Targeted liposomes encapsulated iridium(III) compound greatly enhance anticancer efficacy and induce cell death via ferroptosis on HepG2 cells.

{"full_text": " European Journal of Medicinal Chemistry 265 (2024) 116078\n\n\n Contents lists available at ScienceDirect\n\n\n European Journal of Medicinal Chemistry\n journal homepage: www.elsevier.com/locate/ejmech\n\n\nResearch paper\n\nTargeted liposomes encapsulated iridium(III) compound greatly enhance\nanticancer efficacy and induce cell death via ferroptosis on HepG2 cells\nJing Chen a, 1, Wenlong Li a, 1, Gechang Li a, Xiaoming Liu b, Chunxia Huang a, Hua Nie b, **,\nLijuan Liang a, Yi Wang a, Yunjun Liu a, *\na\n School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, PR China\nb\n Jiaying University, Meizhou, 514031, 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, ligands 2-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline (PIP), 2-(2-nitrophenyl)-1H-imidazo[4,5-\nIridium(III) complex f][1,10]phenanthroline (NPIP), 2-(2-nitronaphthalen-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (NNIP) and\nTargeted liposome their iridium(III) metal compounds [Ir(ppy)2(PIP)](PF6) (ppy = 2-phenylpyridine, 1a), [Ir(ppy)2(NPIP)](PF6)\nRNA-Sequence\n (1b), [Ir(ppy)2(NNIP)](PF6) (1c) were designed and synthesized. The anti-cancer activities of 1a, 1b and 1c on\nFerroptosis\nAnti-tumor in vivo\n BEL-7402, HepG2, SK-Hep1 and non-cancer LO2 were detected using MTT method. 1a shows moderate, 1b and\n 1c display low or no anti-cancer activities. To elevate the anti-cancer effectiveness, encapsulating the compounds\n 1a, 1b and 1c into the ordinary or targeted liposomes to produce 1alip, 1blip, 1clip, or targeted 1aTlip, 1bTlip\n and 1cTlip. The IC50 values of 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip against HepG2 cells are 7.9 \u00b1 0.1, 8.6\n \u00b1 0.2, 16.9 \u00b1 0.5, 5.9 \u00b1 0.2, 7.3 \u00b1 0.1 and 9.7 \u00b1 0.7 \u03bcM, respectively. Specifically, the anti-tumor activity\n assays in vivo found that the inhibitory rates are 23.24 % for 1a, 61.27 % for 1alip, 76.06 % for 1aTlip. It is\n obvious that the targeted liposomes entrapped iridium(III) compound greatly enhance anti-cancer efficacy.\n Additionally, 1alip, 1blip and 1clip or targeted 1aTlip, 1bTlip and 1cTlip can effectively restrain the cell colony\n and proliferation in the G0/G1 period. 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip can increase reactive oxygen\n species (ROS) concentration, arouse a decline in the mitochondrial membrane potential and promote Ca2+\n release. RNA-sequence was applied to examine the signaling pathways. Taken together, the liposomes or targeted\n liposomes encapsulated compounds trigger cell death by way of apoptosis, autophagy, ferroptosis, disruption of\n mitochondrial function and PI3K/AKT/mTOR signaling pathways.\n\n\n\n\n1. Introduction compounds can intensify the effects of photodynamic treatment by up\u00ad\n setting the cellular redox equilibrium [21]. Sun et al. found that\n The serious side-effects of cisplatin stimulate scientist to search other sulfur-ligated organo-iridium(III) compounds could inhibit tumorigen\u00ad\nmetal compounds as an alternative of cisplatin. As potent anti-cancer esis and metastasis in breast cancer by targeting the Wnt/\u03b2-catenin\nreagents, iridium(III) compounds target the mitochondria, proteins, signaling pathway [22]. Yi and co-workers discovered [Ir(ppy)2(BD\u00ad\nDNA, and lysosomes [1,2]. At present, there are two types of iridium(III) PIP)]PF6 triggering A549 cell death through activating PI3K/Akt/mTOR\nanti-cancer compounds, namely, cyclic metal structures, and pathway [23].\nsemi-sandwich structures [3]. These iridium(III) compounds exert Recently, because of its non-apoptotic form of controlled cell death,\nanti-cancer effects via various mechanisms such as inhibiting protein ferroptosis has emerged as a potential target for cancer treatment [24].\nfunction and catalyzing cellular oxidation [4]. In the past decade, Wang et al. discovered that iridium(III) compounds bearing ferrocene\nanti-cancer activities of iridium(III) compounds have aroused great in\u00ad alteration relocate to the lysosome and dramatically impact ferroptosis\nterest and found a lot of iridium(III) compounds with distinctive and cancer immunity pathways [25]. On the other hand, liposomes as\nanti-cancer efficacy [5\u201320]. Zhong et al. reported that iridium nano-drugs carriers are widely applied in drug delivery system, and the\n\n\n * Corresponding author.\n ** Corresponding author.\n E-mail addresses: niehua@jyu.edu.cn (H. Nie), lyjche@gdpu.edu.cn (Y. Liu).\n 1\n These authors contribute equally.\n\nhttps://doi.org/10.1016/j.ejmech.2023.116078\nReceived 18 July 2023; Received in revised form 1 December 2023; Accepted 18 December 2023\nAvailable online 19 December 2023\n0223-5234/\u00a9 2023 Elsevier Masson SAS. All rights reserved.\n\fJ. Chen et al. European Journal of Medicinal Chemistry 265 (2024) 116078\n\n\nliposomes are made up of a bilayer structure constructed by phospho\u00ad BEL-7402, HepG2 and SK-Hep1, while 1alip, 1blip, 1clip or 1aTlip,\nlipids and cholesterol, the structures of liposome are similar to the 1bTlip and 1cTlip exerted excellent anti-cancer activity.\nbiofilm, therefore, all water-soluble and lipid-soluble drugs can be\nencapsulated into the liposome. In the latest studies, numerous com\u00ad 2. Results and discussion\npounds show low or no cytotoxicity, after these compounds have been\nencapsulated in liposomes, the liposome entrapped compounds display 2.1. Chemistry\nhigh cytotoxic activity in vitro and vivo [26\u201328]. Gu et al. reported that\nthe anti-cancer activity of liposome-loaded compound [Ir The absorption and luminescence spectra of 10.0 \u03bcM compounds 1a,\n(Hppy)2(HMNPIP)](PF6) increased by 43.5 times than the compound 1b, 1c were determined. See from Fig. S1a (ESI), the peaks at 273 nm (\u03b5\nalone [29]. = 85,860), 278 nm (\u03b5 = 79,540) and 275 nm (\u03b5 = 84,150) for 1a, 1b and\n As expectation, targeted liposome entrapped compounds will show 1c are allocated to \u03c0\u2013\u03c0* transitions of intraligands. As shown in Fig. S1b\nhigher anti-cancer efficacy than ordinary (non-targeted) liposomes. The (ESI), the compounds 1a, 1b, 1c emit a weak luminescence with a\ndesialic acid glycoprotein receptor (ASGP-R) is an endocytic receptor maximum emission peak at 563 (\u03bbex = 400 nm), 562 (\u03bbex = 400 nm) and\nspecifically expressed mainly on the surface of mammalian hepatocytes, 565 nm (\u03bbex = 275 nm).\nwhile it is rarely found in the non-hepatocytes, and its high binding In PBS solution, UV\u2013Vis spectra were applied to detect the stability of\naffinity with molecules such as N-acetylgalactosamine (GalNAc) or 1a, 1b, 1c, 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip at 0 and 24 h. As\ngalactose or polymers makes it an ideal target for the therapy of hepa\u00ad shown in Fig. S2 (ESI), we found no change in the peak shapes of 1a, 1b,\ntocellular carcinoma [30\u201332]. A breakthrough in the targeted delivery 1c, 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip, indicating a stable\nof compounds has been achieved by using GalNAc as a target head to existence of compounds and liposome-entrapped compounds at room\ntarget hepatocellular carcinoma [33]. In order to gain more insights, temperature.\nfurtherly understanding the anti-cancer mechanism of iridium(III) We determined the purity of 1a, 1b, 1c using high performance liquid\ncompounds, in this article, 2-phenyl-1H-imidazo [4,5-f][1,10[phenan\u00ad chromatography (HPLC) with methanol and water as mobile phase, the\nthroline (PIP), 2-(2-nitrophenyl)-1H-imidazo [4,5-f] [1,10]phenan\u00ad volume ratios of methanol and water are 70:30 for 1a, 60:40 for 1b,\nthroline (NPIP) and 2-(2-nitronaphthalen-1-yl)-1H-imidazo [4,5-f] [1, 80:20 for 1c. As shown in Fig. S3 (ESI), during the 30 min period, we\n10]phenanthroline (NNIP) were prepared (Scheme 1). Iridium(III) discovered only a main peak, the purity values for 1a, 1b, 1c calculated\ncompounds [Ir(ppy)2(PIP)](PF6) (1a), [Ir(ppy)2(NPIP)](PF6) (1b) and from the peak area are 98.41 %, 97.50 % and 96.54 %.