Ligand-free ^99mTc-polyurea dendrimer complexes: nanoradiotheranostics targeting ovarian cancer

Abstract

A folic acid-targeted polyurea (PURE) dendrimer was easily radiolabelled with Technetium-99m (^99mTc-PURE_G4-FA₂) avoiding the use of additional ligands and bioconjugation chemistry. This straightforward strategy is enabled in PURE dendrimers due to their favourable surface terminal groups configuration, showing coordination capabilities and turning these biodendrimers into attractive platforms for nanoradiotheranostics.

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In recent decades, extensive efforts have been made to prevent, diagnose, and treat cancer. In this field, nuclear medicine offers effective tools that integrate imaging and therapy. A non-invasive approach, such as radioimaging allows for the characterization and quantification of biological processes in early cancer diagnosis, targeted drug delivery and monitoring of therapeutic response. In pursuit of this goal, nuclear imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) utilize radionuclides emitting positrons or gamma (γ) photons. Conversely, therapeutic radionuclides that emit alpha (α) or beta (β) particles are crucial for the emergence of molecular radiotheraphy.¹ Moreover, Auger electron therapy displays a huge potential in cancer treatment.²

Due to its unique properties which includes low cost, low absorbed dose per patient, good coordination chemistry and a short half-life time (ca. 6 h), ^99mTc is the most used radionuclide for nuclear imaging.³ In addition, ^99mTc emits Auger electrons with low energy that can be delivered over short distances to tissues, with high linear energy transfer.⁴ Rhenium (Re), a group 7 congener of technetium, has two β⁻ emitting isotopes, ¹⁸⁶Re and ¹⁸⁸Re that are suitable for the development of therapeutic radiopharmaceuticals.⁵ Tc and Re can be considered a “matched pair” suitable for creating radiolabelled compounds for nuclear imaging (^99mTc) or radionuclide therapy (^186/188Re). Based on isostructural Re and Tc compounds theranostic agents can be achieved, which can deliver ionizing particles (¹⁸⁶Re/¹⁸⁸Re) to treat a tumor or provide images (^99mTc) for diagnosis.

Despite advancements in recent decades, numerous radiopharmaceuticals in clinical use still display some limitations, such as poor pharmacokinetics/biodistribution, non-specificity and renal toxicity at relatively high doses.⁶ The exploration of nanotechnology in nuclear medicine enables the development of nanoradiopharmaceuticals, which may overcome some of these limitations and are best suited for theranostic approaches. Multiple advancements have been reported using nanoparticles such as metal oxide nanoparticles, liposomes, and dendrimers, which can be designed and radiolabelled for nanotheranostic applications.⁷ Dendrimers, in particular, have been extensively explored for multimodal imaging and theranostics.⁸ Due to their functional surface (e.g., primary amines), dendrimers can be easily decorated with target molecules, thus enabling molecular recognition⁹ and making them outstanding nanocarriers. PAMAM dendrimers have been reported as suitable systems for radiolabelling with medical radionuclides.¹⁰ However, PAMAM radiolabelling is usually a multistep process that requires the incorporation of chelators for radiometal coordination. Therefore, it is crucial to develop systems that allow simple and straightforward radiolabelling.

Polyurea (PURE) dendrimers are a class of biodendrimers characterized by low cytotoxicity.¹¹ The amine-terminated surface allows further conjugation/targeting (e.g., folic acid)^12,13 and/or complexation (e.g., siRNA),¹⁴ rendering these nanosystems highly attractive from a therapeutic stand point.

PURE dendrimers exhibit a distinct 3D architecture compared to PAMAM. This arrangement is imparted by the tris(2-aminoethyl)amine (TREN) monomer resulting in terminal ends that mimic tridentate ligands suitable for facial coordination, specifically to coordinate the fac-[M(CO)₃]⁺ core (where M = Tc, Re) (Fig. 1).^15,16 This subtle structural feature (apart from the amide/urea distinct backbone linkages) strongly impacts the coordination geometry of the nanoparticle shell.

Fig. 1 Comparison of the terminal branching in PAMAM and PURE dendrimers with relevant tridentate chelators, including the scorpionate type, suitable for a stable coordination of the fac-[M(CO)₃]⁺ (where M = Tc, Re) core.

