Asymmetric rotaxanes as dual-modality supramolecular imaging agents for targeting cancer biomarkers

The study addresses the development of dual-modality imaging agents that combine PET and OFI within a single molecular construct to overcome differences in pharmacokinetics when two separate agents are used. OFI offers real-time intraoperative guidance but suffers from limited tissue penetration and quantification; PET complements OFI with high sensitivity and quantitative capability. Prior dual-labelled probes, particularly PSMA-11 derivatives for prostate cancer, have shown successful PET/OFI imaging and guidance for surgery. Supramolecular systems and mechanically interlocked molecules (MIMs), such as rotaxanes, offer modular architectures and controlled stoichiometry that could be advantageous for multimodal probe design and pharmacokinetic tuning. Building on prior supramolecular PET agents using rotaxanes, the authors aim to create bimodal PET/OFI rotaxane conjugates targeting PSMA by integrating a ⁶⁸Ga-chelate and fluorescein onto a rotaxane axle that is mechanically bonded to a PSMA-targeted β-cyclodextrin host. The work explores two cooperative capture strategies (four-component and six-component CB[6]-mediated azide–alkyne cycloadditions) to access asymmetric [3]- and [4]-rotaxanes, evaluate their stability, photophysics, and in vitro PSMA-targeted uptake.

Dual-modality PET/OFI probes have been developed using small-molecule fluorophores (e.g., coumarin, BODIPY, fluorescein, rhodamine, cyanine) and radiometal chelates. PSMA-11-based dual-labelled ligands have shown nanomolar affinity, strong PSMA-specific uptake, and successful fluorescence-guided surgical applications. Despite advances, most supramolecular imaging probes reported are single-modality. Prior work by the authors introduced rotaxane-based PET tracers (⁸⁹Zr or ⁶⁸Ga) conjugated to antibodies, demonstrating feasibility of supramolecular PET agents. However, earlier asymmetric rotaxane syntheses suffered from low yields due to weak host–guest binding when a longer PEG2 linker was used between fluorescein and a biphenyl guest. This study builds on these findings by optimizing host–guest interactions and cooperative capture to realize dual-modality supramolecular agents.

