Study of Cytotoxic and Photodynamic Activities of Dyads Composed of a Zinc Phthalocyanine Appended to an Organotin

The combination of photodynamic therapy and chemotherapy is a promising strategy to enhance cancer therapeutic efficacy and reduce drug resistance. In this study two zinc(II) phthalocyanine-tin(IV) conjugates linked by a triethylene glycol chain were synthesized and characterized. In these complexes, the zinc(II) phthalocyanine was used as a potential photosensitizer for PDT and the tin complex was selected as cytostatic moiety. The two dyads composed of zinc(II) phthalocyanine and tin complexes exhibited high cytotoxicity, in absence of light stimulation, against MCF-7 human breast cancer cells with low LC50 values in the range of 0.016–0.453 µM. In addition, these complexes showed superior cytotoxicity than their mixture of equimolar component, accompanied with a higher activity towards cancer cells compared to human healthy fibroblasts. However, under irradiation of the zinc phthalocyanine unit (at 650 nm) no photodynamic activity could be detected, due to the most likely quenching of zinc(II) phthalocyanine singlet excited state by the nearby tin complex according to a photoinduced electron transfer process. This study demonstrates the potential of heterometallic anticancer chemotherapeutics composed of a zinc phthalocyanine and tin complex, and it highlights that the development of such conjugates requires that the sensitizer preserves its photophysical properties and in particular its singlet oxygen sensitization ability in the conjugate in order to combine the PDT activity with the cytotoxicity of the anticancer drug.


Introduction
Recently, there has been considerable interest in combining photodynamic therapy (PDT) with a chemotherapeutic anticancer agent [1][2][3][4][5][6]. The combination of different therapeutic approaches that acts on distinct disease pathways has shown several advantages, such as enhanced therapeutic efficacy, reduced side effects, and drug resistance problems. There are generally three approaches to combine PDT and chemotherapy, including consecutive administration of a photosensitizer and an anticancer drug, the use of their covalent and noncovalent conjugates, and co-encapsulation of these agents in a polymeric nanocarrier [7,8]. The combination of a photosensitizer with an appended anticancer drug, cisplatin [9,10], carboplatin [11,12], doxorubicin (DOX) [13][14][15], and organometallic ruthenium(II) complexes [16,17] has been attempted, in order to evaluate the possibility of synergistic anticancer properties between the two entities. Interesting results were obtained showing an enhanced anticancer efficacy at lower drug doses [18] due to the dark chemostatic effect of the drug amplified by the PDT effect generated upon light excitation of the photosensitizer.
Tin organometallic derivatives are important classes of organometallic drugs, as they exhibit a wide spectrum of biological effects and have been widely studied as bactericides, fungicides, acaricides and wood preservatives. In addition, some compounds possess antitumor activity. The cytotoxic activity of these compounds results from the inhibition of macromolecular synthesis, the alteration of energy-producing metabolism in the mitochondria, and the reduction of DNA synthesis. In addition, the cytotoxicity induced also arises from their interaction with the cell membrane which increases the concentration of cytosolic Ca 2+ and induces apoptosis. Phthalocyanines are promising second-generation photosensitizers for PDT, owing to their suitable photo-physical and photo-chemical properties, such as the strong absorption in the tissue-penetrating red visible region and high efficiency in generating singlet oxygen [19][20][21][22][23].
In this work, we have investigated the combination of tin complexes with a zinc phthalocyanine photosensitizer in order to take advantage of the intrinsic cytotoxicity of tin complex in the dark added to the potentially high phototoxicity of zinc phthalocyanine upon light excitation. The two entities were linked with a triethylene glycol chain, which can enhance the biocompatibility of the system (Figure 1). Although the length of this spacer might have an impact on the biological activity, this parameter was not explored in this study. Two dyads are composed of tetrasubstituted zinc phthalocyanine connected to a tin phenyl complex liganded via a malonate anchor. One tin organometallic compound bears two phenyl groups in its coordination sphere (SnPh 2 ), while the other is bound to three phenyls (SnPh 3 ). Phenyl was selected as organometallic moiety around tin(IV), because it was shown in previous studies that this type of complex gives the most potent anticancer activity [24]. The cytotoxicity of the three dyads was assessed on human healthy (fibroblasts) and cancer cell lines (MCF-7) to quantify their therapeutic potential. It was shown that the two dyads ZnPc-SnPh 2 and ZnPc-Sn 2 Ph 6 exhibit superior cytotoxicity than their equimolar mixtures of individual components and they display higher selectivity towards MCF-7 carcinoma cell line than healthy cells. On the other hand, these two dyads showed no photodynamic activity under light irradiation.

