Effect of Alkyl Chain Length on the Photophysical, Photochemical, and Photobiological Properties of Ruthenium(II) Polypyridyl Complexes for Their Application as DNA-Targeting, Cellular-Imaging, and Light-Activated Therapeutic Agents.

www.acsabm.org Article Eﬀect of Alkyl Chain Length on the Photophysical, Photochemical, and Photobiological Properties of Ruthenium(II) Polypyridyl Complexes for Their Application as DNA-Targeting, CellularImaging, and Light-Activated Therapeutic Agents Downloaded via GOVERNMENT COLG LAHORE on September 2, 2021 at 06:10:13 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. Sandra Estalayo-Adrián,* Salvador Blasco, Sandra A. Bright, Gavin J. McManus, Guillermo Orellana, D. Clive Williams, John M. Kelly, and Thorﬁnnur Gunnlaugsson* Cite This: https://doi.org/10.1021/acsabm.1c00284 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: A family of six Ru(II) polypyridyl complexes (1−6) which contain phenanthroline-based ligands functionalized with alkyl chains of diﬀerent lengths (one methyl group, 10 and 21 carbon alkyl chains) and either 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ancillary ligands have been synthesized and characterized. The inﬂuence of the alkyl chain length on their photophysical and photochemical properties as well as in their photobiological applications has been elucidated by monitoring the changes in their MLCT-centered absorption and emission bands. The presence of one methyl group or 10 carbon alkyl chains does not seem to signiﬁcantly aﬀect the photophysical and photochemical properties of the resulting Ru(II) complexes when compared to the well-known [Ru(phen)3]2+ and [Ru(TAP)2phen]2+. However, an eﬀect on their emission properties and in their ability to photosensitize singlet oxygen is observed for the Ru(II) complexes containing 21 carbon alkyl chains. The binding of these complexes to salmon testes DNA (stDNA) was investigated by observing the changes in the photophysical properties. Complexes 1, 2, 4, and 5 all showed changes in their MLCT bands that could be analyzed using conventional ﬁtting methods, such as the Bard equation. In contrast, complexes 3 and 6, possessing long aliphatic chains, gave rise to nonclassic behavior. In addition to these analyses, both thermal denaturation and circular dichroism studies of 1−6 were carried out in the presence of stDNA which conﬁrmed that these complexes bind to DNA. Confocal microscopy and viability studies in HeLa cervical cancer cells reveal an alkyl chain-length dependence on the cellular uptake and cytotoxicity of the resulting Ru(II) complexes due to an enhancement of their lipophilicity with increasing alkyl chain length. Thus, complexes containing 10 and 21 carbon alkyl chains are rapidly taken up into HeLa cells and, in particular, those with 21 carbon alkyl chains show a signiﬁcant phototoxicity against the same cell line. Therefore, this study provides further insight into the possible modulation of the photophysical, photochemical, and photobiological properties of Ru(II) polypyridyl complexes by varying the length of the alkyl chains attached to the polypyridyl ligands coordinated to the Ru(II) center and the nature of the auxiliary groups, which we show has a signiﬁcant eﬀect on photophysical and biological properties. KEYWORDS: ruthenium(II), polypyridyl complexes, luminescence, lipophilicity, anticancer, cellular imaging ■ INTRODUCTION Ruthenium(II) [Ru(II)] polypyridyl complexes have attracted much interest in the past number of decades for their interesting photophysical, photochemical, and redox properties.1−3 Such complexes are characterized by their metal to ligand charge-transfer center (MLCT) absorption of light in the visible region (with λmax from ca. 400 to 450 nm), luminescence in the red spectral range (with λmax from ca. 600 to 650 nm), large Stokes shift, and relatively long luminescence lifetimes (typically from hundreds of nanoseconds to a few microseconds, due to 3MLCT emission). These properties make Ru(II) polypyridyl complexes very attractive candidates for applications in photobiology as light-activated therapeutic © XXXX American Chemical Society agents and imaging probes, not only for targeting DNA but also in a cellular environment.4−11 Therefore, the design of ruthenium-based systems with the ability to be internalized by the cells has become a crucial matter for such biological applications. Received: March 8, 2021 Accepted: July 12, 2021 A https://doi.org/10.1021/acsabm.1c00284 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX ACS Applied Bio Materials www.acsabm.org Article Figure 1. Chemical structures of the Ru(II) polypyridyl complexes [Ru(phen)3]2+ and [Ru(TAP)2phen]2+ and 1−6 studied in this work. resulting Ru(II) complexes and as such inﬂuence their photobiological behavior when evaluated in a cellular environment.1 With this in mind, we report herein, the synthesis and characterization of a family of Ru(II) polypyridyl complexes (1−6) containing diﬀerent lengths of alkylamide functionalized phenanthroline ligands (7−9, one methyl group, 10 and 21 carbon alkyl chains, respectively) and either 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ancillary ligands (Figure 1). The eﬀect of the alkyl chain length on their photophysical and photochemical properties and their ability to bind to DNA as well as the impact on their cellular uptake and application as light-activated therapeutic agents is investigated. No signiﬁcant eﬀect on the photophysical properties of complexes containing one methyl group or 10 carbon alkyl chains was observed when compared to the wellknown parent complexes [Ru(phen) 3 ] 2+ and [Ru(TAP)2phen]2+. However, the extension of the alkyl chain to 21 carbons aﬀects the emission properties of the resulting Ru(II) complexes as well as their ability to produce singlet oxygen. Furthermore, the enhancement of the lipophilicity with the increasing alkyl chain length results in an improvement of the cellular uptake of the complexes and, as such, of their phototoxicity against HeLa cervical cancer cells, suggesting a possible modulation of the toxicity when designing Ru(II) polypyridyl complex-based light-activated therapeutic agents by varying the alkyl chain length. In this contribution we give a full account of our work involving 1−6. Part of this research, dealing with the self-assembly formation of 3 and 6, respectively, and some photophysical and biological proﬁling, was recently communicated.21 Factors such as molecule size, charge, and lipophilicity have been shown to play a key role in the cellular uptake mechanism and subcellular distribution of Ru(II) polypyridyl complexes, and thus, diﬀerent strategies have been developed over the years with the aim of improving the cellular uptake of these complexes. For instance, these complexes have been conjugated to nanoparticles12−17 and biomolecules such as peptides,18−25 carbohydrates,26−28 or lipids.29−31 Another strategy consists of the coordination of lipophilic ligands to the Ru(II) center. In this context, the extension of the surface area of aromatic ligands such as the well-known [2,3h]dipyrido[3,2-a:2′,3′-c]phenazine (dppz) has been shown to confer lipophilicity to the resulting Ru(II) complex and enhance their cellular uptake.32−34 Other lipophilic ligands include 7-diphenyl-1,10-phenanthroline (dip) or alkyl chainbased derivatives which have been shown to increase the intracellular internalization of the complexes when compared to their more hydrophilic analogues.35,36 The incorporation of lipophilic tails to the hydrophilic ruthenium-based core also provides the resulting molecule with an amphiphilic character and a general structural resemblance to biological lipids, thus facilitating their insertion into the lipid bilayer that constitutes the cell membrane.37 Lipid mimetic luminescent Ru(II) polypyridyl complexes have been developed for their potential use in labeling liposomes and phospholipids membranes.38−40 Furthermore, the presence of hydrophobic alkyl chains has been shown to have an eﬀect not only on the lipophilicity of the Ru(II) complexes but also on their ability to form self-assembly supramolecular structures in aqueous solution acting as metallosurfactants. They have been extensively used in recent years in imaging and drug delivery applications.41−43 The presence of metals in the headgroup of these amphiphilic structures confers them with advantageous properties.44 In this context, Ru(II)-based metallosurfactants have shown great potential in several biological applications such as in the imaging of physiological hypoxia, phospholipid membranes labeling, gene therapy, or anticancer agents.40,45−47 The addition of new functionalities to the ligands coordinated to the Ru(II) center can have a dramatic eﬀect on the photophysical and photochemical properties of the ■ RESULTS AND DISCUSSION Synthesis of Ligands and Complexes. The synthesis of the Ru(II) polypyridyl complexes 1−6 was achieved according to a procedure previously used in our laboratory (Scheme 1).12,15,33,34,48 First, ligand 7 was prepared following a method described in the literature based on the amide coupling reaction between 5-amino-1,10-phenanthroline (10) and acetic anhydride (11) in MeCN.49 Ligand 8 was synthesized using the same synthetic strategy reported by us in a previous work for ligand 9, which consists of the amide coupling reaction B https://doi.org/10.1021/acsabm.1c00284 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX ACS Applied Bio Materials www.acsabm.org Article Scheme 1. Synthesis of the Ru(II) Polypyridyl Complexes 1−6a a Conditions: (i) CH3CN, r.t., 2 days; (ii) EDC, DMAP, CH2Cl2, r.t., 2 days; (iii) EtOH/H2O (1:1), microwave reaction, 140 °C, 40 min (1−3) or 10 min (4−6). between 10 and undecanoic acid (12) in the case of 8, or docosanoic acid (13) in the case of 9, using N-ethyl-N′-(3(dimethylamino)propyl)carbodiimide hydrochloride (EDC) in the presence of 4-(dimethylamino)pyridine (DMAP) in dry DCM.48 Ligands 7−9 were obtained as beige solids in 62, 57, and 95% yields, respectively. Ru(II) complexes 1−6 were subsequently prepared by microwave-assisted reaction using the appropriate Ru(II) cis-bispolypyridyl dichloride precursor complex (14 or 15) and the corresponding N-1,10phenanthrolin-5-yl-alkylamide ligand (7−9) in a H2O/EtOH (1:1) mixture at 140 °C for 40 (1−3) or 10 min (4−6). The chloride salts of 1−6 were obtained as red solids in 60, 60, 50, 52, 43, and 42% yields, respectively, after puriﬁcation by column chromatography on neutral alumina using MeCN/ H2O (10:0 to 9:1) as eluent. Ligands 7−9 and complexes 1−6 were fully characterized by conventional methods, including the use of 1H NMR and 13C NMR spectroscopies, elemental analysis, HRMS, melting point analysis, and IR spectroscopy (Figures S1−S22, Supporting Information). Photophysical Characterization. In order to gain a better insight into the impact the alkyl chain length has on the absorption and emission properties of the synthesized Ru(II) complexes, the photophysical properties of 1, 2, 4, and 5 were investigated in 10 mM sodium phosphate-buﬀered aqueous solution at pH 7.4 and 298 K, and the results were compared to those previously reported by us for 3 and 6, and for the wellknown parent complexes [Ru(phen) 3 ] 2+ and [Ru(TAP)2phen]2+, respectively.48 Complexes containing phen as ancillary ligands (1−3) showed absorption spectra similar to that recorded for [Ru(phen)3]2+ (Figure 2a and Table 1) with the π−π* Figure 2. UV−vis absorption and emission spectra of (a) [Ru(phen)3]2+ and 1−3 and (b) [Ru(TAP)2phen]2+ and 4−6 in aerated 10 mM sodium phosphate-buﬀered aqueous solution at pH 7.4 and 298 K (λexc = 440 nm ([Ru(phen)3]2+ and 1−3), 436 nm ([Ru(TAP)2phen]2+), 413 nm (4 and 5), or 418 nm (6)). intraligand transitions of the phen ligand localized at ca. 220 and 260 nm, while the broad band center at ca. 450 nm was attributed to the characteristic MLCT transition in the metallic complexes.50 Likewise, absorption maxima similar to those recorded for [Ru(TAP)2phen]2+ were observed for the complexes containing TAP as ancillary ligands (4−6) (Figure 2b and Table 1).51 Thus, intense bands at ca. 230 and 270 nm are attributed to π−π* intraligand transitions in the TAP ligand, and the broad band cantered at ca. 415 nm corresponds to the MLCT transition of the Ru(II) center. Excitation into the MLCT bands of