In Situ Bioconjugation of a Maleimide-Functionalized Ruthenium-Based Photosensitizer to Albumin for Photodynamic Therapy.

{"full_text": " pubs.acs.org/IC Article\n\n\n\n In Situ Bioconjugation of a Maleimide-Functionalized Ruthenium-\n Based Photosensitizer to Albumin for Photodynamic Therapy\n Robin Vinck,*,# Orsolya D\u00f6m\u00f6t\u00f6r,# Johannes Karges, Marta Jakubaszek, Johanne Seguin,\n Micka\u00ebl Tharaud, Vincent Gu\u00e9rineau, Kevin Cariou, Nathalie Mignet, E\u0301 va A. Enyedy, and Gilles Gasser*\n Cite This: Inorg. Chem. 2023, 62, 15510\u221215526 Read Online\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n ABSTRACT: Maleimide-containing prodrugs can quickly and\n selectively react with circulating serum albumin following their\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 11:22:20 (UTC).\n\n\n\n\n injection in the bloodstream. The drug\u2212albumin complex then\n benefits from longer blood circulation times and better tumor\n accumulation. Herein, we have applied this strategy to a previously\n reported highly phototoxic Ru polypyridyl complex-based photo-\n sensitizer to increase its accumulation at the tumor, reduce off-\n target cytotoxicity, and therefore improve its pharmacological\n profile. Specifically, two complexes were synthesized bearing a\n maleimide group: one complex with the maleimide directly\n incorporated into the bipyridyl ligand, and the other has a\n hydrophilic linker between the ligand and the maleimide group. Their interaction with albumin was studied in-depth, revealing their\n ability to efficiently bind both covalently and noncovalently to the plasma protein. A crucial finding is that the maleimide-\n functionalized complexes exhibited significantly lower cytotoxicity in noncancerous cells under dark conditions compared to the\n nonfunctionalized complex, which is a highly desirable property for a photosensitizer. The binding to albumin also led to a decrease\n in the phototoxicity of the Ru bioconjugates in comparison to the nonfunctionalized complex, probably due to a decreased cellular\n uptake. Unfortunately, this decrease in phototoxicity was not compensated by a dramatic increase in tumor accumulation, as was\n demonstrated in a tumor-bearing mouse model using inductively coupled plasma mass spectrometry (ICP-MS) studies.\n Consequently, this study provides valuable insight into the future design of in situ albumin-binding complexes for photodynamic\n therapy in order to maximize their effectiveness and realize their full potential.\n\n\n \u25a0 INTRODUCTION\n In the past few years, photodynamic therapy (PDT) has gained\n Radachlorin, Purlytin, Lutrin, TOOKAD soluble) with better-\n defined structures, improved aqueous solubility, longer\n growing interest in the fight against cancer. This approved absorption wavelengths, and reduced skin sensitization.\n medical technique involves the use of a photosensitizer (PS) However, these second-generation porphyrin-based com-\n and light in order to induce cellular damage in tumors in a pounds are still far from the ideal of a perfect PS since only\n spatially and temporally controlled manner.1\u22124 a few combine all of the aforementioned advantages.2,4,6\u22128\n Typical PDT procedures start with local or systemic Among non-porphyrin PSs, the use of ruthenium(II)\n administration of the PS, followed after a certain time by (Ru(II)) polypyridyl complexes has recently received much\n local light irradiation of the targeted tissues. Light-mediated interest thanks to their simple synthesis, chemical stability,\n excitation of the PS leads to the generation of reactive oxygen good 1O2 production yields, and generally good aqueous\n species (ROS) and/or singlet oxygen (1O2), following a type I solubility.9\u221218 Notably, TLD-1433 is currently involved in a\n or type II mechanism, respectively, which are responsible for phase II clinical trial for patients with bladder cancer.19,20\n local cellular damage.5\n However, these Ru(II)-based PSs are known to poorly absorb\n Most clinically applied PSs are based on tetrapyrrolic\n scaffolds. First-generation PSs (i.e., Photofrin and hematopor- light in the biological spectral window (600\u2212900 nm).8,21 This\n phyrin derivatives) are a mixture of several chemical entities.\n They have often been associated with slow elimination rates, Received: June 16, 2023\n leading to extended photosensitivity in patients, low aqueous Published: September 14, 2023\n solubility, photodegradation, tedious synthetic procedures, and\n lack of selectivity toward diseased tissues. Some of these\n drawbacks have been addressed to yield second-generation PSs\n (e.g., Photosens, Foscan, Laserphyrin, Redaporphin, Photogem,\n\n \u00a9 2023 American Chemical Society https://doi.org/10.1021/acs.inorgchem.3c01984\n 15510 Inorg. Chem. 2023, 62, 15510\u221215526\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 1. Chemical structures of the Ru(II) complexes studied in this work and putative scheme of a reaction of a maleimide-functionalized PS\nwith the thiol group of cysteine-34 (Cys-34) of albumin.\n\nfeature prevents the treatment of tumors located deeply in the graphene oxide,61\u221263 inorganic materials,34,42,64\u221272 or pro-\ntissues or of large tumors.22 teins.71,73,74 It is worth noting that a substantial proportion of\n To address this drawback, we recently reported the density these studies combine both active and passive targeting to\nfunctional theory (DFT)-guided design and in vitro study of further enhance PS tumor selectivity.\nnew Ru(II) polypyridyl complexes absorbing at longer Among these targeting approaches, the conjugation or\nwavelengths.23 One of these compounds, namely, [Ru- binding of the PS to endogenous blood plasma proteins, and\n(bphen)2dmbipy][PF6]2 (bphen = 4,7-diphenyl-1,10-phenan- more particularly albumin, seems especially attractive to\nthroline, dmbipy = 4,4\u2032-dimethyl-2,2\u2032-bipyridine, Figure 1), increase its blood residence time and promote its accumulation\nwhich has previously been described by others for its cytotoxic specifically within tumors.75,76 Albumin is the most abundant\nproperties,24 showed a great PDT potential on various cancer plasma protein with a concentration of 35\u221250 g/L of human\ncell lines in a two-dimensional (2D) cell monolayer model as plasma.77 This protein has been described to accumulate in\nwell as in a three-dimensional (3D) HeLa multicellular tumor tumors through the EPR effect78\u221280 and also through a gp60\nspheroid (MCTS) model. In contrast to the majority of and caveolin-1-mediated transcytosis, promoting its extrava-\npublished Ru(II) polypyridyl complexes, this compound was sation from the blood circulation to the diseased tissues.81,82\nable to exert a phototoxic effect upon irradiation up to 595 nm, Several attempts to conjugate chemotherapeutic drugs to\nwhich lies at the frontier of the biological spectral window. In albumin in order to increase their tumor accumulation resulted\naddition, [Ru(bphen)2dmbipy][PF6]2 displays little photo- in the clinical success of Abraxane, a now-marketed paclitaxel\nbleaching and good stability in human plasma. formulation with enhanced efficacy and safety compared to the\n PDT features a spatial and temporal control of the classical formulation.83\u221285 While albumin was predominantly\ntherapeutic effect, which intrinsically provides PDT with a investigated as a therapeutic carrier for purely organic drugs,\nfirst level of selectivity toward diseased tissues. However, to there is a growing interest in exploring its potential for\nimprove its therapeutic potential, PDT could benefit from a anticancer metal complexes.86\u221290\nsecond level of selectivity by directly targeting the tumor.25 In particular, this strategy has already been successfully\nOver the years, many strategies have been developed to employed in the case of Ru(II)-based PSs. Shi et al. reported\nincrease the selectivity of chemotherapeutics toward cancer the elaboration of a nanocarrier based on lanthanide-doped\ncells, either through active or passive targeting.26\u221229 Some upconversion nanoparticles coated with human serum albumin\nactive targeting strategies have already been applied to Ru(II)- (HSA) and a Ru(II) polypyridyl complex for both imaging and\nbased PSs, including conjugation of the complex to aptamers,30 PDT purposes. This nanocarrier showed a good cellular uptake\nantibodies and nanobodies,29,31,32 oligonucleotides,33,34 bomb- as well as an enhanced in vitro phototoxicity on HeLa and\nesin,35\u221237 biotin,33,38\u221241 folic acid,42 somatostatin,43 glucose,44 HepG2 cell lines compared to the Ru(II) complex alone.71 In\nmannose,45 tamoxifen,46 cobalamin,47 taurine,48 or trans- an alternative approach, Chakrabortty et al. reported the design\nferrin.34,49,50 Passive targeting strategies exploiting the and in vitro evaluation of an engineered HSA molecule\nenhanced permeation and retention (EPR) effect have also functionalized with mitochondria-targeting groups, solubilizing\nbeen explored through encapsulation or conjugation methods poly(ethylene glycol) (PEG) chains and Ru(II) polypyridyl\nusing polymers,38,51\u221258 liposomes,59,60 carbon nanotubes and complexes. This macromolecule demonstrated an impressive\n 15511 https://doi.org/10.1021/acs.inorgchem.3c01984\n Inorg. Chem. 2023, 62, 15510\u221215526\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 2. 1H NMR spectra recorded for complex 3 at pH 2.8, 9.4, and 7.0 as a function of time; dashed frame: maleimide CH, solid frames:\nmaleamate and at pH 2.8 maleamic acid CH. No differences were observed when the complex was incubated for 5 min or 24 h at pH 2.8 {c = 0.5\nmM; 10% (v/v) D2O}.