Metalloimmunotherapy with Rhodium and Ruthenium Complexes: Targeting Tumor-Associated Macrophages.

Tumor associated macrophages (TAMs) suppress the cancer immune response and are a key target for immunotherapy. The effects of ruthenium and rhodium complexes on TAMs have not been well characterized. To address this gap in the field, a panel of 22 dirhodium and ruthenium complexes were screened against three subtypes of macrophages, triple-negative breast cancer and normal breast tissue cells. Experiments were carried out in 2D and biomimetic 3D co-culture experiments with and without irradiation with blue light. Leads were identified with cell-type-specific toxicity toward macrophage subtypes, cancer cells, or both. Experiments with 3D spheroids revealed complexes that sensitized the tumor models to the chemotherapeutic doxorubicin. Cell surface exposure of calreticulin, a known facilitator of immunogenic cell death (ICD), was increased upon treatment, along with a concomitant reduction in the M2-subtype classifier arginase. Our findings lay a strong foundation for the future development of ruthenium- and rhodium-based chemotherapies targeting TAMs.

Unlocking the Potential of Ru(II) Dual-action Compounds with the Power of the Heavy-atom Effect.

We report the synthesis, photochemical and biological characterization of two new Ru(II) photoactivated complexes based on [Ru(tpy)(Me2 bpy)(L)]2+ (tpy = 2,2':6',2''-terpyridine, Me2 bpy = 6,6'-dimethyl-2,2'-bipyridine), where L = pyridyl-BODIPY (pyBOD). Two pyBOD ligands were prepared bearing flanking hydrogen or iodine atoms. Ru(II)-bound BODIPY dyes show a red-shift of absorption maxima relative to the free dyes and undergo photodissociation of BODIPY ligands with green light irradiation. Addition of iodine into the BODIPY ligand facilitates intersystem crossing, which leads to efficient singlet oxygen production in the free dye, but also enhances quantum yield of release of the BODIPY ligand from Ru(II). This represents the first report of a strategy to enhance photodissociation quantum yields through the heavy-atom effect in Ru(II) complexes. Furthermore, Ru(II)-bound BODIPY dyes display fluorescence turn-on once released, with a lead analog showing nanomolar EC50 values against triple negative breast cancer cells, >100-fold phototherapeutic indexes under green light irradiation, and higher selectivity toward cancer cells as compared to normal cells than the corresponding free BODIPY photosensitizer. Conventional Ru(II) photoactivated complexes require nonbiorthogonal blue light for activation and rarely show submicromolar potency to achieve cell death. Our study represents an avenue for the improved photochemistry and potency of future Ru(II) complexes.

Ru(II)-Based Acetylacetonate Complexes Induce Apoptosis Selectively in Cancer Cells.

The synthesis, chemical and biological characterization of seven Ru(II) polypyridyl complexes containing acetylacetonate (acac) ligands are reported. Electronic absorption spectra were determined and electrochemical potentials consistent with Ru(III/II) couples ranging from +0.60 to +0.73 V vs Ag/AgCl were measured. A series of complexes were screened against MDA-MB-231, DU-145, and MCF-10A cell lines to evaluate their cytotoxicities in cancer and normal cell lines. Although most complexes were either nontoxic or equipotent in cancer cells and normal cell lines, compound 1, [Ru(dpqy)(acac)(py)](PF6), where dqpy is 2,6-di(quinolin-2-yl)pyridine, showed up to 2.5:1.0 selectivity for cancer as compared to normal cells, along with nanomolar EC50 values in MDA-MB-231 cells. Lipophilicity, determined as the octanol/water partition coefficient, log Po/w, ranged from -0.33 (0.06) to 1.15 (0.10) for the complexes. Although cytotoxicity was not correlated with electrochemical potentials, a moderate linear correlation between lipophilicity and toxicities was observed. Cell death mechanism studies indicated that several of the Ru-acac compounds, including 1, induce apoptosis in MDA-MB-231 cells.

Photosensitive Ru(II) Complexes as Inhibitors of the Major Human Drug Metabolizing Enzyme CYP3A4.

