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ARTICLE IN PRESS
Applied Radiation and Isotopes 64 (2006) 333–336
www.elsevier.com/locate/apradiso
Microﬂuidic technology for PET radiochemistry
J.M. Gilliesa,, C. Prenanta,b, G.N. Chimona,b, G.J. Smethursta, B.A. Dekkera, J. Zweita,b
a
Cancer Research-UK/University of Manchester Radiochemical Targeting and Imaging Group, Paterson Institute for Cancer Research,
Manchester, M20 4BX, UK
b
School of Chemical Engineering and Analytical Sciences, University of Manchester, P.O. Box 88, Manchester, M60 1QD, UK
Received 8 December 2004; received in revised form 30 August 2005; accepted 30 August 2005
Abstract
This paper describes the ﬁrst application of a microfabricated reaction system to positron emission tomography (PET) radiochemistry.
We have applied microﬂuidic technology to synthesise PET radiopharmaceuticals using 18F and 124I as labels for ﬂuorodeoxyglucose
(FDG) and Annexin-V, respectively. These reactions involved established methods of nucleophilic substitution on a mannose triﬂate
precursor and direct iodination of the protein using iodogen as an oxidant. This has demonstrated a proof of principle of using
microﬂuidic technology to radiochemical reactions involving low and high molecular weight compounds. Using microﬂuidic reactions,
[18F]FDG was synthesised with a 50% incorporation of the available F-18 radioactivity in a very short time of 4 s. The radiolabelling
efﬁciency of 124I Annexin-V was 40% after 1 min reaction time. Chromatographic analysis showed that such reaction yields are
comparable to conventional methods, but in a much shorter time. The yields can be further improved with more optimisation of the
microﬂuidic device itself and its ﬂuid mixing proﬁles. This demonstrates the potential for this technology to have an impact on rapid and
simpler radiopharmaceutical synthesis using short and medium half-life radionuclides.
r 2005 Elsevier Ltd. All rights reserved.
Keywords: PET radiochemistry; Radiosynthesis; Microfabrication; Microﬂuidics
1. Introduction
Positron emission tomography (PET) allows the study of
in vivo biochemistry and biology underlying disease and
therapeutic intervention (Gambhir, 2002, Massoud and
Gambhir, 2003, Reader and Zweit, 2001). This unique
capability allows a rational assessment of, for example,
anti-cancer drug development in early clinical trials.
Current practice involves the manipulation of macroscopic
quantities of material in the synthesis of various radiopharmaceuticals. Nanotechnology, the miniaturisation of
macroscale processes and devices, offers distinct advantages to PET radiochemistry. In particular, intrinsic
reduction in resources and logistics is required for PET
radiochemical preparations. Microﬂuidic technologies are
capable of controlling and transferring tiny quantities of
liquids which allow chemical and biochemical assays to be
Corresponding author. Tel.: +44 161 446 3150; fax: +44 161 446 3109.
E-mail address: [email protected] (J.M. Gillies).
0969-8043/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.apradiso.2005.08.009
integrated and carried out on a small scale. In the ﬁrst
place, radiochemical reactions on-chip can be easily
shielded and will not require the space and resources
required for conventional hot cell synthesis. Secondly, it
provides a scope for an integrated total system (synthesis,
separation and analysis). Thirdly, due to the efﬁcient and
rapid mixing in miniaturised reactors (Regenfuss et al.,
1985), the speed of radiochemical synthesis and puriﬁcation can be accelerated. Finally, the photolithographic
fabrication of the microfabricated device allows the
manufacture of complex, yet relatively inexpensive and
disposable devices (Mitchell, 2001; Ramsey, 1999). We
demonstrate the rapid radiosynthesis of the PET metabolic
tracer 2-[18F]ﬂuorodeoxyglucose (2-[18F]FDG) and the
radioiodination of the apoptosis marker, [124I]Annexin-V
(Dekker et al., 2005a, 2005b; Keen et al., 2005) using a
simple microreactor. Radiolabelling using the microreactor
demonstrated considerable improvements in speed of
reaction while using reduced reagent volumes and concentrations in comparison to conventional radiosynthesis.
