Marta Jakubaszek

Bruno Goud

Stefano Ferrari

Gilles

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

Mechanisms of Action of Ru(II) Polypyridyl Complexes in Living Cells upon Light Irradiation

published or not. The documents may come

Mechanisms of action of Ru( ii ) polypyridyl complexes in living cells upon light irradiation

Introduction

The earliest reports on the use of light in combination with chemical entities in the field of medicine are more than 100 years old. [START_REF] Kessel | [END_REF] Since then, this medical technique, known as photodynamic therapy (PDT), has evolved to a successful alternative or complimentary treatment to chemotherapy, radiotherapy and surgery. Nowadays PDT is an approved and common treatment in dermatology. It is used to treat acne, psoriasis, keloid scars and port wine stains, helping patients to improve their appearance and quality of life. 2,3 PDT also gives another, new perspective for cancer therapy due to its spatial and temporal control. 4 This treatment modality is currently approved for a wide range of cancer types using commercially available photosensitisers such as Photofrin®, Visudyne®, Foscan® or Levulan® (see Figure 1 for structures). 5,6 PDT usually requires three main components, namely a photosensitiser (PS), molecular oxygen ( 3 O2) and light. After injection/application of the PS into/on the patient, the latter is irradiated at a specific, defined wavelength, allowing the PS to reach its singlet excited state 1 PS * . After intersystem crossing (ISC), the PS reaches an excited state, which has a triplet character ( 3 PS * ).

It might then react in two different electron exchange mechanisms, resulting in the formation of very reactive singlet oxygen 1 O2 (Type II) or radical anions or cations, which can further react with oxygen producing other reactive oxygen species (ROS) like hydrogen peroxide H2O2, superoxide O2 -or hydroxyl radicals • OH (Type I). Both mechanisms, namely Types I and II, lead to the formation of products that impair metabolic pathways and eventually lead to eukaryotic cell or bacteria death. The ratio between these two processes depends on the PS used as well as the concentrations of molecular oxygen and other biological substrates. 7 The most attractive feature of PDT is its subsistent selectivity. Indeed, areas that are affected by PDT treatments are only those where the PS has accumulated and where light is applied.

Additionally, due to the short life of generated 1 O2 (40 ns) and radicals, the area of action is estimated to be only 20 nm. 8 Currently used PSs are based on cyclic tetrapyrrolic structures like porphyrins, phtalocyanines or chlorins. 9 Although they fill the requirements of a PS, they also have a number of drawbacks.

Photofrin®, for example, exhibits poor light penetration into the tumour as well as low clearance from the patients bodies that leads to photosensitivity. 10 There is therefore a need for new PSs that overcome these unwanted effects and that have a higher uptake and selectivity towards cancer cells. 11 Recently designed molecules can be classified in two main classes, namely modified porphyrin-based PSs or porphyrin-free PDT systems. 6 In the second class, inert Ru(II) polypyridyl complexes have raised great interest not only as alternatives to cisplatin but also as a novel PDT PSs because of their favourable photophysical properties (e.g. long excited state lifetimes, visible light absorption and two-photon excitation). 6,[12][13][14][15] One of these compounds, namely TLD-1433, is currently undergoing a human clinical trial against invasive bladder cancer (Figure 2).

Understanding the mechanism(s) of action of these compounds in living cells/mice upon light irradiation is extremely important to establish their therapeutic potential and to design new generation PSs. Unfortunately, to date, there is a scarcity of studies exploring in depth the mode(s) of action of these compounds. 16 In this feature article, we review only biological studies that describe more than just the phototoxicity and the cellular localisation of some Ru(II) complexes, starting from the results obtained with TLD-1433, the PS of the McFarland group currently in clinical trial. To the end of our feature article, we have decided to classify the Ru(II) complexes depending on their cellular localisation. Of note, only coordinatively saturated and substitutionally inert Ru(II) polypyridyl complexes are discussed herein.

TLD-1433 and its derivatives

In 2013, the group of prof. McFarland reported two compounds, namely TLD-1411 and TLD-1433 (see Figure 2). 17 Both molecules were first investigated for photodynamic inactivation (PDI) of pathogenic bacteria. in vitro and in vivo was reported by Lilge and co-workers. 18 For in vitro studies, four cell lines were used, namely CT26 and CT25.26 (respectively wild type and N-nitroso-Nmethylurethane-induced mouse colon carcinoma), U87MG (human glioblastoma cell line) and F98 (rat glioblastoma). The Lethal Dose to kill 50 % of the cell population (LD50) was determined for TLD-1411 and TLD-1433 on all four cell lines in the dark and after light irradiation. Concentration of 4 µM of TLD-1411 and 1 µM of TLD-1433 effectively killed 100% of CT-26 WT and U87MG cells upon light irradiation (green LED emitting at 525 ± 25 nm; 45 J cm -2 ). U87MG cells were chosen to check whether these PSs could be used in hypoxic and normoxic conditions. A photodynamic effect was observed in normoxic conditions with concentrations of 18 µM (70% of cells killed). Unfortunately, TLD-1411 and TLD-1433 did not work in hypoxia conditions in human cell lines. The compounds were also tested in vivo using 8-10 week-old BALB/C mice injected with CT26.WT murine colon carcinoma. The maximum tolerated dose 50 (MTD50) values for TLD-1411 and TLD-1433 were established to be 36 mg . kg -1 and 103 mg . kg -1 , respectively. Mice treated with doses of TLD-1411 higher than MTD50 showed sign of weakness, ataxia and died a couple of days post-injection. On the contrary, TLD-1433 when given at higher doses than MTD50 did not cause death and all behavioural symptoms disappeared 24 h post-injection. Accumulation studies showed that both compounds were detectable in the tumour, liver and brain after 24 h. Tumour concentration of TLD-1411 was lower than the one of TLD-1433 (4.32 µM to 16.1 µM). The efficacy of PDT treatment was also tested using the same mouse model. Mice with grown tumours were injected with compounds and irradiated after 4 h thereafter with 190 J . cm -2 for 32 min in 30 s cycles.

Tumours were significantly reduced when treated with 2 mg . kg -1 of TLD-1411 and displayed a growth delay of 8 days. However, all tumours recurred. A higher dose of 5 mg . kg -1 of TLD-1433 gave a tumour reduction and growth delay of 9 days. The researchers also checked whether continuous wave (cw lasers) or pulsed lasers would give better results with the tested PSs. Cw lasers are regularly used in PDT applications. Pulsed ones have the advantage of lowering down the local tissue heating, keeping the high power density. Mice treated with TLD-1411 and TLD-1433 showed significant increase in survival when higher doses of the compounds as well as cw light source was applied.

