On-demand radiosynthesis of: N -succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) on an electrowetting-on-dielectric microfluidic chip for 18F-labeling of protein

Article abstract of DOI:10.1039/c9ra06158d

An all-electronic, droplet-based batch microfluidic device, operated using the electrowetting on dielectric (EWOD) mechanism was developed for on-demand synthesis of N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB), the most commonly

Full text of DOI:10.1039/c9ra06158d

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PAPER
On-demand radiosynthesis of N-succinimidyl-4-
1
8
18
[
F]fluorobenzoate ([ F]SFB) on an
Cite this: RSC Adv., 2019, 9, 32175
electrowetting-on-dielectric microfluidic chip for
1
8
F-labeling of protein†
abcd
abe
f
Hee-Kwon Kim,
Kirstin A. Zettlitz,
R. Michael van Dam
Muhammad Rashed Javed,
Supin Chen,
abi
ab
abi
gh
Jeffrey Collins, Anna M. Wu, Chang-Jin “C. J.” Kim,
abg
abf
and Pei Yuin Keng
*
An all-electronic, droplet-based batch microfluidic device, operated using the electrowetting on dielectric
1
8
(
(
EWOD) mechanism was developed for on-demand synthesis of N-succinimidyl-4-[ F]fluorobenzoate
18
18
[
F]SFB), the most commonly used F-prosthetic group for biomolecule labeling. In order to facilitate
the development of peptides, and proteins as new diagnostic and therapeutic agents, we have diversified
18
the compact EWOD microfluidic platform to perform the three-step radiosynthesis of [ F]SFB starting
18
from the no carrier added [ F]fluoride ion. In this report, we established an optimal microliter droplet
18
reaction condition to obtain reliable yields and synthesized [ F]SFB with sufficient radioactivity for
subsequent conjugation to the anti-PSCA cys-diabody (A2cDb) and for small animal imaging. The three-
18
step, one-pot radiosynthesis of [ F]SFB radiochemistry was adapted to a batch microfluidic platform
with a reaction droplet sandwiched between two parallel plates of an EWOD chip, and optimized.
Specifically, the ratio of precursor to base, droplet volume, reagent concentration, reaction time, and
18
evaporation time were found be to be critical parameters. [ F]SFB was successfully synthesized on the
18
EWOD chip in 39 ꢀ 7% (n ¼ 4) radiochemical yield in a total synthesis time of ꢁ120 min ([ F]fluoride
18
activation,
[ F]fluorination, hydrolysis, and coupling reaction, HPLC purification, drying and
18
reformulation). The reformulation and stabilization step for [ F]SFB was important to obtain a high
Received 8th August 2019
Accepted 17th September 2019
protein labeling efficiency of 33.1 ꢀ 12.5% (n ¼ 3). A small-animal immunoPET pilot study demonstrated
18
that the [ F]SFB-PSCA diabody conjugate showed specific uptake in the PSCA-positive human prostate
cancer xenograft. The successful development of a compact footprint of the EWOD radiosynthesizer has
the potential to empower biologists to produce PET probes of interest themselves in a standard laboratory.
DOI: 10.1039/c9ra06158d
rsc.li/rsc-advances
1
probes that have been identied. In addition to small molecule
PET probes, biomolecules such as peptides, proteins and anti-
1
. Introduction
Despite the efficacy of positron emission tomography (PET) in bodies as diagnostic and therapeutic agents have received
medicine and life sciences, the majority of PET imaging is increasing interest due to their high specicity and affinity in
1
8
2,3
performed using a single probe, 2-[ F]uoro-2-deoxyglucose targeting distinct cellular antigens in vivo. For clinical appli-
(
1
8
[ F]FDG), while there are more than 1800 F-18 labelled PET cation, the radioisotope F-18 is most attractive for PET imaging
due to the moderate half-life (109.7 min), high positron yield
a
f
Department of Molecular and Medical Pharmacology, University of California, Los
Department of Materials Science and Engineering, National Tsing Hua University,
No. 101, Section 2, Guangfu Road, Hsinchu, Taiwan. E-mail: keng.py@mx.nthu.
edu.tw; Fax: +886-3-5722366; Tel: +886-3-5715131 ext. 33884
Angeles, Los Angeles, CA 90095, USA
b
Crump Institute for Molecular Imaging, University of California, Los Angeles, Los
Angeles, CA 90095, USA
g
Bioengineering Department, University of California, Los Angeles, Los Angeles, CA
90095, USA
Mechanical and Aerospace Engineering Department, University of California, Los
c
Department of Nuclear Medicine, Molecular Imaging & Therapeutic Medicine
Research Center, Chonbuk National University Medical School and Hospital,
h
Jeonju, 54907, Republic of Korea
Angeles, Los Angeles, CA 90095, USA
d
i
Research Institute of Clinical Medicine of Chonbuk National University, Biomedical
Department of Molecular Imaging and Therapy, Beckman Research Institute, City of
Research Institute of Chonbuk National University Hospital, Jeonju, 54907, Republic
Hope, Duarte, CA 91010, USA
of Korea
†
Electronic supplementary information (ESI) available. See DOI:
e
Department of Chemical Engineering, University of Wah, Quaid Avenue, Wah Cantt,
10.1039/c9ra06158d
Pakistan
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(96.7%), higher resolution image due to the short positron the nal coupling reaction with the acylation agent (TSTU or
trajectory (0.5 mm mean), high specic activity, wide avail- HSTU), an additional azeotropic evaporation is performed at
ability, and the ability to transport the radioisotope from the end of the hydrolysis step to achieve a high coupling effi-
4
18–21
a network of cyclotrons to nearby imaging sites. To enable ciency.
The improved synthesis has been implemented on
biologists to label and image a specic biologically important conventional automated radiosynthesizers (e.g., IBA Synthera
1
4,22
mechanism, such as receptor-specic interactions of peptides, and Soe ELYXYS FLEX/CHEM) with 69–85% RCY.
proteins, hormones, antibodies and antibody fragments, a new
1
8
Though [ F]SFB can be convenient reacted with peptides or
labeling technology platform to advance medicine and life proteins to produce imaging probes, it is not widely available
1
8
sciences is critically needed. Previously, our group has demon- due to insufficient demand. The radiosynthesis of [ F]SFB can
strated the rst proof-of-principle batch microuidic radio- only be performed in a specialized radiochemistry laboratory
1
8
18
synthesizer for the radiosynthesis of [ F]FDG, [ F]fallypride and requires an expensive automated synthesizer, and a hot cell
1
8
and [ F]FLT with reliable yield and with sufficient radioactivity for operation of the synthesizer. Due to the high capital
5
–7
18
for imaging multiple mice. Our group has also successfully investment needed for radiochemistry, [ F]SFB is limited to
demonstrated the synthesis of high molar activity radiotracers a relatively small number of groups that have access to a nearby
8
23
using this all-electronic microuidic radiosynthesizer. In this radiochemistry laboratory. Thus, a new microuidic platform
report, we further expand the applications from 2-step nucleo- for radiochemistry that was compact and self-shielded, so that it
philic uorination of aliphatic substrates to a more challenging could be used in any standard laboratory, has the potential to
and complicated reaction, which involves a 3-step nucleophilic overcome this challenge and enable wide availability of [ F]SFB
uorination of an aromatic substrate. [ F]uoroarenes, such as and thus enable F-labeling of a wide range of biomolecules for
1
8
1
8
18