\n\nphototoxic effect in HeLa cells, which was attributed to an\nenhanced 1O2 production yield and a better photostability of\n \u25a0 RESULTS AND DISCUSSION\n The ligand 4-aminomethyl-4\u2032-methyl-2,2\u2032-bipyridine was\nthe complex associated with favorable mitochondrial local- synthesized as previously reported from 4-bromomethyl-4\u2032-\nization.73 Unfortunately, none of these studies investigated the methyl-2,2\u2032-bipyridine with hexamethylenetetramine.101 The\nin vivo potential of their systems. Moreover, these studies precursor [Ru(bphen)2]Cl2 was prepared by the reduction of\ninvolved the ex vivo conjugation of human serum albumin Ru(III)Cl3 and substitution with bphen in the presence of\n(HSA) to the PS, which requires the use of commercially LiCl. Following this, the [Ru(bphen)2dmbipyNH2][PF6]2\navailable HSA. An alternative strategy involves the design of a complex was obtained by a coordination reaction of the\nprodrug incorporating a maleimide (Mal) moiety. Upon precursor with the above-mentioned ligand. The desired\nintravenous (IV) injection of the prodrug, the Mal group [Ru(bphen)2dmbipyMal][PF6]2 complex was then obtained\nreacts quickly and selectively with the circulating albumin\u2019s using maleic anhydride condensation on the amine (Scheme\ncysteine-34 (Cys-34) sulfhydryl group, leading to the in situ S1). In parallel, [Ru(bphen)2dmbipyNH2][PF6]2 was con-\nformation of a drug\u2212albumin conjugate. This method jugated to a PEG5 linker generated as previously de-\ntherefore allows the development of a simple, well-charac- scribed,102\u2212105 which was activated as an NHS-ester using\nterized, low-molecular-weight drug, which is able to benefit standard conditions. The Mal moiety was finally added onto\nfrom the advantages of albumin-bound drugs.91,92 the linker using 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-\n To the best of our knowledge, while this strategy has already dihydro-1H-pyrrol-1-yl)acetate (Scheme S1). The compounds\nbeen successfully applied to chemotherapeutic complexes of were then converted into their chloride salts using a counterion\nplatinum, Ru, and osmium, it has never been applied to exchange resin to yield complexes 2 and 3. The identity of the\nvectorize a metal-based PS for PDT applications.93\u2212100 We compounds was confirmed by 1H, 13C NMR, and high-\nbelieve that Ru(II)-based PSs, and particularly [Ru- resolution mass spectrometry (HRMS) (Figures S1\u2212S10), and\n(bphen)2dmbipy][PF6]2 or its analogue with chlorides as the purity was confirmed by elemental analysis. The control\ncounterions [Ru(bphen)2dmbipy][Cl]2 (1), could greatly complex [Ru(bphen)2dmbipy][PF6]2 was obtained in a\nbenefit from in situ albumin conjugation to improve its previous study and also converted to the dichloride salt\ntumor accumulation. Therefore, in this article, we describe the using a counterion exchange resin to give complex 1.\nsynthesis, in vitro, and in vivo characterization of the first Complexes were used as racemic mixtures of the \u25b3 and \u039b\nRu(II) polypyridyl complexes containing a Mal function for in isomers.\nsitu albumin conjugation. The Mal function was either directly Solubility and Stability. Complexes 1 and 2 showed a\nadded onto the complex or spaced using a hydrophilic good aqueous solubility (\u22651 mM), which decreases signifi-\npoly(ethylene glycol) (PEG) linker to further improve its cantly in buffered media like 15 mM phosphate or phosphate-\naqueous solubility and blood circulation time yielding, buffered saline (PBS) at pH 7.40 or even in the presence of 0.1\nrespectively, [Ru(bphen)2dmbipyMal][Cl]2 (2) (dmbipyMal M KCl. Moderate (ca. 7%) precipitation takes place on the\n= 4-maleimidomethyl-4\u2032-methyl-2,2\u2032-bipyridine) and [Ru- basis of the observed decrease in the UV\u2212visible (UV\u2212vis)\n(bphen)2dmbipyPEGMal][Cl]2 (3) (PEGMal = N-(1-(2,5- absorption spectra (see Figure S11A for 2) even at complex\ndioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15,18-pen- concentrations of 20 \u03bcM after 24 h in PBS buffered samples.\ntaoxa-3-azaicosan-20-yl)-4-oxopentanamidyl). Of note, we We have already observed this behavior with analogous\nhave already described complex 2 in another study, albeit it complexes in previous studies. We hypothesized that a high\nwas used only as an intermediate to the synthesis of a peptide- anion concentration decreases the repulsive interaction\nconjugated PDT PS.37 We show that all three complexes can between the positively charged complexes, leading to their\nbind noncovalently to albumin, and only 2 and 3 exhibit the aggregation.37,40 Additionally, as we previously observed, the\ncapability of covalently binding to the protein. The three presence of HSA in buffered media could prevent the\ncomplexes were then characterized in vitro and in a preliminary precipitation of complexes 1 and 2 for at least 2 days (see\nin vivo experiment in tumor-bearing mice to evaluate the UV\u2212vis spectra in Figure S11B).37,40 In contrast, complex 3,\npotential of this targeting strategy. which includes a hydrophilic PEG spacer, suffers from only a\n 15512 https://doi.org/10.1021/acs.inorgchem.3c01984\n Inorg. Chem. 2023, 62, 15510\u221215526\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nTable 1. n-Octanol/Aqueous Phase Distribution Coefficients at pH 7.40 (Expressed as log D7.40) Determined for 1\u22123,\n[Ru(phen)3]Cl2 (Ru-phen) and [Ru(bpy)3]Cl2 (Ru-bpy) at 25 \u00b0C\n aqueous medium 1 2 3 Ru-phena Ru-bpyb\n H2Oc \u22120.03 \u00b1 0.02d +0.81 \u00b1 0.05 +0.82 \u00b1 0.05 \u22121.6 \u00b1 0.1e <\u22121.7\n 0.1 M NaClc >+3.7 >+3.7 +2.4 \u00b1 0.1\n PBS >+3.7 >+3.7 +2.3 \u00b1 0.1 \u22121.30 \u00b1 0.05f <\u22121.7g\n 15 mM phosphate +1.5 \u00b1 0.1 +2.3 \u00b1 0.1 +1.1 \u00b1 0.1 \u22121.5 \u00b1 0.1 <\u22121.7\na\n log D7.40 = \u22121.09 in 15 mM phosphate and 1 M NaCl. blog D7.40 < \u22121.7 in 15 mM phosphate and 1 M NaCl. cpH between 6.5 and 8.0. dReported\npartition coefficient: log P = + 0.48 in water.24 eReported partition coefficient: log P = \u22121.5 in water.109 fReported partition coefficients: log P =\n\u22121.1 in PBS,110 log P = \u22120.33 solvent not reported.109 gReported partition coefficients: log P = \u22121.21 in PBS,111 log P = \u22120.41 solvent not\nreported.109\n\nrather moderate decrease in its aqueous solubility in the aqueous phase over the nonpolar solvent (log D = \u22120.03)\npresence of PBS components. A concentration of 100 \u03bcM when no extra salt is added in the aqueous fraction, while it\ncomplex 3 was attainable in this medium. Overall, all studied becomes extremely lipophilic in PBS buffer (log D > +3.7). A\ncomplexes displayed an acceptable solubility at a concentration log D = +0.48 was reported by Mazuryk et al. for 1 dissolved in\nof 20 \u03bcM in PBS over a 3 h time span (96% of 1 and 2, and water.108 Experiments were repeated with the two main\n100% of 3 remained in solution). At the same time, pure components of PBS separately, i.e., with 0.1 M NaCl and 15\naqueous stock solutions of 1 and 2 were proven to be stable for mM phosphate. The results showed a more pronounced role of\nat least 3 days based on the 1H NMR measurements (spectra the chloride ions over the phosphate (see Table 1). The same\nnot shown). However, when the pH was set to 7.4, the tendency was observed for complexes 2 and 3. Ion pair\nhydrolysis of the maleimide group in complex 2 took place, as formation most probably takes place between the positively\nsignals corresponding to a maleamate group formed (Figure charged complexes and anions like Cl\u2212 and H2PO4\u2212/HPO42\u2212,\nS12). Analogously, aqueous stock solutions of 3 were stable in leading to a reduced aqueous partition. Phenyl substitution of\nslightly acidic solutions for at least 24 h, according to the 1H the coordinating 1,10-phenanthrolines at positions 4 and 7\nNMR spectra depicted in Figure 2. However, at pH 7.4 and might be responsible for this phenomenon through \u03c0-stacking\n7.0, complex 3 behaved as complex 2 and maleamate formed in since the lipophilicity of the nonsubstituted complexes\na somewhat slower process (Figure 2). A negligible amount of [Ru(phen)3]Cl2 and [Ru(bpy)3]Cl2 do not display a significant\nmaleamate could be detected at neutral pH for the freshly dependency on the electrolyte concentration of the aqueous\nprepared (5 min) sample, while the hydrolysis progressed to phase. Both appear to be rather hydrophilic even in the\n37, 62, and 100% at time points 1.5, 4, and 24 h, respectively. presence of 1 M NaCl and 15 mM phosphate (log D7.40 =\nThe hydrolysis appeared even faster at a higher pH of 9.4, \u22121.09 and <\u2212 1.7, respectively).\nwhich suggests that the hydrolysis rate is pH-dependent, as it Since the log D of a drug is related to the putative\nwas proven for alkyl-maleimides.106 Noteworthily, the partitioning between lipid membrane and blood plasma, the\nhydrolysis of the present compounds is faster (ca. 52%/1 h log D values determined in n-octanol\u2212PBS buffer system can\nfor 2 and 62%/4 h for 3) compared to that of ethylmaleimide be considered as more realistic. The results of lipophilicity\nreported at pH 7.16 and 30 \u00b0C (kobs \u2248 10\u22125 1/s, t1/2 \u2248 19 measurements for complexes 1\u22123 show that they are highly\nh).106,107 Therefore, stock solutions of 3 used for in vitro lipophilic, which suggests that their cellular uptake via passive\nexperiments were prepared in a slightly acidic solution and transport is possible.