Organotin(IV) carboxylate complexes containing polyether oxygen chains with two-photon absorption in the near infrared region and their anticancer activity

Accepted Manuscript

Organotin(IV) carboxylate complexes containing polyether oxygen chains with two-

photon absorption in the near infrared region and their anticancer activity

Rongting Guan, Ziqi Zhou, Mingzhu Zhang, Hui Liu, Wei Du, Xiaohe Tian, Qiong

Zhang, Hongping Zhou, Jieying Wu, Yupeng Tian

PII:

S0143-7208(18)30996-3

10.1016/j.dyepig.2018.05.072

DYPI 6801

DOI:

Reference:

To appear in: Dyes and Pigments

Received Date: 2 May 2018

Revised Date: 30 May 2018

Accepted Date: 30 May 2018

Please cite this article as: Guan R, Zhou Z, Zhang M, Liu H, Du W, Tian X, Zhang Q, Zhou H, Wu J,

Tian Y, Organotin(IV) carboxylate complexes containing polyether oxygen chains with two-photon

absorption in the near infrared region and their anticancer activity, Dyes and Pigments (2018), doi:

10.1016/j.dyepig.2018.05.072.

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ACCEPTED MANUSCRIPT

Graphic abstract

ACCEPTED MANUSCRIPT

Organotin(IV) Carboxylate Complexes Containing Polyether Oxygen Chains

with Two-photon Absorption in the Near Infrared Region and their Anticancer

Activity

Rongting Guan ^a, Ziqi Zhou ^a, Mingzhu Zhang ^a, Hui Liu ^a, Wei Du ^a, Xiaohe Tian ^b,

Qiong Zhang ^a*, Hongping Zhou ^a, Jieying Wu ^a*, Yupeng Tian ^a*

^aDepartment of Chemistry, Anhui Province Key Laboratory of Chemistry for

Inorganic/Organic Hybrid Functionalized Materials, Anhui University, Hefei 230601,

PR China

^bSchool of Life Science, Anhui University, Hefei 230039, PR China

E-mail: jywu1957@163.com, zhangqiong.314@163.com; Fax: +86-551-63861279;

Tel: +86-551-63861279

Two novel organo tin carboxylate complexes (BuSnO)₆L₆and Bu₃SnOL, where L =

a cinnamic acid unit containing polyether chains, were rationally designed for

two-photon fluorescent imaging and anticancer purpose. The organotin (IV) complex

(BuSnO)₆L₆possessed strong one-photon exited fluorescence (1PF) intensity and

moderate two-photon absorption cross-section in the near infrared region. Living cell

evaluation revealed that (BuSnO)₆L₆exhibited good bio-compatibility and

outstanding selectivity in the selected tumor cell lines as well as in the normal cells.

Furthermore, the anti-tumor activity and mechanism of (BuSnO)₆L₆have been

investigated systematically. The results indicated that (BuSnO)₆L₆could induce

apoptotic cell death through a mitochondrial dysfunction, ROS (Reactive oxygen

species produced by cell during metabolism) and Cytochrome c elevation pathway.

This work provides a novel strategy for rationally designing carboxylate organotin

complex with two-photon activity and anti-tumor activity.

1. Introduction

Organometallics compounds have been extensively explored due to their versatile

molecular structures [1-6] and biochemical activity [7-11]. Among organometallics,

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organotin compounds are of great importance for their structures and anti-proliferative

properties against a number of cell lines [12-16]. From all the studied organotin

compounds, the organotin carboxylates [17-19] are no doubt the most intensively

investigated. Carboxylic acid precursors offer a number of distinct advantages such as

coordination ability through the oxygen electron-donating atoms that allows it to

serve as either a multidentate or a bridging building block in structural assemblies

[20-23]. So far, a large variety of organotin complex types, including ladder,

O-capped complex, cyclic trimer, butterfly drum, football cage and hexatin cage, have

been reported [24-29]. However, these studies only provided the structure of

organotin (IV) carboxylate complexes, the fluorescence or anticancer activity has

rarely been reported, which greatly limits the visualization study on the anticancer

mechanism.

In our previous work, several organotin (IV) carboxylate complexes with

one/two-photon fluorescence have been reported [30, 31]. It is well known that

two-photon microscopy (2PM) has become an indispensable tool for biomedical

applications. Compared with one-photon microscopy (1PM), 2PM offers several

advantages, including longer observation time, higher spatial resolution, and deeper

tissue imaging [32-35]. Recently, theranostic pro-drugs with good luminescence

properties to monitor the drug delivery and release process have been actively

investigated [36]. Combining the structural diversity and low cost of the organotin

carboxylate complexes with 2PA properties, it is proposed to develop the complexes

with anti-tumor activity and two-photon absorption properties to be applied in

theranostic medicines.

