Sylvain Patinet

Lina Alzate

Etienne Barthel

Davy Dalmas

Damien Vandembroucq

Veronique Lazarus

Finite size effects on crack front pinning at heterogeneous planar interfaces: Experimental, finite elements and perturbation approaches

Keywords: Interfacial brittle fracture, Toughening, Crack pinning, Finite element method, Perturbation approach

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Introduction

Predicting the threshold for crack propagation is a key issue in material science: it determines the design quality of structures and their durability for a wide range of systems ranging from bulk materials to thin films. In particular it was shown that one of the most efficient mechanisms to increase effective toughness is crack pinning by material heterogeneities [START_REF] Bower | A three-dimensional analysis of crack trapping and bridging by tough particles[END_REF]. For example the macroscopic effective toughness of a material can be increased very significantly by the dispersion of hard particles in the matrix [START_REF] Mower | Experimental investigations of crack trapping in brittle heterogeneous solids[END_REF]. Another system that takes advantage of crack pinning consists of patterned interfaces presenting heterogeneous toughness landscapes. Their technological interest lies in the development of multi-layer materials with high mechanical stability. A promising method to develop new materials consists in designing optimal heterogeneous interfaces with high toughness while maintaining their functional features.

Heterogeneities affect the effective toughness by interfering with crack propagation, deflecting crack fronts and crack surfaces. However, it is difficult to predict the effect of heterogeneities quantitatively and experimental investigation of this toughening mechanism raises several difficulties. For example it is not easy to create well controlled heterogeneity distributions and to observe crack propagation in situ. Also, due to crack deflection, the problem is often three-dimensional and theoretical or numerical developments become quite complex.

In the literature, most of the experimental investigations on crack pinning in heterogeneous materials were statistical approaches and studied post-mortem fracture surfaces [START_REF] Bouchaud | Scaling properties of cracks[END_REF][START_REF] Santucci | Statistics of fracture surfaces[END_REF][START_REF] Ponson | Failure mechanisms and surface roughness statistics of fractured fontainebleau sandstone[END_REF][START_REF] Dalmas | Crack propagation through phase-separated glasses: Effect of the characteristic size of disorder[END_REF][START_REF] Bonamy | Intermittency and roughening in the failure of brittle heterogeneous materials[END_REF]. Experiments with direct visualization of the crack front in situ during propagation are very unfrequent. For example, with an original experimental setup, [START_REF] Schmittbuhl | Direct observation of a self-affine crack propagation[END_REF] were able to observe the crack front morphology during propagation along a disordered heterogeneous interface. This setup has been widely used to obtain statistical information on crack front roughness [START_REF] Delaplace | High resolution description of a crack front in a heterogeneous plexiglas block[END_REF][START_REF] Santucci | Fracture roughness scaling: A case study on planar cracks[END_REF] and stochastic dynamics of propagation [START_REF] Måløy | Dynamical event during slow crack propagation[END_REF][START_REF] Tallakstad | Local dynamics of a randomly pinned crack front during creep and forced propagation: An experimental study[END_REF]. However quantitative predictions seem out of reach, mainly because of the presence of shear on the crack front during loading and the ill-controlled nature of the heterogeneities. Recently, [START_REF] Chopin | Crack front dynamics across a single heterogeneity[END_REF] proposed a new experimental approach with better controlled heterogeneities, but the setup also suffers from mode mixity. In the experiments of [START_REF] Mower | Experimental investigations of crack trapping in brittle heterogeneous solids[END_REF], the front is trapped by second-phase particles introduced in a brittle epoxy. The front shape becomes complicated since it can not penetrate in the particles, and these experiments are difficult to model analytically.

Here, we report on a study of crack propagation measured along a well controlled patterned interface in a classical Double Cantilever Beam (DCB) geometry. The crack propagates at the weakest interface in a stack of thin films deposited on glass, allowing direct visualization of the crack front. This weakest interface is not homogeneous however: a defect strip with a different interfacial toughness lies at the center of the sample. Several values of toughness contrast have been investigated and we have monitored the interaction of the interface crack with these defects. The interface cracks are either trapped or attracted by the defect depending on the sign of the toughness contrast between the defect strip and its homogeneous surrounding medium. This setup [START_REF] Barthel | Asymmetric silver to oxide adhesion in multilayers deposited on glass by sputtering[END_REF][START_REF] Dalmas | Crack front pinning by design in planar heterogeneous interfaces[END_REF] has several advantages: 1) the crack is loaded in pure tensile mode (mode I); 2) the crack propagation is purely interfacial (without deflexion out of the plane of the interface); 3) the toughness contrast can be tuned to keep the deformations of the crack front small.

In the framework of Linear Elastic Fracture Mechanics, propagation criteria are based on the comparison of the Energy Release Rate (ERR) and adhesion energy [START_REF] Griffith | The phenomena of rupture and flow in solids[END_REF] -or equivalently of mode I stress intensity factor (SIF) and toughness [START_REF] Irwin | Analysis of stresses and strains near the end of a crack traversing a plate[END_REF]. The ERR is derived from an elastic analysis, and depends on loading, elastic response of the solid and geometry of both body and crack. Perturbation methods were initiated by [START_REF] Rice | First-Order variation in elastic fields due to variation in location of a planar crack front[END_REF], based on [START_REF] Bueckner | Weight functions and fundamental fields for the penny-shaped and the half-plane crack in three-space[END_REF] weight-function theory, and specifically used by [START_REF] Gao | A First-Order perturbation analysis of crack trapping by arrays of obstacles[END_REF] to study the morphology of cracks trapped in heterogeneous interfaces. They provide analytical predictions for the variation of the ERR when the crack front is slightly perturbed and effectively describe the crack front as an elastic line. However, the first order formula proposed by [START_REF] Rice | First-Order variation in elastic fields due to variation in location of a planar crack front[END_REF] for a half-plane crack in an infinite media does not take into account any finite size effect. In the case of the present measurements, they may provide erroneous predictions for large enough crack front perturbations (deformation or wavelength) in comparison with sample sizes. However several improvements of this formula have been proposed to take into account the finite size of the crack [START_REF] Gao | Nearly circular connections of elastic half spaces[END_REF][START_REF] Gao | Nearly circular shear mode cracks[END_REF][START_REF] Leblond | The tensile tunnel-crack with a slightly wavy front[END_REF][START_REF] Lazarus | In-plane perturbation of the tunnel-crack under shear loading I: bifurcation and stability of the straight configuration of the front[END_REF][START_REF] Lazarus | Perturbation approaches of a planar crack in linear elastic fracture mechanics: A review[END_REF], the finite thickness of the body [START_REF] Legrand | Coplanar perturbation of a crack lying on the mid-plane of a plate[END_REF] and even the interaction between two cracks [START_REF] Pindra | In-plane perturbation of a system of two coplanar slit-cracks-I: case of arbitrarily spaced crack fronts[END_REF].

Our aim here is to assess the use of analytical perturbation theories to model crack propagation in heterogeneous interfaces. To evaluate the merits of these models, we need a reference solution which would provide an exact modeling of the data. In the case of the mildly distorted crack fronts proposed here, Finite Element (FE) simulations can be used to determine the local values of the ERR. In addition, with FE simulations, we can accurately take into account the exact geometry of the sample, including the shape of the crack front which is determined experimentally.

The experimental method is presented first with some details concerning the synthesis of the samples and the cleavage tests (section 2). From the measured crack front deformations and loadings, the ERR pattern is then determined by numerical and theoretical methods. The ERR along the crack front is quantitatively calculated through detailed three-dimensional FE analysis (section 3). Analytical perturbation methods are presented in section 4 and we show how the local ERR contrast can be inferred. Then FE and analytical methods are applied to the different samples and their results compared in section 5. In particular, it is shown that the ERR contrasts calculated by the two approaches are in good agreement if the finite size of the plate is duly taken into account in the analytical perturbation models [START_REF] Legrand | Coplanar perturbation of a crack lying on the mid-plane of a plate[END_REF], in contrast to [START_REF] Rice | First-Order variation in elastic fields due to variation in location of a planar crack front[END_REF]'s model, which is conventionally used for this type of geometry although it can apply only to infinite half-spaces. We finally discuss the crack propagation criterion and show how these results can be used to give quantitative estimates of local adhesion energy values for textured interfaces in section 6.

Experiments

Synthesis of samples

To control and visualize the propagation of a purely interfacial crack along a patterned interface we start from thin film multi-layers deposited by magnetron sputtering [START_REF] Barthel | Asymmetric silver to oxide adhesion in multilayers deposited on glass by sputtering[END_REF] on rectangular glass substrates (thickness h = 700 µm, width b, length L). Schematically, a typical sample is glass/silver/top layer (see Fig. 1). Three types of interfaces are studied, with respectively low toughness contrast (LC), medium contrast (MC) and high contrast (HC). In LC-b40, the defect strip is an organic layer which is directly deposited on glass, inducing lower adhesion. For MC-b40 and MC-b50, the silver layer is deposited on an Si 3 N 4 sublayer. The toughness contrast originates from the difference between Ag/Si 3 N 4 and Si 3 N 4 /Si 3 N 4 adhesion. In HC-b40 the silver layer was deposited directly on the glass substrate, increasing the toughness contrast due to lower adhesion on glass. The sample nature and dimensions are summarized in Tab. 1, ranked from the lowest to the highest adhesion. Underlayer thicknesses vary between 10 and 50 nm.

Cleavage test

To propagate a crack in the multilayer, we use cleavage under ambient conditions: a glass backing of thickness 700 µm is glued on top of the multilayer with an epoxy glue [START_REF] Barthel | Asymmetric silver to oxide adhesion in multilayers deposited on glass by sputtering[END_REF][START_REF] Dalmas | Crack front pinning by design in planar heterogeneous interfaces[END_REF], resulting in a glass/multilayer/glass sandwich. This sandwich is amenable to opening in a DCB setup. The test is carried out by progressive opening of the two glass arms by the gradual introduction of a wedge between the two plates (Fig. 2a). The positioning of the wedge is controlled by an electric jack for precise control of the opening of the two glass plates. With this method, cleavage is displacement controlled and the propagation of the crack is stable: the crack length increases in a controlled way. The propagation of the crack front is performed in a quasi-static manner, i.e. further increment of the opening is applied only after complete arrest of the crack front. The specimen is semi-transparent and we directly observe the crack front propagation during opening. In order to record the data, two high magnifications cameras are used. The first camera monitors the displacement of the glass arms δ, which is measured at the corners of the crack surfaces (see Fig. 2a). The second camera records the crack front shape a(z) (see Fig. 2b). Its position a(z) is measured from the line parallel to z passing through the external corners of the crack surfaces.

Once the wedge penetrates between the two plates, crack propagation takes place at the weakest interface of the multilayer. In order to identify this interface X-Ray photoelectron spectroscopy is used on both fracture surfaces. In these samples, purely interfacial fractures have always been found. As indicated in Fig. 1, the crack takes place at one of the silver layer interfaces for all the configurations. This observation is consistent with the weak adhesion of silver layers and the perfectly interfacial crack path demonstrated earlier [START_REF] Barthel | Asymmetric silver to oxide adhesion in multilayers deposited on glass by sputtering[END_REF]. As the crack propagates in a patterned interface, two fracture interfaces can be defined, one involving the silver layer and the other one in the defect strip (see Tab. 1).