Synthesis of the Compounds
Scheme 1 illustrates the synthetic route used to prepare the two dyads ZnPc-SnPh2 and ZnPc-Sn2Ph6. An excess of phthalonitrile 1 underwent mixed cyclisation with phthalonitrile 2 7 in the presence of Zn(OAc)2.2H2O and 1,8-diazabicyclo

Synthesis of the Compounds
Scheme 1 illustrates the synthetic route used to prepare the two dyads ZnPc-SnPh 2 and ZnPc-Sn 2 Ph 6 . An excess of phthalonitrile 1 underwent mixed cyclisation with phthalonitrile 2 7 in the presence of Zn(OAc) 2 .2H 2 O and 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU) in n-pentanol to give the «3 + 1» unsymmetrical phthalocyanine 3 in 18% yield (Scheme 1). The ethyl esters of the malonate were subsequently hydrolyzed with sodium hydroxide in acetone and then acidified to afford the phthalocyanine 4 with 85% yield. The introduction of tin complex was accomplished by activating the carboxylic acid group by deprotonation with triethylamine (TEA) before being reacted with the tin precursor containing two or one chloro ligands [24]. The complexes were characterized by proton NMR and elemental analysis as well as by high resolution mass spectrometry. The dyads ZnPc-SnPh2, ZnPc-Sn2Ph6 are soluble in CHCl3, DMSO, THF and DMF.

UV-Vis Electronic Absorption Properties
The absorption spectra of the dyads are compared with those of their components, which are displayed in Figure 2 and the spectroscopic data are gathered in Table 1. In chloroform, ZnPc(EG3)4 and the dyads ZnPc-SnPh2 and ZnPc-Sn2Ph6 give typical UV-Vis spectra of non-aggregated phthalocyanines showing intense and sharp Q-bands in the The complexes were characterized by proton NMR and elemental analysis as well as by high resolution mass spectrometry. The dyads ZnPc-SnPh 2 , ZnPc-Sn 2 Ph 6 are soluble in CHCl 3 , DMSO, THF and DMF.

UV-Vis Electronic Absorption Properties
The absorption spectra of the dyads are compared with those of their components, which are displayed in Figure 2 and the spectroscopic data are gathered in Table 1. In chloroform, ZnPc(EG 3 ) 4 and the dyads ZnPc-SnPh 2 and ZnPc-Sn 2 Ph 6 give typical UV-Vis spectra of non-aggregated phthalocyanines showing intense and sharp Q-bands in the red visible region around 680 nm along with the less intense vibronic overtone at 616 nm ( Figure 2A). They also displayed a Soret band at 360 nm. The presence of the appended tin complex does not alter the spectrum of the dyads because: (i) it does not absorb beyond 300 nm (Supplementary Figure S1) and (ii) there is no electronic communication between the malonate group and the zinc phthalocyanine on the ground state.  The spectra were also recorded in water with 1% of DMSO to approach the conditions used in biological tests ( Figure 2B). The shape and the relative intensity of the Q-bands are clearly different in aqueous environment with a broadening and an inversion of the intensity of the Q(0,0) and Q(0,1) bands, which are located respectively around 620 nm and 690 nm. This change witnesses the formation of H-aggregates. Although the solubility of the compounds is maintained in aqueous medium owing to the ethylene glycol chains, the hydrophobicity of phthalocyanine core induces the formation of π-stacked H-aggregates in aqueous environment.

