Microfluidic reactor for the radiosynthesis of PET radiotracers

Abstract

Here we show the first application of a microfabricated reaction system to PET radiochemistry, we term “microfluidic PET”. The short half-life of the positron emitting isotopes and the trace chemical quantities used in radiolabelling make PET radiochemistry amenable to miniaturisation. Microfluidic technologies are capable of controlling and transferring tiny quantities of liquids which allow chemical and biochemical assays to be integrated and carried out on a small scale. Such technologies provide distinct advantages over current methods of PET radiochemical synthesis. To demonstrate “proof of principle” we have investigated the radiohalogenation of small and large molecular weight molecules using the microfluidic device. These reactions involved the direct radioiodination of the apoptosis marker Annexin V using iodine-124, the indirect radioiodination of the anti-cancer drug doxorubicin from a tin-butyl precursor and the radiosynthesis of 2-[¹⁸F]FDG from a mannose triflate precursor and fluorine-18 and hence provide a test bed for microfluidic reactions. We demonstrate the rapid radioiodination of the protein Annexin V (40% radiochemical yield within 1 min) and the rapid radiofluorination of 2-[¹⁸F]FDG (60% radiochemical yield within 4 s) using a polymer microreactor chip. Chromatographic analysis showed that the labelling efficiency of the unoptimised microfluidic chip is comparable to conventional PET radiolabelling reactions.

Introduction

Molecular positron emission tomography (PET) imaging allows the study of molecular and cellular processes associated with diseases including cancer (Gambhir, 2002; Massoud and Gambhir, 2003; Reader and Zweit, 2001). PET is an in vivo molecular imaging technique based on the external detection of biomolecules labelled with positron emitting isotopes. The development of microfluidic PET radiochemistry has the potential to further advance molecular PET imaging, which is emerging as an important technology in the post genomic era of molecular medicine. The short half-life of the positron emitting isotopes and the trace chemical quantities used in radiolabelling make PET radiochemistry amenable to miniaturisation.

Microfluidic, technologies, also known as “lab on a chip” technologies, are capable of controlling and transferring quantities of liquids which allow chemical and biochemical assays to be integrated and carried out on a small scale (Regenfuss et al., 1985; Mitchell, 2001; Ramsey, 1999). Such technologies provide distinct advantages over current methods of PET radiochemical synthesis. Significantly, radiochemical reactions on a microfabricated device (chip) can be easily shielded and will not need the space and resources required for conventional hot cell synthesis. Secondly, it provides scope for an integrated total system (synthesis, purification and analysis). Thirdly, due to the rapid and thorough mixing achieved in miniaturised reactors (Fletcher et al., 2002), the speed and radiochemical yield of radiochemical syntheses could be enhanced. Finally, the photolithographic fabrication of the microfabricated device allows the manufacture of complex, yet relatively inexpensive and disposable devices (Stuernstrom and Roeraade, 1998; Lin et al., 2001; Tsai and Lin, 2001).

Currently, radiosynthesis of compounds labelled with positron emitting radioisotopes are carried out in lead-shielded “hot cells” using automated systems in order to prevent radiation exposure to the operators. Such automated systems carry out a range of operations such as heating, cooling, transfer of liquids and gaseous reagents, mixing, evaporation and distillation. Essentially, they perform all the operations used by radiochemists. There are essentially two types of systems currently in operation (Luthra et al., 1994; Crouzel et al., 1987; Crouzel et al., 1993). These are either static systems, e.g., the GEMS TracerLab MX FDG Synthesizer, in which switching valves and transfer lines made from inert materials are used to transfer reagents around the system, or robotic systems, e.g. SYNTHIA (Bjurling et al., 1996), in which a robot performs some or most of the operations. These systems are generally designed to handle total liquid volumes in the range of 0.2–0.5 mL.

Regardless of the automation approach, all of the existing automated radiosynthesis systems, static and/or robotic, share some common features. To ensure practical transfer of reagents etc. around the system, small volumes of liquids, usually of the order of 500 μL, are used as solvents and, usually, only mg amounts of organic precursors for radiolabelling are used. Carbon-11 and fluorine-18 labelled compounds produced by these systems are usually associated with only micrograms of stable “carrier”. The other hardware used in systems e.g. HPLC pumps, columns, heaters, rotary evaporators etc. are quite space consuming. In contrast to this, however, in all radiosyntheses the number of radioactive atoms or molecules involved is vanishingly small. We are, therefore, faced with the dilemma of having to use a fairly large automated system to handle minute amounts of radioactive materials. The automated systems associated parameters (e.g. volumes, timing, temperatures, etc.), are already very well defined, making the transformation to microfabricated devices relatively straightforward, in principle. Given the small amounts of reagents, especially the starting concentration of radioisotope, involved in the radiosynthesis of compounds of clinical interest, it was hypothesised that miniaturisation of automated radiosynthesis systems is feasible.

Miniaturisation of chemical systems started in the last decade with the development of a number of microanalytical systems. These devices combine sensors, actuators and microfluidic elements to create a micro total analysis system (μTAS) (Manz and Widmer, 1990; Kopp et al., 1998; Hadd et al., 1997). The devices consisted of photolithographically etched microstructures on silicon substrates. Each structure performed an individual task in order to complete the chemical synthesis and analysis, e.g. reagent mixing or separation systems. Each structure was linked with a channel to the next structure in order to allow the flow of reactants and products from one part of the microfabricated device to the next.

At present, the simplest microfabricated systems are microreactors which correspond to a single operation. Most of these operations perform mixing or separation. The majority of the present microfluidic structures actually belong to this group (Ramsey, 1999). There are a number of examples of microreactors used for chemical reactions. For example, Chambers, et.al. has developed a microreactor in which elemental fluorine has been used to allow selective fluorination of organic compounds (Chambers and Spink, 1999).

Microfabrication technology would appear extremely attractive for application in PET radiochemistry, since a number of important advantages could be gained. The possibility of performing multiple radiosyntheses from one batch of labelling agent would be simplified. Potentially, more rapid radiochemistry could be performed using miniaturised devices not only for radiosyntheses, but also for purification and analysis. This would lead to improved specific radioactivity of radioligands and would allow exploration of the chemistry with positron emitters which have extremely short half-lives, e.g. nitrogen-13 (t_1/2=9.96 min) and even oxygen-15 (t_1/2=2.04 min). Such developments could allow the possible radiolabelling of a greatly expanded range of compounds. An additional advantage would be that, since the devices could be mass-produced they could be discarded after use, thus overcoming a major bottleneck by removing the need to clean the system after each radiosynthesis.

However, before the microfabricated synthesis and/or analysis device could be developed, it was necessary to decide what type of radiosynthesis was to be attempted on the microfabricated system. The purpose being to use microfabricated devices to carry out a range of aqueous or organic-based radiolabelling reactions on model compounds, in order to establish a simple radiolabelling and detection of the radiolabelled products as “proof of principle”. The proof of principle being that radiochemistry on a microfabricated device was feasible. Therefore, the aims of this work were to develop a microfabricated device for the radiolabelling of commonly used PET radioligands.