\nused within 12 h. Additionally, stability studies made on Albumin-Binding Studies. In order to evaluate the ability\ncomplex 2 in Eagle\u2019s minimal essential (EMEM) cell culture of the complexes to bind albumin, complexes 1\u22123 were\nmedium at pH 7.4 showed interaction of 2 with EMEM incubated with bovine serum albumin (BSA) in a 2:1 molar\ncomponents within 30 min, and hydrolysis to maleamate was excess. BSA was initially used as a model for HSA since it bears\ninitially suppressed (see Figure S13). These results, however, the same cysteine at position 34. The mixtures were then\nshould be treated with caution because some precipitate also dialyzed against distilled water to remove any unbound\nformed in the NMR tube. Overall, complexes 2 and 3 appear complex and subjected to matrix-assisted laser desorption\nto slowly hydrolyze in an aqueous medium under physiologic ionization time-of-flight (MALDI-TOF) mass spectrometry,\nconditions. However, as we expect the Mal group to react using BSA as a control. While BSA can be observed as a single\nquickly and selectively with the albumin contained both in peak in the 60\u221270 kDa region (mean mass of 66 332 Da), an\ncomplete culture medium and in blood, this instability might additional peak could be observed when complexes 2 and 3\nnot dramatically affect the performances of the complexes. were incubated with BSA (Figure S14). This would suggest\n Lipophilicity. The lipophilic or hydrophilic character is an that the complexes were successfully covalently bound to BSA.\nimportant property influencing the ability of a drug candidate However, the same phenomenon was observed for complex 1.\nto penetrate biological membranes. The shake-flask method The additional peak observed could therefore represent a\nwas used for the determination of the n-octanol/water noncovalent complex between BSA and the respective\npartition of the Ru(II) complexes. The complexes [Ru- compounds, although it is surprising that such a complex\n(phen)3]Cl2 and [Ru(bpy)3]Cl2 were used as references. would resist the ionization process. We therefore cannot\nTable 1 exhibits the logarithm of the distribution coefficient of exclude that ion pairing between BSA and free complexes\nthe complexes determined at pH 7.40 (log D7.40). Interestingly, occurred in plasma during ionization. Thus, a more in-depth\nthe lipophilicity of complexes 1\u22123 appears to be highly study was performed by using spectroscopic techniques. Of\ndependent on the salt content of the aqueous medium as note, HSA was used in the following experiments to better\npreviously observed.40 1 shows a slight preference for the represent what could happen in human patients.\n 15513 https://doi.org/10.1021/acs.inorgchem.3c01984\n Inorg. Chem. 2023, 62, 15510\u221215526\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 3. UV\u2212vis absorbance spectra of 1 in the presence of increasing amounts of HSA (A). Derived difference spectra of the same system (B)\nand absorbance changes of 1 (\u29eb), 2 (\u25b3), and 3 (\u25cf) at 500 nm plotted together with quantitative binding curves corresponding to one (red dashed\nline) and two (red dotted line) binding sites on the protein. {ccomplex = 20 \u03bcM; cHSA = 0\u221280 \u03bcM; pH = 7.4 (PBS); 25 \u00b0C}. Difference spectra are\ncalculated as follows: Absorbance(complex\u2011HSA mixture) \u2212 Absorbance(complex alone) \u2212 Absorbance(HSA alone).\n\n\n\n\nFigure 4. Three-dimensional luminescence spectra of HSA (A), 2 (B), and the HSA\u22122 (1:1) (C) system. Peak intensities (int.) and wavelength\ncoordinates ([\u03bbEX, \u03bbEM]) are indicated in the figure; peaks with symbol # originate (partly) from the second-order diffraction effect of the\nmonochromator {cHSA = ccomplex = 5 \u03bcM; pH = 7.40 (PBS); 25 \u00b0C; spectra are corrected by self-absorbance and inner filter effect}.\n\n To study the interaction of complexes 1\u22123 with albumin, we observation and the slight differences between the octanolic\nrecorded the UV\u2212vis spectra of the complexes in the presence and HSA-bound spectra (Figure S15B for 2) strongly support\nof HSA to follow protein adduct formation processes. Figure 3 an additional binding mode for complexes 2 and 3 via the\nshows a minor difference of 1 upon the addition of increasing maleimide moiety (vide infra). In the case of complex 2, the\namounts of HSA in a wavelength range where the protein does biphasic binding character is clearly seen (Figure S16), while 3\nnot absorb light. The binding process takes place rapidly as the shows a lower binding affinity toward HSA since the same\nequilibrium is reached within 1\u22122 min. The spectrum profile amount of HSA affects the absorbance spectrum of 3 much less\nof the albumin-bound complex is practically identical to the than 1 and 2 (Figure 3C).\none recorded in n-octanol (see Figure S15A). This observation To further study the binding mode between 1\u22123 and\nsuggests that the metal complex is accommodated in one or albumin, we exploited the intrinsic luminescence of these\nmore of the hydrophobic binding pockets of HSA. The Ru(II) polypyridyl complexes.112 As the three-dimensional\ncalculated difference between the UV\u2212vis spectra (Figure 3B) luminescence spectra in Figure 4 show, complex 2 has two\nindicates two isosbestic points at 356 and 482 nm. The excitation maxima at 280 and 460 nm and emits light between\nchanges of the absorbance difference values at 500 nm (Figure 550 and 750 nm. A ca. 6-fold increase of phosphorescence was\n3C) suggest the existence of more than two binding sites on observed in the presence of 1 equiv of albumin. On the\nHSA since quantitative binding curves assuming one (dashed contrary, the emission peak of HSA at \u03bbEM = 320 nm (that\nline) or two (dotted line) binding sites plotted in the figure are originates mainly from the tryptophan at position 214 (Trp-\ncrossed by the collected absorbance values. Complexes 2 and 3 214) situated near site I in subdomain IIA) is significantly\ndisplayed somewhat different behavior (Figures S16 and S17). reduced, which is a result of the binding of complex 2 at site I.\nAlthough spectral changes are similar to those observed for The very same behavior was obtained for 1, while 3 quenched\ncomplex 1, no isosbestic points were observed. This only slightly the intrinsic fluorescence of HSA (Figure S18).\n 15514 https://doi.org/10.1021/acs.inorgchem.3c01984\n Inorg. Chem. 2023, 62, 15510\u221215526\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 5. Phosphorescence spectra of 1 in the presence of increasing amounts of HSA (A). Measured and fitted intensities at 630 nm are plotted\ntogether with the absorbance changes of 1 titrated by HSA (B) {ccomplex = 3 \u03bcM (fluorometry), 20 \u03bcM (UV\u2212vis); \u03bbEX = 445 nm; pH = 7.40 (PBS);\n25 \u00b0C}.\n\n When the Ru(II) complexes are titrated by HSA, the are still able to photosensitize oxygen and thus maintain their\nemission intensity gradually increases, as seen for complex 1 in photodynamic potential even when bound to albumin.\nFigure 5. The emission maximum is gradually shifted toward Binding Location Study. The binding location of the\nshorter wavelengths, and the measured intensities reached metal complexes in HSA was investigated as the next step. The\nsaturation after the addition of ca. 15 equiv of HSA. In Figure binding at site I (subdomain IIA) and site II (subdomain IIIA)\n5B, it can be seen that the fluorometric titration curve is far less was studied via Trp quenching and site marker displacement\nsteep compared to the absorbance-ratio curve; namely, the experiments. Conditional binding constants computed by\nintrinsic fluorescence of complex 1 is not sensitive to all of the HypSpec based on the titration data are listed in Table 2,\nbinding events. The titration data could be fitted well with the and fitted quenching curves are depicted in Figure 6.\nsimple 1:1 binding model; therefore, no more complex binding\nmodel was applied. The binding constant of log K\u2032 = 5.1 \u00b1 0.1\nwas calculated with the computer program HypSpec.100\nComplex 2 showed rather similar behavior in the presence of\nHSA, although a slightly higher binding constant was\ncalculated on the basis of 2\u2212HSA titrations (Table 2).\n\nTable 2. Conditional Binding Constants (log K\u2032) of the\nComplexes at Binding Sites I and II of HSA and Binding\nConstant Calculated Based on the Intrinsic\nPhosphorescence of the Complexes Determined by\nSpectrofluorometric Measurements {pH = 7.40 (PBS); 25\n\u00b0C}c\n Figure 6. Fluorometric quenching curves recorded for the HSA\u22121\n log K\u2032a 1 2 3 (\u25cf), HSA-2 (\u29eb), and HSA\u22123 (\u25b2) systems. Dashed lines are the\nTrp quenching 5.2 \u00b1 0.1b 5.2 \u00b1 0.1 fitted curves calculated on the basis of quenching constants (log K\u2032)\nWF displacement 5.5 \u00b1 0.1 5.5 \u00b1 0.1 listed in Table 2 {cHSA = 1 \u03bcM; \u03bbEX = 295 nm; pH = 7.40 (PBS); 25\nDG displacement 5.4 \u00b1 0.1 5.4 \u00b1 0.1 4.5 \u00b1 0.1\n \u00b0C}.\nintrinsic phosphorescence of the 5.1 \u00b1 0.1 5.4 \u00b1 0.1 4.2 \u00b1 0.1\n complex Calculated constants for complexes 1 and 2 are fairly similar\na\n Data presented are the mean value \u00b1 standard deviation of at least at both sites. The Trp-214 quenching constant for 1 is in good\ntwo independent assays. blog K\u2032 = 5.09 reported by Mazuryk et al.24 agreement with the data reported by Mazuryk et al. (log K\u2032 =\nc\n WF: warfarin and DG: dansylglycine. 