Design, synthesis and biological evaluation of liposome entrapped iridium(III) complexes toward SGC-7901 cells.

{"full_text": " Journal of Inorganic Biochemistry 241 (2023) 112134\n\n\n Contents lists available at ScienceDirect\n\n\n Journal of Inorganic Biochemistry\n journal homepage: www.elsevier.com/locate/jinorgbio\n\n\n\n\nDesign, synthesis and biological evaluation of liposome entrapped iridium\n(III) complexes toward SGC-7901 cells\nYichuan Chen a, 1, Yiying Gu a, 1, Huiyan Hu a, Haimei Liu a, Wenlong Li a, Chunxia Huang a,\nJing Chen a, Lijuan Liang a, Yunjun Liu a, b, *\na\n School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China\nb\n Guangdong Provincial Key Laboratory of Advanced Drug Delivery, Guangdong Provincial Engineering Center of Topical Precise Drug Delivery System, Guangdong\nPharmaceutical University, Guangzhou 510006, PR China\n\n\n\n\nA R T I C L E I N F O A B S T R A C T\n\nKeywords: In this study, two new iridium(III) polypyridyl complexes [Ir(bzq)2(DIPH)](PF6) (bzq = deprotonated benzo[h]\nIridium(III) complexes quinoline, DIPH = 4-(2,5-dibromo-4-(1H-imidazo[4,5-f][1,10]phenanthrolim-2-yl)-4-hydroxybutan-2-one) (Ir1)\nLiposomes and [Ir(piq)2(DIPH)](PF6) (piq = deprotonated 1-phenylisoquinoline) (Ir2) were synthesized and characterized\nMolecular docking\n by elemental analysis, HRMS, 1H and 13C NMR. The cytotoxic activity of Ir1, Ir2, Ir1lipo and Ir2lipo against\nImmunogenic cell death\n cancer cells SGC-7901, HepG2, A549, HeLa, B16 and normal NIH3T3 cells in vitro was evaluated using 3-(4,5-\nEndoplasmic reticulum\n dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) method. Ir1 and Ir2 showed no cytotoxic ac\u00ad\n tivity, but their liposome-entrapped Ir1 (Ir1lipo) and Ir2 (Ir2lipo) showed significant cellular activity, especially\n sensitive to SGC-7901 with IC50 values of 4.7 \u00b1 0.2 and 12.4 \u00b1 0.5 \u03bcM, respectively. The cellular uptake,\n endoplasmic reticulum (ER) localization, autophagy, tubulin polymerization, glutathione (GSH), malondialde\u00ad\n hyde (MDA) and release of cytochrome c were investigated to explore the mechanisms of apoptosis. The cal\u00ad\n reticulin (CRT), heat shock protein 70 (HSP70), high mobility group box 1 (HMGB1) were also explored. Western\n blotting showed that Ir1lipo and Ir2lipo inhibited PI3K (phosphoinositide-3 kinase), AKT (protein kinase B), p-\n AKT and activated Bcl-2 (B-cell lymphoma-2) protein and apoptosis-regulated factor caspase 3 (cysteinyl\n aspartate specific proteinase-3) and cleaving PARP (poly ADP-ribose polymerase). The results demonstrated that\n Ir1lipo and Ir2lipo induce cell apoptosis through targeting the endoplasmic reticulum (ER), cause oxidative\n stress damage, inhibiting PI3K/AKT signaling pathway, immunogenic cell death (ICD) and inhibit the cell growth\n at G2/M phase.\n\n\n\n\n Abbreviations: A549, human lung carcinoma; AKT, protein kinase B; Annexin V-FITC, fluorescein isothiocyanate-labeled 3\u2032 ,6\u2032 -dihydroxy-5-isothiocyanato-3H-\nspiro(isobenzofuran-1,9\u2032 -xanthen)-3-one; B16, Murine melanoma cells; Bad, Bcl-2 associated death promoter; Bax, Bcl-2 associated x protein; BCA, bicinchoninic\nacid; Bcl-2, B-cell lymphoma-2; bzq, benzo[h]quinoline; Calcein AM, Calcein Acetoxymethyl Ester; Caspase 3, cysteinyl aspartate specific proteinase-3; CHO-HP,\ncholesterol; CRT, calreticulin; DAMPs, damage associated molecular patterns; DAPI, 4\u2032 ,6-diamidino-2-phenylindole; DIPH, 4-(2,5-dibromo-4-(1H-imidazo[4,5-f]\n[1,10]phenanthrolim-2-yl)-4-hydroxybutan-2-one); DMEM, Dulbecco\u2019s Modified Eagle Medium; DMSO, dimethylsulfoxide; DSPE-mPEG2000, 1,2-distearoyl-sn-\nglycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol)-2000; ER, endoplasmic reticulum; FAK, Focal adhesion kinase; FBS, Fetal bovine serum; FITC,\nfluorescein isothiocyanate; GSH, glutathione; HeLa, human cervical cancer cells; HepG2, human liver carcinoma cells; HMGB1, high mobility group box1; HSP70,\nheat shock protein 70; ICD, immunogenic cell death; MCTSs, multicellular tumor spheroids; MDA, malondialdehyde; MDC, monodansylcadaverine; MTT, 3-(4,5-\ndimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide; NIH3T3, Mouse fibroblast cells; PARP, poly ADP-ribose polymerase; PBS, phosphate buffer solution; PC-98\nT, Egg yolk lecithin; PCC, Pearson\u2019s correlation coefficients; PDI, Polydispersity index; PI, Propidium iodide; PI3K, phosphatidylinositol 3-kinase; piq, 1-phenyl\u00ad\nisoquinoline; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluoride; RIPA, 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxy\u00ad\ncholate; RLU, Relative luminometer units; RNase, ribonuclease; RPMI, 1640 Roswell Park Memorial Institute 1640; SDS-PAGE, sodium dodecyl sulfate\npolyacrylamide gel electrophoresis; SGC-7901, human gastric cells; TBA, thiobarbituric acid; TBST, 20 mM Tris\u2013HCl, 150 mM NaCl, 0.2% Tween 20; TMS, tetra\u00ad\nmethylsilane; Tris, Tris(hydroxymethyl)aminomethane; Tween, 20 polysorbate 20.\n * Corresponding author at: School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.\n E-mail address: lyjche@gdpu.edu.cn (Y. Liu).\n 1\n These authors ontribute equally to this work.\n\nhttps://doi.org/10.1016/j.jinorgbio.2023.112134\nReceived 17 December 2022; Received in revised form 18 January 2023; Accepted 18 January 2023\nAvailable online 21 January 2023\n0162-0134/\u00a9 2023 Elsevier Inc. All rights reserved.\n\fY. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112134\n\n\n\n\n Scheme 1. Synthetic route of ligand and its complexes Ir1 and Ir2.\n\n\n1. Introduction In the present study, we determined the anticancer activity of Ir1,\n Ir2, Ir1lipo and Ir2lipo on SGC-7901 cells and investigated the cellular\n Cancer has become a major public health problem that poses a signal pathways associated with cell death. The results showed that\nserious threat to human life and health [1,2]. Gastric cancer is one of the complexes-loaded liposomes significantly increased cellular uptake and\nmost common malignancies of the digestive system and is diagnosed in locate at the endoplasmic reticulum (ER), inhibited cellular glutathione\n>1 million people worldwide each year [3,4]. Metastasis is the main (GSH) synthesis, increased malondialdehyde (MDA) levels leading to\ncause of death in cancer patients [5]. However, malignant tumor cells oxidative cellular damage and induced apoptosis. In addition, cytotox\u00ad\noften grow wildly and spread to other tissues or organs without control icity of multicellular tumor spheroids (MCTSs), autophagy activation,\n[6]. Tumor heterogeneity is a fundamental feature of malignancy, and microtubule protein inhibition, cytochrome c, Bcl-2 (B-cell lymphoma-\ngastric cancer poses a challenge for humans to overcome cancer due to 2) family proteins and immunogenic cell death (ICD) were also inves\u00ad\nits highly aggressive nature [7,8]. Chemotherapy remains an effective tigated. Ir1lipo and Ir2lipo induced apoptosis mainly through inhibit\u00ad\nand commonly used method in the clinical treatment for recurrence and ing the PI3K/AKT signaling pathway and further activated the Bcl-2 (B-\nmetastasis of cancer [9\u201312]. Since the discovery of cisplatin by Rosen\u00ad cell lymphoma-2) protein and caspase 3, upregulated the cleaved PARP\nberg et al. in the 1960s, the efficacy of platinum drugs such as carbo\u00ad (poly ADP-ribose polymerase). Furthermore, the results from the calre\u00ad\nplatin and oxaliplatin in cancer chemotherapy has been limited by side ticulin (CRT) exposure, heat shock protein 70 kDa (HSP70) and high\neffects such as drug resistance, neurotoxicity, nephrotoxicity, hepato\u00ad mobility group box 1 (HMGB1) further confirm that Ir1lipo and Ir2lipo\ntoxicity, and bone marrow suppression [13\u201317]. In recent years, many can lead to immune cell death (ICD).