Upregulated receptors or cell surface markers in cancer cells are useful targets for therapeutic agents. Usually, targeting mosaic is conjugated with the complex. It is also common to use the association of the serum or membrane proteins with the active compound in non-covalent manner to improve compound uptake. Ru complexes are known to associate with human serum albumin (HSA) or transferrin. 19,20 Transferrin is a 78 kDa glycoprotein necessary for chelating Fe 3+ from the serum. 21 Cancer cells display upregulated levels of transferrin receptors due to their higher demand for Fe 3+ to grow. 22 In 2016, Lilge et al. confirmed that the uptake of TLD-1433 as well as ROS production upon light irradiation (96 laser diode array light source; 625 nm; 90 ± 6 J cm -2 ) were improved in cell free environment when the complex was mixed with transferrin. 23 TLD-1433 associated with transferrin showed also lower dark cytotoxicity, probably due to enhanced Fe 3+ delivery to the cancer cells, and resistance to photobleaching in contrary to TLD-1433 alone.

Cell localisation of TLD-1433 and its impact on cell metabolism by changing the cellular redox balance was published in a recent study. 24 Colocalisation studies performed by confocal and time-resolved laser scanning microscopy were inconclusive. Additionally, fluorescence signals of the tracking dyes vanished before the TLD-1433 signal could be detected. It is possible that redox reactions and complex activation during laser scanning could be the reason for that unexpected phenomena.

The good results obtained with TLD-1433 led to the preparation of a series of cyclometalated Ru(II) complexes similar to TLD 1433 structure (1-4, Figure 3). 25 Cyclometalated Ru(II) complexes are usually more photostable and their absorption spectra is red-shifted compared to diamine Ru(II) complexes. The (photo-)toxicity of the complexes was checked in two cell lines, namely SK-MEL-28 (melanoma) and CCD-1064Sk (normal skin fibroblasts). Complexes 1-3 were found to be highly cytotoxic in the dark towards melanoma cell line and were affecting much less normal skin fibroblasts. Complex 4 did not show any cytotoxicity in the dark. Upon irradiation with visible light (400-700 nm, 34.7 mW . cm -2 ), all complexes appeared to be extremely cytotoxic to melanoma cells. Particularly, complex 4 had a surprising PI of more than 1100, much higher than the three other complexes. To determine if complexes 1-4 would possibly bind to DNA, a mobility shift assay was performed. Upon light irradiation with visible light, the pUC19 plasmid formed aggregates in the presence of the complexes. No single-strand nor double-strand DNA breaks were observed under these conditions. Ethidium bromide staining with or without light irradiation was impaired, presumably as a result of the intercalation of complexes 1-4 into DNA, or quenching of the ethidium bromide fluorescence. Confocal microscopy and DIC images were taken to assess compounds uptake and cells morphology before and after light treatment (400-700 nm, 34.7 mW . cm -2 , 50 J . cm -2 ). Complexes 1 and 2, which had the highest uptake in melanoma cells, as determined by confocal microscopy, were not taken up by non-cancerous cells. Complexes 3 and 4, despite their lower uptake in melanoma cells, caused impressive changes of cell shape upon light irradiation, contrary to complexes 1 and 2.

Mitochondria targeting compounds

Mitochondria are the cell energy centres and play and important role in the intrinsic apoptotic pathway. DNA damage, metabolic stress or the presence of unfolded proteins might lead to the permeabilisation of mitochondrial outer membrane. The release of mitochondrial proteins into the cytosol (e.g. cytochrome c) activates an apoptotic signalling cascade and finally leads to cell death. 26 Generation of singlet oxygen or other ROS in this organelle might trigger a rapid apoptotic response in the targeted cell, making this cellular compartment an interesting target for PDT photosensitizers.

Two Ru(II) polypyridyl compounds that target mitochondria functionalized with tyrosine and tryptophan were designed in 2013 (Figure 4). 27 Both amino acids were chosen to improve the cellular uptake of the Ru complexes. Cytotoxicity in the dark and upon light irradiation (4 h with visible light source λ~450-480 nm, 10 J cm -2 ) of both compounds was examined in A549 (pulmonary carcinoma) and HCT116 (colon cancer) cell line. Promising phototoxic index (PI) values in A549 cell line (>10 for complex 5 and >10 for complex 6) and in HCT116 (>9 and >10, respectively) encouraged the authors to perform further biological studies.

Singlet oxygen 1 O2 production upon light irradiation was confirmed and was suggested to be responsible for cell death. Fluorescence spectroscopy, UV-Vis absorption and isothermal titration calorimetry experiments showed that the Ru(II) complexes were able to bind CT-DNA in a non-covalent way, probably by intercalation into the DNA groove. Irradiation of pUC19 plasmid with the Ru(II) complexes led to photo-cleavage of the DNA, suggesting this mechanism as the main cause of cell death. This finding was further confirmed by single cell gel electrophoresis, which revealed DNA damage in treated A549 cells upon light irradiation.

Confocal laser scanning microscopy helped identify the cellular localisation of the complexes in A549 cells. Unexpectedly, none of the compounds was found to localise in the cell nucleus.

Signals from Mitotracker Green suggested the presence of the complexes in mitochondria and cell membranes. Microscopy studies after light irradiation would have been an interesting addition to the work since some of the compounds are known to modify their localisation after illumination of the cells. 28 Nevertheless, singlet oxygen is known to alter the mitochondrial trans-membrane potential, which might trigger the apoptotic pathway. To further investigate the molecular mechanism of cell death, western blot analysis was performed. It revealed that caspase-3, a marker of apoptosis, was found to be overexpressed in irradiated cells. The researchers concluded that the mechanism of cell death included the disruption of mitochondria membrane potential that, in turn, triggered the caspase-3-dependent apoptotic pathway.

Biological evaluation on Ru(II) complexes containing pdppz ([2,3-h]dipyrido[3,2-a:2',3'c]phenazine) ligands was published in 2015. 16 Complexes 7 and 8 were expected to bind DNA because of their extended dppz ligand, while complex 9 was used as a control (see Figure 5 for chemical structures). Experiments confirmed that complexes 7 and 8 were able to intercalate DNA in non-cell environment. Incubation of complex 8 with the plasmid pBR322 upon light irradiation (390nm, 2 J cm -2 ) caused single and double breaks in the DNA. Such effect was not seen with complex 7. It was shown that HeLa cells could actively uptake compounds 7 and 8-in a temperature-dependent manner. Confocal microscopy studies of complex 8 demonstrated that this compound colocalised with mitochondria and lysosomes, which clustered near the nucleus. It is possible that small amounts of 8 were also able to localise to the nucleus. Alkaline comet assay revealed DNA damage in treated and irradiated cells. ICP-MS experiments would have been an attractive addition to this study. The triphenylphospine (TPP) present in complexes 12 and 13 adds lipophilic character to the compounds, resulting in better mitochondria targeting abilities. 30 Confocal microscopy with Mitotracker Green in HeLa cell line revealed that complex 13 localises in the mitochondria.

