New Designs for Phototherapeutic Transition Metal Complexes

Abstract In this Minireview, we highlight recent advances in the design of transition metal complexes for photodynamic therapy (PDT) and photoactivated chemotherapy (PACT), and discuss the challenges and opportunities for the translation of such agents into clinical use. New designs for light‐activated transition metal complexes offer photoactivatable prodrugs with novel targeted mechanisms of action. Light irradiation can provide spatial and temporal control of drug activation, increasing selectivity and reducing side‐effects. The photophysical and photochemical properties of transition metal complexes can be controlled by the appropriate choice of the metal, its oxidation state, the number and types of ligands, and the coordination geometry.


Introduction
Nature uses light to promote and control some of the most crucial reactions,f rom photosynthesis,c onverting light and carbon dioxide to oxygen and sugar, to the synthesis of vitamin Dinour skin. In the same way,the power of light can be harnessed for medical applications.T he use of light in medicine (phototherapy) dates back to ancient Egypt, India and China for the treatment of skin diseases.I nt he 19 th century,N iels Ryberg Finsen used light for the treatment of smallpox and lupus vulgaris,which earned him the 1903 Nobel Prize in Physiology. [1] Later in the 20 th century,light was used to stimulate tissue healing (photobiostimulation), opening an ew avenue in phototherapy,b yd eliberately triggering intracellular photobiochemical reactions following light absorption by endogenous chromophores such as porphyrins. [2] In contrast, the use of light in combination with exogenous chemicals,t oc ontrol the therapeutic activity of agents selectively in space and time,h as been explored only in recent decades.P hotofrin, based on oligomers of porphyrin units,w as the first approved photosensitizer for clinical photodynamic therapy,i n1 993. [3] Importantly,t he potential for activating ad rug selectively in the tissue of interest, thus minimising side effects,i st he obvious,b ut not unique advantage of this strategy.B ecause photoactivatable molecules exert their therapeutic action via excited states,t he mechanism of action is often significantly different from traditional chemotherapeutics and less subject to crossresistance with existing pharmaceuticals. [4] This potential of photoactivatable therapeutics,c ombined with the development of laser-a nd LED-based technologies for effective delivery of light to different tissues,h as led to the development and clinical investigation of several photodynamic therapy agents,i ncluding four organic tetrapyrrolic derivatives (Photofrin, Chlorin e6, Visudyne and Foscan), and four metal-based photosensitizers:W ST11 (Pd II, ,a pproved for vascular-targeted of prostate cancer), [5] Lutex (Lu III ,lutetium texaphyrin, for cervical intraepithelial neoplasia), Purlytin (Sn IV ,a ge-related macular degeneration), and TLD1433 (Ru II ,non-muscle invasive bladder cancer). [5,6,8] Although the application of metal-based complexes for phototherapy is at ar elatively early stage compared to organic photosensitizers,t he chemical know-how accumulat-ed over years of experience with cisplatin and other metal-based drugs can accelerate development of photoactivatable complexes.I nt urn, progress in this area provides new scope for the application of bioinorganic chemistry in medicine.Here we review recent advances in the development of metalbased photoactivatable agents for both photodynamic therapy (PDT) and photoactivated chemotherapy (PACT), recognising opportunities and challenges for the clinical translation of these agents.Afew agents from our laboratories will be discussed in detail as examples of promising candidates for PDT and PACT.More general comprehensive reviews are available elsewhere. [6]

PDT Agents
Photodynamic therapy requires the simultaneous presence of three essential components:1 )a photosensitizer, 2) oxygen, and 3) light. Absorption of light excites the photosensitizer from the ground electronic state to an excited singlet state,which can convert to along-lived triplet excited state by intersystem crossing (ISC). Quenching of this state by biomolecules (type Ir eaction) or directly by molecular oxygen 3 O 2 (type II reactions) produces oxygen radicals and singlet oxygen ( 1 O 2 ), respectively.Both these reactive oxygen species (ROS) can induce severe oxidative damage to cellular biological molecules,r esulting in vascular injury and tumor cell death. Owing to the highly reactive nature of singlet oxygen ( 1 D g state 95 kJ mol À1 higher in energy than 3 O 2 ), type-II processes are generally considered as the main photosensitization mechanism in PDT. [7] Importantly,t he photo-Inthis Minireview,w ehighlight recent advances in the design of transition metal complexes for photodynamic therapy( PDT) and photoactivated chemotherapy(PACT), and discuss the challenges and opportunities for the translation of sucha gents into clinical use.N ew designs for light-activated transition metal complexes offer photoactivatable prodrugs with novel targeted mechanisms of action. Light irradiation can provide spatial and temporal control of drug activation, increasing selectivity and reducing side-effects.T he photophysical and photochemical properties of transition metal complexes can be controlled by the appropriate choice of the metal, its oxidation state,the number and types of ligands,and the coordination geometry. sensitizer returns to the ground state upon quenching and becomes newly available for another cycle.T herefore,i n principle,o nly ac atalytic amount of photosensitizer is required to achieve therapeutic efficacy.
