Biotin-Pt(IV)-Ru(II)-Boron-Dipyrromethene Prodrug as "Platin Bullet" for Targeted Chemo- and Photodynamic Therapy.

{"full_text": " pubs.acs.org/IC Article\n\n\n\n Biotin\u2212Pt(IV)\u2212Ru(II)\u2212Boron\u2212Dipyrromethene Prodrug as \u201cPlatin\n Bullet\u201d for Targeted Chemo- and Photodynamic Therapy\n Arpan Bera, Amrita Nepalia, Aarti Upadhyay, Deepak Kumar Saini,* and Akhil R. Chakravarty*\n Cite This: Inorg. Chem. 2024, 63, 17249\u221217262 Read Online\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n ABSTRACT: Using the principle of \u201cMagic Bullet\u201d, a cisplatin-derived\n platinum(IV) prodrug heterobimetallic Pt(IV)\u2212Ru(II) complex, cis,cis,trans-\n [Pt(NH3)2Cl2{Ru(tpy\u2212BODIPY)(tpy\u2212COO)}(biotin)]Cl2 (Pt\u2212Ru\u2212B, 2),\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 11:25:27 (UTC).\n\n\n\n\n having two axial ligands, namely, biotin as water-soluble B-vitamin for\n enhanced cellular uptake and a BODIPY\u2212ruthenium(II) (Ru\u2212B, 1)\n photosensitizer having N,N,N-donor tpy (4\u2032-phenyl-2,2\u2032:6\u2032,2\u2033-terpyridine)\n bonded to boron-dipyrromethene (BODIPY), is developed as a \u201cPlatin\n Bullet\u201d for targeted photodynamic therapy (PDT). Pt\u2212Ru\u2212B exhibited\n intense absorption near 500 nm and emission near 513 nm (\u03bbex = 488 nm) in\n a 10% dimethyl sulfoxide-Dulbecco\u2019s phosphate-buffered saline medium (pH\n 7.2). The BODIPY complex on light activation generates singlet oxygen as\n the reactive oxygen species (ROS) giving a quantum yield (\u03a6\u0394) of \u223c0.64\n from 1,3-diphenylisobenzofuran experiments. Pt\u2212Ru\u2212B exhibited prefer-\n ential cellular uptake in cancer cells over noncancerous cells. The dichlorodihydrofluorescein diacetate assay confirmed the\n generation of cellular ROS. Confocal images revealed its mitochondrial internalization. Pt\u2212Ru\u2212B showed submicromolar\n photocytotoxicity in visible light (400\u2212700 nm) in A549 and multidrug-resistant MDA-MB-231 cancer cells. It remained nontoxic in\n the dark and less toxic in nontumorigenic cells. Cellular apoptosis and alteration of the mitochondrial membrane potential were\n evidenced from the respective Annexin V-FITC/propidium iodide assay and JC-1 dye assay. A wound healing assay using A549 cells\n and Pt\u2212Ru\u2212B revealed inhibition of cancer cell migration, highlighting its potential as an antimetastatic agent.\n\n\n \u25a0 INTRODUCTION\n Cisplatin and related platin analogues are clinically approved\n significantly enhances the photocytotoxicity of the prodrug.16\n A related work showed that binding of Ru(II) to biotin and\n anticancer drugs.1\u22124 Their therapeutic efficacy is largely BODIPY in a heteroleptic complex significantly increases the\n affected by the inherent or acquired drug resistance due to generation of singlet oxygen compared to the Pt(IV)-based\n the absence of desirable selectivity.5\u22127 To circumvent these prodrug.17 This has prompted us to conceptualize a new\n predicaments, six-coordinate kinetically stable 5d6-Pt(IV) heterobimetallic prodrug molecule, namely, {Biotin\u2212Pt(IV)\u2212\n prodrugs derived from cisplatin are developed as alterna- Ru(II)\u2212tpy\u2212BODIPY} where Pt(IV) is bonded to biotin and\n tives.8\u221210 The pseudo-octahedral Pt(IV) complexes with their the {Ru(II)\u2212tpy\u2212BODIPY} moiety (1) as a photosensitizer\n remarkable stability ensure better circulation within the through a tpy\u2212COO\u2212 linker. Thus, a heterobimetallic\n bloodstream, thus minimizing the undesired reactivity that is cis,cis,trans-[Pt(NH3)2Cl2{Ru(tpy\u2212BODIPY)(tpy\u2212COO)}-\n often encountered by planar cisplatin with labile Pt\u2212Cl bonds. (biotin)]Cl2 (Pt\u2212Ru\u2212B, 2) complex is designed and prepared\n Moreover, two axial ligands in these Pt(IV) prodrugs offer a with an aim to release concomitantly two axial ligands from the\n versatile platform for customization, facilitating the incorpo- Pt(IV) prodrug on reduction forming: (i) {Ru(tpy\u2212\n ration of additional functionalities by allowing attachment of a BODIPY)(tpy\u2212COO)} (1, Ru\u2212B) for PDT activity, (ii)\n tumor targeting group for better selectivity, and a photo- biotin as therapeutically active molecule, and (iii) the chemo-\n sensitizer (PS) for type-II PDT (photodynamic therapy) active cisplatin analogue (Figure 1). The {Biotin\u2212Pt(IV)\u00b7\u00b7\u00b7\n activity (Figure 1).11\u221214 Targeted drug delivery being an Ru(II)\u2212tpy\u2212BODIPY} single molecular system in complex 2\n important concept of \u201cMagic Bullet\u201d, coined by Paul Ehrlich in\n 1907 for better efficacy and to reduce unwanted toxicity, is\n applicable for the Pt(IV) prodrug design where a cancer cell Received: July 22, 2024\n targeting group could be tagged in one axial position, while a Revised: August 22, 2024\n photosensitizer is appended at the other axial site for better Accepted: August 26, 2024\n therapeutic efficacy.15 We have recently shown that a Pt(IV) Published: September 5, 2024\n prodrug bearing boron-dipyrromethene (BODIPY) as a\n photosensitizer and vitamin biotin as two axial ligands\n\n \u00a9 2024 American Chemical Society https://doi.org/10.1021/acs.inorgchem.4c03083\n 17249 Inorg. Chem. 2024, 63, 17249\u221217262\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 1. Chemical structure of the complex cis,cis,trans-[Pt(NH3)2Cl2{Ru(tpy\u2212BODIPY)(tpy\u2212COO)}(biotin)]Cl2 (Pt\u2212Ru\u2212B) and the concept\nbehind the design of this prodrug. Pt\u2212Ru\u2212B generated chemo-active cisplatin and the photosensitizer upon reduction. The significant observations\nof this study are given as an inset (key points).\n\nhas two potentially labile axial linkers that can be cleaved photoactive Pt(IV)\u2212Ru(II)\u2212arene complexes, while Gibson et\nreadily by reducing agents with a two-electron Pt(IV) \u2192 Pt(II) al. documented Pt(IV)\u2212Au(I) complexes in their studies.34a,b\nreduction. This molecular design has proven to be significantly Our choice of biotin in 2 is to enhance the solubility and cell\nmore effective than the {Biotin\u2212Ru(II)\u2212tpy\u2212BODIPY} in 1 targeting properties of the complex as biotin-tagged Pt(IV)\nwhere a cleavage of the constituent bonds cannot be effected complexes reported by Guo and co-workers showed targeted\nby an external trigger. activity in breast cancer cells.35 Herein, we present the chemo-\n The choice of a photosensitizer (PS) in PDT is very PDT activity of a biotinylated heterobimetallic Pt(IV)\u2212Ru(II)\nimportant. Cisplatin cores that are covalently linked to prodrug (2) derived from cisplatin and the Ru\u2212B complex 1\nporphyrin/phthalocyanine dyes are used to achieve triple (Scheme 1). Significant results include: (i) photocytotoxicity\naction light-activated cytotoxicity, namely, chemotherapy, at submicromolar complex concentration involving singlet\nPDT, and cancer immunotherapy.18,19 Guo and co-workers oxygen as the ROS, (ii) high prodrug uptake in cancer cells\nreported the conjugation of a red-light active PDT agent with a compared to the normal cells, (iii) preferential mitochondrial\ncisplatin analogue as a chemotherapeutic agent in photo- localization, (iv) high molar extinction coefficient of the visible\nchemotherapy.20 \u201cPhotoactivated Chemotherapy\u201d (PACT) as band and high \u03a6\u0394 value, and (v) observation of antimetastatic\na new methodology is based on a Pt(IV) prodrug that on light activity. Complex 2 presents a rare single molecular platform\nactivation releases two axial ligands generating cytotoxic that rapidly generates three therapeutically active constituents\nspecies in a controlled manner increasing drug efficacy.21 on reduction.