MicroSPET and microPET are increasingly used in oncology, neurology and cardiology to analyse molecular alterations in vivo in animal models of diseases. In particular, microPET is the principal in vivo molecular imaging technique, and is already extensively used for assessing numerous variables related to physiological and pathological states in the mouse. Other methods, too, that are based on different physical principles, such as magnetic resonance imaging and spectroscopy, and bioluminescence techniques, are used for molecular imaging, for research on animal models.

These tremendous developments in instrumentation technology are also paralleled by developments in radiochemistry, radiochemical technology and biotechnology with a continuous attention to micro and nanotechnology. In particular, over the last few months new technological approaches to the synthesis of radiotracers have been proposed by a few research teams while new genetically engineered mice and cellular clones, along with labelling techniques of different reporter products have been described. These new technologies are improving day after day the applications of the imaging methodologies and our ability to understand diseases, trace cellular and molecular processes in vivo, and monitor the efficacy of treatments performed with the most advanced techniques including the use of alpha emitting radionuclides and stem cells.

Micro-reactors nanotechnology for radiochemistry

Continuous-flow micro-reactors have been used for chemical processes on nanoliter to microliter scales. Micro-reactor devices consist of a network of micron-sized channels (typical dimensions in the range 10–300 mm) embedded in a solid substrate. These devices have very small dimensions, 10 millimeters in diameter and 0.1 millimeters in depth, and permit to manipulate and transfer very small quantities of fluid to achieve a chemical synthesis, within an integrated circuit. Miniaturization of radio-syntheses might lead to the use of smaller quantities than presently required of expensive precursors, easier purification processes with greater yield and specific activity. are now emerging as an extremely useful technology for the intensification and miniaturization of chemical processes.

Synthesis of [18F]FDG, in an integrated microfluidic device

Chung-Cheng Lee and colleagues, from a broad range of institutions in California, report the synthesis of [18F]FDG, in an integrated microfluidic device [1]. Five sequential processes - [18F]fluoride concentration, water evaporation, radio-fluorination, solvent exchange, and hydrolytic deprotection -proceeded with high radiochemical yield and purity and with shorter synthesis time relative to conventional automated synthesis. Multiple doses of [18F]FDG for positron emission tomography imaging studies in mice were prepared. They also designed a chemical reaction circuit with the capacity to synthesize large [18F]FDG doses. The chip has a coin-shaped reactor (volume 5 ml) equipped with a vacuum vent. It was used to synthesize 1.74 mCi of [18F]FDG, an amount sufficient for several mouse experiments. From the purified and sterilized product, two doses (375 mCi and 272 mCi) were used for microPET molecular imaging of two mouse models of cancer. The authors conclude that their results, which constitute a proof of principle for automated multistep syntheses at the nanogram to microgram scale, could be generalized to a range of radiolabelled substrates.

Radio-halogenation of small and large molecular weight molecules with a microfluidic device

Gillies et al. in two distinct yet very similar papers [2, 3] report the radio-halogenation of small and large molecular weight molecules using the microfluidic device. These reactions involved the direct radio-iodination of the apoptosis marker Annexin V using Iodine-124, the indirect radio-iodination of the anti-cancer drug doxorubicin from a tin-butyl precursor and the radio-synthesis of [18F]FDG from a mannose triflate precursor and Fluorine-18. They demonstrate the rapid radio-iodination of the protein Annexin V (40% radiochemical yield within 1 min) and the rapid radio-fluorination of [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.

Hydrodynamically-driven micro-reactor to label carboxylic esters with positron-emitters

It should be mentioned that the use of a hydrodynamically-driven micro-reactor, had previously been proposed by Shui-Yu Lu [4] from the National Institutes of Health in Bethesda, Maryland, USA, to label carboxylic esters with one of two short-lived positron-emitters, carbon-11 or fluorine-18. The authors proved the feasibility of various syntheses with different radiochemical yield depending on the infusion rate. Their results already exemplified the advantages of the micro-reactor methodology for producing radiotracers using small quantities of substrates, and achieve a rapid reaction optimisation and easy product purification.

Molecular and cellular biotechnologies for imaging and therapy development

Three papers recently published highlight the synergistic potentials of the rapidly evolving areas of molecular and cellular biotechnology, of animal engineering and of radionuclide based imaging and therapy.

Affibodies: small radiolabelled targeting proteins for visualization of tumors in vivo