The evolution of the crack front morphologies (only for equilibrium positions) during cleavage tests is shown in Fig. 3 in the case of sample HC-b30. In the first regime, the propagation takes place at the homogeneous silver interface. The crack front propagates with the overall curved shape which is characteristic of homogeneous interfaces for finite width samples (Sec. 3.3). This simple curved shape is preserved until the crack comes into contact with the defect strip, where deformation of the crack front begins: it is held back by the defect strip and curves into the outer region. After this transient regime, stationary propagation is recovered: in this third regime, where the crack propagates in the patterned area of the interface, the overall curvature of the crack front is now decorated by an additional modulation: the front lags behind in the high adhesion defect strip.

The crack front positions a(z) and the loadings δ, with the sample geometric dimensions and the elastic constants of the glass (E,ν), serve as input parameters to model the cleavage tests in the next sections by two different ways. We compute the ERR landscape along the crack front first by FE numerical calculations (Sec. 3), second, by theoretical perturbation approaches (Sec. 4) and compare the results (Sec. 5).

ERR obtained numerically by Finite Elements

FE calculations are performed with the aim to analyze the validity of the analytical perturbation approaches proposed in this paper (Sec. 4.2). To directly model the whole experimental pinning cleavage tests and obtain a reference solution of the global and local ERRs, we needed to take into account the whole geometry of the problem, including specimen geometry and the crack morphology (Sec. 3.1). This is the major part of the present FE calculations (Sec. 3.2). Besides, we also consider the case of an homogeneous interface (Sec. 3.3). Even in this case, due to the finite width of the specimen, the front shapes are not straight and are derived here by FE calculations. They are used as reference unperturbed crack geometry for the perturbation methods.

Simulation method

To calculate the local ERR distribution, the static elasticity problem is resolved by FE for each equilibrium position of the crack front. The input parameters for the FE calculations are the geometrical dimensions of the sample (L, b, h, H) (Fig. 2), along with the position of the front a(z) and the associated opening δ, which are measured during the gradual opening of the sample.

The simulations are performed with the FE code CAST3M developed by the French Commissariat à l' Énergie Atomique (CEA). The values of Young's modulus E = 71 GPa and Poisson's ratio ν = 0.22 are those of glass for all the cleavage tests. The multilayer is neglected from a mechanical point of view due to its extremely low thickness compared to the glass plates. The results presented in this work correspond to meshes composed of 144,896 bilinear 8-node parallelepipedic elements and 180,471 nodes. The mesh of the glass plate is inhomogeneous. Its density increases as the displacement gradients, and thus especially in the vicinity of the crack tip. The mesh is refined until the displacement field converges at mechanical equilibrium. The mesh of the experimental crack front shape a(z) = a + δa(z) is obtained by deforming an invariant mesh in the z-direction of a straight crack front around its mean position a (Fig. 4). The mesh around the crack front is constructed to form a kind of regular cylinder around the front which is adapted to the "G-theta" method used to calculated the local ERR at each point of the front. This method developed by [START_REF] Destuynder | Sur une interprétation mathématique de l'intégrale de rice en théorie de la rupture fragile[END_REF], allows us to calculate G accurately and quickly [START_REF] Destuynder | Quelques remarques sur la mécanique de la rupture élastique[END_REF].

As output of the simulation, the 3D displacement fields obtained are used to obtain the local ERR G(z) along the front with the "G-theta" method. This method uses the displacement fields obtained by FE resolution of the elasticity problem. Due to the symmetry of the problem with respect to the xz plane, i.e. the plane in which the crack propagates, only the upper glass plate located above the plane y = 0 is simulated. Symmetry boundary conditions are applied on this boundary. The boundary x = L is clamped. Stress free boundary conditions are applied on the rest of the boundary surfaces. Concerning the loading, a point displacement ∆ in the y-direction is applied on the triangular tip as in the experiments. The value of ∆ is fixed so that the displacement at the corner δ of the sample shown in Fig. 2a converges toward the displacement measured experimentally. The final error between the experimental and numerical value of δ is less than 1%, i.e. less than the uncertainty of experimental measurement, for all crack configurations studied.

Evolution of the ERR during the propagation along the heterogeneous interface

The local ERRs are calculated along the crack fronts for all equilibrium positions of the crack obtained experimentally. In the following we illustrate our results in the case of the multilayer HC-b30 that exhibits the strongest adhesion contrast among the four types of interfaces presented in Sec. 2.

We plot the following results:

• The ERR landscape G(z) of a crack front trapped by the central defect strip (Fig. 5);

• The average ERR G 0 =< G(z) > as a function of the average position a of the crack front (Fig. 6a);

• The average fluctuations ∆G 1 and ∆G 2 of G(z) with respect to G 0 for interface areas situated outside and inside the central defect strip respectively (Fig. 6b).

The evolution of the ERR landscape G(z) when the crack propagates is reported in Fig. 5a. The ERR contrast expected between the defect strip and its surrounding homogeneous medium is well reproduced by the ERR landscape. As reported in Fig. 3, three successive stages can be associated with this ERR landscape: homogeneous propagation (the front has a curved shape characteristic of finite width effect); interaction between the crack front and the central defect strip (the front is progressively deformed); stationary pinning (the deformation stays invariant in the direction of propagation).

A profile of the ERR G(z) obtained for a crack front trapped by the defect strip in the last stationary regime is shown in Fig. 5b. Despite the presence of some fluctuations, the presence of the central defect strip can be clearly identified and characterized by a significant variation of the ERR compared to average ERR G 0 . In the homogeneous and the defect zones, the ERR is nearly constant longitudinally to the crack front in the direction z. It is therefore possible to attribute a characteristic ERR value for the two types of interfaces, homogeneous and defect strip, encountered by the crack. We note G 1 and G 2 their respective average value from which we extract the corresponding mean variations from ∆G 1 = G 1 -G 0 and ∆G 2 = G 2 -G 0 . The signs of ∆G 1 and ∆G 2 are by definition opposed. The notations G 0 , G 1 , G 2 , ∆G 1 and ∆G 2 are reported on Fig. 5b.

Thanks to the FE calculations we compute the global ERR G 0 by calculating the average ERR along the front knowing the local ERRs so that G 0 =< G(z) >. In Fig. 6a, the variation of G 0 shows the three regimes as a function of The contrasts are calculated outside (∆G 1 ) and inside (∆G 2 ) the defect strip.

the average position of the crack in its direction of propagation a. These regimes correspond again to the morphology variations reported in Fig. 3. The average ERR G 0 is almost constant at the beginning and at the end of cleavage test. Between these two ERR values a transient regime is observed. These three regimes correspond chronologically to the propagation of the crack front in a homogeneous medium, its interaction with the strip edges and finally to its stationary pinning by the defect strip. Changes in G 0 reflect the change in the effective toughness of the interface due to the presence of the defect strip during the crack propagation. The increase in G 0 can be associated with a toughening of the interface. This aspect will be discussed in more detail in Sec. 6.

The determination of local ERR values allows us to refine our understanding of the interaction between the crack front and the central defect strip. In Fig. 6b, we represent the variations ∆G 1 and ∆G 2 as a function of the distance between the average position of the crack a and the beginning of the masked zone a 0 in the crack propagation direction x. The three regimes described above, from the change in G 0 , are considerably emphasized. We observe that the variations ∆G i are equal to zero for aa 0 < 0, i.e. before the interaction of the crack with the defect. During the penetration of the strip by the crack front, in the vicinity of aa 0 = 0, ∆G i vary dramatically. The ∆G i finally reach constant values for aa 0 > 0 when the crack front is stationary trapped by the defect. Note that, in absolute value, the variation ∆G 1 is about one order of magnitude lower than ∆G 2 . The width of the homogeneous zone is indeed an order of magnitude greater than that the defect strip one. The signs of variations ∆G i are consistent with the observed variations in G 0 . A defect strip whose adhesion is larger (smaller) than the homogeneous medium produces a variation ∆G 2 > 0 (∆G 2 < 0) and ∆G 1 < 0 (∆G 1 > 0).

Simulation of the overall crack front curvature for an homogeneous interface

Even for a homogeneous pattern-free interface, for a sample of finite width b, the crack fronts are not exactly straight but adopt a curved shape (see wide dashed black lines in Fig. 3). This shape is due to the anticlastic deformation of the bent plates. To our knowledge, there is no simple analytical formula for modeling this contribution to the crack front shape. Therefore we have calculated the crack shape in an equivalent homogeneous medium corresponding to the experimental geometry and loading. Using the FE method we have calculated crack shapes such that G(z) = G 0 along the front where G 0 is the average ERR computed previously by FE. The crack front shape is determined from a mere steepest descent iterative algorithm. The equilibrium shape is reached when the difference between the local ERR G(z) and G 0 is less than 1% at any point of the front. These background curved shapes will then be subtracted from the experimental fronts before analysis of the deformed crack fronts in the heterogeneous samples with the perturbation formulas (Sec. 5).

Analytical approaches

The idea here is to decompose the calculation of the local ERR along the deformed crack front as the sum of its global average value and its local increase along the perturbed configuration. Analytical methods allowing us to estimate the mean value (beam model) and the local relative values (perturbation methods) are presented in sections 4.1 and 4.2 respectively. Our goal is here to be able to determine the ERR landscape analytically in order to compare theoretical predictions with previous FE numerical calculations in sections 5 and 6.

Determination of the average ERR

The first step of our theoretical study is to determine the global ERR, i.e. the mean ERR G 0 . A standard data analysis is to calculate G 0 by a simple beam bending model at fixed displacement (Fig. 2b). According to [START_REF] Kanninen | An augmented double cantilever beam model for studying crack propagation and arrest[END_REF], the ERR in a state of plane stress reads:

G 0 = 3Eh 3 δ 2 16(a + 0.64h) 4 .
(1)

Note that the geometry of the problem meets the assumptions leading to Eq. 1, i.e. a ≫ h and La > 2h, for all the cleavage tests studied in this work. We need to adapt the beam model to pass from a three-dimensional elastic problem to a beam model in one dimension. Kanninen's model is therefore employed using the average crack front position a calculated along the width of the plate in the z direction. This average position is obtained directly from experimental crack front positions a(z).

In practice, we plot the evolution of δ 2 as a function of (a + 0.64h) 4 . In Fig. 7, the case of sample HC-b30 is reported as an example. In this kind of graph, G 0 is simply proportional to the slope of the curve. We can clearly identified the three different regimes described in Sec. 3.2.

We extract the nearly constant value of G 0 in regime III, i.e. where the the pinning is stationary, by a linear regression procedure. In the case of sample HC-b30, we obtain G 0 = 0.95 ± 0.14 J/m 2 as reported in Fig. 7. This quantity will be used to determine adhesion properties in Sec. 6. The comparison with FE numerical calculations in Fig. 6a shows a good agreement. For all the experiments, the variations of G 0 due to the interaction between the crack and the defect are well reproduced by Eq. 1 as a function of the average position of the crack front. G 0 is only slightly underestimated with respect to FE calculations. The maximum relative error between the beam model predictions and the FE numerical calculations remains below 10% for all samples. This result validates the use of Kanninen's model to evaluate G 0 in our system.

Perturbation method

We now present the perturbation approaches to model the cleavage test. Our goal is to evaluate the local ERRs from the knowledge of the crack front morphology a(z), this time using a lightweight analytical method. We first discuss the formulas giving the first order variation of the local ERR due to a perturbation of the crack front position. We then customize them to our experiments: we assume that the ERR landscape is stepwise to reduce the determination of the local ERR to a single parameter, namely the ERR contrast. The usefulness of those formulas appears when applied to the experiments, since they allow to reduce the errors linked to the local noise of measures in the crack front position a(z).

Variation of the local ERR due to small crack front perturbations

Two different cases are considered: 1) when the wavelength λ of the crack front perturbation is small compared to the plate thickness, that is when the medium can be supposed to be infinite and 2) when λ is large, that is when a thin plate model can be used.