\nThe PACT methodology offers further scope of molecular\ndesign by incorporating PDT-active axial ligand, while a tumor\ntargeting moiety could be at the other axial site thus allowing\none to have a dual \u201cChemo-PDT\u201d effect from a single\n \u25a0 RESULTS AND DISCUSSION\n Synthesis and General Properties. The precursor\nmolecular platform on light activation. The effectiveness of complex, cis,cis,trans-[Pt(NH3)2Cl2(OH)2] (Oxoplatin), was\nPDT using organic porphyrin dyes is limited by sluggish prepared using a technique previously described by Dhar et\ndiffusion rates, short lifetimes of the ROS, and the lack of al.36 Following previously published protocols, ligand tpy\u2212\ntargeted delivery at the diseased cells besides skin sensitivity BOD (L1) was synthesized involving a reaction of 2,4-dimethyl\nand hepatotoxicity.22\u221228 We have chosen a BODIPY (boron- pyrrole and tpy\u2212CHO (tpy, terpyridine) in dichloromethane,\ndipyrromethene) dye as a photosensitizer for its remarkable while tpy\u2212COOH (L2) was synthesized from 2-acetylpyridine\nphoto- and redox stability. Moreover, BODIPY cores are and 4-formyl benzoic acid reaction (Schemes S1 and S2,\namenable to modifications allowing one to have optimal Supporting Information).17,37 As shown in Scheme 1, ligand L1\nabsorption and emission properties required for cellular was first reacted with [Ru(DMSO)4Cl2] in an alcoholic\nimaging with high fluorescence quantum yield (\u03a6F) and/or solution, which resulted in the formation of [RuL1(DMSO)-\nsubmicromolar PDT activity with high singlet oxygen quantum Cl2] as an intermediate (Scheme S3, Supporting Information).\nyield (\u03a6\u0394).29\u221231 Inclusion of two heavy metals, namely, Ru(II) This ruthenium intermediate was then treated with L2 to\nand Pt(IV), within a single molecular framework is intended produce the ruthenium\u2212bis-terpyridine complex in moderate\nfor cumulative or potentially cooperative outcomes with high yield. The synthetic procedure for the pseudo-octahedral 5d6-\n\u03a6\u0394 values as recently reported for Pt(IV)\u2212Ru(II) polypyridyl Pt(IV) metallodrug {Pt\u2212Ru\u2212B} is presented in Scheme 1.\nprodrugs by Gasser et al. while simultaneously exhibiting Our attempt for direct substitution of two axially bound\nanticancer and imaging properties with organelle-targeting hydroxyl groups in Oxoplatin by the carboxyl group of biotin\ncapabilities, as evidenced in an Ir(III)\u2212Pt(IV) prodrug and the \u2212OOC\u2212tpy\u2212Ru\u2212B complex gave undesirable\nreported by Sasmal et al.32,33 Zhu et al. reported non- reactivity. Therefore, activation of the carboxyl group for\n 17250 https://doi.org/10.1021/acs.inorgchem.4c03083\n Inorg. Chem. 2024, 63, 17249\u221217262\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nScheme 1. Synthetic Scheme for the Complexes Ru\u2212B (1) peak near \u03b4 6.29, which was assigned to the pyrrole \u2212CH\nand Pt\u2212Ru\u2212B (2): (i) EtOH, 80\u00b0C, N2 Atmosphere and (ii) proton. The Pt(IV)-bonded NH3 protons appeared as a broad\nTBTU, TEA, Dry DMF, 45\u00b0C, 48 ha signal within 6.81\u22126.68 ppm.35 The aliphatic \u2212CH2\u2212 protons\n that are specific to the biotin moiety appeared as a broad signal\n extending from 1.67 to 1.31 ppm. The resonance signals of the\n aromatic carbon were observed in the range of 116\u2212163 ppm.\n At 0.67 ppm, a peak with characteristic features was observed\n in the 11B NMR spectrum. The electrospray ionization mass\n spectrometry (ESI-MS) in methanol of the complexes gave a\n prominent peak assignable to the [M-2Cl]2+ species. The\n isotopic distribution analysis revealed a variation of 0.5 in the\n m/z value suggesting dipositive nature of the species.38\n Furthermore, comparing the experimental isotopic distribution\n to the simulated counterpart has led to the identification of the\n species. The complexes are 1:2 electrolytic giving molar\n conductance values of \u223c140 S m2 mol\u22121 in dimethylformamide\n (DMF) indicating their dipositive nature.39\n Photophysical Properties. The UV\u2212visible absorption\n spectra of prodrug Pt\u2212Ru\u2212B and complex Ru\u2212B in a solvent\n mixture of 10% DMSO and DPBS exhibited a strong\n absorption band centered around 500 nm with high molar\n extinction coefficient values (Figure 2a). The distinctive\n\n\n\n\na\n Reagents used: EtOH, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethy-\nlaminium tetrafluoroborate (TBTU), triethylamine (TEA), and N,N-\ndimethylformamide (DMF).\n Figure 2. (a) UV\u2212visible absorption and (b) emission spectra (\u03bbex =\nboth biotin and the Ru\u2212B unit was achieved using either 1-(3- 488 nm) of complex Ru\u2212B and Pt\u2212Ru\u2212B in a 10% DMSO/DPBS\n(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride medium (pH = 7.2) (color code: black, complex Ru\u2212B and red, Pt\u2212\nand N-hydroxy succinimide or 2-(1H-benzotriazole-1-yl)- Ru\u2212B).\n1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU). Multi-\nnuclear NMR techniques 1H, 13C, and 11B and ESI-MS were electronic absorption arises from a spin-allowed \u03c0\u2212\u03c0*\nused to characterize the prodrug Pt\u2212Ru\u2212B and Ru\u2212B transition within the BODIPY framework, which imparts a\ncomplex. Selected physicochemical data are presented in sharp characteristic to the spectrum.40 Additionally, the\nTable 1 (Figures S1\u2212S14, Supporting Information). The prominent peak includes a Ru(II)-d\u03c0 to terpyridine (tpy)-\u03c0*\npurity of the complexes was ascertained from the elemental (MLCT) transition. This latter appears as a minor hump on\nanalysis data. DMSO-d6 was used for NMR spectroscopy of the the higher-energy region of the UV\u2212visible spectra. The\ncomplexes. On purification by column chromatography, the absorption observed within 300\u2212400 nm is a result of the\ncomplexes were isolated in the form of their chloride salts. The \u03c0\u2212\u03c0* transitions occurring within the polypyridyl subunits.41\nsinglet 1H NMR spectral peaks observed between \u03b4 \u223c2.5 and Using 488 nm excitation, the emission spectra of the\n1.5 were assigned to the pyrrole \u2212CH3 protons, along with a complexes were recorded in a mixture of 10% DMSO/DPBS\n\nTable 1. Selected Physicochemical Data for Complexes Ru\u2212B and Pt\u2212Ru\u2212B\n entry Ru\u2212B (1) Pt\u2212Ru\u2212B (2)\nUV\u2212visible: \u03bbmax/nm (\u03b5/M\u22121 cm\u22121)a 317 (5.42 \u00d7 104), 478 (2.18 \u00d7 104), 500 316 (4.15 \u00d7 104), 477 (2.02 \u00d7 104), 500\n (4.88 \u00d7 104) (4.43 \u00d7 104)\nemission: \u03bbem/nmb(\u03a6F) 512 (<0.01) 513 (<0.01)\n\u03a6\u0394 (1O2)c 0.63 0.64\n1\n H NMR chemical shift (\u03b4, ppm) of \u03b3-proton of BODIPYd 6.29 6.29\n\u039bMe/S m2 mol\u22121 138 141\nCV dataf: Pt(IV)\u2212Pt(II) (Epc/V), BODIPY {Ef/V) [\u0394Ep/mV]}, \u22121.64 V (65 mV), \u22122.06 V (82 mV), \u22121.41 V, \u22121.72 V (75 mV), \u22122.19 V (132\n Ru(III)/Ru(II) (Epa/V) +0.78 V mV), +0.72 V\n\na\n In a 10% DMSO/DPBS medium. sh, shoulder. bEmission wavelength and fluorescence quantum yield (\u03a6F). The excitation wavelength (\u03bbex) =\n630 nm. cSinglet oxygen quantum yield \u03a6\u0394 in DMSO. The value for tpy\u2212BODIPY is 0.06. dIn DMSO-d6. eMolar conductivity in DMF. fEf (V vs\nFc/Fc+), \u0394Ep values at a scan rate of 100 mV s\u22121. Epa and Epc are the anodic and cathodic peak potential, respectively, at a scan rate of 100 mV s\u22121.\nEf is 0.5 \u00d7 (Epa + Epc).