1
8
18
6
-[ F]uoro-3,4-dihydroxy-L-phenylalanine ([ F]FDOPA), N- PET imaging.
succinimidyl-4-[ F]uorobenzoate ([ ]SFB), [ F]altanserin,
1
8
18
18
Leveraging advancements in microfabrication and micro-
F]setoperone and others are commonly found in radiophar- electronics, microuidic technology has the potential of inte-
1
8
[
maceuticals currently used in the clinic due to their high grating the entire PET radiochemistry operation, which
9
metabolic stability in vivo. However, no carrier added (n.c.a) includes synthesis, purication and quality control modules
24
aromatic nucleophilic uorination reaction involves high into a compact and inexpensive radiosynthesizer. Microuidic
temperature synthesis, toxic reagents, long reaction time and devices capable of manipulating liquid in nanoliter to micro-
10
oen suffers from low yield. Successful demonstration of this liter scales allow precise control over reaction conditions and
class of PET probes on a microuidic radiosynthesizer would the rapid mixing, heat and mass transfer enable quicker reac-
further demonstrate the versatility of the EWOD radio- tions that provide distinct advantages for PET probes radio-
25–28
synthesizer for diverse PET probes synthesis needed in the chemistry.
The small volume is commensurate to the
clinic and in preclinical research. Furthermore, the develop- picomole to nanomole quantities of PET probes needed for
ment of such microdroplet radiochemistry methodology can be preclinical and clinical PET imaging. Recently, Kimura and his
translated among other droplet-based radiochemistry platforms colleagues in Japan optimized the three-steps radiosynthesis of
1
8
including an emerging ultra-compact droplet-based micro- [ F]SFB into a single continuous ow microuidic chip with
1
1
29