each complex resulted in broad emission bands centered at 606 and 603 nm for 1 and 2, respectively (Figure 2a). These values are very close to the emission maximum observed for [Ru(phen)3]2+.50 However, as we showed in our previous work,48 a slightly red-shifted emission band at 614 nm was observed for 3 when compared to [Ru(phen)3]2+. Similarly, the emission spectra of 4 and 5 exhibited broad bands centered at 636 nm (Figure 2b), while the emission maximum at 643 nm of 6 was found to be slightly red-shifted compared to that observed for [Ru(TAP)2phen]2+.51 The excitation spectra recorded for these complexes were found to be identical to their absorption spectra (Figure S23, Supporting Information). Luminescence quantum yields (Φem) were determined in aerated 10 mM sodium phosphate-buﬀered aqueous solution C https://doi.org/10.1021/acsabm.1c00284 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX ACS Applied Bio Materials www.acsabm.org Article Table 1. Absorption and Emission Properties of 1, 2, 4, and 5 in 10 mM Sodium Phosphate-Buﬀered Aqueous Solution at pH 7.4 and 298 Ka λmax (nm) (ε, 104 M−1 cm−1)b complex 2+ [Ru(phen)3] 1 2 3f [Ru(TAP)2phen]2+ 4 5 6f π−π* IL e 224, 262 221 (7.83), 262 (9.88) 222 (7.91), 262 (10.0) 222 (6.60), 263 (7.60) 230 (6.12), 272 (6.93)h 232 (5.78), 272 (6.64) 232 (6.07), 273 (7.08) 230 (5.18), 274 (6.06) emission π−π* MLCT e 447 (1.90) 447 (1.67) 448 (1.76) 451 (1.60) 410 (1.65)h 414 (1.56) 413 (1.74) 418 (1.59) λem max (nm) e 604 606 603 614 645h 636 636 643 c Φem (air) τem (ns)d (air) τem (ns)d (N2) PTO2 (air-satd) 0.050 0.057 0.060 0.070 0.029 0.033 0.034 0.017 e e 0.52 0.50 0.49 0.20 0.17 0.15 0.15 0.16 480 644 650 1085g 690h 538 570 587g 990 1300 1270 1354g 835h 636 674 696g a Complexes [Ru(phen)3]2+, 3, [Ru(TAP)2phen]2+ and 6 are included for comparison.48,50,51. bλmax, absorbance. Estimated uncertainty ±1%. cAirsaturated aqueous solution of [Ru(bpy)3]Cl2 as reference (Φem = 0.040).58 Estimated uncertainty ±5%. dIf not otherwise indicated, the luminescence decays are monoexponential. Estimated uncertainty ±10%. eHerman et al.50 The reported values are in aqueous solution. fEstalayoAdrián et al.48 gThe luminescence decays are biexponential; reported data correspond to the pre-exponential weighted mean lifetimes (τM) and were calculated according to τM = ∑(aiτi)/∑ai.56 hOrtmans et al.51 The reported values are in aqueous solution. longer than those reported for 1 and 2. This can be attributed to the excited state of 3, both being less susceptible to nonradiative deactivation and also being protected from oxygen quenching. This gives rise to a lower PTO2 value for 3 when compared to 1. Similar behavior has previously been reported in the literature for related Ru(II) complexes.57 In contrast to what is found for the phen complexes, τM values of 587 and 696 ns were observed for the TAP analogue 6 in both aerated and deoxygenated aqueous solutions, respectively, similar to the values measured for 4 and 5. The PTO2 value displayed by 6 was also comparable to those determined for 4 and 5. These measurements show that overall the long alkyl chain does not have a signiﬁcant eﬀect on either the nonradiative decay or the oxygen quenching of the excited state of 6. To further investigate the eﬀect of the long alkyl chain on the photophysical behavior of 3 and 6, and based on our previous work where we demonstrated the ability of these complexes to self-aggregate in solution into micellar species (critical micelle concentration (cmc) of 114 and 113 μM were determined for 3 and 6, respectively),48 luminescence lifetimes of aerated aqueous solutions of 3 and 6 at diﬀerent concentrations (below and above their cmc) were measured (Figure 3). As expected, biexponential ﬁts were needed for the luminescence decay kinetics at all concentrations and thus τM were calculated (Tables S1 and S2, Supporting Information). Interestingly, the increase of the complex concentration had an opposite eﬀect on the τM values of both 3 and 6. Thus, while the phen complex showed larger τM values when the concentration of the complex was increased, the τM values of the TAP analogue were shorter at larger complex concentrations. The reasons for this (whether due to changes to the nonradiative decay rate, protection from oxygen quenching, or possible self-quenching upon aggregation) will require further detailed study beyond the scope of the current publication. Singlet Oxygen Photosensitization. Singlet oxygen (1O2) photosensitization is one of the possible modes of action (Type II mechanism) of the molecules used in photodynamic therapy (PDT).7 Therefore, quantum yields of 1 O2 production (ΦΔ) of 1, 2, 4, and 5 were determined in O2saturated D2O by direct measurement of 1O2 phosphorescence at 1265 nm (following excitation of the complex at 532 nm). These may be compared to those previously determined for at pH 7.4 and revealed an inﬂuence of the alkyl chain length (Table 1). Complexes 1−3 showed Φem of 0.057, 0.060, and 0.070, respectively, while Φem of 0.033, 0.034, and 0.017 were observed for the TAP analogous complexes 4−6, respectively.58 The values observed for 1 and 2 and for 4 and 5 are similar to those determined for [Ru(phen)3]2+ and [Ru(TAP)2phen]2+, respectively. However, in line with what we showed in our previous work,21 3 exhibits a slightly higher Φem when compared to those displayed by [Ru(phen)3]2+, 1 and 2. In contrast, 6 shows the opposite eﬀect and a lower Φem was observed than those exhibited for [Ru(TAP)2phen]2+, 4, and 5. Complexes 1 and 2 showed luminescence lifetimes (τem) of 644 and 650 ns, respectively, in aerated 10 mM sodium phosphate-buﬀered aqueous solution at pH 7.4. Solution deoxygenation resulted in a signiﬁcant increase in the excitedstate lifetimes with values of 1300 and 1270 ns for 1 and 2, respectively. Similarly, in aerated solution τem of 538 and 570 ns were observed for the TAP analogue complexes 4 and 5, respectively. Only slightly longer τem of 636 and 674 ns, respectively, were observed when solutions containing 4 and 5 were deoxygenated. These results demonstrate that the luminescence of the excited state of TAP complexes is less aﬀected by the presence of dissolved oxygen than that of complexes containing phen as ancillary ligands. This is in agreement with previous work reported in the literature23,52−54 and was further corroborated through calculation of the values of the proportion of triplet excited states quenched by O2 (PTO2) for both phen and TAP complexes.55 Hence, phen derivatives display PTO2 values of ca. 0.50, while the TAP analogues exhibit smaller values of ca. 0.15, showing a larger eﬃciency of quenching of the excited states of the phen complexes by oxygen than those of the TAP complexes. Furthermore, whereas emission decay curves for 1, 2, 4, and 5 could be ﬁtted to a monoexponential function, luminescence lifetime data for 3 and 6, as reported in our previous work,21 required biexponential ﬁts, and as such the values shown here correspond to the pre-exponential weighted mean lifetimes (τM).56 Since these compounds contain long alkyl chains, the presence of diﬀerent emitting species may be due either to the formation of aggregates or possibly to the long alkyl chain interacting with the hydrophobic phen ligand. The phen complex 3 showed τM values of 1085 and 1354 ns in aerated and deoxygenated aqueous solutions, respectively, which are D https://doi.org/10.1021/acsabm.1c00284 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX ACS Applied Bio Materials www.acsabm.org Article This is supported by the values of the fraction of triplet excited states quenched by O2 yielding 1O2 (fTΔ) determined for each complex (Table 2). Thus, phen-containing complexes [Ru(phen)3]2+, 1, and 2 showed fTΔ values of 0.46, 0.60, and 0.51 vs 1.00, 0.93, and 0.79 observed for their TAP analogues [Ru(TAP)2phen]2+, 4, and 5. In contrast, when ΦΔ values were converted from O2saturated D2O to air-saturated H2O conditions, [Ru(phen)3]2+ and 1−3 containing phen as ancillary ligands displayed higher ΦΔ values than their TAP analogues [Ru(TAP)2phen]2+ and 4−6. This is due to a lower eﬃciency of quenching of the excited states of TAP complexes by oxygen in H2O when compared with the phen complexes. The calculated values of the proportion of triplet excited states quenched by O2 (PTO2) in air-saturated H2O conﬁrmed this fact with PTO2 values of ca. 0.50 and 0.20 observed for phen and TAP complexes, respectively, with the exceptions of 3 and 6 (Table 2).55 These complexes containing the 21 carbon alkyl chain show much lower ΦΔ values, which may be attributed to them being in a micellar state, as the direct measurement of the 1O2 phosphorescence requires a high concentration of photosensitizer (ca. 300 μM). This demonstrates that the aggregation state can have an eﬀect on the interaction between the Ru-centered MLCT excited state and molecular oxygen. Therefore, the ability of 3 and 6 to produce 1O2 is concentration dependent, depending on whether they are free in solution or forming micelles. The eﬀective production of 1O2 by 1, 2, 4, and 5 was also demonstrated by using the water-soluble 1O2 chemical probe, 9,10-anthracenediylbis(methylene)dimalonic acid (ABDA).61 This molecule is known to be photo-oxidized by 1O2, resulting in a non-emissive endoperoxide product. Thus, the changes in the emission spectra of ABDA in air-saturated H2O and in the presence of 1, 2, 4, and 5 upon diﬀerent irradiation times (470 nm LED source) were monitored (Figure S25, Supporting Information) and compared to those observed for [Ru(phen)3]2+, 3, [Ru(TAP)2phen]2+, and 6 in our previous work.21 A decrease in the emission intensity of ABDA at 405 nm was observed as the irradiation time increased, consistent with the generation of 1O2 by all of the Ru(II) complexes tested.62 Interestingly, the rate of disappearance of the ABDA ﬂuorescence was aﬀected by the presence of long alkyl chains Figure 3. Evolution of the pre-exponential weighted mean lifetime (τM) of aerated aqueous solutions of 3 and 6 at diﬀerent concentrations, at 298 K. [Ru(phen)3]2+, 3, [Ru(TAP)2phen]2+, and 6 (Table 2 and Figure S24, Supporting Information) and show how the ΦΔ of these complexes are aﬀected by the presence of diﬀerent length alkyl chains.48,59 It should be noted that D2O was used as solvent instead of regular H2O due to the much shorter 1O2 lifetime in H2O compared to its deuterated analogue. However, knowing the ΦΔ values in H2O and in aerated solution is more relevant when extrapolating to biological systems. Thus, the ΦΔ values obtained in O2-saturated D2O were converted into those in air-saturated H2O (see Experimental Section, Tables 2 and S3, Supporting Information). Production of 1O2 was shown to be dependent on the nature of the ancillary ligands (phen or TAP) and on the alkyl chain length. Complexes [Ru(TAP)2phen]2+ and 4−6 containing TAP as ancillary ligands displayed higher ΦΔ values than their phen analogues in O2-saturated D2O. This behavior can be explained by the fact that the excited-state reduction potentials of the TAP complexes are signiﬁcantly higher than those of the phen analogues, resulting in a considerable slowing of the superoxide competing route that requires photoinduced electron transfer (PET) from the Ru(II) complex to O2.55,60 Table 2. Quantum Yields of Singlet Oxygen Production (ΦΔ) of 1, 2, 4, and 5 (A532 ≈ 0.40) Determined by Time-Resolved Near-Infrared Phosphorescence (λexc = 532 nm)a direct detection of 1O2 (time-resolved NIR phosphorescence) indirect detection of 1O2 (1O2 probe ABDA) complex ΦΔ (O2-satd D2O)b ΦΔ (air-satd H2O)c PTO2 (O2-satd D2O)d PTO2 (air-satd H2O)d fTΔ (O2-satd D2O)d ΦΔ (air-satd H2O)e % photobleaching (405 nm, 10 s irradiation) [Ru(phen)3]2+ 1 2 3 [Ru(TAP)2phen]2+ 4 5 6 0.39 0.52 0.44 0.18f 0.72 0.69 0.59 0.14f 0.21 0.31 0.24 0.03f 0.14 0.18 0.16 0.01f 0.84 0.86 0.87 0.50 0.72 0.74 0.75 0.50 0.46 0.50 0.48 0.08 0.14 0.19 0.20 0.08 0.46 0.60 0.51 0.36 1.00 0.93 0.79 0.28 0.22 0.23 0.23 5f 5 4 83f 19f 22 18 73f a Complexes [Ru(phen)3]2+, 3, [Ru(TAP)2phen]2+, and 6 are included for comparison.48. bO2-saturated D2O solution of [Ru(phen)3]Cl2 as reference (ΦΔ = 0.39).59 Estimated errors ±5%. cCalculated from the values obtained in O2-saturated D2O and knowing the emission lifetimes of the complexes in O2- and Ar-saturated D2O, and air- and Ar-saturated H2O (see Experimental Section