Three other compounds were found to moderately localise in that compartment. Localisation results were confirmed by ICP-MS analysis, showing that complexes 10-12 were present in higher amount in the cytoplasm (Figure 7). All four compounds were designed to produce singlet oxygen not only using a one-photon but also a two-photon irradiation process. Confocal microscopy images of Hela cells taken before and after two-photon irradiation (810-830 nm for 3 minutes; 800 J . cm -1 ) with 2,7dichlorodihydro-fluorescein diacetate (DCFH-DA) allowed verifying singlet oxygen production in all samples. Dark and light cytotoxicities of all compounds were tested under one-photon irradiation. Compounds were not toxic under dark conditions (IC50 >100 µM). After irradiation (LED source; 450 nm; 12 J . cm -2 ), complexes 10-12 showed similar cytotoxicity, varying from 12.4 to 15.5 µM. Probably due to its high concentration in mitochondria, complex 13 was found to be the most effective compound tested, with a PI >28. Since monolayer cell cultures are not a good model for tumour treatment, HeLa multicellular tumour spheroids (MCTS) were used for further tests. Diffusion of the compounds (10 µM, 8 h treatment) was examined in 800 µm MCTSs. Treated spheroids were imaged with one-photon and two-photon z-stack microscopy. The luminescence signal of the compounds was found in all depth sections of the spheroids. Two-photon microscopy showed deeper penetration of the complexes through spheroids than one-photon microscopy, probably due to its excitation wavelength in the therapeutic window. This confirmed the high permeability of the complexes through the MCTSs. Singlet oxygen generation with DCFH-DA was also investigated in MCTS.

Enrichment of the singlet oxygen signal was observed in the treated spheroids. The results showed lower signal of produced singlet oxygen in the cores of the spheroids as compared to their surface. Compounds treatment also inhibited MCTSs growth after irradiation with twophoton technique. The best results were obtained again with complex 13. All synthesised compounds exhibited good photodynamic therapy ability against the HeLa cell line. However, further investigations should include healthy cells to establish a possible therapeutic window for these compounds.

An interesting study was recently published by the same group, who designed mitochondrialocalising Ru(II) complexes that can be activated by glutathione (GSH). 31 The aim of the study was to improve the tumour selectivity of the Ru complexes that are used as PDT PSs. Complex 14 is a dinuclear Ru(II) complex, which is bridged by a GSH activating ligand 4,4"-azobis(2,2'bipyridine) (Figure 8). Specific properties of the ligand cause quenching of luminescence of the Ru complex. Since intracellular concentration of GSH in cancer cells are higher than in healthy ones, the authors were hoping that the complex would be activated and transformed into complex 15 (Figure 8), and this mostly in cancer cells. In 2018, Stang, Chao and coworkers prepared a tetrametallic macrocyclic structure containing Ru(II) and Pt(II) atoms, that can be used in two-photon PDT (Figure 9). 32 and irradiated with TP laser (800nm, 50 mW, 20 s mm -1 ) did not exhibit observable weight loss.

The tumour volume of the treated group was reduced by 78%, while control mice groups showed 13-fold increase in tumour mass. Additionally, examination of tumour tissue of the treated group showed pathological changes, which were not observed in other organs like liver, kidney, heart, etc.

Nucleus targeting compounds

Besides mitochondria, another important target for PDT PSs is the nucleus. Generation of singlet oxygen or other ROS, in close proximity to the DNA, might allow for DNA damage, and finally lead to cell death. It is known that dipyridophenazine (dppz) ligands have the ability to intercalate within DNA. 33,34 That is why in 2014 six different [Ru(bpy)2dppz] 2+ derivatives 17-22 were investigated by our groups (Figure 10). Singlet oxygen production study showed that all compounds had a high efficacy for 1 O2 production but only in hydrophobic environment. The excited state of the complexes bearing a dppz ligand are quenched very fast in the presence of water molecules. 36 All synthesised complexes were found to be non-cytotoxic in the dark (IC50 >100 µM) against HeLa and MRC-5 cells (normal lung fibroblast). Light cytotoxicity studies were performed using two different light treatments: 10 min at 350 nm (2.58 J . cm -2 ) and 20 min at 420 nm (9.27 J . cm -2 ). Among all compounds, only complexes 17 and 18 showed an interesting phototoxic effect. The PI value for complex 9 was higher than 150, while for complex 18 it was 42. The cellular uptake of the The first complex was difficult to detect even when cells were treated with high doses of compound. The low luminescence quantum yield is probably responsible for this result. On the other hand, complex 18 was able to accumulate preferentially in the nucleus. Because of the luminescent quenching effect of the complex in aqueous environment mentioned above, fluorescence microscopy localisation was supported by HR-CS AAS. The results showed that both complexes efficiently accumulated in the nucleus (0.43 ± 0.05 and 0.96 ± 0.06 nmol Ru per mg protein). To check if nuclear localisation and binding to DNA might have been the reason of toxicity, DNA photocleavage experiments were conducted. Treatment of pcDNA3 plasmid with complexes and irradiation at 420 nm for 20 min (9.27 J . cm -2 ) showed that both complex 17 and 18 were able to cleave plasmid DNA. Administration of compounds in the dark did not cause cleavage of the plasmid. In a follow up study, our groups further explored the molecular cell death mechanism of complex 18. 37 Mechanistic studies on the outcome of DNA binding led to the conclusion that irradiation of the intercalated compound caused oxidative damage of purines in DNA. Importantly, alkaline comet assay supported these results in living cells. Confocal microscopy images of different cell lines such as U2OS (human bone osteosarcoma), MCF-7 (mammary gland adenocarcinoma) and RPE-1 (normal retina pigmented epithelium) confirmed that the complex was mainly localised in the nucleus 35 were investigated (see Figure 12 for structures). The aim of this study was to investigate if small structural differences could cause significant changes in the biological response. It is worth noting that both complexes were investigated for use in one-photon (OP) and two-photon (TP) PDT. Dark and light cytotoxicity studies on HeLa and MRC-5 cell line monolayers showed that the introduction of -OMe groups on the ligand enhanced toxicity compared to those bearing the -OH groups (decrease of the IC50 value from 16.7 ± 2.6 µM in -OH bearing compound to 3.1 ± 0.6 µM in -OMe compound in HeLa). Both complexes were also much more effective than the positive control aminolevulinic acid (5-ALA), an approved PDT PS. Interestingly, the compounds were also studied on 3D multicellular tumour spheroid to provide a comprehensive overview on how Ru(II) complexes might act in solid tumours. Surprisingly, only complex 23 was active on MCTSs upon light irradiation (LED light source; IC50 32.5 ± 6.8 µM). To further explore the mechanism of action of the complexes, cellular localization and uptake of the compounds were studied. ICP-MS showed that the amount of complex 23 was much higher in HeLa cells than complex 24 (2.4 nmol Ru/mg protein to 0.9 nmol Ru/mg protein). This result might explain the differences between the IC50 values obtained for both complexes in the dark and upon light irradiation.

