Synthesis, characterization and irradiation enhances anticancer activity of liposome-loaded iridium(III) complexes.

{"full_text": " Journal of Inorganic Biochemistry 256 (2024) 112549\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\nSynthesis, characterization and irradiation enhances anticancer activity of\nliposome-loaded iridium(III) complexes\nShuang Tian a, Qianying Nie b, Haomin Chen a, Lijuan Liang a, Huiyan Hu a, Shuanghui Tang a,\nJiawan Yang a, Yunjun Liu a, *, Hui Yin b, *\na\n School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China\nb\n Department of Microbiology and Immunology, Guangdong Pharmaceutical University, Guangzhou 510006, 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: Herein, we synthesized and characterized two novel iridium (III) complexes: [Ir(bzq)2(PPD)](PF6) (4a, with bzq\nIridium(III) complexes = deprotonated benzo[h]quinoline and PPD = pteridino[6,7-f][1,10]phenanthroline-11,13-diamine) and [Ir\nApoptosis (piq)2(PPD)](PF6) (4b, with piq = deprotonated 1-phenylisoquinoline). The anticancer efficacy of these com\u00ad\nFerroptosis\n plexes, 4a and 4b, was investigated using 3-(4,5-dimethylthiazole)-2,5-diphenltetraazolium bromide (MTT).\nPhotodynamic therapy\n Complex 4a exhibited no cytotoxic activity, while 4b demonstrated moderate efficacy against SGC-7901, A549,\nWestern blot\n and HepG2 cancer cells. To enhance their anticancer potential, we explored two strategies: (I) light irradiation\n and (II) encapsulation of the complexes in liposomes, resulting in the formation of 4alip and 4blip. Both stra\u00ad\n tegies significantly increased the ability of 4a, 4b to kill cancer cells. The cellular studies indicated that both the\n free complexes 4a, 4b and their liposomal forms 4alip and 4blip effectively inhibited cell proliferation. The cell\n cycle arrest analysis uncovered 4alip and 4blip arresting cell growth in the S period. Additionally, we investi\u00ad\n gated apoptosis and ferroptosis pathways, observing an increase in malondialdehyde (MDA) levels, a reduction of\n glutathione (GSH), a down-regulation of GPX4 (glutathione peroxidase) expression, and lipid peroxidation. The\n effects on mitochondrial membrane potential and intracellular Ca2+ concentrations were also examined,\n revealing that both light-activated and liposomal forms of 4alip and 4blip caused a decline in mitochondrial\n membrane potential and an enhancement in intracellular Ca2+ levels. In conclusion, these complexes and them\n encapsulated liposomes induce cell death through apoptosis and ferroptosis.\n\n\n\n\n1. Introduction highlighted the numerous iridium (III) complexes demonstrating a\n prominent efficiency in restraining tumor cell growth [5\u201315]. on the\n Cancer, a malignant disease, poses a serious threat to global health other hand, photodynamic therapy (PDT) has been considered as\ndue to its characteristics of uncontrolled proliferation, transformation, another effective method to cure cancer, which uses specific light\nand metastasis. This results in high recurrence and mortality rates, wavelengths to enhance drug efficacy. Studies by Sun and colleagues\nmaking cancer a challenging medical issue worldwide [1]. In the cancer have shown that an iridium(III) complex exhibits minimal cytotoxicity\nchemotherapy, a series of platinum metal drugs have been a corner\u00ad (IC50 > 100 \u03bcM) to HepG2 cells, however, upon light irradiation (425\nstone. Their mechanism involves binding to DNA through the aquation nm), the cytotoxicity significantly increases [16]. Similarly, Liu and\nof Pt\u2013Cl bonds, resulting in intracellular reduction of chloride ion team found [Ir(piq)2(IPPH)](PF6) exhibiting non-cytotoxic y (IC50 >\nconcentration to approximately 20 mM [2]. However, due to the serious 100 \u03bcM) on A549 cells in the absence of light, whereas its cytotoxicity is\nside-effects of cisplatin, a lot of alternative metal-based compounds have markedly enhanced under illumination (IC50 = 0.2 \u00b1 0.05 \u03bcM) [17].\nbeen developed. This has brought about the exploration of non\u2011platinum Many photosensitizers currently undergoing clinical or pre-clinical\nmetal anticancer drugs possessing distinct mechanisms of action. studies predominantly affect the mitochondria and induce apoptosis\nNotably, iridium (III) compounds have shown potential as effective anti- via PDT [18]. Furthermore, liposomes, consisting of phospholipid and\ncancer agents, targeting various cellular components such as mito\u00ad cholesterol bilayers, can encapsulate both hydrophilic and lipophilic\nchondria, proteins, DNA, and lysosomes [3,4]. Recent studies have drugs, significantly enhancing their anticancer efficacy. Our previous\n\n\n * Corresponding authors.\n E-mail addresses: lyjche@gdpu.edu.cn (Y. Liu), huiyin0103@gdpu.edu.cn (H. Yin).\n\nhttps://doi.org/10.1016/j.jinorgbio.2024.112549\nReceived 24 January 2024; Received in revised form 18 March 2024; Accepted 31 March 2024\nAvailable online 2 April 2024\n0162-0134/\u00a9 2024 Elsevier Inc. All rights reserved.