\n\n 17251 https://doi.org/10.1021/acs.inorgchem.4c03083\n Inorg. Chem. 2024, 63, 17249\u221217262\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n(Figure 2b). Notably, the complexes exhibited a subdued dimethyl sulfoxide, methanol, and ethanol. The complexes are\nemission centered at around 513 nm, with a fluorescence moderately soluble in acetonitrile and essentially insoluble in\nquantum yield (\u03a6F) value below 0.01. The quenching in hydrocarbons and diethyl ether. The complexes show low\nBODIPY fluorescence observed in these complexes is solubility in water and an aqueous buffer. The stability of the\nattributed to the facile intersystem crossing (ISC) due to the prodrug Pt\u2212Ru\u2212B under physiological conditions was studied\npresence of heavy metal atoms platinum and ruthenium. by absorption spectroscopy. The complexes in the dark\n Electrochemistry. The Pt(IV)-based prodrugs are known showed good stability in a mixture of 10% dimethyl sulfoxide\nto exhibit a characteristic one-step two-electron transfer (DMSO) and DPBS buffer (pH, 7.2) over a period of 48 h\nprocess leading to the conversion of pseudo-octahedral d6- with negligible reduction of the absorption intensity at 500 nm\nPt(IV) to planar d8-Pt(II) species.42 To investigate the redox (Figure S19, Supporting Information). The photostability of\nbehavior of the present complexes, cyclic voltammetry (CV) the prodrug was investigated with the same solvent mixture,\nexperiments were performed in an anhydrous DMF solvent using a broadband photoreactor of 400\u2212700 nm as the light\nusing tetrabutylammonium hexafluorophosphate (TBAHP) as source, and the complexes showed only a marginal reduction in\nthe supporting electrolyte (Table 1). The Ru\u2212B complex gave absorption intensity, suggesting their good photostability\na distinct and irreversible Ru(II) to Ru(III) oxidation process (Figure S20, Supporting Information). Furthermore, stability\nnear +0.78 V vs Fc/Fc+ similar to those reported for studies were conducted under different pH conditions,\n[Ru(tpy)2]2+.43 The Ru\u2212B complex in the cathodic scan specifically at high pH of 9 and low pH of 4. In both cases,\nexhibited a BODIPY-based quasi-reversible voltammetric prodrug Pt\u2212Ru\u2212B exhibited negligible changes in absorbance\nresponse at \u22121.64 V (\u0394Ep = 65 mV at 100 mV s\u22121) (Figures throughout the 48 h duration. The observed stability under\nS15 and S16, Supporting Information). Another cathodic highly acidic conditions implies the potential for oral\nresponse observed at \u22122.06 V is attributed to the reduction of administration in clinical treatments (Figure S21, Supporting\nthe terpyridyl ligand bound to the Ru(II) center. The Pt\u2212Ru\u2212 Information).\nB complex showed similar redox properties as in Ru\u2212B with Prodrug Activation. The prodrug is designed to release\nthe exception that it exhibited an additional voltammetric three distinct therapeutically important species with specific\nresponse characterized by an irreversible cathodic process functionalities upon intracellular Pt(IV) \u2192 Pt(II) reduction.47\nassignable to a one-step, two-electron transfer Pt(IV) to Pt(II) First species is cisplatin or its analogue, a chemotherapeutic\nreduction (Figure S15, Supporting Information). This redox agent known for its anticancer properties. The second species\nresponse occurred at a potential of \u22121.41 V (vs Fc/Fc+) at a is a ruthenium\u2212BODIPY dyad, which exhibits remarkable\nscan rate of 100 mV s\u22121. The irreversibility of this process is PDT activity, enabling the generation of reactive oxygen\ndue to a structural change resulting from the dissociation of\n species upon light activation. The third species is biotin, which\ntwo axial ligands from the six-coordinate 5d6-Pt(IV) prodrug,\n is tagged to facilitate cancer cell targeting and enhance the\nleading to the formation of a four-coordinate 5d8-Pt(II)\n selectivity of the prodrug toward the cancer cells. Together,\nspecies. The absence of an anodic peak corresponding to the\n they work synergistically to provide a multidimensional\nobserved cathodic peak at various scan rates suggested the\n modality for cancer treatment. The release of the components\ninvolvement of an electron transfer with a simultaneous\nchemical transformation (EC) mechanism in this metal- was studied by ESI-MS spectrometry. To investigate the redox\nmediated redox process.44 While this Pt-based redox process transformation of the prodrug, a DMSO-d6 solution containing\noccurred beyond the biological potential window of +0.4 to the prodrug at a concentration of 100 \u03bcM was subjected to a\n\u22120.4 V (vs SCE), the redox transformation could still be 24 h incubation period in the presence of sodium ascorbate\nachieved through chemical reduction of the complex. (500 \u03bcM), which is a cellular reducing agent. Following the\n Theoretical Studies. Density Functional Theory (DFT) incubation, the resulting solution was analyzed by using ESI-\ncalculations were conducted using Gaussian 09W to elucidate MS in diluted HPLC methanol. The ESI-MS data showed the\nboth the structural characteristics and photophysical properties presence of reduction products such as unbound Ru\u2212bis-\nof the Pt\u2212Ru\u2212B complex.45,46 The Pt(IV) core is surrounded terpyridine complex with BODIPY, biotin, and cisplatin species\nby six ligands adopting a cis,cis,trans pseudo-octahedral (Figure S22, Supporting Information). The prodrug was\narrangement, having two axial Pt\u2212O bonds with a length of incubated at a concentration of 100 \u03bcM with 500 \u03bcM sodium\n2.04 \u00c5 (Figures S17 and S18 and Tables S1 and S2, Supporting ascorbate, and subsequently, the mixture was exposed to visible\nInformation). The boron atom within the BODIPY unit adopts light (400\u2212700 nm, 10 Joules/cm2). Similarly, peaks were\na tetrahedral geometry. HOMO\u2212LUMO diagrams were observed for the unbound ruthenium\u2212bis-terpyridine complex\nconstructed based on optimized geometry. Notably, the alongside its conjugates with BODIPY, biotin, and cisplatin\nHOMO is situated on the biotin moiety, while the LUMO is (Figure S23, Supporting Information). However, when\ncentered on the ruthenium component. The energy difference subjected solely to light exposure (400\u2212700 nm, 10 Joules/\nbetween the HOMO and LUMO states is 1.03 eV. Employing cm2), the mass peak corresponding to the Pt\u2212Ru\u2212B complex\ntime-dependent density functional theory (TD-DFT), the- remained unchanged even after 24 h of continuous\noretical calculations were performed to predict the electronic illumination (Figure S24, Supporting Information). The\ntransitions within the Pt\u2212Ru\u2212B complex. Key findings prodrug seemed to be reduced only in the presence of\nobtained from these calculations are presented in Table S3 chemical reductants. Additionally, to replicate the intracellular\nin the Supporting Information. The Pt\u2212Ru\u2212B complex gave a DNA binding characteristic of the released cisplatin, a\n\u03bbmax value of 486 nm with a good oscillator strength of 0.28 in truncated model of DNA known as 9-ethylguanine (9-EtG)\nDMSO, and this data is consistent with the experimentally was added to a small portion of the sodium ascorbate-\nobserved \u03bbmax value of 500 nm. incubated sample. The analysis of mass spectra in HPLC\n Solubility and Stability. The complexes exhibit good methanol indicated the presence of a Pt(II)-9-EtG adduct,\nsolubility in various solvents, including dimethylformamide, specifically [Pt(NH3)2Cl(9-EtG)]+ (m/z = 444.0695), suggest-\n 17252 https://doi.org/10.1021/acs.inorgchem.4c03083\n Inorg. Chem. 2024, 63, 17249\u221217262\n\fInorganic Chemistry pubs.acs.org/IC Article\n\ning the formation of a complex of cisplatin with 9-EtG (Figure resulting in the production of highly toxic singlet oxygen\nS25, Supporting Information). (1O2).50 The advantage of singlet oxygen in PDT is that there\n Light-Induced Singlet Oxygen Generation. To assess are no naturally occurring enzymes specifically designed to\nthe potential of the complexes in generating singlet oxygen neutralize or counteract this ROS. As a result, singlet oxygen\n(1O2) upon light activation, a trap experiment was conducted produced through this pathway can exert its potent effects\nusing 1,3-diphenylisobenzofuran (DPBF), which serves as a within a confined range, making it an advantageous strategy for\nscavenger that readily reacts with singlet oxygen to form targeted therapeutic applications. To ascertain the type of ROS\nendoperoxide.48 This reaction leads to a reduction in the generated, supercoiled (SC) plasmid DNA served as a probe in\nintensity of the DPBF-based absorption band, typically the experiments. The experimental setup involved utilizing a 10\nobserved at around 417 nm. The singlet oxygen quantum \u03bcM concentration of the Pt\u2212Ru\u2212B complex and a 400\u2212700\nyield (\u03a6\u0394) for both Pt\u2212Ru\u2212B and Ru\u2212B was determined by nm visible light photoreactor as the light source for irradiation.