\nmetal complexes have been synthesized, among which iridium(III)\ncomplexes show great potential as promising anticancer candidates 2. Experimental\n[18\u201330]. Compared to conventional dosage forms, nanodrugs have a\nhigher delivery efficiency and are passively targeted to solid tumor tis\u00ad 2.1. Materials and methods\nsue [31,32]. PEG-based liposomal drug delivery systems are widely\nrecognized for producing extended circulation times and promoting All chemicals and reagents used in the experiments commercially\ntumor accumulation through enhanced permeability and retention ef\u00ad were purchased and used directly without further purification unless\nfects [33\u201336]. In our previous studies [37\u201340], the low solubility of otherwise stated. IrCl3\u22c53H2O was obtained from the Kunming Boren\niridium(III) complexes makes them difficult to enter cells and exert Precious Metals Co., Ltd. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-\nanticancer activity. The liposomes loaded with iridium(III) complexes diphenyl-2H-tetrazolium bromide) was purchased from Sigma\u2013Aldrich\nwere able to greatly alter solubility and modify cellular dynamics and (St. Louis, MO, USA; 98% purity). Human gastric cells SGC-7901,\ncellular uptake to enhance antitumor efficacy. Human liver carcinoma cells HepG2, Human lung carcinoma A549,\n In order to obtain more information on the anticancer activity and Human cervical cells HeLa, Murine melanoma cells B16, Mouse fibro\u00ad\nfurther understanding the anticancer mechanism of iridium(III) com\u00ad blast cells NIH3T3 were gained from the Cell Center of Sun Yat-Sen\nplexes and their liposomes, in this work, two new iridium(III) complexes University (Guangzhou, China). High glucose Dulbecco\u2019s modified Ea\u00ad\n[Ir(bzq)2(DIPH)](PF6) (bzq = deprotonated benzo[h]quinolone, DIPH gle\u2019s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640\n= 2,5-dibromo-4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)-4- were purchased from Corning Corp (New York, USA) and Fetal bovine\nhydroxybutan-2-one, Ir1) and [Ir(piq)2(DIPH)](PF6) (piq = deproto\u00ad serum (FBS) was obtained from Gibco Corporation. PC-98 T (Egg yolk\nnated 1-phenylisoquinoline, Ir2, Scheme 1) were synthesized charac\u00ad lecithin), CHO-HP (cholesterol) and 1,2-distearoyl-sn-glycero-3-phos\u00ad\nterized by elemental analysis, HRMS, 1H and 13C NMR. The Ir1lipo and phoethanolamine-N-methoxy (polyethylene glycol)-2000 (DSPE-\nIr2lipo were characterized by size distribution, zeta potential, and mPEG2000) were purchased from AVT Pharmaceutical Tech Co., Ltd.\nrelease in vitro. The 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetra\u00ad (Shanghai, China). Propidium iodide (PI), Annexin V-FITC Apoptosis\nzolium bromide (MTT) studies demonstrated that Ir1 and Ir2 showed no Detection Kit, Hoechst and ER-Tracker Red were obtained from Beyo\u00ad\nactivity against the selected cancer cells SGC-7901, HepG2, A549, HeLa, time Institute of Biotechnology (Shanghai, China). CRT, HMGB1 and\nB16 and normal NIH3T3 cells. To enhance the in vitro activity of Ir1, HSP70 Rabbit Polyclonal Antibody, FITC-labeled Goat Anti-Rabbit IgG\nIr2, the liposomes Ir1lipo, Ir2lipo were prepared by ethanol injection were purchased from Beyotime Institute of Biotechnology (Shanghai,\nmethod. Surprisingly, Ir1lipo and Ir2lipo showed a strong inhibitory China). Microanalysis (C, H, and N) was carried out with a Perkin\u2013Elmer\neffect on the above tumor cells, especially on SGC-7901 cells. 240Q elemental analyzer. 1H and 13C NMR spectra were recorded on a\n\n 2\n\fY. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112134\n\n\nVarian-500 spectrometer with DMSO\u2011d6 as a solvent and tetrame\u00ad 2.61, N, 6.84%. HRMS (CH3CN): Calcd for C53H36Br2IrN6O2PF6 m/z =\nthylsilane (TMS) as an internal standard at room temperature. The spray 1141.0875 ([M-PF6]+), found: m/z = 1141.0829 ([M-PF6]+) (Fig. S2a,\nvoltage, tube lens offset, capillary voltage and capillary temperature supporting information). IR (KBr, cm\u2212 1, Fig. S2b, supporting informa\u00ad\nwere set at 4.50 kV, 30.00 V, 23.00 V and 200 \u25e6 C, respectively, and the tion): 3425 m, 3042 m, 2922 m, 1075 m, 1606 m, 1575 m, 1539 m,\nquoted m/z values are for the major peaks in the isotope distribution. 1444 s, 1379 m, 1351 s, 1270 m, 1148 m, 1079 m, 1048 s, 843 m, 729 m,\n 557 m. 1H NMR (DMSO\u2011d6, 500 MHz, Fig. S2c, supporting information):\n2.2. Synthesis of complexes The H proton in the imidazole ring was not observed, 9.11 (dd, 2H, Ha,a\u2019,\n J = 3.0 Hz, J = 7.0 Hz), 9.06\u20139.02 (m, 2H, H1,1\u2032 ), 8.44 (s, 1H, Hk),\n2.2.1. Synthesis of the ligand DBIPB 8.41\u20138.39 (m, 2H, Hc,c\u2019), 8.16 (s, 1H, Hn), 7.99 (t, 2H, H11,11\u2032 , J = 7.0\n A mixture of 1,10-phenanthroline-5,6-dione (0.315 g, 1.5 mmol) Hz), 7.90\u20137-83 (m, 8H, H7,7\u2032 ,b,b\u2019,4,4\u2032 ,2,2\u2032 ), 7.45 (t, 2H, H5,5\u2032 , J = 6.5 Hz),\n[41], 2,5-dibromo-1,4-benzenedicarbroxadehyde (0.437 g, 1.5 mol), 7.40\u20137.37 (m, 2H, H6,6\u2032 ), 7.18\u20137.15 (m, 2H, H14,14\u2032 ), 6.97\u20136.93 (m, 2H,\nNH4Ac (3.08 g, 40 mmol) was dissolved in glacial acetic acid (25 mL) H12,12\u2032 ), 6.33 (t, 2H, H13,13\u2032 , J = 7. 0 Hz), 5.28 (t, 1H, Ho, J = 5.5 Hz),\nand refluxed at 130 \u25e6 C for 2 h, then cool to room temperature. A satu\u00ad 4.58 (s, 1H, HOH), 2.70 (d, 2H, Hp, J = 8.5 Hz), 2.19 (s, 3H, H-CH3). 13C\nrated aqueous ammonium solution (25 mL) was added to neutralize the NMR (DMSO\u2011d6, 125 MHz, Fig. S2d, supporting information): 207.98\nsolution to obtain a yellow precipitate, which was filtered and washed (Cq), 158.55 (C9,9\u2032 ), 158.52 (Ch), 150.68 (Ca,a\u2019), 150.65 (C1,1\u2032 ), 149.82\nwith ice water. After dissolution in anhydrous ethanol, the yellow solid (Cl), 149.73 (Ci), 145.91 (Ce,e\u2019,10,10\u2032 ), 142.43 (C3,3\u2032 ), 139.45 (Cn,k),\nwas collected after recrystallization and dried under a vacuum. Yield: 138.39 (Cc,c\u2019), 137.08 (C5,5\u2032 ), 135.75 (C12,12\u2032 ), 134.45 (C14,14\u2032 ), 133.89\n85%. ESI-MS (CH3OH): m/z = 480.87 ([M \u2013 1]). Anal. Calcd for (C8,8\u2032 ), 131.72 (C11,11\u2032 ), 131.51 (C13,13\u2032 ,6,6\u2032 ), 130.59 (C15,15\u2032 ), 128.69\nC20H10Br2N4O: C, 49.82, H, 2.09, N, 11.62%. Found: C, 49.70, H, 2.25, (C4,4\u2032 ), 128.34 (Cd,d\u2019), 128.26 (C7,7\u2032 ), 126.21 (Cf,g), 124.75 (Cb,b\u2019),\nN, 11.81%. IR (KBr, cm\u2212 1): 3355 m, 3064 w, 1783 w, 1687 s, 1618 s, 122.25 (Cm), 121.69 (Cj), 121.30 (C2,2\u2032 ), 70.54 (Co), 57.82 (Cp), 31.60\n1571 s, 1434 w, 1352 w, 1261 w, 1178 s, 1128 w, 1054 s, 804 s, 734 s. (Cr).\n\n2.2.2. Synthesis of [Ir(bzq)2(DIPH)]PF6 (Ir1)\n A mixture of cis-[Ir(bzq)2Cl]2 (0.30 g, 0.25 mmol) [42] and DBIPB 2.3. Preparation of the liposomes\n(0.24 g, 0.5 mmol) in a mixture of 30 mL of dichloromethane and\nmethanol (v/v, 2:1) was heated and refluxed at 40 \u25e6 C for 6 h under argon Liposomes Ir1lipo and Ir2lipo were prepared using the ethanol in\u00ad\nto obtain a yellow solution. After cooling, excessive NH4PF6 powder was jection method [43]. Briefly, 1 mg of Ir1 or Ir2, 500 \u03bcL of PC-98 T (60\nadded to the solution, stirred for 2 h and the filtrate was collected and mg/ml), 8 mg of CHO-HP and 5 mg of DSPE-mPEG2000 were thor\u00ad\ndried under vacuum to obtain a yellow intermediate product [Ir oughly mixed and completely dissolved in anhydrous ethanol. Next, the\n(bzq)2(DBIPB)](PF6). Next, the intermediate product was purified by mixture was slowly injected into the 5 mL PBS (phosphate buffer solu\u00ad\ncolumn chromatography on Al2O3 (pH = 7.05, weak alkaline) with a tion) at 55 \u25e6 C with stirring for 20 min to discard absolute ethanol. After\nmixture of dichloromethane and acetone (v/v, 1:3) as eluent. During the that, the PBS was slowly added to the mixture solution to form a 5 mL\npurification process, the aldehyde group in DBIPB with acetone under\u00ad solution and sonicated using an ultrasonic cell disrupter with the pre\u00ad\nwent an aldehyde and ketone condensation reaction to produce the setting parameters (150 W, 5 min, 1 s on/off) in an ice bath. At the end of\ncomplex [Ir(bzq)2(DIPH)](PF6). Finally, the yellow band was collected. sonication, the solution was centrifuged (10,000 rpm, 4 \u25e6 C) for 10 min\nThe solvent was removed by evaporation under reduced pressure and and the supernatant was collected and stored at 4 \u25e6 C.