Two-Photon Active Organotin(IV) Carboxylate Complexes for

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Two-photon active organotin (IV) carboxylate complexes for visualization of anti-cancer action Hui Wang, Lei Hu, Wei Du, Xiaohe Tian, Qiong Zhang, Zhangjun Hu, Lei Luo, Hongping Zhou, Jieying Wu, and Yupeng Tian ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00786 • Publication Date (Web): 27 Mar 2017 Downloaded from http://pubs.acs.org on April 1, 2017

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ACS Biomaterials Science & Engineering

Two-photon active organotin (IV) carboxylate complexes for visualization of anti-cancer action Hui Wanga, Lei Hua, Wei Dua, Xiaohe Tian*b, Qiong Zhang*a, Zhangjun Huc, Lei Luod, Hongping Zhoua, Jieying Wu*a, Yupeng Tiana,e

[a] Department of Chemistry, Key Laboratory of Functional Inorganic Material Chemistry of Anhui Province, Anhui University, Hefei 230601, P. R. China [b] School of Life Science, Anhui University, Hefei 230601, P.R. China [c]Division of Molecular Surface Physics & Nanoscience, Department of Physics, Chemistry and Biology (IFM), Linköing University, 58183 Linköing, Sweden [d] College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China [e] State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, P. R. China *E-mail: [email protected]; [email protected]; [email protected];

KEYWORDS organotin carboxylate, two-photon property, two-photon microscopy, anticancer agents, crystal structures.

ABSTRACT：It is still a challenge that organotin (IV) carboxylate complexes with high-performance two-photon activity for cancer therapy. At present work, two novel organotin carboxylate complexes LSn1 and LSn2, containing coumarin moiety, were rationally designed for two-photon fluorescent imaging and anticancer purpose. The complexes possessed large two-photon action cross-sections, and high quantum yields. Living cells evaluation revealed that complexes LSn1 and LSn2 exhibited good 1

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biocompatibility and deep-tissue penetration over femtosecond laser with wavelength of 840 nm. Furthermore, the antitumor active and as well as possible mechanism of complexes LSn1 and LSn2 have been investigated systematically. The results indicated that complexes LSn1 and LSn2 could induce apoptotic cell death through a mitochondrial dysfunction and ROS elevation pathway. The present work provides a strategy for rationally designing organotin (IV) carboxylate complexes with two-photon activity and antitumor activity.

1. Introduction Organotin (IV) carboxylates, one type of bioactive organometallics, have gained widespread attention due to their structural diversity and effective antitumor activity.1-7 A number of organotin (IV) complexes show high antitumor activity in vivo and in vitro. However, organotin (IV) carboxylate complexes with high anti-cancer activity have fluorescence were rarely reported, which greatly limit the visualization study on the anti-cancer mechanism. In our previous work,

8-9

several organotin

complexes with strong two-photon absorption (2PA) property were designed and synthesized. However, according to our knowledge, the anti-tumor activities of these complexes are still not clearly understood. Recently, two-photon microscopy (2PM) have become an indispensable tool for biomedical application. Compared with one-photon microscopy (OPM), 2PM offers several advantages including longer observation time, higher spatial resolution, and deeper tissue imaging.10-13 Moreover, developing theranostic prodrugs with good luminescence properties to monitor the drug delivery and release process have been actively investigated.14 Near-infrared 2


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(NIR) fluorophores are preferred signaling subunit due to their strong penetrate ability, minimal damage to tissues and low background interference.15 Combining the structural diversity and low cost of the organotin carboxylate complexes with 2PA properties, significantly, it is proposed to develop the complexes with anti-tumor active and two-photon absorption properties to be applied in theranostic medicines. Herein, a series of organotin carboxylate complexes containing coumarin group were designed (Scheme 1). Firstly, coumarin group with a strong electron donor and good planarity has been widely applied in the development of two-photon chromophores;16 while carboxylic acid possesses strong electron-withdrawing ability and structural diversity.17-18 Secondly, bis(tri-n-butyltin) oxide and triphenyltin hydroxide are low cost with high bioactivity. Moreover, the alkyl chain enhances the solubility of the complex molecules. Thirdly, the carboxylic ligand was designed based on coumarin group, which should possess high quantum yields and large two-photon action cross-sections favoring the application of 2PM. 2PM imaging showed that complexes LSn1 and LSn2 could sufficiently penetrate into the cell, respectively. The cytotoxicity studies demonstrated that complexes LSn1 and LSn2 induced tumor cells apoptosis by acting on mitochondrial-dependent pathways.