Thedesign of photosensitizers has improved significantly in recent years through enhancement of their photophysical and biological properties.F igure 1s hows some of the most promising metal-based photosensitizers recently developed for clinical use,i ncluding tetrapyrrolic derivatives of Pd II (WST11, 1), Lu III (Lutex, 2), and Sn IV (Purlytin, 3). [5,6] Notably,t he presence of aP dc entre in WST11 improves stability and excited state reactivity and, at the same time, greatly enhances the ISC rate of the photosensitizer. [5] Another advantage of introducing an on-essential metal is that it can allow quantification and localization of the photosensitizer in cells and tissues by avariety of techniques, including X-ray absorption spectroscopy (XAS) and inductively coupled plasma-mass spectrometry (ICP-MS). Furthermore,i ft he metal complexes are luminescent, their cellular localization can often be precisely determined by confocal microscopy. [8] Ruthenium(II) polypyridyl complexes,i ncluding TLD1433 (4;F igure 1), the first metal-based photosensitizer without atetrapyrrolic moiety to enter clinical trials for nonmuscle invasive bladder cancer PDT (ClinicalTrials.gov, identifier NCT03053635), [6] are attracting significant attention as PDT photosensitizers,a sa re luminescent polypyridyl complexes of Ir III ,O s II and Re I . [9] In the next sections,w ep resent examples of promising new photosensitizers and consider in more detail their mechanism of action, with particular focus on their interaction with proteins upon irradiation. We also highlight efforts to improve delivery of metal-based photosensitizers to cancer cells by attaching them to serum proteins,and how this can result in targeted delivery and their localization in specific subcellular organelles.

Protein Oxidation by Metal-based Photosensitizers
Owing to their abundant and ubiquitous presence in cells and to their reactivity towards 1 O 2 and other ROS, proteins are generally regarded as the main target of photodynamic therapy,followed by lipids and nucleic acids. [10] In particular, cysteine,m ethionine,h istidine,t yrosine and tryptophan amino acid residues are especially susceptible to oxidative damage by 1 O 2 . [11] Although investigations of the effects of PDT agents on proteins have mainly revolved around organic photosensitizers,s ome reports also discuss interactions with metal-based PDT agents.
Thestrongly luminescent octahedral Ir III complexes 5 and 6 ( Figure 2) exhibit high PDT efficiency via both 1-photon and 2-photon light activation, with their extremely long phosphorescence lifetimes in cancer cells giving rise to ahigh 1 O 2 quantum yield. [12] Notably,t he O^O chelated complex 6 showed low dark toxicity to cells,b ut became potently cytotoxic upon irradiation with very low doses of visible light. Moreover,c omplex 6 exhibited sub-micromolar IC 50 values towards 3D multi-cellular tumor spheroids (models for solid tumors) when irradiated with 750 nm two-photon near infrared light. Their effects on proteins in A549 lung cancer cells, was investigated using liquid chromatography-tandem mass spectrometry,w hich revealed specific oxidation of histidine residues to 2-oxo-His in the key proteins aldose reductase and heat-shock protein-70 (Hsp-70) after photoactivation of complex 6.N otably,t he oxidative stress induced during the photoactivation increased the levels of enzymes involved in the glycolytic pathway.T his might arise from as witch in energy (ATP) production from oxidative phosphorylation (reduction of O 2 to H 2 Oi nm itochondria) to glycolysis (conversion of glucose to pyruvate in the cytoplasm). The results suggest that iridium complexes might target specific proteins in cancer cells and induce oxidation irreversibly upon photoactivation, contributing to efficient PDT.