• Rice (1985)'s formula for a half-plane crack in an infinite medium: In his pioneer work [START_REF] Rice | First-Order variation in elastic fields due to variation in location of a planar crack front[END_REF] has calculated the theoretical expression of the ERR first-order variation δG(z) along a planar crack front due to a small perturbation of the front position δa(z). This expression is valid in principle for a semi-infinite crack in an infinite body. From the perspective of a DCB geometry (see Fig. 2a), such a geometric mapping is acceptable if the characteristic perturbation length λ of the crack front meets the conditions: λ ≪ h and λ ≪ a. The first condition implies that the plate must be very thick with respect to the perturbation wavelength. Provided that these assumptions are met and that the load is invariant along the crack front in the z direction, δG(k) is given in Fourier space by:

δG(k) G 0 = F ∞ (k, a) δa(k) with F ∞ (k, a) = (-|k| + 1 G 0 dG da ), (2)
where λ = 2π/|k| is the wavelength of the front perturbation and G 0 the ERR of the straight crack front of average position a. Note that to first order G 0 corresponds to the average ERR as introduced in the previous sections. The derivative dG/da is calculated in our work from the simple Euler-Bernoulli beam model [START_REF] Tada | The stress analysis of cracks handbook[END_REF] consistent with the plate model presented subsequently. In this case, the variation 1/G 0 .(dG/da) with the crack length a is merely given by -4/a.

• Legrand et al. ( 2011)'s formula for a half-plane crack in a thin plate Rice's method has been extended by [START_REF] Legrand | Coplanar perturbation of a crack lying on the mid-plane of a plate[END_REF] adapting it to the case of a semi-infinite planar crack located in the mid-plane of a thin plate. This system satisfies the conditions: λ ≫ h and h ≪ a. Under these assumptions, the perturbation of the crack can be treated with the Love-Kirchhoff theory for thin plates, which greatly reduces the complexity of the problem. Note that in this case, the first condition implies that the plate must be very thin with respect to the perturbation wavelength. The first order variation of the ERR in Fourier space writes as follows:

δG(k) G 0 = F Plate (ka) δa(k) with F plate (ka) = 2 a 2ka cosh(2ka) -sinh(2ka) 2ka -sinh(2ka) .
(3)

Application to a stepwise ERR landscape

The cleavage experiments can be modeled by assigning characteristic ERRs to the homogeneous area (G 1 ) and defect strip (G 2 ). This assumption seems very reasonable from FE numerical results of Sec. 3.2. In the presence of a defect of known width d, the ERR landscape is modeled by:

G(z) = G 0 + Π 1 (z)∆G 1 + Π 2 (z)∆G 2 with Π 1 = 0, Π 2 = 1 if |z| < d/2 Π 1 = 1, Π 2 = 0 if |z| > d/2 , ( 4
)
where Π i are rectangular functions. The variation of the normalized ERR is:

δG(z) G 0 = ∆G 1 G 0 Π 1 (z) + ∆G 2 G 0 Π 2 (z). ( 5
)
The average G 0 provides a relation linking the ERR of the defect strip and the surrounding homogeneous medium.

Denoting G 1 the ERR in the homogeneous zone and G 2 the ERR in the defect strip, we assume that G 0 is an average along the front line, which we simply write as:

G 0 = G 1 (b -d) + G 2 d b . (6)
We can write Eq. 5 in Fourier space to deduce the front shape δa(z) from Eqs. 2 or 3. Using Eq. 6 to express ∆G 2 as a function of ∆G 1 , we obtain:

δa(k) = 1 F(k, a) ∆G 2 G 0 -d/(b -d) Π 1 (k) + Π 2 (k) , (7)
where F(k, a) is one of the elastic kernels from Eqs. 2 or 3. Eq. 7 establishes a relationship which relates the shape of the crack front with the ERR contrasts between the defect strip and the homogeneous medium in the present geometry.

Determination of ERR contrasts

In the previous sections, we have presented experimental front morphologies in homogeneous and patterned interfaces, along with thorough FE modeling to access both global ERR and ERR distribution over the sample. We now analyze the same data with the analytical formulas derived from the perturbation method.

Corrections of experimental crack front positions

In order to apply the perturbation approaches to calculate the local ERR, it is first necessary to correct the experimental front shapes a(z) Exp = a + δa(z) Exp from extrinsic effects that were not taken into account in the perturbation method. This task is equivalent to determining the unperturbed crack front geometry and its variation with crack length δa(z) in the absence of defect strip. Indeed, the perturbation approaches described in Sec. 4.2 apply only to a solid which is infinite in the lateral (z) direction, i.e. an infinite straight crack front. In the experiments, the finite width of the sample induces a curvature of the front due to the anticlastic effect as presented in Sec. 3.3. In order to take into account this additional finite size effect, we take as unperturbed reference crack front, for a given global G 0 level, the curved front a(z) Anti = a + δa(z) Anti calculated by the FE simulations method presented in Sec. 3.3. Examples of curved fronts obtained by this method for the HC-b30 cleavage test are shown in Fig. 8. These shapes are characteristic of curved crack fronts in homogeneous specimens of finite width [START_REF] Jumel | Crack front curvature in the wedge test[END_REF]. Note that, due to the changes of the ratio a/b, and thus the stress state with the advance of the crack, the curvature of the crack depends on the average crack length as expected. The loading on the triangular tip of the plates also reinforces the crack front curvature variation. This curvature variation shown in Fig. 8 emphasizes that the influence of the width of samples is not constant as assumed in [START_REF] Dalmas | Crack front pinning by design in planar heterogeneous interfaces[END_REF]. It clearly depends on the the mean crack length a. The solutions obtained will serve us as the reference unperturbed solutions in the perturbation approaches presented below.

A second correction comes from a slight rotation around the x-axis of the wedge between the plates during loading which induces an imperfect symmetry with respect to the plane z = 0 at the center of the sample. This lack of symmetry is reflected by a slight linear bias in z of the crack position. For instance, the crack front shape, corrected from the anticlastic effect, in a homogeneous medium is a linear function of z instead of being completely flat with δa(z) = 0 for all points along the front. We choose to correct this load-related effect by subtracting a linear fit δa(z) Lin to the experimental position. This procedure partially restores the symmetry of the problem with respect to the plane z=0 at the center of the specimen as assumed in the model described in the previous theoretical section. Note that it does not affect the ERR contrast calculation, but merely allows a better fit of the elastic line model on experimental data.

The variation of the position of the crack front around its average position a corrected from both anticlastic and rotation effects writes:

δa(z) = δa(z) Exp -δa(z) Anti -δa(z) Lin . (8)
An example of a corrected crack front morphology in regime III for the HC-b30 sample is shown in Fig. 9. The impact of the defect strip on the front morphology is clearly visible. Consistent with the FE analysis of Fig. 5 and6, Figure 9: Variation of the experimental crack front positions (symbols) corrected through Eq. 8 for the cleavage test HC-b30. The dashed and solid curves correspond to the fitted semi-infinite (Eq. 2) and thin plate (Eq. 3) models, respectively. Note that the vertical axis is expanded approximatively ten times compared to the horizontal axis.

an increase in ERR corresponds to the anchoring of the crack. Note that the ordinate axis of Fig. 9 is approximatively expanded ten times in comparison with the axis of abscissa. This observation reflects the fact that the perturbations of the crack front position are small compared to the width of the specimen. The morphologies of experimental crack fronts therefore justify a priori a theoretical treatment based on a first-order perturbative approach presented in Sec. 4.2.

Quantitative analysis of crack front deformations with perturbation methods

In order to reproduce the shape of the experimental crack front with Eq. 7, we have chosen to work in the direct space by adjusting the single free parameter ∆G 2 /G 0 . Indeed, the presence of experimental noise on the crack front position makes the determination of the ERR contrast difficult from a direct use of Eq. 7 in Fourier space. The normalized ERR contrast is determined by minimizing the mean square error between the experimental front shape and the front morphology predicted after a numerical transformation into real space of Eq. 7. This adjustment procedure has fewer degrees of freedom than in the work of [START_REF] Dalmas | Crack front pinning by design in planar heterogeneous interfaces[END_REF] where the width of the defect strip was taken as a free parameter.

The morphologies of the fronts obtained from the best fit of the different elastic kernels are plotted in Fig. 9. Both perturbative approaches satisfactorily reproduce the crack front shapes except near the edges of the specimen due to free surface effects. Fig. 9 shows only a slight better agreement with the experimental results for the thin plate model. In order to get quantitative interpretation, the average mean square error over all the crack configurations for the four samples has been computed for both models. We found that the error on the experimental crack front positions is lower in average for the thin plate model than for the semi-infinite model. It seems difficult however to find a significant difference between the semi-infinite and thin plate formulas on the basis of the front morphology. In the next section we will see that quasi-equivalent front shapes in fact involve very different ERR contrasts allowing us to judge of the applicability of the models.

Comparison between perturbation methods and FE results

The fitting procedure presented above gives us the opportunity to access local ERR contrasts, i.e. to the difference of ERR between the defect strip and its homogeneous environment. From this adjustment we can calculate the normalized contrasts ∆G 2 /G 0 directly through Eq. 7. ∆G 1 /G 0 is then deduced from ∆G 2 /G 0 through Eq. 6. This last step completes the determination of the ERR landscape by the perturbation method, knowing the geometry of the system, especially the crack front morphology and the defect strip width.

As expected from the theoretical work of [START_REF] Legrand | Coplanar perturbation of a crack lying on the mid-plane of a plate[END_REF], the absolute values of ERR contrasts predicted by the semi-infinite model are found to be lower than those predicted by the thin plate model. The ratio between the latter and the former is about 2.5. To discriminate between the two elastic line models, we compare their respective predictions with the numerical FE calculations. Comparisons between the ERR contrasts deduced from theoretical approaches with the FE results are shown in Fig. 10 where the results obtained for all crack fronts of the four samples LC-b40, MC-b40, MC-b50, and HC-b30. Despite some discrepancies, Fig. 10 shows a much better description of the ERR contrasts when the plate model is used. The average relative error on ∆G i /G 0 for all the data of Fig. 10 in comparison with the FE results decreases from 61% when Rice's model is applied directly as for a semi-infinite medium to 16% when the finite thickness of the plate is taken into account.

Despite a correct description of the front shapes of the pinned cracks, Rice's model as applied directly to the present DCB tests systematically underestimates the ERR normalized contrasts. This result comes from the fact that the perturbation wavelengths of the crack front are much larger than the specimen thicknesses. This is often encountered in the literature when thin plate geometries is used to study interfacial crack propagation. Therefore, in such geometries, it is necessary to model the cracks by taking into account the finite thickness of the plate, as shown by [START_REF] Legrand | Coplanar perturbation of a crack lying on the mid-plane of a plate[END_REF]. Using this model, we find quantitative agreement, i.e. within the error bars, with the three dimensional FE calculations.

In the more general case, [START_REF] Legrand | Coplanar perturbation of a crack lying on the mid-plane of a plate[END_REF] have also developed a model that connects both regimes (semiinfinite and thin plate) as a function of the wavelength of the perturbations relative to the thickness of the specimen. This more general kernel is not relevant here since the width of the defect is much larger than the plate thickness h, so that we always stand in the vicinity of the thin plate limit.

