Green Light-Triggered Photocatalytic Anticancer Activity of Terpyridine-Based Ru(II) Photocatalysts.

The relentless increase in drug resistance of platinum-based chemotherapeutics has opened the scope for other new cancer therapies with novel mechanisms of action (MoA). Recently, photocatalytic cancer therapy, an intrusive catalytic treatment, is receiving significant interest due to its multitargeting cell death mechanism with high selectivity. Here, we report the synthesis and characterization of three photoresponsive Ru(II) complexes, viz., [Ru(ph-tpy)(bpy)Cl]PF6 (Ru1), [Ru(ph-tpy)(phen)Cl]PF6 (Ru2), and [Ru(ph-tpy)(aip)Cl]PF6 (Ru3), where, ph-tpy = 4'-phenyl-2,2':6',2″-terpyridine, bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline, and aip = 2-(anthracen-9-yl)-1H-imidazo[4,5-f][1,10] phenanthroline, showing photocatalytic anticancer activity. The X-ray crystal structures of Ru1 and Ru2 revealed a distorted octahedral geometry with a RuN5Cl core. The complexes showed an intense absorption band in the 440-600 nm range corresponding to the metal-to-ligand charge transfer (MLCT) that was further used to achieve the green light-induced photocatalytic anticancer effect. The mitochondria-targeting photostable complex Ru3 induced phototoxicity with IC50 and PI values of ca. 0.7 µM and 88, respectively, under white light irradiation and ca. 1.9 µM and 35 under green light irradiation against HeLa cells. The complexes (Ru1-Ru3) showed negligible dark cytotoxicity toward normal splenocytes (IC50s > 50 µM). The cell death mechanistic study revealed that Ru3 induced ROS-mediated apoptosis in HeLa cells via mitochondrial depolarization under white or green light exposure. Interestingly, Ru3 also acted as a highly potent catalyst for NADH photo-oxidation under green light. This NADH photo-oxidation process also contributed to the photocytotoxicity of the complexes. Overall, Ru3 presented multitargeting synergistic type I and type II photochemotherapeutic effects.

Folate-assisted targeted photocytotoxicity of red-light-activable iron(III) complex co-functionalized gold nanoconjugates (Fe@FA-AuNPs) against HeLa and triple-negative MDA-MB-231 cancer cells.

Photo-redox chemistry resulting from ligand to metal charge transfer in red-light-activable iron(III) complexes could be a potent strategic tool for next-generation photochemotherapeutic applications. Herein, we developed an iron(III) complex and folate co-functionalized gold nanoconjugate (Fe@FA-AuNPs) and thoroughly characterized it with NMR, ESI MS, UV-visible, EPR, EDX, XPS, powder X-ray diffraction, TEM and DLS studies. There was a remarkable shift in the SPR band of AuNPs to 680 nm, and singlet oxygen (1O2) and hydroxyl radicals were potently generated upon red-light activation, which were probed by UV-visible and EPR spectroscopic assays. Cellular uptake studies of the nanoconjugate (Fe@FA-AuNPs) revealed significantly higher uptake in folate(+) cancer cells (HeLa and MDA-MB-231) than folate(-) (A549) cancer cells or normal cells (HPL1D), indicating the targeting potential of the nanoconjugate. Confocal imaging indicated primarily mitochondrial localization. The IC50 values of the nanoconjugate determined from a cell viability assay in HeLa, MDA-MB-231, and A549 cells were 27.83, 39.91, and 69.54 µg mL-1, respectively in red light, while in the dark the values were >200 µg mL-1; the photocytotoxicity was correlated with the cellular uptake of the nanoconjugate. The nanocomposite exhibited similar photocytotoxicity (IC50 in red light, 37.35 ± 8.29 µg mL-1 and IC50 in the dark, >200 µg mL-1). Mechanistic studies revealed that intracellular generation of ROS upon red-light activation led to apoptosis in HeLa cells. Scratch-wound-healing assays indicated the inhibition of the migration of MDA-MB-231 cells treated with the nanoconjugate and upon photo-activation. Overall, the nanoconjugate has emerged as a potent tool for next-generation photo-chemotherapeutics in the clinical arena of targeted cancer therapy.