and Table S3, Supporting Information).59 d Calculated according to PTO2 = 1 − τ/τ0.59 eAir-saturated H2O solution of [Ru(bpy)3]Cl2 as reference (ΦΔ = 0.18).67 fEstalayo-Adrián et al.48 E https://doi.org/10.1021/acsabm.1c00284 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX ACS Applied Bio Materials www.acsabm.org (Figure 4 and Table 2). Thus, the intensity of the ABDA emission band at 405 nm was shown to decrease by ca. 5% 1 Article O2 chemical probe was found to decrease exponentially over time in the presence of the TAP derivatives [Ru(TAP)2phen]2+ and 4−6, in contrast to the linear decrease observed for the phen complexes [Ru(phen)3]2+, 1, and 2 (Figure 4). As was anticipated in our previous work, this behavior can be explained by the remarkably photo-oxidant character of the excited state of Ru(II) complexes containing at least two TAP ligands providing them with the ability to directly photo-oxidize the ABDA.48,63,64 Thus, the coexistence of two competitive processes, that is oxidation of the chemical probe by 1O2 production and/or by the TAP complex, can be presumed with the latter dominating. Oxidation of anthracene derivatives by electron-transfer photosensitizers has been previously described.65 This hypothesis is supported by the redox potentials reported in the literature for [Ru(TAP)2phen]2+ (Ered* = +1.15 V/SCE)51 and 9,10-dimethylanthracene (Eox = +1.05 V/SCE).65,66 From these values it can be concluded that direct photo-oxidation of 9,10-dimethylanthracene by [Ru(TAP)2phen]2+ is slightly thermodynamically favorable (ΔG0 = −0.10). These observations suggest that anthracene-based 1O2 probes are not suitable for photosensitizers with a high photo-oxidant excited state such as TAP-containing complexes, and as such, ΦΔ values for [Ru(TAP)2phen]2+ and 4−6 could not be determined through this method. DNA Binding Studies. Having investigated the photophysical properties of 1−6 in solution, we next investigated their ability to bind to DNA. Ru(II) polypyridyl complexes, of the nature presented herein, are known DNA binders. This is due to their cationic nature, as well as the extended polypyridyl ligands which can partially intercalate both between the DNA bases or though groove binding.3,12,17 The added complexity of our design, i.e., the presence of alkyl chains of diﬀerent lengths, made us curious to investigate if these and, in particular, 3 and 6, would behave as “classic” examples of Ru(II) complexes such as [Ru(phen)3]2+ and [Ru(TAP)2phen]2+ upon interacting with DNA or if their self-assembly nature would overwrite such classic binding behavior, which we might expect to see for 1, 2, 4, and 5. The binding interactions of 1−6 were investigated using salmon testes DNA (stDNA) by observing the changes in their MLCT absorption and emission properties upon gradual addition of aliquots of stDNA to a 10 mM sodium phosphatebuﬀered aqueous solution at pH 7.4 containing the corresponding Ru(II) complex. Changes in the MLCT absorption band as well as in the emission intensity were monitored spectroscopically until no signiﬁcant changes were detected at increasing stDNA concentrations. All titrations were repeated at least three times to ensure reproducibility. Complexes 1 and 2 exhibited a 14% and 33% decrease in the absorption of the MLCT band at 447 and 448 nm, respectively, upon addition of stDNA (Figure 5a,c). However, a more complex behavior was observed for 3, where an initial decrease in the absorption of the MLCT band at 451 nm was observed after the addition of 0.5 equiv of stDNA, followed by a strong increase in this absorption band upon the addition of stDNA up to 2.2 equiv (Figure 5e); these overall changes ended in a slight decrease until the absorption plateau was reached. Concomitantly, a red shift of ca. 15 nm in the MLCT band was observed. Hence, while 1 and 2 give rise to more “typical” binding interactions with DNA, the changes observed for 3 seem to indicate that the long chain modulates these interactions. The changes in the MLCT emission were Figure 4. Emission spectra of ABDA (λexc = 380 nm) in the presence of (a) [Ru(phen)3]2+ and 1−3 and (b) [Ru(TAP)2phen]2+ and 4−6 (A470 ≈ 0.01) in air-saturated aqueous solution before irradiation and after 10 s irradiation using a 470 nm LED source at 298 K. Inset: Rate of decay of ABDA emission band at 405 nm photosensitized by the corresponding Ru(II) complex. after 10 s irradiation in the presence of the phen complexes [Ru(phen)3]2+, 1, and 2 and by ca. 20% when the TAP analogues [Ru(TAP)2phen]2+, 4, and 5 were used as photosensitizers. These ΦΔ values determined for [Ru(phen)3]2+, 1, and 2 were found to be similar to those obtained by time-resolved NIR phosphorescence from 1O2 in air-saturated H2O (Table 2). In contrast to what was found with the other complexes, decreases of 83% and 73% of the ABDA emission band were observed in the presence of those containing the 21 carbon alkyl chain, 3 and 6. The reason for the fast decrease of the ABDA emission band with the irradiation time in the presence of these complexes is unclear and will require further study, although a hydrophobic interaction between the 1O2 chemical probe and the alkyl chain of 3 and 6 should be considered. For this reason, the ΦΔ value of 3 could not be quantiﬁed due to the fast consumption of the ABDA under irradiation in the presence of this complex. In addition, the consumption of the F https://doi.org/10.1021/acsabm.1c00284 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX ACS Applied Bio Materials www.acsabm.org Article Figure 5. Changes in the UV−vis absorption and emission spectra of (a, b) 1 (7.16 μM, λexc = 447 nm), (c, d) 2 (6.94 μM, λexc = 448 nm), and (e, f) 3 (6.79 μM, λexc = 451 nm) with increasing concentration of stDNA (0−309, 0−256, and 0−198 μM, respectively) in 10 mM sodium phosphatebuﬀered aqueous solution at pH 7.4. Inset: Plot of εa (M−1 cm−1) or Ia (a.u.) vs [DNA] (M, bases) using data from absorbance at (a) 447, (c) 448, and (e) 451 nm or integrated MLCT emission intensity (b, d, f) and the best ﬁt of the data, when possible, using a modiﬁcation of the Bard equation. concomitantly recorded upon each addition of stDNA, where it was observed that 1 displayed an enhancement in the MLCT emission intensity centered at 603 nm, together with a blue shift of ca. 6 nm over the course of the titration (Figure 5b). A triphasic behavior was, however, exhibited by 2 for which the emission intensity at 604 nm enhanced upon the addition of 2.1 equiv of stDNA, followed by a decrease up to 7.4 equiv of stDNA, and by an increase again until emission intensity did not experience signiﬁcant changes upon further increasing in the stDNA concentration (Figure 5d). Complex 3 also showed a triphasic behavior starting with a decrease in the emission intensity, centered at 612 nm, after the addition of 0.5 equiv of G https://doi.org/10.1021/acsabm.1c00284 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX ACS Applied Bio Materials www.acsabm.org Article Figure 6. Changes in the UV−vis absorption and emission spectra of (a, b) 4 (7.16 μM, λexc = 414 nm), (c, d) 5 (7.85 μM, λexc = 413 nm), and (e, f) 6 (7.03 μM, λexc = 418 nm) with increasing concentration of stDNA (0−710, 0−666, and 0−90 μM, respectively) in 10 mM sodium phosphatebuﬀered aqueous solution at pH 7.4. Inset: Plot of εa (M−1 cm−1) or Ia (a.u.) vs [DNA] (M, bases) using data from absorbance at (a) 414, (c) 413, and (e) 418 nm or integrated MLCT emission intensity (b, d, f) and the best ﬁt of the data, when possible, using a modiﬁcation of the Bard equation. stDNA, and then an increase up to 1.1 equiv of stDNA (Figure 5f). This was then followed by a decrease in the emission intensity until a plateau was reached. For both complexes, the maximum of emission intensity was blue-shifted by ca. 7 nm. Complexes 4−6, containing the TAP moiety, also exhibited signiﬁcant changes in their photophysical properties upon addition of stDNA (Figure 6). Here, 4 and 5 displayed a 10% and 15% hypochromism in the MLCT band at 414 and 413 nm, respectively (Figure 6a,c). The emission titrations of 4 and 5 with stDNA showed a quenching of the MLCT emission intensity at 636 nm by 53% for both complexes (Figure 6b,d), following the trend observed for Ru(II) polypyridyl complexes containing at least two π-deﬁcient TAP ligands for which their excited state is quenched as a consequence of a photoinduced electron transfer between the guanine moieties contained by the DNA and the MLCT excited state of the complex.51,52,63 As was seen for the phen analogue 3 above, the TAP complex 6 showed an initial decrease in the absorption at the H https://doi.org/10.1021/acsabm.1c00284 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX ACS Applied Bio Materials www.acsabm.org Article Table 3. DNA Binding Parameters of 1, 2, 4, and 5a Calculated from Fits to Absorbance and Emission Data in 10 mM Sodium Phosphate-Buﬀered Aqueous Solution at pH 7.4, at 298 K absorption 4 −1 b complex Kb (10 M ) n (base pairs) 1 2 4 5 42 ± 8 553 ± 99 8.1 ± 0.4 15 ± 2 3.6 ± 0.3 3.5 ± 0.3 3.5c 3.5c emission b −1 b 2 Kb (10 M ) n (base pairs)b R2 0.99 0.99 0.99 0.98 34 ± 2 3.4 ± 0.1 0.99 9.4 ± 0.5 9.7 ± 0.3 3.5 3.5 0.99 0.99 R 4 No results for 3 and 6. bResults correspond to the mean ± SEM. cn (base pairs) is ﬁxed to 3.5, as the weak binding of the complex to DNA did not allow to keep n as a free parameter. a as a result of their complicated spectroscopic behavior in the presence of stDNA. From these titration experiments, it is clear that the presence of the alkyl chain improved the aﬃnity of the complexes for DNA since larger Kb values were observed for 2 and 5 when compared with their short chain analogues 1 and 4. At the same time, 3 and 6 gave rise to more complex binding interactions which could not be elucidated using the classic Bard et al. binding model. These results suggest that additional hydrophobic interaction could also occur and contribute to the DNA binding event of such complexes as it was previously discussed in the literature for cationic surfactants.46,72−76 Thermal Denaturation (Tm) and Circular Dichroism Studies. Having investigated the DNA binding of 1−6 by probing the complexes’ ground- and excited-state properties above, we also examined their ability to stabilize or destabilize the DNA upon their binding by carrying out both thermal denaturation (Tm) and circular dichroism (CD) studies.77 The thermal denaturation studies were carried out using stDNA in 10 mM sodium phosphate aqueous buﬀer at pH 7.4, by monitoring the changes in the absorbance at 260 nm of stDNA (150 μM) in the presence of 1−6, respectively, at DNA phosphate-to-ruthenium dye (P/D) ratios of 50, 20, and 10, while the temperature was gradually increased from 30 to 90 °C. Experiments were repeated at least three times to ensure reproducibility. The resulting thermal denaturation curves recorded in the presence of 1−6 at a P/D ratio of 10 (Figure 7) demonstrate the ability of the complexes to stabilize the DNA helix decrease with increasing alkyl chain length, the Tm being determined as 69.1 °C for stDNA alone. While 1−6 do not contain π-extended aromatic ligands, which normally favor binding to DNA by intercalation, the other binding modes such as groove binding or partial intercalation are still possible and both give rise to stabilization in the DNA double helix. At P/D of 50, the acetamide-based complexes 1 and 4 were shown to slightly increase (less than 1 °C) the Tm of stDNA (Figure S26a,b and Table S4, Supporting Information). However, at a P/D of 20, more appreciable changes were observed, resulting in Tm values of 5.0 and 2.6 °C, respectively. In contrast, at a P/D ratio of 10, these complexes were found to further increase Tm signiﬁcantly to 10.4 and 6.2 °C, respectively, demonstrating their binding to DNA. No signiﬁcant changes in the stDNA Tm were observed for 2, 3, 5, and 6 at P/D ratios of 50 and 20 (Figure S26c−f and Table S4, Supporting Information). However, at a P/D of 10, 2 and 5, containing the 10 carbon alkyl chain, displayed a slight increase in the stDNA Tm of 2.1 and 2.6 °C, respectively. In contrast, 3 and 6 did not induce a signiﬁcant increase in the stDNA Tm and, therefore, it can be concluded that their binding interactions do not stabilize the DNA double-helix structure. This is in agreement with the results obtained from MLCT band upon the addition of 0.3 