We report the synthesis and photochemical and biological characterization of the first selective and potent metal-based inhibitors of cytochrome P450 3A4 (CYP3A4), the major human drug metabolizing enzyme. Five Ru(II)-based derivatives were prepared from two analogs of the CYP3A4 inhibitor ritonavir, 4 and 6: [Ru(tpy)(L)(6)]Cl2 (tpy = 2,2':6',2″-terpyridine) with L = 6,6'-dimethyl-2,2'-bipyridine (Me2bpy; 8), dimethylbenzo[i]dipyrido[3,2-a:2',3'-c]phenazine (Me2dppn; 10) and 3,6-dimethyl-10,15-diphenylbenzo[i]dipyrido[3,2-a:2',3'-c]phenazine (Me2Ph2dppn; 11), [Ru(tpy)(Me2bpy)(4)]Cl2 (7) and [Ru(tpy)(Me2dppn)(4)]Cl2 (9). Photochemical release of 4 or 6 from 7-11 was demonstrated, and the spectrophotometric evaluation of 7 showed that it behaves similarly to free 4 (type II heme ligation) after irradiation with visible light but not in the dark. Unexpectedly, the intact Ru(II) complexes 7 and 8 were found to inhibit CYP3A4 potently and specifically through direct binding to the active site without heme ligation. Caged inhibitors 9-11 showed dual action properties by combining photoactivated dissociation of 4 or 6 with efficient 1O2 production. In prostate adenocarcinoma DU-145 cells, compound 9 had the best synergistic effect with vinblastine, the anticancer drug primarily metabolized by CYP3A4 in vivo. Thus, our study establishes a new paradigm in CYP inhibition using metalated complexes and suggests possible utilization of photoactive CYP3A4 inhibitory compounds in clinical applications, such as enhancement of therapeutic efficacy of anticancer drugs.

Dual-Action Ru(II) Complexes with Bulky π-Expansive Ligands: Phototoxicity without DNA Intercalation.

We report the synthesis and photochemical and biological characterization of Ru(II) complexes containing π-expansive ligands derived from dimethylbenzo[i]dipyrido[3,2-a:2',3'-c]phenazine (Me2dppn) adorned with flanking aryl substituents. Late-stage Suzuki couplings produced Me2dppn ligands substituted at the 10 and 15 positions with phenyl (5), 2,4-dimethylphenyl (6), and 2,4-dimethoxyphenyl (7) groups. Complexes of the general formula [Ru(tpy)(L)(py)](PF6)2 (8-10), where L = 4-7, were characterized and shown to have dual photochemotherapeutic (PCT) and photodynamic therapy (PDT) behavior. Quantum yields for photodissociation of monodentate pyridines from 8-10 were about 3 times higher than that of parent complex [Ru(tpy)(Me2dppn)(py)](PF6)2 (1), whereas quantum yields for singlet oxygen (1O2) production were ∼10% lower than that of 1. Transient absorption spectroscopy indicates that 8-10 possess long excited state lifetimes (τ = 46-50 µs), consistent with efficient 1O2 production through population and subsequent decay of ligand-centered 3ππ* excited states. Complexes 8-10 displayed greater lipophilicity relative to 1 and association to DNA but do not intercalate between the duplex base pairs. Complexes 1 and 8-10 showed photoactivated toxicity in breast and prostate cancer cell lines with phototherapeutic indexes, PIs, as high as >56, where the majority of cell death was achieved 4 h after treatment with Ru(II) complexes and light. Flow cytometric data and rescue experiments were consistent with necrotic cell death mediated by the production of reactive oxygen species, especially 1O2. Collectively, this study confirms that DNA intercalation by Ru(II) complexes with π-expansive ligands is not required to achieve photoactivated cell death.

BODIPY-Caged Photoactivated Inhibitors of Cathepsin B Flip the Light Switch on Cancer Cell Apoptosis.