Ovarian cancer is a silent and highly lethal disease, with only 15% of cases being diagnosed at early stages. Early detection at these stages increases the five-year survival rate by about 90%.¹⁷ Therefore, the development of early diagnosis protocols is crucial. In ovarian cancer, 80% of the epithelial carcinomas display a high concentration of folic acid receptor (FR-α), which correlates with the histological degree of the disease;¹⁸ thus, the use of FR-α as molecular target is a promising theranostic approach.¹⁹

Studies based on FR-α as an imaging molecular target, have been reported using PAMAM dendrimers as nanocarriers. Sand et al. studied a ^99mTc-labelled folate-PAMAM dendrimer modified with 2-hydrazinonicotinic acid (HYNIC) for the coordination of ^99mTc(V), with its tumour targeting ability proved by microSPECT imaging studies in tumour-bearing mice.²⁰ Also, Narmani et al. demonstrated that ^99mTc-PAMAM_G4 can target breast cancer cells.²¹ These nanoradiopharmaceuticals are prepared by multiple steps that include surface modification with the molecular targeting vector, attachment of a linker (e.g., polyethylene glycol, PEG) and the synthesis and conjugation of the adequate chelator for the final radiometal coordination.²² More recently, a non-targeted second-generation citric acid dendrimer conjugated with glutamine, was also successfully radiolabelled with ^99mTc, by reaction with ^99mTcO₄⁻ in the presence of stannous ion as a reducing agent.²³ All these examples of ^99mTc-labelled dendrimers were obtained using labelling strategies that involve well-defined coordination environments around the ^99mTc metal centre, which are difficult to apply to the congener ¹⁸⁶Re and ¹⁸⁸Re therapeutic radionuclides.

In a previous study, it was demonstrated that folate-targeted PURE dendrimers (PURE_G4-FA₂) show good molecular recognition of ovarian cancer cells and can be nano-formulated with selected drugs.²⁴ These results prompted further research in the potential of these nanoparticles in the field of nanoradiotheranostics. To achieve this goal, we have focused on the tricarbonyl approach based on ^99mTc(I) complexes that have gained considerable attention in the development of ^99mTc-by Alberto et al. who introduced a convenient and fully aqueous-based kit preparation method for the organometallic precursor fac-[^99mTc(OH₂)₃(CO)₃]⁺ directly from ^99mTcO₄⁻.^25–27 Similarly to the ^99mTc congeners used in imaging applications, Re(I) tricarbonyl complexes are attractive tools for developing ¹⁸⁶Re- and ¹⁸⁸Re-labelled radiotherapeutics.²⁸ Based on this tricarbonyl approach, the ^99mTc-radiolabelling of a generation four PURE dendrimer, with drug loading capacity and targeted with folic acid (PURE_G4-FA₂), as well as the characterization and in vitro stability evaluation of the resulting radioconjugates is reported.

The synthesis of PURE_G4-FA₂ followed our protocol.¹² The radiolabelling was performed by the addition of a freshly prepared fac-[^99mTc(H₂O)₃(CO)₃]⁺ saline solution (pH 6) to an aqueous solution of PURE_G4-FA₂, followed by a reaction at 100 °C for 30 min. The coordination of the dendrimer to ^99mTc certainly involved the substitution of the water molecules of the ^99mTc(I) tricarbonyl precursor, as previously proposed,²⁹ by the amines at the dendrimer surface, thus forming the radiocomplex ^99mTc-PURE_G4-FA₂ (Fig. 2). As a control, radiolabelling was performed for the non-targeted dendrimer (PURE_G4). The radioactive precursors and the novel radiolabelled polyurea dendrimers were characterized using instant thin-layer chromatography on silica gel impregnated strips (iTLC-SG) with methyl ethyl ketone (MEK) as the eluent. The complexes ^99mTc-PURE_G4 and ^99mTc-PURE_G4-FA₂ showed a retention factor (R_f) close to zero due to high polarity and molecular weight, while the precursors ^99mTcO₄⁻ and fac-[^99mTc(H₂O)₃(CO)₃]⁺ displayed a R_f value close to 1.0. The iTLC-SG analysis, before any purification steps, demonstrated that the radiochemical purity of ^99mTc-PURE_G4 and ^99mTc-PURE_G4-FA₂ were >95% (Fig. S1, ESI†). This result shows that the functionalization of the PURE_G4 dendrimer with the folic acid targeting units did not compromise its coordination capability towards the fac-[^99mTc(CO)₃]⁺ core.

Fig. 2 Coordination complex between the surface of the PURE_G4-FA₂ dendrimer (blue colour), conjugated with folic acid (orange colour), and ^99mTc.