5.09).24 The binding site preference and affinity of 3 for HSA\n were generally different from those of 1 and 2. Although a Trp-\nComplex 3 behaved again somewhat differently since no 214 quenching constant similar to that of 1 could be calculated\nshifting of the emission maximum was observed (Figure S19), for 3 (log K\u2032 = 5.4 \u00b1 0.1), the fluorescence of Trp-214 was\nand a remarkably lower binding constant was calculated (log K\u2032 quenched in only 27% (Figure 6). The warfarin (WF)\n displacement experiment showed even less alteration in the\n= 4.2 \u00b1 0.1) in comparison to those of 1 and 2. The measured fluorescence signal of the HSA\u2212WF system upon the addition\nand calculated data points in Figures 5 and S19 show a strong of 3 (Figure S21). The results of this latter finding and the\nfit, although it should be noted that the binding of a second Trp-214 quenching experiments strongly suggest that the\n(and third) complex on HSA cannot be ruled out. Of note, binding of 3 takes place on a place(s) other than site I. The\nthese results could also be confirmed by following the changes calculated quenching constant (log K\u2032 = 5.4 \u00b1 0.1) cannot be\n handled as a binding constant at site I; therefore, it is not listed\nin the lifetime decay of the metal complexes in the presence\n in Table 2. Most probably, the allosteric effect of binding of\nand absence of HSA (Figure S20). Interestingly, the lifetime of complex 3 can be registered at site I. This phenomenon can\nthe albumin-bound complexes increased in the absence of also be observed in the reverse experiment; namely, the\nmolecular oxygen (Table S1). This suggests that the complexes addition of WF to the 3\u2212HSA system increases the measured\n 15515 https://doi.org/10.1021/acs.inorgchem.3c01984\n Inorg. Chem. 2023, 62, 15510\u221215526\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nemission intensity of the complex (Figure S22). Lifetime\nmeasurements (data in Table S1) reveal that this increase\noriginates mainly from the growing lifetime of bound 3, i.e., the\nclose environment of the bound metal complex changes but\nnot primarily the bound fraction. The other two complexes\nbehaved differently; both 1 and 2 could displace WF from site\nI, and similar binding constants could be calculated from the\ndisplacement data as it was obtained in quenching experiments\n(Table 2). At the same time, WF addition did not affect the\nfluorescence of 1\u2212HSA or 2\u2212HSA systems (Figure S23).\n The binding at site II was investigated in the HSA\u2212\ndansylglycine (DG)\u2212complex 1\u22123 ternary systems (see three- Figure 7. Free Cys-34 SH content of HSA at various complex-to-\ndimensional fluorescence spectra and lifetime data collected for HSA ratios. Symbols denote the following complexes and conditions:\nHSA\u2212DG\u22123 in Figure S24 and Table S2). A relatively low open triangle: 2, 3 h incubation; blue filled triangle: 2, 30 min\nbinding constant of 3 at site II could be computed, which is incubation; blue filled square: 2, 0.5% SDS, 30 min; red filled circle: 3,\nabout 1 order of magnitude lower compared to those of 1 and 30 min; red filled diamond: 3, 0.5% SDS, 30 min; cross symbol: 1 as a\n2 (Table 2). The decrease of HSA-bound 3 fluorescence in the negative control, 30 min {cHSA = 130 \u03bcM; cCys\u201134 = 23 \u03bcM (18% of\npresence of DG seems to originate both from changes of the HSA); pH = 7.00 (100 mM phosphate buffer)}.\nenvironment of the bound form (\u03c42 decreased from 3060 to\n2870 ns) and the slight increase of the bound fraction (\u03b12: 47% Measurements on the sodium dodecyl sulfate (SDS)-\n\u2192 53%). In contrast, 1 and 2 could displace DG, but the denatured protein revealed nearly quantitative interaction of\nphosphorescence of these complexes did not change in the 2 and 3 with the Cys-34 thiol group of HSA. Two scenarios are\nreversed type titrations. possible regarding the interaction with the native protein: (i)\n To summarize, complexes 1 and 2 can effectively displace concurrent binding at other sites in HSA reduces the effective\nWF and DG, apparently occupying sites I and II. On the other concentration of 2 and 3, or (ii) the hydrolysis of the Mal\nhand, 3 can slightly displace DG but not WF. Most probably, moiety competes with the covalent binding of the complexes\nits binding at another additional site could be responsible for with Cys-34, which takes relatively longer on the native\nthe elevated phosphorescence of the complex. This particular protein. Complex 3 seems to react more efficiently with the\nsite seems to be insensitive to any events occurring at sites I or native protein than complex 2, which could be due to its lower\nII in the case of complexes 1 and 2, while the binding site of noncovalent binding.\ncomplex 3 appears to be allosterically connected to sites I and Complexes 2 and 3 were found to react with the Cys-34\nII. thiol group of HSA. Although the interaction is not\n Covalent Binding with Albumin Study. The possible quantitative, a significant binding via the maleimide moiety is\ncovalent interaction of 2 and 3 with the Cys-34 thiol moiety in highly probable in blood, as well.\nalbumin was investigated as well. Molecules containing the Overall, these data suggest that complexes 1 and 2 strongly\nsulfhydryl group can form a stable thioether conjugate with bind noncovalently to sites I and II of albumin, while this\nmaleimide at pH between 6.5 and 7.5. HSA, as the most interaction appears significantly weaker for 3. This could be\nabundant plasma protein having one accessible cysteine (Cys- due to the relatively higher hydrophilicity and steric hindrance\n34), is the most likely target of Mal-linked agents in the blood. provided by the PEG spacer. While both 2 and 3 are able to\nca. 20\u221230% of the Cys-34 thiol groups of HSA are oxidized in bind covalently via their Mal moiety to the Cys-34 of albumin,\nthe human blood (disulfide bridges are formed with small the relatively lower noncovalent binding of 3 is counter-\nsulfhydryl compounds);113 in contrast, commercially available balanced with a relatively higher covalent binding efficiency.\nlyophilized HSA is usually oxidized at a higher extent Cell Studies. Complex 1 has been described previously as\n(depending on the recovering and storage conditions). The cytotoxic even when cells were incubated in the absence of\nconcentration of the free thiol group of Cys-34 of the protein light exposure, which is an undesired property for a PDT\nwas determined via reaction with 4,4\u2032-dithiodipyridine PS.23,24 We therefore compared the cytotoxic potential of\n(DTDP) by spectrophotometry according to our previously complexes 1\u22123 in the dark on noncancerous immortalized\nreported protocol.100 Then, 18 \u00b1 2% of Cys-34 of the albumin human retinal pigment epithelial cells (RPE-1) following 48 h\nstock was determined to be present in its nonoxidized form. of incubation. As shown in Table 3, complex 1 induced a\nSince the reaction of the Mal moiety of 2 and 3 with the thiol significant cytotoxic effect, with an IC50 of about 2 \u03bcM, which\ngroup of Cys-34 decreases the free thiol content of the protein, represents the concentration needed to kill 50% of the cells. Of\nthe DTDP protocol can be used in order to quantify the note, the IC50 of complex 1 was in the same range as the\nsulfhydryl groups of HSA in the presence of the metal cytotoxicity measured by Mazuryk et al. on 4T1 cells following\ncomplexes (see Figure S25 for UV\u2212vis spectra). Figure 7 24 h of incubation.24 In contrast, complexes 2 and 3 were\nshows the changes in free thiol content in HSA as a function of found to be significantly less toxic with IC50 values of 74 and\nthe added equivalents of the complexes. Complex 1 applied as 100 \u03bcM, respectively. However, following their irradiation at\na negative control barely affects the quantity of free thiol 595 nm, complexes 2 and 3 remained highly phototoxic, with\ngroups. On the other hand, the Mal-functionalized complexes IC50 values in the range 4\u22128 \u03bcM on CT26 and RPE-1 cells.\n2 and 3 interact to a significant extent with Cys-34. The Their phototherapeutic index (PI), which represents the ratio\nincubation of 2 with HSA for 3 h (\u25b3) or 30 min (\u25b2) did not between their toxicity in the dark vs their toxicity following\nresult in remarkable differences. In addition, the interaction irradiation, was found to be in the same range as that of\nwith the native protein did not result in quantitative covalent complex 1, if not slightly better. Additional experiments using\nbinding at this site. A similar behavior was observed for 3. higher concentrations of the complexes would be required to\n 15516 https://doi.org/10.1021/acs.inorgchem.3c01984\n Inorg. Chem. 2023, 62, 15510\u221215526\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nTable 3. IC50 Values of Complexes 1\u22123 under Various Conditionsa\n cell line compound A\ufffd48 h cytotoxicity (\u03bcM) B\ufffddark (\u03bcM) C\ufffd595 nm (\u03bcM) PI\n 1 5.35 \u00b1 0.08 0.43 \u00b1 0.07 12\n CT26 2 N.D. >100 8.4 \u00b1 0.6 >12\n 3 >100 4.43 \u00b1 0.02 >23\n 1 2.2 \u00b1 0.1 10.1 \u00b1 0.5 0.8 \u00b1 0.3 13\n RPE-1 2 74 \u00b1 7 >100 4.1 \u00b1 0.1 >24\n 3 100 \u00b1 2 >100 4.4 \u00b1 0.2 >23\na\n Column A: cells were incubated continuously with the complexes for 48 h in the dark. Column B: cells were incubated with the complexes for 4 h\nin the dark, then the medium was exchanged, and cells were incubated for an additional 44 h in the dark. Column C: cells were incubated with the\ncomplexes for 4 h in the dark, then the medium was exchanged, cells were irradiated for 2 h at 595 nm (22.47 J/cm2) and then incubated in the\ndark for an additional 44 h in the dark. The cell viability was determined using a resazurin assay. Results are presented as means \u00b1 standard\ndeviation (SD) of three replicate experiments. PI: phototherapeutic index, N.D.: not determined.