Infra-Red (IR) Spectroscopic Study
Carboxylate ligand can chelate tin(IV) cation according to several potential binding modes ( Figure 3) [25][26][27]. In solid state, the tin(IV) complexes are usually polymeric compounds with cis-trigonal bipyramidal geometry having five-coordinate tin sites under the bridging mode (type A).  The spectra were also recorded in water with 1% of DMSO to approach the conditions used in biological tests ( Figure 2B). The shape and the relative intensity of the Q-bands are clearly different in aqueous environment with a broadening and an inversion of the intensity of the Q(0,0) and Q(0,1) bands, which are located respectively around 620 nm and 690 nm. This change witnesses the formation of H-aggregates. Although the solubility of the compounds is maintained in aqueous medium owing to the ethylene glycol chains, the hydrophobicity of phthalocyanine core induces the formation of π-stacked H-aggregates in aqueous environment.

Infra-Red (IR) Spectroscopic Study
Carboxylate ligand can chelate tin(IV) cation according to several potential binding modes ( Figure 3) [25][26][27]. In solid state, the tin(IV) complexes are usually polymeric compounds with cis-trigonal bipyramidal geometry having five-coordinate tin sites under the bridging mode (type A). π-stacked H-aggregates in aqueous environment.

Infra-Red (IR) Spectroscopic Study
Carboxylate ligand can chelate tin(IV) cation according to several potential binding modes ( Figure 3) [25][26][27]. In solid state, the tin(IV) complexes are usually polymeric compounds with cis-trigonal bipyramidal geometry having five-coordinate tin sites under the bridging mode (type A).  Particularly in solution, the complexes can exist as monomers either under a fivecoordinate chelating bridged mode (type B) or as four-coordinated tin with carboxylate acting as a monodentate ligand (type C). ATR-IR spectra can be used as tools to diagnose the mode of coordination of tin complexes [28]. More specifically, the mode of binding can be deduced by the wavenumber difference (ν = ν as − ν s ) between the carboxylate antisymmetric (ν as ) and symmetric (ν s ) stretches. If this difference is larger than 250 cm −1 , tin(IV) is involved in a tetrahedral geometry (type C binding mode), while values between 250 and 150 cm −1 indicate a bridging polymeric structure (type A). When ν is lower than 150 cm −1 , then carboxylate is bidentate and binds to Sn(IV) under the chelating bridged mode (type B). The strong asymmetric stretching band of the carbonyl group in the acid form of compound 4, situated at 1725 cm −1 has shrunk and is shifted to lower energy (≈1550 cm −1 ) after the reaction with chloro-phenyl tin derivatives, which is a clear indication that complexation of tin by the carboxylate has occurred. The wavenumber values of the stretching bands of the carboxylate before and after complexation of tin(IV) are gathered in Table 2. For the dyads ZnPc-SnPh 2 and ZnPc-Sn 2 Ph 6 , the ν values are less than 150 cm −1 , which indicates that the carboxylate ligand is bidentate and binds to Sn(IV) under the chelating bridged mode.