\nanalyzing the graph of \u0394OD (change in optical density) Upon light exposure of the prodrug in the presence of SC\nplotted against light irradiation time. Rose Bengal (RB) was pUC19 DNA, a significant conversion of the DNA from its\nused as a standard (\u03a6\u0394 \u223c0.76 in DMSO). Using this approach, supercoiled form to \u223c70% nicked circular (NC) form was\nthe \u03a6\u0394 values for Pt\u2212Ru\u2212B and Ru\u2212B were 0.64 and 0.63, observed (Figure 3b). The presence of hydroxyl radicals (such\nrespectively (Figure 3a, Figure S26, Supporting Information). as potassium iodide) and peroxide radical scavengers (catalase)\n resulted in only minor changes in the DNA scission activity of\n the prodrug. However, singlet oxygen quenchers such as\n sodium azide (NaN 3 ), 2,2,6,6-tetramethyl-4-piperidone\n (TEMP), and 1,5-dihydroxynaphthalene (DHN) resulted in\n a significant decrease in the percentage of NC formation. The\n DNA photocleavage data suggest the formation of singlet\n oxygen during the DNA photocleavage process. The addition\n of Tiron and SOD (superoxide radical scavenger) also showed\n some inhibitory effects, indicating their involvement in a type-I\n pathway. The literature reports have shown that photoexcited\n Ru(II)-organic chromophore dyads display similar electron\n transfer reactions with biological substrates.51 Overall, the\n results indicate production of both superoxide and singlet\n oxygen by the prodrug from the respective type-I and type-II\n photosensitization processes.\n In previous reports, the utility and importance of molecular\n oxygen in PDT were observed from an experiment under\n argon that showed a significant decrease in the %NC DNA\n formation. Despite this reduced %NC, photoinduced DNA\n cleavage was still evident from the experiment suggesting the\nFigure 3. (a) Reduction in the absorption spectral intensity of DPBF possibility of cleavage under hypoxic conditions without\nat 417 nm in the presence of Pt\u2212Ru\u2212B (2) (light source: 400\u2212700 involving any ROS but from direct interaction with the\nnm photoreactor). The spectra were recorded at 5 s time intervals. biomolecule.52,53\n(b) Gel electrophoresis image showing photocleavage of pUC19 Cellular Uptake. The cellular uptake of the prodrug in\nDNA by complex 2 (10 \u03bcM) upon 532 nm green diode laser exposure A549 cancer cells was investigated from a flow cytometry\nfor 5 min. Lanes are (1) DNA + 2 in light, (2) DNA control in light, experiment at various time intervals of 2, 4, 5, and 6 h. The\n(3) DNA + 2 in dark, (4) DNA + 2 + NaN3 (6 \u03bcM) in light, (5)\nDNA + 2 + KI (6 \u03bcM) in light, (6) DNA + 2 + Tiron (6 \u03bcM) in light,\n fluorescence was measured by using an excitation wavelength\n(7) DNA + 2 + SOD (4 units) in light, (8) DNA + 2 + TEMP (6 of 495 nm. The molecule\u2019s capability to penetrate and\n\u03bcM) in light, (9) DNA + 2 + DHN (6 \u03bcM) in light, (10) DNA + 2 in accumulate within the cancer cells is vital for its anticancer\nargon under light irradiation, and (11) DNA + 2 + catalase (4 units) activity. A notable increase in the fluorescence intensity of the\nin light. cells treated with the complex provided clear evidence of the\n internalization and accumulation of the metallodrug within the\nThe values are remarkable considering the \u03a6\u0394 value of ligand cancer cells. Furthermore, a significant enhancement in the\ntpy\u2212BODIPY as only 0.06, which is significantly low compared fluorescence intensity was observed with an extended\nto the complexes highlighting the heavy metal effect in singlet incubation time, specifically from 2 to 4 h, suggesting an\noxygen generation and the utility of heavy metal for enhanced increased uptake and retention of the prodrug over time.\nPDT activity.49 Notably, the complex exhibited time-dependent enhancement\n DNA Photocleavage and the Nature of ROS. Photo- in cellular uptake, and the peak of incorporation was achieved\ndynamic therapy (PDT) relies on the generation of reactive following a 4 h incubation period (Figure 4a). To investigate\noxygen species (ROS). There exist two primary mechanistic the cancer cell targeting efficacy of the compound Pt\u2212Ru\u2212B,\npathways, known as type-I PDT and type-II PDT, each the cellular uptake was examined in both cancerous A549 cells\ncontributing differently to the therapeutic effect. The type-I and noncancerous Beas-2B cells. More positive shift in the\nprocess is based on an electron transfer mechanism with histogram, determined from the histogram median value, was\nbiomolecules. This process leads to the formation of peroxides, observed in A549 cells, which signifies a stronger uptake of the\nhydroxyl radicals, and superoxide anion radicals. In a type-II drug for cancer cells in comparison to normal cells (Figure 4a\nprocess, the excited triplet state of the photosensitizer can for A549 cells and Figure 4b for Beas-2B cells). In addition,\ndirectly transfer energy to molecular triplet oxygen (3O2), various experiments were conducted to gain insights into the\n 17253 https://doi.org/10.1021/acs.inorgchem.4c03083\n Inorg. Chem. 2024, 63, 17249\u221217262\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n of 4 h, after which they were subjected to light treatment.\n Simultaneously, a separate set of experiments were conducted\n without light treatment to determine the inherent cytotoxicity\n of the molecules. Subsequently, the cells were incubated for an\n additional 20 h, followed by the determination of the half-\n maximum inhibitory concentration (IC50) using the MTT\n assay. During the light treatment, the photosensitizers were\n activated by a visible light source (400\u2212700 nm photoreactor)\n using a light dose of approximately 5 Joules/cm2 for a duration\n of 15 min. In the tested cell lines, the complexes were nontoxic\n nature in the absence of photoactivation as evidenced from the\n IC50 values exceeding 100 \u03bcM. Upon photoactivation, the\n biotin-conjugated prodrug yielded an IC50 value of \u223c0.022 \u03bcM\n in A549 and \u223c0.050 \u03bcM in MDA-MB-231 cancer cells (Figure\n 5, Table 2). In contrast, under similar conditions, Ru\u2212B\n\n\n\n\nFigure 4. Cellular uptake analysis by flow cytometry for A549 (a) and\nBeas-2B (b) cells that were incubated with Pt\u2212Ru\u2212B (10 \u03bcM) at 37 Figure 5. Cell viability profiles from the MTT assay of Pt\u2212Ru\u2212B in\n\u00b0C for 4 h. The higher shift in the band position for Pt\u2212Ru\u2212B in (a) A549 cells, (b) MDA-MB-231 cells, and (c) Beas-2B cells on light\ncancerous A549 cells in panel a compared to Beas-2B cells in panel b exposure (green light photoreactor of 400\u2212700 nm, dosage: 5 Joules/\nindicates higher cellular uptake in the cancer cells than in the normal cm2) and in darkness (black).