Microfluidic radiolabeling of biomolecules with PET radiometals

Abstract

Introduction

A robust, versatile and compact microreactor has been designed, fabricated and tested for the labeling of bifunctional chelate conjugated biomolecules (BFC-BM) with PET radiometals.

Methods

The developed microreactor was used to radiolabel a chelate, either 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) that had been conjugated to cyclo(Arg-Gly-Asp-DPhe-Lys) peptide, with both ⁶⁴Cu and ⁶⁸Ga respectively. The microreactor radiolabeling conditions were optimized by varying temperature, concentration and residence time.

Results

Direct comparisons between the microreactor approach and conventional methods showed improved labeling yields and increased reproducibility with the microreactor under identical labeling conditions, due to enhanced mass and heat transfer at the microscale. More importantly, over 90% radiolabeling yields (incorporation of radiometal) were achieved with a 1:1 stoichiometry of bifunctional chelate biomolecule conjugate (BFC-BM) to radiometal in the microreactor, which potentially obviates extensive chromatographic purification that is typically required to remove the large excess of unlabeled biomolecule in radioligands prepared using conventional methods. Moreover, higher yields for radiolabeling of DOTA-functionalized BSA protein (Bovine Serum Albumin) were observed with ⁶⁴Cu/⁶⁸Ga using the microreactor, which demonstrates the ability to label both small and large molecules.

Conclusions

A robust, reliable, compact microreactor capable of chelating radiometals with common chelates has been developed and validated. Based on our radiolabeling results, the reported microfluidic approach overall outperforms conventional radiosynthetic methods, and is a promising technology for the radiometal labeling of commonly utilized BFC-BM in aqueous solutions.

Introduction

Over the past decade, interest in the use of radiolabeled peptides for use in positron emission tomography (PET) imaging for preclinical development and clinical diagnostics, and radiation-based therapeutics has increased. Such agents are becoming more prevalent since many peptide-targeted receptors are overexpressed in cancer, and the development of routine methods of solid-phase peptide synthesis has allowed the development of highly specific imaging probes and/or therapeutic agents with the appropriate choice of PET radioisotope [1]. These agents are generally composed of two parts, the biomolecule (BM) responsible for receptor targeting and a bifunctional chelate (BFC) for coordinating the radioisotope.

The cyclic pentapeptide cyclo(− Arg-Gly-Asp-(D)-Phe-Lys-) (cyclo(RGDfK)) is a highly potent and selective inhibitor for the α_vβ₃ receptor, an integrin required for tumor-induced angiogenesis, and is over-expressed in various types of tumors [2]. Many radioligands containing the peptide sequence of cyclo(RGDfK) have been prepared to monitor expression of the α_vβ₃ receptor, and their evaluations have been carried out in both murine tumour models [3], [4], [5] and patient studies [5], [6], with promising results. The preparation of these radioligands requires that cyclo(RGDfK) peptides be labeled with positron-emitting radionuclide such as ¹⁸F, ⁶⁴Cu or ⁶⁸Ga. Among these PET radioisotopes, the distribution of ¹⁸F is limited to relatively small distances from the production cyclotron facility, due to the short half-life of ¹⁸F (t_1/2 = 1.83 hr). On the other hand, ⁶⁴Cu and ⁶⁸Ga allow for wider distribution, either due to relatively long half life (t_1/2 = 12.7 hr for ⁶⁴Cu) or availability of an on-site, self-contained generator (⁶⁸Ge/⁶⁸Ga generator for ⁶⁸Ga), which has drawn increased attention to ⁶⁴Cu and ⁶⁸Ga as promising radionuclides [7], [8], [9], [10], [11], [12], [13]. ⁶⁴Cu can be effectively produced by both reactor-based and accelerator-based methods, and has suitable decay characteristics (β+: 0.653 MeV, 17.4%; β−: 0.578 MeV; 39%) that allow this radiometal to be used as a PET imaging agent [10], [11], [12], and a nuclear therapy agent [12], [14], [15]. ⁶⁸Ga is a positron emitter with a half-life of 67.6 min and decays via positron emission (89%), and its relatively shorter half-life and hydrophilic nature are beneficial for the rapid renal clearance of small peptides labeled with ⁶⁸Ga. In addition, ⁶⁸Ga can be conveniently and economically obtained from the ⁶⁸Ge/⁶⁸Ga generator, and the long half-life of the parent nuclide ⁶⁸Ge (t_1/2 = 280 days) allows PET imaging on-site without the need for a dedicated cyclotron [16]. The ready availability of ⁶⁸Ga for clinical PET has prompted rapid tracer development based on this isotope, and more than 40 centers in Europe have utilized this type of generator, with most of them focusing on either basic or transitional research on radiochemistry of ⁶⁸Ga [12].