\n\fS. Tian et al. Journal of Inorganic Biochemistry 256 (2024) 112549\n\n\nstudies have demonstrated that liposome-encapsulated iridium(III) CH2Cl2-CH3CH2OH (v/v, 5:1) was employed as the eluent, collecting the\ncomplexes exhibit a strong inhibitory effect on cell growth and effec\u00ad yellow band, finally a yellow powder was gained. Yield: 82%. Anal calcd\ntively prevent tumor growth [19\u201321]. Additionally, we have found for C42H26N10PF6Ir: C, 50.05; H, 2.60; N, 13.90%. Found: C, 50.20; H,\niridium (III) complexes with ligands bearing -NH2 group, and their 2.42; N, 13.76%. 1H NMR (DMSO\u2011d6, 500 MHz, Fig. S1, SI): \u03b4 9.92 (d, J\nliposome-loaded counterparts, showing significant ability to inhibit = 8.0 Hz, 1H), 9.47 (d, J = 7.5 Hz, 1H), 8.75 (s, 1H), 8.55 (d, J = 8.0 Hz,\ncancer cell growth [21,22]. To gain more insights into their anticancer 2H), 8.29\u20138.24 (m, 3H), 8.13\u20138.09 (m, 4H), 8.00 (d, J = 8.5 Hz, 4H),\nactivities, this article introduces pteridino[6,7-f][1,10]phenanthroline- 7.90 (d, J = 9.0 Hz, 2H), 7.60 (d, J = 5.0 Hz, 2H), 7.51\u20137.48 (m, 2H),\n11,13-diamine (PPD), featuring two -NH2 substituent groups as a 7.24 (t, J = 7.5 Hz, 2H), 6.33 (d, J = 7.5 Hz, 2H). 13C NMR (DMSO\u2011d6,\nligand, two of its iridium (III) complexes (Scheme 1): [Ir(bzq)2(PPD)] 125 MHz, Fig. S2, SI): 172.48, 164.92, 163.40, 156.76, 156.10, 152.80,\n(PF6) (bzq = deprotonated 2-phenylpyridine) (4a) and [Ir(piq)2(PPD)] 150.93, 149.77, 147.66, 147.16, 144.43, 140.82, 138.08, 135.76,\n(PF6) (piq = deprotonated 1-phenylisoquinoline) (4b) were prepared 135.19, 134.23, 132.92, 131.36, 130.50, 130.18, 129.93, 129.02,\nand characterized. Anticancer efficiency of 4a, 4b, and their liposome- 128.36, 127.15, 126.29, 124.72, 123.26, 120.90. HRMS (CH3CH2OH):\nloaded forms, 4alip and 4blip, was evaluated under various condi\u00ad m/z = 863.1915 ([M \u2013 PF6])+.\ntions, including light irradiation.\n 2.2.2. Synthesis of [Ir(piq)2(PPD)](PF6) (4b)\n2. Experimental This compound was obtained with the same method described for 4a\n with [Ir(piq)2Cl]2 [23] in place of [Ir(bzq)2Cl]2. Yield: 80%. Anal calcd\n2.1. Materials and methods for C46H30N10PF6Ir: C, 50.12; H, 2.85; N, 13.21%. Found: C, 50.33; H,\n 2.97; N, 13.38%. 1H NMR (DMSO\u2011d6, 500 MHz, Fig. S3, SI): \u03b4 9.97 (dd, J\n Dimethyl sulfoxide (DMSO) and RPMI 1640 were procured from = 1.5, J = 7.5 Hz, 1H), 9.55 (dd, J = 2.0, J = 8.0 Hz, 1H), 9.06\u20139.01 (m,\nSigma, and IrCl3\u22c53H2O was obtained from the Kunming Boren Precious 2H), 8.77 (s, 1H), 8.42 (d, J = 8.5 Hz, 2H), 8.30 (s, 1H), 8.15\u20138.05 (m,\nMetals Co.Ltd. Various cell lines including SGC-7901 (gastric cancer), 6H), 7.98 (dd, J = 1.5, J = 5.0 Hz, 1H), 7.92\u20137.87 (m, 5H), 7.54\u20137.48\nB16 (melanoma), A549 (human lung cancer), HCT-116 (colon cancer), (m, 4H), 7.19 (t, J = 8.0 Hz, 2H), 6.98 (t, J = 7.5 Hz, 2H), 6.27 (d, J =\nHepG2 (human hepatocellular cancer), and LO2 (normal human liver) 8.5 Hz, 2H). 13C NMR (DMSO\u2011d6, 125 MHz, Fig. S4, SI): 172.49, 168.27,\nwere acquired from the Chinese Academy of Sciences (CAS) Cell Bank, 164.95, 163.43, 162.80, 156.16, 153.77, 152.22, 150.35, 149.73,\nShanghai, China. 147.08, 145.88, 144.50, 141.63, 137.03, 135.91, 135.31, 132.99,\n Dimethyl sulfoxide served as the solvent and tetramethylsilane 132.53, 132.11, 131.51, 131.05, 130.66, 129.85, 128.43, 128.18,\n(TMS) as the internal standard for conducting nuclear magnetic reso\u00ad 126.91, 126.35, 125.99, 122.89, 122.66. HRMS (CH3CH2OH): m/z =\nnance (NMR) spectroscopy. The NMR data were gained on a Varian-500 915.1718 ([M \u2013 PF6])+.\nspectrometer. Additionally, UV\u2013Vis spectra were measured on a Shi\u00ad\nmadu UV-3101PC spectrophotometer. Molecular weights of the syn\u00ad 2.3. Determination of purity of 4a and 4b\nthesized compounds were precisely examined using high-resolution\nmass spectrometry (HRMS). The purity of the synthesized 4a and 4b was detected using HPLC at\n room temperature. The mobile phases A and B consisted of H2O with\n 0.1% trifluoroacetic acid (TFA) and CH3OH with 0.1% TFA, respec\u00ad\n2.2. Synthesis of complexes 4a and 4b\n tively. The flow rate was maintained at 3 mL/min. The volume ratios of\n H2O to MeOH are 6:94 and 2:98 for 4a and 4b, respectively. Detection of\n2.2.1. Synthesis of [Ir(bzq)2(PPD)](PF6) (4a)\n the complexes was conducted at a wavelength of 254 nm.\n Cis-[Ir(bzq)2Cl]2 (0.233 g, 0.20 mmol) [23] and PPD (0.126 g, 0.40\nmmol) [24] were dissolved in 28 mL CH2Cl2 and 14 mL CH3OH. This\nsolution was refluxed at 40 \u25e6 C under an argon atmosphere. After 6 h, the 2.4. Preparation of 4alip and 4blip\nsolution was cooled and saturated aqueous NH4PF6 was added, the\nabove solution was stirred for an additional 2 h. This process resulted in The liposomal forms of complexes 4a and 4b, designated as 4alip and\nthe formation of a distinct yellow precipitate. The crude product was 4blip, were synthesized using a modified reverse-phase evaporation\nsubsequently purified using neutral alumina column chromatography, method [25]. Initially, 4a or 4b, egg yolk lecithin (PC-98 T), cholesterol\n\n\n\n\n Scheme 1. Synthesis of PPD and 4a and 4b.\n\n 2\n\fS. Tian et al. Journal of Inorganic Biochemistry 256 (2024) 112549\n\n\n(CHO-HP), and DSPE-MPEG 2000 in a weight ratio of 1:30:10:10 (w/w) added to each well. After 4 h, the MTT formazan product was dissolved\nwere combined in a 20 mL vial. These components were dissolved in 8 with a solution (100 \u03bcL) containing 50% dimethylformamide and 20%\nmL of chloroform (6 mL) and H2O (2 mL). The mixture was sonicated for sodium dodecyl sulfate, the optical density was measured at 490 nm.