Kwon et al. have recently reported structure-activity relationships for related cyclometalated Ir III complexes 7-10 ( Figure 3), for one-and two-photon PDT and targeting of the endoplasmic reticulum (ER). [13] This organelle plays an important role in protein synthesis,l ipid manufacture and metabolism, production of steroid hormones,a nd detoxification. ER damage leads to ER stress response,which can cause cell death via apoptosis involving the mitochondrial pathway. Importantly,t he favorable triplet state energy of Ir III complexes and high emission quantum yield of complex 9 resulted in ar emarkably high singlet oxygen quantum yield in water (95 %), along with superoxide production. Confocal microscopy revealed that 9 predominantly localized in the ER, and exhibited along luminescence lifetime (500 ns) in cancer cells. Upon low-dose "sunlight" irradiation (1.0 Jcm À2 ), 9 showed photocytotoxicity toward SK-OV-3o varian and MCF-7 breast cancer cells.T he potential use of 9 as at wo-photon PDT agent was further investigated by evaluating the morphological changes of SK-OV-3c ells,u pon irradiation with an 860 nm laser.Notably,cell shrinkage was observed as afunction of time also in these conditions,demonstrating twophoton-induced phototoxicity of 9.T he specific oxidative reactions arising from attack on endogenous proteins are attributed to both singlet oxygen (oxidation of methionine residues) and superoxide radicals (photo-induced cross-linking of metabolic proteins). Overall, ROSgeneration resulted in protein dysfunction and triggered cancer cell death.

Protein-assisted Delivery of Metal-based Photosensitizers
Thenatural association of metal-based drugs with human serum proteins whose transmembrane transporters are upregulated in malignant tissues,i sawell-known strategy to achieve selective accumulation of metal coordination complexes in tumors rather than in healthy tissues.A ccordingly, deliberate attachment of metal-based photosensitizers to serum proteins,e ither through covalent linking or noncovalent interaction, has been recently investigated as adelivery strategy for PDT agents to target cancerous tissues effectively.
Ruthenium(II) photosensitizers are known to associate with albumin and transferrin in serum, enabling highly www.angewandte.org efficient receptor-mediated transport into cancer cells. [14] Lilge and colleagues have recently developed rutherrin by combining TLD1433 and transferrin, which not only enhances effective cancer targeting, but also improves photosensitizer efficiency,w ith as ignificantly increased absorbance in the green-to-near infrared (NIR) range. [14] Weil and colleagues have reported 11-cHSA-PEO-TPP, ( Figure 4) as ad elivery system for the ruthenium-based photosensitizer 11,based on the blood protein serum albumin (HSA, the most abundant blood serum protein, ca. 0.6 mm)m ade polycationic and decorated with multiple triphenylphosphonium (TPP) groups to target mitochondria, and polyethylene oxide (PEO) chains to improve water solubility. [15] In addition to mitochondrial localization, the 11-cHSA-PEO-TPP conjugate exhibited significantly improved phosphorescence intensity and lifetime,aswell as enhanced 1 O 2 quantum yields compared to the parent Ru II complex (11), using blue (470 nm) light.
To improve the cancer targeting ability and photochemical properties of our organoiridium photosensitizer 12 (Figure 5), we anchored it to the free thiol of HSA at cysteine-34 using maleimide-functionalized ligands.T he conjugate exhibited significant enhancement of phosphorescence intensity compared to the complex alone,t ogether with surprising nuclear localization of the Ir III complex. [16] Titration studies indicated that the iridium(III) complex interacts with histidine,r esulting in an emission enhancement of about 37-fold. In contrast, no significant luminescence enhancement was observed on interaction with other amino acids,i ncluding Cys.T his suggested that, after binding of Ir to Cys 34, one sterically-hindered monodentate maleimide ligand is released, being displaced by ah istidine side-chain, likely nearby His 39. Ir III complexes with weakly bound ligands are known to bind strongly to amino acids/proteins through ligand substitution reactions, especially to histidine and histidine-rich proteins such as HSA. [17] The 12-HSA conjugate exhibited al onger phosphorescence lifetime than complex 12 and remarkably higher 1 O 2 generation quantum yield. Notably,t he 12-HSA conjugate showed little toxicity in the dark, but potent photocytotoxicity with significant selectivity for cancer cells,i ncluding cancer cell spheroids,over healthy cells.Surprisingly,upon uptake into the cells,HSA appears to facilitate delivery of the Ir into the nucleus.H owever,H SA itself cannot penetrate the nuclear membrane,a nd Ir is probably released from the 12-HSA conjugate and then migrates into the nucleus.I n contrast to more traditional Ir-based conjugates, 12-HSA conjugate seems to be the first example of an HSAfunctionalized iridium conjugate which targets cell nuclei.