uidic radiosynthesizer developed by one of us.
64% radiochemical yield. One major downside of such ow-
F-labeling of peptides and biomolecules is typically per- through radiosynthesizer is in their inability to perform
1
8
1
8
formed via a 2-step methodology: (1) n.c.a. [ F]uoride ion solvent exchange reactions, and so part of the process must be
labeling of a prosthetic group at high temperature and (2) carried out in conventional, macroscale apparatus. Bejot and
1
8
conjugation of the F-labelled prosthetic group to the protein co-workers have demonstrated the integration of solvent evap-
1
2
18
of interest under physiological conditions. N-Succinimidyl-4- oration steps and achieved the synthesis of [ F]SFB with 64%
[
1
8
18
F]uorobenzoate ([ F]SFB) is one of the most commonly radiochemical yield within 25 min in a 60 mL batch microreactor
used prosthetic groups for labeling protein due to its versatility with integrated valves and mechanical actuators to control the
21
in labeled amine groups, which oen doesn't require the ow of reagents in and out of the reaction vessel.
introduction of special handles into the protein, as well as its in Previously, our group has develop a dedicated EWOD
While we demonstrated the synthesis of [ F] microuidic radiosynthesizer capable of performing all unit
4,13
18
vivo stability.
SFB as a proof of concept, this technology could also be applied operations involved in the multistep synthesis of [ F]FDG, [ F]
1
8
18
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to synthesize many other prosthetic groups to prepare a wide FLT and [ F]fallypride such as solvent exchange, uorination
range of labelled proteins for PET imaging. The multistep and hydrolysis on a single chip in even smaller reaction
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8
synthesis of [ F]SFB has been reported in conventional volumes (1–16 mL) without the need for bulky mechanical
5
–7,30
macroscale radiosynthesizers with radiochemical yields (RCYs) actuators needed in other microuidic systems.
ranging from 20–77% within 30–90 min.
Herein, we
Over the years, report on two main advancements over our previous publica-
many research studies have been devoted to improving the tions: (1) we expanded the capability of the EWOD microuidic
14–16
12,13,16
18
laborious multistep and multi-pot
synthesis of [ F]SFB, synthesizer to perform aromatic nucleophilic uorination and
ultimately leading to a convenient and shorter three-step, one- (2) we established several critical microscale chemical rule sets
1
4,15,17
18
pot procedure.
Following the uorination step, an to achieve a facile radiosynthesis of [ F]SFB in high and reliable
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8
organic base is used in the hydrolysis step to yield the uo- yield. We further show that [ F]SFB synthesized on the EWOD
robenzoic acid ([ F]FBA) intermediate. Due to the sensitivity of chip could be conjugated to the anti-prostate stem cell antigen
1
8
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PSCA) cys-diabody (A2cDb),
RSC Advances
31,32
(
resulting in sufficient specic chemical and radiochemical purity. The HPLC was equipped
1
8
ꢂ1
activity of the F-labeled PSCA diabody (2.9 mCi mg protein) to with HPLC analysis was carried out using a Phenomenex Luna
conduct a pilot immunoPET study of a prostate cancer xeno- reversed-phase C-18 column (250 ꢃ 4.6 mm) (Torrance, CA).
1
8
gra bearing mouse (Fig. 1). While this paper is focused on the The purication of the nal product [ F]SFB was performed on
development of the droplet radiochemistry on the EWOD chip, a similar analytical HPLC system with an additional Secur-
our collaborators are also currently developing an automated, ityGuard C18 column (Phenomenex) (Torrance, CA).
self-shielded prototype droplet radiosynthesizer equipped with
1
8
a radioactivity concentration, reagent delivery, product collec-
tion and purication platforms.
2
.2 Radiosynthesis of [ F]SFB on EWOD chip
Kryptox 2.2.2 (12 mg; 0.032 mmol) and potassium carbonate
2.4 mg; 0.017 mmol) were dissolved in water (120 mL). An
aliquot of the phase transfer catalyst mixture (7 mL) was mixed
(
2. Materials and methods
1
8
18
with no-carrier-added [ F]uoride/[ O]H
2
O (50 mL; ꢁ185 mBq
2.1 EWOD chip
1
8
to generate the [ F]KF/K222 complex). Separately, the FB
precursor (1.2 mg, 3.40 mmol) was dissolved in 40 mL of anhy-
drous DMSO, and HSTU (5 mg; 0.14 mmol) was dissolved in
Potassium carbonate (K CO ), potassium hydrogen carbonate
2
3
(
(
(
KHCO ), 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclohexacosane
Kryptox 222), tetrapropylammonium hydroxide solution
3
5
0 mL of anhydrous MeCN. A stock solution of 160 mM NPr OH
0 0
4
1 M; NPr OH), O-(N-succinimidyl)-N,N,N ,N -tetramethyluro-
4
was prepared by diluting 16 mL of the 1 M NPr OH in 100 mL of
4
nium hexauorophosphate (HSTU), anhydrous acetonitrile
MeCN, 99.8%), anhydrous dimethyl sulfoxide (DMSO, 99.9%),
H
2
O/DMSO (3 : 1 v/v) mixture.
Six droplets (2 mL each) of the [ F]KF/K222 complex were
loaded onto the EWOD chip using a micropipette through one
of the dedicated loading edges. Each droplet was transported
from the loading edge to the reaction site via EWOD actuation.
One MeCN droplet (3 mL) was then added to the reaction site.
(
1
8
hexanes, ethyl acetate, 1 M acetic acid solution, triuoroacetic
acid, and methanol were purchased from Sigma-Aldrich
Chemical Co. (St. Louis, MO). 4-(Ethoxycarbonyl)-N,N,N-trime-
thylbenzenaminium triate (FB precursor) and the N-succini-
midyl 4-uorobenzoate (SFB) standard compound was
purchased from ABX Advanced Biochemical Compounds
ꢄ
The resulting mixture was heated to 105 C and held at this
temperature for 1 min to remove the water. Subsequently, one
cycle of azeotropic distillation was performed by adding 3
droplets (3 mL each) of MeCN to the dried residue and heating at
(
Radeberg, Germany) and used as received.
1
8
No-carrier-added [ F]uoride ion was obtained from the
UCLA Crump Institute Cyclotron and Radiochemistry Tech-
ꢄ
18
1
05 C for 1 min. To the dried [ F]KF/K222 complex, one 2 mL
1
8
nology Center by irradiation of 97% O-enriched water with an
1 MeV proton beam using an RDS-111 cyclotron (Siemens
droplet of the FB precursor (compound 1) (84 mM) was added.
1
ꢄ
The reaction mixture was gradually heated to 120 C and held at
Medical Solution, Knoxville, TN). Radioactivity was determined
using a calibrated ion chamber (Capintec CRC-15R, Ramsey,
NJ). A radio-thin layer chromatography (radio-TLC) scanner
the set temperature for 4.5 min to perform the uorination
reaction. Aer the uorination reaction, two droplets (3 mL
each) of the NPr OH solution (160 mM) in 3 : 1 mixture of H O/
4
2
(MiniGITA star; Raytest USA, Inc, Wilmington, NC) was used to
1
8
DMSO was added to the ethyl-[ F]uorobenzoate intermediate
compound 2). The saponication reaction was performed by
analyze uorination efficiency, and analytical high performance
liquid chromatography (HPLC; Knauer, Germany) equipped
with a degasser (Model 5050), pump (Model 1000), a variable
wavelength UV detector (254 nm) and a radiometric detector
(
ꢄ
heating the reaction mixture at 105 C for 1.5 min. Aer the
saponication reaction, two droplets (3 mL each) of the HSTU
1
8
solution (280 mM) in MeCN was directly added to the crude [ F]
(Eckert & Ziegler, Washington DC, USA) was used to measure

