Unusual enantioselective cytoplasm-to-nucleus translocation and photosensitization of the chiral Ru(II) cationic complex via simple ion-pairing with lipophilic weak acid counter-anions

Xi-Juan Chao; Chun-Hua Huang; Miao Tang; Zhu-Ying Yan; Rong Huang; Yan Li; Ben-Zhan Zhu

doi:10.1093/nar/gkad155

. 2023 Mar 20;51(7):3041–3054. doi: 10.1093/nar/gkad155

Unusual enantioselective cytoplasm-to-nucleus translocation and photosensitization of the chiral Ru(II) cationic complex via simple ion-pairing with lipophilic weak acid counter-anions

Xi-Juan Chao ^1,^2,², Chun-Hua Huang ^3,², Miao Tang ^4,², Zhu-Ying Yan ⁵, Rong Huang ⁶, Yan Li ⁷, Ben-Zhan Zhu ^8,^9,^✉

¹ State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences; University of Chinese Academy of Sciences, The Chinese Academy of Sciences, Beijing 100085, P.R. China

² Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000, China

³ State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences; University of Chinese Academy of Sciences, The Chinese Academy of Sciences, Beijing 100085, P.R. China

⁴ State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences; University of Chinese Academy of Sciences, The Chinese Academy of Sciences, Beijing 100085, P.R. China

⁵ State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences; University of Chinese Academy of Sciences, The Chinese Academy of Sciences, Beijing 100085, P.R. China

⁶ State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences; University of Chinese Academy of Sciences, The Chinese Academy of Sciences, Beijing 100085, P.R. China

⁷ State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences; University of Chinese Academy of Sciences, The Chinese Academy of Sciences, Beijing 100085, P.R. China

⁸ State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences; University of Chinese Academy of Sciences, The Chinese Academy of Sciences, Beijing 100085, P.R. China

⁹ Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA

^✉

To whom correspondence should be addressed. Tel: +86 10 62849030; Fax: +86 10 62923563; Email: bzhu@rcees.ac.cn

The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.

PMCID: PMC10123113 PMID: 36938880

Abstract

Targeted and enantioselective delivery of chiral diagnostic-probes and therapeutics into specific compartments inside cells is of utmost importance in the improvement of disease detection and treatment. The classical DNA ‘light-switch’ ruthenium(II)-polypyridyl complex, [Ru(DIP)₂(dppz)]Cl₂ (DIP = 4,7-diphenyl-1,10-phenanthroline, dppz = dipyridophenazine) has been shown to be accumulated only in the cytoplasm and membrane, but excluded from its intended nuclear DNA target. In this study, the cationic [Ru(DIP)₂(dppz)]²⁺ is found to be redirected into live-cell nucleus in the presence of lipophilic 3,5-dichlorophenolate or flufenamate counter-anions via ion-pairing mechanism, while maintaining its original DNA recognition characteristics. Interestingly and unexpectedly, further studies show that only the Δ-enantiomer is selectively translocated into nucleus while the Λ-enantiomer remains trapped in cytoplasm, which is found to be mainly due to their differential enantioselective binding affinities with cytoplasmic proteins and nuclear DNA. More importantly, only the nucleus-relocalized Δ-enantiomer can induce obvious DNA damage and cell apoptosis upon prolonged visible-light irradiation. Thus, the use of Δ-enantiomer can significantly reduce the dosage needed for maximal treatment effect. This represents the first report of enantioselective targeting and photosensitization of classical Ru(II) complex via simple ion-pairing with suitable weak acid counter-anions, which opens new opportunities for more effective enantioselective cancer treatment.

Graphical Abstract

INTRODUCTION

The design and development of organometallic transition metal-based complexes capable of targeting nucleus have been increasing ever since some Pt complexes as clinical anti-cancer drugs by forming adduct with DNA (1–17). For ruthenium complexes, they have raised a lot of interest due to their ‘light switch’ effect of being brightly luminescent when intercalated into DNA and virtually non-emissive in aqueous solution (18,19), which is advantageous for use by fluorescence microscopy. In addition to their strong DNA binding ability, their extraordinary photo-physical properties, such as red emission wavelengths, intense luminescence, large Stokes shifts, and good photo-stability (19–21), make them potentially invaluable in the fields of bioassay, bioimaging, photosensitizing, solar energy conversion and photocatalysis (22–30). Therefore, for most of ruthenium complexes, a growing number of biological applications have been explored in the last few years, however, ruthenium complexes capable of targeting nuclear DNA are still rare due to the low lipophilicity of most ruthenium complexes (29–31).

It has been shown that the cellular uptake efficiency of the classical Ru-based DNA-binding [Ru(N^{^}N)₂(dppz)]Cl₂ was dependent on the nature of the ancillary ligands, where more hydrophobic ligands such as 4,7-diphenyl-1,10-phenanthroline (DIP) promoted increased rate of cellular uptake (19). The later work confirmed that the cellular uptake for the most lipophilic [Ru(DIP)₂(dppz)]Cl₂ was an energy-independent process, and passive diffusion was suggested as the potential mechanism (31). However, the accumulation of [Ru(DIP)₂(dppz)]Cl₂ was found to be restricted only in the cytoplasm, without reaching its preferable nuclear DNA target.

Lipophilic drugs are supposed to diffuse readily through cell membranes, thereby leading to their increased cellular uptake. For [Ru(DIP)₂(dppz)]Cl₂ complex that accumulates mainly in the cytoplasm, it is probably because it is trapped by cytosolic components such as proteins and membrane structures, leading to the formation of tightly-bound cytosolic component-[Ru(DIP)₂(dppz)]Cl₂ adducts and thus unable to reach nucleus. Based on our previous researches on the synergistic chemical and biological effects between organic and inorganic compounds (32–38), and recent studies on preferential nuclear delivery of the hydrophilic and cell-impermeable ruthenium complexes such as the typical [Ru(bpy)₂(dppz)]Cl₂ (bpy = 2,2’-bipyridyl) and [Ru(phen)₂(dppz)]Cl₂ (phen = 1,10-phenanthroline) via ion-pairing with lipophilic weak acid counter-anions (39–43), we speculated that the positively charged [Ru(DIP)₂(dppz)]²⁺ should also be neutralized by suitable counter-anions that may compete with the cytosolic components via forming non-charged neutral and relatively stable ion-pairing complex, which might further enable [Ru(DIP)₂(dppz)]Cl₂ to move across cytosol and diffuse directly into nucleus. To test the above hypothesis, a series of weak hydrophobic acids were investigated, and we found that both chlorinated phenols and the widely used non-steroid anti-inflammatory drugs (NSAIDs, fenamic acids) could indeed redirect rac-[Ru(DIP)₂(dppz)]Cl₂ from cytoplasm to live-cell nucleus. More interestingly, further studies with pure enantiomers (Δ- and Λ-) of [Ru(DIP)₂(dppz)]Cl₂ (Figure 1A) found that only the Δ-enantiomer can be translocated into nucleus while the Λ-enantiomer remains trapped in cytoplasm, which is quite different from previous studies on the enantiomers of ruthenium complexes in cells (44–47). As the vital regulator of cell proliferation and growth, nucleus is always considered to be the most sensitive and effective target for most of the therapeutic drugs, especially for treatment of malignant cancers (48–50). A further study showed that the Δ-enantiomer can efficiently induce nucleic acid damage and cell apoptosis upon visible-light irradiation, while the Λ-enantiomer showed a much weaker cancer cell killing ability. Such enantioselective distribution and enantio-specific induction of apoptosis will allow us to employ a small dosage of active drugs for a maximized killing effect.