Confocal microscopy showed that the Ru complexes under study localised in different compartments of the cell. Complex 23 was found to accumulate in the nucleus and mitochondria while 24 localised in the outer cell membranes. Imaging was also performed after light irradiation. Complex 23 changed its localisation and moved completely into the nucleus, probably as result of damage generated by singlet oxygen in membranes, enabling the compound to reach the nucleus. Worthy of note, these Ru complexes might also localise in other compartments that escaped detection by confocal microscopy. Indeed, due to luminescence quenching in aqueous solution of these dppz-containing complexes, their detection is only possible in hydrophobic environment. 38 OP and TP absorption was also used to image both compounds in spheroids. For both complexes, TP imaging gave better results. In this experiment, complex 23 completely permeated the MCTSs, while complex 24 could only be detected in the external parts of the spheroids (Figure 13 and Figure 14). Comparable results with structurally related compounds were obtained by the Glazer group in 2014. 39 Although complexes 25 and 26 have very similar photophysical properties (Figure 15), the differences in their overall charge and hydrophilicities led to distinct biological effects.

While complex 25 localised in the mitochondria, complex 26 did not show specific organelle localisation and was found in the cytosol. Upon irradiation (30 s pulses; >400 nm; 7 J . cm -2 ), complex 25 caused necrotic cell death distinct from complex 26 which turned on the apoptotic pathway. It is possible that the addition of sulfonic acid groups on complex 26 induced a different cell localisation and consequently a different type of cell death. Similar conclusions were brought in 2015, when small changes in the structure of Ru(II)-based PSs cancelled phototoxicity of the complex. 40 In this case, two inert Ru(II) polypyridyl complexes with a nitrile containing ddpz ligand and two bypridine or phenantroline ancillary ligands were tested. In contrast to previously described Ru(II) complexes with dppz ligands, both did not exhibit high singlet oxygen production (20% comparing to 50%-90%). This is probably why these complexes did not display any cytotoxic effect upon light irradiation (RPR 200 Rayonet chamber reactor; 420 nm; 9.27 J . cm -2 ).

A series of four cyclometallated Ru(II) complexes with π-expansive ligands were described by

McFarland in 2015 (Figure 16). nm; 34.2 mW . cm -2 ). On the contrary complex 30 showed an astonishing PI value, namely 1400.

To assess if the complexes can interact with the DNA, a photocleavage assay was used. It was shown that all complexes could impair ethidium bromide staining due to induced DNA aggregation and precipitation. Because complex 30 was the most promising one, further tests were performed with it. Since 30 was generating singlet oxygen very weakly (less than 0.56% relative to [Ru(bpy)3] 2+ , it was suggested that the other ROS is responsible for the phototoxic effect in cells. Indeed, tests with dihydroethidium (DHE) in HL-60 cells confirmed that superoxide O2 •-was responsible for cell death. It was also shown in SK-MEL-28 cells that complex 30 altered its localisation upon light irradiation (from nucleus to cytoplasm) and induced morphology changes in the cells.

Lysosome targeting compounds

In 2015, our group in collaboration with the Chao group introduced highly charged homoleptic complexes that are suitable for TP-PDT (see Figure 17). 41 The compounds were found to be photostable and did not break down in bovine plasma.

Electron paramagnetic resonance (EPR) experiments demonstrated that the main type of ROS generated by the three compounds at 450 nm irradiation was 1 O2. Cellular localisation of complexes 31, 32 and 33 was determined using confocal laser scanning microscopy in HeLa cell line monolayers as well as in HeLa multicellular tumour spheroids. All three complexes were found to localise in the lysosomes, probably entering the cell by endocytosis pathway.

ICP-MS experiments confirmed the microscopy outcomes. All compounds were not cytotoxic in the dark. After OP irradiation (450 nm, 10 J . cm -2 ), complex 31 showed particularly high phototoxicity with IC50 value of 1.5 µM (PI 313). All complexes had a higher phototoxicity than 5-ALA, which was used as a control PS. The same trend was also observed for MCTSs.

Further investigations were performed with complex 31, which was found to be the most promising compound of the series. Calcein AM staining and ROS indicator staining (DCFH-DA) showed that cell death was only limited to the place of irradiation. Morphology studies after TP light treatment showed that cells underwent shrinking and formed bubbles. The cellular localisation of complex 31 was also altered. After irradiation, the compound was found in the cytoplasm, nucleus and nucleoli (see Figure 18). Microscopy analysis indicated that cells died by a necrotic process, bursting their content into the extracellular space. Overall, this investigation revealed that lysosomes might be a good target for future PDT PSs.

Targeting conjugates

The need for new Ru(II) polypyridyl complexes with better selectivity towards cancer cells led to the design of compounds with tumour specific targeting moieties. Such moieties might be antibodies, cell surface receptors, aptamers, etc. 42,43 In 2015, Weil and Rau introduced a Ru(II)based PS that was conjugated to somatostatin, 44 a peptide hormone produced by δ-cells of the pancreas inhibiting the release of insulin and glucagon. 45 Somatostatin receptors are frequently overexpressed in many tumour cancer cells, making them a good target for anticancer agents. 46 In this study, [Ru(byp)3] 2+ derivative was conjugated to the peptide hormone to form complex 34 (Figure 19). The cellular uptake of the conjugate was analysed by laser scanning confocal microscopy in A549 cells, which express different types of somatostatin receptors. The intensity of the compound emission was measured. A hundred times higher uptake of the somatostatin conjugate compared to the control was observed. Tumour selectivity was tested on wild type CHO-K1/Ga15 (Chinese hamster ovarian epithelial cell line expressing Gα15 alpha subunit protein) and cells overexpressing somatostatin receptor 2, CHO-K1/Ga15/SSTR2. Very high selectivity towards receptor overexpressing cells was confirmed by functional calcium flux assay. The IC50 value for cytotoxicity by complex 34, after light irradiation of A549 cells (LED array; 470 nm for 5 min; 6.9 ± 0.9 J . cm 2 ), was 13.2 ± 1.1 µM. Interestingly, the compound did not show any dark cytotoxicity up to 300 µM.

A different approach was utilised by the same research groups two years later, when a Ru(II) complex was conjugated to a protein carrier scaffold containing mitochondria targeting groups to yield complex 35 (see Figure 20 for Ru(II) complex structure). 47 photon absorbing PS offer deeper tissue penetration and better spatial resolution, 48 researchers also looked at the TP properties of complex. Data obtained for 35 showed 5 times higher TP cross-section values for the conjugate than for the Ru(II) complex alone. This nanotransporter platform with enhanced cellular uptake, phototoxicity and specificity against a leukemic cell line is undoubtedly a successful solution for selective delivery of PDT PSs.

In 2018, a biological evaluation of the use of a Ru(II) complex conjugated with tamoxifen as a TP-PDT PS was published (see Figure 21 for structure). 49 The Estrogen Receptor (ER) is highly overexpressed in breast cancer cells, making it a great target for anticancer therapy. 50 For over 30 years, tamoxifen has been an approved drug for the endocrine treatment of oestrogenreceptor-positive breast cancer. 51 (human liver) and COS-7 (monkey kidney) non-cancerous cell lines. Competitive assay with 17ϐ-estradiol (inhibitor of ER) showed that the uptake of complex 36 depended on interaction with the ER. Complex 36 as found to be non-toxic to cells in the dark. Upon light irradiation (450 nm, 12 J . cm -2 ) almost all MCF-7 cells treated with 16 µM of complex 36 were killed (99%) in comparison to the control (complex 37 which is not conjugated to tamoxifen, Figure 21).