Asupramolecular approach to protein-assisted delivery of PDT agents has also recently been explored. Liu and coworkers synthesised ap oly-phenanthroline Ru II photosensitizer with 3p endant cyclodextrin groups which strongly associate with adamantane-functionalized transferrin molecules via non-covalent binding of the adamantane group to the cyclodextrin cavity. [18] Notably,t he resulting supramolecular assembly exhibited selective accumulation in A549 lung cancer cells through transferrin receptor-mediated endocytosis,d isplaying promising PDT efficiency.I nterestingly,g lutathione was found to have ap rotective role in A549 cells during irradiation treatment with this supramolecular system.

Photoactivated Chemotherapy (PACT) Agents
Effective PDT relies on the presence of oxygen in the target tissue.H owever,t umor masses often contain hypoxic regions where,o wing to the suboptimal vascularization, the concentration of oxygen is remarkably low. [19] Photoactivated chemotherapy has the potential to overcome this limitation. [20] In PACT,l ight activation of otherwise inert metal complexes,c an produce cytotoxic species in ac ontrolled manner.F our main classes of PACT agents can be identified based on their mechanism of action, although the chemical complexity of these compounds can rarely be contained by such clear-cut boundaries,a s recently shown for some Ru II complexes. [21] 1) Photoinduced electron transfer:C omplexes with inert low-spin d 6 metal centres such as Pt IV [22] and Co III [23] can be activated by irradiation into their ligand-tometal charge-transfer (LMCT) bands causing the permanent transfer of electrons from the ligand to the metal centre,and ligand dissociation. [4] Thetherapeutic effect can be ascribed to the reduced metal complex, the released ligands,o rb oth. A different type of photoinduced electron transfer is observed for polyazaaromatic Ru II complexes,w here light irradiation oxidizes the metal centre generating an excited-state highlyoxidising Ru III species that can interact with DNAo ro ther biomolecules. [24] 2) Photosubstitution:Light irradiation of inert d 6 Ru II , [25] Rh III [26] and Ir III [27] complexes can create an excited metal-toligand charge-transfer triplet state ( 3 MLCT), which rapidly interconverts to ah ighly dissociative excited metal-centred triplet state ( 3 MC). This results in the release of aligand and generation of reactive species,w hich can both contribute to the therapeutic effect. [21] Complexes with different metals/ configuration, including aPt II -curcumin derivative,have also been investigated for photosubstitution reactions. [28] 3) Bioactivel igand release:S everal complexes based on Mn I ,Re I and Cr III centres can release,upon irradiation, small bioactive molecules such as NO [29] and CO. [30] Ru II complexes acting as photocages for enzyme inhibitors [31] or neurotransmitters [32] have also been investigated. 4) Ligand photocleavage:F or this class of complexes, photoactivation is ligand-centred and results in the formation

Azido Pt IV Agents
Thephotochemistry and photobiology of azido Pt IV PACT complexes have been studied in detail in our laboratory.I n general, octahedral Pt IV complexes are inert low-spin 5d 6 prodrugs for Pt II anticancer agents,b eing reduced by intracellular reduction (e.g.b ya scorbate or thiols such as glutathione). [34] However,e xtracellular reduction (e.g.i n blood) can limit their therapeutic efficacya nd increase side effects.F or example,t he Pt IV prodrug iproplatin has more severe side effects than carboplatin with no enhanced chemotherapeutic effects. [35] Accordingly,g iven also the promising clinical achievement of PDT in light-controlled oncotherapy,there is interest in controlling the reduction and activation of Pt IV prodrugs by light irradiation.
Thep hotoreduction of azido Pt IV complexes was first explored by Vogler who observed light-mediated reduction of trans-[Pt(N 3 ) 2 (CN) 4 ] 2À with release of azidyl radicals. [36] Our group has focused on the design of complexes with the general formula [Pt(N 3 ) 2 (OH) 2 (L)(L')] where La nd L' are a(m)mine ligands which can be cis or trans,w ith trans hydroxide ligands,a rbitrarily defined as axial ligands.Afew of the most studied complexes are shown in Figure 6a. Unexpectedly,c ell viability studies revealed an increased photocytotoxicity of the trans diammine complex 14 compared to the cis isomer 13.
[22a] Related trans complexes were also more active, [37] suggesting that this class of complexes has different modes of action compared to conventional cisdiam(m)ine Pt II drugs.T he potency of trans Pt IV diazido agents increases for complexes bearing ab ulky amine substituent, as in 15 [38] or 16.