Herein, a series of organotin carboxylate complexes containing a cinnamate group

substituted with flexible ether oxygen chains were designed (Scheme 1). Firstly, і)

flexible polyether oxygen chains is introduced for increasing the solubility of the

compounds. The high lipid solubility could be beneficial to associate with lipophilic

region within the cell such as mitochondria and endoplasmic reticulum, which are

membrane rich organelles with high hydrophobicity; ii) the carboxylate unit possesses

strong electron-withdrawing ability and structural diversity [37, 38]. Secondly,

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butyl(hydroxy)stannanone is low cost with high bio-activity. Moreover, the alkyl

chain enhances the solubility of the complex. Thirdly, poly-ligands can be aggregated

within the drum structure organotin (IV) complex to enhance two-photon

cross-sections, which could be ideal for imaging guided cancer therapy. Relying on

the comprehensive studies, (BuSnO)₆L₆was selected for bio-applications due to its

excellent 2PA property. The cytotoxicity studies demonstrated that (BuSnO)₆L₆

induced tumor cell apoptosis by acting on on Reactive oxygen species

(ROS)-mediated mitochondrial dysfunction pathway (Scheme. 2). All these results

suggested that (BuSnO)₆L₆is a potential anti-cancer material for further biomedical

applications in vivo.

Scheme. 1 The synthesis routes of the compounds.

Scheme. 2 Schematic depicting the possible mechanism of (BuSnO)₆L₆-induced

apoptotic pathway in HeLa cells. (BuSnO)₆L₆induces a boost in reactive oxygen

species and evokes mitochondrial dysfunction, which then increases cytochrome c

release, sequentially, initiating process of apoptosis.

2. Experimental section

2.1. Materials and Apparatus

All solvents were dried and purified by usual methods. IR spectra (4000-400 cm^-1),

as KBr pellets, were recorded with a Nicolet FT-IR 170 SX sepctrophotometer. Mass

spectrum was performed with a Micromass MALDI-TOF-MS and Micromass

GCT-MS (ESI source). Elemental analysis was carried out on a Perkin-Elmer 240

1

analyzer. The H-NMR, ¹³C-NMR spectra and ¹¹⁹Sn-NMR (tetramethyltin as the

external standard, ¹¹⁹Sn) spectra (CDCl₃as a solvent)were carried out on Bruker 400

Auance spectrometer, respectively.

2.2. Optical Measurements

SHIMADZU

UV-3600

spectrophotometer

and

HITACHI

F-7000

spectrophotometer were used to record UV-vis absorption spectra and fluorescence

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spectra, respectively. Two-photon absorption (2PA) cross-sections (σ) of all

compounds (0.2 mM) were obtained using two-photon excited fluorescence (2PEF)

method [39] with femtosecond laser pulse and Ti:sapphire system (680-1080 nm, 80

MHz, 140 fs).

2. 3. Synthesis

Synthesis of (E)-3-(3-ethoxy-4-(2-(2-methoxyethoxy)ethoxy)phenyl)acrylic acid

(L):

3-Ethoxy-4-(2-(2-methoxyethoxy)ethoxy)benzaldehyde (Lex) (2.24 g, 10 mmol) and

malonic acid (1.04 g, 10 mmol) were dissolved in pyridine (50 mL) in the flask (150

mL), and several drops of piperidine were added to it. The reaction mixture was

refluxed for 6 h. Resulting mixture was cooled and poured into ice water, and diluted

hydrochloric acid (HCl) was added to adjust the pH under stirring. Resulting mixture

was filtered after the pH to the value of 6. The white solid (L) was obtained (2.11 g,

o

yield 80 %). M.p = 125-127 C. IR (KBr, cm^-1): 3034 (s), 1680 (s), 1620 (s), 1600

(m), 1520 (w), 1513 (s), 1430 (m), 1254 (s). ¹H-NMR (400 MHz, d₆-acetone, ppm): δ

1.41 (s, 3 H), 3.30 (s, 3 H), 3.49~3.52 (t, 2 H, J= 6.0 Hz), 3.67~3.70 (t, 2 H, J= 6.0

Hz), 3.83~3.85 (q, 2 H, J= 4.0 Hz), 4.13~4.16 (t, 2 H, J= 6.0 Hz), 4.18~4.21 (q, 2 H,

J= 6.0 Hz), 6.41 (d, 1 H, J= 16.0 Hz), 7.03 (d, 1 H, J=8.0 Hz), 7.20 (d, 1 H, J= 8.0 Hz),

13

7.32 (s, 1 H), 7.60 (d, 1 H, J=8.0 Hz); C-NMR (100 MHz, d₆-acetone, ppm): δ

15.19, 58.82, 65.17, 69.53, 70.22, 72.70, 113.26, 114.42, 116.68, 123.45, 128.66,

145.66, 150.15, 152.04, 167.98. ESI-MS (m/z): Calcd.: 310.14, Found 333.17

(M+Na⁺). Anal. Calc. for C₁₈H₁₉N₃O: C, 61.92; H, 7.15; Found: C, 61.97; H, 7.16.

