The development of dual-modality imaging agents combining positron emission tomography (PET) and optical fluorescence imaging (OFI) is highly desirable for enhancing clinical diagnosis and intraoperative surgeries. Such agents offer the advantages of high sensitivity and the ability to perform both in vivo and intraoperative monitoring. OFI, while offering real-time imaging capabilities during surgery, suffers from limitations in tissue penetration depth. PET, conversely, has excellent penetration but lacks real-time imaging capabilities. Combining both modalities within a single molecular entity allows for the synergistic advantages of both technologies while avoiding potential pharmacokinetic differences associated with separate agents. Small-molecule fluorophores such as coumarin, BODIPY, fluorescein, rhodamine, and cyanine dyes are frequently used for OFI. Dual-labelled antibody or peptide-based probes, particularly those targeting prostate-specific membrane antigen (PSMA), are well established for prostate cancer imaging. While effective, designing and synthesizing these dual-modality imaging agents remains a challenge. This study explored the potential of supramolecular systems and mechanically interlocked molecules (MIMs), specifically rotaxanes, as a platform for creating efficient dual-modality PET/OFI imaging agents. Previous work by the authors demonstrated the use of rotaxanes for single-modality PET imaging, and this research extends that work to create bimodal agents.

Existing literature highlights the significant interest in dual-modality imaging agents for disease biomarker detection. Dual-modality PET/OFI agents are particularly attractive due to their ability to combine the high sensitivity and deep tissue penetration of PET with the real-time imaging and intraoperative capabilities of OFI. Several studies have successfully demonstrated the use of dual-labeled probes for targeting prostate cancer cells, often utilizing PSMA as the target. PSMA-11, for instance, is a clinically validated ⁶⁸Ga-labeled radiotracer that has shown great promise in preclinical and clinical studies. However, the development of such dual-modality agents remains challenging, requiring careful consideration of both radionuclide and fluorophore properties, as well as the targeting vector. Supramolecular chemistry and MIMs provide unique opportunities in this area, offering potential for controlling pharmacokinetics, metabolism, and stoichiometry of imaging agents. While some research explores single-modality supramolecular agents, the application of supramolecular compounds as dual-modality imaging probes is less explored.

This study focused on the synthesis of asymmetric rotaxanes using two different approaches: a four-component and a six-component cooperative capture strategy. Both strategies utilized a cucurbit[6]uril (CB[6])-mediated alkyne-azide ‘click’ reaction. The four-component approach involved the reaction of a fluorescein-derivatized biphenyl alkyne, β-cyclodextrin (β-CD), CB[6], and an azido derivative of the desferrioxamine B chelate (DFO-azido). The six-component approach involved an additional fluorescein-azido compound and a biphenyl dialkyne, resulting in a more complex [4]rotaxane structure. The binding affinity of the fluorescein-biphenyl alkyne to β-CD was determined through 1H NMR titrations, using Benesi–Hildebrand, Scott, and Scatchard methods. The successful synthesis of the [3]rotaxane and [4]rotaxane structures was confirmed using various techniques, including reverse-phase analytical HPLC, high-resolution electrospray ionization mass spectrometry, and multinuclear NMR spectroscopy. Radiolabeling with ⁶⁸Ga was achieved using standard radiolabeling methods. Radiochemical yields and purities were determined using radio-TLC and radio-HPLC. For the targeted rotaxane, a Glu-urea-Lys-derivatized β-CD macrocycle was used. The stability of the rotaxanes, both before and after metal complexation, was assessed in water, PBS, and human serum. Cellular uptake studies were performed using PSMA-positive (LNCaP) and PSMA-negative (PC-3) prostate cancer cell lines. Cellular binding was measured by gamma counting and normalized to total protein content. Blocking assays and experiments with azide were performed to assess the specificity of binding to PSMA.

The study successfully synthesized three new asymmetric rotaxane-based radiotracers. Both the four-component and six-component strategies proved effective, yielding [3]rotaxanes and [4]rotaxanes, respectively. The four-component strategy yielded a higher isolated yield (72%) than the six-component approach (16%). The binding constant for the 1:1 β-CD inclusion complex with the optimized fluorescein-derivatized biphenyl guest molecule (1) was significantly higher (557 ± 329 M⁻¹) than that obtained with a previous design using a longer PEG2 linker (167 ± 70 M⁻¹), demonstrating the importance of linker design for efficient rotaxane synthesis. The ⁶⁸Ga-radiolabeling of the [3]rotaxane and [4]rotaxane structures was achieved with high radiochemical yields (>98%) and purities (>95%). The synthesized [3]rotaxanes showed stability in aqueous solutions, with the metallated rotaxanes exhibiting significantly enhanced stability compared to their non-metallated counterparts. In cellular uptake assays, the PSMA-targeted [3]rotaxane ([⁶⁸Ga]Ga-9) showed specific binding to PSMA-positive LNCaP cells, with a significant reduction in uptake observed in the presence of azide and with a PSMA-blocking agent. Uptake was significantly lower than that observed for the clinical radiotracer [⁶⁸Ga]Ga-PSMA-11, however. The non-targeted [3]rotaxane ([⁶⁸Ga]Ga-4) displayed non-specific binding to both cell lines. The fluorescence quantum yield of the non-radioactive PSMA-targeted [3]rotaxane ([natGa]-9) was determined to be 0.70 ± 0.04. The PSMA-targeted [3]rotaxane exists as a mixture of mechanically planar chiral diastereomers.

The findings demonstrate the feasibility of using the CB[6]-β-CD-azide-alkyne click reaction for the synthesis of asymmetric rotaxanes as dual-modality imaging agents. The improved design of the fluorescein-biphenyl guest molecule significantly enhanced the efficiency of rotaxane formation. The successful radiolabeling and stability studies validate the use of this platform for developing PET/OFI probes. The specific binding of the PSMA-targeted rotaxane to LNCaP cells, along with the correct biological profile observed in blocking and azide experiments, indicates its potential as a targeted imaging agent. Although the cellular association was lower than the clinical standard ([⁶⁸Ga]Ga-PSMA-11), this study provides a proof-of-concept for the use of rotaxanes as dual-modality probes. The lower cellular uptake can be attributed to the first-generation nature of these supramolecular radiotracers compared to the highly optimized clinical radiotracer. Further optimization of the targeting vector and the rotaxane design can improve the binding affinity and cellular uptake.

This study successfully synthesized three new asymmetric rotaxane-based radiotracers with both PET and OFI capabilities. The CB[6]-β-CD-azide-alkyne click reaction is a versatile approach to developing bimodal imaging agents. The successful creation of a PSMA-targeted bimodal rotaxane-based PET/OFI probe demonstrates the potential of this platform. Future research should focus on optimizing the rotaxane design to improve binding affinity and cellular uptake, as well as exploring different fluorophores and chelates for improved imaging characteristics.

The main limitation of this study is the relatively low cellular uptake of the PSMA-targeted rotaxane compared to the clinical standard [⁶⁸Ga]Ga-PSMA-11. This suggests that further optimization of the targeting moiety and rotaxane structure may be required to enhance the binding affinity to PSMA. Another limitation is the detection of multiple isomers in the radiolabeled PSMA-targeted [3]rotaxane, which may affect its biological activity. Further work is necessary to investigate the impact of these isomers and explore strategies for isomer separation.