\n5 min, then removing the organic phase, the residue was rehydrated IC50 values were gained by plotting the percentage of viable cells\nwith PBS and subjected to vortex mixing for 30 min at 45 \u25e6 C. The final against the logarithm of the concentration and determining the con\u00ad\nstep involved ultrasonication and centrifugation to yield a clear, trans\u00ad centration corresponding to 50% cell viability. Each experiment was\nparent liposome preparation. performed in triplicate. The cell viability was calculated according to the\n equation:\n2.5. Characterization of 4alip and 4blip (( )/ )\n Cell viability% = Asample \u2212 Amedium control (Acell control \u2212 Amedium control ) \u00d7 100\n\n The zeta potentials and particle sizes of both 4alip and 4blip were Whereas Asample, Amedium and Ablank are the absorbance values of\ndetermined using a laser particle sizer. To gain encapsulation efficiency, sample, medium and control.\nthe liposomes were first centrifuged and a clear supernatant was ob\u00ad\ntained. The liposomes were then disrupted with methanol, and then\n 2.9. Cell colony experiment\nmeasuring the absorbance at 254 nm. The concentration of 4a and 4b in\nthe solution was determined based on a pre-established standard curve.\n SGC-7901 cells were co-incubated with the compounds 4a, 4b, and\n The encapsulation efficiency (EE%) of 4alip and 4blip was gained:\n their liposomal formulations, 4alip and 4blip, at their respective IC50\nEE (%) =\n Wencapsulated\n \u00d7 100%. concentrations, throughout this period, the culture medium was\n Wtotal refreshed daily. After one week, the cells were washed and fixed with\n In this equation, Wencapsulated is the drug-loaded content detected in paraformaldehyde. Finally, the crystal violet-stained SGC-7901 cells\nthe supernatant after centrifugation, and Wtotal is the weight of 4a or 4b were photographed to observe the cell morphology and proliferation.\nin the preparation of 4alip and 4blip.\n 2.10. Cell cycle determination\n2.6. Release examination\n Cells of SGC-7901 were co-cultured with 4a, 4b, 4alip, and 4blip\n The release profiles of 4a and 4b from their respective liposomal (IC50 concentration) for 24 h. Afterwards, washing three times with PBS,\nsuspensions, 4alip and 4blip, were determined using a dialysis method the cells were fixed with 70% CH3CH2OH and stored at 4 \u25e6 C overnight.\npreviously described in literature [26]. In this procedure, 5 mL of either Then the cells were again washed with PBS and stained with propidium\n4alip or 4blip suspension, containing 1 mg of 4a or 4b, was enclosed in a iodide (PI). Finally, the cells were analyzed with FACS Calibur flow\ndialysis bag (8\u201314 kDa). This setup was then immersed in 25 mL of PBS cytometry.\nat 37 \u25e6 C, with Tween 80 (0.5%, w/v) in a beaker. At specified time in\u00ad\ntervals, a 3.5 mL sample of the dialysate was removed for analysis, and 2.11. Apoptosis studies\nan equivalent volume of fresh buffer was added into the beaker to\nmaintain constant volume. The concentration of 4a or 4b in the dialysate After exposure to IC50 concentration of 4a, 4b, 4alip, 4blip for 24 h,\nwas quantitatively assessed using a UV\u2013Visible spectrophotometer. SGC-7901 cells were washed with PBS three times and dyed with 200 \u03bcL\nFrom these measurements, the cumulative amount of each compound Annexin V-FITC solution (25 \u03bcg/mL Annexin V-FITC, 2.5 \u03bcg/mL propi\u00ad\nreleased from the liposomes was calculated. The resulting data were dium iodide (PI), the percentage of the cell in the living, early and late\nused to plot release curves, representing the cumulative release con\u00ad apoptosis was measured with flow cytometry.\ncentration of 4a or 4b against time.\n 2.12. Change of the mitochondrial membrane potential\n2.7. Singlet oxygen detection\n After seeded in 12-well plate overnight, SGC-7901 cells were\n The singlet oxygen 1O2 was determined by UV\u2013Vis spectra. A mixture exposed to IC50 concentrations of 4a, 4b, 4alip, 4blip for 24 h. Next, SGC-\nsolution of DPBF (40 \u03bcM) and 4a or 4b (10 \u03bcM) was irradiated with 7901 cells were washed with PBS three times, the cells were dyed with 1\nwhite light (LED lamp, 460 nm, 7.03 J/cm2) in the different time, the \u03bcg/mL of JC-1 (5,5\u2032,6,6\u2032-tetrachloro-1,1\u2032,3,3\u2032-tetraethyl-imidacarbocya\u00ad\nabsorption of DPBF (417 nm) was measured. [Ru(bpy)3]2+ was used as a nineiodide) for 30 min. Then the cells were washed twice with PBS and\nreference. The quantum yield \u03a6 were gained based on the Eq. [27]: observed.\n ( / ) ( / )\n\u03a6sample = \u03a6Ref \u00d7 Ksample KRef \u00d7 FRef Fsample\n 2.13. Ferroptosis studies\n K is the slope, F is calibration factor of the absorbance, F = 1\u201310-OD\n(OD is the absorbance of the photosensitizer at the light source 2.13.1. Intracellular lipid peroxidation\nwavelength). SGC-7901 cells were treated with IC50 concentrations of 4a, 4b,\n 4alip, and 4blip for a period of 24 h. Afterwards, the residual compounds\n2.8. Cell viability and IC50 determination were removed through washing the cells three times using PBS solution.