Development of 68Ga-labelled ultrasound microbubbles for whole-body PET imaging

Article abstract of DOI:10.1039/c9sc00684b

Microbubble (MB) contrast agents have revolutionalised the way ultrasound (US) imaging can be used clinically and pre-clinically. Contrast-enhanced US offers improvements in soft-tissue contrast, as well as the ability to visualise disease processes at th

Full text of DOI:10.1039/c9sc00684b

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Development of 68Ga-labelled Ultrasound Microbubbles for
Whole-body PET Imaging
Received 00th
January 20xx,
Javier Hernández-Gil,*a Marta Braga,b Bethany I. Harriss,a Laurence S. Carroll, b Chee Hau Leow,c
Meng-Xing Tang,c Eric O. Aboagye,*b and Nicholas J. Long*a
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Microbubble (MB) contrast agents have revolutionised the way we understand ultrasound (US) imaging. Contrast-enhanced
US offers improvements in soft-tissue contrast, as well as the ability to visualise disease processes at the molecular level.
However, its inability to provide in vivo whole-body imaging can hamper the development of new MB formulations. Herein,
we describe a fast and efficient method for achieving 68Ga-labelling of MBs after a direct comparison of two different
strategies. The optimised approach produces 68Ga-labelled MBs in good yields through the bioorthogonal inverse-electron-
demand Diel-Alder reaction between a trans-cyclooctene-modified phospholipid and a new tetrazine-bearing HBED-CC
chelator. The ability to noninvasively study the whole-body distribution of 68Ga-labelled MBs was demonstrated in vivo using
positron emission tomography (PET). This method could be broadly applicable to other phospholipid-based formulations,
providing accessible solutions for in vivo tracking of MBs.
development of alternative MB agents for new diagnostic and
therapeutic US applications in patients.
Introduction
Clinical translation, however, is always a long, high-risk and
The development and further refinement of contrast agents
plays a central role in the fields of anatomical and molecular
imaging, acting as a driving force to overcome limitations
inherent to existing imaging modalities.1 In ultrasound (US), the
use of microbubbles (MBs, Scheme 1), which consist of a gas
core stabilised by a phospholipid or polymer shell, has improved
the poor ability of this technique to distinguish between tissues
with similar acoustic responses.2 In the presence of an acoustic
field, these compressible MBs produce strong nonlinear signals,
which can be differentiated from the surrounding tissue,
enhancing soft-tissue contrast and signal-to-noise ratios in
regions of interest. The fact that MBs typically present sizes of
1-5 μm, and therefore are restricted to intravascular targets,
alongside their ability to incorporate disease-targeted ligands,
has recently oriented the use of US imaging towards detecting
and monitoring vascular pathologies at the molecular level.2c In
this regard, different preclinical studies have successfully
demonstrated the capability of MBs to visualise receptors
overexpressed in inflammation, angiogenesis and/or early
tumour formation,3a-c and a phospholipid-based formulation
has even recently entered first in-human clinical trials in various
cancer types.3d-e These examples, together with the proven
capacity of MBs to load drugs3f will further support the
expensive endeavour, and therefore, early evaluation of the
biodistribution and/or pharmacokinetics of MBs could facilitate their
(pre)clinical development.4a Whole-body imaging has long
demonstrated utility in the evaluation of in vivo fates and/or possible
off-target effects.4 Unfortunately, contrast-enhanced US is limited to
local imaging. This limitation has created a demand for new MB
formulations that can provide whole-body imaging and therefore,
offer a better understanding of diagnostic and/or drug activity during
the initial stages of MB development.
a.Department of Chemistry, Imperial College London (United Kingdom), E-mail:
j.hernandez-gil@imperial.ac.uk, n.long@imperial.ac.uk.
b.Department of Surgery & Cancer, Imperial College London (United Kingdom), E-
mail: eric.aboagye@imperial.ac.uk.
c. Department of Bioengineering, Imperial College London (United Kingdom).
Scheme 1. Schematic structure/composition of the developed
microbubbles (MBs), showing two surface conjugation sites (not to
scale).
†
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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Scheme 2. A) “Direct labelling approach”: conjugation of a metal chelator to the headgroup of PE and subsequent labelling and purification. B) “Click
labelling approach”: development of bioorthogonal components for ‘click’ ligation of a 68Ga-chelator-tetrazine and a trans-cyclooctene-modified
phospholipid to yield 68Ga-labelled MBs
Nuclear imaging techniques, namely positron emission and versatile way to radiolabel the shell components of MBs, as well
tomography (PET) and single-photon-emission tomography (SPECT), as to provide reliable validation checkpoints with conventional
provide non-invasive and whole-body imaging with high sensitivity analytical techniques. To test this hypothesis, two 68Ga-labelling
and unlimited penetration depth.5 Hence, a few groups have strategies were initially devised;- (i) direct conjugation of a metal
attempted to develop dual nuclear/US MB agents.6 Most of these chelator to the phospholipid head-groups and (ii) bioorthogonal
studies, however, have used streptavidin-biotin interactions to ‘click’ ligation between a chelator-based prosthetic group and a
radiolabel MBs and/or employed SPECT tracers (iodine-125, complementary functionalised-phospholipid (Scheme 2A-B).
technetium-99m or indium-111).6a-d Although elegant and Through a direct comparison between both approaches, we have
encouraging, these approaches face disadvantages: (i) immunogenic developed a rapid and efficient method for labelling ultrasound MB
streptavidin-coated MBs are not recommended for human use and agents with a generator-produced PET isotope, offering accessible
(ii) SPECT cannot provide quantitative analysis in vivo. PET, unlike solutions for in vivo tracking of MBs.
SPECT, allows determination of tracer concentration in tissue with
higher sensitivity and image quality. Despite these advantages, only
one group has incorporated a 18F-labelled phospholipid onto MBs,
allowing non-invasive assessment of the whole-body MB fates.6e
However, 18F-labelling usually requires time-consuming multi-step
synthesis, and more importantly, depends on expensive cyclotron
facilities. So, there is an unmet need for a rapid and robust method
to generate radiolabelled MBs using broadly accessible PET isotopes.
Gallium-68 (hereafter, 68Ga) is a radiometal that offers aqueous
single-step and high-yielding complexations with high positron
emission (89%) and on-site availability, allowing hospitals
economical production of a high-quality and generator-produced
PET isotope.7 In addition, its short half-life (67.7 min) enables the
perfect monitoring of MBs, which have circulation times of around
10-20 minutes.2 However, the development of 68Ga-labelled MBs
(hereafter, 68Ga-MBs) has never been attempted, which inspired us
to focus on this particular problem in the field of US imaging. We
reasoned that the optimised method should offer a fast, accessible
Results and Discussion
Design and preparation of microbubbles
Here, we focused on phospholipid-shelled MBs, which represent the
most common and versatile US contrast agent.2,3 We recently
demonstrated that a cyanine7.5-modified phospholipid could be
successfully incorporated into a MB formulation of commercially
available 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (poly-
ethylene glycol)-2000] (DSPE-PEG(2000)) phospholipids.8a We
reasoned that 1,2-distearoyl-sn-glycero-3-phosphoethanolamine
(PE), which shares DSPE-PEG(2000) backbone without its PEGylated
moiety, could therefore, be combined with DPPC and DSPE-
PEG(2000), and thus offer an alternative anchor point for the PET
tracer. The absence of a bulky PEG45 polymer on PE also facilitates
the purification and characterisation of resulting products, while
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Figure 1. A) Structure of PE-NOTA (1), PE-DOTA (2), PE-DOTAGA (3) and PE-PEG4-TCO (4). B) Radiochemical yields for the reaction of 68Ga3+ with 1
(black), 2 (green) and 3 (blue) under different phospholipid concentrations (0.05 to 250 µM) and pH values (3.5 and 4.5), after 10 min at 90 ºC.
maintaining DSPE-PEG(2000) available for subsequent conjugation of
To test the feasibility of our first approach (Scheme 2A), we
targeting ligands (Scheme 1). With such a rationale in mind, we functionalised PE with three chelators of known affinity for 68Ga:7a
prepared a new formulation containing DPPC, DSPE-PEG(2000) and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7,10-
PE at 85:5:10 molar ratios, following standard protocols (Table S1).8 tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 2-
Optical microscopy was used to visualise the size and concentration (4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)-
of the new MB formulation and, as shown in Table S2, no significant pentanedioic acid (DOTA-GA). PE-NOTA (1) and PE-DOTA (2) were
difference was observed between PE-containing MBs and our prepared by conjugation of the primary amine headgroup of PE with
previous formulation without PE. The stock MB solution containing a NHS-activated form of each chelator, whereas PE-DOTAGA (3) was
10% of PE yielded a mean bubble diameter of 2.10 ± 1.07 µm and a obtained via reaction of a p-isothiocyanatobenzyl PE intermediate
concentration of (1.22 ± 0.68) x 1010 MBs/mL while the unmodified with an ethylendiamine-modified DOTAGA reagent. 1-3 were
formulation generated bubbles with a mean size of 2.18 ± 1.06 µm purified by preparative thin-layer chromatography (TLC) and
and a concentration of (8.02 ± 0.03) x 109 MB/mL. The acoustic obtained in high yields (Figure 1A and SI).
response of both MBs was also compared. The two formulations To examine whether
were diluted to a concentration of approximately 106 MBs/mL in a 2L amount of generator-produced 68Ga3+
water tank and their US contrast enhancements quantified (Table reacted with over a range of phospholipid concentrations
S2). and two pH values (3.5 and 4.5). All reactions were incubated
Both MBs produced a similar US contrast at 4.5 MHz, indicating that for 10 min at 90 ºC and the radiochemical yields (RCYs) were
1
-
3
can efficiently chelate 68Ga, a fixed
2 MBq) was separately
(

1-3
the new formulation can also be used for US imaging.
measured using radioTLC.