Crack front propagation and material properties

Propagation criterion

Thanks to our experimental procedure, the interpretation of the previous results in terms of material properties, i.e. adhesion, is fairly straightforward. Indeed, due to the quasi-static propagation of the crack, a propagation criterion has to be fulfilled at any point of the observed front. For a given load, the crack front stops at locations where the propagation driving force is no longer sufficient. It is common for brittle fracture to assume that the crack front advance is ruled by the Griffith criterion. In this framework, the equilibrium morphology of the crack front a(z) = a + δa(z) for a given opening δ obeys: where G c (z) is the local adhesion energy. Therefore, we can deduce the fracture energies of both the surrounding homogeneous medium and the defect strip interfaces denoted G 1c and G 2c respectively. In a very similar way, the adhesion energy has been measured for homogeneous interfaces by [START_REF] Barthel | Asymmetric silver to oxide adhesion in multilayers deposited on glass by sputtering[END_REF].

G(z) ≤ G c (z), (9)
While the data of the crack front geometry and the loading geometry can give a direct access to the ERR G(z), the same is not systematically true for the adhesion energy G c (z). It would be the case if the arrest condition for a crack front Eq. 9 was an equality and not only a simple inequality. Thus, the fracture energy can only be deduced from the ERR for stationary crack propagation regimes, i.e. where the materials properties are invariant in the crack propagation direction [START_REF] Roux | Effective toughness of heterogeneous brittle materials[END_REF]. In our system, it is the case for the regimes I and III described previously. Between these two stationary regimes a transient regime (II) is observed where the inequality of Eq. 9 remains and the determination of G c (z) is not possible.

In the first regime (stage I of Fig. 3), the crack propagates in a completely homogeneous zone and G 1 = G 1c along the front. When the crack begins to interact with the obstacle (stage II of Fig. 3), G 2 ≤ G 2c in the defect strip and G 2 increases with the applied load involving a significant change in ∆G i . Once the defect is penetrated by the crack G 2 = G 2c in strip and, for each increment of the opening, the crack advances with ∆G i nearly constant. In this last stationary regime of crack propagation (stage III in Fig. 3) the toughness contrast is invariant in the direction of propagation, and we have equality between the local values of adhesion energy and ERR all along the front. The present methods can then be used to measure interfacial toughness in the defect strip.

Note that it is also possible to simulate the quasi-static propagation of a crack for a given toughness landscape. The adhesion energy of the defect strip and its surrounding homogeneous medium could be obtained by adjusting their values in order to reproduce the shape of the crack front observed experimentally. This procedure is however computationally demanding as it implies to describe the propagation of the crack which involves the calculation of a large number of configurations. In this study, the aim was not to perform propagation simulations of the crack, but to validate the analytical perturbation approach, hence it was enough to take advantage of our experimental setup which offers a direct observation of the crack front equilibrium shape and to perform direct simulations of the elastic problem for each equilibrium configuration.

Determination of the defect strip adhesion

In this last section we leverage the originality of this work by focusing on the determination of the defect strip adhesion G 2c in the framework of crack pinning in heterogeneous interfaces. The measurement of G 2c is carried out in the regime III where the crack front pinning is invariant in the direction of propagation.

In this regime, we compute the fracture energy G 2c as the sum of global ERR plus the average ERR fluctuation in the defect strip G 0 + ∆G 2 . Since G 2c fluctuates due to experimental noise we choose to compute its average G 2c over all the crack front configurations belonging to the last stationary pinning regime (blue diamond symbols in Fig. 6). In the case of FE numerical calculations, G 2c is simply computed from the mean local ERRs G(z) in the defect strip as presented in Sec. 3.2. In the case of analytical approaches, G 0 is computed with Eq. 1 (Sec. 4.1) while ∆G 2 are determined through the fit of the experimental crack front positions by the two perturbation approaches (Sec. 5.2). We finally computed G 2c by averaging G 0 + ∆G 2 .

The results are summarized in Tab. 2. As expected following the conclusions about ERR contrasts of the previous section, the semi-infinite model predictions deviate significantly from the FE results summarized in Tab. 2. In turn, we observe a very satisfactory agreement between the values obtained from perturbative approaches using the plate model and the values calculated by FE. The agreement is even quantitative, i.e. inside the error bars, for the samples LC-b40, MC-b40 and MC-b50. Moreover, nearly identical adhesion energies are found for the MC-b40 and MC-b50 samples due to the identical nature of their cracked interfaces as reported in Tab. 1. The adhesion energy for the defect strip of sample HC-b30 is slightly out of the margins of error calculated. This interface presents however the largest adhesion contrast of the four samples studied. One interpretation could therefore reside in the fact that in the latter case we touched the limits of first-order perturbative approaches.

As a result, our approaches based on the patterning of a weak interface can be considered as a new method to determine the adhesion energy of heterogeneous interfaces for transparent materials by measuring the crack front deformation that it induces.

Summary and perspectives

We have shown how the results of cleavage test experiments can be thoroughly analyzed by FEM. The ERR has been computed for all the measured crack front and a consistent picture of the crack propagation in the heterogeneous interface has been reconstructed. This analysis of the data is particularly illuminating when considering the increasing deformation of the crack front as it hits the edge of the high toughness area as it progresses forward. Due to the increasing curvature of the crack front, the local energy release rate rises until it reaches the toughness of the pinning area which then starts to rupture.

This relation between front curvature and local energy release rate is at the core of the perturbation method proposed by Rice. Fitting the front with a perturbation kernel is a much lighter way of analyzing the data. Here we have demonstrated that plate thickness is a first order parameter in modelling crack front morphology by perturbation methods when the wavelengths of the perturbation are comparable to the plate thickness. In our experimental configuration, the semi-infinite kernel (Rice) gives results which are only qualitatively correct, while the agreement is clearly improved when the finite plate thickness kernel is used.

Our study, combining experiments, numerical simulations and theoretical analysis shows that it is possible to describe quantitatively the local adhesion contrasts only from the observed crack front morphologies taking into account the whole problem geometry. As a result, it can serve as a method to characterize the local toughness of transparent materials.

To improve the description of the depinning threshold for crack propagation in heterogeneous brittle materials two directions of investigation can be naturally considered from this work. The first one is to develop an approach capable of handling the anticlastic effect analytically [START_REF] Jumel | Crack front curvature in the wedge test[END_REF]. Under this condition, our approach could be more effective and avoid any numerical simulations. A second perspective is related to the size of the perturbation induced by the heterogeneity of the material. Elastic line models presented in this paper are indeed limited by a first order perturbative approach. If it seems that it can account for adhesion contrasts up to 4 J/m 2 , we expect a loss of validity as the toughness contrast increases. Approaches including higher orders in perturbation [START_REF] Rice | Weight function theory for three-dimensional elastic crack analysis[END_REF][START_REF] Bower | A three-dimensional analysis of crack trapping and bridging by tough particles[END_REF][START_REF] Lazarus | Brittle fracture and fatigue propagation paths of 3d plane cracks under uniform remote tensile loading[END_REF][START_REF] Favier | Coplanar propagation paths of 3D cracks in infinite bodies loaded in shear[END_REF][START_REF] Leblond | Second-order coplanar perturbation of a semi-infinite crack in an infinite body[END_REF] could be a solution and should allow to treat much higher adhesion contrasts.

Figure 1 :

 1 Figure 1: (a) Schematics of a patterned multilayer deposited on glass exemplified for the HC-b30 sample. (b), (c) and (d): the multi-layers are reinforced by a glass backing prior to cleavage test for the HC-b30, MC-b40 (MC-b50) and LC-b40 samples respectively (see text for details). The plane of fracture is shown in red.

Figure 2 :

 2 Figure 2: (a) Schematic of cleavage test on a double cantilever beam. The opening of the crack δ is imposed by the wedge in order to control the average length of the crack a. (b) Top view sample photography during the cleavage test HC-b30. Thanks to transparency of the glass, interfacial crack is observed. The deformation of the crack comes from the difference of adhesion energies between the silver interface and the defect strip.

Figure 3 :

 3 Figure 3: Evolution of the equilibrium shape of the measured crack front for the HC-b30 sample. The three successive regimes of propagation are distinguished. I: Stationary propagation along the silver homogeneous interface (wide dashed black lines); II: transient deformation of the front when entering the defect strip (red dotted lines); III: stationary propagation of the deformed front in the defect strip (continuous blue lines).

Figure 4 :

 4 Figure 4: Typical mesh of the cleavage test used in the finite element calculations. The unbroken interface is colored in blue. The highest mesh density corresponds to the crack front position. A zoom of the mesh in the vicinity of the crack tip is also reported in red.

Figure 5 :Figure 6 :

 56 Figure 5: Local Energy Release Rate (ERR) computed by Finite Element (FE) for the cleavage test HC-b30. (a) Evolution of the ERR landscape. The three successive stages of propagation are distinguished as in Fig. 3. The ERR contrast expected between the defect strip and its surrounding homogeneous medium is well reproduced by the ERR landscape. (b) Local ERR along a crack front during regime III of Fig. 3. G 0 denotes the average along the front while ∆G 1 and ∆G 2 denote the average variations of the energy release rate compared to G 0 outside and inside the defect strip respectively.

Figure 7 :

 7 Figure 7: Typical data plot according to Kanninen's model for the cleavage test HC-b30. The slope corresponds to the mean Energy Release Rate (ERR) G 0 . The three successive regimes of propagation are distinguished as in Fig. 3. I: stationary propagation along the silver homogeneous interface (black circles); II: transient deformation of the front when entering the defect strip (red squares); III: stationary propagation of the deformed front in the defect strip (blue diamonds). The straight line is a linear regression of the data in the regime III.

Figure 8 :

 8 Figure 8: Variation of the crack front position calculated by the finite element method for an equivalent homogeneous medium whose curvature comes from anticlastic coupling. The obtained shapes are exemplified for the HC-b30 cleavage test for different mean crack front positions. Note that the scale of the axes are different. The front is almost straight.

Figure 10 :

 10 Figure 10: Comparison of normalized Energy Release Rate (ERR) contrasts ∆G i /G 0 calculated by the finite element method with those obtained from the semi-infinite (red open symbols) and the thin plate (black solid symbols) theoretical approaches. The blue dotted lines correspond to the perfect equivalence between these two quantities. The error bars are deduced from statistical dispersions.

Table 1 :

 1 Nature and dimensions of the patterned samples. For each stack the patterned layer is shown in bold.

	Sample	Stack	b (mm) L (mm) d (mm) H (mm) Homog. Interf.	Defect strip
	LC-b40	Gl./Org/Ag/Si 3 N 4	38	64	2.82	3	Glass/Ag	Glass/Organic
	MC-b40 Gl./Si 3 N 4 /Ag/Si 3 N 4	40	65	3.2	3	Si 3 N 4 /Ag	Si 3 N 4 /Si 3 N 4
	MC-b50 Gl./Si 3 N 4 /Ag/Si 3 N 4	49	82.5	3.87	2.5	Si 3 N 4 /Ag	Si 3 N 4 /Si 3 N 4
	HC-b30	Gl./Ag/Si 3 N 4	29.5	36.5	3.2	1.5	Glass/Ag	Glass/Si 3 N 4

Table 2 :

 2 Average adhesion energy of the different defect strip interfaces G 2c = G 0 + ∆G 2 computed by FE analysis, perturbation theory using Rice (semi-infinite geometry) and Legrand (plate geometry) formulas respectively.