\n As a lipid peroxidation sensor, C11-BODIPY 581/591 (2.0 \u03bcM) was\n 3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium bromide (MTT) applied to dye the cells for 30 min. C11-BODIPY 581/591 is commonly\nassay [28] was employed to determine the cell viability and half- used to detect oxidative stress in cells, which is a crucial parameter in\nmaximal inhibitory concentration (IC50) values. The cancer cells (8 \u00d7 understanding the cellular impact of treatments. The stained cells were\n103 cells per well) were seeded in 96-well microassay culture plates then photographed. The imaging was conducted at specific excitation\novernight at 37 \u25e6 C in a 5% CO2 atmosphere. Various concentrations of (\u03bbex) and emission (\u03bbem) wavelengths to detect the fluorescence of C11-\nthe test complex, dissolved in DMSO, were added to the above wells. The BODIPY (\u03bbex = 488 nm, \u03bbem = 510 nm for green, \u03bbex = 581 nm, \u03bbem =\ncontrol wells received 100 \u03bcL of culture medium without the test com\u00ad 591 nm for red). This imaging approach enables the visualization of lipid\npound. The plates were incubated for 48 h at 37 \u25e6 C in a 5% CO2 atmo\u00ad peroxidation within the cells, providing valuable insights into the\nsphere. After incubation, 20 \u03bcL of MTT dye solution (5 mg/mL) was oxidative stress.\n\n 3\n\fS. Tian et al. Journal of Inorganic Biochemistry 256 (2024) 112549\n\n\n2.13.2. Intracellular MDA concentration congruent with theoretical calculations. In the 13C NMR spectra, the\n SGC-7901 cells were co-incubated with 4a, 4b, 4alip, and 4blip (IC50 observed peaks at 172.48 and 164.92 ppm for 4a, and 172.49 and\nconcentrations) for 24 h. After incubation, the cells were washed twice 168.27 ppm for 4b, correspond to the carbon atoms bonded to the -NH2\nwith PBS to remove any residual treatment agents. The cells were then groups.\nlysed using Radio-Immunoprecipitation Assay (RIPA) buffer, a process UV\u2013Vis spectra was applied to examine the stability of complexes 4a\nwhich helps in breaking down the cell membranes and releasing the or 4b, as shown in Fig. S5a (SI), At 0 and 24 h, the peak shapes for 4a and\ncellular contents. The lysates were centrifuged at 4 \u25e6 C for 20 min. This 4b are the same, indicating a stable existence of 4a or 4b. Additionally,\nstep allows for the separation of the soluble supernatant containing the stability of 4a was also determined using HRMS spectra, as shown in\nproteins from the cell debris. The supernatant was then collected for Fig. S5b (SI), the determined molecular weights are the same upon\nfurther analysis. Bicinchoninic Acid (BCA) protein assay was applied to irradiation as that in the dark at 0 h and 48 h, which further affirms that\nmeasure the total protein concentration in each sample. Based on these the complexes are stable.\nconcentrations, the proteins were normalized for subsequent analysis. As depicted in Fig. S5c (SI), during a 20-min analysis period, a sin\u00ad\nNext, following the protocol outlined in the Lipid Peroxidation MDA gular peak was observed for each complex, confirming their purity at\nassay kit, we determined malondialdehyde (MDA) content. MDA is a 97.8% and 96.4% for 4a and 4b.\nmarker for oxidative stress and lipid peroxidation within cells. The MDA Particle sizes of 4alip and 4blip were examined using dynamic light\ncontents were determined with multimode microplate readers at 532 scattering (DLS), revealing average sizes of 85.8 \u00b1 1.7 nm for 4alip and\nnm. 80.9 \u00b1 0.8 nm for 4blip (Fig. S5d, SI). The zeta potentials of \u2212 7.09 mV\n for 4alip and \u2212 12.09 mV for 4blip suggest a moderate electrostatic\n2.13.3. GSH content determination repulsion between particles, contributing to suspension stability and\n After a 24-h incubation of SGC-7901 cells with 4a, 4b, 4alip and resistance to agglomeration [29]. The morphology of 4alip was exam\u00ad\n4blip, the cells were digested, centrifuged, and collected in EP pipes. A ined by transmission electron microscope (TEM), as found from Fig. S5e\nprotein removal reagent was then added to the EP pipes, and the con\u00ad (SI), 4alip displays a spherical particle size.\ntents were thoroughly mixed. The eppendorf tubes underwent two rapid Encapsulation efficiency (EE%) was found to be 89.94% for 4alip and\nfreeze-thaw cycles, followed by a 5-min refrigeration at 4 \u25e6 C. The pipes 85.42% for 4blip. Release kinetics, as shown in Fig. S5f (SI), indicated\nwere then centrifuged for 15 min. The resulting supernatant was care\u00ad initial release concentrations of 29.1% and 15.2% within the first 0.5 h\nfully transferred from the EP pipes to a 96-well plate. Subsequently, for 4alip and 4blip, respectively. Cumulative 24-h release of free 4a and\nGlutathione (GSH) Removal Aid and GSH Removal Solution were added 4b reached 48.7% and 46.5%, respectively, demonstrating a controlled\nto the wells, and the plate was incubated at 25 \u25e6 C for 60 min. Following release profile.