equiv of stDNA, followed by a strong enhancement up to 2.0 equiv of stDNA (Figure 6e). Thereafter, a slight decrease was observed until the absorption reached a plateau. It is possible that this initial drop could be due to breakup of some degree of self-assemblies that are initially formed for 6 upon initial DNA addition. A red shift of ca. 6 nm was concomitantly observed in the MLCT band. The MLCT emission was also monitored, and, unexpectedly, the typical quenching seen for such complexes was not observed for 6 (Figure 6f). In contrast, 6 exhibited a triphasic behavior consisting of an initial strong quenching in the MLCT emission intensity centered at 643 nm after the addition of 0.3 equiv of stDNA, which was then followed by an emission enhancement up to the addition of 2.3 equiv of stDNA. After this a decrease was observed in the emission intensity upon further addition of stDNA until no more changes were observed (i.e., a plateau was observed). These changes were accompanied by a less obvious blue shift of ca. 4 nm. From the above spectroscopic changes, the binding parameters such as the binding constant (Kb) and the binding size (n) for some of these complexes were calculated using a modiﬁed Bard et al. model.68,69 The plots of εa vs [DNA] and the corresponding best ﬁt of the data, when possible, as well as the Ia vs [DNA], are shown as insets in Figures 5 and 6 for the two types of systems. The values of the obtained binding parameters are summarized in Table 3. A ﬁrst observation of the values determined from these titrations revealed a higher aﬃnity of the phen complexes (1 and 2) for stDNA when compared with their TAP analogues (4 and 5), and a diﬀerence of 1 order of magnitude was found for complexes containing the same N-1,10-phenanthrolin-5-yl-alkylamide ligand and either phen or TAP as ancillary ligands. Thus, 1 and 4 exhibited Kb values in the order of 105 and 104 M−1, respectively, while 2 and 5 showed Kb values in the order of 106 and 105 M−1, respectively. This behavior could be anticipated from the fact that TAP complexes displayed a smaller hypochromic eﬀect in their MLCT band upon addition of stDNA than phen complexes and a lower curvature of the titration curve. This might be due to partial intercalation of the phen ligands between the DNA base pairs or their location within the minor groove.70,71 While we were able to determine the binding size n as ca. 3.5 base pairs for the phen-based complexes, in the case of the TAP complexes (due to their relatively weak DNA binding) n could not be kept as a free parameter when the spectroscopic data were analyzed. Hence, it was necessary to ﬁx these as 3.5 by assuming that both the phen and the TAP systems have similar binding sizes. Unfortunately, DNA binding parameters for 3 and 6 containing the 21 carbon alkyl chain could not be determined I https://doi.org/10.1021/acsabm.1c00284 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX ACS Applied Bio Materials www.acsabm.org Article binding, or partial or full intercalation. However, these changes are similar to what we have previously seen in our investigations, which demonstrate that they are due to the association of these complexes with DNA.33,34,69 Similarly, the CD spectra of stDNA obtained in the presence of 2 and 5 showed the evolution of a negative band at 285 and 296 nm, respectively (Figure 8). In addition, the phen derivative 2 also Figure 7. Thermal denaturation curves of stDNA (150 μM) in 10 mM sodium phosphate-buﬀered aqueous solution at pH 7.4, in the absence and presence of (a) 1−3 and (b) 4−6 at a P/D ratio of 10. the ground- and the excited-state investigations, which demonstrated that 3 and 6 interact with stDNA in a nonclassic manner due to their long alkyl chain lengths. Hence, in order to shed more light on the binding of these complexes to DNA, circular dichroism (CD) studies were also carried out. CD titrations of stDNA with 1−6 were performed in 10 mM sodium phosphate aqueous buﬀer at pH 7.4. Here, the concentration of stDNA was kept constant (150 μM) and the concentration of these complexes was varied to give P/D ratios of 50, 20, 10, and 5. All of the titrations were repeated at least three times to ensure reproducibility. Overall, these results showed that the CD spectrum of stDNA was aﬀected by the presence of all of these complexes. However, these changes were mainly within the UV regions, where the absorption bands of complexes and DNA overlap. In the presence of 1 and 4 at higher loadings (e.g., P/D of 5), the appearance of a negative band was observed in the DNA absorption region with a maximum at approximately 293 nm for both complexes, which corresponds to the π−π* intraligand transitions of the ancillary phen and TAP ligands (Figure S27a,b, Supporting Information). Hence, it is diﬃcult to state with certainly if these bands result from an induced CD (ICD) or are due to structural changes in the DNA as a result of these complexes interacting with the double-helix structure through groove Figure 8. Circular dichroism spectra of stDNA (150 μM) in 10 mM sodium phosphate-buﬀered aqueous solution at pH 7.4, in the absence and presence of (a) 2 and (b) 5 at diﬀerent P/D ratios. exhibited a small positive ICD signal at about 475 nm that can be attributed to the typical MLCT absorption band. In contrast to these results, and in agreement with that seen above, only minor changes were observed upon addition of 3 and 6 to stDNA with only a small negative band in the UV region being observed, resulting in an almost complete disappearance of the characteristic DNA CD signal at a P/D ratio of 5 (Figure S27c,d, Supporting Information). Once again, the phen derivative 3 showed a small positive ICD signal at ca. 480 nm in the charge-transfer region. Taking into account the previous spectroscopic studies outlined above, we can conclude that 1, 2, 4, and 5 bind to DNA through interactions that are likely a combination of both electrostatic attraction (between the cationic Ru(II) complex and the negatively charged phosphate backbone of J https://doi.org/10.1021/acsabm.1c00284 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX ACS Applied Bio Materials www.acsabm.org DNA) and a partial intercalation into the DNA base pairs (or insertion into the grooves of the ancillary ligands), as has been demonstrated in the literature.70,71 However, in the case of 3 and 6, it can be anticipated that, in addition to the above binding modes, additional hydrophobic interactions exist. However, as discussed above, both 3 and 6 exhibited complicated spectroscopic behavior upon addition of DNA, which meant that Kb values could not be determined for these complexes, and hence, the strength of their interaction with DNA could not be quantiﬁed. Furthermore, the above DNA denaturation and CD studies have shown that, for both 3 and 6, no signiﬁcant stabilization nor changes in the conformation of the DNA helical structure occur, which would suggesting a low aﬃnity of complexes for both 3 and 6 for stDNA. Lipophilicity and Cellular Uptake Studies. Lipophilicity plays a key role in the cellular uptake mechanism of molecules designed to be used in a cellular environment and can strongly inﬂuence their cytotoxicity and cellular localization.35 In this context, the gradual increase in the length of the alkyl chain is expected to make the complexes herein more lipophilic and improve their ability to cross the cell membrane compared to [Ru(phen)3]2+ or [Ru(TAP)2phen]2+. Therefore, the logarithm of the partition coeﬃcient (log P) between octanol and water was evaluated for 1, 2, 4, and 5 using the “shake-ﬂask” method,78 with the log P values of [Ru(phen)3]2+, 3, [Ru(TAP)2phen]2+, and 6 also included for comparison (Table 4).48 Both the alkyl chain length and the ancillary respectively, showed the highest log P values, 0.49 and −1.97, respectively, both [Ru(TAP)2phen]2+ and 4, containing only one methyl group, displayed the lowest log P values, −2.76 in both cases. Furthermore, complexes containing phen as ancillary ligands were shown to be more lipophilic than those containing TAP. It has been demonstrated that substitution of phen ancillary ligands by 2,2′-bipyridine (bpy) results in a signiﬁcant reduction in the lipophilicity of the complex.80 However, to the best of our knowledge, log P values of TAP-containing complexes have only been reported by us previously,48 although ICP-MS measurements have demonstrated that [Ru(phen)3]2+ tethered to the cellpenetrating peptide TAT exhibited a greater uptake in HeLa cells than its TAP analogue.23 This suggests that TAP complexes are more hydrophilic than their phen analogues, which is in agreement with the results presented here for complexes [Ru(phen)3]2+, [Ru(TAP)2phen]2+, and 1−6. Cellular uptake of 1, 2, 4, and 5 in HeLa cervical cancer cells was then investigated and compared to that observed for [Ru(phen)3]2+, 3, [Ru(TAP)2phen]2+, and 6 in our previous study.48 Thus, HeLa cells were incubated with 1 and 4 (50 μM) at 37 °C for 24 h and 2 and 5 (10 μM) at 37 °C for 2, 4, and 24 h before being imaged using confocal ﬂuorescence microscopy (Figure 9). As expected, an increase of the alkyl chain length has a positive eﬀect on the cellular uptake of these complexes, this being in agreement with the lipophilicity studies discussed above. In the same manner as [Ru(phen)3]2+ and [Ru(TAP)2phen]2+, 1 and 4, containing the acetamide functionalized phen ligand, were not taken up into the cells and luminescence from these complexes could only be imaged in the medium under high-powered laser, with little or no luminescence inside the cells. However, as was previously reported21 for the complexes bearing a 21 carbon alkyl chain, 3 and 6, time-dependent cellular uptake was observed for 2 and 5 with a 10 carbon alkyl chain. These complexes were taken up into the cells after 2 h incubation with an increase of their cellular uptake observed after 4 and 24 h incubation, as evidenced by increasing the red luminescent emission from these complexes within the cytoplasm of the cells (Figure S28, Supporting Information). Furthermore, it should be pointed out that 5 showed a very weak luminescence inside the cells compared to that seen for 2, 3, and 6. A reduction in the emission intensity of the Ru(II) TAP complexes in the cellular environment when compared to their phen analogues has also been reported by other authors in the literature.23,24 It is well-known that Ru(II) complexes containing at least two π-acceptor ligands, such as TAP, are strong enough oxidizing agents to extract an electron from weakly reducing biomolecules (e.g., guanine or tryptophan) through a PET process, resulting in a quenching of the luminescence of the metallic complex.63,64 In this context, it can be presumed that the luminescence of 5 is quenched by a PET process between the Ru(II) moiety and the amino acids residues of the proteins existing within the cell. Although the same behavior could be expected for 6, the longer alkyl chain might prevent the metallic center from being in close proximity to the reducing species and protect its luminescence from being quenched. To further understand the luminescence properties of these complexes in a biological environment, emission studies of the TAP complexes 5 and 6 in the presence of L-tryptophan were carried out. The same studies were conducted for 2 and 3 for comparison. The emission spectra of the four complexes (10 Table 4. log P Values of 1, 2, 4, and 5 at 298 K and Their Corresponding IC50 Values in HeLa Cells Both in the Dark and with Exposure to Lighta complex log Pb IC50 dark (μM)b IC50 light (μM)b PIc [Ru(phen)3]2+d 1 2 3d [Ru(TAP)2phen]2+d 4 5 6d −2.28 ± 0.12 −2.52 ± 0.08 −1.40 ± 0.05 1.07 ± 0.08 −2.76 ± 0.02 −2.76 ± 0.22 −1.97 ± 0.05 0.49 ± 0.07 >100 >100 38 ± 7 13 ± 2 >100 >100 14 ± 4 11 ± 3 >100 >100 9±4 0.47 ± 0.01 31 ± 3 15 ± 2 9±2 2±1 4 27 >3 >7 2 6 a Complexes [Ru(phen)3]2+, 3, [Ru(TAP)2phen]2+ and 6 are included for comparison.48. blog P and IC50 values correspond to the mean ± SEM. cPhototoxic index (PI) is deﬁned as the ratio of the IC50 value in the dark to the IC50 value upon light irradiation. dEstalayo-Adrián et al.48 ligands coordinated to the Ru(II) center showed inﬂuence on the lipophilicity of the complexes. Thus, the log P values obtained for 1−3 containing phen as ancillary ligands exhibited an enhancement of the lipophilicity with the increasing