Synthesis of (BuSnO)₆L₆:

L (0.62 g, 2 mmol) was dissolved in benzene (50 mL) in the flask (150 mL), then

BuSnO(OH) (0.42 g, 2 mmol) was added to it under a nitrogen atmosphere, and

refluxed for 12 h using a Dean−Stark apparatus. Then, the solvent was removed under

reduced pressure. Toluene and petroleum ether were added to disperse solid. The

o

yellow solid was obtained (0.56 g). M.p = 290-291 C. IR (KBr, cm^-1): 2897 (m),

2560 (s), 1693 (s), 1595 (s), 1527 (s), 1432 (s), 1268 (vs), 1133 (s), 926(m),

ν_as(COO), 1595, 1527; ν_s(COO), 1432, 1388; [∆ν = ν_as(COO)–ν_s(COO), 163, 139];

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ν(O–Sn–O), 667; ν(Sn–C), 605; ν(Sn–O), 513. ¹H-NMR (400 MHz, CDCl₃, ppm) δ=

0.95-0.97 (t, 3 H, J = 4.0 Hz), 1.36-1.39 (t, 4 H, J = 6.0 Hz), 1.78-1.82 (t, 2 H, J = 8.0

Hz), 3.56 (s, 3 H), 3.55-3.58 (t, 2 H, J = 6.0 Hz), 3.73-3.76 (t, 2 H, J = 6.0 Hz),

3.88-3.90 (t, 2 H, J = 4.0 Hz), 4.02-4.07 (q, 2 H, J = 6.0 Hz), 4.18-4.21 (t, 2 H, J = 6.0

Hz), 6.35 (d, 1 H, J = 12.0 Hz), 6.85 (d, 1 H, J = 8.0 Hz), 7.02 (t, 2 H, J = 8.0 Hz),

13

7.57 (d, 1 H, J = 16.0 Hz); C-NMR (100 MHz, CDCl₃, ppm) δ = 13.79, 14.81,

26.58, 27.46, 59.09, 64.42, 69.56, 70.86, 71.97, 112.37, 113.37, 120.87, 122.47,

128.47, 145.07, 148.92, 150.34, 173.77; ¹¹⁹Sn-NMR (CDCl₃) = -483.48 ppm.

MALDI-TOF-MS (m/z): Calcd.: 2998.23, Found 2998.35. Anal. Calc. for

C₁₂₀H₁₈₀O₄₂Sn₆: C, 47.66; H, 6.18. Found: C, 47.60; H, 6.16.

Synthesis of Bu₃SnOL:

L (0.62 g, 2 mmol) was dissolved in benzene (50 mL) in the flask (150 mL), then

(Bu₃Sn₂)O (0.60 g, 1 mmol) was added to it under a nitrogen atmosphere, and

refluxed for 12 h using a Dean−Stark apparatus. Then, the solvent was removed under

reduced pressure. Toluene and petroleum ether were added to it to disperse solid. The

o

yellow solid was obtained (0.79 g, yield 68 %). M.p = 185-187 C. IR (KBr, cm^-1):

2926 (vs), 1643 (s), 1508 (m), 1395 (s), 1260 (vs), 1136 (w), ν_as(COO), 1637, 1513;

ν_s(COO), 1254, 1119; [∆ν = ν_as(COO)–ν_s(COO), 383, 394]; ν(O–Sn–O), 680;

ν(Sn–C), 601; ν(Sn–O), 511. ¹H-NMR (400 MHz, CDCl₃, ppm) δ= 0.90-0.92 (t, 9 H,

J=6.0 Hz), 1.30-1.37 (m, 12 H), 1.39-1.42 (t, 3 H, J = 6.0 Hz), 1.42-1.44 (q, 6 H, J =

4.0 Hz), 3.40 (s, 3 H), 3.57-3.59 (t, 2 H, J = 4.0 Hz), 3.75-3.77 (t, 2 H, J = 4.0 Hz),

4.05-4.07 (t, 2 H, J = 4.0 Hz), 4.20-4.22 (t, 2 H, J = 4.0 Hz), 6.35 (d, 1 H, J= 12.0 Hz),

6.88 (d, 1 H, J = 8.0 Hz), 7.05 (d, 2 H, J = 8.0 Hz), 7.54 (d, 1 H, J = 12.0 Hz);

¹³C-NMR (100 MHz, CDCl₃, ppm) δ= 13.78, 14.81, 26.57, 26.87, 27.46, 59.08,

64.43, 68.87, 69.56, 70.85, 71.97, 111.63, 113.42, 120.40, 122.38, 128.49, 144.11,

148.94, 150.36, 173.77; ¹¹⁹Sn-NMR (CDCl₃) = 106.47 ppm. ESI-MS (m/z): Calcd.:

600.24, Found 623.42 (M+Na⁺). Anal. Calc. for C₂₈H₄₈O₆Sn: C, 56.11; H, 8.07.