uorobenzoic acid intermediate (compound 3) for the nal
ꢄ
esterication step. The reaction mixture was heated to 105 C
1
8
and held at the set temperature for 3.5 min to yield [ F]SFB, the
nal product. The crude reaction product was extracted with 40

mL MeCN and 20 mL MeOH. A small aliquot of the crude product
was removed for radio-TLC and radio-HPLC analyses, while the
remainder of the crude product was subjected to HPLC
purication.
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8
2
.3 [ F]SFB purication and reformulation
About 60 mL of crude product collected from the EWOD chip
was loaded onto the analytical radio-HPLC equipped with a 100
mL sample loop. The product was separated via an isocratic
2
elution of MeCN/H O/TFA 50 : 50 : 0.1 (v/v/v) at a ow rate of 1
1
8
Fig. 1 Overall radiosynthetic scheme of [ F]SFB on the EWOD chip
ꢂ1
18
mL min . The [ F]SFB fraction eluted between 15 and 16 min
and the product was collected into a vial containing 3 mL of 1 M
acetic acid. The HPLC mobile phase was evaporated to dryness
18
and the conjugation of the F-labeled prosthetic group to the PSCA-
diabody. The conjugation is performed in sodium-borate buffer (SBB,
0 mmol L , pH 8.7) and incubated for 10 min at 34 C. Cartoon of
ꢂ1
ꢄ
5
ꢄ
diabody is not to scale.
at 77 C under a constant nitrogen ow for 10 min to obtain
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
nal compound for the subsequent radiolabeling reaction. The calculating % ID/g values based on 1% of the injected dose. All
1
8
dried [ F]SFB was transferred from the scintillation vial into procedures performed in studies involving animals were in
a polypropylene Eppendorf tube (200 mL) using ꢁ200 mL of accordance with the Animal Welfare Act and Regulations
MeCN to minimize loss of product on the sides of the wall of the (USDA) and the PHS Policy (NIH/OLAW) and were approved by
larger volume scintillation vial. Then the MeCN was evaporated the University of California Los Angeles (UCLA) Chancellor's
ꢄ
to dryness in a water bath heated at 77 C. The dried and Animal Research Committee (ARC), Los Angeles, USA.
1
8
formulated [ F]SFB was obtained with 39 ꢀ 7% (n ¼ 4) radio-
chemical yield within 120 min. The average radio chemical
purity was 98%.
3
. Results
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8
3.1 Droplet radiosynthesis of [ F]SFB
2
.4 Quality control
Based on the one-pot synthetic methodologies that have been
1
8
reported for [ F]SFB using conventional macroscale radio-
The radio-uorination, hydrolysis and esterication efficiencies
15
1
8
synthesizer, we have further optimized the reaction conditions
on the EWOD chip to achieve a reliable yield. All processes,
of the respective intermediate product and [ F]SFB were
determined by radio-TLC on silica gel plates (60 F254 with
aluminum backing), with 1 : 1 v/v ethyl acetate/hexane mixture
as the mobile phase. The radioactivity distribution was scanned
with a radio-TLC scanner. The retention factors (R ) of the [ F]
f

1
8
which include [ F]uoride complex drying, solvent exchange,
uorination, hydrolysis and coupling reaction, were performed

1
8
on a single EWOD chip. The optimal reaction condition of the
uorination step used 167 nmol of precursor, 216 nmol of
1
8
18
18
uoride ion, [ F]uorobenzoic acid, [ F]SFB and ethyl-4-[ F]
K CO and 390 nmol of K in 2 mL of DMSO. Reacting at
2.2.2
2
3