alkyl chain length. In this manner, 3, containing a 21 carbon alkyl chain, displayed the highest lipophilicity with a log P value of 1.07, followed by 2, with a 10 carbon alkyl chain and a log P value of −1.40. On the other hand, [Ru(phen)3]2+ and 1, containing the acetamide functionalized phen ligand, were found to be the least lipophilic complexes with log P values of −2.28 and −2.52, respectively. Modulation of the lipophilicity of Ru(II) polypyridyl complexes by varying the number of methylene groups contained by the polypyridyl ligands have already been reported by researchers in the literature.79 A similar behavior was observed for the TAP analogues 4−6. Thus, while 6 and 5, with 21 and 10 carbon alkyl chains, K Article https://doi.org/10.1021/acsabm.1c00284 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX ACS Applied Bio Materials www.acsabm.org Article Figure 9. Confocal ﬂuorescence microscopy images of HeLa cells showing the uptake of (a) [Ru(phen)3]2+ (50 μM), 1 (50 μM), 2 (10 μM), and 3 (10 μM) and (b) [Ru(TAP)2phen]2+ (50 μM), 4 (50 μM), 5 (10 μM), and 6 (10 μM) after 24 h incubation. The nucleus is stained blue with Hoechst 33258. to a concentration of 100 μM, and as such, IC50 values could not be determined for these complexes. However, both [Ru(TAP)2phen]2+ and 4 were found to be slightly toxic after light activation with IC50 values of 31 and 15 μM, respectively. It has been demonstrated that light exposure can facilitate the cellular internalization of some Ru(II) complexes as their ability to generate 1O2 can increase the cell membrane permeability.36,81−83 To further study the eﬀect of light in the cellular uptake, HeLa cells were treated with [Ru(TAP)2phen]2+ or 4 (50 μM) before being irradiated for 1 h and incubated for a further 2 h (Figure S31, Supporting Information). HeLa cells, incubated with either complex in the dark for 3 h, were used as a control. No change in the cellular uptake of any of the complexes was observed upon light activation. However, while HeLa cells containing [Ru(TAP)2phen]2+ remained unaltered, evidence of increased toxicity of 4 after light irradiation was found in cell morphology changes compared to the ones in dark conditions. This was in agreement with the lower IC50 value observed for 4 after light activation when compared with that obtained for [Ru(TAP)2phen]2+. These results demonstrate that a photoinduced cell membrane permeabilization is not the reason for the phototoxicity observed for [Ru(TAP)2phen]2+ or 4. Moreover, although both derivatives [Ru(phen)3]2+ and 1, which instead contain phen as ancillary ligands, were shown to generate more 1O2, these complexes did not display any toxicity in HeLa cells after light irradiation. As was suggested for [Ru(TAP)2phen]2+ in our previous study,48 the phototoxicity observed for 4 should not be related to the Type-II photosensitization eﬀect as this complex produce less 1O2 than 1, but maybe due to their additional Type-I photosensitization μM) in the absence or in the presence of L-tryptophan (5 mM) were recorded (Figure S29, Supporting Information). Emission intensities of the phen complexes 2 and 3 decreased by 9 and 35% at 603 and 614 nm, respectively, in the presence of Ltryptophan. Interestingly, both 5 and 6 showed similar luminescence quenching in the presence of L-tryptophan with an 89 and 91% decrease in emission at 636 and 643 nm, respectively. Therefore, it may be presumed that, although both complexes exhibited a decrease of the emission in the presence of the reducing amino acid, the higher lipophilicity of 6, when compared to 5, results in a larger intracellular concentration and, consequently, in a higher emission intensity despite the luminescence quenching by reducing amino acids residues of the proteins within the cell. Cellular Toxicity Studies. In order to elucidate the role of the alkyl chain length in the ability of these complexes to reduce cell viability, the cytotoxic potentials of 1, 2, 4, and 5 in HeLa cells were evaluated using the Alamar Blue assay and compared to those observed for [Ru(phen)3]2+, 3, [Ru(TAP)2phen]2+, and 6 in our previous study.48 Because these complexes were shown to produce singlet oxygen, their ability to induce cytotoxicity after light activation was also studied. Therefore, cells were treated with 1, 2, 4, and 5 for 24 h before being either irradiated with 18 J cm−2 of light for 1 h using a UV-ﬁltered Hg−Xe arc lamp or kept in the dark. Cells were incubated for a further 23 h followed by addition of the Alamar Blue dye and assessment of the cytotoxicity (Table 4 and Figure S30, Supporting Information). As expected from the lack of cellular uptake observed by confocal microscopy and in the same manner as [Ru(phen)3]2+ and [Ru(TAP)2phen]2+, 1 and 4, containing the acetamide functionalized phen ligand, did not induce any cytotoxicity in the HeLa cells in the dark up L https://doi.org/10.1021/acsabm.1c00284 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX ACS Applied Bio Materials www.acsabm.org mechanism (PET) because of its highly oxidizing character in the excited state.63,64 In contrast to these results, 2 and 5, with a 10-carbon alkyl chain, displayed modest IC50 values against HeLa cells of 38 and 14 μM, respectively, after 48 h incubation in the absence of light. Some increase in cytotoxicity was observed after they were exposed to visible light, both complexes showing IC50 values of 9 μM which corresponds to a phototoxic index (PI) of ca. 4 and 2, respectively. Similarly, in our previous work we reported IC50 values of 13 and 11 μM in dark condition for 3 and 6, respectively,48 which contain a 21 carbon alkyl chain. After photoactivation, IC50 values of 0.47 and 2 μM were observed for 3 and 6, respectively, which correspond to PIs of ca. 27 and 6, revealing 3 as a promising light-activated therapeutic agent. As was demonstrated above, having a long alkyl chain increases the lipophilicity of these complexes and makes them more eﬃciently internalized into cells. This results in a larger intracellular concentration of the Ru(II) complex, which explains the increase of the IC50 values with the length of the alkyl chain. Likewise, the photoactivation observed for 2, 3, 5, and 6 is in agreement with their previously demonstrated ability to produce 1O2 or other ROS.48 Therefore, these results demonstrate that the toxicity observed in the dark is due to the increasing length of the alkyl chain, while the ruthenium core provide the resulting complexes with the ability to be photoactivated. probably due to hydrophobic interaction between the highly lipophilic complexes and the probe. The DNA binding ability of these complexes was also evaluated. We show that while 1, 2, 4, and 5 all give rise to classic changes in their photophysical properties, where changes in both the absorption and emission spectra could be analyzed to give their aﬃnity (binding constants) and binding size, those observed for 3 and 6 were complex, so that we were unable to treat the data in a meaningful way to determine binding parameters. In vitro studies of complexes 1−6 in HeLa cervical cancer cells were then performed in order to probe the applicability of these complexes in live systems. Confocal ﬂuorescence microscopy revealed the inﬂuence of the alkyl chain length on the cellular internalization and thus 2, 3, 5, and 6, containing long alkyl chains, were shown to be rapidly taken up into the cells with a preliminary localization in the cell membrane while 1 and 2 were not internalized by the cells as was found for [Ru(phen)3]2+ and [Ru(TAP)2phen]2+. This is in agreement with the log P values determined for these complexes, where an increase of the lipophilicity with the alkyl chain length was observed, 3 and 6 being the most lipophilic complexes. Finally, cellular toxicity studies showed that complexes 2, 5, and 6 displayed moderate phototoxicity against HeLa cells in contrast to 3 which exhibited signiﬁcant light-dependent cytotoxicity with a PI of ca. 27, revealing a potential for its use as a light-activated therapeutic agent. In conclusion, this work demonstrates that the cellular uptake and phototoxicity of Ru(II) polypyridyl complexes can be modulated by carefully selecting the ancillary ligands (phen or TAP) and the alkyl chain length of the lipophilic ligands coordinated to the ruthenium center. ■ CONCLUSIONS In this study, we have reported a family of six Ru(II) polypyridyl complexes in which the metal center was coordinated to either phen (1−3) or TAP (4−6) as ancillary ligands and where the third ligand consisted of a diﬀerent length of alkylamide functionalized phen. The eﬀect of the alkyl chain length on the photophysical, photochemical, and photobiological properties of these complexes was evaluated. No signiﬁcant changes were observed in the absorption and emission properties of the complexes containing either the acetamide functionalized phenanthroline ligand, 1 and 4, or the 10 carbon alkyl chain, 2 and 5, when compared to the wellknown complexes [Ru(phen)3]2+ and [Ru(TAP)2phen]2+. However, the extension of the alkyl chain to 21 carbons seemed to aﬀect both the luminescence quantum yields and lifetimes of complexes 3 and 6, most likely due to their ability to self-assemble in solution as a result of their amphiphilic character. In addition, the luminescence lifetime of 3 was found to rise upon increasing the complex concentration, while the opposite eﬀect was observed for 6, indicating diﬀerent photophysical responses to the self-assembly process by the phen (3) and TAP (6) complexes, due in part to oxygen quenching protection and self-quenching of their excited states. Direct measurement of 1O2 phosphorescence at 1265 nm in O2-saturated D2O revealed an eﬀect of the alkyl chain length on the ability of 1−6 to produce 1O2. Thus, while 1, 2, 4, and 5 showed moderate 1O2 photosensitization in air-saturated H2O, very low quantum yield values of 1O2 were obtained for 3 and 6. Eﬀorts were made to indirectly quantify the 1O2 production by 3 and 6 by monitoring the ﬂuorescence disappearance of the water-soluble 1O2 chemical probe ABDA, as this method allowed one to work in a concentration range where the complexes are expected to behave as free monomers, facilitating their interaction with molecular oxygen. However, no conclusive results were obtained from these experiments ■ Article EXPERIMENTAL SECTION Materials and Instrumentation. All chemicals and solvents were obtained commercially and, unless speciﬁed, used without further puriﬁcation. Solvents for synthetic purposes were used at general purpose reagent (GPR) grade unless otherwise stated. Dry solvents were obtained from a solvent puriﬁcation system (SPS) purchased from Innovative Technology Inc. General Characterization Techniques. All NMR spectra were recorded using either a Bruker Avance III 400 NMR spectrometer operating at 400 MHz for 1H NMR and 101 MHz for 13C NMR or a Bruker Avance II 600 NMR spectrometer operating at 600 MHz for 1 H NMR and 151 MHz for 13C NMR. Chemical shifts (δ) were referenced relative to the internal solvent signals. Electrospray ionization (ESI) mass spectra were recorded on a Micromass LCT spectrometer (calibrated using leucine enkephalin m/z = 556.2771). Matrix-assisted laser desorption/ionization (MALDI) mass spectra were recorded on a MALDI QToF Premier (Waters Corp., Micromass MS Technologies, Manchester, U.K.), and high-resolution (HR) mass spectra were determined by a peak matching method using Glu-Fib as an internal reference (m/z = 1570.677). Infrared spectra were recorded on a PerkinElmer Spectrum One FT-IR spectrometer ﬁtted with a Universal ATR Sampling Accessory for solid samples. Melting points were determined using an IA9000 digital melting point apparatus. Elemental analyses were conducted at the Microanalytical Laboratory, School of Chemistry, University College Dublin (UCD). Photophysical Characterization. UV−vis absorption spectra were recorded on a Varian CARY 50 spectrophotometer. Emission and excitation spectra were recorded on a Varian Carey Eclipse ﬂuorimeter. Luminescence quantum yields (Φem) were determined using an established method.48,58 Luminescence lifetimes were measured by single-photon timing on a Horiba Fluoromax-4TCM https://doi.org/10.1021/acsabm.1c00284 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX ACS Applied Bio Materials www.acsabm.org SPC or a Fluorolog FL 3−22 equipped with a FluoroHub v2.0 singlephoton timing module. Direct Detection of Singlet Oxygen (Time-Resolved NearInfrared Phosphorescence). Quantum yields of singlet oxygen production were measured using an Edinburgh Instruments (U.K.) LP-900 laser kinetic spectrometer system equipped with a frequencydoubled Nd:YAG laser (Minilite II, Continuum, CA) and a Hamamatsu H10330-45 NIR PMT module for the singlet oxygen emission monitoring at 1265 nm (Bentham TM300 monochromator with 600 grooves mm−1 NIR grating). Absorbance matched (A532 ≈ 0.40) solutions of the ruthenium complex and the reference photosensitizer [Ru(phen)3]Cl2 (ΦΔ = 0.39 ± 0.03 in O2-saturated D2O at room temperature). Full details have been previously given.48,59 All 1O2 luminescence decay proﬁles were ﬁtted using a single-exponential function [after excluding the fast (sub-μs) decay due to the residual Ru(II) sensitizer emission even under O2 saturation of the solution].59,84 From the experimental data obtained in D2O, the ΦΔ values in air-equilibrated H2O were calculated taking into account eqs 1 and (2):55 τ POT2 = τkq[O2] = 1 − τ0 (1) treatments performed on three independent days. For photoactivation studies, cells were illuminated with 18 J cm−2 of light for 1 h using a Hamamatsu L2570 200 W Hg−Xe arc lamp equipped with an aqueous solution of NaNO2 as UV ﬁlter. Synthesis and Characterization. 