\n\nprecisely determine their PI. However, while all three to reach cellular organelles, such as the nucleus, the\ncomplexes appeared to be soluble at the highest concentration mitochondria, or the lysosomes, where they could exert the\ntested in culture medium (100 \u03bcM), solubility issues will occur highest effect.115\nat higher concentrations and significantly alter the results. Of Nevertheless, the thioether bond in the thiosuccinimide\nnote, much higher PIs were previously obtained for Ru(II)- product obtained in the reaction between a thiol and a\nbased PSs. For instance, the PI of TLD-1433 was reported to maleimide can be reversible through thiol-exchange reac-\nbe above 9000 on CT26 cells.20 However, many other tions.116 If the albumin complexes are efficient at accumulating\nparameters (e.g., tumor accumulation, irradiation wavelength, and residing at the tumor site, one could hope that the PS will\nand light source) are to be taken into account to evaluate the eventually be released from the protein. On the other hand,\nefficiency of a PS. this phenomenon could also lead to the premature release of\n To rationalize these results, we performed a cellular the PS in blood circulation or in sensitive organs, thus\ninternalization assay on CT26 cells. Cells were incubated increasing off-target toxicity. Such complex processes are\nwith 5 \u03bcM of the complexes 1\u22123 for 4 h. Following a thorough however hard to predict using solely in vitro experiments, and\nwashing procedure, the cells were harvested and digested in in vivo data could help in understanding the fate of complexes\nnitric acid. Subsequently, the digests were analyzed by 1\u22123 following their administration.\ninductively coupled plasma mass spectrometry (ICP-MS) to Animal Studies. We therefore sought to evaluate the\nquantify the amount of Ru internalized by the cells. As can be biodistribution of complexes 1\u22123 in a CT26 tumor-bearing\nseen in Figure 8, complexes 2 and 3 were significantly less mouse model. Complexes 1\u22123 were injected into the tail vein\n at a dose of 2 \u03bcmol/kg (corresponding to 2.04, 2.27, and 3.25\n mg/kg, respectively). After a given time interval (ranging from\n 1 min to 24 h), the mice were euthanized and their blood and\n organs were harvested, digested in nitric acid, and the resulting\n digests were then analyzed by ICP-MS (3 mice/group). It is\n important to mention that this method only provides the\n amount of Ru in tissues but does not give any information\n about its form (initial complex, degradation product,\n metabolites, etc.). Of note, due to solubility issues, complexes\n 1 and 2 required the addition of polysorbate 80 (1%) to\n prevent aggregation, while complex 3 appeared completely\n soluble at this concentration. The formulation of 1 and 2 with\n polysorbate was performed using the film rehydration\nFigure 8. Cellular uptake of complexes 1\u22123 in CT26 cells. The results technique, described in more detail in the Experimental\nare presented as mean and SD of three replicates. (*P < 0.05, ***P < Section, while complex 3 was directly solubilized in 5%\n0.0003, ****P < 0.0001, t-test). glucose. While polysorbate might slightly alter the biodis-\n tribution of the compounds, we first made sure that it does not\ninternalized than complex 1 (3 and 8 times less, respectively). prevent the binding of complex 2 with albumin (see Figure\nIt is well-known that the lipophilicity of a compound can have S26). As shown in Figure 9A, complexes 1 and 2 were quickly\na significant effect on its cellular internalization.114 While this eliminated from the bloodstream within the first 6 h following\ncould explain that the more hydrophilic complex 3 is less injection, and only 0.19 and 0.29 \u03bcM Ru were detected in the\ninternalized than complexes 1 and 2, it does not seem to apply blood 24 h following injection (Figure 9C). In contrast,\nto complex 2, which is highly hydrophobic. However, as complex 3 appeared to be eliminated more slowly in the first\ncomplexes 2 and 3 can covalently bind to albumin, which is hours following injection, and 1.61 \u03bcM Ru was still detected in\npresent in the culture medium, their reduced internalization the blood at 24 h following injection. The comparable\ncould be due to their covalent binding to the bulky protein, elimination half-lives for the three complexes suggest a similar\nwhich would in turn prevent their uptake via passive diffusion. elimination mechanism. Consequently, the area under the\nThis could be an issue for future in vivo applications. If the curve (AUC) for complex 3, which represents the overall\ncomplexes are not released and subsequently internalized by exposure of the animals to the complex, is significantly higher\ncancer cells once vectorized to the tumor, they will not be able than those of complexes 1 and 2. While we cannot exclude that\n 15517 https://doi.org/10.1021/acs.inorgchem.3c01984\n Inorg. Chem. 2023, 62, 15510\u221215526\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 9. Complexes 1\u22123 were injected IV at a 2 \u03bcmol/kg dose of the complexes formulated in 1% polysorbate 80 in PBS (complexes 1 and 2) or\nin 5% glucose (complex 3) (n = 3 mice/time group). In (E) and (F), all three complexes were formulated in 5% glucose. (A) Blood Ru content as a\nfunction of time following the administration of complexes 1\u22123 as determined by ICP-MS. (B) Time-dependent Ru tumor content determined by\nICP-MS. Data are presented as mean and SD of the % of the total injected dose per gram of tumor (*P < 0.05, t-test). (C) Blood Ru kinetic\nparameters using a two-phase exponential decay fit. AUC: area under the curve. (D) Time-dependent biodistribution of complexes 1\u22123 in CT26\ntumor-bearing mice following the IV injection complexes 1\u22123 (n = 3 mice/time group). Results are presented as the mean and SD of the % of the\nRu dose injected per gram of organ. (E) Biodistribution of complexes 1\u22123 in CT26 tumor-bearing mice 48 h following the injection of complexes\n1\u22123. Results are presented as the mean and SD of the % of the total Ru injected dose per gram of the corresponding organ. (F) Ru tumor content\ndetermined by ICP-MS 48 h following the injection of complexes 1\u22123. Data are presented as mean and SD of the % of the total injected dose per\ngram of tumor (*P < 0.05, **P < 0.005, t-test).\n\nthe PEG spacer in complex 3, or the different formulation complex 3 appeared slightly but significantly higher in the\nused, might be fully responsible for this difference in the blood tumor 24 h following injection in comparison to that of\nRu profiles, its slightly better covalent binding efficiency to complex 1. Of note, these results are to be taken with caution,\nalbumin (see Figure 7) might also participate in the extension considering the small number of animals per group and the\nof its blood circulation. Complexes 1\u22123 are distributed in the high variability of the results.\norgans in a similar way and accumulate mainly in the liver and In order to confirm the trend observed previously, the\nthe kidney, suggesting that the elimination of the complexes experiment was repeated on a reduced number of animals to\noccurs both by biliary and renal excretion (Figure 9D). All evaluate the biodistribution at a single time point, 48 h\nthree complexes also appeared to accumulate in the tumors in following administration of complexes 1\u22123. As we realized in\na comparable manner (Figure 9B). The concentration of the meantime that the low solubility of complexes 1 and 2 in\n 15518 https://doi.org/10.1021/acs.inorgchem.3c01984\n Inorg. Chem. 2023, 62, 15510\u221215526\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nsaline and PBS was not due to their low solubility in water but complexes [Ru(phen)3]Cl2 and [Ru(bpy)3]Cl2118 were synthesized\nto the aggregating effect induced by high salt concentrations, as previously reported. The ligand 5-(aminomethyl)-2,2\u2032-bipyridine\nwe formulated the three complexes in 5% glucose. All three was prepared as previously reported.101 Spectral data were in\ncomplexes appeared soluble in this vehicle at the concentration accordance with the literature. The complex [Ru(bphen)2dmbipy]-\n [PF6]2 was obtained in a previous study23 and converted to the\nrequired for IV administration (about 400 \u03bcM). As shown in dichloride salt by elution with MeOH from the ion-exchange resin\nFigure 9E,F, complex 3 demonstrated a modestly (ca. 2-fold) Amberlite IRA-410 to yield [Ru(bphen)2dmbipy][Cl]2 (1). The\nhigher accumulation in the tumor in comparison to complex 1. hydrophilic BOC-protected linker 2,2-dimethyl-4,24-dioxo-\nComplex 2, however, did not accumulate significantly more in 3,8,11,14,17,20-hexaoxa-5,23-diazaheptacosan-27-oic acid was pre-\nthe tumor in comparison to complex 1. These results confirm pared as previously described.102\u2212105 Cells were purchased from the\nthe trend initially observed and show that replacing the ATCC. Sinapic acid (SA, used as the matrix for MALDI-TOF\npolysorbate formulation with 5% glucose did not reverse the experiments, was of the highest grade available and used without\nsuperiority of complex 3 over complex 2. further purification) was purchased from Sigma-Aldrich Co. Human\n Overall, these results suggest that adding a Mal moiety to serum albumin (HSA) containing fatty acids (A8763, essentially\n globulin-free), KCl, NaCl, NaH2PO4, KH2PO4, Na2HPO4, warfarin\ncomplex 1 had a minor effect on its tumor accumulation. Since\n (WF), dansylglycine (DG), 2,2\u2032-dithiodipyridine (DTDP), sodium\nthis strategy has already been proven as efficient in other metal dodecyl sulfate (SDS), and trimethylsilylpropanesulfonate (DSS)\ncomplexes,93\u221295 one could hypothesize that the high hydro- were purchased from Sigma-Aldrich. Milli-Q water was used for\nphobicity of the three compounds leads to their fast capture in sample preparation. Samples for albumin-binding studies were\nthe liver before they had a chance to react with albumin in vivo, prepared in phosphate-buffered saline (PBS) at pH 7.40. Stock\nwhich gives a slight advantage to the more hydrophilic complex solutions of HSA, WF, and DG were prepared as described\n3. Given the overall low tumor accumulation and inferior previously.119 Pure aqueous stock solutions (c = 1 mM) of the\nphototoxicity of complexes 2 and 3 in comparison to that of 1, Ru(II) complex were freshly prepared every day, and their\nwe decided not to evaluate their PDT efficiency in vivo. concentrations and molar absorptivities were determined based on a\n weight-in-volume basis. In the HSA binding experiments, these stock\n\n\u25a0 CONCLUSIONS\nThe in situ conjugation of drug\u2212maleimide prodrugs to\n solutions were diluted with PBS to get the working solutions (c = 50\u2212\n 200 \u03bcM, pH = 7.40). All measurements were carried out at 25.0 \u00b1 0.2\n \u00b0C.