	Sample	Stack	Finite element (J.m -2 ) Rice model (J.m -2 ) Plate model (J.m -2 )
	LC-b40	Gl./Org/Ag/Si 3 N 4	0.35 ± 0.04	0.46 ± 0.19	0.32 ± 0.13
	MC-b40 Gl./Si 3 N 4 /Ag/Si 3 N 4	1.57 ± 0.11	1.13 ± 0.09	1.52 ± 0.14
	MC-b50 Gl./Si 3 N 4 /Ag/Si 3 N 4	1.86 ± 0.12	1.16 ± 0.12	1.75 ± 0.18
	HC-b30	Gl./Ag/Si 3 N 4	4.63 ± 0.31	2.23 ± 0.36	4.09 ± 0.64

Acknowledgements

The support of the ANR Programme SYSCOMM (ANR-09-SYSC-006) is gratefully acknowledged.


Michał Łomzik

Olga Mazuryk

Dorota Rutkowska-Zbik

Grażyna Stochel

Philippe C Gros
email: philippe.gros@univ-lorraine.fr

Małgorzata Brindell
email: brindell@chemia.uj.edu.pl

Jerzy Haber

New ruthenium compounds bearing semicarbazone 2-formylopyridine moiety: Playing with auxiliary ligands for tuning the mechanism of biological activity

Keywords: ruthenium, polypyridine, semicarbazone, anticancer activity, apoptosis, auxiliary ligands

Two ruthenium(II) complexes Ru1 and Ru2 bearing as a one ligand 2,2'-bipyridine substituted by a semicarbazone 2-formylopyridine moiety (bpySC: 5-(4-{4'-methyl-[2,2'-bipyridine]-4-yl}but-1-yn-1-yl)pyridine-2-carbaldehyde semicarbazone) and as the others 2,2'-bipyridine (bpy) and 4,7-diphenyl-1,10-phenanthroline (dip), respectively, as auxiliary ligands have been prepared. Their biological activity has been studied on murine colon carcinoma (CT26) and human lung adenocarcinoma (A549) cell lines. The anti-proliferative activity was dependent on the presence of bpy or dip in the complex, with one order of magnitude higher cytotoxicity for Ru2 (dip ligands). Ru1 (bpy ligands) exhibited a distinct increase in cytotoxicity going from 24 to 72 h of incubation with cells as was not observed for Ru2. Even though both studied compounds were powerful apoptosis inducing agents, the mechanism of their action was entirely different. Ru1-incubated A549 cells showed a notable increase in cells number in the S-phase of the cell cycle, with concomitant decrease in the G2/M phase, while Ru2 promoted a cell accumulation in the G0/G1 phase. In contrast, Ru1 induced marginal oxidative stress in A549 cell lines even upon increasing the incubation time. Even though Ru1 preferably accumulated in lysosomes it triggered the apoptotic cellular death via an intrinsic mitochondrial pathway. Ru1incubated A549 cells showed swelling and enlarging of the mitochondria. It was not observed in case of Ru2 for which mitochondria and endoplasmic reticulum were found as primarily localization site. Despite this the apoptosis induced by Ru2 was caspase-independent. All these findings point to a pronounced role of auxiliary ligands in tuning the mode of biological activity.

Introduction

One of the most important issues in the field of medicinal inorganic chemistry concerns the discovery of more effective and safer anticancer drugs. Many of the currently studied metallodrugs are designed to be prodrugs and selectively activated in the cancer tissue. However, a major advantage of this strategy can also be its main weakness, since several possible active molecules are produced and start to interact with biomolecules in the human body. Investigation of all these interactions is very challenging and is still the focus of many studies. Because of this, the perspective about designing new anti-cancer drugs slowly shifted [1], and an increased attention is paid to ruthenium compounds inert to substitution mainly those with polypyridyl ligands.

Their electronic and structural properties can be tuned easily by the selection of appropriate ligands giving rise to the formation of versatile compounds which might be designed either for therapeutic and/or optical imaging purpose.

The design of potential drugs based on organic compounds usually involves the modification of the lead structure in respect to the site of action, or otherwise new compounds are formed based on the known molecular targets (e.g. receptor proteins or enzymes) [START_REF] Avendano | Medicinal chemistry of anticancer drugs[END_REF]. Even though many active rutheniumbased compounds have been discovered, their biological activity mechanism remains unclear and their targets still unidentified. The lack of this knowledge hampers rational design of new anticancer agents based on these complexes. A new approach recently developed, is to design compounds where one or more polypyridyl ligands around ruthenium center are modified by anchoring an organic molecule with a known biological target or mechanism of action. The choice of the organic targeting molecule is critical since it can have various functions such as i) tuning the lipophilic character of the whole Ru complex for specific subcellular accumulation (mitochondria, reticulum endoplasmic, nucleus, lysosome) [START_REF] Lameijer | [END_REF]4]; ii) targeting the specific

A C C E P T E D M

A N U S C R I P T 4 receptors, enzymes or cellular processes [5,6]; iii) oxygen-dependent accumulation in cells [7,8]; iv) creating synergistic effects between the biologically active ligand systems and the metal centre; v) tuning the photophysical/photochemical properties for an appropriate light activation (photodynamic and photoactivated therapy) [9,10]; vi) combining therapy and diagnosis (theranostics) [11] and others.

In this context, we have designed two new ruthenium(II) polypyridyl complexes in which one bipyridyl ligand is modified by a semicarbazone 2-formylopyridine (Scheme 1). α-N-heterocyclic (thio)semicarbazones and their metal complexes are known as anticancer agents [12][13][14] therefore combining such moiety with Ru(II) polypyridyl complexes might bring additional benefits towards their anticancer activity. The chosen semicarbazone is a structural analogue of 3aminopyridine-2-carboxyaldehyde thiosemicarbazone, which is currently clinically tested as an anticancer drug (Triapine®) [15,16]. The anticancer activity of the Triapine® arises from its ability to inhibit the action of ribonucleotide reductase (RR) [17]. The catalytically active centre of RR comprises a tyrosyl radical stabilized with two iron ions which might be a target for Triapine® having good metal chelating properties [18]. Ribonucleotide reductase is required for DNA synthesis since it catalyses the reduction of ribonucleotides into deoxyribonucleotides.

Consequently the inhibition of this enzyme interrupts cell proliferation. To take an advantage of its chelating properties, semicarbazone 2-formylopyridine moiety was kept away from Ru centre by a spacer based on an alkyne chain (Scheme 1). 2,2'-bipyridine (bpy) and 4,7-diphenyl-1,10phenanthroline (dip) were chosen as auxiliary ligands to evaluate the impact of their diverse lipophilic character and size on the biological activity. The synthesis, photophysical properties and biological evaluation on two cancer cell lines: murine colon carcinoma (CT26) and human lung adenocarcinoma (A549) of the Ru1 and Ru2 complexes are reported. Their cytotoxicity,

A C C E P T E D M

A N U S C R I P T 5 cellular distribution, induction of apoptosis, effect on cell-cycles, mitochondria potential, intracellular calcium concentration as well as reactive oxygen production (ROS) are discussed in relation to the parent ruthenium(II) complexes ([Ru(bpy)3] 2+ and [Ru(dip)2(bpy)] 2+ ) to understand their mechanism of action. Our results prove that the choice of the auxiliary ligands might have a predominate effect on the mode of action of ruthenium complexes having one ligand modified with biologically active organic substituent.

N N N N N N Ru 2+ Me N N N H NH 2 O N N N N N N Ru 2+ Me N N N H NH 2 O Ru1 Ru2
Scheme 1. Ruthenium compounds studied in this work.

Materials and methods

Synthetic procedures

All solvents were at analytical grade and were used without further purification. DMF was stirred over calcium hydride for 12 h, filtrated and distilled from over potassium carbonate [START_REF] Armarego | Purification of Laboratory Chemicals[END_REF].

Diisopropylamine was distilled over NaH and stored under argon [START_REF] Armarego | Purification of Laboratory Chemicals[END_REF]. Reagents were purchased from Sigma-Aldrich (ruthenium(III) chloride, n-butyllithium solution in hexanes, isopropylmagnesium solution in THF, 2,2'-bipyridyl, 4,7-diphenyl-1,10-phenantroline), Alfa under argon conditions at -78°C, a solution of n-butyllithium in hexane (0.75 mL, 1.2 mmol) was added dropwise. The solution was stirred for 30 minutes at -78°C and then warmed up to 0°C. A solution of 4,4'-dimethyl-2,2'-bipyridine (1) (0.184 g, 1 mmol) in dry THF (10 mL) was then added. After 2 h, 3-(trimethylsilyl)propagyl bromide (0.25 mL, 1.5 mmol) was added rapidly.

After another hour, the mixture was warmed to room temperature and quenched with water (30 mL). The organic phase was extracted with diethyl ether (3x20 mL) and the combined organic layers were washed twice with brine and dried over magnesium sulphate. The solvent was removed under reduced pressure to give the pure product 2 (0.276 g, 92%). 2 was found too instable to be stored and was involved immediately in the next stage of the synthesis. S5).

Synthesis of 4-(prop-2-yn-1-yl)-4'-methyl-2,2'-bipyridyl (3).

To a solution of 2 (0.276 g, 0.9 mmol) in methanol (5 mL) was added potassium carbonate (0.205 g, 1.5 mmol). The mixture

A C C E P T E D M A N U S C R I P T 7
was heated under argon at 50ºC for 2 h. After this time, the reaction was cooled down to 0°C, neutralized with 2M HCl and extracted with diethyl ether (3x30 mL). The combined organic layers were washed twice with brine and dried over magnesium sulphate. The solvent was removed under reduced pressure to give the pure product as a brown solid (0.150 g, 72%). To a solution of 3 (0.170 g, 0.75 mmol) in acetonitrile (6 mL), copper (I) iodide (0.05 g, 30 mol%) and Tetrakis(triphenylphosphine)palladium(0) (0.045 g, 5 mol%) were added under argon conditions. Diisopropylamine (6 mL) was added and the mixture was stirred for 15 min. After that, a suspension of 6 (0.210 g, 0.9 mmol) in methanol (1 mL) was added. The mixture was refluxed for 18 hours. Concentrated ammonia (1 mL) was added to the brown suspension and the mixture was filtrated through Celite (followed by 70 mL of acetonitrile). The solvent was removed under reduced pressure and the residue was dissolved in dichloromethane/concentrated ammonia mixture. The organic phase was separated, washed twice with concentrated ammonia (till all copper was removed), once with brine and dried over magnesium sulfate. The solvent was removed under reduced pressure to give the crude product which was dissolved in chloroform (15 mL) and treated with cyclohexane (60 mL). The mixture was added. After 24h at -20ºC, the dark solid obtained was filtrated and washed with cold acetone, twice with diethyl ether and dried under vacuum. The crude product was finally purified by flash chromatography (neutral Al2O3, dichloromethane/methanol (5/95)) to give the pure product.

A C C E P T E D M A N U S C

1. cis-Ru(bpy)2Cl2 (8) [22]

. 8 was obtained using 2,2'-bipyridine as ligand (0.181 g, 38%). ) ppm (Fig. S11).

[HRMS] Calculated for C42H36N10ORu: m/z 399.1052 (M + ), Found 399.1058, (M + ) Anal. Calc.

for C42H36N10ORu: C (58.1%), H (4.2%), N (16.1%), Found: C (58.2%), H (4.4%), N (16.1%).

4. Synthesis of [Ru(dip)2(bpySC)]Cl2 (Ru2).

To a solution of 9 (0.019 g, 0.023 mmol) in absolute ethanol (10 mL) under argon conditions, was added a solution of bpySC (0.010 g, 0.026 mmol) in absolute ethanol (5 mL). The mixture was refluxed for 22 h. After that time the solvent was removed under reduced pressure. The crude product was dissolved in dichloromethane and purified by flash chromatography (Al2O3 neutral, dichloromethane/Methanol(5/95 to 10/90 gradient) to give the pure product with yield (0.013 g, 47%). ) ppm (Fig. S12).