\n[Ir(ppy)2(NNIP)](PF6) (1c) (ppy = 2-phenylpyridine) were successfully The peaks in the IR spectra at 3332, 3373, 3389 and 3329 cm\u2212 1 for\nsynthesized and characterized. The compounds were loaded into the ligand NNIP, 1a, 1b, 1c are allocated to N\u2013H (imidazole ring) stretching\nordinary or targeted liposomes to produce 1alip, 1blip, 1clip or targeted vibration, while the C\u2013H stretching vibration was observed at 3066\n1aTlip, 1bTlip, 1cTlip. 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenylte\u00ad cm\u2212 1 for NNIP, 3040 cm\u2212 1 for 1a, 3041 cm\u2212 1 for 1b and 3043 cm\u2212 1 for\ntrazolium bromide (MTT) assay showed that 1a showed moderate, 1b 1c. The stretching vibration for P\u2013F (PF\u22126 ) bond was found at 853 cm\u2212 1\nand 1c displayed low or no effect on blocking the proliferation of for 1a, 844 cm\u2212 1 for 1b, 854 cm\u2212 1 for 1c, whereas its bending vibration\n\n\n\n\n Scheme 1. Synthetic routes of ligands and compounds 1a, 1b, 1c.\n\n 2\n\fJ. Chen et al. European Journal of Medicinal Chemistry 265 (2024) 116078\n\n\nlocates at 557, 557 and 556 cm\u2212 1 for 1a, 1b and 1c, the stretching and 2.4. Cellular uptake studies\nbending vibration for P\u2013F bond in NNIP were not observed, which in\u00ad\ndicates that the compounds were successfully synthesized. Cell uptake of iridium(III) compounds by HepG2 cells was studied\n In the HRMS assay, we found the molecular weights of 1a, 1b and 1c under a fluorescent microscope to observe whether iridium(III) com\u00ad\nwere consistent between the determined and calculated values. Owing pounds can enter the cells. See from Fig. 1, after a 6 h treatment of\nto probably rapid exchange, the peak of the proton bonded to nitrogen in HepG2 cells with the compounds and their liposomes, the cell nuclei\nthe imidazole ring was not discovered in the 1H NMR spectra, similar were stained blue with 4\u2032,6-diamidino-2-phenylindole (DAPI), while 1a,\nresults were observed in other iridium(III) compounds [7,9]. Addition\u00ad 1b and 1c emit weak green fluorescence, a bright green fluorescence was\nally, we also found that the peaks at 172.59 ppm for 1b, 172.55 ppm for seen in 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip-treated groups. The\n1c are allocated for C atoms bonded with \u2013NO2 in the 13C NMR spectra. merge demonstrated that the liposomes 1alip, 1blip, 1clip, 1aTlip,\n 1bTlip and 1cTlip access the cells and focus on the cell nuclei. Fig. S6\n2.2. Preparation and characterization of liposomes (ESI) indicates that 1alip, 2alip and 3alip cause an increase of green\n fluorescence intensity by 5.30, 3.99 and 1.95 times, the targeted lipo\u00ad\n Liposomes are regarded as a potent drug delivery mechanism due to somes 1aTlip, 1bTlip and 1cTlip induce an increase by 7.52, 4.11 and\ntheir variable structure, lack of immunogenicity, and lack of toxicity. 3.45 times, while 1a, 1b and 1c increased by 5.13, 1.34 and 1.18 times\nFurthermore, the stability of the liposomes is strongly connected to their than the original. Consequently, low cell uptaken amounts of 1b and 1c\nencapsulation rate, polydispersity index (PDI), and particle size poten\u00ad agree with their cytotoxic activity.\ntial. We used thin film dispersion method to prepare ordinary and tar\u00ad\ngeted liposomes. Fig. S4a (ESI) shows that 1alip, 1blip, 1clip, 1aTlip, 2.5. Anti-proliferative ability in vitro\n1bTlip and 1cTlip show light-yellow. The encapsulation rates for 1a, 1b\nand 1c in the 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip are 90.31 %, The antiproliferative ability of compounds 1a, 1b, 1c toward HepG2,\n86.32 %, 84.25 %, 86.54 %, 89.49 % and 89.67 %, respectively. The BEL-7402, SK-Hep1, LO2 was assessed using MTT. After 1.56, 3.125,\nmean particle sizes for 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip are 6.25, 12.5, 25, 50, 100 \u03bcM of 1a, 1b or 1c were added to the above cells\n103.07 \u00b1 1.9, 94.4 \u00b1 0.7, 94.1 \u00b1 0.7, 83.0 \u00b1 1.2, 87.9 \u00b1 2.6 and 93.3 for 48 h, the IC50 values were obtained (Table 1). We observed that 1a\n\u00b1 2.4 nm, respectively (Fig. S4b, ESI). The PDIs for 1alip, 1blip, 1clip, demonstrates moderately antiproliferative active against BEL-7402,\n1aTlip, 1bTlip and 1cTlip are 0.132 \u00b1 0.012, 0.200 \u00b1 0.006, 0.233 \u00b1 HepG2, Sk-Hep1 and non-cancer LO2 cells, whilst 1b and 1c showed\n0.003, 0.231 \u00b1 0.006, 0.287 \u00b1 0.013 and 0.264 \u00b1 0.024. The PDI values non-toxic with IC50 values more than 100 \u03bcM. To explore why 1a\nare less than 0.3, indicating that the liposomes were homogeneous and showed moderate cytotoxic activity, while 1b and 1c displayed no\nstable. The zeta potentials were determined to be \u2212 83.74, \u2212 63.70, cytotoxicity, we determined the partition coefficient (logP), logP values\n\u2212 58.56, \u2212 66.49, \u2212 64.26, and \u2212 60.77 mV for 1alip, 1blip, 1clip, 1aTlip, for 1a, 1b, 1c are 0.276 \u00b1 0.035, \u2212 0.438 \u00b1 0.026, \u2212 0.700 \u00b1 0.042. The\n1bTlip and 1cTlip (Fig. S4c, ESI). In summary, the particle size, low PDI low logP values suggest that compounds 1b and 1c have poor lip\u00ad\nvalues and zeta potentials show that the ordinary or targeted liposomes ophilicity, which results in a less amount of the compounds to enter the\nare very high stability [34,35]. cells. In addition, the cell uptake also confirms the less amounts of 1b\n and 1c to enter the cells. Therefore, 1b and 1c show low antiproliferative\n2.3. pKa and pH measurement ability to inhibit the cell growth. To improve the anti-cancer efficiency,\n compounds 1a, 1b, 1c were loaded in the ordinary and targeted lipo\u00ad\n Compounds 1a, 1b, 1c were dissolved in CH3CN\u2013H2O, using pH somes to produce 1alip, 1blip, 1clip and targeted 1aTlip, 1bTlip and\nmeter to measure the pH values. The pH values of 6.90, 6.64 and 6.81 for 1cTlip. Surprisingly, 1alip, 1blip and 1clip exhibit high or moderate\n1a, 1b and 1c indicate that the compounds show weak acidity. Ac\u00ad anti-cancer effect on BEL-7402, HepG2, SK-Hep1 cells, while the tar\u00ad\ncording to pH partition theory [36,37], once inside cells, the compounds geted liposomes 1aTlip, 1bTlip, 1cTlip show higher antiproliferative\nwith weak acid will enter alkaline compartments mitochondria [38]. activity than ordinary liposomes 1alip, 2alip, 3alip. Particularly, 1aTlip,\nHence, the compounds can enter the mitochondria. Additionally, pka 1bTlip, 1cTlip reveal the highest antiproliferative activity toward\nvalues play an important role in drug properties including distribution, HepG2 cells with a low IC50 value of 5.9 \u00b1 0.2, 7.3 \u00b1 0.1 and 9.7 \u00b1 0.7\nadsorption, metabolism and so on [39]. Manallack reported that the \u03bcM. Hence, the targeted liposomes greatly enhance the antiproliferative\ncompounds may enter cancer cells in the range of 4 < pKa < 10 [40]. ability. 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip display moderate\nFig. S5 (ESI) reveals that the pKa values of 1a, 1b and 1c are 6.85, 6.62 anti-cancer efficacy on non-cancer LO2 cells. Additionally, we also\nand 6.65, respectively. The pKa values fall well within the scope of 4 < discovered that 1alip, 1blip, 1aTlip and 1bTlip exhibit higher anti-\npKa < 10, indicating that the compounds may enter the cells. cancer efficiency than cisplatin and [Ir(bzq)2(DIPH)]PF6-loaded lipo\u00ad\n Note: In the following cell experiments, the excited and emission somes (IC50 = 16.0 \u00b1 1.4 \u03bcM) [41] on HepG2 cells. Owing to 1aTlip,\nwavelengths of dyes are listed as following: DAPI (2-(4-Amidinophenyl)- 1bTlip, 1cTlip showing the highest antiproliferative ability against\n6-indolecarbamidine dihydrochloride): \u03bbex = 340 nm, \u03bbem = 488 nm, HepG2, therefore, in the following cell experiments, we investigate\nDCFH-DA (2\u2032,7\u2032-Dichlorodihydrofluorescein diacetate): \u03bbex = 488 nm, anti-cancer effect of the compounds and compounds-loaded liposomes\n\u03bbem = 525 nm, Mito-Tracker Red: \u03bbex = 579 nm, \u03bbem = 599 nm, Fluo-3 on HepG2 cells.\nAM (Fluo-3-pentaacetoxymethyl ester): \u03bbex = 506 nm, \u03bbem = 526 nm,\nJC-1 monomers: \u03bbex = 514 nm, \u03bbem = 529 nm, JC-1 aggregates: \u03bbex = 2.6. Wound healing, colony formation, cell cycle arrest\n585 nm, \u03bbem = 590 nm, BODIPY 581/591C11 oxidized: \u03bbex = 500 nm,\n\u03bbem = 510 nm, BODIPY 581/591C11 reduced: \u03bbex = 581 nm, \u03bbem = 591 Cancer is characterized by infinite cell growth and metastasis [42].