\nthe yellow powder was obtained after vacuum drying. Yield: 77%. Anal\nCalcd for C49H32Br2IrN6O2PF6: C, 47.70, H, 2.61, N, 6.81%. Found: C,\n47.51, H, 2.84, N, 6.97%. HRMS (CH3CN): Calcd for C49H32Br2Ir\u00ad 2.4. Particle size and surface potential of the liposomes\nN6O2PF6 m/z = 1089.0560 ([M-PF6]+), found: m/z = 1089.1400 ([M-\nPF6]+) (Fig. S1a, supporting information). IR (KBr cm\u2212 1, Fig. S1b, sup\u00ad The mean particle diameter and zeta potential of Ir1lipo and Ir2lipo\nporting information): 3439 s, 3043 m, 2923 m, 1706 m, 1620 m, 1567 were measured using Zetasizer Nano ZS-90 (Malvern, UK). Each sample\nm, 1446 s, 1354 m, 1329 m, 1191 m, 1081 m, 1051 m, 846 s, 722 m, 558 was diluted with ultrapure water (1: 5) tested at 25 \u25e6 C and repeated\nm. 1H NMR (DMSO\u2011d6, 500 MHz, Fig. S1c, supporting information): The three times.\nH proton in the imidazole ring was not observed, 9.10 (d, 1H, Ha, J = 8.0\nHz), 9.05 (d, 1H, Ha\u2019, J = 7.0 Hz), 8.52\u20138.50 (m, 2H, H1,1\u2032 ), 8.13 (s, 1H,\nHk), 8.03 (d, 2H, Hc,c\u2019, J = 4.5 Hz), 7.99\u20137.96 (m, 4H, H3,3\u2032 ,8,8\u2032 ), 7.92 (s, 2.5. Determination of encapsulation efficiency (EE%)\n1H, Hn), 7.88 (d, 2H, H5,5\u2032 , J = 3.0 Hz), 7.85 (d, 2H, H6,6\u2032 , J = 3.5 Hz),\n7.58 (d, 1H, H10, J = 3.0 Hz), 7.56 (d, 1H, H10\u2019, J = 4.0 Hz), 7.47\u20137.42 The Ir1 or Ir2 content in liposomes was quantified via the UV\u2013Vis\n(m, 2H, H9,9\u2032 ), 7.24\u20137.20 (m, 2H, Hb,b\u2019), 6.34 (t, 2H, H2,2\u2032 , J = 6.0 Hz), spectrometer. In brief, the complexes were dissolved in absolute ethanol\n5.30 (t, 1H, Ho, J = 4.0 Hz), 4.57 (s, 1H, HOH), 2.72 (d, 2H, Hp, J = 8.5 to prepare different concentrations (5, 7.5, 10, 12.5, 15 and 20 \u03bcg/mL)\nHz), 2.20 (s, 3H, H-CH3). 13C NMR (DMSO\u2011d6, 125 MHz, Fig. S1d, sup\u00ad of solutions, and the relationship between concentration and absorbance\nporting information): 207.99 (Cq), 158.51 (Ch), 150.68 (Ca,a\u2019), 149.80 values (standard curve) was obtained by UV\u2013Vis spectroscopy of the\n(C1), 149.72 (C1\u2019), 145.92 (C13,13\u2032 ), 142.43 (Cl), 139.45 (Ci), 137.08 (Ce, above six solutions. Next, 1 mL liposome was dissolved in 10 mL abso\u00ad\n lute ethanol under sonication for 3 h to completely extract the incor\u00ad\ne\u2019), 135.75 (C3,3\u2032 ), 134.45 (Cn), 133.89 (C7,7\u2032 ), 131.72 (Ck), 131.50 (Cc,\n porated Ir1 or Ir2. Then the solutions were filtered through a membrane\nc\u2019), 130.59 (C12,12\u2032 ), 128.69 (C5,5\u2032 ,6,6\u2032 ,8,8\u2032 ), 128.36 (C11,11\u2032 ), 128.28\n(C4,4\u2032 ), 126.21 (Cd,d\u2019), 124.75 (C9,9\u2032 ,10,10\u2032 ), 122.75 (Cf,g), 122.25 (Cm), (0.22 \u03bcM diameter) and Ir1 or Ir2 concentration was measured using a\n121.62 (C2,2\u2032 ,b,b\u2019), 121.30 (Cj), 70.55 (Co), 57.83 (Cp), 31.60 (Cr). Perkin Elmer Lambda UV\u2013Vis spectrometer (Perkin-Elmer, UK). The\n encapsulation efficiency (EE) was calculated using the following\n2.2.3. Synthesis of [Ir(piq)2(DIPH)]PF6 (Ir2) equation:\n Complex [Ir(piq)2(DBIPB)](PF6) was synthesized and purified as the Edrug\n EE(%) = \u00d7 100%\nsame method described for [Ir(bzq)2(DBIPB)](PF6), with cis-[Ir Tdrug\n(piq)2Cl]2 (0.38 g, 0.25 mmol) [42] in place of cis-[Ir(bzq)2Cl]2 (0.30 g,\n0.25 mmol). During the process of purification, [Ir(piq)2(DBIPB)](PF6) where Edrug represents the amount of complex encapsulated in the\nwas transferred into [Ir(piq)2(DIPH)](PF6). Yield: 79%. Anal Calcd for liposome and Tdrug represents the amount of total complex initially\nC53H36Br2IrN6O2PF6: C, 49.50, H, 2.82, N, 6.54%. Found: C, 49.84, H, added.\n\n 3\n\fY. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112134\n\n\n2.6. Drug release in vitro from liposome experiments using IBM SPSS Statistics (version 20.0).\n\n In vitro the release of Ir1lipo and Ir2lipo in phosphate buffered 2.11. Cellular uptake\nsaline (PBS, pH = 7.4) containing 0.5% Tween 80 (w/v) was determined\nby the dialysis method. 4 mL of Ir1lipo and Ir2lipo were filtered The well-growing SGC-7901 cells were cultured in 12-well plates (1\nthrough a membrane (0.22 \u03bcM diameter) and placed in a dialysis bag \u00d7 105 cells per well) in an incubator overnight and exposed to IC50\n(8\u201314 kDa molecular weight) in 200 mL PBS solution which was concentrations of Ir1, Ir2, Ir1lipo and Ir2lipo for 24 h. The cell nuclei\ncontinuously stirred at 37 \u25e6 C (100 rpm). Samples (3 mL) were collected were stained with (4\u2032 ,6-diamidino-2-phenylindole) DAPI (5 \u03bcg/mL in\nat the different time intervals (0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, 24, 30, 36, PBS) for 20 min at 37 \u25e6 C followed by washing twice with cold PBS. The\n48, 60, 72 and 84 h) and supplied with an equal amount of fresh PBS cell images were captured under the ImageXpress R Micro XLS System\n(0.5% Tween 80). The concentrations of Ir1 and Ir2 were measured by (MD company, USA) and the uptake extent in SGC-7901 cells of the\nUV\u2013Vis spectroscopy. complexes and liposomes were analyzed using a flow cytometer (Beck\u00ad\n man Instruments, NJ).\n2.7. pKa of the complex determination\n 2.12. Wound healing in vitro\n The pKa values of the complexes were determined by potentiometric\ntitration method using a pH meter (Basic pH Meter PB-10, Sartorius). The in vitro wound healing assay is an effective and convenient\nFirstly, 100 \u03bcM of Ir1 or Ir2 was dissolved in a mixture of acetonitrile method for detecting the collective migration of SGC-7901 cells. The\nand water (v/v, 3:7), HCl (100 mM) and KOH (60 mM) were prepared. cells (5.0 \u00d7 105 cells per well) were seeded in 6-well plates and cultured\nThe pH values were slowly decreased to about pH 2.0 through gradually overnight. After creating a scratch with a sterile pipette tip, the mono\u00ad\ntitrating with 100 mM of HCl, then NaOH solution was gradually added, layer was washed with PBS to remove any residual cell debris and a fresh\nthe change in pH was recorded by slowly dropping a KOH solution (60 medium containing 1% FBS was added. The cells were treated with IC50\nmM) into the above solution. concentration of Ir1, Ir2, Ir1lipo and Ir2lipo for 24 h in an incubator at\n 37 \u25e6 C, 5% CO2 and 95% air. Finally, the cells were photographed at\n2.8. Cell culture 0 and 24 h under an inverted microscope (Olympus Co., Tokyo, Japan).\n\n SGC-7901 cells were cultured with RMPI-1640 medium and HepG2, 2.13. Localization of the complexes in the endoplasmic reticulum\nA549, HeLa, B16, NIH3T3 cells were cultured with DMEM medium\nsupplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco), 100 U/ SGC-7901 cells (1 \u00d7 105 cells/well) were seeded in 12-well plates (1\nmL penicillin and 0.1 mg/mL streptomycin in a humidified incubator mL/well) in RPMI 1640 medium overnight. Thereafter, the IC50 con\u00ad\ncontaining 95% air and 5% CO2 at 37 \u25e6 C. centration of the complexes and liposomes were added and incubated\n for 4 h. After that, the cells were washed two times with cold PBS and\n2.9. Cell viability assay of 3D culture stained with 500 \u03bcL of a solution of ER Tracker Red (1 \u03bcM, Beyotime\n Biotechnology, Shanghai, China) at 37 \u25e6 C for 20 min in the dark. The\n SGC-7901 cells in the exponential growth phase were dissociated by nuclei were stained with Hoechst (10 \u03bcg/mL) for 20 min. Subsequently,\ntrypsinization and centrifuged for 5 min at 1000 g to gain single-cell the cells were washed twice, and the images were acquired under an\nsuspensions. Diluted SGC-7901 cells suspensions (5.0 \u00d7 104, 200 \u03bcL) ImageXpress Micro XLS System as soon as possible.