uorobenzoate were 0.00, 0.00, 0.32, and 0.57, respectively
ꢄ
1
8
120 C for 4.5 min yielded an average uorination efficiency of
(Fig. S-1†). Radiochemical purity of the nal [ F]SFB product
1
8
93
ꢀ
1% (n
¼
6). The saponication of 4-[ F]uo-
was analyzed on the Phenomenex Luna reversed-phase C-18
ꢄ
roethylbenzoate was carried out at 105 C for 1.5 min using
NPr OH (167 mM) in a 3 : 1 v/v mixture of H O/DMSO. Lastly,
column (250 ꢃ 4.6 mm) with isocratic elution of MeCN/H O/
2
ꢂ1
4
2
TFA 50 : 50 : 0.1 (v/v/v) at a ow rate of 0.5 mL min . The
1
8
the coupling step was performed using 1670 nmol of HSTU in
identity of the nal [ F]SFB product was conrmed by
comparing to a chromatogram of SFB reference standard SFB. A
representative HPLC chromatogram (UV and g-detection) of the
ꢄ
a mixture of MeCN and DMSO (6 : 1 v/v) at 105 C for 3.5 min.
1
8
The RCY of [ F]SFB aer HPLC purication was 39 ꢀ 7% (n ¼
4), The total synthesis time was ꢁ120 min, including the on-
1
8
puried and formulated [ F]SFB is shown in Fig. S-2.†
1
8
chip [ F]uoride ion concentration, radiouorination, saponi-
cation, coupling reaction, analytical-scale HPLC purication,
and followed by solvent removal to complete dryness (to facili-
tate subsequent conjugation reaction). In a typical radiosyn-
1
8
2.5 Conjugation of [ F]SFB with PSCA-diabody
1
8
[
F]SFB was resuspended in sodium borate buffer (33 MBq) in
5 mL SBB, 50 mM, pH 8.7 and incubated with A2cDb (50 mg in 50
2
18
18
thesis on the EWOD chip, ꢁ222 MBq of [ F]uoride/[ O]H
2
O
ꢄ
18
mL SBB) for 10 min at 34 C. Conjugated [ F]FB-A2cDb was
puried using a Micro Bio-Spin size exclusion columns (BioRad)
that was pre-blocked with PBS, 1% fetal bovine serum (FBS) as
previously described. Labelling efficiency and radiochemical
purity were determined using ITLC strips (Biodex Medical
Systems) with saline as solvent and analyzed by gamma count-
18
was loaded on the chip and ꢁ37 MBq [ F]SFB was collected
aer the HPLC purication. Upon removal of the HPLC mobile
18
phase and reformulation, ꢁ33 MBq of [ F]SFB was used for the
33
subsequent protein labeling and mice imaging experiment.
Even under the optimized drying condition, ꢁ5% of the radio-
activity was lost during the solvent removal process, which
resulted in only 33 MBq used for subsequent labeling
experiment.
0
ing (Wizard 3 1480 Automatic Gamma Counter, PerkinElmer).
The immunoreactive fraction of radiolabeled A2cDb was
measured by incubation with excess PSCA-positive cells (22Rv1-
PSCA) and control cells (22Rv1) for 1 h at room temperature.
Supernatant and cell-bound fractions were analyzed by gamma
1
8
3
.2 Radiolabeling of A2cDb using [ F]SFB
1
8
34
[
F]SFB radiolabeling of A2cDb and purication of the result-
counting as previously described.
1
8
ing [ F]FB-A2cDb conjugate was completed within 30 min with
a radiolabeling efficiency of 33 ꢀ 13% (n ¼ 3) and radiochemical
purity of 94.6 ꢀ 2.1% (n ¼ 3) as determined by instant thin layer
2
.6 Small animal imaging
A male nude mouse (Jackson Laboratories, JAX002019 NU/J)
bearing subcutaneous 22Rv1-PSCA (right shoulder) and 22Rv1
1
8
chromatography (ITLC). [ F]FB-A2cDb retained antigen-
specic binding to PSCA-expressing cells (immunoreactive
fraction 75.6 ꢀ 6.5%, n ¼ 2).
(
[
le shoulder) xenogras was injected intravenously with 10 mg
1
8
F]FB-A2cDb (30 mCi/1.1 MBq) in 100 mL saline. PET/CT
imaging was performed under 2% isourane anesthesia. A
two-hour dynamic PET acquisition (Inveon, Siemens) was fol-
lowed by a CT scan (MicroCAT, Siemens). PET images were
1
8
3
.3 [ F]FB-A2cDb immunoPET imaging of prostate cancer
xenogra bearing mouse
1
8
reconstructed as ltered back projections (FBP), and PET/CT [ F]FB-A2cDb (10 mg/1.1 MBq) was injected i.v. into a male nude
images are presented as maximum intensity projection (MIP) mice bearing PSCA-positive and PSCA-negative prostate cancer
35
overlays using AMIDE soware. Ex vivo biodistribution was xenogras and a dynamic PET scan (0–120 min) was acquired.
performed by weighing and gamma counting tissues and Antigen-specic accumulation in the PSCA-positive tumor (right
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shoulder) could be observed as early as 30 min post injection observed low uorination and radiochemical yields when
and increased up to 2 h post injection. A small amount of simply down-scaling the reagent volume and amount used in
activity was seen in the PSCA-negative tumor (le shoulder), but macroscale reactions. The focus of this work was to establish
this is potentially blood pool activity as the diabody is not a reliable droplet radiochemistry method for aromatic nucleo-
completely cleared from the blood at 120 post injection. The philic uorination and the subsequent functional group trans-
1
8
[
F]FB-A2cDb showed rapid clearance from the blood and renal formation by investigating the effect of reagent concentrations,
secretion of activity into the urine.
reagent ratios, reaction times, and droplet sizes. During this
development stage, the majority of the operation was performed
manually, which limited the radioactivity used in this work.
While this report focused on the development of a reliable
microscale radiochemistry on the EWOD chip, we are also
currently developing an automated reagent delivery, product
4
. Discussion
4.1 EWOD chip for radiosynthesis
Droplets sandwiched between the two plates of the EWOD chip
are moved by electrowetting force when an electrical potential is
applied to electrodes on the base plate to accomplish operations
38
collection and purication system towards a fully automated
microuidic radiosynthesizer.
36
4.2.1 Optimization of the microdroplet radiochemistry
such as droplet generation, transport, splitting and mixing. The
EWOD electrodes at the reaction site are capable of resistive
heating and thermistic temperature sensing in addition to droplet
4.2.1.1 Precursor to base ratio. Our group adapted the
synthetic methodology developed by Tang and Kabalka et al. in
15
37
the microdroplet radiosynthesis on EWOD chip. Briey, the
ethyl 4-(trimethylammonium triate)benzoate (FB precursor)
movement (Fig. 2). The reaction site electrodes were congured
as four concentric rings (maximum size 12 mm diameter) to
center droplets and accurately monitor and control their
temperature as they shrink. Each of the four rings is indepen-
dently capable of feedback temperature control, which prevents
the regions of faster droplet evaporation from excessive heating.
1
8
was radiouorinated using the activated complex of [ F]KF/
1
8
K
2.2.2 to yield ethyl-4-[ F]uorobenzoate intermediate. High
throughput optimization studies were rst performed using the
lower cost and easily prepared glass-Teon substrates, which
5
mimic the sandwiched conguration of the EWOD chip. The
optimized conditions found on the glass-Teon substrates were
then translated onto the EWOD chip. Such workow enabled
large numbers of optimization experiments in the shortest time
period and at the lowest cost. Based on our rst optimization
study, we found that the highest uorination yield was obtained
by using the phase transfer catalyst (PTC) complex (K /K2.2.2) to
precursor ratio of 2.3 (Fig. 3). The number of moles of the PTC
complex is equal to the number of moles of K2.2.2; the number of
1
8
4
.2 Radiosynthesis of [ F]SFB synthesis on the EWOD chip
5–7
Leveraging our previous work in developing an all-electronic,
compact, easy to use and reliable EWOD microuidic radio-
synthesizer for F-18 PET radiochemistry, in this report we
demonstrated a three-step, one-pot radiosynthesis of [ F]SFB
on the EWOD chip. Similar to our previous reports, we found
that the conditions established in the macroscale are not suit-
able for the microdroplet radiosynthesis. In particular, we
+
1
8
moles of K
2
CO
3
is xed at 0.5ꢃ the amount of K2.2.2. In the
+
macroscopic conditions, the ratios of K /K
complex to
.2.2
2
precursor used in the uorination of the FB precursor typically
ranged between 4 : 1 to 6 : 1. As shown in Fig. 3, uorination
efficiency drastically decreases at molar ratios above 3 in the
microdroplet synthesis.
Fig. 2 (a) EWOD chip base plate design. Inset shows detail of the
multifunctional electrowetting, heating, and temperature sensing
electrodes at the reaction site. (b) Cross-sectional view of EWOD chip.
(
c) Droplet movement on chip by sequential connection of EWOD Fig. 3 Investigation of the effect of the phase transfer catalyst
+
electrodes to actuation voltage.
complex (K /K2.2.2) to precursor ratio.
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4.2.1.2 Concentration and reaction time. Capitalizing the