1 demonstrated the highest labelling
efficiency, with RCYs greater than 80% at moderately low
Evaluation of the first approach: direct conjugation of a metal
chelator to the phospholipid head-groups
chelator concentrations (10
M) for both pH conditions,
corresponding to molar activities of 1-2 MBq·nmol-1 (Figure 1B).
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of small molecules in biological matrices and many SPE
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DOI: 10.1039/C9SC00684B
cartridges (in our case, HybridSPE®, Oasis® Prime and Oasis®
HLB cartridges) contain sorbents that can retain phospholipids.
This effect was clearly observed when using Oasis® Prime
cartridges, retaining 62 ± 15% of the initial loaded activity and,
also noticeable with HybridSPE® and Oasis® HLB cartridges
trapping 32 ± 7 and 17 ± 3%, respectively. Therefore, the sub-
optimal purification yields obtained because of the lipid nature
of
1 alongside its low molar activities prompted us to focus on
the second approach.
Figure 2. A) Structure of HBED-CC-Tetrazine (5) and DOTA-GA-
Tetrazine (6).
Evaluation of the second approach: bioorthogonal ‘click’ ligation
between a chelator-based prosthetic group and a complementary
functionalised-phospholipid
As previously reported, NOTA derivatives show better
affinity for 68Ga3+ than DOTA conjugates as a consequence of
the close fit of the relatively small Ga(III) metal ion in the pseudo
octahedral cavity of the potentially six-coordinate 9-aneN3, with
For the second strategy (Scheme 2B), we hypothesised that a
tetrazine-bearing radioconjugate could combine high molar activities
and straightforward work-up procedures from small-sized chelators
with high-yielding and fast reactivities from ‘click’ ligations. In this
regard, our group and others have been designing new probes suited
for bioorthogonal radiolabelling via the inverse-electron-demand
Diels-Alder (iEDDA) cycloadditions between tetrazines and olefins.10
iEDDA reactions avoid the use of metal catalysts, which could
compete with the far less abundant 68Ga3+ radiometal, as well as
offer rapid kinetics at low concentrations, essential when working
with short-lived radioisotopes.11 Thus, a trans-cyclooctene (TCO)-
attached carbonyl and amide groups.9 We then selected
optimising a 68Ga-labelling method under in vivo conditions.
Purified 68GaCl3 (70-75 MBq) was reacted with
20 nmol) in
acetate buffer (0.1 M, pH 4.0) for 15 min at 90 ºC. The RCY of
[68Ga][Ga(
)], under these conditions, was >85% (non-isolated,
estimated by radio-HPLC). Next, different solid-phase extraction
(SPE) cartridges were used for [68Ga][Ga(
)] purification but,
1 for
1 (
1
1
unfortunately all sorbents tested presented low recovery yields
(Table S3). The best results were obtained with Oasis® HLB
cartridges with an average eluted activity of 27 ± 5%. These all-
purpose cartridges, with a strongly hydrophilic and reversed-
phase polymer, allow acidic, basic and neutral analyte
purifications from pH 0 to 14. Under our labelling conditions, pH
modified phospholipid was synthesised using
a commercially
available TCO-PEG4-NHS ester and PE. TCO was chosen because of its
higher reactivity compared to other strained olefins.12 PE-PEG4-TCO
(4) was purified by preparative-TLC and characterised by 1H NMR
spectroscopy and MALDI-TOF mass spectrometry (Figure 1a and SI).
Compound 5, containing N,N′-bis[2-hydroxy-5-(carboxyethyl)-
= 3.5 - 4.0, [68Ga][Ga(
1)] behaves as a zwitterionic phospholipid
benzyl]ethylenediamine-N,N-diacetic acid (HBED-CC) and
a
with a positively charged Ga-chelating moiety and a negatively
charged phosphate group (pKa ~ 1.5-2.5), meeting a priori the
manufacturer requirements for high recovery yields.
Phospholipids, however, are a major concern for LC-MS analysis
tetrazine, was also prepared (Figure 2 and SI). In a six-step sequence,
the desired product was ultimately obtained by in situ coupling
pH 3.5, 25 ºC
pH 4.5, 25 ºC
pH 5.5, 25 ºC
100
50
0
100
50
0
100
50
0
0.1
1
10
100
500
0.1
1
10
100
500
0.1
1
10
100
500
[chelator] (µM)
[chelator] (µM)
[chelator] (µM)
pH 3.5, 90 ºC
pH 4.5, 90 ºC
pH 5.5, 90 ºC
100
50
0
100
50
0
100
50
0
0.1
1
10
100
500
0.1
1
10
100
500
0.1
1
10
100
500
[chelator] (µM)
[chelator] (µM)
[chelator] (µM)
Figure 3. Radiochemical yields for the reaction of 68Ga3+ with 5 (blue) and 6 (black) under different concentrations of chelator (0.05 to 500 µM), pH
values (3.5, 4.5 and 5.5) and temperatures (25 and 90 ºC), after 10 min.
4 | J. Name., 2012, 00, 1-3
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coupling HATU-activated [Fe(HBED-CC)]- with (4-(6-methyl-1,2,4,5-
Next, we reacted a purified fraction of [68Ga][Ga(
5)] (~ 75
tetrazin-3-yl)phenyl)methanamine. Here, we used HATU, which MBq) with PE-PEG4-TCO (0.107 μmol) at 60 ºC (Figure 4A). After
required a 15-minute activation, instead of the most common 2-day 15 minutes, the radio-HPLC chromatogram showed the
tetrafluorophenol/dicyclohexylcarbodiimide
process.13
The appearance of a new major peak at a slower retention time (τr
formation of the iron complex assures the protection of coordinating = 10.8 min) than the labelled precursor (τr = 5.3-6.4 min), which
phenolic and carboxylate groups while exposing the two propionic corresponded to radiochemical conversions of the
acid arms for amine conjugation. HBED-CC-tetrazine derivative 5 was dihydropyridazine products of
80-85% (non-isolated,
purified by flash chromatography and its monosubstituted nature estimated by radio-HPLC) (Figure 4A-B). Next, 68Ga-labelled PE
confirmed using 1H, 13C NMR spectroscopy and ESI mass (hereafter, 68Ga-PE) was combined with DPPC and DSPE-
spectrometry (see SI). As a reference, a tetrazine-bearing DOTA-GA PEG(2000) and the resulting formulation was activated to form
tracer (6), previously designed by our group,10b was prepared (Figure gas-filled MBs. Excess of unreacted [68Ga][Ga(
2 and SI).
were removed via a centrifugation/washing methodology.17
A head-to-head comparison of the 68Ga-chelating efficiency The RCY of purified 68Ga-MBs was around 40-50%, generating
between 5 and 6 was performed (Figure 3). At room temperature, samples with 30-35 MBq and concentrations of (1.28 ±
5)] and free lipids

the difference between both chelators was remarkable. Whilst 5 0.68)·109 MB/mL, in 40-50 minutes after 68Ga elution from the
performed extremely well, 6 presented poor ability to coordinate generator.
68Ga3+. This suggested that the labelling efficiency of 6 was limited by
the higher kinetic barriers that macrocyclic-based chelators generally
present compared to acyclic ligands.7a,14 5 also showed little pH-
dependence, yielding near-quantitative RCYs at the low
concentration of 5 M for all pH values tested (based on radioTLC
analysis of the reaction solution). At high temperature, both
chelators exhibited a similar efficiency, with conversions higher than
90% at 5 M. At lower concentrations, however,
5 again
demonstrated better performance over 6. At 1 M and pH values of
3.5 and 4.5, RCYs of [68Ga][Ga(5)] were near quantitative while RCYs
of [68Ga][Ga(6)] were lower than 30%. This corresponded to
maximum molar activities for 5 of 15-20 MBq·nmol-1. Next, we
selected 5 to evaluate its 68Ga-labelling efficiency under in vivo
conditions. This time, [68Ga][Ga(5)], obtained after 10 min incubation
at room temperature, could be successfully purified with
conventional reverse-phase cartridges. The entire procedure was
performed in less than 20 min, yielding purities >98% and RCYs >95%
(determined by radio-HPLC analysis of the pure product).