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Scheme 1. Synthetic routes for L and complexes LSn1-LSn2. 2. Experimental sections 2.1 Materials and apparatus All solvents and chemicals were dried and purified before used. 1H NMR, NMR (TMS as the internal standard) and

119

13

C

Sn-NMR (tetramethyltin as the external

standard, 119Sn ) spectra (DMSO as a solvent) were carried out on Bruker 400 Auance spectrometer, respectively. IR spectra (4000-400 cm-1) were carried out on FT-IR spectrometer (KBr pellets). Bruker Autoflex III Smartbeam was used to record MALDI-TOF mass spectra. Elemental analyses were obtained with Perkin-Elmer 240 analyzer. 2.2 Optical measurements SHIMADZU

UV-3600

spectrophotometer

and

HITACHI

F-7000

spectrophotometer were used to record UV-vis absorption spectra and fluorescence spectra, respectively. Fluorescein as the reference was used to determine the ﬂuorescence quantum yields (Φ).19 2PA cross sections (σ) of all compounds (0.2 mM) were obtained using two-photon excited fluorescence (2PEF) method20 with femtosecond laser pulse and Ti: sapphire system (700-1080 nm, 80 MHz, 140 fs). 2.3 Synthesis of L A mixture of 7-(diethylamino)-2-oxo-2H-chromene-3-carbaldehyde21 (5 mmol, 1.2 g)

and malonic acid ( 7.5 mmol, 0.78 g) were dissolved in 50 mL acetonitrile, and

then added piperidine (3 drops), refluxed for 6 h. The mixture was cooled down to room temperature, and then poured into water. Under vigorously stirring, K2CO3 (aq) 4


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was added to adjust the pH to 6. The generated crude product was washed with water and recrystallized from acetonitrile to afford yellow solids (1.24 g). Yield: 87%. 1

H-NMR (d6-DMSO, 400 MHz) δ: 1.140 (t, J=7.0 Hz, 6H), 3.476 (q, 4H), 6.562 (s,

1H), 6.755 (m, 2H), 7.469 (m, 2H), 8.254 (s, 1H), 12.281 (s, 1H).

13

C-NMR

(d6-DMSO, 100 MHz): 12.307, 44.230, 96.111, 108.04, 109.70, 112.82, 118.56, 130.44, 139.24, 145.06, 151.62, 156.18, 159.71, 167.88. IR (KBr, cm-1): 3422, 2978, 2583, 1720, 1672, 1585, 1512, 1417, 1354, 1317, 1280, 1196, 1132, 1076, 1048, 1015, 990, 869, 818, 797, 776, 734, 706, 649, 575, 540, 468. MS (MALDI-TOF) [m/z-H]: 286.477. Anal. Calc. for C16H17NO4: C, 66.89; H, 5.96; N, 4.88. Found: C, 66.84; H, 5.98; N, 4.89. 2.4 Synthesis of complex LSn1 To prepare the benzene solution (60 mL) of L (1 mmol, 0.28 g), bis(tri-n-butyltin) oxide (0.5 mmol, 0.32 g) was added under a nitrogen atmosphere, refluxed for 12 h using a Dean-Stark apparatus. Then, the solvent was removed under reduced pressure to obtain the yellow solid (0.53 g). Yield: 91%. 1H-NMR (d6-DMSO, 400 MHz) δ: 0.868 (t, J=7.3 Hz, 10H), 1.132 (m, 12H), 1.313 (q, 6H), 1.585 (q, 5H), 3.462 (q, J=6.8 Hz, 4H), 6.542 (s, 1H), 6.587 (m, 2H), 6.749 (d, J=12.0 Hz, 1H), 7.268 (d, J=15.8 Hz, 1H), 7.461 (d, J=8.9 Hz, 1H), 8.165 (s, 1H).

