the nanomedicine or interference with its [ Zr]Zr(oxinate) product, involving vigorous vortexing fol-
manufacture.
4
2
lowed by separation and evaporation of the organic layer
In our search for new radiometals to track cells/nanomedi- (CHCl ). Sato et al. recently reported an improved synthetic
3
8
9
89
cines with PET for longer periods of time we turned our atten- method for [ Zr]Zr(oxinate) from [ Zr]ZrCl in aqueous
tion towards Mn (t1/2 = 5.59 days, β = 29.6%). Recently we media, however vortexing of the mixture was still necessary.
4
4
5
2
+
14
111
89
52
3 4 2
Fig. 1 Structures of the radio-ionophore complexes discussed: [ In]In(oxinate) (A), [ Zr]Zr(oxinate) (B) and [ Mn]Mn(oxinate) (C).
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52
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Fig. 2 (A) Radiochemical scheme for the synthesis of [ Mn]Mn(oxinate)
2
, and (B) chart showing the log P values of unchelated Mn and [ Mn]Mn
(
oxinate) in water/octanol. Error bars represent standard deviation (n = 2).
2
nat
Synthesis and characterisation of Mn(oxinate)2
λmax = 255 nm was observed across each pH. In the absence of
oxine, no increase in absorbance was seen, demonstrating that
the absorbance increase is due to metal–ligand binding. At
each pH, a decrease in the absorbance change occurred after
To determine the chemical identity of the species formed
during radiosynthesis we synthesised and characterised the
non-radioactive Mn(oxinate) complex ( Mn = Mn = Mn =
2
naturally occurring Mn). This was achieved by addition of 0.5
equivalents of MnCl to a basic solution of 8-hydroxyquinoline
5
5
55
nat
0
.5 equivalents of MnCl , indicating the preferential formation
2
of the bis-oxine complex. This was particularly prominent with
increasing pH values (e.g. pH 9) which is consistent with the
2
–
deprotonated with one equivalent of potassium hydroxide –
relatively high pK of the hydroxyl group from the oxine ligand
a
resulting in the formation of a pale yellow precipitate.
Formation of the bis-oxinate Mn(II) complex was confirmed
with mass spectrometry (electrospray in positive mode) with a
m/z peak at 344.0348 consistent with a protonated Mn
oxinate) complex (M + 1) and another peak at 687.0684 relat-
2
ing to a protonated 2[Mn(oxinate) ] species (2M + 1) (Fig. S1†).
No peaks were observed with m/z matching a ligand-to-metal
stoichiometric ratio of 3 : 1, suggesting that the oxidation of
3
complex had not occurred.
Additionally, IR spectroscopy showed a reduction of the broad
O–H stretch band at 2700–3400 cm upon metal complexa-
tion – relating to the deprotonation of the hydroxyl associated
with formation of the Mn–O bond (Fig. S2†). Further analysis
of Mn(oxinate) complex was limited by its insolubility in all
the organic solvents tested. Whilst previous work has reported
1
9
(
a
pK = 11.54). Additionally, the absorbance at 255 nm was
plotted as a function of the mole fraction (relative proportion,
or fraction, of a compound in solution) of MnCl added
2
2
0
(
known as a Job plot ). The Job plot in Fig. 3D showed that
(
2
the change in absorption decreased once a mole fraction of
MnCl2 reached 0.33, characteristic of a ML2 complex (where
M = Mn; L = 8-hydroxyquinoline). In the case of Mn(oxinate)
ML ) being preferentially formed, the absorption would be
3
(
3
the metal to form a Mn(oxinate)
seen to decrease at a mole fraction of 0.25 – which we did not
observe. Additionally, electron paramagnetic resonance (EPR)
spectroscopy of a solution of 1 mM oxine (1.46% v/v DMSO in
−
1
1
mM ammonium acetate solution) mixed with 0.5 equivalents
2
+
of MnCl
2
showed the presence of Mn (Fig. S4†). These
2
results lead us to propose that the chemical species formed
5
2
1
6,17
during the radiosynthesis is [ Mn]Mn(oxinate) .
2
the synthesis of Mn(oxinate)
ised the structure of the manganese complex. Li et al. reported
the 1D coordination polymer of [Mn(oxinate) and showed
each Mn ion was bound to the oxine ligand in a severely dis-
2
,
few reports have character-
Another important aspect of the direct cell radiolabelling
strategy is that the compound (i.e. Mn(oxinate) ) must be meta-
2
2 n
]
2
+
stable, with sufficient stability to cross the cell membrane but
unstable enough to decompose inside the cell releasing the
radiometal (Scheme 1). To test the metastability of Mn
1
8
torted N O octahedral geometry. However the high tempera-
2
4
ture (453 K) synthesis method used in this case limits compar-
ability with the compound synthesised herein.