Preparation of 68Ga-labelled MBs
Figure 4A shows the chromatogram of purified [68Ga][Ga(
labelled at room temperature and pH 4.2 0.3. As for other
HBED-based chelators, forms different geometric isomers
5)]

5
when complexed to Ga3+.15 Although a mixture of species can
potentially present different pharmaceutical profiles, a recent
study measuring the cell binding and internalisation properties
of [68Ga]Ga-PSMA-HBED-CC labelled at room temperature
(containing a mixture of species) and at 95 ºC (containing the
thermodynamically most stable isomer) on PSMA-expressing
LNCaP cells showed comparable results for both fractions,
indicating a stereochemical independent receptor-interaction
of the radioligand.16
Figure 4. A) Radio-HPLC chromatograms of (top) [68Ga]Ga-HBED-CC-
tetrazine precursor and (bottom) reaction between 4 and 5, after 15 min
at 60 ºC. B) Scheme of the reaction between TCO-modified PE (4) and
[68Ga]Ga-HBED-CC-tetrazine (5).
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In vivo study of 68Ga-labelled MBs
Conclusions
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DOI: 10.1039/C9SC00684B
Encouraged by the successful 68Ga-labelling of MBs,
comparative dynamic PET imaging and biodistribution studies of
[68Ga][Ga(5)], 68Ga-PE and 68Ga-MBs were undertaken on Balb/c
nude mice (n = 5-6). The optimised labelling approach yielded MB
stock solutions with high activity and concentration, allowing us to
use injected concentrations of approximately 5 x 107 MB/injection
with activities between 0.37-0.74 MBq. These MB concentration
values are in the same order of typical diagnostic doses used in US
imaging, suggesting that this strategy generates 68Ga-MBs that can
also be exploited for complementary US studies.
In conclusion, we have developed an efficient method for
labelling MBs with a generator-produced PET isotope using a rapid
and clean ligation between a TCO-modified phospholipid and a 68Ga-
HBED-CC-tetrazine tracer. Bioconjugation of TCO to phospholipids is
simple and reproducible, and the new HBED-CC-tetrazine chelator
provides efficient and high-yielding 68Ga-labelling, affording
reproducible synthesis of 68Ga-MBs under mild conditions. This method
offers real-time monitoring and the possibility of easily customising
alternative phospholipid-based formulations. We also confirmed
that the resulting 68Ga-MBs allow non-invasive study of the whole-
body distribution of MBs in mice. As contrast-enhanced US has
emerged as a promising imaging modality for clinical applications, we
believe that this strategy is a favourable way of non-invasively
evaluating the pharmacokinetics and off-target accumulation of new
MB formulations in their initial stages of (pre)clinical development.
Therefore, this strategy can be easily implemented across multiple
centres and hospitals, accelerating MB evaluation and development.
Future work will explore the incorporation of targeted-modified
phospholipids into the MB formulation and compare 68Ga-labelled
MB containing targeted and non-targeted vectors.
Kinetic analysis showed that the MBs had a pharmacokinetic
profile different from that of freely-injected PE and 5 (Figures 5 and
S6-S8). Following the injection of free [68Ga][Ga(5)], activity
accumulated in the bladder, gallbladder and intestines within the
first 5 minutes, which confirmed that [68Ga][Ga(5)] was characterised
by a rapid tissue distribution and subsequent clearance through the
urinary route. 68Ga-PE and 68Ga-MBs, on the contrary, presented
longer circulation times in the animals, with high blood pool mean
values persisting for the entire imaging period. Both formulations,
however, presented differences in their biodistribution, which
became more evident at later time points. Between 0 to 2 min post-
injection (PI),68Ga-PE and 68Ga-MBs were mainly present in the heart
and liver, whereas at 5-20 min time interval, 68Ga-PE was mainly
accumulated in the bladder and gallbladder, and 68Ga-MBs were
present in the liver, gallbladder and spleen. The uptake in the liver
and spleen reached equilibrium around 3 min PI at approximately 66
and 40% ID/g, respectively. Previous studies have also indicated the
liver and the spleen as the major organs for the accumulation of MBs
with sizes around 1 - 10 µm.6a,b In addition, 68Ga-MBs showed an
initial peak of activity in the lungs of 43 ± 12% ID/g, which decreased
to 20 ± 5% ID/g at 20 min, indicating that 68Ga-MBs were gradually
redistributed into the systemic circulation after an initial
accumulation in the lungs. Biodistribution data confirmed the PET
results. At 20 min PI, both [68Ga][Ga(5)] and 68Ga-PE were mostly
present in the urine (689 ± 64 and 146 ± 26% ID/g, respectively),
consistent with an efficient urinary excretion route. 68Ga-MBs, on the
other hand, showed a more distributed uptake within the animal
models, with accumulation of radioactivity mainly observed in the
liver (47 ± 4% ID/g), spleen (40 ± 2 %ID/g) and urine (57 ± 11% ID/g).
Experimental section
Materials
All the syntheses were carried out with the following commercially
available reagents used without further purification: 4-
(aminomethyl)benzonitrile hydrochloride (97%, Sigma Aldrich), di-
tert-butyl dicarbonate (≥ 99%, Sigma Aldrich), nickel(II)
trifluoromethanesulfonate (96%, Sigma Aldrich), hydrazine hydrate
(50-60 %, Sigma Aldrich), sodium nitrite (NaNO2, ≥ 97%, Sigma
Aldrich), 3-(4-hydroxyphenyl)propionic acid (98%, Sigma Aldrich),
sodium
hydroxide
(98.5-100.5%,
VWR
International),
ethylenediamine-N,N′-diacetic acid (≥ 98%, Sigma Aldrich),
formaldehyde (37 wt. % in H2O, Sigma Aldrich), hydrochloric acid
(37%, VWR International), trifluoroacetic acid (≥ 99.5%, Tokyo
Chemical Industry Co., Ltd.), N,N-diisopropylethylamine (≥ 99%,
Sigma Aldrich), iron(III) chloride (97%, Sigma Aldrich). 1-
[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate,N-[(dimethylamino)-1H-1,2,3-triazolo-
[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium
hexafluorophosphate N-oxide (HATU, 97%, Sigma Aldrich), glutaric
anhydride (95%, Sigma Aldrich). Triethylamine (≥ 99%, Sigma
Aldrich), N-hydroxysuccinimide (98%, Sigma Aldrich). N,N′-
Dicyclohexylcarbodiimide (DCC, 99%, Sigma Aldrich), trans-
cyclooctene-PEG4-NHS ester (TCO-PEG4-NHS,
Bioscience), trans-cyclooctene-NHS ester (E)-cyclooct-4-enyl-2,5-
dioxo-1-pyrrolidinyl carbonate (TCO-NHS ester, 95%, Jena
>
95%, Jena
>
Bioscience), 4-nitroaniline (≥ 99%, Sigma Aldrich), p-phenylene
diisothiocyanate (98%, Sigma Aldrich), 2,2′,2”-(10-(4-((2-
aminoethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-
tetraazacyclododecane-1,4,7-triyl)triacetic acid (NH2-DOTA-GA,
ChemMtech mdt), 2,2′,2”-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-
oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid
(DOTA-NHS ester, ChemMtech mdt), 2,2′-(7-(2-((2,5-dioxopyrrolidin-
1-yl)oxy)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NOTA-
Figure 5. Small-animal PET imaging of Balb/c nude mice after intravenous
injection of [68Ga][Ga(5)] (left), 68Ga-labelled PE (middle) and, 68Ga-
labelled MBs (right) at early (0-2 min) and late (5-20 min) time intervals.
6 | J. Name., 2012, 00, 1-3
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Chemical Science
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ARTICLE
NHS ester, ChemMtech mdt), sodium acetate (≥ 99%, Sigma Aldrich), produced using a modified formulation described previously.18 1 mL
View Article Online
acetic acid (≥ 99%, Sigma Aldrich), ammonium acetate (≥ 98%, Sigma of the resulting lipid solution (refer to sectioDnO4I:in10t.1h0is39d/oCc9uSmC0e0n6t8f4oBr
Aldrich), decafluorobutane (99%, FluorMed, L.P.), 1,2-dipalmitoyl-sn- further details) was sealed in a 2 mL glass vial and the headspace was
glycero-3-phosphocholine (DPPC, ≥ 99%, Avanti Polar Lipids), 1- then purged with decafluorobutane at room temperature. The
palmitoyl-2-hydroxy-sn-glycero-3 phosphocholine (LPC,
Avanti Polar Lipids), 1,2-distearoyl-sn-glycero-3- HL-AH High Speed Digital Amalgamator Amalgam Capsule Blend
phosphoethanolamine (18:0 PE-NH2, ≥ 99%, Avanti Polar Lipids), 1,2- Mixer HL-AH (4000 rpm for 30s, two cycles). Particle size of non-
distearoyl-sn-glycero-3-phosphoethanolamine-N- radioactive MBs was measured on a bright-field microscope (Nikon
[amino(polyethylene glycol)-2000] (ammonium salt) (DSPE- Eclipse 50i, 40 x objective), 10 µL diluted samples (1:100 dilution
PEG(2000)-NH2, ≥ 99%, Avanti Polar Lipids), primuline (50%, Sigma factor from stock sample) were first introduced into
≥ 99%, microbubbles were produced via mechanical agitation using a dental
a
Aldrich), propylene glycol (≥ 99.5%, Sigma Aldrich), glycerol (99%, haemocytometer and then sized and counted according to our
Sigma Aldrich), Dulbecco’s phosphate-buffered saline (10x, Sigma reported protocol.19 ζ-potential analyses of microbubbles in solution
Aldrich). Solvents were HPLC grade and obtained from VWR (1:1000 dilution factor from stock sample) were measured with a
Chemicals. Anhydrous chloroform and methanol were purified and NanoSizer (Malvern Nano-Zs, UK) and using DTS1070 cuvettes.
dried according to standard methods prior to use and, anhydrous Alternatively, 68Ga-labelled MBs were counted and sized using a
dimethyl sulfoxide was obtained from Sigma Aldrich (≥ 99.5%).