The folate-targeted PURE_G4-FA₂ dendrimer and the corresponding ^99mTc-PURE_G4-FA₂ radiocomplex were analysed by RP-HPLC, using a reverse phase C18 column coupled to a UV detector and a radiometric detector, respectively. The HPLC chromatogram of the non-radioactive dendrimer, PURE_G4-FA₂, demonstrated major peaks between 13 and 16 min (Fig. S2, ESI†). The obtained HPLC profile reflects the statistical distribution of two folate molecules in the dendrimer surface, resulting in the formation of regioisomers. The ¹H NMR analysis of PURE_G4-FA₂ showed that on the average, two folic acid molecules had been conjugated to the dendrimer surface. The HPLC radiochromatogram of the labelled dendrimer, ^99mTc-PURE_G4-FA₂ demonstrated two main peaks (RTs around 14 and 18 minutes), and another prominent peak with a shorter retention time (approximately 6 min) likely corresponding to unbound fac-[^99mTc(H₂O)₃(CO)₃]⁺ (Fig. 3C).

Fig. 3 RP-HPLC radiochromatograms (γ-detection). Radioactive precursors pertechnetate [^99mTcO₄]⁻ (RT = 3.92 min) (A) and tri-carbonyl complex fac-[^99mTc(H₂O)₃(CO)₃]⁺ (RT = 7.62 min) (B). Dendrimer ^99mTc-PURE_G4-FA₂ before (RT = 5.64, 15.20, 16.52 min) (C) and after (RT = 14.93, 16.26 min) (D) purification. The samples were eluted using the conditions described in Table S1 (ESI†): water (0.1% trifluoroacetic acid, TFA) and methanol (0.1% TFA).

The discrepancy observed between the iTLC-SG or HPLC analysis of the radiochemical preparations of ^99mTc-PURE_G4-FA₂ might indicate the presence of fac-[^99mTc(CO)₃]⁺ not coordinated to the terminal triamine and thus loosely bound to the dendrimer. Eventually, it resisted the elution conditions employed in the iTLC-SG analysis but succumbed to the more aggressive elution conditions of the HPLC analysis (0.1% aqueous TFA solution and MeOH gradient). Thus, the preparations of ^99mTc-PURE_G4-FA₂ were purified by HPLC, as detailed in the ESI,† to remove the loosely bound or unreacted fac-[^99mTc(H₂O)(CO)₃]⁺. The fractions corresponding to radiolabelled PURE_G4-FA₂, presented RTs in the range 14 to 18 min (Fig. 3D). The radiolabelling yield assessed by iTLC before any purification was approximately 98% and about 45% after HPLC purification. The specific activity of the purified radiodendrimer was ca. 70 MBq mg⁻¹.

The UV-vis spectrum of the purified ^99mTc-PURE_G4-FA₂ dendrimer was also obtained (Fig. S3, ESI†). The presence of two main species suggests the labelling of two major dendrimer regioisomers.

Further study was conducted on the in vitro evaluation of ^99mTc-PURE_G4-FA₂ using HPLC-purified samples. the collected fractions with RTs in the range 14 to 18 min. The distribution of ^99mTc-PURE_G4-FA₂ between organic and aqueous phases (P_o/w) was studied using the n-octanol/0.1 M PBS pH 7.4 system. The radioconjugate showed a high hydrophilic character with a log P_o/w value of −1.56. To assess its in vitro stability, ^99mTc-PURE_G4-FA₂ was subjected to overnight incubation under physiological (saline solution) conditions and in the presence of cell culture media (RPMI and DMEM culture media supplemented and non-supplemented with 0.2% bovine serum albumin, BSA) (Fig. S4, ESI†). The radioactive dendrimer was found to be stable in all tested conditions as confirmed by iTLC-SG analysis.

In summary, this study demonstrated that PURE dendrimers have surface groups that behave as pseudo chelators suitable for a stable coordination of the fac-[^99mTc(CO)₃]⁺ core. To our knowledge, this is the first report of a dendrimer displaying such intrinsic feature that enables the mimicking of common tridentate N-donor chelators, namely, like those observed in tripodal or scorpionate type. These features, led to the development of a novel class of stable and highly hydrophilic radiolabelled dendrimers useful for diagnostic purposes. These promising results justify future studies into therapeutic potential, particularly with the therapeutic counterpart ¹⁸⁸Re to profit from the matched pair ^99mTc/¹⁸⁸Re within theranostic approaches. Also, the drug loading capacity of generation four PURE dendrimers (PURE_G4) foresees other therapeutic schemes that could significantly impact future nanoradiotheranostics.

We are grateful to Fundação para a Ciência e a Tecnologia (FC&T, Portugal) for funding (PTDC/MEC-ONC/29327/2017).

Conflicts of interest

There are no conflicts to declare.