[HRMS]: m/z: Calculated for C70H52N10ORu: m/z 575.1679 (M + ), Found 575.1658 (M + ).

Spectroscopy measurements

UV-Vis absorption spectra of ruthenium complexes were recorded at 25ºC in water using Perkin Elmer Lambda 35 spectrophotometer. Luminescence measurements were registered on Perkin Elmer LS55 spectrofluorimeter in the range 480 -900 nm upon excitation at the maximum of charge transfer band for each ruthenium complex. The average of three scans was subjected to smoothing. For the determination of the quantum yield of luminescence (Φ), aqueous solutions of [Ru(bpy)3] 2+ with a small amount of DMSO (<0.008% v/v) were used as standards (Φ = 0.028 [24] and 0.042 [25] for air-equilibrated and deoxygenated conditions, respectively). The spectra for the ruthenium complexes were recorded at a concentration that had an absorbance less than 0.05 units at the excitation wavelength. The quantum yield was calculated according to the following equation: [START_REF] Lakowicz | Principles of Fluorescence Spectroscopy[END_REF]

Φ = Φref × [Aref/A] × [I/Iref] × [n 2 /nref 2 ]

where I is the integrated luminescence intensity, A is the optical density, and n is the refractive index, ref refers to the values for the reference. The mean value was calculated from a minimum of three independent experiments.

Luminescence lifetime experiments were performed at room temperature using Fluorolog-3

Horiba Jobin Yvon with single photon counting technique. The excitation wavelength was fixed at 464 nm (NanoLED diode) and average luminescence lifetime was measured at emission maximum. The instrument response functions were measured using a light scattering solution of Ludox (colloidal silica, Sigma-Aldrich). The DAS6 software (HORIBA Scientific) was used for

A C C E P T E D M A N U S C R I P T 12
deconvolution of the obtained decays and for calculation of the lifetime values. Two or three exponential fit were chosen based on χ 2 parameter (the goodness of fit evaluation). A singleexponential fit was found to be an optimal description of the obtained results for the ruthenium compounds.

Computational characterization

To obtain ground state geometries and electronic structures of Ru1 and Ru2 Density Functional Theory (DFT) was applied using hybrid B3LYP functional [START_REF] Dirac | [END_REF][28][29][30][31] and 6-31G(d,p) basis set [32][33][34][35] for all atoms except for ruthenium, for which LANL2DZ basis-set [36,37] was applied. To compute electronic excitation spectra, Time Dependent -Density Functional Theory (TD-DFT)

was used with the same functional and basis sets for light atoms, with LANL2DZ pseudopotential for Ru. Eighty lowest laying electronic excitations were determined. All calculations were done with Gaussian 09 program [START_REF] Frisch | Gaussian, Inc[END_REF], orbitals were visualized using GaussView [START_REF] Dennington | GaussView[END_REF] software.

Cell Culture Conditions

Biological studies were performed using murine colon carcinoma CT26 and human lung adenocarcinoma A549 cell lines. Cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% antibioticspenicillin (100 units/mL) and streptomycin (100 μg/mL). Cells were routinely cultured at 37 °C in a humidified incubator in a 5% CO2 atmosphere.

Cytotoxicity

Cell cycle

A549 cells were seeded into a 6 wells plate with a density of 3 × 10 4 cells per cm 2 . Cells were cultured in the full medium for 1 day. Then, the medium was removed and replaced with a basic medium containing various concentrations of the studied compounds, and incubated for 24 h.

Then, the cells were washed with PBS, detached by trypsin, fixed in cold methanol for 30 min, stained with propidium iodide (PI) for 4 h in the dark, followed by analyzing using a BD LSRFortessa cytometer. The experiment was performed twice.

Apoptosis

The early stage of apoptosis is manifested by the relocation of phosphatidylserine (PS) from the inner side of the cellular membrane to the outer side. This process can be revealed by the staining cells with the labeled Annexin V. Annexin V is a cellular protein that in the presence of Ca 2+ ions, selectively binds to PS. DAPI was used instead of propidium iodide to assess the cell necrosis, due to the overlapping excitation/emission parameter of the propidium iodide and the Ru complexes in live cells [41]. A549 cells were seeded into a 6 wells plate with a density of 3 × 10 4 cells per cm 2 and cultured in the full medium for 1 day. Afterwards, the medium was removed and replaced by a medium containing different amounts of the studied compounds and incubated for 24 h. Then, cells were washed with PBS and binding buffer. The cells were stained with Annexin V-FITC for 10 min in the dark and then, with DAPI (0.5 µM) for 5 min. Cells were analyzed by a BD LSRFortessa cytometer. As a positive control, H2O2 (300 µM) was used.

Mitochondrial morphology

MitoTracker Green (ThermoFisher Scientific) was used to visualize mitochondria of A549 cells.

A549 cells were seeded on a 6 wells plate with a density of 3 × 10 4 cells per cm 2 24 h prior to the experiments. Next, the Ru complexes were added and incubated for 24 h. After the incubation, the Ru compounds were washed and cells were stained with MitoTracker Green (100 nM) for 30 min at 37 °C. After the staining, cells were visualized using an Olympus fluorescence microscope IX51.

Cytosolic calcium homeostasis

Cytosolic calcium concentration was measured using Fluo-8 AM probe (AAT Bioquest). A549

and CT26 cells were seeded on a 96 wells plate with a density of 1.5 × 10 4 cells per cm 2 one day before the experiments. Then, cells were incubated with various concentrations of the compounds

Mitochondria membrane potential

Mitochondria membrane potential was evaluated using JC-1 probe (AAT Bioquest). A549 and CT26 cells were seeded on 96 wells plate with a density of 1.5 × 10 4 cells per cm 2 one day before the experiments. Cells were incubated with various concentrations of the Ru compounds for 24 h in the dark. Then, cells were washed with PBS and stained with JC-1 (10 µM) for 30 min in the dark at 37 °C. Next, cells were washed with PBS and analyzed by a Tecan Infinite 200 microplate reader measuring fluorescence of the probe at 525 nm and 590 nm using 490 nm as an excitation wavelength. Experiment was performed three times in triplicate and the mean values ± standard deviations were calculated.

Total ROS production

The level of the oxidative stress induced in cells after the incubation with the Ru complexes was conducted for A549 and CT26 cell lines using the cyto-ID Hypoxia/Oxidative stress detection kit.

A549 and CT26 cells were seeded with a density of 3 × 10 4 cells per cm 2 , respectively. A day later the compounds at different concentrations in medium without serum were added and incubated in the dark for 24/72 h. Then, cells were washed with PBS, treated with trypsin and analyzed by BD LSR cytometer. As a positive control, pyocyanin (300 µM) was used. The level of the oxidative stress was determined as a percentage of the ROS positive cells of the whole cell population.

A C C E P T E D M

A N U S C R I P T 17

Results and discussion

Synthesis

The preparation of the target complexes Ru1 and Ru2 required the synthesis of ligand bpySC (Scheme 2) that was synthesised by a Sonogashira coupling between 4-(but-3-yn-1-yl)-4'-methyl-2,2'-bipyridine (3) and 5-bromopyridine-2-carbaldehyde semicarbazone (6). 6 was synthesised by formylation of 5-bromo-2-iodopyridinefollowed by treatment with semicarbazide hydrochloride.

3 was synthesised by a selective monolithiation of 4,4'-dimethyl-2,2'-bipyridine (1) using LDA followed by reaction with 3-(trimethylsilyl)propagyl bromide and final deprotection using potassium bicarbonate [START_REF] Song | [END_REF]. respectively. The cis-Ru(NN)2Cl2 complexes were prepared by microwave synthesis from ruthenium(III) chloride and the proper ligand in DMF according to previously described procedures [42,[START_REF] Kharissova | Microwave-assisted synthesis of coordination and organometallic compounds[END_REF]. Interestingly, the coordination of ruthenium occurred exclusively on the bipyridine side of the bpySC and no trace of ruthenium coordination by the SC moiety was observed.

Photophysical properties

The complexes have been characterized by UV-Vis spectroscopy using water as solvent. The photophysical data are collected in Table 1. In order to measure the effect of bpySC on the properties, complexes containing one bpy instead of bpySC were prepared and characterized. As shown in Table 1 all complexes displayed intense 1 MLCT (metal ligand charge transfer) bands in the visible part of the spectrum, around 450 nm. Both Ru1 and Ru2 showed a slight red shift

A C C E P T E D M A N U S C R I P T

19 compared with their unsubstituted counterparts. In contrast, the emission band exhibited a notable blue shift (11 nm for Ru1 and 10 nm for Ru2). The introduction of the bpySC ligand also came with a decrease of the luminescence quantum yield (QY) that were 0.92% and 1.18% for Ru1 and Ru2 respectively under air-equilibrated conditions while argon-equilibrated conditions allowed to increase these values to 1.63%. and 2.62%, respectively. A decay of the MLCT excited state lifetimes was also observed for both Ru1 and Ru2. Thus while globally affecting negatively the luminescence of the complexes, the bpySC ligand allowed to maintain QY and lifetimes exploitable for further use in imaging experiments. data taken from a [24] ; b [25] ; c [44]

A C C E P T E D M A N U S C R I P T

Computational characterization

Cytotoxicity of ruthenium complexes in vitro

The cytotoxic effect of the newly synthesised Ru complexes and their reference compounds was determined on two cancer cell lines: murine colon carcinoma (CT26) and human lung adenocarcinoma cell line (A549). Complexes were tested in serum free conditions (S-) and also in the presence of 2% serum (S+). The obtained results are presented in Table 2. Generally, the presence of serum reduced the cytotoxic effect of both Ru1 and Ru2. Such an effect has been previously described [45] and might be related to a reduction in the Ru uptake due to binding to the proteins present in the serum (see Table S1) impeding complexes accumulation. Ru complexes with dip ligands were much more cytotoxic in comparison with those having bpy ones. Replacing one of the dip ligands with bpy or bpySC ligand decreases slightly the cytotoxicity of the Ru compounds. The observed effect arises from the dependence of the Ru complexes accumulation and therefore their toxicity on their lipophilicity. In agreement with some previous studies on Ru polypyridyl complexes [START_REF] Mazuryk | [END_REF], the poor cellular uptake of [Ru(bpy)3] 2+ or Ru1 can be related to their weak lipophilic character.

Increasing the incubation time from 24 to 72 h resulted in a distinct increase in an antiproliferative activity of Ru1, in particular against CT26 cells, while no significant change was observed in the cytotoxicity of Ru2. [Ru(bpy)3] 2+ remains continuously non cytotoxic even after 72 h of incubation. At the same time a dramatic increase in the toxicity after 72 h was observed for bpySC ligand alone (IC50 ca. 30 times higher). It suggests that this particular ligand might play a crucial role in the anti-proliferative activity of Ru1. Furthermore, it can be assumed that an appropriate selection of auxiliary ligands might lead to a change in action mechanism. For the studied system, switching from two dip ligands to two bpy ones clearly highlighted the cytotoxic

[Ru(bpy)3] 2+ >320 >320 >320 >320 [Ru(dip)3] 2+ 1.2 ± 0.2 - - -

Cell accumulation and cellular targets

A549 cells were used to study the accumulation of the studied ruthenium complexes. A higher cellular uptake was found for complex Ru2 in excellent agreement with the cytotoxicity data.