Countess II FL Automated Cell Counter from Life Technologies. Non-
radioactive controls were run to confirm size and concentration of
MBs presented non-significant differences from both methods used
to count and size MBs. MBs were purified by centrifugation adapting
a reported method using a ROTINA 35 R centrifuge from Hettich
Zentrifugen.17
For experiments working with radioactive substances, water
(Ultrapur, VWR) was used for preparation of buffers, HCl and NaCl
solutions. 5.5 M HCl prepared by dilution of hydrochloric acid 37%
(Emprove,VWR).
68Ga was eluted from an Eckert & Ziegler Radiopharma GmbH
GalliaPharm® 68Ge/68Ga generator (Berlin, Germany) using a fully
automated Modular-Lab system. Aqueous HCl solutions (0.1 M, 5 mL)
was passed through the generator and the eluate was collected in a
single fraction and used directly for 68Ga-labeling reactions (refer to
radiochemistry section below for further details).
Methods
1D 1H and 13C NMR spectra were recorded on either a Bruker AV-500
or on a Bruker AV-400 spectrometer at room temperature using
standard pulse programs. Chemical shifts (δ) are quoted in parts per
million (ppm) and referenced to the appropriate residual solvent
signal. Coupling constants (J) are reported to the nearest 0.5 Hz.
Mass spectra (m/z) of phospholipids were run on a MALDI micro MX
- TOF mass spectrometer from Waters. Samples were spotted 1:1 v/v
with a matrix solution (4-nitroaniline, 10 mg/mL in methanol) and
measured in reflector mode. High resolution mass spectra (m/z)
were recorded on either a VG platform II or VG AutoSpec
spectrometers, with only molecular ions (M+, MH+, MNa+, MK+,
Different purification cartridges were used for radioactive
experiments: silica-based strong cation exchange cartridge (Bond
Elut-SCX, 100 mg, 1 mL) purchased from Agilent (further referred as
SCX cartridge); Sep-Pack® Light tC18 cartridge (145 mg sorbent per
cartridge), Sep-Pack® Plus Light C8 cartridge (145 mg sorbent per
cartridge), Oasis® HLB cartridge (1cc/30 mg) and Oasis® Prime HLB
+
cartridge Plus Light (100 mg) acquired from Waters. HybridSPE®
-
MNH4 ) and major peaks being reported. Flash chromatography (FC)
was performed on silica gel (Merck Kieselgel 60 F254 320-400 mesh).
Thin Layer Chromatography (TLC) was performed using TLC Silica Gel
60 F24 (aluminium sheets 20x20 cm for analytical runs and glass
plates 20x20 cm for preparative TLC purifications of phospholipids)
from Merck. Plates were visualised either by quenching of ultraviolet
fluorescence (λ = 254 and 366 nm) or by charring with 10% KMnO4 in
1M H2SO4. Phospholipids were additionally visualised by charring
with 5 % primuline in acetone:water (8:2 v/v), lipids appearing as
.yellow spots under 365 nm wavelength. Flash purifications were
carried out in a IsoleraTM Spektra System using Biotage® SNAP Ultra
C18 for reverse phase conditions (12, 30 and 60 g sizes) and, Biotage®
SNAP Ultra C18 for normal phase conditions (25, 50 and 100 g sizes).
Gradients are indicated throughout the text. Analytical HPLC
chromatograms were run in a Waters AutoPurificationTM System
equipped with a 2489 UV/vis Dectector and a 3100 Mass Detector,
using a Phenomenex Gemini C18 column (150 mm x 4.6 mm) at a
flow rate of 1.2 mL·min-1 and gradient A: (5- 98% B over 12 min then
98% B for 3 min) Buffer A = H2O (0.1% TFA), Buffer B = MeCN (0.1%
Phospholipid (30 mg/1 mL SPE Tubes) acquired from SUPELCO. iTLC
experiments were measured on a Scan-RAMTM PET/SPECT radio-TLC
Scanner from LabLogic using iTLC-SG
– Glass microfibre
chromatography paper impregnated with silica gel from Agilent
Technologies. Analysis data was performed with Laura 3 software
(LabLogic, Sheffield, UK). Radio-HPLC was carried out on an Agilent
1100 series HPLC system (Agilent Technologies, Stockport, UK)
equipped with a -RAM Model 3 gamma-detector (IN/US Systems
Inc., Florida, USA) and Laura 3 software (LabLogic, Sheffield, UK) was
used for processing all analytical HPLC chromatograms. For tetrazine
chelators, a Phenomenex Germini 5 u C18 (150 X 4.6 mm) was used
and an acidic gradient: 5-95 % B over 15 min, then 95% B for 5 min)
where A = H2O (0.1 % TFA) and B = MeCN (0.1 % TFA). For
phospholipids a Phenomenex Bondclone 10 C18 (150 x 3.90 mm) was
used, as well as a neutral gradient: 5-95 % B over 7 min, then 95% B
for 8 min) where A = 100 mM Ammonium Formate (H2O) and B =
Methanol.
TFA). Supelco Iso-DiscTM Filters PFTE-4-4 (4mm x 0.45 µm) from 68GaCl3 was eluted from a GalliaPharm® 68Ge/68Ga Generator from
Sigma Aldrich were used for sample filtration prior injection.
Eckert & Ziegler Radiopharma GmbH (0.74 GBq) using a fully
automated Modular-Lab system and an adapted NaCl based method
The lipid-coated decafluorobutane-filled microbubbles (MBs) were
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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Page 8 of 13
ARTICLE
Journal Name
reported previously.20 Briefly, 68GaCl3 was eluted in 5 mL of a 0.1 M + COOCH2COH, 7H), 4.36 (s, cis-PO4CH2CH, 1H), 5.19V(ise,wPAOrti4cCleHO2nCliHne,
HCl solution, which was subsequently trapped on a SCX cartridge 1H), 7.09 (d, CH, 2H, 3JH-H = 8.3 Hz), 7.47 (m,DCOHI,: 210H.1).03M9A/CL9DSIC(0m0a6t8r4ixB:
(pre-conditioned with 1 mL 5.5 M HCl and 10 mL water) and the 4-nitroaniline); m/z for [C51H93N3O9PS2]+ ([M+H+CH3CH2OH]+)
activity was then eluted, with minimal loss (2-5%), using a mixture of expected = 986.6, found 986.6.
12.5 L of 5.5 M HCl and 500 L of 5 M NaCl solution. This eluate was
subsequently buffered for direct use in 68Ga-labelling experiments.
Synthesis of PE-iso-DOTA-GA (3)
PE-iso (7.0 mg, 7.45 μmol) was dissolved in
chloroform:methanol mixture (0.5:0.5 v/v) and triethylamine (50 μL).
1 mL of a
ORGANIC SYNTHESES
All the syntheses and experiments containing NHS ester compounds To this solution, 2,2′,2”-(10-(4-((2-aminoethyl)amino)-1-carboxy-4-
were performed under anhydrous conditions and an atmosphere of oxobutyl)-1,4,7,10 tetraazacyclododecane-1,4,7-triyl)triacetic acid
nitrogen in flame-dried glassware. All purified products were kept in (NH2-DOTA-GA, 10.0 mg, 19.28 μmol) was added and the mixture
the freezer and found to be stable for more than six months.
was left to stir for 5 h. After that, the crude was concentrated in
rotavapour and purified by preparative TLC. Rf 0.15-0.25
=
Synthesis of phospholipids
1
(CHCl3:MeOH:H2O 5:5:0.2 v/v). (6.8 mg, 4.65 μmol, 62 %). H NMR
3
(400 MHz, CDCl3): δ (ppm) 0.71 (t, CH3, 6H, JH-H = 6.5 Hz), 1.09 (s,
Synthesis of PE-NOTA (1) and PE-DOTA (2)
CH2, 56H), 1.44 (m, CH2CH2CO, 4H), 1.69 (m, CH2CH2, 2H), 1.93-2.55
(m, NCH2CH2N + NCH2CH2N + CH2CH2CO, 20H), 2.58-2.73 (m,
NCH2COOH, 6H), 2.88 (m, CH2CH2, 2H), 3.62-3.88 (m, CH2CH2N +
CH2CH2N + PO4CH2CH + COOCH2COH + CH2CH2, 8H), 4.17-4.27 (m,
CH2CH2, 2H), 5.06 (s, PO4CH2CH, 1H), 7.21 (s, CH, 4H). MALDI (matrix:
18:0 PE-NH2 (7.5 mg, 10 μmol) was dissolved in 3 mL of a
chloroform:methanol mixture (0.6:0.4 v/v) and triethylamine (50 μL).
