MICROREACTOR-BASED SYSTEM FOR RADIOLABELING OF
BIOMOLECULES WITH METALLIC RADIOISOTOPES
Amit V. Desai1, Dexing Zeng2, Tobias D. Wheeler1, David Ranganathan2, David E. Reichert2, and
Paul J.A. Kenis1*
1
Department of Chemical & Biomolecular Engineering, University of Illinois, Urbana-Champaign, USA and
2
Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, USA
ABSTRACT
Here, we report a robust, versatile, compact microreactor-based system for labeling of biomolecules with metallic radioisotopes or radiometals. The microreactor exploits rapid mixing in grooved (staggered herringbones) microchannels, and efficient heat transfer in micro-incubation chambers to achieve high radiolabeling efficiencies. We demonstrate the application
of the system by labeling RGD, a cancer-targeting biomolecule, with radioisotopes of copper and gallium. More significantly,
the high labeling efficiencies were achieved without using excess of any reagents, thus obviating the need for subsequent purification. The reported microreactor-based system has potential for the synthesis of radiopharmaceuticals for clinical and research applications.
KEYWORDS: Staggered Herringbone Mixers, Radiometals, Cancer Imaging And Therapy, Radiopharmaceutical Synthesis
INTRODUCTION
Radiolabeled biomolecules have potential use in many applications, ranging from study of plant metabolism for biofuel
development to imaging of cancerous tumors. Typical syntheses of radiopharmaceuticals (radiolabeled biomolecules with
clinical applications) involve labeling of targeting biomolecules with radioisotopes, which function as imaging contrast agents
for diagnosis or as radiation sources for killing diseased cells for therapy. The labeling procedure involves mixing and heating of liquid reagents, and are conventionally performed manually or using automated synthesis modules. Since the radioisotope solution is generally available in small quantities (~100 μL), the solution is diluted to larger volumes, due to the inability
to effectively manipulate small reagent volumes using conventional procedures. To compensate for the reduced reaction kinetics resulting from dilution of the radioisotope, 10 to 100-fold excess of the biomolecule is used during labeling [1]. This
use of excess biomolecules necessitates chromatographic purification procedures, which require sophisticated instrumentation
and are cost and time-intensive. Hence, for clinical applications, the purification procedures are avoided, which results in a
high fraction of unlabeled biomolecules, and consequently poor signal-to-noise ratio. Here, we exploit the ability of microfluidics to efficiently handle small reagent volumes to address the above issues.
Most of the previous efforts on application of microfluidics for radiolabeling have focused on labeling with non-metallic
radioisotopes, primarily C11 and F18 [1]. Metallic radioisotopes (radiometals) have the following key advantages over nonmetallic isotopes: (1) radiometals are more easily available or accessible, (2) they often have longer half-lives, and (3) labeling using radiometals proceeds via aqueous chemistries under mild conditions, which is preferred for biomolecules. In addition, radiometal-based labeling proceeds via a generic reaction scheme, which enables development of an universal system for
synthesis of a variety of radiopharmaceuticals. Although, the previously reported microreactors for radiolabeling can be
adapted to labeling with radiometals [1], our microreactor design is advantageous compared to the existing ones with respect
to scalability to handle large volumes (up to 1 mL), and simplicity of design, fabrication and operation. We demonstrate the
utility of our microreactor-based system for labeling a cancer-targeting biomolecule with two different radiometals.
DESIGN, FABRICATION AND OPERATION OF THE MICROREACTOR
Details on the design, fabrication and operation of the microreactor can be found in our previous publication [2]. The microreactor comprises a serpentine microchannel with staggered herringbones for mixing, and a series of reservoirs for incubation of the mixed reagents (Figure 1A). Radiometal-based labeling of biomolecules proceeds via a two-step reaction scheme.
In the first step, the radiometal is bound to a weaker ligand (represented as buffer in Figure 1A) via anion exchange, and in
the second step, the radiometal is chelated to a bifunctional-chelator conjugated to the biomolecule. The three reagent solutions are completely mixed within the serpentine channels by chaotic advection induced by the herringbones [2]. The rapid
and efficient mixing by herringbone structures avoid the need for long mixing channels, and hence, enable high operating flow
rates (up to 1 mL/min,), and consequently high throughput. Once the solutions are thoroughly mixed, the mixture is incubated
in the reservoirs to allow for the chelation reaction. This semi-batch mode of operation has two main advantages over continuous-flow reactors previously reported [1], namely (1) superior control over the time required for labeling, as determined by
chelation kinetics, and (2) scalability to handle large volumes (up to 1 mL), as determined by the size of the reservoirs. Compared to previously reported batch-flow microreactors [2], the fabrication and interfacing of our microreactor-based labeling
system is simple and inexpensive, which is crucial for development of disposable devices for clinical applications.