(
oxinate) , we monitored its absorbance at 255 nm using the
2
same conditions as in the UV-vis titrations. We observed
gradual decomposition of the complex indicated by a decrease
in the absorption (Fig. S5†). This process, however, is slow and
only a small fraction of decomposition is evident after 1 h.
Taking into account that direct cell labelling using ionophores
is fast (<30 min), and that metastability is required for metal
release inside the cells, these results further support the use of
this complex for direct cell labelling.
To gain further insight into the preferential stoichiometry
of the complex being formed in the radiochemical reaction,
UV-vis spectrophotometric titrations were carried out (repre-
sentative spectra in Fig. S3†). Unlike other characterisation
spectroscopic methods such as NMR, the low concentrations
of reactants required for UV-vis spectrometry are comparable
to those in radiochemistry which is always performed at low
radiometal concentrations and metal-to-ligand ratios. Thus,
the change in the absorbance spectrum of a 0.1 mM 8-hydroxy-
quinoline solution (1.46% v/v DMSO in 1 mM ammonium
5
2
In vitro cell labelling with [ Mn]Mn(oxinate)
2
5
2
acetate solution) was monitored upon titration of increasing The cell labelling properties of [ Mn]Mn(oxinate) were
2
2
equivalents of MnCl at pH 7–9 (Fig. 3A–C and S3†). Upon assessed in three different cell lines and in human platelets.
addition of MnCl , an increase in an absorption band with Table 1 summarises the cell labelling efficiencies and cellular
2
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Fig. 3 UV-vis titration (λ = 255 nm) of MnCl
2
into a 0.1 mM solution of oxine (1.46% v/v DMSO in 1 mM ammonium acetate solution) performed at
room temperature at (A) pH 7; (B) pH 8; (C) pH 9; (D) a Job plot of mole fraction (fraction of reagent in solution) of MnCl
2
versus absorbance. Mole
fraction of Mn = [χMn/(χMn + χoxine)] where χMn = total moles of MnCl in solution and χoxine = total moles of oxine in solution.
2
5
2
52
6
2
Table 1 Cell labelling efficiency of [ Mn]Mn(oxinate) and unchelated Mn. Labelling conditions: § – 10 cells incubated for 30 min at 37 °C in
3
6
8
mL cell medium; † – 5×10 cells per mL incubated for 20 min at room temperature; ‡ – 10 cells per mL incubated for 30 min at 37 °C. All values
given as mean ± standard deviation. * – single experiment carried out in triplicate
5
2
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Cell line labelled (LE = labelling efficiency)
[
Mn]Mn(oxinate)
2
LE (%)
Unchelated Mn LE (%)
§
Breast cancer cells (MDA-MB 231)
33.4 ± 9.2 (n = 5)
52.8 ± 3.8*
45.6 ± 29.1 (n = 3)
14.8 ± 1.1*
3.9 ± 3.2 (n = 5)
8.5 ± 0.6*
0.9 ± 0.4 (n = 3)
2.7 ± 0.1*
§
Human embryonic kidney cells (HEK-293T)
Gamma delta (γδ) T-cells
Human blood platelets
†
‡
5
2
52
retention of Mn. Across all cell types, the presence of oxine
Mn in these cells after 24 h, however, was approximately
8
9
resulted in higher cellular uptake compared to that achieved three times lower than that of Zr (27.1 ± 6.8% and 74.9 ±
with the unchelated Mn control, demonstrating the iono- 6.2%, respectively) (Fig. 4A). Further analysis with Mn-
phore properties of oxine for Mn(II). The presence of excess labelled MDA-MB-231 breast cancer cells showed that cellular
amounts of a similar divalent metal such as Ca in the cell efflux of Mn from labelled cells occurs rapidly with over 50%
medium (1.8 mM) during the MDA-MD-231 radiolabeling of the initial intracellular Mn leaving the cells after 4 hours.