\nthe preparation of reagents as per the instructions of the GSH assay kit,\nthe samples in the 96-well plate were mixed well and incubated for an 3.2. Cell viability and IC50 determination\nadditional 5 min. After this incubation, 50 \u03bcL of NADPH solution (0.5\nmg/mL) was added to each well. The absorbance was measured at 412 The cytotoxic efficacy of 4a, 4b, 4alip, and 4blip toward cancer cells\nnm. SGC-7901 and A549 was assessed using the 3-(4,5-dimethylthiazole)-\n 2,5-diphenyltetrazolium bromide (MTT) assay. We employed two stra\u00ad\n2.14. Expression of apoptosis-related protein tegies to enhance the anticancer efficacy of 4a and 4b: (I) light irradi\u00ad\n ation and (II) encapsulation within liposomes. Fig. 1a (I) and (II) display\n Treatment of SGC-7901 cells with 4a, 4b, 4alip and 4blip for 24 h, the the results of treating SGC-7901 cancer cells with varying amounts of 4a\nconcentration of protein in the supernatant was obtained according to and 4b for 48 h, 4b demonstrated a moderate ability to inhibit cell\nBCA (bicinchoninic acid) assay. In the sodium dodecyl sulfate- proliferation, whereas 4a exhibited a weaker effect on killing cancer\npolyacrylamide gel electrophoresis, amount of the proteins in the per cells. Further experiments involved a 4-h treatment of SGC-7901 cells\nlane is equal. After gel electrophoresis, the gels were transferred to with 4a and 4b, followed by 30 min of light irradiation (65 w LED lamp,\npolyvinylidene difluoride membranes (Millipore) and blocked with 5% light dose of 7.03 J\u22c5cm\u2212 2, \u03bb = 460 nm). Post-irradiation, the cell viability\nnon-fat milk in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, reduced compared to non-irradiated controls (Fig. 1a (III) and (IV)). This\npH 8.0) buffer for 3 h. After washing four times with TBST buffer, the indicates that light irradiation enhances the cytotoxic effects of the\nmembranes were incubated with primary antibody solution at 4 \u25e6 C complexes. To optimize the anticancer potential, 4a and 4b were\novernight, washing four times again, the secondary antibodies were then encapsulated into liposomes, creating 4alip and 4blip. Fig. 1a (V-VIII)\nconjugated with horseradish peroxidase (1:5000 dilution) for 70 min at illustrate the enhanced inhibitory effects of 4alip and 4blip on SGC-7901\nroom temperature. Finally, the blots were treated with the Amersham cells, both with and without irradiation. These liposome-encapsulated\nECL Plus western blotting detection reagents. complexes demonstrated superior cytotoxicity compared to their non-\n encapsulated counterparts.\n2.15. Data analysis The IC50 values ranged from 84.1 \u00b1 2.1 to 2.4 \u00b1 0.2 \u03bcM (Table 1), 4a\n shows non-cytotoxic toward SGC-7901, A549 cells (IC50 > 100 \u03bcM), the\n The data in the study were reported as the mean \u00b1 standard devia\u00ad cytotoxic activity of 4a and 4b is lower than cisplatin in the dark.\ntion (SD). t-test was utilized to evaluate the differences between groups. However, under light irradiation, the anticancer efficacy of 4a and 4b\n*p < 0.05 indicates a statistical difference. significantly enhanced, while IC50 values of 4alip and 4blip toward SGC-\n 7901 cells under light irradiation are 4.1 \u00b1 0.2 and 2.4 \u00b1 0.2 \u03bcM. The\n3. Results and discussion cytotoxic activity of 4a and 4b toward SGC-7901 cells is higher than that\n of complex [Ir(bzq)2(DIPH)](PF6) (IC50 = 4.7 \u00b1 0.2 \u03bcM) [19], compa\u00ad\n3.1. Synthesis and characterization rable to the complex [Ir(bzq)2(NDPPZ)](PF6) (IC50 = 1.6 \u00b1 0.3 \u03bcM)\n [30], but lower than that of [Ir(piq)2(NDPPZ)](PF6) (IC50 = 0.8 \u00b1 0.1\n PPD was synthesized via condensation of 1,10-phenanthroline-5,6- \u03bcM, NDPPZ = 11-nitrodipyrido[3,2-a:2\u2032,3\u2032-c]phenazine) [30] and [Ir\ndione with 2,4,5,6-tetraaminopyrimidine sulfate, following the (piq)2(dmdppz)](PF6) (IC50 = 0.6 \u00b1 0.2 \u03bcM, dmdppz = 5,8-dimethox\u00ad\nmethod delineated in existing literature [24]. Complexes 4a and 4b were ylpyrido[3,2-a:2\u2032,3\u2032-c]phenazine) [31]. Generally, upon light irradia\u00ad\nsynthesized through reaction of [Ir(bzq)2Cl]2 or [Ir(piq)2Cl]2 with PPD. tion, the compounds elevate reactive oxygen species (ROS) and 1O2,\nCharacterization of these complexes was conducted using HRMS, 1H while ROS and 1O2 are able to quickly act with adjacent biomolecules,\nNMR, and 13C NMR spectra. HRMS spectra revealed molecular weights further disturbing the functions of normal cell, finally triggering cell\n\n 4\n\fS. Tian et al. Journal of Inorganic Biochemistry 256 (2024) 112549\n\n\n\n\nFig. 1. (a) Cell viability of SGC-7901 cells treated with different concentrations of 4a (I) and 4b (II) (without light), 4a (III) and 4b (IV) upon light irradiation, 4alip\n(V) and 4blip (VI) without light, 4alip (VII) and 4blip (VIII) upon light irradiation, (b) photocatalytic oxidation of DPBF (40 \u03bcM) under white light in the presence of\n10 \u03bcM of 4a and 4b in DMSO. (c) Frontier orbital energy levels and electron cloud distribution of 4a and 4b.\nNote: In all the following cell experiments, 4a (light), 4b (light), 4alip (light) and 4blip (light) stand for a 4-h treatment of SGC-7901 cells with 4a, 4b, 4alip, 4blip,\nfollowed by 30 min of light irradiation (65 w LED lamp, light dose of 7.03 J\u22c5cm\u2212 2, \u03bb = 460 nm).\n\n\n 5\n\fS. Tian et al. Journal of Inorganic Biochemistry 256 (2024) 112549\n\n\nTable 1\nIC50 (\u03bcM) value of complexes and their liposomes with (without) light irradiation toward selected cancer cells for 48 h.