1,4,5,8-Tetraazaphenanthrene (TAP),86 5-amino-1,10-phenanthroline,87 the precursor complexes cis-[Ru(phen)2Cl2] and cis-[Ru(TAP)2Cl2],88 and complexes [Ru(phen)3]2+, [Ru(TAP)2phen]2+, 3, and 6 were synthesized according to procedures previously reported in the literature.48 5-Acetamido-1,10-phenanthroline (7). 5-Amino-1,10-phenanthroline (153 mg, 783 μmol, 1 equiv) and acetic anhydride (0.75 mL, 7.93 mmol, 10 equiv) were mixed in dry MeCN (25 mL). The reaction mixture was stirred in the dark and under an inert atmosphere at room temperature for 2 days. Solvent was removed under reduced pressure, yielding the product as a beige solid which was dried in vacuo (114 mg, 482 μmol, 62%). mp 226−231 °C (decomp.) Lit. mp 230 °C.87 δH (400 MHz, DMSO-d6): 10.14 (1H, s, NH), 9.13 (1H, dd, H2, 3J = 4.2 Hz, 4J = 1.6 Hz), 9.03 (1H, dd, H9, 3J = 4.3 Hz, 4J = 1.7 Hz), 8.63 (1H, dd, H4, 3J = 8.4 Hz, 4J = 1.7 Hz), 8.44 (1H, dd, H7, 3J = 8.2 Hz, 4J = 1.7 Hz), 8.17 (1H, s, H6), 7.82 (1H, dd, H3, 3J = 8.4 Hz, 3J = 4.3 Hz), 7.74 (1H, dd, H8, 3J = 8.2 Hz, 3 J = 4.3 Hz), 6.13 (3H, s, CH3). δC (101 MHz, DMSO-d6): 169.49 (CO), 149.86, 149.29, 145.85 (q), 143.77 (q), 135.81, 131.86 (q), 131.68, 128.10, 124.59 (q), 123.59, 122.84, 119.86, 23.63 (CH3). νmax (ATR)/cm−1: 3200 (amide N−H stretch), 3040 (aromatic C−H stretch), 2924 (alkane C−H stretch), 1688 (CO stretch), 1532 (amide N−H bend), 1476 (aromatic CC stretch), 1420 (C−N stretch). ESI+-HRMS: m/z calc = 238.0980 for C14H12N3O; m/z found = 238.0973 [M + H]+. N-1,10-Phenanthrolin-5-ylundecanamide (8). 5-Amino-1,10-phenanthroline (101 mg, 516 mmol, 1 equiv) was dissolved in dry CH2Cl2 (10 mL), and the solution was cooled to 0 °C before undecanoic acid (108 μL, 516 μmol, 1 equiv), N-ethyl-N′-(3(dimethylamino)propyl)carbodiimide hydrochloride (246 mg, 1.28 mmol, 2.5 equiv), and 4-(dimethylamino)pyridine (62.4 mg, 511 μmol, 1 equiv) were added to the solution. The resulting mixture was stirred under inert atmosphere and at 0 °C for 1 h and then for a further 2 days at room temperature. After removing the solvent under reduced pressure, an orange oil was obtained which was dried in vacuo. The addition of H2O caused precipitation of a beige solid which was isolated by centrifugation and washed several times with more H2O. The resulting solid was re-dispersed in MeCN, collected by centrifugation, and dried in vacuo, yielding the product as a beige solid (107 mg, 295 μmol, 57%). mp 83−86 °C. δH (600 MHz, DMSO-d6): 10.07 (1H, s, NH), 9.12 (1H, dd, H2, 3J = 4.2 Hz, 4J = 1.6 Hz), 9.03 (1H, dd, H9, 3J = 4.2 Hz, 4J = 1.7 Hz), 8.59 (1H, dd, H4, 3J = 8.4 Hz, 4J = 1.6 Hz), 8.44 (1H, dd, H7, 3J = 8.1 Hz, 4J = 1.7 Hz), 8.17 (1H, s, H6), 7.82 (1H, dd, H3, 3J = 8.4 Hz, 3J = 4.2 Hz), 7.74 (1H, dd, H8, 3J = 8.1 Hz, 3J = 4.2 Hz), 2.52 (2H, t, H2′, 3J = 7.3 Hz), 1.69 (2H, m, H3′), 1.31 (14H, m, H4′−H10′), 0.84 (3H, t, H11′, 3J = 7.0 Hz). δC (151 MHz, DMSO-d6): 172.44 (CO), 149.82, 149.26, 145.86 (q), 143.77 (q), 135.75, 131.79 (q), 131.57, 128.09 (q), 124.64 (q), 123.55, 122.78, 119.97, 35.96, 31.30, 28.99, 28.96, 28.81, 28.73, 28.69, 25.22, 22.08, 13.95. νmax (ATR)/cm−1: 3255 (amide N− H stretch), 3046 (aromatic C−H stretch), 2919 and 2849 (alkane C− H stretch), 1655 (CO stretch), 1543 (amide N−H bend), 1467 (aromatic CC stretch), 1424 (C−N stretch). ESI+-HRMS: m/z calc = 386.2203 for C23H29N3NaO; m/z found = 386.2206 [M + Na]+. Bis(1,10-phenanthroline)(N-1,10-phenanthrolin-5-ylacetamide)ruthenium(II) Chloride (1). The precursor complex cis-[Ru(phen)2Cl2] (101 mg, 190 μmol, 1 equiv) and ligand 7 (66.7 mg, 281 μmol, 1.5 equiv) were suspended in EtOH/H2O (1:1, 8 mL). The mixture was deoxygenated by sparging with argon for 15 min and heated at 140 °C for 40 min using microwave irradiation. Solvent was removed at reduced pressure and the resulting solid was puriﬁed by alumina chromatography using MeCN/H2O (10:0 to 9:1) as eluent, yielding the product as a red solid which was dried in vacuo (87.3 mg, 125 μmol, 60%). Calculated for C38H27N7Cl2ORu + 2.3H2O: C, 56.27; H, 3.93; N, 12.09; Cl, 8.74. Found: C, 56.21; H, 3.88; N, 12.12; Cl, 8.20. mp 106−109 °C (decomp.). δH (400 MHz, CD3CN): ΦΔ = ΦTPOT2 fΔT (2) ΦT is considered to be equal to 1 for these types of complexes.55 Assuming that fTΔ is the same in O2-saturated D2O and air-saturated H2O, then ΦΔ values in air-equilibrated H2O were determined using eq 3,: i y i y complex j jj1 − τH2O,air zzz/jjj1 − τD2O,O2 zzz ΦΔcomplex zz jj z ,H2O,air = ΦΔ,D2 O,O2 j jj z j τH2O,Ar τD2O,Ar zz k { k { (3) Indirect Detection of Singlet Oxygen (Photo-oxidation of a Chemical Probe). Singlet oxygen production was also evaluated using the water-soluble 1O2 trap ABDA.48,85 Partition Coeﬃcients (log P). Partition coeﬃcients were determined by the shake-ﬂask method. The log P values were calculated according to a method previously employed in our research group.48 ij c − c water yz zz log P = logjjj t j c water zz k { DNA Binding Studies Techniques. UV−vis absorption and emission titrations were carried out on samples of the dye at (1 ± 0.5) × 10−5 M at 298 K by monitoring changes in the absorption and emission spectra of the complexes in 10 mM sodium phosphate buﬀer (pH 7.4) upon successive additions of aliquots of stDNA. The results are quoted using the concentration of stDNA expressed as a nucleotide phosphate-to-dye ratio (P/D ratio). Binding constants (Kb) and binding size (n) were determined using a reorganization of the original Bard et al. equation. Kb values represent the mean ± SEM of three independent experiments. General Biological Procedures. Cell Culture. HeLa cells were grown in a cell culture ﬂask using Dulbecco’s modiﬁed Eagle medium supplemented with 10% fetal bovine serum, 1% penicillin/ streptomycin, and 0.2% plasmocin at 37 °C in a humidiﬁed atmosphere of 5% CO2. Confocal Microscopy. HeLa cells were seeded at a density of 5 × 104 cells/mL and treated as indicated. Cells were then washed using a known protocol48 and imaged by live microscopy using an Olympus FV1000 point scanning microscope with a 60× oil immersion lens (NA, 1.42). Viability Assays. HeLa cells were seeded at a density of 2.5 × 103 cells/mL in a 96-well plate and treated with diﬀerent concentrations of the appropriate Ru(II) complex. Into each well 20 μL of Alamar Blue (BioSource) was added and left to incubate at 37 °C in the dark for 4 h. Fluorescence was read at 590 nm using a ﬂuorescence microplate reader (SpectraMax Gemini XS, Molecular Devices; λexc = 544 nm). Data points represent the mean ± SEM of triplicate N Article https://doi.org/10.1021/acsabm.1c00284 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX ACS Applied Bio Materials www.acsabm.org 11.61 (1H, s, NH), 9.60 (1H, dd, H4L7, 3J = 8.6 Hz, 4J = 1.1 Hz), 8.64 (1H, s, H6L7), 8.59 (4H, m, Hphen), 8.47 (1H, dd, H7L7, 3J = 8.3 Hz, 4J = 1.1 Hz), 8.25 (2H, s, H5phen or H6phen), 8.24 (2H, s, H5phen or H6phen), 8.09 (1H, dd, H2L7, 3J = 5.2 Hz, 4J = 1.1 Hz), 8.02 (4H, m, Hphen), 7.89 (1H, dd, H9L7, 3J = 5.2 Hz, 4J = 1.1 Hz), 7.62 (5H, m, H3L7 and Hphen), 7.52 (1H, dd, H8L7, 3J = 8.3 Hz, 3J = 5.2 Hz), 2.45 (3H, s, CH3). δC (101 MHz, CD3CN): 171.81 (CO), 154.17, 153.97, 153.85, 153.80, 153.68, 152.27, 148.87 (q), 137.62, 137.59, 136.94, 135.46, 131.93 (q), 131.88 (q), 128.97, 128.91, 126.85, 126.83, 126.76, 126.74, 126,64, 125.76, 119.79, 24.40 (CH3). νmax (ATR)/cm−1: 3234 (amide N−H stretch), 3046 (aromatic C−H stretch), 2994 (alkane C−H stretch), 1683 (CO stretch), 1533 (amide N−H bend), 1480 (aromatic CC stretch), 1424 (C−N stretch). ESI+-HRMS: m/z calc = 699.1321 for C38H27N7ORu; m/z 2+ found = 349.5676 [M] . Bis(1,10-phenanthroline)(N-1,10-phenanthrolin-5ylundecanamide)ruthenium(II) Chloride (2). Complex 2 was synthesized according to the same procedure described for 1 but using cis-[Ru(phen)2Cl2] (164 mg, 307 μmol, 1 equiv) as precursor complex and ligand 8 (109 mg, 300 μmol, 1 equiv), yielding the product as a red solid (161 mg, 99.0 μmol, 60%). Calculated for C47H45N7Cl2ORu + 2.6H2O: C, 59.88; H, 5.37; N, 10.40; Cl, 7.52. Found: C, 59.57; H, 5.08; N, 10.37; Cl, 7.18. mp 220−223 °C (decomp.). δH (400 MHz, CD3CN): 11.33 (1H, s, NH), 9.48 (1H, dd, H4L8, 3J = 8.6 Hz, 4J = 1.0 Hz), 8.64 (1H, s, H6L8), 8.60 (4H, m, Hphen), 8.47 (1H, dd, H7L8, 3J = 8.3 Hz, 4J = 1.0 Hz), 8.25 (4H, 2s, H5phen and H6phen), 8.09 (1H, dd, H2L8, 3J = 5.2 Hz, 4J = 1.0 Hz), 8.02 (4H, m, Hphen), 7.90 (1H, dd, H9L8, 3J = 5.2 Hz, 4J = 1.0 Hz), 7.62 (5H, m, H3L8 and Hphen), 7.53 (1H, dd, H8L8, 3J = 8.3 Hz, 3J = 5.2 Hz), 2.79 (2H, t, H2′ L8, 3J = 7.4 Hz), 1.71 (2H, m, H3′ L8), 1.34 (14H, m, H4′−H10′ L8), 0.84 (3H, t, H11′ L8, 3J = 6.7 Hz). δC (101 MHz, CD3CN): 175.03 (CO), 154.18, 153.99, 153.88, 153.73, 152.34, 148.89 (q) 137.67, 136.99, 136.08 (q), 135.40, 131.93 (q), 129.03, 128.97, 128.13 (q), 126.91, 126.81, 126.71, 125.85, 119.95, 37.41, 32.61, 30.31, 30.22, 30.03, 26.60, 23.36, 14.37. νmax (ATR)/ cm−1: 3243 (amide N−H stretch), 3043 (aromatic C−H stretch), 2922 and 2851 (alkane C−H stretch), 1688 (CO stretch), 1535 (amide N−H bend), 1457 (aromatic CC stretch), 1424 (C−N stretch). ESI+-HRMS: m/z calc = 825.2729 for C47H45N7ORu; m/z 2+ found = 412.6378 [M] . Bis(1,4,5,8-tetraazaphenantherene)(N-1,10-phenanthrolin-5ylacetamide)ruthenium(II) Chloride (4). Complex 4 was synthesized according to the same procedure described for 1 but using cis[Ru(TAP)2Cl2] (90.8 mg, 169 μmol, 1 equiv) as precursor complex and ligand 7 (44.1 mg, 186 μmol, 1.1 equiv), yielding the product as a red solid (67.5 mg, 87.2 μmol, 52%). Calculated for C34H23N11Cl2ORu + 5.2H2O + 0.2NaCl: C, 46.46; H, 3.83; N, 17.53; Cl, 8.87. Found: C, 46.27; H, 3.17; N, 16.89; Cl, 8.66. mp 162−164 °C (decomp.). δH (400 MHz, DMSO-d6): 10.71 (1H, s, NH), 9.06 (5H, m, H4L7 and HTAP), 8.80 (1H, d, H7L7, 3J = 8.2 Hz), 8.66 (5H, s, H6L7, H9TAP and H10TAP), 8.51 (1H, d, HTAP, 3J = 2.8 Hz)), 8.48 (1H, d, HTAP, 3J = 2.8 Hz), 8.30 (1H, d, H2L7, 3J = 5.3 Hz), 8.24 (2H, m, HTAP), 8.17 (1H, d, H9L7, 3J = 5.3 Hz), 7.82 (1H, dd, H3L7, 3J = 5.3 Hz, 3J = 8.5 Hz), 7.73 (1H, dd, H8L7, 3J = 5.3 Hz, 3J = 8.2 Hz), 2.32 (3H, s, CH3). δC (101 MHz, DMSO-d6): 169.82 (C O), 153.94, 152.66, 149.65, 149.50, 148.67, 148.62, 146.82 (q), 144.52 (q), 144.51 (q), 144.48 (q), 144.06 (q), 141.98 (q), 141.93 (q), 141.91 (q), 137.36, 133.92, 132.41, 132.28, 130.34 (q), 126.45 (q), 126.34, 125.64, 119.01, 23.80 (CH3). νmax (ATR)/cm−1: 3220 (amide N−H stretch), 3052 (aromatic C−H stretch), 2979 (alkane C−H stretch), 1681 (CO stretch), 1528 (amide N−H bend), 1485 (aromatic CC stretch), 1383 (C−N stretch). MALDI+-HRMS: m/ z calc = 703.1131 for C34H23N11ORu; m/z found = 703.1135 [M]+. Bis(1,4,5,8-tetraazaphenantherene)(N-1,10-phenanthrolin-5ylundecanamide)ruthenium(II) Chloride (5). Complex 5 was synthesized according to the same procedure described for 1 but using cis-[Ru(TAP)2Cl2] (101 mg, 192 μmol, 1 equiv) as precursor complex and ligand 8 (82.5 mg, 227 μmol, 1.2 equiv), yielding the product as a red solid (73.0 mg, 81.1 μmol, 43%). Calculated for C43H41N11Cl2ORu + 4.6H2O + 0.1NaCl: C, 