\nalbumin has been described as an efficient strategy to improve Instrumentation and Methods. 1H and 13C NMR spectra were\nthe tumor accumulation and the efficiency of antitumor drugs. recorded on a Bruker 400 MHz NMR spectrometer. Chemical shifts\nIn this study, we showed that this strategy is not applicable to (\u03b4) are reported in parts per million (ppm) referenced to\nevery compound. In vitro, the Ru(II) complexes bearing a Mal tetramethylsilane (\u03b4 0.00) ppm using the residual proton solvent\n peaks as internal standards. Coupling constants (J) are reported in\nmoiety described in this work were able to strongly bind hertz (Hz), and the multiplicity is abbreviated as follows: s (singlet), d\nnoncovalently in several hydrophobic pockets of albumin, as (doublet), and m (multiplet). Electrospray ionization mass spectrom-\nwell as covalently to its Cys-34. This property appeared to have etry (ESI-MS) experiments were carried out using an LTQ-Orbitrap\na significant and negative impact on their ability to penetrate XL from Thermo Scientific (Thermo Fisher Scientific, Courtaboeuf,\nthe cells, which led to a decrease in their phototoxicity. France) and operated in positive ionization mode, with a spray\nUnfortunately, this decrease was not compensated by a voltage at 3.6 kV. No Sheath and auxiliary gas were used. The applied\ndramatic increase in tumor accumulation in vivo, although voltages were 40 and 100 V for the ion transfer capillary and the tube\nthe addition of a hydrophilic linker slightly improved the blood lens, respectively. The ion transfer capillary was held at 275 \u00b0C.\ncirculation of the complex. One improvement to this system Detection was achieved in the Orbitrap with a resolution set to\n 100,000 (at m/z 400) and a m/z range between 150 and 2000 in\nwould be to add a cleavable linker between the maleimide profile mode. The spectrum was analyzed using the acquisition\ngroup and the PS, which would enable its release in the tumor software XCalibur 2.1 (Thermo Fisher Scientific, Courtaboeuf,\nmicroenvironment. Interestingly, the ability of complexes 2 France). The automatic gain control (AGC) allowed accumulation\nand 3 to react with Cys-34 of albumin appeared to be inversely of up to 2 \u00d7 105 ions for FTMS scans. Maximum injection time was\ncorrelated with their propensity to be bound in the set to 300 ms, and 1 \u03bcscan was acquired. Ten microliters was injected\nhydrophobic pockets of the protein. This phenomenon may using a Thermo Finnigan Surveyor HPLC system (Thermo Fisher\nbe responsible for a hampered thiol\u2212Mal reaction, which Scientific, Courtaboeuf, France) with a continuous infusion of\nconsequently increases the chances for the Mal moiety to be methanol at 100 \u03bcL/min. Elemental microanalyses were performed\nhydrolyzed before it could react with Cys-34. Thus, Mal- on a Thermo Flash 2000 elemental analyzer. MALDI-TOF experi-\n ments were performed on a MALDI-TOF/TOF UltrafleXtreme mass\nprodrugs that strongly bind noncovalently to albumin are to be\n spectrometer (Bruker Daltonics, Bremen). Mass spectra were\nconsidered with caution. In addition, incorporating a hydro- obtained in a linear positive ion mode. The laser intensity was set\nphilic spacer between the Mal group and the complex appears just above the ion generation threshold to obtain peaks with the\nto be beneficial to both its blood circulation time and its highest possible signal-to-noise (S/N) ratio without significant peak\nalbumin conjugation. Applying the in situ Mal-mediated broadening. All data were processed by using the FlexAnalysis\nalbumin bioconjugation to photosensitizers therefore appears software package (Bruker Daltonics). Fluorescence and phosphor-\nas a promising strategy to improve the efficiency and safety of escence lifetime measurements were carried out on a Fluoromax\nPDT. Nevertheless, a strong investigation into the interaction (Horiba Jobin Yvon) spectrofluorometer equipped with a DeltaHub\nbetween Ru(II) complexes and plasma proteins is needed to time-correlated single photon counting (TCSPC) system using\ndesign the appropriate system. nanoLED light sources N-295, N-350, and N-460 (Horiba Jobin\n Yvon). ICP-MS studies were performed using an Element II HR-ICP-\n\n\u25a0 EXPERIMENTAL SECTION\n Materials. All chemicals were obtained from commercial sources\n MS instrument (Thermo Fisher Scientific).\n Synthesis. [Ru(bphen)2dmbipyNH2][PF6]2. Ru(bphen)2Cl2 (200\n mg, 0.24 mmol, 1.0 equiv) and 5-(aminomethyl)-2,2\u2032-bipyridine (57\nand used without further purification. The Ru(II) complexes cis- mg, 0.29 mmol, 1.2 equiv) were dissolved in a 1:3 mixture of H2O/\ndichlorotetrakis(dimethyl sulfoxide) Ru(II)117 and dichlorobis(4,7- ethanol (50 mL) and refluxed overnight under a N2 atmosphere. The\ndiphenyl-1,10-phenanthroline)Ru(II) (Ru(bphen)2Cl2)102 and the solvent was evaporated, and the residue was dissolved in 10 mL of\n\n 15519 https://doi.org/10.1021/acs.inorgchem.3c01984\n Inorg. Chem. 2023, 62, 15510\u221215526\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nH2O. A saturated aq. NH4PF6 solution was added, and the resulting amount of methanol, and a saturated solution of NH4PF6 was added.\nprecipitate was collected by vacuum filtration. The solid was washed The orange solid was isolated by filtration and washed with cold water\nwith H2O (50 mL) and Et2O (50 mL). The product was dried in a and Et2O. The counterion PF6\u2212 was exchanged to Cl\u2212 by elution with\nhigh vacuum. Yield: 89%. 1H NMR (400 MHz, CD3CN) \u03b4 = 8.59 (s, MeOH from the ion-exchange resin Amberlite IRA-4101 to yield\n1H), 8.44 (s, 1H), 8.29 (d, J = 5.5 Hz, 1H), 8.26 (d, J = 5.5 Hz, 1H), [Ru(bphen)2dmbipyPEGMal][PF6]2 (3) as an orange powder (61\n8.15\u22128.07 (m, 6H), 7.93 (d, J = 5.9 Hz, 1H), 7.74\u22127.70 (m, 3H), mg, 0.040 mmol, 89%). 1H NMR (400 MHz, CD3CN) \u03b4 = 8.79 (s,\n7.62\u22127.47 (m, 22H), 7.39 (dd, J = 5.9, 1.7 Hz, 1H), 7.24 (d, J = 5.7 1H), 8.53 (s, 1H), 8.30 (dd, J = 5.5, 2.8 Hz, 2H), 8.23\u22128.14 (m, 4H),\nHz, 1H), 4.38 (s, 2H), 2.53 (s, 3H); 13C NMR (100 MHz, CD3CN) \u03b4 8.10 (dd, J = 5.5, 3.6 Hz, 2H), 7.76 (dd, J = 5.5, 3.8 Hz, 2H), 7.73 (d,\n= 158.4, 156.6, 152.9, 152.6, 152.4, 152.3, 151.8, 151.3, 149.5, 149.4, J = 5.9 Hz, 1H), 7.70\u22127.52 (m, 23H), 7.28\u22127.17 (m, 3H), 6.78 (s,\n149.4, 148.9, 148.7, 148.6, 142.9, 136.2, 136.2, 130.3, 130.2, 130.0, 2H), 6.68\u22126.60 (m, 1H), 6.00 (s, 1H), 4.58 (dd, J = 17.6, 6.1 Hz,\n130.0, 129.6, 129.5, 129.4, 129.4, 129.3, 127.4, 126.8, 126.7, 126.6, 1H), 4.51 (dd, J = 17.6, 6.1 Hz, 1H), 4.03 (s, 2H), 3.53\u22123.34 (m,\n126.4, 125.6, 124.5, 42.8, 20.8. 20H), 3.31\u22123.19 (m, 4H), 2.58 (s, 3H), 2.52 (s, 4H). 13C NMR (100\n [Ru(bphen)2dmbipyMal][Cl]2 (2). [Ru(bphen)2dmbipyMal]- MHz, CD3OD) \u03b4 = The counterion PF6\u2212 was exchanged to Cl\u2212 by\n[PF6]2 was synthesized as previously described. 1H and 13C NMR elution with MeOH from the ion-exchange resin Amberlite IRA-4101.\nspectra were in accordance with the reported data.37 13\n C NMR (100 MHz, CD3CN) \u03b4 = 175.6, 174.6, 171.9, 169.2, 158.8,\n The counterion PF6\u2212 was exchanged to Cl\u2212 by elution with MeOH 158.3, 153.3, 153.0, 152.5, 152.2, 150.74, 150.68, 149.9, 149.8, 137.1,\nfrom the ion-exchange resin Amberlite IRA-410. Elemental analysis 137.0, 135.7, 131.1, 131.02, 130.94, 130.88, 130.4, 130.3, 130.0, 127.7,\ncalcd for C64H45Cl2N7O2Ru + H2O (%): C 67.78, H 4.18, N 8.65; 127.4, 127.0, 126.8, 71.5, 71.3, 71.2, 70.4, 70.3, 58.3, 43.0, 40.64,\nfound: C 67.73, H 3.94, N 8.36. 40.55, 40.4, 31.7, 31.4, 21.4, 18.4. ESI-HRMS (pos. detection mode):\n [Ru(bphen)2dmbipyPEGNH2][PF6]2. To a solution of 2,2- calcd for C82H78N10O10Ru m/z [M]2+ 732.2467; found: 732.2481.\ndimethyl-4,24-dioxo-3,8,11,14,17,20-hexaoxa-5,23-diazaheptacosan- Elemental analysis calcd for C64H45Cl2N7O2Ru + 5H2O (%): C 60.59,\n27-oic acid (0.150 g, 0.31 mmol, 1.00 equiv) in anhydrous H 5.46, N 8.62; found: C 60.68, H 5.46, N 8.75.\ndichloromethane (DCM) (5 mL) were added N-hydroxysuccinimide Physicochemical Properties. 1H NMR Spectroscopy for\n(0.043 g, 0.37 mmol, 1.2 equiv) and dicyclohexylcarbodiimide (0.076 Stability Studies. 1H NMR spectroscopic studies were carried out\ng, 0.37 mmol, 1.2 equiv). The mixture was agitated for 15 h at room on a Bruker Avance III HD Ascend 500 Plus instrument. Complexes\ntemperature. The solid was filtered off, and the filtrate was evaporated were dissolved in 0.5 mM concentration in water, PBS buffer, 5 mM\nto yield a transparent oil (0.185 g). To a solution of this oil in phosphate (pH 7.4), or EMEM, and 10% (v/v) D2O was added to the\nanhydrous DCM (2 mL) was added a solution of [Ru- samples. Spectra were recorded with the WATERGATE water\n(bphen)2dmbipyNH2][PF6]2 (0.197 mg, 0.16 mmol, 1.00 equiv) suppression pulse scheme using DSS as an internal standard.