Calcein AM and PI staining confirmed these cytotoxicity studies. Annexin V and PI assays showed that the treated and irradiated cells were in late apoptosis or necrosis. ROS generation of the complex 36 was verified by DCFH-DA. Moreover, upon addition of NaN3 (singlet oxygen scavenger), only very week fluorescence of the DCFH-DA was observed. Confocal microscopy studies showed that complex 36 localised in the lysosomes. Acridine orange (AO) staining demonstrated that upon light irradiation complex 36 caused lysosomes disruption. Very importantly, as a further confirmation of the mode of cell death action, complex 36 was found to generate singlet oxygen upon two-photon irradiation (fs, 820 nm) leading to cell death (calcein AM and PI staining).

Nanomaterials like carbon nanotubes or nanodots can also be used as carriers for different therapeutic drugs or diagnostic molecules. 52 In 2015, Zhang et al. developed carbon nanotubes functionalised with TP-absorbing Ru(II) complexes for bimodal photothermal and photodynamic therapy. 53 Two years later, a full biological evaluation of carbon nanodots modified with ruthenium complex was published. 54 This study showed that the combination with Ru(II) polypyridyl complexes might improve their intercellular uptake as well as their features required for PDT. For the studies, two complexes were used: complex 38 alone and complex 38 conjugated to carbon nanodots (see Figure 22 for structure of Ru(II) complex).

These compounds exhibited TP phosphorescence as well as higher 1 O2 production in acidic environment than at neutral pH. Both compounds were taken up by A549 cells as well as normal LO2 cells, as confirmed by ICP-MS. The ruthenium content was estimated to be 10.6 ± 0.3 ng/10 6 cells for complex 38 and 16.2 ± 0.4 ng/10 6 for complex 38 with CDs in A549 cells and 6.0 ±0.2 ng/10 6 and 6.4 ±0.2 ng/10 6 in LO2 cells, indicating that CDs might improve the uptake into the cancerous cells. To assess the type of mechanism causing cell death, the researchers performed multiple experiments. Cell morphology, annexin V staining, protein levels of caspase 3 and 7 as well as ATP levels in irradiated A549 cells confirmed that apoptosis was the main cause of cell death.

This mechanism was likely triggered by the high amounts of The PDT therapeutic potential of a PS is usually dependent on the oxygen levels in the targeted tissue since most of the PSs act with type II mechanism. In 2017, an interesting work on cyclometalated Ru(II) complexes, which act as Type I PSs, was published by Huang and coworkers. 55 The aim of this study was to design new PSs that could exhibit good PDT effects under hypoxia conditions. One of the designed PS contains a coumarin moiety (complex 39)

while the other one does not (complex 40) (see Figure 23 for structures). Coumarins have electron-donating and light-harvesting abilities. In both normoxia and hypoxia conditions, complex 39 showed low dark cytotoxicity and caused fast cell apoptosis after light irradiation in HeLa cells (white light; 400-800 nm, 30 mW cm -2 , 10 min). Cell death was confirmed with flow cytometry and fluorescent microscopy experiments. ROS generation studies confirmed that complex 39 generated high level of ROS under hypoxic and normoxic conditions compared to complex 39 and Ru(byp)3 2+ , which were used as a control. Highlyoxidative hydroxyl radicals were detected after light irradiation. Complex 40 was a far less effective PS compared to complex 39. To further verify the effectiveness of complex 39, in vivo studies (HeLa derived tumours in mice) were performed. Dosage of 5 mg kg -1 of the PS caused tumour growth inhibition and serious tumour cell damage after irradiation (xenon lamp, 250 mW cm -2 , 15 min) (Figure 24). No side-effects during 14 days of treatment were observed.

Histopathology as well as clearing time studies confirmed that complex 39 was not toxic for organs and was not accumulating in the body. Complex 42 was found to localise in the cell nucleus of HeLa and CHO cell lines. On the contrary, complex 41 (Figure 18), which did not have a NLS but was conjugated with cell permeable peptide, remained in the cell cytoplasm. Complex 42 was found to be taken up by cells through an energy-depended transport and to be not cytotoxic in the dark on HeLa cells (IC50 value of 83.4 µM). Light irradiation of treated cells at 440 nm (5 mW . cm -2 , 15 min) lowered the IC50 value to 51.8 µM. Single cell irradiation experiments with cells treated with complex 42 or complex 41, co-stained with nuclear dye DRAQ 7 (which only enters dead or permeabilised cells), demonstrated that the phototoxic effect of complex 42 was a result of its nuclear localisation. CT-DNA binding affinity studies along with photo-cleavage of pUC19 plasmid showed that complex 42 binds strongly DNA and is able to cleave it upon light irradiation. Tests with NaN3 revealed that singlet oxygen was not responsible for DNA cleavage. It was proposed that either Type I mechanism of electron exchange or direct oxidative damage at the guanine bases was the cause of DNA damage.

Conclusions and outlook

In recent years, many Ru(II) polypyridyl complexes were studied as potential PDT PSs. Their strong absorption in the visible light, ability to produce singlet oxygen upon light irradiation, tunable photophysics and lack of cytotoxicity in the dark makes them very attractive candidates.

Unfortunately, not many of them were analysed in-depth from a biological point of view. The mechanism of action of these compounds in living cells, a key factor in order to obtain their approval for a given indication, is very often still unknown or has only been superficially investigated. Worse, as shown in this Feature Article, there are only a few in vivo studies reported to date. However, despite this, one of such compounds has already entered clinical trial as a PDT PS against bladder cancer, clearly emphasising the potential of such complexes in this area of research. Further investigations in field of Ru(II) polypyridyl complexes as PDT PSs are of course needed. There is undoubtedly a necessity for new complexes that will exert their action by Type I mechanism. This is a crucial feature that will help fight very difficult to treat hypoxic tumours. During the designing process of the PDT PSs, adjustments will also need to be made in order to have PSs that can be activated at higher wavelengths. It is known that longer wavelengths will allow for deeper penetration through tissue. As shown in this Feature Article, this can also be obtained by Ru(II) polypyridyl complexes that are activated by two photon irradiation. However, this technique will require further proofs of its suitability for in vivo models, since studies in this field of research, not only with Ru(II) complexes, are for the moment much too scarce. Overall, we are convinced that this field of research is still in its infancy and that very exciting results will be published in the near future.

Figure 1 .

 1 Figure 1. Chemical structures of Photofrin®, Visudyne®, Foscan® and Levulan®.

Figure 2 .

 2 Figure 2. TLD-1411 and TLD-1433

Figure 3 .

 3 Figure 3. Structure of complexes 1-4

Figure 4

 4 Figure 4 Structures of complexes 5 and 6.

Figure 5 .Figure 6 .

 56 Figure 5. Chemical structures of complexes 7, 8, 9.

Figure 7 .

 7 Figure 7. ICP-MS quantification of the internalized Ru by the HeLa cells. Figure taken from ref 30 with permission from Elsevier.

Figure 6 .