Found: C, 56.21; H, 8.04.

3. Results and discussion

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3.1 Crystal structure description

The crystal structures of L and complex Bu₃SnOL and the optimized structure of

(BuSnO)₆L₆were shown in Fig. 1. The relevant crystal data and structural parameters

were summarized in Table S1. The selected bond distances and angles were listed in

Table S2 and Table S3.

Ligand L crystallized in the triclinic crystal system with Pī space group and

complex Bu₃SnOL crystallizes in the monoclinic crystal system with P2₁/n space

group. Fig. 1 (a), the dihedral angle between the carboxylate unit and the vinyl group

is 7.62°, the benzene ring adjoins the vinyl group with the dihedral angle being 4.19°,

indicating that the carboxylate ligand bears good planarity. The bond length of single

bond C–O of carboxylate of L is longer than that of complex Bu₃SnOL, which

proved that the coordination of Sn with L shortens’ the bond length. As shown in Fig.

1 (b), the center Sn (IV) adopts a distorted tetrahedron coordination geometry due to

the bond angles (002)–Sn(01)–C(00F) 89.75°, (002)–Sn(01)–C(00H) 97.24°,

O(002)–Sn(01)–C(00I) 93.56°, which is coordinated by one oxygen atom from the

carboxylic units and three butyl groups. The bond length Sn(01)–O(002) 2.18 Å falls

in the typical Sn–O bond length range, which reveals that between the central tin atom

and carbonyl oxygen atom exists a weak bond and it is a monodentate coordinated

form organic tin and the ligand L.

As shown in Fig. 1 (c), the prismane-like cage centrosymmetric structure is built

around a drum shaped Sn6O6 central stannoxane core that is made up of an upper and

lower Sn3O3 rings and the side faces of six Sn2O2 rings [40]. The two Sn3O3 rings

are connected further by six Sn-O bonds containing µ3-coordinate O atoms. The two

tin atoms in each of the six Sn2O2 rings are bridged by a carboxylato ligand to form a

symmetrical bridge. Each tin atom is hexacoordinated and the coordination

environment is comprised of five oxygen atoms and one carbon. The coordination

geometry around each tin atom is a distorted octahedral. Furthermore, the Sn-O bond

lengths inside the core cage (Sn3-O9 2.086 Å) is comparatively shorter than the Sn-O

bond to the bridging carboxylato ligands (Sn3-O4 2.165 Å), which are both shorter

than the Sn-O bond lengths (2.18 Å) of mononuclear organo tin (IV) complex

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Bu₃SnOL. It indicates the delocalization of multinuclear organo tin (IV) complex is

better than that of mononuclear organo tin (IV) complex.

Fig. 1 (a) The single molecular structure of L. (b) The single molecular structure of

Bu₃SnOL. (c) The optimize structure of (BuSnO)₆L₆. All H atoms have been omitted

for clarity.

3.2 Optical Properties

UV-vis absorption spectra and TD-DFT studies.

The UV-vis absorption spectra and molecular orbitals of the compounds are shown

in Fig. S1, Fig. 2, Table 1 and Table 2. The spectra of the compounds feature an

absorption band in 280-290 nm and a moderately intense absorption in the range of

320-330 nm in all solvents. For L, from the Fig. 2 (a), the high energy band at about

290 nm was assigned to the π benzene→π* benzene transition. The low energy band

about 320 nm with stronger intensity was attributed to the π→π* transition of the

whole ligand molecule mixed with the ICT transition due to the H-1→L+1, which

was further confirmed by TD-DFT. The result of TD-DFT that Bu₃SnOL was similar

to L, indicating that the tributyltin does not participate in the process of charge

transfer, which was consistent with our previous work [31]. While for (BuSnO)₆L₆,

as shown in Fig 2 (b) the first arrangement, the highest occupied molecular orbital

(HOMO) is mainly localized on one ligand and the lowest unoccupied molecular

orbital (LUMO) is mainly localized on another ligand and the centre of framework,

which was attributed to the ligand-ligand charge transfer transition (LLCT). The

second arrangement, the HOMO is mainly localized on the centre of framework and

the LUMO is located on the round of framework and part of ligand, which indicated

that the HOMO → LUMO transition can be assigned as the π→π* transition of the

whole complex molecule mixed with the n(O)→π*(COO) transition.

