Studies of anticancer activity in vitro and in vivo of iridium(III) polypyridyl complexes-loaded liposomes as drug delivery system.

Accepted Manuscript Studies of anticancer activity in vitro and in vivo of iridium(III) polypyridyl complexesloaded liposomes as drug delivery system Wen-Yao Zhang, Fan Du, Miao He, Lan Bai, Yi-Ying Gu, Lin-Lin Yang, Yun-Jun Liu PII: S0223-5234(19)30525-2 DOI: https://doi.org/10.1016/j.ejmech.2019.06.009 Reference: EJMECH 11412 To appear in: European Journal of Medicinal Chemistry Received Date: 3 June 2019 Accepted Date: 3 June 2019 Please cite this article as: W.-Y. Zhang, F. Du, M. He, L. Bai, Y.-Y. Gu, L.-L. Yang, Y.-J. Liu, Studies of anticancer activity in vitro and in vivo of iridium(III) polypyridyl complexes-loaded liposomes as drug delivery system, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/ j.ejmech.2019.06.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT Graphic abstract Two iridium(III) complexes [Ir(ppy)2(HPIP)](PF6) (Ir-1) and [Ir(ppy)2(BHPIP)](PF6) (Ir-2) were synthesized and characterized. The complexes Ir-1 and Ir-2 were RI PT encapsulated in liposomes Ir-1-Lipo and Ir-2-Lipo. The anticancer activity of the complexes and liposomes was investigated by apoptosis, comet assay, ROS, AC C EP TE D M AN U analysis and in vivo antitumor activity. SC mitochondrial membrane potential, release of cytochrome c, tubules and western blot ACCEPTED MANUSCRIPT Submitted to Eur J Med Chem. Studies of anticancer activity in vitro and in vivo of iridium(III) RI PT polypyridyl complexes-loaded liposomes as drug delivery system Wen-Yao Zhanga, Fan Dua, Miao Hea, Lan Baia, Yi-Ying Gua, Lin-Lin Yangb,*, M AN U a SC Yun-Jun Liua,c* School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, P.R. China b Department of Pediatrics, Guangdong Women and Children Hospital, Guangzhou c TE D 510000, P.R. China Guangdong Engineering Research Center for lead compounds & Drug Discovery, AC C EP Guangzhou, 510006, P.R. China *Corresponding author. E-mail address: fy_yanglinlin@126.com (L.L. Yang); 1 ACCEPTED MANUSCRIPT lyjche@gdpu.edu.cn (Y.J. Liu). Abstract: Two iridium(III) polypyridyl complexes [Ir(ppy)2(HPIP)](PF6) (Ir-1), [Ir(ppy)2(BHPIP)](PF6) (Ir-2) and their liposomes Ir-1-LIpo and Ir-2-Lipo were RI PT synthesized and characterized by elemental analysis, IR, 1H NMR and 13C NMR. The anticancer activity in vitro and in vivo was evaluated. The cytotoxic activity in vitro of the complexes and their liposomes Ir-1-Lipo and Ir-2-Lipo against cancer cells SC was investigated by MTT methods. Ir-1 and Ir-2 show no cytotoxic activity, while M AN U Ir-1-Lipo and Ir-2-Lipo exhibit high cytotoxic effect. The IC50 values range from 5.2 ± 0.8 to 22.3 ± 1.8 µM. The apoptosis, reactive oxygen species, the change of mitochondrial membrane potential, intracellular Ca2+ levels and a release of cytochrome c were investigated. The effect of Ir-1-Lipo and Ir-2-Lipo on tubules TE D was also explored. In the C57BL/6 mice model, Ir-1 only displays a tumor inhibitory rate of 23.21%, while lr-1-Lipo exhibits satisfactory in vivo antitumor efficacy with tumor inhibitory rate of 72.55%. This study demonstrates that complexes EP encapsulated in liposomes induce apoptosis in B16 through ROS-mediated AC C lysosomal-mitochondria dysfunction, inhibition of polymerization of microtubules and induce cell cycle arrest at S phase. Keywords: Iridium(III) polypyridyl complexes; Liposomes; Cytotoxicity in vitro and in vivo assays; Microtubules; ROS. 1. Introduction 2 ACCEPTED MANUSCRIPT Cancer remains one of the most challenging and dangerous health burden for human beings worldwide. Although, in the past decades, great efforts have been attempted to overcome cancer, it is still challenging to defeat the disease [1]. The RI PT main cause of cancer-related death is not the tumor itself but the metastasis from the tumor [2]. Chemotherapy is the commonly applied approach for cancer management, but the toxic and side effects make chemotherapy become troublesome [3]. With the SC successful clinical application of cisplatin, the therapeutic value of metal-based drugs M AN U has long been established [4]. However, this platinum-based drug has several adverse side effects, such as renal toxicity, myelosuppression, nausea, vomiting, and cytotoxicity, which frequently limit its use in the clinic [5,6]. In order to overcome these limitations, more attention has been paid to find anti-cancer activities in other TE D metal complexes as alternatives of cisplatin. Among metal complexes, organometallic iridium complexes have recently emerged as promising alternatives and are expected to overcome the limitations of platinum-based drugs [7-9]. It has been reported that EP iridium(III) complexes generate reactive oxygen species to induce apoptosis by acting AC C on mitochondria and exert anticancer effects through interaction with DNA [10-12]. In order to overcome the disadvantages of metal complexes, there is an urgent need to engineer multifunctional transfer systems to improve the biochemical characteristics of iridium(III) complexes. In the past decades, nanocarrier-based drug delivery systems (DDS) have gained increasing attention. It has been widely accepted that nanoparticles with a diameter of 20 to 200 nm can passively target solid tumor tissues [13,14]. Considering the passive targeting ability through enhancing 3 ACCEPTED MANUSCRIPT permeability and retention (EPR) effects, the metal complexes tend to be loaded into nanoparticles [15], micelles [16-18], liposomes [19], and sorts of nanodispersions. And it is very important to choose the right materials or the nanocarriers. Among RI PT various carriers, liposomes with similar properties to biofilms represent the most acceptable form of parenteral delivery system due to their high biocompatibility and safety [20]. Surface conjugation by polyethylene (glycol) (PEG) can improve the SC liposome's systemic circulation [21,22]. And the nano-sized and hydrophilic layer of M AN U PEG will prolong liposome blood circulation. As far as we know, the studies of anticancer activity of iridium(III) complexes encapsulated in liposomes has been paid less attention so far. To obtain more insight of iridium (III) complexes and their liposomes on anticancer complexes TE D activity and mechanism, in this report, we synthesized two new iridium(III) [Ir(ppy)2(HPIP)](PF6) (ppy = 2-phenylpyridine, 2-(4-hydroxy)phenylimidazo[4,5-f][1,10]phenanthroline, (BHPIP EP [Ir(ppy)2(BHPIP)](PF6) Ir-1) HPIP = and = AC C 2-(3-bromo-4-hydroxy)phenylimidazo[4,5-f][1,10]phenanthroline, Ir-2, Scheme 1) and characterized by elemental analysis, IR, ESI-MS, 1H NMR and 13C NMR. To improve the pharmacokinetics and increase the anticancer effect of complexes, Ir-1 and Ir-2-loaded PEGylated liposomes (Ir-1-Lipo, Ir-2-Lipo) were prepared based on reverse-phase-evaporation method. The liposomes Ir-1-Lipo and Ir-2-Lipo were characterized by particle size, drug entrapment and morphology and release in vitro. The cellular trafficking mechanism, cytotoxicity ， reactive oxygen species and 4 ACCEPTED MANUSCRIPT apoptotic effect of Ir-1-Lipo, Ir-2-Lipo on B16 cells were investigated in detail. Finally, the antitumor efficacy in vivo of Ir-1 and Ir-1-Lipo was evaluated in B16 RI PT tumor-bearing mice. 2. Results and discussion and Ir-2-Lipo ligands HPIP and BHPIP were obtained by reacting M AN U The SC 2.1. Synthesis and characterization of complexes Ir-1, Ir-2 and liposomes Ir-1-Lipo 1,10-phenanthroline-5,6-dione and ammonium acetate with 4-hydroxybenzaldehyde or 3-bromo-4-hydroxybenzaldehyde in glacial acetic acid solvent. The complexes [Ir(ppy)2(HPIP)](PF6) (Ir-1) and [Ir(ppy)2(BHPIP)](PF6) (Ir-2) were synthesized by TE D the direct reaction of HPIP or BHPIP and precursor complex cis-[Ir(ppy)2Cl]2 in a mixture of dichloromethane and methanol. The synthesized complexes Ir-1 and Ir-2 were characterized by elemental analysis, ESI-MS, IR, 1H NMR and 13C NMR. The EP liposomes were prepared by reverse-phase-evaporation method. The mole ratio of AC C phospholipid/cholesterol and drug/lipid at 3:1 and 1:30 was used to produce stable liposomes and achieve high drug loading. The average particle size of Ir-1-Lipo and Ir-2-Lipo is 123.6 nm and 113.5 nm (Fig. S1, supporting information). The ζ potentials of Ir-1-Lipo and Ir-2-Lipo are −35.60 ± 1.26 mV and −13.23 ± 1.94 mV, respectively. When the absolute value of zeta potential exceeded 30 mV, the liposomes were regarded as highly stable, while the zeta potential in the range of 10-20 mV indicated the liposomes were relatively stable [23]. These data indicate that 5 ACCEPTED MANUSCRIPT the liposomes are stable. The morphology of the particle was observed via TEM imaging. The particles are perfectly spherical in shape and distributed fairly uniform in the copper grid (Fig. S2, supporting information), which also shows that liposomes RI PT have a stable morphology. Therefore, all these results identified that the Ir-1 and Ir-2 prodrug can be encapsulated in stable and well-defined liposomes. The UV-Vis spectra of 10 µM of Ir-1, Ir-2, Ir-1-Lipo and Ir-2-Lipo in PBS SC solution are shown in Fig. S3 (supporting information). The maximum absorbance of M AN U Ir-1, Ir-2, Ir-1-Lipo and Ir-2-Lipo appears at 208, 204, 202 and 205 nm, respectively. The complexes (1.1 µM) and their liposomes (1.1 µM) can emit luminescence in PBS solution at ambient temperature (Fig. S4, supporting information), with a maximum appearing at 499, 500, 500 and 499 nm for Ir-1, Ir-2, Ir-1-Lipo and Ir-2-Lipo, TE D respectively. 2.2. In vitro drug release studies EP The in vitro drug release was performed in phosphate buffered saline (pH 7.4) AC C containing 1% SDS (w/v) by dialysis method. The release profiles of Ir-1 from Ir-1-Lipo and Ir-2 from Ir-2-Lipo are shown in Fig. 1. The results clearly revealed that the encapsulated Ir-1 and Ir-2 released in a sustained manner without any sign of burst release. Lack of any burst release indicates that all of the drug encapsulated in the lipid bilayer and not present in the outer surface. Fig. 1 shows that Ir-1 released in a fast rate compared to Ir-2. The accumulative release of Ir-1-Lipo was up to 18.8% within 48 h, while Ir-2 released from Ir-2-Lipo was merely 13.8%. A controlled 6 ACCEPTED MANUSCRIPT release of drug from the nanocarrier system might be beneficial for the cancer treatment as it will provide a constant exposure of the anticancer drug to the cancer cells. In addition, the encapsulation efficiency (EE(%)) is 83.0% for Ir-1-Lipo and RI PT 70.0% for Ir-2-Lipo, respectively. 2.3. Cytotoxicity in vitro assays SC The in vitro antiproliferative activities of Ir-1, Ir-2, Ir-1-Lipo and Ir-2-Lipo M AN U toward MCF-7, HeLa, B16, SGC-7901, A549, BEL-7402 and normal LO2 cell lines were investigated after 48 h exposure period using the MTT assays. The anti-tumor proliferative effects induced by the two complexes with subtle changes in chemical structure are also surprisingly similar. As shown in Table 1, the growth inhibition TE D concentration (IC50) values of 50% of the tumor cells obtained by Ir-1 and Ir-2 were both greater than 200 µM, and thus can be considered as inactive. However, it is surprising when complexes are encapsulated in liposomes, the anti-tumor activity is EP significantly improved. The IC50 values of Ir-1-Lipo and Ir-2-Lipo toward HeLa and AC C B16 cells are 9.5 ± 0.9, 5.2 ± 0.8 µM and 6.2 ± 0.4 and 10.8 ± 1.5 µM, respectively. The cell viability of Ir-1 (A, blue), Ir-2 (B, blue), Ir-1-Lipo (A, red) and Ir-2-Lipo (B, red) against B16 cells is depicted in Fig. S5 (supporting information), obviously, all the liposomes exhibited a dose-dependent manner to inhibit the cell growth in B16. In summary, the ability of anti-tumor cell proliferation in vitro showed the following trends: Ir-1-Lipo > Ir-1-Lipo > Ir-1 ≈ Ir-2. The liposomes Ir-1-Lipo and Ir-2-Lipo exhibit higher anticancer activity than complexes Ir-1 and Ir-2, which may be caused 7 ACCEPTED MANUSCRIPT When complexes are encapsulated in liposomes to form a composite, they will be easier to enter into the cell to exert efficacy. RI PT 2.4. Location at the lysosomes and lysosomal permeabilization Acidic organelle lysosomes are involved in a variety of physiological processes in cells, including protein breakdown, autophagy, induction of apoptosis, release of SC hydrolases and degradation of related receptors. It is worth noting that lysosomes in M AN U cancer cells are accompanied by an increase in the number and volume while the stability is decreasing. [24-26]. To study whether the complexes encapsulated liposomes target lysosomes, B16 cells were treated with 5.0 and 10.0 µM of Ir-1-Lipo and Ir-2-Lipo for 0.5 h, the cells were stained with LysoTracker red. It can TE D be seen from Fig. 2 that the green fluorescence emitted by the liposomes Ir-1-Lipo and Ir-2-Lipo after entering the cell overlaps with the red fluorescence of the lysosome stained with LytoTracker red, and there is a perfect merge. This indicates EP that the liposomes Ir-1-Lipo and Ir-2-Lipo interact on the lysosomes of the cells. AC C Lysosome as a key target for anti-tumor therapy, the non-negligible factor is that lysosomal membrane permeabilization leads to protease infiltration into the cytoplasm triggering apoptosis pathway. The lysosomal metachromatic fluorescent dye AO, which represents a protonated oligomer (AOH+) when the lysosomal membrane is stable, emits red fluorescence. However, when lysosomal membrane permeabilization occurs, the dye AO emits green fluorescence while represents in a deprotonated form [27]. As shown in Fig. S6 (supporting information), a number of obvious red 8 ACCEPTED MANUSCRIPT fluorescent points were observed in the control (a). However, after B16 cells were incubated with 5.0 and 10.0 µM of Ir-1-Lipo (b) and Ir-2-Lipo (c) for 24 h, the red fluorescence decreased and the green fluorescence increased, indicating that RI PT Ir-1-Lipo and Ir-2-Lipo can increase lysosomal membrane permeabilization. 