\nnm, Annexin V-FITC: \u03bbex = 488 nm, \u03bbem = 525 nm, propidium iodide To further assess the ability of compounds hindering cell growth and\n(PI): \u03bbex = 535 nm, \u03bbem = 615 nm. migration, we performed cell cloning and scratch tests. As depicted in\n In all the following cell experiments, IC50 concentrations for 1a, 1b, Fig. S7a (ESI), the ordinary liposomes 1alip, 1blip, 1clip and targeted\n1c, 1alip, 1blip, 1clip, 1aTlip, 1bTlip, 1cTlip are 1a (8.0 \u03bcM), 1b (8.6 liposomes 1aTlip, 1bTlip, 1cTlip can validly prevent the cell migration,\n\u03bcM), 1c (17.0 \u03bcM), 1alip (8.0 \u03bcM), 1blip (8.6 \u03bcM), 1clip (17.0 \u03bcM), while 1a, 1b or 1c show a low ability to hinder the cell migration. On the\n1aTlip (6.0 \u03bcM), 1bTlip (7.3 \u03bcM) and 1cTlip (9.7 \u03bcM). other hand, in a variety of human cancers, such as hepatocellular car\u00ad\n cinoma, colon cancer, and breast cancer, overexpressed FAK protein was\n observed. It can mediate a variety of cell function-related signaling,\n including cell proliferation, migration, and adhesion [43,44].\n\n 3\n\fJ. Chen et al. European Journal of Medicinal Chemistry 265 (2024) 116078\n\n\n\n\nFig. 1. Cellular uptake was measured while HepG2 cells were exposed to the compounds and compound-loaded liposomes 1a (8.0 \u03bcM), 1b (8.6 \u03bcM), 1c (17.0 \u03bcM),\n1alip (8.0 \u03bcM), 1blip (8.6 \u03bcM), 1clip (17.0 \u03bcM), 1aTlip (6.0 \u03bcM), 1bTlip (7.3 \u03bcM) and 1cTlip (9.7 \u03bcM) for 6 h and stained with DAPI (\u03bbex = 340 nm).\n\n\n targeted liposomes downregulated the expression of CDK2, which\nTable 1\n further confirms at the G0/G1 period, 1alip, 1blip, 1clip, 1aTlip, 1bTlip\nIC50 values (\u03bcM) of complexes and liposomes toward cancer cells for 48 h.\n and 1cTlip hinder cell growth. Additionally, Fig. 2b demonstrated that\n Compounds BEL-7402 HepG2 SK-Hep1 LO2 ordinary liposomes 1alip, 1blip, 1clip, and targeted liposomes 1aTlip,\n 1a 13.78 \u00b1 0.55 20.9 \u00b1 1.2 22.4 \u00b1 1.2 32.1 \u00b1 0.8 1bTlip and 1cTlip can validly inhibit FAK protein expression, which\n 1b >100 78.2 \u00b1 3.7 >100 >100 suggests that 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip may validly\n 1c >100 >100 >100 >100\n hinder HepG2 cell migration via inhibiting the FAK signaling pathway.\n 1alip 8.9 \u00b1 0.4 7.9 \u00b1 0.1 12.9 \u00b1 0.8 10.8 \u00b1 0.3\n 1blip 12.9 \u00b1 0.5 8.6 \u00b1 0.2 27.8 \u00b1 0.9 16.7 \u00b1 0.5\n 1clip 16.4 \u00b1 0.4 16.9 \u00b1 0.5 22.5 \u00b1 1.5 14.7 \u00b1 0.4 2.7. Exploration of apoptotic mechanism with RNA-sequence assay\n 1aTlip 8.6 \u00b1 0.2 5.9 \u00b1 0.2 9.1 \u00b1 0.1 10.8 \u00b1 0.1\n 1bTlip 11.1 \u00b1 0.2 7.3 \u00b1 0.1 8.1 \u00b1 0.6 9.8 \u00b1 0.3\n To explore the apoptotic mechanism, RNA-sequence assay was car\u00ad\n 1cTlip 10.4 \u00b1 0.3 9.7 \u00b1 0.7 20.3 \u00b1 0.8 11.3 \u00b1 0.6\n cisplatin nd ried out. Using 2 \u00d7 IC50 concentration 1alip and 1aTlip to treat HepG2\n 14.8 \u00b1 3.4 9.4 \u00b1 1.5 18.2 \u00b1 2.6 cells, after 24 h, the RNA was extracted, and the RNA-sequencing was\n analyzed to obtain heatmap (Fig. 3a for 1alip, 3b for 1aTlip), upset and\nnd: not determination.\n venn map (c and d), GO enrichment and KEGG enrichment maps.\n Observing from Fig. 3c and d, in 1alip group, 1462 genes showed sig\u00ad\n To further assess the ability of the compounds and compound-loaded\n nificant changes in the expression, in which the expression of 667 and\nliposomes to prevent HepG2 cell colony, through a continuous seven\n 795 genes was increased and reduced. In 1aTlip-treated group, 1026\nday-treatment of HepG2 with 1a (8.0 \u03bcM), 1b (8.6 \u03bcM), 1c (17.0 \u03bcM),\n genes were dysregulated, including 472 upregulated and 554 down\u00ad\n1alip (8.0 \u03bcM), 1blip (8.6 \u03bcM), 1clip (17.0 \u03bcM), 1aTlip (6.0 \u03bcM), 1bTlip\n regulated genes. Notably, we found that the same expression of 817\n(7.3 \u03bcM) and 1cTlip (9.7 \u03bcM), cells were washed four times and stained.\n genes in 1alip and 1aTlip groups was observed. The above clustering\nAs shown in Fig. S7b (ESI), we observed that the live cells (purple)\n analysis of the dysregulated genes showed that the gene expression\ndecreased in the 1alip-1clip and 1aTlip-1cTlip-treated groups. There\u00ad\n profiles in the control, 1alip and 1aTlip groups were significantly\nfore, ordinary 1alip, 1blip, 1clip and targeted liposomes 1aTlip, 1bTlip\n distinguishable.\nand 1cTlip can validly inhibit the cell colony, while compounds 1a, 1b\n The results of GO Enrichment analysis for 1alip and 1aTlip are shown\nand 1c demonstrate a weak efficacy on inhibiting the cell colony in\n in Fig. 3e and g, differentially expressed genes are heavily enriched\nHepG2 cells.\n mainly in cellular biological processes (BP) involved in tissue develop\u00ad\n By regulating cell cycle, inhibition of cancer cell proliferation is an\n ment, response to stimuli, response to stimulus, response to organic\nimportant strategy for cancer treatment. To assess the efficacy of the\n substance, development of multicellular organism and cellular response\ncompounds and compound-encapsulated liposomes on the progression,\n to chemical stimulus.\nwe measured cell cycle distribution. See from Fig. 2a, at G0/G1 phase, a\n KEGG enrichment analysis showed that 1alip and 1aTlip (Fig. 3f and\n24 h exposure of HepG2 to 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip\n h) induce cell death via the same signaling pathways, including p53\n(IC50 concentration) led to an increase of 19.70, 9.61, 12.12, 21.67,\n signaling pathway, apoptosis, autophagy, MAPK signaling pathway,\n16.64 and 21.47 %, meanwhile, a reduction at S period was also\n glutamate glycine metabolism and cell adhesion molecules. In addition,\ndiscovered. Compounds 1a, 1b and 1c exhibit a poor antiproliferative\n 1aTlip induces cell death through PI3K/AKT signaling pathway.\nability, targeted liposomes 1aTlip, 1bTlip and 1cTlip show the highest\nability to prevent the cell proliferation among the compounds and\n 2.8. Annexin V and PI staining apoptosis and mechanism studies\ncompounds-loaded liposomes. The results show that 1alip, 1blip, 1clip,\n1aTlip, 1bTlip and 1cTlip exert anti-cancer activity through hindering\n Drugs access cell to damage DNA and prevent the replication of DNA,\ncell growth in G0/G1 period.\n leading to apoptosis [47]. Most cytotoxic anti-cancer drugs have been\n On the other hand, cycle protein CDK2 (cyclin-dependent kinase 2)\n reported to cause cell apoptosis [48]. Main aim of cancer chemotherapy\nplays a key role in the progression of cell cycle [45]. Inhibition of CDK2\n is to cause apoptosis in tumor cells after exposure to anti-cancer drugs.\nactivity can validly hinder HepG2 cell proliferation [46]. As shown in\n Apoptotic cells have morphological features such as chromatin\nFig. 2b, we observed that ordinary liposomes 1alip, 1blip, 1clip, and\n condensation, DNA breakage, apoptotic vesicle formation and cell\n\n 4\n\fJ. Chen et al. European Journal of Medicinal Chemistry 265 (2024) 116078\n\n\n\n\nFig. 2. (a) Cell cycle distribution of HepG2 (I) exposed to 1a (8.0 \u03bcM II), 1b (8.6 \u03bcM III), 1c (17.0 \u03bcM IV), 1alip (8.0 \u03bcM V), 1blip (8.6 \u03bcM VI), 1clip (17.0 \u03bcM VII),\n1aTlip (6.0 \u03bcM VIII), 1bTlip (7.3 \u03bcM IX), 1cTlip (9.7 \u03bcM X), (b) expression of FAK and CDK2 after HepG2 cells were exposure to 1a (8.0 \u03bcM), 1b (8.6 \u03bcM), 1c (17.0\n\u03bcM), 1alip (8.0 \u03bcM), 1blip (8.6 \u03bcM), 1clip (17.0 \u03bcM), 1aTlip (6.0 \u03bcM), 1bTlip (7.3 \u03bcM) and 1cTlip (9.7 \u03bcM) for 24 h.\n\nshrinkage, which are associated with endogenous nucleic acid endonu\u00ad caspase 3, cleaving PARP and controlling the expression of Bcl-2 family\nclease activity. Wang et al. found that three newly synthesized iridium proteins.\n(III) compounds exert anti-cancer activity via Furthermore, inhibition of PI3K/AKT/m-TOR can enhance the acti\u00ad\nmitochondria-endoplasmic reticulum pathway [49]. It was found that vation of autophagic signaling and induces tumor cell death [52\u201355].