Fluorescence Spectroscopy
Fluorescence spectra of the dyads ZnPc-SnPh 2 , ZnPc-Sn 2 Ph 6 were compared to those of the reference zinc phthalocyanine ZnPc(EG 3 ) 4 in order to study the possibility of a singlet excited state quenching by the tin complex. As for the electronic absorption spectra, the fluorescence emission measurements were conducted both in CHCl 3 and water with 1% of DMSO ( Figure 4). Whatever the solvent conditions, the fluorescence intensity of the dyads ZnPc-SnPh 2 , ZnPc-Sn 2 Ph 6 decreased compare to the reference phthalocyanine ZnPc(EG 3 ) 4 .
Fluorescence spectra of the dyads ZnPc-SnPh2, ZnPc-Sn2Ph6 were compared to those of the reference zinc phthalocyanine ZnPc(EG3)4 in order to study the possibility of a singlet excited state quenching by the tin complex. As for the electronic absorption spectra, the fluorescence emission measurements were conducted both in CHCl3 and water with 1% of DMSO ( Figure 4). Whatever the solvent conditions, the fluorescence intensity of the dyads ZnPc-SnPh2, ZnPc-Sn2Ph6 decreased compare to the reference phthalocyanine ZnPc(EG3)4. Based on the relative fluorescence intensities, the quenching of ZnPc-SnPh 2 and ZnPc-Sn 2 Ph 6 is estimated at 60% and 36% in CHCl 3 and 70% and 75% in water + 1% DMSO respectively. In aqueous medium, the fluorescence intensity is lower than in CHCl 3 , most probably because the H-aggregates impart a supplementary quenching process. In addition to the formation of H-aggregates, another fluorescence quenching process in the dyads could be attributed to photoinduced electron transfer to the tin complex (formation of «ZnP + -Sn III »). Indeed, energy transfer is unlikely from thermodynamic point of view because the organo-tin complex absorbs in the deep UV, therefore its lowest energy excited state is necessarily higher than that of the singlet excited state of the zinc phthalocyanine (Supplementary Figure S1).
Overall, the fluorescence of the zinc phthalocyanine in these dyads is significantly quenched relative to that of the reference ZnPc(EG 3 ) 4 . The fluorescence inhibition is due to the presence of tin complex and the formation of H-aggregates, particularly in aqueous environment for the latter. The singlet excited state quenching could affect the efficiency of PDT, due to the reduction of the quantum yield of triplet excited state formation by the intervention of a competitive deactivation processes, which depopulate the singlet excited state (see below).

Singlet Oxygen Measurements
In order to directly estimate the impact of the tin complex on the production of singlet oxygen, the singlet oxygen quantum yield of the compounds was determined in CHCl 3 and deuterated water + 1% of DMSO by recording the fluorescence intensity of singlet oxygen emission at 1270 nm. D 2 O was chosen instead of H 2 O since the singlet oxygen lifetime value in D 2 O is higher than in H 2 O. This is known that solvents with high vibrational frequencies are more able to quench 1 O 2 [29]. The data are collected in Table 3. In CHCl 3 , the singlet oxygen quantum yield measurements reflect, within experimental errors, the degree of fluorescence quenching. Moreover, the singlet oxygen quantum yield of ZnPc(EG 3 ) 4 is in agreement with previously reported in DMSO [30]. In D 2 O, Pharmaceuticals 2021, 14, 413 7 of 15 however, the singlet oxygen emission is importantly reduced and could not be detected because the quantum yields is too low for the sensitivity of this technique. However, we cannot exclude that the singlet oxygen quantum yield of ZnPc(EG 3 ) 4 is not null in biological medium as the latter is quite active in PDT (see below).