Notes and references

1. E. Boros and A. B. Packard, Chem. Rev., 2019, 119, 870.
2. A. Ku, J. V. Facca, Z. Cai and R. M. Reilly, EJNMMI Radiopharm. Chem., 2019, 4, 27.
3. P. Martini, M. Pasquali, A. Boschi, L. Uccelli, M. Giganti and A. Duatti, Molecules, 2018, 23, 2039.
4. T. Esteves, C. Xavier, S. Gama, F. Mendes, P. D. Raposinho, F. Marques, A. Paulo, J. C. Pessoa, J. Rino, G. Viola and I. Santos, Org. Biomol. Chem., 2010, 8, 4104.
5. D. R. Melis, A. R. Burgoyne, M. Ooms and G. Gasser, RSC Med. Chem., 2022, 13, 217.
6. T. Xiao, D. Li, X. Shi and M. Shen, Macromol. Biosci., 2020, 20, 1900282.
7. L. Zhao, M. Zhu, Y. Li, Y. Xing and J. Zhao, Molecules, 2017, 22, 1350.
8. J. Zhu, L. Zhao, Y. Cheng, Z. Xiong, Y. Tang, M. Shen, J. Zhao, S. Jinhua and X. Shi, Nanoscale, 2015, 7, 18169.
9. Z. Xiong, M. Shen and X. Shi, Sci. China Mater., 2018, 61, 1387.
10. L. Farzin, S. Sheibani, M. E. Moassesi and M. Shamsipur, J. Biomed. Mater. Res., Part A, 2019, 107A, 251.
11. R. B. Restani, P. I. Morgado, M. P. Ribeiro, I. J. Correia, A. Aguiar-Ricardo and V. D. B. Bonifácio, Angew. Chem., Int. Ed., 2012, 51, 5162.
12. I. Santos, C. Ramos, C. Mendes, C. O. Sequeira, C. S. Tomé, D. G. H. Fernandes, P. Mota, R. F. Pires, D. Urso, A. Hipólito, A. M. M. Antunes, J. B. Vicente, S. A. Pereira, V. D. B. Bonifácio, S. C. Nunes and J. Serpa, Nutrients, 2019, 11, 2523.
13. P. Mota, R. F. Pires, J. Serpa and V. D. B. Bonifácio, Molecules, 2019, 24, 3111.
14. R. B. Restani, J. Conde, P. V. Baptista, M. T. Cidade, A. M. Bragança, J. Morgado, I. J. Correia, A. Aguiar-Ricardo and V. D. B. Bonifácio, RSC Adv., 2014, 4, 54872.
15. E. Boros and A. B. Packard, Chem. Rev., 2019, 119(2), 870.
16. F. Silva, C. Fernandes, M. P. C. Campello and A. Paulo, Polyhedron, 2017, 125, 186.
17. F. Leung, E. P. Diamandis and V. Kulasingam, Adv. Clin. Chem., 2014, 66, 25.
18. S. D. Weitman, R. H. Lark, L. R. Coney, D. W. Fort, V. Frasca, V. R. ZurawskiJr and B. A. Kamen, Cancer Res., 1992, 52, 3396.
19. Y.-S. Yi, Immune Netw., 2016, 16, 337.
20. M. Song, G. Guo, M. Gao, C. Shi, D. Xu, L. You, X. Wu, X. Su, R. Zhuang, W. Pan, T. Liu and X. Zhang, Chem. Biol. Drug Des., 2017, 89, 755.
21. A. Narmani, K. Yavari and J. Mohammadnejad, Colloids Surf., B, 2017, 159, 232.
22. N. Drude, L. Tienken and F. M. Mottaghy, Methods, 2017, 130, 14.
23. S. M. Ghoreishi, A. Khalaj, O. Sabzevari, L. Badrzadeh, P. Mohammadzadeh, S. S. Mousavi Motlagh, A. Bitarafan-Rajabi and M. S. Ardestani, Int. J. Nanomed., 2018, 13, 4671.
24. A. Cruz, P. Mota, C. Ramos, R. F. Pires, C. Mendes, J. Pais, S. C. Nunes, V. D. B. Bonifácio and J. Serpa, Antioxidants, 2020, 9, 133.
25. G. V. Sidorenko, A. E. Miroslavov and M. Yu Tyupina, Coord. Chem. Rev., 2023, 476, 214911.
26. R. Alberto, R. Schibli, A. Egli, A. P. Schubiger, U. Abram and T. A. Kaden, J. Am. Chem. Soc., 1998, 120(31), 7987.
27. R. Alberto, K. Ortner, N. Wheatley, R. Schibli and A. P. Schubiger, J. Am. Chem. Soc., 2001, 123(13), 3135.
28. R. Melis, A. R. Burgoyne, M. Ooms and G. Gasser, RSC Med. Chem., 2022, 13, 217.
29. S. Mundwiler, L. Candreia and R. Alberto, Chimia, 2003, 57, 193.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4cc00892h

‡ Present address: Laboratory of Physics of Materials and Emerging Technologies (LaPMET), Center of Physics and Engineering of Advanced Materials (CeFEMA), Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal.

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