[22b] Notably,a ni nv ivo experi-ment with complex 16 demonstrated the efficacy of this complex in slowing the growth of OE19 oesophageal cancer xenografts in nude mice using low doses of 16 and short irradiation times with blue light. [39] Complex 18 with two pyridine ligands exhibited the highest cytotoxicity upon irradiation. [22d] Ar elatively straightforward pathway was initially proposed for the photoreduction of Pt IV azido complexes via reductive elimination, to release two azidyl radicals (which could form 3m olecules of N 2 )a nd yield as quare-planar Pt II species.A lthough this mechanism was supported by UV-vis measurements showing ar apid decrease in intensity of the band assigned to the Pt ! N 3 LMCT upon irradiation, different analytical techniques revealed that several decomposition pathways are available depending on the structure of the complex and on the solution environment. Thep hotoactivated processes are not limited to photoreduction, but also include photosubstitution and, for cis complexes,photoisomerisation. [40] Pathways for atypical trans photoactivatable Pt IV complex are summarised in Figure 6b.N otably,a ll the observed photoproducts have lost at least one azide ligand. Liberation of the ammine ligand was also observed for L = NH 3 ,but not for bulkier secondary or tertiary amines such as piperidine and pyridine,r espectively. [38,41] Then umber of possible photoreduction pathways seemed somewhat reduced for complexes with more sterically-hindered amines,w ith three photoproducts only observed for 16, [42] and one for 15. [43] Theefficiency of photodecomposition processes for these diazido Pt IV complexes depends intimately on the types of ligands and their stereochemistry.C omplexes with at least one bulky amine are reduced more rapidly, [38] and, notably, trans complexes can be reduced at longer wavelengths and to ag reater extent than their cis analogues. [22a] This was rationalised by DFT calculations,s howing how low-lying excited states have alower energy for trans complexes. [44] Figure 6. a) Chemical structures of selected azido Pt IV complexes, and b) various photoproducts detectedu pon irradiation (the complexes for which aparticular photoproduct has been detected experimentallya re indicated). Charges are omitted for clarity.
Investigating interactions of biomolecules with these complexes upon irradiation is key to understanding the photoproducts that could form inside cells.U nexpectedly, irrespective of the azido Pt IV complex, guanosine monophosphate (GMP) markedly accelerated the photoreduction process,likely by trapping the resulting Pt II species. [43] Similar to formation of bis(GMP) adducts by cisplatin, [Pt(L)(L')-(GMP-N7) 2 ] 2+ readily formed, and more rapidly for the trans than the cis isomers. [45,22a] However,o ther adducts were also formed including the mono adduct [Pt(N 3 )(L)(L')(GMP-N7)] + ,f or 16-18 at short irradiation times. [22b,d] Notably, 17 induced oxidation of guanine upon irradiation, likely via singlet oxygen generation even under an argon atmosphere and, remarkably,f ormation of the ammine complex [Pt-(NH 3 )(py)(MA)(8-OH-GMP)] probably via an itrene intermediate. [46] DNAp latination is observed upon irradiation, but not in the dark, and is slower for complexes carrying bulky ligands. [38,42] Bulky trans complexes generate fewer plasmid DNAi ntrastrand crosslinks than cisplatin, but cause much larger DNAu nwinding. [22b,38, 47] Also they form adducts that are less effectively removed by DNAr epair mechanisms, [22b] and stall RNAp ol II transcription to ag reater extent than cisplatin. [47] Thep hotoreduction of complex 13 in the presence of dimethyl sulphide and 1-methylimidazole (models for methionine and histidine side chains,r espectively), suggested that these complexes might also attack proteins upon irradiation. [40,41] Furthermore,s ulfur oxidation and evolution of O 2 suggested that ROSmay be produced in the process. [41a] High resolution mass spectrometry studies with complex 18 confirm its ability to platinate peptides (subP and bombesin) and proteins (thioredoxin) upon irradiation, and to oxidise some amino acid residues (Met, Tr pa nd Cys). [48] Azidyl radicals liberated by photoreduction of 18 can extract an electron from tryptophan to generate Tr pr adicals which can be trapped and detected by EPR. [49] This suggests that electron transport pathways in cells might be attacked, although the quenching of azidyl radicals by Tr pi tself can have ap rotective effect against phototoxicity in cells.A similar effect was observed for the antioxidant melatonin. [49a]

Mechanisms of Cytotoxicity for Azido Pt IV Complexes
Although initially conceived as prodrugs for cisplatin and related conventional anticancer drugs,diazido Pt IV complexes possess unique mechanisms of action;t he trans complexes have higher potencyc ompared to their cis isomers,a nd lack cross-resistance with cisplatin. Different mechanisms of cytotoxicity are involved especially for trans diazido complexes. [22] In particular for complex 16, trans-[Pt(N 3 ) 2 (OH) 2 -(NH 3 )(pyridine)],s ignificant DNAc ross-linking is observed only at aconcentration of 2 IC 50 . [22b] Importantly,t he lipophilicity of these diazido complexes does not correlate with cellular accumulation of platinum (in the dark), and this,i nt urn, is not correlated with the photocytotoxicity. [50] On the other hand, Pt accumulation in cells upon irradiation appears to correlate with cytotoxicity for complexes 13,and 16-18. [38,47,51] Hence photoexcited state structure-activity relationships and the nature of the photoproducts are probably key to understanding biological potency.