Acquired resistance to apoptotic agents is a long-standing challenge in cancer treatment. Cathepsin B (CTSB) is an enzyme which, among many essential functions, promotes apoptosis during cellular stress through regulation of intracellular proteolytic networks on the minute time scale. Recent data indicate that CTSB inhibition may be a promising method to steer cells away from apoptotic death toward necrosis, a mechanism of cell death that can overcome resistance to apoptotic agents, stimulate an immune response and promote antitumor immunity. Unfortunately, rapid and selective intracellular inactivation of CTSB has not been possible. However, here we report on the synthesis and characterization of photochemical and biological properties of BODIPY-caged inhibitors of CTSB that are cell permeable, highly selective and activated rapidly upon exposure to visible light. Intriguingly, these compounds display tunable photophysical and biological properties based on substituents bound directly to boron. Me2BODIPY-caged compound 8 displays the dual-action capability of light-accelerated CTSB inhibition and singlet oxygen production from a singular molecular entity. The dual-action capacity of 8 leads to a rapid necrotic response in MDA-MB-231 triple negative breast cancer cells with high phototherapeutic indexes (>30) and selectivity vs noncancerous cells that neither CTSB inhibition nor photosensitization gives alone. Our work confirms that singlet oxygen production and CTSB inactivation is highly synergistic and a promising method for killing cancer cells. Furthermore, this ability to trigger intracellular inactivation of CTSB with light provides researchers with a powerful photochemical tool for probing biochemical processes on short time scales.

Batch-to-batch variation in domestic paints: Insights into the newly commercialized recycled paints.

A few recent studies attempted to evaluate the differentiation of paints at a production batch level and reported results depending largely on the paint type. The discrimination from production batches is much more random than brands and/or models levels and subject to many unknowns, which suggests that a particular production batch can suddenly present a substantially different composition than the one produced right before or right after it. To add to this existing complexity, most of the paint companies now propose a range of recycled paints among their products. These recycled paints are composed of wastes collected by recycling plants, sorted by their color and binder type (i.e. latex, alkyds), and mixed together in large tanks to form the basis material for future formulations. Quality controls on these recycled batches are voluntarily less precise, and a higher variation is expected in esthetic and chemical properties of the paint. In this project, we collaborated with a North American paint producer that gave us access to its samples, paint formulations recipes, and a summary of the quality controls and corrections they performed on each production batches. The whole study was conducted blindfold and a final verification was made with the manufacturer to evaluate the accuracy of the results. The data set comprised two models of regular paints and two models of recycled paint. Five different production batches were collected per model for a total of 20 samples analyzed by Microscopy, Infrared spectroscopy, Raman spectroscopy, and Pyrolysis GC/MS.

Catch and Release Photosensitizers: Combining Dual-Action Ruthenium Complexes with Protease Inactivation for Targeting Invasive Cancers.

Dual action agents containing a cysteine protease inhibitor and Ru-based photosensitizer for photodynamic therapy (PDT) were designed, synthesized, and validated in 2D culture and 3D functional imaging assays of triple-negative human breast cancer (TNBC). These combination agents deliver and release Ru-based PDT agents to tumor cells and cause cancer cell death upon irradiation with visible light, while at the same time inactivating cathespin B (CTSB), a cysteine protease strongly associated with invasive and metastatic behavior. In total five Ru-based complexes were synthesized with the formula [Ru(bpy)2(1)](O2CCF3)2 (3), where bpy = 2,2'-bipyridine and 1 = a bipyridine-based epoxysuccinyl inhibitor; [Ru(tpy)(NN)(2)](PF6)2, where tpy = terpiridine, 2 = a pyridine-based epoxysuccinyl inhibitor and NN = 2,2'-bipyridine (4); 6,6'-dimethyl-2,2'-bipyridine (5); benzo[ i]dipyrido[3,2- a:2',3'- c]phenazine (6); and 3,6-dimethylbenzo[ i]dipyrido[3,2- a:2',3'- c]phenazine (7). Compound 3 contains a [Ru(bpy)3]2+ fluorophore and was designed to track the subcellular localization of the conjugates, whereas compounds 4-7 were designed to undergo either photoactivated ligand dissociation and/or singlet oxygen generation. Photochemical studies confirmed that complexes 5 and 7 undergo photoactivated ligand dissociation, whereas 6 and 7 generate singlet oxygen. Inhibitors 1-7 all potently and irreversibly inhibit CTSB. Compounds 4-7 were evaluated against MDA-MB-231 TNBC and MCF-10A breast epithelial cells in 2D and 3D culture for effects on proteolysis and cell viability under dark and light conditions. Collectively, these data reveal that 4-7 potently inhibit dye-quenched (DQ) collagen degradation, whereas only compound 7 causes efficient cell death under light conditions, consistent with its ability to release a Ru(II)-based photosensitizer and to also generate 1O2.