\nwere transferred to 100 \u03bcL Matrigel matrix (Corning) confocal petri dish\nwith RPMI 1640 (200 \u03bcL) containing 10% FBS at 37 \u25e6 C for 30 min. Next, 2.14. Measurement of glutathione (GSH) and malondialdehyde (MDA)\n200 \u03bcL of Matrigel and RPMI 1640 (v/v, 1:9) was slowly added into the content\nconfocal Petri dishes to produce a gel-cell-gel structure. The single cells\nwere cultured at 37 \u25e6 C with 5% CO2 and SGC-7901 multicellular tumor GSH and GSSG contents were measured by GSH and GSSH Assay Kit\nspheroids (MCTSs) appeared on day 4. After treatment with treated with (S0053, Beyotime Biotechnology, Shanghai, China). SGC-7901 cells (5\nIC50 of Ir1, Ir2, Ir1lipo and Ir2lipo for 24 h, the cells were stained with \u00d7 105 cells per well) were seeded in 6-well plates and inoculated over\u00ad\nCalcein-AM (Calcein Acetoxymethyl Ester)/PI (Meilum Biotechnology, night. Next, after treatment with IC50 concentrations of Ir1, Ir2, Ir1lipo\nDalian) and Hoechst for 30 min and imaged under a confocal microscope and Ir2lipo for 24 h, SGC-7901 cells were collected, centrifugated and\n(Leica TCS-SP8 SR, Germany). the supernatant was collected. According to the manufacturer\u2019s in\u00ad\n structions, total glutathione was measured at 412 nm using a microplate\n2.10. Cytotoxicity assay in vitro reader (BioTek, Winooski, USA), and GSH content was obtained by\n subtracting the amount of GSSG from the total glutathione. The MDA\n The cytotoxic activity in vitro of Ir1, Ir2, Ir1lipo and Ir2lipo against levels were quantified using the Lipid Peroxidation MDA Assay Kit\nthe selected cancer cells lines SGC-7901, A549, HepG2, HeLa, B16 and (S0131S, Beyotime Biotechnology, Shanghai, China). In brief, the SGC-\nnormal NIH3T3 cells was evaluated using 3-(4,5-dimethylthiazole-2-yl)- 7901 of supernatant was prepared in lysis buffer and centrifuged\n2,5-biphenyl tetrazolium bromide (MTT) method [44]. The cells (1 \u00d7 (12,000 g, 15 min, 4 \u25e6 C). The samples were treated with TBA (thio\u00ad\n104 cells per well, 100 \u03bcL) were placed in 96-well culture plates barbituric acid) in the boiling water bath for 40 min and the absorbance\n(Corning) and grew overnight at 37 \u25e6 C in a 5% CO2 incubator. When the was measured at 530 nm. MDA levels were expressed as \u03bcmol/g protein.\ncells reached 70% growth, the complexes and liposomes were added to\nthe wells at different concentrations (0\u2013100 \u03bcM) and blank liposomes 2.15. Measurement of apoptosis\nwere added as the control group. After 48 h, the medium in the wells was\nremoved and replaced with 9 mL of medium and 1 mL of MTT dye so\u00ad Annexin V-FITC/PI double staining method was used to assess the\nlution (100 \u03bcL, 5 mg/mL) and incubation was continued for 4 h at 37 \u25e6 C. apoptosis of the SGC-7901 cells. Logarithmic growth phase SGC-7901\nNext, the suspension was replaced with DMSO (100 \u03bcL/well) to solu\u00ad cells (5 \u00d7 105 cells per well) were seeded into 6-well plates and\nbilize the MTT formazan. The absorbance of each well was measured cultured overnight. When the cells reached 70%, the cells were treated\nunder a microplate spectrophotometer (Multiskan FC, Thermo Scienti\u00ad with IC50 concentrations of Ir1, Ir2, Ir1lipo and Ir2lipo at 37 \u25e6 C in 5%\nfic, Shanghai, China) at a wavelength of 490 nm. The IC50 values were CO2 for 24 h. Next, after removing the medium, the cells were washed\nobtained from the analysis of absorbance data from three independent twice with cold PBS and centrifuged at 1000 rpm for 5 min. Finally, the\n\n 4\n\fY. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112134\n\n\ncells were resuspended Annexin V binding buffer (195 \u03bcL) and stained solution and incubated with immunol staining blocking buffer for 70\nwith 1 mg/mL of Annexin V-FITC (5 \u03bcL) and 500 mg/mL of pyridine min. After washing 3 times with immunol staining wash buffer, the cells\niodide (10 \u03bcL) at 37 \u25e6 C in the dark for 20 min. The apoptosis was were incubated with anti-rabbit monoclonal anti-\u03b1-tubulin antibody\nimmediately determined by flow cytometry (Beckman Instruments, NJ) (1:100 dilution) overnight at 4 \u25e6 C followed by anti-rabbit FITC conju\u00ad\nand the obtained results were analyzed with Flow Jo (version 10.0). gated IgG antibody (1:500 dilution) for 1 h. Subsequently, the cells were\n washed with immunol staining wash buffer three times and the cell\n2.16. Analysis of cell cycle arrest nuclei were stained with DAPI (10 mg/mL) for 30 min. Eventually, the\n cells were visualized and imaged by the ImageXpress Micro XLS System.\n To further investigate the effect of complexes and liposomes on the\ninhibition of SGC-7901 cells proliferation, the cell cycle distribution was 2.21. Western blot analysis\nmeasured by flow cytometry analysis. The cells were seeded in 6-well\nplates at a density of 5 \u00d7 105 cells/well and incubated in RPMI 1640 SGC-7901 cells were seeded into 6-well plates at a density of 5.0 \u00d7\nsupplemented with 10% FBS for 24 h. After treatment with IC50 con\u00ad 105 cells per well and incubated overnight. After treatment with Ir1, Ir2,\ncentrations of complexes and liposomes for 24 h, the cells were collected Ir1lipo and Ir2lipo for 24 h, SGC-7901 cells were lysed in RIPA (50 mM\nand fixed in 75% ethanol at 4 \u25e6 C overnight. Next day, the cell pellets Tris pH = 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate)\nwere washed twice with cold PBS and resuspended in 200 \u03bcL of assay buffer containing 1 mM PMSF (phenylmethylsulfonyl fluoride) by\nbuffer with 0.1% Triton X-100, 4 \u03bcL of PI (propidium iodide, 0.02 mg/ centrifugation (12,000 g, 4 \u25e6 C, 15 min) to extract proteins. The protein\nmL) and 4 \u03bcL of RNase A (ribonuclease, 0.2 mg/mL) for 30 min at 37 \u25e6 C concentrations were determined by BCA (bicinchoninic acid) Protein\nin the dark. Analysis of samples was performed using a flow cytometer Assay Kit (P0012S, Beyotime Biotechnology, Shanghai, China). The\n(Beckman Instruments, NJ). protein samples (20 \u03bcL) with equal concentration were separated\n through 10 or 15% sodium dodecyl sulfate polyacrylamide gel electro\u00ad\n2.17. Release of cytochrome c phoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride\n (PVDF) membranes (Millipore Sigma, USA). After three washes with\n SGC-7901 cells were seeded in a 12-well plate (1 \u00d7 105 cells/well) TBST (20 mM Tris\u2013HCl, 150 mM NaCl, 0.2% Tween 20, pH = 8.0), the\nand cultured overnight at 37 \u25e6 C in a 5% CO2 incubator. Then the cells PVDF membranes were incubated with primary antibodies (1:5000\nwere treated with IC50 concentration of the complexes and liposomes for dilution) overnight at 4 \u25e6 C, followed by the corresponding secondary\n24 h. The cells were fixed with cold immunol staining fixed solution for antibodies for 70 min. Finally, after being washed with TBST four times,\n30 min and blocked with immunol staining blocking buffer for 1 h at the PVDF membranes were visualized using BeyoECL Star and the blots\nroom temperature. Next, the cells were treated with the primary anti\u00ad were imaged using a FluorChemE (ProteinSimple, CA, USA). The protein\nbody against cytochrome c (1:50 dilution) overnight at 4 \u25e6 C. Subse\u00ad expression levels were quantified with Image J software and normalized\nquently, after washing three times with immunol staining wash buffer, to the control \u03b2-actin.\nSGC-7901 cells were incubated overnight by Anti-Mouse FITC conju\u00ad\ngated IgG antibody (1:500 dilution) in the dark for 1 h at room tem\u00ad 2.22. Molecular docking studies\nperature. Finally, the images were acquired under the ImageXpress\nMicro XLS system (MD company, USA). The molecular structure of all candidate drug compounds obtained\n from PubChem databank (https://pubchem.ncbi.nlm.nih.gov/) and\n2.18. Measurement of CRT, HSP70, HMGB1 drawn by ChemBioDraw Ultra 17.0. The crystal structures of p53 (PDB\n ID: 4BUZ), CDK1 (PDB ID: 5LQF), p21(PDB ID: 5E0U), PARP (PDB ID:\n SGC-7901 cells (1 \u00d7 105 cells/well) were seeded in the 12-well plate 4PJV), PI3K (PDB ID: 1E8X), Caspase 3 (PDB ID: 1GFW), Bad (PDB ID:\novernight, then the cells were treated with IC50 concentration of Ir1, Ir2, 1G5J), Bax (PDB ID: 2K7W) and Bcl-2 (PDB ID: 4LVT) were downloaded\nIr1lipo and Ir2lipo. After 24 h, the cells were washed with cold PBS from the RCSB protein data bank (PDB) (http://www.rcsb.org/). Auto\u00ad\nthree times, fixed with alcohol (75%) and blocked with immunol dock Vina 1.2.2 was used to detect the binding energy and interaction\nstaining blocking buffer for 1 h at room temperature. Next, after washing patterns between drugs and targets.