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334
Here, we show the ﬁrst application of a microfabricated
reaction system to PET radiochemistry, we term ‘‘microﬂuidic PET’’.
2. Experimental
The design and fabrication of a simple microﬂuidic
reactor, to generate adequate mixing and transfer of
reactants, is shown in Fig. 1 (Stuernstrom and Roeraade,
1998; Lin et al. 2001; Tsai and Lin, 2001). The microreactor
was constructed from three layers of thermally bonded
soda-lime glass plates (15 15 1 mm) using standard
photolithographic techniques. The microreactor disc
(Fig. 1) was 10 mm in diameter and 0.1 mm deep. The
middle and bottom plates were etched using 50% HF
solution to form two 10 mm diameter 100 mm deep discs,
in which the reagents mixing would take place (Fig. 1). This
gave a total internal volume of 16.0 mL. The top plate had
three 1 mm holes drilled to form the inlets. The central
mixing disc was connected via 0.1 mm channels to the three
inlets. The inlets were connected, via fused silica capillaries,
to external reagent reservoirs (PEEK HPLC loops, 200 mL)
linked to a nitrogen gas manifold (51.4 mL min 1 ﬂowrate).
This generated a back pressure of 6.0 104 Nm 2 which
was enough to drive the contents of the three reagent
reservoirs through the microreactor at a ﬂowrate of
250 mL min 1. The middle and bottom plates were then
aligned and a hole (1 mm diameter hole 5 mm deep) was
drilled horizontally into the interface between the middle
and bottom plates, to act as placement for the outlet.
Detection and imaging of apoptosis involves the highly
speciﬁc binding of Annexin-V to phosphatidylserine (PS)
that appears on the extracellular membrane of cells
undergoing apoptosis (Tokita et al., 2003). Direct methods
for the radiolabelling of Annexin-V have been developed
using 124I-iodine (Dekker et al., 2005a, 2005b; Keen et al.,
2005). This method was used in conjunction with the
microfabricated device. In the radioiodination of AnnexinV described here, reservoir 1 contained unlabelled Annexin-V (10 mL in 200 mL PBS at pH 7.0). Reservoir 2
contained 124I, 5 mL (20 MBq) in 200 mL PBS at pH 7.0
and reservoir 3 contained the oxidising agent iodogen
(40 mg in 200 mL acetonitrile). The reaction on the microfabricated device was carried out by ﬂushing all three of the
reagent reservoirs (under a stream of nitrogen) through the
microfabricated device at the same time, at a total ﬂowrate
of 250 mL min 1. The reaction product generated from this
continuous ﬂow was sampled and analysed over a range of
time points (0–100 s) during the course of the reaction, until
all the reagent reservoirs were completely empty. The
unpuriﬁed reaction mixture appearing at the outlet of the
chip was analysed by radioTLC at various time points
(10–120 s) (Fig. 2) with a mobile phase 5% trichloroacetic
acid and analysed using an Instant Imager electronic
autoradiography system (Packard, USA). The labelling
efﬁciency refers to the proportion of radioactivity that is
incorporated into the compound as a fraction of the total
radioactivity used. After 2 min, a 4075% labelling
efﬁciency was obtained for both the microﬂuidic and
conventional reactions (Fig. 2).
3. Discussion
To demonstrate ‘‘proof of principle’’, we have investigated the radioiodination of small and large molecular
weight molecules using the microﬂuidic device. These
reactions involved the direct radioiodination of the
apoptosis marker Annexin-V, and the radioﬂuorination
of the PET radiotracer 2-[18F]FDG.
Top Plate
Fitting Inlets
15 mm
3 mm
Middle Plate
Etched mixing discs
(10 mm diameter,100µm deep)
Vortex Mixer (0.2 mm diameter)
Bottom Plate
Fitting Outlet
Fig. 1. Three-dimensional diagram representing the construction of the
microreactor incorporating a three tier system of inlets, reactor and outlet.
Fig. 2. On-chip radiolabelling of [124I]Annexin-V. Labelling efﬁciency
assessed over the ﬁrst 120 s of the reaction. Samples were analysed using
radioTLC. This graph shows that both the conventional and microﬂuidic
reactions are almost instantaneous and reach the maximum labelling
efﬁciencies within 120 s. Labelling efﬁciency was determined as percentage
of radioactivity incorporated/total radioactivity used.