\ncells. Untreated cells were used as a control.\n Table 2. Photocytotoxicity (in \u03bcM) of Pt\u2212Ru\u2212B Prodrug\ncellular uptake pathway of the prodrug. The cellular uptake of (2) and Ru\u2212B Complex (1)a\nPt\u2212Ru\u2212B was found to be significantly reduced when the cells A549/lighta MDA-MB-231/ Beas-2B/lighta\nwere preincubated with biotin, suggesting the involvement of entry [dark] (PI) lighta [dark] (PI) [dark] (PI)\nmultivitamin transporters (SMVTs) in facilitating the cellular Pt\u2212Ru\u2212B 0.022 \u00b1 0.004 0.050 \u00b1 0.003 1.29 \u00b1 0.20\nincorporation of the prodrug (Figure S27, Supporting [>100] (>4545) [>100] (>2000) [>100] (>77)\nInformation). To emphasize the cancer cell targeting capability Ru\u2212B 0.295 \u00b1 0.060 0.545 \u00b1 0.050 1.88 \u00b1 0.50\n [>100] (>338) [>100] (>183) [>100] (>53)\nof the prodrug, the cellular uptake was examined in both cisplatin + 0.280 \u00b1 0.083 0.537 \u00b1 0.06 1.99 \u00b1 0.04\ncancerous A549 cells and noncancerous Beas-2B cells derived Ru\u2212B + [34.24 \u00b1 1.6] [92.32 \u00b1 1.3] [55.68 \u00b1 2.5]\nfrom the same tissue of origin, specifically the lungs. biotinb (122) (172) (28)\n Photocytotoxicity. The evaluation of cytotoxicity of Pt\u2212 cisplatinc 32.5 \u00b1 2.5\n [35.0 \u00b1 2.2]\nRu\u2212B and Ru\u2212B was carried out using the MTT (3-(4,5- a\ndimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Light source for Pt\u2212Ru\u2212B and Ru\u2212B was a Luzchem photoreactor\nThe cytotoxicity of the complexes induced by light was (\u03bb = 400\u2212700 nm, dose: 5 Joules/cm2). PI is photocytotoxicity index\nassessed on three cell lines: A549 (human lung adenocarcino- [PI = IC50 (dark)/IC50 (light)]. A549 and MDA-MB-231 are the\n cancer cells, while Beas-2B was used as a normal cell. bA mixture of\nma), MDA-MB-231 (human epithelial breast adenocarcino- cisplatin, complex Ru\u2212B, and biotin in a 1:1:1 molar ratio. cData\nma), and Beas-2B (human nontumorigenic lung epithelial cell taken from ref 16.\nline).16 The selection of the A549 cell line was based on its\noverexpression of sodium-dependent multivitamin transporter\n(SMVT) receptors, while the MDA-MB-231 cell line was exhibited relatively higher IC50 values ranging from 0.30 to\nchosen for its inherent multidrug resistance property.54,55 0.55 \u03bcM (Figure S28, Supporting Information). No cytotox-\nAdditionally, to confirm the targeted photodynamic effect of icity was observed in the Beas-2B cell line under dark\nthe prodrug on cancer cells compared to normal cells, the conditions, indicating its inactivity. The photocytotoxicity\ncytotoxicity of the prodrug was also assessed in Beas-2B index (PI), which is determined by calculating the ratio of dark\nnormal cells. and light IC50 values, serves as a measure of the therapeutic\n The experimental procedure involved incubation of the cells efficacy of a drug in PDT. A higher PI value signifies a reduced\nwith increasing concentrations of the complexes for a duration occurrence of off-site toxicity caused by the administered\n 17254 https://doi.org/10.1021/acs.inorgchem.4c03083\n Inorg. Chem. 2024, 63, 17249\u221217262\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 6. Confocal microscopy images of complex Pt\u2212Ru\u2212B in A549 cells recorded after 4 h of incubation using nuclear staining Hoechst dye and\nMito Tracker Red: (i) Hoechst dye, (ii) Mito Tracker Red, (iii) complex Pt\u2212Ru\u2212B, (iv) bright field, (v) merged without bright field, and (vi)\nmerged image with a bright field. The scale bar is 50 \u03bcm. Panel v indicates significant mitochondrial localization of the prodrug.\n\nphotosensitizer. In the cancerous A549 cell line, Pt\u2212Ru\u2212B indicating the potential of the complex to serve as a visualizing\nexhibited a remarkable PI value exceeding 4500. However, in agent. By examining different images captured with the nuclear\nthe nontumorigenic Beas-2B cells, the PI value was staining blue emitting Hoechst dye, the green emission of the\nsignificantly low at \u223c77, implying a reduced phototoxicity. complex, MitoTracker deep red (MTR) as a mitochondrial\nFurthermore, an MTT assay was done on the A549, MDA- marker, lysosome tracker deep red (LTR), and ER tracker\nMB-231, and Beas-2B cells using a combination of cisplatin, deep red (ERR), it became apparent that there is no significant\nbiotin, and Ru\u2212B in a 1:1:1 molar ratio (Figure S28, overlap between the complex emission and nuclear staining\nSupporting Information). The IC50 values observed under dye. However, the Pearson\u2019s correlation coefficients between\nlight conditions were 0.28, 0.54, and 1.98 \u03bcM, respectively, the complex-based fluorescence and mito, lyso and ER tracker\nwhereas under dark conditions, the respective IC50 values were dyes are found to be \u223c0.71, 0.32, and 0.15, respectively (Figure\n34.24, 92.32, and 55.68 \u03bcM. Our previously reported S29, Supporting Information). Pearson\u2019s correlation coefficient\ncompound gave IC50 values between 0.61 and 1.54 \u03bcM in (PCC) value indicates preferential localization of the prodrug\nthe tested cancer cell lines when exposed to light at Pt\u2212Ru\u2212B within the mitochondria with a PCC of \u223c0.71\nwavelengths of 600\u2212720 nm with a light dose of 30 Joules/ (Figure 6).\ncm2 and exhibited a photocytotoxicity index within 65\u2212163. Cellular ROS Generation. PDT involves generation of\nThis significant enhancement in cytotoxicity is attributed to reactive oxygen species (ROS) by type-I and/or type-II\nthe increased singlet oxygen production due to the altered photoprocesses.56 To investigate the in vitro photoinduced\ncoordination environment of the BODIPY photosensitizer, production of ROS by Pt\u2212Ru\u2212B in A549 cells, a 2\u2032,7\u2032-\nspecifically the polypyridyl system of ruthenium rather than its dichlorofluorescein diacetate (DCFDA) assay was carried out.\ndirect coordination to the Pt\u2212OH bond. A related study Upon cellular uptake, this cell-permeable dye undergoes\ninvolving a heteroleptic biotin-containing BODIPY\u2212Ru\u2212bis- hydrolysis by esterase enzymes and subsequently undergoes\nterpyridine complex gave an IC50 value of 0.16 \u03bcM in A549 oxidation by ROS to form fluorescent DCF (2,7-dichloro-\ncancer cells under visible light exposure with a dose of 2.2 fluorescein).57 The prodrug-treated A549 cells were subjected\nJoules/cm2 and a lower PI value \u223c625 when compared to over to incubation with DCFDA. Subsequently, one set of cells was\n4545 obtained in this work. The MTT assay results strongly exposed to visible light (400\u2212700 nm), while the other set was\nsuggest the synergistic action of Pt\u2212Ru\u2212B. left in the dark. The fluorescence intensity measurement\n Intracellular Localization Analysis by Microscopy. exhibited negligible alterations within the dark-maintained set,\nSpecific intracellular localization of a targeted cancer drug while a noteworthy augmentation in fluorescence intensity was\nmolecule to a cellular organelle is crucial for understanding its observed within the light-exposed set of cells, indicating ROS\npossible mechanism of action, efficacy, and therapeutic impact. generation upon light exposure (Figure 7).