In addition to platinum-mediated toxicity,n itrogen/oxygen radical species generated upon irradiation can also contribute to the cytotoxicity of these complexes.I nhibition of thioredoxin following treatment with 18, trans-[Pt(N 3 ) 2 -(OH) 2 (pyridine) 2 ], and irradiation, has been attributed to oxidation rather than platination suggesting an important role for ROSi nt he toxicity. [48b] Furthermore,asignificant production of RNS/ROS is observed for 6hafter irradiation of cancer cells treated with 16 or 18. [43, 48b] Delivery of azide ions to cancer cells might also play arole in the biological activity of these complexes,given the ability of azide to inhibit awide range of heme proteins,b ut this has been little explored so far. [52] Intriguingly,u pon irradiation, 13 trans,trans,cis-[Pt(N 3 ) 2 -(OH) 2 (NH 3 ) 2 ]dramatically altered the morphology of cancer cells causing ballooning followed by cellular shrinkage,loss of contact with neighbouring cells,and detachment from the cell culture flask. Although nuclear packing was observed, the absence of other hallmarks of apoptosis (such as blebbing and cellular fragmentation) suggested ad ifferent mechanism of cell death for this complex. [51] Similarly,compound 16 did not show apoptotic markers such as caspase 3and 7activation, or redistribution of phosphatidyl serine.A lso,i nc ontrast to cisplatin, no arrest of the cell cycle was detected for this compound. Rather, 16 seemed to induce autophagic cell death, showing increased levels of proteins associated with this self-eating process. [39] Involvement of the immune system in the mechanism of action of several metal anticancer complexes has been recently recognised. [53] Notably,oxaliplatin, but not cisplatin, promotes immunogenic cell death in vivo, [54] likely due to its different mechanism of action, relying on generation of ribosome biogenesis stress rather than DNAd amage. [55] The role of immunogenic cell death in the mechanism of action of photoactivatable Pt IV complexes is currently being explored.

Improving Delivery of Azido Pt IV Complexes
Thet wo hydroxido ligands of complexes 13-18 allow for their facile derivatization via ester bond formation. In particular,t he succinate derivative obtained by reaction of those complexes with succinic anhydride is av ery useful intermediate to link such complexes to targeting moieties or delivery platforms in order to improve their accumulation in target tissues.
Exploiting this derivativatisation route,M archansg roup attached complex 18 to ac yclic RGD peptide to target the a v b 3 integrin receptor overexpressed on the surface of several cancer cells and angiogenic vasculature.T he resulting bioconjugate 18-RGD (Figure 7) accumulated selectively and displayed antiproliferative activity in SK-MEL-28 melanoma cancer cells which exhibit high expression of the a v b 3 integrin receptor. [56] Angewandte Chemie Minireviews 68 www.angewandte.org Another derivatization strategy consists in tethering these complexes to nanoparticulate platforms.T his can improve delivery to tumors in vivo,through the enhanced permeation and retention (EPR) effect, and the nanoparticles can be further decorated with cancer-targeting moieties and/or additional cytotoxic agents.P rominent examples of such systems include the near-infrared photoactivatable nanoplatform developed by Xing and co-workers,w here complex 18 was attached, via the succinate moiety,t ou pconversion nanoparticles (UCNP), allowing the Pt IV complex to be activated by near-infrared radiation (l = 980 nm). [57] Interestingly,t his platform also included an apopotosis-sensitive fluorescent peptide thus allowing imaging of the apoptotic process in living cells.