13

C-NMR (d6-DMSO, 100

MHz): 12.313, 13.633, 18.370, 26.449, 27.642, 44.160, 96.147, 108.13, 109.46, 113.97, 123.62, 130.03, 130.10, 135.32, 142.70, 151.08, 155.86, 159.97, 170.38. IR (KBr, cm-1): 2922, 2856, 1714, 1622, 1586, 1519, 1448, 1418, 1379, 1275, 1230, 1192, 1133, 1076, 1012, 988, 863, 821, 768, 709, 677, 645, 505, 468. 5


119

Sn-NMR


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(DMSO): δ=-18.47 ppm. MS (MALDI-TOF) [m/z-butyl]: 520.145. Anal. Calc. for C28H43NO4Sn: C, 58.35; H, 7.52; N, 2.43. Found: C, 58.39; H, 7.51; N, 2.42. 2.5 Synthesis of complex LSn2 This compound was obtained by the same method used for LSn1, using L (1 mmol, 0.28 g) and Ph3SnOH (1 mmol, 0.36 g). Yield: 90% (0.53 g). 1H-NMR (d6-DMSO, 400 MHz) δ: 1.113 (t, J=6.9 Hz, 6H), 3.439 (q, J=6.8 Hz, 4H), 6.520 (s, 1H), 6.577 (d, J=15.8 Hz, 2H), 6.726 (d, J=12.0 Hz, 1H), 7.238 (d, J= 15.8 Hz, 1H), 7.427 (m, 10H), 7.838 (d, J=7.1 Hz, 6H), 8.121 (s, 1H).

13

C-NMR (d6-DMSO, 100

MHz): 12.305, 44.167, 96.126, 108.09, 109.49, 113.68, 122.99, 123.03, 128.18, 136.20, 143.19, 151.16, 155.89, 159.86. IR (KBr, cm-1): 2969, 2924, 1720, 1614, 1569, 1511, 1474, 1333, 1289, 1190, 1131, 1070, 987, 865, 822, 785, 733, 697, 664, 619, 526, 445.

119

Sn-NMR (DMSO): δ=-258.29 ppm. MS (MALDI-TOF) [m/z]:

637.284. Anal. Calc. for C34H31NO4Sn: C, 64.18; H, 4.91; N, 2.20. Found: C, 64.12; H, 4.92; N, 2.19.

3. Results and discussion 3.1 Crystal structures. The crystal structures of complexes LSn1 and LSn2 were displayed in Figure 1. Crystallographic data, selected bond lengths and angles for complexes LSn1 and LSn2 were summarized in Table S1-S3. Both complexes LSn1 and LSn2 crystallize in the monoclinic crystal system with P21/n space group. As shown in Figure 1. The center Sn (IV) adopts a distorted tetrahedron coordination geometry, which is coordinated by one oxygen atom from 6


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the carboxylic units and three carbon atoms from benzene rings or butyl groups. The dihedral angle between the carboxylate unit and the vinyl group is 7.69o (LSn1) and 11.25o (LSn2), respectively, the coumarin group adjoins the vinyl group with the dihedral angle being 4.50o (LSn1) and 7.27o (LSn2), indicating that the carboxylate ligand containing coumarin bears good planar. The dihedral angles between the coumarin group and the SnCO plane (Sn(1)C(28)O(4) for LSn1, or Sn(1)C(16)O(4) for LSn2) are 11.89o and 12.76o, respectively. Those structural features indicate that complexes LSn1 and LSn2 should have high delocalized π-electron system, which favors nonlinear optical response.

Figure 1. Molecule structure of LSn1 (a), and LSn2 (b). All H atoms have been omitted for clarity.