5
2
52
2
+
52
5
2
experiments, compared to the pM-nM concentrations of This result confirms this efflux is fast and not cell-specific, at
5
2
[
Mn]Mn(oxinate) , demonstrates sufficient complex inertness least for the cells tested in this work (Fig. 4B). A possible expla-
2
for direct cell labelling. This was surprising due to the simi- nation for this rapid efflux of the radiometal is cell death due
larity of these two metals and the expected lability of the Mn to the radiolabelling process. However, a cell viability assay
(
oxinate) complex. Furthermore, UV-vis studies demonstrated comparison (trypan blue) showed no different between
2
2
+
52
oxine selectivity for Mn by showing complete formation of MDA-MB-231 cells labelled with [ Mn]Mn(oxinate)
2
(0.05 Bq
nat
Mn(oxinate)
2
even in the presence of a large excess (up to 20 per cell) and non-labelled cells, both immediately and 24 h
2
+
52
eq.) of Ca (Fig. S6†). The cell labelling ability of [ Mn]Mn after labelling (Fig. 4C). To investigate the cellular efflux of
8
9
52
(
oxinate)
2
was directly compared to that of [ Zr]Zr(oxinate)
4
in
showed various time points during the 30 min labelling period of
comparable cell labelling efficiency to [ Zr]Zr(oxinate) (45.6 ± MDA-MB-231 cells and lipophilicity measurements of this
Mn further, aliquots of the cell supernatant were taken at
5
2
gamma-delta T-cells. Interestingly, [ Mn]Mn(oxinate)
2
8
9
4
2
9.1% and 46.6 ± 6.8%, respectively), suggesting that the solution were performed to identify the nature of the radio-
5
2
number of oxine ligands in the primary coordination sphere of active species. At each time point log P values of the Mn
these metal complexes is not an important factor. Retention of species in the supernatant were negative/hydrophilic (ca. −1.5)
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89
Fig. 4 (A) Graph comparing the labelling efficiency (left) and cellular retention after 24 h (right) of [ Mn]Mn(oxinate)
2
(green) with that of [ Zr]Zr
52
(
oxinate)
4
(blue) in gamma-delta (γδ) T-cells. (B) Cellular retention of Mn over 24 h in MDA-MB 231 cells. (C) Cell viability of MDA-MB 231 cells
5
2
6
radiolabelled with [ Mn]Mn(oxinate)
2
(50 KBq per 10 cells) compared to unlabelled controls. All error bars represent standard deviation (n = 3).
philic compound, leaves the cell in a hydrophilic form, and (ii)
the hydrophilic log P values obtained during incubation
5
2
cannot be fully explained by decomposition of [ Mn]Mn
oxinate) in cell medium.
These results together suggest that Mn leaves the cell via
(
2
5
2
a specific cellular pathway. Manganese is a biologically essen-
tial metal found in a variety of tissues. It is particularly impor-
tant for the catalytic activity of the mitochondrial enzyme
superoxide dismutase (Mn-SOD) to detoxify reactive oxygen
2
1,22
2+
species.
Mn is neurotoxic, and Mn can cross the BBB
2
3–25
barrier via the divalent metal transporter-1 (DMT-1).
Hence, human biology has evolved mechanisms to manage
and transport manganese ions efficiently, and calcium chan-
2
+
nels are known to be permeable to Mn . However, little is
2
4
known about the intracellular handling of this element.
Fig. 5 Log P analysis of cell supernatant of MDA-MB 231 cells labelled Similar rapid cellular efflux was previously reported when lab-
5
2
64
with [ Mn]Mn(oxinate)
3
2
. Log P values of supernatant taken during the elling cells with
Cu, using the lipophilic, redox-active
0 min labelling period show a negative (hydrophilic value) compared
with the lipophilicity value of [ Mn]Mn(oxinate)
green bar on left). Error bars represent standard deviation (n = 3) except
complex Cu(PTSM) (PTSM = pyruvaldehyde-bis(N4-methyl-
thiosemicarbazonate)) which is known to release copper biore-
ductively immediately on entry into cells.
52
2
in cell-free medium
(
2
6,27
5
2
In these studies,
2
for the log P of [ Mn]Mn(oxinate) which is a single experiment per-
formed in triplicate.
after using this compound to label C6 glioma cells and murine
Th1 cells, just 38% and 47%, respectively, of the radiolabel
2
6,27
was retained 5 h post-labelling.
has several biological roles and the low retention of Cu may
Copper, like manganese,
6
4
5
2
whereas the log P value for [ Mn]Mn(oxinate)
2
in cell-free also be due to an active cellular process. If radiomanganese
medium was positive/lipophilic (0.88) (Fig. 5). These results leaves the cell due to a cellular process, this may limit the tech-
5
2
52
indicate that: (i) Mn, after entering the cell as part of a lipo- nique of directly labelling cells using
Mn ionophores.
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5
2
Although the tracer may be used to track cells at earlier time of [ Mn]Mn-DOXIL in mice using PET. Whilst previous
points, the rate of efflux means that the long half-life of the work has described the labelling and imaging of DOXIL with
3
8
39
40
isotope – and the resulting radiation dose – cannot alone technetium-99m, rhenium-186, and indium-111, this
5
2
justify use of Mn for this application.