52.24; H, 5.12; N, 15.59; Cl, 7.53. Found: C, 51.89; H, 4.45; N, 15.34; Cl, 6.93. mp 150−151 °C (decomp.). δH (400 MHz, CD3CN): 11.67 (1H, s, NH), 9.71 (1H, d, H4L8, 3J = 8.5 Hz), 8.94 (4H, m, HTAP), 8.69 (1H, s, H6L8), 8.58 (5H, s, H7L8, H9TAP and H10TAP), 8.29 (1H, d, HTAP, 3J = 2.7 Hz), 8.27 (1H, d, HTAP, 3J = 2.7 Hz), 8.20 (1H, d, HTAP, 3J = 2.7 Hz), 8.16 (1H, d, HTAP, 3J = 2.7 Hz), 8.14 (1H, d, H2L8, 3J = 5.1 Hz), 7.99 (1H, d, H9L8, 3J = 5.1 Hz), 7.70 (1H, dd, H3L8, 3J = 5.1 Hz, 3J = 8.5 Hz), 7.60 (1H, dd, H8L8, 3J = 5.1 Hz, 3J = 8.5 Hz), 2.84 (2H, t, H2′ L8, 3J = 7.4 Hz), 1.74 (2H, m, H3′ L8), 1.33 (14H, m, H4′−H10′ L8), 0.86 (3H, t, H11′ L8, 3J = 7.0 Hz). δC (101 MHz, CD3CN): 175.04 (CO), 154.69, 153.19, 150.38, 150.33, 150.30, 150.14, 150.09, 149.84, 149.70, 148.30 (q), 146.38 (q), 146.34 (q), 146.31 (q), 146.28 (q), 145.33 (q), 143.31 (q), 143.28 (q), 143.26 (q), 138.34, 136.99, 136.24 (q), 133.72, 133.55, 132.11 (q), 128.29 (q), 126.90, 125.98, 119.90, 37.31, 32.56, 30.27, 30.17, 30.00, 29.98, 26.55, 23.32, 14.32. νmax (ATR)/cm−1: 3247 (amide N−H stretch), 3048 (aromatic C−H stretch), 2923 and 2852 (alkane C−H stretch), 1671 (CO stretch), 1535 (amide N−H bend), 1485 (aromatic CC stretch), 1422 (C− N stretch). ESI+-HRMS: m/z calc = 829.2539 for C43H41N11ORu; m/z 2+ found = 414.6258 [M] . ■ Article ASSOCIATED CONTENT * Supporting Information sı The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsabm.1c00284. Figures of NMR, MS, and IR spectra and ground and excited-state changes, and cellular imaging, etc.; tables for binding constants, CD, and excited-state lifetimes (PDF) Special Issue Paper This paper missed the Biospecies Sensors Forum special issue. ■ AUTHOR INFORMATION Corresponding Authors Sandra Estalayo-Adrián − School of Chemistry and Trinity Biomedical Sciences Institute (TBSI) and Advanced Materials and BioEngineering Research (AMBER) Centre, Trinity College Dublin, The University of Dublin, Dublin 2, Ireland; Email: estalays@tcd.ie Thorﬁnnur Gunnlaugsson − School of Chemistry and Trinity Biomedical Sciences Institute (TBSI) and Advanced Materials and BioEngineering Research (AMBER) Centre, Trinity College Dublin, The University of Dublin, Dublin 2, Ireland; orcid.org/0000-0003-4814-6853; Email: gunnlaut@tcd.ie Authors Salvador Blasco − School of Chemistry and Trinity Biomedical Sciences Institute (TBSI), Trinity College Dublin, The University of Dublin, Dublin 2, Ireland Sandra A. Bright − School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute (TBSI), Trinity College Dublin, The University of Dublin, Dublin 2, Ireland Gavin J. McManus − School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute (TBSI), Trinity College Dublin, The University of Dublin, Dublin 2, Ireland Guillermo Orellana − Department of Organic Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, E-28040 Madrid, Spain; orcid.org/0000-0002-45726564 D. Clive Williams − School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute (TBSI), Trinity College Dublin, The University of Dublin, Dublin 2, Ireland O https://doi.org/10.1021/acsabm.1c00284 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX ACS Applied Bio Materials www.acsabm.org John M. Kelly − School of Chemistry and Trinity Biomedical Sciences Institute (TBSI), Trinity College Dublin, The University of Dublin, Dublin 2, Ireland; orcid.org/00000002-3706-1777 (14) Zhang, P.; Wang, J.; Huang, H.; Yu, B.; Qiu, K.; Huang, J.; Wang, S.; Jiang, L.; Gasser, G.; Ji, L.; Chao, H. Unexpected high photothemal conversion efficiency of gold nanospheres upon grafting with two-photon luminescent ruthenium(II) complexes: A way towards cancer therapy? Biomaterials 2015, 63, 102−114. (15) Martínez-Calvo, M.; Orange, K. N.; Elmes, R. B. P.; la Cour Poulsen, B.; Williams, D. C.; Gunnlaugsson, T. Ru(ii)-polypyridyl surface functionalised gold nanoparticles as DNA targeting supramolecular structures and luminescent cellular imaging agents. Nanoscale 2016, 8, 563−574. (16) Wumaier, M.; Yao, T.-M.; Hu, X.-C.; Hu, Z.-A.; Shi, S. Luminescent Ru(ii)-thiol modified silver nanoparticles for lysosome targeted theranostics. Dalton Trans. 2019, 48, 10393−10397. (17) Estalayo-Adrián, S.; McManus, G. J.; Dalton, H. L.; Savyasachi, A. J.; Kelly, J. M.; Gunnlaugsson, T. Functionalisation of gold nanoparticles with ruthenium(ii) polypyridyl complexes for their application in cellular imaging. Dalton Trans. 2020, 49, 14158−14168. (18) Puckett, C. A.; Barton, J. K. Fluorescein Redirects a Ruthenium−Octaarginine Conjugate to the Nucleus. J. Am. Chem. Soc. 2009, 131, 8738−8739. (19) Puckett, C. A.; Barton, J. K. Targeting a ruthenium complex to the nucleus with short peptides. Bioorg. Med. Chem. 2010, 18, 3564− 3569. (20) Cosgrave, L.; Devocelle, M.; Forster, R. J.; Keyes, T. E. Multimodal cell imaging by ruthenium polypyridyl labelled cell penetrating peptides. Chem. Commun. 2010, 46, 103−105. (21) Blackmore, L.; Moriarty, R.; Dolan, C.; Adamson, K.; Forster, R. J.; Devocelle, M.; Keyes, T. E. Peptide directed transmembrane transport and nuclear localization of Ru(ii) polypyridyl complexes in mammalian cells. Chem. Commun. 2013, 49, 2658−2660. (22) Joshi, T.; Pierroz, V.; Ferrari, S.; Gasser, G. Bis(dipyridophenazine)(2-(2′-pyridyl)pyrimidine-4-carboxylic acid)ruthenium(II) Hexafluorophosphate: A Lesson in Stubbornness. ChemMedChem 2014, 9, 1419−1427. (23) Marcélis, L.; Kajouj, S.; Ghesquière, J.; Fettweis, G.; Coupienne, I.; Lartia, R.; Surin, M.; Defrancq, E.; Piette, J.; Moucheron, C.; Kirsch-De Mesmaeker, A. Highly DNA-Photoreactive Ruthenium 1,4,5,8-Tetraazaphenanthrene Complex Conjugated to the TAT Peptide: Efficient Vectorization inside HeLa Cells without Phototoxicity − The Importance of Cellular Distribution. Eur. J. Inorg. Chem. 2016, 2016, 2902−2911. (24) Huang, H.; Yu, B.; Zhang, P.; Huang, J.; Chen, Y.; Gasser, G.; Ji, L.; Chao, H. Highly Charged Ruthenium(II) Polypyridyl Complexes as Lysosome-Localized Photosensitizers for Two-Photon Photodynamic Therapy. Angew. Chem., Int. Ed. 2015, 54, 14049− 14052. (25) Burke, C. S.; Byrne, A.; Keyes, T. E. Highly Selective Mitochondrial Targeting by a Ruthenium(II) Peptide Conjugate: Imaging and Photoinduced Damage of Mitochondrial DNA. Angew. Chem., Int. Ed. 2018, 57, 12420−12424. (26) Gottschaldt, M.; Schubert, U. S.; Rau, S.; Yano, S.; Vos, J. G.; Kroll, T.; Clement, J.; Hilger, I. Sugar-Selective Enrichment of a DGlucose-Substituted Ruthenium Bipyridyl Complex Inside HepG2 Cancer Cells. ChemBioChem 2010, 11, 649−652. (27) Wenzel, M.; de Almeida, A.; Bigaeva, E.; Kavanagh, P.; Picquet, M.; Le Gendre, P.; Bodio, E.; Casini, A. New Luminescent Polynuclear Metal Complexes with Anticancer Properties: Toward Structure−Activity Relationships. Inorg. Chem. 2016, 55, 2544−2557. (28) Lameijer, L. N.; Hopkins, S. L.; Brevé, T. G.; Askes, S. H. C.; Bonnet, S. d- Versus l-Glucose Conjugation: Mitochondrial Targeting of a Light-Activated Dual-Mode-of-Action Ruthenium-Based Anticancer Prodrug. Chem. - Eur. J. 2016, 22, 18484−18491. (29) Lo, K. K.-W.; Lee, T. K.-M.; Lau, J. S.-Y.; Poon, W.-L.; Cheng, S.-H. Luminescent Biological Probes Derived from Ruthenium(II) Estradiol Polypyridine Complexes. Inorg. Chem. 2008, 47, 200−208. (30) Dosio, F.; Stella, B.; Ferrero, A.; Garino, C.; Zonari, D.; Arpicco, S.; Cattel, L.; Giordano, S.; Gobetto, R. Ruthenium polypyridyl squalene derivative: A novel self-assembling lipophilic probe for cellular imaging. Int. J. Pharm. 2013, 440, 221−228. Complete contact information is available at: https://pubs.acs.org/10.1021/acsabm.1c00284 Notes The authors declare no competing ﬁnancial interest. ■ ACKNOWLEDGMENTS We thank Science Foundation Ireland (SFI PI Awards 10/45 IN.1/B2999 and 13/IA/1865), Marie Skłodowska-Curie actions (MSCA, to S.B.), and Trinity College Dublin (TCD) for ﬁnancial support. We also thank Drs. Feeney, Hessman, O’Brien, and Ruether for the help with MS and NMR studies. ■ Article REFERENCES (1) Juris, A.; Balzani, V.; Barigelletti, F.; Campagna, S.; Belser, P.; von Zelewsky, A. Ru(II) polypyridine complexes: photophysics, photochemistry, eletrochemistry, and chemiluminescence. Coord. Chem. Rev. 1988, 84, 85−277. (2) Balzani, V.; Juris, A.; Venturi, M.; Campagna, S.; Serroni, S. Luminescent and Redox-Active Polynuclear Transition Metal Complexes. Chem. Rev. 1996, 96, 759−834. (3) Vos, J. G.; Kelly, J. M. Ruthenium polypyridyl chemistry; from basic research to applications and back again. Dalton Trans. 2006, 4869−4883. (4) 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. (5) Mari, C.; Pierroz, V.; Leonidova, A.; Ferrari, S.; Gasser, G. Towards Selective Light-Activated RuII-Based Prodrug Candidates. Eur. J. Inorg. Chem. 2015, 2015, 3879−3891. (6) 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. (7) Poynton, F. E.; Bright, S. A.; Blasco, S.; Williams, D. C.; Kelly, J. M.; Gunnlaugsson, T. The development of ruthenium(ii) polypyridyl complexes and conjugates for in vitro cellular and in vivo applications. Chem. Soc. Rev. 2017, 46, 7706−7756. (8) White, J. K.; Schmehl, R. H.; Turro, C. An overview of photosubstitution reactions of Ru(II) imine complexes and their application in photobiology and photodynamic therapy. Inorg. Chim. Acta 2017, 454, 7−20. (9) Thota, S.; Rodrigues, D. A.; Crans, D. C.; Barreiro, E. J. Ru(II) Compounds: Next-Generation Anticancer Metallotherapeutics? J. Med. Chem. 2018, 61, 5805−5821. (10) Monro, S.; Colón, K. L.; Yin, H.; Roque, J.; Konda, P.; Gujar, S.; Thummel, R. P.; Lilge, L.; Cameron, C. G.; McFarland, S. A. Transition Metal Complexes and Photodynamic Therapy from a Tumor-Centered Approach: Challenges, Opportunities, and Highlights from the Development of TLD1433. Chem. Rev. 2019, 119, 797−828. (11) Shum, J.; Leung, P. K.-K.; Lo, K. K.-W. Luminescent Ruthenium(II) Polypyridine Complexes for a Wide Variety of Biomolecular and Cellular Applications. Inorg. Chem. 2019, 58, 2231−2247. (12) Elmes, R. B. P.; Orange, K. N.; Cloonan, S. M.; Williams, D. C.; Gunnlaugsson, T. Luminescent Ruthenium(II) Polypyridyl Functionalized Gold Nanoparticles; Their DNA Binding Abilities and Application As Cellular Imaging Agents. J. Am. Chem. Soc. 2011, 133, 15862−15865. (13) Rogers, N. J.; Claire, S.; Harris, R. M.; Farabi, S.; Zikeli, G.; Styles, I. B.; Hodges, N. J.; Pikramenou, Z. High coating of Ru(ii) complexes on gold nanoparticles for single particle luminescence imaging in cells. Chem. Commun. 2014, 50, 617−619. P https://doi.org/10.1021/acsabm.1c00284 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX ACS Applied Bio Materials www.acsabm.org (31) Siewert, B.; van Rixel, V. H. S.; van Rooden, E. J.; Hopkins, S. L.; Moester, M. J. B.; Ariese, F.; Siegler, M. A.; Bonnet, S. Chemical Swarming: Depending on Concentration, an Amphiphilic Ruthenium Polypyridyl Complex Induces Cell Death via Two Different Mechanisms. Chem. - Eur. J. 2016, 22, 10960−10968. (32) Schatzschneider, U.; Niesel, J.; Ott, I.; Gust, R.; Alborzinia, H.; Wölfl, S. Cellular Uptake, Cytotoxicity, and Metabolic Profiling of Human Cancer Cells Treated with Ruthenium(II) Polypyridyl Complexes [Ru(bpy)2(N−N)]Cl2 with N−Nbpy, phen, dpq, dppz, and dppn. ChemMedChem 2008, 3, 1104−1109. (33) Cloonan, S. M.; Elmes, R. B. P.; Erby, M.; Bright, S. A.; Poynton, F. E.; Nolan, D. E.; Quinn, S. J.; Gunnlaugsson, T.; Williams, D. C. Detailed Biological Profiling of a Photoactivated and Apoptosis Inducing pdppz Ruthenium(II) Polypyridyl Complex in Cancer Cells. J. Med. Chem. 2015, 58, 4494−4505. (34) Poulsen, B. C.; Estalayo-Adrián, S.; Blasco, S.; Bright, S. A.; Kelly, J. M.; Williams, D. C.; Gunnlaugsson, T. Luminescent ruthenium polypyridyl complexes with extended ‘dppz’ like ligands as DNA targeting binders and cellular agents. Dalton Trans. 2016, 45, 18208−18220. (35) Puckett, C. A.; Barton, J. K. Methods to Explore Cellular Uptake of Ruthenium Complexes. J. Am. Chem. Soc. 2007, 129, 46− 47. (36) Svensson, F. R.; Matson, M.; Li, M.; Lincoln, P. Lipophilic ruthenium complexes with tuned cell membrane affinity and photoactivated uptake. Biophys. Chem. 2010, 149, 102−106. (37) Johnson, I. D., Probes for Lipids and Membranes. In Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies, 11th ed.; Thermo Fisher Scientiﬁc, 2010; Chapter 13. (38) Li, L.; Szmacinski, H.; Lakowicz, J. R. Synthesis and Luminescence Spectral Characterization of Long-Lifetime Lipid Metal−Ligand Probes. Anal. Biochem. 