Os2–Os4 Switch Controls DNA Knotting and Anticancer Activity

Abstract Dinuclear trihydroxido‐bridged osmium–arene complexes are inert and biologically inactive, but we show here that linking dihydroxido‐bridged OsII–arene fragments by a bridging di‐imine to form a metallacycle framework results in strong antiproliferative activity towards cancer cells and distinctive knotting of DNA. The shortened spacer length reduces biological activity and stability in solution towards decomposition to biologically inactive dimers. Significant differences in behavior toward plasmid DNA condensation are correlated with biological activity.

times the U equiv of the parent oxygen. The unlocated hydrogens were placed in the formula so as to give the correct calculated density. The P20 atom was disordered with two orientations about the phosphorus atom and the occupancy of the two components was linked to a free variable which refined to a ratio 77:23 major:minor. The minor component was refined isotropically.
Cell Cultures. A2780, A549 and H596 cells were cultured in RPMI 1640 cell culture medium supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine and 10% fetal bovine serum (all from Sigma).
Determination of IC50 Values. The concentrations of the osmium complexes that inhibit 50% of the proliferation of cancer cells were determined using the sulforhodamine B assay. [5] A2780 cells were seeded in 96-well plate (Falcon) at 5000 cells/well, and incubated for 48 h before the treatment. The complexes were solubilised in DMSO to provide 10 mM stock solutions. These were serially diluted by cell culture media to give concentrations four-fold greater than the final concentrations for the assay. The complexes diluted in cell culture media were added to the 96-well plate with cells in triplicate. The final DMSO concentration in each well was no more than 1% (v/v). The media containing the complexes were removed after 24 h. The cells were washed with phosphate buffered saline once and cell culture medium was added (150 µL/well). The cells were then allowed to grow for a further 72 h. The surviving cells were fixed by adding 150 µL/well of 50% (w/v) trichloroacetic acid and incubated for 1 h in the fridge (277 K). The plates were washed with tap water three times and dried under a flow of warm air, 0.4% sulforhodamine B solution (100 µL/well) was added, followed by washing with 1% acetic acid five times and drying under a flow of warm air. The dye was dissolved in 10 mM Tris buffer (200 µL/well). The absorbance of each well was determined using a Thermo Multiskan Ascent plate reader (Labsystems) at 540 nm. The absorbance of SRB in each well is directly proportional to the cell number. Then the absorbance was plotted against concentration and the IC50 determined by using Origin software.
Circular and Linear Dichroism (CD and LD). CD/LD spectra of ct-DNA (200 µM in bases) modified by the two tetranuclear osmium complexes were recorded at ambient temperature (ca. 293 K) on a CD Jasco J-815 spectropolarimeter adapted for LD spectroscopy. Complexes 1[PF 6 ] 4 and 2[PF 6 ] 4 are achiral and do not exhibit intrinsic CD signals.
DNA-metal complex adducts were prepared as follows: an appropriate volume of the osmium complex (stock solution was 400 M in 5% DMSO/95% sodium cacodylate buffer (1 mM), pH 7.2) was added to a 200 µM ct-DNA solution (dissolved in 1 mM sodium cacodylate buffer, 20 mM NaCl, pH 7.2) at different ratios, keeping the ct-DNA concentration constant in all the experiments. The ct-DNA base to metal complex mol ratios were 20:1, 10:1, 5:1 and 2.5:1). CD spectra were recorded at 293 K at 200 nm/min, data integration time 1 s and 4 accumulated scans, in a quartz cuvette with 0.5 cm pathlength.
Linear dichroism (LD) is directly related to the orientation of DNA relative to the helix axis. This technique was used to study the geometry of DNA adducts of cationic osmium tetramers 1 and 2. [6] Changes in the intensity of the negative LD band at 260 nm might result from either an increase in DNA flexibility or a shortening of the DNA by kinking, bending, compaction or aggregation. [7] The LD experiments were performed with a small volume quartz LD cell with a rotating outer quartz cylinder of internal diameter of 2.9 mm, and stationary inner cylinder, a 2.4 mm diameter quartz rod. [8] The rotation speed used in the experiments was 3000 rpm.
Atomic force microscopy (AFM). Topographic images of pBR322 plasmid DNA (4363 bp) with tetramers 1·[PF6]4 or 2·[PF6]4 were collected on a Veeco Multimode V instrument with a Nanoscope V controller operating in tapping-mode. All solutions were prepared with 18.2 MΩ⋅cm Milli-Q water filtered through 0.2 nm filters and centrifuged once (8000×g) to avoid salt deposits in order to provide a clear background for AFM images. Plasmid DNA stock solutions were kept at 253 K. MgCl2 was employed at a concentration of 0.9 mM in H2O to increase attachment of negatively-charged plasmid molecules to the mica surface. [9] Open-circular (OC) and linear (L) plasmid molecules were obtained after incubating a solution of supercoiled pBR322 plasmid DNA (2.5 g/mL) in HEPES buffer (2 mM, pH 7.2) at 333 K for 30 min. [10] DNA-osmium complex adducts were prepared by adding an appropriate volume of the Os(II) complex in HEPES buffer (DNA base to metal complex ratio 5:1 or 2.5:1 as indicated) to the incubated plasmid DNA (final plasmid concentration = 1.6 g/mL) in 2 mM HEPES buffer (pH 7.2). Samples were prepared for AFM by placing a drop (12 μl) of plasmid DNA solution (1.6 g plasmid/mL) or DNA-metal complex solution onto freshly cleaved mica (9.9 mm diameter, Agar Scientific Ltd.) pre-treated with MgCl2 (0.9 mM aqueous solution) for a firm adsorption of the negatively charged DNA. After adsorption for 5 min at room temperature, the samples were gently rinsed for 10 s with 18.2 MΩ⋅cm Milli-Q water directed onto the mica surface using a squeeze bottle. The samples were blow-dried with compressed dry N2 and the images were obtained in air at room temperature (ca. 293 K). The cantilevers used for imaging were Bruker FMV Si probes (k 2.8 N/m, nominal tip radius 10-12 nm). Two samples from each mixture were imaged in several places to obtain reliable measurements at two different DNA base-to-metal complex ratios. The control plasmid was also imaged for each experiment under the same experimental conditions. Images were processed using WSxM software. [11] Computation. All calculations were performed with the Gaussian 03 (G03) program [12] employing the DFT method, Becke three parameter hybrid functional and Lee-Yang-Parr's gradient corrected correlation functional (B3LYP). [13] The LanL2DZ basis set [14] and effective core potential were used for the Os atom and the 6-31G basis set [15] was used for all other atoms. Geometry optimizations of complexes 1·[PF6]4 and 2·[PF6]4 in the ground state were performed in the gas phase and the nature of all stationary points was confirmed by normal mode analysis. The conductor-like polarizable continuum model method (CPCM) [16] with acetone as solvent was used to calculate the electronic structure and the excited states of 1·[PF6]4 and 2·[PF6]4 in solution. One-hundred and twenty singlet excited states and the corresponding oscillator strengths were determined with a Time-dependent Density Functional Theory (TDDFT) [17] calculation. The electronic distribution and the localization of the singlet excited states were visualized using the electron density difference maps (EDDMs). [18] GaussSum 1.05 [19] was used for EDDMs calculations.