In addition, the absorption deviation in solution can be attributed to the interaction

of the solute with the local molecular environment including solvent molecules and

other surrounding solute molecules. One can see that the band position and width are

slightly different in different solutions, which further identified the effect of solvent

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on absorption behavior. Some interesting results can be observed from Fig. S1, the

UV-vis absorption spectra of (BuSnO)₆L₆exhibited weak blue-shift in DMSO:H₂O

(1:1) by comparison with other organic solvents, which may result from the fact that

the molecule possessing n→π* transition could express blue-shift in large polarity

solvent and the weak negative solvatochromism because of an opposite polar form in

the ground and excited states [41].

Table 1. The photophysical data of all compounds in different solvents.

Fig.2 Molecular orbitals of the compounds.

Table 2. Selected experimental and calculated optical data for all compounds(H: HOMO, L:

LUMO)

One-photon excited fluorescence (1PEF).

From Fig. S2 and Table S4, the emission wavelengths of complexes ((BuSnO)₆L₆

and Bu₃SnOL) show a weak red-shift compared with ligand L, which results in the

Stokes shift (Table S4) of complexes ((BuSnO)₆L₆and Bu₃SnOL) are bigger than L.

Meanwhile, the quantum yield of (BuSnO)₆L₆are bigger than that of L and

Bu₃SnOL. While as shown in Fig. S3 (a), the 1PEF intensity of complex

(BuSnO)₆L₆is stronger than that of its free ligand L and complex Bu₃SnOL. Above

experimental results suggesting that the increase of the numbers of ligand of drum

structure ((BuSnO)₆L₆) extended the π-conjugated system and is more conducive of

electron distribution in the complex molecules, which enhanced the efficiency of the

linear optical effect in unit volume [42]. Besides, from Fig. S3 (b), the fluorescence

lifetime of complex (BuSnO)₆L₆(0.51 ns) is longer than ligand L (< 0.1 ns) and

complex Bu₃SnOL (< 0.1 ns). The short fluorescence lifetime in these compounds is

in accordance with rapid intersystem crossing due to the strong spin−orbital coupling

[43].

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Furthermore, 1PEF spectra of (BuSnO)₆L₆and Bu₃SnOL as shown in Fig. S2,

displayed regular red-shift in DMSO:H₂O (1:1), indicating that this charge separation

increased in the excited state, resulting in a larger dipole moment than that in the

ground state, which explains the sensitivity of the emission spectra of these

compounds to solvent polarity [44]. Generally, most luminescent materials exhibited

low fluorescence intensity in protic solvents [45] while (BuSnO)₆L₆showed

relatively strong fluorescence in absolute DMSO, due to the process that the protic

solvent might stabilize the excited state of (BuSnO)₆L₆[46, 47].

Two-photon excited fluorescence (2PEF) and Two-photon absorption (2PA)

cross-sections.

The 2PEF spectra of L, Bu₃SnOL and (BuSnO)₆L₆in DMSO were obtained from

680 to 900 nm under 500 mW. As displayed in Fig. 3, only (BuSnO)₆L₆has property

of 2PEF, and the emission intensity of them was investigated at 680 nm. Upon the

input laser power increasing, the logarithmic plots have a slope of 1.99, indicating a

two-photon excitation mechanism (Fig. S4). From Fig. S3 (a) and Fig. 3, we can

draw the following conclusions. Firstly, 2PEF intensity of (BuSnO)₆L₆was stronger

than the ligand and complex Bu₃SnOL, as well as the single-photon fluorescence

intensity, and the maximum 2PA cross-section (σ_max) of (BuSnO)₆L₆is 426 GM. The

large 2PA cross-section values in DMSO solution of (BuSnO)₆L₆on account of the

drum structure of organo tin (ΙV) complex enhanced the efficiency of the nonlinear

optical effect of unit volume [42]. Secondly, the fluorescence peak wavelengths of

(BuSnO)₆L₆is evidently red-shifted by 170 nm in comparison with those of 1PEF in

DMSO, which can be explained by the reabsorption effect [46]. This interesting

consequence could be attributed to the novel structure of drum shape complexs, which

is instructive to our work in the future.

Fig. 3 (a) 2PEF spectra of the compounds in DMSO (c = 0.2 mM) at 680 nm. (b) 2PA

across section of (BuSnO)₆L₆in 680-900 nm regions in DMSO (c = 0.2 mM).

3.3. Cytotoxicity and Biological Imaging.

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The antitumor activities of L and complexes (((BuSnO)₆L₆and Bu₃SnOL) were

evaluated against three tumor cell lines including HeLa (human cervicalcarcinoma

cell line), HepG2 (human hepatocellular livercarcinoma cell line), A549 (human

pulmonary adenocarcinoma cell) and one normal cell HELF (human embryoniclung

fibroblast). Cisplatin is a clinical anticancer drug, which was used as positive control.