2.5. Location of the liposomes at mitochondria and change of mitochondrial SC membrane potential cause mitochondrial M AN U The cathepsin released after permeabilization of the lysosomal membrane can membrane permeation, which triggers the lysosomal-mitochondrial apoptosis pathway [28-30]. Mitochondria, the main energy supply site of cells, not only regulate cell growth and cell cycle, but also play an TE D important role in cell signaling and apoptosis [31]. Increased mitochondrial membrane permeability will promote Ca2+ loaded in mitochondria, promote the release of a series of apoptotic factors such as cytochrome c, and induce apoptosis [32]. After B16 EP cells were treated with 5.0 and 10.0 µM of Ir-1-Lipo and Ir-2-Lipo for 1 h, the cells AC C were stained with the dye MitoTracker Red to assess whether the liposomes target to the mitochondria. The red fluorescence emitted by the mitochondria stained by MitoTracker Red and the green fluorescence emitted by the liposomes Ir-1-Lipo and Ir-2-Lipo are clearly seen from Fig. 3. Interestingly, the two-color fluorescence can be perfectly superimposed, indicating that the liposomes Ir-1-Lipo and Ir-2-Lipo are targeted to the mitochondria. Mitochondrial membrane potential is formed during mitochondrial respiratory 9 ACCEPTED MANUSCRIPT oxidation and is closely related to the cellular mitochondrial apoptosis pathway. Once the mitochondrial membrane potential is reduced, the cells will enter an irreversible process of apoptosis. Therefore, the mitochondrial membrane potential was RI PT investigated by using the fluorescent probe JC-1 on BI6 cells treated with the liposomes Ir-1-Lipo and Ir-2-Lipo. It is well known that JC-1 is a lipophilic cationic dye that selectively translocates to mitochondria and undergoes color changes with SC the changes in mitochondrial membrane potential (∆Ψm) [33]. In normal cells with a M AN U higher ∆Ψm, JC-1 exists in the mitochondrial matrix in the form of a polymer (J-aggregates) and emits red fluorescence, and when it is in apoptotic cells, it exists in a monomeric form to emit green fluorescence. As shown in Fig. S7 (supporting information), the red fluorescence in most B16 cells (a) is converted to green TE D fluorescence after 24 h of treatment with Ir-1-Lipo (b, 5 µM) and Ir-2-Lipo (c, 10 µM), indicating the loss of MMP. The depolarization ratio of mitochondria was obtained by analyzing the fluorescence intensity of green/red value of JC-1 by the EP ImageXpress Micro XLS system (Fig. S8, supporting information). The results AC C showed that the liposomes Ir-1-Lipo and Ir-2-Lipo can decrease ∆Ψm and cause mitochondrial dysfunction, which further mediates apoptosis of B16 cells through mitochondrial pathway. 2.6. Intracellular reactive oxygen species (ROS) detection Mitochondria and lysosomes are the main sites of endogenous reactive oxygen species (ROS), in which mitochondria increase ROS levels by altering redox potential. 10 ACCEPTED MANUSCRIPT ROS, a class of single electron reduction products, is considered to be a key factor in apoptosis and inflammatory pathways [34]. The studies have shown that an increase in ROS levels promotes the opening of mitochondrial permeability transition pores RI PT and regulates the expression of some important pro-apoptotic proteins [35]. To gain insight into the anticancer mechanisms of these liposomes, the specific fluorescent probe DCFH-DA was used to investigate the changes in ROS levels in B16 cells. As SC shown in Fig. S9 (supporting information), incubation of B16 cells (a) with Ir-1-Lipo M AN U (b, 5 µM) and Ir-2-Lipo (c, 10 µM) markedly increased intracellular ROS levels as indicated by the increased levels of DCF fluorescence. The fluorescence intensity analysis by the ImageXpress Micro XLS system indicated that the green fluorescence intensity of DCF treated with Ir-1-Lipo and Ir-2-Lipo increased by 10.7 and 10.9 TE D times, respectively, compared with the control group (Fig. S10, supporting information). The results demonstrate that liposomes Ir-1-Lipo and Ir-2-Lipo inhibit EP cancer cell proliferation by significantly increasing the level of endogenous ROS. AC C 2.7. Determination of intracellular Ca2+ levels Ca2+ is essential for normal life activities, especially during cell apoptosis and migration [36]. Subtle changes in Ca2+ distribution in subcellular structures are closely related to mitochondrial function and endogenous ROS levels [37-39]. The level of Ca2+ in mitochondria is significantly increased, which will promote the release of apoptotic factor cytochrome c and induce apoptosis of B16 cells. The results of intracellular Ca2+, as reflected in changes in the fluorescence of the indicator dye 11 ACCEPTED MANUSCRIPT Fluo-3/AM, are shown in Fig. 4A. In the control (a), no obvious green fluorescent points were observed, indicating that the level of intracellular free Ca2+ is very low. However, B16 cells were incubated with Ir-1-Lipo (5.0 µM) and Ir-2-Lipo (10.0 µM) RI PT for 24 h, the green fluorescence intensity increases. To quantitatively analyze the Fluo-3 fluorescence intensity, it was found that the Ca2+ levels in cells treated with Ir-1-Lipo (5.0 µM) and Ir-2-Lipo (10.0 µM) increased by 8.1-fold and 6.0-fold SC compared with the control (Fig. 4B). The data demonstrate that liposomes can levels. 2.8. Detection of cytochrome c level M AN U increase Ca2+ levels through the changes in mitochondrial dysfunction and ROS TE D As a key protein in the mitochondria, cytochrome c has a non-negligible role in redox, energy metabolism, and apoptosis pathways. The changes in the endogenous ROS levels and mitochondrial damage will result in the release of cytochrome c, EP thereby inducing the formation of apoptotic bodies and accelerating the execution of AC C apoptosis [40,41]. As is clear from Fig. 5A, the release of cytochrome c in B16 cells was significantly increased after the treatment of B16 cells with Ir-1-Lipo (5.0 µM) and Ir-2-Lipo (10.0 µM) for 24 h. The green fluorescence intensity was determined, as shown in Fig. 5B, it was found that the release of cytochrome c by liposome showed concentration tolerance, whereas the complexes Ir-1 and Ir-2 showed a slight effect on cytochrome c release. 