exibility of the four concentric multifunctional heaters, the
reaction volume at each step can be varied between 1–17 mL (for
the current chip design and setup) to achieve the optimal
reaction conditions. In this example, the EWOD radiochemistry
1
8
platform can efficiently accommodate larger volume of the [ F]
uoride radioisotope (12 mL) to produce sufficient radioactivity

1
8
of [ F]SFB for protein conjugation and micro-PET imaging of
mice, while performing other reactions in smaller volumes (i.e.,
2
mL) to increase the overall reagent concentrations and thus the
reaction kinetics. Furthermore, by decreasing the reaction
droplet size, only a minute amount of reagent is needed to
achieve the same reagent concentration, a signicant advantage
when using scarce and expensive reagents. More importantly,
the higher concentration of reaction does not affect the down-
stream HPLC purication because the total amount of reagents
remains in the nanomole range and does not saturate the
Fig.
4
Kinetics of the fluorination reaction at different reagent
ꢄ
concentrations. Reactions were performed in 2 mL of DMSO at 120 C.
analytical HPLC column. We have recently demonstrated the The blue trace (diamond marker) represents the optimized concen-
tration, i.e. 12ꢃ the concentration used in the macroscale reaction; the
green trace (square marker) represents 4ꢃ concentrations and the red
trace (circular marker) represents the same concentration as used in
the macroscale.
effect of droplet size and reagent concentration in enhancing
the microdroplet radiosynthesis of [ F]FLT on the EWOD chip.
1
8
6
As a rst step to improve upon our previous results, we reduced
the reaction volume from 6 mL to 2 mL with a concurrent
increase in the reagent concentration. Using the 2 mL reaction
volumes, we next investigated the reaction kinetics of the uo-
rination reaction (4.5, 7 and 10 min) on the Teon-glass
substrate in the presence of 7 mM precursor, and 18 mM PTC
complex (i.e. in optimal PTC : precursor ratio of 2.3 as discussed
step resulted in a low and unreliable performance of the
hydrolysis step, perhaps due to dilution of the reaction, which
reduces the overall concentration of reagents. However, upon
optimization of the uorination reaction using smaller droplet
size (i.e. 2 mL instead of 6 mL), minimal residual DMSO
remained aer uorination and the hydrolysis performed well.
In comparison to the 2 mL reaction droplet, a large pool of
DMSO condensates remained on the heater site using 6 mL of
reaction droplet, which necessitates an additional drying step
before proceeding to the hydrolysis step (Fig. S-3†). Further-
more, the additional heating time to remove the DMSO
condensates aer the uorination step resulted in a large loss of
radioactivity and was time consuming.
ꢄ
previously) at 120 C. The uorination conversion was lower at
4.5 min than longer reaction times and increased with
increasing reaction time as anticipated (Fig. 4, red trace). Upon
increasing the reagent concentrations by 4ꢃ, the uorination
efficiency in the 4.5 min reaction increased from ꢁ40% to
ꢁ
75% (Fig. 4, square green trace). Further enhancement of the
uorination efficiency to 81% was observed by increasing the