 6 Figure 6. Chemical structures of complex 14 and complex 15.

Figure 9 .

 9 Figure 9. Chemical structures of complex 16.

35

 35

Figure 10 .

 10 Figure 10. Chemical structures of [Ru(bpy)2dppz] 2+ derivatives.

  Ru compounds was investigated by High-Resolution Continuum Source Atomic AbsorptionSpectrometry (HR-CS AAS), showing that it correlated well with toxicity studies. The most cytotoxic complexes 17 and 18 had the highest accumulation in the HeLa cells (1.08 and 1.76 nmol Ru per mg protein). Accumulation of the compounds in the MRC-5 cell line was different since only 0.76 and 0.18 nmol Ru per mg protein were determined. This indicates that the complexes penetrated the non-cancerous cell line to a lesser extent than the cancerous line.Cellular localisation of complexes 17 and 18 was investigated using confocal microscopy.

Figure 11 .

 11 Figure 11. Cellular localisation of complex 18. Figure taken from ref 36, with permission from John Wiley and Sons.

  . ICP-MS confirmed these results. The determination of the presence of specific markers of DNA damage response, analysis of DNA content and cytotoxicity studies after irradiation showed that cells underwent cell cycle arrest and loss of viability. Annexin V and PI staining experiments of interphase cells excluded classic apoptotic or necrotic cell death. Further analysis demonstrated that cell death was caused by DNA damage and endoplasmic reticulum-(ER) mediated stress response pathways. On the other hand, treatment and irradiation of mitotic cells caused death according to classic apoptotic pathways, indicating two distinct modes of cells death in interphase or mitosis and pointing to the potential of the use of these compounds in combination with established cancer therapeutics. Further studies on Ru(II) polypyridyl complexes with dppz ligands were performed by our group in collaboration with the Chao group. 28 Two substitutionally inert complexes, namely [Ru(phen)2(dppz-7,8-(OMe)2] 2+ (complex 23) and [Ru(phen)2dppz-7,8(OH)2] 2+ (complex 24)

Figure 12 .

 12 Figure 12. Chemical structures of complex 23 and complex 24.

Figure 13 .

 13 Figure 13. A. OP and TP images of 23 after incubation with HeLa spheroids for 12 h.

Figure

  Figure taken from ref. 29 with permission from John Wiley and Sons.

Figure 14 .

 14 Figure 14. A. OP and TP images of 24 after incubation with HeLa spheroids for 12 h. B stack images of the same HeLa spheroids captured every 5 μm along the Z-axis.

Figure

  Figure taken from ref. 29 with permission from John Wiley and Sons.

Figure 15 .

 15 Figure 15. Chemical structures of complex 25 and 26.

Figure 16 .

 16 Figure 16. Chemical structures of complex 27-30.

Figure 17 .

 17 Figure 17. Chemical structures of highly charged complexes.

Figure 18 .

 18 Figure 18. Micrographs of and ROS generation in HeLa cells incubated with complex 31 after irradiation with a two-photon confocal laser. Figure taken from ref. 42 with permission from John Wiley and Sons22

Figure 19 .

 19 Figure 19. Chemical structure of complex 34.

Figure 20 .

 20 Figure 20. Chemical structure of Ru(II) complex that was conjugated to the protein carrier scaffold.

Figure 21 .

 21 Figure 21. Chemical structure of complex 36 and 37

Figure 22 .

 22 Figure 22. Chemical structure of complex 38.

Figure 23 .

 23 Figure 23. Chemical structure of complex 39 and complex 40.

Figure 24 .

 24 Figure 24.A. Relative tumor volume of different groups after various treatments. B Tumor weights of different groups after 14 days treatments. C. H&E stained tumor slices of different groups.

Figure 42 Figure 25 .

 4225 Figure taken from ref.56 with permission from The Royal Society of Chemistry

  [START_REF] Kessel | [END_REF] O2 produced in lysosomes, which caused lysosomal permeability. This hypothesis was further confirmed with confocal microscopy and flow cytometry analysis. To further investigate if complex 38 and complex 38 with CDs would be efficacious in solid tumours, the researchers performed several experiments on MCTSs. Complex 38 and complex 38 with CDs were found to be able to penetrate 400 µm A549 spheroids. Confocal microscopy studies with calcein AM staining corroborated that spheroids treated with the compounds in the dark condition were viable and that cell death was only limited to the irradiated area. Once again, complex 38 with CDs was found to be better

	than ruthenium complex alone. IC50 values that were obtained on spheroids upon light
	irradiation (20 min; 810 nm; 100 mW; 80 mHz; 100 fs) were 12.0 µM for complex 38 (PI >8.3)
	and 2.2 µM for complex 38 with CDs (PI >45.5). Both compounds were successfully used as
	imaging agents in a living organism, namely zebrafish.

Award from the Swiss Chemical Society, an ERC Consolidator Grant, the Thieme Chemistry Journal Award and the Jucker Award for his contribution to cancer research.

Acknowledgments

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.) and by grants from the Stiftung zur Krebsbekämpfung and the Stiftung für wissenschaftliche Forschung of the University of Zurich (S.F.) and by grants from the Institut Curie and the Centre National de la Recherche Scientifique (CNRS) (B.G.).

Abbreviations

Conflicts of interest

There are no conflicts to declare


Félix Mouhat

David Bousquet

Anne Boutin

Lila Bouëssel

Du Bourg

François-Xavier Coudert

Alain H Fuchs

Lila Bouëssel Du Bourg

Softening upon Adsorption in Microporous Materials: A Counterintuitive Mechanical Response