12 ACCEPTED MANUSCRIPT 2.9. Assay of apoptosis It is well known that the infinite growth of cancer cells is closely related to the inhibition of apoptosis [42]. Therefore, the apoptosis was assayed by flow cytometry. RI PT The results of flow cytometry analysis of apoptosis induced by the complexes and liposomes treatment are shown in Fig. 6. In the control (a), the percentage in the early apoptosis is 0.96%. Simultaneously, it can be seen that after treatment of cells with SC Ir-1 (b, 5.0 µM) and Ir-2 (e, 10.0 µM) for 24 h, the percentage of early apoptosis of M AN U cells was 2.17% and 3.18%, respectively. This means that the complexes Ir-1 and Ir-2 have low effect on the apoptosis of B16 cells. However, B16 cells were exposed to 2.5 (c) and 5.0 µM (d) of Ir-1-Lipo or 5.0 (f) and 10.0 µM (g) of Ir-2-Lipo for 24 h, the percentages in the early apoptosis are 4.79% and 11.30%, 3.10% and 7.97%, TE D respectively. The above results revealed a significant increase in early apoptosis of cells after treatment with liposomes Ir-1-Lipo and Ir-2-Lipo and a concomitant EP concentration tolerance. AC C 2.10. DNA damage studies DNA fragmentation in chromosomes is an important marker of apoptosis, and the degree of damage is explored by single cell gel electrophoresis experiments [43]. As shown in Fig. S11 (supporting information), a red fluorophore having no tailing phenomenon was observed in the control group (a), indicating that it stayed in the nuclear matrix due to the large molecular weight of the undamaged DNA in the cells. Interestingly, when B16 cells were treated with 5.0 µM Ir-1-Lipo (b) and 10.0 µM 13 ACCEPTED MANUSCRIPT Ir-2-Lipo (c) for 24 h, the comet-like fluorophore appeared in a fluorescence microscope. At the same time, when DNA damage in the nucleus is aggravated, it appears that the comet tail will become longer and the fluorescence will increase. The RI PT results show that the Ir-1-Lipo and Ir-2-Lipo can induce DNA fragmentation, providing further evidence of apoptosis. SC 2.11. Cell cycle arrest analysis M AN U In the organism, a series of biological processes such as cell proliferation, growth, and differentiation receive regulation of the cell cycle [44]. The cell cycle-dependent protein kinases and their regulatory factors play an important role in the regulation of the cell cycle. Therefore, targeting the cancer cell proliferation cycle provides a new TE D direction for the development of novel targeted drugs. The results of the periodic flow cytometry in Fig. S12 (supporting information) indicate that after treatment with 5.0 µM of Ir-1-Lipo and 10.0 µM of Ir-2-Lipo treated B16 cells for 24 h, we observed an EP increasing accumulation of cells in S phase and a decrease of cells in G0/G1 and AC C G2/M phase, suggesting that Ir-1-Lipo and Ir-2-Lipo inhibit the cell growth in B16 cells at S phase, whereas Ir-1 and Ir-2 have no obvious effect on the cell cycle distribution. The cyclin-dependent kinase CDK2 is an important factor in ensuring that cells complete the G1 phase and enter the S phase. It is worth noting that overexpression of the protein CDK2 and cyclin A will accelerate the progression of the G1/S phase and induce tumorigenesis [45]. The molecular expression of the protein CDK2/cyclin A after B16 cells were treated with complexes and liposomes for 14 ACCEPTED MANUSCRIPT 24 h is shown in Fig. 7A and 7B. The above results show that Ir-1-Lipo and Ir-2-Lipo can indeed induce S-phase cell cycle arrest. RI PT 2.13. In vitro inhibition of cell invasion Metastasis of tumor cells is one of the main causes of cancer patients' difficulty in eradicating and dying. The destruction of cell cycle regulation mechanism is the SC root cause of cancer cell proliferation and migration. After demonstrating that M AN U liposomes can block cells in the S phase, the effect of liposomes on inhibiting cancer cell invasion is further explored. The invasion and migration ability of B16 cells after being treated by the complexes and liposomes was investigated by transwell chambers. The results of invasion inhibition are shown in Fig. S13 (supporting information), We TE D can clearly observe that these liposomes have an excellent inhibitory effect on B16 cell invasion, and the inhibitory effect is enhanced with increasing concentration. In addition, B16 cells treated with liposome 2.5 (b) and 5.0 µM (c) Ir-1-Lipo and 5.0 (d) EP and 10.0 µM (e) Ir-2-Lipo were quantified to inhibit the number of invading cells for AC C 24 h. It was found that the order of invasive inhibition was Ir-1-Lipo > Ir-2-Lipo >> Ir-2 > Ir-1 as shown in Fig. S14 (supporting information). Therefore, all the above results demonstrate that the liposomes Ir-1-Lipo and Ir-2-Lipo exhibit outstanding invasion inhibition ability to B16 cells. 2.14. Effects on the microtubule networks Rearrangement of intracellular cytoskeletal proteins is a critical step leading to 15 ACCEPTED MANUSCRIPT tumor cell migration and invasion. Silk pseudopodia is not only the main structure of cytoskeleton formation, but also plays an important role in the initial stage of cancer cell invasion [46,47]. The cytoskeleton is a protein fiber network structure in RI PT eukaryotic cells, which can induce apoptosis when its morphology changes. To further understand whether liposomes inhibit the metastatic mechanism of cancer cells by targeting microtubules, microtubule morphology was studied by immunofluorescence SC staining. As shown in Fig. 8, in the control (a) and B16 cells were treated with 2.5 µM M AN U of Ir-1-Lipo (b) and 5.0 µM of Ir-2-Lipo (e), B16 cells were spread out with a well-organized microtubule network. However, after B16 cells were exposed to 5.0 µM of Ir-1-Lipo (c) and 10.0 µM of Ir-2-Lipo (f) for 24 h, it can be seen that the cells contract from the original shuttle to a round, polygonal, or irregular shape. The TE D results indicated that the liposomes Ir-1-Lipo and Ir-2-Lipo triggered cell morphology collapse by inhibiting microtubule polymerization. EP 2.15. The mechanism studies of apoptosis AC C The mitochondrial apoptotic pathway is central to apoptosis during apoptosis [48], which is mainly regulated by proapoptotic proteins (Bax) and anti-apoptotic proteins (Bcl-2) in Bcl-2 family proteins [49,50]. Bcl-2 family proteins can induce mitochondrial membrane permeability and promote the release of apoptotic factor cytochrome c, which in turn leads to activation of caspase cascade and induction of apoptosis [51,52]. Therefore, the expression of apoptosis-related protein levels caused by liposome Ir-1-Lipo and Ir-2-Lipo was analyzed by Western blotting. After treated 16 ACCEPTED MANUSCRIPT with Ir-1-lipo and Ir-2-lipo for 24 h, the liposome could significantly increase the expression of protein Bax and strongly inhibit the expression of protein Bcl-2 as shown in Fig. 9A and 9B. The multiple apoptotic pathways of cells are closely related RI PT to the apoptosis-executing protein caspase-3 [53,54]. Therefore, the expression levels of protein caspase-3 and PARP in the mitochondrial apoptotic pathway are evaluated (Fig. 9A and 9B), and their expression levels are significantly up-regulated after being SC subjected to liposomes Ir-1-Lipo and Ir-2-Lipo. The results indicate that M AN U liposome-induced apoptosis of B16 cells could be carried out by the mitochondrial pathway. 