Anna Orlova et al. [5], from the Affibody AB, Bromma and Department of Oncology, Radiology, and Clinical Immunology, Rudbeck Laboratory, Uppsala University, Sweden, have focussed their study on the detection of cell-bound proteins that are produced due to aberrant gene expression in malignant tumors and that can provide important diagnostic information influencing patient management. They theorize that use of small radiolabelled targeting proteins would enable high-contrast radionuclide imaging of cancers expressing such antigens if adequate binding affinity and specificity could be provided. In their paper they describe a HER2-specific 6kDa Affibody molecule with 22 pmol/L affinity that can be used for the visualization of HER2 expression in tumors in vivo using a gamma camera. They constructed a library for affinity maturation by re-randomization of relevant positions identified after the alignment of first-generation variants of nanomolar affinity (50 nmol/L). One selected Affibody molecule, ZHER2:342 showed a >2,200-fold increase in affinity achieved through a single-library affinity maturation step. When radio-iodinated, the affinity-matured Affibody molecule showed clear, high-contrast visualization of HER2-expressing xenografts in mice as early as 6hours post-injection. The tumor uptake at 4 hours post-injection was improved 4-fold (due to increased affinity) with 9% of the injected dose per gram of tissue in the tumor. They conclude that Affibody molecules represent a new class of affinity molecules that can provide small sized, high affinity cancer-specific ligands, which may be well suited for tumor imaging and that if the molecule was to be tested in the clinic, 123I or 124I could be used. Furthermore they observe that the development of suitable chemistry to label the ZHER2:342 molecule with generator-produced 99mTc or 68Ga could further facilitate future imaging studies.

211At therapy against thyroid carcinoma cell line genetically modified to express NIS (K1-NIS)

In another paper Petrich et al. [6], from the Klinik fur Nuklearmedizin, Medizinische Hochschule Hannover, Germany and the Institut fur Pathologie, GSF-Forschungszentrum fur Umwelt und Gesundheit, Neuherberg, Germany report on the in vivo effects of the high linear energy transfer (LET) emitter radioastatine (211At) on tumor growth and outcome in nude mice. They carried out their study based on the fact that sodium/iodide symporter (NIS) gene is currently explored in several trials to eradicate experimental cancer with 131I by its beta-emission. The same authors have recently characterized NIS-specific cellular uptake of an alternative halide, 211At, which emits high-energy alpha-particles. In the present study they administered 211At in a fractionated therapy scheme to NMRI nude mice harbouring rapidly growing solid tumors established from a papillary thyroid carcinoma cell line genetically modified to express NIS (K1-NIS). Animals were observed over 1 year. Tumor growth, body weight, blood counts, survival, and side effects were measured compared with control groups without therapy and/or lack of NIS expression.

They observed that within 3 months, 211At caused complete primary tumor eradication in all cases of K1-NIS tumor-bearing nude mice (n=25) with no tumor recurrence during1 year follow-up. Survival rates of the K1-NIS/211At group were 96% after 6months and 60% after 1 year, in contrast to those of control groups (maximum survival 40 days).

They conclude that 211At represents a promising substrate for NIS-mediated therapy of various cancers either with endogenous or gene transfer-mediated NIS expression.

In vivo visualization of stem cell survival, proliferation, and migration after cardiac delivery

Finally in a third paper Cao et al. [7], from the Department of Radiology, Bio-X Program, Stanford University School of Medicine, California, USA report on the in vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery. Their study was based on recent findings that have shown that stem cell therapy can promote tissue regeneration. Monitoring stem cells in vivo is a major challenge owing to limitations of conventional histological assays and imaging modalities. They stably transduced murine embryonic stem (ES) cells with a lentiviral vector carrying a novel triple-fusion (TF) reporter gene that consists of firefly luciferase, monomeric red fluorescence protein, and truncated thymidine kinase (fluc-mrfp-ttk). ES cell viability, proliferation, and differentiation ability were not adversely affected by either reporter genes or reporter probes compared with nontransduced control cells. Afterward, ES cells carrying the TF reporter gene (ES-TF) were injected into the myocardium of adult nude rats. Control animals received nontransduced ES cells. At day 4, the bioluminescence and PET signals in study animals were significantly higher than in controls. Both signals increased progressively from week 1 to week 4, which indicated ES cell survival and proliferation in the host. Histological analysis demonstrated the formation of intracardiac and extracardiac teratomas. Finally, animals (n=4) that were treated with intraperitoneal injection of ganciclovir (50 mg/kg) did not develop teratomas when compared with control animals (n=4) treated with saline (1 mL/kg). The authors conclude that this is the first study to characterize ES cells that stably express fluorescence, bioluminescence, and positron emission tomography reporter genes and monitor the kinetics of ES cell survival, proliferation, and migration. They also suggest that this versatile imaging platform should have broad applications for basic research and clinical studies on stem cell therapy. It must be noted however that the authors also raise a concern: that of stem cells misbehaviour. The possibility of teratoma formation after transplantation poses a daunting challenge for clinical application of ES cells. To date, no study has addressed this issue from an imaging standpoint. The authors hypothesize that a PET reporter gene (ttk) could also serve as a suicide gene using ganciclovir treatment.

Conclusions

There is no shortage of challenges and opportunities for molecular imaging nowadays. The growth of nanotechnologies applied to radiochemical syntheses, besides guaranteeing reproducible techniques, opens the way to simpler and more cost effective procedures for molecular imaging. This will result in wider use, appreciation and success of both. Using microfluidic reactions the yields of radiopharmaceutical syntheses using short and medium half-life radionuclides can be further improved by optimisation of the microfluidic devices and of fluid mixing profiles.

What emerges from examples of integration of nano and biotechnology into molecular imaging and therapy studies is a vision of the future, a representation of great opportunities for molecular medicine to exploit the potentials of imaging to develop, pursue and monitor the science of individualized treatment.