\nphosphatidylserine was recognized by Annexin V but not stained by AKT as an essential mediator plays a key role in the PI3K signaling\npropidium iodide (PI) [50]. The IC50 dose of compounds and pathway [56], inhibition of AKT expression will promote apoptosis. As\ncompounds-loaded liposomes were used to treat HepG2 cells, we shown in Fig. 4b, 1alip and 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip\ndetermined the percentage of live cell, early, late apoptosis. As can be downregulated the expression of PI3K, mTOR and AKT. Additionally, we\nseen in Fig. 4a, in 1a, 1b, 1c-treated groups, the proportion of early examined the expression of phosphorylation level of p-mTOR, p-PI3K,\napoptotic cells is 6.37 %, 5.25 % and 5.38 %, respectively. We discov\u00ad see from Fig. 4c, 1alip and 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip also\nered that compounds 1a, 1b, 1c reveal low apoptotic efficiency. How\u00ad reduce the expression of p-mTOR and p-PI3K. These results suggest that\never, ordinary liposomes 1alip, 1blip, 1clip and targeted liposomes the ordinary liposomes or targeted liposomes promote apoptosis by\n1aTlip, 1bTlip and 1cTlip induce an increase of 12.87, 16.47, 6.97, controlling the expression of Bcl-2 family proteins, hindering the signal\n15.67, 17.37 and 13.67 % comparison to the control. The targeted pathway of PI3K/AKT/mTOR.\nliposomes-entrapped compounds 1aTlip, 1bTlip and 1cTlip show higher\napoptotic effect than 1alip, 1blip and 1clip. The data obtained suggest\nthat the targeted liposome-encapsulated compounds show the highest 2.9. Co-location and mitochondrial dysfunction\napoptotic efficacy among these compounds and their liposomes.\n To further investigate the mechanism of 1alip, 1blip, 1clip and tar\u00ad Mitochondria serve as the command-and-control center of apoptosis,\ngeted liposomes 1aTlip, 1bTlip and 1cTlip inducing apoptosis in HepG2 and induction of apoptosis decreases the mitochondrial membrane po\u00ad\ncells, expression of Bcl-2 family proteins was explored. As depicted in tential [57]. As can be seen in Fig. 5a, the mitochondria were stained red\nFig. 4b and 24 h exposure of HepG2 to 1alip, 1blip, 1clip, 1aTlip, 1bTlip with Mito Tracker Deep Red, we found that compounds 1a, 1b, 1c, li\u00ad\nand 1cTlip (IC50 concentration) led to an increase of Bax, caspase 3, and posomes 1alip, 1blip, 1clip, targeted liposomes 1aTlip, 1bTlip and\na decrease of Bcl-2 expression, we also observed that PARP (poly(ADP- 1cTlip colocalized strongly in the mitochondria. The results are consis\u00ad\nribose) polymerase) was cleaved. On the other hand, Bax/Bcl-2 ratio is tent with those obtained from the pH determination. To evaluate the\nproportional to the apoptosis [51]. As expected, it is clear from Fig. 4b correlation, we calculated Pearson\u2019s colocalization coefficients (PCC) to\nthat increase in the expression of Bax and decrease of Bcl-2 resulted in an be 0.9977 for 1a, 0.9981 for 1b, 0.9977 for 1c, 0.9976 for 1alip, 0.9947\nincrease of Bax/Bcl-2 ratio, which further confirms that 1alip, 1blip, for 1blip, 0.9988 for 1clip, 0.9960 for 1aTlip, 0.9940 for 1bTlip, and\n1clip, 1aTlip, 1bTlip and 1cTlip induce apoptosis through revitalizing 0.9954 for 1cTlip, respectively, which hints a positive correlation. The\n obtained results demonstrate that 1a, 1b, 1c, 1alip, 1blip, 1clip, 1aTlip,\n\n 5\n\fJ. Chen et al. European Journal of Medicinal Chemistry 265 (2024) 116078\n\n\n\n\nFig. 3. Analysis of single-cell RNA sequencing heatmap for 1alip (a) and 1aTlip (b), (c and d) upset and venn map for 1alip and 1aTlip. (e and f) GO and KEGG\nenrichment for 1alip, (g and h) GO and KEGG for 1aTlip while HepG2 cells were treated with 2 \u00d7 IC50 concentration 1alip and 1aTlip for 24 h.\n\n\n1bTlip and 1cTlip interact on the mitochondria. fluorescence matching with high MMP, while in the mitochondrial\n To investigate whether iridium(III) compounds and their liposomes damage state, aggregates JC-1 are converted into monomer JC-1 (green\ntrigger apoptotic through mitochondrial signaling pathway, the efficacy fluorescence matching with a low MMP) [9]. Observe from Fig. 5b, the\nof metal compounds and liposome entrapped compounds on the varia\u00ad change of red (control) into the green fluorescence (treated groups)\ntion of mitochondrial membrane potential (\u0394MMP) was examined using indicates that compounds, ordinary liposomes, and targeted liposomes\nan indicator of JC-1 (5,5\u2032,6,6\u2032-tetrachloro-1,1\u2032,3,3\u2032-tetraethylbenzimida\u00ad encapsulated compounds cause a decrease in the MMP. Additionally,\nzolyl carbocyanine iodide). Aggregates of JC-1 produces bright red ratio of red/green fluorescence was quantitatively determined. To\n\n\n 6\n\fJ. Chen et al. European Journal of Medicinal Chemistry 265 (2024) 116078\n\n\n\n\nFig. 4. (a) Apoptosis assays of HepG2 cells (I) incubated with 1a (II), 1b (III), 1c (IV), 1alip (V), 1blip (VI), 1clip (VII), 1aTlip (VIII), 1bTlip (IX), 1cTlip (X) at IC50\nconcentration for 24 h. (b) assay of expression of proteins after HepG2 cells were treated with IC50 concentration 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip for 24\nh, (c) The expression of p-mTOR and p-PI3K while HepG2 cells were treated with IC50 concentration 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip for 24 h.\n\n\neliminate the disruptions of the green fluorescence caused by the com\u00ad 2.10. Determination of intracellular Ca2+ levels\npounds or compound-loaded liposomes, 1a, 1b, 1c, 1alip, 1blip, 1clip,\n1aTlip, 1bTlip and 1cTlip were used as references. As depicted in Fig. S8 In disease and stress states, the cellular environment is disrupted,\n(ESI), 1a, 1b, 1c, 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip caused a leading to an increase in free calcium ions [58]. Calcium ion concen\u00ad\ndecline in the red/green ratio comparison with the control, moreover, trations are closely related to apoptosis, cellular autophagy, and cell\ncompounds 1a, 1b, 1c exhibit a poor effect on the change in MMP. The proliferation. To explore the impact of compounds and\ngained results hint that the liposome-loaded metal compounds act on the compound-loaded liposomes on the change of Ca2+ concentration using\nmitochondria, then cause a reduction of MMP, lastly, lead to mito\u00ad Fluo-3 AM as a probe. In the presence of esterase, Fluo-3 AM is cleaved\nchondrial dysfunction to trigger apoptosis. into Fluo-3, binding of Fluo-3 with Ca2+ will produce a bright fluores\u00ad\n cence. As shown in Fig. S9 (ESI), we found that only an increase by 1.05,\n\n\n 7\n\fJ. Chen et al. European Journal of Medicinal Chemistry 265 (2024) 116078\n\n\n\n\nFig. 5. (a) The location of the compounds and compounds entrapped liposomes in the Mitochondria after HepG2 cells were exposed to 1a, 1b, 1c, 1alip, 1blip, 1clip,\n1aTlip, 1bTlip and 1cTlip for 4 h. (b) The changes of mitochondrial membrane potential while HepG2 cells were exposed to cisplatin (positive control), 1a, 1b, 1c,\n1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip for 24 h. The concentrations of 1a, 1b, 1c, 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip are 8.0, 8.6, 17.0, 8.0, 8.6, 17.0,\n6.0, 7.3 and 9.7 \u03bcM.\n\n\n1.16 and 1.36 times in 1a-1c-treated group comparison with control. 1bTlip and 1cTlip caused an increase of DCF fluorescence by 83.2, 17.5,\nHowever, the calcium ion concentrations in the 1alip, 1blip, 1clip and 7.58, 80.74, 11.7 and 37.8 times than that in the control. The targeted\nthe targeted liposomes, 1aTlip, 1bTlip and 1cTlip-treated groups were liposome 1cTlip show higher efficiency on ROS than ordinary liposomes\nsignificantly increased by 8.61, 3.06,1.84 times, and 8.82, 8.51 and 3.10 1clip. Unexpectedly, liposomes 1blip and 1clip exhibit higher efficacy\ntimes than original. Compounds 1a, 1b, 1c show a weak ability to raise on ROS than their targeted liposomes 1aTlip and 1bTlip. Therefore,\nCa2+ concentration, while the targeted liposomes 1aTlip-1cTlip reveal 1alip-1clip and 1aTlip-1cTlip can enhance ROS content. Taken together,\nthe highest ability to enhance the intracellular Ca2+ concentration. The we consider that liposomes 1alip-1clip and targeted liposomes\nresults demonstrate that ordinary and the targeted liposomes entrapped 1aTlip-1cTlip cause an increase in ROS content.\ncompounds can elevate the intracellular Ca2+ concentration.