1997, 244, 80−85. (39) Svensson, F. R.; Li, M.; Nordén, B.; Lincoln, P. Luminescent Dipyridophenazine-Ruthenium Probes for Liposome Membranes. J. Phys. Chem. B 2008, 112, 10969−10975. (40) Mechler, A.; Stringer, B. D.; Mubin, M. S. H.; Doeven, E. H.; Phillips, N. W.; Rudd-Schmidt, J.; Hogan, C. F. Labeling phospholipid membranes with lipid mimetic luminescent metal complexes. Biochim. Biophys. Acta, Biomembr. 2014, 1838, 2939−2946. (41) KaŠ Č áková, S.; Giuliani, A.; Lacerda, S.; Pallier, A.; Mercère, P.; Tóth, É .; Réfrégiers, M. X-ray-induced radiophotodynamic therapy (RPDT) using lanthanide micelles: Beyond depth limitations. Nano Res. 2015, 8, 2373−2379. (42) Verwilst, P.; Park, S.; Yoon, B.; Kim, J. S. Recent advances in Gd-chelate based bimodal optical/MRI contrast agents. Chem. Soc. Rev. 2015, 44, 1791−1806. (43) Arroyo, I. Z.; Gomez, C.; Alarcon, H.; Jimenez, A.; Pardo, A.; Montaño, G.; Armijos, R. X.; Noveron, J. C. Alkyl Length Effects on the DNA Transport Properties of Cu(II) and Zn(II) Metallovesicles: An In Vitro and In Vivo Study. J. Drug Delivery 2018, 2018, 2851579. (44) Griffiths, P. C.; Fallis, I. A.; Chuenpratoom, T.; Watanesk, R. Metallosurfactants: Interfaces and micelles. Adv. Colloid Interface Sci. 2006, 122, 107−117. (45) Komatsu, H.; Yoshihara, K.; Yamada, H.; Kimura, Y.; Son, A.; Nishimoto, S.-i.; Tanabe, K. Ruthenium Complexes with Hydrophobic Ligands That Are Key Factors for the Optical Imaging of Physiological Hypoxia. Chem. - Eur. J. 2013, 19, 1971−1977. (46) Nagaraj, K.; Murugan, K. S.; Thangamuniyandi, P.; Sakthinathan, S. Nucleic acid binding study of surfactant copper(ii) complex containing dipyrido[3,2-a:2′-3′-c]phenazine ligand as an intercalator: in vitro antitumor activity of complex in human liver carcinoma (HepG2) cancer cells. RSC Adv. 2014, 4, 56084−56094. (47) Lebrón, J. A.; Ostos, F. J.; Moyá, M. L.; López-López, M.; Carrasco, C. J.; López-Cornejo, P. Cooperative interaction between metallosurfactants, derived from the [Ru(2,2′-bpy)3]2+ complex, and DNA. Colloids Surf., B 2015, 135, 817−824. (48) Estalayo-Adrián, S.; Blasco, S.; Bright, S. A.; McManus, G. J.; Orellana, G.; Williams, D. C.; Kelly, J. M.; Gunnlaugsson, T. Watersoluble amphiphilic ruthenium(ii) polypyridyl complexes as potential light-activated therapeutic agents. Chem. Commun. 2020, 56, 9332− 9335. (49) Del Guerzo, A.; Kirsch-De Mesmaeker, A.; Demeunynck, M.; Lhomme, J. Characterisation of bifunctional ruthenium(II) complexes, potential DNA photo-probes. Presence of folded and unfolded conformers. J. Chem. Soc., Dalton Trans. 2000, 1173−1180. (50) Herman, L.; Elias, B.; Pierard, F.; Moucheron, C.; Kirsch-De Mesmaeker, A. Effects of Protonation on the Spectroscopic Properties of Tetrapyridoacridine (TPAC) Mono- and Dinuclear Ru(II) Complexes in Their Ground and 3MLCT Excited States. J. Phys. Chem. A 2007, 111, 9756−9763. (51) Ortmans, I.; Elias, B.; Kelly, J. M.; Moucheron, C.; KirschDeMesmaeker, A. [Ru(TAP)2(dppz)]2+: a DNA intercalating complex, which luminesces strongly in water and undergoes photoinduced proton-coupled electron transfer with guanosine-5′-monophosphate. Dalton Trans. 2004, 668−676. (52) Lecomte, J.-P.; Kirsch-De Mesmaeker, A.; Kelly, J. M.; Tossi, A. B.; Görner, H. Photo-induced electron transfer from nucleotides to ruthenium-tris-1,4,5,8-tetraazaphenanthrene: model for photosensitized DNA oxidation. Photochem. Photobiol. 1992, 55, 681−689. (53) Lecomte, J.-P.; Kirsch-De Mesmaeker, A.; Feeney, M. M.; Kelly, J. M. Ruthenium(II) Complexes with 1,4,5,8,9,12-Hexaazatriphenylene and 1,4,5,8-Tetraazaphenanthrene Ligands: Key Role Played by the Photoelectron Transfer in DNA Cleavage and Adduct Formation. Inorg. Chem. 1995, 34, 6481−6491. (54) Kajouj, S.; Marcélis, L.; Lemaur, V.; Beljonne, D.; Moucheron, C. Photochemistry of ruthenium(ii) complexes based on 1,4,5,8tetraazaphenanthrene and 2,2′-bipyrazine: a comprehensive experimental and theoretical study. Dalton Trans. 2017, 46, 6623−6633. (55) García-Fresnadillo, D.; Georgiadou, Y.; Orellana, G.; Braun, A. M.; Oliveros, E. Singlet-Oxygen (1Δg) Production by Ruthenium(II) complexes containing polyazaheterocyclic ligands in methanol and in water. Helv. Chim. Acta 1996, 79, 1222−1238. (56) Carraway, E. R.; Demas, J. N.; DeGraff, B. A. Luminescence quenching mechanism for microheterogeneous systems. Anal. Chem. 1991, 63, 332−336. (57) García-Fresnadillo, D.; Orellana, G. Interaction of Sulfonated Ruthenium(II) Polypyridine Complexes with Surfactants Probed by Luminescence Spectroscopy. Helv. Chim. Acta 2001, 84, 2708−2730. (58) Suzuki, K.; Kobayashi, A.; Kaneko, S.; Takehira, K.; Yoshihara, T.; Ishida, H.; Shiina, Y.; Oishi, S.; Tobita, S. Reevaluation of absolute luminescence quantum yields of standard solutions using a spectrometer with an integrating sphere and a back-thinned CCD detector. Phys. Chem. Chem. Phys. 2009, 11, 9850−9860. (59) Hergueta-Bravo, A.; Jiménez-Hernández, M. E.; Montero, F.; Oliveros, E.; Orellana, G. Singlet Oxygen-Mediated DNA Photocleavage with Ru(II) Polypyridyl Complexes. J. Phys. Chem. B 2002, 106, 4010−4017. (60) Elias, B.; Kirsch-De Mesmaeker, A. Photo-reduction of polyazaaromatic Ru(II) complexes by biomolecules and possible applications. Coord. Chem. Rev. 2006, 250, 1627−1641. (61) Entradas, T.; Waldron, S.; Volk, M. The detection sensitivity of commonly used singlet oxygen probes in aqueous environments. J. Photochem. Photobiol., B 2020, 204, 111787. (62) In order to exclude any degradation of ABDA upon irradiation, the emission spectrum of ABDA in the absence of the Ru(II) complexes was also recorded at diﬀerent irradiation times. No change in the emission intensity of ABDA at 405 nm was observed with the irradiation time, further demonstrating that the changes observed in the presence of 1, 2, 4, and 5 are due to the 1O2 production by the complexes. (63) Marcélis, L.; Ghesquière, J.; Garnir, K.; Kirsch-De Mesmaeker, A.; Moucheron, C. Photo-oxidizing RuII complexes and light: Targeting biomolecules via photoadditions. Coord. Chem. Rev. 2012, 256, 1569−1582. (64) Estalayo-Adrián, S.; Garnir, K.; Moucheron, C. Perspectives of ruthenium(ii) polyazaaromatic photo-oxidizing complexes photoreactive towards tryptophan-containing peptides and derivatives. Chem. Commun. 2018, 54, 322−337. Q Article https://doi.org/10.1021/acsabm.1c00284 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX ACS Applied Bio Materials www.acsabm.org (65) Kotani, H.; Ohkubo, K.; Fukuzumi, S. Photocatalytic Oxygenation of Anthracenes and Olefins with Dioxygen via Selective Radical Coupling Using 9-Mesityl-10-methylacridinium Ion as an Effective Electron-Transfer Photocatalyst. J. Am. Chem. Soc. 2004, 126, 15999−16006. (66) The oxidation potential of ABDA has not been previously determined and as such the value reported for 9,10-dimethylanthracene was used instead, assuming that the malonic acid moiety in ABDA did not have a strong eﬀect in the oxidation potential of the anthracene derivative. (67) Wessels, J. M.; Foote, C. S.; Ford, W. E.; Rodgers, M. A. J. Photooxidation of Tryptophan: O2(1Δg) versus Electron-Transfer Pathway. Photochem. Photobiol. 1997, 65, 96−102. (68) Carter, M. T.; Rodriguez, M.; Bard, A. J. Voltammetric studies of the interaction of metal chelates with DNA. 2. Tris-chelated complexes of cobalt(III) and iron(II) with 1,10-phenanthroline and 2,2′-bipyridine. J. Am. Chem. Soc. 1989, 111, 8901−8911. (69) Poulsen, B. C.; Estalayo-Adrián, S.; Blasco, S.; Bright, S. A.; Kelly, J. M.; Williams, D. C.; Gunnlaugsson, T. Luminescent ruthenium polypyridyl complexes with extended ‘dppz’ like ligands as DNA targeting binders and cellular agents. Dalton Trans. 2016, 45, 18208−18220. (70) Haworth, I. S.; Elcock, A. H.; Rodger, A.; Richards, W. G. A Binding Mode of A-[tris(1,10-phenanthroline)ruthenium(II)]2+ Exhibiting Preference for Purine-3′,5′-Pyrimidine Sites of DNA. J. Biomol. Struct. Dyn. 1991, 9, 553−569. (71) Eriksson, M.; Leijon, M.; Hiort, C.; Norden, B.; Graeslund, A. Binding of DELTA- and LAMBDA-[Ru(phen)3]2+ to [d(CGCGATCGCG)]2 Studied by NMR. Biochemistry 1994, 33, 5031−5040. (72) Husale, S.; Grange, W.; Karle, M.; Bürgi, S.; Hegner, M. Interaction of cationic surfactants with DNA: a single-molecule study. Nucleic Acids Res. 2008, 36, 1443−1449. (73) Guo, L.; Zhang, Z.; Qiao, H.; Liu, M.; Shen, M.; Yuan, T.; Chen, J.; Dionysiou, D. D. Spectroscopic study on interaction between three cationic surfactants with different alkyl chain lengths and DNA. Spectrochim. Acta, Part A 2015, 151, 237−246. (74) Guo, Q.; Zhang, Z.; Song, Y.; Liu, S.; Gao, W.; Qiao, H.; Guo, L.; Wang, J. Investigation on interaction of DNA and several cationic surfactants with different head groups by spectroscopy, gel electrophoresis and viscosity technologies. Chemosphere 2017, 168, 599− 605. (75) Nagaraj, K.; Velmurugan, G.; Sakthinathan, S.; Venuvanalingam, P.; Arunachalam, S. Influence of self-assembly on intercalative DNA binding interaction of double-chain surfactant Co(iii) complexes containing imidazo[4,5-f][1,10]phenanthroline and dipyrido[3,2-d:2′-3′-f]quinoxaline ligands: experimental and theoretical study. Dalton Trans. 2014, 43, 18074−18086. (76) Nehru, S.; Veeralakshmi, S.; Kalaiselvam, S.; Subin David, S. P.; Sandhya, J.; Arunachalam, S. DNA binding, antibacterial, hemolytic and anticancer studies of some fluorescent emissive surfactantruthenium(II) complexes. J. Biomol. Struct. Dyn. 2021, 39, 2242− 2256. (77) Kelly, J. M.; Tossi, A. B.; McConnell, D. J.; OhUigin, C. A study of the interactions of some polypyridylruthenium (II) complexes with DNA using fluorescence spectroscopy, topoisomerisation and thermal denaturation. Nucleic Acids Res. 1985, 13, 6017− 6034. (78) Comment: It must be noted that since the complexes discussed here do not contain ionizable groups, they are not expected to be protonated/deprotonated at physiological pH, and as such log P values were calculated instead of the logarithm of their distribution coeﬃcient (log D). (79) Gorle, A. K.; Feterl, M.; Warner, J. M.; Wallace, L.; Keene, F. R.; Collins, J. G. Tri- and tetra-nuclear polypyridyl ruthenium(ii) complexes as antimicrobial agents. Dalton Trans. 2014, 43, 16713− 16725. (80) Gill, M. R.; Garcia-Lara, J.; Foster, S. J.; Smythe, C.; Battaglia, G.; Thomas, J. A. A ruthenium(II) polypyridyl complex for direct imaging of DNA structure in living cells. Nat. Chem. 2009, 1, 662− 667. (81) Dobrucki, J. W. Interaction of oxygen-sensitive luminescent probes Ru(phen)32+ and Ru(bipy)32+ with animal and plant cells in vitro: Mechanism of phototoxicity and conditions for non-invasive oxygen measurements. J. Photochem. Photobiol., B 2001, 65, 136−144. (82) Svensson, F. R.; Abrahamsson, M.; Strömberg, N.; Ewing, A. G.; Lincoln, P. Ruthenium(II) Complex Enantiomers as Cellular Probes for Diastereomeric Interactions in Confocal and Fluorescence Lifetime Imaging Microscopy. J. Phys. Chem. Lett. 2011, 2, 397−401. (83) Huang, H.; Yu, B.; Zhang, P.; Huang, J.; Chen, Y.; Gasser, G.; Ji, L.; Chao, H. Highly Charged Ruthenium(II) Polypyridyl Complexes as Lysosome-Localized Photosensitizers for Two-Photon Photodynamic Therapy. Angew. Chem., Int. Ed. 2015, 54, 14049− 14052. (84) Martinez, L. A.; Martínez, C. G.; Klopotek, B. B.; Lang, J.; Neuner, A.; Braun, A. M.; Oliveros, E. Nonradiative and radiative deactivation of singlet molecular oxygen (O2(a1Δg)) in micellar media and microemulsions. J. Photochem. Photobiol., B 2000, 58, 94− 107. (85) González-Béjar, M.; Liras, M.; Francés-Soriano, L.; Voliani, V.; Herranz-Pérez, V.; Duran-Moreno, M.; Garcia-Verdugo, J. M.; Alarcon, E. I.; Scaiano, J. C.; Pérez-Prieto, J. NIR excitation of upconversion nanohybrids containing a surface grafted Bodipy induces oxygen-mediated cancer cell death. J. Mater. Chem. B 2014, 2, 4554−4563. (86) Nasielski-Hinkens, R.; Benedek-Vamos, M.; Hautain, Y.; Nasielski, J. 1H-NMR spectra of pyrazino [2,3-f] quinoxalines and 5,6-diaminoquinoxalines. Bull. Soc. Chim. Belg. 1976, 85, 781−786. (87) Lecomte, J. P.; Kirsch-De Mesmaeker, A.; Demeunynck, M.; Lhomme, J. Synthesis and characterisation of a new DNA-binding bifunctional ruthenium(II) complex. J. Chem. Soc., Faraday Trans. 1993, 89, 3261−3269. (88) Doi, T.; Nagamiya, H.; Kokubo, M.; Hirabayashi, K.; Takahashi, T. Synthesis of a tetrabenzyl-substituted 10-membered cyclic diamide. Tetrahedron 2002, 58, 2957−2963. R Article https://doi.org/10.1021/acsabm.1c00284 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX