Marta Martinez-Alonso
email: m.martinez-alonso@chimieparistech.psl.eu

Gilles Gasser
email: gilles.gasser@chimeparistech.psl.eu

Ruthenium polypyridyl complex-containing bioconjugates

Keywords: Bioconjugates, Bioinorganic Chemistry, Inorganic Chemical Biology, Medicinal Inorganic Chemistry, Ruthenium Polypyridyl Complexes A549: human lung carcinoma cells A549R: cisplatin resistant human lung carcinoma cells Ab: antibody Abs: Absorption ABC: avidin-biotin complex ABDA: 9, 10-anthracenediyl-bi(methylene)-dimalonic acid ABTS leukaemia cancer cells AML: acute myeloid leukaemia amol: attomole AQC: anthraquinone-2-carboxylic acid AQS: anthraquinone-2-sulfonic acid B16F10: murine melanoma cells BBB: blood brain barrier CaSki: human caucasian cervical epidermoid carcinoma circular dichroism Chinese Hamster Ovary cells CLSM: confocal laser scanning microscopy ConA: concavalin cell-penetrating peptide CsA: cyclosporine A CT-DNA: calf thymus DNA cyclic voltammetry DABA: 2, 4-diaminobutyric acid DAPA: 2, 6-Diaminopimelic acid DAPI: 4', 6-Diamidino-2-phenylindole nuclear stain dcbpy: 2, 2′-bipyridine-4, 4′-dicarboxylic acid dcbpy dimyristoylphosphatidylglycerol dpp: 4, 7-diphenyl-1, 10-phenanthroline dppg: dipalmitoylphosphatidylglycerol dppn: Benzo[i]dipyrido-[3, 2- epidermal growth factor receptor ER: endoplasmic reticulum FLIM: fluorescence lifetime imaging FP: fluorescence polarization

Highlights

• Bioconjugation improves some of the features of the Ru(II) polypyridyl complexes.

• The properties of the complexes are not usually altered by the bioconjugation.

• Peptides are among the most common biomolecules used in bioconjugation.

• The bioconjugates were used for different purposes.

Introduction

In the last decades, Ru(II) polypyridyl complexes have gained great attention in different research areas due to their outstanding photophysical properties. The prototype of this family of complexes is [Ru(bpy)3] 2+ , which shows strong absorption in the visible region of the spectra (λmax = 452 nm, 13'000 M -1 cm -1 , MLCT), strong phosphorescence emission (λmax = 620 nm, ΦPL = 0.095 in deoxygenated CH3CN at r.t., 3 MLCT) and long emission lifetime (τ = 930 ns in deoxygenated CH3CN at r.t.). The electrochemical properties of this complex are also relevant with an interesting redox behaviour: reversible oxidation of the Ru(II)/Ru(III) pair at ca. 1 V and three reduction peaks (the first two reversible and the last one quasi-reversible), corresponding to each of the bpy ligands. In addition, the versatility of Ru(II) polypyridyl complexes as energy donors, electron acceptors and electron donors along with their great stability in solution, tunability and ease of synthesis [START_REF] Arias-Rotondo | The photophysics of photoredox catalysis: a roadmap for catalyst design[END_REF] make them excellent candidates as catalysts, [START_REF] Zeng | Exceptionally Efficient Unsymmetrical Ruthenium(II) NNN Complex Catalysts Bearing a Pyridyl-Based Pyrazolyl-Imidazolyl Ligand for Transfer Hydrogenation of Ketones[END_REF][START_REF] Kärkäs | Molecular ruthenium water oxidation catalysts carrying non-innocent ligands: mechanistic insight through structure-activity relationships and quantum chemical calculations[END_REF][START_REF] Johnson | Activating a Low Overpotential CO2 Reduction Mechanism by a Strategic Ligand Modification on a Ruthenium Polypyridyl Catalyst[END_REF] solar dyes, [START_REF] Aghazada | Ruthenium Complexes as Sensitizers in Dye-Sensitized Solar Cells[END_REF][START_REF] Adeloye | Towards the Development of Functionalized PolypyridineLigands for Ru(II) Complexes as Photosensitizers inDye-Sensitized Solar Cells (DSSCs)[END_REF][START_REF] Bomben | Cyclometalated ruthenium chromophores for the dye-sensitized solar cell[END_REF] in light-emitting electrochemical cells [START_REF] Li | A biotinylated ruthenium(II) photosensitizer for tumor-targeted two-photon photodynamic therapy[END_REF][START_REF] Wiederholt | A 2,2"-bipyridine ligand for incorporation into oligodeoxynucleotides: synthesis, stability and fluorescence properties of ruthenium-DNA complexes[END_REF][START_REF] Bideh | Efficient near infrared light emitting electrochemical cell (NIR-LEEC) based on new binuclear ruthenium phenanthroimidazole exhibiting desired charge carrier dynamics[END_REF] and light-emitting diodes, [START_REF] Gong | Trifunctional Light-Emitting Molecules Based on Rhenium and Ruthenium Bipyridine Complexes[END_REF] as well as in many biological applications such as anticancer agents [START_REF] Gong | Trifunctional Light-Emitting Molecules Based on Rhenium and Ruthenium Bipyridine Complexes[END_REF][START_REF] Liu | Harnessing ruthenium(II) as photodynamic agents: Encouraging advances in cancer therapy[END_REF][START_REF] Levina | Recent developments in ruthenium anticancer drugs[END_REF][START_REF] Zeng | The development of anticancer ruthenium(II) complexes: from single molecule compounds to nanomaterials[END_REF][START_REF] Schatzschneider | Cellular Uptake, Cytotoxicity, and Metabolic Profiling of Human Cancer Cells Treated with Ruthenium(II) Polypyridyl Complexes [Ru(bpy)2(N-N)]Cl 2 with N-N=bpy, phen, dpq, dppz, and dppn[END_REF] or photosensitizers for photodynamic therapy (PDT). [START_REF] Notaro | Monomeric and dimeric coordinatively saturated and substitutionally inert Ru(II) polypyridyl complexes as anticancer drug candidates[END_REF][START_REF] Mari | Combination of Ru(II) complexes and light: new frontiers in cancer therapy[END_REF][START_REF] Huang | Highly Charged Ruthenium(II) Polypyridyl Complexes as Lysosome-Localized Photosensitizers for Two-Photon Photodynamic Therapy[END_REF][START_REF] Hess | Evaluation of the Medicinal Potential of Two Ruthenium(II) Polypyridine Complexes as One-and Two-Photon Photodynamic Therapy Photosensitizers[END_REF][START_REF] Heinemann | Critical Overview of the Use of Ru(II) Polypyridyl Complexes as Photosensitizers in One-Photon and Two-Photon Photodynamic Therapy[END_REF] Fig. 1A displays the percentage of publications related to the different applications that Ru(II) polypyridyl complexes have been involved in. Almost half of them are in biological related areas.

State-of-the-art

In 1936, Burstall reported the synthesis of the first Ru(II) polypyridyl complex, namely [Ru(bpy)3]Cl2. [START_REF] Burstall | Optical activity dependent on co-ordinated bivalent ruthenium[END_REF]. In the early 80s, the discovery of the "ruthenium blue dimer" ([(bpy)2Ru(H2O)(µ-O)(H2O)Ru(bpy)2] 4+ ) in water splitting [START_REF] Dau | The Mechanism of Water Oxidation: From Electrolysis via Homogeneous to Biological Catalysis[END_REF] triggered the expansion of the preparation of this kind of complexes. Since then, an important variety of bipyridine-type ligands has been designed and developed to tune the physico-chemical properties of this type of complexes. [START_REF] Vos | Ruthenium polypyridyl chemistry; from basic research to applications and back again[END_REF] In the last 20 years, the number of publications about ruthenium polypyridyl complexes has grown exponentially (Fig. 1B), highlighting their relevance. Regarding their use as anticancer drugs, a ruthenium polypyridyl complex (TLD-1433), developed by McFarland and co-workers [START_REF] Monro | Transition Metal Complexes and Photodynamic Therapy from a Tumor-Centered Approach: Challenges, Opportunities, and Highlights from the Development of TLD1433[END_REF][START_REF] Shi | Ru(II) dyads derived from α -oligothiophenes: A new class of potent and versatile photosensitizers for PDT[END_REF] is currently in Phase II clinical trials as a photodynamic therapy photosensitizer against bladder cancer. [START_REF] Inc | TLD-1433 Clinical Trial[END_REF] This achievement is definitively an encouragement for scientists to working with ruthenium complexes.

Bioconjugation is a strategy to covalently combine two or more molecules, where at least one of the fragments is a biomolecule or a bioactive compound. [START_REF] Hermanson | Introduction to Bioconjugation[END_REF] The discovery of naturally occurring metal complexes like the haem group in haemoglobin (Fe), vitamin B12, cobalamin (Co) [START_REF] Reddy | Coordination compounds in biology[END_REF] and hydrogenases (Ni/Fe and Fe/Fe), [START_REF] Monney | Transition metal bioconjugates with an organometallic link between the metal and the biomolecular scaffold[END_REF] has motivated an increasing interest in the development of new synthetic metal-containing biomolecules/bioconjugates. Some reviews concerning ruthenium-containing bioconjugates have been reported by Gunnlaugsson and coworkers, who reviewed only bioconjugates with in vitro and in vivo applications, by Lo and coworkers, who gathered some examples among luminescent Ruthenium polypyridyl complexes) and by Chen and co-workers, who reported different transition metal complexes as PSs for theranostic applications. [START_REF] Poynton | The development of ruthenium(II) polypyridyl complexes and conjugates for: In vitro cellular and in vivo applications[END_REF][START_REF] Shum | Luminescent Ruthenium(II) Polypyridine Complexes for a Wide Variety of Biomolecular and Cellular Applications[END_REF][START_REF] Li | Transition metal complexes as photosensitizers for integrated cancer theranostic applications[END_REF] However, these reviews are not only exclusively dedicated to bioconjugation. In this review, we intend to discuss the recent advances on the preparation and A B use of ruthenium polypyridyl complex-containing bioconjugates. We have made sure to highlight the applications of these conjugates.

Among the biomolecules used in bioconjugation, DNA and nucleotides are on top of the list of biomolecules conjugates to ruthenium polypyridyl complexes (76 % of publications), as shown in Fig. 2. The second place is for complexes bearing proteins, peptides and amino acids (21 %), and the remaining 3 % (following decreasing order) is shared among lipids, sugars and carbohydrates, antibodies and vitamins. Thus, there is still place for the design, study and development of new bioconjugates, especially with uncommon biomolecules. We note that this review will not gather ruthenium-containing nucleotides and DNA conjugates [START_REF] Wiederholt | A 2,2"-bipyridine ligand for incorporation into oligodeoxynucleotides: synthesis, stability and fluorescence properties of ruthenium-DNA complexes[END_REF][START_REF] Jacquet | Photoadduct between tris(1,4,5,8tetraazaphenanthrene)ruthenium(II) and guanosine monophosphate-a model for a new mode of covalent binding of metal complexes to DNA[END_REF][START_REF] Khan | Solid-Phase Synthesis and Photophysical Properties of DNA Labeled at the Nucleobase with Ru(bpy) 2 (4-m-4'-pa-bpy)2+[END_REF][START_REF] Hurley | Ru(II) and Os(II) Nucleosides and Oligonucleotides: Synthesis and Properties[END_REF][START_REF] Irvoas | Programmable multimetallic linear nanoassemblies of ruthenium-DNA conjugates[END_REF][START_REF] Kahrović | Structural feature of calf thymus deoxyribonucleic acid-ruthenium(III) interaction in aqueous solution by difference Fourier transformed infrared spectroscopy[END_REF] since this was recently reviewed by our group. [START_REF] Flamme | Applications of Ruthenium Complexes Covalently Linked to Nucleic Acid Derivatives[END_REF] We also note that Chao and co-workers recently reported a review on how to design metal complexes to target specific organelles. [START_REF] Qiu | Organelle-targeting metal complexes: From molecular design to bio-applications[END_REF] They previously published another review on the use of ruthenium complexes as anticancer drugs and their potential targets in cells. [START_REF] Zeng | The development of anticancer ruthenium(II) complexes: from single molecule compounds to nanomaterials[END_REF] Fig. 2. Pie chart of number of publications for different Ru(II) polypyridyl bioconjugates. The label presents the search. Searched on June 9 th 2020.

Peptides

There are many different synthetic pathways to covalently attach ruthenium complexes to peptides. This will depend on the target amino acid (AA). Lysine and cysteine are the most common AAs that have been used to attach novel functionalities to peptides. Lysine easily

Ru polypyridyl DNA + nucleotides

Ru polypyridyl proteins + peptides + amino acids

Ru polypyridyl antibodies

Ru polypyridyl lipids Ru polypyridyl sugars + carbohydrates

Ru polypyridyl vitamins

reacts with N-hydroxysuccinimide (NHS), thiocyanate or maleimide through the free amino group from its lateral chain. Maleimide can easily react with cysteines by Michael addition. In addition, cysteines, which are nucleophilic, can react with alkyl bromide, iodoacetamide and epoxides. Amino groups can react with the carboxylic acid of aspartic acid and glutamic acid in the presence of crosslinking carboxydiimide reagents. In the case of metal complex with a leaving group (i.e., a labile molecule like a water molecule), histidine is one of the best options for direct linkage. [START_REF] Lam | Ru(II)-diimine functionalized metalloproteins: From electron transfer studies to light-driven biocatalysis[END_REF] Fig. 3 gathers some examples of structures bearing different reactive moieties that are coupled to amino acids. Meier-Menches and Casini recently reported strategies to prepare metal-peptide bioconjugates. [START_REF] Meier-Menches | Design Strategies and Medicinal Applications of Metal-Peptidic Bioconjugates[END_REF] (bpy) One of the easiest and simplest ways to functionalize Ru(II) polypyridyl complexes is their coupling with an amino acid. Some amino acids were attached to bipyridyl ligands through amid bonds. One of the first examples of these AA-labelled complexes was reported by Magnuson et al. to mimic Photosystem II (PSII) reactions. [START_REF] Magnuson | Mimicking Electron Transfer Reactions in Photosystem II: Synthesis and Photochemical Characterization of a Ruthenium(II) Tris(bipyridyl) Complex with a Covalently Linked Tyrosine[END_REF] In the photosynthesis, once the water oxidation has occurred, the released electrons are delivered to a tyrosine (Tyrosine Z, TyrZ). To check this mechanism, photosensitizers (PS) 1-Ala and 2-Tyr (Fig. 4) bearing alanine and tyrosine, respectively, were synthesised. The two-step photoinduced electron transfer (eT) mechanism (only for 2-Tyr) occurs as follows. Firstly, in the presence of electron acceptors (Methylviologen, MV 2+ and Co(NH3)5Cl 2+ ) and light, the Ru(II) core is oxidized to Ru(III). In the second step, one electron is intramolecularly extracted from the covalently attached tyrosine and transferred back to the Ru(III), restoring the PS. This mechanism successfully met the actual water oxidation in PSII.

With the same purpose, Eswaran et al. described the photoinduced electron transfer reactions of Ru(II) derivatives with alanine (1-Ala), tyrosine (2-Tyr), methionine (3-Met) and phenylalanine (4-Phe) shown in Fig. 4. [START_REF] Eswaran | Photoinduced Electron Transfer Reactions of Ruthenium(II)-Complexes Containing Amino Acid with Quinones[END_REF] In this case, however, different quinones were selected as quenchers. The emission intensities of the complexes were quenched by the quinones following a trend: higher quenching was observed for quinones with higher reduction potentials.

Recently, Ramu et al. prepared and tested compounds 2-Tyr and 5-Trp (with tyrosine and tryptophan, respectively, see Fig. 4) in cells as photodynamic therapy (PDT) agents. [START_REF] Ramu | Photo-induced cytotoxicity and anti-metastatic activity of ruthenium(II)-polypyridyl complexes functionalized with tyrosine or tryptophan[END_REF] Both amino acid derivatives displayed improved water-solubility when compared with the nonconjugated ruthenium complex. Different DNA binding studies (i.e., UV-vis, fluorescence and isothermal titration calorimetry, ITC) revealed that complexes 2-Tyr and 5-Trp are able to bind DNA through groove binding and that the process was entropically driven. The binding affinity constants calculated by the different techniques were Ka = 1.18-3.2 × 10 5 M -1 and Ka = 1.57-5.3 × 10 5 M -1 for 2-Tyr and 5-Trp, respectively. Gel electrophoresis exhibited photoinduced DNA cleavage through singlet oxygen generation ( 1 O2). Additionally, 1 O2 quantum yield values were 0.80 for 2-Tyr and 0.72 for 5-Trp, respectively in acetonitrile. Cytotoxicity was also tested in A549 cells (human lung carcinoma cells) and Hct116 cells (human colon cancer), by incubating cells with the complexes for 4 h in the dark before irradiation (λ ~ 450-480 nm, 10 J/cm 2 ). Both of the compounds were found to be non-toxic in the dark (IC50 > 300 µM). After of the pH (Fig. 5). [START_REF] Geisser | pH-Dependent Excited-State Dynamics of [Ru(bpy)3] 2+ -Modified Amino Acids: Effects of an Amide Linkage and Remote Functional Groups[END_REF] They used DAPA (2,6-Diaminopimelic acid), DABA (2,4diaminobutyric acid), ornithine and lysine as the amino acids, attached through an amid bond to a bipyridine ligand. Thus, the number of carbons increase from 1 in 6-DAPA, to 4 in 9-Lys.

Each complex has three pKa, corresponding to the amide, carboxylic acid and amine groups. At very low and high pH, no difference was found in the UV-vis absorption. This fact suggests the absence of direct electronic interactions between the amino acid and the ruthenium core.

However, regarding the emission at pH 1, it got completely quenched, due to the protonation of the amide bond. At intermediate pH values, between 2 and 8, the emission intensity and the lifetimes increased with the number of methylene groups. These findings revealed that there is no interaction between the amino acids and the ruthenium chromophore in the ground state, but there is in the excited state. This difference of states (i.e., ground and excited state) is smaller for 6-DAPA and 7-DABA. Therefore, the amid linkage could be a key factor to control the photophysical properties of this kind of complexes. Unlike other amino acids, ʟ-proline has been coupled to Ru(II) complexes as a chiral auxiliary for the synthesis and isolation of enantiomerically pure complexes. Although some other chiral molecules were used (2-diphenylphosphino-2′-hydroxy-1,1′-binaphthyl (HO-MOP), 2sulfinylphenols (SO), salicyloxazolines (Salox) or N-acetyl-tert-butanesulfinamide (ASA)), Meggers and co-workers were pioneers in using ʟ-proline as a cheap, simple and efficient strategy to prepare enantiomerically pure complexes. [START_REF] Fu | Proline as Chiral Auxiliary for the Economical Asymmetric Synthesis of Ruthenium(II) Polypyridyl Complexes[END_REF] The reaction of racemic [Ru(bpy)2Cl2] with ʟ-proline in ethylene glycol in the presence of K2CO3 at 190 ºC, yielded both Λ-10 and Δ-10 (Fig. 6) diastereomers in ≥ 20:1 ratio. Although both diastereomers are probably formed in the reaction, at high temperatures, only the Λ derivative is stable, whereas the Δ isomer might release the proline ligand. This turns the Λ isomer into the thermodynamically stable diastereomer. To check the scope of this reaction, the minor isomer (Δ-10) was heated in ethylene glycol at 190 ºC under argon. After 10 min, conversion to the major isomer Λ was observed (≥ 20:1). In addition, the replacement of ʟ-proline by a bipyridine (bpy) or phenanthroline (phen) in acetonitrile in acid conditions (trifluoroacetic acid, TFA) at 110 ºC resulted in the formation of the Λ isomer (99:1 and 98:2 for bpy and phen, respectively).

Ru

Nevertheless, it did not work that well with other Ru complexes. The exposition of the Λ isomer in different solvents (i.e., MeCN, DMF, MeOH) to visible light at room temperature resulted in the racemization of the complex in about 2 days. This rate increased by diminishing time to 10 h, when the evolution was performed at 50 ºC. In dark conditions, however, no racemization was observed. We note that this interesting methodology of asymmetric synthesis is not exclusive for ruthenium complexes and was found to be also successful, for example, with Ir(III) biscyclometalated complexes. [START_REF] Helms | Proline and α-Methylproline as Chiral Auxiliaries for the Synthesis of Enantiopure Bis-Cyclometalated Iridium(III) Complexes[END_REF] In addition, the conversion from Δ to Λ enantiomers A distinct application as a protecting ligand was found for ʟ-proline. Bonnet and co-workers.

in
reported sterically hindered complexes 10, 11a, 11b and 12 (Fig. 6), by systematically adding methyl groups on the bpy ligand(s) [from zero [START_REF] Bideh | Efficient near infrared light emitting electrochemical cell (NIR-LEEC) based on new binuclear ruthenium phenanthroimidazole exhibiting desired charge carrier dynamics[END_REF] to two (11a and 11b) and to four [START_REF] Liu | Harnessing ruthenium(II) as photodynamic agents: Encouraging advances in cancer therapy[END_REF]]. [START_REF] Cuello-Garibo | Influence of the Steric Bulk and Solvent on the Photoreactivity of Ruthenium Polypyridyl Complexes Coordinated to L-Proline[END_REF] All complexes were obtained as racemic mixtures, with the Λ as the major isomer, except for the most hindered 12, which was obtained as the single Λ-isomer. The photoreactivity of the complexes was studied under different conditions and solvents. Thus, 10 showed loss of hydrogen atoms in the proline ligand after irradiation with 493 nm light under air in PBS as seen by UV-vis and MS. The same experiment performed under argon did not display any change, except for the racemization of the complex. With these features, the authors proposed a mechanism of photooxidation and photoisomerization. The same experiments showed no photooxidation for 12. Photoconversion from 11a (kinetic product) to 11b (thermal product) was detected. Photooxidation, but in a slower rate, was also observed. The explanation dwells in the acidity of the NH of the proline and in the number of methyl substituents on the bpy ligands. An increment of the number of methyls causes an increase of the electron density on the ruthenium, as the methyl groups are electron donors, which decreases the acidity of the proline amine. When these experiments were performed in MeCN, non-selective photosubstitution of either the proline or the dmbpy (4,4'-dimethyl-2,2'-bipyridine) by solvent molecules was observed. Thus, this study highlights the value of the solvent in the photoreactivity of ruthenium polypyridyl complexes, especially when studying biological systems and some microenvironments that are more hydrophobic than H2O. The same authors also prepared the first light-activated trans ruthenium-based anticancer prodrug candidates. [START_REF] Van Rixel | Green light-induced apoptosis in cancer cells by a tetrapyridyl ruthenium prodrug offering two: Trans coordination sites[END_REF] The water-soluble complex 13 is based on a tetrapyridyl ligand (6,6′bis[N-(isoquinolyl)-1-amino]-2,2′-bipyridine, biqbpy), leaving two trans positions for the coordination of monodentate ligands, like N-acetyl-ʟ-methionine (Fig. 7). A parent compound was also prepared with the same tetradentate ligand and a chloride and a DMSO molecule in the trans positions. The stability studies of 13 in aqueous solution in the dark displayed the loss of the proton of the carboxylic acid. However, upon green light irradiation (λ = 520 nm), the replacement of one of the methionines by a water molecule occurred. The cytotoxicity was investigated in three cell lines A549 (human lung carcinoma cells), A431 (human epidermoid carcinoma cells) and MRC-5 (noncancerous human foetal lung fibroblasts). The EC50 values showed low toxicity in the dark, but an increase of the phototoxicity upon 60 min irradiation with green light (520 nm, 21 mW cm -2 , 75 J cm -2 ). For A549 cells, for instance, the EC50 decreased from 20 µM to 3.6 µM after 6 h of irradiation, and from 11 µM to 5 µM after 24 h. Furthermore, 13 was less toxic in the dark than its chloride-DMSO derivative. Apoptosis was found as the unique mechanism of cell death after irradiation by flow cytometry analysis. The cellular uptake (ICP-MS) after 6 h showed a lower uptake of 13 with regard to the parent compound. 13 was mainly accumulated in the membrane fraction (i.e., mitochondria, endosomes, lysosomes, etc). Since the singlet oxygen quantum yield was low (2.3 %), and the DNA binding studies revealed just small interactions between the ruthenium complex and DNA -1 O2-based DNA cleavage was not observed -the cell death mechanism is not triggered by a, but through PACT (photoactivated chemotherapy), and DNA represents only one of the possible targets.

Peptides

Peptides are simple models of proteins, and are commonly used as organelle-targeting agents (i.e., cell-penetrating peptides (CPPs), nuclear localization sequences (NLSs) or mitochondrial penetrating peptide (MPP)). [START_REF] Puckett | Exploring the cellular accumulation of metal complexes[END_REF] Furthermore, some cancer cells overexpress some peptide receptors contrary to healthy cells. Therefore, the conjugation of such peptides can help to target tumour cells. [START_REF] Gunnoo | Bioconjugation -using selective chemistry to enhance the properties of proteins and peptides as therapeutics and carriers[END_REF] Among the peptide conjugations used, the classic functionalizations involves the amides linkages on bipyridines, phenanthrolines, imidazo-phenanthrolines and benzimidazoles. Among the different imidazo-phenanthroline derivatives reported so far, Keyes and co-workers prepared a series of Ru(II) peptide conjugates, mainly in view of cell imaging agents directed to different cellular targets. Complexes 14 and 15, shown in Fig. 8, were prepared as first examples of water-soluble arginine labelled cell-penetrating probes. [START_REF] Neugebauer | Ruthenium polypyridyl peptide conjugates: membrane permeable probes for cellular imaging[END_REF][START_REF] Neugebauer | Membrane permeable luminescent metal complexes for cellular imaging[END_REF] These complexes were conjugated to 5 or 8 arginine (Arg, R) residues, respectively. The photophysical properties in PBS of both complexes exhibited no differences, since the arginine chain is far away from the ruthenium core. Two methods were tested for imaging the complexes in the same conditions: fluorescence lifetime imaging and resonance Raman mapping. The luminescence lifetime was oxygen sensitive and pH independent, whereas Raman spectroscopy was pHdependent but not oxygen-dependent. Thus, the authors probed the effectiveness of both methods to independently map two parameters. The cellular uptake was investigated in Sp2/0-Ag 14 myeloma cells and human blood platelets by confocal laser scanning microscopy (CLSM). Complex 15 (Ru-R8) was found to be passively transported through myeloma cells in about 12 min. In the first 2-3 min, the dye accumulates in the membrane and passively diffuses inside the cell. After 10 or 15 min, the process is finished and no more changes are observed.

In platelets, compound 15 showed intense luminescence in the cytoplasm, but complex 14 was not emissive, highlighting the relevance of the chain length. Myeloma cells treated with 15 and incubated for 48 h showed slight toxicity, with 30 % of cell death. In addition, unlike other organic chromophores, complex 15 showed photostability, a key future in cell imaging. As a result, two imaging methods (i.e., resonance Raman and FLIM) can be successfully used to image cells with the ruthenium probes, mainly with the R8 derivative.

A similar complex, 16 (Fig. 8), in which the bipyridyl ligands were replaced by dppz (dipyrido-[3,2-a:2',3'-c]-phenazine), was prepared as a cell membrane probe. [START_REF] Cosgrave | Multimodal cell imaging by ruthenium polypyridyl labelled cell penetrating peptides[END_REF] The dppz ligands make the complex behave as a "molecular light-switch". Moreover, the arginine coupling confers enhanced luminescence properties to 16 (in MeCN/DMSO 9:1), better than those of the corresponding complex without the arginine moiety. However, when increasing amounts of water were added to an acetonitrile solution, the emission got completely quenched at 18 % v/v water/acetonitrile. When the complex was suspended in lipid vesicles of dipalmitoylphosphatidylglycerol (dppg) in PBS at pH 7.4, a strongly switch on of the emission was observed. CLSM under the same conditions (in myeloma cells) as previously reported showed strong emission from the outer and internal membranes of organelles, specifically from endosomal vesicles, which are usually related to endocytosis as the mechanism of internalization. Resonance Raman mapping was recorded following some characteristic Raman bands associated to the complex inside the cell. This technique revealed that 16 crossed the membrane and distributed throughout the cell. This complex acts as a light-switch to stain membranes.

The peptide conjugates 17a and 17b (Fig. 8) were prepared as αIIbβ3 integrin binders. Integrins, which are over-expressed in some kind of tumour cells, have specificity for an RGD (Arg-Gly-Asp) motif binding site. [START_REF] Adamson | RGD Labeled Ru(II) Polypyridyl Conjugates for Platelet Integrin αIIbβ3 Recognition and as Reporters of Integrin Conformation[END_REF] Thus, these complexes have been labelled with this tripeptide. The binding affinity of the complexes towards BSA (bovine serum albumin, a nonspecific protein without RGD recognition site) showed only small increase of the emission intensity -meaning weak interaction -, whereas for αIIbβ3 integrin the increment was around an order of magnitude -suggesting strong interaction. The dissociation constants were in the micromolar range, with a single binding site for 17a (Kd = 0.09 ± 0.01 µM) and a two-step binding for 17b (first Kd = 0.25 ± 0.29 µM and second Kd = 4.37 ± 0.82 µM), being stronger for the former. The interaction of the Ru conjugates with the integrin was found to be through the RGD binding site, since a displacement experiment with Eptifibatide (an integrin inhibitor that specifically binds the RGD motif site with a Kd in the nanomolar range) was performed. The presence of the inhibitor inhibited the binding of the complexes to the integrin. The binding affinity was also checked in the presence of Mn 2+ and DTT (dithiohtreitol), which are able to alter the active-like conformations. The affinity of 17a towards the integrin treated with Mn 2+ or DTT was the same as the non-treated one, whereas for 17b, the binding affinity was much higher. Cellular uptake and localization (confocal imaging) of the conjugates in Chinese Hamster Ovary cells wild type (CHO-WT, not expressing αIIbβ3) and transfected CHO-αIIbβ3 (expressing αIIbβ3) revealed weak emission for the CHO-WT cells but strong staining for CHO-αIIbβ3 after 4 h incubation. This emission was localized at the cell surface, suggesting binding with the integrins located in the cell membrane. The same experiment with the addition of SZ22, an αIIb specific antibody, displayed stronger emission from the cell surface, confirming the co-localization in these cell membrane integrins. All these findings suggest the better ability of 17a to deeper penetrate in the integrin pocket, rather than 17b, due to the smaller size and higher hydrophilicity of the former.

Complexes 18a and 18b (Fig. 9), however, were prepared as targeted delivery dyes to direct them to the nucleus through the attachment of NLS (nuclear localization sequence)

peptides. [START_REF] Blackmore | Peptide directed transmembrane transport and nuclear localization of Ru(II) polypyridyl complexes in mammalian cells[END_REF] The NLS peptide is derived from the transcription factor NF-κB, a transmembrane molecular carrier. In this case, the cellular uptake by CLSM after 16 h incubation in CHO cells showed fast penetration of both conjugates in the cell and also in the nucleus in less than 5 min (co-stained with DAPI, a nuclear dye). Furthermore, 18b was also distributed in the cytoplasm and the nucleolus. Both complexes exhibited nearly complete cytotoxicity at 144 µM in the same cell line after 16 h. Nonetheless, the parent complexes (without the peptide) did not show any toxicity and therefore, the toxicity of the Ru conjugates is not due to interaction with the nuclear material. Thus, the attachment of this peptide allowed to successfully target the nucleus. Keyes and co-workers also prepared the phenanthroline-peptide conjugates. Complexes 19, 20

and 21 (Fig. 10) were prepared as the octaarginine conjugate, endoplasmic reticulum (ER)

targeting peptide and NLS derivatives, respectively, to direct the dyes to the ER and nucleus. [START_REF] Byrne | Precision targeted ruthenium(II) luminophores; highly effective probes for cell imaging by stimulated emission depletion (STED) microscopy[END_REF] Compound 21 behaves as a molecular light-switch due to the dppz ligand. The cellular uptake (CLSM) and co-localization studies were assessed in HeLa cells (human cervical epitheloid carcinoma). For 20, the ER-directed complex, the uptake was fast. After 2 h, the complex was distributed exclusively in the cytoplasm, and after 4 h, localized in the ER.

The polyarginine derivative 19 was also taken up quickly, but located in the cytoplasm, without specificity over time. The NLS conjugate 21 exhibited emission from the membrane after less than 6 h and, after 24 h the complex had crossed the nuclear membrane and bound to DNA.

The images are shown in Fig. 11. STED (stimulated emission depletion) imaging was also performed. This technique allowed to distinguish the tubules in the ER using 20 and the different cell phases through the chromosomes in the cell with 21. The photostability of the ERtargeting complex was found to be comparable to AlexaFluor 532, a well-known ER dye. This complex was not toxic at 100 µM after 24 h incubation, whereas for 19, no cell viability was detected at the same conditions. The NLS derivative showed partial toxicity, with 55 % of cell viability at 200 µM. However, at the working concentration (50-70 µM), viability increased to 73 %, enough so as not to induce excessive cell death. Thus, these complexes are promising candidates as cell imaging dyes for ER and nucleus. nm white light laser and the emission was collected between 590 and 700 nm. 21 was excited using a 470 nm white light laser and the emission was collected between 565 and 700 nm. to nuclear DNA. Cell viability assays unveiled some toxicity of 21 at 50 µM (after 24 h, 50 % cell viability), but acceptable viability at 10 µM (after 24 h, 80 % cell viability). After 15 min irradiation (470 nm, 0.8 µW), cell death was observed. These findings demonstrate the ability of 22 to act as a theranostic probe.

The dinuclear complex 23 (Fig. 10), bearing the same MPP sequence as 22, exhibited quick cellular uptake in HeLa and CHO cells when incubated at 37 ºC. [START_REF] Martin | Peptide-Bridged Dinuclear Ru(II) Complex for Mitochondrial Targeted Monitoring of Dynamic Changes to Oxygen Concentration and ROS Generation in Live Mammalian Cells[END_REF] Initially, the complex was detected in the cytoplasm, which was diffusing towards mitochondria, so that the internalization was complete after 2 h. Nonetheless, when incubated at 4 ºC, the complex was unable to cross the cell membrane. As shown in Fig. 12, incubation of complex 23 with MitoTracker Deep Red showed strong co-localization. Through colour distribution chart, the intracellular O2 concentration was determined to be ≈ 183 µM, which is a common value for normally metabolizing cells. Incubation of the complex with Antimycin A, a classical mitochondrial uncoupler, showed quenching of emission luminescence probably caused by ROS (reactive oxygen species). This dinuclear complex displayed low cell viability (15 %) at 200 µM after 24 h in HeLa cells (IC50 = 47 ± 1.1 µM). A time-dependent experiment at 75 µM showed 40 % viability after 5 h. However, after 2 h, cells were viable. Phototoxicity was induced when the cells (after 2 h incubation with 75 µM of the complex) were irradiated (488 nm, 20 min, 0.64 µW/cm -3 ). Again, the complex was found to be a good mitochondria imaging agent, thanks to the right selection of the directed peptides. Recently, Chen and co-workers reported a Ru-cRGD (cyclic RGD) conjugate 24 (Fig. 13) with theranostic activity against cervical carcinoma. [START_REF] Zhao | Designing luminescent ruthenium prodrug for precise cancer therapy and rapid clinical diagnosis[END_REF] The ruthenium conjugate was found to preferentially accumulate in malignant cells (CaSki: human caucasian cervical epidermoid carcinoma, SiHa: human squamous carcinoma of the cervix and HeLa: human cervical epitheloid carcinoma) over non-cancerous cells (Ect1/E6E7: normal ectocervical cells), even when CaSki were co-cultured with Ect1/E6E7. CaSki Multicellular tumour spheroids (MCTSs) were used as 3D model. After 3 h incubation, the conjugate 24 displayed strong two-photon luminescence and was able to suppress the spheroid growth by 60 %. The metabolic in vivo experiments in mice (intravenous injection for 36 h) showed preferential internalization of 24 in tumour tissues rather than in other organs. The in vivo antitumour activity in mice inoculated with CaSki cells displayed inhibition rates of 58 % and 74 % at intravenous injection doses of 2 µmol/kg and 4 µmol/kg, respectively. In addition, no death or change in the body weight of the mice were observed and no side effects were detected. Staining tissues with 24 also showed preferential accumulation in tumour tissues, with a Pearson's correlation coefficient of 0.85.

Thus, this Ru-bioconjugate is a good candidate as a theranostic probe. Gasser and co-workers reported a series of Ru(II) polypyridyl complex-peptide conjugates 25-28 (Fig. 14), bearing two bipyridine (bpy), phenanthroline (phen) or dipyridylamine (hdpa) ligands, to modulate the lipophilicity of the complexes. [START_REF] Mari | Towards Selective Light-Activated Ru II -Based Prodrug Candidates[END_REF] The peptides were selected to improve the cellular uptake of the Ru(II) complexes. There were a NLS to target the nucleus and bombesin (BBN) to target a membrane receptor overexpressed in certain cancer cells.

Moreover, a photolabile protecting group (PLPG or photo-cage), an o-nitrobenzyl moiety, was chosen as a spacer and to control drug activation by light to give the Ru-PLPG-peptide bioconjugate. Complexes without the photo-cage (25 and 27) were prepared as controls. Upon light irradiation (350 nm) of the bioconjugates 25-28 in PBS solution, a full photo-release of the metal complexes was achieved in 6 to 10 min (light dose: 1.55 -2.58 J cm -2 ). The efficiency in photo-uncaging is similar for both the NLS and BBN series: serie-c > serie-a > serie-b. This means that there is no relevant effect of the peptide on the photo-release. The correlation of uncaging efficiency with the extinction coefficient showed that different energy is needed for full release, being higher for serie-b than for serie-c. The cytotoxicity of the complexes was investigated in HeLa and MRC-5 cells after 4 h incubation, in the dark or after 10 min irradiation at 350 nm. The cellular uptake was assessed after 2 h incubation in HeLa cells. The NLS conjugates 26a and 26b showed cellular internalization and localization in the nucleus, whereas localization for the control derivatives 25a and 25b were detected in the cytoplasm.

Both 25c and 26c were not emissive and therefore were not detected. 26a was not toxic in the dark up to 100 µM, whereas 26b and 26c displayed some toxicity more pronounced in HeLa than in MRC-5 cells. The irradiation of the complexes did not show any improvement of the activity. All the NLS controls (25a, 25b and 25c) were non-toxic on both cell lines in the dark and after irradiation. Regarding the BBN conjugates, 28a showed nuclear localization, while 28b was spread in the cytoplasm. 28c was detected in cells and located weakly in the cytoplasm and strongly in the membrane. 28a exhibited moderate toxicity both with and without light irradiation (IC50 ≈ 50 µM). 28b and 28c showed phototoxicity; IC50 of 28b in the dark was 93.8 µM, and diminished by half upon irradiation, obtaining a PI = 2.2. 28c was non-toxic in the dark (IC50 > 100 µM), but slightly toxic after irradiation (IC50 = 60 µM). Thus, the PI > 1.7.

None of these complexes showed toxicity on the MRC-5 cells. Controls 27a, 27b and 27c

showed weak emission and only 27b was found to localize in the cytoplasm and none of them were found to be toxic. These studies highlight the relevance of the PLPG for the photorelease of the peptides and to achieve the desire toxicity of the conjugates. In a previous study, the same group reported some bombesin, NLS and MLS (mitochondrial localization signal) conjugates Weil and co-workers prepared the first ruthenium somatostatine (SST) conjugate 29 (Fig. 15). [START_REF] Wang | Receptor selective ruthenium-somatostatin photosensitizer for cancer targeted photodynamic applications[END_REF] SST was selected since some somatostatine receptors (SSTRs) are overexpressed in some tumour cells. This photostable conjugate under visible light (λ = 470 nm, P = 50 ± 3 mW, 50 ± 3 mW cm -2 ) was also an efficient 1 O2 generator (> 70 %). Complex 29 was tested in A549 cancer cells, which overexpress SSTRs. After 4h incubation, 29 successfully accumulated inside cells, with a higher uptake compared to the control experiment (the Ru(II) derivative without the SST moiety). The phototoxic activity of 29 in A549 cells revealed non-toxicity up to 300 µM in the dark and IC50 = 13.2 µM after light irradiation (λ = 470 nm, 5 min, 6.9 ± 0.9 J cm -2 ), resulting in a PI > 23. Very recently, the same compound was tested in acute myeloid leukaemia (AML) with promising results. [START_REF] Vegi | Somatostatin receptor mediated targeting of acute myeloid leukemia by photodynamic metal complexes for light induced apoptosis[END_REF] The uptake of bioconjugate 29 was finished in 4 h in OA3 cells. Clonogenic assays after incubation with 29 in different leukaemic cell lines revealed reduction of colony growth of 99 %, 98 % and 92 % for OCI-AML3, HL60 and THP1, respectively, after 6 min light exposure and only 25 % in healthy cord blood cells (CB). This fact emphasizes the selectivity of 29 for AML cells over normal cells. Co-localization experiments in OCI-AML3 spotted 29 mainly in the lysosomes (Pearson's coefficient 0.94), with some detection also in membranes (0.70) and mitochondria (0.60). However, it was poorly taken up by the nucleous (0.35). In addition, ROS production experiments showed 7.4-fold increase in ROS levels in leukaemic cells rather than CB cells. These new findings demonstrate the effective conjugation of SST to a ruthenium-based PDT PS. So far, octaarginine conjugates enters the cell but their distribution is in the cytoplasm, without targeting specific organelles. Puckett and Barton found a way to direct these complexes to the nucleus by attaching a fluorescein moiety to the octaarginine. [START_REF] Puckett | Fluorescein Redirects a Ruthenium-Octaarginine Conjugate to the Nucleus[END_REF] Complexes 30 (Ru-ᴅoctaarginine), 31 (Ru-ᴅ-octaarginine-fluorescein) and 32 (Ru-fluorescein) were synthesised (Fig. 16) and imaged in HeLa cells after 30 min incubation. Complex 30 (5 µM) was found to be distributed, as expected, as intense blebs in the cytoplasm, with exclusion from the nucleus.

An endocytosis mechanism of internalization was unveiled. Conjugate 31 entered the nucleus under the same incubation conditions. It displayed diffuse emission in both the cytoplasm and the nucleus and strong luminescence in the nucleoli and blebs in the cytoplasm. Compound 32, however, did not enter the cells after 30 min, and was poorly internalized even after longer incubation times and a higher concentration (20 µM for 41 h). At 10 µM, 30 showed exclusively blebs in the cytoplasm and, at 15-20 µM, there were mixed stains. For 31, nucleolar and blebs in the cytoplasm were of same intensity and weaker emission was found in the cytoplasm and nucleus. These findings suggest that above 5 µM, the mechanism is non-endocytotic, whereas at lower concentrations, it is endocytotic. The role of the fluorescein moiety is to increase the lipophilicity of the conjugates, enhancing their ability to cross the cell membrane.

When the peptide was shortened in derivatives 33 and 34 (Fig. 16), the uptake of the conjugates in HeLa cells after 2 or 24 h incubation was changed. 33 (5-20 µM) showed non-nuclear staining and only emission in the cytoplasm. [START_REF] Puckett | Targeting a ruthenium complex to the nucleus with short peptides[END_REF] At higher concentrations (30-40 µM), the distribution was heterogeneous and, at 100 µM, 74 % of cells displayed nuclear localization.

Unlike 30, conjugate 33 presented less effective cellular internalization due to the short oligoarginine chain (4 units instead of 8) and needed higher concentrations to accumulate in the nucleus. As for 34, no stain was found in the cells and, at 30 µM, only some emissive blebs were detected in the cytoplasm. The reason could be the length of the peptide and the carboxylic acid terminal group in the fluorescein conjugate, increasing the charge of the compound, whereas, in the non-fluorescein derivative, there is an amide terminal group. A tetraarginine derivative 35 (Fig. 17) was also prepared, with similar uptake as for 33. Since oligoarginines show better cellular uptake than oligolysines, and this might be consequence of the global charge, some complexes were prepared with other peptide sequences, varying the number of arginines. Conjugates 36, with two arginines (less total charge) and 37, bearing lysines (Fig. 17), showed luminescence from the cytoplasm at 40 µM after 2 h. At 100 µM and 4 h, the internalization improved, although still weakly, with higher number of cells with nuclear localization. Emission intensity in cells was higher for 37 than for 36, as a consequence again of the charge. Longer peptide chains (NLS) were attached, forming complexes 38 and 39, respectively (Fig. 17). The overall charge of these complexes was reduced, if compared with the octaarginine derivative. In both cases, the uptake at 10 µM after 2 h was weak, showing exclusively faint blebs without nuclear localization. As a result, none of these strategies achieved an improvement of the uptake over the octaarginine derivatives. Examples of bimetallic complexes with terpyridine bearing different peptide chains (40a-42b)

were reported by Ypsilantis et al. (Fig. 18). [START_REF] Ypsilantis | Stepwise synthesis, characterization, DNA binding properties and cytotoxicity of diruthenium oligopyridine compounds conjugated with peptides[END_REF] They assayed the interactions of complexes 40a and 40b with an oligonucleotide d(5'-CGCGAATTCGCG-3')2. 1 H-NMR studies (298 K,

[complex]:nucleotide 1:1) revealed fast kinetics binding of 40b in the middle part of the sequence, suggesting an orientation of the ptrpy (4′-phenyl-2,2';6',2"-terpyridine) ligand towards the minor groove. However, 40a did not show any specific binding, as the peaks slightly shifted. Cytotoxicity of 40a and 40b was investigated in human lung carcinoma cell lines H1299 and H1437. None of the complexes were found to be cytotoxic in any of the cell lines after 24 h (IC50 > 750 µM), despite the interaction with the oligonucleotide, suggesting a different target than DNA.

Peptoids

Peptoids were firstly explored in the early 90s. They consist on N-alkylated glycine oligomers that can form and mimic secondary structures such as α-helix or β-turns. Furthermore, they are better cellular transporters than the classic peptides due to their increased hydrophobicity and cell permeability. Therefore, they are promising drug candidates. Among the bipyridine-peptoid conjugates, Lee et al. prepared the mono-and bi-metallic complexes 43, 44 and 45 (Fig. 19) as CALI (chromophore-assisted light inactivation)

reagents. [START_REF] Lee | Potent and selective photoinactivation of proteins with peptoid-ruthenium conjugates[END_REF] Peptoid GU40C, a highly selective antagonist of vascular endothelial growth factor (VEGF)-induced activation of the VEGF receptor 2 (VEGFR2) was used in these conjugates. Thus, the activity of 43 was investigated in endothelial cells exposed to VEGF and the activation of VEGFR2 was monitored. Without irradiation, the conjugate was not able to inhibit VEGFR2. Nevertheless, after irradiation (> 380 nm, 10 min), the inhibition was successful. The IC50 of 43 was 49 µM in the dark and 59 nM upon irradiation (> 800-fold increase). Complex 43 was also found to inhibit the formation of vessel-like tube structures in endothelial cells, also when irradiated (IC50 = 50 nM). 43 was also incubated in H441 cells, which express epidermal growth factor receptor (EGFR) and VEGFR2. Cell viability remained unaffected and VEGFR2 was reduced whereas EGFR was not. This highlights the specificity towards VEGFR2. Regarding 44, the complex contains the peptoid GU40C4, where two GU40C units are joined by a linker. 44 was found to bind the VEGFR2 extracellular domain.

Inhibition assays showed 50% inhibition in the dark at 500 nM, similar to that of GU40C4.

Upon irradiation, however, this IC50 decreased to 590 pM, a 1,700-fold increase in potency due to the Ru(II)-peptoid singlet oxygen generation. Compound 45 was prepared as the peptoid RIP1 (which targets an ATPase) conjugate. This derivative was irradiated in HeLa cells and inhibition of peptidolysis was observed. The potency increased after 10 min and 20 min irradiation, from IC50 = 300 nM to 85 nM, respectively, suggesting a dependence of singlet oxygen generation. A dose-dependent assay showed peptidolysis after 30 min irradiation, but no effect was detected in the dark. Comparing both 43 and 45, the binding of the latter was weaker, probably owing to the indole side chain and the adenine molecule.

Proteins

Many articles have reported the binding ability of Ru(II) polypyridyl complexes with proteins (e.g., determination of their binding or dissociation constants). Some of these compounds have been used as probes to determine the hydrodynamics of proteins. [START_REF] Szmacinski | Synthesis and evaluation of Rucomplexes as anisotropy probes for protein hydrodynamics and immunoassays of highmolecular-weight antigens[END_REF][START_REF] Szmacinski | Long-lifetime Ru(II) complexes for the measurement of high molecular weight protein hydrodynamics[END_REF] However, not many articles have reported on the synthesis and isolation of the conjugates. In the 90s, Winkler and Gray studied and gathered the ET mechanisms in aminoruthenium complexes covalently attached to the histidine moieties of some proteins. As for the fluorescence immunoassay, the histidine-tagged recombinant protein (the most common recombinant protein) was immobilized. Afterwards, the antibody (anti-6X His tag)

was added to bind the histidine-tagged protein. Finally, the Ru-containing conjugate was added to bind to the antibody as a universal signal reporter (Fig. 20C). Again, the luminescence intensity showed an 8-fold increment when compared with the negative control, showing its successful ability to be used as a probe for immunoassays. Thordarson and co-workers studied the photoreduction of a series of Ru(II) terpyridine cytochrome c (cyt c) bioconjugates. The complexes were conjugated to cyt c through a Michael addition involving a maleimide with two different spacers: one with a long, flexible alkyl chain to yield 47-LC and another one with a short, rigid aromatic chain to give 47-SC (Fig. 21). [START_REF] Peterson | Photoinduced reduction of catalytically and biologically active Ru(II)bisterpyridine-cytochrome c bioconjugates[END_REF] The maleimide function was selected to selectively attach the single free cysteine (Cys102) of the protein. These spacers did not lead to significant modifications of the photophysical characterization of the complexes. In order to evaluate the catalytic and biological activity of the conjugates, the oxidation of 2,2'-azino-bis[ethyl-benzothiazoline-( 6)-sulfonic acid (ABTS) and cytochrome c oxidase (CCOx) was assessed for 47-LC and 47-SC, respectively. This assay demonstrated that none of the bioconjugates modified the structure or activity of the protein.

The energy/electron transfer rates, measured by time-resolved emission experiments, were 2.78 × 10 5 s -1 and 5.95 × 10 5 s -1 for 47-LC and 47-SC, respectively. UV-vis and TA (transient absorption) measurements allowed to determine the mechanism as an electron transfer pathway.

When comparing the UV-vis spectra of both derivatives, before and after irradiation (480 nm), the changes in the spectra showed the reduction of the haem group from Fe 3+ to Fe 2+ (initially, cyt c is oxidized). These changes also suggested photoinduced electron transfer from the ruthenium moiety as the donor, to cyt c as the acceptor. In addition, TA emission intensity of 47-SC in the reduced form (Fe 2+ ) was higher than that of the initially oxidized form (Fe 3+ ).

Although the photoreduction mechanism is unknown, the authors pointed out glycerol as the ultimate electron source.

The same authors prepared a similar conjugate 48 (Fig. 21) to understand the photoinduced electron transfer (eT) mechanism. [START_REF] Hvasanov | Optimising the synthesis, polymer membrane encapsulation and photoreduction performance of Ru(II)-and Ir(III)-bis(terpyridine) cytochrome c bioconjugates[END_REF] In this case, yeast iso-1 cytochrome c, an electron carrier protein that is light-activatable at room temperature, was used. These studies were performed in mixtures of the parent complex (i.e., the Ru(II) complex with the maleimide moiety, but no protein) and iso-1 cyt c in PBS solutions or encapsulated in a PS140-b-PAA48 (polystyrene140b-poly(acrylic acid)48) membrane. Low concentrations were used to prevent intramolecular electron transfer. In the encapsulated complex, the haem reduction was completed in 50 min.

The quantum efficiency (electrons/absorbed photons) in the encapsulated solution was found to be twice that of 48 (1.1 × 10 -3 %). Thus, the encapsulation increased the eT rate. The effect of the covalent binding ability was determined in the same conditions. When comparing the reduction curve of the bioconjugate with that of the parent compound and cyt c, a dramatic decreased was observed for the latter. This highlights that the link to the protein is crucial to ensure the activation. As in the previous complexes 47, the ruthenium moiety behaves as the donor and cyt c as the acceptor. Nevertheless, it was noticed that encapsulation increased the photoreduction process. Moreover, the addition of EDTA as a sacrificial electron donor was essential to provide the complex with extra electrons for the eT mechanism, since the photoreduction of 48 in PBS was negligible. Larger proteins have been also used. α/β-tubulin is the responsible of microtubules (MTs) formation, a kind of cytoskeletal filaments in cells. Osada et al. prepared a Ru-tubulin conjugate 49 to self-assemble into MTs (Fig. 22). [START_REF] Okeyoshi | Thermo-and photo-enhanced microtubule formation from Ru(bpy)3 2+ -conjugated tubulin[END_REF] To check the capability of self-assembly of the conjugate, a solution of 49 along with free tubulin was heated from 3 to 25 ºC and the changes were tracked by UV-vis spectroscopy. Free tubulin did not show any change, implying that any MT was formed. However, the absorption increased when 49 was added. The self-assembly finished in just 10 min, when the Ru-tubulin/tubulin ratio was over 0.5. When the temperature was decreased from 25 to 3 ºC, a depolymerization reaction was observed, indicating a reversible process. The self-assembly only occurred when a minimum ratio of 0.2 was achieved.

The kinetics was analysed at 0.22 ratio and temperatures from 15 to 37 ºC. The rate of assembly increased with the rise of the temperature, whereas the increment was low for the free tubulin.

The rate increased by 30 times at 37 ºC and the activation energy of MTs formation was 15 times higher than for the non-conjugated tubulin. Photoirradiation of 49 also increased the formation of MTs at 25 and 30 ºC. Thus, this conjugate can behave as a photothermal energy sensitizer. This effect was also observed by cross-polarized light at 29 ºC. The assembly of 49 was enhanced by photo-irradiation, and the process was observed to start from the wall to the central axis of the tubule. Furthermore, this conjugate was capable of inducing parallel orientation of the MTs. The length of MT was found to be 20-50 µm, as measured by fluorescence microscopy. Thus, the conjugate 49 was able to enhance the self-assembly by using both thermal energy and photoenergy, despite the positive charges and the steric hindrance of the metal complex. This curious thermal behaviour is the consequence of the release of non-radiative energy (i.e. heat) from the excited state of the Ru(II). Adapted from reference [START_REF] Chakrabortty | Mitochondria Targeted Protein-Ruthenium Photosensitizer for Efficient Photodynamic Applications[END_REF]. Copyright © 2017 American Chemical Society.

Weil et al. used HSA as a backbone to attach different Ru polypyridyl complexes with the aim to specifically target organelles in view of PDT applications. [START_REF] Chakrabortty | Mitochondria Targeted Protein-Ruthenium Photosensitizer for Efficient Photodynamic Applications[END_REF] HSA was modified with 20 PEO (poly(ethylene oxide)) units to make it water soluble, 34 TPP (triphenylphosphonium) units to target mitochondria and 10 Ru(II) polypyridyl complexes to act as PDT PSs (Fig. 23).

The resulting conjugate 50 was found to be stable and water-soluble. In addition, the photostability of the conjugate was higher than the one of the free Ru(II) complex, even after long irradiation times (65 h). The indirect method (9,10-anthracenediyl-bi(methylene)dimalonic acid (ABDA)) for singlet oxygen production (470 nm, 20 mW/cm 2 , 5 min) revealed efficient generation of 1 O2 for 50, whereas the modified HSA without a Ru(II) complex remained inactive. The emission quantum yield and the lifetime slightly improved when the solution was changed from water (Φ = 6.1 %, τ = 625 ns) to a simulated body fluid (Φ = 6.7 %, τ = 735 ns). The cellular uptake of 50 was imaged in HeLa cells (500 nM, 4h) and colocalization with different stains (i.e., membrane, nucleus, mitochondria and lysosomes) revealed internalization mostly in the mitochondria. The cytotoxicity in HeLa cells after 240 min incubation in the dark (IC50 = 9 µM) and upon light irradiation (470 nm, 20 mW/cm 2 ) (34.9 nM) was measured. An impressive PI of 250 was determined. The phototoxicity was also assessed in other cell lines: CHO (Chinese Hamster Ovary cells), MCF-7 (human breast adenocarcinoma) and A549 (human lung carcinoma cells). All of them showed low IC50 values after light irradiation; IC50 = 135.2 nM for CHO, IC50 = 114.3 nM for MCF-7 and IC50 = 119.1 nM for A549. Clonogenics in acute myeloid leukaemia (AML) OCI-AML3 cells treated with 50 after 2 and 5 min irradiation showed reduction of colony growth (44 % and 84.4 %, respectively) with regard to the non-irradiated cells. Also, remaining colonies were smaller in size. This assay was also performed in healthy BM cells (primary bone marrow) and murine AML cells (AE9a, leukaemia cells). After 2 and 5 min irradiation, the decrease was 37 % and 88 %, respectively, in AE9a cells, whereas the reduction was 10 % and 28 % in healthy BM cells, revealing specificity towards leukaemia cells. Two-photon absorption (TPA) experiments showed the great ability of the conjugate to act as a TP probe, with a TP cross-section 5-times higher than the one of the free Ru(II) complex.

Artificial Metalloproteins

Some proteins include a cofactor, which can be a metal ion, a cluster or a small organic molecule. When the proteins are bound to a metal ion, they are called metalloproteins. A great example of naturally occurring metalloproteins is myoglobin (Mb). [START_REF] Finkelstein | Metalloproteins[END_REF] The similarities between iron and ruthenium instigated the researchers to replace them in the haem group. In In 2006, Ishida et al. reported the synthesis and complete characterization of the first artificial metalloprotein bearing a Ru(II) ion as the metallic core. [START_REF] Ishida | Artificial Metalloproteins with a Ruthenium Tris(bipyridyl) Complex as the Core[END_REF] This artificial metalloprotein, 51 (Fig. 22), was designed with the insertion of unnatural amino acid 5'-amino-2,2'-bypyridine-5carboxylic acid (5Bpy) fragments in the peptide chain, so that the 5Bpy units can fold to accommodate a Ru 2+ atom and lead to the formation of a Ru(bpy)3 2+ -type complex, as shown in Fig. 22. Thus, two -ʟ-Pro-Gly-were introduced between two 5Bpys. ʟ-Ala and ʟ-Phe were placed at the N and C terminal of 5Bpy and the sequence was completed with glycines. The authors precisely characterized the metalloprotein and found similar spectroscopic properties as for Ru(bpy)3 2+ -type complexes. Furthermore, circular dichroism (CD) allowed them to determine the optical isomers for the different fractions obtained in the synthesis. Two metallopeptides, also incorporating bpy in their peptide sequence, were later reported by Gamba et al. [START_REF] Gamba | Custom-Fit Ruthenium(II) Metallopeptides: A New Twist to DNA Binding With Coordination Compounds[END_REF] These dinuclear complexes were prepared as optically-pure isomers and bear similar peptide sequences: Ac-βAla-bpy-βAla-bpy-NH2 for ΛΛ-52 and ΔΔ-52, and Ac-βAlabpy-Gly-βAla-bpy-NH2 for ΛΛ-53 and ΔΔ-53 (Fig. 25). DNA binding was evaluated using ethidium bromide displacement studies. Emission-quenching experiments revealed interaction of the four complexes with DNA. However, higher affinity (i.e., low dissociation constants) was observed for ΛΛ-52 and ΛΛ-53 than for the ΔΔ-enantiomers. Moreover, the affinity was higher for A/T-rich oligonucleotides than for G/C-rich sequences. Curiously, the trends of dissociation constants were different for the 52 and 53 series. A negative effect was detected for ΛΛ-53 compared with ΛΛ-52 (Kd[AAATTT] ≈ 8.2 and 5.8 µM, respectively), whereas the opposite effect was observed for ΔΔ-53 and ΔΔ-52 (Kd[AAATTT] ≈ 31 and 14 µM, respectively). CD and STD (saturation transfer difference) NMR experiments confirmed the previous results. Since these conjugates were not internalized in cells, a derivative with octaarginine 54 was synthesised as the diasteromeric mixture since the synthesis of the optically pure complexes was unsuccessful. Compound 54 displayed the same features than the other complexes and the preference for A/T-rich oligonucleotides was slightly increased. AFM studies of ΔΔ-52 and 54 with DNA showed alteration of the DNA plasmid, with higher affinity for 54. Cellular uptake studies with 54 (25 µM for 30 min) in Vero cells (kidney epithelial cells) confirmed internalization and showed blebs in the cytoplasm and diffuse emission from the nuclei. The cytotoxic activity was evaluated in different cells lines for ΔΔ-52 and 54. In A2780cis (ovarian carcinoma cells), 54 was more toxic than ΔΔ-52 (IC50 ≈ 7 µM vs 286 µM).

Similar results were obtained in MCF-7 (breast cancer cells) and NCI-H460 (lung carcinoma), in which only the octaarginine derivative showed cytotoxicity.

Antibodies

The main strategies to conjugate organic compounds with antibodies or other heavy proteins imply maleimide and NHS (N-hydoxysuccinimide) moieties. Maleimides can easily react with thiol groups from cysteines. This is of interest since the number of sulfhydryl groups in a protein is reduced, in comparison with the number of amino groups. For instance, immunoglobulin G1 (IgG1) has 8 thiol groups from cysteines versus 30 available amino groups from lysines. [START_REF] Jain | Current ADC Linker Chemistry[END_REF] Interestingly, cysteine residues are usually found in the specific binding sites of the protein, making this conjugation more selective. [START_REF] Terpetschnig | Synthesis and Spectral Characterization of a Thiol-Reactive Long-Lifetime Ru(II) Complex[END_REF] NHS moieties, however, react with lysines. Zhou et al. described a detailed protocol to prepare Ru-Ab bioconjugates through NHS linkage. [START_REF] Zhou | Synthesis, labeling and bioanalytical applications of a tris(2,2′-bipyridyl)ruthenium(II)-based electrochemiluminescence probe[END_REF] The conjugation of Ab with metal complexes could improve their applications as chemotherapeutic agents, [START_REF] Del Solar | Metal-based antibody drug conjugates. Potential and challenges in their application as targeted therapies in cancer[END_REF] theranostics, [START_REF] Aghevlian | Panitumumab Modified with Metal-Chelating Polymers (MCP) Complexed to 111 In and 177 Lu-An EGFR-Targeted Theranostic for Pancreatic Cancer[END_REF] or probes. [START_REF] Mortensen | Imidazole carbamate probes for affinity guided azide-transfer to metal-binding proteins[END_REF] Thus, the search for novel metalbased antibody-drug conjugates (ADC) has increased in the last years. Terpetschnig et al. prepared a Ru(II) polypyridyl maleimide probe to detect proteins (HSA, human serum albumin), enzymes (β-galactosidase) and antibodies (IgG, immunoglobulin G). [START_REF] Terpetschnig | Synthesis and Spectral Characterization of a Thiol-Reactive Long-Lifetime Ru(II) Complex[END_REF] The reaction of this complex [Ru(bpy)2(phen-5-maleimide)](PF6)2 with the corresponding proteins was performed at pH 6.5 to avoid side reactions with the amino groups of other amino acids. The interest of these probes lies in the long-lived emission decays (over 1 µs). Complex 55 (Fig. 26) showed an effect of oxygen quenching (45 %) in the analyses by intensity decays in phosphate buffer. These quenching detections allowed the authors to measure the oxygen quenching constant (kq). For an efficient quencher, this constant should approximately be 1 × 10 10 M -1 s -1 . The value found for 55 was 2.93 × 10 9 M -1 s -1 , which corresponds to a 96 % quenching, when compared with the parent maleimide compound.