Biological Effect Analysis of ZnPc(EG 3 ) 4 :Cytotoxicity, PDT and ROS Production
The cytotoxic effect of ZnPc(EG 3 ) 4 on human breast cancer cells (MCF-7) was determined and compared with that on healthy fibroblasts. For this, a dose-response experiment was performed by incubating cells in the darkness with increasing doses of ZnPc(EG 3 ) 4 for 72 h. Then, the viability of cells was quantified and reported in Figure 5a. ZnPc(EG 3 ) 4 exhibited a LC 50 of 16.55 µM on MCF-7 cells and of 63.80 µM on fibroblasts. This difference (4 fold) between these two cell lines demonstrated that this compound was more cytotoxic on cancer cells than healthy cells.
The photodynamic therapeutic potential was also investigated on breast cancer and healthy cells. Several concentrations of compound (from 0.01 to 0.50 µM) were tested. Cells were incubated 24 h with ZnPc(EG 3 ) 4 and submitted or not to laser excitation (650 nm, 20 min, 39 J.cm −2 ). Two days later, the viability was quantified MCF-7 cells demonstrated a significant photosensitivity from 0.05 µM of ZnPc(EG 3 ) 4 ( Figure 5b). Indeed, cells submitted to laser excitation exhibited 31% cell death. This PDT effect increased in accordance with increasing concentration of ZnPc(EG 3 ) 4 to reach 92% cell death when cells were incubated with 0.50 µM of ZnPc(EG 3 ) 4 and submitted to photoexcitation. This effect is very strong and indicates that the reference compound ZnPc(EG 3 ) 4 is quite active in PDT, most reasonably upon production of singlet oxygen. We also investigated this PDT efficiency on healthy cells in order to verify if the discriminatory effect already observed on dark cytotoxicity (Figure 5c) could be reproduced under laser excitation. Unfortunately, this effect is about the same on cancer or healthy cells, with a significant effect from 0.05 µM and a high PDT efficiency at 0.50 µM. However, we could note a difference between the two cell lines at this concentration; indeed, only 8% of cancer cells survived when 28% of fibroblasts survived after irradiation.
To identify the phototoxicity mechanism involved here, ROS quantification was performed and reported in Figure 5d. Cells were incubated with DCFH 2 -DA (2 ,7dichlorodihydrofluorescein diacetate), which is a non-fluorescent molecule. In the presence of ROS this molecule is oxidized into the fluorescent 2 ,7 -dichlorodihydrofluorescein (DCF), whose green luminescence can be detected using a fluorescence microscope. The results showed that in the presence of 0.50 µM of ZnPc(EG 3 ) 4 , light excitation at a wavelength of 650 nm induced green fluorescence inside the cells, mainly on cancer cells but also on healthy cells, thus demonstrating ROS production and confirming cell death induced by PDT effect.
Altogether these data demonstrated that this compound is very efficient for dark cytotoxicity and overall for PDT action, but it is not really discriminant between cancer and healthy cells. Although often, the main limitation of cancer therapy is the poor selectivity of cancer cells inducing a large panel of secondary effects on healthy organs and cells. So we decided to graft organotin complex on ZnPc(EG 3 ) 4 to improve the effect and the selectivity on cancer cells.

Cytotoxic Potential Analysis of the Dyads ZnPc-SnPh 2 and ZnPc-Sn 2 Ph 6 on Cancer and Healthy Cells
In order to determine the therapeutic potential of these new compounds, the cytotoxic activity on MCF-7 cells and also on healthy fibroblasts was studied. Towards this goal, the cells were incubated 72 h in darkness, with increasing concentrations of each compound (from 0.01 to 100 µM). Drug concentrations leading to 50% cell mortality (LC 50 ) were determined using the classical sigmoidal dose-response curves of cytotoxicity obtained when plotted as a logarithmic function of the concentration (µM).
The analysis of the curves in Figure 6, shows that the compound ZnPc-Sn 2 Ph 6 is about 30 times more toxic on MCF-7 cells (LC 50 = 0.016 µM) than the compound ZnPc-SnPh 2 (LC 50 = 0.453 µM). It is also interesting to note that both tin dyads present a higher toxicity on cancer cells compared to healthy cells ( Figure 6). Indeed, the LC 50 are lower on cancer cells than on fibroblasts with LC 50 of 0.016 vs. 0.032 µM for the compound ZnPc-Sn 2 Ph 6 , and LC 50 of 0.453 and 0.513 for the compound ZnPc-SnPh 2 . These results suggest that the ZnPc-SnPh 2 dyad may be a potentially interesting basis to develop targeting cancer cells.