is the first time to the best of our knowledge that it has been
tracked using PET imaging. [ Mn]Mn-DOXIL was prepared
with a labelling efficiency of >80%. At 1 h post-injection
p.i.) of [ Mn]Mn-DOXIL (1 MBq, 5 mg kg doxorubicin
5
2
5
2
Liposome (DOXIL/CAELYX) labelling with [ Mn]Mn(oxinate)
2
for in vivo PET imaging
52
−1
(
In addition of being used to label cells directly, our group has dose) in female B6CBAF1 mice, the majority of the radioac-
recently shown that oxine is an excellent ionophore for radio- tivity was observed in the bloodstream, with high signal in
metal labelling of liposomal nanomedicines that encapsulate the heart and major blood vessels such as the carotid
metal-chelating drugs, including preliminary in vitro work with arteries and descending aorta (Fig. 6A). Some activity in
5
2
2
52
Mn. Interestingly, [ Mn]Mn(oxinate)
labelled the FDA-approved nanomedicine DOXIL/CAELYX® in the liver and spleen. This slow transition from the blood-
PEGylated liposomal doxorubicin) with high efficiency (>80%) stream at early time points to the gradual accumulation in
and high in vitro human serum stabilities (ca. 95% after 72 h the spleen and liver is typical of “stealth” PEGylated lipo-
2
efficiently radio- these regions remained at 24 h p.i., with increasing uptake
(
2
52
at 37 °C). The high retention of Mn inside the doxorubicin- somal nanomedicines such as DOXIL; the stealth properties
loaded liposome is not surprising as Mn has previously been of these liposomes inhibit recognition by the mononuclear
shown to drive the loading of doxorubicin into liposomes by phagocyte system/reticuloendothelial system (MPS/RES).
forming an stable intraliposomal Mn-doxorubicin complex.
2
+
4
1,42
2
8
52
The circulation half-life of [ Mn]Mn-DOXIL was calculated
by measuring radioactivity in blood samples taken at
2
+
Abraham et al. characterised the complexation between Mn
and doxorubicin showing that the metal coordinates with a specific time points (Fig. 6B). The data were fitted to a one-
bidentate site consisting of the carbonyl and hydroxyl groups compartment pharmacokinetic model that allowed the cal-
2
9
on the central aromatic ring system.
The longitudinal imaging of liposomal medicines in vivo curve (AUC). Thus, the calculated circulation half-life (t1/2
not only gives information on their biodistribution, but can be was 33.6 h, typical of a stealth nanomedicine and similar to
culation of the circulation half-life (t1/2) and area under the
)
3
0,31
used to predict the efficacy of new treatments.
lar, imaging can provide information supporting ‘personalised CAELYX (20–30 h).
medicine’ in which the response to liposomal therapies can be ling is stable while in the bloodstream (since the observed
predicted from patient-to-patient. For this, particular focus has biodistribution does not match that of free
been placed on the use of nuclear imaging techniques because below).
In particu- that calculated by other preclinical studies with DOXIL/
3
8,39,42
This also confirms that radiolabel-
5
2
Mn, see
5
2
their high sensitivity gives them the ability to image thera-
Despite the confirmed stability of [ Mn]Mn-DOXIL in cir-
peutic nanomedicines using very low doses which have a culation and observation of the expected biodistribution at
3
2
reduced or no physiological effect (microdosing). The radi- early time points, the PET images at 72 h p.i. (Fig. 6A)
5
2
olabelling technique developed by our group differs from pre- showed a profile characteristic of unchelated Mn (high
4
3,44
vious methods developed in which the radiometal is chelated signal in kidneys, pancreas and salivary glands
to ligands on a modified lipid bilayer,
). This was
as well as direct confirmed by ex vivo biodistribution studies at 72 h p.i.
incorporation of the radio-isotope into the lipid molecule (Fig. 6C) that demonstrated high radioactivity in these organs
3
0,33,34
3
5
−1
−1
itself. The use of radio-ionophore complexes for directly lab- (kidneys (20 ± 3%ID g ), pancreas (11 ± 2%ID g ) and sali-
elling liposomes with PET radionuclides has been previously vary glands (8 ± 2%ID g− )). Conversely, spleen and liver
reported by others with Cu (t1/2 = 12 h; β = 17.8%) and uptake, commonly the organs with higher liposome presence
1
6
4
+
5
2
−1
recently Mn – using a variety of ionophores, including oxine. at this timepoint, were relatively low (9 ± 3%ID g and 11 ±
However, in each case a drying step, involving high tempera- 2%ID g− , respectively) compared to other studies with
1
2
tures and a stream of argon is necessary to remove solvents stealth liposomes of similar physicochemical characteristics.
3
6,37
before incubation with the liposomes.
Additionally, the Image-based quantification of the PET images is in agree-
radiometal was trapped internally by an encapsulated DOTA ment with these findings (Fig. S7†). Release of the contents
chelator; hence modification of the liposomal medicine is of the liposomal nanodrug over time is expected, and indeed
necessary. By taking advantage of the chelating properties of is required for anti-tumour efficacy, and has been previously
some drugs, already-formulated liposomal nanomedicines can observed. For example, previous work from our group noted
easily be labelled and tracked in vivo without the need for increasing activity uptake in the bone over time when track-
8
9
external modification of the nanomedicine or interfering with ing Zr-labelled liposomal nanodrugs – indicative of the free
its manufacture – which may potentially affect the biodistribu- radionuclide that is supposedly released when the liposomes
2
tion of the nanodrug.
extravasate and release their contents.