Figure 1. — [Ru(DIP)₂(dppz)]²⁺ is redirected into nucleus by 3,5-DCP-OH in live cells. (A) Chemical structures of the two positively-charged chiral forms (Δ- and Λ-) of [Ru(DIP)₂(dppz)]Cl₂ (Ru), and 3,5-dichlorophenol (**3,5-DCP-OH**). (**B, C**) LCSM images (B) and flow cytometry analysis (C) of HeLa cells incubated with Ru (30 μM) in the absence or presence of 3,5-DCP-OH (60 μM, 1 h) in complete culture medium containing 10% FBS. Dead cell stain To-Pro-3 (1 μM) was used to exclude or detect dead cells. (D) Co-staining experiment with the membrane-permeable DNA stain Hoechst33342 by confocal microscopy confirmed the nuclear uptake of Ru by 3,5-DCP-OH. Quantitation by line plots shows nuclear uptake of intense luminescence in the nucleus for cells treated with Ru/3,5-DCP-OH.

MATERIALS AND METHODS

Chemicals

The [Ru(DIP)₂(dppz)]Cl₂ complexes were synthesized according to references (51–53). Chlorinated phenols and fenamic acid NSAIDs drugs were purchased from Sigma. To-Pro-3 (TP3) was purchased from Invitrogen. Oligomycin, 2-deoxy-d-glucose, and all other inhibitors were purchased from Sigma-Aldrich.

Preparation of chlorinated phenols solutions

All chlorinated phenols were prepared by dissolving in 0.1 M NaOH solution.

Cell culture

Cells were cultured in DMEM or RPMI 1640 medium with 10% fetal bovine serum (FBS), all with 1% penicillin-streptomycin, at 37°C under a 5% CO₂ atmosphere.

Flow cytometry

HeLa cells were detached from monolayer culture with trypsin, re-suspended in medium with serum, and diluted to 1 × 10⁶ cells/ml. The [Ru(DIP)₂(dppz)]Cl₂ complex (30 μM) (dissolved in DMSO) and 3,5-DCP-OH (50–100 μM, 1 h) or FFA (100 μM, 3 h) were added to the cells, successively. The cells were isolated by centrifugation and rinsed with cold PBS (phosphate buffered saline) for 3 times, dead cells were stained with To-Pro-3 (1 μM), which enters cells with compromised membranes. Temperature dependence studies used cells that had been cooled at 4°C and incubated with 30 μM Ru(II) complexes at 4°C for 1 h in the presence of 3,5-DCP-OH. For inhibitors, cells were previously treated with NH₄Cl, chlorpromazine and colchicine at the stated concentrations for 30 min, and then incubated with Ru(II) complexes/3,5-DCP-OH for 1 h before imaging. Flow cytometry was performed on a BD FACS Caliber using ∼20 000 cells per sample. Luminescence data were obtained by excitation at 488 nm with emission at 600–630 nm for Ru(II) complexes and excitation at 633 nm and emission at 650–670 nm for To-Pro-3.

Microscopic study

After incubated with Ru(II) complexes and chlorinated phenols, cells were rinsed with PBS for 3 times, and were luminescently imaged on a CLSM using 40× oli-immersion lens for slide imaging, or on a super-resolution SIM using 100× oli-immersion lens. The imaging was excited at 488 nm and emission monitored at 600–630 nm. All cells were washed with PBS before imaging. Microscopy was performed on a Leica TCS SP5 CLSM or a super-resolution microscopy Delta Vision OMX SIM, respectively. Live cells were distinguished by their low To-Pro-3 emission with excitation at 633 nm and observation at 650–670 nm. For TEM, cells were co-incubated with [Ru(DIP)₂(dppz)]Cl₂ (30 μM)/3,5-DCP-OH (100 μM, 1 h), then fixed with 2.5% glutaraldehyde and dehydrated using ethanol. TEM samples were sectioned in Araldite resin by microtome (Lecia EM UC6) and examined on aH-7650B instrument operating at 80 kV equipped.

Partitioning study

Organic solvents/aqueous (Tris–HCl buffer,10 mM, pH 7.4) phase partition for Ru(II) complexes in the presence or absence of chlorinated phenols and fenamic acids, were conducted using the ‘shake-flask’ method, with the concentration in each phase determined by UV–vis absorbance(Beckman DU-800).

ICP-MS analysis

Exponentially growing HeLa cells were plated at a density of 2.5 × 10⁶ cells/ml in DMEM medium. [Ru(DIP)₂(dppz)]Cl₂ (30 μM)/3,5-DCP-OH (100 μM) were added to the culture medium and co-incubated for varying amounts of time at 37°C with 5% CO₂. After digestion in trypsin−EDTA solution, HeLa cells were counted and the nuclear and cytoplasm fractions of cells were separated by a nuclear and cytoplasmic protein extraction kit (Pierce). The samples were digested in 60% HNO₃ at room temperature overnight. Each sample was diluted with Milli-Q H₂O to obtain 2% HNO₃ solutions. The standards for calibration were freshly prepared by diluting a RuCl₃ stock solution with 2% HNO₃ in Milli-Q H₂O. The ruthenium concentration in each part was determined by ICP-MS.

DNA agarose gel electrophoresis

The DNA agarose gel electrophoresis experiments were performed at room temperature. [Ru(DIP)₂(dppz)]Cl₂ with different concentration of 3,5-DCP-OH or FFA was added to 20 μl of Tris–HCl buffer with 5 μg/ml plasmid PBR323 to get a final concentration of 50 μM Ru(II). After incubation for 30 min, all samples were placed in ice to end the reaction and then loaded onto 1% agarose gels containing ethidium bromide and electrophoresed for 40 min at 80 V/cm.

Separation and purification of the two enantiomers

The two enantiomers of [Ru(DIP)₂(dppz)]Cl² were separated using a CYCLOBOND I 2000 DMP high-performance liquid chromatography (HPLC) column (250 × 21.2 mm, 5 μm, Sigma) on a Agilent 1260 HPLC. Samples were eluted at a flow rate of 20 ml/min with an isocratic solvent composition of 5/2/93 (v/v/v) 20 mM KPF6(aq.):CH3CN:EtOH. The fractions were monitored at 275 nm. The Δ-enantiomer eluted first, followed by the Λ-isomer (Supplementary Figure S1). The fractions of the two enantiomers were collected manually with heart cutting. Then, the collected solution was evaporated under reduced pressure and re-dissolved in acetone to a saturated solution. The chloride salt of the compound was precipitated from the saturated solution by the addition of an acetone solution of tetra-n-butylammonium chloride (1 M). The solids were filtered off, washed with acetone and diethyl ether, dried (34). Assignment of the two fractions was confirmed by circular dichroism (Supplementary Figure S2) (54). The optical purity of enantiomers was estimated as 98% ee for Δ-enantiomer and 98% ee for Λ-enantiomer by HPLC analysis (250 × 4.6 mm, 5 μm, CYCLOBOND I 2000 DMP, Sigma) (Supplementary Figure S1).

Cultivation of crystals

100 μl [Ru(II)(DIP)₃]Cl₂ (50 mM, in acetonitrile) and 100 μl 2,4,6-TCP (100 mM, in 200 mM NaOH) were mixed in the PB buffer (pH 7.4).After 10 min of vigorous stirring, the resultant brown precipitate was filtered, then washed with 4:1 water/acetonitrile and dried in vacuum. The clean precipitate was then re-dissolved in 3:1 1, 4-dioxane/H₂O. After 3 days, single crystals suitable for X-ray analysis were grown at room temperature.

Single crystal X-ray diffraction

The synthetic complex was determined by single crystal X-ray diffraction. Data collection was performed on an Agilent Gemini A Ultra diffractometer, using graphite-monochromated Cu Kα radiation (λ = 1.5418 Å) for the complex. The determination of crystal class and unit cell parameters was carried out by CrysAlis (Agilent 2011) program package, raw frame data was processed using CrysAlis (Agilent 2011), and the structures were solved by use of SHELX97 program and refined by full-matrix least-squares on F values.

Phosphatidylserine translocation

After co-treatment with [Ru(DIP)₂(dppz)]Cl₂ (30 μM)/3,5-DCP-OH (100 μM) for 1 h or [Ru(DIP)₂(dppz)]Cl₂ (30 μM)/FFA (100 μM) for 3 h, cells were rinsed with PBS for 3 times and refilled with new fresh medium, then cells were exposed either to light irradiation or not for 30 min. After being incubated for another 2–3 h, cells were stained with Annexin-V-flourescein isothiocyanate (FITC) (10 μM) and To-Pro-3 (0.5 μM) for 15 min. Fluorescence was observed by confocal with 488 nm excitation and 525 nm emission for Annexin-V FITC.