Discussion of AFM results:
1. AFM analysis of free pBR322 plasmid DNA: AFM images were recorded 30 min after mixing and again after 24 h of incubation at 310 K. Typical images of the control sample where the free pBR322 plasmid was deposited onto Mg 2+ -treated freshly-cleaved mica (needed for a firm adsorption of the negatively charged DNA) are shown in Figure 3  Reported apparent heights of double-stranded DNA deposited onto mica vary within the range 0.5-2.0 nm (AFM operating in air). [24] Images of the free plasmid were recorded as controls for all the samples under the same experimental conditions; these confirmed the stability of the plasmid after 24 h of incubation at 310 K.

AFM analysis of the interaction of plasmid DNA with tetramer 1:
Topographical images for the interaction between cationic complexes 1 and 2 with pBR322 plasmid DNA (4363 bp) are shown in Figure S14 and Figure 3 (main script). The Os II complexes were added to solutions of pBR322 DNA plasmid at a DNA base: Os 4 ratio of 5:1.The image recorded 30 min after mixing pBR322 plasmid with tetramer 1·[PF 6 ] 4 (the more biologically-active complex) resulted in only a few particles of buffer salts observed on the mica surface ( Fig.  S14a) with an average height of 1.4  0.5 nm (n=22), comparable to those observed in the control samples (Figs. 3a, b, main script). However, no plasmids could be detected on any of the analyzed samples. These results indicate that the DNA adduct formed by the OC or L plasmids with complex 1 (4+ charge) was less negatively charged than the pristine DNA, hence could not readily adsorb onto the positively-charged substrate and thus could not be observed by AFM. The incubation of the solution of the DNA: 1 adduct for 24 h at 310 K gave rise to an identical situation in which only buffer salt particles were observed (Fig. S14b) with a height (1.7  0.3 nm; n=22) similar to that of the particles before and after the incubation period of 24 h (Figs. 3a, b, main script). Surprisingly, it was however possible to image a large network of cross-linked plasmids close to a step edge of the surface on the same sample (Figs. S14c, d). The network appeared to be directly anchored to the step and it was not possible to find any other similar structure on the terraces of the Mg 2+ -treated mica surface. Such a special situation might be due to a different termination or surface charge density of the step with respect to the planar regions of the substrate. The analysis of segments of the DNA strands involved in the network (Fig.  S14c, d) resulted in an average height of 2.2  0.3 nm (n=26), which is ca. three times larger than the value observed for the OC and L forms of the free plasmid (Figs. 3a, b, main script). This is most probably due to the binding of the positively-charged tetramer 1 to the pBR322 DNA plasmid which reduces its electrostatic binding to the substrate and thus increases its height when measured by AFM.