2.16. In vivo antitumor activity of Ir-1 and Ir-1-Lipo TE D In order to further assess the anticancer effect of Ir-1 and Ir-1-Lipo in vivo, antitumor efficacy study was performed in B16 cancer cell bearing tumor xenograft model. Mice were randomly divided into control and experimental groups. The mice EP were intraperitoneal injected different doses of Ir-1-Lipo (4.8 and 9.6 mg/kg) and AC C Ir-1 (6.0 mg/kg) every day for 7 days. As shown in Fig. 10, at the end of the treatment period, in the 4.8 mg/kg and 9.6 mg/kg of Ir-1-Lipo group, tumor volume was decreased 18.86% and 38.87% as compared with Ir-1-treated mice. Inhibition of high dose Ir-1-Lipo on tumor growth is more potent than those of low dose and Ir-1-treated group. Meanwhile, the tumor weight was also measured at the end of experiment. The tumor weight of Ir-1-Lipo (4.8 and 9.6 mg/kg) and Ir-1-treated group was 3.58 g, 1.88 g and 5.26 g, respectively. Inhibitory rate of tumor growth 17 ACCEPTED MANUSCRIPT induced by Ir-1-Lipo (4.8 and 9.6 mg/kg) treated group and Ir-1-treated group was 47.75%, 72.55% and 23.21%, respectively. This shows that the antitumor effect of Ir-1-Lipo is obviously concentration-dependent, and Ir-1 was encapsulated in RI PT liposomes, the anticancer activity in vivo is enhanced. 3. Conclusions SC In this study, we synthesized and characterized two new Ir-1 and Ir-2 complexes. M AN U The complexes show almost no anticancer activity against HeLa, A549, B16, MCF-7, SGC-7901, BEL-7402 and LO2 cells. Therefore, we have designed liposomes as biocompatible nanocarrier to deliver metal complexes. The particles exhibited a controlled release of drug in vitro. After encapsulated in liposomes, Ir-1 and Ir-2 TE D showed high anticancer activity against B16 cells. Ir-1-Lipo and Ir-2-Lipo can cause apoptosis by increasing the level of intracellular reactive oxygen species and reducing mitochondrial membrane potential and further inducing mitochondrial dysfunction. EP On the other hand, Ir-1-Lipo and Ir-2-Lipo can target lysosomes and increase AC C lysosomal permeabilization, target microtubules and inhibit the polymerization of microtubules. Additionally, Ir-1-Lipo and Ir-2-Lipo can cause DNA damage and inhibit the cell growth at S phase. In summary, Ir-1-Lipo and Ir-2-Lipo cause apoptosis through two major pathways (Fig. 11): (I) DNA damage and inhibiting polymerization of microtubules → cell cycle arrest → apoptosis; (II) Increase of intracellular ROS → lysosomal-mitochondrial dysfunction → release of cytochrome c and activation of caspase 3 → cleaved PARP → apoptosis. Most importantly, 18 ACCEPTED MANUSCRIPT Ir-1-Lipo exhibited a significantly superior anticancer effect on the tumor growth in vivo. This work is helpful for designing and synthesizing new iridium complexes as potent anticancer agents. In addition, the liposomal formulation can provide a RI PT promising platform for the delivery of metal-complex into tumor by enhancing therapeutic efficacy. SC 4. Experimental Section The purchased synthetic M AN U 4.1. Materials and methods raw materials (4-hydroxybenzaldehyde, 3-bromo-4-hydroxybenzaldehyde, 2-phenylpyridine, 1,10-phenanthroline) and all reagents were used without further purification unless otherwise specified. Water TE D obtained by purification from the Millipore Milli-Q system was used throughout the experiment. Fluorescent dye kits and related consumables are sourced from Beyotime Biotechnology. FBS and RPMI 1640 were purchased from Gibco company. The EP tumor cell lines SGC-7901, HeLa, BEL-7402, A549, B16, MCF-7 and normal LO2 AC C cells were purchased from the American Type Culture Collection. Prior to each test, all complexes were dissolved in DMSO and the final concentration of DMSO was maintained at 1% (V/V). Elemental analysis of C, H, and N was performed by a PerkinElmer 240Q elemental analyzer. Electrospray ionization mass spectra were recorded by LCQ system (Finnigan MAT, USA) and the major peaks in the isotope distribution were represented by m/z values. 1H NMR and 13C NMR spectra were obtained by analysis on a Varian-500 spectrometer using DMSO-d6 as a solvent at 19 ACCEPTED MANUSCRIPT room temperature. 4.2. Synthesis of complex RI PT 4.2.1. Synthesis of [Ir(ppy)2(HPIP)]PF6 (Ir-1) A mixture of cis-[Ir(ppy)2Cl]2 (0.32 g, 0.3 mmol) [55] and HPIP (0.187 g, 0.6 mmol) [56] in a mixture of 42 mL of dichloromethane and methanol SC (VCH2Cl2:VCH3OH = 2:1) was refluxed under argon for 6 h to give a clear yellow M AN U solution. Upon cooling, a yellow precipitate was obtained by dropwise addition of saturated aqueous NH4PF6 solution with stirring at room temperature for 2 h. The crude product was purified by column chromatography on neutral alumina with a mixture of CH2Cl2/acetone (1:1, v/v) as eluent. The yellow band was collected. The TE D solvent was removed under reduced pressure and a yellow powder was obtained. Yield: 74%. Anal. Calc for C41H28N6OIrPF6: C, 51.40; H, 2.95; N, 8.77. Found: C, 51.63; H, 2.81; N, 8.95. 1HNMR: δ 9.12 (d, 2H, J = 8.5 Hz), 8.25 (d, 2H, J = 8.5 Hz), EP 8.12 (d, 2H, J = 8.5 Hz), 8.07 (d，2H, J = 5.0 Hz), 7.95 (d,4H, J = 5.0 Hz), 7.86 (t, 2H, 13 AC C J = 5.0 Hz), 7.50 (d, 2H, J = 6.5 Hz), 7.06 (t, 2H, J = 7.0 Hz), 7.01 (t, 2H, J = 7.0 Hz). C NMR: 172.64, 167.01, 159.13, 150.78, 149.10, 147.48, 143.50, 138.71, 132.09, 131.31, 130.31, 128.38, 126.52, 125.11, 124.44, 123.89, 122.38, 120.01, 115.81. ESI-MS: m/z = 814 [M + 1]. 4.2.2. Synthesis of complex [Ir(ppy)2(BHIP)]PF6 (Ir-2) A mixture of cis-[Ir(ppy)2Cl]2 (0.32 g, 0.3 mmol) [54] and BHIP (0.23 g, 0.6 20 ACCEPTED MANUSCRIPT mmol) [57] in a mixture of 42 mL of dichloromethane and methanol (VCH2Cl2:VCH3OH = 2:1) was refluxed under argon for 6 h to give a clear yellow solution. Upon cooling, a yellow precipitate was obtained by dropwise addition of RI PT saturated aqueous NH4PF6 solution with stirring at room temperature for 2 h. The crude product was purified by column chromatography on neutral alumina with a mixture of CH2Cl2/acetone (1:1, v/v) as eluent. The yellow band was collected. The SC solvent was removed under reduced pressure and a yellow powder was obtained. M AN U Yield: 72%. Anal. Calcd for C41H27N6OBrIrPF6: C, 47.49; H, 2.63; N, 8.11. Found: C, 47.61; H, 2.80; N, 8.02. 1HNMR: δ 9.11 (d, 2H, J = 7.5 Hz), 8.43 (s, 1H,), 8.25 (d, 2H, J = 8.0 Hz), 8.13 (d, 1H, J = 8.0 Hz), 8.06 (d, 2H, J = 4.5 Hz), 7.95 (t, 4H, J = 7.5 Hz), 7.86 (t, 2H, J = 7.5 Hz), 7.50 (d, 2H, J = 6.0 Hz), 7.06 (t, 4H, J = 7.5 Hz), 6.98 (t, 4H, TE D J = 8.0 Hz), 6.29 (d, 2H, J = 7.0 Hz), 3.55 (s, 1H). 