Immunofluorescence

After co-treatment with Ru (30 μM)/3,5-DCP-OH (100 μM) for 1 h or Ru (30 μM)/FFA (100 μM) for 3 h, cells were rinsed with PBS for 3 times and refilled with new fresh medium, then cells were exposed either to light irradiation or not for 30 min. After being incubated for another hours, cells were fixed with 100% formaldehyde for 5 min at room temperature. Cells were then permeabilized with 0.5% Triton X-100 for 10 min at room temperature, and blocked with 1% bovine serum albumin (BSA) for 30 min. After being washed with PBS, they were incubated with primary antibody phosphate-H2AX for 1 h at room temperature, then with secondary antibody conjugated with Alexflour488 for 1 h at room temperature. To confirm the distribution of foci for gamma-H2AX, the nucleus was stained with Hoechst33342 (1 μg/mL). Images were acquired through confocal.

RESULTS AND DISCUSSION

Redirecting the positively-charged DNA ‘light-switching’ rac-[Ru(DIP)₂(dppz)]Cl₂ from cytoplasm to live-cell nuclei by chlorinated phenols

It has been shown that the lipophilic rac-[Ru(DIP)₂(dppz)]Cl₂ (for simplicity, [Ru(DIP)₂(dppz)]Cl₂ will be used to represent rac-[Ru(DIP)₂(dppz)]Cl₂ thereafter) was able to enter HeLa cells due to its relatively high lipophilicity, but only limited in the cytoplasm without reaching its intended target, the nucleus (18). Similar results were observed in this research (Figure 1B). However, in the presence of 19 chlorinated phenols, [Ru(DIP)₂(dppz)]Cl₂ was found to be readily redirected from cytoplasm into the nucleus, as illustrated by confocal laser scanning microscopy (CLSM) (Figure 1B and Supplementary Figure S3). Among all chlorinated phenols tested, 3,5-dichlorophenol (3,5-DCP-OH) (Figure 1A) was found to be the most effective in facilitating nuclear uptake of [Ru(DIP)₂(dppz)]Cl₂, so 3,5-DCP-OH was chosen as a model compound for further detailed studies. Besides the enhanced nuclear uptake, a higher whole cellular uptake was also observed with addition of 3,5-DCP-OH compared with that of free [Ru(DIP)₂(dppz)]Cl₂ as illustrated by luminescence with flow cytometry (Figure 1C). It should be noted that this is in clear contrast with our previous findings on [Ru(bpy)₂(dppz)]Cl₂ (bpy = 2.2’-bipyridyl), where only a very limited number of highly chlorinated phenols such as 2,3,4,5-TeCP (TeCP: tetrachlorophenol, which is most effective), 3,4,5-TCP (TCP: trichlorophenol) and PCP (pentachlorophenol) at much higher concentrations (300 μM) (Ru:PCPs = 1:3) were effective in facilitating nuclear uptake (Supplementary Figure S4) (39). This is probably due to the much lower lipophilicity of [Ru(bpy)₂(dppz)]Cl₂ (log P_o/w = –2.50), while the log P_o/w value for [Ru(DIP)₂(dppz)]Cl₂ complex is 1.3 (18).

The nuclear uptake of [Ru(DIP)₂(dppz)]Cl₂ was further confirmed by co-staining with the classic live-cell DNA dye Hoechst33342 and tracked them by both CLSM and three dimensional structured illumination microscopy (SIM) with higher spatial resolution (Figure 1D and Supplementary Figure S5A). The blue emission from Hoechst33342 and red emission from [Ru(DIP)₂(dppz)]²⁺/3,5-DCP-O⁻ in the nucleus were almost completely overlaid, indicating the nuclear DNA stain of [Ru(DIP)₂(dppz)]Cl₂ in the presence of 3,5-DCP-OH; meanwhile red luminescence was still observed in the cytoplasm. In contrast, cells treated with [Ru(DIP)₂(dppz)]Cl₂ alone showed only cytoplasmic luminescence. Quantitation by line plots does show nuclear uptake by intense luminescence in the nucleus for cells treated with [Ru(DIP)₂(dppz)]Cl₂ in the presence of 3,5-DCP-OH, even though with cytoplasmic luminescence still being observed (that should be mainly from Λ-enantiomers, vide infra as discussed below) (Figure 1D). Besides, nuclear uptake was further confirmed by both transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS) measurements as shown in Supplementary Figures S5B and S6.

3,5-DCP-OH, used as a disinfectant and herbicide, is a well-known and well characterized toxin (55). So, we first speculated whether it was the toxicity of 3,5-DCP-OH that induced cell damage, thus leading to the nuclear uptake of [Ru(DIP)₂(dppz)]Cl₂. To determine the health of cells after treatments, a series of experiments were performed. First, a standard live/dead cell staining procedure using two commercially available dyes (SYTO 9 and To-Pro-3) was performed (Figure 1B and Supplementary Figure S7). SYTO 9 is a membrane-permeable nucleic acid dye that stains the RNA of living cells (56), negative/weak staining by SYTO 9 indicates cell mortality (Supplementary Figure S7C); while To-Pro-3 is a membrane-impermeable DNA stain, positive staining by To-Pro-3 indicates cell mortality (18) (Supplementary Figure S7B). As shown by co-staining with To-Pro-3 (Figure 1B) and SYTO 9 (Supplementary Figure S7A), cell viability isn’t affected after taking up Ru into nucleus in the presence of 3,5-DCP-OH. In addition, Trypan blue stain assay was also used to determine whether the plasma membrane permeability was affected, positive blue stain indicates cell membrane damage (Supplementary Figure S7D). As we can see from Supplementary Figure S7A, no obvious blue stain is observed for cells treated with either [Ru(DIP)₂(dppz)]Cl₂ alone or together with 3,5-DCP-OH at the stated concentration. At last, the cytotoxicity towards HeLa cells was further evaluated by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) assay, and no obvious cytotoxicity of [Ru(DIP)₂(dppz)]Cl₂ and/or 3,5-DCP-OH towards cells was observed either (Supplementary Figure S8). All the above results demonstrated that both plasma membrane permeability and cell viability were not affected by 3,5-DCP-OH and/or [Ru(DIP)₂(dppz)]Cl₂ under the experimental conditions employed, excluding the initial concern we brought up above.

Interestingly, for cytoplasm-localized [Ru(DIP)₂(dppz)]Cl₂ (cells were first treated with [Ru(DIP)₂(dppz)]Cl₂ alone for certain time, then washed with fresh medium to remove extracellular [Ru(DIP)₂(dppz)]Cl₂), subsequent addition of 3,5-DCP-OH can also direct [Ru(DIP)₂(dppz)]Cl₂ from cytoplasm to nucleus gradually, as can be seen from the gradually increased luminescence intensity in nuclear by CLSM (Supplementary Figure S9). Besides, we also found that cells treated with [Ru(DIP)₂(dppz)]Cl₂/3,5-DCP-OH exhibited a concentration-dependent nuclear uptake of Ru, and nuclear uptake was observed even at much lower concentration of [Ru(DIP)₂(dppz)]Cl₂ (as low as 5 μM) or 3,5-DCP-OH (20 μM) (Supplementary Figure S10). A series of experiments with various specific inhibitors (see Materials and Methods for details) demonstrated that passive diffusion, but not endocytosis and active transport, was the major mechanism of cellular uptake for [Ru(DIP)₂(dppz)]Cl₂/3,5-DCP-OH (Supplementary Figure S11), which is consistent with our previous work on [Ru(bpy)₂(dppz)]²⁺/PCP⁻ (39,40).