Cu(II) flavonoids as potential photochemotherapeutic agents.

Flavonoids, naturally derived polyphenolic compounds, have received significant attention due to their remarkable biochemical properties that offer substantial health benefits to humans. In this work, a series of six Cu(II) flavonoid complexes of the formulation [Cu(L1)(L2)](ClO4) where L1 is 3-hydroxy flavone (HF1, 1 and 4), 4-fluoro-3-hydroxy flavone (HF2, 2 and 5), and 2,6-difluoro-3-hydroxy flavone (HF3, 3 and 6); L2 is 1,10-phenanthroline (phen, 1-3) and 2-(anthracen-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (aip, 4-6) were successfully synthesized, fully characterized and also evaluated for their in vitro photo-triggered cytotoxicity in cancer cells. The single-crystal X-ray diffraction structure of complex 2 shows square pyramidal geometry around the Cu(II) center. The complexes 1-6 showed quasi-reversible cyclic voltammetric responses for the Cu(II)/Cu(I) couple at ∼-0.230 V with a very large ΔEp value of ∼350-480 mV against the Ag/AgCl reference electrode in DMF-0.1 M tetrabutylammonium perchlorate (TBAP) at a scan rate of 50 mV s-1. The complexes were found to have considerable binding propensity for human serum albumin (HSA) and calf thymus DNA (ct-DNA). The complexes displayed remarkable dose-dependent photocytotoxicity in visible light (400-700 nm) in both A549 (human lung cancer) and MCF-7 (human breast cancer) cell lines while remaining significantly less toxic in darkness. They were found to be much less toxic to HPL1D (immortalized human peripheral lung epithelial) normal cells compared to A549 and MCF-7 cancer cells. Upon exposure to visible light, they generate reactive oxygen species, which are thought to be the main contributors to the death of cancer cells. In the presence of visible light, the complexes predominantly elicit an apoptotic mode of cell death. Complex 6 preferentially localizes in the mitochondria of A549 cells.

A Platinum(II) Boron-dipyrromethene Complex for Cellular Imaging and Mitochondria-targeted Photodynamic Therapy in Red Light.

Cisplatin-derived platinum(II) complexes [Pt(NH3 )2 (pacac)](NO3 ) (1, DPP-Pt) and [Pt(NH3 )2 (Acac-RB)](NO3 ) (2, Acacplatin-RB), where Hpacac is 1,3-diphenyl-1,3-propanedione and HAcac-RB is a red-light active distyryl-BODIPY-appended acetylacetone ligand, are prepared, characterized and their photodynamic therapy (PDT) activity studied (RB abbreviated for red-light BODIPY). Complex 2 displayed an intense absorption band at λ=652 nm (ϵ=7.3×104  M-1  cm-1 ) and 601 nm (ϵ=3.1×104  M-1  cm-1 ) in 1 : 1 DMSO-DPBS (Dulbecco's Phosphate Buffered Saline). Its emission profile includes a broad maximum at ~673 nm (λex =630 nm). The fluorescence quantum yield (ΦF ) of HAcac-RB and 2 are 0.19 and 0.07, respectively. Dichlorodihydrofluorescein diacetate and 1,3-diphenylisobenzofuran assay of complex 2 indicated photogeneration of singlet oxygen (ΦΔ : 0.36) as reactive oxygen species (ROS). Light irradiation caused only minor extent of ligand release forming chemo-active cisplatin analogue. The complex showed ~70-100 fold enhancement in cytotoxicity on light exposure in A549 lung cancer cells and MDA-MB-231 multidrug resistant breast cancer cells, giving half maximal inhibitory concentration (IC50 ) of 0.9-1.8 µM. Confocal imaging showed its mitochondrial localization and complex 2 exhibited anti-metastasis properties. Immunostaining of ß-tubulin and Annexin V-FITC/propidium iodide staining displayed complex 2 induced photo-selective microtubule rupture and cellular apoptosis, respectively.