978-0-9798064-4-5/µTAS 2011/$20©11CBMS-0001
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15th International Conference on
Miniaturized Systems for Chemistry and Life Sciences
October 2-6, 2011, Seattle, Washington, USA
Figure 1: (A) Photograph of radiolabeling microreactor. (B) Photograph of shielded syringe pump for manipulating radiometal. The other syringe pumps and computer controls are not shown. (C) Structure of the biomolecule RGD (a cyclic peptide), which is used for tumor imaging. (D) Examples of radiolabeling schemes tested (68Ga-DOTA-RGD not shown).
The microreactor comprised of polydimethylsiloxane microstructures bonded to a glass substrate, and was fabricated using
conventional soft-lithographic procedures. A thin film heater with a temperature controller (Figure 1B) is attached to the microreactor to perform radiolabeling at elevated temperatures. The reagents are manipulated using syringe pumps that are
computer controlled using a LabVIEW interface. The capability for automated labeling not only reduces the errors in manual
handling, but also minimizes the exposure of the operator to ionizing radiations. We also reduced the space occupied by the
„hot-cell‟, which minimizes the bulky, expensive lead-based radiation shielding.
RESULTS AND DISCUSSION
We validated the microreactor by labeling cyclo-(RGDfk), where RGD stands for D-Phenylalanine, Aspartic Acid, Arginine, Lysine (Figure 1C). To demonstrate the versatility of the system, cyclo-(RGDfk) was labeled with two different radiometals, copper-64 (64Cu) and gallium-68 (68Ga), and two different bifunctional chelators, 1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), as shown in Figure 1D. The
comparison of radiolabeling efficiencies for 64Cu-DOTA-RGD, 68Ga-DOTA-RGD and 68Ga-NOTA-RGD between those obtained using the microreactor and conventional procedures is shown in Figure 2A. The higher efficiencies were achieved using the microreactor due to the more effective mixing of small volumes, and enhanced heat transfer. We also observed the
radiolabeling to be more reliable using the microreactor, evident by the lower error bars for microreactor-based labeling. To
verify the hypothesis that effective mixing of small volumes is key, we compared the radiolabeling efficiencies obtained using
conventional procedures for larger volumes (100 μL as opposed to 10 μL) for 64Cu-DOTA-RGD. The efficiencies for conventional labeling with large volumes are similar to those obtained using the microreactor for labeling small volumes.
The improvement in performance of the microreactor is more evident for radiolabeling with 68Ga, as the labeling is more
sensitive to temperature, and the enhanced heat transfer in the microreactor is expected to have a stronger influence on the
labeling kinetics. To demonstrate the application of the microreactor system for screening reaction conditions, we studied the
influence of radiometal concentration and residence time in the reservoirs on the radiolabeling efficiencies (Figure 2B). We
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observed that radiolabeling efficiencies greater than 90% could be achieved for radiometal concentrations greater than 50 μM
within 20 minutes. More importantly, these high efficiencies were achieved without using excess of any reagents, thus avoiding any chromatographic purification. We also observed that a single microreactor operated for more than 720 hours and was
exposed to 20 mCi of radiation before the device leaked, demonstrating robustness of the system.
Figure 2: (A) Comparison of radiolabeling efficiencies for microreactor vs. conventional technique for three different labeling schemes at three different temperatures. (B) Effect of radiometal concentration and residence time on radiolabeling efficiency for the three schemes. For part (B), all labeling was performed on-chip.
CONCLUSIONS
Here, we reported the development of a robust, reliable, versatile, compact microreactor-based system for labeling biomolecules with radiometals. The system was used to label a clinically relevant biomolecule with different radiometals and via
different bi-functional chelators. The microreactor outperformed conventional approaches, particularly in terms of higher radiolabeling efficiencies. This microreactor-based system has potential for labeling biomolecules with clinical applications,
such as cancer imaging, and research applications, such as bio-distribution studies.
ACKNOWLEDGEMENTS
We acknowledge financial support from DOE (Office of Biological and Environmental Research, grant DE-SC0004038).
The production of 64Cu2+ was supported by the National Institutes of Health (grant R24 CA86307).
REFERENCES
[1] A.M. Elizarov, “Microreactors for Radiopharmaceutical Synthesis”, Lab Chip, vol. 9, pp. 1326-1333, 2009.
[2] T.D. Wheeler, D. Zeng, A.V. Desai, B. Onal, D.E. Reichert, P.J.A. Kenis, "Microfluidic labeling of biomolecules with
radiometals for use in nuclear medicine," Lab Chip, vol. 10, no. 24, pp. 3387-3396, 2010.
CONTACT
*Paul J. A. Kenis, tel: +1-217-2650523; [email protected]
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