The formation of neutral, lipophilic and relatively stable ion-pair between [Ru(DIP)₂(dppz)]Cl₂ and 3,5-DCP-OH

The above results clearly showed that 3,5-DCP-OH can redirect [Ru(DIP)₂(dppz)]Cl₂ from the cytoplasm to the live-cell nucleus. Since [Ru(DIP)₂(dppz)]Cl₂ is lipophilic enough to pass through cell membrane but localizes in the cytoplasm, and according to our recent work on nuclear delivery of the more hydrophilic and cell-impermeable [Ru(bpy)₂(dppz)]Cl₂ via ion-pairing with chlorophenolate counter-anions (39–43), we speculated that a neutral, relatively stable and more lipophilic ion-pairing complex was also probably formed between 3,5-DCP-O⁻ and [Ru(DIP)₂(dppz)]²⁺, thus enabling [Ru(DIP)₂(dppz)]Cl₂ to escape the trapping by cytoplasmic components and readily enter the nucleus. To test the above hypothesis, partitioning study was conducted by using different organic solvents/aqueous solution. Since the log P_o/w value for [Ru(DIP)₂(dppz)]Cl₂ complex is 1.3 (18), much higher than that of [Ru(bpy)₂(dppz)]Cl₂ (log P_o/w = –2.50), we expect that [Ru(DIP)₂(dppz)]Cl₂ should be almost completely partitioned into 1-octanol phase, and addition of 3,5-DCP-OH would not clearly enhance its partition into the organic phase. We found this was indeed the case here. However, when ethyl acetate was used as organic solvent, we found that [Ru(DIP)₂(dppz)]Cl₂ can only be partially partitioned into ethyl acetate phase; under this condition, when 3,5-DCP-OH was present, [Ru(DIP)₂(dppz)]Cl₂ was found to be almost completely partitioned into the organic phase (Supplementary Figure S12 and Table S1). Analogous effects were observed by using chloroform as organic solvent (Supplementary Figure S13). These results demonstrated that a new adduct was indeed formed between [Ru(DIP)₂(dppz)]²⁺ and 3,5-DCP-O⁻, which should be neutral and more lipophilic than [Ru(DIP)₂(dppz)]Cl₂ alone.

To further establish whether a neutral adduct was formed between [Ru(DIP)₂(dppz)]²⁺ and 3,5-DCP-O⁻, agarose gel electrophoresis experiment was performed. As we know, when plasmid DNA is subjected to electrophoresis, relatively fast migration would be observed for the intact supercoil form due to the large number of negative charges on DNA. However, the migration should be inhibited if the negative charges of DNA were compromised by the positive-charges of [Ru(DIP)₂(dppz)]²⁺. As expected, we found that the migration of plasmid PBR323 DNA was indeed inhibited by [Ru(DIP)₂(dppz)]Cl₂ (50 μM). However, with the addition of 3,5-DCP-OH into the [Ru(DIP)₂(dppz)]Cl₂/DNA system, the DNA migration was found to be recovered progressively (Figure 2A), which indicates DNA recovers its original negatively-charged characteristic. These results further suggest that the weak hydrophobic acid 3,5-DCP-OH (pK_a = 8.25) (55), which could be readily dissociated to produce its negatively-charged anion (3,5-DCP-O⁻) under physiological pH, may compete with DNA to bind with the positively-charged cationic complex [Ru(DIP)₂(dppz)]²⁺, and the formed adduct [Ru(DIP)₂(dppz)](3,5-DCP-O⁻)₂ should be electrically neutral. It should be noted that the recovery of DNA migration was achieved only when the molar ratios of 3,5-DCP-O⁻/[Ru(DIP)₂(dppz)]²⁺ were relatively high, which suggests that the binding constant for [Ru(DIP)₂(dppz)]Cl₂/3,5-DCP-OH would be much smaller than that for [Ru(DIP)₂(dppz)]Cl₂/DNA. This was confirmed by the fluorescent competition binding experiment (Supplementary Figure S14). In the competition titration experiment with 3,5-DCP-OH against (50 μM) [Ru(DIP)₂(dppz)]Cl₂/ (200 μM) DNA, the 50% inhibitory concentration (IC₅₀) of 3,5-DCP-OH was found to be around 1.5 mM from the established competitive titration curve. A much higher concentration of 3,5-DCP-OH is needed to compete with DNA (200 μM) to bind with [Ru(DIP)₂(dppz)]Cl₂, indicating a smaller binding affinity for [Ru(DIP)₂(dppz)]Cl₂/3,5-DCP-OH.

Figure 2. — Evidence for the formation of neutral and lipophilic ion-pair between Ru complex and 3,5-DCP-O⁻. (A) Agarose gel electrophoresis experiment of [Ru(DIP)₂(dppz)]Cl₂ (50 μM) upon different concentrations of 3,5-DCP-OH was performed. 3,5-DCP-OH can gradually restore the migration of DNA by competing to bind [Ru(DIP)₂(dppz)]Cl₂. (Top) 3,5-DCP-OH was added into the plasmid DNA system after [Ru(DIP)₂(dppz)]Cl₂. (Bottom) [Ru(DIP)₂(dppz)]Cl₂ was added into the plasmid DNA system after 3,5-DCP-OH. (B) Crystal structure of the ion pair complex [Ru(DIP)₃]²⁺(2,4,6-TCP-O⁻)₂ by ORTEP drawing with 30% thermal ellipsoids, The other solvent molecules were omitted for clarity. The interactions between Δ-[Ru(DIP)₃]²⁺ cation and 2,4,6-TCP-O⁻ anion (indicated by the white dashed line) were through the short contact between H atom at 3 or 8 position of the ligand DIP and the O atom of the 2,4,6-TCP-OH.

We also tried to co-crystallize the adduct, but unfortunately, our efforts were not successful (possibly due to the presence of the phenazinenitrogens of dppz ligand). Since chlorinated phenols were also found to enhance the cellular and nuclear uptake of [Ru(DIP)₃]Cl₂ (a close analog of [Ru(DIP)₂(dppz)]Cl₂) (Supplementary Figures S15 and S16), we then extended our studies on the interactions between [Ru(DIP)₃]Cl₂ and chlorinated phenols. Fortunately, a single crystal was obtained after incubation of [Ru(DIP)₃]Cl₂ with 2,4,6-trichlorophenol (2,4,6-TCP-OH) and its solid-state structure was determined by single-crystal X-ray diffraction. The solution of the diffracted data clearly shows that an ion pair is indeed formed between [Ru(DIP)₃]²⁺ and 2,4,6-TCP-O⁻ with a 1:2 stoichiometry (Figure 2B; for detailed crystal data, see Supplementary Tables S2 and S3), which is consistent with our previous study (40). The high ratio of 3,5-DCP-OH/Ru required in partitioning study (Supplementary Figure S12) was probably due to the fact that 3,5-DCP-OH can’t be readily dissociated to produce its negatively-charged anion (3,5-DCP-O⁻) in organic/water solvents than in pure water; while in agarose gel electrophoresis experiment (Figure 2A), the high ratio of 3,5-DCP-OH/Ru needed for recovery can be attributed to the higher binding affinity of [Ru(DIP)₂(dppz)]Cl₂/DNA as we discussed above.

Enantioselective distribution of the two chiral forms (Δ- and Λ-enantiomer) of [Ru(DIP)₂(dppz)]Cl₂ complex in the presence of 3,5-DCP-OH

Our previous study showed that there was a remarkable luminescence difference between the two chiral forms (Δ- and Λ-enantiomer) of [Ru(bpy)₂(dppz)]Cl₂ in live-cell nuclear DNA imaging via ion-pairing method (40). We found that the Δ-[Ru(bpy)₂(dppz)]Cl₂ showed bright emission intensity inside the nucleus, while Λ-[Ru(bpy)₂(dppz)]Cl₂ exhibited a much weaker nuclear luminescence (Supplementary Figure S17). However, quantification by ICP-MS indicated that both enantiomers were facilitated into nucleus, with just a slight smaller amount of Λ-[Ru(bpy)₂(dppz)]Cl₂ than that of Δ-[Ru(bpy)₂(dppz)]Cl₂ (Supplementary Figure S18). The brighter luminescence in nucleus for Δ-enantiomer can be attributed to its longer excited-state lifetime than that for Λ-enantiomer (Supplementary Figure S17), which arises from an overall more favorable binding geometry (44–46). Similar phenomena were also reported by Lincoln group on the two enantiomers of an alkyl ether substituted [Ru(phen)₂(dppz)]Cl₂ in fixed cells, with Δ-enantiomer showing much brighter emission intensity inside the nucleus when compared to Λ-enantiomer due to a longer lifetime of Δ-enantiomer (44–46).