• Guest design and host–guest characterization: A fluorescein-derivatized biphenyl alkyne guest (compound 1) with a short ethylenediamine linker was synthesized (3 steps, overall 20% yield). 1H NMR titrations in D2O quantified inclusion of 1 in β-cyclodextrin (β-CD): Job’s plots confirmed a 1:1 complex; chemical shift analyses (Benesi–Hildebrand, Scott, Scatchard models) yielded binding constants across monitored protons giving an average K ≈ 557 ± 329 M⁻1, higher than a prior PEG2-linked analogue (167 ± 70 M⁻1). Sliding of β-CD along the guest and rapid face inversion were inferred from NMR data.
• Four-component cooperative capture synthesis ([3]rotaxanes): In H2O at 70 °C for 1 min, equimolar 1, β-CD, CB[6], and an azido-derivatized DFO chelate (3) produced asymmetric [3]semirotaxane 4, isolated by semi-preparative HPLC (72% yield). Axle capping by Ga3+ complexation gave natGa-4 and [⁶⁸Ga]Ga-4. Radiosynthesis with [⁶⁸Ga][Ga(H2O)6]Cl3 afforded >98% decay-corrected RCY and >95% RCP, confirmed by radio-TLC (product at Rf 0.0–0.1; ⁶⁸Ga-citrate control at Rf 0.9–1.0) and co-eluting radio-/UV-HPLC peaks.
• Six-component cooperative capture synthesis ([4]rotaxanes): Equimolar biphenyl dialkyne (2), DFO-azido (3), fluorescein-azido (5), β-CD, and 2 equivalents of CB[6] in H2O (70 °C, 1 min) effected two CB[6]-accelerated click reactions to form [4]-species. Products included a previously reported [4]pseudorotaxane with two DFO chelates, a symmetric bisfluorescein [4]rotaxane (7), and the desired asymmetric [4]semirotaxane 6. Compound 6 was purified (16% yield) and characterized by HPLC, HRMS-ESI, and multinuclear NMR. Metallation of 6 gave natGa-6 and [⁶⁸Ga]Ga-6 in >99% chemical yield and >98% radiochemical yield, with >95% purity; radiolabeling was quantitative in <10 min at 23 °C.
• PSMA-targeted [3]rotaxane: Using a Glu-urea-Lys-functionalized β-CD (compound 8), the four-component method (1, 8, 3, CB[6]; H2O, 70 °C, 1 min) yielded PSMA-targeted [3]semirotaxane 9 (38% isolated). Ga3+ complexation provided natGa-9 and [⁶⁸Ga]Ga-9. natGa-9 showed a single HPLC peak (Rt 8.28 min), whereas [⁶⁸Ga]Ga-9 displayed three radiometric peaks (Rt 7.77, 8.05, 8.37 min), attributed to diastereomeric GaDFO complexes, fluorescein equilibria, and mechanically planar chiral epimers arising from the oriented β-CD macrocycle.
• Photophysical characterization: For natGa-9, UV/Vis-excitation/emission maxima were 491/520 nm with a Stokes shift of 29 nm; fluorescence quantum yield Φf = 0.70 ± 0.04 in 0.1 M NaOH (relative to fluorescein Φf = 0.93).
• Stability studies: In water, semirotaxanes 4 and 9 had half-lives of 24.61 ± 2.00 h and 28.85 ± 2.50 h, respectively, indicating dethreading. Metallated [3]rotaxanes natGa-4 and natGa-9 were stable with half-lives of 560.5 ± 55.40 h and 254.7 ± 19.50 h, respectively. [⁶⁸Ga]Ga-4 and [⁶⁸Ga]Ga-9 were stable in PBS and human serum for 2 h at 37 °C with radioactivity retained.
• Cell culture and binding assays: PSMA-positive LNCaP and PSMA-negative PC-3 prostate cancer cells were used. Radiotracers were incubated for 1.5 h at 37 °C; cell-associated activity normalized to protein content was quantified by gamma counting. Controls included [⁶⁸Ga]Ga-PSMA-11 (positive) and [⁶⁸Ga]Ga-4 (non-targeted negative). Specificity assays used 0.1% NaN3 (to inhibit internalization) and blocking with 200 µM free PSMA ligand.

• Host–guest optimization: The fluorescein–biphenyl guest (1) with an ethylenediamine linker formed a stronger 1:1 inclusion complex with β-CD (average K ≈ 557 ± 329 M⁻1 across models/protons) versus a prior PEG2-linked analogue (167 ± 70 M⁻1), a ~3.4-fold increase.
• Efficient four-component assembly: Asymmetric [3]semirotaxane 4 was isolated in 72% yield after a 1 min reaction at 70 °C in water. Subsequent Ga3+ complexation afforded [⁶⁸Ga]Ga-4 with RCY >98% and RCP >95% (radio-TLC product Rf 0.0–0.1; citrate control Rf 0.9–1.0). Co-elution in radio-/UV-HPLC confirmed identity.
• Six-component assembly of asymmetric [4]rotaxanes: The strategy produced three products, including the target asymmetric [4]semirotaxane 6 (16% isolated yield). Metallation yielded natGa-6 and [⁶⁸Ga]Ga-6 in >99% chemical and >98% radiochemical yields with >95% purity; radiolabeling quantitative in <10 min at 23 °C.
• First PSMA-targeted bimodal rotaxane: PSMA-targeted [3]semirotaxane 9 was isolated in 38% yield; Ga-complexation provided natGa-9 and [⁶⁸Ga]Ga-9, with multiple radiolabeled isomers observed by HPLC. natGa-9 showed fluorescein-like photophysics (λex 491 nm, λem 520 nm, Φf = 0.70 ± 0.04).
• Stability: Semi-rotaxanes 4 and 9 were unstable in water (t1/2 24.61 ± 2.00 h and 28.85 ± 2.50 h), consistent with dethreading. Metallated [3]rotaxanes were stable (natGa-4 t1/2 560.5 ± 55.40 h; natGa-9 t1/2 254.7 ± 19.50 h). [⁶⁸Ga]Ga-4 and [⁶⁸Ga]Ga-9 remained intact in PBS and human serum for 2 h at 37 °C.
• Cell uptake and specificity: In PSMA+ LNCaP cells, [⁶⁸Ga]Ga-9 showed 0.92 ± 0.26% cell-associated activity versus 0.48 ± 0.02% for non-targeted [⁶⁸Ga]Ga-4 (P = 0.085). [⁶⁸Ga]Ga-9 uptake decreased with 0.1% NaN3 to 0.57 ± 0.09% (~35% reduction; P = 0.695) and with PSMA blocking to 0.62 ± 0.04% (~30% reduction; P = 0.175). In PSMA− PC-3 cells, [⁶⁸Ga]Ga-9 uptake was 0.32 ± 0.003% (~3-fold lower than LNCaP; P = 0.055). The positive control [⁶⁸Ga]Ga-PSMA-11 showed higher uptake (3.85 ± 0.64% in LNCaP; P = 0.008), validating the assay.