Maleimide: cysteine linkage

Anisotropy decays (fluorescence polarization) were also measured. The Ru-antibody conjugate displayed short rotational correlation time (15 -30 ns). Both results might indicate that the cys residues are mainly exposed to the buffer. Very recently, Gasser, Zarschler and co-workers reported on the synthesis of a Ru-nanobody conjugate as a PS for PDT. [START_REF] Karges | Synthesis and Characterization of an Epidermal Growth Factor Receptor-Selective RuII Polypyridyl-Nanobody Conjugate as a Photosensitizer for Photodynamic Therapy[END_REF] Nanobodies (NBs) are small fragments of an antibody (Ab), which represent the antigen-binding domain of heavy-chain-only Abs. The authors selected the 7C12NB, which specifically binds the epidermal growth factor receptor (EGFR), overexpressed in some types of solid tumours. Thus, complex 56 (Fig. 27) is composed of three building blocks: the [Ru(phen)2(dppz))] 2+ complex, the 7C12NB and a peptide chain containing polyglycines. The photophysical properties of the bioconjugate 56 were similar to those of similar Ru(II) polypyridyl complexes. Singlet oxygen production was determined since the A temperature change (incubation for 1 h at 4 ºC and then 1 h at 37 ºC) was proposed since EFGR internalization is an energy-dependent mechanism, and thus, at 4 ºC, the endocytosis process is supressed. Nevertheless, the complex was non-toxic (IC50 > 35 µM). Moreover, no ROS production was found in A431 cells.

NHS: lysine linkage

Sugars/carbohydrates

The coordination of sugars to metal ions aims to enhance the solubility in biological tissues (i.e., water) and thus, increase the biocompatibility of the metal complexes. There are two main strategies to prepare metal glycoconjugates. [START_REF] Gottschaldt | Prospects of Metal Complexes Peripherally Substituted with Sugars in Biomedicinal Applications[END_REF] The first one consists on the direct attachment of the metal to the carbohydrate. This strategy was widely used with Pd, Pt, Cu, Fe, Mo or W. [START_REF] Allscher | Carbohydrate-Metal ComplexesCarbohydrate-metal complexes : Structural Chemistry of Stable Solution Species[END_REF] The second methodology introduces the sugar in the periphery and mainly impacts on the solubility of the resulting conjugate. Regarding Ru(II) polypyridyl complexes, the latter is more appropriate.

As for the uptake, sugars are involved in the cellular internalization based on two classes of transporters: GLUT (glucose transporters) proteins mediate sugar transport in an energyindependent process (passive), whereas the SGLT (solute carriers) use the Na + /K + /ATP pumps in an active mechanism. Larger molecules with more than one sugar residue require an endocytotic or energy-dependent internalization mechanism through multiple steps to reach the cytoplasm. [START_REF] Gottschaldt | Prospects of Metal Complexes Peripherally Substituted with Sugars in Biomedicinal Applications[END_REF] These transporters are key in cancer therapy since they are overexpressed in some kind of tumours. [START_REF] Calvo | Potential Role of Sugar Transporters in Cancer and Their Relationship with Anticancer Therapy[END_REF]. In addition, there are some special receptor proteins, called lectins, which recognize and reversibly bind to carbohydrates. They play important roles in different biological activities such as cell aggregation, cell signalling or pathogen recognition (inhibition of bacterial and fungal growth). [START_REF] Gottschaldt | Prospects of Metal Complexes Peripherally Substituted with Sugars in Biomedicinal Applications[END_REF][START_REF] Santos | Lectins: Function, structure, biological properties and potential applications[END_REF] Depending on the atom involved in the bond between the carbohydrate and the metal complex, the bioconjugates can be divided into O-, S-(when an O atom has been replaced by S) or Cglycosidic bonds. Changes in the FP experiment of both complexes (20 µM) were tracked after the addition of the different lectins (5 µM). The results were consistent with the previously reported affinities: 60-Gal to PNA and 60-Glc to ConA. When varying the concentrations of these two lectins, it was possible to determine the dissociation constants (Kd). They found lower Kd of the clustered complexes 60-Gal and 60-Glc towards PNA and ConA (Kd = 6.1 × 10 -6 and 1.8 × 10 -5 M, respectively), than the free galactose and glucose (Kd ≈ 10 -3 to 10 -4 M), confirming again the high affinity of the metal complexes. The binding affinity of the conjugates (2 µM) was also tested against tetanus toxin c-fragment (TTC, 0.05 µM), a non-toxic peptide, which is transported from peripheral axons into spinal motoneurons. The polarization increment was almost 7 times higher for 60-Gal, than for 60-Glc, which confirmed the already known good affinity of TTC for galactose. Very recently, Liu et al. reported some imidazo phenanthroline glucocongujates (62a, 62b and 62c shown in Fig. 33) as two-photon absorption (TPA) PDT agents. [START_REF] Liu | Boosting two-photon photodynamic therapy with mitochondria-targeting ruthenium-glucose conjugates[END_REF] A similar complex to 62a without the glucose moiety was prepared as comparison. The TPA cross sections of the complexes were found to be higher than those for other organometallic complexes. Moreover, singlet oxygen quantum yield values ( 1 O2 phosphorescence emission) were high (0.74-0.90), meaning the complexes were suitable as PDT agents. Lipophilicity measurements (logP) as cellular uptake indicators showed a lower lipophilicity for the complexes bearing the glucose moiety and also for 62b, with the phenyl ring in the imidazole. Cellular uptake and cytotoxicity experiments were tested on HeLa (cervical cancer cells), HepG2 (hepatocellular cancer cells), A549 (human pulmonary carcinoma), A549R (cisplatin resistant) and L02 (human hepatic healthy cells, under glucose starvation). The uptake of the glucoconjugates in HeLa cells was the highest and in L02 cells the lowest, highlighting the selectivity for cancerous over noncancerous cells, whereas for the parent compound, no difference was detected. These evidences suggested an uptake mechanism through energy-dependent glucose transporters (GLUTs) for 62a and a passive transport was assigned for the Ru-derivative without the glucose moiety.

O-glycosidic bonds

Both confocal microscopy and ICP-MS showed that the complexes mainly accumulated in the mitochondria (more than 81 %) in HeLa cells. None of the complexes were toxic in the dark in HeLa cells. Nonetheless, after irradiation (450 nm, 12 J/cm 2 ), the toxicity increased dramatically, leading to a PI of 44 for 62a. Under TPA (25 mW, 120 s), 62a was able to generate ROS in HeLa cells. Finally, in vivo experiments in mice with 62a (the most promising PS) showed disappearance of the tumour after 7 days treatment, when two-photon irradiation was used. In addition, no side effects and negligible body weight loss were detected. PDT and/or PACT agents (Fig. 34). [START_REF] Lameijer | Effects of the Bidentate Ligand on the Photophysical Properties, Cellular Uptake, and (Photo)cytotoxicity of Glycoconjugates Based on the [Ru(tpy)(NN)(L)] 2+ Scaffold[END_REF] They also prepared the corresponding chloride complexes ([Ru(tpy)(NN)(Cl)]Cl) as comparison. The water-soluble glucose-containing complexes were photoactivatable (with the exception of complex 63h), so that the thioether ligand is released and replaced by a water molecule upon light irradiation. The authors measured the photosubstitution quantum yield (Φ450) of 63a-g, using blue light (450 or 470 nm)

to irradiate the 1 MLCT band of their complexes. They observed a bathochromic effect on the substitution process, in agreement with the formation of the aqua derivatives. The Φ450 were 0.5-2 %, leading to photosubstitution reactivities (ξ = Φ450 × ε450) of 11-256. Complex 63d had the highest Φ450 value (0.02), whereas 63e had the lowest one, despite being structurally similar.

The azo derivative 63h did not show any ligand dissociation, suggesting a strong electronic effect of the azo ligand on the photoreactivity of the ruthenium core. The singlet oxygen quantum yield (ΦΔ) of the complexes revealed small production of 1 O2 in CD3OD (ΦΔ = 0.002-0.14), except for 63e (ΦΔ = 0.71). Surprisingly, its chloride derivative 63e had a yield of 0.023, highlighting the influence of the thioether ligand. The addition of a phenyl ring in the bidentate ligand of 63e compared with its analogue 63d, lowers the energy of the 3 ππ* of the bidentate ligand. However, for 63d, the 3 ππ* states might be too high in energy or there is a quick conversion to the 3 MC state, making impossible the energy transfer process with molecular oxygen. This is also in agreement with the non-emissive nature of the complexes. The cytotoxicity and phototoxicity (3.2 J cm -2 at 454 nm) of the complexes was tested in two cell lines, namely A549 (human lung carcinoma) and MCF-7 (human breast adenocarcinoma) after 48 h complex incubation (see Table 1). None of the glyconconjugates were photocytotoxic, except 63e. Nonetheless, after modifying the protocol for 63d, they found an improved phototoxicity index (PI, 2.4 and 2.6 for A549 and MCF-7, respectively). The normal protocol implied the replacement of the cell media, so that compound, which did not enter the cell, was removed and only complex inside the cell could be photoactivated. In the new protocol, they did not change the media. Thus, the complex could be activated outside the cell membrane and, after release of the thioether ligand, enter the cell. However, this modification did not show improvement for 63a-c and 63f-g. The cellular uptake (25 µM, A549 cells after 24 h) and the lipophilicity (log Po/w) of complexes showed some correlations. Although the cellular uptake of the glycoconjugates was low, internalization of 63d-f was 10 times higher than for 63a-c, correlating well with the more lipophilic nature of 63d-f. In conclusion, all these complexes are good photocaging agents, but only 63d showed potential as a PACT agent, whereas 63e could act as a photosensitizer for PDT.

In a previous work, complex 63e was studied in depth. [START_REF] Lameijer | d-Versus l-Glucose Conjugation: Mitochondrial Targeting of a Light-Activated Dual-Mode-of-Action Ruthenium-Based Anticancer Prodrug[END_REF] Apart from the ᴅ-glycoconjugate, the ʟ-glycoconjugate was also prepared (ᴅ-63e and ʟ-63e, Fig. 34). The properties of both enantiomers were similar, except the optical density, as expected, and both complexes underwent photoactivation. (Photo)cytotoxicity experiments measured using the same conditions as above (the "normal" protocol with replacement of the media, see above) showed some differences. Complex ʟ-63e exhibited lower toxicity in the dark than ᴅ-63e, but higher toxicity after light irradiation, leading to high PI values . Thus, the chiral nature of the glucoses has a clear effect on the cytotoxicity. Cellular localization was independent of the incubation times and both conjugates showed almost the same cellular localization. Further experiments revealed localization in mitochondria and an energy-independent mechanism of cellular uptake (using NaN3 as all energy-dependent uptake mechanisms). Since DNA is present in mitochondria and Ru(II) complexes with the N^N ligand (dppn, benzo[i]dipyrido-[3,2a:2',3'-c]phenazine) are known as good DNA intercalators, [START_REF] Frasconi | Photoexpulsion of Surface-Grafted Ruthenium Complexes and Subsequent Release of Cytotoxic Cargos to Cancer Cells from Mesoporous Silica Nanoparticles[END_REF][START_REF] Foxon | Electrochemical and Photophysical Properties of DNA Metallo-intercalators Containing the Ruthenium(II) Tris(1-pyrazolyl)methane Unit[END_REF] some experiments to check interaction with DNA were performed. In the presence of CT-DNA and blue light, the emission maximum of ᴅ-63e were red-shifted by 42 nm and the intensity enhanced by up to 10 times. Under these conditions, the respective aqua complex is also formed. In addition, a competitive experiment for DNA intercalation between ethidium bromide (a well-known DNA intercalator, EtBr) and ᴅ-63e showed competition of both compounds for the same binding sites. Finally, the authors worked out two extra conclusions. First, the aqua complex interacts with plasmid DNA with a high base pair/Ru ratio. Second, both the ᴅ-glycoconjugate and the aqua complex generate singlet oxygen, which triggers DNA photocleavage. According to the idea of the photoinduced release of a monodentate ligand, dos Santos et al.

prepared a Ru(II) complex (complex 64, Fig. 35) containing a BODIPY moiety, a nitrogen oxide precursor and two lactose units and its parent compound without the lactose moieties. [START_REF] Santos | Cytotoxicity, cellular uptake, and subcellular localization of a nitrogen oxide and aminopropyl-β-lactose derivative ruthenium complex used as nitric oxide delivery agent[END_REF] Both complexes showed similar spectroscopic features, indicating that the lactose incorporation does not modify these features. The cellular uptake of the lactose derivative 64 (10 µM) in B16F10 cells (murine melanoma) after 1 h showed 99 % of internalization, whereas the parent compound (i.e., without lactose units) only showed 13 %.

Even 30 min were enough for 64 to be internalized in the cytoplasm. The subcellular location was found to be near the mitochondria, while the nucleus was excluded. The reason of the rapid uptake could be the presence of the aminopropyl-β-lactose moieties, easily taken up by lectins.

The cytotoxicity was evaluated in the same cell line. Although the internalization was extremely different, both complexes caused 75 % of cell death and produced NO (nitric oxide), meaning that the parent compound is much more toxic than its lactose conjugate. 150 µM of both 64 and the parent compound produced the maximum concentration of NO after 24 h. Therefore, the cytotoxic activity might be caused by the release of NO. derivatives 65a, 65b and 65c (Fig. 36). [START_REF] Haider | Metallocyclodextrins as building blocks in noncovalent assemblies of photoactive units for the study of photoinduced intercomponent processes[END_REF] Since cyclodextrins are both externally and internally hydrophobic, the guest can bind through non-covalent interactions. Firstly, some quinones, shown in Fig. 35, were used as guests: AQC (anthraquinone-2-carboxylic acid), AQS (anthraquinone-2-sulfonic acid) and BQ (1,4-benzoquinone). The emission intensity of 65b decreased (40 %, 20 % and 25 %, for AQS, AQC and BQ, respectively) when the quinones were added. The rate constants were 5 × 10 8 s -1 for AQS and 3 × 10 8 s -1 for AQC. The equilibrium constants were also determined in saturated conditions. The authors found out that 85 % of AQS and 77 % of AQC were bound to the cyclodextrin appended moiety in 65b (Ka(AQC) = 860 ± 200 M -1 , and Ka(AQS) = 1100 ± 100 M -1 ). This quenching suggests an electron transfer process. For the BQ, it was not possible to obtain a value due to the weak binding of this quinone with CD. Secondly, an osmium metalloguest was prepared [Os(biptpy)(tpy)] 2+ , where biptpy = 4′-(4-biphenyl)-2,2′:6′,2′′-terpyridine (Fig. 37). The photoinduced transfer from the ruthenium host to the osmium guest was measured with steady state and time-resolved spectroscopy. Excess of the Ru derivative was needed to ensure all of the osmium complex is in the cyclodextrin cavities. However, no effect was observed for the energy transfer (ET) mechanism. In the electron transfer (eT) option, the Os(II) is previously oxidized to Os(III), in the presence of cerium(IV) salt, which is used as the oxidizing agent. The lifetime of the Rucyclodextrin was quenched by the Os(III) metalloguest. For the free ruthenium complex, the lifetime was 1.9 ns, whereas for the quenched species, two lifetimes were detected: 100 ± 10 ps (80 %) and 1.9 ns (20 %). Independent binding studies of the osmium guest to free CD performed in the same conditions confirms 80 % of the osmium species are bound to the cyclodextrin cavities mediated by an electron transfer pathway (k = 9.5 × 10 9 s -1 ). This indicates the high efficiency of the process. Complexes decorated with cyclodextrin cavities were also used as probes to detect steroids.