reagent concentration by 12ꢃ (Fig. 4, blue trace). Prolonging the
reaction time to 7, 10 and 12 min did not further improve the
conversion. In contrast, upon lowering the uorination time
from 4.5 to 3.0 min, a drastic reduction in the uorination
conversion (27 ꢀ 13% (n ¼ 6)) was observed (Fig. 4, blue trace).
Therefore, we chose the following optimal uorination condi-
Based on the new method, we investigated two reaction
ꢄ
ꢄ
temperatures (95 C and 105 C) and the reaction times needed to
18
achieve quantitative conversion to the [ F]uorobenzoic acid
intermediate product (Compound 3). We found a high depro-
tions: 4.5 min reaction, 84 mM precursor, 108 mM K
95 mM K2.2.2 in DMSO.
Aer the substitution reaction using n.c.a [ F]uoride ion,
2 3
CO and
ꢄ
tection efficiency at 105 C, while no signicant conversion was
1
ꢄ
observed at lower temperature (95 C) despite prolonging the
1
8
ꢄ
reaction time up to 25 min. At 105 C, the hydrolysis was quan-
the ethyl ester protecting group is typically removed by hydro-
titatively completed in less than 2 min as conrmed by radio-TLC.
In the nal reaction step, the crude tetra-n-propylammo-
lysis using an aqueous base (e.g.: NaOH) or acid (e.g.: HCl) to
1
8
18
form the 4-[ F]uorobenzoic acid ([ F]FBA) intermediate
product (8, 9). However, this method requires an intermediate
purication step to remove the water prior to the nal coupling
step. In this work, we adapted the method developed by the
Kabalka group and the Shen group, which used an organic base
18
nium 4-[ F]uorobenzoate salt (Compound 3) in a mixture of
NPr OH in H O/DMSO was directly treated with HSTU without
4
2
removing the residual water. It is worth noting that conven-
tional macroscopic method typically requires a time-consuming
azeotropic drying step or solid phase extraction (SPE) purica-
tion step in between the hydrolysis and the esterication step
due to the sensitivity of the coupling agent (i.e.: HSTU) to water.
In the microdroplet reaction, we found that the coupling reac-
15,39
for the deprotection reaction.
This method eliminates the
need for the intermediate purication step and thus reduces the
1
8
overall radiosynthetic time. On the EWOD chip, the ethyl 4-[ F]
uorobenzoate intermediate (Compound 2) was hydrolyzed

18
tion of HSTU and the [ F]uorobenzoate salt proceed with
using NPr OH in a mixture of DMSO and water (3 : 1 v/v).
4
quantitative yield in a mixture of solvents (MeCN/DMSO 1/6 v/v)
on EWOD chip, which is consistent with an earlier report by
Initially, we found that residual DMSO from the uorination
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18
Table 1 Optimized radiosynthetic protocol for the preparation of [ F]SFB on EWOD chip with the corresponding times needed for reaction,
evaporation, EWOD transport, and heating. The total heating time refers to the sum of temperature ramping time and the reaction time. Off-chip
a
steps such as product extraction, HPLC purification, drying and formulation are also included for reference
Step
1
Time (min)
Reagents (nmol)
Droplet volume (mL)
1
8
Load [ F]KF/K2.2.2
EWOD actuation
12
6
8
K
2
3
CO (216)
ꢄ
Heat to 105 C
K2.2.2 (390)
2
3
Azeotropic drying
EWOD actuation
Heat to 105 C
MeCN
9
2
1
2
ꢄ
Fluorination
Precursor (167)
Load precursor by EWOD
Reaction at 120 C
Total heating time
Hydrolysis
1
4.5
5
ꢄ
4
5
NPr
4
OH (960)
6
6
Load NPr
4
OH by EWOD
2
1.5
7
ꢄ
Reaction at 105 C
Total heating time
Esterication
HSTU (1668)
Load HSTU by EWOD
Reaction at 105 C
Total heating time
Product extraction
HPLC purication
Drying and formulation
2
3.5
6
10
10
20
ꢄ
6
7
8
n/a
n/a
2500
n/a
a
Some variable steps such as reagent loading, radioactivity measurement, conguring the EWOD soware and etcetera are not included in the
above table.
40
Bannwarth. Similar to the previous optimization studies, the 4.3 Purication, formulation and quality control
reaction kinetics for the ester formation was rst investigated
on Teon-glass substrates, and then the optimal conditions
18
Due to the minute amount of reagents used in the [ F]SFB
synthesis on the EWOD chip, we utilized the analytical column
HPLC for the purication step to achieve >95% radiochemical
purity. Using the analytical HPLC for the nal purication is
highly desirable as the smaller diameter column provides
a better separation and results in a lower volume of mobile
phase in comparison to the semi-preparative scale purication
were later translated to the EWOD chip. Based on the kinetic
studies, the optimal reaction time was found to be 3.5 min at
ꢄ
1
05 C (Fig. S-2†).
Table 1 summarizes the optimal microdroplet reaction
1
8
condition for [ F]SFB on EWOD to achieve an overall radio-
chemical yield of 39 ꢀ 7% (n ¼ 4). The lower radiochemical yield
18
typically used in macroscale radiosynthesis. The puried [ F]
1
8
of [ F]SFB in comparison to the macroscale synthesizer is
SFB was collected in ꢁ2.5 mL and the mobile phase was grad-
1
8
presumably due to the volatilization of the radioactive [ F]u-
ually evaporated to dryness in a hot water bath under a steady
4
1,42
ꢄ
18
oromethane
(bp: ꢂ78 C) and/or the [ F]uoro-4-