\n complex SGC-7901 A549 LO2\n\n IC50dark IC50light PI IC50 (dark) IC50 (light) PI IC50 (dark) IC50 (light) PI\n\n 4a > 100 18.0 \u00b1 1.6 > 100 84.1 \u00b1 2.1 > 100 > 100\n 4b 11.3 \u00b1 0.2 3.2 \u00b1 0.2 3.5 10.5 \u00b1 1.2 9.1 \u00b1 1.5 1.2 52.9 \u00b1 1.8 29.2 \u00b1 4.1 1.8\n 4alip 10.5 \u00b1 0.5 4.1 \u00b1 0.2 2.6 34.1 \u00b1 2.7 8.7 \u00b1 0.2 3.9 54.9 \u00b1 0.8 8.9 \u00b1 0.4 6.2\n 4blip 5.0 \u00b1 0.1 2.4 \u00b1 0.2 2.1 9.7 \u00b1 1.2 4.3 \u00b1 0.1 2.3 29.4 \u00b1 2.6 4.9 \u00b1 0.3 6.0\n Cisplatin 3.4 \u00b1 0.5 5.8 \u00b1 0.4 17.8 \u00b1 2.5\n\n\n\ndemise [32,33]. Based on the literature [17,34,35], we consider that 4a which is line with the trend of antiproliferative activity of these com\u00ad\nand 4b can validly activate 3O2 to produce 1O2, which makes them plexes. The 4alip and 4blip exhibited higher cytotoxic activity against\nhighly validly photosensitizers in photodynamic therapy (PDT). To SGC-7901 cells than against A549 cells, therefore, SGC-7901 cells were\nconfirm the production of 1O2 under light irradiation, we used 1,3- selected for subsequent experimental analyses.\ndiphenylisobenzofuran (DPBF) as a 1O2 scavenger, see from Fig. 1b, To clarify the difference of 4a, 4alip, 4b, 4blip in the cytotoxicity\nwith increase of light irradiation time, absorbance of DPBF at 417 nm activity, we used flow cytometry to determine the cellular uptake, as\nreduced, indicating an increase of 1O2. The quantum yields of 4a and 4b shown Fig. S6 (SI), 4a shows low green fluorescence, the green fluo\u00ad\nwere detected with [Ru(bpy)3]2+ as a reference (\u03a6 = 0.81, methanol) rescence follows the order of 4blip > 4alip > 4b > 4a, therefore, 4a\n[36]. The quantum yields of 4a and 4b was calculated to be 0.51 and shows the lowest cytotoxic activity. In addition, inductively coupled\n0.66, which indicates that 4b can generate 1O2 more efficiently than 4a. plasma mass spectrometry (ICP-MS) was used to quantitatively measure\nNotably, the anticancer efficacy of 4b is higher than those of 4a against the uptaken amounts of 4b and 4blip, after 4 h co-incubation of SGC-\nthe selected cancer cells, this is line with those quantum yields. Addi\u00ad 7901 cells with 4b (10.0 \u03bcM) and 4blip (10.0 \u03bcM), the cellular\ntionally, we also calculated the energy gap between the highest occupied uptaken amounts for 4b and 4blip are 72.47 and 79.74 ng/106 cells.\norbital (HOMO) and the lowest unoccupied orbital (LUMO) by DFT/ Hence, the uptaken amount of 4blip is more than that of 4b, this is\nB3LYP/6-31G(d, p)(C, H, N)/SDD(Ir). The size of the band gap be\u00ad consistent with the cytotoxic activity.\ntween LUMO-HOMO of a molecule is closely related to its chemical PI: phototoxicity index = IC50(dark)/IC50(light)\nactivity. A larger HOMO-LUMO band gap will result in difficult defor\u00ad\nmation of the electron cloud, lower reactivity, and poorer anticancer 3.3. Cell colony and cell cycle distribution studies\nactivity [37,38]. As shown in Fig. 1c, the \u0394E values for 4a and 4b are\n2.6435 eV and 2.5189 eV, The \u0394E value for 4a is larger than that of 4b, Cell colony formation assays are instrumental in further evaluating\nindicating that the cytotoxic activity of 4a is lower than those of 4b, the efficacy of complexes on inhibiting cell growth. Metastasis, a\n\n\n\n\nFig. 2. (a) cell colony, (b) cell cycle arrest, (c) and (d) expression of cycle-related proteins while a 24 h treatment of SGC-7901 cells with IC50 concentration of 4a, 4b,\n4alip, 4blip upon irradiation or without.\n\n 6\n\fS. Tian et al. Journal of Inorganic Biochemistry 256 (2024) 112549\n\n\nprimary cause of cancer mortality, involves the diffusion of cancer cells This data suggests that these complexes inhibit cell proliferation pri\u00ad\nfrom the initial site to other organs, posing a significant challenge in marily during the S phase, particularly upon light irradiation.\ncancer therapy [39]. Fig. 2a indicates that complexes 4a and 4b, in their Cyclin A2, a pivotal protein in the cell cycle, is crucial for regulating\nbasic form, exhibit limited efficacy in inhibiting cell proliferation. the G1/S and G2/M phases, playing an important role in DNA replica\u00ad\nHowever, their effectiveness is markedly enhanced upon light irradia\u00ad tion, transcription, and tumor progression. To unravel the underlying\ntion or encapsulation in liposomes, as both 4a, 4b and their liposomal mechanisms of these complexes and their liposomal forms in hindering\nforms 4alip, 4blip show substantial efficacy in inhibiting cell cell growth, the study examined the expression of cyclin A2, p53, and\nproliferation. CDK4. Fig. 2c and d show that 4a, 4b, 4alip, and 4blip downregulated\n The cell cycle, a critical process for cell division and proliferation, cyclin A2 and CDK4 expression while upregulating p53 protein levels.\noffers insights into cancer mechanisms and treatment strategies. As Therefore, these complexes and their liposomal forms inhibit cell growth\ndepicted in Fig. 2b, complex 4a (II) induced minor changes in SGC-7901 during S phase through modulating cyclin A2, p53, and CDK4\ncell distribution across G0/G1, S, and G2/M phases. In contrast, when expression.\nSGC-7901 cells (I) were incubated for 24 h with IC50 concentrations of\n4b (III), 4a (IV, light), 4b (V, light), 4alip (VI), 4blip (VII), 4alip (VIII,\nlight), and 4blip (IX, light), notable increases were observed in the S 3.4. Superoxide anion, mitochondrial membrane potential, cytochrome c,\nphase: 6.2% for 4b, 11.6% for 4a (light), 10.4% for 4b (light), 9.1% for apoptosis\n4alip, 7.7% for 4blip, 14.2% for 4alip (light), and 12.3% for 4blip (light).