Biotin and boron-dipyrromethene-tagged platinum(IV) prodrug for cellular imaging and mito-targeted photocytotoxicity in red light.

A platinum(IV) prodrug, cis,cis,trans-[Pt(NH3)2Cl2(biotin)(L)] (1), derived from cisplatin, where HL is the PEGylated red-light active boron-dipyrromethene (BODIPY) ligand, was synthesized, characterized and its photocytotoxicity evaluated. The complex showed a near-IR absorption band at 653 nm (ε ∼9.19 × 104 M-1 cm-1) in dimethyl sulfoxide and Dulbecco's phosphate-buffered saline (1 : 1 v/v) at pH 7.2. When excited at 630 nm, it showed an emission band at 677 nm in DMSO with a fluorescence quantum yield of 0.13. The 1,3-diphenylisobenzofuran titration experiment gave a singlet oxygen quantum yield (ΦΔ) of ∼0.32. A mechanistic DNA photocleavage study revealed singlet oxygen as the reactive oxygen species (ROS). The complex with biotin and PEGylated-distyryl-BODIPY showed significantly higher cellular uptake in A549 cancer cells as compared to non-cancerous Beas-2B cells from flow cytometry, indicating selectivity towards cancer cells. A dichlorodihydrofluorescein diacetate assay showed cellular ROS generation. Confocal images revealed predominant internalization in the mitochondria. The prodrug showed remarkable photodynamic therapy (PDT) activity in cancerous A549 and multidrug-resistant MDA-MB-231 cells with a high photocytotoxicity index value (half-maximal inhibitory concentration (IC50): 0.61-1.54 µM in red light), while being non-toxic in the dark. The chemo-PDT activity was significantly less in non-tumorigenic lung epithelial cells (Beas-2B). The prodrug effectively triggered cellular apoptosis, which was confirmed by the Annexin V-FITC/propidium iodide assay, and the alteration of the mitochondrial membrane potential was substantiated by the JC-1 dye assay. The ß-tubulin immunofluorescence assay confirmed that incubating the cells with a light-treated complex resulted in the rapture of the cytoskeletal structure and the formation of apoptotic bodies. The results demonstrate that the prodrug triggered apoptosis via DNA damage, a reduction in mitochondrial function and disruption of the cytoskeletal framework.

Red light-activable biotinylated copper(II) complex-functionalized gold nanocomposite (Biotin-Cu@AuNP) towards targeted photodynamic therapy.

We report the synthesis and characterization of red-light activable gold nanoparticle functionalized with biotinylated copper(II) complex of general molecular formula, [Cu(L3)(L6)]-AuNPs (Biotin-Cu@AuNP), where L3 = N-(3-((E)-3,5-di-tert-butyl-2-hydroxybenzylideneamino)-4-hydroxyphenyl)-5-((3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide, L6 = 5-(1,2-dithiolan-3-yl)-N-(1,10-phenanthrolin-5-yl)pentanamide, which was explored for their photophysical, theoretical and photo-cytotoxic potentials. The nanoconjugate exhibits differential uptake in biotin positive and biotin negative cancer cells as well as normal cells. The nanoconjugate also shows remarkable photodynamic activity against biotin positive A549 (IC50: 13 µg/mL in red light; >150 µg/mL in dark) and HaCaT (IC50: 23 µg/mL in red light; >150 µg/mL in dark) cells under red light (600-720 nm, 30 Jcm-2) irradiation, with significantly high photo-indices (PI>15). The nanoconjugate is less toxic to HEK293T (biotin negative) and HPL1D (normal) cells. Confocal microscopy confirms preferential mitochondrial and partly cytoplasmic localization of Biotin-Cu@AuNP in A549 cells. Several photo-physical and theoretical studies reveal the red light-assisted generation of singlet oxygen (1O2) (Ф (1O2) =0.68) as a reactive oxygen species (ROS) which results in remarkable oxidative stress and mitochondrial membrane damage, leading to caspase 3/7-dependent apoptosis of A549 cells. Overall, the nanocomposite (Biotin-Cu@AuNP) exhibiting red light-assisted targeted photodynamic activity has emerged as the ideal next generation PDT agents.