13C NMR: 172.28, 166.99, 155.98, 150.76, 149.08, 147.49, 144.09, 143.56, 138.69, 132.06, 131.30, 130.93, 130.29, 127.34, 126.53, 125.09, 124.57, 123.88, 122.37, 120.00, 116.85, 110.08. ESI-MS: m/z AC C EP = 892 [M + 1]. 4.3. Preparation of the liposomes Ir-1-loaded PEGylated liposomes (Ir-1-Lipo) were prepared based on reverse-phase-evaporation modification. Briefly, method Ir-1: [58] as previously reported PC-98T:CHO-HP (quality ratio with minor 1:30:10) and PC-98T:DSPE-MPEG2000 (quality ratio 1:5%) were prepared and mixtures dissolving in chloroform and water (ratio of 3:1). Subsequently, the mixtures 21 ACCEPTED MANUSCRIPT sonicated to afford a white/brown emulsion-like mixture. The biphasic suspension was then transferred onto a rotary evaporator for gradual removal of the organic phase. Complete removal of organic solvents was achieved and added double distilled water RI PT at pH 7.4 for hydration, resultant crude liposome suspensions were diluted in double water (pH 7.4) distilled to provide required concentration of liposome concentration. Finally, sonication and centrifugation give the desired liposome. The preparation of M AN U SC Ir-2-Lipo is prepared in a manner identical to that described with Ir-1-Lipo. 4.4. Characterization of the liposomes Ir-1-Lipo and Ir-2-Lipo were characterized by particle size, ζ potential and morphology. Particle size and ζ potential of liposomes were determined by Zetasizer TE D Nano ZS (Malvern, UK). Morphology of liposomes was examined by transmission electron microscopy (TEM) (H-7650, Hitachi, Japan). The above samples were prepared by diluting them with appropriate amount of deionized water. TEM AC C EP micrographs were acquired at the acceleration voltage of 100 kV. 4.5. In vitro drug release The in vitro release of Ir-1 and Ir-2 from Ir-1-Lipo and Ir-2-Lipo was studied in phosphate buffered saline (PBS, pH 7.4) containing 1% SDS (w/v) by dialysis method. To conduct the release study, samples (200 µg) were filled in dialysis membrane (MWCO = 8-10 kDa) and the dialysis membrane was clipped and packed in a Falcon tube containing 200 ml of release medium. The tube as placed in shaking water bath 22 ACCEPTED MANUSCRIPT (Gongyi City Yuhua Instrument CO., LTD, China) and incubated at 37 oC (100 rpm). At predetermined intervals, 2 mL of release medium was withdrawn and replenished with 2 mL of fresh medium. The concentration of Ir-1 and Ir-2 in the medium was RI PT measured using UV-visible spectrophotometer. 4.6. Cell cytotoxicity assay SC The cytotoxic activity of the complexes and liposomes against the selected cancer M AN U cell lines was assayed by MTT method [59]. Cancer cells suspension were seeded in 96-well plates (approximately 1 × 104 cells per well) and grown further to appropriate density in a 37 oC, 5% CO2 incubator. All compounds and liposomes to be tested were dissolved in DMSO and the final concentration range after addition to the wells TE D ranged from 1-100 µM. At the same time, an equal volume of pure DMSO solution was added to the corresponding plate well as a control group. When the 96-well plate was incubated for 48 h, the liquid in the wells was removed and replaced with 90 µL EP of medium and 10 µL of MTT dye solution (20 µL, 5mg·mL-1). The dye-filled AC C 96-well plate was placed in a 37 °C, 5% CO2 incubator for 4 h. After the incubation, each well will be added with 100 µL of DMSO solution to dissolve the formazan produced by MTT. The absorbance of each well was measured by a microplate reader at a wavelength of 490 nm. The IC50 value was determined by plotting the logarithm of the concentration versus the percentage of viable cells and the results of the SPSS software analysis. To ensure the accuracy of the data, each set of experiments will be repeated at least three times and the average will be calculated. 23 ACCEPTED MANUSCRIPT 4.7. Apoptosis assay by flow cytometry When B16 cells are tightly attached in a 6-well plate and grown at a higher RI PT density, the medium removed from the wells will be replaced with medium containing Ir-1 or Ir-2 and different concentrations of Ir-1-Lipo or Ir- 2-Lipo incubation for 24 h. After washing the cells in the wells three times with cold phosphate buffer PBS, SC collection was performed simultaneously with trypsin-EDTA. The supernatant was M AN U removed and washed in a desktop low temperature centrifuge, stained in a flow tube with PBS solution containing 500 mg / mL of pyridine pyridinium (PI) and 1 mg / mL annexin V-FITC in a dark low temperature environment. Fluorescence emission of the dye-treated cells was measured at 530 nm by excitation at 488 nm using a FACS TE D Calibur flow cytometer (Beckman Dickinson & Co., Franklin Lakes, NJ). It is worth noting that sufficient number of cells is a guarantee of the accuracy of sample data. specific fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate AC C The EP 4.8. Reactive oxygen species (ROS) detection (DCHF-DA, 10 µM) was used to investigate changes in intracellular ROS levels induced by liposomes. The cells were seeded at a density of 1.5 × 105 per well in a 12-well plate and incubated overnight. The liquid in the wells was removed while replacing the medium containing the corresponding concentrations of these liposomes and incubated for 24 h. After treatment with these liposomes, the cells in the wells were washed three times with PBS and incubated with fluorescent probes. After 24 ACCEPTED MANUSCRIPT incubation for 30 min, the liquid in the wells was removed and the excess dye was washed with PBS solution. The cells in the 12-well plate were imaged by ImageXpress Micro XLS system (MD Company, US), and the DCF fluorescence RI PT intensity was calculated and analyzed. 4.9. Mitochondrial membrane potential assay SC Widely used mitochondrial membrane potential fluorescent probe JC-1 for M AN U membrane potential detection of B16 cells. Cells (about 2 × 106) were seeded in 12-well plates, followed by treatment with corresponding concentrations of liposomes for 24 h. Then remove the medium from the 12 wells, wash the cells several times with PBS, and stain with the fluorescent probe JC-1. After incubation for 20 min at TE D room temperature, PBS washed the excess dye in the wells and image the cells using ImageXpress Micro XLS system (MD Company, US). EP 4.10. Cell cycle arrest studied AC C B16 cell suspension was seeded in a 6-well plate at a density of 5×105 per well and incubated until tightly attached. After the incubation, discard the liquid in the wells and re-add the medium containing the corresponding concentration of liposomes and incubate for 24 h. After the end of the liposome treatment, the cells obtained by trypsin digestion were washed with PBS on a centrifuge. After being fixed overnight with 75% alcohol, 20 µL of RNAse (0.2 mg / mL) and 20 µL of propidium iodide (0.02 mg / mL) were thoroughly mixed with the cells and incubated for 30 min at 25 ACCEPTED MANUSCRIPT room temperature. The number of cells in the sample is also an important factor influencing the data, while the samples were analyzed using a FACS Calibur flow cytometer. Therefore, we always ensure that the number of cells per sample analyzed RI PT is not less than 10,000 [60]. 