Additionally,
Tc]Tc- and
9
9m
Following on from our previous in vitro work describing previous studies tracking DOXIL using
[
5
2
186
the radiolabeling of DOXIL/CAELYX® ([ Mn]Mn-DOXIL)
[
Re]Re-BMEDA
(N,N-bis(2-mercaptoethyl)-N′,N′-diethyl-
with Mn using oxine, here we describe the evaluation of ethylenediamine) complexes reported increased kidney
5
2
the method to image the in vivo biodistribution and stability uptake after 40 h relating to release of the radioisotope from
Dalton Trans.
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52
Fig. 6 In vivo PET-CT imaging with [ Mn]Mn-DOXIL. (A) Maximum intensity projections and transverse slices through selected organs of PET/CT
52
−1
images of a B6CBAF1 mouse injected with [ Mn]Mn-DOXIL (1 MBq, 5 mg kg doxorubicin dosage) at 1 h post-injection (p.i.), 24 h p.i. and 72 h p.i.
CA = carotid arteries; H = heart; DA = descending aorta; L = liver; K = kidneys; S = spleen; SG = salivary glands; P = pancreas. (B) Blood pharmacoki-
5
2
netics of [ Mn]Mn-DOXIL in B6CBAF1 mice over 72 h and blood half-life of the liposomal nanomedicine. Error bars represent standard deviation (n
52
−1
=
3). (C) Radioactivity distribution of [ Mn]Mn-DOXIL at 72 h p.i. in B6CBAF1 mice. %ID g were calculated from ex vivo gamma counting. Error bars
represent standard deviation (n = 3).
3
8,39
metabolised liposomes.
However, efflux of the radio-
Conclusions
5
2
nuclide from [ Mn]Mn-DOXIL occurs more rapidly and to a
larger extent, with signs of radionuclide efflux at 24 h (high The simplicity, efficiency and effectiveness of 8-hydroxyquino-
uptake in kidneys, Fig. 6A). We hypothesise this may be due line (oxine) as an ionophore for the PET isotope Mn for cell/
to low cellular retention of the isotope within cells compared liposome radiolabelling has been demonstrated. We have pro-
with Zr and the above-mentioned SPECT radionuclides, as vided supporting evidence that the most likely radiometal
our cell labelling results with [ Mn]Mn(oxinate) suggest complex species that enter cells/liposomes is [ Mn]Mn
5
2
8
9
5
2
52
2
5
2
(
2 2
vide supra). Thus, after the accumulation and subsequent (oxinate) . In the context of cell labelling, [ Mn]Mn(oxinate)
destruction of [ Mn]Mn-DOXIL within the liver, the isotope – labels cells with comparable efficiency to [ Zr]Zr(oxinate) ,
4
5
2
89
along with the drug cargo – will be released. Consequently, and does not cause cell death for up to 24 h at the activity
5
2
Mn will return to the blood stream resulting in the free levels used, but the technique is limited by rapid cellular efflux
5
2
52
Mn biodistribution profile observed. Alternatively a similar of Mn. Since manganese is an endogenous essential metal,
5
2
process may occur after [ Mn]Mn-DOXIL is taken up by the rapid efflux is likely to be due to a native cellular efflux
tissue/circulating macrophages. This may explain the lower- pathway. As such, we suggest that direct cell labelling with
5
2
2
than-expected RES uptake observed at 72 h, compared to [ Mn]Mn(oxinate) may be inappropriate for long-term in vivo
8
9
2
52
when tracking liposomes with Zr. Hence we propose that cell tracking that fully exploits the long half-life of Mn.
5
2
52
[
Mn]Mn(oxinate)
2 2
may find applications in imaging liposo- DOXIL labelling with [ Mn]Mn(oxinate) is highly efficient
mal biodistribution, and that the rapid efflux of released and DOXIL radiolabelled in this way has high radiochemical
5
2
Mn from cells, and hence from tissues, makes the distri- stability in vivo whilst in the bloodstream. After 24–72 h post
bution to kidneys, pancreas and salivary glands a useful bio- injection, as the liposomes extravasate and enter tissues/cells,
marker for the subsequent release of the drug cargo from efflux of the radionuclide is evident from the characteristic
liposomes in vivo.