Cytotoxic Potential Analysis of the Dyads ZnPc-SnPh2 and ZnPc-Sn2Ph6 on Cancer and Healthy Cells
In order to determine the therapeutic potential of these new compounds, the cytotoxic activity on MCF-7 cells and also on healthy fibroblasts was studied. Towards this goal, the cells were incubated 72 h in darkness, with increasing concentrations of each compound (from 0.01 to 100 µM). Drug concentrations leading to 50% cell mortality (LC50) were determined using the classical sigmoidal dose-response curves of cytotoxicity obtained when plotted as a logarithmic function of the concentration (µM).
The analysis of the curves in Figure 6, shows that the compound ZnPc-Sn2Ph6 is about 30 times more toxic on MCF-7 cells (LC50 = 0.016 µM) than the compound ZnPc-SnPh2 (LC50 = 0.453 µM). It is also interesting to note that both tin dyads present a higher toxicity on cancer cells compared to healthy cells ( Figure 6). Indeed, the LC50 are lower on cancer cells than on fibroblasts with LC50 of 0.016 vs. 0.032 µM for the compound ZnPc-Sn2Ph6, and LC50 of 0.453 and 0.513 for the compound ZnPc-SnPh2. These results suggest that the ZnPc-SnPh2 dyad may be a potentially interesting basis to develop targeting cancer cells. In order to confirm the therapeutic interest of using a dyad rather than its constituents, we compared the cytotoxicity of tin dyads to the cytotoxic effect of equimolar mixtures of the phthalocyanine ZnPc(EG3)4 and SnPh2 or Sn2Ph6 (Figure 7). For all conditions tested here, and as reported in the Figure 7e, the cytotoxicity induced by dyads is always higher than the equimolar mixture of their constituents. In order to confirm the therapeutic interest of using a dyad rather than its constituents, we compared the cytotoxicity of tin dyads to the cytotoxic effect of equimolar mixtures of the phthalocyanine ZnPc(EG 3 ) 4 and SnPh 2 or Sn 2 Ph 6 ( Figure 7). For all conditions tested here, and as reported in the Figure 7e

Photodynamic Therapy Investigations of Dyads
The PDT efficiency was first studied on cancer cells with the dyads ZnPc-Sn2Ph6 and ZnPc-SnPh2 under laser excitation of the zinc phthalocyanine in its Q-band at 650 nm. Experiments were performed at the concentration of LC50 and below up to a factor of 10. Several concentrations were tested, as reported in the tables below, (Tables 4 and 5) but unfortunately no specific mortality due to the laser was obtained under these conditions. This result can be explained by fluorescence quenching of the zinc phthalocyanine in these dyads, which most probably inhibits the energy transfer process to oxygen.

Photodynamic Therapy Investigations of Dyads
The PDT efficiency was first studied on cancer cells with the dyads ZnPc-Sn 2 Ph 6 and ZnPc-SnPh 2 under laser excitation of the zinc phthalocyanine in its Q-band at 650 nm. Experiments were performed at the concentration of LC 50 and below up to a factor of 10. Several concentrations were tested, as reported in the tables below, (Tables 4 and 5) but unfortunately no specific mortality due to the laser was obtained under these conditions. This result can be explained by fluorescence quenching of the zinc phthalocyanine in these dyads, which most probably inhibits the energy transfer process to oxygen. Another series of experiments was carried out under light excitation 390/420 nm, on MCF-7 cells and also on healthy fibroblasts. Again, no effect could be observed.
Taken together, these results show that these two dyads have a cytotoxic effect on cells and would allow cancer cells to be targeted more effectively than healthy cells. However, they did not show efficacy in PDT under all of the conditions tested most probably because the singlet excited state is quenched by the appended tin complex, that inhibits singlet oxygen production.