BODIPY-Tagged Platinum(II) Curcumin Complexes for Endoplasmic Reticulum-Targeted Red Light PDT.

[Pt(RB)(Cur)]NO3 (RBC), [Pt(IRB)(Cur)]NO3 (IRBC), and [Pt(L)(Cur)]NO3 (PBC), where HCur is curcumin, L is 1-benzyl-2-(2-pyridyl)benzimidazole, and RB and IRB are red-light-active non-iodo and diiodo-BODIPY tagged to L, respectively, were synthesized and characterized, and their anticancer activities were studied (BODIPY, boron-dipyrromethene). RBC and IRBC displayed BODIPY-centered absorption bands within 615-635 nm along with the respective curcumin bands at 445 and 492 nm in 10% dimethyl sulfoxide (DMSO)-Dulbecco's phosphate-buffered saline (DPBS). Emission bands were observed at 723 and 845 nm for RBC and IRBC, respectively, in 10% DMSO-DPBS. RBC (ΦΔ, 0.27) and IRBC (ΦΔ, 0.40) generated singlet oxygen in red light (λ = 642 nm) as evidenced from 1,3-diphenylisobenzofuran (DPBF) titrations. The formation of 1O2 from BODIPY and HO• from the curcumin was evidenced from the mechanistic pUC19 DNA photocleavage studies. The BODIPY complexes showed photocytotoxicity in A549, HeLa, and MDA-MB-231 cells while being less toxic in the dark [IC50: 1.3-6.9 µM, red light; 7.2-12.8 µM, 400-700 nm visible light]. The emissive RBC displayed localization in the endoplasmic reticulum (ER). Apoptotic cell death was evidenced from the Annexin-V/fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay and green fluorescence in red light in the Fluo-4 AM assay due to ER stress, and mitochondrial dysfunction was evidenced from the 5,5,6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) assay in A549 cells.

Photocytotoxic cancer cell-targeting platinum(ii) complexes of glucose-appended curcumin and biotinylated 1,10-phenanthroline.

Mixed-ligand platinum(ii) complexes, [Pt(phen)(pacac)](NO3) (1), [Pt(phen)(cur)](NO3) (2), [Pt(bt-phen)(cur)](NO3) (3) and [Pt(phen)(scur)](NO3) (4), where phen is 1,10-phenanthroline, bt-phen is 5-biotin-1,10-phenanthroline, pacac is 1,3-diphenyl-1,3-propanedioate anion, Hcur is curcumin and Hscur is diglucosylcurcumin, were prepared, characterized and their anticancer activity studied. Complexes 2-4 showed absorption bands within 410-430 nm (ε, 2.1 × 104 to 2.8 × 104 M-1 cm-1) in 10% DMSO-DPBS (Dulbecco's phosphate-buffered saline) and emission bands near 530 nm (λex = 410-430 nm) with a fluorescence quantum yield (ΦF) value of ∼0.02. The curcumin complexes showed stability over a study period of 48 h. The photocytotoxicity was studied using human cervical HeLa, human liver HepG2, human breast cancer MDA-MB 231 and human lung adenocarcinoma A549 cancer cells along with human immortalized lung epithelial HPL1D as normal cells. Complexes 2-4 showed apoptotic photo-induced cell death in light of wavelength 400-700 nm (IC50, half maximal inhibitory concentration: 6-28 µM) by reactive oxygen species (ROS), while remaining inactive in the dark (IC50: 43-95 µM). The selectivity of the complexes 3 and 4 was enhanced significantly towards the cancer cells than towards the normal cells, thus making them targeted photochemotherapeutic agents. The ROS formation and mode of cell death were studied from 2',7'-dichlorofluorescein diacetate (DCFDA) and annexin-V/FITC (fluorescein isothiocyanate)-PI assays, respectively. Preferential nuclear and mitochondrial localization was evidenced from inductively coupled plasma mass spectrometry (ICP-MS) studies.