\n ROS play an important role in cancer cell death and apoptosis, it\n\n\n\n\nFig. 3. (a) Detection of the change of mitochondrial membrane potential, (b) release of cytochrome c, (c) apoptosis (Q2, Q3, Q4 representing late, early and living\ncells), SGC-7901 cells (I) treated with 4a (II), 4a (light) (IV), 4alip (VI) 4alip (light, VIII), 4b (III), 4b (light) (V), 4blip (VII), and 4blip (light) (IX). (d and f) expression\nof apoptosis-related proteins, (e and g) gray values.\n\n 7\n\fS. Tian et al. Journal of Inorganic Biochemistry 256 (2024) 112549\n\n\n\n\n Fig. 3. (continued).\n\n\nincludes hydrogen peroxide, superoxide anion, hydroxyl radicals, etc. In Apoptosis is a type of programmed cell demise [42]. The mito\u00ad\nthis article, superoxide anion O\u2022\u2013 2 levels were measured with dihy\u00ad chondrial dysfunction is usually accompanied by the apoptosis. The\ndroethidium (DHE) as fluorescence probe. In the presence of O\u2022\u2013 2 , DHE ability of the complexes inducing cell apoptosis was investigated. See\ncan be converted to ethidium, which binds RNA or DNA to produce red from Fig. 3c, 4a (II), 4a (light) (IV) and 4alip (VI) exhibit low apoptotic\nfluorescence. The red fluorescence intensity is proportional to the effect, 4b (III), 4b (light) (V), 4blip (VII), 4alip (light) (VIII) and 4blip\nintracellular O\u2022\u2013\n 2 levels. As shown in Fig. S7 (SI), in the control, almost no (light) (IX) show high apoptotic efficacy with an apoptotic percentage of\nred fluorescence was observed. After SGC-7901 cells (a) were incubated 12.41, 16.81, 14.8, 7.44 and 17.34% (early + late apoptosis), respec\u00ad\nwith 2 \u00d7 IC50 concentration of 4alip (b), 4alip (light, c), 4b (d), 4blip (e) tively. These results demonstrate that complex 4b (light), 4blip (light)\nand 4blip (light, f) for 24 h, distinct red fluorescence was discovered, reveal high anticancer activity.\nindicating that the complexes can enhance intracellular O\u2022\u2013 2 levels. The mechanism of apoptosis involves caspases, a family of proteases\n Mitochondria, beyond their role in energy production, are implicated [43]. Western blot analysis, depicted in Fig. 3d-g, revealed that the\nin cell differentiation, signaling, apoptosis, and cell growth and cycle complexes and their liposomes modulate apoptosis by altering Bcl-2\nregulation. They serve as integration points for signals from extrinsic family protein expression. Specifically, they downregulated the\nand intrinsic apoptotic pathways [40,41]. Interaction of compounds expression of p62, caspase 3, and Bcl-2, upregulated the expression of\nwith mitochondria can lead to changes in mitochondrial membrane Bax. This indicates that the complexes and their liposomal forms induce\npotential. This was investigated using JC-1, a fluorescent probe emitting apoptosis through regulating Bcl-2 family protein.\nred or green fluorescence under high or low mitochondrial membrane\npotential (MMP). Fig. 3a demonstrates that 4a and 4b exhibited limited\ncapacity to alter MMP, as indicated by the balance of red and green 3.5. Ferroptosis studies\nfluorescence points. However, a 24-h treatment of SGC-7901 cells with\n4a (light), 4b (light), 4alip, 4blip, 4alip (light) and 4blip (light) resulted As a marker of lipid peroxidation and oxidative stress, malondial\u00ad\nin decreased red and increased green fluorescence, indicating a decline dehyde (MDA) is crucial for assessing cellular oxidative stress and\nin MMP. related disease risks. Fig. 4a shows that complex 4a has a limited ability\n Mitochondrial damage, a consequence of these complex interactions, to increase MDA levels, whereas 4b, 4a (light), 4b (light), 4alip, 4blip,\ncan lead to a release of cytochrome C (cyto C) into the cytosol, an 4alip (light) and 4blip (light) can effectively enhance intracellular MDA\nimportant apoptosis inducer. Fig. 3b shows that control and groups content. This indicates their potential in inducing oxidative stress within\ntreated with 4a and 4b had weak green fluorescence. In contrast, the cells.\ntreatment of SGC-7901 cells with IC50 concentrations of 4a (light), 4b Glutathione (GSH), a key intracellular antioxidant, plays a vital role\n(light), 4alip, 4blip, 4alip (light) and 4blip (light) exhibited increased in antioxidation, protein sulfhydryl protection, and amino acid trans\u00ad\ngreen fluorescence, which indicates an occurrence of cyto C release. port. The GSH to oxidized glutathione (GSSG) ratio is an essential in\u00ad\n dicator of cellular redox status. As demonstrated in Fig. 4b, treating SGC-\n\n 8\n\fS. Tian et al. Journal of Inorganic Biochemistry 256 (2024) 112549\n\n\n\n\n Fig. 3. (continued).\n\n\n7901 cells with 4a (light), 4b (light), 4alip, 4blip, and 4alip (light), 4blip Furthermore, 4a and 4b encapsulated into liposomes as 4alip and 4blip,\n(light) significantly reduces intracellular GSH content and the GSH/ under irradiation, significantly inhibited SGC-7901 cell proliferation.