\nthree times with immunol staining wash buffer, the cells were incubated\nwith primary antibodies (CRT, HSP70 and HMGB1) at 1:70 dilutions at 2.23. Data analysis\n4 \u25e6 C overnight. After that, FITC-labeled Goat Anti-Rabbit IgG was added\nand incubated in dark for 1 h and Hoechst was used to stain the nuclei. All data were expressed as mean \u00b1 SD. Statistical significance was\nFinally, the cells were washed twice with PBS, then photographed evaluated using t-tests. The difference was significant when the *P value\nimmediately under the ImageXpress Micro XLS System. was <0.05.\n\n2.19. Cellular autophagy studies 3. Results and discussion\n\n SGC-7901 cells were placed into a 12-well plate and incubated 3.1. Synthesis and characterization\novernight. Then, the medium was discarded and replaced with fresh\nmedium containing IC50 concentration of Ir1, Ir2, Ir1lipo and Ir2lipo in The ligand DBIPB was obtained by the direct reaction of 1,10-phe\u00ad\nan incubator at 37 \u25e6 C, 5% CO2 and 95% air for 24 h. After that, the cells nanthroline-5,6-dione with 2,5-dibromoterephthalaldehyde in the\nwere washed three times with cold PBS and stained with 50 \u03bcM of MDC glacial acetic acid. The intermediates [Ir(bzq)2(DBIPB)](PF6) and [Ir\n(monodansylcadaverine) at 37 \u25e6 C for 30 min in the dark. Finally, the (piq)2(DBIPB)](PF6) were synthesized with [Ir(bzq)2Cl2]2 or [Ir\ncells were imaged under the ImageXpress Micro XLS system. (piq)2Cl2]2 and DBIPB in a mixture of dichloromethane and methanol\n (v/v, 2:1) as a solvent under argon. Finally, the intermediate product\n2.20. Effect on microtubules networks was purified by column chromatography on Al2O3 (pH = 7.05, weak\n alkaline) with a mixture of dichloromethane and acetone (v/v, 1:3) as\n SGC-7901 cells were grown in 12-well culture plates at a density of 1 eluent. Unexpectedly, in the process of purification, owing to an\n\u00d7 105 cells per well and exposed to the complexes and liposomes with occurrence of aldehyde and ketone condensation reaction, complexes [Ir\nIC50 concentration for 24 h. Next, the cells were washed three times with (bzq)2(DBIPB)](PF6) and [Ir(piq)2(DBIPB)](PF6) were transferred into\ncold PBS and fixed overnight at 4 \u25e6 C with cold immunol staining fix [Ir(bzq)2(DIBH)](PF6) (Ir1) and [Ir(piq)2(DIBH)](PF6) (Ir2).\n\n 5\n\fY. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112134\n\n\n The synthesized complexes Ir1 and Ir2 were characterized by HRMS, Table 1\nUV\u2013Vis, 1HNMR and 13CNMR. In the IR spectra, the peaks at 3355 cm\u2212 1 IC50 (\u03bcM) values of DBIPB, Ir1, Ir2, Ir1lipo and Ir2lipo toward selected cancer\nfor DBIPB, 3439 cm\u2212 1 for Ir1 and 3425 cm\u2212 1 for Ir2 are attributed to the cells for 48 h.\nN\u2013H (imidazole ring) stretching vibration. The peaks of 3064, 3043, Complex SGC- HepG2 A549 HeLa B16 NIH3T3\n3042 cm\u2212 1 for DBIPB, Ir1 and Ir2 are assigned to C\u2013H stretching vi\u00ad 7901\nbration. The peaks of 1783, 1706 and 1705 cm\u2212 1 are assigned to the DBIPB 48.2 \u00b1 > 200 > 200 36.6 \u00b1 78.4 \u00b1 > 200\naldehyde group of DBIPB and carboxyl groups of Ir1 and Ir2, respec\u00ad 3.5 3.8 5.1\ntively, the peaks of 846 cm\u2212 1 for Ir1 and 843 cm\u2212 1 for Ir2 are assigned to Ir1 > 200 > 200 > 200 > 200 > 200 > 200\n Ir1lipo 4.7 \u00b1 16.0 \u00b1 14.5 \u00b1 6.3 \u00b1 7.9 \u00b1 23.4 \u00b1\nthe stretching vibration of P\u2013F bond, and the peaks of 558 cm\u2212 1 for Ir1\n 0.2 1.4 0.4 0.2 0.9 0.7\nand 557 cm\u2212 1 for Ir2 are attributed to the bending vibration of P\u2013F Ir2 > 200 > 200 > 200 > 200 > 200 > 200\nbond (PF\u22126 ). In the HRMS spectra, the determined molecular weights are Ir2lipo 12.4 \u00b1 24.1 \u00b1 22.0 \u00b1 12.5 \u00b1 14.4 \u00b1 24.5 \u00b1\nconsistent with the expected values. 0.5 1.0 0.2 0.4 0.8 1.2\n In the 1H NMR spectra, for the Ir1, the peaks of 8.13 (s, 1H) and 7.92\n(s, 1H) are assigned to the hydrogens of Hk and Hn, the peaks of 5.30 (t,\n can\u2019t cross the cell membrane by passive diffusion [46]. The pH values\n1H), 4.57 (s, 1H), 2.72 (d, 2H) and 2.20 (s, 3H) are attributed to the\n of the complexes Ir1 and Ir2 in water-acetonitrile mixtures are 6.47 and\nhydrogens of Ho, HOH, Hp and Hr (-CH3). For the Ir2, the peaks of 8.44 (s,\n 6.64. The titration curves for Ir1 and Ir2 were shown in Fig. S6 (Sup\u00ad\n1H) and 8.17 (s, 1H) are assigned to the hydrogens of Hk and Hn, the\n porting Information), the pKa values are 6.60 and 6.79 for Ir1 and Ir2,\npeaks of 5.28 (t, 1H), 4.58 (s, 1H), 2.70 (d, 2H) and 2.19 (s, 3H) are\n respectively, indicating that the complexes can cross the cell membrane\nattributed to the hydrogens of Ho, HOH, Hp and Hr (-CH3). While the peak\n and enter the cell. The complexes exhibit weak acidity due to the\nfor the proton on nitrogen atom of the imidazole ring was not observed.\n dissociation of the N\u2013H proton in the imidazole ring [47].\nThis may be caused by metal coordination inducing electron deficiency\nin the ligand, therefore, the NH proton of the imidazole ring is very\nactive and easy to be exchanged between the two imidazole nitrogen 3.4. Cytotoxicity in vitro assays\natoms in solution.\n In the 13C NMR spectra, the peaks of 207.99, 70.55, 57.83 and 31.60 The cytotoxicity of DBIPB, Ir1, Ir2, Ir1lipo and Ir2lipo toward SGC-\nppm for Ir1 are attributed to the carboxyl group Cq, Co, Cp and Cr, 7901, HepG2, A549, HeLa, B16 tumor cells and normal NIH3T3 cells\nrespectively. The peaks of 207.98, 70.54, 57.82 and 31.60 ppm for Ir2 was determined using 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetra\u00ad\nare assigned to the carboxyl group Cq, Co, Cp and Cr, respectively. zolium bromide (MTT) assays. As observed from Table 1, ligand DBIPB,\n The stability of the complexes in PBS solution was determined, as Ir1 and Ir2 showed no inhibitory effect on the cell proliferation against\nshown in Fig. S3 (supporting information), the peak shapes of the the selected cell lines in vitro. Although the pKa values indicated that the\ncomplexes have no change at 0 and 48 h, indicating that the complexes complexes may cross cell membranes, low solubility of Ir1 and Ir2 in\nare stable in PBS solution. PBS inducing the less amounts of complexes entering the cells resulted in\n The ethanol injection method was used with the mole ratio of 5:8:30 no cytotoxic activity. To enhance anticancer activity, the complexes\nof DSPE-mPEG2000, CHO-HP and PC-98 T to produce stable Ir1lipo and were encapsulated in the liposomes by ethanol injection, it is easy for the\nIr2lipo and to achieve high drug loading. As shown in Fig. S4a (sup\u00ad complexes-loaded liposomes to enter the cells, thereby increasing the\nporting information), Ir1lipo and Ir2lipo were transparent and show cellular uptake. As expected, the liposomes Ir1lipo and Ir2lipo dis\u00ad\nlight yellow and orange with an encapsulation efficiency of 95.4% and played noticeable toxicity, especially for SGC-7901 cells with a low IC50\n97.2%, respectively. As can be seen from Fig. S4b (supporting infor\u00ad value of 4.7 \u00b1 0.2 and 12.4 \u00b1 0.5 \u03bcM. The cytotoxic activity of Ir1lipo\nmation), the average particle size of blank liposome, Ir1lipo and Ir2lipo and Ir2lipo is comparable to iridium(III) complex ([Ir(ppy)2(TFPIP)]\nwere 102.0 \u00b1 3.6, 158.4 \u00b1 2.02, 131.6 \u00b1 1.94 nm with PDI of 0.307, (PF6))-loaded liposome (IC50 = 7.6 \u00b1 0.5 \u03bcM, TFPIP = 2-(4\u2032 -tri\u00ad\n0.054, 0.122, respectively. These results indicated that the liposomes are fluoromethyl)-[1,1\u2032 -biphenyl]-4-yl)-1H-imidazo[4,5-f][1,10]phenan\u00ad\nhomogeneous and stable because of the low PDI (Polydispersity index) throline) [24]. Additionally, we also evaluated the cytotoxicity in vitro\nvalue. In addition, the Zeta potential of blank liposome, Ir1lipo and of blank liposomes and we found that the blank liposomes have no\nIr2lipo are \u2212 16.46 \u00b1 2.44, \u2212 17.40 \u00b1 0.92 and \u2212 16.03 \u00b1 2.64 mV, cytotoxic activity against the selected cancer and normal cells. Similar\nrespectively (Fig. S4c, supporting information), which further indicates results can be discovered in our previous work [38], hence, the blank\nthat the liposomes were relatively stable (10\u201320 mV) [45]. liposomes show no interference on the activity of Ir1lipo and Ir2lipo. In\n addition, the cytotoxicity of Ir1, Ir2, Ir1lipo and Ir2lipo follows the\n3.2. In vitro drug release studies of liposomes trend of Ir1lipo > Ir2lipo > Ir1 \u2248 Ir2. Hence, for the complexes with\n low solubility, liposomes are an effective carrier to deliver drug and\n The release of Ir1 and Ir2 from Ir1lipo and Ir2lipo was carried out in increase the anticancer efficacy.