Although widely used conventional methods for labeling with radiometals suffer from several limitations. Due to the radiation emitted by PET radionuclides, the radiolabelings are typically carried out in a heavily lead-shielded fume hood or bunker to minimize radiation exposure to the radiochemists. In addition, radiosyntheses are usually carried out at very low mass amounts (usually less than a few hundred nanograms) of radioactive material. To facilitate convenient handling and efficient mixing, the tiny amounts of radioisotopes need to be dissolved in a relatively large amount of solvent, which results in nanomolar concentrations of the radiometals [17], [18]. To compensate for the slow reaction kinetics resulting from such low concentrations, a very large (~ 100-fold) excess of the non-radioactive precursor (biomolecule conjugate) is used to promote rapid and efficient incorporation of the radioisotope into the PET imaging agent. However, with such a large stoichiometric excess of precursor, issues such as radioisotope pre-concentration (prior to reaction), side reactions (due to possibly more reactive minor impurities), product purification and product analysis can be problematic. To achieve high specific activities using conventional labeling techniques, careful control over reaction conditions and extensive chromatographic purification are required to separate the radiolabeled compounds from the cold precursors. To address the above issues associated with conventional radiosynthesis, we have developed compact microreactors for rapid, efficient labeling without the need for using large excess reagents.

Microfluidic devices, comprising enclosed micro-channels (normally 10–500 μm wide or tall), mixing units, heaters, pumping systems, are able to control and process chemical or biological reactions in a continuous flow manner or batch mode [19], [20], [21], [22]. When used for radiolabeling biomolecules, microreactors can potentially circumvent many of the existing limitations [23], [24], [25], [26]. Microfluidic radiolabeling offers: (1) the ability to manipulate small volumes, which mitigates issues associated with dilution effects; (2) efficient mixing which greatly improves reaction kinetics; (3) the ability for fine level of control over reaction conditions, such as concentrations, temperature, enabling reliable and reproducible labeling; and (4) a much smaller overall footprint of the system, which drastically reduces the volume that requires shielding.

Most microfluidic approaches for the synthesis of radiopharmaceuticals have focused primarily on the synthesis of ¹⁸F- and ¹¹C-labeled agents [22], [27], [28]. For example, Lee et al. [28] developed a poly(dimethylsiloxane) (PDMS)-based microreactor for the multistep synthesis of ¹⁸F-FDG, a radiotracer commonly used to detect cancer via PET. An improved version of this microreactor provided yields of 96% after ~ 15 min of total synthesis time, compared to 75% yield after ~ 45 min using an automated apparatus for conventional synthesis [29]. However, an off-chip purification step was still required to obtain a radiochemical purity of ~ 99%, and loss of ¹⁸F through reaction with or absorption by the PDMS was noted as a significant issue. In other work, Lu et al. [30] developed a hydrodynamically-driven microreactor and investigated the effect of infusion rate on the yields of several ¹⁸F and/or ¹¹C-labeled carboxylic esters using a T-junction, flow-through glass microfluidic system. They observed that incomplete diffusive mixing at lower residence times adversely affected the yield. Recently, our group has developed PDMS-based microreactors for labeling BFC-BM with radiometals [31]. These microreactors were validated with radiosynthesis of ⁶⁴Cu labeled RGD peptide, and demonstrated higher yields compared to conventional techniques under identical reaction conditions. Following up on the validation with ⁶⁴Cu labeling, we report here the versatility of this microreactor-based method by utilizing two commonly used PET radiometals, ⁶⁴Cu and ⁶⁸Ga, to label two common bifunctional chelates, DOTA and NOTA (more specifically S-2-(4-Isothiocyanatobenzyl)-NOTA (p-SCN-Bn-NOTA)), conjugated to a clinically relevant biomolecule, the pentapeptide cyclo(RGDfK). We present a direct comparison between radiolabeling performance of the microreactor and conventional methods, and results on optimization of the labeling conditions using the microreactor.