\nGSSG ratio, as seen in Fig. 4c. This suggests a decrease in cellular anti\u00ad Cell colony and wound healing assays confirmed the heightened efficacy\noxidant capacity, following the order: 4blip (light) > 4b (light) > 4alip of these complexes in inhibiting cell proliferation upon irradiation. Cell\n(light) > 4alip > 4a (light) > 4b > 4a. These findings indicate that 4alip cycle analysis indicated that 4alip and 4blip arrest cell growth in the S\nand 4blip validly hinder GSH production, promote lipid peroxidation, phase. Additionally, both the complexes and their liposome-loaded\nand consequently induce oxidative damage. forms reduced mitochondrial membrane potential and triggered cyto\u00ad\n In the context of ferroptosis, an iron-dependent form of programmed chrome c release, activated caspase 3, led to apoptosis by regulating and\ncell demise different from necrosis and apoptosis, the activity of GPX4 controlling the expression of Bcl-2 family proteins. Irradiation further\n(glutathione peroxidase 4) is an important indicator. Fig. 4d and e show reduced glutathione (GSH) levels and increased malondialdehyde\nthat 4a, 4b, 4alip, and 4blip downregulate GPX4 protein expression, (MDA) and lipid peroxidation, indicating ferroptosis induction by 4a,\nsuggesting their role in inducing ferroptosis. 4b, 4alip, and 4blip. Overall, this research elucidates that the complexes\n Lipid peroxidation, a central mechanism of ferroptosis, was exam\u00ad and their liposome-incorporated forms induce cell death via both\nined using 4,4-difluoro-3a,4a-diaza-sindacene (C11-BODIPY). In the apoptosis and ferroptosis pathways, providing valuable insights for\ncontrol and groups treated with 4a and 4b, weak green fluorescence was finding newly potential anticancer candidate drugs.\nobserved, indicating minimal lipid peroxidation (Fig. 4f). However,\nupon 24-h incubation of SGC-7901 cells with 4a (light), 4b (light), 4alip, Author statement\n4blip, and 4alip (light), 4blip (light), an increment and a reduction in\ngreen and red fluorescence were noted, respectively. This shift signifies a We state that the manuscript has been finished by all authors listed in\ntransition from non-oxidized to oxidized lipids, indicating pronounced the manuscript. The all data are original and real. We agree to be\nlipid peroxidation. Additionally, we also measured the cell viability accountable for all aspects of the work to ensure that questions related to\ninduced by 4alip (light) and 4blip (light) in the presence of Fer-1, as the accuracy or integrity of any part of the work are appropriately\nlisted in Table 2, in the presence of Fer-1, the cell viability increased investigated and resolved.\ncompared with 4alip (light) or 4blip (light) alone. These results collec\u00ad All authors have read the manuscript and approved the manuscript\ntively suggest that 4a, 4b, 4alip, and 4blip can induce ferroptosis. to be submitted to JIB.\n\n4. Conclusions\n CRediT authorship contribution statement\n This study successfully prepared complexes 4a and 4b. Complex 4a\n Shuang Tian: Investigation, Methodology, Writing - original draft.\ndemonstrated negligible cytotoxic activity, while 4b exhibited moderate\n Qianying Nie: Data curation, Software. Haomin Chen: Formal anal\u00ad\ncytotoxicity against selected cancer cells. Notably, upon irradiation, 4a\n ysis. Lijuan Liang: Data curation, Methodology. Huiyan Hu: Formal\ndisplayed moderate and 4b showed high anticancer efficacy.\n analysis. Shuanghui Tang: Software. Jiawan Yang: Data curation.\n\n 9\n\fS. Tian et al. Journal of Inorganic Biochemistry 256 (2024) 112549\n\n\n\n\nFig. 4. Ferroptosis studies (a) MDA content, (b) GSH determination, (c) GSH/GSSG ratio, (d) and (e) expression of GPX4, (f) Assay of lipid peroxidation with C11-\nBODIPY-C11 after SGC-7901 cells were incubated with IC50 concentration of 4a, 4b, 4alip and 4blip upon irradiation or not.\n\n\n Yunjun Liu: Supervision, Project administration, Conceptualization.\nTable 2\n Hui Yin: Conceptualization, Writing \u2013 review & editing.\nCell viability caused by 4alip (light) and 4blip (light) in the presence of Fer-1.\n Control 4alip (light) 4blip (light)\n Declaration of competing interest\n Cell viability (%) (\u2212 ) Fer-1 100 (\u00b1 4.5) 76.2 (\u00b1 3.1) 66.8 (\u00b1 2.4)\n Cell viability (%) (+) Fer-1 99.8 (\u00b1 3.8) 94.1 (\u00b1 3.7) 87.6 (\u00b1 2.7)\n The authors declare no competing interest exists.\n\n Data availability\n\n Data will be made available on request.\n\n 10\n\fS. Tian et al. Journal of Inorganic Biochemistry 256 (2024) 112549\n\n\nAcknowledgements [19] Y.C. Chen, Y.Y. Gu, H.Y. Hu, H.M. Liu, W.L. Li, C.X. Huang, J. Chen, L.J. Liang, Y.\n J. Liu, Design, synthesis and biological evaluation of liposome entrapped iridium\n (III) complexes toward SGC-7901 cells, J. Inorg. Biochem. 241 (2023) 112134.\n We are grateful for National Natural Science Foundation of China [20] J. Chen, W.L. Li, G.C. Li, X.M. Liu, C.X. Huang, H. Nie, L.J. Liang, Y. Wang, Y.J.,\n(No 21877018, 82171700). Liu, targeted liposomes encapsulated iridium(III) compound greatly enhance\n anticancer efficacy and induce cell death via ferroptosis on HepG2 cells, Eur. J.\n Med. Chem. 265 (2024) 116078.\nAppendix A. Supplementary data [21] C.X. Huang, Y.H. Yuan, G.C. Li, S. Tian, H.Y. Hu, J. Chen, L.J. Liang, Y. Wang, Y.\n J. Liu, Mitochondria-targeted iridium(III) complexes encapsulated in liposome\n Supplementary data to this article can be found online at https://doi. induce cell death through ferroptosis and gasdermin-mediated pyroptosis, Eur. J.\n Med. 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