A trackable trinuclear platinum complex for breast cancer treatment

Abstract

Cancer remains a leading cause of death, with triple-negative breast cancer (TNBC) being particularly significant due to limited treatment options. As such, there is interest in anticancer polynuclear platinum(II) complexes, attributed to their unique DNA-binding modes and potential against therapy-resistant cancer phenotypes. However, a persistent challenge with polynuclear compounds is their lack of cellular trackability, hindering their effectiveness and monitoring in clinical settings. Here, we report the preparation of a new azide-appended trinuclear platinum complex, N3-TriplatinNC, and characterize its DNA-targeting, cytotoxicity, and topoisomerase relaxation properties from the nanoscale to the macroscale. Using single-molecule biophysics and in-liquid atomic force microscopy, N3-TriplatinNC was identified as a powerful DNA recognition agent with remarkable potential towards the TNBC cell line, MDA-MB-231. Installation of the azide handle on the polynuclear complex was achieved using a first-in-class approach to produce a complex that retained analogous biological activity to the parent TriplatinNC. Importantly, the azide handle facilitates in situ click chemistry for tracking cellular localization, with subsequent xenograft studies demonstrating in vivo antitumoural potential.

Graphical Abstract

Figure 1.

(A) Worldwide FDA clinically approved platinum(II) drugs cisplatin, carboplatin, and oxaliplatin along with (B) prototypical polynuclear platinum(II) compounds (PPCs) BBR3464 and TriplatinNC. (C) X-ray structures showing the noncovalent DNA-binding modes of TriplatinNC: groove spanning and backbone tracking (PDB: 2DYW). (D) Synthetic route for N₃-TriplatinNC: in route (i) the preparation of N₃-MonoplatinNC is shown while in route (ii) the isolation of the mono-activated di-nuclear platinum(II) 1,1/t,t, complex was achieved. Combining both precursors in step (iii) generates the mono-activated triplatinum(II) precursor to the target N₃-TriplatinNC formed in step (iv). Synthetic route for the preparation of the 1-azido-6-amino hexane linker is shown (inset).

Figure 2.

(A) Summary table of binding data for TriplatinNC and N₃-TriplatinNC along with ethidium bromide displacement assay results for calf thymus, poly A-T and poly G-C DNA. (B) Representative molecular dynamics (MD) structures of TriplatinNC and N₃-TriplatinNC bound to the Dickerson–Drew dodecamer (DDD), obtained via cluster analysis. Both PPCs exhibit similar binding modes, including phosphate clamp interactions with OP1 oxygen atoms and groove-spanning. Importantly, the azide handle in N₃-TriplatinNC positions itself away from the double-helix, enabling post-binding CuAAC. (C) Schematic of the nanofluidic experimental setup. The nanofluidic device with loading reservoirs connecting the two microchannels and the nanochannels (500 μm length, 150 nm width and 100 nm height) spanning between the two microchannels. A representative fluorescence microscopy image is shown from which kymographs can be extracted for each DNA molecule. Kymographs from tens of DNA molecules are used to obtain an intensity versus extension plot, showing the extension and the intensity profile that reflects the underlying DNA sequence. (D) Scatter plot showing DNA extensions for λ-DNA molecules (control) λ-DNA molecules exposed to 0.25, 0.5, and 1 μM concentrations of N₃-TriplatinNC and TriplatinNC. The black lines represent the median DNA extension. (E) Average intensity profile of λ-DNA labelled with YOYO dye and 0.25, 0.5, and 1 μM N₃-TriplatinNC (left) and TriplatinNC (right) compared with λ-DNA labelled with netropsin-YOYO one-step competitive binding (grey). (F) Representative concentric plots from tens of DNA molecules to reveal if the N₃-TriplatinNC/TriplatinNC – YOYO binding creates highly similar intensity patterns (N₃-TriplatinNC) or random intensity patterns (TriplatinNC) across different DNA molecules. The scale bar is 5 μm. (G) Representative image and kymograph for control (untreated) DNA along with 1.5, 2.5, and 5 μM N₃-TriplatinNC (left) and TriplatinNC (right). (H) Representative images and kymographs displaying clustered and branched DNA molecules observed in experiments with N₃-TriplatinNC and DNA concatemers with bright-dark intensity patterns observed in experiments with TriplatinNC. The scale bar is 5 μm.

Figure 3.

MST results for N₃-TriplatinNC. (A) MST results showing normalized MST signal and initial fluorescence using the F-TP (GC-rich) hairpin. (B) Normalized MST signal and initial fluorescence using the F-D6aH (AT-rich) ranging from 0.075 μM to 10 μM of N₃-TriplatinNC DNA hairpins tagged with the Cy5 fluorophore.

Figure 4.

(A, D) Topoisomerase IA relaxtion assay monitored by agarose gel electrophoresis assay as consequence of pre-exposure of pUC19 (400 ng) to either N₃-TriplatinNC or TriplatinNC at the indicated concentration, followed by addition of the enzyme. (B, E) The inhibition of DNA unwinding was probed by high resolution in-liquid AFM colour-coded to specific concentrations used in electrophoretic experiments performed in A and D: untreated control (grey), 0.75 μM N₃-TriplatinNC or 1.0 μM TriplatinNC (light pink), 1 μM N₃-TriplatinNC (pink), 2.50 μM N₃-TriplatinNC or 5.0 μM TriplatinNC (dark pink), and 7.5 μM TriplatinNC (purple). Scale bars: 250 nm, Z-scale: −3 to 4 nm. (C, F) Quantitative analysis of DNA condensation was performed by measuring the smallest bounding area of DNA complexes observed in AFM images using TopoStats [28]. N values are as follows – Control: 113, N₃-TriplatinNC 0.75 μM: 143, 1.0 μM: 42, 2.5 μM: 211, 5.0 μM: 5, TriplatinNC 1.0 μM: 72, 2.5 μM: 25, 5.0 μM: 207, 7.5 μM: 21.

Figure 5.

Intracellular fluorescent labelling of N₃-TriplatinNC through CuAAC chemistry to examine cellular uptake and localization. Confocal microscope images below on 63× oil immersion lens of fixed MDA-MB-231 cells exposed to 5 and 10 μM for 48 and 24 h, respectively, and clicked to alkyne 488 dye (green). Nuclei were stained with NucBlue Fixed (blue), mitochondria were stained with MitoTracker Deep Red (magenta) and nucleoli were visualized by immunofluorescent staining of NPM (red). Scale bars indicate 10 μm (overlay, bottom right corner).

Figure 6.

(A) In vitro cytotoxicity data for N₃-TriplatinNC, TriplatinNC, cisplatin, and carboplatin toward selected breast cancer cell types. (B) NCI-60 LC₅₀ comparing N₃-TriplatinNC versus studied anticancer agents (note: metallodrugs cisplatin, carboplatin, oxaliplatin, bleomycin and arsenic trioxide are marked with an asterisk). (C) Relative tumour volume in mice bearing MDA-MB-231 breast cancer xenografts, highlighting N₃-TriplatinNC’s lower dose (5 mg/kg) to achieve similar effect to carboplatin (40 mg/kg).