\nConfocal imaging is a convenient tool for cellular localization Wound Healing Assay. The antimetastatic potential of\nstudies. The fluorescence property of the prodrug Pt\u2212Ru\u2212B the prodrug Pt\u2212Ru\u2212B in A549 cells was studied using a\n(2) was used as a diagnostic tool for imaging A549 cancer cells scratch-wound healing assay to evaluate the migratory\non 4 h of incubation in the dark, followed by visualization characteristics, including speed, persistence, and polarity, of a\nusing a confocal laser scanning microscope (CLSM). On 488 confluent monolayer of A549 cells. Specifically, the perform-\nnm laser excitation, the cells exhibited a green emission, ance of A549 cells treated with the prodrug for a duration of 12\n 17255 https://doi.org/10.1021/acs.inorgchem.4c03083\n Inorg. Chem. 2024, 63, 17249\u221217262\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n wound area until it eventually disappeared. This observation\n serves as an indication of the notable migratory capacity of\n A549 cells under the given conditions. In contrast, following a\n 12 h treatment with 50 \u03bcM prodrug, a notable suppression of\n A549 cell migration was observed, as evidenced by the reduced\n closure of the wound borders. The results obtained from the\n wound healing assay demonstrated that the prodrug complex\n Pt\u2212Ru\u2212B inhibits cancer cell migration, potentially indicating\n antimetastatic behavior.\n Mitochondrial Dysfunction. The mitochondria, known as\n the cellular powerhouses with ATP (adenosine triphosphate)\n generation, play important role in cellular metabolism.58\n However, an excessive accumulation of ROS within mitochon-\n dria could disrupt mitochondrial function, ultimately leading to\n a decline in ATP content. The loss of mitochondrial\n membrane potential (\u0394\u03a8m) serves as a prominent indicator\n of mitochondrial dysfunction. The modulation of \u0394\u03a8m was\n evaluated using a JC-1 (5,5,6,6\u2032-tetrachloro-1,1\u2032,3,3\u2032-tetrae-\nFigure 7. DCFDA assay to assess the formation of ROS in the A549\ncells upon 10 min photoexposure of Pt\u2212Ru\u2212B (0.5 \u03bcM) after\n thylbenzimidazoyl-carbocyanine iodide) dye assay. Upon entry\nincubation for a duration of 4 h in dark or when maintained in dark. into the mitochondria, the cationic lipophilic JC-1 dye\nThe bars from left are (1) cells alone, (2) cells treated with DCFDA accumulates and forms red-emitting aggregates around the\nand Pt\u2212Ru\u2212B in the dark, (3) cells treated with DCFDA and Pt\u2212 negatively charged mitochondria. However, in apoptotic cancer\nRu\u2212B in light (400\u2212700 nm), and (4) cells treated with H2O2. The cells characterized by a collapsing \u0394\u03a8m, the dye fails to\nDCFDA fluorescent counts were subsequently normalized by cell aggregate into red-emitting clusters and remains as a\nnumber, and w.r.t refers to with respect to. Statistical analysis was monomeric green dye dispersed within the cytoplasm.\nperformed using One Way ANOVA analysis and followed by Tukey\u2019s Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone\nmultiple comparison test (**p < 0.01 and ****p < 0.00001). All the\ndata sets were obtained from three sets of independent experiments.\n (FCCP) was used as a positive control, which induces\n acidification within the mitochondria, resulting in inhibition\n of ATP production. In the absence of photoexposure, the\nh was compared with that of the untreated cells. As shown in prodrug-treated cells exhibited an intense red emission\nFigure 8, the wound borders in the control medium upon the stemming from J-aggregates. Conversely, under the influence\ninitial scratching of the monolayer were distinctly defined. of light exposure, cells incubated with the prodrug displayed a\nHowever, with the progression of time, these borders gradually prominent green emission attributed to the presence of J-\nmigrated toward the center, resulting in a reduction of the monomers (Figure 9a). The observations provide strong\n\n\n\n\nFigure 8. Presented images depict representative scratch assay results performed on A549 cancer cells to observe the impact of treating them with\nPt\u2212Ru\u2212B (50 \u03bcM) on the closure of the scratch. The images captured 0, 4, 8, and 12 h after the introduction of the scratch showcase the\nprogression of scratch closure over time. Wound borders are distinctly defined by blue borders. Scale bar: 200 \u03bcm.\n\n 17256 https://doi.org/10.1021/acs.inorgchem.4c03083\n Inorg. Chem. 2024, 63, 17249\u221217262\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 9. (a) Fluorescence images of the JC-1 dye assay at 10\u00d7 magnification showing red and green fluorescence when A549 cells were incubated\nwith Pt\u2212Ru\u2212B at 0.5 \u03bcM concentration for 4 h at 37 \u00b0C (dark) and on visible light exposure with the 400\u2212700 nm wavelength (5 Joules/cm2 light\ndose). Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) was used as a positive control. Scale bar: 100 \u03bcm. (b) Dot plot of the\nAnnexin V-FITC/propidium iodide assay using A549 cancer cells incubated with the Pt\u2212Ru\u2212B. Cisplatin was used as a positive control. The %\ncell population was given in the respective quadrant: lower left, live cells; lower right, early apoptotic cells; upper left, dead cells; upper right, late\napoptotic cells [D, dark; L. light].\n\nevidence that the complex selectively modulates \u0394\u03a8m upon \u223c75% of the cells stained for Annexin-Propidium Iodide\nlight exposure, suggesting the activation of mitochondrial indicate apoptosis. In contrast, within 1 h of postincubation,\ndriven pathways of the apoptotic process. only \u223c2% of the cells displayed apoptosis when the sample was\n Cellular Apoptosis. The apoptosis induced by prodrug kept in dark (Figure 9b). Positive control cisplatin (at an IC50\nPt\u2212Ru\u2212B on light irradiation was quantified from an Annexin concentration) displayed \u223c9% apoptosis. The results suggest\nV-FITC/propidium iodide dual staining assay. In the initial the Pt\u2212Ru\u2212B induced a PDT effect through the apoptotic\nstages of apoptosis, Annexin V binds to the phosphatidylserine pathway.\nunits on the inner cell membrane, and as the process advances, Western Blotting. To gain a detailed understanding of the\nthe entire complex undergoes translocation toward the outer findings mentioned above, we conducted Western blotting to\ncell membrane. In the late stages of apoptosis, the integrity of examine markers associated with cell death, particularly,\nthe plasma membrane becomes compromised, leading to the cleaved caspase-7 in A549 cells. As an effector caspase\ndisintegration of cells and the formation of apoptotic vesicles facilitating cell death signaling, caspase-7 undergoes cleavage\nor residues. During this progression, impermeable agents such and activation, a process mediated by initiator caspase like\nas propidium iodide gain access to the inner compartments of caspase-1.60 Caspase-7 possessing a catalytic cysteine residue\nthe cell through membrane blebbing.59 The apoptotic cells within its active site enables caspase-7 to cleave various\nwere quantified through a flow cytometry analysis. When cells substrates, including PARP, contributing to the breakdown and\nexposed to light were treated with \u223c0.5 \u03bcM of the prodrug, destruction of the cell.61 Figure 10 shows the presence of\n 17257 https://doi.org/10.1021/acs.inorgchem.4c03083\n Inorg. Chem. 2024, 63, 17249\u221217262\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n findings affirm the complex\u2019s capability to effectively traverse\n the extracellular matrix barrier (ECM) and accumulate within\n the MCTs.\n\n \u25a0 EXPERIMENTAL SECTION\n The chemicals and solvents used in this study were obtained from\n commercial sources and used as such. Oxoplatin as a precursor was\n prepared following a literature procedure.36 The preparations of\n NHS-Bio and Pt-Bio were carried out following reported protocols.35\n Details regarding the instrumentation used are given in the\n Supporting Information.