More recently,c omplex 13 was attached to the PDT photosensitizer chlorin e6 (Ce6) via aP EG linker. [58] Irradiation of the resulting 13-PEG-Ce6 system (Figure 7) not only resulted in the activation of the Pt IV azido PACT agent, but also concomitant production of molecular oxygen, allowing the photosensitizer to maintain its function under hypoxic conditions.Interestingly,owing to its amphiphilic nature, 13-PEG-Ce6 self-assembled into micelles that, when loaded with UCNP,c ould be activated with near-infrared light. This nanocomposite system was tested in vivo in xenograft tumor models,a nd showed effective tumor accumulation and promising anticancer activity upon NIR irradiation.
Finally,h ydrogels have been explored in our group as promising drug delivery systems because of their biocompatibility and tissue-mimicking properties. [59] In particular, there is potential for topical treatment of skin conditions,s uch as non-melanoma skin cancer, at issue especially accessible for PACT.T hrough as elf-assembly reaction, ad opamine derivative of complex 18 was incorporated into aG -quadruplex borate hydrogel, and the resulting material (18-hydrogel, Figure 8) retained the promising antiproliferative activity of its precursor complex. [60] More extensive preclinical testing will be required to verify effective delivery of the Pt IV -azido agent to cancer cells in vivo.

Challenges for Clinical Translation of Photochemotherapeutic Agents
Photoactivation at asuitable wavelength to control depth penetration, is amajor consideration in the clinical translation of metal complexes for phototherapy.I deally the complex should be activated at awavelength whose tissue penetration matches the desired depth of the treatment. Generally,s ince most clinical applications require ap enetration depth > 1mm, light absorbance in the red or NIR regions (including NIR-I:6 50-950 nm, and NIR-II:1000-1350 nm) is advantageous. [1,61] On the other hand, at longer wavelengths (> 900 nm) light absorption by water increases significantly (with l max at 960, 1440 and 1950 nm), thus limiting the range of wavelength available for PDT in the NIR-II. [62] Notably,some polypyridyl ruthenium complexes can achieve high red-light PDT efficiencydue to their low-lying and long-lived emissive intra-ligand triplet excited states,despite their low absorption coefficients at this wavelength. [63] Attempts to achieve longerwavelength photoactivation include attachment to upconversion nanoparticles [64] and multiphoton photosensitization. [65] However,m ultiphoton PDT also has limitations,p osing new challenges for its clinical application. Unlike one photon PDT that can be easily carried out with low power lasers and LEDs, two photon PDT requires higher power lasers for successful photosensitization. [66] Although the area irradiated with 2photon light can be highly specific,t he volume of irradiated tissue is very small ( % 1 mm 3 ), [65] which will necessiate new rastering techniques if wide areas are to be treated. Finally, extremely deep tissue penetration of near-IR light activation would be undesirable for superficial lesions,s uch as gliomas, or non-muscle-invasive bladder cancer (for which TLD1433  in clinical trials with green light PDT), [67] where reducing damage to the underlying healthy tissue is crucial. [68] In conclusion, the ideal photosensitizer for each specific application should exhibit large molar absorption cross sections in the wavelength range that matches tumor invasion depth.