\n 2.12. Autophagy studies\n2.11. Reactive oxygen species assay\n Autophagy is closely associated with a variety of human diseases and\n ROS inducing lipid peroxidation plays a key role in apoptosis, physiology, including hepatocellular carcinoma. Excessive autophagy\nautophagy, and ferroptosis [59]. To eliminate the disruption, in the may lead to autophagic cell death. LC3 is a marker of autophagy, and\nassay of ROS, the compounds and liposome-encapsulated compounds during autophagy formation, the cytoplasmic type LC3-I is converted to\nwere used as references. As shown in Fig. S10 (ESI), compound 1a, 1b the (autophagosome) membrane type LC3-II. Beclin-1 is an essential\nand 1c display a low effect on the ROS content. However, 24 h treatment molecule in autophagosome formation, which mediates the localization\nof HepG2 cells with 1alip, 1blip, 1clip and targeted liposomes 1aTlip, of other autophagic proteins to autophagic vesicles, thus regulating the\n\n 8\n\fJ. Chen et al. European Journal of Medicinal Chemistry 265 (2024) 116078\n\n\nformation and maturation of mammalian autophagosomes [60,61]. As compounds. In addition, the ratio of GSH/GSSG decreased (Fig. 6c), it\ncan be seen from Fig. S11 (ESI), comparing with the control group, LC3-I was caused by the reaction of GSH with ROS to generate GSSG, leading\nin the liposomes and targeted liposomes-treated group was transferred to reduction of GSH content. Additionally, it can be seen from Fig. 6d\ninto LC3-II. Additionally, we also discovered an upregulation in the that the intracellular MDA levels enhanced in 1alip, 1blip, 1clip, 1aTlip,\nBeclin-1 expression. The results show that the liposomes and targeted 1bTlip and 1cTlip-treated groups. The above results implied that 1alip,\nliposomes can validly induce autophagy in HepG2 cells. 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip can lessen GSH amount and raise\n MDA amount and further induce lipid peroxidation.\n2.13. Ferroptosis exploration\n (III) Lipid peroxidation detection\n (I) Assay of number of live cells\n In the ferroptosis, lipid peroxidation was examined using 4,4-\n difluoro-5-(4-phenyl-1,3butadenyl)-4-bora-3a, 4a-diaza-sindacene-3-\n Enhancement of intracellular ROS is closely related to ferroptosis.\n undecanoic acid (C11-BODIPY) as stained reagent. It is well known that\nFerrotin-1 (Fer-1) can inhibit incidence of ferroptosis [62]. To confirm\n red and green fluorescence represent low and high degree of lipid per\u00ad\nwhether the compounds and their liposomes can cause ferroptosis, using\n oxidation, respectively [66,67]. As seen in Fig. 6e and f, in the control,\nordinary or targeted liposomes-loaded compounds as references, num\u00ad\n we observed red fluorescence corresponding to low degree of lipid\nber of live cells was examined with or without Fer-1. See form Fig. 6a,\n peroxidation, while we discovered bright green fluorescence matching\nthe number of live cells (with Fer-1) increased comparison to 1alip,\n with a high degree of lipid peroxidation in 1alip-1clip and targeted\n1blip, 1clip, 1aTlip, 1bTlip and 1cTlip alone (without Fer-1). The\n 1aTlip-1cTlip-treated groups. The results confirm that 1alip-1clip,\nnumber of live cells increased by 9.08, 7.11, 4.04, 11.90, 8.88 and 7.39\n 1aTlip-1cTlip can induce a lipid peroxidation.\ntimes for 1alip + Fer-1, 1blip + Fer-1, 1clip + Fer-1, 1aTlip + Fer-1,\n1bTlip + Fer-1 and 1cTlip + Fer-1, respectively. The results indicate that\n (IV) Ferroptosis-related proteins expression\n1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip can cause ferroptosis.\n Ferroptosis can directly or indirectly prevent the expression of\n (II) Detection of GSH and MDA levels\n glutathione peroxidase 4 (GPX4), resulting in a decrease of intracellular\n antioxidant systems. While ROS generation in mitochondria causes\n A decrease in glutathione (GSH) content in cells can increase the\n cellular malfunction [68]. Ferroptosis is also characterized by the\nsusceptibility of cancer cells to various forms of programmed death and\n accumulation of lipid peroxidation, which can be initiated by the inhi\u00ad\nto chemotherapy [63]. During apoptosis, GSH is oxidized to convert into\n bition of biomarker of iron toxicity GPX4, disrupting the GSH/GSSG\nglutathione disulfide (GSSG), reflecting a decrease in the ratio of GSH\n system and thus killing cells [69]. To determine whether GPX4 is\nversus GSSG, hence, the change in GSH/GSSG redox status can be used\n involved in this process, we examined the expression of GPX4. As shown\nas an indicator of the extent to which apoptosis occurs [64,65].\n in Fig. 6g, the expression of GPX4 was significantly downregulated.\nMalondialdehyde (MDA) is a hallmark by-product of the lipid peroxi\u00ad\n Autophagy can degrade ferritin, which promotes ferroptosis [70].\ndation process. Levels of MDA, a product of lipid peroxidation, increase\n Therefore, we examined the expression of ferritin, as depicted in Fig. 6g,\ndramatically when the cells undergo oxidative stress. To further inves\u00ad\n 1alip and 1blip, 1clip, 1aTlip, 1bTlip, 1cTlip downregulated the\ntigate the mechanism of the liposomes or targeted entrapped compounds\n expression of ferritin, indicating that 1alip, 1blip, 1clip, 1aTlip, 1bTlip,\npromoting apoptosis, the intracellular levels of GSH and MDA were\n 1cTlip can cause ferroptosis through autophagy to degrade the ferritin.\nexamined. As depicted in Fig. 6b, in the liposomes and targeted lipo\u00ad\n Activation of HMGB1 (high mobility group protein 1) has been shown to\nsomes entrapped compounds groups, the GSH content decreased com\u00ad\n increase ROS content, which triggers ferroptosis [71]. Fig. 6g showed\nparison to the control, especially, 1cTlip shows the highest effect on the\n that 1alip, 1blip, 1clip, 1aTlip, 1bTlip, 1cTlip led to a significant\nreduction of GSH among the ordinary and targeted liposomes entrapped\n\n\n\n\nFig. 6. (a) Cell viability assay. (b and c) assay of GSH content and ratio of GSH versus GSSG. (d) measurement of intracellular MDA levels. (e and f) lipid peroxidation\ndetection while HepG2 cells were stained with C11-BODIPY581/591 (\u03bbex = 581 nm for red, \u03bbex = 500 nm for green), (g) the expression of HMGB1, Ferritin, and GPX4,\n*P < 0.05, **P < 0.01. The concentrations of 1a, 1b, 1c, 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip are 8.0, 8.6, 17.0, 8.0, 8.6, 17.0, 6.0, 7.3 and 9.7 \u03bcM.\n\n 9\n\fJ. Chen et al. European Journal of Medicinal Chemistry 265 (2024) 116078\n\n\nincrease in HMGB1 content, which further suggested that 1alip, 1blip, differences in the food consumption, mental status, and physical\n1clip, 1aTlip, 1bTlip, 1cTlip can induce ferroptosis. behavior of the nude mice in each administration group were observed,\n indicating that 1a, 1alip and 1aTlip have no acute toxicity and relatively\n2.14. Antitumor activity in vivo safe. Fig. 7b\u2013d described the shape and size of the tumors and relative\n tumor volume. As can be observed, the tumors in control grew signifi\u00ad\n We further explored the effectiveness of 1a, 1alip and 1aTlip on cantly fast, the relative tumor volume in 1a-treated group was slightly\ntumor suppression, tissue section and immunohistochemical analysis. smaller than the control, while the growth of tumors was validly\nTumor models were successfully established by injecting HepG2 cells inhibited in 1alip and 1aTlip-treated groups. As depicted in Fig. 7e and f,\nsuspension into the axillae of BALB/c nude mice. The body weight of surprisingly, the targeted liposome 1aTlip showed the highest efficacy\nmice and volume of tumors were measured daily. As shown in Fig. 7a, on preventing the tumor growth, the inhibitory tumor growth rates in\nthe body weight of the nude mice in each group basically maintained a 1a, 1alip and 1aTlip-treated groups are 23.24 %, 61.27 % and 76.06 %,\nstable growth trend during the treatment period. Meanwhile, no respectively. The difference of 1a, 1alip and 1aTlip in the inhibitory\n\n\n\n\nFig. 7. (a) Weight change curve of mice. (b) Relative tumor volume. (c) BALB/c mouse bearing HepG2 cell tumor. (d) The sizes of tumors. (e and f) The average\ntumor weight and inhibitory rate (*P < 0.05). (g) H&E staining heart, liver, spleen, lung, kidney, brain, and tumor. (h) assays of \u03b3-H2AX and Ki67 in tumor tissues.\n\n 10\n\fJ. Chen et al. European Journal of Medicinal Chemistry 265 (2024) 116078\n\n\ntumor growth rate lies in the different cytotoxic activity against HepG2 gained from cell cycle, cell colony and wound healing demonstrate that\ncells, moreover, the targeted liposome 1aTlip can target the tumor. 