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Fluid adsorption in nanoporous materials 1,2,3 such as activated carbons, carbon nanotubes or zeolites has been the subject of numerous studies in the past two decades, owing to its practical importance in such processes as separation, ion exchange, catalysis, biosensing and controlled drug delivery. For a long time these materials were considered as sufficiently stiff so that their intrinsic flexibility needed not be taken into account in the models employed to understand and predict equilibrium adsorption properties. 4,5,6 Indeed the magnitude of the adsorption-induced strain in zeolites is usually of the order of 10 -3 to 10 -4 . It was only when transport properties were considered that flexible materials models were tested 7,8 in order to ascertain whether or not it could help in better reproducing and explaining the observed experimental trends. Nonetheless, during these years an important amount of data were collected by the Russian adsorption school of Dubinin and coworkers on the adsorption-induced deformation of microporous carbons and zeolites. These studies were nicely summarized in 2006 in a book by Tvardovskiy. [START_REF] Tvardovskiy | Sorption Deformation[END_REF] These features were at least partly overlooked until the issue of adsorption-deformation in mesoporous systems such as MCM-41, SBA-15, or controlled pore glasses (CPGs) was addressed through the study of the phase behavior of confined fluids, with a specific focus on the capillary condensation phenomenon. [START_REF] Zickler | [END_REF] In such mesoporous materials, a continuous swelling is observed upon fluid adsorption, up to capillary condensation, and this can be understood in terms of the so-called Bangham effect, 11 which qualitatively relies on interfacial energy change upon internal surface adsorption. Models have been developed to rationalize this effect. 12,13 In the case of microporous materials however, the vapor adsorption phenomenon displays a trend different from what happens in mesoporous materials. At low vapor pressure the system undergoes a structural contraction followed by swelling at higher vapor pressure. [START_REF] Tvardovskiy | Sorption Deformation[END_REF] This peculiar non-monotonic behavior been observed experimentally and documented dating back to the 1940's, 14 and was recently reported very clearly by Reichenauer and coworkers [START_REF] Baltzer | Proc. 5th Poromechanics Conference[END_REF] in a series of detailed in situ dilatometry experiments of various gases adsorption in synthetic microporous carbons. This non-monotonic deformation is generic to microporous materials and does not depend on the specifics of host-guest interactions. It contradicts the common intuition that gas adsorption in a confined system increases internal pressures and leads to a continuous volumetric expansion as long as the host framework is flexible (the "sponge" or "balloon picture"). [START_REF] Coudert | [END_REF]17 On the theoretical side, Neimark and coworkers developed a non-local density functional theory, 18,19 that was able to reproduce this contraction-expansion behavior of adsorption strain in zeolites. Density functional theory and Grand Canonical Monte Carlo simulations were also applied to study adsorption deformation in micropores of activated carbons. 20 The concept of adsorption stress enabled to rationalize this phenomenon. 18,21,22 At low gas loading, contraction of the material is driven by the host-guest dispersion attractive interactions, leading to a negative adsorption stress. At higher loading, once the adsorbed phase becomes denser, short-range repulsive interactions come to play which lead to a reversal of the adsorption stress, and the material expands in order to accommodate additional guest molecules. This was a first step towards a comprehensive understanding of the adsorption-deformation coupling phenomena in microporous materials. The next issue to be dealt with is the mechanical stability of the microporous frameworks in the presence of adsorbate species.

Pressure-Induced Amorphization (PIA) was observed in several porous clathrasils and zeolites 23,24,25 as well as in metal-organic frameworks of the ZIF family (Zeolitic Imidazolate Framework). 26,27 This structural transition was attributed to the framework mechanical instability due to softening of low-energy phonon modes. 28 We note in passing that such a mechanism was also proposed for the PIA process in non-porous α-quartz 29 and ice Ih. 30 More recently, PIA in ZIF-8 and some other porous ZIFs was attributed to the framework mechanical instability due to shear mode softening. 31,32 The phenomenon of pressure-induced amorphization is quite sensitive to the presence of molecules inside the pore space. Insertion of guest molecules was shown to either shift amorphization to higher pressure than in the evacuated material, 23,25,31 or even to suppress ("deactivate") PIA as in the case of silicalite-1. 33 It was then deduced that the presence of adsorbate molecules enhances the stability of the microporous frameworks. This was later supported by a Monte Carlo simulation of silicalite-1 which showed an increase of the computed bulk modulus upon CO2 adsorption. 34 It should be mentioned however that there are some scarce indications in the literature of opposite behaviors. For instance, Canepa et al. 35 studied by ab initio simulations the response of MOF-74-Zn when loaded with several molecular gases. They observed that "for almost all adsorbates, the molecular adsorption in the MOF pores induces a substantial increase of the elastic constants -i.e. the MOF looses some of its flexibility". A close examination of their data (Table III of reference 35), shows that, while the bulk modulus increases with the insertion of gas molecules, the individual C12 and C14 elastic constants as well as two of the Young's modulus components decrease when the MOF is loaded with CO2 or CH4 (but not H2O). In many of the cases studied experimentally, the precise value of the gas loading is not well known, and we presume that many of the experiments were performed at high, if not full, loading. Given that the PIA mechanism involves a collapse of the framework structure around the empty pores, 32 it seems clear that the mechanical properties can only be en-hanced when the pore voids are fully filled with adsorbate molecules. This might, however, not be the case at low gas loading. There is thus a great need to better understand the mechanical response of nanoporous materials upon adsorption, and in particular of soft framework materials such as flexible and stimuli-responsive MOFs. 36 In this work we examine the elastic response of two model microporous frameworks upon gas adsorption, with a special focus on the initial stage of the adsorption process, i.e. at low gas loading, in order to try understanding the effect of the non-monotonic volumetric behavior upon adsorption described above on the simultaneous change in the framework mechanical stability. We performed Monte Carlo (MC) simulations of a Lennard-Jones fluid adsorption in pores of two different geometries: a slit-pore which mimics a MOF framework such as ELM-11, 37 and a lozenge-shaped pore which is a simplified model of the MIL-53 family of "breathing" materials 38,39,40 (Figure 1). In both cases the walls of the framework were made of regularly spaced Lennard-Jones particles (more details on the unit cell setup and the forcefield parameters are given in the Supporting Information). These are very simple model systems for flexible nanoporous materials. They exhibit only one order parameter describing the framework deformation, namely the width of the slit-pore (h) and the opening angle of the lozenge-shaped pore (α); all other dimensions are kept constant. It enables to perform accurate direct Monte Carlo simulations in the osmotic statistical ensemble (Nhost, µads, σ, T) where Nhost is the number of host unit cells, µads is the adsorbate chemical potential, T is the temperature, and σ is the mechanical stress (in our case a uniaxial stress) exerted on the system boundaries. It is the appropriate thermodynamic ensemble to treat ad-

h α σ σ
sorption in flexible open porous materials, 41 similar to the Grand isostress ensemble used to describe Surface Force Apparatus (SFA) experiments. 42,43 For each system, simulations of adsorption were performed at increasing values of guest chemical potential μads, and from each osmotic simulation the following quantities were extracted: the loading, or average number of adsorbed particles; the deformation of the unit cell, quantified by the average value of h or α, and which is directly related to the strain; and the elastic modulus (or stiffness constant) along the deformation mode, calculated from the fluctuations of the unit cell (in Voigt notation, C33 for the slit pore, and C66 for the lozenge-shaped pore; see supporting information for details).

We first performed a series of simulations in the case of the slit pore. The width of the empty pore h0 was fixed to the value of 15 Å, which corresponds to a typical microporous pore. This width should not be too small in order to avoid spurious effects due to layering that is known to take place in this particular geometry in very narrow pores. 42,43 We allow the pore width to fluctuate in the osmotic MC simulations and the free energy of the empty host is simply written in the elastic regime as:

Fhost(h) = ½ k (h -h0) 2 (1)
The harmonic constant k is chosen such that the elastic constant of the bare framework C 0 33 (the Young's modulus of the host) is equal to 10 GPa, a rather typical value for MOFs or zeolite microporous materials. The same approach is followed for the lozenge-shaped pore, whose free energy also follows linear elasticity (the mathematical expression for the free energy can be found in a preceding paper 39 ). Changes in the slit-pore width (upper frame) and in the elastic constant C33 (lower frame) as a function of the number of adsorbed molecules. The black curves correspond to the direct osmotic simulations, with uncertainties indicated (when not visible, they are smaller than symbol size). The blue curves to Grand Canonical simulations in which the elastic constant is obtained through the second derivative of Nads with respect to h, using the data of a series of simulations ranging from h = 14 to 15 Å (details in Sup-porting Information). Simulations were performed at 298 K.