Due to their high reliance on oxygen concentration, the presence of hypoxic regions in solid tumors is another major challenge for PDT agents.Combination in the same molecule of aPDT photosensitizer and aP ACTagent able to generate oxygen upon light irradiation provides ap ossible strategy to circumvent this issue,p roducing oxygen-independent PDT photosensitizers,a sr ecently demonstrated for 13-PEG-Ce6. [58] Other strategies that have been pursued include using nanocomposite systems able to catalyse H 2 O 2 decomposition to molecular oxygen in hypoxic cancer cells. [69] In order to reduce PDT-induced hypoxia, photosensitizers able to inhibit cellular respiration have also been used to ensure arelatively high level of intracellular oxygen in the treated areas. [70] Combination therapy with an hypoxia-targeting cytotoxic agent is also able to mitigate the effect of PDT-induced hypoxia, as recently demonstrated by Kim and co-workers. [71] Their novel PDT agent combines aBODIPY photosensitizer with the anti-angiogenesis moiety acetazolamide,i nhibiting carbonic anhydrase IX upregulated in hypoxic cancer cells. [71] Finally,inrecent years,the type-I photosensitization pathway has also attracted attention for PDT in hypoxic regions.T ype-IP DT generates superoxide (CO 2 À )a nd hydroxyl (COH) radicals as well as hydrogen peroxide (H 2 O 2 ). [72] Similar to 1 O 2 ,t he highly reactive radicals COH and CO 2 À can react rapidly with adjacent biomolecules,d isrupting normal cell functions and resulting in cancer cell death. Them ore stable H 2 O 2 can diffuse across cell membranes and generate COH radicals by reaction with cellular ferrous ions (Fenton reaction) to enhance the PDT effect, especially in tumor cells with high iron demand. [73] Tu mor-targeting efficiency is another important issue for both PDT and PACT agents,e specially for systemic administration where non-selective accumulation of these compounds can result in photosensitivity for light-exposed organs such as skin and eyes.Attachment to tumor-targeting vectors such as antibodies,s erum proteins and cancer-targeting peptides,c an greatly increase the clinical utility of these agents by minimising side effects and reducing the amount of agent needed for at herapeutic response.A lthough some cancer-targeting agents have been developed in recent years, [74] as described in this review,their biological evaluation has mostly been limited to in vitro models and their targeting capability in vivo remains to be verified.
Finally,itisimportant to acknowledge that, because of the localised nature of PDT and PACT treatment, their clinical utility is intrinsically limited to localised disease.T ypical indications for photoactivatable agents in oncology include premalignancies,w hich have not yet spread from their primary site,orsmall areas of recurrent or persistent cancer, where this treatment can be combined effectively with surgery and radiotherapy.Afuture challenge is to devise effective strategies which might allow their application to invasive diseases such as metastatic cancer and blood cancer. One way to achieve this might be to remove the need for an external light source by,f or example,i ncorporating ac hemiluminescent fragment into the photoactivatable complex, if chemiluminescence could be triggered at the tumor site.N o examples of this approach have been reported so far.

Conclusions
We have focussed in this Minireview on new designs for photochemotherapeutic transition metal anticancer complexes which have emerged recently,i ncluding octahedral Ir III PDT photosensitizers and Pt IV PACT agents,b oth of which have classical inert low-spin 5d 6 configurations.T his means that after administration, and in the dark, they are likely to reach target sites in tumors intact.
Some tris-chelated Ir III complexes,s uch as complex 6, exhibit long-lived triplet excited states and their phosphorescence not only allows them to be imaged (mapped) in cells, but also to generate destructive singlet oxygen ( 1 O 2 )e fficiently,e ven under relatively hypoxic conditions.T hey are photocatalysts and therefore active at low doses with good selectivity between tumor and normal cells.A sw ell as 1photon activation by visible light, they can be activated by NIR radiation using 2-photon laser methods,w hich increase the range of depth penetration. Surprisingly we discovered that singlet oxygen can cause specific damage to histidine residues in Hsp-70 and aldose reductase,proteins which play important roles in cancer cells.This suggests that it should be possible to damage specific cellular target proteins by designing appropriate photosensitizers which can pre-associate with targets.I nt he case of bis-N,C-chelated Ir III complexes (e.g. complex 12), we have shown that conjugation to the abundant serum protein albumin can have ad ramatic effect on the transport of the photosensitizer into cancer cells,a nd specifically deliver the photosensitizer to the cell nucleus. This is remarkable,a lthough relatively little is currently known about the uptake of albumin into cells.M oreover, conjugation to HSA greatly enhanced phosphorescence of the Ir III complex, which again showed good selectivity between normal and human cells,and could be activated by irradiation with visible light.
Unlike PDT agents,d iazido Pt IV complexes are not catalysts since they decompose when photoactivated, including photoreduction to Pt II and release of azidyl and hydroxyl radicals.They can also form nitrenes and generate oxygen. In contrast to clinical platinum drugs such as cisplatin and carboplatin, the photoactivated trans diazido complexes are more active than their cis analogues.These complexes induce attack on both DNAa nd proteins.S pecific peptide damage includes oxidation of methionine and tryptophan side-chains and interaction of released azidyl radicals with Tr pgenerates Tr pr adicals.H ence their anticancer mechanism of action is quite distinct from that of cisplatin, and they have potential for treating Pt-resistant cancers,e specially surface cancers such as bladder and oesophageal. Derivatization of these complexes,v ia the axial hydroxido ligands allows the introduction of cancer-targeting vectors or the conjugation to other types of delivery systems such as nanoparticles or hydrogels.