1alip-1clip, 1aTlip-1cTlip can validly prevent the cell proliferation in\nTaken together, we consider that targeted liposomes bearing targeted G0/G1 period. Through RNA sequencing studies, we found that 1alip,\nhead GAlNAc can directly deliver compound 1a to reach the tumor 1blip, 1clip and 1aTlip, 1bTlip and 1cTlip cause cell death through p53\nposition, and really realize a targetability on inhibiting hepatocellular and PI3K/AKT/mTOR signaling pathways. Additionally, 1alip-1clip,\ntumor growth. 1aTlip-1cTlip elevate ROS content, lessen mitochondrial membrane\n Hematoxylin is one of the most used protocols for histopathological potential, result in the mitochondrial dysfunction, further promote Ca2+\nanalysis of cells and tissues in cancer diagnosis. Observed from Fig. 7g, release. Through activating Beclin-1 protein, promoting the conversion\nin the 1a, 1alip and 1aTlip-treated groups, the heart, spleen, liver, kid\u00ad from LC3-I into LC3-II, inducing autophagy. Increase of cell viability\nney, and brain tissues showed no significant changes compared to the (with Fer-1), reduction of GSH, increase of MDA, upregulation of\nblank, However, a small area of alveolar wall was found in the lungs ferritin, HMGB1 and downregulation of GPX4, and C11-BODIPY stain\u00ad\nwith mild thickening, a small amount of granulocyte infiltration and ing results reveal that 1alip-1clip, 1aTlip-1cTlip can effectively cause\ncapillary stasis in the alveolar wall, and mild granular degeneration in ferroptosis. Particularly, antitumor activity in vivo confirms that tar\u00ad\nthe hepatocytes. Additionally, cytoplasmic laxity, nucleus fixation and geted liposome entrapped compounds 1a displays very high antitumor\nnecrosis and significant increase in cytoplasmic eosinophilia were efficiency with an inhibitory rate of 76.06 % and really realize the tar\u00ad\nobserved in the tumor tissue. geted liposomes to directly deliver 1a to tumor position. \u03b3-H2AX and\n In addition, we investigated the positive rate of phosphorylated ki67 stained results further confirm that 1alip-1clip, 1aTlip-1cTlip can\nhistone H2AX (\u03b3-H2AX) and Ki67 to confirm apoptosis and inhibiting cause apoptosis and inhibit cell proliferation and tumor growth. In\ncell proliferation and tumor growth. The positive rate of \u03b3-H2AX reflects summary, we consider that 1alip-1clip, 1aTlip-1cTlip induce HepG2 cell\nthe damage degree of DNA [72]. As shown in Fig. 7h, the positive rate in death via four pathways (Fig. 8). In summary, this work can provide help\ncontrol is 18.9 (\u00b11.4)%. A positive increase of 7.9 % for 1a, 12.5 % for for understanding anticancer mechanism and design and synthesis of\n1alip and 17.2 % for 1aTlip indicated an enhancement of apoptotic cells. iridium(III) compounds as potent anticancer candidate drugs.\nHence, 1alip and 1aTlip cause DNA damage and prevent DNA replica\u00ad\ntion, further induce apoptosis, while 1a displays low apoptotic effi\u00ad 4. Experimental\nciency, this is consistent with the apoptotic assay. In the assay of ki67,\nthe positive rate in control is 24.7 (\u00b11.5)%, in 1a, 1alip and 1aTlip-\u00ad 4.1. Materials and methods\ntreated groups, the positive rates are 18.5 (\u00b11.1), 13.4 (\u00b10.8) and\n12.3 (\u00b11.2)%, the decrease in the positive rate shows that 1alip and The cell bank of Shenzhen (Shenzhen, China) offered human hepa\u00ad\n1aTlip can validly prevent the cell proliferation and tumor growth. The tocellular carcinoma SK-Hep1, BEL-7402, HepG2, human normal liver\nresults demonstrate that 1alip and 1aTlip can inhibit the tumor growth, LO2 cells. We purchased 2-phenylpyridine (Hppy), benzaldehyde, 1-\nmoreover, 1aTlip shows higher inhibitory efficacy than 1alip under the nitrobenzaldehyde, 1-nitro-2-naphthalene carboxaldehyde and\nsame experimental conditions. IrCl3\u22c53H2O from Beijing HWRK Chem Co. Ltd. Guangzhou Chemical\n Reagent Factory supplied DMSO and 1,10-phenanthroline (phen). Hy\u00ad\n3. Conclusions drogenated soybean phospholipids (HSPC), distearoyl phosphatidyl\u00ad\n glycerol (DSPG-Na) and cholesterol (CHS) were purchased from the\n In this study, PIP, NPIP, NNIP and iridium(III) metal compounds 1a, Aviator Pharmaceutical Technology Co. Ltd (Shanghai). N-acetylga\u00ad\n1b and 1c were synthesized, ordinary 1alip, 1blip, 1clip and the targeted lactosamine (GalNAc), D-galactosamine (Gal), D-lactitol (D-lactitol)\n1aTlip, 1bTlip and 1cTlip were successfully prepared. Compounds 1a, 1b were obtained from Sigma. Through direct injection, HRMS was tested\nand 1c display low or no cytotoxic activity, while 1alip, 1blip, 1clip and with a Waters Xevo G2-XS QTof mass spectrometer. Using acetone-d6 as\n1aTlip, 1bTlip and 1cTlip show high anticancer activity. The results solvent and tetramethylsilane (TMS) as internal standard, NMR spectra\n\n\n\n\n Fig. 8. The mechanism of 1aTlipo inducing HepG2 cell death.\n\n 11\n\fJ. Chen et al. European Journal of Medicinal Chemistry 265 (2024) 116078\n\n\nwere examined under a Varian-500 spectrometer (500 MHz). 1350 m, 1267 s, 1212 w, 1160 w, 1127 w, 1081 s, 1062 w, 1030 m,\n 962 w, 854 m, 793 s, 754 s, 556 m, 450 m. HRMS (CH3CN): m/z =\n4.2. Preparation of ligands and iridium(III) compounds 892.2159 [(M \u2212 PF6)+]. 1H NMR (Acetone-d6, 500 MHz, Fig. S16,\n ESI): \u03b4 8.96 (dd, J = 1.0 Hz, J = 8.0 Hz, 2H, Ha,a\u2019), 8.71 (d, J = 8.5 Hz,\n4.2.1. Preparation of PIP, NPIP 1H, Ho), 8.26 (d, J = 8.5 Hz, 2H, H1,1\u2019), 8.20 (d, J = 8.5 Hz, 1H, Hl),\n The ligands PIP and NPIP were prepared as the same method as 8.11 (d, J = 8.0 Hz, 1H, Hs), 7.96\u20137.94 (m, 4H, Hc,c\u2019, 7,7\u2019), 7.89\u20137.86\nliterature [9,73]. (m, 4H, H4,4\u2019,b,b\u2019), 7.74\u20137.71 (m, 1H, Hm), 7.67\u20137.62 (m, 2H, Hp,q),\n 7.50 (t, J = 8.0 Hz, 2H, H10,10\u2019), 7.05 (t, J = 6.0 Hz, 2H, H8,8\u2019), 7.01 (t,\n4.2.2. Preparation of NNIP J = 6.0 Hz, 2H, H9,9\u2019), 6.95 (t, J = 7.5 Hz, 2H,H3,3\u2019), 6.33 (dd, J = 1.0\n The mixture of phenanthroline-5,6-dione (0.21 g, 1.0 mmol) [74], Hz, J = 7.5 Hz, 2H, H2,2\u2019). 13C NMR (DMSO\u2011d6, 125 MHz, Fig. S17,\n1-nitro-2-naphthaldehyde (0.15 g, 1.0 mmol) and NH4Ac (1.16 g, 15 ESI): 172.6 (Ce,e\u2019), 167.5 (C5,5\u2019), 167.2 (Ch), 157.1 (C1,1\u2019), 151.9 (Ca,\nmmol) was dissolved in 20 mL of ice acetic acid and refluxed at 130 \u25e6 C. a\u2019), 150.4 (Cn, 6,6\u2019), 149.9 (C3,3\u2019), 149.1 (Ck), 146.1 (Ci), 144.8 (Cc,c\u2019),\nAfter 2 h, using concentrated ammonia to neutralize the solution to 144.5 (Cj), 143.4 (Cl), 139.8 (Co), 138.9 (Cq), 132.6 (Cp), 131.9 (C8),\nproduce a red precipitate. Yield: 86 %. Anal calcd for C23H13N5O2: C, 131.8 (C8\u2019), 130.6 (C10,10\u2019), 129.9 (Cs), 129.3 (11,11\u2019), 128.7 (Cd,d\u2019),\n70.58; H, 3.35; N, 17.89 %. Found: C, 70.77; H, 3.16; N, 17.75 %. IR 127.5 (Cf), 127.1 (C7,7\u2019), 126.1 (C9,9\u2019), 125.5 (C4), 125.0 (C4\u2019), 124.2\n(KBr, cm\u2212 1): 3332 w, 3066 w, 1701 w, 1625 w, 1604 w, 1537 s, 1498 w, (Cg), 122.6 (Cm), 121.2 (Cb,b\u2019), 120.3 (C2,2\u2019).\n1426 m, 1220 m, 1196 m, 1143 m, 1071 s, 1029 s, 864 w, 821 w, 741 w,\n661 m, 448 s. HRMS (CH3OH): m/z = 390.0997 ([M \u2212 H]-). 4.3. Preparation of targeted liposomes\n\n4.2.3. Synthesis of [Ir(ppy)2(PIP)](PF6) (1a), [Ir(ppy)2(NPIP)](PF6) 3,6-dioxo-divinyloctanedioic acid (Dio), 5-cholesten-3b-ol-3,6-\n(1b) and [Ir(ppy)2(NNIP)](PF6) (1c) dioxo-vinyloctanedioate (CHS-Dio) and a targeted ASGP-r receptor\n The precursor cis-[Ir(ppy)2Cl]2 (0.15 mmol, 0.161 g) [75] and PIP glycoligand molecule (CHS-Dio-6-GalNAc) were prepared based on the\n(0.30 mmol, 0.086 g), NPIP (0.30 mmol, 0.102 g), NNIP (0.30 mmol, literature [76]. Through a thin film dispersion method, the targeted li\u00ad\n0.117 g) were dissolved in 30 mL dichloromethane and 15 mL methanol posomes encapsulated 1a, 1b and 1c were obtained via the following\nand refluxed at 40 \u25e6 C under argon, after 7 h, the solution was cooled and procedures: (I) 40 mg of hydrogenated soy phosphatide (HSPC), 10 mg\nadded with saturated aqueous NH4PF6, then stirred for 2 h, the solution of cholesterol (CHS), 4 mg of distearoyl phosphatidylglycerol\nwas filtered to obtain yellow filtrate, then removing the solvent, the (DSPG-Na), 2 mg of 1a, 1b, 1c, 6.5 mg of CHS-PEG2-6-GalNAc together\nyellow solid was gained. Through a neutral alumina column with were placed in a round-bottom flask and mixed thoroughly. (II) dissolve\ndichloromethane and acetone as eluent, the crude product was purified the above mixture in chloroform, heated at 55 \u25e6 C in a water bath and a\nto obtain yellow powder. lipid film was formed. (III) 10 mL distilled water was added into the\n dried lipid film, then heating at 55 \u25e6 C for 1 h. (IV) The suspension of the\n 1a: Yield\uff1a81 %. Anal cacld for C41H28N6PF6Ir: C, 52.28; H, 3.00; N, liposome was ultrasonicated (ultrasonic cell milling machine,\n 8.92 %. Found: C, 52.17; H, 3.06; N, 8.81 %. IR (KBr, cm\u2212 1): 3373 w, Sciente-IID, China) for 10 min. (V) homogenization was performed two\n 3040 w, 1658 w, 1607 s, 1562 s, 1548 m, 1226 s, 1163 s, 1103 w, times at 90 MPa and three times at 180 MPa, respectively. (VI) the\n 1076 m, 1063 m, 1030 s, 951 s, 759 s, 703 m, 557 s, 430 s. HRMS samples were finally stored at 4 \u25e6 C.