Nolte and co-workers synthesised two complexes, 66 and 67 (Fig. 38), separated in their Λ and Δ enantiomers. [START_REF] Nelissen | Switch-on luminescence detection of steroids by tris(bipyridyl)ruthenium(II) complexes containing multiple cyclodextrin binding sites[END_REF] However, owing to the low emission intensity of 66, only 67 was tested as a probe. When directly adding the steroids to a solution of 67, no change in the emission was detected. Therefore, a viologen (N,N'-dinonyl-4,4'-bipyridinium bromide) was firstly used as a guest molecule. The viologen quenched the emission of 67 by 92 % and a 1:1 complex was formed (Ka = 2.8 × 10 5 M -1 ). The interaction of the first bipyridinium was observed to quench the emission of 67, whereas the interaction with the second one had no effect over the emission intensity. Then, different steroids (i.e., ursodeoxycholic acid, lithocholic acid and cholesterol)

were added, so that the emission was recovered, suggesting the displacement of the viologen.

Only ursodeoxycholic acid strongly increased the emission of 67, while lithocholic acid only recovered a 30 % of the intensity and cholesterol showed too weak emission. Thus, this sensor is useful to detect and distinguish steroids. The authors concluded that, like in complex 65b, there is a photoinduced electron transfer process from the Ru core (host) to the viologen (guest).

As an extra advantage, the multiple binding sites available in the molecule prevents the complex to be quenched by oxygen.

Weinheim

Flow cytometry analysis confirmed as well a favourable internalization of 68-Glc, whereas a lower effect was observed for 68-Gal. Both 68-Man and Ru(dmbpy)3Cl2 were undistinguishable by flow cytometry analysis. In order to discard a mechanism of internalization through glucose transporters (GLUT), some glucose (20 mM, 24 h) was added to the cells treated with the glucoconjugate (500 µM, 24 h). No relevant changes were observed when comparing the cells with or without the complex, suggesting again an endocytotic uptake mechanism. The interest in the fructose functionalization of the Ru(II) polypyridyl complexes has been pursued since fructose can take part in many favourable mechanisms. GLUT5 (a glucose transporter), for instance, facilitates fructose uptake and is overexpressed in some breast cancers. Thus, Lau et al. prepared the fructo-conjugates 69a and 69b, with the fructose appended through an amid bond on one bpy (Fig. 41). [START_REF] Lau | Photophysical, Cellular-Uptake, and Bioimaging Studies of Luminescent Ruthenium(II)-Polypyridine Complexes Containing a d-Fructose Pendant[END_REF] Both complexes showed emission intensities comparable to those of the parent compounds (non-fructose derivatives). The lipophilicity of the complexes showed increased lipophilicity for the dpp (4,7-diphenyl-1,10phenanthroline) complex. However, both fructo-conjugates are more hydrophilic than their non-fructose counterparts. Their cytotoxicity was tested in MCF-7 cells. After 48 h incubation, no cytotoxic effect was found for any of the complexes (cell viability > 70 %). The colocalization experiments performed on MCF-7 cells showed intense staining of the cell membrane exclusively for the dpp complex. Oppositely, no staining was observed for 69a, bearing bpy. Other cell lines were selected for further studies with 69b: MDCK (Madin Darby Canine Kidney cells), 3T3 (mouse embryonic fibroblasts) and HeLa (human cervical epitheloid carcinoma). Conjugate 69b (10 µM, 1 h) was found to localize in the cytoplasm in MDCK and 3T3, whereas in HeLa, it was strongly concentrated in the cell membrane. Since the GLUT5 transporter favours the fructose uptake, a competitive experiment between 69b and ᴅ-fructose was carried out in MCF-7 cells. Cells treated with 69b (50 µM, 1 h) showed lower intracellular emission intensity when ᴅ-fructose (50 mM) was added. The same effect was detected in flow cytometry assays, suggesting that the uptake of complex 69b is competitively inhibited by fructose. These assays also showed that cells treated with 69b displayed stronger emission intensity than the untreated cells, highlighting its good internalization. Thereby, these complexes display promising properties as imaging agents.

C-glycosidic bonds

Lipids

Cell membranes are lipophilic. Thus, one of the best strategies to achieve internalization of drugs is by modifying complexes with lipids. Lipid-drug conjugates (LDCs) also present other interesting features. Linking lipids to drugs can improve the performance of orally administrated drugs, enhancing, for example, their gastrointestinal absorption. This approach can be also beneficial in the delivery of anticancer drugs, since tumour cells frequently take up lipids as energy suppliers. In addition, LDCs have been found to overcome drug resistance in anticancer therapy. Small, uncharged and lipid-soluble molecules (i.e. LDCs) are able to cross the blood brain barrier (BBB), a permeable barrier that separates circulating blood from the cranial fluid, by two mechanisms: increasing lipophilicity or targeting membrane receptors capable of transport them through the BBB. [START_REF] Irby | Lipid-Drug Conjugate for Enhancing Drug Delivery[END_REF] From a photophysical point of view, lipids absorb light in the therapeutic window of the nearinfrared (NIR) spectrum. [START_REF] Tsai | Near-infrared Absorption Property of Biological Soft Tissue Constituents[END_REF][START_REF] Hermanson | Introduction to Bioconjugation[END_REF] Since the use of red or NIR light as activation light is a key parameter for some application in PDT and PACT, the attachment of lipids to metal complexes could significantly improve their use as photosensitizers for these techniques.

Fatty acids

One of the first examples of Ru(II) polypyridyl lipid conjugates was reported by Killeen et al. [START_REF] Killeen | Functionalisation of bolaamphiphiles with mononuclear bis(2,2′-bipyridyl)ruthenium(II) complexes for application in self assembled monolayers[END_REF] They synthesised a [Ru(bpy)3] 2+ derivative 70 (Fig. 42), in which one of the bipyridyl ligands has been substituted in 5-position with a bolaamphiphile (i.e., molecules with two hydrophilic ends separated by a long lipophilic chain). Amphiphiles have been widely used to form highly ordered assemblies. However, these membranes are not completely homogeneous since some pores are formed in-between the assemblies. Therefore, the authors proposed the The conjugation of Ru complexes with lipids was proposed to achieve self-aggregates in water and improve the cellular uptake and diffusion. Dosio et al. prepared a probe for cellular imaging 71 (Fig. 42), based on a Ru(II) polypyridyl complex attached to squalene (squalenoylation), a natural organic precursor for the synthesis of all plant and animal sterols. [START_REF] Dosio | Ruthenium polypyridyl squalene derivative: A novel selfassembling lipophilic probe for cellular imaging[END_REF] The authors found out that 71 accumulates inside the cell by passive diffusion, even in multicellular spheroids, unlike the parent compound without the squalene moiety. ICP-MS experiments showed an internalisation of 13 amol/cell for the non-squalenoyl complex and 270 amol of Ru/cell for 71, when MCF-7 cells were incubated with 50 µM of complex, respectively. The cellular concentration for the conjugate was 160 µM, more than 3 times higher than the initial concentration. This evidences the high lipophilicity of 71. Furthermore, the authors found out that the toxicity of both complexes in HT-29 (human colon cancer cells) and MCF-7 cell lines was relatively low (> 100 µM). However, the global emission intensity impressively increased for the squalene-derivative. The emission of 71 could be detected in the nucleus and the cytosol, whereas no emission was observed for the non-squalenoyl compound owing to its slight internalization. Lo et al. reported a series of Ru(II) polypyridyl estradiol conjugates, 72a-b and 73a-b (Fig. 43), successfully designed as biological luminescent probes. [START_REF] Lo | Luminescent Biological Probes Derived from Ruthenium(II) Estradiol Polypyridine Complexes[END_REF] Estradiol belongs to the family of estrogens and has the highest binding affinity to estrogen receptors (ERs). The lipophilicity of the complexes measured as the partition coefficients (log Po/w) revealed an increase for complexes bearing dpp ligands, especially for the estradiol derivative with the longer spacer.

Steroids

Ru

The emission intensities and lifetimes also increased for the conjugates with the dpp ligand in the presence of ERα, evidencing the enhanced binding affinity of the new complexes. However, the binding constants were lower than that of the estradiol, but similar to other estradiolcontaining metal complexes. The cytotoxicity of the complexes, studied in HeLa cells after 48 h incubation was low (IC50 values ranged from 83.1 to 166.6 µM) compared to that of cisplatin (34 µM). Thus, the complexes could be considered as non-toxic. The cellular uptake data showed better internalization for derivatives with dpp, which are in good agreement with the emission intensities and binding affinities. In addition, complex 73b showed distribution mainly inside the cytoplasm of HeLa cells, with higher degree in the perinuclear region, suggesting interaction with hydrophobic organelles such as the Golgi apparatus or the endoplasmic reticulum.

Bonnet and co-workers designed steroid derivatives based on another approach: the photochemical activation and subsequent release of the lipid-containing monodentate ligand, through a thioether bond. This is the same strategy that they have followed for other bioconjugates, which have been previously described (see Section 1.3.1. O-glycosidic bonds, complexes 63a-h). The steroid-thioether moiety coordinates to the Ru(II) polypyridyl core through the S atom. Thus, complexes 74 and 75, shown in Fig. 44, were prepared as cholestanol [START_REF] Bonnet | Rutheniumdecorated lipid vesicles: Light-induced release of [Ru(terpy)(bpy)(OH2)]2+ and thermal back coordination[END_REF] and cholesterol [START_REF] Siewert | Chemical Swarming: Depending on Concentration, an Amphiphilic Ruthenium Polypyridyl Complex Induces Cell Death via Two Different Mechanisms[END_REF] derivatives, respectively. The photochemical activation triggers the substitution of the S-ligands by water molecules, forming the aqua complex. The cholestanol conjugate 74 (Fig. 44) [START_REF] Bonnet | Rutheniumdecorated lipid vesicles: Light-induced release of [Ru(terpy)(bpy)(OH2)]2+ and thermal back coordination[END_REF] was prepared as a first attempt to mimic the behaviour of Ru(II) polypyridyl complexes in the lipid bilayer since 5α-3β-cholestanol is a membrane intercalator. For that purpose, the researchers incorporated the Ru-containing conjugates to lipid vesicles to facilitate the solubility in water and to gain understanding of the behaviour of this kind of complexes in the water-membrane interface. These vesicles (140 -180 nm) were successfully prepared with two lipids (dimyristoylphosphatidylglycerol DMPG, anionic and dimyristoylphosphatidylcholine DMPC, neutral). They found out that the complex interacted both electrostatically and through insertion of the cholestanol in the membrane when complex to the thioether-cholesterol ligand in a negatively charged lipid bilayer, forming 76.

The first step is the adsorption of the metal complex, which is driven by entropy and completed in a few minutes. The second is coordination of the metal complex to the S-ligand, probably electrostatically. This could take hours. [START_REF] Bahreman | Binding of a ruthenium complex to a thioether ligand embedded in a negatively charged lipid bilayer: A twostep mechanism[END_REF]

Vitamins

Vitamins are small molecules essential for any organism. They can be classified as hydrophilic (B and C) and lipophilic (A, D, E and K). The water-soluble vitamins are easily taken up and excreted. Therefore, the conjugation with these vitamins can improve the water solubility of metal complexes and their subsequent cellular internalization. This is the reason why some ruthenium polypyridyl complexes have been functionalized with B vitamins. centre. This is because the pterin ring is slightly more electron rich than the flavin rings. All complexes showed interesting MLCT bands in the absorption spectra, which were pHdependent. Thus, the free ligands had pKa ≈ 9 for the NH of the pyrimidine ring, but after coordination to the metal centre, the acidity of that NH was higher (pKa = 4.3 -5.4). In addition, the absorption spectrum of 77-FA was the same as the one of a similar complex, namely [Ru(bpy)2(1,3-dimethyllumazine)] 2+ , bearing the same chromophore and N^O coordination. Some years later, Scrase et al. demonstrated the coordination of the folate to the ruthenium core through the N^N (77-FA right). [START_REF] Scrase | Folates are potential ligands for ruthenium compounds in vivo[END_REF] The authors explored the synthesis of 77-FA at physiological conditions (37 ºC) and also at higher temperatures (65 ºC). In both cases, the NMR characterization revealed two set of signals, corresponding to the diastereomers (ΛS/ΔR:ΛR/ΔS). The peaks corresponding to the methylene group directly attached to the pterin moiety were crucial to determine the coordination mode of FA, since the protons displayed loss of degeneracy (compared to the free FA), suggesting direct coordination of the non-aromatic amine. Some more evidences of the coordination mode were reported with pH changes. At pH 6 and 9.9, the same products were obtained, while at pH 2.5, the protonation of the NH avoided the coordination of the FA.

Since the content of FA in cells is low, the sequestration of this molecule by an excess of ruthenium complexes could lead to an alteration of the balance of this cofactor. through hydrogen bonding and thus, hindering rotation. 78c, however, showed a normal behaviour at room temperature, whereas, at 185 K, three atropisomers were detected, A, B and C, with C as the most stable one and B the least. In this case, the cooling slowed down the rotation of the NA, making the atropisomers visible. DFT studies supported the experimental results, confirming atropisomer Conf. 1 as the most stable, since -Cl has a higher H-bonding acceptor ability than -I. Due to these properties, these complexes could be interesting as photoactivated drugs to deliver nicotinamide as an antibacterial agent. Biotin (vitamin B7 or vitamin H) in Ru(II) polypyridyl bioconjugates was extensively used.

Bonnet and co-workers prepared some biotin conjugates (79a-c and 80, Fig. 47), following the same strategy used for previous bioconjugates of the type [Ru(tpy)(bpy)(S-biomolecule)] 2+ (see sections 1.3.1. O-glycosidic bonds and 1.4.2. Steroids). [START_REF] Siewert | Synthesis and Avidin Binding of Ruthenium Complexes Functionalized with a Light-Cleavable Free Biotin Moiety[END_REF] As previously shown, this kind of complexes is well-known as photocleavable molecules. The synthesis of the chloride derivative [Ru(tpy)(bpy)Cl] + with the biotin precursor yielded complex 79a (9 %) and a mixture of complexes 79b and 79c (83:17). When the synthetic conditions were modified and the ligand-Ru ratio increased, the formation of 79a was supressed. Moreover, changes in temperature and reaction times yielded an enrichment of 79b vs 79c, suggesting 79b as the thermodynamically stable isomer. Since regioselectivity was not achieved, the separation of both isomers by avidinaffinity column chromatography was performed. Avidin is known to strongly interact with biotin (Ka = 10 13 to 10 15 M -1 ) [START_REF] Diamandis | The biotin-(strept)avidin system: principles and applications in biotechnology[END_REF], forming the avidin-biotin complex (ABC). After 24 h incubation of the 79b/79c mixture with an avidin column, the unbound complex mixture was washed off the column with PBS. Finally, a biotin-loaded PBS solution was used to displace the avidin-bound biotin moiety in 79b with native biotin. Thus, 79b was obtained as a pure complex. Surprisingly, bioconjugate 80 was shown to uniquely coordinate to one biotin moiety.