4.11. Western blot analysis SC When the B16 cells in the 6-well plate were grown in an excellent state, the old M AN U medium was discarded and the medium containing Ir-1, Ir-2 and different concentrations of Ir-1-Lipo or Ir-2-Lipo was separately added. B16 cells after 24 h of incubation were placed in ice cubes, protein suspensions were quickly obtained by cell lysis buffer and then placed in a high speed refrigerated centrifuge for 15 min. TE D After obtaining the supernatant, the protein concentration of each sample obtained by the treatment is determined by the BCA detection method. The obtained protein samples were separately electrophoresed in each lane of sodium dodecyl EP sulfate-polyacrylamide gel by equal volume addition of a microinjector. The isolated AC C gel was transferred to a PVDF membrane and then blocked with TBST buffer containing 5% skim milk powder for 4 h. After washing the skim milk powder with PBST buffer, the PVDF membrane containing the protein molecule was incubated with the specific protein primary antibody at 4 oC overnight. After washing four times with PBST buffer on a shaker, the labeled secondary antibody was incubated with the PVDF membrane bound to the primary antibody for 1 h. Finally, we showed the blot according to the Amersham ECL Plus Western blot detection reagent. To assess the 26 ACCEPTED MANUSCRIPT presence of comparable amount of proteins in each lane, the membranes were stripped finally to detect the β-actin. RI PT 4.12. Comet assay DNA damage was investigated by means of comet assay. B16 cells in culture medium were incubated with 5 µM of the complexes and liposomes at 37 °C for 24 h. SC The cells were harvested by a trypsinization process at 24 h. A total of 100 µL of 0.5% M AN U normal agarose in PBS was dropped gently onto a fully frosted microslide, covered immediately with a coverslip, and then placed at 4 °C for 10 min. The coverslip was removed after the gel has been fixed. 50 µL of the cell suspension (200 cells /µL) was mixed with 50 µL of 1% low melting agarose preserved at 37 °C. A total of 100 µL of TE D this mixture was applied quickly on top of the gel, coated over the microslide, covered immediately with a coverslip, and then placed at 4 °C for 10 min. The coverslip was again removed after the gel has been fixed. A third coating of 50 µL of 0.5% low EP melting agarose was placed on the gel and allowed to place at 4 °C for 15 min. After AC C solidification of the agarose, the coverslips were removed, and the slides were immersed in an ice-cold lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 90 mM sodium sarcosinate, NaOH, pH 10, 1% Triton X-100 and 10% DMSO) and placed in a refrigerator at 4 °C for 2 h. All of the above operations were performed under low lighting conditions to avoid additional DNA damage. After the removal of the lysis solution, the slides were placed horizontally in an electrophoresis chamber. The reservoirs were filled with an electrophoresis buffer (300 mM NaOH, 1.2 mM 27 ACCEPTED MANUSCRIPT EDTA) until the slides were just immersed in the buffer solution, and the DNA was allowed to unwind for 30 min in the electrophoresis solution. Then the electrophoresis was carried out at 25 V and 300 mA for 20 min. After electrophoresis, the slides were RI PT removed, and washed thrice in a neutralization buffer (400 mM Tris, HCl, pH 7.5). Nuclear DNA was stained with 20 µL of EtBr (20 µg/mL) in the dark for 20 min. The slides were washed in chilled distilled water for 10 min to neutralize the excess alkali, SC air-dried and scored for comets by fluorescence microscopy. A total of 10 comets on 4.13. Matrigel invasion assay M AN U each gel were scored. A BD Matrigel invasion chamber was used to investigate cell invasion according TE D to the manufacturer's instructions. B16 cells (4 × 104) in serum free medium containing different concentrations of the complexes and liposomes were seeded into the top chamber of the two-chamber Matrigel system. RPMI 1640 medium (20% FBS) EP was added into the lower chamber. The cells were allowed to invade for 24 h. After AC C incubation, non-invading cells were removed from the upper surface and cells on the lower surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The membranes were photographed and the invading cells were counted under a light microscope. The mean values from three independent assays were calculated. 4.14. Measurement of Intracellular Ca2+ level B16 cells were treated with different concentrations of the complexes and 28 ACCEPTED MANUSCRIPT liposomes for 24 h, the cells were incubated with Fluo-3AM for 30 min at 37 oC in the dark, and washed with PBS three times, then incubated an additional 20 min with PBS at 37 oC to ensure that Fluo-3AM has been completely transformed into Fluo-3, which RI PT can specifically bind to Ca2+ and has a strong fluorescence with an excitation wavelength of 488 nm. The cell nuclei were stained with DAPI at 37 oC. Finally, ImageXpress Micro XLS system was used to observe fluorescence, and Multi SC Wavelength Cell Scoring module was used to analyze the data. The integrated M AN U intensity/cell which represented the fluorescence intensity of each cell was used to measure the levels of Ca2+. The fluorescence intensity of each cell was calculated as the total fluorescence intensity divided by the number of cells. TE D 4.15. Release of cytochrome c studies B16 cells were seeded in a 12-well plate and incubated overnight. Then cells were treated with different concentrations of the complexes and liposomes for 24 h. EP Subsequently, the cells were fixed with ice-cold immunol staining fix solution for 30 AC C min at room temperature. After blocking cells with immunol staining blocking buffer for 1 h, the cells were treated with the primary antibody against cytochrome c (1:50 dilution) overnight at 4 oC. Next, the plate was washed with immunol staining wash buffer three times and probed with Alexa Fluor 488-Labeled Goat Anti-Mouse IgG (1:500 dilution) in the dark for 1 h at room temperature. Finally, the cells were washed with immunol staining wash buffer three times and the cell nuclei were stained with DAPI. The images were obtained using ImageXpress Micro XLS system, 29 ACCEPTED MANUSCRIPT and Multi Wavelength Cell Scoring module was used to analyze the data. The integrated intensity/cell which represents the fluorescence intensity of each cell was used to measure the release of cyto-c. The fluorescence intensity of each cell was RI PT calculated as the total fluorescence intensity divided by the number of cells. 