\n (CH3CN): m/z = 797.1872 [(M \u2212 PF6)+]. 1H NMR (Acetone-d6, 500 The ordinary liposome entrapped iridium(III) compounds 1alip,\n MHz, Fig. S12, ESI): \u03b4 9.21 (d, J = 8.0 Hz, 2H, Ha,a\u2019), 8.34 (d, J = 7.5 1blip and 1clip were prepared with the same method described for the\n Hz, 2H, Hc,c\u2019), 8.28 (d, J = 8.0 Hz, 2H, H1,1\u2019), 8.14 (d, J = 5.0 Hz, 2H, targeted liposomes without CHS-PEG2-6-GalNAc.\n Hj,n), 8.08\u20138.06 (m, 2H, H7,7\u2019), 7.97 (d, J = 7.5 Hz, 2H, Hb,b\u2019), 7.89 (t,\n J = 7.5 Hz, 2H, H10,10\u2019), 7.65 (t, J = 7.5 Hz, 2H, H8,8\u2019), 7.57 (t, J = 4.4. Encapsulation rate determination\n 7.0 Hz, 1H, Hl), 7.52 (d, J = 5.5 Hz, 2H, Hk,m), 7.07 (t, J = 7.0 Hz, 2H,\n H9,9\u2019), 7.01\u20136.95 (m, 4H, H3,3\u2019,4,4\u2019), 6.31 (d, J = 7.0 Hz, 2H, H2,2\u2019). The encapsulation rate (EE%) was determined by UV\u2013Vis spectra\n 13 based on the following procedures: (A) Standard curve of absorbance\n C NMR (DMSO\u2011d6, 125 MHz, Fig. S13, ESI): 167.4 (C5,5\u2019), 153.4\n (Ce,e\u2019), 150.9 (Ch), 149.6 (Ca,a\u2019), 148.8 (C1,1\u2019), 144.6 (C6,6\u2019), 144.5 versus compound concentration: (I) 10 mg 1a, 1b and 1c compounds\n (Ci), 139.2 (Cc,c\u2019,9,9\u2019), 132.7 (Cl), 131.7 (C2,2\u2019,3,3\u2019), 130.8 (Ck,m), were dissolved in anhydrous ethanol to obtain 1 mg mL\u2212 1 solution. (II)\n 130.7 (C7,7\u2019), 130.0 (C11,11\u2019), 129.7 (C10,10\u2019), 127.5 (C8,8\u2019), 127.1 (Cj, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0 mL of 1 mg mL\u2212 1 solutions were diluted\n n), 125.5 (Cd,d\u2019), 122.8 (C4,4\u2019,3,3\u2019), 120.4 (C2,2\u2019,b,b\u2019). with anhydrous ethanol to gain a concentration of 5, 7.5, 10, 12.5, 15\n 1b: Yield: 83 %. Anal calcd for C41H27N7O2PF6Ir: C, 49.90; H, 2.76; and 20 mg mL\u2212 1 solution (5 mL). (III) Determine the maximum absor\u00ad\n N, 9.94 %. Found: C, 49.78; H, 2.86; N, 9.87 %. IR (KBr, cm\u2212 1): 3389 bance values, then plot the standard curve (concentration versus\n w, 3041 w, 1707 w, 1672 w, 1605 s, 1583 s, 1529 s, 1477 m, 1447 s, absorbance value). (B) (I) 1 mL of liposomes were placed in 10 mL of\n 1351 s, 1314 m, 1268 s, 1226 m, 1187 w, 1162 m, 1087 s, 1063 s, volumetric bottle and diluted with absolute ethanol to prepare 10 mL\n 1031 s, 844 s, 809 m, 756 s, 669 m, 557 s, 448. HRMS (CH3CN): m/z solution. (II) Liposomes breakage: 10 mL solution was dissolved in\n = 843.2027 [(M \u2212 PF6)+]. 1H NMR (Acetone-d6, 500 MHz, Fig. S14, anhydrous ethanol at 25 \u25e6 C for 3 h, then filtered through a microporous\n ESI): \u03b4 8.99 (d, J = 8.5 Hz, 2H, Ha,a\u2019), 8.27 (d, J = 8.0 Hz, 2H, H1,1\u2019), membrane (0.22 \u03bcm diameter). (III) By UV\u2013Vis spectroscope, determine\n 8.19 (d, J = 8.0 Hz, 1H, Hj)), 8.08 (d, J = 8.0 Hz, 2H, Hc,c\u2019), 7.99\u20137.95 the maximum absorbance values. (IV) The concentration of compounds\n (m, 5H, H7,7\u2019,b,b\u2019,k), 7.90\u20137.86 (m, 3H, H10,10\u2019,l), 7.71 (s, 1H, Hm), 1a, 1b and 1c was calculated based on the obtained standard curve. (V)\n 7.50 (d, J = 6.5 Hz, 2H, H9,9\u2019), 7.08\u20137.04 (m, 2H, H8,8\u2019), 7.01\u20136.98 The encapsulation efficiency (EE) values were gained via the following\n (m, 4H, H3,3\u2019,4,4\u2019), 6.31 (dd, J = 1.0, J = 7.5 Hz, 2H, H2,2\u2019). 13C NMR equation:\n (DMSO\u2011d6, 125 MHz, Fig. S15, ESI): 172.6 (Ce,e\u2019), 167.5 (C5,5\u2019), 151.3\n Cdrug\n (Ch), 149.5 (Cn), 147.4 (Ca,a\u2019), 144.6 (C1,1\u2019), 143.9 (C6,6\u2019), 139.1 EE% = \u00d7 100%\n Tdrug\n (C3,3\u2019), 132.7 (Ck), 132.2 (Cj), 131.7 (Cc,c\u2019), 131.4 (Cl), 130.7\n (C8,8\u2019,10,10\u2019), 126.9 (C7,7\u2019,9,9\u2019), 125.5 (C11,11\u2019), 124.4 (Ci), 124.3 (Cd, where Cdrug stands for the concentration of the compounds entrapped in\n d\u2019), 122.7 (Ci,m,g,f), 120.4 (C4,4\u2019,2,2\u2019,b,b\u2019). the liposome, Tdrug represents total concentration of the compounds.\n 1c: Yield: 87 %. Anal calcd for C45H29N7O2PF6Ir: C, 52.12; H, 2.82;\n N, 9.46 %. Found: C, 52.33; H, 2.71; N, 9.60 %. IR (KBr, cm\u2212 1): 3329\n w, 3043 w, 1676 w, 1628 m, 1604 s, 1532 s, 1476 s, 1448 m, 1393 w,\n\n 12\n\fJ. Chen et al. European Journal of Medicinal Chemistry 265 (2024) 116078\n\n\n4.5. Examination of particle size, zeta potential and stability observed under a microscope at 0 and 24 h.\n\n The mean particle size and zeta potential of ordinary liposome or\n 4.11. Ca2\uff0bconcentration determination\ntargeted liposome entrapped compounds were detected in a nano\u00ad\nparticle size analyzer (Beckman coulter). According to the literature\n IC50 concentrations of 1a, 1b, 1c, 1alip, 1blip, 1clip, 1aTlip, 1bTlip\n[77], through UV\u2013Vis spectra, the stability of the compounds, ordinary\n and 1cTlip were used to treat HepG2 cells for 24 h. After washing and\nand targeted liposome encapsulated compounds was examined at 0 and\n centrifugation, cells were stained with Fluo-3-pentaacetoxymethyl ester\n48 h in PBS solution. The purity of 1a, 1b, 1c was measured by HPLC\n (Flu-3 AM) and intracellular Ca2+ content was examined by flow\n(mobile phases I: H2O and 0.1 % trifluoroacetic; II methanol and 0.1 %\n cytometry.\ntrifluoroacetic acid).\n\n4.6. Determination of lipid-water partition coefficient (logP) 4.12. Cell cycle arrest\n\n In organic (n-octanol) and H2O phases, the distribution of com\u00ad Placing HepG2 cells in 6-well plate and grown for 20 h, cells were co-\npounds 1a-1c was measured by the shaking bottle method [78]. In brief, incubated for 24 h with IC50 concentration of 1a, 1b, 1c, 1alip, 1blip,\n2 mg of compounds of 1a, 1b or 1c was dissolved in 2 mL octanol and 2 1clip, 1aTlip, 1bTlip and 1cTlip, washing three times, cells were\nmL H2O, the mixed solutions were consecutively shaken for 30 h at collected, lysed, and fixed with 75 % ethanol for 15 h. Then cells were\n37 \u25e6 C. Thereafter, the organic and water phases were separated through discolored with 0.2 % Triton X-100, 15 \u03bcL 1 mg mL\u2212 1 PI and 15 \u03bcL 1 mg\n15 min of centrifugation (1000 rpm). The concentrations (Co) of the mL\u2212 1 RNase for 20 min and analyzed under a flow cytometry.\ncompounds 1a-1c in organic phase were examined using UV\u2013Vis spectra.\nThe mean lipid-water partition coefficient logP = log(Co/Cw). 4.13. Apoptosis studies\n\n4.7. Detection of pKa values 1 \u00d7 105 cells/well of HepG2 cells were seeded into 6-well plate in the\n incubator at 37 \u25e6 C, 5 % of CO2 overnight. Then the cells were exposed to\n 80 mM HCl, 50 mM KOH and 50 \u03bcM compounds 1a, 1b, 1c were IC50 of 1a, 1b, 1c, 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip. After a\nprepared with CH3CN and double distilled H2O (v/v, 4:6). The pKa day, washing cells three times, staining cells 25 min using propidium\nvalues of 1a, 1b and 1c were measured using potentiometric titration iodide (PI) and Annexin V-FITC. Finally, the cells were analyzed under a\nmethod. Through gradually titrating the compound solution with HCl to flow cytometer.\nreduce pH to 2.0. After that, the above solutions were slowly titrated\nwith KOH solution, the pH values were recorded, the pKa values were\ngained according to the curve of pH value versus the volumes of KOH 4.14. Quantitative assay of intracellular ROS\nsolution.\n Placing HepG2 cells into 6-well plate and incubated for 20 h. IC50 of\n 1a, 1b, 1c, 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip were added to\n4.8. In vitro anti-cancer activity assay\n the cells, after 24 h, washing cells four times, using 15 \u03bcM 2\u2032,7\u2032-\n dichlorodihydrofluorescein diacetate to stain the cells for 25 min, ROS\n The anti-cancer activities of compounds and liposome encapsulated\n content was examined.