high uptake of manganese in kidneys, pancreas and salivary
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+
glands, most likely reflecting lysis of liposomes and release of
HRMS (ES, +ve): calculated m/z for [M + H] 344.0352;
+
drug. Since these organs are not usually involved in liposomal found: 344.0348. Calculated m/z for (2[M] + H) = 687.0631;
nanomedicine clearance, the characteristic biodistribution of found: 687.0684. Calculated m/z for [M + H + C
free Mn could be used as a marker of drug release from lipo- 385.0618; found: 385.0805*. Calculated m/z for [M + H +
+
2
H
3
N]
=
5
2
5
2
+
somes radiolabelled using this method. [ Mn]Mn(oxinate)
C H O ] = 432.0876; found: 432.1399*. * = m/z with mass
4 8 2
2
may find other applications in the future as a method to incor- error >10 ppm.
porate tracer amounts of this essential metal inside cells to Elemental analysis: Calc. for C18
study its biological trafficking mechanisms in vitro and in vivo. H, 3.01%; N, 6.97% Found: C, 53.68%; H, 3.88%; N, 6.99%.
H
2 2
12MnN O : C, 53.82%;
2
UV-titration of MnCl and 8-hydroxyquinoline
Within an ultra-micro cuvette a 0.1 mM solution of 8-hydroxy-
quinoline (1 mL, 1.46% v/v DMSO in 1 mM ammonium
Materials and methods
All chemical reagents were purchased from commercial acetate solution pH 7–9) was prepared. The molar equivalents
sources. Water (18.2 MΩ cm) was obtained from an ELGA (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1, 2 and 3 eq.) of an aqueous
Purelab Option-Qsystem. Mass spectrometry (ES-TOF) analysis manganese(II) chloride tetrahydrate solution were then added
was conducted by Lisa Haigh of the Mass Spectrometry sequentially, and the solution mixed with a 1000 μL pipette tip
Service, Imperial College London. IR analysis was carried out and left to stand for 2 min. Absorbance measurements were
using a PerkinElmer Spectrum 100 FT-IR spectrometer. UV taken of the 0.1 mM oxine solution and after each addition of
titrations were carried out using a PerkinElmer Lambda 25 the manganese(II) chloride solution. For the control titrations,
spectrometer, with samples in Brand 70 μL micro cuvettes. manganese(II) chloride were added to a solution containing no
Electron paramagnetic resonance (EPR) spectroscopy was per- 8-hydroxyquinoline.
formed by Enrico Salvadori of the EPR Service, Queen Mary
University London. DOXIL (Caelyx) was kindly provided by the
EPR spectroscopy
Chemotherapy Department at Guys & St Thomas’ Hospital,
A 1 mM solution of 8-hydroxyquinoline (1 mL, 1.46% v/v
DMSO in 1 mM ammonium acetate solution pH 9) was pre-
pared. 0.5 equivalents of an aqueous solution of manganese(II)
chloride tetrahydrate solution was added. EPR measurements
of the resulting solution were performed at 100 K using an
X/Q-band Bruker Elexsys E580 spectrometer (Bruker, Germany)
equipped with a closed-cycle cryostat (Cryogenic Ltd, UK) con-
trolled by a LakeShore temperature controller. Measurements
were carried out in an X-band split-ring resonator module with
5
2
London (UK). [ Mn]MnCl was produced by Jesper Fonslet
2
from the Hevesy Lab, at the Technical University of Denmark.
Radioactivity in samples were measured using CRC-25R dose
calibrator (Capintec). iTLC-SG and SA strips were purchased
from Agilent, UK and scanned using the PerkinElmer Cyclone
Plus Storage Phosphor Imager. Gamma counting was per-
formed using a Wallac 1282 CompuGamma γ counter. The
human biological samples were sourced ethically and their
research use was in accord with the terms of the informed con-
sents under an Institutional Review Board/Ethics Committee
2
mm sample access (ER 4118X-MS2). Samples were loaded on
Suprasil EPR tubes (Wilmad LabGlass) with OD = 1.6 mm, ID =
.1 mm. Baseline spectra of samples containing only the
(
IRB/EC) approved protocol. All animal studies were ethically
reviewed and carried out in accordance with the Animals
Scientific Procedures) Act 1986 and the GSK Policy on the
Care, Welfare and Treatment of Animals.
1
buffer were also collected and used as a reference. All the
spectra presented have been baseline-subtracted.
(
5
2
[
Mn]MnCl production
2
5
2
Synthesis of Mn(oxinate)
2
Mn was prepared according to the procedure described by
4
5
Fonslet et al. In brief, the manganese was produced by
6 MeV proton irradiation of natural chromium. Separation of
An aliquot of 1 M KOH solution (690 μL, 0.69 mmol, 1 eq.) was
added dropwise to a stirring mixture of 8-hydroxyquinoline
1
5
2
the Mn from the chromium target material was performed
by four sequential anion exchange purifications, trapping the
(0.1 g, 0.69 mmol, 1 eq.) in ethanol (10 mL) (1 eq.) at 55 °C.