\n Materials and Methods. Synthesis of cis-Ru(DMSO)4Cl2. The\n synthesis was performed using a modified version of a literature\n procedure.64 In a two-necked round-bottom flask equipped with a\n reflux condenser, 8 mL of dry DMSO was taken, and the solvent was\n degassed using freeze\u2212pump\u2212thaw cycles. Subsequently, 500 mg\n (\u223c1.89 mmol) of RuCl3\u00b7xH2O (39% Ru content) was added, and the\n reaction mixture was heated to 190 \u00b0C while being stirred for 5 min.\n The reaction vessel was cooled gradually to reach an ambient\n temperature. Following this, a volume of 30 mL of anhydrous acetone\n was added to the reaction mixture that was vigorously stirred for a\n duration of 5 min at room temperature. The resulting faint-yellow\n colored solid was separated by filtration, washed with diethyl ether,\n and subjected to vacuum drying process to isolate the product in\nFigure 10. Western blot analysis showing overexpression of cleaved \u223c68% yield (630 mg).\ncaspase-7 and \u03b3-H2AX when A549 cells were incubated with Pt\u2212Ru\u2212 Synthesis of [RuL1(DMSO)Cl2]. A reflux condenser was connected\nB at 0.5 \u03bcM concentration for 4 h at 37 \u00b0C followed by visible light to a 50 mL round-bottom flask containing 20 mL of anhydrous\nexposure with the 400\u2212700 nm wavelength photoreactor and 5 ethanol. Then, the solvent was degassed through a series of freeze\u2212\nJoules/cm2 light dose compared to the untreated controls and dark: pump\u2212thaw cycles to remove any trapped gases. The reaction vessel\n(a) representative Western blots, (b, c) quantitative analysis of was charged with 121 mg of cis-[Ru(DMSO)4Cl2] (0.25 mmol, 1.25\nproteins under different conditions [kDa: kilo Dalton; w.r.t. is with equiv) and 110 mg of ligand L1, tpy\u2212BOD (0.20 mmol, 1 equiv). The\nrespect to]. reaction mixture was refluxed for a period of 12 h, followed by being\n allowed to cool to room temperature. The solvent was removed using\n a rotary evaporator to obtain a residue that was subsequently\n subjected to purification by column chromatography using a neutral\ncleaved caspase-7, which is significantly higher in A549 cells alumina column and chloroform\u2212methanol mixture (9:1 v/v),\ntreated with the drug and exposed to light, in contrast to both yielding 110 mg (\u223c68%) of desired dark brown product.\nthe control group and cells that were maintained in dark. This C36H34BCl2F2N5ORuS (Mw: 805.5388). 1H NMR (500 MHz,\nsuggests activation of apoptosis possibly through the classical DMSO-d6) \u03b4 (ppm): 9.38 (d, 2H, J = 5 Hz), 9.15 (s, 2H), 8.90 (d,\nDNA damage pathway. Ataxia-telangiectasia mutated proteins 2H, J = 5 Hz), 8.52 (d, 2H, J = 10 Hz), 8.04 (td, 2H, J = 5 Hz), 7.73\n(ATM) play a crucial role in signaling responses to DNA (d, 2H, J = 10 Hz), 7.58\u22127.54 (m, 2H), 6.25 (s, 2H), 3.62 (s, 6H),\ndamage.62 Within chromatin, a notable target of ATM is the 2.55 (s, 6H), 1.50 (s, 6H) (s, singlet; d, doublet; td, triplet of doublet;\nhistone protein H2AX, and its phosphorylated form is referred m, multiplet). 13C NMR (125 MHz, DMSO-d6) \u03b4 (ppm): 159.08,\n 158.04, 156.17, 146.75, 143.26, 137.23, 129.35, 128.89, 126.90,\nto as \u03b3-H2AX.63 The elevation in the level of \u03b3-H2AX serves as 124.08, 122.05, 119.22, 45.12, 14.73. 11B NMR (160 MHz, DMSO-\nan indicator of DNA damage and is detectable through d6) \u03b4 (ppm): 0.64. 19F NMR (472 MHz, DMSO-d6) \u03b4 (ppm):\nWestern blotting. As illustrated in Figure 10a\u2212c, the levels of \u03b3- \u2212143.57.\nH2AX in cells treated with the drug under irradiation Synthesis of Complex Ru\u2212B (1). A solution was prepared by\ndemonstrated a substantial increase compared to control dissolving 52 mg of ligand tpy\u2212COOH (L2, 0.15 mmol, 1 equiv) and\ncells. This suggests DNA damage was induced by the prodrug 120 mg of [Ru(L1)(DMSO)Cl2] (0.15 mmol, 1 equiv) in 30 mL of\non light irradiation. dry and degassed ethanol in a 50 mL round-bottom flask under a\n Biological Evaluation in 3D MCTs. Conventional drug nitrogen atmosphere. The resulting mixture was stirred in the dark at\nscreening methods using monolayer cells often face challenges a temperature of 80 \u00b0C for a duration of 24 h. On completion of the\n reaction, the solvent was removed in a rotary evaporator. The\nin replicating the complexities and pathophysiology of tumors. resulting residue was dissolved in methanol, and the solution was\nMulticellular tumor spheroids (MCTs) represent cell aggre- filtered using a Celite bed to separate any undissolved impurities. The\ngates that provide a model intermediate between monolayer resulting filtrate was concentrated and subsequently subjected to\ncells and solid tumors. MCTs offer insights into numerous column chromatographic purification using neutral alumina and a\naspects of solid tumors, including characteristics such as mixture of acetonitrile and methanol (4:1 v/v). The dark brown solid\noxygen and nutrient gradients, cell\u2212cell matrix interactions, thus obtained was washed with diethyl ether, followed by vacuum\ngene expression, and the presence of necrotic cores. To drying in a desiccator containing CaCl2 [Yield: \u223c93 mg (63%)].\ninvestigate the ability of drug complexes to penetrate multiple C56H43BCl2F2N8O2Ru (Mw: 1096.8338). 1H NMR (500 MHz,\nlayers of cells, A549 MCTs with an approximate diameter of DMSO-d6) \u03b4 (ppm): 9.63 (s, 2H), 9.51 (s, 2H), 9.17 (s, 4H), 8.72 (d,\n 2H, J = 10 Hz), 8.36 (d, 2H, J = 10 Hz), 8.16 (d, 2H, J = 10 Hz), 8.07\n200 \u03bcm were subjected to an 8 h incubation with a 30 \u03bcM (s, 4H), 7.84 (d, 2H, J = 5 Hz), 7.59 (t, 4H, J = 5 Hz), 7.31\u22127.25 (m,\nconcentration of complex Pt\u2212Ru\u2212B. Z-stack confocal 4H), 6.29 (s, 2H), 2.52 (s, 6H), 1.56 (s, 6H). 13C NMR (125 MHz,\nmicroscopy was employed to capture images, revealing the DMSO-d6) \u03b4 (ppm): 168.03, 158.59, 158.49, 155.77, 155.43, 152.71,\nemission of green fluorescence from multiple sections within 143.13, 138.54, 138.47, 136.56, 131.12, 130.31, 129.61, 128.88,\nthe MCTs (Figure S30, Supporting Information). These 128.29, 128.11, 126.98, 125.38, 122.07, 121.51, 14.76. 11B NMR (160\n\n 17258 https://doi.org/10.1021/acs.inorgchem.4c03083\n Inorg. Chem. 2024, 63, 17249\u221217262\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nMHz, DMSO-d6) \u03b4 (ppm): 1.00. 19F NMR (472 MHz, DMSO-d6) \u03b4 process and superoxide anion radicals via a type-I electron\n(ppm): \u2212143.64. ESI-MS (m/z) recorded in methanol: Calcd. [M- transfer process in a minor way, as evidenced from the\n2Cl] 2 + : 505.1341; Found: 505.1364. Anal. Calcd. for mechanistic DNA photocleavage studies. The prodrug also\nC56H43BCl2F2N8O2Ru (Mw: 1096.8338) C, 62.23; H, 4.01; N, exhibited DNA photocleavage under hypoxic conditions. The\n10.37. Found: C, 62.41; H, 4.17; N, 10.45. UV\u2212visible spectra in 10%\nDMSO/DPBS at pH = 7.2 [\u03bbmax, nm (\u03b5, M\u22121 cm\u22121)]: 317 (5.42 \u00d7\n metallodrug Pt\u2212Ru\u2212B demonstrated significantly enhanced\n104), 478 (2.18 \u00d7 104), 500 (4.88 \u00d7 104) (DMSO, dimethyl sulfoxide; chemo-PDT activity compared to the chemo-active cisplatin,\nDPBS, Dulbecco\u2019s phosphate-buffered saline). Molar conductivity in the PDT-active hematoporphyrin drug Photofrin, and a 10-fold\nDMF at 298 K (\u039bM/S m2 mol\u22121): 138. increase compared to the dyad complex Ru\u2212B. This prodrug\n Synthesis of Prodrug Pt\u2212Ru\u2212B (2). In an inert nitrogen showed solution stability in the dark but was activated in the\natmosphere, Ru\u2212B (115 mg, 0.11 mmol) and TBTU (2-(1H- presence of cellular reducing agents, resulting in IC50 values in\nbenzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate, 51 the nanomolar concentration in cancer cells while being less\nmg, 0.16 mmol) were added to a 50 mL round-bottom flask active in normal cells. Complex 2 showing significant\ncontaining 10 mL of anhydrous dimethylformamide (DMF). The mitochondrial localization and light-activated apoptotic cell\nresulting mixture was initially stirred at room temperature for 10 min.