A C C E P T E D M

A N U S C R I P T 25 Despite similar luminescence quantum yields for Ru2 and Ru1, much higher concentrations of Ru1 were required to visualize its localization inside the cells. This further confirmed that the accumulation of ruthenium polypyridyl complexes is strongly dependent on their lipophilicity and that in the studied case it is controlled by bpy/dip ligands. Additionally, it points out the superiority of the accumulation/lipophilicity parameters over luminescence parameters of Ru complexes in assessing the potential of the compounds as cellular dyes [START_REF] Mazuryk | [END_REF].

To further investigate the accumulation of the Ru complexes, organelle-specific dyes were used.

Ru2 preferably accumulated into mitochondria and endoplasmic reticulum, as confirmed by overlapping emission of organelle-specific dyes with the studied Ru complex (Fig. 2). Complex

Ru1 exhibited rather small accumulation in those organelles (Fig. S2), while the majority of it is localized within cellular lysosomes (Fig. 3).

A C C E P T E D M

A N U S C R I P T

Cell-cycle arresting properties

The influence of Ru1, Ru2 and bpySC on cell cycle was measured by flow-cytometry (Table 3).

The incubation of A549 cells with Ru1 or bpySC induced a significant increase in the number of cells in the S-phase of the cell cycle, with the corresponding decrease in the percentage of cells in the G2/M phase. During the S-phase of the cell cycle deoxyribonucleotides, which are DNA precursors, are produced. In this phase, corresponding ribonucleotides need to be reduced by ribonucleotide reductase. Inhibition of this enzyme should increase the number of cells arrested in this phase [46,47]. The S-phase arrest caused by Ru1 or bpySC suggests that their cellular activities might be related to the inhibition of ribonucleotide reductase. Contrarily Ru2 caused increased percentage of the cells in the G0/G1 phase, similarly to other Ru complexes containing dip ligands [45]. This further confirms the influence of the auxiliary ligands on the biological activity of Ru compounds.

A C C E P T E D M

A N U S C R I P T 29

Reactive Oxygen Species (ROS) production

The influence of Ru1 and Ru2 on reactive oxygen species (ROS) production in cancer cell lines was evaluated. Ru2 as well as the attached ligand bpySC caused significant concentration dependent increase in ROS production after 24 of h incubation in both A549 and CT26 cell lines (Fig. 4, Fig. S3). Increasing the incubation time 24 h to 72 h also increase the ROS level induced by Ru2. However, Ru1 did not induce oxidative stress in the studied cell lines that could be partially explained by the lower accumulation of this complex. However increasing the incubation time with the compounds from 24 h to 72 h (that will also increase Ru complexes accumulation level) did not induce any increase in ROS production in case of Ru1 as it did with Ru2 (Fig. 4). This strongly suggested that the Ru accumulation level is not linked to the lack of ROS production in case of Ru1 and that completely different action mechanism of action responsible for cytotoxic activity depending on whether bpy or dip ligand is present in the ruthenium complex.

Mitochondrial membrane potential detection

Since the studied Ru complexes either accumulated in mitochondria (Ru2) or changed the mitochondria morphology (Ru1), their influence on alteration in mitochondria function was also studied by analyzing the mitochondria membrane potential. The loss of the mitochondria membrane potential is often considered to be a prelude to apoptosis.

The changes of mitochondrial membrane potential induced by the studied complexes were determined using JC-1 mitochondrial probe. At low mitochondrial membrane potential, JC-1 exists in monomeric form and exhibits green fluorescence, but at high mitochondrial membrane potential JC-1 forms aggregates, which exhibit red fluorescence. Two oligopeptides valinomycin and gramicidin were used as positive controls. Valinomycin is K + ionophore which allows the potassium ions passing freely through the cell membrane and in this way causing the collapse of

Intracellular calcium concentration

Calcium ion is one of the most important signal transducers in cells, involved in numerous physiological and pathological processes. It is known that a variety of metals complexes are able to modify intracellular Ca 2+ signalling and hence induce apoptosis or necrosis [48,49]. Using

6 aesar ( 5 - 2 . 1 . 1 .

 65211 bromo-2-iodopyridine, semicarbazide hydrochloride, 3-(trimethylsilyl)propagyl bromide) concentration of n-butyllithium solution was determined by titration of diphenylacetic acid in dry THF[START_REF] Pirrung | The Synthetic Organic Chemist's Companion[END_REF]. Synthesis of 4-(3-(trimethylsilyl)prop-2-yn-1-yl)-4'-methyl-2,2'-bipyridyl (2). To a solution of freshly distilled diisopropylamine (0.19 mL, 1.3 mmol) in anhydrous THF(5 mL)

2. 1 . 6 .

 16 General procedure for synthesis of cis-Ru(NN)2Cl2. Ruthenium(III) chloride trihydrate (0.262 g, 1 mmol), the ligand (2,2'-bipyridine or 4,7-diphenyl-1,10-phenantroline) (2 mmol) and 2 drops of N-ethylmorpholine were dissolved in degassed N,N-dimethylformamide (10 mL) under argon conditions. The mixture was put into a microwave reactor for 30 min (250W, 160ºC). The solvent was then removed under reduced pressure to c.a. 1 mL and acetone(20 mL)

A 2 . 6 .

 26 C C E P T E D M A N U S C R I P T 13 The evaluation of the cytotoxicity of the Ru complexes on A549 and CT26 cells, was conducted using an Alamar Blue assay. The Alamar Blue test is based on the reduction of blue and nonfluorescent subtrate (resazurin) to a pink and highly fluorescent product (resorufin) by the alive cells. Cells were seeded on a 96 wells plate with a density of 1.5 × 10 4 cells per cm 2 one day before the experiments. Then, cells were incubated with various concentrations of the Ru compounds and synthesized ligand L1 for 24/72 hours in the dark. All compounds were diluted in DMSO and then, added to the appropriate medium with or without 2% FBS to obtain the applied concentrations. The final DMSO concentration was kept constant at 0.1% (v/v) in case of Ru2 and 0.5% (v/v) in case of Ru1 and bpySC. After the incubation, cells were washed with PBS and incubated in the resazurin sodium salt solution (25 µM) for 3 h. The cell viability was quantified at 605 nm using 560 nm excitation light (Tecan Infinite 200 microplate reader). Experiments were performed in triplicate and repeated at least three times to get the mean values ± standard deviation. The viability was calculated with respect to the untreated cells control. The IC50 values were determined using the Hill equation (Origin 9.0) [40] y = y 0 + (𝑦 100 -𝑦 0 )[𝑐] 𝐻 [𝐼𝐶 50 ] 𝐻 + [𝑐] 𝐻 Cellular imaging of accumulated Ru complexes For co-localization experiments, A549 cells were seeded on a 6 well plate at a density of 3 × 10 4 cells per cm 2 24 h prior to the staining. ER-Trackert Blue-White DPX, Mitotracker Green and Lyso-Tracker Blue (Life Technologies) were used to image ER, mitochondria and lysosomes according to the manufacturer's protocols. The Ru complexes were incubated in basic medium for 24 h ([Ru1] = 500 µM, [Ru2] = 2 µM), then rinsed twice with PBS. The chosen concentration of the Ru complexes was not cytotoxic to the cells under these conditions. Images were acquired A C C E P T E D M A N U S C R I P T 14 using a Olympus fluorescence microscope IX51 equipped with an XC10 camera with 470-495 and 530-550 nm emission filters.

  Activation of caspases 3/7, 8 and 9 was examined using luminescent Caspase Glo assays (Promega) according to the manufacturer's manual. A549 and CT26 cells were seeded on a white 96 wells plate with a density of 1.6 × 10 4 cells per cm2 24 h prior to the experiments. Next, the compounds at different concentrations were added to the wells and incubated for 24 h ([Ru1] = 200 µM, [Ru2] = 2 µM, [bpySC] = 100 µM). The luminescence intensity of the cells was measured using a Tecan Infinite 200 plate reader.

for 24 h

 24 in the dark. Next, cells were washed with PBS and stained with Fluo-8 AM (4 µM) for 30 min in the dark at 37 °C. After staining cells were washed twice with PBS and analyzed by a Tecan Infinite 200 microplate reader measuring fluorescence of the probe at 525 nm using 490 excitation wavelength. Experiment was performed three times in triplicate and the mean values ± standard deviations were calculated.

Scheme 2 .

 2 Scheme 2. Synthesis of ligand bpySC Reagents and conditions: (a) (1) LDA, -78ºC, THF, 0.5h

Scheme 3 .

 3 Scheme 3. Synthesis of Ru1 and Ru2.

Ru2Fig. 1 .

 1 Fig. 1. Plots of orbitals responsible for the most intensive absorption bands in the visible range of

  bpySC ligand in Ru1, whereas in Ru2 compound these two auxiliary dip ligands played a key role.

Fig. 2 . 27 Fig. 3 .

 2273 Fig. 2. Fluorescence images of A549 cells showing subcellular localization of Ru2 (2 µM,

31 Fig. 5 .Fig. 6 . 33 Fig. 7 .

 3156337 Fig. 5. Apoptosis of A549 cells upon 24 h exposure to the Ru polypyridyl complexes. Cells were

Fig. 8 .

 8 Fig. 8. Effect of the Ru polypyridyl complexes on mitochondrial membrane potential (ΔΨm) in

Fluo- 8

 8 AM probe the changes in intracellular calcium concentration ([Ca 2+ ]C) in cancer cells after 24 h incubation with the studied compounds were monitored. The results are presented in Fig. 9. Alteration in calcium homeostasis upon treatment with the Ru complexes was cell line dependent. In case of A549 cells, no significant changes in [Ca 2+ ]C after treatment with Ru1 or bpy-SC were observed. Only Ru2 accumulation caused a decrease in cytosolic calcium level, and this effect had been already observed previously with other polypyridyl Ru complexes containing dip ligands [50]. Accumulation of Ru1 in CT26 cell line resulted in concentration dependent decrease in intracellular calcium level, while Ru2 and bpySC caused the opposite effect -increasing the calcium ions concentration. Such an increase in [Ca 2+ ]C can cause a rise in mitochondrial calcium levels since those organelles serves as buffering units and might modulate Ca 2+ feedbackinhibition or activation mechanisms. Increase in the mitochondrial calcium concentration can cause the mitochondrial membrane depolarization and lead to higher amounts of reactive oxygen species production. This can force the opening of the mitochondrial permeability pore causing mitochondrial swelling and releasing cytochrome c and other pro-apoptotic factors, initiating programmed cell death process.

FBS

  fetal bovine serum; FITC -fluorescein isothiocyanate; HOMO -highest occupied molecular orbital; HRMS -high resolution mass spectrometry; IC50concentration causing 50 % decrease in viability; iPrMgCl -Isopropylmagnesium chloride; JC-1 -5,5,6,6-Tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide; LDA -lithium diisopropylamide; LUMO -lowest unoccupied molecular orbital; MeOH -methanol; MLCT -metal ligand charge transfer; NMR -nuclear magnetic resonance; QY -quantum yield; PBS -phosphate buffered saline; PI -propidium iodide; ROS -reactive oxygen species; RR -ribonucleotide reductase; S--in serum free conditions; S+ -in the presence of 2%; THF -tetrahydrofuran; TD-DFT -Time-Dependent DFT.