Synthesis of the Compounds
Generalities 1 H and 13 C NMR spectra were recorded on an AVANCE 300 UltraShield BRUKER and AVANCE 400 BRUKER. Chemical shifts for 1 H NMR spectra are referenced relative to residual proton in the deuterated solvent (CDCl 3 δ = 7.26 ppm for 1 H, DMSO-d 6 δ = 2.50 ppm for 1 H, THF-d 8 δ = 3.57, 1.72 ppm for 1 H) or to an internal reference (TMS, δ = 0 ppm for both 1 H and 13 C). NMR spectra were recorded at room temperature, chemical shifts are written in ppm and coupling constants in Hz. High-resolution mass (HR-MS) spectra were obtained by electrospray ionization coupled with high resolution ion trap orbitrap (LTQ-Orbitrap, ThermoFisher Scientific), working in ion-positive or ion-negative mode. UVvisible absorption spectra were recorded on a Variant Cary 300, using 1 cm path length cells. Emission spectra were recorded on a Fluoromax-4 Horiba Jobin Yvon spectrofluorometer (1 cm quartz cells).
Chemicals were purchased from Sigma-Aldrich or Alfa Aesar and used as received. CHCl 3 is stabilized with EtOH. Thin-layer chromatography (TLC) was performed on silica sheets precoated with Merck 5735 Kieselgel 60F 254 . Column chromatography was carried out with Merck 5735 Kieselgel 60F (0.040-0.063 mm mesh). Reference ZnPc(EG 3 ) 4 was synthesized according to literature [30]. Phthalocyanine 3. A mixture of 2 66 mg (1.52 mmol), phthalonitrile 1 3.1 g (10.68 mmol) and Zn(OAc) 2 .2H 2 O 83 mg (3.81 mmol) in n-pentanol (25 mL) was heated to 100 • C, and a small amount of DBU (0.9 mL) was subsequently added. The mixture was heated at 150 • C for 12 h. After cooling the volatiles were removed under reduced pressure. The residue was dissolved in CHCl 3 and a part of the symmetrical phthalocyanine was separated from the desired compound by flash chromatography using CHCl 3 /MeOH (98:2 v/v) as the eluent. The product was further purified by preparative TLC using the same eluent as before to give a blue green oily compound. Yield: (360 mg, 18%). UV-Vis (DMF, nm) λ max (log /M −1 cm −1 ): 360 (4.83); 616 (4.46); 683 (5.19). 1  3.2.3. Phototoxicity Assay at 650 nm MCF-7 cancer cells were seeded into 96-well plates at a concentration of 1000 cells per well in 100 µL of culture medium and allowed to grow for 24 h. Then, cells were incubated 24 h, with or without various concentrations of compound solutions. After incubation, cells were submitted or not to laser irradiation (650 nm; 39 J·cm -2 ) during 20 min. Two days after irradiation, MTT assay was performed to measure the level of living cells.

Statistical Analysis
Statistical analysis was performed using the Student's t-test to compare paired groups of data. A p value < 0.05 was considered as statistically significant.
Excitation beam is diffracted by a double ruled grating SPEX monochromator (1200 grooves/mm blazed at 330 nm). Emission beam is diffracted by a double ruled grating SPEX monochromator (1200 grooves/mm blazed at 500 nm). Singlet oxygen emission was detected through a double ruled grating SPEX monochromator (600 grooves/mm blazed at 1 µm) and a long-wave pass (780 nm). All spectra were measured in 4 faces quartz cuves. All the emission spectra (fluorescence and singlet oxygen luminescence) have been displayed with the same absorbance (less than 0.2) with the lamp and photomultiplier correction. The standard used for the singlet oxygen quantum yield was the Zinc(II) 2,9,16,23-(tetra-t-butyl)phthalocyanine with a standard quantum yield of 0.25.

Conclusions
Two new dyads ZnPc-SnPh 2 , ZnPc-Sn 2 Ph 6 were prepared, by combining a zinc phthalocyanine sensitizer with a cytotoxic entity such as a tin-based complex. Cytotoxicity studies of the ZnPc-SnPh 2 and ZnPc-Sn 2 Ph 6 dyads on cancer cells (MCF-7) and on healthy fibroblasts show that they exhibit high cytotoxicity. In particular, the dyad ZnPc-Sn 2 Ph 6 containing two tin atoms is highly potent towards cancer cells (LC 50 = 0.16 µM) and is twice less toxic towards healthy human fibroblasts. In contrast, no PDT activity was detected for these two dyads at concentrations close to and below LC 50 , most probably due to quenching of the singlet excited state of phthalocyanine by the tin complex, which decreased the singlet oxygen quantum yield. This result highlights that for the development of conjugates consisting of a sensitizer connected to a chemotherapeutic agent, one must pay attention that the photophysical properties of the sensitizer and in particular its singlet oxygen quantum yield is maintained in the conjugate in order to combine the PDT activity with the cytotoxicity of the anticancer drug. This can be estimated from Stern-Volmer quenching experiments before synthesizing the conjugates. This aspect has been mostly overlooked in many previous studies dealing with similar approach.