To this solution, either DOTA-NHS (10.0 mg, 13.1 μmol) or NOTA-NHS
(10.0 mg, 15.2 μmol) was added and the mixture was left to stir for 5
h. After that, the crude was concentrated in rotavapour and purified
by preparative-TLC. Rf for PE-DOTA = 0.10-0.19 (CHCl3:MeOH:H2O
6:4:0.2 v/v) and, Rf for PE-NOTA = 0.08-0.16 (CHCl3:MeOH:H2O
6:4:0.2 v/v).
4-nitroaniline);
m/z
for
[C72H132N10O18PS2Na2]+
([M-
2H+NH4+2Na+CH3CH2OH]+) expected
=
1565.9, found 1566.0;
[C72H133N10O18PS2Na]+ ([M-H+NH4+Na+CH3CH2OH]+) expected
=
1543.9, found 1544.0.
Synthesis of PE-PEG4-TCO (4) and PE-TCO (7)
PE-DOTA: 9.2 mg, 8.2 μmol, 82%.1H NMR (400 MHz, CDCl3): δ (ppm)
0.87 (t, CH3, 6H, 3JH-H = 6.5 Hz), 1.25 (s, CH2, 56H), 1.57 (m, CH2CH2CO,
4H), 2.27 (m, CH2CH2CO, 4H), 2.41-3.18 (m, NCH2CH2N + NCH2CH2N +
NCH2COOH, 22 H), 3.64-4.43 (m, CH2CH2N + CH2CH2N + PO4CH2CH +
COOCH2COH + COCH2N, 10H), 5.17 (s, PO4CH2CH, 1H). MALDI (matrix:
18:0 PE-NH2 (10 mg, 13.3 μmol) was dissolved in 7.5 mL of a
chloroform:methanol mixture (0.56:0.44 v/v) and triethylamine (50
μL). To this solution, either a solution of TCO-PEG4-NHS (10 mg, 19.43
μmol dissolved in 0.3 mL chloroform) or TCO-NHS (5 mg, 18.71 μmol)
was added and the mixture was left to stir for 5 h. After that, the
crude was concentrated in vacuo and purified by preparative TLC. Rf
for PE-PEG4-TCO = 0.45 (CHCl3:MeOH:H2O 8:2:0.2 v/v) and, Rf for PE-
TCO = 0.50 (CHCl3:MeOH:H2O 8:2:0.2 v/v).
4-nitroaniline);
m/z
for
[C59H118N6O16PNa]+
([M-
H+NH4+Na+CH3CH2OH]+) expected = 1220.8, found 1220.7 and
[C59H117N6O16PNa2]+ ([M-2H+NH4+2Na+CH3CH2OH]+) expected
1242.8, found 1242.6.
=
PE-NOTA: 8.3 mg, 8.0 μmol, 80%. 1H NMR (400 MHz, CDCl3): δ (ppm)
0.87 (t, CH3, 6H, JH-H = 6.6 Hz), 1.25 (s, CH2, 56H), 1.56-1.90 (m,
CH2CH2CO + NCH2CH2N + NCH2CH2N, 16H), 2.26 (m, CH2CH2CO, 4H),
2.64-2-92 (m, NCH2COOH, 6 H), 3.25-3.45 (m, CH2CH2N, 2H), 3.56-
4.41 (m, CH2CH2N + PO4CH2CH + COOCH2COH + 6H), 5.19 (s,
PO4CH2CH, 1H). MALDI (matrix: 4-nitroaniline); m/z for
PE-PEG4-TCO: 13.5 mg, 11.63 μmol, 88%. 1H NMR (400 MHz, CDCl3):
δ (ppm) 0.88 (t, CH3, 6H, 3JH-H = 6.5 Hz), 1.26 (s, CH2, 56H), 1.56-1.64
(m, 2 x CH2CH2CO + 2 x CH2 (8H), 1.66-1.74 (m, CH2 (2H), 1.90-1.98
(m, CH2 (2H), 2.25-2.30 (m, 2 x CH2CH2CO, 4H), 2.33-2.40 (m, CH2
(2H), 2.41-2.52 (m, CH2 (2H), 3.46 (m, CH2CH2N, 2H), 3.52-3.84 (m,
OCH2CH2O + OCH2CH2O, 18H), 4.00 (m, CH2CH2N + COOCH2COH, 4H),
4.14-4.21 (m, trans-PO4CH2CH, 1H), 4.23-4.30 (m, COOCH, 1H), 4.35-
4-41 (m, cis-PO4CH2CH, 1H), 5.23 (m, PO4CH2CH, 1H), 5.37-5.48 (m,
CH, 1H), 5.53-5.64 (m, CH, 1H). MALDI (matrix: 4-nitroaniline); m/z
for [C62H122N3O16PN]+ ([M+NH4+CH3OH]+) expected = 1195.9, found
1195.4
3
[C55H109N5O14PNa]- ([M-3H+NH4+Na+CH3CH2OH]-) expected
=
1117.8, found 1117.5 and [C55H110N5O14P]- ([M-2H+NH4+CH3CH2OH]-
) expected = 1095.7, found 1095.4.
Synthesis of PE-isothiocyanate
18:0 PE-NH2 (50 mg, 66.9 μmol) was dissolved in 15 mL of a
chloroform:methanol mixture (0.8:0.2 v/v) and triethylamine (10 μL).
To this solution, a solution of p-phenylene diisothiocyanate (130 mg,
690.0 μmol, dissolved in 3 mL of chloroform) was dropwise added
and the mixture was left to stir for 5 h. After that, the crude was
concentrated in rotavapour and purified by flash column
chromatography. Rf = 0.5 (CHCl3:MeOH:H2O 8:2:0.2 v/v). (57 mg, 60.2
μmol, 90 %). 1H NMR (400 MHz, CDCl3): δ (ppm) 0.88 (t, CH3, 6H, 3JH-
PE-TCO: 12.0 mg, 12.13 μmol, 91%. 1H NMR (400 MHz, CDCl3): δ
(ppm) 0.88 (t, CH3, 6H, 3JH-H = 6.5 Hz), 1.26 (s, CH2, 56H), 1.58 (m, 2 x
CH2CH2CO, 4H), 1.67 (m, CH2, 4H), 1.83-2.01 (m, CH2, 4H), 2.29 (m,
CH2CH2CO, 4H + CH2, 2H), 3.33 (m, CH2CH2N, 2H), 3.85 (m, CH2CH2N
+ COOCH2COH, 4H), 4.15 (m, trans-PO4CH2CH, 1H), 4.30 (m, COOCH,
1H), 4.38 (m, cis-PO4CH2CH, 1H), 5.20 (m, PO4CH2CH, 1H), 5.53 (m,
CH, 2H). MALDI (matrix: 4-nitroaniline); m/z for [C51H101N2O11P]+
([M+NH4+CH3OH]+) expected = 948.7, found 948.4.