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J.M. Gillies et al. / Applied Radiation and Isotopes 64 (2006) 333–336
Fig. 3. Schematic of the experimental set-up for the production of 2[18F]FDG. FDG was synthesised according to the method of Hamacher et
al. Fluorine-18 was produced from proton bombardment of [18O]H2O
target. In this reaction, two microfabricated devices were linked in
sequence using lengths of fused silica capillary connecting the outlet of
device 1 to inlet of device 2. Reagent reservoirs 1 and 2 connected to device
1 were primed with the following solutions, respectively, [18F]KF/K2.2.2./
K2CO3 in 200 mL DMF and mannose triﬂate (25 mg in 200 mL DMF).
Inlet 3 of both devices were blocked off. A third reagent reservoir was
primed with sodium methanolate in MeOH and connected to inlet 2 of
device 2. The total volume of reactants passed through the chip within 6 s
of the start of reaction and were collected and analysed by radioTLC
(silica gel 60F254: 90% acetonitrile).
The second PET tracer selected for production using the
microfabricated device was 2-[18F]FDG. The current
method of 2-[18F]FDG synthesis is based on the approach
developed by Hamacher et al. (1986). A modiﬁcation of the
Hamacher reaction was carried out using two microfabricated devices linked together in sequence (Fig. 3). The ﬁrst
microfabricated device was designed to carry out the
[18F]ﬂuorination of the protected mannose triﬂate precursor. This was followed by deprotection on the second
microfabricated device using sodium methanolate in
methanol.
Device 1 produced a reaction between 18F(500 MBq)/
KF/Kryptoﬁx.2.2.2/K2CO3 in N,N-dimethylformamide
(DMF) and mannose triﬂate in DMF. This resulted in
the production of the 2-[18F]ﬂuoro-tetra-O-acetyl-mannose
(Fig. 3).
The 2-[18F]ﬂuoro-tetra-O-acetyl-mannose was then
pumped onto the second chip where it mixed with a
solution of 10% sodium methanolate in methanol resulting
in the production of 2-[18F]FDG. Fifty percent of the
available F-18 radioactivity was incorporated as 2[18F]FDG within 6 s. This was determined by analysing a
fraction (1 mL) of the total volume collected at the outlet of
the device. Fig. 4 shows a radiochromatogram of this
unpuriﬁed 2-[18F]FDG fraction. The amount of 2[18F]FDG incorporated F-18 radioactivity was 450%,
with approximately 20–30% of the radioactivity associated
335
Fig. 4. RadioTLC analysis of the products generated from the multichip
synthesis of 2-[18F]FDG.
with the unhydrolysed 2-[18F]ﬂuoro-tetra-O-acetyl-mannose and a further 10–20% was unreacted [18F]F . The
unreacted [18F]F is present due to the incomplete reaction
within device 1 and 2-[18F]ﬂuoro-tetra-O-acetyl-mannose is
present on the radioTLC from the incomplete hydrolysis of
products produced in device 2. The radiochemical purity
that could be expected using this technology will exceed the
495% speciﬁcation stipulated by the European Pharmacopia.. As mentioned earlier, one advantage of microﬂuidic
PET radiochemistry is the feasibility of an integrated
system incorporating synthesis, puriﬁcation and analysis.
In this context, a miniaturised radioHPLC can be
incorporated into the system for on-line analysis. For the
purpose of demonstrating proof of principle, the amount of
18
F used was only 500 MBq. Since the method is based on
continuous ﬂow of reactants, much higher amounts (tens of
GBq) of 18F can be utilised in the reaction.
Taken together, these preliminary results demonstrate
the feasibility of microﬂuidic radiochemistry, and the
preliminary data presented here indicate similar reaction
yields of the two methods. Further studies based on
modelling and experimental validation are necessary for
optimisation of the device.
Acknowledgements
This work is funded by Cancer Research UK. Thanks to
Dr. Graham Cowling for proof reading the manuscript.
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