In Figure 2, we report the evolution of the slit-pore width and the elastic constant C33 as a function of the number of adsorbed molecules (pore loading) at 298 K. A slight increase in the pore width at very low Nads values in the osmotic simulations is observed, which is due to an oscillating solvation pressure effect that is still present, although of small amplitude, in such a 15 Å width slit-pore (as well as in additional simulations performed for widths of 18 Å and 21 Å). This is in keeping with the fact that this effect is not present in the grand canonical simulations. A rather similar feature was observed by Schoen and coworkers in their simulations of pores of several guest molecular diameters. 44 This effect is of small amplitude, and strongly dependent on the relative guest size and pore width h0.

After that low-pressure effect, the volumetric behavior is non-monotonic, as expected, and shows a contraction at low to intermediate gas loading followed by an expansion of the pore at high loading. This is the standard contraction-expansion mechanism commonly observed in microporous materials. [START_REF] Tvardovskiy | Sorption Deformation[END_REF][START_REF] Coudert | [END_REF] What is absolutely new and striking is the observation of a non-monotonic change in the elastic constant upon fluid adsorption. Even in such a simple model as the slit pore, the continuous evolution of elastic stiffness upon adsorption loading has never been reported before. In this particular system, we see that the initial contraction of the porous volume is accompanied by a lowering of the Young's modulus along the z axis, i.e. a softening of the framework. The subsequent expansion at high loading is, on the contrary, accompanied with a stiffening of the framework, as guest molecules fill the micropore. It is worth noting that, on this model whose elastic modulus (10 GPa) is representative of a MOF, the variations in mechanical properties (up to 5 GPa change in C33, i.e. a 50% variation) are quite large, much larger than variations in the pore size itself (0.6 Å in a pore of 15 Å).

To investigate the influence of geometry, we then studied the evolution of elastic constants upon adsorption in the lozenge-shaped pore, from GCMC simulation data at varying pore opening and chemical potential (see Supporting Information). We report in Figure 3 the changes in the order parameter α (lozenge angle) and the elastic constant C66 (shear modulus), as a function of the number of adsorbed molecules at 298 K. Again the nonmonotonic volumetric contraction-expansion behavior is accompanied by a softening-stiffening process upon fluid adsorption. In this particular pore model, adsorptioninduced softening reaches 20% while the transition from softening to stiffening occurs at lower relative loading. The generic features of adsorption-induced softening- The question arises now as to whether or not this behavior of the elastic response of microporous frameworks disclosed here is generic, i.e. whether we can expect to see an adsorption-induced softening of host-guest systems at partial loading. Obviously more work is needed to be able to fully answer this question; for this, experimental studies at low loading are sorely needed. We have begun however to address this question by closely examining the simulation data from our previous Molecular Dynamics (MD) work on series of MOFs. In the case of cubic material ZIF-8, 31 no such behavior was observed at low loading in our simulations of methane adsorption.

The {ZIF-8 + CH4} system exhibits a continuous increase in the computed elastic constants upon methane adsorption. However another ZIF material, which we have previously studied in order to predict the high pressure stability of ZIF frameworks, 32 was observed softening upon CH4 adsorption. The Zn(imidazolate)2 framework of nog topology 45 exhibits the same non-monotonic softening-stiffening behavior as our two model pore systems, upon methane adsorption at 300 K. For this material, we show in Figure 4 the evolution of the softest elastic deformation mode, λmin, as a function of methane loading. It exhibits a softening by a factor of two at low loading, with the modulus going down from 2 GPa to 1 GPa at 20 molecules per unit cell. We conclude from this first systematic study of mechanical properties of microporous materials under adsorption that a non trivial softening of the microporous {guest + host} system can be observed at low loading, paralleling the contraction of the system. This counter-intuitive effect is followed, at higher fluid loading, by the traditionally expected materials stiffening. This phenomenon is rather generic and does not stem from specific interactions, as we have seen it occurring for methane adsorption in pores of various geometries (slit pore, diamondshaped pore, and nog ZIF). However, some microporous frameworks such as ZIF-8 do not show such a behavior for reasons that are not yet clear. It is possible that only some of the components of the framework elastic response experience such a temporary softening in the low loading regime. Work on a larger number of host-guest systems is in progress to clear up this point.

This non-monotic softening-stiffening evolution of mechanical properties, proposed on the basis of molecular simulations, can explain some results from the earlier literature. In particular, it explains the MOF softening upon adsorption found (but not commented upon) by Canepa et al. in their ab initio studies of CO2 and CH4 adsorption in MOF-74-Zn. 35 It can also help explain the recent counter-intuitive experimental results of Alabarse et al., who showed that the presence of water within the pores of AlPO4-54 lead to a decrease in mechanical stability, attested by the occurrence of pressure-induced amorphization at lower pressure than in the guest-free AlPO4-54. 46 This last example shows the importance of better understanding the influence of guest adsorption on mechanical properties of microporous materials, as it can have a drastic impact not only on the modification of the elastic behavior (thus augmenting, through soften- ing, the magnitude of adsorption deformation) but also on the stability under pressure, a key property for many industrial applications.

ASSOCIATED CONTENT

Supporting Information. Full description of the pore models, molecular simulation procedures, and calculation of elastic constants. This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 1 :

 1 Figure 1: Sketch of the two model microporous frameworks. Left: slit-pore; right: lozenge-shaped pore. The red arrows represent the direction of the stress exerted on the solid frameworks, and the unique order parameters of the deformation are represented in green.

Figure 2 .

 2 Figure 2.Changes in the slit-pore width (upper frame) and in the elastic constant C33 (lower frame) as a function of the number of adsorbed molecules. The black curves correspond to the direct osmotic simulations, with uncertainties indicated (when not visible, they are smaller than symbol size). The blue curves to Grand Canonical simulations in which the elastic constant is obtained through the second derivative of Nads with respect to h, using the data of a series of simulations ranging from h = 14 to 15 Å (details in Sup-

Figure 3 .

 3 Figure 3. Changes in the lozenge-shaped pore order parameter α (upper frame) and in the elastic constant C66 (lower frame) as a function of the number of adsorbed molecules.

Figure 4 .

 4 Figure 4. Change in the elastic response of the ZIF nog45 as a function of the number of methane molecules adsorbed species, obtained from isostress-isothermal MD simulations at 300 K.32 The ordinate is the smallest elastic modulus of the material, i.e. the lowest eigenvalue λmin of the elastic stiffness tensor. Uncertainty on each value is of the order of 0.1 GPa (see ref.32).

We thank Alex Neimark and Gudrun Reichenauer for fruitful discussions. We acknowledge funding from Agence Nationale de la Recherche (project ANR-2010-BLAN-0822), and computing time on HPC platforms by GENCI (grant x2015087069).

AUTHOR INFORMATION

Corresponding Author alain.fuchs@cnrs-dir.fr; fx.coudert@chimie-paristech.fr ACKNOWLEDGMENT