The resulting solution was stirred at 55 °C for 0.5 h and then
at room temperature (RT) for 2 h. Manganese(II) chloride tetra-
hydrate (68 mg, 0.34 mmol, 0.5 eq.) in ethanol was then added
dropwise and the mixture stirred for 2 h at RT. The resulting
5
2
Mn out of ethanol–HCl mixtures.
5
2
[
Mn]Mn(oxinate) radiosynthesis
2
5
2
precipitate was then filtered and washed with ethanol and An aliquot of [ Mn]MnCl was made up to 50 μL using chelex-
2
water, then dried in vacuo to leave a pale yellow solid (26 mg, treated water. 8-Hydroxyquinoline was dissolved in DMSO
1
2
2%).
(12.5 mg mL− ) and an aliquot of this solution (4.8 μL) was
IR νmax (cm− ): 3045 w (νCH), 1605 m (νCvN), 1572 vs (νCvC), added to the [ Mn]MnCl
1
52
solution. The mixture was neutral-
2
1
495 vs, 1461 vs, 1424 m, 1386 vs, 1375 vs, 1319 vs (νC–C), 1279 ised via addition of 0.1 M ammonium acetate (pH 7) and the
C–N) s, 1238 m, 1207 w, 1178 w, 1137 w, 1105 vs, 1059 m mixture was then heated at 50 °C for 15 min. iTLC-SA con-
(
(
ν
ν
C–O), 1034 m, 983 w, 960 w, 903 m, 870 m, 824 vs, 806 s, 787 ditions: R
f
= 0.3–0.8, mobile phase = 20% MeOH in CHCl3.
vs, 746 vs, 739 vs, 652 m, 699 m, 583 m, 724 m.
RCY % = 69 ± 20%.
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5
2
For the preparation of the unchelated [ Mn]MnCl
2
solu- natant were then taken and gamma counted, and the labelling
tion, the above procedure was carried out, however DMSO efficiency calculated.
6
(
4.8 μL) was added instead of the 8-hydroxyquinoline solution.
Cellular retention. 2.5 × 10 of the labelled cells were then
= 0, mobile phase = 20% MeOH in added to a 6-well cell culture plate and RPMI medium (3.5 mL)
was then added. The cells were then incubated for 24 h at
iTLC-SA conditions: R
f
CHCl3.
3
5
7 °C. The cell suspensions were then centrifuged at 700g for
min. The supernatant was then removed and the cells were
5
2
MDA-MB 231/HEK-293T cell labelling with [ Mn]Mn(oxinate)
2
5
Tissue culture 6-well plates were seeded with 8 × 10 cells and resuspended in PBS (500 μL) and centrifuged again at 700g for
left for 24 h in Dulbecco’s Modified Eagle Medium (DMEM) 5 min. The supernatants were combined and the cells re-sus-
with 10% fetal bovine serum (FBS). The medium was removed pended in RPMI cell medium (1 mL). Aliquots (100 μL) of the
and replaced with 3 mL of serum-depleted medium (0% FBS). cell suspension and the supernatant were then taken and
5
2
5
2
0 kBq of [ Mn]Mn(oxinate) in serum-depleted medium (0% gamma counted, and the retention calculated.
FBS) were added to the cells (0.05 Bq per cell) which were then
incubated at 37 °C for 30 min. Subsequently, the cell medium
was removed and the cells washed with PBS (2 mL) and trypsin 0.5 MBq of [ Mn]Mn(oxinate)
Cell supernatant log P measurements
5
2
2
in serum-depleted medium
6
(250 μL) was added and the cells incubated for 2 min to allow (0% FBS) were added to 10 cells on tissue culture plates in
trypsinisation. Cell medium (750 μL) was then added and the serum-depleted DMEM (3 mL) which were then incubated at
cells re-suspended. The counts for the resuspended cells and 37 °C for 30 min. At each time point, 5 μL of the cell medium
the cell supernatant plus PBS washes were measured and the was removed and added to water/octanol (1 mL 1 : 1) and the
labelling efficiency calculated.
mixture vortexed for 2 min, then centrifuged for 20 s. An
Cellular retention. The labelled cells were re-plated on a aliquot (200 μL) from each layer was then removed to be
6
-well plate and cell medium (2 mL) was added. After incu- gamma counted.
bation at 37 °C for 4 or 24 h, the cell medium was removed
and the cells washed with PBS (2 mL) and trypsin (250 μL) was
Cell viability
5
2
added and the cells incubated for 2 min to allow trypsinisa- After the labelling of the MDA-MB-231 cells with [ Mn]Mn
tion. Cell medium (750 μL) was then added and the cells re- (oxinate) , the cell medium was removed and the cells washed
2
suspended. The counts for the resuspended cells and the cell with PBS (2 mL). The cell medium and PBS washes were com-
supernatant plus PBS washes were measured in a gamma- bined in a 15 mL falcon and trypsin (250 μL) was added and
counter.