Here in this work, we found that cells treated with Δ-[Ru(DIP)₂(dppz)]Cl₂/3,5-DCP-OH also showed a bright luminescence in the nucleus, however, for cells treated with Λ-[Ru(DIP)₂(dppz)]Cl₂/3,5-DCP-OH, only cytoplasmic luminescence was observed without nuclear luminescence at all (Figure 3A and Supplementary Figure S19). To check whether the differences were also due to the different lifetimes of the two enantiomers, the variation of lifetimes in cells were also measured by fluorescence lifetime imaging microscopy (FLIM). As shown in Figure 3B and Table 1, in the absence of 3,5-DCP-OH, both Δ- and Λ- [Ru(DIP)₂(dppz)]Cl₂ are localized in cytoplasm with similar excited state lifetimes by a biexponential decay, and a short lifetime (τ₂) of ∼10 ns takes a large part of the lifetimes (fraction ≥ 70%) obtained for all rac-, Δ- and Λ-[Ru(DIP)₂(dppz)]Cl₂. But in the presence of 3,5-DCP-OH, a major contribution from a population with an extended lifetime (τ₁) of ∼550 ns is clearly recorded in nucleus for Δ-[Ru(DIP)₂(dppz)]Cl₂, whereas no effective lifetime in nucleus is recorded for Λ-[Ru(DIP)₂(dppz)]Cl₂/3,5-DCP-OH except for a large part of a short lifetime (τ₂) of ∼9 ns in cytoplasm, which is consistent with that of Λ-[Ru(DIP)₂(dppz)]Cl₂ alone present only in cytoplasm. Hence, unlike previous reports that a remarkable luminescence difference between the two chiral forms of Ru complexes such as [Ru(bpy)₂(dppz)]Cl₂ was originated from different lifetimes of the two enantiomers in nucleus, the results by FLIM in this study indicate a selective nuclear uptake of Δ-[Ru(DIP)₂(dppz)]Cl₂ only.

Figure 3. — Cellular uptake of rac-, Δ-, Λ-[Ru(DIP)₂(dppz)]Cl₂ in the absence and presence of 3,5-DCP-OH. (A) Dramatic differences are observed between the two chiral forms in nucleus by SIM, with Δ-enantiomer showing much brighter emission intensity inside the nucleus while Λ-enantiomer showing only cytoplasmic emission. (B) FLIM imaging of rac-, Δ-, Λ-Ru in the absence and presence of 3,5-DCP-OH. (C) ICP-MS assay for Δ- and Λ-Ru distribution in the cell nucleus and cytoplasm (n = 3). Cells treated with Ru (30 μM) in the presence or absence of 3,5-DCP-OH (100 μM). (D) Cellular localization of rac-, Δ-, Λ-Ru characterized by TEM in the presence of 3,5-DCP-OH for 1 h.

Table 1.

Lifetimes recorded in nucleus and cytoplasm for enantiomers of Ru

		Nucleus			Cytoplasm
	τ₁ (ns)	τ₂ (ns)	a ₁:a₂	χ²	τ₁ (ns)	τ₂ (ns)	a ₁:a₂	χ²
rac-Ru	—	—			247	10.5	30:70	1.034
Δ-Ru	—	—			244	10.2	24:76	1.375
Λ-Ru	—	—			264	10.4	25:75	1.504
rac-Ru/3,5-DCP	433	29.3	64:36	1.064	278	16.5	33:67	1.430
Δ-Ru/3,5-DCP	549	44.7	75:25	1.078	—	—
Λ-Ru/3,5-DCP	—	—-			216	8.8	20:80	1.094

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In order to confirm the disappearance of nuclear luminescence is due to the absence of Λ-[Ru(DIP)₂(dppz)]Cl₂ nuclear uptake, ICP-MS was used to quantify the cellular and nuclear uptake of the two chiral forms (Δ- and Λ-enantiomer) of [Ru(DIP)₂(dppz)]Cl₂ in the presence of 3,5-DCP-OH. As shown in Figure 3C, negligible enhancement of nuclear uptake of Λ-enantiomer is observed for cells treated with Λ-enantiomer/3,5-DCP-OH as compared to that with Λ-enantiomer alone; In contrast, a remarkable enhancement of Δ-enantiomer nuclear uptake is observed for Δ-enantiomer/3,5-DCP-OH. Therefore, the ICP-MS study further confirms that 3,5-DCP-OH selectively facilitates nuclear uptake of Δ-enantiomer, but not Λ-enantiomer. Moreover, TEM was also used to reveal the different distribution of the two enantiomers, and the results also show enantioselective nuclear uptake of Δ-enantiomer, but not Λ-enantiomer (Figure 3D).

Possible physicochemical factors for determining the enantioselective distribution of the two chiral forms (Δ- and Λ-enantiomer) of [Ru(DIP)₂(dppz)]Cl₂

What is the molecular mechanism underlying such a dramatically different cellular distribution of the two chiral forms of [Ru(DIP)₂(dppz)]Cl₂ in the presence of 3,5-DCP-OH?

Rac-[Ru(DIP)₂(dppz)]Cl₂ complex alone was found to be mainly accumulated in the cytoplasm, which is probably being trapped by cytoplasmic components to form the tightly-bound cytosolic-[Ru(DIP)₂(dppz)]Cl₂ adducts because of its lipophilic and positively-charged characteristics. Due to the large amount of proteins present in the cytoplasm, proteins should be a main factor needed to be considered in trapping [Ru(DIP)₂(dppz)]Cl₂. In cell-free conditions, we found that addition of bovine serum albumin (BSA, as a protein model) to a solution with rac-[Ru(DIP)₂(dppz)]Cl₂ could result in an obvious luminescence increase (Supplementary Figure S20), suggesting rac-[Ru(DIP)₂(dppz)]Cl₂ indeed can interact with BSA. It should be noted that no obvious luminescence increase was observed for [Ru(bpy)₂(dppz)]Cl₂ and [Ru(phen)₂(dppz)]Cl₂ with BSA under the same experimental conditions (Supplementary Figure S21). As we can see from Supplementary Figure S20, in the presence of BSA, Λ-[Ru(DIP)₂(dppz)]Cl₂ shows relatively higher luminescence than Δ-[Ru(DIP)₂(dppz)]Cl₂. In clear contrast, in the presence of DNA, Δ-[Ru(DIP)₂(dppz)]Cl₂ shows relatively higher luminescence than Λ-[Ru(DIP)₂(dppz)]Cl₂ (Supplementary Figure S22); These results indicate that Λ-[Ru(DIP)₂(dppz)]Cl₂ might be more tending for BSA binding while Δ-[Ru(DIP)₂(dppz)]Cl₂ for DNA binding due to their different structural geometry (57). So after considering all the potential physicochemical factors, we speculate that the different binding affinities of Δ- and Λ-[Ru(DIP)₂(dppz)]Cl₂ with BSA and DNA, along with binding affinity of the ion-pairing complex [Ru(DIP)₂(dppz)]Cl₂/3,5-DCP-OH, might be the major physicochemical factors responsible for the enantio-selective distribution of the two enantiomers. Then we first measured the binding affinities of both Δ- and Λ-[Ru(DIP)₂(dppz)]Cl₂ with BSA and DNA, respectively. The binding affinity between Λ-[Ru(DIP)₂(dppz)]Cl₂ and BSA was found to be (1.19 ± 0.05) × 10⁶ M⁻¹, which is about 2.8-times higher than that between Δ-[Ru(DIP)₂(dppz)]Cl₂ and BSA (4.22 ± 0.23) × 10⁵ M⁻¹ (Supplementary Figure S23) (for details on how to measure binding affinity, see Materials and Methods in Supplementary Materials), which is also consistent with their respective luminescence intensity (Supplementary Figure S20) and previous literature report with analogous Ru complexes (57). For the measurement of binding affinities with DNA, the classical Uv-Vis titration method employed from previous studies was adopted (19, (58). Due to some intrinsically hard-to-be-resolved issues like solubility of [Ru(DIP)₂(dppz)]Cl₂ complex, interferences between closely bound salt ions and especially Ru aggregation around DNA (59), we couldn’t obtain precise, but only estimated, values for Ru–DNA binding affinities. In UV-Vis titration spectra (Supplementary Figure S24), compared with Δ-Ru, Λ-Ru exhibits an unusually larger hypochromism and apparent absence of any change in spectra upon DNA titration, which indicates Λ-Ru itself should be more likely to aggregate around DNA rather than to bind to DNA. This also indirectly indicates the higher Δ-Ru/DNA binding affinity rather than Δ-Ru aggregation itself. Moreover, the more preferred DNA binding for Δ-Ru would also lead to de-aggregation of Δ-Ru aggregates by gradually reducing Δ-Ru concentrations around aggregates due to dynamic equilibrium. Therefore, in general, Δ-Ru has a stronger DNA binding affinity than Λ-Ru, which can also support our discussions in this work.