\nPBS (pH = 7.4) containing 0.5% Tween 80 (w/v), at 37 \u25e6 C and rotated at\n200 rpm by dialysis methods. As shown in Fig. S5 (supporting infor\u00ad 3.5. Uptake of complexes and liposomes\nmation), the percentage of cumulative drug release of Ir1lipo and\nIr2lipo at 4 h reached 13.63 \u00b1 0.81% and 16.34 \u00b1 0.52%, respectively. Liposomes are a new structure for encapsulating and delivering\nAt 84 h, the cumulative rates of Ir1lipo and Ir2lipo reached the highest bioactive agents, which are characterized by high permeability to hy\u00ad\nvalues of 37.53% and 38.72%. During the process of release, the cu\u00ad drophobic drugs and low permeability to hydrophilic drugs, carrying the\nmulative release rate increased gradually, and the cumulative release active molecules to specific sites of action and releasing them slowly\nrate of Ir2lipo was always higher than that of Ir1lipo. The in vitro [48,49]. To verify that liposomes encapsulated complexes can enter\nrelease profile of the liposomes showed that Ir1 and Ir2 from liposomes tumor cells through the cell membrane, SGC-7901 cells were treated\nIr1lipo and Ir2lipo was slowly performed. with IC50 concentration of Ir1, Ir2, Ir1lipo and Ir2lipo for 24 h and the\n cells were stained with DAPI. As seen in Fig. 1a, the cell nuclei were\n3.3. pKa values determination stained blue with DAPI, the complexes or their liposomes emit bright\n green fluorescence, moreover, the green fluorescence emitted by the\n To determine the value of the acid dissociation constant (pKa), the liposomes is stronger than those emitted by Ir1 and Ir2. The overlap of\ncomplexes were titrated in water-acetonitrile mixtures (v/v, 7:3) over a green and blue showed that the complexes and their liposomes can be\npH range of 2 to 10. Acidic drugs (pKa < 4) or basic drugs (pKa > 10) successfully uptaken by the cells. The uptaken amounts of Ir1lipo and\n\n 6\n\fY. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112134\n\n\n\n\nFig. 1. (a) Cellular uptake was assayed after an exposure of SGC-7901 cells to IC50 concentration of Ir1, Ir2, Ir1lipo and Ir2lipo for 24 h and the cells were stained\nwith DAPI. The cellular uptake was investigated using flow cytometry after treatment of SGC-7901 cells (b, I) and NIH3T3 cells (c, I) with IC50 concentration Ir1 (II),\nIr2 (III), Ir1lipo (IV) and Ir2lipo (V) for 24 h.\n\n\nIr2lipo are larger than those of Ir1 and Ir2. Ir1lipo (IV) and Ir2lipo (V) for 24 h, the green fluorescence intensity\n The cellular uptake of complexes and their liposomes was further increased by 18.12% for Ir1, 8.45% for Ir2, 89.78% for Ir1lipo and\ninvestigated using flow cytometry. As illustrated in Fig. 1b, the fluo\u00ad 66.29% for Ir2lipo, respectively, which is consistent with cytotoxicity of\nrescence intensity value in the control (I) was 374,337, the green fluo\u00ad complexes and complex-loaded liposomes toward NIH3T3 cells. The\nrescence intensity only increased by 0.23% and 4.72% in the Ir1 and Ir2- results confirmed that the complexes encapsulated into liposomes have\ntreated groups. However, the fluorescence intensity increased by high cell membrane permeability.\n22.78% and 21.08% in the Ir1lipo and Ir2lipo-treated groups compared\nwith that in the control after 24 h of exposure of SGC-7901 cells to 3.6. Inhibiting cell growth with 3D model\nIr1lipo (IV) and Ir2lipo (V). We also determined the cell uptake of Ir1,\nIr1lipo, Ir2 and Ir2lipo by normal NIH3T3 cells, as shown in Fig. 1c, Cancer cells grown in two-dimensional (2D) monolayer culture\nNIH3T3 cells (I) were exposed to IC50 concentration Ir1 (II), Ir2 (III), usually lose their tissue specificity, while multicellular tumor spheroids\n\n 7\n\fY. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112134\n\n\n\n\nFig. 2. Confocal microscopy images of the 3D SGC-7901 MCTSs after treatment with IC50 concentration of Ir1, Ir2, Ir1lipo and Ir2lipo for 24 h and the live/dead\ncells were stained with Calcein AM/PI.\n\n\n(MCTSs) referred to as 3D in vitro models can reproduce the sophisti\u00ad 3.7. Cell invasion studies\ncated microenvironment of solid tumors in vivo [50]. MCTSs reflect the\nnatural cellular behaviors and metabolic properties of cancer differen\u00ad Malignant tumor cells can infiltrate the lymphatic system and blood\ntiation, invasion and metastasis bridging the limitations of screening the vessels and metastasize to distant organs, resulting in metastatic spread\npathophysiology of anticancer drugs in 2D cell cultures [51,52]. To and treatment failure [53]. In vitro scratch assay is a simple, versatile\nfurther determine the ability of Ir1, Ir2, Ir1lipo and Ir2lipo to prevent and effective method to study collective cell migration and wound\ncell growth, Calcein AM (Calcein Acetoxymethyl Ester) and PI (propi\u00ad healing [54]. Therefore, the scratch assay was used to assess the inhib\u00ad\ndium iodide) were used to stain living and dead cells, respectively. As itory effect of the Ir1, Ir2, Ir1lipo and Ir2lipo on the migratory capacity\nshown in Fig. 2, the cells in the control spheroids and Ir1 and Ir2-treated of SGC-7901 cells. As shown in Fig. S7a (supporting information), Ir1\ngroups grew well with the bright green fluorescence (calcein AM, live and Ir2 attenuated the healing of cellular wounds to a certain extent\ncells). In contrast, in the IC50 concentration of Ir1lipo and Ir2lipo compared to the control, but still did not prevent the tendency of tumor\ngroups, the 3D model showed very low green fluorescence and the cells to migrate. However, the cells were treated with IC50 concentration\nsignificantly enhanced red fluorescence (PI, dead cells), which demon\u00ad of Ir1lipo and Ir2lipo for 24 h, the width of the wound was almost\nstrated that most of the cells were dead in SGC-7901 MCTSs. The results unchanged, and the cell migration was effectively inhibited. Focal\nfurther demonstrated that Ir1lipo and Ir2lipo display higher anticancer adhesion kinase (FAK) is a non-receptor tyrosine kinase with a key role\nactivity than Ir1 and Ir2 under the same conditions. in regulating cell migration, spreading and adhesion and survival of cells\n [55]. In cancer, FAK is a major driver of invasion and metastasis [56].\n Western blot analysis showed that Ir1lipo and Ir2lipo significantly\n\n\n 8\n\fY. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112134\n\n\n\n\nFig. 3. The determination of intracellular MDA (a) and GSH (b) in SGC-7901 treated with IC50 concentration of Ir1, Ir2, Ir1lipo and Ir2lipo for 24 h. The difference\nwas significant when *P < 0.05.\n\n\ninhibited FAK protein expression after an incubation of SGC-7901 cells digested within lysosomes, and recycled to maintain cellular homeo\u00ad\nwith IC50 concentration of Ir1lipo and Ir2lipo for 24 h compared with stasis [57,58]. Dysregulation of autophagy can lead to the development\nthe control (Fig. S7b, supporting information). In summary, Ir1lipo and of various diseases, including tumor development, and higher levels of\nIr2lipo can inhibit the migration, spreading and adhesion of SGC-7901 autophagy induce cell death [59]. Autophagy occurs with the upregu\u00ad\ncells through a downregulation of FAK protein. lation of Beclin-1 expression and the conversion of LC3-I into LC3-II,\n which is a hallmark of autophagy [60]. The extent of autophagy\n occurrence induced by Ir1, Ir2, Ir1lipo and Ir2lipo were evaluated\n3.8. Effect of the liposomes on autophagy using the specific dye monodansylcadaverine (MDC). As shown in\n Fig. S8a (supporting information), In the control or Ir1 and Ir2-treated\n Autophagy is a conserved cellular degradation process whereby groups, weak green fluorescence was found, indicating no occurrence of\ncellular organelles and proteins are engulfed by autophagosomes,\n\n\n\n\n Fig. 4. Assays of microtubules network of SGC-7901 induced by IC50 concentration of Ir1, Ir2, Ir1lipo and Ir2lipo for 24 h.