The photochemical behaviour of this complex was studied, tracking absorption changes under light irradiation (λ = 465 nm) in PBS for 4 h at 298 K. After 80 min, a plateau was reached, leading to a photosubstitution constant of kΦ = 7.1 × 10 -4 s -1 . A photosubstitution quantum yield of 0.011 was also determined. Furthermore, the bioconjugate 80 was found to bind streptavidin as strongly as biotin, suggesting that it could be a good candidate for the ABC-based strategy. determined with three different irradiation wavelengths. The highest efficiency for singlet oxygen production was achieved upon blue light irradiation (463 nm, ΦΔ = 0.75), although to a lesser extent, green light (520 nm, ΦΔ = 0.51) and red light also produce 1 O2 (631 nm, ΦΔ = 0.19). DNA binding constants, determined by absorption and emission, showed similar values as those obtained for typical DNA intercalators (Ka = 6.28 × 10 6 M -1 and 5.8 × 10 6 M -1 , respectively). Thus, the insertion of the biotin moiety did not significantly change the ability of the complex towards DNA binding. Complex 82 was able to photocleave DNA using both blue and green light. However, red light did not cause any cleavage. The biotin conjugate showed lipophilicity (log P0/w = 1.32), possibly favouring cellular uptake. Biological tests were assayed in bacteria and cells. Regarding bacteria, 82 was only active against S. aureus upon blue light irradiation (MIC = 14 µM). Nevertheless, better results were obtained when these compounds were tested as anticancer agents. The cytotoxic activity was evaluated in HaCaT (normal keratinocyte), LS174T (colorectal cancer cells), A549 (lung carcinoma cells) and MCF-7 (breast cancer cells). Whereas the parent complex without biotin did not show any effect, the biotinylated compound 82 caused substantial reduction of cell viability: around 40 % cell viability for A549 at 12.5 µg mL -1 and ca. 50 % for MCF-7 cells at the same concentration.

These results are in line with the fact that both cell lines have highly overexpressed BRs. For the other cell lines, neither toxicity or phototoxicity was observed. Like 81 and as expected for a biotin conjugate, compound 82 also showed avidin affinity. Chen and co-workers reported ruthenium biotin conjugates 83 and 84 (Fig. 49) based on the derivatization from the benzimidazole ligand. [START_REF] Zhao | Cancer-Targeting Functionalization of Selenium-Containing Ruthenium Conjugate with Tumor Microenvironment-Responsive Property to Enhance Theranostic Effects[END_REF] In addition, the authors introduced selenium in the structure of the complex with the aim of increasing their anticancer activity, as they previously showed [START_REF] Lai | Selenium-containing ruthenium complex synergizes with natural killer cells to enhance immunotherapy against prostate cancer via activating TRAIL/FasL signaling[END_REF] that selenium activates ROS production in ER and improves the selectivity between cancer cells and non-cancerous cells. Under hypoxic conditions (acidic, pH 6.86), complex 84 was able to release the pyridylbenzimidazole-biotin ligand, generating the complex [Ru(phenSe)2(H2O)2], which can be considered as the therapeutic drug. Biological assays were performed in biotin receptor-positive HeLa cells and receptor-negative L02 cells.

The bioconjugate 84 was preferentially taken up by HeLa than L02 in a co-cultured model. The number of apoptotic cells in HeLa increased to 20.8 % after drug incubation with 84, whereas in L02, the increment was only 2.1 %. In vivo experiments in mice inoculated with HeLa revealed accumulation of the Se-bioconjugate selectively in the tumour than in other organs. In cancer cells, 84 enters the cell after 6 h, mostly accumulating in mitochondria (Pearson's coefficient 0.9). Thus, the disruption of the mitochondrial membrane potential (MtMP) was tested. 84 induced dose-dependent disruption, whereas 83 only showed a slight variation. High intracellular levels of ROS were detected for 84 after 4 h, whereas 83 generated much lower amounts of ROS. The accumulation of ROS triggers ER stress, and the release of calcium in ER is an indicator of this stress. The calcium levels in cytoplasm after 30 min of incubation with the complexes, showed higher levels for 84 than for 83. All these fact supports the ROSmediated ER stress signal pathway. Finally, in vivo antitumour activity was evaluated in mice.

The tumour inhibition rate after 30 days treatment with 84 was 64.2 % and no body weight change or death was observed. In addition, the complex was found to reduce side effects on liver, lung and kidney and to return blood parameters to normal levels after treatment. Very recently, our group, in collaboration with the one of Zobi, reported the first Ru-cobalamine (vitamin B12) conjugates, 85a and 85b (Fig. 50), modified in the β-position, through an alkyne group. [START_REF] Jakubaszek | Evaluation of the Potential of Cobalamin Derivatives Bearing Ru(II) Polypyridyl Complexes as Photosensitizers for Photodynamic Therapy[END_REF] The non-conjugated ruthenium complexes were poorly singlet oxygen producers.

The biological activity of both the conjugated and non-conjugated complexes in HeLa (cervical cancer cells) and RPE-1 (non-cancerous retina pigmented epithelium cells) revealed surprising results. None of the bioconjugates 85a and 85b were cytotoxic in the dark or after light irradiation. Only the non-conjugated Ru(II) complex modified with the amino groups showed cytotoxicity in the dark (IC50 = 9.33 and 6.08 µM in HeLa and RPE-1, respectively). This toxicity was similar after light irradiation. The cellular uptake experiments by CLSM showed weak emission in HeLa cells, avoiding localization to be determined. As a result, the coupling of cobalamin did not improve the toxicity, probably due to the bulkiness of the conjugate, preventing it to get delivered to cells.

Conclusions

This review highlights the advantages of the conjugation of ruthenium polypyridyl complexes to biomolecules in view of drug delivery and directed targeting. It is remarkable to note that the use of biomolecules is not limited to biology-related applications but also extended to photoinduced electron/energy transfer processes and asymmetric synthesis. This review also focused on the different manners of linking the biomolecules and the ruthenium complexes, which has a direct impact in their behaviours, especially in terms of stability of the resulting bioconjugates. The attachment through a covalent bond between the ruthenium polypyridyl complexes and (small) biomolecules mostly improves (with some exceptions) water solubility, cellular uptake, phototoxicity or binding affinity, without altering the excellent photophysical properties of the metal complexes. The biomolecules are generally linked to ruthenium through the second coordination sphere of ligands (e.g. with a spacer) and rarely directly attached to the ruthenium core in the first coordination sphere. Only some amino acids and vitamins have the correct structure design and available atoms to directly coordinate to ruthenium.

This strategy of bioconjugation has helped to the exponential growth of the ruthenium polypyridyl complexes and allowed to gain understanding in the behaviour of the complexes in biology-related topics. We strongly believe that much more research dedicated to the use of ruthenium polypyridyl complex-containing bioconjugates will be published over the next years, hopefully demonstrating better or even completely new applications, especially in the field of medicinal/biological chemistry. We are convinced that much more in vivo experiments will need to be undertaken to fully unveil the potential of such bioconjugates since the number of articles reporting mice experiments is disappointingly scarce. Regarding the risks associated with the use of such bioconjugates for therapeutic purposes, we believe that it will actually increase the chance of success since the biomolecule should enable lowering the potential toxicity of the metal-based drug.

  cervical epitheloid carcinoma HepG2: human hepatocarcinoma cells HLF-a: primary human lung fibroblasts HSA: human serum albumin HT-29: human colon cancer cells Htme: 2-(methylthio)ethanol IC50: Inhibitory potency; concentration at which a 50 % of cells are death. -PAA48: polystyrene140-b-poly(acrylic acid)48 PSII: photosystem II ptrpy: 4′-phenyl-2,2';6',2"-terpyridine RCA: Ricinus comunnis agglutinin 120 RF: riboflavin RGD: Arginylglycylaspartic acid RNS: reactive nitrogen species ROS: reactive oxygen species SATA: N-succinimidyl S-acetylthioacetate SiHa: human squamous carcinoma of the cervix Sp2/0-Ag 14: myeloma cells SST: somatostatine SSTRs: somatostatine receptors STD: saturation transfer difference STED: stimulated emission depletion TA: transient absorption TCNQ: tetracyanoquinodimethane TFA: trifluoroacetic acid TP: two-photon TPA: two-photon absorption TPP: tripheynlphosphonium tpy: 2,2':6',2''-terpyridine TTC: Tetanus toxin c-fragment VDAC: voltage-dependent anion channel VEGF: vascular endothelial growth factor VEGFR2: vascular endothelial growth factor receptor 2 WGA: Wheat germ agglutinin YDS: yolk disialo oligosaccharide β-CD: β-cyclodextrine

Fig. 1 .

 1 Fig. 1. A) Percentage of publications about different applications of Ru polypyridyl complexes. Searched on April 22 nd 2020. B) Evolution of the number of publications per year related to "Ru polypyridyl" search in Web of Science in the last 20 years. Searched on May 9 th 2020.

Fig. 3 .Fig. 4 .

 34 Fig. 3. Structures of Ru(II)-diimine functionalized complexes that can be covalently linked to proteins. Adapted from reference [40]. © 2015 Elsevier B.V.

Fig. 5 .DAPA, 7 -DABA, 8 -

 578 Fig. 5. Structures of the AA-labelled complexes 6-DAPA, 7-DABA, 8-Orn and 9-Lys.

12 Fig. 6 .

 126 Fig. 6. Structures of ʟ-proline conjugates Λ-10, Δ-10, 11a, 11b and 12.

  aqueous solution of Ru(II) polypyridyl complexes coupled to amino acids were firstly observed by Vagg and Williams in 1981. In this case, the tested complexes used ʟ-serine and ʟ-tryptophan N,O-coordinated to the ruthenium core, instead of ʟ-proline, to yield [Ru(bpy)2(ʟserine)]ClO4, [Ru(phen)2(ʟ-serine)]ClO4, [Ru(bpy)2(ʟ-tryptophan)]ClO4 and [Ru(phen)2(ʟtryptophan)]ClO4, respectively.[START_REF] Vagg | Chiral metal complexes. 1. Photochemical inversion in ternary Ru(II) complexes of diimines and L-tryptophane[END_REF][START_REF] Vagg | Chiral metal complexes 2. Light-catalysed diastereoisomeric equilibration in aqueous solutions of cis-[Ru(phen)2(L-serine)]+ and its 2,2′-bipyridyl analogue[END_REF]

13 Fig. 7 .

 137 Fig. 7. Structure of the tetradentate water-soluble complex 13.

Fig. 8 .Fig. 9 .

 89 Fig. 8. Structures of the peptide conjugates 14, 15, 16, 17a and 17b.

Fig. 10 .

 10 Fig. 10. Structures of the peptide conjugates 19, 20, 21, 22 and 23.

Fig. 11

 11 Fig. 11 Confocal luminescence images of (a-d) 20, (e-h) 19 and (i-l) 21 in live HeLa cells where the ruthenium channel and overlay channel are shown for each complex. Cells were incubated in the absence of light with 70 mM 20 and 19 for 4 h, and 40 mM with 21 for 24 h. The distribution of each complex is shown in a group of cells (column 1 and 2) and then focused on a single HeLa cell (column 3 and 4). 20 and 19 were excited using a 488

  Figure extracted from reference [59], published by The Royal Society of Chemistry, 2016.A recent work from the same authors compared and analysed complexes 21 and 22 (Fig.10) as NLS and MPP.[START_REF] Burke | Highly Selective Mitochondrial Targeting by a Ruthenium(II) Peptide Conjugate: Imaging and Photoinduced Damage of Mitochondrial DNA[END_REF] They were found to intercalate in ctDNA (calf thymus DNA) through the dppz ligand, due to increments in emission intensity and changes in the circular dichroism (CD) spectrum after addition of DNA. Higher binding constants (Ka = 3.6 × 10 7 M -1 and Ka = 2.8 × 10 7 M -1 for 21 and 22, respectively) than those for the parent complexes [Ru(bpy)2(dppz)] 2+ were determined, suggesting an additional electrostatic interaction from the peptide chain. The uptake of the complexes in HeLa cells by CLSM showed fast internalization of 22 (10 µM in the dark). After 1 h, the conjugate reached the cytoplasm and, after 2 h, the mitochondria. As shown before, compound 21 was distributed in the cytoplasm after 2 h, and reached the nucleus after 24 h (40 µM). Both of them showed a temperature-dependent uptake (no permeation was detected at 4 ºC), showing an endocytosis mechanism of internalization. Co-localization studies confirmed nuclear and mitochondria localization for 21 and 22, respectively, with the latter likely to be in mitochondrial substructures (mitochondrial DNA, mtDNA). Luminescent lifetime imaging microscopy (LLIM) was used to confirm the binding of 22 to mtDNA and 21

Fig. 12 ( 24 Fig. 13 .

 122413 Fig. 12 (A,B) Confocal luminescence Imaging of 23 in HeLa cells. (C,D) Confocal luminescence Images of 19 in HeLa cells. (E) Co-localization studies were carried out using 500 nM of the mitochondrial marker MitoTracker Deep Red. Cells were treated with 23 for 2 h and MitoTracker Deep Red for 30 min. 23 is shown in green,

Fig. 14 .

 14 Fig. 14. Structures of complexes 25a-28c (bpy = 2,2'-bipyridine, phen = phenanthroline and hdpa = dipiyridylamine).

29 Fig. 15 .

 2915 Fig. 15. Structure of the Ru-somatostatin bioconjugate 29.

Fig. 16 .Fig. 17 .

 1617 Fig. 16. Structures of the peptide conjugates 30-34.

Fig. 18 .

 18 Fig. 18. Structures of the bimetallic conjugates 40a-42b, where R = H or ph (phenyl).

Fig. 19 .

 19 Fig. 19. Structures of the ruthenium-containing peptoid conjugates 43-45.

Fig. 20 .

 20 Fig. 20. A) Structure of complex 46. B) Schematic of IgG-binding assays of Ru(II)-protein G conjugates. Normal sheep IgG was immobilized on the 96 well plate, and then Ru(II)-protein G conjugates bound to the Fc region of IgG. C. Schematic of Ru(II)-protein G conjugates for detecting histidine-tagged protein. The purified histidinetagged protein BasR was first immobilized on the 96 well plate and then recognized by anti-His antibody. Finally, the Ru(II)-protein G conjugates bound to the Fc region of anti-His antibody. Reproduced from reference [75], © 2012 Lin et al.

Fig. 21 .

 21 Fig. 21. Structures of the protein conjugates 47-SC, 47-LC and 48. (SC = short chain, LC = long chain).

Fig. 22 .

 22 Fig. 22. Structure of Ru-tubulin conjugate 49 and schematic illustration of the formation of photo-enhanced MTs assembled from Ru-conjugated tubulin. Reproduced from Ref. [78] with permission from The Royal Society of Chemistry.

50 Fig. 23 .

 5023 Fig. 23. Scheme of the transporter 50 based on HSA and schematic illustration of a part of the HSA polypeptide sequence exemplary with the PEO, TPP groups attached to, e.g., lysine and Ru conjugated to tyrosine residues.

  1979, Paulson et al. successfully achieved the preparation of RuMb. The studies revealed the coordination of carbon monoxide, as in Mb. Nonetheless, unlike the original Mb, dioxygen irreversibly oxidized RuMb to RuMb + .[START_REF] Paulson | Preparation of ruthenium(II) and ruthenium(III) myoglobin and the reaction of dioxygen, and carbon monoxide, with ruthenium(II) myoglobin[END_REF]

Fig. 24 .

 24 Fig. 24. Molecular design of the artificial metalloprotein with a ruthenium(II) tris(bipyridyl) complex as the core. Adapted from reference [82]. Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

8 Fig. 25 .

 825 Fig. 25. Structures of the dimeric metallopeptides ΛΛ-52 and ΔΔ-52, ΛΛ-53 and ΔΔ-53 and 54.

Fig. 26 .