From Table 3, complexes (BuSnO)₆L₆and Bu₃SnOL exhibited higher cytotoxicity

against these three tumor cell lines than cisplatin in vitro. Furthermore, the result

indicated that the complex (BuSnO)₆L₆revealed higher cytotoxicity and more

appropriate selectivity in the selected tumor cell lines than that in normal cells, while

cisplatin displays similar cytotoxicity in tumor cells as well as normal cells.

Table 3. Inhibitory Concentration IC₅₀(µM) of Complexes (BuSnO)₆L₆, Bu₃SnOL

and Cisplatin against HepG2, HeLa, A549 Cell Lines and HELF.

In consideration of the fact that (BuSnO)₆L₆has a lager 2PA cross-section, we

further explored the potential applications of (BuSnO)₆L₆in imaging-guided

anticancer activity. The hydrolytic stability of complex (BuSnO)₆L₆in aqueous

1

conditions as well as at 37 °C was initially evaluated via H-NMR in CDCl₃. From

Fig. S5, it can be seen that (BuSnO)₆L₆exhibited good stability compared with the

control after 24 hrs incubation at 37 °C.

To evaluate the internalization of (BuSnO)₆L₆in living cells, as shown in Fig. S6,

1PM and 2PM were obtained from HeLa cells incubated with (BuSnO)₆L₆. Within

the short incubation time (30 min), (BuSnO)₆L₆indicated effective uptake toward

living cells and the bright field micrographs showed a good cell shape after cellular

internalization, which highly implies the excellent biocompatibility. In addition,

photo-stability is one of the most important criteria for developing fluorescence

imaging agents. The photo-stability of (BuSnO)₆L₆was examined in living HeLa

cells via photo-bleaching experiments. As shown in Fig. S7, no significant change in

the fluorescence intensity of (BuSnO)₆L₆could be observed after 180 s. The high

photo-stability of (BuSnO)₆L₆makes them favorable for long-term real-time tracking.

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To further demonstrate the specific localization of complex (BuSnO)₆L₆in living

cells, we performed co-localization experiments. Mito-Tracker Deep Red FM (MTR)

and ER-Tracker red dye (ER) was used to co-stain HeLa cells. As shown in Fig. S8,

the fluorescence of (BuSnO)₆L₆overlapped with that of Mito-tracker and ER-Tracker

with a Pearson’s coefficient Rr = 0.48 and 0.65, respectively. It was further verified

that (BuSnO)₆L₆stained cytoplasm within the cell, and could be internalized with

membrane-rich subcellular compartments including mitochondria and endoplasmic

reticulum in an effective manner.

Cellular uptake

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

organotin complexes are critical to its application [48]. The cellular uptake

mechanisms of (BuSnO)₆L₆were also investigated. HeLa cells incubated with

o

(BuSnO)₆L₆at 4 C. Fig. S9 (2) showed that no luminescence, indicating that

(BuSnO)₆L₆enters cells through a temperature-dependent pathway. Pretreatment of

HeLa cells with endocytosis inhibitors (colchicine, chlorpromazine, nocodazole,

2-Deoxy-D-glucose, and NH₄Cl) showed no effect on the uptake of (BuSnO)₆L₆(Fig.

S9). The results demonstrated that (BuSnO)₆L₆penetrates into cells via a

non-endocytotic/non-active transport, but an energy dependent pathway[49].

Mitochondrial Dysfunction and Reactive Oxygen Species (ROS) Levels.

Mitochondria as the bioenergetic center of the cell play an essential role in the

intrinsic apoptotic-signalling pathway [50, 51]. To further evaluate mitochondrial

injury after internalization with the complex, the mitochondrial membrane potential

(MMP：A signal that detects damage to the mitochondria) of HeLa cells was probed

using the membrane-permeable indicator JC-1. As shown in Fig. 4a, cells treated with

(BuSnO)₆L₆show that the red/green ratio signals of JC-1 decreased from 8.3 to 1.3

compared with the control, suggesting mitochondrial depolarization by (BuSnO)₆L₆.

Cytochrome c (cyt c) is an integral membrane protein, which normally locates in the

space between the inner and outer mitochondrial membranes to maintain the natural

physiological function. Once the membrane integrity of mitochondria is damaged, the

pro-death factors are released from mitochondria and initiate cell death signalling-cyt

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c [52]. Herein, the results from the distribution tests showed the visible escape of cyt c

from the mitochondria in the (BuSnO)₆L₆-treated group by the translocation of it

(green from antibody of cyt c) from the mitochondria (red fluorescence from

MitoTracker Red). As shown in Fig. 4b, the fluorescence of cyt c overlapped with

that of Mito-tracker with a Pearson’s coefficient Rr = 0.25, compared with the control

with a Pearson’s coefficient Rr = 0.68. Collecting the above indicated that

(BuSnO)₆L₆provoked the efflux of cyt c following Mitochondrial outer membrane

permeabilization (MOMP) definitely build the apoptosome, which inevitably initiates

lethal apoptosis. Consequently, the introduction of (BuSnO)₆L₆lead to the

mitochondrial depolarization which triggered the release of cyt c to the cytoplasm,

which resulted in the activation of apoptosis or necrosis in return.