\n\n 9\n\fY. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112134\n\n\n\n\n Fig. 5. Images of CRT (a), HMGB1 (b) and HSP70 (c) levels after SGC-7901 cells incubated with IC50 concentration of Ir1, Ir2, Ir1lipo and Ir2lipo for 24 h.\n\n\nautophagy. However, bright green fluorescence was observed after SGC- western blot, as presented in Fig. S8bc (supporting information), Ir1lipo\n7901 cells were exposed to IC50 concentration of lr1lipo and Ir2lipo for and Ir2lipo significantly increased the expression of Beclin-1 and\n24 h. The results showed that Ir1lipo and Ir2lipo significantly induced prompted the conversion of LC3-I into LC3-II. These results further\nautophagy. The expression of Beclin-1 and LC3 proteins was assayed by confirmed that Ir1lipo and Ir2lipo can effectively induce autophagy in\n\n\n 10\n\fY. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112134\n\n\n\n\nFig. 6. (a) Cell cycle distribution of SGC-7901 cells (I) treated with IC50 concentration of Ir1 (II), Ir2 (IV), Ir1lipo (III) and Ir2lipo (V) for 24 h. (b) The expression of\nCyclin B1, p53, CDK1 and p21 proteins after SGC-7901 cells were treated with IC50 concentration of Ir1, Ir2, Ir1lipo and Ir2lipo for 24 h. (c) The quantitative values\nof the expression of Cyclin B1, p53, CDK1, p21 and \u03b2-actin was used as the internal control.\n\n\nSGC-7901 cells. are 0.97 for Ir1, 0.98 for Ir2, 0.98 for Ir1lipo and 0.99 for Ir2lipo,\n indicating an existence of positive correlation. The results reveal that the\n complexes and their liposomes target the endoplasmic reticulum.\n3.9. The location of the liposomes at the endoplasmic reticulum\n\n The endoplasmic reticulum (ER) is the central organelle for the 3.10. Intracellular MDA and GSH detection\nbiosynthesis, folding, assembly and trafficking of secreted proteins and\ntransmembrane proteins, which determines the survival function of cells Oxidative stress is the factor that often contributes to cytotoxicity\n[61,62]. To determine the target organelle of the complexes and lipo\u00ad and apoptosis [63]. To determine the impact of Ir1, Ir2, Ir1lipo and\nsomes, SGC-7901 cells were incubated with IC50 concentration of Ir1, Ir2lipo on oxidative damage, the amount of peroxidation product\nIr2, Ir1lipo and Ir2lipo for 4 h at 37 \u25e6 C and observed under the malondialdehyde (MDA) was measured to estimate the lipid peroxida\u00ad\nImageXpress Micro XLS System. As can be seen from Fig. S9 (supporting tion extent of SGC-7901 cells. As displayed in Fig. 3a, MDA concentra\u00ad\ninformation), the endoplasmic reticulum was stained bright red with ER- tions were elevated significantly after the treatment of SGC-7901 cells\nTracker Red, the cellular nuclei were stained blue with Hoechst, with IC50 concentration of Ir1lipo and Ir2lipo for 24 h, whereas the\nfurthermore, the green fluorescence associated with lr1lipo and Ir2lipo complexes Ir1 and Ir2 showed a slight effect on the lipid peroxidation.\nwas stronger than those of lr1 and lr2. The overlap of red and green Glutathione (GSH) is an intracellular antioxidant that plays an impor\u00ad\nfluorescence indicated that the complexes and their liposomes locate at tant role in maintaining redox homeostasis by protecting cells from\nendoplasmic reticulum. The Pearson\u2019s colocalization coefficients (PCC) oxidative damage and detrimental xenobiotics [64]. As depicted in\nwere obtained by analyzing the red and green fluorescence intensity in Fig. 3b, intracellular GSH content was significantly reduced after 24 h of\n50 cells with the software image pro plus (version 6.0). The PCC values treatment of SGC-7901 with IC50 concentration of the complexes and\n\n 11\n\fY. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112134\n\n\n\n\nFig. 7. The apoptosis percentage of SGC-7901 cells (a) treated with IC50 concentration of Ir1 (b), Ir1lipo (c), Ir2 (d) and Ir2lipo (e) for 24 h. Q2, Q3 and Q4 stand\nforlate, early apoptosis and living cells.\n\n\nliposomes compared to the control, while the ratios of GSH/GSSG 3.12. Immunogenic cell death (ICD) assays\n(glutathione disulfide) in the control, Ir1, Ir2, Ir1lipo and Ir2lipo-\ntreated groups are 6.05 \u00b1 1.58, 5.78 \u00b1 1.73, 5.28 \u00b1 1.13, 4.53 \u00b1 1.41 Immunogenic cell death (ICD) is a unique cell death featured by the\nand 5.11 \u00b1 1.36, respectively. The inhibition of GSH production in cells activation of dying cell immune response and release of damage asso\u00ad\nand reduction of cellular antioxidant capacity follow the order of ciated molecular patterns (DAMPs) [67,68]. ICD is characterized by the\nIr2lipo > Ir1lipo > Ir2 > Ir1. The results suggested that Ir1lipo and high expression level of the calreticulin (CRT), heat shock protein 70\nIr2lipo can effectively inhibit GSH biosynthesis and promote lipid (HSP70), high mobility group box 1 (HMGB1) [69]. To further evaluate\nperoxidation causing oxidative damage to SGC-7901 cells. the ICD effects induced by the ER-targeted complexes and liposomes,\n SGC-7901 cells were incubated with IC50 concentration of Ir1, Ir2,\n3.11. Effect of the complexes and their liposomes on microtubule Ir1lipo and Ir2lipo for 24 h and CRT, HSP70, HMGB1 were explored. As\nnetworks shown in Fig. 5, SGC-7901 cells elicited by Ir1lipo and Ir2lipo treat\u00ad\n ment, translocation of CRT (a) to the cell membrane from ER, release of\n Microtubules are cytoskeletal filaments composed of \u03b1 and \u03b2 tubulin HMGB1 (b) and high expression of HSP70 (c) from the nuclei to the\nsubunits that play an important role in chromosome division and extracellular environment were identified. Overall, the results suggest\ntranslocation as well as cell motility in cancer cells [65]. In addition, that Ir1lipo and Ir2lipo localized at the ER, causing oxidative stress\ntubulin is an established target of various successful anticancer drugs damage which effectively induced immunogenic cell death.\nand disruption of microtubules is a major mechanism for inhibiting the\ngrowth of rapidly dividing cells [66]. As shown in Fig. 4, in the control, 3.13. Cell cycle arrest analysis\nSGC-7901 cells were complete in morphology, showing irregular\npolygonal shape, full of cytoplasm, and microtubules were dispersed Cancer is thought to be a result of cell cycle dysregulation and in\u00ad\nradially to the surrounding. After Ir1 (4.7 \u03bcM) and Ir2 (12.4 \u03bcM) incu\u00ad hibition of cell proliferation is thought to be a common mechanism for\nbation, the cell morphology was almost indistinguishable from that of anti-cancer drugs [70]. To investigate the arresting effect of the cycle of\nthe control and showed no effect on microtubule polymerization. the complexes and liposomes, flow cytometry was used to determine the\nHowever, the morphology of IC50 concentration of Ir1lipo and Ir2lipo- cell cycle phase distribution. As illustrated in Fig. 6a, SGC-7901 cells (I)\ntreated cells was significantly altered, with tumor cells being shuttle- were treated with IC50 concentration of Ir1 (II), Ir1lipo (III), Ir2 (IV)\nshaped, cytoplasm contracting and microtubules becoming blurred. and Ir2lipo for 24 h, compared to the control, a slight increase of 1.54%\nThe results confirmed that Ir1lipo and Ir2lipo can exert antitumor ef\u00ad for Ir1, 3.78% for Ir2 in the percentage of SGC-7901 cells at the G2/M\nfects through inhibiting tubulin polymerization. phase was discovered. However, the liposomes Ir1lipo and Ir2lipo\n induced an increase of 15.20% and 17.53% in the percentage of the cells\n at the G2/M phase, respectively. Hence, Ir1lipo and Ir2lipo showed\n\n 12\n\fY. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112134\n\n\n\n\nFig. 8. (a) The expression levels of PARP, Cleaved-PARP, PI3K, AKT, p-AKT, Caspase 3, Bad, Bax and Bcl-2 in SGC-7901 cells after treatment of SGC-7901 cells with\nIC50 concentration of Ir1, Ir2, Ir1lipo and Ir2lipo for 24 h. (b) Densitometric quantification of the expression of PARP, Cleaved-PARP, PI3K, AKT, p-AKT, Caspase 3,\nBad, Bax and Bcl-2 and \u03b2-actin was used an internal control.