It should be emphasized that the interactions between Ru complexes and DNA caused luminescence enhancement and hypochromicity in the absorption have been considered to be very complex, closely bound metal complexes (aggregation) can give rise to extra hypochromicity in the absorption and further enhanced luminescence (59). Therefore, an estimated binding affinity is usually obtained with these methods to illustrate the binding between Ru complexes and DNA (58, (59). Further investigations are needed to obtain more accurate binding affinity values.

According to our recent structure-activity relationship study on nuclear uptake of the more hydrophilic Ru(II)-polypyridyl complexes (Ru(bpy)₂(dppz)]Cl₂ and Ru(phen)₂(dppz)]Cl₂) with ten different polychlorophenols, both the binding affinities (ion-pair, Ru-DNA and Ru-protein) and lipophilicity of the ion-pairs were found to be the major physicochemical factors determining the cellular distribution of Ru complexes with the ion-pairing method (39, (43). It should be noted that no enantio-selective localizations of the two enantiomers for Ru(bpy)₂(dppz)]Cl₂ or Ru(phen)₂(dppz)]Cl₂ were observed in the presence of 19 different polychlorophenols. Interestingly, in agreement with the above findings, we did not observe any enantio-differential binding of the respective Ru enantiomers with BSA (as a model protein) (Supplementary Figure S21). Since the binding affinity and lipophilicity for the two ion-pairing complexes [Δ-[Ru(DIP)₂(dppz)]Cl₂/3,5-DCP-OH and Λ-[Ru(DIP)₂(dppz)]Cl₂/3,5-DCP-OH] in this study should be the same or similar (Supplementary Figure S25), we speculate that the enantio-differential binding affinities of the two Ru enantiomers with DNA and proteins (BSA as a model protein here) as demonstrated above, might be the major physicochemical factors responsible for the dramatically enantio-selective localization of Δ- and Λ- [Ru(DIP)₂(dppz)]Cl₂ in the presence of 3,5-DCP-OH.

As shown above, the DNA binding affinity for the two enantiomers of [Ru(DIP)₂(dppz)]Cl₂ follows the order: Δ-enantiomer > Λ-enantiomer; in contrast, the BSA binding affinity for the two enantiomers follows the order: Λ-enantiomer > Δ-enantiomer. This would make 3,5-DCP-OH more readily to compete with cytoplasmic proteins for binding with the Δ-enantiomer (rather than for the Λ-enantiomer) to form not only a neutral and lipophilic, but also a relatively strong and stable ion-pairing complex between Δ-[Ru(DIP)₂(dppz)]²⁺ and 3,5-DCP-O⁻, and thus leading to the selective delivery of the Δ-enantiomer into nucleus by 3,5-DCP-OH, while keep Λ-enantiomer still trapped in the cytoplasm. Once the ion-pairing complex between Δ-[Ru(DIP)₂(dppz)]²⁺ and 3,5-DCP-O⁻ reaches nuclear DNA, since DNA has a much stronger binding affinity than 3,5-DCP-O⁻ for the Δ-enantiomer, the ion-pairing complex would be labile enough to be dissociated, and the Δ-enantiomer would then bind tightly with nuclear DNA possibly via intercalation and remains there. The ‘freed’ 3,5-DCP-O⁻ may diffuse back to the cytoplasm or medium, and then transport more Δ-enantiomer into the nucleus, possibly serving as a shuttle carrier. In other words, the cellular uptake and enantio-selective distribution of Δ- and Λ-[Ru(DIP)₂(dppz)]Cl₂ between cytoplasmic proteins and nuclear DNA in the presence of 3,5-DCP-OH can be visualized as several dynamic competing and equilibrium processes. Because of the enantio-differential binding of the two enantiomers with DNA and proteins, the final equilibrium for Δ-enantiomer localization would shift from the cytoplasmic protein to the nuclear DNA side.

It should be emphasized that there might be other currently undiscovered physico-chemical and/or biological factors responsible for the enantioselective nuclear uptake of the Δ-enantiomer, and for selective cytoplasmic trapping for the Λ-enantiomer of [Ru(DIP)₂(dppz)]Cl₂ via ion-pairing method. Therefore, more studies are still needed to fully elucidate this important issue in the future investigation.

Enantioselective apoptotic cell death was induced for cells pretreated with [Ru(DIP)₂(dppz)]Cl₂/3,5-DCP-OH upon prolonged visible-light irradiation

Given the strong binding affinity between DNA and rac-[Ru(DIP)₂(dppz)]Cl₂, and Ru-dppz complexes are known to be photo-activated to form its excited states, which can cause DNA damage either by direct oxidation or by forming singlet oxygen (60, (61), we expect that DNA damage and cell apoptosis should be induced for cells pretreated with [Ru(DIP)₂(dppz)]Cl₂/3,5-DCP-OH after prolonged light irradiation. Indeed, programmed cellular death was observed for such cells after continuous visible light-activation at 488 nm, as demonstrated by Annexin-V-flourescein isothiocyanate (FITC) and To-Pro-3 stain experiments (Figure 4A and Supplementary Figure S26). As expected, DNA double-stand breaks were also observed by immunofluorescence detection of gamma-H2AX (Figure 4B and Supplementary Figure S27), which may be responsible for induction of cell apoptosis. In contrast, no such apoptosis and DNA damage were observed for cells treated with [Ru(DIP)₂(dppz)]Cl₂/3,5-DCP-OH but without light irradiation (Supplementary Figures S26 and S27), which further indicates its photosensitizing effect.

Figure 4. — Enantioselective apoptosis and DNA double-stand breaks was induced to cells pretreated with [Ru(DIP)₂(dppz)]Cl₂ in the presence of 3,5-DCP-OH after prolonged visible light irradiation. After treatment with Ru (30 μM)/3,5-DCP-OH (100 μM) for 1 h, cells were rinsed with PBS for 3 times and refilled with new fresh medium, then cells were exposed to light irradiation (488 nm) for 30 min. (A) Cells were stained with Annexin-V-flourescein isothiocyanate (FITC) (10 μM) and To-Pro-3 (0.5 μM) for 15 min after incubation for another 3 h. (B) DNA double-stand breaks (DSBs) were observed with immunofluorescence detection of gamma-H2AX after incubation for another 3 h. The secondary antibody conjugated with Alexflour488 (green). To confirm the distribution of foci for gamma-H2AX, the nucleus was stained with Hoechst33342 (blue). It shows that Ru by itself can also be used as a DNA imaging agent while acting as a photosensitizing agent. The corresponding experiments without light irradiation were presented in Supplementary Information.

