Over the past decade, theranostic imaging has emerged as a powerful clinical tool in oncology for identifying patients likely to respond to targeted therapies and for monitoring the response of patients to treatment. Herein, we report a theranostic approach to pretargeted radioimmunotherapy (PRIT) based on a pair of radioisotopes of copper: positron-emitting copper-64 (⁶⁴Cu, t_1/2 = 12.7 h) and beta particle-emitting copper-67 (⁶⁷Cu, t_1/2 = 61.8 h). This strategy is predicated on the in vivo ligation between a trans-cyclooctene (TCO)-bearing antibody and a tetrazine (Tz)-based radioligand via the rapid and bioorthogonal inverse electron-demand Diels–Alder reaction. Longitudinal therapy studies were conducted in a murine model of human colorectal carcinoma using an immunoconjugate of the huA33 antibody modified with TCO (huA33-TCO) and a ⁶⁷Cu-labeled Tz radioligand ([⁶⁷Cu]Cu-MeCOSar-Tz). The injection of huA33-TCO followed 72 h later by the administration of 18.5, 37.0, or 55.5 MBq of [⁶⁷Cu]Cu-MeCOSar-Tz produced a dose-dependent therapeutic response, with the median survival time increasing from 68 d for the lowest dose to >200 d for the highest. Furthermore, we observed that mice that received the highest dose of [⁶⁷Cu]Cu-MeCOSar-Tz in a fractionated manner exhibited improved hematological values without sacrificing therapeutic efficacy. Dual radionuclide experiments in which a single administration of huA33-TCO was followed by separate injections of [⁶⁴Cu]Cu-MeCOSar-Tz and [⁶⁷Cu]Cu-MeCOSar-Tz revealed that the positron emission tomography images produced by the former accurately predicted the efficacy of the latter. In these experiments, a correlation was observed between the tumoral uptake of [⁶⁴Cu]Cu-MeCOSar-Tz and the subsequent therapeutic response to [⁶⁷Cu]Cu-MeCOSar-Tz. Significance Radioimmunotherapy is predicated on harnessing the exquisite specificity of antibodies to deliver cytotoxic radiation to tumors. Yet the long circulation time of radioimmunoconjugates leads to high radiation doses to healthy tissues. Pretargeted radioimmunotherapy (PRIT) circumvents this problem by decoupling the antibody and radionuclide, injecting the former prior to the latter, and empowering the two components to combine at the tumor. We have leveraged bioorthogonal click chemistry, a colorectal cancer-targeting antibody, and a pair radioligands bearing radioisotopes of copper—positron-emitting copper-64 and beta particle-emitting copper-67—to create a pretargeting system that enables both theranostic positron emission tomography and PRIT. We believe that the incorporation of theranostic imaging into PRIT regimens could be instrumental in the clinical success of the modality.

Pretargeting-based approaches are being investigated for radioimmunoimaging and therapy applications to reduce the effective radiation burden to the patient. To date, only a few studies have used short-lived radioisotopes for pretargeting of antibodies, and such examples with internalizing antibodies are even rarer. Herein, we have investigated pretargeting methodology using inverse electron-demand Diels-Alder (IEDDA) for tracing two clinically relevant, internalizing monoclonal antibodies, cetuximab and trastuzumab. Bioorthogonal reaction between tetrazine and trans-cyclooctene (TCO) was used for tracing cetuximab and trastuzumab in vivo with a fluorine-18 (t _½ = 109.8 min) labelled tracer. TCO-cetuximab or TCO-trastuzumab was administered 24, 48, or 72 h prior to the injection of tracer to A431 or BT-474 tumour-bearing mice, respectively. With cetuximab, the highest tumour-to-blood ratios were achieved when the lag time between antibody and tracer injections was 72 h. With trastuzumab, no difference was observed between different lag times. For both antibodies, the tumour could be clearly visualized in the PET images with the highest tumour uptake of 3.7 ± 0.1%ID/g for cetuximab and 1.5 ± 0.1%ID/g for trastuzumab as quantified by ex vivo biodistribution. In vivo IEDDA reaction was observed in the blood for both antibodies, but with trastuzumab, this was to a much lower degree than with cetuximab. We could successfully visualize the tumours by using cetuximab and trastuzumab in pretargeted PET imaging despite the challenging circumstances where the antibody is internalized and there is still some unbound antibody circulating in the blood flow. This clearly demonstrates the potential of a pretargeted approach for targeting internalizing antigens and warrants development of pharmacokinetic optimization of the biorthogonal reactants to this end.