\n\n\nhigher inhibitory efficacy on the cell cycle arrest than complexes Ir1 and 3.15. Release of cytochrome c and PI3K/AKT signaling pathway\nIr2 under the same conditions. The results demonstrated that liposomes\nIr1lipo and Ir2lipo significantly inhibited cell proliferation at the G2/M B-cell lymphoma-2 (Bcl-2) family proteins locate in the mitochondria\nphase. through altering mitochondrial membrane permeability and promoting\n When cells damage occurs, the tumor suppressor p53 is activated and leakage of the apoptotic factor cytochrome c, which activates caspases\ncauses high levels of p21 (the cyclin-dependent kinase inhibitor) induces intrinsic apoptosis [75,76]. Caspases are a family of conserved\nexpression, triggering the downregulation of related cell cycle genes and cysteine proteases that play key roles in apoptosis, poly(ADP-ribose)\ninhibiting cell cycle arrest [71]. Cyclin B1 interacts with the cyclin- polymerase (PARP) as a cleavage substrate for Caspase 3, and expres\u00ad\ndependent kinase 1 (CDK1) to form an active heterodimer that guides sion of Cleaved-PARP is thought to be a marker for the onset of apoptosis\nthe G2 phase cells into the M phase [72,73]. To further confirm the cell [77,78]. The release of cytochrome c was detected by immunofluores\u00ad\ncycle arrest, the expression of related proteins was investigated using cence staining. As indicated in Fig. S10 (supporting information),\nwestern blot. As can be seen from Fig. 6b, the expression of p21 and p53 compared to the control, exposure of SGC-7901 cells to IC50 concen\u00ad\nproteins increased after incubation with IC50 concentration of Ir1lipo tration of Ir1 and Ir2 for 24 h resulted in a slight effect on the release of\nand Ir2lipo. Conversely, the expression of Cyclin B1 and CDK1 proteins cytochrome c. However, the fluorescence intensity after a 24 h treatment\nthat promote cell division and proliferation were inhibited, while Ir1 of SGC-7901 cells with IC50 concentration of Ir1lipo and Ir2lipo was\nand Ir2 show no obvious effect on the expression of Cyclin B1, p53, significantly increased, indicating that cytochrome c was efficiently\nCDK1, p21. The quantitative values of the expression of Cyclin B1, p53, released into the cytoplasm. In addition, the protein levels of Bcl-2\nCDK1, p21 and \u03b2-actin are shown in Fig. 6c. The results clearly show that family proteins were determined by western blot. As revealed in\nIr1lipo and Ir2lipo induce cell cycle arrest at the G2/M phase through Fig. 8a, Ir1lipo and Ir2lipo significantly upregulated the expression of\nactivating the p21/p53 signal pathway and inhibition of Cyclin B1/ Bad (Bcl-2-associated death) and Bax (Bcl-2-associated X) proteins and\nCDK1 heterodimer formation in the SGC-7901 cells. downregulated the expression of anti-apoptosis proteins Bcl-2 (B-cell\n lymphoma-2). The expression of Caspase 3 and Cleaved-PARP, which\n were downstream regulatory signals of apoptosis, were significantly\n3.14. Apoptosis studies\n increased.\n To further explore the effects of Ir1, Ir2, Ir1lipo and Ir2lipo on\n Apoptosis is widely known as programmed cell death and plays an\n apoptosis initiation and its possible mechanisms of action, western\nimportant role in cancer [74]. To explore the apoptotic function of Ir1,\n blotting was utilized to detect PI3K (phosphatidylinositol 3-kinase)-AKT\nIr2, Ir1lipo and Ir2lipo, the SGC-7901 cells were stained with FITC\n (protein kinase B) pathway. The results showed that after treatment of\n(fluorescein isothiocyanate)-labeled Annexin V (3\u2032 ,6\u2032 -dihydroxy-5-iso\u00ad\n SGC-7901 cells with IC50 concentration of Ir1lipo and Ir2lipo for 24 h,\nthiocyanato-3H-spiro(isobenzofuran-1,9\u2032 -xanthen)-3-one) and PI (pro\u00ad\n the expression of PI3K was inhibited, the expression of phosphorylated\npidium iodide) and quantified using flow cytometry. As revealed in\n AKT (p-AKT) was significantly reduced, while the expression of AKT\nFig. 7, in the control (a), the percentage of early apoptosis is 2.62%.\n levels was not obviously changed (Fig. 8a and b), Ir1 and Ir2 show weak\nAfter an exposure of 24 h of SGC-7901 cells to IC50 concentration of Ir1\n effect on the Bcl-2 family proteins. Overall, these results demonstrated\n(b), Ir2 (d), Ir1lipo (c) and Ir2lipo (e), an increase of 2.78% for Ir1,\n that Ir1lipo and Ir2lipo induced apoptosis in SGC-7901 via regulating\n6.96% for Ir1lipo, 2.55% for Ir2, 7.88% for Ir2lipo, respectively.\n Bcl-2 family proteins and inhibiting the PI3K/AKT signaling pathway.\nIr1lipo and Ir2lipo show higher apoptotic effect than Ir1 and Ir2 under\nthe identical conditions.\n\n 13\n\fY. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112134\n\n\n\n\n Fig. 9. The binding of the complexes with p53 (PDB: 4BUZ)(Ir1 a and Ir2 b), CDK1 (PDB: 5LQF) (Ir1 c and Ir2 d).\n\n\nTable 2\nThe lowest binding energies of the complexes with proteins.\n Complexes p53 CDK1 p21 PARP PI3K Caspase 3 Bad Bax Bcl-2\n\n Ir1 \u2212 12.50 \u2212 11.05 \u2212 9.16 \u2212 11.68 \u2212 9.04 \u2212 9.04 \u2212 9.08 \u2212 6.73 \u2212 7.68\n Ir2 \u2212 13.07 \u2212 10.26 \u2212 10.03 \u2212 11.79 \u2212 8.37 \u2212 8.37 \u2212 9.43 \u2212 7.05 \u2212 9.05\n\n\n\n3.16. Molecular docking studies p53, CDK1, p21, PARP, PI3K, caspase 3, Bad, Bax and Bcl-2 were ob\u00ad\n tained. As shown in Fig. 9, the complexes Ir1 and Ir2 interact with p53,\n To assess the affinity of liposome loading Ir1 and Ir2 with the target CDK1 through hydrogen bond (The binding of the complexes with p21\nproteins, we performed a molecular docking analysis using Autodock (PDB: 5E0U), PARP (PDB: 4PJV), PI3K (PDB: 1e8x), caspase 3 (PDB:\nVina (version 1.2.2). The method validation was performed using 1GFW), Bad (PDB: 1G5J), Bax (PDB: 2K7W), Bcl-2 (PDB: 4LVT) is shown\ncrystallized and docking ligand. Binding poses, interactions and the in Fig. S11, supporting information). The lowest binding energies are\nbinding energies generated by each interaction of the complexes with listed in Table 2. Complexes Ir1 and Ir2 show large binding affinities\n\n 14\n\fY. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112134\n\n\n\n\n Fig. 10. The molecular mechanism of Ir1lipo and Ir2lipo inducing apoptosis in SGC-7901 cells.\n\n\nwith p53, CDK1, p21 and PARP proteins. All authors have read the manuscript and approved the manuscript\n to be submitted to JIB.\n4. Conclusions\n Declaration of Competing Interest\n In this study, two iridium(III) complexes [Ir(bzq)2(DIPH)](PF6) (Ir1)\nand [Ir(piq)2(DIPH)](PF6) (Ir2) were synthesized and characterized. Ir1 Authors declare no competing interests exist.\nand Ir2 have no cytotoxicity toward SGC-7901, HepG2, HeLa, A549,\nB16 and normal NIH3T3 cells. When the complexes were encapsulated Data availability\ninto the liposomes Ir1lipo and Ir2lipo, Ir1lipo and Ir2lipo exhibit high\ncytotoxic activity against SGC-7901 cells. Compared to Ir1 and Ir2, the Data will be made available on request.\nliposome Ir1lipo and Ir2lipo significantly increased the uptake of SGC-\n7901 cells and inhibited the invasion and metastasis of tumor cells. On Acknowledgments\nthe other hand, Ir1lipo and Ir2lipo inhibit tubulin polymerization and\nregulated the expression cycle of p53 and p21 proteins arresting the cell This work was supported by the National Natural Science Foundation\ncycle at the G2/M phase. The studies on apoptotic mechanism indicated of China (No 21877018).\nthat Ir1lipo and Ir2lipo induced apoptosis through inhibiting the PI3K/\nAKT signaling pathway and triggering Bcl-2 family activation further Appendix A. Supplementary data\npromoting the release of cytochrome c and activation of Caspase 3 to\ncleave PARP. In addition, Ir1lipo and Ir2lipo colocalize at the endo\u00ad Supplementary data to this article can be found online at https://doi.\nplasmic reticulum, generate ER-stress to induce immunogenic cell death org/10.1016/j.jinorgbio.2023.112134.\n(ICD), inhibit GSH biosynthesis, promote lipid peroxidation to impair\nantioxidant capacity (Fig. 10). This work demonstrates that complexes- References\nloaded liposomes greatly enhance the anticancer activity of iridium(III)\ncomplexes and provides a help for the design and synthesis of new [1] R.L. Siegel, K.D. Miller, H.E. Fuchs, A. Jemal, Cancer statistics, CA Cancer J. 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