AFM analysis of the interaction of plasmid DNA with tetramer 2:
The images of the pBR322 plasmid DNA changed drastically upon addition of the less biologically-active complex 2·[PF 6 ] 4 . Interaction with tetramer 2·[PF 6 ] 4 gave rise to the formation of aggregates of plasmid DNA which did stick onto the substrate and appeared as plasmid loops (maximum loop length along the longitudinal DNA axis of ca. 400 nm) emerging from a condensed nucleus, probably generated by the linking of several DNA strands mediated by complex 2 (see Fig. 3c, main script). The height of the plasmid DNA loops was 1.2  0.2 nm (n=19), i.e. larger than the free plasmid but smaller than the DNA: 1 adducts described in the previous section, while the condensed nuclei displayed a height of 5.8  1.2 nm (n=7). Additionally, plasmid molecules with an OC and L structure were observed on the surface (Fig. 3c, main script) with a height of only 0.70  0.06 nm (n=14). These latter molecules were assigned to unreacted free DNA plasmids.
The lateral extension of the plasmid loops became larger and the loops length along the DNA axis increased to values ranging from 600 to 900 nm after incubating the sample for 24 h at 310 K (Fig. 3d, main script). This change can be associated with the cleavage of the tetranuclear structure 2 during the incubation period (loss of bridging ligands), thus partially releasing plasmids from the central core.
The observations that, unlike the aggregates formed with complex 1, the DNA adducts with complex 2 are strongly attached to the Mg 2+ -treated mica and their height is smaller than that of the DNA-1 adducts (i.e. they are subject to a stronger height reduction induced by electrostatic interactions with the substrate), both suggest a larger negative charge density of DNA-2 with respect to DNA-1. Given that both complexes 1 and 2 have a 4+ charge, the difference in charge density is most probably due to the smaller number of bound molecules 2 with respect to 1, caused by the more facile decomposition of 2.
Experiments carried out at a lower DNA base:tetramer 2 mol ratio (2.5:1) showed the formation of similar aggregates with loops emerging from a central condensed nucleus together with OC structures of free plasmids (Fig. S14f). With respect to the 5:1 ratio, the overall surface density of the DNA aggregates increased and the height of the DNA loops was marginally larger, 1.4  0.3 nm (n=18), but still comparable to what observed in Fig. 3c (main  script). Both observations confirm a higher linear density of 2 bound to the DNA strands resulting in the formation of more aggregates with the same shape but a slightly increased height (Fig. S14f) when using a lower DNA base:tetramer 2 ratio. Additionally, more plasmid molecules are attached to the central cores and the extension of some of the aggregate loops is larger (maximum length of ca. 1.4 m along the DNA axis) than observed for the 5:1 ratio.

AFM analysis of the interaction of DNA with pre-incubated tetramers 1 and 2:
AFM images of the adducts of DNA with pre-incubated 1·[PF 6 ] 4 or 2·[PF 6 ] 4 are shown in Figure  S15 at a 30 min time-point. A very low density of plasmids, in short L and OC forms, was found on the mica surface after treatment with pre-incubated 1·[PF 6 ] 4 (Fig. S15a), similarly to that observed without tetramer incubation (Fig. S14a, b). This is consistent with the 1 H NMR studies that showed that after 24 h of incubation only ca. 37% of the 1·[PF 6 ] 4 tetramers decompose (Fig. S9). In fact, the majority of the remaining intact tetranuclear molecules 1 can still bind effectively to the DNA, decreasing its negative charge and therefore preventing its binding to the Mg 2+ -treated mica substrate. As a consequence, the few plasmids attached to the surface (Fig. S15a) should correspond to DNA molecules that did not interact with tetramer 1. This was proven by analyzing their average heights -0.76  0.09 nm (n=3) for OC and 0.73  0.07 nm (n=9) for L plasmidswhich are compatible with the heights measured for the corresponding free DNA forms (Figs. 3a-b, main script).
The conformation of the plasmids did not change significantly after treatment with incubated tetramer 2·[PF6]4 (Fig S15b), for which AFM images closely resemble those of the free plasmid control samples (Fig. 3a, main script). This is consistent with the fact that tetramer 2·[PF6]4 decomposes completely to [Os2(η 6 -p-cym)2( 2 -OH)3] + dimers and free prz linkers after 24 h, as shown by 1 H NMR studies (Figs. 2 and S9), thus confirming the lack of a strong interaction of this biologically-inactive species with DNA. As expected, the height of the DNA plasmids in Fig S15b is also very close to that of the control samples with values of 0.81  0.08 nm (n= 16) and 0.8  0.1 nm (n= 16) measured for the OC and the L forms, respectively. We noticed that a few of the OC plasmids appeared to be partially coiled. These data might indicate a certain degree of interaction between DNA and the monocationic Os II hydroxido-bridged dimers, although such interaction appears to occur to a lesser extent than in the case of Os II tetramers 1 and 2, which yielded DNA adducts at least twice as high.