\nand N,N-diisopropylethylamine (DIPEA) (0.08 mL, 0.48 mmol, 3.0 Lipophilicity and Membrane Permeability. Distribution constants\nequiv) in DCM (3 mL). The mixture was agitated in the dark at room (D) of 1, 2, and 3 were determined by the traditional shake-flask\ntemperature under nitrogen for 15 h. The solvent was evaporated, and method in n-octanol/buffered aqueous solution at pH 7.40 (PBS, or\nthe residue was dissolved in methanol (10 mL). A saturated solution 15 mM phosphate buffer) and in water containing 0 or 0.1 M KCl as\nof NH4PF6 was added, and the orange solid was isolated by filtration, described previously.90,120 The complexes were dissolved in n-octanol\nwashed with cold water and diethyl ether (Et2O), and finally dried presaturated aqueous solution at ca. 20 \u03bcM concentrations. Aqueous\nunder vacuum. Under nitrogen, the solid was dissolved in DCM (1 phases and water-presaturated n-octanolic phases were gently mixed\nmL), and trifluoroacetic acid (0.1 mL) was added. The mixture was in 1:1 or 100:1 volume ratios with a Heidolph Reax 2 overhead shaker\nagitated at room temperature for 15 h. The solvent was evaporated to (\u223c20 rpm) for 2 h. After separation, UV\u2212vis spectra of the\ndryness, the residue was dissolved in a small amount of methanol, and compounds in the aqueous phase were compared to those of the\na saturated solution of NH4PF6 was added. The orange solid was original aqueous solution, and D values of the compounds were\nisolated by filtration and washed with cold water and Et2O. The crude calculated according to the following equation\nproduct was purified by column chromatography on silica gel with a \u00c5\u00c4\u00c5 \u00d1\u00c9\u00d1\n \u00c5\u00c5 Abs(stock. sol.) \u00d1\u00d1 V(aqueous phase)\n \u00c5 1\u00d1\u00d1\u00d1\u00d1 \u00d7\nCH3CN/aq. KNO3 (0.4 M) solution (10:1). The fractions containing \u00c5\n D = \u00c5\u00c5\nthe product were united, and the solvent was removed. The residue \u00c5\u00c5 Abs(aqueous phase after separation) \u00d1\u00d1\u00d1 Vn octanol phase\nwas dissolved in CH3CN, and undissolved KNO3 was removed by \u00c5\u00c7 \u00d6 (1)\nfiltration. The solvent was evaporated, and the product was dissolved An Agilent Cary 8454 diode array spectrophotometer was used to\nin H2O (20 mL). Upon the addition of NH4PF6, the product measure the UV\u2212vis spectra in the interval 200\u2212800 nm.\nprecipitated as a PF6 salt. The solid was obtained by filtration and was Albumin Interaction. MALDI Sample Preparation. Complexes\nwashed with H2O (50 mL) and Et2O (50 mL) to yield [Ru- 1\u22123 from stock solutions in ethanol were added to a 100 \u03bcM solution\n(bphen)2dmbipyPEGNH2][PF6]2 as an orange powder (85 mg, 0.05 of BSA in PBS at a final complex concentration of 200 \u03bcM (final\nmmol, 58%). 1H NMR (400 MHz, CD3CN) \u03b4 = 8.64 (d, J = 1.7 Hz, ethanol concentration: 5%). The mixtures were agitated at 450 rpm,\n1H), 8.48 (d, J = 1.8 Hz, 1H), 8.28 (t, J = 5.5 Hz, 2H), 8.24\u22128.15 (m, 37 \u00b0C for 2 h in a Thermomixer (Eppendorf). The mixtures were then\n4H), 8.11 (t, J = 5.7 Hz, 2H), 7.76 (dd, J = 5.5, 1.4 Hz, 2H), 7.74 (d, J dialyzed against 2 L distilled water for 24 h (3 buffer exchanges) using\n= 6.0 Hz, 1H), 7.71\u22127.53 (m, 21H), 7.26\u22127.21 (m, 2H), 7.17 (t, J = dialysis cassettes (Slide-A-Lyzer MWCO 10 kDa, Thermo Fisher).\n6.1 Hz, 1H), 6.83 (s, 2H), 6.69 (t, J = 5.9 Hz, 1H), 4.56 (d, J = 6.1 The matrix solution was prepared at a concentration of 45 mM in\nHz, 2H), 3.68\u22123.61 (m, 2H), 3.59\u22123.39 (m, 18H), 3.35\u22123.26 (m, H2O/CH3CN/trifluoroacetic acid 1/1/0.1.\n2H), 3.01 (dd, J = 6.1, 4.3 Hz, 2H), 2.61\u22122.54 (m, 4H), 2.51\u22122.46 The samples were prepared by mixing the BSA-complex mixture\n(m, 2H); 13C NMR (100 MHz, CD3CN) \u03b4 = 174.0, 158.2, 157.6, solution with a matrix solution at a volume ratio of 1:9.\n153.2, 153.1, 152.9, 152.6, 152.5, 152.2, 151.6, 149.94, 149.87, 149.85, UV\u2212Visible Spectrophotometric and Circular Dichroism Spec-\n149.5, 149.31, 149.30, 136.71, 136.68, 136.6, 130.8, 130.72, 130.66, troscopic Measurements. UV\u2212visible (UV\u2212vis) spectrophotometry\n130.62, 130.57, 130.11, 130.06, 129.9, 129.3, 127.09, 127.05, 127.0, was utilized to follow the stability of the complexes in various aqueous\n126.3, 126.2, 122.7, 70.84, 70.82, 70.80, 70.6, 70.5, 70.3, 70.2, 70.0, solutions and monitor their interaction with HSA on an Agilent Cary\n67.3, 42.4, 40.6, 39.6, 31.3, 21.3. 8454 diode array spectrophotometer and an Agilent Cary 3500\n [Ru(bphen)2dmbipyPEGMal][Cl]2 (3). Under nitrogen, 2,5- spectrophotometer in the wavelength range between 200 and 800 nm.\ndioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate Samples contained ca. 20 \u03bcM metal complex dissolved in various\n(23 mg, 0.09 mmol, 2.0 equiv) was added to a solution of buffer systems (PBS, phosphate) or in water. In protein binding\n[Ru(bphen)2dmbipyPEGNH2][PF6]2 (73 mg, 0.045 mmol, 1.0 studies, a constant amount of complex was titrated by HSA (0\u221280\nequiv) and DIPEA (80 mg, 0.068 mmol, 1,5 equiv) in DCM (5 \u03bcM).\nmL). The mixture was agitated at room temperature in the dark for 15 The interaction of the metal complexes at the Cys-34 residue of\nh. The solvent was removed, the residue was dissolved in a small HSA was investigated via the DTDP method described in our former\n\n 15520 https://doi.org/10.1021/acs.inorgchem.3c01984\n Inorg. Chem. 2023, 62, 15510\u221215526\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nwork.100 The available cytosine thiol content in HSA was determined GraphPad Prism V6.07 Software. Fluorescence intensities (Y values)\nto be 18%. Complex binding was tested in the following setup: 130 were plotted against compound concentrations (X values). X values\n\u03bcM HSA (23 \u03bcM free thiol) and various amounts of complex (0\u2212120 were transformed into log(X) values. Y values were normalized by\n\u03bcM) were incubated for 3 h or 30 min at pH 7.00 (100 mM setting 100% cell viability for the highest fluorescence intensity and\nphosphate), and the UV\u2212vis spectra were recorded (a), then 110 \u03bcM 0% cell viability for the lowest fluorescence intensity in each data set.\nDTDP was added, and the UV\u2212vis spectra were measured again after IC50 was calculated by nonlinear regression using the algorithm\nanother 40 min waiting time (b). The spectrum of the colored \u201clog(inhibitor) vs normalized response\u201d.\nreaction product 2-thiopyridone (2-TP) was derived by the Phototoxicity Assay. Cells were seeded in triplicate plates in 96-\nsubtraction of the spectrum (a) from the spectrum (b). A blank well plates at a 4000 cells/well density in 100 \u03bcL and were incubated\nexperiment with 1 was carried out in addition to a control experiment. at 37 \u00b0C in 5% CO2 for 24 h. The medium was replaced with\nThe effect of protein unfolding on the thiol binding of 2 and 3 was increasing concentrations of test compounds in 100 \u03bcL of fresh\nstudied by the addition of 0.5% (w/w) SDS to the protein prior to its medium, and cells were incubated at 37 \u00b0C in 5% CO2 for 4 h. The\nreaction with the complex. medium was then replaced with fresh medium, and wells were\n Spectrofluorometric Measurements. Samples were measured in 1 individually irradiated at 595 nm for 2 h (22.47 J/cm2) using a\n\u00d7 1 cm2 cells. PBS buffer was used for sample preparation, and LUMOS-BIO photoreactor (Atlas Photonics). Control plates were\nemission spectra were recorded after 5 min incubation. Four kinds of kept in the dark in a non-CO2 incubator for 4 h. Cells were then\nexperiments were carried out: (i) 1 \u03bcM HSA and various amounts of incubated for an additional 44 h at 37 \u00b0C in 5% CO2. The medium\ncomplex (from 0 to 20 equiv) were used for quenching experiments; was replaced with 100 \u03bcL of fresh medium containing 0.2 mg/mL of\nand (ii) fluorescence of site markers WF and DG was measured in resazurin. After 4 h of incubation at 37 \u00b0C in 5% CO2, the\nsamples containing 1 \u03bcM HSA, 1 \u03bcM marker, and 0\u221220 \u03bcM fluorescence intensity of resorufin was read at 590 nm with an\ncompound; intrinsic fluorescence of the complexes was followed as excitation wavelength of 540 nm using a SpectraMaxM5 microplate\nwell by applying (iii) 5 \u03bcM complex and various HSA concentrations reader (Molecular Devices). Data were fitted using GraphPad Prism\n(0\u2212110 \u03bcM) or (iv) 5 \u03bcM complex, 5 \u03bcM HSA, and various site V6.07 Software. Fluorescence intensities (Y values) were plotted\nmarkers (WF and DG) concentrations (0\u2212160 \u03bcM). Instrumental against compound concentrations (X values). X values were\nparameters are listed in Table S3. The computer program HypSpec121 transformed into log(X) values. Y values were normalized by setting\nwas utilized for the calculation of formation constants for HSA\u2212 100% cell viability for the highest fluorescence intensity and 0% cell\ncompound adducts, as described in our former work.119,122 viability for the lowest fluorescence intensity in each data set. IC50 was\nCorrections for self-absorbance and inner filter effect were done as calculated by nonlinear regression using the algorithm \u201clog(inhibitor)\ndescribed in our former work using the formula suggested by vs normalized response\u201d.