= 6.5 Hz), 1.25 (s, CH2, 56H), 1.53 (m, CH2CH2CO, 4H), 2.26 (m,
H
CH2CH2CO, 4H), 3.70-4.20 (m, CH2CH2N + CH2CH2N + trans-PO4CH2CH
8 | J. Name., 2012, 00, 1-3
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Synthesis of bifunctional chelators
the solution was lyophilized. The remaining solid wasVdieiwssAortlivceledOninlinae
mixture of H2O with 10-15% of dmso and purified by flash
DOI: 10.1039/C9SC00684B
chromatography (IsoleraTM Spektra System). Briefly, the crude was
loaded in a Biotage® SNAP Ultra C18 30 g cartridge and flushed with
25 mL of HCl (1 M) and washed with 5 mL of H2O. The disappearance
of the violet colour indicated the removal of iron(III) species. The
remaining mixture was eluted with the following gradient: A: H2O
with 0.1% TFA, B: CH3CN with 0.1% TFA. A to B: 2 to 30%, 30 CV, 30
to 70%, 5 CV and 70 to 98%, 5 CV. The purified product was
lyophilized and the residue dried in vacuo to give a pink solid as 7 (1.5
mg, 2.1 μmol, 22 %). The product was stable in the freezer for more
than six months. 1H NMR (400 MHz, methanol-d4): δ (ppm) 6.82 (dd,
CH, 3JH-H = 22.3, 8.4 Hz, 2H), 7.18–7.11 (m, CH, 4H), 7.23 (d, CH, 3JH-H
Synthesis of
3,3'-(((ethane-1,2-
diylbis((carboxymethyl)azanediyl))bis(methylene))bis(4-hydroxy-
3,1-phenylene))dipropionic acid (HBED-CC)
HBED-CC was synthesized by modifying a reported method:21
3-(4-Hydroxyphenyl)propionic acid (3.39 g, 20.8 mmol) was dissolved
in 21 mL of a solution of methanol : water : 4.0 N NaOH (0.71:0.19:0.1
v:v:v). To this solution, ethylenediamine-N,N′-diacetic acid (1.80 g,
10.4 mmol) suspended in 5 mL of NaOH (2 N) was added. The pH of
the mixture was adjusted to 6.7 and the solution was cooled in an ice
bath. Then, formaldehyde (aqueous solution at 37 %, 20.8 mmol, 1.5
mL) was added dropwise while keeping the pH of the solution at 6.7
by intermittent addition of NaOH (4 N) and, the suspension was
refluxed for 4 h at 70 ºC. After that, the solution was cooled to room
temperature and methanol was removed on a rotary evaporator. The
remaining aqueous solution was diluted with water (15 mL), acidified
with HCl (4 N) to pH 5.5 and extracted with diethyl ether (15 mL x 3)
to remove unreacted 3-(4-hydroxyphenyl)propionic acid. The
aqueous phase was then adjusted to pH 3 with HCl (6 N) and
unreacted ethylenediamine-N,N′-diacetic acid was filtered off. The
filtrate was then brought to neutrality with NaOH (4 N) and
concentrated in vacuo. The crude was allowed to flash
chromatography using an IsoleraTM Spektra System (Biotage® SNAP
Ultra C18 60 g cartridge, A: H2O with 0.1% TFA, B: CH3CN with 0.1%
3
= 8.2 Hz, 2H), 8.42 (d, CH, JH-H = 8.2 Hz, 2H), 4.42 (s, NHCH2C, 2H),
4.21 (s, NCH2C, 2H), 4.11 (s, NCH2C, 2H), 3.78 (s, NCH2COOH, 2H),
3.64 (s, NCH2COOH, 2H), 3.41 (s, NCH2CH2N, 2H), 3.02 (s, CH3, 3H),
3
2.91 (t, CH2CH2CONH, 3JH-H = 6.8 Hz, 2H), 2.81 (t, CH2CH2CONH, JH-H
3
= 7.5 Hz, 2H), 2.59 (t, CH2CH2CONH, JH-H = 6.8 Hz, 2H), 2.59 (t,
3
CH2CH2CONH, JH-H = 7.5 Hz, 2H). 13C NMR (100 MHz, Methanol-d4):
δ (ppm) 21.1, 31.0, 31.8, 36.9, 38.8, 43.5, 48.4, 48.6, 48.8, 49.0, 49.2,
49.4, 49.6, 51.1, 51.4, 54.2, 116.6, 119.9, 120.5, 128.9, 129.0, 131.7,
131.8, 132.2, 133.2, 133.3, 133.6, 133.7, 144.9, 156.0, 165.2, 168.7,
171.6, 172.0, 175.2, 176.7. HPLC: τr = 8.85 min (gradient A); ESI
(negative mode): m/z for [C36H40N7O9]- ([M-H]-) expected = 714.2888,
found 714.2893.
Synthesis of DOTA-GA-tetrazine (2,2',2''-(10-(1-carboxy-4-((2-(5-((4-
(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-5-
oxopentanamido)ethyl)amino)-4-oxobutyl)-1,4,7,10-
tetraazacyclododecane-1,4,7-triyl)triacetic acid) (5) was carried out
according to a method reported by our group, with some minor
modifications:10b
1
TFA. Gradient: 2-40% B, 55 CV). (277 mg, 0.52 mmol, 6 %). H NMR
(400 MHz, dmso d6): δ (ppm) 2.45 (t, CH2CH2COOH, 3JH-H = 7.8 Hz, 4H),
3
2.70 (t, CH2CH2COOH, JH-H = 7.8 Hz, 4H), 3.20 (s, CH2N, 4H), 3.65 (s,
CH2COOH, 4H), 4.02 (s, CH2N, 4H), 6.79 (d, CHarom, 3JH-H = 8.2 Hz, 2H),
7.07 (m, CHarom, 4H). 13C NMR (100 MHz, dmso- d6): δ (ppm) 29.5 (2C),
35.6 (2C), 49.5 (CH2N, 2C), 52.2 (2C), 52.7 (2C), 115.4 (2C), 118.7 (2C),
129.8 (2C), 131.4 (2C), 131.9 (2C), 154.5 (2C), 170.2 (2C), 173.8 (2C).
Synthesis of tert-butyl (4-cyanobenzyl)carbamate
HPLC/MS τr = 7.70 min (gradient A); m/z for [C26H33N2O10]+ ([M+H]+) 4-(Aminomethyl)benzonitrile hydrochloride (5.0 g, 30.0 mmol) was
expected = 533.2135, found 533.2142.
dissolved in CH2Cl2 (4.0 mL), triethylamine (11.0 mL, 75.0 mmol) and
di-tert-butyl dicarbonate (7.4 g, 33.0 mmol) were added and the
resultant mixture stirred at room temperature for 16 h. The solvent
was removed under reduced pressure, water added (100 mL) and the
product extracted into DCM (3 x 100 mL). The organic layers were
combined, washed with water (1 x 100 mL) and the solvent removed
Synthesis of 3-(3-(((carboxymethyl)(2-((carboxymethyl)(2-hydroxy-
5-(3-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-3-
oxopropyl)benzyl)amino)ethyl)amino)methyl)-4-
hydroxyphenyl)propanoic acid (HBED-CC-Tetrazine, 5)
under
reduced
pressure
to
yield
tert-butyl
(4-
Compound 5 was synthesized by modifying a reported method:13
cyanobenzyl)carbamate (5.8 g, 84 % yield) as a white powder. 1H
NMR (400 MHz, CDCl3): δ (ppm) 1.46 (s, COOC(CH3)3, 9H), 4.37 (d,
CH2, 3JH-H = 6.1 Hz, 2H), 4.97 (sbr, NH, 1H), 7.38 (d, CHarom, 3JH-H = 8.4
Hz, 2H), 7.62 (d, CHarom, 3JH-H = 8.4 Hz, 2H). 13C NMR (100 MHz, CDCl3):
δ (ppm) 28.5 (3C), 44.4 (1C), 80.2 (1C), 111.3 (1C), 118.9 (1C), 127.9
(2C), 132.6 (2C), 144.8 (1C), 156.0 (1C). ESI (positive mode): m/z for
[C13H16N2O2] ([M+H]+) expected = 233.1290, found 233.1296.
HBED-CC (5.3 mg, 9.4 μmol) was dissolved in 0.7 mL of a mixture of
H2O:CH3CN (50:50 v/v) containing 20 μL in N,N-
diisopropylethylamine. To this solution, 190 μL of FeCl3 (0.1 M in
water) was added and the reaction mixture was stirred for 1 h and
then evaporated under reduced pressure to dryness. The resulting
solid was dissolved in CH3CN and evaporated to dryness again to
remove free water. 1.25 mL of anhydrous dmso was then added. To
this solution, a solution of HATU (3.5 mg, 9.5 μmol) in 0.25 mL of
anhydrous dmso and 0.050 mL N,N-diisopropylethylamine was
added dropwise. After 15 min, (4-(6-methyl-1,2,4,5-tetrazin-3-
yl)phenyl)methanamine (2.3 mg, 11.3 μmol) dissolved in 0.5 mL of
anhydrous dmso was added dropwise and the reaction was left to
react for 24 h. The reaction was then quenched with 2 mL of H2O and
Synthesis
yl)benzyl)carbamate
of
tert-butyl
(4-(6-methyl-1,2,4,5-tetrazin-3-
A high pressure reaction tube was treated with tert-butyl (4-
cyanobenzyl)carbamate (464 mg, 2.0 mmol), CH3CN (1050 μL, 20.0
mmol), nickel(II) trifluoromethanesulfonate (356 mg, 1.0 mmol) and
hydrazine hydrate (50-60% NH2NH2) (6.2 mL, 100.0 mmol). The tube
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was sealed and heated to 60°C for 72 h, following which H2O (10 mL) oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid
View Article Online
DOI: 10.1039/C9SC00684B
and sodium nitrite (2.82 g, 40.0 mmol) were added to the mixture. (DOTA-GA-Tetrazine, 6)
HCl (1M) was added dropwise until the pH reached 3 and gases
To
a
stirred solution of 5-((4-(6-methyl-1,2,4,5-tetrazin-3-
stopped evolving, at which point the mixture had turned bright red.