To understand cell oxidative stress caused by (BuSnO)₆L₆treatment, the HeLa cell

line was stained with dichloro-dihydro fluorescein diacetate (DCFH-DA) [53].

DCFH-DA is a non-fluorescent molecule, whereas upon oxidation by reactive oxygen

species (ROS) it is converted to dichlorofluorescein (DCF) which exhibits strong

green fluorescence upon excitation at 488 nm. As displayed in Fig. 4c, compared to

the control, the fluorescence intensity increased by 2.32-fold after treatment with

complex (BuSnO)₆L₆(10 µM), confirming that ROS play a vital role in organotin

(IV)-induced cell death.

Fig. 4 (a) Confocal microscopy imaging of HeLa cells colabeled with JC-1 dye with

or without (BuSnO)₆L₆(10 µM), respectively. (b) Representative fluorescence

micrographs of the translocation of cytochrome c from the mitochondria into cytosol

from treatment by (BuSnO)₆L₆(10 µM) and the control cells. (c) Fluorescence

images of ROS level in HeLa cells after 6 h incubation with (BuSnO)₆L₆(10µM),

followed by DCFH-DA staining for 20 min at 37 °C. Scale bar: 20 µm. One-way

ANOVA was used for statistical analysis, p < 0.005, error bars: SEM.

According to the above-mentioned alterations of the mitochondria, the release of

mitochondrial proteins such as cyt c may lead to apoptosis or necrosis [54, 55]. As

shown in Fig. 5a, it is noticeable that partial (BuSnO)₆L₆was relocated from cell

cytosol to nucleoli during apoptosis and the fluorescence intensity of cell nucleus

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signed by red rings increased ~ 60 fold from 1 h to 6 hrs. Confocal microscopy of

HeLa cells incubated with (BuSnO)₆L₆and propidium iodide (PI) was used to

monitor HeLa cell death. From Fig. 5b, it can be seen that upon the incubation for 6

hrs PI with a red fluorescence signal and (BuSnO)₆L₆with a green fluorescence

gradually entered the cells and stained the nuclei, reflect the plasma membrane and

nuclear membrane permeability increase along the incubation. In addition, the cell

cytoplasm shrinks and membrane bubbling could be found, which was confirmed by

the above experiments.

To the best of our knowledge, during cell apoptosis, cells will show a number of

shape change such as: membrane blebbing, cell shrinkage or rounding, and nuclear

misshape [56,57]. In an attempt to more accurately determine the target of

(BuSnO)₆L₆in the organelles, HeLa cells were incubated with (BuSnO)₆L₆and then

examined by transmission electron microscopy (TEM). HeLa cells stained with

osmium tetroxide (OsO4) were used as control groups to show detailed information of

the intracellular membrane composition including integrity nuclear shape (Fig. 5c). In

contrast, HeLa cells were incubated solely with (BuSnO)₆L₆for 6 hrs, it can be

clearly found that the nuclear region bears a much higher concentration, and nuclear

misshapen was presented. These findings are again in agreement with the two-photon

microscopy imaging, all strongly suggesting that (BuSnO)₆L₆was located on the

position from cell cytosol to nucleoli.

Fig. 5 (a) HeLa cells incubated with (BuSnO)₆L₆(10 µM) after 6 hrs. (b) Confocal

imaging of HeLa cells incubated with (BuSnO)₆L₆(green channel) for different time,

followed by PI stain (red channel) after 8 hrs. Scale bar: 20 µm. (c) TEM images of

HeLa treated with (BuSnO)₆L₆or osmium tetroxide. TEM scale bar = 5 mM.

One-way ANOVA was used for statistical analysis, p < 0.005, error bars: SEM.

To assess the anticancer activity of (BuSnO)₆L₆, we utilized the annexin

V-FITC/PI double staining to quantify the population percentages of apoptotic cells

[58]. From Fig. 6, after treatment with compounds L and (BuSnO)₆L₆(15 µM) for 24

hrs, 31.50 % of early apoptosis cells and 74.05 % of necrotic cells were observed,

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respectively, whereas the viability of the untreated cells remained 90.26 %. complex

(BuSnO)₆L₆was more effective than ligand (L) in inducing PS exposure and the loss

of plasma membrane integrity. The results indicated that complex (BuSnO)₆L₆

induces cell death via apoptotic pathway.

Fig. 6 Annexin V/PI doublestaining analyzed by flow cytometry. HeLa cells were

treated with 15µM L and (BuSnO)₆L₆. One-way ANOVA was used for statistical

analysis, p < 0.005, error bars: SEM.