ow of nitrogen stream. The water bath temperature was set to
ꢄ
ethylbenzoate (bp: 210 C). These losses was suspected to
occur in the rst step of the [ F]SFB synthesis. Despite the
ꢄ
be below the boiling point of MeCN (bp: 82 C) to avoid
bumping and splashing of the solvent mixture. In a typical
evaporation process, ꢁ5% of radioactivity was lost. Aer refor-
1
8
lower radiochemical yield, the EWOD platform and the chem-
1
8
istry reported here reliably produced ꢁ33 MBq of [ F]SFB
18
mulating in 50 mM SBB, a small aliquot of the [ F]SFB was
1
8
starting from 222 MBq of [ F]uoride, which is sufficient for
routine biomolecule labeling and mice imaging. Although the
reaction time for each reaction step on EWOD chip is signi-
cantly faster in comparison to macroscopic module, the overall
synthesis time is only comparable with the conventional
system. During this developmental stage, many operations were
performed manually such as reagent loading, activating droplet
operation, heating and product collection. Our group is
currently developing novel engineering methods for auto-
mating the reagent loading and product extraction, which could
reduce the overall synthesis time to ꢁ80 min.
injected into an analytical HPLC to determine the chemical and
radiochemical purity. In literature, [ F]SFB is known to be
18
stable under acidic conditions and degrades at pH of 10 or
13
higher. In this study, we observed rapid degradation of the
18
[
F]SFB during the drying and reformulation process. Attempts
18
to use the resulting 90–95% pure [ F]SFB for conjugation with
the diabody resulted in an extremely poor conjugation efficiency
18
(
1–2%). To stabilize the puried [ F]SFB during the drying and
reformulation processes, a small amount of aqueous acetic acid
ꢄ
(
bp: 118 C) was added to the HPLC collection vial. Due to the
high boiling point of acetic acid, the overall pH of the nal
mixture remains slightly acidic throughout the entire evapora-
tion process and the resulting pH can be easily buffered with 3
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18
Fig. 5 ImmunoPET imaging of a mouse bearing 22Rv1 (left; “ꢂ”) and 22Rv1-PSCA (right; “+”) subcutaneous tumors. [ F]FB-A2cDb (10 mg/1.11
MBq) was injected via the tail vein. Shown are 10 min frames of a 120 min dynamic scan as 5 mm MIP PET/CT overlay. B ¼ bladder; K ¼ kidney; H
¼
heart.
mL of 1 M acetic acid to achieve the desired pH needed in the This report builds on earlier work on the EWOD radio-
1
8
subsequent conjugation reaction. Using the stabilized [ F]SFB synthesizer, exemplifying the exibility of performing different
1
8
with 95% radiochemical purity, the anti-PSCA-diabody was classes of F-radiochemical reactions, including both aliphatic
labeled with 33.1 ꢀ 12.5% efficiency and radiochemical purity and aromatic nucleophilic uorinations. Furthermore, we
1
8
of 94.6 ꢀ 2.1% (n ¼ 3) as determined by instant thin layer showed that sufficient amount of [ F]SFB was produced for
chromatography (ITLC).
PSCA-diabody labeling and in vivo imaging. The compact EWOD
chip radiosynthesizer could potentially be self-shielded and
automated to enable imaging scientists to routinely synthesize
[ F]SFB in their own laboratories or clinics for labeling
a plethora of biomolecules of interest.
1
8
4.4 Radiolabeling and PET imaging of [ F]FB-A2cDb
1
8
1
8
The anti-PSCA A2cDb was successfully radiolabeled using [ F]
SFB synthesized from the EWOD microuidic radiosynthesizer.
Here, labeling to lysine residues using [ F]SFB and size exclu-
1
8
sion purication of the radiotracer was executed within 30 min
1
8
and resulted in radiolabeled [ F]FB-A2cDb with sufficient
1
8
Conflicts of interest
purity for preclinical in vivo immunoPET imaging. [ F]FB-
A2cDb specically targets human PSCA-expressing prostate
cancer cells in vivo and rapid blood clearance results in high-
contrast PET images at early time points (Fig. 5). The pharma-
cokinetic properties of the 50 kDa cys-diabody format (t1/2 ¼ 2–
Drs van Dam and Keng are consultants to Soe Biosciences. The
Regents of the University of California have licensed technology
to Soe Biosciences that was invented by Drs van Dam, and
Keng, and have taken equity in Soe Biosciences as part of the
licensing transaction.
5
h in mice) make it a good match to the half-life of F-18,
43,44
enabling same-day imaging.
Further advantages of using
a short-lived radionuclide in combination with an antibody
fragment is the reduced radiation dose and exposure for the
patients compared to using radiolabeled full-length antibodies
conjugated to longer-lived isotopes.
Acknowledgements
This work is supported by funds provided in part by the
Department of Molecular and Medical Pharmacology and the
UCLA Foundation from a donation made by Ralph and Marjorie
5. Conclusion
We have demonstrated the capability of the EWOD microuidic Crump for the Crump Institute for Molecular imaging. We
radiosynthesizer to perform a sophisticated 3-step, one-pot thank Dr David Stout at the Crump Cyclotron and Radiochem-
synthesis, which involved a nucleophilic aromatic uorination istry Technology Center and Dr Saman Sadeghi and the staff of
1
8
reaction, saponication and an esterication followed by off- the UCLA Biomedical Cyclotron facility for providing [ F]uo-
chip HPLC purication and reformulation in 39 ꢀ 7% (n ¼ 4) ride ion for these studies. We thank Dr Jia Wang for assistance
radiochemical yield with a total synthesis time of ꢁ120 min. with performing synthesis optimization studies.
32182 | RSC Adv., 2019, 9, 32175–32183
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Aspects, ed. C.-H. Hsieh, InTech, Rijeka, Croatia, 2012, pp.
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- Current Clinical and Research
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