The product was extracted with EtOAc (3 x 40 mL), and the combined
organic layers were washed with H2O (3 x 20 mL), dried over MgSO4,
filtered and concentrated under reduced pressure. The bright pink
crude solid was purified by flash column chromatography (DCM/
Et2O gradient 100:0 v/v to 96:4 v/v) to give tert-butyl (4-(6-methyl-
1,2,4,5-tetrazin-3-yl)benzyl)carbamate (0.37 g, 62% yield) as a bright
yl)benzyl)amino)-5-oxopentanoic acid (10.0 mg, 31.71 mol) in
anhydrous dmso (220 L) and triethylamine (7 L), was added a
solution of N-hydroxysuccinimide (18.8 mg, 162.5 mol) and N,N′-
dicyclohexylcarbodiimide (33.5 mg, 162.5 mol) in anhydrous dmso
(80 L). The resultant mixture was vigorously stirred for 16 h at room
temperature in the absence of light. After that, the solution was
passed through a 0.45 m filter and added to a solution containing
DOTA-GA-NH2 (17 mg, 32 mol) in 100 L of anhydrous dmso. The
reaction was stirred for 5 h at room temperature in the absence of
light. The crude was lyophilized and the residue dried in vacuo to give
a pink oil as crude. The product was purified by flash chromatography
using an IsoleraTM Spektra System (Biotage® SNAP Ultra C18 12 g
cartridge, A: H2O with 0.1% TFA, B: CH3CN with 0.1% TFA. Gradient:
A to B: 2 to 30%, 40 CV, 30 to 70%, 5 CV and 70 to 98%, 5 CV), to yield
1
pink solid. Rf = 0.39 (2% Et2O/ DCM). H NMR (400 MHz, CDCl3): δ
(ppm) 1.46 (s, COOC(CH3)3, 9H), 3.07 (s, CH3, 3H), 4.41 (d, CH2, 3JH-H
=
5.6 Hz, 2H), 5.09 (sbr, NH, 1H), 7.47 (d, CHarom, 3JH-H = 8.4 Hz, 2H), 8.51
(d, CHarom, 3JH-H = 8.4 Hz, 2H). 13C NMR (100 MHz, CDCl3): δ (ppm) 21.2
(1C), 28.5 (3C), 44.5 (1C), 79.9 (1C), 128.1 (2C), 128.3 (2C), 130.9 (1C),
144.1 (1C), 156.1 (1C) 164.0 (1C), 167.3 (1C). ESI (positive mode): m/z
for [C15H20N5O2] ([M+H]+) expected = 302.1617, found 302.1621.
Synthesis
of
(4-(6-methyl-1,2,4,5-tetrazin-3- 6 as a pink solid (19.6 mg, 24.10 mol, 76% yield). HPLC: τr = 7.78 min
yl)phenyl)methanamine
(gradient A). The product was stable in the freezer for more than six
1
months. H NMR (400 MHz, methanol-d4) 1.90-1.98 (m, COCH2CH2,
2H), 2.25 (t, COCH2CH2, 3JH-H = 7.6 Hz, 2H), 2.33 (t, COCH2CH2, 3JH-H
=
Tert-butyl (4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)carbamate (30.4
mg, 0.15 mmol) was treated with TFA/CH2Cl2 (1/1, v/v, 3 mL) and
stirred for 1.5 h at room temperature. The solvent was removed
under reduced pressure to yield the TFA salt of 4-(6-methyl-1,2,4,5-
tetrazin-3- yl)benzylamine 3 (26.4 mg, 84 % yield) as a bright pink
solid. 1H NMR (400 MHz, methanol-d4): δ (ppm) 3.06 (s, CH3, 3H), 4.26
7.6 Hz, 2H), 2.43-3.02 (m, 8H), 3.04 (s, CH3, 3H), 3.12-3.29 (m, 8H),
3.39-4.31 (m, 15H), 4.50 (s, NHCH2C, 2H), 7.55 (d, CHarom, 3JH-H = 8.8
Hz, 2H), 8.51 (d, CHarom,
3JH-H = 8.4 Hz, 2H). 13C NMR (125 MHz,
methanol-d4) δ (ppm) 21.1, 23.2, 28.5, 30.7, 36.2, 36.3, 40.0, 40.2,
43.8, 49.6, 51.1, 54.3, 54.5, 56.2, 57.5, 129.1, 129.3, 132.4, 145.2,
165.2, 168.8, 175.5, 175.5, 175.6, 175.7, 175.7. ESI (positive mode):
m/z calcd for [C36H54N11O11]+ ([M+H]+) 816.4004, found 816.4010.
(s, CH2, 2H), 7.72 (d, CHarom, 3JH-H = 8.4 Hz, 2H), 8.62 (d, CHarom, 3JH-H
8.4 Hz, 2H). 13C NMR (100 MHz, methanol-d4): δ (ppm) 21.1 (1C), 43.9
=
(1C), 129.4 (2C), 130.8 (2C), 134.3 (1C), 138.8 (1C), 164.9 (1C), 169.1
(1C). ESI (positive mode): m/z for [C10H12N5] ([M+H]+) expected = RADIOCHEMISTRY
202.1093, found 202.1083; m/z for [C12H15N6] ([M+H+CH3CN]+)
68GaCl3 was eluted from a GalliaPharm® 68Ge/68Ga Generator from
Eckert & Ziegler Radiopharma GmbH (0.74 GBq) using a fully
Synthesis of 5-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-5- automated Modular-Lab system and an adapted NaCl-based method
expected = 243.138, found 243.1214.
oxopentanoic acid
previously reported.20 Briefly, 68GaCl3 was eluted in 5 mL of a 0.1 M
HCl solution, which was subsequently trapped on a SCX cartridge
(preconditioned with 1 mL 5.5 M HCl and 10 mL water) and the
activity was then eluted, with minimal loss (2-5%), using a mixture of
12.5 L of 5.5 M HCl and 500 L of 5 M NaCl solution. This eluate was
subsequently buffered for direct use in 68Ga-labelling experiments.
A
dry flask was treated with (4-(6-methyl-1,2,4,5-tetrazin-3-
yl)phenyl)methanamine (26.8 mg, 0.13 mmol), glutaric anhydride (76
mg, 10.67 mmol) and THF (2 mL), and the resultant mixture was
heated to 70°C for 4 h. The mixture was cooled to 50°C, and was
stirred at this temperature for a further 16 h. The solvent was
evaporated in vacuo, to give the crude material as a pink oil. The iTLC quantification
product was purified by flash chromatography (IsoleraTM Spektra
Chelators, freshly prepared from stock solutions for each
System with a Biotage® SNAP Ultra C18 12 g cartridge) eluting with a
gradient of 0-50% H2O/CH3CN (15 CV) to give 4 as a red solid (38.9
mg, 0.12 mmol, >90% yield). Rf = 0.2 (10% MeOH/CH2Cl2). HPLC: τr =
6.50 min (gradient A). 1H NMR (500 MHz, methanol-d4): δ (ppm) 1.94
(m, COCH2CH2, 2H), 2.35 (m, 2 x COCH2CH2, 4H), 3.03 (s, CH3, 3H),
experiment, were dissolved in aqueous solutions of sodium acetate
(0.2 M) to provide solutions with chelator concentrations ranging
from 0.05 to 500 M (0.05, 0.5, 5, 50, 100, 250 and 500 M). 68Ga (15
L, approx. 2 MBq in 0.1 M aqueous HCl) was added to chelator
solutions (100 L) and the reaction solution was incubated at either
25 or 90 ºC. The final pH of the reaction solutions was 3.5, 4.5 and
5.5. After 10 min, the reaction solution was analysed by iTLC (glass
microfiber chromatography paper impregnated with silica gel, 100 x
10 mm). As controls, 68Ga3+ (15 L, approx. 2 MBq in 0.1 M aqueous
HCl) was mixed with 100 L of sodium acetate (0.2 M) at the three
tested pHs. The controls were incubated at either 25 or 90 ºC for 10
3
4.49 (s, CH2NH, 2H), 7.54 (d, CH, 3JH-H = 8.8 Hz, 2H), 8.51 (d, CH, JH-H
= 8.5 Hz, 2H). 13C NMR (126 MHz, methanol-d4) (2 Cquat not observed)
δ (ppm) 21.0 22.3, 34.1, 36.0, 43.8, 129.0, 129.3, 132.4, 145.1, 165.2,
168.7, 175.5, 176.8. ESI (positive mode): m/z calcd for [C15H18N5O3]+
([M+H]+) 316.1410, found 316.1414 and, calcd for [C15H17N5O3Na]+
([M+Na]+) 338.1229, found 338.1278.
Synthesis of 2,2',2''-(10-(1-carboxy-4-((2-(5-((4-(6-methyl-1,2,4,5- min and then, analysed by iTLC and Laura 3 software (LabLogic,
tetrazin-3-yl)benzyl)amino)-5-oxopentanamido)ethyl)amino)-4-
Sheffield, UK). The mobile phase used was ammonium acetate (1M
10 | J. Name., 2012, 00, 1-3
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in methanol:water 80:20 v/v) for all chelators. [68Ga(PE-NOTA] Rf = contained unreacted precursor and residual lipids and vesicles, which
View Article Online
0.66 – 0.76; [68Ga(PE-DOTA] Rf = 0.67 – 0.78; [68Ga(PE-iso-DOTAGA] did not form part of the microbubble shell, was discarded. The
DOI: 10.1039/C9SC00684B
Rf = 0.62 – 0.76; [68Ga(DOTAGA-Tetrazine] Rf = 0.67 – 0.77 microbubble cake was re-dispersed to a 1 mL volume of 20 vol%
[68Ga(HBED-CC-Tetrazine] Rf = 0.68 – 0.78; non-chelated 68Ga3+ Rf < glycerol solution in PBS and stored in the same vial with PFB
0.3. iTLC plates were imaged and quantified by digital headspace until further use. The washing step was repeated three
autoradiography using instruments and software described above.
times or until no activity was measured in the infranatant solution.