\ncompounds against BEL-7402, HepG2, SK-Hep1 and LO2 cells were\nassessed by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bro\u00ad\nmide (MTT) method [79]. The cells were incubated with six different 4.15. Ferroptosis studies\nconcentration of compounds 1a, 1b, 1c, 1alip, 1blip, 1clip, 1aTlip,\n1bTlip and 1cTlip dissolved in DMSO (less than 0.05 %) for 48 h, then (I) Ferrotin-1 assay for cell viability\nthe cells were dyed with 10 % MTT dye for 6 h, 100 \u03bcL DMSO was added\nto dissolve the purple formazan product. Ultimately, we recorded the HepG2 cells were treated with the following nineteen groups:\nabsorbance values at 490 nm. ferrotin-1 (Fer-1), IC50 concentrations of 1a, 1a + Fer-1; 1b, 1b + Fer-1;\n 1c, 1c + Fer-1; 1alip, 1alip + Fer-1; 1blip, 1blip + Fer-1; 1clip, 1clip +\n4.9. Measurement of cellular uptake Fer-1; 1aTlip, 1aTlip + Fer-1; 1bTlip, 1bTlip + Fer-1; 1cTlip, 1cTlip +\n Fer-1. After 36 h, 10 % MTT dye was added to the cells for 6 h, then we\n In brief, in 12-well plates, HepG2 cells were cultivated overnight. applied 100 \u03bcL DMSO to dissolve the purple formazan. Ultimately, we\nAfter 6 h of incubation of HepG2 cells with IC50 values of 1a, 1b, 1c, determined the absorbance values (490 nm).\n1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip, HepG2 cells were washed\nseveral times to eliminate the remaining compounds. Ultimately, HepG2 (II) Determination of GSH and MDA content\ncells were immediately observed. (a) glutathione (GSH) content: HepG2 cells were cultivated into\n 6-well plate for 20 h, a treatment of 24 h of HepG2 cells with\n4.10. Cell colony, cell migration research compounds 1a, 1b, 1c, 1alip, 1blip, 1clip, 1aTlip, 1bTlip and\n 1cTlip (IC50 concentration) at 37 \u25e6 C with 5 % CO2, the cells\n (a) cell colony: HepG2 cells growing in 6-well plate were cultured were collected by lysis, then we determined the contents of\nwith 1a, 1b, 1c, 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip at IC50 total glutathione and oxidized glutathione disulfide (GSSG).\nconcentration for a week. During this period, the culture medium was (b) According to the method described in the literature [73],\nreplaced every day. Finally, we used paraformaldehyde to fix the cells we also examined MDA content.\nand applied crystal violet to dye the cells and photographed. (b) cell (III) Lipid peroxidation studies\nmigration: At 37 \u25e6 C, HepG2 (1 \u00d7 104 cells/well) were cultivated in 6-\nwell plate, after the cell density reached about 75 %, using the pipette HepG2 cells were seeded in 6-well plate for 20 h, the cells were\ntip to draw two straight lines to construct artificial wound area. PBS incubated with 1alip, 1blip, 1clip, 1aTlip, 1bTlip, 1clip (IC50 concen\u00ad\nbuffer solution was used to wash dead cells away. IC50 concentration of tration) for 24 h, then the cells were washed three times and dyed with\ncompounds 1a, 1b, 1c, 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip were C11-BODIPY for 30 min and photographed. The red and green fluores\u00ad\nused to treat the wells for 24 h, and the degree of wound healing was cence were examined using flow cytometry.\n\n 13\n\fJ. Chen et al. European Journal of Medicinal Chemistry 265 (2024) 116078\n\n\n4.16. Assay of protein expression recorded the weight and the tumor volume every day. The tumors were\n separated on the 8th day, the inhibitory tumor growth rate was calcu\u00ad\n The assay of protein expression was performed via the following ten lated according to the equation:\nsteps: (a) HepG2 cells were spread evenly overnight in DMEM contain\u00ad\n [(M1 \u2212 M2 ) / M1 ] \u00d7 100%\ning 10 % FBS. (b) HepG2 cells were cultivated with IC50 value of 1a, 1b,\n1c, 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip for 24 h, the culture M1 and M2 represent mean tumor weight in blank and drug-treated\nfluid was dismissed, and the cells were lysed and collected in 1.5 mL EP groups.\ntubes. (c) The cells were centrifuged (12,000 rpm, 13 min) and the su\u00ad\npernatant was collected in a new 1.5 mL EP tube. (d) Concentration of\n 4.20. Drug toxicity test\nprotein was examined based on BCA Protein Concentration Assay Kit. (e)\nProtein from individual sample was added to every lane, sodium dodecyl\n From the different groups, we randomly selected tumor tissues of\nsulfate-polyacrylamide gel electrophoresis was carried out for 2 h. (f)\n brain, kidney, lung, heart, liver, spleen. These tissues were washed with\nThe observed protein bands were transferred to polyvinylidene\n 0.9 % saline and then frozen in paraformaldehyde for 24 h. After fixa\u00ad\ndifluoride (PVDF) membranes, the bands were blocked for 1 h with 5 %\n tion, soaking the tissues in paraffin, then the tissues were cut into slices\nskimmed milk. (VII) After removal of the blocking solution, using TBST\n (5 mm), hematoxylin (H&E) was applied to dye the slices at room\nsolution to wash the strips four times, incubated with the primary\n temperature.\nantibody overnight at 4 \u25e6 C. (g) On the second day, remove the primary\nantibody, using TBST to wash the membrane four times. (h) The sec\u00ad\nondary antibody was used to co-incubate the membrane for 75 min. (i) CRediT authorship contribution statement\nECL chemiluminescence reagent was added and the bands were\nobserved under an Amersham ECL (electrochemiluminescence) kit Jing Chen: Investigation, Methodology, Writing \u2013 original draft.\n(Bainite Biotechnology, China). Wenlong Li: Investigation, Methodology. Gechang Li: Data curation,\n Software. Xiaoming Liu: Investigation. Chunxia Huang: Data curation.\n4.17. RNA sequencing (RNA-seq) studies Hua Nie: Methodology, Project administration. Lijuan Liang: Formal\n analysis. Yi Wang: Software. Yunjun Liu: Conceptualization, Funding\n After 24 h treatment of HepG2 cells with 2 \u00d7 IC50 values of 1alip and acquisition, Project administration, Supervision, Writing \u2013 review &\n1aTlip, washing the cells three times with PBS solution (containing editing.\nDEPC), adding 350 \u03bcL of RNA extract solution to each well, the cells\nwere lysed, using Trizol reagent to separate the total RNA. The Declaration of competing interest\nsequencing libraries were constructed based on the TruSeq RNA sample\npreparation kit. Then the library fragments were purified and quanti\u00ad Authors declare no competing interest exists.\nfied. Ultimately, on the Hiseq platform (illumine), the sequencing li\u00ad\nbrary was sequenced. Data availability\n\n4.18. Co-location and studies on change of mitochondrial membrane The data that has been used is confidential.\npotential\n Acknowledgements\n (I) Co-location assays\n This research was supported by the National Natural Science Foun\u00ad\n The co-location of the compounds at mitochondria was investigated. dation of China (No. 21877018).\nHepG2 cells were co-cultivated with 1a, 1b, 1c, 1alip, 1blip, 1clip,\n1aTlip, 1bTlip and 1cTlip (IC50 concentration) for 6 h, washing the cells Appendix A. Supplementary data\nthree times, using MitoTracker Deep Red (ThermoFisher 15 nM) to stain\nthe cells for 15 min at room temperature, finally, HepG2 cells were Supplementary data to this article can be found online at https://doi.\nphotographed.\n org/10.1016/j.ejmech.2023.116078.\n\n (II) Examination of change in mitochondrial membrane potential\n References\n Use IC50 values of 1a, 1b, 1c, 1alip, 1blip, 1clip, 1aTlip, 1bTlip and [1] X.D. Song, X. Kong, S.F. He, J.X. Chen, J. Sun, B.B. Chen, J.W. Zhao, Z.W. Mao,\n1cTlip to treat HepG2 cells for 24 h, then washing the cells three times, Cyclometalated iridium(III)-guanidinium complexes as mitochondria-targeted\nthe cells were discolored with 5,5\u2032-6,6\u2032-tetrachloro-1,1\u2032-3,3\u2032-tetre\u00ad anticancer agents, Eur. J. Med. Chem. 138 (2017) 246\u2013254.\n [2] T.F. Yang, M.H. Zhu, M. Jiang, F. Yang, Z.L. Zhang, Current status of iridium-based\nthylbenzimidalylcarbocyanine iodide (JC-1), ultimately, the cells were complexes against lung cancer, Front. Pharmacol. 13 (2022), 1025544.\nobserved. The green fluorescence intensity was quantitatively measured [3] M.X. Shao, M.M. Yao, X.C. Liu, C. Gao, W.Y. Liu, J.H. Guo, J.W. Zong, X.Z. Sun,\naccording to the literature [17]. Z. Liu, In vitro and in vivo of triphenylamine-appended fluorescent half-sandwich\n iridium(III) thiosemicarbazones antitumor complexes, Inorg. Chem. 60 (2021)\n 17063\u201317073.\n4.19. In vivo anti-tumor efficacy [4] R.X. Xu, Y.T. Wu, Z. Liu, J.F. 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