More importantly, a more detailed investigation reveals that enantioselective induction of apoptosis and DNA damage were observed for the two chiral form (Δ- and Λ-) of [Ru(DIP)₂(dppz)]Cl₂ complex upon irradiation, with Δ-enantiomer showing much stronger effects compared to Λ-enantiomer (Figure 4 and Supplementary Figures S26 and S27). As we know, nuclear DNA has been a sensitive and effective target for inducing cell death (48–50). Therefore, these results further confirm that DNA is the main target of [Ru(DIP)₂(dppz)]Cl₂ complex in inducing cell damage, and the Δ-enantiomer played a dominating role in killing cells in photodynamic therapy by directly targeting DNA. These results also confirm the above findings that Δ-[Ru(DIP)₂(dppz)]Cl₂ mainly localized in the nucleus, while Λ-[Ru(DIP)₂(dppz)]Cl₂ only in the cytoplasm.

It has been well recognized that the enantiomers of a racemic drug frequently have different physiological activities and pharmacokinetic profiles (62, (63). More importantly, a pair of enantiomers generally shares similar physical and chemical properties under achiral conditions; however, in a chiral environment such as in living organisms, one enantiomer may show the desired biological effects, while the other one exhibits inactivity and even toxic effects. The most widely recognized example is thalidomide, one of the most well-known tragedies in the history of chiral drug development. (R)-Thalidomide is a sedative, while (S)-thalidomide is currently known to lead to birth deformity by initiating its teratogenic effects via binding to cereblon (CRBN), a thalidomide-binding protein, and inhibiting the associated ubiquitin ligase activity (64). The mechanism behind the different activities was found to be different binding affinities to CRBN (65, (66). It was found that (S)-thalidomide displayed a 10-fold stronger binding to CRBN and inhibiton of self-ubiquitylation compared to its (R)-isomer. Thus, the investigation of chiral drugs will allow us to know which target (e.g. DNA) is the main treatment target and which enantiomer is therapeutic versus counterproductive or ineffective, which would further allow us to select the desired enantiomer to increase treatment efficiency and to eliminate unexpected activities as well. Similarly, the two enantiomers of [Ru(DIP)₂(dppz)]Cl₂ here were found to exhibit different cellular impacts, possibly due to their different localizations and targets. It has been suggested that the cytotoxic effects may arise largely from DNA lesions in the nucleus, whereas the protein adducts in the cytoplasm may be linked to the other therapeutic activities. The usage of Δ-[Ru(DIP)₂(dppz)]Cl₂ (which targeted DNA in the nucleus), the effective part of [Ru(DIP)₂(dppz)]Cl₂ complex as photodynamic therapeutic agent for treatment of cancer, should significantly reduce the dosage of [Ru(DIP)₂(dppz)]Cl₂ complex needed for maximal treatment effect, which is also beneficial for the overall cell biocompatibility.

Analogous results were observed when 3,5-DCP-OH was substituted by flufenamic acid, a currently-used non-steroid anti-inflammatory drug (NSAIDs)

As we previously reported, besides chlorophenols, the anthranilic acid class of the NSAIDs currently used as analgesics, antipyretics and anti-inflammatory agents (67) are also effective in facilitating Ru uptake (40). Expectedly, the redirected nuclear uptake of [Ru(DIP)₂(dppz)]Cl₂ was also observed with six fenamic acids (Supplementary Figure S28). Among the six fenamic acid NSAIDs drugs tested, flufenamic acid (FFA) (pK_a: 3.69) (68) showed the most effective ability for Ru nuclear uptake. Analogous biological effects were observed in all other experimental systems where 3,5-DCP-OH was substituted with FFA (Figure 5 and Supplementary Figures S7, S8, S13 and S29). The detailed studies with Δ- and Λ-[Ru(DIP)₂(dppz)]Cl₂ in the presence of FFA were also performed and similar enantioselectivity were obtained in cellular uptake and in inducing cell apoptosis and nucleic acid damage (Supplementary Figures S26, S27 and S30).

Figure 5. — [Ru(DIP)₂(dppz)]Cl₂ was redirected into nucleus by FFA in live cells. (A) Chemical structures of FFA. And flow cytometry analysis of Hela cells incubated with Ru (30 μM) in the absence or presence of FFA (100 μM, 3 h) in complete culture medium containing 10% FBS. **(B)** laser confocal scanning microscopy images of Hela cells incubated with Ru (30 μM) presence of FFA (100 μM, 3 h) in complete culture medium containing 10% FBS. (C) flow cytometry analysis and (D) confocal imaging of live-cell nuclear DNA by the two chiral forms (Δ and Λ).

In addition to HeLa cells, similar nuclear uptake of Ru in the presence of 3,5-DCP-OH or FFA were also observed in other cell-lines, including PC-12, HepG-2, MCF-7 and A549 cells (Supplementary Figures S31 and S32). The above results suggest that the redirected nuclear uptake in a variety of cell lines is a general phenomenon for the cationic [Ru(DIP)₂(dppz)]Cl₂ with two groups of structurally unrelated hydrophobic weak acids, the chlorophenolic compounds and the fenamic acid NSAIDs drugs. Since FFA is a currently used NSAIDs drug, the findings with FFA may be especially important, and therefore would significantly increase the biological significance of this work.

In general, we found that only Δ-[Ru(DIP)₂(dppz)]Cl₂ was selectively translocated from cytoplasm to nucleus via ion-pairing method. Compared with other previously studied metal complexes, [Ru(DIP)₂(dppz)]Cl₂ complex in this research has the following attractive characteristics: (i) selective targets for Δ- and Λ- enantiomer: for the Δ- enantiomer, its main cellular target is nuclear DNA, which could increase its photodynamic efficacy by directly attack DNA since the lifetime of ¹O₂ it produced is extremely short; this further leads to: (ii) it can induce cell apoptosis enantioselectively, with Δ-enantiomer showing much higher cytotoxicity than Λ-enantiomer, which is the most unique and ensures its efficacy with lower dosages of the active enantiomer.

CONCLUSIONS

In this study, we found that the cytoplasm-restricted [Ru(DIP)₂(dppz)]Cl₂ can be redirected into nucleus by all 19 chlorinated phenols and all six NSAIDs drugs tested, via forming ion-pair complexes that are neutral and stable enough to be able to escape cytoplasmic traps, and cross the nuclear membrane to reach nuclear DNA. More thorough studies with the Δ- and Λ-enantiomers indicate it is the Δ-enantiomer mainly being re-localized from cytoplasm to nucleus due to its relatively low binding affinity with cytoplasmic proteins and high binding affinity with nuclear DNA. Enantioselective cytotoxicity of Δ-enantiomers (in nucleus) and Λ-enantiomers (in cytoplasm) further confirms nuclear DNA being a sensitive and effective target in inducing cell damage, thus the pure Δ-enantiomer should improve the anticancer efficiency with a relatively lower dosage. This study represents, for the first time, the enantioselective recognition and re-localization, induction of apoptosis of [Ru(DIP)₂(dppz)]Cl₂ complex in live cells via simple ion-pairing with suitable weak acid counter-anions, which can allow [Ru(DIP)₂(dppz)]Cl₂ to be not only an enantioselective imaging agent for live-cell nuclear DNA, but at the same time also a potential enantioselective photodynamic therapeutic agent for treatment of cancer via enantioselectively targeting nucleus. This novel ion-pairing method and concept might be employed to redirect other potentially bio-medically important chiral metal-complexes to their preferred intracellular targets.

DATA AVAILABILITY

The data underlying this article are available in the article and in its online supplementary material. CCDC 1487349 contains the supplementary crystallographic data for this article. The data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by e-mailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

Supplementary Material

gkad155_Supplemental_File

Click here for additional data file.^{(3.2MB, pdf)}

ACKNOWLEDGEMENTS

We would like to thank the Center for Biological Imaging (CBI), Institute of Biophysics, Chinese Academy of Science, for our Structured Illumination Microscopy work.

Contributor Information

Xi-Juan Chao, State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences; University of Chinese Academy of Sciences, The Chinese Academy of Sciences, Beijing 100085, P.R. China; Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000, China.