\nLakowicz.112 Cellular Uptake. A total of 5 \u00d7 106 CT26 cells (5 \u00d7 106) were\n Time-Resolved Fluorescence Measurements. Ludox (from Sigma- seeded in 10 cm Petri dishes (10 mL/dish) and were incubated at 37\nAldrich) was used as a scatter to obtain the instrument response \u00b0C in 5% CO2. The next day, the medium was replaced with 5 \u03bcM\nfunction. The background (obtained with blank samples) was complex dilution in 10 mL of culture medium, and the dishes were\nsubtracted from the decay. The program DAS6 (version 6.6; Horiba, incubated for 4 h at 37 \u00b0C in 5% CO2. Cells were washed three times\nJobin Yvon) was used for the analysis of the experimental fluorescence with cold PBS, trypsinized, and harvested, and a 10 \u03bcL aliquot of each\ndecays. The fluorescence intensity decay over time is described by a cell suspension was sampled for accurate counting. The cell\nsum of exponentials as the following equation shows suspensions were centrifuged, and the supernatant was discarded.\n The pellets were digested in 100 \u03bcL of 70% HNO3 at 65 \u00b0C for 24 h\n n\n ij t yz\n j zz and then diluted in 5 mL of Milli-Q water (final HNO3 concentration:\n I(t ) = i expj\n jj zz\n i=1 k i { (2) 1.4%). The Ru content in each sample was determined by ICP-MS.\n The amount of Ru detected in the digests was transformed from ppb\nwhere \u03b1i and \u03c4i are the normalized amplitude and lifetime of into ng and normalized using the total number of cells digested.\ncomponent i, respectively. From these parameters, the fraction of Experiments were performed in triplicate.\nemitted light by each component i can be calculated through the In vivo Studies. Animals. This study was carried out in\nequation accordance with the EU regulations and approved by the Ethical\n i i Commission of the faculty of Pharmaceutical and Biological Sciences\n fi = Paris-Descartes (agreement number: E-75-06-02).\n ( i i) (3)\n Eight-week-old Balb/c mice were provided by Janvier Lab and\n The goodness of the fit was judged from a \u03c7R2 value close to 1.0 housed with food and water supplied ad libitum in a 12-h day/night\nand a random distribution of weighted residuals. See details on the cycle.\ninstrument settings for different kinds of fluorophores in Table S3. Formulation in Polysorbate 80. A total of 1.74 \u03bcmol of the test\n In Cellulo Experiments. Cell Culture. CT26 cells were cultured in compound and 50 mg of polysorbate 80 were dissolved in 5 mL of\nDMEM medium (Gibco, Life Technologies) supplemented with 10% anhydrous ethanol. The solution was concentrated to dryness under a\nof fetal calf serum (Gibco) and 100 U/mL penicillin\u2212streptomycin vacuum in a round-bottom flask to yield an orange viscous film that\nmixture (Gibco) and maintained in a humidified atmosphere at 37 \u00b0C was further dried at 40 \u00b0C under a vacuum for 10 min. The film was\nand 5% CO2. RPE-1 cells were cultured in DMEM\u2212F12 medium then rehydrated in 5 mL of PBS (Gibco). The orange solution was\n(Gibco, Life Technologies) supplemented with 10% of fetal calf finally sterile-filtered on a 0.2 \u03bcm cellulose-acetate membrane\nserum (Gibco) and 100 U/mL penicillin\u2212streptomycin mixture (Corning). Test compound concentration was determined by\n(Gibco) and maintained in a humidified atmosphere at 37 \u00b0C and 5% measuring the absorbance of the solution after dilution in acetonitrile\nCO2. at 450 nm in quartz cuvettes using a SpectraMaxM5 (Molecular\n Forty-Eight-Hour Cytotoxicity Assay. Cells were seeded in Devices) spectrophotometer.\ntriplicate plates in 96-well plates at a 4000 cells/well density in 100 [Ru(bphen)2dmbipyMal]Cl2 Conjugation to Albumin Kinetics. A\n\u03bcL and were incubated at 37 \u00b0C in 5% CO2 for 24 h. The medium solution of complex 2 (0.2 mg/mL) formulated in either 5% ethanol\nwas replaced with increasing concentrations of test compounds in 100 in PBS (Gibco) or 1% polysorbate 80 in PBS (Gibco) was added to a\n\u03bcL of fresh medium, and cells were incubated at 37 \u00b0C in 5% CO2 for solution of bovine serum albumin (BSA) (120 mg/mL) in PBS\n48 h. The medium was replaced with 100 \u03bcL of fresh medium (Gibco). The mixture was shaken at 450 rpm at 37 \u00b0C in a\ncontaining 0.2 mg/mL of resazurin. After 4 h of incubation at 37 \u00b0C Thermomixer (Eppendorf). At each time point, 300 \u03bcL of the mixture\nin 5% CO2, the fluorescence intensity of resorufin was read at 590 nm was added to 800 \u03bcL of CH3CN. The mixture was vortexed for 5 s\nwith an excitation wavelength of 540 nm using a SpectraMaxM5 and then centrifugated for 5 min at 10,000g. The absorbance of the\nmicroplate reader (Molecular Devices). Data were fitted using supernatants was recorded at 450 nm in a quartz cuvette using a\n\n 15521 https://doi.org/10.1021/acs.inorgchem.3c01984\n Inorg. Chem. 2023, 62, 15510\u221215526\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nSpectraMaxM5 (Molecular Devices) spectrophotometer. The experi- Johanne Seguin \u2212 Universit\u00e9 Paris Cit\u00e9, UTCBS, INSERM,\nment was performed in triplicates. %conversion was calculated using CNRS, 75006 Paris, France; orcid.org/0000-0001-5689-\nthe following formula 7046\n Abst 0 Abst Micka\u00ebl Tharaud \u2212 Biog\u00e9ochimie a\u0300 l\u2019Anthropoce\u0300ne des\n % conversion = El\u00e9ments et Contaminants Emergents, Institut de Physique du\n Abst 0\n Globe de Paris, 75005 Paris, France; orcid.org/0000-\n Biodistribution Study. Fifteen 10-week-old BALB/c mice were 0001-6131-655X\nimplanted subcutaneously in both flanks with two CT26 tumor Vincent Gu\u00e9rineau \u2212 Institut de Chimie des Substances\nfragments with a diameter of 1 mm, previously extracted from donor Naturelles, CNRS UPR2301, Universit\u00e9 Paris-Sud, Universit\u00e9\nmice. Paris-Saclay, 91198 Gif-sur-Yvette, France\n After 8 days, mice were randomly divided into five groups and Kevin Cariou \u2212 Chimie ParisTech, PSL University, CNRS,\ninjected intravenously in the caudal vein with 2 \u03bcmol/kg of test Institute of Chemistry for Life and Health Sciences, F-75005\ncompound formulated in 1% polysorbate 80 in PBS (complexes 1 and Paris, France\n2) or 5% glucose in water (complex 3). After 1 min, 30 min, 1 h, 6 h,\n Nathalie Mignet \u2212 Universit\u00e9 Paris Cit\u00e9, UTCBS, INSERM,\nand 24 h, mice were sacrificed, and relevant organs, including blood,\ntumors, liver, kidneys, intestine, lungs, and brain, were harvested and CNRS, 75006 Paris, France\nweighed. Organs were then digested in 70% nitric acid at 65 \u00b0C for 24 E\u0301 va A. Enyedy \u2212 MTA-SZTE Lendu\u0308let Functional Metal\nh. Digests were diluted 100 times in 1% HCl, and Ru contents were Complexes Research Group, Department of Molecular and\ndetermined by ICP-MS. The amount of Ru detected in organ digests Analytical Chemistry, University of Szeged, H-6720 Szeged,\nwas transformed from ppb into ng of Ru and expressed as a % of the Hungary; orcid.org/0000-0002-8058-8128\ninjected dose/g of organs. Tumor Ru contents are presented as an\naverage of left and right tumors Ru content. Kinetic parameters were Complete contact information is available at:\ncalculated using GraphPad Prism V6.07 Software by fitting the blood https://pubs.acs.org/10.1021/acs.inorgchem.3c01984\nRu content as a function of time, using a two-phase exponential decay\nequation. The 48 h biodistribution experiment was performed Author Contributions\nsimilarly, but complexes 1\u22123 were all formulated in 5% glucose in #\n R.V. and O.D. contributed equally to this work.\nwater for comparison purposes. Three mice were used for each\ncompound. Notes\n The authors declare no competing financial interest.\n\u25a0\n*\n ASSOCIATED CONTENT\ns\u0131 Supporting Information \u25a0 ACKNOWLEDGMENTS\nThe Supporting Information is available free of charge at This work was financially supported by an ERC Consolidator\nhttps://pubs.acs.org/doi/10.1021/acs.inorgchem.3c01984. Grant Photo-MedMet to G.G. (GA 681679) and has received\n support under the program \u201c\u2018Investissements d\u201dAvenir\u2019\u2019\n Synthesis scheme (Scheme S1); 1H, 13C NMR spectra, launched by the French Government and implemented by\n and HRMS (Figures S1\u2212S10); complex-albumin addi- the ANR with the reference ANR-10-IDEX-0001-02 PSL\n tional figures (Figures S11\u2212S22); binding kinetic of (G.G.) and from a Qlife pr\u00e9maturation funding (G.G. and\n complex 2 formulated in polysorbate 80 or ethanol R.V.). Part of the ICP-MS measurements was supported by the\n (Figure S23); tables containing fluorescence lifetime IPGP multidisciplinary program PARI and by Paris\ufffdIdF\n parameters (Tables S1\u2212S3) (PDF) region SESAME Grant no. 12015908. The support of the\n \u201cLendu\u0308let\u201d Program (ELKH (Hungary), LP2019-6/2019) is\n\n\u25a0 AUTHOR INFORMATION\nCorresponding Authors\n also acknowledged (E.A.E.). O.D. gratefully acknowledges the\n financial support from a J. Bolyai Research Fellowship (bo-\n 125-20) and the U\u0301 NKP-22-5-SZTE-547\ufffdNew National\n Robin Vinck \u2212 Chimie ParisTech, PSL University, CNRS, Excellence Program.\n Institute of Chemistry for Life and Health Sciences, F-75005\n Paris, France; Phone: +33 1 85 78 41 51;\n Email: robin.vinck13@gmail.com; www.gassergroup.com\n Gilles Gasser \u2212 Chimie ParisTech, PSL University, CNRS,\n \u25a0 REFERENCES\n (1) Dolmans, D. E. J. G. J.; Fukumura, D.; Jain, R. K. Photodynamic\n Therapy for Cancer. Nat. Rev. Cancer 2003, 3 (5), 380\u2212387.\n Institute of Chemistry for Life and Health Sciences, F-75005 (2) Bonnet, S. Why Develop Photoactivated Chemotherapy? Dalton\n Paris, France; orcid.org/0000-0002-4244-5097; Trans. 2018, 47 (31), 10330\u221210343.\n Phone: +33 1 85 78 41 51; Email: gilles.gasser@ (3) Dougherty, T. J.; Gomer, C. J.; Henderson, B. W.; Jori, G.;\n chimeparistech.psl.eu; www.gassergroup.com Kessel, D.; Korbelik, M.; Moan, J.; Peng, Q. 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