4. Conclusion

In summary, we have presented two novel organotin (IV) complexes (BuSnO)₆L₆

and Bu₃SnOL, of which (BuSnO)₆L₆possesses excellent optical and biological

properties. (BuSnO)₆L₆exhibited significant 2PA cross-section and substantial

cellular uptake, which favors efficient two-photon imaging and could be applied as

anti-tumor and two-photon real-time imaging agents. Further studies demonstrated

that (BuSnO)₆L₆accumulated in the cytoplasm of HeLa cells and induced apoptosis

via the ROS-mediated mitochondrial dysfunction pathway. Overall, the low cost and

structural diversity of the organotin carboxylate complex with large 2PA cross-section

and antitumor activities provide more hope and opportunity to design and explore the

dual function materials for further biomedical applications.

Acknowledgments

This work was supported by the National Natural Science Foundation of China

(51432001, 21501001, 21602003, 51672002, 51472002). Postdoctoral Science

Foundation of Anhui Province (2016B092), China Postdoctoral Science Foundation

(2017M612050). Anhui Provincial Natural Science Foundation of China

(1708085MC68), and Anhui University Doctor Startup Fund (J01001962), Focus on

returned overseas scholar of Ministry of Education of China, the Higher Education

Revitalization Plan Talent Project (2013).

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Table 1. The photophysical data of all compounds in different solvents.

a

b

c

Comp.

Solvents

Dichloromethane

Tetrshydrofuran

Ethanol

λ_max

logɛ_max

4.34

4.30

4.21

4.27

4.13

4.94

4.89

4.81

4.93

4.90

4.55

4.47

4.45

4.53

4.43

λ_max∆ν(nm)^d

Φ^e

321

319

320

323

320

321

319

317

324

321

319

320

316

320

360

355

353

363

350

380

360

380

410

380

378

370

380

406

39

36

33

40

30

59

61

43

56

89

61

59

50

64

86

0.004

0.003

0.002

0.003

-

L

Dimethyl sulfoxide

DMSO-H₂O

Dichloromethane

Tetrshydrofuran

Ethanol

0.033

0.042

0.025

0.054

0.009

0.005

0.003

0.006

0.008

0.003

(BuSnO)₆L₆

Dimethyl sulfoxide

DMSO-H₂O

Dichloromethane

Tetrshydrofuran

Ethanol

Bu₃SnOL

Dimethyl sulfoxide

DMSO-H₂O

^aPeak position of the longest absorption band in nm. ^bMaximum molar extinction

coefficient. ^cPeak position of SPEF, exited at the maximum wavelength of

absorption. ^dStokes shift in nm. ^eQuantum yield determined by using fluorescein as

standard.

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Table 2. Selected experimental and calculated optical data for all compounds(H: HOMO, L:

LUMO)

Cal. λ_max

(nm)^c

Obs. λ_max

(nm)^d

Characte

r^f

Compd.

OI^a

∆E (eV)^b

f^e

H-1→L+1

H→L

4.18

3.75

3.49

296

320

286

298

318

294

0.1322

0.1571

0.2144

π—π*

L

ICT

H-1→L+1

LLCT

π—π*

(BuSnO)₆L₆

Bu₃SnOL

H→L

3.14

320

324

0.0591

n(O)→π*

(COO)

H-1→L+1

H→L

4.32

294

324

290

320

0.0534

0.368

ICT

3.69

π—π*

a

b

Orbitals involved in the excitation.

Excitation energy (eV).

c

d

e

Calculated peak position of the longest absorption band.

Observed peak position of the longest absorption band in THF.

Oscillator strength. ^fFc instead of Ferrocene; tpa instead of triphenylamine.

Table 3. Inhibitory Concentration IC₅₀(µM) of Complexes (BuSnO)₆L₆, Bu₃SnOL

and Cisplatin against HepG2, HeLa, A549 Cell Lines and HELF.

^aCells were incubated with (BuSnO)₆L₆, Bu₃SnOL and Cisplatin for 24 hrs. Data

were exhibited as the means ± standard deviations (SD).

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O

OH

L

Sn

O Sn

O

Sn

HO

O

Sn

O

Sn

O

Sn

O

Sn

O

Sn

O

Bu₃SnOL

O

(BuSnO)₆L₆

Scheme. 1 The synthesis routes of the compounds.

Scheme. 2 Schematic depicting the possible mechanism of (BuSnO)₆L₆-induced

apoptotic pathway in HeLa cells. (BuSnO)₆L₆induces a boost in reactive oxygen

species and evokes mitochondrial dysfunction, which then increases cytochrome c

release, sequentially, initiating process of apoptosis.

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Fig. 1 (a) The single molecular structure of L. (b) The single molecular structure of

Bu₃SnOL. (c) The optimize structure of (BuSnO)₆L₆. All H atoms have been omitted

for clarity.

Fig. 2 Molecular orbitals of the compounds.

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(a)

(b)