4.16. Targeting microtubules studies SC B16 cells were grown at a density of 106 cells /mL and incubated in the presence M AN U of the complexes and liposomes for 24 h. Subsequently cells were washed twice by PBS, fixed with 2% paraformaldehyde, and incubated with immunostaining blocking solution for 1 h. Cells were then mildly washed with immunostaining wash solution. Subsequently, cells were incubated with antirabbit monoclonal anti-α-tubulin antibody TE D (1:100 dilutions) for 24h at 4 oC, followed by anti-rabbit FITC conjugated IgG antibody (1:500 dilutions) and DAPI (10 µg/mL). After incubation, cells were washed EP with PBS and viewed under a fluorescence microscope. AC C 4.17. Antitumor efficacy of Ir-1 and Ir-1-Lipo in xenograft tumor mice Mice with human tumor xenografts (HOS) were provided by the Laboratory Animal Center of Sun Yat-Sen University. Ir-1 (6.0 mg/kg) and different doses of 4.8 and 9.6 mg/kg of Ir-1-Lipo were injected intraperitoneally into mice of different group (each group contained 6 mice) once a day for seven consecutive days beginning 24 h after inoculation. This dose was the maximum tolerated dose based on our preliminary studies. Control mice were injected with the vehicle. Compounds were 30 ACCEPTED MANUSCRIPT administered by exact body weight, with the injection volume being not more than 0.2 mL. The weights of the animals were recorded every day. All animals were sacrificed on the eighth days after tumor inoculation, and the tumors were excised and weighed. [(C - T)/C] ×100% RI PT The inhibition rate was calculated as follow: T is the average tumor weight of the treated group and C is the average tumor weight M AN U SC of the negative control group [61]. 4.18. Data analysis All data was expressed as means ± SD. Statistical significance was evaluated by a TE D t-test. Differences were considered to be significant when a *P value is less than 0.05. Acknowledgements This work was supported by the National Nature Science Foundation of China (No EP 21877018) and the Natural Science Foundation of Guangdong Province (No AC C 2016A030313728). 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Liu, H.H. Xu, J.H. Zhao, H.F. Wu, X.Y. Li, J.W. Wang, J. Coord. Chem. 65 (2012) 1781-1791. [58] F.D. Szoka, D. Papahadjopoulos, Proc.Natl. Acad. Sci. USA. 75 (1978) 4194-4198. TE D [59] T. Mosmann, J. Immunol. Methods, 65 (1983) 55-63. [60] K.K. Lo, T.K. Lee, J.S. Lau, W.L. Poon, S.H. Cheng. Inorg. Chem. 47 (2008) 200-208. EP [61] R.H. Cao, Q. Chen, X.R. Hou, H.S. Chen, H.J. Guan, Y. Ma, W.L. Peng, A.L. AC C Xu, Bioorg. Med. Chem. 12 (2004) 4613-4623. 36 ACCEPTED MANUSCRIPT Captions for Schemes and Figures Table 1 IC50 values of the complex and liposomes toward selected cancer cell lines RI PT Scheme 1 Structures of complexes Ir-1 and Ir-2 Fig. 1 Percentage of release of Ir-1 and Ir-2 from Ir-1-Lipo and Ir-2-Lipo Fig. 2 Location assay of Ir-1-Lipo (5.0 µM) and Ir-2-Lipo (10.0 µM) in the SC lysosomes after B16 cells were exposed to the liposomes for 0.5 h. Fig. 3 Location assay of Ir-1-Lipo (5.0 µM) and Ir-2-Lipo (10.0 µM) in the M AN U mitochondria after B16 cells were treated with the liposomes for 1 h. Fig. 4 (A) Intracellular Ca2+ levels were assayed after B16 cells were exposed to Ir-1-Lipo (5.0 µM) and Ir-2-Lipo (10.0 µM) for 24 h. (B) The integrated TE D fluorescent intensity/cell was determined after B16 cells were treated with Ir-1 (5.0 µM), Ir-2 (10.0 µM) and different concentration of Ir-1-Lipo and Ir-2-Lipo for 24 h. *P < 0.05 represents significant differences compared with EP control. AC C Fig. 5 (A) The release of cyt-c was examined after B16 cells were exposed to Ir-1-Lipo (5.0 µM) and Ir-2-Lipo (10.0 µM) for 24 h. (B) The integrated fluorescent intensity/cell was determined after B16 cells were incubated with Ir-1 (5.0 µM), Ir-2 (10.0 µM) and different concentration of Ir-1-Lipo and Ir-2-Lipo for 24 h. *P < 0.05 represents significant differences compared with control. Fig. 6 Apoptosis assays after B16 cells (a) were treated with Ir-1 (b, 5.0 µM), Ir-2 (e, 37 ACCEPTED MANUSCRIPT 10.0 µM) and 2.5 and 5.0 µM of Ir-1-Lipo (c and d) and 5.0 and 10.0 µM of Ir-2-Lipo (f and g) for 24 h. Fig. 7 The expression of CDK2 and Cyclin A2 proteins of B16 cells (a) induced by RI PT (A): Ir-1 (b, 5.0 µM), 2.5 and 5.0 µM of Ir-1-Lipo (c and d); (B) Ir-2 (b, 10.0 µM) and 5.0 and 10.0 µM of Ir-2-Lipo (c and d) for 24 h. Fig. 8 Assays of microtubules networks of B16 cells (a) induced by 2.5 and 5.0 µM of SC Ir-1-Lipo (b and c), 5.0 and 10.0 µM of Ir-2-Lipo (e and f) for 24 h. M AN U Fig. 9 The expression of caspase-3, PARP and Bcl-2 family proteins after B16 cells were treated with Ir-1, Ir-1-Lipo, Ir-2 and Ir-2-Lipo for 24 h. Fig. 10 The in vivo antitumor activity of Ir-1-Lipo in B16 xenograft model. (A) Tumor volume growth trend of control group, 6.0 mg/kg of Ir-1, 4.8 mg/kg TE D and 9.6 mg/kg of Ir-1-Lipo groups. Tumor volumes were tracked by the mean tumor volume (cm3) ± SD (n = 6) and calculated as relative tumor growth rate [T/C%] values. (B) Photographs of tumor from treatment groups and vehicle EP group. (C) Tumor weight (mean ± SD) mg after the tumor was treated with AC C Ir-1 or Ir-1-Lipo for 7 days. (D) Inhibiting percentage of tumor growth induced by 6.0 mg/kg Ir-1 and different concentrations of Ir-1-Lipo. *P < 0.05 represents significant differences compared with control. Fig. 11 The mechanism of Ir-1-Lipo and Ir-2-Lipo inducing apoptosis in B16 cells. 38 RI PT ACCEPTED MANUSCRIPT Table 1 IC50 values of the complex and liposomes toward selected cancer cell lines HeLa SGC-7901 MCF-7 Ir-1 Ir-1-Lipo Ir-2 Ir-2-Lipo > 200 5.2 ± 0.8 > 200 10.8 ± 1.5 > 200 9.5 ± 0.9 > 200 6.2 ± 0.4 > 200 8.7 ± 0.3 > 200 8.0 ± 1.2 > 200 13.9 ± 1.6 > 200 21.0 ± 1.6 TE D EP AC C A549 BEL-7402 SC B16 > 200 10.3 ± 0.7 > 200 10.8 ± 0.8 M AN U Complex > 200 12.0 ± 2.4 > 200 11.3 ± 0.5 LO2 > 200 15.0 ± 1.3 > 200 22.3 ± 1.8 AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Highlights Two iridium(III) complexes Ir-1 and Ir-2 and their liposomes Ir-1-Lipo and Ir-2-Lipo were synthesized and characterized. RI PT The cytotoxicity in vitro or in vivo of the complexes and liposomes was investigated. SC The apoptosis and cell cycle distribution were assayed by flow cytometry ROS and mitochondrial membrane potential were studied under fluorescence M AN U microscope. AC C EP TE D The expression of Bcl-2 family proteins was assayed by western blot analysis.