The proliferation of plastics in the environment continues at an alarming rate. Plastic particles have been found to be persistent and ubiquitous pollutants in a variety of environments, including sea water, fresh water, soil, and air. In light of this phenomenon, the scientific and medical communities have become increasingly wary of the dangers posed to human health by chronic exposure to microplastics (< 5 mm diameter) and nanoplastics (< 100 nm diameter). A critical component of the study of the health effects of these pollutants is the accurate determination of their pharmacokinetic behavior in vivo. Herein, we report the first use of molecular imaging to track polystyrene (PS) micro- and nanoplastic particles in mammals. To this end, we have modified PS particles of several sizes—diameters of 20 nm, 220 nm, 1 µm, and 6 µm—with the chelator desferrioxamine (DFO) and radiolabeled these DFO-bearing particles with the positron-emitting radiometal zirconium-89 (⁸⁹Zr; t_1/2 ~ 3.3 d). Subsequently, positron emission tomography (PET) was used to visualize the biodistribution of these radioplastics in C57BL/6J mice at 6, 12, 24, and 48 h after ingestion. The imaging data reveal that the majority of the radioplastics remain in the gastrointestinal tract and are eliminated through the feces by 48 h post-ingestion, a result reinforced by acute biodistribution studies. Ultimately, this work suggests that nuclear imaging—and PET in particular—can be a sensitive and effective tool in the urgent and rapidly growing effort to study the in vivo behavior and potential toxicity of micro- and nanoplastics.

Pretargeted positron emission tomography (PET) imaging based on bioorthogonal chemical reactions has proven its potential in immunoimaging. It may also have great potential in nanotheranostic applications. Here, we report the first successful pretargeted PET imaging of trans-cyclooctene-modified mesoporous silicon nanoparticles, using ¹⁸F-labeled tetrazine as a tracer. The inverse electron-demand Diels–Alder cycloaddition (IEDDA) reaction was fast, resulting in high radioactivity accumulation in the expected organs within 10 min after the administration of the tracer. The highest target-to-background ratio was achieved 120 min after the tracer injection. A clear correlation between the efficiency of the in vivo IEDDA labeling reaction and the injected amount of the tracer was observed. The radioactivity accumulation decreased with the increased amount of the co-injected carrier, indicating saturation in the reaction sites. This finding was supported by the in vitro results. Our study suggests that pretargeted imaging has excellent potential in nanotheranostic PET imaging when using high-specific-activity tracers.

Microplastics and nanoplastics have become ubiquitous environmental pollutants. The threat these plastics pose to human health has fueled research focused on their pathophysiology and toxicology, yet many of their fundamental properties - for example, their in vivo pharmacokinetics - remain poorly understood. In this investigation, we have harnessed positron emission tomography (PET) to track the in vivo fate of micro- and nanoplastics administered to mice intratracheally and intravenously. To this end, 1 μm and 20 nm diameter amine-functionalized polystyrene particles were modified with an isothiocyanate-bearing variant of desferrioxamine (DFO) and radiolabeled with the positron-emitting radiometal [⁸⁹Zr]Zr⁴⁺. Both radioplastics - [⁸⁹Zr]Zr-DFO-PS1000 and [⁸⁹Zr]Zr-DFO-PS20 - were produced in ∼95% radiochemical yield and found to be >85% stable to demetallation over one week at 37 °C in human serum and simulated lung fluid. The incubation of [⁸⁹Zr]Zr-DFO-PS1000 and [⁸⁹Zr]Zr-DFO-PS20 with MH-S cells revealed that the majority of the former were phagocytosed by alveolar macrophages within 4 h, while the latter largely evaded consumption. Finally, the in vivo behavior of the radioplastics was interrogated in mice upon intravenous and intratracheal administration. PET imaging and biodistribution experiments revealed that the intravenously injected plastics accumulated primarily in the liver and spleen, yielding hepatic radioactivity concentrations of 101 ± 48 %ID/g and 92 ± 22 %ID/g at 168 h post-injection for [⁸⁹Zr]Zr-DFO-PS1000 and [⁸⁹Zr]Zr-DFO-PS20_, respectively. In contrast, the mice that received the radioplastics via intratracheal installation displayed the highest uptake in the lungs at the end of one week: 4 ± 2 %ID/g for [⁸⁹Zr]Zr-DFO-PS1000 and 32 ± 6 %ID/g for [⁸⁹Zr]Zr-DFO-PS20. Ultimately, this work illustrates the critical role that the route of exposure plays in the bioaccumulation of plastic particles, reveals that size dramatically influences the pulmonary retention of inhaled particles, and underscores the value of PET imaging as a tool for studying the pharmacokinetics of environmental pollutants.