3.2 Optical properties. The photophysical properties of L, LSn1 and LSn2 were summarized in Table S4. The UV-vis absorption spectra of all the compounds were shown in Figure S1 and Figure 2a. All the compounds displayed two major absorption bands. The high energy band at about 283 nm was assigned to the πcoumarin-πcoumarin* transition. The low energy band about 420-490 nm with stronger intensity was attributed to the π-π* transition of the whole ligand molecule, which 7



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was further confirmed by TD-DFT (Figure S2 and Table S5). As shown in Figure S2, for L, LSn1 and LSn2, the HOMO is mainly localized on the ethyl group, coumarin group and the vinyl group, the LUMO is located on the coumarin group, vinyl group and the carboxylate group, which indicates that the HOMO→LUMO transition can be assigned as a π→π* mixed intraligand charge transfer transition (ICT). From Figure S1 and Table S4 exhibited a weak solvatochromism from the absorption bands, indicating that the dipoles of ground state were little different from that of excited state in diluted solution. As displayed in Figure S3, upon increasing the solvent polarity, the fluorescence maxima of all the compounds showed remarkable red-shifts and the Stokes shifts also show an increasing tendency. From Figure 2b and Table S3, the ligand L and its complexes (LSn1, LSn2) show nearly the same emission wavelengths in all of the solvents. Moreover, the fluorescence intensity of complexes LSn1 and LSn2 is weaker than that of its free ligand L, which is explained that the involvement of the Sn atoms increasing the spin-orbit coupling of fluorescent molecules, reducing the energy level between the singlet and triplet excited states, causing the decrease of fluorescence intensity.9 However, the fluorescence quantum yields of complexes LSn1 and LSn2 are higher than its corresponding free ligand, especially in high polar solvent (DMF). Besides, the fluorescence lifetimes of complexes LSn1 and LSn2 are close to the ligand L (< 1 ns). The short fluorescence lifetime in these compounds is in accordance with rapid inter-system crossing due to the strong spin-orbital coupling.22 8


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Figure 2. (a) Linear absorption spectra of L, LSn1 and LSn2 in DMF (c=10 µM). (b) One- and (c) two-photon fluorescence spectra of L, LSn1 and LSn2 in DMF solution. (d) Two-photon action cross sections (Φσ) of L, LSn1 and LSn2 in DMF solution (c=0.2 mM).

3.3 Two-photon excited fluorescence (2PEF) spectra and 2PA action cross sections. The 2PEF spectra of L and complexes (LSn1 and LSn2) in DMF were obtained from 740 nm to 970 nm under 200 mW. As displayed in Figure S4, the emission intensity of L, LSn1 and LSn2 was investigated at 840 nm. Upon the input laser power increasing, the logarithmic plots have a slope of 2.04 (L), 1.98 (LSn1) and 2.02 (LSn2), respectively, indicating a two-photon excitation mechanism. The two-photon excitation spectra of L, LSn1 and LSn2 in DMF excited at the wavelength of 840 nm were displayed in Figure 2c. The two-photon fluorescence intensity of complexes LSn1 and LSn2 is weaker compared to L, which is similar to 9



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the SPEF. From Figure 2d, the maximum 2PA action cross-sections (Φσmax) are 163 GM for L, 95 GM for LSn1, and 89 GM for LSn2, respectively. The 2PA action cross section values in DMF solution of LSn1 and LSn2 are smaller than the ligand on account of the heavy atom effect of the organotin atom. Importantly, the Φσmax of the complexes is greater than 50 GM, which is favorable for a bright 2PM image without causing appreciable photo-damage to the sample.10 Besides, the Φσmax for the two complexes in the NIR range (about 840 nm), which is favour of biological imaging applications due to the lower optical damaging.

Figure 3. One- and two-photon fluorescence images of live HepG2 cells treated with 10 µM complexes LSn1 and LSn2 for 30 min at 37 oC. Excited wavelength (1P): 405 nm; emission filter: 470-520 nm. Excited wavelength (2P): 840 nm; emission filter: 480-550 nm. Bar=20 µm.