\nTriethylamine (17 mg, 0.16 mmol) was added, and the mixture was\n death with a remarkable photocytotoxicity index value of\nstirred for an additional 15 min. Then, the complex Pt-Bio (64 mg, >4500, antimetastatic properties and accumulation in 3D-\n0.11 mmol), derived from cisplatin with a biotin ligand and axial OH multicellular tumor spheroids, exemplifies a rare and highly\nligand, was added to the reaction mixture, which was further stirred at effective PDT agent signifying the importance of the prodrug-\n45 \u00b0C for a period of 48 h. The solvent was removed in a rotary based approach in next generation PDT drug development.\nevaporator, resulting in a crude mixture, which was dissolved in 10 mL\nof methanol and subjected to column chromatography using neutral\nalumina and a mixture of 4:1 (v/v) acetonitrile and methanol as the\neluent. Yield: \u223c68 mg (38%).\n \u25a0\n *\n ASSOCIATED CONTENT\n s\u0131 Supporting Information\n C66H63BCl4F2N12O5RuPtS (Mw: 1623.1298). 1H NMR (500 The Supporting Information is available free of charge at\nMHz, DMSO-d6) \u03b4 (ppm): 9.65 (s, 2H), 9.55 (s, 2H), 9.15 (d, https://pubs.acs.org/doi/10.1021/acs.inorgchem.4c03083.\n4H, J = 10 Hz), 8.76 (d, 2H, J = 10 Hz), 8.71 (t, 2H, J = 10 Hz), 8.52\n(d, 1H, J = 10 Hz), 8.22 (d, 1H, J = 10 Hz), 8.07 (t, 4H, J = 5 Hz), Detailed experimental procedures, reaction schemes,\n7.86 (d, 2H, J = 10 Hz), 7.62 (d, 2H, J = 5 Hz), 7.58 (d, 2H, J = 5 ESI-MS, NMR and electronic spectral data, cyclic\nHz), 7.28 (s, 4H), 6.75\u22126.72 (m, 6H), 6.44\u22126.36 (m, 2H), 6.29 (s, voltammetry, DPBF titration plots, MTT assay plots,\n2H), 4.30 (t, 1H, J = 10 Hz), 4.15 (d, 1H, J = 5 Hz), 3.11 (d, 1H, J = confocal imaging plots, theoretical calculation results,\n5 Hz), 2.82 (d, 2H, J = 10 Hz), 2.52 (s, 6H), 2.33\u22122.29 (m, 1H), 2.17 and coordinates from theoretical calculations (Schemes\n(d, 1H, J = 10 Hz), 1.61\u22121.34 (m, 12H). 13C NMR (125 MHz, S1\u2212S3; Tables S1\u2212S4; Figures S1\u2212S30) (PDF)\nDMSO-d6) \u03b4 (ppm): 163.19, 158.47, 155.76, 152.77, 143.13, 138.56,\n131.13, 128.88, 128.26, 125.39, 125.11, 122.09, 61.51, 59.68, 55.93,\n28.59, 14.76. 11B NMR (160 MHz, DMSO-d6) \u03b4 (ppm): 0.67. 19F\nNMR (472 MHz, DMSO-d6) \u03b4 (ppm): \u2212143.55. ESI-MS (m/z)\nrecorded in methanol: Calcd. [M-2Cl]2+: 776.1457; Found: 776.1471.\n \u25a0 AUTHOR INFORMATION\n Corresponding Authors\nAnal. Calcd. for C66H63BCl4F2N12O5RuPtS (Mw: 1623.1298): C, Deepak Kumar Saini \u2212 Department of Developmental Biology\n48.84; H, 3.91; N, 10.36. Found: C, 49.07; H, 3.66; N, 10.57. UV\u2212 and Genetics, Indian Institute of Science, Bangalore 560012,\nvisible spectra in 10% DMSO/DPBS at pH = 7.2 [\u03bbmax, nm (\u03b5, M\u22121 India; Department of Bioengineering, Indian Institute of\ncm\u22121)]: 316 (4.15 \u00d7 104), 477 (2.02 \u00d7 104), 500 (4.43 \u00d7 104) Science, Bangalore 560012, India; orcid.org/0000-0001-\n(DMSO, dimethyl sulfoxide; DPBS, Dulbecco\u2019s phosphate-buffered 6671-7256; Phone: +91-80-22932574;\nsaline). Molar conductivity in DMF at 298 K (\u039bM/S m2 mol\u22121): 141. Email: deepaksaini@iisc.ac.in\n Biological Experiments. Comprehensive information regarding the\nDNA photocleavage experiment, MTT assay, confocal imaging, JC-1 Akhil R. Chakravarty \u2212 Department of Inorganic and Physical\nassay, Annexin V-FITC/propidium iodide assay, antimetastatic assay, Chemistry, Indian Institute of Science, Bangalore 560012,\nWestern blot, and 3D MCTs studies is given in the Supporting India; orcid.org/0000-0002-6471-9167; Phone: +91-\nInformation. 80-22932533; Email: arc@iisc.ac.in\n\n\u25a0 CONCLUSIONS\nUsing the concept of \u201cMagic Bullet\u201d in targeted therapy, a\n Authors\n Arpan Bera \u2212 Department of Inorganic and Physical\nheterobimetallic Pt(IV)\u2212Ru(II) system is designed and Chemistry, Indian Institute of Science, Bangalore 560012,\ndeveloped as a prodrug by incorporating biotin (vitamin B7) India\nas an axial ligand linked to a pseudo-octahedral 5d6-Pt(IV) Amrita Nepalia \u2212 Department of Developmental Biology and\ncore, while a 4d6-Ru(II) bis-terpyridine dyad bearing a PDT- Genetics, Indian Institute of Science, Bangalore 560012, India\nactive BODIPY photosensitizer is bound to Pt(IV) as the other Aarti Upadhyay \u2212 Department of Inorganic and Physical\naxial ligand. This single molecular platform exhibited good Chemistry, Indian Institute of Science, Bangalore 560012,\ncellular uptake due to the presence of biotin moiety, while the India\nother axially bound ruthenium(II) complex served as a Complete contact information is available at:\nphotosensitizer generating singlet oxygen as ROS in high https://pubs.acs.org/10.1021/acs.inorgchem.4c03083\nyield. Moreover, the Pt(IV) prodrug, namely, {Biotin\u2212PtIV\u2212\n(O2C\u2212tpy\u2212RuII\u2212tpy\u2212BODIPY)} (2) prepared from cis- Author Contributions\n[Pt(NH3)2Cl2]-derived Oxoplatin precursor is highly suscep- A.B.\ufffdinvestigation (synthesis and chemical/biological as-\ntible to ligand release on one-step irreversible two-electron pects) and writing, A.N.\ufffdinvestigation (cellular), A.U.\ufffd\nPt(IV)\u2192Pt(II) reduction generating all three constituents of investigation (biological), D.K.S.\ufffdreview and editing (con-\nthe prodrug. The complex on photoexposure generated singlet ceptual design and implementation of the cellular studies),\noxygen as the primary ROS through a type-II energy transfer A.R.C.\ufffdoverall project administration (conceptual design and\n 17259 https://doi.org/10.1021/acs.inorgchem.4c03083\n Inorg. Chem. 2024, 63, 17249\u221217262\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nimplementation of the bioinorganic aspects of this study), Photoactive BODIPY for Mitochondria Specific \u201c Chemo-PDT \u201d\nreview, and editing. Activity. J. Inorg. Biochem. 2021, 223, No. 111526.\n (14) Deng, Z.; Wang, N.; Liu, Y.; Xu, Z.; Wang, Z.; Lau, T. C.; Zhu,\nNotes\n G. A Photocaged, Water-Oxidizing, and Nucleolus-Targeted Pt(IV)\nThe authors declare no competing financial interest. Complex with a Distinct Anticancer Mechanism. J. Am. Chem. Soc.\n\n\u25a0 ACKNOWLEDGMENTS\nA.R.C. thanks the Department of Science and Technology\n 2020, 142, 7803\u22127812.\n (15) Strebhardt, K.; Ullrich, A. Paul Ehrlich's magic bullet concept:\n 100 years of progress. Nat. Rev. Cancer 2008, 8, 473\u2212480.\n(DST), Government of India, New Delhi, for financial support (16) Bera, A.; Nepalia, A.; Upadhyay, A.; Saini, D. K.; Chakravarty,\n(CRG/2018/000081) and for a J. C. Bose national fellowship A. R. Biotin and Boron-Dipyrromethene-Tagged Platinum(IV)\n(SR/S2/JCB-26/2007). The authors also thank the Alexander Prodrug for Cellular Imaging and Mito-Targeted Photocytotoxicity\nvon Humboldt (AvH) Foundation, Germany, for donation of in Red Light. Dalton Trans. 2023, 52, 13339\u221213350.\n (17) Paul, S.; Kundu, P.; Kondaiah, P.; Chakravarty, A. R. BODIPY-\nan electrochemical system. D.K.S.\u2019s research group is funded\n Ruthenium(II) Bis-Terpyridine Complexes for Cellular Imaging and\nby DBT and SERB (CRG/2020/000239) grants. A.R.C. Type-I/-II Photodynamic Therapy. Inorg. Chem. 2021, 60, 16178\u2212\nthanks INSA, New Delhi, for Senior Scientist fellowship 16193.\n(INSA/SP/SS/2021). 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