Chun-Hua Huang, State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences; University of Chinese Academy of Sciences, The Chinese Academy of Sciences, Beijing 100085, P.R. China.

Miao Tang, State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences; University of Chinese Academy of Sciences, The Chinese Academy of Sciences, Beijing 100085, P.R. China.

Zhu-Ying Yan, State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences; University of Chinese Academy of Sciences, The Chinese Academy of Sciences, Beijing 100085, P.R. China.

Rong Huang, State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences; University of Chinese Academy of Sciences, The Chinese Academy of Sciences, Beijing 100085, P.R. China.

Yan Li, State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences; University of Chinese Academy of Sciences, The Chinese Academy of Sciences, Beijing 100085, P.R. China.

Ben-Zhan Zhu, State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences; University of Chinese Academy of Sciences, The Chinese Academy of Sciences, Beijing 100085, P.R. China; Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

FUNDING

‘From 0 to 1’ Original Innovation Project; Basic Frontier Scientific Research Program, CAS [ZDBS-LY-SLH027]; NSF China Grants [21836005, 92043301, 22021003, 21777180, 32000910]. Funding for open access charge: NSF China Grants.

Conflict of interest statement. None declared.

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[B25] 25. Liu C., Zhang R., Zhang W., Liu J., Wang Y.L., Du Z., Song B., Xu Z.P., Yuan J.. “Dual-Key-and-Lock” ruthenium complex probe for lysosomal formaldehyde in cancer cells and tumors. J. Am. Chem. Soc. 2019; 141:8462–8472. [DOI] [PubMed] [Google Scholar]

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[B28] 28. Knoll J.D., Turro C.. Control and utilization of ruthenium and rhodium metal complex excited states for photoactivated cancer therapy. Coord. Chem. Rev. 2015; 282–283:110–126. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29. Gill M.R., Thomas J.A.. Ruthenium(II) polypyridyl complexes and DNA–from structural probes to cellular imaging and therapeutics. Chem. Soc. Rev. 2012; 41:3179–3192. [DOI] [PubMed] [Google Scholar]

[B30] 30. Zeng L.L., Gupta P., Chen Y.L., Wang E.J., Ji L.N., Chao H., Chen Z.S.. The development of anticancer ruthenium(II) complexes: from single molecule compounds to nanomaterials. Chem. Soc. Rev. 2017; 46:5771–5804. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B31] 31. Puckett C.A., Barton J.K.. Mechanism of cellular uptake of a ruthenium polypyridyl complex. Biochemistry. 2008; 47:11711–11716. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[B33] 33. Levy S., Shechtman S., Zhu B.Z., Stadtman E.R., Stadler R., Chevion M.. Synergism between the toxicity of chlorophenols and iron complexes. Environ. Toxicol. Chem. 2007; 26:218–224. [DOI] [PubMed] [Google Scholar]

[B34] 34. Sheng Z.G., Li Y., Fan R.M., Chao X.J., Zhu B.Z.. Lethal synergism between organic and inorganic wood preservatives via formation of an unusual lipophilic ternary complex. Toxicol. Appl. Pharm. 2013; 266:335–344. [DOI] [PubMed] [Google Scholar]

[B35] 35. Zhu B.Z., Kalyanaraman B., Jiang G.B.. Molecular mechanism for metal-independent production of hydroxyl radicals by hydrogen peroxide and halogenated quinones. Proc. Natl. Acad. Sci. U.S.A. 2007; 104:17575–17578. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36. Zhu B.Z., Mao L., Huang C.H., Qin H., Fan R.M., Kalyanaraman B., Zhu J.G.. Unprecedented hydroxyl radical-dependent two-step chemiluminescence production by polyhalogenatedquinoid carcinogens and H2O2. Proc. Natl. Acad. Sci. U.S.A. 2012; 109:16046–16051. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B37] 37. Zhu B.Z., Shan G.Q.. Potential mechanism for pentachlorophenol-induced carcinogenicity: a novel mechanism for metal-independent production of hydroxyl radicals. Chem. Res. Toxicol. 2009; 22:969–977. [DOI] [PubMed] [Google Scholar]

[B38] 38. Shao B., Mao L., Qu N., Wang Y.F., Gao H.Y., Li F., Qin L., Shao J., Huang C.H., Xu D.et al.. Mechanism of synergistic DNA damage induced by the hydroquinone metabolite of brominated phenolic environmental pollutants and Cu(II): formation of DNA-Cu complex and site-specifc production of hydroxyl radicals. Free Radic. Biol. Med. 2017; 104:54–63. [DOI] [PubMed] [Google Scholar]

[B39] 39. Chao X.J., Tang M., Huang R., Huang C.H., Shao J., Yan Z.Y., Zhu B.Z.. Targeted live-cell nuclear delivery of the DNA ‘light-switching’ Ru(II) complex via Ion-pairing with chlorophenolate counter-anions: the critical role of binding stability and lipophilicity of the ion-pairing complexes. Nucleic Acids Res. 2019; 47:10520–10528. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Unusual enantioselective cytoplasm-to-nucleus translocation and photosensitization of the chiral Ru(II) cationic complex via simple ion-pairing with lipophilic weak acid counter-anions

Xi-Juan Chao

Chun-Hua Huang

Miao Tang

Zhu-Ying Yan

Rong Huang

Yan Li

Ben-Zhan Zhu

Abstract

Graphical Abstract

Graphical Abstract.

INTRODUCTION

Figure 1.

MATERIALS AND METHODS

Chemicals

Preparation of chlorinated phenols solutions

Cell culture

Flow cytometry

Microscopic study

Partitioning study

ICP-MS analysis

DNA agarose gel electrophoresis

Separation and purification of the two enantiomers

Cultivation of crystals

Single crystal X-ray diffraction

Phosphatidylserine translocation

Immunofluorescence

RESULTS AND DISCUSSION

Redirecting the positively-charged DNA ‘light-switching’ rac-[Ru(DIP)2(dppz)]Cl2 from cytoplasm to live-cell nuclei by chlorinated phenols

The formation of neutral, lipophilic and relatively stable ion-pair between [Ru(DIP)2(dppz)]Cl2 and 3,5-DCP-OH

Figure 2.

Enantioselective distribution of the two chiral forms (Δ- and Λ-enantiomer) of [Ru(DIP)2(dppz)]Cl2 complex in the presence of 3,5-DCP-OH

Figure 3.

Table 1.

Possible physicochemical factors for determining the enantioselective distribution of the two chiral forms (Δ- and Λ-enantiomer) of [Ru(DIP)2(dppz)]Cl2

Enantioselective apoptotic cell death was induced for cells pretreated with [Ru(DIP)2(dppz)]Cl2/3,5-DCP-OH upon prolonged visible-light irradiation

Figure 4.

Analogous results were observed when 3,5-DCP-OH was substituted by flufenamic acid, a currently-used non-steroid anti-inflammatory drug (NSAIDs)

Figure 5.

CONCLUSIONS

DATA AVAILABILITY

Supplementary Material

ACKNOWLEDGEMENTS

Contributor Information

SUPPLEMENTARY DATA

FUNDING

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Redirecting the positively-charged DNA ‘light-switching’ rac-[Ru(DIP)₂(dppz)]Cl₂ from cytoplasm to live-cell nuclei by chlorinated phenols

The formation of neutral, lipophilic and relatively stable ion-pair between [Ru(DIP)₂(dppz)]Cl₂ and 3,5-DCP-OH

Enantioselective distribution of the two chiral forms (Δ- and Λ-enantiomer) of [Ru(DIP)₂(dppz)]Cl₂ complex in the presence of 3,5-DCP-OH

Possible physicochemical factors for determining the enantioselective distribution of the two chiral forms (Δ- and Λ-enantiomer) of [Ru(DIP)₂(dppz)]Cl₂

Enantioselective apoptotic cell death was induced for cells pretreated with [Ru(DIP)₂(dppz)]Cl₂/3,5-DCP-OH upon prolonged visible-light irradiation