2.1.3. Synthesis of 5-bromo-2-formylpyridine (5) [21].

	2.1
	1 H
	NMR (400 MHz, CDCl3): d 1.99 (t, J = 2.6 Hz, 1H), 2.44 (s, 3H), 2.54 (td, J =7.4 and 2.6 Hz,
	2H), 2.94 (t, J = 7.4 Hz, 2H), 7.13 (d, J=4.8 Hz, 1H), 7.20 (dd, J =5.0 and 1.8 Hz, 1H), 8.23 (d, , J =0.8 Hz, 1H), 8.26 (d, J =0.8 Hz, 1H), 8.52 (d, J=5Hz, 1H), 8.58 (d, J=5Hz, 1H) ppm (Fig. S6). 13 C NMR (100 MHz, CDCl3): d 20.41, 21.12, 34.50, 68.87, 83.79, 120.71, 121.63, 124.04, 125.02, 147.99, 148.33, 148.90, 151.03, 155.89, 156.32 ppm. [HRMS -ESI] Calculated for C15H14N2 m/z : 223.1230 (M+H + ), Found: m/z = 223.1239 (M+H + ). 5-bromo-2-formylpyridine was synthesized modifying the procedure described by Song et al. [21]. To a solution of 5-bromo-2-M A N U S C R I P T
	iodopyridine (4) (0.586 g, 2 mmol) in anhydrous THF (10 mL) under argon conditions at -10°C, a solution of isopropylmagnesium chloride (1.5 mL, 2.17 mmol) was added dropwise. After 2 h, dry DMF (0.4 mL, 5.16 mmol) was added rapidly. The mixture was kept at -10°C for another hour and then warmed up to room temperature over 2 h. The reaction was quenched with a saturated aqueous solution of NH4Cl (20 mL) and extracted with diethyl ether (3x25 mL). The combined organic layers were washed twice with brine and dried over magnesium sulphate. The A C C E P T E D
	solvent was removed under reduced pressure to give pure product as light yellow solid (0.306 g,
	80%). 1 H NMR (200 MHz, CDCl3):  7.84 (dd, J = 8.2 and 0.8 Hz, 1H), 8.02 (ddd, J = 8.2, 2.2
	and 0.8 Hz, 1H), 8.85 (d, J=2 Hz, 1H), 10.04 (d, J=0.8 Hz, 1H) ppm.
	8

.4. Synthesis of 2-[(5-bromopyridin-2-yl)methylidene]hydrazinecarboxamide (6). To

	a
	suspension of semicarbazide hydrochloride (0.149 g, 1.3 mmol) in anhydrous ethanol (3 mL), a
	solution of 5 (0.230 g, 1.2 mmol) in ethanol (9 mL) was added. The mixture was refluxed over 3
	h and cooled down to 0°C. The solid was filtrated, washed twice with cold, anhydrous ethanol,
	once with diethyl ether and dried in vacuum to give the pure product as light cream powder
	(0.300 g, 99%). 1 H NMR (400MHz, DMSO-d6)  7.02 (brs, 2H), 7.86 (s, 1H), 8.10 (dd, J = 8.0
	and 1.6 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 8.67 (d, J = 1.6 Hz, 1H), 10.63 (s, 1H) ppm (Fig. S7).
	13 CNMR (100MHz, DMSO-d6): 119.70, 121.57, 137.78, 139.73, 149.26, 152.25, 156.29 ppm
	(Fig. S8). [HRMS-ESI]: Calculated for C7H7N4OBr: m/z 264.9701 (M+Na + ) Found 264.9692
	(M+Na + ).

  CDCl3) d 20.67, 21.22, 34.17, 78.86, 93.77, 119.41, 120.83, 121.33, 122.04, 123.89, 124.83, 138.90, 141.49, 148.23, 148.95, 149.18, 150.04, 151.14, 152.11, 155.79, 156.38, 156.79 ppm (Fig. S10). [HRMS-ESI]: Calculated for C22H20N6O m/z 385.1771 (M+H + ) Found 385.1771

	was kept at 4°C (fridge) and crystals were filtrated, washed with cyclohexane and dried under
	vacuum to give the pure product as a white solid (0.132 g, 45%). 1 H NMR (600 MHz, CDCl3): d
	2.45 (s, 3H), 2.84 (t, J =7 Hz, 2H), 3.03 (t, J = 7 Hz, 2H), 7.14 (dd, J =16.2 and 4.8 Hz, 2H),
	7.64-7.77 (m, 3H), 8.25 (s, 1H), 8.38 (s, 2H), 8.53-8.63 (m, 3H) ppm (Fig. S9). 13 C NMR (150
	MHz,
	R I P T
	9

m/z (M+H + ).

  Hz, 1H), 8.13-8.20 (m, 6H), 8.23 (d, J=5.4 Hz, 1H), 8.26 (d, J=5.4 Hz, 1H), 8.33 (d, J=5.4 and 3.6 Hz, 1H), 8.80 (d, J=12.6 Hz, 1H), 8.90 (d, J=12.6 Hz, 1H

1 H NMR (600 MHz, CD3CN)  2.55 (s, 3H), 2.95 (t, J =6 Hz, 2H), 3.13 (t, J=6 Hz, 2H), 7.04 (s, 1H), 7.22 (d, J=6 Hz, 1H), 7.37 (dd, J=15.6 Hz, 6 Hz, 2H), 7.57-7.63 (m, 20H), 7.67 (d, J=5.4 Hz, 2H), 7.73 (dd, J=5.4 and 2.4 Hz, A C C E P T E D M A N U S C R I P T 11 2H), 7.79 (t, J=5.4

20 Table 1 .

 201 Photophysical properties of the ruthenium(II) complexes in air-equilibrated and deoxygenated aqueous solutions.

					Emission	Emission
		Absorption		(air-equilibrated	(deoxygenated
					conditions)	conditions)
		λmax	ε	λmax
					Φ	τ [µs]	Φ	τ [µs]
		[nm]	[M -1 cm -1 ]	[nm]
					0.37 ±	0.57 ±
	[Ru(bpy)3] 2+			628	0.0280 a	0.0420 b
					0.01	0.01
		452	14 000
		279	88 100
					0.0367 ±	0.76 ±	0.1245 ±	2.51 ±
	[Ru(dip)2(bpy)] 2+ c	438	19 000	613
					0.0004	0.01	0.0004	0.01
		461	18 500
		286	108 600
					0.0092 ±	0.27 ±	0.0162 ±	0.36 ±
	Ru1	427	15 400	617
					0.0009	0.03	0.0002	0.06
		455	18 100
		279	204 800
					0.0118 ±	0.65 ±	0.0262 ±	1.72 ±
	Ru2	435	42 100	623
					0.0003	0.03	0.0004	0.07
		457	43 000

Table 2 .

 2 IC50 of Ru complexes against two cancer cell lines A549 and CT26. Experiments were

	performed in medium without (S-) or with (S+, 2%) serum.
		IC50 (µM) 24h		IC50 (µM) 72 h
		S-	S+	S-	S+
	bpySC	102 ± 59	-	2.8 ± 0.8 3.3 ± 1.2
	Ru1	113 ± 154	202 ± 17	97 ± 14	138 ± 20
	Ru2	6.8 ± 1.2	9.8 ± 2.3 9.0 ± 1.9	16 ± 2
	A549
	[Ru(dip)2(bpy)] 2+	4.0 ± 0.7	-	-	-
	[Ru(bpy)3] 2+	>320	>320	>320	>320
	[Ru(dip)3] 2+	1.2 ± 0.1	-	-	-
	bpySC	96 ± 28	-	1.5 ± 0.3 2.0 ± 0.3
	Ru1	201 ± 25	233 ± 38	31 ± 11	50 ± 7.6
	Ru2	4.6 ± 0.9	5.0 ± 0.1 5.7 ± 1.5	21 ± 5
	CT26
	[Ru(dip)2(bpy)] 2+	3.4 ± 0.7	-	-	-

Table 3 .

 3 Cell cycle distribution of A549 cells after 24 h treatment with the Ru polypyridyl complexes and with ligand bpySC (representative analysis).

		G0/G1	S-phase G2/M
	Control 57.5%	36.0%	6.5%
	bpySC 22.2%	74.0%	3.8%
	Ru1	47.5%	48.4%	4.1%
	Ru2	76.9%	22.5%	0.6%

Acknowledgements

The CNRS and French Ministry of Research are thanked for their support. M. Ł. acknowledges the French Embassy in Poland for providing a cotutelle Ph.D. grant. O. M. acknowledges the financial support from the National Science Center (DEC-2013/11/N/ST5/01606). M. B. acknowledges the financial support from the National Science Center (DEC-2016/21/B/NZ7/01081). This work was carried out using the equipment purchased through financial support from the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (contract no. POIG.02.01.00-12-023/08). This research was supported in part by PL-Grid Infrastructure.

ROS positive cells [%]

24 h 72 h

bpy-SC Ru2 Ru1

Fig. 4. The level of oxidative stress induced in A549 cells after 24 and 72 h treatment with the Ru polypyridyl complexes expressed as percentage of ROS positive cells in the whole cell population (%). Pyocyanin was used as ROS positive control.

Apoptosis inducing properties

The determination of the cellular death mechanism was performed using flow cytometry. A549 cells were double stained with Annexin-V-FITC conjugate and DAPI after incubation with Ru1, Ru2 and bpySC for 24h. Incubation with all studied compounds significantly increased the population of Annexin V-positive cells, indicating cells undergoing early phase of apoptosis (Fig. 5), with no indication of the DAPI-positive cells that would suggest a necrotic cellular death.

Despite lesser accumulation of Ru1, that was previously confirmed by the cytotoxicity and localization study, this compound was found to be powerful apoptosis inducing agent.

Conclusions

A new ligand based on semicarbazone 2-formylopyridine moiety conjugated with 2,2'-bipyridyl (bpySC) by an alkyne spacer has been developed. The two compounds produced as a result of coordination by ruthenium(II) center by bpySC and either two bpy (Ru1) or two dip (Ru2) ligands have been obtained and characterized. They exhibited entirely different biological effects on cancer cell lines. The cytotoxic effect determined after 24 h of treatment with Ru2 was more than an order of magnitude higher than that found for Ru1 as well as bpySC ligand alone against both A549 and CT26 cancer cell lines. The extent of treatment up to 72 h resulted in the increased anti-proliferative activity of both Ru1 and bpySC but not of Ru2. The Ru1 complex accumulated preferentially in lysozyme, while Ru2 in mitochondria and endoplasmic reticulum.

Both Ru complexes and bpySC induced apoptosis. In the case of Ru2 it occurred via caspaseindependent pathway, while Ru1 and bpySC induced apoptosis in cancer cells through

ACCEPTED MANUSCRIPT

A C C E P T E D M

A N U S C R I P T 37 mitochondria signal transduction pathway, even though this organelle was not the primary localization site. Both studied ruthenium complexes had an opposite effect on intracellular calcium concentration. The increased in ROS production was evident upon treatment with Ru2 while negligible for Ru1 even after prolonged incubation time.

Taken together we have demonstrated that changing of bpy into dip ligands has a strong impact on the mechanism of biological activity of the ruthenium(II) complexes. Despite increasing the Ru complexes overall accumulation and cytotoxicity by choosing the more lipophilic dip ligands the role of the semicarbazone 2-formylopyridine moiety in biological activity of the ruthenium complex seems to be marginal. Therefore, a special attention must be paid in choosing appropriate auxiliary ligands in designing new ruthenium complexes to take advantage from conjugation of biologically active molecules to polypyridyl ligands. Appropriately chosen auxiliary ligands can regulate not only cytotoxicity and accumulation of polypyridyl ruthenium complexes, but also determine the mechanism of cellular death and highlight the effects of biologically active molecules.

Abbreviations

Highlights

 New Ru complexes with semicarbazone 2-formylopyridine link to bipyridine were synthesised.  As auxiliary ligands bipyridine and diphenylphenanthroline were chosen.  The studied compounds were powerful apoptosis inducing agents.  The anti-proliferative activity was strongly dependent on auxiliary ligands.  The auxiliary ligands determined effect of the complexes on cell cycle arrest and ROS formation.