 26 Fig. 26. Structure of the ruthenium(II) antibody conjugate 55.

Fig. 27 .

 27 Fig. 27. A) Structure of Ru-NB 56. B) Confocal immunofluorescence microscopy images of A431 cells exposed to 56 for 4, 24, and 48 h at 37 ºC showing specific binding and co-localization of the single-conjugated NB with EGFR. Scale bars:20 mM. C) Amount of cell-associated ruthenium after incubation of EGFR-positive A431 and EGFR-negative MDA-MB-435S cells with 2 or 20 mM of Ru-NB for up to 48 h at 37 ºC. The level of ruthenium in cell lysates of MDA-MB-435S exposed to2 mM of Ru-NB were below the analytical limit and are thus not shown. Figures extracted from reference[90]. © 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  emission lifetime was found to be affected by oxygen (τdegassed = 589 ns and τaerated = 134 ns).Singlet oxygen quantum yield (ΦΔ) evaluated by an indirect method in DMSO or PBS showed low values and quenching in PBS due to hydrogen bonding interactions (ΦΔ = 9 % in DMSO and ΦΔ = 4 % in PBS). The co-localization (by CLSM) of the NB-conjugate with EGFR in A431 cells (human epithelial cells) displayed mainly membrane staining after 48 h incubation at 37 ºC. The cellular uptake was assessed by ICP-MS in EFGR-positive A431 cells and EFGRnegative MDA-MB-435S cells after different incubation times (4, 24 and 48 h) and concentrations in the dark at 37 ºC. A higher metal amount was found in the EGFRoverexpressing cells than in the negative one. Fig.27gathers the co-localization images and cellular uptake graph of 56. These findings suggested that the cellular internalization occurs through an EGFR-mediated process. Conjugate 56 exhibited similar cytotoxicity in A431 cells before and after irradiation (480 nm, 6 × 3.5 min with 15 min gap in between, 6.74 J/cm 2 ) with IC50 > 25 µM in both cases.

Fig. 28 .

 28 Fig. 28. Structure of the ADC 57.

Fig. 29 . 1 and 59 -Fig. 31 .

 2915931 Fig. 29. Structures of glycoconjugates 56-OAc and 56-OH.

Fig. 32 .Fig. 33 .

 3233 Fig. 32. Structures of conjugates 59-Glc and 59-Fru.

Fig. 34 .

 34 Fig. 34. Structures of S-glucoconjugates 63a-h

64 Fig. 35 .

 6435 Fig. 35. Structure of Ru-BODIPY glycoconjugate 64.

Fig. 36 .

 36 Fig. 36. Structure of cyclodextrine (CD) conjugates 65a, 65b and 65c.

Fig. 37 .Fig. 38 .

 3738 Fig. 37. Host-guest interaction for cyclodextrin conjugates 65 (host) with guest quinones (top. AQS = anthraquinone-2-sulfonic acid, AQC = anthraquinone-2-carboxylic acid and BQ = 1,4-benzoquinone) or the Os(III) metalloguest (down) and the electron transfer mechanism of 65b with Os(III) metalloguest (down).

Fig. 39 .

 39 Fig. 39. Structures of S-glycoconjugates 68-Glc, 68-Gal and 68-Man.

Fig. 40 .

 40 Fig. 40. Cellular distribution of glycoconjugated Ru complexes in HepG2 cells (24 h, 500 µM, red). A) Glucose, B) galactose, C) mannose Ru-complex and D) parent compound Ru(dmbpy)3Cl2, Blue: cell nuclei after staining with Hoechst33258. Reproduced from [113]. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA,

Fig. 41 .

 41 Fig. 41. Structure of the fructose conjugates 69a and 69b.

Fig. 42 .

 42 Fig. 42. Structures of the fatty acid conjugates 70 and 71.

Fig. 43 .

 43 Fig. 43. Structures of lipoconjugates 72a-b and 73a-b.

Fig. 44 .

 44 Fig. 44. Structures of the cholestanol 74 and cholesterol bioconjugates 75 and 76.

Fig. 45 .

 45 Fig. 45. Structures of the flavin conjugates 77-LF and 77-RF, and the pterin derivatives 77-FA-NO and 77-FA-NN, where LF = lumiflavin, RF = riboflavin and FA = Folic acid. NO and NN stand for the atom coordination.

Fig. 46 .

 46 Fig. 46. Structures of the nicotinamide conjugates complexes 78a, 78b and 78c (left). Structure and nomenclature of the atropisomers of 78a and 78b (Conf. 1 and Conf. 2) detected at 185 K. The nomenclature to describe the atropisomers of 78c (A, B and C) detected at 185 K (right). H is defined as the head group and T as the tail. Adapted from reference [128] © 2016 The Authors. Published by Elsevier B.V.

80 Fig. 47 .

 8047 Fig. 47. Structures of Ru-biotin derivatives 79a-c and 80.

Fig. 48 .

 48 Fig. 48. Structures of biotin conjugates 81 and 82.

Fig. 49 .

 49 Fig. 49. Structures of bioconjugates 83 and 84.

Fig. 50 .

 50 Fig. 50. Structures of cobalamin conjugates 85a and 85b.

Table 1 .

 1 Cytotoxicity and phototoxicity of complexes 63d and 63e in A549 and MCF-7 cells reported as effective concentrations (EC 50 ). PI is the phototoxicity index, calculated as EC 50dark /EC 50light . Confidence interval values are not shown for clarity.

			A549			MCF-7
	Complex	EC 50 dark EC 50 light	PI	EC 50 dark EC 50 light	PI
	63d	> 100	> 100	-	> 100	> 100	-
	63d a	64	27	2.4	52	20	2.6
	63e (ᴅ-63e)	19	0.72	26	9.6	0.86	11
	63e (ʟ-63e)	50	0.58	86	18	0.61	30

a Modified protocol.

Acknowledgements

This work was financially supported by an ERC Consolidator Grant PhotoMedMet to G.G. (GA 681679) and has received support under the program Investissements d'Avenir launched by the French Government and implemented by the ANR with the reference ANR-10-IDEX-0001-02 PSL (G.G.). M.M. acknowledges the European Commission for the H2020-MSCA-IF-2018 (839499 -OrganometRuPDT) grant.

Abbreviations

3T3: mouse embryonic fibroblasts 5Bpy: 5'-amino-2,2'-bypyridine-5-carboxylic acid A nitrosyl ruthenium complex conjugated with a polyclonal antibody anti-VDAC (anti voltagedependent anion channel), 57 (Fig. 28), was described as a NO delivery anticancer agent by Ramos et al. [START_REF] Bunney | Orexin activation counteracts decreases in nonexercise activity thermogenesis (NEAT) caused by highfat diet[END_REF] This Ab participates on the mitochondria-mediated apoptosis through ROS.

To simplify the measurement, the authors prepared the parent complex cis-[Ru(NO)Cl(dcbpy-

, where dcbpy is 2,2′-bipyridine-4,4′-dicarboxylic acid, and evaluated some of its properties. At different pHs, there are different possible structures. For instance, at physiological pH, cis-[Ru(NO)Cl(dcbpy-H)2] slowly turns into cis-[RuCl(dcbpy)2(NO2)] 4-.

Cyclic voltammetry measurements in aqueous solution (KCl 0.1 M and acetate buffer solution 0.01 M) demonstrated the release of NO in the presence of NADH, through a one-electron reduction centred on the nitric oxide derivative ligand site. NADH plays an important role in the respiratory chain in the mitochondria. The production of ROS/RNS (reactive oxygen / nitrogen species) was evaluated for cis-[Ru(NO)Cl(dcbpy-H)2] in HepG2 cells (human hepatocarcinoma cells). High levels of ROS/RNS were found in succinate-energized mitochondria. Furthermore, at 100 µM of complex, the mitochondrial permeability transition (MPT) inhibitor cyclosporine A (CsA) partially supressed the release of NO. At 50 µM of complex, CsA inhibited almost completely the swelling caused by the complex. These features, along with the inhibition of swelling by a NO scavenger, suggested that NO was an MPT inducer. In addition, this complex was found to decrease the mitochondrial membrane potential.

This means that mitochondria efficiently induced NO release from cis-[Ru(NO)Cl(dcbpy-H)2].

As a mechanism of action, high NO concentrations are known to inhibit mitochondrial respiration, increasing the ROS levels and inducing MPT and finally cell death. Different fractions obtained after purification of conjugate 57 showed cytotoxicity (25 % -90 % cell viability) against HepG2 cells. The cellular uptake determined by ICP-MS showed a Ru content of 10 -8 M, confirming internalisation of the conjugate. Thus, this ADC is able to induce cell death, probably due to release of NO in the mitochondria, targeted thanks to the Ab.

Some of the first examples of Ru(II) bipyridyl glycoconjugate complexes were reported by

Kobayashi and co-workers. [START_REF] Hasegawa | Tris-bipyridine ruthenium complex-based glyco-clusters: amplified luminescence and enhanced lectin affinities[END_REF][START_REF] Hasegawa | Artificial Metalloglycoclusters: Compact Saccharide Shell to Induce High Lectin Affinity as Well as Strong Luminescence[END_REF] They coupled different carbohydrates (α-D-glucose, β-Dgalactose and α-D-mannose) to bpy ligands through amid bonds and a C3 alkyl spacer (Oglyco-bipyridines) to yield complexes 59-Glc, 59-Gal and 59-Man in their corresponding Λ and Δ isomers, except for 59-Gal (Fig. 30A) The stable glycoconjugates exhibited enhanced luminescence, when compared with [Ru(bpy-OH)3]Cl2. This increase could be assigned to the tightly packed saccharide clusters, which could isolate the luminescent core (the Ru centre) from the outer shell, avoiding thermal degradation. In addition, their ability as lectin binders towards concavalin A (ConA) and Ricinus comunnis agglutinin 120 (RCA120) were tested. The three carbohydrate derivatives exhibited high binding affinity to lectins, stronger than the corresponding free p-nitrophenyl glycoderivatives. The ICmin (minimun inhibition concentration) values for each complex showed the highest affinity for the mannocluster Λ-59-Man towards ConA (ICmin = 9.0 × 10 -8 M). Furthermore, no inhibition was observed for 59-Gal and 59-Glc against RCA120, and neither for 59-Gal against ConA. Conversely, the galactoconjugate (as the racemic mixture) inhibited RCA120 (ICmin = 2.5 × 10 -6 M) with moderate affinity. In order to work out the origin of the high affinity, the association constants and the thermodynamic parameters were determined. The association constants (Ka) towards ConA for Λ-59-Man and Λ-59-Glc were 5.4 × 10 6 and 9.5 × 10 5 M -1 , respectively. These are higher than the Ka for monomannoside and other mannosylated compounds. Regarding the thermodynamics (ΔGº = -9.7 and -8.4 kcal mol -1 , for Λ-59-Man and Λ-59-Glc, respectively), ΔHº and ΔSº were all negative, showing that the interaction was favourable in enthalpy and unfavourable in entropy. The stopped motion of the glycoclusters in the binding-sites of the lectins could be the reason of the negative entropy values. The strong luminescence of the using negatively charged lipids. After light irradiation and cleavage of the Ru-S bond, the remaining aqua complex was still interacting with the membrane with electrostatic interactions.

When neutral lipids were used, only the lipophilic tail were inserted in the membrane and, after light irradiation, the aqua derivative formed diffused away.

The Ru-cholesterol conjugate 75 (Fig. 44) [START_REF] Siewert | Chemical Swarming: Depending on Concentration, an Amphiphilic Ruthenium Polypyridyl Complex Induces Cell Death via Two Different Mechanisms[END_REF] was found to be a potent cytotoxic agent in the dark in various cell lines (EC50 = 5.0-6.5 µM), whereas neither the free cholesterol nor the aqua complex showed any toxicity. Also, the cellular uptake was higher for 75 than for the aqua complex. The authors found out that there were two different modes of action for the drug. Concentrations above 3.5 µM led to the formation of aggregates, which were able to form holes in the cell membrane and extract lipids and proteins from it. This caused the permeabilization of the cell membrane, like a detergent, taking the cell into necrosis. At lower concentrations, the conjugate behaved as a monomer and the lipophilic/fatty tail inserted in the cell membrane.

Since the internalization is a slow process, after short incubation times (1 to 6 h), light (λ = 455 nm, 10 min, 6.3 J•cm -2 ) triggers the release of the aqua derivative outside the cell, without affecting it. However, for longer incubation times (24 h), light induces the release of the aqua complex inside the cell, leading to high toxicity. In both cases, the similar mechanisms of action involve PACT principles.

The same authors prepared complex 76 (Fig. 44) and a derivative with 2-(methylthio)ethanol (Hmte) as the S-coordinating ligand, to try to better understand the behaviour of this kind of complexes in a lipid bilayer. When the bpy ligand was replaced by a sterically hindered dcbpy (6,6'-dichloro-2,2'-bipyridine), the corresponding aqua complex and the S-containing complexes were found to be in equilibrium at room temperature in the dark. However, the use of light shifted the equilibrium towards the aqua species. When light was switched off, the initial equilibrium was spontaneously recovered. This process is not thermally dependent and allows a direct control of the complexes over the lipid bilayer. [START_REF] Bahreman | Ruthenium polypyridyl complexes hopping at anionic lipid bilayers through a supramolecular bond sensitive to visible light[END_REF] Kinetic studies of the same complex allowed determining a two-step mechanism for the thermal binding of the aqua Another Ru-biotin conjugate 81 was prepared by Li et al. as a two-photon (TP)-PDT photosensitizer (Fig. 48). [START_REF] Li | A biotinylated ruthenium(II) photosensitizer for tumor-targeted two-photon photodynamic therapy[END_REF] The maximum TPA cross-section at 820 nm was 140.26 GM, much higher than that for the clinical PS tetraphenylporphyrin (H2TPP, 2.2 GM) and the singlet oxygen quantum yield was 0.87 after irradiation at 450 nm. These features make 81 a good PS for TP-PDT. This biotin conjugate showed lipophilicity (log P0/w = 1.77) favouring, a priori, the uptake in cells. The uptake was evaluated in biotin receptor (BR) positive A549R cells and BR negative HLF-a cells (primary human lung fibroblasts). Complex 81 was found to accumulate more in cancerous cells than healthy cells (13.9-fold accumulation in A549R than in HLF-a cells). A competitive uptake assay with biotin pre-treated cells showed that the uptake of BR positive A549R cells is mediated by BRs. Photocytotoxicity was assessed in BR positive HeLa, A549 and A549R cells lines and BR negative HLF-a cells under 820 nm (two-photon) and 450 nm (one-photon). While the complex was found to be non-toxic in the dark, after TP irradiation (820 nm), the toxicity increased in all the cell lines studied in this work, with the best results for A549R cells (IC50 = 3.3 µM). The phototoxicity upon one-photon irradiation (450 nm) was similar to the TP irradiation. 3D multicellular spheroids (MCTSs) were used to assess the behaviour of 81 under TP-PDT. Stronger phosphorescence was observed for 81 in MTCSs of A459R cells than in MTCSs based on HLF-a cells, highlighting again the selectivity towards cancerous cells. To check whether apoptosis was the mechanism of cell death, the activity of caspase-3/7 was evaluated in the dark and upon irradiation. In the dark, caspase activity remained intact, whereas, after light irradiation, caspase activity increased more than 3 times. All these results suggest that singlet oxygen or ROS generation upon light irradiation leads to apoptosis.

Very recently, Oliveira et al. synthesised a Ru(II) complex with an anthracene pendant 82 as PS for PDT (Fig. 48). This complex has three different ligands, a bpy-biotin modified ligand, a dppz ligand and an anthracene-pendant bpy ligand. The singlet oxygen quantum yield (ΦΔ) was

Declaration of Competing Interest

The authors declare no competing financial interests.