3.4 Cytotoxicity and Biological imaging MTT assay. The antitumor activities of complexes LSn1 and LSn2 were evaluated against three tumor cell lines (Hela (human cervical carcinoma cell line), HepG2 10


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(human hepatocellular liver carcinoma cell line) and A549 (human pulmonary adenocarcinoma cell)) and one normal cell HELF (human embryonic lung fibroblast) using MTT assay. Cisplatin4 is a clinical anti-cancer drug, which was used as positive controls. From Table 1, complexes LSn1 and LSn2 exhibited higher cytotoxicity against these three tumor cell lines than cisplatin in vitro. Furthermore, the result indicated that the following order of antitumor activity of the complexes can be seen: LSn2 > LSn1 (except A549 cells). Combined with our previous work,11, 23 we found that Ph3Sn (IV) derivatives exhibit higher activity. Sn-Ph bond cleaved from triphenyltin derivative might due to the weak coordination of Sn and Ph, which is beneficial to form the moieties function as inhibitior.24 Notably, complexes LSn1 and LSn2 exhibited higher cytotoxicity in the selected tumor cell lines than that in normal cell, whereas cisplatin displayed similar cytotoxicity in tumor cells as well as normal cells. These results demonstrated that complexes LSn1 and LSn2 had high selectivity between normal cells and tumor cells.

Table 1. Inhibitory concentration IC50 (µM) of complexes LSn1, LSn2 and Cisplatin against HepG2, Hela, A549 and HELF cell lines. IC50 (µM)a

Compounds

a

HepG2

Hela

A549

HELF

LSn1

4.42±0.05

1.24±0.08

1.10±0.09

18.39±0.10

LSn2

2.33±0.07

0.94±0.06

4.36±0.07

47.29±0.06

Cisplatin

14.62±0.08

9.89±0.11

9.06±0.04

12.47±0.12

Cells were incubated with LSn1, LSn2 and cisplatin for 24 h. Data were exhibited as

the means ±standard deviations (SD).

As a therapeutic and diagnostic agent, the cellular uptake characteristics of 11



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organotin complexes are critical to its application.25 The hydrolytic stability of complexes LSn1 and LSn2 in aqueous conditions as well as at 37 oC were initially evaluated via 1H NMR in d6-DMSO and D2O mixture (v/v, 5/1). From Figure S5 and S6, it can be seen that after 24 h incubation at 37 oC, complexes LSn1 and LSn2 exhibit good stability compared with the control. HepG2 cells as a model were incubated with complexes LSn1 and LSn2, and then imaged using 2PM. As shown in Figure 3, it was clear that LSn1 and LSn2 emitted bright fluorescence throughout cytosol. This implied that complexes LSn1 and LSn2 are membrane permeable and could enter living cells without external vectors. Confocal microscopy of HepG2 cells incubated with LSn1 and LSn2 at different time were also investigated, propidium iodide (PI) was used to monitor HepG2 cell death. From Fig S7-S8, it can be seen that there was no obvious difference observed from the different incubation time. Upon the incubation time increasing, PI with a red fluorescence signal gradually enters the cells and stains the nuclei. After incubation for 6 hours, the cell cytoplasm shrinks and the nuclear membrane is destroyed. The cellular uptake mechanisms of LSn1 and LSn2 were also investigated. HepG2 cells were incubated with LSn1 and LSn2 at 4 oC, respectively. Figure S9 showed that no luminescence was observed, indicating that LSn1 and LSn2 enter cells through a temperature-dependent pathway. Pretreatment of HepG2 cells with endocytosis inhibitors (chloroquine, chlorpromazine and NH4Cl) show no effect on the uptake of LSn1 and LSn2 (Figure S9). The results demonstrated that LSn1 and LSn2 penetrate into cells via an energy-dependent non-endocytic pathway. 12


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To demonstrate the specific localization of complexes LSn1 and LSn2 in living cells, co-localization experiments were performed. MitoTracker® Deep Red FM (MTR) and ER-TrackerTM Green dye (ER) were used to co-stain HepG2 cells. As shown in Figure S10 and S11, partial overlaying of the images between these complexes and MTR/ER were observed. The Pearson’s colocalization coefficients (Rr) between complexes (LSn1 and LSn2) and MTR signal were obtained to be 0.47 and 0.40, respectively, as well as the Rr obtained for LSn1 and LSn2 with ER are 0.36 and 0.34, respectively. It is noteworthy that mitochondrial and endoplasmic reticulum are membrane-rich organelles with high hydrophobicity, as complexes LSn1 and LSn2 are lipophilic molecule with partly overlapping to mitochondrial and ER, so we speculated that complexes LSn1 and LSn2 associate with lipophilic region within the cell.