the cells incubated for 2 min to allow trypsinisation. Cell
medium (750 μL) was then added and the cells re-suspended
and combined with cell medium and PBS washes, then centri-
5
2
Human blood platelet labelling with [ Mn]Mn(oxinate)
2
A Sepharose 4B column was washed with ∼100 mL of HEPES fuged at 200g for 5 min. The supernatant was removed and the
Tyrode’s buffer. 15 mL of donor blood were centrifuged at cell pellet resuspended in cell medium (1 mL). An aliquot
1
20g for 10 min at 25 °C. The plasma layer (cloudy yellow top (10 μL) was taken and mixed with trypan blue solution (10 μL).
layer) was then removed and added to the column and eluted The number of dead and alive cells were then counted on a
with HEPES Tyrode’s buffer. The eluate was collected when it haemocytometer and the % cell viability calculated from the
appeared cloudy (indicating the presence of platelets) and was proportion of the two.
subsequently centrifuged at 400g for 10 min and resuspended
5
2
DOXIL® labelling with [ Mn]Mn(oxinate)
52
2
in PBS (6 mL). 500 μL aliquots were taken and labelled with
5
2
52
1
2 2
00 μL of the [ Mn]Mn(oxinate) and free Mn suspensions [ Mn]Mn(oxinate) (150 μL, 3.2% v/v DMSO in water) was
−1
(in 1 mL serum-depleted medium) both in triplicate. The sus- added to a solution of DOXIL (350 μL, 2 mg mL ) and the
pensions were incubated for 30 min at 37 °C, after which the mixture heated with frequent agitation at 50 °C for 30 min.
suspensions were centrifuged at 120g for 10 min at 25 °C and The mixture was then loaded onto a PD minitrap G-25 size
the supernatant removed. The cell pellet and supernatant were exclusion column (GE Healthcare) following the manufac-
5
2
then gamma counted.
turers’ gravity protocol. The labelled liposomes ([ Mn]Mn-
DOXIL) were then obtained by adding saline (750 μL) to the
column and collecting the eluate.
5
2
Gamma-delta T-cell labelling with [ Mn]Mn(oxinate)
2
5
2
52
[
Mn]Mn(oxinate)
2
and/or unchelated Mn (300 kBq) were
6
PET/CT imaging
added to 5 × 10 γδ T-cells (0.06 Bq per cell) in suspension in
sterile PBS (1 mL). The cells were incubated at room tempera- Animal imaging studies were carried out in accordance with
ture with gentle mixing for 20 min. The cell suspensions were British Home Office regulations governing animal experimen-
then centrifuged at 700g for 5 min. The supernatant was then tation. Female B6CBAF1 mice (9 weeks old) were anesthetised
removed and the cells were resuspended in PBS (500 μL) and with isofluorane (1.5–2%) during all imaging sessions.
5
2
−1
centrifuged again at 700g for 5 min. The supernatants were [ Mn]Mn-DOXIL (1 MBq, 150 μL, 5 mg kg
combined and the cells resuspended in RPMI cell medium dosage) was injected i.v. into the mice (n = 3) at t = 0 h. PET/
1 mL). Aliquots (100 μL) of the cell suspension and the super- CT imaging was performed at t = 1 h, 24 h and 72 h (n = 1).
doxorubicin
(
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PET/CT imaging was performed for 60 min on a nanoScan acknowledge support from the KCL and UCL Comprehensive
in vivo PET/CT preclinical imager (Mediso Medical Imaging Cancer Imaging Centre funded by CRUK and EPSRC in associ-
Systems, Budapest, Hungary). All PET/CT data sets were recon- ation with the MRC and DoH (England). PET scanning equip-
structed using a Monte Carlo based full 3D iterative algorithm ment was funded by an equipment grant from the Wellcome
(Tera-Tomo, Mediso Medical Imaging Systems, Budapest, Trust. J. F. is funded by the European Union Seventh
Hungary). Decay correction to time of injection was applied. Framework Programme FP7/2007–2013 under Grant 602820
CT images were obtained with 55 kVp tube voltage, 1200 ms (Mathias). The research was also supported by the National
exposure time in 360 projections. All the images were analysed Institute for Health Research (NIHR) Biomedical Research
using VivoQuant software (inviCRO, USA).
Centre based at Guy’s and St Thomas’ NHS Foundation Trust
and King’s College London. The views expressed are those of
the author(s) and not necessarily those of the NHS, the NIHR
Ex vivo biodistribution
Biodistribution studies were carried out in accordance with or the Department of Health.
British Home Office regulations governing animal experimen-
tation. Three B6CBAF1 mice were culled by cervical dis-
location whilst under anaesthesia, and the organs of interest
were dissected, weighted and gamma-counted together with
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