68Ga-labelled MBs were counted and sized on a Countess II FL
Automated Cell Counter and stored in the same vial with PFB
headspace until further use.
68Ga-labelling of 1
68GaCl3 eluate (~ 75 MBq) was added to a solution of 2 mL sodium
acetate buffer (0.1 M, pH = 4.0) and 100 µL chelator (0.3 mg·mL-1 in
EtOH). During the labelling, the pH of the reaction mixture inside the
reactor was determined to be 3.7 ± 0.2. After incubating the solution
for 15 min at 90 ºC, the reaction mixture was then diluted with 5 mL
of water and passed through an Oasis® HLB (1cc / 30 mg) cartridge
(preconditioned with 10 mL ethanol and 10 mL water). The cartridge
(*) An alternative dienophile-modified phospholipid without the
PEG4 spacer, named PE-TCO (7), was also tested for reaction with
[68Ga][Ga(5)]. Under the same conditions, 7 showed low conversions
(approx. 10%), probably as a consequence of its poor solubility in
ethanol (data not shown).
was washed with 5 mL of water and the labelled compound was MICROBUBBLE PRODUCTION
eluted with 800 µL of pure ethanol. 1 gave radiochemical conversions
The lipid-coated, decafluorobutane-filled microbubbles (MBs) were
>80%, and the product was obtained in >95% radiochemical purity,
and in 20-30% isolated RCY (n.d.c.). The synthesis was finished within
about 23 min.
manufactured using a modified formulation.18 Matsunaga et al.
prepared MBs containing a lipid mixture of 85 mol % 1,2-dipalmitoyl-
sn-glycero-3-phosphocholine (DPPC), 10 mol
% 1-palmitoyl-2-
68Ga-labelling of 5 and 6ꢀ
hydroxy-sn-glycero-3 phosphocholine (LPC), and 5 mol % 1,2-
dipalmitoyl-sn-glycero-3-phosphoethanolamine-N
68GaCl3 eluate (200-220 MBq) was added to a solution of 4.5 mL
sodium acetate buffer (0.1 M, pH = 4.5) containing 30 µL chelator (1
mg·mL-1 in DMSO). During the labelling, the pH of the reaction
mixture inside the reactor was determined to be 4.2 ± 0.3. After
incubating the solution for 10 min either at room temperature for 5
or at 90 ºC for 6, the 68Ga chloride was quantitatively chelated to the
precursor. The reaction mixture was then diluted with 5 mL of water
and trapped on a Sep-Pack® Light tC18 cartridge (preconditioned with
10 mL ethanol and 10 mL water). The cartridge was washed with 5
mL of water and the labelled compound was eluted with 800 µL of
pure ethanol. Both chelators gave radiochemical conversions >95%,
and the product was obtained in >95% radiochemical purity, and in
70-80% isolated RCY (n.d.c.). The synthesis was finished within about
15 min.
[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000-NH2). In this
paper, we studied the possibility of incorporating 1,2-distearoyl-sn-
glycero-3-phosphoethanolamine (PE-NH2) lipid into the MB shell. For
that reason, we prepared MBs having a final lipid mixture of 85 mol
% DPPC, 10 mol %, (either PE-NH2 or PE-PEG4-TCO) and 5 mol % DSPE-
PEG2000-NH2. In all cases, the total lipid concentration was between
0.86 and 0.88 mg/mL. The mixture of lipids was first dissolved in
chloroform, dried over nitrogen gas, and then further dried in vacuo
overnight to remove residual solvent from the lipid films. Then, 1 mL
of a solution of propylene glycol, glycerol, and phosphate-buffered
saline (PBS) (15:5:80, v/v/v) was used to rehydrate the films. This
created a lipid suspension, which was immediately stirred for 10 min
at 50º-60º C and later, decanted in a sealed 2 mL glass vial. Prior to
activation, the headspace was purged with decafluorobutane at
room temperature and, MBs were produced via mechanical
agitation.
Synthesis of 68Ga-labelled MBsꢀ
An aliquot of purified [68Ga][Ga(5)] (~ 75 MBq) was added to a vial
containing PE-PEG4-TCO (0.13 mg, 0.107 mol). The reaction vial was
incubated for 20 min at 60 ºC under a N2 stream. After that, the
volume of the reaction was reduced to about 100 L and a solution
containing the rest of lipids was added (concretely, 0.62 mg of DPPC
and 0.140 mg DSPE-PEG2000-NH2 dissolved in 1 mL of a solution of
propylene glycol, glycerol, and phosphate-buffered saline (PBS)
(15:5:80, v/v/v)). The vial was sealed and activated to form
microbubbles (see in the next section the experimental details for
this process).
BIOLOGY EXPERIMENTAL PROCEDURES
All animal experiments were conducted by licensed investigators in
accordance with the United Kingdom Home Office Guidance on the
Operation of the Animals (Scientific Procedures) Act 1986 (HSMO,
London, UK, 1990) and with the published guidelines for the Welfare
of Use of Animals in Cancer Research Institute Committee on Welfare
of Animals in Cancer Research.22
PET Imaging
In order to remove unreacted [68Ga][Ga(5)], microbubbles were Dynamic PET imaging of [68Ga(5)], 68Ga-PE and 68Ga-MBs was
purified by centrifugation adapting a reported method.17 The vial performed in non-tumour bearing Balb/c mice (5-6 mice/cohort, 6-8
containing the 68Ga-labelled microbubbles was put upside down and weeks) (Charles River UK Ltd.). Mice were anesthetized with 2.5%
centrifuged at 350 g during 2 min using a ROTINA 35 R centrifuge. isoflurane/O2 and placed in a thermostatically controlled ring in a
Then, the microbubbles were collected into a concentrated cake on dedicated small animal Genisys4 PET scanner (SOFIE Biosciences,
the top of the vial and the remaining suspension (infranatant), which Culver City, USA). Following injection of radioactivity (0.74 MBq in
This journal is © The Royal Society of Chemistry 20xx
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the case of non-MB tracers, and between 0.37-0.74 MBq for 68Ga-
labelled MB, keeping a constant value of 5x107 MB/injection) via
lateral tail vein cannula, PET scans were acquired in a list-mode
format over 0-30 min to give decay-corrected values of radioactivity
accumulation in tissues. The collected data were ordered into 11
time frames (4x15 s, 4x60 s, 3x300 s) and reconstructed with a 3-
dimensional maximum likelihood estimation method (3D ML-EM).
Volumes of interest (VOIs) for each tissue were defined using
Siemens Inveon Research Workplace software (Siemens Molecular
Imaging Inc.l Knoxville, USA) and count densities (counts/min) were
expressed as a percentage of the incubated dose (ID) of radioactivity
and normalized to tissue weight. Tissue kinetics were calculated by
averaging the count densities per timepoint.
C. Woenne, S. Doerk, G. Ladewig, J. W. Jenne, S. Delorme, L.
Grenacher, P. Hallscheidt, G. W. KauffDmOaIn: 1n0,.1W03.V9Sie/eCwm9ASrmtCicl0lee0rO6na8ln4inBde
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9
Biodistribution
Biodistribution studies were carried out on the same cohort of
animals after the end of PET scans (20 min after radioactivity
injection). Tissue samples were quickly collected and radioactivity
content was determined by -counting (LKB Wallac 1282
Compugamma laboratory -counter, PerkinElmer, Massachusetts,
USA). Count densities (counts/min) were expressed as a percentage
of the incubated dose (ID) of radioactivity and normalized to tissue
weight.
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Acknowledgements
This work was supported by the Cancer Research UK (CRUK
Multidisciplinary Award - C53470/A22353).
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a) N. Lazarova, P. W. Causey, J. A. Lemon, S. K. Czorny, J. R.
Forbes, A. Zlitni, A. Genady, F. Stuart Foster and J. F. Valliant,
Nucl. Med. Biol. 2011, 38, 1111-1118; b) J. K. Willmann, Z.
Cheng, C. Davis, A. M. Lutz, M. L. Schipper, C. H. Nielsen and S.
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Page 13 of 13
Chemical Science
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Journal Name
ARTICLE
View Article Online
DOI: 10.1039/C9SC00684B
TOC
We report a rapid and efficient method for labelling ultrasound
microbubble (MB) agents with a generator-produced PET isotope
using
a facile ligation between a trans-cyclooctene-modified
phospholipid and a new 68Ga-HBED-CC-tetrazine tracer. This method
provides accessible solutions for in vivo tracking of MBs.
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