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Figure 4. (a) Confocal microscopy imaging of HepG2 cells co-labelled with JC-1 dye with or without complexes LSn1 and LSn2 (5 µM), respectively. (b) Fluorescence images of ROS level in HepG2 cells after 5 h incubation with complexes LSn1 and LSn2 (5 µM), followed by DCFH-DA staining for 20 min at 37 oC. (c) Effect of complexes LSn1 and LSn2 on the distribution of HepG2 cells in cell cycle populations at 500 nM for 12 h treatment. (d) Hoechst 33342 stained HepG2 cells after incubated with complexes LSn1 and LSn2 (1 µM) after 19 h. (e) Annexin V/PI 14


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double staining analyzed by flow cytometry. HepG2 cells were treated with 10 µM complexes LSn1 and LSn2. Bar=20 µm.

Mitochondrial

dysfunction

and

reactive

oxygen

species

(ROS)

levels.

Mitochondria play an essential component of the intrinsic and extrinsic apoptotic pathways.29-30 We utilize the fluorescence dye JC-1 to monitor the mitochondrial membrane potential (MMP) after incubated with complexes LSn1 and LSn2.29 From Figure 4a, it was an obvious color shift from red to green after HepG2 cells incubation of complexes LSn1 and LSn2 (5 µM), suggesting that the decrease of MMP. Figure S12 showed that the red/green ratio signals of JC-1 decreased from 12.44 to 0.15 (LSn1) and 0.48 (LSn2), respectively. These results suggested that the complexes LSn1 and LSn2 could induce mitochondrial membrane potential decrease. To understand cell oxidative stress caused by complexes LSn1 and LSn2 treatment, DCF (2’, 7’-dichlorodihydro fluorescein) was used to monitored the level of intracellular ROS.31 As displayed in Figure 4b and S13, compared to the control, the fluorescence intensity increased by about 1.61 and 1.78-fold after treated with complexes LSn1 and LSn2 (5 µM), respectively, confirming that ROS play a vital role in organotin (IV)-induced cell death. Induction of cell cycle arrest and apoptosis. To further explore the mechanism of such an anticancer effect, flow cytometry analysis was performed to investigate the cell cycle of HepG2 cells stained with PI (propidium iodide) in the presence of complexes LSn1 and LSn2. From Figure 4c, after the treatment of complex LSn1, 15



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the percentage of HepG2 cells in the S phase (DNA synthesis phase) decreased markedly from 25% to 16%, and there was a sharp increase of cells in the G2M phase (G2 phase follows the successful completion of S phase and prepares the cell for mitosis, consisting of protein synthesis and rapid cell growth. M phase is mitotic phase, which consists of nuclear division (karyokinesis)) from 22% to 29%, indicating that the cell cycle is arrested at the G2M phase. A similar phenomenon was also observed after the treatment of complexes LSn2. These data suggest that the antitumor mechanism induced by complexes LSn1 and LSn2 was acting at G2M phase. Apoptosis cell is characterized by a series of biochemical and morphological events, for instance cell shrinkage, phosphatidylserine (PS) externalization and nuclear fragmentation.32 First, the changes of cell morphology were examined by Hoechst 33342. As shown in Figure 4d, vehicle-treated control cells exhibit a normal overall morphology. After 19 h treatment of complexes LSn1 and LSn2 (1 µM), the majority of HepG2 cells lost their normal morphology and displayed typical apoptosis morphology changes (cell shrinkage, membrane bubbing, chromatin condensation, nuclei fragmentation and the presence of apoptotic bodies). Second, to assess the anticancer activity of complexes LSn1 and LSn2, we utilize the annexin V-FITC/PI double staining to quantify the population percentages of apoptotic HepG2 cells.33-34 From Figure 4e, after treated with complexes LSn1 and LSn2 (10 µM), 42.55% and 73.63% of late apoptosis cells were observed, respectively, whereas the viable of the untreated cells remained 98.93%. Complex 16