The study demonstrates that asymmetric rotaxane architectures assembled via CB[6]-accelerated cooperative capture can integrate both a PET radiometal complex and a fluorophore together with a biological targeting vector in a single construct. Optimizing the guest linker enhanced β-CD inclusion, improving cooperative capture efficiency and enabling higher-yield four-component assembly of [3]rotaxanes relative to prior designs. Both four- and six-component strategies successfully produced asymmetric metallo-rotaxanes with high (radio)chemical yields and purities upon Ga3+ complexation. The PSMA-targeted [3]rotaxane exhibited the expected fluorescein photophysics and sufficient chemical and radiochemical stability for biological evaluation. In cell assays, [⁶⁸Ga]Ga-9 showed specific association with PSMA+ cells relative to PSMA− controls and responded appropriately to internalization inhibition and target blocking, indicating target-mediated uptake. While absolute uptake was lower than that of the optimized clinical standard [⁶⁸Ga]Ga-PSMA-11, these data support the feasibility of rotaxane-based dual-modality agents for targeted imaging. The modularity and stoichiometric precision of the rotaxane platform allow controllable incorporation of multiple functional components, offering a route to fine-tune pharmacokinetics and imaging properties.

The authors efficiently synthesized three stoichiometrically precise asymmetric rotaxane-based radiotracers using CB[6]-β-CD-mediated azide–alkyne click chemistry, enabling incorporation of a PET radiometal chelate and a fluorescein fluorophore. They report the first PSMA-targeted bimodal rotaxane PET/OFI probe and demonstrate its chemical stability, photophysical characteristics, and PSMA-specific cellular association in vitro. The platform is versatile and can be adapted to other fluorophores (to tune optical properties), alternative radiometal chelates, diverse targeting vectors (antibodies, peptides), and even therapeutic payloads to create a range of dual-modality or theranostic agents. Future work should pursue optimization of binding affinity and pharmacokinetics, address isomeric heterogeneity, and evaluate in vivo imaging performance and surgical guidance utility.

• Cellular uptake of the PSMA-targeted rotaxane was modest compared to the clinical standard [⁶⁸Ga]Ga-PSMA-11, suggesting lower affinity and/or suboptimal pharmacokinetics for this first-generation design.
• Radiolabeled products exhibited multiple isomers (diastereomers/geometric and mechanically planar chiral epimers), and isolation of individual isomers was limited by the short half-life of ⁶⁸Ga.
• Semi-rotaxanes were unstable in water (dethreading), necessitating metallated capping for stability.
• The six-component assembly route afforded lower isolated yields (e.g., 16% for [4]semirotaxane 6) compared to the four-component approach.
• Biological evaluation was limited to in vitro cell assays; no in vivo imaging or pharmacokinetic data were reported.