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LSn2 was more effective than all other complexes in inducing PS exposure and the loss of plasma membrane integrity. The results indicated that complexes LSn1 and LSn2 induce cell death via apoptosis pathway. Penetration ability in 3D MCSs. It is noteworthy that various anticancer drugs failed to penetrate into the monolayer cells to in vivo performance, partly due to the limitations of extracellular matrix (ECM). 3D multicellular spheroids (MCSs) as the tissue mode are widely applied to the administration of drug delivery.26-28 Hence, 200 µm 3D MCSs as the in vivo model were utilized to assess the therapeutic outcome of complexes. To investigate the penetration ability of complexes LSn1 and LSn2, 200 µm HepG2 MCSs were incubated with 10 µM complexes LSn1 and LSn2 for 5 h, and then imaged with 2PM (Figure 5). The results revealed that MCSs displayed remarkable fluorescence to a depth over 63 µm (Figure S14-S15), suggesting that in this period they could overcome the ECM barrier and accumulate in MCS.

Figure 5. Two-photon fluorescence images of the 3D multicellular spheroids of HepG2 cells after incubation with 10 µM complexes LSn1 and LSn2 for 5 h, respectively.

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4. Conclusions In summary, we have presented new organotin complexes LSn1 and LSn2, which can be used as anti-tumor and two-photon real-time imaging agents. The design idea was performed by integrating anti-tumor organotin and 2PA active coumarin carboxylates into a single molecule. As expected, complexes LSn1 and LSn2 exhibited significant 2PA action cross-section, substantial cellular uptake, and rapid MCSs penetration, which are favor of efficient two-photon imaging use. Meanwhile, complexes LSn1 and LSn2 exhibited high cytotoxic potency than cisplatin and show high selectivity between cancer cells and non-cancerous cells. Further studies demonstrated that complexes LSn1 and LSn2 accumulate in the cytoplasm of HepG2 cells and induced apoptosis via ROS-mediated mitochondrial dysfunction pathway. These results suggested that complexes LSn1 and LSn2 are promising candidates as chemotherapy agents. Overall, the low cost and structural diversity of the organotin carboxylate complexes with high 2PA and antitumor activities in the NIR region, which provides more hope and opportunity to design and explore the dual functions materials for further biomedical applications. Supporting Information. Crystal data, photophysical properties, confocal microscopy imaging, and characterization data of all the compounds, etc. The material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; [email protected]; [email protected] 18


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Author Contributions H. Wang and L. Hu contributed equally to this work.

Funding Sources This study was supported by grants from the National Natural Science Foundation of China (21602003, 51372003, 51672003, 51432001, 21501001 and 51472002), Ministry of Education Funded Projects Focus on returned overseas scholar, the Higher Education Revitalization Plan Talent Project (2013). Notes The authors declare no competing financial interest. REFERENCES: (1) Noe, A. L.; Habtemariam, A.; Pizarro, A. M.; Sadler, P. J. Designing organometallic compounds for catalysis and therapy. Chem. Commun. 2012, 48, 5219–5246. (2) Zhang, Y. Y.; Zhang, R. F.; Zhang, S. L.; Cheng, S.; Li, Q. L.; Ma, C. L. Syntheses, structures and anti-tumor activity of four new organotin(IV) carboxylates based on 2-thienylsenoacetic acid. Dalton. Trans. 2016, 45, 8412-8421. (3) Hadjikakou, S. K.; Hadjiliadis, N. Antiproliferative and anti-tumor activity of organotin compounds. Coord. Chem. Rev. 2009, 253, 235–249 (4) Shpakovsky, D. B.; Banti, C. N.; Mukhatova, E. M.; Gracheva, Y. A.; Osipova, V. P.; Berberova, N. T.; Albov, D. V.; Antonenko, T. A.; Aslanov, L. A.; Milaeva, E. R.; Hadjikakou, S. K. Synthesis, antiradical activity and in vitro cytotoxicity of novel 19



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Two-photon active organotin (IV) carboxylate complexes for visualization of anti-cancer action Hui Wanga, Lei Hua, Wei Dua, Xiaohe Tian*b, Qiong Zhang*a, Zhangjun Huc, Lei Luod, Hongping Zhoua, Jieying Wu*a, Yupeng Tiana,e


Two-Photon Active Organotin(IV) Carboxylate Complexes for

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